METHODS AND COMPOSITIONS FOR TREATING PROGRANULIN DEFICIENCIES USING iPSC-DERIVED CELLS

Abstract

This disclosure relates to cell therapy approaches for treating progranulin (PGRN) deficiencies with human induced pluripotent stem cell (hiPSC)-derived cells. Advantageously, the hiPSC-derived cells (e.g., microglia progenitor cells) described herein can cross-correct PGRN deficiencies in damaged or diseased cells while reducing the amount of endogenous cell ablation that is needed, as demonstrated by experimental results showing restoration of PGRN levels in GRN mutant cells and brain organoids.

Description

METHODS AND COMPOSITIONS FOR TREATINGPROGRANULIN DEFICIENCIES USING iPSC-DERIVED CELLSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority of U.S. Provisional Application No. 63 / 654,873, filed May 31, 2024, and U.S. Provisional Application No. 63 / 669,633, filed July 10, 2024, the entire contents of which are each incorporated herein by reference.FIELD

[0002] This disclosure generally relates to methods and compositions of cells for treating progranulin deficiencies.BACKGROUND

[0003] Progranulin (PGRN) is a multifunctional glycoprotein encoded by the GRN gene. PRGN plays an important role in several biological processes, including lysosomal processing, inflammation, development, and wound repair. Variations in the gene dosage of GRN are associated with distinct disorders: homozygous mutations in GRN lead to neuronal ceroid lipofuscinosis 11 (CLN11), a severe lysosomal storage disorder, while heterozygous mutations cause Frontotemporal Dementia (FTD-GRN), a neurodegenerative condition.

[0004] Current experimental therapies aimed at restoring PGRN levels include adeno- associated viruses (AAVs) and recombinant protein infusions. However, these approaches face significant limitations. Recombinant proteins often fail to provide a persistent supply of PGRN, thus necessitating repeated administrations. AAV-based therapies can restore progranulin expression only in a subset of cells, which may be insufficient for therapeutic efficacy and can potentially lead to supraphy si ologi cal levels of PGRN in individual cells, posing a risk of adverse effects.SUMMARY

[0005] This disclosure relates generally to cell therapy approaches for addressing progranulin (PGRN) deficiencies. More specifically, this disclosure relates to methods andcompositions involving human induced pluripotent stem cell (hiPSC)-derived cells that can treat conditions associated with low PGRN levels. The hiPSC-derived cells described herein are capable of long-term survival in the central nervous system (CNS) while secreting wildtype levels of PGRN. By ensuring a continuous supply of PGRN, this strategy offers sustained therapeutic benefits, overcoming the limitations of existing therapies such as recombinant protein infusions and adeno-associated virus (AAV) therapies, which often fail to maintain persistent PGRN levels or cause unintended side effects.

[0006] Supporting this approach, this disclosure provides isogenic hiPSC lines that accurately model the PGRN dosage variations seen in conditions like neuronal ceroid lipofuscinosis 11 (CLN11) and Frontotemporal Dementia (FTD-GRN). These hiPSC lines are shown herein to successfully differentiate into cortical neuron progenitor cells (NPCs) and microglia progenitor cells (MPCs). Notably, experimental results demonstrate that MPCs derived from hiPSCs secrete wildtype levels of PGRN and can restore PGRN deficiencies even in GRN mutant cells through metabolic cross-correction. This capability advantageously reduces or eliminates the need for a full ablation of endogenous cells, such as microglia, prior to treatment. Metabolic cross-correction allows normal cells to provide essential factors to deficient cells, thereby correcting metabolic imbalances.

[0007] Accordingly, this disclosure offers a novel and potentially transformative cell therapy approach for treating disorders associated with GRN gene dosage variations. The methods and cell populations described herein provide a promising solution to current therapeutic challenges, paving the way for effective and long-term management of both CLN11 and FTD-GRN.

[0008] In one aspect, this disclosure provides a method of treating a progranulin deficiency in a subject, the method comprising: obtaining a population of microglia precursor cells; and administering said population of microglia precursor cells directly to the central nervous system (CNS) of the subject, wherein the administered microglia precursor cells provide a supply of progranulin to the subject’s PGRN-deficient cells.

[0009] In some embodiments, the population of microglia precursor cells are derived, in vitro, from stem cells. In some embodiments, the stem cells comprise human embryonic stem cells (hESCs) or human induced pluripotent stem cells (hiPSCs).

[0010] In some embodiments, the population of microglia precursor cells express one or more of the following PTPRC (CD45), CD14. ITGAM (CD11B). CX3CR1, MERTK, or P2RY12. In some embodiments, at least about 80% of the population of microglia precursor cells express CD14 and CD45. In some embodiments, at least about 85% of the population of microglia precursor cells express CDllb and CX3CR1. In some embodiments, at least about 70% of the population of microglia precursor cells express CD14, CD45, CDl lb, and CX3CR1.

[0011] In some embodiments, the obtaining comprises: (i) culturing a population of human pluripotent stem cells (PSCs); (ii) inducing said PSCs with BMP-4 for about 2 to about 6 days: (iii) culturing the PSCs induced with BMP -4 for about 2 to about 6 days, wherein said media comprises SCF, VEGF, and bFGF; (iv) culturing the cells from step (iii) in media for about 2 to about 6 days, wherein said media comprises SCF, IL-3, TPO, M-CSF and Flt3 for about 8 days; and (v) culturing the cells from step (iv) in M-CSF, Flt3, and GM- CSF, thereby generating microglial precursor cells.

[0012] In some embodiments, the subject is not treated to ablate endogenous microglial cells prior to administering the population of microglia precursor cells to the central nervous system (CNS) of the subject.

[0013] In some embodiments, the population of microglia precursor cells is administered to the subject via intra-cerebroventricular (ICV) injection.

[0014] In some embodiments, prior to the administering, the subject is diagnosed with neuronal ceroid lipofuscinosis 11 (CLN11) or Frontotemporal Dementia (FTD-GRN).

[0015] In some embodiments, the population of microglia precursor cells comprises between about 25 million and about 500 million microglia precursor cells. In some embodiments, the population of microglia precursor cells comprises between about 100 million and about 300 million microglia precursor cells.

[0016] In some embodiments, the treatment results in improved neurological function in the subject as assessed by standardized cognitive and motor tests.

[0017] In some embodiments, the population of microglia cells are administered to the subject with a pharmaceutically acceptable carrier.

[0018] In another aspect this disclosure provides a therapeutic composition comprising: an effective quantity of a population of microglia precursor cells for treating a progranulin deficiency in a subject; and a cell delivery solution.

[0019] In some embodiments, the population of microglia precursor cells are derived, in vitro, from stem cells. In some embodiments, the stem cells comprise human embryonic stem cells (hESCs) or human induced pluripotent stem cells (hiPSCs).

[0020] In some embodiments, the population of microglia precursor cells express one or more of the following PTPRC (CD45), CD14. ITGAM (CD11B). CX3CR1, MERTK, or P2RY12. In some embodiments, at least about 80% of the population of microglia precursor cells express CD14 and CD45. In some embodiments, at least about 85% of the population of microglia precursor cells express CDllb and CX3CR1. In some embodiments, at least about 70% of the population of microglia precursor cells express CD14, CD45, CDl lb, and CX3CR1.

[0021] In another aspect, this disclosure provides a method of treating a PRGN deficiency in a subject, the method comprising administering a population of microglia precursor cells to said subject. In some embodiments, the microglia precursor cells provide a supply of PRGN to PGRN-deficient cells of the subject. In some embodiments, the microglia precursor cells are wild-type microglia precursor cells. In some embodiments, the microglia precursor cells are generated, in vitro, from human pluripotent stem cells. In some embodiments, at least about 85% of the population of microglia precursor cells express CDllb and CX3CR1. In some embodiments, at least about 70% of the population of microglia precursor cells express CD14, CD45, CDllb, and CX3CR1. In some embodiments, the microglia precursor cells are administered to the subject via intra-cerebroventricular (ICV) injection.BRIEF DESCRIPTION OF DRAWINGS

[0022] FIG. 1 illustrates an exemplary genetic engineering strategy for generating GRN knockout (GRN KO) and R493X Frontotemporal Dementia (FTD-GRN) patient mutation lines using CRISPR / Cas9-mediated editing technology. The GRN gene is depicted in light gray, spanning the genomic region with the coding exons represented by dark gray boxes. The signal peptide for GRN is indicated by a triangle. The location of the guide RNA(gRNA) targeting the first coding exon for generating GRN KO lines is shown. The insertion site for the R493X mutation is highlighted by a gold arrow pointing to the specific location within the 11th coding exon of the GRN gene. The R493X mutation introduces an early stop codon, leading to nonsense-mediated decay and rnRNA destabilization.

[0023] FIG. 2 shows exemplary flow cytometry data demonstrating the successful differentiation of human induced pluripotent stem cells (hiPSCs) into microglia progenitor cells. The differentiated cells were analyzed for the expression of specific microglia markers, including CD45, CD14, CX3CR1, and CDllb.

[0024] FIG. 3 shows exemplary flow cytometry data demonstrating the successful differentiation of human induced pluripotent stem cells (hiPSCs) into cortical neurons. The differentiated cells were analyzed for the expression of specific neuronal markers, including TUBB3, SOX2, PAX6, FOXG1. and TBR1.

[0025] FIGS. 4A and 4B show exemplary heatmap visualizations of gene expression profiles from a bulk RNA sequencing experiment conducted on microglial progenitor cells. The heatmaps include genes associated with progranulin (PGRN) deficiency and related pathways. The rows represent individual genes, while the columns represent a biological replicate for the genotype indicated, including wild-type (WT), R493X / +, and R493X / R493X microglial progenitor cells. In particular, FIG. 4A is a heatmap visualization of relevant transcripts in WT, R493X / +, and R493X / R493X microglia progenitors and FIG. 4B is a heatmap visualization of relevant transcripts in WT, R493X / +, and R493X / R493X microglia progenitors with and without co-culture (CC) with WT microglia progenitors or the addition of recombinant human PGRN (rhPGRN). The color scale indicates the level of gene expression. Genes shown in the heatmap include: APOE, CTSD, CTSL, and CSF1R, which are known to be upregulated in PGRN deficiency; GRN, which is the gene encoding progranulin, demonstrating differential expression in mutant versus wild-type cells; and CXCR1, C l qA, MMP9, C l QB, wtiich are other genes showing significant changes in expression levels.

[0026] FIGS. 5A-5C are exemplary bar graph representations of gene expression from bulk RNA sequencing experiments. The bar graphs illustrate mean expression levels of CTSD (FIG. 5A), LGALS3 (FIG. 5B), and APOE (FIG. 5C) in microglial progenitor cells under the genetic conditions indicated along the x axis. Each circle represent the normalizedcounts for a single replicate. The bars represent the mean expression levels of the indicated genes in wild-type (WT), R493X / +, and R493X / R493X microglial progenitor cells from three replicates.

[0027] FIGS. 6A and 6B show exemplary results of TNF alpha (FIG. 6A) and IL-6 (FIG. 6B) levels in microglial progenitor cells, measured using Homogeneous Time- Resolved Fluorescence (HTRF) assay. These bar graphs compare TNF alpha (FIG. 6A) and IL-6 (FIG. 6B) levels in wild-type (WT), R493X / +, R493X / R493X, GRN-KO / +, and GRN- KO / - microglial progenitor cells over a period of 12 days. The data points show mean values with standard error bars.

[0028] FIGS. 7A-7F show exemplary results from ELISA-based assays measuring intracellular and secreted levels of key proteins in wild-ty pe (WT), R493X / +, and R493X / R493X microglial progenitor cells. The data illustrate the impact of GRN mutations on protein expression and secretion. FIGS. 7A-7C compare levels of intracellular Cathepsin, Galectin-3, and APOE, respectively. FIGS. 7D-7F compare levels of secreted Cathepsin, Galectin-3, and APOE, respectively.

[0029] FIGS. 8A-8C show exemplary results of specific intracellular protein levels in cortical neurons with GRN mutations. These data present the results of ELISA-based assays measuring the intracellular levels of key proteins in wild-type (WT), R493X / +, and R493X / R493X cortical neurons. FIG. 8A compares intracellular levels of Cathepsin D, demonstrating elevated levels of Cathepsin D in R493X / + and R493X / R493X cortical neurons as compared to WT. FIG. 8B compares intracellular levels of LAMP 1, demonstrating R493X / + and R493X / R493X neurons exhibit higher levels of LAMP 1 compared to WT, reflecting changes in lysosomal membrane protein expression. FIG. 8C compares intracellular levels of LAMP2, demonstrating increased levels of LAMP2 in R493X / + and R493X / R493X cortical neurons relative to WT. further indicating alterations in lysosomal function.

[0030] FIGS. 9A-9C show exemplary experimental results taken from neuroburst assays of cortical neurons with GRN mutations. These figures present the results of a neuroburst assays conducted to evaluate neuronal activity7in wild-type (WT), R493X / +, and R493X / R493X cortical neurons. The data highlight the impact of GRN mutations on various aspects of neuronal function. FIG. 9A shows exemplar}7mean correlations of neuronal firing.FIG. 9B shows exemplary results of number of firing neurons. FIG. 9C shows exemplary results of burst strength.

