Compositions and methods for treating Alzheimer's disease

Abstract

The present disclosure features methods and compositions for treating Alzheimer's disease. The disclosed methods include administering to a subject having or suspected of having Alzheimer's disease hematopoietic stem progenitor cells that express at least one neuroprotective agent, such as ApoE2, Trem2, and / or metallothionein.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims the benefits of U.S. Provisional Application No. 62 / 908,913, filed on 1 October 2019, the entire contents of which are incorporated herein by reference. [Background technology]

[0002] Background of the Invention Alzheimer's disease is a neurodegenerative disease characterized by progressive cognitive decline. Recognized as one of the leading causes of death in the United States and a major cause of dementia, Alzheimer's disease is a progressive disease for which no cure or effective treatment is known. It is estimated that more than 5 million Americans have the disease, and it is projected that more than 14 million will be affected by 2060. Neuronal loss in the brain and extracellular deposition of fibrous beta-amyloid (Aβ) protein plaques are prominent pathological features of this disease. Aβ plaques are also widely present in the inner walls of small and medium-sized arteries and arterioles in the cerebral cortex and meninges, as well as in the cerebral microvessel system, and are observed in 85% of Alzheimer's cases in a condition known as cerebral amyloid angiopathy. Because there is no known cure for Alzheimer's disease, there is an urgent need for a treatment for the disease. [Overview of the Initiative]

[0003] As described below, this disclosure features compositions and methods for treating Alzheimer's disease. More specifically, in some embodiments, therapeutic approaches are disclosed that include administering a composition comprising cells expressing at least one therapeutic transgene to a target of interest.

[0004] In one aspect, the present invention is characterized by an expression vector or expression cassette comprising a polynucleotide encoding two or more of the following: apolipoprotein E isoform 2 (ApoE2) polypeptide, triggering receptor 2 (Trem2) polypeptide expressed on myeloid cells, and metallothionein polypeptide, or fragments thereof.

[0005] In some aspects of the above-described situation, the polynucleotide encodes ApoE2 and metallothionein 1G, TREM2 and metallothionein 1G, ApoE2 and TREM2, or ApoE2, TREM2 and metallothionein 1G (MT1G).

[0006] In another aspect, the present invention is characterized by an expression vector or expression cassette comprising polynucleotides encoding the TREM2 polypeptide and the ApoE2 polypeptide or fragments thereof.

[0007] In any of the above scenarios, the vector or cassette contains two or more copies of metallothionein. In any of the above scenarios, the vector contains at least four copies of a polynucleotide encoding MT1G.

[0008] In another aspect, the present invention is characterized by an expression vector comprising an expression cassette for any one of the above aspects.

[0009] In any of the above situations, the vector is a lentiviral vector.

[0010] In any of the above aspects, the vector includes a promoter that drives the expression of the polynucleotide. In some embodiments, the promoter is a human phosphoglycerate kinase promoter. In some embodiments, the promoter is a microglia-specific promoter. In some embodiments, the promoter is a TSPO promoter, an MHC class II promoter, or a CX3CR1 promoter.

[0011] In another aspect, the present invention is characterized by a lentiviral vector comprising a phosphoglycerate kinase (PGK) promoter that drives the expression of a TREM2 polypeptide and an ApoE2 polypeptide, or a polynucleotide encoding a fragment thereof.

[0012] In another aspect, the present invention features a lentiviral vector comprising a microglia-specific promoter that drives the expression of a TREM2 polypeptide and an ApoE2 polypeptide, or a polynucleotide encoding a fragment thereof.

[0013] In any of the above situations, the vector contains one or more copies of a polynucleotide encoding metallothionein.

[0014] In another aspect, the present invention provides cells comprising a vector or cassette of any of the above aspects. In various embodiments, the cassette is inserted into the CX3CR1 or TSPO locus. In some embodiments, the cells are microglia or their progenitor cells, hematopoietic stem cells, hematopoietic stem progenitor cells (HSPCs), or progeny cells of hematopoietic stem cells or hematopoietic stem progenitor cells. In some embodiments, the HSPCs are CD34 + and / or CD38 - and / or CD90 + In some embodiments, the cells are hemizygous for the CX3CR1 gene.

[0015] In another aspect, the present invention provides a method for reducing the level of amyloid-beta in cells or tissues, the method comprising contacting the cells with a polynucleotide encoding two or more of the following: ApoE2 polypeptide, Trem2 polypeptide, and metallothionein polypeptide, or fragments thereof.

[0016] In another aspect, the present invention provides a method for increasing phagocytosis of β-amyloid by cells, the method comprising contacting the cells with a polynucleotide encoding two or more of the ApoE2 polypeptide, Trem2 polypeptide, and metallothionein polypeptide, or fragments thereof.

[0017] In another aspect, the present invention provides a method for treating a subject who has or is prone to developing Alzheimer's disease, the method comprising administering to the subject an effective amount of cells containing a polynucleotide encoding two or more of the ApoE2 polypeptide, Trem2 polypeptide, and metallothionein polypeptide, or fragments thereof.

[0018] In some aspects, Alzheimer's disease is either familial Alzheimer's disease or early-onset Alzheimer's disease.

[0019] In another aspect, the present invention provides a method of treating a subject having or prone to developing neuroinflammation, the method comprising administering to the subject an effective amount of cells comprising a polynucleotide encoding two or more of an ApoE2 polypeptide, a Trem2 polypeptide, and a metallothionein polypeptide, or fragments thereof. In any of the above aspects, the polynucleotide is contained in an expression vector. In some embodiments, the expression vector is a lentiviral vector. In any of the above aspects, the polynucleotide comprises an expression cassette. In any of the above aspects, the polynucleotide encodes ApoE2 and TREM2, ApoE2 and metallothionein 1G, TREM2 and metallothionein 1G, or ApoE2, TREM2, and metallothionein 1G (MT1G). In any of the above aspects, the polynucleotide encodes one or more copies of metallothionein. In any of the above aspects, the polynucleotide encodes at least 4 copies of MT1G. In any of the above aspects, the polynucleotide encodes a Trem2 polypeptide and an ApoE2 polypeptide, or fragments thereof. In any of the above aspects, the polynucleotide further encodes one or more copies of MT1G.

[0020] In any of the above aspects, the polynucleotide comprises a promoter. In any of the above aspects, the polynucleotide comprises a constitutive promoter. In some embodiments, the promoter is a phosphoglycerate kinase promoter. In some embodiments, the promoter is a microglia-specific promoter. In some embodiments, the promoter is a TSPO promoter, an MHC class II promoter, or a CX3CR1 promoter.

[0021] In any of the above aspects, the cells are microglial cells or their progenitor cells, hematopoietic stem cells, hematopoietic stem progenitor cells (HSPCs), or their progeny cells. In some embodiments, the method is performed in vitro or in vivo. In any of the above aspects, the cells are administered intracerebroventricularly, intravenously, or intrathecally.

[0022] In any of the above aspects, the cell is a hematopoietic stem progenitor cell (HSPC). In some embodiments, the HSPC is Lin - , CD34 + , CD38 - , and / or CD90 + . In some embodiments, the HSPC is functionally equivalent to a microglial progenitor cell after transplantation. In some embodiments, the HSPC engrafts in the brain. In some embodiments, the engrafted HSPC is functionally equivalent to a microglial progenitor cell or expresses markers characteristic of microglial progenitor cells.

[0023] In any of the above aspects, the subject undergoes a destructive pretreatment prior to the method. In some embodiments, the destructive pretreatment comprises administering an alkylating agent to the subject. In some embodiments, the alkylating agent is busulfan. In some embodiments, the pretreatment comprises administering a CSF-1R inhibitor. In some embodiments, the inhibitor is PLX3397, PLX5622, or liposomal clodronic acid.

[0024] In any of the above aspects, the HSPC is an allogeneic or autologous cell. In any of the above aspects, the cell is hemizygous for the CX3CR1 gene.

[0025] In any of the above aspects, the method reduces anxiety, improves cognitive function, or increases short-term working memory. In any of the above aspects, the method reduces microglial activation and / or astrocyte response. In any of the above aspects, the method decreases the levels of Iba1 and / or GFAP.

[0026] In another aspect, the present invention provides a pharmaceutical composition containing the HSPC according to claim 15.

[0027] In another aspect, the present invention provides a kit comprising the HSPC described in claim 15 and instructions for delivering it to the target.

[0028] Other features and advantages of the present invention will become apparent from the detailed description and claims.

[0029] definition Unless otherwise defined, all technical and scientific terms used herein have the meanings generally understood by those skilled in the art in the field to which this invention pertains. The following references provide general definitions of many terms used herein: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd edition, 1994); The Cambridge Dictionary of Science and Technology (Walker, ed., 1988); The Glossary of Genetics, 5th edition, R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991). Unless otherwise specified, the following terms have the meanings defined below.

[0030] As used herein, "destructive pretreatment" refers to administering a composition that destroys endogenous microglia to a target.

[0031] "Drugs" refers to small molecule compounds, antibodies, nucleic acid molecules, polypeptides, or fragments thereof.

[0032] "Change" means a change (increase or decrease) in the expression level or activity of a gene or polypeptide, as detected by standard methods known in the art, such as those described herein. As used herein, a change includes changes of 10%, 25%, 40%, and 50% or more in expression level.

[0033] "To improve" means to reduce, suppress, weaken, alleviate, prevent, or stabilize the onset or progression of a disease.

[0034] "Apolipoprotein E isoform 2 (ApoE2) polypeptide" refers to a protein or fragment having at least approximately 85% amino acid sequence identity with GenBank accession number ALQ33369.1 and possessing immunomodulatory activity. An example ApoE2 polypeptide sequence is shown below.

[0035] >ALQ33369.1 Apolipoprotein E isoform 2, partially [Homo sapiens] TIFF0007860891000001.tif26165

[0036] "Apolipoprotein E isoform 2 (ApoE2) polynucleotide" refers to a nucleic acid molecule that encodes the ApoE2 polypeptide. The ApoE2 gene encodes a membrane protein that mediates the binding, internalization, and catabolism of lipoprotein particles. An example ApoE2 polynucleotide sequence is shown below.

[0037] >KU177911.1 Homo sapiens apolipoprotein E isoform 2 (APOE) mRNA, partial cds, alternative splicing type TIFF0007860891000002.tif77165

[0038] A "biological sample" refers to tissue, cells, bodily fluids, or other material derived from an organism.

[0039] In this disclosure, “comprises,” “comprising,” “containing,” and “having” can have the meanings defined in U.S. patent law, and may mean “includes,” “including,” etc.; similarly, “consisting essentially of” or “consists essentially” also have the meanings defined in U.S. patent law, and this term is a non-restrictive term that allows for other entities as long as the basic or novel characteristics of the described are not altered by other entities, but prior art is excluded.

[0040] As used herein, the terms “determine,” “evaluate,” “assay,” “measure,” and “detect” refer to both quantitative and qualitative determinations. Therefore, the term “determine” is interchangeable with “assay,” “measure,” and so on. When quantitative determination is intended, the expression “measure a quantity” of the analyte is used. When qualitative and / or quantitative determination is intended, the expression “measure a level” of the analyte or “detect” the analyte is used.

[0041] "Detecting" refers to confirming the presence, absence, or quantity of the analyte to be detected.

[0042] A “detectable label” means a composition that, when bound to a molecule of interest, makes the latter detectable by spectroscopic, photochemical, biochemical, immunochemical, or chemical means. Useful labels include, for example, radioisotopes, magnetic beads, metal beads, colloidal particles, fluorescent dyes, electron-density reagents, enzymes (e.g., those commonly used in ELISA), biotin, digoxigenin, or haptens.

[0043] "Disease" means any pathological condition or disorder that damages or interferes with the normal function of cells, tissues, or organs. Examples of diseases include neurodegenerative diseases such as Alzheimer's disease.

[0044] The “effective dose” refers to the amount required to improve the symptoms of the disease compared to an untreated patient. The effective dose of the active compound(s) used to carry out the present invention for the therapeutic treatment of the disease varies depending on the method of administration, the age, weight, and general health status of the subject. Ultimately, the attending physician or veterinarian will determine the appropriate dose and dosage plan. Such a dose is called the “effective” dose. In certain embodiments, the effective dose is the amount that alleviates at least one symptom of Alzheimer’s disease, increases cognitive function, or extends survival time.

[0045] An "enhancer" refers to a polynucleotide that increases the transcription of the gene of interest. In one embodiment, the enhancer contains 50 to 1,500 nucleotides. Examples of enhancers useful in the methods of the present invention include, but are not limited to, the following:

[0046] >MPP05A(hTSPO_Upstream Enhancer)("E1.1") TIFF0007860891000003.tif40165

[0047] >MPP05B(hTSPO_Upstream Enhancer)("E1.2") TIFF0007860891000004.tif98165

[0048] >MPP06(hTSPO_Intron Enhancer)("E2") TIFF0007860891000005.tif33165

[0049] In various embodiments, the enhancer is E1, which includes E1.1 and E1.2. In various embodiments, the E1 enhancer includes the sequences E1.1+E1.2 or E1.2+E1.1 in the indicated order from 5' to 3'.

[0050] The following are arrangements that include other modulo elements useful in the method of the present invention:

[0051] >MPP03(hTSPO_Upstream Enhancer + Upstream and Intron Promoter) TIFF0007860891000006.tif128165TIFF0007860891000007.tif70165

[0052] >MPP06-01(hTSPO_proximal 5' promoter + proximal upstream promoter) TIFF0007860891000008.tif84165

[0053] >MPP03(hTSPO_upstream+intron promoter) TIFF0007860891000009.tif188165

[0054] As used herein, "exogenous nucleic acid molecule" refers to a nucleic acid molecule that is not endogenous; that is, a nucleic acid molecule that is not originally present in cells.

[0055] An "expression cassette" refers to the vector elements necessary for gene expression. In one embodiment, the expression cassette includes a promoter, a polynucleotide encoding the polypeptide of interest, and a terminator.

[0056] "Fragment" means a portion of a protein or nucleic acid that is substantially identical to a reference protein or nucleic acid. In some embodiments, the portion retains at least 50%, 75%, or even 80%, 85%, 90%, 95%, or 99% of the biological activity of the reference protein or nucleic acid described herein.

[0057] A "gene locus" refers to a location within the genome where a specific gene sequence is situated.

[0058] "Hematopoietic stem cells (HSCs)" refer to stem cells that produce various types of blood cells.

[0059] "Hematopoietic stem progenitor cells (HSPCs)" refer to cells that are the precursors to hematopoietic stem cells.

