Microglia-selective gene expression vector

Abstract

In the present invention, a promoter on an AAV vector is changed to an Iba1 promoter, and a complementary sequence (miR-9T) of miR-9 and a complementary sequence (miR-129T) of miR-129 are combined. Since it is clearly demonstrated that using this vector makes it possible to express an exogenous gene effectively and specifically on microglia, an AAV vector is found which can express an exogenous gene effectively and specifically on microglia of the central nervous system.

Description

[Technical Field]

[0001] The present invention relates to adeno-associated viral vectors for selective gene expression in microglia. [Background technology]

[0002] In recent years, viral vector-based gene therapy has made rapid progress, demonstrating remarkable efficacy in treating several intractable diseases. Microglia, a type of glial cell present in the central nervous system, have the property of concentrating in neurodegenerative diseases and neuroinflammation, and are deeply involved in the pathological control of these diseases. It was thought that modifying the properties of microglia or expressing therapeutic genes in microglia would be useful in treating neurodegenerative diseases such as Alzheimer's and neuroinflammation such as multiple sclerosis.

[0003] To date, non-pathogenic and safe adeno-associated virus (AAV) vectors have been used in gene therapy for central nervous system diseases. However, the problem with AAV vectors is that they have extremely low affinity for microglia (Non-Patent Document 1), and no AAV vectors have been available to date that can express foreign genes efficiently and specifically in microglia.

[0004] In Non-Patent Document 2, a lentiviral vector expressing the target sequence (miR-9T) of microRNA-9 (miR-9) under the control of the PGK promoter is used to express a foreign gene in striatal microglia. However, the use of lentiviral vectors, which are HIV-derived vectors, is inferior in terms of safety because they have the property of integrating the viral genome into the genome of host cells. Furthermore, lentiviral vectors are relatively large (100 nm in diameter), limiting their spread within the brain parenchyma, resulting in insufficient gene transfer efficiency and specificity in the striatum.

[0005] On the other hand, Patent Document 1 suggests that the use of an ionized calcium-binding adaptor molecule 1 (Iba1) promoter sequence can be used to selectively express genes in microglia, but there are no reports on gene transfer into target cells using a viral vector or on microRNA target sequences. Furthermore, Patent Document 2 discloses an AAV vector containing a microRNA target sequence and cites the Iba1 promoter as an example of a promoter, but there is no suggestion of selectively expressing genes in microglia or using multiple microRNA target sequences. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] Re-table No. 2005-090561 [Patent Document 2] Patent No. 6422897 [Non-patent literature]

[0007] [Non-Patent Document 1] Molecular therapy methods & clinical development:2016;3;16026 [Non-patent document 2] Nature commun.:2013;4;1770 [Non-patent document 3] The Journal of Neuroscience:2013;33(12);5127-5137 [Non-patent document 4] Nature:2016;538;388-391 Summary of the Invention [Problem to be solved by the invention]

[0008] An objective of the present invention is to develop an AAV vector that can express foreign genes efficiently and specifically in microglia in the central nervous system. [Means for solving the problem]

[0009] The present inventors conducted extensive research to solve the above-mentioned problems. In their research, they first used an AAV vector, but were unable to express a target gene in microglia using the same genetic construct as the lentiviral vector described in Non-Patent Document 2. Therefore, they changed the promoter on the AAV vector to the Iba1 promoter and further combined it with the complementary sequence of miR-9 (miR-9T), or they changed the promoter on the AAV vector to the Iba1 promoter and further combined it with the complementary sequence of miR-9 (miR-9T) and the complementary sequence of miR-129 (miR-129T). They found that the use of these vectors enabled efficient and specific expression of foreign genes in microglia.

[0010] In light of this situation, we developed an adeno-associated virus vector for selective gene expression in microglia.

[0011] That is, the present invention is as follows. [1] An adeno-associated virus vector for gene expression in microglia, containing an expression unit containing the Iba1 (ionized calcium-binding adaptor molecule 1) promoter and a complementary sequence of miR-9. [2] The vector according to [1], wherein the expression unit further contains a complementary sequence of miR-129. [3] The vector according to [1] or [2], further comprising a gene of interest placed under the control of the Iba1 promoter. [4] The vector according to [3], wherein the target gene is a green fluorescent protein (GFP) gene. [5] The vector according to any one of [1] to [4], which contains multiple repeats of a complementary sequence of miR-9 and / or a complementary sequence of miR-129. [6] The vector according to any one of [1] to [5], further comprising a woodchuck hepatitis virus-derived post-transcriptional regulatory element (WPRE) gene. [7] The vector according to any one of [1] to [6], which comprises the base sequence shown in SEQ ID NO: 1 or a base sequence having 90% or more identity to the base sequence shown in SEQ ID NO: 1. [8] The vector according to any one of [2] to [6], which comprises the base sequence shown in SEQ ID NO: 2 or a base sequence having 90% or more identity to the base sequence shown in SEQ ID NO: 2. [9] The vector according to any one of [1] to [8], which is used in combination with a capsid gene expression vector.

[10] The vector according to any one of [1] to [9], which is used in combination with a gene expression vector that acts as a helper for adenovirus.

[11] A recombinant virus obtained from the vector according to any one of [1] to

[10] .

[12] A pharmaceutical comprising the vector according to any one of [1] to

[10] or the recombinant virus according to

[11] .

[13] A pharmaceutical for central nervous system diseases as described in

[12] .

[14] A method for introducing a gene into a cell, comprising the step of introducing a target gene into a target cell in vitro using the vector according to any one of [1] to

[10] .

[15] The method according to

[14] , wherein the target cells are central nervous system neurons.

[16] An isolated cell into which the vector according to any one of [1] to

[10] has been introduced. [Effects of the Invention]

[0012] We developed an AAV vector that can efficiently and specifically express foreign genes in microglia in the central nervous system. By injecting this AAV vector into the central nervous system, we were able to achieve microglia-specific expression of foreign genes. Therefore, the vector of the present invention is considered to be promising for basic research targeting microglia, preclinical trials of gene therapy, and gene therapy. [Brief explanation of the drawings]

