Inhibitory neuronal-specific promoters

Abstract

An inhibitory neuron specific promoter sequence is found, the length of the inhibitory neuron specific promoter sequence is shorter than that of an mGAD65 promoter, but the activity of the promoter, the specificity of the inhibitory neuron and the gene expression efficiency are not lost. Specifically, the present invention relates to a promoter comprising DNA having promoter activity, said DNA comprising: a base sequence of a Dlx1 binding site and / or a Dlx2 binding site in a glutamate decarboxylase (GAD) promoter; and a base sequence in a glutamate decarboxylase (GAD) that extends from the 5'terminal of exon 1 to a transcription initiation site (TSS).

Description

Technical Field

[0001] The present invention relates to an inhibitory neuron-specific promoter. Background Art

[0002] The dysfunction of inhibitory neurons or the abnormal balance between excitation and inhibition in the brain are closely related to the onset of epilepsy, autism, schizophrenia, etc. These dysfunctions or imbalances may be treated by introducing therapeutic genes into inhibitory neurons to regulate their functions.

[0003] In recent years, adeno-associated virus (hereinafter referred to as AAV) vectors have been widely used in the study of gene expression in organisms, and are also useful for long-term gene expression in non-dividing cells such as neurons. However, AAV is also the smallest virus among viruses, and the gene size that can be packaged in AAV vectors is as short as about 5.0kb, including the inverted terminal repeat (ITR) sequences at both ends. Therefore, the difficulty lies in the size and number of genes that can be inserted into AAV vectors.

[0004] Until now, it has been reported that the distal-less homeobox (Dlx) gene enhancer sequence (which functions in combination with a minimal promoter located downstream) can be carried in an AAV vector to express a foreign gene specifically in inhibitory neurons of the forebrain (Non-Patent Document 1).

[0005] The present inventors have discovered that the 2.5 kb upstream of exon 1 of the Gad2 gene encoding glutamate decarboxylase (GAD) 65 in the mouse genome functions as an inhibitory neuron-specific promoter (mGAD65 promoter) (Non-Patent Document 2).

[0006] In addition, it was found that the mouse-derived Dlx enhancer (mDlx enhancer) has promoter activity specifically for inhibitory neurons by combining with a minimal promoter, but has promoter activity only in the brain region (forebrain), whereas the mGAD65 promoter has promoter activity in the whole brain (Non-patent Document 2). Compared with the mDlx enhancer + minimal promoter, the mGAD65 promoter has the same promoter activity in the brain region, but is superior in terms of specificity and efficiency for inhibitory neurons.

[0007] On the other hand, in terms of length, the mDlx enhancer + minimal promoter is as short as about 0.7 kb, while the mGAD65 promoter is as long as about 2.5 kb, which is about half of the packaging limit of the AAV vector. The disadvantage is that the usefulness of the AAV vector is greatly lost.

[0008] Prior art literature

[0009] Non-patent literature

[0010] Non-patent literature 1: Nat Neurosci., 2016; 19(12): 1743-1749

[0011] Non-patent literature 2: Mol Brain., 2021; 14(1): 33

[0012] Non-patent literature 3: Nat Biotechnol., 2016; 34(2): 204-209

[0013] Non-patent literature 4: J Comp Neurol., 2018; 526(3): 373-396 Summary of the invention

[0014] Problems to be solved by the invention

[0015] The technical problem of the present invention is to obtain an inhibitory neuron-specific promoter sequence, which is shorter than the mGAD65 promoter but does not lose promoter activity, specificity for inhibitory neurons and gene expression efficiency.

[0016] Solutions for solving problems

[0017] In order to solve the above technical problems, in-depth research was conducted. In the study, first, the genomic regions of the mGAD65 promoter and the mGAD67 promoter were cloned with reference to the mGAD65 promoter described in non-patent literature 2. Next, deletion constructs of various regions of the mGAD65 promoter and the mGAD67 promoter were made, and cross-blood-brain barrier (BBB) ​​AAV (AAV-PHP.eB) vectors expressing green fluorescent protein (GFP) under the control of their respective promoter sequences were made. The obtained AAV vector was administered intravenously to mice, and the expression level of GFP and the expression characteristics on inhibitory neurons were analyzed after 3 weeks.

[0018] In the mGAD65 promoter region, the site to which Dlx1 and Dlx2, which are transcription activators, bind is focused. Dlx1 and Dlx2 bind directly to the GAD promoter and activate transcription. Since the GAD promoter regions reported to be bound by Dlx1 and Dlx2 are Region i and Region ii (hereinafter also referred to as Ri and Rii, respectively), these two regions are retained and the other regions are deleted from the mGAD65 promoter, or either Ri or Rii is retained and deletion treatment is applied to the other regions. It was found that in addition to this deletion treatment, in order to maintain a higher promoter activity, it is particularly important to include a region from the 5' end of exon 1 to the transcription start site (Transcription start site: hereinafter also referred to as TSS) in the Gad1 gene encoding GAD67 or the Gad2 gene encoding GAD65, which is not present in the original mGAD65 promoter (hereinafter also referred to as E TSS region) as a promoter.

[0019] That is, the present invention is as follows.

[0020] [1] A promoter comprising a DNA having promoter activity, the DNA comprising: a base sequence of a Dlx1 binding site and / or a Dlx2 binding site in a glutamate decarboxylase (GAD) promoter; and a region (E) from the 5' end of exon 1 to the transcription start site (TSS) in a glutamate decarboxylase (GAD) promoter. TSS region) base sequence.

