Methods for treating alzheimer's disease

Abstract

The present invention provides methods of alleviating or treating Alzheimer's disease or neurological disorder or condition, or alleviating respective symptoms thereof, comprising administering to a subject an effective amount of (HSPC) or a population of HSPCs, which are optionally genetically modified prior to administration and will differentiate on healthy microglial cells in the brain. These cells are capable of reducing amyloid plaques and inflammation.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

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

[0002] Alzheimer's disease (AD) is the most prevalent cause of dementia and the most common age-related neurodegenerative disorder, and is expected to affect more than 13 million people in the applicant countries by 2050 1，2 As the population ages, 3%-5% of individuals aged 65 years or older may have AD, with an additional 5 to 7 million new cases each year. 3 As a result of AD, dementia develops into severe disability throughout the disease progression, usually leading to death within 5 to 12 years of onset. 4 Pronounced neuronal degeneration, inflammation, and progressive memory and behavioral decline result from the accumulation of extracellular amyloid-β (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, and trigger neuronal dystrophies. 5 Current FDA-approved treatments help manage cognitive impairment, but there are no effective disease-modifying treatments 5，6 Given the complex pathobiology of AD, any potential treatment must target multiple pathological pathways to improve the disease state. Summary of the invention

[0003] Reactive microglia have long been considered a consequence of AD pathology, but are now thought to potentially play a role in disease progression and may be the initiator of disease. 25,27,29,34 However, the role of microglia in AD remains a matter of intense debate (McGeer et al., Neurology 42 (1992)).

[0004] Applicants provide direct evidence herein that microglia play a key role in the progression of Alzheimer's disease and that replacement of diseased App / Psen1 microglia with healthy cells by a single WT HPSC transplantation in 5xFAD mice completely rescued neurocognitive impairment in mice.

[0005] Therefore, a method for alleviating or treating Alzheimer's disease is provided herein. The method includes or is substantially composed of or is composed of applying hematopoietic stem cells or progenitor cells (HSPC) or HSPC colonies to a subject in need systemically or in the subject's hippocampus, and the HSPC or HSPC colonies are optionally genetically corrected before application and will be differentiated on healthy microglia in the brain. These cells can reduce amyloid plaques and inflammation, thereby alleviating or treating AD.

[0006] Also provided is the use of CD34 + Methods and compositions for HSPC gene therapy to correct known familial mutations in AD.

[0007] Methods and compositions are shown using the 5xFAD double transgenic mouse model, which expresses mutant human amyloid beta (A4) precursor protein 695 (APP) with 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), resulting in significantly increased Aβ plaque accumulation in the hippocampus and cortex from 2 to 4 months of age 18，19 These mice showed significant neurocognitive impairment and anxiety-reducing behaviors between 3 and 6 months of age. 20 In addition, 5xFAD mice display the main features of amyloid plaque pathology in AD, including dysfunctional microglia. 18，21 .

[0008] Applicants provide herein that a single systemic transplantation of wild-type HSPCs into adult 5xFAD mice results in preservation of memory and neurocognitive abilities and reduction of Aβ plaque load in the hippocampus and cortex. Donor or donor-derived HSPCs differentiate into microglia with active amyloid plaque clearance potential while also leading to reduced neuroinflammation. This work opens up a new therapeutic approach for the treatment of AD using HSPC gene therapy.

[0009] This article also provides a method for using CD34 + Methods of HSPC transplantation into microglia in the brain and spinal cord and into macrophages in DRG to preserve neuronal and motor function 13. WT HSPC transplantation can lead to the generation of healthy microglia, which can lead to reduced neuroinflammation and Aβ plaque accumulation in the areas of the brain most affected in AD. Alternatively, a patient's own hematopoietic stem and progenitor cells (HSPCs) are used in the following methods, which include or consist essentially of or consist of introduced or modified genes involved in the pathogenesis of AD, and the method is used to treat Alzheimer's disease in the patient by transplanting the genetically modified HSPCs systemically or locally into the patient's hippocampus. The genes to be corrected are PP, MAPT, PSEN1, PSEN2 and Trem2. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1A to Figure 1L: Transplantation of WT HSPC to prevent neurocognitive damage in 5xFAD mice. (Figure 1A) Schematic representation of experimental design and timeline. (Figure 1B to Figure 1D) Memory recognition test evaluated by discrimination index (Figure 1F) and preference index (Figure 1C) in 6-month-old mice. A representative tracking diagram (Figure 1D) of 2-day tests of 5xFAD / 5xFAD HSPC and 5xFAD / WT HSPC mice is shown. (Figure 1E to Figure 1H) Elevated plus maze test in different mouse groups, where the time spent in closed arms (Figure 1E) and open arms (Figure 1F) is expressed in seconds (s), and the ratio of the time spent in the open arms to the total time of the test (Figure 1G). A representative tracking diagram (Figure 1H) of the test of 5xFAD / 5xFAD HSPC and 5xFAD / WT HSPC mice is shown. (FIG. 1I-1L) Open field test, where the distance covered by different groups of mice in the periphery (FIG. 1I), corners (FIG. 1J), and total distance traveled (FIG. 1K) are expressed in meters (m). Representative tracings of the test for 5xFAD / 5xFAD HSPC and 5xFAD / WT HSPC mice are shown (FIG. 1L). All data are expressed as mean ± SEM. **P < 0.005 and ***P < 0.0005 were determined by one-way ANOVA followed by Tukey's multiple comparisons.

[0011] FIG2A-2K : Transplantation of WT HSPCs results in a reduction in Aβ plaque deposition in the cortex and hippocampus of 5xFAD mice. (FIG2A-2D) Representative sagittal sections of the cortex (FIG2A, 2C) and hippocampus (FIG2B, 2D) stained for the Aβ plaque marker 6E10 of 5xFAD / 5xFAD HSPCs (FIG2A, 2B) and 5xFAD / WT HSPCs (FIG2C, 2D) mice. Scale bar, 500 μm. Quantification of the number of plaques (FIG2E, 2H), the area occupied by plaques (FIG2F, 2I), and the percentage of the total area occupied (FIG2G, 2J) in the cortex (FIG2E-2G) and hippocampus (FIG2H-2J). (Fig. 2K) Aβ in the brains of WT (n=3), 5xFAD (n=3), 5xFAD / 5xFAD HSPCs (n=3), and 5xFAD / WT HSPCs (n=3) mice 1-42 Triton-X soluble and guanidine insoluble (ELISA). All data are expressed as mean ± SEM. **P < 0.005, ***P < 0.0005 and ****P < 0.0001 were determined by one-way ANOVA followed by Tukey's multiple comparisons.

[0012] Figure 3A to Figure 3H: Transplanted HSPCs infiltrate into the brains of 5xFAD mice and differentiate into microglia, resulting in reduced microglial activation and Aβ plaque engulfment. (Figure 3A) Representative immunohistochemistry images of sagittal sections from the brains of 5xFAD / WT HSPC mice 4 months after transplantation, showing GFP + Cells, insets show GFP in the hippocampus and cortex + cells. Scale bar, 100 μm (a, inset). (Fig. 3B) Representative images of hippocampal sections immunostained for the microglial marker Iba1, with the inset showing the dentate gyrus region of the hippocampus. Scale bar, 100 μm. (Fig. 3C, Fig. 3D) Immunostained Iba1 in the hippocampus and cortex. + Quantification of the area occupied by cells. Data are mean ± sem, **P < 0.005, ***P < 0.0005 and ****P < 0.0001 determined by one-way ANOVA followed by Tukey's multiple comparisons. (Figure 3E) shows that Iba1 + Microglia (green) and 6E10 +Representative chromogenic images of plaques (purple). Scale bar, 50 μm. (FIG. 3F) Representative 3D reconstructions of immunofluorescence images of brain sections from 5xFAD / 5xFAD HSPC (top) and 5xFAD / WT HSPC mice (bottom), stained from left to right with anti-GFP, anti-Iba1, and anti-6E10 antibodies. The merged image is on the far right. Scale bar, 10 μm or 2 μm (inset). (FIG. 3G) Inset shows the adjacent 6E10 in 5xFAD / 5xFAD HSPC mice. + Plaque inflammatory activity Iba1 + (Figure 3H) The inset shows GFP + Iba1 + Microglia engulf GFP+ 6E10 + Plaque.

[0013] Figures 4A to 4B: RNA-seq reveals changes in the transcriptome profile of microglia and endothelial cells in the cortex and hippocampus, respectively, after transplantation of WT HSPCs in 5xFAD. (Figure 4A) Bar graphs and violin plots of comprehensive gene score analysis of disease-associated microglia (DAM) stage 2 and neurodegeneration-associated endothelial cell gene sets in the cortex and hippocampus of WT (C), 5xFAD / WT HSPCs (T), 5xFAD (D), and 5xFAD / 5xFAD HSPCs (M). The significant P value between T and D (P < 0.05) was determined by Welchs' two-sample t-test with Benjamini-Hochberg correction. (Figure 4B) Heatmap shows the top 60 differentially expressed genes in the cortex and hippocampus. The bar graph and violin plot show 24 microglia-related genes in the cortex and 7 endothelial-related genes in the hippocampus that are significantly differentially expressed between 5xFAD / WTHSPC brains and 5xFAD brains, respectively. The significant P value (P < 0.05) between T and D was determined by Welchs' two-sample t-test with Benjamini-Hochberg correction. The numbers in the upper right corner specify the ROC-AUC values ​​of T, D, and M compared to C, respectively.

[0014] Figures 5A-5B. Transplanted HSPCs infiltrate the brains of 5xFAD mice and differentiate into microglia, resulting in reduced microglial activation and Aβ plaque engulfment. (Figure 5A) Representative images of cortical sections immunostained for the microglial marker Iba1. Scale bar, 100 μm. (Figure 5B) Images showing engulfment of 6E10 + Plaque GFP + Iba1 +Microglia 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 grayscale comparison of antibody staining in Figure 3F). Scale bar, 10 μm. Inset shows GFP + Iba1 + With 6E10 + Colocalization of plaques. Scale bar, 2 μm.

[0015] Figures 6A to 6B: Violin plots of comprehensive gene score analysis of disease-associated microglia (DAM) stage 1 and universal macrophage marker gene sets in the cortex and hippocampus of WT (C), 5xFAD / WT HSPC (T), 5xFAD (D), 5xFAD / 5xFAD HSPC (M). The significant P value between T and D is marked. (Figure 6A) shows disease-associated microglia (stage 1). (Figure 6B) shows universal macrophage markers. The numbers in the upper right corner specify the ROC-AUC values ​​of T, D, and M compared to C, respectively. DETAILED DESCRIPTION

[0016] Throughout the disclosure, various publications, patents, and published patent specifications are cited by identifying citations. The disclosures of these publications, patents, and published patent specifications are hereby incorporated by reference into the present disclosure in their entirety.

