Gene therapy of TREM2 related diseases and disorders

Abstract

Nucleic acids encoding trigger receptor 2 (TREM2) expressed on myeloid cells are described, which are useful in expression constructs, vectors and gene therapy. Also described are methods of using the same for the treatment of TREM2 related diseases and disorders, particularly nervous system diseases such as Alzheimer's disease (AD), white matter disease (ALSP) of adult onset with bulbolithiasis and pigmented glial cells, or Nasu-Harcosa disease (NHD).

Description

[0001] Reference to a Sequence Listing Submitted Electronically

[0002] This disclosure is submitted with a Sequence Listing in ST.26 XML format. The Sequence Listing is provided as a file named "30436_WO", created on September 13, 2023, and having a file size of 74.6 kilobytes (kb). The information of the Sequence Listing in ST.26 XML format is incorporated herein by reference in its entirety. Field of the Invention

[0003] This disclosure generally relates to biology and medicine. More particularly, this disclosure relates to synthetic nucleic acids and their use for treating diseases and disorders associated with triggering receptor expressed on myeloid cells 2 (TREM2), particularly as gene therapy for treating neurological diseases such as Alzheimer's disease (AD), adult-onset leukoencephalopathy with axonal spheroids and pigmented glia (ALSP), or Nasu-Hakola Disease (NHD). Background of the Invention

[0004] TREM2 is a cell surface transmembrane glycoprotein that is primarily expressed in myeloid cells, including microglia. In humans, complete absence of TREM2 has been shown to cause NHD, a rare neurodegenerative disease characterized by late-onset dementia, demyelination, and brain atrophy. See, e.g., Paloneva et al., (2002) Am. J. Hum. Genet. 71:656-662; and Paloneva et al., (2003) J. Exp. Med. 198:669-675.

[0005] Mutations in TREM2 are also associated with the risk of developing AD. See, e.g., Guerreiro et al., (2013) N. Engl. J. Med. 368:117-127. AD is an irreversible, progressive brain disease characterized by the presence of abnormal protein deposits throughout the brain, which inhibit neuronal function, disrupt connections between neurons, and ultimately lead to cell death. These deposits include amyloid-β protein plaques and tangles formed by phosphorylated tau protein. Individuals with mild AD experience memory loss, resulting in confusion, difficulty handling money, repetitive questioning, and changes in personality and behavior. In addition, individuals with moderate AD experience increased memory loss, resulting in disorientation and difficulty recognizing friends and family, inability to learn new things, hallucinations, delusions, and paranoia. Further, individuals with severe AD are unable to communicate and are completely dependent on others for care. Eventually, the protein plaques and tangles spread throughout the brain, resulting in severe tissue atrophy.

[0006] CSF1R mutations can lead to a microgliopathy called colony-stimulating factor 1 receptor (CSF1R)-associated adult-onset leukoencephalopathy (CRL), which is a subclass of ALSP. Interestingly, TREM2 activates a similar signaling pathway, resulting in a phenocopy of the intracellular and functional responses of CSF1R activation. It is believed that increased cellular TREM2 can attenuate CSF1R loss of function and can attenuate CRL-related pathology. See, e.g., Tchessalova et al., (2022) Alzheimer's Dement. 18:e061595.

[0007] There are many known therapeutic methods for increasing TREM2, including the use of small molecules or monoclonal antibodies to modulate downstream signaling of TREM2. More recently, gene therapy methods for increasing TREM2 and / or its activity have even been described. See, e.g., International Patent Application Publication No. WO 2019 / 070894.

[0008] However, additional disease-modifying therapies are needed to increase TREM2 and / or its activity in individuals with TREM2-related diseases and / or conditions. SUMMARY OF THE INVENTION

[0009] To meet this need, the present disclosure describes synthetic nucleic acids for treating TREM2-related diseases and disorders. In one example, the synthetic nucleic acid comprises a nucleotide sequence encoding TREM2 (i.e., a TREM2-encoding transgene), and the 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 to 6. In some examples, the TREM2-encoding transgene can be codon-optimized. Alternatively or additionally, the TREM2-encoding transgene can 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 additional examples, the synthetic nucleic acid can be an expression construct that comprises a first nucleotide sequence of at least one expression control element operably linked to a second nucleotide sequence (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 to 6. As described above, the TREM2-encoding transgene can be codon-optimized. Alternatively or additionally, the TREM2-encoding transgene can 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.

[0011] In some examples, the at least one expression control element can be a promoter, enhancer, post-transcriptional regulatory element or polyadenylation signal. In other examples, the promoter can be a chicken β-actin (CBA) promoter or a CD68 promoter or an F4 / 80 promoter, 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 any one of SEQ ID NOs: 7 to 9. In certain 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.

[0012] In some instances, the enhancer can be the cytomegalovirus enhancer (CMVe) and can include 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 certain instances, the nucleotide sequence of the enhancer is SEQ ID NO: 10.

[0013] In some instances, the post-transcriptional regulatory element can be the woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) and can include 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 certain instances, the nucleotide sequence of the post-transcriptional regulatory element is SEQ ID NO: 11.

[0014] In some instances, the polyadenylation signal can be the bovine growth hormone polyadenylation signal tail (BGHpA) and can include 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 certain instances, the nucleotide sequence of the polyadenylation signal is SEQ ID NO: 12.

[0015] In some instances, the expression construct can include additional nucleotide sequences that encode a second transgene or an inhibitory nucleic acid.

[0016] In another example, the synthetic nucleic acid can be a vector comprising an expression construct described 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 include at least one AAV inverted terminal repeat (ITR) sequence that flanks 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 with SEQ ID NO:13 or its reverse complementary sequence. In other examples, the AAV ITR can be a modified 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 with SEQ ID NO:14 or its reverse complementary sequence. In certain examples, the nucleotide sequence of the AAV ITR is SEQ ID NO:13 or 14 or its reverse complementary sequence.

[0017] In some examples, the vector can also include a TRY region and can include 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 with SEQ ID NO:15. In certain examples, the nucleotide sequence of the TRY region is SEQ ID NO:15.

[0018] In some examples, the vector can also include a nucleotide sequence that helps package the vector in the capsid protein (i.e., a sequence that optimizes the vector size for packaging within the capsid protein, 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 with any one of SEQ ID NO:16 to 18. In certain examples, the nucleotide sequence is SEQ ID NO:16, 17 or 18.

[0019] 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 to 6 (i.e., the TREM2-encoding transgene); and (2) an AAV6 capsid protein.

[0020] In some instances, the rAAV comprises:

[0021] (a) an rAAV vector having a nucleotide sequence in the 5' to 3' order comprising:

[0022] (i) a first AAV ITR or its reverse complementary sequence;

[0023] (ii) a promoter;

[0024] (iii) the TREM2-encoding transgene;

[0025] (iv) a post-transcriptional regulatory element;

[0026] (v) a polyadenylation signal; and

[0027] (vii) a second AAV ITR or its reverse complementary sequence; and

[0028] (b) encapsidated in an AAV6 capsid protein.

[0029] In some instances, the AAV6 capsid protein is a modified AAV6 capsid protein (e.g., AAV6TM capsid protein) comprising 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 certain instances, the amino acid sequence of the AAV6 capsid protein is SEQ ID NO: 19.

[0030] In some instances, 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 to 26. In certain instances, the nucleotide sequence of the rAAV vector is any one of SEQ ID NOs: 21, 22, 23, 24, 25 or 26.

[0031] In some instances, the rAAV vector further comprises a nucleotide sequence (i.e., a stuffing sequence) that aids in packaging the capsid protein with the vector, and the 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 to 18. In certain instances, the nucleotide sequence of the stuffing sequence is SEQ ID NO: 16, 17 or 18.

[0032] In some instances, the rAAV vector further comprises a second nucleic acid having a nucleotide sequence that is reverse complementary to any one of SEQ ID NOs: 21, 22, 23, 24, 25 or 26.

[0033] The present disclosure also describes pharmaceutical compositions comprising the synthetic nucleic acids, expression constructs, vectors or rAAVs herein and a pharmaceutically acceptable carrier.

[0034] In some instances, the pharmaceutical composition can be a formulation comprising the rAAV herein and one or more of the following:

[0035] (a) about 20 mM TRIS (pH 8.0);

[0036] (b) about 1 mM MgCl 2 ;

[0037] (c) about 200 mM NaCl; and

[0038] (d) about 0.005% (w / v) Poloxamer 188.

[0039] In some instances, the concentration of rAAV in the formulation can be from about 1×10 13 vector genomes (vg) to about 7×10 14 vg. In other instances, the concentration of rAAV in the formulation can be about 3.5×10 13 vg, about 7.0×10 13 vg or about 1.4×10 14 vg.

[0040] The present disclosure also describes methods of treating TREM2-related diseases and disorders in an individual. The methods can include at least one step of administering to the individual an effective amount of the synthetic nucleic acids, expression constructs, vectors or rAAVs herein.

[0041] In some instances, the TREM2-related disease or disorder can be AD, ALSP or NHD.

[0042] In some instances, the method may further comprise the steps of: measuring TREM2, CSF1R, neurofilament light chain (NfL), chitinase-3-like protein 1 (Chit1), and / or granulocyte-macrophage colony-stimulating factor (GM-CSF) in plasma and / or cerebrospinal fluid (CSF) and / or urine from an individual, and comparing the values obtained with earlier obtained comparative values or control values to evaluate the effectiveness of the method.

[0043] In some instances, the method may further comprise the step of administering to the individual an effective amount of at least one additional therapeutic agent.

[0044] The present disclosure also describes a composition comprising rAAV, which is used as a medicament for treating diseases and disorders related to TREM2, such as AD, ALSP, or NHD.

[0045] The present disclosure also describes a composition comprising rAAV, which is for treating diseases and disorders related to TREM2, such as AD, ALSP, or NHD.

[0046] The present disclosure also describes the use of rAAV in the preparation of a medicament for treating diseases and disorders related to TREM2, such as AD, ALSP, or NHD, wherein the medicament may optionally comprise an additional therapeutic agent.

[0047] The advantages of the synthetic nucleic acids described herein are that they can increase and / or improve microglial function (i.e., development, maintenance, and / or activation) in AD, ALSP, or NHD by increasing membrane-bound and secreted TREM2 levels and / or by enhancing TREM2 signaling or replacing CSF1R signaling with TREM2 signaling to activate downstream CSF1R effects.

[0048] The advantage of the rAAV described herein is that, compared to rAAV having an AAV9 capsid protein, the rAAV described herein shows a broader central nervous system (CNS) biodistribution and increased microglial transduction. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Other advantages, effects, features, and purposes, in addition to the above, will become more apparent when considering the following detailed description.

[0050] Such detailed description refers to the following drawings, wherein:

[0051] Figure 1 is a schematic diagram depicting a first exemplary rAAV vector for expressing TREM2.

[0052] Figure 2 is a schematic diagram depicting a second exemplary rAAV vector for expressing TREM2.

[0053] Figure 3 It is a schematic diagram depicting the third exemplary rAAV vector for expressing TREM2.

[0054] Figure 4 It is a schematic diagram depicting the fourth exemplary rAAV vector for expressing TREM2.

[0055] Figures 5A - 5B Shows TREM2 mRNA ( Figure 5A ) and protein expression ( Figure 5B ) in the HMC3 cell line when transduced with the exemplary rAAV vector capsidated in the AAV6TM capsid protein compared to the same rAAV vector capsidated in the AAV9 capsid protein (n = 4 / group) (AAV6TM = solid circles; AAV9 = open circles).

[0056] Figures 6A - 6B Shows the expression of TREM2 in the HMC3 cell line when transduced with one of two exemplary rAAV vectors both capsidated in the AAV6TM capsid protein ( Figure 6A = first rAAV vector; Figure 6B = second rAAV vector).

[0057] Figure 7 A - D show the somatosensory cortex ( Figure 7 )、hippocampus ( Figure 7 )、cervical spinal cord ( Figure 7 ) and liver ( Figure 7 ) of mice administered low or high doses of the exemplary rAAV vector capsidated in the AAV6TM capsid protein or the same rAAV vector capsidated in the AAV9 capsid protein (n = 8 - 12 / group).

[0058] Figure 8A -C show the TREM2 protein levels in the CSF ( Figure 8A )、liver ( Figure 8B ) and serum ( Figure 8C ) of mice administered low or high doses of the exemplary rAAV vector capsidated in the AAV6TM capsid protein or the same rAAV vector capsidated in the AAV9 capsid protein (n = 8 - 12 / group; a value of 20.48 pg / mL was set as the detection threshold (dashed line). For all graphs: Statistics were determined using analysis of variance (ANOVA), and then compared to the 5xFAD + vehicle group by Dunnett's test. ( * ) p < 0.1; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001).

