Gene therapy for TREM2-associated diseases and disorders

Abstract

Nucleic acids encoding triggering receptor expressed on myeloid cells 2 (TREM2) and that can be used in gene therapy are described, as are expression constructs, vectors, and methods of using them for the treatment of TREM2-associated diseases and disorders, particularly neurological disorders such as Alzheimer's disease (AD), adult-onset leukoencephalopathy with axonal spheroids and pigmented glia (ALSP), or Nasu-Hakola disease (NHD).

Description

[Technical Field]

[0001] (Reference to electronically submitted sequence listing)

[0001] The present disclosure is filed with a Sequence Listing in ST.26 XML format. The Sequence Listing is provided as a file entitled "30436_WO" created on September 13, 2023, and is 74.6 kilobytes in size. The Sequence Listing information in ST.26 XML format is incorporated herein by reference in its entirety.

[0002] FIELD OF THE INVENTION The present disclosure relates generally to biology and medicine, and more particularly to synthetic nucleic acids and their use as gene therapy for treating diseases and disorders associated with triggering receptor expressed on myeloid cells 2 (TREM2), particularly neurological disorders such as Alzheimer's Disease (AD), Adult-Onset Leukoencephalopathy with Axonal Spheroids and Pigmented Glia (ALSP), or Nasu-Hakola Disease (NHD). [Background technology]

[0003] TREM2 is a cell surface transmembrane glycoprotein that is primarily expressed in myeloid lineage cells, including microglia. In humans, complete absence of TREM2 has been shown to cause NHD, a rare neurodegenerative disease associated with late-onset dementia, demyelination, and cerebral atrophy. See, for example, Paloneva et al. (2002) Am. J. Hum. Genet. 71:656-662 and Paloneva et al. (2003) J. Exp. Med. 198:669-675.

[0004] Mutations in TREM2 are also associated with the risk of developing AD. See, for example, Guerreiro et al. (2013) N. Engl. J. Med. 368:117-127. AD is an irreversible, progressive brain disorder characterized by the presence of abnormal protein deposits throughout the brain, which impair neuronal function, destroy connections between neurons, and ultimately lead to cell death. These deposits consist of amyloid-beta plaques and tangles formed by phosphorylated tau protein. Individuals with mild AD experience memory loss, leading to wandering, difficulty handling money, repetitive questioning, and personality and behavioral changes. Furthermore, individuals with moderate AD exhibit worsening memory loss, difficulty confusing and recognizing friends and family, an inability to learn new things, and hallucinations, delusions, and delusional disorder. Furthermore, individuals with severe AD are unable to communicate and are completely dependent on others for their care. Eventually, protein plaques and tangles spread throughout the brain, leading to significant tissue shrinkage.

[0005] Mutations in CSF1R can lead to a microgliopathy known as Colony Stimulating Factor 1 Receptor (CSF1R)-Related Adult-Onset Leukoencephalopathy (CRL), a subclass of ALSP. Interestingly, TREM2 activates a similar signaling pathway, resulting in intracellular and functional responses that phenocopy CSF1R activation. It is thought that increased cellular TREM2 can attenuate CSF1R loss of function and thus CRL-associated pathology. See, for example, Tchessalova et al. (2022) Alzheimer's Dement. 18:e061595.

[0006] Several therapeutic approaches to increase TREM2 are known, including small molecules or monoclonal antibodies for regulating TREM2 downstream signaling. More recently, gene therapy approaches to increase TREM2 and / or its activity have also been described. See, for example, WO 2019 / 070894.

[0007] However, there is a need for other disease-modifying therapies to increase TREM2 and / or its activity in individuals with TREM2-associated diseases and / or disorders. Summary of the Invention

[0008] To address this need, the present disclosure describes synthetic nucleic acids for use in treating TREM2-associated diseases and disorders. In one example, the synthetic nucleic acid encodes TREM2 (i.e., a TREM2-encoding transgene) and comprises a nucleotide sequence having at least about 90% (e.g., at least about 90%, 91%, 92, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to any one of SEQ ID NOs: 3-6. In some examples, the TREM2-encoding transgene may be codon-optimized. Alternatively, or in addition, the TREM2-encoding transgene may be CpG-minimized or CpG-depleted. In certain examples, the nucleotide sequence of the TREM2-encoding transgene is SEQ ID NO: 3, 4, 5, or 6.

[0009] In another example, the synthetic nucleic acid may be an expression construct comprising a first nucleotide sequence of at least one expression control element operably linked to a second nucleotide sequence encoding TREM2 (i.e., a TREM2-encoding transgene), wherein the second nucleotide sequence has at least about 90% (e.g., at least about 90%, 91%, 92, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to any one of SEQ ID NOs: 3-6. As described above, the TREM2-encoding transgene may be codon-optimized. Alternatively, or in addition, the TREM2-encoding transgene may be CpG-minimized or CpG-depleted. In certain examples, the nucleotide sequence of the TREM2-encoding transgene is SEQ ID NO: 3, 4, 5, or 6.

[0010] In some examples, the at least one expression control element may be a promoter, an enhancer, a post-transcriptional regulatory element, or a polyadenylation signal. In other examples, the promoter may be a chicken β-actin (CBA) promoter, a CD68 promoter, or an F4 / 80 promoter, each of which may comprise a nucleotide sequence having at least about 90% (e.g., at least about 90%, 91%, 92, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to any one of SEQ ID NOs: 7-9. In certain other examples, the nucleotide sequence of the promoter is SEQ ID NO: 7, 8, or 9. In certain other examples, the nucleotide sequence of the promoter is SEQ ID NO: 8.

[0011] In some examples, the enhancer may be a cytomegalovirus enhancer (CMVe) and may comprise a nucleotide sequence having at least about 90% (e.g., at least about 90%, 91%, 92, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to SEQ ID NO: 10. In one particular example, the nucleotide sequence of the enhancer is SEQ ID NO: 10.

[0012] In some examples, the post-transcriptional regulatory element may be a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) and may comprise a nucleotide sequence having at least about 90% (e.g., at least about 90%, 91%, 92, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to SEQ ID NO: 11. In one particular example, the nucleotide sequence of the post-transcriptional regulatory element is SEQ ID NO: 11.

[0013] In some examples, the polyadenylation signal can be a bovine growth hormone polyA (BGHpA) signal tail and can comprise a nucleotide sequence having at least about 90% (e.g., at least about 90%, 91%, 92, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to SEQ ID NO: 12. In one particular example, the nucleotide sequence of the polyadenylation signal is SEQ ID NO: 12.

[0014] In some instances, the expression construct may include an additional nucleotide sequence encoding a second transgene or encoding an inhibitory nucleic acid.

[0015] In another example, the synthetic nucleic acid can be a vector comprising an expression construct herein. In some examples, the vector can be a plasmid or a viral vector. In other examples, the viral vector can be an adeno-associated virus (AAV) vector or a baculovirus vector. When the vector is an AAV vector, it can comprise at least one AAV inverted terminal repeat (ITR) sequence flanking the expression construct. In some examples, the AAV ITR can be a wild-type (WT) AAV2 ITR and can have a nucleotide sequence having at least about 90% (e.g., at least about 90%, 91%, 92, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to SEQ ID NO: 13, or a reverse complement thereof. In other examples, the AAV ITRs may be modified AAV2 ITRs and may have a nucleotide sequence having at least about 90% (e.g., at least about 90%, 91%, 92, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to SEQ ID NO: 14, or a reverse complement thereof. In certain examples, the nucleotide sequence of the AAV ITR is SEQ ID NO: 13 or 14, or a reverse complement thereof.

[0016] In some examples, the vector may also include a TRY region and may have a nucleotide sequence having at least about 90% (e.g., at least about 90%, 91%, 92, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to SEQ ID NO: 15. In one particular example, the nucleotide sequence of the TRY region is SEQ ID NO: 15.

[0017] In some examples, the vector may also include a nucleotide sequence that assists in packaging the vector with capsid proteins (i.e., a sequence that optimizes the size of the vector for packaging within the capsid proteins, also referred to as a stuffer sequence). In some examples, the stuffer sequence has at least about 90% (e.g., at least about 90%, 91%, 92, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to any one of SEQ ID NOs: 16-18. In certain examples, the nucleotide sequence is SEQ ID NO: 16, 17, or 18.

[0018] The present disclosure also describes an rAAV comprising: (i) an rAAV vector comprising a nucleotide sequence having at least about 90% (e.g., at least about 90%, 91%, 92, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to any one of SEQ ID NOs: 3-6 (i.e., a TREM 2-encoding transgene); and (2) an AAV6 capsid protein.

[0019] In some instances, the rAAV (a) In the order from 5' to 3', (i) the first AAV ITR or a reverse complement thereto; (ii) a promoter; (iii) a TREM2-encoding transgene; (iv) post-transcriptional regulatory elements; (v) a polyadenylation signal, and (vii) a rAAV vector having a nucleotide sequence comprising a second AAV ITR or a reverse complement thereof; (b) Encapsidated into AAV6 capsid protein.

[0020] In some examples, the AAV6 capsid protein is a modified AAV6 capsid protein (e.g., an AAV6™ capsid protein) that comprises an amino acid sequence having at least about 95% (e.g., at least about 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to SEQ ID NO: 19. In one particular example, the AAV6™ capsid protein amino acid sequence is SEQ ID NO: 19.

[0021] In some examples, the rAAV vector comprises a nucleotide sequence having at least about 90% (e.g., at least about 90%, 91%, 92, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to any one of SEQ ID NOs: 21-26. In certain examples, the nucleotide sequence of the rAAV vector is any one of SEQ ID NOs: 21, 22, 23, 24, 25, or 26.

[0022] In some examples, the rAAV vector further comprises a nucleotide sequence (i.e., a stuffer sequence) that assists in packaging into the vector a capsid protein having at least about 90% (e.g., at least about 90%, 91%, 92, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to any one of SEQ ID NOs: 16-18. In certain examples, the nucleotide sequence of the stuffer sequence is SEQ ID NO: 16, 17, or 18.

[0023] In some examples, the rAAV vector further comprises a second nucleic acid having a nucleotide sequence having the reverse complement of any one of SEQ ID NOs: 21, 22, 23, 24, 25, or 26.

[0024] The present disclosure also describes pharmaceutical compositions comprising the synthetic nucleic acids, expression constructs, vectors, or rAAV herein and a pharmaceutically acceptable carrier.

[0025] In some examples, the pharmaceutical composition can be a formulation comprising the rAAV herein and one or more of the following: (a) approximately 20 mM TRIS (pH 8.0); (b) about 1 mM MgCl2; (c) about 200 mM NaCl, and (d) Poloxamer 188 at approximately 0.005% (w / v).

[0026] In some instances, the rAAV is about 1 x 10 13 Vector genome (vg) ~ approx. 7 × 10 14 In other examples, the rAAV may be present in the formulation at a concentration of about 3.5 x 10 vg. 13 vg, approx. 7.0×10 13 vg, or approximately 1.4 × 10 14 It may be present in the formulation at a concentration of 0.05g.

[0027] The present disclosure also describes methods of treating TREM2-related diseases and disorders in an individual, which may include administering to the individual at least an effective amount of a synthetic nucleic acid, expression construct, vector, or rAAV described herein.

[0028] In some examples, the TREM2-associated disease or disorder can be AD, ALSP, or NHD.

[0029] In some examples, the method may also include measuring TREM2, CSF1R, neurofilament light chain (NfL), chitotriosidase (Chit1), and / or granulocyte-macrophage colony stimulating factor (GM-CSF) in plasma and / or cerebrospinal fluid (CSF) and / or urine from the individual, and comparing the obtained values ​​to previously obtained comparable or control values ​​to assess the efficacy of the method.

[0030] In some examples, the methods may also include administering to the individual an effective amount of at least one additional therapeutic agent.

[0031] The present disclosure further describes compositions comprising rAAV for use as a medicament for the treatment of TREM2-associated diseases and disorders, such as AD, ALSP, or NHD.

[0032] The present disclosure further describes compositions comprising rAAV for use in treating TREM2-associated diseases and disorders, such as AD, ALSP, or NHD.

[0033] The present disclosure further describes the use of rAAV in the manufacture of a medicament for treating TREM2-associated diseases and disorders, such as AD, ALSP, or NHD, which may optionally include an additional therapeutic agent.

[0034] An advantage of the synthetic nucleic acids described herein is that they can increase and / or improve microglial function (i.e., development, maintenance, and / or activation) in AD, ALSP, or NHD by increasing both membrane-bound and secreted TREM2 levels, and / or by enhancing or replacing CSF1R signaling with TREM2 signaling to activate downstream CSF1R effects.

