Improved microglia-targeting AAV vector

Abstract

In the present invention, there is discovered an AAV vector that enables microglia-specific gene expression for a long period of time and can be used for the treatment of brain diseases. Specifically, it is found that a target gene can be expressed in a microglia-specific manner by using the following vector: an adeno-associated virus (AAV) vector for gene expression in a microglia, the vector including an expression unit that comprises an Iba1 (ionized calcium-binding adaptor molecule 1) promoter, a sequence complementary to miR-9, a sequence complementary to miR-129, and a Woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) gene, wherein the sequence complementary to miR-9 and the sequence complementary to miR-129 are located upstream of the WPRE gene, or the sequence complementary to miR-9 and the sequence complementary to miR-129 are located both upstream and downstream of the WPRE gene.

Description

Improved microglia-targeting AAV vector

[0001] The present invention relates to an adeno-associated virus vector that is highly specific to microglia and allows efficient gene expression in microglia.

[0002] Microglia, immune cells in the central nervous system, have recently been shown to be involved in memory and learning and the onset of neuropsychiatric disorders. Because microglia are cells that fight viruses that have invaded the brain, gene transfer using viral vectors has been extremely difficult.

[0003] In 2019, the present inventors succeeded in enabling selective gene expression in mouse microglia using an adeno-associated virus (AAV) vector (Patent Document 1). The AAV in Patent Document 1 is equipped with the promoter of the macrophage / microglia-specific molecule Iba1 (ionized calcium-binding adaptor molecule 1) and a complementary sequence of a microRNA (miR). The mouse Iba1 promoter used in Patent Document 1 also has a certain degree of promoter activity in neurons. Therefore, in Patent Document 1, to suppress gene expression in neurons, for example, four repeats of the complementary sequence of miR-9 (miR-9.T) and the complementary sequence of miR-129-2-3p (miR-129-2-3p.T), which are endogenously expressed in neurons but not in microglia, were inserted downstream of the woodchuck hepatitis virus-derived posttranscriptional regulator (WPRE) gene. The AAV vector injected into the brain induced mRNA transcription in both microglia and neurons, but in neurons, it bound to endogenously expressed miR-9 and miR-129-2-3p and was degraded. This enabled selective gene expression in microglia one week after AAV vector injection. However, three weeks after AAV vector injection, transgene expression was also observed in neurons. Such undesired gene expression in neurons was particularly evident in the cerebral cortex.

[0004] International Publication No. 2021 / 177116

[0005] An objective of the present invention is to provide an AAV vector that enables long-term microglia-specific gene expression and can be used to treat brain diseases.

[0006] In Patent Document 1, the present inventors disclosed an AAV vector for selective gene expression in microglia, comprising an Iba1 promoter, a complementary sequence of a miR, and a WPRE gene; however, Patent Document 1 does not specify the order in which the complementary sequence of a miR and the WPRE gene are arranged in the AAV vector, and the Examples only disclose vectors in which the complementary sequence of a miR is arranged downstream of the WPRE gene. The present inventors conducted extensive studies to solve the above-mentioned problems, and found that by arranging the miR upstream of WPRE or both upstream and downstream of WPRE in a microglia-specific AAV vector comprising an Iba1 promoter, a complementary sequence of a miR, and the WPRE gene, it is possible to improve the gene expression specificity for microglia and the gene expression efficiency compared to conventional AAV vectors, and thus completed the present invention.

[0007] That is, the present invention is as follows. [1] An adeno-associated virus (AAV) vector for gene expression in microglia, comprising an expression unit including an Iba1 (ionized calcium-binding adaptor molecule 1) promoter, a complementary sequence of miR-9, a complementary sequence of miR-129, and a woodchuck hepatitis virus-derived post-transcriptional regulator (WPRE) gene, wherein the complementary sequence of miR-9 and the complementary sequence of miR-129 are positioned upstream of the WPRE gene; or the complementary sequence of miR-9 and the complementary sequence of miR-129 are positioned both upstream and downstream of the WPRE gene. [2] The vector according to [1], further comprising a gene of interest positioned under the control of the Iba1 promoter. [3] The vector according to [2], wherein the gene of interest is a green fluorescent protein (GFP) gene. [4] The vector according to any one of [1] to [3], which contains multiple repeats of a complementary sequence of miR-9 and a complementary sequence of miR-129. [5] The vector according to any one of [1] to [4], which contains a nucleotide sequence represented by any one of SEQ ID NOs: 1 to 4, or a nucleotide sequence having 90% or more identity to a nucleotide sequence represented by any one of SEQ ID NOs: 1 to 4. [6] The vector according to any one of [1] to [5], which is used in combination with a capsid gene expression vector. [7] The vector according to any one of [1] to [6], which is used in combination with a gene expression vector that performs adenovirus helper function. [8] A recombinant virus obtained from the vector according to any one of [1] to [7]. [9] A pharmaceutical comprising the vector according to any one of [1] to [7], or a recombinant virus obtained from said vector.

[10] A pharmaceutical for a central nervous system disease according to [9].

[11] A method for introducing a gene into microglia, comprising the step of introducing a gene of interest into microglia in vitro using the vector according to any one of [1] to [7].

[12] Isolated microglia into which the vector according to any one of [1] to [7] has been introduced.