[0059] Figures 9A - 9C Shows β-amyloid levels in the hippocampus of mice administered low or high doses of an exemplary rAAV vector capsidated in AAV6TM capsid protein or the same rAAV vector capsidated in AAV9 capsid protein( Figures 9A - 9B ) and X34 in the cortex + plaques( Figure 9C )(n = 8 - 12 / group); Statistical data were determined using analysis of variance (ANOVA) and then compared to the 5xFAD + vehicle group by Dunnett's test. ( * ) p < 0.1; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001).

[0060] Figures 10A - 10B Shows inflammation in the cortex of mice administered low or high doses of an exemplary rAAV vector capsidated in AAV6TM capsid protein or the same rAAV vector capsidated in AAV9 capsid protein( Figure 10A as Iba and Figure 10B as IL-10)(n = 8 - 12 / group); Statistical data were determined using analysis of variance (ANOVA) and then compared to the 5xFAD + vehicle group by Dunnett's test. ( * ) p < 0.1; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001).

[0061] Figure 11 Shows the biodistribution of TREM2 in non-human primates (NHPs) administered low or high doses of an exemplary rAAV vector capsidated in AAV6TM capsid protein (n = 2 / group; vector copies were measured using ddPCR).

[0062] Figures 12A - 12C Shows the levels of TREM2 protein in the liver, spinal cord, and dorsal root ganglia (DRG)( Figure 12A ), CSF( Figure 12B ), and serum( Figure 12C ) of NHPs administered low or high doses of an exemplary rAAV vector capsidated in AAV6TM capsid protein (n = 2 / group; a value of 20.48 pg / mL was set as the detection threshold (dashed line)). Detailed Description

[0063] Overview

[0064] TREM2 is a cell surface transmembrane glycoprotein that is mainly expressed in myeloid cells, including microglia. TREM2 directly binds to amyloid-β, promoting the aggregation of microglia around plaques, limiting plaque accumulation and promoting phagocytosis. TREM2 mutations associated with an increased risk of AD reduce the microglial response to amyloid plaques.

[0065] In addition, CRL is a microgliopathy 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 to maintain and activate microglia. Thus, TREM2 expression can rescue or compensate for CSF1R loss-of-function.

[0066] There are two TREM2 isoforms, where isoform 1 is 230 amino acids (aa) in length (SEQ ID NO:1; see also NCBI Reference Sequence No. (Ref.Seq.No.) NP_061838.1), and where isoform 2 is 219 aa in length (SEQ ID NO:2; see also NCBI Reference Sequence No. NP_001258750.1). Exemplary nucleic acid sequences can be found in NCBI Reference Sequence 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 primate). However, those skilled in the art will understand that other instances of the TREM2 mRNA sequence can be readily obtained using publicly available databases such as GenBank and UniProt.

[0067] The present disclosure describes synthetic nucleic acids for use as gene therapy in treating TREM2-related diseases and disorders, wherein the gene therapy delivers a functional copy of TREM2 encoding TREM2 to an individual in need thereof.

[0068] Abbreviations and Definitions

[0069] 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 invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the methods herein, the preferred methods and materials are described herein.

[0070] In addition, the use of the indefinite article "a" or "an" to refer to an element does not exclude the possibility of there being more than one such element, unless the context clearly dictates that there is one and only one element. Thus, the indefinite article "a" or "an" typically means "at least one".

[0071] In addition, the use of "comprising" and other forms (such as "including, but not limited to", "includes", and "included") is not limiting.

[0072] Some of the abbreviations used herein are as follows:

[0073] "aa" refers to amino acid; "AAV" refers to adeno-associated virus, "AcNPV" refers to Autographa californica nucleopolyhedrovirus; "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 expression vector system; "BBB" refers to blood-brain barrier; "BGHpA" refers to bovine growth hormone polyadenylation 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 colony-stimulating factor 1 receptor gene; "CSF1R" refers to colony-stimulating factor 1 receptor protein; "sCSF1R" refers to 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; "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 familial Alzheimer's disease; "FTD" refers to frontotemporal dementia; "GC" refers to genomic copy; "g" refers to gram; "gDNA" refers to genomic DNA; "GM-CSF" refers to granulocyte-macrophage colony-stimulating factor; "HMC3" refers to human microglial clone 3; "hr" refers to hour; "ICM" refers to intracisternal 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 injection; "kg" refers to kilogram; "min" refers to minute; "mL" refers to milliliter; "MSD" refers to MesoScaleDiscovery; "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 15 ClF 3 N 5); "qRT-PCR" refers to quantitative real-time 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 injection; "sec" refers to second; "SEM" refers to standard error of the mean; "ss" refers to single-stranded; "THB" refers to triethylammonium bicarbonate buffer; "sTREM2" refers to soluble triggering receptor expressed on myeloid cells 2; "TREM2" refers to triggering receptor expressed on myeloid cells 2 gene; "TREM2" refers to 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 posttranscriptional regulatory element; "WT" refers to wild type; and "YAC" refers to yeast artificial chromosome.

[0074] Some of the definitions used herein are as follows:

[0075] As used herein, "AAV6TM" refers to an AAV6TM capsid protein that has at least the following 3 mutations (i.e., a triple mutant) in the amino acid sequence compared to the amino acid sequence of the wild-type (WT; see NCBI Ref.Seq.No. AAB95450.1) AAV6 capsid protein: T492V, Y705F, and Y731F (see, e.g., SEQ ID NO:19).

[0076] As used herein, "about" means within the statistical range of one or more values, e.g., the concentration, length, molecular weight, pH, sequence similarity, time range, temperature, volume, etc. Such values or ranges can be within an order of magnitude of the given value or range, typically within 20%, more typically within 10%, and even more typically within 5%. The allowable variation covered by "about" will depend on the particular system being studied and can be readily understood by those skilled in the art.

[0077] As used herein, "administer" and its various grammatical forms mean to provide 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 of an individual).

[0078] As used herein, "codon-optimized" means, with respect to a nucleotide sequence such as a gene of interest (e.g., TREM2), altering the codons or sequence in the gene or its coding region to reflect the typical codon usage of the host organism (e.g., a mammal such as a human) or its cells, without altering the polypeptide encoded by the nucleotide sequence. Thus, a codon-optimized transgene is optimized for expression in a specific organism, organ, tissue, or cell type, particularly a mammal or a mammalian organ, tissue, or cell type. Alternatively, "codon-optimized" means altering the codons or sequence in a gene to improve protein expression as compared to the unaltered sequence, e.g., by eliminating or altering sites that may be potential splice sites, stop codons, miRNA recognition sequences, etc. The entire nucleotide sequence may be codon-optimized, or only one or more portions, segments, or regions of the nucleotide sequence may be codon-optimized.

[0079] As used herein, "comparison window" refers to a contiguous and specific segment of a nucleotide sequence or an amino acid sequence, wherein the sequence in the comparison window may include additions and / or deletions (i.e., gaps) as compared to a reference sequence (excluding additions and / or deletions) for optimal alignment of the two sequences. Typically, the length of the comparison window is at least 10 contiguous nucleotides / amino acids, and optionally may be 20, 30, 40, 50, 60, 70, 80, 90, or 100 nucleotides / amino acids or longer.

[0080] As used herein, "complementary" refers to the structural relationship between two nucleotides (e.g., on two opposing nucleic acids or on opposing regions of a single nucleic acid strand) that permits the two nucleotides to form base pairs (bp) with each other. For example, a purine nucleotide in one nucleic acid can base pair with a pyrimidine nucleotide in the opposing nucleic acid by forming hydrogen bonds with each other. Complementary nucleotides can base pair in a Watson-Crick manner or in any other manner that permits the formation of a stable duplex. Similarly, two nucleic acids can have regions of multiple nucleotides that are complementary to each other to form complementary regions, as described herein.

[0081] As used herein, "CpG-depleted" with respect to a nucleotide sequence means that all (i.e., 100%) known CpG sites in the nucleotide sequence are eliminated / removed.

[0082] As used herein, "CpG-minimized" with respect to a nucleotide sequence means that some (i.e., < 100%) but not all CpG sites in the nucleotide sequence are eliminated / removed.

[0083] As used herein, "CpG site" etc. refers to a cytosine (C) nucleotide followed by a guanine (G) nucleotide in the linear sequence of bases along a nucleotide sequence in the 5' to 3' direction.

[0084] As used herein, "TREM2-related disease or disorder" refers to a disease or disorder caused by a TREM2 mutation that results in a change in TREM2 expression, TREM2 amount, and / or TREM2 activity / function compared to expected physiology. Examples of such diseases or disorders include, but are not limited to, AD, amyotrophic lateral sclerosis (ALS), ALSP, cognitive deficits, frontotemporal dementia (FTD), NHD, memory loss, multiple sclerosis, spinal cord injury, and traumatic brain injury.

[0085] As used herein, "effective amount" refers to the 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, upon administration of a single or multiple doses to an individual in need, provides a desired effect in the individual being diagnosed or treated (i.e., can produce a clinically measurable difference in the condition of the individual or can prevent deterioration of the individual). A person of ordinary skill in the art can readily determine an effective amount by using known techniques and by observing results obtained in similar circumstances. When determining the effective amount for an individual, a number of factors are considered, including but not limited to the species of mammal, its size, age, and general health; the specific disease, disorder, condition, or symptom involved; the degree or relative degree or severity of the disease, disorder, condition, or symptom; the response of the individual, the therapeutic agent being administered, the mode of administration, the bioavailability characteristics of the formulation being administered, the dosage regimen selected, the use of concomitant medications, and other relevant circumstances.

[0086] As used herein, "expression construct" refers to a nucleotide sequence that is capable of expressing a nucleotide sequence of interest (e.g., a transgene or inhibitory nucleic acid) when transformed, transfected, or transduced into a target cell, tissue, organ, or individual. Exemplary expression constructs are vectors, such as viral vectors, particularly AAV vectors or baculovirus vectors. Here, the expression construct can include at least one expression control element operably linked to the nucleotide sequence of interest, such as a transgene (and / or inhibitory nucleic acid). In this way, the expression construct can be an expression control element that interacts operably with the transgene (and / or inhibitory nucleic acid), such as a promoter, that is capable of directing the expression of the transgene (and / or inhibitory nucleic acid) in a cell, tissue, organ, or individual, particularly in the brain tissue.

[0087] As used herein, an "expression control element" refers to the nucleotide sequence of a promoter, polyadenylation signal, transcription or translation termination sequence, upstream regulatory domain, origin of replication, internal ribosome entry site (IRES), enhancer, etc., which together 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. It is not necessary for all of these control sequences to be present at all times as long as the desired nucleotide sequence can be replicated, transcribed, and translated in the appropriate cell, tissue, organ, or individual.

[0088] As used herein, "administered in combination with" means administering a therapeutic agent (e.g., a nucleic acid, vector, rAAV, or composition herein) simultaneously, sequentially, or in a single combination formulation with one or more additional therapeutic agents.

[0089] As used herein, an "individual" refers to any mammal, including cats, dogs, mice, rats, and primates, particularly humans. Additionally, "subject" or "patient" may be used interchangeably with "individual".

[0090] As used herein, an "individual in need" refers to a mammal, such as a human, having a condition, disease, disorder, or symptom that requires treatment or therapy, including, for example, those listed herein. In particular, the preferred individual to be treated is a human.

[0091] As used herein, an "inhibitory nucleic acid" refers to a nucleic acid molecule capable of attenuating, reducing, or preventing gene or mRNA expression. Exemplary inhibitory nucleic acids include, but are not limited to, shRNA, siRNA, miRNA, amiRNA, etc. An inhibitory nucleic acid herein can be a nucleotide sequence encoding an antisense sequence of a nucleotide sequence of interest.

[0092] As used herein, "nucleic acid" refers to a polymer of nucleotides. Although it can contain any type of nucleotide unit, the term generally applies to nucleotide polymers of deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). Polynucleotides are used to include single-stranded (ss) nucleic acids, double-stranded (ds) nucleic acids, and DNA and RNA made of nucleotide or nucleoside analogs that can be determined by their sequences, which are generally presented in the 5' to 3' direction (as the coding strand), where 5' and 3' represent the linkage formed between the 5'-hydroxyl of one nucleotide and the 3'-hydroxyl of the next nucleotide. For the coding strand present in the 5'-3' direction, its complementary strand (or non-coding strand) is the strand that hybridizes to this sequence according to Watson-Crick base pairing. Thus, as used herein, the complementary strand of a nucleic acid such as a polynucleotide is the same as the "reverse complementary strand" and describes the nucleic acid that will pair with the nucleic acid under discussion in its native form.