[0013] [Figure 1] Selective transduction of microglia in the cerebral cortex, striatum, and cerebellar cortex using an AAV vector incorporating the Iba1 promoter and GFP gene (hereafter referred to as AAV.Iba1). AAV9 vectors were injected into the mouse cerebral cortex (0.5 μL), striatum (1 μL), and cerebellar cortex (10 μL). One week after injection, brain sections were immunostained for GFP and Iba1. (a) Schematic diagram of the AAV genome containing the Iba1 promoter and GFP. (b) Schematic diagram of the cerebral area where immunohistochemical staining was performed. (c-e) Photographs showing the transduction of numerous Iba1-negative non-microglial cells in the cerebral cortex, including the transduction of a small number of microglia (arrowheads). (d) and (e) are magnified photographs of the two boxed areas in (c), respectively. (f, j) Schematic diagrams showing the striatal and cerebellar areas where immunohistochemical staining was performed. Immunohistochemical staining photographs show robust and efficient transduction of microglia, including weak and low-density transduction of Iba1-negative non-microglial cells (arrows) in the striatum (g-i) and cerebellar cortex (k-m). Two boxed areas in (g, k) are enlarged. Scale bars are 100 μm (c, g, k) and 20 μm (d, e, h, i, l, m). (n) Graph showing the specificity of microglial transduction [percentage of Iba1+ cells among GFP+ cells] in three brain regions. The abbreviations in the figure are: Cbm: cerebellar cortex, Ctx: cerebral cortex, GFP: enhanced green fluorescent protein, GL: granule cell layer, Iba1: ionized calcium-binding adaptor molecule 1, ITR: inverted terminal repeat, ML: molecular layer, polyA: polyadenylation signal, Str: striatum, and WPRE: woodchuck hepatitis virus-derived post-transcriptional regulator. [Figure 2]Selective transduction of microglia in the cerebral cortex, striatum, and cerebellar cortex was achieved using an AAV vector incorporating the Iba1 promoter and the complementary sequence of miR-9 (miR-9T) (hereafter referred to as AAV.Iba1.miR-9.T). The combination of the Iba1 promoter and miR-9T partially detargets non-microglial cells in the cerebral cortex. Mouse brains were double immunostained for GFP and Iba1 one week after injection of the AAV vector. (a) Schematic diagram of an AAV construct expressing GFP and 4×miR-9.T driven by the Iba1 promoter. (b) Schematic diagram showing the areas of the cerebrum where immunohistochemical staining was performed. (c-e) Photographs of immunohistochemical staining of the cerebral cortex. (d) and (e) are magnified photographs of the two boxed areas in (c). In contrast to the weak immunolabeling of non-microglial cells, GFP expression was strong in microglia. (f, j) Schematic diagrams of the striatum and cerebellum where immunohistochemical staining was performed. Photographs of immunohistochemical staining in the striatum (g-i) and cerebellar cortex (k-m). Most GFP-expressing cells are Iba1+ microglia. Scale bars are 100 μm (c, g, k) and 20 μm (d, e, h, i, l, m). (n) Graph showing the specificity of microglial transduction [percentage of Iba1+ cells among GFP+ cells] in three brain regions. Abbreviations in the figure represent: Cbm: cerebellar cortex; Ctx: cerebral cortex; GFP: enhanced green fluorescent protein; GL: granule cell layer; Iba1: ionized calcium-binding adaptor molecule 1; ITR: inverted terminal repeat; ML: molecular layer; polyA: polyadenylation signal; Str: striatum; WPRE: woodchuck hepatitis virus-derived posttranscriptional regulator. [Figure 3]Highly efficient and specific microglial targeting was achieved using an AAV vector incorporating the Iba1 promoter, the complementary sequence of miR-9 (miR-9T), and the complementary sequence of miR-129 (miR-129T) (hereafter referred to as AAV.Iba1.miR-9.T.miR-129-2-3p.T). (a) Schematic diagram of an AAV construct containing a quadruplex of the Iba1 promoter, the fluorescent green pigment (GFP) gene, and the complementary sequences of miR-9 and miR-129-2-3p. (b) Schematic diagram of the cerebral area where immunohistochemical staining was performed. (c-e) Photographs of immunohistochemical staining of the cerebral cortex. (d) and (e) are enlarged photographs of the two boxed areas in (c). While weak GFP expression was observed in Iba1-negative non-microglial cells (arrows), strong and efficient GFP expression was observed in microglia. (f, j) Schematic diagrams showing the regions of the striatum and cerebellum where immunohistochemical staining was performed. Photographs of immunohistochemical staining showing highly efficient and specific GFP expression in microglia in the striatum (g-i) and cerebellar cortex (k-m). (h) and (i) are enlarged views of the two squared areas in (g), respectively, and (l) and (m) are enlarged views of the two squared areas in (k). Scale bars are 100 μm (c, g, k) and 20 μm (d, e, h, i, l, m). (n) Graph showing the efficiency of GFP expression in microglia in three brain regions one week after injection of the AAV9 vector in (a). The percentage of microglia (labeled with Iba1) among GFP-expressing cells is shown. The abbreviations in the figure are: cbm: cerebellar cortex, str: striatum, ctx: cerebral cortex, GFP: enhanced green fluorescent protein, GL: granule cell layer, Iba1: ionized calcium-binding adaptor molecule 1, ITR: inverted terminal repeat, ML: molecular layer, polyA: polyadenylation signal, WPRE: woodchuck hepatitis virus-derived post-transcriptional regulator. [Figure 4]Figure 1 shows the efficiency of AAV vector-mediated GFP expression in microglia in three separate brain regions (summarizing the results from Figures 1 to 4). Mice were injected into the cerebral cortex (a), striatum (b), or cerebellum (c) with AAV9 vectors expressing GFP alone or together with microRNAs under the control of the Iba1 promoter or the promoter of the housekeeping gene phosphoglycerate kinase (PGK), as indicated. (a, b) One-way ANOVA with Bonferroni post-hoc test; *: P = 0.003, **: P < 0.001. (c) Kruskal-Wallis test with Dunn's post-hoc test with Bonferroni adjustment for multiple comparisons; ***: P = 0.002. [Figure 5] Measurement of changes in Ca2+ concentration in cerebellar microglia following expression of the intracellular Ca2+ concentration indicator G-CaMP7.09. (a, b) Left panels are confocal images (photographs) of G-CaMP signals expressed using the AAV vector developed in this study in the granule cell layer of acute cerebellar slices. Regions of interest (ROIs 1–4) were set over the large compartment (microglial cell body or its large processes) shown in (a) and the small compartment (microglial fine processes) shown in (b). The right panels of (a) and (b) show changes in Ca2+ signals estimated from the fluorescence of the ROIs shown in the left panels. Perfusion of 100 μM ATP (shown by the black bar) into acute cerebellar slices induced a clear Ca2+ increase in both the cell body and processes of G-CaMP7.09-expressing microglia. (c) Boxplots showing the Ca2+ increase induced in microglia by ATP perfusion. ΔF / Fbasal is the percentage change in Ca2+ concentration increased by ATP administration relative to the basal intracellular Ca2+ concentration; open circles represent individual data points. The horizontal line and box represent the median and interquartile range, respectively. Error bars indicate one standard deviation above and below the mean (black circle). (d) Time-lapse fluorescence images (photograph) showing the movement of a microglial process (shown as an open circle) expressing G-CaMP7.09. Note that no changes in Ca2+ signals were observed in other G-CaMP-expressing microglial compartments outside the open circle. [Figure 6] Microglia can be transduced using the AAV vector developed in this study, even in mice with encephalitis caused by lipopolysaccharide (LPS) treatment. (a) Flowchart showing the experimental protocol. A solution containing AAV, Iba1, miR-9, miR-129-2-3p, and LPS (0.2 μg / μl) was injected into three brain regions on day 0, followed by daily intraperitoneal administration of LPS (1.0 μg / g body weight) until day 7. Mice were sacrificed 6 h after LPS injection on day 7, and acute brain slices were prepared and double immunolabeled for GFP and Iba1. (b-m) Low-, medium-, and high-magnification fluorescence micrographs of the cerebral cortex (b-e), striatum (f-i), and cerebellar cortex (j-m) are shown. The boxed areas in (b), (f), and (j) are enlarged in (c), (g), and (k), respectively. Scale bars: 500 μm (b, f, j), 200 μm (c, g, k), and 20 μm (d, e, h, i, l, m). Abbreviations in the figures are: GFP: enhanced green fluorescent protein (brighter than normal fluorescence), GL: granule cell layer, Iba1: ionized calcium-binding adaptor molecule 1, LPS: lipopolysaccharide, and ML: molecular layer. [Figure 7] These are fluorescence micrographs (photographs) showing significant transduction of microglia using the AAV vector developed in this study in the cerebellum of spinocerebellar ataxia type 1 (SCA1) transgenic mice (hereinafter referred to as SCA1-Tg mice). AAV.Iba1.miR-9.T.miR-129-2-3p.T expressing GFP was injected into the cerebellum of wild-type and SCA1-Tg mice at 24 weeks of age. One week after injection, cerebellar sections were prepared and analyzed by immunohistochemistry. (a-c) GFP immunofluorescence images of sagittal sections of the cerebellum from a wild-type (WT) mouse. (d-f) GFP immunofluorescence images of sagittal sections of the cerebellum from an SCA1-Tg (B05) mouse. The boxed areas in (a) and (d) are enlarged in (b, c) and (e, f), respectively. Scale bars: 500 μm (a, b) and 20 μm (b, c, e, f). DETAILED DESCRIPTION OF THE INVENTION