[13] A method for treating a central nervous system disease, comprising administering the vector according to any one of [1] to [7] or a recombinant virus obtained from the vector to a subject in need thereof.

[14] The vector according to any one of [1] to [7] or a recombinant virus obtained from the vector, for use in treating a central nervous system disease.

[15] Use of the vector according to any one of [1] to [7] or a recombinant virus obtained from the vector in the manufacture of a medicament for treating a central nervous system disease.

[0008] In a microglia-specific AAV vector containing the Iba1 promoter, a complementary sequence of a miR, and a WPRE gene, it was found that by placing the miR upstream of the WPRE or both upstream and downstream of the WPRE, the specificity of gene expression in microglia and the efficiency of gene expression were improved compared to when the miR was placed only behind the WPRE. It was found that microglia specificity was particularly excellent when the miR was placed on both sides of the WPRE. The vector of the present invention allows for the specific and efficient expression of a target protein in microglia.

[0009] Figures 1A-F show the results of selective transduction of microglia in the cerebral cortex using various AAV vectors incorporating the Iba1 promoter, miR complementary sequence, WPRE gene, and GFP gene. AAV9 vectors were injected into the M1 region of the cerebral cortex of C57BL / 6J mice (0.5 μL; 1.0E+12 vg / mL). Three weeks after injection, brain sections were immunostained for GFP and Iba1. Figure 1A shows a schematic diagram of the DNA constructs of various AAV vectors and the administration scheme to mice. Three patterns of miR complementary sequence and WPRE gene placement were examined. (1) the WPRE gene, a complementary sequence of miR-9, and a complementary sequence of miR-129-2-3p (the AAV vector having this arrangement is designated as WPRE-a,b); (2) the complementary sequence of miR-9, a complementary sequence of miR-129-2-3p, and the WPRE gene (the AAV vector having this arrangement is designated as a,b-WPRE); and (3) the complementary sequence of miR-9, a complementary sequence of miR-129-2-3p, the WPRE gene, a complementary sequence of miR-9, and a complementary sequence of miR-129-2-3p (the AAV vector having this arrangement is designated as a,b-WPRE-a,b). Figure 1B shows photographs of immunohistochemical staining (GFP and Iba1) of the cerebral cortex after injection of a,b-WPRE. The lower panel is an enlargement of the boxed area in the upper panel. Scale bars are 50 μm (upper panel) and 25 μm (lower panel). Solid arrows indicate microglia, and dashed arrows indicate neurons. Figure 1C shows photographs of immunohistochemical staining (GFP and Iba1) of the cerebral cortex after injection of a,b-WPRE-a,b. The lower panel is an enlargement of the boxed area in the upper panel. Scale bars are 50 μm (upper panel) and 25 μm (lower panel). Solid arrows indicate microglia. Figure 1D is a graph showing microglial specificity when various AAV vectors are used. Specificity is expressed as the percentage (%) of Iba1-positive cells among GFP-positive cells. In the figure, *** indicates p=0.0005, and **** indicates p<0.0001. Figure 1E is a graph showing the microglial expression efficiency when various AAV vectors are used. The expression efficiency is expressed as the percentage (%) of GFP-positive cells among Iba1-positive cells. In the figure, **** indicates p<0.0001, and n.s. indicates no significant difference.Figure 1F is a graph showing the gene expression intensity in microglia using various AAV vectors. The gene expression intensity in microglia is expressed as the total amount of GFP fluorescence intensity in the Iba1-positive region. In the figure, ns indicates no significant difference. Figures 2A-G show the results of selective microglia transduction in the striatum and cerebellum using various AAV vectors incorporating the Iba1 promoter, miR complementary sequence, WPRE gene, and GFP gene. AAV9 vectors were injected into the mouse striatum or cerebellum (1.0 μL and 10.0 μL, respectively; 1.0E+12 vg / mL). Three weeks after injection, brain sections were immunostained for GFP and Iba1. Figure 2A shows a schematic diagram of the DNA constructs of various AAV vectors and the administration scheme to mice. Two patterns of miR complementary sequence and WPRE gene placement were examined. (1) the WPRE gene, the complementary sequence of miR-9, and the complementary sequence of miR-129-2-3p (the AAV vector having this arrangement is designated as WPRE-a,b); (2) the complementary sequence of miR-9, the complementary sequence of miR-129-2-3p, the WPRE gene, the complementary sequence of miR-9, and the complementary sequence of miR-129-2-3p (the AAV vector having this arrangement is designated as a,b-WPRE-a,b). Figure 2B shows photographs of immunohistochemical staining (GFP and Iba1) of the striatum after injection of WPRE-a,b and a,b-WPRE-a,b. The upper right panel of each panel is an enlarged view of the boxed area in that panel. The scale bar is 50 μm (each panel) and 25 μm (enlarged view). Solid arrows indicate microglia, and dashed arrows indicate neurons. Figure 2C shows photographs of immunohistochemical staining (GFP and Iba1) of the cerebellum after injection of WPRE-a,b and a,b-WPRE-a,b. The upper right image of each panel is an enlargement of the boxed area in that panel. The scale bar is 50 μm (each panel) and 25 μm (enlarged image). Solid arrows indicate microglia. Figure 2D is a graph showing microglia specificity when various AAV vectors are used in the striatum. Specificity is expressed as the percentage (%) of Iba1-positive cells among GFP-positive cells. In the figure, ** indicates p=0.0018.Figure 2E is a graph showing the microglial expression efficiency when various AAV vectors are used in the striatum. Expression efficiency is expressed as the percentage (%) of GFP-positive cells among Iba1-positive cells. In the figure, n.s. indicates no significant difference. Figure 2F is a graph showing microglial specificity when various AAV vectors are used in the cerebellum. Specificity is expressed as the percentage (%) of Iba1-positive cells among GFP-positive cells. In the figure, n.s. indicates no significant difference. Figure 2G is a graph showing microglial expression efficiency when various AAV vectors are used in the cerebellum. Expression efficiency is expressed as the percentage (%) of GFP-positive cells among Iba1-positive cells. In the figure, n.s. indicates no significant difference. Figures 3A to 3F show the results of selective microglial transduction in the cerebral cortex using AAV vectors incorporating the Iba1 promoter, a miR complementary sequence, the WPRE gene, and the GFP gene. The miR complementary sequence and WPRE gene were arranged as follows: miR-9 complementary sequence, miR-129-2-3p complementary sequence, WPRE gene, miR-9 complementary sequence, and miR-129-2-3p complementary sequence (the AAV vector having this arrangement is designated a,b-WPRE-a,b). AAV9 vectors were injected into the mouse cerebral cortex (M1 region) (0.5 μL; 1.0E+12 vg / mL). Three weeks and two months after injection, brain sections were immunostained for GFP and Iba1. Figure 3A shows a schematic diagram of the AAV vector DNA construct and the administration scheme to mice. Figure 3B shows photographs of immunohistochemical staining (GFP and Iba1) of the cerebral cortex 3 weeks after injection of a,b-WPRE-a,b. The lower panel is an enlargement of the boxed area in the upper panel. Scale bars are 50 μm (upper panel) and 25 μm (lower panel). Solid arrows indicate microglia. Figure 3C shows photographs of immunohistochemical staining (GFP and Iba1) of the cerebral cortex 2 months after injection of a,b-WPRE-a,b. The lower panel is an enlargement of the boxed area in the upper panel. Scale bars are 50 μm (upper panel) and 25 μm (lower panel). Solid arrows indicate microglia, and dashed arrows indicate neurons. Figure 3D is a graph showing microglial specificity in the cerebral cortex 3 weeks and 2 months after injection of AAV vectors. Specificity is expressed as the percentage of Iba1-positive cells among GFP-positive cells.In the figure, * indicates p=0.0327. Figure 3E is a graph showing the cumulative percentage of microglia in the cerebral cortex 3 weeks and 2 months after injection of an AAV vector. The horizontal axis indicates the fluorescence intensity of the cell body, and the vertical axis indicates the cumulative percentage (%) of GFP-positive and Iba1-positive cells. Figure 3F is a graph showing the cumulative percentage of neurons in the cerebral cortex 3 weeks and 2 months after injection of an AAV vector. The horizontal axis indicates the fluorescence intensity of the cell body, and the vertical axis indicates the cumulative percentage (%) of GFP-positive and Iba1-negative cells. Figure 4 shows the results of selective microglia transduction in the cerebral cortex of marmosets using an AAV vector incorporating the Iba1 promoter, a complementary sequence of miR, the WPRE gene, and the GFP gene. The AAV vector used was a,b-WPRE-a,b. Photographs of immunohistochemical staining (GFP and Iba1) of the cerebral cortex 4 weeks after injection of an AAV vector. Solid arrows indicate microglia.

