Methods of producing non-human mammals having human neural tissue

Abstract

The present disclosure provides method of producing a non-human mammalian animal model containing human neural tissue, the method including: introducing a first human neural organoid into a central nervous system location of a newborn non-human mammal; introducing a second human neural organoid into the central nervous system location of the newborn non-human mammal to form an assembloid from the first human neural organoid and the second human neural organoid; and allowing the newborn non-human mammal to mature to produce the non-human mammalian animal model containing human neural tissue comprising GABAergic neurons; wherein the human neural tissue integrates into the circuit of the non-human animal and displays complex neural activity. Also provided are non-human mammalian animal models and methods of using the same.

Description

Attorney Docket No: STAN-2232WOClient No: S24-410METHODS OF PRODUCING NON-HUMAN MAMMALS HAVING HUMAN NEURAL TISSUEACKNOWLEDGEMENT OF GOVERNMENT RIGHTS

[0001] This invention was made with Government support under contracts MH019938 and MH1 15012 awarded by the National Institutes of Health. The Government has certain rights in the invention.CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] Pursuant to 35 U.S.C. § 119 (e), this application claims priority to the filing date of United States Provisional Patent Application Serial No. 63 / 727,979 filed December 4, 2024, the disclosure of which application is herein incorporated by reference.SEQUENCING LISTING

[0003] A Sequencing Listing is provided herewith as a Sequencing Listing XML, “STAN- 2232WO_Seq_List” created November 17, 2025 and having a size of 4,696 bytes. The contents of the Sequence Listing XML are incorporated by reference herein in their entirety.INTRODUCTION

[0004] The mammalian cerebral cortex contains glutamatergic neurons produced in the pallium and GABAergic neurons that are mostly born in the subpallium (1-4). After migration throughout the pallium, GABAergic neurons undergo maturation allowing for the development of canonical cortical network activity driven by the interaction of glutamatergic and GABAergic neurons (1 , 5, 6). Imbalances in this network activity are thought to contribute to disease (7, 8), including seizure disorders (9, 10), but lack of experimental access to the developing human brain has limited an understanding of these processes.

[0005] Recent advances in human induced pluripotent stem (hiPS) cell technology combined with self-organizing neural organoid methods facilitate access to features of human cortical development (1 1-15). For example, generation of human cortical organoids (hCO) resembling the pallium recapitulate the intrinsic, sequential generation of neurons and glial cells (16-21 ). However, isolated hCO lack long-range interactions with brain-wide circuits as well as short- range interactions with GABAergic neurons. The human forebrain assembloid (hFA) platform was developed to ameliorate the short-range deficits, whereby hCO are integrated with human subpallial organoids (hSO) that generate GABAergic neurons (16, 22, 23). Transplantation of hiPS cell-derived neurons into rodents may overcome the lack of long-range connectivity: grafted neurons receive neuronal inputs, sensory-evoked electrical activity, and areAttorney Docket No: STAN-2232WOClient No: S24-410 vascularized (24-30). However, the assembly of glutamatergic hCO and GABAergic hSO with a functionally relevant readout has not been achieved in vivo.SUMMARY

[0006] Provided herein are methods for the production of non-human mammalian animal models containing human neural tissue having complex neural activity as the result of transplantation of human derived neural assembloids. Also provided are methods for modeling human epilepsy disorders in non-human mammalian animal models.

[0007] The present disclosure provides a method of producing a non-human mammalian animal model containing human neural tissue having complex neural activity, the method including: introducing a first human neural organoid into a central nervous system location of a newborn non-human mammal; introducing a second human neural organoid into the central nervous system location of the newborn non-human mammal to form an assembloid from the first human neural organoid and the second human neural organoid; and allowing the newborn non-human mammal to mature to produce the non-human mammalian animal model containing human neural tissue comprising GABAergic neurons in an assembloid; wherein the human neural tissue integrates into the circuit of the non-human animal and displays complex neural activity.

[0008] In some cases, the present disclosure provides a method of modeling an epilepsy or an epilepsy-related disorder, the method including introducing a first human neural organoid into a central nervous system location of a newborn non-human mammal; introducing a second human neural organoid into the central nervous system location of the newborn non- human mammal to form an assembloid from the first human neural organoid and the second human neural organoid; allowing the newborn non-human mammal to mature to produce the non-human mammalian animal model containing a first human neural tissue; and characterizing the first human neural tissue to model the epilepsy disorder; wherein the first human neural organoid, the second human neural organoid, or the first and second human neural organoid are: a) produced from a cellular biological sample from a human who has the epilepsy disorder; or b) have a genetic mutation associated with the epilepsy disorder.

[0009] In some cases, the present disclosure provides a method of determining the effectiveness of a candidate agent on an epilepsy disorder, the method comprising administering the candidate agent to the non-human mammalian animal model produced by the methods of the invention; assaying the human neural tissue; and comparing the results of the assaying with mammals administered a control agent that is not the candidate agent.

[0010] Non-human mammalian animal models are also provided. The non-human mammalian animal models of the present disclosure not only serve as models for epilepsyAttorney Docket No: STAN-2232WOClient No: S24-410 disorders but may also be used for the development and screening of therapeutics to treat the epilepsy disorders.

[0011] 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

[0012] The invention is best understood from the following detailed description when read in conjunction with the accompanying drawings. It is emphasized that, according to common practice, 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.

[0013] FIG. 1A-FIG. 1 L. Generation of transplanted human forebrain assembloids. (A) Schematic of hCO and hSO generation, viral labeling, and engraftment of t-hFA into newborn rats. (B) T2-sequences from MR imaging of transplanted hCO (t-hCO) and t-hFA; scale bar = 5 mm. (C) Transplantation success rate for t-hCO (N = 44 rats / 3 hiPS cell lines) and t-hFA (N = 88 rats / 3 hiPS cell lines). (D) MRI-based quantification of t-hCO (N=1 1 rats / 3 hiPS cell lines) and t-hFA (N=32 rats / 3 hiPS cell lines) graft volume at 2, 4 and 6 months (rmANOVA: time main effect p<0.0001 , graft condition main effect p=0.0398, interaction p=0.8438, best-fit linear regression line plotted ±95% confidence interval). (E) Representative immunocytochemistry of t-hFA at 6-months post -transplantation; scale bar = 1 mm. (F) High magnification image of t-hFA graft. Top left, scale bar = 500pm; all other scale bars = 40pm. (G) Quartile distribution of Dlxi1 / 2b::EGFP+ cells along the anteroposterior axis, normalized to the area of the t-hFA (N=5 rats / 3 hiPS cell lines). (H) Uniform manifold approximation and projection (UMAP) dimensional reduction visualization of all clustered high-quality t-hFA human nuclei (n = 4 t- hFA samples, n = 3 hiPS cell lines). CT, corticothalamic cell; ET, extratelencephalic cell; IT, intratelencephalic cell; NP, near-projecting; SP, subplate. (I) UMAP visualization of GABAergic subtypes. (J) Dot plot of GABAergic neuron gene expression (y-axis) in select clusters (x- axis). (K) Gene set enrichment analysis (one-sided Fisher’s exact test) of genes significantly upregulated (adjusted P < 0.05, fold change > 2, expressed in at least 10% of nuclei) in t-hFA GABAergic neurons compared with hSO GABAergic neurons with sets of genes marked by expression in the indicated developmental stages in developing cortical GABAergic neurons obtained from Velmeshev et al. The line denotes Bonferroni-corrected P value of 0.05. (L) GABAergic neuron gene expression (pseudobulk and scaled for each gene) of indicated primary GABAergic neuron developmental genes significantly upregulated in t-hFA GABAergic neurons. Each row represents data from a biological replicate.Attorney Docket No: STAN-2232WOClient No: S24-410

[0014] FIG. 2A-FIG. 2H. t-hFA contain mature and diverse GABA neurons. (A) Dendritic reconstructions of biocytin-labeled neurons from hSO (top) and t-hFA (bottom) groups. Scale bar = 200pm. (B) Representative images of biocytin-labeled neurons co-stained with GABA neuron subtype markers. Scale bar = 40pm. (C) Quantification of total dendritic length in Dlxi1 / 2b::EGFP+ neurons (hSO: N=1 1 cells, 6 organoids, 2 hiPS cell lines; t-hFA N=24 cells, 4 rats, 2 hiPS cell lines; t-test after log transform due to log-distributed residuals). (D) Normalized histogram of dendritic branch order (hSO: N=1 1 cells, 6 organoids, 2 hiPS cell lines; t-hFA N=24 cells, 4 rats, 2 hiPS cell lines; mean ± SEM is plotted). (E) Representative traces from whole-cell electrophysiology of Dlxi1 / 2b::EGFP+ neurons. Micrograph scale bar = 20pm, electrophysiology scale bars = 250ms (horizontal), 20m V (vertical). (F) Quantification of maximal firing rate (hSO N=13 cells, 6 organoids, 2 hiPS cell lines; t-hFA N=32 cells, 4 rats, 2 hiPS cell lines; Mann-Whitney). (G) Quantification of resting membrane potential (hSO N=13 cells, 6 organoids, 2 hiPS cell lines; t-hFA N=30 cells, 4 rats, 2 hiPS cell lines; Welch’s t-test: p = 0.0015). (H) Quantification of capacitance (hSO N=13 cells, 6 organoids, 2 hiPS cell lines; t-hFA N=32 cells, 4 rats, 2 hiPS cell lines; t-test after log-transform due to log-distributed residuals). *" indicates p<0.001 , indicates p<0.0001 .

[0015] FIG. 3A-FIG. 3K. Functional characterization of t-hFA. (A) Schematic of experimental timeline for EEG measurements. (B) Representative co-localization of rendered MRI and CT scans. (C) EEG data from the right somatosensory cortex displayed with multiple timescales of a control rat without a transplant, top- representative EEG voltage traces, bottom- power spectral density (PSD) over time obtained from Short-Time Fourier Transform (STFT). (D) same as (C) for an electrode directly placed into the t-hCO. (E) same as (C) for an electrode directly placed into the t-hFA. (F) Compilation of power spectral density from the electrode placed into the graft for t-hCO (N=6), t-hFA (N=10), and non-transplanted right somatosensory cortex of age-matched RNU nude rats (N=10). Data are summarized over the entire baseline recording. Both the x and y axes are log transformed and mean ± SEM is plotted. (G) EEG power within canonical frequency bands (rmANOVA: frequency band main effect p<0.0001 , graft condition main effect p=0.0059, interaction p<0.0001 ; post-hoc t-test with Holm-Sidak correction). (H) top- schematic of recording configuration targeting putative non-GABAergic Dlxi1 / 2b::EGFP- neurons, bottom- representative trace of sIPSC recording and abolishment of sIPSCs with the GABAR antagonist BMI. (I) Quantification of spontaneous inhibitory postsynaptic currents (sIPSC) frequency (hCO: N=15 cells / 2 rats / 1 hiPS cell line, t-hFA: N=11 cells / 4 rats / 1 hiPS cell line, Mann-whitney). (J) left-schematic of optotagging experiment, middle- example optotagged units. The opsin is expressed only in hCO derived neurons in both conditions, right- example of coincidentally recorded opto-tagged units. (K) Quantification of spontaneous spikes from opto-tagged units over the four-minute recordingAttorney Docket No: STAN-2232WOClient No: S24-410(hCO: N=5 units I 2 rats / 2 hiPS cell lines, t-hFA: N=4 units / 2 rats / 2 hiPS cell lines, Rat cortex: N=18 units / 2 rats, Welch’s ANOVA, p=0.0016, Dunnett’s T3 multiple comparison test). * indicates p<0.05, “ indicates p<0.01 , **** indicates p<0.0001.

[0016] FIG. 4A-FIG. 4G. Modeling mosaic loss of PCDH19. (A) left- Schematic of t-hCO and t-hFA modular combinations to model mosaic loss of PCDH19. right- organoids are transplanted into rat pups, allowed to mature 6-7 months, and hyperthermic seizure induction is performed. (B-C) top- Representative EEG traces as a function of body temperature, bottom- Time-frequency spectrograms derived from STFT. An inset from 41 -42.5°C is shown at the bottom. (B) Representative data for t-hFA WT / WT graft. (C) Representative data for t- hFA mosaic graft. (D) Body temperature plotted as a function of time (Mixed effects model: Time main effect p<0.0001 , graft condition main effect p=0.1345). (E) Kaplan-Meier curves summarizing % of rats in each condition that did not have a seizure as a function of temperature. Only rats in the t-hFA mosaic condition had seizures, so all other groups are depicted with alternating points at 100% across all temperatures (Log-rank test p=0.0093). (F) Proportion of rats in each condition that had a seizure below 42.5 °C (Chi-square test p<0.0001). (G) Duration of seizures across groups (Kruskal-Wallis, p=0.0184, Dunn’s multiple comparison test p = 0.0077, Naive rat vs t-hFA mosaic, all other not significant]. Panel (F) indicates the number of subjects in each condition. * indicates p<0.05, **** indicates p<0.0001 .

[0017] FIG. 5A-FIG. 5D. Baseline behavioral consequences of transplantation. (A) Body weight of rats at 6-7 months post-transplantation (ANOVA: sex main effect p<0.0001 , graft condition main effect p=0.3532, interaction p=0.2087). (B) Activity chamber - distance traveled per minute in Naive rat (N=12), t-hCO (N=1 1 ), and t-hFA (N=14) groups. (rmANOVA time main effect p<0.0001 , graft condition main effect p=0.0015, interaction p=0.1739, asterisks indicate significant post-hoc t-test with Holm-Sidak correction between Naive rat and t-hCO conditions only). (C) Novel object recognition memory task. Discrimination index = [(time with novel object)-(time with familiar object)] / (time spent with both objects) during novel object test (Kruskal-Wallis: p=0.1308). (D) Graft volume of animals that went through the behavioral procedures - this is a subset of rats in Figi D (t-test: p=0.6461 ).

[0018] FIG. 6A-FIG. 6J. Single nucleus RNA-seq data quality and characterization. (A) snRNA-seq quality metrics after filtering showing the distribution of the number of human counts, number of human genes, and mitochondrial (MT) gene fraction per cell in each sample. MT gene fraction plotted as boxplots (horizontal line denotes median; lower and upper hinges correspond to the first and third quartiles; whiskers extend 1.5 times the interquartile range with outliers shown outside this range). Lines denote nuclei quality thresholds. (B) Dot plot of marker gene expression in select clusters. (C) Same integrated UMAP as shown in Fig. 1 H, colored by t-hFA hiPS line combination. (D) Cell type proportions across t-hFA hiPS lineAttorney Docket No: STAN-2232WOClient No: S24-410 combinations colored by clusters. (E) UMAP visualization of label transfer classification scores of reference adult primary human cell populations onto t-hFA snRNA-seq data. (F) Gene Ontology (GO) term enrichment analysis of genes significantly upregulated (adjusted P < 0.05, fold change > 2, expressed in at least 10% of nuclei) in t-hFA GABAergic neurons compared with hSO GABAergic neurons. The line denotes a q value of 0.05. (G) UMAP dimensional reduction visualization of all clustered high-quality hSO nuclei after Seurat integration (n = 4 hSO samples, n = 4 hiPS cell lines). (H) Dot plot of marker gene expression in select clusters. (I) Cell type proportions across hSO samples colored by clusters. (J) UMAP visualization of label transfer classification scores (Seurat) of reference adult primary human GABAergic subclasses onto hSO snRNA-seq data. Sample description: t-hFA1 and t-hFA2 contain hCO from hiPS cell line 1208-2 and hSO from hiPS cell line 81 19-1 ; t-hFA3 contains hCO and hSO from hiPS cell line 8858-3; t-hFA4 contains hCO from hiPS cell line 8119-1 and hSO from hiPS cell line 1208-2. In vitro hSO1 is from 8858-3, in vitro hSO2 is from 1208-2, in vitro hSO3 is from 8119-1 , and in vitro hSO4 is from 0524-1 .

[0019] FIG. 7A-FIG. 7F. Supplemental electrophysiological measurements of Dlxi1 / 2b::EGFP+ neurons. (A) Additional dendritic reconstructions of biocytin-labeled t-hFA neurons, scale bar = 200pm. (B) Diversity of firing properties of Dlxi1 / 2b::EGFP+ neurons in t-hFA as shown by representative traces. (C) Quantification of action potential half width (hSO N=5 cells, 3 organoids, 1 hiPS cell line; t-hFA N=32 cells, 4 rats, 2 hiPS cell lines; Welch’s t- test on log-transformed values, p=0.0013). (D) Quantification of input resistance (hSO N=13 cells, 6 organoids, 2 hiPS cell lines; t-hFA N=32 cells, 4 rats, 2 hiPS cell lines; Mann-Whitney), (E) Quantification of action potential amplitude (hSO N=5 cells, 3 organoids, 1 hiPS cell line; t-hFA N=32 cells, 4 rats, 2 hiPS cell lines; Mann-Whitney p=0.6197). (F) Quantification of spike threshold (hSO N=5 cells, 3 organoids, 1 hiPS cell line; t-hFA N=32 cells, 4 rats, 2 hiPS cell lines; Mann-Whitney p=0.8736). “ indicates p<0.01 , **** indicates p<0.0001.

[0020] FIG. 8A-FIG. 8M. Supplemental baseline EEG measurements. (A-C) Power spectral density across groups for the baseline recording in the (A) contralateral intracranial electrode placed into S1. (B) The contralateral screw electrode over M1 . (C) The ipsilateral screw electrode over M1 . (D-F) EEG power within canonical frequency bands for the baseline recording in the (D) contralateral intracranial electrode placed into S1 (rmANOVA: frequency band main effect p<0.0001 , graft condition main effect p=0.3357, interaction p=0.0047, no post-hoc tests significant. Rat cortex N=10, t-hCO N=6, t-hFA N=10). (E) The contralateral screw electrode over M1 (rmANOVA: frequency band main effect p<0.0001 , graft condition main effect p=0.4671 , interaction p=0.0023, no post-hoc tests significant. Rat cortex N=10, t- hCO N=6, t-hFA N=10). (F) The ipsilateral screw electrode over M1 (rmANOVA: frequency band main effect p<0.0001 , graft condition main effect p=0.6223, interaction p=0.0029, noAttorney Docket No: STAN-2232WOClient No: S24-410 post-hoc tests significant. Rat cortex N=10, t-hCO N=6, t-hFA N=9). (G) Total power from the graft electrode or naive control rat right somatosensory cortex. Total power computed within the frequency bands studied (Welch’s ANOVA, p=0.0022, Dunnett’s T3 multiple comparison test. Rat cortex N=10, t-hCO N=6, t-hFA N=10). (H) Relative power from the graft electrode or naive control rat right somatosensory cortex (rmANOVA: Frequency band main effect p<0.0001 , graft condition main effect p=0.0807, interaction p=0.0476, post-hoc t-test with Holm-Sidak correction). (I) Spontaneous inhibitory postsynaptic currents (sIPSC) frequency before and after treatment with the GABA receptor antagonist BMI. (J) sIPSC amplitude for same events quantified in FIG. 3I: N=1 1 cells, 20.32 ± 2.407 (mean ± SEM). (K) Representative spontaneous excitatory postsynaptic currents (sEPSC) from Dlxi1 / 2b::EGFP+ neurons in t-hFA displayed with multiple timescales. (L) left- sEPSC frequency (left): N=20, 2.245 ± 0.7757 (mean ± SEM) right- sEPSC amplitude: N=20, 16.70 ± 1.475 (mean ± SEM). (M) Example of coincidentally recorded in vivo units from the right rat somatosensory cortex.

[0021] FIG. 9A-FIG. 9D. PCDH-19 KO line generation. (A) genomic annotation of AAVS1 “safe harbor” locus that is target for EGFP and RFP insertion (upper panel), and PCDH19 locus used for generating our-of-frame indels (lower panel). gRNA targeting sequences are shown in green arrows (B) Schematic for the two-step PCDH19 reporter line generation. (C) confirming out-of-frame PCDH19 KO via Sanger sequencing using the hiPS cell genomic DNA. (D) Validation of PCDH19 protein loss in hCO.

[0022] FIG. 10A-FIG. 10D. Additional graft volume and seizure measurements. (A) Transplantation success rate for all rats transplanted with hiPS cell lines genomically engineered to study PCDH19-RE (-85% success rate, N=91 rats, 7 combinations depicted in Fig. 4A, note the WT / WT groups are also included in Figi success rate). (B) Comparison of graft volume in rats grouped by whether the rat demonstrated a seizure (Mann-Whitney: p=0.2268). (C) Spike and wave discharges (SWD) duration during hyperthermia induction (Kruskal-Wallis, p=0.3990). (D) Spike train duration during hyperthermia induction (Kruskal- Wallis, p=0.821 1 ).

[0023] The invention is best understood from the following detailed description when read in conjunction with the accompanying drawings.DEFINITIONS

[0024] 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 (ES) cells, can be cultured over a long period of time while maintaining the ability to differentiate into all types of cells in an organism, but that, unlike ES cells, are derived from differentiated somatic cells, that is, cellsAttorney Docket No: STAN-2232WOClient No: S24-410 that had a narrower, more defined potential and that in the absence of experimental manipulation could not give rise to all types of cells in the organism. hiPSC have a human ES-like morphology, growing as flat colonies with large nucleo-cytoplasmic ratios, defined borders and prominent nuclei. In addition, hiPSC express several pluripotency markers known by one of ordinary skill in the art, including but not limited to alkaline phosphatase, SSEA3, SSEA4, Sox2, Oct3 / 4, Nanog, TRA160, TRA181 , TDGF 1 , Dnmt3b, FoxD3, GDF3, Cyp26a1 , TERT, and zfp42. In addition, the hiPSC are capable of forming teratomas. In addition, they are capable of forming or contributing to ectoderm, mesoderm, or endoderm tissues in a living organism.

[0025] As used herein, “reprogramming factors” refers to one or more, i.e. a cocktail, of biologically active factors that act on a cell to alter transcription, thereby reprogramming a cell to multipotency or to pluripotency. Reprogramming factors may be provided to the cells 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.

[0026] 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.

[0027] 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 organoids 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.

[0028] Genes may be introduced into the somatic cells or the hiPSC derived therefrom 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 geneAttorney Docket No: STAN-2232WOClient No: S24-410 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 is not critical to the practice of the invention.

[0029] 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 individuals that carry a disease-related genotype or are diagnosed with a disease. Moreover, neural, neuromuscular, and epilepsy disorders with less defined or without genetic components can be studied within the model system. 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-line differences in genetic background. Conditions of neurodevelopmental, neuropsychiatric, and neurological disorders that have strong genetic components or are directly caused by genetic or genomic alterations can be modeled with the systems of the invention.

[0030] The methods described herein are associated with the transplantation of brain-region specific organoids into non-human mammals. Brain-region specific organoids are three- dimensional (3D) aggregates of cells that resemble particular regions of the human brain and contain functional neurons that are normally associated with that region of the brain. These organoids are capable of being maintained in suspension culture for long periods of time, e.g. 2 week, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months or more, without adhering to a surface, e.g. a surface of a culture dish. By functional neurons, it is intended to mean that the neurons are capable of forming functional synapses with other neurons, either in the same organoid, in another organoid, or with host neurons. The formation of functional synapses can be revealed using calcium imaging, as described in more details in the Examples.

