Compositions and methods for treating neurocognitive disorders

Abstract

To provide a method and a composition for treating a subject diagnosed as having a neurocognitive disorder (NCD).SOLUTION: A subject having or at risk of developing a neurocognitive disorder may be administered one or more cells that contain a transgene encoding triggering receptor expressed on myeloid cells 2 (TREM2), such as a population of CD34+ hematopoietic stem or progenitor cells that express TREM2, thereby treating or preventing the disorder.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. Said ASCII copy, created on January 29, 2020, is named "51182-018WO2_Sequence_Listing_1.29.20_ST25" and is 22,674 bytes in size.

[0002] The present disclosure relates to compositions and methods for treating neurocognitive disorders such as Alzheimer's disease, Nasu-Hakola disease, frontotemporal lobar degeneration, and Parkinson's disease. [Background technology]

[0003] Neurodegeneration is a pathophysiological process observed in several diseases associated with progressive dementia, such as Alzheimer's disease and Nasu-Hakola disease. A key feature of this process is the massive destruction of brain tissue and the accompanying neurodegeneration and neuronal death that cause a whole range of behavioral deficits, including cognitive decline, language impairment, among others.

[0004] Alzheimer's disease (AD) is a late-onset neurodegenerative disorder responsible for the majority of dementia cases in elderly people. AD patients suffer from progressive cognitive decline characterized by symptoms including insidious loss of short-term and long-term memory, attention deficits, language-specific problems, disorientation, impulse control, social withdrawal, anhedonia, and other symptoms. The distinguishing neuropathological hallmark of AD is the extracellular aggregates of amyloid-β plaques and neurofibrillary tangles composed of hyperphosphorylated microtubule-associated tau protein. The accumulation of these aggregates is associated with neuronal loss and atrophy in several brain regions, including the frontal, temporal, and parietal lobes of the cerebral cortex, as well as subcortical structures such as the basal forebrain cholinergic system and the locus coeruleus in the brainstem. AD ​​is also associated with increased neuroinflammation, characterized by reactive gliosis and elevated levels of proinflammatory cytokines.

[0005] Nasu-Hakola disease, also known as polycystic lipomembranous skeletal dysplasia with sclerosing leukoencephalopathy (PLOSL), is a neurodegenerative disorder characterized by the presence of white matter degeneration, axonal spherules, and cyst-like bone lesions in the upper and lower limbs. PLOSL patients exhibit early-onset dementia and also recurrent fractures. Unlike AD, which widely affects older patients, PLOSL may begin to manifest during adolescence during the osseous stage, during which patients may experience multiple joint pains in the hands, wrists, ankles, and feet. The osseous stage is followed by an early neurological stage, during which patients may exhibit significant personality changes, progressive memory loss, and epileptic seizures. The late neurological stage of PLOSL patients shows significant dementia and motor disability.

[0006] Current treatments for AD and PLOSL seek to ameliorate disease symptoms, and there are no treatments that target the underlying neurodegeneration, thus highlighting the need for new therapeutic avenues. Summary of the Invention

[0007] The present disclosure provides methods for treating neurocognitive disorders (NCDs; e.g., Alzheimer's disease (AD), Nasu-Hakola disease (also known as multicystic lipomembranous dysplasia with sclerosing leukoencephalopathy (PLOSL)), frontotemporal lobar degeneration (FTLD), and Parkinson's disease (PD)) by administering cells comprising a transgene encoding TREM2 ("triggering receptor expressed on myeloid cells 2"), e.g., pluripotent cells (e.g., embryonic stem cells (ESCs) or induced pluripotent stem cells (ISPCs)), multipotent cells (e.g., CD34+ cells, e.g., hematopoietic stem cells (HSCs) or myeloid progenitor cells (MPCs)), blood lineage progenitor cells (BLPCS; e.g., monocytes), macrophages, microglial progenitor cells, or microglia. The cells can be administered to a subject (e.g., a human) with an NCD by one or more of a variety of routes, including directly (e.g., by intraventricular administration) or systemically (e.g., by intravenous administration) into the subject's central nervous system, among others. The disclosure also features compositions containing such cells, as well as kits containing these cells for treating NCDs.

[0008] In a first aspect, the disclosure provides a method of treating a subject diagnosed with an NCD (e.g., AD, PLOSL, FTLD, or PD) by administering to the subject a composition containing a population of cells (e.g., pluripotent cells, ESCs, iPSCs, pluripotent cells, CD34+ cells, HSCs, MPCs, BLPCs, monocytes, macrophages, microglial progenitor cells, or microglia) comprising a transgene encoding TREM2. In some embodiments, the transgene encoding TREM2 may be expressed in macrophages or microglial cells. In some embodiments, the cells express the transgene encoding TREM2.

[0009] In some embodiments, the NCD is a severe NCD. In some embodiments, a severe NCD interferes with a subject's independence and / or normal daily functioning (e.g., social, occupational, or academic functioning, personal hygiene, grooming, dressing, toilet hygiene, functional mobility (e.g., ability to walk, get in and out of bed), and self-care. In some embodiments, a severe NCD is associated with a score obtained by the subject on a cognitive test that is at least 2 standard deviations away from the mean score of a reference population. In some embodiments, the NCD is a mild NCD. In some embodiments, a mild NCD does not interfere with a subject's independence and / or normal daily functioning. In some embodiments, a mild NCD is associated with a score obtained by the subject on a cognitive test that is at least 1-2 standard deviations away from the mean score of a reference population. In some embodiments, the cognitive test is an Eight-item Informant Interview to Differentiate Aging and Dementia (AD8), Annual Wellness Visit (AWV), General Practitioner Assessment of Cognition (GPCOG), Health Risk Assessment (HRA), Memory Impairment Screen (MIS), Mini The NCD is selected from the group consisting of Mental Status Exam (MMSE), Montreal Cognitive Assessment (MoCA), St. Louis University Mental Status Exam (SLUMS), and Short Informant Questionnaire on Cognitive Decline in the Elderly (Short IQCODE). In some embodiments, the NCD is associated with impairment in one or more of complex attention, executive function, learning and memory, language, sensorimotor function, and social cognition. In some embodiments, the NCD is not due to delirium or other psychiatric disorders (e.g., schizophrenia, bipolar disorder, or major depression). In some embodiments, the reference population is the general population. In some embodiments, the reference population is selected based on the subject's age, medical history, education, socioeconomic status, and lifestyle.In some embodiments, the NCD is AD. In some embodiments, the NCD is leukodystrophy. In some embodiments, the NCD is PLOSL. In some embodiments, the NCD is a frontotemporal NCD. In some embodiments, the frontotemporal NCD is FTLD. In some embodiments, the NCD is a movement disorder. In some embodiments, the movement disorder is PD.

[0010] In some embodiments, the TREM2 is a full-length TREM2, e.g., a TREM2 having the amino acid sequence of any one of SEQ ID NOs:1-3, or a variant thereof having at least 85% sequence identity thereto (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto).

[0011] In some embodiments, TREM2 has the amino acid sequence of SEQ ID NO:1 or a variant thereof having at least 85% (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID NO:1.

[0012] In some embodiments, TREM2 has the amino acid sequence of SEQ ID NO:1 or a variant thereof having at least 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO:1.

[0013] In some embodiments, TREM2 has the amino acid sequence of SEQ ID NO:1 or a variant thereof having at least 95% (e.g., at least 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO:1.

[0014] In some embodiments, TREM2 has the amino acid sequence of SEQ ID NO:1.

[0015] In some embodiments, TREM2 has the amino acid sequence of SEQ ID NO:2 or a variant thereof having at least 85% (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID NO:2.

[0016] In some embodiments, TREM2 has the amino acid sequence of SEQ ID NO:2 or a variant thereof having at least 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO:2.

[0017] In some embodiments, TREM2 has the amino acid sequence of SEQ ID NO:2 or a variant thereof having at least 95% (e.g., at least 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO:2.

[0018] In some embodiments, TREM2 has the amino acid sequence of SEQ ID NO:2.

[0019] In some embodiments, TREM2 has the amino acid sequence of SEQ ID NO:3 or a variant thereof having at least 85% (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID NO:3.

[0020] In some embodiments, TREM2 has the amino acid sequence of SEQ ID NO:3 or a variant thereof having at least 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO:3.

[0021] In some embodiments, TREM2 has the amino acid sequence of SEQ ID NO:3 or a variant thereof having at least 95% (e.g., at least 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO:3.

[0022] In some embodiments, TREM2 has the amino acid sequence of SEQ ID NO:3.

[0023] In some embodiments, the transgene encoding TREM2 comprises a TREM2 polynucleotide having the nucleotide sequence of SEQ ID NO:4, or a variant thereof having at least 85% (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO:4.

[0024] In some embodiments, the transgene encoding TREM2 comprises a TREM2 polynucleotide having the nucleotide sequence of SEQ ID NO:4, or a variant thereof having at least 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO:4.

[0025] In some embodiments, the transgene encoding TREM2 comprises a TREM2 polynucleotide having the nucleotide sequence of SEQ ID NO:4, or a variant thereof having at least 95% (e.g., at least 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO:4.

[0026] In some embodiments, the transgene encoding TREM2 comprises a TREM2 polynucleotide having the nucleotide sequence of SEQ ID NO:4.

[0027] In some embodiments, the transgene encoding TREM2 comprises a TREM2 polynucleotide having the nucleotide sequence of SEQ ID NO:5, or a variant thereof having at least 85% (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO:5.

[0028] In some embodiments, the transgene encoding TREM2 comprises a TREM2 polynucleotide having the nucleotide sequence of SEQ ID NO:5, or a variant thereof having at least 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO:5.

[0029] In some embodiments, the transgene encoding TREM2 comprises a TREM2 polynucleotide having the nucleotide sequence of SEQ ID NO:5, or a variant thereof having at least 95% (e.g., at least 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO:5.

[0030] In some embodiments, the transgene encoding TREM2 comprises a TREM2 polynucleotide having the nucleotide sequence of SEQ ID NO:5.

[0031] In some embodiments, the transgene encoding TREM2 comprises a TREM2 polynucleotide having the nucleotide sequence of SEQ ID NO:6, or a variant thereof having at least 85% (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO:6.

[0032] In some embodiments, the transgene encoding TREM2 comprises a TREM2 polynucleotide having the nucleotide sequence of SEQ ID NO:6, or a variant thereof having at least 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO:6.

[0033] In some embodiments, the transgene encoding TREM2 comprises a TREM2 polynucleotide having the nucleotide sequence of SEQ ID NO:6, or a variant thereof having at least 95% (e.g., at least 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO:6.

[0034] In some embodiments, the transgene encoding TREM2 comprises a TREM2 polynucleotide having the nucleotide sequence of SEQ ID NO:6.

[0035] In some embodiments, the transgene encoding TREM2 comprises a TREM2 polynucleotide having the nucleotide sequence of SEQ ID NO:7, or a variant thereof having at least 85% (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID NO:7.

[0036] In some embodiments, the transgene encoding TREM2 comprises a TREM2 polynucleotide having the nucleotide sequence of SEQ ID NO:7, or a variant thereof having at least 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO:7.

[0037] In some embodiments, the transgene encoding TREM2 comprises a TREM2 polynucleotide having the nucleotide sequence of SEQ ID NO:7, or a variant thereof having at least 95% (e.g., at least 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO:7.

[0038] In some embodiments, the transgene encoding TREM2 comprises a TREM2 polynucleotide having the nucleotide sequence of SEQ ID NO:7.

[0039] In some embodiments, the transgene encoding TREM2 comprises a TREM2 polynucleotide having the nucleotide sequence of SEQ ID NO:9, or a variant thereof having at least 85% (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID NO:9.

[0040] In some embodiments, the transgene encoding TREM2 comprises a TREM2 polynucleotide having the nucleotide sequence of SEQ ID NO:9, or a variant thereof having at least 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO:9.

[0041] In some embodiments, the transgene encoding TREM2 comprises a TREM2 polynucleotide having the nucleotide sequence of SEQ ID NO:9, or a variant thereof having at least 95% (e.g., at least 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO:9.

[0042] In some embodiments, the transgene encoding TREM2 comprises a TREM2 polynucleotide having the nucleotide sequence of SEQ ID NO:9.

[0043] In some embodiments, the transgene encoding TREM2 comprises a TREM2 polynucleotide having the nucleotide sequence of SEQ ID NO:11, or a variant thereof having at least 85% (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO:11.

[0044] In some embodiments, the transgene encoding TREM2 comprises a TREM2 polynucleotide having the nucleotide sequence of SEQ ID NO:11, or a variant thereof having at least 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO:11.

[0045] In some embodiments, the transgene encoding TREM2 comprises a TREM2 polynucleotide having the nucleotide sequence of SEQ ID NO:11, or a variant thereof having at least 95% (e.g., at least 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO:11.

[0046] In some embodiments, the transgene encoding TREM2 comprises a TREM2 polynucleotide having the nucleotide sequence of SEQ ID NO:11.

[0047] In some embodiments, the transgene encoding TREM2 may be codon optimized (eg, any one of SEQ ID NO:8, SEQ ID NO:10, or SEQ ID NO:12).

[0048] In some embodiments, the transgene encoding TREM2 comprises a codon-optimized TREM2 polynucleotide sequence of SEQ ID NO:8 or a variant thereof having at least 85% (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO:8.

[0049] In some embodiments, the transgene encoding TREM2 comprises a codon-optimized TREM2 polynucleotide sequence of SEQ ID NO:8 or a variant thereof having at least 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO:8.

[0050] In some embodiments, the transgene encoding TREM2 comprises a codon-optimized TREM2 polynucleotide sequence of SEQ ID NO:8 or a variant thereof having at least 95% (e.g., at least 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO:8.

[0051] In some embodiments, the transgene encoding TREM2 comprises the codon-optimized TREM2 polynucleotide sequence of SEQ ID NO:8.

[0052] In some embodiments, the transgene encoding TREM2 comprises a codon-optimized TREM2 polynucleotide sequence of SEQ ID NO:10, or a variant thereof having at least 85% (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO:10.

[0053] In some embodiments, the transgene encoding TREM2 comprises a codon-optimized TREM2 polynucleotide sequence of SEQ ID NO:10, or a variant thereof having at least 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO:10.

[0054] In some embodiments, the transgene encoding TREM2 comprises a codon-optimized TREM2 polynucleotide sequence of SEQ ID NO:10, or a variant thereof having at least 95% (e.g., at least 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO:10.

[0055] In some embodiments, the transgene encoding TREM2 comprises the codon-optimized TREM2 polynucleotide sequence of SEQ ID NO:10.

[0056] In some embodiments, the transgene encoding TREM2 comprises a codon-optimized TREM2 polynucleotide sequence of SEQ ID NO:12 or a variant thereof having at least 85% (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO:12.

[0057] In some embodiments, the transgene encoding TREM2 comprises a codon-optimized TREM2 polynucleotide sequence of SEQ ID NO:12 or a variant thereof having at least 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO:12.

[0058] In some embodiments, the transgene encoding TREM2 comprises a codon-optimized TREM2 polynucleotide sequence of SEQ ID NO:12 or a variant thereof having at least 95% (e.g., at least 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO:12.

[0059] In some embodiments, the transgene encoding TREM2 comprises the codon-optimized TREM2 polynucleotide sequence of SEQ ID NO:12.

[0060] In some embodiments, the transgene encodes two or more TREM2 proteins (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or more TREM2 proteins). In some embodiments, the transgene encodes between 2 and 10 TREM2 proteins (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 TREM2 proteins). In some embodiments, the transgene encodes between 2 and 5 TREM2 proteins (e.g., 2, 3, 4, or 5 TREM2 proteins). In some embodiments, the transgene encodes two TREM2 proteins. In some embodiments, the TREM2 transgenes are expressed from a single polycistronic expression cassette. In some embodiments, the TREM2 transgenes are separated from each other by one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or more) internal ribosome entry sites (IRES). In some embodiments, the TREM2 transgene is expressed from one or more (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) monocistronic expression cassettes.

[0061] In some embodiments, the transgene encoding TREM2 includes a signal peptide (eg, the TREM2 signal peptide).

[0062] In some embodiments, TREM2 is soluble TREM2 (sTREM2). In some embodiments, TREM2 is a TREM2 C-terminal fragment (TREM2-CTF). In some embodiments, TREM2 is a TREM2 intracellular domain (TREM2-ICD). In some embodiments, TREM2 is a TREM2-A beta-like (TREM-T2β) peptide. In some embodiments, TREM2 does not comprise a functional ectodomain cleavage site. In some embodiments, TREM2 does not comprise a functional intramembrane cleavage site within the TREM2-CTF.

[0063] In some embodiments, TREM2 is a TREM2 fusion protein. In some embodiments, the TREM2 fusion protein comprises the low density lipoprotein receptor family (LDLRf) binding (Rb) domain of apolipoprotein E (ApoE), or a fragment, variant, or oligomer thereof. In some embodiments, the Rb domain of ApoE, or a fragment, variant, or oligomer thereof, is operably linked to the N-terminus of TREM2. In some embodiments, the Rb domain of ApoE, or a fragment, variant, or oligomer thereof, is operably linked to the C-terminus of TREM2. In some embodiments, the TREM2 fusion protein comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) oligomers of the Rb domain of ApoE. In some embodiments, the Rb domain comprises a region of ApoE having at least 70% sequence identity (e.g., at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to residues 25-185 of SEQ ID NO: 13. In some embodiments, the Rb domain comprises a region of ApoE having at least 70% sequence identity (e.g., at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to residues 50-180 of SEQ ID NO: 13. In some embodiments, the Rb domain comprises a region of ApoE having at least 70% sequence identity (e.g., at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to residues 75-175 of SEQ ID NO: 13. In some embodiments, the Rb domain comprises a region of ApoE having at least 70% sequence identity (e.g., at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to residues 100-170 of SEQ ID NO: 13.In some embodiments, the Rb domain comprises a region of ApoE having at least 70% sequence identity (e.g., at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to residues 125-160 of SEQ ID NO: 13. In some embodiments, the Rb domain comprises a region of ApoE having at least 70% sequence identity (e.g., at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to residues 130-150 of SEQ ID NO: 13. In some embodiments, the Rb domain comprises a region of ApoE having at least 70% sequence identity (e.g., at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to residues 148-173 of SEQ ID NO:13 or a portion thereof including residues 159-167, or a variant thereof having at least 70% sequence identity (e.g., at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to residues 159-167 of SEQ ID NO:13. In some embodiments, the Rb domain comprises a region having at least 70% sequence identity (e.g., at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to the amino acid sequence of residues 159-167 of SEQ ID NO:13.

[0064] In some embodiments, the transgene encoding TREM2 further comprises a microRNA (miRNA) targeting sequence (e.g., a miR-126 targeting sequence). In some embodiments, the miRNA targeting sequence (e.g., a miR-126 targeting sequence) is located within the 3'-untranslated region (UTR) of the transgene.

[0065] In some embodiments, TREM2 penetrates the blood-brain barrier (BBB) ​​of a subject.

[0066] In some embodiments, the NCD is a TREM2-associated NCD. In some embodiments, the AD or PLOSL is TREM2-associated AD, PLOSL, FTLD, or PD.

[0067] In some embodiments, the subject suffering from TREM2-related AD or PLOSL has a mutation in the TREM2 gene. The mutation in the TREM2 gene can result in an amino acid substitution (e.g., p.R47H, p.R62H, p.T66M, p.T66M, p.Y38C, p.T96K, p.D87N, p.H157Y, p.R98W, p.T96K, p.D87N, p.L211P, p.R136Q, or p.N68K). In some embodiments, mutations in the TREM2 gene may result from single nucleotide substitutions or deletions (e.g., c.40G>T, c.C97>T, c.132G>A, c.267delGm c.313delG, c.377T>G, c.401A>G, c.482+2T>C, c.558GA).

[0068] In some embodiments, the subject suffering from TREM2-related AD, PLOSL, FTLD, or PD may have any other pathogenic mutation in the TREM2 gene that is known to have a causative role in AD, PLOSL, FTLD, or PD.For example, the pathogenic mutation in the TREM2 gene may be any of the mutations discussed in Guerreiro et al., The New England Journal of Medicine 368:117-27, (2013);Jonsson et al., The New England Journal of Medicine 368:107-16;Ulrich et al., Neuron Review 94:237-48 (2017);and Xing et al., Research and Reports in Biochemistry 5:89-100 (2015), the disclosures of which are incorporated herein by reference as they relate to AD-related and PLOSL-related human TREM2 mutations.

[0069] In some embodiments, the transgene encoding TREM2 comprises a polynucleotide encoding a wild-type human TREM2 polypeptide (e.g., any one of SEQ ID NOs: 1-3). In some embodiments, the transgene encoding TREM2 comprises a polynucleotide encoding a polypeptide having at least 85% sequence identity (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to a polypeptide having the amino acid sequence of any one of SEQ ID NOs: 1-3.

[0070] In some embodiments, the transgene encoding TREM2 comprises a polynucleotide encoding a polypeptide having at least 85% sequence identity (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to a polypeptide having the amino acid sequence of any one of SEQ ID NO:1. In some embodiments, the transgene encoding TREM2 comprises a polynucleotide encoding a polypeptide having at least 90% sequence identity (e.g., at least 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to a polypeptide having the amino acid sequence of any one of SEQ ID NO:1. In some embodiments, the transgene encoding TREM2 comprises a polynucleotide encoding a polypeptide having at least 95% sequence identity (e.g., at least 95%, 96%, 97%, 98%, 99% or more sequence identity) to a polypeptide having the amino acid sequence of any one of SEQ ID NO:1. In some embodiments, the transgene encoding TREM2 comprises a polynucleotide encoding a polypeptide having the amino acid sequence of SEQ ID NO:1.

[0071] In some embodiments, the transgene encoding TREM2 comprises a polynucleotide encoding a polypeptide having at least 85% sequence identity (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to a polypeptide having the amino acid sequence of any one of SEQ ID NO:2. In some embodiments, the transgene encoding TREM2 comprises a polynucleotide encoding a polypeptide having at least 90% sequence identity (e.g., at least 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to a polypeptide having the amino acid sequence of any one of SEQ ID NO:2. In some embodiments, the transgene encoding TREM2 comprises a polynucleotide encoding a polypeptide having at least 95% sequence identity (e.g., at least 95%, 96%, 97%, 98%, 99% or more sequence identity) to a polypeptide having the amino acid sequence of any one of SEQ ID NO:2. In some embodiments, the transgene encoding TREM2 comprises a polynucleotide encoding a polypeptide having the amino acid sequence of SEQ ID NO:2.

[0072] In some embodiments, the transgene encoding TREM2 comprises a polynucleotide encoding a polypeptide having 85% sequence identity (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to a polypeptide having the amino acid sequence of any one of SEQ ID NO:3. In some embodiments, the transgene encoding TREM2 comprises a polynucleotide encoding a polypeptide having at least 90% sequence identity (e.g., at least 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to a polypeptide having the amino acid sequence of any one of SEQ ID NO:3. In some embodiments, the transgene encoding TREM2 comprises a polynucleotide encoding a polypeptide having at least 95% sequence identity (e.g., at least 95%, 96%, 97%, 98%, 99% or more sequence identity) to a polypeptide having the amino acid sequence of any one of SEQ ID NO:3. In some embodiments, the transgene encoding TREM2 comprises a polynucleotide encoding a polypeptide having the amino acid sequence of SEQ ID NO:3.

[0073] In some embodiments, the transgene encoding TREM2 comprises a polynucleotide encoding a polypeptide including one or more amino acid substitutions, e.g., one or more conservative amino acid substitutions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid substitutions, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more conservative amino acid substitutions), compared to a polypeptide having the sequence of any one of SEQ ID NOs: 1-3.

[0074] In some embodiments, the transgene encoding TREM2 comprises a polynucleotide encoding a sTREM2 polypeptide. In some embodiments, the transgene encoding TREM2 comprises a polynucleotide encoding a TREM2-CTF polypeptide. In some embodiments, the transgene encoding TREM2 comprises a polynucleotide encoding a TREM2-ICD polypeptide. In some embodiments, the transgene encoding TREM2 comprises a polynucleotide encoding a TREM2-T2β polypeptide. In some embodiments, the transgene encoding TREM2 comprises a polynucleotide encoding a TREM2 polypeptide that does not include a functional ectodomain cleavage site. In some embodiments, the transgene encoding TREM2 comprises a polynucleotide encoding a TREM2 polypeptide that does not include a functional intramembrane cleavage site in the TREM2-CTF.

[0075] In some embodiments, the cell is a pluripotent cell. In some embodiments, the pluripotent cell is an ESC. In some embodiments, the pluripotent cell is an iPSC. In some embodiments, the cell is a CD34+ cell. In some embodiments, the cell is a pluripotent cell. In some embodiments, the pluripotent cell is a CD34+ cell. In some embodiments, the CD34+ cell is a hematopoietic stem cell. In some embodiments, the CD34+ cell is a myeloid progenitor cell. In some embodiments, the cell is a blood lineage progenitor cell (BLPC). In some embodiments, the BLPC is a monocyte. In some embodiments, the cell is a macrophage. In some embodiments, the cell is a microglial progenitor cell. In some embodiments, the cell is a microglia.

[0076] In some embodiments, the subject's endogenous population of microglia is ablated prior to administering the composition to the subject. In some embodiments, the method includes ablating the subject's endogenous population of microglia prior to administering the composition to the subject. In some embodiments, microglia are ablated using an agent selected from the group consisting of busulfan, PLX3397, PLX647, PLX5622, treosulfan, and clodronate liposomes, by radiation therapy, or a combination thereof.

[0077] In some embodiments, the composition is administered to the subject systemically. In some embodiments, the composition is administered to the subject by intravenous injection. In some embodiments, the composition is administered directly to the central nervous system of the subject. In some embodiments, the composition is administered to the cerebrospinal fluid of the subject. For example, the composition can be administered to the subject by intraventricular injection, intrathecal injection, stereotactic injection, or a combination thereof. In some embodiments, the composition is administered to the subject by intraparenchymal injection.

[0078] In some embodiments, the composition is administered to the subject via bone marrow transplant, hi some embodiments, the composition is administered directly to the subject's bone marrow, for example, by intraosseous injection.

[0079] In some embodiments, the composition is administered to the subject by intraventricular injection. In some embodiments, the composition is administered to the subject by intravenous injection.

[0080] In some embodiments, the composition is administered to the subject by direct administration to the subject's central nervous system and by systemic administration. In some embodiments, the composition is administered to the subject by intraventricular and intravenous injection. In some embodiments, the composition is administered to the subject by intrathecal and intravenous injection. In some embodiments, the composition is administered to the subject by intraparenchymal and intravenous injection.

[0081] In some embodiments, the subject has been diagnosed with an NCD. In some embodiments, the NCD is a severe NCD. In some embodiments, the severe NCD interferes with the subject's independence and / or normal daily functioning (e.g., social, occupational, or academic functioning, personal hygiene, grooming, dressing, toilet hygiene, functional mobility (e.g., ability to walk, get in and out of bed), and self-care). In some embodiments, the severe NCD is associated with a score obtained by the subject on a cognitive test that is at least 2 standard deviations away from the mean score of a reference population. In some embodiments, the NCD is a mild NCD. In some embodiments, the mild NCD does not interfere with the subject's independence and / or normal daily functioning. In some embodiments, the mild NCD is associated with a score obtained by the subject on a cognitive test that is at least 1-2 standard deviations away from the mean score of a reference population. In some embodiments, the cognitive test is selected from the group consisting of AD8, AWV, GPCOG, HRA, MIS, MMSE, MoCA, SLUMS, and Short IQCODE. In some embodiments, the NCD is associated with impairment in one or more of complex attention, executive function, learning and memory, language, sensorimotor function, and social cognition. In some embodiments, the NCD is not due to delirium or other psychiatric disorder (e.g., schizophrenia, bipolar disorder, or major depression). In some embodiments, the reference population is the general population. In some embodiments, the reference population is selected based on the subject's age, medical history, education, socioeconomic status, and lifestyle. In some embodiments, the NCD is AD. In some embodiments, the NCD is a leukodystrophy. In some embodiments, the leukodystrophy is PLOSL. In some embodiments, the NCD is a frontotemporal NCD. In some embodiments, the frontotemporal NCD is FTLD. In some embodiments, the NCD is a movement disorder. In some embodiments, the movement disorder is PD.

[0082] In some embodiments, the method includes administering a population of cells to a subject. In some embodiments, the population of cells is administered to a subject prior to administration of the composition. In some embodiments, the population of cells is administered to a subject after administration of the composition. In some embodiments, the cells are selected from the group consisting of ESCs, iPSCs, CD34+ cells, HSCs, MPCs, BLPCs, microglial progenitor cells, monocytes, macrophages, and microglia. In some embodiments, the cells are not modified to express a transgene encoding TREM2. In some embodiments, the cells are administered systemically to a subject. In some embodiments, the cells are administered to a subject by intravenous injection.

[0083] In some embodiments, endogenous TREM2 is disrupted in cells prior to administration of the composition to a subject.

[0084] In some embodiments, the cell is contacted with a nuclease that catalyzes the cleavage of endogenous TREM2 nucleic acid in the cell to destroy endogenous TREM2.In some embodiments, the nuclease is a CRISPR-associated protein.In some embodiments, the CRISPR-associated protein is CRISPR-associated protein 9.In some embodiments, the CRISPR-associated protein is CRISPR-associated protein 12a.In some embodiments, the nuclease is a transcription activator-like effector nuclease, a meganuclease, or a zinc finger nuclease.

[0085] In some embodiments, endogenous TREM2 is disrupted, for example, by contacting the cell with an inhibitory RNA molecule for a time and in an amount sufficient to disrupt expression of endogenous TREM2. In some embodiments, the inhibitory RNA molecule is a short interfering RNA (siRNA), a short hairpin RNA (shRNA), or a miRNA.

[0086] In some embodiments, endogenous TREM2 is disrupted in a subject prior to administering the composition to the subject. In some embodiments, endogenous TREM2 is disrupted by administering an inhibitory RNA molecule to the subject. In some embodiments, the inhibitory RNA molecule is an siRNA, shRNA, or miRNA. In some embodiments, endogenous TREM2 is disrupted in a population of neurons of a subject prior to administering the composition to the subject. In some embodiments, endogenous TREM2 is disrupted in a population of neurons, for example, by contacting the population of neurons with an inhibitory RNA molecule for a time and amount sufficient to disrupt expression of endogenous TREM2. In some embodiments, the inhibitory RNA molecule is an siRNA, shRNA, or miRNA.

[0087] In some embodiments, the cells are autologous cells. In some embodiments, the cells are allogeneic cells.

[0088] In some embodiments, cells are transduced ex vivo to express TREM2.

[0089] In some embodiments, the cells are transduced with a viral vector selected from the group including adeno-associated virus (AAV), adenovirus, parvovirus, coronavirus, rhabdovirus, paramyxovirus, picornavirus, alphavirus, herpesvirus, poxvirus, and retroviridae viruses.

[0090] In some embodiments, the viral vector is a Retroviridae viral vector. In some embodiments, the Retroviridae viral vector is a lentiviral vector. In some embodiments, the Retroviridae viral vector is an alpharetroviral vector. In some embodiments, the Retroviridae viral vector is a gammaretroviral vector. In some embodiments, the Retroviridae viral vector comprises a central polypurine tract, a woodchuck hepatitis virus post-transcriptional regulatory element, a 5'-LTR, an HIV signal sequence, an HIV Psi signal 5'-splice site, a delta-GAG element, a 3'-splice site, and a 3'-self-inactivating LTR.

[0091] In some embodiments, the viral vector is an AAV selected from the group including AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAVS, AAV9, AAV10, and AAVrh74.

[0092] In some embodiments, the viral vector is a pseudotyped viral vector. In some embodiments, the viral vector is a pseudotyped AAV, pseudotyped adenovirus, pseudotyped parvovirus, pseudotyped coronavirus, pseudotyped rhabdovirus, pseudotyped paramyxovirus, pseudotyped picornavirus, pseudotyped alphavirus, pseudotyped herpesvirus, pseudotyped poxvirus, and pseudotyped retroviridae virus.

[0093] In some embodiments, cells are transfected ex vivo to express TREM2.

[0094] In some embodiments, the cells are transfected using an agent selected from the group including cationic polymers, diethylaminoethyl-dextran, polyethyleneimine, cationic lipids, liposomes, calcium phosphate, activated dendrimers, and magnetic beads; or a technique selected from the group including electroporation, Nucleofection, squeeze-poration, sonoporation, optical transfection, Magnetofection, and impalefection.

[0095] In some embodiments, the expression of TREM2 in cells is mediated using a ubiquitous promoter. Exemplary ubiquitous promoters are elongation factor 1-alpha promoter and phosphoglycerate kinase 1 promoter. In some embodiments, the expression of TREM2 in cells is mediated using a cell lineage-specific promoter. Exemplary cell lineage-specific promoters are CD68 promoter, CD11b promoter, C-X3-C motif chemokine receptor 1 promoter, allograft inflammatory factor 1 promoter, purinergic receptor P2Y12 promoter, transmembrane protein 119 promoter, and colony stimulating factor 1 receptor promoter. In some embodiments, the expression of TREM2 in cells is mediated using a synthetic promoter.