[0060] Cells that may be used in combination with the compositions and methods described herein (e.g., HSCs, HSPCs) include CD34+ cells, CD34+ / CD90+ cells, CD34+CD38- cells, and CD34+ / CD164+ cells. These cells may contain higher proportions of HSCs or HSPCs. These cells are described in WO2015 / 059674; WO2017 / 218948; Radtke et al. Sci. Transl. Med. 9: 1-10, 2017; Radtke et al. Mol Ther Methods Clin Dev.18:679-691, 2020; and Pellin et al. Nat. Comm. 10: 2395, 2019, the contents of which are incorporated herein by reference in their entirety.

[0061] "Hybridization" refers to the hydrogen bonding between complementary nucleic acid bases (which can be Watson-Crick hydrogen bonds, Hoogstin hydrogen bonds, or reverse Hoogstin hydrogen bonds). For example, adenine and thymine are complementary nucleic acid bases that pair up through the formation of hydrogen bonds.

[0062] "Inhibitory nucleic acid" means a double-stranded RNA, siRNA, shRNA, antisense RNA, or a portion thereof, or its mimetic, that, when administered to mammalian cells, results in a reduction in the expression of a target gene (e.g., a 10%, 25%, 50%, 75%, or 90–100% reduction). Typically, nucleic acid inhibitors include at least a portion of the target nucleic acid molecule or its ortholog, or at least a portion of the complementary strand of the target nucleic acid molecule. For example, inhibitory nucleic acid molecules include at least a portion of any or all of the nucleic acids detailed herein.

[0063] The terms “isolated,” “purified,” or “biologically pure” refer to substances that do not contain, to varying degrees, the components normally associated with them as they would be found in nature. “Isolating” means the degree of separation from the source or environment. “Purifying” means a higher degree of separation than isolation. A “purified” or “biologically pure” protein is one from which other substances have been sufficiently removed so that no impurities substantially affect the biological properties of the protein or cause other harmful consequences. That is, the nucleic acids or peptides of this invention are purified if they are substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA technology, or chemical precursors or other chemicals when synthesized chemically. Purity and homogeneity are typically determined using analytical chemistry techniques, such as polyacrylamide gel electrophoresis or high-performance liquid chromatography. The term “purified” may mean that the nucleic acid or protein produces essentially a single band on an electrophoretic gel. For proteins that can undergo modifications such as phosphorylation or glycosylation, differences in modification will result in different isolated proteins, which can be purified separately.

[0064] "Isolated polynucleotide" means a nucleic acid (e.g., DNA) that does not contain adjacent genes in the naturally occurring genome of the organism from which the nucleic acid molecule of the present invention originates. Therefore, this term includes, for example, recombinant DNA that is incorporated into a vector, an autonomously replicating plasmid or virus, or the genomic DNA of a prokaryotic or eukaryotic organism, or a separate molecule independent of other sequences (e.g., cDNA, or a genome or cDNA fragment produced by PCR or restriction endonuclease digestion). Furthermore, this term includes RNA molecules transcribed from DNA molecules, as well as recombinant DNA that is part of a hybrid gene encoding an additional polypeptide sequence.

[0065] "Isolated polypeptide" means the polypeptide of the present invention that has been isolated from its naturally associated components. Typically, the polypeptide is isolated if it is at least 60% by weight and does not contain naturally associated proteins and naturally occurring organic molecules. In some embodiments, the preparation contains at least 75% by weight, at least 90% by weight, or at least 99% by weight of the polypeptide of the present invention. The isolated polypeptide of the present invention can be obtained, for example, by extraction from a natural source, expression of recombinant nucleic acid encoding the polypeptide, or chemical synthesis of a protein. Purity can be measured by any suitable method, for example, column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis.

[0066] A "marker" refers to any protein or polynucleotide whose expression level or activity is altered in relation to a disease or disorder. Examples of markers for Alzheimer's disease include, but are not limited to, tau protein and β-amyloid peptide.

[0067] "Metallothionein 1G (MT1G) polypeptide" refers to a protein or fragment having at least approximately 85% amino acid sequence identity with UniProt accession number P13640-1, and possessing heavy metal binding activity. An example sequence of MT1G polypeptide is shown below.

[0068] >sp|P13640|MT1G_Human Metallothionein 1G OS=Homo sapiens OX=9606 GN=MT1G PE=1 SV=2 TIFF0007860891000010.tif11164

[0069] "Metallothionein 1G (MT1G) polynucleotide" refers to a nucleic acid molecule that codes for the MT1G polypeptide. The MT1G gene codes for a protein that binds to heavy metals. An example sequence of the MT1G polynucleotide is shown below.

[0070] >NM_005950.2 Homo sapiens metallothionein 1G (MT1G), transcription variant 1, mRNA TIFF0007860891000011.tif55165

[0071] "Microglia" refers to immune cells in the central nervous system.

[0072] "Nanoparticles" refer to composite structures with nanoscale dimensions. In particular, nanoparticles are typically particles with a size in the range of about 1 to about 1000 nm and are usually spherical, but different morphologies are possible depending on the composition of the nanoparticles. The portion of the nanoparticle that comes into contact with the external environment is generally identified as the surface of the nanoparticle. In the nanoparticles described herein, the size limit may be limited to two dimensions, and therefore the nanoparticles described herein include composite structures having a diameter of about 1 to about 1000 nm; in this case, the specific diameter depends on the composition of the nanoparticles and the intended use of the nanoparticles by experimental design. For example, nanoparticles used in some therapeutic applications typically have a size of about 200 nm or less, and those used in particular for delivery related to therapeutic drugs typically have a diameter of about 1 to about 100 nm.

[0073] As used herein, “neurodegenerative disease” refers to any group of diseases characterized by progressive loss of neuronal structure and / or function, including neuronal death. Exemplary neurodegenerative diseases include, but are not limited to, Alzheimer’s disease.

[0074] In this specification, "to obtain a drug" includes synthesizing, purchasing, or otherwise acquiring a drug.

[0075] "PLX3397" refers to a colony-stimulating factor 1 receptor (CSF-1R) inhibitor and has the following structure. TIFF0007860891000012.tif62128

[0076] "PLX5622" refers to a colony-stimulating factor 1 receptor (CSF-1R) inhibitor and has the following structure. TIFF0007860891000013.tif37128

[0077] As used herein, terms such as "prevent," "prevention," and "preventive measures" refer to reducing the probability of developing a disorder or condition in subjects who do not currently have a disorder or condition but are at risk of developing one or are prone to developing one.

[0078] A “promoter” means a polynucleotide sufficient to instruct transcription. In some embodiments, the promoter is a translocator protein (TSPO) promoter. In some embodiments, the promoter is a CX3CR1 promoter. In exemplary embodiments, the CX3CR1 promoter contains a sequence or fragment having at least 85% sequence identity to the sequence of GeneBank accession number GQ258357.1. The sequence of GeneBank accession number GQ258357.1 is shown below.

[0079] GeneBank accession number GQ258357.1: TIFF0007860891000014.tif233165TIFF0007860891000015.tif25163

[0080] "To reduce" means a negative change of at least 10%, 25%, 50%, 75%, or 100%.

[0081] "Standard" refers to a standard or comparison condition.

[0082] A "reference sequence" is a defined sequence used as the basis for sequence comparison. A reference sequence can be a subset or the whole of a particular sequence; for example, a segment of a full-length cDNA or gene sequence, or a complete cDNA or gene sequence. In the case of polypeptides, the length of a reference polypeptide sequence is generally, in some embodiments, at least about 16 amino acids, at least about 20 amino acids, at least about 25 amino acids, or about 35 amino acids, about 50 amino acids, or about 100 amino acids, or any integer around or in between. In the case of nucleic acids, the length of a reference nucleic acid sequence is generally at least about 50 nucleotides, at least about 60 nucleotides, at least about 75 nucleotides, at least about 100 nucleotides, or at least about 300 nucleotides, or any integer around or in between.

[0083] Nucleic acid molecules useful in the methods of the present invention include any nucleic acid molecules encoding the polypeptide or a fragment thereof of the present invention. Such nucleic acid molecules do not need to be 100% identical to the endogenous nucleic acid sequence, but will usually exhibit substantial identity. Polynucleotides having "substantial identity" with respect to the endogenous sequence can usually hybridize with at least one strand of a double-stranded nucleic acid molecule. Nucleic acid molecules useful in the methods of the present invention include any nucleic acid molecules encoding the polypeptide or a fragment thereof of the present invention. Such nucleic acid molecules do not need to be 100% identical to the endogenous nucleic acid sequence, but will usually exhibit substantial identity. Polynucleotides having "substantial identity" with respect to the endogenous sequence can usually hybridize with at least one strand of a double-stranded nucleic acid molecule. "Hybridizing" means pairing with a complementary polynucleotide sequence (e.g., the genes described herein) or a portion thereof to form a double-stranded molecule under various stringency conditions (see, for example, Wahl, GM and SL Berger (1987) Methods Enzymol. 152:399; Kimmel, AR (1987) Methods Enzymol. 152:507).

[0084] The terms “nucleotide molecule,” “polynucleotide,” or “nucleic acid sequence” are used interchangeably to refer to molecules containing RNA or DNA. In various embodiments, a nucleotide molecule or polynucleotide contains modified nucleotides (e.g., locked nucleic acid (LNA)). In some embodiments, a nucleotide molecule or polynucleotide contains RNA and DNA. The sugar backbone of a nucleotide molecule is non-limiting and may contain ribose, deoxyribose, or various other suitable sugars. In some embodiments, a nucleic acid molecule contains at least two nucleotides covalently bonded together. In some embodiments, the nucleic acid molecule of the present invention is single-stranded. In some embodiments, the nucleic acid molecule is double-stranded. In some embodiments, the nucleic acid molecule is triple-stranded. In some embodiments, the nucleic acid molecule contains phosphodiester bonds. In some embodiments, the nucleic acid molecule contains single-stranded or double-stranded deoxyribonucleic acid (DNA) or single-stranded or double-stranded ribonucleic acid (RNA). In some embodiments, the nucleic acid molecule contains nucleic acid analogs. In some embodiments, the nucleic acid analog has a skeleton that includes, in addition to and / or in addition to phosphodiester bonds, bonds such as phosphoramide, phosphorothioate, phosphorodithioate, or O-methylphosphoramidite bonds, as non-limiting examples. In some embodiments, the nucleic acid analog is selected from nucleic acid analogs having skeletons selected from positive skeletons, nonionic skeletons, and non-ribose skeletons. In some embodiments, the nucleic acid molecule comprises one or more carbocyclic sugars. In some embodiments, the nucleic acid molecule includes modifications to its ribose-phosphate skeleton. In some embodiments, these modifications are made to facilitate the addition of additional parts, such as labels. In some embodiments, these modifications are made to increase the stability of such molecules in a physiological environment and to extend their half-life.In some aspects, the term “polynucleotide” includes, but is not limited to, sequences containing any of the known base analogs of DNA and RNA, such as: 4-acetylcytosine, 8-hydroxy-N6-methyladenosine, aziridinylcytosine, pseudoisocytosine, 5-(carboxyhydroxyl-methyl)uracil, 5-fluorouracil, 5-bromouracil, 5-carboxymethylaminomethyl-2-thiouracil, 5-carboxymethylaminomethyluracil, dihydrouracil, inosine, N6-isopentenyladenine, 1-methyladenine, 1-methylpseudouracil, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3 -Methylcytosine, 5-methylcytosine, N6-methyladenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxy-amino-methyl-2-thiouracil, β-D-mannosylosine, 5'-methoxycarbonylmethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetate methyl ester, uracil-5-oxyacetic acid, oxybutoxosin, pseudouracil, queosin, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, -uracil-5-oxyacetate methyl ester, uracil-5-oxyacetic acid, pseudouracil, queosin, 2-thiocytosine, and 2,6-diaminopurine.

[0085] For example, stringent salt concentrations are typically less than 750 mM NaCl and 75 mM trisodium citrate, less than 500 mM NaCl and 50 mM trisodium citrate, or less than 250 mM NaCl and 25 mM trisodium citrate. Low-stringency hybridization is obtained in the absence of organic solvents, such as formamide, while high-stringency hybridization is obtained in the presence of at least about 35% formamide, and in some embodiments, at least about 50% formamide. Stringent temperature conditions typically include temperatures of at least about 30°C, at least about 37°C, or at least about 42°C. Various additional parameters, such as hybridization time, the concentration of surfactants, such as sodium dodecyl sulfate (SDS), and the inclusion or exclusion of carrier DNA, are well known to those skilled in the art. By combining these various conditions as needed, various levels of stringency can be obtained. In one embodiment, hybridization is carried out at 30°C in 750 mM NaCl, 75 mM trisodium citrate, and 1% SDS. In another embodiment, hybridization is carried out at 37°C in 500 mM NaCl, 50 mM trisodium citrate, 1% SDS, 35% formamide, and 100 μg / ml denatured salmon sperm DNA (ssDNA). In yet another embodiment, hybridization is carried out at 42°C in 250 mM NaCl, 25 mM trisodium citrate, 1% SDS, 50% formamide, and 200 μg / ml ssDNA. Useful variations of these conditions will be readily apparent to those skilled in the art.

[0086] In most applications, the washing step following hybridization also differs in terms of stringency. Washing stringency conditions can be defined by salt concentration and temperature. As with the above, washing stringency can be increased by lowering the salt concentration or raising the temperature. For example, stringent salt concentrations for the washing step include less than 30 mM NaCl and 3 mM trisodium citrate, or less than 15 mM NaCl and 1.5 mM trisodium citrate. Stringent temperature conditions for the washing step typically include temperatures of at least about 25°C, at least about 42°C, or at least about 68°C. In some embodiments, the washing step is carried out at 25°C in 30 mM NaCl, 3 mM trisodium citrate, and 0.1% SDS. In other embodiments, the washing step is carried out at 42°C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. In other embodiments, the washing step is carried out at 68°C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. Further variations of these conditions will be readily apparent to those skilled in the art. Hybridization techniques are well known to those skilled in the art and are described, for example, in Benton and Davis (Science 196:180, 1977); Grunstein and Hogness (Proc. Natl. Acad. Sci., USA 72:3961, 1975); Ausubel et al. (Current Protocols in Molecular Biology, Wiley Interscience, New York, 2001); Berger and Kimmel (Guide to Molecular Cloning Techniques, 1987, Academic Press, New York); and Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York.

[0087] The term "subject" or "patient" refers to an animal that is the subject of treatment, observation, or experiment. By way of example only, subjects include, but are not limited to, mammals, including human or non-human mammals such as non-human primates, mice, cows, horses, dogs, sheep, cats, etc.

[0088] "Substantially identical" means a polypeptide or nucleic acid molecule that exhibits at least 50% identity to a reference amino acid sequence (e.g., any one of the amino acid sequences described herein) or nucleic acid sequence (e.g., any one of the nucleic acid sequences described herein). In some embodiments, such sequences are identical at the amino acid or nucleic acid level to the sequence used for comparison by at least 60%, 80%, 85%, 90%, 95% or even 99%.

