Vector for expressing predetermined gene in cells in cerebellar or cerebral cortex

Abstract

To provide a vector for expressing a predetermined gene in cells in the cerebellar or cerebral cortex; to provide a vector for treating deficiency of an expression product of a predetermined gene in cells in the cerebellar or cerebral cortex; to provide a vector for treating brain diseases; and to provide a vector for selectively activating or inactivating cells in the cerebellar or cerebral cortex.SOLUTION: The present invention provides a vector for expressing a predetermined gene in cells in the cerebellar or cerebral cortex, the vector comprising a promoter of a CD gene and the predetermined gene.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to vectors, and more specifically, to vectors for expressing a predetermined gene in cells in the cerebellum or cerebral cortex. The present invention also relates to vectors for treating a deficiency of an expression product of a predetermined gene in cells in the cerebellum or cerebral cortex, and to vectors for treating brain diseases. Further, the present invention relates to vectors for selectively activating or inactivating cells in the cerebellum or cerebral cortex.

Background Art

[0002] A major goal in the field of neuroscience is to clarify the roles of each cell in brain function and neural circuits, understand how each cell generates complex brain functions, and how dysfunction of each cell leads to brain diseases. To achieve this, it is necessary to define each cell type and construct gene tools that can selectively label and manipulate cells. In recent years, with the development of technologies such as single-cell RNA sequencing, gene expression profiles of various cell populations in the nervous system have been cataloged, and each cell type has been defined based on genome-wide gene expression. As a result, it has been found that there are actually a wide variety of cell types in the brain. To experimentally investigate the functions and input-output relationships of each cell type in the brain, it is necessary to rely on transgenic mouse lines based on marker genes. However, at present, most of the diverse cell types cannot be individually accessed with existing tools. Adeno-associated virus (AAV) vectors have been shown to be able to introduce any gene into brain cells from mice to humans, and recent developments in virus technology provide next-generation cell type-specific transgenic tools. Furthermore, since many diseases can be treated by approaching specific mutant cells using AAV and performing genetic manipulation, AAV is also expected as a clinical tool. Thus, the construction of transgenic tools using AAV is useful in many fields. It has been shown that the serotype, promoter, enhancer, and microRNA target sequence of AAV affect the infection efficiency of each cell type and gene expression in each cell type, and some cell type-specific AAVs with these elements have been developed [Non-Patent Documents 1 to 6]. However, the types of AAVs that can genetically manipulate cells in a cell type-specific manner are extremely limited.

[0003] One of the fundamental quests in the field of neuroscience is to define the roles of each cell in brain function and neural circuits. This knowledge is key to understanding how each cell generates complex brain functions and how dysfunction of each cell causes various brain diseases. To achieve this goal, it is necessary to define each cell type and generate genetic tools that can be labeled and manipulated in a highly cell-selective manner. Recent technological advancements, such as single-cell RNA sequencing, have facilitated the cataloging of gene expression profiles among various cell populations in the nervous system, enabling the definition of each cell type based on genome-wide gene expression and thereby the identification of diverse cell subtypes in the brain.

[0004] Despite such technological progress, there are significant limitations. Most notably, the current toolset does not enable individual access to most types of cells present in the brain. Recent innovations in viral technology offer promising means to overcome this obstacle. Viral vectors, such as adeno-associated virus (AAV) vectors, are versatile and have the potential to deliver any gene to various types of neurons and glial cells. Furthermore, since AAV can specifically target and genetically manipulate specific mutant cells, it has emerged as a powerful clinical tool for treating a wide range of diseases, thereby enabling the treatment of a wide range of diseases. Therefore, the construction of AAV-based transgenic tools is useful in many fields. Current research has shown that multiple elements, such as AAV serotypes, promoters, enhancers, and microRNA targeting sequences, can potentially affect the infection efficiency and gene expression in specific cell types. Cell-type-specific AAVs equipped with these elements have been partially developed [Non-Patent Documents 1-6], but AAVs that can target specific cell types are very limited.

[0005] The prevalence of autism spectrum disorder (ASD) in the United States is approximately 1 / 54 among 8-year-old children. The prevalence worldwide is thought to affect at least millions of people. The prevalence of attention deficit hyperactivity disorder (ADHD) worldwide varies depending on the literature but is usually about 5-10% in children. This disorder often persists in adults, and the adult prevalence is about 2.5-4%.

Prior Art Documents

Non-Patent Documents

[0006]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

[0007] Accordingly, an object of the present invention is to provide a vector for expressing a predetermined gene in cells in the cerebellum or cerebral cortex. Another object of the present invention is to provide a vector for treating a deficiency of an expression product of a predetermined gene in cells in the cerebellum or cerebral cortex. Further, an object of the present invention is to provide a vector for treating brain diseases. In addition, an object of the present invention is to provide a vector for selectively activating or inactivating cells in the cerebellum or cerebral cortex.

Means for Solving the Problems

[0008] The inventors addressed the above limitations by developing cell-type specific AAVs using the promoters of differentiation cluster (CD) genes. CD numbers are assigned to specific cell surface antigen molecules on leukemia cells and other cells, and the differences in their expression can be utilized to distinguish fine differences in cell types. Various CD genes are also expressed in the brain, providing a unique opportunity to target specific cell types in the brain. The inventors systematically screened AAVs with each CD gene promoter (CD promoter) that specifically induces expression in each cell of the cerebellum and found that some CD promoters can specifically induce gene expression in each cell of the cerebellum. The inventors' approach was successful not only in the cerebellum but also in the cerebral cortex. The inventors identified three CD promoters that specifically express the gene of interest in excitatory neurons, inhibitory neurons, and parvalbumin-positive inhibitory neurons. To evaluate the utility of these AAVs with specific CD promoters, they were applied to calcium imaging and chemogenetic studies. It was revealed that two types of cerebellar interneurons play different roles in social behavior, motor activity, and motor function and a common role in anxiety behavior using AAVs with CD promoters. Furthermore, selective activation of cerebellar molecular layer interneurons by AAVs with specific CD promoters rescued social deficits and motor dysfunction in a mouse model of autism spectrum disorder. The development of AAVs with CD promoters has the potential to revolutionize the understanding of cellular mechanisms in the brain. This includes understanding the roles of each cell type in complex brain functions and diseases, understanding the input-output relationships of these cells, exploring their physiological importance, and ultimately opening the way to new treatments for brain disorders.

[0009] Understanding the roles of individual cells in brain function and the cellular dysfunction leading to brain disorders is a crucial focus of neuroscience research. Recent advances in omics analysis have identified diverse cell types in the brain, but access to these cells remains limited. Here, we report the development of cell-type-specific adeno-associated virus (AAV) vectors in the brain using the promoters of differentiation cluster genes (CD promoters). By screening AAVs with each CD promoter, we newly identified several CD promoters that selectively induce gene expression in specific cell types in the mouse cerebellum and cerebral cortex. We succeeded in enhancing the specificity to each cell type by combining CD promoters with other DNA elements. AAVs equipped with CD promoters enabled in vivo Ca 2+ imaging in specific types of cerebellar cells. Chemogenetic experiments using these AAVs and CD promoters revealed that two types of cerebellar interneurons play different roles in social behavior and motor function and a common role in emotional responses. Furthermore, chemogenetic regulation of cerebellar molecular layer interneurons using CD promoters restored social and motor function deficits in a mouse model of autism spectrum disorder. Our findings demonstrate the usefulness of these AAVs in elucidating the functions of each cell type in brain function and developing novel therapies for brain diseases.

[0010] That is, in this study, we developed cell type-specific AAVs using the promoters of cluster of differentiation (CD) genes. CD molecules are present on the surfaces of various cells, mainly human leukocytes, and CD classification is based on cluster analysis of monoclonal antibodies against these cell surface antigen molecules. Therefore, differences in CD expression can be used to identify fine differences in cell types. Since many CDs have been shown to be expressed in the brain, it was hypothesized that gene manipulation could be achieved specifically in each cell type in the brain by using the promoters of CD genes (CD promoters). When we screened AAVs using each CD promoter that induces specific expression in each cell type of the mouse cerebellum, we found CD promoters that enable specific expression in each cell type of the mouse cerebellum. When this strategy was applied to the mouse cerebral cortex, we discovered three CD promoters that specifically express the gene of interest in excitatory neurons, inhibitory neurons, and parvalbumin-positive inhibitory neurons. To evaluate the utility of AAVs carrying these CD promoters, we applied them to calcium imaging and chemogenetics. We found that by using AAVs carrying CD promoters, two types of cerebellar interneurons play different roles in social behavior, locomotor activity, and motor function, and a common role in anxiety behavior. Furthermore, selective activation of cerebellar molecular layer interneurons by AAVs carrying a specific CD promoter restored social and motor function deficits in an autism spectrum disorder model mouse. AAVs carrying CD promoters are expected to lead to an understanding of the input-output relationships of each cell type, the roles of each cell type in brain function and disease, the interactions between each cell type and their physiological significance, and the establishment of new treatments for brain diseases.

