Compositions and methods for treating or preventing TAR DNA binding protein 43 related diseases

Abstract

A composition for treating or preventing TAR DNA binding protein 43 related diseases, comprising a promoting substance that increases the intracellular amount of a protein having ubiquitination activity.

Description

Cross Reference to Related Applications

[0001] This application is based on and claims the benefit of priority of U.S. Application No. 63 / 493,462, filed March 31, 2023, and U.S. Application No. 63 / 597,559, filed November 9, 2023. The entire contents of these applications are incorporated herein by reference.

[0002] Reference to a Sequence Listing In accordance with WIPO Standard ST.26, the specification refers to a Sequence Listing electronically submitted in the name of “541677WO_ST26.xml.” The.xml file was generated on March 26, 2024, and is 49,152 bytes in size. The entire contents of the Sequence Listing are incorporated herein by reference. BACKGROUND TECHNICAL FIELD

[0003] The present disclosure relates to compositions, methods, etc. for treating or preventing TAR DNA binding protein 43-related diseases.

[0004] BACKGROUND Patent Literature 1 discloses a compound for improving messenger RNA (mRNA) splicing. The same document takes familial autonomic neuropathy as an example of a progressive neurodegenerative disease and discloses a treatment method for the disease.

[0005] Non-Patent Literature 1 focuses on Charcot-Marie-Tooth disease type 2P and describes the inhibition of cell death associated with accumulation of misfolded proteins. The same document discloses that LRSAM1 inhibits accumulation of misfolded luciferase and reduces cytotoxicity.

[0006] Patent Literature 1: US 2018 / 0118748 A1 Non-Patent Literature 1: Mishra R et al., Int J Biochem Cell Biol., 2020; 120: 105697 The entire contents of these publications are incorporated herein by reference. SUMMARY

[0007] One embodiment of the present disclosure is a composition for treating or preventing a TAR DNA binding protein 43-related disease, comprising a promoting substance that increases the intracellular amount of a protein having ubiquitination activity.

[0008] Further, one embodiment of the present disclosure is a composition for improving clearance of TAR DNA binding protein 43 protein, comprising a promoting substance that increases an intracellular amount of a protein having ubiquitination activity.

[0009] Further, one embodiment of the present disclosure is a method for treating or preventing a TAR DNA binding protein 43-related disease, comprising a step of administering an effective amount of a promoting substance to a subject in need thereof, wherein the promoting substance is a substance that increases an intracellular amount of a protein having ubiquitination activity.

[0010] Further, one embodiment of the present disclosure is a use of a promoting substance that increases an intracellular amount of a protein having ubiquitination activity in the manufacture of a composition for treating or preventing a TAR DNA binding protein 43-related disease.

[0011] Further, one embodiment of the present disclosure is a method for inhibiting cell damage, comprising inhibiting occurrence or progression of cell damage in a neuron by contacting the neuron with a promoting substance that increases an intracellular amount of a protein having ubiquitination activity.

[0012] Further, one embodiment of the present disclosure is a method for improving protein clearance, comprising improving clearance of TAR DNA binding protein 43 protein in a neuron by contacting the neuron with a promoting substance that increases an intracellular amount of a protein having ubiquitination activity. SUMMARY A more complete understanding of the present application and the many attendant advantages thereof will be readily understood by referring to the following detailed description when considered in connection with the accompanying drawings, wherein: Figure 1 is a graph showing the degree of cell damage in motor neurons derived from healthy individuals and motor neurons derived from ALS patients using neurite length as an index.

[0014] Figure 2 is a graph showing the cytoplasm / nucleus abundance ratio of TDP-43 protein in motor neurons derived from healthy individuals and motor neurons derived from ALS patients.

[0015] Figure 3 is a graph showing the amount of phosphorylated TDP-43 protein present in the cytoplasm in motor neurons derived from healthy individuals and motor neurons derived from ALS patients.

[0016] Figure 4 is a graph showing the effect of the presence or absence of a promoting substance on neurite length of motor neurons derived from healthy individuals.

[0017] Figure 5is a graph showing the effect of the presence or absence of a promoting substance on the length of neurites of motor neurons derived from ALS patients.

[0018] Figure 6A is a graph showing the cytoplasmic / nuclear abundance ratio of TDP-43 protein due to the presence or absence of a promoting substance in motor neurons derived from healthy individuals.

[0019] Figure 6B is a graph showing the cytoplasmic / nuclear abundance ratio of TDP-43 protein due to the presence or absence of a promoting substance in motor neurons derived from ALS patients.

[0020] Figure 7A is a graph showing the amount of phosphorylated TDP-43 protein present in the cytoplasm of motor neurons derived from healthy individuals.

[0021] Figure 7B is a graph showing the amount of phosphorylated TDP-43 protein present in the cytoplasm of motor neurons derived from ALS patients.

[0022] Figure 8 is a graph showing the effect of splicing in motor neurons derived from healthy individuals and motor neurons derived from ALS patients using the ratio of mRNA containing cryptic exons (CE) to full-length mRNA (FL) of STMN2 as an indicator.

[0023] Figure 9 is a graph showing the effect of the presence or absence of a promoting substance on STMN2 splicing in motor neurons derived from healthy individuals.

[0024] Figure 10 is a graph showing the effect of the presence or absence of a promoting substance on STMN2 splicing in motor neurons derived from ALS patients.

[0025] Figure 11 is a graph showing the effect of the presence or absence of a promoting substance on the amount of CTF35 (C-terminal fragment of TDP-43 protein of about 35 kDa) in motor neurons derived from healthy individuals and motor neurons derived from ALS patients. The ratio of the amount of CTF35 to the total amount of full-length and fragmented TDP-43 protein is used as an indicator.

[0026] Figure 12 is a graph showing the effect of the presence or absence of a promoting substance on the amount of CTF25 (C-terminal fragment of TDP-43 protein of about 25 kDa) in motor neurons derived from healthy individuals and motor neurons derived from ALS patients. The ratio of the amount of CTF25 to the total amount of full-length and fragmented TDP-43 protein is used as an indicator.

[0027] Figure 13 Immunohistofluorescence staining images of TDP-43 protein (A), LRSAM1 (B), MAP2 (C), and DAPI (D) in brain (motor cortex) sections from sporadic ALS patients are shown. MAP2 was used to label the nerve cytoplasm and DAPI to label the nucleus. Figure 13 The arrows in A and 13B indicate the co-localization of TDP-43 and LRSAM1 in the cytoplasm.

[0028] Figure 14 A and 14B show the degree of co-localization of TDP-43 and LRSAM1 in the nerve nucleus and cytoplasm, respectively, using the Pearson correlation coefficient. Figure 14 A shows the distribution of correlation coefficients and cell counts in the histogram. Figure 14 B is a graph comparing the average correlation coefficients of the nucleus and cytoplasm.

[0029] DETAILED DESCRIPTION OF EMBODIMENTS Embodiments will now be described with reference to the accompanying drawings, in which like reference numerals refer to like elements throughout.

[0030] Hereinafter, the present disclosure is described based on embodiments of the present disclosure.

[0031] The disclosures of the patent documents and non-patent documents cited in the present specification are incorporated herein by reference in their entirety.

[0032] GenBank accession numbers and related sequence information and other data available through databases are incorporated herein by reference in their entirety.

[0033] The sequence listing submitted electronically in the present application is incorporated herein by reference in its entirety.

[0034] In addition, among the mRNA base sequences disclosed in the sequence listing of the present application and the accession numbers described in the present specification, uracil (U) is represented as thymine (T). However, the actual mRNA base sequence is applied as "U" together with the "T" reading.

[0035] In one embodiment, the present disclosure relates to a composition. In one embodiment, the composition can be a pharmaceutical composition or a non-pharmaceutical composition, such as an experimental reagent.

[0036] 43 kDa TAR DNA binding protein is an RNA binding protein known as TDP-43, and is normally localized to the nucleus. When abnormal TDP-43 protein (e.g., protein aggregates or fragmented protein) is produced, it can contribute to the development of various diseases. TDP-43-related diseases often exhibit symptoms such as motor dysfunction or cognitive impairment, which can greatly affect quality of life (QOL). However, there is no established effective treatment method leading to the fundamental cure of TDP-43-related diseases.

[0037] In one embodiment, the composition of the present disclosure is used for treating or preventing TAR DNA binding protein 43 (TDP-43)-related diseases. In other words, the composition of one embodiment is a composition for treating or preventing TDP-43-related diseases. When used for this purpose, the composition of the present disclosure is preferably used as a pharmaceutical composition. Details of the composition and its application will be described later.

[0038] In the present specification, "treatment" includes, for example, inhibition of progression, alleviation, reduction, eradication, etc. of a disease, a disorder, or one or more symptoms associated therewith.

[0039] In the present specification, "prevention" includes, for example, prevention or delay of onset of a disease, prevention or delay of recurrence of a disease, etc. The composition of the present disclosure can be used for one or more of therapeutic and preventive purposes.

[0040] In the following description, for the sake of convenience, a human or a human cell as a type of mammal will be described as an example of application of the present disclosure, unless otherwise specified. However, it is similarly applicable to other animal species other than humans or cells thereof, including rodents such as mice and rats, primates such as monkeys, and mammals such as rabbits, as long as the effects of the present disclosure are achieved.

[0041] In the following description, when explaining an amino acid point mutation in any protein, it is expressed in the order of "one-letter abbreviation of the amino acid before substitution, amino acid sequence number from the N-terminus, one-letter abbreviation of the amino acid after substitution". For example, "G294A" means that the 294th glycine from the N-terminus of the amino acid sequence of the given protein has been substituted with alanine.

[0042] In the following description, unless otherwise specified, "nucleic acid" means a polymer in which two or more nucleosides are connected together by an internucleoside bond, regardless of its length or backbone structure. The nucleic acid bases, sugars, and internucleoside bonds forming the polymer can each independently have a natural structure or a non-natural structure in which certain atoms, functional groups, or ring structures, etc. have been added, substituted, or deleted from the natural structure.

[0043] In addition, the nucleic acid in the present specification can exist in the form of a pharmaceutically acceptable salt or ion as long as the effect of the present disclosure is achieved. Examples of the counter ion in the pharmaceutically acceptable salt include cations. Examples of such cations include hydrogen ions and inorganic ions such as metal ions. Examples of the metal ions include alkali metal ions, for example, sodium ions and potassium ions.

[0044] TDP-43, an RNA-binding protein, is involved in the regulation of transcription, splicing, and translation in cells. TDP-43 is a protein encoded by the TDP-43 gene. Human wild-type TDP-43 protein is a protein consisting of 414 amino acids of full length, and is a nuclear protein mainly located in the nucleus. As an example, the mRNA base sequence of human wild-type TDP-43 is shown in SEQ ID NO: 1 (GenBank Accession No: NM_007375.4). In addition, the amino acid sequence of human wild-type TDP-43 protein is shown in SEQ ID NO: 2 (GenBank Accession No: NP_031401.1).

[0045] In TDP-43-related diseases, the production of abnormal TDP-43 proteins is considered to be one of the causes. The abnormal proteins generally become more likely to migrate into the cytoplasm, form protein aggregates, or have changes in the amount and / or type of post-translational modifications such as phosphorylation. As such, the abnormal TDP-43 proteins generally have different properties from the wild-type proteins.

[0046] Examples of the abnormal TDP-43 proteins that can contribute to the development and progression of diseases include, but are not limited to, an N-terminal fragment of human wild-type TDP-43 protein (SEQ ID NO: 3; see Shenouda et al., Front Neurosci., 2022; 16: 868556), a C-terminal fragment protein consisting of amino acid residues 209 to 414 from the N-terminus of human wild-type TDP-43 protein (SEQ ID NO: 4), a C-terminal fragment protein consisting of amino acid residues 90 to 414 from the N-terminus of human wild-type TDP-43 protein (SEQ ID NO: 15), a C-terminal fragment protein consisting of amino acid residues 169 to 414 from the N-terminus of human wild-type TDP-43 protein (SEQ ID NO: 16), a C-terminal fragment protein consisting of amino acid residues 174 to 414 from the N-terminus of human wild-type TDP-43 protein (SEQ ID NO: 17), an amino acid mutant in wild-type TDP-43 protein (see SEQ ID NO: 2), and the like. Examples of the amino acid variants of TDP-43 protein include proteins having point mutations, for example, G294A, G298S, A315T, M337V, Q343R, A382T, and the like, or combinations thereof.

[0047] The TDP-43-related disease in the present disclosure includes a disease that can occur due to abnormal intracellular localization of TDP-43 protein. Examples of the TDP-43-related disease include one or more neuromuscular diseases selected from the group consisting of a neurodegenerative disease and a muscle disease (for example, a disease described in the literature, for example, Riku et al., Int J Mol Sci., 2022 12; 23 (24): 15755, Hu et al., Neurobiol Dis., 2022; 170: 105749, Bede et al., Rev Neurol (Paris), 2022; 178 (3): 196-205., Walker et al., J Neurosci., 2014; 34 (19): 6448-58).

[0048] Specifically, examples of the neurodegenerative disease include Parkinson's disease, amyotrophic lateral sclerosis (ALS), spinal muscular atrophy (SMA), primary lateral sclerosis (PLS), multiple system atrophy (MSA), progressive supranuclear palsy (PSP), Perry syndrome, Alexander disease, Alzheimer's disease, frontotemporal lobar degeneration (FTLD), limbic-predominant age-related TDP-43 encephalopathy (LATE), Huntington's disease, and the like.

[0049] Examples of the muscle disease include inclusion body myositis.

[0050] By targeting these diseases, the TDP-43-related disease can be effectively treated or prevented by addressing a specific pathological condition (for example, abnormal intracellular localization of TDP-43 protein or removal of abnormalities). That is, in one embodiment, the above-mentioned composition can be a composition for improving TDP-43 protein removal.

[0051] From the viewpoint of further improving the effectiveness of disease treatment or prevention, the TDP-43-related disease is preferably a neurodegenerative disease selected from the group consisting of ALS, FTLD, and LATE, and more preferably at least ALS.