[0010] The present invention provides an adeno-associated virus (AAV) vector for gene expression in microglia, which comprises an expression unit including an Iba1 (ionized calcium-binding adaptor molecule 1) promoter, a complementary sequence of miR-9, a complementary sequence of miR-129, and a woodchuck hepatitis virus-derived post-transcriptional regulator (WPRE) gene, wherein the complementary sequence of miR-9 and the complementary sequence of miR-129 are located upstream of the WPRE gene; or the complementary sequence of miR-9 and the complementary sequence of miR-129 are located both upstream and downstream of the WPRE gene.

[0011] "Upstream" means a region before a gene (i.e., on the 5' side), and "downstream" means a region after a gene (i.e., on the 3' side). A gene and a gene located upstream or downstream thereof may be directly linked, or may be linked via one or more bases.

[0012] The expression unit of an AAV vector has inverted terminal repeats (ITRs) at both ends, and may refer to the ITRs and the region sandwiched between them. It may also have an SV40 polyA sequence, and the SV40 polyA sequence may be located upstream of the ITR at the downstream end.

[0013] Those skilled in the art are aware that there are multiple serotypes of adeno-associated virus (AAV). However, 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. For example, AAVs of serotype 9, serotype 1, serotype 6, etc. can be used, and AAV of serotype 9 (AAV9) is preferred. Capsid mutants can also be used. For example, they may be combined with a blood-brain barrier-penetrating capsid mutant or a capsid mutant that is tropic to microglia.

