Reagents and methods for treating headache and migraine pain and co-morbidities thereof

Abstract

Methods for using gene expression changes and mutations in neural organoids to identify and assess effectiveness of treatment methodologies and effective pharmaceutical compositions for treating chronic and neuropathic pain including headache and migraines are disclosed.

Description

REAGENTS AND METHODS FOR TREATING HEADACHE AND MIGRAINE PAIN AND CO-MORBIDITIES THEREOF CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims priority to U.S. Provisional Application No. 63 / 665,241, filed June 27, 2024, which is incorporated herein by reference in its entirety. FIELD OF THE INVENTION

[0002] This invention relates to production and use of human stem cell-derived neural organoids to predict onset risk of headaches including migraines and also when comorbid with dementias in an individual. In particular, the invention provides neural organoid reagents produced from an individual’s induced pluripotent stem cells (iPSCs) for identifying predictive biomarkers and developmental and pathogenic gene expression patterns and dysregulation thereof in disease onset and progression, methods for diagnosing prospectively and concurrently risk of development of headaches including migraines and when comorbid with dementias, and methods for identifying individualized, effective treatments based on such diagnostic methods. The invention also provides reagents and methods for identifying, testing, and validating therapeutic modalities, including chemical and biologic molecules for use as drugs for ameliorating or curing headaches including migraines and when comorbid with dementias. BACKGROUND OF THE INVENTION

[0003] The human brain, and diseases associated with it, has been the object of investigation and study by scientists for decades. Throughout this time, neurobiologists have attempted to increase their understanding of the brain’s capabilities and functions. Neuroscience has typically relied on the experimental manipulation of living brains or tissue samples, but scientific progress has been limited by a number of factors. For ethical and practical reasons, obtaining human brain tissue is difficult while most invasive techniques are impossible to use on live humans. Animal experiments are expensive and time-consuming and many animal experiments are conducted in rodents, which have a brain structure and development that vary greatly from humans. Results obtained in animals must be 1verified in long and expensive human clinical trials and much of the time the animal disease models are not fully representative of disease pathology in the human brain.

[0004] Improved experimental models of the human brain are urgently required to understand disease mechanisms and test potential therapeutics. The ability to detect and diagnose various neurological diseases in their early stages could prove critical in the effective management of such diseases, both at times before disease symptoms appear and thereafter. Neuropathology is a frequently used diagnostic method; however, neuropathology is usually based on autopsy results. Molecular diagnostics in theory can provide a basis for early detection and a risk of early onset of neurological disease. However, molecular diagnostic methods in neurological diseases are limited in accuracy, specificity, and sensitivity. Therefore, there is a need in the art for non-invasive, patient specific molecular diagnostic methods to be developed.

[0005] Consistent with this need, neural organoids hold significant promise for studying neurological diseases and disorders. Neural organoids are developed from cell lineages that have been first been induced to become pluripotent stem cells. Thus, the neural organoid is patient specific. Importantly, such models provide a method for studying neurological diseases and disorders that can overcome previous limitations. Thus, there is a need in the art to develop individual-specific reagents and methods based on predictive biomarkers for diagnosing current and future risk of neurological disease.

[0006] There is no known cure for headache migraine pain (HMP) but treatments to manage its symptoms exist. HMP is a complex headache disorder that affects up to 30% of the population (Lucas, 2021) and a third of those afflicted with migraine experience aura or language disturbance preceding the migraine headache (Lucas, 2021). HMP may be a risk factor for dementias, particularly vascular dementia (Wei Jiang et al., Aging Clin Exp Res 34, 1237–1246, 2022). Genetics is a major contributor to HMP, but environmental factors also play a role (Charlene Bron, C. et al., 2021; Hautakangas, H. et al., 2023).

[0007] Pain-relieving medications that block pain pathways in the brain include Aspirin, Ibuprofen, sumatriptan and rizatriptan. Preventive medications include beta blockers, antidepressants, and anticonvulsants (Alorfi NM, 2023). VYEPTI is the only FDA-approved preventive treatment for migraine (American Headache Society).VYEPTI binds to CGRP and prevents it from activating its receptors, which may play a role in migraine onset.

[0008] Current approaches to identify therapeutics for HMP fail to address the underlying genetic causes that predispose one to HMP and associated comorbidities including dementia, cardiovascular disorders (stroke, myocardial infarction), psychiatric disorders (anxiety, depression, panic disorder), epilepsy, sleep disorders, and inflammatory conditions (Gupta and Gaurkar, 2022). Current approaches fail to account for convergent or divergent upstream drivers for the emergence of subtypes of HMP and / or their severity of clinical manifestations. These drivers include neurodegeneration (Biscetti et al., 2023) and hormonal factors.

[0009] Use of rodent model systems with some behavioral traits that resemble those in migraines largely fail to reflect human migraine etiology (Gupta and Gaurkar, 2022), its heterogeneity, and do not account for known gender differences. For example, nitroglycerin is used as a trigger in rodent models of migraine (Gupta and Gaurkar, 2022). However, the limitations of rodent models become apparent because Familial hemiplegic migraine type 1 (FHM-1), a dominantly inherited subtype of migraine with aura and transient hemiplegia associated with mutations in the CACNA1A gene shows no hypersensitivity to nitric oxide (Hansen et al., 2008). Thus, even though FHM-1 shares many clinical symptoms similar to common types of migraine, the pathophysiological pathways underlying migraine headache in FHM- 1 appears different from the common types of migraine (Hansen et al., 2008).

[0010] Direct electrical stimulation of trigeminal neurons in vivo for activation of the trigeminovascular system has been used as a model of migraine-related pain but involves invasive cannula or electrode placement with adverse effects on cerebrovascular physiology. (Gupta and Gaurkar, 2022).

[0011] Structural genomics (GWAS) have identified genetic variants (Charlene Bron, C. et al., 2021; Hautakangas, H. et al., 2023), but they only incompletely identify the genomic contributors to migraines because of intrinsic limits of such strategies (Amorim et al., 2017) for epistasis or effects of 3D chromatin disruptions of topologically associating domain (Boney and Cavalli, 2016; Anania and Lupiáñe, 2020). This is best illustrated for HMP with comorbid Alzheimer’s disease. GWAS studies have identified only 74 genes with variants for Alzheimer’s disease (Bellenguez et al., 2022) but AD postmortem brain transcriptomics show >2,000 differentially expressed genes (DEGs) (Annese et al., 2018). GWAS studies also failto associate the variants with the etiology of HMP subtypes or identify their upstream drivers.

[0012] In view of the state of the art there is a need to identify predictive biomarkers and developmental and pathogenic gene expression patterns and dysregulation thereof in disease onset and progression specific for an individual, for methods for diagnosing prospectively and concurrently risk of development of headaches including migraines and when comorbid with dementias in such individuals, and for methods for identifying individualized, effective treatments based on such diagnostic methods. SUMMARY OF THE INVENTION

[0013] This invention provides reagents and methods for identifying predictive biomarkers and developmental and pathogenic gene expression patterns and dysregulation thereof in disease onset and progression specific for individuals, methods for diagnosing prospectively and concurrently risk of development of headaches including migraines and when comorbid with dementias in such individuals, and methods for identifying individualized, effective treatments based on such diagnostic methods and the treatments identified thereby.

[0014] The present invention provides neural reagents and methods to identify predictive biomarker gene expression patterns. The present in invention describes methods for diagnosing current and future risk of Headache and Migraine Pain (HMP) and comorbidity onset and identification of effective treatment modalities with an understanding of the predictive diagnostic results.

[0015] In a first aspect, the invention provides a method for reducing or ameliorating disease severity in a patient with headaches including migraines and when comorbid with dementias comprising administering to the individual a therapeutically effective amount of a therapeutic agent capable of reducing or correcting in vitro differentially expressed genes, dysregulated protein expression / function, or dysregulated metabolite production in a neural organoid derived from the patient with HMP compared to a neural organoid derived from a healthy patient and reducing or ameliorating disease severity in the HMP patient thereby.

[0016] In a second aspect, the invention provides a pharmaceutical composition comprising a therapeutically effective amount of a therapeutic agent capable ofreducing or correcting in vitro differentially expressed genes, dysregulated protein expression / function, or dysregulated metabolite production in a neural organoid derived from the patient with HMP compared to a neural organoid derived from a healthy patient, and a pharmaceutically acceptable carrier; wherein the pharmaceutical composition is capable of reducing or ameliorating disease severity in the patient.

[0017] In some embodiments, the differentially expressed genes for which the therapeutic agent is capable of reducing or correcting are set forth in Tables 3-6. In some embodiments, the neural organoid is obtained from an induced pluripotent stem cell that is reprogrammed from a cell sample derived from the patient with HMP. In some embodiments, the neural organoid is procured from minutes to hours up to 15 weeks post inducement.

[0018] In some embodiments, the patient with HMP shows comorbidities listed in Tables 3 and 4. The comorbidities are also associated with the differentially expressed genes listed in Tables 3 and 4.

[0019] In a third aspect, the invention provides a method comprising: a) procuring one or a plurality of cell samples from a patient with HMP or at risk for developing HMP, comprising one or a plurality of cell types; b) reprogramming the one or the plurality of cell samples to produce one or a plurality of induced pluripotent stem cell samples; c) treating the one or the plurality of induced pluripotent stem cell samples to obtain a neural organoid; d) collecting a biological sample from the neural organoid; e) detecting differentially expressed genes, dysregulated protein expression / function, or dysregulated metabolite production in the neural organoid sample compared to a neural organoid sample obtained from a healthy patient; and f) administering one or more therapeutic agents capable of reducing or correcting the differentially expressed genes to the patient with HMP or at risk for developing HMP, wherein the method is capable of treating HMP or preventing development of HMP.

[0020] In some embodiments, the cell types are fibroblasts, buccal cavity cells, or peripheral blood monocytes.

[0021] In certain aspect, step (e) further comprises identifying differentially expressed genes, dysregulated protein expression / function, or dysregulated metabolite production in the neural organoid from the patient with HMP or at risk for developing HMP that are known to associate with HMP, or with clinical symptoms or comorbidities of HMP. The method also further comprises administering one or moretherapeutic agents capable of reducing or correcting the identified differentially expressed genes, dysregulated protein expression / function, or dysregulated metabolite production to the patient with HMP or at risk for developing HMP.

[0022] In some embodiments, the therapeutic agent is a RNA-based therapeutics. The RNA-based therapeutics is antisense oligonucleotide (ASO), Adeno-Associated Virus delivery of small interfering RNA (AAV-RNA), or micro RNA (miRNA). In some embodiments, the differentially expressed genes for which the RNA-based therapeutics is capable of reducing or correcting are set forth in Tables 3-6.

[0023] In some embodiments, at least one cell sample reprogrammed to the induced pluripotent stem cell is a fibroblast. In some embodiments, the neural organoid is about twelve weeks post-inducement and comprises the encoded structures and cell types of the retina, cortex, midbrain, hindbrain, brain stem, and spinal cord. In some embodiments, the neural organoid sample is procured after about one week post inducement, four-weeks post inducement, and / or 12 weeks post inducement. In some embodiments, the neural organoid sample is procured from structures of the neural organoid that mimic the structures in utero at about 5 weeks.

[0024] In a fourth aspect, the invention provides a method comprising: a) collecting a biological sample from a patient with HMP or at risk for developing HMP; b) quantifying production of metabolites from the biological sample that are significantly altered compared to a biological sample collected from a healthy patient; and c) administering one or more therapeutic agents capable of reducing or correcting the altered metabolites to the patient with HMP or at risk for developing HMP, wherein the method is capable of treating HMP or preventing development of HMP. In some embodiments, the biological sample is urine, plasma, or cerebrospinal fluid.

[0025] These and other features and advantages of the present invention will be more fully understood from the following detailed description taken together with the accompanying claims. It is noted that the scope of the claims is defined by the recitations therein and not by the specific discussion of features and advantages set forth in the present description.BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Fig 1A is a diagram setting forth the rationale for producing a neural organoid platform (NNOP) as used herein shown in a micrograph of a 4X dark field image of a NNOP.

