Patent Application
20210284961
This disclosure relates to production and use of human stem cell derived neural organoids to treat autism in a human, using a patient-specific pharmacotherapy. Further disclosed are patient-specific pharmacotherapeutic methods for reducing risk for developing autism-associated co-morbidities in a human. Also disclosed are methods to predict onset risk of autism (and identified comorbidities) in an individual. In particular the 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 autism (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 autism.
The human brain, and diseases associated with it have been the object of investigation and study by scientists for decades. Throughout this time, neurobiologists have attempted to increase their understanding of the brain's capabilities and functions. Neuroscience has typically relied on the experimental manipulation of living brains or tissue samples, but scientific progress has been limited by a number of factors. For ethical and practical reasons, obtaining human brain tissue is difficult while most invasive techniques are impossible to use on live humans. Experiments in animals are expensive and time-consuming and many animal experiments are conducted in rodents, which have a brain structure and development that vary greatly from humans. Results obtained in animals must be verified in long and expensive human clinical trials and much of the time the animal disease models are not fully representative of disease pathology in the human brain.
Improved experimental models of the human brain are urgently required to understand disease mechanisms and test potential therapeutics. The ability to detect and diagnose various neurological diseases in their early stages could prove critical in the effective management of such diseases, both at times before disease symptoms appear and thereafter. Neuropathology is a frequently used diagnostic method; however, neuropathology is usually based on autopsy results. Molecular diagnostics in theory can provide a basis for early detection and a risk of early onset of neurological disease. However, molecular diagnostic methods in neurological diseases are limited in accuracy, specificity, and sensitivity. Therefore, there is a need in the art for non-invasive, patient specific molecular diagnostic methods to be developed.
Consistent with this need, neural organoids hold significant promise for studying neurological diseases and disorders. Neural organoids are developed from cell lineages that have been first been induced to become pluripotent stem cells. Thus, the neural organoid is patient specific. Importantly, such models provide a method for studying neurological diseases and disorders that can overcome previous limitations. Thus, there is a need in the art to develop individual-specific reagents and methods based on predictive biomarkers for diagnosing current and future risk of neurological disease.
This disclosure provides neural reagents and methods for treating autism 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 autism biomarker expression from the patient specific neural organoid sample that are differentially expressed in humans with autism; performing assays on the patient specific neural organoid to identify therapeutic agents that alter the differentially expressed autism biomarkers in the patient-specific neural organoid sample; and administering a therapeutic agent for autism to treat the human.
In one aspect at least one cell sample reprogrammed to the induced pluripotent stem cell is a fibroblast derived from skin or blood cells from humans. In another aspect the fibroblast derived skin or blood cells from humans is identified with the genes identified in Table 1 (Novel Autism Biomarkers), Table 2 (Biomarkers for Autism), Table 5 (Therapeutic Neural Organoid Authentication Genes), or Table 7 (Genes and Acession Numbers for Co-Morbidities Associated with Autism). In yet another aspect, the measured biomarkers comprise nucleic acids, proteins, or metabolites. In another aspect the measured biomarkers comprise one or a plurality of biomarkers identified in Table 1, Table 2, Table 5 or Table 7 or variants thereof. In yet another aspect, a combination of biomarkers is detected, the combination comprising a nucleic acid encoding human TSC1, TSC2, or a TSC2 variant; and one or a plurality of biomarkers comprising a nucleic acid encoding human genes identified in Table 1.
In still another aspect, the neural organoid biological sample is collected after about one hour up to about 12 weeks post inducement. In another aspect the neural organoid sample is procured from structures of the neural organoid that mimic structures developed in utero at about 5 weeks. In yet another aspect the neural organoid at about twelve weeks post-inducement comprises structures and cell types of retina, cortex, midbrain, hindbrain, brain stem, or spinal cord. In a one aspect the neural organoid contains microglia, and one or a plurality of autism biomarkers as identified in Table 1 and Table 7.
In a second embodiment, the disclosure provides methods for treating autism in a human using patient specific pharmacotherapies, 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 autism biomarker expression from the patient specific neural organoid sample that are differentially expressed in humans with autism; performing assays on the patient specific neural organoid to identify therapeutic agents that alter the differentially expressed autism biomarkers in the patient-specific neural organoid sample; and administering a therapeutic agent to treat autism.
In one aspect the measured biomarkers comprise biomarkers identified in Table 1, Table 2, Table 5 or Table 7 and can be genes, proteins, or metabolites encoding the biomarkers identified in Table 1, Table 2, Table 5 or Table 7. In a further aspect the invention provides diagnostic methods for predicting risk for developing autism in a human, comprising one or a plurality subset of the biomarkers as identified in Table 1, Table 2, Table 5, or Table 7. In a third aspect, the subset of measured biomarkers comprise nucleic acids encoding genes or proteins, or metabolites as identified in Table 1, Table 2, Table 5 or Table 7.
