Methods for Treating Alzheimer's Disease

Abstract

Methods for alleviating or treating Alzheimer's disease or a neurological disorder, or alleviating a symptom thereof, are provided, comprising administering to a subject an effective amount of a hematopoietic stem or progenitor cell (HSPC) or population of HSPCs, which are optionally genetically corrected prior to administration and which differentiate into healthy microglial cells in the brain. The cells are capable of reducing amyloid plaques and inflammation. The methods comprise, consist essentially of, or consist of administering to a subject in need thereof, either systemically or to the hippocampus of the subject, a hematopoietic stem or progenitor cell (HSPC) or population of HSPCs, which are optionally genetically corrected prior to administration and which differentiate into healthy microglial cells in the brain.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit under 35 U.S.C. §119(e) and under the Paris Convention of U.S. Provisional Application No. 63 / 328,574, filed April 7, 2022, the contents of which are incorporated herein by reference in their entirety. [Background technology]

[0002] background Alzheimer's disease (AD) is the most common cause of dementia and the most common age-related neurodegenerative disorder, predicted to affect more than 13 million people by 2050 1、2 With the aging population, 3-5% of people aged 65 or older may suffer from AD, with an additional 5-7 million new cases each year. 3 As a result of AD, dementia progresses through disease progression to severe disability and generally results in death within 5-12 years after onset. 4 Marked neuronal degradation, inflammation, and progressive memory and behavioral decline result from the accumulation of extracellular β-amyloid (Aβ) plaques and hyperphosphorylated tau in neurofibrillary tangles (NFTs) throughout the brain, primarily in the hippocampus and cortex. These cardinal features of AD result in increased oxidative stress, inflammation, synaptic loss, and cholinergic dysfunction, leading to neuronal dystrophy. 5 Current FDA-approved treatments help manage cognitive impairment, but there are no effective disease-modifying treatments available. 5、6 Given the complex pathobiology of AD, any potential treatment must target multiple pathological pathways to ameliorate the disease condition. [Prior art documents] [Non-patent literature]

[0003] [Non-Patent Document 1] Long, J.M. and Holtzman, D.M. Cell (2019) 179, 312-339 [Non-Patent Document 2] Vaz, M. and Silvestre, S. European Journal of Pharmacology (2020) 887, 173554 Summary of the Invention [Means for solving the problem]

[0004] Disclosure Summary For many years, reactive microglia were thought to be a consequence of AD pathology, but they are now thought to play a role in disease progression and possibly even initiation. 25、27、29、34 However, the role of microglia in AD remains a subject of intense debate (McGeer et al., Neurology 42 (1992)). Applicants herein provide direct evidence that microglia play a critical role in the progression of Alzheimer's disease and that replacement of diseased App / Psen1 microglia with healthy ones via a single WT hPSC transplant in 5xFAD mice completely rescued neurocognitive dysfunction in mice.

[0005] Accordingly, provided herein are methods for alleviating or treating Alzheimer's disease, the methods comprising, consisting essentially of, or consisting of administering to a subject in need thereof, either systemically or to the subject's hippocampus, hematopoietic stem or progenitor cells (HSPCs) or a population of HSPCs, which have been optionally genetically corrected prior to administration and which differentiate into healthy microglial cells of the brain. The cells reduce amyloid plaques and inflammation, thereby alleviating or treating AD.

[0006] CD34 +Methods and compositions for correcting known familial mutations in AD using HSPC gene therapy are also provided.

[0007] Methods and compositions demonstrate that the use of a 5xFAD double transgenic mouse model expressing mutant human amyloid beta (A4) precursor protein 695 (APP) with the Swedish (K670N, M671L), Florida (I716V), and London (V717I) familial Alzheimer's disease (FAD) mutations and human presenilin 1 (PS1) with two FAD mutations (M146L and L286V) results in robust increases in Aβ plaque accumulation in the hippocampus and cortex by 2 to 4 months of age. 18、19 These mice exhibit significant neurocognitive dysfunction and reduced anxiety behavior between the ages of 3 and 6 months. 20 Furthermore, 5xFAD mice display key features of AD amyloid plaque pathology, including dysfunctional microglia. 18、21 .

[0008] Applicant presents herein that a single systemic wild-type HSPC transplant into adult 5xFAD mice results in the preservation of memory and neurocognitive ability and the reduction of Aβ plaque load in the hippocampus and cortex.Donor or donor-derived HSPC differentiate into microglia, which have active amyloid plaque clearance ability, and at the same time, reduce neuroinflammation.This study paves the way for a new therapeutic approach using HSPC gene therapy for the treatment of AD.

[0009] Furthermore, we report herein the expression of CD34 to microglia in the brain and spinal cord, and to macrophages in the DRG. + Methods are provided for preserving neuronal and motor function using transplantation of HSPCs. 13Transplantation of WT HSPCs may result in the generation of healthy microglia, which may result in reduced neuroinflammation and Aβ plaque accumulation in the most affected areas of the AD brain. Alternatively, the use of a patient's own hematopoietic stem and progenitor cells (HSPCs) is provided for use in a method comprising, consisting essentially of, or consisting of, introducing or correcting genes involved in the pathogenesis of AD in the treatment of Alzheimer's disease in a patient by transplanting genetically modified HSPCs systemically or locally into the hippocampus in the patient. The genes corrected are PP, MAPT, PSEN1, PSEN2, and Trem2. [Brief description of the drawings]

[0010] [Figure 1]Transplantation of WT HSPCs prevents neurocognitive dysfunction in 5xFAD mice. (Figure 1A) Schematic of the experimental design and timeline. (Figures 1B-1D) Memory recognition test assessed by discrimination index (Figure 1F) and preference index (Figure 1C) in 6-month-old mice. Representative tracking plots of 2 days of testing are shown for 5xFAD / 5xFAD HSPC and 5xFAD / WT HSPC mice (Figure 1D). (Figures 1E-1H) Elevated plus maze test in different mouse groups by time spent in closed arms (Figure 1E) and open arms (Figure 1F), expressed in seconds (s), and by the ratio of time spent in open arms vs. total time of testing (Figure 1G). Representative tracking plots of testing are shown for 5xFAD / 5xFAD HSPC and 5xFAD / WT HSPC mice (Figure 1H). (Figure 1I-1L) Open field test with distance traveled by different mouse groups expressed in meters (m) at the edge (Figure 1I), corners (Figure 1J), and total distance traveled (Figure 1K). Representative tracking plots of the test are shown for 5xFAD / 5xFAD HSPC and 5xFAD / WT HSPC mice (Figure 1L). All data are presented as mean ± sem. **P<0.005 and ***P<0.0005 as determined by one-way ANOVA followed by Tukey's multiple comparisons.

[0011] [Diagram 2]Transplantation of WT HSPCs resulted in reduced deposition of Aβ plaques in the cortex and hippocampus of 5xFAD mice. (Figures 2A-2D) Representative sagittal sections of the cortex (Figures 2A-2C) and hippocampus (Figures 2B-2D) stained for the Aβ plaque marker 6E10 from 5xFAD / 5xFAD HSPC mice (Figures 2A-2B) and 5xFAD / WT HSPC mice (Figures 2C-2D). Scale bars: 500 µm. Quantification of plaque number (Figures 2E-2H), area occupied by plaques (Figures 2F-2I), and percentage of total area (Figures 2G-2J) occupying the cortex (Figures 2E-2G) and hippocampus (Figures 2H-2J). (FIG. 2K) Triton®-X soluble and guanidine insoluble Aβ1-42 (ELISA) in brains of WT mice (n=3), 5xFAD mice (n=3), 5xFAD / 5xFAD HSPC mice (n=3), and 5xFAD / WT HSPC mice (n=3). All data are shown as mean ± sem. **P<0.005, ***P<0.0005, and ****P<0.0001 as determined by one-way ANOVA followed by Tukey's multiple comparisons.

[0012] [Diagram 3]Infiltration of transplanted HSPCs into the brains of 5xFAD mice and differentiation into microglia results in reduced microglial activation and phagocytosis of Aβ plaques. (Figure 3A) Representative immunohistochemistry images of sagittal sections of brains from 5xFAD / WT HSPC mice 4 months after transplantation showing GFP+ cells, with insets showing GFP+ cells in the hippocampus and cortex. Scale bar: 100 μm (a, inset). (Figure 3B) Representative images of hippocampal sections immunostained for the microglial marker Iba1, with insets showing the dentate gyrus region of the hippocampus. Scale bar: 100 μm. (Figure 3C, Figure 3D) Quantification of the area occupied by Iba1+ cells in the hippocampus and cortex. Data are mean ± sem, determined as one-way ANOVA followed by Tukey's multiple comparisons, **P<0.005, ***P<0.0005, and ****P<0.0001. (FIG. 3E) Representative chromogenic images showing Iba1+ microglia (green) and 6E10+ plaques (purple). Scale bar: 50 μm. (FIG. 3F) Representative 3D reconstructions of immunofluorescence images of brain sections from 5xFAD / 5xFAD HSPC mice (top panel) and 5xFAD / WT HSPC mice (bottom panel) stained with, from left to right, anti-GFP, anti-Iba1, and anti-6E10 antibodies. The rightmost images are overlaid. Scale bar: 10 μm or 2 μm (inset). (FIG. 3G) Inset showing inflamed, active Iba1+ in close proximity to 6E10+ plaques in 5xFAD / 5xFAD HSPC mice. (FIG. 3H) Inset showing GFP+ Iba1+ microglia phagocytosing 6E10+ plaques engulfed within GFP+.

[0013] [Figure 4]RNA-seq revealed a shift in the microglial and endothelial transcriptome profiles in the cortex and hippocampus, respectively, following transplantation of WT HSPCs in 5xFAD. (Figure 4A) Bar graphs and violin plots for composite gene score analysis of a set of endothelial cell genes associated with disease-associated microglia (DAM) stage 2 and neurodegeneration in the cortex and hippocampus of WT (C), 5xFAD / WT HSPCs (T), 5xFAD (D), 5xFAD / 5xFAD HSPCs (M). Significant P values ​​(P<0.05) between T vs. D as determined by Welch's two-sample t-test with Benjamini-Hochberg correction are shown. (Figure 4B) Heatmap showing the top 60 differentially expressed genes in the cortex and hippocampus. Bar graphs and violin plots showing 24 microglia-related significantly differentially expressed genes in the cortex and 7 endothelium-related significantly differentially expressed genes in the hippocampus between 5xFAD / WT HSPC and 5xFAD brains, respectively. Significant P values ​​(P<0.05) between T vs. D as determined by Welch's two-sample t-test with Benjamini-Hochberg correction are shown. Numbers in the upper right corner identify the ROC-AUC values ​​of T, D, and M compared to C, respectively.

[0014] [Diagram 5] Infiltration of transplanted HSPCs into the brains of 5xFAD mice and differentiation into microglia results in reduced microglial activation and phagocytosis of Aβ plaques. (FIG. 5A) Representative image of a cortical section immunostained for the microglial marker Iba1. Scale bar: 100 μm. (FIG. 5B) Images showing GFP+ Iba1+ microglia phagocytosing 6E10+ plaques and their orthogonal views of brain sections from 5xFAD mice transplanted with WT HSPCs stained with anti-GFP, anti-Iba1, and anti-6E10 antibodies (grayscale; see FIG. 3F for grayscale comparison of antibody staining). Scale bar: 10 μm. Inset shows colocalization of GFP+ Iba1+ with 6E10+ plaques. Scale bar: 2 μm.

[0015] [Figure 6] Violin plots for composite gene score analysis of a set of disease-associated microglia (DAM) stage 1 and universal macrophage marker genes in the cortex and hippocampus of WT (C), 5xFAD / WT HSPCs (T), 5xFAD (D), 5xFAD / 5xFAD HSPCs (M). Significant P-values, T vs. D, are indicated. (Figure 6A) Disease-associated microglia (stage 1). (Figure 6B) Universal macrophage marker. Numbers in the top right corner identify the ROC AUC values ​​of T, D, and M compared to C, respectively. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0016] Detailed Description Throughout this disclosure, various publications, patents, and published patent specifications are referenced by an identifying citation. The disclosures of these publications, patents, and published patent specifications are hereby incorporated by reference in their entireties into this disclosure.

[0017] Before the compositions and methods are described, it is to be understood that this invention is not limited to the particular methodology, protocols, cell lines, assays, and reagents described, as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments of the invention, and is in no way intended to limit the scope of the invention, which is set forth in the appended claims.

