Prevention or treatment of osteoclastogenic diseases and leukoencephalopathy

Abstract

To provide technologies for preventing or treating osteoclastogenic diseases and leukoencephalopathy. [Solution] A preventive or therapeutic agent for osteoclastogenic diseases and leukoencephalopathy, containing an optineurin (OPTN)-related substance as an active ingredient.

Description

Technical Field

[0001] The present invention relates to a pharmaceutical composition for preventing or treating osteoclast disorder diseases and leukodystrophy. Specifically, the present invention relates to a pharmaceutical composition containing an OPTN-related substance containing optineurin (OPTN) or a polynucleotide encoding the same as an active ingredient for preventing or treating osteoclast disorder diseases and leukodystrophy, as well as related screening methods, biomarkers and kits.

Background Art

[0002] Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease that selectively destroys motor neurons in the motor cortex, brainstem and spinal cord. In ALS, it is a highly severe disease in which muscles throughout the body weaken, but currently there is no fundamental treatment method. Most cases of ALS are sporadic, and about 10% have a family history (Non-Patent Documents 1, 2). The present inventors have previously reported that optineurin (OPTN) is one of the causative genes of familial ALS inherited in an autosomal dominant or recessive manner (Non-Patent Document 3). In cases of dominant inheritance, there was an E478G missense mutation, and in cases of recessive inheritance, deletion of exon 5 or Q398* nonsense mutation was involved.

[0003] OPTN is a multifunctional protein, and its dysfunction leads to multiple organ failure. The E50K mutation in OPTN is responsible for adult-onset primary open-angle glaucoma (POAG) (Non-Patent Literature 4), and single nucleotide polymorphisms (SNPs) around the OPTN gene are correlated with Paget's disease (PDB) (Non-Patent Literature 5, 6). Another important function of OPTN is to regulate inflammation via nuclear factor-κB (NF-κB) and receptor-interacting proteins (RIP) (Non-Patent Literature 7, 8). Inflammation is a major cause of several neurodegenerative diseases and exhibits a variety of phenotypes. Recently, inflammation caused by periodontal disease has been reported to be one of the contributing factors to the development of Alzheimer's disease (AD) (Non-Patent Literature 9-11). Osteoclasts play a major role in the development of periodontal disease. In addition to inflammatory effects, periodontal disease-associated salivary microbiota may exacerbate AD pathology through gut-brain axis (GBA) interactions (Non-Patent Literature 12), and GBA damage has been reported not only in AD but also in Parkinson's disease (PD) and ALS (Non-Patent Literature 13).

[0004] Furthermore, microglia play an important role in neuroinflammation and are associated with some neurodegenerative diseases (Non-Patent Literature 14). Osteoclasts and microglia originate from monocytes, and the colony-stimulating factor 1 receptor (CSF1R) is important for monocyte differentiation. CSF1R is known as the causative gene for adult-onset leukoencephalopathy with axonocytes and chromocyte glia (ALSP), which is one of the hereditary leukoencephalopathy caused by microglial dysfunction (Non-Patent Literature 15, 16). [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Mulder DW, Kurland LT, Offord KP, Beard CM. Familial adult motor neuron disease: amyotrophic lateral sclerosis. Neurology. 1986;36(4):511-7. [Non-Patent Document 2] Brown RH, Al-Chalabi A. Amyotrophic Lateral Sclerosis. N Engl J Med. 2017;377(2):162-72.

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[0006] Periodontal disease and leukoencephalopathy, which can be the cause of various diseases, are conditions that may be related to monocyte-derived inflammation. OPTN may be upstream of these changes as an inflammation-related molecule, but the molecules and etiologies involved in these events remain unclear. However, there have been very few effective treatments for diseases related to osteoclasts, which play a major role in the development of periodontal disease, or for leukoencephalopathy, and their development is urgently needed. [Means for solving the problem]

[0007] In order to understand the relationship between factors such as optineurin (OPTN), colony-stimulating factor 1 receptor (CSF1R), diseases related to osteoclasts, and leukoencephalopathy, the inventors of the present invention have conducted intensive research and found that OPTN is related to colony-stimulating factor 1 receptor (CSF1R), and that the normal function of OPTN suitably affects periodontal disease and leukoencephalopathy, thereby completing the present invention. That is, the present invention relates to a technique for preventing or treating osteoclast disorder diseases and leukoencephalopathy.

[0008] That is, the present invention includes the following aspects. <Pharmaceutical composition containing OPTN> [Item 1] A pharmaceutical composition for preventing or treating osteoclast disorder diseases and leukoencephalopathy, containing an OPTN-related substance as an active ingredient. [Item 2] The OPTN-related substance is (a1) OPTN consisting of the amino acid sequence shown in SEQ ID NO: 1; (a2) A mutant OPTN consisting of an amino acid sequence in which one or several amino acid residues are substituted, deleted, inserted and / or added from the amino acid sequence shown in SEQ ID NO: 1, and having an OPTN activity equivalent to that of OPTN; (a3) A mutant OPTN consisting of an amino acid sequence having 80% or more identity to the amino acid sequence shown in SEQ ID NO: 1, and having an OPTN activity equivalent to that of OPTN; (a4) OPTN encoded by the nucleotide sequence shown in SEQ ID NO: 2; (a5) Encoded by a polynucleotide that hybridizes under stringent conditions with a polynucleotide consisting of a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO: 2, and having an OPTN activity equivalent to that of OPTN; (a6) A mutant OPTN encoded by a polynucleotide having 80% identity to the nucleotide sequence shown in SEQ ID NO: 2, and having an OPTN activity equivalent to that of OPTN; and (a7) A mutant OPTN encoded by a nucleotide sequence in which one or more nucleotides are deleted, substituted or added in the nucleotide sequence shown in SEQ ID NO: 2 and having an OPTN activity equivalent to that of OPTN, The pharmaceutical composition according to [Item 1], which is selected from the group consisting of [Item 3] The pharmaceutical composition according to [Item 1] or [Item 2], wherein the osteoclast disorder is selected from the group consisting of osteoporosis, rheumatoid arthritis, bone metastasis of cancer, periodontal disease, Paget's disease of bone, and marble disease.

[0009] <Pharmaceutical composition containing a polynucleotide encoding OPTN> [Item 4] The OPTN-related substance is (b1) A polynucleotide encoding the polypeptide according to [Item 2]; The pharmaceutical composition according to [Item 1], which is [Item 5] The OPTN-related substance is (b2) An OPTN polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 2; (b3) A polynucleotide that hybridizes under stringent conditions with a polynucleotide consisting of a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO: 2 and encodes a polypeptide having an activity equivalent to that of OPTN; (b4) A polynucleotide having 80% identity with the nucleotide sequence shown in SEQ ID NO: 2 and encoding a polypeptide having an OPTN activity equivalent to that of OPTN; and (b5) A polynucleotide consisting of a nucleotide sequence in which one or more nucleotides are deleted, substituted or added in the nucleotide sequence shown in SEQ ID NO: 2 and encoding a polypeptide having an OPTN activity equivalent to that of OPTN; The pharmaceutical composition according to [Item 1], which is a polynucleotide selected from the group consisting of [Item 6] The pharmaceutical composition according to [Claim 4] or [Claim 5], wherein the osteoclastogenic disease is selected from the group consisting of osteoporosis, rheumatoid arthritis, bone metastases of cancer, periodontal disease, Paget's disease of bone, and osteopetrosis. [Section 7] A vector comprising the polynucleotide described in [Clause 4] or [Clause 5].

[0010] <Screening Method> [Section 8] (a) OPTN polynucleotide consisting of the base sequence shown in Sequence ID No. 2, (b) A mutant OPTN polynucleotide containing a nucleotide sequence in which one or more nucleotides are deleted, substituted, or added in the nucleotide sequence shown in Sequence ID No. 2, or (c) Mutant OPTN polynucleotides that hybridize under stringent conditions with a nucleotide sequence complementary to the nucleotide sequence shown in Sequence ID No. 2, It is characterized by using cells into which one of the following has been introduced. A method for screening OPTN agonists by confirming that the expression of the polynucleotide in the cells is enhanced. [Section 9] (1) Prepare candidate compounds, (2) (a) OPTN polynucleotide consisting of the base sequence shown in Sequence ID No. 2, (b) A mutant OPTN polynucleotide containing a nucleotide sequence in which one or more nucleotides are deleted, substituted, or added in the nucleotide sequence shown in Sequence ID No. 2, or (c) Mutant OPTN polynucleotides that hybridize under stringent conditions with a nucleotide sequence complementary to the nucleotide sequence shown in Sequence ID No. 2, Prepare cells into which one of the following has been introduced: (3) The candidate compound is brought into contact with the cells, (4) A method for screening OPTN agonists according to [Clause 8], comprising determining whether the candidate compound enhances the expression of the gene in the cell.

