Methods for treating inherited metabolic disorders

Abstract

The present disclosure provides compositions and methods for treating an inherited metabolic disorder in a subject. Provided herein are compositions and methods for treating a lysosomal storage disease (LSD) in a subject using myeloid lineage cells.

Description

[Technical field]

[0001] The present invention relates to compositions and methods for the treatment of inherited metabolic disorders in a subject. [Background technology]

[0002] Lysosomes are specialized organelles that break down and recycle macromolecules, linking catabolic and anabolic metabolism. Although all tissues require lysosomes to maintain homeostasis, the metabolically demanding brain is especially dependent on lysosomal function, as evidenced by the neurological phenotypes of many lysosomal storage disorders (LSDs). LSDs are inherited disorders characterized by the pathological accumulation of toxic substances in the body's cells due to genetic defects that result in enzyme deficiencies. There are more than 45 types of LSDs, which affect different parts of the body, including the central nervous system. Hurler syndrome is a rare LSD (the most severe form of mucopolysaccharidosis type 1 (MPS1)) characterized by a variety of clinical phenotypes, including cognitive abnormalities and shortened life expectancy. Most LSDs result from an inherited deficiency of a single catabolic enzyme, leading to the accumulation of the enzyme's substrate in the lysosomes. In MPS1, deficiency of alpha-L-iduronidase (IDUA) leads to the accumulation of glycosaminoglycans (GAGs), resulting in cell damage and ultimately cell death.

[0003] Enzyme replacement therapy using recombinant proteins and bone marrow gene therapy to deliver functional genes to hematopoietic stem cells are promising therapeutic candidates, but they are often insufficient to deliver therapeutically relevant levels of enzymes to the central nervous system (CNS). [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Muffat et al. Nat Med. 2016 Nov; 22(11): 1358-1367 [Non-Patent Document 2] Pandaya et al., Nat Neurosci. May 2017; 20(5): 753 - 759 [Non - Patent Document 3] Abud et al., Neuron Apr 19, 2017; 94(2): 278 - 293 [Non - Patent Document 4] Douvaras et al., Stem Cell Reports, Volume 8, Issue 6, 1516 - 1524, June 06, 2017 [Non - Patent Document 5] Van Wilgenburg PLOS ONE, https: / / doi.org / 10.1371 / journal.pone.0071098 - Aug 2013 [Non - Patent Document 6] Haenseler et al., Stem Cell Reports Jun 6, 2017; 8(6): 1727 - 1742 [Non - Patent Document 7] Takata et al., Immunity Jan 18, 2017; 47(1): 183 - 198 [Non - Patent Document 8] Zhu et al. (2016) Toll - like Receptor 4 Deficiency Impairs Motor Coordination. Front. Neurosci. [Non - Patent Document 9] https: / / doi.org / 10.14806 / ej.17.1.200 [Non - Patent Document 10] Perriot, S., Canales, M., Mathias, A. and Du Pasquier, R. Generation of transgene - free human induced pluripotent stem cells from erythroblasts in feeder - free conditions. STAR Protoc 3, 101620(2022) [Non - Patent Document 11] Yoshioka, N. et al. Efficient Generation of Human iPSCs by a Synthetic Self-Replicative RNA. Cell Stem Cell 13, pp. 246 - 254 (2013)

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[0005] Therefore, there is a need for compositions and methods for treating lysosomal dysfunction in human LSDs.Myeloid lineage cells derived from pluripotent stem cells provide an exciting new therapeutic approach to the treatment of some LSDs.This cell + gene therapy is in contrast to enzyme replacement therapy and gene therapy, where each therapy is tailored to a specific genetic defect. [Means for solving the problem]

[0006] In one aspect, the disclosure provides a method of treating a metabolic disorder comprising administering pluripotent stem cell (PSC) derived myeloid cells to the central nervous system of a subject to be treated, allowing the administered PSC derived myeloid cells to engraft and produce an enzyme, monitoring the level of the enzyme in a serum sample or a cerebrospinal fluid sample from the treated subject, and monitoring the amount of substrate in a serum sample, a cerebrospinal fluid (CSF) sample, or a urine sample from the subject to determine progress of the treatment.

[0007] In some embodiments, the metabolic disorder is an inherited metabolic disorder.

[0008] In some embodiments, the inherited metabolic disorder is Hurler syndrome and the enzyme is alpha-L-iduronidase (IDUA).

[0009] In some embodiments, the inherited metabolic disorder is Sly syndrome and the enzyme is beta-glucuronidase (GUSB).

[0010] In some embodiments, the central nervous system consists of the spinal cord, the brain, and cerebrospinal fluid (CSF).

[0011] In some embodiments, the subject is a mouse or a human.

[0012] In some embodiments, the substrate is a glycosaminoglycan.

[0013] In some embodiments, the subject being treated has an accumulation of undegraded substrates in brain cells and all other cells.

[0014] In some embodiments, the enzymes are used to reduce the accumulation of undegraded substrates in the brain cells of the subject being treated.

[0015] In some embodiments, a sustained reduction in total substrate levels of greater than about 20% indicates successful treatment of said metabolic disorder.

[0016] In some embodiments, the amount of enzyme in the serum sample is compared to the amount of enzyme in the serum sample of a healthy subject.

[0017] In some embodiments, the amount of cells injected is about 25×10 6 ~Approx. 1250×10 6 cells, approximately 50 x 10 6 pieces~approx. 1000×10 6 cells, approximately 100 x 10 6 ~About 500×10 6 cells, approximately 100 x 10 6 ~About 300×10 6 cells, more preferably about 150×10 6 ~Approx. 250×10 6 Within the range of cells.

[0018] The patent or application file contains at least 25 drawings executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the U.S. Patent and Trademark Office upon request and payment of the necessary fee. [Brief description of the drawings]

