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

Abstract

To provide compositions and methods for treating diseases and disorders of the central nerve system.SOLUTION: The invention provides compositions and methods for treating or preventing neurological diseases or disorders of the central nervous system (e.g., storage disorder, lysosomal store disorder, neurodegenerative diseases) by reconstitution of brain myeloid cells and microglia upon transplantation of hematopoietic cells with high microglia reconstitution potential. The invention also provides compositions and methods for ablating and reconstituting microglia. The invention also provides a method for delivering a hematopoietic stem cell (HSC) to a subject, which comprises a step of administering the HSC by intracerebral ventricular injection (ICV) in combination with ablation conditioning.SELECTED DRAWING: None

Description

[Technical field]

[0001] STATEMENT OF RIGHTS TO INVETIONS 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 n°[[617162]], and from the Italian Ministry of Health under contract n°GR-2011-02347261. [Background technology]

[0002] 2. Background of the Invention The majority of storage disorders (SD) with central nervous system (CNS) disorders (neuro-SD) have no effective and curative treatment, and patients ultimately die from their destructive disease. In many cases, disease onset occurs in very early childhood and is characterized by subtle signs, leading to diagnosis at a clearly symptomatic, if not progressive, stage. Neuro-SD is also characterized by rapid early disease progression, especially in early-onset variants. For this reason, therapeutic approaches applied with some success in presymptomatic neuro-SD children, including, for example, 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 neuro-SD patients, with benefits mostly only being linked to procedures applied in presymptomatic or early-onset patients. One of the key reasons why these HSC-based approaches have not been successful in ameliorating the 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 diseases. 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 make more rapid this phenomenon are greatly needed. Such strategies may also be therapeutically relevant for some of the acquired neurodegenerative conditions in adulthood, which could benefit from the delivery of therapeutic molecules across the blood-brain barrier by modulating the progeny of transplanted hematopoietic stem and progenitor cells (HSPCs) and / or the activated microglial phenotype that characterizes the majority of these conditions. These disorders, including amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD) and Parkinson's disease (PD), share several common disease / pathogenic mechanisms with neuroLSDs, such as neuroinflammation and the active role of microglia. Thus, 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 by a variety of mechanisms, such as protein delivery or modulation of local inflammation, etc. The present invention provides compositions and methods for one or more of the following: (i) effectively engrafting microglial progenitor cells in CNS cells having or acquiring microglial characteristics, including cells enriched for microglial reconstituting potential, for therapeutic purposes in the conditions listed above; (ii) selectively and exclusively engrafting microglial progenitor cells in CNS genetically modified cells having or acquiring microglial characteristics, including cells enriched for microglial reconstituting 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 targeted to 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, in part renewing the pool of cells when successful, timely, and only in the presence of CNS impairment.

[0004] In one aspect, the invention provides a method of delivery of hematopoietic stem cells (HSCs), comprising administering to a subject the HSCs by intracerebroventricular injection (ICV) in combination with ablative conditioning. In another aspect, the 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 + )

[0005] In another aspect, the present invention provides an isolated hematopoietic stem cell (HSC) transformed with a vector expressing a therapeutic polypeptide or polynucleotide, wherein the HSC is 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 - (For example, 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 - )

[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 injection (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 said subject a kit + , Lin - , Sca1 + , CD150 + , CD48 - , Fdg5 + , CX3CR1 - and CD11b - (For example, 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 injection (ICV) in combination with ablation pretreatment.

[0009] In another aspect, the present invention provides a method for ablating endogenous microglia and reconstituting microglia by HSC engraftment in a subject, 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 by IV or ICV administration to the subject.

[0010] In another aspect, the present invention provides a method of treating a lysosomal storage disorder in a subject, comprising administering to the subject nanoparticles, the nanoparticles containing a cytotoxic agent and one or more trapping molecules covalently linked to a surface of the nanoparticle, the trapping molecules specifically binding 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 injection (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, comprising administering to the subject nanoparticles, the nanoparticles containing a cytotoxic agent and one or more capture molecules covalently linked to a surface of the nanoparticle, the capture molecules specifically binding 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 injection (ICV), wherein the HSCs express the therapeutic polypeptide or polynucleotide.

[0012] In another aspect, the 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 following busulfan bone marrow ablation.

[0013] In another aspect, the invention provides a method for generating sustained hematopoietic mixed chimerism in the brain and tissues outside the 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 invention provides a method for achieving regulated expression of an exogenous gene in engineered microglia, comprising transduction of 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 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 of ablation of microglial cells or their precursors in a subject, comprising administering to the subject nanoparticles, the nanoparticles containing a cytotoxic agent and one or more capture molecules covalently linked to a surface of the nanoparticle, 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 + , 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 - (For example, 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. In 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 play disease, or the like. Selected from isasialic 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 storage 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 palmitoyl protein-thioesterase-1.

[0022] In various embodiments of any aspect described herein, the subject has or is at increased risk of developing a neurodegenerative disease. In 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, HSC is administered in combination with ablation pretreatment.In various embodiments, ablation 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, ablation pretreatment is performed before administering HSC.

[0025] In various embodiments of any aspect described herein, expression of the polypeptide or polynucleotide is by 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 nanoparticle further contains 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 nanoparticle has one or more of an optimized drug loading efficiency, an optimized drug release, and an optimized stability. In various embodiments of any aspect described herein, the nanoparticle comprises one or more capture molecules covalently linked to the surface of the nanoparticle, the capture molecules specifically binding to one or more markers expressed on microglial cells or their precursors. In various embodiments of any aspect described herein, the nanoparticle is administered to the 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 comprises targeted addition of a gene of interest at the TSPO locus in the hematopoietic equivalent of a microglial progenitor. In various embodiments of any aspect described herein, the method comprises administering an engineered autologous population of microglial progenitors to a subject, the subject undergoing ICV brain ablation to selectively reconstitute microglia. In various embodiments of any aspect described herein, the method further comprises administering unmanipulated autologous bone marrow cells. In various embodiments of any aspect described herein, the method further comprises detecting Fdg5 expression to identify brain-resident microglial progenitor cells.

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

[0030] definition Unless otherwise specified, all technical and scientific terms used herein have the meanings commonly understood by those skilled in the art in the field to which the present invention belongs.The following references provide those skilled in the art with a number of general definitions of the terms used in the present 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 Harper Collins Dictionary of Biology (1991).As used herein, the following terms have the meanings ascribed to them below, unless otherwise specified.

[0031] By "agent" is meant any small chemical compound, an antibody, a nucleic acid molecule, or a polypeptide, or fragments 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 material derived from an organism.

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

[0037] As used herein, the terms "determining," "assessing," "assaying," "measuring," and "detecting" refer to both quantitative and qualitative determinations, and thus the term "determining" is used interchangeably herein with "assaying," "measuring," and the like. 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 identifying the presence, absence, or amount of the analyte being detected.

[0039] "Detectable label" refers to a composition that, when linked 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., as 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" refers to the amount required to improve the symptoms of disease compared to untreated patients. The effective amount of the active compound used to implement the present invention for therapeutic treatment of disease varies depending on the method of administration, age, weight and general health of the subject. Ultimately, the attending physician or veterinarian will determine the appropriate amount and dosage regimen. Such amount is called "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 Nos. 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 even 90%, 95%, or 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 various degrees from components that normally accompany it as found in its natural state. "Isolate" refers to some degree of separation from the original source or surroundings. "Purify" refers to a degree of separation greater than isolation. A "purified" or "biologically pure" protein is sufficiently free of other materials such that any impurities do not substantially affect the biological properties of the protein or cause other adverse consequences. That is, the nucleic acids or peptides of the invention are purified when they are substantially free of cellular material, viral material, or culture medium if produced by recombinant DNA techniques, or chemical precursors or other chemicals if chemically synthesized. Purity and homogeneity are usually 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 only one band in an electrophoretic gel. In the case of proteins that may be modified, eg, phosphorylation or glycosylation, different modifications may result in different isolated proteins that can be individually purified.

[0046] By "isolated polypeptide" is meant a polypeptide of the invention separated from components which naturally accompany it. Ordinarily, a polypeptide is isolated when it is at least 60% free, by weight, from the proteins and naturally occurring organic molecules which naturally accompany it. Preferably, a preparation is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight, a polypeptide of the invention. An isolated polypeptide 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, 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 of 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 composite structure of nanoscale dimensions. In particular, nanoparticles are particles that typically range in size from about 1 to about 1000 nm, and are typically spherical, but can have a variety of morphologies depending on the nanoparticle composition. The portion of the nanoparticle that contacts the external environment for the nanoparticle is generally identified as the surface of the nanoparticle. For the nanoparticles described herein, the size limit can be limited to two dimensions, and thus the nanoparticles described herein include composite structures with diameters of about 1 to about 1000 nm, with the specific diameters depending on the nanoparticle composition depending on the experimental design, and the 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 in particular nanoparticles used for delivery with 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] By "enhancing proliferation" is meant 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 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] By "reference" is meant 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 sequences are 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 level or nucleic acid 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 programs). 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 determine the degree of identity, e.g., glycine, alanine, valine, isoleucine, leucine, aspartic acid, glutamic acid, asparagine, glutamine, serine, threonine, lysine, arginine, and phenylalanine, tyrosine. -3 ~e -100 may be used with score probabilities between

[0059] Nucleic acid molecules useful in the methods of the present invention include any nucleic acid molecule that encodes 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 with at least one strand of a double-stranded nucleic acid molecule. "Hybridizing" refers to a pair that forms a double-stranded molecule between complementary polynucleotide sequences (e.g., genes described herein) or portions thereof under various stringent conditions (see, e.g., Wahl, GM and and SL Berger (1987) Methods Enzymol. 152:399; Kimmel, A. (See, R. (1987) Methods Enzymol. 152:507).

[0060] For example, stringent salt concentrations will usually 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 obtained in the absence of organic solvents, such as formamide, while high stringency hybridization can be obtained in the presence of at least about 35% formamide, more preferably at least about 50% formamide. Stringent temperature conditions will usually include a temperature that is 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, concentration of detergent, such as 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 such various conditions as needed. In a preferred embodiment, hybridization is performed in 750 mM NaCl, 75 mM trisodium citrate, and 1% SDS at 30° C. In a more preferred embodiment, hybridization is performed 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 performed 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 one 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 mentioned above, stringency of washing 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, 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 that are 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 steps are performed at 68° C. in 15 mM NaCl, 1.5 mM trisodium citrate and 0.1% SDS. Further variations on these conditions will be readily apparent to those of skill in the art. Hybridization techniques are well known to those of skill in the art and are described, for example, in Benton and Davis (Science 196:180, 1977); Grunstein and Hogness (Proc. Natl. Acad. Sci., USA 72:39; (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 specifically stated 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 to be within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05% or 0.01% of the stated 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 values ​​within the range. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or subranges 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 clearly indicated otherwise. Thus, for example, reference to a "biomarker" includes reference to more than one biomarker.

[0068] Unless specifically stated otherwise or clear from the 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 under U.S. patent law, and "includes," "including," "consisting essentially of," or "consists essentially of" likewise have the meaning ascribed to them under U.S. patent law, and the terms are open-ended, allowing for the presence of other things than what is recited, but excluding prior art embodiments, so long as the basic or novel characteristics of the recited things are not altered by the presence of other things than what is recited. [Brief description of the drawings]