[0011] <Other Inventions> [Section 10] A method for determining the severity of osteoclastogenic diseases and leukoencephalopathy, (a10) A step to measure the amount of OPTN (amount of test biomarker) in the macrophages of the subject, (b10) A step of comparing the amount of the tested biomarker with the amount of OPTN in macrophages of healthy individuals (control biomarker amount), and (c10) A method for determining that a subject is at risk of developing severe osteoclastopathy and leukoencephalopathy if the amount of the tested biomarker is lower than the amount of the control biomarker. [Section 11] A macrophage OPTN biomarker that can be used to assess the severity of osteoclastogenic diseases and leukoencephalopathy. [Section 12] A kit for detecting the biomarker described in [Section 11], comprising a primer set for amplifying OPTN cDNA, a probe that specifically hybridizes to OPTN mRNA, or a substance that specifically binds to OPTN. [Section 13] A method for measuring the activity of OPTN in candidate OPTN substances, (1) Prepare candidate OPTN materials, (2) Convert the OPTN candidate substance into a form that can be introduced into cells, and create a converted OPTN candidate substance. (3) Macrophages were obtained from the bone marrow of Optn knockout mice, (4) The obtained macrophages were then converted from the OPTN candidate substance, (5) Measure the suppression of differentiation of introduced macrophages into osteoclasts. method. [Brief explanation of the drawing]

[0012] [Figure 1A] Figure 1A is a schematic diagram showing the structures of CSF1R and OPTN. [Figure 1B]Figure 1B (left) shows the results of immunoprecipitation demonstrating that the intracellular domain of RCSF1R (CSF1R-ICD) and the full length of CSF1R bind to OPTN. [Figure 1C] Figure 1C shows immunoprecipitation results illustrating the affinity of multiple variants and fragments of the OPTN protein to the CSF1R intracellular domain. OPTN E478 G strongly binds to CSF1R-ICD. Q398* OPTN binds with comparable affinity. OPTN S177 A, an autophagy-deficient variant that binds to CSF1R, is unaffected. The abbreviations in Figure 1 have the following meanings: CC: coiled-coil domain, LZ: leucine zipper domain, LIR: LC3 interaction domain, UBAN: ubiquitin-binding domain between ABIN protein and NEMO, ZF: zinc finger domain, KID: kinase insertion domain, ICD: intracellular domain, FL: full length, IP: immunoprecipitation, IB: immunoblotting.

[0013] [Figure 2A] Figure 2A shows the procedure for the colony formation assay. Nucleated cells (BMNCs) derived from bone marrow were collected from wild-type (WT) mice, Optn knockout (KO) mice, Optn E481G mice, and Optn E50K mice. Optn E481G corresponds to the human OPTN E478G mutation and serves as a model for amyotrophic lateral sclerosis (ALS). E50K is a non-ALS controlled glaucoma model. BMDMs were separated by FACS, and only CSF1R-positive BMNCs were collected. CSF1R-positive BMNCs were stimulated with CSF1, and the number of colonies was counted 10 days after cell seeding. [Figure 2B] Figure 2B is a graph showing the number of BMNC colonies from wild-type (WT) mice and Optn knockout (KO) mice. BMNC from Optn KO mice formed fewer colonies than those from WT mice (n=5 per cohort, P=0.0023, Student's t-test). [Figure 2C]Figure 2C is a graph showing the number of BMNC colonies from wild-type (WT) mice and heterozygous (P = 0.0003) and homozygous (P = 0.0003) Optn E481G mice. Heterozygous and homozygous mutations in Optn E481G reduced CSF1 reactivity (n=5 per cohort, P=0.0003 between WT and + / E481G, P=0.0003 between WT and E481G / E481G, Tukey-Kramer test). [Figure 2D] Figure 2D and Figure 2C are graphs showing the number of BMNC colonies from wild-type (WT) mice, heterozygous (P = 0.0003) and homozygous (P = 0.0003) Optn E50K mice. BMNC from Optn E50K mutant mice formed more colonies (n=5 per cohort, P=0.0014 between WT and E50K / E50K, P=0.0022 between + / E50K and E50K / E50K, Tukey-Kramer test). The abbreviations in Figure 2 mean the following: the graph (box plot) represents the median of each group, and the interquartile range and error bars show the minimum and maximum values. ns: no significant difference, ** P < 0.01, *** P < 0.001. WT: wild-type, KO: knockout.

[0014] [Figure 3A] Figure 3A shows the results of immunohistochemical analysis of OPTN and tartrate-resistant acid phosphatase (TRAP; a marker for osteoclasts). In serial sections of periodontal tissue, TRAP-positive osteoclasts were OPTN-positive. [Figure 3B] Figure 3B shows the results of an in vitro assay for osteoclast differentiation using bone marrow-derived macrophages (BMMs). [Figure 3C]Figure 3C shows that BMMs from Optn knockout (KO) and Optn E481G mice differentiated into more TRAP+ multinucleated cells (MNCs) in response to RANKL compared to BMMs from wild-type (WT) mice (6 mice per cohort, P = 0.0001 between WT and KO, P < 0.0001 between WT and + / E481G, P < 0.0001 between WT and E481G / E481G, Tukey-Kramer test). [Figure 3D] Figure 3D is a photograph showing alveolar bone resorption under ligation-induced inflammation in Optn mutant mice. [Figure 3E] Figure 3E is a graph showing the degree of alveolar bone loss, as assessed by the length from the alveolar bone margin (ABC) to the cemento-enamel junction (CEJ), in WT mice, Optn knockout mice, and Optn E481G mice. The degree of alveolar bone loss was significantly higher in Optn KO and Optn E481G mice compared to WT mice (6 mice per cohort, P = 0.0026 between WT and KO, P = 0.0020 between WT and + / E481G, P < 0.0001 between WT and E481G / E481G, Tukey-Kramer test). The abbreviations in Figure 3 mean the following: The graph (box plot) represents the median of each group, and the interquartile range and error bars show the minimum and maximum values. NS: No significant difference. ** P < 0.01, *** P < 0.001, **** P < 0.0001. TRAP: Tuftrate-resistant acid phosphatase, WT: Wild-type, KO: Knockout, ABC: Alveolar bone margin, CEJ: Cementum-enamel junction.

[0015] [Figure 4A] Figure 4A is a family tree of OPTN E478G mutations associated with amyotrophic lateral sclerosis (ALS), periodontal disease, and leukoencephalopathy. Patient III-2 was not clinically examined. [Figure 4B] Figure 4B shows magnetic resonance imaging (MRI) of the head of patient I-2. The T2-weighted images (T2WI) of the axonal view show diffuse brain atrophy and high signal intensity around the ventricles. [Figure 4C]Figure 4C shows a magnetic resonance imaging (MRI) image of the head of patient I-2. The T1-weighted image (T1WI) shows atrophy of the brain body in the axonal view. [Figure 4D] Figure 4D shows a head MRI of patient II-3. The T1WI axonal image shows atrophy of the brain body. The axonal image shows diffusive brain atrophy and high signal intensity around the ventricles, which worsened over time. [Figure 4E] Figure 4E shows a head MRI of patient II-3. The axonal image is from 10 years after the onset of the disease. From top to bottom, the images are T1WI, T2WI, and fluid-suppressed inversion recovery (FLAIR). g: All of patient II-3's maxillary teeth were lost due to periodontal disease. h: Computed tomography (CT) of the facial bone. [Figure 4F] Figure 4F shows an MRI of the head of patient II-3. The axonal image is from 11 years after the onset of the disease. From top to bottom, the images are T1WI, T2WI, and fluid-suppressed inversion recovery (FLAIR). i and j: Computed tomography (CT) of the facial bones. The alveolar bone of patients II-1(i) and III-1(j) is resorbed and the tooth roots are exposed compared to a healthy male in his 40s (h in Figure 4E). [Figure 5]Figure 5 shows the results of Iba-1 immunostaining in anatomical brains of OPTN-ALS and ALSP cases. In the figure, a through d are representative micrographs of Iba1 immunostaining in the frontal cortex, and e through h are representative micrographs of Iba1 immunostaining in the frontal white matter. a and e are from normal subjects, b and f are from adult-onset leukoencephalopathy (ALSP) patients with axonal globules and pigment cells, c and g are from amyotrophic lateral sclerosis (ALS) patients with the Q398* OPTN mutation, and d and h are micrographs of Iba1 immunostaining from ALS patients with the E478G OPTN mutation. In the frontal cortex, microglia density was reduced in the brains of both ALSP and OPTN-ALS, and the processes of each microglia were thinner, fragmented, and reduced in number. Disrupted frontal white matter from ALSP (f) contained scattered large, round, vacuolated Iba1-positive hypothetical macrophages. Iba-1 immunostained degenerated frontal white matter in ALS brains with both Q398* (g) and E478 G (h) OPTN mutations showed similar findings to those of ALSP (f). In the figure, the scale bars in a to d are 100 μm, and in e to h are 50 μm. ALSP: Adult-onset leukoencephalopathy with axonal globules and pigmented glia. [Figure 6A] Figure 6A shows a photograph of BMM cells from Optn knockout (KO) mice cultured for 10 days after adding RANKL 100 ng / ml and M-CSF 20 ng / ml. It shows differentiation into many TRAP+ multinucleated cells (MNCs). [Figure 6B] Figure 6B is a photograph taken under the same conditions as Figure 6A, but with the addition of AAV-OPTN virus (0.9 x 10⁵ genes / cell). The number of TRAP+ multinucleated cells (MNCs) is reduced and smaller compared to Figure 6A. [Figure 6C] Figure 6C compares A and B, showing that while the number of cells remains unchanged in Vector+ (the group to which the AAV-OPTN virus was added), the surface area decreases, resulting in a reduction in the surface area per cell. [Modes for carrying out the invention]