[0017] Before describing compositions and methods, it should be understood that the present invention is not limited to the specific methodology, protocols, cell lines, assays and reagents described, as these may vary. It should also be understood that the terminology used herein is intended to describe specific embodiments of the present invention and is by no means intended to limit the scope of the present invention as 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", 3rd edition; Ausubel et al., eds. (2007) "Current Protocols in Molecular Biology" series; "Methods in Enzymology" series, (Academic Press, Inc., New York); MacPherson et al. (1991) "PCR 1: A Practical Approach" (IRL Press of 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", 5th edition; 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", 3rd edition (Cold Spring Harbor Laboratory Press (2002)); "Current Protocols In Molecular Biology" (FM Ausubel et al., eds. (1987)); "Methods in Enzymology" series (Academic Press, Inc.); "PCR 2: A Practical Approach" (MJ MacPherson, BD Hames and GR Taylor, eds. (1995)); Harlow and Lane, eds. (1988) "Antibodies, A Laboratory Manual"; Harlow and Lane, eds. (1999) "Using Antibodies, A Laboratory Manual" Manual”; “Animal Cell Culture” (RI Freshney, ed. (1987)); Zigova, Sanberg and Sanchez-Ramos, eds. (2002) “Neural Stem Cells”.;

[0019] All numerical symbols (e.g., pH, temperature, time, concentration, and molecular weight, including ranges) are approximate and, where appropriate, vary in increments of 0.1 or 1, either positively (+) or negatively (-). It should be understood that, although not always explicitly stated, all numerical symbols are preceded by the term "about". The term "about" includes, where appropriate, the exact value of "X", in addition to smaller increments of "X" (such as "X+0.1 or 1" or "X-0.1 or 1"). It should also be understood that, although not always explicitly stated, the agents described herein are exemplary only and that equivalents thereof are known in the art. definition

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

[0021] As used herein, the term "comprising" is intended to mean that compositions and methods include the elements listed, but do not exclude other elements. When used to define compositions and methods, "essentially consisting of ... " should mean excluding other elements that have any basic significance for the combination for the purpose. Therefore, the composition consisting essentially of the elements defined herein will not exclude trace contaminants from separation and purification methods and pharmaceutically acceptable carriers (such as phosphate buffered saline), preservatives, etc. "Essentially consisting of ... " should mean excluding other ingredients exceeding trace elements and substantial method steps or production compositions or process steps for applying the compositions of the present invention or achieving the desired results. The embodiments defined by each of these transitional terms are within the scope of the present invention.

[0022] As used herein, the term "isolated" with respect to cells, nucleic acids such as DNA or RNA refers to molecules separated from other DNA or RNA present in macromolecules of natural origin, respectively. As used herein, the term "isolated" also refers to nucleic acids or peptides that are substantially free of cellular material, viral material or culture medium when produced by recombinant DNA technology, or substantially free of chemical precursors or other chemicals when chemically synthesized. In addition, the term "isolated nucleic acid" means to contain nucleic acid fragments that are not naturally present as fragments and do not exist in the natural state. The term "isolated" is also used herein to refer to cells or polypeptides separated from other cell proteins or tissues. An isolated polypeptide is meant to encompass purified polypeptides and recombinant polypeptides.

[0023] The term "isolated" as used with respect to cells, particularly stem cells, such as mesenchymal stem cells, refers to cells that are separated from other cells or tissues found in their natural tissues in vivo.

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

[0025] The terms "allele" and "allelic variant" are used interchangeably herein and refer to alternative forms of a gene or part thereof. Alleles occupy the same locus or position on homologous chromosomes. When a subject has two identical alleles of a gene, the subject is considered to be homozygous for the gene or allele. When a subject has two different alleles of a gene, the subject is considered to be heterozygous for the gene. The alleles of a particular gene may differ in a single nucleotide or several nucleotides and may include substitutions, deletions, and insertions of nucleotides. The alleles of a gene may also be forms of a gene containing mutations.

[0026] "Cell", "host cell" or "recombinant host cell" are used interchangeably herein. It should be understood that these terms refer not only to the specific subject cell, but also to the progeny or potential progeny of such a cell. Because certain modifications may occur in progeny due to mutations or environmental influences, such progeny may not actually be the same as the parent cell, but are still included within the scope of the term as used herein.

[0027] "Amplification" of a polynucleotide sequence includes methods such as traditional cloning methods, 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. Patent Nos. 4,683,195 and 4,683,202, and Innis et al. (1990) Mol. Cell Biol., Vol. 10, No. 11: 5977-5982 (for PCR); and Wu et al. (1989) Genomics, Vol. 4: 560-569 (for LCR). In general, the PCR procedure describes a method for gene amplification that includes: (i) sequence-specific hybridization of primers to specific genes within a DNA sample (or library); (ii) subsequent amplification, including multiple rounds of annealing, extension, and denaturation using a DNA polymerase; and (iii) screening of PCR products to obtain bands of the correct size. The primers used are oligonucleotides of sufficient length and appropriate sequence to provide for initiation of polymerization, ie, each primer is specifically designed to be complementary to each strand of the genomic locus to be amplified.

[0028] Reagents and hardware for performing PCR are commercially available. Primers for amplifying sequences from specific regions are preferably complementary and specifically hybridized to sequences in the target region or its flanking regions. The nucleotide sequence produced by amplification can be directly sequenced. Alternatively, the amplified sequence can be cloned before sequence analysis. Methods for direct cloning and sequence analysis of genomic fragments for enzymatic amplification are known in the art.

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

[0030] As used herein, the term "gene" or "recombinant gene" refers to a nucleic acid molecule comprising an open reading frame and comprising at least one exon and (optionally) intron sequences. A gene may also refer to a polymorphic or mutant form or allele of a gene.

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

[0032] A polynucleotide or polynucleotide region (or a polypeptide or polypeptide region) having a certain percentage (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99%) of "sequence identity" to another sequence means that when aligned, the percentage of bases (or amino acids) is the same when comparing the two sequences. Such alignments and homology or sequence identity percentages can be determined using software programs known in the art, such as those described in "Current Protocols in Molecular Biology" by Ausubel et al., eds. (2007). Preferably, the alignment is performed using default parameters. One alignment program is BLAST, using default parameters. Specifically, the programs are BLASTN and BLASTP, using the following default parameters: Genetic code = standard; filter = none; strand = both; cutoff = 60; expect = 10; Matrix = BLOSUM62; Descriptions = 50 sequences; sort by = HIGHSCORE; Databases = non-redundant; GenBank + EMBL + DDBJ + PDB + GenBank CDS translations + SwissProtein + SPupdate + PIR. Detailed information on these programs can be found at the following Internet address: http: / / www.ncbi.nlm.nih.gov / blast / Blast.cgi, last accessed on May 21, 2008. Biologically equivalent polynucleotides are polynucleotides having a specified homology percentage and encoding polypeptides having the same or similar biological activity.

[0033] The term "equivalent nucleic acid" refers to a nucleic acid having a nucleotide sequence that has a certain degree of homology to the nucleotide sequence of the nucleic acid or its complement. Homologues of double-stranded nucleic acids are intended to include nucleic acids having a nucleotide sequence that has a certain degree of homology to it or its complement. In one aspect, a homologue of a nucleic acid is capable of hybridizing with a nucleic acid or its complement.

[0034] As used herein, the term "interaction" is meant to include detectable interactions between molecules, such as can be detected using, for example, hybridization assays. The term interaction is also meant to include "binding" interactions between molecules. The interaction can be, for example, protein-protein, protein-nucleic acid, or nucleic acid-nucleic acid interactions in nature.

[0035] "Hybridization" refers to the reaction of one or more polynucleotides to form a hybrid complex stabilized by hydrogen bonds between the bases of the nucleotide residues. Hydrogen bonds can occur by Watson-Crick base pairing, Hoogstein binding, or any other sequence-specific manner. The complex may contain two chains forming a duplex structure, three or more chains forming a multi-chain complex, a single self-hybridizing chain, or any combination of these substances. The hybridization reaction may constitute a step in a broader process, such as the initiation of a PCR reaction or the enzymatic cleavage of a polynucleotide by a ribozyme.

[0036] Hybridization reactions can be performed under conditions of varying "stringency". Typically, low stringency hybridization reactions are performed at about 40°C in about 10×SSC or a solution of equivalent ionic strength / temperature. Moderate stringency hybridizations are typically performed at about 50°C in about 6×SSC, and high stringency hybridization reactions are typically performed at about 60°C in about 1×SSC. Hybridization reactions can also be performed under "physiological conditions" well known to those skilled in the art. Non-limiting examples of physiological conditions are the temperature, ionic strength, pH, and Mg that are typically present 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 described as "complementary." A double-stranded polynucleotide may be "complementary" or "homologous" to another polynucleotide if hybridization can occur between one strand of a first polynucleotide and one strand of a second polynucleotide. "Complementarity" or "homology" (the degree to which one polynucleotide is complementary to another) can be quantified according to the generally accepted base pairing rules in terms of the proportion of bases in opposite strands that are expected to form hydrogen bonds with each other.

[0038] The term "mismatch" refers to hybridizing nucleic acid duplexes that are not 100% homologous. Lack of overall homology may be due to deletions, insertions, inversions, substitutions or frameshift mutations.

[0039] As used herein, the term "oligonucleotide" refers to a polynucleotide, such as deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) where appropriate. The term should also be understood to include equivalents, derivatives, variants and analogs of RNA or DNA prepared by nucleotide analogs, and single-stranded (sense or antisense) and double-stranded polynucleotides suitable for the embodiments. Deoxyribonucleotides include deoxyadenosine, deoxycytidine, deoxyguanosine and deoxythymidine. For clarity, when the nucleotides of nucleic acid are mentioned herein, it can be DNA or RNA, using the terms "adenosine", "cytidine", "guanosine" and "thymidine". It should be understood that if the nucleic acid is RNA, the nucleotides with uracil bases are uridine.

[0040] The terms "polynucleotide" and "oligonucleotide" are used interchangeably and refer to a polymeric form of any length of nucleotides (deoxyribonucleotides or ribonucleotides or their analogs). Polynucleotides may have any three-dimensional structure and may perform any known or unknown function. 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 may include modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modification of the nucleotide structure may be performed before or after polynucleotide assembly. The sequence of nucleotides may be interrupted by non-nucleotide components. Polynucleotides may be further modified after polymerization, such as by conjugation with a labeling component. The term also refers to double-stranded molecules and single-stranded molecules. Unless otherwise stated or required, any embodiment of the invention, ie, a polynucleotide, encompasses a double-stranded form and each of the two complementary single-stranded forms known or predicted to comprise the double-stranded form.

[0041] A polynucleotide is composed of a specific sequence of four nucleotide bases: adenine (A); cytosine (C); guanine (G); thymine (T); when the polynucleotide is RNA, uracil (U) replaces thymine. Therefore, the term "polynucleotide sequence" is an alphabetic representation of a polynucleotide molecule. This alphabetic representation can be entered into a database in a computer with a central processing unit and used for bioinformatics applications such as functional genomics and homology searches. The term "polymorphism" refers to the coexistence of more than one form of a gene or part thereof. A portion 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 the gene." A polymorphic region can be a single nucleotide whose identity varies in different alleles.