[0018] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of tissue culture, immunology, molecular biology, microbiology, cell biology, and recombinant DNA, which are within the skill of the art. See, e.g., Sambrook and Russell eds. (2001) Molecular Cloning: A Laboratory Manual, 3 rdedition;the series Ausubel et al. eds. (2007) Current Protocols in Molecular Biology;the series Methods in Enzymology (Academic Press, Inc., N.Y.);MacPherson et al. (1991) PCR 1: A Practical Approach (IRL Press at Oxford University Press);MacPherson et al. (1995) PCR 2: A Practical Approach;Harlow and Lane eds. (1999) Antibodies, A Laboratory Manual;Freshney (2005) Culture of Animal Cells: A Manual of Basic Technique, 5 thedition; Gait ed. (1984) Oligonucleotide Synthesis; U.S. Patent No. 4,683,195; Hames and Higgins eds. (1984) Nucleic Acid Hybridization; Anderson (1999) Nucleic Acid Hybridization; Hames and Higgins eds. (1984) Transcription and Translation; Immobilized Cells and Enzymes (IRL Press (1986)); Perbal (1984) A Practical Guide to Molecular Cloning; Miller and Calos eds. (1987) Gene Transfer Vectors for Mammalian Cells (Cold Spring Harbor Laboratory); Makrides ed. (2003) Gene Transfer and Expression in Mammalian Cells; Mayer and Walker eds. (1987) Immunochemical Methods in Cell and Molecular Biology (Academic Press, London); Herzenberg et al. eds (1996) Weir’s Handbook of Experimental Immunology; Manipulating the Mouse Embryo: A Laboratory Manual, 3 rdedition (Cold Spring Harbor Laboratory Press (2002)); Current Protocols In Molecular Biology (FM Ausubel, et al. eds., (1987)); the series Methods in Enzymology (Academic Press, Inc.): PCR 2: A Practical Approach (MJ MacPherson, BD Hames and GR Taylor eds. (1995)); Harlow and Lane, eds. (1988) See Antibodies, A Laboratory Manual; Harlow and Lane, eds. (1999) Using Antibodies, A Laboratory Manual; Animal Cell Culture (RI Freshney, ed. (1987)); Zigova, Sanberg and Sanchez-Ramos, eds. (2002) Neural Stem Cells.

[0019] All numerical designations, e.g., pH, temperature, time, concentration, and molecular weight, including ranges, are approximate values ​​that vary (+) or (-) by increments of 0.1 or 1, as appropriate. It is to be understood, although not always explicitly stated, that all numerical designations are preceded by the term "about". The term "about" also includes the exact value "X", as well as small increments of "X", such as "X+0.1 or 1" or "X-0.1 or 1", as appropriate. It is also to be understood, although not always explicitly stated, that the reagents described herein are merely exemplary, and that equivalents of such are known in the art.

[0020] definition As used in this specification and claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a cell" includes a plurality of cells, including mixtures thereof.

[0021] As used herein, the term "comprising" is intended to mean that the compositions and methods include the recited elements but do not exclude others. "Consisting essentially of," when used to define compositions and methods, is intended to mean excluding other elements of any essential importance to the combination for the described purpose. Thus, a composition consisting essentially of elements as defined herein does not exclude trace contaminants from isolation and purification methods, as well as pharma- ceutically acceptable carriers, e.g., phosphate buffered saline, preservatives, and the like. "Consisting of" is intended to mean more than trace elements of other components, and to exclude substantial method steps for administering the compositions of the invention, or process steps for producing the compositions or achieving the intended results. Embodiments defined by each of these transitional phrases are within the scope of the present invention.

[0022] The term "isolated" as used herein with respect to a nucleic acid, such as a cell, DNA or RNA, refers to a molecule separated from other DNA or RNA, respectively, present in the natural source of the macromolecule. The term "isolated" as used herein also refers to a nucleic acid or peptide, when produced by recombinant DNA techniques, that is substantially free of cellular material, viral material, or culture medium, or chemical precursors or other chemicals, when chemically synthesized. Furthermore, "isolated nucleic acid" is intended to include nucleic acid fragments that do not naturally occur as fragments and are not found in the natural context. The term "isolated" is also used herein to refer to a cell or polypeptide that is isolated from other cellular proteins or tissues. Isolated polypeptides are intended to encompass both purified and recombinant polypeptides.

[0023] The term "isolated" when used in reference to cells, particularly stem cells, such as mesenchymal stem cells, refers to cells that are separated from other cells or tissues that are present in the natural tissue within the body.

[0024] "Subject," "individual," or "patient" are used interchangeably herein and refer to a vertebrate, such as a primate, mammal, or preferably a human. Mammals include, but are not limited to, horses, dogs, cows, sheep, mice, rats, monkeys, humans, farm animals, sport animals, and pets.

[0025] The term "allele", which is used interchangeably with "allelic variant" herein, refers to a general form of a gene or a part thereof. Alleles occupy the same locus or position on homologous chromosomes. If a subject has two identical alleles of a gene, the subject is said to be homozygous for the gene or allele. If a subject has two different alleles of a gene, the subject is said to be heterozygous for the gene. The alleles of a particular gene may differ from each other in a single nucleotide or several nucleotides, and may include nucleotide substitutions, deletions, and insertions. An allele of a gene may also be the form of the gene that contains a mutation.

[0026] "Cell", "host cell" or "recombinant host cell" are terms used interchangeably herein. Such terms are understood to refer not only to the particular subject cell, but also to the progeny or potential progeny of such a cell. Because certain modifications may occur in successive generations due to either mutation or environmental influences, such progeny may not actually be identical to the parent cell, but are still included within the scope of the terms as used herein.

[0027] "Amplifying," "amplifying," or "amplification" of a polynucleotide sequence includes methods such as traditional cloning methodologies, PCR, ligation amplification (or ligase chain reaction, LCR), or other amplification methods. These methods are known and practiced in the art. See, for example, U.S. Pat. Nos. 4,683,195 and 4,683,202, and Innis et al., (1990) Mol. Cell Biol. 10(11):5977-5982 (for PCR); and Wu et al. (1989) Genomics 4:560-569 (for LCR). In general, the PCR procedure describes a method of gene amplification that consists of (i) sequence-specific hybridization of primers to specific genes within a DNA sample (or library), (ii) subsequent amplification with multiple rounds of annealing, extension and denaturation using DNA polymerase, and (iii) screening of the PCR products for bands of the correct size. The primers used are oligonucleotides of sufficient length and appropriate sequence to effect initiation of polymerization, i.e., each primer is specifically designed to be complementary to each strand of the genomic locus to be amplified.

[0028] Reagents and hardware for carrying out PCR are commercially available. Primers useful for amplifying sequences from a particular region are preferably complementary to and specifically hybridize to sequences in the target region or its flanking regions. The nucleic acid sequences generated by amplification can be directly sequenced. Alternatively, the amplified sequence(s) can be cloned prior to sequence analysis. Methods for direct cloning and sequence analysis of enzymatically amplified genomic segments are known in the art.

[0029] The term "genotype" refers to the particular allelic composition of a cell as a whole, a particular gene, or a particular polynucleotide region of the genome, while the term "phenotype" refers to the detectable outward manifestation of a particular genotype.

[0030] As used herein, the term "gene" or "recombinant gene" refers to a nucleic acid molecule that contains an open reading frame and includes at least one exon sequence and, optionally, an intron sequence. A gene can also refer to a polymorphic or mutant form or allele of a gene.

[0031] "Homology" or "identity" or "similarity" refers to sequence similarity between two peptides or two nucleic acid molecules. Homology can be determined by comparing positions in each sequence that can be aligned for comparison purposes. If a position in the sequences being compared is occupied by the same base or amino acid, the molecules are homologous at that position. The degree of homology between sequences is a function of the number of matching or homologous positions shared by the sequences. An "unrelated" or "non-homologous" sequence shares less than 40% identity, but preferably less than 25% identity, with one of the sequences of the present invention.

[0032] A polynucleotide or polynucleotide region (or a polypeptide or polypeptide region) has a certain percentage (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99%) of "sequence identity" to another sequence, meaning that, when aligned, that percentage of bases (or amino acids) are the same when comparing the two sequences. This alignment and percent homology or sequence identity can be determined using software programs known in the art, such as those described in Ausubel et al. eds. (2007) Current Protocols in Molecular Biology. Preferably, default parameters are used for the alignment. One alignment program is BLAST using default parameters. In particular, the programs are BLASTN and BLASTP using the following default parameters: genetic code=standard; filter=none; strand=both; cutoff=60; expectation=10; matrix=BLOSUM62; description=50 sequences; sorting=by high score; database=non-redundant, GenBank+EMBL+DDBJ+PDB+GenBank CDS translation+SwissProtein+SPupdate+PIR. Details of these programs can be found at the following internet address: http: / / www.ncbi.nlm.nih.gov / blast / Blast.cgi, last accessed May 21, 2008. Biologically equivalent polynucleotides are polynucleotides that have a particular percent homology and encode polypeptides having the same or similar biological activity.

[0033] The term "equivalent nucleic acid" refers to a nucleic acid having a nucleotide sequence with a certain degree of homology with the nucleotide sequence of a nucleic acid or its complement. A homolog of a double-stranded nucleic acid is intended to include a nucleic acid having a nucleotide sequence with a certain degree of homology with its complement. In one aspect, a homolog of a nucleic acid can hybridize to a nucleic acid or its complement.

[0034] The term "interact" as used herein is intended to include detectable interactions between molecules, such as can be detected using a hybridization assay. The term interact is also intended to include "binding" interactions between molecules. The interactions can be, for example, protein-protein, protein-nucleic acid, or nucleic acid-nucleic acid in nature.

[0035] "Hybridization" refers to a reaction in which one or more polynucleotides react to form a hybridization complex stabilized by hydrogen bonds between the bases of nucleotide residues. Hydrogen bonds can occur by Watson-Crick base pairing, Hoogsteen binding, or in any other sequence-specific manner. The complex can include two strands forming a duplex structure, three or more strands forming a multistranded complex, a single self-hybridizing strand, or any combination of these. A hybridization reaction can constitute a step in a more extensive process, such as the initiation of a PCR reaction, or the enzymatic cleavage of a polynucleotide by a ribozyme.

[0036] Hybridization reactions can be carried out under conditions of different "stringency". Generally, low stringency hybridization reactions are carried out in a solution of about 40°C, about 10xSSC or equivalent ionic strength / temperature. Moderate stringency hybridizations are typically carried out in about 50°C, about 6xSSC, and high stringency hybridization reactions are generally carried out in about 60°C, about 1xSSC. Hybridization reactions can also be carried out under "physiological conditions", as known to those of skill in the art. Non-limiting examples of physiological conditions include the temperature, ionic strength, pH and Mg normally found in cells. 2+ Concentration.

[0037] When hybridization occurs between two single-stranded polynucleotides in an antiparallel configuration, the reaction is called "annealing" and the polynucleotides are referred to as "complementary". When hybridization can occur between one strand of a first polynucleotide and one strand of a second polynucleotide, the double-stranded polynucleotide can be "complementary" or "homologous" to another polynucleotide. "Complementarity" or "homology" (the degree to which one polynucleotide is complementary to another polynucleotide) can be quantified in terms of the ratio of bases in opposite strands that are expected to form hydrogen bonds with each other according to commonly accepted base pairing rules.

[0038] The term "mismatch" refers to a hybridized nucleic acid duplex that is not 100% homologous. The lack of overall homology can be due to deletions, insertions, inversions, substitutions, or frameshift mutations.

[0039] As used herein, the term "oligonucleotide" refers to polynucleotides, such as deoxyribonucleic acid (DNA) and, where appropriate, ribonucleic acid (RNA). The term should also be understood to include single-stranded (sense or antisense) and double-stranded polynucleotides, as equivalents, derivatives, variants, and analogs of either RNA or DNA made from nucleotide analogs, and as applicable to the described embodiments. Deoxyribonucleotides include deoxyadenosine, deoxycytidine, deoxyguanosine, and deoxythymidine. For the purpose of clarity, the terms "adenosine", "cytidine", "guanosine", and "thymidine" are used herein when referring to the nucleotides of a nucleic acid, which may be DNA or RNA. When the nucleic acid is RNA, it is understood that the nucleotide with a uracil base is uridine.

[0040] The terms "polynucleotide" and "oligonucleotide" are used interchangeably and refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides or their analogs. Polynucleotides can have any three-dimensional structure and can perform any function, known or unknown. The following are non-limiting examples of polynucleotides: genes or gene fragments (e.g., probes, primers, EST or SAGE tags), exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, dsRNA, siRNA, miRNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes and primers. Polynucleotides can contain modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure can be imparted before or after assembly of the polynucleotide. The sequence of nucleotides can be interrupted by non-nucleotide components. Polynucleotides can be further modified after polymerization, such as by conjugation with a label component. The term also refers to both double-stranded and single-stranded molecules. Unless otherwise specified or required, any embodiment of the invention that is a polynucleotide encompasses both the double-stranded form and each of the two complementary single-stranded forms that are known or predicted to constitute the double-stranded form.

[0041] A polynucleotide is composed of a specific sequence of the four nucleotide bases: adenine (A); cytosine (C); guanine (G); thymine (T); and, if the polynucleotide is RNA, uracil (U) in place of thymine. Thus, the term "polynucleotide sequence" is an alphabetical representation of a polynucleotide molecule. This alphabetical representation can be entered into a database in a computer having a central processing unit and used for bioinformatics applications such as functional genomics and homology searching. The term "polymorphism" refers to the coexistence of more than one form of a gene or part thereof. A part of a gene in which at least two different forms, i.e., two different nucleotide sequences, exist is referred to as a "polymorphic region of a gene." A polymorphic region can be a single nucleotide, the identity of which differs in different alleles.