[0031] FIG. 10 shows exemplary results of an immunofluorescence analysis of TDP-43 localization in cortical neurons. This figure presents immunofluorescence images showing the localization of TDP-43 in wild-type (WT). R493X / +, and R493X / R493X cortical neurons. The images highlight the impact of GRN mutations on the subcellular distribution of TDP-43, a protein associated with neurodegenerative diseases.

[0032] FIG. 11 is a schematic of a co-culture system for metabolic cross-correction in GRN-KO models. This figure illustrates the experimental setup used to assess the capacity of wild-type (WT) microglia progenitor cells to cross-correct PGRN deficiency in GRN-KO models through metabolic cross-correction. The schematic demonstrates the principle of metabolic cross-correction, whereby healthy WT microglia progenitors secrete PGRN that can be absorbed by GRN-KO cells, potentially restoring PGRN levels and correcting the metabolic imbalance associated with GRN deficiency. This experimental setup is valuable for investigating the therapeutic potential of WT microglia progenitors in treating PGRN-related disorders.

[0033] FIGS. 12A-12C shows exemplary experimental results of cross-correction assays demonstrating PGRN restoration in GRN-KO cells. These figures present the results of crosscorrection assays performed to evaluate the ability of wild-type (WT) microglia progenitor cells to restore PGRN levels in GRN knockout (GRN-KO) cells. The assays were conducted using GRN-KO human induced pluripotent stem cells (hiPSCs), microglia progenitors, and brain organoids. FIG. 12A shows exemplary results of cross-correction in GRN-KO hiPSCs. The schematic shows WT hiPSCs secreting PGRN, which is taken up by GRN-KO hiPSCs. The bar graph compares PGRN levels in GRN-KO hiPSCs co-cultured with WT hiPSCs to GRN-KO hiPSCs alone. FIG. 12B shows exemplary results of cross-correction in GRN-KO microglia progenitors. The schematic illustrates WT microglia progenitors secreting PGRN, which is absorbed by GRN-KO microglia progenitors. The bar graph shows PGRN levels in GRN-KO microglia progenitors co-cultured with WT microglia progenitors compared to GRN-KO microglia progenitors alone. FIG. 12C shows exemplary results of cross-correction in GRN-KO brain organoids. The schematic depicts WT microglia progenitors secreting PGRN, which is taken up by various cell types within the GRN-KO brain organoid, including neurons, astrocytes, oligodendrocytes (Oligos), oligodendrocyte precursor cells (OPCs), andneural progenitor cells (NPCs). The bar graph compares PGRN levels in GRN-KO brain organoids co-cultured with WT microglia progenitors to GRN-KO brain organoids alone.

[0034] FIG. 13 shows exemplary experimental results demonstrating that crosscorrecting with WT microglia progenitors reduces GAL3 accumulation in granulin-deficient microglia cells. In particular FIG. 13 shows a bar graph illustrating GAL3 protein levels, measured by enzyme-linked immunosorbent assay (ELISA) and reported as a percentage of uncorrected control, from cell lysates of granulin-deficient microglia, i.e., granulin-knock out microglia (GRN-KO microglia) and GRN-KO microglia cross-corrected by co-culture with WT microglia progenitor cells.

[0035] FIG. 14 shows exemplary experimental results demonstrating that crosscorrection with wild- ty pe microglia progenitor cells increases GCase activity' in granulin- deficient microglia. In particular, FIG. 14 shows a bar graph illustrating (3-glucocerebrosidase (GCase) enzyme activity, measured by fluorometric assay and reported as a percentage of uncorrected control, in cell lysates of GRN-KO microglia, GRN-KO microglia crosscorrected by co-culture with WT microglia progenitor cells, R493XAmut / mut microglia, and R493XAmut / mut microglia co-cultured with wild-type microglia progenitors.

[0036] FIG. 15 shows exemplary experimental results demonstrating that crosscorrection with WT microglia progenitors modulates mRNA expression levels of inflammatory7genes in granulin-deficient microglia. In particular, FIG. 15 shows a bar graph comparing the expression levels of pro-inflammatory genes (CXCL10, IDO1, IL6, GBP1, IL12B, and TNF), measured by quantitative RT-PCR and expressed as dCt geometric mean (Actin, GAPDH, TBP, FTL), expressed in GRN-KO microglia and GRN-KO microglia co- cultured with WT microglia progenitors.

[0037] FIG. 16 shows exemplary experimental results demonstrating that crosscorrection restores PGRN levels in granulin-deficient cortical NPCs after co-culture with WT microglia progenitor cells. In particular, FIG. 16 shows a bar graph illustrating PGRN protein levels, measured by immunofluorescence and shown as mean intensity as a percentage of WT control, from GRN+ / - neurons, GRN+ / - neurons co-cultured with WT microglia progenitors, GRN- / - neurons, and GRN- / - neurons co-cultured with WT microglia progenitors.

[0038] FIG. 17 shows exemplary experimental results demonstrating that crosscorrection restores PGRN levels in granulin-deficient R493X cortical NPCs after co-culturewith WT microglia progenitor cells. In particular, FIG. 17 show s a bar graph illustrating PGRN protein levels, measured by immunofluorescence and shown as mean intensity as a percentage of WT control, from R493x mutant / + neurons, R493x mutant / + neurons cocultured with WT microglia progenitors, R493x mutant / mutant neurons, and R493x mutant / mutant neurons co-cultured with WT microglia progenitors.DETAILED DESCRIPTION

[0039] This disclosure relates generally to novel cell therapy methods and compositions to address deficiencies in progranulin (PGRN). More specifically, this disclosure provides human induced pluripotent stem cell (hiPSC)-derived cells, and methods thereof, that are capable of treating conditions associated with low PGRN levels. These hiPSC-derived cells are capable of long-term survival in the central nervous system (CNS) while continuously secreting wildtype levels of PGRN. This approach ensures a steady supply of PGRN, addressing the shortcomings of existing therapies like recombinant protein infusions and adeno-associated virus (AAV) therapies, which often fail to provide sustained PGRN levels and can lead to unwanted side effects.

[0040] A key benefit of this approach is the reduced or eliminated need to remove endogenous cells, such as microglia, before treatment. The cross-corrective properties of the hiPSC-derived cells enable them to restore PGRN levels in deficient cells through metabolic cross-correction. This method allows healthy cells to supply necessary7factors to PGRN- deficient cells, correcting metabolic imbalances with or without requiring cell ablation.

[0041] This disclosure includes isogenic hiPSC lines that model the PGRN dosage variations seen in disorders like neuronal ceroid lipofuscinosis 11 (CLN11) and Frontotemporal Dementia (FTD-GRN). These hiPSC lines have been successfully differentiated into cortical neuron progenitor cells (NPCs) and microglia progenitor cells (MPCs). Experimental evidence shows that MPCs derived from these hiPSCs can secrete wildtype levels of PGRN and restore its deficiency in GRN mutant cells via metabolic crosscorrection.

[0042] This cell therapy approach is potentially transformative for treating disorders related to GRN gene dosage variations. The described methods and cell populations offer aninnovative solution to existing therapeutic challenges, enabling effective and long-term management of both CLN 11 and FTD-GRN.I, Definitions

[0043] The following definitions supplement those in the art and are directed to the present disclosure only. The following definitions are not to be imputed to any related or unrelated case, e.g, to any commonly owned patent or patent application. Although some methods and materials similar or equivalent to those described herein can be used to practice features of the disclosure, some preferred materials and methods are described herein. Accordingly, the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0044] Unless otherwise defined herein, scientific and technical terms used in connection with the present application shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.

[0045] It should be understood that this invention is not limited to the particular methodology, protocols, and reagents, etc., described herein and as such may vary. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention, which is defined solely by the claims.

[0046] As used herein, the articles “a,” an." and "the" are used herein to refer to one or to more than one (z.e., to at least one) of the grammatical object of the article. By way of example, “an element’' means one element or more than one element.

[0047] The use of the alternative (e.g. “or”) should be understood to mean either one, both, or any combination thereof of the alternatives.

[0048] As used herein, the terms “ablate,” or “ablation,” and their grammatical equivalents refer to the process of removing, destroying, or eradicating cells in a subject before a cell therapy (e.g, for the administration of a population of microglia progenitor cells). This can be achieved through various methods, including surgical excision, chemical agents, thermal energy (such as radiofrequency or laser), or other physical means. In the context of this disclosure, ablate or ablation refers to the targeted removal or destruction of atleast about 25% of endogenous microglial cells in the central nerv ous system (CNS) prior to administering therapeutic cells.

[0049] As used herein, the term '‘about” or ‘'approximately” refers to a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length that varies by as much as 15%, 10%, 9%, 8%, 7%, 6%. 5%, 4%, 3%, 2% or 1% compared to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length. In some instances, the term “about” or “approximately” refers a range of quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length ± 15%, ± 10%, ± 9%, ± 8%, ± 7%, ± 6%, ± 5%, ± 4%, ± 3%, ± 2%, or ± 1% of a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length. \

[0050] As used herein, the terms “administration,” “administering” and variants thereof refer to the introduction of a composition or therapeutic agent (e.g.. a population of cells) into a subject. Administration includes concurrent and sequential introduction of the composition or therapeutic agent. Administration of the composition or therapeutic agent (e.g, a population of cells) into a subject is by any suitable route, including surgically.Administration of the composition or therapeutic agent (e.g, a population of cells) into a subject can be achieved by injection, for example, by intra-cerebroventricular injection (ICV).

[0051] As used herein, the term “central nervous system (CNS)” refers to the brain and / or spinal cord of a subject.

[0052] As used herein, the terms “cross-correction”, and “metabolic cross-correction” refer to a process whereby healthy cells (e.g, microglial precursor cells) secrete factors (e.g, PGRN) that are taken up by deficient cells, thereby restoring the deficient cells' function.

[0053] As used herein, the term “differentiation,” and its grammatical equivalents, refers to a process by which a stem cell or progenitor cell alters from one cell type to a more specialized cell type. Each specialized cell type in an organism can express a subset of all the genes that constitute the genome of the cell. Each cell type can be defined by its particular pattern of regulated gene expression. Cell differentiation can thus be described as a transition of a cell from one cell type to another cell type coincident with a switch from one pattern of gene expression to another.

[0054] As used herein, the term “endogenous"’ refers to a gene, nucleic acid, polypeptide, protein, cell, etc., that is normally present in a particular cell. For example, an endogenous cell may be a cell that is normally present in the body of a subject.

[0055] As used herein, the terms “enhance” and “increase” are interchangeable and refer to any statistically significant increase in an amount of an agent (e.g., PRGN protein) as compared to a control or reference. For example, an increase in PRGN can refer to an increase of about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% as compared to a control.

[0056] As used herein, the term “frontotemporal dementia (FTD-GRN)” refers to a neurodegenerative condition associated with mutations in the GRN gene, resulting in reduced levels of progranulin.

[0057] As used herein. “GRN” refers to a granulin precursor gene. The GRN gene encodes progranulin, a multifunctional glycoprotein involved in various biological processes including inflammation, cell growth, and lysosomal function. Mutations or deficiencies in the GRN gene are linked to several neurodegenerative and lysosomal storage disorders, such as neuronal ceroid lipofuscinosis 11 (CLN11) and Frontotemporal Dementia (FTD-GRN). An exemplary GRN gene is available as Gene ID: 2896 (e.g., available at the website www[dot]ncbi[dot]nlm[dot]nih[dot]gov / gene / 2896).

[0058] As used herein, the term “human embryonic stem cell,” often abbreviated as “hESC,” and their grammatical equivalents, refers to pluripotent stem cells derived from the inner cell mass of a blastocyst, an early-stage preimplantation embryo. These cells are characterized by their ability to self-renew indefinitely and their capacity to differentiate into any cell type of the three primary germ layers: ectoderm, mesoderm, and endoderm.

[0059] As used herein, the term “human induced pluripotent stem cell,” often abbreviated as “hiPSC,” and their grammatical equivalents, refers to stem cells derived from adult somatic cells that have been reprogrammed to an embryonic stem cell-like state by introducing specific pluripotency-associated genes. These cells are characterized by their ability to self-renew indefinitely and their capacity to differentiate into any cell type of the three primary' germ layers: ectoderm, mesoderm, and endoderm.

[0060] As used herein, the terms “microglia precursor cell(s)'’ and “microglia progenitor cell(s)” refer to early-stage cells that have the potential to differentiate into microglia, the resident immune cells of the central nervous system (CNS), and express markers, including but not limited to, CD45, CDl lb, CD33, CD14, and CX3CR1. Microglia precursor cells can become microglia when delivered into the brain of live animals, and may express canonical microglia markers including but not limited to TMEM119, IBA1, CD163. CX3CR1, CD45, CD206.

[0061] As used herein, the term “neuronal ceroid lipofuscinosis 11 (CLN11)” refers to a lysosomal storage disorder caused by mutations in the GRN gene, leading to progranulin deficiency.

[0062] As used herein, the terms “patient,” “subject,” “individual,” and the like are used interchangeably and refer to any animal, or cells thereof, whether in vitro or in situ, amenable to the compositions and methods described herein. In some instances, the patient, subject or individual is a human.

[0063] As used herein, the term “progranulin,” which is abbreviated as “PGRN,” refers to a glycoprotein encoded by the GRN gene. PGRN is involved in various biological processes including cell growth, wound repair, inflammation, and plays an important role in maintaining lysosomal function and neuronal health. Deficiency or mutation in progranulin is associated with several neurodegenerative and lysosomal storage disorders, such as neuronal ceroid lipofuscinosis 11 (CLN11) and Frontotemporal Dementia (FTD-GRN). Progranulin can be secreted by various cell types, including microglia and microglia precursor cells, in the central nervous system (CNS).