[0014] The present invention provides an adeno-associated virus vector for gene expression in microglia, which comprises an expression unit comprising the Iba1 promoter and a complementary sequence of miR-9. The expression unit may further comprise a complementary sequence of miR-129. The expression unit of an AAV vector has inverted terminal repeats (ITRs) at both ends, and can refer to the ITRs and the region sandwiched between them.

[0015] Those skilled in the art are aware that there are multiple serotypes of adeno-associated virus (AAV), but in the present invention, the serotype of AAV from which the AAV vector is derived is not particularly limited as long as it can express a gene of interest in microglia. That is, the portion other than the expression unit may be derived from any AAV, and for example, AAV of serotype 9, serotype 1, or serotype 6 can be used. Serotype 9 AAV (AAV9) is preferred.

[0016] The Iba1 promoter can be the promoter of the Iba1 gene of mammals such as humans, mice, and rats, and a region of approximately 500 bp, 1 kbp, 1.5 kbp, 2 kbp, or 3 kbp 5' from the transcription start site in each Iba1 gene can be used. The nucleotide sequence of the Iba1 promoter is not particularly limited, and for example, a promoter having the nucleotide sequence shown in SEQ ID NO: 3 or 4 can be used. Alternatively, as long as it can function as an Iba1 promoter, it may have only a portion of the nucleotide sequence shown in SEQ ID NO: 3 or 4, or it may have a sequence that is 90% or more identical to the nucleotide sequence shown in SEQ ID NO: 3 or 4, preferably 95% or more identical, and more preferably 98% or more identical. When the vector of the present invention is used for the purpose of treating humans, it is preferable to use a promoter having the nucleotide sequence shown in SEQ ID NO:4.

[0017] The AAV vector of the present invention can comprise a gene of interest placed under the control of, i.e., linked to, the Iba1 promoter. The type of target gene linked to the Iba1 promoter is not particularly limited, but may be a marker gene such as a GFP gene (including modified forms), a Cre recombinase gene, luciferase, chloramphenicol acetyltransferase, or lacZ; 2+ Examples include genes encoding proteins that emit fluorescence upon sensing the production of intracellular second messengers such as cyclic AMP, photoactivated proteins, and toxin genes.Furthermore, genes used in the treatment of central nervous system disorders may also be used. An example of a GFP gene is shown in SEQ ID NO:5.

[0018] Genes effective in treating central nervous system disorders can be selected appropriately depending on the type of disorder to be treated, and examples of such genes include nucleic acids encoding factors or elements capable of suppressing inflammation in the brain parenchyma in the case of multiple sclerosis. Other examples include nucleic acids encoding anti-inflammatory cytokines that attenuate brain damage, and nucleic acids encoding brain-derived neurotrophic factor (BDNF) or glial cell line-derived neurotrophic factor (GDNF).

[0019] The AAV vector of the present invention may have these target genes already placed under the control of the Iba1 promoter, or may have a multicloning site downstream of the Iba1 promoter for incorporating these target genes.

[0020] The vector of the present invention contains a complementary sequence of miR-9. It may also contain a complementary sequence of miR-129. These may be nucleotide sequences complementary to miR-9 and miR-129, respectively, which may be present in cells into which the vector of the present invention is introduced. The respective nucleotide sequences are not particularly limited, but for example, the complementary sequence of miR-9 is the nucleotide sequence represented by SEQ ID NO: 6, and the complementary sequence of miR-129 is the nucleotide sequence represented by SEQ ID NO: 7. Alternatively, each of these microRNAs may have a sequence that is 90% or more identical to the nucleotide sequence represented by SEQ ID NO: 6 or 7, preferably 95% or more identical, and more preferably 98% or more identical, as long as it is able to exert its function. The complementary sequences of the microRNA genes are used to degrade mRNA transcribed from the AAV vectors of the present invention in cells other than microglia. In other words, because miR-9 and miR-129 are both expressed in astrocytes and neurons, miR-9 and miR-129 bind to these complementary sequences in astrocytes and neurons, causing mRNA degradation. However, because microRNAs are not expressed in microglia, mRNA can be expressed only in microglia.