[0021] [2] The promoter according to [1], wherein the glutamate decarboxylase (GAD) promoter is a mouse-derived GAD65 promoter (mGAD65 promoter), the glutamate decarboxylase (GAD) is a mouse-derived glutamate decarboxylase 65 (mGAD65), the base sequence of the Dlx1 binding site and / or the Dlx2 binding site comprises at least one base sequence selected from the base sequence of Region i of the mGAD65 promoter shown in SEQ ID NO: 1, the base sequence of Region ii of the mGAD65 promoter shown in SEQ ID NO: 2, and a base sequence that is 90% or more identical to the base sequence shown in any of them, and the region (E) from the 5' end of exon 1 to the transcription start site (TSS) in the mGAD65. TSS The base sequence of the region) is the base sequence represented by SEQ ID NO: 3, or a base sequence that is 90% or more identical to the base sequence.

[0022] [3] The promoter according to [1] or [2], comprising a base sequence represented by any one of SEQ ID NOs: 4 to 6, or a base sequence having 90% or more identity with a base sequence represented by any one of SEQ ID NOs: 4 to 6.

[0023] [4] The promoter according to [1], wherein the glutamate decarboxylase (GAD) promoter is a mouse-derived GAD67 promoter (mGAD67 promoter), the glutamate decarboxylase (GAD) is a mouse-derived glutamate decarboxylase 67 (mGAD67), the base sequence of the Dlx1 binding site and / or the Dlx2 binding site comprises at least one base sequence selected from the base sequence of Region i of the mGAD67 promoter shown in SEQ ID NO: 7, the base sequence of Region ii of the mGAD67 promoter shown in SEQ ID NO: 8, and a base sequence that is 90% or more identical to the base sequence shown in any of them, and the region (E) from the 5' end of exon 1 to the transcription start site (TSS) in the mGAD67. TSS region) is the base sequence represented by SEQ ID NO: 9, or a base sequence that is 90% or more identical to the base sequence.

[0024] [5] The promoter according to [1] or [4], comprising a base sequence represented by any one of SEQ ID NOs: 10 to 12, or a base sequence having a sequence that is 90% or more identical to a base sequence represented by any one of SEQ ID NOs: 10 to 12.

[0025] [6] An adeno-associated virus vector for gene expression in inhibitory neurons or a recombinant virus obtained from the adeno-associated virus vector, comprising the promoter according to any one of [1] to [5].

[0026] [7] The adeno-associated virus vector or the recombinant virus obtained from the adeno-associated virus vector according to [6], further comprising a target gene configured under the control of the promoter.

[0027] [8] A medicine comprising the adeno-associated virus vector as described in [7] or a recombinant virus obtained from the adeno-associated virus vector.

[0028] [9] The medicine according to [8], wherein the medicine is used to treat central nervous system diseases caused by dysfunction of inhibitory neurons or abnormal balance between excitation and inhibition.

[0029]

[10] A method for introducing a gene into a cell, comprising the following steps: in vitro, using the adeno-associated virus vector described in [7] or a recombinant virus obtained from the adeno-associated virus vector, to introduce a target gene into a target cell.

[0030]

[11] The method according to

[10] , wherein the target cell is an inhibitory neuron.

[0031]

[12] A method for treating a central nervous system disease caused by dysfunction of inhibitory neurons or abnormal balance between excitation and inhibition, comprising: administering the adeno-associated virus vector as described in [7] or a recombinant virus obtained from the adeno-associated virus vector to a subject in need of the adeno-associated virus vector.

[0032]

[13] The adeno-associated virus vector or the recombinant virus obtained from the adeno-associated virus vector according to [7], wherein the adeno-associated virus vector or the recombinant virus obtained from the adeno-associated virus vector is used to treat central nervous system diseases caused by dysfunction of inhibitory neurons or abnormal balance between excitation and inhibition.

[0033]

[14] Use of an adeno-associated virus vector as described in [7] or a recombinant virus obtained from the adeno-associated virus vector in the manufacture of a medicine for treating central nervous system diseases caused by dysfunction of inhibitory neurons or abnormal balance between excitation and inhibition.

[0034] Effects of the Invention

[0035] In the conventional mGAD65 promoter (about 2.5 kb), it occupies about half of the packaging limit of the AAV vector, which is a big problem. However, according to the present invention, a shortened mGAD65 promoter and mGAD67 promoter can be provided. In particular, compactmGAD65 (also called cmGAD65, mGAD65 (Rii-E TSS )) promoter and compact mGAD67 (also known as cmGAD67, mGAD67 (Rii-E TSS )) promoters have been successfully shortened to less than 0.5 kb. Moreover, by using an AAV vector equipped with the promoter of the present invention, it is possible to express the target gene in inhibitory neurons throughout the central nervous system (CNS) to the same extent or more strongly than the conventional mGAD65 promoter while maintaining the specificity for inhibitory neurons and gene expression efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 It is a schematic diagram of the gene sequence of the shortened mGAD65 promoter series.

[0037] Figure 2 It is a schematic diagram of the gene sequence of the shortened mGAD67 promoter series.

[0038] Figure 3This is a graph showing the relative values ​​of GFP fluorescence intensity when a gene expression experiment in the cerebral cortex was performed using various shortened mGAD promoters.

[0039] Figure 4 This is a diagram showing the specificity of gene expression in a gene expression experiment in the cerebral cortex using various shortened mGAD promoters.

[0040] Figure 5 This is a graph showing the gene expression efficiency in the gene expression experiment in the cerebral cortex using various shortened mGAD promoters.

[0041] Figure 6 This is a graph showing the mRNA expression levels in the cerebral cortex when a gene expression experiment was conducted using various shortened mGAD promoters. DETAILED DESCRIPTION

[0042] <Promoter>

[0043] One embodiment of the present invention is a promoter, which is composed of a DNA having promoter activity, wherein the DNA comprises: a base sequence of a Dlx1 binding site and / or a Dlx2 binding site in a glutamate decarboxylase (GAD) promoter; and a region (E) from the 5' end of exon 1 to the transcription start site TSS in a glutamate decarboxylase (GAD) promoter. TSS region) base sequence.