[0089] Sequence identity is typically measured using sequence analysis software (e.g., the sequence analysis software package of the Genetics Computer Group of the University of Wisconsin Biotechnology Center (1710 University Avenue, Madison, Wis. 53705), BLAST, BESTFIT, GAP, or the PILEUP / PRETTYBOX program). Such software collates identical or similar sequences by assigning a degree of homology to various substitutions, deletions, and / or other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. In an exemplary approach for determining the degree of identity, the BLAST program is used, and e -3 ~e -100 probability scores indicate sequences that are closely related.

[0090] "Subject" means a mammal, including but not limited to human or non-human mammals such as cows, horses, dogs, sheep, cats, etc.

[0091] A “translocator protein promoter” or “TSPO promoter” means a polynucleotide sufficient to instruct the expression of a transgene in microglial cells. In one embodiment, the TSPO promoter responds to inflammation. Examples of promoters useful in the methods of the present invention include, but are not limited to, the following:

[0092] >MPP01(hTSPO_proximal 5' promoter)("P1") TIFF0007860891000016.tif56165

[0093] The P1 promoter contains 635 bp, corresponding to nucleotide residues -562 to +73 (uppercase) of the hTspo immediate-type 5' promoter.

[0094] >MPP02(hTSPO_Intron 5' Promoter)("P2") TIFF0007860891000017.tif99165

[0095] >MPP03(hTSPO_upstream+intron promoter)("P1+P2") TIFF0007860891000018.tif159165

[0096] >MPP04(hTSPO_intron+upstream promoter)("P2+P1") TIFF0007860891000019.tif157165

[0097] >MPP02(hTSPO_Intron 5' Promoter) TIFF0007860891000020.tif128165

[0098] As used herein, terms such as “treat,” “administer,” and “cure” refer to reducing or improving a disorder and / or its associated symptoms. It will be understood, though not to exclude, that treating a disorder or condition does not require the complete elimination of the disorder, condition, or its associated symptoms.

[0099] A "transgene" refers to an exogenous nucleic acid molecule introduced into a host cell that encodes a polypeptide or polynucleotide expressed within the host cell.

[0100] "Triggering receptor 2 (Trem2) polypeptide expressed on myeloid cells" refers to a protein or fragment having at least approximately 85% amino acid sequence identity with UniProt accession number Q9NZC2 and possessing immunomodulatory activity. An example of a Trem2 polypeptide sequence is shown below.

[0101] >sp|Q9NZC2|TREM2_Human triggering receptor 2 expressed on myeloid cells OS=Homo sapiens OX=9606 GN=TREM2 PE=1 SV=1 TIFF0007860891000021.tif26164

[0102] "Triggering receptor 2 (TREM2) polynucleotide expressed on myeloid cells" refers to the nucleic acid molecule encoding the Trem2 polypeptide. The TREM2 gene encodes a membrane protein that forms a receptor signaling complex with the TYRO protein tyrosine kinase-binding protein. An example TREM2 polynucleotide sequence is shown below.

[0103] >AK312215.1 Homo sapiens cDNA, FLJ92504, triggering receptor 2 (TREM2) expressed on Homo sapiens myeloid cells, mRNA TIFF0007860891000022.tif99165

[0104] The ranges provided herein are understood to be abbreviated representations of all values ​​within that range. For example, the range 1–50 is understood to include any number, combination of numbers, or sub-range from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.

[0105] As used herein, terms such as “treat,” “administer,” and “cure” refer to reducing or improving a disorder and / or its associated symptoms. It will be understood, though not to exclude, that treating a disorder or condition does not require the complete elimination of the disorder, condition, or its associated symptoms.

[0106] Unless otherwise specified or it is clear from the context, the term "or" as used herein is understood to be inclusive. Unless otherwise specified or it is clear from the context, the terms "a," "an," and "the" as used herein are understood to be singular or plural.

[0107] Unless otherwise specified or as is evident from the context, the term “about” as used herein is understood to mean within the normal range of acceptance in the art, for example, within two standard deviations of the mean. “About” can be understood to mean within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise evident from the context, all numerical values ​​provided herein are modified by the term “about.”

[0108] In this specification, the enumeration of lists of chemical groups in the definition of a variable element includes the definition of that variable element as any single group or as a combination of the listed groups. In this specification, the enumeration of embodiments of a variation or aspect includes that embodiment as any single embodiment or in combination with any other embodiment or part thereof.

[0109] Any composition or method provided herein may be combined with any one or more of the other compositions and methods provided herein. [Invention 1001] A method for treating a subject who has Alzheimer's disease or is prone to developing Alzheimer's disease, comprising administering to the subject an effective amount of cells comprising an expression vector or expression cassette comprising one or more polynucleotides encoding ApoE2 polypeptide and Trem2 polypeptide. [Invention 1002] An expression vector or expression cassette containing a polynucleotide encoding ApoE2 and metallothionein 1G, TREM2 and metallothionein 1G, ApoE2 and TREM2, or ApoE2, TREM2 and metallothionein 1G (MT1G). [Invention 1003] An expression vector or expression cassette containing polynucleotides encoding the TREM2 polypeptide and the ApoE2 polypeptide or fragments thereof. [Invention 1004] The method of the present invention 1001, or the expression vector or expression cassette of the present invention 1002 or the present invention 1003, wherein the vector or cassette contains two or more copies of metallothionein. [Invention 1005] An expression vector or expression cassette according to the method of Invention 1001 or Invention 1004, or any of Inventions 1002 to 1004, wherein the vector comprises a polynucleotide encoding at least four copies of MT1G. [Invention 1006] The vector comprises a promoter that drives the expression of the polynucleotide, according to any method of Invention 1001 and 1004-1005, or any expression vector or expression cassette according to Invention 1002-1005. [Invention 1007] The method of the present invention 1006, or the expression vector or expression cassette of the present invention 1006, wherein the promoter is a human phosphoglycerate kinase promoter. [Invention 1008] The method of the present invention 1006, or the expression vector or expression cassette of the present invention 1006, wherein the promoter is a microglia-specific promoter. [Invention 1009] The method of the present invention 1008, or the expression vector or expression cassette of the present invention 1008, wherein the promoter is a TSPO promoter, an MHC class II promoter, or a CX3CR1 promoter. [Invention 1010] An expression vector comprising any of the expression cassettes described in invention 1002 to 1009. [Invention 1011] The method of any of Invention 1001 and 1004-1009, or an expression vector or expression cassette of any of Invention 1001-1009, or the vector of Invention 1010, wherein the vector is a lentiviral vector. [Invention 1012] A lentiviral vector comprising a phosphoglycerate kinase (PGK) promoter that drives the expression of polynucleotides encoding the TREM2 polypeptide and the ApoE2 polypeptide or fragments thereof. [Invention 1013] A lentiviral vector comprising a microglia-specific promoter that drives the expression of a polynucleotide encoding the TREM2 polypeptide and the ApoE2 polypeptide or fragments thereof. [Invention 1014] A lentiviral vector according to the present invention 1012 or 1013, further comprising one or more copies of a polynucleotide encoding metallothionein. [Invention 1015] A cell comprising any vector of Invention 1002 to 1014 or any cassette of Invention 1002 to 1009 and 1011. [Invention 1016] The cell of the present invention 1015, wherein the cassette is inserted into the CX3CR1 or TSPO gene locus. [Invention 1017] Cells according to Invention 1015 or Invention 1016, which are microglia or their progenitor cells, hematopoietic stem cells, hematopoietic stem progenitor cells (HSPCs), or progeny cells of hematopoietic stem cells or hematopoietic stem progenitor cells. [Invention 1018] The aforementioned HSPC is CD34 + and / or CD38 - and / or CD90 + The cell according to the present invention 1017. [Invention 1019] A cell according to any of the invention items 1015 to 1018, which is hemizygous for the CX3CR1 gene. [Invention 1020] A method for reducing the level of amyloid-beta in a cell or tissue, comprising contacting the cell with a polynucleotide encoding two or more of the following: ApoE2 polypeptide, Trem2 polypeptide, and metallothionein polypeptide, or fragments thereof. [Invention 1021] A method for increasing phagocytosis of β-amyloid by cells, comprising contacting the cells with a polynucleotide encoding two or more of the following: ApoE2 polypeptide, Trem2 polypeptide, and metallothionein polypeptide, or fragments thereof. [Invention 1022] The method of the present invention 1020 or 1021, wherein the polynucleotide is included in the expression vector. [Invention 1023] The method of the present invention 1022, wherein the expression vector is a lentiviral vector. [Invention 1024] Any method 1020 to 1023 of the present invention, wherein the polynucleotide comprises an expression cassette. [Invention 1025] The method according to any of the present invention 1020 to 1024, wherein the polynucleotide encodes ApoE2 and TREM2, ApoE2 and metallothionein 1G, TREM2 and metallothionein 1G, or ApoE2, TREM2 and metallothionein 1G (MT1G). [Invention 1026] Any method of the present invention 1020 to 1025, wherein the polynucleotide encodes one or more copies of metallothionein. [Invention 1027] The method of the present invention 1026, wherein the polynucleotide encodes at least four copies of MT1G. [Invention 1028] Any method of the present invention 1020 to 1027, wherein the polynucleotide contains a promoter. [Invention 1029] The method of the present invention 1028, wherein the promoter is a phosphoglycerate kinase promoter. [Invention 1030] The method of the present invention 1028, wherein the promoter is a microglia-specific promoter. [Invention 1031] The method of the present invention 1030, wherein the promoter is a TSPO promoter, an MHC class II promoter, or a CX3CR1 promoter. [Invention 1032] The method according to any one of items 1020 to 1031 of the present invention, wherein the cells are microglia, hematopoietic stem cells, hematopoietic stem progenitor cells (HSPCs), or their progeny cells. [Invention 1033] Any method 1020 to 1032 of the present invention, performed in vitro or in vivo. [Invention 1034] A method for treating a subject having Alzheimer's disease or being prone to developing Alzheimer's disease, comprising administering to the subject an effective amount of cells containing a polynucleotide encoding two or more of the following: ApoE2 polypeptide, Trem2 polypeptide, and metallothionein polypeptide, or fragments thereof. [Invention 1035] A method for treating a subject having or prone to developing neuroinflammation, comprising administering to the subject an effective amount of cells containing a polynucleotide encoding two or more of the following: ApoE2 polypeptide, Trem2 polypeptide, and metallothionein polypeptide, or fragments thereof. [Invention 1036] The method of the present invention 1034 or 1035, wherein the polynucleotide is included in the expression vector. [Invention 1037] The method of the present invention 1036, wherein the expression vector is a lentiviral vector. [Invention 1038] Any method 1034 to 1037 of the present invention, wherein the polynucleotide comprises an expression cassette. [Invention 1039] The method according to any one of items 1034 to 1038 of the present invention, wherein the cells are administered into the ventricle, intravenously, or intrathecally. [Invention 1040] The method according to any of the present invention 1034 to 1039, wherein the polynucleotide encodes ApoE2 and TREM2, ApoE2 and metallothionein 1G, TREM2 and metallothionein 1G, or ApoE2, TREM2 and metallothionein 1G (MT1G). [Invention 1041] The method according to any one of the present invention 1034 to 1040, wherein the polynucleotide encodes a TREM2 polypeptide and an ApoE2 polypeptide, or a fragment thereof. [Invention 1042] The method of the present invention 1041, wherein the polynucleotide further codes for one or more copies of MT1G. [Invention 1043] Any method 1034 to 1042 of the present invention, wherein the polynucleotide contains a promoter. [Invention 1044] The method of the present invention 1043, wherein the promoter is a human phosphoglycerate kinase promoter. [Invention 1045] The method of the present invention 1043, wherein the promoter is a microglia-specific promoter. [Invention 1046] The method of the present invention 1045, wherein the promoter is a TSPO promoter, an MHC class II promoter, or a CX3CR1 promoter. [Invention 1047] The method according to any one of items 1034 to 1046 of the present invention, wherein the cells are microglia cells or their progenitor cells, hematopoietic stem cells, hematopoietic stem progenitor cells (HSPCs) or their progeny cells. [Invention 1048] The method of the present invention 1047, wherein the cells are hematopoietic stem progenitor cells (HSPCs). [Invention 1049] The aforementioned HSPC is Lin - CD34 + CD38 - , and / or CD90 + The method of the present invention 1048. [Invention 1050] The method according to any one of the present invention 1047 to 1049, wherein the HSPC is functionally equivalent to microglial progenitor cells after transplantation. [Invention 1051] A method according to any of the present invention 1047 to 1050, wherein the HSPC engrafts in the brain. [Invention 1052] The method of the present invention 1051, wherein the engrafted HSPCs are functionally equivalent to microglial progenitor cells or express markers characteristic of microglial progenitor cells. [Invention 1053] A method according to any one of items 1034 to 1052 of the present invention, wherein the subject undergoes a destructive pretreatment prior to the method. [Invention 1054] The method of the present invention 1053, wherein the destructive pretreatment includes administering an alkylating agent to the subject. [Invention 1055] The method of the present invention 1054, wherein the alkylating agent is busulfan. [Invention 1056] The method of the present invention 1053, wherein the pretreatment includes administering a CSF-1R inhibitor. [Invention 1057] The method of the present invention 1056, wherein the inhibitor is PLX3397, PLX5622, or liposomal clodronate. [Invention 1058] The method according to any of items 1047 to 1052 of the present invention, wherein the HSPC is an allogeneic cell or a self-cell. [Invention 1059] The method according to any of items 1034 to 1058 of the present invention, wherein the cells are hemizygous with respect to the CX3CR1 gene. [Invention 1060] The method of the present invention 1034, wherein Alzheimer's disease is familial Alzheimer's disease or early-onset Alzheimer's disease. [Invention 1061] Any method of the present invention 1034 to 1060 for reducing anxiety, improving cognitive function, or increasing short-term working memory. [Invention 1062] A method according to any one of items 1034 to 1061 of the present invention for reducing microglial activation and / or astrocyte response. [Invention 1063] Any method 1034-1062 of the present invention for reducing the levels of Iba1 and / or GFAP. [Invention 1064] A pharmaceutical composition containing HSPC according to Invention 1017 or Invention 1018. [Invention 1065] A kit comprising an HSPC according to Invention 1017 or Invention 1018, and instructions for delivering it to the target. [Brief explanation of the drawing]