[0071] [Figure 1A]Figures 1A-1H illustrate bone marrow cell reconstitution in the brain after intraventricular 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 bone marrow ablated mice (BU: bone marrow ablated with busulfan treatment; IRR: lethal irradiation). Various time points of analysis are shown. 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 (ICV vs IV and time p < 0.005) in BU and IRR mice. These data indicate rapid and robust bone marrow cell engraftment in the brain following intraventricular injection of HSPCs. Graph bars are shown in triplicate sets of four bars: from left to right: IRR IV (gray), IRR ICV (white), BU IV (dark grey), and BU ICV (darker grey). Figure 1C illustrates a reconstruction of a sagittal section of the brain of a representative ICV-transplanted BU-TX mouse, showing widespread distribution of GFP+ cells 90 days after ICV injection of GFP-transduced HSPCs. Nuclei are shown with GFP (green / gray) and Topro III (TPIII, light blue / light grey). Images were acquired with a Delta Vision Olympus at 20x magnification and processed by Soft Work3.5.0. Reconstructions were performed using Adobe Photoshop CS 8.0 software. FIG. 1D illustrates immunofluorescence analysis for GFP (green / gray) and Iba-1 (red / light gray) in brain slices from BU_TX mice 90 days after ICV transplantation of GFP-transduced HSPCs. M=fusion. 20× and 40× magnification of relevant dotted boxes are shown.Images were acquired with a confocal microscope Radiance2100 (Bio-Rad) Ix70 and processed by Soft Work3.5.0. Figure 1E is an experimental scheme for transplantation of human CD34+ cells (which become HSPCs in humans and are considered as the equivalent population to Lin-cells derived from mice) transduced with LVs encoding GFP or arylsulfatase A (ARSA) in NSG mice or Rag- / -γ-chain- / -As2- / - (RagMLD) mice pretreated with BU 16 mg / kg x 4 days (NSG) or sublethal irradiation (RagMLD). NSG mice also received unmanipulated mononuclear cells from NSG donors. The graph includes four sets of bars. 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 1F illustrates representative dot plots from the analysis of brain mononuclear cells derived from NSG mice transplanted with human CD34+ cells transduced with GFP LV as early as 20 weeks. The abundance of human cells in mouse brain is shown in two representative animals and with two analytical methods (human CD45 for SSC and human CD45 for mouse CD45). These plots also show that human CD45+ cells identified in NSG brains after transplantation express CD11b, CX3Cr1 and GFP. Figure 1G illustrates the abundance of human CD45+CD11b+ cells recovered from the brains of NSG and RagMLD mice transplanted IV or ICV with CD34+ cells derived from umbilical cord blood 12-20 weeks after transplantation (NSG) or sublethally irradiated Rag- / -γ-chain- / -As2- / - (NSG) and 5 weeks after transplantation (Rag- / -γ-chain- / -As2- / -). Values ​​are expressed as fold over IV, which is equal to 3+ / -1.3 in NSG mice and 2.9+ / -0.7 in RagMLD. N>5 mice / group; mean and SD shown. P<0.001 by Student's t-test in NSG mice; p<0.05 by one-way ANOVA with Bonferroni post-hoc test in RagMLD mice.FIG. 1H illustrates results from immunofluorescence analysis for GFP, Iba-1 (costaining), CD11b (costaining), CD68 (no costaining) and CD163 (no costaining) on ​​brain slices from NSG mice 90 days after ICV transfer of GFP-transduced CD34+ cells. In blue, nuclei were stained by TP III. 20× and 40× magnification of relevant dotted boxes are shown. M=confluent. [Figure 1BCD]Figures 1A-1H illustrate bone marrow cell reconstitution in the brain after intraventricular 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 bone marrow ablated mice (BU: bone marrow ablated with busulfan treatment; IRR: lethal irradiation). Various time points of analysis are shown. 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 (ICV vs IV and time p < 0.005) in BU and IRR mice. These data indicate rapid and robust bone marrow cell engraftment in the brain following intraventricular injection of HSPCs. Graph bars are shown in triplicate sets of four bars: from left to right: IRR IV (gray), IRR ICV (white), BU IV (dark grey), and BU ICV (darker grey). Figure 1C illustrates a reconstruction of a sagittal section of the brain of a representative ICV-transplanted BU-TX mouse, showing widespread distribution of GFP+ cells 90 days after ICV injection of GFP-transduced HSPCs. Nuclei are shown with GFP (green / gray) and Topro III (TPIII, light blue / light grey). Images were acquired with a Delta Vision Olympus at 20x magnification and processed by Soft Work3.5.0. Reconstructions were performed using Adobe Photoshop CS 8.0 software. FIG. 1D illustrates immunofluorescence analysis for GFP (green / gray) and Iba-1 (red / light gray) in brain slices from BU_TX mice 90 days after ICV transplantation of GFP-transduced HSPCs. M=fusion. 20× and 40× magnification of relevant dotted boxes are shown.Images were acquired with a confocal microscope Radiance2100 (Bio-Rad) Ix70 and processed by Soft Work3.5.0. Figure 1E is an experimental scheme for transplantation of human CD34+ cells (which become HSPCs in humans and are considered as the equivalent population to Lin-cells derived from mice) transduced with LVs encoding GFP or arylsulfatase A (ARSA) in NSG mice or Rag- / -γ-chain- / -As2- / - (RagMLD) mice pretreated with BU 16 mg / kg x 4 days (NSG) or sublethal irradiation (RagMLD). NSG mice also received unmanipulated mononuclear cells from NSG donors. The graph includes four sets of bars. 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 1F illustrates representative dot plots from the analysis of brain mononuclear cells derived from NSG mice transplanted with human CD34+ cells transduced with GFP LV as early as 20 weeks. The abundance of human cells in mouse brain is shown in two representative animals and with two analytical methods (human CD45 for SSC and human CD45 for mouse CD45). These plots also show that human CD45+ cells identified in NSG brains after transplantation express CD11b, CX3Cr1 and GFP. Figure 1G illustrates the abundance of human CD45+CD11b+ cells recovered from the brains of NSG and RagMLD mice transplanted IV or ICV with CD34+ cells derived from umbilical cord blood 12-20 weeks after transplantation (NSG) or sublethally irradiated Rag- / -γ-chain- / -As2- / - (NSG) and 5 weeks after transplantation (Rag- / -γ-chain- / -As2- / -). Values ​​are expressed as fold over IV, which is equal to 3+ / -1.3 in NSG mice and 2.9+ / -0.7 in RagMLD. N>5 mice / group; mean and SD shown. P<0.001 by Student's t-test in NSG mice; p<0.05 by one-way ANOVA with Bonferroni post-hoc test in RagMLD mice.FIG. 1H illustrates results from immunofluorescence analysis for GFP, Iba-1 (costaining), CD11b (costaining), CD68 (no costaining) and CD163 (no costaining) on ​​brain slices from NSG mice 90 days after ICV transfer of GFP-transduced CD34+ cells. In blue, nuclei were stained by TP III. 20× and 40× magnification of relevant dotted boxes are shown. M=confluent. [Figure 1E]Figures 1A-1H illustrate bone marrow cell reconstitution in the brain after intraventricular 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 bone marrow ablated mice (BU: bone marrow ablated with busulfan treatment; IRR: lethal irradiation). Various time points of analysis are shown. 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 (ICV vs IV and time p < 0.005) in BU and IRR mice. These data indicate rapid and robust bone marrow cell engraftment in the brain following intraventricular injection of HSPCs. Graph bars are shown in triplicate sets of four bars: from left to right: IRR IV (gray), IRR ICV (white), BU IV (dark grey), and BU ICV (darker grey). Figure 1C illustrates a reconstruction of a sagittal section of the brain of a representative ICV-transplanted BU-TX mouse, showing widespread distribution of GFP+ cells 90 days after ICV injection of GFP-transduced HSPCs. Nuclei are shown with GFP (green / gray) and Topro III (TPIII, light blue / light grey). Images were acquired with a Delta Vision Olympus at 20x magnification and processed by Soft Work3.5.0. Reconstructions were performed using Adobe Photoshop CS 8.0 software. FIG. 1D illustrates immunofluorescence analysis for GFP (green / gray) and Iba-1 (red / light gray) in brain slices from BU_TX mice 90 days after ICV transplantation of GFP-transduced HSPCs. M=fusion. 20× and 40× magnification of relevant dotted boxes are shown.Images were acquired with a confocal microscope Radiance2100 (Bio-Rad) Ix70 and processed by Soft Work3.5.0. Figure 1E is an experimental scheme for transplantation of human CD34+ cells (which become HSPCs in humans and are considered as the equivalent population to Lin-cells derived from mice) transduced with LVs encoding GFP or arylsulfatase A (ARSA) in NSG mice or Rag- / -γ-chain- / -As2- / - (RagMLD) mice pretreated with BU 16 mg / kg x 4 days (NSG) or sublethal irradiation (RagMLD). NSG mice also received unmanipulated mononuclear cells from NSG donors. The graph includes four sets of bars. 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 1F illustrates representative dot plots from the analysis of brain mononuclear cells derived from NSG mice transplanted with human CD34+ cells transduced with GFP LV as early as 20 weeks. The abundance of human cells in mouse brain is shown in two representative animals and with two analytical methods (human CD45 for SSC and human CD45 for mouse CD45). These plots also show that human CD45+ cells identified in NSG brains after transplantation express CD11b, CX3Cr1 and GFP. Figure 1G illustrates the abundance of human CD45+CD11b+ cells recovered from the brains of NSG and RagMLD mice transplanted IV or ICV with CD34+ cells derived from umbilical cord blood 12-20 weeks after transplantation (NSG) or sublethally irradiated Rag- / -γ-chain- / -As2- / - (NSG) and 5 weeks after transplantation (Rag- / -γ-chain- / -As2- / -). Values ​​are expressed as fold over IV, which is equal to 3+ / -1.3 in NSG mice and 2.9+ / -0.7 in RagMLD. N>5 mice / group; mean and SD shown. P<0.001 by Student's t-test in NSG mice; p<0.05 by one-way ANOVA with Bonferroni post-hoc test in RagMLD mice.FIG. 1H illustrates results from immunofluorescence analysis for GFP, Iba-1 (costaining), CD11b (costaining), CD68 (no costaining) and CD163 (no costaining) on ​​brain slices from NSG mice 90 days after ICV transfer of GFP-transduced CD34+ cells. In blue, nuclei were stained by TP III. 20× and 40× magnification of relevant dotted boxes are shown. M=confluent. [Figure 1F]Figures 1A-1H illustrate bone marrow cell reconstitution in the brain after intraventricular 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 bone marrow ablated mice (BU: bone marrow ablated with busulfan treatment; IRR: lethal irradiation). Various time points of analysis are shown. 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 (ICV vs IV and time p < 0.005) in BU and IRR mice. These data indicate rapid and robust bone marrow cell engraftment in the brain following intraventricular injection of HSPCs. Graph bars are shown in triplicate sets of four bars: from left to right: IRR IV (gray), IRR ICV (white), BU IV (dark grey), and BU ICV (darker grey). Figure 1C illustrates a reconstruction of a sagittal section of the brain of a representative ICV-transplanted BU-TX mouse, showing widespread distribution of GFP+ cells 90 days after ICV injection of GFP-transduced HSPCs. Nuclei are shown with GFP (green / gray) and Topro III (TPIII, light blue / light grey). Images were acquired with a Delta Vision Olympus at 20x magnification and processed by Soft Work3.5.0. Reconstructions were performed using Adobe Photoshop CS 8.0 software. FIG. 1D illustrates immunofluorescence analysis for GFP (green / gray) and Iba-1 (red / light gray) in brain slices from BU_TX mice 90 days after ICV transplantation of GFP-transduced HSPCs. M=fusion. 20× and 40× magnification of relevant dotted boxes are shown.Images were acquired with a confocal microscope Radiance2100 (Bio-Rad) Ix70 and processed by Soft Work3.5.0. Figure 1E is an experimental scheme for transplantation of human CD34+ cells (which become HSPCs in humans and are considered as the equivalent population to Lin-cells derived from mice) transduced with LVs encoding GFP or arylsulfatase A (ARSA) in NSG mice or Rag- / -γ-chain- / -As2- / - (RagMLD) mice pretreated with BU 16 mg / kg x 4 days (NSG) or sublethal irradiation (RagMLD). NSG mice also received unmanipulated mononuclear cells from NSG donors. The graph includes four sets of bars. 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 1F illustrates representative dot plots from the analysis of brain mononuclear cells derived from NSG mice transplanted with human CD34+ cells transduced with GFP LV as early as 20 weeks. The abundance of human cells in mouse brain is shown in two representative animals and with two analytical methods (human CD45 for SSC and human CD45 for mouse CD45). These plots also show that human CD45+ cells identified in NSG brains after transplantation express CD11b, CX3Cr1 and GFP. Figure 1G illustrates the abundance of human CD45+CD11b+ cells recovered from the brains of NSG and RagMLD mice transplanted IV or ICV with CD34+ cells derived from umbilical cord blood 12-20 weeks after transplantation (NSG) or sublethally irradiated Rag- / -γ-chain- / -As2- / - (NSG) and 5 weeks after transplantation (Rag- / -γ-chain- / -As2- / -). Values ​​are expressed as fold over IV, which is equal to 3+ / -1.3 in NSG mice and 2.9+ / -0.7 in RagMLD. N>5 mice / group; mean and SD shown. P<0.001 by Student's t-test in NSG mice; p<0.05 by one-way ANOVA with Bonferroni post-hoc test in RagMLD mice.FIG. 1H illustrates results from immunofluorescence analysis for GFP, Iba-1 (costaining), CD11b (costaining), CD68 (no costaining) and CD163 (no costaining) on ​​brain slices from NSG mice 90 days after ICV transfer of GFP-transduced CD34+ cells. In blue, nuclei were stained by TP III. 20× and 40× magnification of relevant dotted boxes are shown. M=confluent. [Figure 1G]Figures 1A-1H illustrate bone marrow cell reconstitution in the brain after intraventricular 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 bone marrow ablated mice (BU: bone marrow ablated with busulfan treatment; IRR: lethal irradiation). Various time points of analysis are shown. 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 (ICV vs IV and time p < 0.005) in BU and IRR mice. These data indicate rapid and robust bone marrow cell engraftment in the brain following intraventricular injection of HSPCs. Graph bars are shown in triplicate sets of four bars: from left to right: IRR IV (gray), IRR ICV (white), BU IV (dark grey), and BU ICV (darker grey). Figure 1C illustrates a reconstruction of a sagittal section of the brain of a representative ICV-transplanted BU-TX mouse, showing widespread distribution of GFP+ cells 90 days after ICV injection of GFP-transduced HSPCs. Nuclei are shown with GFP (green / gray) and Topro III (TPIII, light blue / light grey). Images were acquired with a Delta Vision Olympus at 20x magnification and processed by Soft Work3.5.0. Reconstructions were performed using Adobe Photoshop CS 8.0 software. FIG. 1D illustrates immunofluorescence analysis for GFP (green / gray) and Iba-1 (red / light gray) in brain slices from BU_TX mice 90 days after ICV transplantation of GFP-transduced HSPCs. M=fusion. 20× and 40× magnification of relevant dotted boxes are shown.Images were acquired with a confocal microscope Radiance2100 (Bio-Rad) Ix70 and processed by Soft Work3.5.0. Figure 1E is an experimental scheme for transplantation of human CD34+ cells (which become HSPCs in humans and are considered as the equivalent population to Lin-cells derived from mice) transduced with LVs encoding GFP or arylsulfatase A (ARSA) in NSG mice or Rag- / -γ-chain- / -As2- / - (RagMLD) mice pretreated with BU 16 mg / kg x 4 days (NSG) or sublethal irradiation (RagMLD). NSG mice also received unmanipulated mononuclear cells from NSG donors. The graph includes four sets of bars. 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 1F illustrates representative dot plots from the analysis of brain mononuclear cells derived from NSG mice transplanted with human CD34+ cells transduced with GFP LV as early as 20 weeks. The abundance of human cells in mouse brain is shown in two representative animals and with two analytical methods (human CD45 for SSC and human CD45 for mouse CD45). These plots also show that human CD45+ cells identified in NSG brains after transplantation express CD11b, CX3Cr1 and GFP. Figure 1G illustrates the abundance of human CD45+CD11b+ cells recovered from the brains of NSG and RagMLD mice transplanted IV or ICV with CD34+ cells derived from umbilical cord blood 12-20 weeks after transplantation (NSG) or sublethally irradiated Rag- / -γ-chain- / -As2- / - (NSG) and 5 weeks after transplantation (Rag- / -γ-chain- / -As2- / -). Values ​​are expressed as fold over IV, which is equal to 3+ / -1.3 in NSG mice and 2.9+ / -0.7 in RagMLD. N>5 mice / group; mean and SD shown. P<0.001 by Student's t-test in NSG mice; p<0.05 by one-way ANOVA with Bonferroni post-hoc test in RagMLD mice.FIG. 1H illustrates results from immunofluorescence analysis for GFP, Iba-1 (costaining), CD11b (costaining), CD68 (no costaining) and CD163 (no costaining) on ​​brain slices from NSG mice 90 days after ICV transfer of GFP-transduced CD34+ cells. In blue, nuclei were stained by TP III. 20× and 40× magnification of relevant dotted boxes are shown. M=confluent. [Figure 1H]Figures 1A-1H illustrate bone marrow cell reconstitution in the brain after intraventricular 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 bone marrow ablated mice (BU: bone marrow ablated with busulfan treatment; IRR: lethal irradiation). Various time points of analysis are shown. 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 (ICV vs IV and time p < 0.005) in BU and IRR mice. These data indicate rapid and robust bone marrow cell engraftment in the brain following intraventricular injection of HSPCs. Graph bars are shown in triplicate sets of four bars: from left to right: IRR IV (gray), IRR ICV (white), BU IV (dark grey), and BU ICV (darker grey). Figure 1C illustrates a reconstruction of a sagittal section of the brain of a representative ICV-transplanted BU-TX mouse, showing widespread distribution of GFP+ cells 90 days after ICV injection of GFP-transduced HSPCs. Nuclei are shown with GFP (green / gray) and Topro III (TPIII, light blue / light grey). Images were acquired with a Delta Vision Olympus at 20x magnification and processed by Soft Work3.5.0. Reconstructions were performed using Adobe Photoshop CS 8.0 software. FIG. 1D illustrates immunofluorescence analysis for GFP (green / gray) and Iba-1 (red / light gray) in brain slices from BU_TX mice 90 days after ICV transplantation of GFP-transduced HSPCs. M=fusion. 20× and 40× magnification of relevant dotted boxes are shown.Images were acquired with a confocal microscope Radiance2100 (Bio-Rad) Ix70 and processed by Soft Work3.5.0. Figure 1E is an experimental scheme for transplantation of human CD34+ cells (which become HSPCs in humans and are considered as the equivalent population to Lin-cells derived from mice) transduced with LVs encoding GFP or arylsulfatase A (ARSA) in NSG mice or Rag- / -γ-chain- / -As2- / - (RagMLD) mice pretreated with BU 16 mg / kg x 4 days (NSG) or sublethal irradiation (RagMLD). NSG mice also received unmanipulated mononuclear cells from NSG donors. The graph includes four sets of bars. 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 1F illustrates representative dot plots from the analysis of brain mononuclear cells derived from NSG mice transplanted with human CD34+ cells transduced with GFP LV as early as 20 weeks. The abundance of human cells in mouse brain is shown in two representative animals and with two analytical methods (human CD45 for SSC and human CD45 for mouse CD45). These plots also show that human CD45+ cells identified in NSG brains after transplantation express CD11b, CX3Cr1 and GFP. Figure 1G illustrates the abundance of human CD45+CD11b+ cells recovered from the brains of NSG and RagMLD mice transplanted IV or ICV with CD34+ cells derived from umbilical cord blood 12-20 weeks after transplantation (NSG) or sublethally irradiated Rag- / -γ-chain- / -As2- / - (NSG) and 5 weeks after transplantation (Rag- / -γ-chain- / -As2- / -). Values ​​are expressed as fold over IV, which is equal to 3+ / -1.3 in NSG mice and 2.9+ / -0.7 in RagMLD. N>5 mice / group; mean and SD shown. P<0.001 by Student's t-test in NSG mice; p<0.05 by one-way ANOVA with Bonferroni post-hoc test in RagMLD mice.FIG. 1H illustrates results from immunofluorescence analysis for GFP, Iba-1 (costaining), CD11b (costaining), CD68 (no costaining) and CD163 (no costaining) on ​​brain slices from NSG mice 90 days after ICV transfer of GFP-transduced CD34+ cells. In blue, nuclei were stained by TP III. 20× and 40× magnification of relevant dotted boxes are shown. M=confluent.

[0072] [Diagram 2]Figures 2A-2D show short-term monitoring of mice ICV transplanted with GFP+Lin- HSPCs. Figures 2A and B show two graphs showing 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 at each time point; mean and SD are shown. P value < 0.001 at day 4 compared to 1, 3, 6 and 24 hours as analyzed by one-way ANOVA with Bonferroni post-hoc test. Engraftment of ICV transplanted cells was predominantly present 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 injection of transduced Lin- HSPCs (injection represents HSPCs at the time of injection). N > 3 mice at each time point; mean and SD are shown. Two-way anova showed a significant effect of marker and time (p < 0.0001). In Figure 2C, for GFP+ cells, arrows indicate % expression of c-Kit, Sca1, CD34, CXCR4, CD93 and Tie2. In Figure 2C, for GFP- cells, circles are, from top to bottom, CXCR4, CD34, CD93, c-Kit, Sca1 and Tie2 in each row. In Figure 2D, for GFP+ cells, arrows indicate % expression of CD11b, CX3CR1 and CD115. In Figure 2D, for GFP- cells, circles are, from top to bottom, CD11b, CX3CR1 and CD115 in each row. Transplanted cells / their progeny transiently increase expression of hematopoietic stem cells and subsequently improve expression levels of myeloid / microglial markers.