[0019] [Figure 1]FIG. 1: Overview of study design for a pilot, non-GMP, in vivo study to evaluate efficacy of myeloid cell transplantation in the IDUA-KO mouse model of Hurler syndrome. A) Flow cytometric characterization of myeloid cells transplanted into adult mouse brains. B) Flow cytometric characterization of myeloid cells transplanted into young mouse brains. C) Scheme showing the two arms of the efficacy study with compared time points and groups. D) Timeline of in-vivo evaluations and manipulations performed during the "in-life" phase of the efficacy study. [Diagram 2] Graphs showing biochemical analysis of CNS tissues from IDUA-KO mice 1 month after ICV delivery of myeloid cells. A) Significant IDUA enzyme activity can be detected in all CNS tissues of young (at transplant) IDUA-KO animals assessed 1 month after myeloid cell transplant in a dose-dependent manner. B) IDUA enzyme activity in CNS tissues of adult (at transplant) IDUA-KO mice assessed 1 month after myeloid cell transplant. Percentages indicate enzyme activity relative to that of WT littermates. Graph bars represent mean values, dots represent individual samples, and error bars indicate standard deviation. [Diagram 3] Graphs showing quantification of accumulated substrates of IDUA (expressed as total GAGs) in CNS tissues of IDUA-KO animals one month after myeloid cell transplantation. Transplantation of myeloid cells into young (A) or adult (B) IDUA-KO animals reduced the accumulation of toxic substrates in all CNS tissues in a dose-dependent manner compared to vehicle-treated animals. Percentages indicate reduction relative to vehicle-treated animals. Graph bars represent mean values, dots represent individual samples, and error bars indicate standard deviation. [Figure 4]Graphs showing biochemical analysis of CNS tissues from IDUA-KO mice 5 months after ICV delivery of myeloid cells. A) High IDUA enzyme activity can be detected in a dose-dependent manner in all CNS tissues of young (at transplant) IDUA-KO animals assessed 5 months after myeloid cell transplant. B) Significant IDUA enzyme activity in CNS tissues of adult (at transplant) IDUA-KO mice assessed 5 months after myeloid cell transplant. Percentages indicate enzyme activity relative to that of WT littermates. Graph bars represent mean values, dots represent individual samples, and error bars indicate standard deviation. [Figure 5-1] A) Graph showing quantification of accumulated substrates of IDUA (expressed as total GAGs) in CNS tissues of IDUA-KO animals 1, 5, and 8 months after myeloid cell transplantation. Myeloid cell transplantation into young (A) or adult (B) IDUA-KO animals shows a dose-dependent reduction in accumulated toxic substrates in all CNS tissues compared to vehicle-treated animals. This reduction was sustained for 1, 5, and 8 months after myeloid cell transplantation (compare with FIG. 3). Percentages show reduction relative to vehicle-treated animals. Graph bars represent mean values, dots represent individual samples, and error bars show standard deviation. [Figure 5-2] B) Myeloid cells prevent GAG accumulation in the CNS of MPS I and MPS VII mouse models after 1, 5, or 8 months of treatment. Schematic showing the experimental design of adult and juvenile myeloid cell transplantation in MPS I and MPS VII mouse models. GAG accumulation after 1, 5, and 8 months of treatment in the brain, spinal cord, or CSF of juvenile treated MPS I cohorts. Myeloid cells were administered at low (0.4×106) or high (1.4×106) doses. GAG accumulation after 1 and 5 months of treatment in the brain, spinal cord, or CSF of adult treated MPS I cohorts. GAG accumulation after 1 month of treatment in the brain and spinal cord of juvenile treated MPS VII cohorts. GAG accumulation after 1 and 5 months of treatment in the brain and spinal cord of adult treated MPS VII cohorts. [Figure 5-3]C) Enzyme activity levels in CNS tissues of MPS I mice after myeloid cell treatment. Schematic diagram showing experimental design of adult and juvenile myeloid cell transplantation in MPS I mouse model. IDUA enzyme activity levels 1, 5, or 8 months after treatment in brain, spinal cord, or CSF of juvenile treated MPS I cohorts. IDUA enzyme activity levels 1 or 5 months after treatment in brain, spinal cord, or CSF of adult treated MPS I cohorts. Myeloid cells were administered at low (0.4x106) or high (1.4x106) doses. IDUA levels are expressed as a percentage of wild type animals from the same cohorts and time points. [Figure 5-4] D) Myeloid cells prevent GAG accumulation and restore deficient enzyme activity in peripheral tissues of MPS I and MPS VII mice. Schematic diagram showing experimental design of adult and young myeloid cell transplantation in MPS I and MPS VII mouse models. Accumulation of GAG in urine of MPS I mice sampled at regular intervals in 1, 5 or 8 month cohorts of MPS I mice treated with young or adult. Accumulation of GAG in urine of MPS VII mice sampled at regular intervals in 1 or 5 month cohorts of MPS I mice treated with young or adult. IDUA enzyme levels in serum of MPS I mice sampled at regular intervals in 1, 5 or 8 month cohorts of MPS I mice treated with young or adult. IDUA enzyme levels in serum of MPS VII mice sampled at regular intervals in 1 or 5 month cohorts of MPS I mice treated with young or adult. GAG accumulation levels 5 months after treatment in the hearts and livers of MPS I mice or in the livers of adult treated MPS VII mice. [Figure 6]Graph showing the time course of IDUA enzyme activity and substrate quantification in the periphery of adult IDUA-KO animals 1 and 5 months after myeloid cell transplantation. A) IDUA activity can be detected in the peripheral blood of IDUA-KO animals given high doses of myeloid cells by ICV injection. Consistent levels of IDUA activity can be detected 1 and 5 months after myeloid cell transplantation. B) Reduction of total GAG secreted in urine can be detected for IDUA-KO animals given high or low doses of myeloid cells by ICV injection. A sustained reduction in total GAG was observed 1 and 5 months after myeloid cell transplantation. The increase in GAG levels for the vehicle-treated group indicates the progressive nature of the disease. Error bars indicate standard deviation. [Figure 7] Graph showing the time course of IDUA enzyme activity and substrate quantification in the periphery of young IDUA-KO animals 1 and 5 months after myeloid cell transplantation. A) IDUA activity can be detected in the peripheral blood of IDUA-KO animals after receiving high or low doses of myeloid cells by ICV injection. Consistent levels of IDUA activity can be detected 1 and 5 months after myeloid cell transplantation. B) Reduction of total GAG secreted in urine can be detected for IDUA-KO animals receiving high or low doses of myeloid cells by ICV injection. A sustained reduction in total GAG was observed between 1 and 5 months after myeloid cell transplantation. The increase in GAG levels for the vehicle-treated group indicates the progressive nature of the disease. Error bars indicate standard deviation. [Figure 8] Figure 1 shows hindlimb clasping, a behavioral test used as a marker of disease progression in models of neurodegeneration and cerebellar ataxia. The photograph shows mice with different severity scores in the hindlimb clasping test. The table provides further explanation of the scoring. [Figure 9]Figure 1: Myeloid cells prevent ataxia defects in MPS I and MPS VII mice. Schematic showing experimental design of adult and juvenile myeloid cell transplantation in MPS I and MPS VII mouse models. Hindlimb grasping scores after 5 or 8 months of treatment in juvenile MPS I mice. Hindlimb grasping scores after 1, 2, and 5 months of treatment in adult MPS VII mice. Hindlimb grasping scores after 1 month of treatment in juvenile MPS VII mice. [Figure 10-1] A) Schematic showing the experimental design of ICV implantation in MPS I (IDUA-KO) and MPS VII (GUSB-KO) mouse models. [Figure 10-2] B) Immunohistochemistry images of two sagittal sections from an adult IDUA-KO brain one month after ICV delivery of myeloid cells. The top picture is a low magnification stitched image from tiled scans of the lateral (left) or medial (right) sections. Sections were stained for nuclei (DAPI; grey), myeloid marker (CD45; red), and human marker (hKu80, blue). The yellow box indicates the relative portion of the section shown at higher magnification in the bottom panel. Myeloid cells (arrows) are positive for all markers and are found in the choroid plexus and meninges surrounding the brain. [Figure 11] Immunohistochemistry of two sagittal sections from a young IDUA-KO brain one month after ICV delivery of myeloid cells. The top photo is a low magnification stitched image from tiled scans of the lateral (left) or medial (right) sections. Sections were stained for nuclei (DAPI; grey), myeloid marker (CD45; red), and human marker (hKu80, blue) or for nuclei (DAPI; grey), microglial marker (IBA1; red), human marker (hKu80, blue), and myeloid marker (human specific CD163; green). Yellow boxes indicate the relative portion of the section shown at higher magnification in the lower panel. Myeloid cells (arrows) are positive for all markers and are found in the choroid plexus and meninges surrounding the brain. [Figure 12]Immunohistochemistry of two sagittal sections from an adult IDUA-KO brain 5 months after ICV delivery of myeloid cells. The top picture is a low magnification stitched image from tiled scanned images of the lateral (left) or medial (right) sections. Sections were stained for nuclei (DAPI; grey), myeloid markers (CD45; red), and human markers (hKu80, blue). The yellow box indicates the relative portion of the section shown at higher magnification in the lower panel. Myeloid cells (arrows) are positive for all markers and are found in the choroid plexus and meninges surrounding the brain. Some myeloid cells enter the brain parenchyma displaying typical microglial morphology. [Figure 13-1] A) Immunohistochemistry images of lateral sagittal sections from young IDUA-KO brains 5 months after ICV delivery of myeloid cells. The left photo is a low magnification stitched image from tiled scanned images of the lateral section. Sections were stained for nuclei (DAPI; grey), microglial marker (IBA1; red), human marker (hNA; blue), and myeloid marker (human specific CD163; green). Yellow boxes indicate the relative portion of the section shown at higher magnification in the lower panel. Myeloid cells (arrows) are positive for all markers and are found in the choroid plexus and meninges surrounding the brain. [Figure 13-2] B) Myeloid cells are long-term engrafted in the MPS I mouse model. Representative photograph of MPS I brain section 1 month after treatment. C) Representative photograph of MPS I brain section 8 months after treatment showing myeloid cells. Due to autofluorescence in the tissue at this time point, the photograph was developed using DAB-HRP. [Figure 13-3]D) Images showing that the proliferative fraction of myeloid cells decreases with time after engraftment. Representative images of myeloid cells in MPS I mice after 1 month of treatment. Myeloid cells were checked for expression of the cell proliferation marker ki67. E) Graph showing quantification of the percentage of myeloid cells positive for ki67 in adult and juvenile treated cohorts of MPS I mice at 1 and 5 months. F) Representative images of myeloid cells in MPS VII mice after 1 month of treatment. G) Graph showing quantification of the percentage of myeloid cells positive for ki67 in adult cohorts of MPS VII mice after 1 and 5 months of treatment. [Figure 14] Figure 1. Outline of the study design for an exploratory, non-GMP in vivo study to evaluate the efficacy of myeloid cell transplantation in the GUSB-KO mouse model of Sly syndrome. A) Timeline of the "in-life" phase when various samples were collected from the mouse and when hindlimb gripping was performed. B) Coronal section of mouse brain atlas showing the sites of myeloid cell delivery in both ventricles (red arrows). [Figure 15] Graphs depicting biochemical analysis of CNS tissue from GUSB-KO mice one month after ICV delivery of myeloid cells. A) GUSB enzyme activity in CNS tissue of adult GUSB-KO animals was assessed one month after myeloid cell transplantation. B) Quantification of accumulated substrates of GUSB (expressed as total GAGs) in CNS tissue of GUSB-KO animals one month after myeloid cell transplantation. Myeloid cell transplantation into adult GUSB-KO animals reduced accumulation of toxic substrates in the CNS. Percentages indicate reduction in GAGs relative to vehicle-treated animals. Graph bars represent mean values ​​and dots represent individual samples. [Figure 16] Graph showing hindlimb grasping test in adult GUSB-KO animals 1 and 5 months after bone marrow cell transplantation. Bone marrow cell transplantation reduced the behavioral defects observed in GUSB-KO animals, especially 5 months after transplantation. Graph bars represent average values ​​and dots represent individual animals. [Figure 17]Immunohistochemistry images of lateral sagittal sections from adult GUSB-KO brains one month after ICV delivery of myeloid cells. The left photo is a low magnification stitched image from tiled scanned images of the lateral section. Sections were stained for nuclei (DAPI in grey), microglial markers (IBA1 in red), human markers (hNA in blue), and myeloid markers (human-specific CD163 in green). Yellow boxes indicate the relative portions of sections shown at higher magnification in the lower panels. Myeloid cells (arrows) are positive for all markers and are found in the choroid plexus and meninges surrounding the brain. [Figure 18] Graphs showing IDUA enzyme activity in brain lysates of IDUA-KO mice 5 months after transplantation of wild-type (WT) or IDUA-overexpressing (OE) myeloid cells. A) Graph showing IDUA enzyme activity in IDUA-KO brain lysates expressed as a percentage of activity in wild-type animals. B) Quantification of IDUA enzyme activity in brain lysates of IDUA-KO mice after transplantation of microglia that are WT, OE, or KO for the IDUA gene. Transplantation of either WT or OE microglia resulted in significant levels of IDUA in IDUA-KO animal brain lysates. IDUA Het and IDUA WT are heterozygous or wild-type littermates to IDUA-KO animals. Graph bars represent mean values, dots represent individual animals, and error bars indicate standard deviation. [Figure 19-1]A) Graph showing reduction in accumulated substrates of IDUA (expressed as total GAGs) in brain lysates of IDUA-KO mice 5 months after transplantation with wild-type (WT) or IDUA-overexpressing (OE) myeloid cells. Graph showing reduction in GAG levels in brain lysates of IDUA-KO expressed as a percentage of GAG levels in IDUA-KO animals. B) Graph showing reduction in accumulated substrates of IDUA (expressed as total GAGs) in brain lysates of IDUA-KO mice 5 months after transplantation with wild-type (WT) or IDUA-overexpressing (OE) myeloid cells. Quantification of GAG levels in brain lysates of IDUA-KO mice after transplantation with microglia that are WT, OE, or KO for the IDUA gene. Transplantation of either WT or OE microglia results in a significant reduction in GAGs in brain lysates of IDUA-KO animals, approaching levels of IDUA heterozygous animals. IDUA Het and IDUA WT are heterozygous or wild type littermates to IDUA-KO animals. Graph bars represent mean values, dots represent individual animals, and error bars indicate standard deviation. [Figure 19-2] C) Reduction of accumulated substrates of IDUA (expressed as total GAG) in brain lysates of IDUA-KO mice 5 months after transplantation with wild-type (WT) or IDUA-overexpressing (OE) myeloid cells. Myeloid cells prevent GAG accumulation in the CNS and ataxia defects in MPS I and MPS VII mouse models. Schematic showing experimental design of adult and juvenile myeloid cell transplantation in MPS I and MPS VII mouse models. GAG accumulation 5 months after treatment in brain, spinal cord or CSF of adult or juvenile treated MPS I cohorts. Myeloid cells were administered at low (0.4x106) or high (1.4x106) doses. Correlation between CSF and brain GAG levels 5 months after treatment in juvenile MPS I cohorts. GAG accumulation 5 months in brain or spinal cord of adult MPS VII cohorts. Hindlimb grasping scores 5 months after treatment in the juvenile MPS I and adult MPS VII cohorts. [Figure 20-1]A) In vitro repair of toxic substrates accumulated in MG-KO after co-culture with myeloid cells. Knockout microglia (MG-KO) are derived from human iPSC lines in which lysosomal enzymes have been knocked out. In this case, MG-KO were used to model the mucopolysaccharidosis family of LSDs by knocking out IDUA, SGSH, NAGLU or GUSB. These lines are models for the lysosomal storage diseases MPS1 (Hurler syndrome), MPS3A (Sanfilippo A syndrome), MPS3B (Sanfilippo B syndrome), and MPS7 (Sly syndrome), respectively. Co-culture of diseased cells with myeloid cells significantly reduced intracellular GAGs for all tested models of LSD, providing evidence for functional delivery of active lysosomal enzymes from myeloid cells to adjacent diseased cells. [Figure 20-2] B) Myeloid cells restore MPS enzymes to MG-KO cells and reduce glycoprotein accumulation to wild-type levels via a mannose-6-phosphate-dependent process. IDUA and GUSB enzyme activity levels were found in the supernatants of myeloid cells after 96 hours in vitro. Schematic showing the transwell-based system developed to test enzyme cross-repair to MG-KO cells by myeloid cells. Accumulation of GAGs in cell lysates of WT control cells (myeloid cells), MG-KO, and MG-KO co-cultured with myeloid cells for 9-10 days. Each graph shows MG-KO deficient in a different enzyme: IDUA, GUSB, SGSH, and NAGLU. Values ​​are shown as percentage of GAG levels found in untreated MG-KO at the end of the assay. Accumulation of GAGs in cell lysates of MG-KO co-cultured with myeloid cells in the presence and absence of mannose-6-inhibitor. Values ​​are presented as a percentage of GAG levels found in untreated MG-KO at the end of the assay. [Figure 20-3]C) Graph showing that co-culture with myeloid cells restores enzyme levels in enzyme-deficient MG-KO. Enzyme activity of IDUA, GUSB, and NAGLU in cell lysates of myeloid cells and corresponding MG-KO after co-culture alone or with myeloid cells. Quantification of SGSH immunoreactivity by Western blot in cell lysates of myeloid cells, SGSH-KO, and SGSH-KO co-cultured with myeloid cells. [Figure 21] Figure 1 shows in vitro evidence for the transfer of lysosomal enzymes to MG-KO after co-culture with myeloid cells. Knockout microglia (MG-KO) are derived from human iPSC lines in which lysosomal enzymes have been knocked out. In this case, we used MG-KO to model MPS1 (Hurler syndrome), MPS3B (Sanfilippo B syndrome), and MPS7 (Sly syndrome) by knocking out IDUA, NAGLU, or GUSB, respectively. Co-culture of diseased cells with myeloid cells results in variable and detectable intracellular levels of each of the defective lysosomal enzymes, providing evidence for functional delivery of lysosomal enzymes from myeloid cells to adjacent diseased cells. [Figure 22] Figure 1 shows in vitro evidence for the transfer of functional GAA lysosomal enzyme to the MG-KO model of Pompe disease after co-culture with myeloid cells. To model the LSD termed Pompe disease, GAA-KO microglia were derived from human iPSCs in which GAA, the gene responsible for the production of the lysosomal enzyme alpha-glucosidase, was knocked out. Co-culture of GAA-KO cells with myeloid cells results in higher levels of glucosidase and lower levels of glycogen (which is the accumulated substrate for glucosidase in Pompe disease) compared to GAA-KO cells. These results provide evidence for the functional delivery of active glucosidase from myeloid cells to adjacent diseased cells. [Figure 23]Figure 1 shows a cell engineering design for treating MPS, e.g., MPS1. A) CRISPR-Cas9 strategy for insertion of aEF1-IDUA overexpression construct into the AAVS1 locus. IDUA-OE cells produce high levels of functional IDUA. B) CRISPR-Cas9 strategy for creating IDUA knockout in human induced pluripotent stem cells (hiPSCs). IDUA-KO does not produce detectable amounts of IDUA. [Figure 24-1] A) Human induced pluripotent stem cells were used to induce true microglia based on publicly available protocols. Schematic of the differentiation process for myeloid lineage cells. B) Images showing that human induced pluripotent stem cells were used to induce true microglia based on publicly available protocols. Microglial cells express standard microglial markers. [Figure 24-2] C) Graph showing that human induced pluripotent stem cells were used to induce true microglia based on publicly available protocols, with high post-thaw recovery and survival rates. D) Graph showing that human induced pluripotent stem cells were used to induce true microglia based on publicly available protocols. Representative phase contrast image of polarized myeloid cells 12 hours after addition of pHrodo-conjugated E. coli particles. The inset in the top left shows pHrodo particle fluorescence detected after phagocytosis. Quantification of a representative phagocytosis assay. Graph shows pHrodo fluorescence after addition to myeloid cells. Images were acquired every 30 min for 24 hours. [Figure 24-3] E) Human induced pluripotent stem cells were used to induce bona fide microglia based on a publicly available protocol. Expression levels of inflammatory cytokines (TNFα, CXCL10, and IL-6) after polarization of myeloid cells to an M1-like state using LPS and IFNγ. Graphs show mean and SEM. [Figure 24-4]F) Human induced pluripotent stem cells were used to derive bona fide microglia based on publicly published protocols. This bioprocess robustly produced CD45+ microglia in many different cell lines. [Diagram 25] Figure 1: Genetically modified microglia were co-cultured using a transwell culture system to assay their ability for enzyme rescue. The system comprises a regular tissue culture treated dish in which two compartments separated by a microporous membrane can be created to accommodate an insert. A) IDUA-KO microglial cells were co-cultured for 1 week with myeloid lineage cells genetically overexpressing IDUA (IDUA-OE), isolated, and assayed for enzyme activity and substrate accumulation. B) When IDUA c.2 is cultured alone, it shows higher enzyme activity / levels compared to IDUA-KO. However, when IDUA-KO was co-cultured for 7 days (IDUA c.2+IDUA-KO), it showed increased intracellular enzyme activity. When IDUA-KO was co-cultured with IDUA c.2 (IDUA c.2+.IDUA-KO), a reduction in intracellular GAG concentration was observed. C) When cultured alone, higher GAG concentration was observed in the KO control. [Figure 26] 13 is an image showing immunoreactivity for human-specific CD45 in the brain of NSGQ / NSGS mice 3 months after p3 administration of myeloid cells. [Figure 27] FIG. 1 shows immunoreactivity for human-specific CD45 and ku80 in the spinal cord of NSGQ mice 3 months after P3 administration of myeloid cells. [Figure 28] Images showing human-specific CD45 and ku80 immunoreactivity in the brain parenchyma. Branched myeloid lineage cells engrafted in the parenchyma can be seen in the striatum and ventral midbrain, among many other regions. [Figure 29] 13A-13C are images showing immunoreactivity for human-specific TMEM119 and ku80 in the striatum of NSGQ mice 3 months after P3 administration of myeloid cells. [Diagram 30]Images showing immunoreactivity for CD163, IBA1, and human nuclear antigen (hNA) in the brains of NSGQ mice 3 months after P3 administration of myeloid cells. [Diagram 31] A) Graph showing detection (automated counting) of the number of CD45+ / hku80+ cells found in mouse brains. B) Extrapolated count of the number of CD45+ / hku80+ cells found in mouse brains per hemisphere. [Figure 32-1] A) Graph showing myeloid cell derivation and characterization. Total myeloid cells generated in T-75 flasks for the three iPSC lines [N>6]. B) Graph showing myeloid cell derivation and characterization. Viability of myeloid cells after thawing for each of the three iPSC lines [N>6]. C) Images showing myeloid cell derivation and characterization. Representative panel of images showing myeloid cell immunoreactivity for CD45, CD68, P2RY12, TREM2, IB1, TMEM119, and PU.1 after 2 days in culture. [Figure 32-2] D) Derivation and characterization of myeloid cells. Representative flow cytometry plots for CD45 / CD14 and CX3CR1 / CD11B in myeloid cells after thawing [N=3 for each iPSC line]. [Figure 33-1] A) Transcriptome analysis of myeloid cells before and after engraftment. UMAP plot of the Bian et al. 2020 dataset of fetal hematopoietic (CD45+) cells showing total cell origin, Carnegie developmental stage, and cell type grouping. B) Transcriptome analysis of myeloid cells before and after engraftment. Same UMAP plot as in (A) showing expression levels of representative markers for Mac1-3 and Mac4 groups. C) Transcriptome analysis of myeloid cells before and after engraftment. PCA of myeloid cells, hiPSC, and the Bian et al. 2020 dataset. PC plots on the right represent identity scores for each cell when compared to Mac1-3 or Mac4 signatures. Violin plots represent the distribution of results for each sample. [Figure 33-2]D) Transcriptomic analysis of myeloid cells before and after engraftment. Heatmap of differentially expressed markers in each cell group of Bian et al. 2020 compared to all other groups combined. E) Transcriptomic analysis of myeloid cells before and after engraftment. Schematic showing the workflow for bulk RNAseq of myeloid cells and engrafted myeloid cells. F) Transcriptomic analysis of myeloid cells before and after engraftment. Dot plots representing the average expression and percentage of cells expressing Mac4 and Mac1-3 genes in each group. G) Transcriptomic analysis of myeloid cells before and after engraftment. (G) Heatmap of unique genes shown in (F) representing data from bulk RNAseq of engrafted myeloid cells. Green boxes highlight known microglial markers. [Diagram 34] Figure 1 shows pluripotency panel and karyotype analysis of hiPSC lines 6, 82, and 83. A) Pluripotency flow cytometry results for hiPSC line 6. Blue overlay represents experimental unstained control. (B) Phase contrast images from hiPSC line 6 24 hours after thawing at 4x and 10x magnification. (C) Pluripotency flow cytometry results for hiPSC line 82. Blue overlay represents experimental unstained control. (D) Phase contrast images from hiPSC line 82 24 hours and 48 hours after thawing at 4x and 10x magnification. (E) Pluripotency flow cytometry results for hiPSC line 83. Blue overlay represents experimental unstained control. (F) Phase contrast images for hiPSC line 83 24 hours and 48 hours after thawing at 4x and 10x magnification. (G) Karyotypes for hiPSC lines 6, 82, and 83 (G-banded cells analyzed, n = 20). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0020] definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0021] As used herein, the term "about" refers to approximately a + / - 10% variation from a given value.