[0031] The term “neural organoid” as used herein refers to a range of brain-region specific organoids. Neural organoids encompass any organoid that is comprised of neurons from any part of the brain. Cortical organoids, midbrain organoids, striatal organoids, spinal cord / hindbrain organoids, subpallial organoids, and organoids comprising any combination of the aforementioned organoids are encompassed by the term neural organoids. The terms “organoid” and “spheroid” may be used interchangeably.

[0032] The terms “anatomical integration” or “anatomically integrated” as used herein refer to neural tissue that is innervated by host neurons. The human neural tissue present within theAttorney Docket No: STAN-2232WOClient No: S24-410 non-human mammalian animal model comprises neurons originating from the non-human mammalian animal model nervous system and thus the human neural tissue that is anatomically integrated into the non-human mammalian animal model comprises both human neural tissue and non-human mammalian tissue.

[0033] The term “complex neural activity” as used in reference to human neural tissue refers to neural activity that is not statistically significantly different than the neural activity of the non- human mammalian tissue and is significantly different that the neural activity of the organoid or assembloid counterpart of the human neural tissue, e.g., the neural activity of the organoids or assembloids prior to being introduction into the non-human mammal. The complex neural activity may be a range of different neural activities including, without limitation, power spectral density, total power in one or more alpha, beta, delta, and theta spectral bands, spontaneous neural activity of glutamatergic neurons, etc. The complex neural activity may be measured by EEG or any other equivalent technical method. The similarity of the complex neural activity of the human neural tissue may be based on the measurement of the neural activity of non- human mammalian neural activity in a non-human mammal containing the human neural tissue or a non-human mammal that does not contain the human neural tissue. The nonmammalian human neural activity in the human neural tissue is the result of the advanced maturation of the human neurons contained within the human neural tissue due to the transplantation into the non-human mammal.

[0034] The methods and compositions described herein are also associated with assembloids comprising more than one (e.g. two or three or more) of these brain-region specific organoids or the combination of a neural organoid and cells from another lineage (e.g., cortical organoids and microglia, pericytes, etc.). The assembloids described herein resemble multiple regions of the nervous system and contain functional neural circuits between neurons of one organoid (representing one region) and another organoid (representing another region). For example, the cortico-striatal assembloids resemble the cerebral cortex and striatum of the human brain and contain neurons (e.g. human cortical neurons) projecting from the cortical organoid into the striatal organoid, where these neurons are able functionally synapse with human striatal neurons (e.g. medium spiny neurons) of the striatal organoid. Similar to the organoids, these assembloids are also capable of being maintained for long periods of time without adhering to a surface. The assembloids may be assembled prior to transplantation or may be assembled after transplantation, e.g., introducing a first human neural organoid followed by introducing a second human neural organoid into the same central nervous system location.

[0035] Cortex. The adult cerebral cortex contains two main classes of neurons: glutamatergic cortical neurons (also known as pyramidal cells) and GABAergic interneurons. Pyramidal cellsAttorney Docket No: STAN-2232WOClient No: S24-410 are generated in the pallium— the roof of the telencephalon (dorsal forebrain)— and reach their final position by radial migration. In contrast, cortical interneurons are born in the subpallium — the base of telencephalon (ventral forebrain) — and reach the cerebral cortex through a long tangential migration.

[0036] The layers of the cerebral cortex are generated in an “inside-out” sequence, with deep layers being generated first and superficial layer neurons being generated last. In parallel to this process, GABAergic interneurons migrate to the cortical plate, where they disperse tangentially via highly stereotyped routes in the MZ, SP, and lower intermediate zone / subventricular zone (IZ / SVZ). Interneurons then switch from tangential to radial migration to adopt their final laminar position in the cerebral cortex.

[0037] The movement of cortical interneurons is saltatory. First, the cell extends a leading process. Second, the nucleus translocates towards the leading process, a step referred to as nucleokinesis and leads to the net movement of the cell.

[0038] The translocation of the nucleus into the leading process is the mechanism that best defines this type of saltatory neuronal migration. First, a cytoplasmic swelling forms in the leading process, immediately proximal to the nucleus. The centrosome, which is normally positioned in front of the nucleus, moves into this swelling. The centrosome is accompanied by additional organelles, including the Golgi apparatus, mitochondria, and the rough endoplasmic reticulum. Second, the nucleus follows the centrosome. These two steps are repeated producing the typical saltatory movement of migrating neurons.

[0039] Tangentially migrating neurons do not always follow radial glial fibers. In general, tangentially migrating cells can move in clusters or individually. Cellular interactions also differ depending on the nature of the substrate. They can be homotypic, when interactions occur between cells of the same class, or heterotypic, when migrating cells rely on the contact with other cell types for their migration or their substrates. In the most common scenario, neurons respond to cues present in the extracellular matrix or in the surface of other cells to achieve directional migration.

[0040] GABAergic interneurons are inhibitory neurons of the nervous system that play a vital role in neural circuitry and activity. They are so named due to their release of the neurotransmitter gamma-aminobutyric acid (GABA). An interneuron is a specialized type of neuron whose primary role is to modulate the activity of other neurons in a neural network. Cortical interneurons are so named for their localization in the cerebral cortex.

[0041] There are interneuron subtypes categorized based on the surface markers they express, including parvalbumin (PV)-expressing interneurons, somatostatin (SST)-expressing interneurons, VIP-expressing, serotonin receptor 5HT3a (5HT3aR)-expressing interneurons,Attorney Docket No: STAN-2232WOClient No: S24-410 etc. Although these interneurons are localized in their respective layers of the cerebral cortex, they are generated in various subpallial locations.

[0042] Morphologically speaking, cortical interneurons may be described with regard to their soma, dendrites, axons, and the synaptic connections they make. Molecular features include transcription factors, neuropeptides, calcium-binding proteins, and receptors these interneurons express, among many others. Physiological characteristics include firing pattern, action potential measurements, passive or subthreshold parameters, and postsynaptic responses, to name a few.

[0043] The PV interneuron group represents approximately 40% of the GABAergic cortical interneuron population. This population of interneurons possesses a fast-spiking pattern, and fire sustained high-frequency trains of brief action potentials. Additionally, these interneurons possess the lowest input resistance and the fastest membrane time constant of all interneurons. Two types of PV-interneurons make up the PV interneuron group: basket cells, which make synapses at the soma and proximal dendrite of target neurons, and usually have multipolar morphology and chandelier cells, which target the axon initial segment of pyramidal neurons.

[0044] The SST-expressing interneuron group is the second-largest interneuron group. SST- positive interneurons are known as Martinotti cells, and possess ascending axons that arborize layer I and establish synapses onto the dendritic tufts of pyramidal neurons. Martinotti cells are found throughout cortical layers 11- VI, but are most abundant in layer V. These interneurons function by exhibiting a regular adapting firing pattern but also may initially fire bursts of two or more spikes on slow depolarizing humps when depolarized from hyperpolarized potentials. In contrast to PV-positive interneurons, excitatory inputs onto Martinotti cells are strongly facilitating.

[0045] The third group of GABAergic cortical interneurons is designated as the 5HT3aR interneuron group. VIP-expressing interneurons are localized in cortical layers II and III. VIP interneurons generally make synapses onto dendrites, and some have been observed to target other interneurons. Relative to all cortical interneurons, VIP interneurons possess a very high input resistance. In general they possess a bipolar, bitufted and multipolar morphology. Irregular spiking interneurons possess a vertically oriented, descending axon that extends to deeper cortical layers, and have an irregular firing pattern that is characterized by action potentials occurring irregularly during depolarizations near threshold, and express the calcium-binding protein calretinin (CR). Other subtypes include rapid-adapting, fast-adapting neurons IS2, as well as a minor population of VIP-positive basket cells with regular, bursting, or irregular-spiking firing patterns. Of the VIP-negative 5HT3aR group, nearly 80% express the interneuron marker Reelin. Neurogliaform cells are a type of cortical interneuron thatAttorney Docket No: STAN-2232WOClient No: S24-410 belongs to this category: they are also known as spiderweb cells and express neuropeptide Y (NPY), with multiple dendrites radiating from a round soma.

[0046] A transcriptional network plays a role in regulating proper development and specification of GABAergic cortical interneurons, including DLX homeobox genes, LHX6, SOX6 and NKX2-1 , LHX8, GSX1 , GSX2. The DLX family of homeobox genes, specifically DLX1 , DLX2, DLX5, and DLX6, also play a role in the specification of interneuron progenitors, and are expressed in most subpallial neural progenitor cells.

[0047] Glutamatergic neurons. The mature cerebral cortex harbors a heterogeneous population of glutamatergic neurons, organized into a highly intricate histological architecture. So-called excitatory neurons are usually classified according to the lamina where their soma is located, specific combinations of gene expression, by dendritic morphologies, electrophysiological properties, etc.

[0048] Disease relevance. Dysfunction in neural pathways in the forebrain is thought to contribute to severe neuropsychiatric disorders such as epilepsy disorders such as PCDH19- related encephalopathy, CDKL5 deficiency disorder, KCNQ2 developmental and epilepsy encephalopathy, Rett’s syndrome, SCN2A-related epilepsy, Dravet syndrome, GABRA1 - related epilepsy encephalopathy, G4f?RG2-related epilepsy, GRIN2D-related developmental and epilepsy encephalopathy, benign familial neonatal-infantile seizures, benign familial neonatal seizures, childhood absence epilepsy, early infantile epilepsy encephalopathy, febrile seizures, genetic (generalized) epilepsy with febrile seizures plus intractable childhood epilepsy, intractable childhood epilepsy with generalized tonic-clonic seizures, intellectual disability, infantile spasms, juvenile myoclonic epilepsy, Ohtahara syndrome, severe myoclonic epilepsy of infancy. Additionally, epilepsy disorders as a result of mosaicism are of particular relevance. As well as understanding development, the organoids and assembloids described herein are useful to model disorders of the forebrain as well as for testing therapeutics including gene therapy and small molecule drugs. Additionally, epilepsy disorders as a result of mosaicism are of particular relevance. By “mosaicism” it means that some cells in the individual express a mutant or disease associated variant of a protein or do not express the protein whereas other cells produce a healthy or non-disease associate variant of a protein. The mosaicism may be caused by random X inactivation in females, i.e., females that are heterozygous for the mutant or disease associated variant, or mosaic expression in males. Epilepsy disorders associated with mosaicism include epilepsy disorders associated with pathogenic variants of CDKL5, CLCN4, SCN1 A, SCN2A, SLC35A2, GABRA1 , GRIN2B, KCNQ2, STXBP1 , CHD2, SLC2A1 , ARX, MECP2, and PCDH19. In some embodiments, the epilepsy disorder is associated with a channelopathy. A range of different channelopathies find used in the present disclosure. Channelopathies of interest include channelopathiesAttorney Docket No: STAN-2232WOClient No: S24-410 associated with pathogenic variants of a gene selected from the group consisting of: SCN1 A, SCN1 B, SCN2A, SCN3A, SCN8A, KCNQ2, KCNQ3, KCNMA1 , KCNA1 , KCNA2, KCNJII, KCNT1 , CACNA1 H, CACNA1A, CHRNA4, CHRNB2, CHRNA2, GABRA1 , GABRB2, GABRB3, GABRD, and GABRG2.

[0049] PCDH19-related encephalopathy (Also known as Epilepsy and Mental Retardation Limited to Females, EFMR, or Developmental and Epileptic Encephalopathy 9, DEE9) was first described in 1971 by Juberg and Hellman as an early onset seizure disorder triggered by febrile illness, and with female-limited expression. The causative gene was identified in 2008 by Dibbens et al. in a study that involved six new EFMR families, as well as the original EFMR family reported by Juberg and Helman. In the same year, EFMR was further characterized as a neurological disorder with a markedly varied neuropsychiatric profile including intellectual disability (ID), and aggressive, ASD, or obsessive features. In 2009, Depienne et al. identified PCDH19 mutations in sporadic cases with infantile development and epilepsy encephalopathy resembling Dravet syndrome. The hallmark feature of PCDH19-associated epilepsy is that seizures occur in clusters. Seizures typically present as generalized tonic-clonic and / or focal seizures, which may evolve to bilateral, tonic-clonic seizures. An additional unifying feature of PCDH19-related encephalopathy is cellular mosaicism, either due to X-chromosome inactivation in females or early somatic mutation and, as such, somatic mosaicism in males.

[0050] PCDH19-related encephalopathy is associated with a reduction or remission of seizures during adolescence. Unfortunately, neuropsychiatric dysfunction remains, often exacerbating with age and becoming prominent and disabling feature in some patients. ID ranging from mild to profound is present in approximately 70% of the cases. The prevalence of psychiatric comorbidities is unknown; however, reports suggest that ASD is a common feature in both females. PCDH19-related encephalopathy has been described by, for example, Kolc et al. (Mol Psychiatry. 2019 Feb;24(2):241 -251 ), which is specifically incorporated by reference herein.

[0051] CDKL5 deficiency disorder. Pathogenic variants in the X-linked gene encoding cyclin- dependent kinase-like 5 (CDKL5) have been associated with the severe childhood epileptic encephalopathy known as CDKL5 deficiency disorder (CDD). ODD is characterized by a heterogeneous array of clinical symptoms including early-onset seizures, marked hypotonia, autistic features, and severe neurodevelopmental impairment. The disorder predominantly affects young females heterozygous for mutations in the X-linked CDKL5, with an overall incidence of one per forty-two thousand live births, making it one of the most common genetic causes of epilepsy in children. Despite the constellation of phenotypes presented in CDD, the severe, early-onset seizures in particular drastically impact quality of life, with ninety percent of patients displaying seizures by three months of age. Eighty percent of children with CDDAttorney Docket No: STAN-2232WOClient No: S24-410 have daily seizures, and fewer than half report more than two months of seizure freedom. Furthermore, children with epilepsy that presents before 3 years of age have been shown to carry a high burden of behavioral and cognitive comorbidities, and this risk increases with increased seizure incidence. This high burden of refractory seizures in CDD make it a particularly debilitating aspect of the disorder for both patients and their families, highlighting the pressing need to develop effective therapeutics for seizure management.

[0052] KCNQ2 developmental and epileptic encephalopathy. Children with KCNQ2 developmental and epileptic encephalopathy (KCNQ2-DEE) typically present with seizures in the first week of life. Seizures appear as stiffening of the body (tonic) often associated with jerking and changes in breathing or heart rate. The seizures are usually quite frequent (many per day) and often difficult to treat. Typically, the seizures are associated with abnormal brain wave patterns on EEG during this time. The seizures often resolve within months to years, but children have some degree of developmental impairment involving one or more domains (motor, social, language, cognition). This can range from mild to severe depending on several different factors. Some children may also have autism or other neurobehavioral issues. Other, less common presentations have also been reported including later onset seizures, intellectual disability without seizures, infantile spasms and sudden twitching (myoclonus).

[0053] Rett syndrome (RTT) is a pervasive neurological disorder, and it is characterized by compromised brain functions, severe mental retardation, language and learning disabilities, repetitive stereotyped hand movements and developmental regression. RTT is predominantly found in young females, with an incidence of 1 :10,000-20,000 live births, with rare cases reported in males. RTT is considered to be a monogenic neurological disorder, because approximately 90 % cases of classical RTT patients harbor loss-of-function mutations of the X-linked methyl-CpG binding protein 2 (MECP2) gene. Rett syndrome is perhaps the best example for contribution of epigenetic mechanisms in disease pathology, mostly due to the involvement of MeCP2, a key epigenetic modulator in the brain. Epigenetic mechanisms include DNA methylation, histone posttranslational modifications (PTMs) and noncoding RNAs, which regulate gene expression without altering the corresponding DNA sequences. Epigenetic mechanisms are involved in controlling embryonic development, stem cell differentiation and have a high impact in human disease. As a well-studied epigenetic factor, MeCP2 controls gene expression and modulates chromatin architecture through binding to methylated DNA.

[0054] Dravet syndrome (DS) is a devastating epileptic encephalopathy arising in otherwise normal babies in the first year of life, later accompanied by developmental delay, intellectual disability and mood disorders. In the 80% of cases, it is caused by haploinsufficiency of SCN1A gene, that encodes for the alpha subunit of the voltage-gated sodium channelAttorney Docket No: STAN-2232WO Client No: S24-410(VGSC) Nav1 .1. Current pharmacological treatments for DS are ineffective in completely control convulsive attacks or delaying subsequent neurological symptoms, and, thus, a number of gene-based therapeutic strategies are in development. Gene supplementation therapy is hardly feasible in DS, as SCN1A coding sequence largely exceeds the packaging cargo of adeno-associated viral vectors (AAV), that are commonly employed for therapeutic gene delivery in the CNS. In recent years, alternative genetic approaches aiming to restore physiological levels of Nav1.1 to treat DS have been developed. Those strategies rely on boosting the expression of the healthy copy of SCN1A gene at transcriptional or post- transcriptional level to rescue channel haploinsufficiency.

[0055] GRIN2D-re\ated developmental and epileptic encephalopathy (GRIN2D- related DEE) is characterized by mild-to-profound developmental delay or intellectual disability, epilepsy, abnormal muscle tone (hypotonia and spasticity), movement disorders (dystonia, dyskinesia, chorea), autism spectrum disorder, and cortical visual impairment. Additional findings can include sleep disorders and feeding difficulties.

[0056] The terms “astrocytic cell,” “astrocyte,” etc. encompass cells of the astrocyte lineage, i.e. glial progenitor cells, astrocyte precursor cells, and mature astrocytes, which for the purposes of the present invention arise from a non-astrocytic cells (i.e., glial progenitors). Astrocytes can be identified by markers specific for cells of the astrocyte lineage, e.g. GFAP, ALDH1 L1 , AQP4, EAAT1 and EAAT2, etc. Markers of reactive astrocytes include S100, VIM, LCN2, FGFR3 and the like. Astrocytes may have characteristics of functional astrocytes, that is, they may have the capacity of promoting synaptogenesis in primary neuronal cultures; of accumulating glycogen granules in processes; of phagocytosing synapses; and the like. A "astrocyte precursor" is defined as a cell that is capable of giving rise to progeny that include astrocytes.

[0057] Astrocytes are the most numerous and diverse neuroglial cells in the CNS. An archetypal morphological feature of astrocytes is their expression of intermediate filaments, which form the cytoskeleton. The main types of astroglial intermediate filament proteins are glial fibrillary acidic protein (GFAP) and vimentin; expression of GFAP, ALDH1 L1 and / or AQP4P are commonly used as a specific marker for the identification of astrocytes.

[0058] The terms “oligodendrocyte,” “oligodendrocyte progenitor cell,” etc. can encompass cells of the oligodendrocyte lineage, i.e. neural progenitor cells that ultimately give rise to oligodendrocytes, oligodendrocyte precursor cells, and mature and myelinating oligodendrocytes, which for the purposes of the present invention arise from a nonoligodendrocyte cell by experimental manipulation. Oligodendrocytes may have functional characteristics, that is, they may have the capacity of myelinating neurons; and the like. An "oligodendrocyte precursor" or “oligodendrocyte progenitor cell” is defined as a cell that isAttorney Docket No: STAN-2232WOClient No: S24-410 capable of giving rise to progeny that include oligodendrocytes. Oligodendrocytes may be present in the assembloids.

[0059] Oligodendrocytes are the myelin-forming cells of the central nervous system. An oligodendrocyte extends many processes which contact and repeatedly envelope stretches of axons. Subsequent condensation of these wrapped layers of oligodendrocyte membrane form the myelin sheath. One axon may contain myelin segments from many different oligodendrocytes.

[0060] Calcium sensors. Neural activity causes rapid changes in intracellular free calcium, which can be used to track the activity of neuronal populations. Art-recognized sensors for this purpose include fluorescent proteins that fluoresce in the presence of changes in calcium concentrations. These proteins can be introduced into cells, e.g. hiPSC, by including the coding sequence on a suitable expression vector, e.g. a viral vector, to genetically modify neurons generated by the methods described herein. GCaMPs are widely used protein calcium sensors, which are comprised of a fluorescent protein, e.g. GFP, the calcium-binding protein calmodulin (CaM), and CaM-interacting M13 peptide, although a variety of other sensors are also available. Many different proteins are available, including, for example, those described in Zhao et al. (2011) Science 333:1888-1891 ; Mank et al. (2008) Nat. Methods 5(9):805-1 1 ; Akerboom et al. (2012) J. Neurosci. 32(40):13819-40; Chen et al. (2013) Nature 499(7458) :295-300; etc.; and as described in US Patent nos. 8,629,256, 9,518,980 and 9,488,642 and 9,945,844.

[0061] Optogenetics integrates optics and genetic engineering to measure and manipulate neurons. Actuators are genetically-encoded tools for light-activated control of proteins; e.g., opsins and optical switches. Opsins are light-gated ion channels or pumps that absorb light at specific wavelengths. Opsins can be targeted and expressed in specific subsets of neurons, allowing precise spatiotemporal control of these neurons by turning on and off the light source. Channel rhodopsins typically allow the fast depolarization of neurons upon exposure to light through direct stimulation of ion channels. Chlamydomonas reinhardtii Channelrhodopsin-1 (ChR1 ) is excited by blue light and permits nonspecific cation influx into the cell when stimulated. Examples of ChRs from other species include: CsChR (from Chloromonas subdivisa), CoChR (from Chloromonas oogama), and SdChR (from Scherffelia dubia). Synthetic variants have been created, for example ChR2(H134R), C1 V1 (t / t), ChlEF; ChETA, VChR1 , Chrimson, ChrimsonR, Chronos, PsChR2, CoChR, CsChR, CheRiff, and the like. Alternatively, ChR variants that inhibit neurons have been created and identified, for example GtACRI and GtACR2 (from the cryptophyte Guillardia theta), and variants such as iChloC, SwiChRca, Phobos, Aurora. Halorhodopsin, known as NpHR (from Natronomonas pharaoni), causes hyperpolarization of the cell when triggered with yellow light, variants include Halo,Attorney Docket No: STAN-2232WOClient No: S24-410 eNpHR, eNpHR2.0, eNpHR3.0, Jaws. Archaerhodopsin-3 (Arch) from Halorubrum sodomense is also used to inhibit neurons.

[0062] 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.

[0063] 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.DETAILED DESCRIPTION

[0064] 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.

[0065] 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 where either, 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.

[0066] 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 preferredAttorney Docket No: STAN-2232WOClient No: S24-410 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.

[0067] 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 human neural organoid” includes a plurality of such organoids, 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.

[0068] 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.METHODS FOR PRODUCING NON-HUMAN MAMMALIAN ANIMAL MODELS

[0069] As summarized above, methods are provided for producing a non-human mammalian animal model containing human neural tissue, the method including introducing a first human neural organoid into a central nervous system location of a newborn non-human mammal; introducing a second human neural organoid into the central nervous system location of the newborn non-human mammal; and allowing the newborn non-human mammal to mature to produce the non-human mammalian animal model comprising human neural tissue comprising GABAergic neurons; wherein the human neural tissue has complex neural activity.

[0070] The non-human mammalian animal may be any non-human mammalian animal. For example, the non-human mammalian animal includes, without limitation, canines; felines; equines; bovines; ovines; rodentia, such as mice or rats, etc. and primates, e.g., non-human primates, and humans. In a preferred embodiment, the non-human mammalian animal is a rat. In some embodiments, the non-human mammalian animal is newborn animal is between 1 -10 days in age after birth. For instance, the newborn non-human mammalian animal may be at least 1 day old, 2 days old, 3 days old, 4 days old, 5 days old, 6 days old, 7 days, 8 days old, 9 days old, or 10 days old.