[0021] The vector of the present invention is a genetically modified vector that has the basic structure of an AAV vector, and therefore, like AAV vectors, it infects not only microglia but also neurons and astrocytes. The Iba1 promoter is highly selective for microglia and also has promoter activity in neurons and astrocytes, so by using the vectors of the present invention, miR-9T and miR-129-2-3pT are also transcribed in neurons and astrocytes. Importantly, miR-9 and miR-129-2-3p are naturally present in neurons and astrocytes, but these microRNAs (miR-9 and miR-129-2-3p) are almost completely absent in microglia. That is, even if a foreign gene (to which miR-9T and miR-129-2-3pT are attached) introduced by the vector of the present invention is transcribed in neurons or astrocytes, miR-9 and miR-129-2-3p originally present in neurons and astrocytes bind to miR-9T and miR-129-2-3pT contained in the mRNA transcribed from the AAV vector of the present invention, resulting in mRNA degradation. Meanwhile, because miR-9 and miR-129-2-3p are absent in microglia, the mRNA of the introduced foreign gene is not degraded, allowing the foreign gene of interest to be expressed.

[0022] The vector of the present invention may contain multiple repeats of the miR-9 complementary sequence and / or the miR-129 complementary sequence. The number of repeats of each complementary sequence is not particularly limited, but preferably refers to 2 to 6 repeats, more preferably 4 to 6 repeats, and particularly preferably 4 repeats. The number of repeats of the miR-9 complementary sequence and the number of repeats of the miR-129 complementary sequence may be the same or different. The miR-9 complementary sequence and the miR-129 complementary sequence may be arranged alternately.

[0023] The vector of the present invention may further comprise a woodchuck hepatitis virus-derived post-transcriptional regulatory element (WPRE). WPRE prevents poly(A) readthrough, promotes RNA processing and maturation, and enhances RNA nuclear export. It also acts on viral genome transcripts in packaging cells to promote vector packaging and increase viral titer. Furthermore, WPRE promotes mRNA maturation produced by the vector's internal promoter, thereby enhancing target gene expression in transfected target cells.

[0024] A vector may contain a selection marker. A "selection marker" refers to a genetic element that confers a selectable phenotype to a cell into which the selection marker has been introduced, and is generally a gene whose product confers resistance to a drug that inhibits cell growth or kills the cell. Specific examples include the Neo gene, Hyg gene, hisD gene, Gpt gene, and Ble gene. Drugs useful for selecting the presence of a selection marker include, for example, G418 for Neo, hygromycin for Hyg, histidinol for hisD, xanthine for Gpt, and bleomycin for Ble.

[0025] In the vectors of the present invention, the genes to be incorporated into the AAV vector as described above are not limited in terms of the order of the genes or the linking sequences between the genes, as long as they are functionally linked to each other; however, it is preferable that they are arranged in the order of promoter, gene of interest, and miR complementary sequence from the 5' side.

[0026] The vector of the present invention may contain other functional genes, such as enhancers and polyA.

[0027] The vector of the present invention is not particularly limited, and may be, for example, a vector comprising the nucleotide sequence represented by SEQ ID NO: 1 or 2. The nucleotide sequence represented by SEQ ID NO: 1 comprises four repeats of the Iba1 promoter, WPRE, and miR-9 complementary sequence. The nucleotide sequence represented by SEQ ID NO: 2 comprises four repeats of the Iba1 promoter, WPRE, miR-9 complementary sequence, and miR-129 complementary sequence. The vector of the present invention may be a vector consisting only of the nucleotide sequence represented by SEQ ID NO: 1 or 2, or may be a vector further comprising a nucleotide sequence other than the nucleotide sequence represented by SEQ ID NO: 1 or 2. Furthermore, as long as the promoter, gene of interest, and miR complementary sequence function, the vector may also comprise a nucleotide sequence that is 90% or more identical to the nucleotide sequence represented by SEQ ID NO: 1 or 2, preferably 95% or more identical, and more preferably 98% or more identical. The length of the expression unit is usually up to about 4.7 kbp, with the total AAV length being up to about 7.5 kbp.

[0028] The above-mentioned various genes can be incorporated into the AAV vector sequence by methods known to those skilled in the art, including, but not limited to, a method using restriction enzyme treatment.

[0029] As a gene transfer method, a method using a known AAV vector can be used. Specifically, the pAAV plasmid, pRC plasmid, and helper plasmid are introduced into packaging cells such as HEK293 cells to form viruses, and the resulting viruses are then used to infect the target brain cells, preferably glial cells, and more preferably microglial cells.

[0030] The vector of the present invention may be used in combination with a capsid expression vector to protect the target gene from degradation by a degradative enzyme. Examples of capsids include AAV9 capsids, AAV1 capsids, and AAV6 capsids. When the AAV vector used is an AAV9 vector, it is preferable to use an AAV9 capsid.

[0031] The vector of the present invention may be used in combination with a vector for expressing a gene that acts as a helper for adenovirus. The gene responsible for the helper function is not particularly limited as long as it is capable of carrying out the helper function of adenovirus, and examples thereof include E1A, E1B, E2A, E2B, E3, E4, VA, etc. One or more of these genes may be included. The helper function is not particularly limited as long as it is known to those skilled in the art, but for example, E1A has the function of enhancing transcriptional activity, E1B has the function of regulating apoptosis, and E2A and E2B have the function of DNA replication.

[0032] Another aspect of the present invention is a pharmaceutical comprising any of the above vectors or a recombinant virus obtained therefrom. Examples of pharmaceuticals include gene therapy drugs for central nervous system diseases and diseases that cause abnormalities in microglia. The drug is not particularly limited as long as it is a disease involving microglia, but is preferably a drug for central nervous system diseases, such as neurodegenerative diseases such as Alzheimer's disease and neuroinflammation such as multiple sclerosis.

[0033] The content of the active ingredient in the pharmaceutical is not particularly limited as long as a therapeutic effect is obtained, and can be determined appropriately depending on the type of disease, the severity of the disease, the age and weight of the patient, the expression efficiency, and the like.

[0034] The method and dosage of the pharmaceutical of the present invention are not particularly limited as long as a therapeutic effect is obtained, and can be determined appropriately depending on the type of disease, the severity of the disease, the age and weight of the patient, the expression efficiency, and the like.

[0035] The pharmaceutical of the present invention may contain other active ingredients, and for example, may be used in combination with a therapeutic drug for a disease of the central nervous system.

[0036] Another aspect of the present invention is a method for introducing any of the above vectors into target cells in vitro, although the present invention also encompasses a method for introducing any of the above vectors into target cells in vivo in a non-human animal.

[0037] The target cells into which the vector is introduced are preferably central nervous system cells, more preferably microglia, and particularly preferably cerebral cortical microglia.

