Pathological responsive recombinant cells and uses thereof

Abstract

Modified cells expressing, presenting or secreting at least one therapeutic molecule are capable of treating or ameliorating a disease of interest, such as, but not limited to, Alzheimer's disease. In a modified cell, expression of a therapeutic molecule is induced when the modified cell approaches or contacts a pathology associated with a target disease. The present disclosure also relates to compositions and kits comprising the disclosed cells. The present disclosure also relates to methods of treating diseases using the disclosed cells.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 335,585, filed on April 27, 2022, the specification of which is incorporated herein by reference in its entirety.

[0003] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0004] This invention was made with government support under Grant No. DA048813 and Grant No. AG073787 awarded by the National Institutes of Health. The government has certain rights in this invention.

[0005] Reference sequence list

[0006] The contents of the electronic sequence listing (file name: UCI_22_14_PCT.xml; size: 27817 bytes; creation date: April 26, 2023) are hereby incorporated by reference in their entirety. Technical Field

[0007] The present invention relates to cells modified to express at least one therapeutic molecule that treats or ameliorates pathology and / or symptoms associated with a specific disease (e.g., Alzheimer's disease). The present invention also relates to compositions and kits comprising the above cells and methods of using the same to treat Alzheimer's disease. Background Art

[0008] Several common neurodegenerative diseases are characterized by the progressive accumulation of insoluble protein aggregates, including Alzheimer's disease, Parkinson's disease, Huntington's disease, and amyotrophic lateral sclerosis (ALS). In Alzheimer's disease, amyloid β (Aβ) adopts different conformational states, including insoluble fibrillar Aβ plaques and soluble Aβ oligomers, which can drive the development of other downstream pathologies, including neurofibrillary tangles and gliosis. These pathologies collectively lead to the loss of neurons and synapses and brain atrophy, resulting in memory loss, cognitive impairment, behavioral changes, and dementia. Currently, approximately 6 million people over the age of 65 in the United States suffer from Alzheimer's disease. Alzheimer's disease is primarily a late-life disease. Therefore, the incidence of this disease is expected to increase as the population ages. Therefore, it is imperative to develop new therapeutic approaches to meet this growing need. However, one challenge in developing drugs to treat brain diseases is the blood-brain barrier (BBB), which poses a significant obstacle to drug delivery, thereby affecting drug efficacy. Both small molecules and macromolecules have been investigated as effective therapeutic agents for the treatment of brain diseases. However, most macromolecules are unable to penetrate the brain endothelium due to physical limitations of molecules crossing the blood-brain barrier. Therefore, despite decades of efforts to develop safe and effective treatments for brain diseases, delivering drugs across the blood-brain barrier in sufficient concentrations to achieve the target effect remains a fundamental obstacle.

[0009] In recent years, CAR-T cell therapy has demonstrated the ability to use engineered cells to deliver therapeutic agents directly and selectively to cancer pathology sites. For central nervous system diseases, neural and bone marrow stem cells have been explored as vehicles for delivering therapeutic drugs. Unfortunately, human neural stem cells have worrisome tumorigenic potential, and bone marrow stem cells require dangerous pretreatment steps for cell delivery to the brain. Therefore, there is still an urgent need for a safe cell delivery vehicle for the selective delivery of therapeutic drugs directly to the site of central nervous system lesions.

[0010] Microglia are the main innate immune cells of the brain and play a key role in maintaining neuronal homeostasis and detecting pathogenic factors and neuronal damage in the local environment. Recently, a chimeric mouse model was developed to examine the interaction between human iPSC-derived microglia and neuropathology. In this model, after xenotransplantation, human iPSC-derived microglia (xMG) disperse widely, mature into homeostatic microglia, exhibit transcriptional profiles similar to those in vivo, and thrive. The present disclosure describes how to modify such cells to treat Alzheimer's disease and other Aβ-related neurodegenerative diseases. Summary of the invention

[0011] The object of the present invention is to provide a composition and method for treating or preventing a disease or condition (including but not limited to Alzheimer's disease (AD)) associated with beta-amyloid protein-related pathology or improving or delaying symptoms and / or pathological processes associated with beta-amyloid protein-related pathology. In some embodiments, beta-amyloid protein-related pathology is associated with Alzheimer's disease. In some embodiments, beta-amyloid protein-related pathology is associated with Parkinson's disease. In some embodiments, beta-amyloid protein-related pathology is associated with Huntington's disease. In some embodiments, beta-amyloid protein-related pathology is associated with amyotrophic lateral sclerosis (ALS). The present invention is not limited to the aforementioned diseases or conditions associated with beta-amyloid protein pathology.

[0012] The embodiments of the invention can be freely combined with one another insofar as they are not mutually exclusive.

[0013] Although immunodeficient mice with missing Rag2 and IL2 receptor γ (IL2Rγ) genes had been previously used to transplant human cells, initial attempts to generate human microglia chimeric mice using standard immunodeficient mice failed. After multiple crosses and experiments, it was surprisingly found that a mouse model carrying a humanized CSF-1 allele was necessary for long-term engraftment and survival of human xenografted microglia (Hasselmann et al., Neuron, 2019; Supplementary Figure S2). Not only was the generation of this model a challenge, but there were other difficulties, including backcrossing the model to an amyloid-producing line (5×FAD mice) and restoring homozygosity for the Rag2, IL2Rγ, and humanized CSF-1 alleles.

[0014] Studies have shown that mouse microglia are highly sensitive to isolation procedures (Marsh et al., Nat Neurosci, 2022). Despite the challenges, the inventors have developed a method to rapidly purify implanted human microglia with minimal disruption to the implanted human microglia. This was achieved by using a negative magnetic sorting method to eliminate all mouse cells, leaving only untouched but highly pure and viable human microglia. Details and validation of this novel isolation method are provided by Hasselmann et al. in Neuron (2019). This isolation method can isolate human microglia from the brains of chimeric mice to examine gene expression and identify candidate microglial genes that show changes in expression in response to beta-amyloid pathology.

[0015] The above isolation methods enabled single-cell sequencing, which provided a partial list of plaque-reactive microglial genes. However, single-cell sequencing is less sensitive than bulk RNA sequencing and typically only captures the most abundantly expressed transcripts. Despite the challenges, the inventors were able to develop a method to isolate microglia that respond to plaques and microglia that do not respond to plaques from the same chimeric mouse brain, thereby better understanding the human microglial response to plaques. Single-cell sequencing data and subsequent immunohistochemical validation (see Figure 1 A-1P) showed that CD9 and HLA-DRB are highly enriched in plaque-associated human microglia. The inventors also developed a fluorescence activated cell sorting (FACS) method to separate CD9 / HLA-DRB double positive microglia from double negative steady-state microglia. This was achieved by transplanting four independent human microglia samples into mice followed by bulk RNA sequencing. This analysis provides a more complete dataset of plaque-induced human microglial genes as described herein (see Fig. 20 A-20D).

[0016] One aspect of the present disclosure provides a modified cell for treating amyloid-β-related pathologies (e.g., Alzheimer's disease (AD), Parkinson's disease, Huntington's disease, ALS, other neurodegenerative diseases, e.g., other neurodegenerative diseases associated with Aβ) or improving symptoms and / or pathological processes associated with the amyloid-β-related pathologies. The cell is sensitive to amyloid-β-(Aβ)-related pathologies. For example, in some embodiments, when the modified cell approaches or contacts Aβ-related pathologies (also referred to herein as Alzheimer's disease-related pathologies), such as amyloid-β-(Aβ) peptide plaques, soluble Aβ monomers, insoluble Aβ monomers, Aβ oligomers, pyroglutamate Aβ, fibrils, fibers comprising Aβ of different lengths, or a combination thereof, the modified cell expresses, presents, secretes therapeutic molecules, or a combination thereof. In some embodiments, when the modified cell approaches or contacts amyloid-β-related pathologies (e.g., Alzheimer's disease-related pathologies), the modified cell expresses and secretes therapeutic molecules. In some embodiments, when the modified cell is close to or in contact with a beta-amyloid protein-related pathology, the modified cell expresses, presents and secretes a therapeutic molecule. The therapeutic molecule changes at least one aspect of one or more beta-amyloid protein-related pathological phenotypes or beta-amyloid protein-related pathologies. For example, in some embodiments, the therapeutic molecule reduces the size and / or number of Aβ aggregates. The present invention is not limited to the aforementioned examples of changing the beta-amyloid protein-related pathological phenotype.

[0017] β-amyloid protein-related pathological phenotypes or symptoms may include one or more combinations of memory problems, learning disabilities, cognitive problems, vision problems, behavioral changes, personality changes, depression, or epileptic seizures. In some embodiments, Aβ-related pathologies may include β-amyloid protein (Aβ) peptide plaques. In some embodiments, Aβ-related pathologies may include soluble Aβ monomers. In some embodiments, Aβ-related pathologies may include insoluble Aβ monomers. In some embodiments, Aβ-related pathologies may include Aβ oligomers. In some embodiments, Aβ-related pathologies may include fibrils. In some embodiments, Aβ-related pathologies may include fibrils containing Aβ of different lengths. In some embodiments, Aβ-related pathologies may include one of the following or a combination thereof: β-amyloid protein (Aβ) peptide plaques, soluble Aβ monomers, insoluble Aβ monomers, Aβ oligomers, pyroglutamate Aβ, fibrils, fibers containing Aβ of different lengths, or combinations thereof.

[0018] The modified cell may include a nucleic acid sequence encoding a therapeutic molecule. The nucleic acid sequence may be operably linked to a pathological response promoter, for example, a promoter responsive to pathologies associated with Alzheimer's disease, Parkinson's disease, Huntington's disease, ALS, etc., for example, a β-amyloid protein response promoter. As a non-limiting example, a nucleic acid may be operably linked to a promoter responsive to an Aβ peptide, for example, when a cell approaches or contacts an Aβ peptide, the promoter may be activated. In some embodiments, the therapeutic molecule may shear the Aβ peptide. In some embodiments, the therapeutic molecule may reduce the amount of Aβ peptide in the Aβ peptide plaque in the brain of an individual. In some embodiments, the therapeutic molecule may reduce the size or quantity of soluble Aβ monomers, insoluble Aβ monomers, Aβ oligomers, pyroglutamic acid Aβ, fibrils, or fibers comprising different lengths of Aβ. In some embodiments, the therapeutic molecule may reduce the size and / or quantity of Aβ aggregates. In some embodiments, the therapeutic molecule may enhance amyloid hydrolysis. In some embodiments, the therapeutic molecule may enhance the phagocytosis of microglia to β-amyloid protein. The present invention is not limited to the aforementioned mechanism of action for altering the pathological phenotype associated with beta-amyloid protein.

[0019] One aspect is a modified cell comprising a nucleic acid sequence encoding a therapeutic molecule, wherein the nucleic acid sequence is operably linked to a promoter, and when the cell approaches or contacts a beta amyloid (Aβ) peptide plaque, a soluble Aβ monomer, an insoluble Aβ monomer, an Aβ oligomer, a pyroglutamate Aβ, a fibril, or a fiber containing Aβ of different lengths, the promoter activates the transcription of the therapeutic molecule. In some embodiments, the therapeutic molecule shears the Aβ peptide. In some embodiments, the therapeutic molecule reduces the amount of Aβ peptide in the Aβ peptide plaque in the brain of an individual. In some embodiments, the therapeutic molecule reduces the size or quantity of soluble Aβ monomers, insoluble Aβ monomers, Aβ oligomers, pyroglutamate Aβ, a fibril, or a fiber containing Aβ of different lengths. In some embodiments, the therapeutic molecule reduces the size and / or quantity of Aβ aggregates. In some embodiments, the therapeutic molecule enhances amyloid hydrolysis. In some embodiments, the therapeutic molecule enhances the phagocytosis of microglia beta amyloid.

[0020] In some embodiments, the therapeutic molecule is a therapeutic mRNA molecule. In some embodiments, the therapeutic molecule is a therapeutic protein. The therapeutic protein may be a membrane-bound protein or a secreted protein. Secreted therapeutic proteins may include modified proteins. For example, in some embodiments, the therapeutic protein is a protein that has been modified to lack a cytoplasmic domain. In some embodiments, the therapeutic protein is a protein that has been modified to lack a transmembrane domain. In other embodiments, the therapeutic protein is a protein that has been modified to lack a transmembrane domain and a cytoplasmic domain. In some embodiments, the therapeutic molecule comprises an enzyme or an immunomodulatory protein. In some embodiments, the therapeutic molecule comprises TREM2. In some embodiments, the therapeutic molecule comprises an insulin degrading enzyme. In some embodiments, the therapeutic molecule comprises MSR1 (SCARA1). In some embodiments, the therapeutic molecule comprises LRP1. In some embodiments, the therapeutic molecule comprises APOE. In some embodiments, the therapeutic molecule comprises IL4. In some embodiments, the therapeutic molecule comprises IL-10. In some embodiments, the therapeutic molecule comprises endothelin-converting enzyme (ECE). In some embodiments, the therapeutic molecule comprises a protease, such as cathepsin B or cathepsin D. In some embodiments, the therapeutic molecule comprises cathepsin B. In some embodiments, the therapeutic molecule comprises cathepsin D. In some embodiments, the therapeutic molecule comprises a matrix metalloproteinase (MMP), such as MMP2 or MMP9. In some embodiments, the therapeutic molecule comprises matrix metalloproteinase 2 (MMP2). In some embodiments, the therapeutic molecule comprises matrix metalloproteinase 9 (MMP9).Specific therapeutic molecules are described in the following publications, which are incorporated herein by reference in their entirety: Leissring et al., Neuron 2003;40:1087-1093; Eckman et al., J Biol Chem 2001;276:24540-24548; Hernandez-Guillamon et al., J Biol Chem 2015;290(24):15078-15091; Suire and Leissing, J Exp Neurol. 2021;2(1):10-15; Wang et al., Biol Chem 2012;287(47):39843-41; Zhao et al., J Exp Med 2022;219(12):e20212479; Frenkel et al., Nat Commun 2013;4:2030; N'Songo et al., Mol Neurol 2013;287(47):39843-41; Zhao et al., J Exp Med 2022;219(12):e20212479; Frenkel et al., Nat Commun 2013;4:2030; N'Songo et al., Mol Neurol 2013;287(47):39843-41; Zhao et al., J Exp Med 2022;219(12):e20212479; Frenkel et al., Nat Commun 2013;287(47):39843-41; Neurodegene 2013; 8(Supplement 1): P33; Ulrich et al., J Exp Med 2018; 215(4): 1047-1058; Yi et al., Cytotechnology 2020; 72(4): 589-602. In some embodiments, the therapeutic molecule comprises a metalloprotease, which may comprise enkephalinase activity. In some embodiments, the therapeutic molecule comprises enkephalinase. In some embodiments, the therapeutic protein comprises enkephalinase or a derivative thereof. For example, the therapeutic protein may comprise an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3. In some embodiments, the therapeutic protein comprises an amino acid sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3. For example, in some embodiments, the therapeutic protein comprises a membrane-bound enkephalinase as SEQ ID NO: 1. In some embodiments, the therapeutic protein comprises a membrane-bound enkephalinase as SEQ ID NO: 2. In some embodiments, the therapeutic protein comprises a secreted enkephalinase as SEQ ID NO: 3.

[0021] The pathological response promoter can be an endogenous promoter, but it does not have to be an endogenous promoter. In some embodiments, the promoter is an endogenous promoter. In some embodiments, the promoter is not an endogenous promoter. In some embodiments, the promoter is an endogenous promoter, but is also incorporated into the cell in an exogenous manner (alone), for example as a proximal promoter, wherein the therapeutic molecule is operably connected to the proximal promoter. In some embodiments, the promoter is a wild-type (unmodified) form of the promoter. In some embodiments, the promoter is modified (compared to its wild-type form). As a non-limiting example, the CD9 proximal promoter can be produced and integrated within the AAV safe harbor locus.

[0022] The pathological reaction promoter may include a promoter from a gene selected from the group consisting of: DCSTAMP gene, CD9 gene, CD44 gene, LGALS3 gene, SPP1 gene, GPNMB gene, HLA-DRB gene, LPL gene, LIPA, FABP3 gene, MS4A6A gene, CXCR4 gene, CHI3L1 gene, OLR1 gene, CD36 gene, SLAMF8 gene, TREM2 gene, MSR1 gene, B2M gene, ITGAX gene and MITF gene. For example, in some embodiments, the promoter includes a promoter of the CD9 gene. In some embodiments, the promoter includes a promoter of the LGALS3 gene. In some embodiments, the promoter includes a promoter of the HLA-DRB gene. In some embodiments, the promoter includes a promoter of the CD11c (ITGAX) gene. In some embodiments, the promoter includes a promoter selected from the group consisting of: CD9, LGALS3, HLA-DRB and CD11c. In some embodiments, the promoter includes a promoter selected from the following genes: CD9, LGALS3, HLA-DRB, TREM2 and CD11c. In some embodiments, the promoter includes the promoter of the DCSTAMP gene. In some embodiments, the promoter includes the promoter of the CD44 gene. In some embodiments, the promoter includes the promoter of the SPP1 gene. In some embodiments, the promoter includes the promoter of the GPNMB gene. In some embodiments, the promoter includes the promoter of the LPL gene. In some embodiments, the promoter includes the promoter of the LIPA gene. In some embodiments, the promoter includes the promoter of the FABP3 gene. In some embodiments, the promoter includes the promoter of the MS4A6A gene. In some embodiments, the promoter includes the promoter of the CXCR4 gene. In some embodiments, the promoter includes the promoter of the CHI3L1 gene. In some embodiments, the promoter includes the promoter of the OLR1 gene. In some embodiments, the promoter includes the promoter of the CD36 gene. In some embodiments, the promoter includes the promoter of the SLAMF8 gene. In some embodiments, the promoter includes the promoter of the TREM2 gene. In some embodiments, the promoter includes the promoter of the MSR1 gene. In some embodiments, the promoter includes the promoter of the B2M gene. In some embodiments, the promoter includes the promoter of the MITF gene. The present invention is not limited to the aforementioned promoters.Although no data are shown for promoters from DCSTAMP gene, CD44 gene, SPP1 gene, GPNMB gene, LPL gene, LIPA, FABP3 gene, MS4A6A gene, CXCR4 gene, CHI3L1 gene, OLR1 gene, CD36 gene, SLAMF8 gene, TREM2 gene, MSR1 gene, B2M gene and MITF gene, a person skilled in the art would understand that these promoters can be used to replace promoters from CD9, LGALS3, HLA-DRB and CD11c with reasonable expectations of success.

[0023] Other non-limiting examples of pathological response promoters that can be used to generate modified cells of the present disclosure include, but are not limited to, HLA-DRB3, HLA-DRB5, HLA-DRA, CHIT1, DKK2, HLA-DQB1, PLXNA1, CCR7, HLA-DPA1, RGCC, COX6A2, HLA-DRB1, HLA-DPB1, TAFA3, FAIM2, ZNF804A, LGALS1, DOK2, HLA-DOA, GPR153, SLPI, ADGRF5, MYOZ1, HBEGF, BHLHE40, TMEM37, SLC16A8, GRM5, S100A4, GABRB1, LON RF3, PIGR, RAB7B, BIRC7, FABP5, CA2, FCMR, GABRB3, ATF7-NPFF, ANXA2, EGR2, OR9G1, IFNLR1, APOC1, CD200R1, PKD2L1, TDRD6, HLA-DMA, KCNJ5, GLDN, PR EX2, OLFM2, PADI2, COL1A2, RRAGD, ROR2, PTCRA, EDN1, RAMP1, BCL2A1, CD83, CHST2, MAFB, DUSP2, FPR3, DPYD, PHLDA1, PTPRG, IER5L, CPNE8, MPDZ, RNF15 2. PTGER4, MCF2, SDS, GUTA, RGS16, ANGPTL6, PLA2G7, CCL3, ATF3, RAB42, TBX18, STARD13, NR4A3, S100A13, TRDN, GADD45G, ADRA1B, LYPD1, FXYD6, EPHB6 , FERMT2, ART4, TNFSF18, TNFSF15, SLC38A4, CDH6, SCIN, FGR, ZNF385B, FUT9, SORCS1, KLF10, CFD, TRPM1, FLVCR2, NFIL3, MYO1E, ITPRID1, SLC47A1, LIT AF, RASGEF1B, DENND2D, ADCY3, SYTL3, ARL4C, IL4I1, CDCP1, TRERF1, VAT1, INPP1, ARRDC4, ENPP1, GSDME, APOC2, COL4A1, CDKN1A, KCNC4, KCNJ2, USP2, U NC13B, VEGFB, TNFAIP2, HLA-DMB, NAB2, ACP5, CYTL1, FXYD5, ALDH1A2, TMEM176B, TAGLN2, TIMD4, MEP1A, ADGRL2, FAM20C, LRRC39, CD74, RASGRP3, HEG1,TNFRSF14, ADGRE2, FAM110B, GADD45B, TSKU, ID2, DUSP10, TNFSF13B, FKBP5, MYC, BCL6, TNFAIP8L3, ARID5B, APOC4 - APOC2, PMAIP1, P2RY8, CCRL2, Ribonuclease 1 (RNASE1), LSP1, ARHGEF3, CLEC19A, KCNQ5, DTNA, PILRA, WIPF3, CSTB, XYLT1, DBI, SDC2, KCNMA1, ALCAM, TSPAN4, RPS28, KLHDC8B, MBOAT1, FAM20A, CDH8, NAP1L1, SMIM4, ALAS1, PDLIM7, IQGAP2, INPP4B, TRIM58, SLC36A1, TXN, HLA - B, HSPB1, AP1S2, PLAU, HLA - DQA1, CYSTM1, IER5, MS4A7, SPN, CTSD, SELENOP, SDSL, CD14, HLA - F, B3GALNT1, GAPT, RASSF3, GAS7, GAS2L3, GPD2, GPM, SGPP1, LILRA4, NEB, PRXL2C, SNX24, PPARG, CAPG, CD6, INHBA, PTPN7, LYRM9, ATOX1, UGCG, TMSB10, IFI30, M1AP, SH3BGRL3, CMYA5, PRKCH, NIBAN1, GK, HSD3B7, GPR65, LGALS9, RPL36, SH3RF1, WWC3, ALDH5A1, SNTB2, HACD4, LTA4H, SCIMP, SLC11A1, PTPDC1, CPNE2, CALHM2, PRDX1, APOE, IQGAP1, OSBPL3, GRK3, DPEP2, TMEM154, FOXP1, GYPC, SLC15A3, NENF, HPSE, TNFRSF11A, PPM1M, HPCAL1, SLC46A1, SLC1A3, MORC1, GABARAPL1, MGLL, TGIF1, ZYX, MAP4 or MFSD2A.

