Treatment of progressive supranuclear palsy

Abstract

To identify a causative gene for progressive supranuclear palsy to create effective therapeutic methods and therapeutic agents, and to provide useful means for developing the therapeutic methods and therapeutic agents.SOLUTION: In order to solve this problem, a therapeutic agent for progressive supranuclear palsy is provided, which contains a compound that inhibits expression of a filamin A gene. In addition, an evaluation system or the like using cells expressing filamin A is provided, which can be used to search and the like for the therapeutic agents or candidate agents for progressive supranuclear palsy.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to the treatment of progressive supranuclear palsy (PSP), and more particularly to a therapeutic agent for PSP and a means for searching for the same (drug evaluation system). [Background technology]

[0002] PSP is a neurodegenerative disease characterized by the pathological abnormal aggregation of tau protein in neurons and glial cells such as astroglia. Clinical symptoms of PSP vary widely, ranging from motor symptoms such as classic Richardson syndrome and Parkinsonism to psychiatric symptoms such as frontotemporal dementia (Non-Patent Document 1). Because there is no cure for PSP, all patients progress, with most dying within 5 to 10 years of onset (Non-Patent Documents 1 and 2). Furthermore, the motor and psychiatric symptoms of PSP impose a significant burden on families, posing a social problem that must be addressed (Non-Patent Document 3). Human tau protein is broadly classified into two isoforms, 3-repeat tau protein (3R-tau) and 4-repeat tau protein (4R-tau), based on the number of repeats in the microtubule-binding domain. Aggregated tau protein induces cell death due to its toxicity. Diseases characterized by tau protein aggregation are collectively called tauopathies, and include several diseases such as PSP and Alzheimer's disease (AD). While both 3R-tau and 4R-tau aggregate in AD, 4R-tau aggregates predominantly in PSP. The distribution of lesions in PSP differs from that in Alzheimer's disease, with the basal ganglia, midbrain tegmentum, and frontal lobe predominating. Globose-type neurofibrillary tangles (globose-type NFTs) and tufted astroglia (TAs) are 4R-tau aggregate forms characteristic of PSP (Non-Patent Documents 1 and 4). It has been known that the RNA-protein interaction between fused in sarcoma (FUS) and splicing factor proline- and glutamine-rich (SFPQ) regulates the balance between 3R-tau and 4R-tau, and that disruption of this mechanism is involved in the pathology of tauopathy (Non-Patent Document 5). However, the pathological mechanism by which tau protein aggregates remains unknown, and no animal model for PSP has been established.

[0003] PSP is generally sporadic, but rarely occurs in families (Non-Patent Documents 1 and 6). Some familial PSP cases have mutations in the MAPT gene, which encodes the tau protein, and the mutated tau protein has a high aggregation ability (Non-Patent Document 1). [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Boxer AL, Yu JT, Golbe LI, Litvan I, Lang AE, Hoglinger GU. Advances in progressive supranuclear palsy: new diagnostic criteria, biomarkers, and therapeutic approaches. Lancet Neurol 2017; 16: 552-63. [Non-patent document 2] Glasmacher SA, Leigh PN, Saha RA. Predictors of survival in progressive supranuclear palsy and multiple system atrophy: a systematic review and meta-analysis. J Neurol Neurosurg Psychiatry 2017; 88: 402-11. [Non-patent document 3] Uttl B, Santacruz P, Litvan I, Grafman J. Caregiving in progressive supranuclear palsy. Neurology 1998; 51: 1303-1309. [Non-patent document 4] Yoshida M. Astrocytic inclusions in progressive supranuclear palsy and corticobasal degeneration. Neuropathology 2014; 34: 555-70. [Non-patent document 5] Ishigaki S, Fujioka Y, Okada Y, Riku Y, Udagawa T, Honda D et al. Altered Tau Isoform Ratio Caused by Loss of FUS and SFPQ Function Leads to FTLD-like Phenotypes. Cell Rep 2017; 18: 1118-31. [Non-patent document 6] Fujioka S, Algom AA, Murray ME, Strongosky A, Soto-Ortolaza AI, Rademakers R et al. Similarities between familial and sporadic autopsy-proven progressive supranuclear palsy. Neurology 2013; 80: 2076-8 Summary of the Invention [Problem to be solved by the invention]

[0005] Since there are familial PSP families in which no mutations in the MAPT gene are found, it is possible that there may be genetic factors other than the MAPT gene that affect tau protein aggregation. The cause of PSP is unknown, and no effective treatments or drugs exist. Therefore, an objective of the present invention is to break through this current situation by identifying the causative gene for PSP and creating effective treatments or drugs. Another objective is to provide a means useful for the development of treatments or drugs. [Means for solving the problem]

[0006] As a result of investigations aimed at solving the above problems, filamin A (FLNA) was identified as a candidate causative gene through neuropathological analysis of PSP patients and DNA microarray analysis. Further investigations provided evidence supporting the involvement of filamin A in the onset and pathogenesis of PSP and its role as a therapeutic target, and revealed that suppression of filamin A gene expression can have a therapeutic effect. Based on these results, the following inventions are primarily provided. [1] A drug for the treatment of progressive supranuclear palsy that contains a compound that suppresses the expression of the filamin A gene. [1A] An inhibitor of 4-repeat tau expression, comprising a compound that inhibits the expression of the filamin A gene. [1B] A phosphorylated 4-repeat tau inhibitor containing a compound that inhibits the expression of the filamin A gene. [1C] An inhibitor of 4-repeat tau aggregation, which contains a compound that inhibits the expression of the filamin A gene. [2] The therapeutic agent for progressive supranuclear palsy according to [1], wherein the compound is a compound selected from the group consisting of the following (a) to (e): (a) siRNA targeting the filamin A gene; (b) a nucleic acid construct that intracellularly produces siRNA targeting the filamin A gene; (c) a single-stranded RNA having an expression-inhibiting sequence that inhibits the expression of the filamin A gene and a complementary sequence that anneals to the sequence; (d) an antisense nucleic acid targeting the transcript of the filamin A gene; (e) Ribozyme targeting the filamin A gene transcript. [2A] A therapeutic agent for progressive supranuclear palsy according to [1] or [2], characterized in that it is administered to a subject in which expression of filamin A and / or 4-repeat tau is increased in neurons and / or glial cells. [2B] A therapeutic agent for progressive supranuclear palsy according to [1] or [2], characterized in that it is administered to a subject in which 4-repeat tau is overexpressed over 3-repeat tau in neurons and / or glial cells. [3] A method for treating progressive supranuclear palsy in a subject, comprising administering to the subject the progressive supranuclear palsy therapeutic agent described in [1] or [2]. [3A] The method for treating progressive supranuclear palsy described in [3], wherein the subject has increased expression of filamin A and / or 4-repeat tau in neurons and / or glial cells. [3B] The method for treating progressive supranuclear palsy described in [3], wherein the subject has an overexpression of 4-repeat tau over 3-repeat tau in neurons and / or glial cells. [4] A method for evaluating the efficacy of a test substance for progressive supranuclear palsy, comprising the following steps (i) and (ii): (i) contacting a test substance with cells expressing filamin A; (ii) detecting the expression of filamin A, the amount of 4-repeat tau, and / or the amount of phosphorylated tau in the cells, and determining the effectiveness of the test substance based on the detection results, wherein a decrease in the expression of filamin A, a decrease in the amount of 4-repeat tau, and / or a decrease in the amount of phosphorylated tau is an indicator that the test substance is effective. [5] The evaluation method according to [4], wherein the cells are a lymphocyte cell line derived from a patient with progressive supranuclear palsy. [6] A lymphocyte cell line derived from a patient with progressive supranuclear palsy characterized by increased expression of filamin A. [7] A non-human mammal that expresses high levels of filamin A through the introduction of the filamin A gene, resulting in a progressive supranuclear palsy-like condition. [8] The non-human mammal according to [7], which is a transgenic animal. [9] A non-human mammal described in [7] or [8], wherein the progressive supranuclear palsy-like pathology is an increase in 4-repeat tau and / or an increase in phosphorylated tau in neurons and / or glial cells.

[10] The non-human mammal according to any one of [7] to [9], wherein the species (genus) of the non-human mammal is any one selected from the group consisting of mouse, rat, guinea pig, hamster, rabbit, dog, cat and monkey.

[11] The non-human mammal according to any one of [7] to [9], wherein the species (genus) of the non-human mammal is a mouse.

[12] Biomarkers for progressive supranuclear palsy, including filamin A. [Brief explanation of the drawings]