[0038] The method for introducing the vector into the target cells is not particularly limited, but is preferably based on the infectivity that the AAV vector inherently possesses.

[0039] Another aspect of the present invention is an isolated cell transfected with any of the above vectors. The cell is preferably a brain cell, preferably a glial cell, more preferably a microglia, transfected with the above vector. [Example]

[0040] The present invention will be described in more detail below with reference to examples, but it goes without saying that the scope of the present invention is not limited to the examples.

[0041] <Method> Vector construction The target sequences were designed based on microRNA sequences obtained from the miRNA Registry (www.mirbase.org). To construct the AAV.PGK.miR-9.T vector, the corresponding sense 1-2 (S1-S2, SEQ ID NOs: 8-9) and antisense 1-2 (AS1-AS2, SEQ ID NOs: 10-11) oligonucleotides were annealed and inserted into the KpnI and BamHI restriction enzyme sites in the 3' UTR of the transgene expression cassette of the AAV.PGK vector. To construct the AAV.PGK.miR-9.T.miR-129-2-3p.T or AAV.PGK.miR-9.T.miR-136-5p.T vector, sense 1 to 4 (S1 to S4, SEQ ID NOs: 12 to 15 and 20 to 23) and antisense 1 to 4 (AS1 to AS4, SEQ ID NOs: 16 to 19 and 24 to 27) oligonucleotides were annealed and inserted into the KpnI and BamHI restriction enzyme sites in the 3'UTR of the GFP expression cassette of the AAV.PGK vector. The Iba1 promoter was a 1,678-bp genomic region upstream (5'-end) of the first ATG in exon 1 of the mouse Iba1 gene. The Iba1 promoter region was amplified from mouse brain cell-derived genomic DNA by two-step (nested) PCR. The first-step DNA amplification used Iba1-Nest-F (5'-CCTAGAGCCATCTTGTAAGG-3', SEQ ID NO: 28) and Iba1-Nest-R (5'-CGAGGAATTGCTGTTGAG-3', SEQ ID NO: 29). The second-step DNA amplification used Iba1-F (5'-ATGCTCTAGActcgagTACTATAGGATGCATCGTGAAAACC-3', SEQ ID NO: 30) and Iba1-R (5'-CATGGTGGCGaccggtGGCTCCTCAGACGCTGGTTG-3', SEQ ID NO: 31). AAV.Iba1.miR-9.T, AAV.Iba1.miR-9.T.miR-129-2-3p.T, or AAV.Iba1.miR-9.T.miR-136-5p.T was obtained by replacing the PGK promoter in the AAV vector constructed above with the Iba1 promoter: the PGK promoter in the corresponding AAV.PGK vector was replaced with a 1.7 kb mouse Iba1 promoter cloned at the XhoI and AgeI restriction enzyme sites.

[0042] AAV9 vector generation AAV9 vector particles were generated by cotransfecting HEK293T cells with three plasmids: pRC expression plasmid, pHelper (Stratagene, La Jolla, CA, USA), and pAAV9. Viral particles were purified by iodixanol continuous gradient centrifugation, following precipitation with ammonium sulfate or polyethylene glycol 8000, as previously described. The genomic titer of the purified AAV9 vector was determined by quantitative real-time PCR using THUNDERBIRD™ SYBR® qPCR Mix (Toyobo) with primers 5'-CTGTTGGGCACTGACAATTC-3' (SEQ ID NO: 32) and 5'-GAAGGGACGTAGCAGAAGGA-3' (SEQ ID NO: 33), targeting the WPRE sequence. The expression plasmid vector was used as a standard. The concentrated vector expression titer was 2.55 x 10 13 ~2.32×10 14 The range was vg / ml.

[0043] animal C57BL / 6J mice (4–5 weeks old) and SCA1 transgenic mice (28–29 weeks old) were used in this study. All animal procedures were performed in accordance with protocols approved by the Japanese Act on the Welfare and Care of Animals and the Guidelines for Proper Conduct of Animal Experiments issued by the Science Council of Japan.

[0044] Stereotactic injection of AAV9 vectors into the brain parenchyma Mice were anesthetized with an intraperitoneal injection of ketamine (100 mg / kg body weight) and xylazine (10 mg / kg body weight). The depth of anesthesia was monitored throughout the procedure by toe pinch reflex, and additional ketamine and xylazine were injected if necessary. A burr hole was made over the injection site to expose the brain. AAV vectors (AAV.PGK.miR-9.T, AAV.Iba1.miR-9.T, AAV.Iba1.miR-9.T.miR-129-2-3p.T, or AAV.Iba1.miR-9.T.miR-136-5p.T) were injected into the cerebral cortex, striatum, and cerebellum of mice. To reduce the volume of transplantation and increase precision, a 10 μl Hamilton syringe with a 33G needle was used for injection. The following stereotaxic coordinates were used for viral vector injection: cerebral cortex, AP -1.0 mm, ML +1.5 mm, DV +0.9 mm; striatum, AP -1.0 mm, ML +1.75 mm, DV +2.75 mm; cerebellum, AP +6.5 mm, ML 0 mm, DV 2.0 mm (all values ​​relative to bregma). AAV (titer: 1.4 × 10) injected into mouse brains. 13 The volume and infusion rate of the sera (vg / ml) were: 0.5 μl, 10 nl / min in the cerebral cortex; 1 μl, 20 nl / min in the striatum; and 10 μl, 200 nl / min in the cerebellum.

[0045] immunohistochemistry Mice were perfused intracardially with 4% paraformaldehyde in 0.1 M PBS (pH 7.4). The brains were then postfixed for 8 hours and transferred to 1x PBS. Using a microtome (VT1000S; Leica, Germany), the brains were cut into 50 μm-thick coronal slices (cortex and striatum) or 50 μm-thick sagittal slices (cerebellum). The slices were treated with a blocking solution (5% normal donkey serum, 0.5% Triton X-100, and 0.05% NaN3 in PBS) for 1 hour, followed by incubation overnight at 4°C with primary antibodies: rat anti-GFP (Nacalai Tesque) at 1:1,000 or rabbit anti-Iba1 (Wako Pure Chemical Industries) at 1:500. After washing three times with PBS for 15 min, the slices were incubated overnight at 4°C with the following Alexa Fluor 488- or Alexa Fluor 594-conjugated secondary antibodies (Thermo Fisher Scientific): Alexa Fluor 488 donkey anti-rat IgG 1:1,000, or Alexa Fluor 594 donkey anti-rabbit IgG 1:1,000. Finally, the slices were rinsed three times with PBS and mounted using ProLong Diamond Antifade Mountant (Thermo Fisher Scientific).

