Compositions and methods for treating diseases and disorders of the central nervous system

Abstract

The present invention provides compositions and methods for treating or preventing nervous system diseases or disorders of the central nervous system (e.g., storage disorders, lysosomal storage disorders, neurodegenerative diseases, etc.) by reconstituting cerebromyelocytes and microglia upon transplantation of hematopoietic cells enriched in microglia reconstitution potential. The present invention also provides compositions and methods for ablating and reconstituting microglia. The present invention provides, for example, a method of delivering hematopoietic stem cells (HSCs) to a subject, comprising administering the HSCs by intracerebroventricular injection (ICV) in combination with ablation pretreatment.

Description

[Technical Field]

[0001] STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH The work leading to this invention has received funding from the Seventh European Framework Programme for Research and Development (FP7 / 2007-2013) under grant agreement no. [[617162]] and from the Italian Ministry of Health under agreement no. GR-2011-02347261. [Background technology]

[0002] Background of the Invention The majority of storage disorders (SD) associated with central nervous system (CNS) disorders (neuroSD) lack effective and curative treatments, and patients ultimately die from their devastating disease. In many cases, disease onset occurs very early in childhood and is characterized by subtle symptoms, leading to diagnosis when the condition becomes clearly symptomatic, if not progressive. NeuroSD is also characterized by rapid early disease progression, particularly in early-onset variants. For these reasons, therapeutic approaches applied with some success in presymptomatic neuroSD children, including hematopoietic cell transplantation (HCT) in Krabbe disease and adrenoleukodystrophy, or hematopoietic stem cell (HSC) gene therapy (HSC GT) in metachromatic leukodystrophy (MLD), have not benefited the majority of neuroSD patients, with benefits largely associated only with procedures applied in presymptomatic or early-onset patients. One important reason why these HSC-based approaches have not been successful in ameliorating rapidly progressive SD brain disease is the slow rate at which resident CNS tissue macrophages / histiocytes and microglia are replaced by transplanted hematopoietic cell progeny, compared to the rapid progression of primary nervous system disease. Indeed, rapid reconstitution of visceral macrophages by donor-derived cells has been clearly demonstrated after HCT, but a more limited and slower infiltration of the brain parenchyma by donor cells is expected to occur. Thus, strategies that attempt to enhance and more rapid this phenomenon are greatly needed. Such strategies also have the potential to be therapeutically relevant for some acquired neurodegenerative conditions in adulthood, which would suggest that transplanted hematopoietic cells may be a valuable tool for the treatment of these conditions. Hematopoietic stem and progenitor cells Modulation of the progeny of HSPCs and / or the activated microglial phenotype that characterizes most of these conditions can provide the benefit of delivering therapeutic molecules across the blood-brain barrier. These disorders, including amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), and Parkinson's disease (PD), share several common disease / pathogenic mechanisms with neurological disorders, such as neuroinflammation and the active role of microglia. Therefore, new compositions and methods of treatment are urgently needed. Summary of the Invention [Means for solving the problem]

[0003] Summary of the Invention As described below, the present invention features compositions and methods for treating or preventing nervous system diseases or disorders of the central nervous system (e.g., neurodegenerative storage disorders, acquired neurodegenerative diseases, etc.) by establishing CNS bone marrow cell / microglia chimerism using either donor-derived or engineered cells, which can contribute to disease amelioration through various mechanisms, such as protein delivery or modulation of local inflammation. The present invention provides compositions and methods for one or more of the following: (i) effectively engrafting microglial progenitor cells in CNS cells with or that acquire microglial characteristics, including cells enriched for microglial reconstitution potential, for therapeutic purposes in the conditions listed above; (ii) selectively and exclusively engrafting microglial progenitor cells in CNS genetically modified cells with or that acquire microglial characteristics, including cells enriched for microglial reconstitution potential, for therapeutic purposes in the conditions listed above; and (iii) ablating resident myeloid populations, such as cells with proliferative ability, in the brain by CNS-selective methods, which may include nanoparticles that target microglia and / or microglial precursors. This method can be used to achieve selective CNS engraftment and acquisition of myeloid / microglial characteristics of transplanted cells in the brain, to deliver therapeutic molecules and / or modulate myeloid / microglial characteristics, and in the event of successful, timely, and partially renewing the pool of cells in the presence of CNS damage.

[0004] In one aspect, the present invention provides a method for delivering hematopoietic stem cells (HSCs), comprising administering HSCs to a subject by intracerebroventricular infusion (ICV) in combination with ablative conditioning. In another aspect, the present invention provides isolated HSCs transformed with a vector expressing a therapeutic polypeptide or polynucleotide, wherein the HSCs express CD34 + , CD38 - and Fgd5 + (e.g., CD34 + , CD38 - ;CD34 + , CD38 - and Fgd5 + ) is one or more of:

[0005] In another aspect, the present invention provides isolated hematopoietic stem cells (HSCs) transformed with a vector expressing a therapeutic polypeptide or polynucleotide, wherein the HSCs express CD34 + , CD38 - and Fgd5 + (e.g., CD34 + , CD38 - ;CD34 + , CD38 - and Fgd5 + ) is selected with respect to one or more of:

[0006] In another aspect, the present invention provides isolated hematopoietic stem cells (HSCs) transformed with a vector expressing a therapeutic polypeptide or polynucleotide, wherein the HSCs are + , Lin - , Sca1 + , CD150 + , CD48 - , Fdg5 + , CX3CR1 - and CD11b - (e.g., kit + , Lin - , Sca1 + , CD150 + ;kit + , Lin - , Sca1+ , CD150 + , CD48 - ;kit + , Lin - , Sca1 + , CD150 + , CD48 - , Fdg5 + ;kit + , Lin - , Sca1 + , CD150 + , CD48 - , CX3CR1 - ;kit + , Lin - , Sca1 + , CD150 + , CD48 - , Fdg5 + , CX3CR1 - ; and kit + , Lin - , Sca1 + , CD150 + , CD48 - , Fdg5 + , CX3CR1 - and CD11b - ) is one or more of:

[0007] In another aspect, the present invention provides a method of treating a subject having or at increased risk of developing a lysosomal storage disorder or a neurodegenerative disease, comprising administering to a subject a CD34 + , CD38 - and Fgd5 + (e.g., CD34 + , CD38 - ;CD34 + , CD38 - and Fgd5 + ), wherein the HSCs are administered intravenously (IV) or by intracerebroventricular infusion (ICV) in combination with ablation pretreatment.

[0008] In another aspect, the present invention provides a method of treating a subject having or at increased risk of developing a lysosomal storage disorder or a neurodegenerative disease, comprising administering to a subject a kit + , Lin - , Sca1 + , CD150 + , CD48 - , Fdg5 + , CX3CR1 - and CD11b - (e.g., kit + , Lin - , Sca1 + , CD150 + ;kit + , Lin - , Sca1 + , CD150 + , CD48 - ;kit + , Lin - , Sca1 + , CD150 + , CD48 - , Fdg5 + ;kit + , Lin - , Sca1 + , CD150 + , CD48 - , CX3CR1 - ;kit + , Lin - , Sca1 + , CD150 + , CD48 - , Fdg5 + , CX3CR1 - ; and kit + , Lin - , Sca1 + , CD150 + , CD48 - , Fdg5 + , CX3CR1 - and CD11b - ), wherein the HSCs are administered intravenously (IV) or by intracerebroventricular infusion (ICV) in combination with ablation pretreatment.

[0009] In another aspect, the present invention provides a method for ablating endogenous microglia in a subject and reconstituting microglia by HSC engraftment, comprising administering to the subject nanoparticles containing a cytotoxic agent, which nanoparticles can be covalently attached to the surface of one or more capture molecules that specifically bind to one or more markers expressed on microglial cells or their precursors, and combined with HSCs administered to the subject by IV or ICV.

[0010] In another aspect, the present invention provides a method for treating a lysosomal storage disorder in a subject, the method comprising administering to the subject nanoparticles, wherein the nanoparticles contain a cytotoxic agent and one or more trapping molecules covalently linked to the surface of the nanoparticles, and the trapping molecules specifically bind to one or more markers expressed on microglial cells or their precursors, and administering to the subject hematopoietic stem cells (HSCs) by intravenous (IV) or intracerebroventricular infusion (ICV), wherein the HSCs express a therapeutic polypeptide.

[0011] In another aspect, the present invention provides a method of treating a neurodegenerative disease in a subject, the method comprising administering to the subject nanoparticles, wherein the nanoparticles contain a cytotoxic agent and one or more trapping molecules covalently linked to the surface of the nanoparticles, where the trapping molecules specifically bind to one or more markers expressed on microglial cells or their precursors, and administering to the subject hematopoietic stem cells (HSCs) by intravenous (IV) or intracerebroventricular infusion (ICV), wherein the HSCs express a therapeutic polypeptide or polynucleotide.

[0012] In another aspect, the present invention provides a method for generating microglial chimerism in the brain of a subject, independent of extra-CNS hematopoietic tissue chimerism, comprising the step of ICV transplantation of HSPCs and IV transplantation of whole bone marrow cells 0-5 days after busulfan bone marrow ablation.

[0013] In another aspect, the present invention provides a method for generating sustained hematopoietic mixed chimerism in tissues outside the brain and CNS for a short period of time in a subject with ICV and IV transplanted exogenous cells following busulfan bone marrow ablation.

[0014] In another aspect, the present invention provides a method for achieving regulated expression of an exogenous gene in engineered microglia, comprising transducing a hematopoietic equivalent of a microglial precursor with a viral vector encoding a gene of interest under the control of the TSPO promoter.

[0015] In another aspect, the present invention provides a method for functional identification of brain-resident microglial progenitor cells by detecting γH2AX signal, wherein detection of γH2AX signal indicates the presence of brain-resident microglial progenitor cells.

[0016] In another aspect, the invention provides a kit comprising the claimed isolated hematopoietic stem cells (HSCs), which are nanoparticles according to any aspect described herein.

[0017] In another aspect, the present invention provides nanoparticles capable of targeting microglial cells or their precursors.

[0018] In another aspect, the present invention provides a method of delivering nanoparticles to a subject, the method comprising administering the nanoparticles to the subject by intracerebroventricular injection (ICV).

[0019] In another aspect, the present invention provides a method for ablation of microglial cells or their precursors in a subject, comprising administering to the subject nanoparticles, the nanoparticles comprising a cytotoxic agent and one or more capture molecules covalently linked to the surface of the nanoparticles, the capture molecules specifically binding to one or more markers expressed on the microglial cells or their precursors.

[0020] In various embodiments of any aspect described herein, the hematopoietic stem cells (HSCs) (e.g., human) are CD34 + , CD38 - and Fgd5 + (e.g., CD34 + , CD38 - ;CD34 + , CD38 - and Fgd5 + In various embodiments of any aspect described herein, the hematopoietic stem cells (HSCs) (e.g., mouse) are one or more of the following: + , Lin - , Sca1 + , CD150 + , CD48 - , Fgd5 + , CX3CR1 - and CD11b - (e.g., kit + , Lin - , Sca1 + , CD150 + ;kit + , Lin - , Sca1 + , CD150 + , CD48 - ;kit + , Lin - , Sca1 + , CD150 + , CD48 - , Fdg5 + ;kit + , Lin - , Sca1 + , CD150 + , CD48 - , CX3CR1 - ;kit + , Lin - , Sca1 + , CD150 + , CD48 - , Fdg5 + , CX3CR1 - ; and kit + , Lin - , Sca1 + , CD150 +, CD48 - , Fdg5 + , CX3CR1 - and CD11b - In certain embodiments, the human hematopoietic stem cells (HSCs) are one or more of Fgd5 + In various embodiments of any aspect described herein, the hematopoietic stem cells (HSCs) are functionally equivalent to microglial progenitor cells upon transplantation. In various embodiments of any aspect described herein, the HSCs are capable of differentiating into microglial cells. In various embodiments of any aspect described herein, the HSCs are capable of reconstituting ablated microglial cells.

[0021] In various embodiments of any aspect described herein, the subject has or is at increased risk of developing a lysosomal storage disorder, hi various embodiments, the lysosomal storage disorder is adrenoleukodystrophy, activator deficiency / GM2 gangliosidosis, alpha-mannosidosis, aspartylglucosaminuria, cholesteryl ester storage disease, chronic hexosaminidase A deficiency, cystinosis, Danon disease, Fabry disease, Farber disease, fucosidosis, galactosialidosis, Gaucher disease, globoid cell leukodystrophy, GM1 gangliosidosis, I-cell disease / mucolipidosis II, infantile leukodystrophy, or the like. Selected from isosialic acid storage disease / ISSD, juvenile hexosaminidase A deficiency, infantile neuronal ceroid lipofuscinosis, Krabbe disease, lysosomal acid lipase deficiency, metachromatic leukodystrophy, mucopolysaccharidosis, multiple sulfatase deficiency, Niemann-Pick disease, neuronal ceroid lipofuscinosis, Pompe disease / glycogen disease type II, pycnodysostosis, Sandhoff disease, Schindler disease, Salla disease / sialic acid storage disease, Tay-Sachs / GM2 gangliosidosis, and Wolman disease. In various embodiments of any aspect described herein, the lysosomal enzyme is α-glucosidase; glucocerebrosidase; β-galactosidase; β-hexosaminidase A; β-hexosaminidase B; acid sphingomyelinase; galactocerebrosidase; β-galactocerebrosidase; acid ceramidase; arylsulfatase A; α-L-iduronidase; iduronate-2-sulfatase; heparan N-sulfatase; α-N-acetylglucosaminidase; acetonitrile; and one or more of aryl-CoA:α-glucosaminide N-acetyltransferase; N-acetylglucosamine-6-sulfate sulfatase; N-acetylgalactosamine-6-sulfate sulfatase; acid β-galactosidase; arylsulfatase B; β-glucuronidase; acid α-mannosidase; acid β-mannosidase; acid α-L-fucosidase; sialidase; α-N-acetylgalactosaminidase; and palmitoylprotein-thioesterase-1.

[0022] In various embodiments of any aspect described herein, the subject has or is at increased risk of developing a neurodegenerative disease, hi various embodiments, the neurodegenerative disease is selected from amyotrophic lateral sclerosis (ALS), Alzheimer's disease, and Parkinson's disease.

[0023] In various embodiments of any aspect described herein, the therapeutic polypeptide or polynucleotide is an inhibitory nucleic acid or shRNA targeting one or more of lysosomal enzymes, ABCD proteins, miR155, and NOX2 (e.g., in ALS); TREM2; APOE2; and APP alpha (e.g., in Alzheimer's disease).

[0024] In various embodiments of any aspect described herein, the HSCs are administered in combination with ablative pretreatment. In various embodiments, the ablative pretreatment comprises administering a cytotoxic agent to the subject. In various embodiments, the alkylating agent is one or more of busulfan, etoposide, and lomustine. In various embodiments, the ablative pretreatment is performed before administering the HSCs.

[0025] In various embodiments of any aspect described herein, expression of the polypeptide or polynucleotide is via a TSPO promoter. In various embodiments of any aspect described herein, the polypeptide or polynucleotide is expressed from a polynucleotide inserted into the TSPO locus.

[0026] In various embodiments of any aspect described herein, the nanoparticles further contain a cytotoxic agent. In various embodiments, the cytotoxic agent is provided in a fixed dose for delivery of the cytotoxic agent to microglial cells or their precursors. In certain embodiments, the alkylating agent is one or more of an alkylating agent, busulfan, etoposide, and lomustine. In various embodiments of any aspect described herein, the nanoparticles have one or more of optimized drug loading efficiency, optimized drug release, and optimized stability. In various embodiments of any aspect described herein, the nanoparticles comprise one or more trapping molecules covalently linked to the surface of the nanoparticles, wherein the trapping molecules specifically bind to one or more markers expressed on microglial cells or their precursors. In various embodiments of any aspect described herein, the nanoparticles are administered to a subject intravenously (IV) or by intracerebroventricular infusion (ICV).

[0027] In various embodiments of any aspect delineated herein, the exogenous cells are HSCs transplanted ICV and IV on day 0. In various embodiments of any aspect delineated herein, chimerism occurs in minor HLA-mismatched transplant situations.

[0028] In various embodiments of any aspect described herein, the viral vector is a lentiviral vector. In various embodiments of any aspect described herein, the method includes targeted addition of a gene of interest at the TSPO locus in a hematopoietic equivalent of a microglial progenitor. In various embodiments of any aspect described herein, the method includes administering an engineered autologous population of microglial progenitors to a subject, and the subject undergoes intravenous brain ablation to selectively reconstitute microglia. In various embodiments of any aspect described herein, the method further includes administering unmanipulated autologous bone marrow cells. In various embodiments of any aspect described herein, the method further includes detecting Fdg5 expression to identify brain-resident microglial progenitor cells.

[0029] Other features and advantages of the invention will be apparent from the detailed description and claims.

[0030] definition Unless otherwise specified, all technical and scientific terms used herein have the meanings that are commonly understood by those skilled in the art in the technical field to which this invention belongs.The following references provide those skilled in the art with many of the general definitions of the terms used in this invention: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd edition, 1994); The Cambridge Dictionary of Science and Technology (Walker, ed., 1988); The Glossary of Genetics, 5th edition, R. Rieger et al. (ed.), Springer Verlag (1991); and Hale and Marham, The HarperCollins Dictionary of Biology (1991).As used herein, the following terms have the meanings that are ascribed to them below, unless otherwise specified.

[0031] By "agent" is meant any small chemical compound, antibody, nucleic acid molecule, or polypeptide, or fragment thereof.

[0032] By "ameliorate" is meant to slow, inhibit, attenuate, alleviate, arrest or stabilize the onset or progression of a disease.

[0033] The term "antibody," as used herein, refers to an immunoglobulin molecule that specifically binds to an antigen. The term "antibody fragment" refers to a portion of an intact antibody and refers to the antigen-determining variable region of the intact antibody.

[0034] "Alteration" or "change" means improvement or decrease. The alteration may be as low as 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, or may be as high as 40%, 50%, 60%, or even as high as 70%, 75%, 80%, 90%, or 100%.

[0035] By "biological sample" is meant any tissue, cell, fluid, or other substance derived from an organism.

[0036] By "capture reagent" is meant a reagent that specifically binds to a nucleic acid molecule or polypeptide in order to select or isolate the nucleic acid molecule or polypeptide.

[0037] As used herein, the terms "determining," "evaluating," "assaying," "measuring," and "detecting" refer to both quantitative and qualitative determinations, and thus the term "determining" is used interchangeably herein with "assaying," "measuring," etc. Where a quantitative determination is intended, the phrase "determining the amount" of an analyte, etc. is used. Where a qualitative and / or quantitative determination is intended, the phrase "determining the level" of an analyte or "detecting" an analyte is used.

[0038] "Detecting" refers to determining the presence, absence, or amount of the analyte being detected.

[0039] "Detectable label" refers to a composition that, when attached to a molecule of interest, renders the molecule of interest detectable by spectroscopic, photochemical, biochemical, immunochemical, or chemical means. For example, useful labels include radioactive isotopes, magnetic beads, metallic beads, colloidal particles, fluorescent dyes, electron-dense reagents, enzymes (e.g., those commonly used in ELISA), biotin, digoxigenin, or haptens.

[0040] By "disease" is meant any condition or disorder that impairs or interacts with the normal function of a cell, tissue, or organ.

[0041] "Effective amount" means the amount required to improve the symptoms of the disease compared to untreated patients. For the therapeutic treatment of disease, the effective amount of the active compound used to implement the present invention will vary depending on the method of administration, the age, weight and general health of the subject. Ultimately, the attending physician or veterinarian will determine the appropriate amount and dosage regimen. Such an amount is called an "effective" amount.

[0042] By "Fgd5 polypeptide" is meant a protein or fragment thereof having about 85% or higher amino acid sequence identity to NCBI accession numbers NP_689749, NP_001307205, NP_766319, and a protein or fragment thereof having chromatin binding activity or transcriptional regulatory activity. The sequence of an exemplary human Fgd5 protein is provided below: [ka] [ka]

[0043] The sequence of an exemplary mouse Fgd5 protein is provided below: [ka]

[0044] By "fragment" is meant a portion of a protein or nucleic acid that is substantially identical to a reference protein or nucleic acid, in some embodiments, the portion retains at least 50%, 75%, or 80%, or more preferably 90%, 95%, or even 99%, of the biological activity of the reference protein or nucleic acid described herein.

[0045] The terms "isolated," "purified," or "biologically pure" refer to a material that is free, to varying degrees, from components that normally accompany it as found in its natural state. "Isolated" refers to some degree of separation from the original source or surroundings. "Purified" refers to a degree of separation greater than isolation. A "purified" or "biologically pure" protein is sufficiently free of other substances so that any impurities do not substantially affect the biological properties of the protein or cause other adverse effects. That is, a nucleic acid or peptide of the invention is purified when it is substantially free of cellular material, viral material, or culture medium, if produced by recombinant DNA techniques, or from chemical precursors or other chemicals, if chemically synthesized. Purity and homogeneity are typically determined using analytical chemistry techniques, such as polyacrylamide gel electrophoresis or high-performance liquid chromatography. The term "purified" can mean that the nucleic acid or protein gives rise to essentially one band in an electrophoretic gel. In the case of proteins that can be modified, eg, phosphorylated or glycosylated, different modifications can result in different isolated proteins that can be individually purified.

[0046] By "isolated polypeptide" is meant a polypeptide of the invention that has been separated from components that naturally accompany it. Ordinarily, a polypeptide is isolated when it is at least 60% by weight free from the proteins and naturally occurring organic molecules with which it is naturally associated. Preferably, a preparation is at least 75% by weight, more preferably at least 90%, and most preferably at least 99% by weight, a polypeptide of the invention. Isolated polypeptides of the invention can be obtained, for example, by extraction from a natural source, by expression of a recombinant nucleic acid encoding such a polypeptide, or by chemically synthesizing the protein. Purity can be measured by any appropriate method, e.g., column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis.

[0047] As used herein, "storage disorder (SD)" refers to any of a group of diseases resulting from metabolic abnormalities that lead to the accumulation of substrates (e.g., sulfatides, heparan sulfate, glycolipids, ceramides) in lysosomes or other cellular organelles. For example, lysosomal storage disorders (LSDs) are usually caused by lysosomal dysfunction as a result of a deficiency in a single enzyme required for the metabolism of lipids, glycoproteins (sugar-containing proteins), or so-called mucopolysaccharides.

[0048] By "marker" is meant any clinical indicator, protein, metabolite, or polynucleotide having an alteration associated with a disease, disorder, or condition.

[0049] "Microglia" refers to immune cells of the central nervous system.

[0050] "Nanoparticle" refers to a complex structure with nanoscale dimensions. In particular, nanoparticles are particles typically ranging in size from about 1 to about 1,000 nm, typically spherical, although various morphologies are possible depending on the nanoparticle composition. The portion of a nanoparticle that comes into contact with the external environment is generally identified as the nanoparticle's surface. For the nanoparticles described herein, size limitations can be limited to two dimensions. Thus, the nanoparticles described herein include complex structures with diameters of about 1 to about 1,000 nm, with the specific diameter depending on the nanoparticle composition, experimental design, and intended use of the nanoparticle. For example, nanoparticles to be used in some therapeutic applications typically have a size of about 200 nm or less, and nanoparticles used, in particular, for the delivery of therapeutic agents typically have a diameter of about 1 to about 100 nm.

[0051] As used herein, "neurodegenerative disease" refers to any of a group of diseases characterized by the progressive loss of neuronal structure and / or function, including neuronal death. Exemplary neurodegenerative diseases include, but are not limited to, amyotrophic lateral sclerosis and Alzheimer's disease.

[0052] "Enhancing proliferation" means enhancing cell division of cells in vivo or in vitro.

[0053] As used herein, the terms "prevent," "preventing," "prevention," "prophylactic treatment," and the like refer to reducing the likelihood of developing a disorder or condition in a subject who does not have the disorder or condition but is at risk of or susceptible to developing it.

[0054] The term "subject" or "patient" refers to an animal that is the object of treatment, observation, or experiment. By way of example only, a subject includes a mammal, including, but not limited to, a human or a non-human mammal such as a non-human primate, mouse, cow, horse, dog, sheep, or cat.

[0055] By "reduction" is meant a negative alteration of at least 10%, 25%, 50%, 75% or 100%.

[0056] "Reference" means a standard for comparison or control.

[0057] By "substantially identical" is meant a polypeptide or nucleic acid molecule that exhibits at least 50% identity to a reference amino acid sequence (e.g., any one of the amino acid sequences described herein) or nucleic acid sequence (e.g., any one of the nucleic acid sequences described herein). Preferably, such a sequence is at least 60%, more preferably 80% or 85%, and more preferably 90%, 95%, 96%, 97%, 98%, or even 99% or more identical at the amino acid or nucleic acid level to the sequence used for comparison.

[0058] Sequence identity is typically measured using sequence analysis software (e.g., the Sequence Analysis Software Package of the Genetics Computer Group (University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705; BLAST, BESTFIT, GAP), or the PILEUP / PRETTYBOX program). Such software matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions, and / or other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. In an exemplary approach to determining the degree of identity, the BLAST program is used to identify closely related sequences, e.g., -3 ~e -100 can be used with score probabilities between

[0059] Nucleic acid molecules useful in the methods of the present invention include any nucleic acid molecule encoding a polypeptide of the present invention or a fragment thereof. Such nucleic acid molecules need not be 100% identical to an endogenous nucleic acid sequence, but will usually exhibit substantial identity. A polynucleotide having "substantial identity" to an endogenous sequence is usually capable of hybridizing to at least one strand of a double-stranded nucleic acid molecule. "Hybridizing" refers to the pairing of complementary polynucleotide sequences (e.g., genes described herein) or portions thereof to form a double-stranded molecule under various stringent conditions (see, e.g., Wahl, GM and SL Berger (1987) Methods Enzymol. 152:399; Kimmel, AR (1987) Methods Enzymol. 152:507).

[0060] For example, stringent salt concentrations will typically be less than about 750 mM NaCl and less than about 75 mM trisodium citrate, preferably less than about 500 mM NaCl and less than about 50 mM trisodium citrate, and more preferably less than about 250 mM NaCl and less than about 25 mM trisodium citrate. Low stringency hybridization can be achieved in the absence of organic solvents, such as formamide, while high stringency hybridization can be achieved in the presence of at least about 35% formamide, more preferably at least about 50% formamide. Stringent temperature conditions will typically include a temperature of at least about 30°C, more preferably at least about 37°C, and most preferably at least about 42°C. Various additional parameters, such as hybridization time, detergent concentration, e.g., sodium dodecyl sulfate (SDS), and the inclusion or exclusion of carrier DNA, are well known to those skilled in the art. Various levels of stringency can be achieved by combining these various conditions as needed. In a preferred embodiment, hybridization is carried out in 750 mM NaCl, 75 mM trisodium citrate, and 1% SDS at 30° C. In a more preferred embodiment, hybridization is carried out in 500 mM NaCl, 50 mM trisodium citrate, 1% SDS, 35% formamide, and 100 μg / ml denatured salmon sperm DNA (ssDNA) at 37° C. In a most preferred embodiment, hybridization is carried out in 250 mM NaCl, 25 mM trisodium citrate, 1% SDS, 50% formamide, and 200 μg / ml ssDNA at 42° C. Useful variations on these conditions will be readily apparent to those of skill in the art.

[0061] For most applications, the washing steps following hybridization will also vary in stringency. Stringent conditions for washing can be defined by salt concentration and by temperature. As noted above, washing stringency can be increased by decreasing salt concentration or increasing temperature. For example, stringent salt concentrations for washing steps will preferably be less than about 30 mM NaCl and less than about 3 mM trisodium citrate, and most preferably less than about 15 mM NaCl and less than about 1.5 mM trisodium citrate. Stringent temperature conditions for washing steps will usually include temperatures of at least about 25°C, more preferably at least about 42°C, and even more preferably at least about 68°C. In a preferred embodiment, washing steps are performed at 25°C in 30 mM NaCl, 3 mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, washing steps are performed at 42°C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, the washing step is carried out at 68°C in 15mM NaCl, 1.5mM trisodium citrate, and 0.1% SDS. Further variations on these conditions will be readily apparent to those skilled in the art. Hybridization techniques are well known to those skilled in the art and are described, for example, in Benton and Davis (Science 196:180, 1977); Grunstein and Hogness (Proc. Natl. Acad. Sci., USA 72:3961, 1975); Ausubel et al. (Current Protocols in Molecular Biology, Wiley Interscience, New York, 2001); Berger and Kimmel (Guide to Molecular Cloning Techniques, 1987, Academic Press, New York); and Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York.

[0062] By "specifically binds" is meant a compound (e.g., a peptide) that recognizes and binds to a molecule (e.g., a polypeptide) but does not substantially recognize and bind to other molecules in a sample, e.g., a biological sample.

[0063] As used herein, the terms "treat," "treating," "treatment," and the like refer to reducing or ameliorating the disorder and / or symptoms associated therewith. Without limitation, it will be understood that treating a disorder or condition does not require the complete elimination of the disorder, condition, or symptoms associated therewith.

[0064] Unless otherwise specified or clear from the context, the term "about" as used herein is understood to be within the normal range of acceptability in the art, for example, within 2 standard deviations of the mean value.About can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05% or 0.01% of the specified value.Unless otherwise clear from the context, all numerical values ​​presented herein are modified by the term about.

[0065] Ranges provided herein are understood to be shorthand for all of the values ​​within that range. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or subrange from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.

[0066] Any compound, composition or method presented herein can be combined with one or more of any of the other compositions and methods presented herein.

[0067] As used herein, the singular forms "a," "an," and "the" include the plural unless the context clearly indicates otherwise. Thus, for example, reference to a "biomarker" includes reference to more than one biomarker.

[0068] Unless specifically stated otherwise or clear from context, as used herein, the term "or" is understood to be inclusive.

[0069] The term "including" is used herein to mean, and is used interchangeably with, the phrase "including but not limited to."

[0070] As used herein, the terms "comprises," "comprising," "containing," "having," and the like, can have the meaning ascribed to them in U.S. patent law, and "includes," "including," "consisting essentially of," or "consists essentially of" likewise have the meaning ascribed to them in U.S. patent law, and the terms are open-ended, allowing for the existence of things other than those recited, but excluding prior art embodiments, so long as the basic or novel characteristics of the recited things are not altered by the existence of things other than those recited. [Brief explanation of the drawings]