[0052] When the above composition is used for treating or preventing a TDP-43-related disease, the applicable subject is preferably a subject who has developed or can develop a TDP-43-related disease, and the subject can or can not have a specific mutation in a specific gene. That is, the above composition can be used for a subject who does not have a mutation in a specific gene (e.g., a normal homozygote). In addition, the above composition can also be used for a subject who has one or more mutations selected from a specific gene. When the subject has a gene mutation, the gene mutation can each independently be a heterozygous mutation or a homozygous mutation. The subject is preferably a human subject (human living body) or a human-derived cell. The presence or absence of these mutations can be determined using known methods such as PCR.

[0053] Taking ALS, which is a TDP-43-related disease, as an example, the human gene mutation includes one or more of a mutation in the C9orf72 gene, the SOD1 gene, the TBK1 gene, the TARDBP gene, the FUS gene, the NEK1 gene, etc., but is not limited thereto. In one embodiment, the above subject has, for example, a homozygous or heterozygous mutation in the TARDBP gene, but does not have a mutation in the FUS gene and / or the SOD1 gene.

[0054] Examples of the above human gene mutation include one or more selected from the group consisting of an abnormal extension of a 6-base repeat sequence in intron 1 of the C9orf72 gene, a mutation in the SOD1 gene that causes an A4V amino acid point mutation, a mutation in the TBK1 gene that causes a T4A amino acid point mutation, a mutation in the TADDBP gene that causes an A382T amino acid point mutation, a mutation in the FUS gene that causes a P525L amino acid point mutation, a mutation in the NEK1 gene that causes a R261H amino acid point mutation, etc.

[0055] The base sequence of the TARDBP gene can differ between individuals. In the present embodiment, the mRNA base sequence of TDP-43 can be a base sequence that is 80% or higher, 85% or higher, 90% or higher, 95% or higher, 98% or higher, or 100% identical to the base sequence described in SEQ ID NO: 1, as long as it encodes a protein having an equivalent function to the wild-type TDP-43 protein. Examples of the cause of base sequence differences include, but are not limited to, a degenerate codon, etc.

[0056] In addition, the amino acid sequence of the TDP-43 protein can be an amino acid sequence that is 80% or higher, 85% or higher, 90% or higher, 95% or higher, 98% or higher, or 100% identical to the amino acid sequence described in SEQ ID NO: 2, as long as it encodes a protein having an equivalent function to the wild-type TDP-43 protein.

[0057] The search for regions having sequence identity, complementarity, or homology and the calculation of the percentage of complementarity in the present specification can be performed, for example, using publicly known software or methods, such as the BLAST program or the Genetyx software.

[0058] In the present specification, "complementary" or "complementarity" refers to the ability of two nucleic acid bases to form a base pair. For example, adenine is complementary to thymine or uracil, and cytosine is complementary to guanine.

[0059] In one embodiment, the composition of the present disclosure includes a facilitating substance. The facilitating substance has one or more characteristics that increase the amount and / or activity of a predetermined translation product in various cell-applicable targets such as cells, specimens, and living bodies. In the following explanation, the substance is also simply referred to as "facilitating substance".

[0060] As one embodiment, the facilitating substance directly or indirectly facilitates gene expression from a predetermined nucleic acid. Thus, at least the intracellular amount of a predetermined translation product can be increased. In the present specification, "gene expression" or "expression of a gene" includes not only the synthesis (generation) of a translation product based on the information of the base sequence of a predetermined nucleic acid, but also the synthesis (generation) of a transcription product.

[0061] Specifically, the facilitating substance according to one embodiment facilitates at least one selected from the production of a transcription product of a nucleic acid encoding a predetermined protein, the production of a translation product, the function of a transcription product, and the function of a translation product in a cell. Specific examples of the nucleic acid to be facilitated include polymers of deoxyribonucleotides such as endogenous DNA (such as genomic DNA), exogenously introduced DNA, and synthetic DNA (such as complementary DNA); polymers of ribonucleotides such as RNA; and the like.

[0062] Examples of such facilitating substances include, but are not limited to, various organic compounds (including so-called low molecular weight compounds), nucleic acids, proteins, lipids, and combinations thereof. The facilitating substance in the present disclosure can be the translation product itself to be increased. The above-mentioned facilitating substances can each be used independently, or two or more facilitating substances can be used in combination. Preferred embodiments of the facilitating substance will be described later.

[0063] Examples of the transcription product preferably include any RNA synthesized using DNA as a template, with or without RNA processing. Specific examples of the RNA include various polymers of ribonucleotides, such as mRNA precursors, mature mRNAs, and non-coding RNAs such as microRNAs (miRNAs). In one embodiment, the above-mentioned RNA is preferably an mRNA precursor and / or a mature mRNA. By increasing the expression level of a transcription product such as mRNA, the amount of a translation template is increased. Thus, it can contribute to increasing the expression level of a target protein.

[0064] Examples of the translation products include proteins and the like produced by translation from any mRNA, regardless of the presence or absence of post-translational modification.

[0065] These transcription products and translation products can independently be, for example, wild-type products transcribed and translated from the predetermined DNA, or sequence variants such as various splicing variants and base substitution products (including SNPs), and non-wild-type proteins translated from these variants or products.

[0066] The various types of DNA, transcription products, and translation products described above can be independently subjected to one or more well-known extraction or purification steps to be isolated, as needed.

[0067] It is preferable that the transcription products be capable of producing wild-type proteins or proteins having equivalent functions. Furthermore, it is preferable that the translation products be wild-type proteins or proteins having equivalent functions to wild-type proteins. By producing these products, the inherent functions of the cells can be easily maintained or standardized, and the occurrence or progression of cell damage can be easily further reduced. Thus, it can more effectively contribute to the treatment or prevention of diseases.

[0068] To achieve the above-described preferred modes, for example, as shown in the examples described later, methods such as introduction of an exogenous nucleic acid encoding a target protein or a method of facilitating splicing into a mature mRNA capable of translating a target protein can be used, but are not limited to these methods.

[0069] To determine whether the generated translation products have functions equivalent to wild-type, for example, the enzyme activity measured using a well-known method can be compared with the activity of wild-type translation products to make the determination.

[0070] In the present specification, "facilitating" means at least one of: an increase in the abundance (e.g., expression level) of a transcription product and / or a translation product from a predetermined gene in the presence of any substance; and an increase in the function (e.g., activity) of the translation product. For example, in the case where the expression level of a transcription product or a translation product is greater when a substance to be evaluated is in contact with or exposed to a predetermined applicable target than in the absence of the substance to be evaluated, it can be determined that it has been facilitated. In addition to or instead of this, it can also be determined that it has been facilitated by an increase in the activity of the protein itself or an enzyme within the cell.

[0071] As a criterion for determining whether it has been promoted, for example, it can be determined based on the magnitude of a measurement value obtained using any measurement method, or it can be determined based on the magnitude of an arithmetic mean, a geometric mean, or a median value calculated from the measurement values or a ratio thereof, or it can be determined by having a statistically significant difference. In the case of evaluation based on the ratio of measurement values, for example, when the ratio (R2 / R1) of the measurement value (R2) in the experimental group to be determined to the measurement value as a reference or reference value (R1) is, for example, 1.05 times or more, 1.10 times or more, 1.30 times or more, 1.50 times or more, or 2.00 times or more, it can be determined that it has been promoted.

[0072] Examples of promoting the production of a transcription product include one or more of promoting transcription from DNA to mRNA precursor, inhibiting degradation of mRNA precursor, inhibiting degradation of mature mRNA, or controlling or activating RNA processing to form mature mRNA from mRNA precursor, and the like.

[0073] Examples of promoting the production of a translation product include one or more of promoting translation from mature mRNA, inhibiting degradation of a protein produced by translation, and the like.

[0074] To measure the expression level of a transcription product, for example, various measurement methods such as PCR, microarray, and RNA sequencing can be used using a measurement sample such as a cultured cell, a living body, or a sample collected from a living body. These measurement methods are preferably performed in a quantifiable manner. When necessary, a process of extracting a transcription product from the above measurement sample can be performed, or a process of synthesizing complementary DNA (cDNA) by a reverse transcription reaction using a transcription product as a template can be performed, and these products can be used for the above measurement.

[0075] For the measurement of the expression level or activity of a translation product, for example, various measurement methods such as ELISA, Western blotting, flow cytometry, immunostaining, mass spectrometry, intracellular or in vivo accumulation of a substrate labeled with a fluorescent or radioactive substance, and in vitro activity measurement using a substrate can be used using a measurement sample such as a cultured cell, a living body, or a sample collected from a living body. These measurement methods are preferably performed in a quantifiable manner. When necessary, the above process of extracting a translation product from a measurement sample can be performed, and the extract can be used for the above measurement.

[0076] The promoting substance in the present disclosure is preferably a substance that increases ubiquitination activity in a cell. Ubiquitination activity refers to an activity of adding ubiquitin to a target protein, and includes one or more reaction processes in a ubiquitin addition reaction.

[0077] As one embodiment of the promoting substance, for example, it is preferable that the substance increases the intracellular content and / or intracellular activity of a protein having ubiquitination activity. As another embodiment of the promoting substance, for example, it is also preferable that the substance at least increases the intracellular content of a protein having ubiquitination activity. By these embodiments, for example, the state of the cell can be easily improved by increasing the intracellular ubiquitination activity.

[0078] As another embodiment, the promoting substance can be a substance that directly or indirectly promotes the production and / or activity of a translation product that is a protein having ubiquitination activity. Thus, the amount of the translation product that is a protein having ubiquitination activity can be increased.

[0079] To increase a protein having ubiquitination activity in a cell, for example, the production of a transcription product or a translation product can be promoted. For example, the above-described promoting substance can promote the production of a translation product by promoting the translation from a transcription product that encodes a protein having ubiquitination activity. In addition, for example, the above-described promoting substance can promote the production of a target translation product by increasing the expression level of the transcription product, controlling the expression of another gene that controls the expression level of the target translation product, and the like. Other means for achieving this include, for example, a method of introducing the protein having ubiquitination activity itself into a cell. In either case, by these embodiments, for example, the state of the cell can be easily improved by increasing the intracellular ubiquitination activity.

[0080] The protein having the above-described ubiquitination activity includes various ubiquitination-related proteins. Such proteins include, for example, ubiquitin ligases such as LRSAM1 (Leucine-rich repeat and sterile-alpha motif containing protein 1), Gp78 (Glycoprotein 78), CHIP (C-terminal of Hsc70-interacting protein), RNF19A (Ring finger protein 19A), and MGRN1 (mahogunin Ring finger 1). Other ubiquitination-related proteins include, for example, ubiquitin-activating enzymes, ubiquitin-conjugating enzymes, and the like. The above-described TDP-43 is excluded from the protein having ubiquitination activity.

[0081] Whether the produced protein or translation product has ubiquitination activity can be determined, for example, using an increase or decrease in the amount or activity of ubiquitin in the presence or absence of exposure to the promoting substance, such as ELISA, Western blotting, flow cytometry, immunostaining, and the TR TUBE method described below. This determination can utilize, for example, the above-described determination criteria.

[0082] The human mRNA base sequence and the protein amino acid sequence of the above-described ubiquitin ligases are shown below, respectively.

[0083] LRSAM1 : SEQ ID NO: 5 (mRNA base sequence; GenBank Accession No. NM_001005373.4) and SEQ ID NO: 6 (amino acid sequence; GenBank Accession No. NP_001005373.1) Gp78: SEQ ID NO: 7 (mRNA base sequence; GenBank Accession No. NM_001144) and SEQ ID NO: 8 (amino acid sequence; GenBank Accession No. NP_001135.3) CHIP: SEQ ID NO: 9 (mRNA base sequence; GenBank Accession No. NM_005861.4) and SEQ ID NO: 10 (amino acid sequence; GenBank Accession No. NP_005852.2) RNF19A: SEQ ID NO: 11 (mRNA base sequence; GenBank Accession No. NM_183419) and SEQ ID NO: 12 (amino acid sequence; GenBank Accession No. NP_056250.3) MGRN1 : SEQ ID NO: 13 (mRNA base sequence; GenBank Accession No. NM_015246.4) and SEQ ID NO: 14 (amino acid sequence; GenBank Accession No. NP_056061.1) In one embodiment, the promoting substance is preferably a substance that increases at least the intracellular amount of LRSAM1 protein, which is a protein having ubiquitination activity. In one embodiment, the promoting substance is preferably a substance that promotes production of a transcription product and / or a translation product from a nucleic acid encoding LRSAM1 protein, more preferably a substance that promotes production of at least a translation product. Thus, the promoting substance can promote production of a protein having ubiquitination activity such as LRSAM1 protein and increase the intracellular amount of LRSAM1 protein. The above-mentioned LRSAM1 protein is preferably a human LRSAM1 protein, and is also preferably a wild-type LRSAM1 protein, and more preferably a wild-type human LRSAM1 protein.

[0084] LRSAM1 is a gene encoding LRSAM1 protein, which is one type of ubiquitination-related protein. The mRNA base sequence of human LRSAM1 is represented by, for example, SEQ ID NO: 5. The full-length amino acid sequence of human wild-type LRSAM1 protein is represented by, for example, SEQ ID NO: 6.

[0085] The LRSAM1 protein is mainly present in the cytoplasm and has ubiquitination activity on target proteins. The LRSAM1 protein catalyzes the addition of ubiquitin to target proteins (e.g., abnormal proteins). Thus, the ubiquitinated target proteins are promoted to be degraded by the ubiquitin-proteasome system in the cell. As a result, it can help to eliminate abnormal proteins, inhibit cell damage caused by the presence of abnormal proteins, and ultimately treat or prevent TDP-43-related diseases.

