Orthogonally regulated chemical induced differentiation (orchid) and induced microphysiological platforms for accelerated CNS testing (impact) for brain region-specific organoids and assembloids

Abstract

Compositions and methods are provided for the generation of brain organoids comprising diverse cell types, including without limitation neurons, astrocytes, oligodendrocytes, microglia, endothelial cells, etc. The organoids may be region-specific. The invention leverages genome editing, including without limitation CRISPR activation (CRISPRa); and orthogonal chemical induction for directed differentiation of human-induced pluripotent stem cells (iPSCs) to specific brain cell types.

Description

ORTHOGONALLY REGULATED CHEMICAL INDUCED DIFFERENTIATION (ORCHID) AND INDUCED MICROPHYSIOLOGICAL PLATFORMS FOR ACCELERATED CNS TESTING(IMPACT) FOR BRAIN REGION-SPECIFIC ORGANOIDS AND ASSEMBLOIDSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to the filing of United States Provisional Application Serial No. 63 / 750,437, filed January 28, 2025, the disclosure of which application is herein incorporated by reference.BACKGROUND OF THE INVENTION

[0002] Organoid-based models of brain regions have become pivotal for understanding human brain development and disease pathology. However, existing protocols for generating brain organoids face significant limitations, including long maturation times, lack of vasculature, and absence of the complete spectrum of brain cell types, particularly microglia. Approaches like coculture with endothelial cells or transplantation into animal models have shown promise but remain challenging and resource intensive.

[0003] There remains an unmet need for a more comprehensive yet easy to follow method to generate brain organoids that capture the complex cellular diversity of the human brain, including immune system interactions and vasculature. The present invention seeks to address these limitations by introducing a modular approach combining orthogonally regulated chemical- induced differentiation (ORChlD) of induced pluripotent stem cells (iPSCs) and in vitro genetic switch-based organoid reconstitution (INVIGOR).SUMMARY OF THE INVENTION

[0004] A microphysiological system is provided that allows modeling of brain function, testing of hypotheses, and evaluation of drug candidates targeting CNS disorders. The core technology integrates two systems: ORChlD (Orthogonally Regulated Chemical-Induced Differentiation) to selectively drive differentiation into subtypes of neurons, astrocytes, oligodendrocytes, microglia, and endothelial cells; and IMPACT (Induced Microphysiological Platforms for Accelerated CNS Testing) to reconstitute specific ORChlD cell lines to generate brain organoids with controlled cell-type compositions, allowing synchronous and in situ maturation. Region-specific IMPACT organoids can be tailored to different areas of the brain to enable versatile and disease-relevant applications. The methods of the disclosure allow the generation of complex brain organoids with diverse, functional cell types in a fraction of the time required by current methods.

[0005] Compositions and methods are provided for the generation of brain region-specific organoids comprising diverse cell types, including without limitation neurons, astrocytes,oligodendrocytes, microglia, endothelial cells, etc. Methods of the disclosure include genome editing, including without limitation CRISPR activation (CRISPRa); and orthogonal chemical induction for directed differentiation of human-induced pluripotent stem cells (iPSCs) to specific brain cell types. This approach addresses limitations in previously described brain organoid models, which include lack of vasculature, long maturation times, and incomplete cellular composition.

[0006] Brain region-specific organoids are generated using a two-phase workflow. In an embodiment, a first phase comprises generating a plurality of Orthogonally Regulated Chemical Induced Differentiation (ORChlD) cell lines. For these purposes, a pluripotent cell, e.g. an iPSC, is genetically modified by introduction of sequence encoding a “catalytically dead” nuclease fused to a transcriptional activation domain. In some embodiments the nuclease is a Cas nuclease, e.g. Cas12a, Cas9, etc. In an embodiment the nuclease is dCas9, which binds to its target but does not cleave it. In some embodiments the transcriptional activation domain is VPR activation domain. The coding sequence is operably linked to an inducible promoter, e.g. a promoter activated by contact with tetracycline, doxycycline, ecdysone, gibberellic acid, abscisic acid, etc. The inducing agent may be the same or different for each targeted cell type.

[0007] The pluripotent cell is further modified by introduction of sequences for sgRNA targeting transcription factors of interest for differentiation of brain cells. The sgRNA may be introduced on a vector, as protein-RNA complexes, and the like. The loci for sgRNA selection are provided in Table 2. The engineered pluripotent cell may be referred to herein as an ORChlD cell.

[0008] In some embodiments the sgRNA targets are selected from 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10 or more of the cells described in Table 2. In some embodiments, the targeted transcription factor(s) is selected from the factors identified in Table 2. Cells may include one or more or all of the types: neurons, astrocytes, oligodendrocytes, microglia, and endothelial cells; and may comprise one or more of the subtypes set forth in Table 2, e.g. Microglia-1 ; Microglia-2; Microglia-3; Microglia- 4; Astrocytes- 1 ; Astrocytes-2; Astrocytes-3; Astrocytes-4; Oligodendrocytes / OPCs -1 ; Oligodendrocytes / OPCs -2; Endothelial cells; Excitatory neurons -1 ; Excitatory neurons -2; Excitatory neurons -2; Inhibitory neurons-1 ; Inhibitory neurons-2; GABAergic INs (SST + PVALB); Hypothalamus-1 ; Hypothalamus-2; Hypothalamus-3; Hypothalamus-4; Medium Spiny Neurons- 1 ; Medium Spiny Neurons-2; Medium Spiny Neurons-3; Medium Spiny Neurons-4; Medium Spiny Neurons-5; Medium Spiny Neurons-6; Dopaminergic neurons -1 ; Dopaminergic neurons -2; Dopaminergic neurons -3; Dopaminergic neurons -4; Dopaminergic neurons -5; Dopaminergic neurons -6; Dopaminergic neurons -7; Dopaminergic neurons -8; Motor Neurons -1 ; Motor Neurons -2; Motor Neurons -3; Motor Neurons -4; Motor Neurons -5; Motor Neurons -6; Peripheral sensory / DRGs -1 ; Peripheral sensory / DRGs -2; Peripheral sensory / DRGs -3; and Serotonergic neurons.

[0009] The pluripotent cells may also be modified by introduction of sequences encoding cellular identity tags. The cellular identity tags may comprise, without limitation, one or more of epitope tags, fluorescent proteins, etc. The tags may be unique to the targeted cell type; or may be common to all engineered cells. Cellular identity tags may comprise membrane-tethered epitopes, e.g., V5, FLAG, HA, c-Myc, etc. for affinity based identification and sorting. Cellular identity tags may alternatively, or in combination, comprise fluorescent reporter proteins. These tags are useful in cell-type-specific analysis, enabling downstream isolation and characterization.

[0010] In an embodiment a composition is provided of isolated engineered cells of the disclosure e.g. one or a panel of specifically engineered cell lines that can be induced to generate a cell type of interest.

[0011] A selection of ORChlD iPSCs, e.g. a panel of pre-defined cell types are combined to reconstitute embryoid bodies containing the desired proportions of these cells that will be differentiated upon small molecule induction to mimic human brain tissue or to study any combinations of these cell types. This protocol is referred to as Induced Microphysiological Platforms for Accelerated CNS Testing (IMPACT); and allows in situ differentiation of the listed cell types without the need for specialized patterning growth factors or fusing of organoids containing different cell types later in development. The ratio of defined cell types in a culture may also be pre-determined.

[0012] The embryoid body comprising ORChlD cells is induced to express the nuclease- transcription activation fusion protein, thereby activating the sgRNA-targeted transcription factors in each of the ORChlD cell types, and inducing differentiation into the various desired plurality of brain cell types. This orthogonal induction system allows for the generation of cellular diversity, incorporating sub-types of neurons, glial cells, microglia, and endothelial cells. This protocol allows co-culturing of a plurality of cell types for the entire duration of the organoid development, allowing critical cell-cell interactions between neuronal, glial and immune cells akin to the human brain. Time-specific induction of cell differentiation can be detected in IMPACT organoids by comparing organoids that receive an inducing agent vs those cultured in the absence of the inducing agent.

[0013] The modular nature of ORChlD and IMPACT supports both complex brain organoids with all major cell types, and reductionist models comprising a subset of brain cell types, e.g. for studying specific disease mechanisms such as Parkinson's disease with predominantly dopaminergic neurons. The reconstitution model allows for investigation of healthy and diseased cell types within the same organoid if needed. For example, using IMPACT, organoids with healthy neurons and glial ORChlD cells and patient derived / diseased microglial ORChlD cells can be reconstituted to study specific effects of diseased microglia in complex brain organoids.

[0014] The differentiation of the methods of the disclosure accelerates maturation of brain organoids relative to conventional methods. In some embodiments organoids are developed in less than about 45 days, less than about 40 days, less than about 33 days.

[0015] In an embodiment, a composition of embryoid bodies thus generated is provided. In some embodiments the organoids are from about 1 to about 3 mm in diameter. In some embodiments each cell type, or each cell sub-type, expresses a unique cellular identity tag, e.g. one or a combination of fluorescent proteins and epitope tags. Fluorescent tags can be viewed using a fluorescence microscope, flow cytometer, etc., and allow sorting and / or analysis of the cells of each type.

[0016] In some embodiments, methods are provided for determining the activity of a candidate agent on human cells present in an organoid developed by the methods of the disclosure or isolated from such organoids, the method comprising contacting the candidate agent with one or a panel of organoids or purified cells derived therefrom. The cell populations optionally comprise at least one allele encoding a mutation associated or potentially with a neuropsychiatric disease; and determining the effect of the agent on morphological, genetic or functional parameters, including without limitation gene expression profiling. Methods of analysis at the single cell level are also of interest, e.g. migration assays, axonal growth and pathfinding assays, atomic force microscopy, super resolution microcopy, light-sheet microscopy, two-photon microscopy, patch clamping, single cell gene expression (RNA-seq), calcium imaging with pharmacological screens, modulation of synaptogenesis, and the like.