[0024] The nucleic acid sequence encoding the therapeutic molecule can be inserted into the genome of the modified cell (see Example 3). The nucleic acid sequence can be inserted into the genome of the modified cell so that the transcription of the nucleic acid sequence is under the control of the endogenous pathological response promoter in the modified cell genome. The insertion site of the nucleic acid sequence can be located in the gene controlled by the pathological response promoter, upstream or downstream, so that the nucleic acid sequence encoding the therapeutic molecule is in frame with the coding sequence in the gene exon. The nucleic acid sequence can be inserted into the gene controlled by the pathological response promoter so that the nucleic acid sequence encoding the therapeutic molecule is connected to at least a portion of the coding sequence of the gene exon. The exon can be any exon in the gene, including the first or last exon. The first polynucleotide encoding a protease cleavage site, a ribosome skipping sequence or a self-cleaving peptide can be inserted between the coding sequence of the exon and the therapeutic molecule encoding the nucleic acid sequence. For example, in some embodiments, the self-cleaving peptide comprises a P2A sequence. In order to produce secreted therapeutic molecules, a second polynucleotide encoding a secretory peptide signal sequence can be connected to the nucleic acid sequence encoding the therapeutic molecule. In aspects of the present disclosure, where the therapeutic molecule is a therapeutic protein, the coding sequence of the exon and the therapeutic protein encoding nucleic acid sequence can be connected in frame. Similarly, a first polynucleotide encoding a protease cleavage site, a ribosomal skipping sequence, or a self-cleaving peptide and a second polynucleotide encoding a secretory peptide signal sequence can be connected in frame to each other and to the coding sequence of the exon and the nucleic acid sequence encoding the therapeutic protein.

[0025] The modified cells of the present disclosure can be produced using cells from cell lineages that can be differentiated into migratory cells. For example, in some embodiments, modified cells are produced using pluripotent stem cells (PSC). In some embodiments, modified cells are produced using induced pluripotent stem cells (iPSC). In some embodiments, modified cells are produced using myeloid progenitor cells. In some embodiments, modified cells are produced using erythroid bone marrow progenitor cells. In some embodiments, modified cells are produced using hematopoietic stem cells. In some embodiments, modified cells are produced using hematopoietic progenitor cells or precursor cells (HSPC). In some embodiments, modified cells are produced using lymphoid progenitor cells. In some embodiments, modified cells are produced using megakaryocyte-erythrocytes (mk-ery). In some embodiments, modified cells are produced using umbilical cord blood stem cells. In some embodiments, modified cells are produced using embryonic stem cells. In some embodiments, modified cells are produced using myeloid progenitor cells, hematopoietic stem cells or hematopoietic progenitor cells. In some embodiments, the modified cells are produced using pluripotent stem cells (PSC), induced pluripotent stem cells (iPSC), myeloid progenitor cells, erythroid myeloid progenitor cells, hematopoietic stem cells, hematopoietic progenitor or precursor cells (HSPC), lymphoid progenitor cells, megakaryocyte-erythroid (mk-ery), umbilical cord blood stem cells, or embryonic stem cells.

[0026] The modified cells may be microglia-like (MGL) cells, may be induced MGL (iMGL), and may be human induced pluripotent stem cell-derived MGL cells (hiMGL). hiMGL may express P2RY12 and TREM2. hiMGL may express TMEM119 or Iba1. hiMGL may express AXL, STAB1, P2RY6, CCR6, or GPR84 at higher levels than endogenous mature microglia of an individual. hiMGL may express lower levels of CTSL, CTSD, or NPL than endogenous mature microglia of an individual. hiMGL may express lower levels of FFAR2 and COL26A1 than endogenous mature microglia of an individual. hiMGL may express lower levels of Siglec11 and Siglec12 than endogenous mature microglia of an individual. hiMGL may express P2RY13 or OLFML3. hiMGL may be able to phagocytose amyloid beta (Aβ) peptide plaques, soluble Aβ monomers, insoluble Aβ monomers, Aβ oligomers, pyroglutamate Aβ, fibrils and / or fibers comprising Aβ of varying lengths. hiMGL may be able to phagocytose soluble or insoluble forms of Aβ-40 or Aβ-42.

[0027] One aspect of the present disclosure provides a composition comprising a modified cell of the present disclosure.

[0028] One aspect of the present disclosure provides a method for treating Alzheimer's disease in an individual in need thereof, or a method for improving symptoms or pathological processes associated with Alzheimer's disease in the individual. The method comprises administering a modified cell of the present disclosure or a composition of the present disclosure to the individual. The method comprises administering (e.g., transplanting) a modified cell of the present disclosure or a composition of the present disclosure (e.g., hiMGL) to brain tissue of the individual.

[0029] One aspect of the present disclosure provides a method for treating Parkinson's disease in an individual in need thereof, or a method for improving symptoms or pathological processes associated with Parkinson's disease in the individual. The method comprises administering a modified cell of the present disclosure or a composition of the present disclosure to the individual. The method comprises administering (e.g., transplanting) a modified cell of the present disclosure or a composition of the present disclosure (e.g., hiMGL) to brain tissue of the individual.

[0030] One aspect of the present disclosure provides a method for treating Huntington's disease in an individual in need thereof, or a method for ameliorating symptoms or pathological processes associated with Huntington's disease in the individual. The method comprises administering a modified cell of the present disclosure or a composition of the present disclosure to the individual. The method comprises administering (e.g., transplanting) a modified cell of the present disclosure or a composition of the present disclosure (e.g., hiMGL) to brain tissue of the individual.

[0031] One aspect of the present disclosure provides a method for treating amyotrophic lateral sclerosis (ALS) in an individual in need thereof or ameliorating symptoms or pathological processes associated with ALS in the individual. The method comprises administering a modified cell of the present disclosure or a composition of the present disclosure to the individual. The method comprises administering (e.g., transplanting) a modified cell of the present disclosure or a composition of the present disclosure (e.g., hiMGL) to brain tissue of the individual.

[0032] One aspect of the present disclosure provides a method for treating a neurodegenerative disease (e.g., a β-amyloid-related neurodegenerative disease) in an individual in need thereof or ameliorating symptoms or pathological processes associated with ALS in the individual. The method comprises administering a modified cell of the present disclosure or a composition of the present disclosure to the individual. The method comprises administering (e.g., transplanting) a modified cell of the present disclosure or a composition of the present disclosure (e.g., hiMGL) to the brain tissue of the individual.

[0033] One aspect of the present disclosure provides a kit comprising a modified cell or composition of the present disclosure. For example, the kit may comprise a modified cell as described herein, wherein the modified cell comprises a nucleic acid sequence encoding a therapeutic molecule as described herein.

[0034] One aspect of the present disclosure provides a method for reducing the amount of Aβ peptide in Aβ peptide plaques in the brain of an individual; and / or the size or number of soluble Aβ monomers, insoluble Aβ monomers, Aβ oligomers, pyroglutamate Aβ, fibrils, or fibers comprising Aβ of different lengths; the method comprising administering to the individual a modified cell or composition of the present disclosure.

[0035] One aspect of the present disclosure is a method of producing a modified cell of the present disclosure, the method comprising introducing a nucleic acid sequence encoding a therapeutic molecule into the cell, wherein the nucleic acid sequence is operably linked to a promoter responsive to amyloid-β-related pathology, and wherein the therapeutic protein alters at least one aspect of an amyloid-β-related pathology phenotype or an amyloid-β-related pathology.

[0036] Any feature or feature combination described herein is included within the scope of the present invention, provided that the features included in any such combination are not mutually contradictory, which is obvious from the context, this specification and the knowledge of a person of ordinary skill in the art. Other advantages and aspects of the present invention will be apparent from the following detailed description and claims.

[0037] BRIEF DESCRIPTION OF THE DRAWINGS

[0038] The features and advantages of the present invention will become apparent upon consideration of the following detailed description taken in conjunction with the accompanying drawings, in which:

[0039] Figure 1 A-1P shows HLA-DRB, CD9 (tetraspanin), CD11c (ITGAX), and LGALS3 expression and the relative location of amyloid plaques in xMG, which are located proximal and distal to fibrillar amyloid plaques. Figure 1 A, 1E, 1I, and 1M show the expression of amyloid β (Aβ). Figure 1 B, 1F, 1J, and 1N show that xMG expresses cytosolic green fluorescent protein (cytoGFp), which is used as a genetic tag for human cells. Figure 1 C, 1G, 1K, and 1O show the expression of HLA-DRB, CD9, CD11c, and LGALS3 proteins, respectively. Figure 1 A, 1E, 1I, and 1M show the location of fibrillar amyloid plaques. Figure 1 D Display Figure 1 A, superposition of 1B and 1C; Figure 1 H Display Figure 1 Superposition of E, 1F and 1G; Figure 1 L Display Figure 1 Superposition of I, 1J and 1K; Figure 1 P Display Figure 1 Superposition of M, 1N and 1O.

[0040] The brains were fixed with 4% paraformaldehyde and then cut into 40-μm coronal sections using a freezing microtome. We then performed immunofluorescence labeling using antibodies against the indicated proteins and Amylo-Glo to identify fibrillar β-amyloid plaques. Human microglia and amyloid pathology were then visualized using confocal microscopy.

[0041] Figure 2 A-2H and Figure 1 E-1H is similar but shows in closer detail the relative location of CD9 (a tetraspanin) expression and amyloid plaques close to or distant from fibrillar amyloid in xMG. Figure 1 A and 1E show that xMG expresses cytosolic green fluorescent protein (cytoGFp), which is used as a genetic tag for human cells. Figure 1 B and 1F show the expression of CD9 protein. Figure 1 C and 1G show the location of fibrillar amyloid plaques. Figure 1 E, 1F, and 1G show Figure 1 Higher power views of A, 1B, and 1C. Figure 1 D shows Figure 1 A, 1B and 1C are superimposed, and Figure 1 H shows Figure 1 Superposition of E, 1F and 1G.

[0042] Figure 3 A and 3B show designs for modifying the human iPSC genome, where neprilysin (NEP) is inserted downstream of the endogenous CD9 locus using a P2A element, such that CD9 expression remains under the control of the endogenous CD9 promoter and NEP expression is also co-regulated and co-expressed under the control of the CD9 promoter. Figure 3 A shows the NEP membrane-anchored construct. Figure 3 B shows the construct of secreted NEP (sNEP). SP = secretory peptide signal sequence. P2A = self-cleaving peptide used to achieve polycistronic expression.

[0043] Figure 4 A-4C shows that microglia according to the present invention digest and phagocytose fluorescently labeled fibrotic human Aβ42 amyloid in vitro. Figure 4 A shows that membrane-anchored neprilysin-expressing microglia digest and phagocytose digested Aβ. Figure 4 B shows that secretory enkephalinase-expressing microglia digest and phagocytose digested Aβ. Figure 4C shows phagocytosis of Aβ by wild-type (WT) microglia (wild-type cells differentiated from a human parental iPSC line that were not edited by CRISPR to contain a neprilysin construct), membrane-anchored neprilysin-expressing microglia (NEP), and secretory neprilysin-expressing microglia (sNEP). An increase in the Y axis over time indicates increased internalization and phagocytosis of Aβ. As observed in the sNEP and NEP data, a decrease in the slope over time indicates increased degradation of Aβ after internalization.

[0044] Figure 5 A-5C shows the expression of enkephalinase in the cortex and hippocampus of wild-type (WT) or AD mice developing amyloid plaques (5×-MITRG). Figure 5 A and 5B depict the levels of human enkephalinase in soluble cortical and hippocampal brain extracts of mice transplanted with PBS (n=13), wild-type microglia (WT; n=9), membrane-bound enkephalinase-expressing microglia (NEP; n=11), and secretory enkephalinase-expressing microglia (sNEP; n=9), respectively, as analyzed by human enkephalinase DuoSet ELISA. Figure 5 C depicts the levels of neprilysin (monoclonal antibody, SN5C) in soluble hippocampal samples from 5×-MITRG (5×) and MITRG-WT (WT) transplanted mice, normalized to the level of GAPDH per lane (n=3). The antibody preferentially detects membrane-bound NEP but not sNEP. This Western blot demonstrated that NEP levels were significantly upregulated only in 5×-MITRG mice transplanted with CD9-NEP microglial progenitor cells. Data are presented as mean ± SEM. Statistical significance was calculated using a two-way ANOVA test, ns=not significant or p≥0.05; *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.

[0045] Figure 6 A-6D shows the reduction of soluble amyloid species in the brain treated with microglia of the present disclosure. Cortical tissues extracted from mice transplanted with PBS (n=13), wild-type microglia (WT; n=9), membrane-bound enkephalinase-expressing microglia (NEP; n=11), and secretory enkephalinase-expressing microglia (sNEP, n=9) were measured by MSD analysis. Figure 6 A, 6C) and hippocampus ( Figure 6The levels of human Aβ-42 and Aβ-40 peptides were measured in soluble extracts of 6B and 6D). Data are presented as mean ± SEM. Statistical significance was calculated using ANOVA and Tukey post hoc test, ns = not significant or p ≥ 0.05; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

[0046] Figure 7 A-7D shows a reduction in insoluble amyloid species in the brain treated with microglia of the present disclosure. Insoluble amyloid species provide a biochemical measure of amyloid plaques, and Aβ-42 is considered to be the more neurotoxic and aggregation-prone species of Aβ. The cortex of mice transplanted with PBS (n=13), wild-type microglia (WT; n=9), membrane-bound microglia expressing enkephalinase (NEP; n=11), and secretory microglia expressing enkephalinase (sNEP; n=9) were analyzed by (MSD) Aβ multiplex analysis. Figure 7 A, 7C) and hippocampus ( Figure 7 Human Aβ-42 and Aβ-40 peptides in insoluble extracts of B and 7D). Data are presented as mean ± SEM. Statistical significance was calculated using ANOVA and Tukey post hoc test, ns = not significant or p ≥ 0.05; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

[0047] Figure 8 A-8B shows the reduction of soluble Aβ oligomers in the brain treated with microglia of the present disclosure. MSD analysis was performed on the cortex of mice transplanted with PBS (n=13), wild-type microglia (WT; n=9), membrane-bound enkephalinase-expressing microglia (NEP; n=11), and secretory enkephalinase-expressing microglia (sNEP; n=9). Figure 8 A) and hippocampus ( Figure 8 B) Human Aβ oligomers collected in soluble extracts of samples. Data are presented as mean ± SEM. Statistical significance was calculated using ANOVA and Tukey post hoc test, ns = not significant or p ≥ 0.05; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

[0048] Fig. 9 A-9H shows that delivery of neprilysin according to the methods of the present disclosure has no effect on synaptic density in wild-type MITRG mice ( Fig. 9A-9D), but reduced synaptic loss in 5×-MITRG (9E-9H) mice. The levels of soluble cortical (9A, 9C, 9E, 9G) and hippocampal (9B, 9D, 9F, 9H) samples of mice transplanted with PBS (n=13), wild-type microglia (WT; n=9), membrane-bound neprilysin-expressing microglia (NEP; n=11), and secretory neprilysin-expressing microglia (sNEP; n=9) were analyzed by ELISA. Data are presented as mean ± SEM. Statistical significance was calculated using analysis of variance and Tukey post hoc test, ns=not significant or p≥0.05; *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.

[0049] Fig.10 A-10B shows that delivery of enkephalinase according to the methods of the present disclosure can reduce astrogliosis in 5×-MITRG mice. Soluble cortical ( ELISA) was analyzed in 5×-MITRG mice transplanted with PBS (n=13), wild-type microglia (WT; n=9), membrane-bound enkephalinase-expressing microglia (NEP; n=11), and secretory enkephalinase-expressing microglia (sNEP; n=9). Fig.10 A) and hippocampus ( Fig.10 B) Glial fibrillary acidic protein (GFAP) levels in samples. Data are presented as mean ± SEM. Statistical significance was calculated using ANOVA and Tukey post hoc test, ns = not significant or p ≥ 0.05; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

[0050] Fig.11 A-11D shows that targeted induction of enkephalinase in microglia in response to Aβ pathology of the present disclosure limits off-target degradation of additional neuropeptide substrates of enkephalinase in vivo. Soluble cortical ( ELISA) was analyzed in mice transplanted with PBS (n=13), wild-type microglia (WT; n=9), membrane-bound enkephalinase-expressing microglia (NEP; n=11), and secreted enkephalinase-expressing microglia (sNEP; n=9). Fig.11 A, 11C) and hippocampus ( Fig.11 B, 11D) Non-targeted enkephalinase substrate bradykinin in samples Fig.11 A-11B) and somatostatin ( Fig.11C-11D) levels. Data are presented as mean ± SEM. Statistical significance was calculated using ANOVA and Tukey post hoc test, ns = not significant or p ≥ 0.05; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

[0051] Fig.12 A and 12B show in schematic form the design of the in vivo experiments described in this disclosure.

[0052] Fig.13 Showing that amyloid pathology induces CD9 expression within human microglia, resulting in highly localized induction of the enkephalinase payload. Nuclei (Ku80) of multiple transplanted human microglia are shown. However, CD9 expression and the resulting enkephalin induction was restricted to human microglia adjacent to Amylo-Glo positive amyloid beta plaques.

[0053] Fig.14 showed that human microglia expressing sNEP in vivo alleviated hippocampal Aβ pathology.

[0054] Fig.15 The number of medium and large AmyloGlo-positive plaques in vivo was significantly reduced. Statistical significance was calculated using ANOVA and Tukey post hoc test, ns = not significant or p ≥ 0.05; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

[0055] Fig.16 A-16D shows the effect of human iPSC-microglia transplantation on Aβ protein in 5×-MITRG mice. The cortex of mice transplanted with PBS (n=13) and wild-type microglia (WT; n=9) was analyzed by MSD detection. Fig.16 A, 16C) and hippocampus ( Fig.16 Human Aβ-42 (B, 16D) collected from soluble extracts of samples Fig.16 A-16B) and Aβ-40 ( Fig.16 C-16D) peptide levels. Data are presented as mean ± SEM. Statistical significance was calculated using an unpaired T test. ns = not significant or p ≥ 0.05; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

[0056] Fig.17A-17D shows the effect of human iPSC-microglia transplantation on Aβ protein in 5×-MITRG mice. The cortex of mice transplanted with PBS (n=13) and wild-type microglia (WT; n=9) was analyzed by MSD detection. Fig.17 A, 17C) and hippocampus ( Fig.17 Human Aβ-42 (B, 17D) collected from insoluble extracts of samples Fig.17 A-17B) and Aβ-40 ( Fig.17 C-17D) peptide levels. Data are presented as mean ± SEM, and statistical significance was calculated using unpaired T-test. ns = not significant or p ≥ 0.05; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

[0057] Fig.18 A-18D shows the effect of targeted delivery of neprilysin on Aβ protein in 5×-MITRG mice. MSD analysis was performed on the cortex of mice transplanted with wild-type microglia (WT; n=9), membrane-bound neprilysin-expressing microglia (NEP; n=11), and secretory neprilysin-expressing microglia (sNEP; n=9). Fig.18 A, 18C) and hippocampus ( Fig.18 Human Aβ-42 (B, 18D) collected from soluble extracts of samples Fig.18 B) and Aβ-40 ( Fig.18 C-18D) peptide levels. Data are presented as mean ± SEM. Statistical significance was calculated using ANOVA and Tukey post hoc test, ns = not significant or p ≥ 0.05; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

[0058] Fig.19 A-19D shows the effect of targeted delivery of neprilysin on Aβ protein in 5×-MITRG mice. MSD analysis was performed on the cortex of mice transplanted with wild-type microglia (WT; n=9), membrane-bound neprilysin-expressing microglia (NEP; n=11), and secretory neprilysin-expressing microglia (sNEP; n=9). Fig.19 A, 19C) and hippocampus ( Fig.19 Human Aβ-42 (B, 19D) collected from soluble extracts of samples Fig.19 A-19B) and Aβ-40 ( Fig.19C-19D) peptide levels. Data are presented as mean ± SEM. Statistical significance was calculated using ANOVA with Tukey post hoc test, ns = not significant or p ≥ 0.05; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

[0059] Fig. 20 A-20D shows a volcano plot of bulk RNA sequencing from four independent xMG lines. 5×-MITRG mouse pups were transplanted with human microglial progenitors generated from four independent induced pluripotent stem cell lines. At 6 months of age, xMG were isolated from the brains of 5×-MITRG mice and xMG subpopulations were isolated by fluorescence-activated cell sorting (FACS). To enrich for plaque-associated disease-associated microglia (DAMs), xMG were FACS-sorted to isolate CD9 / HLA-DRB double-positive microglia. In contrast, isolated steady-state microglia were negative for both CD9 and HLA-DRB expression. RNA was isolated from each sample and then analyzed by bulk RNA sequencing. Bulk sequencing comparisons between the resulting steady-state and DAM xMG subpopulations revealed a large number of genes that were significantly upregulated within DAM microglia. Nearly all DAM genes previously identified by single-cell sequencing were again identified as enriched in the bulk-sequenced DAM subpopulation. However, due to the greater sequencing depth provided by bulk sequencing, many other significantly enriched DAM genes were also identified, including the genes provided in

[0020] . Fig. 20 A-20D provides a volcano plot of these bulk sequencing comparisons of xMGs derived from each of four independent induced pluripotent stem cell lines, and labels the significantly enriched subsets of genes. This analysis provides a more complete dataset of plaque-induced human microglial genes described herein.

[0060] These plots compare flow cytometry (FAC) sorted CD9+ / HLA-DRB+ double positive plaque-associated microglia to double negative steady-state microglia. The significant subset of genes (FDR≤0.05) with log2(fold change) ≥1 (at least 2-fold increase) when comparing DAM to steady-state subpopulations across patient lines is shown, and the remainder are shown below. DETAILED DESCRIPTION

[0061] The present disclosure relates to compositions and methods for treating diseases or conditions associated with beta-amyloid protein-related pathologies, such as, but not limited to, Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis (ALS), and the like. More specifically, the present disclosure describes cells that have been modified to express (and present or secrete) at least one therapeutic molecule that can change at least one beta-amyloid protein-related pathological phenotype. These cells have been modified so that when the modified cells are in contact with or close to beta-amyloid protein-related pathologies, therapeutic molecules are expressed. Therefore, the embodiments of the present disclosure can generally be practiced by producing cells that express therapeutic molecules when cells are in contact with or close to beta-amyloid protein-related pathologies. Therefore, the present disclosure also describes methods for preparing such cells and methods for treating diseases using such cells.

[0062] Before further describing the present disclosure, it should be understood that the present invention is not limited to the specific embodiments described, and therefore may of course vary. For example, the present invention is not limited to embodiments related to Alzheimer's disease. It should also be understood that the terms used herein are only used to describe specific embodiments and are not intended to be limiting, as the scope of the present invention is limited only by the claims.

[0063] It must be noted that as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. For example, a compound refers to one or more compound molecules. Thus, the terms "a," "an," "one or more," and "at least one" can be used interchangeably. Similarly, the terms "including," "comprising," and "having" can be used interchangeably. It should also be noted that the claims can be drafted to exclude any optional elements. Therefore, this statement is intended to serve as antecedent basis for the use of exclusive terminology such as "only," "only," and the like when reciting claim elements or using a "negative" limitation.

[0064] The publications discussed herein are provided only for their disclosure prior to the filing date of the present application. Nothing herein shall be construed as an admission that the present invention is not entitled to antedate such disclosure by virtue of prior invention. In addition, the publication dates provided may differ from the actual publication dates and may require independent confirmation. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and / or materials associated with the cited publications.

[0065] The present disclosure relates to modified cells that express therapeutic molecules for treating diseases associated with beta-amyloid protein-related pathology when the cells are in contact with or in proximity to beta-amyloid protein-related pathology. As used herein, "modified cells" are cells that have been intentionally altered using, for example, recombinant DNA technology, CRISPR technology, etc. The modified cells of the present disclosure can produce a sequence encoding a therapeutic molecule that can change at least one beta-amyloid protein-related pathology phenotype by, for example, introducing at least one nucleotide into the cell, wherein the nucleic acid sequence is inserted into the cell in such a way that transcription of the nucleotide sequence occurs when the cell is in proximity to or in contact with beta-amyloid protein-related pathology.