[0071] [Figure 1A]Figures 1A–1H illustrate bone marrow cell reconstitution in the brain after intracerebroventricular injection of mouse and human HSPCs. Figure 1A illustrates the experimental scheme for ICV transplantation of lineage- (Lin−) cells (which become HSPCs in mice) in myeloablated mice (BU: myeloablation with busulfan treatment; IRR: lethal irradiation). Various time points of analysis are indicated. Lin− cells were transduced with LV encoding green fluorescent protein (GFP). Figure 1B is a graph illustrating the frequency of GFP+ cells identified within the total bone marrow (CD45+CD11b+) brain compartment at various time points after ICV and IV HSPC transplantation in BU-treated (BU-TX) and irradiated (IRR) mice. N≥5 mice per time point and group; mean and SD are shown. Two-way ANOVA showed a significant effect of route and time of cell administration in BU and IRR mice (ICV vs. IV and time p<0.005). These data demonstrate rapid and robust bone marrow cell engraftment in the brain after intraventricular injection of HSPCs. Graphs are shown in triplicate, four-bar sets. From left to right: IRR IV (gray), IRR ICV (white), BU IV (dark gray), and BU ICV (even darker gray). Figure 1C illustrates a sagittal section reconstruction of the brain of a representative ICV-transplanted BU-TX mouse, demonstrating widespread distribution of GFP+ cells 90 days after ICV injection of GFP-transduced HSPCs. Nuclei are labeled with GFP (green / gray) and Topro III (TPIII, light blue / light gray). Images were acquired with a Delta Vision Olympus at 20x magnification and processed with SoftWork 3.5.0. Reconstructions were performed using Adobe Photoshop CS 8.0 software. Figure 1D illustrates immunofluorescence analysis for GFP (green / gray) and Iba-1 (red / light gray) in brain slices from BU_TX mice at 90 days after ICV transplantation of GFP-transduced HSPCs. M = fused. 20x and 40x magnification of the relevant dotted boxes are shown.Images were acquired using a confocal microscope, Radiance 2100 (Bio-Rad) 1x70, and processed using SoftWork 3.5.0. Figure 1E shows the experimental scheme for transplantation of human CD34+ cells (considered to represent HSPCs in humans and equivalent to mouse-derived Lin-cells) transduced with LVs encoding GFP or arylsulfatase A (ARSA) in NSG mice or Rag- / -γ-chain- / - As2- / - (RagMLD) mice pretreated with BU16 mg / kg for 4 days (NSG) or sublethally irradiated (RagMLD). NSG mice also received unmanipulated mononuclear cells from NSG donors. The graph contains four sets of bars. From the leftmost bar in each set, the bars represent IV only (white), LIN-IV and LIN-ICV (dark gray), KLS-IV and LIN-ICV (medium gray), and BM-IV and LIN-ICV (gray). Figure 1F shows representative dot plots from the analysis of brain mononuclear cells derived from NSG mice transplanted with GFP-LV-transduced human CD34+ cells as early as 20 weeks. The abundance of human cells in the mouse brain is shown in two representative animals and by two analytical methods (human CD45 for SSC and human CD45 for mouse CD45). These plots also show that human CD45+ cells identified in the NSG brain after transplantation express CD11b, CX3Cr1, and GFP. Figure 1G shows the abundance of human CD45+CD11b+ cells recovered from the brains of NSG and RagMLD mice transplanted with IV or ICV CD34+ cells derived from umbilical cord blood 12–20 weeks after transplantation (Burton-Bauer et al., 2014). These plots also show that human CD45+ cells identified in the NSG brain after transplantation express CD11b, CX3Cr1, and GFP. Figure 1G shows the abundance of human CD45+CD11b+ cells recovered from the brains of NSG and RagMLD mice transplanted with BU-treated or sublethally irradiated Rag- / -γ-chain- / -As2- / - (NSG) and 5 weeks after transplantation (Rag- / -γ-chain- / -As2- / -). Values ​​are expressed as fold over IV, where IV is equal to 3 + / - 1.3 in NSG mice and 2.9 + / - 0.7 in RagMLD. N > 5 mice / group; mean and SD are shown. For NSG mice, p < 0.001 by Student's t-test; for RagMLD mice, p < 0.05 by one-way ANOVA with Bonferroni post-hoc test.Figure 1H illustrates the results from immunofluorescence analysis for GFP, Iba-1 (costaining), CD11b (costaining), CD68 (no costaining), and CD163 (no costaining) on ​​brain sections from NSG mice 90 days after ICV transfer of GFP-transduced CD34+ cells. In blue, nuclei were stained with TP III. 20x and 40x magnifications of the relevant dotted boxes are shown. M = fused. [Figure 1BCD]Figures 1A–1H illustrate bone marrow cell reconstitution in the brain after intracerebroventricular injection of mouse and human HSPCs. Figure 1A illustrates the experimental scheme for ICV transplantation of lineage- (Lin−) cells (which become HSPCs in mice) in myeloablated mice (BU: myeloablation with busulfan treatment; IRR: lethal irradiation). Various time points of analysis are indicated. Lin− cells were transduced with LV encoding green fluorescent protein (GFP). Figure 1B is a graph illustrating the frequency of GFP+ cells identified within the total bone marrow (CD45+CD11b+) brain compartment at various time points after ICV and IV HSPC transplantation in BU-treated (BU-TX) and irradiated (IRR) mice. N≥5 mice per time point and group; mean and SD are shown. Two-way ANOVA showed a significant effect of route and time of cell administration in BU and IRR mice (ICV vs. IV and time p<0.005). These data demonstrate rapid and robust bone marrow cell engraftment in the brain after intraventricular injection of HSPCs. Graphs are shown in triplicate, four-bar sets. From left to right: IRR IV (gray), IRR ICV (white), BU IV (dark gray), and BU ICV (even darker gray). Figure 1C illustrates a sagittal section reconstruction of the brain of a representative ICV-transplanted BU-TX mouse, demonstrating widespread distribution of GFP+ cells 90 days after ICV injection of GFP-transduced HSPCs. Nuclei are labeled with GFP (green / gray) and Topro III (TPIII, light blue / light gray). Images were acquired with a Delta Vision Olympus at 20x magnification and processed with SoftWork 3.5.0. Reconstructions were performed using Adobe Photoshop CS 8.0 software. Figure 1D illustrates immunofluorescence analysis for GFP (green / gray) and Iba-1 (red / light gray) in brain slices from BU_TX mice at 90 days after ICV transplantation of GFP-transduced HSPCs. M = fused. 20x and 40x magnification of the relevant dotted boxes are shown.Images were acquired using a confocal microscope, Radiance 2100 (Bio-Rad) 1x70, and processed using SoftWork 3.5.0. Figure 1E shows the experimental scheme for transplantation of human CD34+ cells (considered to represent HSPCs in humans and equivalent to mouse-derived Lin-cells) transduced with LVs encoding GFP or arylsulfatase A (ARSA) in NSG mice or Rag- / -γ-chain- / - As2- / - (RagMLD) mice pretreated with BU16 mg / kg for 4 days (NSG) or sublethally irradiated (RagMLD). NSG mice also received unmanipulated mononuclear cells from NSG donors. The graph contains four sets of bars. From the leftmost bar in each set, the bars represent IV only (white), LIN-IV and LIN-ICV (dark gray), KLS-IV and LIN-ICV (medium gray), and BM-IV and LIN-ICV (gray). Figure 1F shows representative dot plots from the analysis of brain mononuclear cells derived from NSG mice transplanted with GFP-LV-transduced human CD34+ cells as early as 20 weeks. The abundance of human cells in the mouse brain is shown in two representative animals and by two analytical methods (human CD45 for SSC and human CD45 for mouse CD45). These plots also show that human CD45+ cells identified in the NSG brain after transplantation express CD11b, CX3Cr1, and GFP. Figure 1G shows the abundance of human CD45+CD11b+ cells recovered from the brains of NSG and RagMLD mice transplanted with IV or ICV CD34+ cells derived from umbilical cord blood 12–20 weeks after transplantation (Burton-Bauer et al., 2014). These plots also show that human CD45+ cells identified in the NSG brain after transplantation express CD11b, CX3Cr1, and GFP. Figure 1G shows the abundance of human CD45+CD11b+ cells recovered from the brains of NSG and RagMLD mice transplanted with BU-treated or sublethally irradiated Rag- / -γ-chain- / -As2- / - (NSG) and 5 weeks after transplantation (Rag- / -γ-chain- / -As2- / -). Values ​​are expressed as fold over IV, where IV is equal to 3 + / - 1.3 in NSG mice and 2.9 + / - 0.7 in RagMLD. N > 5 mice / group; mean and SD are shown. For NSG mice, p < 0.001 by Student's t-test; for RagMLD mice, p < 0.05 by one-way ANOVA with Bonferroni post-hoc test.Figure 1H illustrates the results from immunofluorescence analysis for GFP, Iba-1 (costaining), CD11b (costaining), CD68 (no costaining), and CD163 (no costaining) on ​​brain sections from NSG mice 90 days after ICV transfer of GFP-transduced CD34+ cells. In blue, nuclei were stained with TP III. 20x and 40x magnifications of the relevant dotted boxes are shown. M = fused. [Figure 1E]Figures 1A–1H illustrate bone marrow cell reconstitution in the brain after intracerebroventricular injection of mouse and human HSPCs. Figure 1A illustrates the experimental scheme for ICV transplantation of lineage- (Lin−) cells (which become HSPCs in mice) in myeloablated mice (BU: myeloablation with busulfan treatment; IRR: lethal irradiation). Various time points of analysis are indicated. Lin− cells were transduced with LV encoding green fluorescent protein (GFP). Figure 1B is a graph illustrating the frequency of GFP+ cells identified within the total bone marrow (CD45+CD11b+) brain compartment at various time points after ICV and IV HSPC transplantation in BU-treated (BU-TX) and irradiated (IRR) mice. N≥5 mice per time point and group; mean and SD are shown. Two-way ANOVA showed a significant effect of route and time of cell administration in BU and IRR mice (ICV vs. IV and time p<0.005). These data demonstrate rapid and robust bone marrow cell engraftment in the brain after intraventricular injection of HSPCs. Graphs are shown in triplicate, four-bar sets. From left to right: IRR IV (gray), IRR ICV (white), BU IV (dark gray), and BU ICV (even darker gray). Figure 1C illustrates a sagittal section reconstruction of the brain of a representative ICV-transplanted BU-TX mouse, demonstrating widespread distribution of GFP+ cells 90 days after ICV injection of GFP-transduced HSPCs. Nuclei are labeled with GFP (green / gray) and Topro III (TPIII, light blue / light gray). Images were acquired with a Delta Vision Olympus at 20x magnification and processed with SoftWork 3.5.0. Reconstructions were performed using Adobe Photoshop CS 8.0 software. Figure 1D illustrates immunofluorescence analysis for GFP (green / gray) and Iba-1 (red / light gray) in brain slices from BU_TX mice at 90 days after ICV transplantation of GFP-transduced HSPCs. M = fused. 20x and 40x magnification of the relevant dotted boxes are shown.Images were acquired using a confocal microscope, Radiance 2100 (Bio-Rad) 1x70, and processed using SoftWork 3.5.0. Figure 1E shows the experimental scheme for transplantation of human CD34+ cells (considered to represent HSPCs in humans and equivalent to mouse-derived Lin-cells) transduced with LVs encoding GFP or arylsulfatase A (ARSA) in NSG mice or Rag- / -γ-chain- / - As2- / - (RagMLD) mice pretreated with BU16 mg / kg for 4 days (NSG) or sublethally irradiated (RagMLD). NSG mice also received unmanipulated mononuclear cells from NSG donors. The graph contains four sets of bars. From the leftmost bar in each set, the bars represent IV only (white), LIN-IV and LIN-ICV (dark gray), KLS-IV and LIN-ICV (medium gray), and BM-IV and LIN-ICV (gray). Figure 1F shows representative dot plots from the analysis of brain mononuclear cells derived from NSG mice transplanted with GFP-LV-transduced human CD34+ cells as early as 20 weeks. The abundance of human cells in the mouse brain is shown in two representative animals and by two analytical methods (human CD45 for SSC and human CD45 for mouse CD45). These plots also show that human CD45+ cells identified in the NSG brain after transplantation express CD11b, CX3Cr1, and GFP. Figure 1G shows the abundance of human CD45+CD11b+ cells recovered from the brains of NSG and RagMLD mice transplanted with IV or ICV CD34+ cells derived from umbilical cord blood 12–20 weeks after transplantation (Burton-Bauer et al., 2014). These plots also show that human CD45+ cells identified in the NSG brain after transplantation express CD11b, CX3Cr1, and GFP. Figure 1G shows the abundance of human CD45+CD11b+ cells recovered from the brains of NSG and RagMLD mice transplanted with BU-treated or sublethally irradiated Rag- / -γ-chain- / -As2- / - (NSG) and 5 weeks after transplantation (Rag- / -γ-chain- / -As2- / -). Values ​​are expressed as fold over IV, where IV is equal to 3 + / - 1.3 in NSG mice and 2.9 + / - 0.7 in RagMLD. N > 5 mice / group; mean and SD are shown. For NSG mice, p < 0.001 by Student's t-test; for RagMLD mice, p < 0.05 by one-way ANOVA with Bonferroni post-hoc test.Figure 1H illustrates the results from immunofluorescence analysis for GFP, Iba-1 (costaining), CD11b (costaining), CD68 (no costaining), and CD163 (no costaining) on ​​brain sections from NSG mice 90 days after ICV transfer of GFP-transduced CD34+ cells. In blue, nuclei were stained with TP III. 20x and 40x magnifications of the relevant dotted boxes are shown. M = fused. [Figure 1F]Figures 1A–1H illustrate bone marrow cell reconstitution in the brain after intracerebroventricular injection of mouse and human HSPCs. Figure 1A illustrates the experimental scheme for ICV transplantation of lineage- (Lin−) cells (which become HSPCs in mice) in myeloablated mice (BU: myeloablation with busulfan treatment; IRR: lethal irradiation). Various time points of analysis are indicated. Lin− cells were transduced with LV encoding green fluorescent protein (GFP). Figure 1B is a graph illustrating the frequency of GFP+ cells identified within the total bone marrow (CD45+CD11b+) brain compartment at various time points after ICV and IV HSPC transplantation in BU-treated (BU-TX) and irradiated (IRR) mice. N≥5 mice per time point and group; mean and SD are shown. Two-way ANOVA showed a significant effect of route and time of cell administration in BU and IRR mice (ICV vs. IV and time p<0.005). These data demonstrate rapid and robust bone marrow cell engraftment in the brain after intraventricular injection of HSPCs. Graphs are shown in triplicate, four-bar sets. From left to right: IRR IV (gray), IRR ICV (white), BU IV (dark gray), and BU ICV (even darker gray). Figure 1C illustrates a sagittal section reconstruction of the brain of a representative ICV-transplanted BU-TX mouse, demonstrating widespread distribution of GFP+ cells 90 days after ICV injection of GFP-transduced HSPCs. Nuclei are labeled with GFP (green / gray) and Topro III (TPIII, light blue / light gray). Images were acquired with a Delta Vision Olympus at 20x magnification and processed with SoftWork 3.5.0. Reconstructions were performed using Adobe Photoshop CS 8.0 software. Figure 1D illustrates immunofluorescence analysis for GFP (green / gray) and Iba-1 (red / light gray) in brain slices from BU_TX mice at 90 days after ICV transplantation of GFP-transduced HSPCs. M = fused. 20x and 40x magnification of the relevant dotted boxes are shown.Images were acquired using a confocal microscope, Radiance 2100 (Bio-Rad) 1x70, and processed using SoftWork 3.5.0. Figure 1E shows the experimental scheme for transplantation of human CD34+ cells (considered to represent HSPCs in humans and equivalent to mouse-derived Lin-cells) transduced with LVs encoding GFP or arylsulfatase A (ARSA) in NSG mice or Rag- / -γ-chain- / - As2- / - (RagMLD) mice pretreated with BU16 mg / kg for 4 days (NSG) or sublethally irradiated (RagMLD). NSG mice also received unmanipulated mononuclear cells from NSG donors. The graph contains four sets of bars. From the leftmost bar in each set, the bars represent IV only (white), LIN-IV and LIN-ICV (dark gray), KLS-IV and LIN-ICV (medium gray), and BM-IV and LIN-ICV (gray). Figure 1F shows representative dot plots from the analysis of brain mononuclear cells derived from NSG mice transplanted with GFP-LV-transduced human CD34+ cells as early as 20 weeks. The abundance of human cells in the mouse brain is shown in two representative animals and by two analytical methods (human CD45 for SSC and human CD45 for mouse CD45). These plots also show that human CD45+ cells identified in the NSG brain after transplantation express CD11b, CX3Cr1, and GFP. Figure 1G shows the abundance of human CD45+CD11b+ cells recovered from the brains of NSG and RagMLD mice transplanted with IV or ICV CD34+ cells derived from umbilical cord blood 12–20 weeks after transplantation (Burton-Bauer et al., 2014). These plots also show that human CD45+ cells identified in the NSG brain after transplantation express CD11b, CX3Cr1, and GFP. Figure 1G shows the abundance of human CD45+CD11b+ cells recovered from the brains of NSG and RagMLD mice transplanted with BU-treated or sublethally irradiated Rag- / -γ-chain- / -As2- / - (NSG) and 5 weeks after transplantation (Rag- / -γ-chain- / -As2- / -). Values ​​are expressed as fold over IV, where IV is equal to 3 + / - 1.3 in NSG mice and 2.9 + / - 0.7 in RagMLD. N > 5 mice / group; mean and SD are shown. For NSG mice, p < 0.001 by Student's t-test; for RagMLD mice, p < 0.05 by one-way ANOVA with Bonferroni post-hoc test.Figure 1H illustrates the results from immunofluorescence analysis for GFP, Iba-1 (costaining), CD11b (costaining), CD68 (no costaining), and CD163 (no costaining) on ​​brain sections from NSG mice 90 days after ICV transfer of GFP-transduced CD34+ cells. In blue, nuclei were stained with TP III. 20x and 40x magnifications of the relevant dotted boxes are shown. M = fused. [Figure 1G]Figures 1A–1H illustrate bone marrow cell reconstitution in the brain after intracerebroventricular injection of mouse and human HSPCs. Figure 1A illustrates the experimental scheme for ICV transplantation of lineage- (Lin−) cells (which become HSPCs in mice) in myeloablated mice (BU: myeloablation with busulfan treatment; IRR: lethal irradiation). Various time points of analysis are indicated. Lin− cells were transduced with LV encoding green fluorescent protein (GFP). Figure 1B is a graph illustrating the frequency of GFP+ cells identified within the total bone marrow (CD45+CD11b+) brain compartment at various time points after ICV and IV HSPC transplantation in BU-treated (BU-TX) and irradiated (IRR) mice. N≥5 mice per time point and group; mean and SD are shown. Two-way ANOVA showed a significant effect of route and time of cell administration in BU and IRR mice (ICV vs. IV and time p<0.005). These data demonstrate rapid and robust bone marrow cell engraftment in the brain after intraventricular injection of HSPCs. Graphs are shown in triplicate, four-bar sets. From left to right: IRR IV (gray), IRR ICV (white), BU IV (dark gray), and BU ICV (even darker gray). Figure 1C illustrates a sagittal section reconstruction of the brain of a representative ICV-transplanted BU-TX mouse, demonstrating widespread distribution of GFP+ cells 90 days after ICV injection of GFP-transduced HSPCs. Nuclei are labeled with GFP (green / gray) and Topro III (TPIII, light blue / light gray). Images were acquired with a Delta Vision Olympus at 20x magnification and processed with SoftWork 3.5.0. Reconstructions were performed using Adobe Photoshop CS 8.0 software. Figure 1D illustrates immunofluorescence analysis for GFP (green / gray) and Iba-1 (red / light gray) in brain slices from BU_TX mice at 90 days after ICV transplantation of GFP-transduced HSPCs. M = fused. 20x and 40x magnification of the relevant dotted boxes are shown.Images were acquired using a confocal microscope, Radiance 2100 (Bio-Rad) 1x70, and processed using SoftWork 3.5.0. Figure 1E shows the experimental scheme for transplantation of human CD34+ cells (considered to represent HSPCs in humans and equivalent to mouse-derived Lin-cells) transduced with LVs encoding GFP or arylsulfatase A (ARSA) in NSG mice or Rag- / -γ-chain- / - As2- / - (RagMLD) mice pretreated with BU16 mg / kg for 4 days (NSG) or sublethally irradiated (RagMLD). NSG mice also received unmanipulated mononuclear cells from NSG donors. The graph contains four sets of bars. From the leftmost bar in each set, the bars represent IV only (white), LIN-IV and LIN-ICV (dark gray), KLS-IV and LIN-ICV (medium gray), and BM-IV and LIN-ICV (gray). Figure 1F shows representative dot plots from the analysis of brain mononuclear cells derived from NSG mice transplanted with GFP-LV-transduced human CD34+ cells as early as 20 weeks. The abundance of human cells in the mouse brain is shown in two representative animals and by two analytical methods (human CD45 for SSC and human CD45 for mouse CD45). These plots also show that human CD45+ cells identified in the NSG brain after transplantation express CD11b, CX3Cr1, and GFP. Figure 1G shows the abundance of human CD45+CD11b+ cells recovered from the brains of NSG and RagMLD mice transplanted with IV or ICV CD34+ cells derived from umbilical cord blood 12–20 weeks after transplantation (Burton-Bauer et al., 2014). These plots also show that human CD45+ cells identified in the NSG brain after transplantation express CD11b, CX3Cr1, and GFP. Figure 1G shows the abundance of human CD45+CD11b+ cells recovered from the brains of NSG and RagMLD mice transplanted with BU-treated or sublethally irradiated Rag- / -γ-chain- / -As2- / - (NSG) and 5 weeks after transplantation (Rag- / -γ-chain- / -As2- / -). Values ​​are expressed as fold over IV, where IV is equal to 3 + / - 1.3 in NSG mice and 2.9 + / - 0.7 in RagMLD. N > 5 mice / group; mean and SD are shown. For NSG mice, p < 0.001 by Student's t-test; for RagMLD mice, p < 0.05 by one-way ANOVA with Bonferroni post-hoc test.Figure 1H illustrates the results from immunofluorescence analysis for GFP, Iba-1 (costaining), CD11b (costaining), CD68 (no costaining), and CD163 (no costaining) on ​​brain sections from NSG mice 90 days after ICV transfer of GFP-transduced CD34+ cells. In blue, nuclei were stained with TP III. 20x and 40x magnifications of the relevant dotted boxes are shown. M = fused. [Figure 1H]Figures 1A–1H illustrate bone marrow cell reconstitution in the brain after intracerebroventricular injection of mouse and human HSPCs. Figure 1A illustrates the experimental scheme for ICV transplantation of lineage- (Lin−) cells (which become HSPCs in mice) in myeloablated mice (BU: myeloablation with busulfan treatment; IRR: lethal irradiation). Various time points of analysis are indicated. Lin− cells were transduced with LV encoding green fluorescent protein (GFP). Figure 1B is a graph illustrating the frequency of GFP+ cells identified within the total bone marrow (CD45+CD11b+) brain compartment at various time points after ICV and IV HSPC transplantation in BU-treated (BU-TX) and irradiated (IRR) mice. N≥5 mice per time point and group; mean and SD are shown. Two-way ANOVA showed a significant effect of route and time of cell administration in BU and IRR mice (ICV vs. IV and time p<0.005). These data demonstrate rapid and robust bone marrow cell engraftment in the brain after intraventricular injection of HSPCs. Graphs are shown in triplicate, four-bar sets. From left to right: IRR IV (gray), IRR ICV (white), BU IV (dark gray), and BU ICV (even darker gray). Figure 1C illustrates a sagittal section reconstruction of the brain of a representative ICV-transplanted BU-TX mouse, demonstrating widespread distribution of GFP+ cells 90 days after ICV injection of GFP-transduced HSPCs. Nuclei are labeled with GFP (green / gray) and Topro III (TPIII, light blue / light gray). Images were acquired with a Delta Vision Olympus at 20x magnification and processed with SoftWork 3.5.0. Reconstructions were performed using Adobe Photoshop CS 8.0 software. Figure 1D illustrates immunofluorescence analysis for GFP (green / gray) and Iba-1 (red / light gray) in brain slices from BU_TX mice at 90 days after ICV transplantation of GFP-transduced HSPCs. M = fused. 20x and 40x magnification of the relevant dotted boxes are shown.Images were acquired using a confocal microscope, Radiance 2100 (Bio-Rad) 1x70, and processed using SoftWork 3.5.0. Figure 1E shows the experimental scheme for transplantation of human CD34+ cells (considered to represent HSPCs in humans and equivalent to mouse-derived Lin-cells) transduced with LVs encoding GFP or arylsulfatase A (ARSA) in NSG mice or Rag- / -γ-chain- / - As2- / - (RagMLD) mice pretreated with BU16 mg / kg for 4 days (NSG) or sublethally irradiated (RagMLD). NSG mice also received unmanipulated mononuclear cells from NSG donors. The graph contains four sets of bars. From the leftmost bar in each set, the bars represent IV only (white), LIN-IV and LIN-ICV (dark gray), KLS-IV and LIN-ICV (medium gray), and BM-IV and LIN-ICV (gray). Figure 1F shows representative dot plots from the analysis of brain mononuclear cells derived from NSG mice transplanted with GFP-LV-transduced human CD34+ cells as early as 20 weeks. The abundance of human cells in the mouse brain is shown in two representative animals and by two analytical methods (human CD45 for SSC and human CD45 for mouse CD45). These plots also show that human CD45+ cells identified in the NSG brain after transplantation express CD11b, CX3Cr1, and GFP. Figure 1G shows the abundance of human CD45+CD11b+ cells recovered from the brains of NSG and RagMLD mice transplanted with IV or ICV CD34+ cells derived from umbilical cord blood 12–20 weeks after transplantation (Burton-Bauer et al., 2014). These plots also show that human CD45+ cells identified in the NSG brain after transplantation express CD11b, CX3Cr1, and GFP. Figure 1G shows the abundance of human CD45+CD11b+ cells recovered from the brains of NSG and RagMLD mice transplanted with BU-treated or sublethally irradiated Rag- / -γ-chain- / -As2- / - (NSG) and 5 weeks after transplantation (Rag- / -γ-chain- / -As2- / -). Values ​​are expressed as fold over IV, where IV is equal to 3 + / - 1.3 in NSG mice and 2.9 + / - 0.7 in RagMLD. N > 5 mice / group; mean and SD are shown. For NSG mice, p < 0.001 by Student's t-test; for RagMLD mice, p < 0.05 by one-way ANOVA with Bonferroni post-hoc test.Figure 1H illustrates the results from immunofluorescence analysis for GFP, Iba-1 (costaining), CD11b (costaining), CD68 (no costaining), and CD163 (no costaining) on ​​brain sections from NSG mice 90 days after ICV transfer of GFP-transduced CD34+ cells. In blue, nuclei were stained with TP III. 20x and 40x magnifications of the relevant dotted boxes are shown. M = fused.

[0072] [Figure 2]Figures 2A–2D show short-term monitoring of mice transplanted with GFP+ Lin- HSPCs via ICV. Figures 2A and 2B show two graphs depicting the abundance of GFP+ cells identified among CD45+ cells in the brain (Figure 2A) and bone marrow (Figure 2B) of BU-treated and transplanted (BU_TX) mice at the indicated time points after ICV injection of Lin- HSPCs transduced with LV encoding GFP. N≥3 mice per time point; mean and SD are shown. Analysis by one-way ANOVA with Bonferroni post-hoc test shows P <0.001 on day 4 compared with 1, 3, 6, and 24 h. Engraftment of ICV-transplanted cells was predominant in the brain, with only a few or no GFP+ cells detected in the bone marrow of transplanted mice. Figures 2C and 2D show, in two graphs, the expression of the indicated hematopoietic stem cell (Figure 2C) and myeloid / microglial (Figure 2D) markers by GFP+ (donor) and GFP- (recipient) CD45+ cells recovered from the brains of BU_TX mice at various time points after ICV infusion of transduced Lin- HSPCs (infusion refers to HSPCs at the time of infusion). N≥3 mice per time point; mean and SD are shown. Two-way anova revealed significant effects of marker and time (p<0.0001). In Figure 2C, for GFP+ cells, arrows indicate the % expression of c-Kit, Sca1, CD34, CXCR4, CD93, and Tie2. In Figure 2C, for GFP- cells, circles indicate, from top to bottom, CXCR4, CD34, CD93, c-Kit, Sca1, and Tie2 in each column. In Figure 2D, for GFP+ cells, arrows indicate the % expression of CD11b, CX3CR1, and CD115. In Figure 2D, for GFP- cells, circles indicate, from top to bottom, CD11b, CX3CR1, and CD115 in each row. Transplanted cells / their progeny transiently increase the expression of hematopoietic stem cells, followed by improved expression levels of myeloid / microglial markers.