[0046] Confocal laser scanning microscope analysis The proportion of myeloid cells in the brain transduced by AAV vectors was investigated by immunohistochemistry. 50-μm-thick sections were prepared from the cerebral cortex, striatum, and cerebellum and double-immunostained with antibodies against GFP and Iba1. Thirty consecutive optical sections (total thickness: 15 μm) spaced at 0.5 μm intervals (field of view: 25, 513.67 μm) were prepared. 2 ) were imaged with a confocal laser scanning microscope (LSM800; Carl Zeiss, Oberkochen, Germany) using a 40× / 0.95 NA objective. Subsequently, maximum intensity projection (MIP) was performed using image processing software (ZEN blue). From each brain slice, 32 non-overlapping MIP images were captured (a total of 816,386.42 μm). 2) and counted the number of GFP-expressing myeloid cells. GFP-positive cells co-labeled with Iba1 were considered brain myeloid cells. Three brain slices per mouse were analyzed from five mice. At least 500 cells were counted.

[0047] Ca in G-CaMP-expressing microglia 2+ Measurement of kinetics and confocal microscopy analysis Confocal Ca analysis from acute cerebellar slices 2+ Parasagittal slices (200–250 μm thick) of the cerebellar vermis were prepared using a VibroSlicer (VT1200S; Leica, Germany) and placed in a solution containing 125 mM NaCl, 2.5 mM KCl, 2 mM CaCl, 1 mM MgCl, 1.25 mM NaHPO, 26 mM NaHCO, and 20 mM D-glucose (artificial cerebrospinal fluid; ACSF) bubbled with 95% O and 5% CO at room temperature for at least 1 h before recording. G-CaMP7.09 regulates intracellular Ca 2+ Recently developed Ca2+ ion channels, which increase their fluorescence with increasing concentrations, 2+ Ca2+ from G-CaMP7.09 expressing cells. 2+ To record the signal, confocal fluorescence images (exposure time 200–300 ms, 512 × 512 pixels, no binning) were acquired every 2 seconds using a high-speed spinning disk confocal unit (CSU-X1, Yokogawa Electric, Tokyo) equipped with a 40x water-immersion objective (LUMPLFLN 40XW, Olympus, Tokyo), a water-cooled CCD camera (iXon3 DU-897E-CS0-#BV-500, Andor, Belfast, Northern Ireland), and an upright microscope (BX51WI, Olympus, Tokyo). A 488 nm light beam from a diode laser module (Stradus488-50, VORTRAN, USA) was used for excitation, and the emitted fluorescence was collected through a bandpass (500–550 nm) filter. During recording, cerebellar slices were maintained at room temperature and perfused with ACSF solution. Intracellular Ca in microglia 2+To induce an increase, 100 μM ATP dissolved in ACSF was applied extracellularly. Image processing and analysis were performed using Andor iQ2 (Andor), NIH imageJ, Igor Pro8 (WaveMetrics), and custom programs. Because a single measurement took 10–20 min, image drift (translational drift) was occasionally observed. In these cases, image drift was corrected using the Image Stabilizer plugin for ImageJ (http: / / www.cs.cmu.edu / ~kangli / code / Image_Stabilizer.html) provided by K. Li. The fluorescence at each pixel at time t was first background-subtracted, and the intracellular Ca concentration was calculated. 2+ The relative increase in fluorescence due to the increase in F was calculated as ΔF / F 基底 It is measured by calculating where F 規定 is the basal fluorescence intensity averaged over the pre-stimulation frames (frames before ATP application), and ΔF = F t -F 規定 The background fluorescence was obtained from an area without cell structures in the same frame. ΔF / F in each region of interest (ROI) 基底 The mean value of γ was calculated for each frame. ROIs were set on cellular structures expressing G-CaMP. Because it was not possible to determine whether adjacent ROIs in a frame were located on the same cell or on different cells, each ROI was referred to as a microglial cell compartment. Microglial processes are usually within a few hundred micrometers, so in this study, we focused on the Ca 2+ Imaging data were collected from 12 different visual fields separated by more than 300 μm in five slices obtained from two mice. 2+ Imaging data were obtained from more than 12 microglia. ATP-induced Ca signaling in G-CaMP-expressing cells 2+ To quantify the signal, ΔF / F 基底 The peak amplitude of the ATP was measured in a time window of 120 seconds after the onset of ATP action.

[0048] statistical analysis Statistical analysis was performed using the nonparametric Kruskal-Wallis test followed by the Steel-Dwass test (SPSS 22.0J version 2.0.1). P < 0.05 was considered statistically significant.

[0049] Comparative Example 1: Targeting of microglia in the striatum and cerebellar cortex using an AAV9 vector incorporating the Iba1 promoter We have been able to target specific cell populations using AAV vectors containing cell type-specific promoters, such as the glial fibrillary acidic protein (GFAP) promoter (astrocytes) (PloS one 11, e0162023 (2016)), the neuronal-specific enolase (NSE) promoter (neurons) (Cerebellum (London, England) 16, 913-922 (2017)), and the L7-6 promoter (cerebellar Purkinje cells) (Molecular therapy. Methods & clinical development 6, 159-170 (2017)). Therefore, we hypothesized that by using a microglia-specific promoter, we might be able to create an AAV vector that expresses foreign genes specifically in microglia. We generated microglia-specific transgenic mice using a 1.9-kb Iba1 genomic region containing exon 1, intron 1, and part of exon 2 (Journal of neuroscience research 81, 357-362 (2005)), and investigated whether a similar region (a 1,678-base pair genomic region upstream of the first ATG of the Iba1 gene) could function as a microglia-specific promoter when incorporated into an AAV vector. An AAV9 vector expressing enhanced green fluorescent protein (GFP) driven by the Iba1 promoter (AAV.Iba1) (Figure 1a) was injected into the cerebral cortex, striatum, and cerebellar cortex (Figure 1b, f, j). Immunohistochemistry of the striatum and cerebellar cortex 1 week after injection revealed numerous transduced microglia (GFP and Iba1 double-positive cells) (Figure 1g-i and k-m), but also some Iba1-negative non-microglial cells (arrows in Figure 1h, i, l, m). In contrast, the majority of GFP-expressing cells in the cerebral cortex were Iba1-negative pyramidal neuron-like cells (Figure 1c-e). Quantitative analysis revealed that the ratios of GFP- and Iba1-double-positive cells (microglia) to GFP-positive cells were 2.1 ± 1.3% (cerebral cortex; 59 of 2,791 GFP-positive cells, n = 5 mice), 68.8 ± 9.6% (striatum; 1,808 of 2,628 GFP-positive cells, n = 5 mice), and 85.7 ± 5.5% (cerebellar cortex; 2,244 of 2,618 GFP-positive cells, n = 5 mice) (Fig. 1n). These results indicate that AAV.Iba1 can efficiently transduce resident microglia in the striatum and cerebellar cortex, but hardly transduce them in the cerebral cortex.