[0064] As used herein, the term “progranulin (PGRN) deficiency” refers to a condition characterized by insufficient levels of progranulin, a protein encoded by the GRN gene, which is implicated in various neurodegenerative and lysosomal storage diseases such as neuronal ceroid lipofuscinosis 11 (CLN11) and Frontotemporal Dementia (FTD-GRN).

[0065] As used herein, the term “pharmaceutically acceptable excipient, carrier or diluent” refers to any material which, when combined with an active ingredient (e.g., a population of cells), allows the ingredient to retain biological activity and is non-reactive with the subject’s immune system. Examples include, but are not limited to, any of the standard pharmaceutical excipients, carriers, or diluents, such as a phosphate buffered saline solution.normal saline, water, emulsions such as oil / water emulsion, and various types of wetting agents.

[0066] As used herein, the term '‘substantially” or “essentially” refers to a quantity7, level, value, number, frequency, percentage, dimension, size, amount, weight or length that is about 90%, 91%. 92%, 93%. 94%. 95%. 96%. 97%. 98%. or 99% or higher compared to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length. In some instances, the terms “essentially the same” or “substantially the same” refer a range of quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length that is about the same as a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length.

[0067] As used herein, the term “treat,” or a grammatical equivalent thereof, refers to a means to reduce the frequency or severity of at least one sign or symptom of a disease or disorder experienced by a subject.

[0068] As used herein, a statement that a cell or population of cells is “positive” for a particular marker, or “expresses” a particular marker, refers to the detectable presence on or in the cell of a particular marker, for example, a surface marker or an intracellular marker, such as transcription factors. When referring to a surface marker, the term refers to the presence of surface expression as detected by flow cytometry, for example, by staining with an antibody that specifically binds to the marker and detecting said antibody, wherein the staining is detectable by flow cytometry at a level substantially above the staining detected carrying out the same procedure with an isotype-matched control or fluorescence minus one (FMO) gating control under otherwise identical conditions and / or at a level substantially similar to that for cell known to be positive for the marker, and / or at a level substantially higher than that for a cell known to be negative for the marker.

[0069] As used herein, a statement that a cell or population of cells is “negative” for a particular marker, or fails to express a particular marker or gene, refers to the absence of substantial detectable presence on or in the cell of a particular marker, such as a surface marker or an intracellular marker, such as transcription factors. When referring to a surface marker, the term refers to the absence of surface expression as detected by flow cytometry, for example, by staining with an antibody that specifically binds to the marker and detecting said antibody, wherein the staining is not detected by flow cytometry at a level substantiallyabove the staining detected carry ing out the same procedure with an isoty pe-matched control or fluorescence minus one (FMO) gating control under otherwise identical conditions, and / or at a level substantially lower than that for cell known to be positive for the marker, or at a level substantially similar as compared to that for a cell known to be negative for the marker.

[0070] As used herein, the terms "progenitor cell(s),” and "precursor cell(s),” and their grammatical equivalents, refer to a descendant of a stem cell that can further differentiate into specialized cell types within a particular cell lineage. These cells are committed to a particular cell lineage and possess the ability' to proliferate and give rise to specialized cells through the process of differentiation.

[0071] As used herein, the term "‘stem cell” refers to a cell with the ability to divide for indefinite periods in culture and to give rise to specialized cells.

[0072] As used herein, the term “substantially” or “essentially” refers to a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length that is about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or higher compared to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length. In some instances, the terms “essentially the same” or “substantially the same” refer a range of quantity', level, value, number, frequency, percentage, dimension, size, amount, weight or length that is about the same as a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, w eight or length.

[0073] As used herein, the term “wild-type” refers to a cell that has not been genetically modified or edited to modulate expression of a metabolite, e.g., progranulin. Wild-type cells can be generated, in vitro, from stem cells as described herein.

[0074] Throughout this disclosure, various aspects of the invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numencal values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example. 1, 2, 2.7, 3. 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.II. Generation of microglia precursor cells from pluripotent stem cells

[0075] Disclosed herein is a serum- and feeder-free protocol to differentiate PSCs (including iPSCs) towards microglia precursor cells. In some embodiments, the microglia precursor cells are generated, in vitro, from human pluripotent stem cells. In some embodiments, the microglia precursor cells that are wild-type microglia precursor cells. In some embodiments, the microglia precursor cells that can provide a supply of progranulin to PGRN-deficient cells of a subject.

[0076] Various methods known in the art can be employed in connection with the present disclosure. Muffat et.al, Nat Med. 2016 Nov; 22(11): 1358- 1367, Pandaya et. al. Nat Neurosci. 2017 May; 20(5): 753-759, Abud et. al, Neuron 2017 Apr 19;94(2):278-293, Douvaras et. al, Stem Cell Reports, Volume 8, Issue 6, P1516-1524, June 06, 2017, Van Wilgenburg PLOS ONE. https: / / doi.org / 10.1371 / joumal.pone.0071098-Aug 2013, Haenseler et.al, Stem Cell Reports 2017 Jun 6;8(6): 1727-1742 and Takata et.al. Immunity 2017 Jul 18;47(1 ): 183-198, each of which are incorporated by reference. Microglia cells arise from myeloid progenitors in the yolk sack during embryonic development. In an attempt to mimic the embryonic development of these myeloid cells, primitive streak-like cells were generated from PSCs, followed by hematopoietic and myelopoietic cocktails in serum-free media. This resulted in the appearance of microglia precursor cells in the supernatant fraction of the culture that expressed markers including but not limited to CD45 (also known as PTPRC), CD14, CX3CR1, CD33, CDllb (also known as ITGAM), MERTK and P2RY12. These microglia precursor cells can continue to be generated in the supernatant fraction of the culture for a considerable amount of time, often reaching 3 to 4 months. Microglia precursor cells are typically collected and frozen around 30 days after initiation of the differentiation protocol (the exact timing of collection is PSC-line dependent and should be experimentally defined based on the day of maximum yield). The yield is usually between 25 and 120 myeloid cells for every starting PSC, and their post-thaw viability’ is 85±10%.

[0077] In some embodiments the present disclosure provides methods for generating microglia precursor cells from pluripotent stem cells. In some embodiments the pluripotent stem cells are from any mammalian species, but preferably from humans. In some embodiments the pluripotent stem cells are either induced pluripotent stem cells (“iPS cells” or ‘’iPSCs”), or embryonic stem cells (“ES cells” or “ESCs”). Such methods involve a stepwhere the pluripotent stem cells are cultured under conditions that induce microglia precursor cell differentiation, leading to the generation of CD 45 + / CD14 + / CX3CR1 + cells. In some embodiments the differentiation medium comprises BMP4, GM- CSF, VEGF, SCF, IL3, TPO, M-CSF and FLT31. In some embodiments the medium further optionally comprises bFGF.

[0078] In some of the embodiments, the pluripotent stem cells are cultured under conditions that induce microglia precursor cell differentiation, leading to the generation of CX3CR1 + microglia precursor cells. In some of the embodiments, the pluripotent stem cells are cultured under conditions that induce microglia precursor cell differentiation, leading to the generation of CD45+ microglia precursor cells. In some embodiments the pluripotent stem cells are cultured or expanded in a bioreactor. In some embodiments the pluripotent stem cells are cultured in a cell factory under active gassing. By “active gassing” is meant exerting or applying a gas mixture pressure gradient in the cell factory' or cell factories. Gas mixtures contemplated by the present disclosure include ratios of about 1 % to about 20% CO2 to about 80% to about 99% air, about 3% CO2 to about 97% air and about 5% CO2 to about 95% air.

[0079] In some of the embodiments of the present disclosure that involve culturing pluripotent stem cells under conditions that induce microglia precursor cell differentiation (leading to the generation of CD45+ / CD14+ / CX3CR1+ microglia precursor cells), a multi- step process is used in which the cells are cultured with different combinations of cytokines and tissue culture media at each stage. Steps in these multi-step processes may result in inducing differentiation of pluripotent stem cells into primitive hemangioblasts, and / or inducing differentiation of primitive hemangioblasts into myeloid progenitors.

[0080] In some embodiments, the methods provided herein for the generation of CD45+ / CD14 + / CX3CR1+ microglia precursor cells from pluripotent stem cells comprise performing one or more of the following steps: First, contacting a cell culture with a first composition comprising BMP4 in a culture medium, wherein when the cell culture is initially contacted with the first composition the cell culture comprises pluripotent stem cells. A small molecule able to activate the same pathway as BMP4 can be used; Second, contacting the cell culture with a second composition comprising one or more of SCF, and VEGF, and optionally bFGF, (for example each of SCF, and VEGF, with or without bFGF) in a hematopoietic cell medium; Third, contacting the cell culture with a third composition comprising one or moreof SCF. IL-3. TPO. M-CSF, and FLT3 ligand ( for example each of SCF, IL-3, TPO, M-CSF, and FLT3 ligand) in a hematopoietic cell medium; and fourth, contacting the cell culture with a fourth composition comprising one or more of M-CSF , FLT3 ligand, and GM-CSF (for example each of M-CSF, FLT3 ligand, and GM-CSF) in a hematopoietic cell medium. In some embodiments all of the above four steps are performed in order. In some of such embodiments the medium used for any of these four steps is a serum free medium. In some of such embodiments the medium used for any of these four steps is a chemically defined medium.

[0081] In the first of the above four steps, in some embodiments a tissue culture medium suitable for maintenance of stem cells is used, while in other embodiments a tissue culture medium suitable for differentiation of stem cells is used. In the last three of the above four steps, any suitable hematopoietic cell medium can be used.

[0082] In some embodiments, when carrying out the methods described above or elsewhere herein for the generation of microglia precursor cells from pluripotent stem cells, instead of discarding the tissue culture supernatant when performing media changes, the supernatant is centrifuged, and the cells present in the supernatant are recovered and added back to the cell cultures. This is advantageous because certain of the key cell types induced during the conversion of pluripotent stem cells to microglia precursor cells are found predominantly in the cell supernatants - as opposed to being in the layer of cells that adheres to the cell culture plates. Thus, in some embodiments, when media is changed cells present in the culture supernatant are recovered and added back to the cell cultures. In some embodiments, for media exchanges performed when the cells are in contact with the third composition or the fourth composition, cells present in the culture supernatant are recovered and added back to the cell cultures. In some embodiments the present disclosure provides myeloid cells or microglial progenitor cells, such as those produced by the methods described herein. In some embodiments the present disclosure provides a “substantially pure’?populations of such cells.

[0083] Accordingly, in certain aspects this disclosure provides methods of generating populations of microglia precursor cells for the treatment of a progranulin deficiency in a subject. In some embodiments, human iPSCs are plated onto Vitronectin (Thermo Scientific, A14700) at 1.0 x 104 cells / cm2 in Essential 8 medium (Thermo Scientific, A1517001) containing about lOpM ofY- 27632 (Tocris, 1254) for about 24 hours. PSCs can be culturedfor an additional 2 days in Essential 8 medium with daily medium changes before being induced by Essential 6 (Thermo Scientific, A1516401) medium supplemented with about 80ng / mL of BMP -4 (R&D Systems, 314E-GMP-050). BMP-4 induction can be continued for about 4 days with daily medium changes before the cultures were changed to StemPro-34 SFM medium (Thermo Scientific, 10639011) (containing IX GlutaMAX Thermo Scientific, 35050061) supplemented with about lOOng / mL of SCF (R&D Systems. 255B-GMP-050), about 80ng / mL of VEGF (R&D Systems, 293-GMP-050) and about 25ng / mL of bFGF (R&D Systems, 233-GMP-025) for 2 days with daily medium changes. On about day 6 and about day 8, the cells can be cultured with StemPro-34 SFM medium containing about 50ng / mL of SCF, about 50ng / mL of IL-3 (R&D Systems, 203-GMP-050). about 50ng / mL of M-CSF (R&D Systems, 216-GMP-500). about 50ng / mL of Flt3 ligand (R&D Systems. 308EGMP- 050) and about 5ng / mL of TPO (R&D Systems, 288-TPE-050). Starting on about day 10, cells from the supernatant fraction can be pelleted, resuspended in the same fresh medium as about day 6 and about day 8 and placed back into their respective vessel. Beginning on about day 14. cells in the supernatant fraction were pelleted, resuspended in StemPro-34 SFM medium containing about 50ng / mL of Flt-3. about 50ng / mL of M-CSF and about 25ng / mL of GM-CSF (R&D Systems, 215- GMP-050) and placed back into their respective vessel. On about Day 14 medium change can be repeated every' other day until the cells in the supernatant reached a concentration of about > 1.0 x 10A6 live cells per mL. Beginning on about day 18 and every’ other day after, cell counts can be performed prior to pelleting the cells in the supernatant for medium exchange to determine the harvest date.

[0084] In some embodiments, the population of microglia precursor cells are cultured from about 1 to about 30 days, from about 2 to about 25 days, from about 2 to about 20 days, from about 2 to about 18 days, from about 2 to about 15 days, from about 2 to about 10 days, from about 2 to about 8 days, from about 2 to about 6 days or from about 2 to about 4 days. In some embodiments, the population of microglia precursor cells are cultured for about 2 days, for about 4 days, for about 6 days or about 8 days, for about 10 days, for about 12 days for about 14 days, for about 16 days, for about 18 days, for about 20 days, for about 22 days, for about 24 days, for about 26 days, for about 28 days or for about 30 days.