[0011] That is, the present invention provides the following. [Aspect 1] A vector for expressing a predetermined gene in cells in the cerebellum or cerebral cortex, the vector comprising a promoter of a CD gene and the predetermined gene. [Aspect 2] The vector according to embodiment 1, wherein the cerebellum is the gray matter of the cerebellar cortex. [Embodiment 3] The vector according to embodiment 1, wherein the cerebellum is the outer molecular layer, the central Purkinje cell layer, or the inner granular cell layer. [Embodiment 4] The vector according to embodiment 1, wherein the cells in the cerebellum or cerebral cortex are cerebellar molecular layer interneurons, inhibitory neurons, excitatory neurons, parvalbumin-positive inhibitory neurons, Golgi cells, Purkinje cells, Lugaro cells, or astrocytes. [Embodiment 5] The vector according to embodiment 4, wherein the astrocyte is Bergmann glia. [Embodiment 6] The vector according to embodiment 1, wherein the CD gene is a CD1 gene, a CD9 gene, a CD24 gene, a CD34 gene, a CD38 gene, a CD44 gene, a CD52 gene, a CD68 gene, a CD74 gene, a CD81 gene, a CD83 gene, a CD164 gene, a CD276 gene, or a CD300 gene. [Embodiment 7] The vector according to embodiment 1, comprising an enhancer. [Embodiment 8] The vector according to embodiment 7, wherein the enhancer is an mscRE16 enhancer, an S5E6 enhancer, or an HGT017 enhancer. [Embodiment 9] The vector according to embodiment 1, comprising a miRNA targeting sequence. [Embodiment 10] The vector according to embodiment 9, wherein the miRNA is miR-133a, miR-1188, miR-1983, or miR-3086-5p. [Embodiment 11] The vector according to embodiment 1, wherein the predetermined gene encodes a metabotropic receptor. [Embodiment 12] The vector according to embodiment 1, wherein the predetermined gene encodes a G protein-coupled receptor. [Embodiment 13] The vector according to embodiment 1, wherein the predetermined gene is an M3Dq gene. [Aspect 14] The vector according to Aspect 1, wherein the vector is a viral vector. [Aspect 15] The vector according to Aspect 1, wherein the viral vector is an adeno-associated viral vector. [Aspect 16] The vector according to Aspect 1, which is a vector for treating the lack of an expression product of a predetermined gene in cells in the cerebellum or cerebral cortex. [Aspect 17] The vector according to Aspect 1, which is a vector for treating brain diseases. [Aspect 18] The vector according to Aspect 1, which is a vector for treating autism spectrum disorder, epilepsy, tuberous sclerosis, spinocerebellar degeneration, schizophrenia, or cerebellar dysfunction. [Aspect 19] The vector according to Aspect 1, which is for use in combination with a substance that interacts with the expression product of a predetermined gene. [Aspect 20] The vector according to Aspect 19, wherein the substance that interacts with the expression product of a predetermined gene is a clozapine metabolite. [Aspect 21] The vector according to Aspect 20, wherein the clozapine metabolite is clozapine N-oxide. [Aspect 22] A pharmaceutical composition comprising the vector according to Aspect 1. [Aspect 23] The vector according to Aspect 1, which is a vector for selectively activating or inactivating cells in the cerebellum or cerebral cortex. [Aspect 24] A pharmaceutical composition for treating autism spectrum disorder, epilepsy, tuberous sclerosis, spinocerebellar degeneration, schizophrenia, or cerebellar dysfunction, comprising the vector according to Aspect 1. [Aspect 25] A pharmaceutical composition for treating autism spectrum disorder, comprising the vector according to Aspect 1, wherein the predetermined gene is the M3Dq gene. [Advantages of the Invention]

[0012] The AAV with the CD promoter in the present invention is expected to lead to the elucidation of the input-output relationships of each cell type, the roles of each cell type in brain function and diseases, the interactions between each cell type and their physiological significance, and the establishment of new treatment methods for brain diseases. In addition, since AAV can be used to approach specific mutant cells and perform genetic manipulation, which may lead to the treatment of many diseases, AAV is also expected as a clinical tool.

[0013] In the present invention, by discovering three CD promoters (hCD1d, mCD24, hCD52) that enable specific gene expression in each cell type of the cerebellum and specifically express the target gene in parvalbumin-positive inhibitory neurons, inhibitory neurons, and excitatory neurons of the cerebral cortex, it may lead to the development of treatment methods targeting the cells in which each promoter specifically functions.

[0014] In the present invention, by using CD promoters (hCD68, hCD9), it was found that two types of cerebellar interneurons play different roles in social behavior, locomotor activity, and motor function, and play a common role in anxiety behavior. Furthermore, when the cerebellar molecular layer interneurons (MLI) are selectively activated by AAV with the hCD68 promoter, it has been shown that the social disorder and motor disorder of autism spectrum disorder model mice are restored, indicating the potential applicability to certain developmental disorder mental diseases. For the development of treatment methods targeting the target cells discovered this time, its utilization as a research tool can also be expected.

[0015] When expressing a predetermined gene in cells in the cerebellum or cerebral cortex using the vector provided by the present invention, the predetermined gene can be expressed with high specificity in specific cells for the purpose of expressing the predetermined gene. For example, when the vector provided by the present invention is used included in a pharmaceutical composition, the accompanying side effects are few.

Brief Description of the Drawings

[0016]

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Modes for Carrying Out the Invention

[0017] The present invention provides a vector for expressing a predetermined gene in cells in the cerebellum or cerebral cortex, the vector comprising a promoter of a CD gene and the predetermined gene. More specifically, the present invention provides a vector for expressing a predetermined gene in cells in the cerebellum or cerebral cortex, the vector comprising a promoter of a CD gene operably linked to the predetermined gene.

[0018] Examples of the cerebellum or cerebral cortex include, for example, the gray and white matter of the cerebral cortex. Examples of the cerebellum include, for example, the outer molecular layer, the central Purkinje cell layer, the inner granule cell layer, and the like.

[0019] Examples of cells in the cerebellum or cerebral cortex include, for example, cerebellar molecular layer interneurons, inhibitory neurons, excitatory neurons, parvalbumin-positive inhibitory neurons, Golgi cells, Purkinje cells, Lugaro cells, astrocytes, and the like. Examples of astrocytes include, for example, Bergmann glia and the like.

[0020] Examples of the CD gene include, for example, the CD1 gene, the CD9 gene, the CD24 gene, the CD34 gene, the CD38 gene, the CD44 gene, the CD52 gene, the CD68 gene, the CD74 gene, the CD81 gene, the CD83 gene, the CD164 gene, the CD276 gene, the CD300 gene, and the like. Examples of the CD1 gene include, for example, the CD1a gene, the CD1b gene, the CD1c gene, the CD1d gene, and the like. Examples of the CD300 gene include, for example, the CD300A gene, the CD300C2 gene, the CD300C gene, the CD300E gene, the CD300B gene, the CD300D3 gene, the CD300D1 gene, the CD300F gene, and the like.

[0021] Examples of CD genes include animal CD genes. Examples of animals include vertebrates and invertebrates. Examples of vertebrates include fish, amphibians, reptiles, birds, mammals, etc. Examples of mammals include mice, rats, rabbits, pigs, cows, monkeys, humans, etc. Examples of invertebrates include arthropods such as insects, crustaceans, spiders, and myriapods, as well as mollusks, etc. Therefore, examples of CD genes include mammalian CD genes, and examples of mammalian CD genes include human CD genes.

[0022] The promoter of the CD gene is, for example, all or part of -100 bp to -10,000 bp, -200 bp to -9,000 bp, -300 bp to -8,000 bp, -400 bp to -7,000 bp, -500 bp to -6,000 bp, -600 bp to -5,000 bp, -610 bp to -4,000 bp, -620 bp to -3,000 bp, -630 bp to -2,900 bp, -640 bp to -2,800 bp, -650 bp to -2,700 bp, -660 bp to -2,600 bp, -670 bp to -2,500 bp, -680 bp to -2,400 bp, -690 bp to -2,300 bp, or -700 bp to -2,200 bp from the transcription start site of the CD gene.