[0017] In some embodiments, one or more such organoids are provided, including without limitation a panel of such in vitro derived organoids are provided, where the panel includes two or more genetically different cells. In some embodiments a panel of such organoids are provided, where the systems can be subjected to a plurality of candidate agents, or a plurality of doses of a candidate agent. Candidate agents include small molecules, i.e. drugs, genetic constructs that increase or decrease expression of an RNA of interest, infectious agents, electrical changes, and the like. In some embodiments a panel refers to a system or method utilizing patient-specific systems from two or more distinct conditions, and may be three or more, four or more, five or more, six or more, seven or more genetically distinct conditions.

[0018] These and other objects, advantages, and features of the invention will become apparent to those persons skilled in the art upon reading the details of the subject methods and compositions as more fully described below.BRIEF DESCRIPTION OF THE DRAWINGS

[0019] The invention is best understood from the following detailed description when read in conjunction with the accompanying drawings. It is emphasized that, according to commonpractice, the various features of the drawings are not to-scale. On the contrary, the dimensions of the various features are arbitrarily expanded or reduced for clarity. Included in the drawings are the following figures.

[0020] FIG. 1 : Schematic representation of the ORChlD protocol for editing iPSCs. Briefly, PiggyBac Transposase and constructs with compatible insertion sites are used to integrate Part 1 and Part 2 synthetic DNA constructs in the iPSCs. See Table 1 for details related to Part 1 and Part 2 construct designs. Edited cells undergo antibiotic selection and single clone isolation by cell sorting. These stable ORChlD lines are then used for IMPACT protocol.

[0021] FIG. 2: Schematic representation of IMPACT protocol. Briefly, the ORChlD lines for the desired cell types can be mixed in required fractions to generate embryoid bodies (EBs) with 10k cells each. The EBs are patterned using small molecules outlined in Figure 3 and differentiation of the different cell types is induced by addition of Doxycycline.

[0022] FIG. 3: Schematic representation of the IMPACT organoid differentiation timeline. SB, DM and XAV are used for dual SMAD inhibition. bFGF and EGF are used for progenitor expansion. Doxycycline is used for induction of dCas9-VPR to mediate cell type specific transcription factor upregulation thereby inducing differentiation of iPSCs to intended cell types like iA (induced astrocytes), iN (induced neurons), iO (induced oligodendrocytes), iM (induced microglia) and iE (induced endothelial cells).

[0023] FIG. 4. Micrographs of various cell types visualized by immunostaining of dissociated cells from IMPACT organoids. A) iAstrocytes labeled with GFAP antibodyB) Excitatory iNeurons labeled with MAP2 antibodyC) Dopaminergic iNeurons labeled with Tyrosine Hydroxylase (TH) antibodyD) iOligodendrocytes labeled with 04 antibodyE) iM icroglia labeled with TMEM119B and IBA1 F) iEndothelial cells labeled with CD31 (PECAM-1).DETAILED DESCRIPTION OF T HE INVENTION

[0024] Before the present compositions and methods are described, it is to be understood that this invention is not limited to particular compositions and methods described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

[0025] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limits of that range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range, and each range whereeither, neither or both limits are included in the smaller ranges is also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[0026] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, some potential and preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. It is understood that the present disclosure supersedes any disclosure of an incorporated publication to the extent there is a contradiction.

[0027] It must be noted that as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a reprogramming factor polypeptide” includes a plurality of such polypeptides, and reference to "the induced pluripotent stem cells" includes reference to one or more induced pluripotent stem cells and equivalents thereof known to those skilled in the art, and so forth.

[0028] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.DEFINITIONS

[0029] By “pluripotency” and pluripotent stem cells it is meant that such cells have the ability to differentiate into all types of cells in an organism. The term “induced pluripotent stem cell” encompasses pluripotent cells, that, like embryonic stem cells (hESC), can be cultured over a long period of time while maintaining the ability to differentiate into all types of cells in an organism. hiPSC have a human hESC-like morphology, growing as flat colonies containing cells with large nucleo-cytoplasmic ratios, defined borders and prominent nuclei. In addition, hiPSC express pluripotency markers known by one of ordinary skill in the art, including but not limited to alkaline phosphatase, SSEA3, SSEA4, SOX2, OCT3 / 4, NANOG, TRA-1-60, TRA-1-81 , etc. In addition, the hiPSC are capable of forming teratomas and are capable of forming or contributing to ectoderm, mesoderm, or endoderm tissues in a living organism.

[0030] As used herein, “reprogramming factors” refers to one or more, i.e. a cocktail, of biologically active factors that act on a cell, thereby reprogramming a cell to multipotency or to pluripotency. Reprogramming factors may be provided to the cells, e.g. cells from an individual with a family history or genetic make-up of interest for heart disease such as fibroblasts, adipocytes, etc.; individually or as a single composition, that is, as a premixed composition, of reprogramming factors. The factors may be provided at the same molar ratio or at different molar ratios. The factors may be provided once or multiple times in the course of culturing the cells of the subject invention. In some embodiments the reprogramming factor is a transcription factor, including without limitation, OCT3 / 4; SOX2; KLF4; c-MYC; NANOG; and LIN-28.

[0031] Somatic cells are contacted with reprogramming factors, as defined above, in a combination and quantity sufficient to reprogram the cell to pluripotency. Reprogramming factors may be provided to the somatic cells individually or as a single composition, that is, as a premixed composition, of reprogramming factors. In some embodiments the reprogramming factors are provided as a plurality of coding sequences on a vector. The somatic cells may be fibroblasts, adipocytes, stromal cells, and the like, as known in the art. Somatic cells or hiPSC can be obtained from cell banks, from normal donors, from individuals having a neurologic or psychiatric disease of interest, etc.

[0032] Following induction of pluripotency, hiPSC are cultured according to any convenient method, e.g. on irradiated feeder cells and commercially available medium. The hiPSC can be dissociated from feeders by digesting with protease, e.g. dispase, preferably at a concentration and for a period of time sufficient to detach intact colonies of pluripotent stem cells from the layer of feeders. The spheroids can also be generated from hiPSC grown in feeder-free conditions, by dissociation into a single cell suspension and aggregation using various approaches, including centrifugation in plates, etc.

[0033] The somatic cells or the hiPSC derived therefrom are engineered as described herein by ORChlD to introduce inducible transcription factors. In addition the cells may be further modified for a variety of purposes, e.g. to replace genes having a loss of function mutation, provide marker genes, etc. Alternatively, vectors are introduced that express antisense mRNA, siRNA, ribozymes, etc. thereby blocking expression of an undesired gene. Other methods of gene therapy are the introduction of drug resistance genes to enable normal progenitor cells to have an advantage and be subject to selective pressure, for example the multiple drug resistance gene (MDR), or anti-apoptosis genes, such as BCL-2. Various techniques known in the art may be used to introduce nucleic acids into the target cells, e.g. electroporation, calcium precipitated DNA, fusion, transfection, lipofection, infection and the like, as discussed above. The particular manner in which the DNA is introduced into the cells, including transient or any form of genetically encoded element is not critical to the practice of the invention.

[0034] Disease-associated or disease-causing genotypes can be generated in healthy hiPSC through targeted genetic manipulation (CRISPR / CAS9, etc) or hiPSC can be derived from individual patients that carry a disease-related genotype or are diagnosed with a disease, e.g.22q11 .2 deletion syndrome etc. Moreover, neural diseases with less defined or without genetic components can be studied within the model system, e.g. hypoxic brain injury, inflammation etc. Conditions of neurodevelopmental and neuropsychiatric disorders and neural diseases that have strong genetic components or are directly caused by genetic or genomic alterations can be modeled with the systems of the invention. Genetic alterations include for example point mutations in genes such as NLGN1 / 3 / 4, NRXN1 / 4, SHANK1 / 2 / 3, GRIN2B / A, FMR1 , or CHD8 that represent risk alleles for autism spectrum disorders, point mutations in or deletions of genes such as CACNA1C, CACNB2, NLGN4X, LAMA2, DPYD, TRRAP, MMP16, NRXN1 or NIPAL3 that are associated with schizophrenia or autism spectrum disorders (ASD), etc, a triplet expansion in the HTT gene that cause to Huntington’s disease (HD), monoallelic mutations in genes such as SNCA, LRRK2 and biallelic mutations in genes such as PINK1 , DJ-1 , or ATP13A2 that predispose to Parkinson disease (PD), single nucleotide polymorphisms (SNPs) in genes such as ApoE, APP, and PSEN1 / 2 that confer risks for developing Alzheimer’s disease (AD) and other forms of dementia, as well as SNPs in genes such as CACNA1C, CACNB3, ODZ4, ANK3 that are associated with bipolar disease (BP); Angelman (UBE3A), Rett (MEPC2), Tuberous sclerosis (TSC1 / 2). Genomic alterations include copy number variations (CNVs) such as deletions or duplications of 1 q21.1 , 7q11 .23, 15q11.2, 15q13.3, 22q11.2 or 16p11 .2, 16p13.3 that are associated with ASD, schizophrenia, intellectual disability, epilepsy, etc; trisomy 21 and Down Syndrome, Fragile X syndrome caused by alteration of the FMR1 gene. Any number of neurodevelopment disorders with a defined genetic etiology can be additionally modeled by introducing mutations in or completely removing disease-relevant gene(s) in control hiPSC using genome editing, e.g. CRISPR. A particular advantage of this method is the fact that edited hiPSC lines share the same genetic background as their corresponding, non-edited hiPSC lines. This reduces variability associated with line to line differences in genetic background.

[0035] Disease relevance. The effect of drugs on brain cells, e.g. neurons, astrocytes, microglia, etc. are of particular interest, where efficacy and toxicity may rest in sophisticated analysis of neuronal migratory and electrical interactions, or the ability of neurons to form functional networks, rather than on simple viability assays. The discrepancy between the number of lead compounds in clinical development and approved drugs may partially be a result of the methods used to generate the leads and highlights the need for new technology to obtain more detailed and physiologically relevant information on cellular processes in normal and diseased states.