[0071] The non-human animals of the present disclosure contain human neural tissue having complex neural activity. The term “complex neural activity” as used in reference to human neural tissue refers to neural activity that is not statistically significantly different than the neural activity of the non-human mammalian tissue and is significantly different that the neuralAttorney Docket No: STAN-2232WOClient No: S24-410 activity of the organoid or assembloid counterpart of the human neural tissue, e.g., the neural activity of the organoids or assembloids prior to being introduction into the non-human mammal. The complex neural activity of the human neural tissue is the result of the advanced maturation of the neurons contained in the human neural tissue following introduction of the first and the second human neural organoid. The neural activity may be a range of different neural activities. For instance, the neural activity may include, without limitation, power spectral density, total power in alpha spectral band, total power in beta spectra band, total power in delta spectral band, total power in theta spectral power, spontaneous neural activity of glutamatergic neurons, etc.

[0072] In some embodiments, the non-human mammalian animal is immunocompromised. In these embodiments, the non-human mammalian animal may be immunocompromised for any reason. For instance, the immunocompromise may be the result of including, without limitation, a genetic mutation, a chemical treatment, etc. When the immunocompromise is the result of a genetic mutation, the genetic mutation may be any mutation or set of mutations that results in immunosuppression. When the immunocompromised non-human mammalian animal is a mouse, the mouse may comprise any genetic mutation or set of genetic mutations that result in immunosuppression. For instance, the immunocompromised mouse includes, without limitation, an athymic nude mouse, a BALB / c nude mouse, CD-1 nude mouse, a Fox Chase SCID mouse, a Fox Chase SCID beige mouse, a hACE2-NCG mouse, a NCG mouse, a NOD SCID mouse, a NIH-III nude mouse, a NU / NU mouse, a SCID hairless congenic, a SCID hairless outbred mouse, a NCI SCID / NCr mouse, etc. When the immunocompromised non- human mammalian animal is a rat, the rat may comprise any genetic mutation or set of genetic mutations that result in immunosuppression. For instance, the immunocompromised rat includes, without limitation, a RNU nude rat, a SRG rat, an athymic rat, etc.

[0073] When the immunocompromise is the result of a chemical treatment, the chemical treatment may be any chemical treatment that results in immunosuppression. For instance, the chemical treatment includes, without limitation, glucocorticoids such as prednisolone, dexamethasone, etc.; cytostatic drugs such as methotrexate, cyclophosphamide, azathioprine, etc.; mycophenolate; immunophilin drugs such as rapamycin, tacrolimus, cyclosporine A, etc.; everolimus; cell therapies directed to the suppression of proliferation of specific cells such as mesenchymal stem cells, regulatory T cells, etc.; antibody treatments such as rituximab anti-thymocyte globulin, anti-lymphocyte globulin, etc.; blockage of costimulatory pathways such as CD28 / B7, etc. Other known chemical treatments have been described in the art, such as in Diehl R. et al. (Cell Mol Immunol. 2017 Feb;14(2):146-179) which has specifically been incorporated by reference.Attorney Docket No: STAN-2232WOClient No: S24-410

[0074] The methods of the present disclosure comprise introducing a first human neural organoid into a non-human mammalian animal. The first human neural organoid may be any human neural organoid deemed useful. For example, the human neural organoid includes, without limitation, striatal organoids, subpallial organoids, cortical organoids, midbrain organoids, spinal organoids, combinations of the aforementioned organoids, etc. In some embodiments, the first human neural organoid is a cortical organoid. In some embodiments, the first human neural organoid is a subpallial organoid.

[0075] The methods of the present disclosure comprise introducing a second human neural organoid into a non-human mammalian animal. The second human neural organoid may be any human neural organoid deemed useful. For example, the human neural organoid includes, without limitation, subpallial organoids, cortical organoids, combinations of the aforementioned organoids, etc. In some embodiments, the second human neural organoid is a cortical organoid. In some embodiments, the second human neural organoid is a subpallial organoid. In some embodiments, either the first human neural organoid or the second neural organoid is subpallial organoid. In some embodiments, the human neural organoids are generated from induced human pluripotent stem cells (hiPSCs). Methods for generating hiPSCs are well known in the art and are also described below.

[0076] Human induced pluripotent stem cells. Initially, hiPSCs can be obtained from any convenient source, or can be generated from somatic cells using art- recognized methods. The hiPSCs are dissociated from feeders into single cells and grown in suspension culture, preferably when dissociated as intact colonies. In certain embodiments the culture is feeder layer free, e.g. when grown on vitronectin coated culture dishes. The culture may further be free on non-human components, i.e. xeno-free. The hiPSCs may be cultured in any medium suitable for the growth and expansion of hiPSCs. For example, the medium may be Essential 8 medium. Suspension growth optionally includes in the culture medium an effective dose of a selective Rho-associated kinase (ROCK) inhibitor 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):1 187-200; Fasudil (HA-1077) HCI (J Clin Invest, 2014, 124(9):3757-66); GSK429286A (Proc Natl Acad Sci U S A, 2014, 1 11 (12):E1 140-8); RKI-1447; AT13148; etc. In particular embodiments the ROCK inhibitor Y-27632 is used. Optionally a WNT pathway inhibitor such as XAV-939 is added.

[0077] Human cortical spheroids. hCS may be generated by the methods previously described, for example in Pasca et al. (2015) Nat. Methods 12(7) :671 -678, entitled “FunctionalAttorney Docket No: STAN-2232WOClient No: S24-410 cortical neurons and astrocytes from human pluripotent stem cells in 3D culture” and in U.S. Patent No. 10,494,602, each herein specifically incorporated by reference.

[0078] For example, a suspension culture of hiPS cells is cultured to provide a neural progenitor spheroid, as described above. After about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days in suspension culture, the floating neural progenitor spheroids are moved to neural media to differentiate the neural progenitors. The media is supplemented with an effective dose of FGF2 and EGF. The growth factors can be provided at a concentration for each of at least 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.

[0079] To promote differentiation of neural progenitors into hCS, comprising glutamatergic neurons, after about 1 week, about 2 weeks, about 3 weeks, about 4 weeks after FGF2 / EGF exposure the neural medium is changed to replace the FGF2 and EGF with an effective dose of BDNF and NT3. The growth factors can be provided at a concentration for each of at least 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. The cortical spheroids comprise functional glutamatergic neurons.

[0080] Human forebrain assembloids. The hSO can be functionally integrated with separately cultured human cortical organoids (hCO), to form forebrain assembloids (hCO-hSO) which include functionally-integrated excitatory glutamatergic and GABAergic neurons. The resulting hCO-hSO contains forebrain circuits and provides for functional integration of these circuits. Functionally integrated cells interact in a physiologically relevant manner, e.g. forming synapses or neuromuscular junctions, transmitting signals, forming multicellular structures, and the like. Human forebrain assembloids may be generated by the methods previously described, for example in Birey F. et al. (2017) Nature. May 4;545(7652):54-59, entitled “Assembly of functionally integrated human forebrain organoids” and U.S. Patent No. 10,676,715, each herein specifically incorporated by reference.

[0081] The cortical organoids are co-cultured with the human subpallial organoids in neural medium under conditions permissive for cell fusion. Condition permissive for cell fusion may include culturing the hSO and hCO in close proximity, e.g. in direct contact with one another.

[0082] Assembly may be performed with organoids after around about 30 days, about 60 days, about 90 days of culture for hStrO; and after around about 30 days, about 60 days, about 90 days of culture for hCO. The hStrO and hCO organoids may be co-cultured for a period of 3 days, 5 days, 8 days, 10 days, 14 days, 18 days, 21 days or more. The resulting cortico-striatal assembloids are demonstrated to contain functional neural circuits, where theAttorney Docket No: STAN-2232WOClient No: S24-410 assembloids comprise glutamatergic neurons projecting from the hCO to the hStrO. The glutamatergic neurons may be unidirectional neurons, for example unidirectional CTIP2 and / or SATB2 expressing neurons. Methods for confirming the functionality of the neurons are known in the art and include optogenetic methods and imaging of calcium activity in neurons, such as those methods described in the examples. In some embodiments, the methods may comprise confirming the functionality of the neurons in the cortico-striatal assembloid.

[0083] The assembloids may be formed prior to or after introduction into the non-human mammal. For instance, the assembloids may be assembled as disclosed above. Additionally, assembloids may be assembled after introduction into the non-human mammal. When assembloids are assembled post-transplantation, a first human neural organoid is introduced to a central nervous system location followed by a second neural organoid being introduced to the same central nervous system location. In some embodiments, the second human neural organoid is introduced immediately after, i.e. , within minutes, the first human neural organoid. In some embodiments, the second human neural organoid is introduced after a delay following the introduction of the first human neural organoid. The delay may be a range of different times. For instance, the delay may be 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, or more than 10 days.

[0084] In some embodiments, the neural organoids of the present disclosure are isolated from an individual who is predicted to have or has been diagnosed with an epilepsy disorder. The epilepsy may be any epilepsy disorder that is deemed suitable for organoid culture. Epilepsy disorders that find use in the present disclosure include, with limitation, PCDH19-related encephalopathy, CDKL5 deficiency disorder, KCNQ2 developmental and epileptic encephalopathy, Rett’s syndrome, SCN2A-related epilepsy, Dravet syndrome, GABRA1 - related epileptic encephalopathy, GABRG2-related epilepsy, GRIN2D-related developmental and epileptic encephalopathy, benign familial neonatal-infantile seizures, benign familial neonatal seizures, childhood absence epilepsy, early infantile epileptic encephalopathy, febrile seizures, genetic (generalized) epilepsy with febrile seizures plus intractable childhood epilepsy, intractable childhood epilepsy with generalized tonic-clonic seizures, intellectual disability, infantile spasms, juvenile myoclonic epilepsy, Ohtahara syndrome, severe myoclonic epilepsy of infancy, etc. Additionally, epilepsy disorders as a result of mosaicism are of particular relevance. By “mosaicism” it means that some cells in the individual express a mutant or disease associated variant of a protein or do not express the protein whereas other cells produce a healthy or non-disease associate variant of a protein. The mosaicism may be caused by random X inactivation in females, i.e., females that are heterozygous for the mutant or disease associated variant, or mosaic expression in males. Epilepsy disordersAttorney Docket No: STAN-2232WOClient No: S24-410 associated with mosaicism include epilepsy disorders associated with pathogenic variants of CDKL5, CLCN4, SCN1 A, SCN2A, SLC35A2, GABRA1 , GRIN2B, KCNQ2, STXBP1 , CHD2, SLC2A1 , ARX, MECP2, and PCDH19. Epilepsy disorders associated with mosaicism are known in the art and have been described by Winawer et al. (Ann Neurol.2018 Jun;83(6):1 133- 1 146), Terzic et al. (Neurobiol Dis.2021 Jan:148:105176), Stosser et al. (Genet Med.2018 Apr;20(4):403-410), Uddin et al. (Neurol Genet. 2017 Dec 18;3(6):e199), Lamar et al. (Front Mol Neurosci. 2018 Jun 15;11 :208), Bartolini et al. (Neurol. Int. 2021 , 13(4), 555-568), Poirier et al. (Hum Genet. 2005 Oct;118(1):45-8), and Zeng et al. (Front Mol Neurosci. 2022 Mar 30;15:809951), each of which are specifically incorporated by reference herein.

[0085] In some embodiments, the epilepsy disorder is associated with a channelopathy. A range of different channelopathies find used in the present disclosure. Channelopathies of interest include channelopathies associated with pathogenic variants of a gene selected from the group consisting of: SCN1 A, SCN1 B, SCN2A, SCN3A, SCN8A, KCNQ2, KCNQ3, KCNMA1 , KCNA1 , KCNA2, KCNJII, KCNT1 , CACNA1 H, CACNA1A, CHRNA4, CHRNB2, CHRNA2, GABRA1 , GABRB2, GABRB3, GABRD, and GABRG2.

[0086] The methods of embodiments of the present disclosure comprise introducing a first human neural organoid into a central nervous system location. The first human neural organoid may be introduced in a variety of ways. In some embodiments, introducing comprises employing an introducer loaded with the neural organoid. The introducer may be any introducer that can be loaded with neural organoid. Introducers that find use in the present disclosure include, without limitation, a syringe, an auto-injector, a tube, a pipette, a pipette tip, a needle, etc.

[0087] In some embodiments, the introducing comprises making access to a central nervous system location. In some embodiments, making access to a central nervous system location comprises performing a craniotomy. In some embodiments, performing a craniotomy further comprises perforating the dura of the brain. In some embodiments, when the dura is perforated, a central nervous system location, e.g. a specific brain region, is contacted with an introducer wherein the introducer is retracted upon contact and a neural organoid is deposited at the site of contact. The first human neural organoid may be introduced into any location within the central nervous system deemed useful including, without limitation, a brain, a spinal cord, etc. When a human neural organoid is introduced into a brain, it may be in any suitable location within the brain. For instance, the human neural organoid may introduced to a specific region of the brain including, without limitation, the frontal cortex, the motor cortex, somatosensory cortex, parietal cortex, occipital cortex, temporal cortex, the cerebellum, spinal cord, etc. In some embodiments, the location within the brain is specific for the type of organoid used. For instance, cortical organoids may be introduced to the somatosensory cortex. InAttorney Docket No: STAN-2232WOClient No: S24-410 some embodiments, the location with the brain is not specific for the type of organoid used. In some embodiments, the first and second human neural organoid is introduced to the same central nervous system location.

[0088] In some embodiments, the methods further comprise introducing a second human neural organoid into the central nervous system location. In some embodiments, the second human neural organoid is the same as the first human neural organoid. For example, if the first human neural organoid is a cortical organoid then the second human neural organoid is a cortical organoid. In some embodiments, the second human neural organoid is different from the first human neural organoid. In this instance, the first human neural organoid may be a cortical organoid and the second human neural organoid may be a midbrain organoid, a striatal organoid, a subpallial organoid or any other neural organoid that is not a cortical organoid. In some embodiments, the central nervous system location is the same as the first central nervous system location. For example, when the first human neural organoid is introduced into the somatosensory cortex then the second human neural organoid is also introduced into the somatosensory cortex.

[0089] Following the introduction of the first and second human neural organoid into the central nervous system location of the newborn non-human mammalian animal, the newborn non-human mammal is allowed to mature thereby producing a non-human mammalian animal model comprising human neural tissue. The newborn non-human mammalian animal may be allowed to mature for any amount of time deemed suitable. Suitable amounts of time to allow for the newborn non-human mammalian animal to mature includes, without limitation, 5-10 days, 10-15 days, 15-20 days, 20-25 days, 25-30 days, 30-35 days, 35-40 days, 40-45 days, 45-50 days, 50-60 days, 60-70 days, 70-80 days, 80-90 days, 90-100 days, 100-110 days, 1 10-120 days, 120-130 days, 130-140 days, 140-150 days, 150-160 days, 160-170 days, 170- 180 days, 180-190 days, 190-200 days, 200-210 days, 210-220 days, 220-230 days, 230-240 days, 240-250 days, or greater than 250 days. The amount of time to allow for the newborn non-human mammalian animal to mature may be at any intervening amount of time within a specific range. For instance, when the amount of time to allow for the newborn non-human mammalian animal to mature is from 80-90 days, the amount of time may be 90 days, 91 days, 92 days, 93 days, 94 days, 95 days, 96 days, 97 days, 98 days, 99 days, or 100 days.

[0090] In some embodiments, the non-human mammalian animal model comprises anatomically integrated human neural tissue. In some embodiments, the anatomical integration of the human neural tissue does not result in discernable locomotor or memory deficits in the non-human mammalian animal model. In some embodiments, the anatomically integrated human neural tissue is vascularized by the non-human mammal’s vasculature. InAttorney Docket No: STAN-2232WOClient No: S24-410 some embodiments, the anatomically integrated human neural tissue comprises GABAergic neurons.

[0091] The human neural tissue of the present disclosure contains GABAergic neurons. The human neural tissue may contain a range of different GABAergic neurons. For instance, the human neural tissue may include, without limitation, medial ganglionic eminence (MGE)- derived parvalbumin positive (PV+) GABAergic neurons, MGE-derived somatostatin positive (SST+) GABAergic neurons, caudal ganglionic eminence (CGE)-derived vasoactive intestinal polypeptide positive (VI P+) GABAergic neurons, CGE-derived lysosomal associated membrane protein 5 positive (LAMP5+) GABAergic neurons, CGE-derived gamma-synuclein positive (SNCG+) GABAergic neurons, etc. In some embodiments, the MGE-derived PV+GABAergic neurons are fast spiking PV+GABAergic neurons. In some embodiments, the PV+GABAergic neurons are morphologically, transcriptionally, and electrophysiologically mature PV+GABAergic neurons. In some embodiments, the mature morphology refers to the length of the dendrites of the PV+GABAergic neurons. In some embodiments, the transcriptionally mature PV+GABAergic neurons have increased expression of genes that are normally upregulated in 3rd trimester or older human cortical GABAergic neurons. In some embodiments, the genes that are normally upregulated in 3rd trimester or older human cortical GABAergic neurons include one or more of VIP, RELN, and CCK. In some embodiments, the genes that are normally upregulated in 3rd trimester or older human cortical GABAergic neurons include one or more of the genes listed in FIG. 1 L. In some embodiments, the electrophysiologically mature PV+GABAergic neurons comprise fast spiking PV+neurons.

[0092] In some embodiments, the PV+, SST+, and the VP+GABAergic neurons have mature morphology. In some embodiments, the mature morphology is an increased total dendrite length when compared to the PV+, SST+, and the VP+GABAergic neurons generated in vitro, i.e., in an organoid that was not transplanted into a non-human mammal. In some embodiments, the PV+GABAergic neurons have a total dendrite length of about 5mm to about 15mm, about 6mm to about 15mm, about 7mm to about 15mm, about 8mm to about 15mm, about 9mm to about 15mm, or about 10mm to about 15mm. In some embodiments, the SST+GABAergic neurons have a total dendrite length of about 2mm to about 6mm, about 3mm to about 6mm, about 4mm to about 6mm, about 5mm to about 6mm, about 2mm to about 5mm, or about 2mm to about 4mm. In some embodiments, the VP+GABAergic neurons have a total dendrite length of about 2mm to about 10mm, about 3mm to about 10mm, about 4mm to about 10mm, about 5mm to about 10mm, about 2mm to about 9mm, about 2mm to about 8mm, about 2mm to about 9mm, about 2mm to about 8mm, about 2mm to about 7mm, about 2mm to about 6mm, or about 2mm to about 6mm.Attorney Docket No: STAN-2232WOClient No: S24-410

[0093] The GABAergic neurons of the human neural tissue may have specific activities. Specific activities that the GABAergic neurons may have include, without limitation, maximal firing rates, resting membrane potential, capacitance, action potential half width, input resistance, spontaneous inhibitory postsynaptic current frequency, etc. For instance, the GABAergic neurons of the human neural tissue may have a maximal firing rate between about 10Hz to about 150Hz such as between about 20Hz to about 120Hz, between about 30Hz to about 120Hz, about 10Hz to about 140Hz, between about 20Hz to about 140Hz, between about 30Hz to about 140Hz, 10Hz to about 130Hz, between about 20Hz to about 130Hz, between about 30Hz to about 130Hz, 10Hz to about 120Hz, between about 20Hz to about 120Hz, or between about 30Hz to about 120Hz.ln some embodiments, the GABAergic may have an average maximal firing rate of about 10Hz, about 15Hz, about 20Hz, about 25Hz, about 30Hz, about 35Hz, about 40Hz, about 45Hz, about 50Hz or greater than about 50Hz.

[0094] In some embodiments, the GABAergic neurons of the human neural tissue may have a resting membrane potential between about -40mV to about -90mV, between about -40mV to about -80mV, between about -40mV to about -70mV, between about -40mV to about - 60mV, between about -50mV to about -90mV, between about -50mV to about -80mV, between about -50mV to about -70mV, between about -60mV to about -90mV, or between about -60mV to about -80mV. In some embodiments, the GABAergic neurons may have an average resting membrane potential of about -55mV, about -60mV, about -65mV, about -70mV, about -75mV, about -80mV, about -85mV, about -90mV, or greater than about -90mV.

[0095] In some embodiments, the GABAergic neurons of the human neural tissue may have a capacitance between about 50pF to about 1500pF, about 60pF to about 1500pF, about 70pF to about 1500pF, about 80pF to about 1500pF, about 90pF to about 1500pF, about 100pF to about 1500pf, about 150pF to about 1500pf, about 200pF to about 1500pf, about 250pF to about 1500pf, about 300pF to about 1500pf, about 350pF to about 1500pf, about 400pF to about 1500pf, about 400pF to about 1500pf, about 450pF to about 1500pf, about 500pF to about 1500pf, 50pF to about 1400pF, about 60pF to about 1400pF, about 70pF to about1400pF, about 80pF to about 1400pF, about 90pF to about 1400pF, about 100pF to about1400pf, about 150pF to about 1400pf, about 200pF to about 1400pf , about 250pF to about1400pf, about 300pF to about 1400pf, about 350pF to about 1400pf , about 400pF to about1400pf , about 400pF to about 1400pf, about 450pF to about 1400pf, about 500pF to about1400pf , 50pF to about 1300pF, about 60pF to about 1300pF, about 70pF to about 1300pF, about 80pF to about 1300pF, about 90pF to about 1300pF, about 10OpF to about 1300pf, about 150pF to about 1300pf , about 200pF to about 1300pf, about 250pF to about 1300pf , about 300pF to about 1300pf, about 350pF to about 1300pf, about 400pF to about 1300pf , about 400pF to about 1300pf, about 450pF to about 1300pf, about 500pF to about 1300pf ,Attorney Docket No: STAN-2232WOClient No: S24-41050pF to about 1200pF, about 60pF to about 1200pF, about 70pF to about 1200pF, about 80pF to about 1200pF, about 90pF to about 1200pF, about 100pF to about 1200pf, about 150pF to about 1200pf, about 200pF to about 1200pf , about 250pF to about 1200pf , about 300pF to about 1200pf, about 350pF to about 1200pf , about 400pF to about 1200pf , about 400pF to about 1200pf, about 450pF to about 1200pf, about 500pF to about 1200pf, 50pF to about 1 10OpF, about 60pF to about 1100pF, about 70pF to about 1 10OpF, about 80pF to about1 10OpF, about 90pF to about 1 10OpF, about 100pF to about 1 10Opf, about 150pF to about1 10Opf, about 200pF to about 110Opf, about 250pF to about 1 10Opf, about 300pF to about1 10Opf, about 350pF to about 110Opf, about 400pF to about 1 10Opf, about 400pF to about1 10Opf, about 450pF to about 110Opf, about 500pF to about 110Opf, 50pF to about 1000pF, about 60pF to about 1000pF, about 70pF to about 1000pF, about 80pF to about 1000pF, about 90pF to about 10OOpF, about 10OpF to about 10OOpf, about 150pF to about 10OOpf, about 200pF to about 10OOpf, about 250pF to about 10OOpf, about 300pF to about 10OOpf , about 350pF to about 10OOpf, about 400pF to about 10OOpf, about 400pF to about 10OOpf , about 450pF to about 10OOpf, or about 500pF to about 10OOpf. In some embodiments, The GABAergic neurons have an average capacitance of about 50pF, about 60pF, about 70pF, about 80pF, about 90pF, about 10OpF, about 110pF, about 120pF, about 130pF, about 140pF, about 150pF, about 160pF, about 170pF, about 180pF, about 190pF, about 200pF, about 210pF, about 220pF, about 230pF, about 240pF, about 250pF, about 260pF, about 270pF, about 280pF, about 290pF, about 300pF, about 310pF, about 320pF, about 330pF, about 340pF, about 350pF, about 360pF, about 370pF, about 380pF, about 390pF, about 400pF, about 410pF, about 420pF, about 430pF, about 440pF, about 450pF, about 460pF, about 470pF, about 480pF, about 490pF, about 500pF, about 510pF, about 520pF, about 530pF, about 540pF, about 550pF, about 560pF, about 570pF, about 580pF, or about 590pF.