[0023] The promoter of the CD gene may be the promoter of the CD gene of a predetermined organism, but it is not essential for it to be the promoter of the CD gene of a predetermined organism. The promoter of the CD gene may be, for example, a deletion, substitution, or addition of bases to the promoter of the CD gene of a predetermined organism.

[0024] Examples of the promoter sequence of the CD1d gene include the sequence shown in SEQ ID NO: 1. Examples of the promoter sequence of the CD9 gene include the sequence shown in SEQ ID NO: 2. Examples of the promoter sequence of the CD24 gene include the sequence shown in SEQ ID NO: 3. Examples of the promoter sequence of the CD34 gene include the sequence shown in SEQ ID NO: 4. Examples of the promoter sequence of the CD38 gene include the sequence shown in SEQ ID NO: 5. Examples of the promoter sequence of the CD44 gene include the sequence shown in SEQ ID NO: 6. Examples of the promoter sequence of the CD52 gene include the sequence shown in SEQ ID NO: 7. Examples of the promoter sequence of the CD68 gene include the sequence shown in SEQ ID NO: 8. Examples of the promoter sequence of the CD74 gene include the sequence shown in SEQ ID NO: 9. Examples of the promoter sequence of the CD81 gene include the sequence shown in SEQ ID NO: 10. Examples of the promoter sequence of the CD83 gene include the sequence shown in SEQ ID NO: 11. Examples of the promoter sequence of the CD164 gene include the sequence shown in SEQ ID NO: 12. Examples of the promoter sequence of the CD276 gene include the sequence shown in SEQ ID NO: 13. Examples of the promoter sequence of the CD300A gene include the sequence shown in SEQ ID NO: 14. In addition, examples of the promoter sequence of the CD gene include sequences amplified using a primer pair whose sequence is shown by two sequences selected from the sequences shown in SEQ ID NOs: 15 to 72. Examples of the two sequences selected from the sequences shown in SEQ ID NOs: 15 to 72 include the sequences shown in SEQ ID NOs: 15 and 16 (for the promoter of the CD1d1 gene), the sequences shown in SEQ ID NOs: 17 and 18 (for the promoter of the CD2 gene), the sequences shown in SEQ ID NOs: 19 and 20 (for the promoter of the CD3E gene), the sequences shown in SEQ ID NOs: 21 and 22 (for the promoter of the CD9 gene), the sequences shown in SEQ ID NOs: 23 and 24 (for the promoter of the CD11b gene), the sequences shown in SEQ ID NOs: 25 and 26 (for the promoter of the Mouse_CD24 gene),The sequences shown in SEQ ID NOs: 27 and 28 (for the promoter of the CD34 gene), the sequences shown in SEQ ID NOs: 29 and 30 (for the promoter of the CD38 gene), the sequences shown in SEQ ID NOs: 31 and 32 (for the promoter of the CD44 gene), the sequences shown in SEQ ID NOs: 33 and 34 (for the promoter of the CD46 gene), the sequences shown in SEQ ID NOs: 35 and 36 (for the promoter of the CD47 gene), the sequences shown in SEQ ID NOs: 37 and 38 (for the promoter of the CD52 gene), the sequences shown in SEQ ID NOs: 39 and 40 (for the promoter of the CD53 gene), the sequences shown in SEQ ID NOs: 41 and 42 (for the promoter of the CD59 gene), the sequences shown in SEQ ID NOs: 43 and 44 (for the promoter of the CD74 gene), the sequences shown in SEQ ID NOs: 45 and 46 (for the promoter of the CD81 gene), the sequences shown in SEQ ID NOs: 47 and 48 (for the promoter of the CD82 gene), the sequences shown in SEQ ID NOs: 49 and 50 (for the promoter of the CD83 gene), the sequences shown in SEQ ID NOs: 51 and 52 (for the promoter of the CD84 gene), the sequences shown in SEQ ID NOs: 53 and 54 (for the promoter of the CD86 gene), the sequences shown in SEQ ID NOs: 55 and 56 (for the promoter of the CD93 gene), the sequences shown in SEQ ID NOs: 57 and 58 (for the promoter of the CD97 gene), the sequences shown in SEQ ID NOs: 59 and 60 (for the promoter of the CD99 gene), the sequences shown in SEQ ID NOs: 61 and 62 (for the promoter of the CD164 gene), the sequences shown in SEQ ID NOs: 63 and 64 (for the promoter of the CD200 gene), the sequences shown in SEQ ID NOs: 65 and 66 (for the promoter of the CD248 gene), the sequences shown in SEQ ID NOs: 67 and 68 (for the promoter of the CD276 gene), the sequences shown in SEQ ID NOs: 69 and 70 (for the promoter of the CD300A gene), and the sequences shown in SEQ ID NOs: 71 and 72 (for the promoter of the CD320 gene), etc.

[0025] As the sequence of the promoter of the CD gene, for example, the homology with one sequence selected from the group consisting of SEQ ID NOs: 1 to 14 is 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 99.9% or more. Also, as the sequence of the promoter of the CD gene, for example, the homology with the sequence amplified using a primer pair whose sequence is shown by two sequences selected from the sequences shown in SEQ ID NOs: 15 to 72 is 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 99.9% or more. Here, the homology can be calculated, for example, using the homology calculation algorithm NCBI BLAST (National Center for Biotechnology Information Basic Local Alignment Search Tool) (Proc. Natl. Acad. Sci. USA 87: 2264-2268, 1990; Proc Natl Acad Sci USA 90: 5873, 1993) under the conditions of expectation value = 10; allowing gaps; filtering = ON; match score = 1; mismatch score = -3. Other algorithms for determining homology include BLASTN and BLASTX (Altschul SF, et al: J Mol Biol 215: 403, 1990), etc. These programs can use their respective default parameters.

[0026] The organism from which the cells in the cerebellum or cerebral cortex are derived and the organism from which the CD gene is derived do not have to be of the same species, but are preferably of the same species. For example, when the cells in the cerebellum or cerebral cortex are derived from a human, the CD gene does not have to be a human CD gene, but is preferably a human CD gene.

[0027] The vector provided by the present invention may contain an enhancer. Examples of the enhancer include the mscRE16 enhancer, the S5E6 enhancer, the HGT017 enhancer, and the like. When the vector provided by the present invention contains an enhancer, when expressing a predetermined gene in cells in the cerebellum or cerebral cortex, the predetermined gene can be expressed with high specificity in specific cells for the purpose of expressing the predetermined gene. For example, when the vector provided by the present invention is included in a pharmaceutical composition and used, side effects can be reduced.

[0028] The vector provided by the present invention may contain an miRNA targeting sequence. Examples of the miRNA include miR-133a, miR-1188, miR-1983, miR-3086-5p, and the like. When the vector provided by the present invention contains an miRNA targeting sequence, when expressing a predetermined gene in cells in the cerebellum or cerebral cortex, the predetermined gene can be expressed with high specificity in specific cells for the purpose of expressing the predetermined gene. For example, when the vector provided by the present invention is included in a pharmaceutical composition and used, side effects can be reduced.

[0029] The predetermined gene encodes, for example, a metabotropic receptor. Further, the predetermined gene encodes, for example, a G protein-coupled receptor. The predetermined gene is, for example, the M3Dq gene. The expression product of the M3Dq gene is a metabotropic receptor, a G protein-coupled receptor (GPCR), and it is known that when the ligand clozapine N-oxide (CNO) binds, it increases the calcium concentration and elevates the activity of nerve cells. Examples of the predetermined gene include a gene encoding a substance that has a function of activating or inactivating cells in the cerebellum or cerebral cortex, either alone or in combination with other substances. Here, examples of the substance in the substance that has a function of activating or inactivating cells in the cerebellum or cerebral cortex include proteins and RNAs. Further, examples of other substances include substances that interact with the expression product of the predetermined gene, such as a ligand of the expression product of the predetermined gene.

[0030] The vector is, for example, a viral vector, and the viral vector is, for example, an adeno-associated viral vector.