[0044] Glutamate decarboxylase (GAD) is an enzyme that synthesizes GABA (gamma-aminobutylic acid), and two isotypes (GAD65 and GAD67) are known to exist. The promoters of these glutamate decarboxylase (GAD) are GAD65 promoter and GAD67 promoter, respectively.

[0045] The animal from which the glutamate decarboxylase (GAD) promoter of the present embodiment is derived is not particularly limited as long as it is a mammal having the promoter, and examples thereof include mice, rats, and marmosets. Hereinafter, the mouse-derived GAD65 promoter and GAD67 promoter are also referred to as mGAD65 promoter and mGAD67 promoter, respectively.

[0046] The base sequences contained in the promoter of the present embodiment preferably include, in order: (1) the base sequence of the Dlx1 binding site and / or the Dlx2 binding site, (2) the E TSS The base sequence of the region may be the reverse order as long as it has promoter activity.

[0047] The promoter of this embodiment only needs to have the base sequence of the Dlx1 binding site and / or the Dlx2 binding site and E TSS The base sequence of the region may be any base sequence, and other regions in the base sequence of glutamate decarboxylase (GAD) may be further included as long as it can function as a promoter. In addition, any base sequence may be further included for the purpose of enhancing promoter activity or stabilizing promoter activity.

[0048] As the mGAD65 promoter, for example, the promoter shown in GenBank no. AB032757 can be used, and as the mGAD67 promoter, for example, the promoter shown in GenBank no. z49978 can be used. As for the sequences of these promoters, as long as the promoter of the present embodiment can function as a promoter, substitution, deletion, addition, or modification of about 10% or less of the base sequence (for example, about 1 to 10 bases or about 1 to 100 bases) may occur.

[0049] The Dlx1 binding site and / or the Dlx2 binding site exist in each of the GAD65 promoter and the GAD67 promoter.

[0050] Although not particularly limited, for example, when the GAD65 promoter is the promoter shown in GenBank No. AB032757, it may be Region i, which is 2294 to 2088 nucleotides upstream of the transcription start site (TSS) of mGAD65, or Region ii, which is 958 to 598 nucleotides upstream of the 5' side.

[0051] Alternatively, when the GAD65 promoter is a promoter having a base sequence shown in sequence number 13, for example, the base sequence of Region i of the mGAD65 promoter may be the base sequence shown in sequence number 1, and the base sequence of Region ii of the mGAD65 promoter may be the base sequence shown in sequence number 2. It should be noted that, as long as the promoter in the present embodiment can function as a promoter, substitution, deletion, addition, or modification of less than about 10% (for example, about 1 to 10 bases or about 1 to 20 bases) of the base sequence may occur.

[0052] Although not particularly limited, for example, when the GAD67 promoter is the promoter shown in GenBank no. z49978, it may be Region i, which is 966 to 692 nucleotides upstream of the transcription start site (TSS) of mGAD67, or Region ii, which is 410 to 142 nucleotides upstream of the 5' side.

[0053] Alternatively, when the GAD67 promoter is a promoter having a base sequence shown in SEQ ID NO: 14, for example, the base sequence of Region i of the mGAD67 promoter may be the base sequence shown in SEQ ID NO: 7, and the base sequence of Region ii of the mGAD67 promoter may be the base sequence shown in SEQ ID NO: 8. It should be noted that, as long as the promoter in the present embodiment can function as a promoter, substitution, deletion, addition, or modification of about 10% or less (for example, 1 to 10 bases or 1 to 20 bases) of the base sequence may occur.

[0054] In this embodiment, when the glutamate decarboxylase (GAD) promoter is a GAD65 promoter (mGAD65) derived from mice, the base sequence of the Dlx1 binding site and / or the Dlx2 binding site may include at least one base sequence selected from the base sequence of Region i of the mGAD65 promoter shown in SEQ ID NO: 1, the base sequence of Region ii of the mGAD65 promoter shown in SEQ ID NO: 2, and a base sequence that is more than 90%, preferably more than 95%, and more preferably more than 98% identical to the base sequence shown in any of them.

[0055] Furthermore, when the glutamate decarboxylase (GAD) is mouse-derived GAD65 (mGAD65), the promoter may be a region (E TSS The base sequence of the region) is the base sequence represented by SEQ ID NO: 3 or a base sequence that is 90% or more, preferably 95% or more, and more preferably 98% or more identical to the base sequence.

[0056] In the promoter of this embodiment, each base sequence preferably includes the following in order: (1) the base sequence of Region i, and (2) the base sequence of Region ii. However, as long as the promoter of this embodiment can function as a promoter, the order may be the opposite.

[0057] In addition, as long as it can function as a promoter, the promoter of the present embodiment may further include other regions in the base sequence of glutamate decarboxylase (GAD) before the base sequence of Region i, between the base sequence of Region i and the base sequence of Region ii, or after the base sequence of Region ii. In addition, for the purpose of enhancing promoter activity, any base sequence may also be further included.

[0058] The promoter of the present embodiment may include a base sequence shown in any one of sequence numbers 4 to 6, or include a base sequence having a sequence that is 90% or more, preferably 95% or more, and more preferably 98% or more identical to the base sequence shown in any one of sequence numbers 4 to 6. In addition, the promoter of the present embodiment may also be composed of a base sequence shown in any one of sequence numbers 4 to 6. The base sequence of sequence number 4 is when the glutamate decarboxylase (GAD) promoter is a mouse-derived GAD65 promoter (mGAD65), indicating Ri-Rii-D-E TSS The base sequence of SEQ ID NO: 5 is a sequence representing Ri-Rii-E when the glutamate decarboxylase (GAD) promoter is a mouse-derived GAD65 promoter (mGAD65). TSS The base sequence of sequence number 6 is a representation when the glutamate decarboxylase (GAD) promoter is a mouse-derived GAD65 promoter (mGAD65). TSS It should be noted that, in this specification, the region upstream of Ri in mGAD65 is sometimes referred to as U (Upstream), the region between Ri and Rii is referred to as M (Middle), and the region downstream of Rii and upstream of the 5' end of exon 1 is referred to as D (Downstream).