[0086] In one embodiment, the promoting substance is further more preferably a substance that promotes the production of a transcription product capable of expressing a wild-type LRSAM1 protein or a protein having an equivalent function to the wild-type protein. In addition or instead, the promoting substance is further more preferably a substance that promotes the production of a wild-type LRSAM1 protein or a protein having an equivalent function to the wild-type protein as a translation product. When a promoting substance capable of expressing a wild-type LRSAM1 protein is used, the amino acid sequence of the protein is preferably a full-length wild-type sequence.

[0087] By increasing the expression level of a transcription product or a translation product from a nucleic acid (e.g., a genomic gene, an exogenously introduced nucleic acid, an mRNA, etc.) encoding a ubiquitination-related protein such as LRSAM1, or by enhancing the function of these products, the intracellular content and / or intracellular ubiquitination activity of the protein such as LRSAM1 can be increased. This can effectively contribute to maintaining or improving cell function. Specifically, by promoting the induction of autophagy, or by promoting the degradation of abnormal proteins that cause the onset or progression of TDP-43-related diseases, the intracellular elimination of abnormal proteins can be easily standardized. Thus, it can help to treat or prevent TDP-43-related diseases. These functions can be particularly significant by expressing a wild-type protein or a protein having an equivalent function to the wild-type protein.

[0088] The base sequence of the gene or nucleic acid encoding the LRSAM1 protein can differ between individuals. Therefore, it does not need to have 100% identity to the above-described sequence as long as it encodes a protein having an equivalent function (e.g., ubiquitination activity) to the wild-type LRSAM1 protein. For example, the base sequence of the human LRSAM1 gene can be a base sequence having 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, or 100% identity to the wild-type base sequence.

[0089] Moreover, the mRNA base sequence of LRSAM1 can be a base sequence identical to the base sequence described in SEQ ID NO: 5 at 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99% or more, or 100%, as long as it encodes a protein having an equivalent function to the wild-type LRSAM1 protein. That is, for the mRNA base sequence of LRSAM1, bases can not be deleted, substituted, or added, and, for example, 1 to 625 (or 1 to 468, 1 to 312, 1 to 156, 1 to 63, 1 to 31) bases can be deleted, substituted, or added. Examples of such mRNA base sequences include, but are not limited to, degenerate codons, and the like.

[0090] Moreover, the amino acid sequence of LRSAM1 protein can be an amino acid sequence identical to the amino acid sequence described in SEQ ID NO: 6 at 80% or more, 85% or more, 90% or more, 95% or more, 99% or more, or 100%, as long as it encodes a protein having an equivalent function to the wild-type LRSAM1 protein. That is, for the LRSAM1 protein, amino acids can not be deleted, substituted, or added, and, for example, 1 to 145 (or 1 to 108, 1 to 72, 1 to 36, 1 to 14, 1 to 7) amino acids can be deleted, substituted, or added.

[0091] To determine whether the generated translation product has an equivalent function to the wild-type LRSAM1 protein, for example, the amount of ubiquitinated protein can be measured using a method such as the trypsin-resistant tandem ubiquitin binding entity (TR-TUBE) method, and compared with the activity or amount of the wild-type translation product to make the determination. As a criterion for determining that it has equivalent function, for example, the ratio to the wild-type result can be set to 70% or more, and preferably 80% or more.

[0092] In one embodiment, the promoting substance is preferably a substance containing a nucleic acid. More specifically, the promoting substance is preferably formed to include one or more selected from an expression-enhancing nucleic acid and an expression vector.

[0093] The expression-enhancing nucleic acid means a nucleic acid for expressing a target gene, and is preferably a nucleic acid exogenously introduced. In one embodiment, the expression-enhancing nucleic acid is preferably a nucleic acid for promoting expression of a target gene. The target of gene expression promotion is, for example, an endogenous nucleic acid (e.g., genomic DNA) encoding one or more of the above-described proteins (e.g., LRSAM1, Gp78, RNF19A, and MGRN1), or a nucleic acid exogenously introduced. The above-described promoting substances can each be used independently, or multiple types of promoting substances can be used in combination.

[0094] By containing such a substance, for example, it is possible to easily control the amount of translation product having ubiquitination activity (for example, an increase in gene expression, such as an increase in production of a transcription product such as mRNA and / or a translation product such as a protein). Thus, intracellular clearance of an abnormal protein such as a TDP-43 mutant protein can be effectively and easily standardized. This is advantageous because by taking the target translation product as the LRSAM1 protein and, for example, taking the target nucleic acid for expression enhancement as the nucleic acid encoding the LRSAM1 protein, ubiquitination of the abnormal protein can be increased and intracellular clearance of the abnormal protein can be further improved.

[0095] Examples of expression-enhancing nucleic acids include, but are not limited to, one or more nucleic acids selected from the group consisting of antisense, non-coding RNA, small activating RNA, and the like. These expression-enhancing nucleic acids have a function capable of expressing a target gene and / or promoting intracellular expression of a gene in a cell. Thus, the intracellular amount of a target translation product can be increased. These nucleic acids can be obtained or manufactured, for example, by screening using generally known methods.

[0096] In one embodiment, the expression-enhancing nucleic acid preferably has fewer bases than the nucleic acid contained in the expression vector described later. In one embodiment, the expression-enhancing nucleic acid has, for example, 5 to 300 bases per strand, preferably 10 to 100 bases per strand.

[0097] Antisense sequences used as expression-enhancing nucleic acids can take the following forms, for example, (a) nucleic acids that control RNA processing, (b) nucleic acids that are partially or completely complementary to miRNA, and (c) nucleic acids that are partially or completely complementary to natural antisense transcripts (NAT). These antisense can complementarily bind to a full-length target nucleic acid, or can complementarily bind to a part of a target nucleic acid sequence.

[0098] The above form (a) complementarily binds to a sequence related to splicing in a pre-mRNA to promote RNA processing, for example, splicing, to produce a desired mature mRNA (for example, an mRNA capable of translating a full-length wild-type protein). This contributes to an increase in expression of a target gene (specifically, an increase in production of a transcription product such as mRNA and / or a translation product such as a protein).

[0099] The above form (b) is complementary to miRNA and inhibits the function of miRNA. miRNA is generally a kind of nucleic acid that is complementary to the 3' UTR region of mRNA. Therefore, miRNA inhibits the expression of the target gene by increasing the degradability of mRNA or inhibiting translation from mRNA. This form weakens or eliminates the function of miRNA by antisense complementary to miRNA, thereby releasing the inhibition of gene expression. Thus, the production of mRNA or translation from mRNA is promoted, thereby contributing to an increase in the expression of the target gene (specifically, an increase in the production of a transcription product such as mRNA and / or a translation product such as a protein).

[0100] The above form (c) is complementary to NAT and inhibits the function of NAT. NAT is a kind of nucleic acid that is produced in a cell and is complementary to any region of a specific mRNA. In other words, NAT increases the degradability of mRNA or inhibits translation from mRNA. This form weakens or eliminates the function of NAT by a mechanism similar to that of the above form (b), thereby releasing the inhibition of gene expression. Thus, the production of mRNA or translation from mRNA is promoted, thereby contributing to an increase in the expression of the target gene (specifically, an increase in the production of a transcription product such as mRNA and / or a translation product such as a protein).

[0101] In addition to these, antisense used as an expression-enhancing nucleic acid includes, for example, a nucleic acid capable of being complementary to a region including a site such as a translation-inhibiting element (TIE) of mRNA, an upstream ORF (uORF), a premature termination codon (PTC), a guanine or adenine repeat sequence, and the like.

[0102] These antisense can contribute to the inhibition of mRNA degradation or the promotion of translation of mRNA, or can contribute to the promotion of the production of mRNA capable of producing a wild-type protein or a protein having an equivalent function to the wild-type protein. This contributes to an increase in the expression of the target gene (specifically, an increase in the production of a transcription product such as mRNA and / or a translation product such as a protein).

[0103] These expression-enhancing nucleic acids contribute to the improvement of the production of the target wild-type protein, which is preferable from the viewpoint of promoting the inherent function of exerting the wild-type protein and improving the elimination of abnormal proteins.

[0104] Furthermore, the antisense used as the expression-enhancing nucleic acid includes, for example, a nucleic acid that is complementary to a regulatory RNA that contributes to suppression of gene expression, a nucleic acid that is complementary to a recognition sequence of an RNA-binding protein that destabilizes mRNA, and the like. In this case, since the function of the RNA or protein for suppressing expression is suppressed, it functions to release the suppression of gene expression. Thus, the production of mRNA or translation from mRNA is promoted, thereby contributing to an increase in the expression of the target gene (specifically, an increase in the production of a transcription product such as mRNA and / or a translation product such as a protein).

[0105] The number of bases in the antisense used as the expression-enhancing nucleic acid is usually 10 to 30 bases per strand, preferably 14 to 25 bases per strand.

[0106] The antisense used as the expression-enhancing nucleic acid has at least 75% or greater, 80% or greater, 85% or greater, 90% or greater, 95% or greater, or 100% complementarity to a region having homology (identity) to the complementary strand of the base sequence of the target nucleic acid, in any of the above forms. For example, in the case where the base sequence of the antisense is 20 bases, when the nucleic acid base sequence in the complementary strand of the base sequence of the antisense and the base sequence of the target nucleic acid are compared, it is possible that no nucleic acid base is deleted, substituted, or inserted, or, for example, it is possible that 1 to 5 (or 1 to 4, 1 to 3, 1 to 2, 1) nucleic acid bases are deleted, substituted, or inserted. Such nucleic acids can be screened, designed, and obtained using, for example, generally known methods.

[0107] Examples of non-coding RNA include nucleic acids such as regulatory RNA and SINE-UP containing a SINE element.

[0108] The regulatory RNA is an RNA having a function of promoting transcription from a promoter of a nucleic acid incorporated in endogenous DNA (e.g., genomic DNA) or exogenously introduced, and promoting transcription into mRNA from a target nucleic acid. At the same time, the production of a target translation product (e.g., LRSAM1 protein, etc.) can be promoted.

[0109] The SINEUP is an RNA having a SINE factor sequence, and is a polyribonucleotide having a domain that promotes translation into a protein and a domain having a sequence complementary to mRNA. By using this, translation from mRNA is promoted (i.e., it mainly contributes to an increase in the production of a translation product).

[0110] The number of bases of the non-coding RNA can be appropriately set depending on the type or function thereof.

[0111] Small activating RNA (saRNA) is a nucleic acid complementary to part or all of a promoter sequence in genomic DNA. Thus, translation from mRNA is facilitated, thereby contributing to an increase in expression of a target gene (specifically, an increase in production of a transcription product such as mRNA and / or a translation product such as a protein).

[0112] The number of bases of the saRNA is usually 10 to 30 bases per strand, preferably 18 to 24 bases per strand. The saRNA is usually single-stranded or double-stranded RNA.

[0113] When formed to contain an expression-enhancing nucleic acid as a facilitator, the expression-enhancing nucleic acid can be, for example, in a single-stranded or double-stranded form, as long as the effects of the present disclosure are achieved. The backbone structure of these nucleic acids can be, for example, deoxyribonucleotides, ribonucleotides, bases containing non-nucleotides such as pyrrole or piperidine, or a combination thereof.

[0114] The structural unit of the nucleic acid in the expression-enhancing nucleic acid can be a natural type or a non-natural type, as long as the effects of the present disclosure are achieved. Non-natural forms include, for example, nucleic acids in which the sugar, base, or atom or molecule constituting the internucleoside bond in the nucleotide is substituted, modified, or deleted from the natural nucleotide.

[0115] In one embodiment, the facilitator can be, for example, an expression vector capable of expressing a target gene. Specifically, the facilitator can be an expression vector containing a nucleic acid including a base sequence encoding a protein or substance that facilitates expression, and capable of expressing a target protein or substance in a cell. Using such an expression vector, for example, a target transcription product and / or translation product can be expressed in a subject, and the amount of the translation product can be increased in various environments, for example, in vivo, in vitro, or ex vivo. This can lead to beneficial effects such as maintenance or improvement of cell function and improvement of clearance of abnormal proteins.

[0116] In the present specification, an "expression vector" refers to a vector containing a nucleic acid including a promoter base sequence and a first base sequence operably linked to the promoter base sequence, and capable of newly producing a nucleic acid or a protein based on the information of the first base sequence in a cell or a test tube. The first base sequence is formed, for example, to be capable of producing mRNA encoding a predetermined protein, or to be capable of producing the above-described expression-enhancing nucleic acid. That is, the above-described expression-enhancing nucleic acid itself is not included in the "expression vector".

[0117] The nucleic acid forming the expression vector can also contain, for example, a replication initiation sequence or a selection marker sequence such as a drug resistance gene in its base sequence for replication in a cell. Examples of the promoter base sequence include, but are not limited to, sequences capable of expressing in mammalian cells such as SV40 and CMV, sequences capable of expressing in Escherichia coli such as trp, lacI, and lacZ, sequences capable of expressing in a test tube such as SP6 and T7, and the like.

[0118] The type of expression vector is not particularly limited, and examples thereof include a lipid particle in which a nucleic acid is encapsulated in a lipid membrane, a plasmid vector, or a viral vector in which a nucleic acid is encapsulated in a capsid, and the like.

[0119] Examples of the viral vector include a DNA-containing viral vector such as an adenoviral vector and an adeno-associated viral vector, an RNA-containing viral vector such as a retroviral vector and a lentiviral vector, and the like. Examples of the structural unit of the polynucleotide contained in the viral vector include a deoxyribonucleotide backbone or a ribonucleotide backbone, and the like. These backbones are each independently preferably a natural type that does not contain a modification or an atomic substitution, or the like.

[0120] In a case where it is configured so that the predetermined protein can be produced with the above-described expression vector, the produced protein can include a mutation, for example, a deletion, a substitution, or an addition of an amino acid in its amino acid sequence, or can not include such a mutation, as long as it has an intended function. The identity between the produced protein or the amino acid sequence in the protein and the protein or the amino acid sequence in the wild-type protein corresponding to the protein can be, for example, 80% or greater, 85% or greater, 90% or greater, 95% or greater, or 100%.