[0031] The vector provided by the present invention can be used, for example, to treat the lack of the expression product of a predetermined gene in cells in the cerebellum or cerebral cortex. Examples of the expression product of a predetermined gene include, in addition to proteins, RNA and the like. The vector provided by the present invention can be used, for example, to treat brain diseases. Purkinje cells are known to cause autism spectrum disorder, spinocerebellar degeneration, tuberous sclerosis, etc., and inhibitory cells are known to regulate the activity of Purkinje cells. Also, it is known that various diseases such as autism spectrum disorder, epilepsy, and schizophrenia occur due to the balance between inhibitory cells and excitatory cells. Therefore, the vector provided by the present invention can be used, for example, to treat autism spectrum disorder, epilepsy, tuberous sclerosis, spinocerebellar degeneration, schizophrenia, or cerebellar dysfunction. Here, examples of treatment include, in addition to treatment, prevention. Examples of treatment include, in addition to complete cure, alleviation of symptoms and the like. The vector provided by the present invention can be used, for example, to selectively activate or inactivate cells in the cerebellum or cerebral cortex. Examples of the vector provided by the present invention include, in addition to vectors for medical acts on humans, vectors not for medical acts on humans. Examples of vectors not for medical acts on humans include vectors for experiments, research, and the like. Examples of the vector provided by the present invention include, in addition to vectors for in vivo use, vectors for in vitro use.

[0032] The vector provided by the present invention may be used in combination with a substance that interacts with the expression product of a predetermined gene, such as a ligand of the expression product of a predetermined gene. Using it in combination with a substance that interacts with the expression product of a predetermined gene, such as a ligand of the expression product of a predetermined gene, may be performed, for example, by using it in combination with a vector that expresses the substance. Examples of substances that interact with the expression product of a predetermined gene, such as a ligand of the expression product of a predetermined gene, include, for example, clozapine metabolites. Examples of clozapine metabolites include, for example, clozapine N-oxide (CNO).

[0033] The vector provided by the present invention may be included in a pharmaceutical composition. The pharmaceutical composition containing the vector provided by the present invention may contain, in addition to the vector provided by the present invention, for example, pharmaceutically acceptable carriers and additives. The pharmaceutically acceptable carrier or additive is not particularly limited and can be appropriately selected according to, for example, the dosage form, etc., and can be any carrier, diluent, excipient, suspending agent, lubricant, adjuvant, medium, delivery system, emulsifier, tablet disintegrating substance, absorbent, preservative, surfactant, coloring agent, flavor or sweetener, etc. The dosage form of the pharmaceutical composition containing the vector provided by the present invention is not particularly limited and can be appropriately selected according to the desired administration method, and examples include injections (solutions, suspensions, solid preparations for in-use dissolution, etc.), solid preparations (tablets, capsules, suppositories, powders, etc.). As the injection, for example, a pH adjuster, buffer, stabilizer, isotonic agent, local anesthetic, etc. can be added to the composition, and injections for subcutaneous, intra-articular, intramuscular, intravenous use, etc. can be produced by a conventional method. Examples of the pH adjuster and the buffer include sodium citrate, sodium acetate, sodium phosphate, etc. Examples of the stabilizer include sodium pyrosulfite, EDTA, thioglycolic acid, thiolactic acid, etc. Examples of the isotonic agent include sodium chloride, glucose, etc. Examples of the local anesthetic include procaine hydrochloride, lidocaine hydrochloride, etc. The solid preparation may be provided with an enteric coating. The pharmaceutical composition containing the vector provided by the present invention may be prepared as a liquid preparation or a solid preparation, and can be formulated into various dosage forms such as injections, jelly preparations, spray preparations, tablets, granules, etc. When preparing as a liquid preparation, sterilized water, physiological saline, glucose aqueous solution, etc. can be used as a carrier, and further, if desired, a bactericide, isotonic agent, stabilizer, etc. can be blended. When preparing as a solid preparation, additives such as starch, lactose, mannitol, inorganic salts, etc. can be blended, and further, if desired, a binder, disintegrant, lubricant, etc. can be blended.

[0034] The pharmaceutical composition containing the vector provided by the present invention can be administered, for example, by any administration method suitable for the dosage form, such as intrathecal administration, oral administration, intravenous administration, subcutaneous administration, transdermal administration, intramuscular administration, intra-articular administration, nasal administration, intraperitoneal administration, direct injection into the target tissue, inhalation administration, enteral administration, enema administration, parenteral nutrition, etc. The dosage of the pharmaceutical composition containing the vector provided by the present invention may vary depending on the age, weight, health condition, etc. of the subject. The pharmaceutical composition containing the vector provided by the present invention may be administered once a day or may be administered multiple times a day, and the administration frequency can be, for example, 1 to 100 times per month.

[0035] The present invention provides a method for expressing a predetermined gene in cells in the cerebellum or cerebral cortex, which includes administering a vector containing a promoter of the CD gene and the predetermined gene to the cells in the cerebellum or cerebral cortex. After administering the above-mentioned vector to the cells in the cerebellum or cerebral cortex, a substance that interacts with the expression product of the predetermined gene, such as a ligand of the expression product of the predetermined gene, may be administered to the cells in the cerebellum or cerebral cortex.

[0036] The present invention provides a method for expressing a predetermined gene in cells in the cerebellum or cerebral cortex of a subject, which includes administering a vector containing a promoter of the CD gene and the predetermined gene to the above-mentioned subject. Examples of the subject include animals. Examples of animals include vertebrates and invertebrates. Examples of vertebrates include fish, amphibians, reptiles, birds, mammals, etc. Examples of mammals include mice, rats, rabbits, pigs, cows, monkeys, humans, etc. Examples of invertebrates include arthropods such as insects, crustaceans, spiders and myriapods, and mollusks, etc. The subject is preferably a mammalian animal, and the mammalian animal is preferably a human. After administering the above-mentioned vector to the above-mentioned subject, a substance that interacts with the expression product of the predetermined gene, such as a ligand of the expression product of the predetermined gene, may be administered to the above-mentioned subject.

[0037] The present invention provides a method for treating a deficiency of an expression product of a predetermined gene in cells in the cerebellum or cerebral cortex, the method comprising administering the vector to cells in the cerebellum or cerebral cortex. After administering the vector to cells in the cerebellum or cerebral cortex, a substance that interacts with the expression product of the predetermined gene, such as a ligand of the expression product of the predetermined gene, may be administered to the cells in the cerebellum or cerebral cortex.

[0038] The present invention provides a method for treating a deficiency of an expression product of a predetermined gene in cells in the cerebellum or cerebral cortex of a subject, the method comprising administering the vector to the subject. After administering the vector to the subject, a substance that interacts with the expression product of the predetermined gene, such as a ligand of the expression product of the predetermined gene, may be administered to the subject.

[0039] The present invention provides a method for treating a brain disease of a subject, the method comprising administering the vector to the subject. After administering the vector to the subject, a substance that interacts with the expression product of the predetermined gene, such as a ligand of the expression product of the predetermined gene, may be administered to the subject.

[0040] The present invention provides a method for treating an autism spectrum disorder, epilepsy, tuberous sclerosis, spinocerebellar degeneration, schizophrenia, or cerebellar dysfunction of a subject, the method comprising administering the vector to the subject. After administering the vector to the subject, a substance that interacts with the expression product of the predetermined gene, such as a ligand of the expression product of the predetermined gene, may be administered to the subject.

[0041] The present invention provides a method for selectively activating or inactivating cells in the cerebellum or cerebral cortex, which includes administering the above-mentioned vector to cells in the above-mentioned cerebellum or cerebral cortex. After administering the above-mentioned vector to cells in the above-mentioned cerebellum or cerebral cortex, a substance that interacts with the expression product of a predetermined gene, such as a ligand of the expression product of the predetermined gene, may be administered to cells in the above-mentioned cerebellum or cerebral cortex.

[0042] The present invention provides a method for selectively activating or inactivating cells in the cerebellum or cerebral cortex of a subject, which includes administering the above-mentioned vector to the above-mentioned subject. After administering the above-mentioned vector to the above-mentioned subject, a substance that interacts with the expression product of a predetermined gene, such as a ligand of the expression product of the predetermined gene, may be administered to the above-mentioned subject.

[0043] In these methods, the administration of a substance that interacts with the expression product of a predetermined gene, such as a ligand of the expression product of the predetermined gene, may be performed, for example, by administering a vector that expresses the substance. Examples of such methods include, in addition to methods performed as medical acts on humans, methods not performed as medical acts on humans. Examples of methods not performed as medical acts on humans include methods performed as experiments, research, etc. Examples of such methods include, in addition to methods performed in vivo, methods performed in vitro.

[0044] The present invention provides the use of a vector containing a promoter of a CD gene and the above-mentioned predetermined gene in the production of a composition for expressing a predetermined gene in cells in the cerebellum or cerebral cortex. The present invention also provides the use of a vector containing a promoter of a CD gene and the above-mentioned predetermined gene, and a substance that interacts with the expression product of a predetermined gene, such as a ligand of the expression product of the predetermined gene, in the production of a composition for expressing a predetermined gene in cells in the cerebellum or cerebral cortex.