[0016] <Pharmaceutical composition containing OPTN> The inventors conducted various experiments to understand the relationship between optineurin (OPTN), colony-stimulating factor 1 receptor (CSF1R), and factors such as osteoclast-related diseases and leukoencephalopathy. First, mutant OPTN was analyzed by mass spectrometry, and related molecules were comprehensively investigated. As a result, it was found that OPTN is associated with colony-stimulating factor 1 receptor (CSF1R). Next, nucleated cells (BMNC) derived from bone marrow were collected from newly established Optn mutant mice, and a colony formation assay with CSF1 was performed, thereby finding that mutations that impair Optn function decrease the number of colonies. On the other hand, one case series of an OPTN mutant family showing periodontal disease and leukoencephalopathy was identified. It was found that OPTN gene mutations increase osteoclast activity and affect periodontal disease. Monocyte changes may cause these abnormalities.

[0017] In one embodiment, the present invention relates to a pharmaceutical composition for preventing or treating osteoclast disorder diseases and leukoencephalopathy, containing an optineurin (OPTN)-related substance as an active ingredient.

[0018] In the present invention, the "optineurin-related substance" or "OPTN-related substance" means a substance having the original activity, action, and function of OPTN, such as an inflammatory response, autophagy regulation, and NF-κB activity inhibitory ability. In the present invention, the OPTN-related substance may be a protein or a polynucleotide having a nucleotide sequence encoding the same. In the present invention, the OPTN-related substance can be referred to as an OPTN agonist, an OPTN-acting substance, or an OPTN-stimulating substance. Examples of the OPTN-related substance include the protein of mutant OPTN described in Neurological Sciences (2022) 43:5391-5396 and the polynucleotide of mutant OPTN encoding the same, that is, the proteins and polypeptides shown in Table 1:

[0019]

Table 1

[0020] In this invention, "osteoclastic disease" refers to a disease in which osteoclasts become abnormal. Osteoclasts are essential cells for maintaining healthy bone, but if they become excessively activated, they can cause pathological bone destruction associated with various diseases such as osteoporosis, rheumatoid arthritis, periodontal disease, and bone metastasis from cancer (Fitoterapia 142 (2020) 104482). Examples of "osteoclastic disease" in this invention include, but are not limited to, osteoporosis, rheumatoid arthritis, bone metastasis from cancer, periodontal disease, Paget's disease of bone, and osteopetrosis. Osteoporosis is a disease in which bone is abnormally destroyed due to increased function of osteoclasts. Rheumatoid arthritis is a disease in which inflammatory T cells called Th17 come into contact with osteoclasts on the bone surface and convert N-type to R-type, causing bone destruction. Bone metastasis of cancer is a disease in which cancer cells spread to the bone, causing abnormal bone destruction.

[0021] Periodontal disease is a disease in which the gums (gingiva) and bone (alveolar bone) that support the teeth are destroyed. External factors such as bacteria and increased activity of osteoclasts are involved. Paget's disease of bone is a chronic skeletal disorder in which abnormalities in the metabolic turnover of certain bones result in the bones in those areas becoming thicker and softer. Bone breakdown and formation increase, causing the bones to become thicker than normal but with reduced strength. Non-patent documents 5 and 6 only show that single nucleotide polymorphism P around the OPTN gene is correlated, but they do not describe whether increasing or decreasing OPTN activity is beneficial. Osteopetrosis is a disease that develops when osteoclasts are congenitally deficient or do not function properly. It is known that the accumulation of old bone makes the bones brittle, and the prognosis for life is extremely poor. In this invention, "leukoencephalopathy" refers to a condition in which the white matter of the cerebrum is damaged. While it can be caused by anticancer drugs, APLS, which is caused by mutations in CSF1R, is a well-known example of a genetic cause and is accompanied by microglial abnormalities.

[0022] In one embodiment of the present invention, an OPTN-related material is (a1) OPTN consisting of the amino acid sequence shown in Sequence ID No. 1; (a2) A mutant OPTN having OPTN activity equivalent to that of OPTN, consisting of an amino acid sequence in which one or more amino acid residues are substituted, deleted, inserted, and / or added from the amino acid sequence shown in Sequence ID No. 1; (a3) A mutant OPTN having an amino acid sequence that is 80% or more identical to the amino acid sequence shown in Sequence ID No. 1, and having OPTN activity equivalent to that of OPTN; (a4) OPTN encoded by the nucleotide sequence shown in Sequence ID No. 2; (a5) A mutant OPTN having OPTN activity equivalent to that of OPTN, encoded by a polynucleotide that hybridizes under stringent conditions with a polynucleotide consisting of a nucleotide sequence complementary to the nucleotide sequence shown in Sequence ID No. 2; (a6) A mutant OPTN encoded by a polynucleotide having 80% identity with the base sequence shown in Sequence ID No. 2, and having OPTN activity equivalent to that of OPTN; and (a7) A mutant OPTN having OPTN activity equivalent to that of OPTN, which is encoded by a nucleotide sequence in which one or more nucleotides are deleted, substituted, or added in the nucleotide sequence shown in Sequence ID No. 2. This invention relates to a pharmaceutical composition for preventing or treating osteoclastogenic diseases and leukoencephalopathy, which contains an OPTN-related substance selected from the group consisting of the following as an active ingredient.

[0023] Sequence ID 1: Amino acid sequence of OPTN: MSHQPLSCLTEKEDSPSESTGNGPPHLAHPNLDTFTPEELLQQMKELLTENHQLKEAAMKLNNQAMKGRFEELSAWTEKQKEERQFFEIQSKEAKERLMALSHENEKLKEELGKLKGKSERSSEDPTDDSRLPRAEAEQEKDQLR TQVVRLQAEKADLLGIVSELQLKLNSSGSSEDSFVEIRMAEGEAEGSVKEIKHSPGPTRTTVSTGTALSKYRSRSADGAKNYFEHEELTVSQLLLCLREGNQKVERLEVALKEAKERVSDFEKKTSNRSEIETQTEGSTEKENDE EKGPETVGSEVEALNLQVTSLFKELQEAHTKLSEAELMKKRLQEKCQALERKNSAIPSELNEKQELVYTNKKLELQVESMLSEIKMEQAKTEDEKSKLTVLQMTHNKLLQEHNNALKTIEELTRKESEKVDRAVLKELSEKLEL AEKALASKQLQMDEMKQTIAKQEEDLETMTILRAQMEVYCSDFHAERAAREKIHEEKEQLALQLAVLLKENDAFEDGGRQSLMEMQSRHGARTSDSDQQAYLVQRGAEDRDWRQQRNIPIHSCPKCGEVLPDIDTLQIHVMDCII

[0024] Sequence ID 2: The base sequence encoding OPTN:

[0025] In this embodiment, (a1) of the present invention is the polypeptide of OPTN itself. The mutant OPTN polypeptides of (a2) and (a3) ​​represent variants of the (a1) OPTN polypeptide. (a4) is a polypeptide identified by the nucleotide sequence corresponding to the OPTN polypeptide. (a5) to (a7) are polypeptides identified by the nucleotide sequences of variants of (a4).

[0026] With respect to (a2), the number of amino acid residues that may be substituted, deleted, inserted and / or added is not particularly limited, as long as the polypeptide of (a2) has OPTN activity equivalent to that of OPTN, but the upper limit may be, for example, 4, 3, 2, or 1. With respect to (a3) ​​and (b3), the amino acid sequence identity may be, for example, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, or 99% or more.

[0027] Regarding (a5), "hybridizing under stringent conditions" means hybridizing under somewhat strict conditions, and those skilled in the art will readily understand that the appropriate stringent conditions for promoting hybridization can be varied. For example, hybridization can be carried out at approximately 45°C with 6.0 × sodium chloride / sodium citrate (SSC), followed by washing with 2.0 × SSC at 50°C. For example, the salt concentration in the washing step can be selected from low stringency of 2.0 × SSC at approximately 50°C to high stringency of 0.2 × SSC at approximately 50°C. The temperature in the washing step can also be increased from room temperature of approximately 22°C under low stringency conditions to approximately 65°C under high stringent conditions. Both temperature and salt can be varied, or other variables can be varied while keeping the temperature and salt concentration constant.

[0028] Regarding (a6), the identity of the base sequences may be, for example, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, or 99% or more.

[0029] Whether a certain polypeptide has OPTN activity equivalent to that of OPTN can be determined, for example, by a method that utilizes the inhibitory effect on NF-κB described in Non-Patent Documents 3 and 7.