[0027] FIG.1B is a photomicrograph of an NNOP illustrated by comparison with a diagram of a 5-week-old fetus and corresponding anatomical structures.

[0028] FIG.2 is a diagram illustrating the strategy for identifying gene expression and metabolic profiles in NNOP related to HMP and when comorbid with ADRD for use in drug development.

[0029] FIG.3 shows that Normal-NNOP express all brain regions and all cell types including microglia. It also Whole Organoid Bright Field Imaging showing the anatomy of the whole organoid with corresponding brain structures.

[0030] FIG.4 shows that Normal-NNOP express all brain regions and all cell types including microglia. It also shows 3D Clarity Immuno-fluorescent staining and Confocal Imaging showing expression of the biomarker protein of the indicated region on the neural organoid.

[0031] FIG.5A shows results of transcriptomic data produced using NNOP from ADRD-HMP donor. Each dot is an RNA expression level of each gene. Each X and Y axis shows Reads Per Kilo base per Million reads (RPKM) which represents a gene expression level of each gene from two randomly selected organoids from hundreds of organoids. Independent NNOP sample transcriptomic data are shown at different time points in development. Plots represent high data reproducibility for ~15,000 genes expressed at 12 weeks in culture (W12) with a variance of <0.95 in independent replicates.

[0032] FIG.5B shows results of transcriptomic data produced using NNOP from ADRD-HMP donor in the presence of CGS-21680 and PDE4 inhibitor, Roflumilast. Each dot is an RNA expression level of each gene. Each X and Y axis shows Reads Per Kilo base per Million reads (RPKM) which represents a gene expression level of each gene from two randomly selected organoids from hundreds of organoids. Independent NNOP sample transcriptomic data are shown at different time points in development. Plots represent high data reproducibility for ~15,000 genes expressed at 12 weeks in culture (W12) with a variance of <0.95 in independent replicates.

[0033] FIG.5C shows results of transcriptomic data produced using healthy patients (normal NNOP). Each dot is an RNA expression level of each gene. Each X and Y axis shows Reads Per Kilo base per Million reads (RPKM) which represents a gene expression level of each gene from two randomly selected organoids from hundreds of organoids. Independent NNOP sample transcriptomic data are shown at different time points in development. Plots represent high data reproducibility for ~15,000 genes expressed at 12 weeks in culture (W12) with a variance of <0.95 in independent replicates.

[0034] FIG.6 shows metabolomics signatures induced by mTOR activator and ADORA2A receptor agonist in combination with a PDE4 inhibitor in ADRD-MHP- NNOP derived from to independent donors performed using 1H-NMR-based metabolomics.

[0035] FIGs.7A and 7B show quantitation of metabolites in NNOP as measured by 1H-NMR. Preliminary data using Normal-NNOP shows physiological sensitivity (peak area replicability): independent sample 1 and sample 2 of Normal-NNOP show reproducibility and feasibility of metabolomics.

[0036] FIG.8 shows quantitation of metabolites in NNOP as measured by 1H- NMR from two randomly selected NNOP samples derived from the same healthy patient. DETAILED DESCRIPTION

[0037] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of ordinary skill in the art. The following references provide one of skill with a general definition of many of the terms used in this disclosure: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed.1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991). These references are intended to be exemplary and illustrative and not limiting as to the source of information known to the worker of ordinary skill in this art. As used herein, the following terms have the meanings ascribed to them below, unless specified otherwise.

[0038] It is noted here that as used in this specification and the appended claims, the singular forms ”a,” ”an,” and ”the” also include plural reference, unless the context clarity dictates otherwise.

[0039] The term “about” or “approximately” means within 25%, such as within 20% (or 5% or less) of a given value or range.

[0040] As used herein, the terms “or” and “and / or” are utilized to describe multiple components in combination or exclusive of one another. For example, “x, y, and / or z” can refer to “x” alone, “y” alone, “z” alone, “x, y, and z,” “(x and y) or z,” “x or (y and z),” or “x or y or z.”

[0041] It is noted that terms like “preferably,” “commonly,” and “typically” are not utilized herein to limit the scope of the claimed invention or to imply that certain features are critical, essential, or even important to the structure or function of the claimed invention. Rather, these terms are merely intended to highlight alternative or additional features that can or cannot be utilized in a particular embodiment of the present invention.

[0042] For the purposes of describing and defining the present invention it is noted that the term “substantially” is utilized herein to represent the inherent degree of uncertainty that can be attributed to any quantitative comparison, value, measurement, or other representation. The term “substantially” is also utilized herein to represent the degree by which a quantitative representation can vary from a stated reference without resulting in a change in the basic function of the subject matter at issue.

[0043] A "neural organoid” means a non-naturally occurring three-dimensional organized cell mass that is cultured in vitro from a human induced pluripotent stem cell (iPSC) and develops analogously to the human nervous system in terms of neural marker expression and structure. Further a neural organoid has two or more regions. The first region expresses cortical or retinal marker or markers. The remaining regions each express markers of the brain stem, cerebellum, and / or spinal cord.

[0044] Neural markers are any protein or polynucleotide expressed consistent with a cell lineage. By "neural marker" it is meant any protein or polynucleotide, the expression of which is associated with a neural cell developmental fate. Exemplaryneural markers include markers associated with the hindbrain, midbrain, forebrain, or spinal cord. One skilled in the art will understand that neural markers are representative of the cerebrum, cerebellum, midbrain, and brainstem regions. Exemplary brain structures that express neural markers include the cortex, hypothalamus, thalamus, retina, medulla, pons, and lateral ventricles. Further, one skilled in the art will recognize that within the brain regions and structures, granular neurons, dopaminergic neurons, GABAergic neurons, cholinergic neurons, glutamatergic neurons, serotonergic neurons, dendrites, axons, neurons, neuronal, cilia, Purkinje fibers, pyramidal cells, spindle cells, express neuronal markers. One skilled in the art will recognize that this list is not exhaustive nor all encompassing and that neural markers are found throughout the central nervous system including other brain regions, structures, and cell types.

[0045] Exemplary spinal cord, cerebellum, hindbrain, midbrain, blood brain barrier, cortex, microglia, retina, and choroid plexus markers include but are not limited to those genes described in FIG.3. Exemplary markers of dopaminergic neurons include but are not limited to tyrosine hydroxylase, vesicular monoamine transporter 2 (VMAT2), dopamine active transporter (DAT) and Dopamine receptor D2 (D2R). Exemplary granular neuron markers include, but are not limited to SOX2, NeuroD1, DCX, EMX2, FOXG1l, and PROX1. Exemplary brain stem markers include, but are not limited to FGF8, INSM1, GATA2, ASCL l, GATA3. Exemplary GABAergic markers include but are not limited to NKCCl or KCC2. Exemplary astrocytic markers include but are not limited to GFAP and those genes in Table 3. Exemplary oligodendrocytic markers include but are not limited to those genes in Table 3. One skilled in the art will understand that the list is exemplary and that additional biomarkers exist.

[0046] Diagnostic or informative alteration or change in a biomarker is meant as an increase or decrease in the expression levels or activity of a gene or gene product as detected by conventional methods known in the art such as those described herein. As used herein, such an alteration can include a 10% change in expression levels, a 25% change, a 40% change, or even a 50% or greater change in expression levels.

[0047] A mutation is meant to include a change in one or more nucleotides in a nucleotide sequence, particularly one that changes an amino acid residue in thegene product. The change may or may not have an impact (negative or positive) on activity of the gene.

[0048] The term "differentially expressed genes” (DEGs) refer to statistically significant changes in gene expression at RNA level in a tested sample compared to a control sample.

[0049] As set forth herein gene expression markers for diseases and disabilities in an individual as a consequence of Headache and Migraine Pain (HMP) are set forth in Tables 3-6 herein. Neural Organoids for Studying Headache and Migraine Pain (HMP)

[0050] Neural organoids are generated in vitro from patient tissue samples. Neural organoids were previously disclosed in U.S. Patent No.11,345,890, incorporated herein in its entirety. A variety of tissues can be used to produce neural organoids including skin cells, hematopoietic cells, or peripheral blood mononuclear cells (PBMCs) or in vivo stem cells directly. One of skill in the art will further recognize that other tissue samples can be used to generate neural organoids. In one embodiment skin cells are collected in a petri dish and induced to an embryonic- like pluripotent stem cell (iPSC) that have high levels of developmental plasticity. iPSCs are grown into neural organoids in said culture under appropriate conditions as set forth herein and the resulting neural organoids closely resemble developmental patterns similar to human brain. In particular, neural organoids develop anatomical features of the retina, forebrain, midbrain, hindbrain, and spinal cord. Importantly, neural organoids express >98% of the about 15,000 transcripts found in the adult human brain. iPSCs also can be derived from the skin or blood cells of humans identified with HMP or comorbid ADRD as well as normal humans (i.e., humans without HMP or comorbid ADRD).

[0051] The about 12-week-old iPSC-derived human neural organoid has ventricles and other anatomical features characteristic of a 35-40 day old neonate. In an additional embodiment the about 12-week-old neural organoid expresses beta 3-tubulin, a marker of axons as well as somato-dendritic Puncta staining for MAP2, consistent with dendrites. In yet another embodiment, at about 12 weeks the neural organoid displays laminar organization of cortical structures. Cells within the laminarstructure stain positive for doublecortin (cortical neuron cytosol), Beta3 tubulin (axons) and nuclear staining. The neural organoid, by 12 weeks, also displays dopaminergic neurons and astrocytes. The neural organoid generated by the method disclosed in U.S. Patent No.11,345,890 has similar anatomy to archetypal brain structure and gene expression profile that corresponds to the various regions and cell types of the brain, namely cerebellum, hindbrain, midbrain, cortex, blood brain barrier, spinal cord, retina, choroid plexus, and microglia (FIG.1 and FIG.3). These brain regions and gene expression patterns are missing in other models of neural organoids described elsewhere; and thus, making the neural organoids described herein provides an advantageous model that closely mimics the physiological nervous system.

[0052] The neural organoids as described herein offer many advantages in studying HMP and with comorbid with Alzheimer’s disease and related dementia (ADRD). HMP is a heritable neurological disorder that affects the nervous system and the parts of the body controlled by the nerves. ADRD is a group of dementia disorders that affect memory, thinking, and behavior.

[0053] Some co-morbidities associated with HMP are described in Table 5. The genes associated with these co-morbidities are also listed in Table 5. Strikingly, the transcriptomic data from the neural organoids obtained from HMP patients with comorbid ADRD (ADRD-HMP-NNOP) statistically overlap with the transcriptomic data from postmortem brain samples of ADRD-HMP patients (Tables 3-6).

[0054] Furthermore, these overlapped biomarkers are also dysregulated in neural organoids obtained from Alzheimer’s disease and related dementia (ADRD) patients, demonstrating that there are common pathways and gene expressions that are altered in HMP and ADRD (Table 3). Treatment with drugs for ADRD-HMP-NNOP rescues expression of these dysregulated genes, further suggesting that these biomarkers are relevant in progression of these diseases (Table 3).