[0059] In this embodiment, when the glutamate decarboxylase (GAD) promoter is a GAD67 promoter (mGAD67) derived from mice, the base sequence of the Dlx1 binding site and / or the Dlx2 binding site may include at least one base sequence selected from the base sequence of Region i of the mGAD67 promoter shown in SEQ ID NO: 7, the base sequence of Region ii of the mGAD67 promoter shown in SEQ ID NO: 8, and a base sequence that is more than 90%, preferably more than 95%, and further preferably more than 98% identical to the base sequence shown in any of them.

[0060] Furthermore, when the glutamate decarboxylase (GAD) is mouse-derived GAD67 (mGAD67), the promoter may be a region (E TSS The base sequence of the region) is the base sequence represented by SEQ ID NO: 9 or a base sequence that is 90% or more, preferably 95% or more, and more preferably 98% or more identical to the base sequence.

[0061] In the promoter of this embodiment, each base sequence preferably includes the following in order: (1) the base sequence of Region i, and (2) the base sequence of Region ii. However, as long as the promoter of this embodiment can function as a promoter, the order may be the opposite.

[0062] In addition, as long as it can function as a promoter, the promoter of the present embodiment may further include other regions in the base sequence of glutamate decarboxylase (GAD) before the base sequence of Region i, between the base sequence of Region i and the base sequence of Region ii, or after the base sequence of Region ii. In addition, for the purpose of enhancing promoter activity, any base sequence may also be further included.

[0063] In addition, in the promoter of this embodiment, Region ii and E TSS Part of the region can overlap, that is, the base sequence of Region ii can be shared with E TSS When the glutamate decarboxylase (GAD) promoter is a mouse-derived GAD67 promoter (mGAD67), it is preferred that Region ii and E TSS The region partially overlaps, that is, the base sequence of Region ii is preferably shared with E TSS A portion of the base sequence of a region.

[0064] The promoter of the present embodiment may include a base sequence shown in any one of sequence numbers 10 to 12, or include a base sequence having a sequence that is 90% or more, preferably 95% or more, and more preferably 98% or more identical to the base sequence shown in any one of sequence numbers 10 to 12. In addition, the promoter of the present embodiment may be composed of a base sequence shown in any one of sequence numbers 10 to 12. The base sequence of sequence number 10 is when the glutamate decarboxylase (GAD) promoter is a mouse-derived GAD67 promoter (mGAD67), indicating Ri-M-Rii-E TSS The base sequence of SEQ ID NO: 11 is a sequence representing Ri-Rii-E when the glutamate decarboxylase (GAD) promoter is a mouse-derived GAD67 promoter (mGAD67). TSS The base sequence of SEQ ID NO: 12 is a sequence indicating Rii-E when the glutamate decarboxylase (GAD) promoter is a mouse-derived GAD67 promoter (mGAD67). TSSIt should be noted that, in this specification, the region upstream of Ri in mGAD67 is sometimes referred to as U (Upstream), the region between Ri and Rii is referred to as M (Middle), and the region downstream of Rii and upstream of the 5' end of exon 1 is referred to as D (Downstream).

[0065] The method for producing the promoter of the present embodiment is not particularly limited, and the promoter can be produced by gene recombination techniques known to those skilled in the art.

[0066] <Vector or recombinant virus obtained from the vector>

[0067] Another embodiment of the present invention is an adeno-associated virus vector for gene expression in inhibitory neurons comprising the above-mentioned promoter, or a recombinant virus obtained from the adeno-associated virus vector.

[0068] It is known to those skilled in the art that there are multiple serotypes of adeno-associated virus (AAV). In the present embodiment, the serotype of AAV from which the AAV vector is derived is not particularly limited as long as it can cause inhibitory neurons to express the target gene.

[0069] The vector of this embodiment may further contain a target gene configured under the control of the above-mentioned promoter, i.e., linked to the above-mentioned promoter. The type of the target gene linked to the promoter is not particularly limited, and examples thereof include: fluorescent protein genes such as GFP (including modified forms), Cre recombinase genes, luciferase, chloramphenicol acetyltransferase, lacZ and other marker genes, Ca sensing genes, and the like. 2+ Or protein genes that produce fluorescence from intracellular second messengers such as cyclic AMP, light-activated proteins, chemical genetic tools such as DREADD, and toxin genes. In addition, it can be a gene for treating central nervous system diseases caused by abnormal function of inhibitory neurons or abnormal balance between excitation and inhibition.

[0070] Examples of central nervous system diseases caused by abnormal function of inhibitory neurons or abnormal balance between excitation and inhibition include epilepsy, autism, schizophrenia, etc. Genes having therapeutic effects on these diseases can be appropriately selected depending on the type of disease to be treated.

[0071] The vector of this embodiment may be a vector in which these target genes are arranged in advance under the control of the above-mentioned promoter, and further, a multicloning site for integrating these target genes may be arranged downstream of the above-mentioned promoter as needed.

[0072] The vector of this embodiment may contain other functional genes, for example, may further contain a post-transcriptional regulatory factor (WPRE) and / or a polyadenylation signal (polyA) sequence derived from woodchuck hepatitis virus.

[0073] WPRE has the function of preventing the read-through of polyA sequences, promoting RNA processing and maturation, and increasing RNA transport outside the nucleus. In addition, WPRE has the function of acting on viral genome transcripts in packaging cells to promote vector packaging and increase viral titer. Moreover, WPRE promotes the maturation of mRNA produced by the internal promoter of the vector, and thus has the function of enhancing the expression of the target gene in the target cell into which the gene is introduced.