[0045] The present invention provides the use of the above-mentioned vector in the manufacture of a composition for expressing a predetermined gene in cells in the cerebellum or cerebral cortex of a subject. The present invention also provides the use of the above-mentioned vector and a substance that interacts with the expression product of a predetermined gene, such as a ligand of the expression product of the predetermined gene, in the manufacture of a composition for expressing a predetermined gene in cells in the cerebellum or cerebral cortex of a subject.

[0046] The present invention provides the use of the above-mentioned vector in the manufacture of a composition for treating a deficiency of the expression product of a predetermined gene in cells in the cerebellum or cerebral cortex. The present invention also provides the use of the above-mentioned vector and a substance that interacts with the expression product of a predetermined gene, such as a ligand of the expression product of the predetermined gene, in the manufacture of a composition for treating a deficiency of the expression product of a predetermined gene in cells in the cerebellum or cerebral cortex.

[0047] The present invention provides the use of the above-mentioned vector in the manufacture of a composition for treating a brain disease of a subject. The present invention also provides the use of the above-mentioned vector and a substance that interacts with the expression product of a predetermined gene, such as a ligand of the expression product of the predetermined gene, in the manufacture of a composition for treating a brain disease of a subject.

[0048] The present invention provides the use of the above-mentioned vector in the manufacture of a composition for treating an autism spectrum disorder, epilepsy, tuberous sclerosis, spinocerebellar degeneration, schizophrenia, or cerebellar dysfunction of a subject. The present invention also provides the use of the above-mentioned vector and a substance that interacts with the expression product of a predetermined gene, such as a ligand of the expression product of the predetermined gene, in the manufacture of a composition for treating an autism spectrum disorder, epilepsy, tuberous sclerosis, spinocerebellar degeneration, schizophrenia, or cerebellar dysfunction of a subject.

[0049] The present invention provides the use of the above-mentioned vector in the production of a composition for selectively activating or inactivating cells in the cerebellum or cerebral cortex. The present invention also provides the use of the above-mentioned vector and a substance that interacts with the expression product of a predetermined gene, such as a ligand of the expression product of a predetermined gene, in the production of a composition for selectively activating or inactivating cells in the cerebellum or cerebral cortex of a subject.

[0050] The present invention provides the use of the above-mentioned vector in the production of a composition for selectively activating or inactivating cells in the cerebellum or cerebral cortex of a subject. The present invention also provides the use of the above-mentioned vector and a substance that interacts with the expression product of a predetermined gene, such as a ligand of the expression product of a predetermined gene, in the production of a composition for selectively activating or inactivating cells in the cerebellum or cerebral cortex of a subject.

[0051] In these uses, the use of a substance that interacts with the expression product of a predetermined gene, such as a ligand of the expression product of a predetermined gene, may be carried out by using a vector that expresses the substance. Examples of the composition include, in addition to compositions for medical treatment of humans, compositions not for medical treatment of humans. Examples of compositions not for medical treatment of humans include, for example, experimental compositions, research compositions, and the like. Examples of the composition include, in addition to compositions for in vivo use, compositions for in vitro use. The composition may be a pharmaceutical composition.

Examples

[0052] Details of the experimental model and the subject: This study was conducted based on the recommendations of the rules regarding animal experiments and related activities at Tokyo Medical and Dental University. All animal experiments were approved by the Genetic Recombination Experiment Committee and the Animal Experiment Committee of Tokyo Medical and Dental University. All animals were group-housed and maintained on a 12-hour:12-hour light-dark cycle. Every effort was made to reduce the number of animals used and minimize animal suffering and pain. In this study, WT mice (ICR and C57BL6N, 8 - 16 weeks old, Sankyolabo service) and TSC1 flox mice (from 3 days after birth to 10 weeks old, Jackson lab, strain number 005680) were used.

[0053] Details of the method: Plasmid: The CD gene promoter sequence was amplified by PCR from mouse and human genomic DNA using the primer pair (SEQ ID NOs: 15 - 72) and inserted into the site of the hSyn promoter in AAV-U6-sgRNA-hSyn-mCherry (Addgene plasmid #87916 from Alex Hewitt) and in AAV-syn-EGFP (Addgene plasmid #50465 from Bryan Roth). The hSyn promoter was removed by ApaI and BaMH1 or XbaI. The promoter, enhancer, and reporter were cloned using the Gibson Cloning Assembly Kit (New England BioLabs) according to the standard procedure. Specifically, for pAAV-L7-6-iCre, the iCre coding sequence was amplified from pCAG-iCre (Addgene plasmid #89573 from Wilson Wong) and cloned into pAAV / L7-6-GFP-WPRE (Addgene plasmid #126462 from Hirokazu Hirai); for AAV-S5E6 and AAV-HGT017, these sequences were amplified from pAAV-S5E6-dTom-nlsdTom (Addgene plasmid #135641 from Jordane Dimidschstein) and CN1253-scAAV-eHGT_017h-minBG-SYFP2-WPRE3-BGHpA (Addgene plasmid #163497 from Allen Institute for Brain Science and Boaz Levi) and cloned into the 5' region of the CD9 promoter; for AAV-mscRE16, the sequence was amplified from AiP1002-pAAV-mscRE16-minBGpromoter-EGFP-WPRE-hGHpA (Addgene plasmid #163486 from Allen Institute for Brain Science and Bosiljka Tasic) and cloned into the 5' region of the CD68 promoter.For AAV-M3Dq-mcherry, the hM3D(Gq)-mCherry sequence was amplified from pAAV-hSyn-DIO-hM3D(Gq)-mCherry (Addgene plasmid # 44361 from Bryan Roth) and cloned into pAAV-mscRE16-CD68 and pAAVHGT017-CD9. In pAAV-HGT017-CD9-mcherry-miR_targeting_sequences (SEQ ID NOs: 73 - 76), four copies of a single miR_targeting sequence were amplified and inserted between the 5' end of the hGH polyA regulatory sequence insert and the 3' end of the mcherry insert in pAAV-HGT017-CD9-mcherry. For AAV containing jGCaMP8s, the mcherry sequence was removed from pAAV-HGT017-CD9-mcherry-miR133a_targeting and the cDNA of jGCaMP8s was inserted into the removed mcherry region of pAAV-HGT017-CD9-mcherry-miR133a_targeting.

[0054] Packaging and injection of AAV: AAVs were produced using standard manufacturing methods. The plasmids required for AAV production were transfected into HEK293 cells with polyethyleneimine. AAV9, PHPeB (Chan et al., 2017), and CAPB10 (Goertsen et al., 2022) were used. Viruses were harvested from both cell lysates and media after 120 hours. A Purification Kit (Takara) was used for purification of virus particles. All batches produced were within the range of 10 10 ~10 11 virus genomes per milliliter.

[0055] The procedure for virus vector injection has been changed from the previous lentivirus protocol (Uesaka et al., 2014). 1-1.5 μL of the virus solution was injected at a rate of 100 nl / min into the vermis of the cerebellum and the S1 cerebral cortex of adult C57BL / 6N mice. 1.5 μL of pAAV-L7-6-iCre and pAAV-L7-6-GFP were injected into the cerebellum of TSC1 flox mice at 3-4 days after birth, and then, at 8-9 weeks after birth, 1.5 μL of pAAV-mscRE16-CD68-M3Dq-mcherry was injected into the cerebellum.

[0056] Immunohistochemistry: The mice were perfused with 4% paraformaldehyde in 0.1 M phosphate buffer, and cerebellar and cerebral sections (100 μm thick) were prepared using a microslicer. After permeabilization and blocking of non-specific binding, the following antibodies were applied at 4°C for 2 days: antibody against Car8 (Car8-GP-Af500 or Car8-Go-Af780, diluted 1:300, Nittobo Medical); antibody against S100b (S100b-GP-Af630, 1:300, Nittobo Medical); antibody against parvalbumin (PV-Go-Af460, 1:300, Nittobo Medical), antibody against RFP (390004, 1:1000, Synaptic Systems or PM005, 1:500, MBL), antibody against neurogranin (AB5620, 1:1000, Merck Millipore), antibody against CaMKII (ab52476, 1:500, abcam), antibody against NeuN (MAB377, 1:1000, Merck Millipore), and / or antibody against GFP (#06083-05, 1:1000, Nacalai Tesque). Sections immunolabeled using secondary antibodies (anti-rat AlexaFluor488, anti-guinea pig Cy3, anti-goat AlexaFluor488, anti-goat Alexa Fluor 647, anti-mouse Cy5, anti-rabbit Cy3, anti-rabbit Alexa Fluor 488) were washed and photographed under a fluorescence microscope (BZ-X700 or BZ-X800, Keyence).