[0121] In a case where the nucleic acid is externally introduced into a cell or a subject to produce a predetermined protein, the identity of the nucleic acid base sequence can be determined based on the designed nucleic acid base sequence, and it is acceptable that other mutations can be inevitably included in the transcription and / or translation process in the cell.

[0122] In one embodiment, the facilitating substance can include a substance that facilitates the function of the translation product. One or more types of such facilitating substances can be included in the composition. By including such a facilitating substance, it is possible to control the expression level or activity of a protein that is decreased or can be decreased due to a disease, and effectively treat or prevent the disease.

[0123] Examples of the substance that facilitates the function of the translation product include, but are not limited to, an allosteric regulator of a protein, a substance that stabilizes a post-translational modification, and the like.

[0124] The above-described facilitating substances can each be independently synthesized or produced, and manufactured, for example, using a method generally known in the technical field.

[0125] Examples of application targets of the promoting substance include living subjects, such as humans and non-human animals, and samples derived from these animals, preferably living humans or samples derived from humans. The application target of the promoting substance can be a healthy subject (whether or not there is a risk of developing a disease), or a subject (e.g., a human patient or a disease model animal) that has developed a disease, or a sample derived from these subjects. That is, the promoting substance can be used in various environments, such as in vivo, which includes or excludes humans, in vitro, which includes humans, or ex vivo, which includes humans. The target is more preferably a human subject, and even more preferably a healthy individual or a human patient.

[0126] Examples of non-human animals include non-human mammals, such as: rodents, e.g., rats, mice, and guinea pigs; monkeys; pigs; dogs; and cats. In one embodiment, the non-human animal is a terrestrial mammal.

[0127] Examples of samples include, but are not limited to, one or more of tissues, cells, and body fluids.

[0128] The promoting substance is applied to a target that includes at least one or more cells. Such application targets can include, for example, isolated or pure cultured cells, samples containing cells, and subjects, such as living bodies formed from cells.

[0129] Examples of tissues include brain regions (such as the cerebrum, midbrain, diencephalon, pons, medulla oblongata, and cerebellum), the spinal cord, the stomach, the pancreas, the kidney, the liver, the adrenal gland, the skin, muscles (such as skeletal muscle and smooth muscle), the lung, the intestine (such as the large intestine and the small intestine), the heart, blood vessels, and the like. These are generally formed from cell aggregates.

[0130] Cells can include differentiated cells, precursor cells, or stem cells that form tissues. Taking brain-derived cells as an example, brain-derived samples include various types of neural cells, such as neurons, glial cells (including astrocytes, microglia, and oligodendrocytes), neural precursor cells, neural stem cells, and the like. Among them, the cells on which the promoting substance of the present application acts are preferably these neural cells. These cells can be used as aggregates of homogenous cells obtained by isolation, pure culture, or the like, or as aggregates of cells containing two or more different types of cells.

[0131] Examples of body fluids include liquid components, such as cerebrospinal fluid, blood, serum, plasma, saliva, urine, and sweat, or extracts thereof.

[0132] These samples can generally be collected from living or dead animals using well-known methods such as biopsy or dissection. When necessary, an extraction step or a separation step (e.g., isolation) can be performed once or multiple times.

[0133] As other forms of cells, various cultured cells, such as primary cultured cells, immortalized cell lines, or pluripotent stem cells, such as ES cells and iPS cells, can be used.

[0134] The above-mentioned promoting substance or the composition containing the same can be in a solid state or a liquid state at 1 atm and 20°C depending on the intended use. Unless otherwise specified, the description related to the state in the present specification refers to the state at 1 atm and 20°C.

[0135] The liquid can be a solution containing a solvent or a dispersion containing a dispersion medium.

[0136] When the promoting substance is used in the form of a composition, the composition can further contain a carrier as necessary. These carriers are preferably pharmaceutically acceptable carriers from the viewpoint of reducing the occurrence of unintended effects on the application target.

[0137] As the carrier, a carrier used in the technical field can be used without particular limitation. As the carrier, for example, an excipient, a disintegrant, a disintegration aid, a binder, a lubricant, a coating agent, a coloring agent, a diluent, a vehicle, a solvent, a solubilizer, an isotonic agent, a pH adjuster, a stabilizer, a propellant, an adhesive, and the like can be used. These carriers can be used independently of each other, or two or more carriers can be used in combination.

[0138] As the vehicle or diluent, for example, various liquids, such as water, water containing electrolytes (for example, saline), monovalent alcohol having 1 to 3 carbon atoms (for example, methanol, ethanol, and propanol), polyvalent alcohol (for example, glycerol), or a culture medium used for cell culture, can be used. These liquids can be used independently of each other, or two or more liquids can be used in combination, as the above-mentioned solvent or dispersion medium that can form a composition.

[0139] The above is the description regarding the promoting substance and the composition. Hereinafter, another embodiment is described. In the following description, the content different from the above-mentioned embodiment will be mainly described, and for the content not specifically described, the content described in the present specification will be appropriately applied. The matters described in the present specification can be applied independently of each other, or two or more matters can be applied in combination.

[0140] In one embodiment, the present disclosure relates to a method for treating or preventing a TDP-43-related disease.

[0141] In one embodiment, the method includes a step of administering a promoting substance as an active ingredient to a subject in need thereof. In one embodiment, the method includes administering an effective amount of an active ingredient to a subject in need thereof. Thus, a TDP-43-related disease can be effectively treated or prevented. Examples of subjects include healthy subjects and subjects who have developed or are likely to develop a TDP-43-related disease, preferably human subjects. Examples of subjects who are likely to develop a TDP-43-related disease include healthy individuals who have not yet developed the disease but are at risk due to factors such as genetic mutations or environmental factors, or patients who have a disease other than a TDP-43-related disease. Subjects who are likely to develop a TDP-43-related disease can be determined, for example, by measuring the presence or absence of a genetic mutation associated with the disease using generally known methods.

[0142] In one embodiment, the method includes inhibiting the occurrence or progression of nerve cell damage by administering an active ingredient to a subject (e.g., various living organisms such as humans). Thus, a TDP-43-related disease can be more effectively treated or prevented.

[0143] In the present specification, a "disorder" of a cell includes one or more of the following cases: a case where the function inherent in a normal cell is reduced or lost while the cell morphology is normal; a case where the cell morphology is changed to a form different from the normal cell morphology; and a case where cell death such as apoptosis or necrosis occurs due to the above cases.

[0144] In the case of neurons, examples of reduced cell function include one or more of the following: reduction or loss of action potential generation, weakening of synapses or synaptic plasticity, reduced or lost energy metabolism, inhibition of neurogenesis, and the like.

[0145] In the case of neurons, examples of changes in cell morphology include one or more of the following: retraction, elongation inhibition, reduction of the structure of the dendrite and axon, demyelination, swelling or contraction of the cell body, and the like.

[0146] The promoting substance can be administered to a subject (e.g., various living organisms such as humans) as it is, or can be administered to a subject in the form of the above-described composition. When two or more promoting substances are administered in combination, the promoting substances can be administered all at once, or the substances can be administered sequentially in any order.

[0147] The method of administration to a subject can be appropriately selected depending on the characteristics of the substance to be used. Examples of the method of administration include various in vivo administrations, such as oral administration and parenteral administration.

[0148] For oral administration, for example, solid dosage forms such as tablets, capsules, powders, fine granules, and granules can be used, and liquid dosage forms such as solutions, syrups, and suspensions can be used.

[0149] Examples of non-oral administration include intravenous, intramuscular, intraperitoneal, intrathecal, subcutaneous or intradermal injection of a liquid; injection or inhalation of a solid or liquid into the gastrointestinal tract; and the like. These administrations can be single rapid administration or multiple rapid administrations, or can be continuous administration, such as infusion.

[0150] The dose to be administered to the subject can be appropriately selected in accordance with the characteristics of the substance to be used and the therapeutically effective amount. When the subject is an adult human with a body weight of 60 kg, the daily dose of each active ingredient can be determined independently, for example, 0.1 ng to 1000 mg of each active ingredient. Furthermore, when the active ingredient is a viral vector, the dose can be, for example, 1 x 10 5 vg / kg to 1 x 10 20 vg / kg / 1 kg of body weight of a human subject.

[0151] The number of doses of the active ingredient per day can be appropriately selected in accordance with the characteristics of the substance to be used and the therapeutically effective amount, and, for example, it can be administered once or twice or more. Furthermore, the administration method during the treatment can be appropriately selected in accordance with the characteristics of the substance to be used and the therapeutically effective amount. With respect to the above-mentioned administration methods, for example, the administration can be performed continuously every day, or can be performed intermittently by setting one or a plurality of non-administration periods of arbitrary days, months, or years.

[0152] Hereinafter, still another embodiment is described. In the following description, matters different from the above-described embodiments will be mainly described, and for matters not specifically described, the matters described in this specification will be appropriately applied. Matters and embodiments described in this specification can each be applied independently, or two or more matters and embodiments can be applied in combination.

[0153] In one embodiment, the present disclosure relates to a method for inhibiting cell damage. Furthermore, in one embodiment, the present disclosure relates to a method for promoting maintenance or improvement of cell function.

[0154] In one embodiment, the method includes a step of contacting a promoting substance with a neuron, which is one of the application targets. This can inhibit the occurrence or progression of cell damage in the neuron, and maintain the autonomous movement of the skeletal muscle. Thus, the disease can be effectively treated or prevented.

[0155] The neuron as the application target is preferably a human neuron, more preferably a human motor neuron.

[0156] In the case of a neuron, examples of normal cell function include one or more of the following: generation of action potential, energy metabolism, and neurogenesis.

[0157] The measurement of cell damage can be performed, for example, by measuring neurite length when a stress stimulus is applied to neurons (preferably neurons derived from ALS patients) as described in the examples below. An example of a stress stimulus is tunicamycin treatment.

[0158] The degree of cell damage when the step of contacting the promoting substance with various cells (e.g., neurons) is included, as compared to when the step of contacting the promoting substance with cells is not included, is preferably 90% or less, more preferably 70% or less, and even more preferably 50% or less, as the degree of inhibition of cell damage by the promoting substance.

[0159] The method of contacting the promoting substance with various cells such as neurons is not particularly limited, and examples thereof include methods of directly or indirectly contacting the promoting substance with cells. The promoting substance can be contacted with cells as is, or the promoting substance can be contacted with cells in the presence of the above-described carrier. An example of a method for indirect contact is a method in which the promoting substance is administered in vivo using the above-described method, and is exposed to body constituent cells such as motor neurons via body fluids such as blood and lymph. That is, the above-described methods can each be independently applied to various environments, such as in vivo, in vitro, and ex vivo.

[0160] Further, another embodiment of the present disclosure relates to a method for improving protein clearance.

[0161] Another embodiment of the present disclosure relates to a method for reducing or inhibiting abnormal localization of a protein.

[0162] Another embodiment of the present disclosure relates to a method for modulating protein phosphorylation. More specifically, one embodiment of the present disclosure relates to a method for reducing or inhibiting protein phosphorylation.

[0163] Another embodiment of the present disclosure relates to a method for promoting protein degradation.

[0164] Another embodiment of the present disclosure relates to a method for inhibiting accumulation of a protein in the cytoplasm.

[0165] In one embodiment, each of the above-described methods includes a step of contacting the promoting substance with neurons as one of the application targets.

[0166] The proteins that are the application targets of the above-described methods are each independently a TDP-43 protein, a protein derived from TDP-43, or the like. Specifically, these proteins are each, for example, the above-described abnormal TDP-43 protein, preferably a TDP-43 protein or a fragment thereof, more preferably a non-wild type TDP-43 protein. A preferred example of a TDP-43 protein fragment is a C-terminal fragment of a TDP-43 protein, which is known to be produced when full-length TDP-43 protein is cleaved by caspase 3 or the like. Examples of the amino acid sequence of the C-terminal fragment include the sequences shown in SEQ ID NO: 4, SEQ ID NO: 15, SEQ ID NO: 16, and SEQ ID NO: 17. Among these, the protein shown in SEQ ID NO: 15 is about 35 kDa (which can be referred to as CTF35), and the protein shown in SEQ ID NO: 16 or SEQ ID NO: 17 is about 25 kDa (which can be referred to as CTF25). The proteins can each be a full-length amino acid sequence or a peptide fragment having any number of residues. The abnormal localization and formation of aggregates of the abnormal protein can be inhibited by inhibiting the translocation of the TDP-43 protein from the nucleus, inhibiting the accumulation of the TDP-43 protein in the cytoplasm, inhibiting the abnormal post-translational modification of the TDP-43 protein, or promoting the degradation of the abnormal TDP-43 protein. Furthermore, the clearance of the abnormal protein can be normally maintained. Thus, the maintenance or improvement of cellular function can be promoted, and thus a TDP-43-related disease can be treated or prevented.

[0167] Furthermore, another embodiment of the present disclosure relates to a method for inhibiting abnormal RNA splicing. Examples of the target to be inhibited in the present method include the STMN2 (Stathmin-2) gene, or a transcription product (e.g., a pre-mRNA) from the gene, described in the examples described later.

[0168] The above-described various methods preferably each independently include a step of bringing the promoting substance into contact with various cells such as neurons. That is, a preferred embodiment of the above-described methods is to maintain the survival of cells such as neurons in the presence of the promoting substance. The method of bringing the promoting substance into contact with various cells such as neurons is not particularly limited, and examples thereof include a method of directly or indirectly bringing the promoting substance into contact with the cells. The promoting substance can be brought into contact with the cells as is, or the promoting substance can be brought into contact with the cells in the presence of the above-described carrier. An example of the method for indirect contact is a method in which the promoting substance is administered in vivo using the above-described method, and is exposed to body constituent cells such as motor neurons via a body fluid such as blood and lymph. Similar to other embodiments, the methods according to the above-described embodiments can each independently be applied to various environments such as in vivo, in vitro, and ex vivo.