[0073] [Figure 3A]Figures 3A-3J demonstrate that optimization of combinatorial transplantation protocols may allow for modulation of the contribution of IV- versus ICV-transplanted cells to donor bone marrow-brain chimerism after transplantation for appropriate clinical applications. Figure 3A is a graph showing brain engraftment of donor (GFP+) Lin- HSPCs IV transplanted on day 0 (24 hours after the last dose of brusulfan) or 5 days later (day 5) within total bone marrow CD45+CD11b+ cells, microglia (μ), transiently expanded μ (TAμ), and CNS macrophages. This graph demonstrates that transplantation of HSPCs at the two time points results in similar brain engraftment in the study population. Figure 3B illustrates the gating strategy for identifying μ, TAμ, and CNS macrophages (CNSmac) as CD45+lowCD11bhigh, CD45+low, CD11b+low, and CD45highCD11bhigh in 3-day-old (pnd) newborn mice, 21- and 60-pnd adult mice, and adult HSPC-transplanted animals at 2 months posttransplant. Figure 3C illustrates the experimental scheme demonstrating the IV and / or ICV transplantation strategy of differentially labeled (LV encoding GFP or ΔNGFR) hematopoietic cells in busulfan-pretreated recipients. The transplanted hematopoietic cells were Lin- HSPCs, c-kit+Sca1+Lin- (KSL) cells, or whole bone marrow (BM). Figure 3D is a graph illustrating the abundance of donor-derived cells (CD45.1 = progeny of IV-transplanted cells, GFP-expressing cells = progeny of ICV-transplanted cells) in the BM of transplanted mice at sacrifice 3 months posttransplant. GFP+ ICV-transplanted cells do not show robust engraftment in the BM. Figure 3E is a graph illustrating the abundance of donor-derived cells (as the sum of ΔNGFR and GFP-expressing cells) within the CD45+CD11b+ cells, μ, TAμ, and CNSmac of transplanted mice, as indicated by color coding, on day 0. Co-delivery of hematopoietic cells by ICV and IV improves brain donor chimerism after transplantation in all combinations tested compared to IV Lin-transplantation alone.From the leftmost bar in each set: IV only (white), LIN IV LIN ICV (dark gray), KLS IV LIN ICV (medium gray), and BM IV LIN ICV (gray). Figure 3F is a graph illustrating the differential abundance of donor-derived ΔNGFR (progeny of IV-transplanted cells) and GFP (progeny of ICV-transplanted cells) within CD45+CD11b+ cells, μ, TAμ, and CNSmac of the indicated transplanted mice on day 0. Lin- ICV delivery leading to IV transplantation of whole BM results in the lowest engraftment of IV-transplanted cells / their progeny in the brain. The sets of bars (four bars per set) are, from left to right: IV only (NGFR), LIN IV LIN ICV (DNGFR), LIN IV LIN ICV (GFP), KLS IV LIN ICV (DNGFR), KLS IV LIN ICV (GFP), BM IV LIN ICV (DNGFR), and BM IV LIN ICV (GFP). Figure 3G illustrates the experimental scheme showing the IV and / or ICV transplantation strategy of differentially labeled (LV encoding GFP or ΔNGFR) hematopoietic cells into busulfan-pretreated recipients. In this case, IV cells were transplanted 5 days after chemotherapy, while ICV cells were transplanted on day 0. The transplanted hematopoietic cells were Lin-HSPCs, KSL cells, or whole BM. Figure 3H is a graph depicting the abundance of donor-derived cells (CD45.1 = progeny of IV-transplanted cells, GFP-expressing cells = progeny of ICV-transplanted cells) among total BM cells recovered from transplanted mice at sacrifice 3 months after transplantation. GFP+ ICV-transplanted cells do not engraft in the BM. Figure 3I is a graph depicting the abundance of donor-derived cells (as the sum of ΔNGFR and GFP-expressing cells) among CD45+CD11b+ cells, μ, TAμ, and CNSmac of transplanted mice, as indicated by color coding, on day 5. Co-delivery of hematopoietic cells by ICV and IV improves brain-donor chimerism after transplantation in all combinations tested compared with IV Lin-transplantation alone. The leftmost bar in each set is IV alone (white), LIN IV LIN ICV (dark gray), KLS IV LIN ICV (medium gray), and BM IV LIN ICV (gray).Figure 3J is a graph illustrating the differential abundance of donor-derived ΔNGFR (progeny of IV-transplanted cells) and GFP (progeny of ICV-transplanted cells) expressing cells within the CD45+CD11b+ cells, μ, TAμ, and CNSmac of the indicated transplanted mice at day 5. Lin- ICV delivery leading to IV transplantation of whole BM results in the lowest engraftment of IV-transplanted cells / their progeny in the brain. The bar sets (4 bars / set) are, from left to right: IV only (NGFR), LIN IV LIN ICV (DNGFR), LIN IV LIN ICV (GFP), KLS IV LIN ICV (DNGFR), KLS IV LIN ICV (GFP), BM IV LIN ICV (DNGFR), and BM IV LIN ICV (GFP). One-way Anova with Bonferroni post-hoc test, *=p<0.05, **=p<0.01, ***=p<0.001, ****=p<0.0001. [Figure 3B]Figures 3A-3J demonstrate that optimization of combinatorial transplantation protocols may allow for modulation of the contribution of IV- versus ICV-transplanted cells to donor bone marrow-brain chimerism after transplantation for appropriate clinical applications. Figure 3A is a graph showing brain engraftment of donor (GFP+) Lin- HSPCs IV transplanted on day 0 (24 hours after the last dose of brusulfan) or 5 days later (day 5) within total bone marrow CD45+CD11b+ cells, microglia (μ), transiently expanded μ (TAμ), and CNS macrophages. This graph demonstrates that transplantation of HSPCs at the two time points results in similar brain engraftment in the study population. Figure 3B illustrates the gating strategy for identifying μ, TAμ, and CNS macrophages (CNSmac) as CD45+lowCD11bhigh, CD45+low, CD11b+low, and CD45highCD11bhigh in 3-day-old (pnd) newborn mice, 21- and 60-pnd adult mice, and adult HSPC-transplanted animals at 2 months posttransplant. Figure 3C illustrates the experimental scheme demonstrating the IV and / or ICV transplantation strategy of differentially labeled (LV encoding GFP or ΔNGFR) hematopoietic cells in busulfan-pretreated recipients. The transplanted hematopoietic cells were Lin- HSPCs, c-kit+Sca1+Lin- (KSL) cells, or whole bone marrow (BM). Figure 3D is a graph illustrating the abundance of donor-derived cells (CD45.1 = progeny of IV-transplanted cells, GFP-expressing cells = progeny of ICV-transplanted cells) in the BM of transplanted mice at sacrifice 3 months posttransplant. GFP+ ICV-transplanted cells do not show robust engraftment in the BM. Figure 3E is a graph illustrating the abundance of donor-derived cells (as the sum of ΔNGFR and GFP-expressing cells) within the CD45+CD11b+ cells, μ, TAμ, and CNSmac of transplanted mice, as indicated by color coding, on day 0. Co-delivery of hematopoietic cells by ICV and IV improves brain donor chimerism after transplantation in all combinations tested compared to IV Lin-transplantation alone.From the leftmost bar in each set: IV only (white), LIN IV LIN ICV (dark gray), KLS IV LIN ICV (medium gray), and BM IV LIN ICV (gray). Figure 3F is a graph illustrating the differential abundance of donor-derived ΔNGFR (progeny of IV-transplanted cells) and GFP (progeny of ICV-transplanted cells) within CD45+CD11b+ cells, μ, TAμ, and CNSmac of the indicated transplanted mice on day 0. Lin- ICV delivery leading to IV transplantation of whole BM results in the lowest engraftment of IV-transplanted cells / their progeny in the brain. The sets of bars (four bars per set) are, from left to right: IV only (NGFR), LIN IV LIN ICV (DNGFR), LIN IV LIN ICV (GFP), KLS IV LIN ICV (DNGFR), KLS IV LIN ICV (GFP), BM IV LIN ICV (DNGFR), and BM IV LIN ICV (GFP). Figure 3G illustrates the experimental scheme showing the IV and / or ICV transplantation strategy of differentially labeled (LV encoding GFP or ΔNGFR) hematopoietic cells into busulfan-pretreated recipients. In this case, IV cells were transplanted 5 days after chemotherapy, while ICV cells were transplanted on day 0. The transplanted hematopoietic cells were Lin-HSPCs, KSL cells, or whole BM. Figure 3H is a graph depicting the abundance of donor-derived cells (CD45.1 = progeny of IV-transplanted cells, GFP-expressing cells = progeny of ICV-transplanted cells) among total BM cells recovered from transplanted mice at sacrifice 3 months after transplantation. GFP+ ICV-transplanted cells do not engraft in the BM. Figure 3I is a graph depicting the abundance of donor-derived cells (as the sum of ΔNGFR and GFP-expressing cells) among CD45+CD11b+ cells, μ, TAμ, and CNSmac of transplanted mice, as indicated by color coding, on day 5. Co-delivery of hematopoietic cells by ICV and IV improves brain-donor chimerism after transplantation in all combinations tested compared with IV Lin-transplantation alone. The leftmost bar in each set is IV alone (white), LIN IV LIN ICV (dark gray), KLS IV LIN ICV (medium gray), and BM IV LIN ICV (gray).Figure 3J is a graph illustrating the differential abundance of donor-derived ΔNGFR (progeny of IV-transplanted cells) and GFP (progeny of ICV-transplanted cells) expressing cells within the CD45+CD11b+ cells, μ, TAμ, and CNSmac of the indicated transplanted mice at day 5. Lin- ICV delivery leading to IV transplantation of whole BM results in the lowest engraftment of IV-transplanted cells / their progeny in the brain. The bar sets (4 bars / set) are, from left to right: IV only (NGFR), LIN IV LIN ICV (DNGFR), LIN IV LIN ICV (GFP), KLS IV LIN ICV (DNGFR), KLS IV LIN ICV (GFP), BM IV LIN ICV (DNGFR), and BM IV LIN ICV (GFP). One-way Anova with Bonferroni post-hoc test, *=p<0.05, **=p<0.01, ***=p<0.001, ****=p<0.0001. [Figure 3CD]Figures 3A-3J demonstrate that optimization of combinatorial transplantation protocols may allow for modulation of the contribution of IV- versus ICV-transplanted cells to donor bone marrow-brain chimerism after transplantation for appropriate clinical applications. Figure 3A is a graph showing brain engraftment of donor (GFP+) Lin- HSPCs IV transplanted on day 0 (24 hours after the last dose of brusulfan) or 5 days later (day 5) within total bone marrow CD45+CD11b+ cells, microglia (μ), transiently expanded μ (TAμ), and CNS macrophages. This graph demonstrates that transplantation of HSPCs at the two time points results in similar brain engraftment in the study population. Figure 3B illustrates the gating strategy for identifying μ, TAμ, and CNS macrophages (CNSmac) as CD45+lowCD11bhigh, CD45+low, CD11b+low, and CD45highCD11bhigh in 3-day-old (pnd) newborn mice, 21- and 60-pnd adult mice, and adult HSPC-transplanted animals at 2 months posttransplant. Figure 3C illustrates the experimental scheme demonstrating the IV and / or ICV transplantation strategy of differentially labeled (LV encoding GFP or ΔNGFR) hematopoietic cells in busulfan-pretreated recipients. The transplanted hematopoietic cells were Lin- HSPCs, c-kit+Sca1+Lin- (KSL) cells, or whole bone marrow (BM). Figure 3D is a graph illustrating the abundance of donor-derived cells (CD45.1 = progeny of IV-transplanted cells, GFP-expressing cells = progeny of ICV-transplanted cells) in the BM of transplanted mice at sacrifice 3 months posttransplant. GFP+ ICV-transplanted cells do not show robust engraftment in the BM. Figure 3E is a graph illustrating the abundance of donor-derived cells (as the sum of ΔNGFR and GFP-expressing cells) within the CD45+CD11b+ cells, μ, TAμ, and CNSmac of transplanted mice, as indicated by color coding, on day 0. Co-delivery of hematopoietic cells by ICV and IV improves brain donor chimerism after transplantation in all combinations tested compared to IV Lin-transplantation alone.From the leftmost bar in each set: IV only (white), LIN IV LIN ICV (dark gray), KLS IV LIN ICV (medium gray), and BM IV LIN ICV (gray). Figure 3F is a graph illustrating the differential abundance of donor-derived ΔNGFR (progeny of IV-transplanted cells) and GFP (progeny of ICV-transplanted cells) within CD45+CD11b+ cells, μ, TAμ, and CNSmac of the indicated transplanted mice on day 0. Lin- ICV delivery leading to IV transplantation of whole BM results in the lowest engraftment of IV-transplanted cells / their progeny in the brain. The sets of bars (four bars per set) are, from left to right: IV only (NGFR), LIN IV LIN ICV (DNGFR), LIN IV LIN ICV (GFP), KLS IV LIN ICV (DNGFR), KLS IV LIN ICV (GFP), BM IV LIN ICV (DNGFR), and BM IV LIN ICV (GFP). Figure 3G illustrates the experimental scheme showing the IV and / or ICV transplantation strategy of differentially labeled (LV encoding GFP or ΔNGFR) hematopoietic cells into busulfan-pretreated recipients. In this case, IV cells were transplanted 5 days after chemotherapy, while ICV cells were transplanted on day 0. The transplanted hematopoietic cells were Lin-HSPCs, KSL cells, or whole BM. Figure 3H is a graph depicting the abundance of donor-derived cells (CD45.1 = progeny of IV-transplanted cells, GFP-expressing cells = progeny of ICV-transplanted cells) among total BM cells recovered from transplanted mice at sacrifice 3 months after transplantation. GFP+ ICV-transplanted cells do not engraft in the BM. Figure 3I is a graph depicting the abundance of donor-derived cells (as the sum of ΔNGFR and GFP-expressing cells) among CD45+CD11b+ cells, μ, TAμ, and CNSmac of transplanted mice, as indicated by color coding, on day 5. Co-delivery of hematopoietic cells by ICV and IV improves brain-donor chimerism after transplantation in all combinations tested compared with IV Lin-transplantation alone. The leftmost bar in each set is IV alone (white), LIN IV LIN ICV (dark gray), KLS IV LIN ICV (medium gray), and BM IV LIN ICV (gray).Figure 3J is a graph illustrating the differential abundance of donor-derived ΔNGFR (progeny of IV-transplanted cells) and GFP (progeny of ICV-transplanted cells) expressing cells within the CD45+CD11b+ cells, μ, TAμ, and CNSmac of the indicated transplanted mice at day 5. Lin- ICV delivery leading to IV transplantation of whole BM results in the lowest engraftment of IV-transplanted cells / their progeny in the brain. The bar sets (4 bars / set) are, from left to right: IV only (NGFR), LIN IV LIN ICV (DNGFR), LIN IV LIN ICV (GFP), KLS IV LIN ICV (DNGFR), KLS IV LIN ICV (GFP), BM IV LIN ICV (DNGFR), and BM IV LIN ICV (GFP). One-way Anova with Bonferroni post-hoc test, *=p<0.05, **=p<0.01, ***=p<0.001, ****=p<0.0001. [Figure 3E]Figures 3A-3J demonstrate that optimization of combinatorial transplantation protocols may allow for modulation of the contribution of IV- versus ICV-transplanted cells to donor bone marrow-brain chimerism after transplantation for appropriate clinical applications. Figure 3A is a graph showing brain engraftment of donor (GFP+) Lin- HSPCs IV transplanted on day 0 (24 hours after the last dose of brusulfan) or 5 days later (day 5) within total bone marrow CD45+CD11b+ cells, microglia (μ), transiently expanded μ (TAμ), and CNS macrophages. This graph demonstrates that transplantation of HSPCs at the two time points results in similar brain engraftment in the study population. Figure 3B illustrates the gating strategy for identifying μ, TAμ, and CNS macrophages (CNSmac) as CD45+lowCD11bhigh, CD45+low, CD11b+low, and CD45highCD11bhigh in 3-day-old (pnd) newborn mice, 21- and 60-pnd adult mice, and adult HSPC-transplanted animals at 2 months posttransplant. Figure 3C illustrates the experimental scheme demonstrating the IV and / or ICV transplantation strategy of differentially labeled (LV encoding GFP or ΔNGFR) hematopoietic cells in busulfan-pretreated recipients. The transplanted hematopoietic cells were Lin- HSPCs, c-kit+Sca1+Lin- (KSL) cells, or whole bone marrow (BM). Figure 3D is a graph illustrating the abundance of donor-derived cells (CD45.1 = progeny of IV-transplanted cells, GFP-expressing cells = progeny of ICV-transplanted cells) in the BM of transplanted mice at sacrifice 3 months posttransplant. GFP+ ICV-transplanted cells do not show robust engraftment in the BM. Figure 3E is a graph illustrating the abundance of donor-derived cells (as the sum of ΔNGFR and GFP-expressing cells) within the CD45+CD11b+ cells, μ, TAμ, and CNSmac of transplanted mice, as indicated by color coding, on day 0. Co-delivery of hematopoietic cells by ICV and IV improves brain donor chimerism after transplantation in all combinations tested compared to IV Lin-transplantation alone.From the leftmost bar in each set: IV only (white), LIN IV LIN ICV (dark gray), KLS IV LIN ICV (medium gray), and BM IV LIN ICV (gray). Figure 3F is a graph illustrating the differential abundance of donor-derived ΔNGFR (progeny of IV-transplanted cells) and GFP (progeny of ICV-transplanted cells) within CD45+CD11b+ cells, μ, TAμ, and CNSmac of the indicated transplanted mice on day 0. Lin- ICV delivery leading to IV transplantation of whole BM results in the lowest engraftment of IV-transplanted cells / their progeny in the brain. The sets of bars (four bars per set) are, from left to right: IV only (NGFR), LIN IV LIN ICV (DNGFR), LIN IV LIN ICV (GFP), KLS IV LIN ICV (DNGFR), KLS IV LIN ICV (GFP), BM IV LIN ICV (DNGFR), and BM IV LIN ICV (GFP). Figure 3G illustrates the experimental scheme showing the IV and / or ICV transplantation strategy of differentially labeled (LV encoding GFP or ΔNGFR) hematopoietic cells into busulfan-pretreated recipients. In this case, IV cells were transplanted 5 days after chemotherapy, while ICV cells were transplanted on day 0. The transplanted hematopoietic cells were Lin-HSPCs, KSL cells, or whole BM. Figure 3H is a graph depicting the abundance of donor-derived cells (CD45.1 = progeny of IV-transplanted cells, GFP-expressing cells = progeny of ICV-transplanted cells) among total BM cells recovered from transplanted mice at sacrifice 3 months after transplantation. GFP+ ICV-transplanted cells do not engraft in the BM. Figure 3I is a graph depicting the abundance of donor-derived cells (as the sum of ΔNGFR and GFP-expressing cells) among CD45+CD11b+ cells, μ, TAμ, and CNSmac of transplanted mice, as indicated by color coding, on day 5. Co-delivery of hematopoietic cells by ICV and IV improves brain-donor chimerism after transplantation in all combinations tested compared with IV Lin-transplantation alone. The leftmost bar in each set is IV alone (white), LIN IV LIN ICV (dark gray), KLS IV LIN ICV (medium gray), and BM IV LIN ICV (gray).Figure 3J is a graph illustrating the differential abundance of donor-derived ΔNGFR (progeny of IV-transplanted cells) and GFP (progeny of ICV-transplanted cells) expressing cells within the CD45+CD11b+ cells, μ, TAμ, and CNSmac of the indicated transplanted mice at day 5. Lin- ICV delivery leading to IV transplantation of whole BM results in the lowest engraftment of IV-transplanted cells / their progeny in the brain. The bar sets (4 bars / set) are, from left to right: IV only (NGFR), LIN IV LIN ICV (DNGFR), LIN IV LIN ICV (GFP), KLS IV LIN ICV (DNGFR), KLS IV LIN ICV (GFP), BM IV LIN ICV (DNGFR), and BM IV LIN ICV (GFP). One-way Anova with Bonferroni post-hoc test, *=p<0.05, **=p<0.01, ***=p<0.001, ****=p<0.0001. [Figure 3F]Figures 3A-3J demonstrate that optimization of combinatorial transplantation protocols may allow for modulation of the contribution of IV- versus ICV-transplanted cells to donor bone marrow-brain chimerism after transplantation for appropriate clinical applications. Figure 3A is a graph showing brain engraftment of donor (GFP+) Lin- HSPCs IV transplanted on day 0 (24 hours after the last dose of brusulfan) or 5 days later (day 5) within total bone marrow CD45+CD11b+ cells, microglia (μ), transiently expanded μ (TAμ), and CNS macrophages. This graph demonstrates that transplantation of HSPCs at the two time points results in similar brain engraftment in the study population. Figure 3B illustrates the gating strategy for identifying μ, TAμ, and CNS macrophages (CNSmac) as CD45+lowCD11bhigh, CD45+low, CD11b+low, and CD45highCD11bhigh in 3-day-old (pnd) newborn mice, 21- and 60-pnd adult mice, and adult HSPC-transplanted animals at 2 months posttransplant. Figure 3C illustrates the experimental scheme demonstrating the IV and / or ICV transplantation strategy of differentially labeled (LV encoding GFP or ΔNGFR) hematopoietic cells in busulfan-pretreated recipients. The transplanted hematopoietic cells were Lin- HSPCs, c-kit+Sca1+Lin- (KSL) cells, or whole bone marrow (BM). Figure 3D is a graph illustrating the abundance of donor-derived cells (CD45.1 = progeny of IV-transplanted cells, GFP-expressing cells = progeny of ICV-transplanted cells) in the BM of transplanted mice at sacrifice 3 months posttransplant. GFP+ ICV-transplanted cells do not show robust engraftment in the BM. Figure 3E is a graph illustrating the abundance of donor-derived cells (as the sum of ΔNGFR and GFP-expressing cells) within the CD45+CD11b+ cells, μ, TAμ, and CNSmac of transplanted mice, as indicated by color coding, on day 0. Co-delivery of hematopoietic cells by ICV and IV improves brain donor chimerism after transplantation in all combinations tested compared to IV Lin-transplantation alone.From the leftmost bar in each set: IV only (white), LIN IV LIN ICV (dark gray), KLS IV LIN ICV (medium gray), and BM IV LIN ICV (gray). Figure 3F is a graph illustrating the differential abundance of donor-derived ΔNGFR (progeny of IV-transplanted cells) and GFP (progeny of ICV-transplanted cells) within CD45+CD11b+ cells, μ, TAμ, and CNSmac of the indicated transplanted mice on day 0. Lin- ICV delivery leading to IV transplantation of whole BM results in the lowest engraftment of IV-transplanted cells / their progeny in the brain. The sets of bars (four bars per set) are, from left to right: IV only (NGFR), LIN IV LIN ICV (DNGFR), LIN IV LIN ICV (GFP), KLS IV LIN ICV (DNGFR), KLS IV LIN ICV (GFP), BM IV LIN ICV (DNGFR), and BM IV LIN ICV (GFP). Figure 3G illustrates the experimental scheme showing the IV and / or ICV transplantation strategy of differentially labeled (LV encoding GFP or ΔNGFR) hematopoietic cells into busulfan-pretreated recipients. In this case, IV cells were transplanted 5 days after chemotherapy, while ICV cells were transplanted on day 0. The transplanted hematopoietic cells were Lin-HSPCs, KSL cells, or whole BM. Figure 3H is a graph depicting the abundance of donor-derived cells (CD45.1 = progeny of IV-transplanted cells, GFP-expressing cells = progeny of ICV-transplanted cells) among total BM cells recovered from transplanted mice at sacrifice 3 months after transplantation. GFP+ ICV-transplanted cells do not engraft in the BM. Figure 3I is a graph depicting the abundance of donor-derived cells (as the sum of ΔNGFR and GFP-expressing cells) among CD45+CD11b+ cells, μ, TAμ, and CNSmac of transplanted mice, as indicated by color coding, on day 5. Co-delivery of hematopoietic cells by ICV and IV improves brain-donor chimerism after transplantation in all combinations tested compared with IV Lin-transplantation alone. The leftmost bar in each set is IV alone (white), LIN IV LIN ICV (dark gray), KLS IV LIN ICV (medium gray), and BM IV LIN ICV (gray).Figure 3J is a graph illustrating the differential abundance of donor-derived ΔNGFR (progeny of IV-transplanted cells) and GFP (progeny of ICV-transplanted cells) expressing cells within the CD45+CD11b+ cells, μ, TAμ, and CNSmac of the indicated transplanted mice at day 5. Lin- ICV delivery leading to IV transplantation of whole BM results in the lowest engraftment of IV-transplanted cells / their progeny in the brain. The bar sets (4 bars / set) are, from left to right: IV only (NGFR), LIN IV LIN ICV (DNGFR), LIN IV LIN ICV (GFP), KLS IV LIN ICV (DNGFR), KLS IV LIN ICV (GFP), BM IV LIN ICV (DNGFR), and BM IV LIN ICV (GFP). One-way Anova with Bonferroni post-hoc test, *=p<0.05, **=p<0.01, ***=p<0.001, ****=p<0.0001. [Figure 3GH]Figures 3A-3J demonstrate that optimization of combinatorial transplantation protocols may allow for modulation of the contribution of IV- versus ICV-transplanted cells to donor bone marrow-brain chimerism after transplantation for appropriate clinical applications. Figure 3A is a graph showing brain engraftment of donor (GFP+) Lin- HSPCs IV transplanted on day 0 (24 hours after the last dose of brusulfan) or 5 days later (day 5) within total bone marrow CD45+CD11b+ cells, microglia (μ), transiently expanded μ (TAμ), and CNS macrophages. This graph demonstrates that transplantation of HSPCs at the two time points results in similar brain engraftment in the study population. Figure 3B illustrates the gating strategy for identifying μ, TAμ, and CNS macrophages (CNSmac) as CD45+lowCD11bhigh, CD45+low, CD11b+low, and CD45highCD11bhigh in 3-day-old (pnd) newborn mice, 21- and 60-pnd adult mice, and adult HSPC-transplanted animals at 2 months posttransplant. Figure 3C illustrates the experimental scheme demonstrating the IV and / or ICV transplantation strategy of differentially labeled (LV encoding GFP or ΔNGFR) hematopoietic cells in busulfan-pretreated recipients. The transplanted hematopoietic cells were Lin- HSPCs, c-kit+Sca1+Lin- (KSL) cells, or whole bone marrow (BM). Figure 3D is a graph illustrating the abundance of donor-derived cells (CD45.1 = progeny of IV-transplanted cells, GFP-expressing cells = progeny of ICV-transplanted cells) in the BM of transplanted mice at sacrifice 3 months posttransplant. GFP+ ICV-transplanted cells do not show robust engraftment in the BM. Figure 3E is a graph illustrating the abundance of donor-derived cells (as the sum of ΔNGFR and GFP-expressing cells) within the CD45+CD11b+ cells, μ, TAμ, and CNSmac of transplanted mice, as indicated by color coding, on day 0. Co-delivery of hematopoietic cells by ICV and IV improves brain donor chimerism after transplantation in all combinations tested compared to IV Lin-transplantation alone.From the leftmost bar in each set: IV only (white), LIN IV LIN ICV (dark gray), KLS IV LIN ICV (medium gray), and BM IV LIN ICV (gray). Figure 3F is a graph illustrating the differential abundance of donor-derived ΔNGFR (progeny of IV-transplanted cells) and GFP (progeny of ICV-transplanted cells) within CD45+CD11b+ cells, μ, TAμ, and CNSmac of the indicated transplanted mice on day 0. Lin- ICV delivery leading to IV transplantation of whole BM results in the lowest engraftment of IV-transplanted cells / their progeny in the brain. The sets of bars (four bars per set) are, from left to right: IV only (NGFR), LIN IV LIN ICV (DNGFR), LIN IV LIN ICV (GFP), KLS IV LIN ICV (DNGFR), KLS IV LIN ICV (GFP), BM IV LIN ICV (DNGFR), and BM IV LIN ICV (GFP). Figure 3G illustrates the experimental scheme showing the IV and / or ICV transplantation strategy of differentially labeled (LV encoding GFP or ΔNGFR) hematopoietic cells into busulfan-pretreated recipients. In this case, IV cells were transplanted 5 days after chemotherapy, while ICV cells were transplanted on day 0. The transplanted hematopoietic cells were Lin-HSPCs, KSL cells, or whole BM. Figure 3H is a graph depicting the abundance of donor-derived cells (CD45.1 = progeny of IV-transplanted cells, GFP-expressing cells = progeny of ICV-transplanted cells) among total BM cells recovered from transplanted mice at sacrifice 3 months after transplantation. GFP+ ICV-transplanted cells do not engraft in the BM. Figure 3I is a graph depicting the abundance of donor-derived cells (as the sum of ΔNGFR and GFP-expressing cells) among CD45+CD11b+ cells, μ, TAμ, and CNSmac of transplanted mice, as indicated by color coding, on day 5. Co-delivery of hematopoietic cells by ICV and IV improves brain-donor chimerism after transplantation in all combinations tested compared with IV Lin-transplantation alone. The leftmost bar in each set is IV alone (white), LIN IV LIN ICV (dark gray), KLS IV LIN ICV (medium gray), and BM IV LIN ICV (gray).Figure 3J is a graph illustrating the differential abundance of donor-derived ΔNGFR (progeny of IV-transplanted cells) and GFP (progeny of ICV-transplanted cells) expressing cells within the CD45+CD11b+ cells, μ, TAμ, and CNSmac of the indicated transplanted mice at day 5. Lin- ICV delivery leading to IV transplantation of whole BM results in the lowest engraftment of IV-transplanted cells / their progeny in the brain. The bar sets (4 bars / set) are, from left to right: IV only (NGFR), LIN IV LIN ICV (DNGFR), LIN IV LIN ICV (GFP), KLS IV LIN ICV (DNGFR), KLS IV LIN ICV (GFP), BM IV LIN ICV (DNGFR), and BM IV LIN ICV (GFP). One-way Anova with Bonferroni post-hoc test, *=p<0.05, **=p<0.01, ***=p<0.001, ****=p<0.0001. [Figure 3I]Figures 3A-3J demonstrate that optimization of combinatorial transplantation protocols may allow for modulation of the contribution of IV- versus ICV-transplanted cells to donor bone marrow-brain chimerism after transplantation for appropriate clinical applications. Figure 3A is a graph showing brain engraftment of donor (GFP+) Lin- HSPCs IV transplanted on day 0 (24 hours after the last dose of brusulfan) or 5 days later (day 5) within total bone marrow CD45+CD11b+ cells, microglia (μ), transiently expanded μ (TAμ), and CNS macrophages. This graph demonstrates that transplantation of HSPCs at the two time points results in similar brain engraftment in the study population. Figure 3B illustrates the gating strategy for identifying μ, TAμ, and CNS macrophages (CNSmac) as CD45+lowCD11bhigh, CD45+low, CD11b+low, and CD45highCD11bhigh in 3-day-old (pnd) newborn mice, 21- and 60-pnd adult mice, and adult HSPC-transplanted animals at 2 months posttransplant. Figure 3C illustrates the experimental scheme demonstrating the IV and / or ICV transplantation strategy of differentially labeled (LV encoding GFP or ΔNGFR) hematopoietic cells in busulfan-pretreated recipients. The transplanted hematopoietic cells were Lin- HSPCs, c-kit+Sca1+Lin- (KSL) cells, or whole bone marrow (BM). Figure 3D is a graph illustrating the abundance of donor-derived cells (CD45.1 = progeny of IV-transplanted cells, GFP-expressing cells = progeny of ICV-transplanted cells) in the BM of transplanted mice at sacrifice 3 months posttransplant. GFP+ ICV-transplanted cells do not show robust engraftment in the BM. Figure 3E is a graph illustrating the abundance of donor-derived cells (as the sum of ΔNGFR and GFP-expressing cells) within the CD45+CD11b+ cells, μ, TAμ, and CNSmac of transplanted mice, as indicated by color coding, on day 0. Co-delivery of hematopoietic cells by ICV and IV improves brain donor chimerism after transplantation in all combinations tested compared to IV Lin-transplantation alone.From the leftmost bar in each set: IV only (white), LIN IV LIN ICV (dark gray), KLS IV LIN ICV (medium gray), and BM IV LIN ICV (gray). Figure 3F is a graph illustrating the differential abundance of donor-derived ΔNGFR (progeny of IV-transplanted cells) and GFP (progeny of ICV-transplanted cells) within CD45+CD11b+ cells, μ, TAμ, and CNSmac of the indicated transplanted mice on day 0. Lin- ICV delivery leading to IV transplantation of whole BM results in the lowest engraftment of IV-transplanted cells / their progeny in the brain. The sets of bars (four bars per set) are, from left to right: IV only (NGFR), LIN IV LIN ICV (DNGFR), LIN IV LIN ICV (GFP), KLS IV LIN ICV (DNGFR), KLS IV LIN ICV (GFP), BM IV LIN ICV (DNGFR), and BM IV LIN ICV (GFP). Figure 3G illustrates the experimental scheme showing the IV and / or ICV transplantation strategy of differentially labeled (LV encoding GFP or ΔNGFR) hematopoietic cells into busulfan-pretreated recipients. In this case, IV cells were transplanted 5 days after chemotherapy, while ICV cells were transplanted on day 0. The transplanted hematopoietic cells were Lin-HSPCs, KSL cells, or whole BM. Figure 3H is a graph depicting the abundance of donor-derived cells (CD45.1 = progeny of IV-transplanted cells, GFP-expressing cells = progeny of ICV-transplanted cells) among total BM cells recovered from transplanted mice at sacrifice 3 months after transplantation. GFP+ ICV-transplanted cells do not engraft in the BM. Figure 3I is a graph depicting the abundance of donor-derived cells (as the sum of ΔNGFR and GFP-expressing cells) among CD45+CD11b+ cells, μ, TAμ, and CNSmac of transplanted mice, as indicated by color coding, on day 5. Co-delivery of hematopoietic cells by ICV and IV improves brain-donor chimerism after transplantation in all combinations tested compared with IV Lin-transplantation alone. The leftmost bar in each set is IV alone (white), LIN IV LIN ICV (dark gray), KLS IV LIN ICV (medium gray), and BM IV LIN ICV (gray).Figure 3J is a graph illustrating the differential abundance of donor-derived ΔNGFR (progeny of IV-transplanted cells) and GFP (progeny of ICV-transplanted cells) expressing cells within the CD45+CD11b+ cells, μ, TAμ, and CNSmac of the indicated transplanted mice at day 5. Lin- ICV delivery leading to IV transplantation of whole BM results in the lowest engraftment of IV-transplanted cells / their progeny in the brain. The bar sets (4 bars / set) are, from left to right: IV only (NGFR), LIN IV LIN ICV (DNGFR), LIN IV LIN ICV (GFP), KLS IV LIN ICV (DNGFR), KLS IV LIN ICV (GFP), BM IV LIN ICV (DNGFR), and BM IV LIN ICV (GFP). One-way Anova with Bonferroni post-hoc test, *=p<0.05, **=p<0.01, ***=p<0.001, ****=p<0.0001. [Figure 3J]Figures 3A-3J demonstrate that optimization of combinatorial transplantation protocols may allow for modulation of the contribution of IV- versus ICV-transplanted cells to donor bone marrow-brain chimerism after transplantation for appropriate clinical applications. Figure 3A is a graph showing brain engraftment of donor (GFP+) Lin- HSPCs IV transplanted on day 0 (24 hours after the last dose of brusulfan) or 5 days later (day 5) within total bone marrow CD45+CD11b+ cells, microglia (μ), transiently expanded μ (TAμ), and CNS macrophages. This graph demonstrates that transplantation of HSPCs at the two time points results in similar brain engraftment in the study population. Figure 3B illustrates the gating strategy for identifying μ, TAμ, and CNS macrophages (CNSmac) as CD45+lowCD11bhigh, CD45+low, CD11b+low, and CD45highCD11bhigh in 3-day-old (pnd) newborn mice, 21- and 60-pnd adult mice, and adult HSPC-transplanted animals at 2 months posttransplant. Figure 3C illustrates the experimental scheme demonstrating the IV and / or ICV transplantation strategy of differentially labeled (LV encoding GFP or ΔNGFR) hematopoietic cells in busulfan-pretreated recipients. The transplanted hematopoietic cells were Lin- HSPCs, c-kit+Sca1+Lin- (KSL) cells, or whole bone marrow (BM). Figure 3D is a graph illustrating the abundance of donor-derived cells (CD45.1 = progeny of IV-transplanted cells, GFP-expressing cells = progeny of ICV-transplanted cells) in the BM of transplanted mice at sacrifice 3 months posttransplant. GFP+ ICV-transplanted cells do not show robust engraftment in the BM. Figure 3E is a graph illustrating the abundance of donor-derived cells (as the sum of ΔNGFR and GFP-expressing cells) within the CD45+CD11b+ cells, μ, TAμ, and CNSmac of transplanted mice, as indicated by color coding, on day 0. Co-delivery of hematopoietic cells by ICV and IV improves brain donor chimerism after transplantation in all combinations tested compared to IV Lin-transplantation alone.From the leftmost bar in each set: IV only (white), LIN IV LIN ICV (dark gray), KLS IV LIN ICV (medium gray), and BM IV LIN ICV (gray). Figure 3F is a graph illustrating the differential abundance of donor-derived ΔNGFR (progeny of IV-transplanted cells) and GFP (progeny of ICV-transplanted cells) within CD45+CD11b+ cells, μ, TAμ, and CNSmac of the indicated transplanted mice on day 0. Lin- ICV delivery leading to IV transplantation of whole BM results in the lowest engraftment of IV-transplanted cells / their progeny in the brain. The sets of bars (four bars per set) are, from left to right: IV only (NGFR), LIN IV LIN ICV (DNGFR), LIN IV LIN ICV (GFP), KLS IV LIN ICV (DNGFR), KLS IV LIN ICV (GFP), BM IV LIN ICV (DNGFR), and BM IV LIN ICV (GFP). Figure 3G illustrates the experimental scheme showing the IV and / or ICV transplantation strategy of differentially labeled (LV encoding GFP or ΔNGFR) hematopoietic cells into busulfan-pretreated recipients. In this case, IV cells were transplanted 5 days after chemotherapy, while ICV cells were transplanted on day 0. The transplanted hematopoietic cells were Lin-HSPCs, KSL cells, or whole BM. Figure 3H is a graph depicting the abundance of donor-derived cells (CD45.1 = progeny of IV-transplanted cells, GFP-expressing cells = progeny of ICV-transplanted cells) among total BM cells recovered from transplanted mice at sacrifice 3 months after transplantation. GFP+ ICV-transplanted cells do not engraft in the BM. Figure 3I is a graph depicting the abundance of donor-derived cells (as the sum of ΔNGFR and GFP-expressing cells) among CD45+CD11b+ cells, μ, TAμ, and CNSmac of transplanted mice, as indicated by color coding, on day 5. Co-delivery of hematopoietic cells by ICV and IV improves brain-donor chimerism after transplantation in all combinations tested compared with IV Lin-transplantation alone. The leftmost bar in each set is IV alone (white), LIN IV LIN ICV (dark gray), KLS IV LIN ICV (medium gray), and BM IV LIN ICV (gray).Figure 3J is a graph illustrating the differential abundance of donor-derived ΔNGFR (progeny of IV-transplanted cells) and GFP (progeny of ICV-transplanted cells) expressing cells within the CD45+CD11b+ cells, μ, TAμ, and CNSmac of the indicated transplanted mice at day 5. Lin- ICV delivery leading to IV transplantation of whole BM results in the lowest engraftment of IV-transplanted cells / their progeny in the brain. The bar sets (4 bars / set) are, from left to right: IV only (NGFR), LIN IV LIN ICV (DNGFR), LIN IV LIN ICV (GFP), KLS IV LIN ICV (DNGFR), KLS IV LIN ICV (GFP), BM IV LIN ICV (DNGFR), and BM IV LIN ICV (GFP). One-way Anova with Bonferroni post-hoc test, *=p<0.05, **=p<0.01, ***=p<0.001, ****=p<0.0001.

[0074] [Figure 4A]Figures 4A-4E demonstrate the presence of a microglial signature in bone marrow cells recovered from the brains of transplanted mice. Figure 4A is a graph illustrating the abundance of μ and TAμ cells in the brains of mice 90 days after transplantation of GFP+ HSPCs via IV or ICV (n = 5 per group). Figure 4B shows representative dot plots showing sorted cell populations for gene expression analysis. Specifically, μ and TAμ, identified by CD45 and CD11b markers, are shown in the brains of naive P10 and adult control (ADULT_CT) animals, busulfan-treated mice, and transplanted mice (BU_TX) at 90 days after HCT. The dot plots within the dotted squares demonstrate both GFP- endogenous cells and GFP+ donor-derived cells within the μ and TAμ populations of a representative transplanted BU-treated mouse. Figure 4C is a graph depicting the fold change (calculated as 2-DDCT) in expression of select microglial genes obtained by real-time PCR in each indicated population recovered from the brains of busulfan-treated IV- and ICV-implanted mice or P10 mice, calculated based on the expression of the same genes in ADULT_CT μ cells. Mean values ​​are shown. Figure 4D is a principal component analysis (PCA), and Figure 4E is a heatmap, both showing expression analysis of genes within samples identified as microglial signatures by Butovsky (Butovsky et al., Nature neuroscience 17:131-143 (2014)) (μ and TAμ recovered from naive, P10, and ADULT_CT and HCT animals) and reported in Gosselin et al. (Gosselin et al., Cell 159:1327-1340 (2014)), including microglia and macrophages (LPM = large peritoneal macrophages; SPM = small peritoneal macrophages; BMDM = bone marrow-derived macrophages; TGEM = thioglycollate-elicited peritoneal macrophages).Overall, these data indicate that cells isolated from the brains of ICV- and IV-implanted mice show expression of these genes at levels similar to those of μ-cells isolated from control mice, rather than macrophages. [Figure 4B]Figures 4A-4E demonstrate the presence of a microglial signature in bone marrow cells recovered from the brains of transplanted mice. Figure 4A is a graph illustrating the abundance of μ and TAμ cells in the brains of mice 90 days after transplantation of GFP+ HSPCs via IV or ICV (n = 5 per group). Figure 4B shows representative dot plots showing sorted cell populations for gene expression analysis. Specifically, μ and TAμ, identified by CD45 and CD11b markers, are shown in the brains of naive P10 and adult control (ADULT_CT) animals, busulfan-treated mice, and transplanted mice (BU_TX) at 90 days after HCT. The dot plots within the dotted squares demonstrate both GFP- endogenous cells and GFP+ donor-derived cells within the μ and TAμ populations of a representative transplanted BU-treated mouse. Figure 4C is a graph depicting the fold change (calculated as 2-DDCT) in expression of select microglial genes obtained by real-time PCR in each indicated population recovered from the brains of busulfan-treated IV- and ICV-implanted mice or P10 mice, calculated based on the expression of the same genes in ADULT_CT μ cells. Mean values ​​are shown. Figure 4D is a principal component analysis (PCA), and Figure 4E is a heatmap, both showing expression analysis of genes within samples identified as microglial signatures by Butovsky (Butovsky et al., Nature neuroscience 17:131-143 (2014)) (μ and TAμ recovered from naive, P10, and ADULT_CT and HCT animals) and reported in Gosselin et al. (Gosselin et al., Cell 159:1327-1340 (2014)), including microglia and macrophages (LPM = large peritoneal macrophages; SPM = small peritoneal macrophages; BMDM = bone marrow-derived macrophages; TGEM = thioglycollate-elicited peritoneal macrophages).Overall, these data indicate that cells isolated from the brains of ICV- and IV-implanted mice show expression of these genes at levels similar to those of μ-cells isolated from control mice, rather than macrophages. [Figure 4C]Figures 4A-4E demonstrate the presence of a microglial signature in bone marrow cells recovered from the brains of transplanted mice. Figure 4A is a graph illustrating the abundance of μ and TAμ cells in the brains of mice 90 days after transplantation of GFP+ HSPCs via IV or ICV (n = 5 per group). Figure 4B shows representative dot plots showing sorted cell populations for gene expression analysis. Specifically, μ and TAμ, identified by CD45 and CD11b markers, are shown in the brains of naive P10 and adult control (ADULT_CT) animals, busulfan-treated mice, and transplanted mice (BU_TX) at 90 days after HCT. The dot plots within the dotted squares demonstrate both GFP- endogenous cells and GFP+ donor-derived cells within the μ and TAμ populations of a representative transplanted BU-treated mouse. Figure 4C is a graph depicting the fold change (calculated as 2-DDCT) in expression of select microglial genes obtained by real-time PCR in each indicated population recovered from the brains of busulfan-treated IV- and ICV-implanted mice or P10 mice, calculated based on the expression of the same genes in ADULT_CT μ cells. Mean values ​​are shown. Figure 4D is a principal component analysis (PCA), and Figure 4E is a heatmap, both showing expression analysis of genes within samples identified as microglial signatures by Butovsky (Butovsky et al., Nature neuroscience 17:131-143 (2014)) (μ and TAμ recovered from naive, P10, and ADULT_CT and HCT animals) and reported in Gosselin et al. (Gosselin et al., Cell 159:1327-1340 (2014)), including microglia and macrophages (LPM = large peritoneal macrophages; SPM = small peritoneal macrophages; BMDM = bone marrow-derived macrophages; TGEM = thioglycollate-elicited peritoneal macrophages).Overall, these data indicate that cells isolated from the brains of ICV- and IV-implanted mice show expression of these genes at levels similar to those of μ-cells isolated from control mice, rather than macrophages. [Figure 4D]Figures 4A-4E demonstrate the presence of a microglial signature in bone marrow cells recovered from the brains of transplanted mice. Figure 4A is a graph illustrating the abundance of μ and TAμ cells in the brains of mice 90 days after transplantation of GFP+ HSPCs via IV or ICV (n = 5 per group). Figure 4B shows representative dot plots showing sorted cell populations for gene expression analysis. Specifically, μ and TAμ, identified by CD45 and CD11b markers, are shown in the brains of naive P10 and adult control (ADULT_CT) animals, busulfan-treated mice, and transplanted mice (BU_TX) at 90 days after HCT. The dot plots within the dotted squares demonstrate both GFP- endogenous cells and GFP+ donor-derived cells within the μ and TAμ populations of a representative transplanted BU-treated mouse. Figure 4C is a graph depicting the fold change (calculated as 2-DDCT) in expression of select microglial genes obtained by real-time PCR in each indicated population recovered from the brains of busulfan-treated IV- and ICV-implanted mice or P10 mice, calculated based on the expression of the same genes in ADULT_CT μ cells. Mean values ​​are shown. Figure 4D is a principal component analysis (PCA), and Figure 4E is a heatmap, both showing expression analysis of genes within samples identified as microglial signatures by Butovsky (Butovsky et al., Nature neuroscience 17:131-143 (2014)) (μ and TAμ recovered from naive, P10, and ADULT_CT and HCT animals) and reported in Gosselin et al. (Gosselin et al., Cell 159:1327-1340 (2014)), including microglia and macrophages (LPM = large peritoneal macrophages; SPM = small peritoneal macrophages; BMDM = bone marrow-derived macrophages; TGEM = thioglycollate-elicited peritoneal macrophages).Overall, these data indicate that cells isolated from the brains of ICV- and IV-implanted mice show expression of these genes at levels similar to those of μ-cells isolated from control mice, rather than macrophages. [Figure 4E]Figures 4A-4E demonstrate the presence of a microglial signature in bone marrow cells recovered from the brains of transplanted mice. Figure 4A is a graph illustrating the abundance of μ and TAμ cells in the brains of mice 90 days after transplantation of GFP+ HSPCs via IV or ICV (n = 5 per group). Figure 4B shows representative dot plots showing sorted cell populations for gene expression analysis. Specifically, μ and TAμ, identified by CD45 and CD11b markers, are shown in the brains of naive P10 and adult control (ADULT_CT) animals, busulfan-treated mice, and transplanted mice (BU_TX) at 90 days after HCT. The dot plots within the dotted squares demonstrate both GFP- endogenous cells and GFP+ donor-derived cells within the μ and TAμ populations of a representative transplanted BU-treated mouse. Figure 4C is a graph depicting the fold change (calculated as 2-DDCT) in expression of select microglial genes obtained by real-time PCR in each indicated population recovered from the brains of busulfan-treated IV- and ICV-implanted mice or P10 mice, calculated based on the expression of the same genes in ADULT_CT μ cells. Mean values ​​are shown. Figure 4D is a principal component analysis (PCA), and Figure 4E is a heatmap, both showing expression analysis of genes within samples identified as microglial signatures by Butovsky (Butovsky et al., Nature neuroscience 17:131-143 (2014)) (μ and TAμ recovered from naive, P10, and ADULT_CT and HCT animals) and reported in Gosselin et al. (Gosselin et al., Cell 159:1327-1340 (2014)), including microglia and macrophages (LPM = large peritoneal macrophages; SPM = small peritoneal macrophages; BMDM = bone marrow-derived macrophages; TGEM = thioglycollate-elicited peritoneal macrophages).Overall, these data indicate that cells isolated from the brains of ICV- and IV-implanted mice show expression of these genes at levels similar to those of μ-cells isolated from control mice, rather than macrophages.