[0030] The polypeptides of (a1) to (a7) can be produced by genetic engineering techniques using the polynucleotide represented by SEQ ID NO: 2. Alternatively, the polypeptides of (a1) to (a7) can also be produced by general chemical synthesis methods of proteins (such as the liquid phase method or the solid phase method).

[0031] <Pharmaceutical composition containing a polynucleotide encoding OPTN> As another embodiment of the present invention, an OPTN-related substance is (b1) a polynucleotide encoding the polypeptides of (a1) to (a7); The present invention relates to a pharmaceutical composition for preventing or treating osteoclast disorder diseases and leukodystrophy, which contains an active ingredient that is

[0032] As yet another embodiment of the present invention, an OPTN-related substance is (b2) an OPTN polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 2; (b3) a polynucleotide that hybridizes under stringent conditions with a polynucleotide consisting of a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO: 2 and encodes a polypeptide having activity equivalent to that of OPTN; (b4) a polynucleotide having 80% identity with the nucleotide sequence shown in SEQ ID NO: 2 and encoding a polypeptide having OPTN activity equivalent to that of OPTN; and (b5) a polynucleotide consisting of a nucleotide sequence in which one or more bases are deleted, substituted or added in the nucleotide sequence shown in SEQ ID NO: 2 and encodes a polypeptide having OPTN activity equivalent to that of OPTN; This invention relates to a pharmaceutical composition for preventing or treating osteoclastogenic diseases and leukoencephalopathy, which contains an OPTN-related substance as an active ingredient, which is a polynucleotide selected from the group consisting of the following.

[0033] In the present invention, if the OPTN-related substance is a polynucleotide, preferably the polynucleotide in the present invention is introduced into a gene therapy vector. Gene therapy often refers to the treatment of genetic disorders, but in the present invention, it means the treatment to prevent or treat osteoclastic disorders and leukoencephalopathy, or to inhibit the progression of osteoclastic disorders and leukoencephalopathy. In the present invention, gene therapy may include in vivo copy insertion of a gene into the cells of a patient with osteoclastic disorders and leukoencephalopathy. The cells are modified to induce differentiation, transdifferentiation, or reprogramming. The cells may also be modified to serve as a vehicle for delivering therapeutic proteins.

[0034] The present invention further provides cells containing the vector of the present invention. The cells of the present invention can be used in cell therapy to prevent or treat osteoclastic diseases and leukoencephalopathy, or to inhibit the progression of osteoclastic diseases and leukoencephalopathy.

[0035] In this specification, “treatment” means a method or process aimed at (1) delaying the onset of osteoclastic disorders and leukoencephalopathy or fibrotic conditions; (2) slowing or halting the progression, exacerbation or worsening of the symptoms of osteoclastic disorders and leukoencephalopathy or fibrotic conditions; (3) achieving remission of the symptoms of osteoclastic disorders and leukoencephalopathy or fibrotic conditions; or (4) curing osteoclastic disorders and leukoencephalopathy or fibrotic conditions. Treatment may be administered as a preventive measure before the onset of the disease or condition, or treatment may be administered after the onset of the disease.

[0036] In this specification, “prevention” means preventing the development of osteoclastogenic diseases and leukoencephalopathy or fibrotic conditions.

[0037] In this invention, a pharmaceutical composition generally refers to an agent used for the treatment or prevention of a disease, or for examination and diagnosis.

[0038] The pharmaceutical composition of the present invention can be formulated by methods known to those skilled in the art. For example, it can be used parenterally in the form of a sterile solution with water or other pharmaceutically acceptable liquid, or as an injectable suspension. For example, it can be formulated by mixing it with a pharmacokinetically acceptable carrier or medium, specifically sterile water, physiological saline, vegetable oil, emulsifier, suspension agent, surfactant, stabilizer, flavoring agent, excipient, vehicle, preservative, binder, etc., in a unit dose form generally accepted for pharmaceutical practice. The amount of active ingredient in these formulations should be set so as to obtain an appropriate volume within the indicated range.

[0039] Sterile compositions for injection can be formulated according to standard compounding procedures using a vehicle such as distilled water for injection.

[0040] Examples of aqueous solutions for injection include physiological saline, glucose, and isotonic solutions containing other adjuvants (e.g., D-sorbitol, D-mannose, D-mannitol, sodium chloride). Suitable solubilizers, such as alcohol (ethanol, etc.), polyalcohols (propylene glycol, polyethylene glycol, etc.), and nonionic surfactants (polysorbate 80™, HCO-50, etc.), may also be used in combination.

[0041] Examples of oily liquids include sesame oil and soybean oil, and benzyl benzoate and / or benzyl alcohol may be used in combination as solubilizers. It may also be combined with buffers (e.g., phosphate buffer and sodium acetate buffer), analgesics (e.g., procaine hydrochloride), stabilizers (e.g., benzyl alcohol and phenol), and antioxidants. The prepared injection solution is usually filled into appropriate ampoules.

[0042] The pharmaceutical composition of the present invention is preferably administered by parenteral administration. For example, it can be in the form of an injectable, nasal, pulmonary, or transdermal formulation. For example, it can be administered systemically or locally by intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection, etc.

[0043] The method of administration can be appropriately selected depending on the patient's age and symptoms. The dosage of the pharmaceutical composition containing polypeptide can be set, for example, in the range of 0.0001 mg to 1000 mg per kg of body weight per dose. Alternatively, for example, the dosage can be set to 0.001 to 100,000 mg per patient, but the present invention is not necessarily limited to these values. The dosage and method of administration will vary depending on the patient's weight, age, symptoms, etc., but those skilled in the art can set an appropriate dosage and method of administration considering these conditions.

[0044] <Screening Method> In another embodiment, the present invention, (a) OPTN polynucleotide consisting of the base sequence shown in Sequence ID No. 2, (b) A mutant OPTN polynucleotide containing a nucleotide sequence in which one or more nucleotides are deleted, substituted, or added in the nucleotide sequence shown in Sequence ID No. 2, or (c) Mutant OPTN polynucleotides that hybridize under stringent conditions with a nucleotide sequence complementary to the nucleotide sequence shown in Sequence ID No. 2, It is characterized by using cells into which one of the following has been introduced. This invention relates to a method for screening OPTN agonists by confirming that the expression of the polynucleotide in the cells is enhanced. Sequence ID 2 in the sequence listing is the nucleotide sequence of the human OPTN gene.

[0045] The isolated gene or nucleic acid is incorporated into a suitable vector to transform eukaryotic and prokaryotic host cells. Furthermore, by introducing a suitable promoter and expression-related sequences into these vectors, mRNA or protein encoded by the target nucleic acid is expressed in the respective host cells. In this invention, plasmids and lambda-based phage vectors can be used as vectors, into which the target nucleic acid is inserted. The plasmid can be either a self-replicating plasmid containing a transcription promoter region or a plasmid that can be incorporated into the chromosomes of animal cells. Vertebrate cell expression vectors usable in this invention typically have a promoter located upstream of the gene to be expressed, an RNA splice site, a polyadenylation site, and a transcription termination sequence, and may further have an origin of replication if necessary. Examples of such expression vectors include, but are not limited to, pSV2dhfr (Mol. Cell. Biol., 1, p.854-864, 1981) which has the initial SV40 promoter, pEF-BOS (Nucleic Acids Res., 18, p.5322, 1990) which has the human extension factor promoter, and pCEP4 (Invitrogen) which has the cytomegalovirus promoter. For example, eukaryotic host cells include cells from vertebrates, insects, and yeast. Examples of vertebrate cells include, but are not limited to, monkey COS cells (Cell, 23, p.175-182, 1981), dihydrofolate reductase-deficient strains of Chinese hamster ovary cells (CHO) (Proc. Natl. Acad. Sci. USA, 77, p.4216-4220, 1980), human fetal kidney-derived HEK293 cells, and 293-EBNA cells (Invitrogen) which are the same cells into which the Epstein-Barr virus EBNA-1 gene has been introduced.

[0046] To confirm the enhancement of polynucleotide expression, this can typically be done using real-time polymerase chain reaction (PCR) or DNA microarray techniques.

[0047] The screening method of the present invention is more specifically as follows: (1) Prepare candidate compounds, (2) (a) OPTN polynucleotide consisting of the base sequence shown in Sequence ID No. 2, (b) A mutant OPTN polynucleotide containing a nucleotide sequence in which one or more nucleotides are deleted, substituted, or added in the nucleotide sequence shown in Sequence ID No. 2, or (c) Mutant OPTN polynucleotides that hybridize under stringent conditions with a nucleotide sequence complementary to the nucleotide sequence shown in Sequence ID No. 2, Prepare cells into which one of the following has been introduced: (3) The candidate compound is brought into contact with the cells, (4) A method for screening OPTN agonists, comprising determining whether the candidate compound enhances the expression of the gene in the cell.