[0096] In some embodiments, the GABAergic neurons of the human neural tissue may have an action potential half width between about 1 ms to about 5ms, about 1 ms to about 4.5ms, about 1 ms to about 4ms, about 1 ms to about 3.5ms, about 1 ms to about 3ms, about 1 ms to about 2.5ms, or about 1 ms to about 2ms. In some embodiments, the GABAergic neurons of the human neural tissue may have an average action potential half width of about 1 ms, about 1 .1 ms, 1.2ms, about 1 .3ms, about 1.4ms, about 1 .5ms, about 1 .6ms, about 1 .7ms, about 1 .8ms, about 1 .9ms, about 2.0ms, about 2.1 ms, about 2.2ms, about 2.3ms, about 2.4ms, about 2.5ms, about 2.6ms, about 2.7ms, about 2.8ms, about 2.9ms, or about 3.0ms.

[0097] In some embodiments, the GABAergic neurons of the human neural tissue may have an input resistance between about 0GQ to about 2GQ, about 0GQ to about 1 .9GQ, about 0GQ to about 1 .8GQ, about 0GQ to about 1 .7GQ, about 0GQ to about 1 .6GQ, about 0GQ to about 1 .5GQ, about 0GQ to about 1 .4GQ, about 0GQ to about 1 .3GQ, about 0GQ to about 1 .2GQ,Attorney Docket No: STAN-2232WOClient No: S24-410 about OGQ to about 1 .1 GQ, about OGQ to about 1.0GQ, about OGQ to about 0.9GQ, about OGQ to about 0.8GQ, about OGQ to about 0.7GQ, about OGQ to about 0.6GQ, about OGQ to about 0.5GQ, or about OGQ to about 0.5GQ. In some embodiments, the GABAergic neurons of the human neural tissue may have an average input resistance of about OGQ, about 0.05 GQ, about 0.1 GQ, about 0.15GQ, about 0.2GQ, about 0.25GQ, about 0.3GQ, about 0.35GQ, about 0.4GQ, about 0.45GQ, about 0.5GQ, about 0.55GQ, about 0.6GQ, about 0.65GQ, about 0.7GQ, about 0.75GQ, about 0.8GQ, about 0.85GQ, about 0.9GQ, or about 0.95GQ.

[0098] In some embodiments, the GABAergic neurons of the human neural tissue may have a spontaneous inhibitory postsynaptic current frequency between about 0.5Hz to about 15Hz, about 1 Hz to about 15Hz, about 2Hz to about 15Hz, about 3Hz to about 15Hz, about 4Hz to about 15Hz, about 5Hz to about 15Hz, about 1 Hz to about MHz, about 1 Hz to about 13Hz, about 1 Hz to about 12Hz, about 1 Hz to about 11 Hz, about 1 Hz to about 10Hz, about 2Hz to about 10Hz, about 3Hz to about 10Hz, about 4Hz to about 10Hz, or about 5Hz to about 10Hz. In some embodiments, the GABAergic neurons of the human neural tissue may have an average spontaneous inhibitory postsynaptic current frequency of about 1 Hz, about 2Hz, about 3Hz, about 4Hz, about 5HZ, about 6 Hz, about 7Hz, about 8Hz, about 9Hz, or about 10Hz.

[0099] Additional methods for producing non-human mammals have human neural tissue are known art and have been described by, for example, Internation Patent Publication WO 2023 / 239483 which is specifically incorporated by reference herein.METHODS FOR MODELING EPILEPSY DISORDERS

[0100] As summarized above, methods are provided for modeling an epilepsy disorder, the method including introducing a first human neural organoid into a central nervous system location of a newborn non-human mammal; introducing a second human neural organoid into the central nervous system location of the newborn non-human mammal; allowing the newborn non-human mammal to mature to produce the non-human mammalian animal model comprising a first human neural tissue; and characterizing the first human neural tissue to model the epilepsy disorder; wherein the first human neural organoid, the second human neural organoid, or the first and second human neural organoid are: a) produced from a cellular biological sample from a human who has the epilepsy disorder; or b) have a genetic mutation associated with the epilepsy disorder.

[0101] To model the epilepsy disorder, the first human neural organoid, the second human neural organoid, or the first and the second human neural may be produced from a cellular biological sample from a human who has the epilepsy disorder or may have a genetic mutation associated with the epilepsy disorder. In some embodiments, only the first human neuronalAttorney Docket No: STAN-2232WOClient No: S24-410 organoid is produced from a cellular biological sample from a human who has the epilepsy disorder or may have a genetic mutation associated with the epilepsy disorder. In some embodiments, only the second human neuronal organoid is produced from a cellular biological sample from a human who has the epilepsy disorder or may have a genetic mutation associated with the epilepsy disorder. In some embodiments, the first human neuronal organoid and the second human neural organoid are produced from a cellular biological sample from a human who has the epilepsy disorder or may have a genetic mutation associated with the epilepsy disorder. When the epilepsy disorder is the result of mosaicism, only the first human neural organoid or only the second human neural organoid is produced from a cellular biological sample from a human who has the epilepsy disorder or may have a genetic mutation associated with the epilepsy disorder such that one neural organoid represents the cells having the genetic background of pathogenic variant of the gene associated with the mosaic epilepsy disorder and one neural organoid represents the cells that do not have the genetic background of pathogenic variant of the gene associated with the mosaic epilepsy disorder.

[0102] The first human neural organoid produced from the cellular biological sample from an individual living with a neuropsychiatric disorder may be produced using any method deemed useful. In some embodiments, the first human neural organoid is produced by converting cells of the cellular biological sample into induced pluripotent stem cells; and differentiating the induced pluripotent stem cells into the first human neural organoid.

[0103] The epilepsy may be any epilepsy disorder that is deemed suitable for organoid culture. Epilepsy disorders that find use in the present disclosure include, with limitation, PCDH19-related encephalopathy, CDKL5 deficiency disorder, KCNQ2 developmental and epileptic encephalopathy, Rett’s syndrome, SCN2A-related epilepsy, Dravet syndrome, GABRA1 -related epileptic encephalopathy, GASRG2-related epilepsy, GRIN2D-related developmental and epileptic encephalopathy, benign familial neonatal-infantile seizures, benign familial neonatal seizures, childhood absence epilepsy, early infantile epileptic encephalopathy, febrile seizures, genetic (generalized) epilepsy with febrile seizures plus intractable childhood epilepsy, intractable childhood epilepsy with generalized tonic-clonic seizures, intellectual disability, infantile spasms, juvenile myoclonic epilepsy, Ohtahara syndrome, severe myoclonic epilepsy of infancy, etc. Additionally, epilepsy disorders as a result of mosaicism are of particular relevance. By “mosaicism” it means that some cells in the individual express a mutant or disease-associated variant of a protein or do not express the protein whereas other cells produce a healthy or non-disease-associated variant of a protein. The mosaicism may be caused by random X inactivation in females, i.e. , females that are heterozygous for the mutant or disease-associated variant, or mosaic expression in males dueAttorney Docket No: STAN-2232WOClient No: S24-410 to early somatic mutation. Epilepsy disorders associated with mosaicism include epilepsy disorders associated with pathogenic variants of CDKL5, CLCN4, SCN1 A, SCN2A, SLC35A2, GABRA1 , GRIN2B, KCNQ2, MECP2, and PCDH19.

[0104] In some embodiments, the epilepsy disorder is associated with a channelopathy. A range of different channelopathies find use in the present disclosure. Channelopathies of interest include channelopathies associated with pathogenic variants of a gene selected from the group consisting of: SCN1 A, SCN1 B, SCN2A, SCN3A, SCN8A, KCNQ2, KCNQ3, KCNMA1 , KCNA1 , KCNA2, KCNJII, KCNT1 , CACNA1 H, CACNA1A, CHRNA4, CHRNB2, CHRNA2, GABRA1 , GABRB2, GABRB3, GABRD, and GABRG2. Epilepsy disorders associated with channelopathies are known in the art and have been described by, for example, Spillane et al. (J Neurol Neurosurg Psychiatry. 2015 Nov 1 1 ;87(1 ):37-48) which is specifically incorporated by reference herein.

[0105] The epilepsy disorders of the present disclosure may be characterized in a variety of ways. In some embodiments, the characterizing comprises measuring the neuronal morphology of the first human neural tissue. In these embodiments, a number of different neuronal morphological features can be measured. For instance, the morphological features that can be measured include, without limitation, soma diameter, dendrite number, dendrite length, dendrite density, dendritic spine number, dendritic spine length, dendritic spine density, axon length, etc. The neuronal morphology may be measured in a variety of ways. For example, the measuring includes, without limitation, histologically staining the first human neural tissue, antibody staining the first human neural tissue, expressing a detectably labeled protein in the first human neural tissue, etc.

[0106] Histological stains that find use in the present disclosure include, without limitation, H&E staining, Nissl staining, Luxol-fast blue staining, Kluver-Barrera staining, Bodian silver staining, Holzer staining, Gallyas-Braak staining, thionine staining, Weil-Myelin staining, Solochrome staining, Peris staining, Fluoro-Jade staining, Congo Red staining, thioflavine S staining, amino cupric silver staining, Neutral Red Counter staining, cupric silver staining, Campbell-Switzer Alzheimer staining, autometallography staining, etc. Antibody stains that find use in the present disclosure include, without limitation, 4G8, 6E10, Ap1-40, Ap1 -42, alpha synuclein, Asyn-pSer129, AT8, BrdU + hematoxylin, calbindin, caspase-3, caspase-9, cathepsin-D, CD68, c-fos, ChAT + Nissl, doublecortin, endoglin, ferritin, GAD-67, GFAP, GFP, HuIgG, Iba1 , Ki-67, LAMP1 , luciferase, MAP-2, MBP, mDectin, NeuN, Nestin, Oligo2, Orexin A, parvalbumin, p-c-jun, P.U.1 , RGMa, S830, SMI-71 , SMI-99, somatostatin, STEM-101 , TDP- 43, TH, TMEM1 19, TPH, etc. The first human neural tissue of the present disclosure may also be antibody stained or immunostained for reasons other than histology. For instance, the first human neural tissue may be antibody stained or immunostained for a number of reasonsAttorney Docket No: STAN-2232WOClient No: S24-410 including, without limitation, to determine the presence or absence of a protein, to determine the differential expression of a protein, to determine the localization of a protein, etc.

[0107] A detectably labeled protein of the present disclosure may be any protein labeled with a detectable moiety. Detectable moieties may include, without limitation, a fluorescent protein, a luminescent protein, etc. For instance, the marker protein may be a fluorescent protein or a luminescent protein. Non-limiting examples of useful fluorescent proteins include but are not limited to GFP, EBFP, Azurite, Cerulean, mCFP, Turquoise, ECFP, mKeima-Red, TagCFP, AmCyan, mTFP, TurboGFP, TagGFP, EGFP, TagYFP, EYFP, Topaz, Venus, mCitrine, TurboYFP, mOrange, TurboRFP, tdTomato, TagRFP, dsRed2, mRFP, mCherry, mPlum mRaspberry, mScarlet, etc. Examples of luminescent proteins, include without limitation, Cypridinia luciferase, Gaussia luciferase, Renilla luciferase, Phontinus luciferase, Luciola luciferase, Pyrophorus luciferase, Phrixothrix luciferase, etc.

[0108] In some embodiments, the characterizing comprises measuring intrinsic electrophysical properties of the first human neural tissue. Any intrinsic electrophysical properties of the first human neural tissue may be measured. Non-limiting examples of electrophysical properties that may be measured include, without limitation, resting membrane potential, depolarization threshold, membrane capacitance, maximal firing rates, minimum firing rates, etc. Intrinsic electrophysical properties may be measured using a multitude of techniques including, without limitation, sharp electrodes, patch-clamp, fluorescent bioelectricity reporters, EEG, etc.

[0109] In some embodiments, the characterizing comprises measuring gene expression in the first human neural tissue. The expression of any gene may be measured, particularly genes related to neural development or neural function. The expression of a single gene, sets of genes, the transcriptome, or the proteome may be measured. The expression of genes may be measured in the form or mRNA or protein.

[0110] In some embodiments, the characterizing comprises axon tracing of the first human neural tissue. The axons may be traced in any way deemed useful. The axon tracing may be retrograde tracing or anterograde tracing. The axon tracing may be performed with the use of a viruses, protein or small molecule. Non-limiting examples of viruses that facilitate anterograde axon tracing include, without limitation, herpes simplex virus 1 (HSV-1), HSV-1 strain H129, rhabdoviruses, etc. Non-limiting examples of viruses that facilitate retrograde axon tracing include, without limitation, rabies, pseudorabies, glycoprotein, deleted rabies, etc. Non-limiting examples of proteins and small molecules that facilitate anterograde axon tracing include, without limitation, Phaseolus vulgaris-leucoagglutinin, wheat germ agglutin, dextran amines, etc. Non-limiting examples of proteins and small molecules that facilitate retrograde axon tracing include, without limitation, horse radish peroxidase (HRP), wheat germ agglutin,Attorney Docket No: STAN-2232WOClient No: S24-410 cholera toxin subunit B, hydroxystilbamidine, Fast Blue, Diamidino Yellow, True Blue, the carbocyanines Dil and DiO, fluorescent lax microspheres, etc. Non-limiting examples of proteins and small molecules that facilitate retrograde axon tracing include, without limitation, horse radish peroxidase (HRP), wheat germ agglutin, cholera toxin subunit B, hydroxystilbamidine, Fast Blue, Diamidino Yellow, True Blue, the carbocyanines Dil and DiO, fluorescent lax microspheres, etc. Other viruses, proteins and small molecules that facilitate axon tracing have been described in the art, for example, in Xu, X. et al. (Neuron. 2020 Sep 23;107(6):1029-1047) and in Saleeba, C. et al. (Front Neurosci. 2019 Aug 27;13:897), each of which herein specifically incorporated by reference.

[0111] In some embodiments, the characterizing includes measuring neural activity using EEG. In some embodiments, the neural activity is power spectral density. In some embodiments, the neural activity is total power of one or more of an alpha, beta, gamma, delta, or theta spectral band of the first human neural tissue. In some embodiments, the neural activity is spontaneous neural activity of glutamatergic neurons.

[0112] In some embodiments, the method of modeling an epilepsy disorder further comprises characterizing the non-human mammalian animal model. In these embodiments, characterizing the non-human mammalian animal model may include, without limitation, assaying behavioral responses to tasks, memory responses to tasks, motor responses to tasks, sensory responses to tasks, etc. Nonlimiting examples of behavioral tests that find use in the present disclosure include, without limitation, Acoustic Startle Response, Elevated Plus Maze, Elevated Zero Maze, Foot Placement Analysis, Forced Exercise I Walking, Forced Swim Test in Mice, Hot Plate Test, Hargreaves Test, Grid Walking Test, Water Maze, Vertical Screen Test, Tail Suspension Test, Rota Rod Test, Resident Intruder, Passive Avoidance Test, Oxymax System, etc. Memory tests that find use in the present disclosure include, without limitation, Water Maze, Passive Avoidance Test, Novel Object Recognition, etc. Nonlimiting examples of motor tests that find use in the present disclosure include, without limitation, Acoustic Startle Response, Balance Beam, Bar Holding Test, Analysis of Locomotion Using CatWalk Test, Foot Placement Analysis, Forced Exercise / Walking, Forced Swim Test in Mice, Grid Walking Test, Vertical Screen Test, Rota Rod Test, Neurological Exam, Locomotor Activity Test, etc. Sensory tests that find use in the present disclosure include, without limitation, Acoustic Startle Response, Cold Plate Test, Hot Plate Test, Hargreaves Test, Neurological Exam, Mechanical Sensitivity (Von Frey), etc. The above disclosed tests are well known in the art and have been previously described in, for example, Buccafusco, J. et al. (Methods of Behavior Analysis in Neuroscience. 2nd edition. Boca Raton (FL): CRC Press / Taylor & Francis; 2009.) in addition to others, herein specifically incorporated by reference.Attorney Docket No: STAN-2232WOClient No: S24-410

[0113] In some embodiments, the method further includes inducing a seizure before or after the characterizing. When a seizure is induced after the characterizing, the characterizing may be repeated following the seizure to determine any changes in the results in the characterizing that may be the result of the seizure. The seizure may be induced by any method that is relevant to the epilepsy disorder. For instance, the seizure may be a temperature-induced seizure, a light-induced seizure, a sound-induced seizure, an electrical-induced seizure, a chemical-induced seizure, an injury-induced seizure, etc. When the seizure is a temperature- induced seizure, the seizure may be induced my elevating the temperature of the non-human animal to a temperature of about 40C, about 40.5C, about 41 C, about 41.5C, about 42C, about 42.5C, about 43C, about 43.50, about 440, about 44.50, about 45C or greater than about 450. When the seizure is a light-induced seizure, the seizure may be induced by using optogenetics stimulation of the human neural tissue or may be induced by subjecting the non- human mammal to strobing lights. When the seizure is a sound-induced seizure, the seizure may be an audiogeneic seizure such as those disclosed in Kandratavicius et al. (Neuropsychiatr Dis Treat. 2014 Sep 9;10:1693-1705) which is specifically incorporated by reference herein. When the seizure is an electrical-induced seizure, the seizure may be due to maximal electroshock, electrical stimulation of the amygdala, etc. When the seizure is a chemical-induced seizure, the chemical-induced seizure includes, without limitation, intraperitoneal injection or caudal vein injection of pentylenetetrazol (PTZ), intravitreal injection of N-methyl-D-aspartic acid (NMDA), inhalation of flurothyl, intraperitoneal injection of NMDA, subcutaneous or intraperitoneal injection of kainic acid (KA), intracerebro-ventricular, intraamygdala, or intrahippocampal injection of KA, subcutaneous injection of KA and lorazepam, graded intraperitoneal injections of KA, intraperitoneal injection of pilocarpine, intrahippocampal or intracerebro-ventricular injection of pilocarpine, intramuscular injection of coriaria lactone, intrahippocampal injection of penicillin, intracortical injection of iron ion, etc. When the seizure is an injury-induced seizure, the injury-induced seizure includes, without limitation, fluid percussion injury, impact-acceleration, closed head injury, intracerebro- ventricular injection of Theiler's virus, intrahippocampal injection of tetanus toxin, controlled cortical impact, hypoxia / ischemia, etc. Methods of inducing seizures in non-human mammals are known in the art and have been described by, for example, Wang et al. (Aging Dis. 2022 Feb 1 ;13(1 ):215-231 ) which is specifically incorporated by reference herein.

[0114] In some embodiments, the method further includes stimulating the human neural tissue. The stimulating can be a range of different stimulations including, without limitation, electrical stimulation, light stimulation, etc. In some embodiments, the stimulation is electrical stimulation. In some embodiments, the stimulating is light stimulation. When the stimulating is light stimulation, the human neural tissue may be stimulated with optical fibers. When theAttorney Docket No: STAN-2232WOClient No: S24-410 stimulating is light stimulation, one or more neurons of the human neural tissue may contain a light activable polypeptide.

[0115] The activity of the human neural tissue may be altered as a result of the activation of the light activatable polypeptide present in the human neural tissue. The light activable polypeptide may either depolarize or hyperpolarize the neurons in the human neural tissue. Non-limiting examples of light activatable polypeptides capable of mediating a hyperpolarizing current can be found, e.g., in International Patent Application No. PCT / US201 1 / 028893; U.S. Patent No. 9,175,095. Non-limiting examples of hyperpolarizing light-activatable polypeptides include NpHr, eNpHr2.0, eNpHr3.0, eNpHr3.l or GtR3. Non-limiting examples of depolarizing light activatable polypeptides include "C1 V1", channel rhodopsin 1 (ChR1 ), VChR1 , channel rhodopsin 2 (ChR2). Additional information regarding other light-activated cation channels, anion pumps, and proton pumps can be found in U.S. Patent Application Publication Nos: 2009 / 0093403; and International Patent Application No: PCT / US2011 / 028893.

[0116] The optical fibers of the present disclosure may be any optical fiber that is capable of transmitting multiple light wavelengths. In some embodiments, the light wavelength is red light from about 625 nm to about 740 nm. In some embodiments, the light wavelength is orange light from about 590 nm to about 625 nm. In some embodiments, the light wavelength is yellow light from about 565 nm to about 590 nm. In some embodiments, the light wavelength is green light from about 520 nm to about 565 nm. In some embodiments, the light wavelength is blue light from about 445 nm to about 520 nm. In some embodiments, the light wavelength is indigo light from about 425 nm to about 445 nm. In some embodiments, the light wavelength is violet light from about 380 nm to about 425 nm.

[0117] In some embodiments, the method further comprises introducing a third human neural organoid and a fourth human neural organoid into a second central nervous system location wherein the third human neural organoid and a fourth human neural organoid is derived from an individual that does not have an epilepsy disorder. In some embodiments, the third human neural organoid and a fourth human neural organoid is the same as the first human neural organoid and the second human neural organoid except that it is derived from an individual that does not have an epilepsy disorder. For example, if the first human neural organoid is a cortical organoid and the second human neural organoid is a subpallial organoid then the third human neural organoid is a cortical organoid and the fourth human neural organoid is a subpallial organoid. In some embodiments, the third and fourth human neural organoid are different from the first and second human neural organoid. In this instance, the first human neural organoid may be a cortical organoid and the second human neural organoid may be a subpallial organoid then the third and fourth human neural organoid may be a midbrain organoid, a striatal organoid, or any other neural organoid that is not a cortical organoid or aAttorney Docket No: STAN-2232WOClient No: S24-410 subpallial organoid. In some embodiments, the second central nervous system location is the same as the first central nervous system location but is in the opposite brain hemisphere. In some embodiments, the second central nervous system location is different from the first central nervous system location. In these embodiments, if the first central nervous system location is the frontal cortex then the second central nervous system location is any other central nervous system location that is not the frontal cortex such as the motor cortex, somatosensory cortex, parietal cortex, occipital cortex, temporal cortex, spinal cord or the cerebellum.

[0118] In embodiments where the third and fourth human neural organoid are introduced into a second central nervous system location, the third and fourth human neural organoid produces a second human neural tissue. In these embodiments, the method further comprises characterizing the second human neural tissue. The second human neural tissue is used as a control or reference to compare to the first human neural tissue. The results of the characterizing of the first human neural tissue may then be compared to the results of characterizing the second human neural tissue such that the differences between the first and second human neural tissue may be determined.METHODS FOR DETERMINING THE EFFECTIVENESS OF A DRUG ON AN EPILEPSY DISORDER

[0119] Also disclosed herein are methods for determining the effectiveness of a candidate agent on an epilepsy disorder, the method comprising administering the candidate agent to the non-human mammalian animal model comprising human neural tissue produced using the methods for modeling an epilepsy disorder; assaying the human neural tissue; and comparing the results of the assaying with mammals administered a control agent that is not the candidate agent. The non-human mammals of the present disclosure not only serve as models for epilepsy disorders but may also be used for the development and screening of therapeutics to treat the epilepsy disorders. A candidate agent may be a chemical, small molecule, a protein, a genetic agent or an antibody. In some embodiments, the candidate agent is administered systemically in the non-human mammalian animal model. In some embodiments, the candidate agent is administered locally at the site of the human neural tissue.