[0007] [Figure 1] A Japanese pair of monozygotic twins with simultaneous PSP. (a) The pedigree shows the unaffected individual (white) and the monozygotic twins with PSP (black, Twin-A and Twin-B). Circles represent females, and squares represent males. All 12 microsatellite marker haplotypes were identical, consistent with Twin-A and Twin-B being monozygotic twins. (b-n) Neuropathological findings in Twin-A and Twin-B are consistent with PSP. Twin-B's frontal lobe was atrophic (b). Coronal sections revealed atrophy of the internal globus pallidus (c, arrow) and the subthalamic nucleus (c, arrowhead). In the midbrain, the tegmental region was atrophic, and the substantia nigra was brownish (d, arrow). Microscopic findings: Low-magnification images of Twin-B show aggregates positive for the 4-repeat tau antibody RD4 (e) and the 3-repeat tau antibody RD3 (f) in the globus pallidus. High-magnification images of Twin-A (g-j) and Twin-B (k-n) are shown. TAs (g-i, k-m) and globose-type NFTs (j-n), characteristic of PSP, are shown. Scale bars are 10 mm (c, d), 20 μm (e, f), and 10 μm (g-n). Photographs show Gallas-Braak (GB) staining (g, k), RD4 antibody staining (e, i, j, m, n), RD3 antibody staining (f), and AT8 antibody staining (h, l). [Figure 2]FLNA gene duplication was identified in identical twins with simultaneous PSP. (a) Whole-exome analysis or chromosomal microarray analysis using eXome Hidden Markov Model (XHMM) showed that Twin-A, Twin-B, and their unaffected female sibling (II-3) possessed a copy number gain region of approximately 0.3 Mb in Xq28. The top row shows the XHMM of Twin-A. The vertical axis indicates the Z-score. The other rows show microarray data for Twin-A, Twin-B, and their unaffected female sibling (II-1, II-2, II-3). The vertical axis indicates the log2 ratio. (b) Enlarged view of the copy number gain region observed in Twin-A's microarray analysis. The dotted box in (a) indicates the region. The figure also indicates the locations of low copy repeats (LCRs) and coding genes. The copy number gain region includes 16 coding genes, with the copy number changing stepwise in the LCRs. Copy numbers calculated by microarray analysis are plotted, showing that the FLNA gene was duplicated to two copies. (c) X chromosome inactivation (XCI) analysis using the methylated region of the FRAXA gene revealed a markedly skewed pattern (XCI ratio = 93:7) in the unaffected female sibling (II-3). (d) Real-time quantitative PCR analysis using cDNA derived from immortalized lymphocytes of Twin-A, Twin-B, and the unaffected female sibling (II-3). The mRNA expression levels of genes within the copy number gain region, including the FLNA gene, were increased in Twin-A and Twin-B but not in II-3. Values ​​were normalized by the housekeeping gene GUSB and are relative values ​​using II-1 as the control. [Figure 3-1] Filamin-A promotes 4R-tau aggregation. (a) Western blot of the frontal lobe of an autopsy brain. Of the 16 coding genes in the copy number gain region of Xq28, five genes (†; Filamin-A (FLNA), RPL10, GDI1, FAM3A, G6PD) were elevated above the median + standard deviation of the healthy control group (Normal-1 to 5) in both Twin-A and Twin-B. GAPDH was used as a loading control. [Figure 3-2]Continued from Figure 3. (b) When Filamin-A, one of the five genes mentioned above, was co-expressed with GFP-tagged 4R-tau (GFP-4R-tau) in HEK293 cells, it statistically significantly increased the expression of GFP-4R-tau compared to the control empty expression (P<0.001, n=5). Tukey-Kramer test was performed. *** indicates P<0.001. Error bars indicate standard error. [Figure 3-3] Continued from Figure 3. (c) Western blot of immortalized lymphocytes. Twin-A and Twin-B had increased expression of Filamin-A and endogenous tau protein compared with their unaffected siblings (II-1, II-2, and II-3). Tau protein was dephosphorylated with protein phosphatase and analyzed with the TAU-5 antibody. (d) Western blot analysis using three types of siRNA that suppress Filamin-A expression revealed that Twin-A's immortalized lymphocytes had decreased expression of not only Filamin-A but also endogenous 4R-tau protein. The RD4 antibody was used. The Tukey-Kramer test was performed. *** indicates P<0.001, and * indicates P<0.05. Error bars indicate standard error. [Figure 3-4] Continued from Figure 3. (e) Western blot of the TBS-soluble fraction (S1) extracted from HEK293 cells expressing Filamin-A and GFP-4R-tau. Using the phospho-tau antibodies AT8 (Ser202 / Thr205) and PHF-1 (Ser396 / Ser404), we observed increased phosphorylation of GFP-4R-tau due to Filamin-A expression. (f) Cycloheximide (CHX) chase experiments demonstrated protein stabilization of GFP-4R-tau due to Filamin-A expression (n=3). HEK293 cells expressing Filamin-A and GFP-4R-tau were treated with CHX, and proteins were collected at the indicated times (n=3). [Figure 3-5]Continued from Figure 3. (g) Western blot performed using homogenate (Ho), TBS-soluble fraction (S1), and sarkosyl-insoluble fraction (P3) of HEK293 cells expressing Filamin-A and GFP-4R-tau. In this experiment, Filamin-A plasmid was transfected at various doses as shown. In Ho, GFP-4R-tau expression increased in a Filamin-A expression-dependent manner (n=3). In S1 and P3, GFP-4R-tau expression increased statistically significantly at maximum Filamin-A expression (P<0.05 for both S1 and P3). The arrow indicates GFP-4R-tau, and the arrowhead indicates endogenous tau. [Figure 3-6] Continued from Figure 3. (h) Co-immunoprecipitation using TAU-5 antibody. In HEK293 cells expressing Filamin-A and GFP-4R-tau, Filamin-A, heat shock proteins HSP90, HSP70, HSP40, and ubiquitin were immunoprecipitated together with GFP-4R-tau. [Figure 4-1] Filamin-A colocalizes with aggregated tau in PSP autopsy brains, and experimentally excessive Filamin-A induces tau aggregation in primary astrocytes. (a) Western blot analysis of the frontal cortex of 34 patients. In both the TBS-soluble fraction (S1) and the sarkosyl-insoluble fraction (P3), Filamin-A expression was statistically significantly elevated in PSP compared with healthy controls and patients with other neurodegenerative diseases (P<0.01 for S1, P<0.05 for P3). GAPDH was used as a loading control. The dotted line indicates the membrane boundary. The Tukey-Kramer test was performed for parametric data in S1, and the Steel-Dwass test was performed for nonparametric data in P3. [Figure 4-2] (b) In P3 brains of 11 PSP autopsy cases (Twin-A, Twin-B, and 9 sporadic PSP cases), the expression level of Filamin-A was positively correlated with the expression level of 4R-tau. The correlation was evaluated by the non-correlation test of the Pearson product-moment correlation coefficient. [Figure 4-3]Continued from Figure 4. (c-e) Fluorescent immunostaining of the frontal cortex for Twin-B (c), PSP-6 (d), and PSP-9 (e). Filamin-A (shown in red in the original image) and phosphorylated tau AT8 (shown in green in the original image) co-localized in TAs and NFTs. Scale bar 5 μm. [Figure 4-4] Continued from Figure 4. (f) Fluorescent immunostaining of rat primary astrocytes co-expressing Filamin-A (shown in red in the original image) and GFP-4R-tau (shown in green in the original image). When Filamin-A was expressed, GFP-4R-tau aggregated in the cell body and proximal processes of astroglia. The dotted box in the low-magnification image is shown next to a high-magnification image. The arrow indicates aggregated GFP-4R-tau. Scale bar: 5 μm. [Figure 4-5] Continued from Figure 4. (g) Western blot of primary astrocytes showed a statistically significant increase in GFP-4R-tau expression upon Filamin-A expression (P<0.01, n=3). Arrowheads indicate nonspecific bands. Student's t-test was performed. ** indicates P<0.01, * indicates P<0.05. Error bars indicate standard error. [Figure 5] Ectopic gray matter in Twin-B. Twin-B showed ectopic gray matter in the anterior horn of the right lateral ventricle (inside the white dotted box, a, b) and the cerebellum (inside the black solid box, c). Scale bars are 10 mm (a), 500 μm (b), and 1 mm (c). Microscopic images are Kluver-Barerra stained (b, c). [Figure 6] Neuroradiological images of Twin-A. (a-d) Brain MRI of Twin-A at age 66. T2-weighted axial images showed cerebral atrophy, mainly in the frontal and temporal lobes (a-c). T1-weighted sagittal images showed atrophy of the midbrain (d, arrow). (e-g) 99mTc-ECD cerebral blood flow SEPCT images of Twin-A at age 66. Decreased blood flow was observed in the frontal and temporal lobes. Rt indicates the right side. [Figure 7]Neuropathological findings in Twin-A. The cerebrum, cerebellum, and brainstem were generally atrophic (a, b). Coronal sections revealed atrophy of the internal globus pallidus (c, arrow) and subthalamic nucleus (c, arrowhead). In the midbrain, the tegmental region was atrophic, and the substantia nigra was brownish (d, arrow). Microscopic findings revealed neuronal loss and gliosis in the subthalamic nucleus (e), internal globus pallidus (f), midbrain tegmental region (g), and substantia nigra (h). Globose-type NFTs (i) were observed in the substantia nigra of the midbrain. Scale bars are 5 mm (c), 10 mm (d), 50 μm (e-g), 100 μm (h), and 5 μm (i). Photographs show hematoxylin-eosin staining (e-i). [Figure 8] Whole exome analysis using XHMM for Twin-A and healthy Japanese men. Z scores for the Xq28 chromosome region were extracted from the XHMM data and graphed. Copy number abnormalities, including those in the FLNA gene, observed in Twin-A were not observed in the 513 healthy Japanese men. [Figure 9] Copy number analysis by real-time quantitative PCR. (a) Chromosomal microarray results for Twin-A and the location of the primer pairs (#1–#5; #2 indicates the FLNA gene region) used for real-time quantitative PCR are shown. The lower part of the figure illustrates the copy number changes. (b) Copy number analysis by real-time quantitative PCR using genomic DNA from Twin-A and Twin-B. As with the microarray results, copy number in a specific region of Xq28 changed stepwise from one copy to three copies. The MECP2 gene, located outside the region of copy number gain in Xq28, was used as the reference gene, and genomic DNA from a male twin sibling (II-2) was used as the control sample. (c) Copy number analysis by real-time quantitative PCR using genomic DNA from sporadic PSP (PSP-1–9). The FLNA gene had two copies in Twin-A and Twin-B, but only one copy in all nine sporadic PSP cases. The MECP2 gene was used as the reference gene, and genomic DNA from PSP-1 was used as the control sample. [Figure 10]The p.Ala39Gly mutation in Filamin-A (FLNAAla39Gly) attenuates the upregulation of 4R-tau by Filamin-A. FLNAAla39Gly was co-expressed with GFP-4R-tau in HEK293 cells. Compared with co-expression of wild-type Filamin-A (FLNAWT), the expression of GFP-4R-tau was statistically significantly reduced (P<0.05, n=3). Student's t-test was performed. * indicates P<0.05. Error bars indicate standard error. [Figure 11] Correlation between TBS-soluble filamin-A and age at onset of PSP. In 11 PSP cases, including Twin-A and Twin-B, a negative correlation was observed between TBS-soluble filamin-A and age at onset of PSP. The correlation was evaluated using a non-correlation test of the Pearson product-moment correlation coefficient. [Figure 12] Clinical course and neuropathological features of Twin-A and Twin-B. NFT: globose-type neurofibrillary tangle, TA: tufted astroglia. The severity of neuronal death / tau pathology was classified as absent (-), mild (+), moderate (++), or severe (+++). [Figure 13] Coding genes within the copy number gain region of Xq28. chrX: chromosome X, CNS: central nervous system, MIM: Mendelian Inheritance in Man. [Figure 14]Clinical information and neuropathological features of 34 autopsy brain cases. PSP: progressive supranuclear palsy, CBD: corticobasal degeneration, AD: Alzheimer's disease, PD: Parkinson's disease, DLB: dementia with Lewy bodies, ALS: amyotrophic lateral sclerosis, bvFTD: behavioral variant frontotemporal dementia, MSA: multiple system atrophy, SjS: Sjögren's syndrome, CIDP: chronic inflammatory demyelinating polyneuropathy, PE: pulmonary embolism, M: male, F: female, PMI: post-mortem interval, ND: not done, AG: argyrophilic grain, CERAD: Consortium to Establish a Registry for Alzheimer's Disease. Asterisks (*) indicate cerebral hemisphere weight. [Figure 15-1] (a) Module structure. Wild-type Filamin-A (FLNAWT) contains an N-terminal actin-binding domain (ABD) and 24 immunoglobulin-like domains (Ig). FLNAABD+Ig1-15+Ig24 is a truncated FLNA consisting of the actin-binding domain (ABD) involved in protein interaction with F-actin, Ig residues 1 to 15, and Ig residue 24, which is involved in FLNA dimerization. FLNAABD+Ig9-15+Ig24 (ΔFLNA) is a truncated FLNA consisting of the ABD, Ig residues 9 to 15, and Ig residue 24. AAV9-ΔFLNA-6xHis is an adeno-associated virus vector type 9 (AAV9) carrying the ΔFLNA cDNA. CBA indicates chicken β-actin promoter, 6xHis indicates 6xHis tagged protein, WRPE indicates woodchuck hepatitis virus posttranscriptional regulatory element, and SpA indicates SV40 poly A. [Figure 15-2](Continued from Figure 15.) (b) Immunoprecipitation using tau antibody (TAU-5 antibody). Each plasmid was transfected into HEK293 cells. ΔFLNA interacts with tau protein similarly to wild-type FLNA (FLNAWT) and FLNAABD+Ig1-15+Ig24. [Figure 15-3] Continued from Figure 15. (c) Fluorescent immunostaining using tau antibody (K9JA) and FLNA antibody. (d) Western blot using 4-repeat tau antibody (RD4) and phosphorylated tau antibody (AT8). Wild-type (WT) mice were injected with AAV9-ΔFLNA-6xHis into the right frontal lobe at 2 months of age and analyzed at 3 months of age. * indicates the injection site. ΔFLNA increased the expression and phosphorylation of endogenous tau in mice. WT mice injected with AAV9-empty-6xHis served as controls. [Figure 15-4] Continued from Figure 15. (e) Fluorescent immunostaining using 4-repeat tau antibody (RD4), 3-repeat tau antibody (RD3), and 6xHis antibody. AAV9-ΔFLNA-6xHis was injected into the right frontal lobe of a genetically engineered mouse (hT-PAC-N) expressing human tau protein at 2 months of age, and analysis was performed at 3 months of age. ΔFLNA increased the expression of both 4-repeat and 3-repeat tau. (f) Western blot. TBS-soluble fraction (S1) and sarkosyl-insoluble fraction (P3) were extracted from brain homogenate (Ho). Increased expression of both 4-repeat and 3-repeat tau was observed in S1 and P3. The control was hT-PAC-N injected with AAV9-empty-6xHis. [Figure 16] Immunostaining of transgenic mice (hFLNA-Tg) in which human Filamin-A (FLNA) expression is induced downstream of the CAG promoter. Eight-month-old mice showed increased expression of FLNA and 4-repeat tau in the hippocampus and frontal cortex. Non-transgenic mice (non-Tg) served as controls. [Figure 17-1](a) Each plasmid was introduced into mouse fetal brains at 14 days of gestation (E14) by in utero electroporation, and immunofluorescence staining was performed at 18 days of gestation (E18). Wild-type FLNA (FLNAWT) induced ectopic gray matter and increased the fluorescence intensity of GFP-4R-tau. In contrast, actin-binding-defective mutant FLNA (Filamin-A p.Ala39Gly mutation: FLNAA39G) did not show these changes. * and ** indicate P values ​​of Tukey's test less than 0.05 and 0.01, respectively. [Figure 17-2] Continued from Figure 17. (b) Each plasmid was introduced into mouse fetal brains at 14 days of gestation (E14) by in utero electroporation, and primary cortical neurons were harvested at 15 days of gestation (E15). Fluorescent immunostaining was performed after 2 days of cell culture (DIV). Administration of 0.1% DMSO (control) increased the expression of AT8-positive phosphorylated tau by wild-type FLNA (FLNAWT), but not by the actin polymerization inhibitor cytochalasin D (CytoD). *** indicates a Tukey P value of less than 0.001. DETAILED DESCRIPTION OF THE INVENTION