[0073] [Figure 3A]Figures 3A-3J show that optimization of combinatorial transplantation protocols allows for modulating the contribution of IV vs. ICV transplanted cells to donor bone marrow brain chimerism post-transplant for appropriate clinical applications. Figure 3A is a graph showing brain engraftment of donor (GFP+) Lin- HSPCs transplanted IV 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. The graph shows that transplantation of HSPCs at the two time points results in similar brain engraftment in the test population. FIG. 3B illustrates the gating strategy for identification of μ, TAμ, and CNS macrophages (CNSmac) as CD45+lowCD11bhigh, CD45+low, CD11b+low, and CD45highCD11bhigh in newborn mice 3 days postnatal (pnd), adult mice 21pnd and 60pnd, and adult HSPC-transplanted animals 2 months after transplantation. FIG. 3C illustrates the experimental scheme showing the IV and / or ICV transplantation strategy of differentially labeled (GFP- or ΔNGFR-encoding LV) hematopoietic cells in busulfan-pretreated recipients. Transplanted hematopoietic cells are Lin- HSPCs, or c-kit+Sca1+Lin- (KSL) cells, or whole bone marrow (BM). FIG. 3D is a graph illustrating the presence 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 after transplantation. GFP+ICV transplanted cells do not show robust engraftment in the BM. Figure 3E is a graph illustrating the percentage of donor-derived cells (as the sum of cells expressing ΔNGFR and GFP) in 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-transplants alone.From the leftmost bar in each set: IV only (white), LIN IV LIN ICV (dark grey), KLS IV LIN ICV (medium grey) and BM IV LIN ICV (grey). 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 at 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 (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). Figure 3G illustrates the experimental scheme showing the transplantation strategy of IV and / or ICV of differentially labeled (LV encoding GFP or ΔNGFR) hematopoietic cells in busulfan pretreated recipients, where IV cells were transplanted 5 days after chemotherapy, whereas ICV cells were transplanted on day 0. The transplanted hematopoietic cells are Lin- HSPCs or KSL cells or whole BM. FIG. 3H is a graph illustrating the prevalence of donor-derived cells (CD45.1=progeny of IV-transplanted cells, GFP-expressing cells=progeny of ICV-transplanted cells) among all BM cells recovered from transplanted mice at sacrifice 3 months after transplantation. GFP+ICV-transplanted cells do not engraft in BM. FIG. 3I is a graph illustrating the prevalence of donor-derived cells (as the sum of ΔNGFR and GFP-expressing cells) among CD45+CD11b+ cells, μ, TAμ, and CNSmac of transplanted mice, indicated by color coding, at day 5. Co-delivery of hematopoietic cells by ICV and IV improves brain donor chimerism after transplantation in all combinations tested compared to IV with Lin-transplant alone. The leftmost bar in each set is IV only (white), LIN IV LIN ICV (dark gray), KLS IV LIN ICV (medium gray), and BM IV LIN ICV (gray).FIG. 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 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 / 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 show that optimization of combinatorial transplantation protocols allows for modulating the contribution of IV vs. ICV transplanted cells to donor bone marrow brain chimerism post-transplant for appropriate clinical applications. Figure 3A is a graph showing brain engraftment of donor (GFP+) Lin- HSPCs transplanted IV 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. The graph shows that transplantation of HSPCs at the two time points results in similar brain engraftment in the test population. FIG. 3B illustrates the gating strategy for identification of μ, TAμ, and CNS macrophages (CNSmac) as CD45+lowCD11bhigh, CD45+low, CD11b+low, and CD45highCD11bhigh in newborn mice 3 days postnatal (pnd), adult mice 21pnd and 60pnd, and adult HSPC-transplanted animals 2 months after transplantation. FIG. 3C illustrates the experimental scheme showing the IV and / or ICV transplantation strategy of differentially labeled (GFP- or ΔNGFR-encoding LV) hematopoietic cells in busulfan-pretreated recipients. Transplanted hematopoietic cells are Lin- HSPCs, or c-kit+Sca1+Lin- (KSL) cells, or whole bone marrow (BM). FIG. 3D is a graph illustrating the presence 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 after transplantation. GFP+ICV transplanted cells do not show robust engraftment in the BM. Figure 3E is a graph illustrating the percentage of donor-derived cells (as the sum of cells expressing ΔNGFR and GFP) in 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-transplants alone.From the leftmost bar in each set: IV only (white), LIN IV LIN ICV (dark grey), KLS IV LIN ICV (medium grey) and BM IV LIN ICV (grey). 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 at 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 (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). Figure 3G illustrates the experimental scheme showing the transplantation strategy of IV and / or ICV of differentially labeled (LV encoding GFP or ΔNGFR) hematopoietic cells in busulfan pretreated recipients, where IV cells were transplanted 5 days after chemotherapy, whereas ICV cells were transplanted on day 0. The transplanted hematopoietic cells are Lin- HSPCs or KSL cells or whole BM. FIG. 3H is a graph illustrating the prevalence of donor-derived cells (CD45.1=progeny of IV-transplanted cells, GFP-expressing cells=progeny of ICV-transplanted cells) among all BM cells recovered from transplanted mice at sacrifice 3 months after transplantation. GFP+ICV-transplanted cells do not engraft in BM. FIG. 3I is a graph illustrating the prevalence of donor-derived cells (as the sum of ΔNGFR and GFP-expressing cells) among CD45+CD11b+ cells, μ, TAμ, and CNSmac of transplanted mice, indicated by color coding, at day 5. Co-delivery of hematopoietic cells by ICV and IV improves brain donor chimerism after transplantation in all combinations tested compared to IV with Lin-transplant alone. The leftmost bar in each set is IV only (white), LIN IV LIN ICV (dark gray), KLS IV LIN ICV (medium gray), and BM IV LIN ICV (gray).FIG. 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 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 / 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 3C-D]Figures 3A-3J show that optimization of combinatorial transplantation protocols allows for modulating the contribution of IV vs. ICV transplanted cells to donor bone marrow brain chimerism post-transplant for appropriate clinical applications. Figure 3A is a graph showing brain engraftment of donor (GFP+) Lin- HSPCs transplanted IV 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. The graph shows that transplantation of HSPCs at the two time points results in similar brain engraftment in the test population. FIG. 3B illustrates the gating strategy for identification of μ, TAμ, and CNS macrophages (CNSmac) as CD45+lowCD11bhigh, CD45+low, CD11b+low, and CD45highCD11bhigh in newborn mice 3 days postnatal (pnd), adult mice 21pnd and 60pnd, and adult HSPC-transplanted animals 2 months after transplantation. FIG. 3C illustrates the experimental scheme showing the IV and / or ICV transplantation strategy of differentially labeled (GFP- or ΔNGFR-encoding LV) hematopoietic cells in busulfan-pretreated recipients. Transplanted hematopoietic cells are Lin- HSPCs, or c-kit+Sca1+Lin- (KSL) cells, or whole bone marrow (BM). FIG. 3D is a graph illustrating the presence 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 after transplantation. GFP+ICV transplanted cells do not show robust engraftment in the BM. Figure 3E is a graph illustrating the percentage of donor-derived cells (as the sum of cells expressing ΔNGFR and GFP) in 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-transplants alone.From the leftmost bar in each set: IV only (white), LIN IV LIN ICV (dark grey), KLS IV LIN ICV (medium grey) and BM IV LIN ICV (grey). 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 at 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 (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). Figure 3G illustrates the experimental scheme showing the transplantation strategy of IV and / or ICV of differentially labeled (LV encoding GFP or ΔNGFR) hematopoietic cells in busulfan pretreated recipients, where IV cells were transplanted 5 days after chemotherapy, whereas ICV cells were transplanted on day 0. The transplanted hematopoietic cells are Lin- HSPCs or KSL cells or whole BM. FIG. 3H is a graph illustrating the prevalence of donor-derived cells (CD45.1=progeny of IV-transplanted cells, GFP-expressing cells=progeny of ICV-transplanted cells) among all BM cells recovered from transplanted mice at sacrifice 3 months after transplantation. GFP+ICV-transplanted cells do not engraft in BM. FIG. 3I is a graph illustrating the prevalence of donor-derived cells (as the sum of ΔNGFR and GFP-expressing cells) among CD45+CD11b+ cells, μ, TAμ, and CNSmac of transplanted mice, indicated by color coding, at day 5. Co-delivery of hematopoietic cells by ICV and IV improves brain donor chimerism after transplantation in all combinations tested compared to IV with Lin-transplant alone. The leftmost bar in each set is IV only (white), LIN IV LIN ICV (dark gray), KLS IV LIN ICV (medium gray), and BM IV LIN ICV (gray).FIG. 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 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 / 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 show that optimization of combinatorial transplantation protocols allows for modulating the contribution of IV vs. ICV transplanted cells to donor bone marrow brain chimerism post-transplant for appropriate clinical applications. Figure 3A is a graph showing brain engraftment of donor (GFP+) Lin- HSPCs transplanted IV 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. The graph shows that transplantation of HSPCs at the two time points results in similar brain engraftment in the test population. FIG. 3B illustrates the gating strategy for identification of μ, TAμ, and CNS macrophages (CNSmac) as CD45+lowCD11bhigh, CD45+low, CD11b+low, and CD45highCD11bhigh in newborn mice 3 days postnatal (pnd), adult mice 21pnd and 60pnd, and adult HSPC-transplanted animals 2 months after transplantation. FIG. 3C illustrates the experimental scheme showing the IV and / or ICV transplantation strategy of differentially labeled (GFP- or ΔNGFR-encoding LV) hematopoietic cells in busulfan-pretreated recipients. Transplanted hematopoietic cells are Lin- HSPCs, or c-kit+Sca1+Lin- (KSL) cells, or whole bone marrow (BM). FIG. 3D is a graph illustrating the presence 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 after transplantation. GFP+ICV transplanted cells do not show robust engraftment in the BM. Figure 3E is a graph illustrating the percentage of donor-derived cells (as the sum of cells expressing ΔNGFR and GFP) in 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-transplants alone.From the leftmost bar in each set: IV only (white), LIN IV LIN ICV (dark grey), KLS IV LIN ICV (medium grey) and BM IV LIN ICV (grey). 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 at 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 (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). Figure 3G illustrates the experimental scheme showing the transplantation strategy of IV and / or ICV of differentially labeled (LV encoding GFP or ΔNGFR) hematopoietic cells in busulfan pretreated recipients, where IV cells were transplanted 5 days after chemotherapy, whereas ICV cells were transplanted on day 0. The transplanted hematopoietic cells are Lin- HSPCs or KSL cells or whole BM. FIG. 3H is a graph illustrating the prevalence of donor-derived cells (CD45.1=progeny of IV-transplanted cells, GFP-expressing cells=progeny of ICV-transplanted cells) among all BM cells recovered from transplanted mice at sacrifice 3 months after transplantation. GFP+ICV-transplanted cells do not engraft in BM. FIG. 3I is a graph illustrating the prevalence of donor-derived cells (as the sum of ΔNGFR and GFP-expressing cells) among CD45+CD11b+ cells, μ, TAμ, and CNSmac of transplanted mice, indicated by color coding, at day 5. Co-delivery of hematopoietic cells by ICV and IV improves brain donor chimerism after transplantation in all combinations tested compared to IV with Lin-transplant alone. The leftmost bar in each set is IV only (white), LIN IV LIN ICV (dark gray), KLS IV LIN ICV (medium gray), and BM IV LIN ICV (gray).FIG. 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 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 / 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 show that optimization of combinatorial transplantation protocols allows for modulating the contribution of IV vs. ICV transplanted cells to donor bone marrow brain chimerism post-transplant for appropriate clinical applications. Figure 3A is a graph showing brain engraftment of donor (GFP+) Lin- HSPCs transplanted IV 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. The graph shows that transplantation of HSPCs at the two time points results in similar brain engraftment in the test population. FIG. 3B illustrates the gating strategy for identification of μ, TAμ, and CNS macrophages (CNSmac) as CD45+lowCD11bhigh, CD45+low, CD11b+low, and CD45highCD11bhigh in newborn mice 3 days postnatal (pnd), adult mice 21pnd and 60pnd, and adult HSPC-transplanted animals 2 months after transplantation. FIG. 3C illustrates the experimental scheme showing the IV and / or ICV transplantation strategy of differentially labeled (GFP- or ΔNGFR-encoding LV) hematopoietic cells in busulfan-pretreated recipients. Transplanted hematopoietic cells are Lin- HSPCs, or c-kit+Sca1+Lin- (KSL) cells, or whole bone marrow (BM). FIG. 3D is a graph illustrating the presence 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 after transplantation. GFP+ICV transplanted cells do not show robust engraftment in the BM. Figure 3E is a graph illustrating the percentage of donor-derived cells (as the sum of cells expressing ΔNGFR and GFP) in 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-transplants alone.From the leftmost bar in each set: IV only (white), LIN IV LIN ICV (dark grey), KLS IV LIN ICV (medium grey) and BM IV LIN ICV (grey). 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 at 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 (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). Figure 3G illustrates the experimental scheme showing the transplantation strategy of IV and / or ICV of differentially labeled (LV encoding GFP or ΔNGFR) hematopoietic cells in busulfan pretreated recipients, where IV cells were transplanted 5 days after chemotherapy, whereas ICV cells were transplanted on day 0. The transplanted hematopoietic cells are Lin- HSPCs or KSL cells or whole BM. FIG. 3H is a graph illustrating the prevalence of donor-derived cells (CD45.1=progeny of IV-transplanted cells, GFP-expressing cells=progeny of ICV-transplanted cells) among all BM cells recovered from transplanted mice at sacrifice 3 months after transplantation. GFP+ICV-transplanted cells do not engraft in BM. FIG. 3I is a graph illustrating the prevalence of donor-derived cells (as the sum of ΔNGFR and GFP-expressing cells) among CD45+CD11b+ cells, μ, TAμ, and CNSmac of transplanted mice, indicated by color coding, at day 5. Co-delivery of hematopoietic cells by ICV and IV improves brain donor chimerism after transplantation in all combinations tested compared to IV with Lin-transplant alone. The leftmost bar in each set is IV only (white), LIN IV LIN ICV (dark gray), KLS IV LIN ICV (medium gray), and BM IV LIN ICV (gray).FIG. 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 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 / 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 show that optimization of combinatorial transplantation protocols allows for modulating the contribution of IV vs. ICV transplanted cells to donor bone marrow brain chimerism post-transplant for appropriate clinical applications. Figure 3A is a graph showing brain engraftment of donor (GFP+) Lin- HSPCs transplanted IV 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. The graph shows that transplantation of HSPCs at the two time points results in similar brain engraftment in the test population. FIG. 3B illustrates the gating strategy for identification of μ, TAμ, and CNS macrophages (CNSmac) as CD45+lowCD11bhigh, CD45+low, CD11b+low, and CD45highCD11bhigh in newborn mice 3 days postnatal (pnd), adult mice 21pnd and 60pnd, and adult HSPC-transplanted animals 2 months after transplantation. FIG. 3C illustrates the experimental scheme showing the IV and / or ICV transplantation strategy of differentially labeled (GFP- or ΔNGFR-encoding LV) hematopoietic cells in busulfan-pretreated recipients. Transplanted hematopoietic cells are Lin- HSPCs, or c-kit+Sca1+Lin- (KSL) cells, or whole bone marrow (BM). FIG. 3D is a graph illustrating the presence 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 after transplantation. GFP+ICV transplanted cells do not show robust engraftment in the BM. Figure 3E is a graph illustrating the percentage of donor-derived cells (as the sum of cells expressing ΔNGFR and GFP) in 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-transplants alone.From the leftmost bar in each set: IV only (white), LIN IV LIN ICV (dark grey), KLS IV LIN ICV (medium grey) and BM IV LIN ICV (grey). 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 at 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 (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). Figure 3G illustrates the experimental scheme showing the transplantation strategy of IV and / or ICV of differentially labeled (LV encoding GFP or ΔNGFR) hematopoietic cells in busulfan pretreated recipients, where IV cells were transplanted 5 days after chemotherapy, whereas ICV cells were transplanted on day 0. The transplanted hematopoietic cells are Lin- HSPCs or KSL cells or whole BM. FIG. 3H is a graph illustrating the prevalence of donor-derived cells (CD45.1=progeny of IV-transplanted cells, GFP-expressing cells=progeny of ICV-transplanted cells) among all BM cells recovered from transplanted mice at sacrifice 3 months after transplantation. GFP+ICV-transplanted cells do not engraft in BM. FIG. 3I is a graph illustrating the prevalence of donor-derived cells (as the sum of ΔNGFR and GFP-expressing cells) among CD45+CD11b+ cells, μ, TAμ, and CNSmac of transplanted mice, indicated by color coding, at day 5. Co-delivery of hematopoietic cells by ICV and IV improves brain donor chimerism after transplantation in all combinations tested compared to IV with Lin-transplant alone. The leftmost bar in each set is IV only (white), LIN IV LIN ICV (dark gray), KLS IV LIN ICV (medium gray), and BM IV LIN ICV (gray).FIG. 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 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 / 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 show that optimization of combinatorial transplantation protocols allows for modulating the contribution of IV vs. ICV transplanted cells to donor bone marrow brain chimerism post-transplant for appropriate clinical applications. Figure 3A is a graph showing brain engraftment of donor (GFP+) Lin- HSPCs transplanted IV 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. The graph shows that transplantation of HSPCs at the two time points results in similar brain engraftment in the test population. FIG. 3B illustrates the gating strategy for identification of μ, TAμ, and CNS macrophages (CNSmac) as CD45+lowCD11bhigh, CD45+low, CD11b+low, and CD45highCD11bhigh in newborn mice 3 days postnatal (pnd), adult mice 21pnd and 60pnd, and adult HSPC-transplanted animals 2 months after transplantation. FIG. 3C illustrates the experimental scheme showing the IV and / or ICV transplantation strategy of differentially labeled (GFP- or ΔNGFR-encoding LV) hematopoietic cells in busulfan-pretreated recipients. Transplanted hematopoietic cells are Lin- HSPCs, or c-kit+Sca1+Lin- (KSL) cells, or whole bone marrow (BM). FIG. 3D is a graph illustrating the presence 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 after transplantation. GFP+ICV transplanted cells do not show robust engraftment in the BM. Figure 3E is a graph illustrating the percentage of donor-derived cells (as the sum of cells expressing ΔNGFR and GFP) in 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-transplants alone.From the leftmost bar in each set: IV only (white), LIN IV LIN ICV (dark grey), KLS IV LIN ICV (medium grey) and BM IV LIN ICV (grey). 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 at 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 (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). Figure 3G illustrates the experimental scheme showing the transplantation strategy of IV and / or ICV of differentially labeled (LV encoding GFP or ΔNGFR) hematopoietic cells in busulfan pretreated recipients, where IV cells were transplanted 5 days after chemotherapy, whereas ICV cells were transplanted on day 0. The transplanted hematopoietic cells are Lin- HSPCs or KSL cells or whole BM. FIG. 3H is a graph illustrating the prevalence of donor-derived cells (CD45.1=progeny of IV-transplanted cells, GFP-expressing cells=progeny of ICV-transplanted cells) among all BM cells recovered from transplanted mice at sacrifice 3 months after transplantation. GFP+ICV-transplanted cells do not engraft in BM. FIG. 3I is a graph illustrating the prevalence of donor-derived cells (as the sum of ΔNGFR and GFP-expressing cells) among CD45+CD11b+ cells, μ, TAμ, and CNSmac of transplanted mice, indicated by color coding, at day 5. Co-delivery of hematopoietic cells by ICV and IV improves brain donor chimerism after transplantation in all combinations tested compared to IV with Lin-transplant alone. The leftmost bar in each set is IV only (white), LIN IV LIN ICV (dark gray), KLS IV LIN ICV (medium gray), and BM IV LIN ICV (gray).FIG. 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 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 / 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 show that optimization of combinatorial transplantation protocols allows for modulating the contribution of IV vs. ICV transplanted cells to donor bone marrow brain chimerism post-transplant for appropriate clinical applications. Figure 3A is a graph showing brain engraftment of donor (GFP+) Lin- HSPCs transplanted IV 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. The graph shows that transplantation of HSPCs at the two time points results in similar brain engraftment in the test population. FIG. 3B illustrates the gating strategy for identification of μ, TAμ, and CNS macrophages (CNSmac) as CD45+lowCD11bhigh, CD45+low, CD11b+low, and CD45highCD11bhigh in newborn mice 3 days postnatal (pnd), adult mice 21pnd and 60pnd, and adult HSPC-transplanted animals 2 months after transplantation. FIG. 3C illustrates the experimental scheme showing the IV and / or ICV transplantation strategy of differentially labeled (GFP- or ΔNGFR-encoding LV) hematopoietic cells in busulfan-pretreated recipients. Transplanted hematopoietic cells are Lin- HSPCs, or c-kit+Sca1+Lin- (KSL) cells, or whole bone marrow (BM). FIG. 3D is a graph illustrating the presence 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 after transplantation. GFP+ICV transplanted cells do not show robust engraftment in the BM. Figure 3E is a graph illustrating the percentage of donor-derived cells (as the sum of cells expressing ΔNGFR and GFP) in 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-transplants alone.From the leftmost bar in each set: IV only (white), LIN IV LIN ICV (dark grey), KLS IV LIN ICV (medium grey) and BM IV LIN ICV (grey). 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 at 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 (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). Figure 3G illustrates the experimental scheme showing the transplantation strategy of IV and / or ICV of differentially labeled (LV encoding GFP or ΔNGFR) hematopoietic cells in busulfan pretreated recipients, where IV cells were transplanted 5 days after chemotherapy, whereas ICV cells were transplanted on day 0. The transplanted hematopoietic cells are Lin- HSPCs or KSL cells or whole BM. FIG. 3H is a graph illustrating the prevalence of donor-derived cells (CD45.1=progeny of IV-transplanted cells, GFP-expressing cells=progeny of ICV-transplanted cells) among all BM cells recovered from transplanted mice at sacrifice 3 months after transplantation. GFP+ICV-transplanted cells do not engraft in BM. FIG. 3I is a graph illustrating the prevalence of donor-derived cells (as the sum of ΔNGFR and GFP-expressing cells) among CD45+CD11b+ cells, μ, TAμ, and CNSmac of transplanted mice, indicated by color coding, at day 5. Co-delivery of hematopoietic cells by ICV and IV improves brain donor chimerism after transplantation in all combinations tested compared to IV with Lin-transplant alone. The leftmost bar in each set is IV only (white), LIN IV LIN ICV (dark gray), KLS IV LIN ICV (medium gray), and BM IV LIN ICV (gray).FIG. 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 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 / 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 show 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 mouse brains at 90 days after transplantation of GFP+ HSPCs IV or ICV (n=5 per group). Figure 4B illustrates representative dot plots showing sorted cell populations for gene expression analysis. In particular, μ and TAμ identified by CD45 and CD11b markers in the brains of naive P10 and adult control (ADULT_CT) animals, and busulfan-treated and transplanted mice (BU_TX) at 90 days after HCT are shown. Dot plots contained within dotted squares show both GFP- endogenous and GFP+ donor-derived cell chimerism within the μ and TAμ populations of a representative transplanted BU-treated mouse. Figure 4C is a graph illustrating the fold change in expression of select microglial genes (calculated as 2-DDCT) obtained by real-time PCR in each of the indicated populations harvested from busulfan-treated IV- and ICV-implanted mouse brains or P10 mice, calculated based on the expression of the same genes in ADULT_CT μ-cells. Mean values ​​are shown. FIG. 4D is a principal component analysis (PCA) and FIG. 4E is a heatmap, both showing expression analysis of genes in samples identified as microglial signatures by Butovsky (Butovsky et al., Nature neuroscience 17:131-143 (2014)) (μ and TAμ harvested 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 show 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 mouse brains at 90 days after transplantation of GFP+ HSPCs IV or ICV (n=5 per group). Figure 4B illustrates representative dot plots showing sorted cell populations for gene expression analysis. In particular, μ and TAμ identified by CD45 and CD11b markers in the brains of naive P10 and adult control (ADULT_CT) animals, and busulfan-treated and transplanted mice (BU_TX) at 90 days after HCT are shown. Dot plots contained within dotted squares show both GFP- endogenous and GFP+ donor-derived cell chimerism within the μ and TAμ populations of a representative transplanted BU-treated mouse. Figure 4C is a graph illustrating the fold change in expression of select microglial genes (calculated as 2-DDCT) obtained by real-time PCR in each of the indicated populations harvested from busulfan-treated IV- and ICV-implanted mouse brains or P10 mice, calculated based on the expression of the same genes in ADULT_CT μ-cells. Mean values ​​are shown. FIG. 4D is a principal component analysis (PCA) and FIG. 4E is a heatmap, both showing expression analysis of genes in samples identified as microglial signatures by Butovsky (Butovsky et al., Nature neuroscience 17:131-143 (2014)) (μ and TAμ harvested 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 show 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 mouse brains at 90 days after transplantation of GFP+ HSPCs IV or ICV (n=5 per group). Figure 4B illustrates representative dot plots showing sorted cell populations for gene expression analysis. In particular, μ and TAμ identified by CD45 and CD11b markers in the brains of naive P10 and adult control (ADULT_CT) animals, and busulfan-treated and transplanted mice (BU_TX) at 90 days after HCT are shown. Dot plots contained within dotted squares show both GFP- endogenous and GFP+ donor-derived cell chimerism within the μ and TAμ populations of a representative transplanted BU-treated mouse. Figure 4C is a graph illustrating the fold change in expression of select microglial genes (calculated as 2-DDCT) obtained by real-time PCR in each of the indicated populations harvested from busulfan-treated IV- and ICV-implanted mouse brains or P10 mice, calculated based on the expression of the same genes in ADULT_CT μ-cells. Mean values ​​are shown. FIG. 4D is a principal component analysis (PCA) and FIG. 4E is a heatmap, both showing expression analysis of genes in samples identified as microglial signatures by Butovsky (Butovsky et al., Nature neuroscience 17:131-143 (2014)) (μ and TAμ harvested 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 show 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 mouse brains at 90 days after transplantation of GFP+ HSPCs IV or ICV (n=5 per group). Figure 4B illustrates representative dot plots showing sorted cell populations for gene expression analysis. In particular, μ and TAμ identified by CD45 and CD11b markers in the brains of naive P10 and adult control (ADULT_CT) animals, and busulfan-treated and transplanted mice (BU_TX) at 90 days after HCT are shown. Dot plots contained within dotted squares show both GFP- endogenous and GFP+ donor-derived cell chimerism within the μ and TAμ populations of a representative transplanted BU-treated mouse. Figure 4C is a graph illustrating the fold change in expression of select microglial genes (calculated as 2-DDCT) obtained by real-time PCR in each of the indicated populations harvested from busulfan-treated IV- and ICV-implanted mouse brains or P10 mice, calculated based on the expression of the same genes in ADULT_CT μ-cells. Mean values ​​are shown. FIG. 4D is a principal component analysis (PCA) and FIG. 4E is a heatmap, both showing expression analysis of genes in samples identified as microglial signatures by Butovsky (Butovsky et al., Nature neuroscience 17:131-143 (2014)) (μ and TAμ harvested 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 show 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 mouse brains at 90 days after transplantation of GFP+ HSPCs IV or ICV (n=5 per group). Figure 4B illustrates representative dot plots showing sorted cell populations for gene expression analysis. In particular, μ and TAμ identified by CD45 and CD11b markers in the brains of naive P10 and adult control (ADULT_CT) animals, and busulfan-treated and transplanted mice (BU_TX) at 90 days after HCT are shown. Dot plots contained within dotted squares show both GFP- endogenous and GFP+ donor-derived cell chimerism within the μ and TAμ populations of a representative transplanted BU-treated mouse. Figure 4C is a graph illustrating the fold change in expression of select microglial genes (calculated as 2-DDCT) obtained by real-time PCR in each of the indicated populations harvested from busulfan-treated IV- and ICV-implanted mouse brains or P10 mice, calculated based on the expression of the same genes in ADULT_CT μ-cells. Mean values ​​are shown. FIG. 4D is a principal component analysis (PCA) and FIG. 4E is a heatmap, both showing expression analysis of genes in samples identified as microglial signatures by Butovsky (Butovsky et al., Nature neuroscience 17:131-143 (2014)) (μ and TAμ harvested 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 transplanted mouse brains exhibit features of microglial maturation. Figure 5A shows functional enrichment of differentially upregulated genes in μCT cells vs. μ-transplanted cells. Figure 5B shows functional enrichment of differentially downregulated genes in μCT cells vs. μ-transplanted cells. Figure 5C shows functional enrichment of differentially upregulated genes in μCT cells vs. TAμ-transplanted cells. Figure 5D shows functional enrichment of differentially downregulated genes in μCT cells vs. TAμ-transplanted cells. A preliminary ranking analysis by gene set enrichment analysis (GSEA) was performed using RNA-Seq differential gene expression data against gene ontology (GO) biological processes (http: / / software.broadinstitute.org / gsea / msigdb / collection) 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 is a graph showing the fold change in expression values ​​of genes normalized by RNA-Seq, whose expression is upregulated in adult mice (Matcovitch-Natan et al., Science. 353:6301 (2016)) in the representation populations recovered from busulfan-treated transplanted mice or P10 mouse brains relative to ADULT_CT μ-cells. Figure 5F is a graph showing fold change in expression values ​​of genes normalized by RNA-Seq that are upregulated in p10 mice (Matcovitch-Natan et al., Science. 353:6301 (2016)) relative to ADULT_CT μ-cells and in the indicated populations recovered from the brains of busulfan-treated transplanted mice or P10 mice. For statistical tests, see Table 2. [Figure 5A-2]Figures 5A-5E show that bone marrow cells derived from transplanted mouse brains exhibit features of microglial maturation. Figure 5A shows functional enrichment of differentially upregulated genes in μCT cells vs. μ-transplanted cells. Figure 5B shows functional enrichment of differentially downregulated genes in μCT cells vs. μ-transplanted cells. Figure 5C shows functional enrichment of differentially upregulated genes in μCT cells vs. TAμ-transplanted cells. Figure 5D shows functional enrichment of differentially downregulated genes in μCT cells vs. TAμ-transplanted cells. A preliminary ranking analysis by gene set enrichment analysis (GSEA) was performed using RNA-Seq differential gene expression data against gene ontology (GO) biological processes (http: / / software.broadinstitute.org / gsea / msigdb / collection) 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 is a graph showing the fold change in expression values ​​of genes normalized by RNA-Seq, whose expression is upregulated in adult mice (Matcovitch-Natan et al., Science. 353:6301 (2016)) in the representation populations recovered from busulfan-treated transplanted mice or P10 mouse brains relative to ADULT_CT μ-cells. Figure 5F is a graph showing fold change in expression values ​​of genes normalized by RNA-Seq that are upregulated in p10 mice (Matcovitch-Natan et al., Science. 353:6301 (2016)) relative to ADULT_CT μ-cells and in the indicated populations recovered from the brains of busulfan-treated transplanted mice or P10 mice. For statistical tests, see Table 2. [Figure 5A-3]Figures 5A-5E show that bone marrow cells derived from transplanted mouse brains exhibit features of microglial maturation. Figure 5A shows functional enrichment of differentially upregulated genes in μCT cells vs. μ-transplanted cells. Figure 5B shows functional enrichment of differentially downregulated genes in μCT cells vs. μ-transplanted cells. Figure 5C shows functional enrichment of differentially upregulated genes in μCT cells vs. TAμ-transplanted cells. Figure 5D shows functional enrichment of differentially downregulated genes in μCT cells vs. TAμ-transplanted cells. A preliminary ranking analysis by gene set enrichment analysis (GSEA) was performed using RNA-Seq differential gene expression data against gene ontology (GO) biological processes (http: / / software.broadinstitute.org / gsea / msigdb / collection) 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 is a graph showing the fold change in expression values ​​of genes normalized by RNA-Seq, whose expression is upregulated in adult mice (Matcovitch-Natan et al., Science. 353:6301 (2016)) in the representation populations recovered from busulfan-treated transplanted mice or P10 mouse brains relative to ADULT_CT μ-cells. Figure 5F is a graph showing fold change in expression values ​​of genes normalized by RNA-Seq that are upregulated in p10 mice (Matcovitch-Natan et al., Science. 353:6301 (2016)) relative to ADULT_CT μ-cells and in the indicated populations recovered from the brains of busulfan-treated transplanted mice or P10 mice. For statistical tests, see Table 2. [Figure 5B]Figures 5A-5E show that bone marrow cells derived from transplanted mouse brains exhibit features of microglial maturation. Figure 5A shows functional enrichment of differentially upregulated genes in μCT cells vs. μ-transplanted cells. Figure 5B shows functional enrichment of differentially downregulated genes in μCT cells vs. μ-transplanted cells. Figure 5C shows functional enrichment of differentially upregulated genes in μCT cells vs. TAμ-transplanted cells. Figure 5D shows functional enrichment of differentially downregulated genes in μCT cells vs. TAμ-transplanted cells. A preliminary ranking analysis by gene set enrichment analysis (GSEA) was performed using RNA-Seq differential gene expression data against gene ontology (GO) biological processes (http: / / software.broadinstitute.org / gsea / msigdb / collection) 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 is a graph showing the fold change in expression values ​​of genes normalized by RNA-Seq, whose expression is upregulated in adult mice (Matcovitch-Natan et al., Science. 353:6301 (2016)) in the representation populations recovered from busulfan-treated transplanted mice or P10 mouse brains relative to ADULT_CT μ-cells. Figure 5F is a graph showing fold change in expression values ​​of genes normalized by RNA-Seq that are upregulated in p10 mice (Matcovitch-Natan et al., Science. 353:6301 (2016)) relative to ADULT_CT μ-cells and in the indicated populations recovered from the brains of busulfan-treated transplanted mice or P10 mice. For statistical tests, see Table 2. [Figure 5C-1]Figures 5A-5E show that bone marrow cells derived from transplanted mouse brains exhibit features of microglial maturation. Figure 5A shows functional enrichment of differentially upregulated genes in μCT cells vs. μ-transplanted cells. Figure 5B shows functional enrichment of differentially downregulated genes in μCT cells vs. μ-transplanted cells. Figure 5C shows functional enrichment of differentially upregulated genes in μCT cells vs. TAμ-transplanted cells. Figure 5D shows functional enrichment of differentially downregulated genes in μCT cells vs. TAμ-transplanted cells. A preliminary ranking analysis by gene set enrichment analysis (GSEA) was performed using RNA-Seq differential gene expression data against gene ontology (GO) biological processes (http: / / software.broadinstitute.org / gsea / msigdb / collection) 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 is a graph showing the fold change in expression values ​​of genes normalized by RNA-Seq, whose expression is upregulated in adult mice (Matcovitch-Natan et al., Science. 353:6301 (2016)) in the representation populations recovered from busulfan-treated transplanted mice or P10 mouse brains relative to ADULT_CT μ-cells. Figure 5F is a graph showing fold change in expression values ​​of genes normalized by RNA-Seq that are upregulated in p10 mice (Matcovitch-Natan et al., Science. 353:6301 (2016)) relative to ADULT_CT μ-cells and in the indicated populations recovered from the brains of busulfan-treated transplanted mice or P10 mice. For statistical tests, see Table 2. [Figure 5C-2]Figures 5A-5E show that bone marrow cells derived from transplanted mouse brains exhibit features of microglial maturation. Figure 5A shows functional enrichment of differentially upregulated genes in μCT cells vs. μ-transplanted cells. Figure 5B shows functional enrichment of differentially downregulated genes in μCT cells vs. μ-transplanted cells. Figure 5C shows functional enrichment of differentially upregulated genes in μCT cells vs. TAμ-transplanted cells. Figure 5D shows functional enrichment of differentially downregulated genes in μCT cells vs. TAμ-transplanted cells. A preliminary ranking analysis by gene set enrichment analysis (GSEA) was performed using RNA-Seq differential gene expression data against gene ontology (GO) biological processes (http: / / software.broadinstitute.org / gsea / msigdb / collection) 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 is a graph showing the fold change in expression values ​​of genes normalized by RNA-Seq, whose expression is upregulated in adult mice (Matcovitch-Natan et al., Science. 353:6301 (2016)) in the representation populations recovered from busulfan-treated transplanted mice or P10 mouse brains relative to ADULT_CT μ-cells. Figure 5F is a graph showing fold change in expression values ​​of genes normalized by RNA-Seq that are upregulated in p10 mice (Matcovitch-Natan et al., Science. 353:6301 (2016)) relative to ADULT_CT μ-cells and in the indicated populations recovered from the brains of busulfan-treated transplanted mice or P10 mice. For statistical tests, see Table 2. [Figure 5C-3]Figures 5A-5E show that bone marrow cells derived from transplanted mouse brains exhibit features of microglial maturation. Figure 5A shows functional enrichment of differentially upregulated genes in μCT cells vs. μ-transplanted cells. Figure 5B shows functional enrichment of differentially downregulated genes in μCT cells vs. μ-transplanted cells. Figure 5C shows functional enrichment of differentially upregulated genes in μCT cells vs. TAμ-transplanted cells. Figure 5D shows functional enrichment of differentially downregulated genes in μCT cells vs. TAμ-transplanted cells. A preliminary ranking analysis by gene set enrichment analysis (GSEA) was performed using RNA-Seq differential gene expression data against gene ontology (GO) biological processes (http: / / software.broadinstitute.org / gsea / msigdb / collection) 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 is a graph showing the fold change in expression values ​​of genes normalized by RNA-Seq, whose expression is upregulated in adult mice (Matcovitch-Natan et al., Science. 353:6301 (2016)) in the representation populations recovered from busulfan-treated transplanted mice or P10 mouse brains relative to ADULT_CT μ-cells. Figure 5F is a graph showing fold change in expression values ​​of genes normalized by RNA-Seq that are upregulated in p10 mice (Matcovitch-Natan et al., Science. 353:6301 (2016)) relative to ADULT_CT μ-cells and in the indicated populations recovered from the brains of busulfan-treated transplanted mice or P10 mice. For statistical tests, see Table 2. [Figure 5D-1]Figures 5A-5E show that bone marrow cells derived from transplanted mouse brains exhibit features of microglial maturation. Figure 5A shows functional enrichment of differentially upregulated genes in μCT cells vs. μ-transplanted cells. Figure 5B shows functional enrichment of differentially downregulated genes in μCT cells vs. μ-transplanted cells. Figure 5C shows functional enrichment of differentially upregulated genes in μCT cells vs. TAμ-transplanted cells. Figure 5D shows functional enrichment of differentially downregulated genes in μCT cells vs. TAμ-transplanted cells. A preliminary ranking analysis by gene set enrichment analysis (GSEA) was performed using RNA-Seq differential gene expression data against gene ontology (GO) biological processes (http: / / software.broadinstitute.org / gsea / msigdb / collection) 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 is a graph showing the fold change in expression values ​​of genes normalized by RNA-Seq, whose expression is upregulated in adult mice (Matcovitch-Natan et al., Science. 353:6301 (2016)) in the representation populations recovered from busulfan-treated transplanted mice or P10 mouse brains relative to ADULT_CT μ-cells. Figure 5F is a graph showing fold change in expression values ​​of genes normalized by RNA-Seq that are upregulated in p10 mice (Matcovitch-Natan et al., Science. 353:6301 (2016)) relative to ADULT_CT μ-cells and in the indicated populations recovered from the brains of busulfan-treated transplanted mice or P10 mice. For statistical tests, see Table 2. [Figure 5D-2]Figures 5A-5E show that bone marrow cells derived from transplanted mouse brains exhibit features of microglial maturation. Figure 5A shows functional enrichment of differentially upregulated genes in μCT cells vs. μ-transplanted cells. Figure 5B shows functional enrichment of differentially downregulated genes in μCT cells vs. μ-transplanted cells. Figure 5C shows functional enrichment of differentially upregulated genes in μCT cells vs. TAμ-transplanted cells. Figure 5D shows functional enrichment of differentially downregulated genes in μCT cells vs. TAμ-transplanted cells. A preliminary ranking analysis by gene set enrichment analysis (GSEA) was performed using RNA-Seq differential gene expression data against gene ontology (GO) biological processes (http: / / software.broadinstitute.org / gsea / msigdb / collection) 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 is a graph showing the fold change in expression values ​​of genes normalized by RNA-Seq, whose expression is upregulated in adult mice (Matcovitch-Natan et al., Science. 353:6301 (2016)) in the representation populations recovered from busulfan-treated transplanted mice or P10 mouse brains relative to ADULT_CT μ-cells. Figure 5F is a graph showing fold change in expression values ​​of genes normalized by RNA-Seq that are upregulated in p10 mice (Matcovitch-Natan et al., Science. 353:6301 (2016)) relative to ADULT_CT μ-cells and in the indicated populations recovered from the brains of busulfan-treated transplanted mice or P10 mice. For statistical tests, see Table 2. [Figure 5D-3]Figures 5A-5E show that bone marrow cells derived from transplanted mouse brains exhibit features of microglial maturation. Figure 5A shows functional enrichment of differentially upregulated genes in μCT cells vs. μ-transplanted cells. Figure 5B shows functional enrichment of differentially downregulated genes in μCT cells vs. μ-transplanted cells. Figure 5C shows functional enrichment of differentially upregulated genes in μCT cells vs. TAμ-transplanted cells. Figure 5D shows functional enrichment of differentially downregulated genes in μCT cells vs. TAμ-transplanted cells. A preliminary ranking analysis by gene set enrichment analysis (GSEA) was performed using RNA-Seq differential gene expression data against gene ontology (GO) biological processes (http: / / software.broadinstitute.org / gsea / msigdb / collection) 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 is a graph showing the fold change in expression values ​​of genes normalized by RNA-Seq, whose expression is upregulated in adult mice (Matcovitch-Natan et al., Science. 353:6301 (2016)) in the representation populations recovered from busulfan-treated transplanted mice or P10 mouse brains relative to ADULT_CT μ-cells. Figure 5F is a graph showing fold change in expression values ​​of genes normalized by RNA-Seq that are upregulated in p10 mice (Matcovitch-Natan et al., Science. 353:6301 (2016)) relative to ADULT_CT μ-cells and in the indicated populations recovered from the brains of busulfan-treated transplanted mice or P10 mice. For statistical tests, see Table 2. [Figure 5EF]Figures 5A-5E show that bone marrow cells derived from transplanted mouse brains exhibit features of microglial maturation. Figure 5A shows functional enrichment of differentially upregulated genes in μCT cells vs. μ-transplanted cells. Figure 5B shows functional enrichment of differentially downregulated genes in μCT cells vs. μ-transplanted cells. Figure 5C shows functional enrichment of differentially upregulated genes in μCT cells vs. TAμ-transplanted cells. Figure 5D shows functional enrichment of differentially downregulated genes in μCT cells vs. TAμ-transplanted cells. A preliminary ranking analysis by gene set enrichment analysis (GSEA) was performed using RNA-Seq differential gene expression data against gene ontology (GO) biological processes (http: / / software.broadinstitute.org / gsea / msigdb / collection) 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 is a graph showing the fold change in expression values ​​of genes normalized by RNA-Seq, whose expression is upregulated in adult mice (Matcovitch-Natan et al., Science. 353:6301 (2016)) in the representation populations recovered from busulfan-treated transplanted mice or P10 mouse brains relative to ADULT_CT μ-cells. Figure 5F is a graph showing fold change in expression values ​​of genes normalized by RNA-Seq that are upregulated in p10 mice (Matcovitch-Natan et al., Science. 353:6301 (2016)) relative to ADULT_CT μ-cells and in the indicated populations recovered from the brains of busulfan-treated transplanted mice 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 have clonal and hematopoietic repopulation potential, as well as microglial reconstitution potential. Figure 6A illustrates the experimental scheme for HCT. CFUs were plated from 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 (#CFC) obtained from tissues of BU and IRR animals. Figure 6C is a graph illustrating the number of colonies (#CFC) and GFP+CFC 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 derived from primary recipients. Mice were sacrificed 4 months after transplantation.