[0022] As used herein, the term "subject" refers to any animal (e.g., mammal), including but not limited to humans, non-human primates, rodents, etc., that will be the recipient of a particular treatment. Typically, the terms "subject" and "patient" are used interchangeably herein with respect to human subjects. As used herein, a "healthy subject" is characterized as being disease-free and / or free of any metabolic disease.

[0023] As used herein, the term "non-human animal" refers to all non-human animals, including, but not limited to, vertebrates such as rodents, non-human primates, ovine, bovine, ruminants, lagomorphs, porcines, caprines, equines, canines, felines, and avian species.

[0024] As used herein, the term "cell culture" refers to any in vitro culture of cells. Included in this term are continuous cell lines (e.g., with an immortal phenotype), primary cell cultures, transformed cell lines, finite cell lines (e.g., non-transformed cells), and any other cell populations maintained in vitro.

[0025] As used herein, the term "in vitro" refers to an artificial environment and to processes or reactions that occur within an artificial environment. In vitro environments can consist of, but are not limited to, test tubes and cell cultures. The term "in vivo" refers to the natural environment (e.g., an animal or a cell) and to processes or reactions that occur within a natural environment.

[0026] As used herein, the term "sample" is used in its broadest sense. In one sense, this is meant to include specimens or cultures obtained from any source, as well as biological and environmental samples. Biological samples can be obtained from animals (including humans) and encompass fluids, solids, tissues, and gases. Biological samples include blood products such as plasma, serum, etc.

[0027] As used herein, the term "pluripotent stem cells" or "PSCs" has its ordinary meaning in the art, i.e., a self-renewing cell that has the ability to develop into endodermal, ectodermal, and mesodermal cells. In some embodiments, the PSCs are human PSCs ("hPSCs"). PSCs include embryonic stem cells (ESCs), induced pluripotent stem cells, and human induced pluripotent stem cells ("iPS cells" or "iPSCs" or "hiPSCs"). The terms ES cells and iPS cells have their ordinary meaning in the art.

[0028] As used herein, terms such as "treat" or "treatment" or "treating" refer to therapeutic measures that cure a diagnosed pathological disease or disorder, restore regenerative function, slow down or alleviate symptoms thereof, and / or halt their progression. Thus, those in need of treatment include those who already have the disorder. In certain embodiments, a subject is successfully "treated" for a disease or disorder when the subject exhibits, for example, total, partial, permanent, or temporary alleviation or elimination of any symptoms associated with the disease or disorder.

[0029] As used herein, the term "myeloid cells" refers to PSC-derived cells that differentiate after hematopoietic differentiation, express myeloid markers (including but not limited to CD45, CD11b, CD33, CD14, CX3CR1) and can perform normal myeloid functions including but not limited to phagocytosis, response to external stimuli, secretion of cytokines, and polarization into pro- or anti-inflammatory states. These myeloid cells become tissue-resident macrophages when delivered to different organs of the body, for example, they become microglia when delivered to the brain of a living animal because they express standard microglial markers including but not limited to TMEM119, IBA1, CD163, CX3CR1, CD45, CD206.

[0030] As used herein, the term "microglial progenitor cells" refers to myeloid lineage cells derived from PSCs.

[0031] As used herein, the term "microglia" refers to myeloid lineage cells derived from PSCs.

[0032] As used herein, the term "substrate" refers to glycosaminoglycans or other undegraded compounds that accumulate as a result of lysosomal storage diseases (LSDs).

[0033] As used herein, the term "central nervous system" refers to the spinal cord, brain, and cerebrospinal fluid (CSF).

[0034] As used herein, the term "KO (knockout)" refers to a cell line or organism that has been genetically engineered to lack one or more specific genes.

[0035] As used herein, the term "WT (wild type)" refers to the typical or most common form, appearance, or lineage occurring in the wild; the normal, unmutated form of a gene common in nature, or the alleles at each locus required to produce the wild-type phenotype.

[0036] As used herein, the term "MG-WT" refers to wild-type myeloid cells.

[0037] As used herein, the term "heterozygous" refers to inheriting different forms of a particular gene from each parent. A heterozygous genotype is in contrast to a homozygous genotype, in which an individual inherits the same form of a particular gene from each parent.

[0038] As used herein, the term "metabolic disorder" or "inherited metabolic disorder" refers to different types of medical conditions caused by genetic defects, most commonly inherited from parents, that interfere with the body's metabolism.

[0039] As used herein, the term "cell" refers to the basic membrane-bound unit that contains the basic molecules of life and makes up all living organisms.

[0040] As used herein, the term "somatic cell" refers to any cell of the body, except sperm and egg cells. Somatic cells are diploid, meaning that they contain two sets of chromosomes, one inherited from each parent. Somatic cells include brain cells. Brain cells include, but are not limited to, neurons, oligodendrocytes, astrocytes, microglia, perivascular macrophages, meningeal macrophages, endothelial cells, pericytes, ependymal cells, and blood cells.

[0041] As used herein, the term "xenograft" refers to the transplantation, implantation, or injection of cells derived from human origin (e.g., hiPSC-derived microglia) into another mammal, e.g., a mouse, into the recipient.

[0042] As used herein, the term "ramified" refers to microglial cells that are found in specific locations throughout the brain and spinal cord. Ramified microglia are in a "resting" state morphology.

[0043] As used herein, the term "heatmap" refers to a two-dimensional table of numerical values ​​as shades of color. Heatmaps are used to depict gene expression and other multivariate data. The dense and intuitive display makes heatmaps well suited for displaying high-throughput data. Heatmaps fundamentally rely on color encoding and meaningful permutations of rows and columns.

[0044] As used herein, the term "gait analysis" refers to an important tool for testing mouse models that provides quantifiable behavioral data about how a given disease, injury, or drug affects an animal's movement.

[0045] Generation of myeloid lineage cells from pluripotent stem cells Disclosed herein are serum-free and feed-free protocols for differentiating PSCs (including iPSCs) into myeloid lineages. A variety of methods known in the art can be used in conjunction 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, Edition 6, Pages 1516-1524, June 6, 2017, Van Wilgenburg PLOS ONE, https: / / doi.org / 10.1371 / journal.pone.0071098--Aug 2013, Haenseler et al., Stem Cell Reports June 6, 2017;8(6):1727~1742 and Takata et al., Immunity 2017 18;47(1):183-198, the disclosures of which are incorporated herein by reference. Microglial cells arise from myeloid progenitors within the yolk sac during embryonic development. In an attempt to mimic the embryonic development of these myeloid cells, we generated primitive streak-like cells from PSCs, followed by hematopoietic and myelopoietic cocktails in serum-free medium. This resulted in the appearance of myeloid cells expressing myeloid markers, including but not limited to CD45, CD14, CX3CR1, CD33, and CD11b, in the supernatant fraction of the culture. These myeloid cells can continue to be generated for a significant period of time in the supernatant fraction of the culture, often reaching 3-4 months. Myeloid cells are typically harvested and frozen approximately 30 days after the start of the differentiation protocol (the exact time of harvest depends on the PSC line and should be empirically defined based on the day of maximum yield). The yield is typically between 25 and 120 myeloid cells per starting PSC, with a post-thaw viability of 85±10%.