[0055] The neural organoids are powerful tools to study HMP or comorbid ADRD in vitro. The neural organoid offers the advantages of replicability, reliability and robustness, as shown herein using replicate neural organoids from the same source of iPSCs. With regard to the advantages provided by the neural organoids provided herein, firstly, the neural organoids obtained from the same patient’s sample (eitherfrom skin or blood sample) are highly uniform and replicable with regards to gene expression. FIG.5 shows that expression of one gene in two independent neural organoids randomly selected from thousands of organoids that are generated from one patient’s sample, are closely matched with an overall variance of over 0.95 for all the genes tested. The transcriptomic data generated from the NNOP are reliable and robust because they corroborate the transcriptomic data obtained from postmortem samples (Table 3-6). Additionally, compared to expensive, labor intensive, and time-consuming animal models, neural organoids can be generated from relatively easy-to-obtain skin or blood samples in live patients. Some of the gene expression patterns also correlate with some clinical features of ADRD-HMP (Table 6). The clinically relevant biomarkers of neural organoids can be used as early diagnostic tools to identify individuals at risk for developing HMP or to identify treatments in patients at early stage of the disease when the symptoms have not yet arisen. Developmental Transcriptomics

[0056] A “transcriptome” is a collection of all RNA, including messenger RNA (mRNA), long non-coding RNAs (lncRNA), microRNAs (miRNA) and, small nucleolar RNA (snoRNA), other regulatory polynucleotides, and regulatory RNA (lncRNA, miRNA) molecules expressed from the genome of an organism through transcription therefrom. Thus, transcriptomics is the study of the mRNA transcripts produced by the genome at a given time in any particular cell or tissue of the organism. Transcriptomics employs high-throughput techniques to analyze genome expression changes associated with development or disease. In certain embodiments, transcriptomic studies can be used to compare gene expression in normal, healthy tissues with diseased tissues. In further embodiments, mutated genes or variants associated with disease or the environment can be identified.

[0057] The aim of developmental transcriptomics is to identify genes associated with, or significant in, organismal development and disease and dysfunctions associated with development. During development, genes undergo up- and down- regulation as the organism develops. Thus, transcriptomics provides insights into cellular processes and the biology of the organism.

[0058] Generally, as used in the methods disclosed herein in certain embodiments, RNA is sampled from the neural organoid described herein within at about one week, about four weeks, or about twelve weeks of development; most particularly RNA from all three time periods are denoted as samples. However, RNA from the neural organoid can be harvested at minutes, hours, days, or weeks after reprogramming. For instance, RNA can be harvested at about 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, and 60 minutes. In a further embodiment the RNA can be harvested 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, or 24 hours. In a further embodiment the RNA can be harvested at 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, or 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks 10 weeks, 11 weeks, 12 weeks or more in culture. The term “reprogram” refers to the process to change the identity of adult cells by returning them to a stem cell-like state that is capable of differentiating into a different cell type. After enriching for RNA sequences, an expressed sequence tag (EST) library can be generated and quantitated using the AmpliSeqTM technique from ThermoFisher; exemplars of alternate technologies include RNASeq and chip-based hybridization methods. Transcript abundance in such experiments is compared in control neural organoids from healthy individuals vs. neural organoids generated from individuals with disease and the fold change in gene expression calculated.

[0059] Furthermore, in one embodiment RNA from neural organoids for ADRD- HMP are converted to DNA libraries and then the representative DNA libraries are sequenced using exon specific primers for 20,814 genes using the AmpliSeqTM technique available commercially from ThermoFisher; exemplars of alternate technologies include RNASeq and chip based hybridization methods. Readout results on the order of <1 cpm are considered background noise. All cpm data are normalized data and the readouts are a direct representation of the abundance of the RNA for each gene.

[0060] Briefly, in one embodiment the array consists of a single gene used to predict risk. In alternative embodiments reads contain a plurality of genes, known tobe associated with HMPrisk. In yet another embodiment the genes on the libraries can be comprised of disease-specific gene sequences as provided in Tables 3-9 or a combination of genes in the tables with alternative disease specific genes. Exemplarily, changes in expression or mutation of disease-specific genes are detected using such sequencing, and differential gene expression detected thereby, qualitatively by detecting a pattern of gene expression or quantitatively by detecting the amount or extent of expression of one or a plurality of disease-specific genes or mutations thereof. Results of said assays using the AmpliSeqTM TM technique can be used to identify genes that can predict disease risk or onset and can be targets of therapeutic intervention. In further embodiments, hybridization assays can be used, including but not limited to sandwich hybridization assays, competitive hybridization assays, hybridization-ligation assays, dual ligation hybridization assays, or nuclease assays. Neural Organoids and Pharmaceutical Testing

[0061] Neural organoids are a productive model for pharmaceutical testing. Currently, drug screening studies including toxicity, safety and or pharmaceutical efficacy, are performed using a combination of in vitro experiments, rodent / primate studies and computer modeling. Collectively, these studies seek to model human responses, in particular physiological responses of the central nervous system.

[0062] Human neural organoids are advantageous over current pharmaceutical testing methods for several reasons. First, neural organoids are easily derived from healthy and diseased patients, mitigating the need to conduct expensive clinical trials. Second, rodent models of human disease are unable to mimic the physiological nuances unique to human growth and development. Third, the use of primates creates ethical concerns. Finally, current methods are indirect indices of drug safety. Alternatively, neural organoids offer a relatively inexpensive, easily accessible model of human brain development. The model allows for a direct, and thus more thorough understanding, of the safety, efficacy and toxicity of pharmaceutical compounds.

[0063] Starting material for neural organoids is easily obtained from healthy and diseased patients. Further, because human organoids are easily grown they can beproduced in mass. This allows for efficiency in screening pharmaceutical compounds.

[0064] Neural organoids are advantageous for identifying biomarkers of a disease or a condition, the method comprising a) obtaining a biological sample from a human patient; and b) detecting whether at least one biomarker is present in the biological sample by contacting the biological sample with an array comprising binding molecules specific for the biomarkers and detecting binding between the at least one biomarker and the specific binding molecules. In further embodiments, the biomarker serves as a gene therapy target or a target for pharmaceutical intervention. Developmental Transcriptomics and Predictive Medicine

[0065] Neural organoids are advantageous for identifying biomarkers of a disease or a condition, the method comprising a) obtaining a biological sample from a human patient; and b) detecting whether at least one biomarker is present in the biological sample by contacting the biological sample with an array comprising binding molecules specific for the biomarkers and detecting binding between the at least one biomarker and the specific binding molecules. In further embodiments, the biomarker serves as a gene therapy target or a target for pharmaceutical intervention.

[0066] Headache and migraine pains (HMP) are neurological disorders characterized by dysfunctions of the brain vasculature and glymphatic system involved in clearance of toxic metabolites through the CSF. Of the conditions that are dominant in one sex, those that create the highest burden, such as depression and headaches, tend to affect women more (Smith K., Women's health research lacks funding - in a series of charts. Nature.2023 May;617(7959):28-29). Despite this huge when ranked by funding amount, diseases that affect mainly women drop down. Migraine, headaches, and anxiety disorders disproportionately affect women, all attract much less funding in proportion to the burden they exert on the US population than do other (Smith K., Women's health research lacks funding - in a series of charts. Nature.2023 May;617(7959):28-29). Migraine is associated with many comorbidities including cardiovascular disorders (stroke, myocardial infarction) (Nasser M Alorfi. Pharmacological Methods of Pain Management: Narrative Reviewof Medication Used. Int J Gen Med.2023; 16: 3247–3256. Buse, D.C., Reed, M.L., Fanning, K.M. et al. Comorbid and co-occurring conditions in migraine and associated risk of increasing headache pain intensity and headache frequency: results of the migraine in America symptoms and treatment (MAST) study. J Headache Pain 21, 23 (2020).

[0067] For comorbid coronary artery disease, for example, success will help save nearly 20,000 life years and almost 40,000 years with disease for women over a 30- year period. (Smith K., Women's health research lacks funding - in a series of charts. Nature.2023 May;617(7959):28-29). Gene mutations are associated with the onset of HMP. Accordingly, identification of gene expression changes in the human neural organoid at about week 1, about week 4 and about week 12 provide insight into the risk of HMP onset later in life.

[0068] The skilled worker will recognize these markers as set forth exemplarily herein to be human-specific marker proteins as identified, inter alia, in genetic information repositories such as GenBank. One skilled in the art will recognize that alternative gene combinations can be used to predict HMP risk.

[0069] One advantage of using a neural organoid is that the risk of HMP onset can be determined using an in vitro model of brain development. In a particular embodiment, the neural organoid is about twelve weeks post-inducement and comprises the encoded structures and cell types of the retina, cortex, midbrain, hindbrain, brain stem, and spinal cord. However, because transcriptomics provides a snapshot in time, in one embodiment the neural organoid is procured after about one-week post inducement, four-week post inducement, and / or twelve-weeks post inducement. In a further embodiment, the neural organoid sample is procured from structures of the neural organoid that mimic structures developed in utero at about five weeks.

[0070] In an alternative embodiment is provided a method for predicting a risk for developing HMP in a human, the method comprising collecting a biological sample; measuring biomarkers in the biological sample; and detecting measured biomarkers from the sample that are differentially expressed in humans with HMP.

[0071] The diagnostic panel can be provided according to the invention as an array of diagnostically relevant portions of one or a plurality of these genes, whereinthe array can comprise any method for immobilizing, permanently or transiently, said diagnostically relevant portions of said one or a plurality of these genes, sufficient for the array to be interrogated and changes in gene expression detected and, if desired, quantified. In alternative embodiments the array comprises specific binding compounds for binding to the protein products of the one or a plurality of these genes. In yet further alternative embodiments, said specific binding compounds can bind to metabolic products of said protein products of the one or a plurality of these genes.

[0072] Another embodiment uses the neural organoids derived from the human patient in the non-diagnostic realm. The neural organoids express markers characteristic of a large variety of neurons and also include markers for astrocytic, oligodendritic, microglial, and vascular cells. The neural organoids from all the major regions of the brain including the retina, cortex, midbrain, brain stem, and the spinal cord in a single brain structure expressing greater than 98% of the genes known to be expressed in the human brain. Such characteristics enable the neural organoid to be used as a platform for drug screening, toxicity, safety, and / or pharmaceutical efficacy studies. Additionally, since the neural organoid is patient specific, pharmaceutical testing using the neural organoid allows for patient specific pharmacotherapy for HMP or comorbid ADRD and related comorbidities. Relevance for mTOR and ADORA2A mediated pathway in HMP mTOR activators

[0073] The data with the NNOP model described herein suggested that an activator of mTOR would be beneficial to treat HMP. Given the central role of mTOR in many pathways, an mTOR inhibitor such as Rapamycin, or its analogues, including everolimus and temsirolimus, however, can be used in conjunction at lower doses to fine tune the activity of an mTOR activator or therapeutic agent.

[0074] In some embodiments, the activators of mTOR include, but are not limited to, MHY1485 (4,6-dimorpholino-N-(4-nitrophenyl)-1,3,5-triazin-2-amine). The chemical structure of MHY1485 is provided below:ADORA2A Agonists inhibitors

[0075] ADORA2A gene is expressed at a lower level in ADRD-HMP-NNOP compared to normal (p<0.05; Table 5), a finding that is consistent with the use of ADORA2A receptor agonists as candidates for disease modifying drugs for HMP. Furthermore, multiple genes in the ADORA2A STRING analysis (Genecards) show epigenetic co-regulation with expression of the ADORA2A gene (Table 5). These data strongly support the hypothesis that ADORA2A agonist would augment the adenosine receptor 2A pathway function. Indeed, CGS-21680 in the presence of the PDE4 inhibitor (to upregulated PDE4 expression), an ADORA2A receptor agonist, rescued the expression of dysregulated HMP genes in ADRD-HMP-SPOR-NNOP as shown in Table 5.

[0076] It has also been contemplated that other strategies using RNA therapeutics to activate mTOR pathway ADORA2A pathway can also have therapeutic benefits. Such RNA therapeutics can target not only mTOR and ADORA2A but also any genes in the network shown in Table 3-6. Examples of RNA therapeutics include, but are not limited to, antisense oligonucleotides (ASO), Adeno-Associated Virus delivery of small interfering RNA (AAV-RNA) or micro RNA (miRNA). As used herein, the term “RNA-based therapeutics” refers to therapeutic approaches using messenger RNA (mRNA), antisense RNA (asRNA), RNA interference (RNAi), or RNA aptamers to manipulate the expression and activity of specific target molecules. The term “therapeutic agents” refer to an agent that is capable of correcting DEGs in vitro in neural organoids derived from patients with HMP or in vivo in patients with HMP, and reducing or ameliorating disease severity in the HMP patient thereby. The agent includes, but is not limited to, small molecules, chemical compounds, or RNA-based therapeutics.Relevance for glymphatic system in HMP

[0077] Nine DEGs that are correlated with genetic biomarkers for migraine (Hautakangas et. al., 2022) are dysregulated in ADRD-HMP-NNOP. These include CACN1A1, PRDM16, WSCD1, YAP1, DOCK4, HMOX2, JAG1, PDE4B, PRKCE and ARG2 that support a role for their functional dysregulation in headache migraine pain and when comorbid in ADRD.

