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WO2021096929A1 - Exosomes humains dérivés de neurones pour la maladie d'alzheimer et les co-morbidités de ceux-ci - Google Patents

Exosomes humains dérivés de neurones pour la maladie d'alzheimer et les co-morbidités de ceux-ci Download PDF

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WO2021096929A1
WO2021096929A1 PCT/US2020/059971 US2020059971W WO2021096929A1 WO 2021096929 A1 WO2021096929 A1 WO 2021096929A1 US 2020059971 W US2020059971 W US 2020059971W WO 2021096929 A1 WO2021096929 A1 WO 2021096929A1
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disease
alzheimer
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Rene ANAND
Susan MCKAY
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    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/48—Reproductive organs
    • A61K35/54—Ovaries; Ova; Ovules; Embryos; Foetal cells; Germ cells
    • A61K35/545—Embryonic stem cells; Pluripotent stem cells; Induced pluripotent stem cells; Uncharacterised stem cells
    • A—HUMAN NECESSITIES
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/30—Nerves; Brain; Eyes; Corneal cells; Cerebrospinal fluid; Neuronal stem cells; Neuronal precursor cells; Glial cells; Oligodendrocytes; Schwann cells; Astroglia; Astrocytes; Choroid plexus; Spinal cord tissue
    • A—HUMAN NECESSITIES
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
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    • C12N2502/00—Coculture with; Conditioned medium produced by
    • C12N2502/13—Coculture with; Conditioned medium produced by connective tissue cells; generic mesenchyme cells, e.g. so-called "embryonic fibroblasts"
    • C12N2502/1323—Adult fibroblasts
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    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • This disclosure relates to production and use of human stem cell derived neural organoids to identify patients with Alzheimer’s disease and Alzheimer's disease patient treatment using patient-specific pharmacotherapy. Further disclosed are patient-specific pharmacotherapeutic methods for reducing risk for developing Alzheimer’s disease- associated co-morbidities in a human. Also disclosed are methods to predict onset risk of Alzheimer's disease (and identified comorbidities) in an individual.
  • inventive processes disclosed herein provide neural organoid reagents produced from an individual's induced pluripotent stem cells (iPSCs) for identifying patient-specific pharmacotherapy, predictive biomarkers, and developmental and pathogenic gene expression patterns and dysregulation thereof in disease onset and progression, and methods for diagnosing prospective and concurrent risk of development or establishment of Alzheimer’s disease (and comorbidities) in the individual.
  • 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 Alzheimer’s disease.
  • 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.
  • the neural organoid is patient specific.
  • such models provide a method for studying neurological diseases and disorders that overcome previous limitations. Accordingly, there is a need in the art to develop patient-specific reagents, therapeutic modalities, and methods based on predictive biomarkers for diagnosing and/or treating current and future risk of neurological diseases including Alzheimer’s disease.
  • This disclosure provides neural reagents and methods for treating Alzheimer’s disease in a human, using patient-specific pharmacotherapies, the methods comprising: procuring one or a plurality of cell samples from a human, comprising one or a plurality of cell types; reprogramming the one or the plurality of cell samples to produce one or a plurality of induced pluripotent stem cell samples; treating the one or the plurality of induced pluripotent stem cell samples to obtain one or more patient specific neural organoids; collecting a biological sample from the patient specific neural organoid; detecting changes in Alzheimer’s disease biomarker expression from the patient specific neural organoid sample that are differentially expressed in humans with Alzheimer’s disease; performing assays on the patient specific neural organoid to identify therapeutic agents that alter the differentially expressed Alzheimer’s disease biomarkers in the patient-specific neural organoid sample; and administering a therapeutic agent for Alzheimer’s disease to treat the human.
  • At least one cell sample reprogrammed to the induced pluripotent stem cell is a fibroblast derived from skin or blood cells from humans.
  • the fibroblast derived skin or blood cells from humans is identified with the genes identified in Table 1 (Novel Alzheimer’s disease Biomarkers), Table 2 (Biomarkers for Alzheimer’s disease), Table 5 (Alzheimer’s disease Therapeutic Neural Organoid Authentication Genes), or Table 7 (Genes and Accession Numbers for Co-Morbidity Susceptibility / Resistance Associated with Alzheimer’s disease).
  • the measured biomarkers comprise nucleic acids, proteins, or their metabolites.
  • the measured biomarkers comprise one or a plurality of biomarkers identified in Table 1 , Table 2, Table 5 or Table 7 or variants thereof.
  • a combination of biomarkers is detected, the combination comprising a nucleic acid encoding human A2M, APR variants and one or a plurality of biomarkers comprising a nucleic acid encoding human genes identified in Table 1.
  • the biomarkers for Alzheimer’s disease include human nucleic acids, proteins, or their metabolites as listed in Table 1.
  • sequence data for the genes listed above can be obtained in publicly available gene databases such as GeneCards, GenBank, Malcard, Uniport and PathCard databases.
  • the neural organoid biological sample is collected after about one hour up to about 12 weeks post inducement.
  • the neural organoid sample is procured from structures of the neural organoid that mimic structures developed in utero at about 5 weeks.
  • the neural organoid at about twelve weeks post-inducement comprises structures and cell types of retina, cortex, midbrain, hindbrain, brain stem, or spinal cord.
  • the neural organoid contains microglia, and one or a plurality of Alzheimer’s disease biomarkers as identified in Table 1 and Table 7.
  • the method is used to detect environmental factor susceptibility including infectious agents that cause or exacerbate Alzheimer's disease, or accelerators of Alzheimer's disease.
  • the method is used to identify nutritional factor deficiency susceptibility or supplements for treating Alzheimer’s disease.
  • the nutritional factor or supplement is for glucose dyshometostasis or other nutritional factors related to pathways (Pathcards database; Weizmann Institute of Science) regulated by genes identified in Tables 1, 2, 5 or 7.
  • fetal cells from amniotic fluid can be used to grow neural organoids and as such nutritional and toxicological care can begin even before birth so that the child develops in utero well.
  • the disclosure provides methods for reducing risk of developing Alzheimer’s disease associated co-morbidities in a human comprising procuring one or a plurality of cell samples from a human, comprising one or a plurality of cell types; reprogramming the one or the plurality of cell samples to produce one or a plurality of induced pluripotent stem cell samples; treating the one or the plurality of induced pluripotent stem cell samples to obtain one or more patient specific neural organoids; collecting a biological sample from the patient specific neural organoid; detecting biomarkers of an Alzheimer’s disease related co-morbidity in the patient specific neural organoid sample that are differentially expressed in humans with Alzheimer’s disease; and administering an anti- Alzheimer’s or anti co-morbidity therapeutic agent to the human.
  • the measured biomarkers comprise biomarkers identified in Table 1, Table 2, Table 5 or Table 7 and can be nucleic acids, proteins, or their metabolites (identifiable in GeneCards and PathCard databases).
  • the invention provides diagnostic methods for predicting risk for developing Alzheimer’s disease in a human, comprising one or a plurality subset of the biomarkers as identified in Table 1, Table 2, Table 5, or Table 7.
  • the subset of measured biomarkers comprise nucleic acids, proteins, or their metabolites as identified in Table 1 , Table 2, Table 5 or Table 7.
  • the biomarkers can be co related to disease onset, progression, and severity and include glucose, and cholesterol mentabolism.
  • the method and/or neural organoid has uses in guided and patient specific toxicology guided by genes from patient’s selective vulnerabililty to infectious agents or to accumulate currently EPA approved safe levels of copper.
  • methods for detecting at least one biomarker of Alzheimer’s disease, the method comprising, obtaining a biological sample from a human patient; and contacting the biological sample with an array comprising specific-binding molecules for the at least one biomarker and detecting binding between the at least one biomarker and the specific binding molecules.
  • the biomaker detected is a gene therapy target.
  • the disclosure provides a kit comprising an array containing sequences of biomarkers from Table 1 or Table 2 for use in a human patient.
  • the kit further contains reagents for RNA isolation and biomarkers for Alzheimer’s disease.
  • the kit further advantageously comprises a container and a label or instructions for collection of a sample from a human, isolation of cells, inducement of cells to become pluripotent stem cells, growth of patient-specific neural organoids, isolation of RNA, execution of the array and calculation of gene expression change and prediction of concurrent or future disease risk.
  • the biomarkers for Alzheimer’s disease include human nucleic acids, proteins, or their metabolites as listed in Table 1.
  • the biomarkers can include biomarkers listed in Table 2.
  • biomarkers can comprise any markers or combination of markers in Tables 1 and 2 or variants thereof.
  • sequence data for the genes listed above can be obtained in publicly available gene databases such as GeneCards, GenBank, Malcard, Uniport and PathCard databases.
  • the disclosure provides a method for detecting one or a plurality of biomarkers from different human chromosomes associated with Alzheimer's disease or Alzheimer's disease comorbidity susceptibility using data analytics that obviates the need for whole genome sequence analysis of a person or patient’s genome.
  • the methods are used to determine gene expression level changes that are used to identifly clinically relevant symptoms and treatments, time of disease onset, and disease severity.
  • the neural organoids are used to identify novel biomarkers that serve as data input for development of algorithm techniques as predictive analytics.
  • the algorithmic techniques include artificial intelligence, machine and deep learning as predictive analytics tools for identifying biomarkers for diagnostic, therapeutic target and drug development process for disease.