[0077] [Figure 7]Figures 7A-E show that ICV co-delivery of HSPCs has therapeutic relevance 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 only routes 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). FIG. 7B is a graph showing ARSA activity (expressed as fold over that measured in tissues of Rag- / -γ-chain- / -As2- / - mice transplanted with ARSA-transduced cells ICV or IV or ICV+IV as indicated) measured in brain and bone marrow (BM). N=3 mice / 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 to IV alone or ICV alone approaches. FIG. 7C is a scheme of transplantation experiments 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 following busulfan bone marrow ablation IV only 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 animals' latency 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 show superior rotarod performance compared to IV only 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 10x106 total BM cells IV and 0.3 or 1x106 Lin- cells (mouse equivalent of human CD34+ cells) ICV. Figure 8B is the Kaplan-Meier survival curve of transplanted animals. At the end of the experiment, the IV+ICV 3e5 on day 0 and IV only groups showed 100% survival in contrast to the IV+ICV 1e6 on day 0 group. Figure 8C is a graph illustrating the chimerism of donor CD45.2 cells in peripheral blood (PB), BM, spleen (Spl) and thymus (Thy) of transplanted mice at the time of sacrifice. FIG. 8D shows the GFP+ cell abundance within donor CD45.2 cells in tissues from transplanted animals, indicating that ICV-transplanted GFP+ cells did not engraft in hematopoietic tissues of transplanted mice. N=5 per group. FIG. 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 sacrifice. FIG. 8F shows the GFP+ cell abundance within 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 situation, 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 10x106 total BM cells IV and 0.3x106 Lin- cells (mouse equivalent of human CD34+ cells) ICV. Figure 9B is the Kaplan-Meier survival curve of 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 PB, BM, Spl and Thy of transplanted mice at the time of sacrifice. Figure 9D shows the GFP+ cell abundance in donor CD45.2 cells in tissues from transplanted animals, indicating that ICV-transplanted GFP+ cells did not engraft in hematopoietic tissues of transplanted mice. N=5 per group. Figure 9E is a graph illustrating the chimerism of donor CD45.2 cells in brain bone marrow populations of transplanted mice at sacrifice. Figure 9F shows the GFP+ cell abundance in donor CD45.2 cells in brain bone marrow cells from transplanted animals, indicating that ICV-transplanted GFP+ cells contributed to increased chimerism in donor brains. 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 in mice compared to ICV+IV and IV only (control IV). 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 row, GFP is at the bottom and Cherry is at the top. N=3-5 per group. IT HSPC delivery may constitute a valuable route to achieve 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 an experimental scheme showing how long-term (LT)-HSCs and precursors within a pool of HSPCs were pre-isolated using c-kit, Sca-1 and lineage-negative staining, as well as SLAM receptor markers CD150 and CD48. The indicated taxonomic populations 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), and subsequently transplanted IV or ICV competitively at their original ratios into busulfan-bone marrow ablated mice. ICV-transplanted animals also received unmanipulated whole BM cells for hematopoietic rescue on day 5 post-transplant. FIG. 11B shows histograms of marker gene expression in in vivo liquid progeny of the indicated LV-transduced transplanted cells in mice described in A. FIG. 11C is a graph illustrating the abundance of cells derived from each of the transplanted KSL subpopulations in 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 row, from bottom to top, LT-HSC (light grey), MPP (dark grey), HPC2 (grey) and HPC1 (even lighter grey). FIG. 11D is a graph illustrating the abundance of cells derived from IV transplanted KSL and NOT-KSL in total bone marrow (CD45+CD11b+) cells, μ and TAμ of the brain of BU_TX mice at 90 days after transplantation. In each row, from bottom to top, KSL (grey) and not-KSL (white). FIG. 11E is a graph illustrating 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).FIG. 11F is a graph illustrating the abundance of cells derived from each of the engrafted KSL subpopulations in the total bone marrow cells, μ, and TAμ of the brains of IV-transplanted busulfan-bone marrow-ablated mice at various time points after HCT. N=10 mice per group. From bottom to top in each row: LT-HSC (light gray), MPP (dark gray), HPC2 (gray), and HPC1 (even light gray). FIG. 11G is a graph illustrating the abundance of cells derived from each of the engrafted KSL subpopulations in the total bone marrow cells, μ, and TAμ of the brains of ICV-transplanted busulfan-bone marrow-ablated mice at various time points after HCT. From bottom to top in each row: LT-HSC (light gray), MPP (dark gray), HPC2 (gray), and HPC1 (even light gray). N=10 mice per group. 11H and 11I show immunofluorescence analysis of brain slices of BU-treated mice IV-transplanted with KSL subpopulations at 90 days after transplantation. In 11H, LT-HSC progeny are GFP+ and MPP progeny are ΔNGFR+ (light grey). Iba1 staining is in the blue channel. Magnification 20×. M=confluent. Right panel shows another representative confluent at 20× (top) and 40× (bottom). In 11I, HPC2 progeny are Cherry+ and MPP progeny are ΔNGFR+ (grey). GFP+ staining was not detected in the absence of ΔNGFR immunofluorescence. Nucleus-related TPIII (dark grey) is shown. Top panel shows 20× magnification. Bottom panel shows another representative confluent at 20× (top) and 40× (bottom). Images were acquired by confocal microscopy (Radiance2100, Bio-Rad) and processed by Soft Work3.5.0.100. 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 an experimental scheme showing how long-term (LT)-HSCs and precursors within a pool of HSPCs were pre-isolated using c-kit, Sca-1 and lineage-negative staining, as well as SLAM receptor markers CD150 and CD48. The indicated taxonomic populations 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), and subsequently transplanted IV or ICV competitively at their original ratios into busulfan-bone marrow ablated mice. ICV-transplanted animals also received unmanipulated whole BM cells for hematopoietic rescue on day 5 post-transplant. FIG. 11B shows histograms of marker gene expression in in vivo liquid progeny of the indicated LV-transduced transplanted cells in mice described in A. FIG. 11C is a graph illustrating the abundance of cells derived from each of the transplanted KSL subpopulations in 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 row, from bottom to top, LT-HSC (light grey), MPP (dark grey), HPC2 (grey) and HPC1 (even lighter grey). FIG. 11D is a graph illustrating the abundance of cells derived from IV transplanted KSL and NOT-KSL in total bone marrow (CD45+CD11b+) cells, μ and TAμ of the brain of BU_TX mice at 90 days after transplantation. In each row, from bottom to top, KSL (grey) and not-KSL (white). FIG. 11E is a graph illustrating 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).FIG. 11F is a graph illustrating the abundance of cells derived from each of the engrafted KSL subpopulations in the total bone marrow cells, μ, and TAμ of the brains of IV-transplanted busulfan-bone marrow-ablated mice at various time points after HCT. N=10 mice per group. From bottom to top in each row: LT-HSC (light gray), MPP (dark gray), HPC2 (gray), and HPC1 (even light gray). FIG. 11G is a graph illustrating the abundance of cells derived from each of the engrafted KSL subpopulations in the total bone marrow cells, μ, and TAμ of the brains of ICV-transplanted busulfan-bone marrow-ablated mice at various time points after HCT. From bottom to top in each row: LT-HSC (light gray), MPP (dark gray), HPC2 (gray), and HPC1 (even light gray). N=10 mice per group. 11H and 11I show immunofluorescence analysis of brain slices of BU-treated mice IV-transplanted with KSL subpopulations at 90 days after transplantation. In 11H, LT-HSC progeny are GFP+ and MPP progeny are ΔNGFR+ (light grey). Iba1 staining is in the blue channel. Magnification 20×. M=confluent. Right panel shows another representative confluent at 20× (top) and 40× (bottom). In 11I, HPC2 progeny are Cherry+ and MPP progeny are ΔNGFR+ (grey). GFP+ staining was not detected in the absence of ΔNGFR immunofluorescence. Nucleus-related TPIII (dark grey) is shown. Top panel shows 20× magnification. Bottom panel shows another representative confluent at 20× (top) and 40× (bottom). Images were acquired by confocal microscopy (Radiance2100, Bio-Rad) and processed by Soft Work3.5.0.100. 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 an experimental scheme showing how long-term (LT)-HSCs and precursors within a pool of HSPCs were pre-isolated using c-kit, Sca-1 and lineage-negative staining, as well as SLAM receptor markers CD150 and CD48. The indicated taxonomic populations 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), and subsequently transplanted IV or ICV competitively at their original ratios into busulfan-bone marrow ablated mice. ICV-transplanted animals also received unmanipulated whole BM cells for hematopoietic rescue on day 5 post-transplant. FIG. 11B shows histograms of marker gene expression in in vivo liquid progeny of the indicated LV-transduced transplanted cells in mice described in A. FIG. 11C is a graph illustrating the abundance of cells derived from each of the transplanted KSL subpopulations in 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 row, from bottom to top, LT-HSC (light grey), MPP (dark grey), HPC2 (grey) and HPC1 (even lighter grey). FIG. 11D is a graph illustrating the abundance of cells derived from IV transplanted KSL and NOT-KSL in total bone marrow (CD45+CD11b+) cells, μ and TAμ of the brain of BU_TX mice at 90 days after transplantation. In each row, from bottom to top, KSL (grey) and not-KSL (white). FIG. 11E is a graph illustrating 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).FIG. 11F is a graph illustrating the abundance of cells derived from each of the engrafted KSL subpopulations in the total bone marrow cells, μ, and TAμ of the brains of IV-transplanted busulfan-bone marrow-ablated mice at various time points after HCT. N=10 mice per group. From bottom to top in each row: LT-HSC (light gray), MPP (dark gray), HPC2 (gray), and HPC1 (even light gray). FIG. 11G is a graph illustrating the abundance of cells derived from each of the engrafted KSL subpopulations in the total bone marrow cells, μ, and TAμ of the brains of ICV-transplanted busulfan-bone marrow-ablated mice at various time points after HCT. From bottom to top in each row: LT-HSC (light gray), MPP (dark gray), HPC2 (gray), and HPC1 (even light gray). N=10 mice per group. 11H and 11I show immunofluorescence analysis of brain slices of BU-treated mice IV-transplanted with KSL subpopulations at 90 days after transplantation. In 11H, LT-HSC progeny are GFP+ and MPP progeny are ΔNGFR+ (light grey). Iba1 staining is in the blue channel. Magnification 20×. M=confluent. Right panel shows another representative confluent at 20× (top) and 40× (bottom). In 11I, HPC2 progeny are Cherry+ and MPP progeny are ΔNGFR+ (grey). GFP+ staining was not detected in the absence of ΔNGFR immunofluorescence. Nucleus-related TPIII (dark grey) is shown. Top panel shows 20× magnification. Bottom panel shows another representative confluent at 20× (top) and 40× (bottom). Images were acquired by confocal microscopy (Radiance2100, Bio-Rad) and processed by Soft Work3.5.0.100. Figure 11J shows histogram plots showing differential levels of CXCR4 expression in KSL and NOT-KSL cells and KSL subpopulations upon transplantation. [Fig. 11DEFG]Figures 11A-11J show post-transplant brain bone marrow cells derived from early hematopoietic stem / progenitor cells. Figure 11A illustrates an experimental scheme showing how long-term (LT)-HSCs and precursors within a pool of HSPCs were pre-isolated using c-kit, Sca-1 and lineage-negative staining, as well as SLAM receptor markers CD150 and CD48. The indicated taxonomic populations 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), and subsequently transplanted IV or ICV competitively at their original ratios into busulfan-bone marrow ablated mice. ICV-transplanted animals also received unmanipulated whole BM cells for hematopoietic rescue on day 5 post-transplant. FIG. 11B shows histograms of marker gene expression in in vivo liquid progeny of the indicated LV-transduced transplanted cells in mice described in A. FIG. 11C is a graph illustrating the abundance of cells derived from each of the transplanted KSL subpopulations in 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 row, from bottom to top, LT-HSC (light grey), MPP (dark grey), HPC2 (grey) and HPC1 (even lighter grey). FIG. 11D is a graph illustrating the abundance of cells derived from IV transplanted KSL and NOT-KSL in total bone marrow (CD45+CD11b+) cells, μ and TAμ of the brain of BU_TX mice at 90 days after transplantation. In each row, from bottom to top, KSL (grey) and not-KSL (white). FIG. 11E is a graph illustrating 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).FIG. 11F is a graph illustrating the abundance of cells derived from each of the engrafted KSL subpopulations in the total bone marrow cells, μ, and TAμ of the brains of IV-transplanted busulfan-bone marrow-ablated mice at various time points after HCT. N=10 mice per group. From bottom to top in each row: LT-HSC (light gray), MPP (dark gray), HPC2 (gray), and HPC1 (even light gray). FIG. 11G is a graph illustrating the abundance of cells derived from each of the engrafted KSL subpopulations in the total bone marrow cells, μ, and TAμ of the brains of ICV-transplanted busulfan-bone marrow-ablated mice at various time points after HCT. From bottom to top in each row: LT-HSC (light gray), MPP (dark gray), HPC2 (gray), and HPC1 (even light gray). N=10 mice per group. 11H and 11I show immunofluorescence analysis of brain slices of BU-treated mice IV-transplanted with KSL subpopulations at 90 days after transplantation. In 11H, LT-HSC progeny are GFP+ and MPP progeny are ΔNGFR+ (light grey). Iba1 staining is in the blue channel. Magnification 20×. M=confluent. Right panel shows another representative confluent at 20× (top) and 40× (bottom). In 11I, HPC2 progeny are Cherry+ and MPP progeny are ΔNGFR+ (grey). GFP+ staining was not detected in the absence of ΔNGFR immunofluorescence. Nucleus-related TPIII (dark grey) is shown. Top panel shows 20× magnification. Bottom panel shows another representative confluent at 20× (top) and 40× (bottom). Images were acquired by confocal microscopy (Radiance2100, Bio-Rad) and processed by Soft Work3.5.0.100. 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 an experimental scheme showing how long-term (LT)-HSCs and precursors within a pool of HSPCs were pre-isolated using c-kit, Sca-1 and lineage-negative staining, as well as SLAM receptor markers CD150 and CD48. The indicated taxonomic populations 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), and subsequently transplanted IV or ICV competitively at their original ratios into busulfan-bone marrow ablated mice. ICV-transplanted animals also received unmanipulated whole BM cells for hematopoietic rescue on day 5 post-transplant. FIG. 11B shows histograms of marker gene expression in in vivo liquid progeny of the indicated LV-transduced transplanted cells in mice described in A. FIG. 11C is a graph illustrating the abundance of cells derived from each of the transplanted KSL subpopulations in 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 row, from bottom to top, LT-HSC (light grey), MPP (dark grey), HPC2 (grey) and HPC1 (even lighter grey). FIG. 11D is a graph illustrating the abundance of cells derived from IV transplanted KSL and NOT-KSL in total bone marrow (CD45+CD11b+) cells, μ and TAμ of the brain of BU_TX mice at 90 days after transplantation. In each row, from bottom to top, KSL (grey) and not-KSL (white). FIG. 11E is a graph illustrating 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).FIG. 11F is a graph illustrating the abundance of cells derived from each of the engrafted KSL subpopulations in the total bone marrow cells, μ, and TAμ of the brains of IV-transplanted busulfan-bone marrow-ablated mice at various time points after HCT. N=10 mice per group. From bottom to top in each row: LT-HSC (light gray), MPP (dark gray), HPC2 (gray), and HPC1 (even light gray). FIG. 11G is a graph illustrating the abundance of cells derived from each of the engrafted KSL subpopulations in the total bone marrow cells, μ, and TAμ of the brains of ICV-transplanted busulfan-bone marrow-ablated mice at various time points after HCT. From bottom to top in each row: LT-HSC (light gray), MPP (dark gray), HPC2 (gray), and HPC1 (even light gray). N=10 mice per group. 11H and 11I show immunofluorescence analysis of brain slices of BU-treated mice IV-transplanted with KSL subpopulations at 90 days after transplantation. In 11H, LT-HSC progeny are GFP+ and MPP progeny are ΔNGFR+ (light grey). Iba1 staining is in the blue channel. Magnification 20×. M=confluent. Right panel shows another representative confluent at 20× (top) and 40× (bottom). In 11I, HPC2 progeny are Cherry+ and MPP progeny are ΔNGFR+ (grey). GFP+ staining was not detected in the absence of ΔNGFR immunofluorescence. Nucleus-related TPIII (dark grey) is shown. Top panel shows 20× magnification. Bottom panel shows another representative confluent at 20× (top) and 40× (bottom). Images were acquired by confocal microscopy (Radiance2100, Bio-Rad) and processed by Soft Work3.5.0.100. 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 an experimental scheme showing how long-term (LT)-HSCs and precursors within a pool of HSPCs were pre-isolated using c-kit, Sca-1 and lineage-negative staining, as well as SLAM receptor markers CD150 and CD48. The indicated taxonomic populations 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), and subsequently transplanted IV or ICV competitively at their original ratios into busulfan-bone marrow ablated mice. ICV-transplanted animals also received unmanipulated whole BM cells for hematopoietic rescue on day 5 post-transplant. FIG. 11B shows histograms of marker gene expression in in vivo liquid progeny of the indicated LV-transduced transplanted cells in mice described in A. FIG. 11C is a graph illustrating the abundance of cells derived from each of the transplanted KSL subpopulations in 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 row, from bottom to top, LT-HSC (light grey), MPP (dark grey), HPC2 (grey) and HPC1 (even lighter grey). FIG. 11D is a graph illustrating the abundance of cells derived from IV transplanted KSL and NOT-KSL in total bone marrow (CD45+CD11b+) cells, μ and TAμ of the brain of BU_TX mice at 90 days after transplantation. In each row, from bottom to top, KSL (grey) and not-KSL (white). FIG. 11E is a graph illustrating 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).FIG. 11F is a graph illustrating the abundance of cells derived from each of the engrafted KSL subpopulations in the total bone marrow cells, μ, and TAμ of the brains of IV-transplanted busulfan-bone marrow-ablated mice at various time points after HCT. N=10 mice per group. From bottom to top in each row: LT-HSC (light gray), MPP (dark gray), HPC2 (gray), and HPC1 (even light gray). FIG. 11G is a graph illustrating the abundance of cells derived from each of the engrafted KSL subpopulations in the total bone marrow cells, μ, and TAμ of the brains of ICV-transplanted busulfan-bone marrow-ablated mice at various time points after HCT. From bottom to top in each row: LT-HSC (light gray), MPP (dark gray), HPC2 (gray), and HPC1 (even light gray). N=10 mice per group. 11H and 11I show immunofluorescence analysis of brain slices of BU-treated mice IV-transplanted with KSL subpopulations at 90 days after transplantation. In 11H, LT-HSC progeny are GFP+ and MPP progeny are ΔNGFR+ (light grey). Iba1 staining is in the blue channel. Magnification 20×. M=confluent. Right panel shows another representative confluent at 20× (top) and 40× (bottom). In 11I, HPC2 progeny are Cherry+ and MPP progeny are ΔNGFR+ (grey). GFP+ staining was not detected in the absence of ΔNGFR immunofluorescence. Nucleus-related TPIII (dark grey) is shown. Top panel shows 20× magnification. Bottom panel shows another representative confluent at 20× (top) and 40× (bottom). Images were acquired by confocal microscopy (Radiance2100, Bio-Rad) and processed by Soft Work3.5.0.100. 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 presence of donor cells (CD45.2+) among brain bone marrow CD11b+ cells in mice ICV-transplanted with Fgd5 cells after busulfan and irradiation pretreatment. N>4 per group. Figure 12D is a graph illustrating 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 μ, TAμ, and CNSmac populations among donor-derived cells in ICV-transplanted busulfan-pretreated mice. N>4 per group. [Fig. 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 presence of donor cells (CD45.2+) among brain bone marrow CD11b+ cells in mice ICV-transplanted with Fgd5 cells after busulfan and irradiation pretreatment. N>4 per group. Figure 12D is a graph illustrating 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 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 cells and negative cells to brain-marrow chimerism. Figure 13A is a series of graphs showing bone marrow characteristics of CX3CR1-GFP mice, specifically, the expression of GFP in the various bone marrow subpopulations, as indicated. From bottom to top in each row, GFP neg (light grey), GFP low (dark grey) and GFP high (light grey). Figure 13B illustrates the experimental setup used to generate chimeric mice using reporter mice, CX3CR1-GFP mice, and cells isolated from chimerisms generated in the brain. Mice receiving GFP+ / highLin-HSPCs did not engraft with CX3CR1 CD45.2 donor cells in the brain (representative dot plots are shown in the left box), whereas transplantation with whole bone marrow not sorted for CX3CR1 showed 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 cells and negative cells to brain-marrow chimerism. Figure 13A is a series of graphs showing bone marrow characteristics of CX3CR1-GFP mice, specifically, the expression of GFP in the various bone marrow subpopulations, as indicated. From bottom to top in each row, GFP neg (light grey), GFP low (dark grey) and GFP high (light grey). Figure 13B illustrates the experimental setup used to generate chimeric mice using reporter mice, CX3CR1-GFP mice, and cells isolated from chimerisms generated in the brain. Mice receiving GFP+ / highLin-HSPCs did not engraft with CX3CR1 CD45.2 donor cells in the brain (representative dot plots are shown in the left box), whereas transplantation with whole bone marrow not sorted for CX3CR1 showed 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 cells and negative cells to brain-marrow chimerism. Figure 13A is a series of graphs showing bone marrow characteristics of CX3CR1-GFP mice, specifically, the expression of GFP in the various bone marrow subpopulations, as indicated. From bottom to top in each row, GFP neg (light grey), GFP low (dark grey) and GFP high (light grey). Figure 13B illustrates the experimental setup used to generate chimeric mice using reporter mice, CX3CR1-GFP mice, and cells isolated from chimerisms generated in the brain. Mice receiving GFP+ / highLin-HSPCs did not engraft with CX3CR1 CD45.2 donor cells in the brain (representative dot plots are shown in the left box), whereas transplantation with whole bone marrow not sorted for CX3CR1 showed 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]14A-D show the contribution of human LT-HSCs, defined as CD34+ and CD38-, to the development of brain-myeloid progeny cells. FIG. 14A is a representative dot plot showing the gating strategy for the identification of human CD34+CD38+ (progenitor) and CD34+CD38- (stem-enriched cells) from human mobilized peripheral blood. Cells were differentially transduced with LV encoding GFP and tag-BFP at the indicated abundances and mixed into NSG myeloablated mice for transplantation. FIG. 14B is a graph showing the abundances of cells marked by GFP (CD38-) or tag-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. Means and SEM are shown. In each column, from bottom to top, CD38- (gray) and CD38+ (white). Figure 14C is a representative dot plot showing the gating strategy for the identification of long-term and short-term human HSCs and progenitors by expression of markers CD38 and CD90 on CD34+ cells recovered from human mobilized peripheral blood. Cells were differentially transduced with LV 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 abundances 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+ (grey), CD38-CD90-low (light grey), CD38+CD90- (medium grey) and CD38+CD90+high (even lighter grey). N>5 mice / group. Means and SEM are shown. Overall, CD34+CD38- was shown to be the largest contributor 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 at day 5 after one or four doses of busulfan, an increase in the fraction of apoptotic annexin+ cells was detected in the hematopoietic bone marrow and, most importantly, in CD45+c-kit+ cells. From the leftmost bar in each set, CO (white), 1xBU (dark grey) and 4xBU (black). Figure 15B shows representative FACS plots of γH2AX+ cells within vital CD45+ brain cells of control animals and BU-treated mice. Figure 15C shows the γH2AX marker distribution in the brains of mice analyzed at day 1 after busulfan pretreatment or control untreated mice (CTR). Insets are representative laser scanning confocal microscopy micrographs co-immunostained for γH2AX, neurons (NeuN), microglia (Iba1) or astrocytes (GFAP). Scale bar = 10 μm. Images were acquired with a confocal microscope Radiance2100 (Bio-Rad) Ix70 at a magnification of 20× and processed by Soft Work3.5.0. Reconstruction was performed with Adobe Photoshop CS8.0 software. Inset, magnification is 40×. [Figure 15B]Figures 15A-15C illustrate the identification of brain-resident putative μ precursors by BU sensitivity. Figure 15A shows that at day 5 after one or four doses of busulfan, an increase in the fraction of apoptotic annexin+ cells was detected in the hematopoietic bone marrow and, most importantly, in CD45+c-kit+ cells. From the leftmost bar in each set, CO (white), 1xBU (dark grey) and 4xBU (black). Figure 15B shows representative FACS plots of γH2AX+ cells within vital CD45+ brain cells of control animals and BU-treated mice. Figure 15C shows the γH2AX marker distribution in the brains of mice analyzed at day 1 after busulfan pretreatment or control untreated mice (CTR). Insets are representative laser scanning confocal microscopy micrographs co-immunostained for γH2AX, neurons (NeuN), microglia (Iba1) or astrocytes (GFAP). Scale bar = 10 μm. Images were acquired with a confocal microscope Radiance2100 (Bio-Rad) Ix70 at a magnification of 20× and processed by Soft Work3.5.0. Reconstruction was performed with Adobe Photoshop CS8.0 software. Inset, magnification is 40×. [Figure 15C]Figures 15A-15C illustrate the identification of brain-resident putative μ precursors by BU sensitivity. Figure 15A shows that at day 5 after one or four doses of busulfan, an increase in the fraction of apoptotic annexin+ cells was detected in the hematopoietic bone marrow and, most importantly, in CD45+c-kit+ cells. From the leftmost bar in each set, CO (white), 1xBU (dark grey) and 4xBU (black). Figure 15B shows representative FACS plots of γH2AX+ cells within vital CD45+ brain cells of control animals and BU-treated mice. Figure 15C shows the γH2AX marker distribution in the brains of mice analyzed at day 1 after busulfan pretreatment or control untreated mice (CTR). Insets are representative laser scanning confocal microscopy micrographs co-immunostained for γH2AX, neurons (NeuN), microglia (Iba1) or astrocytes (GFAP). Scale bar = 10 μm. Images were acquired with a confocal microscope Radiance2100 (Bio-Rad) Ix70 at a magnification of 20× and processed by Soft Work3.5.0. Reconstruction was performed with Adobe Photoshop CS8.0 software. Inset, magnification is 40×.