[0036] IMPACT organoids containing neurons, astrocytes, oligodendrocytes, microglia, and vasculature, or any desired combination of these cell types, offer a comprehensive platform for studying a wide range of neurological, developmental, and vascular conditions. These organoids can be applied to model neurodegenerative disorders such as Alzheimer’s disease, by investigating amyloid-beta plaque deposition, tau pathology, and glial-neuronal interactions; Parkinson’s disease, through modeling dopaminergic neuron loss and glial responses; Huntington’s disease, by exploring mechanisms of neuronal degeneration and toxic glial interactions; and multiple sclerosis, to understand demyelination and potential re-myelination therapies mediated by oligodendrocytes. They are equally valuable in studying neurovascular and ischemic injuries, such as stroke and reperfusion injury, by examining the role of vascular systems, microglia, and neuroinflammatory responses, as well as for understanding combined neuronal, glial, and vascular interactions.

[0037] In the field of neurodevelopmental disorders, these organoids allow modeling of autism spectrum disorders (ASD) by examining synaptic dysfunction, microglial pruning, and astrocyte- mediated neuronal development, as well as schizophrenia, by exploring altered neuronal-glial signaling and vascular dysfunction. They can also help investigate hypoxia-related injury mechanisms and therapeutic approaches in cerebral palsy and preterm brain injuries. In the domain of neurovascular diseases, the organoids can model vascular dementia to study bloodbrain barrier (BBB) dysfunction and vascular contributions to cognitive decline, as well as rare genetic syndromes involving neurovascular dysfunction. For infectious diseases, these organoids are ideal for studying the neuropathological effects of conditions like Zika virus infection, which causes microcephaly through vascular and neuronal interactions, and HIV-associated neurocognitive disorders (HAND), by exploring microglial activation and neuroinflammation.

[0038] Additionally, the inclusion of vasculature and immune-relevant cells like microglia allows the organoids to simulate tumor microenvironments for glioblastoma multiforme (GBM), providing insights into tumor invasion, therapeutic resistance, and interactions with astrocytes and vasculature. They are also applicable in modeling metastatic brain cancer to understand the interplay between vascular systems and cancer cells. These organoids are invaluable for drug discovery and toxicity testing, offering platforms for high-throughput drug screening targeting neuronal, glial, and vascular components. They enable studies on neuroinflammation, BBB permeability, and personalized medicine approaches to treatment. Moreover, they can support the development and testing of gene-editing therapies, such as CRISPR, for neurodegenerative and vascular diseases, and facilitate cell replacement therapies targeting neuronal, glial, and vascular repair.

[0039] These organoids provide a novel avenue to study rare genetic disorders like lysosomal storage diseases, leukodystrophies, metabolic disorders, Copy number variant diseases, andother neurovascular syndromes, advancing our understanding of their underlying mechanisms and testing potential interventions. By incorporating these diverse applications into the patent, the organoids’ ability to replicate the complexity of the human brain at cellular and systemic levels positions them as a groundbreaking tool for advancing research and developing therapeutics across multiple clinical domains.

[0040] In addition, a number of important clinical conditions are associated with altered function of neurons, glia (astrocytes and oligodendrocytes), microglia, vasculature etc, including neurodegenerative disorders (Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), and amyotrophic lateral sclerosis (ALS), or psychiatric conditions such as schizophrenia and other psychoses, bipolar disorders, mood disorders, intellectual disability or autism spectrum disorders.

[0041] The models may incorporate cells with genetic changes, including genotypes that affect migratory, or synaptic function in disorders such as schizophrenia, autism spectrum disorders, monogenic disorders such as Timothy Syndrome, or polygenic diseases 22q11.2 deletion syndrome etc. In other embodiments effects of genotypes and agents on neural development can be assessed using neural cultures of the invention during the process of differentiation and migration. Thus, neurodevelopmental effects can be tested that either immediately affect function, maturation, and viability of developing cells, or exhibit long-term effects emerging as phenotypes in mature neural cultures.

[0042] A number of neuropsychiatric disorders may arise from an alteration or disruption in the balance of excitation and inhibition in the cerebral cortical circuitry. Additionally, a number of studies have shown that the lack of proper cortical interneuron specification may play a significant role in the development of neuropsychiatric disorders (schizophrenia, autism spectrum disorders, epilepsy and other seizure disorders). This may entail a deviation from either the course of interneuron development, or aberrant transcriptional regulation in the cortical interneuron specification process. Understanding the effects of both GABAergic neurotransmission, alterations in inhibitory cortical circuits, and how they may be responsible for the clinical features observed in schizophrenia or autism are paramount to this field of research. Conditions of interest may also include DISC1 -related disorders, Rett syndrome, Fragile X, Alexander's disease, and others.

[0043] Autism spectrum disorders (ASDs) are neurodevelopmental disorders characterized by varying degrees of impaired social interaction and communication and the presence of repetitive and stereotypical behaviors. Some models of ASD emphasize the idea that abnormal synapse development underlies many features of the disease and postulate abnormalities in excitatory- inhibitory balance (E / l ratio). A better understanding of neuronal interactions in ASDs will shed light on pathogenesis and the development of new treatment strategies.

[0044] The terms "treatment", "treating", "treat" and the like are used herein to generally refer to obtaining a desired pharmacologic and / or physiologic effect. The effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and / or may be therapeutic in terms of a partial or complete stabilization or cure for a disease and / or adverse effect attributable to the disease. "Treatment" as used herein covers any treatment of a disease in a mammal, particularly a human, and includes: (a) preventing the disease or symptom from occurring in a subject which may be predisposed to the disease or symptom but has not yet been diagnosed as having it; (b) inhibiting the disease symptom, i.e., arresting its development; or (c) relieving the disease symptom, i.e., causing regression of the disease or symptom.

[0045] The terms "individual," "subject," "host," and "patient," are used interchangeably herein and refer to any mammalian subject for whom diagnosis, treatment, or therapy is desired, particularly humans.

[0046] Fluorescent proteins. Protein-encoded fluorophores are genetically encoded fluorescent proteins derived primarily from marine organisms and engineered for diverse applications in biological imaging. The most well-known is green fluorescent protein (GFP), originally isolated from Aequorea victoria, which fluoresces green under blue or ultraviolet light. Variants of GFP, such as enhanced GFP (EGFP), have been optimized for higher brightness and photostability. Expanding the palette, blue fluorescent protein (BFP) and cyan fluorescent protein (CFP) are engineered from GFP to emit in the blue and cyan spectra, respectively. Yellow fluorescent protein (YFP) is another GFP derivative with a shifted emission toward yellow, useful in Forster resonance energy transfer (FRET) studies. A notable addition to the green spectrum is mNeonGreen, a brighter and more photostable variant often preferred in advanced imaging techniques. These fluorescent proteins are particularly useful for high-resolution imaging modalities such as confocal microscopy.

[0047] Infrared and Red fluorescent proteins, such as DsRed from Discosoma species, and its derivatives like mCherry and tdTomato, provide options for red-shifted imaging. Infrared fluorophores, such as miRFP670nano, derived from bacterial phytochromes, enable deeper tissue imaging due to reduced autofluorescence in this spectrum. These properties make miRFP670nano particularly valuable for intravital imaging and tissue-level studies using multiphoton microscopy. Furthermore, novel variants such as mScarlet-l offer improved brightness and photostability in the red spectrum, making them ideal for single-molecule tracking, live-cell imaging, and applications requiring long-term observation with minimal phototoxicity. Additionally, photoactivatable and photoconvertible proteins, such as PA-GFP, Dendra2, andKaede, allow spatiotemporal tracking of protein dynamics through light-induced fluorescence changes, making them suitable for super-resolution imaging techniques like STORM and PALM.

[0048] Blue Fluorescent proteins. Blue-emitting proteins, such as TagBFP, are optimized for high photostability and brightness, facilitating imaging in the blue spectrum and serving as reliable markers for cell sorting and analysis in FACS. The compatibility of these proteins with diverse imaging platforms, including confocal and widefield microscopy, FACS, and super-resolution microscopy, enhances their utility in both basic research and clinical diagnostics.

[0049] Genome editing. Gene editing, or genome editing, is a type of genetic engineering in which DNA is inserted, replaced, or removed from a genome using nucleases. The nucleases may be artificially engineered. Alternately, the nucleases may be found in nature. The nucleases create specific double-stranded breaks (DSBs) at desired locations in the genome, but the “dead” forms specifically bind without inducing a break. Nucleases include, for example, Zinc Finger Nucleases (ZFNs), Transcription Activator-Like Effector Nucleases (TALENs), CRISPR, (e.g., the CRISPR / Cas system), and engineered meganuclease re-engineered homing endonucleases. CRISPR nucleases include for example a Cas nuclease, a Cpf 1 nuclease, a C2c1 nuclease, a C2c3 nuclease, and a C2c3 nuclease. Cas9d is of particular interest as a dead nuclease.

[0050] In an embodiment, the nuclease comprises a CRISPR / Cas system. The CRISPR (clustered regularly interspaced short palindromic repeats) locus, which encodes RNA components of the system, and the Cas (CRISPR-associated) locus, which encodes proteins (Jansen et al., 2002. Mol. Microbiol. 43: 1565-1575; Makarova et al., 2002. Nucleic Acids Res.30: 482-496; Makarova et al., 2006. Biol. Direct 1 : 7; Haft et al., 2005. PLoS Comput. Biol. 1 : e60) make up the gene sequences of the CRISPR / Cas nuclease system. CRISPR loci in microbial hosts contain a combination of CRISPR-associated (Cas) genes as well as non-coding RNA elements capable of programming the specificity of the CRISPR-mediated nucleic acid cleavage.

[0051] In certain embodiments, a Cas protein is a “derivative” of a naturally occurring Cas protein.A “functional derivative” of a native sequence polypeptide is a compound having a qualitative biological property in common with a native sequence polypeptide. The term “derivative” encompasses both amino acid sequence variants of polypeptide, covalent modifications, and fusions thereof. Suitable derivatives of a Cas polypeptide or a fragment thereof include but are not limited to mutants, fusions, covalent modifications of Cas protein or a fragment thereof. Cas protein, which includes Cas protein or a fragment thereof, as well as derivatives of Cas protein or a fragment thereof, may be obtainable from a cell or produced in vitro or by a combination of these two procedures. The cell may be a cell that naturally produces Cas protein or a cell that naturally produces Cas protein and is genetically engineered to produce the endogenous Casprotein at a higher expression level or to produce a Gas protein from an exogenously introduced nucleic acid, which encodes a Gas that is the same as or different from the endogenous Gas. In some cases, the cell does not naturally produce Gas protein and is genetically engineered to produce a Gas protein.