[0066] Cells used to generate modified cells of the present disclosure can be obtained commercially (i.e., purchased), obtained from cell culture (e.g., previously stored cells), or can be obtained from individuals. As used herein, the terms "individual," "subject," and "patient" are well known in the art and are used interchangeably herein to refer to any person or other animal for which the cells of the present disclosure can be used for treatment. Examples include, but are not limited to, humans and other primates, including non-human primates, such as chimpanzees and other apes and monkeys; farm animals, such as cattle, sheep, pigs, seals, goats, and horses; domestic mammals, such as dogs and cats; and laboratory animals, including rodents, such as mice, rats, and guinea pigs. The terms "individual," "subject," and "patient" do not themselves indicate a specific age, sex, race, etc. Therefore, the present disclosure is intended to cover individuals of any age, whether male or female, and includes, but is not limited to, the elderly, adults, children, infants, young children, and toddlers. Likewise, unless otherwise indicated, the cells and methods of the present disclosure can be from and / or applied to any race, including, for example, Caucasian (white), African American (black), Native American, Native Hawaiian, Hispanic, Latino, Asian, African (e.g., African-American), and European.

[0067] When the cell approaches or contacts Alzheimer's disease-related pathology, any type of cell that can be modified to express therapeutic molecules can be used to produce modified cells of the present disclosure. In some aspects, cells from cell lineages that can differentiate into migratory cells are used to produce modified cells. Examples of cells that can be used to produce modified cells of the present disclosure include, but are not limited to, pluripotent stem cells (PSC), induced pluripotent stem cells (iPSCS), myeloid progenitor cells or precursor cells, erythrocyte myeloid progenitor cells, hematopoietic stem cells and progenitor cells (HSPC), cord blood stem cells, lymphoid progenitor cells, and megakaryocyte-erythrocyte (mk-ery) progenitor cells. In some aspects, the cell used to produce the modified cell of the present disclosure can be a pluripotent stem cell (PSC), including induced PSC (iPSC), progenitor cells, embryonic stem cells, or cells derived therefrom. "Pluripotent stem cells" (PSC) used herein refers to cells with the ability to self-renew by division, and can develop into three primary germ cell layers of early embryos, thereby developing into all cells of the human body. "Induced pluripotent stem cells" or "iPSC" used herein refers to a pluripotent stem cell that can be directly produced from somatic cells. In some aspects, the cells used to generate the modified cells of the present disclosure can be circulating blood progenitor cells (eg, myeloid progenitor cells, CD34+ hematopoietic progenitor cells, or monocytes).

[0068] In some aspects, the cells used to produce the modified cells of the present disclosure are microglia-like cells (MGL), including induced MGL (iMGL) cells, such as human iMGL (hiMGL) or microglia progenitor cells. "Microglia progenitor cells" used herein refer to cells that can differentiate into microglia or microglia-like cells. Microglia progenitor cells can include hematopoietic progenitor cells, erythrocyte progenitor cells, primitive macrophages, etc. Microglia progenitor cells can also be derived from pluripotent stem cells (PSC), including induced pluripotent stem cells (iPSC) and embryonic stem cells (ESC). iMGL is further described in US20200239844 and Abud et al. Neuron, 2017: PMID: 28426964, and its disclosure is incorporated herein by reference in its entirety. In some embodiments, iMGL expresses microglia marker proteins, such as P2RY12 and TREM2. In some embodiments, the expression of AXL, P2RY6, CCR6 or GPR84 of iMGL is higher than that of adult microglia. In some embodiments, the expression of CTSL, CTSD or NPL of iMGL is lower than that of adult microglia. In some embodiments, iMGL expresses mRNA sequences indicating the gene ontology of mitotic cell cycle process. In some embodiments, iMGL reduces the expression of FFAR2 and COL26A1 when cultured with rat hippocampal neurons. In some embodiments, iMGL increases the expression of Siglec11 and Siglec12 when cultured with rat hippocampal neurons. In some embodiments, iMGL differentiates in vitro. In some embodiments, iMGL expresses P2RY13 and OLFML3. In some embodiments, iMGL is able to phagocytose human synaptosomes. In some embodiments, iMGL has a lower phagocytic activity on E. coli particles than macrophages. iMGL is able to phagocytose amyloid Aβ fibers or tau oligomers. In some embodiments, IFNγ induces iMGL to secrete TNFα, IL-8, CCL2 and CCL4. In some embodiments, IL-1β induces iMGL to secrete TNFα, IL-8, CCL3, CCL4 and CXCL10. In some embodiments, ADP induces calcium to flow into iMGL transiently. In some embodiments, iMGL migrates in response to ADP. In some embodiments, iMGL migrates into human brain organoids (BORG). In some embodiments, iMGL extends a branching process connected to BORG. In some embodiments, iMGL gathers near the site of injury in BORG. In some embodiments, iMGL is produced by contacting human induced hematopoietic progenitor cells (iHPCs) with a microglia differentiation medium containing CSF-1, IL-34 and TGFβl or CSF-1, IL-34 and TGFβ mimetics.In some embodiments, iMGLs are produced by seeding human induced hematopoietic progenitor cells (iHPCs) on a basement membrane protein-coated dish; and contacting the human iHPCs with a microglia differentiation medium comprising CSF-1; IL-34; CSF-1, IL-34, and TGFβ1; or CSF-1, IL-34, and a TGFβ mimetic.

[0069] As used herein, "microglia-like cells" or "iMGLs" refer to microglia-like cells similar to fetal and adult microglia, which can be derived from pluripotent stem cells (PSCs), including iPSCs and embryonic stem cells (ESCs). As used herein, "microglia" refers to innate immune cells resident in the central nervous system that play a role in synaptic plasticity, neurogenesis, homeostatic function, and immune activity.

[0070] Progenitor cells can be generated from PSCs (including iPSCs) using methods known in the art. Such progenitor cells may include, but are not limited to, hematopoietic progenitor cells, erythroid progenitor cells, or primitive macrophages.

[0071] As used herein, "iPSC-derived microglial lineage cells" refer to human microglia-like cells (iMGLs) or microglial progenitor cells that can be derived from iPSCs.

[0072] In various aspects, iMGL or microglia progenitor cells of the present disclosure can be produced from PSC using methods known in the art. In some aspects, iMGL or microglia progenitor cells of the present disclosure can be produced from iPSC or ESC using methods known in the art. In some aspects, microglia progenitor cells may include hematopoietic progenitor cells, erythrocyte progenitor cells or primitive macrophages. In some aspects, iMGL may be derived from microglia progenitor cells.

[0073] In some aspects, iMGLs of the present disclosure can be generated by the following steps: (i) differentiating PSCs using a medium supplemented with hematopoietic differentiation factors to produce induced hematopoietic progenitor cells (iHPCs); (ii) isolating CD43+iHPCs; (iii) differentiating CD43+iHPCs into human iMGLs using a microglia differentiation medium; and (iv) maturing iMGLs. In some aspects, HPC generation techniques allow for the collection of precursor-rich culture medium and for (iii) without the need to isolate CD43+iHPCs.

[0074] In some aspects, human microglia-like cells (hiMGLs) of the present disclosure can be generated by: (i) differentiating PSCs using a medium supplemented with hematopoietic differentiation factors; and (ii) differentiating CD43+ iHPCs into iMGLs using a microglia differentiation medium.

[0075] In some aspects, human microglia-like cells (hiMGL) of the present disclosure can be generated from a first type of cell, comprising the steps of: (i) differentiating the first type of cell into iHPC; and (ii) differentiating the iHPC to produce iMGL. In some aspects, the first type of cell is not a PSC or an ESC.

[0076] In some aspects, human microglia-like cells (hiMGLs) of the present disclosure can be produced by a method comprising the step of differentiating iHPCs to produce iMGLs.

[0077] In some aspects, human microglia-like cells (hiMGLs) of the present disclosure can be produced by a method comprising the steps of differentiating engineered iHPCs to produce engineered hiMGLs, wherein the engineered iHPCs and engineered hiMGLs express therapeutic molecules (e.g., membrane-bound enkephalinase, soluble enkephalinase, etc.).

[0078] The iMGL produced by any method described herein can express any factor or combination of any factor expressed by typical typical microglia. In some aspects, the iMGL produced is c-kit- / CD45+. In some aspects, c-kit- / CD45+iMGL is detected using flow cytometry, immunofluorescence microscopy, qPCR, RNA-seq or proteomics. In some aspects, other cell types are detected using flow cytometry, immunofluorescence microscopy, qPCR, RNA-seq or proteomics. In some aspects, the iMGL produced comprises two independent iMGL colonies: (1) CD45+ / CX3CR1- and (2) CD45+ / CX3CR1+. In some aspects, the iMGL produced is CD43+, CD235a+ or CD41+. In some aspects, the iMGL produced is CD43+ / CD235a+ / CD41+.

[0079] In some aspects, TRIM14, CABLES1, MMP2, SIGLEC 11 and 12, MITF and / or SLC2A5 mRNA and / or protein expression can be enriched in the iMGL produced. In certain aspects, COMT, EGR2, EGR3 and / or FFAR2 mRNA and / or protein expression is enriched in the iMGL produced.

[0080] In some aspects, iMGLs expressing specific gene profiles can be provided. Any iMGL described herein may include a gene expression profile similar to that of typical microglia. In some aspects, any iMGL composition described herein includes expression of any of the following genes: RUNX1, PU.1, CSF1R, CX3CR1, TGFBR1, RSG10, GAS6, PROS1, P2RY12, GPR34, C1Q, CR3, CABLES1, BHLHE41, TREM2, ITAM, APOE, SLCO2B1, SLC7A8, PPARD, C9orf72, GRN, LRRK2, TARDBP, and CRYBB1. Any iMGL disclosed herein may comprise expression of any of these genes in any combination: RUNX1, SPI1, CSF1R, CX3CR1, TGFBR1, RSG10, GAS6, MERTK, PSEN2, PROS1, P2RY12, P2RY13, OLFML3, GPR34, C1Q, CR3, CABLES1, BHLHE41, TREM2, TYROBP, ITGAM, APOE, SLCO2B1, SLC7A8, PPARD, TMEM119, GPR56, C9orf72, GRN, LRRK2, TARDBP, and CRYBB1.

[0081] In some aspects, in any iMGL composition described herein, TREM2 and P2RY12 can be co-expressed. In some aspects, any iMGL composition described herein may not express any one or more of the genes KLF2, TREM1, MPT, ITGAL, and ADGRE5.

[0082] The cells used to produce the modified cells of the present disclosure can be autologous cells, allogeneic cells, or xenogeneic cells. The term "autologous" as used herein refers to cells or tissues from a subject, wherein the subject can be both a donor subject and a recipient subject. "Allogeneic" as used herein refers to cells of the same species that are genetically different from the cells being compared. The term "xenogeneic" as used herein refers to cells from a different species than the recipient. In certain embodiments, the modified cells can be autologous. In certain embodiments, the modified cells can be allogeneic. In certain embodiments, the modified cells can be xenogeneic.

[0083] In certain embodiments, the method comprises administering (e.g., transplanting) a modified cell of the present disclosure or a composition of the present disclosure (e.g., hiMGL) to brain tissue of an individual. The methods disclosed herein may involve obtaining and modifying cells from a subject, and then administering (e.g., transplanting) the modified cells (e.g., hiMGL) to the same subject. In other embodiments, the method may involve obtaining and modifying cells from a donor subject and administering (e.g., transplanting) the modified cells (e.g., hiMGL) into a recipient subject. In addition, in some embodiments, the method may include obtaining and modifying cells from a donor species and administering (e.g., transplanting) the modified cells (e.g., hiMGL) into a recipient species.

[0084] As used herein, "nucleic acid molecule", "nucleic acid", "nucleic acid sequence" and the like refer to polynucleotides, such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), oligonucleotides, fragments produced by polymerase chain reaction (PCR), and fragments produced by any of ligation, cleavage, endonuclease action and exonuclease action. Nucleic acid molecules or sequences can be composed of naturally occurring nucleotide monomers (such as DNA and RNA) or naturally occurring nucleotide analogs (for example, α-enantiomeric forms of naturally occurring nucleotides) or a combination of the two. Nucleic acid molecules or sequences can be single-stranded or double-stranded.

[0085] Nucleic acid transfer methods known in the art can be used to introduce nucleic acid molecules or sequences into cells to produce modified cells. In some respects, transfection can be used to introduce nucleic acid molecules or sequences into cells, including viral vector-mediated transfection, combination, electroporation, liposome-mediated gene transfer, transduction and direct transfer methods, such as microinjection or particle bombardment. Nucleic acid molecules or sequences can be double-stranded or single-stranded linear nucleic acid molecules or sequences or circular nucleic acid molecules or sequences. In some respects, nucleic acid molecules or sequences can be located in a carrier, such as a plasmid or a viral vector.

[0086] In some respects, nucleic acid or sequence include nucleic acid vectors.Terms "nucleic acid vector", "vector" etc. are used herein to refer to nucleic acid molecules or sequences that can transfer or carry another nucleic acid molecule or sequence.Nucleic acid to be transferred can usually be connected to a carrier nucleic acid molecule or sequence, such as inserted into a carrier nucleic acid molecule or sequence.The vector may include a sequence that instructs autonomous replication in the cell, or may include enough sequences to allow integration into host cell DNA.Useful vectors may include, for example, plasmids (e.g., DNA or RNA plasmids), transposons, cosmids, bacterial artificial chromosomes and viral vectors.Useful viral vectors may include, for example, replication-defective retroviruses and slow viruses.In some respects, the nucleic acid vector comprising a nucleic acid molecule or sequence may be retained in the cytoplasm of a modified cell.

[0087] In some aspects, the nucleic acid sequence can be inserted into the genome of the modified cell. In some aspects, the nucleic acid sequence can be inserted into the genome of the cell to form a "synthetic allele" of the cell gene. The term "synthetic allele" refers to an allele of a gene in the genetic material of a cell, which is modified relative to one or two alleles of the same gene in the genetic material of a reference cell from the same subject. In a non-limiting example, the reference cell is a diploid germline cell from the same subject; in another non-limiting example, according to the present disclosure, the reference cell is a cell collected from a subject before the therapeutic intervention. Synthetic alleles can be created by gene editing techniques known in the art, and can be genetically (e.g., on its nucleic acid sequence) and / or epigenetically (e.g., on its DNA methylation state, histone acetylation state, chromatin structure, or in other aspects that do not substantially change the coding sequence) with non-synthetic alleles of the same gene in the reference cell from the subject. Synthetic alleles can be modified in their coding and / or non-coding sequences. In some aspects, the nucleic acid sequence can be inserted into the genome of the modified cell so that it is under the control of an endogenous promoter in the genome of the modified cell. In some aspects, the nucleic acid sequence can be inserted into or modified in the cell genome, inside or downstream (i.e., after the 3' end) of a certain locus, so that the nucleic acid sequence in the inserted nucleic acid sequence is controlled by the locus promoter. "Locus" used herein refers to the position of a specific gene in the genome, which may include one or more exons and / or introns. Therefore, the terms "locus" and "gene" can be used interchangeably herein. In some aspects, the nucleic acid sequence is inserted into a gene or replaces all or part of a gene. In some aspects, the nucleic acid sequence includes a nucleic acid sequence encoding a therapeutic protein, and the nucleic acid sequence is inserted into the locus so that the nucleic acid sequence meets the reading frame with the coding sequence of the exon in the locus. In some aspects, the nucleic acid sequence is inserted into the locus so that the nucleic acid sequence meets the reading frame with any exon producing the desired result in the locus. For example, in some aspects, the exon may be the first exon in the locus. In some aspects, the exon may be the last exon in the locus. As used herein, "in frame with the coding sequence of the exon", "in frame with the exon", etc. means that when the exon sequence is connected to the nucleic acid sequence encoding the protein (e.g., therapeutic protein), the connected sequence forms a single open reading frame (ORF). In some aspects, a first polynucleotide encoding a protease cleavage sequence or a self-cleavage peptide (e.g., a 2A peptide) can be inserted between the exon sequence and the nucleic acid sequence encoding the therapeutic protein. The resulting encoded protein will contain a protease cleavage sequence or a self-cleavage peptide between the amino acid sequence encoded by the exon sequence and the therapeutic protein, thereby enabling the therapeutic protein to be separated from the amino acid sequence encoded by the exon sequence or produced independently.In some aspects, the second polynucleotide encoding a secretory peptide signal sequence (SP) can be connected to the nucleic acid sequence encoding the therapeutic protein so that the sequence encoding the signal peptide is in frame with the nucleic acid sequence encoding the therapeutic protein. In some aspects, the nucleotide sequence of one or more domains (e.g., cytoplasmic domain and / or transmembrane domain) encoding the therapeutic protein can be deleted so that the therapeutic protein lacks such domains.

[0088] Suitable gene editing techniques for inserting nucleic acid sequences into the genome of modified cells may include any gene editing system known in the art. In some aspects, suitable gene editing techniques may include conventional genome editing systems, such as conventional homologous recombination, ssODNs homologous recombination; chemical systems, such as peptide NA systems; protein-based nuclease systems, such as giant nuclease systems, zinc finger nuclease systems, and TALEN systems; homing endonuclease (HE) systems, such as adeno-associated virus (AAA) systems; and RNA-protein-based systems, such as CRISPR systems, PRIME editing, etc.

[0089] As used herein, "therapeutic molecules" refer to molecules that reduce and / or eliminate and / or improve and / or treat and / or prevent at least one amyloid-β-related pathological phenotype when applied to an individual or expressed in an individual. In some aspects, therapeutic molecules can treat the main cause of the disease. For example, therapeutic molecules can reduce the number or amount of soluble or insoluble amyloid-β (Aβ) peptide oligomers, pyroglutamate Aβ, fibrils, fibers, and / or total plaque load. In some embodiments, therapeutic molecules can enhance amyloid hydrolysis. In some embodiments, therapeutic molecules can enhance the phagocytosis of amyloid-β by microglia. The present invention is not limited to the mechanism by which therapeutic molecules affect (e.g., reduce, eliminate, improve, treat, prevent, etc.) amyloid-β-related pathological phenotypes. When producing modified cells disclosed herein, any type of therapeutic molecule can be used. The therapeutic molecule can be a therapeutic RNA molecule, or it can be a therapeutic protein, which can be a membrane-bound protein or a secreted protein. Therapeutic proteins that can be used to produce modified cells of the present disclosure include, but are not limited to, enzymes, peptides or protein binding domains (e.g., antibodies or fragments thereof), nucleic acid binding proteins, chimeric proteins (e.g., chimeric antigen receptors), anti-inflammatory proteins, thrombolytic proteins, immunomodulatory molecules, proteases, and metalloendopepeptidases, one example of which is enkephalinase. In some aspects, the therapeutic protein is selected from the group consisting of enzymes, peptides or protein binding domains, nucleic acid binding proteins, chimeric proteins (e.g., chimeric antigen receptors), anti-inflammatory proteins, thrombolytic proteins, proteases, and metalloendopepeptidases. In some aspects, the therapeutic protein is a metalloendopepidases. In some aspects, the therapeutic protein is an enkephalinase, which can be a membrane-bound enkephalinase or a secreted form of enkephalinase.

[0090] The therapeutic molecule can be therapeutic RNA. The therapeutic RNA that can be used to produce the modified cell of the present disclosure includes but is not limited to mRNA translation inhibitors (for example, antisense molecules), molecules (for example, RNAi) of interfering RNA, catalytic activity RNA molecules (for example, ribozymes) and RNA (for example, aptamers) of binding proteins and other ligands. The method of producing such molecules is known to those skilled in the art, and is also disclosed in U.S. Patent Publication No. 2014 / 0303073, U.S. Patent Publication No. 2012 / 0232128, U.S. Patent Publication No. 2011 / 0118334, U.S. Patent Publication No. 2011 / 0033859, U.S. Patent Publication No. 2006 / 0089323, U.S. Patent Publication No. 2012 / 0263782, U.S. Patent Publication No. 2012 / 0301449 and U.S. Patent Publication No. 2004 / 0137429, and the full disclosures of these patents are incorporated herein by reference.

[0091] In some aspects, the therapeutic molecules disclosed herein can affect (e.g., reduce) at least one amyloid-β-related pathological phenotype. The term "Alzheimer's disease-related phenotype" as used herein refers to any observable features or characteristics of Alzheimer's disease, such as behavior, which may include memory problems, cognitive problems, vision problems, behavioral changes, personality changes, depression and epilepsy. In some aspects, therapeutic molecules can change (e.g., reduce, eliminate, prevent, improve, etc.) at least one aspect of Alzheimer's disease-related pathology. "Pathology" as used herein refers to anatomical and / or physiological changes caused by the disease. In Alzheimer's disease, it is believed that amyloid-β (Aβ) plays a vital role in the development of the disease. For example, fragments of amyloid-β (Aβ) peptides can aggregate to form plaques, which can be observed in the brains of Alzheimer's patients. The presence of such Aβ plaques may be considered as a pathology associated with Alzheimer's disease. Similarly, it has been observed that an Aβ called Aβ oligomer (AβO) is significantly increased in the early stages of Alzheimer's disease and is located in or within synapses. The presence of such AβOs may also be considered a pathology associated with Alzheimer's disease. In addition, another hallmark of Alzheimer's disease patients is the presence of neurofibrillary tangles, which are composed of tau protein threads. The presence of such neurofibrillary tangles may also be considered a pathology associated with Alzheimer's disease. Thus, in some aspects, at least one aspect of Alzheimer's disease-associated pathology may include the formation or presence of Aβ plaques, AβOs, and / or neurofibrillary tangles.

[0092] As used herein, the term "β-amyloid-related pathology phenotype" refers to any observable features or characteristics of a disease or condition associated with β-amyloid-related pathology, such as behavior, which may include memory problems, cognitive problems, vision problems, behavioral changes, personality changes, depression, and epilepsy. In some aspects, a therapeutic molecule can alter (e.g., reduce, eliminate, prevent, improve, etc.) at least one aspect of β-amyloid-related pathology.

[0093] In some aspects, the therapeutic molecule is a therapeutic protein. Such a protein may have enzymatic and / or anti-inflammatory activity. In some aspects, the therapeutic protein may have protease activity. In some aspects, the therapeutic protein may be a metalloproteinase, an example of which is enkephalinase. In some aspects, the therapeutic protein may be an enkephalinase. In some aspects, the therapeutic protein comprises or consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97% or at least 99% identical to SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3, wherein the therapeutic protein has enkephalinase activity. In some aspects, the therapeutic protein comprises or consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97% or at least 99% identical to SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3, wherein the therapeutic protein has enkephalinase activity and wherein the difference in sequence is due to conservative amino acid substitutions. In some aspects, the therapeutic protein comprises or consists of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3. In some aspects, the therapeutic protein comprises SEQ ID NO: 1. In some aspects, the therapeutic protein comprises SEQ ID NO: 2. In some aspects, the therapeutic protein comprises SEQ ID NO: 3. In some aspects, the therapeutic protein comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97% or at least 99% identical to SEQ ID NO: 1. In some aspects, the therapeutic protein comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identical to SEQ ID NO: 2. In some aspects, the therapeutic protein comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identical to SEQ ID NO: 3.