In another embodiment are methods of pharmaceutical testing for drug screening, toxicity, safety, and/or pharmaceutical efficacy studies using patient-specific neural organoids.
In a third embodiment, methods are provided for detecting at least one biomarker of autism, 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.
In a fourth embodiment, the biomaker detected is a gene therapy target.
In a fifth embodiment the disclosure provides a kit comprising an array containing sequences of biomarkers from Table 1 or Table 2 for use in a human patient. In one aspect the kit further contains reagents for RNA isolation and biomarkers for tuberous sclerosis genetic disorder. In a further aspect, 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.
In a sixth embodiment the biomarkers for autism include human nucleic acids, proteins, or metabolites as listed in Table 1. These are biomarkers that are found within small or large regions of the human chromosome that change and are associated with autism, but within which chromosomal regions specific genes with mutations have not be identified as causative for autism.
In a further embodiment, the biomarkers can include biomarkers listed in Table 2. In another embodiment, biomarkers can comprise any markers or combination of markers in Tables 1 and 2 or variants thereof.
In another embodiment of the first aspect, the measured biomarkers include human nucleic acids, proteins, or metabolites of Table 1 or variants thereof.
In another embodiment the method is used to detect environmental factors that cause or exacerbate autism, or accelerators of autism. In a further aspect the method is used to identify nutritional factors or supplements for treating autism. In a further aspect the nutritional factor or supplement is zinc, manganese, or cholesterol or other nutritional factors related to pathways regulated by genes identified in Tables 1, 2, 5 or 7.
In yet another embodiment the methods are used to determine gene expression level changes that are used to identify clinically relevant symptoms and treatments, time of disease onset, and disease severity. In yet another aspect the neural organoids are used to identify novel biomarkers that serve as data input for development of algorithm techniques as predictive analytics. In one aspect 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.
In a seventh embodiment the invention provides methods for predicting risk of co-morbidity onset that accompanies autism. Said methods first determines gene expression changes in neural organoids from a normal human individual versus an autistic human individual. Genes that change greater than 1.4 fold are associated with co-morbidities as understood by those skilled in the art.
In an eighth embodiment, the invention provides kit for predicting the risk of current or future onset of autism. 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 autism.
In a ninth embodiment, the invention provides methods for identifying therapeutic agents for treating autism. 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 autism.
In a tenth embodiment, the invention provides methods for predicting a risk for developing autism 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 autism. In certain embodiments, the at least one cell sample reprogrammed to the induced pluripotent stem cell is a fibroblast. In certain embodiments, the measured biomarkers comprise nucleic acids, proteins, or metabolites. In certain embodiments, the measured biomarker is a nucleic acid encoding human TSC1, TSC2 or a TSC2 variant. In certain embodiments, the measured biomarkers comprise one or a plurality of genes as identified in Tables 1, 2, 5 or 6. In certain embodiments, the neural organoid sample is procured from minutes to hours up to 15 weeks post inducement. In certain embodiments, the biomarkers to be tested are one or a plurality of biomarkers in Table 6 (Diagnostic Neural Organoid Authentication Genes).
These and other data findings, features, and advantages of the present invention will be more fully understood from the following detailed description taken together with the accompanying claims. It is noted that the scope of the claims is defined by the recitations therein and not by the specific discussion of features and advantages set forth in the present description.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of ordinary skill in the art. The following references provide one of skill with a general definition of many of the terms used in this disclosure: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991). These references are intended to be exemplary and illustrative and not limiting as to the source of information known to the worker of ordinary skill in this art. As used herein, the following terms have the meanings ascribed to them below, unless specified otherwise.
It is noted here that as used in this specification and the appended claims, the singular forms “a,” “an,” and “the” also include plural reference, unless the context clarity dictates otherwise.
The term “about” or “approximately” means within 25%, such as within 20% (or 5% or less) of a given value or range.
As used herein, the terms “or” and “and/or” are utilized to describe multiple components in combination or exclusive of one another. For example, “x, y, and/or z” can refer to “x” alone, “y” alone, “z” alone, “x, y, and z,” “(x and y) or z,” “x or (y and z),” or “x or y or z.”
It is noted that terms like “preferably,” “commonly,” and “typically” are not utilized herein to limit the scope of the claimed invention or to imply that certain features are critical, essential, or even important to the structure or function of the claimed invention. Rather, these terms are merely intended to highlight alternative or additional features that can or cannot be utilized in a particular embodiment of the present invention.
For the purposes of describing and defining the present invention, it is noted that the term “substantially” is utilized herein to represent the inherent degree of uncertainty that can be attributed to any quantitative comparison, value, measurement, or other representation. The term “substantially” is also utilized herein to represent the degree by which a quantitative representation can vary from a stated reference without resulting in a change in the basic function of the subject matter at issue.