[0110] [Figure 1]Figure 1 shows the expression of transgenes by the method of this disclosure. Figure 1 is a Western blot image showing the expression of Trem2 and ApoE2 in 293T cells and BV-2 microglia cells that were pre-transduced with lentiviral vectors encoding human ApoE2 and Trem2 cDNA. [Figure 2A] Figures 2A–2E are schematic diagrams, annotated sequences, peptide domain maps, and bar graphs related to the preparation and testing of lentiviral vectors. Figure 2A is a schematic diagram showing the structure of the Trem2 protein on the cell membrane. ECS: Extracellular space; ICS: Intracellular space. [Figure 2B] Figures 2A–2E show schematic diagrams, annotated sequences, peptide domain maps, and bar graphs related to the construction and testing of lentiviral vectors. Figure 2B shows the annotated amino acid sequence of Trem2. Shaded fragments indicate the functional domains of Trem2, and bold or shaded letters indicate single amino acid mutations in Trem2 associated with Alzheimer's disease (AD). [Figure 2C] Figures 2A–2E show schematic diagrams, annotated sequences, peptide domain maps, and bar graphs related to the construction and testing of lentiviral vectors. Figure 2C provides peptide domain maps for ApoE variants, namely ApoE2, ApoE3, and ApoE4. ApoE3 is the dominant "neutral" variant in the majority of people. ApoE4 is associated with an increased incidence of Alzheimer's disease (AD), while ApoE2 carriers exhibit a certain level of resistance to Alzheimer's disease (AD). [Figure 2D] Figures 2A–2E show schematic diagrams, annotated sequences, peptide domain maps, and bar graphs related to the preparation and testing of lentiviral vectors. Figure 2D is a schematic diagram of an in vitro model for evaluating the phagocytic ability of the microglial cell line BV2 cells to Aβ using a fluorescence imaging system. [Figure 2E]Figures 2A–2E show schematic diagrams, annotated sequences, peptide domain maps, and bar graphs related to the preparation and testing of lentiviral vectors. Figure 2E is a bar graph showing the quantification of AB oligomer uptake by transduced and control cells. Mean ± SD. Asterisks indicate significance in the Kruskal-Wallis test, "*" indicates p<0.05, and "****" indicates p<0.0001. [Figure 3A] Figures 3A and 3B are bar graphs and Western blot images showing the transduction of 5xFAD hematopoietic stem cells (HSCs) with therapeutic vectors. Lin-hematopoietic stem progenitor cells (HSPCs) were isolated from 5xFAD donors and transduced with therapeutic ApoE2, Trem2, and MT1G lentiviral vectors (LV). Figure 3A is a bar graph showing the transduction efficiency of 5xFAD HSPCs with the indicated vectors, expressed as mean vector copy number (VCN) ± SD. The mean vector copy number was measured in liquid culture progeny of HSPCs transduced with the lentiviral vectors indicated for in vivo transplantation. Figure 3B provides Western blots demonstrating the expression of human ApoE2 and human Trem2 in liquid culture progeny of transduced HSCs. HSCs were transduced using the indicated lentiviral vectors. HepG cells expressing hApoE2 were used as a positive control. [Figure 3B] See the explanation in Figure 3A. [Figure 4A]Figures 4A–4F are bar graphs and plots showing the phenotype of 5xFAD mice in behavioral studies. 5xFAD mice (untreated and mock-transplanted) and age-matched wild-type controls (untreated and mock-transplanted) underwent monthly behavioral studies from 4 to 12 months of age using novel object recognition tests (Figure 4A), Y-maze (Figure 4B), and elevated cross maze (Figure 4C); the latter demonstrated statistically significant behavioral defects in 5xFAD animals from 6 months of age. At 12 months of age, mice underwent a Morris water maze test and showed clear phenotypic differences by multiple parameters including probe trial latency (Figure 4D), latency to reach the hidden platform (Figure 4E), and reverse trial latency (Figure 4F). Mean ± SEM (standard error of the mean); asterisks indicate significance in multiple t-tests for repeated measures. [Figure 4B] See the explanation in Figure 4A. [Figure 4C] See the explanation in Figure 4A. [Figure 4D] See the explanation in Figure 4A. [Figure 4E] See the explanation in Figure 4A. [Figure 4F] See the explanation in Figure 4A. [Figure 5A] Figures 5A–5D are bar graphs and plots showing the phenotype of treated 5xFAD mice during behavioral testing. Treated 5xFAD mice and age-matched controls (untreated and mock-transplanted) underwent monthly behavioral testing of an elevated cross maze (Figure 5A) and a Morris water maze-probe trial (Figure 5B), latency to reach a hidden platform (Figure 5C), and reverse trial latency (Figure 5D). Mean ± SEM (standard error of the mean); asterisks indicate significance in two-way ANOVA with Dunnett's post-hoc test for 5xFAD in Figure 5A, two-way ANOVA with Tukey's post-hoc test for wild-type in Figure 5C, two-way ANOVA with Tukey's post-hoc test for 5xFAD in Figure 5D, and one-way ANOVA with Tukey's post-hoc test for WT in Figure 5B. [Figure 5B] See the explanation in Figure 5A. [Figure 5C] See the explanation in Figure 5A. [Figure 5D] See the explanation in Figure 5A. [Figure 6A] Figures 6A–6H are bar graphs showing the phenotype of treated 5xFAD mice at histological evaluation. Treated 5xFAD mice and age-matched controls (untreated and mock-transplanted) were sacrificed at over 12 months of age, and their brains were processed for staining and signal quantification for Iba1 (Figure 6A - cortex, Figure 6B - hippocampus), GFAP (Figure 6C - cortex, Figure 6D - hippocampus), and Ab (Figure 6E - cortex, Figure 6F - hippocampus; Figure 6G - cortex of mice with vector copy number (VCN) > 0.5 in the brain, Figure 6H - hippocampus of mice with vector copy number (VCN) > 0.5 in the brain). Mean ± SEM (standard error of the mean); asterisks indicate significance in one-way ANOVA using Dunnett's post-hoc test for WT in Figure 6A and for 5xFAD in Figure 6H. [Figure 6B] See the explanation in Figure 6A. [Figure 6C] See the explanation in Figure 6A. [Figure 6D] See the explanation in Figure 6A. [Figure 6E] See the explanation in Figure 6A. [Figure 6F] See the explanation in Figure 6A. [Figure 6G] See the explanation in Figure 6A. [Figure 6H] See the explanation in Figure 6A. [Modes for carrying out the invention]

[0111] Detailed description of the invention The present invention features compositions and methods useful for reconstituting microglia after HSPC transplantation, and for treating and preventing Alzheimer's disease.

[0112] As will be reported in more detail below, the present invention is at least in part based on the finding that behavioral symptoms present in a mouse model of Alzheimer's disease were improved after transplantation of HSPCs expressing ApoE2. Accordingly, the present invention provides compositions and methods for treating Alzheimer's disease using HSPCs that overexpress ApoE2 and / or other therapeutic agents, such as TREM2 and metallothionein.

[0113] In particular, the present invention provides an approach for the effective genetic engineering of central nervous system (CNS) microglia and myeloid cells for delivering therapeutic agents to the brain for the treatment of neurodegenerative diseases. In gene therapy clinical trials, the replacement of microglia after transplantation of genetically modified hematopoietic stem cells (HSCs) has been demonstrated to potentially suppress neuropathy in monogenic neurodegenerative diseases. While we do not wish to be bound by theory, it is conceivable that, in the early stages of development, a group of myeloid hematopoietic progenitor cells integrate into the central nervous system (CNS) to provide lifelong support. Indeed, recent advances in hematopoietic stem cell transplantation (HCT) have provided evidence that, after myelostomy and HSC infusion, new microglia-like cells develop and colonize the central nervous system (CNS). These cells can integrate locally and functionally and, in addition to providing therapeutic molecules to surrounding cells, may actively contribute to the neuronal environment by determining and reforming neural networks, maintaining neuronal homeostasis, pruning synaptic spines, and, of course, becoming a functional part of the immune system in terms of both surveillance and phagocytosis. Gene therapy clinical trials have been developed to target monogenic neurodegenerative diseases in which paracrine release of key lysosomal enzymes has shown therapeutic efficacy. Therefore, manipulating microglia through intracellular transplantation of genetically modified hematopoietic stem cells (HSCs) may offer a route of medical intervention for currently incurable neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS) and Alzheimer's disease (AD).

[0114] Therapeutic agents: ApoE2, TREM2, and metallothionein This invention relates to apolipoprotein E (ApoE) and triggering receptor 2 expressed on myeloid cells. T riggering R eceptor E xpressed on M yeloid cells 2 It is characterized by cells that overexpress APOE (Trem2). ApoE, the major apolipoprotein of the central nervous system (CNS), is generally known to be involved in lipid metabolism in various organs. However, ApoE is also synthesized and secreted by astrocytes, microglia, and, to a lesser extent, neurons in the CNS. In the brain, ApoE is involved not only in damage repair through lipid redistribution but also in regulating neurite outgrowth and cerebral vascular integrity. ApoE is deeply involved in the pathogenesis of late-onset Alzheimer's disease, with the APOE4 allele associated with a markedly increased risk of developing AD, while APOE2 has neuroprotective effects (reducing AD risk by 50% and delaying disease onset) (Serrano-Pozo et al., Ann Neurol 2015; 10:371). Consistent with this latter observation, direct CNS gene delivery of APOE2 in an AD mouse model showed neuroprotective effects, reducing Aβ levels and amyloid plaques (Zhao et al., Neurobiol Aging 2016;44:159).

[0115] TREM2 (Triggering receptor expressed on myeloid cells 2) is a cell surface receptor in microglia, and its deficiency or haploinsufficiency increases Aβ accumulation due to cellular dysfunction, which leads to apoptosis (Wang et al., Cell 2015; 160(6):1061) (Guerreiro & Hardy, 2014). Restoring or increasing TREM2 function in microglia is likely to improve the clearance of neurotoxic Aβ in AD. Upregulation of TREM2 in the brain of AD mice via a viral vector improved AD-related neuropathology, including Aβ deposition, neuroinflammation, and neuronal and synaptic loss, and was also accompanied by improved spatial cognitive function (Jiang et al., Neuropsycopharmacology 2014; 39(13):2949). Interestingly, staining with anti-Aβ antibody revealed relatively widespread amyloid deposition in the same region where increased binding of TSPO-selective radioligands was detected. This is consistent with the proposed link between amyloid pathology and reactive microgliosis. Therefore, the present invention provides, in particular, a gene therapy strategy based on the transplantation of hematopoietic stem progenitor cells (HSPCs) transduced by a lentiviral vector (LV), aimed at generating microglial progeny genetically engineered to express TREM2 and / or APOE2 in a controlled manner under the control of the TSPO promoter. This would enable the specific release of therapeutic factors by newly generated microglia recruited to the vicinity of disease sites (amyloid plaques), potentially allowing for more efficient and specific removal of misfolded proteins at sites of neuronal death. In various aspects of the present invention, overexpression of TREM2 and / or APOE2 in brain myeloid / microglia progeny derived from transplanted hematopoietic stem progenitor cells (HSPCs) is a valuable alternative to already tested central nervous system (CNS) gene delivery approaches because the present invention can deliver protective factors more broadly throughout the brain parenchyma.Metallothionein (MT) belongs to a group of intracellular cysteine-rich metal-binding proteins and is involved in homeostasis of essential metals such as zinc and copper, detoxification of harmful metals such as cadmium, and protection from oxidative stress. Furthermore, MT is involved in the processes of neuroprotection and nerve regeneration in several pathological conditions, including Alzheimer's disease (AD) (Juarez-Rebollar, Rios, Nava-Ruiz, & Mendez-Armenta, 2017; Ruttkay-Nedecky et al., 2013). In addition, overexpression of metallothionein (such as MT1G) contributes to neuroprotection in LSD mouse models of infantile ceroid lipofuscinosis and Krabbe disease. Therefore, metallothionein may also be beneficial in Alzheimer's disease (AD).

[0116] HSC transplantation in Alzheimer's disease Neurodegenerative diseases such as Alzheimer's disease are characterized by the progressive loss of functional neurons. In some neurodegenerative diseases, a relationship between microglia in the central nervous system and neurodegeneration has been observed. For example, activation of glial cells in the brain of patients is involved in the spread of disease to other areas of the central nervous system, and abnormal activation of microglial cells in Alzheimer's patients may promote a neurotoxic environment, potentially contributing to the characteristic loss of motor neurons.

[0117] This disclosure features a composition comprising hematopoietic stem progenitor cells (HSPCs) overexpressing ApoE2 and / or other therapeutic agents, such as TREM2 and metallothionein, which are useful for transplantation into the brains of subjects suffering from Alzheimer's disease that have undergone destructive preconditioning that disrupts endogenous microglia. This preconditioning improves the engraftment of the transplanted HSPCs. In some embodiments, the HSPCs are delivered directly to the brain via intraventricular infusion (ICV). In other embodiments, the HSPCs are provided by intravenous (IV) injection. HSPCs used for reconstitution of the microglial population are modified to express ApoE2, TREM2 and / or metallothionein.

[0118] ICV delivery after destructive conditioning is superior to conventional methods of treating neurodegenerative diseases using hematopoietic stem cell (HSC) transplantation; conventional methods are generally ineffective because the replacement of resident microglia with the progeny of transplanted cells is slow. Conventional HSC transplantation methods include the use of whole bone marrow, apheresis products, or umbilical cord blood, or, in the case of autologous gene therapy, the use of hematopoietic stem progenitor cells (HSPCs). In contrast, the present invention provides a cell population rich in cells with microglial regrowth activity. Furthermore, direct delivery of hematopoietic stem progenitor cells (HSPCs) or fractions of the HSPC pool to the brain by intracerebrovascular infusion (ICV) has been found to improve the rate and extent of microglial rearrangement by transplanted donor cells and increase the delivery of therapeutic proteins to the brain compared to a single intravenous (IV) transplant. This ICV approach is therapeutically beneficial and enhances microglial replacement by transplanted cells. Furthermore, this disclosure envisions the molecular engineering of microglia for therapeutic gene expression (e.g., expression of ApoE2, TREM2, and / or metallothionein).

[0119] Regeneration of genetically engineered microglia in the central nervous system Microglia have a different developmental origin from bone marrow-derived myelomonocytes (Ginhoux et al. Science 330, 841-845 (2010); its contents are incorporated herein by reference in their entirety). However, cells with a microglia-like phenotype can be induced from transplanted donor hematopoietic stem cells (HSPCs). HSPCs capable of generating microglia-like cells after transplantation into bone marrow-disrupted recipients are retained within human and mouse long-term hematopoietic stem cells (HSCs), thereby providing a reservoir of pluripotent cells that can differentiate into therapeutic microglia for the treatment of Alzheimer's disease.

[0120] HSPCs expressing ApoE2, TREM2, and / or metallothionein can be systemically administered to a subject, migrate to the brain, and differentiate into microglia-like cells, thereby replacing dead or damaged microglia. However, as described herein, an alternative method of administering HSPCs, intracerebrospinal fluid, results in faster and more extensive microglia rearrangement. Therefore, in some embodiments of this disclosure, HSPCs are administered into the ventricles of the subject's brain. In some embodiments, HSPCs are delivered into the cerebrospinal fluid of the ventricles. This administration route avoids the inefficiencies associated with systemically administered compositions having to cross the blood-brain barrier. In some embodiments of this disclosure, intracerebrospinal fluid administration results in faster establishment of progeny cells in the recipient's brain compared to systemic administration. In some embodiments, this direct delivery method results in more extensive microglia cell replacement.