[0093] As used herein, "nucleoside" refers to a nucleobase-sugar combination, where the nucleobase portion is generally a heterocyclic base. The two most common classes of such heterocyclic bases are purines and pyrimidines. The sugar is generally a pentose, such as ribose or deoxyribose (e.g., 2'-deoxyribose).

[0094] As used herein, "nucleotide" refers to an organic molecule having a nucleoside (a nucleobase, such as adenine, cytosine, guanine, thymine, or uracil; and a pentose, such as ribose or 2'-deoxyribose) and a phosphate group, which can serve as a monomer unit of nucleic acid polymers such as DNA and RNA.

[0095] As used herein, "oligonucleotide" refers to a short nucleic acid molecule (e.g., having a length of less than about 100 nucleotides). Oligonucleotides can be ss or ds.

[0096] As used herein, "operably linked" and the like mean that the elements of an expression construct (or other nucleic acid construct) are configured to perform their normal functions (i.e., under the influence of an expression control element). Thus, an expression control element (e.g., a promoter) that is operably linked to a desired nucleotide sequence (e.g., a transgene or an inhibitory nucleic acid) can effect the expression of the desired nucleic acid. The control element need not be adjacent to the desired nucleotide sequence, as long as it can direct its expression (i.e., maintain the correct reading frame). Thus, for example, there can be an intervening sequence that is not translated but is transcribed between the promoter and the desired nucleotide sequence, and the promoter can still be considered to be "operably linked" to the desired nucleotide sequence.

[0097] As used herein, "pharmaceutically acceptable" when referring to a material such as a carrier or diluent means that it does not abrogate the biological activity or properties of a therapeutic agent (e.g., a nucleic acid, an expression construct, a vector, an rAAV, or a composition herein), and is relatively non-toxic (i.e., the material can be administered to an individual without causing adverse biological effects or interacting in a harmful manner with any component of the composition containing it).

[0098] As used herein, "pharmaceutically acceptable carrier" means a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, stabilizer, dispersing agent, suspending agent, diluent, excipient, thickening agent, solvent, or encapsulating material, involved in carrying or transporting a therapeutic agent within or to an individual so that it can perform its intended function. Additional components that can be included in the pharmaceutical compositions for use in the practice of the present invention are known in the art and are described, for example, in Remington’s Pharmaceutical Sciences, 21st Edition, University of the Sciences in Philadelphia, PA (2006).

[0099] As used herein, "pharmaceutical composition" means a composition or therapeutic agent (e.g., a nucleic acid, an expression construct, a vector, an rAAV, or a 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, thickening agent, excipient, etc.

[0100] As used herein, "recombinant adeno-associated virus", "recombinant AAV", and "rAAV" refer to viral particles that contain an rAAV vector encapsidated by AAV capsid proteins.

[0101] As used herein, "recombinant adeno-associated virus vector", "recombinant AAV vector", and "rAAV vector" refer to synthetic polynucleotide vectors that contain one or more heterologous sequences (i.e., nucleic acid sequences not of AAV origin), the heterologous sequences flanked by at least one AAV ITR sequence. When present in a host cell that has been infected with a suitable helper virus (or expresses suitable helper functions), such an rAAV vector can replicate and be packaged into infectious viral particles, the host cell expressing the AAV rep and cap gene products (i.e., AAV Rep and Cap proteins).

[0102] As used herein, "sequence identity" in the context of two nucleotide sequences or two amino acid sequences means that the residues in the two sequences are the same when aligned to obtain maximum correspondence over a specified comparison window.

[0103] As used herein, "synthetic" refers to nucleic acids or other molecules or compounds that are synthetically produced (e.g., using a machine, such as a solid-phase nucleic acid synthesizer) or recombinantly produced (i.e., not naturally derived from a natural source that normally produces nucleic acids or other compounds).

[0104] As used herein, "transgene" refers to a nucleotide sequence that is introduced into a cell and is capable of being transcribed into RNA and optionally, translated and / or expressed under appropriate conditions. A transgene confers a desired property on the cell into which it is introduced, or otherwise produces a desired therapeutic or diagnostic result. The transgenes herein can be nucleotide sequences encoding a polypeptide of interest, such as TREM2.

[0105] As used herein, "treatment" and its various grammatical forms refer to a process that can slow, control, delay or stop the progression of a disease or disorder disclosed herein, or improve the symptoms of the disease or disorder, but does not necessarily mean complete elimination of all symptoms of the disease or disorder. Treatment, etc. includes administering the nucleic acids, expression constructs, vectors, rAAV or compositions herein to treat a disease or disorder in an individual, particularly a human.

[0106] As used herein, "TREM2" refers to human triggering receptor expressed on myeloid cells 2 (also known as PLOSL2, TREM-2, Trem2a, Trem2b or Trem2c).

[0107] As used herein, "vector" refers to a recombinant plasmid or recombinant virus that includes an oligonucleotide or polynucleotide to be delivered to a host cell 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).

[0108] 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 nucleopolyhedrovirus (AcNPV) vector).

[0109] Composition

[0110] Synthetic nucleic acid

[0111] TREM2-Encoding Transgene: The synthetic nucleic acids herein can be transgenes (i.e., TREM2-encoding transgenes) that include a nucleotide sequence encoding TREM2. The TREM2-encoding transgene can be codon-optimized and / or CpG-minimized and / or CpG-depleted. Although CpG-depleted nucleotide sequences eliminate / remove 100% of CpG sites, CpG-minimized nucleotide sequences eliminate / remove <100% of CpG sites. For example, a CpG-minimized nucleotide sequence can remove 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 CpG sites. Alternatively, a CpG-minimized nucleotide sequence can eliminate / remove 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 CpGs. Alternatively, a CpG-minimized nucleotide sequence can eliminate / remove 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 CpG sites.

[0112] In some instances, the TREM2-encoding transgene includes 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 certain instances, the nucleotide sequence of the TREM2-encoding transgene is SEQ ID NO:3, which is a codon-optimized sequence.

[0113] In other instances, the TREM2-encoding transgene includes 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 certain instances, the nucleotide sequence of the TREM2-encoding transgene is SEQ ID NO:4, which is not only a codon-optimized sequence but also a CpG-depleted sequence.

[0114] In some instances, the TREM2-encoding transgene comprises a nucleotide sequence having at least about 90% sequence identity with 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 with SEQ ID NO:5. In certain instances, the nucleotide sequence of the TREM2-encoding transgene is SEQ ID NO:5, which is not only a codon-optimized sequence but also a 10% CpG-minimized sequence.

[0115] In some instances, the TREM2-encoding transgene comprises a nucleotide sequence having at least about 90% sequence identity with 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 with SEQ ID NO:6. In certain instances, the nucleotide sequence of the TREM2-encoding transgene is SEQ ID NO:6, which is not only a codon-optimized sequence but also a 25% CpG-minimized sequence.

[0116] The synthetic nucleic acids herein can exist alone or can be part of an expression construct, vector, or rAAV, as further described herein.

[0117] Expression construct: As described above, synthetic nucleic acids such as the TREM2-encoding transgene can be incorporated into an expression construct. In some instances, the expression construct can include at least one expression control element operably linked to the TREM2-encoding transgene having a nucleotide sequence of any one of SEQ ID NOs: 3 to 6 (or a nucleotide sequence having at least about 90% to about 99% sequence identity therewith). In certain instances, the nucleotide sequence of the TREM2-encoding transgene is SEQ ID NO:3, 4, 5, or 6.

[0118] 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 splicing site, at least one post-transcriptional regulatory element, at least one polyadenylation signal, or a combination thereof.

[0119] When at least one expression control element is a promoter, the promoter can be a CBA promoter or a CD68 promoter or an F4 / 80 promoter. In some instances, the promoter is a CBA promoter and has a nucleotide sequence with at least about 90% sequence identity to SEQ ID NO:7. In other instances, the promoter is a CD68 promoter and has a nucleotide sequence with at least about 90% sequence identity to SEQ ID NO:8. In other instances, the promoter is an F4 / 80 promoter and has a nucleotide sequence with at least about 90% sequence identity to SEQ ID NO:9 (see also, International Patent Application Publication No. WO2006 / 122141). In certain instances, the nucleotide sequence of the promoter is SEQ ID NO:8.

[0120] When at least one expression control element is an enhancer, the enhancer can be CMVe and has a nucleotide sequence with at least about 90% sequence identity to SEQ ID NO:10. In certain instances, the nucleotide sequence of the enhancer is SEQ ID NO:10

[0121] When at least one expression control element is a post-transcriptional regulatory element, the post-transcriptional regulatory element can be WPRE and has a nucleotide sequence with at least about 90% sequence identity to SEQ ID NO:11. In certain instances, the nucleotide sequence of the post-transcriptional regulatory element is SEQ ID NO:11

[0122] When at least one expression control element is a polyadenylation signal, the polyadenylation signal can be a BGHpA tail and has a nucleotide sequence with at least about 90% sequence identity to SEQ ID NO:12. In certain instances, the nucleotide sequence of the polyadenylation signal is SEQ ID NO:12.

[0123] In some instances, the expression construct can also include the nucleotide sequences of one or more internal ribosome entry sites (IRES), self-cleaving peptide coding sequences such as the T2A peptide coding sequence.

[0124] In some instances, the expression construct includes at least one additional nucleotide sequence for another transgene and / or inhibitory nucleic acid.

[0125] The expression constructs herein can exist alone, or can be part of a vector or even exist as rAAV further described herein.

[0126] Vectors: As described above, synthetic nucleic acids or expression constructs can be integrated into vectors, particularly viral vectors such as rAAV vectors. The rAAV vector can contain the "plus strand" or "minus strand" of the rAAV vector. In some instances, the rAAV vector is ss (e.g., ss DNA or ss RNA). In other instances, the rAAV vector is ds (e.g., ds DNA or ds RNA).

[0127] To aid expression, the rAAV vector includes ITRs flanking the nucleotide sequence of interest (e.g., the TREM2-encoding transgene). In some instances, the ITR sequence is full-length (i.e., about 145 nt in length and contains a functional Rep binding site (RBS) and a terminal resolution site (trs)), and is the WT AAV2 ITR, which includes a nucleotide sequence having at least about 90% sequence identity with SEQ ID NO:13 or its reverse complementary sequence. In certain instances, the nucleotide sequence of the WT AAV2 ITR is SEQ ID NO:13 or its reverse complementary sequence. In other instances, the ITR is a modified ITR (i.e., includes additions, deletions, substitutions, etc.), and is a modified AAV2 ITR, which includes a nucleotide sequence having at least about 90% sequence identity with SEQ ID NO:14 or its reverse complementary sequence. In certain instances, the nucleotide sequence of the modified AAV2 ITR is SEQ ID NO:13 or 14 or its reverse complementary sequence.

[0128] 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., 5' ITR) and the TREM2-encoding transgene. In some instances, the TRY region includes a nucleotide sequence having at least about 90% sequence identity with SEQ ID NO:15. In certain instances, the nucleotide sequence of the TRY region is SEQ ID NO:15.

[0129] Additional sequences can be included in the rAAV vector to aid in packaging the rAAV vector into the capsid protein (i.e., sequences that optimize the size of the vector for packaging within the capsid protein; "filler sequences"). In some instances, such filler sequences include nucleotide sequences having at least about 90% sequence identity with SEQ ID NOs:16 to 18. In certain instances, the nucleotide sequence of the filler sequence is SEQ ID NO:16, 17, or 18.

[0130] The vectors herein can exist alone or can be part of rAAV, as further described herein.

[0131] rAAV

[0132] As described above, the vector can be incorporated into rAAV (i.e., the rAAV vector is encapsidated in the AAV capsid protein). The rAAV herein includes a capsid protein that is prone to diffuse through the CNS, especially 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 AAV6, AAV9, or AAVrh.10 serotypes.

[0133] Particularly of interest herein is rAAV that can infect microglia through an AAV6-based capsid protein, particularly the AAV6TM capsid protein. In some examples, the AAV6TM capsid protein includes an amino acid sequence having at least about 95% sequence identity with SEQ ID NO: 19. In certain examples, the amino acid sequence of the AAV6TM capsid protein is SEQ ID NO: 19.

[0134] In this way, the rAAV herein includes (a) an AAV vector comprising a TREM2-encoding transgene and (b) an AAV6 capsid protein.

