Polynucleotides, vectors, cells, pharmaceutical compositions and screening methods for treating neurodegenerative diseases

Abstract

The purpose of the present invention is to provide a novel method for increasing the number of newly generated neurons in the adult brain, and to provide a polynucleotide, a vector, and a pharmaceutical composition for use in the method.　The present invention provides: a polynucleotide characterized by including (A) the nucleic acid sequence of the Plagl2 gene, (B) an miR-shRNA (microRNA adapted short hairpin RNA) nucleic acid sequence for the Dyrk1a gene, and (C) a promoter sequence operatively connected to the nucleic acid sequences; a vector including the polynucleotide; and a pharmaceutical composition including the polynucleotide and the vector.

Description

[Technical Field]

[0001] The present invention relates to polynucleotides, vectors, cells, pharmaceutical compositions and screening methods for treating neurodegenerative diseases. [Background technology]

[0002] It has become clear that neural stem cells and neural progenitor cells exist in the adult brain of mammals, including humans, although their absolute numbers are small, and that neurogenesis occurs in some brain regions. However, the majority of neural stem cells in the adult brain are quiescent and have low proliferation and neurogenesis capacities. This proliferation and neurogenesis capacity further declines with age, resulting in almost no new neurons being born in the aging brain. A significant decrease in newborn neurons is thought to be a cause of declines in brain function such as dementia, as well as neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease.

[0003] Under these circumstances, attempts have been made to promote functional recovery by amplifying and differentiating the neural stem cells and neural progenitor cells present in small quantities in the adult brain through stimulation with growth factors and other methods (see Non-Patent Document 1). However, the effectiveness of these attempts has not been sufficient. Furthermore, active research has been conducted on cell transplantation therapy models using neural stem cells and neural progenitor cells derived from fetal brain, ES cells, and iPS cells. However, it is unclear whether cell transplantation therapy in the brain can reconstruct normal neural circuits, and there is a significant risk of side effects. Furthermore, although many attempts have been made to increase the number of newly generated neurons in the adult brain, the effectiveness of these attempts has been limited (see Non-Patent Documents 1-5). [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Nagahara AH et al. (2009) Neuroprotective effects of brain-derived neurotrophic factor in rodent and primate models of Alzheimer's disease, Nat.Med.,vol.15(3),pp.331-337. [Non-patent document 2] Henriette van Praag et al.,Exercise enhances learning and hippocampal neurogenesis in aged mice,J Neurosci.,2005 Sep 21;25(38):8680-5 [Non-patent document 3] Benedetta Artegiani et al.,Overexpression of cdk4 and cyclinD1 triggers greater expansion of neural stem cells in the adult mouse brain. J Exp Med.2011 May 9;208(5):937-48 [Non-patent document 4] Benraiss, A.et al.(2013) Mobilization of endogenous progenitor cells regenerates functionally-integrated medium spiny striopallidal projection neurons and delays disease progression in a transgenic model of Huntington's disease. Cell Stem Cell 12, 787-799 [Non-patent document 5] Berdugo-Vega, G.et al.(2020). Increasing neurogenesis refines hippocampal activity rejuvenating navigational learning strategies and contextual memory throughout life. Nat. Commun. 11, 135 Summary of the Invention [Problem to be solved by the invention]

[0005] The present invention aims to provide a novel method for increasing the number of new neurons in the adult brain, as well as to provide polynucleotides, vectors, transformed cells, and pharmaceutical compositions to be used in this method, and a method for screening for substances that increase the number of new neurons in the adult brain. [Means for solving the problem]

[0006] In light of this situation, the inventors conducted extensive research and found that by introducing a specific gene set into the brains of aged mice via lentivirus, endogenous neural stem cells were efficiently activated and proliferated, producing a large number of neurons. Aged neural stem cells returned to their adolescent or younger state, producing a large number of neurons and improving memory and learning abilities. Furthermore, when introduced into the brains of Alzheimer's disease model mice, endogenous neural stem cells were efficiently activated and produced a large number of neurons, improving memory and learning abilities. The gist of the present invention is as follows.

[0007] [1] (A) The nucleic acid sequence of the Plagl2 gene; (B) Nucleic acid sequence of miR-shRNA (microRNA adapted short hairpin RNA) for the Dyrk1a gene, and (C) a promoter sequence operably linked to the nucleic acid sequence. A polynucleotide comprising: [2] The polynucleotide according to [1], wherein the Plagl2 gene and the Dyrk1a gene are mammalian genes. [3] The polynucleotide according to [1] or [2], wherein the promoter is selected from the group consisting of Hes5 promoter, GFAP promoter, Sox2 promoter, and Lfng promoter. [4] A vector comprising the polynucleotide according to any one of [1] to [3]. [5] The vector according to [4], wherein the vector is a lentivirus vector, an adeno-associated virus vector, an adenovirus vector, or a plasmid vector. [6] A pharmaceutical composition containing the vector according to [4] or [5]. [7] (a) a vector comprising a nucleic acid sequence of a Plagl2 gene and a promoter sequence operably linked to the nucleic acid sequence of the Plagl2 gene; and (b) a vector comprising a nucleic acid sequence of a microRNA adapted short hairpin RNA (miR-shRNA) against the Dyrk1a gene and a promoter sequence operably linked to the nucleic acid sequence of the miR-shRNA, or a vector comprising an siRNA or miRNA against the Dyrk1a gene. A pharmaceutical composition comprising: [8] A cell transformed with the vector according to [4] or [5]. [9] A pharmaceutical composition comprising the cells described in [8].

[10] The pharmaceutical composition according to [6], [7] or [9], which is used for the treatment of a neurodegenerative disease or an inner ear disease.

[11] The pharmaceutical composition of

[10] , which is administered to a subject by intraventricular injection, intrathecal bolus injection or infusion, intraganglionic injection, intraneuronal injection, subcutaneous injection, or intratympanic injection.

[12] (1) A step of applying a test substance to neural stem cells or inner ear supporting cells and measuring the expression of Plagl2 gene and Dyrk1a gene; (2-1) selecting a test substance having the ability to enhance Plagl2 gene expression and the ability to suppress Dyrk1a gene expression from the results of the above step (1); or (2-2) A step of selecting a test substance capable of enhancing Plagl2 gene expression and a test substance capable of suppressing Dyrk1a gene expression based on the results of the above step (1). A method for screening for a substance for treating a neurodegenerative disease or an inner ear disease, comprising: [Effects of the Invention]

[0008] According to the present invention, by introducing a specific gene set into the adult or aged brain, it is possible to efficiently activate and proliferate endogenous neural stem cells, thereby producing a large number of neurons. Neural stem cells in the aged brain can be restored to their adolescent or younger state, producing a large number of neurons and improving memory and learning abilities. This method is also expected to be effective in treating neurodegenerative diseases such as Alzheimer's disease, as it efficiently activates endogenous neural stem cells, producing a large number of neurons, and improving memory and learning abilities. Furthermore, the specific gene set of the present invention can differentiate not only neural stem cells in the brain but also supporting cells in the inner ear into hair cells, suggesting its effectiveness in treating inner ear diseases. [Brief explanation of the drawings]