[0035] An advantage of the rAAV described herein is that it exhibits broader central nervous system (CNS) biodistribution and increased microglial transduction compared to rAAV with the AAV9 capsid protein. [Brief explanation of the drawings]

[0036] Further advantages, benefits, features and objects will become more readily apparent from a consideration of the following detailed description, which refers to the following drawings. [Figure 1]FIG. 1 is a schematic diagram showing a first exemplary rAAV vector for expressing TREM2. [Figure 2] FIG. 1 is a schematic diagram showing a second exemplary rAAV vector for expressing TREM2. [Figure 3] FIG. 1 is a schematic diagram showing a third exemplary rAAV vector for expressing TREM2. [Figure 4] FIG. 1 is a schematic diagram showing a fourth exemplary rAAV vector for expressing TREM2. [Figure 5A] TREM2 mRNA (FIG. 5A) and protein expression (FIG. 5B) in HMC3 cell lines when transduced with an exemplary rAAV vector encapsidated in AAV6TM capsid protein compared to the same rAAV vector encapsidated in AAV9 capsid protein (n=4 / group) (AAV6TM=filled circles, AAV9=open circles). [Figure 5B] TREM2 mRNA (FIG. 5A) and protein expression (FIG. 5B) in HMC3 cell lines when transduced with an exemplary rAAV vector encapsidated in AAV6TM capsid protein compared to the same rAAV vector encapsidated in AAV9 capsid protein (n=4 / group) (AAV6TM=filled circles, AAV9=open circles). [Figure 6A] TREM2 expression in the HMC3 cell line is shown when transduced with one of two exemplary rAAV vectors, both encapsidated in the AAV6™ capsid protein (Figure 6A = first rAAV vector, Figure 6B = second rAAV vector). [Figure 6B] TREM2 expression in the HMC3 cell line is shown when transduced with one of two exemplary rAAV vectors, both encapsidated in the AAV6™ capsid protein (Figure 6A = first rAAV vector, Figure 6B = second rAAV vector). [Figure 7]A-D show the biodistribution of TREM2 in the somatosensory cortex (Figure 7A), hippocampus (Figure 7B), cervical spinal cord (Figure 7C), and liver (Figure 7D) of mice administered low or high doses of an exemplary rAAV vector encapsidated in an AAV6TM capsid protein or the same rAAV vector encapsidated in an AAV9 capsid protein (n = 8-12 / group). [Figure 8] Figures 8A-C show TREM2 protein levels in the CSF (Figure 8A), liver (Figure 8B), and serum (Figure 8C) of mice administered low or high doses of an exemplary rAAV vector encapsidated in AAV6TM capsid protein or the same rAAV vector encapsidated in AAV9 capsid protein (n = 8-12 / group; a value of 20.48 pg / mL was set as the detection threshold (dotted line). For all graphs, statistics were determined using ANOVA followed by Dunnett's test compared to the 5xFAD + vehicle group. (*) p < 0.1, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001). [Figure 9] Figures 9A-C show amyloid-β levels in the hippocampus (Figures 9A-9B) and X34+ plaques in the cortex (Figure 9C) of mice administered low or high doses of an exemplary rAAV vector encapsidated in an AAV6™ capsid protein or the same rAAV vector encapsidated in an AAV9 capsid protein (n=8-12 / group, compared to the 5xFAD+vehicle group, statistics determined using ANOVA followed by Dunnett's test; (*) p<0.1, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). [Figure 10]A-B show inflammation in the cortex (FIG. 10A for Iba and FIG. 10B for IL-10) of mice administered low or high doses of an exemplary rAAV vector encapsidated in an AAV6™ capsid protein or the same rAAV vector encapsidated in an AAV9 capsid protein (n=8-12 / group, compared to the 5xFAD + vehicle group, statistics determined using ANOVA followed by Dunnett's test; (*) p<0.1, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). [Figure 11] FIG. 1 shows the biodistribution of TREM2 in non-human primates (NHPs) administered low or high doses of an exemplary rAAV vector encapsidated in AAV6™ capsid protein (n=2 / group, vector copies were measured using ddPCR). [Figure 12] Figures 12A-C show TREM2 protein levels in the liver, spinal cord, and dorsal root ganglion (DRG) (Figure 12A), CSF (Figure 12B), and serum (Figure 12C) of NHPs administered an exemplary rAAV vector encapsidated in a low or high dose of AAV6™ capsid protein (n=2 / group; a value of 20.48 pg / mL was set as the detection threshold (dotted line)). DETAILED DESCRIPTION OF THE INVENTION

[0037] overview TREM2 is a cell surface transmembrane glycoprotein expressed primarily in myeloid lineage cells, including microglia. TREM2 directly binds to amyloid beta, facilitating microglial assembly around plaques, limiting plaque accumulation, and promoting phagocytosis. TREM2 mutations, associated with increased AD risk, reduce the microglial response to amyloid plaques.

[0038] Additionally, CRL is a microglial disease that can be caused by mutations in the kinase domain of CSF1R and represents the most common genetic form of ALSP. Interestingly, CSF1R and TREM2 share a common convergent signaling pathway for maintaining and activating microglia. Thus, TREM2 expression can rescue or compensate for CSF1R loss of function.

[0039] Two TREM2 isoforms exist: isoform 1 is 230 amino acids (aa) long (SEQ ID NO: 1; NCBI Reference SEQ ID NO: NP 061838.1) and isoform 2 is 219 aa long (SEQ ID NO: 2; NCBI Reference SEQ ID NO: NP_001258750.1). Exemplary nucleic acid sequences can be found in NCBI Reference SEQ ID NOs: NM_018965.4 and NM_001271821.2 (human), NM_031254.3 and NM_001272078.1 (mouse), XP_006244486.1 and XP_006244487.1 (rat), and XP_001117305.2 and XP_001174118.2 (non-human primates). However, one of skill in the art will appreciate that further examples of TREM2 mRNA sequences are readily available using publicly available databases such as, for example, GenBank and UniProt.

[0040] The present disclosure describes synthetic nucleic acids for use as gene therapy in the treatment of TREM2-associated diseases and disorders, which deliver a functional copy of TREM2 encoding TREM2 to an individual in need thereof.

[0041] Abbreviations and Definitions Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described herein.

[0042] In addition, reference to an element by the indefinite article "a" or "an" does not exclude the possibility that more than one element is present, unless the context clearly requires that there is one and only one element. Thus, the indefinite article "a" or "an" normally means "at least one."

[0043] Furthermore, the use of "including" and other forms such as "including but not limited to," "include," "includes," and "included" is not limiting.

[0044] Certain abbreviations used herein are as follows: "aa" refers to amino acid. "AAV" refers to adeno-associated virus. "AcNPV" refers to Autographa californica nuclear polyhedrosis. "AD" refers to Alzheimer's disease. "ALS" refers to amyotrophic lateral sclerosis. "ALSP" refers to adult-onset leukoencephalopathy with axonal spheroids and pigmented glia. "BAC" refers to bacterial artificial chromosome. "BEYS" refers to baculovirus vector expression system. "BBB" refers to the blood-brain barrier. "BGHpA" refers to bovine growth hormone polyA signal tail. "bp" refers to base pair. "CBA" refers to chicken-β actin. "Chit1" refers to chitotriosidase. "CNS" refers to central nervous system. "CSF" refers to cerebrospinal fluid. "CMVe" refers to cytomegalovirus enhancer. "CpG" refers to cytosine-phosphate-guanine. "CRL" refers to Colony Stimulating Factor 1 Receptor-Related Adult-Onset Leukoencephalopathy."CSF1R" refers to the colony stimulating factor 1 receptor gene. "CSF1R" refers to the colony stimulating factor 1 receptor protein. "sCSF1R" refers to the soluble colony stimulating factor 1 receptor protein. "DAM" refers to disease-associated microglia. "ddPCR" refers to droplet digital polymerase chain reaction. "DEA" refers to diethylamine. "DNA" refers to deoxyribonucleic acid, and "DRG" ​​refers to dorsal root ganglion. "ds" refers to double-stranded. "EDTA" refers to ethylenediaminetetraacetic acid. "EGTA" refers to ethylene glycol tetraacetic acid. "ELISA" refers to enzyme-linked immunosorbent assay. "FA" refers to formic acid. "FAD" refers to familiar Alzheimer's disease. "FTD" refers to frontotemporal dementia. "GC" refers to genome copy. "g" refers to gram. "gDNA" refers to genomic DNA. "GM-CSF" refers to granulocyte-macrophage colony-stimulating factor (human microglial clone 3). "HMC3" refers to human microglial clone 3. "hr" refers to hour."ICM" refers to intra-cisterna magna. "ICV" refers to intracerebroventricular. "iPSC" refers to induced pluripotent stem cell. "IRES" refers to internal ribosome entry site. "ITR" refers to inverted terminal repeat. "IV" refers to intravenous. "kg" refers to kilogram. "min" refers to minute. "mL" refers to milliliter. "MSD" refers to MesoScale Discovery. "Nab" refers to neutralizing antibody. "NfL" refers to neurofilament light chain. "NHD" refers to Nasu-Hakola Disease. "NHP" refers to non-human primate. "nt" refers to nucleotide. "pg" refers to picogram. "PLX" refers to PLX3397 (also known as pexidartinib; 5-((5-chloro-1H-pyrrolo[2,3-b]pyridin-3-yl)methyl)-N-((6-(trifluoromethyl)pyridin-3-yl)methyl)pyridin-2-amine or C). 20 H 15ClF3N5. "qRT-PCR" refers to real-time quantitative reverse transcription polymerase chain reaction. "rAAV" refers to recombinant adeno-associated virus. "RBS" refers to Rep binding site. "RNA" refers to ribonucleic acid. "SC" refers to subcutaneous. "sec" refers to second. "SEM" refers to standard error of mean. "ss" refers to single-stranded. "THB" refers to triethylammonium bicarbonate buffer. "sTREM2" refers to soluble triggering receptor expressed on myeloid cells 2 protein. "TREM2" refers to the triggering receptor expressed on myeloid cells 2 gene. "TREM2" refers to the triggering receptor expressed on myeloid cells 2 protein. "trs" refers to terminal resolution site. "μL" refers to microliter. "VC" refers to vector copy. "vg" refers to vector genome. "WPRE" refers to woodchuck hepatitis virus post-transcriptional regulatory element. "WT" refers to wild-type."YAC" refers to yeast artificial chromosome.

[0045] Certain definitions used herein are defined as follows: As used herein, "AAV6TM" refers to an AAV6TM capsid protein (AAV6 capsid protein amino acid sequence: T492V, Y705F, and Y731F (see, e.g., SEQ ID NO: 19)) having at least the following three mutations in its amino acid sequence (i.e., a triple mutant) compared to wild-type (WT; NCBI reference sequence AAB95450.1).

[0046] As used herein, "about" means within a statistically significant range of a value(s), e.g., a specified concentration, length, molecular weight, pH, sequence similarity, time frame, temperature, volume, etc. Such values ​​or ranges may be within an order of magnitude, typically within 20%, more typically within 10%, and even more typically within 5% of a given value or range. The allowable variation encompassed by "about" will depend on the particular system under study and can be readily appreciated by one of ordinary skill in the art.

[0047] As used herein, "administer," "administering," "administration," and the like refer to providing a substance (e.g., a synthetic nucleic acid, expression construct, vector, or rAAV herein) to an individual in a pharmacologically useful manner (e.g., to treat a disease, disorder, condition, or symptom in the individual).

[0048] As used herein, "codon optimization," with respect to a nucleotide sequence such as a gene of interest (e.g., TREM2), refers to the alteration of codons or sequences in the gene or coding region therein to reflect the typical codon usage of a host organism (e.g., a mammal, such as a human) or cells thereof, without altering the polypeptide encoded by the nucleotide sequence. Thus, a codon-optimized transgene is optimized for expression in a particular organism, organ, tissue, or cell type, particularly a mammal, or mammalian organ, tissue, or cell type. Alternatively, "codon optimization" refers to the alteration of codons or sequences in a gene to improve protein expression compared to a sequence lacking the alteration, for example, by eliminating or altering sites that may be cryptic splice sites, stop codons, miRNA recognition sequences, etc. The entire nucleotide sequence may be codon-optimized, or only one or more portions, parts, or regions of the nucleotide sequence may be codon-optimized.

[0049] As used herein, a "comparison window" refers to a specific contiguous segment of a nucleotide or amino acid sequence, where the sequence in the comparison window may contain additions and / or deletions (i.e., gaps) compared to a reference sequence (which does not contain additions and / or deletions) for optimal alignment of the two sequences. Generally, the comparison window is at least 10 contiguous nucleotides / amino acids in length, and optionally can be 20, 30, 40, 50, 60, 70, 80, 90, or 100 nucleotides / amino acids in length, or longer.

[0050] As used herein, "complementary" refers to a structural relationship between two nucleotides (e.g., on two opposing nucleic acids or on opposing regions of a single nucleic acid strand) that allows the two nucleotides to form base pairs (bp) with each other. For example, purine nucleotides of one nucleic acid that are complementary to pyrimidine nucleotides of an opposing nucleic acid may base pair with each other by forming hydrogen bonds. Complementary nucleotides may base pair in a Watson-Crick manner or in any other manner that allows the formation of a stable duplex. Similarly, two nucleic acids may have regions of multiple nucleotides that are complementary to each other and form a region of complementarity, as described herein.

[0051] As used herein, "CpG-depleted" in reference to a nucleotide sequence means that all (ie, 100%) known CpG sites are eliminated / removed in the nucleotide sequence.

[0052] As used herein, "CpG minimized" in reference to a nucleotide sequence means that some, but not all (i.e., less than 100%) CpG sites are eliminated / removed in the nucleotide sequence.

[0053] As used herein, a "CpG site" or the like means a cytosine (C) nucleotide followed by a guanine (G) nucleotide in a linear sequence of bases along the 5' to 3' direction in a nucleotide sequence.

[0054] As used herein, "TREM2-associated disease or disorder associated" refers to a disease or disorder resulting from a mutation in TREM2 that causes alterations in TREM2 expression, TREM2 amount, and / or TREM2 activity / function relative to its expected physiological function. Examples of such diseases or disorders include, but are not limited to, AD, amyotrophic lateral sclerosis (ALS), ALSP, cognitive impairment, frontotemporal dementia (FTD), NHD, memory loss, multiple sclerosis, spinal cord injury, and traumatic brain injury.

[0055] As used herein, "effective amount" refers to an amount, concentration, or dose of a therapeutic agent (e.g., a nucleic acid, expression construct, vector, or rAAV herein), or a pharmaceutical composition thereof, that, when administered in single or multiple doses to an individual in need thereof, provides the desired effect in such an individual under diagnosis or treatment (i.e., is likely to make a clinically measurable difference in the individual's condition or prevent deterioration in the individual). An effective amount can be readily determined by one of ordinary skill in the art by the use of known techniques and by observing results obtained under similar circumstances. In determining an effective amount for an individual, numerous factors are taken into consideration, including, but not limited to, the species of mammal, its size, age, and general health; the particular disease, disorder, condition, or symptom involved; the extent, involvement, or severity of the disease, disorder, condition, or symptom; the individual's response; the therapeutic agent administered; the mode of administration; the bioavailability characteristics of the administered preparation; the selected dosage regimen; the use of concomitant medications; and other relevant circumstances.