[0078] The glymphatic system is a brain-wide perivascular pathway driven by aquaporin-4 on the endfeet of astrocytes, which deliver nutrients and active substances to the brain parenchyma through periarterial cerebrospinal uid (CSF)in ux pathway and remove metabolic wastes. The glymphatic system is an emergingpathway of removing metabolic waste products and toxic solutes from the brain tissue. It is made of a network of perivascular spaces, filled in cerebrospinal and interstitial fluid, encompassing penetrating and pial vessels and communicating with the subarachnoid space. It is separated from vessels by the blood brain barrier and from brain tissue by the endfeet of the astrocytes rich in aquaporin 4, a membrane protein which controls the water flow along the perivascular space. The SYK pathway is involved in regulating the glymphatic pathway and SYK inhibitors are expected to be additionally beneficial for HMP due to dysregulation of the glymphatic pathway (Mocsal et al., The SYK tyrosine kinase: a crucial player in diverse biological functions. Nat Rev Immunol.2010, 10, 387-402; Abtahian et al., Regulation of blood and vascular separation by signaling proteins SLP-76 and Syk. 2003, 299, 247-251. Vittorini, M.G., Sahin, A., Trojan, A. et al. The glymphatic system in migraine and other headaches. J Headache Pain 25, 34 (2024)). Throughout the body, lymphatic fluid movement supports critical functions including clearance of excess fluid and metabolic waste. The glymphatic system is the analog of the lymphatic system in the CNS.

[0079] In keeping with the expectation that the glymphatic system genes would be changed, we found that the gene for the protein that regulates SYK expression, PTPN6 is normalized by the ADORA2A agonist when used in combination with the PDE4 inhibitor Roflumilast. An endogenous ligand of CLEC-2 is the membrane protein podoplanin (PDPN), which is expressed on the surface of certain types of tumor cells and lymphatic endothelial cells (LECs).

[0001] C-type lectin-like receptor 2 (CLEC-2, also known as CLEC-1b) which binds to various ligandsincluding the mucin-like protein podoplanin (PDPN). Interestingly, we also see normalization of CLEC1b by the ADORA2A agonist when used in combination with the PDE4 inhibitor Roflumilast. Methods of treating HMP and pharmaceutical compositions

[0080] Provided herein is a method for reducing or ameliorating disease severity in an individual having HMP by administering to the individual a therapeutically effective amount of a pharmaceutical composition comprising a drug capable of reducing or correcting dysfunctional expression of genes associated with HMP in vitro in a neural organoid platform and accordingly reducing or ameliorating disease severity in such individuals.

[0081] In some embodiments, a drug capable of reducing or correcting dysfunctional expression of genes associated with HMP in vitro in a neural organoid platform is an mTOR activator or a ADORA2A receptor agonist CGS-21680 (in the presence of the PDE4 inhibitor Roflumilast inhibitor). In some embodiments, the mTOR activator is MHY1485. In some embodiments, the ADORA2A receptor agonist is apadenoson (ATL-146e). The genes that are dysfunctionally expressed and associated with HMP include the genes listed in Tables 3-6. The neural organoids can be generated from biological samples obtained from HMP patients as described throughout this application.

[0082] As used herein, the term “inhibitor” refers to a molecule / compound that decreases the activity of a biological molecule target. For example, a PDE inhibitor is a compound that reduces PDE esterase enzyme activity and influences signaling activity downstream of it pathways. In contrast, an “activator” refers to a molecule / compound that increases the activity of a biological molecule target. For example, an mTOR activator is a compound that enhances mTOR kinase activity and influences signaling activity downstream of mTOR.

[0083] The compounds described herein can be administered to an individual as a pharmaceutical composition comprising a pharmaceutically acceptable carrier. In some embodiments, pharmaceutical compositions are lyophilized. In other cases, pharmaceutical compositions as provided herein contain auxiliary substances such as wetting or emulsifying agents, pH buffering agents, gelling or viscosity enhancingadditives, preservatives, flavoring agents, colors, and the like, depending upon the route of administration and the preparation desired. Pharmaceutical compositions can be formulated according to conventional pharmaceutical practice (see, e.g., Remington: The Science and Practice of Pharmacy, 20th edition, 2000, ed. A. R. Gennaro, Lippincott Williams & Wilkins, Philadelphia, and Encyclopedia of Pharmaceutical Technology, eds. J. Swarbrick and J. C. Boylan, 1988-1999, Marcel Dekker, New York).

[0084] The term “therapeutically effective amount” of a drug for treating HMP will be understood to include dosage amounts that provide improvements or reduction in impairments or progression thereof of symptoms or comorbidities associated with HMP. The term “comorbidities” refers to simultaneous presence of two or more medical conditions or symptoms in a patient, often co-occurring with a primary condition. As used herein, the term “dysfunctional gene expression” or ”differential gene expression” will be understood to mean statistical differences in gene expression as shown in the transcriptomic study in samples obtained or derived from HMP or those comorbid with ADRD patients or treated samples with drugs compared to normal patients or untreated samples respectively. As used herein, the term “reducing or correcting dysfunctional gene expression” refers to changes in expression of certain genes in NNOP in response to addition of therapeutically effective amounts of a drug a disclosed herein, wherein dysfunctional gene expression has the meaning set forth above, and degree of effect on the NNOP will be understood to reduce the extent of the dysfunction in expression of said genes. Patient-specific pharmacotherapy using neural organoids and method of predicting risk of HMP

[0085] As discussed above, the neural organoid model used as disclosed herein allows a patient-specific pharmacotherapy approach. Particularly, information regarding differentially expressed genes (DEGs) specific to the patient can be obtained from the neural organoids and guide treatments targeting these DEGs. Thus, provided herein is a method of treating HMP or preventing development of HMP, using a patient-specific pharmacotherapy by a) procuring one or a plurality of cell samples from a patient with HMP or at risk for developing HMP, comprising one or a plurality of cell types including but not limited to fibroblasts, buccal cavity cells, or peripheral blood monocytes; b) reprogramming the one or the plurality of cellsamples to produce one or a plurality of induced pluripotent stem cell samples; c) treating the one or the plurality of induced pluripotent stem cell samples to obtain a neural organoid; d) collecting a biological sample from the neural organoid; e) detecting genes from the neural organoid sample that are differentially expressed compared to a neural organoid sample obtained from a healthy patient; f) administering one or more therapeutic agents capable of reducing or correcting the differentially expressed genes to the patient with HMP or at risk for developing HMP.

[0086] Additionally, information regarding DEGs specific to the patient in step e) can be compared to the DEGs in postmortem brains of HMP patient, Genome-Wide Association Study (GWAS) of HMP population in general, biomarker profile of plasma / cerebrospinal fluid / urine of HMP patients (Table 3), or genes associated with clinical symptoms or comorbidities of HMP (Table 5), to assist diagnosis and determination of HMP treatment or risk.

[0087] The data disclosed herein demonstrate that the DEGs in neural organoids derived from HMP patients encode dysregulated proteins or are associated with changes in metabolites that are known in HMP (Tables 3-5). Thus, determination of DEGs from the neural organoid model is an effective tool to predict or identify new biomarkers in HMP. Alternatively, step e) can involve detecting protein expression / function or metabolite generation from the neural organoid sample that are dysregulated compared to a neural organoid sample obtained from a healthy patient; and step f) can involve administering one or more therapeutic agents capable of correcting the dysregulated protein expression / function or metabolite production to the patient with HMP or at risk for developing HMP.

[0088] In some embodiments, the protein expression / function or metabolite production can be detected directly in biological samples obtained from a patient with HMP or at risk for developing HMP using conventional assays informed by the identities of such genes or metabolites disclosed herein. In some embodiments, one or more therapeutic agents capable of correcting the dysregulated protein expression / function or metabolite production in the biological samples can also be identified and used to treat HMP or prevent development of HMP. The biological samples can be urine, plasma, or cerebrospinal fluid (CSF).

[0089] As used herein, the term ”correcting” refers to the capacity to bring about changes in gene expression, protein expression / function, or metabolite production to a level that is closer to the level in control healthy condition.

[0090] A particular class of drugs provided herein are agonists of adenosine A2a receptor. (Genbank Accession No. NP_001265429.1) encoded by ADORA2A (Genbank Accession No. NM_001278497.2). In specific embodiments, such drugs include methyl (1R,4r)-4-(3-(6-amino-9-((2R,3R,4S,5S)-5-(ethylcarbamoyl)-3,4- dihydroxytetrahydrofuran-2-yl)-9H-purin-2-yl)prop-2-yn-1-yl)cyclohexane-1- carboxylate (known in the art as apadenoson). In other specific embodiments, suchdrugs include 2-p-(2-Carboxyethyl) phenethylamino-5 -N-ethylcarboxamidoadenosine hydrochloride hydrate (known in the art as CGS-21680).

[0091] In specific embodiments the pharmaceutical compositions comprise drugs that are agonists of adenosine A2a receptor. (Genbank Accession No. NP_001265429.1) encoded by ADORA2A (Genbank Accession No. NM_001278497.2). In specific embodiments, such drugs include methyl (1R,4r)-4-(3- (6-amino-9-((2R,3R,4S,5S)-5-(ethylcarbamoyl)-3,4-dihydroxytetrahydrofuran-2-yl)- 9H-purin-2-yl)prop-2-yn-1-yl)cyclohexane-1-carboxylate (known in the art as apadenoson). In other specific embodiments, such drugs include 2-p-(2-Carboxyethyl) phenethylamino-5 -N-ethylcarboxamidoadenosine hydrochloridehydrate (known in the art as CGS-21680). In addition to such particular and specific embodiments this disclosure encompasses related drug molecules that are agonists of adenosine A2a receptors encoded by ADORA2A and allelic variants thereof, particularly such variants associated with diseases and disabilities in an individual as a consequence of HMP associated with Alzheimer’s Disease, Alzheimer’s Disease Related Dementia (ADRD).

[0092] Additional embodiments of the methods and pharmaceutical compositions disclosed herein comprise combinations of adenosine A2a receptor agonists as disclosed herein as well as combinations of these drugs with other medicaments useful for reducing or ameliorating disease severity in an individual having diseases and disabilities in an individual with chronic pain or neuropathic pain, specifically including but not limited to Alzheimer’s Disease, Alzheimer’s Disease Related Dementia (ADRD) or Mixed Etiology Dementia (MED). In specific embodiments such combinations include methyl (1R,4r)-4-(3-(6-amino-9-((2R,3R,4S,5S)-5-(ethylcarbamoyl)-3,4-dihydroxytetrahydrofuran-2-yl)-9H-purin-2-yl)prop-2-yn-1- yl)cyclohexane-1-carboxylate (known in the art as apadenoson), 2-p-(2-Carboxyethyl) phenethylamino-5 -N-ethylcarboxamidoadenosine hydrochloridehydrate (known in the art as CGS-21680), in therapeutically useful combinations and dosages thereof, as well as combinations with alternative medicaments capable of reducing or ameliorating disease severity in an individual having diseases and disabilities in an individual with chronic pain or neuropathic pain and when comorbid with Alzheimer’s Disease Related Dementia (AD / ADRD).