[0009] [Figure 1] FIG. 1 shows a process for screening genes that contribute to the activation of NSCs (neural stem cells). [Figure 2] FIG. 2 shows the results of in vitro screening for NSC-activating genes by overexpression of genes highly expressed in embryos. [Figure 3] FIG. 3 shows the results of in vivo screening of NSC-activating genes by overexpression of genes highly expressed in embryos. [Figure 4-1] FIG. 4-1 shows the results of in vitro screening for NSC-activating genes by knockdown of genes highly expressed in adults. [Figure 4-2] FIG. 4-2 shows the results of in vitro screening for NSC-activating genes by knockdown of genes highly expressed in adults. [Figure 5] FIG. 5 shows the results of in vivo screening of NSC-activating genes. [Figure 6] FIG. 6 shows the results of in vivo screening of NSC-activating genes. [Figure 7] Figure 7 shows the long-term activation of neurogenesis in the aged mouse brain by iPaD. The combination of Plagl2 overexpression and Dyrk1a knockdown was named "iPaD (inducing Plagl2 and anti-Dyrk1a)." [Figure 8] FIG. 8 shows long-term activation of neurogenesis in the aged mouse brain by iPaD. [Figure 9-1] FIG. 9-1 shows the decline in neurogenesis with aging (wild-type hippocampus). [Figure 9-2] Figure 9-2 shows the age-related decline in neurogenesis and the rejuvenation of aged NSCs by iPaD. [Figure 10-1] FIG. 10-1 shows the schedule for the Barnes maze test. [Figure 10-2] Figure 10-2 shows the effect of iPaD on improving cognitive function in aged mice (Barnes maze test). [Figure 10-3] Figure 10-3 shows the effect of iPaD on improving cognitive function in aged mice (Barnes maze test). [Figure 11] FIG. 11 shows age-related changes in the number of MCM2-positive cells in the hippocampal dentate gyrus of Alzheimer's disease model mice (5×FAD mice) and normal mice (wild-type littermate mice). [Figure 12] FIG. 12 shows age-related changes in the number of DCX-positive cells in the hippocampal dentate gyrus of Alzheimer's disease model mice (5×FAD mice) and normal mice (wild-type littermate mice). [Figure 13] FIG. 13 shows age-related increases in the number of amyloid β plaques in the hippocampal dentate gyrus of Alzheimer's disease model mice (5×FAD mice). [Figure 14]FIG. 14 shows the results of a comparison of cognitive functions between Alzheimer's disease model mice (5×FAD mice) and normal mice (wild-type littermate mice) (Barnes circular maze test). [Figure 15] FIG. 15 shows the results of a comparison of cognitive functions between Alzheimer's disease model mice (5×FAD mice) and normal mice (wild-type littermate mice) (contextual fear conditioning test). [Figure 16] FIG. 1 shows the ameliorative effect of iPaD on Alzheimer's disease (number of MCM2-positive cells). [Figure 17] FIG. 1 shows the ameliorative effect of iPaD on Alzheimer's disease (number of DCX-positive cells). [Figure 18] FIG. 1 shows the ameliorative effect of iPaD on Alzheimer's disease (number of amyloid β plaques). [Figure 19] FIG. 19 shows the effect of iPaD on improving cognitive function in Alzheimer's disease model mice (5×FAD mice) (Barnes circular maze test). [Figure 20] FIG. 20 shows the effect of iPaD on improving cognitive function in Alzheimer's disease model mice (5×FAD mice) (contextual fear conditioning test). [Figure 21] FIG. 21 shows activation of supporting cells in the inner ear and regeneration of hair cells by iPaD. DETAILED DESCRIPTION OF THE INVENTION

[0010] The present invention will be described in detail below. In this specification, unless otherwise specified, molecular biological techniques can be performed by methods described in general experimental manuals known to those skilled in the art or methods similar thereto. Furthermore, unless otherwise specified, the terms used in this specification should be interpreted as having the meanings commonly used in the technical field.

[0011] According to the present disclosure, by forcibly expressing genes that are highly expressed in embryos and low expressed in adult neural stem cells in target cells such as neural stem cells, and by suppressing the expression of genes that are low expressed in embryos and high expressed in adult neural stem cells, these cells can be efficiently activated and proliferated. That is, the present application provides polynucleotides, vectors, transformed cells containing specific gene sets that can efficiently activate and proliferate neural stem cells and the like present in adult brains, aging brains, diseased brains, etc., and produce a large number of neurons, as well as pharmaceutical compositions containing them, and methods for treating neurodegenerative diseases and the like using them.

[0012] <Polynucleotide> A first embodiment of the polynucleotide of the present disclosure is a polynucleotide to be introduced into a target cell such as a neural stem cell, and includes: (A) a nucleic acid sequence of a gene that is highly expressed in an embryo and lowly expressed in an adult neural stem cell; (B) a nucleic acid sequence that suppresses the expression of a gene that is lowly expressed in an embryo and highly expressed in an adult neural stem cell (e.g., a nucleic acid sequence of a microRNA-adapted short hairpin RNA (miR-shRNA) against the gene); and (C) a promoter sequence operably linked to the nucleic acid sequence.

[0013] A second embodiment of the polynucleotide of the present disclosure is a combination of (a) a polynucleotide to be introduced into a target cell such as a neural stem cell, the polynucleotide comprising the nucleic acid sequence of a gene that is highly expressed in an embryo and lowly expressed in an adult neural stem cell, and a promoter sequence operably linked to the nucleic acid sequence of the gene, and (b1) a polynucleotide comprising a nucleic acid sequence that suppresses the expression of a gene that is lowly expressed in an embryo and highly expressed in an adult neural stem cell (e.g., the nucleic acid sequence of a microRNA-adapted short hairpin RNA (miR-shRNA) for the gene), and a promoter sequence operably linked to a nucleic acid sequence that suppresses the expression of the gene (e.g., the nucleic acid sequence of a miR-shRNA).

[0014] A third embodiment of the polynucleotide of the present disclosure is a combination of (a) a polynucleotide containing the nucleic acid sequence of a gene that is highly expressed in embryos and lowly expressed in adult neural stem cells, and a promoter sequence operably linked to the nucleic acid sequence of the gene, which is introduced into target cells such as neural stem cells, and (b2) a nucleic acid sequence that suppresses the expression of a gene that is lowly expressed in embryos and highly expressed in adult neural stem cells (e.g., a polynucleotide of siRNA or miRNA against the gene).

[0015] As used herein, the term "polynucleotide" is used interchangeably with "nucleic acid," "gene," or "nucleic acid molecule," and refers to a polymer of nucleotides. As used herein, the term "nucleotide sequence" is used interchangeably with "nucleic acid sequence" or "base sequence," and is represented as a sequence of deoxyribonucleotides (abbreviated as A, G, C, and T). For example, a "polynucleotide comprising the nucleotide sequence of SEQ ID NO: 1 or a fragment thereof" refers to a polynucleotide or a fragment thereof comprising the sequence represented by each of the deoxynucleotides A, G, C, and / or T of SEQ ID NO: 1.

[0016] Target cells for introduction of the polynucleotide of the present disclosure include neural stem cells, inner ear supporting cells, tissue stem cells (somatic stem cells) such as hematopoietic stem cells, mesenchymal stem cells, skeletal muscle stem cells, and dental pulp stem cells, retinal Müller glial cells, and tissue progenitor cells. Of these, neural stem cells and inner ear supporting cells are preferred, as their activation and proliferation are promoted by introduction of the polynucleotide of the present disclosure.

[0017] (Regarding the first embodiment) A first embodiment of the polynucleotide of the present disclosure to be introduced into target cells such as neural stem cells is a polynucleotide comprising: (A) a nucleic acid sequence of a gene that is highly expressed in embryos and lowly expressed in adult neural stem cells; (B) a nucleic acid sequence that suppresses the expression of a gene that is lowly expressed in embryos and highly expressed in adult neural stem cells (e.g., a nucleic acid sequence of a microRNA-adapted short hairpin RNA (miR-shRNA) against the above gene); and (C) a promoter sequence operably linked to the above nucleic acid sequence.

[0018] [(A) Nucleic acid sequences of genes highly expressed in embryos and low expressed in adult neural stem cells] (A) The nucleic acid sequence may be any nucleic acid sequence of a gene that is highly expressed in embryos but lowly expressed in adult neural stem cells. However, it is preferably a nucleic acid sequence of a gene whose overexpression in adult neural stem cells promotes the proliferation and activation of adult neural stem cells. Specific examples include nucleic acid sequences of Gsx2, Dmrt3, Cdk4, Plagl2, Sox21, Ascl1, Tgif2, Plagl1, and Hmga2. Of these, the nucleic acid sequence of the Plagl2 gene is more preferred due to its superior ability to promote the proliferation and activation of adult neural stem cells. The Plagl2 gene (pleomorphic adenoma gene like-2) encodes the transcription factor PLAGL2, which has a C2H2 zinc finger domain. It is also known as ZNF900. PLAGL2 functions as a positive regulator of transcription and is known to be localized in the nucleus. As described in detail in the Examples, in the present disclosure, differential expression of nuclear factors was identified in the transcriptome of G1 / G0 NSCs derived from the ganglionic eminence and dorsal cortex of E14 mouse embryos (DDBJ BioProject Accession: PRJDB9010) compared to largely quiescent NSCs derived from the LV-SVZ (SAMD00192826) and DG-SGZ (SAMD00192827) of 2-3 month old mice.

[0019] In the present disclosure, genes that are highly expressed in embryos and lowly expressed in adult neural stem cells, such as the Plagl2 gene, are derived from mammals such as humans, monkeys, pigs, horses, cows, rabbits, sheep, goats, cats, dogs, and guinea pigs, and are preferably derived from humans, monkeys, or mice, with humans being more preferred.