[0075] [Figure 5A-1]Figures 5A-5E show that bone marrow cells derived from the brains of transplanted mice exhibit the features of microglial maturation. Figure 5A shows the functional enrichment of differentially upregulated genes in μCT cells versus μ-transplanted cells. Figure 5B shows the functional enrichment of differentially downregulated genes in μCT cells versus μ-transplanted cells. Figure 5C shows the functional enrichment of differentially upregulated genes in μCT cells versus TAμ-transplanted cells. Figure 5D shows the functional enrichment of differentially downregulated genes in μCT cells versus TAμ-transplanted cells. A preliminary ranking analysis by gene set enrichment analysis (GSEA) was performed using RNA-Seq differential gene expression data (http: / / software.broadinstitute.org / gsea / msigdb / collection) against Gene Ontology (GO) biological processes using default parameters. Semantic similarity of GO (GOSemSim) was used to cluster significantly enriched GOs (GOs with FDR<0.05 for upregulation and FDR<0.001 for downregulation were selected to enhance the clarity of the representation). Figure 5E shows the fold change in expression values ​​of genes normalized by RNA-Seq, where the expression of genes is upregulated in adult mice (Matcovitch-Natan et al., Science. 353:6301 (2016)) relative to ADULT_CT μ-cells in the representation populations recovered from busulfan-treated transplanted mice or P10 mouse brains. Figure 5F is a graph showing fold change in expression values ​​of genes normalized by RNA-Seq, where expression of genes is upregulated in p10 mice (Matcovitch-Natan et al., Science. 353:6301 (2016)) relative to ADULT_CT μ-cells in the indicated populations recovered from the brains of busulfan-treated transplanted or P10 mice. For statistical tests, see Table 2. [Figure 5A-2]Figures 5A-5E show that bone marrow cells derived from the brains of transplanted mice exhibit the features of microglial maturation. Figure 5A shows the functional enrichment of differentially upregulated genes in μCT cells versus μ-transplanted cells. Figure 5B shows the functional enrichment of differentially downregulated genes in μCT cells versus μ-transplanted cells. Figure 5C shows the functional enrichment of differentially upregulated genes in μCT cells versus TAμ-transplanted cells. Figure 5D shows the functional enrichment of differentially downregulated genes in μCT cells versus TAμ-transplanted cells. A preliminary ranking analysis by gene set enrichment analysis (GSEA) was performed using RNA-Seq differential gene expression data (http: / / software.broadinstitute.org / gsea / msigdb / collection) against Gene Ontology (GO) biological processes using default parameters. Semantic similarity of GO (GOSemSim) was used to cluster significantly enriched GOs (GOs with FDR<0.05 for upregulation and FDR<0.001 for downregulation were selected to enhance the clarity of the representation). Figure 5E shows the fold change in expression values ​​of genes normalized by RNA-Seq, where the expression of genes is upregulated in adult mice (Matcovitch-Natan et al., Science. 353:6301 (2016)) relative to ADULT_CT μ-cells in the representation populations recovered from busulfan-treated transplanted mice or P10 mouse brains. Figure 5F is a graph showing fold change in expression values ​​of genes normalized by RNA-Seq, where expression of genes is upregulated in p10 mice (Matcovitch-Natan et al., Science. 353:6301 (2016)) relative to ADULT_CT μ-cells in the indicated populations recovered from the brains of busulfan-treated transplanted or P10 mice. For statistical tests, see Table 2. [Figure 5A-3]Figures 5A-5E show that bone marrow cells derived from the brains of transplanted mice exhibit the features of microglial maturation. Figure 5A shows the functional enrichment of differentially upregulated genes in μCT cells versus μ-transplanted cells. Figure 5B shows the functional enrichment of differentially downregulated genes in μCT cells versus μ-transplanted cells. Figure 5C shows the functional enrichment of differentially upregulated genes in μCT cells versus TAμ-transplanted cells. Figure 5D shows the functional enrichment of differentially downregulated genes in μCT cells versus TAμ-transplanted cells. A preliminary ranking analysis by gene set enrichment analysis (GSEA) was performed using RNA-Seq differential gene expression data (http: / / software.broadinstitute.org / gsea / msigdb / collection) against Gene Ontology (GO) biological processes using default parameters. Semantic similarity of GO (GOSemSim) was used to cluster significantly enriched GOs (GOs with FDR<0.05 for upregulation and FDR<0.001 for downregulation were selected to enhance the clarity of the representation). Figure 5E shows the fold change in expression values ​​of genes normalized by RNA-Seq, where the expression of genes is upregulated in adult mice (Matcovitch-Natan et al., Science. 353:6301 (2016)) relative to ADULT_CT μ-cells in the representation populations recovered from busulfan-treated transplanted mice or P10 mouse brains. Figure 5F is a graph showing fold change in expression values ​​of genes normalized by RNA-Seq, where expression of genes is upregulated in p10 mice (Matcovitch-Natan et al., Science. 353:6301 (2016)) relative to ADULT_CT μ-cells in the indicated populations recovered from the brains of busulfan-treated transplanted or P10 mice. For statistical tests, see Table 2. [Figure 5B]Figures 5A-5E show that bone marrow cells derived from the brains of transplanted mice exhibit the features of microglial maturation. Figure 5A shows the functional enrichment of differentially upregulated genes in μCT cells versus μ-transplanted cells. Figure 5B shows the functional enrichment of differentially downregulated genes in μCT cells versus μ-transplanted cells. Figure 5C shows the functional enrichment of differentially upregulated genes in μCT cells versus TAμ-transplanted cells. Figure 5D shows the functional enrichment of differentially downregulated genes in μCT cells versus TAμ-transplanted cells. A preliminary ranking analysis by gene set enrichment analysis (GSEA) was performed using RNA-Seq differential gene expression data (http: / / software.broadinstitute.org / gsea / msigdb / collection) against Gene Ontology (GO) biological processes using default parameters. Semantic similarity of GO (GOSemSim) was used to cluster significantly enriched GOs (GOs with FDR<0.05 for upregulation and FDR<0.001 for downregulation were selected to enhance the clarity of the representation). Figure 5E shows the fold change in expression values ​​of genes normalized by RNA-Seq, where the expression of genes is upregulated in adult mice (Matcovitch-Natan et al., Science. 353:6301 (2016)) relative to ADULT_CT μ-cells in the representation populations recovered from busulfan-treated transplanted mice or P10 mouse brains. Figure 5F is a graph showing fold change in expression values ​​of genes normalized by RNA-Seq, where expression of genes is upregulated in p10 mice (Matcovitch-Natan et al., Science. 353:6301 (2016)) relative to ADULT_CT μ-cells in the indicated populations recovered from the brains of busulfan-treated transplanted or P10 mice. For statistical tests, see Table 2. [Figure 5C-1]Figures 5A-5E show that bone marrow cells derived from the brains of transplanted mice exhibit the features of microglial maturation. Figure 5A shows the functional enrichment of differentially upregulated genes in μCT cells versus μ-transplanted cells. Figure 5B shows the functional enrichment of differentially downregulated genes in μCT cells versus μ-transplanted cells. Figure 5C shows the functional enrichment of differentially upregulated genes in μCT cells versus TAμ-transplanted cells. Figure 5D shows the functional enrichment of differentially downregulated genes in μCT cells versus TAμ-transplanted cells. A preliminary ranking analysis by gene set enrichment analysis (GSEA) was performed using RNA-Seq differential gene expression data (http: / / software.broadinstitute.org / gsea / msigdb / collection) against Gene Ontology (GO) biological processes using default parameters. Semantic similarity of GO (GOSemSim) was used to cluster significantly enriched GOs (GOs with FDR<0.05 for upregulation and FDR<0.001 for downregulation were selected to enhance the clarity of the representation). Figure 5E shows the fold change in expression values ​​of genes normalized by RNA-Seq, where the expression of genes is upregulated in adult mice (Matcovitch-Natan et al., Science. 353:6301 (2016)) relative to ADULT_CT μ-cells in the representation populations recovered from busulfan-treated transplanted mice or P10 mouse brains. Figure 5F is a graph showing fold change in expression values ​​of genes normalized by RNA-Seq, where expression of genes is upregulated in p10 mice (Matcovitch-Natan et al., Science. 353:6301 (2016)) relative to ADULT_CT μ-cells in the indicated populations recovered from the brains of busulfan-treated transplanted or P10 mice. For statistical tests, see Table 2. [Figure 5C-2]Figures 5A-5E show that bone marrow cells derived from the brains of transplanted mice exhibit the features of microglial maturation. Figure 5A shows the functional enrichment of differentially upregulated genes in μCT cells versus μ-transplanted cells. Figure 5B shows the functional enrichment of differentially downregulated genes in μCT cells versus μ-transplanted cells. Figure 5C shows the functional enrichment of differentially upregulated genes in μCT cells versus TAμ-transplanted cells. Figure 5D shows the functional enrichment of differentially downregulated genes in μCT cells versus TAμ-transplanted cells. A preliminary ranking analysis by gene set enrichment analysis (GSEA) was performed using RNA-Seq differential gene expression data (http: / / software.broadinstitute.org / gsea / msigdb / collection) against Gene Ontology (GO) biological processes using default parameters. Semantic similarity of GO (GOSemSim) was used to cluster significantly enriched GOs (GOs with FDR<0.05 for upregulation and FDR<0.001 for downregulation were selected to enhance the clarity of the representation). Figure 5E shows the fold change in expression values ​​of genes normalized by RNA-Seq, where the expression of genes is upregulated in adult mice (Matcovitch-Natan et al., Science. 353:6301 (2016)) relative to ADULT_CT μ-cells in the representation populations recovered from busulfan-treated transplanted mice or P10 mouse brains. Figure 5F is a graph showing fold change in expression values ​​of genes normalized by RNA-Seq, where expression of genes is upregulated in p10 mice (Matcovitch-Natan et al., Science. 353:6301 (2016)) relative to ADULT_CT μ-cells in the indicated populations recovered from the brains of busulfan-treated transplanted or P10 mice. For statistical tests, see Table 2. [Figure 5C-3]Figures 5A-5E show that bone marrow cells derived from the brains of transplanted mice exhibit the features of microglial maturation. Figure 5A shows the functional enrichment of differentially upregulated genes in μCT cells versus μ-transplanted cells. Figure 5B shows the functional enrichment of differentially downregulated genes in μCT cells versus μ-transplanted cells. Figure 5C shows the functional enrichment of differentially upregulated genes in μCT cells versus TAμ-transplanted cells. Figure 5D shows the functional enrichment of differentially downregulated genes in μCT cells versus TAμ-transplanted cells. A preliminary ranking analysis by gene set enrichment analysis (GSEA) was performed using RNA-Seq differential gene expression data (http: / / software.broadinstitute.org / gsea / msigdb / collection) against Gene Ontology (GO) biological processes using default parameters. Semantic similarity of GO (GOSemSim) was used to cluster significantly enriched GOs (GOs with FDR<0.05 for upregulation and FDR<0.001 for downregulation were selected to enhance the clarity of the representation). Figure 5E shows the fold change in expression values ​​of genes normalized by RNA-Seq, where the expression of genes is upregulated in adult mice (Matcovitch-Natan et al., Science. 353:6301 (2016)) relative to ADULT_CT μ-cells in the representation populations recovered from busulfan-treated transplanted mice or P10 mouse brains. Figure 5F is a graph showing fold change in expression values ​​of genes normalized by RNA-Seq, where expression of genes is upregulated in p10 mice (Matcovitch-Natan et al., Science. 353:6301 (2016)) relative to ADULT_CT μ-cells in the indicated populations recovered from the brains of busulfan-treated transplanted or P10 mice. For statistical tests, see Table 2. [Figure 5D-1]Figures 5A-5E show that bone marrow cells derived from the brains of transplanted mice exhibit the features of microglial maturation. Figure 5A shows the functional enrichment of differentially upregulated genes in μCT cells versus μ-transplanted cells. Figure 5B shows the functional enrichment of differentially downregulated genes in μCT cells versus μ-transplanted cells. Figure 5C shows the functional enrichment of differentially upregulated genes in μCT cells versus TAμ-transplanted cells. Figure 5D shows the functional enrichment of differentially downregulated genes in μCT cells versus TAμ-transplanted cells. A preliminary ranking analysis by gene set enrichment analysis (GSEA) was performed using RNA-Seq differential gene expression data (http: / / software.broadinstitute.org / gsea / msigdb / collection) against Gene Ontology (GO) biological processes using default parameters. Semantic similarity of GO (GOSemSim) was used to cluster significantly enriched GOs (GOs with FDR<0.05 for upregulation and FDR<0.001 for downregulation were selected to enhance the clarity of the representation). Figure 5E shows the fold change in expression values ​​of genes normalized by RNA-Seq, where the expression of genes is upregulated in adult mice (Matcovitch-Natan et al., Science. 353:6301 (2016)) relative to ADULT_CT μ-cells in the representation populations recovered from busulfan-treated transplanted mice or P10 mouse brains. Figure 5F is a graph showing fold change in expression values ​​of genes normalized by RNA-Seq, where expression of genes is upregulated in p10 mice (Matcovitch-Natan et al., Science. 353:6301 (2016)) relative to ADULT_CT μ-cells in the indicated populations recovered from the brains of busulfan-treated transplanted or P10 mice. For statistical tests, see Table 2. [Figure 5D-2]Figures 5A-5E show that bone marrow cells derived from the brains of transplanted mice exhibit the features of microglial maturation. Figure 5A shows the functional enrichment of differentially upregulated genes in μCT cells versus μ-transplanted cells. Figure 5B shows the functional enrichment of differentially downregulated genes in μCT cells versus μ-transplanted cells. Figure 5C shows the functional enrichment of differentially upregulated genes in μCT cells versus TAμ-transplanted cells. Figure 5D shows the functional enrichment of differentially downregulated genes in μCT cells versus TAμ-transplanted cells. A preliminary ranking analysis by gene set enrichment analysis (GSEA) was performed using RNA-Seq differential gene expression data (http: / / software.broadinstitute.org / gsea / msigdb / collection) against Gene Ontology (GO) biological processes using default parameters. Semantic similarity of GO (GOSemSim) was used to cluster significantly enriched GOs (GOs with FDR<0.05 for upregulation and FDR<0.001 for downregulation were selected to enhance the clarity of the representation). Figure 5E shows the fold change in expression values ​​of genes normalized by RNA-Seq, where the expression of genes is upregulated in adult mice (Matcovitch-Natan et al., Science. 353:6301 (2016)) relative to ADULT_CT μ-cells in the representation populations recovered from busulfan-treated transplanted mice or P10 mouse brains. Figure 5F is a graph showing fold change in expression values ​​of genes normalized by RNA-Seq, where expression of genes is upregulated in p10 mice (Matcovitch-Natan et al., Science. 353:6301 (2016)) relative to ADULT_CT μ-cells in the indicated populations recovered from the brains of busulfan-treated transplanted or P10 mice. For statistical tests, see Table 2. [Figure 5D-3]Figures 5A-5E show that bone marrow cells derived from the brains of transplanted mice exhibit the features of microglial maturation. Figure 5A shows the functional enrichment of differentially upregulated genes in μCT cells versus μ-transplanted cells. Figure 5B shows the functional enrichment of differentially downregulated genes in μCT cells versus μ-transplanted cells. Figure 5C shows the functional enrichment of differentially upregulated genes in μCT cells versus TAμ-transplanted cells. Figure 5D shows the functional enrichment of differentially downregulated genes in μCT cells versus TAμ-transplanted cells. A preliminary ranking analysis by gene set enrichment analysis (GSEA) was performed using RNA-Seq differential gene expression data (http: / / software.broadinstitute.org / gsea / msigdb / collection) against Gene Ontology (GO) biological processes using default parameters. Semantic similarity of GO (GOSemSim) was used to cluster significantly enriched GOs (GOs with FDR<0.05 for upregulation and FDR<0.001 for downregulation were selected to enhance the clarity of the representation). Figure 5E shows the fold change in expression values ​​of genes normalized by RNA-Seq, where the expression of genes is upregulated in adult mice (Matcovitch-Natan et al., Science. 353:6301 (2016)) relative to ADULT_CT μ-cells in the representation populations recovered from busulfan-treated transplanted mice or P10 mouse brains. Figure 5F is a graph showing fold change in expression values ​​of genes normalized by RNA-Seq, where expression of genes is upregulated in p10 mice (Matcovitch-Natan et al., Science. 353:6301 (2016)) relative to ADULT_CT μ-cells in the indicated populations recovered from the brains of busulfan-treated transplanted or P10 mice. For statistical tests, see Table 2. [Figure 5EF]Figures 5A-5E show that bone marrow cells derived from the brains of transplanted mice exhibit the features of microglial maturation. Figure 5A shows the functional enrichment of differentially upregulated genes in μCT cells versus μ-transplanted cells. Figure 5B shows the functional enrichment of differentially downregulated genes in μCT cells versus μ-transplanted cells. Figure 5C shows the functional enrichment of differentially upregulated genes in μCT cells versus TAμ-transplanted cells. Figure 5D shows the functional enrichment of differentially downregulated genes in μCT cells versus TAμ-transplanted cells. A preliminary ranking analysis by gene set enrichment analysis (GSEA) was performed using RNA-Seq differential gene expression data (http: / / software.broadinstitute.org / gsea / msigdb / collection) against Gene Ontology (GO) biological processes using default parameters. Semantic similarity of GO (GOSemSim) was used to cluster significantly enriched GOs (GOs with FDR<0.05 for upregulation and FDR<0.001 for downregulation were selected to enhance the clarity of the representation). Figure 5E shows the fold change in expression values ​​of genes normalized by RNA-Seq, where the expression of genes is upregulated in adult mice (Matcovitch-Natan et al., Science. 353:6301 (2016)) relative to ADULT_CT μ-cells in the representation populations recovered from busulfan-treated transplanted mice or P10 mouse brains. Figure 5F is a graph showing fold change in expression values ​​of genes normalized by RNA-Seq, where expression of genes is upregulated in p10 mice (Matcovitch-Natan et al., Science. 353:6301 (2016)) relative to ADULT_CT μ-cells in the indicated populations recovered from the brains of busulfan-treated transplanted or P10 mice. For statistical tests, see Table 2.

[0076] [Figure 6]Figures 6A-6D show that hematopoietic cells associated with the brain parenchyma of naive or post-transplant mice possess clonal and hematopoietic repopulation potential, as well as microglial reconstitution potential. Figure 6A illustrates the experimental scheme for HCT. CFUs were plated from the bone marrow (BM) and brain of naive (UT), BU-treated, and irradiated (IRR) mice, as well as from mice previously transplanted with CD45.2 GFP-transduced HSPCs (BU-HCT). Figure 6B is a graph illustrating the number of colonies (#CFCs) obtained from tissues of BU and IRR animals. Figure 6C is a graph illustrating the number of colonies (#CFCs) and GFP+ CFCs obtained from tissues of BU-HCT animals. Figure 6D is a graph illustrating the abundance of GFP+ cells (and, in the case of BM, lineage differentiation) recovered by FACS analysis in the BM and brain of secondary recipient mice receiving BM or brain cells or peripheral blood mononuclear cells from primary recipients. Mice were sacrificed 4 months after transplantation.

[0077] [Figure 7]Figures 7A-7E demonstrate the therapeutic relevance of ICV co-delivery of HSPCs in two LSD animal models. Figure 7A illustrates the experimental scheme of the transplantation protocol for human CB CD34+ HSPCs transplanted by either IV, IV + ICV, or ICV alone into sublethally irradiated (Sub-L-IRR) Rag2- / - γ-chain- / - As2- / - newborn mice, an immunodeficient animal model of metachromatic leukodystrophy (MLD). Prior to transduction, HSPCs were transduced with LV encoding arylsulfatase (ARSA) (Sessa et al., Lancet 2016). Figure 7B is a graph showing ARSA activity (expressed as fold over that measured in tissues from Rag- / -γ-chain- / - As2- / - mice) measured in the brain and bone marrow (BM) of Rag- / -γ-chain- / - As2+ / + wild-type mice transplanted with ARSA-transduced cells ICV, IV, or ICV+IV, as indicated. N=3 mice per group. Transplanted mice were sacrificed 5 weeks after transplantation. Overall, co-delivery of transduced HSPCs (IV+ICV) resulted in greater ARSA delivery to the brain compared with IV-only or ICV-only approaches. Figure 7C is a schematic of a transplantation experiment in mice deficient in iduronate sulfatase activity (IDS- / -, an animal model of mucopolysaccharidosis type II, or MPS II) of Lin-HSPCs derived from wild-type (IDS+ / +) donors. Wild-type cells were administered to 2-month-old IDS- / - mice after busulfan myeloablation via IV alone or IV + ICV. Transplanted mice were followed for 180 days by behavioral studies. Figures 7D-7E are graphs showing the performance of transplanted and control IDS- / - mice in the rotarod test. Figure 7D shows the latency of the animals to the rotarod. Figure 7E shows the difference in latency to the rotarod between day 4 (last trial) and day 1 (first trial). ICV + IV transplanted mice demonstrate superior rotarod performance compared to IV alone and control mice. Means and SEM are shown. N = 3-8 mice per cohort.

[0078] [Figure 8] Figures 8A-8F show HLA-minor antigen-mismatched HSPC IV+ICV transplantation in mice. Figure 8A illustrates the experimental scheme for performing HLA-minor antigen-mismatched HSPC IV+ICV transplantation in mice. Mice received 10 x 106 whole BM cells IV and 0.3 or 1 x 106 Lin- cells (the mouse equivalent of human CD34+ cells) ICV. Figure 8B shows Kaplan-Meier survival curves for transplanted animals. At the end of the experiment, the IV+ICV 3e5 and IV-only groups on day 0 showed 100% survival, in contrast to the IV+ICV 1e6 group on day 0. Figure 8C is a graph illustrating chimerism of donor CD45.2 cells in the peripheral blood (PB), BM, spleen (Spl), and thymus (Thy) of transplanted mice at the time of sacrifice. Figure 8D shows the abundance of GFP+ cells among donor CD45.2 cells in tissues from transplanted animals, indicating that ICV-transplanted GFP+ cells did not engraft into the hematopoietic tissues of transplanted mice. N=5 per group. Figure 8E is a graph illustrating the chimerism of donor CD45.2 cells in the brain bone marrow population (total CD45+CD11b+ cells) of transplanted mice at the time of sacrifice. Figure 8F shows the abundance of GFP+ cells among donor CD45.2 cells in brain bone marrow cells from transplanted animals, indicating that ICV-transplanted GFP+ cells contributed to increased chimerism in the donor brain. N=5 per group.

[0079] [Figure 9]Figures 9A-9F show MHC-mismatched HSPC IV+ICV transplantation in mice. In this setting, the additive effect of ICV delivery on brain donor chimerism is maintained. Figure 9A illustrates the experimental scheme for performing HLA minor antigen-mismatched HSPC IV+ICV transplantation in mice. Mice received 10 x 106 total BM cells IV and 0.3 x 106 Lin- cells (the mouse equivalent of human CD34+ cells) ICV. Figure 9B shows Kaplan-Meier survival curves for transplanted animals. At the end of the experiment, the IV+ICV 3e5 group on day 0 showed 100% survival, in contrast to the IV-only group. Figure 9C is a graph illustrating the chimerism of donor CD45.2 cells in the PB, BM, Spl, and Thy of transplanted mice at the time of sacrifice. Figure 9D shows the abundance of GFP+ cells among donor CD45.2 cells in tissues from transplanted animals, indicating that ICV-transplanted GFP+ cells did not engraft into the hematopoietic tissues of transplanted mice. N=5 per group. Figure 9E is a graph illustrating the chimerism of donor CD45.2 cells in the brain bone marrow population of transplanted mice at the time of sacrifice. Figure 9F shows the abundance of GFP+ cells among donor CD45.2 cells in brain bone marrow cells from transplanted animals, indicating that ICV-transplanted GFP+ cells contributed to increased chimerism in the donor brain. N=5 per group.

[0080] [Figure 10] Figures 10A-10B show intrathecal (IT) HSPC transplantation in mice. Figure 10A illustrates the experimental scheme for differentially labeled HSPC transplantation IT+IV compared with ICV+IV and IV only (control IV) in mice. Figure 10B shows a graph illustrating donor cell chimerism, generated by the sum of GFP+ and Cherry+ cell engraftment in the BM, brain, and spinal cord of transplanted mice at the time of sacrifice. In each column, GFP is at the bottom and Cherry is at the top. N=3-5 per group. IT HSPC delivery may constitute a valuable route for achieving robust hematopoietic and CNS chimerism.