[0135] In some examples, the rAAV includes:

[0136] (a) An rAAV vector having the following nucleotide sequence in the 5' to 3' order:

[0137] (i) A first AAV ITR or its reverse complementary sequence;

[0138] (ii) A promoter;

[0139] (iii) A TREM2-encoding transgene;

[0140] (iv) A post-transcriptional regulatory element;

[0141] (v) A polyadenylation signal; and

[0142] (vi) A second AAV ITR or its reverse complementary sequence; and

[0143] (b) Encapsidated in the AAV6 capsid protein.

[0144] In other examples, the rAAV includes:

[0145] (a) An rAAV vector having the following nucleotide sequence in the 5' to 3' order:

[0146] (i) A first AAV2 ITR having the nucleotide sequence of SEQ ID NO: 13 or 14 or its reverse complementary sequence;

[0147] (ii) A promoter having the nucleotide sequence of SEQ ID NO:8;

[0148] (iii) A TREM2-encoding transgene having the nucleotide sequence of SEQ ID NO:3;

[0149] (iv) A post-transcriptional regulatory element having the nucleotide sequence of SEQ ID NO:11;

[0150] (v) A polyadenylation signal having the nucleotide sequence of SEQ ID NO:12; and

[0151] (vi) A second AAV2 ITR having the nucleotide sequence of SEQ ID NO:13 or 14 or its reverse complementary sequence; and

[0152] (b) Encapsidated in an AAV6 capsid protein, particularly the AAV6TM capsid protein having the amino acid sequence of SEQ ID NO:19.

[0153] In other additional examples, the rAAV comprises:

[0154] (a) An rAAV vector having the following nucleotide sequence in the 5' to 3' order:

[0155] (ii) A first AAV2 ITR having the nucleotide sequence of SEQ ID NO:13 or 14 or its reverse complementary sequence;

[0156] (ii) A promoter having the nucleotide sequence of SEQ ID NO:8;

[0157] (iii) A TREM2-encoding transgene having the nucleotide sequence of SEQ ID NO:4;

[0158] (iv) A post-transcriptional regulatory element having the nucleotide sequence of SEQ ID NO:11;

[0159] (v) A polyadenylation signal having the nucleotide sequence of SEQ ID NO:12; and

[0160] (vi) A second AAV2 ITR having the nucleotide sequence of SEQ ID NO:13 or 14 or its reverse complementary sequence; and

[0161] (b) Encapsidated in an AAV6 capsid protein, particularly the AAV6TM capsid protein having the amino acid sequence of SEQ ID NO:19.

[0162] In other additional examples, the rAAV comprises:

[0163] (a) An rAAV vector having the following nucleotide sequences in 5' to 3' order:

[0164] (i) A first AAV2 ITR having the nucleotide sequence of SEQ ID NO:13 or 14 or its reverse complementary sequence;

[0165] (ii) A promoter having the nucleotide sequence of SEQ ID NO:8;

[0166] (iii) A TREM2-encoding transgene having the nucleotide sequence of SEQ ID NO:5;

[0167] (iv) A post-transcriptional regulatory element having the nucleotide sequence of SEQ ID NO:11;

[0168] (v) A polyadenylation signal having the nucleotide sequence of SEQ ID NO:12; and

[0169] (vi) A second AAV2 ITR having the nucleotide sequence of SEQ ID NO:13 or 14 or its reverse complementary sequence; and

[0170] (b) Encapsidated in an AAV6 capsid protein, particularly the AAV6TM capsid protein having the amino acid sequence of SEQ ID NO:19.

[0171] In additional other examples, the rAAV comprises:

[0172] (a) An rAAV vector having the following nucleotide sequences in 5' to 3' order:

[0173] (i) A first AAV2 ITR having the nucleotide sequence of SEQ ID NO:13 or 14 or its reverse complementary sequence;

[0174] (ii) A promoter having the nucleotide sequence of SEQ ID NO:8;

[0175] (iii) A TREM2-encoding transgene having the nucleotide sequence of SEQ ID NO:6;

[0176] (iv) A post-transcriptional regulatory element having the nucleotide sequence of SEQ ID NO:11;

[0177] (v) A polyadenylation signal having the nucleotide sequence of SEQ ID NO:12; and

[0178] (vi) A second AAV2 ITR having the nucleotide sequence of SEQ ID NO:13 or 14 or its reverse complementary sequence; and

[0179] (b) Encapsidated in an AAV6 capsid protein, particularly the AAV6TM capsid protein having the amino acid sequence of SEQ ID NO:19.

[0180] Any of the above rAAVs may also include a filler sequence having the nucleotide sequences of SEQ ID NOs: 16 to 18. In some instances, the filler sequence is located between the polyadenylation signal and the second AAV2 ITR.

[0181] In certain instances, the rAAV includes an rAAV vector having the nucleotide sequence of SEQ ID NO:21, which is encapsidated in an AAV6 capsid protein, particularly the AAV6TM capsid protein having the amino acid sequence of SEQ ID NO:19. In some instances, the rAAV includes a nucleotide sequence that is reverse complementary to SEQ ID NO:21.

[0182] In certain other instances, the rAAV includes an rAAV vector having the nucleotide sequence of SEQ ID NO:22, which is encapsidated in an AAV6 capsid protein, particularly the AAV6TM capsid protein (e.g., VP1) having the amino acid sequence of SEQ ID NO:19. In some instances, the rAAV includes a nucleotide sequence that is reverse complementary to SEQ ID NO:22.

[0183] In certain other instances, the rAAV includes an rAAV vector having the nucleotide sequence of SEQ ID NO:23, which is encapsidated in an AAV6 capsid protein, particularly the AAV6TM capsid protein having the amino acid sequence of SEQ ID NO:19. In some instances, the rAAV includes a nucleotide sequence that is reverse complementary to SEQ ID NO:23.

[0184] In certain other instances, the rAAV includes an rAAV vector having the nucleotide sequence of SEQ ID NO:24, which is encapsidated in an AAV6 capsid protein, particularly the AAV6TM capsid protein having the amino acid sequence of SEQ ID NO:19. In some instances, the rAAV includes a nucleotide sequence that is reverse complementary to SEQ ID NO:24.

[0185] In certain other instances, the rAAV includes an rAAV vector having the nucleotide sequence of SEQ ID NO:25, which is encapsidated in an AAV6 capsid protein, particularly the AAV6TM capsid protein having the amino acid sequence of SEQ ID NO:19. In some instances, the rAAV includes a nucleotide sequence that is reverse complementary to SEQ ID NO:25.

[0186] In certain other instances, the rAAV comprises an rAAV vector having the nucleotide sequence of SEQ ID NO: 26, which is encapsidated in an AAV6 capsid protein, particularly in an AAV6TM capsid protein having the amino acid sequence of SEQ ID NO: 19. In some instances, the rAAV comprises a nucleotide sequence that is reverse complementary to SEQ ID NO: 26.

[0187] Pharmaceutical composition

[0188] The synthetic nucleic acids (i.e., expression constructs or vectors) described herein or the rAAV described herein can be formulated into a pharmaceutical composition comprising the synthetic nucleic acid or rAAV and a pharmaceutically acceptable carrier. The pharmaceutical composition can be prepared by methods well known in the art (e.g., Remington: The Science and Practice of Pharmacy, 22nd Edition (Pharmaceutical Press, 2013)).

[0189] In some instances, the pharmaceutical composition can be in the form of a formulation that includes not only the rAAV herein but also one or more of the following: (a) about 10 mM to about 30 mM TRIS buffer, (b) about 0.5 mM to about 1.5 mM MgCl 2 , (c) about 100 mM to about 300 mM NaCl, and (d) about 0.001% (w / v) to about 0.01% (w / v) poloxamer 188.

[0190] In some instances, the rAAV comprises an rAAV vector having the nucleotide sequence of SEQ ID NO: 21, which is encapsidated in an AAV6 capsid protein, particularly in an AAV6TM capsid protein (such as VP1) having the amino acid sequence of SEQ ID NO: 19. In some instances, the rAAV comprises a nucleotide sequence that is reverse complementary to SEQ ID NO: 21. In other instances, the rAAV comprises an rAAV vector having the nucleotide sequence of SEQ ID NO: 22, which is encapsidated in an AAV6 capsid protein, particularly in an AAV6TM capsid protein having the amino acid sequence of SEQ ID NO: 19. In some instances, the rAAV comprises a nucleotide sequence that is reverse complementary to SEQ ID NO: 22. In other instances, the rAAV comprises an rAAV vector having the nucleotide sequence of SEQ ID NO: 23, which is encapsidated in an AAV6 capsid protein, particularly in an AAV6TM capsid protein having the amino acid sequence of SEQ ID NO: 19. In some instances, the rAAV comprises a nucleotide sequence that is reverse complementary to SEQ ID NO: 23. In other instances, the rAAV comprises an rAAV vector having the nucleotide sequence of SEQ ID NO: 24, which is encapsidated in an AAV6 capsid protein, particularly in an AAV6TM capsid protein having the amino acid sequence of SEQ ID NO: 19. In some instances, the rAAV comprises a nucleotide sequence that is reverse complementary to SEQ ID NO: 24. In other instances, the rAAV comprises an rAAV vector having the nucleotide sequence of SEQ ID NO: 25, which is encapsidated in an AAV6 capsid protein, particularly in an AAV6TM capsid protein having the amino acid sequence of SEQ ID NO: 19. In some instances, the rAAV comprises a nucleotide sequence that is reverse complementary to SEQ ID NO: 25. In other instances, the rAAV comprises an rAAV vector having the nucleotide sequence of SEQ ID NO: 26, which is encapsidated in an AAV6 capsid protein, particularly in an AAV6TM capsid protein having the amino acid sequence of SEQ ID NO: 19. In some instances, the rAAV comprises a nucleotide sequence that is reverse complementary to SEQ ID NO: 26.

[0191] In some instances, the concentration of the TRIS buffer can be from about 10 mM to about 30 mM. In other instances, the concentration of the TRIS buffer can be from about 15 mM to about 25 mM, or about 20 mM. In still other instances, the concentration of the TRIS buffer can be 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.

[0192] In some instances, the concentration of MgCl 2 can be from about 0.5 mM to about 1.5 mM. In other instances, the concentration of MgCl 2 can be from about 0.6 mM to about 1.4 mM, from about 0.7 mM to about 1.3 mM, from about 0.8 mM to about 1.2 mM, from about 0.9 mM to about 1.1 mM, or about 1.0 mM. In still other instances, the concentration of MgCl 2 can be 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.

[0193] In some instances, the concentration of NaCl can be from about 100 mM to about 300 mM. In other instances, the concentration of NaCl can be from about 125 mM to about 275 mM, from about 150 mM to about 250 mM, from about 175 mM to about 225 mM, or about 200 mM. In still other instances, the concentration of NaCl can be 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.

[0194] In some instances, the concentration of poloxamer 188 can be from about 0.001% (w / v) to about 0.01% (w / v). In other instances, the concentration of poloxamer 188 can be from about 0.002% (w / v) to about 0.009% (w / v), from about 0.003% (w / v) to about 0.008% (w / v), from about 0.004% (w / v) to about 0.007% (w / v), or from about 0.005% (w / v) to about 0.006% (w / v). In still other instances, the concentration of poloxamer 188 can be 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).

[0195] In certain instances, the formulation can include the rAAV herein and (a) about 20 mM TRIS (pH 8.0), (b) about 1 mM MgCl 2 , (c) about 200 mM NaCl, and (d) about 0.005% (w / v) poloxamer 188.

[0196] In some instances, the titer of the effective amount can be from about 10 9 genome copies (GC) / kg to about 10 14 GC / kg (e.g., about 10 9 GC / kg, about 10 10 GC / kg, about 10 11 GC / kg, about 10 12 GC / kg, about 10 13 GC / kg, or about 10 14 GC / kg). In some instances, a high titer (e.g., >10 12 GC / kg rAAV) is administered to an individual by injection into the CSF space, particularly by ICM.

[0197] In other instances, the dosage range of the effective amount can be from about 1×10 12 VG to about 1×10 15 VG or from about 1×10 13 VG to about 7×10 14 VG. In other instances, the dosage can be about 3.5×10 13 vg, about 7.0×10 13 vg, or about 1.4×10 14 vg. In still other instances, the dosage can be about 1×10 14vg, about 2.0×10 14 vg or about 4.0×10 14 vg. Alternatively, the dose can be about 2×10 13 vg, about 3×10 13 vg, about 4×10 13 vg, about 5×10 13 vg, about 6×10 13 vg, about 7×10 13 vg, about 8×10 13 vg, about 9×10 13 vg, about 1×10 14 vg or about 2×10 14 vg. In certain instances, the dose is 7.0×10 13 vg or 1.4×10 14 vg.