[0094] Table 1: Non-limiting examples of therapeutic proteins.

[0095]

[0096]

[0097]

[0098] * indicates a stop codon; "secretion signal" and "signal peptide" as used herein are used interchangeably.

[0099] In the modified cells of the present disclosure, the nucleic acid sequence encoding the therapeutic molecule is operably connected to the pathological response promoter. For example, the expression of the therapeutic molecule encoded by the operably connected promoter may be induced by the presence of amyloid-β-related pathology. "Induced by the presence of amyloid-β-related pathology", "amyloid-β-related pathological response", "pathological response" and the like used herein refer to the expression of the therapeutic molecule when the modified cell approaches or contacts the amyloid-β-related pathology. Without being bound by theory, it is believed that the activation of the promoter operably connected to the therapeutic molecule encoding nucleic acid sequence occurs in response to endogenous and / or exogenous signals generated by cell contact with pathology or contact with molecules generated by the presence of pathological range. An example of such a signal is a signal that induces an "activated" or "disease-related microglia" phenotype. In some aspects, the expression of the encoded therapeutic molecule depends on the modified cell approaching or contacting the amyloid-β-related pathology. In some aspects, the expression of the encoded therapeutic molecule depends on the contact of the modified cell with the amyloid-β-related pathology. In some aspects, expression of the encoded therapeutic molecule is dependent upon the modified cell being in proximity or contact with β-amyloid (Aβ) peptide plaques, soluble Aβ monomers, insoluble Aβ monomers, Aβ oligomers, pyroglutamate Aβ, fibrils, or fibers composed of Aβ of varying lengths.

[0100] In some aspects, the nucleic acid sequence encoding the therapeutic molecule can be operably connected to the pathological response promoter. As used herein, the term "operably connected" refers to two or more nucleic acid sequences or partial sequences being positioned so that they functionally interact to achieve their intended function. For example, if the promoter can control or regulate the transcription of the nucleic acid sequence in the cis position in the nucleic acid sequence, the promoter can be functionally connected to the nucleic acid (e.g., coding) molecule. Generally speaking, but not necessarily, the functionally connected nucleic acid sequences are close to each other. Although the functionally connected promoter is usually located upstream of the coding sequence, it is not necessarily close to the coding sequence. For example, the nucleic acid sequence encoding the therapeutic molecule can be inserted into a gene comprising multiple exons so that the transcription of the nucleic acid sequence is under the control of the gene promoter. If the nucleic acid sequence encoded by the therapeutic molecule is connected to the last exon of the gene in a manner that meets the reading frame, the nucleic acid sequence will be controlled by the gene promoter, but may be far away from the gene promoter. Enhancers do not need to be close, as long as they can assist the transcription of the nucleic acid sequence. For this purpose, they can be located upstream and / or downstream of the nucleic acid sequence, and may be a distance away from the nucleic acid sequence. A polyadenylation site is functionally linked to a polynucleotide sequence if it is located at the 3' end of the polynucleotide sequence and transcription proceeds through the coding sequence to the polyadenylation signal. Thus, two or more functionally linked nucleic acid sequences may or may not be in direct contact (i.e., immediately adjacent to each other in the viral vector genome).

[0101] As used herein, "β-amyloid protein-related pathological response promoter", "pathological response promoter", etc. refer to a promoter in a cell, and when the cell approaches or contacts β-amyloid protein-related pathology, the promoter activates the transcription of a nucleotide sequence operably connected to the promoter. Therefore, when the cell approaches or contacts β-amyloid protein-related pathology, a nucleic acid sequence encoding a disease treatment molecule and operably connected to the pathological response promoter in the cell is transcribed. Any promoter can be regarded as a pathological response promoter, as long as the transcription of the promoter is activated when the cell containing the promoter approaches or contacts β-amyloid protein-related pathology. In some aspects, the pathological response promoter is exogenous to the modified cell. In some aspects, the pathological response promoter is endogenous to the modified cell. In some aspects, the therapeutic molecule encoding nucleic acid sequence is inserted into the genome of the modified cell so that the transcription of the therapeutic molecule encoding sequence is controlled by the pathological response promoter in the genome.Examples of pathological response promoters that can be used to generate modified cells of the present disclosure include, but are not limited to, the dendritic cell expressed seven transmembrane protein (DCSTAMP) gene (Gene ID: 81501; MIM: 605933) promoter, CD9 (tetraspanin) gene (Gene ID: 928; MIM: 143030) promoter, CD44 gene (Gene ID: 960; MIM: 107269) promoter, galectin 3 (LGALS3) gene (Gene ID: 3958; MIM: 153619) promoter, secretory phosphoprotein 1 (SPP1) promoter gene (Gene ID: 81502; MIM: 607106) promoter, glycoprotein Nmb (GPNMB) gene (Gene ID: 10457; MIM: 604368) promoter, major histocompatibility complex, class II, DR Beta (HLA-DRB1) gene (Gene ID: 3123; MIM: 142857) promoter, lipoprotein lipase (LPL) gene (Gene ID: 4023; MIM: 609708) promoter, lipase A (LIPA) gene (Gene ID: 3988; MIM: 613497) promoter, fatty acid binding protein 3 (FABP3) gene (Gene ID: 2170; MIM: 134651) promoter, transmembrane 4 domain A6A (MS4A6A) gene (Gene ID: 64231; MIM: 606548) promoter, CXC motif chemokine receptor 4 (CXCR4) gene (Gene ID: 7852; MIM: 162643) promoter, chitinase-3-like protein 1 (CHI3L1) gene (Gene ID: 1116; MIM: 134651) promoter. MIM:601525) promoter, oxidized low-density lipoprotein receptor 1 (OLR1) gene (Gene ID: 4973; MIM: 602601) promoter, CD36 gene (Gene ID: 948; MIM: 173510) promoter, signaling lymphocyte activation molecule (SLAM) F8 (SLAMF8) gene (Gene ID: 56833; MIM: 606620) promoter, triggering receptor myeloid cell expressed 2 (TREM2) gene (Gene ID: 54209; MIM: 605086) promoter, macrophage scavenger receptor 1 (MSR1) gene (Gene ID: 4481; MIM: 153622) promoter, beta-2 microglobulin (B2M) gene (Gene ID: 567; MIM: 109700) promoter, integrin subunit Alpha X (ITGAX) gene (Gene ID: 3687; MIM: 151510) promoter and melanocyte inducing transcription factor (MITF) gene (Gene ID: 4286; MIM: 156845) promoter.

[0102] Other non-limiting examples of pathological response promoters that can be used to generate modified cells of the present disclosure include, but are not limited to, HLA-DRB3, HLA-DRB5, HLA-DRA, CHIT1, DKK2, HLA-DQB1, PLXNA1, CCR7, HLA-DPA1, RGCC, COX6A2, HLA-DRB1, HLA-DPB1, TAFA3, FAIM2, ZNF804A, LGALS1, DOK2, HLA-DOA, GPR153, SLPI, ADGRF5, MYOZ1, HBEGF, BHLHE40, TMEM37, SLC16A8, GRM5, S100A4, GABRB1, LONR F3, PIGR, RAB7B, BIRC7, FABP5, CA2, FCMR, GABRB3, ATF7-NPFF, ANXA2, EGR2, OR9G1, IFNLR1, APOC1, CD200R1, PKD2L1, TDRD6, HLA-DMA, KCNJ5, GLDN, PRE X2, OLFM2, PADI2, COL1A2, RRAGD, ROR2, PTCRA, EDN1, RAMP1, BCL2A1, CD83, CHST2, MAFB, DUSP2, FPR3, DPYD, PHLDA1, PTPRG, IER5L, CPNE8, MPDZ, RNF152 , PTGER4, MCF2, SDS, CIITA, RGS16, ANGPTL6, PLA2G7, CCL3, ATF3, RAB42, TBX18, STARD13, NR4A3, S100A13, TRDN, GADD45G, ADRA1B, LYPD1, FXYD6, EPHB6 , FERMT2, ART4, TNFSF18, TNFSF15, SLC38A4, CDH6, SCIN, FGR, ZNF385B, FUT9, SORCS1, KLF10, CFD, TRPM1, FLVCR2, NFIL3, MYO1E, ITPRID1, SLC47A1, LIT AF, RASGEF1B, DENND2D, ADCY3, SYTL3, ARL4C, IL4I1, CDCP1, TRERF1, VAT1, INPP1, ARRDC4, ENPP1, GSDME, APOC2, COL4A1, CDKN1A, KCNC4, KCNJ2, USP2, U NC13B, VEGFB, TNFAIP2, HLA-DMB, NAB2, ACP5, CYTL1, FXYD5, ALDH1A2, TMEM176B, TAGLN2, TIMD4, MEP1A, ADGRL2, FAM20C, LRRC39, CD74, RASGRP3, HEG1,TNFRSF14, ADGRE2, FAM110B, GADD45B, TSKU, ID2, DUSP10, TNFSF13B, FKBP5, MYC, BCL6, TNFAIP8L3, ARID5B, APOC4 - APOC2, PMAIP1, P2RY8, CCRL2, Ribonuclease 1, LSP1, ARHGEF3, CLEC19A, KCNQ5, DTNA, PILRA, WIPF3, CSTB, XYLT1, DBI, SDC2, KCNMA1, ALCAM, TSPAN4, RPS28, KLHDC8B, MBOAT1, FAM20A, CDH8, NAP1L1, SMIM4, ALAS1, PDLIM7, IQGAP2, INPP4B, TRIM58, SLC36A1, TXN, HLA - B, HSPB1, AP1S2, PLAU, HLA - DQA1, CYSTM1, IER5, MS4A7, SPN, CTSD, SELENOP, SDSL, CD14, HLA - F, B3GALNT1, GAPT, RASSF3, GAS7, GAS2L3, GPD2, CPM, SGPP1, LILRA4, NEB, PRXL2C, SNX24, PPARG, CAPG, CD6, INHBA, PTPN7, LYRM9, ATOX1, UGCG, TMSB10, IFI30, M1AP, SH3BGRL3, CMYA5, PRKCH, NIBAN1, GK, HSD3B7, GPR65, LGALS9, RPL36, SH3RF1, WWC3, ALDH5A1, SNTB2, HACD4, LTA4H, SCIMP, SLC11A1, PTPDC1, CPNE2, CALHM2, PRDX1, APOE, IQGAP1, OSBPL3, GRK3, DPEP2, TMEM154, FOXP1, GYPC, SLC15A3, NENF, HPSE, TNFRSF11A, PPM1M, HPCAL1, SLC46A1, SLC1A3, MORC1, GABARAPL1, MGLL, TGIF1, ZYX, MAP4 or MFSD2A (see, Fig. 20 A to Fig. 20 D).

[0103] In some aspects, the pathological response promoter includes a CD9 gene promoter, or a functional variant thereof. As used herein, a "functional variant" refers to a promoter having a nucleotide sequence at least 95% identical to a native promoter, and having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 97% or at least 99% of the activity of the native promoter. The present invention is not limited to the aforementioned pathological response promoter.

[0104] To date, modified cells encoding therapeutic molecules whose expression is controlled by pathological response promoters have been generally described. A detailed embodiment of such a cell will now be described. It should be understood that this description is not intended to limit the present disclosure to a specific embodiment, but is merely to help illustrate one possible embodiment using specific elements of the present disclosure.

[0105] One aspect of the present disclosure is modified microglia-like (iMGL) cells, which include nucleic acid sequences encoding therapeutic molecules, and the therapeutic molecules can be therapeutic proteins, wherein the nucleic acid sequences are operably linked to pathological response promoters, and wherein the therapeutic proteins can reduce at least one amyloid-β-related pathological phenotype. In some aspects, the pathological response promoter includes a promoter selected from the following genes: DCSTAMP gene, CD9 gene, CD44 gene, LGALS3 gene, SPP1 gene, GPNMB gene, HLA-DRB gene, LPL gene, LIPA, FABP3 gene, MS4A6A gene, CXCR4 gene, CHI3L1 gene, OLR1 gene, CD36 gene, SLAMF8 gene, TREM2 gene, MSR1 gene, B2M gene, ITGAX gene and MITF gene. In some aspects, the nucleic acid sequence can be inserted into a gene selected from the following genes: DCSTAMP gene, CD9 gene, CD44 gene, LGALS3 gene, SPP1 gene, GPNMB gene, HLA-DRB gene, LPL gene, LIPA, FABP3 gene, MS4A6A gene, CXCR4 gene, CHI3L1 gene, OLR1 gene, CD36 gene, SLAMF8 gene, TREM2 gene, MSR1 gene, B2M gene, ITGAX gene and MITF gene. In some aspects, the nucleic acid sequence can be inserted into the genome of the iMGL cell so that the nucleic acid sequence is operably linked to an endogenous promoter of a gene selected from the following group: DCSTAMP gene, CD9 gene, CD44 gene, LGALS3 gene, SPP1 gene, GPNMB gene, HLA-DRB gene, LPL gene, LIPA, FABP3 gene, MS4A6A gene, CXCR4 gene, CHI3L1 gene, OLR1 gene, CD36 gene, SLA F8 gene, TREM2 gene, MSR1 gene, B2M gene, ITGAX gene and MITF gene. In some aspects, the nucleotide sequence can be inserted into a gene selected from the following genes: DCSTAMP gene, CD9 gene, CD44 gene, LGALS3 gene, SPP1 gene, GPNMB gene, HLA-DRB gene, LPL gene, LIPA, FABP3 gene, MS4A6A gene, CXCR4 gene, CHI3L1 gene, OLR1 gene, CD36 gene, SLAMF8 gene, TREM2 gene, MSR1 gene, B2M gene, ITGAX gene and MITF gene, so that the nucleotide sequence encoding therapeutic protein is connected to the exon of gene and conforms to the reading frame. In some aspects, the nucleotide sequence encoding therapeutic protein is connected to the first exon of gene and conforms to the reading frame. In some aspects, the nucleotide sequence encoding therapeutic protein is connected to the last exon of gene and conforms to the reading frame.In some aspects, the first polynucleotide encoding a protease cleavage sequence or a self-cleaving peptide can be inserted between an exon and a nucleic acid sequence encoding a therapeutic protein, so that the first polynucleotide is in frame with the exon and the nucleic acid sequence encoding the therapeutic protein. In some aspects, a second polynucleotide encoding a secretory peptide signal sequence (SP) can be connected to a nucleic acid sequence encoding a therapeutic protein, wherein the second polynucleotide can be inserted between the first polynucleotide and the nucleic acid sequence encoding the therapeutic protein, so that the encoded signal peptide is in frame with the exon and the nucleic acid sequence encoding the therapeutic protein. In some aspects, the therapeutic protein can be a metalloproteinase. In some aspects, the therapeutic protein can be a neprilysin, which can be a secreted form of neprilysin or a membrane-bound form of neprilysin. In some aspects, the therapeutic protein can include or consist of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97% or at least 99% identical to SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3, wherein the encoded therapeutic protein has neprilysin activity. In some aspects, the encoded therapeutic protein may comprise or consist of an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identical to SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3, wherein the encoded therapeutic protein has enkephalinase activity, and wherein the difference between the amino acid sequence of the therapeutic protein and SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3 is due to conservative amino acid substitutions. In some aspects, the encoded protein may comprise or consist of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.

[0106] One aspect of the present disclosure is a modified microglia-like (iMGL) cell, comprising a nucleic acid sequence comprising a nucleic acid sequence encoding a therapeutic protein, wherein the nucleic acid sequence is operably linked to a pathological response promoter, and wherein the therapeutic protein can reduce at least one amyloid-β-related pathology. In some aspects, the pathological response promoter is a CD9 gene promoter. In some aspects, the nucleic acid sequence can be inserted into the genome of the iMGL cell so that the nucleic acid sequence is operably linked to an endogenous CD9 promoter. In some aspects, the nucleic acid sequence can be inserted into the CD9 locus. In some aspects, the nucleic acid sequence can be inserted into the CD9 locus so that the nucleic acid sequence encoding the therapeutic protein is connected to the exon of the CD9 gene and conforms to the reading frame. In some aspects, the nucleic acid sequence encoding the therapeutic protein is connected to the first exon of the CD9 gene and conforms to the reading frame. In some aspects, the nucleic acid sequence encoding the therapeutic protein is connected to the last exon of the CD9 gene and conforms to the reading frame. In some aspects, the first polynucleotide encoding a protease cleavage sequence or a self-cleaving peptide can be inserted into a CD9 exon and a nucleic acid sequence encoding a therapeutic protein, so that the first polynucleotide and the CD9 exon and the nucleic acid sequence encoding the therapeutic protein meet the reading frame. In some aspects, the second polynucleotide encoding a secretory peptide signal sequence (SP) can be connected to the nucleic acid sequence encoding the therapeutic protein, wherein the second polynucleotide can be inserted between the first polynucleotide and the nucleic acid sequence encoding the therapeutic protein, so that the encoded signal peptide and the CD9 exon and the nucleic acid sequence encoding the therapeutic protein meet the reading frame. In some aspects, the therapeutic protein can be a metalloproteinase. In some aspects, the therapeutic protein can be an enkephalinase, which can be a secretory form of enkephalinase or a membrane-bound form of enkephalinase. In some aspects, the therapeutic protein may comprise or consist of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identical to SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3, wherein the encoded therapeutic protein has enkephalinase activity. In some aspects, the encoded therapeutic protein may comprise or consist of an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identical to SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3, wherein the encoded therapeutic protein has enkephalinase activity, and wherein the difference between the amino acid sequence of the therapeutic protein and SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3 is due to conservative amino acid substitutions. In some aspects, the encoded protein may comprise or consist of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3. In some aspects, the encoded therapeutic protein comprises SEQ ID NO: 1.In some aspects, the encoded therapeutic protein comprises SEQ ID NO: 2. In some aspects, the encoded therapeutic protein comprises SEQ ID NO: 3. In some aspects, the encoded therapeutic protein comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identical to SEQ ID NO: 1. In some aspects, the encoded therapeutic protein comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identical to SEQ ID NO: 2. In some aspects, the encoded therapeutic protein comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identical to SEQ ID NO: 3.

[0107] One aspect of the present disclosure is a modified microglia-like (iMGL) cell, which comprises: a nucleic acid sequence encoding a therapeutic protein, wherein the nucleic acid sequence is inserted into the CD9 locus of the cell genome so that the therapeutic protein encoding nucleic acid sequence is in frame with at least a portion of the coding sequence of the CD9 gene exon; and, a first polynucleotide encoding a protease cleavage sequence or a self-cleavage peptide, which is inserted between at least a portion of the coding sequence of the CD9 gene exon and the therapeutic protein encoding nucleic acid sequence, so that the encoded protease cleavage site or the self-cleavage peptide is in frame with at least a portion of the coding sequence of the CD9 gene exon and the therapeutic protein encoding nucleic acid sequence; wherein transcription from the CD9 promoter produces an mRNA encoding a mixed protein, the mixed protein comprising an amino acid sequence CD9 gene, a protease cleavage sequence or a self-cleavage peptide and a therapeutic protein encoded by at least a portion of the coding sequence of the first exon. In some aspects, the carboxyl terminus of at least a portion of the encoded CD9 amino acid sequence is connected to the amino terminus of the protease cleavage sequence or the self-cleavage peptide sequence. In some aspects, the carboxyl terminus of the protease cleavage sequence or the self-cleavage peptide sequence is connected to the amino terminus of the therapeutic protein. In some aspects, the modified MGL cell comprises a second polynucleotide encoding a secretory peptide signal sequence (SP) linked to a nucleic acid sequence encoding a therapeutic protein, wherein the encoded signal peptide is in frame with the nucleic acid sequence encoding the therapeutic protein. In some aspects, the modified MGL cell comprises a second polynucleotide encoding a secretory peptide signal sequence (SP) located between the first polynucleotide and the nucleic acid sequence encoding the therapeutic protein, wherein the encoded signal peptide is in frame with the nucleic acid sequence encoding the therapeutic protein. Figure 2A and 2B show examples of such aspects. In some aspects, the therapeutic protein can be a metalloproteinase. In some aspects, the therapeutic protein can be an enkephalinase, which can be a secreted form of enkephalinase or a membrane-bound form of enkephalinase. In some aspects, the therapeutic protein can comprise or consist of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97% or at least 99% identical to SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3, wherein the encoded therapeutic protein has enkephalinase activity. In some aspects, the encoded therapeutic protein may comprise or consist of an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identical to SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3, wherein the encoded therapeutic protein has enkephalinase activity, and wherein the difference between the amino acid sequence of the therapeutic protein and SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3 is due to conservative amino acid substitutions. In some aspects, the encoded protein may comprise or consist of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3. In some aspects, the encoded protein includes SEQ ID NO: 1. In some aspects, the encoded therapeutic protein includes SEQ ID NO: 2. In some aspects, the encoded therapeutic protein includes SEQ ID NO: 3. In some aspects, the encoded therapeutic protein comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identical to SEQ ID NO: 1. In some aspects, the encoded therapeutic protein comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identical to SEQ ID NO: 2. In some aspects, the encoded therapeutic protein comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identical to SEQ ID NO: 3.

[0108] One aspect of the present disclosure is a therapeutic composition comprising an effective amount of modified cells of the present disclosure. Such compositions can be formulated for administration to individuals suffering from a disease that can be treated by a therapeutic protein expressed by the modified cells in the composition. In some aspects, the compositions of the present disclosure can be used to treat and / or improve and / or prevent diseases associated with pathologies associated with amyloid beta.

[0109] In some aspects, the compositions of the present disclosure may include modified cells, and the modified cells include a nucleotide sequence, and the nucleotide sequence includes a nucleotide sequence encoding a therapeutic protein, wherein the nucleotide sequence is operably connected to a pathological response promoter, and wherein the therapeutic protein can reduce the pathological phenotype associated with at least one amyloid beta protein. In some aspects, the nucleotide sequence may be inserted into a gene selected from the following genes: DCSTAMP gene, CD9 gene, CD44 gene, LGALS3 gene, SPP1 gene, GPNMB gene, HLA-DRB gene, LPL gene, LIPA, FABP3 gene, MS4A6A gene, CXCR4 gene, CHI3L1 gene, OLR1 gene, CD36 gene, SLAMF8 gene, TREM2 gene, MSR1 gene, B2M gene, ITGAX gene and MITF gene. In some aspects, the nucleic acid sequence can be inserted into the genome of the iMGL cell so that the nucleic acid sequence is operably linked to an endogenous promoter of a gene selected from the following group: DCSTAMP gene, CD9 gene, CD44 gene, LGALS3 gene, SPP1 gene, GPNMB gene, HLA-DRB gene, LPL gene, LIPA, FABP3 gene, MS4A6A gene, CXCR4 gene, CHI3L1 gene, OLR1 gene, CD36 gene, SLAMF8 gene, TREM2 gene, MSR1 gene, B2M gene, ITGAX gene and MITF gene. In some aspects, the nucleotide sequence can be inserted into the gene selected from the group consisting of DCSTAMP gene, CD9 gene, CD44 gene, LGALS3 gene, SPP1 gene, GPNMB gene, HLA-DRB gene, LPL gene, LIPA, FABP3 gene, MS4A6A gene, CXCR4 gene, CHI3L1 gene, OLR1 gene, CD36 gene, SLAMF8 gene, TREM2 gene, MSR1 gene, B2M gene, ITGAX gene and MITF gene, so that the nucleotide sequence encoding therapeutic protein is connected to the exon of gene and conforms to the reading frame. In some aspects, the nucleotide sequence encoding therapeutic protein is connected to the first exon of gene and conforms to the reading frame. In some aspects, the nucleotide sequence encoding therapeutic protein is connected to the last exon of gene and conforms to the reading frame. In some aspects, the first polynucleotide encoding protease cleavage sequence or self-cleavage peptide can be inserted between exon and the nucleotide sequence encoding therapeutic protein, so that the first polynucleotide and exon and the nucleotide sequence encoding therapeutic protein conform to the reading frame. In some aspects, a second polynucleotide encoding a secretory peptide signal sequence (SP) can be linked to a nucleic acid sequence encoding a therapeutic protein, wherein the second polynucleotide can be inserted between the first polynucleotide and the nucleic acid sequence encoding the therapeutic protein such that the encoded signal peptide is in frame with the exon and the nucleic acid sequence encoding the therapeutic protein.