[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 mouse 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 in 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 mouse 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 in 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 mouse 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 in 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 mouse 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 in 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 to CD45+CD11b+ cells (Figure 17C), and is in the range of CD45+Edu+ cells compared to CD45+Edu- cells (Figure 17D). Figure 17E shows representative epifluorescence micrographs of rhodamine-labeled NP signal (red / light grey) and DAPI nuclear staining (blue / dark grey) in sagittal brain slices from mice analyzed 3 days after ICV injection of NPs. Images were acquired on a Delta Vision Olympus at 20x magnification and processed by Soft Work3.5.0. 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 % 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) close to the subventricular (SVZ) zone and rostral migratory stream (RMS) (right panel) in a sagittal slice of the brain from a mouse analyzed 3 days after ICV injection of NPs, as well as representative confocal microscopy images of rhodaminylated NP signals (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 and is a representative laser scanning confocal microscopy photomicrograph of Iba1+, F4 / 80, rhodamine and co-immunostaining for the proliferation marker Edu. Rh+NPs can be detected in Iba1+ (arrows) and Iba1- (arrowheads) proliferating cells. Figure 17I is a reconstruction of Edu and rhodamine signaling retrieved by fluorescence microscopy on slices from the brain of NP-injected mice showing co-localization of Edu and NP signals.Images were acquired using a confocal microscope Radiance2100 (Bio-Rad) Ix70 and processed by 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 to CD45+CD11b+ cells (Figure 17C), and is in the range of CD45+Edu+ cells compared to CD45+Edu- cells (Figure 17D). Figure 17E shows representative epifluorescence micrographs of rhodamine-labeled NP signal (red / light grey) and DAPI nuclear staining (blue / dark grey) in sagittal brain slices from mice analyzed 3 days after ICV injection of NPs. Images were acquired on a Delta Vision Olympus at 20x magnification and processed by Soft Work3.5.0. 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 % 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) close to the subventricular (SVZ) zone and rostral migratory stream (RMS) (right panel) in a sagittal slice of the brain from a mouse analyzed 3 days after ICV injection of NPs, as well as representative confocal microscopy images of rhodaminylated NP signals (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 and is a representative laser scanning confocal microscopy photomicrograph of Iba1+, F4 / 80, rhodamine and co-immunostaining for the proliferation marker Edu. Rh+NPs can be detected in Iba1+ (arrows) and Iba1- (arrowheads) proliferating cells. Figure 17I is a reconstruction of Edu and rhodamine signaling retrieved by fluorescence microscopy on slices from the brain of NP-injected mice showing co-localization of Edu and NP signals.Images were acquired using a confocal microscope Radiance2100 (Bio-Rad) Ix70 and processed by Soft Work 3.5.0. [Diagram 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 to CD45+CD11b+ cells (Figure 17C), and is in the range of CD45+Edu+ cells compared to CD45+Edu- cells (Figure 17D). Figure 17E shows representative epifluorescence micrographs of rhodamine-labeled NP signal (red / light grey) and DAPI nuclear staining (blue / dark grey) in sagittal brain slices from mice analyzed 3 days after ICV injection of NPs. Images were acquired on a Delta Vision Olympus at 20x magnification and processed by Soft Work3.5.0. 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 % 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) close to the subventricular (SVZ) zone and rostral migratory stream (RMS) (right panel) in a sagittal slice of the brain from a mouse analyzed 3 days after ICV injection of NPs, as well as representative confocal microscopy images of rhodaminylated NP signals (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 and is a representative laser scanning confocal microscopy photomicrograph of Iba1+, F4 / 80, rhodamine and co-immunostaining for the proliferation marker Edu. Rh+NPs can be detected in Iba1+ (arrows) and Iba1- (arrowheads) proliferating cells. Figure 17I is a reconstruction of Edu and rhodamine signaling retrieved by fluorescence microscopy on slices from the brain of NP-injected mice showing co-localization of Edu and NP signals.Images were acquired using a confocal microscope Radiance2100 (Bio-Rad) Ix70 and processed by 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 to CD45+CD11b+ cells (Figure 17C), and is in the range of CD45+Edu+ cells compared to CD45+Edu- cells (Figure 17D). Figure 17E shows representative epifluorescence micrographs of rhodamine-labeled NP signal (red / light grey) and DAPI nuclear staining (blue / dark grey) in sagittal brain slices from mice analyzed 3 days after ICV injection of NPs. Images were acquired on a Delta Vision Olympus at 20x magnification and processed by Soft Work3.5.0. 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 % 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) close to the subventricular (SVZ) zone and rostral migratory stream (RMS) (right panel) in a sagittal slice of the brain from a mouse analyzed 3 days after ICV injection of NPs, as well as representative confocal microscopy images of rhodaminylated NP signals (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 and is a representative laser scanning confocal microscopy photomicrograph of Iba1+, F4 / 80, rhodamine and co-immunostaining for the proliferation marker Edu. Rh+NPs can be detected in Iba1+ (arrows) and Iba1- (arrowheads) proliferating cells. Figure 17I is a reconstruction of Edu and rhodamine signaling retrieved by fluorescence microscopy on slices from the brain of NP-injected mice showing co-localization of Edu and NP signals.Images were acquired using a confocal microscope Radiance2100 (Bio-Rad) Ix70 and processed by Soft Work 3.5.0.