[0046] In some embodiments, the present disclosure provides a method for generating myeloid lineage cells from pluripotent stem cells. In some such embodiments, the pluripotent stem cells are from any mammalian species, but preferably from human. 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 include culturing the pluripotent stem cells under conditions that induce myeloid differentiation, resulting in the generation of CD45+ / CD14+ / CX3CR1+ myeloid lineage 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.

[0047] In some embodiments, pluripotent stem cells are cultured under conditions that induce myeloid differentiation, resulting in the generation of CX3CR1+ myeloid cells. In some embodiments, pluripotent stem cells are cultured under conditions that induce myeloid differentiation, resulting in the generation of CD45+ myeloid cells. In some embodiments, pluripotent stem cells are cultured or expanded in a bioreactor. In some embodiments, pluripotent stem cells are cultured in a cell factory under active gassing. By "active gassing" is meant the application or application of a pressure gradient of a gas mixture within the cell factory or cell factories. Gas mixtures contemplated by the present disclosure include those containing between about 1% and about 20% CO. 2 Ratio of about 80% to about 99% to air, about 3% CO 2 Ratio of about 97% to air, and about 5% CO 2 This includes a ratio of about 95% to air.

[0048] In some embodiments of the present disclosure that involve culturing pluripotent stem cells under conditions that induce myeloid differentiation (resulting in the generation of CD45+ / CD14+ / CX3CR1+ myeloid cells), a multi-step process is used in which the cells are cultured with different combinations of cytokines and tissue culture media at each step. These steps in the multi-step process can result in the induction of differentiation of pluripotent stem cells into primitive hemangioblasts and / or the induction of differentiation of primitive hemangioblasts into myeloid progenitor cells.

[0049] In some embodiments, the methods provided herein for generating CD45+ / CD14+ / CX3CR1+ myeloid cells from pluripotent stem cells include performing one or more of the following steps: first, contacting a cell culture with a first composition comprising BMP4 in a culture medium, wherein the cell culture comprises pluripotent stem cells when the cell culture is initially contacted with the first composition. A small molecule that can 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 (e.g., each of SCF and VEGF, with or without bFGF); third, contacting the cell culture with a third composition comprising one or more of SCF, IL-3, TPO, M-CSF, and FLT3 ligand (e.g., 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 (e.g., 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 sequence. In some such embodiments, the medium used in any of these four steps is a serum-free medium. In some such embodiments, the medium used in any of these four steps is a chemically defined medium.

[0050] In the first of the above four steps, in some embodiments, tissue culture medium suitable for stem cell maintenance is used, while in other embodiments, tissue culture medium suitable for stem cell differentiation is used. In the last three of the above four steps, any suitable hematopoietic cell medium may be used.

[0051] In some embodiments, when performing the methods described above or elsewhere herein for generating myeloid cells from pluripotent stem cells, instead of discarding the tissue culture supernatant when performing a medium exchange, the supernatant is centrifuged and the cells present in the supernatant are collected and returned to the cell culture. This is beneficial because some of the major cell types induced during the conversion of pluripotent stem cells to myeloid cells are found primarily in the cell supernatant as opposed to the layer of cells that adhere to the cell culture plate. Thus, in some embodiments, when changing the medium, the cells present in the culture supernatant are collected and returned to the cell culture. In some embodiments, for medium exchanges performed when the cells are in contact with the third composition or the fourth composition, the cells present in the culture supernatant are collected and returned to the cell culture. 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" population of such cells.

[0052] Treatment of metabolic disorders Disclosed herein is a method for treating patients with LSD using a cell + gene therapy approach. Myeloid cells are generated from pluripotent stem cells derived from healthy donors. PSC-derived myeloid cells are injected into the central nervous system of the patient to be treated. In one embodiment, myeloid cells are delivered systemically by intravenous (IV) administration. In another embodiment, myeloid cells are delivered to the central nervous system by intracerebroventricular (ICV) injection or directly into the cerebrospinal fluid (CSF) of the subject to be treated. In another embodiment, myeloid cells are administered by intracerebroventricular (ICV) injection into the lateral ventricles of the brain, which is the site where cerebrospinal fluid (CSF) is produced and where CSF flow begins. Direct injection of myeloid cells into the cerebrospinal fluid (CSF) is used to bypass the blood-brain barrier. Alternatively, the present disclosure contemplates intraparenchymal administration of myeloid cells. In intraparenchymal administration, myeloid cells are delivered directly into the brain parenchyma, and representative sites of parenchymal administration include, but are not limited to, the striatum, forebrain, and hippocampus. The amount of myeloid cells administered via any of the above routes is approximately 25×10 6 ~Approx. 1250×106 cells, approximately 50 x 10 6 ~About 1000×10 6 cells, approximately 100 x 10 6 ~About 500×10 6 cells, approximately 100 x 10 6 ~About 300×10 6 cells, more preferably about 150×10 6 ~Approx. 250×10 6 These specific delivery methods ensure engraftment of the injected microglial progenitor / myeloid cells, and delivery of the enzyme of interest within the CNS.

[0053] As the PSC-derived myeloid cells become the resident myeloid cells of the tissue, the level of enzyme production stabilizes for long periods of time, providing a steady and continuous supply of the missing enzyme. By "long-term stable" 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 enzyme is constantly and consistently secreted from the transplanted cells and enters the diseased cells of the host, and in the case of Hurler and Sly syndromes, repairs the pathological accumulation of toxic substrates such as glycosaminoglycans, thereby repairing the underlying pathophysiology in the CNS tissue. Some amount of enzyme produced within the CNS by the engrafted PSC-derived myeloid cells also enters the bloodstream, delivering the therapeutic enzyme to the periphery as well, reducing the total amount of accumulated substrate as evidenced by a reduction in total GAGs in the urine. After transplantation, the enzyme levels are monitored in serum, urine or cerebrospinal fluid samples of the patient at different time points. In one embodiment, the enzyme levels are monitored 1 month, 6 months, 12 months and 24 months after transplantation and annually thereafter. The amount of substrate or glycosaminoglycan is monitored in serum, urine or cerebrospinal fluid samples of the patient 1 month, 6 months, 12 months and 24 months after transplantation and annually thereafter to determine the progress of treatment. A sustained reduction in total substrate levels of greater than about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80% or about 90% compared to the individual patient's pre-treatment levels indicates successful treatment of said metabolic disorder. In Hurler syndrome, the enzyme is alpha-L-iduronidase (IDUA). In Sly syndrome, the enzyme is beta-glucuronidase (GUSB).

[0054] In accordance with the present disclosure, the treatment of metabolic disease may be indicated by measuring the enzyme level or the substrate level, or by both the enzyme and substrate levels. In one embodiment, successful treatment of Hurler syndrome is indicated if the level of IDUA increases in the serum, urine and / or cerebrospinal fluid (CSF) of the patient compared to the state before treatment begins. Successful treatment of Hurler syndrome is also indicated if the level of glycosaminoglycans (GAGs) decreases in conventional body fluids, including the urine, blood or cerebrospinal fluid of the patient compared to the state before treatment begins.

[0055] In another embodiment, successful treatment of Sly Syndrome is indicated when the level of GUSB is increased in the serum and cerebrospinal fluid (CSF) of the patient compared to the state before the start of treatment. Successful treatment of Sly Syndrome is also indicated when the level of glycosaminoglycan (GAG) is decreased in conventional body fluids including urine, blood, or cerebrospinal fluid of the patient compared to the state before the start of treatment. When the subject is a young person, the evaluation of the effectiveness of the treatment disclosed herein may be indicated by an improvement in gross motor function as measured by the Peabody Developmental Motor Scale or Gross Motor Function Measure at 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 15 months, 2 years, 3 years, 4 years after the treatment. Appropriate clinical standard evaluations can be evaluated by a skilled physician and can be utilized without undue experimentation when the subject is not a young person. These clinical assessments are applicable at 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 15 months, 2 years, 3 years, 4 years and up to 25 years after treatment.

[0056] In accordance with the present disclosure, the success of the metabolic disease treatment provided herein is recognized by a skilled physician by neurocognitive and gross motor assessments measured over a period of time. Neurocognitive assessment of treated subjects is also recognized by halting or slowing the progression of neurocognitive decline using standard clinically approved neurocognitive tests.

[0057] Pluripotent stem cell-derived myeloid cells offer an exciting new therapeutic approach for the treatment of some LSDs, in contrast to enzyme replacement therapy and gene therapy, where each therapy is tailored to the specific genetic defect.

[0058] In another embodiment, lysosomal storage diseases treatable by the methods of the present disclosure include MPS I (Hurler syndrome), MPS IIIA (Sanfilippo A), MPS IIIB (Sanfilippo B), MPS VII (Sly syndrome), Krabbe disease, Pompe (glycogen storage disease type II), GM1-gangliosidosis, GM2-gangliosidosis (Sandhoff / Tay-Sachs), metachromatic leukodystrophy, MPS II (Hunter), MPS IIIC (Sanfilippo C), MPS IIID (Sanfilippo D), MPS IVA (Morquio syndrome A), MPS IX, MPS VI (Maroteaux-Lamy), Niemann-Pick disease types A and B, adrenoleukodystrophy (ALD), Niemann-Pick disease type C2, Niemann-Pick disease type C1, sphingolipid activator deficiency, galactosialidosis, GM2-gangliosidosis, (GM2-activator deficiency), GM3-gangliosidosis, Fabry disease, Gaucher disease types II and III, α-mannosidosis, β-mannosidosis, fucosidosis, aspartylglucosaminuria, Schindler disease, sialidosis galactosialidosis, mucolipidosis II (I cell disease), mucolipidosis III, Danon disease, Salla disease, mucolipidosis IV, multiple sulfatase deficiency, MCD, and neuronal ceroid lipofuscinosis.

[0059] Those skilled in the art will understand that the effective amount of myeloid cells used in the disclosed method can be determined by routine experimentation. Based on in vitro and in vivo mouse data on Hurler syndrome, human data from the literature (Hurler syndrome), mouse-human scaling, and consideration of changes in brain-to-CSF-GAG levels, a system-based dose prediction model framework was developed. This model was then utilized to estimate the human equivalent dose (number of myeloid cells) of myeloid cells that release IDUA to metabolize increased CSF-GAG levels from disease to healthy range (Table 1 and Table 2).

[0060] [Table 1]

[0061] [Table 2]

[0062] The present disclosure contemplates, for example, that for mouse-to-human brain surface area scaling, a dose for a 50% reduction from disease baseline (10 μg / ml disease CSF-GAG) would require approximately 46 million myeloid cells, a dose for an 80% reduction would require approximately 77 million myeloid cells, and a dose for a 90% reduction would require approximately 92.5 million myeloid cells.

[0063] The present disclosure contemplates, for example, that for mouse-to-human brain volume scaling, a dose for a 50% reduction from disease baseline (10 μg / ml disease CSF-GAG) would require approximately 42 million myeloid cells, a dose for an 80% reduction would require approximately 70 million myeloid cells, and a dose for a 90% reduction would require approximately 83.5 million myeloid cells.

[0064] The myeloid cells may be administered alone or in a preformulated dosage, optionally with pharma- ceutically acceptable carriers and excipients. Administration of an effective amount of the myeloid cells results in an increase in lysosomal enzyme activity in the patient's cells sufficient to ameliorate disease symptoms.

[0065] In another embodiment, the present disclosure provides for the treatment of lysosomal storage diseases by co-culturing wild-type myeloid cells with diseased cells.

[0066] xenograft Disclosed herein are methods for xenotransplantation of human myeloid cells, such as microglial cells, using the NSG mouse model. The immunodeficient background of NSG (NOD / SCID / IL2R gamma) mice allows for the evaluation of allogeneic and xenogeneic cell transplantation experiments without daily administration of immunosuppressants to prevent graft rejection. NSG mice have no mature T or B cells, lack functional natural killer (NK) cells, and have reduced numbers of lymphocytes and myeloid dendritic cells. Human growth factors have been reported to support the survival and stable engraftment of human myeloid cells, including microglia. The NSG-SGM3 (Coughlan, 2016) and NSG-Q (Svoboda, 2019) mouse strains were developed to provide the human growth factors necessary to support bone marrow survival and engraftment.

[0067] The NSG-SGM3 mice used in this disclosure were developed on an NSG background. They are genetically immunodeficient. NSG-SGM3 mice contain three human transgenes; human stem cell factor (SCF), human granulocyte / macrophage-colony stimulating factor 2 (GM-CSF), and human interleukin (IL-3). Upon administration of human cells, the triple transgenic NSG-SGM3 mice constitutively produce adequate circulating levels of these myelosupportive human cytokines that restore the lack of xenoactivity and provide a stable environment for the engraftment of human myeloid cells such as microglia by providing signals for cell proliferation and survival over time.