[0020] In the present disclosure, the nucleic acid sequence of a gene, such as the Plagl2 gene, that is highly expressed in embryos and lowly expressed in adult neural stem cells includes the coding region (CDS) within the mRNA sequence of the gene. The nucleic acid sequence of the gene can be a known sequence as the coding region (CDS) of the mRNA sequence of each of the above mammalian genes, such as a sequence registered in the GenBank sequence database. The nucleic acid sequence of the Plagl2 gene in the present disclosure is identical to the sequence registered in the GenBank sequence database (SEQ ID NO: 1), or has at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 98%, and particularly preferably at least 99% identity, and the encoded peptide has the transcription factor activity of PLAGL2. The most preferred nucleic acid sequence of the Plagl2 gene in the present disclosure is SEQ ID NO: 1.

[0021] (B) A nucleic acid sequence that suppresses the expression of a gene that is expressed at low levels in embryos and at high levels in adult neural stem cells. (B) The nucleic acid sequence is not particularly limited as long as it suppresses the expression of a gene that is underexpressed in embryos and overexpressed in adult neural stem cells. However, such genes that are underexpressed in embryos and overexpressed in adult neural stem cells are preferably genes whose knockdown can efficiently activate quiescent neural stem cells, such as Cdkn1a, Prkcz, Dyrk1a, Zbtb7a, Dusp22, Cidea, Rasd1, Nr4a1, Nfe2l2, Stat6, and Tsc22d3. Of these, Cdkn1a, Prkcz, Dyrk1a, and Zbtb7a are more preferred, with Dyrk1a being even more preferred. The Dyrk1a gene encodes an enzyme that phosphorylates serine / threonine and tyrosine and is thought to be involved in the development of Down syndrome, which is caused by trisomy of chromosome 21. As described in detail in the Examples, in the present disclosure, it was found to be one of the 11 genes whose knockdown in targeted neural stem cells efficiently activated quiescent NSCs.

[0022] In the present disclosure, genes such as the Dyrk1a gene that are lowly expressed in embryos and highly expressed in adult neural stem cells are derived from mammals such as humans, monkeys, pigs, horses, cows, rabbits, sheep, goats, cats, dogs, and guinea pigs, and are preferably derived from humans, monkeys, or mice, with humans being more preferred.

[0023] In the present disclosure, the nucleic acid sequence of a gene, such as the Dyrk1a gene, that is lowly expressed in embryos and highly expressed in adult neural stem cells includes the coding region (CDS) within the mRNA sequence of the gene. The nucleic acid sequence of the gene can be a known sequence as the coding region (CDS) of the mRNA sequence of each of the above mammalian genes, and can be, for example, a sequence registered in the GenBank sequence database. The nucleic acid sequence of the Dyrk1a gene in the present disclosure is identical to the sequence registered in the GenBank sequence database (SEQ ID NO: 2), or has 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and particularly preferably 99% or more homology thereto, and the encoded peptide has the enzymatic activity of Dyrk1a. The most preferred nucleic acid sequence of the Dyrk1a gene in the present disclosure is the sequence of SEQ ID NO: 2.

[0024] In the present disclosure, the nucleic acid sequence that suppresses the expression of a gene that is lowly expressed in embryos and highly expressed in adult neural stem cells is not particularly limited as long as it has a nucleic acid sequence that can suppress the expression of the gene using a conventionally known method, and examples thereof include miR-shRNA (microRNA adapted short hairpin RNA) for the gene. The miR-shRNA for the gene is an shRNA (a hairpin RNA sequence used for gene silencing by RNA interference) that can suppress the expression of the gene. The miR-shRNA is not particularly limited as long as it has a nucleic acid sequence that can suppress the expression of the gene, and can be appropriately designed to match the sequence of the gene. A preferred example of the nucleic acid sequence of such an shRNA is the nucleic acid sequence of SEQ ID NO: 3, which is the nucleic acid sequence of a miR-shRNA for Dyrk1a. However, nucleic acid sequences that have 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and particularly preferably 99% or more homology to the nucleic acid sequence of SEQ ID NO: 3 and that can suppress the expression of the Dyrk1a gene can also be used as the nucleic acid sequence of the miR-shRNA (microRNA adapted short hairpin RNA) for the Dyrk1a gene of the present disclosure.

[0025] [(C) a promoter sequence operably linked to the nucleic acid sequence] In the present disclosure, a nucleic acid sequence of a gene that is highly expressed in embryos and lowly expressed in adult neural stem cells, and a nucleic acid sequence that suppresses the expression of a gene that is lowly expressed in embryos and high expressed in adult neural stem cells (e.g., a nucleic acid sequence of a microRNA-adapted short hairpin RNA (miR-shRNA) for the above gene) are operably linked to a (C) promoter sequence. The (C) promoter sequence used in this embodiment may be a promoter sequence that functions in neural cells such as neural stem cells and inner ear supporting cells. Examples of such promoter sequences include the Hes5 promoter (SEQ ID NO: 4), which functions in neural stem cells, the GFAP promoter, and the Sox2 promoter and Lfng promoter, which function in inner ear supporting cells.

[0026] As used herein, "operably linked" refers to a juxtaposition wherein the described components are in a relationship permitting them to function in their intended manner. In one embodiment, the term refers to a functional linkage between a nucleic acid expression control sequence (e.g., a promoter and / or enhancer) and a polynucleotide sequence of interest, which may be direct or indirect (when there is another intervening polynucleotide sequence). In the present disclosure, a promoter sequence directs transcription of a linked polynucleotide of interest.

[0027] In this embodiment, examples of polynucleotides comprising (A) a nucleic acid sequence of a gene that is highly expressed in embryos and lowly expressed in adult neural stem cells, (B) a nucleic acid sequence that suppresses the expression of a gene that is lowly expressed in embryos and highly expressed in adult neural stem cells (e.g., a nucleic acid sequence of a miR-shRNA (microRNA adapted short hairpin RNA) for the above gene), and (C) a promoter sequence operably linked to the above nucleic acid sequence include (A) a nucleic acid sequence of a Plagl2 gene, (B) a nucleic acid sequence that suppresses the expression of a Dyrk1a gene (e.g., a miR-shRNA (microRNA adapted short hairpin RNA) for the Dyrk1a gene), and (C) a promoter sequence operably linked to the above nucleic acid sequence. Preferred are polynucleotides comprising (A) a nucleic acid sequence of a nucleotide ... Furthermore, preferred polynucleotides in the present invention include, for example, polynucleotides that can hybridize to the complementary sequence of SEQ ID NO: 5 under stringent hybridization conditions.

[0028] Here, "stringent conditions" may refer to low stringency conditions, moderate stringency conditions, or high stringency conditions. "Low stringency conditions" are, for example, 5x SSC, 5x Denhardt's solution, 0.5% SDS, 50% formamide, and 32°C. "Medium stringency conditions" are, for example, 5x SSC, 5x Denhardt's solution, 0.5% SDS, 50% formamide, and 42°C. "High stringency conditions" are, for example, 5x SSC, 5x Denhardt's solution, 0.5% SDS, 50% formamide, and 50°C. Under these conditions, it is expected that DNA with higher homology can be obtained more efficiently as the temperature is increased. However, several factors can affect the stringency of hybridization, including temperature, probe concentration, probe length, ionic strength, time, and salt concentration, and a person skilled in the art can achieve similar stringency by appropriately selecting these factors.

[0029] The polynucleotide of this embodiment may further contain known sequences such as an enhancer sequence that assists in mRNA transcription, protein translation, etc., a Kozak sequence, and an appropriate polyadenylation signal sequence.

[0030] The method for introducing the polynucleotide of this embodiment into target cells is not particularly limited, and examples include the use of vectors such as viruses, plasmids, and artificial chromosomes, or techniques such as lipofection, liposomes, and microinjection. Introducing the polynucleotide of this embodiment into target neural stem cells and transforming them can result in the forced expression of genes that are highly expressed in embryos and lowly expressed in adult neural stem cells, such as the Plagl2 gene, and knockdown of genes that are lowly expressed in embryos and high expressed in adult neural stem cells, such as the Dyrk1a gene. Target cells (neural stem cells, etc.) transformed in this manner can be activated, proliferate, and differentiate to fully exert cellular functions, such as producing numerous neurons. The polynucleotide of this embodiment can be suitably used for neural stem cells present in adult brains, aging brains, diseased brains, etc., and supporting cells present in the inner ear.