[0056] As used herein, "expression construct" refers to a nucleotide sequence that, when transformed, transfected, or transduced into a target cell, tissue, organ, or individual, is capable of expressing a nucleotide sequence of interest (e.g., a transgene or inhibitory nucleic acid). An exemplary expression construct is a vector, such as a viral vector, particularly an AAV vector or a baculovirus vector. Here, the expression construct can comprise at least one expression control element operably linked to a nucleotide sequence of interest, such as a transgene (and / or inhibitory nucleic acid). Thus, the expression construct can be an expression control element, such as a promoter, operably interacting with a transgene (and / or inhibitory nucleic acid) that can direct expression of the transgene (and / or inhibitory nucleic acid) in a cell, tissue, organ, or individual, particularly brain tissue.

[0057] As used herein, "expression control elements" refers to nucleotide sequences such as promoters, polyadenylation signals, transcriptional or translational termination sequences, upstream regulatory domains, origins of replication, internal ribosome entry sites (IRES), enhancers, and the like, which collectively provide for the replication, transcription, and / or translation of a desired nucleic acid (e.g., a transgene or inhibitory nucleic acid) in a cell, tissue, organ, or individual. Not all of these control sequences need always be present so long as the desired nucleotide sequence can be replicated, transcribed, and translated in the appropriate cell, tissue, organ, or individual.

[0058] As used herein, "in combination with" means that a therapeutic agent (e.g., a nucleic acid, vector, rAAV, or composition herein) is administered either simultaneously, sequentially, or in a single combined formulation with one or more additional therapeutic agents.

[0059] As used herein, "individual" means any mammal, including cats, dogs, mice, rats, and primates, particularly humans. Furthermore, "subject" or "patient" may be used interchangeably with "individual."

[0060] As used herein, "an individual in need thereof" means a mammal, such as a human, having a condition, disease, disorder, or symptom that requires treatment or therapy, including, for example, those listed herein. Specifically, the preferred individual to be treated is a human.

[0061] As used herein, "inhibitory nucleic acid" refers to a nucleic acid molecule that can attenuate, reduce, or prevent the expression of a gene or mRNA. Exemplary inhibitory nucleic acids include, but are not limited to, shRNA, siRNA, miRNA, amiRNA, etc. Here, the inhibitory nucleic acid may be a nucleotide sequence that encodes an antisense sequence to a nucleotide sequence of interest.

[0062] As used herein, "nucleic acid" refers to a polymer of nucleotides. The term can contain any type of nucleotide unit but generally applies to nucleotide polymers of deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). Polynucleotide is used to include single-stranded (ss) nucleic acids, double-stranded (ds) nucleic acids, and DNA and RNA made from nucleotide or nucleoside analogs that can be identified by their sequence, which are generally presented in a 5' to 3' direction (as the coding strand), with 5' and 3' indicating the bond formed between the 5' hydroxyl group of one nucleotide and the 3'-hydroxyl group of the next nucleotide. For a coding strand presented in a 5'-3' direction, its complement (or non-coding strand) is the strand that hybridizes to that sequence according to Watson-Crick base pairing. Thus, as used herein, the complement of a nucleic acid, e.g., a polynucleotide, is the same as a "reverse complement" and describes a nucleic acid that, in its natural form, base-pairs with the nucleic acid in question.

[0063] As used herein, "nucleoside" refers to a nucleobase-sugar combination, where the nucleobase moiety is typically a heterocyclic base. The two most common classes of such heterocyclic bases are purines and pyrimidines. The sugar is typically a pentose sugar, such as ribose or deoxyribose (e.g., 2'-deoxyribose).

[0064] As used herein, "nucleotide" means an organic molecule having a nucleoside (a nucleic acid base, e.g., adenine, cytosine, guanine, thymine, or uracil, and a pentose sugar, e.g., ribose or 2'-deoxyribose) and a phosphate group, which can serve as a monomer unit of nucleic acid polymers, e.g., DNA and RNA.

[0065] As used herein, "oligonucleotide" refers to a short nucleic acid molecule (e.g., less than about 100 nucleotides in length). Oligonucleotides can be ss or ds.

[0066] As used herein, "operably linked" and the like means that the elements of an expression construct (or other nucleic acid construct) are configured to perform their normal function (i.e., under the influence of expression control elements). Thus, an expression control element (e.g., a promoter) operably linked to a desired nucleotide sequence (e.g., a transgene or inhibitory nucleic acid) is capable of effecting expression of the desired nucleic acid. Control elements need not be contiguous with the desired nucleotide sequence, so long as they function to direct its expression (i.e., maintain the proper reading frame). Thus, for example, intervening untranslated but transcribed sequences can be present between the promoter and the desired nucleotide sequence, and the promoter can still be considered "operably linked" to the desired nucleotide sequence.

[0067] As used herein, "pharmaceutically acceptable," when referring to a material such as a carrier or diluent, means that it does not abolish the biological activity or properties of a therapeutic agent (e.g., a nucleic acid, expression construct, vector, rAAV, or composition herein) and is relatively non-toxic (i.e., the material can be administered to an individual without causing undesired biological effects or interacting in a deleterious manner with any of the components of the composition in which it is included).

[0068] As used herein, "pharmaceutically acceptable carrier" means a pharmaceutically acceptable material, composition, or carrier, such as a liquid or solid filler, stabilizer, dispersing agent, suspending agent, diluent, excipient, thickener, solvent, or encapsulating material, which is involved in carrying or transporting a therapeutic agent within or to an individual so that the therapeutic agent may perform its intended function. Additional ingredients that may be included in pharmaceutical compositions used in the practice of the present invention are known in the art and are described, for example, in Remington's Pharmaceutical Sciences, 21 st Edition, University of the Sciences in Philadelphia, PA (2006).

[0069] As used herein, "pharmaceutical composition" means a composition or therapeutic agent (e.g., a nucleic acid, expression construct, vector, rAAV, or composition herein) mixed with at least one pharmaceutically acceptable chemical component, such as, but not limited to, a carrier, stabilizer, diluent, dispersing agent, suspending agent, thickener, excipient, etc.

[0070] As used herein, "recombinant adeno-associated virus," "recombinant AAV," and "rAAV" refer to a viral particle containing a rAAV vector encapsidated by AAV capsid proteins.

[0071] As used herein, "recombinant adeno-associated virus vector," "recombinant AAV vector," and "rAAV vector" refer to a synthetic polynucleotide vector that contains one or more heterologous sequences (i.e., nucleic acid sequences not of AAV origin) flanked by at least one AAV ITR sequence. Such rAAV vectors can be replicated and packaged into infectious viral particles when present in a host cell infected with (or expressing appropriate helper functions from) a suitable helper virus that expresses the AAV rep and cap gene products (i.e., AAV Rep and Cap proteins).

[0072] As used herein, "sequence identity," in the context of two nucleotide sequences or two amino acid sequences, means the residues in the two sequences that are the same when aligned for maximum correspondence over a particular comparison window.

[0073] As used herein, "synthetic" means a nucleic acid or other molecule or compound that is artificially synthesized (e.g., using a machine such as a solid phase nucleic acid synthesizer) or recombinantly produced (i.e., not naturally occurring from a natural source that normally produces the nucleic acid or other compound).

[0074] As used herein, "transgene" means a nucleotide sequence that can be introduced into a cell, transcribed into RNA, and optionally translated and / or expressed under appropriate conditions. The transgene confers a desired characteristic on the cell into which it is introduced, or otherwise brings about a desired therapeutic or diagnostic result. Here, the transgene can be, for example, a nucleotide sequence encoding a polypeptide of interest, such as TREM2.

[0075] As used herein, "treat," "treatment," or "treating" refers to a process that may slow, control, retard, or halt the progression of a disease or disorder disclosed herein, or may ameliorate disease or disorder symptoms, but does not necessarily indicate the complete disappearance of all disease or disorder symptoms. Treatment and the like includes the administration of a nucleic acid, expression construct, vector, rAAV, or composition described herein to treat a disease or disorder in an individual, particularly a human.

[0076] As used herein, "TREM2" means human triggering receptor-2 expressed in myeloid cells (also known as PLOSL2, TREM-2, Trem2a, Trem2b, or Trem2c).

[0077] As used herein, "vector" refers to a recombinant plasmid or virus containing an oligonucleotide or polynucleotide that is delivered to a host cell either in vitro or in vivo. Examples of vectors include, but are not limited to, bacterial artificial chromosomes (BACs), cosmids, phagemids, plasmids, viral vectors, and yeast artificial chromosomes (YACs).

[0078] As used herein, "viral vector" refers to a vector derived from a naturally occurring or modified virus, particularly an rAAV vector or a baculovirus vector (e.g., an Autographa californica nuclear polyhedrosis (AcNPV) vector).

[0079] composition synthetic nucleic acid TREM2-encoding transgene: The synthetic nucleic acid herein can be a transgene (i.e., a TREM2-encoding transgene) comprising a nucleotide sequence encoding TREM2. The TREM2-encoding transgene can be codon-optimized and / or CpG-minimized and / or CpG-depleted. A CpG-depleted nucleotide sequence has 100% of the CpG sites eliminated / removed, while a CpG-minimized nucleotide sequence has less than 100% of the CpG sites eliminated / removed. For example, a CpG-minimized nucleotide sequence may have about 1% to about 99%, about 10% to about 90%, about 20% to about 80%, about 30% to about 70%, about 40% to about 60%, or about 50% of the CpG sites removed. Alternatively, the CpG-minimized nucleotide sequence may have about 5% to about 10%, about 10% to about 20%, about 20% to about 30%, about 40% to about 50%, about 50% to about 60%, about 60% to about 70%, about 70% to about 80%, about 80% to about 90%, or about 90% to about 99% of the CpGs eliminated / removed. Alternatively, the CpG-minimized nucleotide sequence may have about 1%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 99% of the CpG sites eliminated / removed.

[0080] In some examples, the TREM2-encoding transgene comprises a nucleotide sequence having at least about 90% sequence identity to SEQ ID NO: 3. Alternatively, the nucleotide sequence has at least about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% sequence identity to SEQ ID NO: 3. In one particular example, the nucleotide sequence of the TREM2-encoding transgene is SEQ ID NO: 3, which is a codon-optimized sequence.

[0081] In other examples, the TREM2-encoding transgene comprises a nucleotide sequence having at least about 90% sequence identity to SEQ ID NO: 4. Alternatively, the nucleotide sequence has at least about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% sequence identity to SEQ ID NO: 4. In one particular example, the nucleotide sequence of the TREM2-encoding transgene is SEQ ID NO: 4, which is not only codon-optimized but also CpG-depleted.

[0082] In some examples, the TREM2-encoding transgene comprises a nucleotide sequence having at least about 90% sequence identity to SEQ ID NO: 5. Alternatively, the nucleotide sequence has at least about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% sequence identity to SEQ ID NO: 5. In one particular example, the nucleotide sequence of the TREM2-encoding transgene is SEQ ID NO: 5, which is not only codon-optimized but also 10% CpG-minimized.

[0083] In some examples, the TREM2-encoding transgene comprises a nucleotide sequence having at least about 90% sequence identity to SEQ ID NO: 6. Alternatively, the nucleotide sequence has at least about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% sequence identity to SEQ ID NO: 6. In one particular example, the nucleotide sequence of the TREM2-encoding transgene is SEQ ID NO: 6, which is not only codon-optimized but also 25% CpG-minimized.

[0084] The synthetic nucleic acids herein can exist by themselves or can exist as part of an expression construct, vector, or rAAV as further described herein.

[0085] Expression Construct: As described above, synthetic nucleic acids, such as a TREM2-encoding transgene, can be incorporated into an expression construct. In some examples, the expression construct can include at least one expression control element operably linked to a TREM2-encoding transgene having the nucleotide sequence of any one of SEQ ID NOs: 3-6 (or a nucleotide sequence having at least about 90% to about 99% sequence identity thereto). In certain examples, the nucleotide sequence of the TREM2-encoding transgene is SEQ ID NO: 3, 4, 5, or 6.

[0086] The at least one expression control element can be at least one transcription factor binding site, at least one repressor binding site, at least one promoter, at least one enhancer, at least one intron splice site, at least one post-transcriptional regulatory element, at least one polyadenylation signal, or a combination thereof.

[0087] When the at least one expression control element is a promoter, the promoter can be a CBA promoter, a CD68 promoter, or an F4 / 80 promoter. In some examples, the promoter is a CBA promoter and has a nucleotide sequence having at least about 90% sequence identity to SEQ ID NO:7. In other examples, the promoter is a CD68 promoter and has a nucleotide sequence having at least about 90% sequence identity to SEQ ID NO:8. In other examples, the promoter is an F4 / 80 promoter and has a nucleotide sequence having at least about 90% sequence identity to SEQ ID NO:9 (see also WO 2006 / 122141). In one particular example, the nucleotide sequence of the promoter is SEQ ID NO:8.

[0088] When the at least one expression control element is an enhancer, the enhancer can be CMVe and have a nucleotide sequence having at least about 90% sequence identity to SEQ ID NO: 10. In one particular example, the nucleotide sequence of the enhancer is SEQ ID NO: 10.

[0089] When at least one expression control element is a post-transcriptional regulatory element, the post-transcriptional regulatory element can be a WPRE and has a nucleotide sequence having at least about 90% sequence identity to SEQ ID NO: 11. In one particular example, the nucleotide sequence of the post-transcriptional regulatory element is SEQ ID NO: 11.

[0090] When the at least one expression control element is a polyadenylation signal, the polyadenylation signal can be a BGHpA tail and has a nucleotide sequence having at least about 90% sequence identity to SEQ ID NO: 12. In one particular example, the nucleotide sequence of the polyadenylation signal is SEQ ID NO: 12.

[0091] In some examples, the expression construct may also include one or more nucleotide sequences of an internal ribosome entry site (IRES), a self-cleaving peptide coding sequence, for example, a T2A peptide.

[0092] In some instances, the expression construct comprises at least one additional nucleotide sequence of another transgene and / or inhibitory nucleic acid.

[0093] The expression constructs herein can exist by themselves or can exist as part of a vector or even an rAAV as further described herein.

[0094] Vector: As described above, the synthetic nucleic acid or expression construct can be incorporated into a vector, particularly a viral vector such as a rAAV vector. The rAAV vector can comprise either the "plus strand" or "minus strand" of the rAAV vector. In some examples, the rAAV vector is ss (e.g., ss DNA or ss RNA). In other examples, the rAAV vector is ds (e.g., ds DNA or ds RNA).