[0081] [Figure 11A]Figures 11A-11J show post-transplant brain bone marrow cells derived from early hematopoietic stem / progenitor cells. Figure 11A illustrates the experimental scheme showing how long-term (LT)-HSCs and progenitors within a pool of HSPCs were pre-isolated using c-kit, Sca-1, and lineage-negative staining, as well as the SLAM receptor markers CD150 and CD48. The indicated subpopulations were then differentially transduced with lentiviral vectors (LV) encoding GFP (KSL) and ΔNGFR (NOT-KSL), and GFP (LT-HSC), ΔNGFR (MPP), tag-BFP (HPC-1), and CHERRY (HPC-2). Subsequently, they were competitively transplanted IV or ICV at their original ratios into busulfan-bone marrow-ablated mice. ICV-transplanted animals also received unmanipulated whole BM cells for hematopoietic rescue 5 days after transplantation. Figure 11B shows histograms of marker gene expression in the in vivo liquid progeny of the indicated LV-transduced and transplanted cells in the mice described in A. Figure 11C is a graph depicting the abundance of cells derived from each of the transplanted KSL subpopulations within total CD45+ hematopoietic BM cells, myeloid (CD11b), and lymphoid (CD3 and B220) lineages of busulfan-treated transplanted (BU_TX) mice at the time of sacrifice. N = 10 mice / group. In each column, from bottom to top, LT-HSC (light gray), MPP (dark gray), HPC2 (gray), and HPC1 (even lighter gray). Figure 11D is a graph depicting the abundance of cells derived from IV-transplanted KSL and NOT-KSL within total bone marrow (CD45+CD11b+) cells, μ, and TAμ in the brains of BU_TX mice at 90 days post-transplant. In each column, from bottom to top, KSL (gray) and not-KSL (white). Figure 11E is a graph showing the abundance of ICV-transplanted KSL- and NOT-KSL-derived cells in total bone marrow (CD45+CD11b+) cells, μ and TAμ of the brain of BU_TX mice at 90 days after transplantation. In each column, from bottom to top, KSL (gray) and not-KSL (white).Figure 11F is a graph depicting the abundance of cells derived from each of the engrafted KSL subpopulations within total bone marrow cells, μ, and TAμ in the brains of IV-transplanted busulfan-myeloablated mice at various time points after HCT. N=10 mice per group. In each column, from bottom to top, LT-HSC (light gray), MPP (dark gray), HPC2 (gray), and HPC1 (even lighter gray). Figure 11G is a graph depicting the abundance of cells derived from each of the engrafted KSL subpopulations within total bone marrow cells, μ, and TAμ in the brains of ICV-transplanted busulfan-myeloablated mice at various time points after HCT. In each column, from bottom to top, LT-HSC (light gray), MPP (dark gray), HPC2 (gray), and HPC1 (even lighter gray). N=10 mice per group. Figures 11H and 11I show immunofluorescence analysis of brain slices from BU-treated mice intravenously transplanted with KSL subpopulations at 90 days post-transplant. In Figure 11H, progeny of LT-HSCs are GFP+, and progeny of MPPs are ΔNGFR+ (light gray). Iba1 staining is in the blue channel. 20x magnification. M = merged. The right panels show other representative merged images at 20x (top) and 40x magnification (bottom). In Figure 11I, progeny of HPC2s are Cherry+, and progeny of MPPs are ΔNGFR+ (gray). GFP+ staining was not detected in the absence of ΔNGFR immunofluorescence. Nuclear TPIII (dark gray) is shown. The top panel shows 20x magnification. The bottom panel shows other representative merged images at 20x (top) and 40x magnification (bottom). Images were acquired by confocal microscopy (Radiance2100, Bio-Rad) and processed by SoftWork3.5.0.100. Figure 11J shows histogram plots showing differential levels of CXCR4 expression in KSL and NOT-KSL cells and KSL subpopulations upon transplantation. [Figure 11B]Figures 11A-11J show post-transplant brain bone marrow cells derived from early hematopoietic stem / progenitor cells. Figure 11A illustrates the experimental scheme showing how long-term (LT)-HSCs and progenitors within a pool of HSPCs were pre-isolated using c-kit, Sca-1, and lineage-negative staining, as well as the SLAM receptor markers CD150 and CD48. The indicated subpopulations were then differentially transduced with lentiviral vectors (LV) encoding GFP (KSL) and ΔNGFR (NOT-KSL), and GFP (LT-HSC), ΔNGFR (MPP), tag-BFP (HPC-1), and CHERRY (HPC-2). Subsequently, they were competitively transplanted IV or ICV at their original ratios into busulfan-bone marrow-ablated mice. ICV-transplanted animals also received unmanipulated whole BM cells for hematopoietic rescue 5 days after transplantation. Figure 11B shows histograms of marker gene expression in the in vivo liquid progeny of the indicated LV-transduced and transplanted cells in the mice described in A. Figure 11C is a graph depicting the abundance of cells derived from each of the transplanted KSL subpopulations within total CD45+ hematopoietic BM cells, myeloid (CD11b), and lymphoid (CD3 and B220) lineages of busulfan-treated transplanted (BU_TX) mice at the time of sacrifice. N = 10 mice / group. In each column, from bottom to top, LT-HSC (light gray), MPP (dark gray), HPC2 (gray), and HPC1 (even lighter gray). Figure 11D is a graph depicting the abundance of cells derived from IV-transplanted KSL and NOT-KSL within total bone marrow (CD45+CD11b+) cells, μ, and TAμ in the brains of BU_TX mice at 90 days post-transplant. In each column, from bottom to top, KSL (gray) and not-KSL (white). Figure 11E is a graph showing the abundance of ICV-transplanted KSL- and NOT-KSL-derived cells in total bone marrow (CD45+CD11b+) cells, μ and TAμ of the brain of BU_TX mice at 90 days after transplantation. In each column, from bottom to top, KSL (gray) and not-KSL (white).Figure 11F is a graph depicting the abundance of cells derived from each of the engrafted KSL subpopulations within total bone marrow cells, μ, and TAμ in the brains of IV-transplanted busulfan-myeloablated mice at various time points after HCT. N=10 mice per group. In each column, from bottom to top, LT-HSC (light gray), MPP (dark gray), HPC2 (gray), and HPC1 (even lighter gray). Figure 11G is a graph depicting the abundance of cells derived from each of the engrafted KSL subpopulations within total bone marrow cells, μ, and TAμ in the brains of ICV-transplanted busulfan-myeloablated mice at various time points after HCT. In each column, from bottom to top, LT-HSC (light gray), MPP (dark gray), HPC2 (gray), and HPC1 (even lighter gray). N=10 mice per group. Figures 11H and 11I show immunofluorescence analysis of brain slices from BU-treated mice intravenously transplanted with KSL subpopulations at 90 days post-transplant. In Figure 11H, progeny of LT-HSCs are GFP+, and progeny of MPPs are ΔNGFR+ (light gray). Iba1 staining is in the blue channel. 20x magnification. M = merged. The right panels show other representative merged images at 20x (top) and 40x magnification (bottom). In Figure 11I, progeny of HPC2s are Cherry+, and progeny of MPPs are ΔNGFR+ (gray). GFP+ staining was not detected in the absence of ΔNGFR immunofluorescence. Nuclear TPIII (dark gray) is shown. The top panel shows 20x magnification. The bottom panel shows other representative merged images at 20x (top) and 40x magnification (bottom). Images were acquired by confocal microscopy (Radiance2100, Bio-Rad) and processed by SoftWork3.5.0.100. Figure 11J shows histogram plots showing differential levels of CXCR4 expression in KSL and NOT-KSL cells and KSL subpopulations upon transplantation. [Figure 11C]Figures 11A-11J show post-transplant brain bone marrow cells derived from early hematopoietic stem / progenitor cells. Figure 11A illustrates the experimental scheme showing how long-term (LT)-HSCs and progenitors within a pool of HSPCs were pre-isolated using c-kit, Sca-1, and lineage-negative staining, as well as the SLAM receptor markers CD150 and CD48. The indicated subpopulations were then differentially transduced with lentiviral vectors (LV) encoding GFP (KSL) and ΔNGFR (NOT-KSL), and GFP (LT-HSC), ΔNGFR (MPP), tag-BFP (HPC-1), and CHERRY (HPC-2). Subsequently, they were competitively transplanted IV or ICV at their original ratios into busulfan-bone marrow-ablated mice. ICV-transplanted animals also received unmanipulated whole BM cells for hematopoietic rescue 5 days after transplantation. Figure 11B shows histograms of marker gene expression in the in vivo liquid progeny of the indicated LV-transduced and transplanted cells in the mice described in A. Figure 11C is a graph depicting the abundance of cells derived from each of the transplanted KSL subpopulations within total CD45+ hematopoietic BM cells, myeloid (CD11b), and lymphoid (CD3 and B220) lineages of busulfan-treated transplanted (BU_TX) mice at the time of sacrifice. N = 10 mice / group. In each column, from bottom to top, LT-HSC (light gray), MPP (dark gray), HPC2 (gray), and HPC1 (even lighter gray). Figure 11D is a graph depicting the abundance of cells derived from IV-transplanted KSL and NOT-KSL within total bone marrow (CD45+CD11b+) cells, μ, and TAμ in the brains of BU_TX mice at 90 days post-transplant. In each column, from bottom to top, KSL (gray) and not-KSL (white). Figure 11E is a graph showing the abundance of ICV-transplanted KSL- and NOT-KSL-derived cells in total bone marrow (CD45+CD11b+) cells, μ and TAμ of the brain of BU_TX mice at 90 days after transplantation. In each column, from bottom to top, KSL (gray) and not-KSL (white).Figure 11F is a graph depicting the abundance of cells derived from each of the engrafted KSL subpopulations within total bone marrow cells, μ, and TAμ in the brains of IV-transplanted busulfan-myeloablated mice at various time points after HCT. N=10 mice per group. In each column, from bottom to top, LT-HSC (light gray), MPP (dark gray), HPC2 (gray), and HPC1 (even lighter gray). Figure 11G is a graph depicting the abundance of cells derived from each of the engrafted KSL subpopulations within total bone marrow cells, μ, and TAμ in the brains of ICV-transplanted busulfan-myeloablated mice at various time points after HCT. In each column, from bottom to top, LT-HSC (light gray), MPP (dark gray), HPC2 (gray), and HPC1 (even lighter gray). N=10 mice per group. Figures 11H and 11I show immunofluorescence analysis of brain slices from BU-treated mice intravenously transplanted with KSL subpopulations at 90 days post-transplant. In Figure 11H, progeny of LT-HSCs are GFP+, and progeny of MPPs are ΔNGFR+ (light gray). Iba1 staining is in the blue channel. 20x magnification. M = merged. The right panels show other representative merged images at 20x (top) and 40x magnification (bottom). In Figure 11I, progeny of HPC2s are Cherry+, and progeny of MPPs are ΔNGFR+ (gray). GFP+ staining was not detected in the absence of ΔNGFR immunofluorescence. Nuclear TPIII (dark gray) is shown. The top panel shows 20x magnification. The bottom panel shows other representative merged images at 20x (top) and 40x magnification (bottom). Images were acquired by confocal microscopy (Radiance2100, Bio-Rad) and processed by SoftWork3.5.0.100. Figure 11J shows histogram plots showing differential levels of CXCR4 expression in KSL and NOT-KSL cells and KSL subpopulations upon transplantation. [Figure 11DEFG]Figures 11A-11J show post-transplant brain bone marrow cells derived from early hematopoietic stem / progenitor cells. Figure 11A illustrates the experimental scheme showing how long-term (LT)-HSCs and progenitors within a pool of HSPCs were pre-isolated using c-kit, Sca-1, and lineage-negative staining, as well as the SLAM receptor markers CD150 and CD48. The indicated subpopulations were then differentially transduced with lentiviral vectors (LV) encoding GFP (KSL) and ΔNGFR (NOT-KSL), and GFP (LT-HSC), ΔNGFR (MPP), tag-BFP (HPC-1), and CHERRY (HPC-2). Subsequently, they were competitively transplanted IV or ICV at their original ratios into busulfan-bone marrow-ablated mice. ICV-transplanted animals also received unmanipulated whole BM cells for hematopoietic rescue 5 days after transplantation. Figure 11B shows histograms of marker gene expression in the in vivo liquid progeny of the indicated LV-transduced and transplanted cells in the mice described in A. Figure 11C is a graph depicting the abundance of cells derived from each of the transplanted KSL subpopulations within total CD45+ hematopoietic BM cells, myeloid (CD11b), and lymphoid (CD3 and B220) lineages of busulfan-treated transplanted (BU_TX) mice at the time of sacrifice. N = 10 mice / group. In each column, from bottom to top, LT-HSC (light gray), MPP (dark gray), HPC2 (gray), and HPC1 (even lighter gray). Figure 11D is a graph depicting the abundance of cells derived from IV-transplanted KSL and NOT-KSL within total bone marrow (CD45+CD11b+) cells, μ, and TAμ in the brains of BU_TX mice at 90 days post-transplant. In each column, from bottom to top, KSL (gray) and not-KSL (white). Figure 11E is a graph showing the abundance of ICV-transplanted KSL- and NOT-KSL-derived cells in total bone marrow (CD45+CD11b+) cells, μ and TAμ of the brain of BU_TX mice at 90 days after transplantation. In each column, from bottom to top, KSL (gray) and not-KSL (white).Figure 11F is a graph depicting the abundance of cells derived from each of the engrafted KSL subpopulations within total bone marrow cells, μ, and TAμ in the brains of IV-transplanted busulfan-myeloablated mice at various time points after HCT. N=10 mice per group. In each column, from bottom to top, LT-HSC (light gray), MPP (dark gray), HPC2 (gray), and HPC1 (even lighter gray). Figure 11G is a graph depicting the abundance of cells derived from each of the engrafted KSL subpopulations within total bone marrow cells, μ, and TAμ in the brains of ICV-transplanted busulfan-myeloablated mice at various time points after HCT. In each column, from bottom to top, LT-HSC (light gray), MPP (dark gray), HPC2 (gray), and HPC1 (even lighter gray). N=10 mice per group. Figures 11H and 11I show immunofluorescence analysis of brain slices from BU-treated mice intravenously transplanted with KSL subpopulations at 90 days post-transplant. In Figure 11H, progeny of LT-HSCs are GFP+, and progeny of MPPs are ΔNGFR+ (light gray). Iba1 staining is in the blue channel. 20x magnification. M = merged. The right panels show other representative merged images at 20x (top) and 40x magnification (bottom). In Figure 11I, progeny of HPC2s are Cherry+, and progeny of MPPs are ΔNGFR+ (gray). GFP+ staining was not detected in the absence of ΔNGFR immunofluorescence. Nuclear TPIII (dark gray) is shown. The top panel shows 20x magnification. The bottom panel shows other representative merged images at 20x (top) and 40x magnification (bottom). Images were acquired by confocal microscopy (Radiance2100, Bio-Rad) and processed by SoftWork3.5.0.100. Figure 11J shows histogram plots showing differential levels of CXCR4 expression in KSL and NOT-KSL cells and KSL subpopulations upon transplantation. [Figure 11HI]Figures 11A-11J show post-transplant brain bone marrow cells derived from early hematopoietic stem / progenitor cells. Figure 11A illustrates the experimental scheme showing how long-term (LT)-HSCs and progenitors within a pool of HSPCs were pre-isolated using c-kit, Sca-1, and lineage-negative staining, as well as the SLAM receptor markers CD150 and CD48. The indicated subpopulations were then differentially transduced with lentiviral vectors (LV) encoding GFP (KSL) and ΔNGFR (NOT-KSL), and GFP (LT-HSC), ΔNGFR (MPP), tag-BFP (HPC-1), and CHERRY (HPC-2). Subsequently, they were competitively transplanted IV or ICV at their original ratios into busulfan-bone marrow-ablated mice. ICV-transplanted animals also received unmanipulated whole BM cells for hematopoietic rescue 5 days after transplantation. Figure 11B shows histograms of marker gene expression in the in vivo liquid progeny of the indicated LV-transduced and transplanted cells in the mice described in A. Figure 11C is a graph depicting the abundance of cells derived from each of the transplanted KSL subpopulations within total CD45+ hematopoietic BM cells, myeloid (CD11b), and lymphoid (CD3 and B220) lineages of busulfan-treated transplanted (BU_TX) mice at the time of sacrifice. N = 10 mice / group. In each column, from bottom to top, LT-HSC (light gray), MPP (dark gray), HPC2 (gray), and HPC1 (even lighter gray). Figure 11D is a graph depicting the abundance of cells derived from IV-transplanted KSL and NOT-KSL within total bone marrow (CD45+CD11b+) cells, μ, and TAμ in the brains of BU_TX mice at 90 days post-transplant. In each column, from bottom to top, KSL (gray) and not-KSL (white). Figure 11E is a graph showing the abundance of ICV-transplanted KSL- and NOT-KSL-derived cells in total bone marrow (CD45+CD11b+) cells, μ and TAμ of the brain of BU_TX mice at 90 days after transplantation. In each column, from bottom to top, KSL (gray) and not-KSL (white).Figure 11F is a graph depicting the abundance of cells derived from each of the engrafted KSL subpopulations within total bone marrow cells, μ, and TAμ in the brains of IV-transplanted busulfan-myeloablated mice at various time points after HCT. N=10 mice per group. In each column, from bottom to top, LT-HSC (light gray), MPP (dark gray), HPC2 (gray), and HPC1 (even lighter gray). Figure 11G is a graph depicting the abundance of cells derived from each of the engrafted KSL subpopulations within total bone marrow cells, μ, and TAμ in the brains of ICV-transplanted busulfan-myeloablated mice at various time points after HCT. In each column, from bottom to top, LT-HSC (light gray), MPP (dark gray), HPC2 (gray), and HPC1 (even lighter gray). N=10 mice per group. Figures 11H and 11I show immunofluorescence analysis of brain slices from BU-treated mice intravenously transplanted with KSL subpopulations at 90 days post-transplant. In Figure 11H, progeny of LT-HSCs are GFP+, and progeny of MPPs are ΔNGFR+ (light gray). Iba1 staining is in the blue channel. 20x magnification. M = merged. The right panels show other representative merged images at 20x (top) and 40x magnification (bottom). In Figure 11I, progeny of HPC2s are Cherry+, and progeny of MPPs are ΔNGFR+ (gray). GFP+ staining was not detected in the absence of ΔNGFR immunofluorescence. Nuclear TPIII (dark gray) is shown. The top panel shows 20x magnification. The bottom panel shows other representative merged images at 20x (top) and 40x magnification (bottom). Images were acquired by confocal microscopy (Radiance2100, Bio-Rad) and processed by SoftWork3.5.0.100. Figure 11J shows histogram plots showing differential levels of CXCR4 expression in KSL and NOT-KSL cells and KSL subpopulations upon transplantation. [Figure 11J]Figures 11A-11J show post-transplant brain bone marrow cells derived from early hematopoietic stem / progenitor cells. Figure 11A illustrates the experimental scheme showing how long-term (LT)-HSCs and progenitors within a pool of HSPCs were pre-isolated using c-kit, Sca-1, and lineage-negative staining, as well as the SLAM receptor markers CD150 and CD48. The indicated subpopulations were then differentially transduced with lentiviral vectors (LV) encoding GFP (KSL) and ΔNGFR (NOT-KSL), and GFP (LT-HSC), ΔNGFR (MPP), tag-BFP (HPC-1), and CHERRY (HPC-2). Subsequently, they were competitively transplanted IV or ICV at their original ratios into busulfan-bone marrow-ablated mice. ICV-transplanted animals also received unmanipulated whole BM cells for hematopoietic rescue 5 days after transplantation. Figure 11B shows histograms of marker gene expression in the in vivo liquid progeny of the indicated LV-transduced and transplanted cells in the mice described in A. Figure 11C is a graph depicting the abundance of cells derived from each of the transplanted KSL subpopulations within total CD45+ hematopoietic BM cells, myeloid (CD11b), and lymphoid (CD3 and B220) lineages of busulfan-treated transplanted (BU_TX) mice at the time of sacrifice. N = 10 mice / group. In each column, from bottom to top, LT-HSC (light gray), MPP (dark gray), HPC2 (gray), and HPC1 (even lighter gray). Figure 11D is a graph depicting the abundance of cells derived from IV-transplanted KSL and NOT-KSL within total bone marrow (CD45+CD11b+) cells, μ, and TAμ in the brains of BU_TX mice at 90 days post-transplant. In each column, from bottom to top, KSL (gray) and not-KSL (white). Figure 11E is a graph showing the abundance of ICV-transplanted KSL- and NOT-KSL-derived cells in total bone marrow (CD45+CD11b+) cells, μ and TAμ of the brain of BU_TX mice at 90 days after transplantation. In each column, from bottom to top, KSL (gray) and not-KSL (white).Figure 11F is a graph depicting the abundance of cells derived from each of the engrafted KSL subpopulations within total bone marrow cells, μ, and TAμ in the brains of IV-transplanted busulfan-myeloablated mice at various time points after HCT. N=10 mice per group. In each column, from bottom to top, LT-HSC (light gray), MPP (dark gray), HPC2 (gray), and HPC1 (even lighter gray). Figure 11G is a graph depicting the abundance of cells derived from each of the engrafted KSL subpopulations within total bone marrow cells, μ, and TAμ in the brains of ICV-transplanted busulfan-myeloablated mice at various time points after HCT. In each column, from bottom to top, LT-HSC (light gray), MPP (dark gray), HPC2 (gray), and HPC1 (even lighter gray). N=10 mice per group. Figures 11H and 11I show immunofluorescence analysis of brain slices from BU-treated mice intravenously transplanted with KSL subpopulations at 90 days post-transplant. In Figure 11H, progeny of LT-HSCs are GFP+, and progeny of MPPs are ΔNGFR+ (light gray). Iba1 staining is in the blue channel. 20x magnification. M = merged. The right panels show other representative merged images at 20x (top) and 40x magnification (bottom). In Figure 11I, progeny of HPC2s are Cherry+, and progeny of MPPs are ΔNGFR+ (gray). GFP+ staining was not detected in the absence of ΔNGFR immunofluorescence. Nuclear TPIII (dark gray) is shown. The top panel shows 20x magnification. The bottom panel shows other representative merged images at 20x (top) and 40x magnification (bottom). Images were acquired by confocal microscopy (Radiance2100, Bio-Rad) and processed by SoftWork3.5.0.100. Figure 11J shows histogram plots showing differential levels of CXCR4 expression in KSL and NOT-KSL cells and KSL subpopulations upon transplantation.

[0082] [Figure 12A]Figures 12A-12E show that Fgd5+ HSCs generate microglia-like progeny in the brain upon both ICV and IV transplantation. Figure 12A illustrates the experimental scheme in which Fgd5+ HSCs (Lin- ckit+ Sca-1+ Flk2- CD34-) were isolated from CD45.2 Fdg5- Green donor mice. Fgd5+ HSCs (n = 500) were transplanted IV or ICV into busulfan-bone marrow-ablated or lethally irradiated CD45.1 recipient mice. Transplanted animals also received unmanipulated CD45.1 whole BM cells for hematopoietic rescue 5 days after transplantation. Figure 12B is a graph illustrating the prevalence of donor cells (CD45.2+) among brain bone marrow CD11b+ cells in mice IV-transplanted with Fgd5 cells after busulfan and irradiation pretreatment. N = 4 per group. Figure 12C is a graph illustrating the abundance of donor cells (CD45.2+) among brain bone marrow CD11b+ cells in mice that received ICV transplants of Fgd5 cells after busulfan and irradiation pretreatment. N≧4 per group. Figure 12D is a graph illustrating the μ, TAμ, and CNSmac populations among donor-derived cells in IV transplanted, busulfan-pretreated mice. N≧4 per group. Figure 12E is a graph illustrating the μ, TAμ, and CNSmac populations among donor-derived cells in ICV transplanted, busulfan-pretreated mice. N≧4 per group. [Figure 12BCDE]Figures 12A-12E show that Fgd5+ HSCs generate microglia-like progeny in the brain upon both ICV and IV transplantation. Figure 12A illustrates the experimental scheme in which Fgd5+ HSCs (Lin- ckit+ Sca-1+ Flk2- CD34-) were isolated from CD45.2 Fdg5- Green donor mice. Fgd5+ HSCs (n = 500) were transplanted IV or ICV into busulfan-bone marrow-ablated or lethally irradiated CD45.1 recipient mice. Transplanted animals also received unmanipulated CD45.1 whole BM cells for hematopoietic rescue 5 days after transplantation. Figure 12B is a graph illustrating the prevalence of donor cells (CD45.2+) among brain bone marrow CD11b+ cells in mice IV-transplanted with Fgd5 cells after busulfan and irradiation pretreatment. N = 4 per group. Figure 12C is a graph illustrating the abundance of donor cells (CD45.2+) among brain bone marrow CD11b+ cells in mice that received ICV transplants of Fgd5 cells after busulfan and irradiation pretreatment. N≧4 per group. Figure 12D is a graph illustrating the μ, TAμ, and CNSmac populations among donor-derived cells in IV transplanted, busulfan-pretreated mice. N≧4 per group. Figure 12E is a graph illustrating the μ, TAμ, and CNSmac populations among donor-derived cells in ICV transplanted, busulfan-pretreated mice. N≧4 per group.

[0083] [Figure 13A]Figures 13A-13B describe the contribution of CX3CR1-expressing and negative cells to brain-marrow chimerism. Figure 13A is a series of graphs showing the bone marrow characteristics of CX3CR1-GFP mice, specifically, GFP expression in the various bone marrow subpopulations indicated. From bottom to top in each column, GFP negative (light gray), GFP low (dark gray), and GFP high (light gray). Figure 13B illustrates the experimental setup used to generate chimeric mice using reporter CX3CR1-GFP mice and cells isolated from chimerism generated in the brain. Mice receiving GFP+ / highLin-HSPCs failed to engraft with CX3CR1 CD45.2 donor cells in the brain (representative dot plots are shown in the left box). Transplantation of whole bone marrow not sorted for CX3CR1 demonstrated sustained engraftment of donor cells, which robustly expressed GFP upon microglial differentiation (representative dot plots are shown in the left box). This indicates that GFP-cells (which do not express CX3CR1) are transplanted to establish brain-marrow chimerism. [Figure 13B-1]Figures 13A-13B describe the contribution of CX3CR1-expressing and negative cells to brain-marrow chimerism. Figure 13A is a series of graphs showing the bone marrow characteristics of CX3CR1-GFP mice, specifically, GFP expression in the various bone marrow subpopulations indicated. From bottom to top in each column, GFP negative (light gray), GFP low (dark gray), and GFP high (light gray). Figure 13B illustrates the experimental setup used to generate chimeric mice using reporter CX3CR1-GFP mice and cells isolated from chimerism generated in the brain. Mice receiving GFP+ / highLin-HSPCs failed to engraft with CX3CR1 CD45.2 donor cells in the brain (representative dot plots are shown in the left box). Transplantation of whole bone marrow not sorted for CX3CR1 demonstrated sustained engraftment of donor cells, which robustly expressed GFP upon microglial differentiation (representative dot plots are shown in the left box). This indicates that GFP-cells (which do not express CX3CR1) are transplanted to establish brain-marrow chimerism. [Figure 13B-2]Figures 13A-13B describe the contribution of CX3CR1-expressing and negative cells to brain-marrow chimerism. Figure 13A is a series of graphs showing the bone marrow characteristics of CX3CR1-GFP mice, specifically, GFP expression in the various bone marrow subpopulations indicated. From bottom to top in each column, GFP negative (light gray), GFP low (dark gray), and GFP high (light gray). Figure 13B illustrates the experimental setup used to generate chimeric mice using reporter CX3CR1-GFP mice and cells isolated from chimerism generated in the brain. Mice receiving GFP+ / highLin-HSPCs failed to engraft with CX3CR1 CD45.2 donor cells in the brain (representative dot plots are shown in the left box). Transplantation of whole bone marrow not sorted for CX3CR1 demonstrated sustained engraftment of donor cells, which robustly expressed GFP upon microglial differentiation (representative dot plots are shown in the left box). This indicates that GFP-cells (which do not express CX3CR1) are transplanted to establish brain-marrow chimerism.

[0084] [Figure 14]Figures 14A-14D show the contribution of human LT-HSCs, defined as CD34+ and CD38-, to the development of brain-to-myeloid progeny cells. Figure 14A is a representative dot plot showing the gating strategy for identifying human CD34+CD38+ (progenitor) and CD34+CD38- (stem-enriched) cells from human mobilized peripheral blood. Cells were differentially transduced with LV encoding GFP and tagged-BFP at the indicated abundances and mixed into NSG myeloablated mice for transplantation. Figure 14B is a graph showing the abundance of cells marked with GFP (CD38-) or tagged-BFP (CD38+) within the human CD45+ cell fraction recovered in the brains of NSG mice transplanted with the cells shown in A. N≥5 mice / group. Mean and SEM are shown. From bottom to top, each column is CD38- (gray) and CD38+ (white). Figure 14C is a representative dot plot showing the gating strategy for identifying long-term and short-term human HSCs and progenitors by expression of the markers CD38 and CD90 on CD34+ cells recovered from human mobilized peripheral blood. Cells were differentially transduced with LVs encoding GFP, Cherry, tag-BFP (cyan), and mO2 (orange) at the indicated abundances and mixed into NSG-pretreated mice for transplantation. Figure 14D is a graph showing the abundance of cells marked by the indicated markers within the human CD45+ cell fraction recovered in the brains of NSG mice transplanted with the cells shown in 14A. From bottom to top: CD38-CD90+ (gray), CD38-CD90-low (light gray), CD38+CD90- (medium gray), and CD38+CD90+high (even lighter gray). N≧5 mice / group. Mean and SEM are shown. Overall, CD34+CD38- showed the greatest contribution to brain-myeloid cell chimerism.

[0085] [Figure 15A]Figures 15A-15C illustrate the identification of brain-resident putative μ precursors by BU sensitivity. Figure 15A shows that on day 5 after one or four doses of busulfan, an increased fraction of apoptotic Annexin+ cells was detected in the hematopoietic bone marrow, and most importantly, within CD45+ c-kit+ cells. From the leftmost bar in each set, CO (white), 1x BU (dark gray), and 4x BU (black) are shown. Figure 15B shows representative FACS plots of γH2AX+ cells within vital CD45+ brain cells from control animals and BU-treated mice. Figure 15C shows the distribution of γH2AX markers in the brains of mice analyzed on day 1 after busulfan pretreatment or control untreated mice (CTR). Insets are representative laser-scanning confocal micrographs co-immunostained for γH2AX, neurons (NeuN), microglia (Iba1), or astrocytes (GFAP). Scale bar = 10 μm. Images were acquired with a confocal microscope Radiance 2100 (Bio-Rad) Ix70 at 20× magnification and processed with SoftWork 3.5.0. Reconstruction was performed using Adobe Photoshop CS8.0 software. Inset: 40× magnification. [Figure 15B]Figures 15A-15C illustrate the identification of brain-resident putative μ precursors by BU sensitivity. Figure 15A shows that on day 5 after one or four doses of busulfan, an increased fraction of apoptotic Annexin+ cells was detected in the hematopoietic bone marrow, and most importantly, within CD45+ c-kit+ cells. From the leftmost bar in each set, CO (white), 1x BU (dark gray), and 4x BU (black) are shown. Figure 15B shows representative FACS plots of γH2AX+ cells within vital CD45+ brain cells from control animals and BU-treated mice. Figure 15C shows the distribution of γH2AX markers in the brains of mice analyzed on day 1 after busulfan pretreatment or control untreated mice (CTR). Insets are representative laser-scanning confocal micrographs co-immunostained for γH2AX, neurons (NeuN), microglia (Iba1), or astrocytes (GFAP). Scale bar = 10 μm. Images were acquired with a confocal microscope Radiance 2100 (Bio-Rad) Ix70 at 20× magnification and processed with SoftWork 3.5.0. Reconstruction was performed using Adobe Photoshop CS8.0 software. Inset: 40× magnification. [Figure 15C]Figures 15A-15C illustrate the identification of brain-resident putative μ precursors by BU sensitivity. Figure 15A shows that on day 5 after one or four doses of busulfan, an increased fraction of apoptotic Annexin+ cells was detected in the hematopoietic bone marrow, and most importantly, within CD45+ c-kit+ cells. From the leftmost bar in each set, CO (white), 1x BU (dark gray), and 4x BU (black) are shown. Figure 15B shows representative FACS plots of γH2AX+ cells within vital CD45+ brain cells from control animals and BU-treated mice. Figure 15C shows the distribution of γH2AX markers in the brains of mice analyzed on day 1 after busulfan pretreatment or control untreated mice (CTR). Insets are representative laser-scanning confocal micrographs co-immunostained for γH2AX, neurons (NeuN), microglia (Iba1), or astrocytes (GFAP). Scale bar = 10 μm. Images were acquired with a confocal microscope Radiance 2100 (Bio-Rad) Ix70 at 20× magnification and processed with SoftWork 3.5.0. Reconstruction was performed using Adobe Photoshop CS8.0 software. Inset: 40× magnification.

[0086] [Figure 16A-1]Figures 16A-16D illustrate the molecular engineering of microglia for regulated therapeutic gene expression. Figure 16A illustrates a representative example and sequence of the mouse TSPO promoter. Figure 16B shows the LV obtained by cloning 2.7 kbp upstream of the Tspo gene into a SIN LV plasmid upstream of the GFP cDNA. We transduced BV-2 cells (a murine microglial cell line) with this vector to evaluate expression driven by this promoter. TSPO expression is known to be stimulated in microglial cells in response to stress and can be mimicked by LPS injection in vivo. Figure 16C is a histogram showing GFP-positive transduced BV2 cells and the shift in GFP mean fluorescence intensity after LPS stimulation. Figure 16D is a graph showing the mean fluorescence intensity (MFI) of GFP (normalized to vector content) of transduced BV2 cells under basal conditions upon LPS stimulation (n = 4: mean ± SEM). *=p<0.0001 Student's T-test. [Figure 16A-2] Figures 16A-16D illustrate the molecular engineering of microglia for regulated therapeutic gene expression. Figure 16A illustrates a representative example and sequence of the mouse TSPO promoter. Figure 16B shows the LV obtained by cloning 2.7 kbp upstream of the Tspo gene into a SIN LV plasmid upstream of the GFP cDNA. We transduced BV-2 cells (a murine microglial cell line) with this vector to evaluate expression driven by this promoter. TSPO expression is known to be stimulated in microglial cells in response to stress and can be mimicked by LPS injection in vivo. Figure 16C is a histogram showing GFP-positive transduced BV2 cells and the shift in GFP mean fluorescence intensity after LPS stimulation. Figure 16D is a graph showing the mean fluorescence intensity (MFI) of GFP (normalized to vector content) of transduced BV2 cells under basal conditions upon LPS stimulation (n = 4: mean ± SEM). *=p<0.0001 Student's T-test. [Figure 16BC]Figures 16A-16D illustrate the molecular engineering of microglia for regulated therapeutic gene expression. Figure 16A illustrates a representative example and sequence of the mouse TSPO promoter. Figure 16B shows the LV obtained by cloning 2.7 kbp upstream of the Tspo gene into a SIN LV plasmid upstream of the GFP cDNA. We transduced BV-2 cells (a murine microglial cell line) with this vector to evaluate expression driven by this promoter. TSPO expression is known to be stimulated in microglial cells in response to stress and can be mimicked by LPS injection in vivo. Figure 16C is a histogram showing GFP-positive transduced BV2 cells and the shift in GFP mean fluorescence intensity after LPS stimulation. Figure 16D is a graph showing the mean fluorescence intensity (MFI) of GFP (normalized to vector content) of transduced BV2 cells under basal conditions upon LPS stimulation (n = 4: mean ± SEM). *=p<0.0001 Student's T-test. [Figure 16D] Figures 16A-16D illustrate the molecular engineering of microglia for regulated therapeutic gene expression. Figure 16A illustrates a representative example and sequence of the mouse TSPO promoter. Figure 16B shows the LV obtained by cloning 2.7 kbp upstream of the Tspo gene into a SIN LV plasmid upstream of the GFP cDNA. We transduced BV-2 cells (a murine microglial cell line) with this vector to evaluate expression driven by this promoter. TSPO expression is known to be stimulated in microglial cells in response to stress and can be mimicked by LPS injection in vivo. Figure 16C is a histogram showing GFP-positive transduced BV2 cells and the shift in GFP mean fluorescence intensity after LPS stimulation. Figure 16D is a graph showing the mean fluorescence intensity (MFI) of GFP (normalized to vector content) of transduced BV2 cells under basal conditions upon LPS stimulation (n = 4: mean ± SEM). *=p<0.0001 Student's T-test.

[0087] [Figure 17A]Figures 17A-17I illustrate nanoparticle (NP) characteristics and biodistribution upon ICV delivery. Figure 17A shows the size of the first-generation rhodamine nanoparticles used. Figures 17B-17D show the brain distribution of ICV-injected first-generation nanoparticles assessed by flow cytometry. Nanoparticle uptake by CD45+ cells is increased in the presence of mannitol (Figure 17B), is higher in CD45+c-kit+ cells compared with CD45+CD11b+ cells (Figure 17C), and is within the range of CD45+Edu+ cells compared with CD45+Edu- cells (Figure 17D). Figure 17E shows representative epifluorescence micrographs of rhodamine-conjugated NP signal (red / light gray) and DAPI nuclear staining (blue / dark gray) in a sagittal brain slice from a mouse analyzed 3 days after ICV NP injection. Images were acquired with a Delta Vision Olympus at 20x magnification and processed with SoftWork 3.5.0. Reconstruction was performed using Adobe Photoshop CS8.0 software. Figure 17F is a graph showing the distribution of nanoparticles in the indicated brain regions, expressed as a percentage of the total area that is Rho+. Figure 17G is an inset of Figure 17E highlighting the prominent distribution of NPs (red, DAPI nuclei in blue) near the subventricular zone (SVZ) and rostral meridional stream (RMS) (right panel) in a sagittal brain slice from a mouse analyzed 3 days after ICV injection of NPs, as well as representative confocal microscopy images of rhodaminylated NP signal (red), Iba1 (green), ki67 (proliferation marker, blue), and DAPI nuclear staining (light blue). NPs are located within Ki67+ microglial cells. Figure 17H ​​shows that NPs are internalized by proliferating cells. Representative laser-scanning confocal micrographs of co-immunostaining for Iba1+, F4 / 80, rhodamine, and the proliferation marker Edu are shown. Rh+ NPs can be detected in Iba1+ (arrows) and Iba1- (arrowheads) proliferating cells. Figure 17I shows reconstructions of Edu and rhodamine signaling recovered by fluorescence microscopy on slices from the brain of NP-injected mice, showing colocalization of Edu and NP signals.Images were acquired using a confocal microscope Radiance2100 (Bio-Rad) Ix70 and processed using Soft Work 3.5.0. [Figure 17BCD]Figures 17A-17I illustrate nanoparticle (NP) characteristics and biodistribution upon ICV delivery. Figure 17A shows the size of the first-generation rhodamine nanoparticles used. Figures 17B-17D show the brain distribution of ICV-injected first-generation nanoparticles assessed by flow cytometry. Nanoparticle uptake by CD45+ cells is increased in the presence of mannitol (Figure 17B), is higher in CD45+c-kit+ cells compared with CD45+CD11b+ cells (Figure 17C), and is within the range of CD45+Edu+ cells compared with CD45+Edu- cells (Figure 17D). Figure 17E shows representative epifluorescence micrographs of rhodamine-conjugated NP signal (red / light gray) and DAPI nuclear staining (blue / dark gray) in a sagittal brain slice from a mouse analyzed 3 days after ICV NP injection. Images were acquired with a Delta Vision Olympus at 20x magnification and processed with SoftWork 3.5.0. Reconstruction was performed using Adobe Photoshop CS8.0 software. Figure 17F is a graph showing the distribution of nanoparticles in the indicated brain regions, expressed as a percentage of the total area that is Rho+. Figure 17G is an inset of Figure 17E highlighting the prominent distribution of NPs (red, DAPI nuclei in blue) near the subventricular zone (SVZ) and rostral meridional stream (RMS) (right panel) in a sagittal brain slice from a mouse analyzed 3 days after ICV injection of NPs, as well as representative confocal microscopy images of rhodaminylated NP signal (red), Iba1 (green), ki67 (proliferation marker, blue), and DAPI nuclear staining (light blue). NPs are located within Ki67+ microglial cells. Figure 17H ​​shows that NPs are internalized by proliferating cells. Representative laser-scanning confocal micrographs of co-immunostaining for Iba1+, F4 / 80, rhodamine, and the proliferation marker Edu are shown. Rh+ NPs can be detected in Iba1+ (arrows) and Iba1- (arrowheads) proliferating cells. Figure 17I shows reconstructions of Edu and rhodamine signaling recovered by fluorescence microscopy on slices from the brain of NP-injected mice, showing colocalization of Edu and NP signals.Images were acquired using a confocal microscope Radiance2100 (Bio-Rad) Ix70 and processed using Soft Work 3.5.0. [Figure 17EF]Figures 17A-17I illustrate nanoparticle (NP) characteristics and biodistribution upon ICV delivery. Figure 17A shows the size of the first-generation rhodamine nanoparticles used. Figures 17B-17D show the brain distribution of ICV-injected first-generation nanoparticles assessed by flow cytometry. Nanoparticle uptake by CD45+ cells is increased in the presence of mannitol (Figure 17B), is higher in CD45+c-kit+ cells compared with CD45+CD11b+ cells (Figure 17C), and is within the range of CD45+Edu+ cells compared with CD45+Edu- cells (Figure 17D). Figure 17E shows representative epifluorescence micrographs of rhodamine-conjugated NP signal (red / light gray) and DAPI nuclear staining (blue / dark gray) in a sagittal brain slice from a mouse analyzed 3 days after ICV NP injection. Images were acquired with a Delta Vision Olympus at 20x magnification and processed with SoftWork 3.5.0. Reconstruction was performed using Adobe Photoshop CS8.0 software. Figure 17F is a graph showing the distribution of nanoparticles in the indicated brain regions, expressed as a percentage of the total area that is Rho+. Figure 17G is an inset of Figure 17E highlighting the prominent distribution of NPs (red, DAPI nuclei in blue) near the subventricular zone (SVZ) and rostral meridional stream (RMS) (right panel) in a sagittal brain slice from a mouse analyzed 3 days after ICV injection of NPs, as well as representative confocal microscopy images of rhodaminylated NP signal (red), Iba1 (green), ki67 (proliferation marker, blue), and DAPI nuclear staining (light blue). NPs are located within Ki67+ microglial cells. Figure 17H ​​shows that NPs are internalized by proliferating cells. Representative laser-scanning confocal micrographs of co-immunostaining for Iba1+, F4 / 80, rhodamine, and the proliferation marker Edu are shown. Rh+ NPs can be detected in Iba1+ (arrows) and Iba1- (arrowheads) proliferating cells. Figure 17I shows reconstructions of Edu and rhodamine signaling recovered by fluorescence microscopy on slices from the brain of NP-injected mice, showing colocalization of Edu and NP signals.Images were acquired using a confocal microscope Radiance2100 (Bio-Rad) Ix70 and processed using Soft Work 3.5.0. [Figure 17GHI]Figures 17A-17I illustrate nanoparticle (NP) characteristics and biodistribution upon ICV delivery. Figure 17A shows the size of the first-generation rhodamine nanoparticles used. Figures 17B-17D show the brain distribution of ICV-injected first-generation nanoparticles assessed by flow cytometry. Nanoparticle uptake by CD45+ cells is increased in the presence of mannitol (Figure 17B), is higher in CD45+c-kit+ cells compared with CD45+CD11b+ cells (Figure 17C), and is within the range of CD45+Edu+ cells compared with CD45+Edu- cells (Figure 17D). Figure 17E shows representative epifluorescence micrographs of rhodamine-conjugated NP signal (red / light gray) and DAPI nuclear staining (blue / dark gray) in a sagittal brain slice from a mouse analyzed 3 days after ICV NP injection. Images were acquired with a Delta Vision Olympus at 20x magnification and processed with SoftWork 3.5.0. Reconstruction was performed using Adobe Photoshop CS8.0 software. Figure 17F is a graph showing the distribution of nanoparticles in the indicated brain regions, expressed as a percentage of the total area that is Rho+. Figure 17G is an inset of Figure 17E highlighting the prominent distribution of NPs (red, DAPI nuclei in blue) near the subventricular zone (SVZ) and rostral meridional stream (RMS) (right panel) in a sagittal brain slice from a mouse analyzed 3 days after ICV injection of NPs, as well as representative confocal microscopy images of rhodaminylated NP signal (red), Iba1 (green), ki67 (proliferation marker, blue), and DAPI nuclear staining (light blue). NPs are located within Ki67+ microglial cells. Figure 17H ​​shows that NPs are internalized by proliferating cells. Representative laser-scanning confocal micrographs of co-immunostaining for Iba1+, F4 / 80, rhodamine, and the proliferation marker Edu are shown. Rh+ NPs can be detected in Iba1+ (arrows) and Iba1- (arrowheads) proliferating cells. Figure 17I shows reconstructions of Edu and rhodamine signaling recovered by fluorescence microscopy on slices from the brain of NP-injected mice, showing colocalization of Edu and NP signals.Images were acquired using a confocal microscope Radiance2100 (Bio-Rad) Ix70 and processed using Soft Work 3.5.0.

[0088] [Figure 18] FIG. 18 shows the preparation of self-assembled NPs and chemotherapeutic drug conjugates.