[0048] From the cells obtained as described above, various methods can be employed to select a cell line transfected with the target nucleic acid. These include methods for directly confirming the presence of nucleic acid, and methods for selecting a cell line that expresses mRNA. In step (3), to "determine whether the candidate compound suppresses the expression of the gene or nucleic acid," for example, the method described in Foster et al., 2019, Cell 179, 895-908 can be used.

[0049] <Biomarkers, etc.> In another embodiment, the present invention provides a method for determining the severity of osteoclastogenic diseases and leukoencephalopathy, (a10) A step to measure the amount of OPTN (amount of test biomarker) in the macrophages of the subject, (b10) A step of comparing the amount of the tested biomarker with the amount of OPTN in macrophages of healthy individuals (control biomarker amount), and (c10) A method for determining that a subject is at risk of developing severe osteoclastopathy and leukoencephalopathy when the amount of the tested biomarker is lower than the amount of the control biomarker. In connection therewith, the present invention, in yet another embodiment, provides a macrophage OPTN biomarker that can determine the severity of osteoclastogenic diseases and leukoencephalopathy. Furthermore, the present invention provides the use of OPTN as a biomarker that can determine the severity of osteoclastogenic diseases and leukoencephalopathy.

[0050] Macrophages from subjects and / or healthy individuals can be collected as monocytes from the blood using methods well known to those skilled in the art.

[0051] The amount of OPTN in macrophages can be measured by immunological methods if antibodies against OPTN are available. For example, it can be measured by the ELISA method, which is well known to those skilled in the art. Detection of mRNA against OPTN can be performed, for example, by RNA scoping (Advanced Cell Diagnostics), a highly sensitive in situ hybridization method. In RNA scoping, if the mRNA of the target molecule is expressed, it can be detected as dots using a probe specific to that molecule. RNA can also be recovered from macrophages and measured by real-time RT-PCR.

[0052] In one embodiment, the present invention provides a biomarker detection kit comprising a primer set for amplifying OPTN cDNA, a probe that specifically hybridizes to OPTN mRNA, or a substance that specifically binds to the OPTN protein.

[0053] In the kit of this embodiment, the primer set is not particularly limited as long as it can amplify the cDNA of the OPTN gene of the animal species to be diagnosed. Similarly, the probe that specifically hybridizes to OPTN mRNA is not particularly limited as long as it specifically hybridizes to the mRNA of the OPTN gene. The probe may be immobilized on a carrier to form a DNA microarray or the like. The specific binding substance is the same as described above. The specific binding substance may be immobilized on a carrier to form a protein chip or the like.

[0054] In another embodiment, the present invention relates to a method for measuring the activity of OPTN in an OPTN candidate substance, (1) Prepare candidate OPTN materials, (2) Convert the OPTN candidate substance into a form that can be introduced into cells, and create a converted OPTN candidate substance. (3) Macrophages were obtained from the bone marrow of Optn knockout mice, (4) The obtained macrophages were then converted from the OPTN candidate substance, (5) Measure the suppression of differentiation of introduced macrophages into osteoclasts. Regarding the method. In this context, an OPTN candidate substance refers to a mutant OPTN that is assumed to possess OPTN activity, regardless of whether it is in the form of a protein, DNA, or RNA. A PTN candidate substance converted into a form that can be introduced into O cells refers to any convert that can be introduced into macrophages, such as a PTN candidate substance incorporated into a viral vector (such as adeno-associated virus) when the PTN candidate substance is RNA, or a convert in which the PTN candidate substance is embedded in lipid nanoparticles (LNPs).

[0055] The suppression of differentiation of introduced macrophages into osteoclasts can be measured by the number and surface area of ​​TRAP-positive cells.

[0056] All references cited herein, including publications and patent documents, are incorporated herein by reference to the same extent as if they were individually and specifically referenced and their entire contents were specifically described herein.

[0057] The present invention will be described in more detail below with reference to examples, but it should be noted that these are merely illustrative examples and do not limit the scope of the present invention. [Examples]

[0058] Example 1 Interaction between OPTN and CSF1R To comprehensively investigate molecules associated with OPTN, tagged OPTN proteins expressed in HEK293T cells were isolated (pull-down), and the precipitated protein complexes were analyzed by mass spectrometry. One of the binding proteins was CSF1R. CSF1R is involved in the development and maintenance of innate immune cells, including macrophages and microglia.

[0059] Next, we narrowly identified the protein domain to which CSF1R binds to OPTN. OPTN is a cytoplasmic protein, while CSF1R contains an intracellular domain. The structures of these two proteins are shown in Figure 1A (17-20). To investigate whether OPTN binds to the intracellular domain of CSF1R (CSF1R-ICD), CSF1R-ICD and full-length CSF1R (CSF1R-FL) were extracted from overexpressing HeLa cells and co-immunoprecipitation was performed with FLAG-tagged OPTN. Immunoprecipitation with anti-FLAG affinity gel and immunoblotting with CSF1R antibody resulted in the observation of binding between CSF1R-ICD and FLAG-OPTN (Figure 1B, left). Since CSF1R-ICD was saturated in detecting CSF1R-FL, CSF1R-FL was immunoprecipitated separately, and as a result, binding between CSF1R-FL and FLAG-OPTN was also detected (Figure 1B, right). The observation that OPTN binds to CSF1R-ICD is consistent with the fact that OPTN is a cytoplasmic protein.

[0060] Next, we investigated the affinity of multiple OPTN protein variants and fragments to the CSF1R intracellular domain (Figure 1C). The dominant-negative form, OPTN E478G, strongly bound to CSF1R-ICD, suggesting that strong binding may influence CSF1R signaling. In contrast, the truncated form of OPTN, Q398*, bound with comparable affinity. We also examined the affinity of the autophagy-impaired variant OPTN S177A to CSF1R, but the binding of the S177A variant was unaffected. Fragments containing the leucine zipper / LC3 interaction domain (LZ / LIR; 141-209) and the coiled-coil domain (CC; 202-444) of the OPTN protein showed higher affinity for binding to CSF1R. In other words, CSF1R strongly binds to the leucine zipper / LC3 interaction region (LZ / LIR; 141-200) and the coiled-coil region (CC; 202-444) of the OPTN protein.

[0061] Example 2 CSF1-mediated colony formation assay using myeloid nuclear cells from ALS mutant Optn mice. To confirm the in vitro biochemical results in Example 1, a colony formation assay was performed using bone marrow cells (Figure 2A). CSF1R is a cytokine receptor for the cytokines CSF1 (M-CSF) and IL-34, and is expressed on the cell surface of mouse bone marrow cells. To investigate whether Optn functions downstream of CSF1R, the responsiveness of bone marrow cells to CSF1, the ligand of CSF1R, was examined. CSF1R-positive cells were separated, and the exact response of CSF1R to its ligand was examined. CSF1R-positive bone marrow cells from previously established Optn knockout (KO) mice (21) showed fewer colonies (P = 0.0023) than cells from wild-type mice in minimal medium containing CSF1, indicating that loss of Optn function impairs CSF1R signaling (Figure 2B).

[0062] We also investigated whether the Optn E481G mutation, which corresponds to the OPTN E478G mutation, affects the responsiveness of myeloid cells derived from newly established Optn E481G single nucleotide mutation (SNV) mice. Both heterozygous (P = 0.0003) and homozygous (P = 0.0003) Optn E481G mice showed reduced responsiveness of CSF1R-positive myeloid cells compared to wild-type (WT) mice, indicating that CSF1R signaling is affected by the E481G mutation and supporting the idea that the E478G mutation functions dominantly negatively (Figure 2C). In contrast to the E478G mutation, myeloid cells derived from newly established Optn E50K SNV-induced mice formed significantly more colonies (Figure 2D). The homozygous model formed more colonies compared to the WT (P = 0.0014) and heterozygous (P = 0.0022) models. The E50K mutation causes glaucoma in human and dominant genetic models, but only the homozygous model enhanced colony formation. The mechanism by which the heterozygous model did not enhance colony formation and the homozygous model responded in the opposite direction to the ALS model remains unclear. Overall, bone marrow nucleated cells derived from ALS mutant Optn mice formed fewer colonies in a colony formation assay using CSF1.

[0063] Example 3 Induction of periodontal disease and leukoencephalopathy by OPTN mutations To investigate the involvement of OPTN in osteoclasts of periodontal tissue, immunohistochemical analysis of OPTN and tartolate-resistant acid phosphatase (TRAP), a marker for osteoclasts, was performed. In periodontal tissue, TRAP-positive osteoclasts were also positive for OPTN (Figure 3A). This finding indicates that osteoclasts express OPTN and suggests that the formation of OPTN and TRAP-positive osteoclasts is related to bone resorption.

