Compositions and methods for the treatment or prevention of TAR DNA-binding protein 43-related diseases

Abstract

A composition for use in the treatment or prevention of TAR DNA-binding protein 43-related diseases, comprising a substance that promotes the increase of intracellular amounts of proteins having ubiquitination activity.

Description

Technical Field

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

[0002] Reference to Sequence Listing This specification refers to the sequence listing electronically submitted as an.xml file named "541677WO_ST26.xml" in accordance with WIPO Standard ST.26. The.xml file was created on March 26, 2024 and has a size of 49,152 bytes. The entire text of the sequence listing is incorporated herein by reference.

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

Background Art

[0004] Patent Document 1 discloses compounds that improve splicing of messenger RNA (mRNA). The same document takes familial dysautonomia as an example of a progressive neurodegenerative disease and discloses a treatment method therefor. The same document takes Charcot-Marie-Tooth disease type 2P as an example and describes suppressing cell death associated with the accumulation of misfolded proteins. The same document discloses that LRSAM1 suppresses the accumulation of misfolded luciferase and reduces cytotoxicity.

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

Prior Art Documents

Patent Documents

[0006]

Patent Document 1

[0007] [Non-Patent Document 1] Non-patent document 1: Mishra R et al., Int J Biochem Cell Biol. 2020; 120:105697

[0008] The entire contents of these publications are incorporated herein by reference. [Overview of the project]

[0009] One embodiment of the present disclosure is a composition for use in the treatment or prevention of TAR DNA-binding protein 43-related diseases, comprising a promoter that increases the intracellular amount of a protein having ubiquitination activity. Another embodiment of the present disclosure is a composition for improving the clearance of TAR DNA-binding protein 43, comprising a promoter that increases the intracellular amount of a protein having ubiquitination activity. Furthermore, one embodiment of the present disclosure includes the step of administering an effective amount of the promoting substance to a subject requiring it, A method for treating or preventing TAR DNA-binding protein 43-related disease, wherein the promoting substance is a substance that increases the intracellular amount of a protein having ubiquitination activity. Furthermore, one embodiment of this disclosure relates to treating or preventing TAR DNA-binding protein 43-related diseases. The use of a promoter that increases the intracellular amount of ubiquitinating proteins in the production of the composition. Another embodiment of the present disclosure is a method for suppressing cell damage, which involves bringing a neuron into contact with a substance that increases the intracellular amount of a protein having ubiquitination activity, thereby suppressing the occurrence or progression of cell damage in the neuron. Another embodiment of the present disclosure is a method for improving protein clearance, which involves bringing a neuron into contact with a substance that increases the intracellular amount of a protein having ubiquitination activity, thereby improving the clearance of the TAR DNA-binding protein 43 protein in the neuron.

[0010] To fully understand the present invention and its many advantages, it is easy to do so by referring to the following detailed description in conjunction with the accompanying drawings. [Brief explanation of the drawing]

[0011] [Figure 1] Figure 1 is a graph showing the degree of cellular damage in motor neurons derived from healthy individuals and motor neurons derived from ALS patients, using neurite length as an indicator. [Figure 2] Figure 2 is a graph showing the cytoplasmic / nuclear abundance ratio of TDP-43 protein in motor neurons derived from healthy individuals and motor neurons derived from ALS patients. [Figure 3] Figure 3 is a graph showing the amount of phosphorylated TDP-43 protein present in the cytoplasm of motor neurons derived from healthy individuals and motor neurons derived from ALS patients. [Figure 4] Figure 4 is a graph showing the effect of the presence or absence of stimulating substances on neurite length in motor neurons derived from healthy individuals. [Figure 5] Figure 5 is a graph showing the effect of the presence or absence of stimulating substances on neurite length in motor neurons derived from ALS patients. [Figure 6A] Figure 6A is a graph showing the cytoplasmic / nuclear abundance ratio of TDP-43 protein in motor neurons derived from healthy individuals, with and without the presence of a stimulating substance. [Figure 6B] Figure 6B is a graph showing the cytoplasmic / nuclear abundance ratio of TDP-43 protein in motor neurons derived from ALS patients, with and without the presence of a stimulating substance. [Figure 7A] Figure 7A is a graph showing the amount of phosphorylated TDP-43 protein present in the cytoplasm of motor neurons derived from healthy individuals. [Figure 7B] Figure 7B is a graph showing the amount of phosphorylated TDP-43 protein present in the cytoplasm of motor neurons derived from ALS patients. [Figure 8] Figure 8 is a graph showing the effect on splicing, using as an index the ratio of mRNA containing cryptic exons (CE) to full-length mRNA (FL) of STMN2, in motor neurons derived from healthy individuals and motor neurons derived from ALS patients. [Figure 9] Figure 9 is a graph showing the effect on STMN2 splicing with or without a promoting substance in motor neurons derived from healthy individuals. [Figure 10] Figure 10 is a graph showing the effect on STMN2 splicing with or without a promoting substance in motor neurons derived from ALS patients. [Figure 11] Figure 11 is a graph showing the effect on the amount of approximately 35 kDa C-terminal fragment of TDP-43 protein (CTF35) with or without a promoting substance 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 fragment of TDP-43 protein was used as an index. [Figure 12] Figure 12 is a graph showing the effect on the amount of approximately 25 kDa C-terminal fragment of TDP-43 protein (CTF25) with or without a promoting substance 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 fragment of TDP-43 protein was used as an index. [Figure 13] Figure 13 is an immunohistofluorescence staining image of TDP-43 protein (A), LRSAM1 (B), MAP2 (C), and DAPI (D) in a section of the brain (motor cortex) of a sporadic ALS patient. MAP2 is used to label the neuronal cytoplasm, and DAPI is used to label the nucleus. The arrows in Figures 13A and B indicate the co-localization of TDP-43 and LRSAM1 in the cytoplasm. [Figure 14]Figures 14A and B show the degree of co-localization of TDP-43 and LRSAM1 in the neuronal nucleus and cytoplasm, respectively, as Pearson correlation coefficients. In Figure 14A, the distribution of the correlation coefficient and the number of cells are shown as a histogram. Figure 14B is a graph comparing the average correlation coefficients of the nucleus and cytoplasm.

Mode for Carrying Out the Invention

[0012] Hereinafter, embodiments will be described with reference to the accompanying drawings. The same reference numerals in the drawings indicate corresponding or identical elements in each figure.

[0013] Hereinafter, the present disclosure will be described based on its embodiments. The descriptions of the patent documents and non-patent documents described in this specification are all incorporated herein by reference. GenBank accession numbers and related sequence information and other data available through the database are all incorporated herein by reference. The sequence listing in electronic format submitted together with this specification is all incorporated herein by reference. The mRNA base sequences disclosed in the sequence listing of the present application and the accession numbers described in this specification are represented with uracil (U) as thymine (T). However, the actual mRNA base sequence is applied with T read as U.