[0074] The vector 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 is introduced, and is generally a gene whose gene product inhibits cell proliferation or confers resistance to a cell-killing agent. Specifically, for example, Neo gene, Hyg gene, hisD gene, Gpt gene and Ble gene can be listed. As drugs useful for selecting the presence of a selection marker, for example, G418 can be listed for Neo, hygromycin can be listed for Hyg, histidinol can be listed for hisD, xanthine can be listed for Gpt, and bleomycin can be listed for Ble.

[0075] The vector of the present embodiment may contain inverted terminal repeat (ITR) sequences at both ends of the expression unit containing the above-mentioned primers, target gene, WPRE, polyA and other base sequences.

[0076] The vector of the present embodiment may be a vector consisting only of the base sequence shown in sequence numbers 15 to 20, or a vector further comprising a base sequence other than the base sequence shown in sequence numbers 15 to 20. In addition, as long as the promoter and the target gene function, the vector may be a vector comprising a base sequence having a sequence that is 90% or more identical to the base sequence shown in sequence numbers 15 to 20, or a vector comprising a base sequence having a sequence that is preferably 95% or more identical, and more preferably 98% or more identical to the base sequence shown in sequence numbers 15 to 20. It should be noted that the base sequence shown in sequence numbers 15 to 20 is shown from the 5' end to the 3' end, starting from the base sequence of the promoter, so that the plasmid is surrounded by one circle.

[0077] The method of integrating the above-mentioned various genes into the AAV vector sequence can be carried out by methods known to those skilled in the art. There is no particular limitation, for example, various genes can be integrated into the vector sequence by a method using restriction enzyme treatment.

[0078] As a gene introduction method, a known method using an AAV vector can be used.

[0079] Specifically, pAAV plasmids, pRC plasmids, and auxiliary plasmids can be introduced into packaging cells such as HEK293 cells, cultured for a specified period of time, and after forming recombinant viruses, recombinant virus particles can be recovered from the culture supernatant. The culture supernatant containing the recombinant virus particles itself can also be used as a recombinant virus, and can also be used as a recombinant virus by further undergoing a concentration and / or purification process. The recombinant virus can be used as a recombinant virus solution in a state dissolved in a solvent. The recombinant virus obtained in this way can infect the inhibitory neurons of the target.

[0080] <Medicine>

[0081] Another embodiment of the present invention is a medicine comprising any of the above-mentioned adeno-associated viral vectors or a recombinant virus obtained from the adeno-associated viral vector.

[0082] The medicine of this embodiment can be used to treat central nervous system diseases caused by abnormal function of inhibitory neurons or abnormal balance between excitation and inhibition. Examples of central nervous system diseases caused by abnormal function of inhibitory neurons or abnormal balance between excitation and inhibition include epilepsy, autism, schizophrenia, etc.

[0083] The content of the active ingredient in the medicine of this embodiment is not particularly limited as long as a therapeutic effect can be obtained, and can be appropriately determined according to the type of disease, the severity of the disease, the age and weight of the patient, the expression efficiency, and the like.

[0084] The usage and dosage of the medicine of this embodiment are not particularly limited as long as a therapeutic effect can be obtained, and can be appropriately determined according to the type of disease, the severity of the disease, the age, weight, expression efficiency, etc. of the patient.

[0085] The medicine of this embodiment may contain other active ingredients, and may be used in combination with a drug for treating a disease of the central nervous system, for example.

[0086] As another embodiment of the present invention, for example, the following can be cited.

[0087] A method for treating central nervous system diseases caused by dysfunction of inhibitory neurons or abnormal balance between excitation and inhibition, comprising: administering any of the above-mentioned adeno-associated virus vectors or recombinant viruses obtained from the adeno-associated virus vectors to a subject in need of the adeno-associated virus vector.

[0088] An adeno-associated viral vector of any of the above or a recombinant virus obtained therefrom, wherein the adeno-associated viral vector or the recombinant virus obtained therefrom is used in the treatment of central nervous system diseases caused by dysfunction of inhibitory neurons or abnormal balance between excitation and inhibition.

[0089] Use of any of the above-mentioned adeno-associated virus vectors or a recombinant virus obtained from the adeno-associated virus vector in the manufacture of a medicine for treating central nervous system diseases caused by dysfunction of inhibitory neurons or abnormal balance between excitation and inhibition.

[0090] <Gene transfer method>

[0091] Another embodiment of the present invention is a method for introducing a target gene into a target cell in vitro using any of the above-mentioned adeno-associated viral vectors or a recombinant virus obtained from the adeno-associated viral vector. This also includes a method for introducing any of the above-mentioned adeno-associated viral vectors or a recombinant virus obtained from the adeno-associated viral vector into a target cell in vivo in a non-human animal.

[0092] The target cells for introduction of the adeno-associated viral vector or a recombinant virus obtained from the adeno-associated viral vector are inhibitory neurons.

[0093] The method for introducing an adeno-associated viral vector or a recombinant virus obtained from the adeno-associated viral vector into a target cell is not particularly limited, but a method based on the infectivity inherently possessed by the adeno-associated viral vector or a recombinant virus obtained from the adeno-associated viral vector is preferred.

[0094] Example

[0095] The present invention will be described in more detail by way of the following examples, but it goes without saying that the scope of the present invention is not limited to the examples.

[0096] <Method>

[0097] [Promoter]

[0098] The plasmid having the template sequence described later was prepared by cutting with a restriction enzyme (for the plasmid used in this example, an XhoI site was configured upstream of the promoter, and an AgeI site was configured downstream of the promoter), amplifying the desired region (expected to function as a promoter) in the plasmid as a template using upstream (Forward) and downstream (Reverse) primers designed to match the cut surface, and performing an InFusion reaction as a method known to those skilled in the art as needed. The promoter sequence was confirmed using an Applied Biosystems SeqStudio Genetic Analyzer (Thermo Fisher Scientific; Waltham, MA, USA) after sequencing reaction using a BigDye (trademark) Terminatorv3.1 Cycle Sequencing Kit (Thermo Fisher Scientific; Waltham, MA, USA) or a SupreDye (trademark) Cycle Sequencing Kit (EdgeBio; San Jose, CA, USA). The thermal cycle program used in the sequencing reaction was implemented with an initial denaturation step of 96°C for 2 minutes and 25 cycles of 96°C for 10 seconds, 50°C for 5 seconds, and 60°C for 4 minutes. The specific preparation method is as follows.