[0089] [Figure 19]Figures 19A-19E illustrate the in vivo and in vivo effects of NP-encapsulated chemotherapeutic agents. Figure 19A shows representative flow cytometry analysis and immunofluorescence staining for γH2AX in a BV2 microglia-like cell line exposed to busulfan. The graph shows the percentage of γH2AX+ cells (determined by flow cytometry) after exposure to various nanoparticle formulations with or without busulfan loading (histograms represent mean + / - SEM of n >= 3 independent experiments). Figure 19B shows the results of an MTT cell viability assay on a BV2 microglia-like cell line exposed to BU-NPs, highlighting the cytotoxicity exerted by exposure to NP-encapsulated busulfan. Figure 19C is a graph illustrating the abundance of γH2AX+ microglial cells in mouse brains (by flow cytometry) assessed 3 days after ICV NP administration (histograms represent mean + / - SEM for n=4 animals / group). Figures 19D and 19E are two graphs showing the viability of BV2 microglial cells exposed to NPs (50- and 100-nm-sized self-assembling formulations, and PLC formulations) containing or not the etoposide chemotherapeutic agent (chmetherapic) and etoposized as free formulations at the indicated concentrations. Viability was assessed using an MTT assay. Viability was measured after 48 and 72 hours of incubation. Delivery of etopodise into NPs increases its ability to kill cells compared to the free formulation. In Figures 19D and 19E, the leftmost bars in each set are self-assembled NPs (50 nm) (dark gray), self-assembled NPs (100 nm), PCL NPs (100 nm) (gray), and etoposide (medium gray).

[0090] [Figure 20AB]Figures 20A-20E show etoposide-containing NPs administered after inducing microglial progenitor proliferation and expanding the CD45+c-kit+ cell pool-induced apoptosis of CD45+c-kit+ and CD45+CD11b+ cells. Figures 20A and 20C show the experimental design used to induce microglial progenitor proliferation with a single busulfan dose or oral administration of a CSF 1R inhibitor (Elmore et al., Neuron. 82(2):380-397 (2014)) for 10 days, followed by intravenous administration of etoposide-containing NPs in adult wild-type mice. Figures 20B and 20D are graphs showing the % of annexin+ early apoptotic cells measured by flow cytometry for brain samples obtained 5 days after NP administration. The % annexin+ cells are calculated based on the indicated cell subfractions. Empty NPs were used as controls. Figure 20E shows the % of CD45+c-kit+ cells in the same samples. Overall, enhanced apoptosis was detected in animals receiving etoposide-charged NPs in CD45+c-kit+ and CD45+CD11b+ cells, consistent with that observed in control mice receiving four busulfan doses. [Figure 20CDE]Figures 20A-20E show etoposide-containing NPs administered after inducing microglial progenitor proliferation and expanding the CD45+c-kit+ cell pool-induced apoptosis of CD45+c-kit+ and CD45+CD11b+ cells. Figures 20A and 20C show the experimental design used to induce microglial progenitor proliferation with a single busulfan dose or oral administration of a CSF 1R inhibitor (Elmore et al., Neuron. 82(2):380-397 (2014)) for 10 days, followed by intravenous administration of etoposide-containing NPs in adult wild-type mice. Figures 20B and 20D are graphs showing the % of annexin+ early apoptotic cells measured by flow cytometry for brain samples obtained 5 days after NP administration. The % annexin+ cells are calculated based on the indicated cell subfractions. Empty NPs were used as controls. Figure 20E shows the % of CD45+c-kit+ cells in the same samples. Overall, enhanced apoptosis was detected in animals receiving etoposide-charged NPs in CD45+c-kit+ and CD45+CD11b+ cells, consistent with that observed in control mice receiving four busulfan doses. DETAILED DESCRIPTION OF THE INVENTION

[0091] Detailed Description of the Invention The present invention features compositions and methods useful for reconstituting microglia upon transplantation of HSPCs, and for the treatment and prevention of nervous system diseases or disorders of the central nervous system (e.g., storage disorders including lysosomal storage disorders, neurodegenerative diseases, etc.).

[0092] The present invention is based, at least in part, on several discoveries described herein. Current methods for treating storage diseases and neurodegenerative diseases using HSC transplantation have limited effect on CNS disease symptoms due to the slow replacement of resident microglia by the progeny of the transplanted cells. Current methods of HSC transplantation into patients suffering from SD and neurodegenerative disorders involve the use of whole bone marrow or apheretic products or umbilical cord blood in the case of autologous gene therapy, or Hematopoietic stem and progenitor cells These include the use of HSPCs (selective for CD34 expression). Here, cell populations enriched for microglial repopulation activity can be identified within these cell sources, and their use can ultimately improve donor microglial reconstitution after transplantation. Furthermore, they can be delivered directly to the brain using intracerebroventricular injection (ICV). Hematopoietic stem and progenitor cells The use of HSPCs, or fractions of the HSPC pool, improves the rate and extent of microglial reconstitution by transplanted donor cells and enhances therapeutic protein delivery to the brain compared to single intravenous (IV) transplantation approaches. This approach therefore offers significant therapeutic potential and can be optimized to obtain broad microglial contributions from ICV-transplanted cells for advanced strategies aimed at replacing exclusively microglia, rather than hematopoietic tissue, with transplanted cells. This is highly relevant in conjunction with the novel strategy developed herein for molecular engineering of microglia for regulated therapeutic gene expression in response to neuroinflammatory or neurodegenerative stimuli.

[0093] As demonstrated herein, HSPC transplantation can generate transcription-dependent de novo microglia through a stepwise process reminiscent of postnatal physiological microglial maturation. Hematopoietic cells capable of generating new microglia upon transplantation into bone marrow-ablated recipients are maintained within long-term hematopoietic stem cells (HSCs) in humans and mice. Similar transcription-dependent de novo microglial cells can also be generated by intraventricular delivery of HSPCs. Importantly, this novel pathway is associated with clinically relevant, more rapid, and more extensive microglial replacement compared to systemic infusion of HSPCs. Thus, the following has been shown: · Intravenous (IV) and intracerebroventricular (ICV) transplantation of HSPCs in mice and humans generates cerebromyeloprogeny. ICV delivery of mouse and human HSPCs results in the generation of progeny myeloid cells in the brain at a more rapid rate and in greater quantities than IV. While ICV-infused HSPCs engraft and expand in the brain, they do not engraft in hematopoietic organs. The contribution of ICV-infused HSPCs to brain-marrow chimerism can, in certain conditions, outweigh the contribution of IV-coinfused HSPCs. The contribution of IV and ICV infused HSPCs to brain-marrow chimerism may be equivalent under certain conditions. · Progeny of both IV and ICV transplanted HSPCs have transcriptional profiles consistent with microglia. In the brain, the progeny of ICV-injected HSPCs more closely resemble microglia than the progeny of IV-injected HSPCs. · After transplantation, hematopoietic cells associated with the brain parenchyma of mice have clonogenic and hematopoietic repopulation potential, as well as microglial reconstitution potential. The combination of ICV+IV delivery of engineered HSPCs has therapeutic relevance for two representative LSDs. · The combination of ICV+IV delivery of HSPCs is feasible in the allogeneic transplant setting. · Intrathecal (IT) delivery of HSPCs can contribute to brain and hematopoietic chimerism in the context of combinatorial HSPC transplantation strategies. Specific nanoparticles target c-kit receptors in targeted areas of the brain. + and nestin + It can be uploaded by myeloproliferative cells. The nanoparticles can efficiently encapsulate etoposide. Etoposide is more effective at killing cells when encapsulated in NPs than in standard formulations. Eto-NPs inhibited the proliferation of microglial precursors and c-kit in the mouse brain. + CD45 + It is taken up by cells. Eto-NPs inhibited the proliferation of microglial precursors and c-kit in the mouse brain. + CD45 + and CD45 + It can induce early apoptosis of cells.

[0094] Previous studies have hypothesized that CNS-resident microglial precursors exist, and that their ablation prior to HCT is essential for the establishment of microglial reconstitution. Herein, the use of novel tools is proposed for novel methods of identifying and transplanting these cells to generate systemic and CNS chimerism, or selective CNS chimerism by transplanted cell progeny. Thus, the present invention provides novel tools for: Identification of cells within the HSPC pool that can be used to generate brain-marrow chimerism and transcription-dependent de novo microglia upon transplantation (defined as "functional equivalents of microglial precursors"). · Optimal pathways and conditions to be used to perform transplantation of functional equivalents of microglial precursors when sustained brain and hematopoietic organ chimerism is required for disease treatment. · Optimal pathways and conditions to be used to perform transplantation of functional equivalents of microglial precursors when selective brain chimerism is sufficient and / or required for disease treatment. · Localization and targeting of microglial precursors using nanocarriers. · Targeted delivery of ablative drugs to microglial precursors for selective brain preconditioning. · Implementation of novel therapeutic strategies in the context of advanced HSC transplantation protocols for bone marrow CNS cell / microglia reconstitution for neurodegenerative diseases.

[0095] The results described herein demonstrate that optimized HSC transplantation protocols can be used to treat nervous system or central nervous system disorders, including, for example, storage disorders and neurodegenerative diseases, by replacing diseased microglia with novel cells that provide novel / therapeutic functions. To this end, molecular targets have been identified for use in treating neurodegenerative diseases via optimized microglial reconstitution approaches. Thus, the following have been shown: · Mouse and human LT-HSCs can generate cerebromyeloprogeny upon both IV and ICV delivery. · The less mature KSL fraction contributes to the development of cerebromyeloprogeny upon ICV delivery. The functional equivalent of the μ precursor within the HSPC pool is contained in LT_HSCs (both mouse and human cells). The functional equivalent of the μ precursor within the mouse HSPC pool is Fdg5+. The functional equivalent of the mu precursor within the mouse HSPC pool is CD11b negative. The functional equivalent of the mu precursor within the mouse HSPC pool is CX3Cr1 negative. The functional equivalent of the μ precursor is contained within CD34+ human HSPCs. Functional equivalents of μ precursors are enriched within the CD38− fraction of CD34+ human HSPCs.

[0096] Hematopoietic cell transplantation (HCT) Recent preclinical and clinical evidence suggests that Hematopoietic stem and progenitor cells It has been shown that HSPCs and / or their progeny can contribute to the turnover of myeloid cell populations in the brain and act as vehicles for the delivery of therapeutic molecules across the blood-brain barrier. However, it remains to be established whether the differentiation and functional characteristics of the reconstituted cells after transplantation, particularly bona fide microglia, can be reconstituted by the progeny of donor cells after transplantation. Over the last 30 years, hematopoietic cell transplantation (HCT) and hematopoietic stem cell (HSC)-based gene therapy have been applied with some benefit to patients suffering from non-hematological and non-oncological diseases affecting the nervous system, such as peroxisomal and lysosomal storage diseases (LSDs) (Cartier et al., Science 326:818-823 (2009); Biffi et al., Science 341:1233-158 (2013); Sessa et al., Lancet 388:476-487 (2016)) and neurodegenerative diseases (Simard et al., Neuron 49:489-502 (2006)). These early clinical evidence, along with supporting preclinical data, have led to a number of promising outcomes. Hematopoietic stem and progenitor cellsIt has been suggested that HSPCs, and / or their progeny, can act as vehicles for the delivery of therapeutic molecules across the blood-brain barrier (BBB). Indeed, HSPCs and / or their progeny could potentially include microglia, whose crucial role in the progression and outcome of these disorders has been extensively described (Jeyakumar et al., Brain 126, 974-987 (2003); Wada et al., Proc. Natl. Acad. Sci. USA 97, 10954-10959 (2000); Ohmi et al., Proc. Natl. Acad. Sci. USA 100, 1902-1907 (2003); Eichler et al., Ann Neurol 63, 729-742 (2008)), including microglia, which play a key role in the turnover of myeloid cell populations in the brain (Ajami et al., Nat. Neurosci 10, 1538-1543 (2007); Ajami et al., Nat. Neurosci 14, pp. 1142-1149 (2011); Biffi et al. J. Clin. Invest. 116, pp. 3070-3082 (2006); Mildner et al. Nat Neurosci. 10, pp. 1544-1553 (2007); Capotondo et al. Proc Natl Acad Sci USA. 109, pp. 15018-15023 (2012)). Importantly, transplant-derived cells have been shown to favorably influence the local environment once integrated into the affected tissue, i.e., by releasing therapeutic molecules into the brain of the transplanted mouse or patient. This concept was demonstrated in patients with demyelinating LSD metachromatic leukodystrophy treated with HSC gene therapy (Biffi et al., Science 341, 1233-158 (2013); Sessa et al., Lancet 388, 476-487 (2016)).Normal or supranormal activity and expression of the defective arylsulfatase A enzyme in patients was induced by a lentiviral vector (LV) integrated into the patient's HSCs and their progeny and measured in the cerebrospinal fluid (CSF) of treated children long after treatment (Biffi et al., Science 341, 1233-158 (2013); Sessa et al., Lancet 388, 476-487 (2016)). Of note, the enzyme cannot efficiently cross the BBB itself (Biffi et al., J. Clin. Invest. 116, 3070-3082 (2006); Matzner et al., Human Molecular Genetics 14, 1139-1152 (2005)). These findings, coupled with significant clinical benefit in patients treated at a presymptomatic stage, formally demonstrate that the patient's brain is seeded by genetically corrected HSPC progeny. However, it remains to be established whether the differentiation and functional characteristics of transplant-derived cells in the brain, particularly bona fide microglia, can be reconstituted by the progeny of donor cells after HCT as documented (Ajami et al., Nat Neurosci 10:1538-1543 (2007); Capotondo et al., Proc Natl Acad Sci USA 109:15018-15023 (2012); Bennett et al., Proc Natl Acad Sci USA 113:E1738-1746 (2016)).

[0097] Although microglia have a distinct developmental origin from bone marrow-derived myelomonocytic cells (Ginhoux et al., Science 330:841-845 (2010)), we and others have recently demonstrated that under certain experimental conditions, donor-sourced cells exhibiting a microglia-like phenotype and expressing some microglial surface markers can be successfully generated in the brains of mice transplanted with donor HSPCs. Essential for this to occur reproducibly and at a high rate is the administration of a conditioning regimen based on the alkylating agent busulfan prior to transplantation, which can ablate functionally defined, brain-resident microglial precursors (Capotondo et al., Proc Natl Acad Sci USA 109:15018-15023 (2012); Wilkinson et al., Mol Ther 21:868-876 (2013)). In this situation, donor-source cells found in the brains of transplanted animals were shown to be derived from the local proliferation and differentiation of HSPCs that migrated into the brain immediately after transplantation.

[0098] This study marks a major step toward better understanding these phenomena and improving their potential clinical translation for the treatment of neurological disorders. Indeed, we demonstrate for the first time that donor-derived bone marrow cells emerging in the brains of mice receiving HSPCs after busulfan-based preconditioning not only share microglial morphology and surface markers, but also a highly similar transcriptional profile. Furthermore, using genome-wide expression analysis, we show that transplanted HSPCs generate microglia-like progeny cells through a process that recapitulates some aspects of physiological postnatal microglial maturation. Importantly, we also clearly demonstrate that posttransplant microgliosis originates from early hematopoietic stem cells / progenitors in mice and humans, whose CXCR4 expression may favor their transport to the brain. Finally, the generation of donor-sourced microglia-like cells is also achieved here for the first time when HSPCs are administered directly into the lateral ventricles of the brains of pretreated mice, instead of intravenously. Notably, this novel delivery route, which allows for clinically relevant, rapid, and more widespread replacement of microglia compared to systemic injection, confirms that microgliosis can result from independent seeding of the brain by intravenously transplanted HSPCs (Capotondo et al., Proc Natl Acad Sci USA. 109:15018-15023 (2012)).

[0099] Overall, this study supports the validity and feasibility of using HSPCs to renew brain myeloid and microglial cells with new populations capable of exerting therapeutic effects in the central nervous system (CNS), and identifies novel modalities, such as transplantation of enriched stem cell fractions and direct delivery of HSPCs to the brain, to increase the actual contribution of transplanted cells to microgliosis.

[0100] Storage disease (SD) Storage disorders (SD) comprise a class of genetic disorders characterized by disruption of normal lysosomal function, resulting in the accumulation of incompletely degraded substrates targeted for degradation after endocytosis or autophagy. The subsequent accumulation of the substrates themselves or products of alternative metabolic pathways in lysosomes affects cellular structure and function, leading to cellular dysfunction or death. Furthermore, primary defects are frequently exacerbated by secondary responses. This is particularly relevant in the central nervous system (CNS), where neuroinflammation occurs and manifests as a primary response to substrate accumulation within microglia and astrocytes, and / or an inflammatory response to injury in primary neurons or oligodendrocytes.

[0101] Examples of SDs include lysosomal storage diseases (LSDs) (such as GM1 and GM2 gangliosidosis), alpha-mannosidosis, globoid cell leukodystrophy (GLD), neuronal ceroid lipofuscinosis (NCL), metachromatic leukodystrophy (MLD), mucopolysaccharidosis disorders (MPS), multiple sulfatase deficiency (MSD), Niemann-Pick disease, and peroxisome storage disorders (such as adrenoleukodystrophy). Approximately 50% of LSDs have CNS inovement, as in the examples listed above. A non-limiting list of exemplary SDs and their associated defective proteins is provided in Table 1. [Table 1]

[0102] The information provides some of the specific relevant LSDs for use of the HSC transplantation protocols described in the present invention.

[0103] Metachromatic leukodystrophy (MLD) Metachromatic leukodystrophy (MLD), a demyelinating LSD due to mutations in the arylsulfatase A (ARSA) gene, is the prototypical example of an LSD, with progressive accumulation of undegraded substrates in the nervous system and secondary neuroinflammation and degeneration. Genetic transmission of MLD is autosomal recessive, and its overall incidence is estimated to be between 1:40,000 and 1:100,000.

[0104] Clinical symptoms consist of severe and unrelenting motor and cognitive impairment, and disease progression is more severe in early-onset clinical variants, usually resulting in death within the first decade of life. A correlation between the phenotype of MLD patients and the type of ARSA mutation they harbor has recently been demonstrated (Cesani et al., Hum Mutat 30:E936-945 (2009); Cesani et al., Ann Neurol 75:127-137 (2014)). HSC gene therapy, using lentiviral vectors to transduce autologous HSCs and exposure to systemic busulfan pretreatment, has been shown to be effective in preventing or attenuating disease symptoms in children affected by the most severe MLD variants and in children treated before the onset of symptoms (Biffi et al., Science 341, 1233-158 (2013); Sessa et al., Lancet 388, 476-487 (2016)).

[0105] Globoid cell leukodystrophy (GLD) Globoid cell leukodystrophy (GLD), also known as Krabbe disease, is an autosomal recessive LSD caused by a deficiency of the lysosomal enzyme galactocerebrosidase (GALC), which catalyzes the catabolism of galactosylceramide (GalCer), a critical myelin component. GLD occurs in approximately 1 in 100,000 births. It usually develops during infancy and follows a rapidly fatal course, although rarer forms with slower onset also exist. The devastating neurodegenerative disorder is due to an alteration in glycosphingolipid catabolism caused by GALC deficiency. The resulting accumulation of incompletely metabolized GalCer leads to a progressive white matter brain disease affecting both the CNS and peripheral nervous system (PNS). Galactosylsphingosine (or psychosine), a substrate of GALC, is also thought to play an important role in pathogenesis. Children with GLD can be treated presymptomatically and before age 4 years with HCT from healthy matched donors, which delays disease onset and attenuates symptoms (Escolar et al., N Engl J Med. 352:2069-2081 (2005)). HSC gene therapy has also proven potentially effective in preclinical models of GLD (Gentner et al., Sci Transl Med 2 (2010)).

[0106] Mucopolysaccharidosis (MPS) Mucopolysaccharidoses (MPS) are a group of lysosomal disorders caused by the absence or dysfunction of lysosomal enzymes necessary for the breakdown of glycosaminoglycans. MPS I is classified into three subtypes based on the severity of symptoms. The three types result from the absence or insufficient levels of the enzyme alpha-L-iduronidase. MPS IH (also known as Hurler syndrome or alpha-L-iduronidase deficiency) is the most severe of the MPS I subtypes, while MPS IS, or Scheie syndrome, is the mildest form of MPS I. MPS I HS (Hurler-Scheie syndrome) is less severe than Hurler syndrome alone. MPS II, Hunter syndrome, or iduronate sulfatase deficiency, is caused by a deficiency of the enzyme iduronate sulfatase. MPS III, or Sanfilippo syndrome, is characterized by severe neurological symptoms. There are four distinct types of Sanfilippo syndrome, each caused by the alteration of a different enzyme required to completely break down the sugar chains of heparin sulfate. Sanfilippo A is the most severe form of MPS III disorder and is caused by the loss or modification of the enzyme heparan N-sulfatase. Children with Sanfilippo A have the lowest survival rate among those with MPS III disorder. Sanfilippo B is caused by the loss or deficiency of the enzyme alpha-N-acetylglucosaminase. Sanfilippo C is caused by the loss or modification of the enzyme acetyl-Co alpha-glucosaminide acetyltransferase. Sanfilippo D is caused by the loss or deficiency of the enzyme N-acetylglucosamine 6-sulfatase.

[0107] Morquio syndrome, MPS IV, results from the loss or deficiency of the enzymes N-acetylgalactosamine 6-sulfatase (type A) or beta-galactosidase (type B), which are required to break down the carbohydrate chains of keratan sulfate. Marote-Lamy syndrome, MPS VI, shares many of the physical symptoms seen in Hurler syndrome and is caused by a deficiency of the enzyme N-acetylgalactosamine 4-sulfatase. Sly syndrome, MPS VII, one of the least common forms of mucopolysaccharidoses, is caused by a deficiency of the enzyme beta-glucuronidase.

[0108] Some MPS patients have been shown to benefit from HCT from healthy matched donors, while HSC GT strategies have been optimized for some MPS cases (Visigalli et al., Blood 116:5130-5139 (2010)).

[0109] Neuronal ceroid lipofuscinosis (NCL) Neuronal ceroid lipofuscinosis is a class of inherited storage disorders that leads to progressive neurological degeneration. Some variants, such as infantile NCL (INCL), are caused by deficiencies of lysosomal enzymes. INCL is caused by mutations in the CLN2 gene, leading to deficiency of PPT1, a lysosomal enzyme responsible for membrane protein degradation. Similarly, late-infantile NCL (LINCL) is caused by deficiency of the lysosomal enzyme TPP1. Neurons are particularly susceptible to lysosomal accumulation of this storage material, and individuals with INCL and LINCL experience widespread progressive neurodegeneration in all parts of the brain, resulting in a vegetative state and death by age 8–12.

[0110] X-linked adrenoleukodystrophy (X-ALD) X-linked adrenoleukodystrophy (X-ALD) is a metabolic genetic disorder characterized by progressive inflammatory demyelination in the brain, with a prevalence of 1:17,000 in males. Mutations in the ABCD1 gene, located on chromosome Xq28, determine the loss of function of the associated ALD protein, which in turn leads to the accumulation of unbranched very-long-chain saturated fatty acids (VLCFAs) within phospholipid fractions, such as lysophagidylcholine (LPC), particularly in the brain and adrenal cortex. Phenotypic variation in X-ALD appears to be linked to brain inflammation, which causes progressive neurological decline, most often in children but also in adults with X-ALD. The onset of brain demyelination may be directly linked to the amount of VLCFAs in complex lipids and their insufficient degradation by microglial cells. Thus, although perivascular macrophages have been shown to closely follow the leading edge of demyelinating lesions and play a key role in clearing myelin remnants, microglial cells behave differently, being largely absent from the same area and becoming apoptotic in the surrounding area (Eichler et al., Ann Neurol 63:729–742 (2008)). Eichler et al. speculated that microglia are unable to degrade VLCFAs in this area, which may lead to microglial activation and apoptosis. Microglial loss and / or dysfunction may play an important role in the early stages of demyelination, primarily due to altered production of proinflammatory cytokines (CCL2, CCL4, IL-1a, CXCL8) and their ability to provide neuroprotective factors against oligodendrocyte loss. In this scenario, microglial cells may be an appropriate target for intervention in X-ALD patients with evidence of cerebral demyelination. In this light, HSC transplantation (Aubourg et al., N Engl J Med 322, 1860-1866 (1990)) and, more recently, gene therapy (Cartier et al., Science 326, 818-823 (2009); Eichler et al., N Engl J Med doi:10.1056 / NEJMoa1700554 (2017)) have been investigated as treatment options for X-ALD.

[0111] Neurodegenerative diseases Neurodegenerative disease is a class of nervous system diseases characterized by the progressive loss of neuronal structure and / or function and / or neuronal cell death.Inflammation is involved in the role of some neurodegenerative diseases.The progressive loss of motor and sensory neurons and mental ability refers to the sensory information of external objects, which is affected by various types of neurodegenerative diseases.Neurodegenerative disease non-limiting examples include ALS, for example, familial ALS or sporadic ALS, and Alzheimer's disease.

[0112] A relationship between microglia and neurodegeneration has been observed. Glial cell activation in ALS plays an important role in disease progression and the spread of pathology to other CNS regions. Abnormal microglial activation in ALS reorganizes the neurotoxic environment. Activated microglial cells are found in close proximity to Aβ plaques in AD brains.

[0113] A medical professional can diagnose a subject as having a neurodegenerative disease by evaluating one or more symptoms of the neurodegenerative disease in the subject. Non-limiting symptoms of a neurodegenerative disease in a subject include difficulty lifting the front of the legs and toes; weakness in the arms, legs, feet, or ankles; weakness or limpness in the hands; slurred speech; difficulty swallowing; muscle spasms; twitching of the arms, shoulders, and tongue; difficulty chewing; difficulty breathing; muscle paralysis; partial or complete loss of vision; double vision; tingling or pain in body parts; electric shock sensations with head movement; tremors; unsteady gait; fatigue; dizziness; memory loss; disorientation; and misunderstanding of spatial relationships. Symptoms of neurodegenerative disorders include: difficulty in reading and writing; difficulty concentrating and thinking; difficulty making judgments and decisions; difficulty planning and performing familiar tasks; depression; anxiety; social withdrawal; emotional lability; irritability; aggression; changes in sleep habits; wandering; dementia; loss of automatic movements; impaired posture and balance; muscle rigidity; bradykinesia; slow and abnormal eye movements; involuntary shaking or twisting movements (chorea); involuntary, persistent muscle contractures (dystonia); inflexibility; lack of impulse control; and appetite changes. A medical professional may also base a diagnosis in part on a subject's family history of neurodegenerative disorders. A medical professional may diagnose a subject as having a neurodegenerative disorder when the subject presents to a health care facility (e.g., a clinic or hospital). In some cases, a medical professional may diagnose a subject as having a neurodegenerative disorder while the subject is hospitalized at a health care facility. Typically, a doctor diagnoses a neurodegenerative disorder in a subject after one or more symptoms appear.

[0114] Treatment method The present invention provides a method of treating a disease and / or disorder, or a symptom thereof, comprising administering to a subject (e.g., a mammal, such as a human) a therapeutically effective amount of a pharmaceutical composition comprising the microglial precursors described herein. Accordingly, one embodiment is a method of treating a subject suffering from or susceptible to a disease or disorder, or a symptom thereof. The method comprises administering to the mammal a therapeutic amount of the cells herein sufficient to treat the disease or disorder, or a symptom thereof, under conditions such that the disease or disorder is treated.

[0115] The methods herein include administering to a subject (including a subject identified as in need of such treatment) an effective amount of a cell described herein or a composition described herein to produce such an effect. Identification of a subject in need of such treatment may be the judgment of the subject or a medical professional and may be subjective (e.g., opinion) or objective (e.g., measurable by a test or diagnostic method).

[0116] The engraftment of transplanted cells results in the expression or activity of polypeptides or other therapeutic agents. For example, lysosomal storage disorders are caused by the lack or loss of function of lysosomal enzymes. Transplanted hematopoietic cells expressing therapeutic proteins (e.g., enzymes) either intravenously or by recombinant methods can engraft and differentiate into microglia, thereby treating enzyme deficiencies. Furthermore, transplanted cells can serve as a vehicle for therapeutic polypeptides in neurodegenerative diseases.

[0117] In certain embodiments, engraftment is enhanced by ablating existing microglia (e.g., with alkylating agents). In particular, nanoparticle delivery of alkylating agents can be effective in creating an environment that allows engraftment of microglial precursors derived exclusively from transplanted cells in the brain. Furthermore, delivery of bone marrow-derived microglial precursor-enriched populations by standard or innovative routes allows sustained reconstitution of brain microglia with donor or engineered cells.

[0118] The methods herein include administering to a subject (including a subject identified as needing such treatment) an effective amount of a compound described herein or a composition described herein to produce such an effect. Identification of a subject in need of such treatment may be the judgment of the subject or a medical professional and may be subjective (e.g., opinion) or objective (e.g., measurable by a test or diagnostic method). Such treatment would be preferably administered to subjects, particularly those suffering from, having, susceptible to, or at risk for a disease, disorder, or symptom thereof. Determination of such "at risk" subjects can be made objectively or subjectively by diagnostic testing or the opinion of the subject or medical provider (e.g., genetic testing, enzyme or protein markers, Markers (as defined herein), family history, etc.). The compounds herein can also be used to treat any other disorder, including multiple sclerosis, that may involve deficiency or loss of myelination.

[0119] The present invention provides a method for delivering nanoparticles comprising a cytotoxic agent and / or ablating microglial cells or their precursors, the method comprising administering nanoparticles comprising a cytotoxic agent to a subject (e.g., a mammal such as a human).

[0120] Generally, the term "nanoparticle" refers to any particle having a diameter of less than 1000 nm. In certain preferred embodiments, the nanoparticles of the present invention have a maximum dimension (e.g., diameter) of 500 nm or less. In other preferred embodiments, the nanoparticles of the present invention have a maximum dimension ranging from 25 nm to 200 nm. In other preferred embodiments, the nanoparticles of the present invention have a maximum dimension ranging from 100 nm or less. In other preferred embodiments, the nanoparticles of the present invention have a maximum dimension ranging from 35 nm to 60 nm. Nanoparticles encompassed by the present invention can be provided in various forms, for example, as solid nanoparticles (e.g., metals such as silver, gold, iron, and titanium), non-metallic, lipid-based solids, polymers, nanoparticle suspensions, or combinations thereof. Metallic, dielectric, and semiconductor nanoparticles, as well as hybrid structures (e.g., core-shell nanoparticles), can also be prepared. Nanoparticles composed of semiconductor materials can also be labeled quantum dots if they are small enough (typically less than 10 nm) that quantization of electronic energy levels occurs. Such nanoscale particles are used in biomedical applications as drug carriers or imaging agents and may be adapted for similar purposes of the present invention. Semisolid and soft nanoparticles have been produced and are within the scope of the present invention. The prototype nanoparticle of semisolid natural products is the liposome. Various types of liposomal nanoparticles are currently used clinically as delivery systems for anticancer drugs and vaccines. Nanoparticles with half hydrophilic and half hydrophobic, called Janus particles, are particularly effective in stabilizing emulsions. They can self-assemble at the water / oil interface and act as solid surfactants. In one embodiment, nanoparticles based on self-assembling bioadhesive polymers are contemplated, all of which are applicable to oral, intravenous, and nasal drug delivery to the brain. Other embodiments, such as oral absorption and ocular delivery of hydrophobic drugs, are also contemplated.Molecular envelope technology involves engineered polymer envelopes that are protected and delivered to the site of disease (Mazza et al., ACS Nano 7, 1016-1026 (2013); Siew et al., Mol Pharm 9, 14-28 (2012); Lalatsa et al., J Control Release 161, 523-536 (2012); Lalatsa et al., Mol Pharm 9, 1665-1680 (2012); Garrett et al., J Biophotonics 5, 458-468 (2012); Uchegbu, Expert Opin Drug Deliv 3, 629-640 (2006); Uchegbu et al., Int J Pharm 224, 185-199 (2001); Qu et al., Biomacromolecules 7, pp. 3452-3459 (2006).

[0121] Several types of particle delivery systems and / or formulations are known to be useful for a diverse range of biomedical applications. Generally, a particle is defined as a small object that, as a whole, behaves as a unit with respect to its transport and properties. Particles are further classified according to their diameter. Coarse particles range between 2,500 and 10,000 nanometers. Fine particles are between 100 and 2,500 nanometers in size. Ultrafine particles, or nanoparticles, are generally between 1 and 100 nanometers in size. The basis for the 100 nm threshold is that novel properties that distinguish particles from bulk materials typically develop at a critical length scale below 100 nm.

[0122] As used herein, a particle delivery system / formulation is defined as any biological delivery system / formulation comprising particles according to the present invention. A particle according to the present invention is any entity having a maximum dimension (e.g., diameter) of less than 100 microns (.mu.m). In some embodiments, particles of the present invention have a maximum dimension of less than 10 p.m. In some embodiments, particles of the present invention have a maximum dimension of less than 2000 nanometers (nm). In some embodiments, particles of the present invention have a maximum dimension of less than 1000 nanometers (nm). In some embodiments, particles of the present invention have a maximum dimension of less than 900 nm, 800 nm, 700 nm, 600 nm, 500 nm, 400 nm, 300 nm, 200 nm, or 100 nm. Typically, particles of the present invention have a maximum dimension (e.g., diameter) of 500 nm or less. In some embodiments, particles of the present invention have a maximum dimension (e.g., diameter) of 250 nm or less. In some embodiments, particles of the present invention have a maximum dimension (e.g., diameter) of 200 nm or less. In some embodiments, particles of the present invention have a maximum dimension (e.g., diameter) of 150 nm or less. In some embodiments, particles of the present invention have a maximum dimension (e.g., diameter) of 100 nm or less. Smaller particles, for example, having a maximum dimension of 50 nm or less, are used in some embodiments of the present invention. In some embodiments, particles of the present invention have a maximum dimension ranging between 25 nm and 200 nm.

[0123] Characterization of particles (including, for example, characterization of morphology, size, etc.) is carried out using a variety of different techniques. Common techniques are electron microscopy (TEM, SEM), atomic force microscopy (AFM), dynamic light scattering (DLS), X-ray photoelectron spectroscopy (XPS), X-ray powder diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF), ultraviolet-visible spectroscopy, dual polarization interferometry and nuclear magnetic resonance (NMR). Characterization (size measurement) is carried out on the original particle (i.e., before loading) or after loading cargo (as used herein, cargo refers to, for example, one or more components of CRISPR-Cas system, such as CRISPR enzyme or mRNA, or guide RNA, or any combination thereof, and can include additional carriers and / or additives), and results in particles of optimal size for delivery for any of the in vivo, ex vivo and / or in vivo applications of the present invention. In certain preferred embodiments, particle size (eg, diameter) characterization is based on measurements using dynamic laser scattering (DLS).

[0124] Particulate delivery systems within the scope of the present invention may be provided in any form, including but not limited to, solids, semi-solids, emulsions, or colloidal particles. Thus, any of the delivery systems described herein may be provided as particulate delivery systems within the scope of the present invention.

[0125] antibody As reported herein, antibodies that specifically bind to markers (e.g., of microglial cells or their precursors) are useful in methods of the present invention, including therapeutic methods. In certain embodiments, the present invention provides a method of ablating microglia, comprising contacting microglia with nanoparticles having a capture molecule that specifically binds to a marker of microglial cells and containing a cytotoxic agent (e.g., an alkylating agent).

[0126] Antibodies can be intact immunoglobulins derived from natural or recombinant sources, or immunoreactive portions of intact immunoglobulins. Antibodies are usually tetramers of immunoglobulin molecules. Tetramers can be naturally occurring or can be reconstituted from single-chain antibodies or antibody fragments. As used herein, the term "antibody" refers not only to intact antibody molecules but also to fragments of antibody molecules that retain antigen-binding ability. Such fragments are also well known in the art and are regularly used both in vivo and in vivo. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments consisting of either the VL or VH domains that exhibit sufficient affinity for the target; single-domain antibodies such as linear antibodies, scFv antibodies, and camelid antibodies (Riechmann, 1999, Journal of Immunological Methods 231:25-38); and multispecific antibodies formed from antibody fragments.