[0014] In one embodiment, the present disclosure relates to a composition. In one embodiment, the composition may be a pharmaceutical composition or a non-pharmaceutical composition such as an experimental reagent.

[0015] TAR DNA-binding Protein of 43kDa is an RNA-binding protein known as TDP-43 It is normally localized in the nucleus. When abnormal TDP-43 proteins (e.g., protein aggregates or protein fragments) are produced, they can contribute to the development of various diseases. TDP-43-related diseases generally manifest as symptoms such as motor dysfunction and cognitive impairment, significantly impacting the quality of life (QOL). It could have a significant impact. However, there is no established effective treatment method that leads to a cure for TDP-43-related diseases.

[0016] In one embodiment, the composition of the present disclosure is used to treat or prevent TAR DNA-binding protein 43 (TDP-43) related diseases. In other words, the composition according to one embodiment is a composition for the treatment or prevention of 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 applications will be described later.

[0017] In this specification, “treatment” means, for example, one or more diseases, disorders, or related conditions. This includes suppressing, reducing, alleviating, and curing the progression of the condition. In this specification, “prevention” includes, for example, preventing or delaying the onset of a disease, and preventing or delaying the recurrence of a disease. The compositions of this disclosure can be used for one or more purposes of treatment and prevention.

[0018] For the sake of clarity, unless otherwise specified, the following explanation will use humans or human cells, which are a type of mammal, as examples of the subjects of this disclosure. However, to the extent that the effects of this disclosure are achieved, it will also apply to other animal species other than humans, including rodents such as mice and rats, primates such as monkeys, and mammals such as rabbits, as well as their cells and orthologs. In the following explanation, when describing an amino acid point mutation in any protein, we will use the following format: "single letter notation of the amino acid before substitution, amino acid sequence number from the N-terminus, and a sentence notation of the amino acid after substitution." The order of representation is "Am(s)" followed by "Am(s)". For example, if it is written as "G294A", it means the amino acids of the specified protein. This means that the glycine at position 294 from the N-terminus of the acid sequence has been replaced with alanine.

[0019] In the following explanation, unless otherwise specified, "nucleic acid" refers to a polymer in which two or more arbitrary nucleosides are linked to each other by nucleoside bonds, regardless of its length or structural framework. The nucleic acid bases, sugars, and nucleoside bonds constituting this polymer may each independently be in their natural form, or they may be in a non-natural form in which specific atoms, functional groups, cyclic structures, etc., are added, substituted, or deleted from the natural form. Furthermore, nucleic acids in this specification may also exist in the form of pharmaceutically acceptable salts or ions, insofar as the effects of this disclosure are achieved. Examples of counterions in pharmaceutically acceptable salts include cations. Examples of such cations include hydrogen ions and inorganic ions such as metal ions. Examples of metal ions include alkali metal ions such as sodium ions and potassium ions.

[0020] TDP-43, an RNA-binding protein, plays a vital role in transcription, splicing, and translation within cells. It is involved in the regulation of [something]. TDP-43 is a protein encoded by the TARDBP gene. The human wild-type TDP-43 protein is a protein consisting of 414 amino acids in its entirety and is mainly localized in the nucleus. It is a nuclear protein. As an example, the mRNA sequence of human wild-type TDP-43 is sequence number 1 This is shown in (GenBank accession number: NM_007375.4). Additionally, the amino acid sequence of the human wild-type TDP-43 protein is shown in Sequence ID No. 2 (GenBank accession number: NP_031401.1).

[0021] In TDP-43-related diseases, the production of abnormal TDP-43 proteins is considered one of the causes. These abnormal proteins typically exhibit characteristics such as increased translocation to the cytoplasm, easier formation of protein aggregates, and altered amounts and / or types of post-translational modifications, such as phosphorylation. Thus, abnormal TDP-43 proteins often have properties that differ from those of the wild type. Examples of abnormal TDP-43 proteins that may contribute to disease onset and progression include the N-terminal fragment of the human wild-type TDP-43 protein (SEQ ID NO: 3; see Shenouda et al., Front Neurosci. 2022; 16: 868556.), the C-terminal fragment protein consisting of amino acid residues 209-414 from the N-terminus of the human wild-type TDP-43 protein (SEQ ID NO: 4), and the N-terminus of the human wild-type TDP-43 protein. C-terminal fragment protein consisting of amino acid residues 90 to 414 from the end (SEQ ID NO: 15), 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), human wild-type TDP-43 protein from the N-terminus 174 to 414 C-terminal fragment protein consisting of amino acid residues of the eye (SEQ ID NO: 17), wild-type TDP-43 protein Examples include amino acid variants in quality (see SEQ ID NO: 2), but are not limited to these. Examples of amino acid variants of the TDP-43 protein include proteins with point mutations such as G294A, G298S, A315T, M337V, Q343R, A382T, or combinations thereof.

[0022] In this disclosure, TDP-43-related diseases include diseases that may arise from abnormal intracellular localization of the TDP-43 protein. Examples of TDP-43-related diseases include neuromuscular diseases, one or more selected from neurodegenerative diseases and muscle diseases (for example, diseases described in literature such as 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., and Walker et al., J Neurosci. 2014 ;34(19):6448-58.). Specifically, neurodegenerative diseases include, for example, 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, and frontotemporal syndrome. Examples include holophyllic disease (FTLD), limbic-dominant late-stage TDP-43 encephalopathy (LATE), and Huntington's disease. Examples of muscle diseases include inclusion body myositis. By targeting these diseases, we can effectively treat or prevent TDP-43-related diseases by addressing specific pathological conditions such as abnormal intracellular localization and clearance abnormalities of the TDP-43 protein. This can be achieved. In other words, in one embodiment, the above-described composition can also be described as a composition for improving the clearance of the TDP-43 protein. From the viewpoint of further enhancing the effectiveness of treating or preventing the disease, the TDP-43-related disease is preferably a neurodegenerative disease selected from ALS, FTLD, and LATE, and is more preferably at least ALS.

[0023] When the above-described composition is used to treat or prevent TDP-43-related disease, the subject to which it is applied is preferably a subject who has developed or is likely to develop TDP-43-related disease, and may or may not have specific mutations in a particular gene. In other words, the above-described composition can be used on subjects that do not have mutations in a particular gene (e.g., homozygous normal). Furthermore, the above-described composition can also be used on subjects that have one or more mutations selected from a particular gene. If the subject has gene mutations, each of these gene mutations may be heterozygous or homozygous, independently of the others. The subject is preferably a human subject (a living human organism) or human-derived cells. The presence or absence of these mutations can be determined by known methods such as PCR.