  • the neural neural organoid along with confirmatory data, and novel data can be used to develop signature algorithms with machine learning, artifiical intelligence and deep learning.
  • the method is used for diagnostic, therapeutic target discovery and drug action discovery for Alzheimer’s disease and Alzheimer’s disease related comorbidites as listed in Table 7.
  • the inventive model neural organoid data is corroborated in post mortem tissues from idiopathic patients and extensively identifies known biomarkers for Alzheimer’s disease and comorbidities.
  • the method can be used with induced pluripotent stem cells from any skin cell, tissue, or organ from the human body allowing for an all encompassing utility for diagnostics, therapeutic target discovery, and drug development.
  • the invention provides methods for predicting a risk comorbidity onset that accompanies Alzheimer’s disease. Said methods first determines gene expression changes in neural organoids from a normal human individual versus a human individual with Alzheimer's disease. Genes that change greater than 1.4 fold are associated with co-morbidities as understood by those skilled in the art.
  • kits for predicting the risk of current or future onset of Alzheimer’s disease provide kits for predicting the risk of current or future onset of Alzheimer’s disease. Said kits provide reagents and methods for identifying from a patient sample gene expression changes for one or a plurality of disease- informative genes for individuals without a neurological disease that is Alzheimer’s disease.
  • the invention provides methods for identifying therapeutic agents for treating Alzheimer’s disease. Such embodiments comprise using the neural organoids provided herein, particularly, but not limited to said neural organoids from iPSCs from an individual or from a plurality or population of individuals.
  • the inventive methods include assays on said neural organoids to identify therapeutic agents that alter disease-associated changes in gene expression of genes identified as having altered expression patterns in disease, so as to express gene expression patterns more closely resembling expression patterns for disease-informative genes for individuals without a neurological disease that is Alzheimer's disease.
  • the invention provides methods for predicting a risk for developing Alzheimer’s disease in a human, comprising procuring one or a plurality of cell samples from a human, comprising one or a plurality of cell types; reprogramming the one or the plurality of cell samples to produce one or a plurality of induced pluripotent stem cell samples; treating the one or the plurality of induced pluripotent stem cell samples to obtain one or more patient specific neural organoids; collecting a biological sample from the patient specific neural organoid; measuring biomarkers in the neural organoid sample; and detecting measured biomarkers from the neural organoid sample that are differentially expressed in humans with Alzheimer’s disease.
  • the at least one cell sample reprogrammed to the induced pluripotent stem cell is a fibroblast.
  • the measured biomarkers comprise nucleic acids, proteins, or their metabolites.
  • the measured biomarker is a nucleic acid encoding human A2M and APP variants.
  • the measured biomarkers comprise one ora plurality of genes as identified in Tables 1, 2, 5 or 6.
  • the neural organoid sample is procured from minutes to hours up to 15 weeks post inducement.
  • the biomarkers to be tested are one or a plurality of biomarkers in Tables 5 or 6 (Alzheimer’s disease Diagnostic Neural Organoid Authentication Genes).
  • Fig 1A is a micrograph showing a 4X dark field image of Brain Organoid Structures typical of approximately 5-week in utero development achieved in 12 weeks in vitro. Average size: 2-3 mm long. A brain atlas is provided for reference (left side).
  • FIG. 1 B shows immuno-fluorescence images of sections of iPSC-derived human brain organoid after approximately 12 weeks in culture.
  • Z-stack of thirty-three optical sections, 0.3 microns thick were obtained using laser confocal imaging with a 40X lens. Stained with Top panel: beta III tubulin (green: axons); MAP2 (red: dendrites); Hoechst (blue: nuclei); Bottom panel: Doublecortin (red).
  • FIG. 2 is a micrograph showing immunohistochemical staining of brain organoid section with the midbrain marker tyrosine hydroxylase.
  • Paraformaldehyde fixed sections of a 8- week old brain organoid was stained with an antibody to tyrosine hydroxylase and detected with Alexa 488 conjugated secondary Abs (green) and counter stained with Hoechst to mark cell nuclei (blue).
  • FIG. 3 Spinning disc confocal image (40X lens) of section. Astrocytes stained with GFAP (red) and mature neurons with NeuN (green).
  • FIG. 4 is a schematic showing in the upper panel a Developmental Expression Profile for transcripts as Heat Maps ofNKCC 1 and KCC2 expression at week 1, 4 and 12 of organoid culture as compared to approximate known profiles (lower panel).
  • NKCCI Na(+)- K(+)-CI(-) cotransporter isoform 1.
  • KCC2 K(+)-CI(-) cotransporter isoform 2.
  • Fig 5A is a schematic showing GABAergic chloride gradient regulation by NKCC 1 and KCC2.
  • FIG. 5B provides a table showing a representative part of the entire transcriptomic profile of brain organoids in culture for 12 weeks measured using a transcriptome sequencing approach that is commercially available (AmpliSeqTM).
  • the table highlights the expression of neuronal markers for diverse populations of neurons and other cell types that are comparable to those expressed in an adult human brain reference (HBR; Clontech) and the publicly available embryonic human brain (BRAINS CAN) atlas of the Allen Institute database.
  • HBR adult human brain reference
  • BRAINS CAN publicly available embryonic human brain
  • FIG. 5C provides a table showing AmpliSeq Tm gene expression data comparing gene expression in an organoid (column 2) at 12 weeks in vitro versus Human Brain Reference (HBR; column 3). A concordance of greater than 98% was observed.
  • FIG. 5D provides a table showing AmpliSeqTM gene expression data comparing organoids generated during two independent experiments after 12 weeks in culture (column
  • FIG. 6A is a schematic showing results of developmental transcriptomics. Brain organoid development in vitro follows KNOWN Boolean logic for the expression pattern of transcription factors during initiation of developmental programs of the brain. Time Points: 1, 4, and 12 Weeks. PITX3 and NURRI (NR4A) are transcription factors that initiate midbrain development (early; at week 1), DLKI, KLHLI, PTPRU, and ADH2 respond to these two transcription factors to further promote midbrain development (mid; at week 4 &12), and TH, VMAT2, DAT and D2R define dopamine neuron functions mimicking in vivo development expression patterns.
  • PITX3 and NURRI are transcription factors that initiate midbrain development (early; at week 1), DLKI, KLHLI, PTPRU, and ADH2 respond to these two transcription factors to further promote midbrain development (mid; at week 4 &12), and TH, VMAT2, DAT and D2R define dopamine neuron functions mimicking in vivo
  • the organoid expresses genes previously known to be involved in the development of dopaminergic neurons (Blaess S, Ang SL. Genetic control of midbrain dopaminergic neuron development. Wiley Interdiscip Rev Dev Biol. 2015 Jan 6. doi: 10.1002/wdev. I69).
  • FIGs. 6B-6D are tables showing AmpliSeqTM gene expression data for genes not expressed in organoid (column 2 in 6B, 6C, and 6D) and Human Brain Reference (column
  • FIG. 7 includes schematics showing developmental heat maps of transcription factors (TF) expressed in cerebellum development and of specific Markers GRID 2.
  • FIG. 8 provides a schematic and a developmental heat map of transcription factors expressed in Hippocampus Dentate Gyms.
  • FIG. 9 provides a schematic and a developmental heat map of transcription factors expressed in GABAergic Intemeuron Development. GABAergic Intemeurons develop late in vitro.
  • FIG. 10 provides a schematic and a developmental heat map of transcription factors expressed in Serotonergic Raphe Nucleus Markers of the Pons.
  • FIG. 11 provides a schematic and a developmental heat map of transcription factor transcriptomics (FIG. 11 A). Hox genes involved in spinal cord cervical, thoracic, and lumbar region segmentation are expressed at discrete times in utero. The expression pattern of these Hoxgene in organoids as a function of in vitro developmental time (1 week; 4 weeks; 12 weeks; ; Figures 11 B and 11C)
  • FIG. 12 is a graph showing the replicability of brain organoid development from two independent experiments. Transcriptomic results were obtained by Ampliseq analysis of normal 12-weekoti brain organoids. The coefficient of determination was 0.6539.
  • FIG. 13 provides a schematic and gene expression quantification of markers for astrocytes, oligodendrocytes, microglia, and vasculature cells.
  • FIG. 14 shows developmental heat maps of transcription factors (TF) expressed in retina development and other specific Markers. Retinal markers are described, for example, in Farkas et al. BMC Genomics 2013, 14:486.
  • FIG. 15 shows developmental heat maps of transcription factors (TF) and Markers expressed in radial glial cells and neurons of the cortex during development
  • FIG. 16 is a schematic showing the brain organoid development in vitro.
  • iPSC stands for induced pluripotent stem cells.
  • NPC stands for neural progenitor cell.
  • FIG. 17 is a graph showing the replicability of brain organoid development from two independent experiments.
  • FIGS. 18A and 18B are tables showing the change in the expression level of certain genes in APP gene duplication organoid.
  • FIG. 19 is human genetic and postmortem brain analysis published data that independently corroborate biomarkers predicted from the Alzheimer’s disease neural organoid derived data, including novel changes in microglial functions increasing susceptibility to infectious agents in Alzheimer's disease.
  • the terms “or” and “and/or * are utilized to describe multiple components in combination or exclusive of one another.
  • “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.”
  • “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.
  • 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 and develops similaly 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.