[0042] A "composition" typically contemplates a combination of an active agent, e.g., stem cells or populations thereof, with a pharma- ceutically acceptable carrier, inert (e.g., detectable agent or label) or active, naturally occurring or non-naturally occurring carrier, e.g., adjuvants, diluents, binders, stabilizers, buffers, salts, lipid-soluble solvents, preservatives, adjuvants, and the like. Carriers also include pharmaceutical excipients and additives proteins, peptides, amino acids, lipids, and carbohydrates (e.g., sugars, including monosaccharides, dioligosaccharides, trioligosaccharides, tetraoligosaccharides, and oligosaccharides; derivatized sugars, e.g., alditols, aldonic acids, esterified sugars, and the like; and polysaccharides or sugar polymers), which may be present alone or in combination and comprise 1-99.99% by weight or volume, alone or in combination. Exemplary protein excipients include serum albumins, e.g., human serum albumin (HSA), recombinant human albumin (rHA), gelatin, casein, and the like. Representative amino acid components that may also function in a buffering capacity include alanine, arginine, glycine, arginine, betaine, histidine, glutamic acid, aspartic acid, cysteine, lysine, leucine, isoleucine, valine, methionine, phenylalanine, aspartame, etc. Carbohydrate excipients are also contemplated to be within the scope of the technology, examples of which include, but are not limited to, monosaccharides such as, for example, fructose, maltose, galactose, glucose, D-mannose, sorbose, etc.; disaccharides such as, for example, lactose, sucrose, trehalose, cellobiose, etc.; polysaccharides such as, for example, raffinose, melezitose, maltodextrin, dextran, starch, etc.; and alditols such as, for example, mannitol, xylitol, maltitol, lactitol, xylitol sorbitol (glucitol), and myo-inositol.

[0043] Compositions used according to the present disclosure, including cells, treatments, therapies, agents, drugs, and pharmaceutical preparations, may be packaged in dosage unit form for ease of administration and uniformity of dosage. The term "unit dose" or "dosage" refers to a physically discrete unit suitable for use in a subject, each unit containing a predetermined amount of the composition calculated to produce a desired response in association with its administration, i.e., the appropriate route and regimen. The amount administered, both according to the number of treatments and the unit dose, depends on the desired outcome and / or protection. The precise amount of the composition also depends on the physician's judgment and is unique to each individual. Factors affecting the dosage include the subject's physical and clinical condition, the route of administration, the intended purpose of the treatment (relief of symptoms vs. cure), and the efficacy, stability, and toxicity of the particular composition. Once formulated, the solution is administered in a manner compatible with the dosage formulation and in an amount that is therapeutically or prophylactically effective. The formulations are easily administered in a variety of dosage forms, such as the types of injectable solutions described herein.

[0044] As used herein, the term "administer" or "administration" or "administering" is intended to mean the delivery of a substance to a subject, such as an animal or human. Administration can be accomplished in one dose, continuously or intermittently throughout the course of treatment. Methods for determining the most effective means and dosage of administration are known to those of skill in the art and vary with the composition used for treatment, the purpose of the treatment, and the age, health, or sex of the subject being treated. Single or multiple administrations can be carried out with the dose level and pattern selected by the treating physician, or in the case of pets or animals, the treating veterinarian. Suitable dosage formulations and methods for administering agents are known in the art. Routes of administration can also be determined, and methods for determining the most effective route of administration are known to those of skill in the art and vary with the composition used for treatment, the purpose of the treatment, the health or disease stage of the subject being treated, and the target cell or tissue. Non-limiting examples of routes of administration include intrahippocampal, intravenous, intraarterial, intramuscular, intracardiac, intrathecal, subventricular, epidural, intracerebral, intraventricular, subretinal, intravitreal, intraarticular, intraocular, intraperitoneal, intrauterine, intradermal, subcutaneous, transdermal, transmucosal, and inhalation.

[0045] The agents of the present disclosure can be administered for treatment by any suitable route of administration. It will also be understood that the most suitable route will vary with the condition and age of the recipient, as well as the disease being treated.

[0046] "Gene delivery vehicle" is defined as any molecule that can carry polynucleotide to be inserted into host cell.Examples of gene delivery vehicle are liposome, biocompatible polymers including natural and synthetic polymers; lipoproteins; polypeptides; polysaccharides; lipopolysaccharides; artificial virus envelopes; metal particles; and bacteria or viruses such as baculovirus, adenovirus and retrovirus, bacteriophage, cosmids, plasmids, fungal vectors, and other recombinant vehicles typically used in the art, which are described for expression in various eukaryotic and prokaryotic hosts, and can be used for gene therapy and simple protein expression.

[0047] "Gene delivery", "gene transfer" and the like, as used herein, are terms that refer to the introduction of an exogenous polynucleotide (sometimes referred to as a "transgene") into a host cell, regardless of the method used for the introduction. Such methods include a variety of well-known techniques, such as vector-mediated gene transfer (e.g., by viral infection, sometimes referred to as transduction), transfection, transformation, or various other protein-based or lipid-based gene delivery complexes, as well as techniques that facilitate the delivery of "naked" polynucleotides (e.g., electroporation, "gene gun" delivery, and various other techniques used for the introduction of polynucleotides). Unless otherwise indicated, the terms transfected, transduced, or transformed may be used interchangeably herein to indicate the presence of an exogenous polynucleotide or a polypeptide expressed therefrom in a cell. The introduced polynucleotide may be stably or transiently maintained in the host cell. Stable maintenance typically requires that the introduced polynucleotide contains an origin of replication compatible with the host cell or is integrated into a host cell replicon, such as an extrachromosomal replicon (e.g., a plasmid), or a nuclear or mitochondrial chromosome. Several vectors are known that are capable of mediating the transfer of genes into mammalian cells, as known in the art and as described herein.

[0048] The term "express" refers to the production of a gene product. In some embodiments, the gene product is a polypeptide or a protein. In some embodiments, the gene product is an mRNA, tRNA, rRNA, miRNA, dsRNA, or siRNA.

[0049] A cell that "stably expresses" an exogenous polypeptide is one that continues to express a polypeptide encoded by an exogenous gene introduced into the cell after replication if the cell is dividing, or for more than a day, up to about a week, up to about two weeks, up to three weeks, up to four weeks, several weeks, up to a month, up to two months, up to three months, several months, up to a year, or longer.

[0050] A "viral vector" is defined as a recombinantly produced virus or virus particle that contains a polynucleotide to be delivered to a host cell either in vivo, ex vivo or in vitro. Examples of viral vectors include retroviral vectors, lentiviral vectors, adenoviral vectors, adeno-associated viral vectors, alphaviral vectors, and the like. Alphaviral vectors, such as Semliki Forest virus-based vectors and Sindbis virus-based vectors, have also been developed for use in gene therapy and immunotherapy. See Schlesinger and Dubensky (1999) Curr. Opin. Biotechnol. 5:434-439 and Ying, et al. (1999) Nat. Med. 5(7):823-827.

[0051] In an embodiment where gene transfer is mediated by a retroviral vector, the vector construct refers to a polynucleotide that includes a retroviral genome or a portion thereof and a therapeutic gene. As used herein, "retroviral-mediated gene transfer" or "retroviral transduction" have the same meaning and refer to the process whereby a gene or nucleic acid sequence is stably transferred into a host cell based on a virus that enters the cell and integrates its genome into the host cell genome. The virus can enter the host cell through its normal infection mechanism, or it can be modified to bind to a different host cell surface receptor or ligand to enter the cell. Retroviruses carry their genetic information in the form of RNA; however, when the virus infects a cell, the RNA is reverse transcribed into a DNA form that is integrated into the genomic DNA of the infected cell. The integrated DNA form is called a provirus. As used herein, retroviral vector refers to a viral particle that can introduce exogenous nucleic acid into a cell by a viral or viral-like entry mechanism. A "lentiviral vector" is a type of retroviral vector known in the art that has certain advantages in transducing non-dividing cells compared to other retroviral vectors. See Trono D. (2002) Lentiviral vectors, New York: Spring-Verlag Berlin Heidelberg.

[0052] In the embodiment where gene transfer is mediated by DNA virus vectors such as adenovirus (Ad) or adeno-associated virus (AAV), vector construct refers to a polynucleotide that includes the virus genome or a part thereof and transgene. Adenovirus (Ad) is a relatively well-characterized homogenous group of viruses that includes more than 50 serotypes. See, for example, International PCT Publication No. WO95 / 27071. Ad does not require integration into host cell genome. Recombinant Ad-derived vectors have also been constructed, particularly those that reduce the possibility of recombination and generation of wild-type virus. See International PCT Application Nos. WO95 / 00655 and WO95 / 11984. Wild-type AAV has high infectivity and specificity to be integrated into the genome of host cell. See Hermonat and Muzyczka (1984) Proc. Natl. Acad. Sci. USA 81:6466-6470, and Lebkowski, et al. (1988) Mol. Cell. Biol. 8:3988-3996.

[0053] Vectors that contain both a promoter and a cloning site to which a polynucleotide can be operatively linked are well known in the art. Such vectors can transcribe RNA in vitro or in vivo and are commercially available from sources such as Stratagene (La Jolla, CA) and Promega Biotech (Madison, WI). To optimize expression and / or in vitro transcription, it may be necessary to remove, add, or modify the 5' and / or 3' untranslated parts of the clone to eliminate extra, cryptic, inappropriate, alternative translation initiation codons, or other sequences that may interfere with or reduce expression at the transcription or translation level. Alternatively, a consensus ribosome binding site may be inserted immediately 5' to the initiation codon to enhance expression.

[0054] "Under transcriptional control" is a term well understood in the art and indicates that transcription of a polynucleotide sequence, usually a DNA sequence, is dependent on the sequence being operably linked to elements that contribute to or facilitate the initiation of transcription. "Operably linked" intends that the polynucleotide is positioned in a manner that allows it to function within the cell.

[0055] Gene delivery vehicles also include some non-viral vectors, including DNA / liposome complexes and targeted viral protein-DNA complexes.Liposomes that also contain targeting antibodies or fragments thereof can be used in the methods of the present invention.In order to enhance delivery to cells, the nucleic acid or protein of the present invention can be conjugated to antibodies or binding fragments thereof that bind cell surface antigens, such as cell surface markers found on stem cells.

[0056] "Probe", when used in the context of polynucleotide manipulation, refers to an oligonucleotide that serves as a reagent for detecting a target potentially present in a sample of interest by hybridizing with the target. Usually, a probe includes a label or a means for binding a label either before or after the hybridization reaction. Suitable labels are described and exemplified herein.

[0057] A "primer" is a short polynucleotide that generally has a free 3'-OH group that hybridizes with the target, thereby binding to a target or "template" potentially present in a sample of interest, and then facilitating the polymerization of a polynucleotide complementary to the target. "Polymerase chain reaction" ("PCR") is a reaction in which a duplicate copy is made from a target polynucleotide using a "pair of primers" or "set of primers" consisting of an "upstream" and a "downstream" primer, and a catalyst for polymerization, such as DNA polymerase and typically a thermostable polymerase enzyme. Methods for PCR are well known in the art and are taught, for example, in M. MacPherson et al. (1991) PCR: A Practical Approach, IRL Press at Oxford University Press. Collectively, the entire process of producing duplicate copies of a polynucleotide, such as PCR or gene cloning, is referred to herein as "replication." Primers can also be used as probes in hybridization reactions, such as Southern or Northern blot analysis. Sambrook et al., supra. Primers can optionally contain detectable labels, as exemplified and described herein.

[0058] As used herein, the term "label" refers to a directly or indirectly detectable compound or composition, e.g., a protein such as a polynucleotide or an antibody, conjugated directly or indirectly to the composition to be detected to produce a "labeled" composition. The term also includes sequences conjugated to a polynucleotide that provide a signal after expression of the inserted sequence, e.g., green fluorescent protein (GFP). The label may be detectable itself (e.g., radioisotope label or fluorescent label) or, in the case of an enzymatic label, may catalyze a chemical alteration of a substrate compound or composition that is detectable. The label may be suitable for small-scale detection or more suitable for high-throughput screening. Thus, suitable labels include, but are not limited to, radioisotopes, fluorescent dyes, chemiluminescent compounds, dyes, and proteins, including enzymes. The label may be simply detectable or quantifiable. A simply detected response generally includes a response whose presence is merely confirmed, whereas a quantified response generally includes a response that has a quantifiable (e.g., numerically reportable) value, such as intensity, polarization, and / or other property. In luminescent or fluorescent assays, the detectable response can be generated directly, using a luminophore or fluorophore associated with the assay component actually responsible for binding, or indirectly, using a luminophore or fluorophore associated with another (e.g., reporter or indicator) component.

[0059] Examples of luminescent labels that produce signals include, but are not limited to, bioluminescence and chemiluminescence. Detectable luminescent responses generally include a change in or the appearance of a luminescent signal. Suitable methods and luminophores for luminescently labeling assay components are known in the art and are described, for example, in Haugland, Richard P. (1996) Handbook of Fluorescent Probes and Research Chemicals (6th ed). Examples of luminescent probes include, but are not limited to, aequorin and luciferase.

[0060] Examples of suitable fluorescent labels include, but are not limited to, fluorescein, rhodamine, tetramethylrhodamine, eosin, erythrosine, coumarin, methyl-coumarin, pyrene, Malachite Green, stilbene, Lucifer Yellow, Cascade Blue™, and Texas Red. Other suitable optical dyes are described in Haugland, Richard P. (1996) Handbook of Fluorescent Probes and Research Chemicals (6th ed.).