[0052] Vectors are known and used in the art, for example see Chavez et al Nat Methods. 2015 Mar 2. doi: 10.1038 / nm, herein specifically incorporated by reference, for introducing an inducible CRISPR system useful in the ORChlD system. In an embodiment the nuclease component is dCas9, which binds to its target but does not cleave it. In some embodiments the transcriptional activation domain is VPR activation domain. The coding sequence is operably linked to an inducible promoter, e.g. a promoter activated by contact with tetracycline, doxycycline, ecdysone, gibberellic acid, abscisic acid, etc. The inducing agent may be the same or different for each targeted cell type.

[0053] The method also includes introducing single-guide RNAs (sgRNAs) into the cell or the organism. An sgRNA is selected to target the promoter region of one or more transcription factors of interest, as set forth in Table 2. The guide RNAs (sgRNAs) include nucleotide sequences that are complementary to the target chromosomal DNA. The sgRNAs can be, for example, engineered single chain guide RNAs that comprise a crRNA sequence (complementary to the target DNA promoter sequence) and a common tracrRNA sequence, or as crRNA-tracrRNA hybrids. The sgRNAs can be introduced into the cell or the organism as a DNA (with an appropriate promoter), as an in vitro transcribed RNA, or as a synthesized RNA. The sgRNAs are usually ubiquitously expressed, e.g. with a U6 promoter, hEF1 ot promoter or other constitutive promoter.

[0054] The pluripotent cells may also be modified by the introduction of sequences encoding cellular identity tags. The cellular identity tags may comprise, without limitation, one or more of epitope tags, fluorescent proteins, etc. The tags may be unique to the targeted cell type; or may be common to all engineered cells. Cellular identity tags may comprise membrane-tethered epitopes, e.g., V5, FLAG, HA, c-Myc, His, GST, etc. for affinity based identification and sorting. Cellular identity tags may alternatively, or in combination, comprise fluorescent reporter proteins. These tags are useful in cell-type-specific analysis, enabling downstream isolation and characterization. These tags may be coordinately expressed with the sgRNA

[0055] The sgRNA construct may be introduced into the pluripotent cell by any convenient method, including, for example PiggyBac genomic insertion via nucleofection. Such vectors are known and used in the art, see for example Kramme et al. Cell Rep Methods. 2021 Sep 27;1 (6): 100082, herein specifically incorporated by reference.Methods of Generating Organoids

[0056] Methods are provided for the obtention and use of in vitro cell cultures of IMPACT organoids and cells comprised therein, including, for example neural progenitors, astrocytes and neurons, oligodendrocytes , microglia, endothelial cells, etc., where the cells are engineered as described herein, and then differentiated from the ORChlD modified induced human pluripotent stem cells (hiPSC).

[0057] Generation of IMPACT organoids and cells comprised therein, including, for example neurons, astrocytes, etc. utilizes a multi-step process. Initially, hiPSC can be obtained from any convenient source, or can be generated from somatic cells using art-recognized methods. The cells are engineered and combined by the ORChlD and IMPACT processes to generate an initial embryoid body. The hiPSC are dissociated using EDTA and cultured, preferably as intact colonies. In certain embodiments the culture is feeder layer free, e.g. when grown on vitronectin coated vessels. The culture may further be free on non-human components, i.e. xeno-free. Suspension growth optionally includes in the culture medium an effective dose of a selective Rho- associated kinase (ROCK) inhibitor, apoptosis inhibitor, ER stress inhibitor, polyamines, for the initial period of culture, for up to about 6 hours, about 12 hours, about 18 hours, about 24 hours, about 36 hours, about 48 hours, (see, for example, Watanabe et al. (2007) Nature Biotechnology 25:681 686). Inhibitors useful for such purpose include, without limitation, Y-27632; Thiazovivin (Cell Res, 2013, 23(10):1187-200; Fasudil (HA-1077) HCI (J Clin Invest, 2014, 124(9):3757-66); GSK429286A (Proc Natl Acad Sci USA, 2014, 111 (12):E1140-8); RKI-1447, AT13148; chroman 1 , emricasan, polyamines, trans-ISRIB (Nature Protocols volume 18, pages58-80 (2023)) etc.

[0058] Following embryoid body formation the culture is subjected to dual SMAD inhibition to promote neural induction by suppressing the activity of SMAD signaling pathways downstream of TGF-|3 (Transforming Growth Factor Beta) and BMP (Bone Morphogenetic Protein) signaling. An effective dose of these two inhibitors is added to the culture medium. In some embodiments the inhibitors are small molecules. For TGF-p / Activin / Nodal signaling inhibition, inhibitors may include SB431542, A-83-01 , etc. For BMP signaling inhibition, agents include Noggin, LDN- 193189, dorsomorphin, etc., which block BMP receptors and prevent SMAD1 / 5 / 8 phosphorylation. By simultaneously suppressing both pathways, dual SMAD inhibition creates a signaling environment conducive to neural lineage commitment. Dorsomorphin (DM, also known as compound C; 5 pM final dissolved in DMSO), SB431542 (SB, 10 pM final, dissolved in ethanol) and XAV-939 (XAV, 2.5 pM dissolved in DMSO) can be added for the first five days for dual SMAD inhibition.

[0059] After about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days in suspension culture, the floating spheroids are moved to neural media to and induced to express the targeted transcription factors. The media may be supplemented with an effective dose of FGF2 and EGF. The growth factors can be provided at a concentration for each of atleast about 0.5 ng / ml, at least about 1 ng / ml, at least about 5 ng / ml, at least about 10 ng / ml, at least about 20 ng / ml, up to about 500 ng / ml, up to about 250 ng / ml, up to about 100 ng / ml.

[0060] Populations of cells can be isolated from the organoid by any convenient method, including flow cytometry, magnetic immunoselection, immunopanning, etc. The cells thus isolated can be resuspended in an acceptable medium and maintained in culture, frozen, analyzed for parameters of interest; transplanted into a human or animal model; and the like.

[0061] In some embodiments the hiPSC are derived from somatic cells obtained from neurologically normal individuals. In other embodiments the hiPSC are derived from somatic cells obtained from an individual comprising at least one allele encoding a mutation associated with a neural disease. In some embodiments a panel of such organoids are provided, where the panel includes two or more genetically different organoids. In some embodiments a panel of such organoids are provided, where the organoids are subjected to a plurality of candidate agents or other therapeutic intervention, or a plurality of doses of a candidate agent or other therapeutic intervention. Candidate agents include without limitation small molecules, i.e. drugs, genetic constructs that increase or decrease expression of an RNA of interest, electrical changes, and the like.

[0062] Methods are also provided for determining the activity of a candidate agent on a diseaserelevant cell, the method comprising contacting the candidate agent with one or a panel of cells differentiated from human pluripotent stem cells, e.g. differentiated from ES cells or from hiPSC, where the pluripotent stem cells optionally comprise at least one allele encoding a mutation associated with a neural disease; and determining the effect of the agent on morphologic, genetic or functional parameters, including without limitation gene expression profiling.Screening Assays

[0063] In screening assays for the small molecules, the effect of adding a candidate agent to an organoid, or to isolated cells to determine the effect on migration, synapse formation, etc. in culture is tested with one or a panel of cellular environments, where the cellular environment includes one or more of: electrical stimulation including alterations in ionicity, stimulation with a candidate agent of interest, contact with other cells including without limitation neurons and neural progenitors, contact with infectious agents, e.g. Zika virus, and the like, and where cells may vary in genotype, in prior exposure to an environment of interest, in the dose of agent that is provided, etc. Usually at least one control is included, for example a negative control and a positive control. Culture of cells is typically performed in a sterile environment, for example, at 37°C in an incubator containing a humidified 92-95% air / 5-8% CO2atmosphere. Cell culture may be carried out in nutrient mixtures containing undefined biological fluids such as fetal calf serum, or media which is fully defined and serum free. The effect of the altering of the environment isassessed by monitoring multiple output parameters, including morphological, functional and genetic changes.

[0064] Examples of analytic methods comprise, for example, assessing the synaptic integration of migrated neurons by using array tomography to detect pre- and post- synaptic proteins in such as the presence of gephyrin (GPHN), a postsynaptic protein localized to GABAergic synapses. To further examine these synaptic puncta ‘synaptograms’ consisting of a series of high-resolution sections through a single synapse may be obtained. Whole-cell voltage clamp recordings of synaptic responses can be performed on slices of the organoid, and to distinguish between excitatory postsynaptic currents (EPSCs, downward deflecting) and IPSCs (upward deflecting), a low Cl-solution may be used in the patch pipette with cells held at -40 mV.

[0065] Live imaging of cells, including during cell migration, may be performed and cells modified to express a detectable marker. Calcium sensitive dyes can be used, e.g. Fura-2 calcium imaging; Fluo-4 calcium imaging, GCaMP6 calcium imaging, voltage imaging using voltage indicators such as voltage-sensitive dyes (e.g. di-4-ANEPPS, di-8-ANEPPS, and RH237) and / or genetically-encoded voltage indicators (e.g. ASAP1 , Archer) can be used on the intact spheroids, or on cells isolated therefrom.

[0066] Methods of analysis at the single cell level are also of interest, e.g. as described above:live imaging (including confocal or light-sheet microscopy), single cell gene expression or single cell RNA sequencing, calcium imaging, immunocytochemistry, patch-clamping, HD-MEA, flow cytometry and the like. Various parameters can be measured to determine the effect of a drug or treatment on the organoid or cells derived therefrom.