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 similarly 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. By “neural 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. One skilled in the art will understand that 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. Further, one skilled in the art will recognize that within the brain regions and structures, granular neurons, dopaminergic neurons, GABAergic neurons, cholinergic neurons, glutamatergic neurons, serotonergic neurons, dendrites, axons, neurons, neuronal, cilia, purkinje fibers, pyramidal cells, spindle cells, express neuronal markers. One skilled in the art will recognize that this list is not 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, NeuroD1, DCX, EMX2, FOXG1I, and PROX1. Exemplary brain stem markers include, but are not limited to FGF8, INSM1, GATA2, ASCL I, GATA3. Exemplary spinal cord markers include, but are not limited to homeobox genes including but not limited to HOXA1, HOXA2, HOXA3, HOXB4, HOXA5, HOXCS, or HOXDI3. Exemplary 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. In one embodiment 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. As used herein, such an alteration can include a 10% change in expression levels, a 25% change, a 40% change, or even a 50% or greater change in expression levels.
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 WO2017123791A1 (https://patents.google.com/patent/WO2017123791Alten), 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. iPSCs are grown into neural organoids in said culture under appropriate conditions as set forth herein and the resulting neural organoids closely resemble developmental patterns similar to human brain. In particular, neural organoids develop anatomical features of the retina, forebrain, midbrain, hindbrain and spinal cord. Importantly, neural organoids express >98% of the about 15,000 transcripts found in the adult human brain. iPSCs can be derived from the skin or blood cells of humans identified with the genes listed in Table 1 (Novel Markers of Autism), Table 2 (Markers of Autism), Table 5 (Neural Organoid Autism Authenticating Genes) and Table 7 (Comorbidities of Autism).
In one embodiment, the about 12-week old iPSC-derived human neural organoid has ventricles and other anatomical features characteristic of a 35-40 day old neonate. In an additional embodiment the about 12 week old neural organoid expresses beta 3-tubulin, a marker of axons as well as somato-dendritic Puncta staining for MAP2, consistent with dendrites. In yet another embodiment, at about 12 weeks the neural organoid displays laminar organization of cortical structures. Cells within the 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.
Accordingly as noted, 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. Thus, transcriptomics is the study of the mRNA transcripts produced by the genome at a given tie in any particular cell or tissue of the organism. Transcriptomics employs high-throughput techniques to analyze genome expression changes associated with development or disease. In certain embodiments, transcriptomic studies can be used to compare normal, healthy tissues and diseased tissue gene expression. In further embodiments, mutated genes or variants associated with disease or the environment can be identified.
Consistent with this, the aim of developmental transcriptomics is identifying genes associated with, or significant in, organismal development and disease and dysfunctions associated with development. During development, genes undergo up- and down-regulation as the organism develops. Thus, transcriptomics provides insight into cellular processes, and the biology of the organism.
Generally, in one embodiment 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. However, RNA from the neural organoid can be harvested at minutes, hours, days or weeks after reprogramming. For instance, RNA can be harvested at about 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes and 60 minutes. In a further embodiment the RNA can be harvested 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, or 24 hours. In a further embodiment the RNA can be harvested at 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, or 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks 10 weeks, 11 weeks, 12 weeks or more in culture. After enriching for RNA sequences, an expressed sequence tag (EST) library is generated and quantitated using the AmpliSeg™ technique from ThermoFisher. Exemplars of alternate technologies include RNASeq and chip based hybridization methods. Transcript abundance in such experiments is compared in control neural organoids from healthy individuals vs. neural organoids generated from individuals with disease and the fold change in gene expression calculated and reported.
Furthermore, in one embodiment RNA from neural organoids for autism, are converted to DNA libraries and then the representative DNA libraries are sequenced using exon-specific primers for 20,814 genes using the AmpliSeg™ 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.
Briefly, in one embodiment, the array consists of one or a plurality of genes used to predict risk. In an alternative embodiment reads contain a plurality of genes, known to be associated with autism. In yet another embodiment the genes on the 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. Exemplarily, changes in expression or mutation of disease-specific genes are detected using such sequencing, and differential gene expression detected thereby, qualitatively by detecting a pattern of gene expression or quantitatively by detecting the amount or extent of expression of one or a plurality of disease-specific genes or mutations thereof. Results of said assays using the AmpliSeg™ technique can be used to identify genes that can predict disease risk or onset and can be targets of therapeutic intervention. In further embodiments, hybridization assays can be used, including but not limited to sandwich hybridization assays, competitive hybridization assays, hybridization-ligation assays, dual ligation hybridization assays, or nuclease assays.
Neural organoids are useful for pharmaceutical testing. Currently, 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 the organoids are easily derived from healthy and diseased patients, mitigating the need to conduct expensive clinical trials. Second, rodent models of human disease are unable to mimic the physiological nuances unique to human growth and development. Third, the use of primates creates ethical concerns. Finally, current methods are indirect indices of drug safety. Alternatively, neural organoids offer an inexpensive, easily accessible model of human brain development. The model permits direct, and thus more thorough, understanding of the safety, efficacy and toxicity of pharmaceutical compounds.