[0024] Taking ALS, a type of TDP-43-related disease, as an example, gene mutations in humans include those in the C9orf72, SOD1, TBK1, TARDBP, FUS, and NEK1 genes. Examples include, but are not limited to, one or more gene mutations. In one embodiment, the subject described above has, for example, a homozygous or heterozygous mutation in the TARDBP gene and FUS gene The child does not have a mutation in the SOD1 gene or / or the SOD1 gene. The aforementioned gene mutations in humans include, for example, one or more selected from the following: abnormal elongation of a 6-base repeat sequence within intron 1 of the C9orf72 gene; mutations in the SOD1 gene that cause A4V amino acid point mutations; mutations in the TBK1 gene that cause T4A amino acid point mutations; mutations in the TARDBP gene that cause A382T amino acid point mutations; mutations in the FUS gene that cause P525L amino acid point mutations; and mutations in the NEK1 gene that cause R261H amino acid point mutations.

[0025] The base sequence of the TARDBP gene may vary from individual to individual. In this embodiment, as long as it encodes a protein having the same function as the wild-type TDP-43 protein, the base sequence of TDP-43 mRNA is 80% or more, 85% or more, 90% or more, and 95% or more of the base sequence described in Sequence ID No. 1. The above may also be base sequences with 98% or more or 100% identity. Examples of what can cause this include, but are not limited to, degenerate codons. Furthermore, insofar as it encodes a protein having the same function as the wild-type TDP-43 protein, the amino acid sequence of the TDP-43 protein may be an amino acid sequence that is 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, or 100% identical to the amino acid sequence described in Sequence ID No. 2. Searching for sites exhibiting sequence identity, complementarity, or homology, and calculating complementarity percentages, as described herein, can be performed using known software or methods, such as the BLAST program or Genetyx software. In this specification, "complementary" or "complementarity" means that a base pair is formed between two nucleic acid bases. It means the ability to complement each other. For example, adenine is complementary to thymidine or uracil, and cytosine is complementary to guanine.

[0026] In one embodiment, the composition of the present disclosure includes a promoting agent. The promoting agent has one or more characteristics that increase the amount and / or activity of a predetermined translation product in an application, including various cells such as cells, specimens, and living organisms. In the following description, the substance will also be simply referred to as the "promoting agent." In one embodiment, the promoting substance directly or indirectly promotes gene expression from a predetermined nucleic acid. This increases the intracellular amount of at least a predetermined translation product. In this specification, "gene expression" includes not only the synthesis (production) of translation products based on information of a given nucleic acid base sequence, but also the synthesis (production) of transcripts. In detail, the promoting substance according to one embodiment promotes at least one of the following within a cell: the generation of a transcript from a nucleic acid encoding a predetermined protein, the generation of a translation product, the function of the transcript, and the function of the translation product. Specific examples of nucleic acids to be promoted include genomic DNA. These can include polymers of deoxyribonucleotides such as endogenous DNA, introduced DNA, and synthetic DNA such as complementary DNA; and polymers of ribonucleotides such as RNA. Examples of such promoting 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 promoting substance in this disclosure may be the translation product itself that is to be increased. The above-mentioned promoting substances may be used individually or in combination of two or more. Preferred embodiments of the promoting substances will be described later.

[0027] Preferably, the transcript is any RNA synthesized using DNA as a template. Whether or not loss occurs is irrelevant. Specific examples of RNA include mRNA precursors, mature mRNA, and myRNA. Polyribonucleotides of various types, such as non-coding RNAs like chloro RNA (miRNA). Mer is an example. In one embodiment, the above-mentioned RNA is preferably an mRNA precursor, and / or This is mature mRNA. An increase in the expression level of transcripts such as mRNA increases the amount of translational template, which can contribute to an increase in the expression level of the target protein. Examples of translation products include proteins produced through translation from any mRNA, regardless of whether or not they have undergone post-translational modifications. These transcripts and translation products are independently produced, for example, from a given DNA. This may include wild-type products that are translated, various splicing variants, sequence variants such as base substitutions (including SNPs), and non-wild-type proteins translated from them. That's fine. The various DNAs, transcripts, and translation products mentioned above may be made publicly available, independently, as needed. The knowledge may be extracted or purified in one or more steps to be isolated.

[0028] The transcript is preferably capable of producing a wild-type protein or a protein with equivalent function. Similarly, the translation product is preferably a wild-type protein or a protein with equivalent function. Producing these products facilitates the maintenance or normalization of the cell's inherent functions, further reducing the occurrence or progression of cell damage. As a result, it can contribute more effectively to the treatment or prevention of disease. To achieve the above-mentioned preferred embodiments, methods such as introducing foreign nucleic acids encoding the target protein or promoting splicing of the target protein into translatable mature mRNA, as shown in the examples described later, are possible, but are not limited to these. To determine whether the generated translation product has the same function as the wild type, for example, the enzyme activity measured by a known method can be compared with the activity of the wild type translation product.

[0029] In this specification, "enhancement" means at least one of the following: an increase in the abundance (e.g., expression level) of a transcript and / or translation product from a given gene, and an increase in the function (e.g., activity) of the translation product, in the presence of any substance. For example, enhancement can be determined if the expression level of the transcript or translation product when the substance under evaluation is brought into contact with or exposed to a given target is greater than the expression level of the transcript or translation product in the absence of the substance under evaluation. In addition to or instead of this, enhancement can also be determined by an increase in the activity of the protein itself or an enzyme within the cell. The criteria for determining whether or not something is promoting it may be, for example, based on the magnitude of the numerical values ​​of the measured values ​​themselves obtained by any measurement method, or the arithmetic mean calculated from the measured values ​​or their ratios. The determination may be based on the magnitude of the mean or median, or on whether there is a statistically significant difference. When evaluating based on the ratio of measured values, for example, the ratio of the measured value R2 in the experimental group being evaluated to the reference measured value or reference value R1 (R2 / R1) is an example. For example, if the increase is 1.05 times or more, for instance 1.10 times or more, for instance 1.30 times or more, for instance 1.50 times or more, or for instance 2.00 times or more, it can be determined that the increase has been accelerated.

[0030] To promote the production of transcripts, for example, by promoting the transcription from DNA to mRNA precursors, or mRNA This includes one or more of the following: inhibition of precursor degradation, inhibition of mature mRNA degradation, or regulation or activation of RNA processing that forms mature mRNA from mRNA precursors. One or more methods for promoting the production of translation products include, for example, promoting translation from mature mRNA and suppressing the degradation of proteins produced through translation.

[0031] The expression level of transcripts can be measured using various methods such as PCR, microarrays, and RNA sequencing, with a sample such as cultured cells, a living organism, or a specimen collected from such organism. These measurement methods are preferably quantitative. If necessary, the transcript can be extracted from the sample, or complementary DNA (cDNA) can be synthesized using the transcript as a template via a reverse transcription reaction, and these can be used in the measurement described above. It may be provided. The expression level or activity of translation products can be measured using, for example, cultured cells, living organisms, or specimens collected from such organisms, using ELISA, Western blotting, flow cytometry, etc. Various measurement methods can be used, such as immunohistochemistry, mass spectrometry, intracellular or in vivo accumulation of substrates with added fluorescent or radioactive substances, and in vitro activity measurement using substrates. The method is preferably one that allows for quantitative analysis. If necessary, the translation product may be extracted from the measurement sample described above, and the extract may be used for the measurement described above.