[0121] Engraftment and differentiation of HSPCs can be challenging in environments containing endogenous microglia. Endogenous microglia may be able to outcompete transplanted HSPCs, and neuroinflammation associated with dying microglia may create an unfavorable environment for HSPC engraftment (e.g., increased inflammation). To overcome these barriers to HSPC engraftment, in some embodiments, existing microglia are destroyed by exposure to agents that can eliminate endogenous microglia. For example, pre-transplant administration of pre-treatment regimens with alkylating agents is an effective means of destroying endogenous microglial progenitor cells (Capotondo et al. (2012); Wilkinson et al. Mol Ther 21, 868-876 (2013); the contents thereof are incorporated herein by reference in their entirety). In some embodiments of this disclosure, the alkylating agent is busulfan. In addition to alkylating agents such as busulfan, CSF-1R inhibitors (e.g., PLX3397 and PLX5622) and liposomal clodronate may also be used (Han et al. Molecular Brain, 10:25, 2017), and optionally, they may be used in combination with nanoparticles, for example, as described in WO2019191650 incorporated herein.

[0122] Generally, the term nanoparticles refer to particles having a diameter of less than 1000 nm. In certain preferred embodiments, the nanoparticles of the present invention have a maximum dimension (e.g., diameter) of 500 nm or less. In other preferred embodiments, the nanoparticles of the present invention have a maximum dimension in the range of 25 nm to 200 nm. In other preferred embodiments, the nanoparticles of the present invention have a maximum dimension of 100 nm or less. In other preferred embodiments, the nanoparticles of the present invention have a maximum dimension in the range of 35 nm to 60 nm. The nanoparticles included in the present invention may be provided in various forms, for example, as solid nanoparticles (e.g., metals such as silver, gold, iron, and titanium; nonmetals; lipid-based solids; polymers), suspensions of nanoparticles, or combinations thereof. Metallic, dielectric, and semiconductor nanoparticles, as well as hybrid structures (e.g., core-shell nanoparticles), can be fabricated. Nanoparticles made from semiconductor materials can also be classified as quantum dots if they are small enough (usually 10 nm or less) that electron energy level quantization occurs. Such nanoscale particles have been used in biomedical applications as drug carriers or imaging agents, and the present invention may be suitable for similar purposes. Semi-solid and soft nanoparticles have been manufactured and are included within the scope of this invention. A semi-solid prototype nanoparticle is a liposome. Currently, various types of liposome nanoparticles are clinically used as delivery systems for anticancer drugs and vaccines. Nanoparticles that are half hydrophilic and half hydrophobic are called Janus particles and are particularly effective in stabilizing emulsions. They can self-assemble at the water / oil interface and act as solid surfactants. In one embodiment, nanoparticles based on self-assembling bioadhesive polymers are envisioned, which can be applied to oral delivery of drugs, intravenous delivery of drugs, and nasal delivery of drugs (all to the brain). Other embodiments, such as oral absorption and ocular delivery of hydrophobic drugs, are also envisioned.Molecular envelope technology includes artificial polymer envelopes that are protected and delivered to the disease site (Mazza et al. ACS Nano 7, 1016-1026 (2013); Siew et al. Mol Pharm 9, 14-28 (2012); Lalatsa et al. J Control Release 161, 523-536 (2012); Lalatsa et al. Mol Pharm 9, 1665-1680 (2012); Garrett et al. J Biophotonics 5, 458-468 (2012); Uchegbu, Expert Opin Drug Deliv 3, 629-640 (2006); Uchegbu et al. Int J Pharm 224, 185-199 (2001); Qu et al. Biomacromolecules 7, 3452-3459). (2006).

[0123] Several types of particle delivery systems and / or formulations are known to be useful in a wide variety of biomedical applications. Generally, particles are defined as small objects that act as a whole unit in terms of their transport and properties. Particles are further classified by diameter. Coarse particles cover the range of 2,500 to 10,000 nanometers. Fine particles are 100 to 2,500 nanometers in size. Ultrafine particles, or nanoparticles, are generally 1 to 100 nanometers in size. The rationale for this 100 nm limit is the fact that novel properties that distinguish particles from bulk materials usually occur at critical length scales of less than 100 nm.

[0124] As used herein, a particle delivery system / formulation is defined as any biological delivery system / formulation comprising the particles according to the present invention. The particles according to the present invention are any entity having a maximum dimension (e.g., diameter) of less than 100 microns (μm). In some embodiments, the particles of the present invention have a maximum dimension of less than 10 p.m. In some embodiments, the particles of the present invention have a maximum dimension of less than 2000 nanometers (nm). In some embodiments, the particles of the present invention have a maximum dimension of less than 1000 nanometers (nm). In some embodiments, the particles of the present invention have a maximum dimension of less than 900 nm, 800 nm, 700 nm, 600 nm, 500 nm, 400 nm, 300 nm, 200 nm, or less than 100 nm. Typically, the particles of the present invention have a maximum dimension (e.g., diameter) of 500 nm or less. In some embodiments, the particles of the present invention have a maximum dimension (e.g., diameter) of 250 nm or less. In some embodiments, the particles of the present invention have a maximum dimension (e.g., diameter) of 200 nm or less. In some embodiments, the particles of the present invention have a maximum dimension (e.g., diameter) of 150 nm or less. In some embodiments, the particles of the present invention have a maximum dimension (e.g., diameter) of 100 nm or less. Smaller particles, for example, particles with a maximum dimension of 50 nm or less, are used in some embodiments of the present invention. In some embodiments, the particles of the present invention have a maximum dimension in the range of 25 nm to 200 nm.

[0125] Particle characterization (including characterization such as morphology and dimensionality) is performed using a variety of different techniques. Common techniques include electron microscopy (TEM, SEM), atomic force microscopy (AFM), dynamic light scattering (DLS), X-ray photoelectron spectroscopy (XPS), powder X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF), ultraviolet-visible spectroscopy, dual polarization interferometry, and nuclear magnetic resonance (NMR). Characterization (dimensional measurement) is performed with respect to the natural particles (i.e., before loading) or after loading of the cargo (in this specification, cargo refers to, for example, one or more components of a CRISPR-Cas system, e.g., CRISPR enzymes or mRNA or guide RNA, or any combination thereof, and may include additional carriers and / or additives) to provide particles of an optimal size for delivery for in vitro, ex vivo, and / or in vivo applications of the present invention. In certain preferred embodiments, the characterization of particle dimensions (e.g., diameter) is based on measurements using dynamic laser scattering (DLS).

[0126] Particle delivery systems within the scope of the present invention may be provided in any form, including but not limited to solid, semi-solid, emulsion, or colloidal particles. Therefore, any delivery system described herein may be provided as a particle delivery system within the scope of the present invention.

[0127] Alkylating agents can be delivered to the target by any method known in the art. For example, alkylating agents can be delivered orally, intravenously, intra-arterially, intraperitoneally, intramuscularly, subcutaneously, intrathecally, by perfusion via a local catheter, or by any other means known in the art.

[0128] In some embodiments, HSPCs expressing ApoE2, TREM2, and / or metallothionein are administered to the subject after removal of endogenous microglia or their progenitor cells. In some embodiments, HSPCs are delivered intravenously after destruction of endogenous microglia or their progenitor cells. In some embodiments, HSPCs are delivered intravascularly after destruction of endogenous microglia or their progenitor cells.

[0129] Modified cells Several aspects of this disclosure provide cells modified to express one or more exogenous nucleic acid molecules (e.g., ApoE2, TREM2, and / or metallothionein). In some embodiments, the exogenous nucleic acid molecules encode neuroprotective substances (e.g., ApoE2, TREM2, and / or metallothionein) that improve neuronal loss or dementia in subjects with or suspected of having Alzheimer's disease. In some embodiments, cells are provided that express neuroprotective substances (e.g., ApoE2, TREM2, and / or metallothionein) that suppress neurodegeneration.

[0130] The modified cells of this disclosure are, in some embodiments, HSPCs. In some embodiments, the cells are microglial cells derived from modified HSPCs. HSPCs can be isolated from subjects with Alzheimer's disease, suspected subjects, or subjects prone to developing it. The cells are then modified to express ApoE2, TREM2, and / or metallothionein, cultured, and administered to the subjects. These autologous cells are less likely to elicit an immune response after administration to the subjects compared to allogeneic cells. However, in some embodiments, HSPCs are isolated from healthy donors. Methods for isolating HSPCs from donors are known in the art.

[0131] ApoE2 In some aspects of this disclosure, the cells of this disclosure are modified to express apolipoprotein E (ApoE). In some aspects of this disclosure, the modified cells express ApoE2 from an exogenous nucleic acid molecule. For example, HSPCs are modified to express ApoE2 from an exogenous nucleic acid molecule and then administered to a subject to introduce neuroprotective substances into the brain of a subject with Alzheimer's disease, a subject suspected of having it, or a subject predisposed to developing it. In some aspects, cells derived from the modified HSPC express ApoE2. For example, in some aspects, microglia or microglia-like cells express ApoE2 from an exogenous nucleic acid molecule.

[0132] Trem2 Some aspects of this disclosure provide modified cells expressing Trem2. The modified cells may be HSPCs administered to subjects with Alzheimer's disease, suspected Alzheimer's disease, or a predisposition to developing such disease. In some aspects, the cells expressing Trem2 are cells derived from HSPCs. For example, in some aspects, the modified cells derived from HSPCs are microglia or microglia-like cells.

[0133] metallothionein Some aspects of this disclosure provide modified cells expressing metallothionein encoded by an exogenous nucleic acid molecule. In some aspects, the metallothionein is MT1, MT2, MT3, or MT4 metallothionein. In some aspects, the metallothionein is MT1. In some aspects, the MT1 metallothionein is MT1A, MT1B, MT1E, MT1F, MT1G, MT1H, MT1L, MT1M, or MT1X metallothionein. In some aspects, the cell expresses two or more metallothionein from an exogenous nucleic acid molecule. In some aspects, the cell expresses MT1G from an exogenous nucleic acid molecule. In some aspects, the cell is an HSPC or its offspring. In some aspects, the cell is a microglia or a microglia-like cell. Metallothioneine is described in International Application Numbers PCT / US2018 / 013908 and PCT / US2018 / 013909, the contents of which are incorporated herein by reference in their entirety.

[0134] Several aspects of this disclosure provide modified cells expressing ApoE2 or a fragment thereof encoded by an exogenous nucleic acid molecule and metallothionein. In some aspects, the metallothionein is MT1, MT2, MT3, or MT4 metallothionein. In some aspects, the modified cells express MT1A, MT1B, MT1E, MT1F, MT1G, MT1H, MT1L, MT1M, or MT1X metallothionein and ApoE2 or a fragment thereof. In some aspects, the modified cells express MT1G and ApoE2 or a fragment thereof from an exogenous nucleic acid molecule. In some aspects, the cells are HSPCs or their offspring. In some aspects, the cells are microglia or microglia-like cells.

[0135] Several aspects of this disclosure provide modified cells expressing Trem2 or a fragment thereof encoded by an exogenous nucleic acid molecule and metallothionein. In some aspects, the metallothionein is MT1, MT2, MT3, or MT4 metallothionein. In some aspects, the modified cells express MT1A, MT1B, MT1E, MT1F, MT1G, MT1H, MT1L, MT1M, or MT1X metallothionein and Trem2 or a fragment thereof. In some aspects, the modified cells express MT1G and Trem2 or a fragment thereof from an exogenous nucleic acid molecule. In some aspects, the cells are HSPCs or their offspring. In some aspects, the cells are microglia or microglia-like cells.

[0136] Some aspects of this disclosure provide modified cells expressing ApoE2, Trem2, or fragments thereof encoded by an exogenous nucleic acid molecule, and metallothionein. In some aspects, the metallothionein is MT1, MT2, MT3, or MT4 metallothionein. In some aspects, the modified cells express MT1A, MT1B, MT1E, MT1F, MT1G, MT1H, MT1L, MT1M, or MT1X metallothionein, ApoE2, and Trem2 or fragments thereof. In some aspects, the modified cells express MT1G, ApoE2, and Trem2, or fragments thereof from an exogenous nucleic acid molecule. In some aspects, the cells are HSPCs or their offspring. In some aspects, the cells are microglia or microglia-like cells.

[0137] Hemizygous CX3CR1 cells CX3CR1, also known as the fractalkine receptor, is a seven-transmembrane domain receptor belonging to the G protein-coupled receptor family. As a G protein-coupled receptor, CX3CR1's role is predominantly repressive, acting to suppress cAMP production and prevent the initiation of secondary messenger-mediated signaling cascades. Intracellular pathways regulated by CX3CR1 signaling primarily involve the regulation of PLC, PI3K, and ERK, which regulate cell migration, adhesion, proliferation, and survival. It is expressed in several cell types (e.g., monocytes, natural killer cells, T cells, and smooth muscle cells). Microglia are the only cells in the central nervous system that express CX3CR1, and it is expressed at high levels, particularly during development and in response to brain injury / pathology.

[0138] Fractalkine (CX3CL1) is a unique ligand for the chemokine receptor CX3CR1 and is expressed either as a membrane-bound molecule or in a soluble form. Cleavage of fractalkine is mediated by at least two enzymes, ADAM10 and ADAM17, which are active in homeostatic and inflammatory states, respectively. In its membrane-bound form, fractalkine primarily acts as an adhesion molecule, while in its soluble form it exhibits chemotaxis towards CX3CR1. Local production and membrane expression of CX3CL1 and CX3CR1 are regulated by other cytokines, such as TNFα, IL-1, IFNγ, NO, and hypoxic conditions.

[0139] Activation of the CX3CR1-CX3CL1 axis is linked to the maintenance of microglia in a quiescent state and the maintenance of homeostasis in neural networks. Under physiological conditions, CX3CL1 appears to suppress microglial activation, but under certain conditions, it may paradoxically promote inflammatory responses. Neurons are the larger producers of CX3CL1 in the brain, and this axis is important for communication with microglial cells. Astrocytes (GFAP) +) also exhibits constitutive mRNA expression of CX3CL1. Endothelial cells of the brain and spinal cord, in contrast to endothelial cells in other regions, do not show constitutive CX3CL1 expression on their surface; this suggests that it is rather dependent on their activation. CX3CL1 and CX3CR1 are also expressed in the choroid plexus.