[0096] In some embodiments, the composition is administered to a subject in an amount sufficient to increase the amount of M2 microglia in the subject's brain relative to the amount of M1 microglia in the subject's brain, reduce the level of one or more pro-inflammatory cytokines in the subject's brain, increase the level of one or more anti-inflammatory cytokines in the subject's brain, improve the cognitive processing ability of the subject, improve motor function of the subject, reduce neuronal loss in the subject, and / or reduce levels of amyloid-beta and neurofibrillary tau protein or aggregates thereof in the subject.

[0097] In some embodiments, the subject is a human.

[0098] In another aspect, the disclosure provides a composition containing a population of cells expressing a transgene encoding TREM2.

[0099] In some embodiments of the preceding aspects, the cell is a pluripotent cell. In some embodiments, the pluripotent cell is an ESC. In some embodiments, the pluripotent cell is an iPSC. In some embodiments, the cell is a CD34+ cell. In some embodiments, the cell is a pluripotent cell. In some embodiments, the pluripotent cell is a CD34+ cell. In some embodiments, the CD34+ cell is a hematopoietic stem cell. In some embodiments, the CD34+ cell is a myeloid progenitor cell. In some embodiments, the cell is a blood lineage progenitor cell (BLPC). In some embodiments, the BLPC is a monocyte. In some embodiments, the cell is a macrophage. In some embodiments, the cell is a microglia.

[0100] In some embodiments, cells are transduced ex vivo to express TREM2. In some embodiments, cells are transfected ex vivo to express TREM2.

[0101] In some embodiments, the TREM2 is a full-length TREM2, e.g., a TREM2 having the amino acid sequence of any one of SEQ ID NOs:1-3, or a variant thereof having at least 85% sequence identity thereto (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto).

[0102] In some embodiments, TREM2 has the amino acid sequence of SEQ ID NO:1 or a variant thereof having at least 85% (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID NO:1.

[0103] In some embodiments, TREM2 has the amino acid sequence of SEQ ID NO:1 or a variant thereof having at least 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO:1.

[0104] In some embodiments, TREM2 has the amino acid sequence of SEQ ID NO:1 or a variant thereof having at least 95% (e.g., at least 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO:1.

[0105] In some embodiments, TREM2 has the amino acid sequence of SEQ ID NO:1.

[0106] In some embodiments, TREM2 has the amino acid sequence of SEQ ID NO:2 or a variant thereof having at least 85% (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID NO:2.

[0107] In some embodiments, TREM2 has the amino acid sequence of SEQ ID NO:2 or a variant thereof having at least 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO:2.

[0108] In some embodiments, TREM2 has the amino acid sequence of SEQ ID NO:2 or a variant thereof having at least 95% (e.g., at least 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO:2.

[0109] In some embodiments, TREM2 has the amino acid sequence of SEQ ID NO:2.

[0110] In some embodiments, TREM2 has the amino acid sequence of SEQ ID NO:3 or a variant thereof having at least 85% (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID NO:3.

[0111] In some embodiments, TREM2 has the amino acid sequence of SEQ ID NO:3 or a variant thereof having at least 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO:3.

[0112] In some embodiments, TREM2 has the amino acid sequence of SEQ ID NO:3 or a variant thereof having at least 95% (e.g., at least 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO:3.

[0113] In some embodiments, TREM2 has the amino acid sequence of SEQ ID NO:3.

[0114] In some embodiments, the transgene encoding TREM2 comprises a TREM2 polynucleotide having the nucleotide sequence of SEQ ID NO:4, or a variant thereof having at least 85% (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO:4.

[0115] In some embodiments, the transgene encoding TREM2 comprises a TREM2 polynucleotide having the nucleotide sequence of SEQ ID NO:4, or a variant thereof having at least 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO:4.

[0116] In some embodiments, the transgene encoding TREM2 comprises a TREM2 polynucleotide having the nucleotide sequence of SEQ ID NO:4, or a variant thereof having at least 95% (e.g., at least 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO:4.

[0117] In some embodiments, the transgene encoding TREM2 comprises a TREM2 polynucleotide having the nucleotide sequence of SEQ ID NO:4.

[0118] In some embodiments, the transgene encoding TREM2 comprises a TREM2 polynucleotide having the nucleotide sequence of SEQ ID NO:5, or a variant thereof having at least 85% (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO:5.

[0119] In some embodiments, the transgene encoding TREM2 comprises a TREM2 polynucleotide having the nucleotide sequence of SEQ ID NO:5, or a variant thereof having at least 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO:5.

[0120] In some embodiments, the transgene encoding TREM2 comprises a TREM2 polynucleotide having the nucleotide sequence of SEQ ID NO:5, or a variant thereof having at least 95% (e.g., at least 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO:5.

[0121] In some embodiments, the transgene encoding TREM2 comprises a TREM2 polynucleotide having the nucleotide sequence of SEQ ID NO:5.

[0122] In some embodiments, the transgene encoding TREM2 comprises a TREM2 polynucleotide having the nucleotide sequence of SEQ ID NO:6, or a variant thereof having at least 85% (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO:6.

[0123] In some embodiments, the transgene encoding TREM2 comprises a TREM2 polynucleotide having the nucleotide sequence of SEQ ID NO:6, or a variant thereof having at least 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO:6.

[0124] In some embodiments, the transgene encoding TREM2 comprises a TREM2 polynucleotide having the nucleotide sequence of SEQ ID NO:6, or a variant thereof having at least 95% (e.g., at least 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO:6.

[0125] In some embodiments, the transgene encoding TREM2 comprises a TREM2 polynucleotide having the nucleotide sequence of SEQ ID NO:6.

[0126] In some embodiments, the transgene encoding TREM2 comprises a TREM2 polynucleotide having the nucleotide sequence of SEQ ID NO:7, or a variant thereof having at least 85% (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID NO:7.

[0127] In some embodiments, the transgene encoding TREM2 comprises a TREM2 polynucleotide having the nucleotide sequence of SEQ ID NO:7, or a variant thereof having at least 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO:7.

[0128] In some embodiments, the transgene encoding TREM2 comprises a TREM2 polynucleotide having the nucleotide sequence of SEQ ID NO:7, or a variant thereof having at least 95% (e.g., at least 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO:7.

[0129] In some embodiments, the transgene encoding TREM2 comprises a TREM2 polynucleotide having the nucleotide sequence of SEQ ID NO:7.

[0130] In some embodiments, the transgene encoding TREM2 comprises a TREM2 polynucleotide having the nucleotide sequence of SEQ ID NO:9, or a variant thereof having at least 85% (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID NO:9.

[0131] In some embodiments, the transgene encoding TREM2 comprises a TREM2 polynucleotide having the nucleotide sequence of SEQ ID NO:9, or a variant thereof having at least 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO:9.

[0132] In some embodiments, the transgene encoding TREM2 comprises a TREM2 polynucleotide having the nucleotide sequence of SEQ ID NO:9, or a variant thereof having at least 95% (e.g., at least 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO:9.

[0133] In some embodiments, the transgene encoding TREM2 comprises a TREM2 polynucleotide having the nucleotide sequence of SEQ ID NO:9.

[0134] In some embodiments, the transgene encoding TREM2 comprises a TREM2 polynucleotide having the nucleotide sequence of SEQ ID NO:11, or a variant thereof having at least 85% (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO:11.

[0135] In some embodiments, the transgene encoding TREM2 comprises a TREM2 polynucleotide having the nucleotide sequence of SEQ ID NO:11, or a variant thereof having at least 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO:11.

[0136] In some embodiments, the transgene encoding TREM2 comprises a TREM2 polynucleotide having the nucleotide sequence of SEQ ID NO:11, or a variant thereof having at least 95% (e.g., at least 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO:11.

[0137] In some embodiments, the transgene encoding TREM2 comprises a TREM2 polynucleotide having the nucleotide sequence of SEQ ID NO:11.

[0138] In some embodiments, the transgene encoding TREM2 may be codon optimized (eg, any one of SEQ ID NO:8, SEQ ID NO:10, or SEQ ID NO:12).

[0139] In some embodiments, the transgene encoding TREM2 comprises a codon-optimized TREM2 polynucleotide sequence of SEQ ID NO:8 or a variant thereof having at least 85% (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO:8.

[0140] In some embodiments, the transgene encoding TREM2 comprises a codon-optimized TREM2 polynucleotide sequence of SEQ ID NO:8 or a variant thereof having at least 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO:8.

[0141] In some embodiments, the transgene encoding TREM2 comprises a codon-optimized TREM2 polynucleotide sequence of SEQ ID NO:8 or a variant thereof having at least 95% (e.g., at least 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO:8.

[0142] In some embodiments, the transgene encoding TREM2 comprises the codon-optimized TREM2 polynucleotide sequence of SEQ ID NO:8.

[0143] In some embodiments, the transgene encoding TREM2 comprises a codon-optimized TREM2 polynucleotide sequence of SEQ ID NO:10, or a variant thereof having at least 85% (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO:10.

[0144] In some embodiments, the transgene encoding TREM2 comprises a codon-optimized TREM2 polynucleotide sequence of SEQ ID NO:10, or a variant thereof having at least 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO:10.

[0145] In some embodiments, the transgene encoding TREM2 comprises a codon-optimized TREM2 polynucleotide sequence of SEQ ID NO:10, or a variant thereof having at least 95% (e.g., at least 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO:10.

[0146] In some embodiments, the transgene encoding TREM2 comprises the codon-optimized TREM2 polynucleotide sequence of SEQ ID NO:10.

[0147] In some embodiments, the transgene encoding TREM2 comprises a codon-optimized TREM2 polynucleotide sequence of SEQ ID NO:12 or a variant thereof having at least 85% (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO:12.

[0148] In some embodiments, the transgene encoding TREM2 comprises a codon-optimized TREM2 polynucleotide sequence of SEQ ID NO:12 or a variant thereof having at least 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO:12.

[0149] In some embodiments, the transgene encoding TREM2 comprises a codon-optimized TREM2 polynucleotide sequence of SEQ ID NO:12 or a variant thereof having at least 95% (e.g., at least 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO:12.

[0150] In some embodiments, the transgene encoding TREM2 comprises the codon-optimized TREM2 polynucleotide sequence of SEQ ID NO:12.

[0151] In some embodiments, the transgene encodes two or more TREM2 proteins (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or more TREM2 proteins). In some embodiments, the transgene encodes between 2 and 10 TREM2 proteins (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 TREM2 proteins). In some embodiments, the transgene encodes between 2 and 5 TREM2 proteins (e.g., 2, 3, 4, or 5 TREM2 proteins). In some embodiments, the transgene encodes two TREM2 proteins. In some embodiments, the TREM2 transgenes are expressed from a single polycistronic expression cassette. In some embodiments, the TREM2 transgenes are separated from each other by one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or more) IRESs. In some embodiments, the TREM2 transgene is expressed from one or more (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) monocistronic expression cassettes.

[0152] In some embodiments, the TREM2 comprises a signal peptide, hi some embodiments, the signal peptide is a TREM2 signal peptide.

[0153] In some embodiments, TREM2 is sTREM2. In some embodiments, TREM2 is a TREM-CTF. In some embodiments, TREM2 is a TREM2-ICD. In some embodiments, TREM2 is a TREM2-T2β peptide. In some embodiments, TREM2 does not contain a functional ectodomain cleavage site. In some embodiments, TREM2 does not contain a functional intramembrane cleavage site within the TREM2-CTF.

[0154] In some embodiments, the TREM2 is a TREM2 fusion protein. In some embodiments, the TREM2 fusion protein comprises the Rb domain of ApoE. In some embodiments, the Rb domain comprises a portion of ApoE having an amino acid sequence of residues 25-185, 50-180, 75-175, 100-170, 125-160, or 130-150 of SEQ ID NO:13. In some embodiments, the Rb domain comprises a region having at least 70% sequence identity to the amino acid sequence of residues 159-167 of SEQ ID NO:13.

[0155] In some embodiments, the transgene encoding TREM2 further comprises a miRNA targeting sequence in the 3'-UTR. In some embodiments, the miRNA targeting sequence is a miR-126 targeting sequence.

[0156] In some embodiments, endogenous TREM2 is disrupted in the cell.

[0157] In some embodiments, the composition is formulated for systemic administration to a subject. In some embodiments, the composition is formulated for administration to a subject by intravenous injection. In some embodiments, the composition is formulated for administration to the cerebrospinal fluid of a subject. In some embodiments, the composition is formulated for administration to a subject by intraventricular injection, intrathecal, stereotactic injection, or a combination thereof. In some embodiments, the composition is formulated for administration by intraparenchymal injection. In some embodiments, the composition is formulated for administration directly to the bone marrow of a subject. In some embodiments, the composition is formulated for administration to a subject by intraosseous injection. In some embodiments, the composition is formulated for administration to a subject by a bone marrow transplant comprising the composition. In some embodiments, the composition is formulated for administration to a subject by intraventricular and intravenous injection.

[0158] In another aspect, the present disclosure provides a pharmaceutical composition comprising a composition according to any of the above aspects and embodiments, further comprising one or more pharma- ceutically acceptable carriers, diluents, or excipients.

[0159] In an additional aspect, the present disclosure provides a kit comprising a composition according to any of the above aspects and embodiments and a package insert, in some embodiments, the package insert instructs a user of the kit to carry out a method according to any of the above aspects and embodiments.

[0160] Additional embodiments of the present invention are described in the paragraphs listed below.

[0161] E1. 1. A method of treating a subject diagnosed with a neurocognitive disorder (NCD), comprising administering to the subject a composition comprising a population of cells (e.g., pluripotent cells, ESCs, iPSCs, pluripotent cells, CD34+ cells, HSCs, MPCs, BLPCs, microglial progenitor cells, monocytes, macrophages, or microglia) that comprises a transgene encoding a triggering receptor expressed in one or more myeloid cells (TREM2) protein having an amino acid sequence that is at least 85% identical to the amino acid sequence of any one of SEQ ID NOs: 1-3.

[0162] E2. The method according to E1, wherein said NCD is a severe NCD.

[0163] E3. The method of E2, wherein said severe NCD interferes with said subject's independence and / or normal daily functioning.

[0164] E4. The method of E2 or E3, wherein said severe NCD is associated with a score obtained by said subject on a cognitive test that is at least 2 standard deviations away from the mean score of a reference population.

[0165] E5. The method according to E1, wherein said NCD is a mild NCD.

[0166] E6. The method of E5, wherein said mild NCD does not interfere with said subject's independence and / or normal daily functioning.

[0167] E7. The method of E5 or E6, wherein said mild NCD is associated with a score obtained by said subject on a cognitive test that is 1 to 2 standard deviations away from the mean score of a reference population.

[0168] E8. The method of E4 or E7, wherein the reference population is the general population.

[0169] E9. The method of E4, E7, or E8, wherein the cognitive test is selected from the group consisting of Eight-item Informant Interview to Differentiate Aging and Dementia (AD8), Annual Wellness Visit (AWV), General Practitioner Assessment of Cognition (GPCOG), Health Risk Assessment (HRA), Memory Impairment Screen (MIS), Mini Mental Status Exam (MMSE), Montreal Cognitive Assessment (MoCA), St. Louis University Mental Status Exam (SLUMS), and Short Informant Questionnaire on Cognitive Decline in the Elderly (Short IQCODE).

[0170] E10. The method of any one of E1-E9, wherein the NCD is associated with impairment in one or more of complex attention, executive function, learning and memory, language, sensorimotor function, and social cognition.

[0171] E11. The method of any one of E1-E10, wherein said NCD is not due to delirium or other psychiatric disorder.

[0172] E12. The method of any one of E1 to E11, wherein said NCD is Alzheimer's disease (AD).

[0173] E13. The method according to any one of E1 to E11, wherein said NCD is a leukodystrophy.

[0174] E14. The method of E13, wherein said leukodystrophy is Nasu-Hakola disease (PLOSL).

[0175] E15. The method according to any one of E1 to E14, wherein the transgene comprises a polynucleotide encoding a TREM2 protein having an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO:1.

[0176] E16. The method of E15, wherein the transgene comprises a polynucleotide encoding a TREM2 protein having an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:1.

[0177] E17. The method of E15, wherein the transgene comprises a polynucleotide encoding a TREM2 protein having an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:1.

[0178] E18. The method of E17, wherein the transgene comprises a polynucleotide encoding a TREM2 protein having the amino acid sequence of SEQ ID NO:1.

[0179] E19. The method according to any one of E1 to E18, wherein the transgene comprises a polynucleotide encoding a TREM2 protein having an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO:2.

[0180] E20. The method of E19, wherein the transgene comprises a polynucleotide encoding a TREM2 protein having an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:2.

[0181] E21. The method of E20, wherein the transgene comprises a polynucleotide encoding a TREM2 protein having an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:2.

[0182] E22. The method of E21, wherein the transgene comprises a polynucleotide encoding a TREM2 protein having the amino acid sequence of SEQ ID NO:2.

[0183] E23. The method according to any one of E1 to E22, wherein the transgene comprises a polynucleotide encoding a TREM2 protein having an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO:3.

[0184] E24. The method of E23, wherein the transgene comprises a polynucleotide encoding a TREM2 protein having an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:3.

[0185] E25. The method of E24, wherein the transgene comprises a polynucleotide encoding a TREM2 protein having an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:3.

[0186] E26. The method of E25, wherein the transgene comprises a polynucleotide encoding a TREM2 protein having the amino acid sequence of SEQ ID NO:3.

[0187] E27. The method of any one of E1 to E26, wherein said TREM2 is full-length TREM2.

[0188] E28. The method according to any one of E1 to E27, wherein the TREM2 comprises a signal peptide.

[0189] E29. The method of E28, wherein the signal peptide is a TREM2 signal peptide.

[0190] E30. The method according to any one of E1 to E29, wherein the TREM2 is soluble TREM2 (sTREM2).

[0191] E31. The method according to any one of E1 to E29, wherein the TREM2 is a TREM2 C-terminal fragment (TREM2-CTF).

[0192] E32. The method according to any one of E1 to E29, wherein the TREM2 is the TREM2 intracellular domain (TREM2-ICD).

[0193] E33. The method according to any one of E1 to E29, wherein said TREM2 is a TREM2-A β-like (TREM2-T2β) peptide.

[0194] E34. The method according to any one of E1 to E33, wherein said TREM2 does not contain a functional ectodomain cleavage site.

[0195] E35. The method of E31, wherein the TREM2-CTF does not contain a functional intramembrane cleavage site.

[0196] E36. The method of any one of E1 to E35, wherein the transgene comprises a polynucleotide encoding two or more TREM2 proteins.

[0197] E37. The method of E36, wherein the transgene comprises a polynucleotide encoding 2 to 10 TREM2 proteins.

[0198] E38. The method of E37, wherein the transgene comprises a polynucleotide encoding two to five TREM2 proteins.

[0199] E39. The method of E38, wherein the transgene comprises a polynucleotide encoding two TREM2 proteins.

[0200] E40. The method of any one of E36 to E39, wherein said TREM2 transgene is expressed from a single polycistronic expression cassette.

[0201] E41. The method according to any one of E36 to E40, wherein the TREM2 transgenes are separated from each other by one or more internal ribosome entry sites (IRES).

[0202] E42. The method of any one of E36 to E39, wherein said TREM2 transgene is expressed from one or more monocistronic expression cassettes.

[0203] E43. The method of any one of E1 to E42, wherein said transgene comprises a polynucleotide having at least 85% sequence identity to the nucleic acid sequence of SEQ ID NO:4.

[0204] E44. The method of E43, wherein the transgene comprises a polynucleotide having at least 90% sequence identity to the nucleic acid sequence of SEQ ID NO:4.

[0205] E45. The method of E44, wherein the transgene comprises a polynucleotide having at least 95% sequence identity to the nucleic acid sequence of SEQ ID NO:4.

[0206] E46. The method of E45, wherein the transgene comprises a polynucleotide having the nucleic acid sequence of SEQ ID NO:4.

[0207] E47. The method of any one of E1 to E46, wherein the transgene comprises a polynucleotide having at least 85% sequence identity to the nucleic acid sequence of SEQ ID NO:5.

[0208] E48. The method of E47, wherein the transgene comprises a polynucleotide having at least 90% sequence identity to the nucleic acid sequence of SEQ ID NO:5.

[0209] E49. The method of E48, wherein the transgene comprises a polynucleotide having at least 95% sequence identity to the nucleic acid sequence of SEQ ID NO:5.

[0210] E50. The method of E49, wherein the transgene comprises a polynucleotide having the nucleic acid sequence of SEQ ID NO:5.

[0211] E51. The method of any one of E1 to E50, wherein the transgene comprises a polynucleotide having at least 85% sequence identity to the nucleic acid sequence of SEQ ID NO:6.

[0212] E52. The method of E51, wherein the transgene comprises a polynucleotide having at least 90% sequence identity to the nucleic acid sequence of SEQ ID NO:6.

[0213] E53. The method of E52, wherein the transgene comprises a polynucleotide having at least 95% sequence identity to the nucleic acid sequence of SEQ ID NO:6.

[0214] E54. The method of E53, wherein the transgene comprises a polynucleotide having the nucleic acid sequence of SEQ ID NO:6.

[0215] E55. The method of any one of E1 to E54, wherein the transgene comprises a polynucleotide having at least 85% sequence identity to the nucleic acid sequence of SEQ ID NO:7.

[0216] E56. The method of E55, wherein the transgene comprises a polynucleotide having at least 90% sequence identity to the nucleic acid sequence of SEQ ID NO:7.

[0217] E57. The method of E56, wherein the transgene comprises a polynucleotide having at least 95% sequence identity to the nucleic acid sequence of SEQ ID NO:7.

[0218] E58. The method of E57, wherein the transgene comprises a polynucleotide having the nucleic acid sequence of SEQ ID NO:7.

[0219] E59. The method of any one of E1 to E58, wherein said transgene comprises a polynucleotide having at least 85% sequence identity to the nucleic acid sequence of SEQ ID NO:9.

[0220] E60. The method of E59, wherein the transgene comprises a polynucleotide having at least 90% sequence identity to the nucleic acid sequence of SEQ ID NO:9.

[0221] E61. The method of E60, wherein the transgene comprises a polynucleotide having at least 95% sequence identity to the nucleic acid sequence of SEQ ID NO:9.

[0222] E62. The method of E61, wherein the transgene comprises a polynucleotide having the nucleic acid sequence of SEQ ID NO:9.

[0223] E63. The method of any one of E1 to E62, wherein said transgene comprises a polynucleotide having at least 85% sequence identity to the nucleic acid sequence of SEQ ID NO:11.

[0224] E64. The method of E63, wherein the transgene comprises a polynucleotide having at least 90% sequence identity to the nucleic acid sequence of SEQ ID NO:11.

[0225] E65. The method of E64, wherein the transgene comprises a polynucleotide having at least 95% sequence identity to the nucleic acid sequence of SEQ ID NO:11.

[0226] E66. The method of E65, wherein the transgene comprises a polynucleotide having the nucleic acid sequence of SEQ ID NO:11.

[0227] E67. The method of any one of E1 to E66, wherein said transgene is a codon-optimized TREM2 transgene.

[0228] E68. The method of E67, wherein the codon-optimized TREM2 transgene comprises a polynucleotide having at least 85% sequence identity to the nucleic acid sequence of SEQ ID NO:8.

[0229] E69. The method of E68, wherein the codon-optimized TREM2 transgene comprises a polynucleotide having at least 90% sequence identity to the nucleic acid sequence of SEQ ID NO:8.

[0230] E70. The method of E69, wherein the codon-optimized TREM2 transgene comprises a polynucleotide having at least 95% sequence identity to the nucleic acid sequence of SEQ ID NO:8.

[0231] E71. The method of E70, wherein the codon-optimized TREM2 transgene comprises the polynucleotide of SEQ ID NO:8.

[0232] E72. The method of any one of E67 to E71, wherein the codon-optimized TREM2 transgene comprises a polynucleotide having at least 85% sequence identity to the nucleic acid sequence of SEQ ID NO:10.

[0233] E73. The method of E72, wherein the codon-optimized TREM2 transgene comprises a polynucleotide having at least 90% sequence identity to the nucleic acid sequence of SEQ ID NO:10.

[0234] E74. The method of E73, wherein the codon-optimized TREM2 transgene comprises a polynucleotide having at least 95% sequence identity to the nucleic acid sequence of SEQ ID NO:10.

[0235] E75. The method of E74, wherein the codon-optimized TREM2 transgene comprises the polynucleotide of SEQ ID NO:10.

[0236] E76. The method of any one of E67 to E75, wherein the codon-optimized TREM2 transgene comprises a polynucleotide having at least 85% sequence identity to the nucleic acid sequence of SEQ ID NO:12.

[0237] E77. The method of E76, wherein the codon-optimized TREM2 transgene comprises a polynucleotide having at least 90% sequence identity to the nucleic acid sequence of SEQ ID NO:12.

[0238] E78. The method of E77, wherein the codon-optimized TREM2 transgene comprises a polynucleotide having at least 95% sequence identity to the nucleic acid sequence of SEQ ID NO:12.

[0239] E79. The method of E78, wherein the codon-optimized TREM2 transgene comprises the polynucleotide of SEQ ID NO:12.

[0240] E80. The method of any one of E1 to E79, wherein the TREM2 is a TREM2 fusion protein.

[0241] E81. The method of E80, wherein the TREM2 fusion protein comprises the receptor binding (Rb) domain of apolipoprotein E (ApoE).

[0242] E82. The method of E81, wherein the Rb domain comprises a portion of ApoE having an amino acid sequence of residues 25-185, 50-180, 75-175, 100-170, 125-160, or 130-150 of SEQ ID NO:13.

[0243] E83. The method of E81 or 82, wherein the Rb domain comprises a region having at least 70% sequence identity to the amino acid sequence of residues 159 to 167 of SEQ ID NO:13.

[0244] E84. The method of any one of E1 to E83, wherein said transgene encoding TREM2 further comprises a microRNA (miRNA) targeting sequence in the 3'-UTR.

[0245] E85. The method of E84, wherein the miRNA targeting sequence is a miR-126 targeting sequence.

[0246] E86. The method of any one of E1 to E85, wherein upon administration of the composition to the subject, the TREM2 penetrates the blood-brain barrier of the subject.

[0247] E87. The method according to any one of E12 to E86, wherein the AD or PLOSL is TREM2-related AD or PLOSL.

[0248] E88. The method according to any one of E1 to E87, wherein the cell is an ESC.

[0249] E89. The method according to any one of E1 to E87, wherein the cell is an iPSC.

[0250] E90. The method according to any one of E1 to E87, wherein said cells are CD34+ cells.

[0251] E91. The method of E90, wherein the CD34+ cells are HSCs.

[0252] E92. The method of E90, wherein the CD34+ cells are MPCs.

[0253] E93. The method of any one of E1 to E92, wherein a population of endogenous microglia is depleted in said subject prior to administration of said composition.

[0254] E94. The method of any one of E1 to E92, comprising removing a population of endogenous microglia in the subject prior to administering the composition to the subject.

[0255] E95. The method of E93 or E94, wherein the microglia are ablated using an agent selected from the group consisting of busulfan, PLX3397, PLX647, PLX5622, treosulfan, and clodronate liposomes, by radiation therapy, or a combination thereof.

[0256] E96. The method of any one of E1 to E95, wherein the composition is systemically administered to the subject.

[0257] E97. The method of E96, wherein the composition is administered to the subject by intravenous injection.

[0258] E98. The method of any one of E1 to E95, wherein the composition is administered directly into the central nervous system of the subject.

[0259] E99. The method of E98, wherein the composition is administered to the subject by direct administration into cerebrospinal fluid.

[0260] E100. The method of E99, wherein the composition is administered to the subject by intraventricular injection, intrathecal injection, stereotactic injection, or a combination thereof.

[0261] E101. The method of E98, wherein the composition is administered to the subject by intraparenchymal injection.

[0262] E102. The method of any one of E1 to E95, wherein said composition is administered directly to the bone marrow of said subject.

[0263] E103. The method of E102, wherein the composition is administered to the subject by intraosseous injection.

[0264] E104. The method of any one of E1 to E95, wherein the composition is administered to the subject by bone marrow transplantation comprising the composition.

[0265] E105. The method of any one of E1 to E95, wherein the composition is administered to the subject by intraventricular injection.

[0266] E106. The method of any one of E1 to E95, wherein the composition is administered to the subject by intrathecal injection.

[0267] E107. The method of any one of E1-95, wherein the composition is administered to the subject by intraparenchymal injection.

[0268] E108. The method of any one of E1 to E95, wherein the composition is administered to the subject by intravenous injection.

[0269] E109. The method of any one of E1 to E95, wherein the composition is administered to the subject by direct administration to the central nervous system of the subject and by systemic administration.

[0270] E110. The method of E109, wherein the composition is administered to the subject by intraventricular and intravenous injection.

[0271] E111. The method of E109, wherein the composition is administered to the subject by intrathecal and intravenous injection.

[0272] E112. The method according to E109, wherein the composition is administered to the subject by intraparenchymal and intravenous injection.

[0273] E113. The method of any one of E1 to E112, further comprising administering to said subject the population of cells.

[0274] E114. The method of E105, wherein the population of cells is administered to the subject prior to administration of the composition.

[0275] E115. The method of E113, wherein the population of cells is administered to the subject after administration of the composition.

[0276] E116. The method according to any one of E113 to E115, wherein the cells are selected from the group consisting of pluripotent cells, ESCs, iPSCs, pluripotent cells, HSCs, MPCs, BLPCs, monocytes, microglial progenitor cells, macrophages, and microglia.

[0277] E117. The method of any one of E113 to E116, wherein the cells have not been modified to express a transgene encoding TREM2.

[0278] E118. The method according to any one of E113 to E117, wherein the cells are systemically administered to the subject.

[0279] E119. The method of E118, wherein the cells are administered to the subject by intravenous injection.

[0280] E120. The method of any one of E1 to E119, wherein endogenous TREM2 is disrupted in the cells prior to administration of the composition to the subject.

[0281] E121. The method of any one of E1 to E120, wherein endogenous TREM2 is disrupted in the subject prior to administration of the composition to the subject.

[0282] E122. The method of E121, wherein endogenous TREM2 is disrupted in a population of neurons in the subject prior to administration of the composition to the subject.

[0283] E123. The method of E120, wherein the endogenous TREM2 is destroyed by contacting the cell with a nuclease that catalyzes cleavage of an endogenous TREM2 nucleic acid in the cell.

[0284] E124. The method of E123, wherein the nuclease is a clustered regularly interspaced short palindromic repeats (CRISPR) associated protein.

[0285] E125. The method of E124, wherein the CRISPR-associated protein is CRISPR-associated protein 9 (Cas9).

[0286] E126. The method of E124, wherein the CRISPR-associated protein is CRISPR-associated protein 12a (Cas12a).

[0287] E127. The method of E123, wherein the nuclease is a transcription activator-like effector nuclease, a meganuclease, or a zinc finger nuclease.

[0288] E128. The method according to any one of E120 to E122, wherein the endogenous TREM2 is disrupted by administering an inhibitory RNA molecule to the cell, the subject, or the population of neurons.

[0289] E129. The method of E128, wherein the inhibitory RNA molecule is a short interfering RNA, a short hairpin RNA, or an miRNA.

[0290] E130. The method according to any one of E1 to E129, wherein said cells are autologous cells.

[0291] E131. The method according to any one of E1 to E129, wherein said cells are allogeneic cells.

[0292] E132. The method of any one of E1 to E131, wherein the cells are transduced ex vivo to express the TREM2.

[0293] E133. The method of E132, wherein the cells are transduced with a viral vector selected from the group consisting of adeno-associated virus (AAV), adenovirus, parvovirus, coronavirus, rhabdovirus, paramyxovirus, picornavirus, alphavirus, herpesvirus, poxvirus, and retroviridae virus.

[0294] E134. The method according to E133, wherein said viral vector is a retroviridae viral vector.

[0295] E135. The method according to E134, wherein the Retroviridae viral vector is a lentiviral vector.

[0296] E136. The method according to E134, wherein said Retroviridae viral vector is an alpharetroviral vector.

[0297] E137. The method according to E134, wherein said Retroviridae viral vector is a gamma retroviral vector.

[0298] E138. The method according to any one of E134 to E137, wherein the Retroviridae viral vector comprises a central polypurine tract, a Woodchuck Hepatitis Virus post-transcriptional regulatory element, a 5'-LTR, an HIV signal sequence, an HIV Psi signal 5'-splice site, a delta-GAG element, a 3'-splice site, and a 3'-self-inactivating LTR.

[0299] E139. The method of E133, wherein the viral vector is an AAV selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, and AAVrh74.

[0300] E140. The method according to any one of E133 to E139, wherein the viral vector is a pseudotyped viral vector.

[0301] E141. The method of E140, wherein the pseudotyped viral vector is selected from the group consisting of pseudotyped AAV, pseudotyped adenovirus, pseudotyped parvovirus, pseudotyped coronavirus, pseudotyped rhabdovirus, pseudotyped paramyxovirus, pseudotyped picornavirus, pseudotyped alphavirus, pseudotyped herpesvirus, pseudotyped poxvirus, and pseudotyped retroviridae viruses.