  • Neural markers are any protein or polynucleotide expressed consistent with a cell lineage.
  • “heural marker” it is meant any protein or polynucleotide, the expression of which is associated with a neural cell fate.
  • Exemplary neural markers include markers associated with the hindbrain, midbrain, forebrain, or spinal cord.
  • neural markers are representative of the cerebrum, cerebellum and brainstem regions.
  • Exemplary brain structures that express neural markers include the cortex, hyopthalamus, thalamus, retina, medulla, pons, and lateral ventricles.
  • neuronal markers 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.
  • this list is not all encompassing and that neural markers are found throughout the central nervous system including other brain regions, structures, and cell types.
  • Exemplary cerebellar markers include but are not limited to ATOH1 , PAX6, SOX2, LHX2, and GRID2.
  • 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 cortical markers include, but are not limited to, doublecortin, NeuN, FOXP2, CNTN4, and TBR1.
  • Exemplary retinal markers include but are not limited to retina specific Guanylate Cyclases (GUY2D, GUY2F), Retina and Anterior Neural Fold Homeobox (RAX), and retina specific Amine Oxidase, Copper Containing 2 (RAX).
  • Exemplary granular neuron markers include, but are not limited to SOX2, NeuroDI , DCX, EMX2, FOXG1I, and PROX1.
  • Exemplary brain stem markers include, but are not limited to FGF8, INSM1 , GATA2, ASCLI, GATA3.
  • Exemplary spinal cord markers include, but are not limited to homeobox genes including but not limited to HOXA1 ,
  • GABAergic markers include, but are not limited to NKCCI or KCC2.
  • Exemplary astrocytic markers include, but are not limited to GFAP.
  • Exemplary oliogodendrocytic markers include, but are not limited to OLIG2 or MBP.
  • Exemplary microglia markers include, but are not limited to AIF1 or CD4.
  • the measured biomarkers listed above have at least 70% homology to the sequences in the Appendix. One skilled in the art will understand that the list is exemplary and that additional biomarkers exist.
  • Diagnostic or informative alteration or change in a biomarker is meant as an increase or decrease in expression level or activity of a gene or gene product as detected by conventional methods known in the art such as those described herein.
  • 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.
  • 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 the gene product.
  • the change may or may not have an impact (negative or positive) on activity of the gene.
  • Neural organoids are generated in vitro from patient tissue samples. Neural organoids were previously disclosed in WO2017123791 A1 (https://patents.google.com/patent/WO2017123791A1/en), incorporated herein, in its entirety. A variety of tissues can be used 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. Use of neural organoids permits study of neural development in vitro. 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.
  • iPSC embryonic-like pluripotent stem cell
  • 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.
  • neural organoids develop anatomical features of the retina, forebrain, midbrain, hindbrain, and spinal cord.
  • neural organoids express >98% of the about 15,000 transcripts found in the adult human brain.
  • iPSCs can be derived from the skin or blood cells of humans identified with the genes listed in Table 1 (Novel Markers of Alzheimer's disease), Table 2 (Markers of Alzheimer’s disease), Table 5 (Neural Organoid Alzheimer’s disease Authenticating Genes) and Table 7 (Comorbidities of Alzheimer’s disease).
  • the about 12-week old iPSC-derived human neural organoid has ventricles and other anatomical features characteristic of a 35-40 day old neonate.
  • 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.
  • the neural organoid displays laminar organization of cortical structures. Cells within the laminar structure 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.
  • neural organoids permit study of human neural development in vitro. Further, the neural organoid offers the advantages of replicability, reliability and robustness, as shown herein using replicate neural organoids from the same source of iPSCs.
  • a “transcriptome” is a collection of all RNA, including messenger RNA (mRNA), long non-coding RNAs (IncRNA), microRNAs (miRNA) and, small nucleolar RNA snoRNA), other regulatory polynucleotides, and regulatory RNA (IncRNA, miRNA) molecules expressed from the genome of an organism through transcription therefrom.
  • mRNA messenger RNA
  • IncRNA long non-coding RNAs
  • miRNA microRNAs
  • small nucleolar RNA snoRNA small nucleolar RNA snoRNA
  • IncRNA, miRNA regulatory RNA
  • transcriptomics employs high-throughput techniques to analyze genome expression changes associated with development or disease.
  • transcriptomic studies can be used to compare normal, healthy tissues and diseased tissue gene expression.
  • mutated genes or variants associated with disease or the environment can be identified.
  • 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 samples.
  • 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.
  • 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.
  • 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.
  • an expressed sequence tag (EST) library is generated and quantitated using the AmpliSeqTM technique from ThermoFisher.
  • 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 and reported.
  • RNA from neural organoids for Alzheimer’s disease 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. Reads in cpm ⁇ 1 are considered background noise. All cpm data are normalized data and the reads are a direct representation of the abundance of the RNA for each gene.
  • the array consists of one or a plurality of genes used to predict risk of Alzheimer’s disease.
  • reads contain a plurality of genes that are used to treat Alzheimer’s disease in a human, using patient-specific pharmacotherapy known to be associated with Alzheimer's disease.
  • the gene libraries can be comprised of disease-specific gene as provided in Tables 1 and 2 or a combination of genes in Table 1 or Table 2 with alternative disease specific genes.
  • 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.
  • 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 are useful for pharmaceutical testing.
  • drug screening studies including toxicity, safety and or pharmaceutical efficacy, are performed using a combination of in vitro work, rodent / primate studies and computer modeling. Collectively, these studies seek to model human responses, in particular physiological responses of the central nervous system.
  • 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 physiological nuances unique to human growth and development.
  • Third, use of primates creates ethical concerns.
  • Third, current methods are indirect indices of drug safety.
  • neural organoids offer an inexpensive, easily accessible model of human brain development. This model permits direct, and thus more thorough, understanding of the safety, efficacy, and toxicity of pharmaceutical compounds.
  • 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.
  • the biomarker serves as a gene therapy target.
  • AD Alzheimer's Disease
  • the disease is a common form of dementia, is associated with memory loss and interferes with other intellectual abilities that complicate daily life.
  • Alzheimer's disease accounts for 60 to 80 percent of dementia cases.
  • Disease onset occurs most often for individuals in their mid-60s and is estimated to affect approximately five million individuals at present.
  • disease onset occurs many years prior to physical expression of symptoms.
  • the cost to society currently exceeds $270 billion and no effective treatment currently exists.
  • AD Alzheimer's disease
  • Plaques are deposits of beta- amyloid protein fragments that build up in the spaces between nerve cells, while tangles are twisted fibers of tau, a protein that builds up inside cells.
  • anatomical examination reveals a loss of neuronal connections in most AD patients. The result is a loss of cognitive function and the ability to perform easily normal daily activities. Thus, AD patients need extensive caregiver assistance. As a result AD is a significant financial, physical and emotional burden and one of the top causes of death in the United States.
  • AD diagnosis often occurs after the onset of physical symptoms. Individuals at risk for AD would benefit from earlier detection of the disease. In addition, early detection of AD would permit development of pharmaceutical and related treatments to improve AD-related outcomes and delay disease onset.
  • This disclosure provides, in a first embodiment, neural reagents and methods for treating Alzheimer’s disease in a human, using patient-specific pharmacotherapies, the methods comprising: procuring one or a plurality of cell samples from a human, comprising one ora plurality of cell types; reprogramming the one or the plurality of cell samples to produce one or a plurality of induced pluripotent stem cell samples; treating the one or the plurality of induced pluripotent stem cell samples to obtain one or more patient specific neural organoids; collecting a biological sample from the patient specific neural organoid; detecting changes in Alzheimer's disease biomarker expression from the patient specific neural organoid sample that are differentially expressed in humans with Alzheimer’s disease; performing assays on the patient specific neural organoid to identify therapeutic agents
  • At least one cell sample reprogrammed to the induced pluripotent stem cell is a fibroblast derived from skin or blood cells from humans.
  • the fibroblast derived skin or blood cells from humans is identified with the genes identified in Table 1 (Novel Alzheimer’s disease Biomarkers), Table 2 (Biomarkers for Alzheimer’s disease), Table 5 (Therapeutic Neural Organoid Authentication Genes), or Table 7 (Genes and Acession Numbers for Co-Morbidities Associated with Alzheimer’s disease).
  • the measured biomarkers comprise nucleic acids, proteins, or their metabolites.
  • the measured biomarkers comprise one or a plurality of biomarkers identified in Table 1 , Table 2, Table 5 or Table 7 or variants thereof.
  • a combination of biomarkers is detected, the combination comprising a nucleic acid encoding human A2M, APP variants; and one or a plurality of biomarkers comprising a nucleic acid encoding human genes identified in Table 1.
  • the biomarkers for Alzheimer’s disease include human nucleic acids, proteins, or their metabolites as listed in Table 1. These are biomarkers that are found to change along with numerous others ones that are extensively correlated with postmortem brains from Alzheimer’s disease patients.
  • the neural organoid biological sample is collected after about one hour up to about 12 weeks post inducement.
  • the neural organoid sample is procured from structures of the neural organoid that mimic structures developed in utero at about 5 weeks.
  • the neural organoid at about twelve weeks post-inducement comprises structures and cell types of retina, cortex, midbrain, hindbrain, brain stem, or spinal cord.