[0061] In another embodiment, the fluorescent label is functionalized to facilitate covalent attachment to cellular components present within or on the surface of a cell or tissue, such as a cell surface marker. Suitable functional groups include, but are not limited to, isothiocyanate groups, amino groups, haloacetyl groups, maleimides, succinimidyl esters, and sulfonyl halides, all of which can be used to attach the fluorescent label to a second molecule. The choice of functional group of the fluorescent label depends on the site of attachment to either the linker, drug, marker, or second labeling agent.

[0062] Attachment of the fluorescent label can be direct to the cellular component or compound, or alternatively, can be via a linker. Suitable binding pairs for use in indirectly linking the fluorescent label to an intermediate include, but are not limited to, antigen / antibody, e.g., rhodamine / anti-rhodamine, biotin / avidin, and biotin / strepavidin.

[0063] The expression "solid support" refers to a non-aqueous surface such as a "culture plate", "gene chip" or "microarray". Such gene chips or microarrays can be used for diagnostic and therapeutic purposes by several techniques known to those skilled in the art. In one technique, oligonucleotides are attached and aligned to gene chips for determining DNA sequences by hybridization approaches, such as those outlined in U.S. Pat. Nos. 6,025,136 and 6,018,041. The polynucleotides of the invention can be modified into probes, which can then be used to detect gene sequences. Such techniques are described, for example, in U.S. Pat. Nos. 5,968,740 and 5,858,659. Probes can also be attached or affixed to electrode surfaces for electrochemical detection of nucleic acid sequences, as described by Kayem et al. in U.S. Pat. No. 5,952,172 and Kelley et al. (1999) Nucleic Acids Res. 27:4830-4837.

[0064] A variety of "gene chip" or "microarray" and similar technologies are known in the art. Examples of these include the LabCard (ACLARA Bio Sciences. Inc.); GeneChip (Affymetric, Inc.); LabChip (Caliper Technologies Corp); low density arrays with electrochemical sensing (Clinical Micro Sensors); LabCD System (Gamera Bioscience Corp.); Omni Grid (Gene Machines); Q Array (Genetix Ltd.); high throughput automated mass spectrometry system with liquid phase expression technology (Gene Trace Systems, Inc.); thermal jet spotting system (Hewlett Packard Company); Hyseq HyChip (Hyseq. Inc.); BeadArray (Illumina, Inc.); GEM (Incyte Microarray Systems); high throughput microarray system capable of distributing 12 to 64 spots on multiple glass slides (Intelligent Bio-Instruments); Molecular Biology Workstation and These include, but are not limited to, the NanoChip (Nanogen, Inc.); microfluidic glass chips (Orchid Biosciences, Inc.); BioChip Arrayer with four PiezoTip piezoelectric drop-on-demand chips (Packard Instruments, Inc.); FlexJet (Rosetta Inpharmatic, Inc.); MALDI-TOF mass spectrometer (Sequnome); ChipMaker 2 and ChipMaker 3 (TeleChem International, Inc.); and GenoSensor (Vysis, Inc.).Examples of "gene chips" or "microarrays" are also described in U.S. Patent Application Publication Nos. 2007 / 0111322, 2007 / 0099198, 2007 / 0084997, 2007 / 0059769, and 2007 / 0059765, as well as U.S. Patent Nos. 7,138,506, 7,070,740, and 6,989,267.

[0065] In one embodiment, a "gene chip" or "microarray" is prepared containing probes or primers homologous to the polynucleotides described herein. A suitable sample is obtained from the patient, and genomic DNA, RNA, protein, or any combination thereof is extracted and amplified as necessary. The sample is contacted with the gene chip or microarray panel under conditions suitable for hybridization of the gene of interest or gene product to the probe or primer contained in the gene chip or microarray. The probe or primer can be detectably labeled, thereby identifying the sequence of interest. Alternatively, chemical or biological reactions can be used to identify the probe or primer that has hybridized with the DNA or RNA of the gene of interest. The patient's genotype or phenotype is then determined with the aid of the aforementioned devices and methods.

[0066] A "pharmaceutical composition" is intended to include a combination of an active agent with a carrier, inert or active, that renders the composition suitable for diagnostic or therapeutic use in vitro, in vivo or ex vivo.

[0067] As used herein, the term "pharmaceutical acceptable carrier" includes any of the standard pharmaceutical carriers, such as phosphate buffered saline solution, water, and emulsions such as oil / water or water / oil emulsions, as well as various types of wetting agents. The composition may also include stabilizers and preservatives. For examples of carriers, stabilizers and adjuvants, see Martin (1975) Remington's Pharm. Sci., 15th Ed. (Mack Publ. Co., Easton).

[0068] For topical use, the pharma- ceutically acceptable carrier is suitable for preparing creams, ointments, jellies, gels, solutions, suspensions, etc. Such carriers are conventional in the art for topical administration, for example, using polyethylene glycol (PEG). These formulations may optionally contain additional pharma- ceutically acceptable ingredients, such as diluents, stabilizers, and / or adjuvants.

[0069] "Substantially homogeneous" refers to a population of cells in which more than about 50%, or alternatively more than about 60%, or alternatively more than 70%, or alternatively more than 75%, or alternatively more than 80%, or alternatively more than 85%, or alternatively more than 90%, or alternatively more than 95% of the cells are of the same or similar phenotype. The phenotype can be determined by expression of a preselected cell surface marker or other marker, such as myosin or actin, or a gene or protein, such as a calcium handling protein, a t-tubule protein, or alternatively a calcium pump protein. In another embodiment, a substantially homogeneous population has a normal expression level that is reduced (e.g., less than about 95%, or alternatively less than about 90%, or alternatively less than about 80%, or alternatively less than about 75%, or alternatively less than about 70%, or alternatively less than about 65%, or alternatively less than about 60%, or alternatively less than about 55%, or alternatively less than about 50%) than a wild-type counterpart cell or tissue.

[0070] "Neurodegenerative diseases" are conditions in which cells are lost in the brain and spinal cord. Examples of neurodegenerative diseases include, but are not limited to, Alzheimer's disease.

[0071] An "effective amount" is an amount sufficient to effect a beneficial or desired result. An effective amount can be administered in one or more administrations, applications, or dosages, and can be determined empirically by one of skill in the art.

[0072] As used herein, "treating" and "treatment" of a disease in a subject refer to (1) preventing symptoms or disease from occurring in a subject who is susceptible to the disease or who has not yet shown symptoms; (2) inhibiting or arresting the onset of the disease; or (3) relieving or causing regression of the disease or symptoms of the disease. As understood in the art, "treatment" is an approach to obtain beneficial or desired results, including clinical results. For the purposes of the present technology, beneficial or desired results can include, but are not limited to, one or more of the following: alleviation or amelioration of one or more symptoms, whether detectable or undetectable, reduction in the severity of a condition (including disease), stabilized (i.e., not worsening) status of a condition (including disease), delay or slowing of progression of a condition (including disease), remission or remission of the status of a condition (including disease), and remission (whether partial or total).

[0073] A "control" is an alternative subject or sample used in an experiment for comparison purposes. A control can be "positive" or "negative". For example, if the purpose of an experiment is to determine the correlation of a mutated allele with a particular phenotype, it is generally preferable to use a positive control (a sample from a subject that has such a mutation and exhibits the desired phenotype) and a negative control (a subject or a sample from a subject that lacks the mutated allele and lacks the phenotype).

[0074] As used herein, "stem cells" define cells that have the ability to divide indefinitely in culture and give rise to specialized cells. At this point, and for convenience, stem cells are classified as somatic (adult) or embryonic. Somatic stem cells are undifferentiated cells found in differentiated tissues that can regenerate themselves (clonal) and differentiate (with certain limitations) to give rise to all of the specialized cell types of the tissue from which they were derived. Embryonic stem cells are primitive (undifferentiated) cells derived from an embryo that have the potential to become a wide range of specialized cell types. Embryonic stem cells are cells that have been cultured under in vitro conditions that allow them to grow without differentiation for months to years. Non-limiting examples of embryonic stem cells are the HES2 (also known as ES02) cell line available from ESI, Singapore, and the H1 (also known as WA01) cell line available from WiCells, Madison, WI. Pluripotent embryonic stem cells can be distinguished from other types of cells by the use of markers including, but not limited to, Oct-4, alkaline phosphatase, CD30, TDGF-1, GCTM-2, Genesis, germ cell nuclear factor, SSEA1, SSEA3, and SSEA4.

[0075] "Mesenchymal stem cells" or MSCs are multipotent stem cells that can differentiate into a variety of cell types. The designation MSC also refers to the term "bone marrow stromal cells." Cell types into which MSCs have been shown to differentiate in vitro or in vivo include osteoblasts, chondrocytes, myocytes, and adipocytes. Mesenchyme is the embryonic connective tissue that originates from the mesoderm and differentiates into hematopoietic and connective tissues, but MSCs do not differentiate into hematopoietic cells. Stromal cells are connective tissue cells that form the supporting structure in which the functional cells of the tissue reside. Although this is an accurate description of one function of MSCs, the term does not convey the relatively recently discovered role of MSCs in tissue repair.

[0076] Hematopoietic stem and progenitor cells (HSPCs) are intended precursor cells with the capacity for self-renewal and multilineage differentiation. In the bone marrow (BM), HSPCs are responsible for blood cell homeostasis. In addition, they also replenish tissue-resident myeloid cells and they can directly participate in the innate immune response upon homing to peripheral tissues. The cells can be isolated from mobilized peripheral blood or umbilical cord blood. See, for example, Bujko et al. (2019) “Hematopoietic Stem and Progenitor Cells (HSPCs)” Adv. Exp. Med. Biol. Adv Exp Med Biol. 2019;1201:49-77. doi: 10.1007 / 978-3-030-31206-0_3. PMID: 31898781. The cells are characterized by the marker profile CD34 + The method of expanding and culturing such cells is known in the art.See, for example, Yadav et al. (2020), Int. J. Stem Cells, Vol. 13(3):326-334, available at https: / / www.ncbi.nlm.nih.gov / pmc / articles / PMC7691860 / .

[0077] "Neural or neuronal stem cells" as used herein refer to cells that have the ability to self-renew and give rise to multiple specialized cell types of the nervous system. In some embodiments, neural stem cells are multipotent neural stem Y cells in the subventricular zone (SVZ) of the forebrain lateral ventricle (LV).

[0078] A clone or "clonal population" is a line of cells genetically identical to an origin cell, in this case a stem cell. "Progenitor" or "progenitor cell" is intended to mean a cell that has the ability to differentiate into a specific type of cell. A progenitor cell may be a stem cell. A progenitor cell may be more specific than a stem cell. A progenitor cell may be unipotent or multipotent. Compared to adult stem cells, progenitor cells may be at a more advanced stage of cell differentiation. Progenitor cells are often found in adult organisms, where they act as a repair system for the body. Examples of progenitor cells include, but are not limited to, satellite cells found in muscle, intermediate progenitor cells formed in the subventricular zone, bone marrow stromal cells, periosteal progenitor cells, pancreatic progenitor cells, and hemangioblasts or endothelial progenitor cells. Examples of progenitor cells may also include, but are not limited to, ependymal cells and neural stem cells derived from the forebrain lateral ventricle (LV).

[0079] The term "propagating" means growing or changing the phenotype of a cell or population of cells. The term "growing" refers to the proliferation of cells in the presence of a support medium, nutrients, growth factors, support cells, or any chemical or biological compounds necessary to obtain a desired number of cells or cell type. In one embodiment, the growth of cells results in the regeneration of a tissue.

[0080] The term "culturing" refers to the in vitro propagation of cells or organisms on or in various types of media. It is understood that the progeny of cells grown in culture may not be completely identical (i.e., morphologically, genetically or phenotypically) to the parent cell. By "expanded" is meant any proliferation or division of cells.

[0081] "Clonal expansion" refers to the growth of a population of cells by the successive division of a single cell into two identical daughter cells and / or populations of identical cells.

[0082] As used herein, the "lineage" of a cell defines the inheritance of the cell, i.e., its predecessors and progeny. The lineage of a cell places the cell within a genetic scheme of development and differentiation.

[0083] A derivative of a cell or cell population is a daughter cell of an isolated cell or cell population. Derivatives include expanded clonal or differentiated cells cultured and propagated from an isolated stem cell or stem cell population. Derivatives also include previously derived stem cells or stem cell populations. In one aspect, the cell or population is described as "donor-derived."

[0084] "Differentiation" refers to the process by which unspecialized cells acquire the characteristics of specialized cells, such as cardiac, hepatic or muscular cells. "Directed differentiation" refers to the manipulation of stem cell culture conditions to induce differentiation into a specific cell type. "Dedifferentiated" defines a cell that reverts to a less committed position within the lineage of a cell. As used herein, the term "differentiating or differentiated" defines a cell that assumes a more committed ("differentiated") position within the lineage of a cell. As used herein, "a cell that differentiates into a mesodermal (or ectodermal or endodermal) lineage" defines a cell that becomes committed to a specific mesodermal, ectodermal or endodermal lineage, respectively. Examples of cells that differentiate into mesodermal lineages or give rise to specific mesodermal cells include, but are not limited to, cells that are adipogenic, leiomyogenic, chondrogenic, cardiogenic, dermal, hematopoetic, hemangiogenic, myogenic, nephrogenic, urogenitogenic, osteogenic, pericardiogenic or interstitial.