[0198] The pharmaceutical composition can be administered by any route, including, for example, intraarterial, intradermal, intramuscular, intrathecal, intravenous (IV), intraventricular, parenteral, subcutaneous (SC) or transdermal administration. Routes that are particularly contemplated are IV administration (e.g., systemic intravenous injection), and / or direct administration to the affected site (e.g., intracisternal magna (ICM) injection, intraventricular (ICV) injection) or a combination thereof.

[0199] Generally, the most suitable route of administration will depend on a variety of factors, including but not limited to the nature of the agent (e.g., its stability in its administration environment and / or the intended target) and / or the condition of the individual (e.g., whether the subject can tolerate oral administration). In some instances, synthetic nucleic acids, rAAV or the pharmaceutical composition are suitable for administration to the CNS of an individual by, for example, intrathecal, ICM, ICV or a combination thereof.

[0200] Kit

[0201] In some instances, the synthetic nucleic acids (i.e., expression constructs or vectors) or rAAV of the present disclosure can be included in a kit, which includes the synthetic nucleic acid or rAAV and instructions for its use. In other instances, 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 instances, the kit contains in a suitable container or other holding device the synthetic nucleic acid or rAAV, one or more controls, and various buffers, reagents, enzymes, and other standard components for the synthetic nucleic acid. In some instances, the container comprises at least one vial, well, test tube, flask, bottle, syringe, or other container device in which the synthetic nucleic acid, rAAV, or other therapeutic oligonucleotide is placed and, in some instances, appropriately aliquoted. In those instances where additional components are provided, the kit includes additional containers in which the components are placed. The kit can also include means for containing the synthetic nucleic acid or rAAV and any other reagents, and the kits are sealed for commercial sale. Such containers can include injection or blow molded plastic containers that house the required vials. The container and / or the kit can include a label with instructions for use and / or warnings.

[0202] In some instances, the kit includes the synthetic nucleic acid or rAAV and a pharmaceutically acceptable carrier, or a pharmaceutical composition comprising the synthetic nucleic acid, rAAV, or other therapeutic oligonucleotide, and instructions for treating a neurodegenerative disease or delaying the progression of a neurodegenerative disease in an individual in need thereof.

[0203] In some instances, the kit includes the synthetic nucleic acid or rAAV and a pharmaceutically acceptable carrier or a pharmaceutical composition comprising the synthetic nucleic acid or rAAV, and instructions for administering the synthetic nucleic acid or rAAV or the pharmaceutical composition.

[0204] Method

[0205] Preparation method

[0206] Methods for preparing 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 instances, rAAV can be produced in a baculovirus vector expression system (BEVS). Preparation 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 Application Nos. 8,945,918 and 9,879,282, International Patent Application Publications WO 2017 / 184879 and WO 2022 / 082017. Alternatively, rAAV can be produced in human embryonic kidney (e.g., HEK293) cells (see, for example, International Patent Application Publication Nos. WO 2020 / 210689 and WO 2022 / 035900). However, rAAV can be produced using any suitable method (e.g., using recombinant rep and cap genes).

[0207] Therapeutic and usage methods

[0208] The nucleic acids, expression constructs, vectors, rAAV, or pharmaceutical compositions described herein can be used in methods for treating neurological diseases such as AD, ALS, ALSP, cognitive deficits, FTD, memory loss, NHD, spinal cord injury, traumatic brain injury, or multiple sclerosis.

[0209] The methods can include the steps described herein, and these steps can, but do not have to, be performed in the order described. However, other orders are also conceivable. In addition, single or multiple steps can be performed in parallel and / or overlap in time and / or be performed separately or in multiple repeated steps. Furthermore, the method can include additional, unspecified steps.

[0210] Herein, synthetic nucleic acids, rAAV, or pharmaceutical compositions containing the same can be used in methods for treating TREM2 - related diseases and disorders, wherein such methods at least include the step of administering to an individual in need of such treatment an effective amount of the synthetic nucleic acid, rAAV, or pharmaceutical composition containing the same.

[0211] 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.

[0212] When it is rAAV, the titer of the effective amount can be about 10 9 genome copies (GC) / kg to about 10 14GC / kg (e.g., about 10 9 GC / kg, about 10 10 GC / kg, about 10 11 GC / kg, about 10 12 GC / kg, about 10 13 GC / kg or about 10 14 GC / kg). In some instances, a high titer (e.g., >10 12 GC / kgrAAV) is administered to an individual by injection into the CSF space, particularly by ICM.

[0213] In other instances, the dose range of the effective amount can be from about 1×10 12 VG to about 1×10 15 VG or about 1×10 13 VG to about 7×10 14 VG. In other instances, the dose can be about 3.5×10 13 vg, about 7.0×10 13 vg or about 1.4×10 14 vg. In still other instances, the dose can be about 1×10 14 vg, about 2.0×10 14 vg or about 4.0×10 14 vg. Alternatively, the dose can be about 2×10 13 vg, about 3×10 13 vg, about 4×10 13 vg, about 5×10 13 vg, about 6×10 13 vg, about 7×10 13 vg, about 8×10 13 vg, about 9×10 13 vg, about 1×10 14 vg or about 2×10 14 vg. In certain instances, the dose is 7.0×10 13 vg or 1.4×10 14 vg.

[0214] In some instances, rAAV or a composition comprising rAAV can be administered to a subject one or more times (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 20 or more times).

[0215] In some instances, the individual has or is suspected of having a disease or disorder associated with TREM2, particularly AD, ALSP or NHD.

[0216] In some instances, the individual is from about 1 month to 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 instances, the individual is from about 10 years to about 20 years old (e.g., about 10 years, 11 years, 12 years, 13 years, 14 years, 15 years, 16 years, 17 years, 18 years, 19 years, 20 years, or any age in between). In other instances, the individual is greater than 20 years old (e.g., about 21 years, 22 years, 23 years, 24 years, 25 years, 26 years, 27 years, 28 years, 29 years, 30 years, or any age in between), greater than 30 years old (e.g., about 31 years, 32 years, 33 years, 34 years, 35 years, 36 years, 37 years, 38 years, 39 years, 40 years, or any age in between), greater than 40 years old (e.g., about 41 years, 42 years, 43 years, 44 years, 45 years, 46 years, 47 years, 48 years, 49 years, 50 years, or any age in between), or even greater than 50 years old (e.g., about 51 years, 52 years, 53 years, 54 years, 55 years, 56 years, 57 years, 58 years, 59 years, 60 years, 70 years, 80 years, 90 years, or any age in between).

[0217] In some instances, the individual has a pathogenic CSF1R mutation.

[0218] In some instances, the method may further comprise the steps of: measuring TREM2 in plasma and / or CSF and / or urine from the individual, and comparing the value obtained with a comparable value obtained earlier or with a control value to evaluate the effectiveness of the method. In some instances, TREM2 is soluble TREM2 (sTREM2).

[0219] In other instances, the method may further comprise the steps of: measuring CSF1R in plasma and / or CSF and / or urine from the individual, and comparing the value obtained with a comparable value obtained earlier or with a control value to evaluate the effectiveness of the method. In some instances, CSF1R is soluble CSF1R (sCSF1R).

[0220] In other instances, the method may further comprise the steps of: measuring NfL in plasma and / or CSF and / or urine from the individual, and comparing the value obtained with a comparable value obtained earlier or with a control value to evaluate the effectiveness of the method.

[0221] In other instances, the method may further comprise the steps of: measuring Chit1 in plasma and / or CSF and / or urine from an individual and comparing the value obtained with a comparable value obtained earlier or with a control value to evaluate the effectiveness of the method.

[0222] In other instances, the method may further comprise the steps of: measuring GM-CSF in plasma and / or CSF and / or urine from an individual and comparing the value obtained with a comparable value obtained earlier or with a control value to evaluate the effectiveness of the method.

[0223] Use

[0224] The rAAV of the present disclosure or a pharmaceutical composition comprising the same can be used for or is suitable for treating an individual (e.g., a human) suffering from or suspected of suffering from a disease or disorder associated with TREM2 such as AD, ALSP, or NHD. Accordingly, there is provided an rAAV or a pharmaceutical composition comprising rAAV for use in or suitable for treating an individual suffering from or suspected of suffering from a disease or disorder associated with TREM2 such as AD, ALSP, or NDH. In addition, there is provided an rAAV or a pharmaceutical composition comprising the same for use in or suitable for preparing a medicament or a pharmaceutical composition for treating a disease or disorder associated with TREM2 such as AD, ALSP, or NHD.

[0225] Nucleic acids, vectors, rAAVs, or pharmaceutical compositions for treatment are also described. In addition, nucleic acids, vectors, rAAVs, or pharmaceutical compositions for treating neurological diseases such as AD, ALS, ALSP, cognitive deficits, FTD, memory loss, NHD, spinal cord injury, traumatic brain injury, or multiple sclerosis are described herein.

[0226] The use of nucleic acids, vectors, rAAVs, or pharmaceutical compositions in the preparation of a medicament for treating neurological diseases such as AD, ALS, cognitive deficits, FTD, memory loss, NHD, spinal cord injury, traumatic brain injury, or multiple sclerosis is also described.

[0227] Examples

[0228] The following examples are provided for illustrative purposes and not for limitation.

[0229] Example 1: Generation of rAAV

[0230] Method: Cells, such as HEK293 cells or Sf9 insect cells, are used to generate rAAV vectors expressing various TREM2-encoding transgenes (see, e.g., International Patent Application Nos. WO 2008 / 024988, 2017 / 184879, and WO 2022 / 082017). ITR sequences flank an expression construct having a transgene promoter / enhancer element, a 3' polyadenylation signal, and a post-translational signal such as a WPRE element.

[0231] The rAAV vectors are encapsidated in an AAV6TM capsid protein (SEQ ID NO:19) or an AAV9 capsid protein (SEQ ID NO:20).

[0232] In vitro studies

[0233] Example 2: In vitro transduction of human microglial cell line with TREM-2 rAAV

[0234] Method: An rAAV vector having a TREM2 transgene (SEQ ID NO:3) is used to transduce the HMC3 microglial cell line at a series of MOIs, and the transgene is encapsidated in an AAV6TM capsid protein (SEQ ID NO:19) or an AAV9 capsid protein (SEQ ID NO:20). At 72 hours post-transduction, the supernatant is collected to obtain protein, and the cells are lysed to obtain RNA.

[0235] TREM2 mRNA is quantified using qRT-PCR and normalized with GAPDH. TREM2 protein is quantified using the MSD assay.

[0236] Results: Figures 5A - 5B It is shown that TREM2 mRNA expression ( Figure 5A ) and protein expression ( Figure 5B ) are both observed in rAAV; however, the rAAV vector encapsidated in the AAV6TM capsid protein performs better than the same rAAV vector encapsidated in the AAV9 capsid protein.

[0237] Example 3: In vitro transduction of human microglial cell line with alternative TREM-2 rAAV

[0238] Method: At 1.09×10 5 to 3.50×10 6Within the MOI range of vg / cell, HMC3 cells were transduced with a first rAAV vector having a TREM2 transgene (SEQ ID NO: 25) or a second rAAV vector having a TREM2 transgene (SEQ ID NO: 26), and the transgene was encapsidated in the AAV6TM capsid protein (SEQ ID NO: 19). Three days after infection, the supernatant was collected for ELISA.

[0239] Results: Both rAAVs effectively transduced HMC3 cells in vitro, resulting in potent, dose-dependent expression and secretion of TREM2 ( Figures 6A - 6B , where Figure 6A is the first rAAV vector, Figure 6B is the second rAAV vector). In contrast, the lowest level of TREM2 (78 pg / mL) was detected in the negative control group treated with only the vehicle.

[0240] Example 4: Determination of the in vitro potency of TREM2 in human induced pluripotent stem cell (iPSC)-derived microglial cells

[0241] Method: Human iPSC-derived microglial cells were either treated with the vehicle (negative control) or transduced with an rAAV vector having a TREM2 transgene (SEQ ID NO: 26), and the TREM2 transgene was encapsidated in the AAV6TM capsid protein (SEQ ID NO: 19) at an MOI of 2.0×10 3 to 2.0×10 6 vg / cell. Seven days after transduction, the cells were lysed for ELISA or processed for RNA extraction. All iPSC-derived cells were obtained from FujiFilm Cell Dynamics, Inc. (Madison, WI).