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

[0089] [Figure 19]19A-19E illustrate the in vivo and in vivo effects of NP-encapsulated chemotherapeutic agents. FIG. 19A is a representative flow cytometric analysis and immunofluorescence staining for γH2AX in a BV2 microglia-like cell line esposed to busulfan. The graph shows the percentage of γH2AX+ cells (determined by flow cytometry) following exposure to various nanoparticle formulations with or without busulfan loading (histograms represent mean + / - SEM of n>=3 independent experiments). FIG. 19B is a graph illustrating 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. FIG. 19C is a graph illustrating the presence of γH2AX+ microglial cells in mouse brains (by flow cytometry) assessed 3 days after ICV administration of NPs (histograms represent mean + / - SEM of n=4 animals / group). FIG. 19D and 19E are two graphs showing the viability of BV2 microglial cells exposed to NPs (50 and 100 nm sized self-assembled formulations, and PLC formulations) with or without etoposide chemotherapeutic agent (chmetherapic) and etoposized as free formulation at the indicated concentrations. Viability was assessed using 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 grey), self-assembled NPs (100 nm), PCL NPs (100 nm) (grey) and etoposide (medium grey).

[0090] [Fig. 20AB]Figures 20A-20E show etoposide containing NPs administered after induction of proliferation of microglial precursors and expansion of 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 precursor proliferation with a single busulfsn dose or oral administration of a CSF 1R inhibitor (Elmore et al., Neuron. 82(2):380-397 (2014)) for 10 days, followed by IV administration of etoposide containing NPs in adult wild-type mice. Figures 20B and 20D show graphs showing % Annexin+ early apoptotic cells measured by flow cytometry for brain samples obtained 5 days after NP administration. % Annexin+ cells are calculated based on the cell subfractions indicated. 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. [Fig. 20CDE]Figures 20A-20E show etoposide containing NPs administered after induction of proliferation of microglial precursors and expansion of 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 precursor proliferation with a single busulfsn dose or oral administration of a CSF 1R inhibitor (Elmore et al., Neuron. 82(2):380-397 (2014)) for 10 days, followed by IV administration of etoposide containing NPs in adult wild-type mice. Figures 20B and 20D show graphs showing % Annexin+ early apoptotic cells measured by flow cytometry for brain samples obtained 5 days after NP administration. % Annexin+ cells are calculated based on the cell subfractions indicated. 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 PREFERRED EMBODIMENTS

[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 and neurodegenerative diseases using HSC transplantation have limited effect on CNS disease manifestations due to the slow replacement of resident microglia by the progeny of transplanted cells. Current methods of HSC transplantation into patients suffering from SD and neurodegenerative disorders include whole bone marrow or apheretic generation in the case of autologous gene therapy. These include the use of hematopoietic stem and progenitor cells (HSPCs selective for CD34 expression) from donor or umbilical cord blood, whereby cell populations enriched for microglial repopulating activity can be identified within these cell sources, the use of which can ultimately improve donor microglial reconstitution following transplantation. Furthermore, hematopoietic stem and progenitor cells (HSPCs), or fractions of the HSPC pool, delivered directly to the brain using intracerebroventricular injection (ICV) 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. Thus, this approach offers great therapeutic potential and can be optimized to obtain extensive microglial contributions of ICV transplanted cells for advanced strategies aimed at replacing exclusively microglia rather than hematopoietic tissues with transplanted cells. This is highly relevant in conjunction with the novel strategy developed herein of molecular engineering of microglia for regulated therapeutic gene expression in response to neuroinflammatory or neurodegenerative stimuli.

[0093] As demonstrated herein, HSPC transplantation can generate transcriptionally-dependent new 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 in long-term hematopoietic stem cells (HSCs) in humans and mice. Similar transcriptionally-dependent new 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 generates progeny bone marrow cells in the brain at a more rapid rate and in greater quantities compared to IV. While ICV-injected 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 co-infused 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 a transcriptional profile 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 mouse brain parenchyma 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 the c-kit receptor in targeted areas of the brain. + and Nestin + It can be uploaded by myeloproliferative cells. The nanoparticles can efficiently encapsulate etoposide. · Etoposide, when encapsulated in NPs, is more effective at killing cells than in the standard formulation. Eto-NPs upregulate the c-kit endothelial cell proliferation in mouse brain by inducing proliferation of microglial precursors. + CD45 + It is taken up by cells. Eto-NPs upregulate the c-kit endothelial cell proliferation in mouse brain by inducing proliferation of microglial precursors. + CD45 + and CD45 + It can induce early apoptosis of cells.

[0094] Previous studies have hypothesized that the existence of CNS-resident microglial precursors, the ablation of which prior to HCT is essential for the establishment of microglial reconstitution. Herein, the use of novel tools is proposed as novel methods to identify these cells and transplant them to generate systemic and CNS chimerism, or selective CNS chimerism by transplanted cell progeny. Thus, the present invention provides novel tools to: Identification of cells within the pool of HSPCs that can be used to induce brain-bone marrow chimerism and transcription-dependent de novo microglia generation 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 indicate that an optimized protocol of HSC transplantation can be used to treat nervous system diseases or disorders of the central nervous system, 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 to be used to treat neurodegenerative diseases by an optimized microglial reconstitution approach. Thus, the following has 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 μ precursors within the HSPC pool is contained in LT_HSCs (both mouse and human cells). The functional equivalent of μ precursors within the mouse HSPC pool is Fdg5+. The functional equivalent of μ precursors within the mouse HSPC pool is CD11b negative. · The functional equivalent of μ precursors 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 indicates that hematopoietic stem and progenitor cells (HSPCs) and / or their progeny can contribute to the turnover of myeloid cell populations in the brain and thus 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 reconstituted cells after transplantation, in particular, bona fide microglia, can be reconstituted by the progeny of donor cells after transplantation, can be assessed. In the last three decades, hematopoietic cell transplantation (HCT) and hematopoietic stem cell (HSC)-based gene therapy approaches 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 evidences, together with supporting preclinical data, suggest that hematopoietic stem and progenitor cells (HSPCs) and / or their progeny may serve as vehicles for the delivery of therapeutic molecules across the blood-brain barrier (BBB). Indeed, HSPCs and / or their progeny may potentially act as vehicles for the delivery of therapeutic molecules across the blood-brain barrier (BBB), including microglia, whose critical 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)), turnover of myeloid cell populations in the brain (Ajami et al. Nat Neurosci 10:1538-1543 (2007); Ajami et al. Nat Neurosci 14:1142-1149 (2011); Biffi et al. J. Clin. Invest. 116:3070-3082 (2006); Mildner et al. Nat Neurosci 10:1544-1553 (2007); Capotondo et al. Proc Natl Acad Sci USA. 109, 15018-15023 (2012) Importantly, cells derived from transplants, once integrated into the diseased tissue, have been shown to favorably influence the local environment, 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 of the arylsulfatase A enzyme, which is deficient in the patients, and its expression were induced by lentiviral vectors (LV) integrated into the patients' HSCs and their progeny, and were 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, 3 pp. 070-3082 (2006); Matzner et al. Human Molecular Genetics, Vol. 14 , 1139-1152 (2005)). These findings, associated with a significant clinical benefit in patients treated at the 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, in particular bona fide microglia, can be reconstituted by the progeny of donor cells after HCT as demonstrated (Ajami et al. Nat Neurosci 10, 1538-1543 (2007); Capotondo et al. Proc Natl Acad Sci USA. 109, 15018-15023 (2008)). 2012); Bennett et al. Proc Natl Acad Sci USA vol. 113, E1738-17 46 pages (2016)).

[0097] Although microglia have a developmental origin distinct from that of bone marrow-derived myelomonocytic cells (Ginhoux et al. Science 330:841-845 (2010)), others and the present inventors have recently demonstrated that under certain experimental conditions, donor-sourced cells that display a microglia-like phenotype and express some microglia surface markers can be successfully generated in the brains of mice transplanted with donor HSPCs. Prerequisite for this to occur reproducibly and at a high rate is the pre-transplant administration of a conditioning regimen based on the alkylating agent busulfan, which is capable of ablating 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, it was shown that the donor-source cells found in the brains of transplanted animals were derived from the local proliferation and differentiation of HSPCs that had migrated to the brain shortly after transplantation.

[0098] With the present study, a major step has been taken forward towards better understanding these events and increasing their potential for clinical translation for the treatment of neurological diseases. Indeed, it is demonstrated for the first time that donor-derived bone marrow cells appearing in the brains of mice receiving HSPCs after busulfan-based pretreatment not only share microglial morphology and surface markers, but also share a highly similar transcriptional profile. Furthermore, by using genome-wide expression analysis, it has been shown that transplanted HSPCs generate microglia-like progeny cells by a process that recapitulates some aspects of physiological postnatal microglial maturation. Importantly, it has also been clearly demonstrated that post-transplant microgliosis originates from mouse and human early hematopoietic stem / progenitors, whose trafficking to the brain may be favored by CXCR4 expression. Finally, the generation of donor-sourced microglia-like cells is also obtained here for the first time when HSPCs are administered directly into the lateral ventricles of the brains of pretreated mice, instead of endovenously. Notably, this novel delivery route, which allows for clinically relevant, rapid and more extensive 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] Collectively, 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 delivery of HSPCs directly 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 diseases characterized by the disruption of normal lysosomal function, resulting in the accumulation of incompletely degraded substrates targeted for degradation following endocytosis or autophagy. 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 of primary neurons or oligodendrocytes.

[0101] Examples of SD include lysosomal storage diseases (LSDs) (such as GM1 and GM2 gangliosidosis), alpha-mannosidosis, globoid cell leukodystrophy (GLD), neuronal ceroid lipozygoskinosis (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 typical SDs and their associated defective proteins is presented in Table 1. [Table 1]

[0102] The information provides some of the specific relevant LSDs for use with 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 prototypic 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 carry has recently been demonstrated (Cesani et al. Hum Mutat 30, E936-945 (2009); Cesani et al. Ann Neurol 75, 127-137 (2014)). Autotransferases HSC gene therapy using lentiviral vectors to transduce explanted HSCs and exposure to systemic busulfan pretreatment has been shown to be effective in preventing or attenuating signs of disease 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 constituent. GLD occurs in approximately 1 in 100,000 births. It usually occurs during infancy and follows a rapid, fatal path, although rare 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 that affects both the CNS and the peripheral nervous system (PNS). Galactosylsphingosine (or psychosine) is also a substrate for GALC and is thought to play an important role in pathogenesis. Children with GLD can be treated presymptomatically and before age 4 years by 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 to be potentially effective in preclinical models of GLD (Gentner et al. Sci Transl Med 2 (2010)).

[0106] Mucopolysaccharidoses (MPS) Mucopolysaccharidoses (MPS) are a group of LSDs caused by the absence or dysfunction of lysosomal enzymes required to break down glycosaminoglycans. MPS I is classified into three subtypes based on the severity of symptoms. The three types are due to the absence or insufficient levels of the enzyme alpha-L-iduronidase. MPS IH (also called Hurler syndrome or α-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 MPS II, Hunter syndrome or iduronate sulfatase deficiency, is caused by a deficiency of the enzyme iduronate sulfatase. MPS III, 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 carbohydrate chains of heparin sulfate. Sanfilipo A is the most severe form of MPS III disorder and is caused by the loss or alteration of the enzyme heparan N-sulfatase. Children with Sanfilipo A have the poorest survival rates of those with MPS III disorder. Sanfilipo B is caused by the loss or deficiency of the enzyme alpha-N-acetylglucosaminase. Sanfilipo C is due to the loss or alteration of the enzyme acetyl-Co alpha-glucosaminide acetyltransferase. Sanfilipo 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 sugar chains of keratan sulfate. Marotele-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 lead to progressive neurological degeneration. Some variants, such as infantile NCL (INCL), are caused by deficiencies in lysosomal enzymes. INCL is caused by mutations in the CLN2 gene that lead to deficiency of PPT1, a lysosomal enzyme responsible for the degradation of membrane proteins. Similarly, late infantile NCL (LINCL) is due to deficiency of the lysosomal enzyme TPP1. Neurons are particularly sensitive to lysosomal accumulation of this storage material, and individuals with INCL and LINCL have widespread progressive neurodegeneration in all parts of the brain, progressing to a vegetative state and death by age 8-12 years.

[0110] X-linked adrenoleukodystrophy (X-ALD) X-linked adrenoleukodystrophy (X-ALD) is a metabolic genetic disease with a frequency of 1:17.000 in males, characterized by progressive inflammatory demyelination in the brain. 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) in phospholipid fractions such as lysophagidylcholine (LPC), especially in the brain and adrenal cortex. The phenotypic variation of X-ALD seems to be linked to brain inflammation that causes progressive neurological decline, mostly in children but also in adults with X-ALD. The onset of brain demyelination can 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 the clearance of 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 in this area, microglia may be unable to degrade VLCFAs, which in turn may lead to microglial activation and apoptosis. Microglial loss and / or microglial dysfunction may be primarily due to the upregulation of proinflammatory cytokines (CCL2, CCL4, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-31, IL-32, IL-33, IL-34, IL-35, IL-46, IL-47, IL-48, IL-59, IL-59, IL-59, IL-59, IL-60, IL-61, IL-62, IL-73, IL-74, IL-8, IL-9, IL-11, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-19, IL-20, IL-19, IL-21, IL-19, IL-22, IL-1 1a, CXCL8) and their altered ability to supply neuroprotective factors against oligodendrocyte loss. In this scenario, microglial cells may be a suitable target for intervention in X-ALD patients with evidence of cerebral demyelination. In this view, 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 N Engl J Med doi:10.1056 / NEJMoa1700554 (2017)) It has been investigated as a treatment option for LD.

[0111] Neurodegenerative diseases Neurodegenerative diseases are a class of nervous system diseases characterized by the progressive loss of neuronal structure and / or function and / or cell death of neurons.Inflammation is implicated in the role in some neurodegenerative diseases.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 diseases include non-limiting examples of ALS, such as familial 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 activation of microglial cells 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 leg and toes; weakness in the arms, legs, feet or ankles; weakness or lameness 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 sensation with head movement; tremors; unsteady gait; fatigue; dizziness; memory loss; disorientation; misunderstanding spatial relationships. Symptoms of neurodegenerative disorders include: difficulty in reading and writing; difficulty concentrating and thinking; difficulty in judging and making decisions; difficulty planning and performing familiar tasks; depression; anxiety; social withdrawal; emotional lability; irritability; aggression; changes in sleep habits; migratory behavior; dementia; loss of motility; impaired posture and balance; muscle rigidity; bradykinesia; slow and abnormal eye movements; involuntary shaking or twisting movements (chorea); involuntary continuous muscle contractures (dystonia); lack of flexibility; lack of impulse control; and appetite changes. A medical professional may also base a diagnosis on, in part, the subject's family history of a neurodegenerative disorder. 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 during the subject's stay at a health care facility. Typically, a physician 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 microglial precursors as described herein. Thus, 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, deficiency or loss of function of lysosomal enzymes results in lysosomal storage disorders. Transplanted hematopoietic cells expressing therapeutic proteins (e.g., enzymes) either intravenously or by recombinant methods can engraft and differentiate into microglia, thereby treating enzyme deficiency. In addition, transplanted cells can act 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 may be effective in creating an environment that allows engraftment of microglial precursors derived exclusively from transplanted cells in the brain. Furthermore, delivery of enriched populations of bone marrow-derived microglial precursors 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 in need of 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 will be preferably administered to subjects, particularly those suffering from or having, susceptible to, or at risk for a disease, disorder, or symptoms thereof. Determination of such "at risk" subjects may be made objectively or subjectively, by diagnostic tests, or by the opinion of the subject or medical provider (e.g., genetic tests, enzyme or protein markers, Markers (as defined herein), family history, etc.). The compounds herein may also be used to treat any other disorder, which may involve myelination deficiency or loss, including multiple sclerosis.