[0085] In some embodiments, the population of microglia precursor cells are at least about 70% CD45+, at least about 75% CD45+, at least about 80% CD45+. at least about 85% CD45+, at least about 90% CD45+, at least 91 % CD45+, at least 92% CD45+, at least 93%CD45+, at least 94% CD45+, or at least 95% CD45+. In some embodiments, the population of microglia precursor cells are at least about 70% CD45+. In some embodiments, the population of microglia precursor cells are at least about 75% CD45+. In some embodiments, the population of microglia precursor cells are at least about 80% CD45+. In some embodiments, the population of microglia precursor cells are at least about 85% CD45+. In some embodiments, the population of microglia precursor cells are at least about 90% CD45+.

[0086] In some embodiments, the population of microglia precursor cells are at least about 75% CD14+, at least about 80% CD14+, at least about 85% CD14+, at least about 90% CD14+, at least 91 % CD14+, at least 92% CD14+. at least 93% CD14+. at least 94% CD14+, or at least 95% CD14+. In some embodiments, the population of microglia precursor cells are at least about 75% CD14+. In some embodiments, the population of microglia precursor cells are at least about 80% CD14+. In some embodiments, the population of microglia precursor cells are at least about 85% CD14+. In some embodiments, the population of microglia precursor cells are at least about 90% CD14+.

[0087] In some embodiments, the population of microglia precursor cells are at least about 75% CDl lb+, at least about 80% CDllb+, at least about 85% CDllb+, at least about 90% CDllb+, at least 91 % CDllb+, at least 92% CDl lb+, at least 93% CDllb+, at least 94% CDllb+, or at least 95% CDllb+. In some embodiments, the population of microglia precursor cells are at least about 75% CDllb+. In some embodiments, the population of microglia precursor cells are at least about 80% CDllb+. In some embodiments, the population of microglia precursor cells are at least about 85% CDl lb+. In some embodiments, the population of microglia precursor cells are at least about 90% CDllb+.

[0088] In some embodiments, the population of microglia precursor cells are at least about 75% CX3CR1+. at least about 80% CX3CR1+. at least about 85% CX3CR1+. at least about 90% CX3CR1+, at least 91 % CX3CR1+, at least 92% CX3CR1+, at least 93% CX3CR1+, at least 94% CX3CR1+, or at least 95% CX3CR1+. In some embodiments, the population of microglia precursor cells are at least about 75% CX3CR1+. In some embodiments, the population of microglia precursor cells are at least about 80% CX3CR1+. In some embodiments, the population of microglia precursor cells are at least about 85% CX3CR1+. In some embodiments, the population of microglia precursor cells are at least about 90% CX3CR1+.

[0089] In some embodiments, the population of microglia precursor cells are at least about 75% CD14+ / CD45+, at least about 80% CD14+ / CD45+, at least about 85% CD14+ / CD45+, at least about 90% CD14+ / CD45+, at least 91% CD14+ / CD45+, at least 92% CD14+ / CD45+, at least 93% CD14+ / CD45+, at least 94% CD14+ / CD45+, or at least 95% CD14+ / CD45+. In some embodiments, the population of microglia precursor cells are at least about 70% CD14+ / CD45+. In some embodiments, the population of microglia precursor cells are at least about 75% CD14+ / CD45+. In some embodiments, the population of microglia precursor cells are at least about 80% CD14+ / CD45+. In some embodiments, the population of microglia precursor cells are at least about 85% CD14+ / CD45+. In some embodiments, the population of microglia precursor cells are at least about 90% CD14+ / CD45+.

[0090] In some embodiments, the population of microglia precursor cells are at least about 75% CDllb+ / CX3CRl+, at least about 80% CDllb+ / CX3CRl+, at least about 85% CDl lb+ / CX3CRl+, at least about 90% CDllb+ / CX3CRl+, at least 91% CDllb+ / CX3CRl+, at least 92% CDllb+ / CX3CRl+, at least 93% CDl lb+ / CX3CRl+, at least 94% CDllb+ / CX3CRl+, or at least 95% CDllb+ / CX3CRl+. In some embodiments, the population of microglia precursor cells are at least about 75% CDllb+ / CX3CRl+. In some embodiments, the population of microglia precursor cells are at least about 80% CDllb+ / CX3CRl+. In some embodiments, the population of microglia precursor cells are at least about 85% CDllb+ / CX3CRl+. In some embodiments, the population of microglia precursor cells are at least about 90% CDllb+ / CX3CRl+.

[0091] In some embodiments, the population of microglia precursor cells are at least about 72% CD45+ / CD14+ / CX3CR1+, at least about 73% CD45+ / CD14+ / CX3CR1+, at least about 74% CD45+ / CD14+ / CX3CR1+, at least about 75% CD45+ / CD14+ / CX3CR1+, at least about 80% CD45+ / CD14+ / CX3CR1+, or at least about 85% CD45+ / CD14+ / CX3CR1+, at least about 90% CD45+ / CD14+ / CX3CRI+ or at least about 95% CD45+ / CD14+ / CX3CR1+. In some embodiments, the population of microglia precursor cells are at least about 65% CD45+ / CD14+ / CX3CR1+. In some embodiments, the population of microglia precursor cells are at least about 70% CD45+ / CD14+ / CX3CR1+. In some embodiments, the population of microglia precursor cells are at least about 75% CD45+ / CD14+ / CX3CR1+. In some embodiments, the population of microglia precursor cells are at least about 80% CD45+ / CD14+ / CX3CR1+.

[0092] In some embodiments, the population of microglia precursor cells are at least about 60% CDl lb+ / CD45+ / CD14+ / CX3CRl+ at least about 65% CDllb+ / CD45+ / CD14+ / CX3CRl+, at least about 68% CDllb+ / CD45+ / CD14+ / CX3CRl+, at least about 69% CDllb+ / CD45+ / CD14+ / CX3CRl+, at least about 70% CDllb+ / CD45+ / CD14+ / CX3CRl+, or at least about 71% CDl lb+ / CD45+ / CD14+ / CX3CRl+, at least about 72% CDl lb+ / CD45+ / CD14+ / CX3CRl+, or at least about 75% CDl lb+ / CD45+ / CD14+ / CX3CRl. In some embodiments, the population of microglia precursor cells are at least about 60% CDllb+ / CD45+ / CD14+ / CX3CRl+. In some embodiments, the population of microglia precursor cells are at least about 65% CDllb+ / CD45+ / CD14+ / CX3CRl+. In some embodiments, the population of microglia precursor cells are at least about 70% CDllb+ / CD45+ / CD14+ / CX3CRl+. In some embodiments, the population of microglia precursor cells are at least about 75% CDllb+ / CD45+ / CD14+ / CX3CRl+.

[0093] Microglia precursor cells can be isolated and counted on an automated cell counter NC-200 (Chemometec) prior to freezing. Cells can be resuspended in BamBanker (Waka Chemicals, 30214681)) or STEM-CELLBANKER GMP grade (amsbio, 11924) freezing medium and transferred to cry ogenic vials (Thermo Scientific). Cryogenic vials with cells can be frozen with a controlled rate freezer (CBS CRF2101). Cells can be transferred to liquid nitrogen (gas phase) for long-term storage. To thaw microglia precursor cells, cryogenic vials can be transferred to a 37°C water bath for -2 minutes until a small ice crystal remained. In a biosafety cabinet, cells from the cry ogenic vial can be transferred to a centrifuge tube and quenched with 1 mL of either RPMI-1640 (Thermo Scientific, 11-875- 101) or StemPro-34 SFM medium in a drop-wise fashion. The cells can be counted on the NC-200 and quenched with an additional 3mL of its respective medium. Cells can be centrifuged at 250g for 5 minutes and resuspended in the appropriate assay or culture medium.Ill, Compositions

[0094] Certain aspects of the disclosure provide therapeutic compositions that include a population of microglia precursor cells. In some embodiments, the therapeutic compositions includes an effective quantity of a population of microglia precursor cells to treat a progranulin deficiency. In some embodiments, the therapeutic compositions include aneffective quantity of a population of microglia precursor cells to treat neuronal ceroid lipofuscinosis 11 (CLN11).

[0095] In some embodiments, the therapeutic composition includes a population of microglia precursor cells that are generated, in vitro, from human pluripotent stem cells (e.g., by a method described herein). In some embodiments, the therapeutic composition includes a population of microglia precursor cells that are wild-type microglia precursor cells. In some embodiments, the therapeutic composition includes a population of microglia precursor cells that can provide a supply of progranulin to PGRN-deficient cells of a subj ect.

[0096] In some embodiments, the therapeutic compositions include an effective quantity of a population of microglia precursor cells to treat Frontotemporal Dementia (FTD-GRN). In some embodiments, the therapeutic composition includes a population of microglia precursor cells that are at least about 70% CD45+. at least about 75% CD45+, at least about 80% CD45+, at least about 85% CD45+, at least about 90% CD45+. at least 91 % CD45+, at least 92% CD45+, at least 93% CD45+, at least 94% CD45+, or at least 95% CD45+. In some embodiments, the therapeutic composition includes a population of microglia precursor cells that are at least about 70% CD45+. In some embodiments, the therapeutic composition includes a population of microglia precursor cells that are at least about 75% CD45+. In some embodiments, the therapeutic composition includes a population of microglia precursor cells that are at least about 80% CD45+. In some embodiments, the therapeutic composition includes a population of microglia precursor cells that are at least about 85% CD45+. In some embodiments, the therapeutic composition includes a population of microglia precursor cells that are at least about 90% CD45+.

[0097] In some embodiments, the therapeutic composition includes a population of microglia precursor cells that are at least about 75% CD14+, at least about 80% CD14+, at least about 85% CD14+. at least about 90% CD14+, at least 91 % CD14+, at least 92% CD14+, at least 93% CD14+, at least 94% CD14+, or at least 95% CD14+. In some embodiments, the therapeutic composition includes a population of microglia precursor cells that are at least about 75% CD14+. In some embodiments, the therapeutic composition includes a population of microglia precursor cells that are at least about 80% CD14+. In some embodiments, the therapeutic composition includes a population of microglia precursor cells that are at least about 85% CD14+. In some embodiments, the therapeutic composition includes a population of microglia precursor cells that are at least about 90% CD14+.

[0098] In some embodiments, the therapeutic composition includes a population of microglia precursor cells that are at least about 75% CD1 lb+. at least about 80% CD1 lb+. at least about 85% CD1 lb+, at least about 90% CD1 lb+, at least 91 % CD1 lb+, at least 92% CDllb+, at least 93% CDllb+, at least 94% CDllb+, or at least 95% CDllb+. In some embodiments, the therapeutic composition includes a population of microglia precursor cells that are at least about 75% CD1 lb+. In some embodiments, the therapeutic composition includes a population of microglia precursor cells that are at least about 80% CDl lb+. In some embodiments, the therapeutic composition includes a population of microglia precursor cells that are at least about 85% CDllb+. In some embodiments, the therapeutic composition includes a population of microglia precursor cells that are at least about 90% CDllb+.

[0099] In some embodiments, the therapeutic composition includes a population of microglia precursor cells that are at least about 75% CX3CR1+, at least about 80% CX3CR1+. at least about 85% CX3CR1+. at least about 90% CX3CR1+. at least 91 % CX3CR1+ at least 92% CX3CR1+, at least 93% CX3CR1+, at least 94% CX3CR1+, or at least 95% CX3CR1+. In some embodiments, the therapeutic composition includes a population of microglia precursor cells that are at least about 75% CX3CR1+. In some embodiments, the therapeutic composition includes a population of microglia precursor cells that are at least about 80% CX3CR1+. In some embodiments, the therapeutic composition includes a population of microglia precursor cells that are at least about 85% CX3CR1+. In some embodiments, the therapeutic composition includes a population of microglia precursor cells that are at least about 90% CX3CR1+.

[0100] In some embodiments, the therapeutic composition includes a population of microglia precursor cells that are at least about 75% CD14+ / CD45+, at least about 80% CD14+ / CD45+, at least about 85% CD14+ / CD45+, at least about 90% CD14+ / CD45+, at least 91% CD14+ / CD45+, at least 92% CD14+ / CD45+, at least 93% CD14+ / CD45+, at least 94% CD14+ / CD45+, or at least 95% CD14+ / CD45+. In some embodiments, the therapeutic composition includes a population of microglia precursor cells that are at least about 70% CD14+ / CD45+. In some embodiments, the therapeutic composition includes a population of microglia precursor cells that are at least about 75% CD14+ / CD45+. In some embodiments, the therapeutic composition includes a population of microglia precursor cells that are at least about 80% CD14+ / CD45+. In some embodiments, the therapeutic composition includes a population of microglia precursor cells that are at least about 85% CD14+ / CD45+. In someembodiments, the therapeutic composition includes a population of microglia precursor cells that are at least about 90% CD14+ / CD45+.