[0110] In some aspects, the Alzheimer's disease-related pathological reaction promoter is a CD9 gene promoter. In some aspects, a nucleic acid sequence can be inserted into the genome of an iMGL cell so that the nucleic acid sequence is operably linked to an endogenous CD9 promoter. In some aspects, a nucleic acid sequence can be inserted into a CD9 locus. In some aspects, a nucleic acid sequence can be inserted into a CD9 locus so that a nucleic acid sequence encoding a therapeutic protein is connected to the exon of a CD9 gene and conforms to a reading frame. In some aspects, a nucleic acid sequence encoding a therapeutic protein is connected to the first exon of a CD9 gene and conforms to a reading frame. In some aspects, a nucleic acid sequence encoding a therapeutic protein is connected to the last exon of a CD9 gene and conforms to a reading frame. In some aspects, a first polynucleotide encoding a protease cleavage sequence or a self-cleavage peptide can be inserted between a CD9 exon and a nucleic acid sequence encoding a therapeutic protein so that the first polynucleotide conforms to a reading frame with a CD9 exon and a nucleic acid sequence encoding a therapeutic protein. In some aspects, a second polynucleotide encoding a secretory peptide signal sequence (SP) can be connected to a nucleic acid sequence encoding a therapeutic protein, wherein the second polynucleotide can be inserted between the first polynucleotide and the nucleic acid sequence encoding the therapeutic protein so that the encoded signal peptide is in frame with the CD9 exon and the nucleic acid sequence encoding the therapeutic protein. In some aspects, the therapeutic protein can be a metalloproteinase. In some aspects, the therapeutic protein can be a neprilysin, which can be a secretory form of neprilysin or a membrane-bound form of neprilysin. In some aspects, the therapeutic protein can include or consist of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97% or at least 99% identical to SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3, wherein the encoded therapeutic protein has neprilysin activity. In some aspects, the encoded therapeutic protein may comprise or consist of an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identical to SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3, wherein the encoded therapeutic protein has enkephalinase activity, and wherein the difference between the amino acid sequence of the therapeutic protein and SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3 is due to conservative amino acid substitutions. In some aspects, the encoded protein may comprise or consist of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3. In some aspects, the encoded therapeutic protein comprises SEQ ID NO: 1. In some aspects, the encoded therapeutic protein comprises SEQ ID NO: 2. In some aspects, the encoded therapeutic protein comprises SEQ ID NO: 3.In some aspects, the encoded therapeutic protein comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identical to SEQ ID NO: 1. In some aspects, the encoded therapeutic protein comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identical to SEQ ID NO: 2. In some aspects, the encoded therapeutic protein comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identical to SEQ ID NO: 3.

[0111] Any approach suitable for the treated disease can be used to administer the therapeutic composition. The example of the route of administration that can be used includes but is not limited to oral administration, parenteral administration, intravascular administration, intravenous administration, intramuscular administration, stereotactic administration, intracerebral administration, intracranial administration, intraventricular administration and intradermal administration. In some respects, it may be advantageous to apply the pharmaceutical composition as described above by intravenous injection or direct injection into the target tissue. For example, for systemic administration, it is preferred to give priority to intravenous, intravascular, intramuscular, stereotactic, intracerebral parenchyma or intraventricular treatment. More local application can be subcutaneous, intradermal, intradermal, intralobar, intramedullary, or directly in or near the tissue to be treated. According to the required treatment duration and effect, the composition according to the present invention can be administered once or repeatedly, or it can be administered intermittently, for example, administered every day, for several days, weeks or months, and can be administered with different dosages.

[0112] In various embodiments, the therapeutic compositions described herein are administered on a monthly dosage schedule. In other embodiments, the therapeutic compositions are administered once every two weeks. In yet other embodiments, the therapeutic compositions are administered weekly. In certain embodiments, the therapeutic compositions are administered daily. In selected embodiments, the therapeutic compositions are administered twice daily. In certain embodiments, the therapeutic compositions are administered for at least 3 months, at least 6 months, at least 12 months, or longer. In some embodiments, the therapeutic compositions are administered for at least 18 months, 2 years, 3 years, or longer.

[0113] In some aspects, the target tissue comprises brain tissue. In some aspects, the target tissue comprises one or more target brain regions. In some aspects, the target tissue comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or more target brain regions.

[0114] In some aspects, the target brain region includes the cerebral cortex. In some aspects, the target brain region includes one or more subregions of the cerebral cortex. In some aspects, the target brain region includes the medial prefrontal cortex or a subregion thereof. In some aspects, the target brain region includes the anterior cingulate cortex or a subregion thereof. In some aspects, the target brain region includes the primary sensory cortex or the sensory association cortex.

[0115] In some aspects, the target brain region includes a ventricle. In some aspects, the ventricle includes a lateral ventricle, a third ventricle, a fourth ventricle.

[0116] In some aspects, the target brain region includes the hippocampus or a subregion thereof. In some aspects, the target brain region includes the CA1 region of the hippocampus. In some aspects, the target brain region includes the CA3 region of the hippocampus. In some aspects, the target brain region includes the dentate gyrus of the hippocampus. In some aspects, the target brain region includes the CA2 region of the hippocampus. In some aspects, the target brain region includes the septum region or fornix of the hippocampus. In some aspects, the target brain region includes the entorhinal cortex. In some aspects, the target brain region includes the amygdala or a subregion thereof.

[0117] In some aspects, the target brain region includes the basal ganglia. In some aspects, the target brain region includes the basal ganglia nuclei. In some aspects, the basal ganglia nuclei include the globus pallidus. In some aspects, the basal ganglia nuclei include the substantia nigra compacta or substantia nigra reticulata. In some aspects, the basal ganglia nuclei include the striatum. In some aspects, the basal ganglia nuclei include the caudate putamen. In some aspects, the basal ganglia nuclei include the subthalamic nucleus.

[0118] The mode of administration, dosage and optimal formulation can be determined based on criteria that are usually considered in tailoring treatment to an individual, such as age or weight, severity of general condition, tolerance to treatment and noted side effects.

[0119] The formulations injected into the body fluid system require appropriate isotonicity and pH buffering to achieve the corresponding body fluid levels. Isotonicity can be appropriately adjusted with sodium chloride and other salts as needed. Suitable solvents can be used to increase the solubility of the ingredients in the formulation and the stability of the liquid formulation. Further additives that can be used in the composition of the present invention include, but are not limited to, glucose, conventional antioxidants, and conventional chelating agents. Parenteral dosage forms must also be sterilized before use.

[0120] One aspect is a method for treating Alzheimer's disease in an individual, comprising administering to the individual a modified cell of the present disclosure or a composition comprising a modified cell of the present disclosure. "Treatment of Alzheimer's disease", "treatment of Alzheimer's disease", etc. as used herein refers to reducing the frequency or severity of at least one aspect of at least one Alzheimer's disease-related phenotype and / or Alzheimer's disease-related pathology. In some aspects, treating Alzheimer's disease in an individual includes reducing the incidence or severity of at least one observable characteristic or feature selected from the group consisting of memory problems, learning disabilities, cognitive problems, vision problems, behavioral changes, personality changes, depression, and epilepsy. In some aspects, treating Alzheimer's disease in an individual may include reducing the amount of Aβ peptides in the brain of the individual. In some aspects, treating Alzheimer's disease in an individual may include reducing the size or number of Aβ peptide plaques or soluble or insoluble Aβ proteins, oligomers, pyroglutamate Aβ, fibrils or fibrils in the brain of the individual, or reducing the amount of Aβ peptides in Aβ peptide plaques in the brain of the individual.

[0121] One aspect is a method for treating a disease or condition associated with beta-amyloid protein-related pathology in an individual, comprising administering to an individual a modified cell of the present disclosure or a composition comprising a modified cell of the present disclosure. As used herein, "treating a disease or condition associated with beta-amyloid protein-related pathology", "treating beta-amyloid protein-related pathology", etc. refers to reducing the frequency or severity of at least one aspect of at least one beta-amyloid protein-related pathological phenotype and / or beta-amyloid protein-related pathology. In some aspects, treating a disease or condition associated with beta-amyloid protein-related pathology in an individual includes reducing the incidence or severity of at least one observable characteristic or feature selected from memory problems, learning disabilities, cognitive problems, vision problems, behavioral changes, personality changes, depression, and epileptic seizures. In some aspects, treating a disease or condition associated with beta-amyloid protein-related pathology in an individual may include reducing the brain of an individual. In some aspects, treating a disease or condition associated with amyloid-β-related pathology in an individual may include reducing the size or number of Aβ peptide plaques or soluble or insoluble Aβ protein, oligomers, pyroglutamate Aβ, fibrils or fibrils in the brain of the individual, or reducing the amount of Aβ peptide in Aβ peptide plaques in the brain of the individual.

[0122] One aspect is to treat a disease or condition associated with beta amyloid protein related pathology in an individual, including applying a modified cell comprising a nucleic acid sequence encoding a therapeutic protein to an individual, wherein the nucleic acid sequence is operably linked to a pathological response promoter, and wherein the therapeutic protein can reduce at least one beta amyloid protein related pathological phenotype or pathology. In some aspects, the pathological response promoter includes a promoter from a gene selected from: DCSTAMP gene, CD9 gene, CD44 gene, LGALS3 gene, SPP1 gene, GPNMB gene, HLA-DRB gene, LPL gene, LIPA, FABP3 gene, MS4A6A gene, CXCR4 gene, CHI3L1 gene, OLR1 gene, CD36 gene, SLAMF8 gene, TREM2 gene, MSR1 gene, B2M gene, ITGAX gene and MITF gene. For example, in some embodiments, the promoter includes the promoter of the CD9 gene. In some embodiments, the promoter includes the promoter of the LGALS3 gene. In some embodiments, the promoter includes the promoter of the HLA-DRB gene. In some embodiments, the promoter includes the promoter of the CD11c (ITGAX) gene. In some embodiments, the promoter includes a promoter selected from the following genes: CD9, LGALS3, HLA-DRB and CD11c. In some embodiments, the promoter includes a promoter selected from the following genes: CD9, LGALS3, HLA-DRB, TREM2 and CD11c. In some embodiments, the promoter includes the promoter of the DCSTAMP gene. In some embodiments, the promoter includes the promoter of the CD44 gene. In some embodiments, the promoter includes the promoter of the SPP1 gene. In some embodiments, the promoter includes the promoter of the GPNMB gene. In some embodiments, the promoter includes the promoter of the LPL gene. In some embodiments, the promoter includes the promoter of the LIPA gene. In some embodiments, the promoter includes the promoter of the FABP3 gene. In some embodiments, the promoter includes the promoter of the MS4A6A gene. In some embodiments, the promoter includes the promoter of the CXCR4 gene. In some embodiments, the promoter includes the promoter of the CHI3L1 gene. In some embodiments, the promoter includes the promoter of the OLR1 gene. In some embodiments, the promoter includes the promoter of the CD36 gene. In some embodiments, the promoter includes the promoter of the SLAMF8 gene. In some embodiments, the promoter includes the promoter of the TREM2 gene. In some embodiments, the promoter includes the promoter of the MSR1 gene. In some embodiments, the promoter includes the promoter of the B2M gene. In some embodiments, the promoter includes the promoter of the MITF gene. As previously described, the present invention is not limited to the aforementioned promoters.

[0123] In some aspects, the nucleic acid sequence can be inserted into a gene selected from the following: DCSTAMP gene, CD9 gene, CD44 gene, LGALS3 gene, SPP1 gene, GPNMB gene, HLA-DRB gene, LPL gene, LIPA, FABP3 gene, MS4A6A gene, CXCR4 gene, CHI3L1 gene, OLR1 gene, CD36 gene, SLAMF8 gene, TREM2 gene, MSR1 gene, B2M gene, ITGAX gene, MITF gene. In some aspects, the nucleic acid sequence can be inserted into the genome of the iMGL cell so that the nucleic acid sequence is operably linked to an endogenous promoter of a gene selected from the following group: DCSTAMP gene, CD9 gene, CD44 gene, LGALS3 gene, SPP1 gene, GPNMB gene, HLA-DRB gene, LPL gene, LIPA, FABP3 gene, MS4A6A gene, CXCR4 gene, CHI3L1 gene, OLR1 gene, CD36 gene, SLAMF8 gene, TREM2 gene, MSR1 gene, B2M gene, ITGAX gene and MITF gene. In some aspects, the nucleotide sequence can be inserted into a gene selected from the group consisting of: DCSTAMP gene, CD9 gene, CD44 gene, LGALS3 gene, SPP1 gene, GPNMB gene, HLA-DRB gene, LPL gene, LIPA, FABP3 gene, MS4A6A gene, CXCR4 gene, CHI3L1 gene, OLR1 gene, CD36 gene, SLAMF8 gene, TREM2 gene, MSR1 gene, B2M gene, ITGAX gene and MITF gene, so that the nucleotide sequence encoding therapeutic protein is connected to the exon of the gene and conforms to the reading frame. In some aspects, the nucleotide sequence encoding therapeutic protein is connected to the first exon of the gene and conforms to the reading frame. In some aspects, the nucleotide sequence encoding therapeutic protein is connected to the last exon of the gene and conforms to the reading frame. In some aspects, the first polynucleotide encoding protease cleavage sequence or self-cleavage peptide can be inserted between exons and the nucleotide sequence encoding therapeutic protein, so that the first polynucleotide conforms to the reading frame with exons and the nucleotide sequence encoding therapeutic protein. In some aspects, the second polynucleotide encoding a secretory peptide signal sequence (SP) can be connected to the nucleic acid sequence encoding a therapeutic protein, wherein the second polynucleotide can be inserted between the first polynucleotide and the nucleic acid sequence encoding the therapeutic protein, so that the encoded signal peptide and the exon and the nucleic acid sequence encoding the therapeutic protein meet the reading frame. In some aspects, the Alzheimer's disease-related pathological reaction promoter is a CD9 gene promoter. In some aspects, the nucleic acid sequence can be inserted into the genome of the iMGL cell so that the nucleic acid sequence is operably connected to the endogenous CD9 promoter. In some aspects, the nucleic acid sequence can be inserted into the CD9 locus.In some aspects, a nucleic acid sequence can be inserted into the CD9 locus so that the nucleic acid sequence encoding the therapeutic protein is connected to the exon of the CD9 gene and conforms to the reading frame. In some aspects, the nucleic acid sequence encoding the therapeutic protein is connected to the first exon of the CD9 gene and conforms to the reading frame. In some aspects, the nucleic acid sequence encoding the therapeutic protein is connected to the last exon of the CD9 gene and conforms to the reading frame. In some aspects, the first polynucleotide encoding the protease cleavage sequence or the self-cleavage peptide can be inserted between the CD9 exon and the nucleic acid sequence encoding the therapeutic protein so that the first polynucleotide and the CD9 exon and the nucleic acid sequence encoding the therapeutic protein conform to the reading frame. In some aspects, the second polynucleotide encoding the secretory peptide signal sequence (SP) can be connected to the nucleic acid sequence encoding the therapeutic protein, wherein the second polynucleotide can be inserted between the first polynucleotide and the nucleic acid sequence encoding the therapeutic protein so that the encoded signal peptide and the CD9 exon and the therapeutic protein encoding nucleic acid sequence. In some aspects, the therapeutic protein can be a metalloproteinase. In some aspects, the therapeutic protein can be an enkephalinase, which can be a secreted form of enkephalinase or a membrane-bound form of enkephalinase. In some aspects, the therapeutic protein may comprise or consist of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identical to SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3, wherein the encoded therapeutic protein has enkephalinase activity. In some aspects, the encoded therapeutic protein may comprise or consist of an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identical to SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3, wherein the encoded therapeutic protein has enkephalinase activity, and wherein the difference between the amino acid sequence of the therapeutic protein and SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3 is due to conservative amino acid substitutions. In some aspects, the encoded protein may comprise or consist of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3. In some aspects, the therapeutic protein comprises SEQ ID NO: 1. In some aspects, the therapeutic protein comprises SEQ ID NO: 2. In some aspects, the therapeutic protein comprises SEQ ID NO: 3. In some aspects, the therapeutic protein comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identical to SEQ ID NO: 1. In some aspects, the therapeutic protein comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identical to SEQ ID NO: 2.In some aspects, the therapeutic protein comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identical to SEQ ID NO:3.

[0124] One aspect of the present disclosure is a kit comprising a modified cell of the present disclosure, wherein the modified cell comprises a nucleic acid sequence encoding a therapeutic protein, wherein the nucleic acid sequence is operably linked to a pathologically responsive promoter, and wherein the therapeutic protein can reduce at least one pathological phenotype associated with beta-amyloid protein. The kit may also include other components, such as but not limited to buffers, labels, containers, tubes, vials, syringes, instructions for administering the modified cells of the present disclosure, and the like.

[0125] The present invention may include treatment of neurodegenerative diseases, disorders or conditions (including but not limited to Alzheimer's disease) associated with beta amyloid pathology and methods for alleviating related symptoms or pathological processes. These methods may include administering to individuals in need of such treatment a therapeutic composition comprising a sufficient number of modified cells of the present disclosure or modified cells as described herein. Modified cells and / or therapeutic compositions may be delivered directly to the brain of an individual or at least one specific target brain region. In some embodiments, the target brain region may include the cerebral cortex or its subregion, the hippocampus or its subregion, the basal ganglia or its basal ganglia nuclei, the ventricles, or a combination thereof. In addition, the target brain region may include specific subregions, such as the anterior cingulate cortex, the entorhinal cortex, the dentate gyrus, the CA1, CA3 or CA2 regions of the hippocampus, the fornix, the primary sensory cortex, the sensory association cortex, the septum, the globus pallidus, the substantia nigra compacta, the substantia nigra reticularis, the striatum, the caudate putamen or the subthalamic nucleus.

[0126] In some embodiments, modified cells and / or therapeutic compositions can be administered after Aβ disease pathology occurs. In certain embodiments, administration can be performed after detecting beta amyloid (Aβ) peptide plaques, soluble or insoluble Aβ monomers, Aβ oligomers, pyroglutamate Aβ, fibrils, or fibers comprising Aβ or its fragments in an individual. Modified cells and / or therapeutic compositions can be injected directly into the brain of an individual by stereotactic injection.

[0127] The present invention may also include methods for preventing or alleviating the onset of neurodegenerative diseases or disorders (such as but not limited to Alzheimer's disease) associated with beta amyloid pathology. These methods may include administering to individuals in need of such treatment a therapeutic composition comprising a sufficient number of modified cells of the present disclosure or modified cells as described herein. Modified cells and / or therapeutic compositions may be delivered directly to the brain or at least one specific target brain region of an individual. In some embodiments, the target brain region may include the cerebral cortex or its subregion, the hippocampus or its subregion, the ventricle, or a combination thereof. In addition, the target brain region may include specific regions, such as the anterior cingulate cortex, the entorhinal cortex, the dentate gyrus, the CA1, CA3 or CA2 regions of the hippocampus, the fornix, the primary sensory cortex, the sensory association cortex, the septum, the globus pallidus, the substantia nigra compacta, the substantia nigra reticularis, the striatum, the caudate putamen, or the subthalamic nucleus.

[0128] In order to effectively prevent or delay the occurrence of neurodegenerative disease pathology, modified cells and / or therapeutic compositions can be applied before the occurrence of Aβ disease pathology. In certain embodiments, application can be performed before detecting beta amyloid protein (Aβ) peptide plaques, soluble or insoluble Aβ monomers, Aβ oligomers, pyroglutamate Aβ, fibrils, or fibers comprising Aβ or its fragments in an individual. The application of modified cells and / or therapeutic compositions can be directly injected into the brain of an individual by stereotaxic injection, and can be performed before adulthood in some embodiments.

[0129] Individuals at risk of developing a neurodegenerative disease or condition, or individuals with gene mutations associated with a neurodegenerative disease, may benefit from the methods disclosed herein. Therefore, in some embodiments, individuals include subjects at risk of developing a neurodegenerative disease or condition. Or individuals may contain gene mutations associated with a neurodegenerative disease (e.g., apolipoprotein E (APOE4), presenilin 1 and 2, amyloid precursor (APP) or TREM2 (a triggering receptor expressed on myeloid cells 2)). In certain embodiments, a neurodegenerative disease or condition may be Alzheimer's disease. In some embodiments, the disease or condition is Parkinson's disease. In some embodiments, the disease or this disease is Huntington's disease. In some embodiments, the disease or condition is amyotrophic lateral sclerosis (ALS). In some embodiments, the disease or condition is another neurodegenerative disease associated with amyloid beta. The present invention is not limited to the aforementioned diseases or conditions.

[0130] The present invention may also provide a composition (e.g., a therapeutic composition) comprising a sufficient number of modified cells described herein for use in a method of treating a neurodegenerative disease or condition. In some embodiments, the present invention relates to a composition (e.g., a therapeutic composition) comprising a sufficient number of modified cells described herein for use in a method of treating a disease or condition associated with beta-amyloid pathology.

[0131] In some embodiments, the invention features the use of modified cells described herein to make a therapeutic composition for treating a neurodegenerative disease or disorder. In other embodiments, the invention features the use of modified cells described herein to make a therapeutic composition for treating Alzheimer's disease. In other embodiments, the invention features the use of modified cells described herein to make a therapeutic composition for treating Parkinson's disease. In other embodiments, the invention features the use of modified cells described herein to make a therapeutic composition for treating Huntington's disease. In other embodiments, the invention features the use of modified cells described herein to make a therapeutic composition for treating ALS.

[0132] As described herein, the invention features a human induced microglia-like (iMGL) cell that expresses and presents a therapeutic molecule comprising membrane-bound enkephalinase or expresses and secretes a therapeutic molecule comprising secreted enkephalinase.

[0133] As described herein, the present invention features a human induced microglia-like (iMGL) cell that expresses and presents or expresses and secretes a therapeutic molecule comprising: membrane-bound enkephalinase, secreted enkephalinase, TREM2, APOE, LRP1, insulin degrading enzyme, endothelial cell converting enzyme, plasminogen activator, angiotensin converting enzyme, or matrix metalloproteinase.