[0014] The Iba1 promoter can be a promoter of the Iba1 gene of a mammal, such as a human, mouse, or rat. In each Iba1 gene, a region of approximately 500 bp, 1 kbp, 1.5 kbp, 2 kbp, or 3 kbp 5' from the transcription start site can be used. The nucleotide sequence of the Iba1 promoter is not particularly limited, and a promoter having the nucleotide sequence represented by SEQ ID NO: 5 or 6 can be used, for example. Alternatively, as long as it can function as an Iba1 promoter, it may have only a portion of the nucleotide sequence represented by SEQ ID NO: 5 or 6, or it may have a sequence that is 90% or more identical to the nucleotide sequence represented by SEQ ID NO: 5 or 6, 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 represented by SEQ ID NO: 6.

[0015] The woodchuck hepatitis virus-derived post-transcriptional regulatory element (WPRE) prevents poly(A) read-through, promotes RNA processing and maturation, and enhances RNA export from the nucleus. WPRE also acts on viral genome transcripts in packaging cells to promote vector packaging and increase viral titer. Furthermore, WPRE promotes the maturation of mRNA produced by the vector's internal promoter, thereby enhancing expression of the target gene in transfected target cells. The nucleotide sequence of WPRE is not particularly limited, but for example, a nucleotide sequence represented by SEQ ID NO: 7 can be used.

[0016] The AAV vector of the present invention can contain a gene of interest placed under the control of the Iba1 promoter, i.e., linked to the Iba1 promoter. The type of gene of interest is not particularly limited, and examples thereof include a green fluorescent protein (GFP) gene (including modified forms), a Cre recombinase gene, a marker gene such as luciferase, chloramphenicol acetyltransferase, or lacZ, a Ca ２＋ Examples of genes that can be used include genes encoding proteins that sense the production of intracellular second messengers such as cyclic AMP and emit fluorescence, as well as genes encoding photoactivated proteins and toxins. The GFP gene can be, for example, one having the base sequence set forth in SEQ ID NO: 8. The gene of interest may also be a gene used in the treatment of central neuropathic diseases. Genes with therapeutic effects on central neuropathic diseases can be appropriately selected depending on the type of disease to be treated. For example, in the case of multiple sclerosis, nucleic acids encoding factors or elements capable of suppressing inflammation in the brain parenchyma can be used. 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) can also be used. In the AAV vector of the present invention, these genes of interest may be pre-placed under the control of the Iba1 promoter, or a multicloning site for incorporating these genes of interest may be placed downstream of the Iba1 promoter.

[0017] The vectors of the present invention contain a complementary sequence of miR-9 and a complementary sequence of miR-129. These may be any nucleotide sequences complementary to miR-9 and miR-129 present in cells into which the vectors of the present invention are introduced, and the respective nucleotide sequences are not particularly limited. For example, the complementary sequence of miR-9 is the nucleotide sequence represented by SEQ ID NO: 9, and the complementary sequence of miR-129 is the nucleotide sequence represented by SEQ ID NO: 10. Alternatively, each of these microRNAs may have a sequence that is 90% or more identical to the nucleotide sequence represented by SEQ ID NO: 9 or 10, 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.

[0018] The vectors of the present invention are genetically modified vectors based on the AAV vector structure, and thus, like AAV vectors, they infect not only microglia but also neurons and astrocytes. The Iba1 promoter is a promoter highly selective for microglia and also has promoter activity in neurons and astrocytes. Therefore, by using the vectors of the present invention, miR-9.T and miR-129-2-3p.T are also transcribed in neurons and astrocytes. It is important to note that, while miR-9 and miR-129-2-3p are endogenously present in neurons and astrocytes, neither of these microRNAs (miR-9 nor miR-129-2-3p) is present in microglia. That is, even if a foreign gene (to which miR-9.T and miR-129-2-3p.T are attached) introduced by the vector of the present invention is transcribed in neurons or astrocytes, miR-9 and miR-129-2-3p present endogenously in neurons and astrocytes bind to miR-9.T and miR-129-2-3p.T contained in the mRNA transcribed from the AAV vector of the present invention, resulting in mRNA degradation. On the other hand, because miR-9 and miR-129-2-3p are not present in microglia, the mRNA of the introduced foreign gene is not degraded, allowing the foreign gene of interest to be expressed.

[0019] The vector of the present invention may contain the complementary sequence of miR-9 and the complementary sequence of miR-129 only once, or may contain multiple repeats of each. The term "multiple repeats" is not particularly limited, and may refer to, for example, two to eight repeats of each, preferably two to six repeats of each, and more preferably four repeats of each. 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. Alternatively, the complementary sequence of miR-9 may be arranged after the complementary sequence of miR-129, or the complementary sequence of miR-9 may be arranged after the complementary sequence of miR-129. For example, the complementary sequence of miR-9 may be repeated four times, followed by four repeats of the complementary sequence of miR-129.

[0020] In the vector of the present invention, the complementary sequence of miR-9 and the complementary sequence of miR-129 may be placed upstream of the WPRE gene, or the complementary sequence of miR-9 and the complementary sequence of miR-129 may be placed both upstream and downstream of the WPRE gene.