Starting material for neural organoids is easily obtained from healthy and diseased patients. Further, because human organoids are easily grown they can be produced en mass. This permits efficient screening 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. In further embodiments, the biomarker serves as a gene therapy target.
Changes in gene expression of specific genes when compared to those from non-diseased samples by >1.4 fold identify candidate genes correlating with a disease. Further searches of these genes in data base searches (e.g. Genecard, Malacard, Pubmed SFARI gene data base (https://gene.sfari.org/database/gene-scoring/); Human Protein Atlas (https://www.proteinatlas.org/ENSG00000115091-ACTR3/pathology) identify known diseases correlated previously with the disease state. In one embodiment AmpliSeg™ quantification of fold expression change allows for determination of fold change from control.
Autism and autism spectrum disorder are development disorders that negatively impact social interactions and day-to-day activities. The disorder is characterized by repetitive and unusual behaviors and reduced tolerance for sensory stimulation and gastrointestinal distress. The signs of autism occur early in life, usually around age 2 or 3. Autism affects approximately 1 in 68 children in the United States and approximately one third of people with autism remain non-verbal for their entire life. Many autism-predictive genes are associated with brain development, growth, and/or organization of neurons and synapses.
Early detection of autism is critical to providing therapy and tailored learning to minimize the effects of autism. The current inventive process, in one particular embodiment is a method for predicting a risk for developing autism in a human, the method comprising: procuring one or a plurality of cell samples from the 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 a neural organoid; collecting a biological sample from the 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 autism.
In a further particular embodiment, at least one cell sample such as a fibroblast is reprogrammed to become a pluripotent stem cell. In one embodiment the fibroblast is a skin cell that is induced to become a neural organoid after being reprogrammed to become a pluripotent stem cell. In a particular embodiment the neural organoid is harvested at about 1 week. In an alternate embodiment the neural organoid is harvested at about 4 weeks, and about 12 weeks. In another aspect the neural organoid can be harvested at days or weeks after reprogramming. At each time point the RNA is isolated and the gene biomarkers measured. The measured biomarkers comprise nucleic acids, proteins, or metabolites. In a particular embodiment the measured biomarker is a nucleic acid encoding human TSC1, TSC2 or a TSC2 variant.
In one embodiment the measured biomarker for human TSC1, TSC2, or a TSC2 variant means any nucleic acid sequence encoding a human TSC1 or TSC2 polypeptide having at least 70% homology to the sequence for human TSC1 or TSC2.
In a further embodiment additional measured biomarkers are nucleic acids encoding human genes, proteins, and metabolites as provided in Tables 1 and 2.
Although expression of multiple genes is altered in autism, in one embodiment lead candidate genes can be used to predict risk of autism onset later in life. In a particular embodiment a combination of biomarkers is detected, the combination comprising a nucleic acid encoding human TSC1, TSC2 or a TSC2 variant; and one or a plurality of biomarkers comprising genes, proteins, or metabolites as presented in Table 2. In a further embodiment the measured biomarkers mean any nucleic acid sequence encoding the respective polypeptide having at least 70% homology to the gene accession numbers listed in Table 2. Genes in Table 1 have specific mutations identified with them for autism and constitute likely causative biomarkers for autism.
The skilled worker will recognize these markers as set forth exemplarily herein to be-specific marker proteins as identified, inter alia, in genetic information repositories such as GenBank or the SFARI database. One skilled in the art will recognize that Accession Numbers are obtained using GeneCards, the NCBI database, or SFARI for example. One skilled in the art will recognize that alternative gene combinations can be used to predict autism. In addition autism risk can be predicted using detection of a combination of biomarkers the combination comprising a nucleic acid encoding human TSC1, TSC2, or a TSC2 variant; and one or a plurality of biomarkers comprising comprise human nucleic acids, proteins, or metabolites as listed in Tables 1 and 2.
In a further embodiment a combination of biomarkers is detected, the combination comprising human TSC1, TSC2, or a variant of TSC2; and one or a plurality of biomarkers comprising the biomarkers provided in Table 2 or a variant thereof.
In a further embodiment the combination comprises a nucleic acid encoding human TSC1, TSC2, or a TSC2 variant; and one or a plurality of biomarkers comprising a nucleic acid encoding biomarkers listed in Table 2 or variants thereof. The lead genes noted set forth herein are not exhaustive. One skilled in the art will recognize that other gene combinations can also be used to predict the risk of future autism onset.
One significant inventive advantage/advance in medicine demonstrated herein is the use of a neural organoid for a process to determine the risk of autism onset at birth and detection of environmental factors (e.g. heavy metals, infectious agents or biological toxins) and nutritional factors (e.g. nutritional factor, vitamin, mineral, and supplement deficiencies) that are causes or accelerators of autism. An accelerator of autism is an environmental or nutritional factor that specifically interactions with an autism specific biomarker to affect downstream process related to these biomarkers biological function such that a subclinical or milder state of autism becomes a full blown clinical state earlier or more severe in nature. These can be determined, without whole genome sequence analysis of patient genomes, solely from comparative differential gene expression analyses of in vitro neural organoids as models of brain development, only in conjunction with an inventive process that reproducibly and robustly promotes development of all the major brain regions and cell types.