[0095] To facilitate expression, the rAAV vector includes ITRs flanking the nucleotide sequence of interest (e.g., a TREM2-encoding transgene). In some examples, the ITR sequences are full-length (i.e., approximately 145 nt in length and containing a functional Rep binding site (RBS) and terminal resolution site (trs)) and are WT AAV2 ITRs comprising a nucleotide sequence having at least about 90% sequence identity to SEQ ID NO: 13, or a reverse complement thereof. In one particular example, the nucleotide sequence of the WT AAV2 ITR is SEQ ID NO: 13, or a reverse complement thereof. In other examples, the ITRs are modified ITRs (i.e., including additions, deletions, substitutions, etc.) and are modified AAV2 ITRs comprising a nucleotide sequence having at least about 90% sequence identity to SEQ ID NO: 14, or a reverse complement thereof. In one particular example, the nucleotide sequence of the modified AAV2 ITR is SEQ ID NO: 13 or 14, or a reverse complement thereof.

[0096] The rAAV vector can also include a TRY region as described in Francois et al. (2005) J. Virol. 79:11082-11094, which can be located between the ITR (e.g., the 5' ITR) and the TREM2-encoding transgene. In some examples, the TRY region includes a nucleotide sequence having at least about 90% sequence identity to SEQ ID NO: 15. In one particular example, the nucleotide sequence of the TRY region is SEQ ID NO: 15.

[0097] To aid in packaging of the rAAV vector into capsid proteins, additional sequences (i.e., sequences that optimize the size of the vector for packaging into capsid proteins; "stuffer sequences") can be included in the rAAV vector. In some examples, such stuffer sequences comprise a nucleotide sequence having at least about 90% sequence identity to SEQ ID NOs: 16-18. In certain examples, the nucleotide sequence of the stuffer sequence is SEQ ID NO: 16, 17, or 18.

[0098] The vectors herein can exist by themselves or can exist as part of a rAAV as further described herein.

[0099] rAAV As described above, the vector can be incorporated into rAAV (i.e., an rAAV vector encapsidated in an AAV capsid protein). Here, the rAAV contains a capsid protein that readily spreads through the CNS, particularly when introduced into the CSF space or directly into the brain parenchyma. Examples of capsid proteins that can cross the blood-brain barrier include, but are not limited to, capsid proteins having the AAV6, AAV9, or AAVrh.10 serotypes.

[0100] Of particular interest herein are rAAVs that can infect microglia with AAV6-based capsid proteins, particularly AAV6™ capsid proteins. In some examples, the AAV6™ capsid protein comprises an amino acid sequence having at least about 95% sequence identity to SEQ ID NO: 19. In one particular example, the amino acid sequence of the AAV6™ capsid protein is SEQ ID NO: 19.

[0101] Thus, the rAAV herein comprises (a) an AAV vector containing a TREM2-encoding transgene, and (b) AAV6 capsid proteins.

[0102] In some instances, the rAAV (a) In the order from 5' to 3', (i) the first AAV ITR or a reverse complement thereto; (ii) a promoter; (iii) a TREM2-encoding transgene; (iv) post-transcriptional regulatory elements; (v) a polyadenylation signal, and (vi) a second AAV ITR, the reverse complement thereof, (b) Encapsidated into AAV6 capsid protein.

[0103] In other examples, the rAAV is (a) In the order from 5' to 3', (i) a first AAV2 ITR having the nucleotide sequence of SEQ ID NO: 13 or 14, or a reverse complement thereof; (ii) a promoter having the nucleotide sequence of SEQ ID NO: 8; (iii) a TREM2-encoding transgene having the nucleotide sequence of SEQ ID NO: 3; (iv) a post-transcriptional regulatory element having the nucleotide sequence of SEQ ID NO: 11; (v) a polyadenylation signal having the nucleotide sequence of SEQ ID NO: 12, and (vi) a second AAV2 ITR having the nucleotide sequence of SEQ ID NO: 13 or 14, or the reverse complement thereof; (b) encapsidated into an AAV6 capsid protein, particularly an AAV6TM capsid protein having the amino acid sequence of SEQ ID NO:19.

[0104] In yet another example, the rAAV is (a) In the order from 5' to 3', (ii) a first AAV2 ITR having the nucleotide sequence of SEQ ID NO: 13 or 14, or the reverse complement thereof; (ii) a promoter having the nucleotide sequence of SEQ ID NO: 8; (iii) a TREM2-encoding transgene having the nucleotide sequence of SEQ ID NO: 4; (iv) a post-transcriptional regulatory element having the nucleotide sequence of SEQ ID NO: 11; (v) a polyadenylation signal having the nucleotide sequence of SEQ ID NO: 12, and (vi) a second AAV2 ITR having the nucleotide sequence of SEQ ID NO: 13 or 14, or the reverse complement thereof; (b) encapsidated into an AAV6 capsid protein, particularly an AAV6TM capsid protein having the amino acid sequence of SEQ ID NO:19.

[0105] In yet another example, the rAAV is (a) In the order from 5' to 3', (i) a first AAV2 ITR having the nucleotide sequence of SEQ ID NO: 13 or 14, or a reverse complement thereof; (ii) a promoter having the nucleotide sequence of SEQ ID NO: 8; (iii) a TREM2-encoding transgene having the nucleotide sequence of SEQ ID NO: 5; (iv) a post-transcriptional regulatory element having the nucleotide sequence of SEQ ID NO: 11; (v) a polyadenylation signal having the nucleotide sequence of SEQ ID NO: 12, and (vi) a second AAV2 ITR having the nucleotide sequence of SEQ ID NO: 13 or 14, or the reverse complement thereof; (b) encapsidated into an AAV6 capsid protein, particularly an AAV6TM capsid protein having the amino acid sequence of SEQ ID NO:19.

[0106] In yet another example, the rAAV is (a) In the order from 5' to 3', (i) a first AAV2 ITR having the nucleotide sequence of SEQ ID NO: 13 or 14, or a reverse complement thereof; (ii) a promoter having the nucleotide sequence of SEQ ID NO: 8; (iii) a TREM2-encoding transgene having the nucleotide sequence of SEQ ID NO: 6; (iv) a post-transcriptional regulatory element having the nucleotide sequence of SEQ ID NO: 11; (v) a polyadenylation signal having the nucleotide sequence of SEQ ID NO: 12, and (vi) a second AAV2 ITR having the nucleotide sequence of SEQ ID NO: 13 or 14, or the reverse complement thereof; (b) encapsidated into an AAV6 capsid protein, particularly an AAV6TM capsid protein having the amino acid sequence of SEQ ID NO:19.

[0107] Any of the above rAAVs can further comprise a stuffer sequence having the nucleotide sequence of SEQ ID NOs: 16 to 18. In some examples, the stuffer sequence is located between the polyadenylation signal and the second AAV2 ITR.

[0108] In certain examples, the rAAV comprises an rAAV vector having the nucleotide sequence of SEQ ID NO: 21 encapsidated in an AAV6 capsid protein, particularly an AAV6™ capsid protein having the amino acid sequence of SEQ ID NO: 19. In some examples, the rAAV comprises the reverse complementary nucleotide sequence to SEQ ID NO: 21.

[0109] In certain other examples, the rAAV comprises an rAAV vector having the nucleotide sequence of SEQ ID NO: 22 encapsidated in an AAV6 capsid protein, particularly an AAV6TM capsid protein (e.g., VP1) having the amino acid sequence of SEQ ID NO: 19. In some examples, the rAAV comprises the reverse complementary nucleotide sequence to SEQ ID NO: 22.

[0110] In certain other examples, the rAAV comprises an rAAV vector having the nucleotide sequence of SEQ ID NO: 23 encapsidated in an AAV6 capsid protein, particularly an AAV6™ capsid protein having the amino acid sequence of SEQ ID NO: 19. In some examples, the rAAV comprises the reverse complementary nucleotide sequence to SEQ ID NO: 23.

[0111] In certain other examples, the rAAV comprises an rAAV vector having the nucleotide sequence of SEQ ID NO: 24 encapsidated in an AAV6 capsid protein, particularly an AAV6™ capsid protein having the amino acid sequence of SEQ ID NO: 19. In some examples, the rAAV comprises the reverse complementary nucleotide sequence to SEQ ID NO: 24.

[0112] In certain other examples, the rAAV comprises an rAAV vector having the nucleotide sequence of SEQ ID NO: 25 encapsidated in an AAV6 capsid protein, particularly an AAV6™ capsid protein having the amino acid sequence of SEQ ID NO: 19. In some examples, the rAAV comprises the reverse complementary nucleotide sequence to SEQ ID NO: 25.

[0113] In certain other examples, the rAAV comprises an rAAV vector having the nucleotide sequence of SEQ ID NO: 26 encapsidated in an AAV6 capsid protein, particularly an AAV6™ capsid protein having the amino acid sequence of SEQ ID NO: 19. In some examples, the rAAV comprises the reverse complementary nucleotide sequence to SEQ ID NO: 26.

[0114] Pharmaceutical Composition The synthetic nucleic acids (i.e., expression constructs or vectors) described herein or the rAAV described herein can be formulated as pharmaceutical compositions comprising the synthetic nucleic acid or rAAV and a pharmaceutically acceptable carrier. Pharmaceutical compositions can be prepared by methods well known in the art (see, for example, Remington: The Science and Practice of Pharmacy, 22 nd ed.(Pharmaceutical Press, 2013).

[0115] In some examples, the pharmaceutical composition can be in the form of a formulation that includes not only the rAAV described herein, but also one or more of: (a) about 10 mM to about 30 mM TRIS buffer; (b) about 0.5 mM to about 1.5 mM MgCl; (c) about 100 mM to about 300 mM NaCl; and (d) about 0.001% (w / v) to about 0.01% (w / v) poloxamer 188.

[0116] In some examples, the rAAV comprises an rAAV vector having the nucleotide sequence of SEQ ID NO: 21 encapsidated in an AAV6 capsid protein, particularly an AAV6TM capsid protein (e.g., VP1) having the amino acid sequence of SEQ ID NO: 19. In some examples, the rAAV comprises a reverse complementary nucleotide sequence to SEQ ID NO: 21. In other examples, the rAAV comprises an rAAV vector having the nucleotide sequence of SEQ ID NO: 22 encapsidated in an AAV6 capsid protein, particularly an AAV6TM capsid protein having the amino acid sequence of SEQ ID NO: 19. In some examples, the rAAV comprises a reverse complementary nucleotide sequence to SEQ ID NO: 22. In other examples, the rAAV comprises an rAAV vector having the nucleotide sequence of SEQ ID NO: 23 encapsidated in an AAV6 capsid protein, particularly an AAV6TM capsid protein having the amino acid sequence of SEQ ID NO: 19. In some examples, the rAAV comprises a reverse complementary nucleotide sequence to SEQ ID NO: 23. In other examples, the rAAV comprises an rAAV vector having the nucleotide sequence of SEQ ID NO: 24 encapsidated in an AAV6 capsid protein, particularly an AAV6TM capsid protein having the amino acid sequence of SEQ ID NO: 19. In some examples, the rAAV comprises a reverse complementary nucleotide sequence to SEQ ID NO: 24. In other examples, the rAAV comprises an rAAV vector having the nucleotide sequence of SEQ ID NO: 25 encapsidated in an AAV6 capsid protein, particularly an AAV6TM capsid protein having the amino acid sequence of SEQ ID NO: 19. In some examples, the rAAV comprises a reverse complementary nucleotide sequence to SEQ ID NO: 25. In other examples, the rAAV comprises an rAAV vector having the nucleotide sequence of SEQ ID NO: 26 encapsidated in an AAV6 capsid protein, particularly an AAV6TM capsid protein having the amino acid sequence of SEQ ID NO: 19. In some examples, the rAAV comprises a reverse complementary nucleotide sequence to SEQ ID NO: 26.

[0117] In some examples, the TRIS buffer may have a concentration of about 10 mM to about 30 mM. In some examples, the TRIS buffer may have a concentration of about 15 mM to about 25 mM, or about 20 mM. In still other examples, the TRIS buffer may have a concentration of about 10 mM, about 11 mM, about 12 mM, about 13 mM, about 14 mM, about 15 mM, about 16 mM, about 17 mM, about 18 mM, about 19 mM, about 20 mM, about 21 mM, about 22 mM, about 23 mM, about 24 mM, about 25 mM, about 26 mM, about 27 mM, about 28 mM, about 29 mM, or about 30 mM.

[0118] In some examples, MgCl2 can be at a concentration of about 0.5 mM to about 1.5 mM. In other examples, MgCl2 can be at a concentration of about 0.6 mM to about 1.4 mM, about 0.7 mM to about 1.3 mM, about 0.8 mM to about 1.2 mM, about 0.9 mM to about 1.1 mM, or about 1.0 mM. In still other examples, MgCl2 can be at a concentration of about 0.5 mM, about 0.6 mM, about 0.7 mM, about 0.8 mM, about 0.9 mM, about 1.0 mM, about 1.1 mM, about 1.2 mM, about 1.3 mM, about 1.4 mM, or about 1.5 mM.

[0119] In some examples, NaCl can be at a concentration of about 100 mM to about 300 mM. In other examples, NaCl can be at a concentration of about 125 mM to about 275 mM, about 150 mM to about 250 mM, about 175 mM to about 225 mM, or about 200 mM. In still other examples, NaCl can be at a concentration of about 100 mM, about 110 mM, about 120 mM, about 130 mM, about 140 mM, about 150 mM, about 160 mM, about 170 mM, about 180 mM, about 190 mM, about 200 mM, about 210 mM, about 220 mM, about 230 mM, about 240 mM, about 250 mM, about 260 mM, about 270 mM, about 280 mM, about 290 mM, or about 300 mM.

[0120] In some examples, poloxamer 188 can be at a concentration of about 0.001% (w / v) to about 0.01% (w / v). In other examples, poloxamer 188 can be at a concentration of about 0.002% (w / v) to about 0.009% (w / v), 0.003% (w / v) to about 0.008% (w / v), about 0.004% (w / v) to about 0.007% (w / v), or about 0.005% (w / v) to about 0.006% (w / v). In yet other examples, the poloxamer 188 can be at a concentration of about 0.001% (w / v), about 0.002% (w / v), about 0.003% (w / v), about 0.004% (w / v), about 0.005% (w / v), about 0.006% (w / v), about 0.007% (w / v), about 0.008% (w / v), about 0.009% (w / v), or about 0.01% (w / v).