[0134] With reference to appropriate embodiments herein, in some embodiments, the promoter is configured to activate transcription of a therapeutic molecule when (i) the modified cell is close to a β-amyloid protein (Aβ) peptide plaque, a soluble Aβ monomer, an insoluble Aβ monomer, an Aβ oligomer, pyroglutamate Aβ, a fibril, or a fiber comprising Aβ or a fragment thereof; or (ii) the modified cell is in contact with a β-amyloid protein (Aβ) peptide plaque, a soluble Aβ monomer, an insoluble Aβ monomer, an Aβ oligomer, pyroglutamate Aβ, a fibril, or a fiber comprising Aβ or a fragment thereof. In some embodiments, the cell expresses P2RY12 and TREM2.

[0135] With reference to the appropriate embodiments herein, in some embodiments, the cell comprises a nucleic acid sequence encoding the therapeutic molecule; and a promoter selected from a CD9 gene promoter, a LGALS3 gene promoter, a HLA-DRB gene promoter, or a CD11c gene promoter, wherein the promoter is operably linked to a nucleic acid sequence encoding the therapeutic molecule. In some embodiments, the promoter comprises a CD9 gene promoter. In some embodiments, the promoter comprises a LGALS3 gene promoter. In some embodiments, the promoter comprises an HLA-DRB gene promoter. In some embodiments, the promoter comprises a CD11c gene promoter. In some embodiments, the promoter is one of the aforementioned promoters, and the therapeutic molecule comprises a membrane-bound enkephalinase or a secreted enkephalinase. In some embodiments, the promoter is one of the aforementioned promoters, and the therapeutic molecule comprises one of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3. In some embodiments, the promoter is one of the aforementioned promoters, and the therapeutic molecule comprises TREM2. In some embodiments, the promoter is one of the aforementioned promoters, and the therapeutic molecule comprises an insulin degrading enzyme.

[0136] With reference to the appropriate embodiment herein, in some embodiments, the nucleic acid sequence is inserted into the genome of the cell. In some embodiments, the nucleic acid sequence is inserted into the downstream of the locus controlled by the promoter so that the nucleic acid sequence meets the reading frame with the coding sequence in the locus exon. In some embodiments, the nucleic acid sequence is inserted into the locus controlled by the promoter so that the nucleic acid sequence meets the reading frame connection with at least part of the coding sequence of the locus. In some embodiments, the first polynucleotide encoding protease cleavage site, ribosome skipping sequence or self-cleavage peptide is inserted between the coding sequence of the exon and the nucleic acid sequence encoding therapeutic protein. In some embodiments, the self-cleavage peptide is P2A. In some embodiments, the second polynucleotide encoding the secretory peptide signal sequence is inserted into the 5' end of the nucleic acid sequence encoding the therapeutic protein.

[0137] With reference to the appropriate embodiments herein, in some embodiments, therapeutic proteins affect one or more amyloid beta (Aβ) related pathologies. In some embodiments, Aβ related pathologies include amyloid beta (Aβ) peptide plaques, soluble Aβ monomers, insoluble Aβ monomers, Aβ oligomers, pyroglutamate Aβ, fibrils or fibers comprising Aβ of different lengths. In some embodiments, therapeutic molecules reduce the amount of Aβ peptides in Aβ peptide plaques in the brain of an individual. In some embodiments, therapeutic molecules reduce the size or quantity of soluble Aβ monomers, insoluble Aβ monomers, Aβ oligomers, pyroglutamate Aβ, fibrils or fibers comprising Aβ of different lengths. In some embodiments, therapeutic molecules enhance amyloid hydrolysis. In some embodiments, therapeutic molecules enhance the phagocytosis of microglia to amyloid beta.

[0138] As described herein, the invention features a modified cell that expresses, presents or secretes a therapeutic molecule (Aβ)-associated pathology when the cell is in proximity or contact with beta-amyloid protein, wherein the therapeutic molecule reduces or eliminates the Aβ-associated pathology phenotype or improves the symptoms of Aβ-associated pathology.

[0139] As described herein, the invention features a modified cell that expresses, presents, or secretes a therapeutic molecule when the cell is in proximity or contact with amyloid beta (Aβ)-associated pathology, wherein the therapeutic molecule reduces or eliminates at least one aspect of the Aβ-associated pathology.

[0140] As described herein, the invention features a modified cell that expresses, presents or secretes a therapeutic molecule when the cell is in proximity to or in contact with amyloid-β (Aβ) aggregates, wherein the therapeutic molecule reduces or eliminates Aβ-associated pathology or ameliorates a symptom thereof.

[0141] As described herein, the invention features a modified cell that expresses, presents or secretes a therapeutic molecule when the cell is in proximity to or in contact with amyloid β (Aβ) aggregates, plaques, oligomers or fibrils, wherein the therapeutic molecule reduces or eliminates amyloid β-associated pathology or ameliorates a symptom thereof.

[0142] With reference to appropriate embodiments herein, in some embodiments, the amyloid-β-related pathological phenotype comprises a phenotype selected from the group consisting of memory problems, cognitive problems, vision problems, behavioral changes, personality changes, depression, and epilepsy.

[0143] With reference to appropriate embodiments herein, in some embodiments, the cell comprises a nucleic acid sequence encoding a therapeutic molecule, wherein the nucleic acid sequence is operably linked to a promoter responsive to amyloid-β-associated pathology.

[0144] As described herein, the present invention is characterized by a modified cell that expresses and presents or secretes a therapeutic molecule, the modified cell comprising: a nucleic acid sequence encoding a therapeutic molecule, the therapeutic molecule cleaving β-amyloid protein, binding to β-amyloid protein, enhancing amyloid protein hydrolysis, enhancing amyloid protein hydrolysis, enhancing the phagocytosis of β-amyloid protein by microglia, or a combination thereof; and a promoter selected from a CD9 gene promoter, a LGALS3 gene promoter, a HLA-DRB gene promoter, a TREM2 gene promoter or a CD11c gene promoter, the promoter being operably linked to the nucleic acid sequence encoding the therapeutic molecule.

[0145] As described herein, the present invention is characterized by a modified cell that expresses and presents or secretes a therapeutic molecule, the modified cell comprising: a nucleic acid sequence encoding a therapeutic molecule that cleaves β-amyloid protein, binds β-amyloid protein, enhances amyloid protein hydrolysis, enhances amyloid protein hydrolysis, enhances microglia phagocytosis of β-amyloid protein, or a combination thereof; and a promoter selected from the group consisting of a DCSTAMP gene promoter, a CD9 gene promoter, a CD44 gene promoter, a LGALS3 gene promoter, a SPP1 gene promoter, and a GPNMB gene promoter. The present invention relates to a promoter of a gene promoter, a HLA-DRB gene promoter, a LPL gene promoter, a LIPA gene promoter, a FABP3 gene promoter, a MS4A6A gene promoter, a CXCR4 gene promoter, a CHI3L1 gene promoter, an OLR1 gene promoter, a CD36 gene promoter, a SLAMF8 gene promoter, a TREM2 gene promoter, a MSR1 gene promoter, a B2M gene promoter, an ITGAX gene promoter and a MITF gene promoter; the promoter is operably linked to a nucleic acid sequence encoding a therapeutic molecule.

[0146] As described herein, the present invention features a modified cell that expresses and presents or secretes a therapeutic molecule comprising membrane-bound enkephalinase or secreted enkephalinase, the modified cell comprising: a nucleic acid sequence encoding the therapeutic molecule; and a promoter operably linked to the nucleic acid sequence encoding the therapeutic molecule, the promoter being configured to activate transcription of the therapeutic molecule when: (i) the cell is in proximity to β-amyloid protein (Aβ) peptide plaques, soluble Aβ monomers, insoluble Aβ monomers, Aβ oligomers, pyroglutamate Aβ, fibrils, or fibers comprising Aβ or fragments thereof; or (ii) the cell is in contact with β-amyloid protein (Aβ) peptide plaques, soluble Aβ monomers, insoluble Aβ monomers, Aβ oligomers, pyroglutamate Aβ, fibrils, or fibers comprising Aβ or fragments thereof.

[0147] As described herein, the present invention features a modified cell that expresses and presents or secretes a therapeutic molecule including membrane-bound enkephalinase, secreted enkephalinase, TREM2 or insulin-degrading enzyme, the modified cell comprising: a nucleic acid sequence encoding the therapeutic molecule; and a promoter operably linked to the nucleic acid sequence encoding the therapeutic molecule, the promoter being configured to activate transcription of the therapeutic molecule when (i) the cell is close to amyloid β (Aβ) peptide plaques, soluble Aβ monomers, insoluble Aβ monomers, Aβ oligomers, pyroglutamate Aβ, fibrils or fibers comprising Aβ or fragments thereof; or (ii) the cell is contacted with amyloid β (Aβ) peptide plaques, soluble Aβ monomers, insoluble Aβ monomers, Aβ oligomers, pyroglutamate Aβ, fibrils or fibers comprising Aβ or fragments thereof.

[0148] As described herein, the present invention features a microglia-like (MGL) cell that expresses and presents or secretes a therapeutic molecule that cleaves amyloid β, binds to amyloid β, enhances amyloid proteolysis, enhances microglial phagocytosis of amyloid β, or a combination thereof, the cell comprising: a nucleic acid sequence encoding the therapeutic molecule; and a promoter operably linked to the nucleic acid sequence encoding the therapeutic molecule, the promoter being configured to activate transcription of the therapeutic molecule when (i) the cell approaches amyloid β (Aβ) peptide plaques, soluble Aβ monomers, insoluble Aβ monomers, Aβ oligomers, pyroglutamate Aβ, fibrils, or fibers comprising Aβ or fragments thereof; or (ii) the cell is brought into contact with amyloid β (Aβ) peptide plaques, soluble Aβ monomers, insoluble Aβ monomers, Aβ oligomers, pyroglutamate Aβ, fibrils, or fibers comprising Aβ or fragments thereof.

[0149] With reference to appropriate embodiments herein, in some embodiments, the promoter is configured to activate transcription of a therapeutic molecule when (i) a cell is in proximity to β-amyloid protein (Aβ) peptide plaques, soluble Aβ monomers, insoluble Aβ monomers, Aβ oligomers, pyroglutamate Aβ, fibrils, or fibers comprising Aβ or fragments thereof; or (ii) a cell is contacted with β-amyloid protein (Aβ) peptide plaques, soluble Aβ monomers, insoluble Aβ monomers, Aβ oligomers, pyroglutamate Aβ, fibrils, or fibers comprising Aβ or fragments thereof. In some embodiments, the cell expresses P2RY12 and TREM2.

[0150] With reference to the appropriate embodiments herein, in some embodiments, the cell is a migratory cell or is produced by a cell lineage that can be differentiated into a migratory cell. In some embodiments, the cell is a pluripotent stem cell (PSC), an induced pluripotent stem cell (iPSC), a myeloid progenitor cell, a red myeloid progenitor cell, a hematopoietic stem cell, a hematopoietic progenitor cell, a lymphoid progenitor cell, a megakaryocyte-erythroid (mk-ery), an umbilical cord blood stem cell or an embryonic stem cell. In some embodiments, the cell is a monocyte. In some embodiments, the cell is a hematopoietic precursor cell of bone marrow origin. In some embodiments, the cell is a neural stem cell. In some embodiments, the cell is an iPSC-derived microglia. In some embodiments, the cell is an iPSC-derived hematopoietic precursor cell. In some embodiments, the cell is a hematopoietic precursor cell. In some embodiments, the cell is a hematopoietic precursor cell. In some embodiments, the cell is a microglia-like (MGL) cell. In some embodiments, the cell is a microglia-like cell (hiMGL) induced by humans. In some embodiments, MGL cells are MGL cells derived from human induced pluripotent stem cells. In some embodiments, the cell is a microglia-like (MGL) cell capable of engulfing human synaptosomes. In some embodiments, the cell is a microglia-like (MGL) cell capable of engulfing amyloid Aβ fibers. In some embodiments, the cell is a microglia-like (MGL) cell capable of migrating to the site of injury.

[0151] With reference to the appropriate embodiments herein, in some embodiments, the therapeutic protein is a membrane-bound protein. In some embodiments, the therapeutic protein is a secreted protein. In some embodiments, the therapeutic protein is a metalloproteinase. In some embodiments, the therapeutic protein comprises enkephalinase activity. In some embodiments, the therapeutic molecule comprises a membrane-bound enkephalinase or a secreted enkephalinase. In some embodiments, the therapeutic molecule comprises a peptide according to SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3. In some embodiments, the therapeutic molecule comprises TREM2, APOE, LRP1, or insulin degrading enzyme, endothelin-converting enzyme, plasminogen activator, angiotensin-converting enzyme, or matrix metalloproteinase.

[0152] With reference to the appropriate embodiment herein, in some embodiments, promoter is selected from CD9 gene promoter, LGALS3 gene promoter, TREM2 gene promoter, HLA-DRB gene promoter or CD11c gene promoter.In some embodiments, the promoter is selected from: DCSTAMP gene promoter, CD9 gene promoter, CD44 gene promoter, LGALS3 gene promoter, SPP1 gene promoter, GPNMB gene promoter, HLA-DRB gene promoter, LPL gene promoter, LIPA gene promoter, FABP3 gene promoter, MS4A6A gene promoter, CXCR4 gene promoter, CHI3L1 gene promoter, OLR1 gene promoter, CD36 gene promoter, SLAMF8 gene promoter, TREM2 gene promoter, MSR1 gene promoter, B2M gene promoter, ITGAX gene promoter and MITF gene promoter.In some embodiments, nucleic acid sequence is inserted into the genome of cell. In some embodiments, the nucleic acid sequence is inserted into the downstream of the locus controlled by the promoter so that the nucleic acid sequence is in frame with the coding sequence in the locus exon. In some embodiments, the nucleic acid sequence is inserted into the locus controlled by the promoter so that the nucleic acid sequence is connected to at least part of the coding sequence of the locus in frame. In some embodiments, the first polynucleotide encoding a protease cleavage site, a ribosomal skipping sequence or a self-cleaving peptide is inserted between the coding sequence of the exon and the nucleic acid sequence encoding the therapeutic protein. In some embodiments, the self-cleaving peptide is P2A. In some embodiments, the second polynucleotide encoding the secretory peptide signal sequence is inserted into the 5' end of the nucleic acid sequence encoding the therapeutic protein. In some embodiments, the therapeutic protein affects one or more amyloid beta (Aβ) related pathologies. In some embodiments, Aβ related pathologies include amyloid beta (Aβ) peptide plaques, soluble Aβ monomers, insoluble Aβ monomers, Aβ oligomers, pyroglutamate Aβ, fibrils or fibers containing Aβ of different lengths. In some embodiments, the therapeutic molecule reduces the amount of Aβ peptides in Aβ peptide plaques in the brain of an individual. In some embodiments, the therapeutic molecule reduces the size or number of soluble Aβ monomers, insoluble Aβ monomers, Aβ oligomers, pyroglutamate Aβ, fibrils, or fibers comprising Aβ of varying lengths; enhances proteolysis of amyloid; enhances phagocytosis of amyloid β by microglia, or a combination thereof.

[0153] As described herein, the invention features a composition comprising a cell of any of the embodiments disclosed herein.

[0154] As described herein, the invention features a kit comprising a cell of any embodiment disclosed herein or a composition of any embodiment disclosed herein.

[0155] As described herein, the invention features a composition for use in a method of treating a disease or condition associated with a beta-amyloid-related pathology or ameliorating a symptom or pathological process associated with a disease or condition associated with a beta-amyloid-related pathology, the composition comprising a cell of any embodiment disclosed herein.

[0156] As described herein, the invention features a method of treating an individual having a disease or condition associated with beta-amyloid-related pathology, the method comprising administering a cell of any embodiment disclosed herein or a composition of any embodiment disclosed herein to at least one target brain region in the brain of the individual.

[0157] As described herein, the invention features a method of reducing the amount of Aβ peptide in Aβ peptide plaques; and / or the size or number of soluble Aβ monomers, insoluble Aβ monomers, Aβ oligomers, pyroglutamate Aβ, fibrils, or fibers comprising Aβ of varying lengths in the brain of an individual; comprising administering a cell of any embodiment disclosed herein, or a composition of any embodiment disclosed herein, to at least one target brain region in the brain of the individual.

[0158] As described herein, the invention features a method of reducing neuronal or synaptic loss in a subject in need thereof, the subject having amyloid-β-related pathology, including Aβ plaques in brain tissue, the method comprising administering a cell of any embodiment disclosed herein or a composition of any embodiment disclosed herein to at least one target brain region in the brain of the individual.

[0159] As described herein, the invention features a method of preventing or attenuating a neurodegenerative disease or disorder associated with beta-amyloid pathology, the method comprising administering a cell of any embodiment disclosed herein or a composition of any embodiment disclosed herein to at least one target brain region in the brain of an individual.

[0160] With reference to the appropriate embodiments herein, in some embodiments, at least one target brain region includes cerebral cortex or its subregion, hippocampus or its subregion, ventricle, basal ganglia or its basal ganglia nucleus, entorhinal cortex, medial prefrontal cortex or its subregion, anterior cingulate cortex or its subregion, primary sensory cortex or sensory association cortex, fornix, septum, or a combination thereof. In some embodiments, hippocampus or its subregion includes CA1 region of hippocampus, CA3 region of hippocampus, dentate gyrus of hippocampus, CA2 region of hippocampus, septum or fornix of hippocampus. In some embodiments, ventricle includes lateral ventricle, third ventricle, fourth ventricle or a combination thereof. In some embodiments, basal ganglia nucleus includes globus pallidus, substantia nigra compacta, substantia nigra reticularis, striatum, caudate putamen, subthalamic nucleus.

[0161] With reference to the appropriate embodiments herein, in some embodiments, beta amyloid protein-related pathology is Alzheimer's disease. In some embodiments, beta amyloid protein-related pathology is Parkinson's disease. In some embodiments, beta amyloid protein-related pathology is Huntington's disease. In some embodiments, beta amyloid protein-related pathology is amyotrophic lateral sclerosis (ALS). In some embodiments, the method suppresses the expansion or progression of beta amyloid protein-related pathology. In some embodiments, the method suppresses the expansion of Abeta fibers. In some embodiments, the method reduces synaptic loss.

[0162] With reference to the appropriate embodiments herein, in some embodiments, cells are administered before Aβ-related pathology begins. In some embodiments, cells are administered before the occurrence of beta amyloid protein (Aβ) peptide plaques, soluble Aβ monomers, insoluble Aβ monomers, Aβ oligomers, pyroglutamate Aβ, fibrils, or fibers comprising Aβ or its fragments in individuals. In some embodiments, cells are administered after the presence of beta amyloid protein (Aβ) peptide plaques, soluble Aβ monomers, insoluble Aβ monomers, Aβ oligomers, pyroglutamate Aβ, fibrils, or fibers is detected to include Aβ or its fragments in individuals. In some embodiments, cells are injected into the brain of an individual by stereotaxic injection. In some embodiments, cells are administered to an individual before adulthood. In some embodiments, an individual has a risk of suffering from a neurodegenerative disease or illness. In some embodiments, an individual comprises a genetic mutation associated with a neurodegenerative disease.

[0163] With reference to the appropriate embodiments herein, in some embodiments, the amount of Aβ peptide or Aβ peptide plaques is determined using β-amyloid PET imaging, histological methods, immunoblotting methods, β-amyloid staining methods, or a combination thereof. In some embodiments, the immunoblotting method detects synaptic markers. In some embodiments, the staining method detects β-amyloid. In some embodiments, the staining method includes Golgi staining for measuring the number of synapses. In some embodiments, magnetic resonance imaging (MRI) is used to determine the amount of Aβ peptide in Aβ peptide plaques.

[0164] As described herein, the invention features the use of any embodiment of the cell disclosed herein, any embodiment of the composition disclosed herein, or any embodiment of the kit disclosed herein to reduce the amount of Aβ peptide in Aβ peptide plaques; and / or reduce the size or number of soluble Aβ monomers, insoluble Aβ monomers, Aβ oligomers, pyroglutamate Aβ, fibrils, or fibers containing Aβ of different lengths. In some embodiments, the use is a method comprising transplanting hiMGL into brain tissue of an individual.

[0165] As described herein, the present invention features the use of a cell of any embodiment disclosed herein, a composition of any embodiment disclosed herein, or a kit of any embodiment disclosed herein to treat a disease associated with β-amyloid-related pathology, or to improve a symptom or pathological process associated with β-amyloid-related pathology.

[0166] As described herein, the present invention is characterized by a human induced microglia-like (iMGL) cell that expresses and presents or expresses and secretes a therapeutic molecule, the therapeutic molecule comprising membrane-bound enkephalinase, secreted enkephalinase, TREM2, APOE, LRP1, insulin degrading enzyme, endothelial cell converting enzyme, plasminogen activator, angiotensin converting enzyme or matrix metalloproteinase. In some embodiments, when the cell contacts beta amyloid (Aβ) peptide plaques, soluble Aβ monomers, insoluble Aβ monomers, Aβ oligomers, pyroglutamate Aβ, fibrils or fibers containing Aβ of different lengths, the cell expresses and presents or expresses and secretes the therapeutic molecule. In some embodiments, the cell comprises: a nucleic acid sequence encoding the therapeutic molecule; and a promoter selected from a CD9 gene promoter, a LGALS3 gene promoter, a HLA-DRB gene promoter or a CD11c gene promoter, wherein the promoter is operably linked to a nucleic acid sequence encoding the therapeutic molecule.

[0167] With reference to suitable embodiments herein, in some embodiments, the therapeutic molecule binds to the Aβ peptide.With reference to suitable embodiments herein, in some embodiments, the therapeutic molecule cleaves the Aβ peptide.

[0168] In some embodiments, the Aβ peptide is Aβ-40. The cell of claim 4, wherein the Aβ peptide is Aβ-42.

[0169] With reference to appropriate embodiments herein, in some embodiments, the therapeutic molecule cleaves amyloid beta, binds to amyloid beta, enhances amyloid proteolysis, enhances amyloid proteolysis, enhances phagocytosis of amyloid beta by microglia, or a combination thereof.

[0170] As described herein, the invention is characterized in that cells, compositions and methods, wherein the nucleic acid sequence is inserted into the genome of the cell. For any suitable embodiment herein, in some embodiments, the nucleic acid sequence is inserted into the downstream of the locus controlled by the promoter, so that the nucleic acid sequence meets the reading frame with the coding sequence in the locus exon. In some embodiments, the nucleic acid sequence is inserted into the locus controlled by the promoter, so that the nucleic acid sequence meets the reading frame connection with at least part of the coding sequence of the locus. In some embodiments, the exon is the first exon in the locus. In some embodiments, the exon is the last exon in the locus.

[0171] As described herein, the invention features the use of a cell of any embodiment or claim herein, a composition of any embodiment or claim herein, or a kit of any embodiment or claim herein to treat a disease or condition associated with a beta-amyloid-related pathology or to ameliorate a symptom or pathological process associated with a disease or condition associated with a beta-amyloid-related pathology.

[0172] As described herein, the invention features a method of treating a neurodegenerative disease or disorder comprising administering to a subject a cell of any embodiment or claim herein or a composition of any embodiment or claim herein.