[0099] mGAD65 (Ri-Rii-D-E TSS ) was prepared by amplifying the DNA of Ri-Rii-D from the template sequence shown in sequence number 21 using upstream and downstream primers (sequence numbers 22 and 23), and amplifying the DNA of E from the template sequence shown in sequence number 24 using upstream and downstream primers (sequence numbers 25 and 26). TSS The DNAs are amplified and then fused by performing an InFusion reaction.

[0100] mGAD65 (Ri-Rii-E TSS ) was prepared by amplifying the DNA of Ri-Rii from the template sequence shown in sequence number 27 using upstream and downstream primers (sequence numbers 28 and 29), and amplifying the DNA of E from the template sequence shown in sequence number 24 using upstream and downstream primers (sequence numbers 25 and 26). TSS The DNA was amplified and then fused by performing an InFusion reaction.

[0101] mGAD65 (Rii-E TSS) was prepared by using upstream and downstream primers (SEQ ID NOs: 31 and 32) to perform Rii-E from the template sequence shown in SEQ ID NO: 30 TSS Amplification of DNA.

[0102] mGAD67 (Ri-M-Rii-E) shown in sequence number 10 TSS ) was prepared by using upstream and downstream primers (SEQ ID NOs: 34 and 35) to perform Ri-M-Rii-E from the template sequence shown in SEQ ID NO: 33. TSS Amplification of DNA.

[0103] mGAD67 (Ri-Rii-E TSS ) was prepared by amplifying the DNA of Ri from the template sequence shown in sequence number 36 using upstream and downstream primers (sequence numbers 37 and 38), and amplifying the DNA of Rii-E from the template sequence shown in sequence number 39 using upstream and downstream primers (sequence numbers 40 and 41). TSS The DNAs are amplified and then fused by performing an InFusion reaction.

[0104] mGAD67 (Rii-E) shown in sequence number 12 TSS ) was prepared by using upstream and downstream primers (SEQ ID NOs: 43 and 44) ​​to perform Rii-E from the template sequence shown in SEQ ID NO: 42 TSS Amplification of DNA.

[0105] [Viral vectors and viruses]

[0106] As a viral vector, AAV-PHP.eB (non-patent document 3) that crosses the BBB type was used. The expression plasmid pAAV containing a polyadenylation signal (polyA) sequence and a post-transcriptional regulator (WPRE) derived from woodchuck hepatitis virus was designed to express GFP under the control of a mouse genomic region upstream of the mouse Gad2 gene encoding glutamate decarboxylase (GAD) 65. The short-chained mGAD65 promoter was inserted upstream of the GFP gene at the restriction enzyme sites of XhoI and AgeI in the pAAV plasmid. pAAV-PHP.eB was constructed based on the pAAV2 / 9 plasmid provided by Dr. James M. Wilson of the University of Pennsylvania (non-patent document 3).

[0107] As for the recombinant single-chain AAV-PHP.eB vector, it is manufactured by simultaneously transfecting three plasmids (i.e., the above-mentioned expression plasmid, pHelper (Stratagene, LaJolla, CA, USA) and packaging plasmid (pAAV-PHP.eB)) into HEK293T cells (HCL4517; Thermo Fisher Scientific; Waltham, MA, USA) using methods known to those skilled in the art.

[0108] To briefly explain the process, first, three plasmids (expression plasmid [pAAV / shortened mGAD65 promoter-EGFP-WPRE-SV40], pHelper (Stratagene, LaJolla, CA, USA), and packaging plasmid (pAAV-PHP.eB)) were transfected into HEK293T cells cultured in Dulbecco's modified Eagle's medium (D-MEM; D5796-500Ml, Sigma-Aldrich, StLouis, MO, USA) supplemented with 8% fetal bovine serum (Sigma-Aldrich) using polyethyleneimine. Viruses were collected from the culture medium 6 days after transfection and concentrated by precipitation using 8% polyethylene glycol 8000 (Sigma-Aldrich) and 500mM sodium chloride. The precipitated AAV-PHP.eB particles were resuspended in D-PBS and purified by continuous gradient centrifugation with iodixanol (OptiPrep; Axis-Shield Diagnostics, Dundee, Scotland). The viral solution was further concentrated in D-PBS using Vivaspin 20 (100,000 MWCO PES, Sartorius, Gottingen, Germany). The genomic titer of the viral vector was determined by real-time quantitative PCR using Power SYBR Green qPCR Mix (Toyobo, Osaka, Japan) with primers 5′-CTG TTG GGC ACT GAC AAT TC-3′ (SEQ ID NO: 45) and 5′-GAA GGG ACG TAG CAGAAG GA-3′ (SEQ ID NO: 46) targeting the WPRE sequence. The expression plasmid was used as a standard.

[0109] [animal]

[0110] Wild-type mice with C57BL / 6 background and VGAT-tdTomato mice (non-patent document 4) were used. Animals were kept and handled in accordance with the laws related to the care and management of animals and the guidelines for the proper implementation of animal experiments proposed by the Japan Academy of Sciences. In addition, when conducting animal experiments, efforts were made to minimize the suffering of animals and reduce the number of animals used.