[0302] E142. The method of any one of E1 to E141, wherein the cells are transfected ex vivo to express the TREM2.

[0303] E143. The method of E142, wherein the cells are transfected using: a) an agent selected from the group consisting of cationic polymers, diethylaminoethyl-dextran, polyethyleneimine, cationic lipids, liposomes, calcium phosphate, activated dendrimers, and magnetic beads; or b) a technique selected from the group consisting of electroporation, Nucleofection, squeeze-poration, sonoporation, optical transfection, Magnetofection, and impalefection.

[0304] E144. The method according to any one of E1 to E143, wherein expression of said TREM2 in said cell is mediated by a ubiquitous promoter.

[0305] E145. The method according to E144, wherein the ubiquitous promoter is selected from the group consisting of elongation factor 1-alpha promoter and phosphoglycerate kinase 1 promoter.

[0306] E146. The method according to any one of E1 to E143, wherein expression of said TREM2 is mediated by a cell lineage specific promoter.

[0307] E147. The method of E146, wherein the cell lineage specific promoter is selected from the group consisting of TREM2 promoter, CD68 promoter, CD11b promoter, C-X3-C motif chemokine receptor 1 promoter, allograft inflammatory factor 1 promoter, purinergic receptor P2Y12 promoter, transmembrane protein 119 promoter, and colony stimulating factor 1 receptor promoter.

[0308] E148. The method of any one of E1 to E143, wherein expression of said TREM2 in said cell is mediated by a synthetic promoter.

[0309] E149. The method of any one of E1-E148, wherein the composition is administered to the subject in an amount sufficient to: a) increase the amount of M2 microglia in the brain of the subject relative to the amount of M1 microglia in the brain of the subject; b) reduce the level of one or more pro-inflammatory cytokines in the brain of the subject; c) increase the level of one or more anti-inflammatory cytokines in the brain of the subject; d) improve the cognitive performance of the subject; e) improve the motor function of the subject; f) reduce neuronal loss in the subject; and / or g) reduce the level of amyloid beta and neurofibrillary tau protein, or aggregates thereof, in the subject.

[0310] E150. The method according to any one of E1 to E149, wherein the subject is a human.

[0311] E151. A composition comprising a population of cells comprising a transgene encoding TREM2 (e.g., a transgene capable of being expressed in macrophages or microglial cells).

[0312] E152. The composition of E151, wherein the TREM2 is full-length TREM2.

[0313] E153. The composition described in E151 or E152, wherein the TREM2 or its variant has an amino acid sequence having at least 85% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 1 to 3.

[0314] E154. The composition of E153, wherein the TREM2 has an amino acid sequence having at least 85% sequence identity to SEQ ID NO:1.

[0315] E155. The composition of E154, wherein the TREM2 has an amino acid sequence having at least 90% sequence identity to SEQ ID NO:1.

[0316] E156. The composition of E155, wherein the TREM2 has an amino acid sequence having at least 95% sequence identity to SEQ ID NO:1.

[0317] E157. The composition described in E156, wherein the TREM2 has the amino acid sequence of SEQ ID NO:1.

[0318] E158. The composition according to any one of E151 to E157, wherein the TREM2 has an amino acid sequence having at least 85% sequence identity to SEQ ID NO:2.

[0319] E159. The composition of E158, wherein the TREM2 has an amino acid sequence having at least 90% sequence identity to SEQ ID NO:2.

[0320] E160. The composition of E159, wherein the TREM2 has an amino acid sequence having at least 95% sequence identity to SEQ ID NO:2.

[0321] E161. The composition described in E160, wherein the TREM2 has the amino acid sequence of SEQ ID NO:2.

[0322] E162. The composition according to any one of E151 to E161, wherein the TREM2 has an amino acid sequence having at least 85% sequence identity to SEQ ID NO:3.

[0323] E163. The composition of E162, wherein the TREM2 has an amino acid sequence having at least 90% sequence identity to SEQ ID NO:3.

[0324] E164. The composition of E163, wherein the TREM2 has an amino acid sequence having at least 95% sequence identity to SEQ ID NO:3.

[0325] E165. The composition described in E164, wherein the TREM2 has the amino acid sequence of SEQ ID NO:3.

[0326] E166. The composition described in any one of E151 to E165, wherein the TREM2 comprises a signal peptide.

[0327] E167. The composition described in E166, wherein the signal peptide is a TREM2 signal peptide.

[0328] E168. The composition according to any one of E151 to E167, wherein the TREM2 is sTREM2.

[0329] E169. The composition according to any one of E151 to E167, wherein the TREM2 is TREM2-CTF.

[0330] E170. The composition described in E169, wherein the TREM2 is TREM2-ICD.

[0331] E171. The composition described in E169, wherein the TREM2 is a TREM2-T2β peptide.

[0332] E172. The composition described in any one of E151 to E167, wherein the TREM2 does not contain a functional ectodomain cleavage site.

[0333] E173. The composition of E169, wherein the TREM2-CTF does not contain a functional intramembrane cleavage site.

[0334] E174. The composition according to any one of E151 to E173, wherein the transgene encodes two or more TREM2 proteins.

[0335] E175. The composition of E174, wherein the transgene encodes 2 to 10 TREM2 proteins.

[0336] E176. The composition of E175, wherein the transgene encodes two to five TREM2 proteins.

[0337] E177. The composition described in E176, wherein the introduced gene encodes two TREM2 proteins.

[0338] E178. The composition of any one of E174 to E177, wherein the TREM2 transgene is expressed from a single polycistronic expression cassette.

[0339] E179. The composition according to any one of E174 to E178, wherein the TREM2 transgenes are separated from each other by one or more IRES.

[0340] E180. The composition of any one of E174 to E177, wherein the TREM2 transgene is expressed from one or more monocistronic expression cassettes.

[0341] E181. The composition according to any one of E151 to E180, wherein the transgene comprises a polynucleotide having at least 85% sequence identity to the nucleic acid sequence of SEQ ID NO:4.

[0342] E182. The composition of E181, wherein the transgene comprises a polynucleotide having at least 90% sequence identity to the nucleic acid sequence of SEQ ID NO:4.

[0343] E183. The composition of E182, wherein the transgene comprises a polynucleotide having at least 95% sequence identity to the nucleic acid sequence of SEQ ID NO:4.

[0344] E184. The composition described in E183, wherein the introduced gene comprises a polynucleotide having the nucleic acid sequence of SEQ ID NO:4.

[0345] E185. The composition according to any one of E151 to E184, wherein the transgene comprises a polynucleotide having at least 85% sequence identity to the nucleic acid sequence of SEQ ID NO:5.

[0346] E186. The composition described in E185, wherein the introduced gene comprises a polynucleotide having at least 90% sequence identity to the nucleic acid sequence of SEQ ID NO:5.

[0347] E187. The composition described in E186, wherein the introduced gene comprises a polynucleotide having at least 95% sequence identity to the nucleic acid sequence of SEQ ID NO:5.

[0348] E188. The composition described in E187, wherein the introduced gene comprises a polynucleotide having the nucleic acid sequence of SEQ ID NO:5.

[0349] E189. The composition according to any one of E151 to E188, wherein the transgene comprises a polynucleotide having at least 85% sequence identity to the nucleic acid sequence of SEQ ID NO:6.

[0350] E190. The composition of E189, wherein the introduced gene comprises a polynucleotide having at least 90% sequence identity to the nucleic acid sequence of SEQ ID NO:6.

[0351] E191. The composition of E190, wherein the introduced gene comprises a polynucleotide having at least 95% sequence identity to the nucleic acid sequence of SEQ ID NO:6.

[0352] E192. The composition described in E191, wherein the introduced gene comprises a polynucleotide having the nucleic acid sequence of SEQ ID NO:6.

[0353] E193. The composition according to any one of E151 to E192, wherein the transgene comprises a polynucleotide having at least 85% sequence identity to the nucleic acid sequence of SEQ ID NO:7.

[0354] E194. The composition of E193, wherein the transgene comprises a polynucleotide having at least 90% sequence identity to the nucleic acid sequence of SEQ ID NO:7.

[0355] E195. The composition of E194, wherein the transgene comprises a polynucleotide having at least 95% sequence identity to the nucleic acid sequence of SEQ ID NO:7.

[0356] E196. The composition described in E195, wherein the introduced gene comprises a polynucleotide having the nucleic acid sequence of SEQ ID NO:7.

[0357] E197. The composition according to any one of E151 to E196, wherein the transgene comprises a polynucleotide having at least 85% sequence identity to the nucleic acid sequence of SEQ ID NO:9.

[0358] E198. The composition of E197, wherein the transgene comprises a polynucleotide having at least 90% sequence identity to the nucleic acid sequence of SEQ ID NO:9.

[0359] E199. The composition of E198, wherein the transgene comprises a polynucleotide having at least 95% sequence identity to the nucleic acid sequence of SEQ ID NO:9.

[0360] E200. The composition described in E199, wherein the introduced gene comprises a polynucleotide having the nucleic acid sequence of SEQ ID NO:11.

[0361] E201. The composition according to any one of E151 to E200, wherein the introduced gene comprises a polynucleotide having at least 85% sequence identity to the nucleic acid sequence of SEQ ID NO:11.

[0362] E202. The composition of E201, wherein the introduced gene comprises a polynucleotide having at least 90% sequence identity to the nucleic acid sequence of SEQ ID NO:11.

[0363] E203. The composition of E202, wherein the transgene comprises a polynucleotide having at least 95% sequence identity to the nucleic acid sequence of SEQ ID NO:11.

[0364] E204. The composition of E203, wherein the introduced gene comprises a polynucleotide having the nucleic acid sequence of SEQ ID NO:11.

[0365] E205. The composition described in any one of E151 to E204, wherein the transgene is a codon-optimized TREM2 transgene.

[0366] E206. The composition of E205, wherein the codon-optimized TREM2 transgene comprises a polynucleotide having at least 85% sequence identity to the nucleic acid sequence of SEQ ID NO:8.

[0367] E207. The composition of E206, wherein the codon-optimized TREM2 transgene comprises a polynucleotide having at least 90% sequence identity to the nucleic acid sequence of SEQ ID NO:8.

[0368] E208. The composition of E207, wherein the codon-optimized TREM2 transgene comprises a polynucleotide having at least 95% sequence identity to the nucleic acid sequence of SEQ ID NO:8.

[0369] E209. The composition of E208, wherein the codon-optimized TREM2 transgene comprises the polynucleotide of SEQ ID NO:8.

[0370] E210. The composition of any one of E205 to E209, wherein the codon-optimized TREM2 transgene comprises a polynucleotide having at least 85% sequence identity to the nucleic acid sequence of SEQ ID NO:10.

[0371] E211. The composition of E210, wherein the codon-optimized TREM2 transgene comprises a polynucleotide having at least 90% sequence identity to the nucleic acid sequence of SEQ ID NO:10.

[0372] E212. The composition of E211, wherein the codon-optimized TREM2 transgene comprises a polynucleotide having at least 95% sequence identity to the nucleic acid sequence of SEQ ID NO:10.

[0373] E213. The composition of E212, wherein the codon-optimized TREM2 transgene comprises the polynucleotide of SEQ ID NO:10.

[0374] E214. The composition of any one of E205 to E213, wherein the codon-optimized TREM2 transgene comprises a polynucleotide having at least 85% sequence identity to the nucleic acid sequence of SEQ ID NO:12.

[0375] E215. The composition of E214, wherein the codon-optimized TREM2 transgene comprises a polynucleotide having at least 90% sequence identity to the nucleic acid sequence of SEQ ID NO:12.

[0376] E216. The composition of E215, wherein the codon-optimized TREM2 transgene comprises a polynucleotide having at least 95% sequence identity to the nucleic acid sequence of SEQ ID NO:12.

[0377] E217. The composition of E216, wherein the codon-optimized TREM2 transgene comprises the polynucleotide of SEQ ID NO:12.

[0378] E218. The composition according to any one of E151 to E217, wherein the TREM2 is a TREM2 fusion protein.

[0379] E219. The composition described in E218, wherein the TREM2 fusion protein comprises the Rb domain of ApoE.

[0380] E220. The composition of E219, wherein the Rb domain comprises a portion of ApoE having an amino acid sequence of residues 25-185, 50-180, 75-175, 100-170, 125-160, or 130-150 of SEQ ID NO:13.

[0381] E221. The composition of E219 or E220, wherein the Rb domain comprises a region having at least 70% sequence identity to the amino acid sequence of residues 159 to 167 of SEQ ID NO:13.

[0382] E222. The composition described in any one of E151 to E221, wherein the introduced gene encoding TREM2 further comprises an miRNA targeting sequence in the 3'-UTR.

[0383] E223. The composition described in E222, wherein the miRNA targeting sequence is a miR-126 targeting sequence.

[0384] E224. The composition according to any one of E151 to E223, wherein the cells are ESCs (eg, ESCs that have differentiated into macrophages or microglia).

[0385] E225. The composition according to any one of E151 to E223, wherein the cell is an iPSC (eg, an iPSC that has differentiated into a macrophage or microglia).

[0386] E226. The composition according to any one of E151 to E223, wherein the cells are CD34+ cells.

[0387] E227. The composition of E226, wherein the CD34+ cells are HSCs.

[0388] E228. The composition described in E226, wherein the CD34+ cells are MPCs.

[0389] E229. The composition described in any one of E151 to E228, wherein the cells are transfected ex vivo to express the TREM2.

[0390] E230. The composition of any one of E151 to E228, wherein the cells are transduced ex vivo to express TREM2.

[0391] E231. The composition of any one of E151 to E230, formulated for systemic administration to a human subject.

[0392] E232. The composition of any one of E231 to E245, formulated for administration to a human subject by intravenous injection.

[0393] E233. The composition of any one of E151 to E229, formulated for administration to a human subject directly to the nervous system of said subject.

[0394] E234. The composition of E233, formulated for administration to a human subject into cerebrospinal fluid.

[0395] E235. The composition of E233 or E234, formulated for administration to a human subject by intraventricular injection, intrathecal injection, stereotactic injection, or a combination thereof.

[0396] E236. The composition of E233, formulated for administration by intraparenchymal injection.

[0397] E237. The composition of any one of E151 to E230, formulated for administration directly to the bone marrow of a human subject.

[0398] E238. The composition of E237, formulated for administration to a human subject by intraosseous injection.

[0399] E239. The composition of any one of E151 to E230, formulated for administration to a human subject by bone marrow transplantation comprising said composition.

[0400] E240. The composition of any one of E151 to E230, formulated for administration to said subject by direct administration to the central nervous system of said subject and by systemic administration.

[0401] E241. The composition of E240, which is formulated for administration by intraventricular and intravenous injection.

[0402] E242. The composition of E240, which is formulated for administration by intrathecal and intravenous injection.

[0403] E243. The composition of E240, which is formulated for administration by intraparenchymal and intravenous injection.

[0404] E244. The composition of any one of E231 to E243, wherein the subject has been diagnosed with an NCD.

[0405] E245. The composition according to E244, wherein said NCD is a severe NCD.

[0406] E246. The composition of E245, wherein the severe NCD interferes with the subject's independence and / or normal daily functioning.

[0407] E247. The composition of E244 or E245, wherein said severe NCD is associated with a score obtained by said subject on a cognitive test that is at least 2 standard deviations away from the mean score of a reference population.

[0408] E248. The composition according to E244, wherein said NCD is a mild NCD.

[0409] E249. The composition of E248, wherein said mild NCD does not interfere with the subject's independence and / or normal daily functioning.

[0410] E250. The composition of E248 or E249, wherein said mild NCD is associated with a score obtained by said subject on a cognitive test that is at least 1-2 standard deviations away from the mean score of a reference population.

[0411] E251. The composition of E247 or E250, wherein the reference population is a general population.

[0412] E252. The composition of E247, E250, or E251, wherein the cognitive test is selected from the group consisting of AD8, AWV, GPCOG, HRA, MIS, MMSE, MoCA, SLUMS, and Short IQCODE.

[0413] E253. The composition of any one of E244 to E252, wherein the NCD is associated with impairment in one or more of complex attention, executive function, learning and memory, language, sensorimotor function, and social cognition.

[0414] E254. The composition according to any one of E244 to E253, wherein the NCD is not due to delirium or other psychiatric disorder.

[0415] E255. The composition according to any one of E244 to E254, wherein the NCD is Alzheimer's disease (AD).

[0416] E256. The composition according to any one of E244 to E254, wherein the NCD is a leukodystrophy.

[0417] E257. The composition described in E256, wherein the leukodystrophy is Nasu-Hakola disease (PLOSL).

[0418] E258. A kit comprising the composition according to any one of E151 to E257, or the pharmaceutical composition according to E258, and an accompanying document.

[0419] E259. The kit described in E251, wherein the package insert instructs a user of the kit to carry out a method according to any one of E1 to E150.

[0420] E260. The method according to any one of E1 to E150, wherein said NCD is a frontotemporal NCD.

[0421] E261. The method of claim 20, wherein said frontotemporal NCD is FTLD.

[0422] E262. The method according to any one of E1 to E150, wherein said NCD is a movement disorder.

[0423] E263. The method according to E262, wherein said movement disorder is PD.

[0424] E264. The method according to any one of E1 to E150, wherein the cell is a pluripotent cell (e.g., ESC, iPSC), a multipotent cell (e.g., a CD34+ cell, such as an HSC or MPC), a BLPC, a monocyte, a macrophage, a microglial progenitor cell, or a microglia.

[0425] E265. The method according to any one of E1 to E150, wherein said transgene is capable of being expressed in macrophages or microglial cells.

[0426] E266. The composition according to any one of E151 to E257, wherein the NCD is a frontotemporal NCD.

[0427] E267. The composition of E264, wherein the frontotemporal NCD is FTLD.

[0428] E268. The composition according to any one of E151 to E257, wherein the NCD is a movement disorder.

[0429] E269. The composition according to any one of E151 to E257, wherein the movement disorder is PD.

[0430] E270. The composition of any one of E151 to E257, wherein the cell is a pluripotent cell (e.g., an ESC, an iPSC), a multipotent cell (e.g., a CD34+ cell, such as an HSC or MPC), a BLPC, a monocyte, a macrophage, a microglial progenitor cell, or a microglia.

[0431] E271. The composition according to any one of E151 to E257, wherein the transgene can be expressed in a macrophage or a microglial cell. [Brief description of the drawings]

[0432] [Figure 1]Western blot showing expression of triggering receptor expressed on human myeloid cells 2 (TREM2) protein in mouse macrophages transduced with a lentiviral vector encoding TREM2. Cell lysates were generated from RAW mouse macrophage cells transduced with the MND.TREM2 viral vector (MND.TREM2), the MND.green fluorescent protein (GFP) viral vector (MND.GFP) or from non-transduced control (NTC) cells at a multiplicity of infection (MOI) of 10, 50, 100, or 200. TREM2 expression was assessed using an antibody raised against human TREM2 (Figure 1). [Diagram 2] Western blot showing human TREM2 protein expression in mouse microglial cells transduced with a lentiviral vector encoding TREM2. Cell lysates were generated from primary mouse microglia that were either non-transduced (NT) or transduced with the MND.TREM2 viral vector (MND-TREM2) or MND.GFP viral vector (MND-GFP). TREM2 expression was assessed using an antibody raised against human TREM2 (Figure 2). [Diagram 3] Western blot showing human TREM2 protein expression in lineage negative (Lin-) cells transduced with a lentiviral vector encoding TREM2. Cell lysates from Lin- mouse cells transduced with the MND.TREM2 viral vector (Lenti TREM2) or the MND.GFP viral vector. TREM2 expression was assessed using an antibody raised against human TREM2 (Figure 3). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0433] definition As used herein, the terms "remove", "removing", "removal", and the like refer to the depletion of one or more cells in a population of cells in vivo or ex vivo. In some embodiments of the present disclosure, it may be desirable to remove endogenous cells in a subject (e.g., a subject undergoing treatment for a disease described herein, e.g., an NCD (e.g., Alzheimer's disease (AD), Nasu-Hakola disease (also known as multicystic lipomembranous dysplasia with sclerosing leukoencephalopathy (PLOSL)), frontotemporal lobar degeneration (FTLD), or Parkinson's disease (PD))) before administering a therapeutic population of cells (e.g., pluripotent cells, embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), pluripotent cells, CD34+ cells, hematopoietic stem cells (HSCs), myeloid progenitor cells (MPCs), blood lineage progenitor cells (BLPCs), monocytes, macrophages, microglial progenitor cells, or microglia) to said subject. This may be advantageous, for example, to provide newly administered cells with an environment in which the cells can engraft. Removal of a population of cells can be performed in a manner that selectively targets a specific cell type, for example, using an antibody-drug conjugate that binds to an antigen expressed on the target cell and subsequently causes the death of the target cell. Additionally or alternatively, removal can be performed in a non-specific manner using cytotoxins that do not focus on a particular cell type, but instead can exert their cytotoxic effects on a variety of different cells. Exemplary agents that can be used to remove a population of endogenous cells in a subject, such as a population of endogenous microglia or microglia precursor cells in a subject being treated for the treatment of an NCD, are busulfan, PLX3397, PLX647, PLX5622, treosulfan, clodronate liposomes, and combinations thereof. Examples of depletion include depletion of at least 5% (e.g., at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50% or more) of cells in a population of cells in vivo or in vitro.Quantification of the number of cells in a sample of cells can be performed using a variety of cell counting techniques, for example, by use of a counting chamber, a Coulter counter, flow cytometry, or other cell counting methods known in the art.

[0434] As used herein, "administration" refers to providing or giving to a subject, by any effective route, a therapeutic agent (e.g., a cell described herein) that includes a transgene encoding a triggering receptor expressed in one or more myeloid cells 2 (TREM2) protein (e.g., a transgene that can be expressed in macrophages or microglia). Exemplary routes of administration are described herein and below (e.g., intracerebroventricular (ICV) injection, intrathecal (IT) injection, intraparenchymal (IP) injection, intravenous (IV) injection, and stereotactic injection).

[0435] As used herein, "allogeneic" refers to cells, tissues, DNA, or factors taken or derived from a different subject of the same species. For example, in the situation where transduced TREM2-expressing cells are administered to a subject to treat NCD, the allogeneic cells may be cells obtained from a non-subject and then transduced or transfected with a vector directing the expression of TREM2. The phrase "directing expression" refers to a polynucleotide that comprises a sequence that codes for a molecule to be expressed. The polynucleotide may comprise additional sequences that enhance the expression of the molecule of interest.

[0436] As used herein, "Alzheimer's disease" and "AD" refer to a late-onset neurodegenerative disorder that manifests as cognitive decline, insidious loss of short-term and long-term memory, attention deficits, language-specific problems, disorientation, impulse control, social withdrawal, anhedonia, and other symptoms. Brain tissue from AD patients exhibits neuropathological features such as extracellular condensations of amyloid beta protein and neurofibrillary tangles of hyperphosphorylated microtubule-associated tau protein. The accumulation of these aggregates is associated with neuronal loss and atrophy in several brain regions, including the frontal, temporal, and parietal lobes of the cerebral cortex, as well as subcortical structures such as the basal forebrain cholinergic system and the locus coeruleus in the brainstem. AD ​​is also associated with increased neuroinflammation, characterized by reactive glia and elevated levels of proinflammatory cytokines.

[0437] As used herein, "autologous" refers to cells, tissues, DNA, or factors taken or derived from an individual's own tissues, cells, or DNA. For example, in the context of administering transduced TREM2-expressing cells to a subject to treat an NCD, the autologous cells may be cells obtained from the subject and then transduced or transfected with a vector directing the expression of TREM2.

[0438] As used herein, the term "ApoE" refers to apolipoprotein E, a member of a class of proteins involved in lipid transport. Apolipoprotein E is a fat-binding protein (apolipoprotein) that is part of chylomicrons and intermediate density lipoproteins (IDL). They are essential for normal processing (catabolism) of triglyceride-rich lipoproteins. ApoE is encoded by the APOE gene. The term "ApoE" also refers to variants of wild-type ApoE protein, such as proteins having at least 85% identity (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.9% identity or more) to the amino acid sequence of wild-type ApoE shown in SEQ ID NO: 13.

[0439] As used herein, the term "blood lineage progenitor cells" or "BLPCs" refers to any cell (e.g., mammalian cells) that can differentiate into one or more (e.g., 2, 3, 4, 5 or more) types of hematopoietic (i.e., blood) cells. BLPCs can differentiate into red blood cells, white blood cells (e.g., granulocytes (e.g., basophils, eosinophils, neutrophils, and mast cells) or agranulocytes (e.g., lymphocytes and monocytes), etc.), or platelets. BLPCs can also include differentiated blood cells (e.g., monocytes) that can further differentiate into another blood cell type (e.g., macrophages).

[0440] As used herein, the term "cell type" refers to a group of cells that share a statistically distinguishable phenotype based on gene expression data. For example, cells of a common cell type may share similar structural and / or functional characteristics, such as similar gene activation patterns and antigen presentation profiles. Cells of a common cell type may include those isolated from a common tissue (e.g., epithelial, nervous, connective, or muscle tissue) and / or those isolated from a common organ, tissue system, blood vessel, or other structure and / or region of the body.

[0441] As used herein, the term "cistron" refers to a segment of a DNA or RNA sequence that encodes a single protein or polypeptide product.

[0442] As used herein, "codon optimization" refers to the process of modifying a nucleic acid sequence according to the principle that the frequency of occurrence of synonymous codons (e.g., codons that code for the same amino acid) in coding DNA is biased in different species. Such codon degeneracy allows the same polypeptide to be encoded by a variety of nucleotide sequences. Sequences modified in this manner are referred to herein as "codon optimization." This process may be performed on any of the sequences described herein to enhance expression or stability. Codon optimization may be performed, for example, in the manner described in U.S. Patent Nos. 7,561,972, 7,561,973, and 7,888,112, each of which is incorporated herein by reference in its entirety. The sequence surrounding the translation start site may be converted to a consensus Kozak sequence according to known methods. See, for example, Kozak et al, Nucleic Acids Res. 15: 8125-8148, which is incorporated herein by reference in its entirety. Multiple stop codons may be incorporated.

[0443] As used herein, the term "cognitive test" refers to a test that can be performed by a skilled practitioner to evaluate the cognitive abilities of humans and other animals. Cognitive tests can be used to evaluate inductive reasoning skills, IQ, cognitive development, memory, knowledge organization, metacognition, thinking, and mental time measurement. Cognitive tests can be used to evaluate the processing abilities of subjects across multiple cognitive domains, including, but not limited to, executive function, learning and memory, language, sensory-motor function, and social cognition. Examples of cognitive tests include, but are not limited to, the Eight-item Informant Interview to Differentiate Aging and Dementia (AD8), Annual Wellness Visit (AWV), General Practitioner Assessment of Cognition (GPCOG), Health Risk Assessment (HRA), Memory Impairment Screen (MIS), Mini Mental Status Exam (MMSE), Montreal Cognitive Assessment (MoCA), St. Louis University Mental Status Exam (SLUMS), and Short Informant Questionnaire on Cognitive Decline in the Elderly (Short IQCODE). Those skilled in the art will recognize that other cognitive tests known in the art can also be used to assess cognitive function in a subject.

[0444] As used herein, the term "complex attention" refers to a cognitive function that describes a subject's (e.g., a human subject's) ability to hold information in memory for a short period of time and to manipulate that information (e.g., mental arithmetic). Impairments in complex attention can result in difficulties with focusing on conversations, difficulties with blocking out unwanted information, problems with prospective memory (e.g., memory for remembering something later), and inefficient memory for new information.

[0445] As used herein, the terms "pretreatment" and "pretreating" refer to a process in which a subject is prepared to receive a graft containing cells (e.g., pluripotent cells, ESCs, iPSCs, pluripotent cells, CD34+ cells, HSCs, MPCs, BLPCs, monocytes, macrophages, microglial progenitor cells, or microglia). Such procedures promote engraftment of the cell graft, for example, by selectively depleting endogenous microglia or HSCs, thereby creating voids that are filled by the exogenous cell graft. According to the methods described herein, a subject can be pretreated for cell transplantation therapy by administering to the subject one or more agents capable of ablating endogenous microglia and / or hematopoietic stem or progenitor cells (e.g., busulfan, treosulfan, PLX3397, PLX647, PLX5622, and clodronate liposomes), radiation therapy, or a combination thereof. The pretreatment can be myeloablative or non-myeloablative. Other cell depletion agents and methods known in the art (eg, antibody-drug conjugates) can also be used.

[0446] As used herein, the terms "conservative mutation," "conservative substitution," and "conservative amino acid substitution" refer to the replacement of one or more amino acids with one or more different amino acids that exhibit similar physicochemical properties, such as polarity, electrostatic charge, and steric bulk, which are summarized in Table 1 below for each of the 20 naturally occurring amino acids.

[0447] [Table 1] TIFF2025032191000003.tif26152

[0448] From this table it can be seen that conservative amino acid families include: (i) G, A, V, L, and I; (ii) D and E; (iii) C, S, and T; (iv) H, K, and R; (v) N and Q; and (vi) F, Y, and W. Thus, a conservative variation or substitution is one that substitutes one amino acid for a member of the same amino acid family (e.g., Thr for Ser, Arg for Lys).

[0449] As used herein, the term "delirium or other psychiatric disorder" refers to a condition such as delirium (i.e., a syndrome involving impaired attention, consciousness, and cognition that develops over a short period of time (e.g., hours to days)) or another mental disorder (e.g., schizophrenia, bipolar disorder, and major depression) that is distinct from neurocognitive disorders and does not exhibit cognitive dysfunction as a core symptom. For example, a condition such as delirium or another psychiatric disorder may differ from an NCD in that cognitive dysfunction may be a symptom associated with the disease but is not a core feature of the disease. Delirium or another psychiatric disorder may differ from an NCD in terms of time to onset (e.g., hours to days for delirium versus months to years for NCD), etiology (e.g., substance-induced delirium), duration of symptoms (e.g., delirium may last days to hours, while NCDs may last for years), and resolution (e.g., delirium may resolve completely, while NCDs often do not).

[0450] As used herein, the term "disrupt" with respect to a gene refers to preventing the formation of a functional gene product. A gene product is functional if it fulfills its normal (wild-type) function. Disruption of a gene prevents the expression of a functional factor encoded by the gene and includes the insertion, deletion, or substitution of one or more bases in the sequence encoded by the gene and / or the promoter and / or operator required for the expression of the gene in the animal. The gene to be disrupted may be, for example, by removing at least a portion of the gene from the genome of the animal, modifying the gene to prevent the expression of the functional factor encoded by the gene, expressing a dominant negative factor by an interfering RNA, or an exogenous gene. Materials and methods for genetically modifying a cell to disrupt the expression of one or more genes are described in detail in US8,518,701; US9,499,808; and US2012 / 0222143, the disclosures of each of which are incorporated herein by reference in their entirety (in case of conflict, the present specification takes precedence).

[0451] As used herein, the terms "effective amount," "therapeutically effective amount," and "sufficient amount" of a composition, vector construct, viral vector, or cell described herein refer to an amount sufficient to produce a beneficial or desired result, including a clinical result, when administered to a subject, including a mammal, e.g., a human. Thus, an "effective amount" or its synonyms will depend on the context in which it is applied. For example, in the context of treating an NCD (e.g., AD, PLOSL, FTLD, or PD), it is the amount of the composition, vector construct, viral vector, or cell sufficient to achieve a treatment response compared to the response obtained when the composition, vector construct, viral vector, or cell is not administered. The amount of a given composition described herein that corresponds to such an amount will vary depending on various factors, such as a given drug, pharmaceutical formulation, route of administration, type of disease or disorder, subject identity (e.g., age, sex, weight) or recipient to be treated, but can nevertheless be determined by one of skill in the art. Also, as used herein, a "therapeutically effective amount" of a composition, vector construct, viral vector, or cell of the present disclosure is an amount that produces a beneficial or desired result in a subject compared to a control. As defined herein, a therapeutically effective amount of a composition, vector construct, viral vector, or cell of the present disclosure can be readily determined by one of ordinary skill in the art by methods known in the art. Dosage regimens can be adjusted to obtain the optimal therapeutic response.

[0452] As used herein, the terms "embryonic stem cell" and "ES cell" refer to embryo-derived totipotent or pluripotent stem cells derived from the inner cell mass of a blastocyst that can be maintained in in vitro culture under appropriate conditions. ES cells can differentiate into cells of any of the three vertebrate germ layers, e.g., endoderm, ectoderm, and mesoderm. ES cells are also characterized by their ability to grow indefinitely under appropriate in vitro culture conditions. See, e.g., Thomson et al., Science 282:1145 (1998).

[0453] As used herein, the term "endogenous" describes a molecule (e.g., a polypeptide, nucleic acid, or cofactor) that is found naturally in a particular organism (e.g., a human) or in a particular location within an organism (e.g., an organ, tissue, or cell, such as a human cell).