[0008] 1. Treatment of progressive supranuclear palsy (PSP) A first aspect of the present invention is based on the finding that filamin A is involved in the onset and pathogenesis of PSP, and relates to a therapeutic agent for PSP (hereinafter referred to as "the medicament of the present invention") containing a compound that suppresses filamin A gene expression, preferably a therapeutic agent for PSP containing a compound that suppresses filamin A gene expression as an active ingredient. PSP is one of a group of neurodegenerative diseases (tauopathies) accompanied by abnormal tau pathology. In PSP, for example, neurons in the globus pallidus, subthalamic nucleus, cerebellar dentate nucleus, red nucleus, substantia nigra, and brainstem tegmentum are lost, and abnormally phosphorylated tau protein accumulates in neurons and glial cells. The cause and mechanism of onset remain unknown, and currently no effective treatment is available.

[0009] As used herein, the term "therapeutic drug" refers to a pharmaceutical that exhibits a therapeutic or preventive effect against a target disease or pathology (i.e., PSP). Therapeutic effects include alleviating (alleviating) symptoms characteristic of the target disease / pathology or accompanying symptoms, and preventing or delaying the worsening of symptoms. The latter can be considered a type of preventive effect in that it prevents the condition from becoming severe. As such, therapeutic effects and preventive effects are concepts that partially overlap. A typical example of a preventive effect is preventing or delaying the recurrence of symptoms characteristic of the target disease / pathology. A drug that exhibits some kind of therapeutic effect or preventive effect, or both, against the target disease / pathology is considered a therapeutic drug for the target disease / pathology.

[0010] Filamin is an actin filament cross-linking protein, and three types of filamin (A, B, and C) are known. Filamin A and B are expressed in various organs, but filamin C is only expressed in muscle. Filamin consists of a dimer of subunits with a molecular weight of approximately 280 kD that self-associate at the C-terminus, and cross-links actin filaments in a lattice pattern using the actin-binding domain on the N-terminus, forming a gel structure. Mutations in the filamin A gene have been reported to be involved in periventricular heterotopic gray matter and familial valvular dystrophy. The sequences of filamin A isoform 1 and the gene encoding it (transcript variant 1) are shown in SEQ ID NO: 1 (DEFINITION: filamin-A isoform 1 [Homo sapiens]. ACCESSION: NP_001447. VERSION: NP_001447.2) and SEQ ID NO: 2 (DEFINITION: Homo sapiens filamin A (FLNA), transcript variant 1, mRNA. ACCESSION: NM_001456. VERSION: NM_001456.3), respectively. The sequences of filamin A isoform 2 and the gene encoding it (transcript variant 2) are shown in SEQ ID NO: 3 (DEFINITION: filamin-A isoform 2 [Homo sapiens]. ACCESSION: NP_001104026. VERSION: NP_001104026.1) and SEQ ID NO: 4 (DEFINITION: Homo sapiens filamin A (FLNA), transcript variant 2, mRNA. ACCESSION: NM_001110556. VERSION: NM_001110556.2).

[0011] A compound that suppresses the expression of the filamin A gene is a compound that suppresses the process of filamin A gene expression (including transcription, post-transcriptional regulation, translation, and post-translational regulation). Such a compound may be a compound identified by the screening described below.

[0012] In one embodiment of the present invention, the compound that suppresses the expression of the filamin A gene is an isolated nucleic acid, which may be chemically modified by one or more of the following methods, for example, to prevent degradation by hydrolases such as nucleases. (1) At least some of the phosphate residues of nucleotides [phosphodiester; -OP(=O)(O - )-O-] to phosphorothioate [-OP(=O)(S - )-O-], methylphosphonate [-OP(=O)(CH3)-O-], phosphorodithioate [-OP(=S)(S - )-O-], boranophosphate [-OP(=O)(BH3 - )-O-], phosphotriester [-OP(=O)(OR)-O- (R represents, for example, -CH2CH2CN, etc.)], phosphoramidate [-NH-P(=O)(O - )-O-] and the like. (2) The sugars of at least some of the nucleotides may be substituted with morpholine, and the phosphate residues may be substituted with phosphorodiamidate [-P(=O)(NR2)-O- (R represents, for example, -CH3)]. (3) The hydroxyl group at the second position of the sugar (ribose) of at least some of the ribonucleotides may be substituted with -OR (R represents, for example, -CH3, -CH2CH2OCH3, -CH2CH2NHC(NH)NH2, -CH2CONHCH3, -CH2CH2CN, etc.). (4) At least some of the nucleotide bases (pyrimidines and purines) may be chemically modified, for example, by introducing a methyl group or a cationic functional group into the 5-position of the pyrimidine base, or by substituting a thiocarbonyl group for the carbonyl group at the 2-position. (5) The phosphate moiety or hydroxyl moiety of at least some of the nucleotides may be modified with, for example, biotin, an amino group, a lower alkylamine group, an acetyl group, or the like. (6) At least some of the ribonucleotides may be substituted with BNA, LNA, ENA, etc., in which the sugar conformation is fixed to N-type by bridging the 2' oxygen and 4' carbon of the sugar. (7) At least some of the nucleotides may be substituted with non-nucleotide nucleic acid analogs such as PNA. (8) It may be conjugated with sterols such as cholesterol; vitamins such as α-tocopherol and folic acid; N-acetylgalactosamine; fatty acids; or polymers such as polyethylene glycol, polyamines, and cell membrane-penetrating peptides.

[0013] Examples of compounds that suppress the expression of the filamin A gene are as follows: In the present invention, "suppression of expression" may be either transient suppression or constant suppression. (a) siRNA targeting the filamin A gene (b) A nucleic acid construct that produces siRNA targeting the filamin A gene in cells. (c) a single-stranded RNA having an expression-inhibiting sequence that inhibits the expression of the filamin A gene and a complementary sequence that anneals to the sequence; (d) an antisense nucleic acid targeting the transcript of the filamin A gene; (e) Ribozymes targeting the filamin A gene transcript.

[0014] The compounds (a) and (b) above are compounds used for expression suppression by so-called RNAi (RNA interference). In other words, the pharmaceutical composition of the present invention containing the compound (a) or (b) above can suppress the expression of the filamin A gene by RNAi. RNAi is a process of sequence-specific post-transcriptional gene silencing that can be induced in eukaryotic cells. RNAi in mammalian cells uses short double-stranded RNA (siRNA) whose sequence corresponds to the sequence of the target mRNA. Typically, siRNAs are 15 or more, 16 or more, 17 or more, 18 or more, 19 or more, 20 or more, or 21 or more base pairs long, and 32 or less, 31 or less, 30 or less, 29 or less, 28 or less, 27 or less, 26 or less, 25 or less, 24 or less, or 23 or less base pairs long. For example, siRNAs are 21 to 23 base pairs long. Mammalian cells are known to have two pathways affected by double-stranded RNA (dsRNA): a sequence-specific pathway and a sequence-nonspecific pathway. In the sequence-specific pathway, relatively long dsRNA is cleaved into short interfering RNAs (i.e., siRNAs). On the other hand, the sequence-nonspecific pathway is thought to be triggered by any dsRNA of a certain length, regardless of its sequence. In this pathway, dsRNA activates two enzymes: PKR, which activates and phosphorylates the translation initiation factor eIF2, thereby terminating all protein synthesis, and 2',5' oligoadenylate synthase, which is involved in the synthesis of RNAase L-activating molecules. To minimize progression along this sequence-nonspecific pathway, it is preferable to use double-stranded RNA (siRNA) shorter than about 30 base pairs (see Hunter et al. (1975) J Biol Chem 250: 409-17; Manche et al. (1992) Mol Cell Biol 12: 5239-48; Minks et al. (1979) J Biol Chem 254: 10180-3; and Elbashir et al. (2001) Nature 411: 494-8).

[0015] To induce target-specific RNAi, siRNA consisting of a sense RNA homologous to a portion of the mRNA sequence of the filamin A gene (e.g., the sequence shown in SEQ ID NO: 2 or 4) and an antisense RNA complementary thereto may be introduced into cells or expressed in cells. The compound (a) above corresponds to the former method, and the compound (b) above corresponds to the latter method.

[0016] siRNA targeting the filamin A gene is typically a double-stranded RNA hybridized with a sense RNA consisting of a sequence homologous to a continuous region in the mRNA sequence of the gene and an antisense RNA consisting of its complementary sequence. The length of the "continuous region" here is typically 15 to 30 bases, preferably 18 to 23 bases, and more preferably 19 to 21 bases.

[0017] It is known that double-stranded RNAs with overhangs of several bases at their ends exert a strong RNAi effect. Therefore, it is preferable to use siRNAs with such structures in the present invention. The length of the overhang is not particularly limited, but is preferably two bases long (e.g., TT, UU).

[0018] siRNAs made of modified RNA may also be used, such as phosphorothioated RNA and modified bases (e.g., fluorescently labeled bases).

[0019] In one embodiment of the present invention, the sense RNA of the siRNA has a nucleotide sequence that is, for example, 80% or more, preferably 85% or more, more preferably 90% or more, and even more preferably 95% or more identical to the nucleotide sequence set forth in any of SEQ ID NOs: 9 to 11. The "identity" of the nucleotide sequence can be calculated using the National Center for Biotechnology Information (NCBI) homology algorithm BLAST (Basic local alignment search tool) (http: / / www.ncbi.nlm.nih.gov / BLAST / ) with default (initial setting) parameters.

[0020] siRNAs can be designed and prepared by conventional methods. siRNAs are usually designed using sequences (contiguous sequences) specific to the target sequence. Programs and algorithms have been developed to select appropriate target sequences.

[0021] The "nucleic acid construct that generates siRNA in cells" in (b) above refers to a nucleic acid molecule that, when introduced into a cell, generates the desired siRNA (an siRNA that induces RNAi against the filamin A gene) through an intracellular process. One example of such a nucleic acid construct is shRNA (short hairpin RNA). shRNA has a structure (hairpin structure) in which sense RNA and antisense RNA are linked via a loop structure, and the loop structure is cleaved in the cell to form double-stranded siRNA, thereby exerting the RNAi effect. The length of the loop structure is not particularly limited, but is usually 3 to 23 bases.