[0050] Comparative Example 2: Non-selective transduction with AAV.PGK.miR-9.T When a lentiviral vector expressing four copies of miR-9.T under the PGK promoter was injected into the striatum of adult rats, immunohistochemical analysis confirmed that approximately 75% of the transduced cells were microglia expressing Iba1 (Nature communications 4, 1770 (2013)). To verify whether similar results could be obtained using AAV vectors instead of lentiviral vectors, we engineered AAV9 vectors incorporating essentially the same transgene cassette as the lentiviral vector, consisting of the PGK promoter, GFP, and four complementary miR-9 sequences (AAV.PGK.miR-9.T). We then injected the AAV9 vectors into the cerebral cortex, striatum, and cerebellar cortex of adult mice. One week after injection, brain slices were prepared and double immunolabeled with antibodies against GFP and Iba1. We found GFP-expressing cells in all three regions examined, some of which were Iba1-positive. However, the majority of GFP-expressing cells were not co-immunolabeled with Iba1 and were not considered to be microglia. The ratios of Iba1-positive cells to GFP-positive cells in the cerebral cortex, striatum, and cerebellar cortex were 10.2 ± 3.7% (319 of 2,656 GFP-positive cells, n = 5 mice), 7.4 ± 3.4% (191 of 2,576 GFP-positive cells, n = 5 mice), and 34.4 ± 10.8% (901 of 2,620 GFP-positive cells, n = 5 mice), respectively (data not shown). These results demonstrate that selective targeting of microglia with AAV.PGK.miR-9.T is difficult.

[0051] Example 1: Combining the Iba1 promoter and miR-9.T sequence increases microglia targeting efficiency As shown in Figure 2a, four miR-9.T sequences were added downstream of the WPRE of the GFP-expressing AAV.Iba1. The viral vector (AAV.Iba1.miR-9.T) was injected into three brain regions, and the injected brains were similarly analyzed by immunohistochemistry. Clear GFP-expressing microglia were observed in all three brain regions (Figure 2b-m). However, in the cerebral cortex, although microglia-selective transduction was observed in regions distant from the viral injection site (Figure 2c, d), transduction of numerous Iba1-negative non-microglial cells characterized by large cell bodies was observed around the viral injection site (Figure 2c, e). Quantitative analysis revealed that the ratios of Iba1-positive cells to GFP-positive cells were 27.3 ± 2.4% (cerebral cortex; 753 of 2,760 GFP-positive cells, n = 5 mice), 94.0 ± 2.0% (striatum; 2,663 of 2,833 GFP-positive cells, n = 5 mice), and 100 ± 0% (cerebellar cortex; 2,726 of 2,726 GFP-positive cells, n = 5 mice) (Figure 2n). Thus, although the addition of four miR-9.T sequences to AAV.Iba1 (AAV.Iba1.miR-9.T) enabled exogenous gene expression in microglia in three brain regions, detargeting of non-microglial cells in the cerebral cortex was insufficient, resulting in the failure of microglia-selective transformation.

[0052] Example 2: Detargeting from neurons by adding miR-129-2-3p.T to AAV.Iba1.miR-9.T Although incorporating the Iba1 promoter and miR-9.T into AAV9 vectors was effective in targeting microglia, it was still insufficient to suppress transgene expression in non-microglial cells, particularly in the cerebral cortex. To promote the degradation of transgene mRNA in non-microglial cells, we incorporated an additional quadruplex targeting microRNA-129-2-3p (miR-129-2-3p.T) (Figure 3a) (AAV.Iba1.miR-9.T.miR-129-2-3p.T) or a microRNA-136-5p (miR-136-5p.T) (AAV.Iba1.miR-9.T.miR136-5p.T) (data not shown). Similar to miR-9, both miR-129-2-3p and miR-136-5p are reported to be abundant in neurons, whereas their expression in microglia is low (Non-Patent Document 3). Therefore, it is thought that transgene mRNA containing the complementary target sequence of miR-129-2-3p or miR-136-5p is degraded in neurons. Three different brain regions (cerebral cortex, striatum, and cerebellar cortex) were similarly injected with AAV.Iba1.miR-9.T.miR-129-2-3p.T or AAV.Iba1.miR-9.T.miR-136-5p.T and analyzed by immunohistochemistry one week later. Brain slices injected with AAV.Iba1.miR-9.T.miR-129-2-3p.T showed microglia-specific transduction in all three brain regions (Figures 3b-m). Quantitative analysis revealed that the ratios of Iba1-positive cells to GFP-positive cells were 86.5 ± 5.6% (cerebral cortex; 2750 cells out of 3180 GFP-positive cells, n = 5 mice), 99.6 ± 0.46% (striatum; 3127 cells out of 3140 GFP-positive cells, n = 5 mice), and 100.0% (cerebellar cortex; 2712 cells out of 2712 GFP-positive cells, n = 5 mice) (Fig. 3n). In contrast to the addition of miR-129-2-3p.T to AAV.Iba1.miR-9.T, the addition of miR-136-5p.T to AAV.Iba1.miR-9.T failed to detarget non-microglial cells in the cerebral cortex (data not shown). Therefore, as summarized in Figure 4, the addition of miR-129-2-3p.T to AAV.Iba1.miR-9.T (AAV.Iba1.miR-9.T.miR-129-2-3p.T) significantly improved non-microglial detargeting in the cerebral cortex compared with the use of AAV.PGK.miR-9.T, AAV.Iba1, and AAV.Iba1.miR-9.T, and achieved highly specific transduction of resident microglia in the cerebral cortex, striatum, and cerebellar cortex.

[0053] There are various types of microRNAs, and it has been known that the expression levels of microRNAs vary depending on the cell type. In particular, it has been known that in microglia, not only the expression levels of microRNA-9 (miR-9) and microRNA-129 (miR-129) but also the expression levels of miR-124, miR-127, miR-135, miR-136, miR-137, miR-153, miR-204, miR-325, miR-335, and miR-384 are low (Non-Patent Document 3). The present inventors have found that the combination of microRNA-9 (miR-9) and microRNA-129 (miR-129) is important for microglia-specific gene expression.

[0054] The vector of the present invention enables highly efficient and specific expression of foreign genes in microglia. Specifically, when the vector of the present invention was injected into the cerebellum or striatum, nearly 100% of the cells expressing the foreign gene were microglia, and when injected into the cerebral cortex, approximately 85% of the cells expressing the foreign gene were microglia.