[0454] As used herein, the terms "engraft" and "engraftment" refer to the process of repopulating tissues with hematopoietic stem and progenitor cells (such cells either produced endogenously within the body or transplanted using any of the administration methods described herein (e.g., intravenous injection, intraventricular injection, intraosseous injection, and / or bone marrow transplantation). The terms encompass all events relating to or resulting in engraftment, such as tissue homing of cells and colonization of cells within a tissue of interest.

[0455] As used herein, the term "executive function" refers to a set of cognitive functions that facilitate cognitive control of behavior in a subject (e.g., human).Executive function includes, for example, goal-directed behavior selection and monitoring, attention control, cognitive inhibition, inhibitory control, working memory, and cognitive flexibility.Individuals normally acquire or perfect executive function throughout life, but this process may be derailed by the onset of NCD in a subject, which may adversely affect executive function.

[0456] As used herein, the term "express" refers to one or more of the following events: (1) production of an RNA template from a DNA sequence (e.g., by transcription); (2) processing of the RNA transcript (e.g., by splicing, editing, 5' capping, and / or 3' end processing); (3) translation of the RNA into a polypeptide or protein; and (4) post-translational modification of the polypeptide or protein. Expression of a gene of interest in a subject can be revealed, for example, by detecting an increase in the amount or concentration of mRNA encoding the corresponding protein (e.g., assessed using RNA detection procedures described herein or known in the art, such as quantitative polymerase chain reaction (qPCR) and RNA seq techniques), an increase in the amount or concentration of the corresponding protein (e.g., assessed using protein detection methods described herein or known in the art, such as enzyme-linked immunosorbent assay (ELISA), among others), and / or an increase in the activity of the corresponding protein (e.g., in the case of an enzyme, assessed using enzyme activity assays described herein or known in the art) in a sample obtained from the subject.

[0457] As used herein, the term "exogenous" describes a molecule (e.g., a polypeptide, nucleic acid, or cofactor) that is not naturally found in a particular organism (e.g., a human) or in a particular location within an organism (e.g., an organ, tissue, or cell such as a human cell). Exogenous materials include those provided to an organism from an outside source or extracted from an organism in culture.

[0458] As used herein, the term "functional ectodomain cleavage site" when referring to a TREM2 ectodomain cleavage site refers to amino acid residues within a full-length TREM2 peptide that undergo proteolytic cleavage by an extracellular protease (e.g., a disintegrin and metalloprotease family) ectodomain to generate soluble TREM2, as well as a TREM2 C-terminal fragment. The TREM2 ectodomain cleavage site may be non-functional, for example, as a result of a mutation in the TREM2 gene that alters the amino acid sequence within the ectodomain cleavage site so as to sterically protect the ectodomain cleavage site from proteolytic cleavage, or that affects the three-dimensional protein structure.

[0459] As used herein, the term "functional intramembrane cleavage site" when referring to a TREM2 C-terminal fragment intramembrane cleavage site refers to amino acid residues within the TREM2 C-terminal fragment that undergo proteolytic cleavage by the γ-secretase complex to generate the TREM2 intracellular domain and the TREM2-A β-like peptide. The TREM2 C-terminal fragment intramembrane cleavage site may be non-functional, for example, as a result of a mutation in the TREM2 gene that alters the amino acid sequence within the intramembrane cleavage site so as to sterically protect the intramembrane cleavage site from proteolytic cleavage or that affects the three-dimensional protein structure.

[0460] As used herein, the term "functional potential" in relation to stem cells, such as hematopoietic stem cells, refers to functional properties of stem cells including: 1) multipotency (referring to the ability to differentiate into multiple different blood cell lineages, including, but not limited to, granulocytes (e.g., promyelocytes, neutrophils, eosinophils, basophils), erythrocytes (e.g., reticulocytes, red blood cells), platelets (e.g., megakaryoblasts, thrombocytic megakaryocytes, platelets), monocytes (e.g., monocytes, macrophages), dendritic cells, microglia, osteoclasts, and lymphocytes (e.g., NK cells, B cells, and T cells)); 2) self-renewal (referring to the ability of stem cells to give rise to daughter cells that have the same potential as the mother cell and that may arise repeatedly throughout an individual's life without this potential being exhausted); and 3) the ability of the stem cell or its progeny, when reintroduced into a transplant recipient, to home to the stem cell niche and re-establish productive, sustained cell growth and differentiation.

[0461] As used herein, the term "general population" refers to the entire population of individuals with a particular characteristic of interest (e.g., age, medical history, education, socioeconomic status, or lifestyle, among others). Alternatively, the term "general population" may refer to a subset of the entire population of individuals with a particular characteristic of interest, such as, for example, a random sample with a defined sample size. In the methods disclosed herein, the general population can serve as a working standard (e.g., a reference population) against which a measured variable can be compared. For example, subjects who have been diagnosed with NCD can be evaluated for their cognition using a cognitive test disclosed herein, and the score obtained by the subject on the test can be compared to the performance of individuals in the general population (e.g., the entire general population or a random sample of the general population) on the same test. The size of the random sample of the general population can be determined by a skilled practitioner using methods well known in the art. For example, a skilled practitioner can perform a power analysis before collecting data (e.g., before administering a cognitive test to the subject) to determine the minimum sample required to detect a statistically significant effect with a desired level of confidence.

[0462] As used herein, the terms "hematopoietic stem cells" and "HSCs" refer to immature blood cells that have the ability to self-renew and differentiate into mature blood cells of various cell lineages, including, but not limited to, granulocytes (e.g., promyelocytes, neutrophils, eosinophils, basophils), erythrocytes (e.g., reticulocytes, red blood cells), platelets (e.g., megakaryoblasts, thrombocytic megakaryocytes, platelets), monocytes (e.g., monocytes, macrophages), dendritic cells, microglia, osteoclasts, and lymphocytes (e.g., NK cells, B cells, and T cells). It is known in the art that such cells may or may not include CD34+ cells. CD34+ cells are immature cells that express the CD34 cell surface marker. In humans, CD34+ cells are believed to include a subpopulation of cells with stem cell characteristics as defined above, while in mice, HSCs are CD34-. In addition, HSCs are also referred to as long-term repopulating HSCs (LT-HSCs) and short-term repopulating HSCs (ST-HSCs). LT-HSCs and ST-HSCs are differentiated based on functional potential and expression of cell surface markers. For example, human HSCs are CD34+, CD38-, CD45RA-, CD90+, CD49F+, and lin- (negative for mature cell lineage markers including CD2, CD3, CD4, CD7, CD8, CD10, CD11B, CD19, CD20, CD56, CD235A). In mice, bone marrow LT-HSCs are CD34-, SCA-1+, C-kit+, CD135-, Slamf1 / CD150+, CD48-, and lin- (negative for mature lineage markers including Ter119, CD11b, Gr1, CD3, CD4, CD8, B220, IL-7ra), whereas ST-HSCs are CD34+, SCA-1+, C-kit+, CD135-, Slamf1 / CD150+, and lin- (negative for mature lineage markers including Ter119, CD11b, Gr1, CD3, CD4, CD8, B220, IL-7ra). In addition, ST-HSCs are less quiescent (i.e., more active) and more proliferative than LT-HSCs under homeostatic conditions.However, LT-HSC has a higher self-renewal capacity (i.e., it can survive through adulthood and be continuously transplanted through successive recipients), whereas ST-HSC has a limited self-renewal capacity (i.e., it only survives for a limited period of time and does not have the ability to be continuously transplanted). Any of these HSCs can be used in any of the methods described herein. Optionally, ST-HSC is useful because it is highly proliferative and therefore can generate differentiated progeny more quickly.

[0463] As used herein, the term "HLA-matched" refers to a donor-recipient pair in which there is no mismatch of any of the HLA antigens between the donor and the recipient, such as a donor providing a hematopoietic stem cell graft to a recipient in need of hematopoietic stem cell transplantation therapy. HLA-matched (i.e., all six alleles matched) donor-recipient pairs have a lower risk of graft rejection because endogenous T and NK cells are less likely to recognize the foreign graft as foreign and therefore less likely to mount an immune response against the graft.

[0464] As used herein, the term "HLA-mismatched" refers to a donor-recipient pair in which at least one HLA antigen is mismatched between the donor and the recipient, particularly with respect to HLA-A, HLA-B, HLA-C, and HLA-DR, such as a donor providing a hematopoietic stem cell graft to a recipient in need of hematopoietic stem cell transplantation therapy. In some embodiments, one haplotype is matched and the other is mismatched. HLA-mismatched donor-recipient pairs may be at higher risk of graft rejection compared to HLA-matched donor-recipient pairs, since endogenous T cells and NK cells are more likely to recognize the foreign graft as foreign and therefore such T cells and NK cells are more likely to mount an immune response against the graft.

[0465] As used herein, the phrase "independence and / or normal daily functioning" refers to a subject's ability to successfully perform daily activities without the assistance of a caregiver or social worker. Non-limiting examples of activities that allow an individual to independently perform daily functions include, for example, social, professional, or academic functions, personal hygiene, grooming, dressing, toilet hygiene, functional mobility (e.g., walking ability, getting in and out of bed), and self-feeding. Subjects diagnosed with severe NCDs may have difficulty independently performing normal daily functions, while subjects diagnosed with mild NCDs may not have difficulty independently performing normal daily functions.

[0466] As used herein, the terms "induced pluripotent stem cell", "iPS cell" and "iPSC" refer to pluripotent stem cells that can be derived directly from differentiated somatic cells. Human iPS cells can be generated by introducing a specific set of reprogramming factors into non-pluripotent cells, which can include, for example, Oct3 / 4, Sox family transcription factors (e.g., Sox1, Sox2, Sox3, Sox15), Myc family transcription factors (e.g., c-Myc, 1-Myc, n-Myc), Krüppel-like family (KLF) transcription factors (e.g., KLF1, KLF2, KLF4, KLF5), and / or related transcription factors such as NANOG, LIN28 and / or Glis1. Human iPS cells can also be generated by the use of, for example, miRNAs, small molecules that mimic the action of transcription factors, or cell lineage specifiers. Human iPS cells are characterized by their ability to differentiate into any of the three vertebrate germ layers, such as endoderm, ectoderm, or mesoderm. Human iPS cells are also characterized by their ability to grow indefinitely under appropriate in vitro culture conditions. See, for example, Takahashi and Yamanaka, Cell 126:663 (2006).

[0467] As used herein, the term "IRES" refers to internal ribosome entry site. In general, IRES sequence is a function that allows eukaryotic ribosomes to bind to mRNA transcripts and start translation without binding to the 5' cap end. The mRNA that contains IRES sequence generates two translation products, one that starts from the 5' end of the mRNA and the other that starts from the internal translation mechanism mediated by IRES.

[0468] As used herein, the term "language" refers to the cognitive ability of a subject to learn and use complex systems of communication or to describe the rules governing these systems or the set of utterances that can be produced from such rules. Language abilities may be impaired in a subject with an NCD if the subject exhibits, for example, a limited vocabulary, an inability to produce complex grammar, frequent lexical errors, or aphasia, among other things.

[0469] As used herein, the phrase "learning and memory" refers to cognitive abilities that encompass the acquisition of skills or knowledge and the expression of acquired skills or knowledge (e.g., learning to say new words and speaking new words, respectively). "Learning and memory" can refer to two independent processes: 1) the acquisition of new skills or knowledge (i.e., learning); and 2) the processing, storage, and recall of learned skills or knowledge (i.e., remembering), which can differ by time scale (learning is generally slower and more effortful than memory recall or execution of learned skills) and neurobiological basis. Subjects diagnosed with NCDs may have impaired learning and memory compared to healthy subjects.

[0470] As used herein, the term "leukodystrophies" refers to a group of dominant genetic disorders characterized by the degeneration of white matter in the brain, which may result from defects in the myelin sheath that insulates neuronal axons.Leukodystrophies generally occur during childhood and early childhood and may be characterized by hyperexcitability, environmental sensitivity, muscle rigidity, head tilt, reduced or lost hearing and vision, and epilepsy.Non-limiting examples of leukodystrophies include Nasu-Hakola disease, metachromatic leukodystrophy, Krabbe disease, X-linked adrenoleukodystrophy, Canavan disease, and Alexander disease.

[0471] As used herein, the term "macrophage" refers to a type of white blood cell that engulfs and digests cell debris, foreign material, microorganisms, cancer cells, and anything else that does not have the types of proteins on its surface that are specific to healthy body cells, in a process called phagocytosis. Macrophages are found in essentially all tissues, where they patrol for potential pathogens by amoeboid-like movement. They take on various forms (with various names) throughout the body (e.g., histiocytes, Kupffer cells, alveolar macrophages, microglia, etc.), but are all part of the mononuclear phagocyte system. Besides phagocytosis, they play an important role in non-specific defense (innate immunity) and also help initiate specific defense mechanisms (adaptive immunity) by recruiting other immune cells, such as lymphocytes. For example, they are important as antigen presenters for T cells. Besides increasing inflammation and stimulating the immune system, macrophages also play an important anti-inflammatory role and can dampen immune responses through the release of cytokines.

[0472] As used herein, the term "microglia" or "microglial cells" refers to a type of resident macrophage cell and glial cell found in the brain and spinal cord that serves as the mainstay of immune defense in the central nervous system. The primary functions of microglial cells include immunosurveillance, phagocytosis, extracellular signaling (e.g., production and release of cytokines, chemokines, prostaglandins, and reactive oxygen species), antigen presentation, and promoting tissue repair and regeneration.

[0473] As used herein, the term "microglial progenitor cells" refers to progenitor cells that give rise to microglial cells. Microglial progenitor cells arise in the yolk sac during a limited period of embryonic development, infiltrate the brain mesenchyme, and perpetually renew themselves throughout life.

[0474] As used herein, the term "miRNA targeting sequence" refers to a nucleotide sequence located in the 3'-UTR of a target mRNA molecule that is hybridizable and complementary to a specific miRNA molecule (e.g., miR-126) and promotes RNA-induced silencing complex-dependent and Dicer-dependent mRNA destabilization and / or cleavage, thereby preventing expression of the mRNA transcript.

[0475] As used herein, the term "monocistronic" refers to an RNA or DNA construct that contains coding sequences for a single protein or polypeptide product.

[0476] As used herein, the term "monocyte" refers to a type of white blood cell (i.e., leukocyte) that can differentiate into macrophages and myeloid lineage dendritic cells. Monocytes constitute an important component of the vertebrate adaptive immune response. Three distinct types of monocytes are known to exist, including classical monocytes (i.e., CD14++CD16-), characterized by strong expression of the CD14 cell surface receptor and no expression of CD16, non-classical monocytes (CD14+CD16++), which show low levels of CD14 expression and co-expression of C16, and intermediate monocytes (CD14++CD16+), which show high levels of CD14 expression and low levels of C16 expression. Monocytes perform a variety of functions that serve the immune system, including phagocytosis, antigen presentation, and cytokine secretion.

[0477] As used herein, the term "pluripotent cells" refers to cells that have the capacity to develop into multiple (e.g., 2, 3, 4, 5, or more) differentiated cell types, although not all of them. Non-limiting examples of pluripotent cells include cells of the hematopoietic lineage (e.g., granulocytes (e.g., promyelocytes, neutrophils, eosinophils, basophils), erythroid (e.g., reticulocytes, erythrocytes), platelets (e.g., megakaryoblasts, thrombocytic megakaryocytes, platelets), monocytic (e.g., monocytes, macrophages), dendritic cells, microglia, osteoclasts, and lymphoid (e.g., NK cells, B cells, and T cells). An example of a pluripotent cell is a CD34+ cell.

[0478] As used herein, the term "mutation" refers to a change in the nucleotide sequence of a gene. Mutations in genes can occur naturally, for example, as a result of DNA replication errors, DNA repair, radiation, and exposure to carcinogens, or mutations can be induced as a result of administration of a transgene that expresses a mutated gene. Mutations can result from single nucleotide substitutions or deletions. Nomenclature for mutations and sequence variations uses a "reference sequence code" format, where the reference sequence can be "c" for coding DNA, and the code can include symbols including ">" for single nucleotide substitutions, "del" for deletions, or "a+b" for substitutions occurring within an intron, where x indicates a number corresponding to a nucleotide in the coding DNA sequence (e.g., a nucleotide within an exon of the coding DNA sequence), and y corresponds to the number of nucleotides 3' to x. For example, a TREM2 variant associated with a substitution described as c.482+2T>C has a T to C substitution at two nucleotides 3' to the nucleotide at position 482 of the coding DNA sequence. Mutations may also result in the replacement of a single amino acid in the peptide chain. The nomenclature for describing mutations resulting from amino acid replacement uses the "p.AnB" format, where "p" indicates the change at the protein level, "A" indicates the amino acid found in the wild-type variant of the protein, "n" indicates the number of the amino acid in the peptide chain, and "B" indicates the new amino acid resulting from the replacement. For example, the p.R47H variant of the TREM2 gene corresponds to a change in the protein at amino acid 47, where arginine is replaced by histidine.

[0479] As used herein, the term "myeloablative" or "myeloablation" refers to a conditioning regimen that substantially damages or destroys the hematopoietic system, typically by exposure to cytotoxic drugs (e.g., busulfan) or radiation. Myeloablation includes complete bone marrow destruction brought about by high doses of cytotoxic agents or total body irradiation that destroy the hematopoietic system.

[0480] As used herein, "Nasu-Hakola disease" and "PLOSL" refer to a neurodegenerative disorder characterized by the presence of white matter degeneration, axonal spherules, and cyst-like bone lesions in the upper and lower limbs. PLOSL patients exhibit early-onset dementia as well as recurrent fractures. PLOSL generally progresses through four distinct stages, including a latent stage in childhood followed by a bone symptom stage in adolescence during which patients may experience multiple joint pains in the hands, wrists, ankles, and feet. The bone symptom stage is followed by an early neurological symptom stage during which patients may exhibit prominent personality changes, progressive memory deficits, and epileptic seizures. The late neurological symptom stage of PLOSL patients exhibits prominent dementia and motor disability. Histopathological features of PLOSL include demyelination, axonal loss, the appearance of axonal spherules, fibrillary gliosis, and accumulation of lipid granules around blood vessels and within the neural tissue parenchyma. PLOSL patients also exhibit accumulation of lipid-laden macrophages and free fatty acids in the brain, along with vascular abnormalities in the frontal and temporal cerebral cortical regions. For a comprehensive overview of the clinical, pathological, and cellular features of PLOSL, see Bianchin et al., Cellular and Molecular Neurobiology 24:1-24 (2004).

[0481] As used herein, the term "neurocognitive disorder" or "NCD" refers to a set of clinical disorders or syndromes in which the primary clinical impairment is cognitive function, such as, for example, deficits in complex attention, executive function, learning and memory, language, sensorimotor function, and social cognition. NCDs are characterized as acquired conditions, not developmental conditions. For example, NCDs are conditions in which disrupted cognition has not been evident since birth or very early childhood, thus requiring that cognitive function in NCDs has declined from previously acquired levels. NCDs are distinguished from other disorders in which patients exhibit cognitive impairment, and NCDs include only disorders in which the core impairment is cognition. NCDs can be "severe NCDs" or "mild NCDs." "Severe NCDs" are characterized by significant cognitive decline that interferes with an individual's independence and / or normal daily functioning and is not due to delirium or other psychiatric disorders. Mild NCDs are characterized by moderate cognitive decline that does not interfere with an individual's independence and / or normal daily functioning and is not due to delirium or other psychiatric disorders. Severe and mild NCDs can also be distinguished based on quantitative cognitive testing of any one of the specific cognitive functions described above. For example, severe NCDs can be characterized by a score obtained on a cognitive test by a subject identified as having or at risk of developing an NCD being more than two standard deviations away from the mean score of a reference population (e.g., the mean score of the general population) or being in the third percentile of the distribution of scores of the reference population. Mild NCDs can be characterized by a score obtained on a cognitive test by a subject identified as having or at risk of developing an NCD being one to two standard deviations away from the mean score of the reference population or being between the third and sixteenth percentiles of the distribution of scores of the reference population. Non-limiting examples of cognitive tests that can be used to classify NCD patients as having either severe or mild NCD include AD8, AWV, GPCOG, HRA, MIS, MMSE, MoCA, SLUMS, and Short IQCODE. Additionally, NCDs include syndromic subtypes that represent specific etiologies of NCDs, such as AD or PLOSL.As used herein, the terms "NCD due to Alzheimer's disease" and "NCD due to leukodystrophy" correspond to NCDs resulting from AD and leukodystrophy (e.g., PLOSL), respectively.

[0482] As used herein, the terms "non-myeloablative" or "myelosuppressive" refer to conditioning regimens that do not eliminate substantially all hematopoietic cells of host origin.

[0483] As used herein, the term "sensory-motor function" refers to cognitive abilities that allow a subject (e.g., a human) to interact with the subject's environment using sensory and motor skills. Sensory-motor function encompasses the coordination of sensory and motor skills that allows a person to perform movements in response to the environmental context in which the subject is located. Sensory-motor function may include, but is not limited to, body awareness, spatial awareness, directional awareness, and time measurement. Specific manifestations of sensory-motor function may include, among others, throwing, catching, kicking, bouncing, swinging, cutting, tying laces, hammering, buttoning, pouring, naming, pointing, identifying, moving, performing tasks with body parts, searching, locating, comparing, walking, running, rolling, placing, balancing, clapping, hitting or chasing moving objects, matching visual and motor responses. Subjects diagnosed with NCD may exhibit impaired sensory-motor function compared to healthy subjects.

[0484] As used herein, the term "pluripotent cell" refers to a cell that has the capacity to develop into more than one differentiated cell type, such as hematopoietic (e.g., granulocytic (e.g., promyelocytes, neutrophils, eosinophils, basophils), erythroid (e.g., reticulocytes, red blood cells), platelets (e.g., megakaryoblasts, thrombocytic megakaryocytes, platelets), monocyte (e.g., monocytes, macrophages), dendritic cells, microglia, osteoclasts, and lymphocytic (e.g., NK cells, B cells, and T cells) cell types. Examples of pluripotent cells are ESCs and iPSCs.

[0485] As used herein, the term "plasmid" refers to an extrachromosomal circular double-stranded DNA molecule into which additional DNA segments can be ligated. A plasmid is a type of vector, a nucleic acid molecule that can transport another nucleic acid to which it is linked. Certain plasmids can autonomously replicate in a host cell into which they are introduced (e.g., bacterial plasmids with a bacterial origin of replication and episomal mammalian plasmids). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and are thereby replicated along with the host genome. Certain plasmids can direct the expression of genes to which they are operably linked.

[0486] As used herein, the term "polycistronic" refers to an RNA or DNA construct that contains coding sequences for at least two protein or polypeptide products.

[0487] As used herein, the term "promoter" refers to a recognition site on DNA to which RNA polymerase binds. The polymerase drives the transcription of the transgene. Exemplary promoters suitable for use in the compositions and methods described herein are described, for example, in Sandelin et al., Nature Reviews Genetics 8:424 (2007), which is incorporated by reference herein as its disclosure relates to nucleic acid regulatory elements. In addition, the term "promoter" can refer to synthetic promoters, which are regulatory DNA sequences that do not naturally occur in biological systems. Synthetic promoters include parts of naturally occurring promoters in combination with non-naturally occurring polynucleotide sequences and can be optimized to express recombinant DNA using a variety of transgenes, vectors, and target cell types.

[0488] "Percent sequence identity" with respect to a reference polynucleotide or polypeptide sequence is defined as the percentage of nucleic acids or amino acids in a candidate sequence that are identical to the nucleic acids or amino acids in the reference polynucleotide or polypeptide sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determining percent nucleic acid and amino acid sequence identity can be accomplished in a variety of ways that are within the ability of one of skill in the art, for example, using publicly available computer software such as BLAST, BLAST-2, or Megalign software. One of skill in the art can determine appropriate parameters for aligning sequences, including any algorithms required to obtain maximum alignment over the entire length of the sequences being compared. For example, percent sequence identity values ​​can be generated using the sequence comparison computer program BLAST. As an example, the percent sequence identity of a given nucleic acid or amino acid sequence A to, with, or against a given nucleic acid or amino acid sequence B (which may alternatively be expressed as a given nucleic acid or amino acid sequence A having a certain percent sequence identity to, with, or against a given nucleic acid or amino acid sequence B) is calculated as follows: Multiply by 100(fraction X / Y) where X is the number of nucleotides or amino acids in a programmatic alignment of A and B that are scored as identical matches by a sequence alignment program (e.g., BLAST), and Y is the total number of nucleic acids in B. It will be appreciated that if the length of nucleic acid or amino acid sequence A is not equal to the length of nucleic acid or amino acid sequence B, then the percent sequence identity of A to B will not equal the percent sequence identity of B to A.

[0489] As used herein, the term "pharmacologically acceptable" refers to compounds, materials, compositions and / or dosage forms that are suitable for contact with the tissues of a subject, such as a mammal (e.g., a human), without undue toxicity, irritation, allergic response, or other problematic complications, commensurate with a reasonable benefit / risk ratio.

[0490] As used herein, the term "pharmacologically acceptable" refers to compounds, materials, compositions and / or dosage forms that are suitable for contact with the tissues of a subject, such as a mammal (e.g., a human), without undue toxicity, irritation, allergic response, or other problematic complications, commensurate with a reasonable benefit / risk ratio.

[0491] As used herein, the potent "ApoE-derived receptor binding peptide (Rb)" has the ability to translocate proteins across the BBB to the brain when engineered as a fusion protein. Thus, this method can function to selectively open the BBB for therapeutic agents (e.g., soluble TREM2) when engineered as a fusion protein. Because this peptide utilizes the Rb domain of ApoE, rather than the entire ApoE protein, it can be easily conjugated to diagnostic or therapeutic agents without compromising or interfering with the important biological functions of ApoE. This route is also an alternative uptake route that can facilitate further / secondary brain distribution of agents after they reach the CNS due to the widespread expression of LDLRf members in the brain parenchyma. An exemplary Rb domain can be found in the N-terminus of ApoE. For example, an Rb domain useful in conjunction with the compositions and methods described herein is a polypeptide having the amino acid sequence of residues 1-191 of SEQ ID NO:13, residues 25-185 of SEQ ID NO:13, residues 50-180 of SEQ ID NO:13, residues 75-175 of SEQ ID NO:13, residues 100-170 of SEQ ID NO:13, or residues 125-165 of SEQ ID NO:13, as well as variants thereof, such as polypeptides having at least 85% sequence identity (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) with any of these sequences. An exemplary Rb domain is a region of ApoE having the amino acid sequence of residues 159-167 of SEQ ID NO:13.

[0492] As used herein, the term "regulatory sequence" includes promoters, enhancers and other expression control elements (e.g., polyadenylation signals) that control the transcription or translation of antibody chain genes. Such regulatory sequences are described, for example, in Perdew et al., Regulation of Gene Expression (Humana Press, New York, NY, (2014)), which is incorporated herein by reference.

[0493] As used herein, the term "sample" refers to a specimen isolated from a subject (e.g., blood, blood components (e.g., serum or plasma), urine, saliva, amniotic fluid, cerebrospinal fluid, tissue (e.g., placenta or skin), pancreatic juice, chorionic villus samples, and cells).

[0494] As used herein, the term "signal peptide" refers to a short (usually 16-30 amino acids) peptide region that directs the translocation of a translated protein from the host cytoplasm to a lipid membrane for anchorage. Such signal peptides are generally placed at the amino terminus of the newly translated protein. In some embodiments, the signal peptide is linked to the amino terminus. Typically, the signal peptide is cleaved during transit through the endoplasmic reticulum. Cleavage is not essential as long as the protein retains its desired activity. Exemplary signal peptides include the TREM2 signal peptide.

[0495] As used herein, the term "social cognition" refers to cognitive function encompassing a set of skills that determine how a subject (e.g., a human) processes, remembers, and adapts to information about other conspecific subjects (e.g., other humans) and social situations. Non-limiting examples of social cognition include, for example, emotional responses to social stimuli, processing abilities for theory of mind tasks, the ability to recognize faces, impulse control in social situations, and joint attention. Subjects who have been diagnosed with an NCD may exhibit impaired social cognition compared to healthy subjects.

[0496] As used herein, the term "splice variant" refers to the transcription products (i.e., RNA) of a single gene that can be processed to produce different mRNA molecules as a result of the alternative inclusion or exclusion of specific exons (e.g., exon skipping) within the precursor mRNA. The proteins produced from the translation of specific splice variants can differ in their structure and biological activity.

[0497] As used herein, the terms "stem cell" and "undifferentiated cell" refer to cells in an undifferentiated or partially differentiated state that have the developmental potential to differentiate into multiple cell types. Stem cells can proliferate while maintaining their functional potential to give rise to more such stem cells. Stem cells can divide asymmetrically, known as obligate asymmetric differentiation, with one daughter cell retaining the functional potential of the parent stem cell and the other daughter cell expressing another specific function, phenotype and / or developmental potential that is somewhat different from the parent cell. The daughter cells themselves can be induced to proliferate while also retaining one or more cells with the developmental potential of the parent, and to give rise to progeny that later differentiate into one or more mature cell types. Differentiated cells may themselves be derived from pluripotent cells, etc., that are themselves derived from pluripotent cells. Alternatively, some of the stem cells in a population can divide symmetrically into two stem cells. Thus, the term "stem cell" refers to any subset of cells that, under certain circumstances, have the developmental potential to differentiate into a more specialized or differentiated phenotype, and, under certain circumstances, retain the ability to proliferate without substantial differentiation. In some embodiments, the term stem cell generally refers to a naturally occurring parent cell whose descendants (progeny cells) specialize by differentiation, often in different directions, by acquiring completely different characteristics, as occurs in the gradual diversification of embryonic cells and tissues. Some differentiated cells also have the ability to give rise to cells of greater developmental potential. Such ability may be natural or may be artificially induced by treatment with various factors. Cells that begin as stem cells can progress to a differentiated phenotype, but can also be "reverted" and then induced to re-express the stem cell phenotype, a term often referred to by those skilled in the art as "dedifferentiation" or "reprogramming" or "reverse differentiation."

[0498] As used herein, the term "transfection" refers to any of a wide variety of techniques commonly used to introduce exogenous DNA into prokaryotic or eukaryotic host cells, such as electroporation, lipofection, calcium phosphate precipitation, DEAE-dextran transfection, Nucleofection, squeeze-poration, sonoporation, optical transfection, Magnetofection, impalefection, and the like.

[0499] As used herein, the term "transgene" refers to a recombinant nucleic acid (e.g., DNA or cDNA) that encodes a gene product (e.g., TREM2). The gene product may be an RNA, a peptide, or a protein. In addition to the coding region for the gene product, the transgene may include or be operably linked to one or more elements that facilitate or enhance expression, such as a promoter, enhancer(s), destabilization domain(s), response element(s), reporter element(s), insulator element(s), polyadenylation signal(s), and / or other functional elements. Embodiments of the present disclosure may utilize any known suitable promoter, enhancer(s), destabilization domain(s), response element(s), reporter element(s), insulator element(s), polyadenylation signal(s), and / or other functional elements.

[0500] As used herein, the terms "triggering receptor 2 expressed in myeloid cells" and "TREM2" refer to a transmembrane glycoprotein belonging to the immunoglobulin variable domain receptor family. Its gene is located on human chromosome 6p21.1. The terms "triggering receptor 2 expressed in myeloid cells" and "TREM2" also refer to splice variants resulting from alternative splicing of the TREM2 primary transcript, e.g., variant proteins having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.9% identity or more) to the amino acid sequence of the wild-type TREM2 peptide (e.g., any one of SEQ ID NOs: 1-3), or wild-type TREM2. It refers to variants of wild-type TREM2 peptides and nucleic acids encoding same, including polynucleotides having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.9% identity or more) to the nucleic acid sequence of the gene (e.g., any of the nucleic acid sequences selected from SEQ ID NOs: 4-6, 7, 9), provided that the encoded TREM2 isoform maintains the therapeutic function of wild-type TREM2. The terms "triggering receptor 2 expressed in myeloid cells" and "TREM2" may also refer to the TREM2 protein in which the native signal peptide is present. Additionally, the terms "triggering receptor 2 expressed in myeloid cells" and "TREM2" can refer to all products of TREM2 proteolytic cleavage, including soluble TREM2 (sTREM2), TREM2 C-terminal fragment (CTF), TREM2 intracellular domain (TREM2-ICD), and TREM2-A β-like peptide (T2β). TREM2 cleavage occurs when the mature polypeptide is translocated to the membrane after post-translational processing in the endoplasmic reticulum and is mediated by members of the disintegrin and metalloprotease (ADAM) family.The full-length TREM2 peptide is initially cleaved at the ectodomain to generate an extracellular sTREM2 peptide and a transmembrane TREM2-CTF, the latter of which can be further cleaved by the γ-secretase complex to generate a cytoplasmic TREM2-ICD and an extracellular TREM-T2β peptide. The terms "triggering receptor 2 expressed in myeloid cells" and "TREM2" can refer to a TREM2 protein that does not contain a functional ectodomain cleavage site. The terms "triggering receptor 2 expressed in myeloid cells" and "TREM2" can also refer to a TREM2 protein that does not contain a functional intramembrane cleavage site in the TREM2-CTF. In addition, the terms "triggering receptor 2 expressed in myeloid cells" and "TREM2" can refer to a "TREM2 fusion protein," which is a protein in which TREM2 is operably linked to another polypeptide, half-life modifying agent, or therapeutic agent, such as an ApoE Rb domain (e.g., an Rb domain having an amino acid sequence of residues 25-185, 50-180, 75-175, 100-170, 125-160, or 130-150 of SEQ ID NO: 13). As used herein, "TREM2" can refer to either the peptide or the gene encoding this protein, as appropriate, as will be appreciated by one of skill in the art.