Autism is difficult to diagnose before twenty-four months of age using currently available methods. An advantage of the current method is the identification of individuals susceptible to or having autism shortly after birth. The detection of novel biomarkers, as presented in Table 1 and/or Tables 2, 5, and 6 can be used to identify individuals who should be provided prophylactic treatment. In one aspect such treatments can include avoidance of environmental stimuli and accelerators that exacerbate autism. In a further aspect early diagnosis can be used in a personalized medicine approach to identify new patient specific pharmacotherapies for autism based on biomarker data. In a further aspect, the neural organoid model can be used to test the effectiveness of currently utilized autism therapies. For instance, the neural organoid can be used to test the effectiveness of currently utilized autism pharmacological agents such as Balovaptan (antagonist of vasopressin 1A receptor) and Aripiprazole (antagonist for 5-HT2A receptor). In one aspect the neural organoid could be used to identify the risk and/or onset of autism and additionally, provide patient-specific insights into the efficacy of using known pharmacological agents to treat autism. This allows medical professionals to identify and determine the most effective treatment for an individual autism patient, before symptoms arise. Furthermore, one skilled in the art will recognize that the effectiveness of additional FDA-approved, as well as novel drugs under development could be tested using the methods disclose herein. In a further aspect the method allows for development and testing of non-individualized, global treatment strategies for mitigating the effects and onset of autism.
An accelerator of autism is an environmental or nutritional factor that specifically interactions with an autism specific biomarker to affect downstream process related to this biomarker biological function such that a subclinical or milder state of autism becomes a full blown clinical state earlier or more severe in nature. In a particular embodiment, the neural organoid is about twelve weeks post-inducement and comprises the encoded structures and cell types of the retina, cortex, midbrain, hindbrain, brain stem, and spinal cord. However, because transcriptomics provides a snapshot in time, in one embodiment the neural organoid is procured after about one-week post inducement, four-week post inducement, and/or 12 weeks post inducement. However, the tissues from a neural organoid can be procured at any time after reprogramming. In a further embodiment, the neural organoid sample is procured from structures of the neural organoid that mimic structures developed in utero at about 5 weeks.
Gene expression measured in autism can encode a variant of a biomarker alteration encoding a nucleic acid variant associated with autism. In one embodiment 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.
In an alternative embodiment the method for predicting a risk for developing autism 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 autism wherein the measured biomarkers comprise those biomarkers listed in Table 2.
In a further embodiment the measured biomarker is a nucleic acid encoding human biomarkers or variants listed as listed in Table 1.
In yet another embodiment a plurality of biomarkers comprising a diagnostic panel for predicting a risk for developing autism in a human, comprising biomarkers listed in Tables 1 and 2, or variants thereof. In one aspect of the embodiment 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.
In yet an alternative embodiment the measured biomarker is a nucleic acid panel for predicting risk of autism in humans. The genes encoding the biomarkers listed in Table 1 or variants thereof.
Said panel can be provided according to the invention as an array of diagnostically relevant portions of one or a plurality of these genes, wherein the array can comprise any method for immobilizing, permanently or transiently, said diagnostically relevant portions of said one or a plurality of these genes, sufficient for the array to be interrogated and changes in gene expression detected and, if desired, quantified. In alternative embodiments the array comprises specific binding compounds for binding to the protein products of the one or a plurality of these genes. In yet further alternative embodiments, said specific binding compounds can bind to metabolic products of said protein products of the one or a plurality of these genes. In one aspect the presence of autism is detected by detection of one or a plurality of biomarkers as identified in Table 6.
Another alternative embodiment of the invention disclosed herein uses the neural organoids derived from the human patient in the non-diagnostic realm. The neural organoids express markers characteristic of a large variety of neurons and also include markers for astrocytic, oligodendritic, microglial, and vascular cells. The neural organoids 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. In one aspect measured biomarkers comprise biomarkers in Table 2, further wherein the measured biomarker is a gene, protein, or metabolite.
In yet another alternative embodiment neural organoids can be used to detect environmental factors as causes or accelerators of autism. The neural organoid can also be used in predictive toxicology to identify factors as causes or accelerators of autism. Examples in Table 1, Table 5, Table 7 include, but are not limited to lead, infectious agents or biological toxins. In still another aspect the method can be used to identify treatments that are causes or accelerators of autism and nutritional factors/supplements for treating autism. Examples in Table 1, Table 5, Table 7 include, but are not limited to nutritional factors, vitamins, minerals, and supplements such as zinc, manganese, or cholesterol. One of skill in the art will recognize that this list is not exhaustive and can include other known and unknown nutritional factors, vitamins, minerals, and supplements.