[0101] In some embodiments, the therapeutic composition includes a population of microglia precursor cells that are at least about 75% CDl lb+ / CX3CRl+, at least about 80% CDl lb+ / CX3CRl+, at least about 85% CDllb+ / CX3CRl+, at least about 90% CDllb+ / CX3CRl+, at least 91% CDllb+ / CX3CRl+, at least 92% CDllb+ / CX3CRl+, at least 93% CDl lb+ / CX3CRl+, at least 94% CDllb+ / CX3CRl+, or at least 95% CDllb+ / CX3CRl+. In some embodiments, the therapeutic composition includes a population of microglia precursor cells that are at least about 75% CDllb+ / CX3CRl+. In some embodiments, the therapeutic composition includes a population of microglia precursor cells that are at least about 80% CDllb+ / CX3CRl+. In some embodiments, the therapeutic composition includes a population of microglia precursor cells that are at least about 85% CDllb+ / CX3CRl+. In some embodiments, the therapeutic composition includes a population of microglia precursor cells that are at least about 90% CDllb+ / CX3CRl+.

[0102] In some embodiments, the therapeutic composition includes a population of microglia precursor cells that are at least about 72% CD45+ / CD14+ / CX3CR1+, at least about 73% CD45+ / CD14+ / CX3CRI+, at least about 74% CD45+ / CD14+ / CX3CR1+, at least about 75% CD45+ / CD14+ / CX3CR1+, at least about 80% CD45+ / CD14+ / CX3CR1+, or at least about 85% CD45+ / CD14+ / CX3CR1+, at least about 90% CD45+ / CD14+ / CX3CR1+, or at least about 95% CD45+ / CD14+ / CX3CR1+. In some embodiments, the therapeutic composition includes a population of microglia precursor cells that are at least about 65% CD45+ / CD14+ / CX3CR1+. In some embodiments, the therapeutic composition includes a population of microglia precursor cells that are at least about 70% CD45+ / CD14+ / CX3CR1+. In some embodiments, the therapeutic composition includes a population of microglia precursor cells that are at least about 75% CD45+ / CD14+ / CX3CR1+. In some embodiments, the therapeutic composition includes a population of microglia precursor cells that s are at least about 80% CD45+ / CD14+ / CX3CR1+.

[0103] In some embodiments, the therapeutic composition includes a population of microglia precursor cells that are at least about 60% CDllb+ / CD45+ / CD14+ / CX3CRl+, at least about 65% CDllb+ / CD45+ / CD14+ / CX3CRl+, at least about 68%CDllb+ / CD45+ / CD14+ / CX3CRl+, at least about 69% CDllb+ / CD45+ / CD14+ / CX3CRl+,at least about 70% CDllb+ / CD45+ / CD14+ / CX3CRl+, or at least about 71% CDl lb+ / CD45+ / CD14+ / CX3CRl+, at least about 72% CDllb+ / CD45+ / CD14+ / CX3CRl+, or at least about 75% CD1 lb+ / CD45+ / CD14+ / CX3CRl. In some embodiments, the therapeutic composition includes a population of microglia precursor cells that are at least about 60% CDllb+ / CD45+ / CD14+ / CX3CRl+. In some embodiments, the therapeutic composition includes a population of microglia precursor cells that are at least about 65% CDllb+ / CD45+ / CD14+ / CX3CRl+. In some embodiments, the therapeutic composition includes a population of microglia precursor cells that are at least about 70% CDllb+ / CD45+ / CD14+ / CX3CRl+. In some embodiments, the therapeutic composition includes a population of microglia precursor cells that are at least about 75% CD1 lb+ / CD45+ / CD14+ / CX3CRl+.

[0104] In some embodiments, the therapeutic composition comprises a population of microglia precursor cells, the population comprising about 25 x 10A6 to about 1250 x 10A6 cells, about 50 x 10A6 to about 1000 x 10A6 cells, about 100 x 10A6 to about 500 x 10A6 cells, about 100 x 10A6 to about 300 x 10A6 cells and about 150 x 10A6 to about 250 x 10A6 cells.

[0105] In some embodiments, the therapeutic composition includes a population of microglia precursor cells, and a delivery solution that includes one or more energy components, one or more pH buffers, one or more salts, one or more stabilizing agents, or any combination thereof, for example, a delivery solution as described in International Application No PCT / US23 / 21961, which is incorporated by reference.

[0106] In some embodiments, the therapeutic composition includes a population of microglia precursor cells (e.g., a population of microglia precursor cells described herein), and a delivery solution that includes one or more energy components. An energy' component is any component that can provide chemical energy to one or more cells. In some embodiments, the one or more energy components include a sugar. Exemplary sugars that can be included in the delivery solution include, but are not limited to, dextrose, fructose, galactose, glucose, lactose, maltose, and sucrose. In some embodiments, the sugar is dextrose. The delivery solution can include any amount of the one or more energy sources that achieve a desired effect (e.g., treatment of a progranulin deficiency). In some embodiments, the delivery solution includes one or more energy sources at a concentration of about 24.5 mM to about 24.8 mM, about 24.4 mM to about 24.9 mM, about 24.3 mM to about 25.0 mM, about 24.1 mM to about 25.2 mM, about 23.9 mM to about 25.4 mM, about23.7 mM to about 25.6 rnM. In some embodiments, the delivery solution includes one or more energy sources at a concentration of about 23.5 mM, about 23 mM. about 22 mM, about 21 mM, about 20 rnM or less. In some embodiments, the delivery solution includes one or more energy sources at a concentration of about 26 mM, about 27 mM, about 28 mM, about 29 mM, or about 30 rnM or more.

[0107] In some embodiments, the therapeutic composition includes a population of microglia precursor cells (e.g., a population of microglia precursor cells described herein), and a delivery solution that includes one or more pH buffers. The one or more pH buffers can include any suitable buffering agent, such as a zwitterionic organic chemical buffering agent, examples of which include, but are not limited to, 4-(2-hy droxy ethyl)- 1- piperazineethanesulfonic acid (HEPES), sodium bicarbonate, 4-Morpholinepropanesulfonic acid, 3 -propanesulfonic acid (MOPS), and 2-(N-morpholino)ethanesulfonic acid (MES). The delivery' solution can include any amount of the one or more pH buffers that achieves a desired effect (e.g., treatment of A progranulin deficiency in a subject). In some embodiments, the delivery solution includes one or more pH buffers at a concentration of about 10.6 rnM to about 10.9 mM, about 10.5 mM to about 11.0 mM, about 10.4 mM to about 11.1 mM, about 10.2 rnM to about 11.3 rnM, about 10.0 mM to about 11.5 mM, or about 9.8 mM to about 11.7 mM. In some embodiments, the delivery solution includes one or more pH buffers at a concentration of about 9.5 mM. about 9 mM. about 8 mM. about 7 rnM, about 6 rnM or less, about 12 mM, about 13 mM, about 14 mM, about 15 mM, or about 16 mM. In some embodiments, the one or more pH buffers include one or more of 4-(2- hy droxy ethyl)- 1 -piperazineethanesulfonic acid (HEPES), sodium bicarbonate, 4- Morpholinepropanesulfonic acid, 3 -propanesulfonic acid (MOPS), and 2-(N- morpholinojethanesulfonic acid (MES).

[0108] In some embodiments, the therapeutic composition includes a population of microglia precursor cells (e.g.. a population of microglia precursor cells described herein), and a delivery solution that includes one or more salts. In some embodiments, the one or more salts include one or more of calcium chloride, magnesium chloride, potassium chloride, sodium phosphate monobasic, or sodium chloride.

[0109] In some embodiments, the one or more salts include calcium chloride. The delivery solution can include calcium chloride in any amount suitable to achieve the desired effect (e.g. , treatment of A progranulin deficiency). In some embodiments, the delivery'solution includes calcium chloride at a concentration of about 1.6 mM to about 1.9 mM, about 1.5 mM to about 2.0 mM, about 1.4 mM to about 2. 1 mM, about 1.3 mM to about 2.2 mM, about 1.1 mM to about 2.4 mM, about 0.9 mM to about 2.6 mM, or about 0.7 mM to about 2.8 mM. In some embodiments, the delivery solution includes calcium chloride at a concentration of about 0.5 mM, about 0.3 mM, or about 0. 1 mM or less. In some embodiments, the delivery solution includes calcium chloride at a concentration of about 3 mM, about 3.5 mM, about 4 mM, or about 5 mM or more.

[0110] In some embodiments, the one or more salts include magnesium chloride, delivery solution can include magnesium chloride in any amount suitable to achieve the desired effect (e.g. , treatment of A progranulin deficiency). In some embodiments, the delivery solution includes magnesium chloride at a concentration of about 0.7 mM to about 1.0 mM, about 0.6 mM to about 1.1 mM, about 0.5 mM to about 1.2 mM, about 0.4 mM to about 1.4 mM, about 0.3 mM to about 1.6 mM, about 0.2 mM to about 1.9 mM, or about 0. 1 mM to about 2.2 mM. In some embodiments, the delivery solution includes magnesium chloride at a concentration of about 0.05 mM or less, about 2.5 mM, or about 3.0 mM or more.

[0111] In some embodiments, the one or more salts include potassium chloride. The delivery solution can include potassium chloride in any amount suitable to achieve the desired effect (e.g. , treatment of A progranulin deficiency). In some embodiments, the deliver}' solution includes potassium chloride at a concentration of about 5.1 mM to about 5.4 mM, about 5.0 mM to about 5.5 mM, about 4.9 mM to about 5.7 mM, about 4.8 mM to about 5.8 mM, about 4.6 mM to about 6.0 mM, about 4.4. mM to about 6.2 mM, or about 4.2 mM to about 6.4 mM. In some embodiments, the delivery solution includes calcium chloride at a concentration of about 4.0 mM, about 3.5 mM, or about 3.0 mM or less. In some embodiments, the delivery solution includes calcium chloride at a concentration of about 6.5 mM, about 7.0 mM, about 7.5 mM, or about 8.0 mM or more.

[0112] In some embodiments, the one or more salts include sodium phosphate monobasic. The deliver}' solution can include sodium phosphate monobasic in any amount suitable to achieve the desired effect (e.g.. treatment of A progranulin deficiency). In some embodiments, the delivery solution includes sodium phosphate monobasic at a concentration of about 0.88 mM to about 0.91 mM, about 0.87 mM to about 0.92 mM, about 0.86 mM to about 0.93 mM, about 0.85 mM to about 0.94 mM, about 0.83 mM to about 0.96 mM, orabout 0.81 mM to about 0.98 mM. In some embodiments, the delivery solution includes sodium phosphate monobasic at a concentration of about 0.8 mM, about 0.75 mM, or about 0.7 mM or less. In some embodiments, the delivery solution includes sodium phosphate monobasic at a concentration of about 1.0 mM, about 1.05 mM, or about 1.1 mM or more.

[0113] In some embodiments, the one or more salts include sodium chloride. The delivery solution can include sodium chloride in any amount suitable to achieve the desired effect (e.g. , treatment of A progranulin deficiency). In some embodiments, the delivery solution includes sodium chloride at a concentration of about 119 mM to about 122 mM, about 118 mM to about 123 mM, about 117 mM to about 124 mM, about 115 mM to about 126 mM. or about 113 mM to about 128 mM. In some embodiments, the delivery solution includes sodium chloride at a concentration of about 110 mM, about 105 mM, or about 100 mM or less. In some embodiments, the delivery’ solution includes sodium chloride at a concentration of about 130 mM, about 135 mM, or about 140 mM or more. In some embodiments, the delivery solution includes sodium chloride at a concentration of about 74 mM to about 77 mM, about 73 mM to about 78 mM, about 72 mM to about 79 mM, about 70 mM to about 81 mM, about 68 mM to about 83 mM, or about 66 mM to about 85 mM. In some embodiments, the delivery solution includes sodium chloride at a concentration of about 65 mM, about 60 mM. or about 55 mM or less. In some embodiments, the delivery solution includes sodium chloride at a concentration of about 90 mM, about 95 mM. or about 100 mM or more. In some embodiments, the delivery' solution includes sodium chloride at a concentration of about 93 mM to about 96 mM, about 92 mM to about 97 mM, about 91 mM to about 98 mM, about 90 mM to about 99 mM, about 88 mM to about 101 mM, about 85 mM to about 103 mM. or about 83 mM to about 105 mM. In some embodiments, the delivery solution includes sodium chloride at a concentration of about 80 mM, about 75 mM, or about 70 mM or less. In some embodiments, the delivery solution includes sodium chloride at a concentration of about 105 mM, about 110 mM or about 115 mM or more.

[0114] In some embodiments, the therapeutic composition includes a population of microglia precursor cells (e.g., a population of microglia precursor cells described herein), and a delivery7solution that includes one or more stabilizing agents. A stabilizing agent is to any component that can act to reduce or prevent degradation of other solution components. The one or more stabilizing agents can include any suitable stabilizing agent, such, but not limited to one or more of a protein, such as one or more albumins, such as recombinantalbumin (rHSA), Dextran (including Dextran 40, as one example), Poloxamer (including Poloxamer 188 as one example). Additionally, the one or more stabilizing agents can include one or more of the following, which also may be an excipient of the delivery solution: polyethylene glycol, carboxymethyl cellulose, hyaluronic acid, starches, acrylates, methacrylates, polyvinyl alcohols, polyethylene oxides, polypropylene oxides, polyacrylates, polyvinylpyrrolidone, polymethacrylate, poly lactic-co-gly colic acids, polyacrylamides, polylactides, chitosans, gums, guar gums, xantham gums, carrageenans, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, hydroxyethyl cellulose, cyclodextrin derivatives, beta-cyclodextrin derivatives, alginates, calcium alginates, and stearates. In some embodiments, the one or more stabilizing agents include one or more of recombinant albumin (rHSA), Dextran, and Poloxamer.