[0242] Results: By day 7, rAAV transduction led to potent, dose-dependent expression of TREM2, which was detected in the groups treated with 2.0×10 5 and 2.0×10 6 vg / cell, at approximately 1000 pg / mL and 1250 pg / mL, respectively. In contrast, TREM2 at a concentration of approximately 625 pg / mL was detected in iPSC-derived microglial cells treated with the vehicle. In addition, rAAV transduction led to a dose-dependent increase in TREM2 mRNA expression in all dose groups, while TREM2 mRNA was not detected in microglial cells treated with the vehicle.

[0243] Example 5: Use of TREM2 rAAV to enhance cell viability in a pharmacological CSF1R inhibition assay in human iPSC-derived microglial cells

[0244] Method: iPSC-derived microglia as described in Example 4 were either treated with excipient (negative control) or transduced with an rAAV vector having a TREM2 transgene (SEQ ID NO: 26), the TREM2 transgene being encapsidated in an AAV6TM capsid protein (SEQ ID NO: 19) at an MOI of 2.0×10 3 vg / cell. Seven days after transduction, a 0.1% DMSO solution of 150 nM or 200 nM PLX3397 was added to the cell culture medium and incubated for an additional 8 hours at 37°C and 5% CO 2 2. After incubation at this stage, cell viability was detected using a colorimetric kit (Abcam).

[0245] Results: PLX treatment caused cytotoxicity and reduced the total number of microglia (i.e., approximately 80% cell death); rAAV transduction significantly reduced PLX-induced toxicity to 50% compared to control levels.

[0246] In Vivo Study

[0247] Example 6: In Vivo Study of AAV Capsid Proteins in Rodents

[0248] Method: Thirty-six 1-month-old 5xFAD mice were divided into 3 groups (n = 12 / group) and treated with the same rAAV as in Example 2 or excipient (20 mM Tris pH 8.0, 200 mM NaCl, and 1 mM MgCl 2 2 + 0.001% Pluronic F68)) by ICV injection (10 μl - 5 μl bilaterally per mouse) to determine efficacy. A group of non-transgenic, age-matched littermates (i.e., WT, n = 12) was similarly injected with excipient 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 evaluated by ddPCR.

[0249] In addition, efficacy endpoints were examined, including protein levels, amyloid-β levels (biochemistry and immunohistochemistry), and inflammation. TREM2 protein and amyloid-β levels were measured using the MSD assay. Iba + cells were measured by immunohistochemistry.

[0250] In vivo blood collection: Before the start of treatment, at 2, 3, 4, and 5 months of age (5 time points), in vivo blood was collected from the facial vein / arterial plexus by mandibular bleeding without anesthesia. Then the collected blood was 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.

[0251] Tissue sampling: At 6 months, mice were finally anesthetized by intraperitoneal injection of pentobarbital (600 mg / kg), and CSF, blood, brain, several organs (gonads, kidneys, heart, liver, lungs, and spleen), and spinal cord were collected for biochemical, immunochemical, and / or histological analysis.

[0252] Immunohistology: At each incubation, 5 sections (one section at each of the 2nd, 4th, 6th, 8th, and 10th levels) were randomly selected evenly and systematically from each mouse among all animals in each group. All sections were counterstained with the nuclear dye DAPI. A highly cross-absorbed secondary antibody was used to observe the binding of the primary antibody.

[0253] Imaging: Whole slide scanning of the stained sections was recorded using a Zeiss AxioScan Z1 automated microscope equipped with a large aperture lens, which is equipped with a Zeiss Axiocam 506 mono and a Hitachi 3CCD HV-F202SCL camera and Zeiss ZEN 3.3 software.

[0254] Sample preparation - homogenization: The hippocampus and somatosensory cortex from all animals were homogenized in 14 volumes of PBS at 55 Hz for 50 seconds using a UPHO bead mill (Geneye), and three aliquots were produced (30 μL for RNA / DNA isolation, 50 μL for soluble and insoluble protein isolation, and the rest).

[0255] Sample preparation - DNA and RNA isolation: DNA and RNA were isolated from the hippocampus and somatosensory cortex of all animals from 30 μL PBS aliquots using an Ambion Trizol kit. The quantity and quality of the total extracted RNA were evaluated by UV-VIS spectrometry using a NanoDrop 1000 spectrophotometer. RNA (1 μg per sample) was reverse transcribed using an iScript gDNA Clear cDNA Synthesis Kit.

[0256] Preparation and measurement of amyloid-β40 and amyloid-β42 samples (soluble and insoluble): The second aliquot of 50 μl hippocampal and cortical samples was replaced with an equal volume of 2×THB buffer (2×THB; 500 mM sucrose, 2 mM EDTA, 2 mM EGTA, 40 mM Tris pH 7.4) (including 1× protease inhibitor (Calbiochem)), vortexed and incubated on ice for 15 minutes. The THB homogenate was then processed to extract soluble and deposited proteins from the brain homogenate for analysis of amyloid-β40 and amyloid-β42. To extract 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 minutes at 4°C, and the supernatant was neutralized with 1 / 10 volume of 0.5 M Tris-HCl, pH 6.8 and briefly vortexed. Aliquots were stored at -80°C. To extract deposited proteins, 30 μL of the THB homogenate was mixed with 2.2 parts cold FA, sonicated on ice for 30 seconds, and centrifuged at 20,000×g for 120 minutes at 4°C. The supernatant was neutralized with 19 parts FA neutralization solution (1 M Tris, 0.5 M Na 2 HPO 4 , 0.05% NaN 3 ). Aliquots were stored at -80°C. Then, soluble and insoluble fractions of hippocampal and somatosensory cortex samples from all animals were analyzed by immunosorbent assay from Mesoscale Discovery (amyloid-β40 peptide (6E10) kit and amyloid-β42 peptide (6E10) kit) according to the manufacturer's instructions. The amyloid-β levels in the study samples were evaluated by comparison with the calibration curves provided in the kits and expressed as pg / mg brain.

[0257] Statistics: Statistical analysis was performed in GraphPad Prism 9. The normality of the data was tested using the Kolmogorow-Smirnow-test. If normal distribution was confirmed, single or two-way ANOVA / mixed effects analysis was used, followed by Bonferroni, Dunnett or multiple comparison tests to test for differences between groups. If the data were not normally distributed, the differences between groups were tested using 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 presented as mean + or + / − SEM.

[0258] Results: Figure 7A-7D showed that both capsid proteins led to extensive biodistribution in the cortex, hippocampus, cervical spinal cord, and liver.

[0259] Figures 8A - 8C Both capsid proteins led to TREM2 protein levels in the CSF, liver, and serum, with the AAV6TM capsid protein showing higher TREM2 protein levels in the CSF than the AAV9 capsid protein.

[0260] Figures 9A - 9C The rAAV vector encapsulated in the AAV6TM capsid protein significantly reduced amyloid-β in the hippocampus and cortex, while the rAAV vector encapsulated in the AAV9 capsid protein did not reduce amyloid-β in the hippocampus and cortex.

[0261] Figures 10A - 10B The rAAV vector reduced inflammatory markers in the cortex, independent of the capsid protein.

[0262] In summary, rAAV vector administration led to extensive biodistribution, and for both capsids, there was a dose-dependent increase in TREM2 protein levels; however, a significant reduction in cortical disease burden was only observed in mice treated with the AAV6TM capsid protein.

[0263] Example 7: In Vivo Studies of TREM2 rAAV in Rodents

[0264] Methods: Two different doses of either the excipient or one of the two TREM2 rAAVs from Example 3 were administered by ICV injection to 1-month-old WT C57BL / 6 mice (n = 10 / group). rAAV was injected at doses of 5.68×10 10 vg (1.42×10 11 vg / g brain) or 1.8×10 11 vg (4.49×10 11 vg / g brain). Based on an adult mouse brain weight of 400 mg, the number of vector particles per gram of brain weight was calculated.

[0265] CNS tissues and the liver were collected for biodistribution analysis by ddPCR. CSF, liver, and serum were collected for TREM2 expression analysis.

[0266] Biodistribution was determined by measuring the presence of vg using ddPCR (greater than 50 vg / 1 μg gDNA was defined as positive).

[0267] Results: Mice receiving rAAV were positive for vg in the cortex, indicating that ICV administration successfully produced similar transduction in the brain. Vector design had no significant effect on biodistribution (all p>0.05). ICV administration also resulted in the presence of vg in the spinal cord and liver.

[0268] TREM2 expression in CSF and serum was measured using MSD. ICV injection of rAAV at both doses increased TREM2 in the CSF. These data indicate that there was no difference in the level of TREM2 expression produced by rAAV, consistent with the biodistribution data. Additionally, ICV injection of rAAV at 1.8×10 11 vg significantly increased the level of TREM2 in the blood. When comparing the doses, a dose-dependent effect in the serum was observed.

[0269] Example 8: Evaluation of the in vivo efficacy of TREM2 rAAV in a CSF1R-inhibited mouse model of CRL

[0270] Methods: Two different doses of either the excipient or TREM2 rAAV from Example 3 (i.e., the rAAV vector of SEQ ID NO:26 encapsulated in the AAV6TM capsid protein (SEQ ID NO:19)) were administered by ICV injection to 1-month-old WT C57BL / 6 mice (n = 10). rAAV was injected at doses of 1.8×10 10 vg (4.49×10 10 vg / g brain) or 1.8×10 11 vg (4.49×10 11 vg / g brain). The number of vector particles per gram of brain weight was based on an adult mouse brain weight of 400 mg. At 8 weeks of age (i.e., 4 weeks after ICV injection), one excipient-treated group and two dose-treated groups were fed mouse chow containing PLX. Mice were fed PLX at 185 mg / kg in the chow for 1 week and then sacrificed at 9 weeks of age (i.e., 5 weeks after ICV injection).

[0271] Biodistribution was determined by measuring the presence of vg using ddPCR (greater than 50 vg / 1 μg gDNA was defined as positive).

[0272] Results: Mice receiving rAAV were vg-positive in all tested brain tissues, indicating that ICV administration successfully led to transduction in the brain. ICV administration also resulted in the presence of vg in the spinal cord, and lower levels of transduction were observed in the liver. The reduced biodistribution in the liver was reflected in lower levels of TREM2 in this tissue.

[0273] The inhibition of CSF1R by PLX in mice was evident at both the mRNA and protein levels. Compared to mice treated with vehicle + PLX, CSF1R expression was statistically significantly increased at the mRNA level in mice treated with two doses of rAAV + PLX. In the CSF of animals treated with two doses of rAAV + PLX, the level of the sCSF1R fragment showed a positive trend pattern towards the vehicle level. Thus, rAAV treatment ameliorated the negative effects of PLX in the CSF1R inhibition model.

[0274] In addition, administration of vehicle + PLX to mice led to almost complete depletion of microglia throughout the brain. In fact, the number of IBA1+ microglia was statistically significantly increased in mice treated with either dose of rAAV + PLX compared to mice treated with vehicle + PLX. This observation was further confirmed by analyzing the expression level of the gene AIF1 mRNA encoding IBA1 using qRT-PCR. In addition, the AIF1 mRNA levels were higher in mice treated with either dose + PLX compared to the vehicle + PLX treatment group, indicating significant therapeutic benefits of rAAV.

[0275] In addition, PLX treatment decreased the expression of genes associated with homeostatic microglia, such as P2RY12 and HEXB, which are known to be stably expressed in homeostatic microglia. Significantly higher expression of both P2RY12 and HEXB was observed in mice treated with rAAV + PLX.

[0276] Consistent with the PLX model effects observed in housekeeping and homeostatic genes, disease-associated microglia (DAM) genes (e.g., CCL12, CD48, CD68, LYZ1, and PTPRC) also showed a significant decrease in expression in the vehicle + PLX treatment group. In contrast, the expression of these genes was fully or, in some cases, partially increased by rAAV compared to animals treated with vehicle alone, and in most cases reached normal levels.

[0277] Example 9: Dose range of TREM2 rAAV in the CSF1R inhibitory mouse model of CRL

[0278] Method: Vehicle or the TREM2 rAAV of Example 3 (i.e., the rAAV vector of SEQ ID NO: 26 encapsulated in the AAV6TM capsid protein (SEQ ID NO: 19)) was administered by ICV injection to 4-week-old WT C57BL / 6 mice (n = 16 / group). The TREM2 rAAV was injected at the following 3 doses: 1.33×10 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). The number of vector particles per gram of brain weight was based on an adult mouse brain weight of 400 mg.

[0279] At 8 weeks of age (i.e., 4 weeks after ICV injection), one vehicle-treated group and 1.33×10 11 vg- and 2.44×10 9 -treated groups were treated with PLX3397. Mice were fed with PLX3397 at 185 mg / kg for 1 week and then sacrificed at 9 weeks of age (i.e., 5 weeks after ICV injection).