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. As such, 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.
In a further embodiment is a method by which 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 patterns 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. Consistent with this, 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
In one embodiment the exosomes can be collected at minutes to days after the neural organoid is generated. In a further embodiment, 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.
In a further embodiment, exosomes can be isolated at minutes, hours, days, or weeks after reprogramming. For instance, exosomes can be harvested at about 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, and 60 minutes. In a further embodiment 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. In yet a further embodiment 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, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks 10 weeks, 11 weeks, 12 weeks or more in culture.
Exosomes collected at a wide range of time points, referred to as scaled harvesting herein, allow for insights and data related to regulatory RNA changes that are indicative of disease onset. In one embodiment, the scaled harvesting allows for enrichment of specific biomarkers collected at specific time points from the normal human exosome. Moreover, 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. In one embodiment the neural organoid can be used to establish an exosome profile database (See APL Bioeng. 2019 March; 3(1)) that can be utilized for determining biomarkers characteristic of disease onset and timing of disease onset. In another embodiment 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.
In yet another embodiment, the 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.
In yet another embodiment, 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. In an initial step the exosome from a healthy individual is isolated, more specifically, 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, APP, and associated variants. The biomarkers may further comprise one or a plurality of genes as identified in Tables 1, 2, 5 or 6.
In a further embodiment 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. The use of 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.
These and other data findings, features, and advantage of the present disclosure will be more fully understood from the detailed description taken together with the accompanying claims. It is noted that the scope of the claims is defined by the recitations therein and not by the specific discussion of features and advantages set forth in the present description
The Examples that follow are illustrative of specific embodiments of the invention, and the use thereof. It is set forth for explanatory purposes only and is not taken as limiting the invention. In particular, the example demonstrates the effectiveness of neural organoids in predicting future disease risk.
The neural organoids described above were developed using the following materials and methods.
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 autism.
The invention advantageously provides many uses, including but not limited to a) early diagnosis of these diseases at birth from new born skin cells; b) Identification of biochemical pathways that increase environmental and nutritional deficiencies in new born 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. WT 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).
One skilled in the art will recognize that additional formulations of media and supplements can be used to culture, induce and maintain pluripotent stem cells and neural organoids.
Experimental protocols required the use of multiple media compositions including MEF Media, IPSO Media, EB Media, Neural Induction Media, and Differentiation Medias 1, 2, and 3.
Mouse embryonic fibroblast (MEF) was used in cell culture experiments. MEF Media comprised DMEM media supplemented with 10% Feta Bovine Serum, 100 units/ml penicillin, 100 microgram/ml streptomycin, and 0.25 microgram/ml Fungizone.
Induction media for pluripotent stem cells (IPSO Media) comprised DMEM/F12 media supplemented with 20% Knockout Replacement Serum, 3% Fetal Bovine Serum with 2 mM 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 2 mM Glutamax, IX Minimal Essential Medium containing Nonessential Amino Acids, 55 microM beta-mercaptoethanol, and 4 ng/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’
Three differentiation medias were used to produce and grow neural organoids. Differentiation Media 1 contained DMEM/F12 media and Neurobasal media in a 1:1 dilution. Each media is commercially available from Invitrogen. The base media was supplemented with a 1:200 dilution N2 supplement, a 1:100 dilution B27−vitamin A, 2.5 microgram/ml insulin, 55microM beta-mercaptoethanol kept under nitrogen mask and frozen at −20° C., 100 units/ml penicillin, 100 microgram/ml streptomycin, and 0.25 microgram/ml Fungizone.
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, 2.5 microgram/ml Insulin, 55 umicroMolar beta-mercaptoethanol kept under nitrogen mask and frozen at −20° C., 100 units/ml penicillin, 100 microgram/m1 streptomycin, and 0.25 microgram/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), 2.5 microgram/ml insulin, 55microMolar beta-mercaptoethanol kept under nitrogen mask and frozen at −20° C., 100 units/ml penicillin, 100 microgram/ml streptomycin, 0.25 microgram/ml Fungizone, TSH, and Melatonin.
The equipment used in obtaining, culturing and inducing differentiation of pluripotent stem cells is provided in Table 4 (Equipment used in Experimental Procedures). One skilled in the art would recognize that the list is not at all exhaustive but merely exemplary.
Human induced pluripotent stem cell-derived neural organoids were generated according to the following protocol, as set forth in International Application No. PCT/US2017/013231 incorporated herein by reference. Briefly, irradiated murine embryonic fibroblasts (MEF) were plated on a gelatin coated substrate in MEF media (Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% Feta Bovine Serum, 100 units/ml penicillin, 100 microgram/ml streptomycin, and 0.25 microgram/ml Fungizone) at a density of 2×105 cells per well. The seeded plate was incubated at 37° C. overnight.