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

[0120] In general, "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 between 25 nm and 200 nm. In other preferred embodiments, the nanoparticles of the present invention have a maximum dimension of 100 nm or less. In other preferred embodiments, the nanoparticles of the present invention have a maximum dimension ranging between 35 nm and 60 nm. Nanoparticles encompassed in the present invention can be provided in a variety of forms, for example, as solid nanoparticles (e.g., metals such as silver, gold, iron, titanium, etc.), non-metallic, lipid-based solids, polymers), suspensions of nanoparticles, or combinations thereof. Metallic, dielectric and semiconductor nanoparticles, as well as hybrid structures (e.g., core-shell nanoparticles) may be prepared. Nanoparticles made of semiconductor materials can also be labeled quantum dots if they are small enough (usually 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. Semi-solid and soft nanoparticles have been produced and are within the scope of the present invention. The prototype nanoparticle of semi-solid 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 are called Janus particles and 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, which are all applicable for oral delivery of drugs to the brain, intravenous delivery of drugs, and nasal delivery of drugs. Other embodiments, such as oral absorption and ocular delivery of hydrophobic drugs, are also contemplated.Molecular envelopment 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, 1665-1. 680 (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, 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. In general, a particle is defined as a small body that behaves as a unit with respect to its transport and properties as a whole. 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 of the 100 nm cutoff is that novel properties that distinguish particles from bulk materials usually 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 that includes particles according to the present invention. A particle according to the present invention is any entity that has a maximum dimension (e.g., diameter) of less than 100 microns (.mu.m). In some embodiments, a particle of the present invention has a maximum dimension of less than 10 p.m. In some embodiments, a particle of the present invention has a maximum dimension of less than 2000 nanometers (nm). In some embodiments, a particle of the present invention has a maximum dimension of less than 1000 nanometers (nm). In some embodiments, a particle of the present invention has 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, a particle of the present invention has a maximum dimension (e.g., diameter) of 500 nm or less. In some embodiments, a particle of the present invention has a maximum dimension (e.g., diameter) of 250 nm or less. In some embodiments, a particle of the present invention has a maximum dimension (e.g., diameter) of 200 nm or less. In some embodiments, the particles of the present invention have a maximum dimension (e.g., diameter) of 150 nm or less. In some embodiments, the 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, the 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, dimensions, etc.) is performed 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), powder X-ray 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 (dimension measurement) is performed on native particles (i.e., pre-loaded) or after loading of cargo (herein cargo refers to, for example, one or more components of the CRISPR-Cas system, e.g., CRISPR enzyme or mRNA, or guide RNA, or any combination thereof, which may include additional carriers and / or additives), and may be performed in accordance with the in vivo, ex vivo, and / or ex vivo methods of the present invention. This results in particles of optimal size for delivery for either vivo and / or in vivo applications. In certain preferred embodiments, characterization of particle size (e.g., diameter) is based on measurements using dynamic laser scattering (DLS).

[0124] A particulate delivery system within the scope of the present invention may be provided in any form, including but not limited to a solid, semi-solid, emulsion, or colloidal particle. Thus, any of the delivery systems described herein may be provided as a particulate delivery system 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 the methods of the invention, including therapeutic methods. In certain embodiments, the invention provides a method of ablation of 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] An antibody may be an intact immunoglobulin derived from natural or recombinant sources, or may be an immunoreactive portion of an intact immunoglobulin. An antibody is usually a tetramer of immunoglobulin molecules. The tetramer may be natural or may 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 used regularly, 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 VL or VH domains, linear antibodies, scFv antibodies, single domain antibodies such as camelid antibodies (Riechmann, 1999, Journal of Immunological Methods 231:25-38), which exhibit sufficient affinity for the target, 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). York; Houston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879 ~5883; Bird et al., 1988, Science 242:423-426). However, F(ab')2 and Fab fragments, which lack the Fc fragment of 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)). The antibodies 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 have 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 a remarkable 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 provide 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 of about 60-100 kDa in size achieve faster blood clearance and faster tissue uptake. Power et al. (Generation of recombinant multimeric antibody fragments for tumor diagnosis and therapy. Methods Mol Biol, vol. 207, 3 35-50, 2003); and Wu et al. (Anti-carcinoembryonic antigen (CEA) diabody for rapid tumor targeting and imaging. Tumor Targeting, vol. 4, pp. 47- (see p. 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 90:64 (J. Immunol. 147:60, 1991). Single-chain Fv polypeptide antibodies have been described by Huston et al. (Proc. Nat. Acad. Sci. USA 85:5879-5883, 1988). Another strategy for making bispecific antibody fragments by the use of 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 have been described by Huston et al. (Proc. Nat. Acad. Sci. USA 85:5879-5883, 1988). ) V is directly linked by or through a linker encoding a peptide as described above. H - and V L -Covalently linked VH::VL heterodimers that can be expressed from a nucleic acid, including a coding sequence. 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 antigen-binding sites of the intact antibody. 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 antigen-binding sites of the intact antibody. 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 the 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 made by any of the methods known in the art that utilize soluble polypeptides or immunogenic fragments thereof as immunogens. One way to obtain 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 an immunogenic fragment thereof 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 an immunogenic fragment thereof can be expressed in a cell in vivo, and then the polypeptide is isolated and administered to a suitable host where the antibody is grown.

[0133] Alternatively, antibodies may be derived from an antibody phage display library, if desired. Bacteriophages 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 made 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 different antibodies 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-immunoglobulins).

[0135] Antibodies can be conveniently produced from hybridoma cells that have been engineered to express the antibody. Methods for making hybridomas are well known in the art. Hybridoma cells can be cultured in a suitable medium, and the spent medium can be used as a source of antibody. Polynucleotides encoding the antibody of interest can then be obtained from the hybridoma producing the antibody, and the antibody can then be produced synthetically or recombinantly from these DNA sequences. For the production of large amounts of antibodies, it is generally more convenient to obtain ascites fluid. The method of increasing ascites fluid generally involves injecting hybridoma cells into immunologically naive histocompatible or immune-tolerant mammals, particularly mice. The mammal may be prepared for ascites fluid 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 an antibody in which at least a portion of the 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., mouse) antibodies are produced. Examples of methods for humanizing mouse 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 a suitable 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 comprise a nucleotide sequence encoding a polypeptide or a fragment thereof operably linked to regulatory sequences, such as promoter sequences, terminator sequences, etc., as defined herein. Recombinant vectors capable of expressing genes or nucleic acids contained therein are called "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. The cells that are stably transformed by the introduced DNA can be selected by introducing one or more markers that allow the selection of host cells that contain, for example, 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. It will be clear to one skilled in the art which additional elements to use.

[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 recipient cells that do not contain the vector, the desired copy number of the vector 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, and the like.

[0142] After the introduction of one or more vectors, the host cells are usually grown in a selective medium that selects for the growth of vector-containing cells. The expression of recombinant protein can be detected by immunoassays, including Western blot analysis, immunoblots and immunofluorescence. Purification of recombinant protein can be performed by any conventional procedure, including any of the methods known in the art or described herein, such as extraction, precipitation, chromatography and electrophoresis. A further purification procedure that can be used to purify proteins is affinity chromatography, using a monoclonal antibody that binds to the target protein. Generally, a crude preparation containing recombinant protein is passed through a column on which a suitable monoclonal antibody is immobilized. The protein usually 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 evaluated, 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 methods of the 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 number of cells 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 of skill 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 include measuring DNA synthesis during cell replication. In one embodiment, DNA synthesis is measured by 3H ]-Thymidine or 5-Bromo-2 * -detection is performed using labeled DNA precursors, such as deoxyuridine [BrdU], adding these to cells (or animals) and then detecting incorporation of these precursors into genomic DNA during the S phase (replication) of the cell cycle (Ruefli-Brasse et al., Science 302(5650):1581-4, 2003; Gu et al., Science 302(5644):1581-4, 2003). Issue): pp. 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 ablation pretreatment of endogenous microglial cells.

[0146] In some embodiments, the kit includes a sterile container that contains 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 agent of the present invention is provided with instructions for administering the agent to a subject having or at risk of developing a nervous system disease or a central nervous system disorder. The instructions generally include information on the use of the composition for treating or preventing the disease or disorder. In other embodiments, the instructions include at least one of the following: description of the therapeutic agent, dosage schedule and administration for treating or preventing a nervous system disease or its symptoms, cautions, 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 the art. Such techniques may be found 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 (PCR) (Handbook of Experimental Immunology) (Weir, 1996). "Chain Reaction" (Mullis, 1994); "Current Protocols in Immunology" (Coligan, 1991) and other sources. These techniques may be applied 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 presented 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. EXAMPLES

[0150] Example 1 Intracerebroventricular injection of mouse HSPCs results in rapid and robust engraftment of bone marrow cells in the brain. Recent results (Capotondo et al., PNAS 2012) have demonstrated that myeloid cells are involved in the development of inflammatory bowel disease in the brain after HCT. It has been proposed that engraftment and persistent donor chimerism in the bone marrow hematopoietic niche are not necessarily required to obtain reconstitution of myeloid and microglia-like cells. Based on these findings, we assessed whether reconstitution of myeloid and microglia-like cells occurs upon direct transplantation of HSPCs into the ventricular space in preconditioned mice. - )HSPC(3×10 5 Cells (1000 x 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 increased expression of CD45 + CD11b + In the cerebrospinal compartment, GFP chimerism, which is thought to result from local proliferation of transplanted cells, was highly and gradually increased (Figure 1B). + Control mice IV-transplanted with HSPCs served as a comparison condition. Notably, the rate of microglial reconstitution was much more rapid, and GFP + The degree of GFP chimerism was higher when HSPCs were transplanted ICV compared to IV (Figure 1B) (significant effects of route and time of cell administration shown by two-way ANOVA analysis). This is surprising considering the lower numbers of cells transplanted ICV compared to IV. As with IV infusion, recipient mice pretreated with busulfan showed higher brain-spinal donor chimerism when transplanted with ICV cells compared to irradiated animals.

[0151] Immunofluorescence analysis of sagittal brain sections from contralateral sides of 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 populations were recovered in the olfactory bulb, hypothalamic regions, basal ganglia, subventricular zone and periventricular regions, striatum, and in the pons (Figure 1D). + The morphology of the cells resembled that of intraparenchymal 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 injection of human HSPCs results in rapid and robust bone marrow 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 pretreated immunodeficient animal model. + Cells were isolated from umbilical cord blood transduced with LV encoding GFP and were designated NOD.Cg-Prkdc scid Il2rg tm1WjI In addition, to determine the actual role of ICV cell transplantation in increasing the engraftment potential for delivering therapeutic molecules to the brain, we used 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 by IV or ICV infusion alone, or by a combination of IV and ICV routes (Biffi et al., Science 341, 1233-158 (2013); Sessa et al., Lancet 388, 476-487 (2014)). 016) (Figure 1E).

[0153] Interestingly, a 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 (Figure 1F). 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). 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 Iba1 and CD11b (Figure 1H) markers, 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, where progeny of HSPCs from ICV transplanted mice were identified, namely 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) and subsequently upregulated 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 modulating the contribution of IV versus ICV transplanted cells for appropriate clinical applications. The development of ICV cell delivery for cell and gene therapy in pre-treated recipients requires optimization of transplant conditions according to the different expected target diseases. Different options are applicable that can be adapted to different goals according to the desired 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) Reconstitution of microglia 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 settings to renew the cerebrospinal fluid population with cells of donor origin, we performed IV transplantation (origin, and Lin) at various times (IV and ICV transplantation delivered on the same day or IV delivery 5 days after ICV). - , c kit + Sca1 + Lin - (KSL) cells and total BM) and ICV transplantation (Lin - 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., in the BM (Figures 3D and 3H). Importantly, combined ICV delivery of HSPCs and / or KSL, or whole BM, resulted in the differential labeling of Lin in each of the tested situations. -Compared to the control condition of IV alone, brain-myeloid cell chimerism was increased. Additionally: 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 Lin. - Combining HSPCs was achieved by transplanting cells on the same day at both sites; similar results were obtained by Lin et al. - While a slightly lower chimerism was obtained by combining ICV with IV of whole BM, in all tested conditions, a slightly lower chimerism was accompanied by 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 combination / co-transplantation approach intended to grow the brain engraftment zone of transplanted cells / their progeny, we envisage 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-injected HSPCs to brain-bone marrow chimerism exceeds that of IV-coinjected HSPCs under certain conditions. Notably, the lowest contribution to brain chimerism by IV cell progeny was observed in ICV-transplanted Lin-CSFs at day 5. -This was realized in the context of high chimerism values ​​in the combination of ICV cells with IV transplanted whole BM cells; this protocol allows obtaining almost exclusively chimerism of ICV cell progeny, thus applying 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 a high and robust engraftment exclusively in the brain of the transplanted engineered cells / their progeny, we envisage the following options: for each disease, autologous CD34 cells transduced with a vector encoding the gene of interest, or ... + While the cells are transplanted ICV, unmanipulated autologous bone marrow / apheretic product will be 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 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 Progeny of both IV and ICV transplanted HSPCs have a transcriptional profile consistent with microglia. Donor reconstitution of brain bone marrow cells after HCT was shown to result from a fraction of early brain HSPC transplants undergoing local expansion and differentiation to find favorable conditions in the myeloablated recipient brain (Capotondo et al. Proc Natl Acad Sci USA. 109:15018-15023 (2012)). The transplanted brain bone marrow cells share antigenic characteristics with bone marrow cells isolated from newborn mouse brains (Figures 3B and 4B). Indeed, in both situations, we observed the expression of CD45, previously described by the inventors as a transiently expanding microglia (TAμ). + 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 cells (Capotondo et al. Proc Natl Acad Sci USA. 109:15018-15023 (2012)). Interestingly, we found that CD45 + 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 the former to TA μ cells (Figure 4A). To interpret these findings and further characterize donor-derived cells, we used GFP-GFP expression vectors after brusulfan pretreatment. + μ and TAμ cells (total population and / or GFP) from mice that were ICV or IV transplanted 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, Vol. 113, E 1738-1746 (2016); Butovsky et al., Nature neuroscience 17, 131-143 (2014); Chiu et al., Cell Rep 4, 385-401 (201 3; Hickman et al., Nature neuroscience 16, 1896-1905 (2013) Grommes et al., Journal of neuroimmune pharmacology, vol. 3, pp. 130-140 (2008)). Cells isolated from the brains of ICV- and IV-transplanted mice were compared to microglial gene expression profiles, with ANOVA P values ​​obtained with Tukey's post-hoc tests. 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 than that of TAμ. + The offspring were adult control μ and (GFP) mice from IV transplantation. - and GFP + The expression levels of the selected genes were very similar to those of the IV-implanted mice (particularly GFP). + ) TAμ populations and at slightly lower levels in TAμ isolated from p10 mice, all indicating 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 display 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 naïve animals, genome-wide expression analysis was 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 naïve mice, using the Illumina RNA-Seq platform (Figures 4D and 4E). To test for differences in their gene expression, the resulting expression datasets were isolated together with those from Gosselin and coworkers (Gosselin et al., Cell 159:1327-1340 (2014)), with a particular focus 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 close to each other (Figures 4D and 4E), confirming that reconstituted μ and TAμ cells after transplantation shared a pattern of gene expression consistent with that of microglia. GSEA pre-ranked analysis (Subramanian et al., Proc Natl Acad Sci USA vol. 102, 155 Differential gene expression linked to the TAμ (2005) was performed on the RNA-Seq data (Figure 5A-5F). Oncology biological processes of the resulting genes enriched in μ from adult controls (Figure 5A) and in TAμ from transplanted mice (Figure 5C) were related to immune cell differentiation, immune responses, DNA / RNA processes, and DNA methylation, specification of control μ to mature immune functions. Meanwhile, processes enriched in μ cells from transplanted mice (Figure 5B) spanned a range of neuron-related processes such as neuronal migration, differentiation, and regulation of synaptic plasticity (Colonna and Butovsky, Annu Rev Immunol, (2017); Tay et al., J Physiol 595, 1929-1945 (2017)). Among the processes, we also found glial cell differentiation, gliosis, as well as 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 TAμ enrichment process (Figure 5D) presents a more robust neurofunctional signature underlying different putative stages of maturation for μ cells, consistent with the notion that microglia require different functions depending on their maturation state (Matcovitch-Natan et al., Science 353, aad8670 (2016)). Interestingly, the transplanted TAμ enrichment process (Figure 5D) presents a more robust neurofunctional signature underlying different putative stages of maturation for μ cells, consistent with the notion that microglia require different functions depending on their maturation state (Matcovitch-Natan et al., Science 353, aad8670 (2016)). μ and TAμ cells from mice express genes that have been shown to be robustly modulated during microglial development (Matcovitch-Natan et al., Science vol. 353, aad8 670 (2016). Of note, while μ from transplanted animals matched control μ with respect to expression levels of four of five selected genes related to mature microglial function, distinct pathways were observed in TA μ from transplanted mice (Figure 5E and Table 2) and in genes related to the nascent stage (Figure 5F and Table 2), potentially related to 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 in extramedullary tissues, thought to be transiently localized at those sites, and can proliferate locally, giving rise to tissue-resident myeloid cells, preferentially dendritic cells. Clonogenic potential was ascribed to the presence of these cells in the brain, but upon establishment of the parabiotic pair, chimerism between the two animals was not observed in the brain, unlike that observed in other extramedullary tissues. This may suggest that brain clonogenic activity may be attributable to fixed tissue cells that are not compatible for chimerism in the parabiotic pair, as hypothesized in the case of microglial precursors. Therefore, we attempted to better characterize cells with clonogenic potential in the brain, and to assess whether they are compatible for transplantation in pretreated mice, and for ablation during pretreatment and replacement during HSC transplantation. Further studies are also intended to determine whether these cells indeed have microglial repopulation potential. Mononuclear cells were isolated from bone marrow and brain (upon Percoll enrichment of hematopoietic lineage cells) of naïve mice and animals that underwent lethal bone marrow ablation (by pretreatment 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). After 14 days of plating, discrete hematopoietic colonies were recovered from bone marrow and from brain cultures derived from naïve mice, as expected (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 was restored on ice and in both bone marrow and brain recovered from pretreatment upon HSPC transplantation (Figure 6C). GFP at both sites was significantly higher than that of HSPCs in both bone marrow and brain. +In colonies, CFU output was highly chimeric, indicating that HSPC transplants can contribute to immobilized brain hematopoietic clonogenic precursors. Bone marrow and brain Percoll-enriched mononuclear cells from primary transplant recipients were also used for secondary transplants into busulfan-pretreated recipients (Figure 6A). Strikingly, GFP-GFP cells derived from primary recipients were not detected in the primary recipients. + The cells were identified in the hematopoietic tissues and brain of the secondary recipients long-term after transplantation (FIG. 6D). + While the cells showed multilineage marker expression, 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 molecules to be delivered 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 by a combination of IV and ICV routes (Biffi et al., Science 341, 1233-158 (2013); Sessa et al., Lancet 388, 476-487 (2013)). 6 years) (Figure 1D and Figure 6A). Interestingly, well-defined human bone marrow (CD45 + CD11b + ) cell progeny were identified in the brains of transplanted mice for extended periods following 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 assessed whether greater human cell-bone marrow chimerism in the mouse brain determines greater delivery of ARSA enzyme to the brain. Importantly, the increased contribution of ICV transplanted human HSPCs to brain-bone marrow chimerism led to greater ARSA enzyme delivery to the brain of transplanted mice (Figure 1G). This was especially evident in the case of combined IV+ICV delivery, which resulted in greater As2 + / + The ARSA activity reached levels indistinguishable from controls. Sixteen weeks after transplantation, similar supranormal ARSA activity levels 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 the combined transplantation approach results in greater enzyme delivery to the brain compared to the standard IV transplantation approach, thus appearing as a promising strategy to improve the overall therapeutic potential of HSC gene therapy for SD with CNS disorders. Furthermore, ICV delivery of HSPCs alone has been shown to be sufficient to deliver the same amount of therapeutic enzyme to MLD brain 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 indicates that the contribution of ICV-delivered cells was largely restricted to the brain, rather than to extra-CNS hematopoietic populations.