[0127] Antibodies in the present invention can exist in a variety of forms, including, for example, polyclonal antibodies, monoclonal antibodies, Fv, Fab and F(ab')2, as well as single-chain antibodies (scFv), humanized antibodies and human antibodies (Harlow et al., 1999, In: Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989, In: Antibodies: A Laboratory Manual, Cold Spring Harbor, New York; Houston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al., 1988, Science 242:423-426). For example, F(ab')2 and Fab fragments, which lack the Fc fragment of an intact antibody, are cleared more rapidly from the circulation and may have less nonspecific tissue binding than intact antibodies (Wahl et al., J. Nucl. Med. 24:316-325 (1983)). Thus, antibodies of the present invention include, but are not limited to, whole natural antibodies, bispecific antibodies; chimeric antibodies; Fab, Fab', single-chain V-region fragments (scFv), fusion polypeptides, and unconventional antibodies.

[0128] Non-conventional antibodies include, but are not limited to, nanobodies, linear antibodies (Zapata et al., Protein Eng. 8(10):1057-1062, 1995), single-domain antibodies, single-chain antibodies, and antibodies with multiple valencies (e.g., diabodies, tribodies, tetrabodies, and pentabodies). Nanobodies are the smallest fragments of natural heavy-chain antibodies that have evolved to be fully functional in the absence of light chains. Nanobodies possess the affinity and specificity of conventional antibodies, but they are only half the size of single-chain Fv fragments. The result of this unique structure, combined with exceptional stability and a high degree of homology to human antibody frameworks, is that nanobodies can bind to therapeutic targets inaccessible to conventional antibodies. Recombinant antibody fragments with multiple valencies offer high binding affinity and unique target specificity to cancer cells. These scFv multimers (e.g., diabodies, tetrabodies) offer an improvement over parental antibodies, as small molecules approximately 60-100 kDa in size achieve faster blood clearance and rapid tissue uptake. See Power et al. (Generation of recombinant multimeric antibody fragments for tumor diagnosis and therapy. Methods Mol Biol 207:335-50, 2003); and Wu et al. (Anti-carcinoembryonic antigen (CEA) diabody for rapid tumor targeting and imaging. Tumor Targeting 4:47-58, 1999).

[0129] Various techniques for making and using unconventional antibodies have been described. Bispecific antibodies generated using leucine zippers have been described by Kostelny et al. (J. Immunol. 148(5):1547-1553, 1992). Diabody technology has been described by Hollinger et al. (Proc. Natl. Acad. Sci. USA 90:6444-6448, 1993). Another strategy for making bispecific antibody fragments using single-chain Fv (sFv) dynamides has been described by Gruber et al. (J. Immunol. 152:5368, 1994). Trispecific antibodies have been described by Tutt et al. (J. Immunol. 147:60, 1991). Single chain Fv polypeptide antibodies can be prepared by directly linking V polypeptides with or without a peptide-encoding linker as described by Huston et al. (Proc. Nat. Acad. Sci. USA, 85:5879-5883, 1988). H - and V L - Including covalently linked VH::VL heterodimers that can be expressed from nucleic acids, including coding sequences. See also U.S. Patent Nos. 5,091,513, 5,132,405, and 4,956,778; and U.S. Patent Publication Nos. 20050196754 and 20050196754.

[0130] In various embodiments, the antibody is monoclonal. Alternatively, the antibody is a polyclonal antibody. The preparation and use of polyclonal antibodies are also known to those skilled in the art. The present invention also encompasses hybrid antibodies, in which a pair of heavy and light chains is obtained from a first antibody, while the other pair of heavy and light chains is obtained from a different second antibody. Such hybrids can also be formed using humanized heavy and light chains. Such antibodies are often referred to as "chimeric" antibodies.

[0131] Intact antibodies are generally said to contain "Fc" and "Fab" regions. The Fc region is involved in complement activation and not antigen binding. Antibodies from which the Fc' region has been enzymatically cleaved or which have been produced without the Fc' region (designated "F(ab')2" fragments) retain both of the intact antibody's antigen-binding sites. Similarly, antibodies from which the Fc region has been enzymatically cleaved or which have been produced without the Fc region (designated "Fab'" fragments) retain one of the intact antibody's antigen-binding sites. Fab fragments consist of a covalently bound antibody light chain and a portion of the antibody heavy chain (designated "Fd"). The Fd fragment is the primary determinant of antibody specificity (a single Fd fragment can bind up to 10 different light chains without altering antibody specificity). Isolated Fd fragments retain the ability to specifically bind to an immunogenic epitope.

[0132] Methods for preparing antibodies are well known to those skilled in the science of immunology. Antibodies can be generated by any method known in the art, using a soluble polypeptide or its immunogenic fragment as an immunogen. One method for obtaining antibodies is to immunize a suitable host animal with an immunogen and follow standard procedures for producing polyclonal or monoclonal antibodies. The immunogen will promote the presentation of the immunogen on the cell surface. Immunization of a suitable host can be carried out in several ways. A nucleic acid sequence encoding a polypeptide or its immunogenic fragment can be provided to the host in a delivery vehicle that is taken up by the host's immune cells. The cells then express the polypeptide, thereby generating an immunogenic response in the host. Alternatively, a nucleic acid sequence encoding a human polypeptide or its immunogenic fragment can be expressed in cells in vivo, followed by isolating the polypeptide and administering it to a suitable host where antibodies will be raised.

[0133] Alternatively, antibodies may be derived from an antibody phage display library, if desired. Bacteriophage are capable of infecting and growing within bacteria, which can be engineered to combine with human antibody genes and display human antibody proteins. Phage display is the process by which phage are engineered to "display" human antibody proteins on their surface. Genes from a human antibody gene library are inserted into a population of phages. Each phage carries genes for a different antibody and thus displays a different antibody on its surface.

[0134] Antibodies produced by any method known in the art can then be purified from the host. Antibody purification methods can include salt precipitation (e.g., with ammonium sulfate), ion exchange chromatography (e.g., on a cation or anion exchange column, preferably performed at neutral pH and eluted with a step gradient of increasing ionic strength), gel filtration chromatography (including gel filtration HPLC), and chromatography on affinity resins (such as protein A, protein G, hydroxyapatite, and anti-immunoglobulin).

[0135] Antibodies can be conveniently produced from hybridoma cells engineered to express the antibody. Methods for producing hybridomas are well known in the art. Hybridoma cells can be cultured in a suitable medium, and the spent medium can be used as an antibody source. Polynucleotides encoding the antibody of interest can then be obtained from the antibody-producing hybridoma, and the antibody can then be produced synthetically or recombinantly from these DNA sequences. For large-scale production of antibodies, it is generally more convenient to obtain ascites fluid. Methods for increasing ascites fluid generally involve injecting hybridoma cells into immunologically naive, histocompatible, or immunotolerant mammals, particularly mice. The mammal may be primed for ascites production prior to administration of a suitable composition (e.g., pristane).

[0136] The monoclonal antibodies (Mabs) produced by the methods of the present invention can be "humanized" by methods known in the art. A "humanized" antibody is one in which at least a portion of its sequence has been altered from its initial form to make it more human immunoglobulin-like. Techniques for humanizing antibodies are particularly useful, especially when non-human animal (e.g., murine) antibodies are produced. Examples of methods for humanizing murine antibodies are provided in U.S. Patent Nos. 4,816,567, 5,530,101, 5,225,539, 5,585,089, 5,693,762, and 5,859,205.

[0137] Recombinant Polypeptide Expression To express the polypeptide of the present invention, the DNA molecule obtained by any of the methods described herein or known in the art can be inserted into an appropriate expression vector by techniques known in the art. For example, double-stranded DNA can be cloned into a suitable vector by restriction enzyme ligation, including the use of synthetic DNA linkers, or by blunt-end ligation. DNA ligase is usually used to ligate DNA molecules, and undesired ligation can be avoided by treatment with alkaline phosphatase.

[0138] Thus, the present invention includes vectors (e.g., recombinant plasmids) that contain the nucleic acid molecules (e.g., genes, or recombinant nucleic acid molecules encoding genes) described herein. The term "recombinant vector" includes vectors (e.g., plasmids, phages, phasmids, viruses, cosmids, fosmids, or other purified nucleic acid vectors) that have been altered, modified, or engineered to contain more, less, or different nucleic acid sequences than are contained in the native or naturally occurring nucleic acid molecule from which the recombinant vector is derived. For example, a recombinant vector can contain a nucleotide sequence encoding a polypeptide, or a fragment thereof, operably linked to regulatory sequences, e.g., promoter sequences, terminator sequences, etc., as defined herein. Recombinant vectors that permit expression of the genes or nucleic acids they contain are referred to as "expression vectors."

[0139] In some of the molecules of the present invention described herein, one or more DNA molecules having a nucleotide sequence encoding one or more polypeptides of the present invention are operably linked to one or more regulatory sequences that allow the desired DNA molecule to be incorporated into a prokaryotic host cell. Cells that have been stably transformed by the introduced DNA can be selected, for example, by introducing one or more markers that allow for the selection of host cells containing an expression vector. The selectable marker gene can be directly linked to the nucleic acid sequence to be expressed or can be introduced into the same cell by co-transfection. Additional elements may also be required for optimal synthesis of the proteins described herein. The use of these additional elements will be apparent to those skilled in the art.

[0140] Important factors in selecting a particular plasmid or viral vector include, but are not limited to, the ease with which recipient cells containing the vector can be recognized and selected from those that do not contain the vector, the desired vector copy number in a particular host, and whether it is desirable to be able to "shuttle" the vector between host cells of different species.

[0141] Once a vector has been constructed to contain a DNA sequence for expression, it may be introduced into a suitable host by one or more of a variety of suitable methods known in the art, including, but not limited to, transformation, transfection, conjugation, protoplast fusion, electroporation, calcium phosphate precipitation, direct microinjection, etc.

[0142] After introduction of one or more vectors, host cells are typically grown in a selective medium that selects for the growth of vector-containing cells. Recombinant protein expression can be detected by immunoassays, including Western blot analysis, immunoblots, and immunofluorescence. Purification of recombinant proteins can be performed by any conventional procedure known in the art or described herein, including extraction, precipitation, chromatography, and electrophoresis. A further purification procedure that can be used to purify proteins is affinity chromatography, which uses a monoclonal antibody that binds to the target protein. Typically, a crude preparation containing a recombinant protein is passed through a column to which a suitable monoclonal antibody is immobilized. The protein typically binds to the column via the specific antibody, while impurities pass through. After washing the column, the protein is eluted from the gel, for example, by changing the pH or ionic strength.

[0143] Methods for assessing therapeutic efficacy In one approach, the efficacy of treatment is assessed, for example, by measuring the biological function (e.g., neuronal function) of the treated organ. Such methods are standard in the art and are described, for example, in Textbook of Medical Physiology (10th ed.) (Guyton et al., WB Saunders Co., 2000). In particular, the method of the present invention improves the biological function of tissue or organ by at least 5%, 10%, 20%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, or even 300%, 400%, or 500%. Preferably, the tissue is neuronal tissue, and preferably, the organ is the brain.

[0144] In another approach, the therapeutic efficacy of the method of the present invention is assayed by measuring the increase in cell number in a treated tissue or organ compared to a corresponding control tissue or organ (e.g., a tissue or organ that has not received treatment). Preferably, the cell number in the tissue or organ is increased by at least 5%, 10%, 20%, 40%, 60%, 80%, 100%, 150%, or 200% relative to the corresponding tissue or organ. Methods for assaying cell proliferation are known to those skilled in the art and are described, for example, in Bonifacino et al. (Current Protocols in Cell Biology Loose-leaf, John Wiley and Sons, Inc., San Francisco, Calif.). For example, an assay for cell proliferation may involve measuring DNA synthesis during cell replication. In one embodiment, DNA synthesis is measured by [ 3H ]-thymidine or 5-bromo-2 * It is detected using labeled DNA precursors, such as -deoxyuridine [BrdU], which are added to cells (or animals) and then the incorporation of these precursors into genomic DNA during the S phase (replication) of the cell cycle is detected (Ruefli-Brasse et al., Science 302(5650):1581-4, 2003; Gu et al., Science 302(5644):445-9, 2003).

[0145] kit The present invention provides kits for the treatment or prevention of nervous system diseases or disorders of the central nervous system (e.g., storage disorders, lysosomal storage disorders, neurodegenerative diseases, etc.). In one embodiment, the kit comprises a composition containing isolated hematopoietic stem cells expressing a therapeutic polypeptide. In another embodiment, the kit comprises nanoparticles for pre-ablation of endogenous microglial cells.

[0146] In some embodiments, the kit includes a sterile container containing the therapeutic or prophylactic cell composition. Such a container may be a box, an ampoule, a bottle, a vial, a tube, a bag, a pouch, a blister pack, or other suitable container form known in the art. Such a container may be made of plastic, glass, laminated paper, metal foil, or other material suitable for holding pharmaceuticals.

[0147] If desired, the agents of the present invention are supplied with instructions for administering the agent to a subject having or at risk of developing a nervous system disease or central nervous system disorder. The instructions generally include information regarding the use of the composition for the treatment or prevention of the disease or disorder. In other embodiments, the instructions include at least one of the following: a description of the therapeutic agent, dosage schedules and administration for the treatment or prevention of a nervous system disease or its symptoms, precautions, warnings, indications, counter-indications, overdose information, adverse reactions, animal pharmacology, clinical reviews, and / or references. The instructions may be printed directly on the container (if provided), or may be affixed as a label to the container, or may be a separate sheet, pamphlet, card, or holder in or associated with the container.

[0148] The practice of the present invention employs, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are well within the skill of those in the art. Such techniques are fully explained in such publications as "Molecular Cloning: A Laboratory Manual," 2nd Edition (Sambrook, 1989); "Oligonucleotide Synthesis" (Gait, 1984); "Animal Cell Culture" (Freshney, 1987); "Methods in Enzymology," "Handbook of Experimental Immunology" (Weir, 1996); "Gene Transfer Vectors for Mammalian Cells" (Miller and Calos, 1987); "Current Protocols in Molecular Biology" (Ausubel, 1987); "PCR: The Polymerase Chain Reaction" (Mullis, 1994); and "Current Protocols in Immunology" (Coligan, 1991). These techniques are applicable to the production of the polynucleotides and polypeptides of the invention and thus may be considered in making and practicing the invention. Particularly useful techniques for certain embodiments are described in the sections that follow.

[0149] The following examples are put forward so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the assay, screening and treatment methods of the present invention, and are not intended to limit the scope of what the inventors regard as their invention. [Example]

[0150] Example 1 Intraventricular injection of mouse HSPCs leads to rapid and robust engraftment of bone marrow cells in the brain. Recent results (Capotondo et al., PNAS 2012) suggest that engraftment and persistent donor chimerism in the bone marrow hematopoietic niche are not necessarily required to obtain myeloid cell reconstitution in the brain after HCT. Based on these findings, we evaluated whether myeloid and microglia-like cell reconstitution occurs upon direct transplantation of HSPCs into the ventricular space in pre-treated mice. Mouse strain-negative (Lin - )HSPC(3×10 5 The cells (1000 cells) were labeled with a lentiviral vector (LV) encoding GFP and transplanted by ICV injection into mice after exposure to a myeloablative busulfan dose or lethal irradiation (Figure 1A). Interestingly, transplantation resulted in a significant increase in CD45 expression in ICV-transplanted mice. + CD11b + In the cerebrospinal compartment, GFP chimerism, likely due to local proliferation of transplanted cells, was highly and gradually increased (Fig. 1B). + Control mice IV-transplanted with HSPCs served as a comparison condition. Notably, the rate of microglial reconstitution was much more rapid, with GFP + The degree of GFP chimerism was higher when HSPCs were transplanted ICV compared with IV (Figure 1B) (significant effects of route and time of cell administration were demonstrated by two-way ANOVA analysis). This is surprising considering the lower number of cells transplanted ICV compared with IV. As with IV infusion, recipient mice pretreated with busulfan showed higher cerebrospinal donor chimerism after ICV cell transplant compared with irradiated animals.

[0151] Immunofluorescence analysis of sagittal brain sections from the contralateral side of the cell injection in ICV-transplanted mice consistently demonstrated abundant donor-derived cells expressing GFP distributed throughout the recipient mouse brain (Figure 1C). + Iba1 + Branching parenchymal cells are often grouped into small clusters and show the highest GFP +Cell abundance was recovered in the olfactory bulb, hypothalamic region, basal ganglia, subventricular zone and periventricular region, striatum, and pons (Figure 1D). Importantly, GFP + The cell morphology resembled that of parenchymal microglial cells, with branched structures and thin processes emerging from the cell body already after a relatively short period of transplantation (45-60 days).

[0152] Example 2 Intraventricular infusion of human HSPCs results in rapid and robust myeloid cell engraftment in the brain. To gain insight into the clinical relevance of this phenomenon, we tested the ability of human HSPCs to generate microglia-like cells upon ICV delivery in a pre-treated immunodeficient animal model. + Cells were isolated from umbilical cord blood transduced with LV encoding GFP and designated NOD.Cg-Prkdc scid Il2rg tm1WjI We further investigated the role of ICV cell transplantation in increasing the engraftment potential for therapeutic molecules to be delivered to the brain by using a newly generated mouse model, Rag, which recapitulates the lysosomal disease metachromatic leukodystrophy (MLD) due to arylsulfatase A (ARSA) deficiency in an immunocompromised background. - / - γ-chain - / - As2 - / These mice received human umbilical cord blood CD34+ cells transduced with LV encoding ARSA via IV or ICV infusion alone, or via a combination of IV and ICV routes (Biffi et al., Science 341:1233-158 (2013); Sessa et al., Lancet 388:476-487 (2016)) (Figure 1E).

[0153] Interestingly, a well-defined human bone marrow (CD45 + CD11b +Cell progeny were identified in the brains of transplanted mice for long periods after both IV and ICV transplantation (Figure 1F). ICV cell delivery resulted in greater human cell engraftment in the brain compared with IV delivery (Figure 1G). ICV cell delivery combined with IV resulted in even greater human cell engraftment in the brain (Figure 1G). In all tested transplantation conditions, human cells significantly expressed the microglial markers CX3CR1 and CD11b in cytofluorimetric analysis (Figure 1F). Engrafted cells were distributed within the brain parenchyma and displayed morphological characteristics of microglial cells. Engrafted cells also expressed the markers Iba1 and CD11b (Figure 1H), but did not express CD68 and CD163, which are mostly associated with macrophages (Figure 1H). In the case of ICV delivery, progeny cells were identified and typically sorted into small clusters within the same region; in this case, progeny of HSPCs from ICV-transplanted mice were identified in the subventricular zone.

[0154] Example 3 While ICV-injected HSPCs engraft and expand in the brain, they do not engraft in hematopoietic organs. GFP-labeled Lin - Short-term flow cytometric monitoring of mice transplanted with HSPCs IV or ICV demonstrated the presence, persistence, and modest expansion of ICV-delivered cells in the brains of recipient animals (Figure 2A). In contrast, negligible amounts of GFP were detected in the bone marrow of ICV-transplanted mice. + Only GFP cells were detected (<1%) (Figure 2B). + The cells transiently upregulated early hematopoietic markers (Figure 2C), followed by upregulation of CD11b, CX3CR1, and CD115 microglial markers to levels similar to endogenous microglia (Figure 2D). - CD45 + Endogenous cells transiently and slightly downregulated CD115 (Fig. 2D), a possible effect of busulfan treatment.

[0155] Example 4 Optimization of combinatorial transplantation protocols will allow for modulation of IV versus ICV transplanted cell contribution for appropriate clinical use. The development of ICV cell delivery for cell and gene therapy in pre-treated recipients requires optimization of transplant conditions according to the various anticipated target diseases. Various options are available that can be adapted to the various goals depending on the intended disease. In particular, the following two scenarios can be considered: A) To target efficient and rapid microglial reconstitution, reconstitution of both hematopoietic tissues (including extra-CNS bone marrow populations) and microglia with i) autologous gene-corrected or ii) post-transplant HSCs from healthy donors for the treatment of SD with systemic and neurological disorders; examples of diseases amenable to this approach include MLD, GLD, MPS I, MPS II, and MPS III. B) Microglial reconstitution with autologous gene-corrected HSCs after transplantation to treat SD with exclusively neurological or neurodegenerative diseases; examples of diseases amenable to this approach include INCL, GM1 gangliosidosis, PD, ALS, and AD.

[0156] To develop an effective protocol to be used in these two situations to renew the cerebrospinal fluid population with cells of donor origin, we performed IV transplants (of origin, and Lin) at various timings (IV and ICV transplants delivered on the same day or IV delivery 5 days after ICV). - , c kit + Sca1 + Lin - (KSL) cells and whole BM) and ICV transplantation (Lin - Differentially labeled cells (HSPCs) (Figures 3B and 3G). In these situations, the progeny of ICV-transplanted cells remained restricted to the CNS and were not detected at significant levels in hematopoietic tissues, i.e., the BM (Figures 3D and 3H). Importantly, combined ICV delivery of HSPCs with HSPCs, or KSL, or whole BM, resulted in the differentiation of Lin in each of the tested situations. -Compared to the control condition of IV alone, brain-myeloid cell chimerism was increased. A) The contribution of IV- and ICV-infused HSPCs to brain-bone marrow chimerism was equal in certain conditions; in particular, greater chimerism of cells at the brain and BM level due to brain chimerism consisting of both ICV and IV cell progeny was observed in both IV and ICV-infused HSPCs. - Combining HSPCs was achieved by transplanting cells at both sites on the same day; similar results were obtained by Lin et al. - While chimerism was achieved by combining ICV with IV of whole BM, in all tested conditions, slightly lower chimerism was associated with the use of KSL IV; thus, this protocol could be applied to conditions such as MLD or MPS II, as described in Figure 6. Regarding the clinical translation of this protocol as a combined / co-transplantation approach intended to develop a brain engraftment zone for transplanted cells / their progeny, we envision the following options: in the case of gene therapy, autologous CD34 cells transduced with vectors encoding the gene of interest for each disease, + Lin, the cell - The human equivalent of cells is transplanted both ICV and IV on the same day. When allogeneic healthy donor cells are used, donor CD34 + Cells are administered ICV, while either unmanipulated bone marrow or the apheretic product is transplanted IV on the same day as the ICV transplant. B) The contribution of ICV-infused HSPCs to brain-bone marrow chimerism exceeds that of IV-coinfused HSPCs under certain conditions. Notably, the lowest contribution to brain chimerism by IV cell progeny was observed in ICV-transplanted Lin cells on day 5. -This was realized in the context of high chimerism values ​​in the combination of IV-transplanted whole BM cells with ICV cell progeny; this protocol allows for almost exclusively chimerism of ICV cell progeny and is thus applicable to conditions related exclusively to CNS disorders, such as INCL, as described in Figures 7A-7E. Regarding the clinical translation of this protocol as a combination / co-transplantation approach intended to promote robust engraftment of the transplanted engineered cells / their progeny exclusively in the brain, we envision the following options: for each disease, autologous CD34 cells transduced with a vector encoding the gene of interest; + While the cells are transplanted ICV, unmanipulated autologous bone marrow / apheretic product is infused IV either on the same day or, ideally, 5 days after ICV injection to further reduce competition with the ICV transplanted cells; we do not anticipate the use of this protocol in the allogeneic setting, where hematopoietic engraftment of the transplanted cells is required for establishment of donor tolerance.

[0157] Example 5 The progeny of both IV and ICV transplanted HSPCs have transcriptional profiles consistent with microglia. Donor reconstitution of brain bone marrow cells after HCT has been shown to result from local expansion and differentiation of a portion of the early brain HSPC transplant, finding favorable conditions in the myeloablated recipient brain (Capotondo et al., Proc Natl Acad Sci USA. 109:15018-15023 (2012)). Transplanted brain bone marrow cells share antigenic characteristics with bone marrow cells isolated from the brains of newborn mice (Figures 3B and 4B). Indeed, in both situations, CD45, previously described by the inventors as a transiently expanding microglia (TAμ), is expressed. + CD11b low Interestingly, TAμ cells derived from transplanted mouse brains were highly and preferentially enriched for donor elements during the early post-transplant phase (Figure 4B), whereas only during the late post-transplant phase were they identified as microglia (μ) based on antigenic and morphological characteristics, expressing CD45 + CD11b+ / high Similar high levels of donor chimerism are observed in the cells (Capotondo et al., Proc Natl Acad Sci USA. 109:15018-15023 (2012)). Interestingly, we found that CD45 expression of ICV-infused HSPCs was significantly higher than that of the control group. + CD11b high GFP + We observed that μ progeny were more abundant than μ progeny of IV-injected cells in the presence of similar, but overall higher contributions of TA μ cells (Fig. 4A). To interpret these findings and further characterize donor-derived cells, we performed GFP-positive and GFP-negative TA μ progeny after brusulfan pretreatment. + μ and TAμ cells (total population and / or GFP) from mice transplanted ICV or IV with HSPCs 90 days earlier, as well as from adult and p10 control animals + vs. GFP -Fractions) (Figure 4B) were sorted by FACS. Previously identified microglial genes were amplified by real-time PCR (Tmem119, Tgfbr1, P2ry13, Mertk, Olfml3) on sorted brain bone marrow cells and on bone marrow macrophages from control adult animals (Bennet et al., Proceedings of the National Academy of Sciences of the United States of America 113:E1738-1746 (2016); Butovsky et al., Nature neuroscience 17:131-143 (2014); Chiu et al., Cell Rep 4:385-401 (2013); Hickman et al., Nature neuroscience 16:1896-1905 (2013); Grommes et al., Journal of neuroimmune pharmacology 3:130-140 (2008)). Cells isolated from the brains of ICV- and IV-transplanted mice were compared with microglial gene expression profiles, and ANOVA P values ​​with Tukey's post-hoc test were obtained. Interestingly, cells isolated from the brains of ICV- and IV-transplanted mice showed expression of these genes at levels similar to those of μ cells isolated from control mice, rather than macrophages (Figure 4C). Furthermore, within the μ and TAμ fractions, GFP expression of ICV-transplanted HSPCs was significantly higher. + Offspring were adult control μ and (GFP) mice from IV transplanted mice. - and GFP + The expression levels of selected genes were very similar to those of IV-implanted mice (especially GFP). + ) and at slightly lower levels in TAμ populations isolated from p10 mice, all of which indicate that i) the progeny of both IV- and ICV-transplanted HSPCs have a transcriptional profile consistent with microglia, and ii) the progeny of ICV-injected HSPCs in the brain are more similar to microglia than the progeny of IV-injected HSPCs.

[0158] Example 6 Bone marrow cells derived from transplanted mouse brains exhibit functional characteristics similar to those of mature microglia. To analyze transcriptome differences between μ and TAμ cells from transplanted mice and mature μ recovered from control naive animals, genome-wide expression analyses were performed on sorted μ and TAμ populations from mice transplanted 3 months earlier with GFP-expressing HSPCs, as well as from adult and p10 control naive mice, using the Illumina RNA-Seq platform (Figures 4D and 4E). To examine their gene expression differences, the resulting expression datasets were isolated alongside those from Gosselin and colleagues (Gosselin et al., Cell 159:1327-1340 (2014)), focusing specifically on the 239 genes identified by Butovsky (Butovsky et al., Nature neuroscience 17:131-143 (2014)) (Figures 4D and 4E). Interestingly, all microglial samples included in this analysis clustered closely together (Figures 4D and 4E), confirming that the reconstituted μ and TAμ cells after transplantation shared gene expression patterns consistent with those of microglia. Differential gene expression, coupled with GSEA pre-rank analysis (Subramanian et al., Proc Natl Acad Sci USA 102:15545–15550 (2005)), was performed on the RNA-Seq data (Figures 5A–5F). The resulting genes enriched in μ from adult controls versus μ (Figure 5A) and TAμ from transplanted mice (Figure 5C) were associated with oncological biological processes, including immune cell differentiation, immune response, DNA / RNA processes, and DNA methylation, while control μ cells were assigned to mature immune functions. On the other hand, mu cell-enriched processes from transplanted mice (Figure 5B) spanned a range of neuron-related processes, including regulation of neuronal migration, differentiation, and synaptic plasticity (Colonna and Butovsky, Annu Rev Immunol, (2017); Tay et al., J Physiol 595, 1929–1945 (2017)).Among the enriched processes, we also found glial differentiation, gliosis, and metabolism and cellular respiration, supporting the idea that transplant-derived μ cells are more likely to interact with / affect the neuronal environment, a process that consumes significant amounts of energy (Miyamoto et al., Front Cell Neurosci 7, 70 (2013)). The post-transplant TA μ enrichment process (Figure 5D) displays a stronger neurofunctional signature underlying various putative stages of μ cell maturation, consistent with the notion that microglia require different functions depending on their maturation state (Matcovitch-Natan et al., Science 353, aad8670 (2016)). Interestingly, μ and TAμ cells from transplanted mice express genes shown to be robustly modulated during microglial development (Matcovitch-Natan et al., Science 353:aad8670 (2016)). Notably, μ from transplanted animals matched control μ with respect to the expression levels of four of five selected genes associated with mature microglial function, while distinct pathways were observed in TAμ from transplanted mice (Figure 5E and Table 2) and in genes associated with the neogenesis stage (Figure 5F and Table 2), potentially associated with distinct and dynamic maturation stages. [Table 2]

[0159] Example 7 Hematopoietic cells associated with the brain parenchyma of naive or transplanted mice have clonogenic and hematopoietic repopulation potential, as well as microglial reconstitution potential. HSPCs are identified within extramedullary tissues, appear to be transiently localized in those sites, and are capable of local proliferation and give rise to tissue-resident myeloid cells, predominantly dendritic cells. Clonogenic potential was attributed to the presence of these cells in the brain; however, unlike that observed in other extramedullary tissues, chimerism between the two animals was not observed in the brain upon establishment of parabiotic pairs. This may suggest that brain clonogenic activity may be attributable to fixed tissue cells that are not capable of chimerism in parabiotic pairs, as hypothesized for microglial precursors. Therefore, we sought to better characterize cells with clonogenic potential in the brain and assess their transplantability in pretreated mice, as well as their ablation potential during pretreatment and replacement during HSC transplantation. Further studies are also intended to determine whether these cells indeed possess microglial repopulation potential. Mononuclear cells were isolated from the bone marrow and brain (at Percoll enrichment of hematopoietic lineage cells) of naive mice and animals undergoing lethal bone marrow ablation (pretreated with busulfan or irradiation) without or with the addition of GFP-LV-transduced lineage HSPCs (Figure 6A). These cells were plated in methylcellulose supplemented with cytokines to obtain colony-forming units (CFUs). As expected, discrete hematopoietic colonies were recovered from bone marrow and brain cultures derived from naive mice 14 days after plating (Figure 6B). A dramatic reduction in CFU output was observed from both bone marrow and brain tissues upon busulfan and irradiation pretreatment, with a greater effect of busulfan noted for brain CFU output. Importantly, CFU output recovered on ice in both bone marrow and brain from pretreatment at the time of HSPC transplantation (Figure 6C). GFP in both sites was significantly reduced from pretreatment. +In the colonies, the CFU output was highly chimeric, indicating that HSPC transplants can contribute to immobilized brain hematopoietic clonogenic precursors. Percoll-enriched mononuclear cells from bone marrow and brain from primary transplant recipients were also used for secondary transplants into busulfan-pretreated recipients (Figure 6A). Strikingly, GFP-positive cells derived from primary recipients were not detected. + Cells were identified in the hematopoietic tissues and brain of secondary recipients long-term after transplantation (Figure 6D). + The cells showed multilineage marker expression, while brain-resident cells were mostly CD11b-expressing.

[0160] Example 8 Combined ICV+IV delivery of engineered HSPCs has therapeutic relevance for metachromatic leukodystrophy, a representative LSD with neurodegenerative and extra-CNS features. To determine the actual role of ICV cell transplantation in increasing the engraftment potential for therapeutic molecule delivery to the brain, we developed a newly generated mouse model, Rag, which recapitulates the lysosomal disease ML (ML) due to ARSA deficiency in an immunocompromised background. - / - γ-chain - / - As2 - / These mice received human umbilical cord blood CD34+ cells transduced with lentivirus (LV) encoding ARSA, either IV or ICV alone, or a combination of IV and ICV routes (Biffi et al., Science 341, 1233-158 (2013); Sessa et al., Lancet 388, 476-487 (2016)) (Figure 1D and Figure 6A). Interestingly, well-defined human myeloid (CD45 + CD11b + ) cell progeny were identified in the brains of transplanted mice for a long period of time after both IV and ICV transplantation (Figures 1D-1G). ICV cell delivery resulted in greater human cell engraftment in the brain compared to IV delivery (Figure 1G). ICV cell delivery combined with IV resulted in even greater human cell engraftment in the brain (Figure 1G). Next, Rag - / - γ-chain- / - As2 - / - We evaluated whether greater human cell-bone marrow chimerism in the mouse brain dictates greater delivery of ARSA enzyme to the brain. Importantly, the increased contribution of ICV-transplanted human HSPCs to brain-bone marrow chimerism resulted in greater ARSA enzyme delivery to the brains of transplanted mice (Figure 1G). This was particularly evident in the case of combined IV+ICV delivery, which resulted in greater As2 + / + The ARSA activity reached levels indistinguishable from those of controls. Sixteen weeks after transplantation, similar, supranormal levels of ARSA activity were measured in the bone marrow of mice transplanted with transduced cells IV alone or IV + ICV (Figure 6B). Interestingly, recovery of enzyme activity was also measured in the brains of transplanted mice, with a favorable trend toward increased activity approaching wild-type levels in animals receiving combined IV and ICV transduced cells (Figure 6B). This data demonstrates that combined transplantation approaches result in greater enzyme delivery to the brain compared with standard IV transplantation approaches, and thus appear to be a promising strategy for improving the overall therapeutic potential of HSC gene therapy for SD, a CNS disorder. Furthermore, ICV delivery of HSPCs alone has been shown to be sufficient to deliver the same amount of therapeutic enzyme to MLD brains as IV cell delivery.

[0161] Co-transplantation did not increase enzyme activity in the hematopoietic system, and without intending to be bound by theory, this suggests that the contribution of ICV-delivered cells was largely restricted to the brain, rather than to hematopoietic populations outside the CNS.

[0162] The short survival interval of this animal model and the limited severity of its phenotype in the short term prevented evaluation of the phenotypic effects of increased brain ARSA delivery. However, LV-transduced HSCs (HSC gene therapy) have been successfully used in MLD mice (Biffi et al., J. Clin. Invest. 116:3070-3082 (2006); Biffi et al., J. Clin. Invest. 113:1118-1129 (2004)) and patients (Biffi et al., Science 341:1233-158 (2013); Sessa et al., Lancet 388:476-487 (2016)), as well as other lysosomal storage disease models (Gentner et al., Sci Transl Med 2:58ra84 (2010); Visigalli et al., Blood 116, pp. 5130-5139 (2010)), a dose-effect relationship exists, demonstrating that the greater the reconstitution of enzyme activity in hematopoietic cells and the brain, the greater the therapeutic efficacy in controlling CNS disease symptoms. Thus, the use of ICV cell delivery has the potential to enhance the therapeutic efficacy of transplantation of genetically corrected HSPCs.