[0093] The invention also provides pharmaceutical compositions of drugs capable of reducing or ameliorating HMP severity in an individual having diseases and disabilities in the individual as a consequence of specifically but not limited to Alzheimer’s Disease, Alzheimer’s Disease Related Dementia (AD / ADRD) wherein a particular class of drugs provided herein are antagonists of adenosine A2b receptor (Genbank Accession No. NP_0006667.1) encoded by ADORA2B (Genbank Accession No. NM 000676.4). Specific embodiments of drugs comprising these adenosine A2b receptor antagonists include but are not limited to PBF-1129 and MRS-1706. In addition to such particular and specific embodiments this disclosure encompasses related drug molecules that are antagonists of adenosine A2b receptors encoded by ADORA2B and allelic variants thereof, particularly such variants associated with diseases and disabilities in an individual as a consequence of comorbidities of HMP including but not limited to Alzheimer’s Disease Related Dementia (AD / ADRD). Relevance for adenosine receptors in HMP and Comorbidities

[0094] Certain studies in the prior art suggest the possibility of a role for adenosine receptors in the progression of the neuropathological changes that are observed in ADRD-HMP (Kinney et al., 2018, Alzheimer's & Dementia: Translational Research & Clinical Interventions, Alzheimer’s and Dementia: Translational Research and Clinical Intervention 4: 575-590). For example, an association between a polymorphism of the ADORA2A gene with hippocampal volume in mild cognitive impairment and ADRD-HMP has been reported (Horgusluoglu-Moloch et al., 2017, Targeted neurogenesis pathway-based gene analysis identifies ADORA2A associated with hippocampal volume in mild cognitive impairment and Alzheimer’s disease, Neurobiol. Aging 60: 92–103). Adenosine receptors have differentfunctions, and the A2A receptor has a broader anti-inflammatory effect throughout the body, additionally (Hasko and Pather, 2008, A2A receptors in inflammation and injury: lessons learned from transgenic animals, J. Leucocyte Biol.83: 447-455). Both receptors also regulate the release of dopamine and glutamate in the brain (Sun and Hwang, 2016, Adenosine A2B Receptor: From Cell Biology to Human Diseases, Front. Chem.24: 37; Fuxe et al., 2007, Adenosine receptor-dopamine receptor interactions in the basal ganglia and their relevance for brain function, Physiol Behav 92:210-7; Schiffmann et al., 2007, Adenosine A2A receptors and basal ganglia physiology, Prog Neurobiol 83: 277-92; and Cunha et al., 2008, How does adenosine control neuronal dysfunction and neurodegeneration?, J. Neurobiol. 139:1019-1055). Set forth herein is evidence for a more general role for adenosine receptors in biological a

[0095] The Adenosine A2A Receptor (ADORA2A) gene is expressed at a lower level compared to normal (p<0.05; Table 3) in gene expression analyses using NNOP from individuals with AD / ADRD and shared biomarkers with Progeria (Werner’s syndrome). Furthermore, multiple genes in the STRING analysis (ADORA1; ACTN1, ACTN3, CALM3; GRM5, NECAB2, SYNGR1 and FURIN) show epigenetic co-regulation with the ADORA2A gene. These data strongly suggest that A2A agonists could augment ADORA2A anti-inflammatory pathway function. Multiple agonists have been characterized (CGS21680; DPMA; HE-NECA; ATL-146e; and CVT-3146). As set forth herein this invention utilizes two of them, CGS-21680 and Apadenoson (ATL-146e), as therapeutic compositions for treating ADRD / MED.

[0096] Another adenosine A2A receptor agonist, apadenoson (ATL-146e), chemical name methyl (1R,4r)-4-(3-(6-amino-9-((2R,3R,4S,5S)-5-(ethylcarbamoyl)- 3,4- dihydroxytetrahydrofuran-2-yl)-9H-purin-2-yl)prop-2-yn-1-yl)cyclohexane-1- carboxylate; CAS Number: 250386-15-3 Molecular Weight: 486.529; Soluble in DMSO. MedKoo Biosciences, Inc., is a selective adenosine A2A receptor agonist and a potent inhibitor of inflammation. Apadenoson is tested using a range of concentrations (dose range: 100nM-1microM; EC50 of 110 nM).

[0097] To enhance the effects of adenosine agonist experiments with CGS-21680 were performed in the presence of Roflumilast, a PDE4 inhibitor. Previous work indicated that Roflumilast can upregulate PDE4 expression (Susuki-Miyata et al.,Cross-talk between PKA-C and p65 mediates synergistic induction of PDE4B byroflumilast and NTH. PNAS, E1800–E1809, 2015) to compensate for lower PDE4B expression in ADRD-HMP-SPOR-NNOP when compared to controls (base mean = 491.67; Log2 fold change = 1.49; p-vallue 1.29E-02) .

[0098] A positive Log 2 value indicates PDE4B expression is higher in the normal-NNOP compared to in ADRD-HMP-SPOR-NNOP.

[0099] This invention provides methods for reducing or ameliorating disease severity in an individual having diseases and disabilities in an individual as a consequence of biological aging including but not limited to Alzheimer’s Disease, Alzheimer’s Disease Related Dementia (ADRD) or Mixed Etiology Dementia (MED) comprising administering to the individual a therapeutically effective amount of a pharmaceutical composition comprising a drug capable of reducing or correcting dysfunctional expression of genes associated with chronic pain and neuropathic pain in diseases and disabilities in an individual in vitro in a neural organoid platform and accordingly reduces or ameliorates pain severity in such individuals. Specifically, the methods provided herein rely upon identification of genes associated with chronic pain and neuropathic pain in diseases and disabilities diseases and disabilities in an individual that show dysfunctional or altered gene expression in neural organoid platforms in vitro for which the drugs disclosed herein are capable of reducing this dysfunctional expression. In particular embodiments these genes are set forth in Tables 3-6.

[0100] As used herein, the terms “reducing or ameliorating” are intended to be understood to include improvements or reduction in impairments or progression thereof of symptoms of chronic pain and neuropathic pain in diseases and disabilities diseases and disabilities in an individual, particularly cognitive symptoms of dementia, specifically including but not limited to Alzheimer’s Disease, Alzheimer’s Disease Related Dementia (ADRD) or Mixed Etiology Dementia (MED).

[0101] As used herein, the term “therapeutically effective amount” of a drug for treating chronic pain and neuropathic pain in diseases and disabilities diseases and disabilities in an individual including but not limited to dementia, specifically including but not limited to Alzheimer’s Disease, Alzheimer’s Disease Related Dementia (ADRD) or Mixed Etiology Dementia (MED) will be understood to include dosageamounts that provide improvements or reduction in impairments or progression thereof of symptoms, particularly cognitive symptoms,

[0102] As used herein, the term “dysfunctional gene expression” will be understood to mean differences in gene expression in NOP as shown herein produced from skin cells, inter alia by methods for producing induced pluripotent stem cells that are then differentiated into NNOP s set forth in U.S. Patent No. 11,345, 890, incorporated herein in its entirety, between individuals having chronic pain and neuropathic pain including but not limited to diseases and disabilities diseases and disabilities in an individual with Alzheimer’s Disease, Alzheimer’s Disease Related Dementia (ADRD) or Mixed Etiology Dementia (MED) or a genetic propensity for developing Alzheimer’s Disease (AD) or Alzheimer’s Disease Related Dementia (ADRD) or Mixed Etiology Dementia (MED) and individuals without Alzheimer’s Disease Related Dementia (ADRD) or Mixed Etiology Dementia (MED) or a genetic propensity for developing Alzheimer’s Disease Related Dementia (ADRD) or Mixed Etiology Dementia (MED) or cancer. Useful NOPs as set forth herein can be generated as disclosed herein from adult skin cells of individuals having genetic traits including those of sporadic Alzheimer’s disease (identified as SPOR herein), amyloid plaque disorders (APP), mutations in ApoE4 (APOE), or presenilin (PSEN2).

[0103] In addition to such particular and specific embodiments this disclosure encompasses related drug molecules that are agonists of adenosine A2a receptors encoded by ADORA2A. EXAMPLES

[0104] The Examples that follow are illustrative of specific embodiments of the invention, and the use thereof. It is set forth for explanatory purposes only and is not taken as limiting the invention. In particular, the example demonstrates the effectiveness of neural organoids in predicting future disease risk.MATERIALS AND METHODS

[0105] The neural organoids described above were developed using the following materials and methods. Summary of Methods:

[0106] Neural Organoids derived from induced pluripotent stem cells derived from adult skin cells of ADRD patients were grown in vitro for 4 weeks as previously described in PCT Application PCT / US2017 / 13231 (and U.S. Patent Number US 11,345,890B2). Transcriptomic data from these neural organoids were obtained. The differences in expression of 20,814 genes expressed in the human genome was determined between these neural organoids and those from neural organoids from a normal person (not having HMP). Detailed data analysis using Gene Card and Pubmed data bases was performed. Genes that were expressed at >1.4 fold were found to be highly significant because a vast majority were correlated with genes previously associated with a multitude of neurodevelopmental and neurodegenerative diseases as well as those found to be dysregulated in postmortem patient brains. These genes comprise a suite of biomarkers for Headache and Migraine Pain (HMP).

[0107] Cells used in these methods include human iPSCs, feeder-dependent (System Bioscience. WT SC600A-W) and CF-1 mouse embryonic fibroblast feeder cells, gamma-irradiated (Applied StemCell, Inc #ASF- 1217)

[0108] Growth media, or DMEM media, used in the examples contained the supplements as provided in Table 1. Table 1: Growth Media and Supplements used in Examples

[0109] One skilled in the art will recognize that additional formulations of media and supplements can be used to culture, induce and maintain pluripotent stem cells and neural organoids.

[0110] Experimental protocols required the use of multiple media compositions including MEF Media, IPSC Media, EB Media, Neural Induction Media, and Differentiation Medias 1, 2, and 3.

[0111] Mouse embryonic fibroblast (MEF) was used in cell culture experiments. MEF Media comprised DMEM media supplemented with 10% Feta Bovine Serum, 100 units / ml penicillin, 100 microgram / ml streptomycin, and 0.25 microgram / ml Fungizone.

[0112] Induction media for pluripotent stem cells (IPSC Media) comprised DMEM / F12 media supplemented with 20% Knockout Replacement Serum, 3% FetalBovine Serum with 2mM Glutamax, IX Minimal Essential Medium Nonessential Amino Acids, and 20 nanogram / ml basic Fibroblast Growth Factor

[0113] Embryoid Body (EB) Media comprised Dulbecco's Modified Eagle's Medium (DMEM) (DMEM) / Ham's F-12 media, supplemented with 20% Knockout Replacement Serum, 3% Fetal Bovine Serum containing 2mM Glutamax, IX Minimal Essential Medium containing Nonessential Amino Acids, 55microMolar beta- mercaptoethanol, and 4ng / ml basic Fibroblast Growth Factor.

[0114] Neural Induction Media contained DMEM / F12 media supplemented with: a 1:50 dilution N2 Supplement, a 1:50 dilution GlutaMax, a 1:50 dilution MEM-NEAA, and 10 microgram / ml Heparin`

[0115] Three differentiation medias were used to produce and grow neural organoids. Differentiation Media 1 contained DMEM / F12 media and Neurobasal media in a 1:1 dilution. Each media is commercially available from Invitrogen. The base media was supplemented with a 1:200 dilution N2 supplement, a 1:100 dilution B27 - vitamin A, 2.5microgram / ml insulin, 55 microM beta-mercaptoethanol kept under nitrogen mask and frozen at -20°C, 100 units / ml penicillin, 100 microgram / ml streptomycin, and 0.25 microgram / ml Fungizone.

[0116] Differentiation Media 2 contained DMEM / F12 media and Neurobasal media in a 1:1 dilution supplemented with a 1:200 dilution N2 supplement, a 1:100 dilution B27 containing vitamin A, 2.5microgram / ml Insulin, 55uM beta- mercaptoethanol kept under nitrogen mask and frozen at -20°C, 100units / ml penicillin, 100microgram / ml streptomycin, and 0.25microgram / ml Fungizone.

[0117] Differentiation Media 3 consisted of DMEM / F12 media: Neurobasal media in a 1:1 dilution supplemented with 1:200 dilution N2 supplement, a 1:100 dilution B27 containing vitamin A), 2.5microgram / ml insulin, 55microMolar beta- mercaptoethanol kept under nitrogen mask and frozen at -20°C, 100 units / ml penicillin, 100 microgram / ml streptomycin, 0.25microgram / ml Fungizone, TSH, and Melatonin.