[0115] In some embodiments, the one or more stabilizing agents include recombinant albumin (rHSA). The delivery solution can include rHSA in any amount suitable to achieve the desired effect (e.g.. treatment of A progranulin deficiency). In some embodiments, the delivery solution includes rHSA at a concentration of about 0.07 w / w% to about 0.09 w / w%, about 0.06 w / w% to about 0.1 w / w%, about 0.05 w / w% to about 0.11 w / w%, about 0.04 w / w% to about 0. 12 w / w%, about 0.03 w / w% to about 0. 13 w / w%, about 0.02 w / w% to about 0.15 w / w%, or about 0.01 w / w% to about 0.17 w / w%. In some embodiments, the delivery solution includes rHSA at a concentration of about 0.005 w / w% or less, about 0.2 w / w%, or about 0.25 w / w% or more. In some embodiments, the delivery solution includes rHSA at a concentration of about 0.08 w / w% to about 0. 11 w / w%, about 0.07 w / w% to about 0. 12 w / w%, about 0.06 w / w% to about 0. 13 w / w%, about 0.05 w / w% to about 0. 14 w / w%, about 0.04 w / w% to about 0.16 w / w%, about 0.03 w / w% to about 0.18 w / w%, about 0.02 w / w% to about 0.2 w / w%, about 0.01 w / w% 10 about 0.22 w / w%, or about 0.05 w / w% or less. In some embodiments, the delivery solution includes rHSA at a concentration of about 0.25 w / w%, or about 0.3 w / w%, or about 0.35 w / w% or more. In some embodiments, the delivery solution includes rHSA at a concentration of about 6.50 w / w% to about 6.8 w / w%, about 6.4 w / w% to about 6.9 w / w%, about 6.3 w / w% to about 7.0 w / w%, about 6.1 w / w% to about 7.2 w / w%, about 5.9 w / w% to about 7.4 w / w%, or about 5.7 w / w% to about 7.6 w / w%. In some embodiments, the delivery7solution includes rHSA at a concentration of about 5.5 w / w%, about 5.0 w / w%, or about 4.5 w / w% or less. In some embodiments, the delivery solution includes rHSA at a concentration of about 8.0 w / w%, or about 8.5 w / w%, or about 9.0 w / w% or more.

[0116] In some embodiments, the one or more stabilizing agents include Dextran. The delivery solution can include Dextran in any amount suitable to achieve the desired effect (e.g. , treatment of A progranulin deficiency). In some embodiments, the delivery solution includes Dextran at a concentration of about 17.27 w / w% to about 17.30 w / w%, about 17.26 w / w% to about 17.31 w / w%, about 17.24 w / w% to about 17.33 w / w%, about 17.2 w / w% to about 17.35 w / w%, about 17.1 w / w% to about 17.4 w / w%, or about 17.0 w / w% to about 17.5 w / w%. In some embodiments, the delivery solution includes Dextran at a concentration of about 16.5 w / w%, about 16.0 w / w%, or about 15.0 w / w% or less. In some embodiments, the delivery solution includes Dextran at a concentration of about 18.0 w / w%, about 18.5 w / w%, or about 19.0 w / w% or more. In some embodiments, the delivery solution includes Dextran at a concentration of about 13.02 w / w% to about 13.05 w / w%, about 13.0 w / w% to about 13.1 w / w%, about 12.9 w / w% to about 13.2 w / w%, about 12.7 w / w% to about 13.4 w / w%, about 12.5 w / w% to about 13.5 w / w%, or about 12.2 w / w% to about 13.8 w / w%. In some embodiments, the delivery solution includes Dextran at a concentration of about 12.0 w / w%, about 11.5 w / w%, or about 11.0 w / w% or less. In some embodiments, the delivery solution includes Dextran at a concentration of about 14.0 w / w%, about 14.5 w / w%, or about 15.0 w / w% or more.

[0117] In some embodiments, the one or more stabilizing agents include Poloxamer. The delivery solution can include Poloxamer in any amount suitable to achieve the desired effect (e.g., treatment of a progranulin deficiency). In some embodiments, the delivery solution includes Poloxamer at a concentration of about 0.07 w / w% to about 0.09 w / w%, about 0.06 w / w% to about 0.1 w / w%, about 0.05 w / w% to about 0.11 w / w%, about 0.04 w / w% to about 0.12 w / w%, about 0.03 w / w% to about 0.13 w / w%. about 0.02 w / w% to about 0.15 w / w%, or about 0.01 w / w% to about 0.17 w / w%. In some embodiments, the delivery solution includes Poloxamer at a concentration of about 0.005 w / w% or less, about 0.2 w / w%, or about 0.25 w / w% or more.

[0118] In certain embodiments, the therapeutic composition includes a population of microglia precursor cells (e.g., a population of microglia precursor cells described herein), a delivery solution, and a cryoprotectant. Exemplary cryoprotectants include, but are not limited to, dimethylsulfoxide (DMSO), glycerol, polyethylene glycol, sucrose, trehalose, dextrose, or combinations thereof.

[0119] In certain embodiments, the therapeutic composition includes a population of microglia precursor cells (e.g.. a population of microglia precursor cells described herein), a delivery solution, and a biocompatible scaffold or matrix. In some embodiments, the biocompatible scaffold or matrix includes one or more of extracellular matrix material, synthetic polymers, cytokines, collagen, polypeptides or proteins, polysaccharides including fibronectin, laminin, keratin, fibrin, fibrinogen, hyaluronic acid, heparin sulfate, chondroitin sulfate, agarose or gelatin, and hydrogel.

[0120] In some embodiments, there are several components that are not included in the delivery solution and can be excluded wholly or below detectable limits. Some examples of components that can be excluded are: certain components of animal origin; certain stabilizing agents such as human serum albumin (HSA); certain salts such as zinc sulfate, sodium bicarbonate, and ferric nitrate; certain pH indicators such as Phenol Red; certain sources of energy such as sodium pyruvate: certain amino acids such as Glycine, L-Alanine, L-Arginine hydrochloride, L-Asparagine-H2O, L-Glutamine, L-Cysteine, L-Histidine hydrochloride- H2O, L-Isoleucine, L-Leucine, L-Lysine hydrochloride, L-Methionine, L-Phenylalanine, L- Proline, L-Serine, L-Threonine, L-Try ptophan, L-Tyrosine, and L-Valine; and certain vitamins such as Ascorbic Acid, Choline Chloride, D-Calcium pantothenate, Folic Acid, Niacinamide, Pyridoxal hydrochloride, Riboflavin, Thiamine hydrochloride, Vitamin B12 and i-Inositol.

[0121] In some embodiments, the cell delivery' solution includes an energy source component, such as, D-Glucose (Dextrose), one or more pH buffers, such as, HEPES, a poloxamer. such as, Poloxamer 188. a dextran, such as, dextran 40. and recombinant albumin, in combination with one or more salts. In some embodiments, the one or more salts are selected from calcium chloride, magnesium chloride, potassium chloride, sodium chloride, and sodium phosphate monobasic. In some embodiments, the one or more salts include calcium chloride, magnesium chloride, potassium chloride, sodium chloride, and sodium phosphate monobasic. For example, in one embodiment, the cell delivery solution comprises D-Glucose (Dextrose) at a concentration of about 24 mM, Poloxamer 188 at a concentration of about 0.08% w / w, dextran 40 at a concentration of about 17% w / w, HEPES at a concentration of about 10 mM, recombinant human serum albumin at a concentration of about 0.08% w / w, calcium chloride at a concentration of about 2 mM, magnesium chloride at a concentration of about 0.8 mM, potassium chloride at a concentration of about 5 mM,sodium chloride at a concentration of about 83 mM, and sodium phosphate monobasic at a concentration of about 0.89 mM. In another embodiment, the cell delivery solution comprises Neurobasal Medium, L-Glutamine at a concentration of about 2 mM, human serum albumin at a concentration of 0.1% w / w, and L-Ascorbic Acid at a concentration of about 200 pM.IV. Methods of treatment

[0122] Disclosed herein are methods and populations of cells (z.e., microglia precursor cells) for treating subj ects having a progranulin (PRGN) deficiency.

[0123] Populations of microglia precursor cells are generated from pluripotent stem cells, derived from a healthy donor. In some embodiments, the microglia precursor cells are generated, in vitro, from human pluripotent stem cells (e.g., by a method described herein). In some embodiments, the microglia precursor cells are wild-type microglia precursor cells. Populations of the PSC-derived microglia precursor cells are injected into the central nervous system (i.e., tissue in and / or around the brain and / or spinal cord) of a subject to be treated. In some embodiments, the microglia precursor cells provide a supply of progranulin to PGRN- deficient cells of the subject.

[0124] In one embodiment, microglia precursor cells are delivered systemically by intravenous (IV) administration. In another embodiment, microglia precursor cells are delivered to the Central Nervous System via intra-cerebroventricular (ICV) injection or directly into the cerebrospinal fluid (CSF) of the subject to be treated. In another embodiment, the microglia precursor cells are administered via intra-cerebroventricular (ICV) injection into the lateral ventricles of the brain, the site where the cerebrospinal fluid (CSF) is produced, and the CSF flow starts. The direct injection of microglia precursor cells into the cerebrospinal fluid (CSF) is used to bypass the blood-brain barrier. Alternatively, the disclosure contemplates intraparenchymal administration of microglia precursor cells. For intraparenchymal administration, the microglia precursor cells are delivered directly into the brain parenchyma, that includes but is not limited to the striatum, forebrain and hippocampus, as representative sites of parenchymal administrations.

[0125] The amount of microglia precursor cells to be administered via any of the aforementioned routes is in the range of about 25 x 10A6 to about 1250 x 10A6 cells, about 50x 10A6 to about 1000 x 10A6 cells, about 100 x 10A6 to about 500 x 10A6 cells, about 100 x 10A6 to about 300 x 10A6 cells and about 150 x 10A6 to about 250 x 10A6 cells.

[0126] These specific deliver}7methods ensure engraftment of the injected microglial progenitor cells / microglia precursor cells and delivery of the enzyme of interest in the CNS. The levels of PGRN production are stable for long periods of time as the PSC-derived microglia precursor cells become resident microglia cells of the tissue, providing a steady and continuous supply of the missing enzyme. By “stable for long periods of time” is meant about 5 months to about 25 years, about 10 months to about 25 years, about 15 months to about 25 years, about 20 months to about 25 years, about 2 years to about 25 years, about 5 years to about 25 years and about 10 years to about 25 years. Thus, the missing PGRN is constantly and consistently secreted from the transplanted cells and is entering the host’s damaged or diseased cells, correcting the pathologic impact of deficient PGRN levels, thereby, resulting in correction of the underlying pathophysiology in the CNS tissues. Some amount of the PGRN produced in the CNS by the engrafted PSC-derived microglia precursor cells also accesses the blood stream, delivering the PGRN to the periphery as well. After transplantation, the level of the PGRN can be monitored at different time points in a serum sample, a urine sample or a cerebrospinal fluid sample of the subject. In one embodiment the level of PGRN is monitored at 1 month, 6 months, 12 months and 24 months after transplantation and annually thereafter. A sustained increase in total PRGN levels of more than about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80% or about 90% is indicative of successful treatment of said progranulin deficiency.

[0127] In some embodiments, assessment of the efficacy of the treatment disclosed herein can be show n by improvement of gross motor function as measured by the Peabody Developmental Motor Scale or Gross Motor Function Measure 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 15 months, 2 years, 3 years.4 years post-treatment. Appropriate clinical standard assessments are appreciated by the skilled practitioner.

[0128] In some embodiments, this disclosure provides methods of treating a progranulin deficiency by administering a population of microglia precursor cells to a subject, wherein the subject is not treated by ablation prior to the administration of the population of microglia precursor cells. Advantageously, this approach significantly simplifies the treatment processand reduces potential complications associated with ablative procedures. Specifically, the subject retains a substantial proportion of their endogenous microglial population at the time of treatment.

[0129] In some embodiments, this disclosure provides a method of treating a progranulin deficiency by administering a population of microglia precursor cells following only a partial ablation of the subject’s endogenous microglial cells, distinguishing it from a full ablation approach. Partial ablation refers to the selective removal or reduction of a portion of the endogenous microglial population, rather than completely eradicating all existing microglial cells. This partial ablation can be achieved through targeted therapies that specifically reduce the number of dysfunctional or overactive microglia, while preserving a significant proportion of the healthy microglial population. In some embodiments, the subject, at the time of treatment, retains a substantial fraction of their endogenous microglial cells — typically at least about 25%, at least about 30%, at least about 40%, or at least about 50%, at least about 60%, at least about 70%. at least about 80%. at least about 90%, at least about 95%, or more — ensuring that essential microglial functions such as immune surveillance and neural support are maintained. Following the partial ablation, microglia precursor cells derived from human induced pluripotent stem cells (hiPSCs) or human embryonic stem cells (hESCs) are administered to the central nervous system (CNS) of the subject.

[0130] Accordingly, in some embodiments, the population of microglia precursor cells are administered to a subject who, at the time of treatment, maintain at least about 30%, at least about 40%, at least about 50%, at least about 60%. at least about 70%, at least about 80%. at least about 90%. at least about 95%, or more, of their endogenous microglial cells. This retention of endogenous microglia allows the natural microglial population to continue performing its essential functions, such as immune surveillance and maintenance of neural homeostasis, while the administered microglia precursor cells supplement the deficient progranulin levels.