[0022] Another example of a nucleic acid construct is a vector capable of expressing a desired siRNA. Such vectors include vectors (called stem-loop or short hairpin types) that express shRNA (inserted with a sequence encoding shRNA) that will be converted into siRNA by a subsequent process, and vectors (called tandem types) that express sense and antisense RNAs separately. These vectors can be prepared by those skilled in the art using standard methods (see, for example, Brummelkamp TR et al. (2002) Science 296:550-553; Lee NS et al. (2001) Nature Biotechnology 19:500-505; Miyagishi M & Taira K (2002) Nature Biotechnology 19:497-500; Paddison PJ et al. (2002) Proc. Natl. Acad. Sci. USA 99:1443-1448; Paul CP et al. (2002) Nature Biotechnology 19:505-508; Sui G et al. (2002) Proc Natl Acad Sci USA 99(8):5515-5520; Paddison PJ et al. (2002) Genes Dev. 16:948-958). Currently, various RNAi vectors are available. The vectors of the present invention may be constructed using such known vectors. In this case, an insert DNA encoding a desired RNA (e.g., shRNA) is prepared and then inserted into the cloning site of the vector to form an RNAi expression vector (see, for example, Meng X et al. (2004) J. Biol. Chem. 279(7):6098-6105).

[0023] The origin and structure of the vector are not limited, as long as it functions to intracellularly generate siRNA that exerts RNAi activity against the filamin A gene. Therefore, various viral vectors (adenoviral vectors, adeno-associated viral vectors, retroviral vectors, lentiviral vectors, herpes viral vectors, Sendai viral vectors, etc.) and non-viral vectors (liposomes, positively charged liposomes, etc.) can be used. Examples of promoters that can be used in vectors include the U6 promoter, H1 promoter, and tRNA promoter. These promoters are RNA polymerase III-based promoters, and are expected to have high expression efficiency.

[0024] It has been reported that single-stranded RNAs with a specific structure are useful for suppressing the expression of target genes (e.g., WO 2012 / 005368, JP 2013-55913 A, JP 2013-138681 A, JP 2013-153736 A). Therefore, in one embodiment of the present invention, single-stranded RNA (above (c)) is used to suppress the expression of the filamin A gene through a mechanism similar to that of expression suppression by siRNA (i.e., RNA interference). The single-stranded RNA of the present invention has an expression-suppressing sequence corresponding to the filamin A gene and a complementary sequence capable of annealing to said sequence. The order in which the expression-suppressing sequence and the complementary sequence are linked is not particularly limited. Furthermore, the expression-suppressing sequence and the complementary sequence may be linked directly or via a linker region. The linker region may be composed of nucleotide residues or non-nucleotide residues (e.g., consisting of a structure such as polyalkylene glycol, a pyrrolidine skeleton, or a piperidine skeleton). Examples of the form of the single-stranded RNA of the present invention include a molecule in which the 5'-region and the 3'-region are intramolecularly annealed to form one double-stranded structure (stem structure) (Example 1), and a molecule in which the 5'-region and the 3'-region are each intramolecularly annealed separately to form two double-stranded structures (stem structures) (Example 2).

[0025] The expression-inhibitory sequence is a sequence that exhibits the activity of suppressing the expression of the filamin A gene when the single-stranded RNA of the present invention is introduced into a cell. Typically, a sequence that causes expression inhibition by siRNA (i.e., RNA interference) is used as the expression-inhibitory sequence. For example, the sequence of the RNA (antisense RNA) that constitutes the above-mentioned siRNA (above (a)) can be used as the expression-inhibitory sequence. The length of the expression-inhibitory sequence is not particularly limited, but is, for example, 18 to 32 bases long, preferably 19 to 30 bases long, and more preferably 19 to 21 bases long.

[0026] The length of the single-stranded RNA of the present invention is not particularly limited. The total number of bases constituting the single-stranded RNA (total length) has a lower limit of, for example, 38 bases, preferably 42 bases, more preferably 50 bases, even more preferably 51 bases, and particularly preferably 52 bases, and an upper limit of, for example, 300 bases, preferably 200 bases, more preferably 150 bases, even more preferably 100 bases, and particularly preferably 80 bases. When the single-stranded RNA of the present invention contains a linker region, the total number of bases excluding the linker region has a lower limit of, for example, 38 bases, preferably 42 bases, more preferably 50 bases, even more preferably 51 bases, and particularly preferably 52 bases, and an upper limit of, for example, 300 bases, preferably 200 bases, more preferably 150 bases, even more preferably 100 bases, and particularly preferably 80 bases.

[0027] When designing or preparing the single-stranded RNA of the present invention, past reports such as the above-mentioned patent publications can be used as reference.

[0028] The compound (d) above is a compound used for expression inhibition by the antisense method. In other words, the pharmaceutical composition of the present invention containing the compound (d) above can inhibit the expression of the filamin A gene by the antisense method. When inhibiting expression by the antisense method, for example, an antisense construct is used that, when transcribed in target cells, generates RNA complementary to a specific portion of the mRNA encoding the filamin A gene. Such an antisense construct is introduced into target cells, for example, in the form of an expression plasmid. Alternatively, an oligonucleotide probe can be used as the antisense construct, which, when introduced into target cells, hybridizes with the mRNA and / or genomic DNA sequence encoding the filamin A gene and inhibits its expression. Such an oligonucleotide probe is preferably resistant to endogenous nucleases such as exonucleases and / or endonucleases.

[0029] The sequence of the antisense nucleic acid is not particularly limited as long as it exhibits the activity of suppressing the expression of the filamin A gene. The antisense nucleic acid may bind to the mRNA encoding the filamin A gene or its precursor (pre-mRNA) to induce degradation by an RNase such as RNase H, or may bind to a splicing regulatory site of the pre-mRNA (e.g., an exon / intron boundary region or a purine-rich region within an exon) to induce exon skipping or exon inclusion. The length of the sequence of the antisense nucleic acid is, for example, 12 or more bases, 13 or more bases, 14 or more bases, or 15 or more bases, and, for example, 50 or less bases, 45 or less bases, 40 or less bases, or 35 or less bases.

[0030] An example of an antisense nucleic acid is a gapmer. A gapmer typically has a structure in which a central region (gap) is located between two terminal regions (wings). The wings are composed of chemically modified nucleotides (e.g., BNA, LNA, ENA, ribonucleotides in which the hydroxyl group at position 2 is substituted with -OCH3, or the like, in which the sugar moiety is chemically modified), and the gap is composed of unmodified nucleotides, in which the phosphate residue of each nucleotide is phosphorothioated. The gap can function as a substrate for RNase. The length of the wing sequence is not particularly limited, but may be, for example, 2 or more bases, preferably 3 or more bases, and for example, 5 or less bases. The length of the gap sequence is, for example, 5 or more bases, preferably 6 or more bases, and for example, 10 or less bases. It should be noted that antisense nucleic acids are not limited to gapmers and may also be headmers, tailmers, mixmers, blockmers, totalmers, etc. (see, for example, U.S. Patent Application Publication No. 2012 / 322851, etc.).

[0031] When a DNA molecule is used as an antisense nucleic acid, an oligodeoxyribonucleotide derived from a region including the translation initiation site (for example, the region from -10 to +10) of mRNA encoding the filamin A gene is preferred.

[0032] The complementarity between antisense nucleic acid and target nucleic acid is preferably strict, but some mismatch may exist.The hybridization ability of antisense nucleic acid to target nucleic acid generally depends on both the degree of complementarity and the length of both nucleic acids.Usually, the longer the antisense nucleic acid used, the more mismatches it can have, but still be able to form a stable duplex (or triplex) with target nucleic acid.Those skilled in the art can use standard techniques to confirm the acceptable degree of mismatch.

[0033] Antisense nucleic acids may be DNA, RNA, chimeric mixtures thereof, or derivatives or modified forms thereof. They may be single-stranded or double-stranded. The stability, hybridization ability, etc. of antisense nucleic acids can be improved by modifying the base moiety, sugar moiety, or phosphate backbone moiety. Furthermore, substances that promote cell membrane transport (see, e.g., Letsinger et al., 1989, Proc. Natl. Acad. Sci. USA 86:6553-6556; Lemaitre et al., 1987, Proc. Natl. Acad. Sci. 84:648-652; PCT Publication No. W088 / 09810, published December 15, 1988) or substances (e.g., ligands) that increase affinity for specific cells may be added to the antisense nucleic acids.

[0034] Antisense nucleic acids can be synthesized by standard methods, for example, using a commercially available automated DNA synthesizer (e.g., Applied Biosystems, Inc.). For the preparation of modified nucleic acids and derivatives, see, for example, Stein et al. (1988), Nucl. Acids Res. 16:3209 and Sarin et al. (1988), Proc. Natl. Acad. Sci. USA 85:7448-7451.

[0035] To enhance the effect of antisense nucleic acids in target cells, strong promoters such as pol II and pol III can be used. That is, by introducing a construct containing an antisense nucleic acid placed under the control of such a promoter into target cells, the promoter can ensure sufficient transcription of the antisense nucleic acid.

[0036] The expression of the antisense nucleic acid can be controlled by any promoter (inducible or constitutive) known to function in mammalian cells (preferably human cells), such as the SV40 early promoter region (Bernoist and Chambon, 1981, Nature 290:304-310), the promoter derived from the 3'-terminal region of Rous sarcoma virus (Yamamoto et al., 1980, Cell 22:787-797), or the herpes thymidine kinase promoter (Wagner et al., 1981, Proc. Natl. Acad. Sci. USA 78:1441-1445).

[0037] In one embodiment of the present invention, expression suppression by ribozymes is utilized (in the case of the compound (e) above). While target mRNAs can be destroyed using ribozymes that cleave mRNAs at site-specific recognition sequences, hammerhead ribozymes are preferred. For details on constructing hammerhead ribozymes, see, for example, Haseloff and Gerlach, 1988, Nature, 334:585-591.

[0038] As in the case of using antisense methods, ribozymes may be constructed using modified oligonucleotides, for example, for the purpose of improving stability or targeting ability. To produce an effective amount of ribozyme in target cells, it is preferable to use a nucleic acid construct in which DNA encoding the ribozyme is placed under the control of a strong promoter (e.g., pol II or pol III).

[0039] Without being bound by any particular theory, based on the results of the test examples described below, it is believed that compounds that suppress the expression of the filamin A gene can treat PSP through the following mechanism: filamin A is an actin-binding protein that crosslinks F-actin and is a molecule responsible for the cytoskeleton. Because tau protein has multiple F-actin-binding motifs within its microtubule-binding domain (References 34, 35), it is believed that when filamin A is abundant or its function is enhanced, tau protein is abnormally stabilized via F-actin. Therefore, when filamin A is abundant or its function is enhanced, i.e., in the pathological condition of PSP, it is believed that suppressing the expression level or function of filamin A can normalize tau protein and thereby treat PSP.

[0040] The medicament of the present invention may contain a compound that suppresses the expression of the filamin A gene as a single active ingredient. Furthermore, the medicament of the present invention may contain only one type of compound that suppresses the expression of the filamin A gene, or may contain two or more types. The medicament of the present invention may contain other active ingredients and may be administered in combination with other medicaments (simultaneously, sequentially, or alternately). Examples of other active ingredients or medicaments include Parkinson's disease treatment drugs such as levodopa and amantadine; and depression treatment drugs such as amitriptyline and tandospirone. The other active ingredients or medicaments may be used alone or in combination of two or more types.

[0041] The pharmaceutical preparation of the present invention can be prepared by a conventional method. When prepared as a pharmaceutical preparation, other pharmaceutically acceptable ingredients (e.g., carriers, excipients, disintegrants, buffers, emulsifiers, suspending agents, soothing agents, stabilizers, preservatives, antiseptics, physiological saline, etc.) may be contained.

[0042] Carriers include, but are not limited to, cationic liposomes such as Lipofectin™, Lipofectamine 2000™, and Oligofectamine™; and cationic polymers such as poly(L-lysine), DEAE-dextran, polyethyleneimine, and chitosan.