[0085] As used herein, "pluripotent cell" defines a less differentiated cell that can give rise to at least two distinct (genotypically and / or phenotypically) further differentiated progeny cells. In another embodiment, "pluripotent cell" includes induced pluripotent stem cells (iPSCs), which are stem cells artificially derived from non-pluripotent cells, typically adult somatic cells, and are produced by inducing the expression of one or more stem cell-specific genes. Such stem cell-specific genes include, but are not limited to, the family of octamer transcription factors, i.e., Oct-3 / 4; the family of Sox genes, i.e., Sox1, Sox2, Sox3, Sox15 and Sox18; the family of Klf genes, i.e., Klf1, Klf2, Klf4 and Klf5; the family of Myc genes, i.e., c-myc and L-myc; the family of Nanog genes, i.e., OCT4, NANOG and REX1; or LIN28. Examples of iPSCs are described in Takahashi K. et al. (2007) Cell advance online publication 20 November 2007; Takahashi K. & Yamanaka S. (2006) Cell 126:663-76; Okita K. et al. (2007) Nature 448:260-262; Yu, J. et al. (2007) Science advance online publication 20 November 2007; and Nakagawa, M. et al. (2007) Nat. Biotechnol. Advance online publication 30 November 2007.

[0086] "Multilineage stem cells" or "multipotent stem cells" refer to stem cells that regenerate themselves and at least two further differentiated progeny cells from distinct developmental lineages. The lineages can be from the same germ layer (i.e., mesoderm, ectoderm, or endoderm) or from different germ layers. Examples of two progeny cells with distinct developmental lineages derived from the differentiation of a multilineage stem cell are myogenic cells and adipogenic cells (both of mesodermal origin but give rise to different tissues). Another example is neurogenic cells (of ectodermal origin) and adipogenic cells (of mesodermal origin).

[0087] A population of cells contemplates a collection of more than one cell that may be phenotypically and / or genotypically identical (clonal) or non-identical.

[0088] Amyloid beta precursor protein (APP) is an integral membrane protein expressed in many tissues and concentrated at neuronal synapses. It functions as a cell surface receptor and has been implicated as a regulator of synapse formation, neuroplasticity, antimicrobial activity, and iron efflux. APP is the precursor molecule whose proteolysis gives rise to amyloid beta (Aβ), a polypeptide containing 37-49 amino acid residues, the amyloid fibril form of which is the main constituent of amyloid plaques found in the brains of Alzheimer's disease patients. The protein sequence can be found in UniProt P05067 (human) and P12023 (mouse), or under NCBI reference numbers NP000745 (human), NP001129488 (human), or NP001185752 (mouse), or NP001185753 (mouse). The mRNA encoding this protein can be found under NCBI reference numbers NM201414 or NM000484 (human) or NM001198823 or NM001198824 (mouse), all last accessed on April 1, 2023.

[0089] The gene for microtubule-associated protein tau (MAPT) provides instructions for making a protein called tau, which is found throughout the nervous system, including nerve cells (neurons) in the brain. Tau is involved in the assembly and stabilization of microtubules, which are rigid hollow fibers that make up the structural framework (cytoskeleton) of cells. Microtubules help cells maintain their shape, aid in the process of cell division, and are essential for the transport of materials within cells. Six different versions (isoforms) of tau protein are produced in the adult brain. The isoforms vary in length, from 352 to 441 protein building blocks (amino acids). The region of the protein called the microtubule-binding domain, the part of the protein that attaches (binds) to microtubules, also varies between isoforms. In three of the isoforms, the microtubule-binding domain contains three repeated segments. In the other three isoforms, this domain contains four repeated segments. Typically, the brain has almost equal amounts of the 3-repeat and 4-repeat isoforms. This balance is thought to be essential for normal functioning of neurons. See https: / / medlineplus.gov / genetics / gene / mapt / , last accessed April 1, 2023.

[0090] The gene encoding the MAPT transcript undergoes complex regulated alternative splicing, giving rise to several mRNA species. MAPT transcripts are differentially expressed in the nervous system depending on the maturation stage of neurons and the type of neurons. Mutations in the MAPT gene have been associated with several neurodegenerative disorders, such as Alzheimer's disease, Pick's disease, frontotemporal dementia, corticobasal degeneration, and progressive supranuclear palsy. Amino acid and polynucleotide sequence information can be found at https: / / www.ncbi.nlm.nih.gov / gene / 4137, last accessed April 1, 2023.

[0091] The presenilin 1 (PSEN1) protein is one part (subunit) of a complex called gamma-(γ-)secretase. Presenilin 1 performs the main function of the complex, which is to split (cleave) other proteins into smaller pieces called peptides. This process is called proteolysis, and presenilin 1 is described as the proteolytic subunit of γ-secretase. The γ-secretase complex plays a role in the processing of amyloid precursor protein (APP) made in the brain and other tissues. γ-secretase cleaves APP into smaller peptides, including soluble amyloid precursor protein (sAPP) and several versions of amyloid-beta (β) peptide. Evidence suggests that sAPP has growth-promoting properties and may play a role in the formation of nerve cells (neurons) in the brain both before and after birth. Other functions of sAPP and amyloid-β peptide are under investigation. See https: / / medlineplus.gov / genetics / gene / psen1 / . The amino acid and polynucleotide sequences of the protein and gene can be found at https: / / www.ncbi.nlm.nih.gov / nuccore / ?term=Presenilin+1+(PSEN1), last accessed April 1, 2023.

[0092] Triggering receptor expressed on myeloid cells 2 (Trem2) is a protein that in humans is encoded by the TREM2 gene. It is expressed in immune cells in the central nervous system: macrophages, immature monocyte-derived dendritic cells, osteoclasts, and microglia. In the liver, TREM2 is expressed by several cell types, including macrophages, that respond to injury. In the intestine, TREM2 is expressed by bone marrow-derived dendritic cells and macrophages. TREM2 is overexpressed in many tumor types and has anti-inflammatory activity. The amino acid and polynucleotide sequences of the protein and gene can be found at https: / / www.ncbi.nlm.nih.gov / nuccore / ?term=Triggering+receptor+expressed+on+myeloid+cells+2+(Trem2), last accessed April 1, 2023.

[0093] Forms for carrying out disclosure

[0094] Applicant provides a method for treating a neurodegenerative disease, neurodegenerative disorder, or symptom thereof in a subject in need thereof, by administering to the subject an effective amount of hematopoietic stem and progenitor cells (HSPCs) or a population of HSPCs, thereby treating the neurodegenerative disease, neurodegenerative disorder, or symptom in the subject, wherein the neurodegenerative disease, neurodegenerative disorder, or symptom is selected from the group of: alleviating or treating Alzheimer's Disease (AD) associated with microglial inflammation; reducing Aβ plaque burden in the hippocampus and cortex; promoting or differentiating microglia; promoting differentiation of donor hematopoietic stem and progenitor cells, or a donor cell population comprising hematopoietic stem and progenitor cells, into microglia; or reducing neuroinflammation. The HSPCs or population thereof, in one embodiment, are "donor" or donor-derived HSPCs or population thereof, in that they are delivered or donated to the subject.

[0095] As used herein, the term "neurodegenerative disease or disorder" refers to a condition in which cells of the brain and spinal cord are lost. Examples of neurodegenerative diseases include Alzheimer's disease, Parkinson's disease, prion disease, amyotrophic lateral sclerosis, motor neuron disease, Huntington's disease, spinocerebellar ataxia, and dementia. By alleviating or treating a disease, we mean reducing, or in some embodiments eradicating, clinical markers or characteristics of the disease. By treating a neurodegenerative condition, we mean physical or psychological problems experienced by a subject that may indicate that the subject has or is suffering from a disease or condition. The symptoms may not be identified or manifested by medical tests. Non-limiting examples of symptoms of neurological disease include dementia, memory loss, balance, numbness, or other cognitive decline.

[0096] Provided herein is a method of reducing or treating Alzheimer's disease (AD) associated with microglial inflammation in a subject in need thereof, comprising, consisting essentially of, or consisting of administering to the subject hematopoietic stem or progenitor cells (HSPCs) or a population of HSPCs, thereby reducing or treating AD associated with microglial inflammation. In one embodiment, an effective amount of HSPCs or a population thereof is administered.

[0097] Alzheimer's disease (AD) is the most common cause of dementia and the most common age-related neurodegenerative disorder. AD results in dementia that progresses through disease progression to severe disability and typically results in death within 5-12 years of onset. Symptoms of AD include neuronal degradation, inflammation, and progressive memory and behavioral decline resulting from the accumulation of extracellular β-amyloid (Aβ) plaques and hyperphosphorylated tau in neurofibrillary tangles (NFTs) throughout the brain, primarily in the hippocampus and cortex. These key features of AD result in increased oxidative stress, inflammation, synaptic loss, and cholinergic dysfunction, leading to neuronal dystrophy.

[0098] As will be clear to those skilled in the art, the therapeutic methods of the present disclosure can be combined with diagnostic methods for initial evaluation of the subject to be treated and then to determine whether the treatment has been successful. Symptoms that can be used for initial evaluation and to monitor treatment include personality or behavioral changes, memory dysfunction, cognitive decline, mental status step, neuropsychological tests, and laboratory tests, such as amyloid and tau protein tests that can be measured in cerebrospinal fluid. The ratio of these proteins can help determine whether Alzheimer's disease is present. Alzheimer's disease can result in progressive loss of brain cells, which can be evaluated with brain scans such as magnetic resonance imaging (MRI), computed tomography (CT), and positron emission tomography (PET). PET scans use radioactive substances known as tracers to detect substances in the body. There are various types of PET scans. The most commonly used PET scan is the fluorodeoxyglucose (FDG) PET scan. This scan can identify brain areas with reduced glucose metabolism. Patterns of metabolic changes can distinguish between different types of degenerative brain diseases. PET scans have recently been developed that detect clusters of amyloid proteins (plaques) or tau (neurofibrillary tangles) associated with Alzheimer's dementia. These types of PET scans are typically used in research settings.

[0099] In another embodiment, the HSPC or population of HSPCs are wild type. + In other embodiments, the HSPC or population of HSPCs is genetically modified to correct genes associated with neurodegenerative disorders. The cells in the population are substantially homogenous HSPCs or clonal populations, such as CD34 + They may be HSPCs. They may be autologous or allogeneic to the subject being treated. Methods for preparing substantially homogeneous or clonal populations are known in the art.

[0100] In another embodiment, in the population of HSPC cells, at least 80%, or at least 90%, or at least 95%, or at least 97% of the cells in the population are CD34+ HSPCs.

[0101] The HSPCs or populations thereof may be administered as a composition together with one or more pharma- ceutically acceptable carriers.

[0102] In further embodiments, the HSPC or population of HSPCs is autologous or allogeneic to the subject.

[0103] In one embodiment, Alzheimer's disease is caused by a mutation in a known causative gene, possibly a gene encoding amyloid beta precursor protein (APP), microtubule-associated protein tau (MAPT), presenilin 1 (PSEN1), or triggering receptor 2 (Trem2) expressed in myeloid cells. In another embodiment, HSPCs or a population of HSPCs are modified to correct known familial mutations in APP, MAPT, PSEN1, and TREM2, etc. Gene correction can be performed by a method selected from gene addition, optionally by using a vector such as an adeno-associated virus vector or lentivirus vector, CRISPR / Cas9 technology, or prime editing, i.e., by correcting a single base pair in the defective gene.

[0104] In embodiments, the HSPCs comprise one or more of exogenous or wild-type genes selected from genes encoding amyloid beta precursor protein (APP), microtubule-associated protein tau (MAPT), presenilin 1 (PSEN1), or triggering receptor 2 expressed in myeloid cells (Trem2). The cells can be modified by any method known in the art, such as those described herein.

[0105] In one aspect of the method, the method reduces disorientation, loss of physical function, neuroinflammation, or cognitive impairment.

[0106] In some embodiments, the cell or a gene that is altered within the cell, such as genes encoding amyloid beta precursor protein (APP), microtubule-associated protein tau (MAPT), presenilin 1 (PSEN1), or triggering receptor 2 (Trem2) expressed in myeloid cells, is detectably labeled.

[0107] The cell or cell population can be administered systemically or locally. In one embodiment, HSPC or HSPC population is administered via the hippocampus. In another embodiment, they are administered systemically. The cell or cell population can be combined with a carrier, such as a pharmaceutically acceptable carrier, for the purpose and route of administration. The dosage, dosing schedule, and administration route can be determined by the treating physician or veterinarian, and can be determined taking into account the health, age, and sex of the subject.

[0108] The subject to be treated can be a mammal, for example, a mouse, a dog, a cat, a cow, a horse, or a human patient.If the subject is a non-human mammal, the method is useful for treating the non-human mammal or for use as an animal model for testing new combination therapy.As will be clear to those skilled in the art, a control animal that has not been treated can be used as a reference point.The method can be combined with other treatments for neurological disorders, such as Alzheimer's disease.