[0501] As used herein, a subject suffering from "triggering receptor expressed in myeloid cells 2-related AD" and "TREM2-related AD or PLOSL" is a subject who has been diagnosed with AD or PLOSL and who also contains a deleterious mutation in the TREM2 gene. Over 40 mutations have been reported in the human TREM2 gene, which have various effects on downstream signaling, trafficking, ligand binding, and cell surface expression. TREM2 mutations are discussed in Guerreiro et al., The New England Journal of Medicine 368:117-27 (2013); Jonsson et al., The New England Journal of Medicine 368:107-16 (2013); Ulrich et al., Neuron Review 94:237-48 (2017); and Xing et al., Research and Reports in Biochemistry, 5:89-100 (2015), the disclosures of which are incorporated by reference herein as they relate to human TREM2 mutations in AD and PLOSL.

[0502] As used herein, the term "subject" or "patient" refers to an animal (e.g., a mammal, such as a human). The subject treated according to the methods described herein may be a subject who has been diagnosed with an NCD or who is at risk for developing these conditions. Diagnosis may be performed by any method or technique known in the art. One of skill in the art will understand that the subject treated according to the present disclosure may have undergone standard testing or may have been identified as at risk by the presence of one or more risk factors associated with a disease or condition without testing.

[0503] As used herein, the terms "transduction" and "transducing" refer to the method of introducing a viral vector construct, or a portion thereof, into a cell and the subsequent expression in the cell of a transgene encoded by the vector construct, or a portion thereof.

[0504] As used herein, "treatment" or "treating" refers to an approach to obtain a beneficial or desired result, e.g., a clinical result. Beneficial or desired results include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions, whether detectable or undetectable; reduction in the extent of the disease or condition; stabilization (i.e., not worsening) of the disease, disorder, or condition; prevention of the progression of the disease or condition; delay or slowing of the progression of the disease or condition; amelioration or palliation of the disease or condition; and remission (whether partial or complete). "Ameliorating" or "alleviating" a disease or condition means that the extent and / or undesirable clinical signs of the disease, disorder, or condition are reduced and / or the time course of progression is slowed or prolonged, compared to the extent or time course in the absence of treatment. "Treatment" can also mean prolonging survival, compared to the expected survival in the absence of treatment. Those in need of treatment include those already with the condition or disorder, as well as those susceptible to having the condition or disorder, or those in need of prevention of the condition or disorder.

[0505] As used herein, the term "vector" includes nucleic acid vectors, e.g., DNA vectors such as plasmids, RNA vectors, viruses, or other suitable replicons (e.g., viral vectors). A variety of vectors have been developed for the delivery of polynucleotides encoding exogenous proteins into prokaryotic or eukaryotic cells. Examples of such expression vectors are disclosed, for example, in WO1994 / 011026, which is incorporated herein by reference as it relates to vectors suitable for expressing genes of interest. Expression vectors suitable for use in the compositions and methods described herein include polynucleotide sequences as well as additional sequence elements used, for example, for the expression of proteins and / or the integration of these polynucleotide sequences into the genome of mammalian cells. Certain vectors that can be used for the expression of TREM2 described herein include plasmids that include regulatory sequences that direct gene transcription, such as promoter and enhancer regions. Other useful vectors for the expression of TREM2 include polynucleotide sequences that increase the translation rate of these genes or improve the stability or nuclear export of mRNA resulting from gene transcription. These sequence elements include, for example, 5' and 3' untranslated regions, internal ribosome entry sites (IRES), and polyadenylation signal sites to direct efficient transcription of genes carried in the expression vector. Expression vectors suitable for use in the compositions and methods described herein may also contain a polynucleotide encoding a marker for selecting cells containing such a vector. Examples of suitable markers are genes encoding resistance to antibiotics such as ampicillin, chloramphenicol, kanamycin, nourseothricin, or zeocin.

[0506] Detailed Description Described herein are compositions and methods for treating a neurocognitive disorder (NCD), such as Alzheimer's disease (AD) or Nasu-Hakola disease, also known as polycystic lipomembranous skeletal dysplasia with sclerosing leukoencephalopathy (PLOSL), in a subject (such as a mammalian subject, e.g., a human). The compositions and methods described herein can be used to treat an NCD (e.g., AD (e.g., triggering receptor expressed on myeloid cells 2 (TREM2)-associated AD), PLOSL (e.g., TREM2-associated PLOSL), frontotemporal lobar degeneration (FTLD; e.g., TREM2-associated FTLD), or Parkinson's disease (PD; e.g., TREM2-associated PD)) in a subject (e.g., a human subject) by administering a cell (e.g., a pluripotent cell, an embryonic stem cell (ESC), an induced pluripotent stem cell (iPSC), a pluripotent cell, a CD34+ cell, a hematopoietic stem cell (HSC), a myeloid progenitor cell (MPC), a blood lineage progenitor cell (BLPC), a monocyte, a macrophage, a microglial progenitor cell, or a microglia) that includes a transgene encoding TREM2 (e.g., a transgene that can express TREM2 in macrophages or microglial cells). For example, compositions containing cells that have been modified ex vivo to express TREM2 are described herein. The next section describes in further detail compositions and methods useful for treating NCDs.

[0507] Neurocognitive disorders NCDs are defined as a collection of disorders characterized by cognitive impairment as a core symptom and showing cognitive decline (e.g., acquired impairment) compared to a previous higher level of cognition, rather than developmental impairment. NCDs are broadly divided into severe or mild syndromes (e.g., severe NCD and mild NCD) based on the degree of impairment diagnosed in the subject. Furthermore, NCDs can be classified based on their etiology. For example, non-limiting examples of NCDs can include NCD due to AD, NCD due to leukodystrophy (e.g., PLOSL), vascular NCD, NCD with Lewy bodies, NCD due to Parkinson's disease, frontotemporal NCD, NCD due to traumatic brain injury, NCD due to HIV infection, substance / drug induced NCD, NCD due to Huntington's disease, NCD due to prion disease, NCD due to another medical condition, NCD due to multiple etiologies, and unspecified NCD. The compositions and methods disclosed herein are useful for treating NCDs.

[0508] Alzheimer's disease AD is an NCD characterized by progressive neuronal loss in the frontal, temporal, and parietal lobes of the cerebral cortex, as well as in subcortical structures such as the basal forebrain cholinergic system and the locus coeruleus in the brainstem. Clinical symptoms of AD are progressive declines in several cognitive functions, including short-term and long-term memory, spatial navigation, verbal fluency, impulse control, anhedonia, and social withdrawal. Neuronal atrophy in the brains of AD patients is associated with the accumulation of extracellular and intracellular protein inclusions. Aggregates of insoluble amyloid-β (Aβ) protein are often found in the extracellular space, while neurofibrillary tangles (NFTs) of hyperphosphorylated tau protein are usually found in the intracellular compartment of affected neurons. These neuropathologies are deemed important in the pathogenesis of AD.

[0509] The likelihood of developing AD is strongly influenced by genetic factors. Known mutations in genes encoding the amyloid precursor protein (APP) or the proteolytic enzymes that cleave APP, such as presenilin-1 (PSEN1) and presenilin-2 (PSEN2), have been established as risk factors for early-onset AD. These mutations mediate the production of pathogenic Aβ, the main component of Aβ deposits in the brain. 42 It is associated with increased accumulation of the isoform. An increased risk for late-onset AD is strongly associated with alterations in the ε4 allele of apolipoprotein-E (APOE).

[0510] Nasu Hakola Disease Nasu-Hakola disease (PLOSL) is a rare autosomal recessive leukodystrophy characterized by the presence of white matter degeneration, loss of axons and myelin, the presence of axonal spherules, and even cystic bone lesions in the distal extremities. Clinical symptoms in PLOSL patients include early-onset dementia and even recurrent fractures. Unlike AD, which affects older patients more widely, PLOSL may begin to show symptoms during adolescence during the bone symptom stage, during which patients may experience multiple joint pains in the hands, wrists, ankles, and feet. The bone symptom stage is followed by an early neurological symptom stage, during which patients may exhibit prominent personality changes, progressive memory deficits, and generalized epileptic seizures. The late neurological symptom stage in PLOSL patients corresponds to prominent dementia, motor disability, and ultimately death.

[0511] Myeloid cell-expressed triggering receptor 2-associated Alzheimer's disease Recent studies based on genome and exome sequencing have revealed TREM2 as a significant risk factor in the development of NCDs (e.g., AD, PLOSL, FTLD, or PD). Since then, multiple variants in the TREM2 gene have been associated with an increased risk for AD, with the most common variant being the rs75932628 single nucleotide polymorphism, which results in an arginine to histidine substitution at amino acid 47 (R47H). This mutation is believed to have a strong impact on the ligand-binding properties of TREM2. Other TREM2 variants have been found to result in protein truncation and / or misfolding, disrupted trafficking, reduced cell surface expression, and increased or reduced activation of downstream signaling pathways. TREM2 is a transmembrane protein expressed on mononuclear phagocytes (e.g., microglia, osteoclasts, and alveolar macrophages) and can be activated by lipids or lipoproteins on its extracellular domain. Containing no intracellular signaling domain, TREM2 exerts its effects on multiple intracellular signaling pathways through interaction with the transmembrane adaptor protein DAP12. Furthermore, membrane-bound TREM2 can be cleaved by extracellular proteases to generate soluble TREM2 (sTREM2) and transmembrane C-terminal fragments (TREM2-CTFs), which can be further cleaved by the γ-secretase complex to generate cytoplasmic TREM2 intracellular domain (TREM2-ICD) and extracellular TREM2-A β-like (TREM2-T2β) peptides. TREM2 activity is thought to be important for several intracellular functions, including control of phagocytosis, suppression of inflammatory signals, and cell survival. AD- and PLOSL-associated mutations in TREM2 are associated with attenuated or enhanced TREM2 activity, implicating the importance of TREM2 homeostasis in the pathogenesis of AD and PLOSL. Furthermore, AD patients with the R47H TREM2 variant have reduced microglial recruitment to Aβ plaques, suggesting that altered TREM2 activity may impair normal microglial functioning.Similarly, PLOSL is associated with the histopathological presence of lipid-laden macrophages, suggesting immune dysregulation.Proteolytic processing of TREM2 may also be altered in AD. Levels of sTREM2 appear to be elevated in the cerebrospinal fluid of AD patients and show a correlation with the levels of phosphorylated tau protein in the brain. TREM2 involvement in NCDs (e.g., AD and PLOSL) is discussed in detail in Ulrich et al., Neuron Reviews 94:237-48 and Xing et al., Research and Reports in Biochemistry 5:89-100 (2015), the disclosures of which relate to human TREM2 signaling in AD and are incorporated herein by reference.

[0512] Clinical management of NCDs employs pharmacological and behavioral interventions to alleviate disease symptoms. For example, acetylcholinesterase inhibitors are used to increase acetylcholine levels in the brain as a means to ameliorate cognitive impairment in AD, since this neurotransmitter is found to be deficient in AD patients. In addition, atypical antipsychotics are commonly prescribed to AD patients for behavioral management. Similarly, anticonvulsants are administered to PLOSL patients to control spontaneous occurrence of epileptic seizures. However, this strategy targets to ameliorate the symptoms of the disease without addressing its onset and progression. Unlike these treatments, the compositions and methods described herein offer the advantage of treating another biochemical phenomenon that may underlie the development of NCDs. Thus, the compositions and methods described herein target the physiological causes of the disease and represent a potential curative treatment.

[0513] The compositions and methods described herein can be used to treat NCDs by administering cells (e.g., pluripotent cells, ESCs, iPSCs, pluripotent cells, CD34+ cells, HSCs, MPCs, BLPCs, monocytes, macrophages, microglial progenitor cells, or microglia) that contain a transgene encoding TREM2 (such as a transgene that can be expressed in macrophages or microglial cells). These compositions and methods can be used to treat NCDs of any etiology, e.g., genetic mutation, environmental toxins, or sporadic. These compositions and methods can also be used to treat subjects with TREM2-associated AD or PLOSL. The compositions and methods described herein can be used to treat subjects with normal TREM2 activity, reduced TREM2 activity, and subjects whose TREM2 mutation status and / or TREM2 activity level is unknown. The compositions and methods described herein can also be administered as a preventative treatment to subjects at risk of developing an NCD, such as subjects with TREM2 mutations, subjects with reduced TREM2 activity, and subjects with mutations in one or more of the genes associated with an NCD.

[0514] TREM2-encoding constructs that can be used in conjunction with the compositions and methods described herein include polynucleotides encoding wild-type TREM2 (any one of the amino acid sequences set forth as SEQ ID NOs: 1-3) or variants thereof, such as polynucleotides encoding proteins having at least 85% sequence identity (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to any of the amino acid sequences of SEQ ID NOs: 1-3.

[0515] In some embodiments, TREM2 has the amino acid sequence of SEQ ID NO:1 or a variant thereof having at least 85% (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID NO:1.

[0516] In some embodiments, TREM2 has the amino acid sequence of SEQ ID NO:1 or a variant thereof having at least 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO:1.

[0517] In some embodiments, TREM2 has the amino acid sequence of SEQ ID NO:1 or a variant thereof having at least 95% (e.g., at least 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO:1.

[0518] In some embodiments, TREM2 has the amino acid sequence of SEQ ID NO:1.

[0519] In some embodiments, TREM2 has the amino acid sequence of SEQ ID NO:2 or a variant thereof having at least 85% (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID NO:2.

[0520] In some embodiments, TREM2 has the amino acid sequence of SEQ ID NO:2 or a variant thereof having at least 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO:2.

[0521] In some embodiments, TREM2 has the amino acid sequence of SEQ ID NO:2 or a variant thereof having at least 95% (e.g., at least 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO:2.

[0522] In some embodiments, TREM2 has the amino acid sequence of SEQ ID NO:2.

[0523] In some embodiments, TREM2 has the amino acid sequence of SEQ ID NO:3 or a variant thereof having at least 85% (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID NO:3.

[0524] In some embodiments, TREM2 has the amino acid sequence of SEQ ID NO:3 or a variant thereof having at least 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO:3.

[0525] In some embodiments, TREM2 has the amino acid sequence of SEQ ID NO:3 or a variant thereof having at least 95% (e.g., at least 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO:3.

[0526] In some embodiments, TREM2 has the amino acid sequence of SEQ ID NO:3.

[0527] In some embodiments, the transgene encoding TREM2 comprises a TREM2 polynucleotide having the nucleotide sequence of SEQ ID NO:4, or a variant thereof having at least 85% (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO:4.

[0528] In some embodiments, the transgene encoding TREM2 comprises a TREM2 polynucleotide having the nucleotide sequence of SEQ ID NO:4, or a variant thereof having at least 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO:4.

[0529] In some embodiments, the transgene encoding TREM2 comprises a TREM2 polynucleotide having the nucleotide sequence of SEQ ID NO:4, or a variant thereof having at least 95% (e.g., at least 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO:4.

[0530] In some embodiments, the transgene encoding TREM2 comprises a TREM2 polynucleotide having the nucleotide sequence of SEQ ID NO:4.

[0531] In some embodiments, the transgene encoding TREM2 comprises a TREM2 polynucleotide having the nucleotide sequence of SEQ ID NO:5, or a variant thereof having at least 85% (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO:5.

[0532] In some embodiments, the transgene encoding TREM2 comprises a TREM2 polynucleotide having the nucleotide sequence of SEQ ID NO:5, or a variant thereof having at least 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO:5.

[0533] In some embodiments, the transgene encoding TREM2 comprises a TREM2 polynucleotide having the nucleotide sequence of SEQ ID NO:5, or a variant thereof having at least 95% (e.g., at least 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO:5.

[0534] In some embodiments, the transgene encoding TREM2 comprises a TREM2 polynucleotide having the nucleotide sequence of SEQ ID NO:5.

[0535] In some embodiments, the transgene encoding TREM2 comprises a TREM2 polynucleotide having the nucleotide sequence of SEQ ID NO:6, or a variant thereof having at least 85% (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO:6.

[0536] In some embodiments, the transgene encoding TREM2 comprises a TREM2 polynucleotide having the nucleotide sequence of SEQ ID NO:6, or a variant thereof having at least 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO:6.

[0537] In some embodiments, the transgene encoding TREM2 comprises a TREM2 polynucleotide having the nucleotide sequence of SEQ ID NO:6, or a variant thereof having at least 95% (e.g., at least 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO:6.

[0538] In some embodiments, the transgene encoding TREM2 comprises a TREM2 polynucleotide having the nucleotide sequence of SEQ ID NO:6.

[0539] In some embodiments, the transgene encoding TREM2 comprises a TREM2 polynucleotide having the nucleotide sequence of SEQ ID NO:7, or a variant thereof having at least 85% (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID NO:7.

[0540] In some embodiments, the transgene encoding TREM2 comprises a TREM2 polynucleotide having the nucleotide sequence of SEQ ID NO:7, or a variant thereof having at least 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO:7.

[0541] In some embodiments, the transgene encoding TREM2 comprises a TREM2 polynucleotide having the nucleotide sequence of SEQ ID NO:7, or a variant thereof having at least 95% (e.g., at least 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO:7.

[0542] In some embodiments, the transgene encoding TREM2 comprises a TREM2 polynucleotide having the nucleotide sequence of SEQ ID NO:7.

[0543] In some embodiments, the transgene encoding TREM2 comprises a TREM2 polynucleotide having the nucleotide sequence of SEQ ID NO:9, or a variant thereof having at least 85% (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID NO:9.

[0544] In some embodiments, the transgene encoding TREM2 comprises a TREM2 polynucleotide having the nucleotide sequence of SEQ ID NO:9, or a variant thereof having at least 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO:9.

[0545] In some embodiments, the transgene encoding TREM2 comprises a TREM2 polynucleotide having the nucleotide sequence of SEQ ID NO:9, or a variant thereof having at least 95% (e.g., at least 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO:9.

[0546] In some embodiments, the transgene encoding TREM2 comprises a TREM2 polynucleotide having the nucleotide sequence of SEQ ID NO:9.

[0547] In some embodiments, the transgene encoding TREM2 comprises a TREM2 polynucleotide having the nucleotide sequence of SEQ ID NO:11, or a variant thereof having at least 85% (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO:11.

[0548] In some embodiments, the transgene encoding TREM2 comprises a TREM2 polynucleotide having the nucleotide sequence of SEQ ID NO:11, or a variant thereof having at least 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO:11.

[0549] In some embodiments, the transgene encoding TREM2 comprises a TREM2 polynucleotide having the nucleotide sequence of SEQ ID NO:11, or a variant thereof having at least 95% (e.g., at least 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO:11.

[0550] In some embodiments, the transgene encoding TREM2 comprises a TREM2 polynucleotide having the nucleotide sequence of SEQ ID NO:11.

[0551] In some embodiments, the transgene encoding TREM2 may be codon optimized (eg, any one of SEQ ID NO:8, SEQ ID NO:10, or SEQ ID NO:12).

[0552] In some embodiments, the transgene encoding TREM2 comprises a codon-optimized TREM2 polynucleotide sequence of SEQ ID NO:8 or a variant thereof having at least 85% (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO:8.

[0553] In some embodiments, the transgene encoding TREM2 comprises a codon-optimized TREM2 polynucleotide sequence of SEQ ID NO:8 or a variant thereof having at least 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO:8.

[0554] In some embodiments, the transgene encoding TREM2 comprises a codon-optimized TREM2 polynucleotide sequence of SEQ ID NO:8 or a variant thereof having at least 95% (e.g., at least 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO:8.

[0555] In some embodiments, the transgene encoding TREM2 comprises the codon-optimized TREM2 polynucleotide sequence of SEQ ID NO:8.

[0556] In some embodiments, the transgene encoding TREM2 comprises a codon-optimized TREM2 polynucleotide sequence of SEQ ID NO:10, or a variant thereof having at least 85% (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO:10.

[0557] In some embodiments, the transgene encoding TREM2 comprises a codon-optimized TREM2 polynucleotide sequence of SEQ ID NO:10, or a variant thereof having at least 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO:10.

[0558] In some embodiments, the transgene encoding TREM2 comprises a codon-optimized TREM2 polynucleotide sequence of SEQ ID NO:10, or a variant thereof having at least 95% (e.g., at least 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO:10.

[0559] In some embodiments, the transgene encoding TREM2 comprises the codon-optimized TREM2 polynucleotide sequence of SEQ ID NO:10.

[0560] In some embodiments, the transgene encoding TREM2 comprises a codon-optimized TREM2 polynucleotide sequence of SEQ ID NO:12 or a variant thereof having at least 85% (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO:12.

[0561] In some embodiments, the transgene encoding TREM2 comprises a codon-optimized TREM2 polynucleotide sequence of SEQ ID NO:12 or a variant thereof having at least 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO:12.

[0562] In some embodiments, the transgene encoding TREM2 comprises a codon-optimized TREM2 polynucleotide sequence of SEQ ID NO:12 or a variant thereof having at least 95% (e.g., at least 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO:12.

[0563] In some embodiments, the transgene encoding TREM2 comprises the codon-optimized TREM2 polynucleotide sequence of SEQ ID NO:12.

[0564] In some embodiments, the transgene encodes two or more TREM2 transgenes (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or more TREM2 transgenes). In some embodiments, the transgene encodes between 2 and 10 TREM2 transgenes (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 TREM2 transgenes). In some embodiments, the transgene encodes between 2 and 5 TREM2 transgenes (e.g., 2, 3, 4, or 5 TREM2 transgenes). In some embodiments, the transgene encodes two TREM2 transgenes. In some embodiments, the TREM2 transgenes are expressed from a single polycistronic expression cassette. In some embodiments, the TREM2 transgenes are separated from each other by one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or more) IRESs. In some embodiments, the TREM2 transgene is expressed from one or more (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) monocistronic expression cassettes.

[0565] In some embodiments, the polynucleotide encoding TREM2 encodes sTREM2. In some embodiments, the polynucleotide encoding TREM2 encodes a TREM2-CTF. In some embodiments, the polynucleotide encoding TREM2 encodes a TREM2-ICD. In some embodiments, the polynucleotide encoding TREM2 encodes a TREM2-T2β peptide. In some embodiments, the polynucleotide encoding TREM2 encodes a TREM2 polypeptide that does not include a functional ectodomain cleavage site. In some embodiments, the polynucleotide encoding TREM2 encodes a TREM2 polypeptide that does not include a functional intramembrane cleavage site in the TREM2-CTF. In some embodiments, the polynucleotide encoding wild-type TREM2 may be a codon-optimized polynucleotide to confer resistance to degradation by endogenous TREM2-directed nucleases and inhibitory RNAs, as described in more detail below.

[0566] Wild-type human TREM2 may have the canonical amino acid sequence of the following (UniProt identifier number: Q9NZC2-1): MEPLRLLILLFVTELSGAHNTTVFQGVAGQSLQVSCPYDSMKHWGRRKAWCRQLGEKGPCQRVVSTHNLWLLSFLRRWNGSTAITDDTLGGTLTITLRNLQPHDAGLYQCQSLHG SEADTLRKVLVEVLADPLDHRDAGDLWFPGESESFEDAHVEHSISRSLLEGEIPFPPTSILLLLACIFLIKILAASALWAAAWHGQKPGTHPPSELDCGHDPGYQLQTLPGLRDT (SEQ ID NO:1)

[0567] Additionally or alternatively, human TREM2 may have the amino acid sequence of the following (UniProt Identifier Number: Q9NZC2-2): MEPLRLLILLFVTELSGAHNTTVFQGVAGQSLQVSCPYDSMKHWGRRKAWCRQLGEKGPCQRVVSTHNLWLLSFLRRWNGSTAITDDTLGGTLTITLRNLQPHDAGLYQ CQSLHGSEADTLRKVLVEVLADPLDHRDAGDLWFPGESESFEDAHVEHSISRAERHVKEDDGRKSPGEVPPGTSPACILATWPPGLLVLLWQETTLPEHCFSWTLEAGTG (SEQ ID NO:2)

[0568] Additionally or alternatively, human TREM2 may have the amino acid sequence of the following (UniProt Identifier Number: Q9NZC2-3): MEPLRLLILLFVTELSGAHNTTVFQGVAGQSLQVSCPYDSMKHWGRRKAWCRQLGEKGPCQRVVSTHNLWLLSFLRRWNGSTAITDDTLGGTLTITLRNLQPHDAGLYQCQ SLHGSEADTLRKVLVEVLADPLDHRDAGDLWFPGESESFEDAHVEHSISRPSQGSHLPSCLSKEPLGRRNPLPTHFHPSPPGHLSHQDSSSQRPLGCSLAWTEARDTSTQ (SEQ ID NO:3)

[0569] The polynucleotide encoding TREM2 may have the nucleic acid sequence of the following (Ensembl Identifier Number: ENST00000373113.7): (SEQ ID NO:4)

[0570] Additionally or alternatively, human TREM2 may have the nucleotide sequence of the following (Ensembl Identifier Number: ENST00000338469.3): GGGCAGCGCCTGACATGCCTGATCCTTCTTTTCTGCAGTTCAAGGGAAAGACGAGATCTTGCACAAGGCACTCTGCTTCTGCCCTTGGCTGGGGAAGGGTGGCATGGAGCCTCTCCGGCTGCTCATCTTACTCTTTGTCACAGAGCTGTCCGGAGCCCACAACACCACAGTGTTCCAGGGCGTGGCGGGCCAGTCCCTGCAGGTGTCTTGCCCC TATGACTCCATGAAGCACTGGGGGAGGCGCAAGGCCTGGTGCCGCCAGCTGGGAGAGAAGGGCCCATGCCAGCGTGTGGTCAGCACGCACAACTTGTGGCTGCTGTCCTTCCTGAGGAGGTGGAATGGGAGCACAGCCATCACAGACGATACCCTGGGTGGCACTCTCACCATTACGCTGCGGAATCTACAACCCCATGATGCGGGTCTCTACCA GTGCCAGAGCCTCCATGGCAGTGAGGCTGACACCCTCAGGAAGGTCCTGGTGGAGGTGCTGGCAGACCCCCTGGATCACCGGGATGCTGGAGATCTCTGGTTCCCCGGGGAGTCTGAGAGCTTCGAGGATGCCCATGTGGAGCACAGCATCTCCAGGGCTGAGAGACACGTGAAGGAAGATGATGGGAGGAAAAGCCCAGGAGAAGTCCCACCAG GGACCAGCCAGCCTGCATACTTGCCACTTGGCCACCAGGACTCCTTGTTCTGCTCTGGCAAGAGACTACTCTGCCTGAACACTGCTTCTCCTGGACCCTGGAAGCAGGGACTGGTTGAGGGAGTGGGGAGGTGGTAAGAACACCTGACAACTTCTGAATATTGGACATTTTAAACACTTACAAATAAATCCAAGACTGTCATATTTAGCTGGAT (SEQ ID NO:5)

[0571] Additionally or alternatively, human TREM2 may have the nucleotide sequence of the following (Ensembl Identifier Number: ENST00000373122.8): (SEQ ID NO:6)

[0572] Additionally, or alternatively, a polynucleotide encoding TREM2 may have the following sequence: ATGGAGCCTCTCCGGCTGCTCATCTTACTCTTTGTCACAGAGCTGTCCGGAGCCCACAACACCACAGTGTTCCAGGGCGTGGCGGGCCAGTCCCTGCAGGTGTCTTGCCCTATGACTCCATGAAGCACTGGGGGAGGCGCAAGGCCTGGTGCCGCCAGCTGGGAGAGAAGGGC CCATGCCAGCGTGTGGTCAGCACGCACAACTTGTGGCTGCTGTCCTTCCTGAGGAGGTGGAATGGGAGCACAGCCATCACAGACGATACCCTGGGTGGCACTCTCACCATTACGCTGCGGAATCTACAACCCCATGATGCGGGTCTCTACCAGTGCCAGAGCCTCCATGGCAGT GAGGCTGACACCCTCAGGAAGGTCCTGGTGGAGGTGCTGGCAGACCCCCTGGATCACCGGGATGCTGGAGATCTCTGGTTCCCCGGGGAGTCTGAGAGCTTCGAGGATGCCCATGTGGAGCACAGCATCTCCAGGAGCCTCTTGGAAGGAGAAATCCCCTTCCCACCCACTTCC ATCCTTCTCCTCCTGGCCTGCATCTTTCTCATCAAGATTCTAGCAGCCAGCGCCCTCTGGGCTGCAGCCTGGCATGGACAGAAGCCAGGGACACATCCACCCAGTGAACTGGACTGTGGCCATGACCCAGGGTATCAGCTCCAAACTCTGCCAGGGCTGAGAGACACGTGATGA (SEQ ID NO:7)

[0573] Additionally, or alternatively, a polynucleotide encoding TREM2 may have the codon-optimized nucleotide sequence of SEQ ID NO:8: ATGGAGCCTCTGAGACTGCTGATTCTGCTGTTTGTCACTGAACTGAGCGGCGCACATAATACCACTGTCTTCCAGGGCGTCGCTGGGCAGTCTCTGCAGGTGAGCTGCCCCTACGACTCTATGAAGCACTGGGGCCGGAGAAAGGCATGGTGCCGGCAGCTGGGAGAGAAGGGA CCTTGTCAGAGAGTGGTGAGCACCCACAACCTGTGGCTGCTGTCCTTCCTGAGGCGCTGGAATGGCTCTACAGCCATCACCGACGATACACTGGGCGGCACCCTGACAATCACCCTGAGGAACCTGCAGCCTCACGACGCAGGCCTGTATCAGTGCCAGTCCCTGCACGGCTCT GAGGCCGATACACTGAGGAAGGTGCTGGTGGAGGTGCTGGCCGACCCTCTGGATCACAGGGACGCAGGCGATCTGTGGTTCCCAGGCGAGAGCGAGTCCTTTGAGGATGCCCACGTGGAGCACTCTATCAGCCGGTCCCTGCTGGAGGGAGAGATCCCATTCCCCCCTACCAGC ATCCTGCTGCTGCTGGCCTGTATCTTTCTGATCAAGATCCTGGCAGCATCCGCCCTGTGGGCAGCAGCCTGGCACGGACAGAAGCCAGGAACACACCCACCATCCGAGCTGGATTGCGGACATGACCCCGGCTACCAGCTGCAGACACTGCCTGGCCTGAGGGATACATGATGA (SEQ ID NO:8)

[0574] Additionally, or alternatively, a polynucleotide encoding TREM2 may have the following nucleotide sequence: ATGGAGCCTCTCCGGCTGCTCATCTTACTCTTTGTCACAGAGCTGTCCGGAGCCCACAACACCACAGTGTTCCAGGGCGTGGCGGGCCAGTCCCTGCAGGTGTCTTGCCCTATGACTCCATGAAGCACTGGGGGAGGCGCAAGGCCTGGTGCCGCCAGCTGGGA GAGAAGGGCCCATGCCAGCGTGTGGTCAGCACGCACAACTTGTGGCTGCTGTCCTTCCTGAGGAGGTGGAATGGGAGCACAGCCATCACAGACGATACCCTGGGTGGCACTCTCACCATTACGCTGCGGAATCTACAACCCCATGATGCGGGTCTCTACCAGTGCC AGAGCCTCCATGGCAGTGAGGCTGACACCCTCAGGAAGGTCCTGGTGGAGGTGCTGGCAGACCCCCTGGATCACCGGGATGCTGGAGATCTCTGGTTCCCCGGGGAGTCTGAGAGCTTCGAGGATGCCCATGTGGAGCACAGCATCTCCAGGGCTGAGAGACACGT GAAGGAAGATGATGGGAGGAAAAGCCCAGGAGAAGTCCCACCAGGGACCAGCCAGGCCTGCATACTTGCCACTTGGCCACCAGGACTCCTTGTTCTGCTCTGGCAAGAGACTACTCTGCCTGAACACTGCTTCTCCTGGACCCTGGAAGCAGGGACTGGTTGATGA (SEQ ID NO:9)

[0575] Additionally, or alternatively, a polynucleotide encoding TREM2 may have the codon-optimized nucleotide sequence of SEQ ID NO: 10: ATGGAGCCTCTGCGGCTGCTGATCCTGCTGTTCGTGACCGAGCTGTCCGGCGCCCACAACACCACAGTGTTTCAGGGAGTGGCAGGACAGTCCCTGCAGGTGTCTTGCCCATACGACTCTATGAAGCACTGGGGCCGGAGAAAGGCATGGTGCAGGCAGCTGGGA GAGAAGGGACCATGTCAGCGCGTGGTGTCTACACACAACCTGTGGCTGCTGAGCTTCCTGAGGCGCTGGAATGGCTCCACAGCCATCACCGACGATACACTGGGCGGCACCCTGACAATCACCCTGAGGAATCTGCAGCCACACGACGCCGGCCTGTATCAGTGTC AGAGCCTGCACGGCTCCGAGGCAGATACCCTGCGGAAGGTGCTGGTGGAGGTGCTGGCCGACCCCCTGGATCACAGAGACGCAGGCGATCTGTGGTTCCCTGGCGAGAGCGAGTCCTTTGAGGATGCCCACGTGGAGCACTCTATCAGCCGGGCCGAGAGACACGT GAAGGAGGACGATGGAAGGAAGTCTCCTGGAGAGGTGCCACCTGGAACCAGCCCAGCATGCATCCTGGCAACATGGCCACCAGGCCTGCTGGTGCTGCTGTGGCAGGAGACAACACTGCCCGAGCACTGTTTTTCCTGGACCCTGGAGGCCGGCACAGGCTGATGA (SEQ ID NO:10)

[0576] Additionally or alternatively, a polynucleotide encoding TREM2 may have the nucleotide sequences of SEQ ID NO:7 and SEQ ID NO:9, separated by an IRES sequence and which together have the following nucleotide sequence: (SEQ ID NO:11)

[0577] Additionally or alternatively, a polynucleotide encoding TREM2 may have the nucleotide sequences of SEQ ID NO:8 and SEQ ID NO:10, separated by an IRES sequence and which together have the following nucleotide sequence: (SEQ ID NO:12)

[0578] According to the methods described herein, a subject can be administered a cell containing a transgene comprising a polynucleotide encoding a polypeptide having any one of SEQ ID NOs: 1-3, or a polynucleotide encoding a polypeptide having at least 85% sequence identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to any one of the amino acid sequences of SEQ ID NOs: 1-3, or a polynucleotide encoding a polypeptide containing one or more conservative amino acid substitutions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more conservative amino acid substitutions) to any one of SEQ ID NOs: 1-3, provided that the encoded TREM2 analog maintains the therapeutic function of wild-type TREM2. TREM2 activity is important for normal microglial phagocytic capacity and regulation of inflammatory cytokine production. Loss of TREM2 results in altered neuroimmune responses and neurodegeneration.