[0067] Parameters are quantifiable components of cells, particularly components that can be accurately measured, desirably in a high throughput system. A parameter can also be any cell component or cell product including cell surface determinant, receptor, protein or conformational or posttranslational modification thereof, lipid, carbohydrate, organic or inorganic molecule, nucleic acid, e.g. mRNA, DNA, etc. or a portion derived from such a cell component or combinations thereof. While most parameters will provide a quantitative readout, in some instances a semi-quantitative or qualitative result will be acceptable. Readouts may include a single determined value, or may include mean, median value or the variance, etc. Variability is expected and a range of values for each of the set of test parameters will be obtained using standard statistical methods with a common statistical method used to provide single values.

[0068] Parameters of interest include detection of cytoplasmic, cell surface or secreted biomolecules, frequently biopolymers, e.g. polypeptides, polysaccharides, polynucleotides, lipids, etc. Cell surface and secreted molecules are a preferred parameter type as these mediate cell communication and cell effector responses and can be more readily assayed. In one embodiment, parameters include specific epitopes. Epitopes are frequently identified usingspecific monoclonal antibodies or receptor probes. In some cases the molecular entities comprising the epitope are from two or more substances and comprise a defined structure; examples include combinatorically determined epitopes associated with heterodimeric integrins. A parameter may be detection of a specifically modified protein or oligosaccharide. A parameter may be defined by a specific monoclonal antibody or a ligand or receptor binding determinant.

[0069] Candidate agents of interest are biologically active agents that encompass numerous chemical classes, primarily organic molecules, which may include organometallic molecules, inorganic molecules, genetic sequences, etc. An important aspect of the invention is to evaluate candidate drugs, select therapeutic antibodies and protein-based therapeutics, with preferred biological response functions. Candidate agents comprise functional groups necessary for structural interaction with proteins, particularly hydrogen bonding, and typically include at least an amine, carbonyl, hydroxyl or carboxyl group, frequently at least two of the functional chemical groups. The candidate agents often comprise cyclical carbon or heterocyclic structures and / or aromatic or polyaromatic structures substituted with one or more of the above functional groups. Candidate agents are also found among biomolecules, including peptides, polynucleotides, saccharides, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs or combinations thereof.

[0070] Included are pharmacologically active drugs, genetically active molecules, etc.Compounds of interest include chemotherapeutic agents, anti-inflammatory agents, hormones or hormone antagonists, ion channel modifiers, and neuroactive agents. Exemplary of pharmaceutical agents suitable for this invention are those described in, “The Pharmacological Basis of Therapeutics,” Goodman and Gilman, McGraw-Hill, New York, New York, (1996), Ninth edition, under the sections: Drugs Acting at Synaptic and Neuroeffector Junctional Sites; Cardiovascular Drugs; Vitamins, Dermatology; and Toxicology, all incorporated herein by reference.

[0071] Test compounds include all of the classes of molecules described above, and may further comprise samples of unknown content. Of interest are complex mixtures of naturally occurring compounds derived from natural sources such as plants. While many samples will comprise compounds in solution, solid samples that can be dissolved in a suitable solvent may also be assayed. Samples of interest include environmental samples, e.g. ground water, sea water, mining waste, etc.; biological samples, e.g. lysates prepared from crops, tissue samples, etc.; manufacturing samples, e.g. time course during preparation of pharmaceuticals; as well as libraries of compounds prepared for analysis; and the like. Samples of interest include compounds being assessed for potential therapeutic value, i.e. drug candidates.

[0072] The term samples also includes the fluids described above to which additional components have been added, for example components that affect the ionic strength, pH, totalprotein concentration, etc. In addition, the samples may be treated to achieve at least partial fractionation or concentration. Biological samples may be stored if care is taken to reduce degradation of the compound, e.g. under nitrogen, frozen, or a combination thereof. The volume of sample used is sufficient to allow for measurable detection, usually from about 0.1 to 1 ml of a biological sample is sufficient.

[0073] Compounds, including candidate agents, are obtained from a wide variety of sources including libraries of synthetic or natural compounds. For example, numerous means are available for random and directed synthesis of a wide variety of organic compounds, including biomolecules, including expression of randomized oligonucleotides and oligopeptides. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available or readily produced. Additionally, natural or synthetically produced libraries and compounds are readily modified through conventional chemical, physical and biochemical means, and may be used to produce combinatorial libraries. Known pharmacological agents may be subjected to directed or random chemical modifications, such as acylation, alkylation, esterification, amidification, etc. to produce structural analogs.

[0074] As used herein, the term “genetic agent” refers to polynucleotides and analogs thereof, which agents are tested in the screening assays of the invention by addition of the genetic agent to a cell. The introduction of the genetic agent results in an alteration of the total genetic composition of the cell. Genetic agents such as DNA can result in an experimentally introduced change in the genome of a cell, generally through the integration of the sequence into a chromosome, for example using CRISPR mediated genomic engineering (see for example Shmakov et al. (2017) Nature Reviews Microbiology 15:169). Genetic changes can also be transient, where the exogenous sequence is not integrated but is maintained as an episomal agents. Genetic agents, such as antisense oligonucleotides, can also affect the expression of proteins without changing the cell’s genotype, by interfering with the transcription or translation of mRNA. The effect of a genetic agent is to increase or decrease expression of one or more gene products in the cell.

[0075] Introduction of an expression vector encoding a polypeptide can be used to express the encoded product in cells lacking the sequence, or to over-express the product. Various promoters can be used that are constitutive or subject to external regulation, where in the latter situation, one can turn on or off the transcription of a gene. These coding sequences may include full- length cDNA or genomic clones, fragments derived therefrom, or chimeras that combine a naturally occurring sequence with functional or structural domains of other coding sequences. Alternatively, the introduced sequence may encode an anti-sense sequence; be an anti-sense oligonucleotide; RNAi, encode a dominant negative mutation, or dominant or constitutively active mutations of native sequences; altered regulatory sequences, etc.

[0076] Antisense and RNAi oligonucleotides can be chemically synthesized by methods known in the art. Preferred oligonucleotides are chemically modified from the native phosphodiester structure, in order to increase their intracellular stability and binding affinity. A number of such modifications have been described in the literature, which alter the chemistry of the backbone, sugars or heterocyclic bases. Among useful changes in the backbone chemistry are phosphorothioates; phosphorodithioates, where both of the non-bridging oxygens are substituted with sulfur; phosphoroamidites; alkyl phosphotriesters and boranophosphates. Achiral phosphate derivatives include 3’-O’-5’-S-phosphorothioate, 3’-S-5’-O-phosphorothioate, 3’-CH2- 5’-O-phosphonate and 3’-NH-5’-O-phosphoroamidate. Peptide nucleic acids replace the entire ribose phosphodiester backbone with a peptide linkage. Sugar modifications are also used to enhance stability and affinity, e.g. morpholino oligonucleotide analogs.

[0077] Agents are screened for biological activity by adding the agent to at least one and usually a plurality of cells, in one or in a plurality of environmental conditions, e.g. following stimulation with an agonist, following electric or mechanical stimulation, etc. The change in parameter readout in response to the agent is measured, desirably normalized, and the resulting screening results may then be evaluated by comparison to reference screening results, e.g. with cells having other mutations of interest, normal astrocytes, astrocytes derived from other family members, and the like. The reference screening results may include readouts in the presence and absence of different environmental changes, screening results obtained with other agents, which may or may not include known drugs, etc.

[0078] The agents are conveniently added in solution, or readily soluble form, to the medium of cells in culture. The agents may be added in a flow-through system, as a stream, intermittent or continuous, or alternatively, adding a bolus of the compound, singly or incrementally, to an otherwise static solution. In a flow-through system, two fluids are used, where one is a physiologically neutral solution, and the other is the same solution with the test compound added. The first fluid is passed over the cells, followed by the second. In a single solution method, a bolus of the test compound is added to the volume of medium surrounding the cells. The overall concentrations of the components of the culture medium should not change significantly with the addition of the bolus, or between the two solutions in a flow through method.

[0079] Preferred agent formulations do not include additional components, such as preservatives, that may have a significant effect on the overall formulation. Thus, preferred formulations consist essentially of a biologically active compound and a physiologically acceptable carrier, e.g. water, ethanol, DMSO, etc. However, if a compound is liquid without a solvent, the formulation may consist essentially of the compound itself.

[0080] A plurality of assays may be run in parallel with different agent concentrations to obtain a differential response to the various concentrations. As known in the art, determining the effectiveconcentration of an agent typically uses a range of concentrations resulting from 1 :10, or other log scale, dilutions. The concentrations may be further refined with a second series of dilutions, if necessary. Typically, one of these concentrations serves as a negative control, i.e. at zero concentration or below the level of detection of the agent or at or below the concentration of agent that does not give a detectable change in the phenotype.

[0081] Various methods can be utilized for quantifying the presence of selected parameters, in addition to the functional parameters described above. For measuring the amount of a molecule that is present, a convenient method is to label a molecule with a detectable moiety, which may be fluorescent, luminescent, radioactive, enzymatically active, etc., particularly a molecule specific for binding to the parameter with high affinity fluorescent moieties are readily available for labeling virtually any biomolecule, structure, or cell type. Immunofluorescent moieties can be directed to bind not only to specific proteins but also specific conformations, cleavage products, or site modifications like phosphorylation. Individual peptides and proteins can be engineered to fluoresce, e.g. by expressing them as green fluorescent protein chimeras inside cells (for a review see Jones et al. (1999) Trends Biotechnol. 17(12):477-81). Thus, antibodies can be genetically modified to provide a fluorescent dye as part of their structure

[0082] Depending upon the label chosen, parameters may be measured using other than fluorescent labels, using such immunoassay techniques as radioimmunoassay (RIA) or enzyme linked immunosorbance assay (ELISA), homogeneous enzyme immunoassays, and related non- enzymatic techniques. These techniques utilize specific antibodies as reporter molecules, which are particularly useful due to their high degree of specificity for attaching to a single molecular target. U.S. Pat. No. 4,568,649 describes ligand detection systems, which employ scintillation counting. These techniques are particularly useful for protein or modified protein parameters or epitopes, or carbohydrate determinants. Cell readouts for proteins and other cell determinants can be obtained using fluorescent or otherwise tagged reporter molecules. Cell based ELISA or related non-enzymatic or fluorescence-based methods enable measurement of cell surface parameters and secreted parameters. Capture ELISA and related non-enzymatic methods usually employ two specific antibodies or reporter molecules and are useful for measuring parameters in solution. Flow cytometry methods are useful for measuring cell surface and intracellular parameters, as well as shape change and granularity and for analyses of beads used as antibody- or probe-linked reagents. Readouts from such assays may be the mean fluorescence associated with individual fluorescent antibody-detected cell surface molecules or cytokines, or the average fluorescence intensity, the median fluorescence intensity, the variance in fluorescence intensity, or some relationship among these.