[0111] [Intravenous injection]

[0112] Mice aged 7 to 14 weeks were used. After deep anesthesia was induced by intraperitoneal injection of ketamine (100 mg / kg BW) and xylazine (10 mg / kg BW), AAV-PHP.eB (5.0 × 10 12 vg / mL) was injected into the retro-orbital sinus within 30-40 seconds. Three weeks after the injection of AAV-PHP.eB vector, the mice were deeply anesthetized and perfused from the heart with 4% paraformaldehyde in phosphate buffer (pH 7.4). The brain was removed and immersed in 4% paraformaldehyde in 0.1 M phosphate buffer at 4°C for 6 hours.

[0113] [Statistical Analysis]

[0114] Statistical analysis was performed using GraphPad PRISM version 7 (GraphPad Software, San Diego, CA, USA). Data are presented as mean ± standard error.

[0115] <Relative value of GFP fluorescence intensity in cerebral cortex in Example 1>

[0116] Bright field images and native GFP fluorescence images of the whole brain were obtained using a fluorescent stereomicroscope (VB-700; KEYENCE, Osaka, Japan). To measure the GFP fluorescence intensity of the cerebral cortex, the edge of the corresponding part of the brain excluding the olfactory bulb, superior / inferior tubercles, and brainstem was traced on the bright field image. The GFP fluorescence intensity in the enclosed area was measured using ImageJ software (Fiji). The autofluorescence of the brain of 8 VGAT-tdTomato mice that were not injected with the AAV vector was measured, and the average value was subtracted from the GFP fluorescence value of all samples. Furthermore, the data were normalized to the average value of the cerebral cortex from VGAT-tdTomato mice that were administered with AAV-PHP.eB expressing GFP under the control of the shortened mGAD65 promoter. It should be noted that in the case of VGAT-tdTomato mice, tdTomato is specifically expressed in inhibitory neurons throughout the central nervous system (CNS), so it is possible to determine whether the introduced (GFP-expressing) cells are inhibitory GABAergic neurons without immunohistochemistry.

[0117] The results are shown in Figure 3 (Mean±SEM (standard error) (n=3-5), **: P<0.01, 1-way ANOVA, Dunnett's test). The GFP fluorescence intensity was measured when various shortened mGAD65 promoters or shortened mGAD67 promoters were used. Among the shortened mGAD65 promoters, there were promoters that showed the same or stronger GFP expression than the original mGAD65 promoter, but there was no significant difference in either case ( Figure 3 In the figure, mGAD65 (Ri-Rii-D-E TSS )、mGAD65(Ri-Rii-E TSS )、mGAD65(Rii-E TSS )). On the other hand, there is also a promoter that shows the same degree or stronger GFP expression than the original mGAD65 promoter ( Figure 3 In the figure, mGAD67 (Ri-M-Rii-E TSS )、mGAD67(Ri-Rii-E TSS )、mGAD67(Rii-E TSS )), especially in the mGAD67 promoter, the shortest promoter cmGAD67 (mGAD67 (Rii-E TSS ))A significant increase in fluorescence intensity was observed.

[0118] <Example 2 Specificity and efficiency of gene expression in the cerebral cortex>

[0119] To investigate the specificity and efficiency of gene expression using the shortened mGAD65 promoter, fluorescence images of GFP and tdTomato were acquired from sagittal sections of the brain (thickness 50 μm) using a fluorescence microscope (BZ-X800; KEYENCE) with a 20× objective lens. The number of GFP(+) and / or tdTomato(+) cells was manually counted.

[0120] The specificity and efficiency of gene expression in inhibitory neurons using various shortened mGAD65 promoters were studied. Confocal microscopy image analysis confirmed that in VGAT-tdTomato mice treated with AAV-PHP.eB expressing GFP under the control of various shortened mGAD65 promoters, fluorescence overlap between GFP and tdTomato was observed in some neurons.

[0121] First, specific quantitative analysis of gene expression was performed using the following formula.

[0122] Specificity (%) = (number of cells double-labeled with GFP and tdTomato / total number of GFP-positive cells) × 100

[0123] This formula represents the ratio of VGAT-positive cells consistent with GFP expression relative to GFP-expressing cells derived from AAV vectors (excluding cells expressing only GFP), that is, it represents the ratio of GFP specifically expressed by AAV vector inhibitory neurons.

[0124] The specific quantitative analysis results of gene expression are shown in Figure 4 (Mean±SEM (standard error) (n=4-5), ns: no significant difference, 1-way ANOVA, Dunnett's test). It was shown that more than 95% of cells expressing GFP under the control of the original mGAD65 promoter were double-labeled with tdTomato (95.4%±1.00%, n=4-5. In addition, it was shown that the shortest promoter cmGAD65 (i.e., mGAD65 (Rii-E TSS The specificity of gene expression was 95.3%±1.02% when the mGAD67 promoter was shortened and the shortest promoter cmGAD67 (i.e., mGAD67 (Rii-E TSS )) the specificity of gene expression was 91.6%±0.97%.

[0125] Next, the gene expression efficiency was quantitatively analyzed using the following formula.

[0126] Expression efficiency (%) = (number of cells double-labeled with GFP and tdTomato / total number of tdTomato-positive cells) × 100

[0127] This formula represents the ratio of VGAT-positive cells with consistent expression of GFP relative to VGAT-positive cells (excluding cells that are only VGAT-positive), that is, the efficiency of AAV vectors in expressing GFP in inhibitory neurons is expressed as a ratio.

[0128] The results of quantitative analysis of gene expression efficiency are shown in Figure 5 (Mean±SEM (standard error) (n=4-5), ns: no significant difference, *: P<0.05, 1-way ANOVA, Dunnett's test). 64.7%±0.99% of cells expressing GFP under the control of the original mGAD65 promoter were double-labeled with tdTomato (n=4-5). In addition, the shortest promoter cmGAD65 (i.e., mGAD65 (Rii-E TSS The specificity of gene expression was 61.4%±2.42% when the mGAD67 promoter was shortened and the shortest promoter cmGAD67 (i.e., mGAD67 (Rii-E TSS )) the specificity of gene expression was 71.3%±2.74.