[0032] The promoting substance in this disclosure is preferably a substance that increases intracellular ubiquitination activity. Ubiquitination activity refers to the activity of adding ubiquitin to a target protein and includes one or more reaction steps of a ubiquitination reaction. One embodiment of the promoting substance is preferably a substance that increases the intracellular amount and / or intracellular activity of proteins having ubiquitination activity. Another embodiment of the promoting substance is preferably a substance that at least increases the intracellular amount of proteins having ubiquitination activity. These embodiments make it possible to improve the state of cells by increasing intracellular ubiquitination activity, for example. In another embodiment, the promoting substance is a substance that directly or indirectly promotes the production and / or activity of a translation product, assuming that the translation product is a protein having ubiquitination activity. This makes it possible to increase the amount of the translation product as a protein having ubiquitination activity.

[0033] To increase the amount of ubiquitinating protein in a cell, for example, this can be done by promoting the production of transcripts or translation products. For example, the promoting substance may promote the production of translation products by promoting translation from transcripts encoding ubiquitinating proteins. Alternatively, the promoting substance may promote the production of target translation products by increasing the expression level of transcripts or by controlling the expression of other genes that control the expression level of the target translation product. Other means of achieving this include, for example, introducing the ubiquitinating protein itself into the cell. In any case, these embodiments can, for example, increase intracellular ubiquitinating activity and make it easier to improve the state of the cell.

[0034] The proteins with ubiquitination activity described above are various ubiquitination-related proteins. Examples of such proteins include ubiquitin ligases such as LRSAM1 (leucine rich repeat and sterile alpha motif containing 1), Gp78 (Glycoprotein 78), CHIP (Carboxyl terminus 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 and ubiquitin-conjugating enzymes. However, TDP-43, mentioned above, is excluded from the list of proteins that possess ubiquitinating activity. To determine whether the generated protein or translation product has ubiquitinating activity, methods such as ELISA, Western blotting, flow cytometry, immunohistochemistry, or the TR-TUBE method described later can be used to determine the increase or decrease in ubiquitin content or activity with or without exposure to a promoting substance. For example, the criteria described above can be used for this determination.

[0035] The human mRNA base sequence and protein amino acid sequence encoding the aforementioned ubiquitin ligase are shown below, for example. • LRSAM1: Sequence ID No. 5 (mRNA base sequence; GenBank accession number NM_001005373.4), and Sequence ID No. 6 (amino acid sequence; GenBank accession number NP_001005373.1) Gp78: Sequence ID 7 (mRNA base sequence; GenBank accession number NM_001144), and Sequence ID 8 (amino acid sequence; GenBank accession number NP_001135.3) CHIP: Sequence ID 9 (mRNA base sequence; GenBank accession number NM_005861.4), and Sequence ID 10 (amino acid sequence; GenBank accession number NP_005852.2) • RNF19A: Sequence ID 11 (mRNA base sequence; GenBank accession number NM_183419), and Sequence ID 12 (amino acid sequence; GenBank accession number NP_056250.3) • MGRN1: Sequence ID No. 13 (mRNA base sequence; GenBank accession number NM_015246.4), and sequence number No. 14 (amino acid sequence; GenBank accession number NP_056061.1)

[0036] In one embodiment, the promoting substance is preferably a protein having ubiquitination activity that increases the intracellular amount of LRSAM1 protein. In one embodiment, the promoting substance is preferably a substance that promotes the production of transcripts and / or translation products from nucleic acids encoding LRSAM1 protein, and more preferably a substance that promotes the production of translation products. As a result, the promoting substance can increase the intracellular amount of LRSAM1 protein by at least promoting the production of proteins having ubiquitination activity, such as LRSAM1 protein. The LRSAM1 protein described above is preferably human LRSAM1 protein, preferably wild-type LRSAM1 protein, and more preferably wild-type human LRSAM1 protein.

[0037] LRSAM1 is the gene that encodes the LRSAM1 protein, a type of ubiquitination-related protein. The mRNA sequence of human LRSAM1 is represented, for example, by SEQ ID NO: 5. The full-length amino acid sequence of human wild-type LRSAM1 protein is represented, for example, by SEQ ID NO: 6. The LRSAM1 protein is mainly located in the cytoplasm and has ubiquitination activity for target proteins. The LRSAM1 protein ubiquitinates target proteins (e.g., abnormal proteins). This process catalyzes the addition of a compound. As a result, the ubiquitinated target protein is degraded by the ubiquitin-proteasome system within the cell. Consequently, it may contribute to the promotion of clearance of abnormal proteins, the suppression of cytotoxicity caused by the presence of abnormal proteins, and ultimately, the treatment or prevention of TDP-43-related diseases.

[0038] In one embodiment, it is more preferable that the promoting agent is a substance that promotes the production of a transcript capable of expressing wild-type LRSAM1 protein or a protein having equivalent function to the wild-type protein. In addition to or instead of this, it is more preferable that the promoting agent is a substance that promotes the production of wild-type LRSAM1 protein or a protein having equivalent function to the wild-type protein as a translation product. When using a promoting agent capable of expressing wild-type LRSAM1 protein, the protein The amino acid sequence of the protein is preferably the full-length wild-type sequence.

[0039] By increasing the expression levels of transcripts and translation products from nucleic acids encoding ubiquitination-related proteins such as LRSAM1 (e.g., genomic genes, introduced nucleic acids, and mRNA), or by enhancing the function of these products, the intracellular quantity of LRSAM1 and / or the intracellular activity of ubiquitination can be increased. This effectively contributes to the maintenance or improvement of cellular function. Specifically, it can facilitate the normalization of intracellular clearance of abnormal proteins by promoting the induction of autophagy or by promoting the degradation of abnormal proteins that contribute to the onset or progression of TDP-43-related diseases through ubiquitination. As a result, it may contribute to the treatment or prevention of TDP-43-related diseases. These functions may be particularly pronounced by expressing wild-type proteins or proteins with equivalent function.