[0057] In vivo calcium imaging: The animals were placed on a warm blanket and anesthetized with a mixture of midazolam (4 mg / kg body weight (BW)), butorphanol (5 mg / kg BW), and medetomidine (0.3 mg / kg BW). The depth of anesthesia was constantly monitored by observing the forelimb withdrawal reflex induced by pinching. The skin and muscle on the skull were removed, and a metal plate was fixed on lobules 5 - 7 of the cerebellar vermis with dental acrylic cement. A craniotomy with a diameter of 3 mm was performed, and the dura mater was carefully removed. A sterile circular glass with a diameter of 3 mm was placed directly on the dura mater and fixed in place with a surgical adhesive (Alon-Alpha A, Sankyo). A custom-made metal head plate with a 5-mm circular imaging well was fixed to the skull over the cranial window using dental cement (Super-Bond, Sun Medical, Japan). In vivo calcium imaging was performed using a two-photon microscope (Nikon, AX R MP) equipped with a ×16 objective lens (Nikon, CFI75 LWD 16X W) and an ultra-fast laser (Axon 920-2 TPC). During recording, the animals lay on a warm blanket to maintain their body temperature at 37°C. The laser output was maintained at <20 mW in the sample. Images were acquired in a 2D plane (512×512 pixels, 890 μm×890 μm) using Nikon NIS-Elements software (frame rate, 1 Hz).

[0058] To extract fluorescence changes from the calcium imaging video data, the raw video was first converted to the ΔF / Fb wave (Tsutsumi et al., 2015). ΔF / Fb is expressed as (F - Fb) / (Fb), where Fb is the baseline fluorescence in the absence of calcium fluctuations. Fb was defined as follows. First, the mean + 1SD of the fluorescence video acquisition period for each pixel was set as the threshold, and the fluorescence below the threshold was averaged to obtain Fb. Next, the ΔF / F wave for each pixel was high-pass filtered at 0.1 Hz to remove slow drifts due to photobleaching effects or glial signals.

[0059] Behavior: Male mice at 2 - 3 months old, 7 - 15 days after AAV injection, were used in the behavioral tests. Before the experiment, the mice were acclimated to the test area for at least 30 minutes. The behavior of the mice was recorded through a video camera installed in the laboratory. The excitatory DREADD, hM3Dq, was selectively activated using the DREADD agonist CNO (Catalog number 4936, Tocris, UK). Forty minutes before the behavioral test, 1 mg / kg of CNO was injected intraperitoneally into each mouse. The open field test was performed for 10 minutes in an open field box of 50 cm x 50 cm x 40 cm (width x depth x height). The total distance the mouse moved and the time in the central area and the peripheral area were automatically analyzed by ImageJ software (MouBeAT) (Bello - Arroyo et al., 2018).

[0060] The sociality test was carried out in an open field apparatus (50 x 50 x 40 cm, width x depth x height). The test consisted of a 10 - minute acclimation session and a 10 - minute test session. In the acclimation session, the mice were allowed to freely explore the open field. In the test session, two slit - shaped square cages (8 cm x 8 cm, height 18 cm) were placed in two adjacent corners. One cage contained a novel mouse (8 - week - old male C57BL6N), and the other contained a mouse - shaped doll. First, the test mouse was placed in the outer area of the open field arena, the farthest from these cages. The time spent around the two cage areas (diameter 12 cm) was automatically analyzed using ImageJ software (MouBeAT). The sociality index was evaluated by the following equation: Sociality index = (Tmouse) / (Tdoll), where Tmouse and Tdoll are the residence times around the mouse cage and the doll cage, respectively.

[0061] To evaluate motor function and motor learning, a rotarod test was performed on mice. The rotarod (model LE8205, Panlab) test was conducted continuously for 3 days, with two trials per day at 10-minute intervals (30 and 40 minutes after CNO injection). In each trial, the rotarod was linearly accelerated from 4 rpm to 40 rpm over 300 seconds. The time from the start of rotation to falling was measured.

[0062] The grooming test was performed in a cylindrical cage. The mice were placed in a new empty cage without bedding. The total recording time was 10 minutes. Spontaneous grooming behavior was recorded through a video camera installed in the laboratory. Subsequently, the video was analyzed.

[0063] Statistical analysis: All data are presented as mean ± SEM. Statistical significance was evaluated by the Mann–Whitney U test for comparing two independent samples. To compare two independent samples from two different categories for one dependent variable, two-way ANOVA with post hoc test (Bonferroni correction) was used as shown below. Statistical analysis was performed using the GraphPad Prism program. A difference between groups was considered significant when the p-value was less than 0.05. *, **, ***, ****, ns indicate p < 0.05, p < 0.01, p < 0.001, p < 0.0001, and not significant, respectively.

[0064] Example 1 Screening of CD promoters in the mouse cerebellum: To identify CD promoters that enable gene expression in specific types of cells, we focused on the mouse cerebellum (Figure 1A) and detected 31 CD promoters that are expressed in the cerebellum from previous microarray data. In the analysis of AAV using these CD promoters, the mouse and human genomes were used to amplify approximately -700 bp to -2200 bp from the transcription start site of each CD promoter by PCR, and these promoter sequences were cloned into the AAV backbone using the mcherry or EGFP reporter (Figure 1B). Virus particles were packaged with the capsid AAV9, and these AAVs were injected into the cerebellum of adult mice, respectively (Figure 1B). One to two weeks later, the brains were fixed and immunohistochemistry was performed. All of these AAVs equipped with each CD promoter induced the expression of the reporter gene in the cerebellum. The gray and white matter of the cerebellar cortex is divided into three layers: the outer molecular layer, the central Purkinje cell layer, and the inner granular cell layer (Figure 1A). Each layer contains different types of cells. To examine in which cerebellar cell layer the AAVs with each CD promoter induce gene expression, the cerebellar cell layers where reporter-labeled cells are present were analyzed. It was found that AAVs equipped with any of the human CD52 (hCD52), hCD68, hCD74, hCD83, and hCD164 promoters mainly induced gene expression in the molecular layer and had sparse activity in the granular cell layer (Figure 1C, 1D, and Figure 8). In contrast, most of the cells labeled by the AAV equipped with the hCD9 promoter were in the granular cell layer where Golgi cells seemed to be labeled, and a very small part of the labeled cells were Purkinje cells (Figure 1E, F). Similarly, with the hCD81 promoter, Golgi cells were mainly labeled, but some labeled cells were present in the molecular layer and the others were Purkinje cells. AAVs equipped with either the hCD44 or hCD276 promoter labeled cells in both the molecular layer and the granular cell layer (Figure 1G and 1H). The hCD300 promoter expressed EGFP in the cells of the molecular layer and the granular cell layer where Lugaro cells seemed to be labeled (Figure 8C and 8D).Most of the cells labeled by AAV with the hCD38 promoter were Purkinje cells, and the other cells were present in the granular cell layer where Golgi cells were presumably labeled (Figs. 1I and 1J). The hCD34 promoter was highly selective for cells expressing S100β, a marker of Bergmann glia, a special type of astrocyte in the cerebellum (Figs. 1K, 1L, and 8E). These data indicate that specific CD promoters induce gene expression in different layers and cell types within the mouse cerebellum.

[0065] Example 2 Cell type specificity enhanced by combinations of promoters, enhancers, and microRNA targeting sequences: To examine and optimize the cell-type specificity of the CD promoter, the hCD68 and hCD9 promoters were focused on. The immunoreactivity of parvalbumin (PV) as a marker for molecular layer interneurons and the immunoreactivity of neurogranin (NG) as a marker for Golgi cells were used. The inventors found that the hCD68 promoter selectively labels PV-positive molecular layer interneurons in the molecular layer (>94%) (Figs. 2A and 2E). However, a small number of cells in the granule cell layer were also labeled (Fig. 2D). To further enhance the specificity of the hCD68 promoter for molecular layer interneurons, the hCD68 promoter was combined with an enhancer. In several previous studies, enhancer sequences for cell-type specific gene expression have been identified. The inventors selected various enhancers and analyzed the expression patterns of reporter genes regulated by these enhancers in the cerebellum. The mscRE16 enhancer [Non-Patent Document 1] mainly induced the expression of the reporter gene in molecular layer interneurons and slightly induced the expression of the reporter gene in the granule cell layer (Figs. 9A - D). Based on these findings, a new AAV construct (AAV-RE16_CD68-mcherry) combining both the hCD68 promoter and the mscRE16 enhancer was created. AAV-PHP.eB was used to enhance gene expression. AAV-RE16_CD68-mcherry had better specificity for molecular layer interneurons than AAV containing either mscRE16 or hCD68 alone (Figs. 2B, 2C, 2E). Furthermore, the new AAV containing hCD68 and mscRE16 had fewer labeled cells in the granule cell layer compared to the AAV using only the hCD68 promoter (Fig. 2D).