[0129] In addition, the mRNA expression levels when the gene expression experiment in the cerebral cortex was carried out using various shortened mGAD promoters are shown in FIG. Figure 6 (Mean±SEM (standard error) (n=6), ns: no significant difference, **: P<0.01, 1-way ANOVA, Dunnett's test). The results showed that mGAD67 (Rii-E TSS ) mRNA expression was significantly increased.

[0130] Three weeks after VGAT-tdTomato mice were injected with AAV-PHP.eB expressing GFP under a designed shortened mGAD65 or 67 promoter, mRNA was isolated from the cerebral cortex tissue using the RNeasy Mini Kit (QIAGEN, Hilden, Germany). Reverse transcription was performed using ReverTra Ace quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) Master Mix with gDNA Remover (Toyobo, Osaka, Japan), and cDNA was synthesized from the cerebral cortex mRNA sample, and real-time quantitative PCR was performed using Power SYBR Green qPCR Mix (Toyobo, Osaka, Japan). The upstream (F) and downstream (R) primers (GAPDH and GFP) used were the following sequences. qRTPCR-mGAPDH-F: 5'-GTGTTCCTACCCCCAATGTG-3' (SEQ ID NO. 47); qRTPCR-mGAPDH-R: 5'-GGTGGAAGAGTGGGAGTTGCTG-3' (SEQ ID NO. 48); qPCR-AAV-EGFP-F: 5'-GGACGAGCTGTACAAGTAAAG-3' (SEQ ID NO. 49); qPCR-AAV-WPRE-R: 5'-GGGAAGCAATAGCATGATACAAAGG-3' (SEQ ID NO. 50). The thermal cycle program used in the real-time quantitative PCR was implemented with an initial denaturation step of 95°C for 10 minutes and 40 cycles of 95°C for 15 seconds and 60°C for 1 minute. The obtained data were analyzed by the ΔΔCt method, and the expression levels of cmGAD65 and cmGAD67 promoters were shown when the GFP expression level of the mGAD65 (U-Ri-M-Rii-D) promoter was set to 1.

[0131] The above shows that the mGAD65 promoter, which is as long as 2.5 kb, can be shortened as disclosed in the present invention, and can be compacted to 0.5 kb when cmGAD65 and cmGAD67 are used. Furthermore, in the case of cmGAD67, the promoter activity can be increased by about 2 times.

Claims

1. A promoter, which consists of a DNA having promoter activity, wherein the DNA comprises: The base sequence of the Dlx1 binding site and / or the Dlx2 binding site in the glutamate decarboxylase GAD promoter; and The region from the 5' end of exon 1 to the transcription start site TSS in glutamate decarboxylase GAD is called E TSS The base sequence of the region.

2. The promoter according to claim 1, wherein The glutamate decarboxylase GAD promoter is a GAD65 promoter derived from mice, namely, mGAD65 promoter; the glutamate decarboxylase GAD is a glutamate decarboxylase 65 derived from mice, namely, mGAD65; The base sequence of the Dlx1 binding site and / or the Dlx2 binding site comprises at least one base sequence selected from the group consisting of the base sequence of Region i of the mGAD65 promoter shown in SEQ ID NO: 1, the base sequence of Region ii of the mGAD65 promoter shown in SEQ ID NO: 2, and a base sequence that is 90% or more identical to the base sequence shown in any of them, and The region from the 5' end of exon 1 to the transcription start site TSS in mGAD65 is E TSS The base sequence of the region is the base sequence represented by SEQ ID NO: 3, or a base sequence that is 90% or more identical to the base sequence.

3. The promoter according to claim 1, wherein The base sequence comprises the base sequence represented by any one of SEQ ID NOs: 4 to 6, or a base sequence having 90% or more identity with the base sequence represented by any one of SEQ ID NOs: 4 to 6.

4. The promoter according to claim 1, wherein The glutamate decarboxylase GAD promoter is a GAD67 promoter derived from mice, namely, mGAD67 promoter; the glutamate decarboxylase GAD is a glutamate decarboxylase 67 derived from mice, namely, mGAD67; The base sequence of the Dlx1 binding site and / or the Dlx2 binding site comprises at least one base sequence selected from the group consisting of the base sequence of Region i of the mGAD67 promoter shown in SEQ ID NO: 7, the base sequence of Region ii of the mGAD67 promoter shown in SEQ ID NO: 8, and a base sequence that is 90% or more identical to the base sequence shown in any of them, and The region from the 5' end of exon 1 to the transcription start site TSS in mGAD67 is E TSS The base sequence of the region is the base sequence represented by SEQ ID NO: 9, or a base sequence represented by a base sequence that is 90% or more identical to the base sequence.

5. The promoter according to claim 1, wherein The base sequence comprises the base sequence represented by any one of SEQ ID NOs: 10 to 12, or a base sequence having 90% or more identity with the base sequence represented by any one of SEQ ID NOs: 10 to 12. 6 . An adeno-associated virus vector for gene expression in inhibitory neurons or a recombinant virus obtained from the adeno-associated virus vector, comprising the promoter according to any one of claims 1 to 5 .

7. The adeno-associated virus vector or the recombinant virus obtained from the adeno-associated virus vector according to claim 6, wherein: Also included is a target gene placed under the control of the promoter.

8. A medicine comprising the adeno-associated virus vector according to claim 7 or a recombinant virus obtained from the adeno-associated virus vector.

9. The medicine according to claim 8, wherein The medicine is used for treating central nervous system diseases caused by dysfunction of inhibitory neurons or abnormal balance between excitation and inhibition.

10. A method for introducing a gene into a cell, comprising the following steps: in vitro, using the adeno-associated virus vector according to claim 7 or a recombinant virus obtained from the adeno-associated virus vector to introduce a target gene into a target cell.

11. The method according to claim 10, wherein: The target cells are inhibitory neurons.

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