[0031] (Regarding the second embodiment) A second embodiment of the polynucleotide of the present disclosure to be introduced into target cells such as neural stem cells is a combination of (a) a polynucleotide comprising the nucleic acid sequence of a gene that is highly expressed in embryos and lowly expressed in adult neural stem cells, and a promoter sequence operably linked to the nucleic acid sequence of the gene, and (b1) a polynucleotide comprising a nucleic acid sequence that suppresses the expression of a gene that is lowly expressed in embryos and high in adult neural stem cells (e.g., a nucleic acid sequence of a microRNA-adapted short hairpin RNA (miR-shRNA) for the gene), and a promoter sequence operably linked to the nucleic acid sequence that suppresses the expression of the gene. This polynucleotide combination of this embodiment is used to separately transform target cells such as neural stem cells by, for example, introducing the gene that is highly expressed in embryos and lowly expressed in adult neural stem cells and the nucleic acid sequence that suppresses the expression of a gene that is lowly expressed in embryos and high in adult neural stem cells (e.g., miR-shRNA for the Dyrk1a gene) into separate vectors.

[0032] The explanation for (A) the nucleic acid sequence of a gene that is highly expressed in an embryo and low expressed in an adult neural stem cell in the first embodiment can be applied as is to (a) the nucleic acid sequence of a gene that is highly expressed in an embryo and low expressed in an adult neural stem cell in this embodiment. Furthermore, the explanation for (C) the promoter sequence operably linked to the nucleic acid sequence of the gene in the first embodiment can also be applied to the promoter sequence operably linked to the nucleic acid sequence of the gene. Specific examples include the Hes5 promoter (SEQ ID NO: 4) that functions in neural stem cells, the GFAP promoter, the Sox2 promoter that functions in inner ear supporting cells, and the Lfng promoter.

[0033] In this embodiment, the polynucleotide comprising (a) the nucleic acid sequence of a gene that is highly expressed in embryos and lowly expressed in adult neural stem cells, and a promoter sequence operably linked to the nucleic acid sequence of the gene, is preferably a polynucleotide comprising (a) the nucleic acid sequence of the Plagl2 gene, and a promoter sequence operably linked to the nucleic acid sequence of the Plagl2 gene, and a more preferred example is one comprising the nucleic acid sequence of SEQ ID NO: 6. This polynucleotide may further comprise known sequences such as an enhancer sequence that assists in mRNA transcription, protein translation, etc., a Kozak sequence, and an appropriate polyadenylation signal sequence.

[0034] The explanation for (B) Nucleic acid sequence that suppresses the expression of a gene that is lowly expressed in an embryo and highly expressed in an adult neural stem cell in the first embodiment can be applied to (b1) a nucleic acid sequence that suppresses the expression of a gene that is lowly expressed in an embryo and highly expressed in an adult neural stem cell (e.g., a nucleic acid sequence of a microRNA-adapted short hairpin RNA (miR-shRNA) for the gene) in this embodiment. Furthermore, the explanation for (C) Promoter sequence that is operably linked to the nucleic acid sequence in the first embodiment can be applied to the promoter sequence operably linked to the nucleic acid sequence. Specific examples include the Hes5 promoter (SEQ ID NO: 4) that functions in neural stem cells, the GFAP promoter, and the Sox2 promoter and Lfng promoter that function in inner ear supporting cells.

[0035] In this embodiment, the polynucleotide comprising (b1) a nucleic acid sequence that suppresses the expression of a gene that is lowly expressed in embryos and highly expressed in adult neural stem cells (e.g., the nucleic acid sequence of a miR-shRNA (microRNA adapted short hairpin RNA) against the Dyrk1a gene) and a promoter sequence operably linked to the nucleic acid sequence is preferably a polynucleotide comprising (b1) the nucleic acid sequence of a miR-shRNA (microRNA adapted short hairpin RNA) against the Dyrk1a gene and a promoter sequence operably linked to the nucleic acid sequence of the miR-shRNA, with a more preferred example being one comprising the nucleic acid sequence of SEQ ID NO: 7. This polynucleotide may further comprise known sequences such as an enhancer sequence that assists in mRNA transcription, protein translation, etc., a Kozak sequence, or an appropriate polyadenylation signal sequence.

[0036] The method for introducing the polynucleotide of this embodiment into target cells is not particularly limited, and examples include the use of vectors such as viruses, plasmids, and artificial chromosomes, or techniques such as lipofection, liposomes, and microinjection. The two polynucleotides of this embodiment can be introduced independently into target cells. For example, by incorporating each polynucleotide into a separate vector and introducing it into target neural stem cells or the like to transform them, it is possible to forcibly express a gene that is highly expressed in embryos and lowly expressed in adult neural stem cells, and to knock down a gene that is lowly expressed in embryos and highly expressed in adult neural stem cells. Target cells (neural stem cells, etc.) transformed in this way can be activated and proliferate, producing numerous neurons, and fully exerting their functions. The polynucleotide of this embodiment can be suitably used for neural stem cells present in adult brains, aging brains, diseased brains, etc., and supporting cells present in the inner ear.

[0037] (Regarding the third embodiment) A third embodiment of the polynucleotide of the present disclosure to be introduced into target cells such as neural stem cells is a combination of (a) a polynucleotide comprising the nucleic acid sequence of a gene that is highly expressed in embryos and lowly expressed in adult neural stem cells, and a promoter sequence operably linked to the nucleic acid sequence of the gene, and (b2) a polynucleotide of siRNA or miRNA for a gene that is lowly expressed in embryos and highly expressed in adult neural stem cells. In this embodiment, the (a) polynucleotide is introduced into a vector, and the (b2) polynucleotide (siRNA or miRNA) is used together with a transfection reagent or the like to transform the target cells.

[0038] The explanation for (A) the nucleic acid sequence of a gene that is highly expressed in an embryo and low expressed in an adult neural stem cell in the first embodiment can be applied as is to (a) the nucleic acid sequence of a gene that is highly expressed in an embryo and low expressed in an adult neural stem cell in this embodiment. Furthermore, the explanation for (C) the promoter sequence that is operably linked to the nucleic acid sequence of the gene in the first embodiment can be applied as is to (c) the promoter sequence that is operably linked to the nucleic acid sequence in the first embodiment. Specific examples include the Hes5 promoter (SEQ ID NO: 4) that functions in neural stem cells, the GFAP promoter, the Sox2 promoter that functions in inner ear supporting cells, and the Lfng promoter.

[0039] In this embodiment, the polynucleotide comprising (a) the nucleic acid sequence of a gene that is highly expressed in embryos and lowly expressed in adult neural stem cells, and a promoter sequence operably linked to the nucleic acid sequence of the gene, is preferably a polynucleotide comprising (a) the nucleic acid sequence of the Plagl2 gene, and a promoter sequence operably linked to the nucleic acid sequence of the Plagl2 gene, and a more preferred example is one comprising the nucleic acid sequence of SEQ ID NO: 6.

[0040] The polynucleotide (b2) in this embodiment is not particularly limited as long as it is an siRNA or miRNA of a gene that is lowly expressed in embryos and highly expressed in adult neural stem cells, and has a sequence that can suppress (knock down) the expression of the gene. The siRNA or miRNA of the gene can be designed from the sequence of the gene. For example, the siRNA or miRNA of the Dyrk1a gene can be designed from the sequence of the Dyrk1a gene, and a specific example is the polynucleotide of SEQ ID NO: 8.

[0041] The method for introducing the polynucleotide of this embodiment into target cells is not particularly limited, and examples include the use of vectors such as viruses, plasmids, and artificial chromosomes, or techniques such as lipofection, liposomes, and microinjection. For example, (a) target cells (e.g., neural stem cells) can be transformed with a vector incorporating the polynucleotide of this embodiment to forcibly express a gene such as Plagl2, and (b2) siRNA or miRNA of the Dyrk1a gene or the like as a polynucleotide can be transfected into the target cells to suppress expression of the Dyrk1a gene or the like. Transfection methods known to those skilled in the art can be used. Target cells (e.g., neural stem cells) transformed in this manner can be activated and proliferate, producing numerous neurons and fully fulfilling their cellular functions. The polynucleotide of this embodiment can be suitably used for neural stem cells present in adult brains, aging brains, diseased brains, etc., and supporting cells present in the inner ear.

[0042] <Vector> The present disclosure also includes a vector comprising the polynucleotide of the present disclosure described above. The explanation in the section on polynucleotides above is applicable to the polynucleotide of the present disclosure contained in the vector of the present disclosure.