[0140] To enhance the ability of HSPCs to generate microglia-like progeny after transplantation, we used a mouse model (B6.129P-CX3CR1tm1Litt / J) that was hemizygous for CX3CR1 and in which one CX3CR1 allele was replaced with a GFP reporter gene. We demonstrated that (i) transplantation of whole bone marrow or HSPCs from haploinsufficient donor mice for the CX3CR1 gene resulted in more and faster emergence of microglia-like donor cells in the recipient brain compared to standard wild-type donors, and (ii) in competitive transplantation, cells derived from haploinsufficient donors contributed significantly to the regrowth of hematopoietic organs and brain-bone marrow compartments in the recipient compared to wild-type donor cells. In branching studies performed on engrafted cells, CX3CR1 + / GFP The cells were also shown to have acquired a more mature, microglia-like morphology. CX3CR1 hemizygous mice did not exhibit a clear phenotype.

[0141] Accordingly, this disclosure intends to isolate HSPCs and knock out one allele of CX3CR1 to produce hemizygous cells; these cells can be cultured to obtain a population of cells that are hemizygous for CX3CR1. Such cells can be administered to subjects that require them. This disclosure also intends to modify CX3CR1 hemizygous HSPCs to incorporate nucleic acid sequences encoding ApoE2, TREM2, and / or metallothionein proteins into the deficient CX3CR1 allele locus. In this way, the hemizygous HSPCs are engineered to express therapeutic agents. Alternatively, isolated HSPCs can also be edited to remove one copy of CX3CR1 to produce hemizygous HSPCs. Editing a single copy of CX3CR1 involves, in some embodiments, replacing the CX3CR1 allele with an exogenous nucleic acid molecule encoding a therapeutic agent. Such editing can be performed using any method known in the art.

[0142] Cell modification Any approach known in the art can be used to produce any of the CX3CR1 hemizygous cells described herein. For example, cells can be modified by editing endogenous genes such as CX3CR1. Gene editing is a major interest in biomedical research, blurring the lines between basic and clinical science. Gene editing tools can manipulate the DNA sequence of a cell at a specific chromosomal locus without introducing mutations to other parts of the genome. This technology allows researchers to effectively manipulate the genome of cells in vitro or in vivo.

[0143] In one embodiment, gene editing involves targeting an endonuclease to a specific site in the genome to induce a double-strand break at that specific location. When a donor DNA molecule (e.g., plasmid or oligonucleotide) is introduced, an interaction can occur between the nucleic acid containing the double-strand break and the introduced DNA, especially if these two nucleic acids share homologous sequences. In this case, a process called "gene targeting" occurs, in which the DNA ends of the chromosome enter the homologous sequence of the donor DNA through homologous recombination. By using the donor plasmid sequence as a template for homologous recombination, seamless knockout of the gene of interest can be achieved. Importantly, if the donor DNA molecule contains a deletion within the target gene (e.g., CX3CR1), the donor sequence is introduced into the chromosome via double-strand break repair through homologous recombination, resulting in the deletion being introduced into that chromosomal locus. The concept is to utilize the formation of double-strand breaks to promote homologous recombination by targeting a nuclease to a genomic site containing the target gene, thereby replacing the functional target gene with its deletion variant. The advantage of homologous recombination pathways is that they can potentially generate gene knockouts seamlessly in place of the previous wild-type allele.

[0144] Genome editing tools can use double-strand breaks to enhance the genetic manipulation of cells. In such methods, zinc finger nucleases (e.g., U.S. Patent Nos. 6,534,261; 6,607,882; 6,746,838; 6,794,136; 6,824,978; 6,866,997; 6,933,113; 6,979,539; 7,013,219; 7,030,215; 7,220,719; 7,241,573; 7,241,574; 7,585,849; 7,595,376; 6,903,185;) are used. and 6,479,626; and as described in U.S. Patent Publication Nos. 20030232410 and U.S.2009020314, which are incorporated herein by reference); transcription activator-like effector nucleases (TALENs; for example, as described in U.S. Patent Nos. 8,440,431; 8,440,432; 8,450,471; 8,586,363; and 8,697,853; and as described in U.S. Patent Publication Nos. 20110145940; 20120178131; 20120178169; 20120214228; 20130122581; 20140335592; and 20140335618, which are incorporated herein by reference); and CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) / Cas9 system (e.g., U.S. Patent Nos. 8,697,359; 8,771,945; 8,795,965; 8,871,445; 8,889,356; 8,906,616; 8,932,814; 8,945,839; 8,993,233; and 8,999,641; and U.S. Patent Publication Nos. 20140170753; 20140227787; 20140179006; 20140189896; 20140273231; 20140242664; 20140273232; 20150184139; The following can be used (as described in 20150203872; 20150031134; 20150079681; 20150232882; and 20150247150, which are incorporated herein by reference).For example, the DNA sequence recognition ability and specificity of zinc finger nucleases can be unpredictable. Similarly, TALENs and CRISPR / Cas9 often cleave not only the target site but also other "off-target" sites. These methods have significant problems that can lead to the induction of off-target double-strand breaks and the potential for harmful mutations such as indels, genomic rearrangements, and chromosomal rearrangements associated with these off-target effects. Zinc finger nucleases and TALENs require the use of modular sequence-specific DNA-binding proteins to achieve specificity for a sequence of approximately 18 base pairs in the genome.

[0145] Genome editing mediated by RNA-guided nucleases based on the Type 2 CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat) / Cas (CRISPR Associated) system offers a valuable approach to genome modification. Briefly, the nuclease Cas9, induced by a single guide RNA (sgRNA), binds to a target genomic locus adjacent to a protospacer facing motif (PAM), causing a double-strand break. The zinc finger nuclease is then repaired by non-homologous end joining, resulting in insertion / deletion (indel) mutations, or by homology-directed repair, which requires an exogenous template and can produce precise modifications at the target locus (Mali et al., Science, Feb 15, 2013; 339 (6121): 823-6; its contents are incorporated herein by reference in their entirety). Unlike gene therapies that add a functional or partially functional copy of a gene to target cells but retain the original, dysfunctional copy of that gene, this system can eliminate the defects of the dysfunctional copy. Gene modification using modified nucleases has been demonstrated in tissue culture cells and rodent models of rare diseases.

[0146] CRISPR has been used in a wide range of organisms, including baker's yeast (S. cerevisiae), zebrafish, nematodes (such as C. elegans), plants, mice, and several other species. Furthermore, CRISPR has been modified to create programmable transcription factors that allow scientists to target specific genes and activate or silence them. Currently, libraries of tens of thousands of guide RNAs are available. By inserting a plasmid containing the cas gene and a specially designed CRISPR, it is possible to cleave the genome of an organism at any desired location.

[0147] CRISPR repeats range in size from 24 to 48 base pairs. They typically exhibit some dyad symmetry, suggesting the formation of hairpin-like secondary structures, but they are not true palindromes. Repeats are separated by spacers of similar length; some CRISPR spacer sequences precisely match sequences from plasmids and phages, while others match prokaryotic genomes (self-targeting spacers). New spacers can be rapidly added in response to phage infection.

[0148] CRISPR-related (cas) genes are often associated with CRISPR repeat-spacer arrays. As of 2013, more than 40 different Cas protein families had been described. Of these protein families, Cas1 appears to be ubiquitous across various CRISPR / Cas systems. Using specific combinations of cas genes and repeat structures, eight CRISPR subtypes (Ecoli, Ypest, Nmeni, Dvulg, Tneap, Hmari, Apern, and Mtube) have been defined, some of which are associated with additional gene modules encoding repeat-associated mysterious proteins (RAMPs). Multiple CRISPR subtypes can exist within a single genome. The scattered distribution of CRISPR / Cas subtypes suggests that the system has undergone horizontal gene transfer during microbial evolution.

[0149] Exogenous DNA appears to be processed into small elements (approximately 30 base pairs in length) by proteins encoded by the Cas gene, and then inserted into CRISPR loci near the leader sequence. RNA from the CRISPR loci is constitutively expressed and processed by Cas proteins into small RNAs containing individual exogenous sequence elements with adjacent repeat sequences. These RNAs then guide other Cas proteins to silence the exogenous genetic elements at the RNA or DNA level. Functional diversity among CRISPR subtypes is suggested by evidence. The Cse (Cas subtype Ecoli) protein (called CasA-E in E. coli) forms a functional complex called Cascade, which processes CRISPR RNA transcripts into spacer repeat units held by Cascade. In other prokaryotes, Cas6 processes CRISPR transcripts. Interestingly, CRISPR-based phage inactivation in E. coli requires Cascade and Cas3, but not Cas1 or Cas2. The Cmr (Cas RAMP module) protein, found in Pyrococcus furiosus and other prokaryotes, forms a functional complex with a small CRISPR RNA, which recognizes and cleaves a complementary target RNA. RNA-guided CRISPR enzymes are classified as type V restriction enzymes. See also U.S. Patent Publication 2014 / 0068797, the contents of which are incorporated herein by reference in their entirety.

[0150] As an RNA-guided protein, Cas9 requires an RNA molecule to direct the recognition of a DNA target. However, Cas9 preferentially interrogates DNA sequences containing protospacer-adjacent motif (PAM) sequences (i.e., NGG). However, the Cas9-gRNA complex requires substantial complementarity between the guide RNA (gRNA) and the target nucleic acid sequence to cause double-strand breaks. Synthetic gRNAs can be designed to combine the RNA sequences essential for Cas9 targeting into a single RNA expressed by the RNA polymerase type 2I promoter U6. Synthetic gRNAs are at least slightly over 100 nucleotides long and contain a region that targets the 20 protospacer nucleotides immediately preceding the PAM sequence NGG.

[0151] One approach involves modifying HSPC cells to delete or inactivate the CX3CR1 allele using the CRISPR-Cas system. Cas9 can be used to target the CX3CR1 gene. Upon recognizing the target, Cas9 induces a double-strand break in the CX3CR1 target gene. Homologous recombination repair (HDR) at the double-strand break site allows for insertion of an inactive or deletion of the CX3CR1 sequence. In some embodiments, homologous recombination repair (HDR) at the double-strand break site may allow for insertion of the expression cassette of the present invention.

[0152] One approach involves modifying HSPC cells to delete or inactivate the TSPO allele using the CRISPR-Cas system. Cas9 can be used to target the TSPO gene. Upon recognizing the target, Cas9 induces a double-strand break in the TSPO target gene. Homologous recombination repair (HDR) at the double-strand break site allows for insertion of an inactive or deleted version of the TSPO sequence. In some embodiments, homologous recombination repair (HDR) at the double-strand break site may allow for insertion of the expression cassette of the present invention.

[0153] The following U.S. patents and patent publications are incorporated herein by reference: Patent No. 8,697,35; 20140170753; 20140179006; 20140179770; 20140186843; 20140186958; 20140189896; 20140227787; 20140242664; 20140248702; 20140256046; 20140273230; 20140273233; 20140273234; 20140295556; 20140295557; 20140310830; 20140356956; 20140356959; 20140357530; 20150020223; 20150031132; 20150031133; 20150031134; 20150044191; 20150044192; 20150045546; 20150050699; 20150056705; 20150071898; 20150071899; 20150071903; 20150079681; 20150159172; 20150165054; 20150166980; and 20150184139.

[0154] Development of exogenous therapeutic agents ApoE2, TREM2, and / or metallothionein are therapeutic polypeptides useful for the treatment of Alzheimer's disease. Polynucleotides encoding such proteins are inserted into expression vectors by techniques known in the art. For example, double-stranded DNA can be cloned into a suitable vector by restriction enzyme ligation, including the use of synthetic DNA linkers, or by blunt-end ligation. Typically, DNA ligases are used for ligation of DNA molecules, and undesirable binding can be avoided by alkaline phosphatase treatment.

[0155] This disclosure also includes vectors (e.g., recombinant plasmids) containing nucleic acid molecules encoding ApoE2, TREM2, and / or metallothionein as described herein. The term “recombinant vector” includes vectors (e.g., plasmids, phages, phagemids, viruses, cosmids, fosmids, or other purified nucleic acid vectors) that have been modified, altered, or manipulated to contain more, fewer, or different nucleic acid sequences than those contained in the natural or native nucleic acid molecule from which the recombinant vector originates. For example, a recombinant vector may include nucleotide sequences encoding polypeptides or fragments thereof, functionally linked to regulatory sequences such as promoter sequences, terminator sequences, long terminal repeat sequences, untranslated regions, and enhancers as defined herein. A recombinant expression vector enables the expression of a gene or nucleic acid contained therein. In certain embodiments, a promoter is described in U.S. Provisional Application No. 62 / 908,966, the contents of which are incorporated herein by reference in their entirety. In some embodiments, a promoter includes a translocator protein (TPSO) promoter. In some embodiments, the regulatory sequence comprises a translocator protein (TPSO) promoter combined with one or more enhancers. In some embodiments, the regulatory sequence comprises P1, P2, P1+P2, or P2+P1 alone or in combination with one or more of E1, E2, E1.1, and E1.2. In some embodiments, the regulatory sequence comprises P1+P2, P1, or E1+P1, where the sequence to the left of the "+" is 5' relative to the sequence to the right of the "+".

[0156] In some aspects of this disclosure, one or more DNA molecules having nucleotide sequences encoding one or more polypeptides or polynucleotides described herein can be functionally ligated to one or more regulatory sequences so that the desired DNA molecules can be incorporated into eukaryotic cells. Cells stably transfected or transduced with the introduced DNA (e.g., HSPCs, microglia) can be selected, for example, by introducing one or more markers that enable selection of host cells containing their expression vector. The selection marker gene may be directly ligated to the nucleic acid sequence to be expressed, or it may be introduced into the same cell by simultaneous transfection or simultaneous transduction. Additional elements necessary for the optimal synthesis of the polynucleotides or polypeptides described herein will be apparent to those skilled in the art.

[0157] In some embodiments, HSPCs can be modified by introducing exogenous nucleic acid molecules into the cells. The exogenous nucleic acid may include a transgene encoding a therapeutic agent for Alzheimer's disease. In some embodiments, the exogenous nucleic acid includes a regulatory element for expressing the transgene. For example, the exogenous nucleic acid molecule may include a transgene encoding a therapeutic agent for Alzheimer's disease and a promoter for expressing the transgene. In some embodiments, the promoter is a constitutively active promoter, such as a cytomegalovirus (CMV) or Simianvirus 40 (SV40) promoter. In some embodiments, the promoter may be a tissue-specific promoter, in which case the transgene is expressed after engraftment and differentiation of the HSPC. For example, tetracycline is a drug that can be used to activate a tetracycline-sensitive promoter. In some embodiments, the neuron-specific promoter is a synapsin (Syn) promoter. In some embodiments, the promoter may be an inductive promoter, in which case the transgene is expressed only in the presence or absence of a specific compound. In some embodiments, microglia or microglia-like cells derived from HSPCs containing a brain-specific promoter-driven transgene, transplanted into the target brain, will express the transgene. In some embodiments, the exogenous nucleic acid molecule may include, in addition to the transgene, a detectable label or other marker that enables the identification of cells successfully modified to express the transgene, or cells derived from such cells.