[0043] Examples of excipients that can be used include lactose, starch, sorbitol, D-mannitol, and sucrose. Examples of disintegrants that can be used include starch, carboxymethylcellulose, and calcium carbonate. Examples of buffers that can be used include phosphates, citrates, and acetates. Examples of emulsifiers that can be used include gum arabic, sodium alginate, and tragacanth. Examples of suspending agents that can be used include glycerin monostearate, aluminum monostearate, methylcellulose, carboxymethylcellulose, hydroxymethylcellulose, and sodium lauryl sulfate. Examples of soothing agents that can be used include benzyl alcohol, chlorobutanol, and sorbitol. Examples of stabilizers that can be used include propylene glycol, diethylin sulfite, and ascorbic acid. Examples of preservatives that can be used include phenol, benzalkonium chloride, benzyl alcohol, chlorobutanol, and methylparaben. Examples of antiseptics that can be used include benzalkonium chloride, parahydroxybenzoic acid, and chlorobutanol.

[0044] The dosage form of the formulation is not particularly limited. Examples of dosage forms include tablets, powders, fine granules, granules, capsules, syrups, injections, and inhalants. The medicament of the present invention contains a compound (or active ingredient) that suppresses filamin A gene expression in an amount (i.e., a therapeutically effective amount) necessary to achieve the expected therapeutic (or preventive) effect. The content of the compound (or the amount of the active ingredient) in the medicament of the present invention generally varies depending on the dosage form, but is set, for example, within the range of about 0.01% to about 95% by weight so as to achieve the desired dosage. The medicament of the present invention is administered to a subject by oral or parenteral administration (intravenous, intraarterial, subcutaneous, intradermal, intramuscular, or intraperitoneal injection, transdermal, nasal, transmucosal, intracerebral, or intrathecal injection, etc.) depending on the dosage form. Examples of intracerebral administration include administration via a catheter, implantation of a sustained-release formulation into the brain, and introduction into brain cells by electroporation. These administration routes are not mutually exclusive, and any two or more of them can be used in combination (for example, by intravenous injection simultaneously with oral administration or after a predetermined time has elapsed). When the compound that suppresses the expression of the filamin A gene is a nucleic acid construct (for example, in an embodiment that utilizes RNAi), it is not limited to in vivo administration, but ex vivo administration can also be adopted.

[0045] The "subject" to which the pharmaceutical agent of the present invention is administered is typically a human, but application to non-human mammals (including pet animals, livestock, and laboratory animals, specifically, for example, mice, rats, guinea pigs, hamsters, monkeys, cows, pigs, goats, sheep, dogs, and cats) is also contemplated. The subject may, for example, be a subject in which the expression of filamin A and / or 4-repeat tau is elevated in neurons and / or glial cells, or a subject in which 4-repeat tau is overexpressed relative to 3-repeat tau in neurons and / or glial cells. The dosage varies depending on the symptoms, age, sex, and weight of the subject (e.g., patient), but a person skilled in the art can appropriately determine an appropriate dosage. The dosage schedule can be determined taking into account the symptoms of the subject (e.g., patient) and the duration of the pharmaceutical agent's effect.

[0046] As is clear from the above description, the present application also provides a method for treating PSP, comprising administering a therapeutically effective amount of the pharmaceutical agent of the present invention to a patient with PSP. Here, "treatment," like "therapeutic agent," is used to encompass both therapeutic and prophylactic methods, and also encompasses methods for preventing or delaying the worsening of symptoms. The present application also provides an inhibitor of 4-repeat tau expression containing a compound that inhibits the expression of the filamin A gene; an inhibitor of phosphorylated 4-repeat tau containing a compound that inhibits the expression of the filamin A gene; and an inhibitor of 4-repeat tau aggregation containing a compound that inhibits the expression of the filamin A gene. The phosphorylated 4-repeat tau inhibitor includes an agent that reduces the amount or concentration of phosphorylated 4-repeat tau in cells expressing filamin A. The ingredients, their contents, administration modes, etc. of these agents are the same as those described for "therapeutic agents."

[0047] 2. Search for compounds effective in the treatment of progressive supranuclear palsy (PSP) (evaluation and screening) Another aspect of the present invention relates to the discovery (evaluation, screening) of compounds effective in treating PSP. Studies by the present inventors have identified filamin A as the causative gene for PSP and found that increased filamin A expression leads to increased 4-repeat tau and increased phosphorylated tau. In this aspect, "increased filamin A expression" includes increased mRNA encoding filamin A, and "phosphorylated tau" includes phosphorylated 4-repeat tau. Based on this finding, we provide a method for evaluating the efficacy of a test substance against PSP using increased filamin A expression, increased 4-repeat tau associated with increased filamin A expression, or increased phosphorylated tau associated with increased filamin A expression as an index (hereinafter referred to as the "evaluation method of the present invention"). The evaluation method of the present invention is useful for the discovery (i.e., screening) of candidate therapeutic agents for PSP.

[0048] The evaluation method of the present invention includes the following steps (i) and (ii). (i) contacting a test substance with cells expressing filamin A (ii) detecting the expression of filamin A, the amount of 4-repeat tau, and / or the amount of phosphorylated tau in the cells, and determining the effectiveness of the test substance based on the detection results, wherein a decrease in the expression of filamin A (index 1), a decrease in the amount of 4-repeat tau (index 2), and / or a decrease in the amount of phosphorylated tau (index 3) is an indicator that the test substance is effective.

[0049] In step (i), cells expressing filamin A are prepared. The type of cells expressing filamin A is not particularly limited, and may be in vivo cells, cells collected from a living body, or cultured cells. Cells expressing filamin A are preferably cells highly expressing filamin A. In one embodiment of the present invention, the filamin A-expressing cells are cells derived from a PSP patient, preferably a cell line, and more preferably a lymphocyte cell line. The cells are preferably immortalized cells; for example, lymphocytes from a PSP patient can be collected and immortalized to form a lymphocyte cell line, which can be used in the evaluation method of the present invention. In a preferred embodiment, the immortalization method involves collecting peripheral blood from a PSP patient and infecting B lymphocytes with Epstein-Barr virus. Alternatively, the evaluation method of the present invention can use induced pluripotent stem (iPS) cells derived from PSP patients (iPS cells prepared using cells collected from the patient), or iPS cells differentiated into neurons, glial cells, or brain organoids (PSP patient-derived neurons, glial cells, or brain organoids). Alternatively, the cells used in the evaluation method of the present invention can also be prepared by genetic modification using, for example, gene targeting or genome editing techniques (ZFN, TALEN, CRISPER / Cas9, etc.). Cells that can be subjected to genetic modification include, for example, fibroblasts, cardiac muscle cells, smooth muscle cells, adipocytes, bone cells, chondrocytes, osteoclasts, parenchymal cells, epidermal keratinocytes, epithelial cells (skin epidermal cells, corneal epithelial cells, conjunctival epithelial cells, oral mucosal epithelial cells, hair follicle epithelial cells, oral mucosal epithelial cells, respiratory tract mucosal epithelial cells, intestinal mucosal epithelial cells, etc.), endothelial cells (corneal endothelial cells, vascular endothelial cells, etc.), nerve cells, glial cells, splenocytes, pancreatic beta cells, mesangial cells, Langerhans cells, hepatocytes, bone marrow cells, blood cells (leukocytes), their precursor cells or stem cells, and established cell lines (e.g., HeLa cells, CHO cells, Vero cells, HEK293 cells, HepG2 cells, COS-7 cells, NIH3T3 cells, Sf9 cells). Preferably, human cells are used, but this does not preclude the use of cells from other animal species (monkey, cow, horse, rabbit, mouse, rat, guinea pig, hamster, etc.).Immortalized lymphocytes derived from PSP patients, which exhibit elevated filamin A expression, are not only useful for the evaluation method of the present invention, but also can be used for PSP research and the development of therapeutic drugs, and are therefore of great value. In one embodiment of the present invention, the filamin A-expressing cells are cells, preferably cell lines, derived from transgenic animals (e.g., transgenic mice) into which a filamin A gene, such as the human filamin A gene, has been introduced. The cells are preferably immortalized primary neural stem cells or primary glial cells isolated from fetal brain tissue of the transgenic animals. Examples of immortalization methods include introducing the SV40 T antigen gene, HPV E6E7 gene, v-abl gene, myc gene, human telomere reverse transcriptase (hTERT) gene, or a combination thereof into the cells. The virus used as a vector for introducing the gene is not particularly limited, but is preferably a lentivirus, adenovirus, or retrovirus. Furthermore, the cells derived from the transgenic animal are preferably fetal fibroblasts (MEFs) obtained from the fetal tissue mass of the transgenic animal. These cells have high proliferation potential and can be used as stable cell lines.

[0050] When the cells are in vivo cells, the "contact" in step (i) is typically carried out by administering the test substance to the organism. Alternatively, when the cells are cells collected from an organism or cultured cells, the "contact" is typically carried out by adding the test substance to the culture solution (medium) during culture. The timing of adding the test substance is not particularly limited. Therefore, the test substance may be added at a certain point after starting cell culture in a medium not containing the test substance, or the cell culture may be started in a medium containing the test substance in advance. Standard culture conditions for the cells used may be adopted.

[0051] Organic or inorganic compounds of various molecular sizes can be used as test substances. Examples of organic compounds include nucleic acids, peptides, proteins, lipids (simple lipids, complex lipids (phosphoglycerides, sphingolipids, glycosylglycerides, cerebrosides, etc.)), prostaglandins, isoprenoids, terpenes, steroids, polyphenols, catechins, and vitamins (B1, B2, B3, B5, B6, B7, B9, B12, C, A, D, E, etc.). Existing or candidate components of pharmaceuticals and nutritional foods are also preferred test substances. Plant extracts, cell extracts, culture supernatants, etc. can also be used as test substances. Existing drugs (e.g., libraries of drugs approved by the U.S. Food and Drug Administration (FDA)) can also be used as test substances. Various compound libraries (e.g., Ligand A compound library (such as a compound library box) is available (for example, from Asinex or Namiki Shoji Co., Ltd.), and such a compound library may be used. The test substance may be derived from a natural product or may be synthetic. In the latter case, an efficient screening system can be constructed using, for example, combinatorial synthesis techniques. Furthermore, two or more test substances may be added simultaneously to examine interactions, synergistic effects, etc. between the test substances.

[0052] The time for contacting the test substance can be set arbitrarily. For example, when the cells are cells collected from a living organism or cultured cells, the contact period is, for example, 10 minutes to 1 week, preferably 1 hour to 3 days. The contact may be performed multiple times.

[0053] In step (ii) following step (i), the expression of filamin A (when filamin A expression is used as an indicator), the amount of 4-repeat tau (when the amount of 4-repeat tau is used as an indicator), and / or the amount of phosphorylated tau (when the amount of phosphorylated tau is used as an indicator) in the cells contacted with the test substance are detected, and the effectiveness of the test substance is determined based on the detection results. That is, in the present invention, the following three types of indicators are used. Index 1: Decreased expression of filamin A Index 2: Reduction of 4-repeat tau amount Indicator 3: Reduction of phosphorylated tau levels These indices 1 to 3 are not mutually exclusive, and two or more can be used in combination. By using two or more indices in combination, more useful determination results can be obtained. Therefore, it is preferable to use two or more indices in combination, and more preferably to use all of indices 1 to 3 in combination.

[0054] When Index 1 is used, a test substance is determined to be effective if the expression of filamin A is reduced. When Index 2 is used, a test substance is determined to be effective if the amount of 4-repeat tau is reduced. Similarly, when Index 3 is used, a test substance is determined to be effective if the amount of phosphorylated tau is reduced. The strength (level) of the action / effect of the test substance may be determined based on the degree (level) of reduction in filamin A expression (in the case of Index 1), the degree (level) of reduction in 4-repeat tau (in the case of Index 2), or the degree (level) of reduction in phosphorylated tau (in the case of Index 3). When multiple test substances are used, the strength (level) of the action / effect of each test substance may be compared and evaluated based on the degree (level) of reduction in filamin A expression (in the case of Index 1), the degree (level) of reduction in 4-repeat tau (in the case of Index 2), or the degree (level) of reduction in phosphorylated tau (in the case of Index 3).