  • the neural organoid contains microglia, and one or a plurality of Alzheimer’s disease biomarkers as identified in Table 1 and Table 7.
  • the method is used to detect environmental factors such as infectious agents that cause or exacerbate Alzheimer’s disease, or accelerators of Alzheimer’s disease.
  • An accelerator of Alzheimer’s disease is an environmental or nutritional factor that specifically interacts with an Alzheimer’s disease specific biomarker to affect downstream process related to these biomarkers biological function such that a subclinical or milder state of Alzheimer’s disease becomes a full blown clinical state earlier or more severe in nature.
  • the detection of novel biomarkers can be used to identity individuals who should be provided prophylactic treatment for Alzheimer’s disease.
  • such treatments can include avoidance of environmental stimuli and accelerators that exacerbate Alzheimer’s disease.
  • early diagnosis can be used in a personalized medicine approach to identify new patient specific pharmacotherapies for Alzheimer’s disease based on biomarker data.
  • the neural organoid model can be used to test the effectiveness of currently utilized Alzheimer’s disease therapies.
  • the neural organoid can be used to identify the risk and/or onset of Alzheimer’s disease and additionally, provide patient-specific insights into the efficacy of using known pharmacological agents to treat Alzheimer’s disease.
  • the method allows for development and testing of non-individualized, global treatment strategies for mitigating the effects and onset of Alzheimer’s disease.
  • the method is used to identify nutritional factors or supplements for treating Alzheimer’s disease.
  • the nutritional factor or supplement is thiamine or glucose homeostasis or other nutritional factors related to pathways regulated by genes identified in Tables 1 , 2, 5 or 7.
  • the disclosure provides methods for reducing risk of developing Alzheimer’s disease associated co-morbidities in a human comprising procuring one or a plurality of cell samples from a human, comprising one or a plurality of cell types; reprogramming the one or the plurality of cell samples to produce one or a plurality of induced pluripotent stem cell samples; treating the one or the plurality of induced pluripotent stem cell samples to obtain one or more patient specific neural organoids; collecting a biological sample from the patient specific neural organoid; detecting changes in Alzheimer’s disease biomarker expression from the patient specific neural organoid sample that are differentially expressed in humans with Alzheimer’s disease; and administering a therapeutic agent to treat Alzheimer’s disease.
  • the measured biomarkers comprise biomarkers identified in Table 1, Table 2, Table 5 or Table 7 and can be genes, proteins, or their metabolites.
  • the disclosure provides diagnostic methods for predicting risk for developing Alzheimer’s disease in a human, comprising one or a plurality subset of the biomarkers as identified in Table 1 , Table 2, Table 5, or Table 7.
  • the subset of measured biomarkers comprise nucleic acids, proteins, or their metabolites as identified in Table 1 , Table 2, Table 5 or Table 7.
  • a fourth embodiment are methods of pharmaceutical testing for Alzheimer’s disease drug screening, toxicity, safety, and/or pharmaceutical efficacy studies using patient-specific neural organoids.
  • methods for detecting at least one biomarker of Alzheimer’s disease, the method comprising, obtaining a biological sample from a human patient; and contacting the biological sample with an array comprising specific-binding molecules for the at least one biomarker and detecting binding between the at least one biomarker and the specific binding molecules.
  • the biomaker detected is a gene therapy target.
  • the disclosure provides a kit comprising an array containing sequences of biomarkers from Table 1 or Table 2 for use in a human patient.
  • the kit further contains reagents for RNA isolation and biomarkers for tuberous sclerosis genetic disorder.
  • the kit further advantageously comprises a container and a label or instructions for collection of a sample from a human, isolation of cells, inducement of cells to become pluripotent stem cells, growth of patient-specific neural organoids, isolation of RNA, execution of the array and calculation of gene expression change and prediction of concurrent or future disease risk.
  • the biomarkers can include biomarkers listed in Table 2.
  • biomarkers can comprise any markers or combination of markers in Tables 1 and 2 or variants thereof.
  • the disclosure provides a method for detecting one or a plurality of biomarkers from different human chromosomes associated with Alzheimer’s disease or Alzheimer's disease comorbidity susceptibility using data analytics that obviates the need for whole genome sequence analysis of patient genomes.
  • the methods are used to determine gene expression level changes that are used to identity clinically relevant symptoms and treatments, time of disease onset, and disease severity.
  • the neural organoids are used to identity novel biomarkers that serve as data input for development of algorithm techniques as predictive analytics.
  • the algorithmic techniques include artificial intelligence, machine and deep learning as predictive analytics tools for identifying biomarkers for diagnostic, therapeutic target and drug development process for disease.
  • Gene expression measured in Alzheimer’s disease can encode a variant of a biomarker alterations encoding a nucleic acid variant associated with Alzheimer’s disease.
  • the nucleic acid encoding the variant is comprised of one or more missense variants, missense changes, or enriched gene pathways with common or rare variants.
  • the method for predicting a risk for developing Alzheimer's disease in a human 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 Alzheimer’s disease wherein the measured biomarkers comprise those biomarkers listed in Table 2.
  • the measured biomarker is a nucleic acid encoding human biomarkers or variants listed as listed in Table 1.
  • a plurality of biomarkers comprising a diagnostic panel for predicting a risk for developing Alzheimer’s disease in a human, comprising biomarkers listed in Tables 1 and 2, or variants thereof.
  • a subset of marker can be used, wherein the subset comprises a plurality of biomarkers from 2 to 200, or 2-150, 2-100, 2-50, 2-25, 2-20, 2-15, 2- 10, or 2-5 genes.
  • the measured biomarker is a nucleic acid panel for predicting risk of Alzheimer's disease in humans.
  • Said panel can be provided according to the invention as an array of diagnostically relevant portions of one ora plurality of these genes, wherein the array can comprise any method for immobilizing, permanently or transiently, said diagnostically relevant portions of said one ora plurality of these genes, sufficient for the array to be interrogated and changes in gene expression detected and, if desired, quantified.
  • the array comprises specific binding compounds for binding to the protein products of the one or a plurality of these genes.
  • said specific binding compounds can bind to metabolic products of said protein products of the one or a plurality of these genes.
  • the presence of Alzheimer’s disease is detected by detection of one or a plurality of biomarkers as identified in Table 6 (Alzheimer's disease Diagnostic Biomarkers).
  • 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 form 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 biological platform / device for drug screening, toxicity, safety, and/or pharmaceutical efficacy studies understood by those having skill in the art. Additionally, since the neural organoid is patient specific, pharmaceutical testing using the neural organoid allows for patient specific pharmacotherapy.
  • an eighth embodiment provides methods for predicting a risk for developing Alzheimer's disease in a human, the method comprising procuring one or a plurality of cell samples from a human, comprising one ora plurality of cell types; reprogramming the one or the plurality of cell samples to produce one or a plurality of induced pluripotent stem cell samples; treating the one or the plurality of induced pluripotent stem cell samples to obtain one or more patient specific neural organoids; collecting a biological sample from the patient specific neural organoid; measuring biomarkers in the neural organoid sample; and detecting measured biomarkers from the neural organoid sample that are differentially expressed in humans with Alzheimer’s disease.
  • the one cell sample reprogrammed to the induced pluripotent stem cell is a fibroblast.
  • the measured biomarkers comprise nucleic acids, proteins, or their metabolites.
  • the measured biomarker is a nucleic acid encoding human A2M and APP-variant.
  • the measured biomarkers comprise one or a plurality of genes as identified in Tables 1, 2, 5 or 6.
  • the neural organoid sample is procured from minutes to hours up to 15 weeks post inducement, wherein the the biomarkers to be tested are one or a plurality of biomarkers in Tables 5 or 6 (Diagnostic Neural Organoid Authentication Genes).
  • Exosomes are extracellular vesicles that are released from cells upon fusion of the multivesicular body with the plasma membrane.
  • the extracellular vesicles contain proteins and RNA packets containing micro and messenger RNAs that are transferred between cells.
  • the composition of the exosome reflects the origin cell. This property allows for the use of exosomes to predict disease onset, as well as novel therapeutic agents.
  • in one embodiment is a method for growing and isolating exosomes from healthy individuals.
  • Such individuals are free from diseases including, but not limited to Alzheimer's disease, autism, Parkinson's disease, and cancer.
  • the harvesting of exosomes from healthy individuals allows for the isolation of exosome-based RNA and proteins that serve as biomarkers and therapeutic agents for treating disease conditions such as Alzheimer's disease, autism, Parkinson's disease, and cancer.
  • the embodiment comprises procurement of one or a plurality of cell samples from a healthy human, reprogramming the one or the plurality of cell samples to produce one or a plurality of induced pluripotent stem cell samples; treating the one or the plurality of induced pluripotent stem cell samples to obtain one or more therapeutic patient specific healthy neural organoids; and collecting exosomes, and exosome nucleic acids, proteins and metabolites from a plurality of the therapeutic, patient specific healthy neural organoid.
  • exosome RNA and proteins from healthy individuals are utilized in concert with exosome RNA and proteins isolated from a non-healthy individual at predefined time points, noted herein as scaled harvesting, to predict disease onset while also being therapeutic targets.