[0158] Methods for introducing exogenous nucleic acid molecules into cells are known in the art. For example, eukaryotic cells can take up nucleic acid molecules from the environment by transfection (e.g., calcium phosphate-mediated transfection). Transfection does not use viruses or viral vectors to introduce exogenous nucleic acids into recipient cells. Stable transfection of eukaryotic cells involves the integration of the transfected nucleic acid into the genome of the recipient cell, and the nucleic acid may then be inherited by the offspring of the recipient cell.

[0159] Eukaryotic cells (i.e., HSPCs) can be modified by transduction, in which a virus or viral vector stably introduces an exogenous nucleic acid molecule into the recipient cell. Transduction delivery systems for eukaryotic cells are known in the art. Transduction of most cell types can be achieved using retroviruses, lentiviruses, adenoviruses, adeno-associated viruses, and avian virus systems, and such systems are well known in the art. Generally, retrovirus systems are not suitable for transduction of nerve cells, but lentiviruses are a genus of retroviruses that are well suited for transduction of stem cells and nerve cells. Therefore, in some aspects of this disclosure, the viral vector system is a lentivirus system. In some aspects, the viral vector system is an avian virus system, e.g., the avian virus vector systems described in US8642570, DE102009021592, PCT / EP2010 / 056757, and EP2430167, the contents of which are incorporated herein by reference in their entirety. In some embodiments, the viral vector is assembled or packaged in a packaging cell before contact with the intended recipient cell. In some embodiments, the vector system is a self-inactivating system, in which case the viral vector is assembled in the packaging cell, but after contact with the recipient cell, the viral vector cannot be produced within the recipient cell.

[0160] While the components of a viral vector are encoded in plasmids, the efficiency of transduction decreases as plasmid size increases; therefore, multiple plasmids with different viral sequences required for packaging may be necessary. For example, in a lentiviral vector system, the first plasmid contains nucleotide sequences encoding a group antigen (gag) and / or reverse transcriptase (pol) gene, and the second plasmid encodes a virion protein expression regulator (rev) and / or envelope (env) gene. The exogenous nucleic acid molecule containing the transgene is packaged in the vector and delivered to recipient cells, where the transgene is integrated into the recipient cell's genome. Furthermore, the transgene may be packaged using split packaging systems described in US8642570, DE102009021592, PCT / EP2010 / 056757, and EP2430167.

[0161] After introducing one or more vectors, host cells are cultured before administration to a target. In some embodiments, recombinant protein expression can be detected by immunoassays such as Western blotting, immunoblotting, and immunofluorescence. Purification of recombinant proteins can be carried out by any conventional method known in the art or described herein, e.g., extraction, precipitation, chromatography, and electrophoresis. A further purification method that can be used for protein purification is affinity chromatography using monoclonal antibodies that bind to the target protein. Generally, a crude preparation containing the recombinant protein is passed through a column immobilized with a suitable monoclonal antibody. Typically, the protein binds to the column via the specific antibody, while impurities pass through. After washing the column, the protein is eluted from the gel, for example, by changing the pH or ionic strength.

[0162] Pharmaceutical composition The compositions contemplated in this disclosure include pharmaceutical compositions containing cells expressing neuroprotective substances. In some embodiments, the neuroprotective substances are ApoE2, Trem2, and / or metallothionein. The pharmaceutical compositions may include autologous or allogeneic cells modified to express therapeutic agents.

[0163] The hematopoietic stem progenitor cells (HSPCs) described herein can be administered as therapeutic compositions (e.g., as pharmaceutical compositions). The cell compositions described herein can be provided as sterile liquid preparations, such as isotonic aqueous solutions, suspensions, emulsions, dispersions, or viscous compositions, which may be buffered to a predetermined pH. Liquid preparations may be easier to prepare than gels, other viscous compositions, and solid compositions. Furthermore, liquid compositions may be more convenient to administer (i.e., by injection). On the other hand, viscous compositions may be formulated within a suitable viscosity range to allow for a longer contact time with specific tissues. The liquid or viscous composition may contain a carrier, which may be a solvent or dispersion medium, for example, water, saline, phosphate-buffered saline, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.), and suitable mixtures thereof.

[0164] Sterile injectable solutions can be prepared by adding the cells described herein to a sufficient amount of a suitable diluent. Such compositions may be mixed with a suitable carrier or excipient, such as sterile water, saline, glucose, dextrose, or another carrier or excipient suitable for delivery to living cells. The composition may also be freeze-dried. Depending on the route of administration and the desired formulation, the composition may contain auxiliary substances such as wetting agents, dispersants, emulsifiers (e.g., methylcellulose), pH buffers, gelling agents or thickeners, preservatives, flavoring agents, and coloring agents. A standard text, such as Remington's Pharmaceutical Science, 17th edition, 1985, incorporated herein by reference, can be consulted for preparing suitable formulations without excessive experimentation.

[0165] Additives that enhance the stability and sterility of the cell composition may be added, such as antimicrobial preservatives, antioxidants, chelating agents, and buffering agents. Microbial activity can be reliably prevented by antimicrobial or antifungal agents, including but not limited to parabens, chlorobutanol, phenol, and sorbic acid. However, according to this disclosure, any vehicle, diluent, or additive used must be compatible with the cells.

[0166] The compositions may be isotonic; that is, they have the same osmotic pressure as blood and cerebrospinal fluid. The desired isotonicity of the compositions of the present invention can be achieved using sodium chloride or a pharmaceutically acceptable substance, such as dextrose, boric acid, sodium tartrate, propylene glycol, or other inorganic or organic solutes. Sodium chloride is suitable for buffer solutions containing sodium ions.

[0167] The viscosity of the composition can be maintained at a predetermined level, if necessary, using a pharmaceutically acceptable thickener. In some embodiments, the thickener is methylcellulose, which is readily and economically available and easy to handle. Other suitable thickeners include, but are not limited to, xanthan gum, carboxymethylcellulose, hydroxypropylcellulose, and carbomer. The concentration of the thickener depends on the selected agent and the amount used. Suitable carriers and other additives can be selected according to the route of administration and the nature of the dosage form (for example, a liquid dosage form can be formulated as a solution, suspension, gel, or another liquid form, e.g., a time-release formulation or a liquid-filled form).

[0168] The effective dose of cells administered may vary depending on the target being treated. In one embodiment, approximately 10 4 ~about 10 8 In another configuration, approximately 10 5 ~about 10 7 Individual cells are administered to the target.

[0169] One of ordinary skill in the art can readily determine the amount of cells in the administered composition and the amount of any additives, vehicles, and / or carriers. In one aspect, any additives (other than the cells) are present in an amount of about 0.001% to about 50% (by weight) solution in phosphate buffered saline, and the active ingredient is present on the order of micrograms to milligrams, for example about 0.0001% to about 5 wt%. In another aspect, the active ingredient is present at about 0.0001% to about 1 wt%. In yet another aspect, the active ingredient is present at about 0.0001% to about 0.05 wt%. In still other aspects, the active ingredient is present at about 0.001% to about 20 wt%. In some aspects, the active ingredient is present at about 0.01% to about 10 wt%. In another aspect, the active ingredient is present at about 0.05% to about 5 wt%. In the case of compositions administered to animals or humans, and for specific administration methods, toxicity can be determined by measuring the lethal dose (LD) and LD 50 in a suitable animal model, such as rodents like mice. Also, the dosage of the composition that elicits an appropriate response, the concentration of the components therein, and the timing of administering the composition can be determined. Such determinations do not require undue experimentation in light of the knowledge of one of ordinary skill in the art, this disclosure, and the references cited herein. The duration of continuous administration can also be determined without undue experimentation.

[0170] Treatment method A medical professional can diagnose a subject having a neurodegenerative disease by evaluating one or more symptoms of the disease. Non-specific symptoms of a subject's neurodegenerative disease include: difficulty lifting the front of the foot and toes; weakness of the arms, legs, feet, or ankles; weakness or clumsiness of the hands; slurred speech; difficulty swallowing; muscle spasms; twitching of the arms, shoulders, and tongue; difficulty chewing; difficulty breathing; muscle paralysis; partial or complete vision loss; double vision; tingling or pain in parts of the body; electric shock sensations associated with head movements; tremors; unsteady gait; fatigue; dizziness; memory loss; and disorientation. Symptoms include: impairment; misrecognition of spatial relationships; difficulty reading and writing; decreased concentration and thinking ability; inability to judge or make decisions; difficulty planning and performing familiar tasks; depression; anxiety; social withdrawal; mood swings; excitability; aggression; changes in sleep patterns; wandering; dementia; loss of automatic movements; postural and balance disorders; muscle rigidity; bradykinesia; delayed or abnormal eye movements; involuntary movements of spasms or writhing (chorea); involuntary, sustained muscle contractions (dystonia); lack of flexibility; lack of impulse control; and changes in appetite. In addition, medical professionals may, in some cases, make a diagnosis based on the subject's family history of neurodegenerative disease. Medical professionals may diagnose a subject with neurodegenerative disease when the subject comes to a medical facility (e.g., a clinic or hospital). In some cases, medical professionals may diagnose a subject with neurodegenerative disease while the subject is residing in a care facility. Generally, physicians diagnose a subject with neurodegenerative disease after one or more symptoms have appeared.

[0171] The present disclosure provides a method of treating Alzheimer's disease or its symptoms, the method comprising administering to a subject (e.g., a mammal such as a human) a pharmaceutical composition containing cells that express a neuroprotective substance, such as ApoE2, Trem2, MT1G, or a combination thereof, in a therapeutically effective amount. In some embodiments, the cells are hematopoietic stem progenitor cells. In some embodiments, the cells are microglial progenitor cells. Thus, the method in some embodiments comprises administering to a subject a therapeutically effective amount of the cells described herein under conditions such that Alzheimer's disease is treated.

[0172] The methods herein comprise administering to a subject (including a subject identified as needing such treatment) an effective amount of the cells described herein or a composition containing such cells described herein to effect such an outcome. Such treatment would be suitably administered to a subject suffering from, having, being susceptible to, or at risk of Alzheimer's disease or its symptoms, particularly a human. In some embodiments, the methods herein comprise administering to a subject (including a subject identified as needing such treatment) an effective amount of a compound described herein or a composition described herein to effect such an outcome.

[0173] In some embodiments, the cells or the composition containing the cells are administered to the subject in a targeted manner. For example, in some embodiments, a composition containing cells that express ApoE2, Trem2, or a metalloprotein, or a combination thereof, is administered directly to the subject's brain. In some embodiments, the composition is delivered directly to the brain by intracerebroventricular administration. In some embodiments, the composition is delivered to the lateral ventricle of the subject's brain by this method. Methods of administration useful in the present disclosure are described, for example, in International Application No. PCT / US2020 / 045106, which is incorporated herein by reference in its entirety.

[0174] Alternatively, the composition may be delivered systemically, such as by intravenous administration. Cells administered in this manner need to cross the blood-brain barrier before engrafting in the target brain. Other methods of administration (including parenteral, mucosal, implant, intraperitoneal, intradermal, transcutaneous, intramuscular, intracerebroventricular injection, infusion and / or bolus injection, and subcutaneous) are generally known in the art. In some embodiments, cells are administered to the target in an injection-suitable medium, such as phosphate-buffered saline. Intracerebroventricular administration may be advantageous because the cells administered to the target are intended for the regrowth of microglial cells, and other routes of administration require crossing the blood-brain barrier.

[0175] When transplanted cells engraft in the target brain, they result in a population of cells that express the therapeutic agent. However, since the transplanted cells are to replace endogenous cells (i.e., microglia), in certain embodiments, a method of treating a subject with, susceptible to, or at risk of having Alzheimer's disease further includes administering an agent to the subject to destroy the endogenous microglia before administering the therapeutic agent-expressing HSPCs. In some embodiments, the agent is an alkylating agent. In particular, nanoparticle delivery of alkylating agents may be effective in creating an environment suitable for the engraftment of transplanted HSPCs, as described in International Application No. PCT / US2017 / 056774 (the contents of which are incorporated herein by reference in their entirety).

[0176] kit This disclosure envisions a kit for the treatment or prevention of Alzheimer's disease. In some embodiments, the kit comprises a composition containing a modified HSPC expressing a neuroprotective substance. In some embodiments, the neuroprotective protein is ApoE2, Trem2, or metallothionein, or a combination thereof. The kit may include instructions for a treatment protocol, reagents, equipment (test tubes, reaction vessels, needles, syringes, etc.), and reference materials for calibrating or performing the treatment protocol. Instructions provided with the kit according to this disclosure may describe appropriate operating parameters in the form of detectable labels or separate inserts. Optionally, the kit may further include standards or control information so that a test sample can be compared to a control information standard to determine whether consistent results are obtained. In some embodiments, the kit comprises nanoparticles for the destructive pretreatment of endogenous microglia cells.

[0177] In some embodiments, the kit comprises a sterile container containing a therapeutic or prophylactic cell composition; such containers may be boxes, ampoules, bottles, vials, tubes, bags, pouches, blister packs, or other suitable container forms known in the art. Such containers may be made of plastic, glass, laminated paper, metal foil, or other materials suitable for holding pharmaceuticals.

[0178] If necessary, the agents of the present invention are provided with instructions for administering the agents to subjects who have or are at risk of developing a neurological disorder or impairment of the central nervous system. The instructions generally include information on the use of the composition for the treatment or prevention of the disease or impairment. In other embodiments, the instructions include at least one of the following: a description of the therapeutic agent; a dosing plan and administration for the treatment or prevention of a neurological disorder or its symptoms; precautions; warnings; indications; contraindications; information on overdose; side effects; animal pharmacology; clinical trials; and / or reference materials. The instructions may be printed directly on the container (if any), as a label affixed to the container, or as a separate sheet, pamphlet, card, or folder enclosed with the container.

[0179] The implementation of this invention will utilize conventional techniques of molecular biology (including recombination techniques), microbiology, cell biology, biochemistry, and immunology, unless otherwise indicated, which are well within the scope of those skilled in the art. Such techniques are well described in the following literature: “Molecular Cloning: A Laboratory Manual”, 2nd edition (Sambrook, 1989); “Oligonucleotide Synthesis” (Gait, 1984); “Animal Cell Culture” (Freshney, 1987); “Methods in Enzymology” “Handbook of Experimental Immunology” (Weir, 1996); “Gene Transfer Vectors for Mammalian Cells” (Miller and Calos, 1987); “Current Protocols in Molecular Biology” (Ausubel, 1987); “PCR: The Polymerase Chain Reaction” (Mullis, 1994); “Current Protocols in Immunology” (Coligan, 1991). These techniques are applicable to the production of polynucleotides and polypeptides of the present invention and can therefore be considered when carrying out and implementing the present invention. Techniques particularly useful in specific embodiments will be described in the following sections.