After incubation, the MEFs were washed with pre-warmed sterile phosphate buffered saline (PBS). The MEF media was replaced with 1 mL per well of induced pluripotent stem cell (iPSC) media containing Rho-associated protein kinase (ROCK) inhibitor. A culture plate with iPSCs was incubated at 37° C. The iPSCs were fed every other day with fresh iPSC media containing ROCK inhibitor. The iPSC colonies were lifted, divided, and transferred to the culture wells containing the MEF cultures so that the iPSC and MEF cells were present therein at a 1:1 ratio. Embryoid bodies (EB) were then prepared. Briefly, a 100 mm culture dish was coated with 0.1% gelatin and the dish placed in a 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 pre-warmed PBS lacking Ca2+/Mg2+. A pre-warmed cell detachment solution of proteolytic and collagenolytic enzymes (1 mL/well) was added to the iPSC/MEF cells. The culture dishes were incubated at 37° C. for 20 minutes until cells detached. Following detachment, pre-warmed iPSC media was added to each well and gentle agitation used to break up visible colonies. Cells and media were collected and additional pre-warmed media added, bringing the total volume to 15 mL. Cells were placed on a gelatin-coated culture plate at 37° C. and incubated for 60 minutes, thereby allowing MEFs to adhere to the coated surface. The iPSCs present in the cell suspension were then counted.
The suspension was then centrifuged at 300×g for 5 minutes at room temperature, the supernatant discarded, and cells re-suspended in EB media supplemented with ROCK inhibitor (50 uM final concentration) and 4 ng/ml basic Fibroblast Growth Factor to a volume of 9,000 cells/150 μL. EB media is a mixture of DMEM/Ham's F-12 media supplemented with 20% Knockout Replacement Serum, 3% Fetal Bovine Serum (2 mM Glutamax), 1× Minimal Essential Medium Nonessential Amino Acids, and 55 μM beta-mercaptoethanol. The suspended cells were plated (150 μL) 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. For the first four days of culture, the EB media was supplemented with 50 uM ROCK inhibitor and 4 ng/ml bFGF. During the remaining two to three days the embryoid bodies were cultured, no ROCK inhibitor or bFGF was added.
On the sixth or seventh day of culture, the embryoid bodies were removed from the LIPIDURE® 96 well plate and transferred to two 24-well plates containing 500 μL/well Neural Induction media, DMEM/F12 media supplemented with a 1:50 dilution N2 Supplement, a 1:50 dilution GlutaMax, a 1:50 dilution MEM-Non-Essential Amino Acids (NEAA), and 10 μg/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 5 cm×7 cm) 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 μL 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.
Frozen Matrigel matrix aliquots (500 μL) were thawed on ice until equilibrated at 4° C. A single embryoid body was transferred to each dimple of the film. A single 7 cm×5 cm rectangle holds approximately twenty (20) embryoid bodies. Twenty microliter (20 μL) aliquots of Matrigel were transferred onto the embryoid bodies after removing extra media from the embryoid body with a pipette. The Matrigel was incubated at 37° C. for 30 min until the Matrigel polymerized. The 20 μL droplet of viscous Matrigel was found to form an optimal three dimensional environment that supported the proper growth of the neural organoid from embryoid bodies by sequestering the gradients of morphogens and growth factors secreted by cells within the embryoid bodies during early developmental process. However, the Matrigel environment permitted exchange of essential nutrients and gases. Gentle oscillation by hand twice a day for a few minutes within a tissue culture incubator (37° C./5% CO2) further allowed optimal exchange of gases and nutrients to the embedded embryoid bodies.
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 μg/mL insulin, 55 microM beta-mercaptoethanol kept under nitrogen mask and frozen at −20° C., 100 units/mL penicillin, 100 μg/mL streptomycin, and 0.25 μg/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° C. for 16 hours. After incubation, the embryoid body/Matrigel droplets were transferred from the film to the culture dishes containing media. Static culture at 37° C. was continued for 4 days until stable neural organoids formed.
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 μg/mL insulin, 55microM beta-mercaptoethanol kept under nitrogen mask and frozen at −20° C., 100 units/mL penicillin, 100 μg/mL streptomycin, and 0.25 μg/mL Fungizone. The flasks were placed on an orbital shaker rotating at 40 rpm within the 37° C./5% CO2 incubator.
The media was changed in the flasks every 3-4 days to provide sufficient time for morphogen and growth factor gradients to act on targets within the recipient cells forming relevant structures of the brains. Great care was taken when changing media so as to avoid unnecessary perturbations to the morphogen/secreted growth factor gradients developed in the outer most periphery of the organoids as the structures grew into larger organoids.