[0120] The assaying of the human neural tissue may involve determining the effect a candidate agent has on the neuronal morphology, intrinsic electrophysical properties, gene expression, immunostaining, axon tracing, power spectral density, total power of one or more of an alpha, beta, gamma, delta, or theta spectral band of the first human neural tissue, and / or intracellular calcium levels of the neurons within the human neural tissue. As describedAttorney Docket No: STAN-2232WOClient No: S24-410 herein, the human neural tissue is anatomically integrated into the non-human mammalian animal model and this human neural tissue and the neurons contained therein are functional. The assaying may therefore involve determining whether a candidate agent is able to alter the neuronal morphology, intrinsic electrophysical properties, gene expression, immunostaining and / or axon tracing of the human neural tissue derived from an individual having a neuropsychiatric disorder such that these features more closely resemble that of human neural tissue that is derived from an individual that does not have a neuropsychiatric disorder. In some embodiments, the assay may include inducing a seizure as discussed above.

[0121] As also described herein, various diseases and disorders are associated with neural dysfunction. Accordingly, the assays described herein may find particular utility where the human neural tissue is derived from an individual having an epilepsy disorder, such as PCDH19-related encephalopathy, CDKL5 deficiency disorder, KCNQ2 developmental and epilepsy encephalopathy, Rett’s syndrome, SCN2A-related epilepsy, Dravet syndrome, GABRA1 -related epilepsy encephalopathy, GABF?G2-related epilepsy, GRIN2D-related developmental and epilepsy encephalopathy, benign familial neonatal-infantile seizures, benign familial neonatal seizures, childhood absence epilepsy, early infantile epilepsy encephalopathy, febrile seizures, genetic (generalized) epilepsy with febrile seizures plus intractable childhood epilepsy, intractable childhood epilepsy with generalized tonic-clonic seizures, intellectual disability, infantile spasms, juvenile myoclonic epilepsy, Ohtahara syndrome, severe myoclonic epilepsy of infancy, epilepsy disorders associated with mosaicism, epilepsy disorders associated with channelopathies, etc., etc. Candidate agents that are able to restore the functionality of defects or abnormalities identified in the human neural tissue comprising the neuropsychiatric disorder may have therapeutic utility in the treatment of said disorder.

[0122] Neural activity causes rapid changes in intracellular free calcium. Calcium imaging assays that exploit this can therefore be used to determine the functionality of the anatomically integrated neuronal circuits of the human neural tissue. For example, detected changes in calcium levels with a cell or cluster of cells would indicate a change in activity in said cells. This may involve modifying the human neural organoids that the human neural tissue was derived from to contain genetically-encoded calcium indicator proteins, such those proteins that include the fluorophore sensor GCaMP and imaging those cells. GCaMP comprises a circularly permuted green fluorescent protein, a calcium-binding protein calmodulin (CaM) and CaM-interacting M13 peptide, where brightness of the GFP increases upon calcium binding. Further details about calcium imaging assays are described in Chen et al. (2013) Nature 499(7458): 295-300. Other calcium imaging assays include Fura-2 calcium imaging; Fluo-4 calcium imaging, and Cal-590 calcium imaging.Attorney Docket No: STAN-2232WOClient No: S24-410

[0123] In some embodiments, the methods further comprise assaying the non-human mammalian animal model comprising human neural tissue to determine the effect the candidate agent has on the behavioral responses to tasks, memory responses to tasks, motor responses to tasks, sensory responses to tasks. The assaying may also therefore involve determining whether a candidate agent is able to alter the behavioral responses to tasks, memory responses to tasks, motor responses to tasks, sensory responses to tasks such that these responses more closely resemble that of a non-human mammalian animal model comprising human neural tissue that is derived from an individual that does not have a neuropsychiatric disorder.

[0124] In some embodiments, the non-human mammalian animal model comprises human neural tissue derived from a third and a fourth human neural organoid introduced into the second central nervous system location wherein the third and fourth human neural organoid derived from an individual that does not have an epilepsy disorder. In these embodiments, the human neural tissue that is produced from the first and second human neural organoid that is derived from an individual having an epilepsy disorder is referred to as the first human neural. In these embodiments, the results of the assaying of the human neural tissue derived from an individual having an epilepsy disorder may be compared to results of assaying the human neural tissue, e.g. the second human neural tissue, that is derived from an individual that does not have an epilepsy disorder. The results of the assaying of the first human neural tissue may then be compared to the results of assaying the second human neural tissue such that the differences between the first and second human neural tissue may be determined both in response to the candidate agent and to a control agent.

[0125] 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.

[0126] Included are pharmacologically active drugs, genetically active molecules, etc. Compounds of interest include chemotherapeutic agents, anti-inflammatory agents, hormonesAttorney Docket No: STAN-2232WOClient No: S24-410 or hormone antagonists, ion channel modifiers, and neuroactive agents. Exemplary of pharmaceutical agents suitable forthis 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.

[0127] 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.

[0128] The term samples also include the fluids described above to which additional components have been added, for example components that affect the ionic strength, pH, total protein 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.

[0129] 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.

[0130] 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 geneticAttorney Docket No: STAN-2232WOClient No: S24-410 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.

[0131] Introduction of an expression vector encoding a polypeptide can be used to express the encoded product in human neural tissue 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. The expression vector may be a viral vector, e.g. adeno-associated virus, adenovirus, herpes simplex virus, retrovirus, lentivirus, alphavirus, flavivirus, rhabdovirus, measles virus, Newcastle disease virus, poxvirus and picornavirus vectors.

[0132] 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.

[0133] 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 multipleAttorney Docket No: STAN-2232WOClient No: S24-410 parameters e.g. the neuronal morphology, intrinsic electrophysical properties, gene expression, immunostaining and / or axon tracing of the human neural tissue. The data will include the results from assay combinations with the agent(s), and may also include one or more of a control human neural tissue (i.e. human neural tissue produced from a human neural organoid derived from an individual that does not have a neuropsychiatric disorder, the human neural tissue derived from an individual having a neuropsychiatric disorder), 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.

[0134] The dataset may be prepared from values obtained by measuring parameters in the presence and absence of different stimuli, e.g. a visual stimuli, a touch stimuli, a taste stimuli, a smell stimuli , a auditory stimuli, 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, neuronal morphology, intrinsic electrophysical properties, gene expression, immunostaining and / or axon tracing of the human neural tissue. Desirably, the results are normalized against a standard, usually a "control value or state," to provide a normalized data set such as results obtained from human neural tissue derived from an individual that does not have a neuropsychiatric disorder or unstimulated human neural tissue derived from an individual that has a neuropsychiatric disorder. 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 human neural tissues and assay conditions.

[0135] 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.

[0136] 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.

[0137] Mathematical systems can be used to compare datasets, and to provide quantitative measures of similarities and differences between them. For example, the datasets can beAttorney Docket No: STAN-2232WOClient No: S24-410 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.

[0138] 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.

[0139] 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 human neural tissue 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 human neural tissue 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.

[0140] 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.

[0141] 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 human neural tissues of the same type. A data point may be quantitative, semi-quantitative, or qualitative, depending on the nature of the parameter.Attorney Docket No: STAN-2232WOClient No: S24-410

[0142] 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 reference readout. 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.

[0143] 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.

[0144] 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.

[0145] The data from human neural tissue treated with specific drugs known to interact with particular targets or pathways provide a more detailed set of classification readouts. Data generated from human neural tissues that are genetically modified using over-expression techniques and anti-sense techniques, permit testing the influence of individual genes on the phenotype.

[0146] A preferred knowledge database contains reference data from optimized panels of human neural tissues, 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 human neural tissue types of interest are excluded or included or quantitatively altered in, for example, concentration or time of exposure, etc.

[0147] 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, and neurobiology. With respect to tissue culture and embryonic stem cells, the reader may wish to refer to T eratocarcinomas 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).Attorney Docket No: STAN-2232WOClient No: S24-410

[0148] 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 & Loewy eds., 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.

[0149] Each publication cited in this specification is hereby incorporated by reference in its entirety for all purposes.

[0150] 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.EXPERIMENTALIntroduction

[0151] The mammalian cerebral cortex contains glutamatergic neurons produced in the pallium and GABAergic neurons that are mostly born in the subpallium (1-4). After migration throughout the pallium, GABAergic neurons undergo maturation allowing for the development of canonical cortical network activity driven by the interaction of glutamatergic and GABAergic neurons (1 , 5, 6). Imbalances in this network activity are thought to contribute to disease (7, 8), including seizure disorders (9, 10), but lack of experimental access to the developing human brain has limited an understanding of these processes.

[0152] Recent advances in human induced pluripotent stem (hiPS) cell technology combined with self-organizing neural organoid methods facilitate access to features of human cortical development (11-15). For example, generation of human cortical organoids (hCO) resembling the pallium recapitulate the intrinsic, sequential generation of neurons and glial cells (16-21). However, isolated hCO lack long-range interactions with brain-wide circuits as well as short-Attorney Docket No: STAN-2232WOClient No: S24-410 range interactions with GABAergic neurons. The human forebrain assembloid (hFA) platform was developed to ameliorate the short-range deficits, whereby hCO are integrated with human subpallial organoids (hSO) that generate GABAergic neurons (16, 22, 23). Transplantation of hiPS cell-derived neurons into rodents may overcome the lack of long-range connectivity: grafted neurons receive neuronal inputs, sensory-evoked electrical activity, and are vascularized (24-30). However, the assembly of glutamatergic hCO and GABAergic hSO with a functionally relevant readout has not been achieved in vivo. Given that GABAergic neurons undergo maturation and defects in cortical microcircuits contribute to epilepsy and other excitation / inhibition ratio imbalances (1 , 25), an in vivo preclinical model containing a mature human cortical microcircuit could facilitate advanced interrogation of network activity and disease modeling. Here, we develop a transplanted human forebrain assembloid (t-hFA) model through engraftment of both glutamatergic hCO and GABAergic hSO. We demonstrate reliable t-hFA growth, maturation of intrinsic cellular properties, and advanced in vivo electrophysiological dynamics. Critically, the t-hFA platform facilitated modeling of mosaic PCDH19 loss in human neurons in vivo, revealing an increase in seizure susceptibility to hyperthermia.Results

[0153] Characterization of transplanted human forebrain assembloids. To generate assembloids in vivo and investigate the interaction between human-derived cortical glutamatergic and GABAergic neurons in vivo, hSO were virally labeled with a lentiviral construct driving EGFP expression under a validated Dlxi1 / 2b enhancer (16, 31 , 32). An intact hSO was stereotaxically transplanted into the primary somatosensory cortex (S1) of athymic rats followed by an intact hCO, in the same injection (FIG. 1 A). This procedure was performed at P3-7 because innervation of rat barrel cortex is still ongoing (33); indeed, it was previously showed that transplantation of hCO at P3-7 allows integration into the host’s cortical and subcortical circuitry (34). To quantify the success rate of transplantation, T2-weighed magnetic resonance imaging (MRI) was performed 2 months post-transplantation of rats with t-hFA or transplanted hCO (t-hCO, both groups received a total of two organoids) (FIG. 1 B). Successful transplantation was observed for -77% t-hCO surgeries (N=44 rats using organoids from 3 genetically distinct hiPS cell lines) and -83% t-hFA surgeries (N=88 rats using organoids form 3 genetically distinct hiPS cell lines, FIG. 1 C). The graft volume in t-hCO condition is larger than t-hFA, but both grafts grow at a similar rate (-10-fold increase from 2 to 6 months posttransplant, FIG. 1 D, no ANOVA interaction). Despite the dramatic growth of the graft, body weight measurements, open field activity, and the novel object recognition memory task did not demonstrate adverse effects of transplantation on the rat hosts, although a mild increaseAttorney Docket No: STAN-2232WOClient No: S24-410 in locomotion in the t-hCO condition was observed during minutes 2 and 3 (FIG. 5). To characterize the cytoarchitecture and spatial distribution of grafted GABAergic neurons, immunohistochemistry was performed at 6-months post-transplantation. In histological cryosections, the t-hFA graft is positive for human nuclear antigen (HNA+) and contains hSO- derived Dlxi1 / 2b::EGFP+ cells (FIG. 1 E-F). Immunostaining within the graft revealed host- derived vasculature (rat endothelial cell antigen 1 : RECA-1 ) and HNA-, host-derived microglia (ionized calcium-binding adaptor molecule 1 : IBA1 , FIG. 1 F). The density of Dlxi1 / 2b::EGFP+ cells was quantified along the anterior-posterior axis of the t-hFA (FIG. 1G). The density of Dlxi1 / 2b::EGFP+ cells roughly followed a normal distribution, with -100 cells per mm2 of graft within the middle quartiles.

[0154] To comprehensively characterize the cellular composition of t-hFA, single-nucleus RNA sequencing (snRNA-seq) was performed at around 8 months post-differentiation. Quality filtering yielded 30,705 human single-nucleus profiles (FIG. 1 H and FIG. 6A-D). Transcriptom ic reference mapping with adult cortical cell types (35), along with expression patterns of canonical cell-type markers, identified clusters of major cortical cell classes, including both deep and superficial layer glutamatergic neurons, cycling glial progenitors, oligodendrocyte and astrocyte lineage cells, as expected from our prior characterization of t- hCO (FIG. 1 H, FIG. 6B,D,E). Importantly, all t-hFA samples (derived from n = 3 hiPS cell lines) contained clusters of cells expressing markers of telencephalic GABAergic neurons (GAD2, FOXG1 , and DLX1 / 2: FIG. 6D), that were not present in t-hCO (24). snRNA-seq of stage- matched hSO also yielded telencephalic GABAergic neurons, in addition to oligodendrocyte and astrocyte lineage cells (FIG. 6G-I).

[0155] Maturation and diversification of GABAergic neurons in t-hFA. To evaluate the effect of transplantation on hSO-derived GABAergic neurons of the t-hFA, stage- and cell line- matched GABAergic neurons were compared from in vitro hSO and t-hFA. Differential gene expression analysis highlighted over 400 upregulated genes (fold-change >2 and adjusted p- value <0.05) in t-hFA GABAergic neurons compared to hSO, which were enriched in gene sets associated with neuronal maturation, such as synapse and dendrite localization as well as voltage-gated channels (FIG. 6F). To further assess how the advanced maturation state of t-hFA GABAergic neurons relates to the developing human brain, transcriptom ic comparisons to recent work were performed characterizing the molecular signatures of cortical GABAergic neurons across developmental stages (37). Genes upregulated in t-hFA compared to hSO GABAergic neurons were enriched for sets of genes whose expression began later in development, including genes upregulated in 3rd trimester and early postnatal human cortical GABAergic neurons (FIG. 1 K). Interestingly, these included canonical GABAergic neuron subtype markers such as VIP, RELN, and CCK (FIG. 1 L), suggesting further refinement ofAttorney Docket No: STAN-2232WOClient No: S24-410GABAergic neuron identity in vivo. To further explore this, transcriptomic mapping of t-hFA GABAergic neurons to adult cortical GABA neuron subtypes was performed (35), and found clusters of t-hFA GABAergic neurons that contained high similarity to most canonical subclasses including the MGE-derived PVALB and SST subtypes and the CGE-derived VIP, LAMP5, and SNOG subtypes (FIG 1 l-J). The existence of PVALB, SST, and VIP subtypes in t-hFA was verified through immunohistochemistry (FIG. 2A-B). Importantly, hSO GABAergic neurons appeared more limited in subtype identity (FIG. 6I), suggesting transplantation enhances the molecular maturation state, and consequently diversity, of GABAergic neurons.

[0156] It was tested whether functional maturation of cellular features accompanied the transcriptional maturation. The morphology of stage-matched Dlxi1 / 2b::EGFP+ neurons from intact, in vitro hSO and acute slices of t-hFA were compared through reconstruction of biocytin- f i lied cells. It was found that transplantation increased the total length (>5 fold) and complexity of dendritic morphology (FIG. 2A-D, FIG. 7A) leading to study of the electrophysiological properties of Dlxi1 / 2b::EGFP+ neurons from intact in vitro hSO and t-hFA acute slices using whole-cell patch clamp electrophysiology (FIG. 2E, FIG. 7A). Compared to in vitro, Dlxi1 / 2b::EGFP+ cells from transplanted samples have a significantly increased maximal firing rate, decreased action potential half-width, a more hyperpolarized resting membrane potential, decreased input resistance, and increased capacitance (FIG. 2F-H, FIG. 7B-F). Interestingly, these cellular measures tend to progress in the same direction over the course of development; for example, the maximal firing rate and capacitance increase while input resistance and resting membrane potential decrease in mouse neocortical fast-spiking cells (38). Together, these data demonstrate that transplantation of hFA enhances the transcriptomic, structural, and electrophysiological maturation state of GABAergic neurons.

[0157] In vivo electrophysiological readouts of t-hFA. Having characterized the properties of single neurons at the level of transcriptom ics, electrophysiology, and structure, it was sought to obtain a neurophysiological readout of grafted hiPS cell-derived neurons at the level of brain networks. At ~6 months post transplantation, T2 weighted MRI was performed to localize the graft. One day later, the graft was stereotaxically targeted and contralateral somatosensory rat cortex with intracranial electrodes, and bilateral frontal screw electrodes were placed over motor cortex as a within-animal control. One day after surgery, the localization of the electrodes was confirmed through micro-computed tomography (CT) scanning and MRI / CT colocalization (FIG. 3A-B). An identical procedure in t-hFA, t-hCO, and non-transplanted rat controls was performed and allowed rats to recover 7-10 days before a 4-hour baseline recording session (FIG. 3C-E). The absolute power spectral density (PSD) was calculated from the electrode directly placed into the t-hCO, t-hFA, or control rat cortex (FIG. 3F), and then quantified the total power in canonical frequency bands (FIG. 3G). Compared to ratAttorney Docket No: STAN-2232WOClient No: S24-410 cortex, the total power in the alpha and beta bands is significantly lower in t-hCO, but not t- hFA, and both transplanted groups display less power than rat cortex in the gamma band. Critically, this effect on oscillations was specific to the electrode within the graft - there were no main effect of graft condition between groups in any of the other electrodes (FIG. 8A-F).

[0158] The differences between oscillatory activity between rat cortex and t-hCO, but not t- hFA (FIG. 3G, FIG. 8G), along with the known role of GABAergic neurons in cortical oscillations (6, 39-41 ) prompted the measurement of whether there was a difference in GABA tone between t-hCO and t-hFA. Whole-cell patch clamp recordings of spontaneous inhibitory postsynaptic currents (sIPSCs) were performed in putative non-GABAergic Dlxi1 / 2b::EGFP- cells (FIG. 3H). An increased frequency of sIPSCs was found in t-hFA (median = 4.625 Hz) as compared to t-hCO (median = 0.026 Hz); critically, the GABA receptor antagonist bicuculline (BMI) blocked the sIPSCs (FIG. 3I, FIG. 8). Both t-hCO (24) and t-hFA neurons receive glutamatergic inputs (FIG. 8K,L), but functional GABAergic inputs are present only in t-hFA. Given the increase in GABA tone ex vivo in t-hFA, it was tested whether the in vivo spontaneous activity of glutamatergic neurons differed between t-hCO and t-hFA (FIG. 3J). To selectively record from human glutamatergic neurons in vivo, acute opto-tagging experiments (42) of the t-hCO and t-hFA grafts were performed. In these experiments, an opsin was only expressed in hCO-derived neurons of both conditions, and only units that demonstrated a short-latency response to light stimulation were used for further analysis. Thus, spontaneous in vivo spike rates were directly compared between human glutamatergic neurons of both conditions to putative glutamatergic neurons of naive rats. Compared to rat cortex, t-hCO, but not t-hFA, glutamatergic neurons exhibited increased spiking (FIG. 3K). Taken together, these data suggest GABAergic inputs are present in t-hFA show and this is associated with different patterns of neural activity as compared to t-hCO.

[0159] t-hFA as a platform to model disease-related features. The ability to obtain global electrical readouts from t-hFA led to the investigation of whether this system may be applied to study human neurodevelopmental disorders that show a seizure component. Protocadherin-19-related epilepsy (PCDH19-RE) was focused on, an early-onset disease with an atypical sex-linked inheritance pattern in which only females are affected (43, 44) and where mosaic expression of PCDH19 is thought to contribute to pathophysiology (45). PCDH19-RE has been challenging to model in vitro and in vivo. Modeling in female hiPS cells is problematic due to persistent X-chromosome inactivation on one of the X alleles and frequent erosion, leading to the inability to mimic random X chromosome inactivation (46), and animal models do not fully recapitulate clinical features of disease (47-49).

[0160] Here, the t-hFA platform was applied in combination with CRISPR / Cas9 gene editing on a male hiPS cell line targeting exon 1 of PCDH19 for out of frame indels (FIG. 9). TheAttorney Docket No: STAN-2232WOClient No: S24-410 resulting PCDH19 wild-type (WT) and knockout (KO) hiPS cell lines allowed the transplantation of all combinations of organoid pairs: t-hCO (WT / WT, WT / KO i.e. mosaic, and KO / KO, representing the genotype of each hCO in the transplanted pair) and t-hFA (WT / WT, [WT / KO and KO / WT] i.e. mosaic, and KO / KO - representing the genotype of hCO first and hSO second; FIG. 4A). This design allowed to manipulate PCDH19 in glutamatergic and / or GABAergic neurons in vivo. Following the procedure developed for EEG recordings from the grafted human cells (Fig. 3A-B), a cardinal feature of PCDH19-RE was modeled that has proven difficult to demonstrate in animal models - febrile seizures (47-50). Rats were placed in a heating apparatus while performing EEG recordings (FIG. 4A-D), and recordings were terminated either once a seizure occurred or body temperature reached 42.5°C (51 , 52). Body temperature increased at a similar rate in all groups (FIG. 4D), but we only observed electrographic and behavioral seizures in the mosaic t-hFA condition (FIG. 4B-G). In the t-hFA mosaic condition, four out of eight rats experienced electrographic seizures below the 42.5°C threshold, in contrast, none of the twenty-eight rats in the other experimental conditions exhibited seizures. By behavioral criteria, of the four rats that exhibited seizures, three exhibited Racine stage 5, generalized tonic-clonic seizures, while one displayed a stage 3-4 seizure. Surprisingly, rats in the t-hCO mosaic condition did not demonstrate seizure, while deletion of PCDH19 in either the hCO or hSO component of the t-hFA (50% seizure rate in both groups) sensitized the rat to hyperthermia-induced seizures (FIG. 4C-E). Graft volume did not affect susceptibility to seizures (FIG. 10B). As an internal positive control indicating that increasing the body temperature affects neural activity, rats across multiple conditions nonspecifically exhibited mild electrographic abnormalities such as slow wave discharges and train of spikes (FIG. 10C,D). These data imply that the interaction between glutamatergic and GABAergic neurons may be important when considering the effect of mosaic loss of PCDH19, a homophilic cell-adhesion molecule (53). Taken together, the t-hFA platform allows for the detection of in vivo electrophysiological and behavioral readouts from disease-related hiPS cell lines.Discussion

[0161] Advanced maturation and circuit relevance of organoids are achieved through assembly and functional integration of multiple organoids into assembloids (16, 54, 36) or the transplantation of neural organoids into living animals (24), both of which can support more complex models of human neural development and disease. Here, these approaches are combined and co-transplanted hCO and hSO into the developing rat cortex to generate t-hFA. GABAergic neurons within t-hFA disperse throughout the graft and mature to a greater extent than their in vitro counterparts. This results in in vivo electrophysiological readouts that captureAttorney Docket No: STAN-2232WOClient No: S24-410 the collective activity of hiPS cell-derived neurons, potentially enabling enhanced pre-clinical modeling of seizures - a phenotype unattainable in vitro. In this study the modular nature of the t-hFA platform was utilized to discover that mosaic loss of PCDH19 in t-hFA, but not t- hCO, leads to enhanced propensity for hyperthermia-induced seizures.