[0064] Therefore, we hypothesized that the mutant OPTN promotes osteoclast generation. To determine whether the E481G OPTN mutation promotes osteoclast generation in osteoclast precursor cells, we performed an in vitro osteoclast differentiation assay using bone marrow-derived macrophages (BMMs). BMMs from Optn KO and Optn E481G SNV-transformed mice responded to the nuclear factor κB ligand (RANKL) receptor activator more effectively than BMMs from WT mice, exhibiting increased TRAP + It differentiated into multinucleated cells (MNCs) (Figure 3B). This is due to TRAP. + This was confirmed by quantitatively measuring the number of MNCs (P = 0.0001 between WT and KO, P < 0.0001 between WT and + / E481G, and P < 0.0001 between WT and E481G / E481G) (Figure 3C). Therefore, using a common model of ligation-induced periodontal disease, we investigated the effects of OPTN mutations on periodontal disease in Optn KO and Optn E481 G mice in vivo. Optn KO and Optn E481 G mice showed significant alveolar bone resorption under ligation-induced inflammatory conditions compared to wild-type mice (Figure 3D). The degree of alveolar bone loss was significantly higher in Optn knockout and Optn E481 G mice compared to wild-type mice (P = 0.0026 between WT and KO, P = 0.0020 between WT and + / E481G, and P < 0.0001 between WT and E481G / E481G) (Figure 3E).

[0065] Taken together, these results indicate that the creation of Optn knockout and Optn E481G mice successfully mimicked the patient phenotype of severe alveolar bone loss. These results suggest that Optn E481G is pathogenic and causes severe periodontal disease. Overall, OPTN regulates the differentiation of bone resorbing cells, and Optn mutant mice tend to be more susceptible to periodontal disease.

[0066] Example 4 A case of OPTN E478G family with ALS, periodontal disease, and leukoencephalopathy. We identified a new case series of OPTN E478G mutation ALS families and investigated monocyte-induced complications. Monocyte-induced complications were found in periodontal disease and leukoencephalopathy, suggesting abnormalities in osteoclasts and microglia (Figure 4A). Patient I-2 was E478G heterozygous. Limb flexion was observed, but ALS had not developed. She developed dementia with white matter lesions at age 80 and died at age 91. Her head magnetic resonance imaging (MRI) showed diffuse brain atrophy, periventricular hyperintensity (Figure 4B), and corpus callosum thinning (Figure 4C), which are also characteristic of ALSP. Patient II-3 was a case of E478G homozygous ALS. She first presented with dysarthria at age 53, and subsequently developed limb flexion, dementia with white matter lesions, and periodontal disease. She died at age 67. Her MRI scans at 10 years (Figure 4D) and 11 years (Figure 4E) after the onset of symptoms showed progressively worsening cortical and subcortical atrophy, periventricular hyperintensity, and thinning of the corpus callosum (Figure 4F). Her periodontal disease was very severe, and all of her maxillary teeth were lost (g in Figure 4E). Patients II-1 and III-1 were E478G heterozygotes and had developed periodontal disease. Patient II-1 also developed ALS. Computed tomography (CT) of their facial bones showed resorbed alveolar bone compared to healthy individuals (h in Figure 4E) (i, j in Figure 4F). Unfortunately, patient III-2 was not clinically examined.

[0067] In another OPTN mutation, a woman with the OPTN K258Q(c.772A>C) heterozygous mutation developed periodontal disease in her 20s and required dentures in her late 30s. Subsequently, at age 77, she developed ALS, initially presenting with upper limb weakness. Pathogenicity was examined using an in silico prediction tool, and the results were as follows: Sorting Intolerant From Tolerant: Tolerant, PolyPhen-2: Potential Damage, Mutation Taster: Disease-Caused, Combined Annotation-Dependent Depletion: 17.22. Two East Asians were registered in gnomAD, but the allele frequency was not high, at 4.5 × 10⁶. -5This SNV was potentially pathogenic, but since only one case was identified, it was not conclusive. Nevertheless, the results suggested a link between periodontal disease and OPTN. Furthermore, a man with the OPTN V161M (c.481 G>A) heterozygous mutation developed ALS at age 34, with upper limb weakness as the initial symptom. At age 59, he was able to eat with assistance and did not use a gastrostomy tube. He wore a ventilator only at night or when he had difficulty breathing, and could speak during the day using a voice cannula. He had no cavities, but several teeth were missing, and it was clear that he had periodontal disease, although the timing of its onset was unknown.

[0068] A follow-up study was conducted on the oral cavity and tooth loss status of OPTN-ALS patients, but due to the indifference of patients, their families, and even the physicians themselves, valuable information could not be obtained.

[0069] Example 5 Microglial morphological changes due to OPTN mutations in the frontal lobe cortex Considering the possible interaction between OPTN and CSF1R, and MRI showing white matter lesions, we re-examined previously anatomically validated microglial changes in the brains of OPTN Q398* and E478G mutant ALS cases. In normal control brains, scattered Iba1 immunoreactive branched microglia were observed in the frontal cortex (Figure 5a). In contrast, in ALSP with CSF1R mutations, the number of microglia was significantly reduced (Figure 5b). In ALS with Q398* and E478G OPTN mutations, the number of microglia was reduced to a similar degree as in ALSP (Figures 5c, d). The morphology of microglia in the brains of both mutant OPTN-ALS differed from that of branched or reactive microglia, indicating a finely fragmented process similar to that of ALSP. In the frontal white matter, branched microglia were scattered even in the normal control group (Figure 5e), but in the degenerative areas of the brains of ALSP and both OPTN-ALS patients, microglia were either absent or extremely small (Figure 5 fh). Instead, both ALSP and OPTN-ALS patients commonly observed large, round, Iba1-positive cells indistinguishable from macrophages. These results suggest that OPTN Q398* and E478G mutations are associated with microglia- or macrophage-related leukoencephalopathy.

[0070] Example 6 Genome screening for leukoencephalopathy in CSF1R mutation-negative individuals To investigate the association between OPTN mutations and leukoencephalopathy, we performed genetic analysis of leukoencephalopathy cases. We used 49 cases from 107 CSF1R mutation-negative cases identified in previous studies (22). Using next-generation sequencing, we examined for the presence of OPTN mutations and identified one case with an OPTN E478 G heterozygous mutation. This suggests that OPTN mutations can cause leukoencephalopathy even in patients with normal CSF1R levels.

[0071] Example 7 Correction effect of OPTN Macrophages were isolated from the bone marrow of Optineurin (OPTN)-KO mice and suspended in alpha-modified Eagle Minimum Essential Medium containing mouse 100 ng / mL RANKL and 20 ng / mL M-CSF (BioLegend). The cell suspensions were divided into 2 x 10⁶ units. 5 The cells were added to 96-well plates to a cell / well ratio and cultured for 7 days. The culture medium was replaced with fresh medium containing mouse 100 ng / mL RANKL and 20 ng / mL M-CSF (BioLegend). 10 μL of adenovirus vector alone or adenovirus vector (AAV-OPTN) solution containing the normal OPTN gene (1.81 x 10⁶) was added. 12 Genetic copies ( / mL) were added to each well and allowed to infect for 3 days. Cells were stained using a TRAP staining kit (Wako) and observed.

[0072] The results obtained are shown in Figure 6. Macrophages isolated from the bone marrow of OPTN-KO mice excessively differentiated into osteoclasts and became enlarged, but this was corrected by infection with AAV-OPTN. Infection with AAV-OPTN is effective in correcting macrophage-derived cells collected from the bone marrow of OPTN-KO mice, and is considered useful in treating periodontal disease, leukoencephalopathy, Paget's disease of bone, and amyotrophic lateral sclerosis caused by mutations in the OPTN gene.

[0073] material and method animal Optn E481G (c.1442A>G) knock-in (KI) mice were established as follows: C57BL / 6 mice and platinum transcription activator-like nucleotide enzymes (TALENs) (34) were used, along with single-strand oligoDNA (ssODN). To introduce the ALS mutation, the 32nd base A of exon 12 was replaced with G. For genotyping, in addition to single nucleotide variants (SNVs), silent PstI cleavage sites were introduced. The TCA at bases 16-18 of exon 12 was replaced with AGT. The sequence of the right-hand TALEN was TGGAGGTGTACTGCTCA (SEQ ID NO: 3), the sequence of the left-hand TALEN was AGAGCAGCAAGAGAGA (SEQ ID NO: 4), and the spacer sequence was GATTTTCACGCTGAG (SEQ ID NO: 5).

[0074] Optn E50K (c.148G>A) KI mice were established as follows: Clustered, regularly spaced, short-interval palindromic repeats (CRISPR) / Cas9 using ssODN (35, 36). The specific single-stranded guide RNA sequence is TCAGCTGGTGGTTCTCACCAGG (SEQ ID NO: 6). The cleavage site was designed to cleave the base one level above the SNV. A glaucoma mutation was introduced by substituting G for A at base 159 of exon 2, and a silent sacI cleavage site was introduced for genotyping by substituting A for G at base 149. In both cases, purified protein and ssODN were microinjected into fertilized eggs. Germline transmission was confirmed by Zanger sequencing.

[0075] mass spectrometry Human FLAG-tagged OPTN was expressed in HEK293 T cells, immunoprecipitated with anti-FLAG antibody, and subjected to high-sensitivity direct nanoflow liquid chromatography / tandem mass spectrometry. Mass spectrometry was performed as previously reported (37).