[0068] NSG-Q mice are a widely used model for microglial xenotransplantation because they are immunodeficient and have stable expression of key myeloid-supportive human cytokines (CSF1, CSF2, KITLG, and IL3) associated with the survival of microglia and other myeloid cells (Svoboda, 2019).

[0069] The present disclosure is further illustrated by the following non-limiting examples. EXAMPLES

[0070] Example 1 Generation of knockout cell lines Ten LSD-related genes were targeted to generate knockout cell lines (null KO) by CRISPR / Cas9-based editing: these genes are IDUA, GUSB, SGSH, NAGLU, GAA, GALC, GLB1, ARSA, HEXA, and HEXB. These clones have been confirmed as null KO by genotype via sequencing, and null protein KO via either ELISA or Western blot, and enzyme activity assays. CRISPR / Cas9 allowed the generation of iPSC clones from a single parental line: knock-in of an overexpression construct of the IDUA enzyme for payload delivery, and knock-out of IDUA for use as a target for cross-repair of the enzyme and as a negative control in animal studies. Genotype was verified by Sanger sequencing and protein expression was confirmed by ELISA, resulting in minimal expression of the targeted knockout or increased expression in the overexpression lines. (Figure 23).

[0071] Example 2 In vitro cross-repair assay Wild-type microglial cells (MG) were cultured in 6-well bottoms and knockout cells (PSC or MG) were co-cultured in transwells for approximately 2 weeks. Cells were harvested or lysed in the wells with lysis buffer (1% Triton-X in HBSS, 1x Halt protease inhibitor). Total protein in each lysate was measured by Pierce BCA Protein Assay Kit (Thermo Fisher). Natural substrate accumulation was measured for each sample (GAG or glycogen assay) and, where possible, enzyme activity was also measured for each sample.

[0072] These transwell culture results (in the case of Hurler syndrome) show that healthy microglia release the IDUA enzyme into the culture medium, and that the released IDUA is taken up by IDUA-KO microglia, leading to reduced toxic accumulation of glycosaminoglycans (GAGs) in recipient IDUA knockout microglia. These results demonstrate that wild-type donor microglia are able to cross-repair the enzyme defect in defective cells found in LSD.

[0073] IDUA-OE and IDUA-KO hiPSC lines were robustly differentiated into microglia. To demonstrate the ability of overexpressing cells to functionally repair diseased cells lacking a single lysosomal enzyme, IDUA-KO microglia were co-cultured with IDUA-OE microglia in a transwell system. An increase in intracellular IDUA enzyme activity was observed in IDUA-KO microglia when co-cultured with the overexpressing line, with a corresponding reduction in the accumulation of GAG substrates. The enzyme activity and total GAG accumulation results demonstrate that overexpression of IDUA can metabolically cross-repair and restore function by providing the appropriate missing enzyme (Figure 25). Additional experimental data show that this general mechanism of cross-repair also applies to several other LSDs, thus identifying it as an ideal cellular therapy for LSDs.

[0074] Example 3 In vivo efficacy assay IDUA (alpha-L-iduronidase) knockout mice for Hurler syndrome and GUSB (beta-glucuronidase) knockout mice for Sly syndrome were purchased from Jackson Labs.

[0075] Hurler syndrome Microglial cells were transplanted into the brains of young and adult IDUA knockout mice. 1.4 × 10 6 A high dose of 0.4 × 10 cells was used. 6 A low dose of cells was used. At different time points (1 and 5 months), body fluids (serum, CSF, urine) and tissues (brain, spinal cord) were collected and tested for IDUA enzyme and GAG (glycosaminoglycan) amounts. Figure 1 shows a schematic overview of the non-GMP, in vivo study design to evaluate the efficacy of myeloid cell transplantation in the IDUA-KO mouse model of Hurler syndrome.

[0076] Figure 2 shows biochemical analysis of CNS tissues from IDUA-KO mice 1 month after ICV delivery of myeloid cells. IDUA enzyme levels were rescued 1 month after transplantation in a dose-dependent manner in young and adult IDUA KO mice. IDUA activity was increased to 10-15% of WT levels. Figure 3 shows quantification of accumulated substrates of IDUA (expressed as total GAGs) in CNS tissues of IDUA-KO animals 1 month after myeloid cell transplantation. Myeloid cell transplantation into young or adult IDUA-KO animals dose-dependently reduced accumulation of toxic substrates in all CNS tissues compared to vehicle-treated animals. GAGs were reduced by 20-85% from IDUA KO mice compared to vehicle-treated animals.

[0077] Figure 4 shows biochemical analysis of CNS tissue from IDUA-KO mice 5 months after ICV delivery of myeloid cells, with similar results as after 1 month (Figure 2). Figure 5 shows quantification of accumulated substrates of IDUA (expressed as total GAGs) in CNS tissue from IDUA-KO animals 1, 5, and 8 months after myeloid cell transplantation, with similar results as after 1 month (Figure 3).

[0078] Figure 6 shows rescue of serum IDUA and reduction of urinary GAG levels in an adult mouse model, and Figure 7 shows rescue of serum IDUA and reduction of urinary GAG levels in a young mouse model.

[0079] The photographs shown in Figure 8 show mice with different severity scores in the hindlimb grasping test. The table in Figure 8 provides further explanation of the scoring. Hindlimb grasping is a behavioral test used as a marker of disease progression in models of neurodegenerative and cerebellar ataxia (Zhu et al. (2016) Toll-like Receptor 4 Deficiency Impairs Motor Coordination. Front. Neurosci.). Figure 9 shows the hindlimb grasping test in young IDUA-KO animals 6 and 9 months after bone marrow cell transplantation. Both high and low doses of bone marrow cells were able to reverse the behavioral deficits observed in IDUA-KO animals.

[0080] Figures 10 and 11 show immunohistochemistry of two sagittal sections from adult and young IDUA-KO brains, respectively, one month after ICV delivery of myeloid cells. The results show that both adult and young IDUA-KO have CD45+ myeloid cells distributed throughout the brain after one month in vivo.

[0081] Figures 12 and 13 show immunohistochemistry of two sagittal sections from adult and young IDUA-KO brains, respectively, 5 months after ICV delivery of myeloid cells. The results show that at 5 months in vivo, both adult and young IDUA-KO have similar biodistribution of myeloid cells as the 1 month cohorts.

[0082] Figure 18 shows that the brains of IDUA-KO mice transplanted with MG-WT have 2.8% of the IDUA activity of WT mice. Figure 19A shows that the brains of IDUA KO mice transplanted with MG-WT have 66% reduced GAG levels compared to IDUA KO animals. Figure 19B shows that the brains, spinal cords and CSF of IDUA KO mice transplanted with MG-WT have significantly reduced GAG levels compared to IDUA KO animals at 1 and 5 months (brain, spinal cord and CSF) and 8 months (brain and spinal cord). These results demonstrate the ability to reverse Hurler syndrome in vivo.

[0083] Sly Syndrome Figure 14 shows a schematic outlining the non-GMP, in vivo study design to evaluate the efficacy of myeloid cell transplantation in the GUSB-KO mouse model of Sly syndrome. Myeloid cells are transplanted into the brains of 9-week-old adult IDUA knockout mice. 1.4 x 10 6 A high dose of cells is used. A coronal section of the mouse brain atlas shows the sites of myeloid cell delivery in both ventricles (red arrows). The timeline of the "in-life" phase indicates when the various samples were taken from the mouse and when hindlimb gripping was performed.

[0084] Figure 15 shows biochemical analysis of CNS tissue from adult GUSB-KO mice one month after ICV delivery of myeloid cells. Myeloid cell transplantation into adult GUSB-KO animals reduced the accumulation of toxic substrates (expressed as total GAG) in the CNS. Percentages show the reduction of GAG relative to vehicle-treated animals.

[0085] Figure 16 shows the hindlimb grasping test in adult GUSB-KO animals 2 and 3 months after bone marrow cell transplantation. Bone marrow cell transplantation reduced the behavioral deficits observed in GUSB-KO animals, especially 3 months after transplantation.

[0086] Figure 17 shows immunohistochemistry of a lateral sagittal section from an adult GUSB-KO brain one month after ICV delivery of myeloid cells. Myeloid cells (arrows) were positive for all markers and were found in the choroid plexus and meninges surrounding the brain.

[0087] Example 4 Figure 23 shows the generation of engineered hiPSC lines overexpressing IDUA to deliver a payload carrier. The IDUA-OE cell line can contain a cassette insert containing the aEF1-IDUA cassette into the AAVS1 locus, resulting in sustained high-level expression of IDUA in two distinct clones (Figure 18A). An IDUA knockout line was simultaneously engineered to serve as a target for repair in vitro and as a transplantation control in vivo (Figure 18B).

[0088] Example 5 NSG-Q mice were developed by crossing NSG-SGM3 with NSG-CSF1 mice, which express human M-CSF (colony-stimulating factor 1 (macrophage)) and a triple transgenic SGM3 transgene, each with a human cytomegalovirus promoter / enhancer sequence. The CSF1 and SGM3 transgenes encode human cytokines that function to support stable engraftment of human myeloid lineages and regulatory T cell populations. The NSG-Q line is an immunodeficient mouse model that produces human cytokines that can be used to support the survival of human induced pluripotent stem cells (hiPSCs). Brains from NSG-Q mice transplanted with hiPSC-derived microglia generate cells that express microglial morphology and gene expression patterns similar to human primary microglia. In some brain regions, donor cells represent up to 50% of the total microglial population.

[0089] Myeloid cells were transplanted into 3-day-old NSG-Q mice. Pups were anesthetized and administered intracerebroventricularly with 140k cells per hemisphere for a total of 280k cells per pup; NSG-SGM3 were used as a control. Pups were left to mature for 3 months to allow time for proper engraftment of myeloid cells.

[0090] Immunofluorescence staining of NSG-Q and NSGS revealed successful delivery of myeloid cells into the lateral ventricles (Figure 26). As expected based on previous experiments with other mouse models, myeloid cells engrafted in the meninges and choroid plexus. However, engraftment in NSG-Q was more extensive in the parenchyma and was seen throughout the entire extent of the hemisphere.

[0091] Myeloid cell engraftment in NSG-Q spans the entire length of the spinal cord, and engrafted myeloid cells can be detected in the thoracic, lumbar, and sacral regions of the spinal cord (Figure 27).

[0092] Engraftment of myeloid cells in the brain parenchyma results in a branched, "quiescent" morphology of the resulting microglia and high expression of CD45. Branched myeloid cells engrafted in the parenchyma can be found in the striatum and ventral midbrain, among many other regions (Figure 28). Myeloid cells in the brain parenchyma also express the canonical microglial marker TMEM119, suggesting that they have adapted to the niche and adopted the identity of quiescent microglia (Figure 29). Myeloid cells are also positive for IBA1, a known microglial marker. When found in the meninges or choroid plexus, myeloid cells also express the canonical meningeal macrophage marker CD163 (Figure 30).

[0093] Automated counting methods detected the number of CD45+ / hku80+ myeloid cells found in the mouse brain. There was a 10-fold difference in the number of myeloid cells detected in NSG-Q compared to NSGS. Extrapolation was an estimate of what would have been expected to be present in the entire hemisphere using the sampling counts as the basis for calculation (Figure 31A and Figure 31B).

[0094] The myeloid differentiation protocol is highly efficient, producing an average of 43 myeloid cells for each iPSC, and is highly reproducible. All three lines tested produced an average of 3 × 10 myeloid cells in T-75 flasks. 7 The hiPSCs produced 1000 myeloid cells (Figure 32A). Myeloid cells recovered from cryopreservation showed high viability across the three hiPSC lines (Figure 32B) and displayed canonical microglial markers including CD45, CD14, CD11B and CX3CR1 (Figure 32D). Upon plating, the myeloid cells displayed typical microglial morphology and were immunoreactive for microglial markers such as CD45, CD68, TMEM119, IBA1, P2RY12, Pu.1, and TREM2 (Figure 32C).

[0095] Microglial progenitor cells were isolated and counted with an automated cell counter NC-200 before freezing. Cells were resuspended in BamBanker or STEM-CELLBANKER GMP grade freezing medium and transferred to cryogenic storage vials. Cryogenic storage vials containing cells were frozen using a controlled rate freezer (CBS CRF2101) using an optimized freezing program. After the freezing program was completed, cells were transferred to liquid nitrogen (vapor phase) for long-term storage.

[0096] To thaw the microglial progenitor cells, the cryovial containing the cells was transferred to a 37°C water bath for approximately 2 min until small ice crystals remained. In a biosafety cabinet, the cells were transferred from the cryovial to a centrifuge tube and quenched with 1 mL of RPMI-1640 or StemPro-34 medium dropwise to reduce osmotic shock. The cells were measured in an NC-200 and quenched with an additional 3 mL of the respective medium. The cells were centrifuged at 250 g for 5 min and resuspended in the appropriate assay medium.