[0066] Next, the hCD9 promoter was improved. The hCD9 promoter induced the expression of fluorescent proteins selectively in new granin-positive Golgi cells (>97%) in the granule cell layer (Fig. 2K), but also labeled a small part of Purkinje cells. Combinations of the hCD9 promoter and enhancers were attempted. The inventors found that the S5E6 enhancer [Non-Patent Document 6] induced the expression of the reporter gene in both the molecular layer and the granule cell layer, but not in Purkinje cells (Figs. 9E - G). AAV.CAP-B10 was used as the AAV capsid, and an AAV equipped with both the hCD9 promoter and the S5E6 enhancer was generated. The AAV mainly labeled Golgi cells, but also labeled a part of Purkinje cells (Figs. 2G, 2I, 2K). Next, a new AAV construct containing both hCD9 and the HGT_017 enhancer was created. When the hCD9 promoter and the HGT_017 enhancer were combined, the targeting specificity for cells in the granule cell layer was significantly improved compared to the hCD9 promoter alone or the S5E6 enhancer (86.0 ± 6.3% for HGT017_hCD9, 77.9 ± 6.9% for hCD9, 69.2 ± 8.9% for S5E6_hCD9, Figs. 2H, 2J). Furthermore, the specificity for neurogranin-positive Golgi cells was high in all of hCD9, hCD9 containing S5E6, and hCD9 containing HGT_017 (>97%, Fig. 2K).

[0067] The inventors further attempted to enhance the specificity of AAV vectors for Golgi cells using the hCD9 promoter. The inventors employed microRNA (miRNA) targeting sequences aimed at suppressing gene expression in Purkinje cells. The inventors searched for miRNAs expressed in Purkinje cells reported to date and detected candidate miRNAs (miR-133a, miR-1188, miR-1983, miR-3086-5p) [Non-Patent Document 7]. The inventors designed an AAV vector incorporating four copies of the hCD9 promoter, the HGT017 enhancer, and each miRNA targeting sequence. The AAV-HGT017_hCD9-mcherry vector (miR-133aT) containing the miR-133a targeting sequence effectively reduced the proportion of Purkinje cells expressing mcherry and showed a high level of specificity for cells in the granule cell layer (Figures 3A-B). AAV-HGT017_hCD9-mcherry equipped with other miRNA targeting sequences did not change the proportion of Purkinje cells and cells in the granule cell layer (Figures 3C-H). Immunohistochemical analysis using a neurogranin antibody showed that 95.5% of the cells in the granule cell layer labeled with mcherry of AAVHGT017_hCD9-mcherry containing miR-133aT were neurogranin-positive Golgi cells (Figures 3I-J). These results suggest that the miR-133a targeting sequence enhances specificity for Golgi cells by reducing gene expression in Purkinje cells.

[0068] Example 3 Identification of cell type-specific CD promoters in the mouse cerebral cortex: Next, a CD promoter that induces cell-type specific expression in the mouse cerebral cortex was searched for. The inventors screened CD promoters targeting inhibitory and excitatory neurons in the mouse sensory cerebral cortex. AAVs equipped with the CD promoter were directly injected into the mouse cerebral cortex, and cell-type specificity was evaluated through immunohistochemical assays using PV (inhibitory cell marker) and CaMK2 (excitatory cell marker) antibodies. Injection of AAV containing the hCD1d promoter selectively labeled PV-positive cortical interneurons with a specificity exceeding 94% (Figs. 4A, 4G). Furthermore, AAV having the hCD1d promoter induced little gene expression in CaMK2-positive cortical excitatory neurons (Figs. 4B, 4H). This indicates that when AAV having the hCD1d promoter is directly injected into the cerebral cortex, the hCD1d promoter functions specifically in cortical PV-positive inhibitory neurons. AAV equipped with the mouse CD24 (mCD24) promoter showed 78.5% specificity for PV-positive neurons in the mouse cerebral cortex and very low expression (<1%) in CaMKII-positive excitatory neurons. This suggests that mCD24 is selective for PV-positive neurons and other types of inhibitory neurons (Figs. 4C, 4D, 4G, 4H). Finally, the CD52 promoter induced EGFP expression with a specificity of over 86% in CaMKII-positive excitatory neurons in the cerebral cortex but had low expression in PV-positive neurons (<9%) (Figs. 4E-H). These data suggest that CD promoters can be effectively utilized to promote cell-type specific gene expression in both the cerebellum and cerebral cortex.

[0069] Example 4 In vivo calcium imaging of cerebellar Golgi cells: The present inventors examined the usefulness of an AAV vector containing a CD promoter in research for detecting neural activity patterns. Using an AAV vector containing HGT017_hCD9 and miR133a_targeting sequences, expression of jGCaMP8s in cerebellar Golgi cells was induced. One week after AAV injection, jGCaMP8s signals were selectively observed in Golgi cells expressing neuropeptide (Figure 5A). These jGCaMP8s signals were not present in Car8-positive Purkinje cells and the molecular layer (Figure 5B), confirming the specificity of jGCaMP8s expression for Golgi cells. When in vivo calcium imaging was performed one week after AAV injection, it was found that Golgi cells expressing jGCaMP8s showed significant fluorescence changes indicating spontaneous Ca2 + fluctuations (Figures 5C–D). These results indicate that an AAV vector equipped with a CD promoter is an effective tool for monitoring neural activity.

[0070] Example 5 Regulation of behavior by manipulation of specific cell types in the cerebellum using CD promoter-based AAV: The present inventors investigated the functional usefulness of these AAV vectors. The cerebellum is essential for higher cognitive functions including motor control, emotion, and social behavior. The roles of molecular layer interneurons and Golgi cells in these cerebellar functions were examined. Motor tests were performed using AAV-RE16_CD68-M3Dq-mcherry and AAV-HGT017_CD9-DIOM3Dq-mcherry. Intraperitoneal injection of CNO into mice infected with AAV-RE16_CD68-M3Dq-mcherry significantly decreased the time spent in the center of the box compared to injection of PBS into mice infected with the same virus (control mice), suggesting increased anxiety due to activation of molecular layer interneurons in the cerebellum (Figure 6A). In mice infected with AAVRE16_CD68-M3Dq-mcherry, intraperitoneal injection of CNO significantly increased sociability (Figure 6B). Injection of CNO into mice with RE16-CD68-M3Dqmcherry improved motor ability, presumably due to enhanced motor learning compared to control mice (Figure 6C).

[0071] In mice infected with AAV-HGT017_CD9-M3Dq-mcherry, CNO administration caused a decrease in locomotor activity and an increase in anxiety, as indicated by a decrease in the total distance traveled over 10 minutes and a decrease in the time spent at the center of the box, compared to PBS injection into mice infected with AAV-HGT017_CD9-M3Dq-mcherry (Figure 6D). Furthermore, CNO injection into mice with HGT017-CD9-M3Dqmcherry significantly decreased sociability (Figure 6E). CNO in HGT017-CD9-M3Dqmcherry mice improved motor ability in the first trial of the rotarod test but decreased performance in the last trial compared to control mice (Figure 6F). These results potentially highlight the diverse roles of molecular layer interneurons and Golgi cells in the cerebellum.