[0579] host cell Cells that can be used in conjunction with the compositions and methods described herein include cells that can undergo further differentiation (e.g., pluripotent cells, ESCs, iPSCs, CD34+ cells, HSCs, MPCs, BLPCs, monocytes, or microglial progenitors) or differentiated cells (e.g., macrophages or microglia). For example, one type of cell that can be used in conjunction with the compositions and methods described herein is a pluripotent cell. A pluripotent cell is a cell that has the ability to develop into more than one differentiated cell type. Examples of pluripotent cells are ESCs and iPSCs. ESCs and iPSCs have the ability to differentiate into cells of the ectoderm that gives rise to the skin and nervous system, the endoderm that forms the digestive tract and airway, the endocrine glands, the liver, and the pancreas, and the mesoderm that forms bone, cartilage, muscle, connective tissue, and most of the circulatory system. Another type of cell that can be used in conjunction with the compositions and methods described herein is a pluripotent cell. A pluripotent cell is a cell that has the ability to differentiate into multiple cell types, but not all of them. A non-limiting example of a pluripotent cell is a CD34+ cell (eg, HSC or MPC).

[0580] The cells that can be used in conjunction with the compositions and methods described herein include HSCs and MPCs. HSCs are immature blood cells that have the ability to self-renew and differentiate into mature blood cells, including a variety of cell lineages, including, but not limited to, granulocytes (e.g., promyelocytes, neutrophils, eosinophils, basophils), erythrocytes (e.g., reticulocytes, erythrocytes), platelets (e.g., megakaryoblasts, platelet-producing megakaryocytes, platelets), monocytes (e.g., monocytes, macrophages), dendritic cells, microglia, osteoclasts, and lymphocytes (e.g., NK cells, B cells, and T cells). Human HSCs are CD34+. In addition, HSCs also refer to long-term repopulating HSCs (LT-HSCs) and short-term repopulating HSCs (ST-HSCs). Any of these HSCs can be used in conjunction with the compositions and methods described herein.

[0581] HSCs can differentiate into myeloid progenitor cells that are also CD34+. Myeloid progenitor cells can further differentiate into granulocytes (e.g., promyelocytes, neutrophils, eosinophils, basophils), erythrocytes (e.g., reticulocytes, erythrocytes), platelets (e.g., megakaryoblasts, platelet-producing megakaryocytes, and platelets), monocytes (e.g., monocytes and macrophages), dendritic cells, and microglia. Common myeloid progenitor cells can be characterized by cell surface molecules, including lin-, SCA1-, c-kit+, CD34+, and CD16 / 32. mid It is known that.

[0582] HSCs and myeloid progenitor cells can be obtained from blood products. Blood products are products obtained from the body or body organs that contain cells of hematopoietic origin. Such sources include unfractionated bone marrow, umbilical cord, placenta, peripheral blood, or mobilized peripheral blood. All of the aforementioned crude or unfractionated blood products can be enriched for cells with characteristics of HSCs or myeloid progenitor cells in several ways. For example, more mature differentiated cells can be selected based on the cell surface molecules they express. Blood products can be fractionated by positively selecting for CD34+ cells, which include a subpopulation of hematopoietic stem cells that are capable of self-renewal, multipotency, and that can home into the hematopoietic stem cell niche when reintroduced into the transplant recipient to re-establish productive and sustained hematopoiesis. Such selection is accomplished, for example, using commercially available magnetic anti-CD34 beads (Dynal, Lake Success, NY). Myeloid progenitor cells can also be isolated based on the markers they express. Unfractionated blood products can be obtained directly from donors or retrieved from cryopreservation stores. HSCs and myeloid progenitor cells can also be obtained by differentiation of ES cells, iPS cells, or other reprogrammed mature cell types.

[0583] Cells that can be used in conjunction with the compositions and methods described herein include allogeneic and autologous cells. All of the aforementioned cell types can be differentiated into microglia. The cells described herein can also be differentiated into microglial progenitor cells or microglial stem cells. Differentiation can occur ex vivo or in vivo. Methods for ex vivo differentiation of human ESCs and iPSCs are known to those of skill in the art and are described in Muffat et al., Nature Medicine 22:1358-1367 (2016) and Pandya et al., Nature Neuroscience (2017) (epub ahead of print), which are incorporated herein by reference for their disclosures regarding methods for differentiating pluripotent cells into microglia.

[0584] Microglia Cells that can be used in conjunction with the compositions and methods described herein include those that can differentiate into microglia or are differentiated microglia. Microglia are bone marrow-derived cells that function as immune cells or resident macrophages of the central nervous system. Microglia are genetically and functionally very similar to macrophages and share the ability to dynamically shift between pro-inflammatory and anti-inflammatory states. The pro-inflammatory state is known as classical activation, or M1, and the anti-inflammatory state is called alternative activation, or M2. Microglia can be shifted between the two states by extracellular signals, such as signals from neighboring neurons or astrocytes, cellular debris, toxins, infection, ischemia, and trauma, among others. M1 microglia are often observed in diseased brains, especially in diseases involving neuroinflammation, such as AD. The classical activation M1 phenotype is also observed in mouse models of AD, such as double transgenic APP / PS1 mice. Although it is unclear whether M1 microglia are a cause or a consequence of neuroinflammation, classical activation of microglia can secrete proinflammatory cytokines, such as TNF-α, IL-1β, and IL-6, chemokines, and nitric oxide, which can lead to persistent inflammation, neuronal damage, and further activation of M1 microglia. This positive feedback loop can be detrimental to brain tissue, and thus methods to reduce M1 activation and / or increase M2 activation may help subjects with diseases characterized by neuroinflammation, such as AD, PLOSL, FTLD, or PD.

[0585] Expression of TREM2 in mammalian cells In some patients with AD or PLOSL, TREM2 activity is reduced, and AD brains contain classically activated M1 microglia. In addition, microglia from PLOSL patients appear to have a delayed but enhanced inflammatory response compared to healthy controls. The compositions and methods described herein target these dysfunctions by administering cells (e.g., pluripotent cells, ESCs, iPSCs, pluripotent cells, CD34+ cells, HSCs, MPCs, BLPCs, monocytes, macrophages, microglial progenitors, or microglia) that contain a transgene encoding TREM2 (e.g., a transgene that can be expressed in macrophages or microglial cells). To utilize these agents in therapeutic applications in the treatment of NCDs, these agents can be targeted to the interior of the cell, in certain instances to specific organelles or plasma membranes. A wide range of methods have been established for delivering such proteins to mammalian cells and for stably expressing genes encoding such proteins in mammalian cells.

[0586] Polynucleotides encoding TREM2 One platform that can be used to achieve a therapeutically effective intracellular concentration of TREM2 in mammalian cells (e.g., pluripotent cells, ESCs, iPSCs, pluripotent cells, CD34+ cells, HSCs, MPCs, BLPCs, monocytes, macrophages, microglial progenitor cells, or microglia) is by stable expression of genes encoding these agents (e.g., by integration into the nuclear or mitochondrial genome of mammalian cells). These genes are polynucleotides that code for the primary amino acid sequence of the corresponding protein. To introduce such foreign genes into mammalian cells, these genes can be incorporated into vectors. Vectors can be introduced into cells by a variety of methods, including transformation, transfection, direct uptake, projectile bombardment, and by encapsulation of the vector in liposomes. Examples of suitable methods for transfecting or transforming cells are calcium phosphate precipitation, electroporation, microinjection, infection, lipofection, and direct uptake. Such methods are described in further detail in, for example, Green et al., Molecular Cloning: A Laboratory Manual, Fourth Edition (Cold Spring Harbor University Press, New York (2014)); and Ausubel et al., Current Protocols in Molecular Biology (John Wiley & Sons, New York (2015)), the disclosures of each of which are incorporated herein by reference.

[0587] TREM2 can also be introduced into mammalian cells by targeting a vector containing a gene encoding such an agent to cell membrane phospholipids. For example, the vector molecule can be linked to the VSV-G protein, a viral protein that has affinity for all cell membrane phospholipids, to target the vector to phospholipids on the extracellular surface of the cell membrane. Such constructs can be generated using methods well known to those skilled in the art.

[0588] The recognition and binding of the polynucleotide encoding TREM2 by mammalian RNA polymerase is important for gene expression. It may therefore contain sequence elements within the polynucleotide that exhibit high affinity for transcription factors that recruit RNA polymerase and promote the assembly of a transcription complex at the transcription initiation site. Such sequence elements include, for example, mammalian promoters whose sequences can be recognized and bound by specific transcription initiation factors and ultimately RNA polymerase. Examples of mammalian promoters are described in Smith et al., Mol. Sys. Biol., 3:73 online publication, the disclosure of which is incorporated herein by reference.

[0589] Suitable polynucleotides for use in the compositions and methods described herein also include those encoding TREM2 downstream of a mammalian promoter. Useful promoters for expression of TREM2 in mammalian cells include, for example, the elongation factor 1-alpha (EF1α) promoter, the phosphoglycerate kinase 1 (PGK) promoter, the CD68 molecule (CD68) promoter (see Dahl et al., Molecular Therapy 23:835 (2015), which is incorporated herein by reference as it relates to the use of PGK and CD68 promoters to express TREM2), the C-X3-C motif chemokine receptor 1 (CX3CR1) promoter, the CD11b promoter, the allograft inflammatory factor 1 (AIF1) promoter, the purinergic receptor P2Y12 (P2Y12) promoter, the transmembrane protein 119 (TMEM119) promoter, and the colony stimulating factor 1 receptor (CSF1R) promoter. Alternatively, promoters derived from viral genomes can also be used for stable expression of these agents in mammalian cells. Examples of functional viral promoters that can be used to drive mammalian expression of these agents are the adenovirus late promoter, the vaccinia virus 7.5K promoter, the simian virus 40 (SV40) promoter, the cytomegalovirus promoter, the tk promoter of herpes simplex virus (HSV), the mouse mammary tumor virus (MMTV) promoter, the long terminal repeat (LTR) promoter of human immunodeficiency virus (HIV), the promoter of Moloney virus, the Epstein-Barr virus (EBV), the Rous sarcoma virus (RSV), and the cytomegalovirus (CMV) promoter. Alternatively, synthetic promoters optimized for use in mammalian cells can be used for stable expression of TREM2.

[0590] Once a polynucleotide encoding TREM2 has been integrated into the nuclear DNA of a mammalian cell, transcription of the polynucleotide can be induced by methods known in the art. For example, expression can be induced by exposing the mammalian cell to an external chemical reagent, such as an agent that modulates the binding of transcription factors and / or RNA polymerase to the mammalian promoter to regulate gene expression. The chemical reagent serves to promote the binding of RNA polymerase and / or transcription factors to the mammalian promoter, for example by removing repressor proteins bound to the promoter. Alternatively, the chemical reagent can serve to increase the affinity of the mammalian promoter for RNA polymerase and / or transcription factors, such that the transcription rate of genes placed downstream of the promoter increases in the presence of the chemical reagent. Examples of chemical reagents that enhance polynucleotide transcription by the aforementioned mechanisms are tetracycline and doxycycline. These reagents are commercially available (Life Technologies, Carlsbad, CA) and can be administered to mammalian cells to promote gene expression according to established protocols.

[0591] Other DNA sequence elements that can be included in polynucleotides for use in the compositions and methods described herein are enhancer sequences. Enhancers represent another class of regulatory elements that induce conformational changes in polynucleotides, including genes of interest, such that the DNA adopts a three-dimensional orientation favorable for the binding of transcription factors and RNA polymerase at the transcription start site. Thus, polynucleotides for use in the compositions and methods described herein include those that encode TREM2 and additionally include a mammalian enhancer sequence. Many enhancer sequences are currently known from mammalian genes, examples being enhancers derived from genes encoding mammalian globin, elastase, albumin, alpha-fetoprotein, and insulin. Enhancers for use in the compositions and methods described herein also include those derived from the genetic material of viruses capable of infecting eukaryotic cells. Examples are the SV40 enhancer on the late side of the replication origin (bp 100-270), the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers. Additional enhancer sequences that induce activation of eukaryotic gene transcription are disclosed in Yaniv et al., Nature 297:17 (1982). The enhancer can be spliced, for example, at the 5' or 3' position of the gene into the vector containing the polynucleotide encoding the water-forming NADH oxidase. In a preferred orientation, the enhancer is located 5' of the promoter, which is then positioned 5' to the polynucleotide encoding TREM2.

[0592] Cell-specific gene expression Interfering RNA (RNAi) has been widely used to knock down the expression of endogenous genes by delivering small interfering RNA (siRNA) to cells to cause the degradation of complementary mRNA. An additional application is to exploit the diversity of endogenous microRNAs (miRNAs) to negatively regulate the expression of exogenously introduced transgenes tagged with artificial miRNA target sequences. These miRNA target tagged transgenes can be negatively regulated depending on the activity of a given miRNA, which can be specific to tissue, cell lineage, activation, or differentiation stage. These artificial miRNA target sequences (miRTs) can be recognized as targets by specific miRNAs to induce post-transcriptional gene silencing. While strong transgene expression in targeted cells can result in beneficial therapeutic outcomes, off-target expression, such as ectopic or unregulated transgene expression in HSPCs or other progenitor cells, can have cytotoxic effects and result in counter-selection of transgene-containing cells leading to altered cell behavior and reduced therapeutic efficacy. Incorporation of miRT for miRNAs that are broadly expressed in HSPCs and progenitor cells, but not in cells of myeloid lineage, allows for repression of transgene expression in HSPCs and other progenitor cells, allowing for silent, long-term storage of transgene-containing hematopoietic progeny, while allowing robust transgene expression in mature differentiated target cells. miR-126 is highly expressed in HSPCs, other progenitor cells, and cells of the erythroid lineage, but is absent in cells of the myeloid lineage (e.g., macrophages and microglia) (Gentner et al., Science Translational Medicine. 2:58ra34 (2010)). For example, miR-126 targeting sequences incorporated within a transgene allow for targeted expression of the transgene in cells of the myeloid lineage and repression of expression in HSPCs and other progenitor cells, thus minimizing off-target cytotoxic effects. In some embodiments, a transgene encoding a TREM2 agent may include a miR-126 targeting sequence.

[0593] Signal peptide The polynucleotide encoding TREM2 may include one or more polynucleotides encoding a signal peptide. The signal peptide may have an amino acid sequence between 16 and 30 residues in length and may be located upstream (i.e., 5') of the polynucleotide encoding TREM2. These signal peptides allow recognition of the nascent polypeptide during synthesis by a signal recognition particle, resulting in translocation to the ER, packaging into a transport vesicle, and translocation to a target cell compartment, lipid membrane, or extracellular space. Exemplary signal peptides for protein translocation are those derived from TREM2, IGF-II, alpha-1 antitrypsin, IL-2, IL-6, CD5, immunoglobulins, trypsinogen, serum albumin, prolactin, elastin, tissue plasminogen activator signal peptide (tPA-SP), and insulin. In some embodiments, cells (e.g., pluripotent cells, ESCs, iPSCs, pluripotent cells, CD34+ cells, HSCs, MPCs, BLPCs, monocytes, macrophages, microglial progenitor cells, or microglia) containing a transgene encoding TREM2 can be utilized as a therapeutic strategy to correct a protein deficiency (e.g., TREM2) by injecting the missing protein into the bloodstream. As blood perfuses the patient's tissues, TREM2 is taken up by the cells and transported to its site of action.

[0594] ApoE tagging of secreted TREM2b for blood-brain barrier penetration In some embodiments, TREM2 (e.g., a TREM2 fusion protein) is modified to penetrate the blood-brain barrier (BBB). Modifications to mediate BBB penetration are well known in the art. An exemplary modification is the use of a tag that includes the Rb domain of ApoE (amino acid residues 148-173 of SEQ ID NO: 13). The complete ApoE peptide sequence is shown below. MKVLWAALLVTLAGCQAKVEQAVETEPEPELRQQTEWQSGQRWELALGRFWDYLRWVQTLSEQVQEELLSSQVTQELRALMDETMKELKAYKSELEEQLTPVAEETRARLSKELQAAQARLGADMEDVCGRLVQYRGEVQAMLGQSTEELRVRLASH LRKLRKRLLRDADDLQKRLAVYQAGAREGAERGLSAIRERLGPLVEQGRVRAATVGSLAGQPLQERAQAWGERLRARMEEMGSRTRDRLDEVKEQVAEVRAKLEEQAQQIRLQAEAFQARLKSWFEPLVEDMQRQWAGLVEKVQAAVGTSAAPVPSDNH (SEQ ID NO:13)

[0595] ApoE is a key protein involved in lipid transport, and its cellular internalization is mediated by several members of the low-density lipoprotein (LDL) receptor gene family, including the LDL receptor, the very low-density lipoprotein receptor (VLDLR), and the LDL receptor-related proteins (LRPs, including LRP1, LRP2, and LRP8). LDL receptors have been found to be highly expressed in brain capillary endothelial cells (BCECs), with down-regulation of expression observed in peripheral blood vessels. Restricted expression of LRPs and VLDLRs has also been noted in the liver and brain, when detected in BCECs, neurons, and glial cells. Except for LDLR, several members of the low-density lipoprotein receptor family (LDLRf) proteins, including LRP1 and VLDLR, are highly expressed in BCECs forming the BBB. These proteins may bind to ApoE and promote their transcytosis to the abluminal side of the BBB.

[0596] In addition, receptor-associated proteins (RAPs), antagonists and ligands of both LRP1 and VLDLR, have been shown to be more permeable across the BBB than transferrin in vivo and in vitro (Pan et al., J. Cell Sci. 117:5071-8 (2004)), indicating that these lipoprotein receptors (LDLRfs) may be efficient BBB delivery targets, despite their lower expression than the transferrin receptor. As described herein, the potent Rb peptide derived from ApoE, when engineered as a fusion protein, has the ability to translocate proteins across the BBB to the brain. Thus, this method, when engineered as a fusion protein, may function to selectively open the BBB for therapeutic agents (e.g., soluble TREM2). Because this peptide utilizes the Rb domain of ApoE, rather than the entire ApoE protein, it can be easily conjugated to diagnostic or therapeutic agents without compromising their biological functions or interfering with the important biological functions of ApoE. This route is also an alternative uptake route that can facilitate further / secondary brain distribution after the drug reaches the CNS due to the widespread expression of LDLRf members in the brain parenchyma. Regardless of the administration strategy, e.g., enzyme replacement therapy or cell-based gene-based therapy, both the amount and distribution of therapeutic agent in the brain parenchyma should have a significant impact on the clinical outcome of disease treatment. A detailed description of the development and use of the Rb domain of ApoE in targeted delivery of proteins across the BBB can be found in U.S. Patent Application Publication No. 20140219974, the entirety of which is incorporated herein by reference.

[0597] In some embodiments, the TREM2 fusion protein has a peptide sequence comprising the LDLRf Rb domain of SEQ ID NO: 13, or a fragment, variant, or oligomer thereof. Exemplary receptor binding domains can be found at the N-terminus of ApoE, e.g., between amino acid residues 1-191 of SEQ ID NO: 13, between amino acid residues 25-185 of SEQ ID NO: 13, between amino acid residues 50-180 of SEQ ID NO: 13, between amino acid residues 75-175 of SEQ ID NO: 13, between amino acid residues 100-170 of SEQ ID NO: 13, or between amino acid residues 125-165 of SEQ ID NO: 13. Exemplary receptor binding domains have the amino acid sequence of residues 159-167 of SEQ ID NO: 13.

[0598] In some embodiments, the peptide sequence comprising the receptor binding domain of ApoE may comprise at least one amino acid mutation, deletion, addition, or substitution. In some embodiments, the amino acid substitution may be a combination of two or more mutations, deletions, additions, or substitutions. In some embodiments, at least one substitution is a conservative substitution. In some embodiments, at least one amino acid addition comprises the addition of a selected sequence already found in the Rb domain of ApoE. Those skilled in the art will recognize the appropriate modifications that can be made to the sequence while retaining some degree of biochemical activity for transport across the BBB.

[0599] Vector for expression of TREM2 In addition to achieving high transcription and translation rates, stable expression of exogenous genes in mammalian cells (e.g., pluripotent cells, ESCs, iPSCs, pluripotent cells, CD34+ cells, HSCs, MPCs, BLPCs, monocytes, macrophages, microglial progenitor cells, or microglial cells) can be achieved by integrating a polynucleotide containing the gene into the nuclear genome of the mammalian cells. Various vectors have been developed for delivering and integrating polynucleotides encoding exogenous proteins into the nuclear DNA of mammalian cells. Examples of expression vectors are disclosed, for example, in WO1994 / 011026, which is incorporated herein by reference. Expression vectors for use in the compositions and methods described herein include polynucleotide sequences encoding TREM2, as well as additional sequence elements used, for example, for the expression of these agents and / or for the integration of these polynucleotide sequences into the genome of mammalian cells. Certain vectors that can be used for expression of TREM2 include plasmids that contain regulatory sequences that direct gene transcription, such as promoter and enhancer regions. Other vectors useful for expressing TREM2 include polynucleotide sequences that increase the translation rate of these genes or improve the stability or nuclear export of the mRNA resulting from gene transcription. These sequence elements include, for example, 5' and 3' untranslated regions, IRES, and polyadenylation signal sites to direct efficient transcription of the genes carried on the expression vector. Expression vectors suitable for use in the compositions and methods described herein may also include polynucleotides encoding markers for selecting cells containing such vectors. Examples of suitable markers are genes encoding resistance to antibiotics such as ampicillin, chloramphenicol, kanamycin, nourseothricin, etc.

[0600] Viral vector for expressing TREM2 Viral genomes provide a rich source of vectors that can be used for efficient delivery of exogenous genes into mammalian cells (e.g., pluripotent cells, ESCs, iPSCs, pluripotent cells, CD34+ cells, HSCs, MPCs, BLPCs, monocytes, macrophages, microglial progenitor cells, or microglial cells). Viral genomes are particularly useful vectors for gene delivery because polynucleotides contained within such genomes are typically integrated into the nuclear genome of mammalian cells by generalized or specialized transduction. These processes occur as part of the natural viral replication cycle and do not require additional proteins or reagents to induce gene integration. Examples of viral vectors are retroviruses (e.g., Retroviridae viral vectors), adenoviruses (e.g., Ad5, Ad26, Ad34, Ad35, and Ad48), parvoviruses (e.g., adeno-associated viruses), coronaviruses, negative strand RNA viruses such as orthomyxoviruses (e.g., influenza viruses), rhabdoviruses (e.g., rabies and vesicular stomatitis viruses), paramyxoviruses (e.g., measles and Sendai), positive strand RNA viruses such as picornaviruses and alphaviruses, as well as double-stranded DNA viruses including adenoviruses, herpesviruses (e.g., herpes simplex virus types 1 and 2, Epstein-Barr virus, cytomegalovirus), and poxviruses (vaccinia, modified vaccinia Ankara (MVA), fowlpox, canarypox). Other viruses include, for example, Norwalk virus, togavirus, flavivirus, reovirus, papovavirus, hepadnavirus, human papillomavirus, human foamy virus, and hepatitis virus.Examples of retroviruses are avian leukemia sarcoma, avian C virus, mammalian C, B, D virus, oncoretrovirus, HTLV-BLV group, lentivirus, alpharetrovirus, gammaretrovirus, spumavirus (Coffin, JM, Retroviridae: The viruses and their replication, Virology, Third Edition (Lippincott-Raven, Philadelphia, (1996))). Other examples are murine leukemia virus, murine sarcoma virus, mouse mammary tumor virus, bovine leukemia virus, feline leukemia virus, feline sarcoma virus, avian leukemia virus, human T-cell leukemia virus, baboon endogenous virus, gibbon monkey leukemia virus, Mason-Pfizer monkey virus, simian immunodeficiency virus, simian sarcoma virus, Rous sarcoma virus and lentivirus. Other examples of vectors are described, for example, in McVey et al., (US 5,801,030), the teachings of which are incorporated herein by reference.

[0601] Retroviral Vectors The delivery vector used in the methods and compositions described herein may be a retroviral vector. One type of retroviral vector that can be used in the methods and compositions described herein is a lentiviral vector. A subset of retroviruses, lentiviral vectors (LVs), transduce a wide range of dividing and non-dividing cell types with high efficiency, resulting in stable, long-term expression of transgenes. An overview of the optimization strategy for packaging and transducing LVs is provided in Delenda, The Journal of Gene Medicine 6: S125 (2004), the disclosure of which is incorporated herein by reference.

[0602] The use of lentivirus-based gene transfer techniques relies on the in vitro generation of recombinant lentiviral particles carrying a highly deleted viral genome in which the transgene of interest is housed. In particular, recombinant lentiviruses are recovered by in trans co-expression in permissive cell lines of (1) a packaging construct, i.e., a vector expressing the Gag-Pol precursor together with Rev (alternatively expressed in trans); (2) a vector expressing an envelope receptor, generally of heterologous nature; and (3) a transfer vector, a viral cDNA in which all open reading frames have been removed but which maintains sequences necessary for replication, encapsidation, and expression, and into which the sequences to be expressed have been inserted.

[0603] The LV used in the methods and compositions described herein may include one or more of the following: 5'-long terminal repeat (LTR), HIV signal sequence, HIV Psi signal 5'-splice site (SD), delta-GAG element, Rev responsive element (RRE), 3'-splice site (SA), elongation factor (EF) 1-alpha promoter, and 3'-self-inactivating LTR (SIN-LTR). The lentiviral vector optionally includes a central polypurine tract (cPPT) and a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), as described in US 6,136,597, the disclosure of which is incorporated herein by reference as it relates to the WPRE. The lentiviral vector may further include a pHR' backbone, which may include, for example, as shown below.

[0604] The lentigene LV described in Lu et al., Journal of Gene Medicine 6:963 (2004) can also be used to express DNA molecules and / or transduce cells. The LV used in the methods and compositions described herein may include a 5'-long terminal repeat (LTR), an HIV signal sequence, an HIV Psi signal 5'-splice site (SD), a delta-GAG element, a Rev responsive element (RRE), a 3'-splice site (SA), an elongation factor (EF) 1-alpha promoter, and a 3'-self-inactivating LTR (SIN-LTR). Optionally, it will be readily apparent to one of skill in the art that one or more of these regions may be replaced with another region that performs a similar function.

[0605] TREM2 needs to be expressed at a sufficiently high level. The expression of the transgene is driven by a promoter sequence. Optionally, the LV comprises a CMV promoter. The promoter may be an EF1α or PGK promoter. In another embodiment, the promoter is a microglia-specific promoter, such as a CD68 promoter, a CX3CR1 promoter, an ITGAM promoter, an AIF1 promoter, a P2Y12 promoter, a TMEM119 promoter, or a CSF1R promoter. Those skilled in the art will be familiar with many promoters that are suitable in the vector constructs described herein.

[0606] Enhancer elements can be used to increase expression of modified DNA molecules or to enhance lentiviral integration efficiency. LVs used in the methods and compositions described herein may include a nef sequence. LVs used in the methods and compositions described herein may include a cPPT sequence that enhances vector integration. The cPPT acts as a second origin of (+)-strand DNA synthesis and introduces a partial strand overlap in the middle of the native HIV genome. Introducing a cPPT sequence into the transfer vector backbone significantly increased nuclear transport and the total amount of genome integrated into the DNA of the target cell. LVs used in the methods and compositions described herein may include a woodchuck posttranscriptional regulatory element (WPRE). The WPRE functions at the transcriptional level by promoting nuclear export of transcripts and / or by increasing the efficiency of polyadenylation of nascent transcripts, thus increasing the total amount of mRNA in the cell. Addition of the WPRE to the LV significantly improves transgene expression levels from several different promoters both in vitro and in vivo. LVs used in the methods and compositions described herein may include both a cPPT sequence and a WPRE sequence. The vector may also contain an IRES sequence, which allows expression of multiple polypeptides from a single promoter.

[0607] In addition to IRES sequences, other elements that allow for the expression of multiple polypeptides are useful. The vectors used in the methods and compositions described herein may contain multiple promoters that allow for the expression of more than one polypeptide. The vectors used in the methods and compositions described herein may contain protein cleavage sites that allow for the expression of more than one polypeptide. Examples of protein cleavage sites that allow for the expression of more than one polypeptide are described in Klump et al., Gene Ther.;8:811 (2001), Osborn et al., Molecular Therapy 12:569 (2005), Szymczak and Vignali Expert Opin Biol Ther. 5:627 (2005), and Szymczak et al. Nat Biotechnol. 22:589 (2004), which are incorporated herein by reference as their disclosures relate to protein cleavage sites that allow for the expression of more than one polypeptide. It will be readily apparent to one of skill in the art that other elements that allow for the expression of multiple polypeptides identified in the future will be useful and can be utilized in vectors suitable for use in the compositions and methods described herein.

[0608] The vectors used in the methods and compositions described herein may be clinical grade vectors.

[0609] Viral Regulatory Elements The viral regulatory element is a component of the delivery vehicle used to introduce the nucleic acid molecule into the host cell (e.g., pluripotent cell, ESC, iPSC, pluripotent cell, CD34+ cell, HSC, MPC, BLPC, monocyte, macrophage, microglial progenitor cell, or microglial cell). The viral regulatory element is optionally a retroviral regulatory element. For example, the viral regulatory element may be the LTR and gag sequence from HSC1 or MSCV. The retroviral regulatory element may be derived from a lentivirus or may be a heterologous sequence identified from other genomic regions. Those skilled in the art will also understand that other viral regulatory elements may be used with the nucleic acid molecules described herein as they are identified.

[0610] Adeno-associated virus vectors for nucleic acid delivery The nucleic acids of the compositions and methods described herein can be incorporated into rAAV vectors and / or virions to facilitate their introduction into cells (e.g., pluripotent cells, ESCs, iPSCs, pluripotent cells, CD34+ cells, HSCs, MPCs, BLPCs, monocytes, macrophages, microglial progenitor cells, or microglial cells). AAV vectors can be used in the central nervous system, and suitable promoters and serotypes are discussed in Pignataro et al., J Neural Transm (2017) (epub ahead of print), the disclosure of which is incorporated herein by reference as it relates to promoters and AAV serotypes useful in CNS gene therapy. rAAV vectors useful in the compositions and methods described herein are recombinant nucleic acid constructs that include (1) a heterologous sequence to be expressed (e.g., a polynucleotide encoding TREM2) and (2) viral sequences that facilitate integration and expression of the heterologous gene. The viral sequences may include sequences of AAV required in cis for DNA replication and packaging into virions (e.g., functional ITRs). Such rAAV vectors may also contain marker or reporter genes. Useful rAAV vectors are deleted in whole or in part from one or more of AAV WT genes, but retain functional adjacent ITR sequences. AAV ITRs may be of any serotype suitable for a particular application. Methods for using rAAV vectors are described, for example, in Tai et al., J. Biomed. Sci. 7:279 (2000), and Monahan and Samulski, Gene Delivery 7:24 (2000), the disclosures of which are incorporated herein by reference as they relate to AAV vectors for gene delivery.