[0042] A "composition" generally means a combination of an active agent (e.g., stem cells or a population thereof) and a naturally occurring or non-naturally occurring carrier (inert (e.g., a detectable agent or marker) or active, such as an adjuvant, diluent, binder, stabilizer, buffer, salt, lipophilic solvent, preservative, adjuvant, etc.), and comprises a pharmaceutically acceptable carrier. Carriers also include pharmaceutical excipients and additives proteins, peptides, amino acids, lipids, and carbohydrates (e.g., sugars, including monosaccharides, di-oligosaccharides, tri-oligosaccharides, tetra-oligosaccharides, and other oligosaccharides; derivatized sugars, such as sugar alcohols, aldonic acids, esterified sugars, etc.; and polysaccharides or sugar polymers), which may be present alone or in combination, and the content alone or in combination is 1%-99.99% by weight or volume. Exemplary protein excipients include serum albumin, such as human serum albumin (HSA), recombinant human albumin (rHA), gelatin, casein, and the like. Representative amino acid components (which may also act as buffers) include alanine, arginine, glycine, arginine, betaine, histidine, glutamic acid, aspartic acid, cysteine, lysine, leucine, isoleucine, valine, methionine, phenylalanine, aspartame, etc. Carbohydrate excipients are also within the scope of the present technology, examples of which include, but are not limited to, monosaccharides such as fructose, maltose, galactose, glucose, D-mannose, sorbitol, etc.; disaccharides such as lactose, sucrose, trehalose, cellobiose, etc.; polysaccharides such as raffinose, melezitose, maltodextrin, dextran, starch, etc.; and sugar alcohols such as mannitol, xylitol, maltitol, lactitol, xylitol, sorbitol (glucitol), and inositol.

[0043] Compositions used according to the present disclosure, including cells, treatments, therapies, medicaments, drugs and pharmaceutical preparations, can be packaged in dosage unit form for ease of administration and uniform dosage. The term "unit dose" or "dose" refers to physically separated units suitable for subjects, each unit containing a composition calculated to produce a predetermined amount of the desired response associated with its administration (i.e., appropriate route and scheme). Depending on the number of treatments and the unit dose, the amount of administration depends on the desired result and / or protection. The precise amount of the composition also depends on the judgment of the practitioner and is unique to each individual. Factors affecting dosage include the physical and clinical state of the subject, the route of administration, the intended therapeutic goal (symptom relief and cure) and the efficacy, stability and toxicity of the specific composition. After preparation, the solution will be administered in a manner compatible with the dosage formulation, and administered in an amount such as effective for treatment or effective for prevention. The preparation is easy to administer in various dosage forms, such as the type of injectable solution described herein.

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

[0045] The agents of the present disclosure may be administered by any suitable route of administration for therapy. It should also be understood that the optimal route will vary with the condition and age of the recipient and the disease being treated.

[0046] "Gene delivery vehicle" is defined as any molecule that can carry the inserted polynucleotide into the host cell. Examples of gene delivery vehicles are liposomes, biocompatible polymers, including natural polymers and synthetic polymers; lipoproteins; polypeptides; polysaccharides; lipopolysaccharides; artificial viral envelopes; metal particles; and bacteria or viruses (such as baculovirus, adenovirus and retrovirus), phage, cosmid, plasmid, fungal vectors and other recombinant vectors commonly used in the art that have been described for expression in a variety of eukaryotic and prokaryotic hosts and can be used for gene therapy as well as for simple protein expression.

[0047] As used herein, "gene delivery", "gene transfer" and the like refer to terms for introducing exogenous polynucleotides (sometimes referred to as "transgenes") into host cells, regardless of the method used for introduction. Such methods include a variety of well-known techniques, such as vector-mediated gene transfer (via, for example, 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 (such as electroporation, "gene gun" delivery, and various other techniques for introducing polynucleotides). Unless otherwise indicated, the terms transfection, transduction, or transformation are used interchangeably herein to indicate the presence of exogenous polynucleotides or polypeptides expressed therefrom in cells. The introduced polynucleotides may be stably or transiently maintained in the host cell. Stable maintenance generally requires that the introduced polynucleotides either contain a replication origin compatible with the host cell, or are integrated into a replicon of the host cell, such as an extrachromosomal replicon (e.g., a plasmid) or a nuclear or mitochondrial chromosome. Many vectors are known to be able to mediate the transfer of genes to mammalian cells, as known in the art and described herein.

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

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

[0050] "Viral vector" is defined as a recombinantly produced virus or viral particle containing a polynucleotide to be delivered into a host cell 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 vectors based on Semliki Forest virus and vectors based on Sindbis virus, have also been developed for gene therapy and immunotherapy. See Schlesinger and Dubensky (1999) Curr. Opin. Biotechnol., Vol. 5: pp. 434-439, and Ying et al. (1999) Nat. Med., Vol. 5 (Issue 7): pp. 823-827.

[0051] In aspects where gene transfer is mediated by a retroviral vector, a vector construct refers to a polynucleotide comprising 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 of stably transferring a gene or nucleic acid sequence into a host cell by the virus entering the cell and integrating 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 different host cell surface receptors or ligands to enter the cell. Retroviruses carry their genetic information in the form of RNA; however, once the virus infects the cell, the RNA is reverse transcribed into a DNA form, which is integrated into the genomic DNA of the infected cell. The integrated DNA form is called a provirus. As used herein, a retroviral vector refers to a viral particle capable of introducing exogenous nucleic acids into a cell through a viral or virus-like entry mechanism. "Slow virus vector" is a class of retroviral vectors well known in the art, which 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 aspects where gene transfer is mediated by a DNA viral vector such as adenovirus (Ad) or adeno-associated virus (AAV), a vector construct refers to a polynucleotide comprising a viral genome or a portion thereof and a transgene. Adenovirus (Ad) is a relatively well-characterized homologous virus group, including more than 50 serotypes. See, for example, International PCT Application No. WO 95 / 27071. Ad does not need to be integrated into the host cell genome. Recombinant Ad-derived vectors have also been constructed, particularly those that reduce the potential for recombination and generation of wild-type viruses. See International PCT Application Nos. WO 95 / 00655 and WO 95 / 11984. Wild-type AAV with high infectivity and specificity is integrated into the host cell genome. See Hermonat and Muzyczka (1984) Proc. Natl. Acad. Sci. USA, Vol. 81: pp. 6466-6470, and Lebkowski et al., (1988) Mol. Cell. Biol. Vol. 8: pp. 3988-3996.

[0053] Vectors containing both promoters and polynucleotides to which cloning sites can be operably 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, California) and Promega Biotech (Madison, Wisconsin). In order to optimize expression and / or in vitro transcription, it may be necessary to remove, add or change the 5' and / or 3' non-translated portion of the clone to eliminate additional, potentially inappropriate alternative translation initiation codons or other sequences that may interfere or reduce expression at the transcription or translation level. Alternatively, a consensus ribosome binding site may be inserted directly into the 5' of the start codon to enhance expression.

[0054] "Under transcriptional control" is a term well known in the art and indicates that the transcription of a polynucleotide sequence (usually a DNA sequence) is dependent upon its operative linkage to an element that facilitates the initiation of transcription or promotes transcription. "Operably linked" means that the polynucleotides are arranged in a manner that allows them to function in a cell.

[0055] Gene delivery vehicles also include several 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 acids or proteins of the present invention can be conjugated with antibodies or binding fragments thereof that bind to cell surface antigens (e.g., cell surface markers found on stem cells).

[0056] When used in the context of polynucleotide manipulation, "probe" refers to an oligonucleotide provided as a reagent for detecting a target by hybridization with a target potentially present in a sample of interest. Typically, the probe will contain a label or a device to which a label can be attached before or after the hybridization reaction. Suitable labels are described and illustrated herein.

[0057] A "primer" is a short polynucleotide, usually with a free 3'-OH group, which binds to a target or "template" potentially present in a sample of interest by hybridizing with the target and thereafter promotes polymerization of a polynucleotide complementary to the target. A "polymerase chain reaction" ("PCR") is a reaction in which a "primer pair" or "primer set" consisting of an "upstream" and a "downstream" primer and a polymerization catalyst (such as a DNA polymerase, usually a thermostable polymerase) are used to prepare replicate copies from a target polynucleotide. PCR methods are well known in the art and are taught in, for example, M. MacPherson et al., (1991) "PCR: A Practical Approach" (IRLPress, Oxford University Press). All methods of producing replicate copies of polynucleotides (such as PCR or gene cloning) are collectively 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 may optionally contain a detectable label and are exemplified and described herein.

[0058] As used herein, the term "label" means a directly or indirectly detectable compound or composition, which is directly or indirectly conjugated to a composition to be detected (e.g., a polynucleotide or protein such as an antibody) to produce a "labeled" composition. The term also includes sequences conjugated to polynucleotides that provide signals when the inserted sequence is expressed, such as green fluorescent protein (GFP), etc. The label itself can be detectable (e.g., radioisotope labeling or fluorescent labeling), or in the case of enzyme labeling, can catalyze the chemical change of a detectable substrate compound or composition. The label can be suitable for small-scale detection or more suitable for high-throughput screening. Therefore, suitable labels include, but are not limited to, radioisotopes, fluorescent dyes, chemiluminescent compounds, dyes and proteins, including enzymes. The label can be simply detected, or it can be quantified. The response of a simple detection generally includes a response in which its presence is only confirmed, and a quantitative response generally includes a response with a quantifiable (e.g., numerically reportable) value (such as intensity, polarization and / or other characteristics). In luminescent or fluorescent assays, a detectable response can be generated directly using a luminophore or fluorophore associated with the detection component that actually participates in binding, or indirectly using a luminophore or fluorophore associated with another (e.g., reporter or indicator) component.

[0059] Examples of luminescent labels that generate signals include, but are not limited to, bioluminescence and chemiluminescence. Detectable luminescent responses generally include changes or occurrences of luminescent signals. Suitable methods and luminophores for measuring components of luminescent labels are known in the art, and are for example described in Haugland, Richard P. (1996) "Handbook of Fluorescent Probes and Research Chemicals" (6th edition). 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, methylcoumarin, pyrene, malachite green, stilbene, fluorescent yellow, Cascade Blue.TM. and Texas Red. Other suitable optical dyes are described in Haugland, Richard P. (1996) "Handbook of Fluorescent Probes and Research Chemicals" (6th edition).

[0061] In another aspect, the fluorescent label is functionalized to promote covalent attachment to the cell components present in or on the surface of the cell or tissue, such as cell surface markers. Suitable functional groups include but are not limited to isothiocyanate groups, amino groups, haloacetyl groups, maleimide, succinimide esters and sulfonyl halides, all of which can be used to attach the fluorescent label to the second molecule. The selection of the fluorescent label's functional group will depend on the attachment site with a joint, an agent, a marker or a second labeling agent.

[0062] The fluorescent label may be directly attached to the cellular component or compound, or alternatively, may be attached via a linker. Suitable binding pairs for indirectly linking the fluorescent label to the intermediate include, but are not limited to, antigen / antibody, such as rhodamine / anti-rhodamine, biotin / avidin, and biotin / streptavidin.

[0063] The phrase "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 many techniques known to those skilled in the art. In one technique, oligonucleotides are attached and arranged on a gene chip for use in determining DNA sequences by hybridization methods, such as those outlined in U.S. Patent Nos. 6,025,136 and 6,018,041. The polynucleotides of the present invention can be modified into probes, which in turn can be used to detect genetic sequences. Such techniques have been described, for example, in U.S. Patent Nos. 5,968,740 and 5,858,659. Probes can also be attached or fixed to electrode surfaces for electrochemical detection of nucleic acid sequences, such as described in Kayem et al., U.S. Patent No. 5,952,172, and Kelley et al. (1999) Nucleic Acids Res., Vol. 27: pp. 4830-4837.