[0040] The base sequence of the gene or nucleic acid encoding the LRSAM1 protein may vary from individual to individual. Therefore, as long as the encoded protein has equivalent function to the wild-type LRSAM1 protein (e.g., ubiquitination activity), it does not need to be 100% identical to the above sequence. For example, the base sequence of the human LRSAM1 gene may be a base sequence that is 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, or 100% identical to the wild-type base sequence. . Furthermore, insofar as it encodes a protein having the same function as the wild-type LRSAM1 protein, the mRNA sequence of LRSAM1 may be a sequence that is 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99% or more, or 100% identical to the sequence described in Sequence ID No. 5. In other words, the mRNA sequence of LRSAM1 does not have to contain any deletions, substitutions, or additions of bases; for example, it may contain deletions, substitutions, or additions of 1 to 625 bases (or 1 to 468, 1 to 312, 1 to 156, 1 to 63, or 1 to 31 bases). Examples of such mRNA sequences include, but are not limited to, degenerate codons. Furthermore, insofar as it encodes a protein having equivalent function to the wild-type LRSAM1 protein, the amino acid sequence of the LRSAM1 protein may be an amino acid sequence that is 80% or more, 85% or more, 90% or more, 95% or more, 99% or more, or 100% identical to the amino acid sequence described in SEQ ID NO: 6. In other words, the LRSAM1 protein does not have to have any amino acid deletions, substitutions, or additions, for example, 1 to 145 (or 1 to 108, 1 to 72, 1 to 36, 1 to 14, 1 to 7) amino acids. The no acids may be deleted, substituted, or added. To determine whether the generated translation product has equivalent function to the wild-type LRSAM1 protein, for example, the Trypsin-Resistant Tandem Ubiquitin-Binding Entity (TR-TUBE) method can be used. By measuring the ubiquitination activity or the amount of ubiquitinated protein, the determination can be made by comparing it with the activity or amount in the wild-type translated product. A suitable determination criterion with equivalent functionality is, for example, a percentage of 70% or more, preferably 80% or more, relative to the wild-type result.

[0041] In one embodiment, the promoting substance is preferably a substance containing nucleic acid. More specifically, the promoting substance is preferably composed of one or more substances selected from expression-enhancing nucleic acids and expression vectors. The expression-enhancing nucleic acid refers to a nucleic acid used to express a target gene, and is preferably an introduced nucleic acid. In one embodiment, the expression-enhancing nucleic acid is preferably a nucleic acid used to promote the expression of a target gene. The target of gene expression promotion is, for example, one or more proteins such as LRSAM1, Gp78, CHIP, RNF19A, and MGRN1 mentioned above. These are endogenous nucleic acids (e.g., genomic DNA) or exogenously introduced nucleic acids. The aforementioned promoting substances alone They may be used individually, or multiple types may be used in combination. By including such substances, it becomes easier to control the amount of translation products with ubiquitination activity (for example, increased gene expression such as increased production of transcripts such as mRNA and / or translation products such as proteins). As a result, abnormal proteins such as TDP-43 mutant proteins This method can effectively normalize the intracellular clearance of abnormal proteins. This is advantageous because, by using the LRSAM1 protein as the target translation product and, for example, using the nucleic acid encoding the LRSAM1 protein as the target nucleic acid for increased expression, it is possible to enhance the ubiquitination of abnormal proteins and further improve their intracellular clearance.

[0042] Examples of nucleic acids used to enhance expression include antisense RNA, non-coding RNA, and small molecule active RNA. Examples include, but are not limited to, one or more nucleic acids selected from sexually transmitted RNA, etc. These gene expression-enhancing nucleic acids have the function of enabling the expression of a target gene within a cell and / or promoting the expression of said gene within a cell. This allows for an increase in the amount of the target translation product within the cell. These nucleic acids can be obtained or manufactured, for example, by screening using known methods. In one embodiment, it is preferable that the expression-enhancing nucleic acid has fewer bases than the nucleic acid contained in the expression vector described later. In one embodiment, it is preferable that the expression-enhancing nucleic acid has a base count in the range of, for example, 5 to 300 bases per strand, and for example, in the range of 10 to 100 bases per strand. It's nice.

[0043] Antisense nucleic acids used as expression-enhancing nucleic acids can take the following forms: (a) nucleic acids that regulate RNA processing, (b) nucleic acids that are partially or fully complementary to miRNA, and (c) nucleic acids that are partially or fully complementary to Natural Antisense Transcript (NAT). These antisense nucleic acids may bind complementaryly to the entire length of the target nucleic acid, or complementaryly to a portion of the sequence of the target nucleic acid.

[0044] The above embodiment (a) involves binding complementaryly to the splicing-involved sequence in the mRNA precursor, RNA processing, such as splicing, is promoted to generate the desired mature mRNA (e.g., mRNA capable of translating the full-length wild-type protein). This allows the target gene to be generated. It contributes to increased expression (specifically, increased production of transcripts such as mRNA and / or translation products such as proteins). The above embodiment (b) binds complementaryly to miRNA and inhibits the function of miRNA. miRNA is a type of nucleic acid that generally binds complementaryly to the 3'UTR region of mRNA. Therefore, miRNA increases the degradation of mRNA or suppresses translation from mRNA, thereby inhibiting the function of the target gene. It works to suppress expression. In this embodiment, the antisense that binds complementary to miRNA Therefore, the function of miRNA is weakened or eliminated, and the repression of gene expression is released. This promotes mRNA production and translation from mRNA, thereby contributing to increased expression of the target gene (specifically, increased production of transcripts such as mRNA and / or translation products such as proteins). The above embodiment (c) binds complementaryly to NAT and inhibits the function of NAT. NAT is a type of nucleic acid that is produced in cells and binds complementaryly to any region of a specific mRNA. In other words, NAT increases the degradation of mRNA or suppresses translation from mRNA. This embodiment is the above embodiment (b) The same mechanism as in (b) reduces or eliminates the function of NAT, thereby releasing the repression of gene expression. This promotes mRNA production and translation from mRNA, contributing to increased expression of the target gene (specifically, increased production of transcripts such as mRNA and / or translation products such as proteins).

[0045] In addition to these, antisense proteins used as expression-enhancing nucleic acids include, for example, mRNA translation inhibitory elements (TIEs), upstream ORFs (uORFs), and immature stop codons (PTCs). Examples include nucleic acids that can bind complementarily to regions containing various sites, such as repeating sequences of guanine and adenine. These antisense proteins may contribute to the suppression of mRNA degradation, the promotion of translation from mRNA, or the promotion of mRNA production capable of generating wild-type proteins or proteins with equivalent function. This can lead to increased expression of the target gene (specifically...) It contributes to increased production of transcripts such as mRNA and / or translation products such as proteins. These expression-enhancing nucleic acids are preferable because they contribute to increasing the production of the target wild-type protein, thereby facilitating the wild-type protein's inherent function and improving the clearance of abnormal proteins.

[0046] Other antisense nucleic acids used to enhance gene expression include, for example, nucleic acids that bind complementaryally to regulatory RNAs that contribute to gene expression repression, and RNA-binding proteins that destabilize mRNA. Examples include nucleic acids that bind complementaryally to the recognition sequence of a protein. In this case, the function of RNA or protein that acts to repress gene expression is suppressed, thus releasing the repression of gene expression. This promotes mRNA production and translation from mRNA, contributing to increased expression of the target gene (specifically, increased production of transcripts such as mRNA and / or translation products such as proteins).