[0043] In the present disclosure, the vector used for forced expression of a gene highly expressed in embryonic but low expressed in adult neural stem cells (e.g., Plagl2 gene) and / or for suppressing (knockdown) the expression of a gene lowly expressed in embryonic but highly expressed in adult neural stem cells (e.g., Dyrk1a gene) is not particularly limited as long as it can efficiently deliver genes to target cells (e.g., neural stem cells, inner ear supporting cells). Examples of such vectors include viral vectors such as lentivirus vectors, adeno-associated virus vectors, adenovirus vectors, herpesvirus vectors (e.g., herpes simplex virus vectors), poxvirus vectors, baculovirus vectors, papillomavirus vectors, and papovavirus vectors (e.g., SV40); plasmid vectors; phagemid vectors; cosmid vectors; and bacteriophages such as lambda phage and M13 phage. Of these, lentiviral vectors, adeno-associated viral vectors, adenoviral vectors, and plasmid vectors are preferred, lentiviral vectors and adeno-associated viral vectors are more preferred, and lentiviral vectors are even more preferred.

[0044] Examples of specific expression vectors include pLenti4 / V5-DEST™, pLenti6 / V5-DEST™, and pLenti6.2 / V5-GW / lacZ (Invitrogen) for lentivirus-mediated gene transfer and expression in mammalian cells; pClneo vector (Promega) for expression in mammalian cells; and the like.

[0045] The vector can contain regulatory sequences such as a promoter, enhancer, ribosome binding sequence, terminator, polyadenylation site, etc. to enable expression of the gene of interest. Furthermore, if necessary, the vector can contain selectable marker sequences such as drug resistance genes (e.g., kanamycin resistance genes, ampicillin resistance genes, puromycin resistance genes, etc.), thymidine kinase genes, diphtheria toxin genes, etc., reporter gene sequences such as mCherry (red fluorescent protein), green fluorescent protein (GFP), β-glucuronidase (GUS), FLAG, etc.

[0046] <Pharmaceutical Composition> The present disclosure also includes pharmaceutical compositions containing the vectors of the present disclosure. The present disclosure also includes pharmaceutical compositions containing (a) a vector comprising a nucleic acid sequence of a gene (e.g., the Plagl2 gene) that is highly expressed in embryos and lowly expressed in adult neural stem cells, and a promoter sequence operably linked to the nucleic acid sequence of the gene; and (b) a vector comprising a nucleic acid sequence that suppresses the expression of a gene (e.g., the Dyrk1a gene) that is lowly expressed in embryos and high in adult neural stem cells (e.g., the Dyrk1a gene) (e.g., a nucleic acid sequence of a miR-shRNA (microRNA adapted short hairpin RNA) against the gene) and a promoter sequence operably linked to the nucleic acid sequence that suppresses the expression of the gene, or an siRNA or miRNA against the gene. The present disclosure also includes pharmaceutical compositions containing transformed cells of the present disclosure. The pharmaceutical compositions of the present disclosure may contain at least one selected from the group consisting of pharmaceutically acceptable adjuvants, excipients, carriers, and diluents. The descriptions in the respective sections apply to the vectors and transformed cells of the present disclosure. These pharmaceutical compositions of the present disclosure are used for the treatment of neurodegenerative diseases or inner ear diseases.

[0047] The present disclosure also includes pharmaceutical compositions for treating neurodegenerative diseases or inner ear diseases, which contain, as active ingredients, compounds capable of enhancing the expression of genes highly expressed in embryos and low expressed in adult neural stem cells (e.g., Plagl2 gene) and suppressing the expression of genes low expressed in embryos and high expressed in adult neural stem cells (e.g., Dyrk1a gene), or prodrugs or pharmaceutically acceptable salts thereof. Furthermore, the present disclosure also includes pharmaceutical compositions for treating neurodegenerative diseases or inner ear diseases, which contain, as active ingredients, compounds capable of enhancing the expression of genes highly expressed in embryos and low expressed in adult neural stem cells (e.g., Plagl2 gene), or prodrugs or pharmaceutically acceptable salts thereof, and compounds capable of suppressing the expression of genes low expressed in embryos and high expressed in adult neural stem cells (e.g., Dyrk1a gene), or prodrugs or pharmaceutically acceptable salts thereof.

[0048] The expression of the gene that is highly expressed in embryos and lowly expressed in adult neural stem cells (e.g., Plagl2 gene) and the expression of the gene that is lowly expressed in embryos and highly expressed in adult neural stem cells (e.g., Dyrk1a gene) are preferably the expression of each gene in neural stem cells or inner ear supporting cells. Furthermore, the compound, its prodrug, or its pharmaceutically acceptable salt may be a low-molecular-weight compound, a medium-molecular-weight compound, or a high-molecular-weight compound.

[0049] The pharmaceutical compositions of the present disclosure are administered to a subject by intraventricular injection, intrathecal bolus injection or infusion, intraganglionic injection, intraneuronal injection, subcutaneous injection, or intratympanic injection.

[0050] The dosage of the pharmaceutical composition of the present disclosure is not particularly limited, and an appropriate dosage can be selected depending on various conditions such as the type of disease, the age and symptoms of the patient, the administration route, the purpose of treatment, and the presence or absence of concomitant medications.

[0051] <Treatment method> The present disclosure also includes a method for treating neurodegenerative diseases, characterized by forcibly expressing in neural stem cells a gene (e.g., Plagl2 gene) that is highly expressed in embryos and low expressed in adult neural stem cells, and suppressing the expression of a gene (e.g., Dyrk1a gene) that is low expressed in embryos and high expressed in adult neural stem cells. The present disclosure also includes a method for treating inner ear diseases, characterized by forcibly expressing in inner ear supporting cells a gene (e.g., Plagl2 gene) that is highly expressed in embryos and low expressed in adult neural stem cells, and suppressing the expression of a gene (e.g., Dyrk1a gene) that is low expressed in embryos and high expressed in adult neural stem cells. Methods for forcibly expressing a gene (such as the Plagl2 gene) that is highly expressed in embryos and lowly expressed in adult neural stem cells in target cells such as neural stem cells and inner ear supporting cells, and for suppressing the expression of a gene (such as the Dyrk1a gene) that is lowly expressed in embryos and highly expressed in adult neural stem cells, include methods using the polynucleotides, vectors, transformed cells, and pharmaceutical compositions disclosed herein; the descriptions in the sections on polynucleotides, vectors, and pharmaceutical compositions above are applicable for specific explanations of each method.

[0052] <Transformed cells> The present disclosure also includes target cells into which the polynucleotides of the present disclosure have been introduced. For example, the present disclosure also includes cells transformed with the vectors of the present disclosure, and cells obtained by transformation with the vectors of the present disclosure and transfection with the polynucleotides of the present disclosure. The transformed cells of the present disclosure can be prepared in vitro and administered to a subject in need of treatment. In this case, the transformed cells of the present disclosure can be administered as a pharmaceutical composition together with pharmaceutically acceptable adjuvants, excipients, carriers, and diluents. Furthermore, the present disclosure also encompasses cells transformed by treatment with a compound capable of enhancing the expression of a gene highly expressed in embryos and low in adult neural stem cells (e.g., Plagl2 gene) or suppressing the expression of a gene low expressed in embryos and high in adult neural stem cells (e.g., Dyrk1a gene), or a prodrug or pharmaceutically acceptable salt thereof. It also encompasses cells transformed by treatment with a compound capable of enhancing the expression of a gene highly expressed in embryos and low in adult neural stem cells (e.g., Plagl2 gene), or a prodrug or pharmaceutically acceptable salt thereof, or a compound capable of suppressing the expression of a gene low expressed in embryos and high in adult neural stem cells (e.g., Dyrk1a gene), or a prodrug or pharmaceutically acceptable salt thereof. Among such cells, preferably, the transformed cells are neural stem cells or inner ear supporting cells. Furthermore, the compound, prodrug, or pharmaceutically acceptable salt thereof may be a low-molecular-weight compound, a medium-molecular-weight compound, or a high-molecular-weight compound.