[0169] An example of a method for measuring abnormal protein localization is a method for measuring the ratio between the expression level of TDP-43 protein in the cytoplasm and in the nucleus using cultured cells, cells derived from ALS patients, and the like, as described in the screening method for TDP-43 protein nuclear export inhibitors described later. In this method, a stress stimulus such as tunicamycin can be applied to cultured cells, cells derived from ALS patients, and the like.

[0170] As the degree of reduction or inhibition of abnormal protein localization by the promoting substance, for example, when a step of contacting the promoting substance with various types of cells such as neurons is included, the ratio between the expression level of TDP-43 protein in the cytoplasm and in the nucleus is 90% or less, preferably 70% or less, more preferably 50% or less, as compared to when a step of contacting the promoting substance with the cells is not included.

[0171] An example of a method for measuring protein phosphorylation is a method for measuring the phosphorylation level of, for example, TDP-43 protein using cultured cells, cells derived from ALS patients, and the like, using a conventional method such as immunofluorescence staining or Western blotting, and the like, as described in the examples described later. In this method, a stress stimulus such as tunicamycin can be applied to cultured cells, cells derived from ALS patients, and the like.

[0172] As the degree of reduction or inhibition of protein phosphorylation by the promoting substance, for example, when a step of contacting the promoting substance with various types of cells such as neurons is included, the phosphorylation level of TDP-43 protein is 90% or less, preferably 70% or less, more preferably 50% or less, as compared to when a step of contacting the promoting substance with the cells is not included.

[0173] An example of a method for measuring protein degradation is a method for measuring the expression level of the C-terminal fragment of TDP-43 protein using cultured cells, cells derived from ALS patients, and the like, using a conventional method such as Western blotting, and the like. In this method, a stress stimulus such as tunicamycin can be applied to cultured cells, cells derived from ALS patients, and the like.

[0174] As the degree of inhibition of protein degradation by the promoting substance, for example, when a step of contacting the promoting substance with various types of cells such as neurons is included, the expression level of the C-terminal fragment of TDP-43 protein is 90% or less, preferably 70% or less, more preferably 50% or less, as compared to when a step of contacting the promoting substance with the cells is not included.

[0175] An example of the method for measuring abnormal RNA splicing is a method of measuring the ratio between the expression levels of STMN2 CE and STMN2 FL of the STMN2 gene (STMN2 CE / STMN2 FL) using cultured cells, cells derived from ALS patients, and the like, using a conventional method, for example, using absolute quantitative qPCR (see Examples). In this method, a stress stimulus, for example, tunicamycin, can be applied to the cultured cells, cells derived from ALS patients, and the like.

[0176] As the degree of inhibition of abnormal RNA splicing by the promoting substance, for example, when including a step of contacting the promoting substance with various types of cells such as neurons, the ratio between the expression levels of STMN2 CE and STMN2 FL is 90% or less, preferably 70% or less, more preferably 50% or less, compared to when not including a step of contacting the promoting substance with cells.

[0177] In addition to the above description, as one embodiment, the present specification also discloses matters related to the use of a substance in the production of a composition for treating or preventing a TDP-43-related disease. The substance is, for example, the above-described promoting substance. The composition in the present embodiment preferably contains the above-described promoting substance.

[0178] Furthermore, the present specification also discloses matters related to a substance or a composition for treating or preventing a TDP-43-related disease. The substance is the above-described promoting substance. The composition in the present embodiment preferably contains the above-described promoting substance.

[0179] Another embodiment of the present disclosure relates to a screening method for a TDP-43 protein phosphorylation inhibitor or a screening method for a TDP-43 protein nuclear export inhibitor. The proteins each serving as an application target of the above-described method are each independently a wild-type TDP-43 protein, a non-wild-type TDP-43 protein, a protein derived from TDP-43, and the like. Specifically, these proteins are each, for example, the above-described abnormal TDP-43 protein, preferably a TDP-43 protein or a fragment thereof, more preferably a non-wild-type TDP-43 protein. A preferred example of the TDP-43 protein fragment is a C-terminal fragment of the TDP-43 protein.

[0180] The screening method for a TDP-43 protein phosphorylation inhibitor can include: a step of contacting a test substance with a TDP-43 protein; a step of measuring the phosphorylation level of the TDP-43 protein contacted with the test substance; a step of comparing the measured phosphorylation level with the phosphorylation level measured for the TDP-43 protein not contacted with the test substance; and a step of selecting the test substance that reduces the phosphorylation level of the TDP-43 protein.

[0181] The method of contacting the test substance with the TDP-43 protein is not particularly limited, and examples thereof include a method of directly or indirectly contacting the test substance with the TDP-43 protein. An example of the method for direct contact is a method in which a commercially available recombinant TDP-43 protein or a recombinant TDP-43 protein prepared using a conventional method is used. An example of the method for indirect contact is a method in which the test substance is administered to various types of cells such as neurons to expose the cells, and the method is applicable to various environments such as in vitro, in vivo, and ex vivo. Examples of the cells include motor neurons differentiated from iPS cells derived from a patient with a TDP-43-related disease, preferably motor neurons differentiated from iPS cells derived from an ALS patient, more preferably motor neurons differentiated from iPS cells derived from an ALS patient with a TDP-43 gene mutation. In addition, these cells can further include a step of administering a stress inducer, for example, tunicamycin.

[0182] The phosphorylation level of the TDP-43 protein can be measured using a conventional method, for example, immunofluorescence staining (see Example 1) or Western blotting, etc.

[0183] By comparing the measured phosphorylation level with the phosphorylation level of the TDP-43 protein not contacted with the test substance, it can be determined whether the test substance is a TDP-43 protein phosphorylation inhibitor. When the phosphorylation level of the TDP-43 protein contacted with the test substance is lower than the phosphorylation level of the TDP-43 protein not contacted with the test substance, it can be determined that the test substance is a TDP-43 protein phosphorylation inhibitor, and can be selected. For example, when the phosphorylation level of the TDP-43 protein contacted with the test substance is 70% or less, preferably 50% or less, more preferably 25% or less, compared to the phosphorylation level of the TDP-43 protein not contacted with the test substance, it can be determined that the test substance is a TDP-43 protein phosphorylation inhibitor.

[0184] The screening method for TDP-43 protein nuclear export inhibitors can comprise the steps of: contacting a test substance with a cell expressing TDP-43 protein; measuring the expression level of TDP-43 protein in the nucleus and / or cytoplasm of the cell exposed to the test substance; comparing the measured expression level of TDP-43 protein in the nucleus and cytoplasm and / or the ratio between the expression level of TDP-43 protein in the cytoplasm and the nucleus (calculated by dividing the expression level in the cytoplasm by the expression level in the nucleus) with the expression level of TDP-43 protein in the nucleus and cytoplasm and / or the ratio between the expression level of TDP-43 protein in the cytoplasm and the nucleus in a cell not exposed to the test substance; and selecting a test substance that increases the expression level of TDP-43 protein in the nucleus or decreases the expression level of TDP-43 protein in the cytoplasm and / or the ratio between the expression level of TDP-43 protein in the cytoplasm and the nucleus.

[0185] The method for contacting a test substance with a cell expressing TDP-43 protein is not particularly limited, and examples thereof are methods of administering a test substance to various types of cells such as neurons to expose the cells, and are suitable for various environments such as in vitro, in vivo, and ex vivo. Examples of the cells include motor neurons differentiated from iPS cells derived from a patient with a TDP-43-related disease, preferably motor neurons differentiated from iPS cells derived from an ALS patient, more preferably motor neurons differentiated from iPS cells derived from an ALS patient with a TDP-43 gene mutation. Furthermore, these cells can also include a step of administering a stress inducer such as tunicamycin.

[0186] The expression level of TDP-43 protein in the nucleus and / or cytoplasm can be measured using a conventional method, for example, by immunofluorescence staining (see Example 1) or by Western blotting of nuclear and / or cytoplasmic proteins extracted from the cells.

[0187] By comparing the measured expression level of TDP-43 protein in the nucleus and cytoplasm, and / or the ratio between the expression levels in the cytoplasm and nucleus, with the expression level of TDP-43 protein in the nucleus and cytoplasm, and / or the ratio between the expression levels in the cytoplasm and nucleus in cells not exposed to the test substance, it can be determined whether the test substance is a nuclear export inhibitor. By comparing with the expression level of TDP-43 protein in the nucleus and cytoplasm, and / or the ratio between the expression levels of TDP-43 protein in the cytoplasm and nucleus when not exposed to the test substance, when the expression level of TDP-43 protein in the nucleus is increased, and when the expression level of TDP-43 protein in the cytoplasm and / or the ratio between the expression levels of TDP-43 protein in the cytoplasm and nucleus is decreased upon exposure to the test substance, it can be determined that the test substance is a TDP-43 protein nuclear export inhibitor, and the test substance can be selected. For example, when the ratio between the expression levels of TDP-43 protein in the cytoplasm and nucleus is 80% or less, preferably 50% or less, more preferably 25% or less upon exposure to the test substance compared to when not exposed to the test substance, it can be determined that the test substance is a TDP-43 protein nuclear export inhibitor.

[0188] The TDP-43 protein phosphorylation inhibitor and the TDP-43 protein nuclear export inhibitor obtained by the screening method according to one embodiment of the present disclosure can be used as an active ingredient for treating or preventing a TDP-43-related disease. In particular, these inhibitors can be used as an active ingredient for preventing ALS or treating or preventing a TDP-43-related disease.

[0189] Another embodiment of the present disclosure is a composition comprising a substance that enhances the binding between the LRSAM1 protein and the TDP-43 protein to treat or prevent a TDP-43-related disease (which can be referred to as a binding-promoting substance).

[0190] The binding-promoting substance is a substance that enhances the binding or interaction between the LRSAM1 protein and the TDP-43 protein, or between their fragments, examples of which include a PROTAC (proteolysis targeting chimera) compound or an MGD (molecular glue degrader) compound. A preferred example of the LRSAM1 protein is wild type. The TDP-43 proteins are each independently wild type TDP-43 protein, non-wild type TDP-43 protein, a protein derived from TDP-43, and the like. Specifically, these proteins are each, for example, the abnormal TDP-43 protein described above, preferably the TDP-43 protein or a fragment thereof, more preferably the non-wild type TDP-43 protein. A preferred example of the TDP-43 protein fragment is a C-terminal fragment of the TDP-43 protein.

[0191] A PROTAC compound is a compound containing a TDP-43 protein target ligand, a LRSAM1 (E3 ubiquitin ligase) target ligand, and a linker.

[0192] A PROTAC compound can be prepared as follows. The TDP-43 protein target ligand and the LRSAM1 (E3 ubiquitin ligase) target ligand can be obtained by screening existing ligands, simulation based on X-ray crystallography, screening using a library of compounds, biochemical evaluation (e.g., surface plasmon resonance (SPR) method, etc.), and / or using an artificial intelligence program (e.g., Alphafold2) for protein structure prediction. The obtained ligand can be connected to a linker using methods generally known in the art to prepare a compound that can be used as a PROTAC candidate compound. Whether a PROTAC candidate compound binds to the TDP-43 protein and the LRSAM1 protein and enhances the interaction therebetween can be evaluated by binding a probe to each of the TDP-43 protein and the LRSAM1 protein and competitively measuring the interaction. Specifically, for example, the evaluation can be performed using a method such as fluorescence polarization (FP), time-resolved fluorescence resonance energy transfer (TR-FRET), AlphaScreen / AlphaLISA technology, etc. In this case, when a strong interaction is observed in the presence of the PROTAC candidate compound compared to the absence of the PROTAC candidate compound, it can be determined that the compound is a PROTAC candidate compound. In addition, whether a PROTAC candidate compound has the ability to degrade the TDP-43 protein can be evaluated using cells. The amount of the TDP-43 protein can be quantified by introducing a plasmid in which the TDP-43 protein or a fragment of the TDP-43 protein (C-terminal fragment of the TDP-43 protein) is fluorescently labeled into a neural cell, for example, a Neuro2a cell, using a method well known in the art, and measuring the fluorescence intensity. The PROTAC candidate compound is added to the cell, and when the fluorescence intensity decreases, it can be determined that the compound has the ability to degrade the TDP-43 protein and thus is a PROTAC compound. In this way, a protein PROTAC compound consisting of a TDP-43 protein target ligand, a LRSAM1 (E3 ubiquitin ligase) target ligand, and a linker can be obtained.

[0193] An MGD compound is a compound that enhances the interaction between the TDP-43 protein and the LRSAM1 (E3 ubiquitin ligase) protein by fitting into a pocket formed when the TDP-43 protein and the LRSAM1 protein interact with each other. The MGD compound can be prepared as follows.

[0194] The interaction between the TDP-43 protein or a fragment thereof (C-terminal fragment of TDP-43 protein) and the LRSAM1 protein can be explored to see if there is a pocket that a compound can fit into using three-dimensional structure analysis with X-ray crystallography or nuclear magnetic resonance (NMR) spectroscopy and / or an artificial intelligence program (e.g., Alphafold2) for protein structure prediction. This allows visualization and / or prediction of the binding interface or pocket between the TDP-43 protein or a fragment thereof and the LRSAM1 (E3 ubiquitin ligase) protein. MGD candidate compounds that can fit into the pocket can be generated by virtual screening from a compound library through structure-based docking or simulation and / or by compound design based on the structure information of the pocket. Whether the obtained MGD candidate compounds bind to and enhance the interaction between the TDP-43 protein or a fragment thereof and the LRSAM1 protein when they interact can be evaluated by quantitatively measuring protein-protein interactions using a method such as time-resolved fluorescence resonance energy transfer (TR-FRET). In this case, when a stronger interaction is observed in the presence of an MGD candidate compound than in the absence of the MGD candidate compound, it can be determined that the compound is an MGD compound. In addition, whether the MGD candidate compound has the ability to degrade the target protein can be evaluated using the same evaluation method as the PROTAC candidate compound described above, and when it is determined that the compound has the ability to degrade the TDP-43 protein, it can be determined that it is an MGD compound. In this way, an MGD compound that enhances the interaction between the target protein and LRSAM1 can be obtained.