[0131] In some embodiments, the microglia precursor cells used in this method are derived from human induced pluripotent stem cells (hiPSCs) or human embryonic stem cells (hESCs) and are capable of differentiating into fully functional microglia once administered into the central nervous system (CNS) of the subject. These precursor cells are administered to the subject to provide sufficient levels of progranulin secretion to thereby address the PRGN deficiency without necessitating the removal of existing microglia. This approachoffers a significant advantage in treating progranulin-related disorders, such as neuronal ceroid lipofuscinosis 11 (CLN11) and Frontotemporal Dementia (FTD-GRN), by providing a continuous supply of progranulin through the newly introduced microglia precursor cells, while the existing microglia continue to support neural health and function.

[0132] In accordance with the present disclosure successful treatment of the progranulin deficiency is provided herein and is recognized by the skilled practitioner by neurocognitive assessment and gross motor assessment measured over a period of time. Neurocognitive assessment of the treated subject is also recognized by halting or delaying the progression of neurocognitive decline using standard clinically approved neurocognition tests.

[0133] The methods and compositions described in this disclosure are designed to treat a range of diseases and disorders associated with a progranulin (PGRN) deficiency. These conditions include, but are not limited to, neuronal ceroid lipofuscinosis 11 (CLN11), a lysosomal storage disorder characterized by the accumulation of lipofuscin in neural tissues, leading to neurodegeneration and severe neurological symptoms. Additionally, the methods are applicable for the treatment of Frontotemporal Dementia associated with GRN mutations (FTD-GRN), a neurodegenerative condition marked by the progressive loss of neurons in the frontal and temporal lobes of the brain, resulting in cognitive decline, behavioral changes, and motor dysfunction. Other potential applications include treating neuroinflammatory conditions and other neurodegenerative diseases where PGRN levels are implicated in the pathogenesis, such as Alzheimer’s disease, Parkinson’s disease, and amyotrophic lateral sclerosis (ALS). By restoring normal PGRN levels through the administration of microglia precursor cells, these methods offer a promising therapeutic strategy to mitigate the symptoms and progression of these debilitating disorders.EXAMPLESExample 1: Generation and Differentiation of hiPSC Lines

[0134] This Example describes experimental work that was conducted to generate and characterize human induced pluripotent stem cell (hiPSC) lines with specific GRN gene mutations. This Example also describes experimental work that w as conducted to differentiate engineered hiPSC lines into microglia progenitor cells and cortical neurons. The aim of this work was to create cellular models that recapitulate certain genetic variationsassociated with neuronal ceroid lipofuscinosis 11 (CLN11) and Frontotemporal Dementia (FTD-GRN).

[0135] In particular, hiPSCs were engineered to model GRN homozygous and heterozygous knockouts. These GRN knockout (KO) hiPSC lines were generated using CRISPR / Cas9-mediated editing technology with a guide RNA (gRNA) targeting the first coding exon of the GRN gene.

[0136] FIG. 1 outlines an exemplary' engineering strategy for generating model cell lines.

[0137] An R493X Frontotemporal Dementia (FTD-GRN) patient hiPSC mutation line was generated using CRISPR / Cas-mediated editing technology with a gRNA targeting the 11th coding exon of the GRN gene. A single-stranded oligodeoxynucleotide (ssODN) template containing the R493X single nucleotide polymorphism (SNP) flanked by homology’ sequences was used. The R493X knock-in SNP introduced an early stop codon, leading to nonsense-mediated decay and mRNA destabilization.

[0138] Through the use of CRISPR / Cas technology, hiPSC lines were generated that recapitulate various genotypes relevant to both FTD-GRN and neuronal ceroid lipofuscinosis 11 (CLN11).

[0139] Subsequent to engineering, the hiPSC lines were differentiated into either microglial progenitor cells or cortical neurons. The differentiation of hiPSCs into microglial progenitor cells was performed as described in International Application No PCT / US2023 / 011916 (published as WO2023150089), which is incorporated herein by reference. The differentiation into cortical neurons was performed as described in Ciceri. G., Baggiolini. A., Cho, H.S. et al. An epigenetic barrier sets the timing of human neuronal maturation. Nature 626, 881-890 (2024), which is incorporated herein by reference.

[0140] FIG. 2 shows exemplary experimental results demonstrating the successful differentiation of PGRN-deficient hiPSCs into microglial progenitors. The successful differentiation is evidenced by high expression levels of microglia-specific markers, such as CD45, CD14, CX3CR1, and CDllb, indicating a robust microglial phenotype.

[0141] FIG. 3 shows exemplary experimental results demonstrating that PRGN-deficient hiPSCs successfully differentiated into cortical neurons. This differentiation is evidenced bythe expression of neuronal markers TUBB3, SOX2, PAX6, F0XG1, and TBR1, highlighting the cells’ neuronal identity and maturation.

[0142] Taken together, these data show the development of hiPSC lines, and their differentiation into relevant cell types, which can provide a reliable and reproducible model system. This model system can be used to study disease mechanisms and test novel therapeutic strategies for treating conditions such as CLN 11 and FTD-GRN.Example 2: Transcriptional analysis of R493X microglia progenitors

[0143] This Example describes experiments conducted to evaluate the transcriptional output of R493X microglial progenitor cells. Transcriptomic analysis of these cells confirmed the upregulation of several transcripts know n to be associated w ith PGRN deficiency, including APOE. CTSD, CTSL, and CD68, among others.

[0144] FIGS. 4A and 4B show heatmap visualizations of relevant transcripts in wild-type (WT), R493X / +, and R493X / R493X microglia based on bulk RNA sequencing experiments. In particular, FIG. 4A shows a heatmap visualization of relevant transcripts in WT, R493X / +, and R493X / R493X microglia progenitors. These data illustrate significant transcriptional changes in R493X mutant microglia across three biological replicates as compared to WT, highlighting the impact of the R493X mutation on gene expression. FIG. 4B show s a heatmap visualization of relevant transcripts in WT, R493X / +, and R493X / R493X microglia progenitors with and without co-culture with WT microglia progenitors or the addition of recombinant human PGRN (rhPGRN). The addition of rhPGRN to R493Xmut / + cells and the co-culture of R493Xmut / mut cells with WT cells appear to partially rescue some gene expression patterns, particularly for certain microglia and inflammation markers.

[0145] FIGS. 5A-5C provide exemplary experimental results showing the expression levels of Cathepsin D, Galectin-3, and APOE in WT, R493X / +, and R493X / R493X microglia. The data demonstrate that R493X mutant microglial progenitors exhibit increased levels of Cathepsin D, a protease that cleaves PGRN, Galectin-3, a protein involved in microglial activation, and APOE, which is involved in lipid metabolism.

[0146] These findings underscore the relevance of the R493X mutation in altering the transcriptional landscape of microglial progenitor cells. The transcriptional changes observedin R493X microglial progenitors align with known pathogenic processes in PGRN-related disorders.

[0147] Overall, these data highlight the utility of R493X microglial progenitor cells as a robust model for studying the transcriptional consequences of PGRN deficiency and for evaluating the efficacy of potential therapeutic interventions aimed at restoring normal gene expression and cellular function.Example 3: GRN mutations result in phenotypic differences in microglia progenitors and cortical neurons

[0148] This Example describes experiments conducted to assess the phenotypic impact of GRN mutations in microglial progenitors and cortical neurons. The study focused on comparing R493X / +, R493X / R493X, and wild-type microglial progenitor cells, as well as neural cortical cells, to evaluate their output of key inflammatory markers and proteins involved in neuronal function and lysosomal integrity.

[0149] R493X / +, R493X / R493X, and wild-type microglial progenitor cells were assessed for their output ofTNF-alpha, IL-6, Cathepsin D, Galectin-3, and APOE.

[0150] FIGS. 6A and 6B show exemplary' experimental results for TNF-alpha (FIG. 6 A) and IL-6 (FIG. 6B) levels. These data demonstrate the increased production of certain pro- inflammatory’ cytokines in R493X mutant cells as compared to wild-type cells.

[0151] FIGS. 7A-7F show exemplary’ experimental results for intracellular (FIGS. 7A- 7C) and secreted (FIGS. 7D-7F) forms of Cathepsin D, Galectin-3, and APOE. These results demonstrate significant upregulation of Cathepsin D, Galectin-3, and APOE in both intracellular and secreted forms in R493X mutant cells. These results also indicate that both R493X / + and R493X / R493X microglial progenitors exhibit elevated levels of these proteins compared to wild-type cells. Increased Cathepsin D, Galectin-3, and APOE levels are associated with PGRN deficiency and highlight the impact of GRN mutations on microglial function and activation.

[0152] In addition, R493X / +, R493X / R493X, and wild-type neural cortical cells were evaluated for their output of Cathepsin D, LAMP1, and LAMP2, as well as their respective neural activities as assessed using Neuroburst assays (Incucyte).

[0153] FIGS. 8A-8C show exemplary experimental results for intracellular forms of Cathepsin D. LAMP1, and LAMP2. These data indicate that R493X cortical neurons have an increased intracellular ratio of mature to pro forms of Cathepsin D, and elevated levels of LAMP1 and LAMP2, which are associated with lysosomal integrity and function.

[0154] FIGS. 9A-9C provide exemplary’ experimental results from neuroburst assays, demonstrating impaired neuronal activity in R493X / + and R493X / R493X neural precursor cells compared to wild-type cells. As demonstrated in FIG. 9A, WT neurons show a higher mean correlation, indicating synchronized neuronal activity7, while R493X / + and R493X / R493X neurons exhibit reduced mean correlation, suggesting impaired neuronal network connectivity. As demonstrated in FIG. 9B, WT neurons display a greater number of active neurons compared to R493X / + and R493X / R493X neurons, indicating that GRN mutations reduce neuronal firing capacity. As demonstrated in FIG. 9C, WT neurons exhibit higher burst strength, reflecting robust neuronal activity. In contrast, R493X / + and R493X / R493X neurons show significantly reduced burst strength, indicating weakened neuronal signaling and network function. In summary, the R493X mutant cells exhibit lower mean correlation, fewer firing neurons, and reduced burst strength, indicating compromised neuronal function.

[0155] FIG. 10 shows exemplary image data of TDP-43 expression and localization in R493X / +, R493X / R493X, and wild-ty pe neural cortical cells. The data show mislocalized TDP-43 in R493X mutant cells, with higher signal intensity in the cytoplasmic compartment compared to isogenic control cells. Mis-localization of TDP-43 is associated with neurodegenerative diseases and underscores the pathological consequences of GRN mutations.

[0156] Overall, these experiments highlight the phenotypic differences between GRN mutant and wild-type cells, demonstrating the broad impact of GRN mutations on microglial activation, lysosomal integrity, and neuronal function.Example 4: In vitro cross-correction of PGRN deficiency in hiPSCs, microglia progenitors, and brain organoids by co-culture with wild-type (WT) microglia progenitor cells

[0157] This Example describes experiments conducted to assess the capacity of wild-type microglia progenitor cells to cross-correct for PGRN deficiency in GRN-KO cells.

[0158] FIG. 11 illustrates an exemplary cross-correction assay. Metabolic crosscorrection was performed using a transwell-based system, which allowed wild-type cells to be co-cultured with GRN-KO cells, separated by a 0.4 pm microporous membrane. This setup enabled the exchange of soluble factors without direct cell-to-cell contact.

[0159] In this assay, wild-type microglia progenitor cells were cultured at the bottom of tissue culture dishes, while GRN-KO cells were co-cultured in corresponding transwells. Following a period of co-culture, cells were either harvested or lysed in-well using Lysis buffer (1% Triton-X in HESS, with lx Halt Protease Inhibitor). The total PGRN protein in each lysate was measured using ELISA.

[0160] FIGS. 12A-12C show exemplary experimental results from cross-correction assays performed on hiPSCs (FIG. 12A), microglia progenitor cells (FIG. 12B), and brain organoids (FIG. 12C). As demonstrated in FIG. 12A, the results show a significant increase in PGRN levels in GRN-KO hiPSCs when co-cultured with WT hiPSCs. As demonstrated in FIG. 12B, the results indicate a substantial restoration of PGRN levels in GRN-KO microglia progenitors in the presence of WT microglia progenitors. As demonstrated in FIG. 12C, the results show a significant increase in PGRN levels in the organoids co-cultured with WT microglia progenitors. In summary, these data demonstrate that PGRN was detectable in GRN-KO hiPSCs, microglia progenitors, and brain organoids after co-culturing with wildtype cells for 10 days, compared to stand-alone GRN-KO cells.

[0161] The results indicate that healthy, wild-type cells release PGRN into the culture media, which is then taken up by GRN-KO cells, leading to the restoration of PGRN levels in these deficient cells. Accordingly, these data suggest that PGRN can be effectively transferred from wild-type cells to PGRN-deficient hiPSCs, microglia progenitors, and brain organoids.

[0162] Taken together, these results highlight the potential of hiPSC-derived microglia progenitors as an alternative therapy for FTD-GRN. This approach offers a long-lasting and persistent supply of PGRN to a PGRN-deficient central nervous system (CNS), along with the possibility of additional benefits from the supportive functions of healthy microglia to diseased CNS cells. This innovative therapeutic strategy holds promise for addressing theunderlying cellular deficiencies in GRN-related disorders, paving the way for effective and durable treatments.Example 5: Cross-correction of PGRN deficiency in microglia by co-culture with wildtype (WT) microglia progenitors

[0163] This Example describes experiments conducted to evaluate the ability of WT microglia progenitor cells to cross-correct defects in PGRN-deficient microglia cells.