[0611] The nucleic acids and vectors described herein can be incorporated into rAAV virions to facilitate the introduction of the nucleic acid or vector into cells. The capsid protein of AAV constitutes the outer non-nucleic acid portion of the virion and is encoded by the AAV cap gene. The cap gene encodes three viral coat proteins, VP1, VP2, and VP3, required for the assembly of the virion. The construction of rAAV virions is described, for example, in US5,173,414; US5,139,941; US5,863,541; US5,869,305; US6,057,152; and US6,376,237, the disclosures of which are incorporated herein by reference as they relate to AAV vectors for gene delivery, as well as in Rabinowitz et al., J. Virol. 76:791 (2002) and Bowles et al., J. Virol. 77:423 (2003).

[0612] rAAV virions useful in conjunction with the compositions and methods described herein include those derived from various AAV serotypes, including AAV 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, and rh74. For targeting cells located in or delivered to the central nervous system, AAV2, AAV9, and AAV10 may be particularly useful. The construction and use of AAV vectors of various serotypes and AAV proteins are described, for example, in Chao et al., Mol. Ther. 2:619 (2000); Davidson et al., Proc. Natl. Acad. Sci. USA 97:3428 (2000); Xiao et al., J. Virol. 72:2224 (1998); Halbert et al., J. Virol. 74:1524 (2000); Halbert et al., J. Virol. 75:6615 (2001); and Auricchio et al., Hum. Molec. Genet. 10:3075 (2001), the disclosures of each of which are incorporated by reference herein as they relate to AAV vectors for gene delivery.

[0613] Pseudotyped rAAV vectors are also useful in conjunction with the compositions and methods described herein. Pseudotyped vectors include AAV vectors of a given serotype pseudotyped with a capsid gene from a serotype other than the given serotype (e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, and AAV10, among others). Techniques for the construction and use of pseudotyped rAAV virions are known in the art and are described, for example, in Duan et al., J. Virol. 75:7662 (2001); Halbert et al., J. Virol. 74:1524 (2000); Zolotukhin et al., Methods, 28:158 (2002); and Auricchio et al., Hum. Molec. Genet. 10:3075 (2001).

[0614] AAV virions with mutations in the virion capsid can be used to infect specific cell types more effectively than non-mutated capsid virions. For example, suitable AAV mutants may have ligand insertion mutations to facilitate targeting of AAV to specific cell types. The construction and characterization of AAV capsid mutants, including insertion mutants, alanine screening mutants, and epitope tag mutants, are described in Wu et al., J. Virol. 74:8635 (2000). Other rAAV virions that can be used in the methods described herein include capsid hybrids generated by molecular breeding of viruses and even by exon shuffling. See, for example, Soong et al., Nat. Genet., 25:436 (2000) and Kolman and Stemmer, Nat. Biotechnol. 19:423 (2001).

[0615] Methods for delivering exogenous nucleic acids to target cells Techniques that can be used to introduce polynucleotides, such as codon-optimized DNA or RNA (e.g., mRNA, tRNA, siRNA, miRNA, shRNA, chemically modified RNA), into mammalian cells (e.g., pluripotent cells, ESCs, iPSCs, pluripotent cells, CD34+ cells, HSCs, MPCs, BLPCs, monocytes, macrophages, microglial progenitor cells, or microglial cells) are well known in the art. For example, electroporation can be used to permeabilize mammalian cells (e.g., human target cells) by applying an electrostatic potential to the cells of interest. Mammalian cells, such as human cells, subjected to an external electric field in this manner are then susceptible to uptake of exogenous nucleic acids. Electroporation of mammalian cells is described in detail, for example, in Chu et al., Nucleic Acids Research 15:1311 (1987), the disclosure of which is incorporated herein by reference. A similar technique, Nucleofection™, utilizes an applied electric field to stimulate uptake of exogenous polynucleotides into the nucleus of eukaryotic cells. Nucleofection™ and protocols useful for carrying out this technique are described in detail, for example, in Distler et al., Experimental Dermatology 14:315 (2005), as well as US2010 / 0317114, the disclosures of each of which are incorporated herein by reference.

[0616] Another useful technique for transfection of target cells is squeeze-poration. This technique induces rapid mechanical deformation of cells to stimulate the uptake of exogenous DNA through membrane pores that form in response to applied stress. This technique is advantageous in that no vector is required to deliver nucleic acid to cells, such as human target cells. Squeeze-poration is described in detail, for example, in Sharei et al., Journal of Visualized Experiments 81:e50980 (2013), the disclosure of which is incorporated herein by reference.

[0617] Lipofection is another technique useful for transfection of target cells. This method involves loading nucleic acid into liposomes that often present cationic functional groups, such as quaternary or protonated amines, toward the exterior of the liposome. This promotes electrostatic interactions between the liposome and the cell due to the anionic nature of the cell membrane, ultimately leading to the uptake of exogenous nucleic acid, for example, by direct fusion of the liposome with the cell membrane or by endocytosis of the complex. Lipofection is described in detail, for example, in U.S. Pat. No. 7,442,386, the disclosure of which is incorporated herein by reference. A similar technique that utilizes ionic interactions with the cell membrane to induce the uptake of exogenous nucleic acid is to contact cells with cationic polymer-nucleic acid complexes. Exemplary cationic molecules that associate with polynucleotides to impart a positive charge favorable for interaction with cell membranes are activated dendrimers (e.g., as described in Dennig, Topics in Current Chemistry 228:227 (2003), the disclosure of which is incorporated herein by reference), polyethyleneimine, and diethylaminoethyl (DEAE)-dextran, the use of which as transfection agents is described in detail, for example, in Gulick et al., Current Protocols in Molecular Biology 40:1:9.2:9.2.1 (1997), the disclosure of which is incorporated herein by reference. Magnetic beads are another tool that can be used to transfect target cells in a gentle and efficient manner, since this method utilizes an applied magnetic field to direct the uptake of nucleic acids. This technology is described in detail, for example, in US2010 / 0227406, the disclosure of which is incorporated herein by reference.

[0618] Another useful tool for inducing the uptake of exogenous nucleic acids by target cells is laser transfection, also called phototransfection, a technique that involves exposing cells to electromagnetic radiation of specific wavelengths in order to gently permeabilize the cells and allow polynucleotides to penetrate the cell membrane. The biological activity of this technique appears to be similar to, and in some cases superior to, electroporation.

[0619] Impalefection is another technique that can be used to deliver genetic material to target cells. It relies on the use of nanomaterials such as carbon nanofibers, carbon nanotubes, and nanowires. Needle-like nanostructures are synthesized perpendicular to the surface of a substrate. DNA containing the genes intended for intracellular delivery is attached to the nanostructure surface. A tip with an array of these needles is then pressed against a cell or tissue. Cells impaled with the nanostructures can express the delivered gene(s). An example of this technique is described in Shalek et al., PNAS 107:25 1870 (2010), the disclosure of which is incorporated herein by reference.

[0620] Magnetofection can also be used to deliver nucleic acids to target cells. The principle of magnetofection is to associate nucleic acids with cationic magnetic nanoparticles. The magnetic nanoparticles are made of iron oxide, which is completely biodegradable, and are coated with specific cationic proprietary molecules that vary depending on the application. Their association with gene vectors (DNA, siRNA, viral vectors, etc.) is achieved by salt-induced colloidal aggregation and electrostatic interactions. The magnetic particles are then concentrated in the target cells under the influence of an external magnetic field generated by a magnet. This technology is described in detail in Scherer et al., Gene Therapy 9:102 (2002), the disclosure of which is incorporated herein by reference.

[0621] Another useful tool for inducing the uptake of exogenous nucleic acid by target cells is sonoporation, which is a technique that uses sound (typically ultrasonic frequencies) to change the permeability of cell plasma membrane, making cells permeable and allowing polynucleotides to penetrate the cell membrane.This technique is described in detail, for example, in Rhodes et al., Methods in Cell Biology 82:309 (2007), the disclosure of which is incorporated herein by reference.

[0622] Microvesicles represent another potential carrier that can be used to modify the genome of target cells according to the methods described herein.For example, microvesicles induced by the simultaneous overexpression of glycoprotein VSV-G and genome modification proteins, such as nucleases, can be used to efficiently deliver proteins that later catalyze the site-specific cleavage of endogenous polynucleotide sequences to cells, preparing the genome of cells for the covalent integration of a polynucleotide of interest, such as a gene or regulatory sequence.The use of such vesicles, also called gesicles, in the genetic modification of eukaryotic cells is described in detail, for example, in Quinn et al., Genetic Modification of Target Cells by Direct Delivery of Active Protein [abstract]: Methylation changes in early embryonic genes in cancer [abstract] : Proceedings of the 18th Annual Meeting of the American Society of Gene and Cell Therapy;2015 May 13, Abstract No. 122.

[0623] Modulating gene expression using gene editing techniques Disruption of endogenous TREM2 In some embodiments, endogenous TREM2 is disrupted (e.g., in a subject undergoing treatment, such as in a population of neurons in a subject undergoing treatment, or in cells administered to the subject). An exemplary method for disrupting endogenous TREM2 expression is administering an inhibitory RNA molecule to a subject or contacting the population of neurons of the subject or the population of cells administered to the subject. The inhibitory RNA molecule may function to disrupt endogenous TREM2 expression, for example, acting via an RNA interference (RNAi) pathway. The inhibitory RNA molecule may reduce the expression level (e.g., protein level or mRNA level) of endogenous TREM2. For example, inhibitory RNA molecules include short interfering RNA, short hairpin RNA, and / or miRNA that target full-length endogenous TREM2. siRNAs are double-stranded RNA molecules typically having a length of about 19-25 base pairs. shRNAs are RNA molecules that contain a hairpin turn that reduce expression of a target gene via RNAi. shRNA can be delivered to cells, for example, by transfection, electroporation, or transduction, in the form of a plasmid, for example, a viral or bacterial vector. miRNA is a non-coding RNA molecule that typically has a length of about 22 nucleotides. miRNA binds to a target site on an mRNA molecule and silences the mRNA, for example, by causing cleavage of the mRNA, destabilization of the mRNA, or inhibition of translation of the mRNA. Inhibitory RNA molecules can be modified to include modified nucleotides, for example, 2'-fluoro, 2'-o-methyl, 2'-deoxy, unlocked nucleic acid, 2'-hydroxy, phosphorothioate, 2'-thiouridine, 4'-thiouridine, 2'-deoxyuridine. Without being bound by theory, it is believed that certain modifications can increase nuclease resistance and / or serum stability, or reduce immunogenicity.

[0624] In some embodiments, the inhibitory RNA molecule reduces the level and / or activity or function of endogenous TREM2. In embodiments, the inhibitory RNA molecule inhibits expression of endogenous TREM2. In other embodiments, the inhibitory RNA molecule increases the degradation of endogenous TREM2 and / or reduces the stability of endogenous TREM2. The inhibitory RNA molecule can be chemically synthesized or in vitro transcribed.

[0625] In some embodiments, endogenous TREM2 is disrupted in cells comprising a TREM2 transgene, e.g., using gene editing techniques described herein. In some embodiments, endogenous TREM2 is disrupted globally in a subject, e.g., using gene editing techniques described herein. In some embodiments, endogenous TREM2 is disrupted in a population of neurons in a subject, e.g., using gene editing techniques described herein. In some embodiments, disruption of endogenous TREM2 in a subject, neurons, and / or cells comprising a TREM2 transgene is performed prior to administering the cells to a subject.

[0626] The generation and use of inhibitory therapeutics based on non-coding RNA, such as ribozymes, RNAse P, siRNA, miRNA, etc., are also known in the art, as described, for example, in Sioud, RNA Therapeutics: Function, Design, and Delivery (Methods in Molecular Biology). Humana Press 2010.

[0627] Nuclease-Mediated Gene Regulation Another useful tool for disruption and / or integration of target genes into the genome of a cell is the clustered regularly interspaced short palindromic repeats (CRISPR) / Cas system, which originally evolved as an adaptive defense mechanism in bacteria and archaea against viral infection. The CRISPR / Cas system comprises palindromic repeat sequences in plasmid DNA and CRISPR-associated proteins (Cas; e.g., Cas9 or Cas12a). This DNA and protein assembly directs site-specific DNA cleavage of a target sequence by first integrating foreign DNA into the CRISPR locus. Polynucleotides containing these foreign sequences and the repeat-spacer elements of the CRISPR locus can then be transcribed into the host cell to generate guide RNAs that can then be annealed to the target sequence and localize Cas nuclease to this site. Thus, highly site-specific Cas-mediated DNA cleavage can be engineered in foreign polynucleotides, since the interaction that brings Cas into close proximity to the target DNA molecule is governed by RNA:DNA hybridization. As a result, the CRISPR / Cas system can theoretically be designed to cleave any target DNA molecule of interest (e.g., endogenous TREM2). This technology has been utilized to edit eukaryotic genomes (Hwang et al. Nature Biotechnology 31:227 (2013), the disclosure of which is incorporated herein by reference) and can be used as an efficient means to site-specifically edit cellular genomes to cleave DNA prior to the incorporation of a gene encoding a target gene. The use of CRISPR / Cas to regulate gene expression is described, for example, in US8,697,359, the disclosure of which is incorporated herein by reference. Alternative methods for disrupting target DNA by site-specific cleavage of genomic DNA prior to the incorporation of a gene of interest into a cell include the use of zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs).Unlike CRISPR / Cas systems, these enzymes do not contain a guided polynucleotide to localize to a specific target sequence. Instead, target specificity is controlled by a DNA-binding domain within these enzymes. The use of ZFNs and TALENs in genome editing applications is described, for example, in Urnov et al. Nature Reviews Genetics 11:636 (2010); and Joung et al. Nature Reviews Molecular Cell Biology 14:49 (2013), the disclosures of both of which are incorporated herein by reference. In some embodiments, endogenous TREM2 can be disrupted in cells that contain a TREM2 transgene using these gene editing techniques described herein.

[0628] Transposon-mediated gene regulation In addition to viral vectors, various additional tools have been developed that can be used to incorporate exogenous genes into cells (e.g., pluripotent cells, ESCs, iPSCs, pluripotent cells, CD34+ cells, HSCs, MPCs, BLPCs, monocytes, macrophages, microglial progenitor cells, or microglia). One such method that can be used to incorporate a polynucleotide encoding a target gene into a cell involves the use of a transposon. A transposon is a polynucleotide that encodes a transposase enzyme and contains a polynucleotide sequence or gene of interest flanked by 5' and 3' excision sites. Once the transposon is delivered to a cell, expression of the transposase gene begins, resulting in an active enzyme that cleaves the gene of interest from the transposon. This activity is mediated by site-specific recognition of the transposon excision site by the transposase. In certain cases, these excision sites may be terminal repeats or inverted terminal repeats. Once excised from the transposon, the gene of interest can be integrated into the genome of a mammalian cell by transposase-catalyzed cleavage of a similar excision site present in the nuclear genome of the cell. This allows the gene of interest to be inserted into the nuclear DNA cleaved at the complementary excision site, after which the integration process is completed with covalent ligation of a phosphodiester bond that connects the gene of interest to the DNA of the mammalian cell genome. In certain cases, the transposon may be a retrotransposon, such that the gene encoding the target gene is first transcribed into an RNA product and then reverse transcribed into DNA before integration into the mammalian cell genome. Transposon systems include piggyback transposons (described in detail, for example, in WO2010 / 085699) and Sleeping Beauty transposons (described in detail, for example, in US2005 / 0112764), the disclosures of each of which are incorporated herein by reference.

[0629] Diagnostic methods Methods well known in the art, such as those described in The Diagnostic and Statistical Manual of Mental Disorders, Fifth Edition and the International Classification of Diseases, 11 thUsing methods such as those described in Revision, a subject can be diagnosed as having an NCD (e.g., AD, PLOSL, FTLD, or PD). For example, a diagnosis of an NCD in a subject can be guided by a neuropsychological test that evaluates the degree of cognitive impairment in the subject. A subject's cognitive function can be evaluated by performing a cognitive test that evaluates processing ability across one or more cognitive domains, including but not limited to complex attention, executive function, learning and memory, language, sensorimotor function, and social cognition. A subject's cognitive function can be compared to a standard (e.g., a reference population, such as the general population) appropriate for the subject's age, medical history, education, socioeconomic status, and lifestyle to determine a diagnosis of an NCD in the subject. A subject can be diagnosed as having a severe NCD or a mild NCD. A severe NCD is characterized by a significant cognitive decline that interferes with an individual's independence and / or normal daily functioning and is not due to delirium or other psychiatric disorders. A mild NCD is characterized by a moderate cognitive decline that does not interfere with a subject's independence and / or normal daily functioning and is not due to delirium or other psychiatric disorders. Severe NCDs may be characterized by a score obtained by a subject on a cognitive test that is more than two standard deviations away from the mean score of a reference population (e.g., the mean score of the general population) or that is in the third percentile of the distribution of scores in the reference population. Mild NCDs may be characterized by a score obtained by a subject on a cognitive test that is one to two standard deviations away from the mean score of a reference population (e.g., the mean score of the general population) or that is in the third to sixteenth percentile of the distribution of scores in the reference population.Non-limiting examples of cognitive tests include the Eight-item Informant Interview to Differentiate Aging and Dementia (AD8), Annual Wellness Visit (AWV), General Practitioner Assessment of Cognition (GPCOG), Health Risk Assessment (HRA), Memory Impairment Screen (MIS), Mini Mental Status Exam (MMSE), Montreal Cognitive Assessment (MoCA), St. Louis University Mental Status Exam (SLUMS), and Short Informant Questionnaire on Cognitive Decline in the Elderly (Short IQCODE).In addition, or alternatively, the use of F18-fluorodeoxyglucose PET scans or MRI scans can also be used to determine the presence of neurodegeneration in subjects with NCD.

[0630] Furthermore, the subject can be examined for the presence of biomarkers specific to the particular NCD of interest. For example, the subject can be examined for the presence of biomarkers that indicate the subject has AD, such as the presence of Aβ plaques or NFTs of hyperphosphorylated tau protein in the subject's forebrain, the presence of mutations in the APP, PSEN1, PSEN2, and / or TREM2 genes in the subject, and even changes in the ε4 allele of APOE. To determine whether a subject has PLOSL, the subject can also be examined for the presence of lipid-laden macrophages, the presence of axonal spherules, loss of axon and myelin, white matter degeneration, and / or mutations in the TREM2 gene. Furthermore, PLOSL patients are known to exhibit cystic bone lesions during early disease stages, the presence of which can be used to guide the diagnosis of patients with PLOSL.

[0631] Treatment Method Selecting a target The subject that can be treated as described herein is a subject who has or is at risk of developing an NCD (e.g., AD, PLOSL, FTLD, or PD). The type of NCD can be a TREM2-associated NCD (e.g., TREM2-associated AD, PLOSL, FTLD, or PD), a sporadic NCD (e.g., sporadic AD, PLOSL, FTLD, or PD), an NCD caused by an environmental factor, or an NCD associated with a non-TREM2 mutation, e.g., a mutation in one or more of the genes associated with AD or PLOSL. The compositions and methods described herein can be used to treat subjects with normal TREM2 activity, reduced TREM2 activity, and subjects whose TREM2 mutation status and / or TREM2 activity level is unknown. The compositions and methods described herein can also be administered as a preventative treatment to subjects at risk of developing an NCD, e.g., subjects with a TREM2 mutation, subjects with reduced TREM2 activity, subjects with one or more mutations in the genes associated with NCDs, or subjects exposed to environmental toxins associated with NCDs. A subject at risk for an NCD may exhibit early symptoms of an NCD, but may not yet exhibit symptoms when treatment is administered.

[0632] In some embodiments, the methods and compositions described herein can be administered to subjects with TREM2 mutations, including, for example, single amino acid substitutions (e.g., p.R47H, p.R62H, p.T66M, p.T66M, p.Y38C, p.T96K, p.D87N, p.H157Y, p.R98W, p.T96K, p.D87N, p.L211P, p.R136Q, or p.N68K). In addition, the methods and compositions described herein can be administered to subjects with TREM2 mutations, including, for example, single nucleotide substitutions or deletions (e.g., c.40G>T, c.C97>T, c.132G>A, c.267delGm c.313delG, c.377T>G, c.401A>G, c.482+2T>C, c.558GA). In some embodiments, the methods and compositions described herein can be administered to subjects with any other pathogenic mutation in the TREM2 gene.For example, the pathogenic mutation in the TREM2 gene can be any of the mutations discussed in Guerreiro et al., The New England Journal of Medicine 368, 117-27, (2013), Jonsson et al., The New England Journal of Medicine, 368(2), 107-16, Ulrich et al., Neuron Review 94, 237:48, (2017), and Xing et al., Research and Reports in Biochemistry, 5, 89-100, (2015), the disclosures of which are incorporated herein by reference as they relate to AD-related or PLOSL-related human TREM2 mutations.

[0633] Route of administration The cells and compositions described herein can be administered to a subject with an NCD (e.g., AD, PLOSL, FTLD, or PD) by a variety of routes, such as intraventricular, intrathecal, intraparenchymal, stereotactic, intravenous, intraosseous, or by bone marrow transplantation. In some embodiments, the cells and compositions described herein can be administered systemically (e.g., intravenously), directly to the central nervous system (CNS) (e.g., intraventricular, intrathecal, intraparenchymal, or stereotactic), or directly to bone marrow (e.g., intraosseous). In some embodiments, the cells and compositions described herein are administered to a subject intraventricularly into the lateral cerebral ventricles (a description of this method can be found in Capotondo et al., Science Advances 3:e1701211 (2017), which is incorporated herein by reference, as it relates to intraventricular infusion of hematopoietic stem and progenitor cells into the lateral cerebral ventricles in a mouse model). The optimal route of administration in any given case depends on the particular cells or compositions administered, the subject, the pharmaceutical formulation method, the method of administration (e.g., time of administration and route of administration), the age, weight, sex of the subject, the severity of the disease being treated, the diet of the subject, and the excretion rate of the subject. Multiple routes of administration can be used to treat a single subject, for example, intraventricular or stereotactic injection and intravenous injection, intraventricular or stereotactic injection and intraosseous injection, intraventricular or stereotactic injection and bone marrow transplant, intraventricular or stereotactic injection and intraparenchymal injection, intrathecal injection and intravenous injection, intrathecal injection and intraosseous injection, intrathecal injection and bone marrow transplant, intrathecal injection and intraparenchymal injection, intraparenchymal injection and intravenous injection, intraparenchymal injection and intraosseous injection, or intraparenchymal injection and bone marrow transplant. Multiple routes of administration can be used to treat a single subject at one time, or the subject can be treated via one route of administration initially and then via another route of administration at a second visit, for example, one week, two weeks, one month, six months, or one year later. For treatment of an NCD, the cells may be administered to a subject once, or the cells may be administered to a subject one or more times (eg, 2-10 times) weekly, monthly, or yearly.

[0634] Pretreatment It may be advantageous to deplete or remove endogenous microglia and / or hematopoietic stem and progenitor cells prior to administration of cells (e.g., pluripotent cells, ESCs, iPSCs, pluripotent cells, CD34+ cells, HSCs, MPCs, BLPCs, monocytes, macrophages, microglial progenitor cells, or microglia) or compositions. Microglia and / or hematopoietic stem and progenitor cells can be removed using chemical agents (e.g., busulfan, treosulfan, PLX3397, PLX647, PLX5622, or clodronate liposomes), irradiation, or combinations thereof. Agents used for cell removal may be BBB-permeable (e.g., busulfan) or may lack the ability to cross the BBB (e.g., treosulfan). Exemplary microglia and / or hematopoietic stem and progenitor cell ablative agents are busulfan (Capotondo et al., PNAS 109:15018 (2012), which is incorporated by reference as the disclosure thereof pertains to the use of busulfan to ablate microglia), treosulfan, PLX3397, PLX647, PLX5622, or clodronate liposomes. Other agents for depleting endogenous microglia and / or hematopoietic stem and progenitor cells include cytotoxins that can be covalently attached to an antibody or antigen-binding fragment thereof capable of binding to an antigen expressed by hematopoietic stem cells to form an antibody-drug conjugate. Cytotoxins suitable for antibody drug conjugates include DNA intercalating agents (e.g., anthracyclines), agents capable of disrupting the mitotic spindle (e.g., vinca alkaloids, maytansine, maytansinoids, and their derivatives), RNA polymerase inhibitors (e.g., amatoxins such as a-amanitin and its derivatives), agents capable of interfering with protein biosynthesis (e.g., agents exhibiting rRNA N-glycosidase activity, such as saporin and ricin A chain), among others known in the art.The depletion may eliminate all microglia and / or hematopoietic stem and progenitor cells or may reduce the number of microglia and / or hematopoietic stem and progenitor cells by at least 5% (e.g., at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more). The agent or agents that deplete microglia and / or hematopoietic stem and progenitor cells can be administered at least one week (e.g., 1, 2, 3, 4, 5, or 6 weeks or more) prior to administration of the cells or compositions described herein. Cells administered in the manner described herein can replace the depleted microglia and / or hematopoietic stem and progenitor cells and can repopulate the brain after intraventricular, stereotactic, intravenous, or intraosseous injection, or after bone marrow transplantation. Cells administered intravenously, intraosseously, or by bone marrow transplantation can cross the blood-brain barrier, enter the brain, and differentiate into microglia. Cells administered to the brain, for example intracerebroventricularly or stereotactically, may differentiate into microglia in vivo or may differentiate into microglia ex vivo.

[0635] Stem Cell Recapture The methods described herein may include administering a population of cells (e.g., pluripotent cells, ESCs, iPSCs, pluripotent cells, CD34+ cells, HSCs, MPCs, BLPCs, monocytes, macrophages, microglial progenitor cells, or microglia) to a subject. The cells may be cells that have not been modified to include a transgene encoding TREM2 (e.g., a transgene that can be expressed in macrophages or microglia). The cells may have endogenous TREM2 disrupted. After conditioning as described herein, the cells may be administered systemically (e.g., intravenously) or by bone marrow transplantation to reconstitute the bone marrow compartment. For example, the cells may migrate into a stem cell niche and increase the amount of cells of the hematopoietic lineage at such sites to, for example, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 35%, 40%, 45%, 50%, 55%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, 310%, 320%, 330%, 340%, 350%, 360%, 370%, 380%, 390%, 400%, 410%, 420%, 430%, 440%, 450%, 460%, 470%, 480%, 490%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61 %, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, or more. Administration can be performed before or after administration of a composition described herein.

[0636] Donor cell selection In some embodiments, the subject is a donor. In such a case, the removed cells (e.g., pluripotent cells, ESCs, iPSCs, pluripotent cells, CD34+ cells, HSCs, MPCs, BLPCs, monocytes, macrophages, microglial progenitors, or microglia) can be reinjected into the subject (after modification (e.g., integration of a transgene encoding TREM2 and / or disruption of endogenous TREM2)), and the cells will then home to hematopoietic tissue and establish productive hematopoiesis, thereby expanding or repopulating the defective or missing cell line (e.g., a population of microglia) in the subject. In this scenario, the transplanted cells are least likely to undergo graft rejection, since the injected cells are derived from the subject and express the same HLA class me and class II antigens as the subject expresses. Alternatively, the subject and the donor can be different. In some embodiments, the subject and the donor can be related, e.g., HLA-matched. As described herein, HLA-matched donor-recipient pairs have a lower risk of graft rejection because endogenous T cells and NK cells in the transplant recipient are less likely to recognize foreign hematopoietic stem or progenitor cells as foreign and therefore less likely to mount an immune response to the transplant. An exemplary HLA-matched donor-recipient pair is a genetically related donor and recipient, such as a familial donor-recipient pair (e.g., a sibling donor-recipient pair). In some embodiments, the subject and donor are HLA-mismatched, which occurs when at least one HLA antigen is mismatched between the donor and the recipient, particularly with respect to HLA-A, HLA-B, and HLA-DR. For example, one haplotype may be matched between the donor and the recipient, and the other may be mismatched, to reduce the likelihood of graft rejection.

[0637] Pharmaceutical Compositions and Dosages The number of cells administered to a subject to treat an NCD (e.g., AD, PLOSL, FTLD, or PD (e.g., TREM2-associated AD, PLOSL, FTLD, or PD)) as described herein can depend, for example, on the expression level of TREM2, the subject, the pharmaceutical formulation method, the method of administration (e.g., time and route of administration), the age, weight, and sex of the subject, the severity of the disease being treated, and whether the subject has been treated with an agent that eliminates endogenous microglia. The number of cells administered can range from, for example, 1×10 6 cells / kg~1×10 12 cells / kg, or more (e.g., 1 x 10 7 cells / kg, 1×10 8 cells / kg, 1×10 9 cells / kg, 1×10 10 cells / kg, 1×10 11 cells / kg, 1×10 12 The dosage may be 1×10 cells / kg or more). The cells may be administered in an undifferentiated state or after partial or complete differentiation into microglia. The number of cells may be administered at any suitable dose after pretreatment. A non-limiting example of a dose is about 1×10 5 Approximately 1 x 10 cells / kg of recipient 7 Cells / kg (e.g., approximately 2 x 10 5 cells / kg ~ approx. 9×10 6 cells / kg, approximately 3×10 5 cells / kg ~ approx. 8×10 6 cells / kg, approximately 4×10 5 cells / kg~about 7×10 6 cells / kg, approximately 5×10 5 cells / kg~about 6×10 6 cells / kg, approximately 5×10 5 cells / kg ~ approx. 1×10 7 cells / kg, approximately 6×10 5 cells / kg ~ approx. 1×10 7 cells / kg, approximately 7×10 5 cells / kg ~ approx. 1×10 7 cells / kg, approximately 8×10 5 cells / kg ~ approx. 1×10 7 cells / kg, approximately 9×10 5 cells / kg ~ approx. 1×107 cells / kg, and approximately 1 x 10 6 cells / kg ~ approx. 1×10 7 Additional exemplary doses are, inter alia, about 1×10 10 Approximately 1 x 10 cells / kg of recipient 12 Cells / kg (e.g., approximately 2 x 10 10 cells / kg ~ approx. 9×10 11 cells / kg, approximately 3×10 10 cells / kg ~ approx. 8×10 11 cells / kg, approximately 4×10 10 cells / kg~about 7×10 11 cells / kg, approximately 5×10 10 cells / kg~about 6×10 11 cells / kg, approximately 5×10 10 cells / kg ~ approx. 1×10 12 cells / kg, approximately 6×10 10 cells / kg ~ approx. 1×10 12 cells / kg, approximately 7×10 10 cells / kg ~ approx. 1×10 12 cells / kg, approximately 8×10 10 cells / kg ~ approx. 1×10 12 cells / kg, approximately 9×10 10 cells / kg ~ approx. 1×10 12 cells / kg, and approximately 1 x 10 11 cells / kg ~ approx. 1×10 12 cells / kg).

[0638] The cells and compositions described herein can be administered in an amount sufficient to ameliorate one or more pathological features in NCDs. Administration of the cells or compositions described herein can increase the amount of M2 microglia in the brain of a subject relative to the amount of M1 microglia in the brain of a subject, decrease the level of proinflammatory cytokines in the brain of a subject, increase the level of anti-inflammatory cytokines in the brain of a subject, improve cognitive performance of a subject, improve motor function of a subject, decrease amyloid beta and neurofibrillary tau protein levels or aggregations thereof in a subject, decrease demyelination, decrease the amount or size of axonal spheroids, decrease the occurrence or severity of epileptic seizures, reduce pain in the distal extremities (e.g., ankle, foot, wrist, or hand), reduce osteocysts, decrease bone fractures, reduce motor impairment, decrease vascular pathology, decrease accumulation of lipid-containing macrophages or free fatty acids in the brain, and / or decrease brain tissue loss in a subject. The numbers of M1 and M2 microglia were assessed using ELISA to compare the levels of cytokines, chemokines, and other pro- and anti-inflammatory mediators in the cerebrospinal fluid (CSF) of subjects before and after treatment, and translocator protein-activating protein (TSPO), a protein highly expressed in classically activated M1 microglia, was assayed using, for example, TSPO radioligand. 11The effects of C-(R)-PK11195 can be assessed by using PET imaging to observe or by analyzing the levels of M1 and M2 associated genes and proteins in tissue samples using standard techniques, such as Western blot analysis, immunohistochemistry, or quantitative RT-PCR. Cognitive and motor function can be assessed using standard neurological tests before and after treatment, and amyloid beta and tau proteins can be detected in plasma and CSF using ELISA. Neurodegeneration can be assessed using F18-fluorodeoxyglucose PET scans or MRI scans. Subjects can be evaluated 1, 2, 3, 4, 5, 6 or more months after administration of the population of cells, depending on the route of administration used for the treatment. Depending on the results of the evaluation, the subject can receive additional treatment.