[0121] In one particular example, the formulation may include an rAAV herein and (a) about 20 mM TRIS (pH 8.0), (b) about 1 mM MgCl, (c) about 200 mM NaCl, and (d) about 0.005% (w / v) poloxamer 188.

[0122] In some instances, the effective amount is about 10 9 Genome copies (GC) / kg ~ approx. 10 14 GC / kg (e.g., about 10 9 GC / kg, approx. 10 10 GC / kg, approx. 10 11 GC / kg, approx. 10 12 GC / kg, approx. 10 13 GC / kg, or approximately 10 14 In some instances, individuals may receive high titers (e.g., 10 GC / kg) by injection into the CSF space, particularly by ICM. 12 GC / kg of rAAV) will be administered.

[0123] In other examples, the effective amount is about 1×10 12 vg~approx. 1×10 15 vg or approximately 1 x 10 13 vg~approx. 7×10 14 In another example, the dose may be in the range of about 3.5 x 10 13vg, approx. 7.0×10 13 vg, or approximately 1.4 × 10 14 In yet another example, the dose may be about 1 x 10 14 vg, approx. 2.0×10 14 vg, or approximately 4.0 × 10 14 Alternatively, the dose may be about 2 x 10 13 vg, approx. 3×10 13 vg, approx. 4×10 13 vg, approx. 5×10 13 vg, approx. 6×10 13 vg, approx. 7×10 13 vg, approx. 8×10 13 vg, approx. 9×10 13 vg, approx. 1×10 14 vg, or approximately 2 × 10 14 In one particular example, the dose may be 7.0 x 10 13 vg or 1.4 x 10 14 vg.

[0124] The pharmaceutical compositions can be administered by any route, including, for example, intraarterial, intradermal, intramuscular, intrathecal, intravenous (IV), intracerebroventricular, parenteral, subcutaneous (SC), or transdermal. Specifically contemplated routes are IV administration (e.g., systemic intravenous injection), and / or direct administration to the affected site (e.g., intracisternal (ICM) injection, intracerebroventricular (ICV) injection), or a combination thereof.

[0125] Generally, the most appropriate route of administration will depend on various factors, including, but not limited to, the properties of the agent (e.g., its administration and / or its stability in the intended target environment) and / or the condition of the individual (e.g., whether the subject can tolerate oral administration). In some examples, the synthetic nucleic acid, rAAV, or pharmaceutical composition is suitable for administration to the CNS of an individual, for example, by intrathecal, ICM, ICV, or a combination thereof.

[0126] kit In some examples, the synthetic nucleic acids (i.e., expression constructs or vectors) or rAAVs herein can be included in a kit that includes the synthetic nucleic acid or rAAV and instructions for its use. In other examples, the kit includes the synthetic nucleic acid or rAAV and a package insert containing instructions for use of the kit and / or any of its components. In still other examples, the kit includes, in a suitable container or other means for containment, the synthetic nucleic acid or rAAV, one or more controls, and various buffers, reagents, enzymes, and other standard components well known in the art. In some examples, the container includes at least one vial, well, test tube, flask, bottle, syringe, or other container means into which the synthetic nucleic acid, rAAV, or other therapeutic oligonucleotide is placed, and in some examples, appropriately aliquoted. If additional components are provided, the kit includes additional containers into which the components can be placed. The kit can also include means for containing the synthetic nucleic acid or rAAV and any other reagents in close confinement for commercial sale. Such containers can include injection- or blow-molded plastic containers into which the desired vials are retained. The container and / or kit may include instructions and / or warning labels.

[0127] In some examples, the kit includes a synthetic nucleic acid or rAAV and a pharmaceutically acceptable carrier, or a pharmaceutical composition including the synthetic nucleic acid, rAAV, or other therapeutic oligonucleotide, and instructions for treating or slowing the progression of a neurodegenerative disease in an individual in need thereof.

[0128] In some examples, the kit includes a synthetic nucleic acid or rAAV, a pharmaceutically acceptable carrier or pharmaceutical composition comprising the synthetic nucleic acid or rAAV, and instructions for administering the synthetic nucleic acid or rAAV, or the pharmaceutical composition.

[0129] method Production method Methods for producing rAAV are described, for example, in Samulski et al. (1989) J. Virol. 63:3822-3828 and Wright (2009) Hum. Gene Ther. 20:698-706. In some examples, rAAV can be produced in a baculovirus vector expression system (BEVS). Production of rAAV using BEVS is described, for example, in Urabe et al. (2002) Hum. Gene Ther. 13:1935-1943, Smith et al. (2009) Mol. Ther. 17:1888-1896, and U.S. Patent Nos. 8,945,918 and 9,879,282, and International Publication Nos. 2017 / 184879 and 2022 / 082017. Alternatively, rAAV can be produced in human embryonic kidney (e.g., HEK293) cells (see, e.g., WO 2020 / 210689 and WO 2022 / 035900). However, rAAV can be produced using any suitable method (e.g., using recombinant rep and cap genes).

[0130] Treatment Methods and Uses The nucleic acids, expression constructs, vectors, rAAVs, or pharmaceutical compositions described herein can be used to treat neurological disorders such as, for example, AD, ALS, ALSP, cognitive impairment, FTD, memory loss, NHD, spinal cord injury, traumatic brain injury, or multiple sclerosis.

[0131] The method may include the steps described herein, which may, but need not, be performed in the order described. However, other orders are contemplated. Furthermore, individual or multiple steps may be performed in parallel and / or overlapping time, and / or individually or in multiple repeated steps. Furthermore, the method may include additional, unspecified steps.

[0132] Here, the synthetic nucleic acid, rAAV, or pharmaceutical composition comprising same may be used in a method for treating TREM2-related diseases and disorders, such a method comprising at least the step of administering to an individual in need of such treatment an effective amount of the synthetic nucleic acid, rAAV, or pharmaceutical composition comprising same.

[0133] In some instances, the synthetic nucleic acid, rAVV, or pharmaceutical composition is administered by IV injection. In other instances, the synthetic nucleic acid, rAVV, or pharmaceutical composition is administered by ICM injection of the individual.

[0134] For rAAV, the effective dose is approximately 10 9 Genome copies (GC) / kg ~ approx. 10 14 GC / kg (e.g., about 10 9 GC / kg, approx. 10 10 GC / kg, approx. 10 11 GC / kg, approx. 10 12 GC / kg, approx. 10 13 GC / kg, or approximately 10 14 In some instances, individuals may receive high titers (e.g., 10 GC / kg) by injection into the CSF space, particularly by ICM. 12 GC / kg of rAAV) will be administered.

[0135] In other examples, the effective amount is about 1×10 12 vg~approx. 1×10 15 vg or approximately 1 x 10 13 vg~approx. 7×10 14 In another example, the dose may be in the range of about 3.5 x 10 13 vg, approx. 7.0×10 13 vg, or approximately 1.4 × 10 14 In yet another example, the dose may be about 1 x 10 14 vg, approx. 2.0×10 14 vg, or approximately 4.0 × 10 14 Alternatively, the dose may be about 2 x 10 13 vg, approx. 3×10 13 vg, approx. 4×10 13 vg, approx. 5×10 13 vg, approx. 6×1013 vg, approx. 7×10 13 vg, approx. 8×10 13 vg, approx. 9×10 13 vg, approx. 1×10 14 vg, or approximately 2 × 10 14 In one particular example, the dose may be 7.0 x 10 13 vg or 1.4 x 10 14 vg.

[0136] In some examples, the rAAV or a composition comprising it may be administered to a subject one or more times (e.g., two, three, four, five, six, seven, eight, nine, ten, twenty or more times).

[0137] In some instances, the individual has or is suspected of having a TREM2-related disease or disorder, particularly AD, ALSP, or NHD.

[0138] In some examples, the individual is between about 1 month and about 10 years old (e.g., about 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, 24 months, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, or any age in between). In other examples, the individual is between about 10 years old and about 20 years old (e.g., about 10 years old, 11 years old, 12 years old, 13 years old, 14 years old, 15 years old, 16 years old, 17 years old, 18 years old, 19 years old, 20 years old, or any age in between). In other examples, the individual is over 20 years old (e.g., about 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or any age in between), over 30 years old (e.g., about 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, or any age in between), over 40 years old (e.g., about 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or any age in between), or even over 50 years old (e.g., about 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 70, 80, 90, or any age in between).

[0139] In some instances, the individual has a pathogenic CSF1R mutation.

[0140] In some examples, the method may also include measuring TREM2 in plasma and / or CSF and / or urine from the individual and comparing the obtained values ​​with previously obtained comparable or control values ​​to assess the effectiveness of the method. In some examples, the TREM2 is soluble TREM2 (sTREM2).

[0141] In other examples, the method may also include measuring CSF1R in plasma and / or CSF and / or urine from the individual and comparing the obtained value with a previously obtained comparable value or control value to evaluate the effectiveness of the method. In some examples, the CSF1R is soluble CSF1R (sCSF1R).

[0142] In other examples, the method may also include measuring NfL in plasma and / or CSF and / or urine from an individual and comparing the obtained values ​​with previously obtained comparable or control values ​​to assess the effectiveness of the method.

[0143] In other examples, the method may also include measuring Chit1 in plasma and / or CSF and / or urine from an individual and comparing the obtained values ​​with previously obtained comparable or control values ​​to assess the effectiveness of the method.

[0144] In other examples, the method may also include measuring GM-CSF in plasma and / or CSF and / or urine from an individual and comparing the obtained values ​​with previously obtained comparable or control values ​​to assess the effectiveness of the method.

[0145] use The rAAV herein or pharmaceutical compositions comprising same can be used or adapted for use to treat individuals (e.g., humans) having or suspected of having a TREM2-associated disease or disorder, such as AD, ALSP, or NHD. Thus, the rAAV or pharmaceutical compositions comprising same are provided or adapted for use to treat individuals having or suspected of having a TREM2-associated disease or disorder, such as AD, ALSP, or NDH. The rAAV or pharmaceutical compositions comprising same can also be provided or adapted for use in the manufacture of a medicament or pharmaceutical composition for treating a TREM2-associated disease or disorder, such as AD, ALSP, or NHD.

[0146] Also described herein are nucleic acids, vectors, rAAVs, or pharmaceutical compositions for use in therapy.Furthermore, described herein are nucleic acids, vectors, rAAVs, or pharmaceutical compositions for use in treating neurological disorders, such as AD, ALS, ALSP, cognitive impairment, FTD, memory loss, NHD, spinal cord injury, traumatic brain injury, or multiple sclerosis.

[0147] Also described is the use of a nucleic acid, vector, rAAV, or pharmaceutical composition in the manufacture of a medicament for the treatment of a neurological disorder, such as AD, ALS, ALSP, cognitive impairment, FTD, memory loss, NHD, spinal cord injury, traumatic brain injury, or multiple sclerosis. [Example]

[0148] The following non-limiting examples are offered by way of illustration and not limitation.

[0149] Example 1: Generation of rAAV Methods: rAAV vectors expressing various TREM2-encoding transgenes were generated using cells such as HEK293 cells or Sf9 insect cells (see, e.g., WO 2008 / 024988, WO 2017 / 184879, and WO 2022 / 082017). ITR sequences flank the expression construct, which contains promoter / enhancer elements for the transgene, a 3' polyA signal, and post-translational signals such as a WPRE element.

[0150] The rAAV vectors were encapsidated in either the AAV6™ capsid protein (SEQ ID NO: 19) or the AAV9 capsid protein (SEQ ID NO: 20).

[0151] In vivo studies Example 2: In vitro transduction of human microglial cell lines with TREM-2 rAAV Methods: HMC3 microglial cell line was transduced with rAAV vectors carrying the TREM2 transgene (SEQ ID NO: 3) encapsidated in AAV6™ capsid protein (SEQ ID NO: 19) or AAV9 capsid protein (SEQ ID NO: 20) at various MOIs. 72 hours after transduction, supernatants were collected for protein and cells were lysed for RNA.

[0152] TREM2 mRNA was quantified using qRT-PCR and normalized to GAPDH. TREM2 protein was quantified using the MSD assay.

[0153] Results: Figures 5A-5B show that TREM2 mRNA expression (Figure 5A) and protein expression (Figure 5B) were observed with both rAAV vectors. However, the rAAV vector encapsidated with the AAV6™ capsid protein performed better than the same rAAV vector encapsidated with the AAV9 capsid protein.

[0154] Example 3: In vitro transduction of human microglial cell lines with alternative TREM-2 rAAVs Method: HMC3 cells were treated with 1.09 × 10 5 ~3.50×10 6The cells were transduced with either a first rAAV vector carrying a TREM2 transgene (SEQ ID NO: 25) or a second rAAV vector carrying a TREM2 transgene (SEQ ID NO: 26) encapsidated in the AAV6™ capsid protein (SEQ ID NO: 19) over a range of MOIs of vg / cell. Three days post-infection, supernatants were collected for ELISA.

[0155] Results: Both rAAVs effectively transduced HMC3 cells in vitro, resulting in robust dose-dependent expression and secretion of TREM2 (Figures 6A-6B, Figure 6A for the first rAAV vector and Figure 6B for the second rAAV vector). In contrast, minimal levels of TREM2 (78 pg / mL) were detected in the negative control group treated with vehicle alone.

[0156] Example 4: Determining the in vitro potency of TREM2 in human induced pluripotent stem cell (iPSC)-derived microglial cells Methods: Human iPSC-derived microglia were treated with vehicle (negative control) or 2.0 × 10 3 ~2.0×10 6 The cells were either transduced with a rAAV vector carrying a TREM2 transgene (SEQ ID NO: 26) encapsidated into the AAV6™ capsid protein (SEQ ID NO: 19) at an MOI ranging from 0.01 to 0.01 vg / cell. Seven days after transduction, cells were lysed for ELISA or processed for RNA. All iPSC-derived cells were obtained from FujiFilm Cellular Dynamics, Inc. (Madison, WI).