[0280] Biodistribution, TREM2 expression levels, microglial gene expression, and safety were evaluated by histopathology. Biodistribution was determined using ddPCR, which was developed according to the FDA guideline "Long-Term Follow-up After Administration of Human Gene Therapy Products" (2020; limit of quantification <50 copies / μg gDNA). TREM2 expression in CSF was measured using the MSD assay (using sTREM2 as a representative of tissue protein production as described above).

[0281] Results: All mice receiving TREM2 rAAV were vg-positive in all tested brain regions, indicating that ICV administration successfully led to widespread transduction in the brain. Additionally, compared to the brain, ICV administration of TREM2 rAAV resulted in reduced vector transduction in the liver.

[0282] ICV administration of TREM2 rAAV strongly increased TREM2 levels in CSF at all 3 doses. The biodistribution of TREM2 rAAV in peripheral tissues was low, which was reflected in lower TREM2 levels in the liver and lower TREM2 levels in serum. When comparing the 1.33×10 11 vg and 2.44×10 9 vg doses, a dose-dependent effect was observed in serum.

[0283] Among the 3 doses tested in this study, 2.44×10 9The vg dose did not show effective benefits (i.e., sub-therapeutic dose) in almost any microglial gene tested. Compared to mice treated with vehicle + PLX, the other two doses showed significant improvement in the microglial gene expression profile in TREM2 rAAV + PLX-treated mice, with a model effect in almost 90% of the genes.

[0284] In summary, 1.33×10 11 vg and 1.8×10 10 vg doses inhibited PLX-induced microglial damage in the brainstem of the CSF1R inhibitory mouse model. This effect was also observed in different regions of the brain including the hippocampus, cerebral cortex, and cerebellum.

[0285] Example 10: In Vivo Study of TREM2 rAAV in Non-Human Primates (NHP)

[0286] Method: An rAAV vector with the TREM2 transgene of SEQ ID NO:3 or vehicle was administered to female cynomolgus monkeys aged 2 - 4 years by ICM injection, and the transgene was encapsulated in the AAV6TM capsid protein (SEQ ID NO:19). One month after ICM injection, the vg, presence, and safety of TREM2 were evaluated.

[0287] Results: Figure 11 It was shown that the AAV6TM capsid protein led to widespread biodistribution in NHP.

[0288] Figures 12A - 12C It was shown that the AAV6TM capsid protein led to an increase in TREM2 protein levels in the liver, spinal cord, CSF, and serum.

[0289] In summary, in the NHP safety study, all animals survived, and in any group administered by single injection into the ICM, there were no changes related to the test sample in mortality, clinical signs, body weight / weight gain, body temperature, and neurological parameters. Examinations of general posture, behavior, motor function, proprioception, and postural responses revealed no findings. Clinical pathology showed minimal changes in hematology, coagulation, and clinical chemistry. Histopathological findings were limited and similar to those of the AAV9 capsid protein in previous NHP studies. At high doses, microscopic examination found mild to moderate infiltration of mononuclear cells in the brain and DRG.

[0290] Both AAV9 capsid protein and AAV6TM capsid protein transduce microglia with the rAAV vector encoding TREM2 in vitro; however, compared with AAV9 capsid protein, AAV6TM capsid protein drives higher TREM2 mRNA and protein expression. In vivo, both rAAVs show extensive biodistribution and a dose-dependent increase in TREM2 protein levels. Additionally, in rodent studies, AAV6TM capsid protein shows a biodistribution similar to that of AAV9 capsid protein in key brain regions while showing substantially less hepatotropism.

[0291] Example 11: NHP In Vivo Study of TREM2 rAAV

[0292] Methods: Vehicle or the TREM2 rAAV from Example 3 (rAAV vector of SEQ ID NO: 25 encapsulated in AAV6TM capsid protein (SEQ ID NO: 19)) or solvent was administered to female cynomolgus monkeys via ICM (n = 2 / group). TREM2 rAAV was injected at the following 2 doses: 3.32×10 12 vg (4.49×10 10 vg / g brain) or 1.05×10 13 vg (1.42×10 11 vg / g brain). NHPs were sacrificed at 29 days post-injection to detect potential early toxicity of TREM2 rAAV, with the expectation of capturing potential toxicity when the biodistribution in the whole brain and peripheral organs was expected to be near maximum.

[0293] Biodistribution, TREM2 expression levels, microglial gene expression, and safety were evaluated by histopathology. Biodistribution was determined using ddPCR as described in Example 9. TREM2 expression in CSF was measured using the MSD assay (using sTREM2 as a representative of protein production within tissues as described above).

[0294] Results: All NHPs survived until day 29. In either group that received a single injection of TREM2 rAAV at the 3.32×10 12 dose or 1.05×10 13 total vg dose into the ICM, no changes in mortality, clinical signs, body weight / weight gain, body temperature, or neurological signs related to the test article were observed.

[0295] Slight changes in hematological and coagulation parameters were observed in NHPs treated with the 1.05×10 13 vg dose. These changes included an increase in neutrophil counts on days 15 and 29 and an increase in fibrinogen levels on day 15 compared to baseline values, indicating a potential acute-phase response.

[0296] There were no macroscopic observations related to TREM2 rAAV. Microscopic observations related to the test article included mild to moderate infiltration of single-nucleated cells in the brains of all NHPs administered ≥ 3.32×10 12 vg of NHPS. The mild to moderate infiltration of single-nucleated cells was perivascularly distributed and also present in the PIA. The single-nucleated cells were mainly lymphocytes, and the distribution and severity were greater in animals receiving the 3.32×10 12 vg dose.

[0297] Considering that the only treatment-related findings were a slight increase in neutrophils and fibrinogen and mild to moderate infiltration of single-nucleated cells in the brain and ganglia, it was determined that ICM injection of TREM2 rAAV at any dose was well tolerated in NHPs.

[0298] Regarding biodistribution, all tested tissues were positive in NHPs administered TREM2 rAAV, indicating widespread distribution in the CNS and peripherally.

[0299] In the CSF, administration of 1.05×10 13 vg of TREM2 rAAV increased TREM2. In contrast, a dose-dependent effect on TREM2 was observed for the 2 doses in serum and liver.

[0300] In summary, the results showed widespread distribution throughout the brain, comparable to the effective levels shown in the mouse model. This transduction led to elevated TREM2 in the brain. Additionally, all NHPs survived, and autopsy pathology analysis showed a small increase in neutrophils and fibrinogen related to the test article, as well as mild to moderate infiltration of single-nucleated cells in the brain and ganglia. Therefore, TREM2 rAAV exhibited generally favorable safety characteristics in NHPs.

[0301] Example 12: In Vivo Study of TREM2 rAAV in NHPs

[0302] Method: An excipient or TREM2 rAAV of Example 3 (rAAV vector of SEQ ID NO: 26 encapsidated in the AAV6TM capsid protein (SEQ ID NO: 19)) or vehicle was administered to cynomolgus monkeys (male and female) (n = 6 / group) via ICM. TREM2 rAAV was injected at one of the following 3 doses: 3.32×10 12 vg (4.49×10 10 vg / g brain), 1.05×10 13 vg (1.42×10 11vg / g brain) or 3.32×10 13 vg (4.49×10 11 vg / g brain). After injection, the NHP will be followed up for 29 days or 26 weeks to evaluate tolerance and safety.

[0303] This study will also evaluate the peak vector distribution in the brain approximately 4 weeks after administration, as well as the post-peak assessment 183 days after administration. Control NHP will receive ICM administration of the same volume of formulated buffer excipient, which includes 20 mM Tris (pH 8.0), 1 mM MgCl 2 and 200 mM NaCl, containing 0.005% (w / v) poloxamer 188. All NHP will be screened for AAV6 neutralizing antibodies (NAb) with a titer cut-off of 1:20.

[0304] Sequence Listing

[0305] The following nucleic acid and / or amino acid sequences are mentioned in the above disclosure and are provided for reference as follows.

[0306] SEQ ID NO:1 – Human TREM2 protein isoform 1 (230aa)

[0307] MEPLRLLILLFVTELSGAHNTTVFQGVAGQSLQVSCPYDSMKHWGRRKAWCRQLGEKGPCQRVVSTHNLWLLSFLRRWNGSTAITDDTLGGTLTITLRNLQPHDAGLYQCQSLHGSEADTLRKVLVEVLADPLDHRDAGDLWFPGESESFEDAHVEHSISRSLLEGEIPFPPTSILLLLACIFLIKILAASALWAAAWHGQKPGTHPPSELDCGHDPGYQLQTLPGLRDT

[0308] SEQ ID NO:2 – Human TREM2 protein isoform 2 (219aa)

[0309] MEPLRLLILLFVTELSGAHNTTVFQGVAGQSLQVSCPYDSMKHWGRRKAWCRQLGEKGPCQRVVSTHNLWLLSFLRRWNGSTAITDDTLGGTLTITLRNLQPHDAGLYQCQSLHGSEADTLRKVLVEVLADPLDHRDAGDLWFPGESESFEDAHVEHSISRAERHVKEDDGRKSPGEVPPGTSPACILATWPPGLLVLLWQETTLPEHCFSWTLEAGTG

[0310] SEQ ID NO:3 - Artificial Sequence (TREM2 Transgene 1; 690 nt)

[0311] atggagcccctgcgcctgctgatcctgctgttcgtgaccgagctgagcggcgcccacaacaccaccgtgttccagggcgtggccggccagagcctgcaggtgagctgcccctacgacagcatgaagcactggggccgccgcaaggcctggtgccgccagctgggcgagaagggcccctgccagcgcgtggtgagcacccacaacctgtggctgctgagcttcctgcgccgctggaacggcagcaccgccatcaccgacgacaccctgggcggcaccctgaccatcaccctgcgcaacctgcagccccacgacgccggcctgtaccagtgccagagcctgcacggcagcgaggccgacaccctgcgcaaggtgctggtggaggtgctggccgaccccctggaccaccgcgacgccggcgacctgtggttccccggcgagagcgagagcttcgaggacgcccacgtggagcacagcatcagccgcagcctgctggagggcgagatccccttcccccccaccagcatcctgctgctgctggcctgcatcttcctgatcaagatcctggccgccagcgccctgtgggccgccgcctggcacggccagaagcccggcacccacccccccagcgagctggactgcggccacgaccccggctaccagctgcagaccctgcccggcctgcgcgacacc

[0312] SEQ ID NO:4 - Artificial sequence (TREM2 transgenic 2; 690 nt)

[0313] atggagcccctgaggctgctcatcctgctgtttgtgacagaactgtctggagcccacaacaccacagtgttccagggagttgctggccagtctctgcaagtgtcttgcccctatgacagcatgaagcactggggaaggaggaaggcttggtgtaggcagctgggagagaaaggaccttgccagagggtggtgagcacacacaacctgtggctgctgagcttcctcagaaggtggaatggctctacagccatcacagatgacaccctgggtggcaccctcaccatcaccttaaggaacctgcagcctcatgatgctggcctgtaccaatgccagagcctgcatggctctgaggctgataccctcaggaaggtgttggtggaggtgctggctgatcctctggatcacagggatgctggagacctgtggttcccaggagagtctgagagctttgaggatgcccatgtggagcacagcatcagcaggtctcttctggagggagagatccccttccctcccacaagcatcctgttgctgcttgcctgcatcttcctgatcaagatccttgctgcttctgctctttgggctgctgcctggcatggccagaaacctggaacacatcctccctctgaactggactgtggccatgaccctggctaccagttgcaaaccttgcctggcttgagggacacc

[0314] SEQ ID NO:5 - Artificial sequence (TREM2 transgenic 3; 690 nt)

[0315] atggagcccctgaggctgctgatcctgctgtttgtgacagaactgtctggagcccacaacaccacagtgttccagggagttgctggccagtctctgcaagtgtcttgcccctatgacagcatgaagcactggggaaggaggaaggcttggtgtaggcagctgggagagaaaggaccttgccagagggtggtgagcacacacaacctgtggctgctgagcttcctcagaaggtggaatggctctacagccatcacagatgacaccctgggtggcaccctcaccatcaccctgaggaacctgcagcctcatgatgctggcctgtaccaatgccagagcctgcatggctctgaggctgataccctcaggaaggtgttggtggaggtgctggctgatcctctggatcacagggatgctggagacctgtggttcccaggagagtctgagagctttgaggatgcccatgtggagcacagcatcagcaggtctcttctggagggagagatccccttccctcccacaagcatcctgttgctgcttgcctgcatcttcctcatcaagatccttgctgcttctgctctttgggctgctgcctggcatggccagaaacctggaacacatcctccctctgaactggactgtggccatgaccctggctaccagttgcaaaccttgcctggcttgagggacacc