[0173] With reference to any appropriate embodiment herein, in some embodiments, the neurodegenerative disease or disorder is associated with a beta-amyloid-related pathology. In some embodiments, the beta-amyloid-related pathology is Alzheimer's disease. In some embodiments, the beta-amyloid-related pathology is Parkinson's disease. In some embodiments, the beta-amyloid-related pathology is Huntington's disease. In some embodiments, the beta-amyloid-related pathology is amyotrophic lateral sclerosis (ALS).

[0174] With reference to any appropriate embodiment herein, at least one target brain region includes the cerebral cortex or a subregion thereof. In some embodiments, at least one target brain region includes the hippocampus or a subregion thereof. In some embodiments, at least one target brain region includes the CA1 region of the hippocampus. In some embodiments, at least one target brain region includes the CA3 region of the hippocampus. In some embodiments, at least one target brain region includes the dentate gyrus of the hippocampus. In some embodiments, at least one target brain region includes the CA2 region of the hippocampus. In some embodiments, at least one target brain region includes the septal region or fornix of the hippocampus. In some embodiments, at least one target brain region includes a ventricle. In some embodiments, the ventricle includes the lateral ventricle, the third ventricle, the fourth ventricle, or a combination thereof. In some embodiments, at least one target brain region includes the basal ganglia or its basal ganglia nucleus. In some embodiments, the basal ganglia nucleus includes the globus pallidus. In some embodiments, the basal ganglia nucleus includes the substantia nigra compacta or the substantia nigra reticularis. In some embodiments, the basal ganglia nucleus includes the striatum. In some embodiments, the basal ganglia nucleus includes the caudate putamen. In some embodiments, the basal ganglia nucleus includes the subthalamic nucleus. In some embodiments, at least one target brain region includes the entorhinal cortex. In some embodiments, at least one target brain region includes the medial prefrontal cortex or a subregion thereof. In some embodiments, at least one target brain region includes the anterior cingulate cortex or a subregion thereof. In some embodiments, at least one target brain region includes the primary sensory cortex or the sensory association cortex. In some embodiments, at least one target brain region includes the cerebral cortex or a subregion thereof, the hippocampus or a subregion thereof, the ventricle, or a combination thereof. In some embodiments, at least one target brain region includes the anterior cingulate cortex, the entorhinal cortex, the dentate gyrus, the CA1 region of the hippocampus, the CA3 region of the hippocampus, the fornix, the primary sensory cortex, the sensory association cortex, the septum, or the CA2 region of the hippocampus.

[0175] The present invention also provides a method for producing a therapeutically modified cell, comprising introducing a nucleic acid sequence encoding a therapeutic molecule into a cell, wherein the nucleic acid sequence is operably linked to a promoter responsive to a β-amyloid protein-associated pathology, and wherein the therapeutic protein alters a β-amyloid protein-associated pathology phenotype or at least one aspect of a β-amyloid protein-associated pathology.

[0176] The present invention also provides a method for producing a cell population comprising human microglia-like cells (iMGLs), the method comprising: contacting human induced hematopoietic progenitor cells (iHPCs) with a microglia differentiation medium comprising CSF-1, IL-34 and TGFβ1 or CSF-1, IL-34 and TGFβ mimetics to differentiate the iHPCs into iMGLs, and introducing a nucleic acid encoding a protease that cleaves amyloid Aβ fibers into the iMGLs.

[0177] The present invention also relates to a method for producing a cell population comprising human microglia-like cells (iMGLs), the method comprising: contacting human induced hematopoietic progenitor cells (iHPCs) with a microglia differentiation medium comprising CSF-1, IL-34 and TGFβ1 or CSF-1, IL-34 and a TGFβ mimetic to differentiate the iHPCs into iMGLs, and introducing a nucleic acid encoding a therapeutic molecule that affects at least one aspect of β-amyloid protein-related pathology into the iMGLs.

[0178] The present invention also provides a method for producing a therapeutic cell population comprising human microglia-like cells (iMGLs), the method comprising: seeding human induced hematopoietic progenitor cells (iHPCs) on a basement membrane protein-coated culture dish; and contacting the human iHPCs with a microglia differentiation medium comprising CSF-1 or IL-34 to differentiate the iHPCs into iMGLs; and introducing a nucleic acid encoding a therapeutic molecule that affects at least one aspect of β-amyloid protein-related pathology into the iMGLs.

[0179] The present invention also provides a method for producing a therapeutic cell population comprising human microglia-like cells (iMGL), the method comprising: seeding human induced hematopoietic progenitor cells (iHPC) on a culture dish coated with basement membrane protein; contacting the human iHPC with a microglia differentiation medium comprising CSF-1 or IL-34 to differentiate the iHPC into iMGL; and introducing a nucleic acid encoding a protease that cleaves amyloid Aβ fibers into the iMGL.

[0180] For any appropriate embodiment, in some embodiments, the therapeutic molecule binds to Aβ peptide, cleaves Aβ peptide, reduces the amount of Aβ peptide in Aβ peptide plaques, reduces the size or number of soluble Aβ monomers, insoluble Aβ monomers, Aβ oligomers, pyroglutamate Aβ, fibrils, or fibers comprising Aβ of different lengths, enhances amyloid protein hydrolysis, enhances phagocytosis of β-amyloid protein by microglia, or a combination thereof.

[0181] For any appropriate embodiment, in some embodiments, the cell comprises a nucleic acid sequence encoding a therapeutic molecule, wherein the nucleic acid sequence is operably linked to a promoter responsive to amyloid-β-associated pathology.

[0182] For any suitable embodiment, in some embodiments, the promoter is an endogenous promoter.For any suitable embodiment, in some embodiments, the promoter is a non-endogenous promoter.

[0183] As described herein, the present invention features a method of treating an individual suffering from a disease or condition associated with beta-amyloid-related pathology, the method comprising administering a cell of any embodiment or claim herein, or a composition of any embodiment or claim herein, to at least one target brain region in the brain of the individual. The present invention also provides a method of reducing the amount of Aβ peptide in Aβ peptide plaques in the brain of an individual; and / or the size or number of soluble Aβ monomers, insoluble Aβ monomers, Aβ oligomers, pyroglutamate Aβ, fibrils, or fibers containing Aβ of different lengths; comprising administering a cell of any embodiment or claim herein, or a composition of any embodiment or claim herein, to at least one target brain region in the brain of an individual. The present invention also provides a method of reducing neuronal or synaptic loss in a subject in need thereof, the subject having beta-amyloid-related pathology, including Aβ plaques in brain tissue, the method comprising administering a cell of any embodiment or claim herein, or a composition of any embodiment or claim herein, to at least one target brain region in the brain of the individual. The present invention also provides a method for preventing or alleviating a neurodegenerative disease or condition associated with beta-amyloid pathology, the method comprising administering a cell of any embodiment or claim herein to at least one target brain region in the brain of an individual, or a composition of any embodiment or claim herein. In some embodiments, beta-amyloid-related pathology is Alzheimer's disease. In some embodiments, beta-amyloid-related pathology is Parkinson's disease. In some embodiments, beta-amyloid-related pathology is Huntington's disease. In some embodiments, beta-amyloid-related pathology is amyotrophic lateral sclerosis (ALS). In some embodiments, beta-amyloid-related pathology is Alzheimer's disease, Parkinson's disease, Huntington's disease or ALS. In some embodiments, beta-amyloid-related pathology is a different nervous system disease. In some embodiments, the method inhibits the expansion or progression of beta-amyloid-related pathology. In some embodiments, the method inhibits the expansion of amyloid beta fibers. In some embodiments, the method reduces synaptic loss. In some embodiments, the individual is a subject at risk of a neurodegenerative disease or condition. In some embodiments, the individual comprises a genetic mutation associated with a neurodegenerative disease. In some embodiments, the cells are administered prior to adulthood.

[0184] Imaging (e.g., amyloid beta PET imaging, MRI), histological methods, immunoblotting, amyloid beta staining, or a combination thereof can be used to detect or monitor the level of amyloid beta peptides and / or plaques. In some embodiments, the immunoblotting method detects synaptic markers. In some embodiments, the staining method detects amyloid beta. In some embodiments, the staining method includes Golgi staining for measuring the number of synapses. In some embodiments, magnetic resonance imaging (MRI) is used to determine the amount of Aβ peptides in Aβ peptide plaques.

[0185] As described herein, the present invention features a method of treating an individual suffering from a disease or condition associated with beta-amyloid-related pathology, the method comprising administering a cell of any embodiment or claim herein, or a composition of any embodiment or claim herein, to at least one target brain region in the brain of the individual. The present invention also provides a method of reducing the amount of Aβ peptide in Aβ peptide plaques in the brain of an individual; and / or the size or number of soluble Aβ monomers, insoluble Aβ monomers, Aβ oligomers, pyroglutamate Aβ, fibrils, or fibers containing Aβ of different lengths; comprising administering a cell of any embodiment or claim herein, or a composition of any embodiment or claim herein, to at least one target brain region in the brain of an individual. The present invention also provides a method of reducing neuronal or synaptic loss in a subject in need thereof, the subject suffering from beta-amyloid-related pathology, including Aβ plaques in brain tissue, the method comprising administering a cell of any embodiment or claim herein, or a composition of any embodiment or claim herein, to at least one target brain region in the brain of an individual. The present invention also provides a method for preventing or alleviating a neurodegenerative disease or condition associated with beta-amyloid pathology, the method comprising applying a cell of any embodiment or claim herein to at least one target brain region in the brain of an individual, or a composition of any embodiment or claim herein. In some embodiments, at least one target brain region includes the cerebral cortex or its subregion, the hippocampus or its subregion, the ventricle, the basal ganglia or its basal ganglia nucleus, the entorhinal cortex, the medial prefrontal cortex or its subregion, the anterior cingulate cortex or its subregion, the primary sensory cortex or the sensory association cortex, the fornix, the septum, or a combination thereof. In some embodiments, the hippocampus or its subregion includes the CA1 region of the hippocampus, the CA3 region of the hippocampus, the dentate gyrus of the hippocampus, the CA2 region of the hippocampus, the septum or the fornix of the hippocampus. In some embodiments, the ventricle includes the lateral ventricle, the third ventricle, the fourth ventricle, or a combination thereof.

[0186] In some embodiments, the basal ganglia nuclei include the globus pallidus, substantia nigra pars compacta, substantia nigra pars reticulata, corpus striatum, caudate putamen, and subthalamic nucleus.

[0187] This written description uses embodiments to disclose the invention, including the best mode, and enables those skilled in the art to practice the invention, including making and using any device or system and performing any incorporated method. The patent scope of the invention is defined by the claims and may include other embodiments that occur to those skilled in the art. If these other embodiments have structural elements that are indistinguishable from the literal language of the claims, or they include equivalent structural elements that are insubstantially different from the literal language of the claims, then these other exemplary embodiments are intended to fall within the scope of the claims.

[0188] Example

[0189] The following are non-limiting examples of the present invention. It should be understood that the examples are not intended to limit the present invention in any way. Equivalents or substitutes are within the scope of the present invention.

[0190] Example 1. Mouse Model Description

[0191] Microglia are the major innate immune cells of the brain and play a key role in maintaining neuronal homeostasis and detecting pathogenic factors and neuronal damage in the local environment. Recently, we developed a chimeric mouse model that allows the examination of the interaction between human iPSC-derived microglia and neuropathology (Hasselmann et al., Neuron, 2019). This model contains deletions of the Rag2 and il2rγ genes and a humanized CSF-1 allele, which has been shown to be necessary and sufficient to achieve long-term engraftment and survival of xenografted human microglia in the mouse brain. This model was further crossed with 5×FAD mice (Oakley et al., J Neurosci, 2006), which overexpress familial Alzheimer's disease (FAD) co-integrated transgenic mutant APP (Swedish, Florida, and London) and mutant FAD PS1 (M146L and L286V) and develop extensive amyloid plaque pathology. To generate chimeric mice, mouse pups were transplanted with human pluripotent stem cell-derived microglia within 5 days of birth, resulting in 60-80% human microglial chimerism in the forebrain (Hasselmann et al., Neuron, 2019). In this model, after xenotransplantation, human iPSC-derived microglia (xMG) were widely dispersed, matured into homeostatic microglia, exhibited in vivo-like transcriptional characteristics and thrived.

[0192] Example 2. Transplanted iMGLs migrate to and respond to Aβ pathology

[0193] Understanding how iPSC-derived microglia respond to amyloid-β-related pathologies, such as those associated with Alzheimer's disease (AD), will aid in the rational design of gene expression modules to regulate the expression of therapeutic agents. To this end, 5×fAD transgenic mice were crossed with MITRG (M-CSF h ,IL-3 / GM-CSF h , and TPO h Rag2 tm1.1Flv Il2rγ tm1.1Flv ) background to establish a xenograft-compatible model that produces extensive Aβ pathology as well as synaptic and neuronal loss, termed 5×-MITRG (5×FAD x M-CSF h ,IL-3 / GM-CSF h and TPO h Rag2 tm1.1Flv Il2rγ tm1.1Flv ). This model demonstrated that iPSC-derived microglia migrate toward Aβ pathology and express unique gene signatures when encountering Aβ plaques (Hasselmann et al., Neuron, 2019). Human xenograft microglia (xMG) were isolated from the brains of 9-10 month old 5×-MITRG mice using a negative magnetic sorting method to deplete all mouse cells. The isolated xMG were then subjected to single-cell RNA sequencing using the 10X Genomics Chromium platform and used to distinguish disease-associated microglia (DAM) populations associated with Aβ pathology in 5×-MITRG mice and identify mRNAs enriched in human DAM microglia. The localization of these DAM gene subsets was then validated and examined at the protein level using immunohistochemistry and confocal microscopy. It was observed that the tetraspanin CD9 was highly and specifically expressed only in microglia that were in direct contact with Aβ plaques ( Figure 1 A-1P and Figure 2 A-2F), which is consistent with mouse models and human microglia from AD patient brains. These indicate that iPSC-derived microglia can be transplanted into AD mouse models and that iPSC-derived microglia associated with Aβ pathology express CD9 in detail.

[0194] Example 3. Construction of iPSC-derived microglial cells expressing enkephalin (NEP) under the control of the CD9 promoter.

[0195] This example illustrates the construction of iPSC-derived microglia expressing NEP under the control of the CD9 endogenous promoter.Constructs were generated for membrane-anchored enkephalinase and a secreted form of NEP. Figure 3A and 3B show schematic diagrams of the design of the CD9 locus containing the coding sequences for the membrane-anchored and secreted forms of NEP, respectively.

[0196] iPSCs were collected after digestion with Accutase for 3 minutes at 37°C. 250,000 cells were suspended in a Nucleofector from Human Stem Cells. TM Kit 2 (Lonza) was used to prepare 100 μL of nucleofection buffer. CD9:NEP or CD9:sNEP plasmid template (2 μg; sequence is shown in Table 2) and the cells were incubated with Alt- SpHiFi Cas9 nuclease V3 (50 μg; IDTDNA) and RNP complexes formed by fusion of crRNA:tracrRNA (IDTDNA, double strand) were combined with cell suspension and nucleofected using AmaxaNucleofector program B-016. TM -E8 TM 0.25 μM Thiazovivin (STEMCELL Technologies) and CloneR were added to the Matrigel-coated plates in the culture medium. TM (STEMCELL Technologies) overnight to recover. The next day, cells were mechanically replated onto 96-well Matrigel-coated plates in the presence of 0.25 μM thiazolin and CloneR TM TeSR Supplement TM -E8 TM Clonal isolation and expansion were performed in culture medium. The culture medium was replenished with fresh medium every day. After five days, single clone wells in the culture plate were screened visually. After 10 days, visible clone wells were passaged with ReLeSR. Cell pellets were collected from each well, genomic DNA was extracted using Extracta DNA prep, and then PCR amplified using Taq PCR Master Mix (Thermo Fisher Scientific) to confirm bi-allelic integration of the CD9:NEP or CD9:sNEP cassette.

[0197] Both constructs were then used to differentiate into microglia, using a fully defined and scalable microglial production from a human pluripotent cell line (WO2018160496A1).

[0198] Table 2: Shows the sequences used to generate CD9:NEP and CD9:sNEP iPSC lines. The NEP and sNEP templates include the following left and right homology arms, with the NEP and sNEP DNA sequences placed between the left and right homology arms.

[0199]

[0200]

[0201]

[0202] Table 3: Shows non-limiting examples of constructions used herein and their parts.

[0203]

[0204]

[0205]

[0206] * indicates a stop codon; "secretion signal" and "signal peptide" as used herein are used interchangeably.

[0207] Example 4. Designed iPSC-derived microglia expressing neprilysin in vitro

[0208] The digestion and phagocytic properties of engineered iPSC-derived microglia were determined. The IncuCyte S3 Live Cell Analysis System (Sartorius) was used to examine the expression of isogenic WT, CD9:NEP( Figure 4 A) and CD9:sNEP( Figure 4 B) Phagocytic activity of iPS-derived microglia. iPS-microglia were seeded on Matrigel-coated 96-well plates, with 40K cells per well (6 wells per row). 45 minutes after inoculation, 500ng / ml, 1ug / ml and 2ug / ml 488-labeled fibrotic-Aβ(1-42) (Anaspec) were inoculated into the wells for recording. Using IncuCyte2020B software, the image mask of the fluorescent signal (phagocytosis of 488-labeled fibrotic Aβ) was normalized to the cell body area. Figure 4 As shown in C, significant phagocytosis of cells expressing and secreting enkephalinase was observed ( Figure 4 B). Furthermore, cells expressing tethered enkephalinase had higher levels of phagocytosis compared to wild-type microglia ( Figure 4 A and 4C). These studies demonstrated that engineered microglia were able to express the therapeutic payload neprilysin and that the expressed neprilysin increased the phagocytosis of Aβ aggregates.

[0209] Example 5. Demonstration of engineered iPSC-derived microglia expressing neprilysin in vivo.

[0210] As used herein, the term "xMG" refers to cells that have been xenotransplanted or re-isolated from the brain. The term "iMGL" refers to cells in vitro or prior to transplantation.

[0211] The activity of transplanted xMG according to the present disclosure was explored in a series of transplantation studies conducted in MITRG and 5×-MITRG mice, which were designed to Fig.12 This is shown schematically in A. Briefly, human iPSC-derived microglial progenitors were transplanted into the hippocampus and cortex of 2-month-old WT and 5×-MITRG mice. At 6.5 months of age, mice were sacrificed, and the hippocampus and cortex were microdissected and examined using an ELISA that detects endogenous mouse and human enkephalinase and is more sensitive to soluble, secreted NEP than to membrane-bound NEP. Figure 5 As shown in A-5B, the xMG of the present disclosure specifically expressed soluble (sNEP) and membrane-bound (NEP) when transplanted into 5×-MITRG mice but not WT-MITRG mice ( Figure 5 C) species, where soluble material was detectable above background in soluble extracts from the cortex and hippocampus, and membrane-bound material was detectable in soluble extracts from hippocampal samples. sNEP levels were significantly increased only in 5×-MITRG mice but not in WT mice, which is due to CD9-regulated sNEP expression, which is specifically induced by proximal amyloid pathology (see Figure 1 A-1P and Figure 2 A-2H).

[0212] The inventors observed that if Figure 6 A-6D, Figure 7 A-7D, Fig.18 A-18D and Fig.19 As shown in A-19D, human Aβ-42 and Aβ-40 peptides were statistically significantly reduced in the soluble and insoluble fractions of the cortex and hippocampus of 5×-MITRG mice treated with iMGL expressing NEP or sNEP. Microglia expressing NEP significantly reduced soluble Aβ-42 levels within the cortex (ANOVA with Tukey post hoc test, p<0.05). A trend towards reduced Aβ was also observed in the hippocampus where NEP and sNEP were expressed. Figure 8 As shown in A-8B, statistically significant reductions in soluble human Aβ oligomers in hippocampal and cortical fractions were also observed in NEP-treated animals as measured by MSD. These measurements also showed a trend toward reductions in sNEP-treated animals, but did not reach statistical significance in the samples examined. Nonetheless, overall, these data suggest that both NEP- and sNEP-expressing iMGLs are able to reduce at least some AD-associated Aβ species in vivo.

[0213] The reduction of Aβ species is complementary to the reduction of phenotypic signals associated with AD. Fig. 9 As shown in A-9H, sNEP-treated 5×-MITRG mice showed an increase in presynaptic and postsynaptic markers, suggesting that synaptic loss is reduced or reversed in untreated 5x-fAD mice and human patients. A decrease in the expression of the astrogliosis marker GFAP was observed, as shown in Fig.10 This was also observed in NEP- and sNEP-treated 5xMITRG mice (A-10B). Together, these data suggest that iMGL is able to reduce or correct AD-associated cellular and molecular phenotypes in vivo.

[0214] Crucially, Figure 5 The reduction in AD-related phenotypic signatures shown in A-10B was not accompanied by significant off-target effects of enkephalinase expression ( Fig.16 A-16D and Fig.17 A-17D). Fig.11 As shown in A-11D, the levels of two non-Aβ substrates, enkephalinase, bradykinin, and somatostatin, remained essentially unchanged in cortical and hippocampal samples of sham-treated (PBS), WT, NEP, and sNEP-treated 5xMITRG mice, indicating that according to the present disclosure, systemic enkephalinase activity is not increased by administration of iMGL expressing NEP or sNEP to a level that would result in off-target degradation.

[0215] Taken together, these data provide strong evidence that the systems and methods of the present disclosure deliver pharmaceutically effective doses of therapeutic payloads to sites of AD pathology with high specificity, and that the systems and methods of the present disclosure can effectively treat, alleviate, or prevent amyloid-β-related pathologies, such as Alzheimer's disease-related pathologies.

[0216] Example 6. Amyloid pathology induces CD9 expression in human microglia, leading to highly localized neprilysin induction.

[0217] Two-month-old 5×-MITRG mice were transplanted into the hippocampus and upper cortex with PBS vehicle, unmodified human microglial progenitors, CD9-NEP-modified human microglial progenitors, or CD9-sNEP-modified human microglial progenitors. At 6.5 months of age, mice were sacrificed and brains examined using immunohistochemistry and confocal microscopy. To determine whether enkephalinase expression was induced in plaque-associated human microglia under the control of the CD9 promoter, sections were labeled with Ku80 to detect human microglial nuclei, Amylo-Glo to detect β-amyloid plaques, CD9, and enkephalinase.

[0218] As shown, Ku80+ human microglial nuclei were dispersed throughout the tissue. However, CD9 expression was upregulated only in human Ku80+ microglia adjacent to Amylo-Glo positive amyloid plaques. Furthermore, enkephalinase expression was highly correlated with CD9 expression and was observed only in microglia adjacent to plaques. Thus, these immunohistochemical images demonstrate that CD9-regulated enkephalinase expression is highly restricted to human microglia adjacent to plaques only. Thus, these data demonstrate that human microglia modified to introduce NEP and sNEP sequences under the control of the CD9 promoter are able to function in a specifically designed manner.

[0219] As shown, Ku80+ human microglial nuclei were dispersed throughout the tissue. However, CD9 expression was upregulated only in human Ku80+ microglia adjacent to Amylo-Glo positive amyloid plaques. Furthermore, enkephalinase expression was highly correlated with CD9 expression and was observed only in microglia adjacent to plaques. Thus, these immunohistochemical images demonstrate that CD9-regulated enkephalinase expression is highly restricted to human microglia adjacent to plaques only. Thus, these data demonstrate that human microglia modified to introduce NEP and sNEP sequences under the control of the CD9 promoter are able to function in a specifically designed manner.