  • the method comprises procuring one or a plurality of condition-specific samples from a sample including, but not limited to Alzheimer's disease, autism, Parkinson's disease, or cancer; reprogramming the one or the plurality of cell samples to produce one or a plurality of induced pluripotent stem cell samples; treating the one or the plurality of induced pluripotent stem cell samples to obtain one or more condition-specific, patient specific, neural organoids; collecting exosome nucleic acid and protein from a plurality of the condition-specific patient specific neural organoids; detecting changes in the disease-specific exosome nucleic acids and proteins that are differentially expressed; performing assays on the condition-specific exosome nucleic acids and proteins to identify therapeutic agents that alter the differentially expressed in the condition specific versus healthy human exosome nucleic acids and protein profile; and administering a therapeutic agent to the individual.
  • the neural organoid of the current application is novel in that it allows for a scaled harvesting of exosomes at time points from minutes to hours to up to 15 weeks post inducement.
  • the scaled harvesting of exosomes allows for identification of changes in exosome gene and protein biomarker expression patters that are indicative of disease onset.
  • the presence of exosome gene and protein expression patterns indicative of disease onset subsequently can serve as therapeutic targets.
  • exosome nucleic acid and protein biomarkers from healthy individuals are harvested, fractionated, and/or enriched for specific biomarkers altered in the exosomes of Alzheimer's Disease, autism .Parkinson's disease, or cancer and used directly as therapeutic agents [0094]
  • the exosomes can be collected at minutes to days after the neural organoid is generated.
  • the exosome is isolated from the neural organoid and the nucleic acids and proteins harvested up to 15 weeks after induction of the neural organoid.
  • exosomes can be isolated at minutes, hours, days, or weeks after reprogramming.
  • exosomes can be harvested at about 10 minutes
  • the exosomes 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.
  • the exosome 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,
  • Exosomes collected at a wide range of time points allow for insights and data related to regulatory RNA changes that are indicative of disease onset.
  • the scaled harvesting allows for enrichment of specific biomarkers collected at specific time points from the normal human exosome.
  • exosomes can be fractionated and/or enriched to increase yields or enhance therapeutic and predictive responses.
  • the numerous time points are invaluable in predicting disease occurrence / onset and also provide a novel mechanism for therapeutic agents in numerous conditions, including but not limited to Alzheimer's disease, Parkinson’s Disease, malignant and cancerous tumors, autism, and associated co-morbidities.
  • the neural organoid can be used to establish an exosome profile database (See APL Bioeng. 2019 Mar; 3(1)) that can be utilized for determining biomarkers characteristic of disease onset and timing of disease onset.
  • the effectiveness of treatment strategies and therapeutic agents for a wide range of conditions can be evaluated, based on changes in neuronal organoid derived exosomes.
  • nucleic acids and proteins isolated from the exosome of the neural organoid from the healthy human are utilized to construct a biomarker library and evaluate disease onset and predict disease risk.
  • the alterations in exosome RNA and protein expression can be used to predict the risk of developing Alzheimer's disease, autism, Parkinson's disease, or cancer or tumor in a human.
  • the exosome from a healthy individual is isolated, more specifcally, the method comprises; procuring one or a plurality of cell samples from a healthy human, comprising one or a plurality of cell types; reprogramming the one or the plurality of cell samples to produce one or a plurality of induced pluripotent stem cell samples; treating the one or the plurality of induced pluripotent stem cell samples to obtain one or more therapeutic patient specific healthy neural organoids; and collecting exosome nucleic acids and proteins from a plurality of the therapeutic, patient specific healthy neural organoid.
  • the method further comprises procuring one or a plurality of cell samples from a human with Alzheimer's disease, autism, Parkinson's disease, or cancer or tumor, comprising one or a plurality of cell types; reprogramming the one or the plurality of cell samples to produce one or a plurality of induced pluripotent stem cell samples; treating the one or the plurality of induced pluripotent stem cell samples to obtain one or more Alzheimer's disease, autism, Parkinson's disease, or cancer or tumor patient specific, neural organoids; collecting exosome nucleic acid and protein from the patient specific neural organoids; detecting changes in Alzheimer's disease, autism, Parkinson's disease, or cancer or tumor disease exosome nucleic acid and proteins that are differentially expressed in humans with the condition; performing assays on the Alzheimer's disease, autism, Parkinson's disease, or cancer or tumor disease exosome nucleic acids and proteins to identify therapeutic agents that alter the differentially expressed exosome nucleic acids and protein; and administering a therapeutic agent to the human.
  • the exosome biomarkers used in the prediction and treatment of a condition comprise nucleic acids, proteins, or their metabolites and may include A2M, APR, and associated variants.
  • the biomarkers may further comprise one or a plurality of genes as identified in Tables 1 , 2, 5 or 6.
  • neural organoids can be used to identify novel biomarkers that serve as data input for development of algorithm techniques such artificial intelligence, machine and deep learning, including biomarkers for diagnostic, therapeutic target and drug development process for disease.
  • algorithm techniques such artificial intelligence, machine and deep learning, including biomarkers for diagnostic, therapeutic target and drug development process for disease.
  • data analytics for relevant biomarker analysis permits detection of autism and comorbidity susceptibility, thereby obviating the need for whole genome sequence analysis of patient genomes.
  • Neural Organoids derived from induced pluripotent stem cells derived from adult skin cells of patients were grown in vitro for 4 weeks as previous described in our PCT Application (PCT/US2017/013231). Transcriptomic data from these neural organoids were obtained. Differences in expression of 20,814 genes expressed in the human genome were determined between these neural organoids and those from neural organoids from a normal individual human. Detailed data analysis using Gene Card and Pubmed data bases were performed. Genes that were expressed at greater than 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 post mortem patient brains. These genes comprise a suite of biomarkers for Alzheimer’s disease.
  • the invention advantageously provides many uses, including but not limited to a) early diagnosis of these diseases at birth from new bom skin cells; b) Identification of biochemical pathways that increase environmental and nutritional deficiencies in new bom infants; c) discovery of mechanisms of disease mechanisms; d) discovery of novel and early therapeutic targets for drug discovery using timed developmental profiles; e) testing of safety, efficacy and toxicity of drugs in these pre-clinical models.
  • Cells used in these methods include human iPSCs, feeder-dependent (System Bioscience. Wf SC600A-W) and CF-1 mouse embryonic fibroblast feeder cells, gamma- irradiated (Applied StemCell, Inc #ASF- 1217)
  • Growth media, or DMEM media, used in the examples contained the supplements as provided in Table 3 (Growth Media and Supplements used in Examples).
  • 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.
  • Induction media for pluripotent stem cells comprised DMEM/F12 media supplemented with 20% Knockout Replacement Serum, 3% Fetal Bovine Serum with 2mM Glutamax, IX Minimal Essential Medium Nonessential Amino Acids, and 20 nanogram/ml basic Fibroblast Growth Factor
  • 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, 55microM beta-mercaptoethanol, and 4ng/ml basic Fibroblast Growth Factor.
  • DMEM Dulbecco's Modified Eagle's Medium
  • 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
  • 55microM beta-mercaptoethanol 55microM beta-mercaptoethanol
  • 4ng/ml basic Fibroblast Growth Factor 4ng/ml basic Fibroblast Growth Factor.
  • 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 '
  • 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, 55microM beta-mercaptoethanol kept under nitrogen mask and frozen at -20 e C, 100 units/ml penicillin, 100 microgram/ml streptomycin, and 0.25microgram/ml Fungizone.
  • 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, 55umicroMolar beta-mercaptoethanol kept under nitrogen mask and frozen at -20°C, 100units/ml penicillin, 100microgram/ml streptomycin, and 0.25microgram/ml Fungizone.
  • 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.
  • Example 1 Generation of human induced pluripotent stem cell-derived neural organoids.
  • Human induced pluripotent stem cell-derived neural organoids were generated according to the following protocol, as set forth in International Application No.
  • MEF murine embryonic fibroblasts
  • 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
  • the seeded plate was incubated at 37°C overnight.
  • the MEFs were washed with pre-warmed sterile phosphate buffered saline (PBS).
  • PBS pre-warmed sterile phosphate buffered saline
  • the MEF media was replaced with 1 mL per well of induced pluripotent stem cell (iPSC) media containing Rho-associated protein kinase (ROCK) inhibitor.
  • iPSC induced pluripotent stem cell
  • ROCK Rho-associated protein kinase
  • 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.
  • a 100 mm culture dish was coated with 0.1% gelatin and the dish placed in a 37°C 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 prewarmed PBS lacking Ca 2+/ Mg 2+ .
  • 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.
  • 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 ⁇ beta- mercaptoethanol.
  • the suspended cells were plated (150 ⁇ L) in a LIPIDURE® low- attachment U-bottom 96-well plate and incubated at 37°C.
  • 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.
  • 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.
  • the embryoid bodies were removed from the LIPIDURE® 96 well plate and transferred to two 24-well plates containing 500 ⁇ L/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 pg/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.
  • 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 200 ⁇ L 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.
  • the 20 ⁇ L 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.
  • 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%C02) further allowed optimal exchange of gases and nutrients to the embedded embryoid bodies.
  • 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 pg/mL insulin, 55 microM beta-mercaptoethanol kept under nitrogen mask and frozen at -20°C, 100 units/mL penicillin, 100 pg/mL streptomycin, and 0.25 pg/mL Fungizone, was added to a 100 mm 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 e 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 e C was continued for 4 days until stable neural organoids formed.