[0047] The number of base pairs in the antisense nucleic acid used as an expression-enhancing nucleic acid is typically 10 to 30 base pairs per strand, preferably 14 to 25 base pairs per strand. The antisense used as an expression-enhancing nucleic acid, in any of the above-described embodiments, has at least 75%, 80%, 85%, 90%, 95%, or 100% complementarity in the region having homology (identity) with the complementary strand of the target nucleic acid base sequence. It may have. For example, if the antisense base sequence is 20 bases, the nucleic acid base sequence of the antisense may not have any deletions, substitutions, or insertions when compared to the nucleic acid base sequence in the complementary strand of the target nucleic acid base sequence, or it may have, for example, 1 to 5 (or 1 to 4, 1 to 3, 1 to 2, or 1) nucleic acid bases deleted, substituted, or inserted. Such nucleic acids can be screened, designed, and obtained, for example, by known methods.

[0048] Non-coding RNAs include nucleic acids such as regulatory RNA and SINE element-containing translation upregulator (SINEUP). Regulatory RNA is an RNA that promotes transcription from promoters incorporated into endogenous DNA such as genomic DNA or introduced nucleic acids, thereby facilitating transcription from target nucleic acids to mRNA. This promotes the production of the target translation product (e.g., LRSAM1 protein). It is possible. SINEUP is an RNA molecule containing a SINE factor sequence and a domain that promotes translation into proteins. It is a polyribonucleotide having a domain with a sequence complementary to mRNA. By using this, translation from mRNA is promoted (i.e., it mainly contributes to the increased production of translation products). The number of base pairs in non-coding RNA can be set appropriately depending on its type and function.

[0049] Small activated RNA (saRNA) is a part or all of the promoter sequence in genomic DNA. It is a nucleic acid complementary to the base sequence. This promotes translation from mRNA and contributes to increased expression of the target gene (specifically, increased production of transcripts such as mRNA and / or translation products such as proteins). The number of base pairs in saRNA is typically 10 to 30 per strand, preferably 18 per strand. It consists of approximately 24 base pairs. saRNA is typically single-stranded or double-stranded RNA.

[0050] When the expression-enhancing nucleic acid is included as a promoting substance, the expression-enhancing nucleic acid can be, for example, single-stranded or double-stranded, insofar as the effects of this disclosure are achieved. The structural framework of these nucleic acids may be, for example, deoxyribonucleotides, ribonucleotides, non-nucleotides containing bases such as pyrrolidine or piperidine, or combinations thereof. The constituent units of nucleic acids in expression-enhancing nucleic acids are natural, insofar as the effects of this disclosure are achieved. It may be a native form or a non-native form. Examples of non-native forms include nucleic acids in which sugars, bases, or atoms or molecules constituting the nucleoside bond in the nucleotide are substituted, modified, or deleted from the native nucleotide.

[0051] In one embodiment, the promoting substance is, for example, an expression vector configured to express a target gene. More specifically, the promoting substance is an expression vector comprising a nucleic acid containing a base sequence encoding a protein or substance whose expression is promoted, and capable of expressing the target protein or substance in a cell. Such an expression vector can express a target transcript and / or translation product in a subject and increase the amount of the translation product in various environments such as in vivo, in vitro, or ex vivo. This can lead to beneficial effects such as maintaining or improving cell function or improving the clearance of abnormal proteins.

[0052] In this specification, "expression vector" means a vector comprising a nucleic acid including a promoter base sequence and a first base sequence operably linked to the promoter base sequence, capable of generating a new nucleic acid or protein in a cell or in vitro based on the information of the first base sequence. The first base sequence may, for example, be configured to generate mRNA encoding a predetermined protein, or to generate the expression-enhancing nucleic acid described above. In other words, the expression-enhancing nucleic acid itself is not included in the "expression vector". The nucleic acids constituting the expression vector may further contain, for example, a replication origin sequence for replication in cells or a selection marker sequence such as a drug resistance gene within their base sequence. Promoter base sequences may include sequences expressible in mammalian cells such as SV40 and CMV, sequences expressible in E. coli such as trp, lacI, and lacZ, and sequences expressible in vitro such as SP6 and T7. These are some examples, but are not limited to them.

[0053] The type of expression vector is not particularly limited and examples include lipid particles or plasmid vectors in which nucleic acids are encapsulated in a lipid membrane, or viral vectors in which nucleic acids are encapsulated in a capsid. The aforementioned viral vectors include, for example, DNA-containing viral vectors such as adenovirus vectors and adeno-associated virus vectors, as well as retroviral vectors and lentiviral vectors. Examples include RNA-containing viral vectors such as the following. Polynucleotides contained in viral vectors Examples of the constituent units of a rheotide include a deoxyribonucleotide skeleton or a ribonucleotide skeleton. It is preferable that each of these skeletons is in its natural form, free from modified nucleic acids or atomic substitutions.

[0054] When the expression vector described above is configured to produce a predetermined protein, the produced protein may or may not contain mutations in its amino acid sequence, such as deletions, substitutions, or additions of amino acids, as long as it possesses the intended function. The identity between the produced protein or the amino acid sequence of the protein and the amino acid sequence of the protein or the corresponding wild-type protein is, for example, 80% or more. It can be 85% or more, 90% or more, 95% or more, or 100%. Furthermore, when nucleic acids are introduced externally into cells or subjects for the purpose of producing a specific protein, the identity of the nucleic acid base sequence can be determined based on the designed nucleic acid base sequence, and it is acceptable for other mutations to inevitably be included during the transcription and / or translation process within the cell.

[0055] In one embodiment, the promoting substance may include a substance that enhances the function of the translation product. One or more such promoting substances may be included in the composition. By including such promoting substances, the expression level or activity of proteins that have been reduced or may be reduced by the disease can be controlled to increase, thereby effectively treating or preventing the disease. Substances that promote the function of translation products include, but are not limited to, allosteric regulators of proteins and stabilizers of post-translational modifications.

[0056] Each of the aforementioned promoting substances can be produced independently, for example, by methods known in the art.

[0057] The substances that can be applied to the promoting agent include, for example, living subjects such as humans and non-human animals, and samples derived from these animals, preferably living humans or samples derived from humans. The substances that can be applied to the promoting agent may be healthy subjects (regardless of whether or not they are at risk of developing a disease), subjects in which a disease has already developed (for example, human patients or disease model animals), or samples derived from these subjects. In other words, the promoting agent can be used in various environments such as in vivo (including or excluding humans), in vitro (including humans), or ex vivo (including humans). The subjects that can be applied are more preferably human subjects, and even more preferably healthy individuals or human patients. Examples of non-human animals include rodents such as rats, mice, and guinea pigs; monkeys; pigs; dogs; and mammals other than humans, such as cats. In one embodiment, the non-human animal is a terrestrial mammal. Examples of samples include, but are not limited to, one or more types of tissue, cells, and bodily fluids. The promoting substance is preferably applied to an object containing at least one cell. Such objects may be, for example, isolated or pure-cultured cells themselves, or to a sample containing cells, or to a subject such as a living organism composed of cells.