[0064] Various "gene chips" or "microarrays" and similar technologies are known in the art. Examples of this class include, but are not limited to, 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 expression technology (Gene Trace Systems, Inc.); thermal jet spotting system (Hewlett Packard Company); Hyseq HyChip (Hyseq, Inc.); BeadArray (Illumina, Inc.); GEM (Incyte Microarray Systems); a high-throughput microarray system that can distribute 12 to 64 spots to multiple slides (Intelligent Bio-Instruments); Molecular Biology Workstation and NanoChip (Nanogen, Inc.); microfluidic glass chip (Orchid Biosciences, Inc.); BioChip Arrayer with four PiezoTip piezoelectric drop-on-demand tips (Packard Instruments, Inc.); FlexJet (Rosetta Inpharmatic, Inc.); MALDI-TOF mass spectrometer (Sequnome); ChipMaker 2 and ChipMaker 3 (TeleChem International, Inc.); and GenoSensor (Vysis, Inc.) identified and described in Heller (2002) Annu. Rev. Biomed. Eng., Vol. 4: pp. 129-153. Examples of "gene chips" or "microarrays" are also described in U.S. Patent Publication Nos. 2007 / 0111322, 2007 / 0099198, 2007 / 0084997, 2007 / 0059769, and 2007 / 0059765, and U.S. Patent Nos. 7,138,506, 7,070,740, and 6,989,267.

[0065] In one aspect, a "gene chip" or "microarray" containing probes or primers homologous to the polynucleotides described herein is prepared. A suitable sample is obtained from the patient, genomic DNA, RNA, protein, or any combination thereof is extracted, and amplified if necessary. The sample is contacted with the gene chip or microarray plate under conditions suitable for hybridization of the target gene or gene product with the probe or primer contained on the gene chip or microarray. The probe or primer can be detectably labeled to identify the target sequence. Alternatively, a chemical or biological reaction can be used to identify the probe or primer hybridized with the DNA or RNA of the target gene. The genotype or phenotype of the patient is then determined by means of the above-mentioned device and method.

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

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

[0068] For topical application, pharmaceutically acceptable carriers are suitable for making creams, ointments, gels, solutions, suspensions, etc. Such carriers are common in the art, for example, for topical application with polyethylene glycol (PEG). These preparations may optionally contain additional pharmaceutically acceptable ingredients, such as diluents, stabilizers and / or adjuvants.

[0069] "Substantially homologous" describes a cell population 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 have the same or similar phenotype. The phenotype can be determined by pre-selected cell surface markers or other markers, such as myosin or actin, or the expression of genes or proteins, such as calcium handling proteins, t-tubulin, or alternatively calcium pump proteins. In another aspect, a substantially homologous 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%) compared to a wild-type corresponding cell or tissue.

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

[0071] An "effective amount" is an amount sufficient to produce beneficial or desired results. An effective amount can be administered in one or more administrations, applications, or dosages, and can be determined empirically by one skilled in the art.

[0072] As used herein, "treatment" of a disease in a subject refers to (1) preventing symptoms or disease from occurring in a subject who is susceptible to or does not yet show symptoms of the disease; (2) inhibiting a disease or preventing its development; or (3) ameliorating or causing regression of a disease or disease symptoms. As understood in the art, "treatment" is a method for obtaining beneficial or desired results (including clinical results). For the purposes of the present technology, beneficial or desired results may include one or more, but are not limited to, relief or improvement of one or more symptoms, reduction in the extent of a condition (including a disease), stabilization of the condition (including a disease) state (i.e., not worsening), delay or slowing of a condition (including a disease), progression, improvement or alleviation of a condition (including a disease), state, and relief (whether partial or complete), whether detectable or undetectable.

[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, when the purpose of an experiment is to determine the correlation between a mutated allele and a particular phenotype, it is generally preferred to use a positive control (a sample from a subject carrying such a mutation and exhibiting the desired phenotype) and a negative control (a subject or sample from a subject lacking the mutated allele and lacking the phenotype).

[0074] As used herein, "stem cell" is defined as a cell with the ability to divide indefinitely in culture and produce specialized cells. At this point, for convenience, stem cells are classified as somatic cells (adults) or embryonic cells. Somatic stem cells are undifferentiated cells present in differentiated tissues, which can self-renew (clone) and (under certain restrictions) differentiate to produce all specialized cell types of its source tissue. Embryonic stem cells are primitive (undifferentiated) cells from embryos, which have the potential to become a variety of specialized cell types. Embryonic stem cells are cells that can be cultured under in vitro conditions and can proliferate without differentiation in months to years. Non-limiting examples of embryonic stem cells are HES2 (also referred to as ES02) cell lines available from ESI in Singapore and H1 (also referred to as WA01) cell lines available from WiCells in Madison, Wisconsin. By using markers, including but not limited to Oct-4, alkaline phosphatase, CD30, TDGF-1, GCTM-2, Genesis, germ cell nuclear factor, SSEA1, SSEA3 and SSEA4, pluripotent embryonic stem cells can be distinguished from other types of cells.

[0075] "Mesenchymal stem cells" or MSCs are multipotent stem cells that can differentiate into a variety of cell types. The name 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 embryonic connective tissue that is derived from the mesoderm and differentiates into hematopoietic and connective tissues, whereas MSCs do not differentiate into hematopoietic cells. Stromal cells are connective tissue cells that form a support structure in which the functional cells of a tissue reside. While this is an accurate description of one function of MSCs, the term fails to convey the relatively newly discovered role of MSCs in tissue repair.

[0076] Hematopoietic stem cell progenitor cells and progenitor cells (HSPC) refer to precursor cells with self-renewal and multi-lineage differentiation capabilities. In the bone marrow (BM), HSPCs ensure blood cell homeostasis. In addition, once they develop into peripheral tissues, they can also replenish tissue-resident myeloid cells and directly participate in the innate immune response. These 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, Vol. 1201: pp. 49-77. doi: 10.1007 / 978-3-030-31206-0_3. PMID: 31898781. These cells are characterized by marker distribution CD34 +Methods for expanding and culturing such cells are known in the art. See, for example, Yadav et al. (2020), Int. J. Stem Cells, Vol. 13, No. 3: pp. 326-334, available at https: / / www.ncbi.nlm.nih.gov / pmc / articles / PMC7691860 / .

[0077] As used herein, "neural or neuronal stem cells" refer to cells with self-replication ability and the generation of a variety of specialized cell types of the nervous system. In some aspects, the neural stem cell is a multipotent neural stem cell Ycell in the subventricular zone (SVZ) of the lateral ventricle (LV) of the forebrain.

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

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

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

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

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

[0083] Derivatives of cells or cell colonies are daughter cells of isolated cells or cell colonies. Derivatives include cloned cells or differentiated cells that are cultured and propagated from isolated stem cells or stem cell colonies. Derivatives also include stem cells or stem cell colonies that have been derived. In one aspect, the cells or colonies are described as "donor-derived".

[0084] "Differentiation" describes the process in which non-specialized cells acquire the characteristics of specialized cells such as heart, liver or muscle cells. "Directed differentiation" refers to operating stem cell culture conditions to induce differentiation into specific cell types. "Dedifferentiation" is defined as the return of cells to a less stable position within a cell lineage. As used herein, the term "differentiation" is defined as a cell occupying a more stable ("differentiation") position in a cell lineage. As used herein, "differentiation into cells of a mesoderm (or ectoderm or endoderm) lineage" is defined as cells that are dedicated to specific mesoderm, ectoderm or endoderm lineages, respectively. The example of differentiating into a mesoderm lineage or producing a specific mesoderm cell includes but is not limited to adipogenic cells, smooth muscle cells, chondrogenic cells, cardiogenic cells, skin-derived cells, hematopoietic cells, angiogenic cells, myogenic cells, renal cells, urogenital cells, osteoblasts, pericardial cells or stromal cells.

[0085] As used herein, "pluripotent cells" are defined as less differentiated cells that can produce at least two different (genotype and / or phenotype) further differentiated daughter cells. In another aspect, "pluripotent cells" include induced pluripotent stem cells (iPSCs), which are artificially derived stem cells from non-pluripotent cells (usually adult somatic cells) 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 octamer transcription factor family, i.e., Oct-3 / 4; the Sox gene family, i.e., Sox1, Sox2, Sox3, Sox15, and Sox18; the Klf gene family, i.e., Klf1, Klf2, Klf4, and Klf5; the Myc gene family, i.e., c-myc and L-myc; the Nanog gene family, i.e., OCT4, NANOG, and REX1; or LIN28. Examples of iPSCs are described in Takahashi K. et al. (2007), Cell, published online ahead of print on November 20, 2007: Takahashi K. and Yamanaka S. (2006) Cell, Vol. 126: pp. 663-76; Okita K. et al. (2007) Nature, Vol. 448: pp. 260-262; Yu, J. et al. (2007), Science, published online ahead of print on November 20, 2007; and Nakagawa, M. et al. (2007), Nat. Biotechnol., published online ahead of print on November 30, 2007.

[0086] "Multi-lineage stem cells" or "pluripotent stem cells" refer to self-propagating stem cells and at least two further differentiated daughter cells from different developmental lineages. These lineages can be from the same germ layer (i.e., mesoderm, ectoderm, or endoderm), or from different germ layers. An example of two daughter cells with different developmental lineages differentiated from multi-lineage stem cells is myogenic cells and adipogenic cells (both of which are of mesoderm origin, but produce different tissues). Another example is neurogenic cells (ectoderm origin) and adipogenic cells (mesoderm origin).

[0087] A cell population refers to a collection of more than one cell that is phenotypically and / or genotypically identical (clonal) or different.

[0088] Amyloid beta precursor protein (APP) is an integral membrane protein expressed in many tissues and concentrated in the synapses of neurons. It acts as a cell surface receptor and is considered a regulator of synapse formation, neuroplasticity, antimicrobial activity and iron export. APP is a precursor molecule whose proteolysis produces amyloid beta (Aβ), a polypeptide containing 37 to 49 amino acid residues, whose amyloid fibril form is the main component of amyloid plaques present in the brains of patients with Alzheimer's disease. The protein sequence can be found in UniProt P05067 (human) and P12023 (mouse), or NCBI Ref. NP000745 (human), NP001 129488 (human) or NP001185752 (mouse) or NP001185753 (mouse). The mRNA encoding this protein can be found in NCBI Ref. NM201414 or NM000484 (human) or NM001198823 or NM001198824 (mouse), all of which were last accessed on April 1, 2023.

[0089] The microtubule-associated protein tau (MAPT) gene provides instructions for making a protein called tau, which is present throughout the nervous system, including in nerve cells (neurons) in the brain. It is involved in the assembly and stabilization of microtubules, which are rigid hollow fibers that make up the structural framework of cells (cytoskeleton). Microtubules help cells maintain their shape, assist in the cell division process, and are essential for the transport of substances within cells. Six different variants (isoforms) of tau protein are produced in the adult brain. The lengths of these isoforms vary from 352 to 441 protein building blocks (amino acids). The protein region known as the microtubule binding domain is the part of the protein that is attached (bound) to the microtubules, and it also varies between isoforms. In three isoforms, the microtubule binding domain contains three repeated segments. In the other three isoforms, the domain contains four repeated segments. Normally, the brain has about the same amount of three-repeat isomers and four-repeat isomers. This balance seems to be necessary for the normal function 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, resulting in several mRNA species. MAPT transcripts are differentially expressed in the nervous system, depending on the neuronal maturation stage and neuronal type. Mutations in the MAPT gene are associated with several neurodegenerative diseases, 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 part (subunit) of a complex called gamma (γ) secretase. Presenilin 1 performs the main function of the complex, which is to cut (cleave) other proteins into smaller fragments called peptides. This process is called proteolysis, and presenilin 1 is described as a proteolytic subunit of γ-secretase. The γ-secretase complex plays a role in processing the amyloid precursor protein (APP), which is produced in the brain and other tissues. γ-secretase cuts APP into smaller peptides, including soluble amyloid precursor protein (sAPP) and several variants of amyloid-beta (β) peptides. There is evidence 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 peptides are under investigation. See https: / / medlineplus.gov / genetics / gene / psen1 / . The amino acid and polynucleotide sequences of the protein and gene are available at https: / / www.ncbi.nlm.nih.gov / nuccore / ?term=Presenilin+1+(PSEN1), last accessed on April 1, 2023.