After approximately 12 weeks of in vitro culture, transcriptomic and immunohistochemical analysis indicated that 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 (
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
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 (
Tyrosine hydroxylase, an enzyme used in the synthesis of dopamine, was observed in the organoids using immunocytochemistry (
In sum, 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 2-3 mm 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
Neural organoids were evaluated at weeks 1, 4 and 12 by transcriptomics. The organoid was reproducible and replicable (
Gene expression patterns were analyzed using whole genome transcriptomics as a function of time in culture. Results reported herein indicate that within the neural organoid known developmental order of gene expression in vivo occurs, but on a somewhat slower timeline. For example, the in vitro temporal expression of the transcription factors NURRI and PITX3, genes uniquely expressed during midbrain development, replicated known in vivo gene expression patterns (
Tuberous sclerosis complex (TSC) is a genetic disorder that causes non-malignant tumors to form in multiple organs, including the brain. TSC negatively impacts quality of life, with patients experiencing seizures, developmental delay, intellectual disability, gastrointestinal distress and autism. 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.
Using methods as set forth in Example 1, a human neural organoid from iPSCs was derived from a patient with a gene variant of the TSC2 gene (ARG 1743GLN) from iPSCs (Cat #GM 25318 Coriell Institute Repository, NJ). This organoid served as a genetic model of a TSC2 mutant.
Both normal and TSC2 mutant models were subject to genome-wide transcriptomic analysis using the Ampliseg™ analysis (ThermoFisher) to assess changes in gene expression and how well changes correlated with the known TSC clinical pathology (
Whole genome transcriptomic data showed that of all the genes expressed (13,000), less than a dozen showed greater than two-fold variance in the replicates for both Normal N)) and TSC2. This data supported the robustness and replicability of the human neural organoids at week 1 in culture.
Clinically TSC patients present with tumors in multiple organs including the brain, lungs, heart, kidneys and skin (Harmatomas). In comparison of WT and TSC2, the genes expressed at two-fold to 300-fold differences, included those correlated with 1) tumor formation and 2) autism mapped using whole genome and exome sequencing strategies (SFARI site data base) (
Thus, the transcriptomic data disclosed herein correlated well with known clinical phenotypes of tumors, autism and other clinical symptoms in TSC patients and demonstrated the usefulness of the human neural organoid model.
Autism and autism 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 autism-predictive genes are associated with brain development, growth, and/or organization of neurons and synapses.
Autism has a strong genetic link with DNA mutations comprising a common molecular characteristic of autism. Autism encompasses a wide range of genetic changes, most often genetic mutations. The genes commonly identified as playing a role in autism include novel markers provided in Table 1 and autism 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 autism risk. In a further aspect mutations in the genes disclosed can be determined at hours, days or weeks after reprogramming.
In a second embodiment, 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 autism using above described methods for calculating risk. One skilled in the art would recognize that additional biomarker combinations expressed in the human neural organoid can also be used to predict future autism onset.
The model used herein is validated and novel in that data findings reconcile that the model expresses sixty seven markers of autism that reflect the genes mutated in the genome of humans with autism (SFARI database of biomarkers, 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 autism markers early in development including at birth.
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.
The human neural organoid model data findings can be used in the prediction of comorbiditity onset or risk associated with autism including at birth.(https://en.wikipedia.org/wiki/Conditions_comorbid_to_autism_spectrum_disorders). In detecting comorbidities, genes associated with one or more of these diseases are detected from the patient's grown neural organoid. Such genes include, comorbidities and related accession numbers include, those listed in Table 7:
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. One having skill in the art will recognize that variants derive from the full length gene sequence. Thus, the data findings and sequences in Table 7 encode the respective polypeptide having at least 70% homology to other variants, including full length sequences.
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 autism disease can be elucidated. For instance, Rapamycin has been shown to be beneficial in autism (Caban et al., 2017, Genetics of tuberous sclerosis complex: implications for clinical practice, Appl Clin Genet. 10: 1-8). Consistent with this, a human neural organoid from a patient with tuberous sclerosis was used to determine changes in gene expression following rapamycin treatment. The changes in gene expression provided insights into gene expression alterations that are beneficial and those that are detrimental for autism risk and onset. Neural organoids as provided herein can be used for testing candidate pharmaceutical agents, as well as testing whether any particular pharmaceutical agent inter alia for autism 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.
From the foregoing description, it will be apparent that variations and modifications can be made to the invention described herein to adopt it to various usages and conditions. Such embodiments are also within the scope of the following claims.
The recitation of a listing of elements in any definition of a variable herein includes definitions of that variable as any single element or combination (or sub-combination) of listed elements. The recitation of an embodiment herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.
All patents and publications mentioned in this specification are herein incorporated by reference to the same extent as if each independent patent and publication was specifically and individually indicated to be incorporated by reference.
Having described the invention in detail and by reference to specific aspects and/or embodiments thereof, it will be apparent that modifications and variations are possible without departing from the scope of the invention defined in the appended claims. More specifically, although some aspects of the present invention may be identified herein as particularly advantageous, it is contemplated that the present invention is not limited to these particular aspects of the invention. Percentages disclosed herein can vary in amount by ±10, 20, or 30% from values disclosed and remain within the scope of the contemplated invention.
| Number | Date | Country | |
|---|---|---|---|
| 62933957 | Nov 2019 | US |