[0021] The order of the genes to be incorporated into the AAV vector of the present invention and the binding sequences between the genes are not limited, as long as they are functionally linked to each other. For example, the vector may contain a sequence consisting of, from the 5' end, the Iba1 promoter, the gene of interest, a complementary sequence of miR-9 (4 repeats), a complementary sequence of miR-129 (4 repeats), and the WPRE gene; or the vector may contain a sequence consisting of, from the 5' end, the Iba1 promoter, the gene of interest, a complementary sequence of miR-9 (4 repeats), a complementary sequence of miR-129 (4 repeats), the WPRE gene, a complementary sequence of miR-9 (4 repeats), and a complementary sequence of miR-129 (4 repeats).

[0022] The vector of the present invention may contain a selection marker. A selection marker is a genetic element that provides a selectable phenotype to a cell into which the selection marker has been introduced, and is generally a gene whose product confers resistance to drugs that inhibit cell growth or kill 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.

[0023] The vector of the present invention may also contain other functional genes, such as enhancers.

[0024] The vector of the present invention is not particularly limited, and may be, for example, a vector comprising a nucleotide sequence represented by any one of SEQ ID NOs: 1 to 4. The nucleotide sequences represented by SEQ ID NOs: 1 and 2 comprise, in order from the 5' end, an Iba1 promoter, a gene of interest, a complementary sequence of miR-9 (four repeats), a complementary sequence of miR-129 (four repeats), and a WPRE gene. The nucleotide sequence represented by SEQ ID NO: 1 has two recognition sequences for the restriction enzyme Not-I, and the nucleotide sequence represented by SEQ ID NO: 2 has one recognition sequence for the restriction enzyme Not-I. Furthermore, the nucleotide sequences represented by SEQ ID NOs: 3 and 4 comprise, in order from the 5' end, an Iba1 promoter, a gene of interest, a complementary sequence of miR-9 (four repeats), a complementary sequence of miR-129 (four repeats), a WPRE gene, a complementary sequence of miR-9 (four repeats), and a complementary sequence of miR-129 (four repeats). The nucleotide sequence represented by SEQ ID NO: 3 is a sequence having two recognition sequences for the restriction enzyme Not-I, and the nucleotide sequence represented by SEQ ID NO: 4 is a sequence having one recognition sequence for the restriction enzyme Not-I. The vector of the present invention may be a vector consisting solely of a nucleotide sequence represented by any one of SEQ ID NOs: 1 to 4, or may be a vector further comprising a nucleotide sequence other than a nucleotide sequence represented by any one of SEQ ID NOs: 1 to 4. Furthermore, as long as the promoter, gene of interest, and miR complementary sequence function, the vector may also comprise a nucleotide sequence having 90% or more identity to a nucleotide sequence represented by any one of SEQ ID NOs: 1 to 4, preferably 95% or more identity, and more preferably 98% or more identity. The length of the expression unit is typically up to about 4.7 kbp, and up to about 7.5 kbp for the entire AAV.

[0025] 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.

[0026] A known method using an AAV vector can be used as a gene transfer method. Specifically, a pAAV plasmid, a pRC plasmid, and a helper plasmid are introduced into packaging cells such as HEK293 cells to form a virus, and the resulting virus is then used to infect target brain cells, preferably glial cells, and more preferably microglial cells.

[0027] The vector of the present invention may be used in combination with a vector for capsid expression. Examples of the capsid include AAV9 capsid, AAV1 capsid, and AAV6 capsid. For example, pAAV derived from serotype 2 may be used to produce an AAV9 capsid.

[0028] The vector of the present invention may be used in combination with a vector for expressing a gene that has the helper function of adenovirus. The gene that has the helper function is not particularly limited as long as it has the helper function of adenovirus, and examples thereof include E1A, E1B, E2A, E2B, E3, E4, and VA. 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, and examples thereof include E1A, which has the function of enhancing transcriptional activity, E1B, which has the function of regulating apoptosis, and E2A and E2B, which have the function of DNA replication.

[0029] Another aspect of the present invention is a recombinant virus obtained from any of the above vectors.

[0030] Another aspect of the present invention is a pharmaceutical comprising any of the above vectors or a recombinant virus obtained therefrom. The pharmaceutical is, for example, a gene therapy drug for central nervous system diseases or diseases causing abnormalities in microglia. This pharmaceutical allows for specific and efficient introduction of a target gene into microglia in the cerebral cortex, striatum, cerebellum, etc. The disease to be treated is a central nervous system disease, and is not particularly limited as long as it involves microglia. Examples of central nervous system diseases include neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, and spinocerebellar ataxia; neuroinflammation such as multiple sclerosis; and psychiatric diseases.

[0031] The content of the active ingredient in the pharmaceutical, the method of use and dosage of the pharmaceutical, etc. 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, etc.

[0032] The pharmaceutical of the present invention may contain other active ingredients, and for example, may be used in combination with other therapeutic agents for diseases of the central nervous system.

[0033] Another aspect of the present invention is a method for introducing any of the above vectors into microglia in vitro. However, the present invention also encompasses a method for introducing any of the above vectors into microglia in vivo in non-human animals. Microglia may be derived from the cerebral cortex, striatum, or cerebellum. The method for introducing a vector into microglia is not particularly limited, but is preferably based on the infectivity inherent in AAV vectors.

[0034] Another aspect of the present invention is isolated microglia introduced with any of the above vectors.