[0055] Example 3 Live Ca2+ signaling by G-CaMP expressed in cerebellar microglia using the AAV vector of the present invention 2+Imaging> We investigated whether the microglia-selective gene expression method of the present invention is effective for the fluorescent calcium-sensitive molecule, G-CaMP7.09 (Non-Patent Document 4). Seven to 12 days after injection of AAV.Iba1.miR-9.T.miR-129-2-3p.T expressing G-CaMP7.09 into the cerebellum, transduced microglia were identified in the granule cell layer of acute cerebellar slices. When cerebellar slices were perfused with ATP (100 μM), calcium was detected not only in large intracellular compartments of G-CaMP-expressing microglia (Figure 5a, microglial cell bodies or larger microglial processes) but also in smaller compartments (Figure 5b, microglial microprocesses). 2+ ATP-induced Ca increase 2+ Signal change (ΔF / F 基底 The mean peak amplitude of the ATP-induced Ca signal (see Methods) was 3.36 ± 0.19 (Fig. 5c, subcellular compartments in five cerebellar slices from two mice, n = 91). These results demonstrate that microglia express purinergic receptors and can upregulate ATP-induced Ca signals on the order of seconds. 2+ Typical microglial Ca responses reported previously 2+ Furthermore, some G-CaMP-positive cells showed the movement of microglial processes (Fig. 5d), and some of them increased their motility after ATP application. These results are also consistent with the morphological and dynamic characteristics of microglia. From the above results, it can be seen that the use of the AAV vector of the present invention can enhance the Ca 2+ The sensitive molecule G-CaMP was expressed in microglia to measure intracellular Ca 2+ It was revealed that the dynamics of the gene expression can be monitored by live imaging. This suggests that the method for microglia-specific gene expression using AAV vectors of the present invention can also be applied to the expression of various functional genes.

[0056] Example 4: Transduction of reactive microglia by AAV.Iba1.miR-9.T.miR-129-2-3p.T in LPS-treated mice Although miR-9 is rarely produced in microglia and monocytes, treatment with lipopolysaccharide (LPS) has been reported to induce miR-9 production. Therefore, it is possible that transgene mRNA containing miR-9.T is degraded in microglia after LPS treatment. To verify that AAV.Iba1.miR-9.T.miR-129-2-3p.T is available in LPS-activated reactive microglia, we injected AAV.Iba1.miR-9.T.miR-129-2-3p.T together with LPS (0.2 μg / μl) into adult mice, followed by daily intraperitoneal injections of LPS (0.2 μg / g body weight) for one week (Figure 6a). The mice were then sacrificed for immunohistochemical staining to observe gene-expressing cells in the AAV vector-injected brain regions. We found strong GFP expression specifically in microglia in all three brain regions (Figure 6b-m). Transduced microglia in LPS-treated mice were more numerous and displayed a morphology characterized by shorter and thicker processes compared to mice without LPS treatment. These results demonstrate that AAV.Iba1.miR-9.T.miR-129-2-3p.T can also be used to target gene expression to reactive microglia.

[0057] Example 5: Transduction of microglia in the cerebellum of SCA1-Tg mice with AAV.Iba1.miR-9.T.miR-129-2-3p.T Microglia recognize specific structures of LPS via Toll-like receptor 4, leading to the release of pro-inflammatory cytokines. Microglia in neurodegenerative tissues are activated via scavenger receptors, which induce phagocytosis of apoptotic cell debris and the release of anti-inflammatory cytokines. Therefore, reactive microglia in neurodegenerative tissues may produce different microRNAs due to a different activation pattern than LPS-exposed microglia. Therefore, we investigated whether AAV.Iba1.miR-9.T.miR-129-2-3p.T could be used to transduce microglia in neurodegenerative tissues. As a neurodegenerative disease model, we selected spinocerebellar ataxia type 1 (SCA1) transgenic (SCA1-Tg) mice expressing ATXN1 with an abnormally expanded polyglutamine tract under the control of the Purkinje cell-specific L7 promoter. AAV.Iba1.miR-9.T.miR-129-2-3p.T was injected into the cerebellum of 24-week-old ataxic SCA1-Tg mice and their wild-type littermates. Three weeks after viral vector injection, cerebellar sections were prepared and immunolabeled with antibodies against GFP and Iba1. Confocal laser scanning microscopy revealed efficient and specific transduction of microglia in both SCA1-Tg mice and their wild-type littermates (Figure 7). Notably, the microglia population in SCA1-Tg mice was significantly larger than that in wild-type littermates. These results demonstrate that AAV.Iba1.miR-9.T.miR-129-2-3p.T is also useful for targeting gene expression specifically to microglia localized in neurodegenerative tissues.

Claims

1. An adeno-associated virus vector for gene expression in microglia, comprising an expression unit comprising the Iba1 (ionized calcium-binding adaptor molecule 1) promoter, a complementary sequence of miR-9, and a complementary sequence of miR-129.

2. The vector of claim 1 , further comprising a gene of interest placed under the control of the Iba1 promoter.

3. The vector according to claim 2 , wherein the target gene is a green fluorescent protein (GFP) gene.

4. The vector according to any one of claims 1 to 3, comprising multiple repeats of a complementary sequence of miR-9 and / or a complementary sequence of miR-129.

5. The vector according to any one of claims 1 to 4, further comprising a woodchuck hepatitis virus-derived post-transcriptional regulatory element (WPRE) gene.

6. The vector according to any one of claims 1 to 5, comprising a base sequence represented by SEQ ID NO: 1 or a base sequence having 90% or more identity to the base sequence represented by SEQ ID NO:

1.

7. The vector according to any one of claims 1 to 5, comprising a base sequence represented by SEQ ID NO: 2 or a base sequence having 90% or more identity to the base sequence represented by SEQ ID NO:

2.

8. The vector according to any one of claims 1 to 7, which is used in combination with a vector for expressing a capsid gene.

9. The vector according to any one of claims 1 to 8, which is used in combination with a gene expression vector that acts as a helper for adenovirus.

10. A recombinant virus obtained from the vector according to any one of claims 1 to 9.

11. A pharmaceutical comprising the vector according to any one of claims 1 to 9 or the recombinant virus according to claim 10.

12. A pharmaceutical composition according to claim 11 for treating a central nervous system disease.

13. A method for introducing a gene into a cell, comprising the step of introducing a target gene into a target cell in vitro using the vector according to any one of claims 1 to 9.

14. The method of claim 13, wherein the target cell is a central nervous system neuron.

15. An isolated cell into which the vector according to any one of claims 1 to 9 has been introduced.

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