[0097] Figure 33 shows that engrafted myeloid cells upregulate microglial signature genes. To delve deeper into the molecular profile of myeloid cells before transplantation, we performed single-cell transcriptome analysis. As myeloid cells are derived from a protocol that mimics microglial development from primitive hematopoietic progenitors, we compared the bone marrow single-cell data with a recent dataset of CD45+ fetal hematopoietic cells (Bian et al., 2020). In the original report, 15 clusters were annotated according to the expression of marker genes, with various representations of Carnegie developmental stages and anatomical origin of the samples (Figures 33A and 33B). Among them, we found four macrophage clusters (clusters 1-3 were treated as one to simplify the analysis). Mac4 shared a macrophage molecular signature with Mac1–3, but added distinctive expression of SALL1, as well as higher expression of TMEM119, TREM2, MERTK, and P2R1Y12, suggesting a clearly defined microglial profile.

[0098] Using principal component analysis (PCA), myeloid cells were observed to cluster closely with Mac1-3 and Mac4, while fetal non-macrophage cells (all other cell identities combined) were observed to be located further along the PC2 axis. hiPSCs clustered separately from all other cells along the PC1 axis (Figure 33C). Using cell signatures derived from the original clusters published in Bian et al. 2020 to score the identity of myeloid cells, they were found to be most similar to Mac1-3, with some cells showing slight similarity to Mac4.

[0099] To assess changes following engraftment, we utilized differentially expressed genes in the Mac4 cluster (Figure 33D) and combined them with differential expression analysis of myeloid cells before and after transplantation in the NSG-Quad (NSG-Q) mouse model, an immunodeficient mouse line expressing human cytokines and growth factors (i.e., CSF1, CSF2, SCF, IL-3) that support human myeloid cell survival and engraftment17 (Figure 33E). After engraftment, we detected upregulation of Mac4 markers such as SALL1, TMEM119, MERTK, P2RY12, and TREM2 (Figure 33F), supporting the microglial identity (and increased similarity to Mac4) of myeloid cells following engraftment.

[0100] In parallel, changes in known microglial transcripts and genes of interest were examined between pre- and post-engraftment myeloid cells (Figure 33G). Increased expression was found in several microglial genes, such as HEXB and PU.1 [SPI1]. Several markers, such as IBA1 [AIF1] or PTPRC [CD45], already highly expressed in myeloid cells, showed similar or slightly reduced levels after engraftment. Lysosomal enzymes (Neuronopathic MPS enzymes) were also present at either equivalent or increased levels after engraftment, supporting our therapeutic strategy (Figure 33G).

[0101] A major concern for PSC-derived therapies is potential residual PSCs. In both pre- and post-transplant data sets, POU5F1[OCT4] transcripts were not detectable. To address this further, we examined proliferation markers and observed a 4-fold reduction in MKi67 transcript levels (Figure 33G).

[0102] RNA was isolated from microdissected fixed tissue sections using the RNeasy FFPE kit and analyzed on an Agilent 2100 Bioanalyzer using the Agilent 6000 RNA Nano Kit. cDNA libraries for sequencing were generated using the Illumina Collibri 3' mRNA Library Prep Kit. Libraries were quantified using the Collibri Library Quantification Kit and evaluated on an Agilent 2100 Bioanalyzer using the Agilent High Sensitivity DNA Kit. Libraries were sequenced on an Illumina NextSeq 500 using the NextSeq 500 / 550 High Output Kit (75 cycles). Reads were filtered and trimmed using Cutadapt v3.4 (https: / / doi.org / 10.14806 / ej.17.1.200) and aligned to both hg38 and mm39 using STAR v2.7.8a (PMID:23104886). Aligned reads were sorted and indexed using SAMtools v1.12 (PMID:19505943) and filtered to detect only human-specific reads using XenofilteR (PMID:30286710). Reads aligned to expressed genes were counted using HTSeq v0.13.5 (PMID:35311944) and differential expression analysis was performed using DESEQ2 (PMID:25516281).

[0103] To assess the efficacy of myeloid cell treatment in a juvenile (7 day old) immunodeficient mouse model of Krabbe disease, the reduction in toxic accumulation of undegraded substrate (psychosine) is measured. Analysis of psychosine is performed by Nucrotechnics using mass spectrometry. Tissues for analysis include brain, spinal cord, CSF, and urine, and results are reported in μg / mL.

[0104] For gait analysis, performed 34 days after implantation, animals (mice) are placed on a clear plexiglass track opposite a dark "goal box". Animals are allowed 1 min to walk across the track to reach the "goal box". Immediately after the first trial, animals are removed from the track apparatus and returned to the beginning of the track to complete a second walking trial. All trials are recorded from underneath the track, and tracking of the animal's body and forepaws is performed by DeepLab Cut. Ataxic gait is assessed by the width between the front and hind paws, the position of the print, stride length, speed, and step duration.

[0105] Example 6 Generation of cell lines Generation and maintenance of human iPSC lines hiPSC lines 82 and 83 were obtained by reprogramming peripheral blood mononuclear cells (PBMCs) into induced pluripotent stem cells. PBMCs were sourced from healthy donors through Be The Match BioTherapies (Minneapolis, MN), an FDA-registered cell collection facility that complies with HCT / P regulations. Erythroblasts were isolated and cultured from PBMCs according to Perriot et al. (Perriot, S., Canales, M., Mathias, A., and Du Pasquier, R. Generation of transgene-free human induced pluripotent stem cells from erythroblasts in feeder-free conditions. STAR Protoc 3, 101620 (2022). Erythroblasts were isolated and cultured according to Yoshioka and colleagues (Yoshioka, N. et al. Efficient Generation of Human iPSCs by a Synthetic Self-Replicative RNA. Cell Stem Cell Electroporation was performed using a synthetic self-replicating Venezuelan Equine Encephalitis (VEE) RNA replicon as described by Baghbaderani, BA et al. cGMP-Manufactured Human Induced Pluripotent Stem Cells Are Available for Pre-clinical and Clinical Applications. Stem Cell Reports 5, 647-659 (2015). The VEE replicon is a positive-sense, single-stranded RNA that does not utilize a DNA intermediate, so there is no possibility of genomic integration, and the absence of viral structural genes renders the RNA incompetent for viral packaging and pathology. Human iPSC line 6 was obtained from Lonza (Baghbaderani, BA et al. cGMP-Manufactured Human Induced Pluripotent Stem Cells Are Available for Pre-clinical and Clinical Applications. Stem Cell Reports 5, 647-659 (2015).iPSCs were maintained using Essential 8 medium (Thermo Fisher Scientific, A26559-01) with recombinant laminin (Biolamina, LN521-05) as a hiPSC attachment substrate. Cell passage was performed using EDTA as a non-enzymatic method (Thermo Fisher Scientific, AM9260G). Dissociated cells were plated on laminin 521-coated tissue culture vessels in Essential 8 medium supplemented with 10 μM Y-27632 (Bio-Techne, TB1254-GMP) for the first 24 h.

[0106] Generation of hiPSC knockout cell lines hiPSC lines lacking IDUA, GUSB, NAGLU, and SGSH were generated to test the ability of myeloid cells to cross-repair this enzyme deficiency in vitro. CRISPR / Cas9 single guide RNAs (sgRNAs) targeting the coding exons of each of these four genes were designed and tested. The sequences of these sgRNAs are listed in Table 3. Vials of parental hiPSC lines were thawed and transfected with individual Cas9:sgRNA ribonucleoprotein (RNP) complexes targeting each of the four genes to generate functional knockouts. Transfected cells were plated on vitronectin-coated tissue culture vessels in Essential 8 medium containing Y-27632 (10 μM). Clones were manually isolated by picking and then screened for biallelic KOs using PCR primers flanking the sgRNA target sites, followed by Sanger sequencing. The PCR primers used for genotyping are listed in Table 4. Due to the high editing efficiency observed in similar experiments, only 96 or fewer clones for each gene were screened using this method. Multiple clones of various KO genotypes were identified for each gene, and a single KO clone was selected for myeloid cell differentiation. The final genotype for each KO line is shown in the "Genotype" column of Table 3.

[0107] [Table 3]

[0108] [Table 4]

[0109] Myeloid cell differentiation protocol Human iPSCs were cultured at 1.0 × 10 in Essential 8 medium (Thermo Scientific, A1517001) containing 10 μM Y-27632 (Tocris, 1254). 4 Cells / cm 2PSCs were plated on vitronectin (Thermo Scientific, A14700) for 24 h at 37 °C for 24 h. PSCs were cultured in Essential 8 medium for an additional 2 days with daily medium changes and then induced with Essential 6 (Thermo Scientific, A1516401) medium supplemented with 80 ng / mL BMP-4 (R&D Systems, 314E-GMP-050). BMP-4 induction was continued for 4 days with daily medium changes, after which cultures were switched to StemPro-34 SFM medium (Thermo Scientific, 10639011) (containing 1× GlutaMAX Thermo Scientific, 35050061) supplemented with 100 ng / mL SCF (R&D Systems, 255B-GMP-050), 80 ng / mL VEGF (R&D Systems, 293-GMP-050) and 25 ng / mL bFGF (R&D Systems, 233-GMP-025) for 2 days with daily medium changes. On days 6 and 8, cells were cultured in StemPro-34 SFM medium containing 50 ng / mL SCF, 50 ng / mL IL-3 (R&D Systems, 203-GMP-050), 50 ng / mL M-CSF (R&D Systems, 216-GMP-500), 50 ng / mL Flt3 Ligand (R&D Systems, 308E-GMP-050), and 5 ng / mL TPO (R&D Systems, 288-TPE-050). Starting on day 10, cells from the supernatant fraction were pelleted and resuspended in the same fresh medium as on days 6 and 8 and returned to their respective vessels. Starting on day 14, cells in the supernatant fraction were pelleted and resuspended in StemPro-34 SFM medium containing 50 ng / mL Flt-3, 50 ng / mL M-CSF, and 25 ng / mL GM-CSF (R&D Systems, 215-GMP-050) and returned to their respective vessels. The medium change on day 14 was performed when the cells in the supernatant reached 1.0×10 cells per mL. 6Repeated every other day until a concentration of > 100 viable cells was reached. Starting on day 18 and every other day thereafter, cell counts were performed followed by pelleting the cells in the supernatant for a medium change to determine the harvest date. In some embodiments, the myeloid cells are cultured for about 1 to about 30 days, about 2 to about 25 days, about 2 to about 20 days, about 2 to about 18 days, about 2 to about 15 days, about 2 to about 10 days, about 2 to about 8 days, about 2 to about 6 days, or about 2 to about 4 days. In some embodiments, the myeloid cells are cultured for at least 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 myeloid cells are cultured for about 2 days, about 4 days, about 6 days, or about 8 days, about 10 days, about 12 days, about 14 days, about 16 days, about 18 days, about 20 days, about 22 days, about 24 days, about 26 days, about 28 days, or about 30 days. In some embodiments, the myeloid cells are cultured for about 2 days, about 4 days, about 6 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 14 days, about 16 days, about 18 days, about 20 days, about 22 days, about 24 days, about 26 days, about 28 days, or about 30 days. In some embodiments, the myeloid cells are cultured for about 2 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, about 15 days, about 16 days, about 18 days, or about 17 days. + / CD14 + / CX3CR1 + , at least about 71% CD45 + / CD14 + / CX3CR1 + , 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 + It is.

[0110] Cryopreservation and thawing of bone marrow cells Myeloid cells were isolated and counted with an automated cell counter NC-200 (Chemometec) before freezing. Cells were resuspended in BamBanker (Wako Chemicals, 30214681) or STEM-CELLBANKER GMP grade (amsbio, 11924) freezing medium and transferred to cryogenic storage vials (Thermo Scientific). Cryogenic storage vials containing cells were frozen in a controlled rate freezer (CBS CRF2101). Cells were transferred to liquid nitrogen (vapor phase) for long-term storage. To thaw myeloid cells, cryogenic storage vials were transferred to a 37°C water bath for approximately 2 minutes until small ice crystals remained. In a biosafety cabinet, cells were transferred from the cryogenic storage vials to centrifuge tubes and quenched by dropwise addition of 1 mL of RPMI-1640 (Thermo Scientific, 11-875-101) or StemPro-34 SFM medium. Cells were counted in an NC-200 and quenched with 3 mL of their respective medium. Cells were centrifuged at 250 g for 5 minutes and resuspended in the appropriate assay or culture medium.