[0035] Other embodiments of the present invention can include, for example, the following: A method for treating a central nervous system disease, comprising administering any of the vectors described above or a recombinant virus obtained from the vector to a subject in need thereof. Any of the vectors described above or a recombinant virus obtained from the vector, for use in treating a central nervous system disease. Use of any of the vectors described above or a recombinant virus obtained from the vector in the manufacture of a medicament for treating a central nervous system disease.

[0036] 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.

[0037] <Vector Construction> The vector of the present invention was constructed using a method similar to the vector construction method described in Patent Document 1. The sequence was designed based on microRNA sequences obtained from the miRNA Registry (www.mirbase.org). To construct the vector, sense 1-4 (S1-S4, SEQ ID NOs: 11-14) and antisense 1-4 (AS1-AS4, SEQ ID NOs: 15-18) oligonucleotides were annealed and inserted into the restriction enzyme site of the gene expression cassette of the AAV.PGK vector using a restriction enzyme treatment method known to those skilled in the art. The Iba1 promoter was a 1,678-bp genomic region upstream (5'-side) of the first ATG in exon 1 of the mouse Iba1 gene. The Iba1 promoter region was amplified from genomic DNA derived from mouse brain cells by two-step (nested) PCR. Iba1-nest-F (5'-CCTAGAGCCATCTTGTAAGG-3', SEQ ID NO:19) and Iba1-nest-R (5'-CGAGGAATTGCTGTTGAG-3', SEQ ID NO:20) were used for the first-stage DNA amplification, and Iba1-F (5'-ATGCTCTAGAActcgagTACTATAGGATGCATCGTGAAAACC-3', SEQ ID NO:21) and Iba1-R (5'-CATGGTGGCGaccggtGGCTCCTCAGACGCTGGTTG-3', SEQ ID NO:22) were used for the second-stage DNA amplification. The vector containing the Iba1 promoter of the present invention was obtained by replacing the PGK promoter in the AAV.PGK vector constructed above with the Iba1 promoter. Briefly, 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.

[0038] AAV9 vector production: AAV9 vector particles were produced by co-transfecting HEK293T cells with three plasmids: pRC expression plasmid, pHelper (Stratagene, La Jolla, CA, USA), and pAAV9. Viral particles were purified by precipitation with ammonium sulfate or polyethylene glycol 8000 and iodixanol continuous gradient centrifugation. The genomic titer of the purified AAV9 vector was determined by quantitative real-time PCR using PowerSYBR® Green PCR Master Mix (Thermo Fisher Scientific) with primers 5′-CTGTTGGGCACTGACAATTC-3′ (SEQ ID NO: 23) and 5′-GAAGGGACGTAGCAGAAGGA-3′ (SEQ ID NO: 24) targeting the WPRE sequence.

[0039] <Animals> C57BL / 6J mice (Examples 1-3) and marmosets (Example 4) 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.

[0040] Stereotactic injection of AAV9 vector into brain parenchyma In Examples 1 to 3, mice were anesthetized by intraperitoneal injection of ketamine (100 mg / kg body weight) and xylazine (10 mg / kg body weight). The depth of anesthesia was monitored throughout the surgery 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. The AAV vector of the present invention was injected into the cerebral cortex, striatum, and cerebellum of the mice. To reduce the injection volume and increase the accuracy of the transplant, 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 +1.0 mm, DV +0.8 mm; striatum, AP -1.0 mm, ML +1.75 mm, DV +2.75 mm; cerebellum, AP +6.5 mm, ML 0 mm, DV +1.8 mm (all values ​​are relative to bregma). The volume and injection rate of AAV (titer: 1.0E+12 vg / mL) injected into the mouse brain were: cerebral cortex, 0.5 μL, 25 nL / min; striatum, 1 μL, 50 nL / min; and cerebellum, 10 μL, 500 nL / min. The stereotaxic injection into the marmoset cerebral cortex in Example 4 was also performed using a similar method. Note that the marmoset cerebral cortex was injected with a titer of 5×10 １２ 1 μL (5 × 10 ９ vg) was administered.

[0041] <Immunohistochemistry> In Examples 1 to 3, mice were perfused intracardially with 4% paraformaldehyde in 0.1 M PBS (pH 7.4), and the extracted brains were further fixed overnight and transferred to 1x PBS solution. Using a microtome (VT1000S; Leica, Germany), the brains were cut into 50 μm-thick coronal slices (cerebral cortex and striatum) or 50 μm-thick sagittal slices (cerebellum). The slices were then blocked with a blocking solution (PBS containing 5% normal donkey serum, 0.5% Triton X-100, and 0.05% NaN ３The slices were incubated overnight at 4°C with primary antibodies: rat anti-GFP (Nacalai Tesque) 1:1,000 and rabbit anti-Iba1 (Wako Pure Chemical Industries) 1:500, mixed with blocking solution. After washing five times with PBS, the slices were immersed in PBS and shaken for 15 minutes. Subsequently, the slices were incubated for 3 hours at room temperature with anti-IgG secondary antibodies: Alexa Fluor Plus donkey anti-rat 488 and Alexa Fluor Plus donkey anti-rabbit 555, mixed with blocking solution at a ratio of 1:2,000. Finally, the slices were washed five times with PBS, immersed in PBS and shaken for 15 minutes, and then mounted using ProLong Diamond Antifade Mountant (Thermo Fisher Scientific). Immunohistochemistry of the marmoset cerebral cortex in Example 4 was also performed using a similar technique. For the marmoset cerebral cortex, anti-GFP antibody (rat, 1:1,000, 04404-84, Nacalai Tesque) and anti-Iba1 antibody (rabbit, 1:500, 019-19741, Fujifilm Wako Pure Chemical Industries) were used as primary antibodies, and ProLong Glass Antifade Mountant with NucBlue Stain (P36983, ThermoFisher Scientific) was used as the mounting medium.