[0092] Triggering receptor 2 expressed on myeloid cells (Trem2) is a protein encoded by the TREM2 gene in humans. It is expressed on macrophages, immature monocyte-derived dendritic cells, osteoclasts, and microglia, which are immune cells in the central nervous system. In the liver, TREM2 is expressed by several cell types in response to injury, including macrophages. In the intestine, TREM2 is expressed by myeloid-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 on April 1, 2023.

[0093] Modes for carrying out the present disclosure

[0094] Applicants provide methods for treating a neurodegenerative disease, a disorder, or a symptom thereof in a subject in need thereof, wherein the neurodegenerative disease, disorder, or symptom is selected from: alleviating or treating Alzheimer's disease (AD) associated with microglial inflammation; reducing Aβ plaque load in the hippocampus and cortex; promoting or differentiating microglial ganglia; promoting donor hematopoietic stem cells or progenitor cells or donor cell populations comprising the hematopoietic stem cells or progenitor cells to differentiate into microglia; or reducing neuroinflammation; treating the subject's neurodegenerative disease, disorder, or symptom by administering an effective amount of hematopoietic stem cells or progenitor cells (HSPC) or HSPC populations to the subject. HSPC or its population is in one aspect a "donor" or donor-derived HSPC or its population because they are delivered or donated to the subject.

[0095] As used herein, the term "neurodegenerative disease or disorder" means a disorder of cell loss in the brain and spinal cord. Examples of neurodegenerative diseases include Alzheimer's disease, Parkinson's disease, prion disease, amyotrophic lateral sclerosis, motor neuron disease, Huntington's chorea, spinocerebellar ataxia, and dementia. Alleviating or treating a disease means reducing or in some aspects eradicating the clinical markers or features of the disease. Treating neurodegenerative symptoms means physical or mental problems experienced by a subject, which may indicate that the subject has or is experiencing a disease or disorder. Symptoms cannot be seen and will not show up in medical tests. Non-limiting examples of neurological disease symptoms include dementia, memory loss, sense of balance, numbness, or other cognitive decline.

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

[0097] Alzheimer's disease (AD) is the most common cause of dementia and the most common age-related neurodegenerative disorder. As a result of AD, dementia develops into severe disability throughout the disease progression, usually leading to death within 5 to 12 years after onset. Symptoms of AD can include neuronal degeneration, inflammation, and progressive memory and behavioral decline, which are caused by the accumulation of hyperphosphorylated tau in extracellular amyloid beta (Aβ) plaques and neurofibrillary tangles (NFTs) throughout the brain (mainly in the hippocampus and cortex). These main features of AD can lead to increased oxidative stress, inflammation, synaptic loss and cholinergic dysfunction, and trigger neuronal malnutrition.

[0098] It is obvious to those skilled in the art that the treatment methods disclosed herein can be combined with diagnostic methods for preliminary evaluation of the subject to be treated and then determining whether the treatment is successful. Symptoms that can be used for preliminary evaluation and monitoring of treatment include changes in personality or behavior, memory impairment; cognitive decline, mental status steps, neuropsychological tests, and laboratory tests, such as detection of measurable amyloid and tau proteins in cerebrospinal fluid. The ratio of these proteins can help determine whether Alzheimer's disease is present. Alzheimer's disease can also lead to progressive loss of brain cells, which can be assessed with brain scans such as magnetic resonance imaging (MRI), computed tomography (CT), and positron emission tomography (PET). PET scans use radioactive substances called tracers to detect substances in the body. There are different types of PET scans. The most commonly used PET scan is a fluorodeoxyglucose (FDG) PET scan. This scan can identify brain areas with reduced glucose metabolism. The pattern of metabolic changes can distinguish different types of degenerative brain diseases. PET scans have recently been developed to detect clusters of amyloid (plaques) or tau (neurofibrillary tangles) associated with Alzheimer's disease dementia. These types of PET scans are often used in research settings.

[0099] In another aspect, the HSPC or HSPC population is wild type. In another aspect, the cells are CD34 + In other aspects, the HSPC or HSPC population is genetically modified to correct a gene associated with a neurodegenerative disorder. The cells in the population can be a substantially homogenous HSPC or clonal population, such as CD34 + HSPCs. They can be autologous or allogeneic to the subject being treated. Methods for preparing substantially homogenous or clonal populations are known in the art.

[0100] In another aspect, the HSPC cell population comprises at least 80%, or at least 90%, or at least 95%, or at least 97% of the CD34+ HSPC cell population.

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

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

[0103] In one aspect, Alzheimer's disease is caused by known pathogenic gene mutations, which are optionally genes encoding presenilin 1 (PSEN1) or triggering receptor 2 (Trem2) expressed on myeloid cells. In another aspect, HSPC or HSPC colonies are modified to correct known familial mutations, such as mutations in APP, MAPT, PSEN1 and TREM2. Gene correction can be performed by a method selected from the following: gene addition (optionally by using a vector, such as an adeno-associated virus vector or a lentiviral vector), CRISPR / Cas9 technology, or by guiding editing--correcting a single base pair in a defective gene.

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

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

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

[0107] Cells or cell colonies can be administered systemically or topically. In one aspect, HSPC or HSPC colonies are administered by the hippocampus. In another aspect, they are administered systemically. For the purpose and approach of administration, cells or cell colonies can be combined with a carrier such as a pharmaceutically acceptable carrier. Dosage, dosage regimen, and route of administration will be determined by a treating physician or veterinarian, and may be determined taking into account the health, age, and sex of the subject.

[0108] 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 can be used to treat the non-human mammal or as an animal model to test new combination therapies. It will be apparent to those skilled in the art that untreated control animals can be used as reference points. The method can be combined with other treatments for neurological disorders such as Alzheimer's disease.

[0109] The applicant provides a method for treating or alleviating AD symptoms of a subject by administering an effective amount of hematopoietic stem cells or progenitor cells (HSPC) or HSPC colonies to a subject in need. HSPC or its colonies are "donors" or donor-derived HSPC or their colonies in one aspect because they are delivered or donated to the subject. Administration can be local or systemic. These cells can be autologous or allogeneic for the subject being treated, and can be wild-type HSPC. HSPC or colonies are CD34+ in one aspect, and can optionally be delivered in a pharmaceutically acceptable carrier. When a colony is administered, it can be substantially homologous or cloned HSPC or CD34+HSPC. The method can be combined with other treatments or diagnostic methods known in the art or as described herein. Those skilled in the art can monitor the therapy and assess when the therapy is effective based on clinical or observational criteria. Effective therapy will vary with the subject being treated and the purpose of the therapy.

[0110] The applicant provides a method for treating or alleviating AD symptoms of a person or mammal by administering an effective amount of hematopoietic stem cells or progenitor cells (HSPC) or HSPC colonies to a person or mammal in need, respectively. HSPC or its colony is a "donor" or donor-derived HSPC or its colony in one aspect, because they are delivered or donated to a person or mammal. Administration is systemic, and these cells are autologous for the person or mammal being treated. HSPC or colony is CD34+, and can optionally be delivered in a pharmaceutically acceptable carrier. When a colony is administered, it can be substantially homologous or cloned HSPC or CD34+HSPC. The method can be combined with other treatments or diagnostic methods known in the art or as described herein. Those skilled in the art can monitor the therapy, and evaluate when the therapy is effective based on clinical or observational criteria. Effective therapy will vary with the person or mammal being treated and the purpose of therapy.

[0111] Applicants provide methods for reducing Aβ plaque load in the hippocampus and cortex by administering an effective amount of hematopoietic stem or progenitor cells (HSPCs) or HSPC populations to a subject. The HSPCs or populations thereof are in one aspect "donor" or donor-derived HSPCs or populations thereof because they are delivered or donated to a subject.

[0112] Applicants provide methods for reducing the promotion or differentiation of microglial ganglia by administering an effective amount of hematopoietic stem cells or progenitor cells (HSPCs) or HSPC populations to a subject. HSPCs or populations thereof are in one aspect "donor" or donor-derived HSPCs or populations thereof because they are delivered or donated to a subject.

[0113] Applicants provide a method for promoting the differentiation of donor hematopoietic stem cells or progenitor cells or donor cell populations comprising the hematopoietic stem cells or progenitor cells into microglia by administering an effective amount of hematopoietic stem cells or progenitor cells (HSPC) or HSPC populations to a subject. HSPC or its population is in one aspect a "donor" or donor-derived HSPC or its population because they are delivered or donated to a subject.

[0114] Applicants provide methods of reducing neuroinflammation by administering an effective amount of hematopoietic stem or progenitor cells (HSPCs) or HSPC populations to a subject. HSPCs or populations thereof are in one aspect "donor" or donor-derived HSPCs or populations thereof because they are delivered or donated to a subject.

[0115] In each of these methods, the HSPC or HSPC population is wild type. In another aspect, the cells are CD34 + HSPC. The cells in the population can be a substantially homogenous HSPC or clonal population, such as CD34 + HSPCs. They can be autologous or allogeneic to the subject being treated. Methods for preparing substantially homogenous or clonal populations are known in the art.

[0116] In another aspect, the HSPC cell population comprises at least 80%, or at least 90%, or at least 95%, or at least 97% of the CD34+ HSPC cell population.

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

[0118] These methods can be combined with other treatments or diagnostic methods known in the art or as described herein. One skilled in the art can monitor the therapy and assess when the therapy is effective based on clinical or observational criteria. Effective therapy will vary with the subject being treated and the purpose of the therapy.

[0119] In another aspect, 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 can be used to treat the non-human mammal or as an animal model to test new combination therapies. It will be apparent to those skilled in the art that untreated control animals can be used as a reference point.

[0121] Also provided is a kit comprising genetically modified HSPCs and instructions for use, wherein the genetically modified HSPCs contain one or more exogenous or wild-type genes selected from the following: beta-amyloid precursor protein (APP), microtubule-associated protein tau (MAPT), genes encoding presenilin 1 (PSEN1) or triggering receptor 2 (Trem2) expressed on myeloid cells.

[0122] experiment

[0123] Without being bound by theory, the applicant hypothesizes that wild-type (WT) HSPC transplantation can lead to the generation of healthy microglia, which can lead to reduced neuroinflammation and Aβ plaque accumulation in the areas of the brain most affected in AD. To explore this hypothesis, the applicant used the 5xFAD double transgenic mouse model, which expresses mutant human amyloid beta (A4) precursor protein 695 (App) with Swedish (K670N, M671L), Florida (1716V), 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, Aβ plaque accumulation was observed in the hippocampus and cortex of 5xFAD mice 13，14 These mice showed significant neurocognitive impairment and anxiety-altering behaviors between 3 and 6 months of age. 15 Furthermore, 5xFAD mice display the main features of amyloid plaque pathology in AD, including dysfunctional microglia. 13，16 .