[0111] Replating and polarization of myeloid cells Myeloid cells were cultured at 1.0 × 10 per well of a 96-well tissue culture treated plate in 50 μL of RPMI-1640 (Thermo Scientific, 11-875-101) containing 1× GlutaMAX supplemented with 100 ng / mL IL-34 (R&D Systems, 5265-IL-010 / CF) and 10 ng / mL GM-CSF (maturation medium). 5Cells were plated at 100x the volume of M0-like microglia (M(GM-CSF, IL34), quiescent rather than polarized microglia / macrophages) and 50x the volume of maturation medium was added directly on top of the cells. For M1-like microglia (M(LPS, IFNg), inflammatory microglia / macrophages), an additional 50x the volume of maturation medium supplemented with 200ng / mL LPS (Sigma-Aldrich, L4391) and 200ng / mL IFNg (R&D Systems, 285-GMP) was added directly on top of the cells.

[0112] Cytokine release assay (FRET) Spent media harvested from 48-h polarized microglia was centrifuged at 250g for 5 min and the supernatant was frozen at -80°C. Supernatants were thawed at room temperature and 16ul was transferred to a small volume 96-well plate for the addition of donor and acceptor antibodies from either IL-6, TNFα, or CXCL10 kits (Cisbio, 62HIL06PEG, 62HTNFAPEG, 62HCX10PEG). Samples were incubated for 2-24 hours, processed with standards, and analyzed on a Clariostar microplate reader (BMG Labtech) using Cisbio's recommended parameters according to the manufacturer's instructions. Data were interpreted by calculating the ratio of acceptor and donor luminescence signals and determining the delta ratio (ratio of standard or sample minus standard 0).

[0113] Flow cytometry analysis Myeloid cells were thawed in 2 mL of maturation medium as described above and cell counts were performed on a NucleoCounter. Based on viable cell counts per mL, 5.0 x 10 5Cells were transferred to 5 mL FACS tubes (Falcon). Aliquot samples of cells were prepared and placed into separate tubes for unstained and viability dye controls. Additional maturation medium was added to the cells at a 1:5 dilution to further dilute the cryopreservation medium. Cells were pelleted by centrifugation at 250g for 5 minutes, the supernatant was carefully removed, and the cells were resuspended in 100 μL of surface marker staining cocktail as shown in Table 5. Unstained samples were resuspended in 100 μL of 1×DPBS (Thermo Scientific, 14190250) and viability dye (Thermo Scientific, NC0476349) controls were resuspended in 100 μL of 1×DPBS containing 0.33 μL of viability dye. Samples were incubated at 4° C. for 30 minutes, washed with 2 mL of 1×DPBS, and pelleted by centrifugation at 200g for 5 minutes. Samples were resuspended in 200 ul of FACs buffer (Table 5) and then analyzed on a CytoFLEX LX (Beckman Coulter) and FCS files were exported and gated with FlowJo software.

[0114] [Table 5]

[0115] Phagocytosis of Escherichia coli pHrodo bioparticles Myeloid cells were cultured at 1.0 x 10 per well of a 96-well tissue culture plate. 5Cells were plated at 10 × 10 cells and polarized towards either M0- or M1-like microglia. After 2 days, a medium change was performed by including pHrodo Red E. coli bioparticles (Thermo Scientific, P35361) in both M0- and M1-like microglia maturation medium (see polarization section) at a final concentration of 45.45 μg / mL per well. Cells on the plate were placed in an IncuCyte S3 (Sartorius) in a 37°C incubator with 5% CO2 and imaged every 2 hours for phagocytic activity. One well had M0 medium (no cells) containing only pHrodo Red E. coli bioparticles, and 10 × 10 3 A single well with only M0 media (no pHrodo) containing myeloid cells was also imaged as a control.

[0116] Immunofluorescence staining for imaging Cells were fixed with cold 4% paraformaldehyde (Thermo Scientific, AAJ61899AK) for 10 min at room temperature (RT). After fixation, cells were washed with 3 rounds of 1xDPBS (Thermo Scientific, 14190250) for 5 min at RT, then permeabilized with 3 washes of 1xDPBS containing 0.2% Triton X-100 (Sigma-Aldrich, T8787) for 10 min each at RT. Next, 10% normal donkey serum (Jackson ImmunoResearch, 017-000-121) in DPBS was added to cells for 1 h at RT, and then cells were stained with primary antibody cocktail in blocking solution overnight at 4°C. Cells were subjected to three rounds of washing with 1x DPBS containing 0.2% Triton X-100 (DPBS-T) for 10 min each at RT, after which the following morning secondary antibody cocktail in DPBS was added for 1 h at RT. A further round of three 10 min DPBS-T washes was performed, followed by incubation with DAPI (Invitrogen, D21490) in DPBS for 20 min at RT. Finally, cells were washed with DPBS and stored at 4°C.

[0117] Metabolic cross-repair in vitro Wild-type and enzyme-deficient (MG-KO) microglia were thawed separately in maturation medium. Wild-type microglia were thawed at 1.0 × 10 in 2 mL of maturation medium. 6 MG-KO were plated at 5.0 × 10 in 1 mL of maturation medium into another 6-well tissue culture-treated plate holding a 0.4 μM porous clear polyester membrane insert (Falcon, 353090). 5 The MG-KOs were plated in 100% CO2 medium (2 mL of maturation medium was added to the lower well to prevent the membrane from drying out). After 48 h, the MG-KOs on the inserts were transferred and placed on top of the wild-type wells to initiate co-culture. Medium changes were performed at the start of co-culture and every other day for 10–14 days. Independent MG-KOs on inserts were cultured alone in parallel to serve as disease controls. To measure metabolic cross-repair, wild-type and enzyme-deficient microglia were collected separately from the inserts and wells, lysed with 1% TritonX-100, and intracellular enzyme activity and total GAG content were assessed.

[0118] Dose formulation for animal transplantation Myeloid cells were thawed and transferred to a centrifuge tube. 1 mL of StemPro-34 SFM medium supplemented with 50 ng / mL Flt-3 Ligand, 50 ng / mL M-CSF, and 25 ng / mL GM-CSF (SP34-d14) was added dropwise directly on top before cell counting was performed on the NucleoCounter. An additional 3 mL of StemPro-34 SFM-d14 medium was added on top to further dilute the freezing medium. Cells were centrifuged at 250 g for 5 min at room temperature and the supernatant aspirated. Cell pellet volume was determined using a 20-200 μL single channel pipette and transferred to a 1.5 mL microcentrifuge tube. Based on viable cell count per mL, 7 × 10 cells per μL were obtained. 4 (high dose) or 2 × 10 4 The cell pellet was diluted with additional StemPro-34 SFM-d14 medium for the final live cell formulation (low dose). The prepared cells were kept on ice until ready for transplantation.

[0119] Enzyme activity assay To determine enzyme activity, cells from membrane inserts were lysed in 1% Triton-X 100 containing protease inhibitors, followed by evaluation of enzyme activity and GAG levels. The method was adapted from Ou et al., 2014 (Ou, L., Herzog, TL, Wilmot, CM, and Whitley, CB Standardization of α-L-iduronidase enzyme assay with Michaelis-Menten kinetics. Mol. Genet. Metab. 111, 113-115 (2014)). Enzyme catalytic activity for each lysosomal enzyme was determined by quantifying 4-methylumbelliferyl (4-MU), a fluorescent moiety generated after cleavage of an artificial fluorescent substrate specific to each enzyme (Table 6). The resulting fluorescence of the cleaved substrate was read on a CLARIOstar with excitation set at 355 nm and emission at 460 nm. Enzyme levels and activity were interpolated using a 4-MU standard curve. For animal studies, frozen tissues were incubated at 4°C for 1 h in HBSS (Cytiva, SH30588.01) with 1% Triton X-100 and Halt protease inhibitor (Thermo The lysates were homogenized using a 50% PBS (Biotin Scientific, 87786). A BCA protein assay was performed on the resulting lysates to estimate the amount of isolated protein. Enzyme activity and GAG levels were measured from brain lysates, spinal cord lysates, and serum using up to 50 μg of total protein. For CSF, 1 μl of sample was used in the assay and results were later normalized to total protein concentration.

[0120] [Table 6]

[0121] Quantification of GAGs Frozen tissues were homogenized as described above. Quantification of GAGs from various cell lysates was performed using the cationic dye 1,9 dimethylmethylene blue (DMB) (Sigma Aldrich, 341088-1G), which binds highly charged sulfated GAGs. The resulting absorbance at 525 nm is proportional to the concentration of GAGs in the sample. Values ​​were interpolated using a GAG standard curve.

[0122] Animals and Husbandry The experiment consisted of a comparison group (Prkdc(scid)IDUA - / - and their wild-type Prkdc(scid)IDUA + / + Littermates (strain #: 004083, Jackson Laboratory) and Prkdc(scid)GUSB - / - and their wild-type Prkdc(scid)GUSBs + / + Male and female mice (3 and 8 weeks old; n = 6–8 / age group / genotype) with littermates (Jackson Laboratory) were used. Animals were maintained at 22–25°C on a 12-h light / dark cycle (lights on at 6:00 a.m.) with free access to food and water. Mice were housed 2–5 per cage. All experiments were performed in accordance with the guidelines of the Institutional Animal Care and Use Committee.

[0123] Stereotactic surgery Thirty minutes before surgery, animals were injected subcutaneously with 4 mg / kg meloxicam SR (Putney, RXMELOXICAM-INJ) and anesthetized by inhalation of approximately 2% isoflurane (Attane, RXISO-250) in oxygen throughout the surgical procedure. Anesthetized animals were placed in a stereotaxic frame (Kopf instruments) and injected with 10 μL of a low dose of myeloid cells (0.4 × 10 6 cells / brain), high dose myeloid cells (1.4 × 10 6Either 0.01 mg / mL Flt-3 (0.01 mg / mL Flt-3; 0.01 mg / mL M-CSF; 0.01 mg / mL GM-CSF) or vehicle (StemPro-34 SFM medium containing 50 ng / mL Flt-3, 50 ng / mL M-CSF, and 25 ng / mL GM-CSF; 0.01 mg ...) was injected bilaterally into the lateral ventricles using the following coordinates from bregma with a 27-gauge blunted needle attached to a 10 μL Model 701 RN Hamilton syringe: i) 8-week-old mice: AP -0.3, ML + / - 1.4, DV 2.34 at 10° angle ii) 3 week old mouse: AP+0.1, ML+ / -1.4, DV-2.34 at 10° angle.

[0124] Hindlimb grasping test Depending on the cohort, behavioral testing was performed at 1, 5, and / or 8 months post-surgery. Briefly, animals were removed from their cages and suspended by their tails for approximately 10-15 seconds, and their hindlimb grasping ability was monitored by video recording. Hindlimb grasping was scored on a scale of 0-3 based on: i) Score 0: Both hind legs are spread outward and away from the abdomen ii) Score 1: One hind limb is retracted towards the abdomen for more than 50% of the time the mouse is suspended by its tail. iii) Score 2: Both hind limbs are partially retracted towards the abdomen for more than 50% of the time that the mouse is suspended by its tail. iv) Score 3: Both hind limbs are fully retracted and touching the abdomen for more than 50% of the time that the mouse is suspended by its tail.

[0125] Tissue collection and analysis Depending on the cohort, final tissue collection was performed 1, 5, or 8 months after myeloid cell transplantation. Briefly, animals were anesthetized by inhalation of approximately 2% isoflurane in oxygen and a small incision was made along the back of the neck to expose the muscle tissue. The muscle tissue was then carefully removed to expose the cisterna magna and a 20 um glass pulled pipette was inserted into the cisterna magna to collect approximately 3-8 μl of cerebrospinal fluid (CSF). After CSF collection, blood was collected by intracardiac puncture and then the animals were transcardially perfused with cold phosphate buffered saline (PBS). The brains were cut into two hemispheres and the left hemisphere was placed in cold 4% paraformaldehyde (PFA) for 48 hours for histological analysis and the right hemisphere was snap frozen for biochemical analysis. One spinal column from each group was harvested and placed in 4% PFA for 48 hours for histological analysis and the remaining spinal cord was extruded by PBS flush and snap frozen for biochemical analysis.

[0126] Histology and immunofluorescence Tissues harvested for histological evaluation were fixed in 4% PFA for 48 hours. The left hemisphere was transferred to 20% sucrose in PBS (MP Biomedicals, 0219474705) for 24 hours and then to 30% sucrose in PBS for 24 hours. The brain was then embedded in OCT (Sakura, 4583) / 30% sucrose solution (1:1) and snap frozen in an ethanol bath on dry ice. 20 μm thick sections were then obtained in the sagittal direction using a cryostat (LEICA; CM3050 S) and mounted on positively charged microscope slides. The slides were air-dried for approximately 1 hour and then stored at -80°C. The spinal column was washed with PBS and then the spinal cord was separated from the vertebrae. The extracted spinal cord followed the same sucrose gradient immersion as for the brain hemispheres above. After the last sucrose solution, the spinal cord was sectioned into cervical, thoracic, and lumbar regions, embedded in OCT solution, and stored at -80°C. The spinal cord was sectioned into 20 μm serial slices on a cryostat, mounted on positively charged slides, and stored at -80°C. For immunofluorescence staining, slides were incubated for 30 min at RT, permeabilized with 0.2% Triton-X in PBS (PBS-T), blocked for 1 h with 10% donkey serum in PBS-T, and incubated with primary antibodies (Table 7 below) in blocking solution overnight at 4°C. The next day, slides were washed 3 times with PBS-T and incubated with secondary antibodies (Table 7 below) for 1 h at RT. Slides were then washed 3 times with PBS-T, incubated with 300 nM DAPI in PBS for 30 min, and mounted with Fluorsave™ reagent (MilliporeSigma, 345789) for imaging. Slides were imaged with an Axioscan 7 microscope (Zeiss).