[0195] As one embodiment of the present disclosure, the binding-promoting substance can be used as an active ingredient for treating or preventing a TDP-43-related disease by promoting the degradation of the TDP-43 protein or a fragment of the TDP-43 protein (e.g., a C-terminal fragment of the TDP-43 protein) through the ubiquitin-proteasome system of the LRSAM1 protein. In particular, the binding-promoting substance can be used as an active ingredient for preventing ALS or treating or preventing a TDP-43-related disease.

[0196] With regard to the above matters, matters described in the present specification should be appropriately applied to matters not specifically described.

[0197] With regard to the above embodiment, the present specification further discloses the following embodiments.

[0198] <1> A composition comprising a binding-promoting substance.

[0199] <2> The composition according to <1>, wherein the binding-promoting substance is a substance having at least one, preferably two, more preferably three, of the following (i) to (iii): (i) increasing the intracellular amount of a protein having ubiquitination activity; (ii) increasing ubiquitination activity in a cell; and (iii) promoting production of a transcription product or a translation product in a cell.

[0200] <3> The composition according to <1> or <2> above for use in the treatment or prevention of a TAR DNA binding protein 43 (TDP-43)-related disease.

[0201] <4> The composition according to <3> above, wherein the TDP-43-related disease is one or more selected from the group consisting of a neurodegenerative disease and a muscle disease.

[0202] <5> The composition of <4> above, wherein the neurodegenerative disease is selected from the group consisting of Parkinson's disease, amyotrophic lateral sclerosis (ALS), spinal muscular atrophy (SMA), primary lateral sclerosis (PLS), multiple system atrophy (MSA), progressive supranuclear palsy (PSP), Perry syndrome, Alexander disease, Alzheimer's disease, frontotemporal lobar degeneration (FTLD), and limbic-predominant age-related TDP-43 encephalopathy (LAT), and the muscle disease is inclusion body myositis.

[0203] <6> The composition according to <5> above, wherein the neurodegenerative disease is one or more selected from the group consisting of amyotrophic lateral sclerosis (ALS), frontotemporal lobar degeneration (FTLD), and limbic-predominant age-related TDP-43 encephalopathy (LAT), preferably amyotrophic lateral sclerosis (ALS).

[0204] <7> The composition according to any one of <1> to <6> above for use in improving clearance of TDP-43 protein.

[0205] <8> The composition according to any one of <1> to <7> above for use in inhibiting cell damage.

[0206] <9> The composition according to any one of <1> to <8> above for use in inhibiting cell damage caused by TDP-43 protein.

[0207] <10> The composition according to any one of <1> to <9> above for use in reducing or inhibiting abnormal localization of a protein.

[0208] <11> The composition according to any one of <1> to <10> above for use in promoting degradation of a protein.

[0209] <12> The composition according to any one of <1> to <11> above for use in modulating phosphorylation of a protein.

[0210] <13> The composition according to any one of the above <10> to <12>, wherein the protein is a full-length TDP-43 protein or a fragment thereof.

[0211] <14> The composition according to the above <13>, wherein the protein is a C-terminal fragment of TDP-43 protein.

[0212] <15> The composition according to any one of the above <1> to <14>, for use in inhibiting abnormal splicing of RNA, preferably mRNA.

[0213] <16> The composition according to any one of the above <1> to <15>, for use in a subject or a cell having a mutation in the TARDBP gene.

[0214] <17> The composition according to the above <16>, for use in a subject or a cell having a heterozygous mutation in the TARDBP gene.

[0215] <18> The composition according to the above <16> or <17>, wherein the subject or the cell is a human subject or a cell of human origin.

[0216] <19> The composition according to any one of the above <1> to <18>, wherein the promoting substance is a substance containing one or more selected from the group consisting of an organic compound, a nucleic acid, a protein, a lipid, and a protein, and preferably a nucleic acid.

[0217] <20> The composition according to the above <19>, wherein the promoting substance is a substance containing a nucleic acid, and the promoting substance preferably contains one or more selected from an expression-enhancing nucleic acid and an expression vector.

[0218] <21> The composition according to the above <20>, wherein the promoting substance comprises an expression-enhancing nucleic acid.

[0219] <22> The composition according to the above <21>, wherein the expression-enhancing nucleic acid is one or more selected from the group consisting of an antisense, a non-coding RNA, and a small activating RNA.

[0220] <23> The composition according to the above <22>, wherein the antisense is one or more selected from the following (a) to (e): (a) a nucleic acid that controls RNA processing; (b) a nucleic acid that is partially or completely complementary to a full-length or a partial region of a miRNA; (c) a nucleic acid that is partially or completely complementary to a full-length or a partial region of a natural antisense transcript (NAT); (d) a nucleic acid partially or completely complementary to all or a part of one or more regions selected from the group consisting of a translational inhibitory element (TIE), an upstream ORF (uORF), and a premature termination codon (PTC) of mRNA; and (e) a nucleic acid partially or completely complementary to all or a part of a full-length or partial region of a regulatory RNA that contributes to the suppression of gene expression.

[0221] <24> The composition according to any one of the above <21> to <23>, wherein the expression-enhancing nucleic acid has 5 to 300 bases per strand, and preferably 10 to 100 bases per strand.

[0222] <25> The composition according to the above <20>, wherein the promoting substance comprises an expression vector.

[0223] <26> The composition according to any one of the above <1> to <25>, wherein the promoting substance is a substance that increases the intracellular content of a protein having ubiquitination activity, and the protein having ubiquitination activity is an LRSAM1 protein.

[0224] <27> The composition according to the above <26>, wherein the protein having ubiquitination activity is a human LRSAM1 protein.

[0225] <28> The composition according to the above <26> or <27>, wherein the protein having ubiquitination activity is a wild-type LRSAM1 protein.

[0226] <29> The composition according to any one of the above <1> to <28>, wherein the promoting substance is a substance that promotes the production of a transcription product and / or a translation product from a nucleic acid encoding a protein having ubiquitination activity.

[0227] <30> The composition according to any one of the above <1> to <29>, wherein the promoting substance is a substance that promotes the production of a transcription product and / or a translation product from a nucleic acid encoding a full-length wild-type protein having ubiquitination activity.

[0228] <31> The composition according to any one of the above <1> to <30>, wherein the promoting substance is a substance that promotes the production of a translation product, which is a protein having ubiquitination activity, and by promoting the production of the protein, increases the intracellular amount of the protein, and / or increases the intracellular ubiquitination activity.

[0229] <32> The composition according to any one of the above <1> to <31>, wherein the promoting substance is a substance that increases the intracellular content of a protein or a translation product having ubiquitination activity, and the protein or the translation product is one or more proteins selected from the group consisting of LRSAM1, Gp78, RNF19A, and MGRN1.

[0230] <33> The composition according to any one of the above <29> to <32>, wherein the protein having ubiquitination activity is a LRSAM1 protein.

[0231] <34> The composition according to any one of the above <1> to <32>, wherein the promoting substance is a substance that promotes the following (i) and / or (ii): (i) production of a transcription product that is capable of expressing a wild-type LRSAM1 protein or a protein having an equivalent function to the wild-type LRSAM1 protein; and (ii) production of a translation product that is a wild-type LRSAM1 protein or a protein having an equivalent function to the wild-type LRSAM1 protein.

[0232] <35> The composition according to any one of the above <1> to <34>, wherein the cell is a neural cell, preferably a neuron, more preferably a human neuron, even more preferably a human motor neuron.

[0233] <36> The composition according to any one of the above <1> to <35>, further comprising a pharmaceutically acceptable carrier.

[0234] <37> The composition according to any one of the above <1> to <36>, which is a pharmaceutical composition.

[0235] <38> The composition according to any one of the above <1> to <37>, for use in a subject who has developed or is likely to develop a TDP-43-related disease.

[0236] <39> The composition according to any one of the above <1> to <38>, for use in any of the following contexts: in vivo, including or excluding humans; in vitro, including humans; or ex vivo, including humans.

[0237] <a1>A method for treating or preventing a TDP-43 related disease, comprising the step of administering to a subject in need thereof an effective amount of a promoting substance.

[0238] <a2>A method for improving TDP-43 protein clearance, comprising the step of administering to a subject in need thereof an effective amount of a facilitating substance.

[0239] <a3>A method of inhibiting cell damage, comprising the step of administering to a subject in need thereof an effective amount of a promoting substance.

[0240] <a4>A method for promoting the maintenance or improvement of cell function, comprising the step of administering to a subject in need thereof an effective amount of a promoting substance.

[0241] <a5>A method of reducing or inhibiting abnormal localization of a protein, comprising the step of administering to a subject in need thereof an effective amount of a facilitating substance.

[0242] <a6>A method for promoting protein degradation, comprising the step of administering to a subject in need thereof an effective amount of a promoting substance.

[0243] <a7>A method for modulating protein phosphorylation comprising the step of administering to a subject in need thereof an effective amount of a facilitating substance.

[0244] <a8>A method of inhibiting aberrant splicing of RNA, preferably mRNA, comprising the step of administering to a subject in need thereof an effective amount of a facilitating substance.

[0245] <a9>According to <a1>The method, wherein the occurrence or progression of nerve cell damage is inhibited by administering the promoting substance to the subject, thereby treating or preventing the disease.

[0246] <a10>According to the above <a5>To <a7>The method of any one of the preceding items, wherein the protein is a full-length TDP-43 protein or a fragment thereof.

[0247] <a11>According to the above <a10>A method of diagnosing a disease or disorder associated with TDP-43 proteinopathy in a subject, comprising detecting the presence of a C-terminal fragment of TDP-43 protein in a sample from the subject.

[0248] <a12>According to the above <a1>To <a11>The method of any one of the preceding items, for a subject who has developed or is likely to develop a TDP-43 related disease.

[0249] <a13>According to the above <a1>To <a12>The method of any one of the preceding items, for use in a subject who has developed or is likely to develop one or more diseases selected from amyotrophic lateral sclerosis (ALS), frontotemporal lobar degeneration (FTLD), and limbic-predominant age-related TDP-43 encephalopathy (LATE) as TDP-43-related diseases.

[0250] <a14>According to the above <a1>To <a13>The method of any one of the preceding items, wherein the subject is a human subject.

[0251] <a15>According to the above <a1>To <a14>The method of any one of the preceding items, wherein the promoting substance is a substance having at least one, preferably two, more preferably three, of the following (i) to (iii): (i) increasing the intracellular amount of a protein having ubiquitination activity; (ii) increasing the intracellular ubiquitination activity; and (iii) promoting the production of a transcription product or a translation product in a cell.

[0252] <a16>According to the above <a1>To <a15>The method according to any one of the above <1> to <39>, wherein the promoting substance is administered in the form of a composition according to any one of the above <1> to <39>.

[0253] <b1>Use of a promoting substance that increases the intracellular amount of a protein having ubiquitination activity in the manufacture of a composition for treating or preventing a TDP-43-related disease.

[0254] <b2>Use of a promoting substance for increasing the intracellular amount of a protein having ubiquitination activity in the manufacture of a composition for increasing intracellular ubiquitination activity.

[0255] <b3>According to the above <b1>or <b2>for use in the treatment of a disease, wherein said protein is LRSAM1 protein.

[0256] <b4>According to the above <b1>To <b3>Use according to any one of the preceding items, wherein the promoting substance is a substance comprising a nucleic acid, and the promoting substance preferably comprises one or more selected from the group consisting of an expression enhancing nucleic acid and an expression vector.

[0257] <b5>According to the above <b1>To <b4>Use according to any one of the preceding items, wherein the cell is a neural cell, preferably a neuron, more preferably a human neuron, even more preferably a human motor neuron.

[0258] <b6>According to the above <b1>To <b5>Use according to any one of the preceding items, wherein the composition is a composition according to any one of <1> to <39> above.

[0259] <c1>A method for inhibiting cell damage, comprising inhibiting the occurrence or progression of cell damage in a neuron by contacting a promoting substance with the neuron.

[0260] <c2>A method for improving clearance of a protein, comprising improving clearance of a TDP-43 protein in a neuron by contacting a facilitating substance with the neuron.

[0261] <c3>A method for promoting maintenance or improvement of neuronal function by contacting a promoting substance with neurons.

[0262] <c4>A method of reducing or inhibiting abnormal localization of a protein in a neuron by contacting a facilitating substance with the neuron.

[0263] <c5>A method of promoting protein degradation in a neuron by contacting a promoting substance with the neuron.

[0264] <c6>A method for modulating protein phosphorylation in neurons by contacting a facilitating substance with the neurons.

[0265] <c7>A method of inhibiting aberrant splicing of RNA, preferably mRNA, in a neuron by contacting the neuron with a facilitating substance.

[0266] <c8>According to the above <c1>To <c7>The method of any one of the preceding items, wherein the neurons are motor neurons, preferably human motor neurons.

[0267] <c9>According to the above <c1>To <c8>The method of any one of the preceding items, wherein the promoting substance is a substance having at least one, preferably two, more preferably three, of the following (i) to (iii): (i) increasing the intracellular amount of a protein having ubiquitination activity; (ii) increasing the intracellular ubiquitination activity; and (iii) promoting the production of a transcription product or a translation product in a cell.

[0268] <c10>According to the above <c1>To <c9>The method of any one of the preceding items, wherein the facilitating substance is a nucleic acid-containing substance, and the facilitating substance preferably contains one or more selected from the group consisting of an expression-enhancing nucleic acid and an expression vector.

[0269] <c11>According to the above <c1>To <c10>The method according to any one of the above <1> to <39>, wherein the promoting substance is used in the form of the composition according to any one of the above <1> to <39>.

[0270] Examples Hereinafter, the present disclosure is described in further detail based on examples. However, the scope of the present disclosure is not limited to such examples. Unless otherwise specified, "% v / v" means "volume / volume %". In addition, in the figures, groups represented by groups represented by "*" show statistical significance at P<0.01. In the figures, groups represented by "*" show statistical significance at P<0.05.