[0316] SEQ ID NO:6 - Artificial Sequence (TREM 2 Transgene 4; 690 nt)

[0317] atggagcccctgagactgctcatcctgctgtttgtgacagaactgtctggggcccacaacaccacagtgttccagggggtggctggccagtccctccaggtgtcctgcccctatgactccatgaagcactgggggagaagaaaggcttggtgtagacagctgggggagaaagggccttgccagagagtggtgtccacacacaacctgtggctgctgtccttcctgagaagatggaatggctccacagccatcacagatgacaccctggggggcaccctcaccatcaccctgagaaacctgcagcctcatgatgctggcctgtaccagtgccagtccctgcatggctctgaggctgataccctgagaaaggtgctggtggaggtgctggctgatcctctggatcacagagatgctggggacctgtggttcccaggggagtctgagtcctttgaggatgcccatgtggagcactccatctccagatccctgctggagggggagatccccttccctcccacatccatcctgctgctgctggcctgcatcttcctcatcaagatcctggctgcttctgctctgtgggctgctgcctggcatggccagaaacctgggacacatcctccctctgaactggactgtggccatgaccctggctaccagctgcagaccctgcctggcctgagagacacc

[0318] SEQ ID NO:7 - Chicken β-actin promoter (283 nt)

[0319] catggtcgaggtgagccccacgttctgcttcactctccccatctcccccccctccccacccccaattttgtatttatttattttttaattattttgtgcagcgatgggggcggggggggggggggggcgcgcgccaggcggggcggggcggggcgaggggcggggcggggcgaggcggagaggtgcggcggcagccaatcagagcggcgcgctccgaaagtttccttttatggcgaggcggcggcggcggcggccctataaaaagcgaagcgcgcggcgggcg

[0320] SEQ ID NO:8 - CD68 promoter (715 nt)

[0321] gatatcaaactgcctgtttgggcttctcatttcttacctccccttccctctcccacctgctactgggtgcatctctgctccccccttccccagcagatggttacctttgggctgttgctttcttgtcaccatctgagttctcagacgctggaaagccatgttctcggctctgtgaatgacaatgctgactggagtgctgcccctctgtaaagggctgggtgtggatggtcacaagcccctcacatgcctcagccaagaggaagtagtacaggggtcagcccagaggtccaggggaaaggagtggaaaccgatttccccaccaagggaggggcctgtacctcagctgttcccatagcttacttgccacaactgccaagcaagtttcgctgagtttgacacatggatccctgtggatcaactgccctaggactccgtttgcacccatgtgacactgttgactttgccctgacgaagcagggccaacagtcccctaacttaattacaaaaactaatgactaagagagaggtggctagagctgaggcccctgagtcaggctgtgggtgggatcatctccagtacaggaagtgagactttcatttcctcctttccaagagagggctgagggagcagggttgagcaactggtgcagacagcctagctggactttgggtgaggcggttcagccatatcgaattctgctggggctactggcag

[0322] SEQ ID NO:9 – F4 / 80 promoter (2068 nt)

[0323]

[0324] SEQ ID NO: 10 - Cytomegalovirus enhancer (352 nt)

[0325] aatagtaatcaattacggggtcattagttcatagcccatatatggagttccgcgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgccaatagggactttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgaccttatgggactttcctacttggcagtacatctacgtattagtcatcgctattac

[0326] SEQ ID NO: 11 - Woodchuck hepatitis virus post - transcriptional regulatory element (594 nt)

[0327] ttatcgataatcaacctctggattacaaaatttgtgaaagattgactggtattcttaactatgttgctccttttacgctatgtggatacgctgctttaatgcctttgtatcatgctattgcttcccgtatggctttcattttctcctccttgtataaatcctggttgctgtctctttatgaggagttgtggcccgttgtcaggcaacgtggcgtggtgtgcactgtgtttgctgacgcaacccccactggttggggcattgccaccacctgtcagctcctttccgggactttcgctttccccctccctattgccacggcggaactcatcgccgcctgccttgcccgctgctggacaggggctcggctgttgggcactgacaattccgtggtgttgtcggggaaatcatcgtcctttccttggctgctcgcctgtgttgccacctggattctgcgcgggacgtccttctgctacgtcccttcggccctcaatccagcggaccttccttcccgcggcctgctgccggctctgcggcctcttccgcgtcttcgccttcgccctcagacgagtcggatctccctttgggccgcctccc

[0328] SEQ ID NO:12 - Bovine growth hormone polyadenylation signal tail (235 nt)

[0329] cgactagagctcgctgatcagcctcgactgtgccttctagttgccagccatctgttgtttgcccctcccccgtgccttccttgaccctggaaggtgccactcccactgtcctttcctaataaaatgaggaaattgcatcgcattgtctgagtaggtgtcattctattctggggggtggggtggggcaggacagcaagggggaggattgggaagacaatagcaggcatgctgggga

[0330] SEQ ID NO:13 - Wild - type AAV2 ITR (145 nt)

[0331] aggaacccctagtgatggagttggccactccctctctgcgcgctcgctcgctcactgaggccgggcgaccaaaggtcgcccgacgcccgggctttgcccgggcggcctcagtgagcgagcgagcgcgcagagagggagtggccaa

[0332] SEQ ID NO:14 - Modified AAV2 ITR (141 nt)

[0333] cctgcaggcagctgcgcgctcgctcgctcactgaggccgcccgggcaaagcccgggcgtcgggcgacctttggtcgcccggcctcagtgagcgagcgagcgcgcagagagggagtggccaactccatcactaggggttcct

[0334] SEQ ID NO:15 - Artificial sequence (TRY region; 60 nt)

[0335] agctctgggtatttaagcccgagtgagcacgcagggtctccattttgaagcgggaggtta

[0336] SEQ ID NO:16 - Artificial sequence (padding sequence 1; 1229 nt)

[0337]

[0338] SEQ ID NO: 17 - Artificial Sequence (Padding Sequence 2; 1229 nt)

[0339]

[0340] SEQ ID NO: 18 - Artificial sequence (padding sequence 3; 1254 nt)

[0341]

[0342] SEQ ID NO:19 - AAV6TM capsid protein (736aa)

[0343] MAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQQKQDDGRGLVLPGYKYLGPFNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLRYNHADAEFQERLQEDTSFGGNLGRAVFQAKKRVLEPFGLVEEGAKTAPGKKRPVEQSPQEPDSSSGIGKTGQQPAKKRLNFGQTGDSESVPDPQPLGEPPATPAAVGPTTMASGGGAPMADNNEGADGVGNASGNWHCDSTWLGDRVITTSTRTWALPTYNNHLYKQISSASTGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTTNDGVTTIANNLTSTVQVFSDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLNRTQNQSGSAQNKDLLFSRGSPAGMSVQPKNWLPGPCYRQQRVSKVKTDNNNSNFTWTGASKYNLNGRESIINPGTAMASHKDDKDKFFPMSGVMIFGKESAGASNTALDNVMITDEEEIKATNPVATERFGTVAVNLQSSSTDPATGDVHVMGALPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPPAEFSATKFASFITQYSTGQVSVEIEWELQKENSKRWNPEVQYTSNFAKSANVDFTVDNNGLYTEPRPIGTRFLTRPL

[0344] SEQ ID NO:20 - AVV9 capsid protein (736aa)

[0345] MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLGPGNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQPAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGVGSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTDNNGVKTIANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQAVGRSSFYCLEYFPSQMLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLSKTINGSGQNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWPGASSWALNGRNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADKVMITNEEEIKTTNPVATESYGQVATNHQSAQAQAQTGWVQNQGILPGMVWQDRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFNKDKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNVEFAVNTEGVYSEPRPIGTRYLTRNL

[0346] SEQ ID NO:21 - Artificial Sequence (Vector 1; 8305 nt)

[0347]

[0348] SEQ ID NO: 22 - Artificial Sequence (Vector 2; 8305 nt)

[0349]

[0350] SEQ ID NO: 23 - Artificial Sequence (Vector 3; 8305 nt)

[0351]

[0352] SEQ ID NO:24 - Artificial Sequence (Vector 4; 8305 nt)

[0353]

[0354] SEQ ID NO: 25 - Artificial Sequence (Vector 5; 2804 nt)

[0355]

[0356] SEQ ID NO: 26 - Artificial Sequence (Vector 6; 4048 nt)

[0357]

Claims

1. A recombinant adeno-associated virus (rAAV) comprising: (a) a nucleic acid comprising a nucleotide sequence of an expression construct, the expression construct comprising a microglia-specific promoter operably linked to a TREM2-encoding transgene, wherein the transgene comprises a nucleotide sequence selected from any one of SEQ ID NO: 3 to 6; and (b) a modified AAV6 capsid which, compared to the wild-type AAV6 capsid, comprises an amino acid sequence containing T492V, Y705F and Y731F mutations.

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

19.

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

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

8.

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

6. The rAAV according to claim 5, wherein the nucleotide sequence of the filler sequence is selected from SEQ ID NO: 16 to 18.

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

9. The rAAV according to 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, and wherein each ITR sequence flanks the expression construct.

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

11. The rAAV according to any one of claims 1 to 10, wherein the nucleic acid is a nucleotide sequence selected from SEQ ID NO: 21 to 26.

12. A nucleic acid comprising a nucleotide sequence of an expression construct, the expression construct comprising a microglia-specific promoter operably linked to a transgene encoding a human TREM2 protein, wherein the transgene comprises a nucleotide sequence selected from any one of SEQ ID NO: 3 to 6.

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

14. The nucleic acid according to 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 filler sequence.

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

17. The nucleic acid according to any one of claims 12 to 16, wherein the nucleic acid further comprises the nucleotide sequence of the 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 the bovine growth hormone polyadenylation signal tail (BGHpA).

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

20. The nucleic acid according to claim 19, wherein each ITR sequence is SEQ ID NO: 13 or 14 or its reverse complementary sequence.

21. A nucleic acid, which comprises the following nucleotide sequences in the 5' to 3' order: (a) 5' AAV ITR or its reverse complementary sequence; (b) A microglia-specific promoter; (c) A TREM2-encoding transgene, wherein the transgene comprises a nucleotide sequence selected from any one of SEQ ID NOs: 3 to 6; (d) A post-transcriptional regulatory element; (e) A polyadenylation signal tail; and (f) 3' AAV ITR or its reverse complementary sequence.

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

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

8.

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

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

26. The nucleic acid according to any one of claims 21 to 25, which further comprises the nucleotide sequence of a filler sequence, and wherein the nucleotide sequence of the filler sequence is selected from SEQ ID NOs: 16 to 18.

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

28. The nucleic acid according to claim 20, wherein the nucleotide sequence is selected from SEQ ID NOs: 21 to 26.

29. A vector, which comprises the nucleic acid according to any one of claims 12 to 28.

30. An rAAV, which comprises: the nucleic acid according to any one of claims 12 to 28 or the vector according to claim 29; and an AAV capsid.

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

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

19.

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

34. A method of treating a triggering receptor 2 (TREM2)-related disease or disorder expressed on myeloid cells in an individual, the method comprising the steps of: administering to the individual an effective amount of the rAAV according to any one of claims 1 to 11 or claims 30 to 32, the nucleic acid according to any one of claims 12 to 28, the vector according to claim 29, or the pharmaceutical composition according to claim 33.

35. The method according to claim 34, wherein the TREM2-related disease or disorder is selected from 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 deficit, memory loss, spinal cord injury, traumatic brain injury, and multiple sclerosis.

36. The method according to claim 34 or 35, wherein the administration is by intracisternal magna (ICM) injection.

37. The method according to claim 34 or 35, wherein the administration is by intravenous (IV) injection.

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

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

40. The rAAV, nucleic acid, or vector for use in the use according to claim 39, wherein the TREM2-related disease or disorder is selected from 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 deficit, memory loss, spinal cord injury, traumatic brain injury, and multiple sclerosis.

41. 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 disease or disorder associated with triggering receptor expressed on myeloid cells 2 (TREM2).

42. Use according to claim 31, wherein the TREM2-related disease or disorder is selected from 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 deficit, memory loss, spinal cord injury, traumatic brain injury, and multiple sclerosis.

43. A nucleic acid encoding triggering receptor expressed on myeloid cells 2 (TREM2), having a nucleotide sequence with at least about 95% sequence similarity to any one of SEQ ID NO: 3, 4, 5, or 6.

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