[0127] [Table 7]

[0128] statistical analysis All statistical analyses were performed using Prism 9.4.1 (GraphPad Software LLC). For all graphs: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. For hindlimb grasping, a minimum of 5 animals per group was used across all studies. Because the scoring from hindlimb grasping was not normally distributed, data were analyzed using the non-parametric Kruskal-Wallis test, and Conover-Iman post-hoc tests were used when significant main effects were observed by Kruskal-Wallis. All data are expressed as medians. For enzyme activity and GAG readouts, data were tested for normality using the Shapiro-Wilk normality test. If normally distributed, it was analyzed by one-way ANOVA with Dunnett's multiple comparison test, comparing all groups to the vehicle group. If not normally distributed, it was analyzed by Kruskal-Wallis test followed by Dunn's multiple comparison test. Data are presented as mean and SEM. For urine and serum samples, data were analyzed by Dunnett's multiple comparison test after fitting a mixed effects model, comparing all groups with the vehicle group. Data are presented as mean and SEM.

[0129] Bulk RNA sequencing RNA was isolated from microdissected fixed tissue sections using the RNeasy FFPE kit (Qiagen) and analyzed on an Agilent 2100 Bioanalyzer using the Agilent 6000 RNA Nano kit (Agilent Technologies). cDNA libraries for sequencing were generated using the Collibri 3' mRNA Library Prep Kit for Illumina (ThermoFisher). Libraries were quantified using the Collibri Library Quantification Kit (ThermoFisher) and evaluated on an Agilent 2100 Bioanalyzer using the Agilent High Sensitivity DNA Kit (Agilent Technologies). Libraries were sequenced on an Illumina NextSeq 500 using the NextSeq 500 / 550 High Output Kit (75 cycles). Reads were filtered and trimmed using Cutadapt v3.4 (Martin, M. Cutadapt removes adapter sequences from high-throughput sequencing reads. EMBnet. journal 17, 10-12 (2011)) and aligned to both hg38 and mm39 with STAR v2.7.8a (Dobin, A. et al. STAR: ultrafast universal RNA-seq aligner. Bioinforma. Oxf. Engl. 29, 5-21 (2013). Aligned reads were analyzed using SAMtools v1.12. 44(Li, H. et al. The Sequence Alignment / Map format and SAMtools. Bioinforma. Oxf. Engl. 25, 2078-2079 (2009)) were used to sort and index, filter and detect only human-specific reads using XenofilteR (Kluin, RJC et al. XenofilteR: computational deconvolution of mouse and human reads in tumor xenograft sequence data. BMC Bioinformatics 19, 366 (2018)). Reads aligned to expressed genes were analyzed using HTSeq v0.13.5 (Putri, GH, Anders, S., Pyl, PT, Pimananda, JE and Zanini, F. Analysing high-throughput sequencing data in Python with HTSeq 2.0. Bioinforma. Oxf. Engl. btac166 (2022) doi:10.1093 / bioinformatics / btac166) were used to count the number of

[0130] Single-cell RNA sequencing and analysis Fetal hematopoietic (CD45+) single-cell data from Bian et al., 2020 (Bian, Z. et al. Deciphering human macrophage development at single-cell resolution. Nature 582, 571-576 (2020)) were downloaded from NCBI Gene Expression Omnibus, accession number GSE133345. For downstream analyses, primitive macrophage subpopulations (Mac_1, Mac_2, and Mac_3) were grouped together. UMAP plots were generated using Scanpy 48All cells were color coded by origin, Carnegie developmental stage, and cell type grouping using (SCANPY: Large-scale single-cell gene expression data analysis | Genome Biology | Full Text. https: / / genomebiology.biomedcentral.com / articles / 10.1186 / s13059-017-1382-0). Single-cell RNA sequencing was performed on myeloid cells (n=3 independent biological replicates) and hiPSCs using 10X Genomics. The raw data for each dataset was the "Filtered Feature Barcode Matrix" output by the alignment and deconvolution pipeline of Cell Ranger (Massively Parallel Digital Transcriptional Profiling of Single Cells | Nature Communications. https: / / www.nature.com / articles / ncomms14049). Each dataset was imported and annotated for each cell with mitochondrial content, gene counts by counts, and total counts. Cells with excess mitochondrial numbers were removed using a knee locator-based method (Satopaa, V., Albrecht, J., Irwin, D., and Raghavan, B. Finding a 'Kneedle' in a Haystack: Detecting Knee Points in System Behavior. (2010)). Data were then filtered to include only cells with a minimum of 1000 non-zero transcripts. Double detection and removal was performed so that the dataset subset contained only 5000 cells (so that each dataset had the same cell number in the analysis). Each dataset was iteratively joined with the other datasets included in the analysis. Joining was performed on an inner-join basis against Ensembl IDs (only genes common to both datasets are included in the resulting data object). The myeloid cell and hiPSC data were then joined to the Bien et al. data by Ensembl IDs.Finally, the total counts per cell were normalized to 1,000,000 (CPM normalized) and log2 transformed. PCA was performed on the concatenated dataset using genes with high variability. Differential gene expression was performed using Scanpy's rank_gene_group function, filtering based on a minimum log fold change = 4 and a threshold percentage of cells expressing the gene inside and outside the group = 0.5. Genes enriched in the Mac1-3 and Mac4 groups were used for rarefaction-based scoring. For scoring, the normalized expression matrix was binarized and the rarefaction of the matrix was calculated. This matrix was subset to contain genes from the group of interest (Mac1-3 or Mac4). Gene set scores for each cell were calculated as the sum of the binarized expression data for gene groups multiplied by the rarefaction and normalized by the maximum score to have values ​​ranging from 0 to 1. Differential gene expression was performed on the Bian et al. dataset (same settings as above) based on cell type grouping of all cells to find markers unique to each group. Expression data from this dataset was normalized between 0 and 1 and shown as a heatmap. Unique genes found in the Mac4 and Mac1-3 groups were displayed as dot plots using the single-cell RNA-seq dataset and as a heatmap using the engrafted myeloid cell bulk RNAseq expression data.

[0131] Various software Figures were created and assembled in Affinity Designer 1.10. Various figures were generated using BioRender.com.

Claims

1. A pharmaceutical for use in a method for treating a metabolic disorder involving myeloid cells, comprising: The method comprises: administering myeloid cells to the central nervous system of a subject to be treated; allowing the administered myeloid cells to engraft and produce enzymes; monitoring the level of the enzyme in a serum, urine, or cerebrospinal fluid sample from the treated subject; monitoring the amount of the substrate in the subject's serum, urine, or cerebrospinal fluid sample to determine progress of the treatment; Pharmaceuticals, including

2. The pharmaceutical composition of claim 1, wherein the myeloid cells are myeloid cells derived from pluripotent stem cells (PSCs).

3. The pharmaceutical composition of claim 1, wherein the myeloid cells are myeloid cells derived from non-pluripotent stem cells (PSCs).

4. The pharmaceutical composition of claim 3, wherein the myeloid cells derived from non-pluripotent stem cells (PSCs) are peripheral blood mononuclear cells (PBMCs).

5. The pharmaceutical according to claim 1, wherein the metabolic disorder is a hereditary metabolic disorder.

6. The pharmaceutical composition according to claim 5, wherein the inherited metabolic disorder is Hurler syndrome.

7. The pharmaceutical composition of claim 3, wherein the enzyme is alpha L-iduronidase (IDUA).

8. The pharmaceutical composition of claim 5, wherein the inherited metabolic disorder is Sly syndrome.

9. The pharmaceutical composition of claim 5, wherein the enzyme is beta-glucuronidase (GUSB).

10. The pharmaceutical composition of claim 1, wherein the central nervous system is selected from the group consisting of the intraventricular (ICV) space, the brain, the spinal cord, the cerebrospinal fluid (CSF), and the brain parenchymal space.

11. The pharmaceutical according to claim 1, wherein the subject is a mouse or a human.

12. The pharmaceutical according to claim 1, wherein the substrate is a glycosaminoglycan.

13. The pharmaceutical composition of claim 1, wherein the subject being treated has an accumulation of undegraded substrates in the cells of the subject.

14. The pharmaceutical of claim 1, wherein the enzyme is used to reduce undegraded substrates accumulated in brain cells of a subject being treated.

15. The pharmaceutical composition of claim 1, wherein a sustained reduction in total substrate levels of more than about 20% indicates successful treatment of the metabolic disorder.

16. The pharmaceutical of claim 1, wherein the amount of the enzyme in the serum sample is compared with the amount of the enzyme in the serum sample of a healthy subject.

17. The pharmaceutical of claim 1, wherein the activity of the enzyme in the serum sample is compared with the activity of the enzyme in the serum of a healthy subject.

18. The amount of cells injected into a human is approximately 25 x 10 6 ~Approx. 1250×10 6 The pharmaceutical composition of claim 1, wherein the range of the number of cells is within 100.

19. The amount of cells injected into a human is approximately 50 x 10 6 ~About 1000×10 6 The pharmaceutical composition of claim 1, wherein the range of the number of cells is within 100.

20. The amount of cells injected into a human is approximately 100 x 10 6 ~Approx. 500×10 6 The pharmaceutical composition of claim 1, wherein the range of the number of cells is within 100.

21. The amount of cells injected into a human is approximately 100 x 10 6 ~Approx. 300×10 6 The pharmaceutical composition of claim 1, wherein the amount of the active ingredient is within the range of 100 mg / kg.

22. The amount of cells injected into a human is approximately 150 x 10 6 ~Approx. 250×10 6 The pharmaceutical composition of claim 1, wherein the range of the number of cells is within 100.

23. The pharmaceutical composition of claim 13, wherein the cell is a brain cell.

24. 24. The pharmaceutical composition of claim 23, wherein the brain cells are selected from the group consisting of neurons, oligodendrocytes, astrocytes, microglia, perivascular macrophages, meningeal macrophages, endothelial cells, pericytes, ependymal cells, and blood cells.

25. The pharmaceutical according to claim 5, wherein the inherited metabolic disorder is a lysosomal storage disease.

26. 26. The pharmaceutical composition of claim 25, wherein the lysosomal storage disease is selected from the group consisting of MPS I (Hurler syndrome), MPS IIIA (Sanfilippo A), MPS IIIB (Sanfilippo B), MPS VII (Sly syndrome), Krabbe disease, Pompe (glycogen storage disease type II), GM1-gangliosidosis, GM2-gangliosidosis (Sandhoff / Tay-Sachs), and metachromatic leukodystrophy.

27. 1. A method for generating myeloid cells, comprising: (i) obtaining human iPSCs (pluripotent stem cells); (ii) culturing the pluripotent stem cells (PSCs) in a medium for about 1 to about 4 days; (iii) inducing the cells from step (ii) with BMP-4; (iv) culturing the cells from step (iii) in a medium comprising StemPro-34 SFM, SCF, VEGF, and bFGF for about 2 to about 6 days; (v) culturing the cells from step (iv) in a medium comprising StemPro-34 SFM, SCF, IL-3, TPO, M-CSF, and Flt3 for about 2 to about 6 days; and (vi) culturing the cells from step (v) in M-CSF, Flt3, and GM-CSF, thereby generating myeloid cells, and recovering said cells from the supernatant fraction; A method comprising:

28. 28. The method of claim 27, wherein the PSCs are obtained from frozen stocks prior to step (i).

29. 28. The method of claim 27, further comprising expanding the myeloid cells in a bioreactor.

30. 28. The method of claim 27, further comprising freezing the myeloid cells in cryopreservation medium, wherein the concentration of the myeloid cells in the medium is at least about 1 million cells / ml.

31. Myeloid cells are at least 90% CD45 + 28. The method of claim 27, wherein:

32. Myeloid cells are at least 95% CD45 + 28. The method of claim 27, wherein:

33. Myeloid cells are at least 70% CD45 + / CD14 + / CX3CR1 + 28. The method of claim 27, wherein:

34. Myeloid cells are at least 75% CD45 + / CD14 + / CX3CR1 + 28. The method of claim 27, wherein:

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