[0118] The equipment used in obtaining, culturing and inducing differentiation of pluripotent stem cells is provided in Table 2. One skilled in the art would recognize that the list is not all exhaustive.Table 2: Equipment used in Experimental procedures.Example 1: Generation of human induced pluripotent stem cell-derived neural organoids.

[0119] Human induced pluripotent stem cell-derived neural organoids were generated according to the following protocol, as set forth in International Application No. PCT / US2017 / 013231 (and U.S. Patent Number US 11,345,890 B2) incorporated herein by reference in their entirety. Briefly, irradiated murine embryonic fibroblasts (MEF) were plated on a gelatin coated substrate in MEF media (Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% Feta Bovine Serum, 100 units / ml penicillin, 100 microgram / ml streptomycin, and 0.25 microgram / ml Fungizone) at a density of 2 x 105cells per well. The seeded plate was incubated at 37°C overnight.

[0120] After incubation, the MEFs were washed with pre-warmed sterile phosphate buffered saline (PBS). The MEF media was replaced with 1 mL per well of induced pluripotent stem cell (iPSC) media containing Rho-associated protein kinase (ROCK) inhibitor. A culture plate with iPSCs was incubated at 37°C. The iPSCs were fed every other day with fresh iPSC media containing ROCK inhibitor.The iPSC colonies were lifted, divided, and transferred to the culture wells containing the MEF cultures so that the iPSC and MEF cells were present therein at a 1:1 ratio. Embryoid bodies (EB) were then prepared. Briefly, a 100 mm culture dish was coated with 0.1% gelatin and the dish placed in a 37oC incubator for 20 minutes, after which the gelatin-coated dish was allowed to air dry in a biological safety cabinet. The wells containing iPSCs and MEFs were washed with pre-warmed PBS lacking Ca2+ / Mg2+. A pre-warmed cell detachment solution of proteolytic and collagenolytic enzymes (1 mL / well) was added to the iPSC / MEF cells. The culture dishes were incubated at 37°C for 20 minutes until cells detached. Following detachment, pre-warmed iPSC media was added to each well and gentle agitation used to break up visible colonies. Cells and media were collected and additional pre- warmed media added, bringing the total volume to 15 mL. Cells were placed on a gelatin-coated culture plate at 37°C and incubated for 60 minutes, thereby allowing MEFs to adhere to the coated surface. The iPSCs present in the cell suspension were then counted.

[0121] The suspension was then centrifuged at 300xg for 5 minutes at room temperature, the supernatant discarded, and cells re-suspended in EB media supplemented with ROCK inhibitor (50uM final concentration) and 4ng / ml basic Fibroblast Growth Factor to a volume of 9,000 cells / 150 μL. EB media is a mixture of DMEM / Ham's F-12 media supplemented with 20% Knockout Replacement Serum, 3% Fetal Bovine Serum (2mM Glutamax), 1X Minimal Essential Medium Nonessential Amino Acids, and 55 uM beta-mercaptoethanol. The suspended cells were plated (150 μL) in a LIPIDURE® low-attachment U-bottom 96-well plate and incubated at 37°C.

[0122] The plated cells were fed every other day during formation of the embryoid bodies by gently replacing three fourths of the embryoid body media without disturbing the embryoid bodies forming at the bottom of the well. Special care was taken in handling the embryoid bodies so as not to perturb the interactions among the iPSC cells within the EB through shear stress during pipetting. For the first four days of culture, the EB media was supplemented with 50uM ROCK inhibitor and 4ng / ml bFGF. During the remaining two to three days the embryoid bodies were cultured, no ROCK inhibitor or bFGF was added.

[0123] On the sixth or seventh day of culture, the embryoid bodies were removed from the LIPIDURE® 96 well plate and transferred to two 24-well plates containing 500 μL / well Neural Induction media, DMEM / F12 media supplemented with a 1:50 dilution N2 Supplement, a 1:50 dilution GlutaMax, a 1:50 dilution MEM-Non- Essential Amino Acids (NEAA), and 10 ug / ml Heparin. Two embryoid bodies were plated in each well and incubated at 37°C. The media was changed after two days of incubation. Embryoid bodies with a "halo" around their perimeter indicate neuroectodermal differentiation. Only embryoid bodies having a "halo" were selected for embedding in matrigel, remaining embryoid bodies were discarded.

[0124] Plastic paraffin film (PARAFILM) rectangles (having dimensions of 5cm x 7cm) were sterilized with 3% hydrogen peroxide to create a series of dimples in the rectangles. This dimpling was achieved, in one method, by centering the rectangles onto an empty sterile 200μL tip box press, and pressing the rectangles gently to dimple it with the impression of the holes in the box. The boxes were sprayed with ethanol and left to dry in the biological safety cabinet.

[0125] Frozen Matrigel matrix aliquots (500 μL) were thawed on ice until equilibrated at 4°C. A single embryoid body was transferred to each dimple of the film. A single 7cm x 5cm rectangle holds approximately twenty (20) embryoid bodies. Twenty microliter (20μL) aliquots of Matrigel were transferred onto the embryoid bodies after removing extra media from the embryoid body with a pipette. The Matrigel was incubated at 37°C for 30 min until the Matrigel polymerized. The 20μL droplet of viscous Matrigel was found to form an optimal three dimensional environment that supported the proper growth of the neural organoid from embryoid bodies by sequestering the gradients of morphogens and growth factors secreted by cells within the embryoid bodies during early developmental process. However, the Matrigel environment permitted exchange of essential nutrients and gases. Gentle oscillation by hand twice a day for a few minutes within a tissue culture incubator (37°C / 5%CO2) further allowed optimal exchange of gases and nutrients to the embedded embryoid bodies.

[0126] Differentiation Media 1, a one-to-one mixture of DMEM / F12 and Neurobasal media supplemented with a 1:200 dilution N2 supplement, a 1:100 dilution B27 - vitamin A, 2.5 ug / mL insulin, 55 microM beta-mercaptoethanol kept under nitrogen mask and frozen at -20°C, 100 units / mL penicillin, 100 ug / mLstreptomycin, and 0.25 ug / mL Fungizone, was added to a 100mm tissue culture dish. The film containing the embryoid bodies in Matrigel was inverted onto the 100 mm dish with differentiation media 1 and incubated at 37°C for 16 hours. After incubation, the embryoid body / Matrigel droplets were transferred from the film to the culture dishes containing media. Static culture at 37°C was continued for 4 days until stable neural organoids formed.

[0127] Organoids were gently transferred to culture flasks containing differentiation media 2, a one-to-one mixture of DMEM / F12 and Neurobasal media supplemented with a 1:200 dilution N2 supplement, a 1:100 dilution B27 + vitamin A, 2.5 ug / mL insulin, 55 μM beta-mercaptoethanol kept under nitrogen mask and frozen at -20°C, 100 units / mL penicillin, 100 ug / mL streptomycin, and 0.25 ug / mL Fungizone. The flasks were placed on an orbital shaker rotating at 40 rpm within the 37°C / 5% CO2incubator.

[0128] The media was changed in the flasks every 3-4 days to provide sufficient time for morphogen and growth factor gradients to act on targets within the recipient cells forming relevant structures of the brains. Great care was taken when changing media so as to avoid unnecessary perturbations to the morphogen / secreted growth factor gradients developed in the outer most periphery of the organoids as the structures grew into larger organoids.

[0129] Based on transcriptomic analysis, iPSC cells form a body of cells after 3D culture, which become neural progenitor cells (NPC) after neural differentiation media treatment. Neurons were observed in the cell culture after about one week. After about four (4) weeks, neurons of multiple lineage appeared. After about twelve (12) weeks, the organoid developed to a stage having different types of cells, including microglia, oligodendrocyte, astrocyte, neural precursor, neurons, and interneurons. Example 2: Human induced pluripotent stem cell-derived neural organoids express characteristics of human brain development and the reproducibility, replicability, and robustness of the NNOP model.

[0130] After ~12 weeks of in vitro culture, transcriptomic and immunohistochemical analysis indicated that organoids were generated according tothe methods delineated in Example 1. Specifically, the organoids contained cells expressing markers characteristic of neurons, astrocytes, oligodendrocytes, microglia, and vasculature (Fig.3) and all major brain structures of neuroectodermal derivation. Morphologically identified by bright field imaging, the organoids included readily identifiable neural structures including cerebral cortex, cephalic flexure, and optic stalk (compare, Grey's Anatomy Textbook). The gene expression pattern in the neural organoid was >98 % concordant with those of the adult human brain reference (Clontech, #636530). The organoids also expressed genes in a developmentally organized manner described previously (e.g. for the midbrain mesencephalic dopaminergic neurons, Blaese et al., 2015, Wiley Interdiscip Rev Dev Biol 4(2): 113-34). The structures also stained positive for multiple neural specific markers (dendrites, axons, nuclei), cortical neurons (Doublecortin), midbrain dopamine neurons (Tyrosine Hydroxylase), and astrocytes (GFAP) as shown by immunohistology).

[0131] All human neural organoids were derived from iPSCs of fibroblast origin (from Coriell Biorepository, NJ or other commercial sources). The development of a variety of brain structures was characterized in the organoids. Biomarkers specific for particular regions of human brain were detected as set forth in International Application No. PCT / US2017 / 013231 (and U.S. Patent Number US 11,345,890 B2), incorporated by reference in their entirety herein.

[0132] FIG.3 shows transcriptomic data of about 13,000 genes of NNOP obtained from ADRD-HMP and normal patients in controlled clinical studies. Neural organoids were cultured in media after neural differentiation was initiated with addition of retinoic acid as per the published protocol (International Patent Application (PCT) Publication Number WO2017123791A1). The cultures were replenished with fresh media every week. Organoids were harvested after the third day (D3), Week 1 and Week 4, W12 in culture. Each dot represented RNA expression level of a gene measured in two randomly selected NNOP out of hundreds of NNOP. The variance was as low as <0.95 showing that there was little variability between independent organoid replicates, which demonstrated the reproducibility, replicability, and robustness of the NNOP model. Thus, NNOP models were demonstrated to be a reliable model to study HMP, and comparative data analysis from NNOP platform between normal and ADRD patient-derivedsamples permitted identification of therapeutic targets and individualization of treatment decisions. Example 3: Human Neural Organoid is a suitable model to study HMP and predict risk of comorbid dementia disease onset

[0133] Development of HMP therapeutics has primarily relied on rodent models (Lama et al., Neuronal Signaling 2021; 5(4):NS20210026). These models have limited utility because they neither reflect the pathophysiology of HMP completely nor are able to properly reflect the differences due to human genetic diversity. Human neural organoids have emerged as promising preclinical models. However, all incomplete organoids are unreliable pre-clinical models. Examples 1-3 established that the disclosed neural organoids expressed characteristics of human brain development and the data generated from these organoids showed reproducibility, replicability, and robustness.

[0134] Accordingly, the experiments set forth herein were performed to characterize the transcriptomic profile of HMP patient-derived neural organoids and how gene expression and comorbidities of HMP in clinical settings were recapitulated.

[0135] In the performance of these experiments, skin cells from HMP and healthy patients were collected and reprogrammed to induced pluripotent stem cells (iPSC). iPSC was cultured into neural organoids as outlined in summary of the methods described above. After 12 weeks, whole neural organoids were collected and RNAs extracted therefrom for RNA sequencing with Ampliseq. Designation of neurons, astrocytes, oligodendrocytes, or microglia as shown in Table 3 were determined by matching gene expression patterns from published HMP patient postmortem data in which single cell analysis was performed and the biomarkers were used to benchmark the data to the cell types.