[0058] Examples of tissues include brain regions such as the cerebrum, midbrain, diencephalon, pons, medulla oblongata, and cerebellum; the spinal cord; muscles such as the stomach, pancreas, kidneys, liver, adrenal glands, skin, skeletal muscle, and smooth muscle; the lungs, intestines such as the large and small intestines; the heart; and blood vessels. These are typically composed of aggregates of cells. Examples of cells include differentiated cells, progenitor cells, or stem cells that constitute tissues. Taking brain-derived cells as an example of a sample derived from the brain, examples include neurons, glial cells such as astrocytes, microglia, and oligodendrocytes, neural progenitor cells, and various other nervous system cells such as neural stem cells. Among these, it is preferable that the cells on which the promoting substance of this disclosure acts are these nervous system cells. These cells may be used as aggregates of the same type of cells obtained by isolation or pure culture, or as aggregates of cells containing two or more different types of cells. Examples of bodily fluids include cerebrospinal fluid, blood, serum, plasma, saliva, urine, sweat, and other liquid components or extracts thereof. These samples can typically be collected from living or dead animals by known methods such as biopsy or dissection. If necessary, one or more extraction or separation steps, such as isolation, may be performed.

[0059] Other forms of cells that can be used include, for example, primary cultured cells, immortalized cell lines, or diverse stem cells such as ES cells and iPS cells.

[0060] The above-mentioned promoting substance or composition containing the substance is solid at 1 atmosphere and 20°C, depending on its mode of use. Alternatively, it may be in the form of a liquid. Unless otherwise specified, descriptions of properties in this specification refer to conditions at 1 atmosphere and 20°C. The liquid may be a solution containing a solvent, or a dispersion containing a dispersion medium.

[0061] When the above-mentioned promoting substance is used in a compositional form, the composition may further contain a carrier as needed. These carriers must be pharmaceutically acceptable carriers for the target application. This is preferable from the viewpoint of reducing the occurrence of unintended effects. Any carrier used in this art can be used without particular limitation. Examples of carriers include excipients, disintegrants, disintegration aids, binders, lubricants, coatings, dyes, diluents, bases, solvents, solubilizers, isotonic agents, pH adjusters, stabilizers, propellants, and adhesives. These carriers can be used individually or in combination of two or more.

[0062] As a base material or diluent, various liquids can be used, such as water, electrolyte-containing water including saline solution, monohydric alcohols with 1 to 3 carbon atoms including methanol, ethanol, and propanol, polyhydric alcohols including glycerin, or culture media for cell culture. These liquids can be used individually or in combination of two or more as the solvent or dispersion medium described above to constitute the composition.

[0063] The above description pertains to the promoting substance and composition. Below, another embodiment will be described. The following description will primarily focus on aspects that differ from the embodiments described above. For aspects not specifically described, the provisions described herein will apply as appropriate. The provisions described herein can be applied individually or in combination of two or more.

[0064] In one embodiment, this disclosure relates to a method for treating or preventing TDP-43-related diseases. In one embodiment, the method includes administering a promoting substance as an active ingredient to a subject who requires it. In another embodiment, the method administers an effective amount of the active ingredient to a subject who requires it. This makes it possible to effectively treat or prevent TDP-43-related disease. Subjects include, for example, healthy individuals and subjects who have developed or are at risk of developing TDP-43-related disease, and preferably human subjects. Subjects who are at risk of developing TDP-43-related disease include, for example, healthy individuals who do not currently have the disease but are at risk due to factors such as genetic mutations or environmental factors, or patients with diseases other than TDP-43-related disease. Subjects who are at risk of developing TDP-43-related disease can be determined, for example, by measuring the presence or absence of genetic mutations associated with the disease using a known method. In one embodiment, the method involves administering the active ingredient to a subject (e.g., various living organisms such as humans) to suppress the onset or progression of nerve cell damage. This makes it possible to treat or prevent TDP-43-related diseases more effectively.

[0065] In this specification, "cell disorder" includes one or more cases in which the morphology of a cell is normal but the functions inherent in a normal cell are reduced or lost, the morphology of a cell changes to a form different from that of a normal cell, and cell death such as apoptosis or necrosis resulting from these conditions. Examples of cellular dysfunction, taking neurons as an example, include one or more types such as a reduction or absence of action potential generation, weakening of synapses and synaptic plasticity, a decrease or absence of energy metabolism, and suppression of neurogenesis. Examples of morphological changes in cells, taking neurons as an example, include one or more types such as dendrite and axon retraction, inhibition of elongation, reduction of spine structure, shedding of myelin sheath, and swelling or shrinkage of the cell body.

[0066] The promoting substance may be administered to the subject (e.g., various living organisms such as humans) as is, or it may be administered to the subject in the form of the composition described above. When two or more promoting substances are administered in combination, they may be administered all at once, or each substance may be administered sequentially in any order.

[0067] The method of administration to the subject can be appropriately selected depending on the characteristics of the substance used. Examples of administration methods include various types of in vivo administration, such as oral and parenteral administration. . For oral administration, solid dosage forms such as tablets, capsules, powders, granules, and other solid forms may be used, as well as liquid dosage forms such as solutions, syrups, and suspensions. Parenteral administration methods include, for example, intravenous, intramuscular, intraperitoneal, intrathecal, subcutaneous, or intradermal injection of liquids, infusion of solids or liquids into the gastrointestinal tract, or inhalation. These administrations may be single or multiple rapid doses, or continuous infusion such as intravenous drip.

[0068] The dosage administered to subjects can be appropriately selected according to the characteristics of the substance used and the therapeutically effective dose. For a human adult weighing 60 kg, the daily dose of each active ingredient can be independently set to, for example, 0.1 ng to 1000 mg per active ingredient. Furthermore, when the active ingredient is a viral vector, for example, 1 × 10⁻¹⁶ per kg of human subject body weight. 5 Vector genome (vg) / kg ~ 1 × 10⁻⁶ 20 It can be expressed as vg / kg. The number of times the active ingredient is administered per day can be appropriately selected according to the characteristics of the substance used and the effective therapeutic dose; for example, it may be once or twice or more. Furthermore, the method of administration during the treatment period can be appropriately selected according to the characteristics of the substance used and the effective therapeutic dose. The above-mentioned method of administration may be, for example, carried out continuously every day, or intermittently with a non-administration period of any number of days, months, or years of one or more days.

[0069] Further embodiments of this disclosure are described below. In the following descriptions, the differences from the embodiments described above will be the main points to be discussed, and for matters not specifically described, the provisions described herein will apply as appropriate. The provisions and embodiments described herein may be applied one or more in combination.