[0042] Confocal Laser Scanning Microscopy Analysis In Examples 1 to 3, the proportion of cells 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. Images of areas where GFP fluorescence could be observed were taken at regular intervals using a confocal laser scanning microscope (LSM800; Carl Zeiss, Oberkochen, Germany) at the optima setting using a 10× / 0.95NA or 20× / 0.95NA objective lens. Subsequently, maximum intensity projection (MIP) images were performed using image processing software (ZEN blue). Non-overlapping MIP images were captured from each brain slice, and the number of GFP-expressing cells was counted. GFP-positive cells colabeled with Iba1 were considered microglial cells. At least 30 cells were counted. A similar method was used for the confocal laser microscope analysis of the marmoset cerebral cortex in Example 4. 100 μm thick sections were prepared from the marmoset cerebral cortex and photographed using an all-in-one fluorescence microscope (Keyence Corporation: BZ-X800).

[0043] <Statistical Analysis> Statistical analysis was performed using unpaired Student's t-test or one-way ANOVA for the specificity of gene expression, expression efficiency, and fluorescence intensity in Figure 1F. Statistical analysis was performed using cumulative plots and the Kolmogorov-Smirnov test for Figures 3E and 3F.

[0044] Example 1: AAV vectors in which miR complementary sequences are located both upstream and downstream of the WPRE gene exhibit high microglia specificity in the cerebral cortex. Using the method described above, various AAV vector DNA constructs containing the Iba1 promoter, miR complementary sequences, the WPRE gene, and the like were prepared (FIG. 1A). The following three patterns of miR complementary sequences and WPRE gene locations were prepared. The miR-9 complementary sequence and the miR-129-2-3p complementary sequence were each repeated four times. (1) the WPRE gene, a complementary sequence of miR-9, and a complementary sequence of miR-129-2-3p (AAV vectors having these arrangements are designated as WPRE-a, b (control)); (2) a complementary sequence of miR-9, a complementary sequence of miR-129-2-3p, and the WPRE gene (AAV vectors having these arrangements are designated as a, b-WPRE); and (3) a complementary sequence of miR-9, a complementary sequence of miR-129-2-3p, the WPRE gene, a complementary sequence of miR-9, and a complementary sequence of miR-129-2-3p (AAV vectors having these arrangements are designated as a, b-WPRE-a, b). Various AAV vectors were injected into the cerebral cortex of mice. Three weeks after injection, brain slices were prepared and analyzed immunohistochemically by double immunolabeling with antibodies against GFP and Iba1. Microglia expressing GFP and Iba1 were observed in both a,b-WPRE (Fig. 1B) and a,b-WPRE-a,b (Fig. 1C). Furthermore, both a,b-WPRE and a,b-WPRE-a,b demonstrated significantly higher microglial specificity and expression efficiency than the control AAV vector, WPRE-a,b (Fig. 1D-E). Furthermore, microglial specificity was significantly higher in a,b-WPRE-a,b compared to a,b-WPRE (Fig. 1E). The gene expression intensity in microglia was comparable when a,b-WPRE was used and when a,b-WPRE-a,b was used (Fig. 1F).These results demonstrate that the use of an AAV vector in which the miR complementary sequence is located upstream of the WPRE gene or both upstream and downstream of the WPRE gene results in high microglial specificity and high microglial expression efficiency in gene expression in the cerebral cortex. Furthermore, it has been shown that among such AAV vectors, the AAV vector in which the miR complementary sequence is located both upstream and downstream of the WPRE gene is particularly excellent in terms of enhancing microglial specificity.

[0045] Example 2: AAV vectors with miR complementary sequences located both upstream and downstream of the WPRE gene exhibit high microglia specificity in other brain regions. We investigated whether AAV vectors with miR complementary sequences located both upstream and downstream of the WPRE gene exhibit high microglia specificity in brain regions other than the cerebral cortex (striatum and cerebellum). The following two types of AAV vectors were used in Example 1 (Figure 2A): (1) WPRE-a,b (control) (2) a,b-WPRE-a,b. Various AAV vectors were injected into the striatum and cerebellum of mice. Three weeks after injection, brain slices were prepared and analyzed immunohistochemically by double immunolabeling with antibodies against GFP and Iba1. Microglia expressing GFP and Iba1 were observed in both the striatum (Figure 2B) and cerebellum (Figure 2C) when WPRE-a,b or a,b-WPRE-a,b was used. Furthermore, in the striatum, microglia specificity was significantly higher when a,b-WPRE-a,b was used compared with the control AAV vector WPRE-a,b (Figure 2D). Furthermore, in the cerebellum, microglia specificity was sufficiently high (>90%) when the control AAV vector WPRE-a,b was used, but remained unchanged when a,b-WPRE-a,b was used (Figure 2F). Furthermore, in either the striatum or cerebellum, no significant difference was observed in the efficiency of microglial expression between the control AAV vectors WPRE-a,b and a,b-WPRE-a,b (Figures 2E and 2G). These results demonstrate that the use of AAV vectors in which miR complementary sequences are located both upstream and downstream of the WPRE gene results in high microglial specificity in gene expression in brain regions other than the cerebral cortex (striatum and cerebellum).