[0220] Fig.13 Results shown show that amyloid pathology induces CD9 expression in human microglia, leading to a highly localized induction of enkephalinase. Ku80 was used to visualize the nuclei of transplanted human microglia. Importantly, CD9 expression and the consequent induction of enkephalin were only observed in human microglia localized in the vicinity of Amylo-Glo positive amyloid-β plaques.

[0221] also, Fig.14 demonstrated that human microglia expressing sNEP in vivo can alleviate hippocampal Aβ pathology, suggesting that the use of genetically modified human microglia has therapeutic potential for treating Alzheimer's disease. Fig.15 As shown, upon induction of neprilysin, the number of medium-sized and large AmyloGlo-positive plaques in vivo was significantly reduced, further highlighting the potential of this approach as a therapeutic strategy for Alzheimer's disease.

[0222] Example 7: Use of the modified cells described herein to prevent the development of Alzheimer's disease.

[0223] A 45-year-old subject underwent genetic testing and discovered that she was at higher risk for developing Alzheimer's disease and consulted her physician for professional advice. The physician recommended a new therapy involving IPSC-derived human microglia (iMGLs), which express and release therapeutic molecules (e.g., neprilysin) when in proximity to Alzheimer's-associated pathology (e.g., α-β plaques). The physician explained that the therapeutic molecule (e.g., neprilysin) would hopefully prevent any Aβ pathology from forming. The subject agreed to undergo treatment, but first was required to undergo a battery of cognitive tests. After receiving treatment, the subject was able to successfully complete annual cognitive testing at physical checkups. No adverse side effects were reported.

[0224] Example 8: Use of the modified cells described herein to ameliorate Alzheimer's disease pathology.

[0225] A 65-year-old man was recently diagnosed with Alzheimer's disease after undergoing multiple cognitive tests and a biopsy, which revealed beta-amyloid plaques. Together with his family, they consulted with a doctor to explore possible treatment options. The doctor recommended a treatment composition consisting of modified iMGL cells. The doctor explained that a sample of the subject's cells would be obtained and modified to incorporate a therapeutic molecule with enkephalin activity. After modification, the cells would be infused into the subject's hippocampus. After agreeing to the treatment, the subject would need to undergo two surgeries, one to remove the cells and another to infuse the modified cells. The subject then underwent regular cognitive testing and biopsies to assess the effectiveness of the treatment. Six months after treatment, the subject's cognitive test results improved, and their biopsies showed a reduction in amyloid plaques. The doctor plans to monitor the subject every six months for the next 5 years and annually thereafter. No adverse side effects have been reported.

[0226] Although preferred embodiments of the present invention have been shown and described, it will be readily apparent to those skilled in the art that modifications may be made thereto without departing from the scope of the appended claims. Therefore, the scope of the present invention is limited only by the following claims. In some embodiments, the figures presented in this patent application are drawn to scale, including angles, dimensional ratios, etc. In some embodiments, the drawings are representative only and the claims are not limited by the size of the drawings. In some embodiments, the description of the invention described herein using the phrase "comprising" includes embodiments that may be described as "consisting essentially of" or "consisting of", and therefore satisfies the written description requirements for claiming protection of one or more embodiments of the present invention using the phrase "consisting essentially of" or "consisting of".

Claims

1. Human induced microglia-like (iMGL) cells that express and present a therapeutic molecule comprising membrane-bound enkephalinase or express and secrete a therapeutic molecule comprising secreted enkephalinase.

2. Human induced microglia-like (iMGL) cells that express and present or express and secrete a therapeutic molecule comprising: membrane-bound enkephalinase, secreted enkephalinase, TREM2, APOE, LRP1, insulin degrading enzyme, endothelin-converting enzyme, plasminogen activator, angiotensin-converting enzyme, or matrix metalloproteinase.

3. A cell according to claim 1 or 2, wherein the promoter is configured to activate transcription of the therapeutic molecule when (i) the cell is close to β-amyloid protein (Aβ) peptide plaques, soluble Aβ monomers, insoluble Aβ monomers, Aβ oligomers, pyroglutamate Aβ, fibrils, or fibers comprising Aβ or its fragments; or (ii) the cell is in contact with β-amyloid protein (Aβ) peptide plaques, soluble Aβ monomers, insoluble Aβ monomers, Aβ oligomers, pyroglutamate Aβ, fibrils, or fibers comprising Aβ or its fragments.

4. The cell of claim 3, wherein the cell expresses P2RY12 and TREM2.

5. The cell according to claim 1 or 2, wherein the cell comprises a nucleic acid sequence encoding the therapeutic molecule; and a promoter selected from a CD9 gene promoter, a LGALS3 gene promoter, a HLA-DRB gene promoter or a CD11c gene promoter, wherein the promoter is operably linked to the nucleic acid sequence encoding the therapeutic molecule.

6. The cell of claim 5, wherein the promoter comprises a CD9 gene promoter.

7. The cell of claim 5, wherein the promoter comprises a LGALS3 gene promoter.

8. The cell of claim 5, wherein the promoter comprises an HLA-DRB gene promoter.

9. The cell according to claim 5, wherein the promoter comprises a CD11c gene promoter.

10. The cell of any one of claims 6-9, wherein the therapeutic molecule comprises membrane-bound enkephalinase or secreted enkephalinase.

11. The cell of any one of claims 6-9, wherein the therapeutic molecule comprises one of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO:

3.

12. The cell of any one of claims 6-9, wherein the therapeutic molecule comprises TREM2.

13. The cell of any one of claims 6-9, wherein the therapeutic molecule comprises an insulin degrading enzyme.

14. The cell according to any one of claims 1-13, wherein the nucleic acid sequence is inserted into the genome of the cell.

15. The cell of claim 14, wherein the nucleic acid sequence is inserted downstream of a gene locus controlled by a promoter such that the nucleic acid sequence is in frame with a coding sequence in an exon of the gene locus.

16. The cell of claim 14, wherein the nucleic acid sequence is inserted into a locus under the control of a promoter such that the nucleic acid sequence is linked in frame with at least a portion of a coding sequence of the locus.

17. The cell according to any one of claims 15-16, wherein a first polynucleotide encoding a protease cleavage site, a ribosomal skipping sequence or a self-cleaving peptide is inserted between the coding sequence of the exon and the nucleic acid sequence encoding the therapeutic protein.

18. The cell of claim 17, wherein the self-cleaving peptide is P2A.

19. The cell of claim 17, wherein a second polynucleotide encoding a secretory peptide signal sequence is inserted at the 5' end of the nucleic acid sequence encoding the therapeutic protein.

20. The cell of any one of claims 1-19, wherein the therapeutic protein affects one or more amyloid beta (Aβ)-related pathologies.

21. The cell of claim 20, wherein the Aβ-related pathology comprises amyloid β (Aβ) peptide plaques, soluble Aβ monomers, insoluble Aβ monomers, Aβ oligomers, pyroglutamate Aβ, fibrils, or fibers comprising Aβ of varying lengths.

22. The cell of any one of claims 20-21, wherein the therapeutic molecule reduces the amount of Aβ peptide in Aβ peptide plaques in the brain of the individual.

23. The cell of any one of claims 20-21, wherein the therapeutic molecule reduces the size or amount of soluble Aβ monomers, insoluble Aβ monomers, Aβ oligomers, pyroglutamate Aβ, fibrils, or fibers comprising Aβ of varying lengths.

24. The cell of any one of claims 20-21, wherein the therapeutic molecule enhances amyloid proteolysis.

25. The cell of any one of claims 20-21, wherein the therapeutic molecule enhances phagocytosis of amyloid-β by microglia.

26. A modified cell that expresses and presents or secretes a therapeutic molecule when the cell is close to or in contact with amyloid-β (Aβ)-related pathology, wherein the therapeutic molecule reduces or eliminates Aβ-related pathological phenotypes or improves Aβ-related pathological symptoms.

27. A modified cell that expresses and presents or secretes a therapeutic molecule when the cell is in proximity or contact with amyloid beta (Aβ)-associated pathology, wherein the therapeutic molecule reduces or eliminates at least one aspect of the Aβ-associated pathology.

28. A modified cell that expresses and presents or secretes a therapeutic molecule when the cell is in proximity to or in contact with β-amyloid protein (Aβ) aggregates, wherein the therapeutic molecule reduces or eliminates β-amyloid protein-related pathology or improves its symptoms.

29. A modified cell that expresses and presents or secretes a therapeutic molecule when the cell is close to or in contact with β-amyloid protein (Aβ) aggregates, plaques, oligomers or fibrils, wherein the therapeutic molecule reduces or eliminates β-amyloid protein-related pathology or improves its symptoms.

30. The cell of any one of claims 26-29, wherein the amyloid-β-related pathological phenotype comprises a phenotype selected from the group consisting of memory problems, cognitive problems, vision problems, behavioral changes, personality changes, depression, and epilepsy.

31. The cell of any one of claims 26-30, wherein the cell comprises a nucleic acid sequence encoding the therapeutic molecule, wherein the nucleic acid sequence is operably linked to a promoter responsive to amyloid-β-related pathology.

32. A modified cell that expresses and presents or secretes a therapeutic molecule, the modified cell comprising: a. a nucleic acid sequence encoding a therapeutic molecule that cleaves amyloid beta, binds to amyloid beta, enhances amyloid proteolysis, enhances amyloid proteolysis, enhances the phagocytosis of amyloid beta by microglia, or a combination thereof; and b. A promoter, wherein the promoter is selected from a CD9 gene promoter, a LGALS3 gene promoter, a HLA-DRB gene promoter, a TREM2 gene promoter or a CD11c gene promoter, and the promoter is operably linked to a nucleic acid sequence encoding the therapeutic molecule.

33. A modified cell that expresses and presents or secretes a therapeutic molecule, the modified cell comprising: a. a nucleic acid sequence encoding a therapeutic molecule that cleaves amyloid beta, binds to amyloid beta, enhances amyloid proteolysis, enhances amyloid proteolysis, enhances the phagocytosis of amyloid beta by microglia, or a combination thereof; and b. a promoter selected from the group consisting of a DCSTAMP gene promoter, a CD9 gene promoter, a CD44 gene promoter, a LGALS3 gene promoter, a SPP1 gene promoter, a GPNMB gene promoter, a HLA-DRB gene promoter, a LPL gene promoter, a LIPA gene promoter, a FABP3 gene promoter, a MS4A6A gene promoter, a CXCR4 gene promoter, a CHI3L1 gene promoter, an OLR1 gene promoter, a CD36 gene promoter, a SLAMF8 gene promoter, a TREM2 gene promoter, a MSR1 gene promoter, a B2M gene promoter, an ITGAX gene promoter, and a MITF gene promoter; the promoter is operably linked to a nucleic acid sequence encoding the therapeutic molecule.

34. A modified cell that expresses and presents or secretes a therapeutic molecule comprising membrane-bound enkephalinase or secreted enkephalinase, the modified cell comprising: a. a nucleic acid sequence encoding the therapeutic molecule; and b. A promoter operably linked to a nucleic acid sequence encoding the therapeutic molecule, the promoter being configured to activate transcription of the therapeutic molecule when (i) the cell is close to β-amyloid protein (Aβ) peptide plaques, soluble Aβ monomers, insoluble Aβ monomers, Aβ oligomers, pyroglutamate Aβ, fibrils, or fibers comprising Aβ or its fragments; or (ii) the cell is in contact with β-amyloid protein (Aβ) peptide plaques, soluble Aβ monomers, insoluble Aβ monomers, Aβ oligomers, pyroglutamate Aβ, fibrils, or fibers comprising Aβ or its fragments.

35. A modified cell that expresses and presents or secretes a therapeutic molecule comprising membrane-bound enkephalinase, secreted enkephalinase, TREM2, or insulin-degrading enzyme, the modified cell comprising: a. a nucleic acid sequence encoding the therapeutic molecule; and b. A promoter operably linked to a nucleic acid sequence encoding the therapeutic molecule, the promoter being configured to activate transcription of the therapeutic molecule when (i) the cell is close to β-amyloid protein (Aβ) peptide plaques, soluble Aβ monomers, insoluble Aβ monomers, Aβ oligomers, pyroglutamate Aβ, fibrils, or fibers comprising Aβ or its fragments; or (ii) the cell is in contact with β-amyloid protein (Aβ) peptide plaques, soluble Aβ monomers, insoluble Aβ monomers, Aβ oligomers, pyroglutamate Aβ, fibrils, or fibers comprising Aβ or its fragments.

36. A microglia-like (MGL) cell that expresses and presents or secretes a therapeutic molecule that cleaves amyloid β, binds to amyloid β, enhances amyloid proteolysis, enhances the phagocytosis of amyloid β by microglia, or a combination thereof; the cell comprises: a. a nucleic acid sequence encoding the therapeutic molecule; and b. A promoter operably linked to a nucleic acid sequence encoding the therapeutic molecule, the promoter being configured to activate transcription of the therapeutic molecule when (i) the cell is close to β-amyloid protein (Aβ) peptide plaques, soluble Aβ monomers, insoluble Aβ monomers, Aβ oligomers, pyroglutamate Aβ, fibrils, or fibers comprising Aβ or its fragments; or (ii) the cell is in contact with β-amyloid protein (Aβ) peptide plaques, soluble Aβ monomers, insoluble Aβ monomers, Aβ oligomers, pyroglutamate Aβ, fibrils, or fibers comprising Aβ or its fragments.

37. A cell according to any one of claims 31-36, wherein the promoter is configured to activate transcription of the therapeutic molecule when (i) the cell is close to a β-amyloid protein (Aβ) peptide plaque, a soluble Aβ monomer, an insoluble Aβ monomer, an Aβ oligomer, pyroglutamate Aβ, a fibril, or a fiber comprising Aβ or a fragment thereof; or (ii) the cell is in contact with a β-amyloid protein (Aβ) peptide plaque, a soluble Aβ monomer, an insoluble Aβ monomer, an Aβ oligomer, pyroglutamate Aβ, a fibril, or a fiber comprising Aβ or a fragment thereof.

38. The cell of claim 37, wherein the cell expresses P2RY12 and TREM2.

39. The cell of any one of claims 31-35, wherein the cell is a migratory cell or is derived from a cell lineage that can differentiate into a migratory cell.

40. The cell of any one of claims 31-35, wherein the cell is a pluripotent stem cell (PSC), an induced pluripotent stem cell (iPSC), a myeloid progenitor cell, an erythroid myeloid progenitor cell, a hematopoietic stem cell, a hematopoietic progenitor cell, a lymphoid progenitor cell, a megakaryocyte-erythroid cell (mk-ery), an umbilical cord blood stem cell, or an embryonic stem cell.

41. The cell of any one of claims 31-35, wherein the cell is a monocyte.

42. The cell of any one of claims 31-35, wherein the cell is a bone marrow-derived hematopoietic precursor cell.

43. The cell of any one of claims 31-35, wherein the cell is a neural stem cell.

44. The cell of any one of claims 31-35, wherein the cell is an iPSC-derived microglia.

45. The cell of any one of claims 31-35, wherein the cell is an iPSC-derived hematopoietic precursor cell.

46. ​​The cell of any one of claims 31-35, wherein the cell is a hematopoietic precursor cell.

47. The cell of any one of claims 31-35, wherein the cell is a microglia-like (MGL) cell.

48. The cell of claim 47, wherein the cell is a human induced microglia-like cell (hiMGL).

49. The cell of claim 47, wherein the MGL cell is a human induced pluripotent stem cell-derived MGL cell.

50. The cell of any one of claims 31-36, wherein the cell is a microglia-like (MGL) cell capable of phagocytosing human synaptosomes.

51. The cell according to any one of claims 31-36, wherein the cell is a microglia-like (MGL) cell capable of phagocytosing amyloid Aβ fibers.

52. The cell of any one of claims 31-36, wherein the cell is a microglia-like (MGL) cell capable of migrating to a site of injury.

53. The cell of any one of claims 31-52, wherein the therapeutic protein is a membrane-bound protein.

54. The cell of any one of claims 31-52, wherein the therapeutic protein is a secreted protein.

55. The cell of any one of claims 31-52, wherein the therapeutic protein is a metalloprotease.

56. The cell of any one of claims 31-52, wherein the therapeutic protein comprises neprilysin activity.

57. The cell of any one of claims 31-52, wherein the therapeutic molecule comprises membrane-bound enkephalinase or secreted enkephalinase.

58. The cell of any one of claims 31-52, wherein the therapeutic molecule comprises a peptide as described in SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO:

3.

59. The cell of any one of claims 31-33 or 35-36, wherein the therapeutic molecule comprises TREM2, APOE, LRP1, or insulin degrading enzyme, endothelin-converting enzyme, plasminogen activator, angiotensin-converting enzyme, or matrix metalloproteinase.

60. The cell according to any one of claims 31-59, wherein the promoter is selected from a CD9 gene promoter, a LGALS3 gene promoter, a TREM2 gene promoter, a HLA-DRB gene promoter or a CD11c gene promoter.

61. The cell according to any one of claims 31 or 34-36, wherein the promoter is selected from: DCSTAMP gene promoter, CD9 gene promoter, CD44 gene promoter, LGALS3 gene promoter, SPP1 gene promoter, GPNMB gene promoter, HLA-DRB gene promoter, LPL gene promoter, LIPA gene promoter, FABP3 gene promoter, MS4A6A gene promoter, CXCR4 gene promoter, CHI3L1 gene promoter, OLR1 gene promoter, CD36 gene promoter, SLAMF8 gene promoter, TREM2 gene promoter, MSR1 gene promoter, B2M gene promoter, ITGAX gene promoter and MITF gene promoter.

62. The cell of any one of claims 31-61, wherein the nucleic acid sequence is inserted into the genome of the cell.

63. The cell of claim 62, wherein the nucleic acid sequence is inserted downstream of a locus controlled by a promoter such that the nucleic acid sequence is in frame with a coding sequence in an exon of the locus.

64. The cell of claim 62, wherein the nucleic acid sequence is inserted into a locus under the control of a promoter such that the nucleic acid sequence is linked in frame to at least a portion of a coding sequence of the locus.

65. The cell of any one of claims 63-64, wherein a first polynucleotide encoding a protease cleavage site, a ribosomal skipping sequence, or a self-cleaving peptide is inserted between the coding sequence of the exon and the nucleic acid sequence encoding the therapeutic protein.

66. The cell of claim 65, wherein the self-cleaving peptide is P2A.

67. The cell of claim 65, wherein a second polynucleotide encoding a secretory peptide signal sequence is inserted 5' to the nucleic acid sequence encoding the therapeutic protein.

68. The cell of any one of claims 26-35, wherein the therapeutic protein affects one or more amyloid beta (Aβ)-related pathologies.

69. The cell of claim 68, wherein the Aβ-related pathology comprises amyloid β (Aβ) peptide plaques, soluble Aβ monomers, insoluble Aβ monomers, Aβ oligomers, pyroglutamate Aβ, fibrils, or fibers comprising Aβ of varying lengths.

70. The cell of any one of claims 68-69, wherein the therapeutic molecule reduces the amount of Aβ peptide in Aβ peptide plaques in the brain of the individual.

71. The cell of any one of claims 68-69, wherein the therapeutic molecule reduces the size or number of soluble Aβ monomers, insoluble Aβ monomers, Aβ oligomers, pyroglutamate Aβ, fibrils, or fibers comprising Aβ of varying lengths; enhances amyloid proteolysis; enhances phagocytosis of amyloid β by microglia, or a combination thereof.

72. A composition comprising the cells of any one of claims 1-71.

73. A kit comprising the cell of any one of claims 1-71 or the composition of claim 72.

74. A composition for use in a method for treating a disease or condition associated with a beta-amyloid-related pathology or ameliorating a symptom or pathological process associated with a disease or condition associated with a beta-amyloid-related pathology, the composition comprising the modified cell of any one of claims 1-71.

75. A method of treating an individual suffering from a disease or condition associated with beta-amyloid-related pathology, the method comprising administering the cell of any one of claims 1-71 or the composition of claim 72 to at least one target brain region in the brain of the individual.

76. Ways to reduce: i) the amount of Aβ peptide in the Aβ peptide plaques; and / or ii) the size or amount of soluble Aβ monomers, insoluble Aβ monomers, Aβ oligomers, pyroglutamate Aβ, fibrils, or fibrils comprising Aβ of varying lengths; Comprising administering the cell of any one of claims 1-71 or the composition of claim 72 to at least one target brain region in the brain of an individual.

77. A method of reducing neuronal or synaptic loss in a subject in need thereof, the subject having amyloid-β-related pathology, including Aβ plaques in brain tissue, the method comprising administering the cell of any one of claims 1-71 or the composition of claim 72 to at least one target brain region in the brain of the individual.

78. A method of preventing or attenuating a neurodegenerative disease or condition associated with beta-amyloid pathology, the method comprising administering the cell of any one of claims 1-71 or the composition of claim 72 to at least one target brain region in the brain of an individual.

79. The method of any one of claims 75-78, wherein at least one target brain region comprises the cerebral cortex or a subregion thereof, the hippocampus or a subregion thereof, the ventricles, the basal ganglia or their basal ganglia nuclei, the entorhinal cortex, the medial prefrontal cortex or a subregion thereof, the anterior cingulate cortex or a subregion thereof, the primary sensory cortex or the sensory association cortex, the fornix, the septum, or a combination thereof.

80. The method of any one of claims 75-78, wherein the amyloid-beta-related pathology is Alzheimer's disease, Parkinson's disease, Huntington's disease, or amyotrophic lateral sclerosis (ALS).

81. The method of any one of claims 75-79, wherein the cells are administered prior to the onset of Aβ-related pathology.

82. The method of any one of claims 75-79, wherein the cells are administered prior to the presence of amyloid beta (Aβ) peptide plaques, soluble Aβ monomers, insoluble Aβ monomers, Aβ oligomers, pyroglutamate Aβ, fibrils, or fibers comprising Aβ or fragments thereof in the individual.

83. The method of any one of claims 75-79, wherein the cells are administered after the presence of amyloid beta (Aβ) peptide plaques, soluble Aβ monomers, insoluble Aβ monomers, Aβ oligomers, pyroglutamate Aβ, fibrils, or fibers comprising Aβ or fragments thereof are detected in the individual.

84. The method of any one of claims 75-79, wherein the cells are administered into the brain of the individual by stereotactic injection.

85. The method according to any one of claims 75-84, wherein the amount of Aβ peptide or Aβ peptide plaques is determined using β-amyloid PET imaging, MRI, histological methods, immunoblotting, β-amyloid staining, or a combination thereof.

86. The cell of any one of claims 1-71, the composition of claim 72, or the kit of claim 73 for: i) reducing the amount of Aβ peptide in Aβ peptide plaques; ii) reducing the size or amount of soluble Aβ monomers, insoluble Aβ monomers, Aβ oligomers, pyroglutamate Aβ, fibrils or Aβ fibers comprising deposits of Aβ peptides of varying lengths.

87. The use according to claim 86, wherein the use is for a method comprising transplanting the hiMGL into brain tissue of an individual.

88. Use of the cell according to any one of claims 1-71, the composition according to claim 72, or the kit according to claim 73 in treating a disease associated with beta-amyloid protein-related pathology or ameliorating a symptom or pathological process associated with beta-amyloid protein-related pathology.

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