[0053] <Method for screening a substance for treating neurodegenerative disease or inner ear disease> The present disclosure provides: (1) a step of applying a test substance to neural stem cells or inner ear supporting cells, and measuring the expression of genes that are highly expressed in embryos and low in adult neural stem cells (e.g., Plagl2 gene) and genes that are low in expression in embryos and highly expressed in adult neural stem cells (e.g., Dyrk1a gene); (2-1) A step of selecting, from the results of the above step (1), a test substance having the ability to enhance the expression of a gene (such as the Plagl2 gene) that is highly expressed in embryos and lowly expressed in adult neural stem cells, and the ability to suppress the expression of a gene (such as the Dyrk1a gene) that is lowly expressed in embryos and highly expressed in adult neural stem cells; or (2-2) A step of selecting, based on the results of the above step (1), a test substance capable of enhancing the expression of a gene (such as the Plagl2 gene) that is highly expressed in embryos and lowly expressed in adult neural stem cells, and a test substance capable of suppressing the expression of a gene (such as the Dyrk1a gene) that is lowly expressed in embryos and highly expressed in adult neural stem cells. The present invention also includes a method for screening for a substance for treating a neurodegenerative disease or an inner ear disease, comprising:

[0054] Substances selected by the screening method disclosed herein can efficiently activate and proliferate neural stem cells, producing a large number of neurons. Therefore, it is expected that neural stem cells in the aging brain can be restored to their adolescent or younger state, producing a large number of neurons and improving memory and learning ability. They are also expected to be effective in treating neurodegenerative diseases such as Alzheimer's disease, efficiently activating endogenous neural stem cells to produce a large number of neurons and improving memory and learning ability. Furthermore, substances selected by the screening method disclosed herein can differentiate not only neural stem cells in the brain but also supporting cells in the inner ear into hair cells, and are therefore expected to be effective in treating inner ear diseases. [Example]

[0055] The present disclosure will be specifically described in the following examples, but the present disclosure should not be construed as being limited by these examples.

[0056] 1. Screening for neural stem cell (NSC) activating genes We reasoned that the expression of multiple genes must be altered to stimulate and proliferate aged NSCs. Because adult NSCs are largely quiescent, we compared the transcriptomes of G1 / G0 NSCs derived from the ganglionic eminence and dorsal cortex of E14 mouse embryos (DDBJ BioProject Accession: PRJDB9010) with those derived from the LV-SVZ (SAMD00192826) and DG-SGZ (SAMD00192827) of 2-3 month-old mice, which are largely quiescent. We identified differentially expressed nuclear factors and their known modulators.

[0057] First, we identified genes that are highly expressed in embryonic stem cells but are low in adult NSCs ("embryonic-high"). Second, we investigated the effects of bFGF and BMP on the quiescence (EdU) state of NSCs. -These genes were overexpressed in vitro in NSCs maintained in a 24-well platelet-free medium (Fig. 1). Two days later, the percentage of proliferating cells that incorporated EdU was measured to identify genes capable of proliferating and activating NSCs. In this study, each of the top 80 genes highly expressed in embryos was overexpressed in quiescent NSCs in vitro, and NSC proliferation and activation were monitored. The results are shown in Fig. 2. Among the genes tested, Gsx2, Dmrt3, Cdk4, Plagl2, Sox21, Ascl1, Tgif2, Plagl1, and Hmga2 efficiently activated quiescent NSCs.

[0058] Further in vivo testing was performed on Gsx2, Dmrt3, Cdk4, Plagl2, Sox21, Ascl1, Tgif2, Plagl1, and Hmga2. These nine genes were individually cloned into lentiviruses under the control of the Hes5 promoter (SEQ ID NO: 9), which specifically operates in NSCs and astrocytes. Each lentivirus was injected into the hippocampal dentate gyrus of 6-month-old mice, and brain sections were examined one week later (Figure 3). The combination of Cdk4 and cyclin D1 (D1K4) was used as a positive control because it is known to activate NSCs in the adult brain. Among the genes tested, Plagl2 (SEQ ID NO: 10; the sequence of the Hes5 promoter and Plagl2 ligated is SEQ ID NO: 11) significantly increased MCM2 expression in the hippocampal dentate gyrus of 6-month-old mice compared to control mice. + It most efficiently increased the number of mitotic cells (activated NSCs, likely intermediate progenitor cells).

[0059] Next, to identify genes whose expression can be suppressed to proliferate and activate NSCs, we targeted genes with low expression in embryos but high expression in adult NSCs. By introducing siRNA of the target gene into quiescent NSCs, we performed knockdown of genes expressed at high levels in adult NSCs ("adult-high") and their modulator genes. The results of the in vitro knockdown screening are shown in Figures 4-1 and 4-2. Of the 124 genes tested, we selected 11 genes whose knockdown efficiently activated quiescent NSCs.

[0060] Using lentivirus, shRNAs of the 11 selected genes were induced in vivo along with Plagl2 under the control of the Hes5 promoter (SEQ ID NO: 9). Each lentivirus was injected into the hippocampal dentate gyrus of 6-, 18-, or 20-month-old mice, and brain sections were examined 2 or 4 weeks later. The results showed that shRNAs of Cdkn1a, Prkcz, and Dyrk1a downregulated MCM2 in 18-month-old mice. + The number of activated NSCs / IPCs increased (Figure 5, top). Note that "control" in Figure 5 shows the results when only mCherry was linked to the Hes5 promoter, and "Scramble" shows the results when mCherry, Plagl2, and a random sequence were linked to the Hes5 promoter. + The increase or decrease in the number of activated NSCs / IPCs was judged by comparison with "Scramble." In addition, shRNA (SEQ ID NO: 13) of Dyrk1a (SEQ ID NO: 12) increased DCX in 6-month-old and 19-month-old (18 months + 4 weeks) mice. + dendrites + The number of cells increased more efficiently than the other cells (Fig. 5 middle, Fig. 5 bottom, Fig. 6). + DCX in 6- and 19-month-old (18 months + 4 weeks) mice induced with shRNA expression of Prkcz, Dyrk1a, and Zbtb7a showed an increased number of activated NSCs / IPCs. + dendrites +The numbers of IPCs are shown. The IPCs are intermediate progenitor cells.

[0061] These data indicate that the combination of Plagl2 overexpression and Dyrk1a knockdown most efficiently stimulates neurogenesis in the dentate gyrus of the hippocampus in both 6-month-old and 19-month-old mice. This combination of Plagl2 overexpression and Dyrk1a knockdown was named iPaD (inducing Plagl2 and anti-Dyrk1a activity). This combination of Plagl2 overexpression and Dyrk1a knockdown was also shown to be highly potent in the dentate gyrus of the hippocampus. + GFAP + BrdU + Activated NSCs and DCX + dendrites + BrdU + MCM2 containing immature neurons + These results suggest that Plagl2 overexpression and Dyrk1a knockdown in endogenous neural stem cells may efficiently activate and proliferate these neural stem cells, leading to the generation of numerous neurons and improving memory and learning abilities in the adult and aged brains.

[0062] 2. Long-term effects of iPaD on neurogenesis To examine the long-term effects of iPaD lentivirus on neurogenesis in the aged brain, we transduced iPaD lentivirus into the dentate gyrus of 18-month-old mice. At this age, activated NSCs (MCM2) were expressed. + ;DCX - ), IPC(MCM2 + ;DCX + / - ), immature neurons (MCM2 - ;DCX + ;dendrite +) were present in only small numbers in this region, and control lentivirus did not affect their numbers (Figure 7, Control / 8wpi). In contrast, iPaD lentivirus efficiently activated NSCs and induced the formation of IPCs and immature neurons 1 month after infection (19 months of age) (Figure 7, iPaD / 4wpi). Similar effects were observed at 8 and 12 weeks after infection (20 and 21 months of age, respectively) (Figure 7, iPaD / 8wpi, iPaD / 12wpi). This suggests that in the aged brain, Plagl2 overexpression and Dyrk1a knockdown in endogenous neural stem cells by iPaD lentivirus sustained activation of neurogenesis. Plagl2 alone also activated neurogenesis, but not as efficiently as iPaD lentivirus (Figure 8). Combining Plagl2 overexpression with Dyrk1a knockdown produced significant effects on neural stem cell activation and neurogenesis. These results suggest that iPaD lentivirus-activated NSCs continuously proliferate and are not exhausted even after 3 months. + Or DCX + The number of these cells significantly decreased with age and almost disappeared by 18 months of age (Fig. 9-1). However, iPaD lentivirus increased the number of these cells in the dentate gyrus of the hippocampus of aged brains (19 months of age) to levels comparable to those of 9-month-old or even younger wild-type mice (Fig. 9-2), suggesting that neurogenesis in the aged brain can be effectively reactivated by iPaD lentivirus.

[0063] To confirm the therapeutic efficacy of iPaD lentivirus in treating Alzheimer's disease, we transfected iPaD lentivirus into the hippocampal dentate gyrus of Alzheimer's disease model mice and performed the same analysis described above. The results showed that iPaD lentivirus efficiently activated NSCs and induced the formation of IPCs and immature neurons in Alzheimer's disease model mice. Furthermore, amyloid-β deposition was suppressed, and memory improvement was observed in fear conditioning experiments. Details are described in the section "4. Therapeutic Effect of iPaD on Alzheimer's Disease" below.