[0072] Example 6 Rescue of social deficits in ASD model mice by CD promoter-based AAV: Since it was found that injecting CNO into mice with AAV-RE16_CD68-M3Dq-mcherry improved social behavior (Figure 6), the therapeutic potential of CD promoter-based AAV vectors in improving social deficits and motor impairments in autism spectrum disorder (ASD) model mice was investigated. To generate ASD model mice and control mice, on the 3rd to 4th day after birth, AAV carrying L7-iCre or L7-EGFP respectively was injected into the cerebellum of TSC1flox mice. At 8 - 9 weeks of age, these mice were further administered AAV-RE16_CD68-M3Dq-mcherry into the cerebellum. The behaviors of three mouse groups were compared: 1) control mice (TSC1flox mice infected with AAVL7-EGFP and AAV-RE16_CD68-M3Dq-mcherry, PBS applied before behavioral tests), 2) TSC1 conditional knockout mice (infected with AAV-L7-iCre and AAVRE16_CD68-M3Dq-mcherry, PBS applied before behavioral tests), 3) TSC1 conditional knockout mice with M3Dq activation (infected with L7-iCre and RE16_CD68-M3Dq-mcherry, CNO administered before behavioral tests). Our results confirmed that TSC1 conditional knockout mice had a reduced preference for unfamiliar mice compared to control mice (Figures 7A, 7B). We investigated the effect of M3Dq activation of molecular layer interneurons on the social behavior of TSC1 conditional knockout mice. M3Dq activation by injecting CNO into TSC1 conditional knockout mice resulted in a partial but significant improvement in social deficits compared to TSC1 conditional knockout mice administered PBS (Figures 7A, 7B). Furthermore, attempts were also made to rescue the motor impairments seen in TSC1 conditional knockout mice. The rotarod test was used to confirm the motor function deficits in TSC1 conditional knockout mice (Figure 7C). The motor functions between TSC1 conditional knockout mice with and without M3Dq activation of molecular layer interneurons in the cerebellum were compared.M3Dq activation in TSC1 conditional knockout mice significantly improved motor function compared to mice without M3Dq activation (Figure 7C). These results suggest that activation of molecular layer interneurons improves social and motor deficits in TSC1 conditional knockout mice.

[0073] Discussion: In the present invention, we successfully developed cell-type specific adeno-associated virus (AAV) with a differentiation cluster (CD) gene promoter targeting specific cell types in the mouse cerebellum and cerebral cortex. Our data highlight the utility of these CD promoter-based AAVs for elucidating the unique contributions of different cell types to various neural functions such as social interaction, motor ability, and emotional processing. Furthermore, we propose these special AAVs as potential therapeutic agents for treating brain diseases such as autism spectrum disorder (ASD).

[0074] Our CD promoter-based AAVs showed remarkable cell-type specificity. For example, the CD68 promoter provided more than 90% specificity in inducing reporter gene expression in cerebellar molecular layer interneurons. The level of specificity is comparable to that achieved by the GAD65 promoter [Non-Patent Document 3]. Similarly, the CD1d and CD52 promoters in the cerebral cortex showed high specificity equivalent to established promoters and enhancers such as the GAD65 promoter [Non-Patent Document 3], the Scn1a enhancer [Non-Patent Document 6], and the CaMKII promoter. Combining CD promoters with enhancer elements and miRNA targeting sequences further improved cell-type specificity. Such composite regulatory DNA sequences can significantly enhance the accuracy of AAV-mediated gene manipulation in target cell types, enhancing the applicability and utility of our approach.

[0075] In contrast to transgenic animals, in the studies of the present inventors, AAV vectors are utilized as a simpler and more cost-effective alternative. This will accelerate the establishment of input-output relationships between different cell types, the roles of each cell type in brain function and development, the dynamics of cell-cell interactions, and new therapies for brain diseases. However, there are limitations to AAV vectors. AAV vectors can only accommodate genetic material of limited size. In this invention, a CD promoter in the range of 700 bp to 2000 bp in length was utilized. In particular, a 2000-bp promoter is relatively long for AAV vector packaging. Researchers need to carefully consider the size and number of genes to be expressed when selecting an appropriate vector system for research or therapeutic purposes. When attempting to express a large gene or multiple genes with a single vector, the limited size of the genetic material can be a challenge. Recent strategies have been reported to avoid size constraints, such as using multiple vectors or fragmenting large genes into smaller genes for packaging in independent vectors and then reconstructing them within target cells.

[0076] Using CD promoter-based AAV, the different and common roles of molecular layer interneurons and Golgi cells in the regulation of cerebellar functions such as anxiety, sociability, and motor control were revealed. The different roles may arise from the types of cells targeted by each interneuron. Molecular layer interneurons can directly regulate cerebellar output by suppressing Purkinje cells, while Golgi cells can suppress input to the cerebellum by regulating granule cells. Activation of molecular layer interneurons may enhance the reward value or salience of social stimuli and increase social motivation. The increase in anxiety due to activation of molecular layer interneurons may reflect an elevated state of alertness or arousal that promotes social and motor behaviors. Conversely, activation of Golgi cells may cause an increase in anxiety and a decrease in motor activity, which may reflect a state of reduced motivation and exploratory drive that has an adverse effect on social and motor behaviors.

[0077] The inventors have demonstrated that the activation of molecular layer interneurons in the cerebellum improves social deficits in ASD model mice. This finding is consistent with previous studies suggesting that cerebellar dysfunction is involved in the pathophysiology of ASD. Activation of molecular layer interneurons may restore the balance of excitation and inhibition in the cerebellum, thereby enhancing communication with other brain regions important for social behavior. It is worth noting that the specificity of CD promoter-based AAVs may vary between different animal models and human patients. Further optimization and validation steps are required before considering clinical applications.

[0078] Furthermore, the inventors' research expands the utility of the CD promoter by demonstrating its scalability in targeting specific cell types in the cerebellum and cerebral cortex. The CD gene is mainly known as a cell surface molecule of leukocytes and other immune cells, and has broad applicability considering the diversity of CD antigens (over 370 CD antigens have been identified in humans). Since the CD gene is also expressed in other organs such as blood cells and immune cells, AAVs using the CD promoter can affect not only neuroscience but also many fields such as immunology and hematology.

[0079] In conclusion, the inventors designed a series of AAVs equipped with CD promoters that can accurately target specific cell populations. These CD promoter-based AAV vectors can be further customized by incorporating various enhancers and miRNA targeting sequences. The inventors' research paves the way for accessing and manipulating specific cell types in both basic and translational research.

Claims

1. A vector for expressing a predetermined gene in cells in the cerebellum or cerebral cortex, the vector comprising a promoter of a CD gene and the predetermined gene.

2. The vector according to claim 1, wherein the cerebellum is the gray matter of the cerebellar cortex.

3. The vector according to claim 1, wherein the cerebellum is the outer molecular layer, the central Purkinje cell layer, or the inner granule cell layer.

4. The vector according to claim 1, wherein the cells in the cerebellum or cerebral cortex are cerebellar molecular layer interneurons, inhibitory neurons, excitatory neurons, parvalbumin-positive inhibitory neurons, Golgi cells, Purkinje cells, Lugaro cells, or astrocytes.

5. The vector according to claim 4, wherein the astrocyte is Bergmann glia.

6. The vector according to claim 1, wherein the CD gene is a CD1 gene, a CD9 gene, a CD24 gene, a CD34 gene, a CD38 gene, a CD44 gene, a CD52 gene, a CD68 gene, a CD74 gene, a CD81 gene, a CD83 gene, a CD164 gene, a CD276 gene, or a CD300 gene.

7. The vector according to claim 1, comprising an enhancer.

8. The vector according to claim 7, wherein the enhancer is an mscRE16 enhancer, an S5E6 enhancer, or an HGT017 enhancer.

9. The vector according to claim 1, comprising a miRNA targeting sequence.

10. The vector according to claim 9, wherein the miRNA is miR-133a, miR-1188, miR-1983, or miR-3086-5p.

11. The vector according to claim 1, wherein the predetermined gene encodes a metabotropic receptor.

12. The vector according to claim 1, wherein the predetermined gene encodes a G protein-coupled receptor.

13. The vector according to claim 1, wherein the predetermined gene is an M3Dq gene.

14. The vector according to claim 1, wherein the vector is a viral vector.

15. The vector according to claim 1, wherein the viral vector is an adeno-associated viral vector.

16. The vector according to claim 1, for treating a deficiency of an expression product of a predetermined gene in cells in the cerebellum or cerebral cortex.

17. The vector according to claim 1, which is a vector for treating brain diseases.

18. The vector according to claim 1, which is a vector for treating autism spectrum disorder, epilepsy, tuberous sclerosis, spinocerebellar degeneration, schizophrenia, or cerebellar dysfunction.

19. The vector according to claim 1, which is for use in combination with a substance that interacts with the expression product of a predetermined gene.

20. The vector according to claim 19, wherein the substance that interacts with the expression product of a predetermined gene is a clozapine metabolite.

21. The vector according to claim 20, wherein the clozapine metabolite is clozapine N-oxide.

22. A pharmaceutical composition comprising the vector according to claim 1.

23. The vector according to claim 1, which is a vector for selectively activating or inactivating cells in the cerebellum or cerebral cortex.

24. A pharmaceutical composition for treating autism spectrum disorder, epilepsy, tuberous sclerosis, spinocerebellar degeneration, schizophrenia, or cerebellar dysfunction, comprising the vector according to claim 1.

25. A pharmaceutical composition for treating autism spectrum disorder, comprising the vector according to claim 1, wherein the predetermined gene is the M3Dq gene.

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