[0076] Co-immunoprecipitation Cultured HeLa cells were transfected with an expression vector using lipofectamine 2000 (11668-019, Thermo Fisher Scientific, Waltham, MA, USA). 24 hours after transfection, cells were harvested in NP-40 buffer containing a protease inhibitor cocktail (05892970001, Roche, Basel, Switzerland). Protein A-Sepharose CL-4B (17-0780-01, GE HealthCare Technologies, Inc. Chicago, IL, USA) was added to the cell lysate and incubated at 4°C for 1 hour. After incubation, the cell lysate was incubated with Anti-FLAG M2 Affinity gel (A2220, MilliporeSigma, Burlington, MA, USA) to precipitate FLAG-tagged proteins. Finally, the proteins were separated on an SDS-PAGE gel and analyzed by immunoblotting using anti-CSF1R antibody (C-20) (sc-692, Santa Cruz, Dallas, TX, USA).

[0077] Colony formation assay Bone marrow cells (BMCs) were extracted from the femurs and tibias of E481G mice using S-clone® SF-03 medium (Iwai North America, Inc., Signal Hill, CA, USA). Nucleated bone marrow cells (BMNCs) were isolated from the BMCs using the density gradient medium Lymphoprep (Stem Cell Technology, Vancouver, Canada) and resuspended in S-clone® SF-03 medium. BMNCs were stained with PE-labeled anti-mouse CSF1R antibody (BioLegend, San Diego, CA, USA), and CSF1R-positive cells were collected using a FACSAria II cell sorter (Becton, Dickinson and Company, Franklin Lakes, NJ, USA). CSF1R-positive cells were resuspended in Methocult M3231 (Stem Cell Technologies, Vancouver, Canada), a methylcellulose semi-solid medium, and cultured in 35 mm culture dishes (5 × 10 cells per dish) in the presence of mouse M-CSF (R&D Systems, Minneapolis, USA) at a concentration of 10 ng / mL. 3 The substance was added to cells and cultured. After 10 days of culture, the number of colonies was counted using an inverted microscope. The experiment was performed with 5 individuals for each genotype group, and the average value of 3 to 6 experiments was adopted.

[0078] Analysis of osteoclasts BMM (Bone marrow metabolite) extracted from mouse bone marrow was mixed in 2 × 10¹⁶ α-modified Eagle Minimum Essential Medium containing 15% fetal bovine serum. 5Cells were seeded in 96-well plates at a cell / well density. To evaluate osteoclast generation, BMMs were cultured for 7 days with 100 ng / mL mouse RANKL (577102, BioLegend, San Diego, CA, USA) and 20 ng / mL mouse macrophage colony-stimulating factor (M-CSF, 576402, BioLegend, San Diego, CA, USA). Subsequently, the medium was replaced with fresh medium containing soluble RANKL (sRANKL) and M-CSF, and the cells were cultured for a further 3 days. The resulting cells were stained using a TRAP staining kit (TRAP / ALP stain kit, FUJIFILM Wako Chemicals, Tokyo, Japan). TRAP-positive cells with three or more nuclei were identified. + The cells were considered osteoclasts. Experiments were conducted with 6 individuals for each genotype group, and the average value of 3 to 5 experiments was adopted.

[0079] Immunohistochemistry of osteoclasts Antibodies for immunohistochemistry were obtained from Cayman (Ann Arbor, MI, USA) and Abcam (Cambridge, MA, USA). Paraffin-embedded mouse tissue was fragmented into 6 μm fragments. The resulting fragments were deparaffinized with xylene, rehydrated by sequentially decreasing the amount of ethanol, and washed with distilled water. The fragments were incubated with 3% hydrogen peroxide to block endogenous peroxidase. The resulting fragments were washed with Tris-buffered saline (TBS, pH 7.2) and then treated with 0.1% bovine serum albumin (MilliporeSigma, St. Louis, MO, USA) to prevent nonspecific binding. The primary antibody was diluted with DAKO antibody diluent (Agilent Technologies, Santa Clara, CA, USA) and incubated at room temperature for approximately 1 hour. After incubation with the primary antibody, the fragments were washed with TBS and incubated with ENVISION (Agilent Technologies, Santa Clara, CA, USA) in a humid chamber for 30 minutes. The antibody conjugates were visualized using 3,3'-diaminobenzidine (DAB) substrate, washed with distilled water, and pair-stained with hematoxylin. As a negative control, samples were incubated with normal rabbit IgG instead of the primary antibody.

[0080] Periodontal disease induction by ligation and micro-CT analysis Periodontitis was induced in 8-week-old male and female mice by ligating 5-0 silk thread (Ethicon) to the maxillary second molar for 7 days. The maxilla was scanned using the Skyscan 1176 system (Bruker). Three-dimensional reconstruction was performed using CTVOX software. Experiments were conducted with 6 individuals for each genotype group.

[0081] Human specimens Experiments were conducted using postmortem specimens from two ALSP cases with confirmed CSF1R mutations neuropathologically, two ALS patients (3, 38-40) with Q398* and E478G OPTN mutations, and two normal control individuals. Written informed consent was obtained from all subjects. Procedures involving human specimens followed the ethical guidelines of Wakayama Medical University and the Declaration of Helsinki.

[0082] Brains were fixed in 10% neutral buffered formalin for several weeks and sliced. The frontal lobe was excised, embedded in paraffin, and cut into 6 μm thick sections. For immunohistochemical analysis, these sections were dewaxed and antigen recovery was performed by heating / autoclaving (120°C for 20 minutes in 10 mM sodium citrate buffer (pH 6.0)). The resulting fragments were then incubated overnight at 4°C with rabbit polyclonal antibody against Iba1 (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) at a dilution of 1:200. 3,3'-diaminobenzidine tetrahydrochloride was used as a chromogenic agent, and the bound antibody was detected using N-Histofine Simple Stain MAX PO (Nichirei Biosciences, Tokyo, Japan).

[0083] Genome screening DNA from CSF1R mutation-negative leukoplakia patients was provided by Shinshu University and screened for OPTN mutations using the Ion PGM® system (Thermo Fisher Scientific, Waltham, MA, USA). The identified mutations were also confirmed by Sanger sequencing. This study was approved by the Ethics Committee of Hiroshima University.

[0084] statistical analysis Data analysis was performed using JMP Pro version 16.0.0 (SAS Institute Inc., Carey, NC, USA). Differences between two groups were statistically analyzed using the Student t-test. Differences between two or more groups were statistically analyzed using one-way ANOVA, followed by the Tukey-Kramer test. A p-value of 0.05 or less was considered statistically significant. [Industrial applicability]

[0085] According to the present invention, it is possible to provide a technology for preventing or treating osteoclastogenic diseases and leukoencephalopathy.