  • 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 pg/mL insulin, 55microM beta-mercaptoethanol kept under nitrogen mask and frozen at -20°C, 100 units/mL penicillin, 100 pg/mL streptomycin, and 0.25 pg/mL Fungizone.
  • the flasks were placed on an orbital shaker rotating at 40 rpm within the 37°C/5% CO2 incubator.
  • FIG.16 illustrates neural organoid development in vitro. Based on transcriptomic analysis, iPSC cells form a body of cells alter 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 or before, neurons of multiple lineage appeared. At about twelve (12) weeks or before, the organoid developed to a stage having different types of cells, including microglia, oligodendrocyte, astrocyte, neural precursor, neurons, and intemeurons.
  • NPC neural progenitor cells
  • Example 2 Human induced pluripotent stem cell-derived neural organoids express characteristics of human brain development.
  • organoids were generated according to the methods delineated in Example 1. Specifically, the organoids contained cells expressing markers characteristic of neurons, astrocytes, oligodendrocytes, microglia, and vasculature (FIGs. 1-14) 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 developmental ⁇ organized manner described previously (e.g. for the midbrain mesencephalic dopaminergic neurons; Blaese et al., Genetic control of midbrain dopaminergic neuron development. Rev Dev Biol. 4(2): 113-34, 2015).
  • 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).
  • All human neural organoids were derived from iPSCs of fibroblast origin (from System Biosciences, Inc). The development of a variety of brain structures was characterized in the organoids. Retinal markers are shown in FIG.15. Doublecortin (DCX), a microtubule associated protein expressed during cortical development, was observed in the human neural organoid (FIG. 1A and FIG. 1B, and FIG. 16). Midbrain development was characterized by the presence of tyrosine hydroxylase (FIG. 2). In addition, transcriptomics revealed expression of the midbrain markers DLKI, KLHL I, and PTPRU (FIG. 6A).
  • FIG. 5A A schematic of the roles of NKCCI and KCC2 is provided in FIG. 5A.
  • FIG. 5B indicates that a variety of markers expressed during human brain development are also expressed in the organoids described in Example 1.
  • Markers expressed within the organoids were consistent with the presence of excitatory, inhibitory, cholinergic, dopaminergic, serotonergic, astrocytic, oligodendritic, microglial, vasculature cell types. Further, the markers were consistent with those identified by the Human Brain Reference (HBR) from Clontech (FIG. 5C) and were reproducible in independent experiments (FIG. 5D). Non-brain tissue markers were not observed in the neural organoid (FIG. 6B).
  • HBR Human Brain Reference
  • FIG. 5B delineates the expression of markers characteristic of cerebellar development.
  • FIG. 8 provides a list of markers identified using transcriptomics that are characteristic of neurons present in the hippocampus dentate gyrus. Markers characteristic of the spinal cord were observed after 12 weeks of in vitro culture.
  • FIG. 8 provides a list of markers identified using transcriptomics that are characteristic of neurons present in the hippocampus dentate gyrus. Markers characteristic of the spinal cord were observed after 12 weeks of in vitro culture.
  • FIG. 9 provides a list of markers identified using transcriptomics that are characteristic of GABAergic intemeuron development.
  • FIG. 10 provides a list of markers identified using transcriptomics that are characteristic of the brain stem, in particular, markers associated with the serotonergic raphe nucleus of the pons.
  • FIG. 11 lists the expression of various Hox genes that are expressed during the development of the cervical, thoracic and lumbar regions of the spinal cord.
  • FIG. 12 shows that results are reproducible between experiments.
  • the expression of markers detected using transcriptomics is summarized in FIG. 13.
  • the results reported herein support the conclusion that the invention provides an in vitro cultured organoid that resembles an approximately 5 week old human fetal brain, based on size and specific morphological features with great likeness to the optical stock, the cerebral hemisphere, and cephalic flexure in a 2-3mm organoid that can be grown in culture.
  • High resolution morphology analysis was carried out using immunohistological methods on sections and confocal imaging of the organoid to establish the presence of neurons, axons, dendrites, laminar development of cortex, and the presence of midbrain marker.
  • This organoid includes an interactive milieu of brain circuits as represented by the laminar organization of the cortical structures in Fig. 13 and thus supports formation of native neural niches in which exchange of miRNA and proteins by exosomes can occur among different cell types.
  • Neural organoids were evaluated at weeks 1 , 4 and 12 by transcriptomics.
  • the organoid was reproducible and replicable (FIGs. 5C, 5D, FIG. 12, and FIG. 18).
  • Brain organoids generated in two independent experiments and subjected to transcriptomic analysis showed >99% replicability of the expression pattern and comparable expression levels of most genes with ⁇ 2-fold variance among some of the replicates.
  • Example 3 Tuberous sclerosis complex model
  • Tuberous sclerosis complex is a genetic disorder that causes non- malignant tumors to form in multiple organs, including the brain. TSC negatively affects quality of life, with patients experiencing seizures, developmental delay, intellectual disability, gastrointestinal distress and Alzheimer’s disease.
  • TSC Tuberous sclerosis complex
  • Two genes are associated with TSC: (1) the TSC1 gene, located on chromosome 9 and also referred to as the hamartin gene and (2) the TSC2 gene located on chromosome 16 and referred to as the tuberin gene.
  • a human neural organoid from iPSCs was derived from a patient with a gene variant of the TSC2 gene (ARG I743GLN) from iPSCs (Cat# GM25318 Coriell Institute Repository, NJ). This organoid served as a genetic model of a TSC2 mutant.
  • Alzheimer's disease and Alzheimer’s disease spectrum disorder is a development disorder that negatively impacts social interactions and day-to-day activities. In some cases, the disease can include repetitive and unusual behaviors and reduced tolerance for sensory stimulation. Many of the Alzheimer’s disease-predictive genes are associated with brain development, growth, and/or organization of neurons and synapses. [00147] Alzheimer's disease has a strong genetic link with DNA mutations comprising a common molecular characteristic of Alzheimer’s disease. Alzheimer’s disease encompasses a wide range of genetic changes, most often genetic mutations. The genes commonly identified as playing a role in Alzheimer’s disease include novel markers provided in Table 1 and Alzheimer's disease markers provided in Table 2.
  • Expression changes and mutations in the noted genes disclosed herein from the neural organoid at about week 1 , about week 4 and about week 12 are used in one embodiment to predict future Alzheimer’s disease risk.
  • mutations in the genes disclosed can be determined at hours, days or weeks after reprogramming.
  • mutations in Table 1 in the human neural organoid at about week 1 , about week 4, and about week 12 are used to predict the future risk of Alzheimer’s disease using above described methods for calculating risk.
  • additional biomarker combinations expressed in the human neural organoid can also be used to predict future Alzheimer’s disease onset.
  • the model used herein is validated and novel in that data findings reconcile that the model expresses four hundred and seventy two markers of Alzheimer’s disease patient post mortem brains and databases (Table 2), as shown in Table 5.
  • Table 2 as shown in Table 5.
  • the model is novel in that it uses, as starting material, an individual’s iPSCs originating from skin or blood cells as the starting material to develop a neural organoid that allows for identification of Alzheimer’s disease markers early in development including at birth
  • sequence data for the genes listed above can be obtained in publicly available gene databases such as GeneCards, GenBank, Malcard, Uniport and PathCard databases.
  • sequence data for the genes listed above can be obtained in publicly available gene databases such as GeneCands, GenBank, Malcard, Uniport and PathCard databases.
  • Example 5 Predicting Risk of Disease Onset from Neural Organoid Gene Expression
  • Gene expression in the neural organoid can be used to predict disease onset. Briefly, gene expression is correlated with Gene Card and Pubmed database genes and expression compared for dysregulated expression in diseased vs non-disease neural organoid gene expression.
  • Example 6 Prediction of Co-Morbidities Associated with Alzheimer’s disease
  • the human neural organoid model data findings can be used in the prediction of comorbidity onset or risk associated with Alzheimer’s disease including at birth (Reference: European Bioinformatic Institute (EBI) and ALLEN INSTITUTE databases) and in detecting comorbidities, genes associated with one or more of these diseases are detected from the patient’s grown neural organoid.
  • EBI European Bioinformatic Institute
  • ALLEN INSTITUTE databases genes associated with one or more of these diseases are detected from the patient’s grown neural organoid.
  • genes include, comorbidities and related accession numbers include, those listed in Table 7:
  • Table 7 Genes and Co-Morbidity Susceptibility/Resistance Associated with Alzheimer’s Disease
  • ACTN2 Cardiomyopathy Dilated, 1Aa, With Or Without Left Ventricular Noncompaction and Atrial Standstill 1.
  • ADAMTS2 Ehlers-Danlos Syndrome, Dermatosparaxis Type and Ehlers-Danlos Syndrome.
  • ADAMTS3 Hennekam Lymphangiectasia-Lymphedema Syndrome 3 and Hennekam Syndrome.
  • AKR1C2 46 Xy Sex Reversal 8 and Perrault Syndrome 1.
  • ANK1 Spherocytosis Type 1 and Hereditary Spherocytosis.
  • ARHGEF9 Epileptic Encephalopathy, Early Infantile, 8 and Hyperekplexia.
  • ARMC4 Ciliary Dyskinesia, Primary, 23 and Kartagener Syndrome.
  • ATP2B3 Spinocerebellar Ataxia, X-Linked 1 and Muscular Atrophy.