[0055] Filamin A expression can be detected using real-time quantitative PCR, microarrays, RNA-Seq, immunological techniques such as Western blot and ELISA, and proteomic analysis using mass spectrometry. Furthermore, 4-repeat tau can be detected using immunological techniques such as Western blot and ELISA, and proteomic analysis using mass spectrometry. Similarly, phosphorylated tau can be detected using immunological techniques such as Western blot and ELISA, and proteomic analysis using mass spectrometry.

[0056] Typically, cells not contacted with the test substance (other conditions being the same) (hereinafter referred to as "control cells") are prepared as a comparison control, and the expression of filamin A (when indicator 1 is used), the amount of 4-repeat tau (when indicator 2 is used), and / or the amount of phosphorylated tau (when indicator 3 is used) in the control cells are also detected. The effectiveness of the test substance is then determined by comparing the detection results with those of the control cells (preferably, a quantitative determination is made rather than a qualitative determination). In this way, determining the action or effect of the test substance by comparison with the control can provide more reliable determination results.

[0057] As mentioned above, the evaluation method of the present invention is useful for searching for candidate therapeutic agents for PSP, i.e., for screening. In other words, the present invention enables the identification of candidate or lead compounds as active ingredients of therapeutic agents. When the evaluation method of the present invention is used for screening, an effective test substance is selected based on the determination results in step (ii). If the selected substance has sufficient efficacy, the substance can be used as is as an active ingredient in a therapeutic agent for PSP. On the other hand, if the substance does not have sufficient efficacy, it can be modified, such as by chemical modification, to enhance its efficacy and then used as an active ingredient in a therapeutic agent for PSP. Of course, even if the substance has sufficient efficacy, similar modifications can be made to further enhance its efficacy.

[0058] 3. Progressive supranuclear palsy (PSP) disease model A further aspect of the present invention relates to a non-human mammal that reproduces the pathology of PSP. The non-human mammal of the present invention is useful as a disease model (model animal) for PSP. A typical example of the non-human mammal of the present invention is a transgenic animal (hereinafter also referred to as a "TG animal"), but is not limited to this. For example, various genetically modified animals produced using genome editing techniques (e.g., gene knock-in) or viral vectors (e.g., gene expression induction using an adeno-associated viral vector) can also be considered as non-human mammals serving as disease models for PSP. Note that a "transgenic non-human mammal (TG animal)" refers to a non-human mammal into which exogenous DNA has been introduced early in development, so that all of its constituent cells carry the exogenous DNA, or its progeny (which carry the exogenous gene).

[0059] The model animal of the present invention is not particularly limited to a mammalian species (genus), and includes mice, rats, guinea pigs, hamsters, rabbits, dogs, cats, monkeys, etc. Rodents such as mice and rats are preferred, and mice are most preferred.

[0060] Typically, the exogenous DNA of the present invention contains the human filamin A gene (e.g., having the sequence of SEQ ID NO: 2 or SEQ ID NO: 4) as a transgene. A homolog, ortholog, or mutant of the human filamin A gene may be used as the transgene, as long as its forced expression leads to an increase in the amount of 4-repeat tau or phosphorylated tau via increased filamin A expression. The term "mutant" as used herein refers to a gene that has a sequence identical or homologous to a portion of the human filamin A gene, but whose entire sequence differs from that of the human filamin A gene. Examples of human filamin A gene mutants include DNA sequences that contain one or more base substitutions, deletions, insertions, and / or additions relative to the DNA sequence of the human filamin A gene. Mutants may be naturally occurring or artificially constructed using genetic engineering techniques. The copy number of the transgene is not particularly limited, but may be, for example, 1 to 100.

[0061] It is preferable that the exogenous DNA contains an enhancer that activates the transcription of the introduced gene. An "enhancer" refers to a sequence that acts directly or indirectly on a promoter to enhance its transcriptional activity. An enhancer generally acts on a promoter from a distant position. The position of the enhancer in the exogenous DNA may be upstream or downstream of the promoter. There are no particular limitations on the enhancer, as long as it can act on the promoter used in the exogenous DNA to enhance its transcriptional activity.

[0062] The non-human mammal of the present invention (typically a TG animal) carries the above-mentioned exogenous gene in a heterozygous or homozygous form. In other words, the genotype for the above-mentioned exogenous gene is heterozygous or homozygous.

[0063] Methods for producing TG animals, which are typical examples of non-human mammals of the present invention, include the microinjection method in which DNA is directly injected into the pronucleus of a fertilized egg, a method using a retroviral vector, a method using ES cells, etc. Below, the microinjection method using mice will be described as a specific example of a method for producing TG animals of the present invention.

[0064] In the microinjection method, fertilized eggs are first collected from the oviduct of a female mouse after mating has been confirmed, and after culturing, the desired DNA construct (exogenous DNA) is injected into the pronucleus. The form of the DNA construct is not particularly limited, but linear or circular forms are preferred from the viewpoint of transfer efficiency. It is particularly preferred to use a linear DNA construct. The DNA construct is prepared so that the gene to be transferred is efficiently integrated into the chromosome and its good expression is ensured. The DNA construct contains a transgene (typically the human filamin A gene) and a promoter (and, if necessary, an appropriate enhancer sequence, selection marker, replication origin, terminator sequence, etc.).

[0065] After the injection procedure, the fertilized eggs are transplanted into the oviducts of pseudopregnant mice, and the transplanted mice are raised for a predetermined period to obtain offspring (F0). To confirm that the transgene has been properly integrated into the chromosomes of the offspring, DNA is extracted from the tails of the offspring and subjected to Southern hybridization analysis, slot blot (dot blot) analysis, PCR analysis, etc.

[0066] The identified transgenic individuals are then mated with wild-type mice to obtain heterozygous transgenic mice (carrying the exogenous DNA in a heterozygous form). Homozygous transgenic mice (carrying the exogenous DNA in a homozygous form) can be obtained by mating male and female heterozygous transgenic mice obtained in this manner. For breeding or maintenance, male and female homozygous transgenic mice can be mated.

[0067] The non-human mammals of the present invention (typically TG animals) reproduce the pathology of PSP. Typically, they exhibit a phenotype of increased 4-repeat tau and / or increased phosphorylated tau in neurons and glial cells. This characteristic makes the non-human mammals of the present invention useful for discovering and verifying the effectiveness of therapeutic agents for PSP. For example, substances that improve (including cure) the characteristic phenotype (pathology) exhibited by the non-human mammals of the present invention can be identified as candidate therapeutic agents for PSP. Furthermore, in the non-human mammals of the present invention, increased amounts of 4-repeat tau and / or increased amounts of phosphorylated tau form the basis of the phenotype (pathology). Therefore, it is possible to detect the amount of 4-repeat tau or phosphorylated tau (e.g., by fluorescent bioimaging, Western blotting, immunostaining, etc.) and use the changes in the amount as an index to assess the efficacy of a test compound.

[0068] 4. Biomarkers for Progressive Supranuclear Palsy (PSP) The biomarker for progressive supranuclear palsy (PSP) of the present invention includes filamin A. For example, when the expression level of filamin A in neurons and / or glial cells derived from a subject is higher than the expression level of filamin A in neurons and / or glial cells derived from a healthy subject, for example, when the expression level is two or more times or three or more times higher, the subject can be determined to have PSP. [Example]

[0069] The following studies were conducted to create a new treatment strategy for progressive supranuclear palsy (PSP). 1. Method (1) Analysis target We analyzed 32 cases from a Japanese family including identical twins (Twin-A and Twin-B) who developed PSP simultaneously, as well as from the Aichi Medical University Institute of Aging Brain Bank. The 32 cases included nine cases of PSP (PSP-1–9), three cases of corticobasal degeneration (CBD) (CBD-1–3), three cases of AD (AD-1–3), four cases of Parkinson's disease (PD) (PD-1–4), three cases of dementia with Lewy bodies (DLB) (DLB-1–3), five cases of amyotrophic lateral sclerosis (ALS) (ALS-1–5), and five healthy controls (Normal-1–5). Pathological diagnoses were based on the diagnostic criteria for each disease (References 7–12). Age-related changes were assessed using Braak NFT staging (grades 0, I-VI) (Reference 13), AT8 staging (grades 0, I-VI) (Reference 14), argyrophilic grain (AG) staging (grades 0, I-III) (Reference 15), and the Consortium to Establish a Registry for Alzheimer's Disease (CERAD) score (grade 0, AC) (Reference 16). Written informed consent was obtained from the subjects or their relatives for analysis. The research plan for this study was reviewed and approved by the ethics committees of Nagoya University, Aichi Medical University, and Yokohama City University.

[0070] (2) Microsatellite markers To confirm the identity of the genomic DNA of Twin-A and Twin-B, 12 microsatellite markers were genotyped using fluorescent primers from the ABI PRISM Linkage Mapping Set version 2.5 (Applied Biosystems) to assess the relatedness of the monozygotic twins.

[0071] (3) Human neuropathological analysis The autopsy brains were fixed in 20% formalin. The brain specimens were embedded in paraffin and sectioned at 4.5 μm thickness. The sections were stained with hematoxylin and eosin (H&E), Kluver-Barrera staining (KB), and Gallyas-Braak staining. The primary antibodies used were 3R-tau antibody (RD3), 4R-tau antibody (RD4), and phosphorylated tau antibody (AT8). The ENVISION kit (DAB) (Wako) was used for staining.

[0072] (4) Immortalized lymphocytes Peripheral blood samples were collected from Twin-A, Twin-B, and three siblings (II-1, II-2, and II-3), and B lymphocytes were immortalized by Epstein-Barr virus infection. These immortalized lymphocytes were cultured in RPMI 1640 medium (Gibco) supplemented with 10% fetal bovine serum (FBS) at 37°C and 5% CO2.

[0073] (5) Sanger sequencing of the MAPT gene Sanger sequencing was performed on exon 10 of the MAPT gene and its neighboring introns using a 3730xl DNA analyzer (Applied Biosystems). Polymerase chain reaction (PCR) was performed using a Multiplex PCR Assay Kit (Takara). The primers used were as follows: forward 5'-GGATGTGACTCAACCTCCCG-3' (SEQ ID NO: 5), reverse 5'-CGGGCTACATTCACCCAGAG-3' (SEQ ID NO: 6).

[0074] (6) Whole exome analysis Genomic DNA was extracted from Twin-A's peripheral blood and whole-exome analysis was performed using the SureSelect Human All Exon V6 kit (Agilent Technologies). Captured libraries were sequenced using a HiSeq2500 (Illumina). Reads were aligned to the human reference sequence GRCh37 using Novoalign, and duplicate reads were removed using Picard. Variants were called using the Genome Analysis Toolkit (GATK) and annotated using ANNOVAR. The average coverage depth was 69.7×, with 95.1% of the coding region covered by ≥20 reads.

[0075] (7) Copy number analysis Copy number was calculated from whole-exome analysis data using eXome Hidden Markov Model v1.0 (XHMM) (References 17, 18). First, BAM files for Twin-A and 513 healthy Japanese males were generated. Then, using GATK DepthOfCoverage, the average depth of each target region was calculated from the BAM files for each sample and integrated into a samples-by-target matrix. Targets with outliers in size, depth, and GC content, as well as samples with outliers in the mean and standard deviation of depth, were excluded. The depth of the integrated matrix was centered by the mean for each target and used for principal component analysis. Z-scores were calculated for each target for each sample, and copy number was calculated from the Z-scores using the XHMM algorithm. Z-scores were visualized using SignalMap Version 1.9.0.05 (Roche Nimblegen). Copy number aberrations detected by XHMM were reassessed using microarray and real-time quantitative PCR, as described below.