[0083] Both single cell multiparameter and multicell multiparameter multiplex assays, where input cell types are identified and parameters are read by quantitative imaging and fluorescenceand confocal microscopy are used in the art, see Confocal Microscopy Methods and Protocols (Methods in Molecular Biology Vol. 122.) Paddock, Ed., Humana Press, 1998. These methods are described in U.S. Patent no. 5,989,833 issued Nov. 23, 1999.

[0084] Neuronal activity parameters. Of particular interest for the disclosed neuronal screening system are parameters related to the electrical properties of the cells and therefore directly informative about neuronal function and activity. Methods to measure neuronal activity may sense the occurrence of action potentials (spikes). The characteristics of the occurrence of a single spike or multiple spikes either in timely clustered groups (bursts) or distributed over longer time (spike train) of a single neuron or a group of neurons indicate neuronal activation patterns and thus reflect functional neuronal properties, which can be described my multiple parameters. Such parameters can be used to quantify and describe changes in neuronal activity in the systems of the invention.

[0085] Neuronal activity parameters include, without limitation, total number of spikes (per recording period); mean firing rate (of spikes); inter-spike interval (distance between sequential spikes); total number of bursts (per recording period); burst frequency; number of spikes per burst; burst duration (in milliseconds); inter-burst interval (distance between sequential bursts); burst percentage (the portion of spikes occurring within a burst); total number of network bursts (spontaneous synchronized network activity); network burst frequency; number of spikes per network burst; network burst duration; inter-network-burst interval; inter-spike interval within network bursts; network burst percentage (the portion of bursts occurring within a network burst); salutatory migration, etc.

[0086] Quantitative readouts of neuronal activity parameters may include baseline measurements in the absence of agents or a pre-defined genetic control condition and test measurements in the presence of a single or multiple agents or a genetic test condition. Furthermore, quantitative readouts of neuronal activity parameters may include long-term recordings and may therefore be used as a function of time (change of parameter value). Readouts may be acquired either spontaneously or in response to or presence of stimulation or perturbation of the complete neuronal network or selected components of the network. The quantitative readouts of neuronal activity parameters may further include a single determined value, the mean or median values of parallel, subsequent or replicate measurements, the variance of the measurements, various normalizations, the cross-correlation between parallel measurements, etc. and every statistic used to a calculate a meaningful and informative factor.

[0087] Comprehensive measurements of neuronal activity using electrical or optical recordings of the parameters described herein may include spontaneous activity and activity in response totargeted electrical or optical stimulation of all neuronal cells or a subpopulation of neuronal cells within the organoid. Furthermore, spontaneous or induced neuronal activity can be measured in the self-assembled functional environment and circuitry of the neural culture or under conditions of selective perturbation or excitation of specific subpopulations of neuronal cells as discussed above.

[0088] In the provided assays, comprehensive measurements of neuronal activity can be conducted at different time points along neuronal maturation and usually include a baseline measurement directly before contacting the neural culture with the agents of interest and a subsequent measurement under agent exposure. Moreover, long-term effects of agents on neural maturation and development can be assessed by contacting the immature neural culture at an early time point with agents of interest and acquiring measurements of the same cultures after further maturation at a later time point compared to control cultures without prior agent exposure.

[0089] In some embodiments, standard recordings of neuronal activity of mature neural cultures are conducted after about 2 weeks, after about 3 weeks, after about 4 weeks, after about 6 weeks, after about 8 weeks following fusion (i.e. after mixing the different subdomain components of the culture). Recordings of neuronal activity may encompass the measurement of additive, synergistic or opposing effects of agents that are successively applied to the cultures, therefore the duration recording periods can be adjusted according to the specific requirements of the assay. In some embodiments the measurement of neuronal activity is performed for a predetermined concentration of an agent of interest, whereas in other embodiments measurements of neuronal activity can be applied for a range of concentrations of an agent of interest.

[0090] In some embodiments the provided assays are used to assess maturation of the neural culture or single components including GABAergic interneurons, glutamatergic neurons, astrocytes, oligodendrocytes, microglia, endothelial cells etc. Maturation of neuronal cells can be measured based on morphology by optically assessing parameters such as dendritic arborization, axon elongation, total area of neuronal cell bodies, number of primary processes per neuron, total length of processes per neuron, number of branching points per primary process as well as density and size of synaptic puncta stained by synaptic markers such as synapsin-1 , synaptophysin, bassoon, PSD95, and Homer. Moreover, general neuronal maturation and differentiation can be assessed by measuring expression of marker proteins such as MAP2, TUJ- 1 , NeuN, Tau, PSA-NCAM, and SYN-1 alone or in combination using FACS analysis, immunoblotting, or fluorescence microscopy imaging, patch clamping. Maturation and differentiation of neuronal subtypes can further be tested by measuring expression of specific proteins. For excitatory neuronal cells this includes staining for e.g. VGLUT1 / 2, GRIA1 / 2 / 3 / 4,GRIN1 , GRIN2A / B, GPHN etc. For inhibitory neuronal cells this includes staining for e.g. GABRA2, GABRB1 , VGAT, and GAD67.

[0091] The results of an assay can be entered into a data processor to provide a dataset.Algorithms are used for the comparison and analysis of data obtained under different conditions. The effect of factors and agents is read out by determining changes in multiple parameters. The data will include the results from assay combinations with the agent(s), and may also include one or more of the control state, the simulated state, and the results from other assay combinations using other agents or performed under other conditions. For rapid and easy comparisons, the results may be presented visually in a graph, and can include numbers, graphs, color representations, etc.

[0092] The dataset is prepared from values obtained by measuring parameters in the presence and absence of different cells, e.g. genetically modified cells, cells cultured in the presence of specific factors or agents that affect neuronal function, as well as comparing the presence of the agent of interest and at least one other state, usually the control state, which may include the state without agent or with a different agent. The parameters include functional states such as synapse formation and calcium ions in response to stimulation, whose levels vary in the presence of the factors. Desirably, the results are normalized against a standard, usually a "control value or state," to provide a normalized data set. Values obtained from test conditions can be normalized by subtracting the unstimulated control values from the test values, and dividing the corrected test value by the corrected stimulated control value. Other methods of normalization can also be used; and the logarithm or other derivative of measured values or ratio of test to stimulated or other control values may be used. Data is normalized to control data on the same cell type under control conditions, but a dataset may comprise normalized data from one, two or multiple cell types and assay conditions.

[0093] The dataset can comprise values of the levels of sets of parameters obtained under different assay combinations. Compilations are developed that provide the values for a sufficient number of alternative assay combinations to allow comparison of values.

[0094] A database can be compiled from sets of experiments, for example, a database can contain data obtained from a panel of assay combinations, with multiple different environmental changes, where each change can be a series of related compounds, or compounds representing different classes of molecules.

[0095] Mathematical systems can be used to compare datasets, and to provide quantitative measures of similarities and differences between them. For example, the datasets can be analyzed by pattern recognition algorithms or clustering methods (e.g. hierarchical or k-means clustering, etc.) that use statistical analysis (correlation coefficients, etc.) to quantify relatedness.These methods can be modified (by weighting, employing classification strategies, etc.) to optimize the ability of a dataset to discriminate different functional effects. For example, individual parameters can be given more or less weight when analyzing the dataset, in order to enhance the discriminatory ability of the analysis. The effect of altering the weights assigned each parameter is assessed, and an iterative process is used to optimize pathway or cellular function discrimination.

[0096] The comparison of a dataset obtained from a test compound, and a reference dataset(s) is accomplished by the use of suitable deduction protocols, Al systems, statistical comparisons, etc. Preferably, the dataset is compared with a database of reference data. Similarity to reference data involving known pathway stimuli or inhibitors can provide an initial indication of the cellular pathways targeted or altered by the test stimulus or agent.

[0097] A reference database can be compiled. These databases may include reference data from panels that include known agents or combinations of agents that target specific pathways, as well as references from the analysis of cells treated under environmental conditions in which single or multiple environmental conditions or parameters are removed or specifically altered. Reference data may also be generated from panels containing cells with genetic constructs that selectively target or modulate specific cellular pathways. In this way, a database is developed that can reveal the contributions of individual pathways to a complex response.

[0098] The effectiveness of pattern search algorithms in classification can involve the optimization of the number of parameters and assay combinations. The disclosed techniques for selection of parameters provide for computational requirements resulting in physiologically relevant outputs. Moreover, these techniques for pre-filtering data sets (or potential data sets) using cell activity and disease-relevant biological information improve the likelihood that the outputs returned from database searches will be relevant to predicting agent mechanisms and in vivo agent effects.

[0099] For the development of an expert system for selection and classification of biologically active drug compounds or other interventions, the following procedures are employed. For every reference and test pattern, typically a data matrix is generated, where each point of the data matrix corresponds to a readout from a parameter, where data for each parameter may come from replicate determinations, e.g. multiple individual cells of the same type. As previously described, a data point may be quantitative, semi-quantitative, or qualitative, depending on the nature of the parameter.

[0100] The readout may be a mean, average, median or the variance or other statistically or mathematically derived value associated with the measurement. The parameter readout information may be further refined by direct comparison with the corresponding referencereadout. The absolute values obtained for each parameter under identical conditions will display a variability that is inherent in live biological systems and also reflects individual cellular variability as well as the variability inherent between individuals.

[0101] Classification rules are constructed from sets of training data (i.e. data matrices) obtained from multiple repeated experiments. Classification rules are selected as correctly identifying repeated reference patterns and successfully distinguishing distinct reference patterns. Classification rule-learning algorithms may include decision tree methods, statistical methods, naive Bayesian algorithms, and the like.