[0157] Results: By day 7, rAAV transduction was achieved at 2.0 x 10 ng / mL, approximately 1000 pg / mL and 1250 pg / mL, respectively. 5 and 2.0 x 10 6 rAAV transduction resulted in a robust dose-dependent increase in TREM2 mRNA expression, detected in the groups treated with 1000 mg / vg / cell. In contrast, TREM2 was detected in vehicle-treated iPSC-derived microglia at a concentration of approximately 625 pg / mL. Furthermore, rAAV transduction resulted in a dose-dependent increase in TREM2 mRNA expression in all dose groups, whereas TREM2 mRNA was not detected in vehicle-treated microglia.

[0158] Example 5: Improved cell viability using TREM2 rAAV in a pharmacological CSF1R inhibition assay in human iPSC-derived microglial cells Methods: iPSC-derived microglia as described in Example 4 were treated with vehicle (negative control) or 2.0 x 10 3 Cells were either transduced with a rAAV vector carrying a TREM2 transgene (SEQ ID NO:26) encapsidated into the AAV6™ capsid protein (SEQ ID NO:19) at an MOI of 10 ...

[0159] Results: PLX treatment caused cytotoxicity and reduced the total number of microglial cells (i.e., approximately 80% cell death), whereas rAAV transduction significantly reduced PLX-induced toxicity by 50% compared to control levels.

[0160] In vivo studies Example 6: In vivo rodent studies of AAV capsid proteins Methods: Thirty-six 1-month-old 5xFAD mice were assigned to three groups (n=12 / group) and treated with the same rAAV or vehicle (20 mM Tris pH 8.0, 200 mM NaCl, and 1 mM MgCl2 + 0.001% Pluronic F68) as in Example 2 via ICV injection (10 μl to 5 μl per mouse, bilaterally) to determine efficacy. A group of non-transgenic age-matched littermates (i.e., WT) (n=12) was similarly injected with vehicle as a control. The treatment dose was 5.68×10 10 vg / animal or 1.8 × 10 11 vg / animal. Five months after ICV injection, the presence of vg was assessed by ddPCR.

[0161] Additionally, efficacy endpoints were examined, including protein levels, amyloid-β levels (both biochemical and immunohistochemical), and inflammation. TREM2 protein and amyloid-β levels were measured using the MSD assay. + Cells were measured by immunohistochemistry.

[0162] In vivo blood collection: In vivo blood was collected by submandibular bleeding from the facial vein / arterial plexus without anesthesia before treatment initiation and at 2, 3, 4, and 5 months of age (five time points). The collected blood was then transferred to serum gel clot activator microtubes. After incubation at room temperature for at least 20 minutes (maximum 60 minutes), serum was prepared from the samples by centrifugation (10,000 × g, 5 minutes, room temperature). After centrifugation, the serum was frozen on dry ice and stored at -80°C.

[0163] Tissue Sampling: At 6 months, mice were terminally anesthetized by i.p. injection of pentobarbital (600 mg / kg), and CSF, blood, brain, several organs (gonads, kidney, heart, liver, lung, and spleen), and spinal cord were collected for biochemical, immunochemical, and / or histological analysis.

[0164] Immunohistology: For each incubation, a uniform, systematically random set of five sections per mouse was selected from all animals per group (one section each from levels 2, 4, 6, 8, and 10). All sections were counterstained with the nuclear dye DAPI. Primary antibody binding was visualized using a highly cross-absorbed secondary antibody.

[0165] Imaging: Whole slide scans of stained sections were recorded on a Zeiss automated microscope AxioScan Z1 with a high aperture lens equipped with a Zeiss Axiocam 506 mono and Hitachi 3CCD HV-F202SCL camera and Zeiss ZEN 3.3 software.

[0166] Sample preparation - Homogenization: Hippocampus and somatosensory cortex from all animals were homogenized in 14 volumes of PBS using a UPHO bead mill (Geneye) at 55 Hz for 50 seconds, generating three aliquots (30 μL for RNA / DNA isolation, 50 μL for soluble-insoluble protein isolation and the remainder).

[0167] Sample preparation - DNA and RNA isolation: DNA and RNA were isolated from the hippocampus and somatosensory cortex of all animals from 30 μL aliquots of PBS using the Ambion TriZOL kit. The quantity and quality of extracted total RNA was assessed by UV-VIS spectroscopy using a NanoDrop 1000 spectrophotometer. RNA (1 μg per sample) was reverse transcribed using the iScript gDNA Clear cDNA Synthesis Kit.

[0168] Preparation and measurement of amyloid-β40 and amyloid-β42 samples (soluble and insoluble): A second aliquot of 50 μl of hippocampal and cortical samples was replaced with the same volume of 2× THB buffer (2× THB; 500 mM sucrose, 2 mM EDTA, 2 mM EGTA, 40 mM Tris, pH 7.4) containing 1× protease inhibitor (Calbiochem) and incubated on ice for 15 min. The THB homogenate was then processed for extraction of soluble and deposited proteins from the brain homogenate for analysis of amyloid-β40 and amyloid-β42. For extraction of non-plaque-associated proteins, 50 μl of the THB homogenate was mixed with 1 part DEA solution (0.4% DEA, 100 mM NaCl). The mixture was centrifuged at 20,000 × g for 120 min at 4 °C. The supernatant was neutralized with 1 / 10 volume of 0.5 M Tris-HCl, pH 6.8, and vortexed briefly. Aliquots were stored at -80°C. For extraction of deposited proteins, 30 μL of THB homogenate was mixed with 2.2 volumes of cold FA, sonicated on ice for 30 seconds, and centrifuged at 20,000 × g and 4°C for 120 minutes. The supernatant was mixed with 19 volumes of FA neutralization solution (1 M Tris, 0.5 M NaHPO, 0.05% NaN). Aliquots were stored at -80°C. The soluble and insoluble fractions of hippocampal and somatosensory cortex samples from all animals were then analyzed by immunosorbent assay using Mesoscale Discovery (Amyloid-β40 Peptide (6E10) Kit and Amyloid-β42 Peptide (6E10) Kit) according to the manufacturer's instructions. Amyloid-β levels in study samples were assessed by comparison with the calibration curve provided in the kit and are expressed as pg / mg brain.

[0169] Statistics: Statistical analysis was performed using GraphPad Prism 9. Data were tested for normality using the Kolmogorow-Smirnow test. If normal distribution was confirmed, differences between groups were tested with one- or two-way ANOVA / mixed-effects analysis, followed by Bonferroni, Dunnett, or Sidak multiple comparison tests. If data were not normally distributed, differences between groups were tested with the Kruskal-Wallis test, followed by Dunn's post-hoc test for multiple comparisons. The 5xFAD vehicle group was used as the reference group. Data are expressed as mean + or + / - SEM.

[0170] Results: Figures 7A-7D show that both capsid proteins resulted in widespread biodistribution in the cortex, hippocampus, cervical spinal cord, and liver.

[0171] Figures 8A-8C show that both capsid proteins resulted in TREM2 protein levels in the CSF, liver, and serum, with the AAV6™ capsid protein showing higher TREM2 protein levels in the CSF than the AAV9 capsid protein.

[0172] Figures 9A-9C show that rAAV vectors encapsidated in AAV6TM capsid proteins significantly reduced amyloid-β in the hippocampus and cortex, whereas rAAV vectors encapsidated in AAV9 capsid proteins did not reduce amyloid-β in the hippocampus and cortex.

[0173] Figures 10A-B show that rAAV vectors reduced inflammatory markers in the cortex regardless of capsid protein.

[0174] Overall, rAAV vector administration resulted in widespread biodistribution and a dose-dependent increase in TREM2 protein levels for both capsids, although a significant reduction in cortical disease burden was only observed in mice treated with the AAV6™ capsid protein.

[0175] Example 7: In vivo rodent studies of TREM2 rAAV Methods: Two different doses of vehicle or one or the other of the TREM2 rAAV from Example 3 were administered to 1-month-old WT C57BL / 6 mice by ICV injection (n=10 / group). rAAV was administered at 5.68 x 10 10 vg(1.42×10 11 vg / g brain) or 1.8 × 10 11 vg(4.49×10 11 The vector particles per gram of brain weight were calculated based on an adult mouse brain weight of 400 mg.

[0176] CNS tissues and liver were collected to analyze biodistribution by ddPCR.CSF, liver, and serum were collected to analyze TREM2 expression.

[0177] Biodistribution was determined by measuring the presence of vg using ddPCR (>50 vg / 1 μg gDNA was defined as positive).

[0178] Results: Mice receiving rAAV were positive for vg in the cortex, indicating that ICV administration achieved comparable transduction success in the brain. There was no significant effect of vector design on biodistribution (all p>0.05). ICV administration also resulted in the presence of vg in the spinal cord and liver.

[0179] Using MSD, TREM2 expression was measured in CSF and serum. ICV injection of rAAV increased TREM2 in CSF at both doses. These data indicate no difference in the TREM2 expression levels produced by either rAAV, consistent with the biodistribution data. In addition, ICV injection of rAAV increased TREM2 expression in 1.8 x 10 11 vg significantly increased blood TREM2 levels. A dose-dependent effect was observed in serum.

[0180] Example 8: Evaluation of in vivo efficacy of TREM2 rAAV in a CSF1R-inhibited mouse model for CRL Methods: Vehicle or two different doses of the TREM2 rAAV of Example 3 (i.e., the rAAV vector of SEQ ID NO: 26 encapsidated in the AAV6™ capsid protein (SEQ ID NO: 19)) were administered by ICV injection to 1-month-old WT C57BL / 6 mice (n=10). The rAAV was administered at 1.8 x 10 10 vg(4.49×10 10 vg / g brain) or 1.8 × 10 11 vg(4.49×10 11 Mice were injected at either a dose of 185 mg / kg PLX chow or 185 mg / kg PLX chow (vg / g brain). Vector particles per gram of brain weight were based on an adult mouse brain weight of 400 mg. At 8 weeks of age (i.e., 4 weeks after ICV injection), one vehicle-treated group and both dose-treated groups were fed PLX-containing mouse chow. Mice were fed 185 mg / kg PLX chow for 1 week and then sacrificed at 9 weeks of age (i.e., 5 weeks after ICV injection).

[0181] Biodistribution was determined by measuring the presence of vg using ddPCR (>50 vg / 1 μg gDNA was defined as positive).

[0182] Results: Mice receiving rAAV were positive for vg in all brain tissues tested, indicating successful transduction in the brain with ICV administration. ICV administration also resulted in the presence of vg in the spinal cord, with lower levels of transduction observed in the liver. The reduced biodistribution in the liver was reflected in lower TREM2 levels in this tissue.

[0183] Inhibition of CSF1R by PLX treatment was evident in mice at both the mRNA and protein levels. There was a statistically significant improvement in CSF1R expression at the mRNA level in mice treated with both doses of rAAV plus PLX compared with mice treated with vehicle plus PLX. The levels of sCSF1R fragments in the CSF of animals treated with both doses of rAAV plus PLX showed a positive trend pattern relative to vehicle levels. Thus, rAAV treatment ameliorates the negative effects of PLX in the CSF1R inhibition model.

[0184] Furthermore, administering vehicle plus PLX to mice resulted in a near-complete depletion of microglia throughout the brain. Indeed, IBA1 expression was significantly higher in mice treated with either dose of rAAV plus PLX compared to mice treated with vehicle plus PLX. + There was a statistically significant increase in the number of microglia. This observation was further confirmed by analysis of AIF1 mRNA expression levels, the gene encoding IBA1, using qRT-PCR. Furthermore, the significant therapeutic benefit of rAAV was evident in higher AIF1 mRNA levels in mice treated with either dose plus PLX compared with the vehicle plus PLX treatment group.

[0185] Furthermore, PLX treatment reduced the expression of homeostatic microglia-related genes, such as P2RY12 and HEXB, which are known to be stably expressed in homeostatic microglia under homeostatic conditions. Significantly higher expression of both P2RY12 and HEXB was observed in mice treated with rAAV+PLX.

[0186] Consistent with the observed PLX model effects in ubiquitous and homeostatic genes, disease-associated microglial (DAM) genes (e.g., CCL12, CD48, CD68, LYZ1, and PTPRC) also showed significant decreases in expression in the vehicle + PLX-treated group. In contrast, expression of these genes was fully, or in some cases partially, elevated by rAAV to normal levels in most cases compared with vehicle-only treated animals.

[0187] Example 9: TREM2 rAAV dose range in the CSF1R-inhibited mouse model of CRL Methods: Vehicle or the TREM2 rAAV of Example 3 (i.e., the rAAV vector of SEQ ID NO: 26 encapsidated in the AAV6™ capsid protein (SEQ ID NO: 19)) was administered by ICV injection to 4-week-old WT C57BL / 6 mice (n=16 / group). TREM2 rAAV was injected at three doses: 1.33 x 10 11 vg(3.33×10 11vg / g brain), 1.8 × 10 10 vg(4.49×10 10 vg / g brain), or 2.44 × 10 9 vg(6.10×10 9 vg / g brain). Vector particles per gram of brain weight was based on an adult mouse brain weight of 400 mg.

[0188] At 8 weeks of age (i.e., 4 weeks after ICV injection), one vehicle-treated group and 1.33 x 10 11 vg and 2.44×10 9 Both treatment groups were treated with PLX3397. Mice were fed 185 mg / kg PLX3397 chow for 1 week and then sacrificed at 9 weeks of age (i.e., 5 weeks after ICV injection).

[0189] Biodistribution, TREM2 expression levels, microglial panel gene expression, and safety were assessed by histopathology. Biodistribution was determined using ddPCR developed in accordance with the FDA guidance "Long-Term Follow-up After Administration of Human Gene Therapy Products" (2020; limit of quantification of gDNA less than 50 copies / µg). TREM2 expression in CSF was measured using the MSD assay (as described above, sTREM2 was used as a surrogate for protein production in tissues).

[0190] Results: All mice receiving TREM2 rAAV were positive for vg in all brain regions tested, indicating that ICV administration successfully transduced widespread brain tissue. In addition, ICV administration of TREM2 rAAV resulted in reduced vector transduction in the liver compared with the brain.