[0086] References 17. Ryan TA, Tumbarello DA. Optineurin: A Coordinator of Membrane-Associated Cargo Trafficking and Autophagy. Front Immunol. 2018;9:1024. 18. Ying H, Yue BY. Optineurin: The autophagy connection. Exp Eye Res. 2016;144:73-80. 19. Hu B, Duan S, Wang Z, Li X, Zhou Y, Zhang X, et al. Insights Into the Role of CSF1R in the Central Nervous System and Neurological Disorders. Front Aging Neurosci. 2021;13:789834. 20. Miura T, Mezaki N, Konno T, Iwasaki A, Hara N, Miura M, et al. Identification and functional characterization of novel mutations including frameshift mutation in exon 4 of CSF1R in patients with adult-onset leukoencephalopathy with axonal spheroids and pigmented glia. J Neurol. 2018;265(10):2415-24. 21. Kurashige T, Kuramochi M, Ohsawa R, Yamashita Y, Shioi G, Morino H, et al. Optineurin defects cause TDP43-pathology with autophagic vacuolar formation. Neurobiol Dis. 2021;148:105215. 22. Kondo Y, Matsushima A, Nagasaki S, Nakamura K, Sekijima Y, Yoshida K. Factors predictive of the presence of a CSF1R mutation in patients with leukoencephalopathy. Eur J Neurol. 2020;27(2):369-75. 23. Freischmidt A, Wieland T, Richter B, Ruf W, Schaeffer V, Muller K, et al. Haploinsufficiency of TBK1 causes familial ALS and fronto-temporal dementia. Nat Neurosci. 2015;18(5):631-6. 24. Fingert JH, Darbro BW, Qian Q, Van Rheeden R, Miller K, Riker M, et al. TBK1 and flanking genes in human retina. Ophthalmic Genet. 2014;35(1):35-40. 25. Ritch R, Darbro B, Menon G, Khanna CL, Solivan-Timpe F, Roos BR, et al. TBK1 gene duplication and normal-tension glaucoma. JAMA Ophthalmol. 2014;132(5):544-8. 26. Morton S, Hesson L, Peggie M, Cohen P. Enhanced binding of TBK1 by an optineurin mutant that causes a familial form of primary open angle glaucoma. FEBS Lett. 2008;582(6):997-1002. 27. Liu Z, Li H, Hong C, Chen M, Yue T, Chen C, et al. ALS-Associated E478G Mutation in Human OPTN (Optineurin) Promotes Inflammation and Induces Neuronal Cell Death. Front Immunol. 2018;9:2647. 28. Hu WB, Wang X, Pang ZL, Duan R, Hong CG, Liu ZZ. Neutralizing peripheral circulating IL1β slows the progression of ALS in a lentivirus-infected OPTN(E478G) mouse model. Animal Model Exp Med. 2023;6(1):18-25. 29. Spiller KJ, Restrepo CR, Khan T, Dominique MA, Fang TC, Canter RG, et al. Microglia-mediated recovery from ALS-relevant motor neuron degeneration in a mouse model of TDP-43 proteinopathy. Nat Neurosci. 2018;21(3):329-40. 30. Martinez‐Muriana A, Mancuso R, Francos-Quijorna I, Olmos‐Alonso A, Osta R, Perry VH, et al. CSF1R blockade slows the progression of amyotrophic lateral sclerosis by reducing microgliosis and invasion of macrophages into peripheral nerves. Sci Rep. 2016;6:25663. 31. Xue P, Hu X, Chang E, Wang L, Chen M, Wu TH, et al. Deficiency of optineurin enhances osteoclast differentiation by attenuating the NRF2-mediated antioxidant response. Exp Mol Med. 2021;53(4):667-80. 32. Mizuno N, Iwata T, Ohsawa R, Ouhara K, Matsuda S, Kajiya M, et al. Optineurin regulates osteoblastogenesis through STAT1. Biochem Biophys Res Commun. 2020;525(4):889-94. 33. Hu X, Foster BL, Zhao B, Tseng HC, Wu YC, Ko CC. Optineurin regulates osteoblast function in an age-dependent fashion in a mouse model of Paget's disease of bone. Bone. 2023;177:116929. 34. Sakuma T, Ochiai H, Kaneko T, Mashimo T, Tokumasu D, Sakane Y, et al. Repeating pattern of non-RVD variations in DNA-binding modules enhances TALEN activity. Sci Rep. 2013;3:3379. 35. Chang N, Sun C, Gao L, Zhu D, Xu X, Zhu X, et al. Genome editing with RNA-guided Cas9 nuclease in zebrafish embryos. Cell Res. 2013;23(4):465-72. 36. Ran FA, Hsu PD, Wright J, Agarwala V, Scott DA, Zhang F. Genome engineering using the CRISPR-Cas9 system. Nat Protoc. 2013;8(11):2281-308. 37. Natsume T, Yamauchi Y, Nakayama H, Shinkawa T, Yanagida M, Takahashi N, et al. A direct nanoflow liquid chromatography-tandem mass spectrometry system for interaction proteomics. Anal Chem. 2002;74(18):4725-33. 38. Ito H, Nakamura M, Komure O, Ayaki T, Wate R, Maruyama H, et al. Clinicopathologic study on an ALS family with a heterozygous E478G optineurin mutation. Acta Neuropathol. 2011;122(2):223-9. 39. Kamada M, Izumi Y, Ayaki T, Nakamura M, Kagawa S, Kudo E, et al. Clinicopathologic features of autosomal recessive amyotrophic lateral sclerosis associated with optineurin mutation. Neuropathology. 2014;34(1):64-70. 40. Ayaki T, Ito H, Komure O, Kamada M, Nakamura M, Wate R, et al. Multiple Proteinopathies in Familial ALS Cases With Optineurin Mutations. J Neuropathol Exp Neurol. 2018;77(2):128-38.

Claims

1. A pharmaceutical composition containing an optineurin (OPTN)-related substance as an active ingredient for the prevention or treatment of osteoclastogenic diseases and leukoencephalopathy.

2. OPTN-related substances, (a1) OPTN consisting of the amino acid sequence shown in Sequence ID No. 1; (a2) A mutant OPTN having OPTN activity equivalent to that of OPTN, consisting of an amino acid sequence in which one or more amino acid residues are substituted, deleted, inserted and / or added from the amino acid sequence shown in Sequence ID No. 1; (a3) A mutant OPTN having an amino acid sequence that is 80% or more identical to the amino acid sequence shown in Sequence ID No. 1, and having OPTN activity equivalent to that of OPTN; (a4) OPTN encoded by the nucleotide sequence shown in Sequence ID No. 2; (a5) A mutant OPTN having OPTN activity equivalent to that of OPTN, encoded by a polynucleotide that hybridizes under stringent conditions with a polynucleotide consisting of a base sequence complementary to the base sequence shown in Sequence ID No. 2; (a6) A mutant OPTN encoded by a polynucleotide having 80% identity with the base sequence shown in Sequence ID No. 2 and having OPTN activity equivalent to that of OPTN; and (a7) A mutant OPTN having OPTN activity equivalent to that of OPTN, which is encoded by a nucleotide sequence in which one or more nucleotides are deleted, substituted or added in the nucleotide sequence shown in Sequence ID No.

2. A pharmaceutical composition according to claim 1, selected from the group consisting of the following.

3. The pharmaceutical composition according to claim 2, wherein the osteoclastogenic disease is selected from the group consisting of osteoporosis, rheumatoid arthritis, bone metastases of cancer, periodontal disease, Paget's disease of bone, and osteopetrosis.

4. OPTN-related substances, (b1) A polynucleotide encoding the polypeptide according to claim 2; The pharmaceutical composition according to claim 1.

5. OPTN-related substances, (b2) OPTN polynucleotide consisting of the base sequence shown in Sequence ID No. 2; (b3) A polynucleotide that hybridizes under stringent conditions with a polynucleotide having a base sequence complementary to the base sequence shown in Sequence ID No. 2, and which encodes a polypeptide having activity equivalent to that of OPTN; (b4) A polynucleotide having 80% identity with the base sequence shown in Sequence ID No. 2, and encoding a polypeptide having OPTN activity equivalent to that of OPTN; and (b5) A polynucleotide encoding a polypeptide having OPTN activity equivalent to that of OPTN, comprising a nucleotide sequence in which one or more bases are deleted, substituted, or added in the nucleotide sequence shown in Sequence ID No. 2; The pharmaceutical composition according to claim 1, wherein the polynucleotide is selected from the group consisting of the following.

6. The pharmaceutical composition according to claim 4 or 5, wherein the osteoclastogenic disease is selected from the group consisting of osteoporosis, rheumatoid arthritis, bone metastases of cancer, periodontal disease, Paget's disease of bone, and osteopetrosis.

7. A vector comprising the polynucleotide according to claim 4 or 5.

8. (a) OPTN polynucleotide consisting of the base sequence shown in Sequence ID No. 2, (b) A mutant OPTN polynucleotide containing a nucleotide sequence in which one or more nucleotides are deleted, substituted, or added in the nucleotide sequence shown in Sequence ID No. 2, or (c) Mutant OPTN polynucleotides that hybridize under stringent conditions with a nucleotide sequence complementary to the nucleotide sequence shown in Sequence ID No. 2, It is characterized by using cells into which one of the following has been introduced. A method for screening OPTN agonists by confirming that the expression of the polynucleotide in the cells is enhanced.

9. (1) Prepare candidate compounds, （２） (a) OPTN polynucleotide consisting of the base sequence shown in Sequence ID No. 2, (b) A mutant OPTN polynucleotide containing a nucleotide sequence in which one or more nucleotides are deleted, substituted, or added in the nucleotide sequence shown in Sequence ID No. 2, or (c) Mutant OPTN polynucleotides that hybridize under stringent conditions with a nucleotide sequence complementary to the nucleotide sequence shown in Sequence ID No. 2, Prepare cells into which one of the following has been introduced: (3) Contact the cells with the candidate compound, (4) A method for screening OPTN agonists according to claim 8, comprising determining whether the candidate compound enhances the expression of the gene in the cell.

10. A method for determining the severity of osteoclastogenic diseases and leukoencephalopathy, (a10) A step of measuring the amount of OPTN (amount of test biomarker) in the macrophages of the subject, (b10) A step of comparing the amount of the test biomarker with the amount of OPTN in macrophages of healthy individuals (control biomarker amount), and (c10) A method for determining that a subject is at risk of developing severe osteoclastopathy and leukoencephalopathy when the amount of the tested biomarker is lower than the amount of the control biomarker.

11. A macrophage OPTN biomarker that can be used to assess the severity of osteoclastogenic diseases and leukoencephalopathy.

12. A biomarker detection kit according to claim 11, comprising a primer set for amplifying OPTN cDNA, a probe that specifically hybridizes to OPTN mRNA, or a substance that specifically binds to OPTN.

13. A method for measuring the activity of OPTN in candidate OPTN substances, (1) Prepare candidate OPTN materials, (2) Convert the OPTN candidate substance into a form that can be introduced into cells, and create a converted OPTN candidate substance. (3) Obtain macrophages from bone marrow derived from Optn knockout mice, (4) The obtained macrophages are then converted from the OPTN candidate substance, (5) Measure the suppression of differentiation of introduced macrophages into osteoclasts. method.

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