[0102] A knowledge database will be of sufficient complexity to permit novel test data to be effectively identified and classified. Several approaches for generating a sufficiently encompassing set of classification patterns, and sufficiently powerful mathematical / statistical methods for discriminating between them can accomplish this.

[0103] The data from cells treated with specific drugs known to interact with particular targets or pathways provide a more detailed set of classification readouts. Data generated from cells that are genetically modified using over-expression techniques and anti-sense techniques, permit testing the influence of individual genes on the phenotype.

[0104] A preferred knowledge database contains reference data from optimized panels of cells, environments and parameters. For complex environments, data reflecting small variations in the environment may also be included in the knowledge database, e.g. environments where one or more factors or cell types of interest are excluded or included or quantitatively altered in, for example, concentration or time of exposure, etc.

[0105] For further elaboration of general techniques useful in the practice of this invention, the practitioner can refer to standard textbooks and reviews in cell biology, tissue culture, embryology, stem cell biology, human development and neurobiology. With respect to tissue culture and embryonic stem cells, the reader may wish to refer to Teratocarcinomas and embryonic stem cells: A practical approach (E. J. Robertson, ed., IRL Press Ltd. 1987); Guide to Techniques in Mouse Development (P. M. Wasserman et al. eds., Academic Press 1993); Embryonic Stem Cell Differentiation in Vitro (M. V. Wiles, Meth. Enzymol. 225:900, 1993); Properties and uses of Embryonic Stem Cells: Prospects for Application to Human Biology and Gene Therapy (P. D. Rathjen et al., Reprod. Fertil. Dev. 10:31 , 1998).

[0106] General methods in molecular and cellular biochemistry can be found in such standard textbooks as Molecular Cloning: A Laboratory Manual, 3rd Ed. (Sambrook et al., Harbor Laboratory Press 2001); Short Protocols in Molecular Biology, 4th Ed. (Ausubel et al. eds., John Wiley & Sons 1999); Protein Methods (Bollag et al., John Wiley & Sons 1996); Nonviral Vectors for Gene Therapy (Wagner et al. eds., Academic Press 1999); Viral Vectors (Kaplift & Loewyeds., Academic Press 1995); Immunology Methods Manual (I. Lefkovits ed., Academic Press 1997); and Cell and Tissue Culture: Laboratory Procedures in Biotechnology (Doyle & Griffiths, John Wiley & Sons 1998). Reagents, cloning vectors, and kits for genetic manipulation referred to in this disclosure are available from commercial vendors such as BioRad, Stratagene, Invitrogen, Sigma-Aldrich, and ClonTech.

[0107] Each publication cited in this specification is hereby incorporated by reference in its entirety for all purposes.

[0108] It is to be understood that this invention is not limited to the particular methodology, protocols, cell lines, animal species or genera, and reagents described, as such may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which will be limited only by the appended claims.

[0109] As used herein the singular forms "a", "and", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a cell" includes a plurality of such cells and reference to "the culture" includes reference to one or more cultures and equivalents thereof known to those skilled in the art, and so forth. All technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this invention belongs unless clearly indicated otherwise.

[0110] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of howto make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g. amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Centigrade, and pressure is at or near atmospheric.EXPERIMENTALRecapitulation of Cellular Diversity in 3D Brain Region-Specific Organoids and Assembloids

[0111] Current brain organoid models suffer from long maturation times, lack of vasculature, and incomplete cellular representation, particularly regarding microglia. Existing approaches, such as co-culturing with microglia or endothelial cells, or transplanting organoids into animal models, partially address these limitations but fail to replicate the full complexity of human brain tissue. The time required to obtain organoids with mature cell types can range from 100 days to 600 days.

[0112] A two-phase workflow for generating brain organoids that more accurately reflect the cellular composition of the human brain is provided herein.

[0113] Generating ORChlD iPSC lines. ORChlD employs genetically engineered iPSC lines with dead Cas9 (dCas9) fused to the VPR activation domain for transcriptional activation of endogenous transcription factors required for differentiation. This activation is controlled by the widely used small molecule doxycycline (DOX) allowing for orthogonal and inducible differentiation of specific cell types in parallel. Each desired cell type has a unique DNA cassette with sgRNAs targeting the required transcription factors, a fluorescent protein and a peptide tag detectable at the cell surface using a hybrid CD52 receptor. The ORChlD lines have Puromycin and Hygromycin resistance to enable selection of cells containing the engineered elements. The method to generate the ORChlD lines is as follows.

[0114] TRE-dCas9-VPR insertion: The iPSC lines are first edited with the PB-TRE-dCas9-VPR construct using PiggyBac genomic insertion via nucleofection. Cells with successful integration of TRE-dCas9-VPR are selected using the antibiotic Hygromycin and sorted using FACS to obtain single cell clones. The dCas9-VPR expression is under the control of doxycycline mediated TRE promoter activation.

[0115] Cell type specific sgRNA and tag insertion: The iPSC clones with optimal insertion of TRE- dCas9-VPR are edited using PiggyBac genomic insertion via nucleofection to integrate sgRNA(s) targeting cell type specific transcription factors and unique fluorescent protein and CD52-peptide tags. Cells with successful insertion of the sgRNA and tag cassette are selected by the antibiotic Puromycin and sorted using FACS to obtain single cell clones.

[0116] A schematic representation of generation of ORChlD lines is shown in FIG. 1. Table 1 summarizes the details of the different constructs used for making the ORChlD. Table 2 summarizes the transcription factors, and their combinations targeted to achieve differentiation of the intended cell types.Table 1Part 1 : PiggyBac insertion of dCas9 VPR and responsive elementsPart 2: PiggyBac insertion of Fluorescent marker and small peptide for FACS sorting using Megagate and sgRNA for TF induction for respective cell linesPart 3: Transposase expression>Design features and molecular information of the plasmid DNA constructs used for editing iPSCs to generate ORChlD lines. Part 1 construct is used for expression of Doxycycline inducible dCas9-VPR for CRISPRa. Part 2 constructs deliver the sgRNAs against target transcription factors specific to the cell type as indicated, and the unique cellular identity tags as indicated in column 4.Table 2List of transcription factors targeted for cell type specific differentiation using ORChlD iPSC lines.

[0117] The RefSeqGene accession number for each human gene referenced in Table 2 is as follows (in parentheses). In genes notated with a “P1” or “P2”, reference is to the different promoters at that gene. Guide RNAs are targeted to the promoter region found in each of the genes. ASCL1 (NG_053159.1); ATOH1 (NG_030030.1); CEBPA (NG_012022.1); CEBPB (NG 030043.1); CTIP2 (NG_029141.1 ) ; DLX2 (NG_030010.1); EBF1 (NG_029015.1); EN1 (NG_007123.1); ETV2 (NG_053154.1); FEV (NG_030030.1); FLI1 (NG_053153.1); FOXA2 (NG_011881.1); GATA2 (NG_029334.1); GSX2 (NG_053229.1); IRF5-P1 (NG_030043.1); IRF5- P2 (NG_012831.2); IRF8 (NG_030032.1); ISL1-P1 (NG_023040.1); ISL1-P2 (NG_023040.1); ISL2 (NG_053075.1); KIF7 (NG_030338.1); LHX3-P1 (NG_008097.1); LHX3-P2 (NG_008097.1); LHX6 (NG_029683.1); LMX1 A (NG_030033.1); LMX1 B (NG_008320.1); MAFB (NG_033816.1); MEF2C (NG_053153.1); MNX1 (NG_013212.1); MYT1L (NG_053161.1); NEUROG1 (NG_053157.1); NEUROG2 (NG_053156.1); NEUROG2 (NG_053156.1); NFIA (NG_016335.1); NFIB (NG_053155.1); NKX2.1 (NG_013340.1); NKX6-2 (NG_051187.1); NR4A2-P1 (NG 011684.1); NR4A2-P2 (NG_011684.1); OLIG2 (NG_029013.1); PHOX2A (NG_012015.1); PITX3 (NG_008147.1); POU3F2 (NG_053160.1); POU4F1 (NG_030034.1);RUNX2-P1 (NG_008174.1); RUNX2-P2 (NG_008174.1); SOX10 (NG_008101 .1); SOX9 (NG_012490.1); SPI1 (NG_030361.1).

[0118] Generating IMPACT organoids. IMPACT is the reconstitution of organoids by mixing different proportions of the ORChlD iPSCs to create organoids with preset fractions of neuronal, glial, microglial, and endothelial populations. IMPACT enables in situ differentiation of different cell types, offering flexibility for studying cell-cell interactions in both healthy and diseased states. The cells in these organoids can be identified uniquely with membrane-tethered epitopes and fluorescent reporters for downstream cell-type-specific analysis and isolation upon dissociation.

[0119] Embryoid Body Reconstitution Reconstitute embryoid bodies with varying proportions of ORChlD iPSCs, creating organoids with preset fractions of neurons, glial cells, microglia, and endothelial cells. Figure 2 illustrates reconstitution of iA (induced astrocytes), iN (induced neurons), iO (induced oligodendrocytes), iM (induced microglia) and iE (induced endothelial cells).

[0120] Differentiation of cell types: Reconstituted IMPACT embryoid bodies are patterned using previously described small molecules for the first 25 days along with Doxycycline induction to achieve differentiation of all cell types in parallel in situ. The organoids are cultured without any cell type specific cues. The timeline for differentiation is illustrated in FIG. 3.

[0121] Using the above-mentioned protocols, we have established the ORChlD iPSC lines.Using the iN, iO, iDN, iM, iE and iA ORChlD iPSCs, we made IMPACT organoids with equal proportions of each of the cell types. After following the differentiation scheme indicated in Figure 3, we obtained IMPACT organoids with successful in situ differentiation of the iN, iO, iDN, iM, iE and iA ORChlD lines into neurons (immunopositive for MAP2 antibody), iDN (immunopositive for TH antibody), oligodendrocytes (immunopositive for 04 antibody), iM (immunopositive for Iba1 and TMEM119), iE (immunopositive for CD31), and astrocytes (immunopositive for GFAP antibody), respectively. The representative micrographs of primary cultures obtained from dissociating the IMPACT organoids at day 33 and immunostained with the above-mentioned antibodies can be found in FIG. 4.Methods

[0122] Cloning. The constructs listed in Table 1 were cloned using the Megagate protocol and the sgRNAs were cloned using Sapl golden gate cloning protocol. Golden gate assembly was also done for multiplexed sgRNA array with individual U6 promoters using Sapl wherever more than one sgRNAs were required to be delivered.