[0124] Here, the applicant demonstrated that a single WT HSPC transplant into adult 5xFAD mice resulted in preservation of memory and neurocognitive abilities and reduced Aβ plaque load in the hippocampus and cortex. Donor HSPCs differentiated into microglia with active amyloid plaque clearance potential while also resulting in reduced neuroinflammation. This work opens up new therapeutic avenues for the use of HSPC gene therapy to treat AD, neurodegenerative disorders, and symptoms associated with each of them.

[0125] Materials and methods

[0126] Animals. All mice were of C57BL6 background. The applicant used 5xFAD transgenic mouse model (Tg6799, C57BL6-Tg(APPSwFlLon, PSEN1*M146L*L286V)6799Vas, Jackson Laboratory) 13. Standard PCR reactions were performed using the following primers to identify mice expressing all 5 mutations: 5′-ACCCCCATGTCAGAGTTCCT-3′ (normal forward), 5′-CGGGCCTCTTCGCTATTAC-3′ (mutant reverse) and 5′-TATACAACCTTGGGGGATGG-3′ (WT reverse). GFP transgenic mice that widely express enhanced GFP complementary DNA under the control of the chicken β-actin promoter were used as donors for HSPC transplantation experiments (C57Bl / 6-Tg (CAG-EGFP) 1Osb / J; 003291, Jackson Laboratory). Mice were housed 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 at the University of California, San Diego (UCSD), and all protocols were approved by the UCSD Animal Care and Use Committee.

[0127] HSPC isolation, transduction, transplantation, and implantation. Bone marrow cells were extracted from the femurs of 5xFAD (GFP transgenic mice) aged 6 to 8 weeks. HSPCs were isolated by immunomagnetic separation using anti-Sca1 antibody conjugated to magnetic beads (Miltenyi Biotec). 2×10 resuspended in 100 ml phosphate-buffered saline (PBS) were injected via the tail vein. 6 Scal + HSPCs were directly transplanted into lethally irradiated (7 Gray; X-Rad 320; PXi) 5xFAD mice. + In mice with HSPCs, bone marrow cell engraftment of transplanted cells was measured in peripheral blood 2 months after transplantation; blood samples freshly collected from the tail were treated with red blood cell lysis buffer (eBioscience) and subsequently analyzed by flow cytometry (BD Accuri C6, BD Biosciences) to determine GFP expression. + The proportion of cells.

[0128] Behavioral testing. Mice were tested at 6 months of age using three major behavioral tests to assess neurocognitive and motor activity. Prior to behavioral testing, mice were exposed to the testing apparatus and testing room to acclimate to the environment. Applicants then performed the tests and analyzed NORT as previously published. 55,56. Briefly, during the familiarization phase, all mice had free access to two replicates of the same object for 10 min and were then returned to the home cage. After 24 h, one object was retained as the familiar object, while the other was replaced by a novel object. The entire process was recorded by an overhead camera, and the object study time was manually recorded by a blinded researcher. The elevated plus maze test is one of the most widely used tests for measuring anxiety-like behavior in mice. Applicants used the protocol previously published to perform the EPM 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 on the central area and allowed to explore the maze for 5 minutes. The time the mouse spent in the open arms and closed arms was recorded. Each animal was placed in the center of an open field (50 cm×50 cm) and allowed to move freely for 30 minutes while the movements were recorded by an overhead camera. The video recordings were then analyzed by an automatic tracking system (ANY-Maze).

[0129] Immunohistochemical analysis. Brains were collected from euthanized mice and fixed with 10% formalin, embedded in paraffin, and sectioned at 5 μm thickness by standard methods. Sections were dewaxed and stained with mouse anti-6E10 (1:30,000; 803001, Biolegend), rabbit anti-GFP (1:600; ab183734, Abcam), and rabbit anti-Iba-1 (1:3000; SAG4318, Wako) antibodies. Slides were stained on Ventana Discovery Ultra (Ventana Medical Systems). Antigen retrieval was performed at 95°C for 40 minutes using CC1 (Tris-EDTA based; pH 8.6). The primary antibody was incubated at 37°C for 32 minutes. For 6E10, an additional rabbit anti-mouse IgG antibody (1:6000; ab133469, Abcam) was used to minimize nonspecific staining of mouse to mouse. All rabbit antibodies were incubated on sections using HRP-conjugated goat anti-rabbit (OmniMap system; Ventana, catalog number 760-4315) at 37°C for 12 minutes and then developed 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 mosaic images of tissue sections. ImageProPremier software (Media Cybemetics) was used for quantification of Aβ plaques and Iba1.

[0130] Immunofluorescence and image acquisition. Sections were dewaxed and transferred to preheated antigen retrieval solution, kept at 95°C for 30 minutes, cooled at room temperature for 20 minutes, and incubated in blocking solution (0.25% Triton X-100 and 3% BSA in Tris-buffered saline). Sections were then incubated with the following primary antibodies overnight at 4°C: rabbit anti-GFP (1:200; ab290, Abcam), chicken anti-Iba1 (1:200; 234006, Synaptic Systems), and mouse anti-6E10 (1:250; 805701, BioLegend). Appropriate Alexa Fluor-conjugated secondary antibodies (Invitrogen) were used for visualization of antigens. Images were acquired using a Leica SP8 confocal microscope to obtain high-resolution mosaic images of tissue sections. Confocal image stacks and 3D view images were analyzed with Imaris software (Bitsplane, Oxford Instruments).

[0131] Aβ 1-42 ELISA. The brain tissue was homogenized in a 0.15% triton in PBS solution containing a protease inhibitor cocktail using a homogenizer and centrifuged at 200,000×g for 20 minutes at 4°C. The supernatant was removed and defined as the soluble fraction. Guanidine-HCl was added to 0.5 M (final concentration) before use in enzyme-linked immunosorbent assay (ELISA). The precipitate was dissolved in 6 M guanidine-HCl buffer containing a protease inhibitor cocktail by ultrasonic treatment. The dissolved precipitate was centrifuged at 200,000×g for 20 minutes at 4°C and used as the insoluble fraction. The amount of Aβ1-42 in each fraction was determined by a sandwich amyloid β-protein 42 ELISA kit (KHB3441, Invitrogen).

[0132] RNA-seq. Sample preparation. Use previously published protocols 58 Hippocampus and cortex were dissected from the brain and subsequently frozen at -80°C in RNA form. Total RNA was isolated using the RNeasy Tissue kit (Qiagen) according to the manufacturer's instructions. RNA quality was assessed using an Agilent Tapestation 4200 and RNA was isolated using a 400 μl (10 μl, 10 μl) kit according to the manufacturer's instructions. Stranded mRNA Prep (Illumina) used 50 ng RNA (RNA integrity number (RIN) greater than 8.0) to generate RNA sequencing libraries. 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] RNA-seq data analysis. RNASeq data were processed using Kallisto (version 0.45.0) and Mus musculus genome GRCm38 Ensembl version 94 annotations (Mus_musculus GRCm38.94chr_patch_hapl_scaff.gtf). Gene-level TPM values ​​and gene annotations were calculated using tximport and biomaRt R packages. Data were organized using custom python scripts and log2(TPM+1) was used to reduce log. Comprehensive feature analysis was performed and a comprehensive score for a set of genes was calculated. Genes were first normalized and averaged. 59 The threshold-centered modified Z-score method was used to normalize the gene expression values ​​(formula = (expr-SThr) / 3*stddev). The samples were ranked based on the final comprehensive score. 60 Differential expression analysis was performed. The false discovery rate was controlled by setting the adjusted P value threshold to 0.1.

[0134] Statistics. Applicants did not exclude any animals from the experiments. Experimenters were blinded to the genotype of specific samples whenever possible. In all experiments, cohorts were age-matched and gender-balanced. Non-objective computational measurements, such as immunostaining quantification by scanning equipment, 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 are comparable to previous publications using the 5xFAD model. 35The sample sizes reported in are similar. One-way analysis of variance (ANOVA) was used, followed by Tukey's multiple comparisons to compare multiple groups. Statistical significance was set at P < 0.05. For RNA-seq data, gene signatures were used to classify sample categories, and the performance of the multi-category classification was measured by ROC-AUC (area under the receiver operating characteristic curve) values. Color-coded bar graphs were 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 Welch's two-sample t-test (unpaired, unequal variances (equal_var=False), and unequal sample sizes) parameters. Multiple hypothesis correction was performed by adjusting the P value with statsmodels.stats.multitest.multipletests (fdr_bh: Benjamini / Hochberg principles). The results were independently verified using R statistical software (R version 3.6.1; 2019-07-05). Violin plots, bee swarm plots, and bubble plots were created using the python seaborn package version 0.10.1.

[0135] Code availability. The code used to perform the RNA sequencing analyses is publicly available at the following links: https: / / github.com / sahoo00 / BoNE; https: / / github.com / sahoo00 / Hegemon.

[0136] result

[0137] Transplantation of WT mouse HSPCs restores neurocognitive abilities in 5xFAD mice

[0138] To investigate the potential of WT HSPC transplantation for the treatment of AD, we used ScA1 cells isolated from WT green fluorescent protein (GFP) transgenic mice (5xFAD / WT HSPC; n=10, 4F and 6M). +HSPCs were injected intravenously into lethally irradiated 2-month-old 5xFAD mice. HSPC implantation of donor origin was measured by flow cytometry, and the results were expressed as the percentage of GFP-positive blood cells in peripheral blood, and the range was 25% to 90%. As a control, the applicant analyzed WT littermates (WT; n = 9, 5F and 4M), lethally irradiated WT littermates transplanted with WT mouse HSPCs (WT / WT HSPCs; n = 10, 5F and 5M), untreated 5xFAD littermates (5xFAD; n = 10, 5F and 5M), lethally irradiated 5xFAD mice transplanted with 5xFAD HSPCs (5xFAD / 5xFAD HSPCs; n = 10, 5F and 5M).

[0139] To determine the effect of WT HSPC transplantation on neurocognitive function in 5xFAD mice, Applicants performed a battery of well-characterized behavioral tests in 6-month-old mice, 4 months after transplantation. No differences between males and females were observed in any of the tests (data not shown). In addition, myeloablative radiation had no effect on the activity of both WT and 5xFAD mice, as no differences were observed in any of the tests when compared to WT / WT HSPC and 5xFAD / 5xFAD HSPC, respectively (Figure 1). Memory function in 5xFAD mice began to deteriorate at 4 to 5 months of age. 17，18 To determine whether WT HSPC transplantation could prevent memory loss, we used the well-validated hippocampal-dependent novel object recognition task (NORT). 19,20 . During training, mice were briefly exposed to two identical objects (F1 and F2), and in the test phase 24 hours later, mice were exposed to a new object (N) instead of F2; their discrimination index was then calculated. On the day of adaptation, the distance traveled by mice in all groups was equal (data not shown). On the day of testing, WT, WT / WT HSPC and 5xFAD / WT HSPC mice spent more time exploring new objects, indicating that mice transplanted with 5xFAD / WT HSPC remembered familiar objects and had normal ability to distinguish between new and familiar objects. In contrast, mice transplanted with untreated 5xFAD and 5xFAD / 5xFAD HSPC did not show significant changes in the time spent exploring new or familiar objects (Figures 1B to 1D), indicating impaired memory function.