[0191] ICV administration of TREM2 rAAV robustly increased TREM2 levels in the CSF at all three doses. The lower biodistribution of TREM2 rAAV in peripheral tissues was reflected in lower TREM2 levels in the liver as well as in serum. 11vg and 2.44×10 9 When comparing vg doses, a dose-dependent effect was observed in serum.

[0192] Of the three doses tested in this study, 2.44 × 10 9 The vg dose showed no significant benefit (i.e., subtherapeutic dose) in most of the microglial genes tested. The other two doses showed significant improvements in microglial gene expression profiles in TREM2 rAAV+PLX-treated mice compared to vehicle+PLX-treated mice in nearly 90% of the genes with model effects.

[0193] In summary, 1.33 x 10 11 vg and 1.8×10 10 The vg dose suppressed PLX-induced microglial damage in the brainstem of a CSF1R-inhibited mouse model, and this effect was also observed in different brain regions, including the hippocampus, cortex, and cerebellum.

[0194] Example 10: In vivo non-human primate (NHP) studies of TREM2 rAAV Methods: Two- to four-year-old female cynomolgus monkeys were administered an rAAV vector carrying the TREM2 transgene of SEQ ID NO:3 encapsidated in the AAV6™ capsid protein (SEQ ID NO:19) or vehicle via intravenous injection. One month after ICM injection, vg, TREM2 presence, and safety were assessed.

[0195] Results: Figure 11 shows that the AAV6TM capsid protein resulted in widespread biodistribution in NHPs.

[0196] Figures 12A-C show that the AAV6TM capsid protein resulted in increased TREM2 protein levels in the liver, spinal cord, CSF, and serum.

[0197] Overall, in the NHP safety study, all animals survived, and there were no test-substance-related changes in mortality, clinical signs, body weight / weight gain, body temperature, or neurological parameters in any group administered a single injection into the ICM. There were no findings from examinations of general demeanor, behavior, motor function, proprioception, or postural responses. Clinical pathology revealed minimal changes in hematology, coagulation, and clinical chemistry. Histopathological findings were limited and comparable to those of AAV9 capsid proteins in previous NHP studies. At the higher doses, there was microscopic evidence of minimal to mild mononuclear cell infiltration in the brain and DRG.

[0198] Both AAV9 and AAV6™ capsid proteins transduced microglial cells in vitro with rAAV vectors encoding TREM2; however, AAV6™ capsid protein drove higher TREM2 mRNA and protein expression compared to AAV9 capsid protein. In vivo, both rAAVs demonstrated broad biodistribution and a dose-dependent increase in TREM2 protein levels. In addition, AAV6™ capsid protein showed comparable biodistribution to AAV9 capsid protein in key brain regions in rodent studies, while exhibiting substantially lower liver tropism.

[0199] Example 11: TREM2 rAAV TREM2NHP studies Methods: Vehicle or the TREM2 rAAV of Example 3 (rAAV vector of SEQ ID NO: 25 encapsidated in the AAV6™ capsid protein (SEQ ID NO: 19)) or vehicle was administered by ICM to female cynomolgus monkeys (n=2 / group). TREM2 rAAV was injected at two doses: 3.32 x 10 12 vg(4.49×10 10 vg / g brain) or 1.05 × 10 13 vg (1.42 x 10 vg / g brain). NHPs were sacrificed 29 days after injection to detect potential early toxicity of TREM2 rAAV, anticipating capturing potential toxicity at a time when biodistribution throughout the brain and peripheral organs was expected to be near its maximum.

[0200] Biodistribution, TREM2 expression levels, microglial panel gene expression, and safety by histopathology were assessed. Biodistribution was determined using ddPCR as described in Example 9. TREM2 expression in CSF was measured using the MSD assay (sTREM2 was used as a surrogate for protein production in tissues, as described above).

[0201] Results: All NHPs survived to day 29. 3.32 × 10 12 Dose or 1.05 x 10 13 There were no test article-related changes in mortality, clinical signs, body weight / weight gain, temperature, or neurological signs in any of the groups receiving a single injection of TREM2 rAAV into the ICM at the total vg dose.

[0202] The minimal change in hematology and coagulation parameters was 1.05 x 10 13 These changes consisted of increased fibrinogen levels on day 15, as well as increased neutrophil counts on days 15 and 29 compared to baseline values, indicating a potential acute phase response.

[0203] There were no TREM2 rAAV-associated macroscopic observations. Test article-associated microscopic observations included 3.32 × 10 12 All NHPS administered at doses above vg contained minimal to mild mononuclear cell infiltration in the brain. Minimal to mild mononuclear cell infiltration was present in perivascular areas and in the pia mater. The mononuclear cells were primarily lymphocytes, and the distribution and severity ranged from 3.32 × 10 12 was greater in animals receiving the vg dose.

[0204] Given that the only treatment-related findings were minimal increases in neutrophils and fibrinogen, and minimal to mild mononuclear infiltrates in the brain and ganglia, we determined that ICM injections of either dose of TREM2 rAAV were well tolerated in NHPs.

[0205] With regard to biodistribution, all tissues tested were positive in NHPs administered TREM2 rAAV, indicating widespread distribution throughout the CNS and periphery.

[0206] In the CSF, TREM2 rAAV administration resulted in 1.05 × 10 13 In contrast, a dose-dependent effect of the two doses on TREM2 was observed in serum and liver.

[0207] Collectively, the results demonstrated widespread distribution throughout the brain, comparable to levels shown to be effective in mouse models. This transduction resulted in elevated TREM2 expression in the brain. Furthermore, all NHPs survived, and postmortem pathology analysis demonstrated minimal test-substance-associated increases in neutrophils and fibrinogen, as well as minimal to mild mononuclear infiltration in the brain and ganglia. Consequently, TREM2 rAAV demonstrated an overall favorable safety profile in NHPs.

[0208] Example 12: In vivo NHP studies of TREM2 rAAV Methods: Male and female cynomolgus monkeys (n=6 / group) are administered vehicle or the TREM2 rAAV of Example 3 (rAAV vector of SEQ ID NO:26 encapsidated in the AAV6™ capsid protein (SEQ ID NO:19)) by ICM. TREM2 rAAV is injected at one of three doses: 3.32 x 10 12 vg(4.49×10 10 vg / g brain), 1.05 × 10 13 vg(1.42×10 11 vg / g brain), or 3.32 × 10 13 vg(4.49×10 11 vg / g brain). NHPs will be followed for 29 days or 26 weeks after injection to assess tolerability and safety.

[0209] This study also evaluates peak vector distribution in the brain approximately 4 weeks after administration, as well as post-peak assessment 183 days after administration. Control NHPs receive an ICM administration of the same volume of formulation buffer vehicle containing 20 mM Tris (pH 8.0), 1 mM MgCl2, and 200 mM NaCl containing 0.005% (w / v) poloxamer 188. All NHPs are screened for AAV6 neutralizing antibodies (NAbs) with a titer cutoff of 1:20.

[0210] Sequence Listing The following nucleic acid and / or amino acid sequences are mentioned in the above disclosure and are provided below for reference.

[0211] SEQ ID NO:1 - Human TREM2 protein isoform 1 (230 aa) MEPLRLLILLFVTELSGAHNTTVFQGVAGQSLQVSCPYDSMKHWGRRKAWCRQLGEKGPCQRVVSTHNLWLLSFLRRWNGSTAITDDTLGGTLTITLRNLQPHDAGLYQCQSLHG SEADTLRKVLVEVLADPLDHRDAGDLWFPGESESFEDAHVEHSISRSLLEGEIPFPPTSILLLLACIFLIKILAASALWAAAWHGQKPGTHPPSELDCGHDPGYQLQTLPGLRDT

[0212] SEQ ID NO:2 - Human TREM2 protein isoform 2 (219 aa) MEPLRLLILLFVTELSGAHNTTVFQGVAGQSLQVSCPYDSMKHWGRRKAWCRQLGEKGPCQRVVSTHNLWLLSFLRRWNGSTAITDDTLGGTLTITLRNLQPHDAGLYQ CQSLHGSEADTLRKVLVEVLADPLDHRDAGDLWFPGESESFEDAHVEHSISRAERHVKEDDGRKSPGEVPPGTSPACILATWPPGLLVLLWQETTLPEHCFSWTLEAGTG

[0213] SEQ ID NO: 3 - Artificial sequence (TREM2 transgene 1; 690 nt) atggagcccctgcgcctgctgatcctgctgttcgtgaccgagctgagcggcgcccacaacaccaccgtgttccagggcgtggccggccagagcctgcaggtgagctgcccctacgacagcatgaagcactggggccgccgcaaggcctggtgccgccagctgggcgagaagggcccctgccagcgcgtggtgagcacccacaacctgtggctgctgagcttcctgcgccgctggaacggcagcaccgccatcaccgacgacaccctgggcggcaccctgaccatcaccctgcgcaacctgcagccccacgacgccggcctgtaccagtgccagagcctgcacggcagcgaggccgacaccctgcgcaaggtgctggtggaggtgctggccgaccccctggaccaccgcgacgccggcgacctgtggttccccggcgagagcgagagcttcgaggacgcccacgtggagcacagcatcagccgcagcctgctggagggcgagatccccttcccccccaccagcatcctgctgctgctggcctgcatcttcctgatcaagatcctggccgccagcgccctgtgggccgccgcctggcacggccagaagcccggcacccacccccccagcgagctggactgcggccacgaccccggctaccagctgcagaccctgcccggcctgcgcgacacc

[0214] SEQ ID NO: 4 - Artificial sequence (TREM2 transgene 2; 690 nt) atggagcccctgaggctgctcatcctgctgtttgtgacagaactgtctggagcccacaacaccacagtgttccagggagttgctggccagtctctgcaagtgtcttgcccctatgacagcatgaagcactggggaaggaggaaggcttggtgtaggcagctgggagagaaaggaccttgccagagggtggtgagcacacacaacctgtggctgctgagcttcctcagaaggtggaatggctctacagccatcacagatgacaccctgggtggcaccctcaccatcaccttaaggaacctgcagcctcatgatgctggcctgtaccaatgccagagcctgcatggctctgaggctgataccctcaggaaggtgttggtggaggtgctggctgatcctctggatcacagggatgctggagacctgtggttcccaggagagtctgagagctttgaggatgcccatgtggagcacagcatcagcaggtctcttctggagggagagatccccttccctcccacaagcatcctgttgctgcttgcctgcatcttcctgatcaagatccttgctgcttctgctctttgggctgctgcctggcatggccagaaacctggaacacatcctccctctgaactggactgtggccatgaccctggctaccagttgcaaaccttgcctggcttgagggacacc

[0215] SEQ ID NO: 5 - Artificial sequence (TREM2 transgene 3; 690 nt) atggagcccctgaggctgctgatcctgctgtttgtgacagaactgtctggagcccacaacaccacagtgttccagggagttgctggccagtctctgcaagtgtcttgcccctatgacagcatgaagcactggggaaggaggaaggcttggtgtaggcagctgggagagaaaggaccttgccagagggtggtgagcacacacaacctgtggctgctgagcttcctcagaaggtggaatggctctacagccatcacagatgacaccctgggtggcaccctcaccatcaccctgaggaacctgcagcctcatgatgctggcctgtaccaatgccagagcctgcatggctctgaggctgataccctcaggaaggtgttggtggaggtgctggctgatcctctggatcacagggatgctggagacctgtggttcccaggagagtctgagagctttgaggatgcccatgtggagcacagcatcagcaggtctcttctggagggagagatccccttccctcccacaagcatcctgttgctgcttgcctgcatcttcctcatcaagatccttgctgcttctgctctttgggctgctgcctggcatggccagaaacctggaacacatcctccctctgaactggactgtggccatgaccctggctaccagttgcaaaccttgcctggcttgagggacacc

[0216] SEQ ID NO: 6 - Artificial sequence (TREM2 transgene 4; 690 nt) atggagcccctgagactgctcatcctgctgtttgtgacagaactgtctggggcccacaacaccacagtgttccagggggtggctggccagtccctccaggtgtcctgcccctatgactccatgaagcactgggggagaagaaaggcttggtgtagacagctgggggagaaagggccttgccagagagtggtgtccacacacaacctgtggctgctgtccttcctgagaagatggaatggctccacagccatcacagatgacaccctggggggcaccctcaccatcaccctgagaaacctgcagcctcatgatgctggcctgtaccagtgccagtccctgcatggctctgaggctgataccctgagaaaggtgctggtggaggtgctggctgatcctctggatcacagagatgctggggacctgtggttcccaggggagtctgagtcctttgaggatgcccatgtggagcactccatctccagatccctgctggagggggagatccccttccctcccacatccatcctgctgctgctggcctgcatcttcctcatcaagatcctggctgcttctgctctgtgggctgctgcctggcatggccagaaacctgggacacatcctccctctgaactggactgtggccatgaccctggctaccagctgcagaccctgcctggcctgagagacacc

[0217] SEQ ID NO:7 - Chicken β-actin promoter (283 nt) catggtcgaggtgagccccacgttctgcttcactctccccatctcccccccctccccacccccaattttgtatttatttattttttaattattttgtgcagcgatgggggcggggggggggggggggcgcgcgccaggcggggcggggcggggcgaggggcggggcggggcgaggcggagaggtgcggcggcagccaatcagagcggcgcgctccgaaagtttccttttatggcgaggcggcggcggcggcggccctataaaaagcgaagcgcgcggcgggcg

[0218] SEQ ID NO:8 - CD68 promoter (715 nt) gatatcaaactgcctgtttgggcttctcatttcttacctccccttccctctcccacctgctactgggtgcatctctgctccccccttccccagcagatggttacctttgggctgttgctttcttgtcaccatctgagttctcagacgctggaaagccatgttctcggctctgtgaatgacaatgctgactggagtgctgcccctctgtaaagggctgggtgtggatggtcacaagcccctcacatgcctcagccaagaggaagtagtacaggggtcagcccagaggtccaggggaaaggagtggaaaccgatttccccaccaagggaggggcctgtacctcagctgttcccatagcttacttgccacaactgccaagcaagtttcgctgagtttgacacatggatccctgtggatcaactgccctaggactccgtttgcacccatgtgacactgttgactttgccctgacgaagcagggccaacagtcccctaacttaattacaaaaactaatgactaagagagaggtggctagagctgaggcccctgagtcaggctgtgggtgggatcatctccagtacaggaagtgagactttcatttcctcctttccaagagagggctgagggagcagggttgagcaactggtgcagacagcctagctggactttgggtgaggcggttcagccatatcgaattctgctggggctactggcag