[0123] Cell culture. Protocols for iPSC culture and generation of brain organoids were used as reported previously. The method for generating embryoid bodies for brain organoids used hereis described, for example, in U.S. Patent no. 10,494,602, herein specifically incorporated by reference, see FIG. 3. For example, the desired combination of modified pluripotent stem cells were cultured in the Essential 8 cell culture media (Invitrogen).

[0124] hiPSC were dissociated into a single cell suspension using Accutase (ICT) for 5-7 min.The single cell suspension was aggregated by centrifugation in microwells and maintained in Essential 8 media: DMEM / F12 (1 :1) (Invitrogen) with the CEPT cocktail (Tocris) for 24 hours (day 0). Next day (day 1), spheroids are transferred to low attachment plates in Essential 6 medium:Essential 8 medium (1 :1) (Invitrogen) with Dorsomorphin (Sigma, 5 pM), SB-431542 (Tocris, 10 pM) and XAV-939 (Tocris, 2.5 pM) and Doxycycline hyclate (Sigma, 2 uM). No media change is performed the next day (day 2), but from day 3 to day 6, spheroids are maintained in Essential 6 medium (Invitrogen) with Dorsomorphin (Sigma, 5 pM), SB-431542 (Tocris, 10 pM) and XAV-939 (Tocris, 2.5 pM) and Doxycycline hyclate (Sigma, 2 uM), with daily media changes. On day 7, spheroids are transferred to Neurobasal / B27 medium supplemented with the growth factors FG2 (20 ng / ml) and EGF (20 ng / ml) and Doxycycline hyclate (Sigma, 2 uM).References1. Velasco, S., Paulsen, B. & Arlotta, P. Annual Review of Neuroscience 3D Brain Organoids: Studying Brain Development and Disease Outside the Embryo. (2020) doi : 10.1146 / annurev-neuro-070918.2. Yoon, S. J. et al. Reliability of human cortical organoid generation. Nat. Methods 2018 161 16, 75-78 (2018).3. Pasca, A. M. et al. Functional cortical neurons and astrocytes from human pluripotent stem cells in 3D culture. Nat. Methods 2015 127 12, 671-678 (2015).4. Yoon, S. J. et al. Reliability of human cortical organoid generation. Nat. Methods 2018 161 16, 75-78 (2018).5. Cakir, B. et al. Engineering of human brain organoids with a functional vascular-like system. Nat. Methods 2019 1611 16, 1169-1175 (2019).6. Xu, R. et al. Developing human pluripotent stem cell-based cerebral organoids with a controllable microglia ratio for modeling brain development and pathology. Stem Cell Rep. 16, 1923-1937 (2021).7. Schafer, S. T. et al. An in vivo neuroimmune organoid model to study human microglia phenotypes. Cell 186, 2111-2126.e20 (2023).8. Revah, O. et al. Maturation and circuit integration of transplanted human cortical organoids. Nature 610, 319-326 (2022).9. Michno, W. P. et al. Adrenomedullin promotes interneuron migration in a dual human model for hypoxic interneuronopathy of prematurity. bioRxiv 2023.05.01.538334 (2023) doi:10.1101 / 2023.05.01 .538334.

[0125] The preceding merely illustrates the principles of the invention. It will be appreciated that those skilled in the art will be able to devise various arrangements which, although not explicitly described or shown herein, embody the principles of the invention and are included within its spirit and scope. Furthermore, all examples and conditional language recited herein are principally intended to aid the reader in understanding the principles of the invention and the concepts contributed by the inventors to furthering the art, and are to be construed as being without limitation to such specifically recited examples and conditions. Moreover, all statements herein reciting principles, aspects, and embodiments of the invention as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. Additionally, it is intended that such equivalents include both currently known equivalents and equivalents developed in the future, i.e., any elements developed that perform the same function, regardless of structure. The scope of the present invention, therefore, is not intended to be limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of present invention is embodied by the appended claims.

Claims

TH T WHICH IS CLAIMED IS:1 A method for generating brain region-specific organoids comprising the steps of: (a) modifying a pluripotent cell by introduction of a genetic sequence encoding a dead nuclease fused to a transcriptional activation domain, which sequence is operably linked to a promoter activated by an inducer;(b) modifying a plurality of cells obtained in step (a) by introduction of sequences encoding sgRNA targeting transcription factors of interest for differentiation of brain cells, and optionally sequences encoding cellular identity tags;(c) combining two or more cells obtained in step (b) to form an embryoid body;(d) contacting the embryoid body of step (c) with an agent that activates the inducible promoter for a period of time sufficient to induce expression of the transcription factors of interest, and to induce differentiation of the cells into a brain region-specific organoid.

2. The method of claim 1 , wherein step (b) comprises introduction of sgRNA targeting transcription factors for differentiation of one or more cell types selected from Astrocytes-1 ; Astrocytes-2; Astrocytes-3; Astrocytes-4; Dopaminergic neurons -1 ; Dopaminergic neurons -2; Dopaminergic neurons -3; Dopaminergic neurons -4; Dopaminergic neurons -5; Dopaminergic neurons -6; Dopaminergic neurons -7; Dopaminergic neurons -8; Endothelial cells; Excitatory neurons -1 ; Excitatory neurons -2; Excitatory neurons -2; GABAergic INs (SST + PVALB); Hypothalamus-1 ; Hypothalamus-2; Hypothalamus-3; Hypothalamus-4; Inhibitory neurons-1 ; Inhibitory neurons-2; Medium Spiny Neurons-1 ; Medium Spiny Neurons-2; Medium Spiny Neurons-3; Medium Spiny Neurons-4; Medium Spiny Neurons-5; Medium Spiny Neurons-6; Microglia-1 ; Microglia-2; Microglia-3; Microglia-4; Motor Neurons -1 ; Motor Neurons -2; Motor Neurons -3; Motor Neurons -4; Motor Neurons -5; Motor Neurons -6; Oligodendrocytes / OPCs -1 ; Oligodendrocytes / OPCs -2; Peripheral sensory / DRGs -1 ; Peripheral sensory / DRGs -2; Peripheral sensory / DRGs -3; and Serotonergic neurons.

3. The method of claim 1 or claim 2, wherein the sgRNA targets the promoter region of one or more factors selected from ASCL1 (NG_053159.1); ATOH1 (NG_030030.1); CEBPA (NG_012022.1); CEBPB (NG_030043.1); CTIP2 (NG_029141.1); DLX2 (NG_030010.1); EBF1 (NG 029015.1); EN1 (NG_007123.1); ETV2 (NG_053154.1); FEV (NG_030030.1); FLI1 (NG 053153.1); FOXA2 (NG_011881.1); GATA2 (NG_029334.1); GSX2 (NG_053229.1); IRF5-P1 (NG_030043.1); IRF5-P2 (NG_012831.2); IRF8 (NG_030032.1); ISL1-P1 (NG_023040.1); ISL1-P2 (NG_023040.1); ISL2 (NG_053075.1); KIF7 (NG_030338.1); LHX3-P1 (NG_008097.1); LHX3-P2 (NG_008097.1); LHX6 (NG_029683.1); LMX1A (NG_030033.1); LMX1 B (NG_008320.1); MAFB (NG_033816.1); MEF2C (NG_053153.1); MNX1 (NG_013212.1); MYT1 L(NG_053161.1); NEUR0G1 (NG_053157.1); NEUR0G2 (NG_053156.1); NEUR0G2 (NG_053156.1); NFIA (NG_016335.1); NFIB (NG_053155.1); NKX2.1 (NG_013340.1); NKX6-2 (NG_051187.1); NR4A2-P1 (NG_011684.1); NR4A2-P2 (NG_011684.1); OLIG2 (NG_029013.1); PHOX2A (NG_012015.1); PITX3 (NG_008147.1); POU3F2 (NG_053160.1); POU4F1 (NG_030034.1); RUNX2-P1 (NG_008174.1); RUNX2-P2 (NG_008174.1); SOX10 (NG_008101.1); SOX9 (NG_012490.1); and SPI1 (NG_030361 .1).

4. The method of any of claims 1-3, wherein the embryoid body comprises two or more cell types selected from neurons, astrocytes, oligodendrocytes, microglia, and endothelial cells.

5. The method of any of claims 1-3, wherein the embryoid body comprises at least one cell sub-type from neurons, astrocytes, oligodendrocytes, microglia, and endothelial cells.

6. The method of any of the previous claims, wherein the cellular identity tags are one or both of a fluorescent protein, and a cell surface epitope tag.

7. The method of any of the previous claims, wherein the dead nuclease fused to a transcriptional activation domain is dCas9 fused to VPR activation domain.

8. The method of any of the previous claims, wherein the agent that activates the inducible promoter is doxycycline.

9. The method of any of the previous claims, wherein the embryoid body comprises two or more cell types combined in a pre-determined ratio or concentration.

10. An isolated pluripotent stem cell modified by (a) introduction of a genetic sequence encoding a dead nuclease fused to a transcriptional activation domain, which sequence is operably linked to a promoter activated by an inducer; and (b) introduction of sequences encoding sgRNA targeting transcription factors of interest for differentiation of brain cells.

11. The isolated pluripotent stem cell of claim 10, further modified by introduction of sequences encoding cellular identity tags.

12. A brain region-specific organoid obtained by the method of any of claims 1-9.

13. A method determining the effect of a candidate agent on a human brain, the method comprising: contacting the candidate agent with one or a panel of brain region-specific organoids differentiated from induced human pluripotent stem cells (hiPSC) according to the method of any of claims 1-8; and determining the effect of the agent on morphologic, genetic or functional parameters.

14. The method of Claim 12, wherein a panel of organoids comprises at least two organoids having differing genotypes or under differing environmental conditions.

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