[0076] (8) Chromosomal microarray As previously described, high-resolution chromosomal microarray analysis was performed using a CytoScan HD Array (Affymetrix) with genomic DNA extracted from Twin-A, Twin-B, and three siblings (II-1, II-2, and II-3). Data were analyzed using Chromosome Analysis Suite software v1.2.0.225 (Affymetrix). 250 ng of genomic DNA purified by ethanol precipitation was digested with the restriction enzyme Nsp1 and ligated to the adaptor using T4 DNA ligase. PCR amplification was then performed using primers targeting the adaptor sequence and Titanium Taq DNA Polymerase (Affymetrix). The PCR products were purified using magnetic beads, fragmented with DNase I, and biotin-labeled with terminal deoxynucleotidyl transferase. The labeled DNA was hybridized to a CytoScan HD Chip using a Gene Chip Hybridization Oven 640 (Affymetrix). After washing, the chip was scanned using a GeneChip Fluidics Station 450 (Affymetrix).

[0077] (9) RNA reverse transcription RNA was extracted from cells using the miRNeasy Mini Kit (Qiagen) and purified using ImProm-II. TM Complementary DNA (cDNA) was prepared from 1.0 μg of RNA using the Reverse Transcription System (Promega).

[0078] (10) Real-time quantitative PCR Thunderbird SYBR qPCR Mix (TOYOBO) and the CFX96 system (BioRad) were used. For genomic DNA primers, MECP2, TKTL1, G6PD, the intergenic region between CTAG1B and CTAG2, and GAB3, the sequences were the same as previously reported (Reference 19). FLNA was newly designed with the following sequences: forward 5'-AAGGGGGAGTACACACTGGT-3' (SEQ ID NO: 7), reverse 5'-CACCACAACGCGGTAGGG-3' (SEQ ID NO: 8). For cDNA primers, FLNA, RPL10, ATP6AP1, GDI1, and GUSB, the sequences were the same as previously reported (Reference 19). PCR conditions were 95°C for 3 minutes, followed by 40 cycles of 95°C for 10 seconds and 55°C for 30 seconds. Relative gene expression levels were calculated using the 2-ΔΔCt method, with a housekeeping gene used as a reference and a control sample used as a calibrator for each experiment.

[0079] (11)X chromosome inactivation analysis Genomic DNA was digested with the methylation-sensitive restriction enzymes HpaII and HhaI (Takara), and the CAG repeat region of the FRAXA gene was amplified using primers with a fluorescent probe (Reference 20). PCR products were analyzed using an ABI PRISM 3500 Genetic Analyzer (Applied Biosystems). X-chromosome inactivation ratios of less than 80:20 were considered random patterns, 80:20 or greater were considered skewed patterns, and 90:10 or greater were considered markedly skewed patterns (Reference 20). Data were processed using Peak Scanner software 2 (Applied Biosystems).

[0080] (12) DNA plasmid mCherry-FilaminA-N-9 (Addgene, plasmid 55047) contains a mutation at base 7876 of the FLNA cDNA sequence. This was replaced with the wild-type FLNA sequence using the KOD Plus Mutagenesis Kit (TOYOBO) and used as the mCherry-FLNA vector. To eliminate actin-binding ability, a mutation was introduced at base 116 of the FLNA cDNA sequence based on a previously reported mCherry-FLNA vector. Ala39Gly We constructed a vector (Reference 32). Furthermore, the entire FLNA cDNA sequence of the mCherry-FLNA vector was deleted, resulting in the mCherry-empty vector, which served as a negative control. Subcloned cDNA sequences of various genes were inserted into the mCherry-empty vector using In-Fusion HD (Takara) to construct the mCherry-RPL10, mCherry-GDI1, mCherry-FAM3A, and mCherry-G6PD vectors. We also inserted a GFP-tagged human 0N4R tau sequence into the pDEST12.2 or pLenti CMV Neo vectors, and overexpressed GFP-tagged 4R-tau (GFP-4R-tau) in cultured cells (Reference 5).

[0081] (13) Transfection HEK293 cells were cultured in 10% FBS-supplemented DMEM medium (Nakarai Tesque) at 37°C and 5% CO2. Lipofectamine 2000 (Invitrogen) was used to transfect DNA plasmids into HEK293 cells, and Lipofectamine 3000 (Invitrogen) was used to transfect primary astrocytes. Cells were harvested 48 hours after transfection and used for the respective analyses.

[0082] (14) Small interfering RNA Small interfering RNAs (siRNAs) were purchased from Invitrogen. Their ID numbers are as follows: FLNA siRNA #1 (s5275, sense RNA sequence: SEQ ID NO: 9, antisense RNA sequence: SEQ ID NO: 10), FLNA siRNA #2 (s5276, sense RNA sequence: SEQ ID NO: 11, antisense RNA sequence: SEQ ID NO: 12), FLNA siRNA #3 (s5276, sense RNA sequence: SEQ ID NO: 13, antisense RNA sequence: SEQ ID NO: 14), and control siRNA (Silencer Negative Control siRNA No. 1, 4390843). Each siRNA was transfected into immortalized lymphocytes by electroporation (Neon, Invitrogen). The transfection conditions were one pulse, 30 ms, and 1350 V. Cells were harvested 24 hours later and used for the respective analyses.

[0083] (15) Cycloheximide chase experiment To verify the stability of tau protein in cultured cells, we performed a cycloheximide chase experiment (Reference 21). HEK293 cells were expressing GFP-4R-tau and mCherry-FLNA, and cycloheximide (100 μg / ml) was added to inhibit de novo protein synthesis. Cells were harvested at each observation time point and used for Western blotting. GFP-4R-tau expression levels were normalized to GAPDH expression levels at the start of the addition.

[0084] (16) Sarkosyl-insoluble tau Sarkosyl-insoluble tau was recovered from GFP-4R-tau-expressing HEK293 cells and human autopsy brain (Reference 22). Each sample was dissolved in 10 volumes of TBS buffer [50 mM Tris / HCl (pH 8.0), 274 mM NaCl, 5 mM KCl, protease inhibitor cocktail (Roche, product code 04693159001), and phosphatase inhibitor (Roche, product code 04906837001)] to prepare the homogenate (Ho). The Ho was ultracentrifuged (27,000 × g, 4°C, 20 minutes), and the supernatant was designated the TBS-soluble fraction (S1). The precipitate was dissolved in high salt / sucrose buffer [0.8 M NaCl, 10% sucrose, 10 mM Tris / HCl (pH 7.4), 1 mM EDTA, protease inhibitor cocktail, and phosphatase inhibitor] and ultracentrifuged under the same conditions as above. The supernatant was dissolved in sarkosyl (final concentration 1%), incubated at 37°C for 1 hour, and then ultracentrifuged (150,000 × g, 4°C, 1 hour). The precipitate was dissolved in TE buffer [10 mM Tris / HCl (pH 8.0), 1 mM EDTA] and designated as sarkosyl-insoluble fraction P3.

[0085] (17) Co-immunoprecipitation HEK293 cells were transfected with 8 μg of each of vectors expressing GFP-4R-tau and mCherry-FLNA on a 100 mm plate. After 48 hours, cells were harvested using trypsin-EDTA (Gibco), washed four times with PBS, and lysed in Cell Lysis Buffer M (Wako) [20 mM Tris-HCl (pH 7.4), 200 mM NaCl, 0.05% Nonidet P-40, 2.5 mM MgCl2]. Immunoprecipitation was performed using 5 μg of tau antibody (TAU-5, ab80579, Abcam) and the Dynabeads Protein G Immunoprecipitation Kit (Invitrogen), and the precipitates were used for various Western blotting analyses.

[0086] (18) Lentivirus The packaging vector and lentiviral vector were transfected into HEK293T cells using Lipofectamine 2000 (Invitrogen) to produce lentiviral particles (Reference 5). 48 hours after transfection, the lentivirus-containing supernatant was collected and frozen at -80°C for storage.

[0087] (19) Primary rat astroglia Cerebral cortices were harvested from day 1 Wistar rats and incubated at 37°C for 15 minutes in Hanks' balanced salt solution (HBSS) supplemented with 0.25% trypsin and DNase I (Reference 23). Mixed glia were cultured in a T75 flask in DMEM medium supplemented with 20% FBS, with the medium replaced every 3 days. Once the cells reached confluence, they were shaken at 37°C and 200 rpm in a thermostatic shaker for 24 hours to remove microglia and oligodendrocytes. Animal experiments were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Nagoya University Animal Care and Use Committee.

[0088] (20) Western blotting Western blotting was performed as previously reported (References 24, 25). The primary antibodies were as follows: FLNA antibody (Santa Cruz) Biotechnology (SCB), sc-17749, sc-28284), EMD antibody (SCB, sc-25284), RPL10 antibody (Abcam, ab138978), DNASE1L1 antibody (SCB, sc-134320), TAZ antibody (SCB, sc-293183), ATP6AP1 antibody (Abnova, H00000) 537-M01), GDI1 antibody (GeneTex, GTX54148), FAM50A antibody (SCB, sc-100967), PLXNA3 antibody (SCB, sc-37 4662), LAGE3 antibody (SCB, sc-515776), UBL4A antibody (Proteintech, 14253-1-AP), SLC10A3 antibody (Novus Biologicals, NBP1-79316), FAM3A antibody (R&D, MAB2865-SP), G6PD antibody (SCB, sc-373886), IKBKG antibody (SCB, sc-8032), CTAG1B antibody (SCB, sc-53869), GAPDH antibody (Abcam, ab8 245), mCherry antibody (Abcam, ab167453), GFP antibody (MBL, 598), Tau-5 antibody (Abcam, ab80579), RD4 antibody (Millipore, 05-804), AT8 antibody (Invitrogen, MN1020), PHF-1 antibody (Peter provided by Dr. Davie), HSP90 antibody (Cell Signaling Antibodies used were: Cell Signaling Technology (CST, 4874), HSP70 antibody (CST, 4872), HSP40 antibody (CST, 4871), and Ubiquitin antibody (CST, 3933). Images were taken using the LAS3000 imaging system (Fujifilm). Signals were quantified using IMAGE GAUGE software version 4.22 (Fujifilm) and used to compare protein expression levels.

[0089] (21) Fluorescent immunostaining For astroglial staining, sections were fixed with 4% paraformaldehyde (PFA) for 30 minutes and permeabilized with 1% Triton X-100 (Sigma) for 5 minutes. Then, they were blocked with Tris-NaCl-blocking (TNB) buffer (PerkinElmer). For human autopsy brain tissue staining, formalin-fixed paraffin-embedded sections were deparaffinized, microwaved for 15 minutes in 50 mM citrate buffer (pH 6.0), and blocked with TNB buffer. The primary antibodies used were mCherry (Abcam, ab167453), AT8 (Invitrogen, MN1020), FLNA (SCB, sc-28284), and GFAP (Abcam, ab4674). Secondary antibodies were from the Alexa Fluor series (Invitrogen). The sections were mounted with ProLong gold antifade reagent (Invitrogen, P36930). A confocal laser microscope (LSM710, Carl Zeiss) was used for imaging.

[0090] (22) Statistical analysis R software (ver. 3.5.1) was used for analysis. Student's t-test was used for comparison between two groups. Tukey-Kramer test was used for parametric comparison between multiple groups, and Steel-Dwass test was used for nonparametric comparison between multiple groups. Correlation was evaluated using the Pearson product-moment correlation coefficient non-correlation test. Values ​​are expressed as median ± standard error of the mean (SEM). P values ​​less than 0.05 were considered significant and are indicated in figures as follows: ***P < 0.001, **P < 0.01, *P < 0.05.

[0091] (23) Transgenic mice The transgene (SEQ ID NO: 15) was composed of the full-length human FLNA cDNA sequence and a FLAG tag sequence at its 3' end, and was designed to be expressed under the control of the CAG promoter. This was injected into fertilized eggs of C57BL / 6J strain mice to generate transgenic mice.