[0162] The t-hFA platform has several advantages that may ultimately enable new applications. First, t-hFA allows for interactions among different cell lineages within an in vivo context, facilitating the experimental manipulation of a diverse set of hiPS cell-derived neurons. Second, these diverse cell-types mature morphologically and display some characteristic electrophysiological features; most notably, fast-spiking PV+ neurons exhibit firing rates of up to 1 15 Hz in the t-hFA. Third, transplantation of intact organoids provides stereotaxic access to collect electrophysiological readouts from a functional unit of human tissue, which includes both glutamatergic and GABAergic neurons, and is associated with a different pattern of activity in t-hFA as compared to t-hCO. This approach makes translationally-relevant network and behavioral readouts, such as seizures, accessible directly from human cells.

[0163] Taken together, t-hFA offers experimental access to a unit of human tissue assembled in vivo out of both cortical glutamatergic and GABAergic neurons. This approach holds promise in studying the assembly of human circuits, modeling disease, and evaluating therapeutics on human cells in vivo - particularly important for neurological and psychiatric disorders defined by neurophysiological and behavioral dysfunction, such as seizures.Methods

[0164] Generation of hCOs and hSOs from hiPS cells and lentivirus infection. hCO generation and culture was performed from hiPS cells as previously described (17, 21 ). Four hiPS cell lines were used in the study- 0524-1 , 1208-2, 81 19-1 , 8858-3. In brief, hiPS cells were incubated with Accutase (Innovate Cell Technologies, AT-104) at 37 °C for 7 min and dissociated into single cells. The single-cell suspension was then collected in a 50-ml Falcon tube and a cell pellet was obtained via centrifugation at 200 g for 4 min. Cell counting was performed after resuspending the cell pellets. Approximately 3 x 106 cells in Essential 8 medium (Life Technologies, A1517001 ) supplemented with ROCK inhibitor Y-27632 (10 pM; Selleckchem; Cat. No.: S1049) were added per well of the AggreWell 800 plate (STEMCELL Technologies; Cat. No.: 34815). Plates were then centrifuged at 100 g for 3 min to capture the cells in the microwells and incubated at 37 °C with 5% CO2 (day -1 ). 24h after the cell aggregation (day 0), we collected spheroids from each microwell by firmly pipetting (with a cut end of a P1000 tip) medium in the well up and down and transferring it into ultra-low attachment plastic dishes (Corning, 3262) in Essential 6 medium (Life Technologies,Attorney Docket No: STAN-2232WOClient No: S24-410A1516401 ) supplemented with dorsomorphin (DM; 2.5 pM; Sigma-Aldrich; Cat. No.: P5499) and SB-431542 (SB; 10 pM; Tocris; Cat. No.:1614). From day 2 to day 5, Essential 6 medium was changed every day and supplemented with dorsomorphin and SB-431542. On the sixth day in suspension, neural spheroids were transferred to neural media consisting of Neurobasal A (Life Technologies; Cat. No.: 10888), B-27 supplement without vitamin A (B27; Life Technologies, Cat. No.: 12587), GlutaMax (1 :100, Life Technologies; Cat. No.: 35050) and 10 U / ml Penicillin-Streptomycin (Gibco; Cat. No. : 15140122). From day 6 to day 24, The neural medium was supplemented with 20 ng ml-1 epidermal growth factor (EGF; R&D Systems, Cat. No.: 236-EG) and 20 ng ml-1 basic fibroblast growth factor (FGF; R&D Systems; Cat. No.: 233-FB), with medium changed daily from day 6 to day 17 and every other day till day 24. From day 25 to day 42, the neural medium contained 20 ng ml-1 brain-derived neurotrophic factor (BDNF; Peprotech; Cat. No.: 450-02) and 20 ng ml-1 NT3 (Peprotech; Cat. No.:450-03), with medium changes every other day. From day 43 onward, the hCOs were cultured with only neural medium without growth factors with medium changes every 4 days. For hSO generation, the protocol was modified from hCO generation as followed: from day 0 to 23, additional Wnt pathway inhibitor XAV-939 (XAV; 1.25 pM; Tocris; Cat. No.: 3748) was added, and from day 12-23, Smoothened Agonist (SAG; 100 nM; EMD Millipore; Cat. No.: 566660) was added.

[0165] For viral labeling, all organoids were infected at least 1 week before transplant, most commonly between day 35 and day 40 of differentiation. Organoids were transferred to a 1 .5- ml microcentrifuge Eppendorf tube containing 100 pL neural medium with lentivirus and incubated overnight. The next day, neural organoids were transferred into fresh neural medium in 24-well ultra-low attachment plates with fresh medium changed daily to wash out any ambient virus particles. All lentiviruses were generated by VectorBuilder with previously used vector maps and published sequences: pLV[Exp]-Dlxi1 / 2b::EGFP (i.e. Dlxi1 / 2b enhancer driving EGFP), pLV[Exp]-SYN1 -ChR2(H134R) / EYFP, pLV[Exp]-SYN1 -ChRmine / EYFP (32, 55, 56).

[0166] Generation of PCDH19 KO hiPS cell reporter lines The parental hiPS cell line 8858- 3 was generated at Stanford and previously described (57). The Cas9, gRNA-S1 and EGFP donor plasmids were obtained from Addgene (plasmids #42230, #41818 and #52344, respectively). To generate the RFP donor plasmid, firstly, the EGFP donor plasmid (Addgene #52344) was digested with Hindi (NEB, Cat. No. R0103S) and EcoRV (NEB, Cat. No. R0195S). The resulting 9025-bp backbone was purified from the agarose gel, blunted with Klenow Fragment (Thermo Fisher Scientific, Cat. No. EP0054), and dephosphorylated with Shrimp Alkaline Phosphatase (Thermo Fisher Scientific, Cat. No. 783901000UN). Next, the Addgene plasmid pK029.CAG-loxP-stop-loxP-RFP-ires-tTA-WPRE (#69138) was digestedAttorney Docket No: STAN-2232WOClient No: S24-410 with Pmll and Hpal, resulting in a 1676-bp fragment that was then purified and inserted to the abovementioned dephosphorylated backbone using the T4 DNA ligase (Thermo Fisher Scientific, Cat. No. EL001 1 ). For PCDH19 targeting, gRNA was generated by annealing primer pairs 5’- CACCGCGAGAGGCGGGCTTCGCGC-3’ (SEQ ID NO: 01) and 5’- AAACGCGCGAAGCCCGCCTCTCGC-3’ (SEQ ID NO: 02) and inserting the annealed oligos into backbone plasmid (addgene #53188) digested with Bbsl (NEB, Cat. No. R3539L). The resulting gRNA, here named AA16_pgR-PCDH19.8, was used for PCDH19 targeting.

[0167] The generation of PCDH19 KO reporter lines involves two steps: inserting the EGFP or RFP into the AAVS1 “safe harbor” locus and disrupting PCDH19 exon 1 in the reporter populations. To generate the EGFP and RFP cell populations, 3 pg Cas9, 1 pg gRNA-S1 and 1 pg donor plasmids (EGFP and RFP) were used. On the day of nucleofection, hiPS cells were washed with DPBS and incubated with 1 mL Accutase at 37°C for 10 min, after which 9 mL Essential 8 Medium was added to the well for resuspension. After cell counting, the single cell suspension containing 3 x106 cells was centrifuged. The cell pellet was used for nucleofection using the P3 Primary Cell 4D-NucleofectorTM X Kit L (Lonza, V4XP-3024), a 4D-nucleofector core unit and X unit (Lonza) using the nucleofection program DC100. After nucleofection, the cells were immediately seeded into a well of a 6-well plate that was precoated with vitronectin and contained pre-warmed Essential 8 Medium supplemented with the ROCK inhibitor Y-27632 (10 pM; Selleckchem, S1049). While the nucleofected cells recovered to reach 70%-80% confluency, 1 pg ml-1 of puromycin was applied for 5 days, after which the media was switched back to StemFlex media. Puromycin-resistant clones became visible after 7 days. Clones were pooled together, expanded and cryopreserved.

[0168] To generate the PCDH19 KO cells in EGFP populations, 2824 ng peSpCas9(1 .1 ).gRNA (Addgene #71814), 1168ng AA16_pgR-PCDH19.8 and 1000ng pAAV.CAG.RFP (Addgene #22910) was used. To generate PCDH19 KO cells in RFP populations, 2824 ng peSpCas9(1 ,1 ).gRNA (Addgene #71814), 1168ng AA16_pgR-PCDH19.8 and 1000ng pCMV.EGFP (provided in the nuleofection kit) were used. 2 days post nucleofection, cells were sorted into 96-well plates to ensure single clone formation by seeding one cell per well. The sorting was performed on a BD Aria II (Stanford Shared FACS Facility). Around 2 weeks post-sorting, colonies were visible and expanded. Genotyping was performed by Sanger sequencing, using primer pairs 5’-CCTCCGCCCTCTTTACCTTC-3’ (SEQ ID NO: 03) & 5’-TCTTCTGCTTGGTGACCAGC-3’ (SEQ ID NO: 04) and Phire Tissue PCR mix (Thermo Fisher Scientific, Cat. No. F170L). Clones containing frame-shifting indels were further expanded together with clones containing no indels.

[0169] Validation of knockout was performed by Western blot analysis, as follows, day 25 hCO were lysed using a modified Radio Immunoprecipitation Assay (RIPA) buffer (50 mMAttorney Docket No: STAN-2232WOClient No: S24-410Tris-HCI, 150 mM NaCI, 1 mM EDTA, 1 % NP-40, and 1% TX-100 in H2O with pH 7.4 and protease inhibitors). Lysates were incubated on ice for 30 minutes followed by centrifugation at 15,000 RPM for 15 minutes at 4°C. The supernatant was transferred to a new tube, and the pellet was saved at -80°C. Protein concentration in the supernatant was determined by BCA assay. 1 g / L protein was prepared with 4x LDS buffer and 10% DTT and samples were incubated at 95°C for 5 minutes. Samples were run on a BOLT 4-12% Bis-Tris PAGE Gel for 1 -2 hours at 130V. Proteins were transferred to a PVDF membrane using the iBlot2 (Invitrogen). Membranes were blocked with 5% skim milk in TBST for 1 hour at RT and then washed once with TBST for 5 minutes at RT. Membranes were incubated for 48 hours at 4°C with primary antibody against PCDH19 (rabbit, 1 :500, Abeam) and 24 hours at 4°C with primary antibody against GAPDH (mouse, 1 :5000, GeneTex). Membranes were washed three times with TBST and then incubated with near-infrared fluorophore-conjugated species-specific secondary antibodies: goat anti-mouse IgG polyclonal antibody (IRDye 680RD, 1 :10,000, Ll- COR Biosciences, cat. no. 926-68070) and goat anti-rabbit IgG polyclonal antibody (IRDye 800CW, 1 :10,000, LI-COR Biosciences, cat. no. 926-32211 ) for 1 hour at RT. Following incubation with secondary antibodies, membranes were washed three times with TBST, once with TBS, and imaged using a LI-COR Odyssey CLx imaging system (LI-COR).

[0170] Organoid transplantation. All procedures were performed in accordance with NIH guidelines and with prior approval of the Stanford University Administrative Panel on Laboratory Animal Care (APLAC). Organoid transplantation procedures were performed using a modified version of a previously established protocol (58). Briefly, organoids at 30-60 DIV were transplanted into 3-7 day old athymic rat (FOXN1- / -) pups. Rats were anesthetized with isoflurane (5% induction, 2-3% maintenance) and received Ethiqa-XR (0.65 mg / kg) before being mounted on a stereotaxic frame with a neonatal insert (RWD) and heating pad. While maintaining an intact dura, a craniotomy was performed over the somatosensory cortex and organoids were deposited at a rate of 10 pL / min. The number of organoids transplanted in each condition was equated (i.e. 2 hCO in the t-hCO groups and [1 hCO +1 hSO] in the t-hFA group).

[0171] MRI monitoring of graft development. Growth of transplanted organoids was monitored using serial T2-weighted MRI beginning at 2- months post-transplantation, as previously described (3). An actively shielded Bruker 3T (TE = 81.0) or 1 1.7T (TE = 54.0) horizontal bore scanner (Bruker Corp., Billerica, MA) was used to acquire MRI sequences. The 3T scanner was equipped with a B-GA105S HP gradient insert (450 mT / m, 4200 T / m / s) and interfaced to ParaVision 360 v3.4 with a 82mm volume coil. Slice thickness for all 3T images was 1 -mm. The 7T scanner was equipped with a BGA-12S gradient insert (660 mT / m, 4570 T / m / s) and interfaced to ParaVision 360 V. 3.5. Slice thickness for all 7T images was 1 -Attorney Docket No: STAN-2232WOClient No: S24-410 mm. The 11 .7T scanner was equipped with a B-GA9S HP gradient insert (380 mT / m, 3420 T / m / s) and interfaced to ParaVision 360 v2. Slice thickness for all 11 ,7T images was 0.5-mm. Given different slice thicknesses were used, the images were subsampled to 1 mm slice thickness and interpolated. Respiration rate was monitored via pneumatic pad placed under the animal (SA instruments, NY). Graft volume was quantified using Imaris and 3D Slicer. Transplantation was defined as successful if all of the following criteria were satisfied: animal survived surgery, graft was located within the somatosensory cortex, and a contiguous area of signal was observed on the first MRI follow-up.

[0172] EEG placement. Rats were anesthetized with 5% isoflurane for induction and subsequently maintained on 1 -3% isoflurane during surgery. After shaving the head and back, animals were placed in a stereotactic frame (RWD) above a regulated heating pad. Ethiqa XR (Fidelis) was administered subcutaneously at a dose of 0.65 mg / kg for analgesia, and PuraLube Vet Ointment (Dechra Veterinary Products) was applied to the eyes. The scalp was next sterilized with betadine and 70% ethanol, and a midline incision was made to expose the skull. Six burr holes were made with a dental drill, and stainless-steel screws (J. I. Morris, FF00CE125) were placed bilaterally over the frontal cortices (relative to Bregma: AP +2.76 mm, lateral 2.6 mm). Silver wires (A-M Systems, 791400) were positioned over the bilateral somatosensory cortices on stereotactic coordinates obtained from preoperative MR imaging and any existing craniotomies from initial organoid transplantation. The most common coordinates for somatosensory cortices were (AP -1 .72 mm, lateral 5.0 mm). Reference and ground screws were placed over the cerebellum. Screws and electrodes were secured to custom-made headpiece adapters Mill-Max headpiece adapters (ED90267-ND, Digi-Key Electronics), and the implant was secured to the skull with dental cement (Metabond, S399, S371 , S398; Jet Set4 Liquid, Lang Dental, 3802X6). Lidocaine and triple-antibiotic ointment were then applied to the incision site. Following the implantation procedure, animals received a subcutaneous injection of warm saline, then were housed singly. Clavamox antibiotic were added to cage water bottles post-surgery. Electrode placement was verified by CT imaging.

[0173] CT image acquisition and reconstruction. All CT imaging was performed using a T riFoil explore CT 120 Small-Animal X-ray CT scanner with Hamamatsu L10321 Xray source, XIMEA MH1 10XC-KK-TP camera, and Al 0.5mm filter or Al+Cu filters. CT image reconstruction was performed using the SkyScan NRecon (Version 2.0) and Data Viewer software platforms. Briefly, the density histogram in the NRecon ‘preview slice’ feature was adjusted to optimize projection data for visualization of skull landmarks and EEG leads. Reconstruction was then performed, and the resulting file was opened in DataViewer. The volume of interest (VOI) was next defined across three orthogonal planes prior to export.Attorney Docket No: STAN-2232WOClient No: S24-410

[0174] Colocalization of MR and CT images for reconstruction of EEG array and lead locations. T2-weighted MR and Micro-CT images were co-localized using FIJI / lmageJ (Imaged 1 ,53q) and Imaris (9.8.2). Initially, images were converted from DICOM to IMS format via the Imaris File Converter (9.8.2). For Micro-CT images, the series were compiled into a single image stack using FIJI / lmageJ, then converted into IMS format using the Imaris File Converter (9.8.2). Consistency between units of Micro-CT and MR images was verified prior to further processing in Imaris. Corresponding MR and Micro-CT images were opened in Imaris. Colocalization of the two images was performed by alternating between the '3D view’ and ‘section’ view, with the latter used to translate and rotate images to achieve alignment. Proper alignment was confirmed by assessing colocalization in the XY, YZ, and ZX planes in the ‘section’ view. Landmarks, including the skull, brain surface, and eyes, were used to verify colocalization. The co-localized composite image was then used to determine the position of individual EEG electrodes with respect to the rat brain. To generate a representative visualization of the EEG array and rat brain, the "surface" function in Imaris was used.

[0175] EEG acquisition and analysis. Baseline recordings were performed 7-10 days after EEG placement. Head mounts were first connected to the head stage, which contained a digitizer and amplifier board (Intan Technologies, C3334), then tethered to an acquisition board (Open Ephys) via lightweight SPI interface cables (Intan Technologies, C3206). Continuous real-time EEG data were acquired using Open Ephys software (version 0.5.3.0, https: / / open-ephys.org), with data sampled at 2 kHz and bandpass filtered between 1 Hz and 300 Hz. Simultaneously, video was recorded using high-resolution color cameras to capture behavioral data. EEG and video recordings were performed during a similar time of day (between 10:00 and 18:00) for all animals. A blinded reviewer then used custom MATLAB software (version R2019b) to visually inspect the EEG recordings for artifacts, which were marked and documented. Spectral power and coherence were calculated using an adapted version of MATLAB's coherence software (https: / / www.mathworks.com / help / wavelet / ref / wcoherence.html). The custom software used for quantitative EEG analysis is available on GitHub (https: / / github.com / huguenardlab / EEG). In summary, artifact-free EEG data were filtered to remove 60 Hz noise and its harmonics using MATLAB's sine fit function and then down sampled to 500 Hz. Power spectral density (PSD) was computed for every 2-second window using the periodogram method, and coherence was calculated using MATLAB’s magnitude-squared coherence function (mscohere). EEG power was analyzed across specific frequency bands: delta (1-4 Hz), theta (4-8 Hz), alpha (8-12 Hz), beta (12-30 Hz), and gamma (30-59). Total power within frequency bands was calculated as the area under the curve of the power spectrum density x frequencies investigated. Total absolute power was calculated as the sum of squares of all power valuesAttorney Docket No: STAN-2232WOClient No: S24-410 within a given band, while relative power was expressed as the percentage of total EEG power within the investigated frequency band. The results of the MRI / CT co-localization informed exclusion of data from M1 ipsilateral screw electrode in cases where the screw was directly over the graft. For seizure induction experiments, EEG signals were categorized as normal (similar in amplitude and frequency to baseline recordings), spiking (transient, isolated bursts of high-amplitude activity occurring at less than one event per second), spike-and-wave discharges (SWDs; bursts of activity lasting more than 1 second), train of spikes (at least 2 spikes per second), ictal (seizure activity characterized by sustained high-frequency and high- amplitude activity lasting more than 5 seconds, corresponds to stage 3-5 of the Racine scale), and post ictal (following a seizure). Video EEG recordings were manually synchronized with corresponding temperature changes using time stamps to ensure accurate analysis of the data.

[0176] Hyperthermia-induced seizures. The hyperthermia-induced seizure procedure was adapted from a previous study (59). Briefly, rats were anesthetized, and a rectal temperature probe (RET-4, Physitemp Instruments Inc.) was carefully inserted and secured to the tail with tape. The probe was connected to a temperature monitor (Physitemp Instruments Inc.) to monitor core body temperature. After recovery from anesthesia, the animals were acclimated to the rectal probe and recording chamber for at least 5 minutes. Subsequently, baseline EEG with simultaneous video and body temperature were recorded for an additional 5 minutes without the heat lamp. Rats were then transferred to a pre-heated chamber (40°C) with an infrared heat lamp (250-watt, HL-1 , Physitemp Instruments Inc.) positioned overhead. Body temperature was continuously monitored. The seizure severity is scored using Racine scale (60) from 1 -5: (1 ) Mouth and facial movements. (2) Head nodding. (3) Forelimb clonus. (4) Rearing. (5) Rearing and falling. The experiment was terminated upon observing a stage 3-5 seizure or when core body temperature reached 42.5°C. Generalized tonic-clonic (GTC) was defined as a Racine score 5 seizure - bilateral forelimb clonus with rearing and falling. After heat exposure, rats were immediately placed on ice and provided chilled, subcutaneous saline until their core temperature returned to 37°C. The severity and duration of seizures were evaluated using continuous EEG and video monitoring. Experimenters were blinded to the genotype during recording and analysis. The endpoint temperature of 42.5°C was selected based on a pilot study in which significant morbidity occurred when body temperature continued to rise above ~42.7°C. In addition, 42.5°C is a threshold reported in the literature (51 , 52).

[0177] Ex vivo transplanted assembloid slice electrophysiology. Rats were anesthetized using isoflurane and brains extracted and immersed in ice-cold, sucrose-based slicing solution containing 234 mM sucrose, 11 mM glucose, 26 mM NaHCO3, 2.5 mM KOI, 1.25 mMAttorney Docket No: STAN-2232WOClient No: S24-410NaH2PO4, 10 mM MgSO4 and 0.5 mM CaCI2 (approximately 310 mOsm). Coronal sections containing t-hCO or t-hFA were collected using a vibratome (Leica VT 1200) at a thickness of 400 pm prepared as previously described (61 ). Slices were then moved to a continuously oxygenated slice chamber at room temperature, which contained aCSF made with: 10 mM glucose, 26 mM NaHCO3, 2.5 mM KCI, 1.25 mM NaHPO4, 1 mM MgSO4, 2 mM CaCI2 and 126 mM NaCI (-300 mOsm). Slice recordings were performed in a submerged chamber continuously perfused with aCSF bubbled with 95% 02 and 5% CO2 at room temperature. The graft area was targeted visually at low-magnification and target cells were visually identified at high magnification by the presence of EGFP driven by the Dlxi1 / 2b enhancer (FIG. 2E). Borosilicate glass pipettes (3-7MQ) were used for whole cell recordings and 0.5% biocytin (Sigma-Aldrich B4261 ) was included in the internal solution to later visualize morphology. For recordings of spontaneous inhibitory postsynaptic currents (sIPSC), a cesium-based, low Cl-internal solution containing 126 mM CsMeSO3, 10 mM HEPES, 1 mM EGTA, 2 mM QX-314 chloride, 0.1 mM CaCI2, 4 mM MgATP, 0.3 mM Na3GTP, 8 mM Na2- phosphocreatine (280-290 mOsm, pH 7.3 with CsOH) was used and held cells at the reversal potential of ionotropic glutamate receptors, liquid junction potential was calculated based on the Nernst-Planck equation which revealed (62)). All other recording were done with a potassium-based internal solution containing 135 mM KMeSO3, 5 mM KCI, 10 mM HEPES, 1 mM EGTA, 0.1 mM CaCI2, 4 mM MgATP, 0.3 mM Na2GTP (285-290 mOsm, pH 7.2-7.4). Data were acquired with a MultiClamp 700B Amplifier (Molecular Devices) and a Digidata 1550B Digitizer (Molecular Devices), low-pass filtered at 2 kHz, digitized at 20 kHz and analyzed using Clampfit (Molecular Devices), Easy Electrophysiology (RRID:SCR_021190), and custom MATLAB (Mathworks) software. Electrophysiological properties were analyzed according to (24, 63, 64). For maximal firing rate analysis, transplanted neurons underwent an extended range of current injections (due to a more mature, lower input resistance after transplantation) with steps ranging from 10 to 50pA starting at -200pA until firing rate plateaued or decreased over 5 sweeps. The recordings using the potassium internal solution were corrected with a liquid junction potential of -9.68 mV. sIPSCs and sEPSCs were analyzed using Easy Electrophysiology Software v2.6.3 template matching algorithm - first fitting the template to clear events, then analyzing all traces, and finally manually curating each trace.