[0064] To examine whether iPaD lentivirus can activate non-NSCs, we injected it into brain regions lacking neural stem cells. Although the Hes5 promoter was also active in astrocytes, neither of these cells expressed MCM2 or DCX, suggesting that iPaD lentivirus-induced activation is specific to NSCs.

[0065] 3. Improvement of cognitive function with iPaD (Barnes maze test) To investigate whether activated neurogenesis improves cognitive function in aged mice, spatial learning and memory were analyzed using the Barnes maze test. Using this test, we previously demonstrated that spatial learning and memory depend on neurogenesis in the dentate gyrus of the hippocampus (Imayoshi, I., et al. Roles of continuous neurogenesis in the structural and functional integrity of the adult forebrain. Nat Neurosci 11, 1153-1161 (2008)). Older mice were shown to require longer distances and longer latency to reach the target hole during training sessions, demonstrating poorer performance than younger mice (Shoji, H. & Miyakawa, T. Age-related behavioral changes from young to old age in male mice of a C57BL / 6J strain maintained under a genetic stability program. Neuropsychopharmacol. Rep. 39, 100-118 (2019)).

[0066] Nineteen-month-old mice were divided into two groups and injected with control lentivirus or iPaD lentivirus one month prior (Figure 10-1). The Barnes maze test was performed on these mice as follows. Ten to twelve male mice per group were used for each behavioral analysis. Automatic tracking of mice was performed using ANY Maze software (Stoellting) to detect multiple body points. One day before virus injection, P7C3-A20 (MedKoo) was administered intraperitoneally at 10 mg / kg once daily until sacrifice to allow recovery from the surgical damage. P7C3-A20 was dissolved in 5% dextrose, 3% DMSO, and 10% Cremaphor EL (Nacalai) solution. Spatial memory was measured using a Barnes circular maze (92 cm diameter, 12 holes, Brain Science Idea). The behavioral testing space was surrounded by a black curtain, and four representative cues were placed at least 30 cm from the edge of the maze. A 5-min habituation session was conducted at one of the escape holes. One day after the habituation phase, each mouse underwent two training sessions per day to memorize the location of one escape hole, which was randomized among mice but fixed for each individual mouse. At the beginning of the training session, the mouse was placed inside a white cylinder in the center of the maze for 30 s, and the cylinder was removed when recording began. Each recording session automatically stopped after a maximum of 5 min, either when the mouse entered the escape hole or after 10 s of hovering around the escape hole. If the mouse failed to find the escape hole during the 5-min training session, the experimenter gently guided the mouse to the escape hole. After entering the escape hole, the mouse was left undisturbed in the escape hole for 60 s. After the end of training, the first 3-min probe test was conducted at a daily interval. During training, a camera installed directly above the maze recorded the total distance traveled, the number of times the mice circled the correct and misunderstood holes, and the latency to enter the goal hole. During the probe test, the total distance traveled around the correct and misunderstood holes, the number of visits, and the duration of stay were recorded. The light intensity was 100 lux on the first day of training and increased to approximately 30 lux per day. The maze was thoroughly cleaned with a 70% ethanol solution and allowed to dry between tests.

[0067] The results showed that mice injected with iPaD lentivirus reached the target hole in a shorter distance, made fewer errors, and had a shorter latency than mice injected with control lentivirus (Figure 10-2). In the first probe test (1 day after training), iPaD mice stayed in the target hole longer than control mice (Figure 10-3). These data indicate that iPaD lentivirus injection improved cognitive function in aged mice in the Barnes maze test.

[0068] 4. Comparison of Alzheimer's disease model mice and normal mice (1) Comparison of the number of MCM2-positive cells, DCX-positive cells, and amyloid beta deposition in the hippocampal dentate gyrus 5×FAD mice were used as a mouse model of Alzheimer's disease. 5×FAD mice (Tg6799 line) were developed by introducing mutant human amyloid precursor protein (APP) (Swedish mutation: K670N, M671L; Florida mutation: I716V; London mutation: V717I) and mutant human presenilin 1 (PS1) (M146L; L286V) genes under the control of the mouse Thy1 promoter. These mice exhibit amyloid-β deposition in brain regions and reduced neurogenesis, resulting in impaired memory and learning abilities. 5×FAD mice (B6 / SJL genetic background) were purchased from the Jackson Laboratory and crossed with C57BL / 6J wild-type mice for more than two generations. Wild-type littermate mice served as controls. All mice were handled in accordance with the "Regulations for the Conduct of Animal Experiments at Kyoto University." The experimental protocol was approved by the Animal Experimentation Committee of the Institute for Virus Research and Frontier Medical Sciences, Kyoto University.

[0069] Sections of the hippocampal dentate gyrus were prepared from 4-, 6-, and 8-month-old Alzheimer's disease model mice (5xFAD mice) and normal mice (wild-type littermate mice). The numbers of MCM2- and DCX-positive cells were counted by immunostaining to confirm age-related changes. The results are shown in Figure 11 (MCM2-positive cells) and Figure 12 (DCX-positive cells). Quantitative evaluation of labeled cells was performed according to the method described in the "Quantitative Evaluation of Labeled Cells" section. Sections of the hippocampal dentate gyrus were also prepared from 4-, 6-, and 8-month-old Alzheimer's disease model mice (5xFAD mice) to confirm age-related changes in amyloid-β deposition. The results are shown in Figure 13. Quantitative evaluation of amyloid-β deposition was performed according to the method described in the "Quantitative Evaluation of Amyloid-β Deposition" section.

[0070] (Quantitative evaluation of labeled cells) The dentate gyrus of three or more mice in each experiment was analyzed. For each animal, 50-μm-thick serial sections were prepared from the dentate gyrus anterior to the iPaD lentivirus injection point, and every seventh section was evaluated. All sections, except for the lesion section, were used for immunostaining. After immunostaining, z-stack images were acquired using a confocal microscope (LSM780) with a 10x or 20x objective. Stained cells were counted using Imaris software (Bitplane) or ImageJ, and 1 mm of the dentate gyrus was analyzed. 3 The number of cells was quantified per 100 cells.

[0071] (Quantitative assessment of amyloid beta deposition) Every seventh brain section from each mouse was labeled with a mouse anti-β-amyloid primary antibody (Clone 6E10; 1:500; BioLegend 9320) and detected with a fluorescently labeled secondary antibody. After immunostaining, z-stack images were acquired using a confocal microscope (LSM780) with a 10x objective. The number of Aβ plaques was quantified using ImageJ after extensive thresholding and noise reduction. Results were obtained from 1 mm of the dentate gyrus of the hippocampus. 3 The number of plaques was expressed as the number of plaques per 1000 cells.

[0072] As shown in Figure 11, the number of cells positive for MCM2, a marker of activated NSCs, was approximately 1,000 cells / mm in 4-month-old normal mice. 3 However, this decreased with age, reaching approximately 700 cells / mm at 6 months of age. 3 At 8 months of age, the number of cells is approximately 680 cells / mm 3 On the other hand, in Alzheimer's disease model mice (5×FAD mice), the number of MCM2-positive cells was approximately 350 cells / mm even at 4 months of age. 3 The number was small, and remained small with no significant change as the baby got older.

[0073] As shown in Figure 12, the number of cells positive for DCX, an undifferentiated neural marker, was approximately 2700 cells / mm in 4-month-old normal mice. 3 However, this decreased with age, reaching approximately 1500 cells / mm at 6 months of age. 3 At 8 months of age, the number of cells is approximately 800 cells / mm 3 On the other hand, in Alzheimer's disease model mice (5×FAD mice), the number of DCX-positive cells was approximately 1200 cells / mm even at 4 months of age. 3 This number decreases further with age, reaching approximately 800 cells / mm at 6 months of age. 3 At 8 months of age, the number of cells is approximately 150 / mm 3 This is what happened.

[0074] As shown in Figure 13, in Alzheimer's disease model mice (5xFAD mice), amyloid β deposition (amyloid β plaques) significantly increased with age, reaching approximately 1,000 plaques / mm at 4 months of age. 3 At 6 months of age, the number of cells was approximately 2400 / mm 3 At 8 months of age, the number is approximately 3700 cells / mm 3 This is what happened.

[0075] (2) Behavioral tests Alzheimer's disease model mice (5xFAD mice) and normal mice (wild-type littermate mice) were subjected to behavioral tests (Barnes circular maze test, contextual fear conditioning test) according to the methods described below. The results are shown in Figure 14 (Barnes circular maze test) and Figure 15 (contextual fear conditioning test).