[0092] 2.Results The following tests were carried out according to the method described in 1 above. Test Case 1: A pair of identical twins with simultaneous PSP We first performed pathological analysis of a pair of identical twins who developed PSP simultaneously. Sanger sequencing revealed no pathogenic mutations in the MAPT gene, and microsatellite markers confirmed that the twins were identical (Figure 1a). Both twins had been employed after graduating from high school, but at age 45, they exhibited depression and disinhibited behaviors and took leave from work, meeting the clinical diagnosis of frontotemporal dementia, behavioral variant (Reference 26). Higher brain function progressively declined, and in the advanced stages, they exhibited supranuclear vertical oculomotor disorder, trunk-predominant rigidity, and gait instability. Both died of pneumonia at age 67. Their three siblings (II-1, II-2, and II-3) were neurologically normal. Autopsy revealed macroscopic atrophy of the frontal lobe, globus pallidus, and midbrain (Figure 1b–d). Microscopic examination revealed neuronal loss, gliosis, and 4R-tau-positive globose-type NFTs and TAs in the subthalamic nucleus, internal segment of the globus pallidus, midbrain tegmental area, and cerebellar dentate nucleus (Figure 1g-n). Based on the diagnostic criteria, a pathological diagnosis of PSP was made (Reference 7). However, compared with previously reported PSP cases (48 PSP cases with a brain weight of 1.1 ± 0.02 kg (Reference 27)), the brain weights of Twin-A and Twin-B were smaller (970 g and 775 g, respectively). The severity of clinical symptoms and neuropathological findings (Figure 12) was greater in Twin-B than in Twin-A, and heterotopic gray matter was observed in Twin-B in the anterior horn of the lateral ventricle and cerebellum (Figure 5). Additional neuropathological findings and neuroradiological images are shown in Figures 6 and 7.

[0093] Test Case 2: Identification of FLNA gene duplication in identical twins with PSP The "exome-first" approach, which primarily employs whole-exome sequencing (XHMM), has identified not only sequence abnormalities but also copy number abnormalities in diseases of unknown etiology (References 28-31). Based on the hypothesis that the aforementioned genomic abnormalities in monozygotic twins are involved in PSP pathogenesis, we performed whole-exome sequencing of Twin-A using XHMM. Although we did not identify sequence abnormalities, including those in the MAPT gene, we detected a region of copy number abnormalities approximately 0.3 Mb in the Xq28 chromosome region (Figure 2a). Copy number abnormalities in this region were not observed in 513 healthy Japanese males (Figure 8). Next, we performed chromosomal microarray analysis on Twin-A, Twin-B, and their three siblings. As shown in Figure 2b, we identified a copy number gain region between 153.561 Mb and 153.878 Mb on Xq28 in Twin-A, Twin-B, and one unaffected female (II-3). The copy number varied stepwise in the low-copy repeat (LCR) region: two copies from 153.561 Mb to 153.878 Mb, three copies from 153.624 Mb to 153.783 Mb, and two copies from 153.792 Mb to 153.868 Mb. The copy number changes were also confirmed by real-time quantitative PCR (Figure 9). A family with X-linked mental retardation carrying a similar copy number abnormality in Xq28 has been reported, and X chromosome inactivation in asymptomatic female carriers has been reported (Reference 19). X chromosome inactivation analysis showed a markedly skewed pattern in II-3 of the asymptomatic female carrier, indicating that the abnormal X chromosome allele was inactivated by DNA methylation (Figure 2c). mRNA expression analysis of immortalized lymphocytes was performed by real-time quantitative PCR. The mRNA expression levels of FLNA, RPL10, ATP6AP1, and GDI1, which are contained in the copy number abnormality region, were more than twice as high in Twin-A and Twin-B as in control case II-1, but were comparable in II-3 (Figure 2d).

[0094] Test Example 3. Filamin-A promotes phosphorylation of 4R-tau, protein stability, and sarkosyl insolubility Sixteen genes were encoded within the identified copy number aberration region (Fig. 13). Western blot analysis of the frontal lobe revealed increased expression of Filamin-A, RPL10, GDI1, FAM3A, and G6PD in two twins compared with healthy controls (Normal-1 to 5) (Fig. 3a). To examine the contribution of these five proteins to 4R-tau pathology, we created mCherry-tagged expression constructs for each protein and transfected them into HEK293 cells together with a GFP-tagged wild-type 4R-tau construct (GFP-4R-tau), followed by Western blot analysis. Expression of Filamin-A, unlike the other four, statistically significantly increased GFP-4R-tau protein expression (P<0.001, Fig. 3b). Furthermore, in immortalized lymphocytes derived from twins, expression of not only Filamin-A but also tau protein was increased (Fig. 3c). When three siRNAs targeting FLNA were transfected into immortalized lymphocytes derived from Twin-A, all siRNAs reduced the expression of not only Filamin-A but also tau protein (Fig. 3d). These results suggest that, among the 16 genes in the copy number aberration region, FLNA is a regulator of tau protein expression. Furthermore, Western blot analysis using the major phospho-tau antibodies AT8 and PHF-1 revealed increased phosphorylation of GFP-4R-tau in HEK293 cells expressing Filamin-A and GFP-4R-tau (Fig. 3e). A cycloheximide administration chase experiment showed that the protein half-life of GFP-4R-tau transfected into HEK293 cells was prolonged upon Filamin-A expression (Fig. 3f). Soluble fractions (S1) and sarkosyl-insoluble fractions (P3) were extracted from HEK293 cells expressing Filamin-A and GFP-4R-tau. In both S1 and P3, high expression of Filamin-A significantly increased the expression of GFP-4R-tau (P<0.05, Fig. 3g). Immunoprecipitation was performed using the tau antibody TAU-5 in HEK293 cells expressing Filamin-A and GFP-4R-tau, and the interaction of tau protein with Filamin-A was identified (Fig. 3h).Furthermore, expression of Filamin-A induced the expression of heat shock proteins HSP90, HSP70, and HSP40, as well as ubiquitin, suggesting that the interaction between tau and Filamin-A exerts stress on cells. The protein structure of Filamin-A contains an actin-binding domain (ABD) at its N-terminus, and a p.Ala39Gly mutation within this ABD region is known to abolish binding to filamentous actin (F-actin) (Reference 32). Expression of p.Ala39Gly mutant Filamin-A in HEK293 cells did not result in increased expression of GFP-4R-tau protein, unlike wild-type Filamin-A, suggesting that Filamin-A affects tau via F-actin (Figure 10).

[0095] Test Example 4. Compounds that inhibit the expression of the Filamin-A gene reduce the expression level of tau protein The test was carried out in the same manner as in the test using siRNA in Test Example 3, except that the siRNA was replaced with an antisense nucleic acid. Antisense nucleic acids can also reduce the expression levels of not only Filamin-A but also tau protein.

[0096] Test Case 5. Increased protein expression of Filamin-A and its co-localization with 4R-tau in autopsy brains of PSP patients We analyzed the expression of filamin-A protein by Western blot in autopsy brains from 32 cases: 11 cases of PSP (including twin-A, twin-B, and nine sporadic cases), three cases of CBD, three cases of AD, four cases of PD, three cases of DLB, five cases of ALS, and five healthy controls. Case details are shown in Figure 14. Genomic DNA extracted from the PSP autopsy brains was analyzed for FLNA gene copy number using real-time quantitative PCR. No cases other than twin cases (twin-A and twin-B) showed increased FLNA gene copy number (Figure 9). Previous studies have shown that TA, a pathological hallmark of PSP, frequently appears in the frontal lobe (Reference 33). Based on this, we sampled the frontal lobe for this Western blot. The results showed that the expression of filamin-A protein in the PSP autopsy brains was statistically significantly increased in both the TBS-soluble fraction S1 and the sarkosyl-insoluble fraction P3 (Figure 4a). In S1, the expression level of Filamin-A protein and the age at onset of PSP showed a statistically significant negative correlation (Fig. 11). In P3, the expression levels of Filamin-A protein and 4R-tau protein showed a statistically significant positive correlation (Fig. 4b). Fluorescent immunostaining was performed on the frontal cortex of Twin-B (Fig. 4c) and a sporadic PSP case (Fig. 4d, e). Filamin-A colocalized with AT8 antibody-positive TA and globose-type NFTs.

[0097] Test Example 6. Filamin-A expression induces aggregation of 4R-tau in primary astroglia We expressed Filamin-A in primary astrocytes from the rat cerebral cortex and examined its effect on 4R-tau aggregation. In primary astrocytes co-expressing Filamin-A and GFP-4R-tau, GFP-4R-tau aggregated in the proximal parts of cell processes and in the cell body (Fig. 4f). The distribution of these tau aggregates resembled that of TA in PSP. Western blot analysis showed that Filamin-A expression significantly increased the expression of GFP-4R-tau in primary astrocytes (P<0.01, n=3).

[0098] Test Example 7. Increased expression and phosphorylation of 4R-tau occur when ΔFLNA is transfected To examine the effect of ΔFLNA on 4R-tau pathology, we constructed an mCherry-tagged expression construct and co-transfected it with a GFP-tagged wild-type 4R-tau construct (GFP-4R-tau) into HEK293 cells. Western blot analysis was performed. Expression of ΔFLNA increased GFP-4R-tau expression (Figure 15b). To examine the in vivo effects, we injected AAV9-ΔFLNA-6xHis into the right frontal cortex of wild-type (WT) mice and genetically engineered mice expressing human tau protein (hT-PAC-N) at 2 months of age and analyzed the results at 3 months of age. The expression of 4R-tau was increased (Figure 15c-f).

[0099] Test Example 8. Increased expression of 4R-tau occurs in transgenic mice (hFLNA-Tg) in which human FLNA expression is induced We analyzed 8-month-old transgenic mice (hFLNA-Tg) and found increased expression of FLNA and 4-repeat tau in the hippocampus and frontal cortex (Figure 16).

[0100] Test Case 9: Actin-binding mutant FLNA does not cause ectopic gray matter We transfected mouse fetal brains at 14 days of gestation (E14) with a mutant FLNA that lacks actin binding, and these mice did not develop ectopic gray matter or show increased expression of 4R-tau (Fig. 17a).

[0101] Test Example 10. Effect of actin polymerization inhibitors FLNA was transfected into mouse fetal brains on day 14 of gestation (E14). Compared to treatment with 0.1% DMSO, treatment with the actin polymerization inhibitor cytochalasin D (CytoD) did not result in an increase in the expression of phosphorylated tau (Figure 17b).

[0102] 3. Discussion This study revealed that Filamin-A promotes 4R-tau aggregation and is involved in the pathogenesis of PSP. Identical twins without MAPT gene mutations developed PSP simultaneously, and duplication of the FLNA gene, encoding Filamin-A, was identified in their genomic DNA. Pathological analysis using brain bank samples revealed that Filamin-A expression was increased in autopsy brains with PSP and colocalized with aggregated tau protein. Furthermore, biochemical analysis revealed that Filamin-A promotes phosphorylation, protein stability, and sarkosyl insolubility of 4R-tau, which are precursors to its aggregation, suggesting that Filamin-A may be upstream of 4R-tau in the pathogenesis of PSP.

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[0104] The present invention provides a new treatment strategy for PSP, for which no effective treatment or therapeutic agent exists. The PSP treatment agent of the present invention targets filamin A and exerts its effect through a unique mechanism of action. Meanwhile, the cells (e.g., lymphocyte cell lines derived from PSP patients) or transgenic non-human mammals with high filamin A expression provided by the present invention are useful for research and development of treatment or therapeutic agents for PSP. In other words, the use of the present invention is expected to lead to the creation of further treatment or therapeutic agents for PSP.

[0105] The present invention is not limited to the above-described embodiments and examples. Various modifications within the scope of the claims and within the scope that can be easily conceived by a person skilled in the art are also included in the present invention. The contents of papers, published patent applications, patent publications, and other publications explicitly stated in this specification are incorporated herein by reference in their entirety.

Claims

[Claim 1] The invention described herein.

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