[0136] R-Analysis was performed on the RNA sequencing data according to Yamada et al. (Yamada et al., 2021, J Hum Genet 66, 93–102) to address both the intra-experimental and inter-experimental quality. FIG.6 and FIG.7 shows how the high quality of data across ~13-15,000 genes expressed in the disclosed NNOP model (with R2variance of <0.95). Log2 fold change with a positive value and a negative value indicated a decreaseand an increase in expression, respectively, of an ADRD-HMP-NNOP gene when compared to Normal-NNOP. Thousands of gene expression patterns were found to be significantly altered in ADRD-HMP-NNOPs; however, only a subset of these genes are shown in Tables 3-6. Table 3. Differentially expressed genes (DEG) in NNOP derived from ADRD patients (ADRD-HMP-NNOP) compared to NNOP derived from healthy patients (Normal NNOP). Each table shows ADRD-HMP-NNOP DEGs that are corroborated by ADRD genetics, postmortem data and / or clinical or biochemical pathways. A positive Log 2value indicates expression is higher in the normal-NNOP compared to in ADRD-HMP- SPOR-NNOP and vice versa for a negative Log 2 value.Table 3: AD Pathology: alpha-2 macroglobulin (A2M), a major component of the innate immune system and higher baseline serum A2M concentration is associated with an almost threefold greater risk of progression to clinical symptoms of AD in men (Varma et al., Molecular Psychiatry volume 22,13–23 (2017). BACE 2 is involved in the proteolytic processing of the amyloid precursor protein (APP) (Yan et al., Nature. 1999; 402:533-7; Hussain et al., Mol Cell Neurosci.2000;16:609-19). CP, Ceruloplasmin is involved in the peroxidation of Fe (II) transferrin to Fe (III) transferrin. Mutations in this gene cause aceruloplasminemia, which results in iron accumulation and neurologic abnormalities. Ferroptosis is dependent cell death mechanism has been implicated in the pathogenesis of AD (Chen et al., Front Cell Dev Biol.2021; 9: 704298). Example 4: Neural Organoids for Testing Drug Efficacy

[0137] Neural organoids hold significant promise in pharmaceutical testing, safety, efficacy, and toxicity profiling studies. Specifically, using pharmaceuticals and human neural organoids, beneficial and detrimental genes and pathways associated with a disease can be elucidated.(A) mTOR activator (MHY1485) as a disease modifying drug with human ADRD-HMP-NNOP platform

[0138] Time-resolved transcriptomic data from ADRD-HMP-NNOP identified mTOR mediated pathway as an important upstream driver of HMP DEGs (Table 4). Experiments are set out to test the utility of MHY1485, an mTOR activator, as adisease modifying drug with an ADRD-HMP-NNOP model derived from a sporadic HMP patient.

[0139] In the performance of these experiments, MHY1485 (4,6-dimorpholino-N- (4-nitrophenyl)-1,3,5-triazin-2-amine; solubility in DMSO > 19.4 mg / ml) is a selective MTOR activator (Park et al., Tissue Eng Regen Med 21, 159–169 (2024)). A range of concentrations (dose range: 1-10 μM) EC50 of 2 μM) is tested for a period of 1 week. The DEG results are then statistically analyzed, using as controls vehicle alone (DMSO at 0.1%). The disease biomarker modifying efficacy is expected to have a mechanistic basis in epigenetics and trackable by transcriptomic DEG analysis in response to chronic exposure of MHY1458. Differentially expressed genes (DEGs) are identified to further discern the gene regulatory network responses to 1-10 μM MHY 1485 in human ADRD-HMP-NNOP models. Three replicates from three independent experiments at different doses of MHY1458 will be tested to identify dose range. The experiments are to be performed at Week 4 and 12 of development in culture. Organoids are then harvested after 1 week of exposure.

[0140] A complete description of culture production and characterization of neural organoids has been provided in International (PCT) Application Publication Number WO2017123791A1, which is incorporated by reference in its entirely herein. Changes in gene expression following chronic exposure to dose-dependent levels of MHY1485 are determined in order to identify reliably HMP disease DEG modifying efficacy. MHY1485 is lipophilic and dissolved in DMSO as a vehicle; controls are vehicle alone (DMSO at 0.1%). MHY1458 is tested at dose ranges of 1- 10 μM, with experiments performed at week 4 and 12 in culture. Organoids are harvested after 1 week of exposure (at week 5 and Week 13). Organoids are subject to 10X Genomics single cell analysis.

[0141] RNA from organoids in culture are isolated and processed for library construction and analyzed by AmpliSeq (ThermoFisher) to measure changes in mRNA levels (whole genome gene expression analysis). These assays are performed at time points of 1, 4, and 12 weeks. Changes in gene expression levels will be normalized as CPM (Counts Per Kilo Base per Million reads). Values of <1 are considered background.

[0142] Preliminary quality biostatistical assessment of the Ampliseq data (MA plot), normalization between the samples and identification of the differentially expressed genes are conducted using the Bioconductor packages DESeq2 and edgeR (Love et al.2014, Genome Biol.15(12):550.; Robinson et al., 2010, Bioinformatics 26: 139-140). Differentially expressed genes with p<0.05 are considered significant and further analyzed using the topGO package from Bioconductor (Alexa et al., 2021, Cancers 13: 1478). Heat maps for these differentially expressed genes and other custom visualizations will be created using the ggplot2package in R.

[0143] Time dependent transcriptomics provides a Quality Control measure of reproducibility for each batch of organoid development starting at W1 until maturity is reached at W12. If these QC measures are not met in a batch of organoids, it provides a rationale for rejection of drug-dependent changes if the random sampling of replicates at W1 show variance with an R2 co-efficient of < 0.94-0.95 in any batch.

[0144] Anticipated Results:

[0145] Per statistical analysis (DESeq2 and edgeR – see above), differentially expressed genes with p<0.05 will be significant for MHY 1485. Among these genes we expect to find those that overlap with both GWAS studies and ADRD postmortem studies (Annese et al.2018). These DEGs will serve as HMP IND enabling lead validation results for future clinical trials. Table 4. Differentially expressed genes (DEG) related to mTOR mediated pathway in NNOP derived from ApoE4 patients (ADRD-HMP-NNOP) compared to NNOP derived from healthy patients (Normal NNOP). A positive Log 2 value indicates expression is higher in the normal-NNOP compared to in ADRD-HMP-APOE-NNOP and vice versa for a negative Log 2 value.. Table 5. Differentially expressed genes (DEG) related to mTOR mediated pathway in NNOP derived from ApoE4 patients (ADRD-HMP-NNOP) compared to NNOP derived from healthy patients (Normal NNOP). A positive Log 2 value indicates expression is higher in the normal-NNOP compared to in ADRD-HMP-APOE-NNOP and vice versa for a negative Log 2 value(B) Adenosine receptor 2a agonists with PDE 4 inhibitor with human ADRD-HMP-NNOP platform as disease biomarker modifying drugs

[0146] HMP and Alzheimer’s disease and related dementia (ADRD) have been known to share some biological pathways and clinical symptoms. Indeed, some DEGs in ADRD-HMP overlapped with those of NNOP derived from a patient with sporadic ADRD-HMP compared to healthy patients (Table 3). Particularly, the log2 Fold change values were both negative in ADRD-HMP-SPOR-NNOP, indicating anincrease in expression; or both positive, indicating a decrease in gene expression). The associations of these genes with particular clinical symptoms and / or biological pathways linked to chronic pain, neuropathic pain or HMP are shown in Table 6. Table 6: Differentially expressed genes (DEG) related to pain mediated pathway in NNOP derived from sporadic ADRD patients (ADRD-HMP-SPOR-NNOP) compared to NNOP derived from healthy patients (NORM-NNOP). Migraine: Key migraine biomarkers are GJA1, TGFBR2, GATA3, HPSE2, HEY2, EIF3F, and RERE. Italic Log2 values are the difference between normal and ADRD-HMP-SPOR-NNOP. Bold Log2 values are the difference between ADRD-HMP-SPOR-NNOP control and treated with the CGS-21680 and Roflumilast.

[0147] Migraines also involve changes in brain chemicals and blood vessel activity. Vasodilation in the brain’s blood vessels is one of the factors that can contribute to migraine pain (D’Andrea et al., Neurol Sci 43, 2745–2749 (2022). The dysfunctional glymphatic system lead to accumulation of metabolic waste and can trigger a migraine attack. During a migraine attack, the activation of the trigeminal system’s receptors on the vessel wall leads to the release of nitric oxide from the endothelium. This release causes vasodilation and stretching of the vascular trigeminal system, which promotes the pain associated with a migraine.(C) Confirmation of changes in protein expression level and metabolic profiledriven by DEGs in ADRD-HMP-NNOP

[0148] To confirm that changes in RNA levels also reflect changes at the protein level, expression of protein encoded by DEGs in ADRD-MHP-NNOP with and without drug treatment are measured using Western capillary immunoassays. An example of the experiment is shown in FIG.4. Samples are separated through a stacking and separation matrix. Proteins are immobilized via a photo-activated capture chemistry. Proteins are identified with primary antibody and HRP-conjugated secondary Ab, and chemiluminescent substrate / signal is quantitated.

[0149] Additionally, the metabolomics signatures induced by mTOR activator and ADORA2A receptor agonist in combination with a PDE4 inhibitor in ADRD-MHP- NNOP derived from various donors are performed using 1H-NMR-based metabolomics (FIG.5). Multiple lines of evidence suggest that arginine metabolism may be an important driver of migraine pathology (D’Andrea, G et al., Neurol Sci 43, 2745–2749, 2022). The DEG data from ADRD-HMP-NNOP shows dysregulation of gene expression for ARG2. The change in ARG2 gene expression was mechanistically consistent with changes with altered arginine levels reported in ADRD-HMP patient brains as a consequence of changes in the corresponding protein levels and their functions and validates the use of ADRD-HMP-NNOP for clinical diagnostic milestones of metabolic biomarkers for HMP and for tracking efficacy of therapeutic agent efficacy.Other Embodiments:

[0150] From the foregoing description, it will be apparent that variations and modifications may be made to the invention described herein to adopt it to various usages and conditions. Such embodiments are also within the scope of the following claims.

[0151] The recitation of a listing of elements in any definition of a variable herein includes definitions of that variable as any single element or combination (or sub- combination) of listed elements. The recitation of an embodiment herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.

[0152] All patents and publications mentioned in this specification are herein incorporated by reference to the same extent as if each independent patent and publication was specifically and individually indicated to be incorporated by reference.

[0153] was specifically and individually indicated to be incorporated by reference.