[0271] Example 1 Using ALS as an example of a TDP-43-related disease, the following method was used to construct a pathological model for evaluating cell damage First, human induced pluripotent stem (iPS) cells were induced from cells derived from an ALS patient and cells derived from a healthy individual, respectively, using a conventional method. The cells derived from an ALS patient used were known to have an ALS risk mutation as a heterozygous mutation in the TARDBP gene. Next, each iPS cell was differentiated into a motor neuron (hereinafter, a motor neuron is also simply referred to as a "neuron") using a conventional method.

[0272] In order to long-term preserve the cells, the neurons were subjected to freezing treatment 2 to 7 days after the differentiation treatment, if necessary. The neurons that have been subjected to freezing treatment are also referred to as "frozen neurons".

[0273] Subsequently, the neurons were cultured under the conditions shown below using a 48-well culture plate (Corning, 3548).

[0274] Coating of the culture plate was performed in advance before cell seeding. Specifically, a 0.02% poly-L-omithine solution (Sigma-Aldrich, P4957) was added to the wells of the culture plate and left to stand for 2 hours in a 37°C 5% CO2 incubator. After that, the wells were washed and a 20 pg / mL laminin solution (Thermo Fisher Scientific, 23017015) was used and left to stand for another 2 hours in a 37°C 5% CO2 incubator. After that, the frozen neurons were thawed using a conventional method and the thawed neurons were seeded into the wells at 50,000 cells / well and the neurons were cultured in the presence of a culture medium having the following composition.

[0275] The culture solution used was a mixture having the following composition.

[0276] ​DEM / F 12 (Thermo Fisher Scientific, 21331-020): 50% v / v Neurobasal Medium (Thermo Fisher Scientific, 21103-049): 50% v / v Glutamax Supplement (Thermo Fisher Scientific, 35050061): 1% v / v Penicillin-Streptomycin (10000 units / mL) (Thermo Fisher Scientific, 15140-148): 0.5% v / v Component N1 (Elixirgen): 3% v / v Component A (Elixirgen): 0.1% v / v Component D4 (Elixirgen): 0.1% v / v Component P (Elixirgen): 0.05% v / v Treatments were performed in which, after 7 days of culture, the culture solution in which a solution of dimethyl sulfoxide (final concentration: tunicamycin 0.1 pg / mL, DMSO 0.01% v / v) as stress inducer was dissolved was brought into contact with the neurons and the occurrence of cell damage over time was evaluated. In addition, a group was prepared without stress inducer (to which the same concentration of dimethyl sulfoxide was added).

[0277] To evaluate cell damage, live cell imaging was performed according to the attached protocol using IncuCyte S3 (Sartorius) and the neurite length per unit area (unit: mm / mm2) was analyzed over time, which was used as an evaluation indicator. Smaller values of neurite length indicate that cell damage has occurred. 2

[0278] Cell damage evaluation (1) Figure 1 The results of the neurite length in each experimental group are shown in the graph below, calculated as a ratio based on the results at the start of the stress inducer treatment (0 hours). The smaller the value on the vertical axis, the more the neurite length decreases over time, indicating that cell damage has occurred. Figure 1 The experimental groups in are as follows.

[0279] • Healthy-DMSO: group using neurons derived from healthy individuals without stress inducer • Healthy-Tunicamycin: group using neurons derived from healthy individuals with stress inducer • ALS-DMSO: group using neurons derived from ALS patients without stress inducer​ ALS- tunicamycin: group using neurons derived from ALS patients with stress inducer Neurons derived from ALS patients showed a significant decrease in neurite length compared to neurons derived from healthy individuals. Furthermore, when neurons derived from healthy individuals and neurons derived from ALS patients were treated with tunicamycin, neurite length in neurons derived from ALS patients became significantly shorter than neurite length in untreated neurons, indicating the occurrence of cellular damage. In contrast, a significant decrease in neurite length was not observed in neurons derived from healthy individuals Figure 1

[0280] TDP-43 localization assessment (1) Intracellular localization of TDP-43 protein was assessed by immunofluorescent staining of neurons. Cultured neurons were washed with phosphate buffered saline (PBS) and fixed with 4% paraformaldehyde (PFA). After blocking with PBS containing 5% fetal bovine serum (FBS) and 0.1% Triton-X, primary antibody dilution containing anti-TDP-43 antibody and anti-beta-III tubulin antibody was added and incubated overnight at 4°C. Anti-beta-III tubulin antibody was added for visualization of cell bodies of neurons. The next day, after washing with PBS, secondary antibody dilution conjugated with Alexa dye was added and incubated for 1 hour at room temperature. After further nuclear staining, washing with PBS, and imaging under a fluorescence microscope. Collected images were analyzed using Matlab (Mathworks) to calculate the fluorescence intensity of TDP-43 in the nuclear and cytoplasmic regions, respectively.

[0281] Intracellular localization results of TDP-43 protein are shown in Figure 2 . Figure 2 Experimental groups in

[0282] Healthy- tunicamycin: group using neurons derived from healthy individuals with stress inducer Healthy- tunicamycin: group using neurons derived from healthy individuals with stress inducer ALS- tunicamycin: group using neurons derived from ALS patients with stress inducer ALS- tunicamycin: group using neurons derived from ALS patients with stress inducer Figure 2 Intracellular localization of TDP-43 protein shown in Figure 2 "cytoplasmic / nuclear ratio" in ) is expressed as the ratio of TDP-43 intensity in the cytoplasm to TDP-43 fluorescence intensity in the nucleus. The higher the value of this ratio, the higher the proportion of TDP-43 protein localized in the cytoplasm, which means that abnormal localization occurs.​

[0283] Two-way analysis of variance revealed the fact that neurons derived from ALS patients and treated with stress inducers interact to significantly promote abnormal localization of TDP-43 protein in the cytoplasm. This abnormal localization is a phenomenon similar to that observed in brain tissue of ALS patients.

[0284] Assessing the degree of phosphorylation of TDP-43 (1) The degree of phosphorylation of TDP-43 protein was assessed by immunofluorescence staining of neurons. In addition to the first antibody used for the above-described immunofluorescence staining, in the procedure for the above-described immunofluorescence staining, an anti-phosphorylated TDP-43 (S409) antibody was used instead of an anti-TDP-43 antibody. Other than that, the fluorescence intensity was calculated in the same manner as the above-described procedure.

[0285] Phosphorylation of TDP-43 protein is considered to indicate the following intracellular state. Specifically, it is reported that TDP-43 that is not solubilized in the cytoplasm is phosphorylated. The non-solubilization of TDP-43 in the cytoplasm can cause inhibition of the protein degradation system, mitochondrial toxicity, and the like. In addition, non-solubilized TDP-43 can inhibit the normal nuclear translocation of TDP-43 protein and promote TDP-43 dysfunction in the nucleus. That is, highly phosphorylated TDP-43 protein refers to a state in which the normal function of TDP-43 protein is inhibited and cell damage can occur.

[0286] Figure 3 The abundance of phosphorylated TDP-43 protein (pTDP-43) in the cytoplasm (arithmetic mean of fluorescence intensity) is shown. Figure 3 The experimental groups in Figure 2 are the same as those shown in

[0287] As Figure 3 shown, neurons derived from ALS patients have significantly higher expression of phosphorylated TDP-43 in the cytoplasm compared to neurons derived from healthy individuals. In addition, in the presence of stress inducers, phosphorylation of TDP-43 in neurons derived from ALS patients significantly increased. From this, it was inferred that the normal function of TDP-43 was inhibited.

[0288] Based on the above, this model is considered to be an appropriate experimental model capable of reflecting the pathological condition of ALS patients.

[0289] Example 2 Using the above-described experimental model, the effect of the promoting substance on the cell damage in neurons was evaluated. The target of the promotion of gene expression was LRSAM1. LRSAM1 is one of the proteins having intracellular ubiquitination activity. The promoting substance was a lentiviral vector containing a nucleic acid encoding a wild-type human LRSAM1 protein (SEQ ID NO: 6) (see the CDS base sequence in SEQ ID NO: 5), and the vector was prepared using a conventional method. This promoting substance was formed to promote the production of a transcription product and / or a translation product of LRSAM1 in a cell, and to increase the intracellular amount of the LRSAM1 protein.

[0290] Separately, a lentiviral vector not containing a nucleic acid encoding a wild-type LRSAM1 protein was prepared. This lentiviral vector does not correspond to the promoting substance in the present specification, and even when it is applied to a cell, it does not increase the intracellular amount of a protein having ubiquitination activity.

[0291] In the above-described experimental model, on the third day of the culture, the above-described lentiviral vector was added to the neuronal culture plate at a predetermined multiplicity of infection (MOI) (for example, MOI 0.01 or 0.03), and the culture was continued. Then, on the seventh day of the culture, a stress inducer (or dimethyl sulfoxide alone) was added to achieve the above-described final concentration, and the culture was further continued in the presence of the stress inducer for up to 96 hours.

[0292] Cell damage evaluation (2) Using the same method as in Example 1, the results of the evaluation of the occurrence of cell damage in neurons by cell morphology analysis are shown in Figure 4 and 5 . Figure 4 Results using neurons derived from a healthy individual are shown in Figure 5 Results using neurons derived from an ALS patient are shown in

[0293] As shown in these figures, in neurons derived from an ALS patient, the occurrence or progression of cell damage was significantly inhibited in the group exposed to the promoting substance (see Figure 5 ; MOI 0.03 group). In addition, in neurons derived from a healthy individual, no significant difference was observed in terms of the expression of LRSAM1 protein (see Figure 4 ). In the experimental groups shown in Figure 5 , the inventors confirmed that the expression level of LRSAM1 in neurons had increased.

[0294] TDP-43 localization evaluation (2) Using the same method as in Example 1, the results of the calculation of the cytoplasmic / nuclear ratio of TDP-43 in the presence or absence of the promoting substance are shown in Figure 6A and 6B . Figure 6A and 6B The experimental groups shown are as follows. Figure 6A and Figure 6B In this study, the value of the empty DMSO group in each neuron was set to 100%, and the result for each group was expressed as a percentage of that value.

[0295] • Empty DMSO: A group containing no stress inducers or promoters. • Empty-coated cytokines: A group containing stress inducers but not stress promoters. • LRSAM1-DMSO: The group containing no stress inducers but containing promoting substances. • LRSAM1-Chlamydiaein: A group containing both stress inducers and promoters. like Figure 6A and 6B As shown, by promoting the production of LRSAM1 protein, a translation product, and thereby increasing intracellular ubiquitination activity, the cytoplasm / nucleus ratio was reduced in both neurons derived from healthy individuals and neurons derived from ALS patients. Furthermore, the reduction in the cytoplasm / nucleus ratio was more significant in neurons derived from ALS patients compared to neurons derived from healthy individuals. Therefore, by using a promoter, the abnormal localization and clearance of TDP-43 can be improved, thereby allowing cells such as motor neurons to perform their inherent functions.

[0296] Assess the phosphorylation level of TDP-43(2) Regarding the abundance of cytoplasmic pTDP-43, results derived from neurons of healthy individuals with or without the promoting substance are shown in... Figure 7A In, and the results derived from neurons of ALS patients are shown in Figure 7B middle. Figure 7A and Figure 7B The experimental group and Figure 6A and Figure 6B The experimental groups shown are the same. Figure 7A and Figure 7B In this study, the value of the empty DMSO group in each neuron was set to 100%, and the result for each group was expressed as a percentage of that value.

[0297] As these figures illustrate, the expression of phosphorylated TDP-43 is inhibited by a promoting substance, the expression of which is increased by tunicamycin treatment. As mentioned above, phosphorylation of TDP-43 can lead to inhibition of the proteolytic system and mitochondrial toxicity, and can also cause dysfunction of TDP-43 in the cell nucleus. Therefore, regulating phosphorylation by a promoting substance to inhibit the expression of phosphorylated TDP-43 results in the aforementioned abnormal inhibition. Thus, the promoting substance can be modified to allow cells such as motor neurons to perform their inherent functions.

[0298] Improvement by promoting substance evaluation splicing regulation Focusing on STMN2, which is one of the genes whose splicing is regulated by TDP-43, the amount of production of STMN2 transcription products was evaluated using the following method. Specifically, RNA was extracted from cultured neurons using Quick-RNA Micro Kit (Zymo Research). After measuring the RNA concentration, a certain amount of RNA was reverse-transcribed to synthesize cDNA using Superscript IV Master Mix (ThermoFisher Scientific). Absolute quantitative qPCR was performed by generating a calibration curve using nucleic acids identical to the sequence amplified by qPCR and having a known copy number. By absolute quantitative qPCR, the amount of production of transcription products of full-length wild-type mRNA of STMN2 (hereinafter referred to as STMN2FL, included in the graph) and the amount of production of transcription products of mRNA containing cryptic exon of STMN2 (hereinafter referred to as STMN2CE, included in the graph) were measured.

[0299] Cryptic exon refers to a sequence originally located in an intron region but inadvertently included as an exon, which occurs due to abnormal splicing. A high ratio of the amount of production of STMN2CE to the amount of production of STMN2FL (STMN2CE / STMN2FL) means that the amount of production of non-wild-type transcription products is relatively high, and abnormal splicing is more likely to occur. That is, a high STMN2CE / STMN2FL ratio means that the normal function of TDP-43 is weakened or lost.

[0300] The results are shown in Figure 8 . Figure 8 The experimental groups shown in

[0301] • Healthy - empty - DMSO: a group using neurons from a healthy individual, containing no stress inducer and no promoting substance.

[0302] • Healthy - empty - tunicamycin: a group using neurons from a healthy individual, containing a stress inducer but no promoting substance.

[0303] • Healthy - LRSAM1 - DMSO: a group using neurons from a healthy individual, containing no stress inducer but containing a promoting substance.

[0304] • Healthy - LRSAM1 - tunicamycin: a group using neurons from a healthy individual, containing a stress inducer and a promoting substance.