[0076] For each behavioral analysis, 8–12 male mice were used per group. Automated tracking of mice was performed using ANY-maze software (Stoelting) to detect multiple body points. For virus injection studies, P7C3-A20 (MedKoo) was administered intraperitoneally at 10 mg / kg once daily for one week, starting one day before virus injection, to allow recovery from surgical damage. P7C3-A20 was dissolved in 5% dextrose, 3% DMSO, and 10% Cremaphor EL (Nacalai) solution.

[0077] Spatial memory was confirmed using the Barnes maze test described above in "3. Improvement of cognitive function with iPaD (Barnes maze test)."

[0078] Contextual memory was assessed using a contextual fear conditioning test, modified from a previously reported protocol (Walgrave, H. et al., Cell Stem Cell 28, 1805-1821.e1808, doi:10.1016 / j.stem.2021.05.001 (2021)). Twenty-four stainless steel rods wired to an electric shock generator were attached to the floor of the chamber (17 × 10 × 10 cm). Testing was performed in a sound-attenuating cubicle with a 60 dB background white noise. On day 1, each mouse was placed in a chamber (Context A, floor: stainless steel rods, walls: transparent) and allowed to explore for 3 min. A 2-s foot shock of 80 mA was then administered. The mouse remained in the chamber for an additional 1 min. The chamber was disinfected with 70% ethanol for each animal and allowed to dry. After 24 and 48 h, the mouse was placed back into Context A, and the same procedure as on day 1 was repeated. Fear responses were measured during the first 3 minutes using TimeFZ2 (O'HARA & CO., LTD, Japan).

[0079] In this example, data were expressed as mean ± standard error (SEM). Statistical analysis was performed using R software. Statistical differences were examined using one-way or two-way analysis of variance, followed by post-hoc testing using the Tukey-Kramer or Bonferroni method. A P value of <0.05 was considered significant.

[0080] As shown in Figures 14 and 15, it can be seen that the Alzheimer's disease model mice (5xFAD mice) are inferior to normal mice in both spatial memory ability and contextual memory ability.

[0081] 5. Therapeutic effect of iPaD on Alzheimer's disease To investigate whether iPaD lentivirus has a therapeutic effect on Alzheimer's disease, control or iPaD lentivirus was transfected into the dentate gyrus of 5-month-old 5xFAD mice. Four weeks later (6 months of age) and 12 weeks later (8 months of age), 50-μm-thick serial sections were prepared in the dentate gyrus anterior to the lentiviral injection site, and every seventh section was examined for the number of MCM2-positive cells, DCX-positive cells, and amyloid-β deposition. Four weeks after iPaD lentivirus transfection, behavioral tests (Barnes circular maze test and contextual fear conditioning test) were also performed. The results are shown in Figure 16 (MCM2-positive cells), Figure 17 (DCX-positive cells), Figure 18 (Amyloid-β deposition), Figure 19 (Barnes circular maze test), and Figure 20 (contextual fear conditioning test). Quantitative evaluation of labeled cells was performed according to the method described in the above section "Quantitative evaluation of labeled cells." Quantitative evaluation of amyloid-β deposition was performed according to the method described in the above section "Quantitative evaluation of amyloid-β deposition." The Barnes circular maze test was performed according to the method described in the above section "3. Improvement of cognitive function by iPaD (Barnes maze test)." The contextual fear conditioning test was performed according to the method described in "(2) Behavioral tests" in the above section "4. Comparison of Alzheimer's disease model mice and normal mice."

[0082] As shown in Figure 16, in mice transfected with iPaD lentivirus, the number of cells positive for MCM2, a marker for activated NSCs, significantly increased at 4 weeks after administration. At 12 weeks after administration, the number decreased from that at 4 weeks, but was still significantly higher than in the control group.

[0083] As shown in Figure 17, in mice transfected with iPaD lentivirus, the number of cells positive for DCX, a marker for undifferentiated neurons, significantly increased at 4 weeks after administration. At 12 weeks after administration, the number decreased from 4 weeks after administration, but was still significantly higher than in the control group.

[0084] As shown in Figure 18, four weeks after transfection with the iPaD lentivirus, no difference in amyloid-β deposition was observed compared to the control lentivirus, but a significant inhibitory effect was confirmed by 12 weeks after transfection. That is, the number of amyloid-β plaques in the control mice increased by more than two-fold compared to the fourth week, whereas the increase in the iPaD lentivirus-transfected mice was limited to approximately 1.2-fold.

[0085] As shown in Figures 19 and 20, in mice into which the iPaD lentivirus had been introduced, spatial memory and contextual memory were restored to the levels of normal mice shown in Figures 14 and 15, respectively.

[0086] As described above, it was found that the introduction of iPaD lentivirus significantly improved the symptoms of Alzheimer's disease and provided excellent therapeutic effects.

[0087] 6. iPaD induces differentiation of inner ear supporting cells into hair cells iPaD was expressed in supporting cells (Hensen cells) of the inner ear of one-month-old mice for two weeks. + The cluster shown in this figure consisted of 11 BrdU+ cells (Figure 21). It is believed that one supporting cell divided into 11 cells. Of these, three cells were mCherry+ (numbers 2, 3, and 7). Because the Hes5 promoter is weakly active only in supporting cells in the inner ear but not in other cells, the eight mCherry-negative cells may have transformed from supporting cells into other cell types. One of these cells (number 11) became Myosin6-positive, suggesting that it had differentiated into a hair cell. Because supporting cells at this stage have already lost their proliferation potential, iPaD may efficiently restore this proliferation potential and allow at least some of them to differentiate into hair cells. [Industrial Applicability]

[0088] According to the present disclosure, by introducing a specific gene set into the adult or aged brain, it is possible to efficiently activate and proliferate endogenous neural stem cells, producing a large number of neurons. Neural stem cells in the aged brain can be restored to their adolescent or younger state, producing a large number of neurons and improving memory and learning abilities. This is also expected to be effective in treating neurodegenerative diseases such as Alzheimer's disease, as it can efficiently activate endogenous neural stem cells, produce a large number of neurons, and improve memory and learning abilities. Furthermore, the specific gene set disclosed herein can differentiate not only neural stem cells in the brain but also supporting cells in the inner ear into hair cells, suggesting its effectiveness in treating inner ear diseases.

Claims

1. (A) Nucleic acid sequence of the Plagl2 gene; (B) Nucleic acid sequence of miR-shRNA (microRNA adapted short hairpin RNA) against the Dyrk1a gene, and (C) a promoter sequence operably linked to the nucleic acid sequence. A polynucleotide comprising:

2. The polynucleotide of claim 1, wherein the Plagl2 gene and the Dyrk1a gene are mammalian genes.

3. 3. The polynucleotide of claim 1, wherein the promoter is a promoter selected from the group consisting of Hes5 promoter, GFAP promoter, Sox2 promoter and Lfng promoter.

4. A vector comprising the polynucleotide of any one of claims 1 to 3.

5. The vector of claim 4 , wherein the vector is a lentiviral vector, an adeno-associated viral vector, an adenoviral vector, or a plasmid vector.

6. A pharmaceutical composition comprising the vector according to claim 4 or 5.

7. (a) a vector comprising a nucleic acid sequence of a Plagl2 gene and a promoter sequence operably linked to the nucleic acid sequence of the Plagl2 gene; and (b) a vector comprising a nucleic acid sequence of a microRNA adapted short hairpin RNA (miR-shRNA) against the Dyrk1a gene and a promoter sequence operably linked to the nucleic acid sequence of the miR-shRNA, or a vector comprising an siRNA or miRNA against the Dyrk1a gene. A pharmaceutical composition comprising:

8. A cell transformed with the vector according to claim 4 or 5.

9. A pharmaceutical composition comprising the cells of claim 8.

10. 10. The pharmaceutical composition according to claim 6, 7 or 9, which is used for the treatment of a neurodegenerative disease or an inner ear disease.

11. 11. The pharmaceutical composition of claim 10, wherein the composition is administered to a subject by intraventricular injection, intrathecal bolus injection or infusion, intraganglionic injection, intraneuronal injection, subcutaneous injection, or intratympanic injection.

12. (1) A step of treating neural stem cells or inner ear supporting cells in vitro with a test substance and measuring the expression of the Plagl2 gene and the Dyrk1a gene; (2-1) selecting a test substance having the ability to enhance Plagl2 gene expression and the ability to suppress Dyrk1a gene expression from the results of the above step (1); or (2-2) A step of selecting a test substance capable of enhancing Plagl2 gene expression and a test substance capable of suppressing Dyrk1a gene expression based on the results of the above step (1). A method for screening for a substance for treating a neurodegenerative disease or an inner ear disease, comprising:

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