[0140] Although impaired memory function is a hallmark of AD, changes in anxiety have also been shown to be a significant complication and are associated with cognitive impairment in AD patients. 21，22It has been reported that 5xFAD mice have altered anxiety-like behaviors in the elevated plus maze (EPM) associated with more time spent in the open arms 17 In fact, the aversion to open arms has been described as an innate fear of heights. 23 , and rodents prefer closed arms because they provide protection from potential predators 24 . Both 5xFAD mice and 5xFAD / 5xFAD HSPC mice spent more time in the open exposed arms, indicating that the anxiety and risk perception of these mice have changed (Figure 1E to Figure 1H). On the contrary, the more time spent by WT controls and 5xFAD / WT HSPC in closed arms is at similar levels (Figure 1E to Figure 1H). Then, the applicant tested the motor activity of mice in an open field. In the open field, compared with WT, WT / WT HSPC and 5xFAD / WT HSPC, the general motor function represented by speed (data not shown) and distance is slightly increased in 5xFAD and 5xFAD / 5xFAD HSPC (Figure 1K). In addition, compared with WT controls and 5xFAD / WT HSPC, 5xFAD and 5xFAD / 5xFAD HSPC show significantly higher levels of peripheral / corner activity (Figure 1I to Figure 1L), indicating that the 5xFAD mice processed by WT HSPC retain normal anxiety-like behavior and activity. Taken together, these results demonstrate that transplantation of WT HSPCs in 5xFAD mice leads to preservation of their memory, risk perception, normal anxiety levels, and motor activity.

[0141] Wild-type HSPC transplantation reduces amyloid-β deposition in the brains of 5xFAD mice

[0142] In AD, Aβ plaques are most abundant in the hippocampus and cortical regions of the brain 15 Aβ deposits were also found in the hippocampus and cortex of 6-month-old 5xFAD mice. 17 . Applicants quantified Aβ plaques in the hippocampus and cortex of 5xFAD mice using the 6E10 antibody against amino acids 1-16 of Aβ after immunohistochemical analysis. In both the hippocampus and cortex, Aβ levels in 5xFAD mice transplanted with 5xFAD HSPCs were equivalent to those of untreated 5xFAD mice (Figures 2A to 2B). In contrast, 5xFAD mice transplanted with WT HSPCs showed a significant reduction in the number, size, and total area of ​​plaques in both the cortex and hippocampus, respectively (Figures 2A to 2J). These results prompted the applicants to test whether the improved cognitive ability after reduced plaque burden was associated with reduced levels of soluble (Triton fraction) and insoluble (guanidine fraction) Aβ. Therefore, in all groups, Aβ was measured by ELISA. 1-42Applicants found that soluble and insoluble Aβ levels were significantly increased in 5xFAD mice 4 months after transplantation with WT HSPCs compared to untreated mice and mice treated with 5xFAD HSPCs. 1-42 Thus, these results demonstrate that transplantation of WT HSPCs has a significant beneficial effect on reducing the Aβ plaque burden in the cortex and hippocampus regions of 5xFAD mice.

[0143] Implanted WT HSPCs differentiate into microglia and participate in Aβ plaque reduction in 5xFAD mice

[0144] The applicant then studied WT GFP + HSPC engraftment and differentiation in the brain of 5xFAD mice, and GFP expression + HSPC-derived cells were engrafted into both the cortex and hippocampus of all treated mice (Fig. 3A). These cells were immunoreactive with Ibal, characterizing them as microglia-like cells (Fig. 3A, Fig. 3F). Recent reports suggest that microglial dysfunction promotes AD pathology and is associated with Aβ plaque clearance 25，26 . Compared with WT mice, both 5xFAD mice and 5xFAD / 5xFAD HSPC mice showed significantly higher immunoreactive areas for Iba1 staining in the hippocampus and cortex (Figure 3C, Figure 3D). In contrast, 5xFAD treated with WT HSPCs showed significantly reduced levels of Ibal positive cells in both the hippocampus and cortex compared with the 5xFAD control group. In addition, the microglia were mostly amoeboid and activated, indicating active inflammation in the hippocampus and cortex of 5xFAD and 5xFAD / 5xFAD mice (Figure 3b), as previously described for AD25, 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 by studying the phenotype of microglia adjacent to Aβ plaques using dual staining with Iba1 / 6E10. + The cells were active and inflammatory in 5xFAD mice transplanted with 5xFAD HSPCs, whereas they had a ramified and multipolar morphology and reduced plaque size in the brains of 5xFAD mice transplanted with WT HSPCs, as reflected by the clearance of the plaque halo (Figure 3E). +Microglia were derived from transplanted HSPCs. We performed immunofluorescence staining on brain sections from 5xFAD / WT HSPC mice and 5xFAD / 5xFAD HSPCs as controls and observed Iba1 in contact with plaques. + Microglia are also GFP + (Figure 3F). In addition, Aβ plaques were larger and more fibrotic in 5xFAD controls compared to 5xFAD / WT HSPC mice, and when GFP was expressed in 5xFAD / WT HSPC brains, Aβ plaques were more fibrotic and more fibrotic in 5xFAD controls compared to 5xFAD / WT HSPC mice. + When surrounded by microglia, plaque size decreased (Fig. 3F and Fig. 5). Taken together, these results indicate that amyloid plaques are engulfed by microglia derived from transplanted WT HSPCs, which accumulate around plaques and reduce amyloid burden by actively engulfing amyloid fibrils.

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

[0146] To decipher the relative contribution of HSPC transplantation to AD disease remission, Applicants examined transcriptome profiles by RNA sequencing (RNA-seq) in the hippocampus and cortex of WT (n=2), 5xFAD (n=3), 5xFAD / 5xFAD HSPC (n=3), and 5xFAD / WT HSPC (n=3) mice. Recent GWAS in AD patients have identified risk genes that are highly expressed in microglia and amplify the proinflammatory state. 6，27-29 Since we observed that transplanted WT HSPCs differentiated into microglia in the brain, resulting in a reduction in inflammatory microglia in the hippocampus and cortex of 5xFAD mice, we hypothesized that there would be different myeloid expression profiles between treated 5xFAD / WT HSPC mice and the disease group. We therefore evaluated the myeloid expression profiles of WT HSPCs in a previously published dataset of signature genes associated with AD. 29,30A comprehensive gene scoring analysis was performed, analyzing the transcriptome profiles of the hippocampus and cortex. Applicants evaluated two previously disclosed sequential but distinct stages of "disease-associated microglia" (DAMs) in AD. Stage 1 represents the first stage of DAM activation; it is Trem2-independent and involves activation of the gene set Cx3cr1, P2ry12, Tmem119, Tyrobp, Ctsb, Ctsd, Apoe, B2m, Fth1, Lyz2. The second stage of DAM activation, Stage 2, includes the induction of genes related to lipid metabolism and phagocytic pathways associated with Aβ clearance (Trem2, AXL, Cst7, CtsI, LPL, Cd9, Csf1, Ccl6, Itgax, Clec7a, Lirb4, Timp2) 4 In addition, the applicants also evaluated previously published universal macrophage markers (Tyorbp, Fcer1g) from human and mouse tissues and found that they were differentially expressed in AD. 31 . Comprehensive gene analysis revealed that DAM markers and universal macrophage markers of stage 1 and stage 2 had higher expression levels in the cortex of 5xFAD and 5xFAD / 5xFAD HSPC mice compared to 5xFAD / WT HSPC and WT mice, because there were outliers in 5xFAD / 5xFAD HSPC, so the difference was only significant in 5xFAD mice (Figure 4A, Figure 6A, Figure 6B). These data show that microglial inflammatory activation in 5xFAD mice was rescued by WT HSPC transplantation. This was confirmed by gene expression analysis (DEG), which revealed that among the 61 genes differentially expressed in the cortex between 5xFAD / WT HSPC mice and 5xFAD mice, 49 genes were upregulated in 5xFAD mice compared to 5xFAD / WT HSPC mice, and 24 of them were associated with microglia (Figure 4B).

[0147] Interestingly, the distribution of microglia in the hippocampus did not differ between groups. Further gene scoring analysis revealed that the expression of neurodegeneration-related endothelial cell genes in the hippocampus of 5xFAD mice and 5xFAD / 5xFAD HSPC mice was significantly increased compared to 5xFAD mice transplanted with WT HSPCs (Figure 4A). DEG analysis also revealed that the top 7 differentially expressed genes in the hippocampus of 5xFAD / WT HSPC mice were associated with endothelial cells compared to 5xFAD controls (Figure 4B). This phenomenon is unique to the hippocampus, as the endothelial cell gene profiles in the cortex did not differ between groups.

[0148] Experimental Discussion

[0149] Alzheimer's disease is the most common cause of dementia and the most common age-related neurodegenerative disorder. With an aging population, AD is expected to affect 131.5 million people worldwide by 2050, with devastating effects. The cost of AD is expected to soar from $1 trillion to $2 trillion by 2030, placing a heavy burden on patients, families and public health systems. 32,33 Over the past few decades, a great deal of work has been done to develop disease-modifying drugs that halt, prevent, or slow the progression of AD. Unfortunately, although hundreds of therapies from multiple pharmacological classes have shown preclinical efficacy in ameliorating cognitive impairment and disease burden in animal models, no therapy has been shown to be effective in human clinical trials. 33 Therefore, there is an urgent unmet medical need for the treatment of AD.

[0150] Reactive microglia have long been considered a consequence of AD pathology, but are now thought to potentially play a role in disease progression and may be the initiator of disease. 25,27,29,34 However, the role of microglia in AD remains a matter of intense debate. 35 Our study provides direct evidence that microglia play a key role in disease progression and that replacing diseased App / Psen1 microglia with healthy cells in 5xFAD mice by a single WT HPSC transplant completely rescued neurocognitive impairment in mice. As Aβ deposition is associated with cognitive changes, we examined Aβ plaque levels in different groups of animals. Consistent with previous reports linking Aβ reduction with functional and cognitive improvements, the effects of WTHSPC transplantation on behavior were accompanied by a decrease in 6E10 in the hippocampus and cortex. + Plaques and soluble Aβ 1-42 Without being bound by theory, the 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. Specifically, early studies have shown that plaques form over time in amyloid mouse models and AD patients. 36,37 and are surrounded by microglia that appear to be reactive and ineffective in phagocytosis and clearance of Aβ 38-41 In contrast, microglia derived from transplanted WT HSPCs were similar to 6E10 cells, which appeared smaller. + Plaques colocalized, indicating that WT microglia in 5xFAD / WT HSPC mice actively engulfed Aβ plaques. This is consistent with recent reports that observed that Trem2 involved in microglia-dependent phagocytosis increased Aβ deposition. 42,43In addition, studies have shown that when they are young and healthy, they can wrap around Aβ plaques and form a barrier and engulf these plaques. 25 , while microglia can participate in the spread of Aβ aggregates by carrying and releasing phagocytosed Aβ seeds in the brain, thereby forming new plaques, as d'Erico et al. 35 Applicants show that this beneficial or detrimental effect of microglia on Aβ burden depends on their ability to process Aβ peptides and that transplantation of WT HSPCs to replace App / Psen1 mutant microglia shifts the balance to reduce Aβ burden. Therefore, this data represents direct evidence for the involvement of microglia in Aβ burden control and proliferation.