[0109] Applicant provides a method for treating or alleviating symptoms of AD in a subject in need thereof by administering to the subject an effective amount of hematopoietic stem and progenitor cells (HSPCs) or a population of HSPCs. The HSPCs or populations, in one embodiment, are "donor" or donor-derived HSPCs or populations, in that they are delivered or donated to the subject. Administration may be local or systemic. The cells may be autologous or allogeneic to the subject being treated and may be wild-type HSPCs. The HSPCs or populations, in one embodiment, are CD34+ and may be delivered in a pharma- ceutically acceptable carrier, if desired. When a population is administered, the population may be substantially homogenous or clonal for HSPCs or CD34+ HSPCs. The method may be combined with other therapeutic or diagnostic methods known in the art or described herein. One skilled in the art may monitor the treatment and assess when the treatment has been effective based on clinical or observational criteria. Effective treatments vary depending on the subject being treated and the purpose of the treatment.

[0110] Applicant provides a method for treating or alleviating symptoms of AD in a human or mammal in need thereof by administering to the subject an effective amount of hematopoietic stem and progenitor cells (HSPCs) or a population of HSPCs, respectively, to the human subject or mammal. The HSPCs or populations, in one embodiment, are "donor" or donor-derived HSPCs or populations, in that they are delivered or donated to the human or mammal. The administration is systemic and the cells are autologous to the human or mammal being treated. The HSPCs or populations are CD34+ and can be delivered in a pharma- ceutically acceptable carrier, if desired. When a population is administered, the population can be substantially homogenous or clonal for HSPCs or CD34+ HSPCs. The method can be combined with other therapeutic or diagnostic methods known in the art or described herein. The skilled artisan can monitor the treatment and assess when the treatment has been effective based on clinical or observational criteria. Effective treatments vary depending on the human or mammal being treated and the purpose of the treatment.

[0111] Applicants provide methods for reducing Aβ plaque burden in the hippocampus and cortex by administering to the subject an effective amount of hematopoietic stem and progenitor cells (HSPCs) or a population of HSPCs, which in one embodiment are "donor" or donor-derived HSPCs or a population of HSPCs, in that they are delivered or donated to the subject.

[0112] Applicants provide methods for promoting or reducing differentiating microglia by administering to the subject an effective amount of hematopoietic stem and progenitor cells (HSPCs) or a population of HSPCs, which in one embodiment are "donor" or donor-derived HSPCs or a population of HSPCs, in that they are delivered or donated to the subject.

[0113] Applicants provide methods for promoting the differentiation of hematopoietic stem or progenitor cells (HSPCs) of a donor, or a donor cell population comprising hematopoietic stem or progenitor cells, into microglia by administering to the subject an effective amount of the HSPCs or population of HSPCs to the subject. The HSPCs or population of HSPCs, in one embodiment, are "donor" or donor-derived HSPCs or population of HSPCs, in that they are delivered or donated to the subject.

[0114] Applicants provide methods for reducing neuroinflammation by administering to a subject an effective amount of hematopoietic stem and progenitor cells (HSPCs) or a population of HSPCs, which in one embodiment are "donor" or donor-derived HSPCs or a population of HSPCs, in that they are delivered or donated to the subject.

[0115] In each of these methods, the HSPC or population of HSPCs is wild type. + The cells within the population are substantially homogenous HSPCs or clonal populations, e.g., CD34 +They may be HSPCs. They may be autologous or allogeneic to the subject being treated. Methods for preparing substantially homogeneous or clonal populations are known in the art.

[0116] In another embodiment, in the population of HSPC cells, at least 80%, or at least 90%, or at least 95%, or at least 97% of the cells in the population are CD34+ HSPCs.

[0117] The HSPCs or populations thereof may be administered as a composition together with one or more pharma- ceutically acceptable carriers.

[0118] The method can be combined with other therapeutic or diagnostic methods known in the art or described herein. Those skilled in the art can monitor the treatment and evaluate when the treatment is effective based on clinical or observational criteria. Effective treatments vary depending on the subject being treated and the purpose of the treatment.

[0119] In further embodiments, the HSPC or population of HSPCs is autologous or allogeneic to the subject.

[0120] In each of these methods, the subject to be treated can be a mammal, such as a mouse, dog, cat, cow, horse, or human patient. When the subject is a non-human mammal, the method is useful for treating the non-human mammal or for use as an animal model for testing new combination therapies. As will be apparent to those skilled in the art, a control animal that has not been treated can be used as a reference point.

[0121] Kits are also provided that include genetically modified HSPCs that contain one or more exogenous or wild-type genes selected from genes encoding amyloid beta precursor protein (APP), microtubule-associated protein tau (MAPT), presenilin 1 (PSEN1), or triggering receptor expressed in myeloid cells 2 (Trem2), and instructions for use. EXAMPLES

[0122] experiment

[0123] Without being bound by theory, applicants hypothesized that transplantation of wild-type (WT) HSPCs may result in the generation of healthy microglia that may result in reduced neuroinflammation and Aβ plaque accumulation in the most affected areas of the AD brain. To examine this hypothesis, applicants used a 5xFAD double transgenic mouse model expressing mutant human amyloid beta (A4) precursor protein 695 (App) with the Swedish (K670N, M671L), Florida (I716V), and London (V717I) familial Alzheimer's disease (FAD) mutations and human presenilin 1 (Psen1) with two FAD mutations (M146L and L286V). By 2 to 4 months of age, accumulation of Aβ plaques in the hippocampus and cortex of 5xFAD mice is observed. 13、14 These mice exhibit significant neurocognitive dysfunction and anxiety-related behavioral changes between 3 and 6 months of age. 15 Furthermore, 5xFAD mice display key features of AD amyloid plaque pathology, including dysfunctional microglia. 13、16 .

[0124] Here, applicants demonstrate that a single WT HSPC transplant into adult 5xFAD mice resulted in the preservation of memory and neurocognitive abilities and a reduction in Aβ plaque burden in the hippocampus and cortex. Donor HSPC differentiate into microglia, which have the ability to clear active amyloid plaques, while also reducing neuroinflammation. This study paves the way for new therapeutic approaches using HSPC gene therapy for the treatment of AD, its respective associated neurodegenerative disorders and symptoms.

[0125] material and method

[0126] Animals. All mice were on a C57BL6 background. Applicants used the 5xFAD transgenic mouse model (Tg6799, C57BL6-Tg(APPSwFlLon, PSEN1 * M146L * L286V)6799 Vas, Jackson Laboratory) 13 Standard PCR reactions were performed using the following primers: 5'-ACCCCCATGTCAGAGTTCCT-3' (common forward), 5'-CGGGCCTCTTCGCTATTAC-3' (mutant reverse), and 5'-TATACAACCTTGGGGGATGG-3' (WT reverse) to identify mice expressing all five mutations. GFP transgenic mice, ubiquitously expressing enhanced GFP complementary DNA under the control of the chicken beta-actin promoter, were used as donors for HSPC transplantation experiments (C57Bl / 6-Tg(CAG-EGFP)1Osb / J; 003291, Jackson Laboratory). Mice were maintained in a temperature- and humidity-controlled animal facility with a 12-hour light / dark cycle and free access to water and food. Both male and female mice were used in all experiments. All mice were housed in the University of California, San Diego (UCSD) vivarium, and all protocols were approved by the UCSD Institutional Animal Care and Use Committee.

[0127] Isolation, transduction, transplantation, and engraftment of HSPCs. Bone marrow cells were flushed from the femurs of 6- to 8-week-old 5xFAD GFP transgenic mice. HSPCs were isolated by immunomagnetic separation using anti-Sca1 antibodies conjugated to magnetic beads (Miltenyi Biotec). 2 × 10 6-HT1C1 cells resuspended in 100 ml of phosphate-buffered saline (PBS) were cultured at 37 °C for 1 h. 6 Sca1 cells were transduced by tail vein injection into lethally irradiated (7 Gray; X-Rad 320, PXi) 5xFAD mice. + HSPCs were directly transplanted. WT GFP +In mice that received HSPCs, myeloid cell engraftment of transplanted cells was measured in peripheral blood 2 months after transplantation. Freshly collected blood samples from the tail were treated with red blood cell lysis buffer (eBioscience) and then analyzed by flow cytometry (BD Accuri C6, BD Biosciences) to detect GFP-GFP cells. + The percentage of cells was determined.

[0128] Behavioral testing. Mice were tested at 6 months of age using three main behavioral tests to assess neurocognitive and locomotor activity. Prior to behavioral testing, mice were exposed to the testing apparatus and testing room to acclimate to the environment. Applicants then performed and analyzed the NORT as previously published. 55、56 Briefly, all mice were exposed ad libitum to two replicates of the same object for 10 min during the familiarization phase and then returned to their home cage. After 24 h, one object remained the familiar object while the other was replaced with a novel object. The whole process was recorded by an overhead video camera and object investigation times were recorded manually by a blinded investigator. The elevated plus maze test is one of the most widely used tests to measure anxiety-like behavior in mice. Applicant conducted this EPM using a protocol as previously published 57 Briefly, the elevated plus maze has a "+" shape with two alternating open arms and two alternating closed arms extending from a central platform. Each mouse was placed in the central field and allowed to explore the maze for 5 min. The time spent in the open and closed arms was recorded. Each animal was placed in the center of the open field arena (50 cm x 50 cm) and allowed to move freely for 30 min, with movement recorded by an overhead camera. The footage is then analyzed by an automated tracking system (ANY-Maze).

[0129] Immunohistochemistry. Brains were collected from euthanized mice, fixed using 10% formalin, embedded in paraffin wax, and sectioned at 5 μm thickness by standard methods. Sections were deparaffinized and stained with mouse anti-6E10 antibody (1:30,000, 803001, BioLegend), rabbit anti-GFP antibody (1:600, ab183734, Abcam), and rabbit anti-Iba-1 antibody (1:3000, SAG4318, Wako). Slides were stained with Ventana Discovery Ultra (Ventana Medical Systems). Antigen retrieval was performed using CC1 (Tris-EDTA-based, pH 8.6) at 95°C for 40 min. Primary antibodies were incubated at 37°C for 32 min. For 6E10, an additional rabbit anti-mouse IgG antibody (1:6000, ab133469, Abcam) was utilized to minimize mouse-on-mouse nonspecific staining. All rabbit antibodies were detected using HRP-conjugated goat anti-rabbit antibody (OmniMap system, Ventana, Cat. No. 760-4315) incubated on sections for 12 min at 37°C and visualized using diaminobenzidine as a chromogen followed by hematoxylin as a counterstain. Slides were rinsed, dehydrated through alcohol and xylene, and coverslipped. Images were acquired by a Keyence BZ-X710 digital microscope system for high-resolution stitching of tissue section images. ImagePro Premier software (Media Cybernetics) was used for quantification of Aβ plaques and Iba1.

[0130] Immunofluorescence and image acquisition. Sections were deparaffinized, transferred to pre-warmed 95°C antigen retrieval solution for 30 min, cooled at room temperature for 20 min, and incubated in blocking solution (0.25% Triton®-X-100 and 3% BSA in Tris-buffered saline). Sections were then incubated overnight at 4°C with the following primary antibodies: rabbit anti-GFP antibody (1:200, ab290, Abcam), chicken anti-Iba1 antibody (1:200, 234006, Synaptic Systems), and mouse anti-6E10 antibody (1:250, 805701, BioLegend). Antigens were visualized using appropriate Alexa Fluor-conjugated secondary antibodies (Invitrogen). Images were acquired using a Leica SP8 confocal microscope for high-resolution stitching of tissue section images. Confocal image stacks and 3D view images were analyzed with Imaris software (Bitplane, Oxford Instruments).

[0131] Aβ 1-42 ELISA of Aβ. Brain tissue was homogenized in 0.15% Triton in PBS containing protease inhibitor cocktail with a homogenizer and centrifuged at 200,000×g for 20 min at 4°C. The supernatant was removed and defined as the soluble fraction. Guanidine-HCl was added to 0.5m (final concentration) before performing enzyme-linked immunosorbent assay (ELISA). The pellet was solubilized in 6m guanidine-HCl buffer containing protease inhibitor cocktail by sonication. The solubilized pellet was centrifuged at 200,000×g for 20 min at 4°C and used as the insoluble fraction. Aβ in each fraction was analyzed by ELISA. 1-42 The amount of was determined by sandwich amyloid beta 42 ELISA kit (KHB3441, Invitrogen).

[0132] RNA-Seq. Sample preparation. Hippocampi and cortices were dissected from brains using previously published protocols. 58The samples were frozen in RNA later at -80°C for 10 min. Total RNA was isolated using the RNeasy Tissue kit (Qiagen) according to the manufacturer's instructions. RNA was assessed for quality using an Agilent Tapestation 4200, and 50 ng of RNA with an RNA Integrity Number (RIN) of >8.0 was used to generate RNA sequencing libraries using Illumina® Stranded mRNA Prep (Illumina) according to the manufacturer's instructions. The resulting libraries were multiplexed and sequenced on an Illumina NovaSeq 6000 with 100 base pair (bp) paired end reads (PE100) to a depth of approximately 25 million reads per sample. Samples were demultiplexed using bcl2fastq conversion software (Illumina).