[0219] SEQ ID NO:9 - F4 / 80 promoter (2068 nt)

[0220] SEQ ID NO:10 - Cytomegalovirus enhancer (352 nt) aatagtaatcaattacggggtcattagttcatagcccatatatggagttccgcgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgccaatagggactttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgaccttatgggactttcctacttggcagtacatctacgtattagtcatcgctattac

[0221] SEQ ID NO:11 - Woodchuck hepatitis virus post-transcriptional regulatory element (594 nt) ttatcgataatcaacctctggattacaaaatttgtgaaagattgactggtattcttaactatgttgctccttttacgctatgtggatacgctgctttaatgcctttgtatcatgctattgcttcccgtatggctttcattttctcctccttgtataaatcctggttgctgtctctttatgaggagttgtggcccgttgtcaggcaacgtggcgtggtgtgcactgtgtttgctgacgcaacccccactggttggggcattgccaccacctgtcagctcctttccgggactttcgctttccccctccctattgccacggcggaactcatcgccgcctgccttgcccgctgctggacaggggctcggctgttgggcactgacaattccgtggtgttgtcggggaaatcatcgtcctttccttggctgctcgcctgtgttgccacctggattctgcgcgggacgtccttctgctacgtcccttcggccctcaatccagcggaccttccttcccgcggcctgctgccggctctgcggcctcttccgcgtcttcgccttcgccctcagacgagtcggatctccctttgggccgcctccc

[0222] SEQ ID NO:12 - Bovine growth hormone polyA signal tail (235 nt) cgactagagctcgctgatcagcctcgactgtgccttctagttgccagccatctgttgtttgcccctcccccgtgccttccttgaccctggaaggtgccactcccactgtcctttcctaataaaatgaggaaattgcatcgcattgtctgagtaggtgtcattctattctggggggtggggtggggcaggacagcaagggggaggattgggaagacaatagcaggcatgctgggga

[0223] SEQ ID NO: 13 - Wild-type AAV2 ITR (145 nt) aggaacccctagtgatggagttggccactccctctctgcgcgctcgctcgctcactgaggccgggcgaccaaaggtcgcccgacgcccgggctttgcccgggcggcctcagtgagcgagcgagcgcgcagagagggagtggccaa

[0224] SEQ ID NO: 14 - Modified AAV2 ITR (141 nt) cctgcaggcagctgcgcgctcgctcgctcactgaggccgcccgggcaaagcccgggcgtcgggcgacctttggtcgcccggcctcagtgagcgagcgagcgcgcagagagggagtggccaactccatcactaggggttcct

[0225] SEQ ID NO: 15 - Artificial sequence (TRY region; 60 nt) agctctgggtatttaagcccgagtgagcacgcagggtctccattttgaagcgggaggtta

[0226] SEQ ID NO: 16 - Artificial sequence (stuffer sequence 1; 1229 nt)

[0227] SEQ ID NO: 17 - Artificial sequence (stuffer sequence 2; 1229 nt)

[0228] SEQ ID NO: 18 - Artificial sequence (stuffer sequence 3; 1254 nt)

[0229] SEQ ID NO: 19 - AAV6™ capsid protein (736 aa) MAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQQKQDDGRGLVLPGYKYLGPFNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLRYNHADAEFQERLQEDTSFGGNLGRAVFQAKKRVLEPFGLVEEGAKTAPGKKRPVEQSPQEPDSSSGIGKTGQQPAKKRLNFGQTGDSESVPDPQPLGEPPATPAAVGPTTMASGGGAPMADNNEGADGVGNASGNWHCDSTWLGDRVITTSTRTWALPTYNNHLYKQISSASTGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTTNDGVTTIANNLTSTVQVFSDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLNRTQNQSGSAQNKDLLFSRGSPAGMSVQPKNWLPGPCYRQQRVSKVKTDNNNSNFTWTGASKYNLNGRESIINPGTAMASHKDDKDKFFPMSGVMIFGKESAGASNTALDNVMITDEEEIKATNPVATERFGTVAVNLQSSSTDPATGDVHVMGALPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPPAEFSATKFASFITQYSTGQVSVEIEWELQKENSKRWNPEVQYTSNFAKSANVDFTVDNNGLYTEPRPIGTRFLTRPL

[0230] SEQ ID NO:20 - AVV9 capsid protein (736aa) MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLGPGNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQPAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGVGSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTDNNGVKTIANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQAVGRSSFYCLEYFPSQMLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLSKTINGSGQNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWPGASSWALNGRNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADKVMITNEEEIKTTNPVATESYGQVATNHQSAQAQAQTGWVQNQGILPGMVWQDRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFNKDKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNVEFAVNTEGVYSEPRPIGTRYLTRNL

[0231] SEQ ID NO: 21 - Artificial sequence (Vector 1; 8305 nt)

[0232] SEQ ID NO: 22 - Artificial sequence (Vector 2; 8305 nt)

[0233] SEQ ID NO: 23 - Artificial sequence (Vector 3; 8305 nt)

[0234] SEQ ID NO: 24 - Artificial sequence (Vector 4; 8305 nt)

[0235] SEQ ID NO: 25 - Artificial sequence (Vector 5; 2804 nt)

[0236] SEQ ID NO: 26 - Artificial sequence (Vector 6; 4048 nt)

Claims

1. A recombinant adeno-associated virus (rAAV), comprising: (a) a nucleic acid comprising the nucleotide sequence of an expression construct comprising a microglia-specific promoter operably linked to a TREM2-encoding transgene, the TREM2-encoding transgene comprising a nucleotide sequence selected from any one of SEQ ID NOs: 3-6; (b) a modified AAV6 capsid comprising an amino acid sequence that includes T492V, Y705F, and Y731F mutations compared to a wild-type AAV6 capsid.

2. The rAAV of claim 1, wherein the amino acid sequence of the modified AAV6 capsid is SEQ ID NO:

19.

3. The rAAV of claim 1 or 2, wherein the microglia-specific promoter is a CD68 promoter.

4. The rAAV of claim 3, wherein the nucleotide sequence of the CD68 promoter is SEQ ID NO:

8.

5. The rAAV of any one of claims 1 to 4, wherein the nucleic acid further comprises a nucleotide sequence of a stuffer sequence.

6. The rAAV of claim 5, wherein the nucleotide sequence of the stuffer sequence is selected from the group consisting of SEQ ID NOs: 16-18.

7. The rAAV of any one of claims 1 to 6, wherein the nucleic acid further comprises a nucleotide sequence of a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE).

8. The rAAV of any one of claims 1 to 7, wherein the nucleic acid further comprises a nucleotide sequence of a bovine growth hormone polyA signal tail.

9. The rAAV of any one of claims 1 to 8, wherein the nucleic acid further comprises two nucleotide sequences of adeno-associated virus inverted terminal repeat (ITR) sequences, each ITR sequence flanking the expression construct.

10. 10. The rAAV of claim 9, wherein each ITR sequence is a wild-type AAV2 ITR, a modified AAV2 ITR, or a reverse complementary sequence thereto.

11. The rAAV of any one of claims 1 to 10, wherein the nucleic acid is a nucleotide sequence selected from the group consisting of SEQ ID NOs: 21 to 26.

12. A nucleic acid comprising the nucleotide sequence of an expression construct comprising a microglia-specific promoter operably linked to a transgene encoding a human TREM2 protein, the transgene comprising a nucleotide sequence selected from any one of SEQ ID NOs: 3-6.

13. The nucleic acid of claim 12, wherein the microglia-specific promoter is the CD68 promoter.

14. The nucleic acid of claim 12 or 13, wherein the nucleotide sequence of the microglia-specific promoter is SEQ ID NO:

8.

15. The nucleic acid according to any one of claims 12 to 14, wherein the nucleic acid further comprises a nucleotide sequence of a stuffer sequence.

16. The nucleic acid of claim 15, wherein the nucleotide sequence of the stuffer sequence is selected from the group consisting of SEQ ID NOs: 16 to 18.

17. The nucleic acid of any one of claims 12 to 16, wherein the nucleic acid further comprises a nucleotide sequence of a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE).

18. The nucleic acid according to any one of claims 12 to 17, wherein the nucleic acid further comprises the nucleotide sequence of bovine growth hormone polyA signal tail (BGHpA).

19. 19. The nucleic acid of any one of claims 12 to 18, wherein the nucleic acid further comprises two nucleotide sequences of adeno-associated virus inverted terminal repeat (ITR) sequences, each ITR sequence flanking the expression construct.

20. 20. The nucleic acid of claim 19, wherein each ITR sequence is SEQ ID NO: 13 or 14, or a reverse complement thereof.

21. A nucleic acid comprising, in 5' to 3' order: (a) the 5′ AAV ITR or a reverse complement thereto; (b) a microglia-specific promoter; (c) a TREM2-encoding transgene, the TREM2-encoding transgene comprising a nucleotide sequence selected from any one of SEQ ID NOs: 3-6; (d) post-transcriptional regulatory elements; (e) a polyA signal tail, and (f) a 3′ AAV ITR or a reverse complement thereto; A nucleic acid comprising the nucleotide sequence of

22. 22. The nucleic acid of claim 21, wherein the microglia-specific promoter is the CD68 promoter.

23. 23. The nucleic acid of claim 21 or 22, wherein the microglia-specific promoter comprises SEQ ID NO:

8.

24. 24. The nucleic acid of any one of claims 21 to 23, wherein the post-transcriptional regulatory element is a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE).

25. The nucleic acid of any one of claims 21 to 24, wherein the polyA signal tail is a bovine growth hormone polyA signal tail (BGHpA).

26. 26. The nucleic acid of any one of claims 21 to 25, further comprising a nucleotide sequence of a stuffer sequence, wherein the nucleotide sequence of the stuffer sequence is selected from the group consisting of SEQ ID NOs: 16 to 18.

27. 27. The nucleic acid of any one of claims 21 to 26, wherein the 5' AAV ITR and the 3' AAV ITR are AAV2 ITRs, and the AAV2 ITRs are SEQ ID NO: 13 or 14, or the reverse complement thereof.

28. 21. The nucleic acid of claim 20, wherein the nucleotide sequence is selected from the group consisting of SEQ ID NOs: 21-26.

29. A vector comprising the nucleic acid according to any one of claims 12 to 28.

30. rAAV, A nucleic acid according to any one of claims 12 to 28 or a vector according to claim 29; and an AAV capsid.

31. The rAAV of claim 30, wherein the AAV capsid is a modified AAV6 capsid comprising an amino acid sequence including T492V, Y705F, and Y731F mutations compared to a wild-type AAV6 capsid.

32. The rAAV of claim 31, wherein the amino acid sequence of the modified AAV6 capsid is SEQ ID NO:

19.

33. 1. A pharmaceutical composition comprising: An rAAV according to any one of claims 1 to 11 or claims 30 to 32, a nucleic acid according to any one of claims 12 to 28, or a vector according to claim 29; and a pharmaceutically acceptable carrier.

34. 1. A method of treating a triggering receptor expressed on myeloid cells 2 (TREM2)-associated disease or disorder in an individual, comprising:

34. A method comprising administering to the individual an effective amount of the rAAV of any one of claims 1 to 11 or 30 to 32, the nucleic acid of any one of claims 12 to 28, the vector of claim 29, or the pharmaceutical composition of claim 33.

35. 35. The method of claim 34, wherein the TREM2-associated disease or disorder is selected from the group consisting of Alzheimer's disease (AD), adult-onset leukoencephalopathy with axonal spheroids and pigmented glia (ALSP), Nasu-Hakola disease (NHD), frontotemporal dementia, amyotrophic lateral sclerosis (ALS), cognitive impairment, memory loss, spinal cord injury, traumatic brain injury, and multiple sclerosis.

36. 36. The method of claim 34 or 35, wherein the administering step is by intracisternal magna (ICM) injection.

37. 36. The method of claim 34 or 35, wherein the administering step is by intravenous (IV) injection.

38. 32. An rAAV according to any one of claims 1 to 11 or claims 30 to 32, a nucleic acid according to any one of claims 12 to 28, or a vector according to claim 29 for use in therapy.

39. 30. The rAAV of any one of claims 1 to 11 or claims 30 to 32, the nucleic acid of any one of claims 12 to 28, or the vector of claim 29, for use in treating a triggering receptor expressed on myeloid cells 2 (TREM2)-associated disease or disorder.

40. 40. The rAAV, nucleic acid, or vector for use according to claim 39, wherein the TREM2-associated disease or disorder is selected from the group consisting of Alzheimer's disease (AD), adult-onset leukoencephalopathy with axonal spheroids and pigmented glia (ALSP), Nasu-Hakola disease (NHD), frontotemporal dementia, amyotrophic lateral sclerosis (ALS), cognitive impairment, memory loss, spinal cord injury, traumatic brain injury, and multiple sclerosis.

41. 30. Use of an rAAV according to any one of claims 1 to 11 or claims 30 to 32, a nucleic acid according to any one of claims 12 to 28, or a vector according to claim 29 in the manufacture of a medicament for the treatment of a triggering receptor expressed on myeloid cells 2 (TREM2)-associated disease or disorder.

42. 32. The use of claim 31, wherein the TREM2-related disease or disorder is selected from the group consisting of Alzheimer's disease (AD), adult-onset leukoencephalopathy with axonal spheroids and pigmented glia (ALSP), Nasu-Hakola disease (NHD), frontotemporal dementia, amyotrophic lateral sclerosis (ALS), cognitive impairment, memory loss, spinal cord injury, traumatic brain injury, and multiple sclerosis.

43. A nucleic acid encoding triggering receptor expressed in myeloid cells 2 (TREM2), having a nucleotide sequence comprising at least about 95% sequence similarity to any one of SEQ ID NOs: 3, 4, 5, or 6.

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