[0305] • ALS - empty - DMSO: a group using neurons from an ALS patient, containing no stress inducer and no promoting substance.

[0306] ALS-LRSAMl-Empty: a group using neurons from ALS patients, containing neither stress inducer nor facilitator substance.

[0307] ALS-LRSAMl-DMSO: a group using neurons from ALS patients, containing neither stress inducer nor facilitator substance.

[0308] ALS-LRSAMl-Empty: a group using neurons from ALS patients, containing neither stress inducer nor facilitator substance.

[0309] As shown in Figure 8 , neurons derived from ALS patients have significantly higher STMN2CE / STMN2FL ratio than neurons derived from healthy individuals (comparison between Healthy-Empty-DMSO and ALS-Empty-DMSO in Figure 8 ). This indicates that abnormal splicing is more likely to occur in TDP-43 related diseases such as ALS, and the normal function of TDP-43 is weakened or lost.

[0310] In neurons derived from ALS patients, the group treated with tunicamycin has a significantly higher STMN2CE / STMN2FL ratio (comparison between ALS-Empty-DMSO and ALS-Empty-Tunicamycin in Figure 10 ). This indicates that tunicamycin treatment exacerbates abnormal splicing. Furthermore, in the group treated with tunicamycin, expressing LRSAMl with a facilitator substance significantly reduces the STMN2CE / STMN2FL ratio (comparison between ALS-Empty-Tunicamycin and ALS-LRSAMl-Tunicamycin in Figure 10 ). On the other hand, in neurons derived from healthy individuals, no significant change in the STMN2CE / STMN2FL ratio was observed in the groups ( Figure 9 ).

[0311] From the above findings, the normal function of TDP-43 is restored by the facilitator substance in neurons derived from ALS patients.

[0312] Example 3 The effect of the facilitator substance on the degradation of the C-terminal fragment of the TDP-43 protein was evaluated. The C-terminal fragments of the TDP-43 protein of about 35 kDa and about 25 kDa are known. Hereinafter, they will be referred to as CTF35 and CTF25, respectively.

[0313] Similar to the experimental model used in Example 2, on the third day of the neuronal culture, lentiviral vectors were added to the culture plates to achieve a predetermined MOI (e.g., MOI 0.03) and the culture was continued. On the seventh day of the culture, stress inducer (or DMSO only) was added to achieve a predetermined final concentration (e.g., 0.1 pg / mL) and the culture was continued in the presence of the stress inducer for 96 hours. Tunicamycin was used as the stress inducer.

[0314] The cultured cells were lysed on ice using 8M urea, 50mM Tris-HCl solution containing 1x protease / phosphatase inhibitors to obtain cell lysate. The protein concentration in the cell lysate was measured and, after denaturation, the quantification of the C-terminal fragment of TDP-43 protein was performed using the fully automated capillary electrophoresis immunoassay system Wes (Simple Protein). In the automated capillary electrophoresis immunoassay, similar to the Western blot method, the amount of target protein can be quantified by analyzing the detected bands.

[0315] The results of the evaluation of the C-terminal fragment proteins CTF35 and CTF25 of the TDP-43 protein are shown in Figure 11 and 12 . Figure 11 and Figure 12 The experimental groups shown in

[0316] • Healthy - empty - DMSO: group using neurons from healthy individuals, without stress inducer and promoting substance.

[0317] • Healthy - empty - tunicamycin: group using neurons from healthy individuals, containing stress inducer but not promoting substance.

[0318] • Healthy - LRSAM1 - DMSO: group using neurons from healthy individuals, without stress inducer but containing promoting substance.

[0319] • Healthy - LRSAM1 - tunicamycin: group using neurons from healthy individuals, containing stress inducer and promoting substance.

[0320] • ALS - empty - DMSO: group using neurons from ALS patients, without stress inducer and promoting substance.

[0321] • ALS - empty - tunicamycin: group using neurons from ALS patients, containing stress inducer but not promoting substance.

[0322] • ALS - LRSAM1 - DMSO: group using neurons from ALS patients, without stress inducer but containing promoting substance.

[0323] ALS-LRSAM1-tunicam: using neurons from ALS patients, containing a set of stress inducers and promoting substances.

[0324] Wes includes Compass for SW software to quantify the band intensities of full-length TDP-43, CTF35 and CTF25. After that, the quantification values of full-length TDP-43, CTF35 and CTF25 proteins are divided by the sum of TDP-43 quantification values, and the results are shown in Figure 11 and 12 Therefore, in neurons derived from ALS patients, the amount of CTF35 and CTF25 is significantly higher than that in neurons derived from healthy individuals Figure 11 and 12 Comparison between healthy-empty-DMSO and ALS-empty-DMSO in Figure 11 and 12 Comparison between ALS-empty-DMSO and ALS-empty-tunicam in

[0325] Using brain tissue sections from sporadic ALS patients, the degree of co-localization between LRSAM1 protein and TDP-43 protein was evaluated using immunohistochemical staining method. The tissue samples were obtained by TARGET ALS MULTICENTER POSTMORTEM TISSUE CORE.

[0326] From paraffin sections of the brain motor cortex tissue, deparaffinization and dehydration were performed using a conventional method. After that, the cell membrane was permeabilized with PBS containing 0.5% Triton-X, and blocking was performed using Blocking One (Nacalai). Next, after dilution with signal enhancer A (Nacalai), the above-described anti-TDP-43 antibody and LRSAM1 antibody (Sigma) and anti-MAP2 antibody (Abeam) were added as a primary antibody, and incubated overnight at 4°C. The anti-MAP2 antibody was used to visualize neurons. The next day, after washing with PBS, a secondary antibody diluent conjugated with Alexa dye and a DAPI solution for nuclear staining (Invitrogen) were added, and incubated for 1 hour at room temperature. After washing with PBS, the sample was sealed with a coverslip using a mounting solution. The sample was imaged using a fluorescence microscope, and the captured images were analyzed using Matlab (Mathworks). As an analysis method, after surrounding the nucleus and cytoplasm in the image, the signals of TDP-43 and LRSAM1 in the nucleus or cytoplasm of each single cell were calculated, and the degree of colocalization of these signals was calculated as a Pearson correlation coefficient. The higher this value, the higher the degree of colocalization, and it was possible to compare the degree of localization of TDP-43 and LRSAM1 in the nucleus and cytoplasm of neurons.

[0327] The results of the intracellular localization of TDP-43 protein and LRSAM1 in the brains of ALS patients are shown in Figure 13 . The images in Figure 13 are fluorescence staining images of TDP-43 (A), LRSAM1 (B), MAP2 indicating the cytoplasm of neurons (C), and DAPI indicating the nucleus (D), respectively. Figure 13 The dotted lines in A and 13B indicate the positions of the nuclei created from Figure 13 D. Figure 13 A shows that TDP-43 leaks out of the nucleus in neurons in the brain of a sporadic ALS patient, as previously reported. In addition, comparing Figure 13 A and Figure 13 B, colocalization of TDP-43 and LRSAM1 in the cytoplasm was observed, as indicated by arrows.

[0328] Brain samples from six patients were stained and imaged using the above-described method, and the nucleus and cytoplasm were defined from the captured images, and the correlation coefficient was calculated as described above. Figure 14 (A) is a histogram showing the distribution of the correlation coefficient. Comparing the correlation coefficient between TDP-43 and LRSAM1 in the nucleus and cytoplasm, it was found that more cells showed a higher correlation in the cytoplasm than in the nucleus Figure 14 (A)). In addition, when the average correlation coefficient of the nucleus and the cytoplasm was calculated, the correlation coefficient in the cytoplasm was statistically significantly higher (B). In other words, it was shown that extranuclear TDP-43 co-localizes with LRSAM1 at a high rate. Figure 14

[0329] The above results show that a promoting substance targeting a predetermined target (e.g., a target contributing to an increase in ubiquitination activity, such as LRSAM1) can contribute to maintaining or improving normal cellular functions. Therefore, a promoting substance can contribute to favorable effects, such as suppression of cellular damage, improvement of intracellular TDP-43 clearance, proper control of post-translational modification (e.g., phosphorylation), and treatment or prevention of TDP-43-related diseases. In addition, it is assumed that a technique that increases the intracellular amount of a protein having ubiquitination activity, such as LRSAM1, and thereby increases intracellular ubiquitination activity can improve the function and localization of TDP-43 in the normal direction. Therefore, the technique can be a suitable method for treating or preventing TDP-43-related diseases. These problems are new findings that become clear by completing the present disclosure, and can be used, for example, to protect neural cells such as motor neurons.

[0330] In addition, according to the results of the present embodiment, it will be clear to those skilled in the art that the same effects can be obtained even when the expression-enhancing nucleic acid according to the present embodiment is used as a promoting substance targeting a predetermined target (e.g., LRSAM1).

[0331] Focusing on ALS, a TDP-43-related disease, it is known that its pathology is mainly TDP-43 (Ling SC et al., Neuron., 2013; 79(3):416-38). In addition, it is also known that the pathology associated with TDP-43 and FUS is mutually exclusive (Guo L et al., Cold Spring Harb Perspect Med., 2017; 7(9): a024554). In this case, the use of a promoting substance to reduce intracellular abnormal localization of TDP-43 or increase clearance of abnormal TDP-43 protein can reduce cellular damage in motor neurons and the like, which is beneficial for treating or preventing TDP-43-related diseases. In addition, since it has been reported that TDP-43 is the main pathological entity of ALS, by increasing the intracellular clearance of TDP-43 protein, the amount of the main pathological protein can be reduced. Therefore, it will be clear to those skilled in the art that a promoting substance is particularly useful for treating or preventing ALS.

[0332] ​Furthermore, the ALS patient-derived neurons used in the present embodiment have a heterozygous mutation in the TARDBP gene encoding the TDP-43 protein. Thus, in the neurons, roughly, two types of proteins can be produced: normal TDP-43 protein (wild-type TDP-43 protein) and mutant TDP-43 protein. As in the present embodiment, by using the facilitator substance targeting a predetermined target (e.g., LRSAM1), the abundance ratio of normal TDP-43 protein in the cell can be increased. Furthermore, the intrinsic function of TDP-43 can be enhanced, which can contribute to the improvement of normal cell function. In particular, since the mutant TDP-43 protein tends to accumulate in the cytoplasm, the mutant TDP-43 protein is more likely to be degraded by the LRSAM1 protein present in the cytoplasm, whereas the normal TDP-43 protein present in the nucleus is able to exert its intrinsic function in the cell.

[0333] In Patent Literature 1 and Non-Patent Literature 1, there is no discussion on TDP-43 protein and TDP-43-related diseases.

[0334] Non-Patent Literature 1 describes the effects of stress inducer treatment on cell death. However, the cells used in Non-Patent Literature 1 are non-neuronal cells, which have different properties from those of the neuronal cells used in the present embodiment. This is supported by, for example, the fact that the intracellular localization of TDP-43 mutants differs between non-neuronal cells and neuronal cells (Shenouda M et al., Front Neurosci., 2022; 16:868556.). Therefore, the results of the present embodiment using neuronal cells such as motor neurons represent one of the new findings that became clear at the time of completion of the present disclosure, and suggest a new therapeutic or preventive effect against neurological diseases.

[0335] According to the present embodiment, the maintenance or improvement of cell function can be facilitated. This is useful in the field of pharmaceuticals.

[0336] It is clear that many modifications and variations of the present application are possible in light of the above teachings. Therefore, it is to be understood that within the scope of the appended claims, the application can be practiced otherwise than as specifically described herein.

Claims

1. A composition for treating or preventing a TAR DNA binding protein 43-related disease, comprising a promoting substance that increases an intracellular amount of a protein having ubiquitination activity.

2. A composition for improving clearance of TAR DNA binding protein 43 protein, comprising a promoting substance that increases an intracellular amount of a protein having ubiquitination activity.

3. The composition according to claim 1 or 2, for use in a subject who has developed or is likely to develop a TAR DNA binding protein 43-related disease.

4. The composition according to claim 3, wherein the TAR DNA binding protein 43-related disease is amyotrophic lateral sclerosis, frontotemporal lobar degeneration, or age-related TDP-43 encephalopathy predominantly in the limbic system.

5. The composition according to claim 1 or 2, wherein the promoting substance is a substance that increases an intracellular amount of a LRSAM1 protein as a protein having ubiquitination activity.

6. The composition according to claim 5, wherein the protein is a wild-type LRSAM1 protein.

7. The composition according to claim 1 or 2, wherein the promoting substance is a substance that increases an intracellular amount of a LRSAM1 protein by promoting production of a translation product from a nucleic acid encoding the LRSAM1 protein.

8. The composition according to claim 7, wherein the protein is a wild-type LRSAM1 protein.

9. The composition according to claim 1 or 2, wherein the promoting substance is a substance containing a nucleic acid.

10. The composition according to claim 9, wherein the promoting substance comprises one or more selected from the group consisting of an expression-enhancing nucleic acid and an expression vector.

11. The composition according to claim 1 or 2, wherein the cell is a nervous system cell.

12. A method for treating or preventing a TAR DNA binding protein 43 associated disease comprising: a step of administering to a subject in need thereof an effective amount of a promoting substance, wherein the promoting substance is a substance that increases an intracellular amount of a protein having ubiquitination activity.

13. The method according to claim 12, wherein the disease is treated or prevented by administering the promoting substance to the subject to inhibit occurrence or progression of neuronal cell damage.

14. Use of a promoting substance that increases an intracellular amount of a protein having ubiquitination activity in the manufacture of a composition for treating or preventing a TAR DNA binding protein 43-related disease.

15. A method for inhibiting cell damage, comprising inhibiting occurrence or progression of cell damage in a neuron by contacting the neuron with a promoting substance that increases an intracellular amount of a protein having ubiquitination activity.

16. A method for improving clearance of a protein, comprising improving clearance of TAR DNA binding protein 43 protein in a neuron by contacting the neuron with a promoting substance that increases an intracellular amount of a protein having ubiquitination activity.

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