  • CABYR Suppurative Thyroiditis.
  • CACNA1E NFAT and Cardiac Hypertrophy
  • CACNB1 Headache and Malignant Hyperthermia
  • CACNB4 Episodic Ataxia, Type 5 and Epilepsy, Idiopathic Generalized 9.
  • CACNG2 Mental Retardation, Autosomal Dominant 10 and Autosomal Dominant Non-Syndromic Intellectual Disability.
  • CACNG8 Dilated Cardiomyopathy.
  • CADM3 Cleft Lip/Palate-Ectodermal Dysplasia Syndrome.
  • CCDC103 Ciliary Dyskinesia, Primary, 17 and Ciliary Dyskinesia, Primary, 1.
  • CCDC65 Ciliary Dyskinesia, Primary, 27 and Primary Ciliary Dyskinesia.
  • CD14 Mycobacterium Chelonae and Croup. CD163 Rosai-Dorfman Disease and Non-Langerhans-Cell Histiocytosis.
  • CD1C Mycobacterium Malmoense and Foramen Magnum Meningioma.
  • CD34 Dermatofibrosarcoma Protuberans and Gastrointestinal Stromal Tumor.
  • CDCA5 Cornelia De Lange Syndrome.
  • CD01 Hepatoblastoma and Esophagus Adenocarcinoma.
  • CHRM2 Major Depressive Disorder and Intestinal Schistosomiasis
  • CHRNA3 Smoking As A Quantitative Trait Locus 3 and Autosomal Dominant Nocturnal Frontal Lobe Epilepsy.
  • CHST3 Spondyloepiphyseal Dysplasia With Congenital Joint Dislocations and Multiple Joint Dislocations, Short Stature, And Craniofacial Dysmorphism With Or Without Congenital Heart Defects.
  • CNNM1 Urofadal Syndrome 1.
  • CNTNAP3B Exstrophy Of Bladder.
  • CPT1B Carnitine Palmitoyltransferase I Deficiencyand Visceral Steatosis.
  • CRABP2 Embryonal Carcinoma and Basal Cell Carcinoma.
  • CREB3L3 Hyperlipoproteinemia Type V and Hepatocellular Carcinoma.
  • CSF1 Pigmented Villonodular Synovitis and Tenosynovial Giant Cell Tumor.
  • CYP1B1 Glaucoma 3 Primary Congenital, A and Anterior Segment Dysgenesis 6.
  • CYP26B1 Radiohumeral Fusions With Other Skeletal And Craniofacial Anomalies and Occipital Encephalocele.
  • DCX Lissencephaly, X-Linked, 1 and Subcortical Band Heterotopia DCX Lissencephaly, X-Linked, 1 and Subcortical Band Heterotopia.
  • DNAH11 Ciliary Dyskinesia, Primary, 7 and Primary Ciliary Dyskinesia.
  • DNAI1 Ciliary Dyskinesia, Primary, 1 and Kartagener Syndrome.
  • DNASE1L1 Human Monocytic Ehrlichiosis and Xerophthalmia Human Monocytic Ehrlichiosis and Xerophthalmia.
  • DSC2 Arrhythmogenic Right Ventricular Dysplasia, Familial, 11 and Familial Isolated Arrhythmogenic Ventricular Dysplasia, Right Dominant Form.
  • DSG2 Arrhythmogenic Right Ventricular Dysplasia, Familial, 10 and Cardiomyopathy, Dilated, 1Bb.
  • EEF1A2 Epileptic Encephalopathy, Early Infantile, 33 and Mental Retardation, Autosomal Dominant 38
  • EGF Hypomagnesemia 4 Renal and Familial Primary Hypomagnesemia With Normocalciuria And Normocalcemia.
  • EPSTI1 Lupus Erythematosus and Systemic Lupus Erythematosus.
  • EYA4 Cardiomyopathy Dilated, 1J and Deafness, Autosomal Dominant 10.
  • FAM5C Tongue Squamous Cell Carcinoma and Myocardial Infarction.
  • FAM64A Suppurative Periapical Periodontitisand Clonorchiasis.
  • FGF17 Hypogonadotropic Hypogonadism 20 With Or Without Anosmia and Normosmic Congenital Hypogonadotropic Hypogonadism.
  • FXYD5 Leukemia Acute Myeloid.
  • GABBR2 Epileptic Encephalopathy, Early Infantile, 59 and Neurodevelopmental Disorder With Poor Language And Loss Of Hand Skills.
  • GAL3ST4 Pectus Excavatum.
  • GDAP1 Charcot-Marie-Tooth Disease, Type 4A and Charcot-Marie-Tooth Disease, Recessive Intermediate A.
  • GLT1D1 Hepatocellular Carcinoma.
  • GLYATL2 Mitochondrial acyltransferase which transfers the acyl group to the N- terminus of glycine. Conjugates numerous substrates, such as arachidonoyl-CoA and saturated medium and long-chain acyl-CoAs ranging from chain-length C8:0-CoA to C18:0-CoA, to form a variety of N- acylglycines.
  • GNA14 Kaposiform Hemangioendothelioma and Angioma, Tufted.
  • GPD1 Hypertriglyceridemia, Transient Infantileand Brugada Syndrome.
  • GRIA2 Status Epilepticus and Lateral Sclerosis.
  • GRM1 Spinocerebellar Ataxia, Autosomal Recessive 13 and Spinocerebellar Ataxia 44.
  • GRM4 Epilepsy, Idiopathic Generalized 10 and Schizophrenia.
  • GRM7 Age-Related Hearing Loss and Lubs X-Linked Mental Retardation Syndrome.
  • GSC Short Stature, Auditory Canal Atresia, Mandibular Hypoplasia, And Skeletal Abnormalities and Synostosis.
  • GYLTL1B Interstitial Myocarditis and Muscular Dystrophy-Dystroglycanopathy , Type
  • HAVCR2 Hepatitis A and Hepatitis.
  • HESX1 Septooptic Dysplasia and Pituitary Stalk Interruption Syndrome.
  • HIP1R Cataract 8 Multiple Types and Parkinson Disease, Late-Onset.
  • HIVEP2 Mental Retardation, Autosomal Dominant 43 and Hivep2-Related Intellectual Disability.
  • HLA-DRA Graham-Little-Piccandi-Lassueur Syndrome and Heart Lymphoma.
  • HMGCR Hyperlipidemia, Familial Combined and Marek Disease.
  • HNF1B Renal Cysts And Diabetes Syndrome and Diabetes Mellitus, Noninsulin- Dependent.
  • HPD Tyrosinemia Type lii and Hawkinsinuria.
  • HPGD Digital Clubbing Isolated Congenitaland Hypertrophic Osteoarthropathy, Primary, Autosomal Recessive, 1.
  • HSPG2 Schwartz-Jampel Syndrome, Type 1 and Dyssegmental Dysplasia, Silverman-Handmaker Type.
  • HTR2A Major Depressive Disorder and Obsessive-Compulsive Disorder.
  • HTR2C Anxiety and Premature Ejaculation.
  • IFNA1 Hepatitis C and Hepatitis.
  • IGF1 Insulin-Like Growth Factor I and Pituitary Gland Disease.
  • IGFBP2 Malignant Ovarian Cyst and Insulin-Like Growth Factor I.
  • IRF5 Systemic Lupus Erythematosus 10 and Inflammatory Bowel Disease 14.
  • IRF6 Popliteal Pterygium Syndrome and Van Der Woude Syndrome 1.
  • IRX5 Hamamy Syndrome and Griscelli Syndrome Type 3.
  • JMJD6 Deep Angioma and Intramuscular Hemangioma JMJD6 Deep Angioma and Intramuscular Hemangioma.
  • KCNA4 Episodic Ataxia, Type 1 and Episodic Ataxia.
  • KCNIP2 Spinocerebellar Ataxia Type 19/22 and Brugada Syndrome.
  • KIFAP3 Progressive Bulbar Palsy and Amyotrophic Lateral Sclerosis 1.
  • KLF10 Hemoglobinopathy and Pancreatic Cancer KLF10 Hemoglobinopathy and Pancreatic Cancer.
  • KLHL1 Spinocerebellar Ataxia 8.
  • KPNA2 Malignant Germ Cell Tumor and Ovarian Endodermal Sinus Tumor.
  • MAGEA5 Melanoma and Dyskeratosis Congenita.
  • MAGI2 Nephrotic Syndrome 15 and Chromosome 1P36 Deletion Syndrome are MAGI2 Nephrotic Syndrome 15 and Chromosome 1P36 Deletion Syndrome.
  • MAPK8 Fatty Liver Disease and Renal Fibrosis MAPK8IP1 Diabetes Mellitus, Noninsulin-Dependent and Sarcomatoid Squamous Cell Skin Carcinoma.
  • MEGF10 Myopathy Areflexia, Respiratory Distress, And Dysphagia, Early-Onset and Dysphagia.assodated with schizophrenia, Areflexia, Respiratory Distress, And Dysphagia, Early-Onset and Dysphagia.
  • MLC1 Megalencephalic Leukoencephalopathy With Subcortical Cysts and Mld- Related Megalencephalic Leukoencephalopathy With Subcortical Cysts.
  • NPAS3 Holoprosencephaly 8 and Schizophrenia.
  • PCDH18 Hemophagocytic Lymphohistiocytosis and Patent Foramen Ovale.