[0151] Applicants noted strong changes in the phenotype and density of microglial populations following transplantation of WT HSPCs in 5xFAD. Proliferation and activation of microglia in the brain are important features in AD, and Applicants confirmed these features in 5xFAD mice that were either untreated or transplanted with App / Psen1 HSPCs. Notably, in mice transplanted with WT HSPCs, Iba1 + The number and morphological changes of microglia were significantly reduced. This key feature also plays a direct role in the neurocognitive preservation of 5xFAD mice. In fact, chronically activated microglia continuously release inflammatory mediators and amplify the chronic inflammatory environment of AD, providing minimal protection and even leading to synaptic and neuronal loss by engulfing synapses and secreting inflammatory factors that damage neurons. 9，41，44 . Applicants show for the first time that replacing microglia carrying mutations in the App and Psen1 genes with healthy microglia results in a reduction in the number of microglia and a reduced inflammatory state. Applicants also show that this is sufficient to prevent neurocognitive impairment in 5xFAD mice, strongly suggesting that microglia-associated neuroinflammation in AD is a major feature of neurodegeneration in AD and that modulation of this phenotype represents an important therapeutic target. In summary, the data suggest that healthy microglia have a therapeutic effect on AD by enhancing engulfment of plaques, limiting the spread of aggregates, and preventing chronic neuroinflammation.

[0152] To further assess changes in the gene profiles of brain cell populations, the applicants used RNAseq analysis to examine the effects of WTHSPC transplantation on the cortex and hippocampus of 5xFAD mice. Studies have shown that plaque-associated microglia activate specific signaling pathways, and Keren-Shaul et al. identified DAMs that increase with disease progression in 5xFAD mice and in human postmortem brains. 29Indeed, DAM in AD is associated with the expression of genes, many of which were discovered in human genome-wide association studies (GWAS), including Trem2, a receptor required for DAM activation. 29,45 Specifically, DAMs are significantly overexpressed in late AD, when increased phagocytic and inflammatory activity leads to more damage to surrounding cell populations. However, Applicants showed that 5xFAD mice transplanted with WT HSPCs exhibited a significant reduction in DAMs in the cortex, while the newly introduced microglia retained the ability to phagocytose and engulf plaques. 31 Universal macrophage markers that are increased in neurological diseases, including gliomas, were also reduced in 5xFAD / WT HSPC mice. Together, these data demonstrate for the first time that the pathways triggered by App and Psen1 mutations can be blocked in the absence of these mutations in microglia.

[0153] Surprisingly, the distribution of microglia in the hippocampus did not differ between the groups. Interestingly, however, microglia have been reported to be involved in other neurodegenerative disorders. 30 The neurodegeneration-associated endothelial cell gene expression profile that was increased in AD was also found to be increased in this tissue compartment in 5xFAD mice, and this was corrected in 5xFAD / WT HSPC mice. In AD, age-dependent degeneration of the endothelial blood-brain barrier (BBB) ​​occurs in the human hippocampus, and severe impairment of the BBB transport machinery has been reported to occur late in the disease and contribute to hippocampus-dependent cognitive impairment. 46-49 In addition, there is increasing evidence that endothelial dysfunction plays a key role in the development of AD. 50 In AD, the accumulation of Aβ plaques may impair normal endothelial function and cause endothelium-dependent vasoconstriction 50 On the other hand, it has also been noted that endothelial cells participate in Aβ clearance when microglial clearance mechanisms are unable to function due to higher plaque loads. However, unlike microglia, endothelial cells are unable to proliferate efficiently. Therefore, sustained endothelial cell exposure to debris such as amyloid plaques can trigger cell death, dysfunction, decreased blood flow, and increased inflammatory responses in AD. 51 Therefore, we hypothesize that WT HSPC-derived microglia protect the endothelium of the hippocampus by clearing plaques as well as reducing inflammation, which further leads to AD rescue. These findings support previous findings that microglia and endothelial cells are essential for maintaining cognitive function in AD. 52-54 .

[0154] In summary, Applicants' findings demonstrate that transplantation of WT HSPCs into the 5xFAD mouse model results in engraftment and differentiation of these cells into microglia, thereby rescuing the AD disease phenotype. This report provides answers to the long-standing debate as to whether restoration of microglia can modulate Aβ load during disease progression and whether this is sufficient to prevent neurocognitive impairment in 5xFAD mice, demonstrating for the first time that microglia alone can modulate Aβ load and rescue AD. Therefore, this work strongly supports this strategy as a therapeutic approach to treat AD and represents evidence for the use of HSPC gene therapy to correct known familial mutations in AD, opening new promising therapeutic prospects for this incurable condition.

[0155] Incorporated by Reference

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

[0157] Equivalent

[0158] Unless defined otherwise, 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 present technology exemplarily described herein can be suitably implemented in the absence of any one or more elements, any one or more limitations not specifically disclosed herein. Thus, for example, the terms "comprising", "including", "containing", etc. should be understood broadly and not restrictively. In addition, the terms and expressions adopted herein have been used as descriptive terms rather than limiting terms, and when using such terms and expressions, it is not intended to exclude any equivalents of the features shown and described or parts thereof, but it should be recognized that various modifications are possible within the scope of the present technology claimed.

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

[0161] The technology has been described broadly and generically herein. Each narrower species and subgeneric grouping falling within the generic disclosure also forms part of the technology. This includes the generic description of the technology with a proviso or negative limitation deleting any subject matter from the genus, regardless of whether the deleted material is specifically recited herein.

[0162] In addition, where features or aspects of the technology are described in terms of Markush groups, those skilled in the art will recognize that the technology is also thereby described in terms of any individual member or subgroup of members of the Markush group.

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

[0164] Other aspects are set out in the following claims. References 1 Sun, 2 Jia,J.et al. The cost of Alzheimer's disease in China and re-estimation of costs worldwide. Alzheimers Dement 14, 483-491, doi:10.1016 / j.jalz.2017.12.006(2018). 3 Robinson,M.,Lee,BY&Hane,FTRecent Progress in Alzheimer'sDisease Research,Part 2:Genetics and Epidemiology.J Alzheimers Dis 57,317-330,doi:10.3233 / JAD-161149(2017). 4 Vermunt, L. et al. Duration of preclinical, prodromal, and dementia stages of Alzheimer's disease in relation to age, sex, and APOEgenotype. 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al.Kidney preservation by bone marrow celltransplantation in hereditary nephropathy.Kidney Int 79,1198-1206,doi:10.1038 / ki.2010.537(2011). 17 Syres,K.et al.Successful treatment of the murine model ofcystinosis using bone marrow cell transplantation.Blood 114,2542-2552,doi:10.1182 / blood-2009-03-213934(2009). 18 Oakley,H.et al.Intraneuronal beta-amyloid aggregates,neurodegeneration,and neuron loss in transgenic mice with five familialAlzheimer's disease mutations:potential factors in amyloid plaque formation.JNeurosci 26,10129-10140,doi:10.1523 / JNEUROSCI.1202-06.2006(2006). 19 Richard,B.C.et al.Gene Dosage Dependent Aggravation of theNeurological Phenotype in the 5XFAD Mouse Model of Alzheimer's Disease.JAlzheimers Dis 45,1223-1236,doi:10.3233 / JAD-143120(2015). 20 Jawhar,S.,Trawicka,A.,Jenneckens,C.,Bayer,T.A.&Wirths,O.Motordeficits,neuron loss,and reduced anxiety coinciding with axonal degenerationand intraneuronal Abeta aggregation in the 5XFAD mouse model of Alzheimer'sdisease.Neurobiol Aging 33,196e129-140,doi:10.1016 / j.neurobiolaging.2010.05.027(2012). 21 Wendt,S.et al.Changes in phagocytosis and potassium channelactivity in microglia of 5xFAD mice indicate alterations in purinergicsignaling in a mouse model of Alzheimer's disease.Neurobiol Aging 58,41-53,doi:10.1016 / j.neurobiolaging.2017.05.027(2017). 22 Jawhar,S.,Trawicka,A.,Jenneckens,C.,Bayer,T.A.&Wirths,O.Motordeficits,neuron loss,and reduced anxiety coinciding with axonal degenerationand intraneuronal Aβaggregation in the 5XFAD mouse model of Alzheimer'sdisease.Neurobiol Aging 33,196.e129-140,doi:10.1016 / j.neurobiolaging.2010.05.027(2012). 23 Cotel,M.C.,Jawhar,S.,Christensen,D.Z.,Bayer,T.A.&Wirths,O.Environmental enrichment fails to rescue working memory deficits,neuronloss,and neurogenesis in APP / PS1KI mice.Neurobiol Aging 33,96-107,doi:10.1016 / j.neurobiolaging.2010.02.012(2012). 24 Reed,J.M.&Squire,L.R.Impaired recognition memory in patients withlesions limited to the hippocampal formation.Behav Neurosci 111,667-675,doi:10.1037 / / 0735-7044.111.4.667(1997). 25 Wang,H.,Ferguson,G.D.,Pineda,V.V.,Cundiff,P.E.&Storm,D.R.Overexpression of type-1 adenylyl cyclase in mouse forebrain enhancesrecognition memory and LTP.Nat Neurosci 7,635-642,doi:10.1038 / nn1248(2004). 26 Burke,A.D.,Goldfarb,D.,Bollam,P.&Khokher,S.Diagnosing and TreatingDepression in Patients with Alzheimer's Disease.Neurol Ther 8,325-350,doi:10.1007 / s40120-019-00148-5(2019). 27 Kaiser,N.C.et al.Differences in anxiety among patients with early-versus late-onset Alzheimer's disease.J Neuropsychiatry Clin Neurosci 26,73-80,doi:10.1176 / appi.neuropsych.12100240(2014). 28 Mendez,M.F.Erratum:The Relationship Between Anxiety and Alzheimer's Disease.J Alzheimers Dis Rep 5,563,doi:10.3233 / ADR-219003(2021). 29 Pentkowski,N.S.,Rogge-Obando,K.K.,Donaldson,T.N.,Bouquin,S.J.&Clark,B.J.Anxiety and Alzheimer's disease:Behavioral analysis and neuralbasis in rodent models of Alzheimer's-related 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Claims

1. A method for treating a neurodegenerative disease, disorder or symptom thereof in a subject in need thereof, wherein the neurodegenerative disorder or symptom is selected from: a) Alleviating or treating Alzheimer's disease (AD) associated with microglial inflammation; b) reduced Aβ plaque burden in the hippocampus and cortex; c) promote or differentiate microglial ganglia; d) promoting differentiation into microglia; or e) reduce neuroinflammation; The method comprises administering to the subject an effective amount of hematopoietic stem or progenitor cells (HSPCs) or a population of HSPCs, thereby treating the neurodegenerative disease, disorder or symptom in the subject.

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

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

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

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

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

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

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

9. The method according to claim 8, wherein the known familial mutation is corrected by gene addition, optionally by using 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 according to 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 HSPC population is detectably labeled.

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

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

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

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

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 and instructions for use, wherein the genetically modified HSPCs contain one or more exogenous or wild-type genes selected from the group consisting of beta-amyloid precursor protein (APP), microtubule-associated protein tau (MAPT), genes encoding presenilin 1 (PSEN1) or triggering receptor expressed on myeloid cells 2 (Trem2).

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