[0639] kit The compositions described herein can be provided in a kit for use in treating NCD (e.g., AD, PLOSL, FTLD, or PD). The compositions can include host cells described herein (e.g., pluripotent cells, ESCs, iPSCs, pluripotent cells, CD34+ cells, HSCs, MPCs, BLPCs, monocytes, macrophages, microglial progenitor cells, or microglia) that include a transgene encoding TREM2 (e.g., a transgene that can be expressed in macrophages or microglia) and that can optionally have endogenous TREM2 disrupted. The cells can be cryopreserved, for example, in dimethylsulfoxide (DMSO), glycerol, or another cryoprotectant. The kit can include a package insert that instructs a user of the kit, such as a physician, to carry out the methods described herein. The kit can optionally include a syringe or other device for administering the composition. EXAMPLES

[0640] The following examples are set forth to provide one of ordinary skill in the art with an illustration of how the compositions and methods described herein can be used, made, and evaluated, and are intended to be merely illustrative of the disclosure and are not intended to limit the scope of what the inventors regard as the disclosure.

[0641] Example 1. Generation of cells containing a transgene encoding triggering receptor 2 expressed in myeloid cells An exemplary method for generating cells (e.g., pluripotent cells, ESCs, iPSCs, pluripotent cells, CD34+ cells, HSCs, MPCs, BLPCs, monocytes, macrophages, microglial progenitor cells, or microglia) containing a transgene encoding triggering receptor expressed in myeloid cells 2 (TREM2) for use in the compositions and methods described herein is by transduction. Retroviral vectors (e.g., lentiviral, alpharetroviral, or gammaretroviral vectors) containing a microglia-specific promoter, such as the CD68 promoter, and a polynucleotide encoding TREM2 can be engineered using standard techniques known in the art. After engineering the retroviral vector, the retrovirus can be used to transduce cells to generate a population of cells expressing TREM2.

[0642] An additional exemplary method for generating cells containing a transgene encoding TREM2 for use in the compositions and methods described herein is transfection. Using molecular biology techniques known in the art, plasmid DNA can be generated that contains a promoter, such as a microglia-specific promoter (e.g., CD68 promoter), and a polynucleotide encoding TREM2. For example, the TREM2 gene can be amplified from a human cell line using PCR-based techniques known in the art, or the gene can be synthesized, for example, using solid-phase polynucleotide synthesis procedures. The TREM2 gene and promoter can then be ligated into a plasmid of interest, for example, using an appropriate restriction endonuclease-mediated cleavage and ligation protocol. After the plasmid DNA has been manipulated, the plasmid can be used to transfect cells, for example, using electroporation or another transfection technique described herein, to generate a population of cells expressing TREM2. In both exemplary methods described herein, TREM2 can be expressed as a TREM2 fusion protein. The TREM2 fusion protein can include a peptide sequence that includes the LDLRf Rb domain of ApoE to allow penetration of the TREM2 fusion protein across the blood-brain barrier.

[0643] Example 2. Administration of a population containing a transgene encoding TREM2 to a subject suffering from a neurocognitive disorder According to the methods disclosed herein, a practitioner can treat a subject, such as a human subject, to reduce or alleviate symptoms of NCD, such as Alzheimer's disease (AD), Nasu-Hakola disease (PLOSL), frontotemporal lobar degeneration (FTLD), or Parkinson's disease (PD). To this end, a practitioner can administer a population of cells (e.g., pluripotent cells, ESCs, iPSCs, pluripotent cells, CD34+ cells, HSCs, MPCs, BLPCs, monocytes, macrophages, microglial progenitor cells, or microglia) containing a transgene encoding TREM2 (e.g., a transgene that can be expressed in macrophages or microglia) to a human subject. Using techniques described herein or known in the art, cells can be transduced or transfected ex vivo to express TREM2. A population of cells containing a transgene encoding TREM2 can be administered to a subject, for example, systemically (e.g., intravenously), directly to the central nervous system (CNS) (e.g., intraventricularly or stereotactically), or directly to the bone marrow (e.g., intraosseously) to treat an NCD. Cells can also be administered to a patient by multiple routes of administration, for example, intravenously and intraventricularly. Cells can be administered at a concentration of 1×10 6 cells / kg~1×10 12 cells / kg or more (e.g., 1 x 10 7 cells / kg, 1×10 8 cells / kg, 1×10 9 cells / kg, 1×10 10 cells / kg, 1×10 11 cells / kg, 1×10 12 The cells are administered in a therapeutically effective amount, such as 100,000 cells / kg, or more.

[0644] Prior to administering the population of cells to the subject, one or more agents, such as busulfan, treosulfan, PLX3397, PLX647, PLX5622, and / or clodronate liposomes, may also be administered to the subject to ablate the subject's endogenous microglia and / or hematopoietic stem and progenitor cells. Other cell ablation methods known in the art, such as irradiation, may be used alone or in combination with one or more of the aforementioned agents to ablate the subject's microglia and / or hematopoietic stem and progenitor cells. These agents and / or treatments may ablate at least 5% (e.g., at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 99%, or more) of the endogenous microglia and / or hematopoietic stem and progenitor cells as assessed by PET imaging techniques known in the art. After removal of the microglia, the population of cells is administered to the subject, whereby the cells repopulate the brain and differentiate into microglia. The population of cells can be administered to the subject, for example, one week to one month (e.g., one week, two weeks, three weeks, four weeks) or more after removal of the microglia.

[0645] Following removal of the subject's endogenous microglia and / or hematopoietic stem and progenitor cells, a population of cells can be administered to the subject systemically (e.g., intravenously) or by bone marrow transplantation to reconstitute the bone marrow compartment. The number of cells can be administered at any suitable dose after conditioning. A non-limiting example of a dose is about 1×10 5 Approximately 1 x 10 cells / kg of recipient 7 cells / kg (e.g., about 2×10 5 cells / kg ~ approx. 9×10 6 cells / kg, approximately 3×10 5 cells / kg ~ approx. 8×10 6 cells / kg, approximately 4×10 5 cells / kg~about 7×10 6 cells / kg, approximately 5×10 5 cells / kg~about 6×10 6 cells / kg, approximately 5×10 5 cells / kg ~ approx. 1×10 7 cells / kg, approximately 6×105 cells / kg ~ approx. 1×10 7 cells / kg, approximately 7×10 5 cells / kg ~ approx. 1×10 7 cells / kg, approximately 8×10 5 cells / kg ~ approx. 1×10 7 cells / kg, approximately 9×10 5 cells / kg ~ approx. 1×10 7 cells / kg, or approximately 1 x 10 6 cells / kg ~ approx. 1×10 7 The dose is 100 mg / kg of cells. Administration can be before or after administration of cells containing a transgene encoding TREM2. The population of cells can be administered to the subject in an amount sufficient to treat one or more of the pathological features of NCD. For example, the population of cells can be administered in an amount sufficient to increase the amount of M2 microglia in the subject's brain compared to the amount of M1 microglia in the subject's brain. The relative increase can be measured using conventional techniques known in the art, for example by performing ELISA on the subject's CSF before and after treatment to evaluate the levels of pro-inflammatory and anti-inflammatory cytokines secreted by M1 and M2 microglia at both time points. Standard neurological tests can also be performed by a physician before and after treatment to evaluate changes in cognitive and motor function. The subject can also be evaluated, for example, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months or more after administration of the population of cells, depending on the route of administration used for the treatment. Observation of a decrease in pro-inflammatory cytokines, an increase in anti-inflammatory cytokines, a decrease in amyloid beta and / or neurofibrillary tau protein levels or aggregation thereof, a decrease in the occurrence or severity of epileptic seizures, relief of pain in the distal extremities (e.g., ankle, foot, wrist, or hand), a decrease in the occurrence of fractures, and / or improved cognitive or motor function following administration of a population of cells comprising a transgene encoding TREM2 indicates that the treatment is successfully treating the NCD.

[0646] Example 3. Disruption of endogenous TREM2 in cells prior to administration to a subject suffering from a neurocognitive disorder In any of the methods disclosed herein, cells (e.g., pluripotent cells, ESCs, iPSCs, pluripotent cells, CD34+ cells, HSCs, MPCs, BLPCs, monocytes, macrophages, microglial progenitor cells, or microglia) can be treated to destroy endogenous TREM2 before administration to a subject (e.g., a subject diagnosed with an NCD, such as AD, PLOSL, FTLD, or PD). An exemplary method of destroying endogenous TREM2 in a cell is to induce one or more double-strand breaks (DSBs) using a CRISPR / Cas system (e.g., CRISPR / Cas9 or CRISPR / Cas12a) containing a TREM2-specific guide RNA (gRNA). Following non-homologous end joining (NHEJ) to repair the DSB, the presence of the newly formed indel mutations results in the destruction of endogenous TREM2. Alternative methods for disrupting endogenous TREM2 by site-specific cleavage of genomic DNA prior to integrating a TREM2 transgene into cells include the use of zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs). Unlike the CRISPR / Cas system, these enzymes do not contain guide polynucleotides that localize to specific target sequences, but instead rely on internal DNA binding domains within the enzyme to mediate target specificity. In an exemplary embodiment, cells are engineered ex vivo with nucleases to reduce or decrease endogenous TREM2 expression by 5% or more (e.g., 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more).

[0647] Example 4. Generation of mammalian cell lines expressing TREM2 To evaluate the ability of the lentivirus-encoded codon-optimized TREM2 transgene to stably express in mammalian cell lines, mouse RAW macrophage cell lines, mouse primary microglia, and mouse lineage negative (Lin-) negative cells were transduced in vitro. In the first experiment, mouse RAW macrophage cells were transduced with lentiviral vectors containing transgenes encoding human TREM2 protein (MND.TREM2) or GFP (MND.GFP) at a multiplicity of infection (MOI) of 10, 50, 100, or 200. Another set of control cells was not transduced (NTC). TREM2 expression was assessed using an antibody raised against human TREM2. Stable expression of human TREM2 was observed in mouse macrophages (Figure 1).

[0648] In a separate experiment, mouse primary microglia were transduced with lentiviral vectors carrying transgenes encoding the human TREM2 protein (MND-TREM2) or GFP (MND-GFP). Another set of control cells was not transduced (NT). TREM2 expression was assessed using an antibody raised against human TREM2. Stable expression of human TREM2 was observed in mouse primary microglia (Figure 2).

[0649] In a separate experiment, mouse Lin- cells were transduced with a lentiviral vector carrying a transgene encoding the human TREM2 protein (Lenti TREM2) or GFP (Lenti GFP). TREM2 expression was assessed using an antibody raised against human TREM2. Stable expression of human TREM2 was observed in mouse Lin- cells (Figure 3).

[0650] Taken together, the results demonstrate that stable expression of codon-optimized human TREM2 protein can be achieved in vitro using lentiviral vectors, resulting in elevated levels of TREM2 in immortalized mouse macrophages, primary microglia, and Lin- cells, where human TREM2 is normally absent. These findings demonstrate a potential therapeutic approach for diseases caused by or associated with mutations in the TREM2 gene.

[0651] Other embodiments Various modifications and variations of the above disclosure will be apparent to those skilled in the art without departing from the scope and spirit of the disclosure. Although the disclosure has been described in relation to specific embodiments, it should be understood that the disclosure as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the disclosure that are apparent to those skilled in the art are intended to be within the scope of the disclosure.

[0652] Other embodiments are found in the claims.

[0653] JPEG2025032191000004.jpg223158JPEG2025032191000005.jpg223158JPEG2025032191000006.jpg223158 JPEG2025032191000007.jpg223158JPEG2025032191000008.jpg223158JPEG2025032191000009.jpg223158 JPEG2025032191000010.jpg223158JPEG2025032191000011.jpg223158JPEG2025032191000012.jpg223158 JPEG2025032191000013.jpg223158JPEG2025032191000014.jpg223158JPEG2025032191000015.jpg223158

Claims

1. 1. A method of treating a subject diagnosed with a neurocognitive disorder (NCD), comprising administering to the subject a composition comprising a population of cells that comprises a transgene encoding a triggering receptor expressed in one or more myeloid cells 2 (TREM2) protein having an amino acid sequence that is at least 85% identical to the amino acid sequence of any one of SEQ ID NOs: 1-3.

2. 2. The method of claim 1, wherein the NCD is a severe NCD.

3. 3. The method of claim 2, wherein the severe NCD interferes with the subject's independence and / or normal daily functioning.

4. 4. The method of claim 2 or 3, wherein said severe NCD is associated with a score obtained by said subject on a cognitive test that is at least 2 standard deviations away from the mean score of a reference population.

5. 2. The method of claim 1, wherein the NCD is a mild NCD.

6. 6. The method of claim 5, wherein the mild NCD does not interfere with the subject's independence and / or normal daily functioning.

7. 7. The method of claim 5 or 6, wherein said mild NCD is associated with a score obtained by said subject on a cognitive test that is 1-2 standard deviations away from the mean score of a reference population.

8. The method of claim 4 or 7, wherein the reference population is the general population.

9. The cognitive tests include Eight-item Informant Interview to Differentiate Aging and Dementia (AD8), Annual Wellness Visit (AWV), General Practitioner Assessment of Cognition (GPCOG), Health Risk Assessment (HRA), Memory Impairment Screen (MIS), Mini Mental Status Exam (MMSE), Montreal Cognitive Assessment (MoCA), St.

9. The method of claim 4, 7, or 8, wherein the said test is selected from the group consisting of: Louis University Mental Status Exam (SLUMS), and Short Informant Questionnaire on Cognitive Decline in the Elderly (Short IQCODE).

10. 10. The method of any one of claims 1-9, wherein the NCD is associated with impairment in one or more of complex attention, executive function, learning and memory, language, sensorimotor function, and social cognition.

11. 11. The method of any one of claims 1 to 10, wherein the NCD is not due to delirium or other psychiatric disorder.

12. The method of any one of claims 1 to 11, wherein the NCD is Alzheimer's disease (AD).

13. The method of any one of claims 1 to 11, wherein the NCD is a leukodystrophy.

14. 14. The method of claim 13, wherein the leukodystrophy is Nasu-Hakola disease (PLOSL).

15. 15. The method of any one of claims 1 to 14, wherein the transgene encodes a TREM2 protein having an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO:1, optionally wherein the TREM2 protein has an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:1, optionally wherein the TREM2 protein has an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:1, optionally wherein the TREM2 protein has the amino acid sequence of SEQ ID NO:

1.

16. 16. The method of any one of claims 1 to 15, wherein the transgene encodes a TREM2 protein having an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO:2, optionally wherein the TREM2 protein has an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:2, optionally wherein the TREM2 protein has an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:2, optionally wherein the TREM2 protein has the amino acid sequence of SEQ ID NO:

2.

17. 17. The method of any one of claims 1 to 16, wherein the transgene encodes a TREM2 protein having an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO:3, optionally wherein the TREM2 protein has an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:3, optionally wherein the TREM2 protein has an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:3, optionally wherein the TREM2 protein has the amino acid sequence of SEQ ID NO:

3.

18. The method of any one of claims 1 to 17, wherein the TREM2 is full-length TREM2.

19. The method of any one of claims 1 to 18, wherein the TREM2 comprises a TREM2 signal peptide.

20. The method of any one of claims 1 to 17, wherein the TREM2 is soluble TREM2 (sTREM2), TREM2 C-terminal fragment (TREM2-CTF), TREM2 intracellular domain (TREM2-ICD), or TREM2-A β-like (TREM2-T2β) peptide.

21. The method of any one of claims 1 to 20, wherein the TREM2 does not contain a functional ectodomain cleavage site or a functional intramembrane cleavage site.

22. The method of any one of claims 1 to 21, wherein the transgene encodes two or more TREM2 proteins.

23. 23. The method of any one of claims 1 to 22, wherein the transgene comprises a polynucleotide having at least 85% sequence identity to the nucleic acid sequence of SEQ ID NO:4, optionally wherein the transgene comprises a polynucleotide having at least 90% sequence identity to the nucleic acid sequence of SEQ ID NO:4, optionally wherein the transgene comprises a polynucleotide having at least 95% sequence identity to the nucleic acid sequence of SEQ ID NO:4, optionally wherein the transgene comprises a polynucleotide having the nucleic acid sequence of SEQ ID NO:

4.

24. 24. The method of any one of claims 1 to 23, wherein the transgene comprises a polynucleotide having at least 85% sequence identity to the nucleic acid sequence of SEQ ID NO:5, optionally wherein the transgene comprises a polynucleotide having at least 90% sequence identity to the nucleic acid sequence of SEQ ID NO:5, optionally wherein the transgene comprises a polynucleotide having at least 95% sequence identity to the nucleic acid sequence of SEQ ID NO:5, optionally wherein the transgene comprises a polynucleotide having the nucleic acid sequence of SEQ ID NO:

5.

25. 25. The method of any one of claims 1 to 24, wherein the transgene comprises a polynucleotide having at least 85% sequence identity to the nucleic acid sequence of SEQ ID NO:6, optionally wherein the transgene comprises a polynucleotide having at least 90% sequence identity to the nucleic acid sequence of SEQ ID NO:6, optionally wherein the transgene comprises a polynucleotide having at least 95% sequence identity to the nucleic acid sequence of SEQ ID NO:6, optionally wherein the transgene comprises a polynucleotide having the nucleic acid sequence of SEQ ID NO:

6.

26. 26. The method of any one of claims 1 to 25, wherein the transgene comprises a polynucleotide having at least 85% sequence identity to the nucleic acid sequence of SEQ ID NO:7, optionally wherein the transgene comprises a polynucleotide having at least 90% sequence identity to the nucleic acid sequence of SEQ ID NO:7, optionally wherein the transgene comprises a polynucleotide having at least 95% sequence identity to the nucleic acid sequence of SEQ ID NO:7, optionally wherein the transgene comprises a polynucleotide having the nucleic acid sequence of SEQ ID NO:

7.

27. 27. The method of any one of claims 1 to 26, wherein the transgene comprises a polynucleotide having at least 85% sequence identity to the nucleic acid sequence of SEQ ID NO:9, optionally wherein the transgene comprises a polynucleotide having at least 90% sequence identity to the nucleic acid sequence of SEQ ID NO:9, optionally wherein the transgene comprises a polynucleotide having at least 95% sequence identity to the nucleic acid sequence of SEQ ID NO:9, optionally wherein the transgene comprises a polynucleotide having the nucleic acid sequence of SEQ ID NO:

9.

28. 28. The method of any one of claims 1 to 27, wherein the transgene comprises a polynucleotide having at least 85% sequence identity to the nucleic acid sequence of SEQ ID NO:11, optionally wherein the transgene comprises a polynucleotide having at least 90% sequence identity to the nucleic acid sequence of SEQ ID NO:11, optionally wherein the transgene comprises a polynucleotide having at least 95% sequence identity to the nucleic acid sequence of SEQ ID NO:11, optionally wherein the transgene comprises a polynucleotide having the nucleic acid sequence of SEQ ID NO:

11.

29. 29. The method of any one of claims 1 to 28, wherein the transgene is a codon-optimized TREM2 transgene having at least 85% sequence identity to the nucleic acid sequence of any one of SEQ ID NOs: 8, 10, or 12, optionally wherein the codon-optimized TREM2 transgene comprises a polynucleotide having at least 90% sequence identity to the nucleic acid sequence of any one of SEQ ID NOs: 8, 10, or 12, optionally wherein the codon-optimized TREM2 transgene comprises a polynucleotide having at least 95% sequence identity to the nucleic acid sequence of any one of SEQ ID NOs: 8, 10, or 12, optionally wherein the codon-optimized TREM2 transgene comprises a polynucleotide having the nucleic acid sequence of any one of SEQ ID NOs: 8, 10, or 12.

30. 30. The method of any one of claims 1 to 29, wherein the TREM2 is a TREM2 fusion protein.

31. 31. The method of claim 30, wherein the TREM2 fusion protein comprises the receptor binding (Rb) domain of apolipoprotein E (ApoE).

32. 32. The method of claim 31 , wherein the Rb domain comprises a portion of ApoE having an amino acid sequence of residues 25-185, 50-180, 75-175, 100-170, 125-160, or 130-150 of SEQ ID NO:

13.

33. The method of claim 31 or 32, wherein the Rb domain comprises a region having at least 70% sequence identity to the amino acid sequence of residues 159-167 of SEQ ID NO:

13.

34. 34. The method of any one of claims 1 to 33, wherein the transgene encoding TREM2 further comprises a microRNA (miRNA)-126 (miR-126) targeting sequence in the 3'-UTR.

35. The method of any one of claims 12 to 34, wherein the AD or PLOSL is TREM2-associated AD or PLOSL.

36. The method of any one of claims 1 to 35, wherein the cell is a pluripotent or multipotent cell.

37. 37. The method of claim 36, wherein the pluripotent cells are CD34+ cells.

38. 38. The method of claim 37, wherein the CD34+ cells are hematopoietic stem cells (HSCs) or myeloid progenitor cells (MPCs).

39. 37. The method of claim 36, wherein the pluripotent cell is an embryonic stem cell (ESC) or an induced pluripotent stem cell (iPSC).

40. 36. The method of any one of claims 1 to 35, wherein the cell is a blood lineage progenitor cell (BLPC), a microglial progenitor cell, a monocyte, a macrophage, or a microglia.

41. 41. The method of claim 40, wherein the BLPCs are monocytes.

42. 42. The method of any one of claims 1 to 41, wherein the subject has a population of endogenous microglia depleted prior to administration of the composition.

43. 42. The method of any one of claims 1 to 41, comprising removing a population of endogenous microglia in the subject prior to administering the composition to the subject.

44. 44. The method of claim 42 or 43, wherein the endogenous microglia are ablated using an agent selected from the group consisting of busulfan, PLX3397, PLX647, PLX5622, treosulfan, and clodronate liposomes, by radiation therapy, or a combination thereof.

45. 45. The method of any one of claims 1-44, wherein the composition is administered to the subject by systemic administration, by direct administration to the subject's central nervous system, by direct administration to the subject's bone marrow, or by bone marrow transplantation comprising the composition.

46. The method of any one of claims 1 to 45, further comprising administering to the subject a population of cells.

47. 47. The method of claim 46, wherein the population of cells is administered to the subject prior to administration of the composition or after administration of the composition.

48. 47. The method of claim 45 or 46, wherein the cell is a pluripotent or multipotent cell.

49. 49. The method of claim 48, wherein the pluripotent cells are CD34+ cells.

50. 50. The method of claim 49, wherein the CD34+ cells are HSCs or MPCs.

51. 49. The method of claim 48, wherein the pluripotent cells are ESCs or iPSCs.

52. The method of any one of claims 46 to 51, wherein the cell is a BLPC, a microglial progenitor cell, a monocyte, a macrophage, or a microglia.

53. 53. The method of claim 52, wherein the BLPCs are monocytes.

54. 54. The method of any one of claims 46-53, wherein the cells have not been modified to express a transgene encoding TREM2.

55. 55. The method of any one of claims 1-54, wherein endogenous TREM2 is disrupted in the cell, in the subject, or in a population of neurons in the subject prior to administration of the composition to the subject.

56. 56. The method of claim 55, wherein the endogenous TREM2 is destroyed by contacting the cell with a nuclease that catalyzes cleavage of an endogenous TREM2 nucleic acid in the cell.

57. 57. The method of claim 56, wherein the nuclease is CRISPR-associated protein 9 (Cas9), CRISPR-associated protein 12a (Cas12a), a transcription activator-like effector nuclease, a meganuclease, or a zinc finger nuclease.

58. 58. The method of any one of claims 55-57, wherein the endogenous TREM2 is disrupted by administering an inhibitory RNA molecule to the cell, the subject, or the population of neurons.

59. 59. The method of claim 58, wherein the inhibitory RNA molecule is a short interfering RNA, a short hairpin RNA, or a miRNA.

60. 60. The method of any one of claims 1 to 59, wherein the cells are autologous or allogeneic cells.

61. 61. The method of any one of claims 1 to 60, wherein the cells are transfected or transduced ex vivo to express the TREM2.

62. 62. The method of claim 61, wherein the cells are transduced with a viral vector selected from the group consisting of adeno-associated virus (AAV), adenovirus, parvovirus, coronavirus, rhabdovirus, paramyxovirus, picornavirus, alphavirus, herpesvirus, poxvirus, and retroviridae viruses.

63. 63. The method of claim 62, wherein the viral vector is a retroviridae viral vector.

64. 64. The method of claim 63, wherein the Retroviridae viral vector is a lentiviral vector, an alpharetroviral vector, or a gammaretroviral vector.

65. 65. The method of any one of claims 62 to 64, wherein the Retroviridae viral vector comprises a central polypurine tract, a woodchuck hepatitis virus post-transcriptional regulatory element, a 5'-LTR, an HIV signal sequence, an HIV Psi signal 5'-splice site, a delta-GAG element, a 3'-splice site, and a 3'-self-inactivating LTR.

66. 63. The method of claim 62, wherein the viral vector is an AAV selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, and AAVrh74.

67. The method of any one of claims 62 to 66, wherein the viral vector is a pseudotyped viral vector.

68. 68. The method of claim 67, wherein the pseudotyped viral vector is selected from the group consisting of pseudotyped AAV, pseudotyped adenovirus, pseudotyped parvovirus, pseudotyped coronavirus, pseudotyped rhabdovirus, pseudotyped paramyxovirus, pseudotyped picornavirus, pseudotyped alphavirus, pseudotyped herpesvirus, pseudotyped poxvirus, and pseudotyped Retroviridae virus.

69. 69. The method of any one of claims 1 to 68, wherein expression of TREM2 in the cell is mediated by a ubiquitous promoter, a cell lineage specific promoter, or a synthetic promoter.

70. 70. The method of claim 69, wherein the ubiquitous promoter is selected from the group consisting of elongation factor 1-alpha promoter and phosphoglycerate kinase 1 promoter.

71. 70. The method of claim 69, wherein the cell lineage specific promoter is selected from the group consisting of TREM2 promoter, CD68 promoter, CD11b promoter, C-X3-C motif chemokine receptor 1 promoter, allograft inflammatory factor 1 promoter, purinergic receptor P2Y12 promoter, transmembrane protein 119 promoter, and colony stimulating factor 1 receptor promoter.

72. A composition comprising a population of cells expressing a transgene encoding TREM2.

73. 73. The composition of claim 72, wherein the TREM2 is full-length TREM2.

74. The composition of claim 72 or 73, wherein the TREM2 or variant thereof has an amino acid sequence having at least 85% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 1-3.

75. 75. The composition of claim 74, wherein the TREM2 has an amino acid sequence having at least 85% sequence identity to SEQ ID NO:1, optionally wherein the TREM2 protein has an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:1, optionally wherein the TREM2 protein has an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:1, optionally wherein the TREM2 protein has the amino acid sequence of SEQ ID NO:

1.

76. The composition of claim 74 or 75, wherein the TREM2 has an amino acid sequence having at least 85% sequence identity to SEQ ID NO:2, optionally wherein the TREM2 protein has an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:2, optionally wherein the TREM2 protein has an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:2, optionally wherein the TREM2 protein has the amino acid sequence of SEQ ID NO:

2.

77. 77. The composition of any one of claims 74-76, wherein the TREM2 has an amino acid sequence having at least 85% sequence identity to SEQ ID NO:3, optionally wherein the TREM2 protein has an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:3, optionally wherein the TREM2 protein has an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:3, optionally wherein the TREM2 protein has the amino acid sequence of SEQ ID NO:

3.

78. The composition of any one of claims 72-77, wherein the TREM2 comprises a TREM2 signal peptide.

79. The composition of any one of claims 72-78, wherein the TREM2 is sTREM2, TREM2-CTF, TREM2-ICD, or TREM2-T2β peptide.

80. The composition of any one of claims 72-79, wherein the TREM2 does not contain a functional ectodomain cleavage site or a functional intramembrane cleavage site.

81. The composition of any one of claims 72-80, wherein the transgene encodes two or more TREM2 transgenes.

82. 82. The composition of any one of claims 72-81, wherein the transgene comprises a polynucleotide having at least 85% sequence identity to the nucleic acid sequence of SEQ ID NO:4, optionally wherein the transgene comprises a polynucleotide having at least 90% sequence identity to the nucleic acid sequence of SEQ ID NO:4, optionally wherein the transgene comprises a polynucleotide having at least 95% sequence identity to the nucleic acid sequence of SEQ ID NO:4, optionally wherein the transgene comprises a polynucleotide having the nucleic acid sequence of SEQ ID NO:

4.

83. 83. The composition of any one of claims 72-82, wherein the transgene comprises a polynucleotide having at least 85% sequence identity to the nucleic acid sequence of SEQ ID NO:5; optionally, the transgene comprises a polynucleotide having at least 90% sequence identity to the nucleic acid sequence of SEQ ID NO:5; optionally, the transgene comprises a polynucleotide having at least 95% sequence identity to the nucleic acid sequence of SEQ ID NO:5; optionally, the transgene comprises a polynucleotide having the nucleic acid sequence of SEQ ID NO:

5.

84. 84. The composition of any one of claims 72-83, wherein the transgene comprises a polynucleotide having at least 85% sequence identity to the nucleic acid sequence of SEQ ID NO:6, optionally wherein the transgene comprises a polynucleotide having at least 90% sequence identity to the nucleic acid sequence of SEQ ID NO:6, optionally wherein the transgene comprises a polynucleotide having at least 95% sequence identity to the nucleic acid sequence of SEQ ID NO:6, optionally wherein the transgene comprises a polynucleotide having the nucleic acid sequence of SEQ ID NO:

6.

85. 85. The composition of any one of claims 72-84, wherein the transgene comprises a polynucleotide having at least 85% sequence identity to the nucleic acid sequence of SEQ ID NO:7; optionally, the transgene comprises a polynucleotide having at least 90% sequence identity to the nucleic acid sequence of SEQ ID NO:7; optionally, the transgene comprises a polynucleotide having at least 95% sequence identity to the nucleic acid sequence of SEQ ID NO:7; optionally, the transgene comprises a polynucleotide having the nucleic acid sequence of SEQ ID NO:

7.

86. 9. The composition of any one of claims 72-85, wherein the transgene comprises a polynucleotide having at least 85% sequence identity to the nucleic acid sequence of SEQ ID NO:9; optionally, the transgene comprises a polynucleotide having at least 90% sequence identity to the nucleic acid sequence of SEQ ID NO:9; optionally, the transgene comprises a polynucleotide having at least 95% sequence identity to the nucleic acid sequence of SEQ ID NO:9; optionally, the transgene comprises a polynucleotide having the nucleic acid sequence of SEQ ID NO:

9.

87. 87. The composition of any one of claims 72-86, wherein the transgene comprises a polynucleotide having at least 85% sequence identity to the nucleic acid sequence of SEQ ID NO:11, optionally wherein the transgene comprises a polynucleotide having at least 90% sequence identity to the nucleic acid sequence of SEQ ID NO:11, optionally wherein the transgene comprises a polynucleotide having at least 95% sequence identity to the nucleic acid sequence of SEQ ID NO:11, optionally wherein the transgene comprises a polynucleotide having the nucleic acid sequence of SEQ ID NO:

11.

88. 82. The composition of any one of claims 72-81, wherein the transgene is a codon-optimized TREM2 transgene.

89. 89. The composition of claim 88, wherein the codon optimized TREM2 transgene comprises a polynucleotide having a nucleic acid sequence having at least 85% sequence identity to the nucleic acid sequence of any one of SEQ ID NOs: 8, 10, or 12; optionally, the codon optimized TREM2 transgene comprises a polynucleotide having at least 90% sequence identity to the nucleic acid sequence of any one of SEQ ID NOs: 8, 10, or 12; optionally, the codon optimized TREM2 transgene comprises a polynucleotide having at least 95% sequence identity to the nucleic acid sequence of any one of SEQ ID NOs: 8, 10, or 12.

90. The composition of any one of claims 72-89, wherein the TREM2 is a TREM2 fusion protein.

91. 91. The composition of claim 90, wherein the TREM2 fusion protein comprises the Rb domain of ApoE.

92. 92. The composition of claim 91, wherein the Rb domain comprises a portion of ApoE having an amino acid sequence of residues 25-185, 50-180, 75-175, 100-170, 125-160, or 130-150 of SEQ ID NO:

13.

93. The composition of claim 91 or 92, wherein the Rb domain comprises a region having at least 70% sequence identity to the amino acid sequence of residues 159-167 of SEQ ID NO:

13.

94. The composition of any one of claims 72 to 93, wherein the transgene encoding TREM2 further comprises a miR-126 targeting sequence in the 3'-UTR.

95. The composition of any one of claims 72 to 94, wherein the cell is a pluripotent or multipotent cell.

96. The composition of claim 95, wherein the pluripotent cells are CD34+ cells.

97. The composition of claim 96, wherein the CD34+ cells are HSCs or MPCs.

98. 96. The composition of claim 95, wherein the pluripotent cells are ESCs or iPSCs.

99. The composition of any one of claims 72 to 94, wherein the cell is a BLPC, a microglial progenitor cell, a macrophage, or a microglia.

100. 100. The composition of claim 99, wherein the BLPC is a monocyte.

101. The composition of any one of claims 72-100, wherein the cells are transfected or transduced ex vivo to express the TREM2.

102. A pharmaceutical composition comprising the composition of any one of claims 72 to 101, further comprising a pharma- ceutically acceptable carrier, diluent, or excipient.

103. A kit comprising the composition of any one of claims 72 to 101 or the pharmaceutical composition of claim 102 and a package insert, said package insert instructing a user of the kit to carry out the method of any one of claims 1 to 71.

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