[0162] The short survival interval of this animal model and the limited severity of its phenotype in the short term prevented the evaluation of the phenotypic effects of increasing brain ARSA delivery. However, the administration of LV-transduced HSCs (HSC gene therapy) to MLD mice (Biffi et al., J. Clin. Invest. 116 vol., pp. 3070-3082 (2006); Biffi et al., J. Clin. Invest. vol. 113, 1118-1129 (2004)) and patients (Biffi et al., Science 341, 1233-158 (2013); Sessa et al., Lancet 388, 476-487 (201 6)), as well as other lysosomal storage disease models (Gentner et al., Sci Transl Med vol. 2 , 58ra84 (2010); Visigalli et al., Blood 116:5130-5139 (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 manifestations. 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, especially the actual role of ICV cell transplantation in increasing the likelihood of HSPC engraftment to deliver therapeutic molecules to the brain and to exert benefits against LSDs with both brain and systemic disorders, we performed a mouse model of mucopolysaccharidosis type II (MPS II), in which 2-month-old bone marrow-ablated iduronate sulfatase (IDS) mice were cultured in mice. - / - To the recipient, Lin - HSPCs and whole BM were transplanted from wild-type donors (Figure 6C). Mice with IV alone had wild-type IDS + / + They received whole BM exclusively via IV from the donor. Controls were left untreated. After 180 days, the behavior of treated and control mice was tested by rotarod. Interestingly, both treatments improved the performance of the mice over the four trials. Mice's rotarod performance improved over time (FIG. 6D), with both IV and ICV+IV treated mice spending increased time on the rod in the fourth trial compared to the first trial (FIG. 6E). However, the performance of ICV+IV treated mice exceeded that of IV alone transplanted animals. 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 transplants 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. The applicability of the ICV HSPC transplantation approach in the context of allogeneic transplantation in patients was evaluated, specifically the combination of ICV and IV transplantation in the context of both minor antigen and MCH mismatched transplant situations. The impact of ICV delivery of allogeneic HSPCs on overall survival and CNS microglial engraftment in mice receiving hematopoietic cell transplants from mismatched donors was examined. MCH-mismatched (BALB / cJ CD45.2 donors into B6.SJL CD45.1 recipients; an ICV dose of one cell was tested, 3 × 10 5 cells / mouse), and minor antigen mismatched (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 (1000 Rad / mouse) transplant setting. Total body irradiation (TBI) (1000 Rad) was used to pre-treat recipient mice on day -1. Mice received either fresh whole bone marrow (tBM) alone or ICV-injected GFP-transduced Lin -After transplantation, the mice were followed for up to 9-10 weeks (minor antigen mismatched) and 16 weeks (MHC mismatched). Rare inter-current death (ICD) was observed in the recipients and was not related to any specific treatment (Figs. 8B and 9B). All surviving animals had a stable gain in weight and good overall health. Cytometric analysis of PB, BM, spleen and thymus (the latter thymus in case of minor antigen mismatched transplantation) demonstrated successful donor cell engraftment (Figs. 8C and 9C). Importantly, GFP-GFP, a progeny of ICV transplanted HSPCs, was detected in the recipients. + No cells were detected in the hematopoietic tissues examined (Figures 8D and 9D). Compared to IV-only recipients, IV+ICV minor antigen-mismatched animals demonstrated dose-dependent engraftment from donor-derived cells in the brain. In both minor antigen- and MHC-mismatched animals, ICV delivery of HSPCs provided an advantage in donor-derived microglial chimerism in the CNS compared to IV-only transplantation. In conclusion, delivery of allogeneic hematopoietic stem / progenitor cells to the lateral ventricle is feasible in 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 assessed 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, cells were delivered IV (1.0 × 10 6cells / mouse), either ICV8 (0.3×10^6 cells / mouse) or intrathecal (IT) (0.3×10 6 HSPCs (CD45.2 GFP) were transplanted at 100x the normal concentration (100x the normal concentration) in 100% HSPC-treated mice. 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). + We demonstrated good BM engraftment of CD45.1 BM-transplanted cells, whereas in IT-transplanted mice, the same cells presumably out-grown CD45.2 GFP cells transplanted on day 0. + We showed that little engraftment occurred due to competition with HSPCs. CD45.2 GFP engrafted in the cerebrospinal compartment in all groups. + HSPCs were analyzed (middle graph in Figure 10B), and ICV+IV injected mice showed the highest donor chimerism compared to other groups. + CD45.1 cell progeny were observed in ICV transplanted mice. Engrafted donor (GFP+mCherry) cells were analyzed in the spinal cord bone marrow compartment of all groups (right graph in Figure 10B), and ICV+IV injected mice showed the lowest GFP chimerism compared to other groups. Compared to IT mice, ICV transplanted mice showed the lowest mCherry chimerism. +An increase in the percentage of CD45.1 BM transplanted 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-only deliveries are also associated with significant spinal cord chimerism with the donor. IT transplanted with donor cells was also exclusively observed in peripheral blood and bone marrow, indicating that IT is not retained in the CNS upon transplantation. Therefore, this transplantation procedure can be considered, especially in the case of such diseases that have 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 generate TAμ and μ in mouse brain upon IV transplantation, whereas the Lin fraction is not double positive for c-kit and Sca-1 (Not-KSL). - Progenitor cells do not (Capotondo et al., Proc Natl Acad Sci USA 109:15018-1502 3 (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 evaluate the possible differential contribution of transplanted cells to bone marrow brain cell turnover (Figs. 11A, 11D and 11E). KSL cells up-regulated 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, CNSmac (Capotondo et al., Proc Natl Acad Sci USA vol. 109, pp. 15018-15023 (2013)). 12). KSL and not-KSL cells instead contributed equally to cerebromyelocellular reconstitution when infused ICV (Fig. 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 cerebromyelocellular engraftment (Fig. 11A, 11B). Upon competitive IV transplantation, CD48 - / CD150 + Long-term HSCs (LT-HSCs) and CD48 - / CD150 - Compared with multipotent progenitors (MPPs), these showed the greatest ability to reconstitute the cerebromyeloblast compartment compared to other injected populations (Figure ​(Figure11F). In contrast, the more highly committed CD48 + CD150 - Cells also contributed to reconstitution of cerebromyeloblast populations in mice transplanted ICV (Figure 11G). Hematopoietic reconstitution of transplanted mice is shown in Figure 11C. Histology on frozen slices of brains from IV and ICV transplanted mice confirmed these results (Figures 11H and 11I). Consistent with previous findings, even in these situations, donor-derived cells displayed a 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 investigated the expression of the CXCR4 receptor, which is known to be involved in HSC mobilization and homing to the BM (Dar et al., Experimental hematology 34:967-98). ~975 (2006); Rettig et al., Leukemia 26:34-53 (2012); Sugiyama et al., Immunity 25:977-988 (2006)) were analyzed for KSL, not-KSL cells and the four KSL subpopulations mentioned above (cells were analyzed at the end of transduction at the time of transplantation). Interestingly, the reconstitution potential of microglia-like cells in the IV injection situation, i.e. cells enriched in KSL, LT-HSC and MPP expressed higher levels of CXCR4 compared to not-KSL and HPC-1 and HPC-2 (Figure 11J). Without intending to be bound by theory, this finding suggests that cells expressing CXCR4 at higher levels may favor early recruitment to the brain upon IV injection and therefore the ability to contribute to brain-myeloid cell chimerism. This cannot be applied to ICV delivery where different signals may be important.

[0168] To more rigorously assess whether bona fide HSCs are capable of generating 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 -Cells were isolated from CD45.2 Fgd5-Zsgreen donors and transplanted IV or ICV into busulfan-pretreated or lethally irradiated CD45.1 recipient mice along with unmanipulated whole bone marrow CD45.1 supports (Figure 12A). IV cell delivery led to robust hematopoietic donor chimerism, whereas ICV-injected cells did not contribute to hematopoiesis in the peripheral blood. Interestingly, ICV-transplanted animals did not exhibit peripheral blood chimerism, but rather donor-derived CD45.2 + Cells were identified in the brain in both transplantation situations (Figure 12B and C). Reconstitution of brain bone marrow cells was confirmed to be less efficient in irradiated rather than busulfan-ablated recipients, as described above (Capotondo et al., Proc Natl Acad Sci. USA vol. 109, pp. 15018-15023 (2012)). Donor-derived cells were C. They expressed D45 and CD11b and were present in the majority of the TAμ cell compartment (FIGS. 12D and 12E).

[0169] These data demonstrate that bona fide HSCs gave rise to 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 allow 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. In particular, 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, confirming that a fraction of the HSPC pool expressed CX3CR1, but no actual expression could be detected 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. + No progeny cells could be detected in the bone marrow and brain of repopulated mice 1.5 months after transplantation (FIG. 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 (FIG. 13B). These data indicate that the bone marrow equivalent of μ precursors is retained in LT-HSC 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 differentially labeled CD34 + CD38 + (defined as precursor based on 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 according to 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 carrying various reporter genes) (Figure 14C). Interestingly, consistent with what was observed in the mouse context, it was confirmed that the population with increased stem cell potential had a higher capacity 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 implanted 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 have been developed based on integrating vector-mediated gene transfer and targeted gene addition that can be applied in certain situations where sustained but regulated transgene expression may be required.

[0173] Neurodegenerative diseases of adulthood, such as amyotrophic lateral sclerosis (ALS) or Alzheimer's disease (AD), and neurodegenerative SD, are characterized by an elevated neuroinflammatory response sustained by microglial activation, and one of the molecules upregulated by activated microglia in the course of pathological events is the 18 kDa translocator protein (TSPO), as shown by others and by us using a selective translocator protein (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 main 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 novel engineered microglia in response to tissue injury and inflammation. We are developing innovative tools that allow engineering of brain microglia, targeting TSPO, by HSC induction for regulated delivery of therapeutic molecules to the brain, including potential applications in neurodegenerative diseases and neuroLSDs. In particular, brain microglia are reconstituted in diseased brains by "sensor" cells that are engineered to express genes of interest upon cell activation in response to local neurodegeneration and neuroinflammation using the advanced transplantation protocol detailed above (this situation would also be compatible with the use of preconditioning regimens directing μ-precursors).

[0174] To this end, we use the TSPO promoter sequence to drive transgene expression, including the evidence-based selected TSPO promoter sequence (Wang et al., Cell Tissue Res 350:261-275 (2012)) in front of a marker or therapeutic gene in the context of advanced generation LVs that will be used for HSC transduction. Various strategies have been tested, which can be achieved by inserting a therapeutic gene before / within the first intron / exon of the TSPO gene utilizing its promoter by targeted gene addition using CRISPR-Cas technology. TSPO knockout mice provide a testimony for the feasibility of this latter strategy (Banati et al. Nat Commun 5, 5452 (2014)). In the context of third generation LV The selected TSPO promoter sequence (FIG. 16B) was inserted in front of GFP as a marker gene in the mouse model (Wang et al., Cell Tissue Res 350:261-275 (2013)). The transduced and / or edited cells were then used to exclusively reconstitute brain-myeloid cells in recipient mice by advanced protocols.

[0175] One working hypothesis is that upon transplantation of HSCs genetically modified / edited at the TSPO locus, a population of brain myeloid cells and microglia will arise that express genes that upon activation of cells in a neuroinflamed and degenerative environment will allow for accurate molecular monitoring of the neuroinflammatory response and / or contribute to therapeutic activity and amelioration of the disease phenotype. Reporter genes to characterize the tools under development will be used in the ALS mouse model (SOD1.G93A mice) (Peviani et al., CNS Neurol Disord Drug Targets Vol. 9). , 491-503 (2010)) and in animal models of globoid cell leukodystrophy (GLD) (Visigalli et al. Neurobiol Dis 34, 51-62 (2009) The approach has been validated in animal models characterized by microglial activation and TSPO upregulation, such as serotonin-dependent inflammatory cytokines (SEQ ID NO: 1), which are characterized by microglial activation and TSPO upregulation. The approach has been validated for its functionality and its potential for therapeutic action 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 i) the existence of a functionally defined microglial precursor population located in the brain that is susceptible to ablation upon administration of busulfan-based pretreatment and that may arise simultaneously with endogenous CNS-resident microglial precursors (μPs). Phosphorylated histone 2AX (γH2AX) is a sensitive and specific biomarker that tracks cells targeted by busulfan in the brain. Thus, functional brain-resident μPs can be identified by utilizing γH2AX, a pharmacological marker of busulfan and its cytotoxicity in these cells. γH2AX was investigated as a biomarker of busulfan cytotoxicity. In particular, the presence, distribution and cellular localization of γH2AX was examined by flow cytometry (FC) and immunofluorescence (IF) on brain slices from mice treated with systemic busulfan pretreatment. FC was specifically determined by the expression of viable CD45+ cells in busulfan-treated versus control untreated animals on days 1 and 5 after the last busulfan dose. + In cells, γH2AX signaling was enhanced (Fig. 15B). At the same time, early apoptosis after busulfan treatment was also observed, particularly with 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 the almost absent neuronal cells, mainly located in the hippocampus. +Except for the lesions, they were barely detectable in control mice (Figure 16C, top photo), and a correlation between physiological neuronal activity and H2AX phosphorylation has previously been 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 migratory stream (RMS) (Figure 15C, bottom photograph). γH2AX + The lesions were localized in both neurons (Fig. 15C inset) and glial cells, namely microglia and astrocytes (Fig. 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. Thus, 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 markers of hematopoietic stem cells and microglia-specific genes, 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 to develop efficient turnover of brain microglia with donor-derived cells. A similar, but CNS-restricted, regimen protects patients with CNS-restricted diseases (e.g., INCL, PD, ALS, etc.) from the side effects of systemic preconditioning with bone marrow ablation. In preliminary studies, we investigated the possibility of intracerebral administration of busulfan by a cannula implanted in the lateral ventricle in mice. Various drug formulations of busulfan were tested, including clinical grade busulfan formulations. However, ICV administration could not guarantee exposure of brain-resident μPs to busulfan levels comparable to those achieved by systemic drug administration, nor could it favor transplanted HSPC engraftment (data not shown). It was rather associated with widespread neurotoxicity and local inflammation. Therefore, a targeted drug delivery strategy using nanoparticles was implemented. Without intending to be bound by theory, nanoparticles may have the potential to enable efficient and selective delivery of ablative drugs to functionally defined μPs.

[0178] Recently, polymeric nanoparticles (NPs) have attracted great interest as a promising means to improve the pharmacological profile of drugs, including chemotherapeutic agents. NPs allow for tuning of material composition, surface functionalization and degradation rate, allowing i) high selectivity in targeting cells, reducing the risk of side effects of drugs formulated with NPs compared to the same drug without NPs; ii) multiple drug delivery; and iii) controlled drug release over time. NPs are made from artificial or natural polymers and have sizes between 10 and 400 nm. Various biodegradable polymers such as chitosan, poly(ε-caprolactone) (PCL), poly(alkyl-cyanoacrylate) (PACA), poly-lactic 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 affecting 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 obtain enhanced CNS biodistribution or target cell specificity. Modification of lipophilicity, nanoparticle core structure and composition allow for tuning of the biodegradation time of the material to optimize drug loading and release profiles.

[0179] A novel drug delivery biodegradable and biocompatible nanocarrier (based on PCL NPs) (Figure 17A) was validated to selectively target microglia / macrophages after intraparenchymal administration in a spinal cord injury mouse model. The biodistribution of such microglia-targeted NPs after ICV administration in mice was validated, confirming efficient uptake and widespread distribution by microglia / macrophages in various CNS regions (Figures 17A-17I). Cytofluorimetric analysis on injected mouse brains revealed that the NPs, especially when injected ICV with mannitol to favor BBB penetration, upregulate CD45 + c-kit + Cells (Figure 17C) and proliferative 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 rate, and modulate drug release over time. Interestingly, immunofluorescence confocal analysis confirmed these findings and showed that NPs were concentrated in brain regions, such as the SVZ and RMS, that were first colonized by donor-derived HSPCs immediately after transplantation, during the process leading to brain microglial reconstitution in areas of intense γH2AX-positive signal after busulfan administration. Thus, representative regions are likely enriched in μPs (FIG. 17G). Of note, the rhodamine-containing NPs + Iba + Bone marrow cells were also proliferative prior to sacrifice and staining, as suggested by both the ki67 positive signal (FIG. 16G) and Edu administration (FIG. 17H). + Rhodamine + The cells were also positive for hematopoietic bone marrow markers and displayed both ramified and round microglia-like morphology. Importantly, rhodamine + Iba1 + The cells were also identified as early / stem cell myeloma. NPs sometimes expressed -canestin (Figure 17H, right panel). The presence and distribution of rhodamine signals in NP-injected brains was interestingly consistent with that of Edu signals, suggesting that NPs occur preferentially in proliferating cells, which may include cells with microglial precursor characteristics (Figure 17I).

[0180] Next, we optimized novel NPs by de novo chemical synthesis to allow loading with busulfan and etoposide by exploiting two different chemical moieties, selected to be compatible with the drugs (referred to as "SP" and "QMS") covalently grafted to a poly-(2-hydroxy-ethyl methacrylate) backbone (herein referred to as BK-510). This allowed us to achieve encapsulation of biologically relevant amounts of drugs in 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 chemical therapeutic drugs (such as busulfan or etoposide) was further improved by introducing a so-called "self-assembly" approach, which allows the formation of NPs starting from lyophilized material (Figure 18). This makes the nanoparticle material suitable for scale-up synthesis, long-term storage of lyophilized pharmaceutical products without loss of potency and consistency between different batches.

[0181] 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 h of incubation, indicating that the inclusion of BU in the NPs did not impair drug efficacy. Busulfan-loaded (NP-BUS) or empty NPs in the lateral ventricles of mice increased the uptake of brain microglia (CD45 + / CD11b +) induced a significant increase in γH2AX signals in cells (Figure 19C).

[0182] To facilitate the development of an effective brain pretreatment regimen, NP loading was optimized with other drugs such as etoposide and lomustine, which showed preliminary supporting results (Figures 19D-19E). Cell proliferation assays were performed on 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 ug / ml. Etoposide-loaded NPs or non-encapsulated 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 ug / ml). As a control, blank NPs (without drug) were tested. In this case, the final NP concentration in the medium was matched to that used to administer 25ug / ml of etoposide encapsulated in the NPs. As can be observed in Figures 20D-20E, all three NP formulations are equally effective at delivering etoposide, thus determining cell death. During short incubation periods (self-assembled 100nm NPs, 48 ​​hour time point), the empty NPs are neither toxic (pre-assembled 100nm PCL NPs, and self-assembled 50nm NPs) nor only mildly toxic. However, cell viability in the case of the empty NPs is always greater than that of the etoposide-loaded NPs, suggesting that the effect observed with the etoposide-loaded NPs is due to the release of the drug, rather than the NPs themselves. Interestingly, the effect observed in the case of the etoposide-loaded NPs tested at a final drug concentration of 1.6ug / ml is more pronounced than that observed with the unencapsulated drug. This difference was even more pronounced at an incubation time of 72 h, determining that encapsulation of etoposide in NPs enhanced its efficacy and improved the cytotoxicity of already very low concentrations of the drug.

[0183] These NP formulations were administered i) at a single dose of 25 mg / kg busulfan or ii) in the brain to induce proliferation of microglial precursors and / or CD45 + c-kit + CSF 1R inhibitors have been shown to expand cells (Elmore et al., Neuron. 82 Etoposide was injected ICV into the brains of wild-type adult animals pre-treated with oral administration of NPs (Vol. 2: 380-397 (2014)) for 10 days (Figures 19A, 19C, 19D). Five days after NP injection, animals were analyzed for the occurrence of etoposide-mediated death of microglial cells and / or their precursors, using busulfan standard treatment (four doses of 25 mg / kg) as a reference for ablation of brain microglial precursors. Interestingly, etoposide-loaded NPs significantly reduced the CD45 expression level, consistent with that observed upon busulfan treatment with standard ablation. + c-kit + and CD45 + CD11b + Apoptosis was induced in the cells (by Annexin V positive staining in flow cytometry) (FIGS. 19B and 19E).

[0184] Overall, the NP formulations tested here achieved targeted delivery of ablation drugs to microglial precursors required for selective brain conditioning.

[0185] Example 13 A novel transplantation modality to generate de novo microglia via transcriptional reliance from hematopoietic stem and progenitor cells Using a multifaceted approach, we identified novel strategies to improve donor turnover of bona fide cerebromyelocytes after HCT, a phenomenon that can result in robust and rapid engraftment of new transcriptionally dependent microglia through a process that can recapitulate the physiological maturation of postnatal cerebromyelocytes.

[0186] First, we applied transcriptional profiling analysis to newly formed myeloid cells (identified as μ and TAμ cells) recovered from the brains of busulfan-treated chimeric mice. This analysis revealed that the gene expression patterns of donor-derived cerebromyeloma cells, sorted for expression of CD45, CD11b, and donor cell markers, closely resembled those of endogenous microglial cells in naive mice at various developmental ages. It was noted that the newly formed cells expressed typical microglial markers such as Tmem119, Tgfbr1, P2ry13, Olfml3, and Mertk. Interestingly, these cells clustered in a completely separate manner compared to macrophages, confirming that the reconstituted brain cells after transplantation were transcriptionally distinct from bone marrow-resident or circulating myeloid cells and closely resembled the endogenous cerebromyeloma population (Gosselin et al., Cell 159:1327-1340 (2014)). Bennett and coworkers (Bennett et al. Proc Natl Acad Sci USA vol. 113, E1738-1746 (2016)) showed that cells in the adult CNS derived from whole bone marrow transplants do not express Tmem119, which was considered by the above authors as a marker that could distinguish microglia from other myeloid cells. These findings, which are apparently at odds with the data showing a robust Tmem119 expression in donor-derived brain bone marrow cells, can be inte...

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