[0164] WT, granulin knockout (GRN-KO), and R493X microglia progenitor cells were generated in vitro from human induced pluripotent stem cells (hiPSCs) as described herein. Cross-correction assays were performed using a transwell-based co-culture system as described in Example 4, which allowed WT microglia progenitors to be co-cultured with GRN-KO and R493X microglia progenitors while separated by a microporous membrane. This setup enabled the exchange of soluble factors, including PGRN. without direct cell-to- cell contact.

[0165] Following co-culture, cells were harvested and analyzed for GAL3 protein levels using ELISA, GCase enzyme activity was assessed using a fluorometric assay, and inflammatory gene expression was assessed by quantitative RT-PCR.

[0166] FIG. 13 shows exemplary experimental results demonstrating cross-correction with WT microglia progenitors reduces Galectin-3 (GAL3) accumulation in granulin- deficient microglia cells. The bar graph shows GAL3 protein, measured by ELISA from GRN-KO microglia cell lysates, have elevated GAL3 as compared with GRN-KO microglia that w ere co-cultured with WT microglia progenitors, which indicates partial rescue of PGRN deficiency through metabolic cross-correction.

[0167] FIG. 14 shows exemplary experimental results demonstrating that crosscorrection with WT microglia progenitors increases GCase activity in granulin-defi cient microglia. In particular, FIG. 14 illustrates (3-glucocerebrosidase (GCase) enzyme activity measured from cell lysates, as a percentage of uncorrected control, showed GRN-KO microglia have reduced GCase activity, while GRN-KO microglia co-cultured with WT microglia progenitors demonstrate increased GCase activity. Similarly, R493XAmut / mut microglia show ed reduced GCase activity, while R493XAmut / mut microglia co-cultured with WT microglia progenitors demonstrated increased GCase activity.

[0168] FIG. 15 shows exemplary experimental results demonstrating that crosscorrection with WT microglia progenitors modulates mRNA expression levels of inflammatory genes in granuhn-deficient microglia. In particular, FIG. 15 illustrates the expression levels of pro-inflammatory genes (CXCL10, IDO1, IL6, GBP1, IL12B, and TNF) measured by quantitative RT-PCR and expressed as dCt geometric mean (Actin, GAPDH, TBP, FTL). GRN-KO microglia show baseline expression levels of inflammatory’ markers. GRN-KO microglia co-cultured with WT microglia progenitors demonstrated reduced levels of expression of most inflammatory genes, including CXCL10, IDO1, IL6, GBP1, IL12B, and TNF, compared to GRN-KO microglia.

[0169] These experimental results demonstrate that WT microglia progenitor cells can cross-correct certain aspects of PGRN deficiency in affected microglia, including reducing pathological protein accumulation, restoring lysosomal enzyme function, and modulating inflammatory responses.Example 6: Cross-correction of PGRN deficiency in cortical neurons by co-culture with wild-type (WT) microglia progenitors

[0170] This Example describes experiments conducted to evaluate the capacity of WT microglia progenitor cells to cross-correct PGRN deficiency in cortical neurons derived from GRN-KO and R493X mutant hiPSC lines.

[0171] WT microglia progenitor cells and GRN-deficient (GRN+ / -, GRN- / -, R493XAmut / +, and R493XAmut / mut) cortical neural precursor cells (NPCs) were generated as described herein. WT microglia progenitors were co-cultured with GRN-KO and R493X cortical NPCs. Following co-culture, NPCs were identified by staining with a neural cellspecific antibody to distinguish the NPCs from the microglia progenitor cells. Subsequently, PGRN levels were assessed by fluorescence imaging using fluorescent antibodies against PGRN.

[0172] FIG. 16 shows exemplary experimental results demonstrating that crosscorrection restores PGRN levels in granuhn-deficient neurons after co-culture with WT microglia progenitor cells. In particular, FIG. 16 shows a bar graph of exemplary results illustrating PGRN protein levels measured by fluorescence and expressed as mean intensity as a percentage of wild-type (WT) control. GRN+ / - neurons showed reduced PGRN levels, while GRN+ / - neurons co-cultured with WT microglia progenitors demonstrated restoredPGRN levels. Similarly, GRN- / - neurons alone showed reduced PGRN levels, while GRN- / - neurons co-cultured with WT microglia progenitors demonstrated increased PGRN levels. These results demonstrate that WT microglia progenitors can effectively cross-correct PGRN deficiency in GRN-deficient neurons.

[0173] FIG. 17 shows exemplary experimental results demonstrating that crosscorrection restores PGRN levels in granulin-deficient R493X neurons after co-culture with WT microglia progenitor cells. In particular, FIG. 17 shows a bar graph of exemplary experimental results that illustrates PGRN protein levels measured by immunofluorescence and expressed as PGRN intensity per neuron as a percentage of WT control. R493XAmut / + neurons showed reduced PGRN levels, while R493XAmut / + neurons co-cultured with WT microglia progenitors demonstrate restored PGRN levels. Similarly, R493XAmut / mut neurons showed reduced PGRN levels, while R493XAmut / mut neurons co-cultured with WT microglia progenitors demonstrated increased PGRN levels. These results demonstrate that WT microglia progenitors can effectively cross-correct PGRN deficiency in both heterozygous and homozygous R493X mutant neurons.EQUIVALENTS AND SCOPE, INCORPORATION BY REFERENCE

[0174] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments described herein. It is understood that modifications which do not substantially affect the activity of the various embodiments of this disclosure are also provided within the description of the disclosure provided herein. The scope of the present disclosure is not intended to be limited to the above description, but rather is as set forth in the appended claims.

[0175] In the claims articles such as “a,” "an." and “the” may mean one or more than one unless indicated to the contrary or otherwise evident from the context. Claims or descriptions that include “or” between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The disclosure includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The disclosure also includes embodiments in which more than one, or all of the group members, are present in, employed in, or otherwise relevant to a given product or process.

[0176] Furthermore, it is to be understood that the disclosure encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, descriptive terms, etc., from one or more of the claims or from relevant portions of the description is introduced into another claim. For example, any claim that is dependent on another claim can be modified to include one or more limitations found in any other claim that is dependent on the same base claim. Furthermore, where the claims recite a composition, it is to be understood that methods of using the composition for any of the purposes disclosed herein are included, and methods of making the composition according to any of the methods of making disclosed herein or other methods known in the art are included, unless otherwise indicated or unless it w ould be evident to one of ordinary skill in the art that a contradiction or inconsistency would arise.

[0177] Where elements are presented as lists, e.g., in Markush group format, it is to be understood that each subgroup of the elements is also disclosed, and any element(s) can be removed from the group. It should be understood that, in general, where the disclosure, or aspects of the embodiments, is / are referred to as comprising particular elements, features, steps, etc., certain embodiments of the disclosure or aspects of the embodiments consist, or consist essentially of, such elements, features, steps, etc. Thus, for each embodiment of the disclosure that comprises one or more elements, features, steps, etc., the disclosure also provides embodiments that consist or consist essentially of those elements, features, steps. etc.

[0178] Where ranges are given, endpoints are included. Furthermore, it is to be understood that unless otherwise indicated or otherwise evident from the context and / or the understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value within the stated ranges in different embodiments of the disclosure, to the tenth of the unit of the low er limit of the range, unless the context clearly dictates otherwise. It is also to be understood that unless otherwise indicated or otherwise evident from the context and / or the understanding of one of ordinary skill in the art, values expressed as ranges can assume any subrange within the given range, w herein the endpoints of the subrange are expressed to the same degree of accuracy as the tenth of the unit of the lower limit of the range.

[0179] In addition, it is to be understood that any particular embodiment of the present disclosure may be explicitly excluded from any one or more of the claims. Where ranges aregiven, any value within the range may explicitly be excluded from any one or more of the claims. Any embodiment, element, feature, application, or aspect of the compositions and / or methods of the disclosure, can be excluded from any one or more claims. For purposes of brevity, all of the embodiments in which one or more elements, features, purposes, or aspects is excluded are not set forth explicitly herein.

[0180] Throughout this disclosure various publications, patents, and sequence database entries are mentioned. The disclosures of these publications, patents, and sequence database entries, including those items listed above, are hereby incorporated by reference in their entirety as if each individual publication or patent was specifically and individually indicated to be incorporated by reference. In case of conflict, the present application, including any definitions herein, will control.

[0181] Although the disclosure has been described with reference to the examples provided above, it should be understood that various modifications can be made without departing from the scope of the disclosure. Accordingly, the above examples are intended to illustrate but not limit the present disclosure.

Claims

CLAIMSWhat is claimed is:

1. A method of treating a progranulin (PRGN) deficiency in a subject, the method comprising: obtaining a population of microglia precursor cells; and administering said population of microglia precursor cells directly to the central nervous system (CNS) of the subject, wherein the administered microglia precursor cells provide a supply of progranulin to the subjects PGRN-deficient cells.

2. The method of claim 1, wherein the population of microglia precursor cells are derived, in vitro, from stem cells.

3. The method of claim 2, wherein the stem cells comprise human embryonic stem cells (hESCs) or human induced pluripotent stem cells (hiPSCs).

4. The method of any of claims 1 to 3, wherein the population of microglia precursor cells express one or more of the following PTPRC (CD45). CD14, ITGAM (CD11B), CX3CR1 , MERTK, or P2RY12.

5. The method of any of claims 1 to 4, wherein at least about 80% of the population of microglia precursor cells express CD 14 and CD45.

6. The method of any of claims 1 to 5, wherein at least about 85% of the population of microglia precursor cells express CDllb and CX3CR1.

7. The method of any of claims 1 to 6, wherein at least about 70% of the population of microglia precursor cells express CD 14, CD45, CDllb, and CX3CR1.

8. The method of any of claims 1 to 7, wherein the obtaining comprises:(i) culturing a population of human pluripotent stem cells (PSCs);(ii) inducing said PSCs with BMP-4 for about 2 to about 6 days;(iii) culturing the PSCs induced with BMP-4 for about 2 to about 6 days, wherein said media comprises SCF, VEGF. and bFGF;(iv) culturing the cells from step (iii) in media for about 2 to about 6 days, wherein said media comprises SCF, IL-3, TPO, M-CSF and Flt3 for about 8 days; and(v) culturing the cells from step (iv) in M-CSF, Flt3, and GM-CSF, thereby generating microglial precursor cells.

9. The method of any of claims 1 to 8, wherein the subject is not treated to ablate endogenous microglial cells prior to administering the population of microglia precursor cells to the central nervous system (CNS) of the subject.

10. The method of any of claims 1 to 9, wherein the population of microglia precursor cells is administered to the subject via intra-cerebroventricular (ICV) injection.

11. The method of any of claims 1 to 10, wherein, prior to the administering, the subject is diagnosed with neuronal ceroid lipofuscinosis 11 (CLN11) or Frontotemporal Dementia (FTD-GRN).

12. The method of any of claims 1 to 11, wherein the population of microglia precursor cells comprises between about 25 million and about 500 million microglia precursor cells.

13. The method of claim 12, wherein the population of microglia precursor cells comprises betw een about 100 million and about 300 million microglia precursor cells.

14. The method of any of claims 1 to 13, wherein the treatment results in improved neurological function in the subject as assessed by standardized cognitive and motor tests.

15. The method of any of claims 1 to 14, wherein the population of microglia cells are administered to the subject with a pharmaceutically acceptable carrier.

16. A therapeutic composition comprising: an effective quantity of a population of microglia precursor cells for treating a progranulin deficiency in a subject; and a cell delivery solution.

17. The composition of claim 16, wherein the population of microglia precursor cells are derived, in vitro, from stem cells.

18. The composition of claim 17, wherein the stem cells comprise human embry onic stem cells (hESCs) or human induced pluripotent stem cells (hiPSCs).

19. The composition of any of claims 16 to 18, wherein the population of microglia precursor cells express one or more of the following PTPRC (CD45), CD14, ITGAM (CD11B), CX3CR1, MERTK, or P2RY12.

20. The composition of any of claims 16 to 19, wherein at least about 80% of the population of microglia precursor cells express CD14 and CD45.

21. The composition of any of claims 16 to 20, wherein at least about 85% of the population of microglia precursor cells express CDl lb and CX3CR1.

22. The composition of any of claims 16 to 21, wherein at least about 70% of the population of microglia precursor cells express CD14, CD45, CDl lb, and CX3CR1.

23. A method for treating a progranulin (PRGN) deficiency in a subject, the method comprising: administering said population of microglia precursor cells directly to the central nervous system (CNS) of the subject.

24. The method of claim 23, wherein the microglia precursor cells provide a supply of PRGN to PGRN-defi cient cells of the subject.

25. The method of claim 23 or claim 24. wherein the microglia precursor cells are wildtype microglia precursor cells.

26. The method of any one of claims 23-25, wherein, the microglia precursor cells are generated, in vitro, from human pluripotent stem cells.

27. The method of any one of claims 23-26, wherein at least about 85% of the population of microglia precursor cells express CDllb and CX3CR1.

28. The method of any one of claims 23-27, wherein at least about 70% of the population of microglia precursor cells express CD14, CD45, CDllb, and CX3CR1.

29. The method of any one of claims 23-28. wherein the microglia precursor cells are administered to the subject via intra-cerebroventricular (ICV) injection.

Images