[0133] Analysis of RNA-seq data. RNASeq data were processed using Kallisto (version 0.45.0), Mus musculus genome GRCm38 Ensembl version 94 annotation (Mus_musculus GRCm38.94 chr_patch_hapl_scaff.gtf). Gene-level TPM values ​​and gene annotations were calculated using tximport and biomaRt R packages. Data were systematized and log reduced using log2(TPM+1) using custom python scripts. Genes were first normalized and averaged to perform composite signature analysis and calculate a composite score for gene sets. StepMiner 59 Gene expression values ​​were normalized according to a modified Z-score approach to center around a threshold (formula = (expr-SThr) / 3 * The samples were ordered based on the final composite score. Applicants performed differential expression analysis using DESeq2. 60 The false discovery rate was controlled by setting the corrected P-value threshold at 0.1.

[0134] Statistics. Applicants did not exclude any animals from the experiments. Experimenters were blinded to the genotype of the specific samples whenever possible. In all experiments, cohorts were age-matched and sex-balanced. Non-objective computational measurements, such as immunostaining quantification by scanning devices, did not require blinding. All data are expressed as mean ± sem and statistics and graphs were performed using GraphPad software Prism v.9.0. No statistical methods were used to predetermine sample size, but sample sizes were similar to those reported in previous publications using the 5xFAD model. 35 Multiple groups were compared using one-way analysis of variance (ANOVA), followed by Tukey's multiple comparisons. Statistical significance was set at P<0.05. For RNA-seq data, gene signatures are used to classify sample categories, and the performance of multiclass classification is measured by the value of ROC-AUC (area under the receiver operating characteristic curve). Color-coded bar charts are combined with violin plots to visualize classification based on gene signatures. All statistical tests were performed using R version 3.2.3 (2015-12-10). Standard t-tests were performed using the python scipy.stats.ttest_ind package (version 0.19.0) with the Welch's two-sample t-test (unpaired, unequal variances (equal_var=False), and unequal sample sizes) parameters. Multiple hypothesis correction was performed by correcting P values ​​with statsmodels.stats.multitest.multipletests (fdr_bh: Benjamini / Hochberg principle). Results were independently verified using R statistical software (R version 3.6.1; 2019-07-05). Violin plots, swarm plots, and bubble plots were created using the python seaborn package version 0.10.1.

[0135] Availability of code. The codes used to perform the RNA sequencing analyses are publicly available at the following links: https: / / github.com / sahoo00 / BoNE, https: / / github.com / sahoo00 / Hegemon.

[0136] result

[0137] Transplantation of WT mouse HSPCs results in normal neurocognitive performance in 5xFAD mice

[0138] To examine the feasibility of transplanting WT HSPCs for the treatment of AD, Applicants transfected lethally irradiated 2-month-old 5xFAD mice with Sca1 HSPCs isolated from WT green fluorescent protein (GFP) transgenic mice (5xFAD / WT HSPCs, n=10, 4 females and 6 males). + HSPCs were injected intravenously. Engraftment of donor-derived HSPCs was measured by flow cytometry and expressed as the percentage of GFP-positive blood cells in peripheral blood, ranging from 25 to 90%. As controls, applicants analyzed WT littermates (WT, n=9, 5 females and 4 males), lethally irradiated WT littermates transplanted with HSPCs from WT mice (WT / WT HSPC, n=10, 5 females and 5 males), untreated 5xFAD littermates (5xFAD, n=10, 5 females and 5 males), and lethally irradiated 5xFAD mice transplanted with 5xFAD HSPCs (5xFAD / 5xFAD HSPC, n=10, 5 females and 5 males).

[0139] To determine the impact of transplantation of WT HSPCs on neurocognitive function in 5xFAD mice, applicants performed a battery of well-characterized behavioral tests in 6-month-old mice, i.e., 4 months after transplantation. No differences were observed between males and females in any of the tests (data not shown). In addition, myeloablative irradiation did not have any effect on the activity of WT and 5xFAD mice, since no differences were observed in any of the tests when compared to WT / WT HSPCs and 5xFAD / 5xFAD HSPCs, respectively (Figure 1). Memory function in 5xFAD mice begins to deteriorate between 4 and 5 months of age 17、18 To determine whether transplantation of WT HSPCs prevents the loss of memory function, Applicants used the well-validated hippocampal-dependent novel object recognition task (NORT). 19、20 Mice were briefly exposed to two identical objects (F1 and F2) during the training session, and 24 h later, during the test phase, mice were exposed to a novel object (N) instead of F2. Their discrimination index was then calculated. On the habituation day, the distance traveled by all mouse groups was equal (data not shown). On the test day, WT, WT / WT HSPC, and 5xFAD / WT HSPC mice spent significantly more time exploring the novel object, indicating that mice transplanted with 5xFAD / WT HSPCs remembered the familiar object and had a normal ability to discriminate between novel and familiar objects. In contrast, untreated 5xFAD mice and 5xFAD / 5xFAD HSPC-transplanted mice showed no significant change in the time to explore the novel or familiar objects (Figure 1B-D), indicating a decline in memory function.

[0140] Although memory decline is a hallmark of AD, altered anxiety is also a significant complication and has been shown to correlate with cognitive impairment in AD patients. 21、22 It was reported that changes in anxiety-like behavior correlated with 5xFAD mice spending more time in the open arms in the elevated plus maze (EPM).17 In fact, the aversive nature of the open arm has been described as an innate fear of heights. 23 ,Rodents prefer closed arms because they provide protection from potential predation. 24 Both 5xFAD and 5xFAD / 5xFAD HSPC mice spent more time in the exposed open arms, suggesting an altered perception of anxiety and danger in these mice (Figure 1E-H). In contrast, WT controls as well as 5xFAD / WT HSPCs spent more time in the closed arms at similar levels (Figure 1E-H). Next, applicants tested the locomotor activity of mice in the open field. General motor function, expressed as speed (data not shown) and distance, was slightly elevated in 5xFAD and 5xFAD / 5xFAD HSPCs compared to WT, WT / WT HSPCs, and 5xFAD / WT HSPCs in the open field (Figure 1K). In addition, 5xFAD and 5xFAD / 5xFAD HSPCs showed significantly higher levels of edge / corner activity compared to WT controls and 5xFAD / WT HSPCs (Figure 1I-L), indicating preservation of normal anxiety-like behavior and activity in 5xFAD mice treated with WT HSPCs. Taken together, these results demonstrate that transplantation of WT HSPCs in 5xFAD mice resulted in preservation of their memory, danger perception, normal anxiety levels, and locomotor activity.

[0141] Transplantation of wild-type HSPCs reduced amyloid-β burden in the brains of 5xFAD mice

[0142] In AD, Aβ plaques are most abundant in the hippocampal and cortical regions of the brain 15 Aβ deposits are also widely found in the hippocampus and cortex of 6-month-old 5xFAD mice. 17Applicants quantified Aβ plaques in the hippocampus and cortex of 5xFAD mice after immunohistochemical analysis using the 6E10 antibody, which targets amino acids 1-16 of Aβ. Levels of Aβ in 5xFAD mice transplanted with 5xFAD HSPCs were equivalent to untreated 5xFAD mice in both the hippocampus and cortex (Figures 2A-B). In contrast, 5xFAD mice transplanted with WT HSPCs showed a significant reduction in plaque number, size, and total area in both the cortex and hippocampus, respectively (Figures 2A-J). Given these results, Applicants tested whether the improvement in cognitive performance following reduction in plaque burden could be associated with a reduction in the levels of soluble (Triton fraction) and insoluble (guanidine fraction) Aβ. Thus, in all groups, Aβ 1-42 Applicants found that levels of soluble and insoluble Aβ were significantly lower in 5xFAD mice 4 months after transplantation of WT HSPCs compared to untreated and 5xFAD HSPC-treated mice. 1-42 We found reduced levels of Aβ (FIG. 2K). Thus, these results demonstrated that transplantation of WT HSPCs had a significant beneficial effect on reducing Aβ plaque burden in the cortical and hippocampal compartments of 5xFAD mice.

[0143] Engrafted WT HSPCs differentiate into microglia and are involved in reducing Aβ plaques in 5xFAD mice

[0144] Applicants next investigated the expression of WT GFP in the brains of 5xFAD mice. + We examined HSPC engraftment and differentiation and expressed GFP in both the cortex and hippocampus of all treated mice. + We found substantial engraftment of HSPC-derived cells (Figure 3A). These cells were immunoreactive with Iba1, characterizing them as microglia-like cells (Figure 3A, F). Recent reports have shown that microglial dysfunction contributes to AD pathology and is associated with Aβ plaque clearance. 25、26Both 5xFAD and 5xFAD / 5xFAD HSPC mice show significantly higher immunoreactive areas for Iba1 staining in the hippocampus and cortex compared to WT mice (Figure 3C,D). In contrast, 5xFAD treated with WT HSPCs showed a significant reduction in the levels of Iba1 positive cells in both the hippocampus and cortex compared to the 5xFAD control group. In addition, microglia were mostly amoeboid and activated, suggesting active inflammation in the hippocampus and cortex of 5xFAD and 5xFAD / 5xFAD mice, as previously described for AD (Figure 3b). 25、26 In contrast, Iba1 in 5xFAD / WT HSPC mice + Microglia displayed a more ramified, "quiescent" phenotype (Figure 3B), suggesting reduced inflammation in the brains of treated mice. This was confirmed using the dual chromogenic stain Iba1 / 6E10 to examine the phenotype of microglia in close proximity to Aβ plaques. Plaque-associated Iba1 + The cells were active and inflamed in 5xFAD mice transplanted with 5xFAD HSPCs, whereas in the brains of 5xFAD mice transplanted with WT HSPCs, they had a branched, multipolar morphology, accompanied by a reduction in plaque size, reflected primarily by the clearance of plaque halos (Figure 3E). + To prove that the microglia were derived from the transplanted HSPCs, Applicant performed immunofluorescence staining of brain sections from 5xFAD / WT HSPC mice and 5xFAD / 5xFAD HSPCs as controls, and found that Iba1 + Microglia express GFP + We also observed that Aβ plaques were larger and more fibrillar in 5xFAD control compared to 5xFAD / WT HSPC mice, and plaque size was significantly greater in 5xFAD / WT HSPC brains than in GFP-positive mice (Figure 3F). +When surrounded by microglia, it was reduced (Figure 3F and Figure 5). Taken together, these results suggested phagocytosis of amyloid plaques by microglia derived from transplanted WT HSPCs, which clustered around the plaques and reduced the amyloid burden by actively phagocytosing amyloid fibrils.

[0145] RNA sequencing analysis reveals profound differences in the transcriptome profiles of microglia in the cortex and endothelial cells in the hippocampus following WT HSPC transplantation in 5xFAD mice

[0146] To elucidate the relative contribution of HSPC transplantation to AD disease alleviation, applicants examined the transcriptome profiles by RNA sequencing (RNA-seq) of the hippocampus and cortex of WT mice (n=2), 5xFAD mice (n=3), 5xFAD / 5xFAD HSPC mice (n=3), and 5xFAD / WT HSPC mice (n=3). Recent GWAS in AD patients have identified risk genes that are highly expressed in microglia and amplify a pro-inflammatory state. 6、27~29 Because Applicants observed that in 5xFAD mice, transplanted WT HSPCs differentiated into microglia in the brain, resulting in a reduction of inflammatory microglia in the hippocampus and cortex, Applicants hypothesized diverse myeloid expression profiles between treated 5xFAD / WT HSPC mice and disease groups. Applicants therefore analyzed the transcriptome profiles of the hippocampus and cortex by composite gene score analysis of previously published datasets of signature genes associated with AD. 29、30Applicants evaluated two previously published sequential but distinct stages of "disease-associated microglia" (DAM) in AD. Stage 1 corresponds to the first stage of DAM activation. It is Trem2-independent and involves activation of a set of genes Cx3cr1, P2ry12, Tmem119, Tyrobp, Ctsb, Ctsd, Apoe, B2m, Fth1, Lyz2. The second phase of DAM activation, stage 2, involves induction of genes related to lipid metabolism and phagocytosis pathways associated with Aβ clearance (Trem2, AXL, Cst7, CtsI, LPL, Cd9, Csf1, Ccl6, Itgax, Clec7a, Lirb4, Timp2). 4 In addition to this, applicants will also evaluate previously published universal macrophage markers (Tyorbp, Fcer1g) for human and mouse tissues, which have also been found to be differentially expressed in AD. 31 Combined gene analysis revealed that stage 1 and stage 2 DAM markers and universal macrophage markers in the cortex of 5xFAD and 5xFAD / 5xFAD HSPC mice had higher expression levels compared to 5xFAD / WT HSPC and WT mice, with the difference being significant only in 5xFAD mice due to outliers in 5xFAD / 5xFAD HSPCs (Figure 4A, Figure 6A, Figure 6B). These data indicate that the inflammatory activation of microglia in 5xFAD mice is rescued by transplantation of WT HSPCs. This was confirmed by gene expression analysis (DEGs), which revealed that of 61 genes differentially expressed in the cortex between 5xFAD / WT HSPC and 5xFAD mice, 49 were upregulated in 5xFAD compared to 5xFAD / WT HSPC mice, and 24 of these were related to microglia (Figure 4B).