[0180] The following examples are intended to provide those skilled in the art with a complete description of the methods of carrying out and using the compositions and therapeutic methods of the present disclosure, and are not intended to limit the scope of what the inventors consider to be the invention. [Examples]

[0181] Example 1: Preparation and characterization of lentiviral vectors Lentiviral vectors containing nucleic acids encoding human ApoE2 (hApoE2) and Trem2 (hTrem2) were constructed under transcriptional regulation by the human PGK promoter, and the expression of target molecules in BV-2 cells was tested (Figure 1 and Figures 2A-2C). Trem2 is an immune receptor in the brain, and is expressed on the cell membrane so that its N-terminus is exposed to the extracellular space.

[0182] Trem2 and ApoE2 vectors were used in in vitro experiments with BV-2 microglia cells. Correct cell surface expression was confirmed by flow cytometry of BV-2 cells transfected with Trem2 (not shown).

[0183] The ability of mock-treated BV2 cells and BV2 cells transfected with Trem2_LV and APoE2-LV to phagocytose amyloid-beta (Aβ) was evaluated using a fluorescence imaging system. Stable transfected BV2 cells and control BV2 cells were treated with appropriately processed 5-FAM-tagged amyloid-beta (Aβ) monomers (150 μM, in 1×PBS) and oligomers (150 μM, in 1×PBS, incubated at 37°C for 72 hours), and then read using a fluorescence microplate reading system (Figure 2D). As expected, BV-2 microglia took up abundant amyloid-beta (Aβ) oligomers compared to control 293T cells (Figure 2E). Interestingly, Trem2-transfected BV-2 cells showed increased amyloid-beta (Aβ) oligomer phagocytosis compared to control untransfected BV-2 cells (Figure 2E).

[0184] Example 2: Hematopoietic stem cell gene therapy in 5xFAD mice We designed experiments to investigate the use of hematopoietic stem cell (HSC) gene therapy using lentiviral vectors (LV) overexpressing ApoE2 and Trem2+ / - metallothionein (MT) in a reliable animal model of Alzheimer's disease (AD), namely 5XFAD mice. Because Alzheimer's disease (AD) affects only the central nervous system (CNS), we employed an innovative transplantation protocol based on the delivery of transduced hematopoietic stem progenitor cells (HSPCs) into the lateral ventricle of recipient mice, linking rescue from busulfan myeloablative conditioning to a backup of non-transduced hematopoietic cells. The overall objective of this study was to evaluate the efficacy of an intracerebral gene therapy approach based on the transplantation of mouse hematopoietic stem progenitor cells (HSPCs) transduced with lentiviral vectors (LV) encoding the cDNA of human ApoE2 or Trem2 genes in combination with metallothionein (MT), and to select the most promising transcripts for future development.

[0185] Numerous transgenic mouse models have been created over the past few years to characterize the pathological and behavioral changes underlying Alzheimer's disease (AD). 5xFAD transgenic mice carry five mutations that cause overproduction of Aβ42; these mice exhibit amyloid plaque pathology similar to that seen in Alzheimer's disease (AD), although other transgenic models develop amyloid plaques more slowly. Accumulation of Aβ42 in neurons in the 5XFAD brain begins at 6 weeks of age. These mice develop early-stage AD disease at approximately 6 weeks of age. Survival rates of 5xFAD mice have been reported to decline after 10 months.

[0186] Lineage-negative (Lin-) cells were purified from the bone marrow of donor mice (5XFAD), and one of the following vectors, namely APOE2 / TREM2 or a combination of them with MT1G, was introduced according to a standard transduction protocol at a multiple of infection (MOI) of 75–100. Good and comparable transduction efficiency was obtained in the preparation of all samples in which approximately 10 vector copies were measured in in vitro liquid culture (Figure 3A). Expression of hApoE2 and Trem2 was confirmed by Western blotting in liquid culture offspring of transduced cells (Figure 3B).

[0187] These transduced cells (0.3 × 10⁶ to 0.5 × 10⁶ lentiviral vector (LV)-transduced Lin-cells) were intraventricularly (ICV) injected into the brains of busulfan-treated 5XFAD recipient mice. Bone marrow support (2.0 × 10⁶ whole bone marrow cells, transduced or untransduced, with or without a lentiviral vector (LV) containing a reporter gene such as green fluorescent protein (GFP)) was administered 3–5 days post-transplant to rescue peripheral hematopoiesis. Each treatment cohort contained 10–15 animals. Controls included untreated 5xFAD mice or 5xFAD mice transplanted with untransduced / GFP-transduced 5xFAD Lin-cells, and untreated wild-type animals or wild-type animals transplanted with untransduced / GFP-transduced wild-type Lin-cells; all were age-matched to the treated animals. Diazepam was administered in drinking water for 9 days as seizure prophylaxis. Baytril was administered orally for two weeks, followed by sulfatrim, as a prophylactic antibiotic. After transplantation, mice were monitored by collecting clinical signs to identify inter-current deaths (ICDs) early until the scheduled final evaluation.

[0188] The 5XFAD mouse model exhibited age-dependent motor phenotypes, in addition to reduced working memory impairment on alternating response tasks and decreased anxiety levels evident in the elevated cusp maze task. For phenotypic evaluation, behavioral tests (novel object recognition, open field test, and elevated cusp maze) were performed monthly from 4 months to 12 months of age to monitor phenotypic changes and assess the beneficial effects of gene therapy in transplanted mice; however, the Morris water maze (MWM) test was performed at 12 months of age, when the study endpoint was reached, to record cognitive impairment. Of the tests performed, the elevated cusp maze demonstrated clear phenotypic progression of Alzheimer's disease (AD) in control animals (green fluorescent protein (GFP) transplanted and untreated 5xFAD mice), while the other tests (novel object recognition, open field test) provided no information in the experimental setting (Figures 4A–4F). The Morris water maze (MWM) revealed a pathological phenotype characterized by a decreased ability to reach the hidden inverse platform over multiple trials and a smaller distance traveled within the target quadrant (NW) (probe trials) (Figures 4A-4F).

[0189] These tests were also used to evaluate the phenotype of the treated mice. Interestingly, in the elevated cruciform maze test to assess anxiety, at 12 months, Trem2-implanted mice spent less time in the open arm compared to affected control mice, and the pathological phenotype observed in controls of the same age was prevented (Figure 5A). Similar results were observed in the Morris water maze (MWM) test (Figures 5B-D). In probe trials, all 5xFAD control mice showed a significant decrease in the distance traveled within the target quarter (NW), while wild-type and Trem2-implanted animals covered more distance, indicating that they had improved memory retention or retrieval compared to 5xFAD controls. Furthermore, Trem2-implanted animals showed a significant improvement in latency to find the hidden platform after 5 trials. In addition, the cognitive flexibility deficit observed in 5xFAD during reverse learning in the Morris water maze (MWM) task was prevented by transplantation of Trem2-introduced cells. In fact, the average latency to reach the platform was significantly shorter in Trem2-treated mice than in 5xFAD control mice, suggesting cognitive flexibility as good as that observed in wild-type mice. Conversely, ApoE2-treated mice did not show significant differences from 5xFAD controls in behavioral tests. Transduction of transplanted cells with MT1G lentiviral vectors (LV) in addition to vectors encoding Trem2 or ApoE2 did not affect the outcomes of behavioral tests in their respective cohorts (data not shown).

[0190] After the behavioral tests, the treated and control animals underwent a final evaluation, including a complete necropsy, after cardiac perfusion with saline and tissue collection for molecular and histological evaluation. Digital droplet PCR (ddPCR) performed on the brains of transplanted animals demonstrated engraftment of transduced cells (vector copy number (VCN) measured in their liquid culture progeny is shown in Figure 3A), with VCN values in the range of 0.5 - 1.5 copies of LV / mouse genome, and the highest VCN measured in the APoE2+MT1G cohort. Engraftment of transduced cells in the bone marrow could not be confirmed by ddPCR (data not shown).

[0191] Pathological neuroinflammation is associated with neurodegeneration and is primarily mediated by microglia, the commensal immune cells of the central nervous system. Microglia play a crucial role in sensing environmental changes, responding to harmful stimuli, and phagocytosing debris and apoptotic neurons. Neuroinflammation was assessed using specific markers of microglial activation (Iba1) and astrocyte response (GFAP) (Figures 6A-6D). As expected, Iba1 and GFAP expression was higher in 5xFAD animals compared to wild-type animals. The expression levels of these markers were highly heterogeneous in treated mice, likely due to differences in transdextrin / offspring engraftment rates. In particular, Iba1 and GFAP signaling in the brains (cerebral cortex and hippocampus) of treated mice was reduced by 4–70% compared to the signals measured in 5xFAD controls; however, in cohort-wide comparisons, significance was observed only for Iba1 signaling in the cerebral cortex region. Amyloid-beta (Aβ) aggregates induce a range of reactions, including neuronal cell death, neuroinflammation, and gliosis, ultimately leading to cognitive impairment. Histological analysis of the brains of 5xFAD controls revealed intracellular accumulation of plaques containing Aβ deposits (Figure 6E-6H). Interestingly, treated animals showed a dose-dependent significant reduction in Aβ levels in the cerebral cortex and hippocampus; specifically, higher rates of amyloid-beta (Aβ) reduction were observed in animals where the vector copy number (VCN) in brain tissue exceeded the mean of 0.5 for the study cohort. These effects on neuroinflammation and amyloid-beta (Aβ) deposition were more pronounced in Trem2-treated animals than in ApoE2-treated animals and were unaffected by the use of MT1G vectors in addition to vectors encoding Trem2 or ApoE2.

[0192] A hematopoietic stem cell (HSC) gene therapy strategy for Alzheimer's disease (AD) was validated in the most studied animal model of the disease (5xFAD mouse). This gene therapy strategy was based on lentiviral vector (LV)-mediated hematopoietic stem cell (HSC) genetic engineering to express (i) Trem2 or (ii) ApoE2 in microglia-like tissue offspring. In both settings, the potential synergistic effect of these two therapeutic constructs with MT1G expression was investigated.

[0193] Novel lentiviral vectors were developed to express therapeutic transcripts that may influence the major molecular mechanisms of Alzheimer's disease (AD) in the central nervous system (CNS) of Alzheimer's disease (AD) patients. In particular, Trem2 and ApoE2 were expressed. The vectors were constructed in the form of a vesicular stomatitis virus G (VSV-G) pseudotyped lentiviral vector (LV) for transduction of microglia cell lines and hematopoietic stem progenitor cells (HSPCs). In vitro transduction of BV-2 cells with the Trem2-encoding lentiviral vector (LV) demonstrated enhanced phagocytosis of amyloid-beta (Aβ) oligomers, thereby supporting the selection of this transgene.

[0194] These vectors were used in a study of in vivo CNS (central nervous system) hematopoietic stem cell (HSC) gene therapy in 5xFAD mice, aiming to evaluate whether either of these two strategies influences disease progression. 5xFAD hematopoietic stem cells (HSCs) transduced with two therapeutic lentiviral vectors (LVs) engrafted in the brains of myelosophical 5xFAD mice transplanted intraventricularly (ICV). Behavioral and histological analyses of these mice compared to mock-treated controls indicated that these constructs, particularly Trem2, may have a positive impact on disease onset and early stages. Improved behavior, reduced neuroinflammation, and a dose-dependent reduction in amyloid-beta (Aβ) deposits were observed. In all assessments, some variability exists due to technical issues, such as generally lower-than-target engraftment of transduced cells in the CNS across the entire cohort.

[0195] Animals treated with Trem2 showed promising results, as the treatment reduced the clinical symptoms of the disease and disease-related histological abnormalities as observed in behavioral studies.

[0196] Other aspects From the foregoing description, it will be apparent that the invention described herein can be modified and altered to suit various uses and conditions. Such embodiments are also included in the following claims.

[0197] In this specification, the enumeration of lists of elements in the definition of a variable element includes the definition of that variable element as any single element or as a combination (or partial combination) of the listed elements. In this specification, the enumeration of embodiments includes that embodiment as any single embodiment or as a combination with any other embodiment or part thereof.

[0198] All patents and publications described herein are incorporated herein by reference to the same extent as when each individual patent and publication is specifically and individually indicated as being incorporated by reference. This application may relate to PCT / US2020 / 045106 and PCT / US2017 / 05677, as well as PCT applications filed on 1 October 2020, entitled “MICROGLIA SPECIFIC PROMOTERS AND METHODS OF USE THEREFORE” and “COMPOSITIONS AND METHODS FOR TREATING AMYOTROPHIC LATERAL SCLEROSIS” (claiming priority to the following provisional applications 62 / 908,966 and 62 / 908,942, respectively), all of which are incorporated herein by reference.

Claims

1. A composition for treating subjects having or being prone to developing Alzheimer's disease, comprising (i) a TREM2 polypeptide and metallothionein 1G (MT1G) polypeptide, or (ii) an effective amount of the subject's own hematopoietic stem progenitor cells (HSPCs) comprising an expression vector or expression cassette containing one or more polynucleotides encoding ApoE2 polypeptide, TREM2 polypeptide, and MT1G polypeptide.

2. The composition according to claim 1, wherein the vector or cassette contains two or more copies of metallothionein.

3. The composition according to claim 1 or 2, wherein the vector comprises a polynucleotide encoding at least four copies of MT1G.

4. The composition according to any one of claims 1 to 3, wherein the vector comprises a promoter that drives the expression of the polynucleotide.

5. The composition according to claim 4, wherein the promoter is a human phosphoglycerate kinase promoter, or the promoter is a microglia-specific promoter.

6. The composition according to claim 5, wherein the promoter is a TSPO promoter, an MHC class II promoter, or a CX3CR1 promoter.

7. The composition according to any one of claims 1 to 6, wherein the vector is a lentiviral vector.

8. The composition according to any one of claims 2 to 6, wherein the cassette is inserted into the CX3CR1 or TSPO gene locus.

9. The aforementioned HSPC is CD34 ＋ and / or CD38 － and / or CD90 ＋ The composition according to claim 8.

10. The composition according to claim 8 or claim 9, wherein the HSPC is hemizygous with respect to the CX3CR1 gene.

11. A composition for treating a subject having or being prone to developing Alzheimer's disease, comprising (i) a TREM2 polypeptide and an MT1G polypeptide, or (ii) an effective amount of the subject's own hematopoietic stem progenitor cells (HSPCs) comprising a polynucleotide encoding ApoE2 polypeptide, TREM2 polypeptide, and an MT1G polypeptide.

12. The composition according to claim 11, for use in intraventricular administration, for use in intravenous administration, or for use in intrathecal administration.

13. The aforementioned HSPC is Lin － CD34 ＋ CD38 － , and / or CD90 ＋ The composition according to claim 11.

14. The composition according to claim 11, wherein the HSPC is hemizygous with respect to the CX3CR1 gene.

15. A kit comprising the composition according to claim 9 and instructions for delivering it to a target.

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