  • PCGF5 Interleukin-7 Receptor Alpha Deficiency PCGF5 Interleukin-7 Receptor Alpha Deficiency.
  • PDGFRL Colorectal Cancer and Hepatocellular Carcinoma
  • PIEZ01 Dehydrated Hereditary Stomatocytosis 1 With Or Without Pseudohyperkalemia And/Or Perinatal Edema and Lymphedema, Hereditary, lii.
  • PLA2G1B Distal Hereditary Motor Neuropathy, Type liand Neurodegeneration With Brain Iron Accumulation 2B.
  • PLA2G7 Platelet-Activating Factor Acetylhydrolase Deficiency and Atopy.
  • PLB1 PLB1 include Amyotrophic Lateral Sclerosis 3 and Opportunistic Mycosis.
  • PLP1 Pelizaeus-Merzbacher Disease and Spastic Paraplegia 2, X- Linked. include Spastic Paraplegia 2, X-Linked and Pelizaeus-Merzbacher Disease, myelin sheaths, as well as in oligodendrocyte development and axonal survival
  • PRKCB Papillary Glioneuronal Tumors and Choidoid Glioma.
  • PRODH Hyperprolinemia Type I and Schizophrenia 4.
  • PRRX1 Agnathia-Otocephaly Complex and Dysgnathia Complex.
  • PTK2B Cone-Rod Dystrophy 5 and Transient Cerebral Ischemia.
  • RASIP1 Enamel Erosion and Tooth Erosion.
  • RASL12 Nemaline Myopathy 6 and Zika Fever.
  • REEP1 Spastic Paraplegia 31 Autosomal Dominant and Neuronopathy, Distal Hereditary Motor, Type Vb.a Neurodegenerative Disorder.
  • RNASE2 Peripheral Demyelinating Neuropathy, Central Dysmyelination, Waardenburg Syndrome, And Hirschsprung Disease and Lacrimoauriculodentodigital Syndrome
  • ROBG3 Gaze Palsy Familial Horizontal, With Progressive Scoliosis, 1 and Horizontal Gaze Palsy With Progressive Scoliosis.
  • SCN2A Seizures, Benign Familial Infantile, 3 and Epileptic Encephalopathy, Early Infantile, 11.
  • SCN2B Atrial Fibrillation, Familial, 14and Familial Atrial Fibrillation.
  • SLC1A3 Episodic Ataxia, Type 6 and Episodic Ataxia.
  • SLC26A2 Achondrogenesis, Type lb and Epiphyseal Dysplasia, Multiple, 4.
  • SLC34A2 Pulmonary Alveolar Microlithiasis and Testicular Microlithiasis.
  • SLC6A1 Myoclonio-Atonic Epilepsy and Myoclonic-Astastic Epilepsy.
  • STAB1 Bacillary Angiomatosis and Histiocytosis.
  • TAC1 Bronchitis and Neurotrophic Keratopathy.
  • TANK Vaccinia TAS2R16 Alcohol Dependence and Alcohol Use Disorder.
  • TET2 Myelodysplastic Syndrome and Refractory Anemia.
  • Thrombocythemia 1 THPO Thrombocythemia 1 and Essential Thrombocythemia.
  • THSD1 Intracranial Aneurysm and Cerebral Arterial Disease.
  • TNFSF10 Malignant Glioma and Ulceroglandular Tularemia.
  • TRAPPC3 Tietz Albinism-Deafness Syndrome and Cardiac Tamponade.
  • TRIP13 Mosaic Variegated Aneuploidy Syndrome 3 and Mosaic Variegated Aneuploidy Syndrome.
  • TSPAN2 Focal demyelination associated with amyloid plaque formation in Alzheimer's disease
  • TSPAN7 X-Linked Non-Specific Intellectual Disability and Acute Apical Periodontitis.
  • VAMP2 Tetanus and Primary Bacterial Infectious Disease.
  • VASH2 Angiogenesis inhibitor VASH2 Angiogenesis inhibitor.
  • VIL1 Type 1 Diabetes Mellitus 13 and Dacryoadenitis VIL1 Type 1 Diabetes Mellitus 13 and Dacryoadenitis.
  • VLDLR Cerebellar Ataxia Mental Retardation, And Dysequilibrium Syndrome 1 and Cerebellar Hypoplasia.
  • VPREB1 ConRJiobolomycosis and Mu Chain Disease.
  • WNT9A Gastric cancer WT1 Wilms Tumor 1 and Denys-Drash Syndrome.
  • ZDBF2 Nasopalpebral Lipoma-Coloboma Syndromeand Coloboma Of Macula.
  • MAK Retinitis Pigmentosa 62 and Mak-Related Retinitis Pigmentosa are MAK Retinitis Pigmentosa 62 and Mak-Related Retinitis Pigmentosa.
  • GNG2 Hemiplegic Migraine.
  • sequence data for the genes listed above can be obtained in publicly available gene databases such as GeneCards, GenBank, Malcard, Uniport and PathCard databases.
  • 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; Accession Number for these markers are set forth in exemplary fashion in Table 7.
  • variants derive from the full length gene sequence.
  • the data findings and sequences in Table 7 encode the respective polypeptide having at least 70% homology to other variants, including full length sequences.
  • Example 7 Neural Organoids for Testing Drug Efficacy.
  • Neural organoids can be used for pharmaceutical testing, safety, efficacy, and toxicity profiling studies. Specifically, using pharmaceuticals and human neural organoids, beneficial and detrimental genes and pathways associated with Alzheimer's disease can be elucidated. Neural organoids as provided herein can be used fortesting candidate pharmaceutical agents, as well as testing whether any particular pharmaceutical agent inter alia for Alzheimer's disease should be administered to a particular individual based on responsiveness, alternation, mutation, or changes in gene expression in a neural organoid produced from cells from that individual or in response to administration of a candidate pharmaceutical to said individual’s neural organoid.

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Abstract

L'invention concerne des procédés d'utilisation de changements d'expression génique et de mutations dans des organoïdes neuraux pour identifier des réseaux neuronaux qui prédisent l'apparition de la maladie d'Alzheimer et l'ARN d'exosomes qui peut être utilisé pour prédire l'apparition et agir en tant que cibles thérapeutiques.
PCT/US2020/059971 2019-11-11 2020-11-11 Exosomes humains dérivés de neurones pour la maladie d'alzheimer et les co-morbidités de ceux-ci Ceased WO2021096929A1 (fr)

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CN114150057A (zh) * 2021-12-21 2022-03-08 贾龙飞 一种诊断阿尔茨海默病的外泌体蛋白及其用途
CN116334210A (zh) * 2023-03-06 2023-06-27 中国人民解放军总医院 Aldh1a1和amigo2基因在制备房颤诊断和干预产品中的应用
CN116949169A (zh) * 2023-07-31 2023-10-27 山东大学齐鲁医院 Smek1在缺血性脑卒中诊治中的应用
CN118718105A (zh) * 2024-07-29 2024-10-01 哈尔滨医科大学 促心梗后心肌再血管化的肿瘤源外泌体的制备方法及应用
WO2025059281A1 (fr) * 2023-09-14 2025-03-20 Mayo Foundation For Medical Education And Research Enrichissement de vésicules extracellulaires spécifiques de neurones, et leur utilisation dans l'identification et le traitement de sujets atteints de troubles neurodégénératifs
WO2025080972A1 (fr) * 2023-10-11 2025-04-17 Regents Of The University Of Minnesota Produits d'exosomes dérivés d'organoïdes, procédés de fabrication et procédés d'utilisation
EP4481035A4 (fr) * 2022-02-18 2025-06-18 Panexo Biotech Sg Pte.Ltd Cellule souche pluripotente humaine induite surexprimant tlx et son utilisation

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Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114150057A (zh) * 2021-12-21 2022-03-08 贾龙飞 一种诊断阿尔茨海默病的外泌体蛋白及其用途
CN114150057B (zh) * 2021-12-21 2024-04-26 贾龙飞 一种诊断阿尔茨海默病的外泌体蛋白及其用途
EP4481035A4 (fr) * 2022-02-18 2025-06-18 Panexo Biotech Sg Pte.Ltd Cellule souche pluripotente humaine induite surexprimant tlx et son utilisation
CN116334210A (zh) * 2023-03-06 2023-06-27 中国人民解放军总医院 Aldh1a1和amigo2基因在制备房颤诊断和干预产品中的应用
CN116949169A (zh) * 2023-07-31 2023-10-27 山东大学齐鲁医院 Smek1在缺血性脑卒中诊治中的应用
CN116949169B (zh) * 2023-07-31 2024-02-06 山东大学齐鲁医院 Smek1在缺血性脑卒中诊治中的应用
WO2025059281A1 (fr) * 2023-09-14 2025-03-20 Mayo Foundation For Medical Education And Research Enrichissement de vésicules extracellulaires spécifiques de neurones, et leur utilisation dans l'identification et le traitement de sujets atteints de troubles neurodégénératifs
WO2025080972A1 (fr) * 2023-10-11 2025-04-17 Regents Of The University Of Minnesota Produits d'exosomes dérivés d'organoïdes, procédés de fabrication et procédés d'utilisation
CN118718105A (zh) * 2024-07-29 2024-10-01 哈尔滨医科大学 促心梗后心肌再血管化的肿瘤源外泌体的制备方法及应用

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