[0147] Interestingly, the microglial profile did not differ between the different groups in the hippocampus. Further gene score analysis revealed a significant increase in the expression of endothelial cell genes associated with neurodegeneration in the hippocampus of 5xFAD and 5xFAD / 5xFAD HSPC mice compared to 5xFAD transplanted WT HSPCs (Figure 4A). DEG analysis also revealed that the top 7 differentially expressed genes in the hippocampus of 5xFAD / WT HSPC mice compared to 5xFAD controls were associated with endothelial cells (Figure 4B). This phenomenon is specific to the hippocampus, since the profile of endothelial cell genes in the cortex did not differ between the different groups.

[0148] Experimental Considerations

[0149] Alzheimer's disease is the most common cause of dementia and the most common age-related neurodegenerative disorder. With an increasing aging population, AD will have a devastating impact, projected to affect 131.5 million people worldwide by 2050. The cost of AD will accelerate from 1 trillion to an estimated 2 trillion by 2030, which will pose a huge burden to patients, families, and public health systems. 32、33 Significant efforts have been made over the last few decades to generate disease-modifying drugs that would halt, prevent, or slow AD. Unfortunately, although hundreds of treatments from various pharmacological classes have shown preclinical efficacy in animal models to reduce cognitive impairment and disease burden, none have proven effective in human clinical trials. 33 Thus, there is an urgent unmet medical need for treatment of AD.

[0150] For many years, reactive microglia were thought to be a consequence of AD pathology, but they are now thought to play a role in disease progression and may even be at the beginning. 25、27、29、34 However, the role of microglia in AD remains a hotly debated topic. 35Applicant's study provided direct evidence that microglia play a key role in disease progression and that replacement of diseased App / Psen1 microglia with healthy ones via a single WT HSPC transplant in 5xFAD mice fully rescued neurocognitive dysfunction in mice. As Aβ deposition has been associated with cognitive changes, Applicant examined Aβ plaque levels in different animal groups. Consistent with previous reports correlating Aβ reduction with functional and cognitive improvements, the effect of WT HSPC transplantation on behavior included an increased upregulation of 6E10 in the hippocampus and cortex. + Plaques and soluble Aβ 1-42 Without being bound by theory, Applicants hypothesize that WT HSPC-derived microglia directly assist in the clearance of toxic Aβ in disease mouse models in which microglia-mediated clearance pathways are dysregulated. In particular, previous studies have demonstrated that plaques form over time in amyloidosis mouse models and in AD patients. 36、37 , which showed that Aβ is surrounded by microglial cells that appear to be responsive but inefficient in phagocytosis and clearance of Aβ. 38~41 In contrast, microglia derived from transplanted WT HSPCs were 6E10 + Colocalization with and smaller appearance of Aβ plaques suggests active phagocytosis of Aβ plaques by WT microglia in 5xFAD / WT HSPC mice, consistent with a recent report in which Trem2, involved in microglia-dependent phagocytosis, was observed to enhance Aβ deposition. 42、43 In addition, they have been shown to be capable of encapsulating, forming a barrier, and phagocytosing Aβ plaques when young and healthy. 25 On the other hand, microglia may be involved in spreading Aβ aggregates to form new plaques by retaining and releasing phagocytosed Aβ species in the brain, as observed by d'Erico et al. 35Applicants show that this beneficial or detrimental effect of microglia on Aβ burden depends on their ability to process Aβ peptides, and that replacement of App / Psen1 mutant microglia with WT HSPC transplants can switch the balance towards reducing Aβ burden. Thus, the data provide direct evidence for the involvement of microglia in the control and augmentation of Aβ burden.

[0151] Applicants noted robust changes in the phenotype and density of microglial populations following WT HSPC transplantation in 5xFAD. Microglial proliferation and activation in the brain are key features in AD, and Applicants confirmed these features in 5xFAD mice that were untreated or transplanted with App / Psen1 HSPCs. Notably, Iba1 in WT HSPC-transplanted mice was significantly increased in AD. + There was a clear reduction in the number of microglia and changes in their morphology. This important feature also plays a direct role in the preservation of neurocognition in 5xFAD mice. Indeed, chronically activated microglia continuously release inflammatory mediators, amplifying the chronic inflammatory environment of AD, providing minimal protection, and even participating in the loss of synapses and neurons through phagocytosis of synapses, and the secreted inflammatory factors damage neurons. 9、41、44 Applicants have shown for the first time that replacement of microglia with mutations in the App and Psen1 genes by healthy microglia reduces the number of microglia and reduces the inflammatory state. Applicants also show that this replacement was sufficient to prevent neurocognitive dysfunction in 5xFAD mice, strongly suggesting that microglia-associated neuroinflammation in AD is a major feature of neurodegeneration in AD and that modulating this phenotype represents an important therapeutic target. Taken together, this data indicates that healthy microglia have therapeutic impacts on AD by enhancing phagocytosis of plaques, limiting the spread of aggregates, and preventing chronic neuroinflammation.

[0152] To further evaluate the changes in the genetic profiles of brain cell populations, applicants used RNAseq analysis to examine the effects of transplantation of WT HSPCs into the cortex and hippocampus of 5xFAD mice. Plaque-associated microglia have been shown to activate specific signaling pathways, and Keren-Shaul et al. identified DAMs that increased with disease progression not only in 5xFAD mice but also in human postmortem brains. 29 Indeed, DAM in AD has been linked to the expression of genes, many of which have been found in genome-wide association studies (GWAS) in humans, including Trem2, a receptor required for DAM activation. 29、45 In particular, DAMs are significantly overexpressed in later stages of AD, where increased phagocytic and inflammatory activity leads to further damage to surrounding cell populations. However, applicants showed that 5xFAD mice transplanted with WT HSPCs showed a significant reduction in DAMs in the cortex, while the newly introduced microglial cells retained the ability to phagocytose and engulf plaques. A universal macrophage marker increased in neurological diseases, including AD. 31 was also decreased in 5xFAD / WT HSPC mice. Overall, these data demonstrate for the first time that pathways triggered by mutations in the App and Psen1 genes can be prevented in the absence of these mutations in microglia.

[0153] Surprisingly, the microglial profile in the hippocampus did not differ between the different groups. Interestingly, however, expression profiling of endothelial cell genes associated with neurodegeneration that have been reported to be increased in other neurodegenerative disorders showed similar results. 30 was also found to be increased in this tissue compartment in 5xFAD mice, and this was corrected in 5xFAD / WT HSPC mice. In AD, an age-dependent deterioration of the endothelial blood-brain barrier (BBB) ​​occurs in the human hippocampus, and severe dysfunction of BBB transport mechanisms has been reported in advanced stages of the disease, resulting in hippocampus-dependent cognitive impairment. 46~49Furthermore, increasing evidence suggests that endothelial dysfunction plays a central role in the pathogenesis of AD. 50 In AD, Aβ plaque accumulation may impair normal endothelial function and result in endothelium-dependent vasoconstriction. 50 On the other hand, endothelial cells have also been implicated in Aβ clearance when the microglial clearance mechanism is overwhelmed by a higher plaque load. However, unlike microglia, endothelial cells do not proliferate efficiently. As a result, persistent exposure of endothelial cells to debris, such as amyloid plaques, leads to cell death, dysfunction, reduced blood flow, and increased inflammatory responses in AD. 51 Therefore, we hypothesize that WT HSPC-derived microglia protect the endothelium within the hippocampus by clearing plaques and reducing inflammation, which further leads to AD rescue. These findings support previous findings that asserted that microglia and endothelial cells are essential for preserving cognitive function in AD. 52~54 .

[0154] In conclusion, our findings demonstrate that transplantation of WT HSPCs into the 5xFAD mouse model results in the engraftment and differentiation of these cells into microglia, rescuing the AD disease phenotype. This report provides an answer to the long-standing debate about whether restoring microglial cells can modulate Aβ burden during the course of disease progression, and was sufficient to prevent neurocognitive dysfunction in 5xFAD mice, demonstrating for the first time that microglia alone were able to modulate Aβ burden and rescue AD. Thus, this study strongly supports this strategy as a therapeutic approach to treat AD, providing evidence for using HSPC gene therapy to correct known familial mutations in AD, opening new promising therapeutic perspectives for this incurable disorder.

[0155] Incorporation by Reference

[0156] All publications, patents, and patent applications mentioned in this specification and any attached supplemental documents are incorporated herein by reference to the same extent as if each individual publication, patent, patent application, or supplemental document was specifically and individually indicated to be incorporated by reference. Throughout the specification, Arabic numerals refer to technical publications, and all bibliographic citations are provided in the References section immediately preceding the claims.

[0157] Equivalent

[0158] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this technology belongs.

[0159] The technology illustratively described herein may suitably be practiced in the absence of any element(s), limitation(s) not specifically disclosed herein. Thus, for example, the terms "comprising," "including," "containing," and the like, should be interpreted broadly and without limitation. In addition, the terms and expressions used herein are used as terms of description and not of limitation, and in the use of such terms and expressions, it is not intended to exclude any equivalents of the features shown and described or portions thereof, and it is recognized that various modifications are possible within the scope of the technology described in the claims.

[0160] Accordingly, it should be understood that the materials, methods, and examples provided herein are representative of preferred embodiments and exemplary, and are not intended as limitations on the scope of the technology.

[0161] The present technology is described broadly and generically herein. Each of the narrower species and subgeneric groupings that fall within the generic disclosure also form part of the present technology. This includes conditional generic descriptions of the present technology, or negative limitations that remove any subject matter from the genus, regardless of whether the excised material is specifically recited herein.

[0162] In addition, where features or aspects of the technology are described in terms of a Markush group, one of skill in the art will recognize that the technology is also hereby described in terms of any individual members or subgroups of members of the Markush group.

[0163] All publications, patent applications, patents, and other references mentioned herein are expressly incorporated herein by reference in their entirety to the same extent as if each was individually incorporated by reference. In case of conflict, the present specification, including definitions, will control.

[0164] Other aspects are within the scope of the following claims. References [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4]

Claims

1. 1. A method for treating a neurodegenerative disease, neurodegenerative disorder, or symptom thereof in a subject in need thereof, comprising administering to a subject a therapeutically effective amount of a neurodegenerative disease, neurodegenerative disorder, or symptom ... a) reducing or treating Alzheimer's disease (AD) associated with microglial inflammation; b) reducing Aβ plaque burden in the hippocampus and cortex; c) promoting or differentiating microglia; d) promoting differentiation into microglia, or e) Reducing neuroinflammation is selected from the group administering to the subject an effective amount of hematopoietic stem and progenitor cells (HSPCs) or a population of HSPCs, thereby treating the neurodegenerative disease, disorder, or condition in the subject. method.

2. The method of claim 1 , wherein the HSPC is a wild-type HSPC or a population of HSPC.

3. The method of claim 1, wherein the HSPC or population of HSPCs has been genetically modified to correct a gene associated with the neurodegenerative disorder.

4. 4. The method of any one of claims 1 to 3, wherein the neurodegenerative disorder is Alzheimer's disease resulting from a mutation in a known causative gene, optionally in a gene encoding amyloid beta precursor protein (APP), microtubule associated protein tau (MAPT), presenilin 1 (PSEN1), or triggering receptor 2 expressed in myeloid cells (Trem2).

5. A population of HPSCs is administered to the subject, and at least 80% of the HPSCs within the population are CD34 ＋ The method according to any one of claims 1 to 4, wherein the cell is a HPSC.

6. The method of any one of claims 1 to 5, wherein the HSPC or population of HSPCs is autologous or allogeneic to the subject.

7. 7. The method of any one of claims 1 to 6, wherein the neurodegenerative disorder is Alzheimer's disease and the HSPC or population of HSPCs comprises one or more of exogenous or wild-type genes selected from genes encoding amyloid beta precursor protein (APP), microtubule associated protein tau (MAPT), presenilin 1 (PSEN1), or triggering receptor expressed in myeloid cells 2 (Trem2).

8. 7. The method of any one of claims 1 to 6, wherein the neurodegenerative disorder is Alzheimer's disease and the HSPC or population of HSPCs is modified to correct a known familial mutation in one or more of the familial mutations selected from APP, MAPT, PSEN1, or TREM2.

9. 9. The method of claim 8, wherein the known familial mutation is corrected by addition of a gene, optionally by use of a vector such as an adeno-associated viral vector or a lentiviral vector.

10. The method of claim 8, wherein the known familial mutation is corrected by a method comprising CRISPR / Cas9 technology.

11. The method of any one of claims 1 to 10, wherein the neurodegenerative disorder is selected from disorientation, loss of physical function, neuroinflammation, or cognitive impairment.

12. The method of any one of claims 1 to 11, wherein the HSPC or population of HSPCs is detectably labeled.

13. The method of any one of claims 7 to 12, wherein the one or more of APP, MAPT, PSEN1, or Trem2 are detectably labeled.

14. The method of any one of claims 1 to 13, wherein the HSPC or population of HSPCs is administered systemically or locally.

15. The method of any one of claims 1 to 13, wherein the HSPC or population of HSPCs is administered via the hippocampus.

16. The method of any one of claims 1 to 15, wherein the subject is a mammal.

17. 17. The method of claim 16, wherein the mammal is a murine, canine, feline, bovine, equine, or human patient.

18. A kit comprising genetically modified HSPCs containing one or more exogenous or wild-type genes selected from genes encoding amyloid beta precursor protein (APP), microtubule associated protein tau (MAPT), presenilin 1 (PSEN1), or triggering receptor expressed in myeloid cells 2 (Trem2), and instructions for use.

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