[0178] In vitro human subpallial organoid electrophysiology. Stage and cell-line matched in vitro hSOs were infected with lentivirus encoding Dlxi1 / 2b::EGFP at the same age as t-hFA samples. For 1 -2 weeks prior to recording, hSOs were placed on cell culture inserts (Corning, Cat. No. 353090) in the wells of 6-well plates. 1 .5 ml NPC media was added into the wells to create the air-liquid interface supporting hSOs. Flattened organoids were gently detached from the insert and recordings proceeded as described above. For maximal firing rate analysis,Attorney Docket No: STAN-2232WOClient No: S24-410 neurons underwent current injections steps ranging from 5 to 10pA until firing rate plateaued or decreased over 5 sweeps.

[0179] Acute in vivo extracellular electrophysiology. Rats were transplanted with either two hCO or one hCO and one hSO organoids in the same manner described above. The hCO was infected with lentivirus carrying either hSyn-ChR2-EYFP or hSyn-ChRmine-EYFP at least 1 week prior to transplantation for two reasons 1 .) to allow for any ambient lentivirus to be thoroughly washed out of solution before transplantation and 2.) to select organoids with extensive expression of the opsin, as indicated by the fluorophore. We performed opto-tagging (42) to directly compare of human glutamatergic units within t-hCO and t-hFA. When organoids were older than 240 days post-differentiation (6-7months post-transplant), rats were anesthetized with 5% isoflurane for induction and 1-3% isoflurane during surgery. Animals were placed into a stereotactic frame (Kopf) and buprenorphine SR and dexamethasone were administered subcutaneously. The skull was exposed and cleaned. A stainless-steel ground screw was fastened to the skull. To target t-hCO / t-hFA, stereotactic coordinates were determined based on images acquired with MRI. A circular craniotomy (~1 cm diameter) was made with a high-speed drill directly above the t-hCO / t-hFA. Once the bone was as thin as possible, but before drilling all the way through the bone, the remaining intact bone disk was removed using forceps to reveal the underlying t-hCO / t-hFA. For rat cortical recordings, electrodes were positioned in primary somatosensory cortex. Single units were recorded using either 32- or 64-channel high-density silicon probes (Cambridge Neurotech) grounded to the ground screw and pre-amplified with an RHD amplifier (Intan). Electrodes were lowered through the craniotomy into the target site using a manipulator and the craniotomy was filled with sterile saline. Data acquisition was performed at 30 kHz with an Open Ephys acquisition system. For opto-tagging experiments in t-hCO or t-hFA a 200 pm diameter fiber-optic patch cord (Doric), coupled to a 473 nm laser (Omicron) or 635nm laser (CNI) was connected to a 200 pm diameter optical fiber that was positioned above the craniotomy. Immediately before this, the power output from the patch cord was adjusted to 20 mW. 10 pulses of 473nm or 635 nm light (1 -2 Hz, 10 ms pulse width) were delivered at the start of the recording. Laser timing was controlled by a Master-8 pulse generator (AMPI). Light-responsive units were defined as units that displayed a spiking response within 15 ms of light onset on > 70% of trials. Recordings of rat cortex were not opto-tagged.

[0180] For analysis, spikes were sorted using Kilosort2 and units were manually curated using Phy2 (65). For visualization, raw voltages were bandpass filtered (300-8000Hz). For recordings in rat cortex, putative parvalbumin-expressing neurons (units with a trough to peak latency of <0.5 ms) were excluded from analysis to facilitate comparison to excitatory neuronsAttorney Docket No: STAN-2232WOClient No: S24-410 in t-hCO / t-hFA (66). Spike counts were computed as the number of spikes for each recorded unit over a 4-minute recording window (starting 1 s after laser offset where applicable).

[0181] Tissue preparation and immunohistochemistry. Animals were anesthetized and transcardially perfused with phosphate-buffered saline followed by 4% paraformaldehyde (PFA). Post-fixation with PFA and sucrose solutions was performed, followed by cryosectioning (Leica CM1860) at a thickness of 50 pm. Immunolabeling for cell types of interest was then performed. Specifically, free-floating tissue sections were washed 3 times with PBS, then blocked and permeabilized for 1 -hour at room temperature PBS-Triton 0.3% with 10% normal donkey serum.

[0182] For 50 pm sections, primary antibodies were diluted in blocking buffer and incubated with sections overnight at 4°C. After 3 washes with PBS, sections were incubated with a solution of corresponding Alexa Fluor secondary antibodies for 2 hours at room temperature. Samples mounted on microscope slides using Fluoromount-G Mounting Medium (Southern Biotech).

[0183] For acute electrophysiology, sections and in vitro hSOs, primary antibodies were diluted in blocking buffer and incubated with sections for 5 days at 4°C. After 3 washes with PBS, samples were incubated with a solution of corresponding Alexa Fluor secondary antibodies overnight at 4°C. Samples were mounted between two 22mm x 50mm glass coverslips using Fluoromount-G Mounting Medium (Southern Biotech). A previously published protocol served as the basis for our approach (67).

[0184] Primary antibodies used were: anti-parvalbumin (guinea pig, 1 :1000; gp72, Swant), anti-somatostatin (rat, 1 :50; MAB354, EMD Millipore), anti-human nuclear antigen (mouse, 1 :100; ab191 181 , abeam), anti-vasoactive intestinal peptide (rabbit, 1 :500; 20077, Immunostar), anti-GFP (rabbit, 1 :500; a21311 , Life Technologies), anti-RECA-1 (mouse, 1 :50; ab9774, abeam), anti-IBA1 (goat, 1 :100; ab5076, abeam), and streptavidin, DyLight 549 (1 :1000; SA-5549, Vector Labs).

[0185] Distribution of Dlxi1 / 2b::EGFP cells within transplanted assembloids. Immunostained 50 pm thick sections were imaged on a confocal microscope (Leica TCS SP8). Images were then separated by channel and log transformed. Cell segmentation, classification and quantification were performed using QuPath (v.0.3.2) (68) with a CellPose extension (69). Input parameters for the pre-trained generalist algorithm “Cyto3” were first optimized for our dataset. Deep learning using a “human-in-the-loop” approach was then performed to establish a custom model for our dataset based on the optimized “Cyto3” algorithm. The custom model was additionally trained across grafts from different animals to ensure accuracy. Quantification was performed along the anterior-posterior axis of each t-hFA graft, as defined by rat neuroanatom ical landmarks. Density of Dlxi1 / 2b::EGFP+ cells was normalized to totalAttorney Docket No: STAN-2232WOClient No: S24-410 surface area of the graft, and graft border was delineated using the HNA stain. Quartile binning was performed based on the total anterior-posterior extent of an individual graft.

[0186] Dendritic reconstruction and analysis of dendritic morphology. Whole-cell patch clamp was performed using an internal solution with 0.2% biocytin. Sections and in vitro hSOs were then immunostained, as described above, to examine dendritic morphology of recorded cells and apply molecular markers for cell type determination. Z-stack images of biocytinpositive neurons were acquired using a confocal microscope (Leica TCS SP8) with z- compensation enabled.

[0187] The confocal laser microscopy system only permitted 4-channel imaging, and three channels were occupied by streptavidin, GFP, and HNA. Therefore, one to two GABAergic neuronal markers were assayed for per sample based on the most likely cell type from corresponding electrophysiological data. When biocytin-labeled cells were negative for the targeted subtype markers, they were classified as “unknown”.

[0188] Deconvolution was first performed based on a theoretical point-spread function using Huygens Software (Scientific Volume Imaging). Tracing was performed using neuTubel .O (70). Axons were identified based on distinctive visual cues, namely their tortuous, aspiny appearance and constant diameter. Axons were excluded from traces and quantification. Traces were then analyzed using the TREES Toolbox package (71 ) in MATLAB to obtain total path length and branch order.

[0189] Single nucleus RNA-seq (10x Chromium). Single nuclei isolation was performed as previously described using sucrose gradient buffers. In brief, flash frozen hSO and t-hFA (at around day 250) were homogenized using a low sucrose cell lysis buffer containing 0.1% Triton-X with a 2 ml glass tissue grinder (Sigma-Aldrich / KIMBLE, D8938) on ice. Crude nuclei were then filtered through a 40 pm filter and centrifuged at 320g (Eppendorf 581 OR) in a 50- ml Falcon tube for 10 min at 4 °C. After removing the supernatant, nuclei pellets were resuspended in 3 ml low sucrose buffer. Then, a 12.5 ml high sucrose solution was gently laid beneath the cell resuspension, followed by a final centrifugation step at 350g for 20 min at 4 °C with no brake. After removing the supernatant, samples were resuspended in 0.04% BSA / PBS with 0.2 U / pl RNAse inhibitor (40 U / pl, Ambion, AM2682). A targeted recovery of 8,000 nucleus per sample were loaded onto a Chromium Next GEM Chip G. Dual index snRNA-seq libraries were generated with the Chromium Single cell 3' GEM, Library & Gel Bead Kit v3.1 (10x Genomics). Libraries from different samples were pooled in equal molar and sequenced by Admera Health and CZ biohub on a NovaSeq 6000 S4 2x150 (Illumina) then trimmed to 28x10x10x90nt.

[0190] Single-nucleus RNA-seq analysis. Gene expression levels were quantified for each putative nuclei barcode using the 10x Genomics CellRanger analysis software suite (versionAttorney Docket No: STAN-2232WOClient No: S24-4107.1 .0). Specifically, reads were mapped to a combined human (GRCh38, Ensemble release 98) and rat (Rnor_6.0, Ensemble release 100) reference genome created using the mkref command and quantified using the count command with -include-introns=TRUE to include reads mapping to intronic regions and -force-cells=10000. For t-hFA samples, human nuclei were identified based on a conservative requirement of at least 95% of total mapped reads aligning to the human genome, as described previously (16, 24). All subsequent analyses were performed on the filtered barcode matrices outputted from CellRanger using the R (version 4.3.2) package Seurat (version 5.0.1 ) (72).

[0191] To ensure that only high-quality nuclei were included for downstream analyses, an iterative filtering process was implemented for each sample. First, low-quality nuclei with less than 1 ,000 unique genes detected and with mitochondrial counts accounting for greater than 5% of the total counts were identified and removed. Subsequently, raw gene count matrices were normalized by regularized negative binomial regression using the sctransform function (vst.flavor=”v2”), which also identified the top 3,000 highly variable genes using default parameters. Dimensionality reduction using principal component analysis (RCA) on the top variable genes was performed, and clusters of nuclei were identified in RCA space by shared nearest-neighbor graph construction and modularity detection implemented by the FindNeighbors and FindClusters functions using a dataset dimension of 30 (dims = 30 chosen based on visual inspection of elbow plot and used for all samples and integration analyses) with default parameters. Iterative rounds of clustering (resolution = 2) was performed to identify and remove clusters of putative low-quality cells based on outlier low gene counts (median below the 10th percentile) and outlier high-fraction mitochondrial genes (median above the 95th percentile). T-hFA samples (n = 4) and hSO samples (n = 4) were each separately integrated using the IntegrateData function with the above parameters. Further rounds of quality filtering were subsequently performed on the integrated datasets as described above. Integrated datasets were further subsetted and reclustered by broad cell classes: GABAergic neurons (GAD1 and GAD2 containing clusters), excitatory neurons (SLC17(16)A7, NEUROD6, BCL1 1 B, and / or SATB2), and non-neuronal cells (all remaining clusters). Putative doublet clusters were then identified in each subsetted dataset by co-expression of broad cell class marker genes and removed.

[0192] Following low quality cell removal, datasets were clustered (FindClusters function; resolution = 2) and embedded for visualization purposes with Uniform Manifold Approximation and Projection (UMAP). Major cell classes were identified and categorized through a combination of marker gene expression (16) and annotation via reference mapping using Seurat’s TransferData workflow to classify organoid cells based on a well-annotated adult cortical snRNA-seq dataset (35). Specifically, progenitor clusters were identified by theAttorney Docket No: STAN-2232WOClient No: S24-410 expression of MKI67, TOP2A, and EGFR. Astrocyte clusters expressed high levels of SLC1 A3 and AQP4 and mapped to adult astrocytes. OPCs expressed PDGFRA and SOX10, whereas oligodendrocytes expressed markers of myelination (MOG and MYRF). Glutamatergic neurons clusters were further classified into subclasses by mapping to annotations defined by the reference adult dataset except for L6b neurons which also contained markers of subplate (SP) (ST18) and therefore labeled L6b / SP. A small cluster of glutamatergic neurons did not obviously map to populations in the reference dataset and were labeled “other.GluN”. GABAergic neurons were subsetted and further subclustered and annotated in a similar fashion. GABAergic neurons that expressed MEIS2 did not obviously map to GABAergic neuron subclasses in the adult dataset and were labeled “MEIS2”. Choroid plexus-like cells were defined by expression of cilia-associated genes and absence of expression of neuronal genes.

[0193] Differential gene expression analysis between t-hFA and hSO GABAergic neurons was performed using a pseudobulk approach across sample replicates, implemented with the Libra R package (version 1 .0.0) (73). Specifically, the edgeR (version 4.0.15, R package) loglikelihood ratio test was performed between groups on gene counts summed across cells for a given cell class for each sample replicate. For heat map visualizations, counts per million (CPM) normalized expression values were calculated using edgeR (cpm() function) and scaled (to achieve mean = 0, standard deviation = 1 ). Gene Ontology (GO) enrichment analyses among significantly upregulated t-hFA GABAergic neuron genes (Benjamini- Hochberg-adjusted P values less than 0.05, expressed in at least 10% of GABAergic neurons, and fold change increase of at least 2) were performed using the ToppGene Suite (https: / / toppgene.cchmc.org / ) (74). We used the ToppFun application with default parameters and reported Benjamini-Hochberg-adjusted P values calculated from hypergeometric tests from GO annotations.

[0194] To assess the global transcriptom ic maturation state of t-hFA GABAergic neurons, upregulated t-hFA versus hSO GABAergic neuron genes were compared to gene sets obtained across developing stages of human cortical GABAergic neuron development (37). Specifically, genes that were identified as specific to GABAergic neurons were classified by the age of onset of gene expression (50% of the maximum expression, as defined by Data S2 in Velmeshev et al. (37). To increase power to detect developing stage associations, all GABAergic subtype genes were aggregated together by developing stage in addition to aggregating genes associated with stages beyond 4 years of age. In total, there were 187 genes with onset in 2nd trimester, 104 genes with onset in 3rd trimester, 108 genes with onset between 0-1 years, 75 genes with onset between 1 -4 years, and 42 genes with onset betweenAttorney Docket No: STAN-2232WOClient No: S24-4104-20 years in developing GABAergic neurons. Gene set enrichment analyses using these gene sets were performed using a one-sided Fisher’s exact test.

[0195] Behavioral studies. Male and female rats were used in approximately equal proportions for all behavioral experiments.

[0196] Open Field Activity Chamber. The locomotor assessment took place in an Open Field Activity Arena (Med Associates Inc., St. Albans, VT. Model ENV-515) mounted with three planes of infrared detectors and within a custom-designed sound-attenuating chamber (Med Associates Inc., St. Albans, VT. MED-017M-027). The arena was 43 cm (L) x 43 cm (W) x 30 cm (H), and the sound-attenuating chamber was 74 cm (L) x 60 cm (W) x 60 cm (H). Rats were placed in the corner of the testing arena and allowed to explore the arena for 10 minutes while being tracked by an automated tracking system. Male rats were tested first, followed by testing of females. Parameters, including distance moved, vertical counts, vertical time, time spent in the center of arena and latency to center of arena, were analyzed. The periphery was defined as the zone 10 cm away from the arena wall. The arena was cleaned with a 1 % Virkon solution at the end of each trial. Animals were tested after 5 days of handling.

[0197] Novel Object Recognition (NOR) Test. The Novel Object Recognition task was conducted in a black square plastic arena (50 cm x 50 cm x 45 cm). A 50 mL falcon tube was used during the habituation session, and two different objects (green tower and white bottle) were used during training and testing sessions. All objects were generally consistent in height and volume, but had different shapes, textures, and appearances. The experiment took place over the course of 3 days, with males being tested first, followed by testing of females on separate days. During the habituation phase, rats were allowed to freely explore the arena for 5 minutes with the habituation objects (two 50mL falcon tubes). The 50mL falcon tubes were only used during the habituation phase and were not used during the training or testing phases. The training and testing phases took place approximately 24 hours after habituation. During the training session, rats were first re-introduced to the arena with two, new identical objects (either green towers or white bottles) placed diagonally in opposite corners, 15 cm from the respective corners. Animals were allowed to freely explore for 5 minutes. After training was complete, rats were returned to their home cage for 5 minutes before beginning the testing phase. For the testing session, one of the two objects used during Training was replaced with a novel object, and the animals were allowed to freely explore for 5 minutes. Throughout the experiment, rats were tracked with an automated tracking system (Noldus Information Technology, Wageningen, the Netherlands) and object interaction was manually scored by the experimenter. Object interaction was defined as a rat physically interacting with the object or pointing its nose toward an object within a 2 cm radius. Objects for training and testing and the location of these objects were pseudorandomized to avoid object and locationAttorney Docket No: STAN-2232WO Client No: S24-410 bias. The arena and objects were cleaned with 1% Virkon between experimental sessions. Animals were tested in NOR after being tested in the Open Field Activity Chamber.

[0198] Statistics. All numerical data are presented as means ± SEM unless otherwise noted. Whenever possible, experiments and analyses were performed in a blinded manner. Prior to statistical modeling, data were examined to guide use of the appropriate parametric / linear models (75). A parametric model was applied if residuals had a normal distribution. When residuals did not lie on the identity line of the Normal QQ plot, and were not considered normally distributed based on the Shapiro-Wilk test, then data were log-transformed prior to performing parametric analysis. In cases where a parametric model was not appropriate (i.e. QQplot and Shapiro-Wilk demonstrated an invalid assumption of normally distributed residuals), then a non-parametric test was used. Statistical analyses were performed using Prism 9 (GraphPad). A full model (column, row, and column x row interaction) was fit when possible. If GraphPad cannot, then a main effects model (column and row) was used. When appropriate, repeated measures ANOVA (rmANOVA) was used with the Geisser- Greenhouse’s epsilon correction for sphericity and Holm-Sidak correction for multiple comparisons. Multiple comparisons were always conducted to account for all of the comparisons (i.e. GraphPad’s “conservative” option of one family for all comparisons). ANOVAs were two-way, unless otherwise indicated. All t-tests were two-tailed.REFERENCES1 . A. Kepecs, G. Fishell, Interneuron cell types are fit to function. Nature 505, 318-326 (2014).2. C. P. Wonders, S. A. Anderson, The origin and specification of cortical interneurons. Nature Reviews Neuroscience 7, 687-696 (2006).3. J. C. Silbereis, S. Pochareddy, Y. Zhu, M. Li, N. Sestan, The cellular and molecular landscapes of the developing human central nervous system. Neuron 89, 248-268 (2016).4. J. S. Hu, D. Vogt, M. Sandberg, J. L. Rubenstein, Cortical interneuron development: a tale of time and space. Development 144, 3867-3878 (2017).5. G. Bartolini, G. 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[0199] In at least some of the previously described embodiments, one or more elements used in an embodiment can interchangeably be used in another embodiment unless such a replacement is not technically feasible. It will be appreciated by those skilled in the art that various other omissions, additions and modifications may be made to the methods and structures described above without departing from the scope of the claimed subject matter.Attorney Docket No: STAN-2232WOClient No: S24-410All such modifications and changes are intended to fall within the scope of the subject matter, as defined by the appended claims.

[0200] It will be understood by those within the art that, in general, terms used herein, and especially in the appended claims (e.g., bodies of the appended claims) are generally intended as “open” terms (e.g., the term “including” should be interpreted as “including but not limited to,” the term “having” should be interpreted as “having at least,” the term “includes” should be interpreted as “includes but is not limited to,” etc.). It will be further understood by those within the art that if a specific number of an introduced claim recitation is intended, such an intent will be explicitly recited in the claim, and in the absence of such recitation no such intent is present. For example, as an aid to understanding, the following appended claims may contain usage of the introductory phrases “at least one” and “one or more” to introduce claim recitations. However, the use of such phrases should not be construed to imply that the introduction of a claim recitation by the indefinite articles “a” or “an” limits any particular claim containing such introduced claim recitation to embodiments containing only one such recitation, even when the same claim includes the introductory phrases “one or more” or “at least one” and indefinite articles such as “a” or “an” (e.g., “a” and / or “an” should be interpreted to mean “at least one” or “one or more”); the same holds true for the use of definite articles used to introduce claim recitations. In addition, even if a specific number of an introduced claim recitation is explicitly recited, those skilled in the art will recognize that such recitation should be interpreted to mean at least the recited number (e.g., the bare recitation of “two recitations,” without other modifiers, means at least two recitations, or two or more recitations). Furthermore, in those instances where a convention analogous to “at least one of A, B, and C, etc.” is used, in general such a construction is intended in the sense one having skill in the art would understand the convention (e.g., “ a system having at least one of A, B, and C” would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). In those instances where a convention analogous to “at least one of A, B, or C, etc.” is used, in general such a construction is intended in the sense one having skill in the art would understand the convention (e.g., “ a system having at least one of A, B, or C” would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). It will be further understood by those within the art that virtually any disjunctive word and / or phrase presenting two or more alternative terms, whether in the description, claims, or drawings, should be understood to contemplate the possibilities of including one of the terms, either of the terms, or both terms. For example, the phrase “A or B” will be understood to include the possibilities of “A” or “B” or “A and B.”Attorney Docket No: STAN-2232WOClient No: S24-410

[0201] In addition, where features or aspects of the disclosure are described in terms of Markush groups, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group.

[0202] As will be understood by one skilled in the art, for any and all purposes, such as in terms of providing a written description, all ranges disclosed herein also encompass any and all possible sub-ranges and combinations of sub-ranges thereof. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc. As will also be understood by one skilled in the art all language such as “up to,” “at least,” “greater than,” “less than,” and the like include the number recited and refer to ranges which can be subsequently broken down into sub-ranges as discussed above. Finally, as will be understood by one skilled in the art, a range includes each individual member. Thus, for example, a group having 1 -3 articles refers to groups having 1 , 2, or 3 articles. Similarly, a group having 1 -5 articles refers to groups having 1 , 2, 3, 4, or 5 articles, and so forth.