[0154] Having described the invention in detail and by reference to specific aspects and / or embodiments thereof, it will be apparent that modifications and variations are possible without departing from the scope of the invention defined in the appended claims. More specifically, although some aspects of the present invention may be identified herein as particularly advantageous, it is contemplated that the present invention is not limited to these particular aspects of the invention. Percentages disclosed herein may vary in amount by ±10, 20, or 30% from values disclosed and remain within the scope of the contemplated invention.REFERENCES 1. Buse, D.C., Reed, M.L., Fanning, K.M. et al. Comorbid and co-occurringconditions in migraine and associated risk of increasing headache pain intensity and headache frequency: results of the migraine in America symptoms and treatment (MAST) study. J Headache Pain 21, 23 (2020). 2. Jatin Gupta and Sagar S Gaurkar. Migraine: An Underestimated NeurologicalCondition Affecting Billions. Cureus.2022 Aug; 14(8): e28347. 3. Women’s health research lacks funding – these charts show how4. Lucas C. Migraine with aura. Rev Neurol (Paris). 2021 Sep;177(7):779-784.5. Charlene Bron, Heidi G Sutherland and Lyn R Griffith. Exploring theHereditary Nature of Migraine. Neuropsychiatr Dis Treat.2021; 17: 1183–1194. 6. Heidi Hautakangas et. al., Genome-wide analysis of 102,084 migraine casesidentifies 123 risk loci and subtype-specific risk alleles. Nat Genet.2022 Feb;54(2):152-160. 7. Nasser M Alorfi. Pharmacological Methods of Pain Management: NarrativeReview of Medication Used. Int J Gen Med.2023; 16: 3247–3256. 8. Eptinezumab-jjmr (VYEPTI™) Approved By FDA for Migraine Prevention9. Leonardo Biscetti, Elena Cresta, Letizia Maria Cupini, Paolo Calabresi, PaolaSarchielli. The putative role of neuroinflammation in the complex pathophysiology of migraine: From bench to bedside. Neurobiol Dis.2023 May:180:106072. 10. Jatin Gupta and Sagar S Gaurkar. Animal models of migraine andexperimental techniques used to examine trigeminal sensory processing. Cureus. 2022 Aug; 14(8): e28347. 11. J M Hansen, L L Thomsen, J Olesen, M Ashina. Familial hemiplegic migrainetype 1 shows no hypersensitivity to nitric oxide. Cephalalgia.2008 May;28(5):496-505.12. Carlos Eduardo G. Amorim , Ziyue Gao,Zachary Baker, José FranciscoDiesel, Yuval B. Simons, Imran S. Haque,Joseph Pickrell ,Molly Przeworski. The population genetics of human disease: The case of recessive, lethal mutations. PLOS Genetics 14(7): e1007499.13. Boyan Bonev & Giacomo Cavalli. Organization and function of the 3Dgenome. Nature Reviews Genetics volume 17, 661–678 (2016).14. Chiara Anania, Darío G Lupiáñe. Order and disorder: abnormal 3D chromatinorganization in human disease. Briefings in Functional Genomics, Volume 19, Issue 2, March 2020, 128–138.15. Bellenguez, C., Küçükali, F., Jansen, I.E. et al. New insights into the geneticetiology of Alzheimer’s disease and related dementias. Nat Genet 54, 412–436 (2022).16. Annese A, Manzari C, Lionetti C, Picardi E, Horner DS Chiara M, CaratozzoloMF, Tullo A, Fosso B, Pesole G, D'Erchia AM. Whole transcriptome profiling of Late-Onset Alzheimer's Disease patients provides insights into the molecular changes involved in the disease. Sci Rep.2018 Mar 9;8(1):4282.17. Andrews MG and Kriegstein, AR. Challenges of Organoid Research. AnnuRev Neurosci.2022 Jul 8:45:23-39.18. Abhirami Ratnakumar, Samuel E Zimmerman, Bryen A Jordan, Jessica CMar Estrogen activates Alzheimer’s disease genes. Alzheimers Dement (N Y). 2019 Dec 9:5:906-917.

Claims

WHAT IS CLAIMED IS:

1. A method for reducing or ameliorating disease severity in a patient withHeadache and Migraine Pain (HMP) or glymphatic system dysfunction comprising administering to the individual a therapeutically effective amount of a therapeutic agent capable of reducing or correcting in vitro differentially expressed genes, dysregulated protein expression / function, or dysregulated metabolite production in a neural organoid derived from the patient with HMP compared to a neural organoid derived from a healthy patient and reducing or ameliorating disease severity in the HMP patient thereby.

2. The method of claim 1, wherein the differentially expressed genes for whichthe therapeutic agent is capable of reducing or correcting are set forth in Tables 3-6.

3. The method of claim 1, wherein the neural organoid is obtained from inducedpluripotent stem cell that is reprogrammed from a cell sample derived from the patient with HMP.

4. The method of claim 3, wherein the neural organoid is procured from minutesto hours up to 15 weeks post inducement.

5. The method of claim 1, wherein the therapeutic agent is an mTOR activator.

6. The method of claim 5, wherein the mTOR activator is MHY1485.

7. The method of claim 5, wherein the mTOR activator is co-administered withan mTOR inhibitor.

8. The method of claim 7, wherein the mTOR inhibitor is rapamycin, everolimus,temsirolimus, or other rapalogues.

9. The method of claim 1, wherein the therapeutic agent is a ADORA2Areceptor agonist.

10. The method of claim 1, wherein the therapeutic agent is a PDE4 inhibitor.

11. The method of claim 1, wherein the therapeutic agent is a PDE4 inhibitor used with a ADORA2A receptor agonist.

12. The method of claim 1, wherein the therapeutic agent is an RNA-based therapeutics.

13. The method of claim 12, wherein the RNA-based therapeutics is antisense oligonucleotide (ASO), Adeno-Associated Virus delivery of small interfering RNA (AAV-RNA), or micro RNA (miRNA).

14. The method of claim 13, wherein the differentially expressed genes for which the RNA-based therapeutics is capable of reducing or correcting are set forth in Table 3-6.

15. The method of claim 1, wherein the patient with HMP shows comorbidities listed in Table 4.

16. The method of claim 15, wherein the comorbidities are associated with the differentially expressed genes listed in Table 3.

17. A pharmaceutical composition comprising a therapeutically effective amount of a therapeutic agent capable of reducing or correcting in vitro differentially expressed genes, dysregulated protein expression / function, or dysregulated metabolite production in a neural organoid derived from the patient with MHP compared to a neural organoid derived from a healthy patient, and a pharmaceutically acceptable carrier; wherein the pharmaceutical composition is capable of reducing or ameliorating disease severity in the patient.

18. The pharmaceutical composition of claim 17, wherein the differentially expressed genes for which the therapeutic agent is capable of reducing or correcting are set forth in Tables 3-6.

19. The pharmaceutical composition of claim 17, wherein the neural organoid is obtained from induced pluripotent stem cell that is reprogrammed from a cell sample derived from the patient with MHP.

20. The pharmaceutical composition of claim 19, wherein the neural organoid is procured from minutes to hours up to 15 weeks post inducement.

21. The pharmaceutical composition of claim 17, wherein the therapeutic agent is an mTOR activator.

22. The pharmaceutical composition of claim 21, wherein the mTOR activator is MHY1485.

23. The pharmaceutical composition of claim 21, further comprising an mTOR inhibitor.

24. The pharmaceutical composition of claim 23, wherein the mTOR inhibitor is rapamycin, everolimus, temsirolimus, or other rapalogues.

25. The pharmaceutical composition of claim 17, wherein the the drug is methyl (1R,4r)-4-(3-(6-amino-9-((2R,3R,4S,5S)-5-(ethylcarbamoyl)-3,4- dihydroxytetrahydrofuran-2-yl)-9H-purin-2-yl)prop-2-yn-1-yl)cyclohexane-1- carboxylate (apadenoson).

26. The pharmaceutical composition of claim 17, wherein the drug is 2-p-(2- Carboxyethyl) phenethylamino-5 -N-ethylcarboxamidoadenosinehydrochloride hydrate (CGS-21680).

27. The pharmaceutical composition of claim 17, wherein the ADORA2A receptor agonist is co-administered with an PDE4 inhibitor that is Roflumilast.

28. The pharmaceutical composition of claim 17, wherein the therapeutic agent is a RNA-based therapeutics.

29. The method of claim 28, wherein the RNA-based therapeutics is antisense oligonucleotide (ASO), Adeno-Associated Virus delivery of small interfering RNA (AAV-RNA), or micro RNA (miRNA).

30. The method of claim 29, wherein the differentially expressed genes for which the RNA-based therapeutics is capable of reducing or correcting are set forth in Table 3-6.

31. The pharmaceutical composition of claim 17, wherein the patient with ADRD shows comorbidities listed in Table 3.

32. The pharmaceutical composition of claim 31, wherein the comorbidities are associated with the differentially expressed genes listed in Table 3-6.

33. A method comprising: a) procuring one or a plurality of cell samples from a patient with HMP or at risk for developing HMP, comprising one or a plurality of cell types; b) reprogramming the one or the plurality of cell samples to produce one or a plurality of induced pluripotent stem cell samples; c) treating the one or the plurality of induced pluripotent stem cell samples to obtain a neural organoid; d) collecting a biological sample from the neural organoid; e) detecting differentially expressed genes, dysregulated protein expression / function, or dysregulated metabolite generation in the neural organoid sample compared to a neural organoid sample obtained from a healthy patient; and f) administering one or more therapeutic agents capable of reducing or correcting the differentially expressed genes, dysregulated protein expression / function, or dysregulated metabolite generation to the patient with HMP or at risk for developing HMP, wherein the method is capable of treating HMP or preventing development of HMP.

34. The method of claim 33, wherein the cell types are fibroblasts, buccal cavity cells, or peripheral blood monocytes.

35. The method of claim 34, wherein step (e) further comprises identifying differentially expressed genes, dysregulated protein expression / function, or dysregulated metabolite generation in the neural organoid from the patient with HMP or at risk for developing HMP that are known to associate with HMP, or with clinical symptoms or comorbidities of HMP.

36. The method of claim 35, further comprising administering one or more therapeutic agents capable of reducing or correcting the identified differentially expressed genes, dysregulated protein expression / function, or dysregulated metabolite generation to the patient with HMP or at risk for developing HMP.

37. The method of claim 35, further comprising administering one or more therapeutic agents capable of reducing or correcting the identified differentially expressed genes, dysregulated protein expression / function, or dysregulated metabolite generation to the patient with chronic or neuropathic pain associated with cancer.

38. The method of claim 33, wherein the differentially expressed genes for which the one or more therapeutic agents is capable of reducing or correcting are set forth in Tables 3-6.

39. The method of claim 33, wherein the therapeutic agent is an mTOR activator.

40. The method of claim 38, wherein the mTOR activator is MHY1485.

41. The method of claim 33, wherein the mTOR activator is co-administered with an mTOR inhibitor is rapamycin, everolimus, temsirolimus or other rapalogues.

42. The method of claim 33, wherein the mTOR activator is co-administered with an mTOR inhibitor43. The method of claim 33, wherein the drug is methyl (1R,4r)-4-(3-(6-amino-9- ((2R,3R,4S,5S)-5-(ethylcarbamoyl)-3,4-dihydroxytetrahydrofuran-2-yl)-9H- purin-2-yl)prop-2-yn-1-yl)cyclohexane-1-carboxylate (apadenoson).

44. The method of claim 33, wherein the drug is 2-p-(2-Carboxyethyl) phenethylamino-5 -N-ethylcarboxamidoadenosine hydrochloride hydrate(CGS-21680).

45. The method of claim 33, wherein the ADORA2A receptor agonist is co- administered with an PDE4 inhibitor.

46. The method of claim 33, wherein the ADORA2A receptor agonist is co- administered with an PDE4 inhibitor that is Roflumilast.

47. The method of claim 33, wherein the ADORA2A receptor agonist is co- administered with an PDE4 inhibitor that is AWD-12-281, Elbion, Tofimilast, UK-500,001, GSK256066, SCH900182, SCH900182, Almirall, Lotamilast, Ensifentrine / RPL554, or CHF 6001, 48. The method of claim 33, wherein the therapeutic agent is a RNA-based therapeutics.

49. The method of claim 46, wherein the RNA-based therapeutics is antisense oligonucleotide (ASO), Adeno-Associated Virus delivery of small interfering RNA (AAV-RNA), or micro RNA (miRNA). 50 The method of claim 47, wherein the differentially expressed genes for which the RNA-based therapeutics is capable of reducing or correcting are set forth in Table 3-6.

51. The method of claim 33, wherein the at least one cell sample reprogrammed to the induced pluripotent stem cell is a fibroblast. 5452. The method of claim 33, wherein the neural organoid is about twelve weeks post-inducement and comprises the encoded structures and cell types of the retina, cortex, midbrain, hindbrain, brain stem, and spinal cord.

53. The method of claim 33, wherein the neural organoid sample is procured after about one week post inducement, four-weeks post inducement, and / or 12 weeks post inducement.

54. The method of claim 33, wherein the neural organoid sample is procured from structures of the neural organoid that mimic the structures in utero at about 5 weeks.

55. A method comprising: a) collecting a biological sample from a patient with HMP or at risk for developing HMP; b) quantifying production of metabolites from the biological sample that are significantly altered compared to a biological sample collected from a healthy patient; and c) administering one or more therapeutic agents capable of reducing or correcting the altered metabolites to the patient with HMP or at risk for developing HMP, wherein the method is capable of treating HMP or preventing development of HMP.

56. The method of claim 55, wherein the biological sample is urine, plasma, or cerebrospinal fluid.

Images