[0163] Example 9 Combined ICV+IV delivery of wild-type HSPCs has therapeutic relevance in mucopolysaccharidosis type II, a representative LSD with neurodegenerative and extra-CNS features. To confirm these findings, particularly the actual role of ICV cell transplantation in increasing the likelihood of HSPC engraftment to deliver therapeutic molecules to the brain and exert benefits against LSDs with both brain and systemic disorders, we performed a mouse model of mucopolysaccharidosis type II (MPS II), myeloablated iduronate sulfatase (IDS), 2-month-old mice. - / - To the recipient, Lin - HSPCs and whole BM were transplanted from wild-type donors (Figure 6C). Mice receiving IV alone had wild-type IDS. + / +The donors received whole BM exclusively via IV. Controls were left untreated. After 180 days, the behavior of treated and control mice was tested using the rotarod. Interestingly, both treatments improved the mice's rotarod performance over four trials (Figure 6D), and mice treated with both IV and ICV + IV increased the time they spent on the rod in the fourth trial compared to the first trial (Figure 6E). However, the performance of mice treated with ICV + IV exceeded that of animals transplanted with IV alone. Without intending to be bound by theory, this indicates that the use of ICV cell delivery has the potential to enhance the therapeutic efficacy of wild-type HSPC transplantation in MPS II.

[0164] Example 10 HLA minor and MHC antigen-mismatched HSPC IV+ICV transplantation in mice is feasible and associated with high brain donor chimerism. We evaluated the applicability of the ICV HSPC transplantation approach in the context of allogeneic transplantation in patients, specifically the combination of ICV and IV transplantation in the context of both minor antigen and MCH-mismatched transplant situations. We tested the impact of ICV delivery of allogeneic HSPCs on overall survival and CNS microglial engraftment in mice receiving hematopoietic cell transplants from mismatched donors. An ICV dose of 1 cell was tested, 3 × 10 cells, into MCH-mismatched (BALB / cJ CD45.2 donors) recipients. 5 cells / mouse), and minor antigen mismatch (MHC matched; BALB_B CD45.2 donor to B6.SJL CD45.1 recipient; two ICV cell doses were tested, 3 × 10 5 and 1 × 10 6 ICV+IV HCT was applied in the context of a transplant setting (1000 Rad / mouse). On day -1, whole body irradiation (TBI) (1000 Rad) was used to condition recipient mice. Mice received either fresh whole bone marrow (tBM) alone or ICV-injected GFP-transduced Lin. -The donor cells were integrated with the transplanted HSPCs (Figures 8A and 9A). After transplantation, the mice were followed for up to 9-10 weeks (minor antigen mismatched) and 16 weeks (MHC mismatched). Rare intercurrent deaths (ICDs) were observed in the recipients and were not related to any specific treatment (Figures 8B and 9B). All surviving animals had stable weight gain and good overall health. Cytometric analysis of the PB, BM, spleen, and thymus (the latter in the case of minor antigen mismatched transplants) demonstrated successful donor cell engraftment (Figures 8C and 9C). Importantly, GFP-positive progeny of ICV-transplanted HSPCs were detected. + No cells were detected in the hematopoietic tissues examined (Figures 8D and 9D). Compared with IV-only recipients, IV+ICV minor antigen-mismatched animals demonstrated dose-dependent engraftment of donor-derived cells in the brain. In both minor antigen- and MHC-mismatched animals, ICV delivery of HSPCs provided benefits in donor-derived microglial chimerism in the CNS compared with IV-only transplantation. In conclusion, delivery of allogeneic hematopoietic stem / progenitor cells to the lateral ventricle is feasible using a combined delivery method and can enhance donor-derived chimerism in the brain in both minor-mismatched and MHC-mismatched settings. This data supports the applicability of ICV transplantation in the context of allogeneic hematopoietic cell transplantation procedures to enhance CNS-related benefits.

[0165] Example 11 Intrathecal delivery of HSPCs can contribute to brain and hematopoietic chimerism in the context of combinatorial HSPC transplantation strategies. We evaluated whether intrathecal delivery of HSPCs could contribute to CNS donor chimerism similar to ICV cell delivery. Lin-HSPCs were isolated from CD45.2 donor mice and transduced with LV encoding GFP. After transduction, the cells were injected IV (1.0 × 10) into CD45.1 myeloablated recipients. 6cells / mouse), and intrathecal (ICV) (0.3×10^6 cells / mouse) or intrathecal (IT) (0.3×10 6 HSPCs (CD45.2 GFP) were transplanted. Five days after transplantation, ICV and IT transplanted mice received whole BM cells from CD45.1 donors transduced with LV encoding mCherry (Figure 10A). Mice were sacrificed 45 days after transplantation. + High and comparable engraftment of CD45.2 GFP was observed in the BM of IV and IT transplanted mice, whereas CD45.2 GFP was not observed in the BM of ICV transplanted mice. + No (or very low) cells were observed (Figure 10B, left graph). + CD45.1 BM-transplanted cells showed good BM engraftment, whereas in IT-transplanted mice, the same cells likely out-grafted CD45.2 GFP cells transplanted on day 0. + The CD45.2 GFP cells engrafted in the cerebrospinal compartment in all groups were poorly engrafted due to competition with HSPCs. + HSPCs were analyzed (Figure 10B, middle graph), and ICV+IV injected mice showed the highest donor chimerism compared to other groups. + CD45.1 cell progeny were observed in ICV-transplanted mice. Analysis of engrafted donor (GFP+mCherry) cells in the spinal cord bone marrow compartment of all groups (right graph in Figure 10B) revealed that ICV+IV-injected mice showed the lowest GFP chimerism compared to other groups. Compared to IT-transplanted mice, ICV-transplanted mice showed the lowest mCherry chimerism. +An increase in the percentage of CD45.1 BM transplant cells was observed. Overall, these data indicate that IT can be used as an additional administration route for HSPCs to achieve bone marrow cell reconstitution both in the brain and in the spinal cord of transplant recipients. ICV and IV delivery alone were also associated with significant spinal cord chimerism with the donor. IT transplanted with donor cells was also observed exclusively in the peripheral blood and bone marrow, indicating that IT are not retained in the CNS upon transplantation. Therefore, this transplantation procedure can be considered, especially in cases of diseases with both CNS and systemic disorders.

[0166] Example 12 Post-transplant brain bone marrow cells derived from early hematopoietic stem / progenitor cells c-kit in HSPCs + , Sca-1 + , Lin - The (KSL) fraction can induce TAμ and μ in the mouse brain upon IV transplantation, but not c-kit and Sca-1 (Not-KSL) double-positive Lin - This is not the case for progenitor cells (Capotondo et al., Proc Natl Acad Sci USA 109:15018-15023 (2012)). This finding was further investigated in a strict competition situation by co-transplanting differentially labeled KSL and not-KSL cells in individual animals using both IV (FIG. 11D) and ICV (FIG. 11E) routes to assess the possible differential contribution of transplanted cells to bone marrow-brain cell turnover (FIGS. 11A, 11D, and 11E). KSL cells, when injected IV with not-KSL, were CD45 + CD11b + Myeloid brain cells; CD45 + CD11b high μ cells; CD45 + CD11b + / low Transiently amplified μ-TAμ cells; CD45 high CD11b highThey contributed almost exclusively to a diverse brain myeloid cell population (Figure 11D), identified as CNS-associated macrophages, or CNSmac (Capotondo et al., Proc Natl Acad Sci USA 109:15018-15023 (2012)). KSL and not-KSL cells instead contributed equally to brain myeloid cell reconstitution when infused ICV (Figure 11E). We addressed the ability of differentially labeled subpopulations identified within the KSL compartment by differential expression of the SLAM markers CD150 and CD48 to contribute to brain myeloid cell engraftment (Figures 11A, 11B). Upon competitive IV transplantation, CD48 - / CD150 + Long-term HSCs (LT-HSCs) and CD48 - / CD150 - Compared to multipotent progenitors (MPPs), these showed the greatest ability to reconstitute the cerebromyeloblast compartment compared to other injected populations (Figure 11F). In contrast, the more highly committed CD48 + CD150 - These cells also contributed to the reconstitution of cerebromyeloblastic populations in ICV-transplanted mice (Figure 11G). Hematopoietic reconstitution in transplanted mice is shown in Figure 11C. Histology of frozen brain slices from IV- and ICV-transplanted mice confirmed these results (Figures 11H and 11I). Consistent with previous findings, even in these settings, donor-derived cells displayed branched morphology with thin processes emanating from the cell body and expressed the myeloid markers Iba-1 and CD11b (Figures 11H and 11I).

[0167] To interpret these findings, we analyzed the expression of the CXCR4 receptor, known to be involved in HSC mobilization and homing to the BM (Dar et al., Experimental Hematology 34:967-975 (2006); Rettig et al., Leukemia 26:34-53 (2012); Sugiyama et al., Immunity 25:977-988 (2006)). KSL, not-KSL cells, and the four KSL subpopulations were analyzed (cells were analyzed at the end of transduction at the time of transplantation). Interestingly, in the IV infusion setting, the potential for microglia-like cell reconstitution, i.e., cells enriched in KSL, LT-HSC, and MPP, expressed CXCR4 at higher levels than not-KSL, HPC-1, and HPC-2 (Figure 11J). While not intending to be bound by theory, this finding suggests that cells expressing high levels of CXCR4 may be advantageous for early recruitment to the brain upon IV infusion and therefore for their ability to contribute to brain-myeloid cell chimerism, which may not be applicable to ICV delivery, where different signals may be important.

[0168] To more rigorously assess whether bona fide HSCs can generate new microglia-like cells upon ICV transplantation in the absence of competition, LT-HSCs were isolated by surrogate markers and functional signatures of Fgd5 expression in Fgd5-Zsgreen animals (Gazit et al., J Exp Med 211:1315-1331 (2014)). Indeed, the Fgd5-green reporter line allows for the reliable isolation of cells highly enriched for HSC activity. 500 bona fide HSCs were isolated by Zsgreen + ,system - , c-kit + , Sca1 + , Flkt-2 - , CD34 -CD45.2 Fgd5-Zsgreen cells were isolated from donors and transplanted IV or ICV into busulfan-pretreated or lethally irradiated CD45.1 recipient mice, along with unmanipulated whole bone marrow CD45.1 support (Figure 12A). IV cell delivery led to robust hematopoietic donor chimerism, whereas ICV-infused cells did not contribute to hematopoiesis in the peripheral blood. Interestingly, ICV-transplanted animals did not exhibit peripheral blood chimerism, but the donor-derived CD45.2 + Cells were identified within the brain in both transplantation settings (Figures 12B and 12C). Reconstitution of brain bone marrow cells was confirmed to be less effective in irradiated recipients than in busulfan-ablated recipients, as previously described (Capotondo et al., Proc Natl Acad Sci USA 109:15018-15023 (2012)). Donor-derived cells expressed CD45 and CD11b and were present in the majority of the TAμ cell compartment (Figures 12D and 12E).

[0169] These data demonstrate that bona fide HSCs generated microglia-like progeny in the brain upon ICV or IV transplantation, but this process was much more efficient when the early precursors were also administered ICV.

[0170] To further identify cells within HSCs that confer μ-reconstituting potential with relevant markers, we used CX3CR1-GFP reporter mice, which express a GFP reporter gene under the control of the seven-transmembrane receptor CX3CR1, a specific receptor for the novel CX3C chemokine fractalkine highly expressed on microglia and myeloid-lineage hematopoietic cells. Specifically, we assessed whether low but detectable levels of CX3CR1 expression could identify cells with microglia-reconstituting potential within the HSC pool. To this end, we characterized the bone marrow and HSPC pools of CX3CR1-GFP heterozygous mice and confirmed that, although a fraction of the HSPC pool expressed CX3CR1, actual expression was undetectable within bona fide LT-HSCs (Figure 13A). GFP in the Lin-HSPC compartment + and high CXCR1-GFP cells were sorted and these cells were transplanted into busulfan-treated recipients along with unmanipulated whole bone marrow from normal (non-CXCR1-GFP) donors as support. + Progeny cells could not be detected in the bone marrow and brain of repopulated mice 1.5 months after transplantation (Figure 13B). Rather, GFP-expressing μ cells could be identified in the brain of control mice transplanted with unsorted whole bone marrow from CX3CR1-GFP mice (Figure 13B). These data indicate that the bone marrow equivalent of μ precursors is retained in LT-HSCs and does not express CX3Cr1.

[0171] labeled CD34 + Cells and subfractions were cultured in immunodeficient NOD / LtSz-scid IL2Rγ null Further experiments were performed in the human context by transplanting NSG mice. In particular, differentially labeled CD34 + CD38 + (defined as precursors based on the literature) and CD34 + CD38 - We performed co-transplantation of cells sorted by CD38 (enriched for long-term stem cell activity) (Figure 14A). Interestingly, in humans, the CD38 -Only a fraction of the cells correlated with the appearance of labeled bone marrow human cells in the brains of transplanted mice (Figure 14B). In this context, similar to what was done in the mouse context, we went ahead and further dissected the HSPC fraction by CD38 and CD90 expression. - CD90 + HSC (labeled with GFP), CD38 - CD90 - MPP (transduced with LV encoding mO2) and CD38 + CD90 - and CD38 + CD90 + Committed progenitor cells (labeled with Cherry and Tag-BFP LVs, respectively) were isolated and labeled (with LVs bearing various reporter genes) (Figure 14C). Interestingly, consistent with what was observed in the mouse context, populations with increased stem cell potential were found to have a greater ability to contribute to chimerism of human brain cells with microglia-associated cells (Figure 14D).

[0172] Example 13 Molecular engineering of microglia for regulated therapeutic gene expression. Molecular engineering of either IV or ICV transplanted cells in the context of the innovative protocols described herein is necessary to address the precise requirements of efficacy, safety, and specificity / regulation of therapeutic gene expression. In this regard, two different strategies based on integrating vector-mediated gene transfer and targeted gene addition have been developed that can be applied in certain situations where sustained, yet regulated, transgene expression may be required.

[0173] Adult neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), and neurodegenerative diseases (SD), are characterized by heightened neuroinflammatory responses sustained by microglial activation. One of the molecules upregulated by activated microglia during pathological events is the 18-kDa selective translocator protein (TSPO), as shown by us and others using a TSPO radioligand for positron emission tomography (Visigalli et al., Neurobiol Dis 34:51-62 (2009); Turner et al., Neurobiol Dis 15:601-609 (2004)). Microglial cells are the primary cell type responsible for increased TSPO signaling. Therefore, TSPO is a useful and sensitive marker for monitoring microglia-associated neuroinflammation in the brain, and its promoter sequence could be used as an optimal regulatory sequence for marker or therapeutic protein production by newly engineered microglia in response to tissue injury and inflammation. We are developing innovative tools that allow for the engineering of brain microglia by targeting TSPO through HSC induction for the regulated delivery of therapeutic molecules to the brain, including potential applications in neurodegenerative diseases and neuroLSDs. In particular, brain microglia are reconstituted in the diseased brain by "sensor" cells engineered to express genes of interest upon cell activation in response to local neurodegeneration and neuroinflammation using the advanced transplantation protocols detailed above (this situation is also adaptable for the use of preconditioning regimens that direct μ precursors).

[0174] To this end, the TSPO promoter sequence is used to drive transgene expression. This can be achieved by including evidence-based selected TSPO promoter sequences (Wang et al., Cell Tissue Res 350:261-275 (2012)) before a marker or therapeutic gene in the context of advanced-generation LVs to be used for HSC transduction, and by inserting a therapeutic gene before or within the first intron / exon of the TSPO gene, utilizing its promoter, through targeted gene addition using CRISPR-Cas technology; various strategies have been tested. TSPO knockout mice provide evidence of the feasibility of this latter strategy (Banati et al., Nat Commun 5:5452 (2014)). The selected TSPO promoter sequence (Wang et al., Cell Tissue Res 350:261-275 (2012)) (Figure 16A) inserted in front of GFP as a marker gene in the context of third-generation LVs (Figure 16B) was confirmed to drive sustained transgene expression in microglial cells (in the microglial cell line BV2) and in response to stimulation with bacterial LPS, as expected by the naive sequence (Figures 16C and 16D). The transduced and / or edited cells were then used to exclusively reconstitute brain and bone marrow cells in recipient mice using advanced protocols.

[0175] One working hypothesis is that upon transplantation of genetically modified / edited HSCs at the TSPO locus, a population of brain myeloid cells and microglia will emerge that express genes that allow for precise molecular monitoring of neuroinflammatory responses and / or contribute to therapeutic activity and amelioration of disease phenotypes upon cellular activation in a neuroinflammed and degenerative environment. Reporter genes for characterizing the developmental approach have been tested in animal models characterized by microglial activation and TSPO upregulation, such as the ALS mouse model (SOD1.G93A mouse) (Peviani et al., CNS Neurol Disord Drug Targets 9:491-503 (2010)) and the animal model of globoid cell leukodystrophy (GLD) (Visigalli et al., Neurobiol Dis 34:51-62 (2009)). The approach has been confirmed for its functionality, and its potential for therapeutic action has been tested in available animal models.

[0176] Example 14 Identification of microglial precursors by busulfan toxicity and use of the γH2AX marker in Fgd5 reporter mice. Our findings provide evidence for the existence of a functionally defined microglial precursor population located in the brain that is susceptible to ablation upon administration of busulfan-based pretreatment, potentially co-occurring with endogenous CNS-resident microglial precursors (μPs). Phosphorylated histone 2AX (γH2AX) is a sensitive and specific biomarker for tracking cells targeted by busulfan in the brain. Therefore, functional brain-resident μPs can be identified by utilizing γH2AX, a pharmacological marker of busulfan and its cytotoxicity within these cells. γH2AX was investigated as a biomarker of busulfan cytotoxicity. Specifically, the presence, distribution, and cellular localization of γH2AX were examined by flow cytometry (FC) and immunofluorescence (IF) on brain slices from mice treated with systemic busulfan pretreatment. FC specifically increased viable CD45 expression in busulfan-treated versus control untreated animals on days 1 and 5 after the last busulfan dose. + Intracellular γH2AX signaling was enhanced (Fig. 15B). At the same time, early apoptosis after busulfan treatment was also observed, particularly CD11b + and c-kit + In cells, CD45 + γH2AX was detected by Annexin V staining in brain cells (Fig. 15A). By IF, γH2AX was found in few neuronal cells, mainly located in the hippocampus. + Except for lesions, they were barely detectable in control mice (Fig. 16C, top photo), demonstrating a correlation between physiological neuronal activity and H2AX phosphorylation, as previously shown. In contrast, in busulfan-treated mice, γH2AX + The signal was enhanced in the nuclei of cells lining the lateral ventricles, subventricular zone (SVZ), and rostral meridian stream (RMS) (Fig. 15C, bottom panel). +The lesions were localized to both neurons (Figure 15C, inset) and glial cells, namely, microglia and astrocytes (Figure 15C, inset). Collectively, these data indicate that both neuronal and non-neuronal cells localized in well-defined regions of the brain, including the CNS stem cell niche, are sensitive to busulfan. Therefore, these results have the potential to identify microglial progenitor cells. Similar experiments have been performed in mice expressing reporter genes driven by the promoters of hematopoietic stem cell- and microglia-specific gene markers, such as Fgd5 or Cx3Cr1, to better track busulfan-sensitive cells.

[0177] Example 15 Selective brain pretreatment targeting microglial precursors using nanocarriers for targeted delivery. Systemic administration of busulfan helps develop efficient turnover of brain microglia with donor-derived cells. A similar, but CNS-restricted, regimen protects patients with CNS-restricted diseases (e.g., incomplete myeloablative encephalopathy, PD, and ALS) from the side effects of systemic preconditioning with bone marrow ablation. In preliminary studies, we investigated the feasibility of intracerebral administration of busulfan via a cannula implanted in the lateral ventricle in mice. Various drug formulations of busulfan, including clinical-grade busulfan formulations, were tested. However, ICV administration could not guarantee exposure of brain-resident μPs to busulfan levels comparable to those achieved by systemic drug administration, nor did it favor engraftment of transplanted HSPCs (data not shown). Rather, it was associated with widespread neurotoxicity and local inflammation. Therefore, we implemented a targeted drug delivery strategy using nanoparticles. Without intending to be bound by theory, nanoparticles may have the potential to efficiently and selectively deliver ablative drugs to functionally defined μPs.

[0178] Recently, polymeric nanoparticles (NPs) have attracted significant interest as a promising means of improving the pharmacological profile of drugs, including chemotherapeutic agents. NPs allow for tunable material composition, surface functionalization, and degradation rate, enabling: i) high cell targeting selectivity, which reduces the risk of side effects of drugs formulated with NPs compared to the same drug without NPs; ii) multiplexed drug delivery; and iii) controlled drug release over time. NPs are fabricated from artificial or natural polymers and range in size from 10 to 400 nm. Various biodegradable polymers, such as chitosan, poly(ε-caprolactone) (PCL), poly(alkyl-cyanoacrylate) (PACA), polylactic acid (PLA), or poly(lactic-co-glycolic acid) (PLGA), can be used as the core matrix. Surface functionalization with hydrophilic polymers, such as poly(ethylene glycol) (PEG), is used to improve biocompatibility, aqueous solubility, and NP stability. The surface properties of NPs determine the selectivity of uptake by targeting cells, thus influencing biodistribution and half-life in biological fluids. Meanwhile, the physicochemical properties of the NP core are responsible for the drug loading capacity and drug release profile. Functionalization of the NP surface with targeting moieties, including antibodies, can determine preferential binding to receptors or transporters expressed at the blood-brain barrier (BBB) ​​or on specific cell types to enhance CNS biodistribution or target cell specificity. Modification of lipophilicity, nanoparticle core structure, and composition can control the biodegradation time of the material, optimizing the drug loading and release profile.

[0179] A novel drug delivery system (based on biodegradable and biocompatible PCL NPs) (Figure 17A) was validated to selectively target microglia / macrophages after intraparenchymal administration in a mouse model of spinal cord injury. The biodistribution of these microglia-targeted NPs after ICV administration in mice was validated, confirming efficient uptake by microglia / macrophages and widespread distribution in various CNS regions (Figures 17A-17I). Cytofluorimetric analysis of injected mouse brains revealed that the NPs, especially when injected ICV with mannitol to favor BBB penetration, selectively target microglia / macrophages and CD45 NPs. + c-kit + cells (Figure 17C) and proliferating EdU + Uptake by cells was predominant (Fig. 17D), and CD45 + These microglia-targeted NPs were based on FDA-approved biodegradable materials, including low-molecular-weight PEG chains that ensure NP stability, regulate degradation rates, and modulate drug release over time. Interestingly, immunofluorescence confocal analysis confirmed these findings, showing that NPs concentrated in brain regions, such as the SVZ and RMS, that were first colonized by donor-derived HSPCs immediately after transplantation, during a process that led to cerebral microglial reconstitution in areas of intense γH2AX-positive signal after busulfan administration. Thus, representative regions are likely enriched in μPs (Figure 17G). Notably, the rhodamine-containing NPs + Iba + Bone marrow cells were also proliferative before sacrifice and staining, as suggested by both the ki67 positive signal (Figure 16G) and Edu administration (Figure 17H). + Rhodamine + The cells were also positive for hematopoietic bone marrow markers and displayed both ramified and rounded microglia-like cell morphologies. Importantly, rhodamine + Iba1 +Cells also occasionally expressed the early / stem cell marker nestin (Figure 17H, right panel). The presence and distribution of rhodamine signal in NP-injected brains interestingly coincided with that of Edu signal, suggesting that NPs occur preferentially in proliferating cells, which may include cells with microglial precursor characteristics (Figure 17I).

[0180] Next, we optimized novel NPs via de novo chemical synthesis to enable loading with busulfan and etoposide by utilizing two distinct chemical moieties selected for compatibility with drugs (termed "SP" and "QMS") covalently grafted onto a poly(2-hydroxyethyl methacrylate) backbone (herein referred to as BK-510). This enabled us to achieve biologically relevant amounts of drug encapsulation within the NPs (i.e., in the case of busulfan, ranging from 210.7 ± 5.3 to 257.6 ± 7.2 μg / ml). The formulation of such nanoparticles loaded with chemotherapeutic drugs (such as busulfan or etoposide) was further improved by introducing a so-called "self-assembly" approach, which allows for the formation of NPs starting from lyophilized material (Figure 18). This makes the nanoparticle material suitable for scale-up synthesis and long-term storage of lyophilized pharmaceutical products without loss of potency and consistency between different batches.

[0181] By using γH2AX as a reliable pharmacodynamic marker of busulfan-associated genotoxicity, + The increase in cell numbers was highlighted by immunofluorescence and flow cytometry analysis after in vivo exposure to busulfan-loaded NPs (Figures 19A and 19B). This was further confirmed by cell viability assays performed on the BV2 microglia-like cell line, highlighting the specific cytotoxicity of BU-loaded NPs after 72 hours of incubation and indicating that BU inclusion in NPs did not impair drug efficacy. Busulfan-loaded (NP-BUS) or empty NPs in the lateral ventricles of mice significantly increased the number of brain microglia (CD45) upon exposure to NP-BUS. + / CD11b+ ) induced a significant increase in γH2AX signals in cells (Figure 19C).

[0182] To facilitate the development of an effective brain preconditioning regimen, NP loading was optimized with other drugs, such as etoposide and lomustine, which showed preliminary supportive results (Figures 19D-19E). Cell proliferation assays were performed on the BV2 cell microglia-like cell line. Etoposide was loaded into various NP formulations (pre-assembled 100 nm PCL NPs; self-assembled 100 nm NPs; or self-assembled 50 nm NPs) at an apparent drug concentration of 275 μg / ml. Etoposide-loaded NPs or unencapsulated drug were added to cell culture medium, and cell viability was then measured by CellTiter cell proliferation MTS assay at 37°C 48 and 72 hours after drug administration. Various drug concentrations were tested (1.6, 6.25, and 25 μg / ml). Empty NPs (containing no drug) were tested as a control. In this case, the final NP concentration in the medium matched the concentration used to administer 25 μg / ml of encapsulated etoposide in the NPs. As can be seen in Figures 20D-20E, all three NP formulations were equally effective at delivering etoposide, thus determining cell death. During short-term incubation (self-assembled 100 nm NPs, 48 ​​h time point), empty NPs were neither toxic (pre-assembled 100 nm PCL NPs and self-assembled 50 nm NPs) nor only mildly toxic. However, cell viability with empty NPs consistently exceeded that of etoposide-loaded NPs, suggesting that the observed effect with etoposide-loaded NPs was due to drug release rather than the NPs themselves. Interestingly, the effect observed with etoposide-loaded NPs tested at a final drug concentration of 1.6 μg / ml was more pronounced than that observed with unencapsulated drug. This difference was even more pronounced at an incubation time of 72 h, confirming that encapsulating etoposide in NPs enhanced its efficacy and improved the cytotoxicity of already very low concentrations of the drug.

[0183] These NP formulations induce proliferation of microglial precursors and / or CD45 expression in the brain, similar to i) a single dose of busulfan at 25 mg / kg or ii) a single dose of busulfan at 25 mg / kg. + c-kit + The NPs were inject...

Claims

1. A composition comprising human hematopoietic stem / progenitor cells (HSPCs), characterized in that the composition is delivered to a subject by intracerebroventricular injection (ICV) in combination with ablation pretreatment, wherein the human HSPCs are CD34 ＋ and CD38 － , CX3CR1 － and Fgd5 ＋ The composition is one or more of:

2. The human HSPC is ＋ , Lin － , Sca1 ＋ , CD150 ＋ , CD48 － and CD11b － 10. The composition of claim 1, wherein the composition is one or more of:

3. The human HSPCs are ＋ 2. The composition of claim 1, wherein:

4. 4. The composition of any one of claims 1 to 3, wherein the subject has or is at increased risk of developing a lysosomal storage disorder or a neurodegenerative disease.

5. The lysosomal storage disorder is selected from the group consisting of adrenoleukodystrophy, activator deficiency / GM2 gangliosidosis, alpha-mannosidosis, aspartylglucosaminuria, cholesteryl ester storage disease, chronic hexosaminidase A deficiency, cystinosis, Danon disease, Fabry disease, Farber disease, fucosidosis, galactosialidosis, Gaucher disease, globoid cell leukodystrophy, GM1 gangliosidosis, I-cell disease / mucolipidosis II, infantile free sialic acid storage disease / I 5. The composition of claim 4, wherein the disease is selected from SSD, juvenile hexosaminidase A deficiency, infantile neuronal ceroid lipofuscinosis, Krabbe disease, lysosomal acid lipase deficiency, metachromatic leukodystrophy, mucopolysaccharidosis disorders, multiple sulfatase deficiency, Niemann-Pick disease, neuronal ceroid lipofuscinosis, Pompe disease / Glycogen storage disease type II, Pycnodysostosis, Sandhoff disease, Schindler disease, Salla disease / sialic acid storage disease, Tay-Sachs / GM2 gangliosidosis, and Wolman disease.

6. 6. The composition of claim 5, wherein the neurodegenerative disease is selected from amyotrophic lateral sclerosis (ALS), Parkinson's disease, and Alzheimer's disease.

7. 7. The composition of any one of claims 1 to 6, wherein the ablation pretreatment is performed prior to administration of the human HSPCs.

8. The composition of claim 1 , wherein the ablation pretreatment comprises administration of a cytotoxic agent to the subject.

9. The composition of claim 8 , wherein the cytotoxic agent is an alkylating agent.

10. 10. The composition of claim 9, wherein the alkylating agent is busulfan.

11. 1. A composition for treating a subject having or at increased risk of developing a lysosomal storage disorder or a neurodegenerative disease, said composition comprising: ＋ and CD38 － and Fgd5 ＋ 1. A composition comprising human hematopoietic stem and progenitor cells (HSPCs), wherein the human hematopoietic stem and progenitor cells (HSPCs) are one or more of the following:

12. 1. A composition for treating a subject having or at increased risk of developing a lysosomal storage disorder or a neurodegenerative disease, said composition comprising: ＋ And kit ＋ , Lin － , Sca1 ＋ , CD150 ＋ , CD48 － , Fdg5 ＋ , CX3CR1 － and CD11b － 10. A composition comprising human HSPCs, wherein the human HSPCs are one or more of the following:

13. 1. A composition for ablating endogenous microglia in a subject and reconstituting said microglia by engraftment of HSPCs, comprising: (a) a nanoparticle comprising a cytotoxic agent and one or more capture molecules covalently linked to the surface of the nanoparticle, wherein the capture molecules specifically bind to one or more markers expressed on microglial cells or their precursors; and (b) human HSPCs, wherein the human HSPCs are administered to the subject intravenously (IV) and by intracerebroventricular infusion (ICV). wherein the human HSPCs are CD34 ＋ And kit ＋ , Lin － , Sca1 ＋ , CD150 ＋ , CD48 － , Fdg5 ＋ , CX3CR1 － and CD11b － The composition is one or more of:

14. 1. A composition for treating a lysosomal storage disorder in a subject, comprising: (a) a nanoparticle comprising a cytotoxic agent and one or more capture molecules covalently linked to the surface of the nanoparticle, wherein the capture molecules specifically bind to one or more markers expressed on microglial cells or their precursors; and (b) human HSPCs, wherein the human HSPCs are CD34 ＋ and is administered to the subject by intracerebroventricular infusion (ICV), wherein the human HSPCs express a therapeutic polypeptide. A composition comprising:

15. The lysosomal storage disorder is selected from the group consisting of adrenoleukodystrophy, activator deficiency / GM2 gangliosidosis, alpha-mannosidosis, aspartylglucosaminuria, cholesteryl ester storage disease, chronic hexosaminidase A deficiency, cystinosis, Danon disease, Fabry disease, Farber disease, fucosidosis, galactosialidosis, Gaucher disease, globoid cell leukodystrophy, GM1 gangliosidosis, I-cell disease / mucolipidosis II, infantile free sialic acid storage disease / I 15. The composition of claim 14, wherein the disease is selected from SSD, juvenile hexosaminidase A deficiency, infantile neuronal ceroid lipofuscinosis, Krabbe disease, lysosomal acid lipase deficiency, metachromatic leukodystrophy, mucopolysaccharidosis disorders, multiple sulfatase deficiency, Niemann-Pick disease, neuronal ceroid lipofuscinosis, Pompe disease / Glycogen storage disease type II, Pycnodysostosis, Sandhoff disease, Schindler disease, Salla disease / sialic acid storage disease, Tay-Sachs / GM2 gangliosidosis, and Wolman disease.

16. The composition of claim 15, wherein the therapeutic polypeptide is a lysosomal enzyme or an ABCD protein.

17. The lysosomal enzymes are α-glucosidase; glucocerebrosidase; β-galactosidase; β-hexosaminidase A; β-hexosaminidase B; acid sphingomyelinase; galactocerebrosidase; β-galactocerebrosidase; acid ceramidase; arylsulfatase A; α-L-iduronidase; iduronate-2-sulfatase; heparan N-sulfatase; α-N-acetylglucosaminidase; acetyl CoA; α-glucosaminide N-acetase; 17. The composition of claim 16, wherein the enzyme is one or more of: N-acetylglucosamine-6-sulfate sulfatase; N-acetylgalactosamine-6-sulfate sulfatase; acid β-galactosidase; arylsulfatase B; β-glucuronidase; acid α-mannosidase; acid β-mannosidase; acid α-L-fucosidase; sialidase; α-N-acetylgalactosaminidase; and palmitoylprotein-thioesterase-1.

18. 1. A composition for treating a neurodegenerative disease in a subject, comprising: (a) a nanoparticle comprising a cytotoxic agent and one or more capture molecules covalently linked to the surface of the nanoparticle, wherein the capture molecules specifically bind to one or more markers expressed on microglial cells or their precursors; and (b) human HSPCs, wherein the human HSPCs are CD34 ＋ and is administered to the subject by intracerebroventricular infusion (ICV), wherein the human HSPCs express a therapeutic polypeptide or polynucleotide. A composition comprising:

19. 19. The composition of claim 18, wherein the neurodegenerative disease is selected from amyotrophic lateral sclerosis (ALS) and Alzheimer's disease.

20. The composition of claim 18 or 19, wherein the therapeutic polypeptide or polynucleotide is one or more of an inhibitory nucleic acid or shRNA targeting one or more of miR155 and NOX2; TREM2; APOE2; and APP alpha.

21. 21. The composition of any one of claims 18 to 20, wherein expression of the polypeptide or polynucleotide is under the control of a TSPO promoter.

22. 22. The composition of any one of claims 18 to 21, wherein the polypeptide or polynucleotide is expressed from a polynucleotide inserted into the TSPO locus.

23. 1. A composition comprising human HSPCs for use in a method for generating microglial chimerism in the brain of a subject independent of extra-CNS hematopoietic tissue chimerism, the method comprising ICV transplantation of human HSPCs and IV transplantation of whole bone marrow cells 0-5 days after busulfan bone marrow ablation, wherein the human HSPCs express CD34 ＋ and CD38 － , kit ＋ , Lin － , Sca1 ＋ , CD150 ＋ , CD48 － , Fdg5 ＋ , CX3CR1 － and CD11b － The composition is one or more of:

24. 1. A composition comprising exogenous cells for use in a method for generating sustained hematopoietic mixed chimerism in tissues outside the brain and CNS in a subject for a short period of time, the method comprising ICV and IV transplantation of the exogenous cells following busulfan bone marrow ablation, the exogenous cells being human HSPCs transplanted ICV and IV on day 0, the human HSPCs expressing CD34 ＋ and CD38 － , kit ＋ , Lin － , Sca1 ＋ , CD150 ＋ , CD48 － , Fdg5 ＋ , CX3CR1 － and CD11b － The composition is one or more of:

25. 25. The composition of claim 23 or 24, wherein the chimerism occurs in a minor HLA-mismatched transplant situation.

26. A composition comprising human HSPCs, wherein the composition is delivered to a subject by intracerebroventricular injection (ICV) in combination with ablation pretreatment, and wherein the human HSPCs are ＋ , Lin － , Sca1 ＋ , CD150 － , CD48 ＋ and the human HSPCs are CD34 ＋ and CD38 － , CX3CR1 － and Fgd5 ＋ The composition is one or more of:

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