[0203] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it is readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

[0204] Accordingly, 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. Moreover, nothing disclosed herein is intended to be dedicated to the public regardless of whether such disclosure is explicitly recited in the claims.

[0205] 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 presentAttorney Docket No: STAN-2232WOClient No: S24-410 invention is embodied by the appended claims. In the claims, 35 U.S.C. §112(f) or 35 U.S.C. §112(6) is expressly defined as being invoked for a limitation in the claim only when the exact phrase "means for" or the exact phrase "step for" is recited at the beginning of such limitation in the claim; if such exact phrase is not used in a limitation in the claim, then 35 U.S.C. § 112 (f) or 35 U.S.C. §112(6) is not invoked.

[0206] Notwithstanding the appended claims, the disclosure set forth herein is also described by the following clauses:

[0207] 1 . A method of producing a non-human mammalian animal model, the method comprising: introducing a first human neural organoid into a central nervous system location of a newborn non-human mammal; introducing a second human neural organoid into the central nervous system location of the newborn non-human mammal to form an assembloid from the first human neural organoid and the second human neural organoid; and allowing the newborn non-human mammal to mature to produce the non- human mammalian animal model comprising human neural tissue comprising GABAergic neurons; wherein the human neural tissue integrates into the circuit of the non-human animal and displays complex neural activity.

[0208] 2. The method of clause 1 , wherein the first human neural organoid, the second human neural organoid, or both the first neural and the second neural organoid are organoids generated from induced human pluripotent stem cells (hiPSCs).

[0209] 3. The method of clauses 1 or 2, wherein the first human neural organoid is a cortical organoid or a subpallial organoid.

[0210] 4. The method of any of clauses 1 -3, wherein the first human neural organoid is a cortical organoid.

[0211] 5. The method of any of clauses 1 -4, wherein the second human neural organoid is a subpallial organoid.

[0212] 6. The method of any of clauses 1 -5, wherein the central nervous system location is selected from the group consisting of the frontal cortex, motor cortex, somatosensory cortex, parietal cortex, occipital cortex, temporal cortex, striatum, spinal cord, thalamus, and the cerebellum.

[0213] 7. The method of any of clauses 1 -6, wherein the central nervous system location is the somatosensory cortex.Attorney Docket No: STAN-2232WOClient No: S24-410

[0214] 8. The method of any of clauses 1 -7, wherein the complex neural activity is total power in an alpha spectral band.

[0215] 9. The method of clauses 1 -8, wherein the complex neural activity is total power in a beta spectral band.

[0216] 10. The method of any of clauses 1 -9, wherein the complex neural activity is total power in the alpha, delta, gamma, and theta spectral bands.

[0217] 11. The method of any of clauses 1 -9, wherein the complex neural activity is total power in the beta, delta, gamma, and theta spectral bands.

[0218] 12. The method of any of clauses 1 -11 , wherein the complex neural activity is total power in the alpha, beta, delta, gamma, and theta spectral bands.

[0219] 13. The method of any of clauses 1 -12, wherein the complex neural activity is spontaneous neural activity of glutamatergic neurons.

[0220] 14. The method of any of clauses 1 -13, wherein the human neural tissue comprises human medial ganglionic eminence (MGE)-derived parvalbumin positive (PV+) GABAergic neurons.

[0221] 15. The method of clause 14, wherein the PV+GABAergic neurons comprise fast spiking PV+GABAergic neurons.

[0222] 16. The method of clauses 14 or 15, wherein the PV+GABAergic neurons are morphologically, transcriptionally, and electrophysiologically mature PV+GABAergic neurons.

[0223] 17. The method of any of clauses 1 -14, wherein the human neural tissue comprises human MGE-derived somatostatin positive (SST+) GABAergic neurons.

[0224] 18. The method of any of clauses 1 -17, wherein the human neural tissue comprises human caudal ganglionic eminence (CGE)-derived vasoactive intestinal polypeptide positive (VI P+) GABAergic neurons.

[0225] 19. The method of any of clauses 1 -18, wherein the human neural tissue comprises human CGE-derived lysosomal associated membrane protein 5 positive (LAMP5+) GABAergic neurons.

[0226] 20. The method of any of clauses 1 -19, wherein the human neural tissue comprises human CGE-derived gamma-synuclein positive (SNCG+) GABAergic neurons.

[0227] 21 . The method of any of clauses 1 -20, wherein the GABAergic neurons have a maximal firing rate of about 10Hz to about 130Hz.

[0228] 22. The method of any of clauses 1 -21 , wherein the GABAergic neurons have an resting membrane potential of about -40mV to about -80mV.Attorney Docket No: STAN-2232WOClient No: S24-410

[0229] 23. The method of any of clauses 1 -22, wherein the GABAergic neurons have an average capacitance of about 250pF.

[0230] 24. The method of any of clauses 1 -23, wherein the GABAergic neurons have an average action potential half width of about 2 ms.

[0231] 25. The method of any of clauses 1 -24, wherein the GABAergic neurons have an average input resistance of about 2 GO.

[0232] 26. The method of any of clauses 1 -25, wherein the GABAergic neurons have an average spontaneous inhibitory postsynaptic current frequency of about 5 Hz.

[0233] 27. The method of any of clauses 1 -26, wherein the newborn non-human mammal is a rodent.

[0234] 28. The method of clause 27, wherein the rodent is a rat.

[0235] 29. The method of clause 27, wherein the rodent is a mouse.

[0236] 30. The method of any clauses 1-26, wherein the newborn non-human mammal is a primate.

[0237] 31 . The method of any of clauses 1 -30, wherein the newborn non-human mammal is an immunocompromised non-human mammal.

[0238] 32. The method of clause 31 , wherein the immunocompromised non- human mammal comprises a genetic mutation.

[0239] 33. The method of clause 31 , wherein the immunocompromised non- human mammal is immunocompromised as a result of a chemical or genetic treatment.

[0240] 34. The method of any of clauses 1 -33, wherein the newborn non-human mammal is from 1 to 10 days old.

[0241] 35. The method of any of clauses 1 -34, wherein the first and / or second organoid are produced from a cellular biological sample from a human having an epilepsy disorder.

[0242] 36. The method of clause 35, wherein the epilepsy disorder is associated with mosaicism.

[0243] 37. The method of clause 36, wherein the epilepsy disorder is associated with a pathogenic variant of a gene selected from the group consisting of: CDKL5, CLCN4, SCN1A, SCN2A, SLC35A2, GABRA1 , GRIN2B, KCNQ2, STXBP1 , CHD2, SLC2A1 , ARX, MECP2, and PCDH19.

[0244] 38. The method of clause 35, wherein the epilepsy disorder is associated with a channelopathy.Attorney Docket No: STAN-2232WOClient No: S24-410

[0245] 39. The method of clause 38, wherein the channelopathy is associated with a pathogenic variant of a gene selected from the group consisting of: SCN1 A, SCN1 B, SCN2A, SCN3A, SCN8A, KCNQ2, KCNQ3, KCNMA1 , KCNA1 , KCNA2, KCNJII, KCNT1 , CACNA1 H, CACNA1A, CHRNA4, CHRNB2, CHRNA2, GABRA1 , GABRB2, GABRB3, GABRD, and GABRG2.

[0246] 40. A method of modeling a human epilepsy disorder, the method comprising: introducing a first human neural organoid into a central nervous system location of a newborn non-human mammal; introducing a second human neural organoid into the central nervous system location of the newborn non-human mammal to form an assembloid from the first human neural organoid and the second human neural organoid; allowing the newborn non-human mammal to mature to produce the non- human mammalian animal model comprising a first human neural tissue; and characterizing the first human neural tissue to model the epilepsy disorder; wherein the first human neural organoid, the second human neural organoid, or the first and second human neural organoid are: a) produced from a cellular biological sample from a human who has the epilepsy disorder; or b) have a genetic mutation associated with the epilepsy disorder.

[0247] 41 . The method of clause 40, wherein the first human neural organoid or the second human neural organoid are: a) produced from a cellular biological sample from a human who has the epilepsy disorder; or b) have a genetic mutation associated with the epilepsy disorder.

[0248] 42. The method of clause 40, wherein the first human neural organoid and the second neural organoid are: a) produced from a cellular biological sample from a human who has the epilepsy disorder; or b) have a genetic mutation associated with the epilepsy disorder.

[0249] 43. The method of any of clauses 40-42, wherein the first human neural organoid, the second human neural organoid, or both the first neural and the second neural organoid are organoids generated from induced human pluripotent stem cells (hiPSCs).Attorney Docket No: STAN-2232WOClient No: S24-410

[0250] 44. The method of clauses 40 or 43, wherein the first human neural organoid is selected from the group consisting of: a cortical organoid, a striatal organoid, a midbrain organoid, and a subpallial organoid.

[0251] 45. The method of any of clauses 40-44, wherein the first human neural organoid is a cortical organoid.

[0252] 46. The method of any of clauses 40-45, wherein the second human neural organoid is a subpallial organoid.

[0253] 47. The method of any of clauses 40-46, wherein the central nervous system location is selected from the group consisting of the frontal cortex, motor cortex, somatosensory cortex, parietal cortex, occipital cortex, temporal cortex, striatum, spinal cord, thalamus, and the cerebellum.

[0254] 48. The method of any of clauses 40-47, wherein the central nervous system location is the somatosensory cortex.

[0255] 49. The method of clause 40-48, wherein the epilepsy disorder is associated with mosaicism.

[0256] 50. The method of clause 49, wherein the epilepsy disorder is associated with a pathogenic variant of a gene selected from the group consisting of: CDKL5, CLCN4, SCN1A, SCN2A, SLC35A2, GABRA1 , GRIN2B, KCNQ2, STXBP1 , CHD2, SLC2A1 , ARX, MECP2, and PCDH19.

[0257] 51 . The method of clause 40-48, wherein the epilepsy disorder is associated with a channelopathy.

[0258] 52. The method of clause 51 , wherein the channelopathy is associated with a pathogenic variant of a gene selected from the group consisting of: SCN1 A, SCN1 B, SCN2A, SCN3A, SCN8A, KCNQ2, KCNQ3, KCNMA1 , KCNA1 , KCNA2, KCNJII, KCNT1 , CACNA1 H, CACNA1A, CHRNA4, CHRNB2, CHRNA2, GABRA1 , GABRB2, GABRB3, GABRD, and GABRG2.

[0259] 53. The method of any of clauses 40-52, wherein the characterizing comprises measuring the power spectral density of the first human neural tissue.

[0260] 54. The method of any of clauses 40-53, wherein the characterizing comprises measuring the total power of one or more of an alpha, beta, gamma, delta, or theta spectral band of the first human neural tissue.

[0261] 55. The method of any of clauses 40-54, wherein the characterizing comprises measuring spontaneous neural activity of glutamatergic neurons.

[0262] 56. The method of any of clauses 40-55, wherein the characterizing comprises measuring spontaneous inhibitory postsynaptic currents.Attorney Docket No: STAN-2232WOClient No: S24-410

[0263] 57. The method of any of clauses 40-56, wherein the characterizing comprises measuring neuronal morphology of the first human neural tissue.

[0264] 58. The method of any of clauses 40-57, wherein the characterizing comprises measuring intrinsic electrophysiological properties of the first human neural tissue.

[0265] 59. The method of any of clauses 40-58, wherein the characterizing comprises measuring maximal firing rates of the first human neural tissue.

[0266] 60. The method of any of clauses 40-59, wherein the characterizing comprises measuring gene expression in the first human neural tissue.

[0267] 61 . The method of any of clauses 40-60, wherein the characterizing comprises immunostaining the first human neural tissue.

[0268] 62. The method of any of clauses 40-61 , wherein the characterizing comprises axon tracing of the first human neural tissue.

[0269] 63. The method of any of clauses 40-62, wherein the characterizing comprises measuring intracellular calcium levels or voltage.

[0270] 64. The method of any of clauses 40-63, further comprising characterizing the non-human mammalian animal model.

[0271] 65. The method of clause 64, wherein the characterizing comprises assaying the non-human mammalian animal models behavioral responses to tasks.

[0272] 66. The method of clause 64 or 65, wherein the characterizing comprises assaying the non-human mammalian animal models memory responses to tasks.

[0273] 67. The method of any of clauses 64-66, wherein the characterizing comprises assaying the non-human mammalian animal models motor responses to tasks.

[0274] 68. The method of any of clauses 64-67, wherein the characterizing comprises assaying the non-human mammalian animal models sensory responses to tasks.

[0275] 69. The method of any of clauses 40-68, further comprising inducing a seizure before the characterizing.

[0276] 70. The method of any of clauses 40-68, further comprising inducing a seizure after the characterizing and repeating the characterizing.

[0277] 71 . The method of clauses 69 or 70, wherein the seizure is selected from the group consisting of: a temperature-induced seizure, a light-induced seizure, a sound-induced seizure, an electrical-induced seizure, a chemical-induced seizure, and an injury-induced seizure.Attorney Docket No: STAN-2232WOClient No: S24-410

[0278] 72. The method of any of clauses 40-71 , wherein the characterizing is performed using EEG.

[0279] 73. The method of any of clauses 40-72, further comprising stimulating the human neural tissue before the characterizing.

[0280] 74. The method of clause 73, wherein the stimulating is electrical stimulation.

[0281] 75. The method of clause 73, wherein the stimulating is light stimulation.

[0282] 76. The method of clause 75, wherein one or more neurons of the human neural tissue comprises a light-activatable polypeptide.

[0283] 77. The method of clause 76, wherein the light-activatable polypeptide hyperpolarizes the one or more neurons of the human neural tissue.

[0284] 78. The method of any of clauses 40-72, further comprising introducing a second and a third human neural organoid to a second central nervous system location of the newborn non-human mammal and characterizing a second human neural tissue that is produced from the second human neural organoid; wherein the third and the fourth human neural organoid is derived from an individual that does not have an epilepsy disorder.

[0285] 79. The method of clause 76, wherein the third and the fourth human neural organoids are the same as the first and the second human neural organoid except that it is derived from an individual that does not have an epilepsy disorder.

[0286] 80. The method of clause 78 or 79, wherein the third and fourth human neural organoids are introduced to the same central nervous system location to which the first human neural organoid is introduced except in the opposite brain hemisphere.

[0287] 81 . The method of any of clauses 40-80, wherein the newborn non-human mammal is a rodent.

[0288] 82. The method of clause 81 , wherein the rodent is a rat.

[0289] 83. The method of clause 81 , wherein the rodent is a mouse.

[0290] 84. The method of any clauses 40-72, wherein the newborn non-human mammal is a primate.

[0291] 85. The method of any of clauses 40-72, wherein the newborn non-human mammal is an immunocompromised non-human mammal.

[0292] 86. The method of clause 85, wherein the immunocompromised non- human mammal comprises a genetic mutation.

[0293] 87. The method of clause 85, wherein the immunocompromised non- human mammal is immunocompromised as a result of a chemical treatment.Attorney Docket No: STAN-2232WOClient No: S24-410

[0294] 88. The method of any of clauses 40-87, wherein the newborn non-human mammal is from 1 to 10 days old.

[0295] 89. A method of determining the effectiveness of a candidate agent on an epilepsy disorder, the method comprising: administering the drug to the non-human mammalian animal model produced by any of the methods of clauses 40-88; assaying the first human neural tissue; and comparing the results of the assaying with mammals administered a control agent that is not the candidate agent.

[0296] 90. The method of clause 89, wherein the epilepsy disorder is associated with mosaicism.

[0297] 91 . The method of clause 90, wherein the epilepsy disorder is associated with a pathogenic variant of a gene selected from the group consisting of: CDKL5, CLCN4, SCN1A, SCN2A, SLC35A2, GABRA1 , GRIN2B, KCNQ2, STXBP1 , CHD2, SLC2A1 , ARX, MECP2, and PCDH19.

[0298] 92. The method of clause 89, wherein the epilepsy disorder is associated with a channelopathy.

[0299] 93. The method of clause 92, wherein the channelopathy is associated with a pathogenic variant of a gene selected from the group consisting of: SCN1 A, SCN1 B, SCN2A, SCN3A, SCN8A, KCNQ2, KCNQ3, KCNMA1 , KCNA1 , KCNA2, KCNJII, KCNT1 , CACNA1 H, CACNA1A, CHRNA4, CHRNB2, CHRNA2, GABRA1 , GABRB2, GABRB3, GABRD, and GABRG2.

[0300] 94. The method of any of clauses 89-93, wherein the candidate agent is selected from the group consisting of a chemical, a small molecule, a gene therapy, and an antibody.

[0301] 95. The method of any of clauses 89-94, wherein the candidate agent is administered systemically.

[0302] 96. The method of any of clauses 89-95, wherein the candidate agent is administered locally at the site of the human neural tissue.

[0303] 97. The method any of clauses 89-95, wherein the assaying comprises inducing a seizure in the non-human mammal.

[0304] 98. The method of clause 97, wherein the seizure is selected from the group consisting of: a temperature-induced seizure, a light-induced seizure, a sound- induced seizure, an electrical-induced seizure, a chemical-induced seizure, and an injury-induced seizure.Attorney Docket No: STAN-2232WOClient No: S24-410

[0305] 99. The method of any of clauses 89-98, wherein the assaying comprises measuring neuronal morphology of the human neural tissue.

[0306] 100. The method of any of clauses 89-99, wherein the assaying comprises measuring intrinsic electrophysiological properties of the human neural tissue.

[0307] 101. The method of any of clauses 89-100, wherein the assaying comprises measuring maximal firing rates of the human neural tissue.

[0308] 102. The method of any of clauses 89-101 , wherein the assaying comprises measuring gene expression in the human neural tissue.

[0309] 103. The method of any of clauses 89-102, wherein the assaying comprises immunostaining the human neural tissue.

[0310] 104. The method of any of clauses 89-103, wherein the assaying comprises axon tracing of the human neural tissue.

[0311] 105. The method of any of clauses 89-104, wherein the characterizing comprises measuring intracellular calcium levels.

[0312] 106. The method of clause 89-105, wherein the human neural tissue comprises a calcium sensor.

[0313] 107. The method of clause 89-106, wherein the calcium sensor isGCaMP6s.

[0314] 108. A non-human mammal according to any of clauses 1 -88.

Claims

Attorney Docket No: STAN-2232WOClient No: S24-410 WHICH IS CLAIMED IS:1 . A method of modeling a human epilepsy disorder, the method comprising: introducing a first human neural organoid into a central nervous system location of a newborn non-human mammal; introducing a second human neural organoid into the central nervous system location of the newborn non-human mammal to form an assembloid from the first human neural organoid and the second human neural organoid; allowing the newborn non-human mammal to mature to produce a non- human mammalian animal model comprising a first human neural tissue; and characterizing the first human neural tissue to model the epilepsy disorder; wherein the first human neural organoid, the second human neural organoid, or the first and second human neural organoid: a) are produced from a cellular biological sample from a human who has the epilepsy disorder; or b) have a genetic mutation associated with the epilepsy disorder.

2. The method of claim 1 , wherein the first human neural organoid is selected from the group consisting of: a cortical organoid, a striatal organoid, a midbrain organoid, and a subpallial organoid.

3. The method of any of claims 1 -2, wherein the first human neural organoid is a cortical organoid and the second neural organoid is a subpallial.

4. The method of any of claims 1 -3, wherein the central nervous system location is selected from the group consisting of the frontal cortex, motor cortex, somatosensory cortex, parietal cortex, occipital cortex, temporal cortex, striatum, spinal cord, thalamus, and the cerebellum.

5. The method of claim 1 -4, wherein the epilepsy disorder is associated with mosaicism.

6. The method of claim 5, wherein the epilepsy disorder is associated with a pathogenic variant of a gene selected from the group consisting of: CDKL5, CLCN4, SCN1 A, SCN2A, SLC35A2, GABRA1 , GRIN2B, KCNQ2, STXBP1 , CHD2, SLC2A1 , ARX, MECP2, and PCDH19.Attorney Docket No: STAN-2232WOClient No: S24-4107. The method of claims 1 -6, wherein the epilepsy disorder is associated with a channelopathy associated with a pathogenic variant of a gene selected from the group consisting of: SCN1A, SCN1 B, SCN2A, SCN3A, SCN8A, KCNQ2, KCNQ3, KCNMA1 , KCNA1 , KCNA2, KCNJII, KCNT1 , CACNA1 H, CACNA1A, CHRNA4, CHRNB2, CHRNA2, GABRA1 , GABRB2, GABRB3, GABRD, and GABRG2.

8. The method of any of claims 1 -7, wherein the characterizing comprises one or more of: measuring the power spectral density of the first human neural tissue, measuring the total power of one or more of an alpha, beta, gamma, delta, or theta spectral band of the first human neural tissue, measuring spontaneous neural activity of glutamatergic neurons, measuring spontaneous inhibitory postsynaptic currents, measuring neuronal morphology of the first human neural tissue, measuring intrinsic electrophysiological properties of the first human neural tissue, measuring maximal firing rates of the first human neural tissue, measuring gene expression in the first human neural tissue, immunostaining the first human neural tissue, axon tracing of the first human neural tissue, and measuring intracellular calcium levels or voltage.

9. The method of any of claims 1-8, further comprising characterizing the nonhuman mammalian animal model, wherein the characterizing comprises one or more of: assaying the non-human mammalian animal models behavioral responses to tasks, assaying the non-human mammalian animal models memory responses to tasks, assaying the non- human mammalian animal models motor responses to tasks, assaying the non-human mammalian animal models motor responses to tasks, and assaying the non-human mammalian animal models sensory responses to tasks.

10. The method of any of claims 1 -9, further comprising inducing a seizure before the characterizing or inducing a seizure after the characterizing and repeating the characterizing, wherein the seizure is selected from the group consisting of: a temperature- induced seizure, a light-induced seizure, a sound-induced seizure, an electrical-induced seizure, a chemical-induced seizure, and an injury-induced seizure.11 . The method of any of claims 1 -10, wherein the characterizing is performed using EEG.

12. The method of any of claims 1 -11 , further comprising stimulating the humanAttorney Docket No: STAN-2232WOClient No: S24-410 neural tissue before the characterizing.

13. A method of determining the effectiveness of a candidate agent on an epilepsy disorder, the method comprising: administering the drug to the non-human mammalian animal model produced by any of the methods of claims 1 -12; assaying the first human neural tissue; and comparing the results of the assaying with mammals administered a control agent that is not the candidate agent.

14. The method of claim 13, wherein the candidate agent is selected from the group consisting of a chemical, a small molecule, a gene therapy, and an antibody.

15. The method of claims 13 or 14, wherein the candidate agent is administered systemically or locally at the site of the human neural tissue.

16. The method of any of claims 13-15, wherein the assaying comprises inducing a seizure in the non-human mammal.

17. The method of claim 16, wherein the seizure is selected from the group consisting of: a temperature-induced seizure, a light-induced seizure, a sound-induced seizure, an electrical-induced seizure, a chemical-induced seizure, and an injury-induced seizure.

18. The method of any of claims 13-17, wherein the assaying comprises one or more of: measuring neuronal morphology of the human neural tissue, measuring intrinsic electrophysiological properties of the human neural tissue, measuring maximal firing rates of the human neural tissue, measuring gene expression in the human neural tissue, immunostaining the human neural tissue, axon tracing of the human neural tissue, and measuring intracellular calcium levels.

19. The method of claims 13-18, wherein the human neural tissue comprises a calcium sensor.

20. A non-human mammal according to any of claims 1 -19.

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