[0046] Example 3: AAV Vectors with miR Complementary Sequences Placed Both Upstream and Downstream of the WPRE Gene Maintain High Microglia Specificity for a Long Period (2 Months) We investigated whether AAV vectors with miR complementary sequences placed both upstream and downstream of the WPRE gene exhibit high microglia specificity for a long period in the cerebral cortex. Among the AAV vectors used in Example 1, a,b-WPRE-a,b was used (Figure 3A). This AAV vector was injected into the cerebral cortex of mice. Three weeks and two months after injection, brain slices were prepared and analyzed immunohistochemically by double immunolabeling with antibodies against GFP and Iba1. As a result, microglia expressing GFP and Iba1 were observed at both three weeks (Figure 3B) and two months (Figure 3C) after AAV vector injection. Furthermore, when a,b-WPRE-a,b was used, microglia specificity decreased somewhat over time, but remained high at both 3 weeks and 2 months after AAV vector injection (Figure 3D). Furthermore, the cumulative percentage of microglia in the cerebral cortex after AAV vector injection showed no significant difference at either 3 weeks or 2 months (Figure 3E), while the cumulative percentage of neurons showed a significant difference at both time points (Figure 3F). These results demonstrate that the use of AAV vectors with miR complementary sequences located both upstream and downstream of the WPRE gene results in high microglia specificity in gene expression in the cerebral cortex over a long period of at least 2 months.

[0047] It is widely known that microglia are deeply involved in the pathology of central nervous system diseases. However, because it has been impossible to efficiently express foreign genes selectively in microglia, gene therapy targeting microglia has not been performed, and research with gene therapy in mind has made little progress. It is expected that the use of the AAV vector of the present invention will lead to widespread use of gene therapy targeting microglia in the future.

[0048] Example 4 Demonstration that an AAV vector in which a miR complementary sequence is located both upstream and downstream of the WPRE gene is capable of expressing a target gene in microglia in the cerebral cortex of a primate We confirmed whether an AAV vector in which a miR complementary sequence is located both upstream and downstream of the WPRE gene is capable of expressing a target gene in microglia in the cerebral cortex of a primate. Of the AAV vectors used in Example 1, a,b-WPRE-a,b was used. 5 x 10 miRs were injected into the cerebral cortex of a marmoset (individual number H292: Izumo, 1.6 years old, weighing 509 g). １２ 1 μL (5 × 10 ９ vg). 29 days later (4 weeks later), the marmosets were sacrificed and the brains were removed. The body weight at the time of sacrifice was 532 g. 100 μm-thick slices were prepared, immunofluorescently stained with anti-GFP and anti-Iba1 antibodies, and then photographed with an all-in-one fluorescence microscope (Keyence Corporation: BZ-X800). As a result, microglia expressing GFP and Iba1 were observed. In other words, it was confirmed that, even in primates, target genes can be expressed in microglia in the cerebral cortex by using an AAV vector in which the miR complementary sequence is located both upstream and downstream of the WPRE gene.

Claims

1. An adeno-associated virus (AAV) vector for gene expression in microglia, comprising an expression unit including the Iba1 (ionized calcium-binding adaptor molecule 1) promoter, a complementary sequence of miR-9, a complementary sequence of miR-129, and the woodchuck hepatitis virus-derived post-transcriptional regulator (WPRE) gene, wherein the complementary sequence of miR-9 and the complementary sequence of miR-129 are positioned upstream of the WPRE gene; or wherein the complementary sequence of miR-9 and the complementary sequence of miR-129 are positioned both upstream and downstream of the WPRE gene.

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 gene of interest is the green fluorescent protein (GFP) gene.

4. The vector according to claim 1, which comprises multiple repeats of a complementary sequence of miR-9 and a complementary sequence of miR-129.

5. The vector according to claim 1, comprising a base sequence represented by any one of SEQ ID NOs: 1 to 4, or a base sequence having 90% or more identity to a base sequence represented by any one of SEQ ID NOs: 1 to 4.

6. The vector according to claim 1, which is used in combination with a vector for expressing a capsid gene.

7. The vector according to claim 1, which is used in combination with a gene expression vector that acts as a helper for adenovirus.

8. A recombinant virus obtained from a vector according to any one of claims 1 to 7.

9. A pharmaceutical comprising the vector according to any one of claims 1 to 7 or a recombinant virus obtained from said vector.

10. A medicine for central nervous system diseases according to claim 9.

11. A method for introducing a gene into microglia, comprising the step of introducing a target gene into microglia in vitro using the vector according to any one of claims 1 to 7.

12. Isolated microglia into which the vector according to any one of claims 1 to 7 has been introduced.

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