[0070] In one embodiment, this disclosure relates to a method for suppressing cell damage. In another embodiment, this disclosure relates to a method for facilitating the maintenance or improvement of cell function. In one embodiment, the method includes the step of bringing a promoting substance into contact with a neuron, which is one of the targets of application. This suppresses the occurrence or progression of neuronal cell damage and maintains voluntary movement of skeletal muscle. As a result, diseases can be effectively treated or prevented. The neurons to which this method is applied are preferably human neurons, and more preferably human motor neurons. Examples of normal cell functions, taking neurons as an example, include one or more functions such as the generation of action potentials, energy metabolism, and neurogenesis. Cell damage can be measured, for example, by measuring the length of neurites when stress stimuli are applied to neurons, preferably neurons derived from ALS patients, as described in the examples below. Yes, it is possible. An example of a stress stimulus would be tunicamycin treatment. The degree of suppression of cell damage by the promoting substance is such that the degree of cell damage when the process includes contact between the promoting substance and various cells such as neurons is 90% or less, preferably 70% or less, and more preferably 50% or less, compared to the case where the process does not include contact between the promoting substance and the cells. .

[0071] The method of contact between the promoting substance and various cells such as neurons is not particularly limited, and methods include direct or indirect contact between the promoting substance and cells. The promoting substance may be brought into contact as is, or in the presence of the carrier described above. As an indirect contact method, for example, the promoting substance may be administered intra vivo using the method described above, exposing the cells of the body, such as motor neurons, to bodily fluids such as blood or lymph. In other words, the above methods can each be applied independently in various environments such as in vivo, in vitro, and ex vivo.

[0072] Another embodiment in this disclosure relates to a method for improving protein clearance. . Another embodiment in this disclosure relates to a method for reducing or suppressing abnormal localization of proteins. Another embodiment in this disclosure relates to a method for regulating protein phosphorylation. More specifically, one embodiment in this disclosure relates to a method for reducing or inhibiting protein phosphorylation. Another embodiment in this disclosure relates to a method for accelerating protein degradation. Another embodiment in this disclosure relates to a method for suppressing the accumulation of proteins in the cytoplasm. In one embodiment, each of the methods described above includes a step of bringing a promoting substance into contact with a neuron, which is one of the targets of application. The proteins to which the above-described method is applied include, independently, TDP-43 protein or proteins derived from TDP-43. Specifically, these proteins include, for example, the abnormal TDP-43 proteins mentioned above, preferably TDP-43 protein or its fragments, and more preferably non-wild-type TDP-43 protein. As for TDP-43 protein fragments, they are produced when full-length TDP-43 protein is cleaved by caspase 3, etc. A preferred example is the C-terminal fragment of the TDP-43 protein, which is known to have the following properties. The amino acid sequence of the C-terminal fragment is shown, for example, in SEQ ID NOs: 4, 15, 16, and 17. The sequences are listed below. Of these, the protein shown in sequence number 15 is approximately 35 kDa (sometimes referred to as CTF35), and the protein shown in sequence number 16 or sequence number 17 is approximately 25 kDa (sometimes referred to as CTF25). The proteins may be full-length amino acids or peptide fragments having any number of residues. By inhibiting the translocation of TDP-43 protein out of the cell nucleus, inhibiting the accumulation of TDP-43 protein in the cytoplasm, inhibiting abnormal post-translational modifications of TDP-43 protein, or promoting the degradation of abnormal TDP-43 proteins, it is possible to suppress abnormal localization of abnormal proteins and the formation of aggregates. Furthermore, it is possible to maintain normal clearance of abnormal proteins. As a result, it is possible to maintain or improve cell function, and consequently, treat or prevent TDP-43-related diseases.

[0073] Another embodiment in this disclosure relates to a method for suppressing RNA splicing abnormalities. The target of this method is the STMN2 (Stathmin-2) gene, as shown in the examples described later. Examples include offspring, or transcripts derived from the gene in question (e.g., mRNA precursors).

[0074] The various methods described above each preferably include a step of bringing a promoting substance into contact with various cells such as neurons. In other words, a preferred embodiment of the above-described method is to keep cells such as neurons alive in the presence of a promoting substance. The method of contact between the promoting substance and various cells such as neurons is not particularly limited and includes methods of direct or indirect contact between the promoting substance and the cells. The promoting substance may be brought into contact as is, or it may be brought into contact in the presence of the carrier described above. As an indirect contact method, for example, the promoting substance may be administered in vivo by the method described above and exposed to biological constituent cells such as motor neurons via bodily fluids such as blood or lymph. The methods according to the above-described embodiments, like the other embodiments, can each be applied independently in various environments such as in vivo, in vitro, and ex vivo. Methods for measuring abnormal protein localization include, for example, cultured cells and cells derived from ALS patients. For example, a method is used to measure the ratio of TDP-43 protein expression levels in the cytoplasm and nucleus, as described in the screening method for TDP-43 protein extranuclear translocation inhibitors described later. In this method, cultured cells, cells derived from ALS patients, etc. are injected with tunicamycin or other substances. You may give a response stimulus. The degree to which the promoting substance reduces or suppresses abnormal protein localization is, for example, when the step of bringing the promoting substance into contact with various cells such as neurons, the ratio of TDP-43 protein expression levels in the cytoplasm and nucleus is 90% compared to when the step of bringing the promoting substance into contact with the cells is not included. It is less than or equal to %; preferably 70% or less; more preferably 50% or less. Methods for measuring protein phosphorylation include, for example, cultured cells and cells derived from ALS patients. For example, the phosphorylation level of the TDP-43 protein can be measured by conventional methods, such as immunofluorescence staining or Western blotting, as described in the examples below. In this method, cultured cells, cells derived from ALS patients, etc., are subjected to stress such as tunicamycin. You may stimulate it. The degree to which the promoting substance reduces or suppresses protein phosphorylation is, for example, when the phosphorylation level of the TDP-43 protein is 90% or less compared to when the step of contacting the promoting substance with various cells such as neurons is included, compared to when the step of contacting the promoting substance with the cells is not included. The percentage should be 70% or less, and more preferably 50% or less. Methods for measuring protein degradation include, for example, using cultured cells, cells derived from ALS patients, etc. For example, the expression level of the C-terminal fragment of the TDP-43 protein can be measured by a conventional method, for example, Western Methods for measurement include blotting. In this method, cultured cells and ALS patients You may apply stress stimuli such as tunicamycin to the target cells, etc. The degree to which the promoting substance suppresses protein degradation is, for example, determined by the expression level of the C-terminal fragment of the TDP-43 protein when the promoting substance is brought into contact with various cells such as neurons. Compared to a case that does not include the step of bringing the promoting substance into contact with the cells, the efficiency is 90% or less, preferably 70% or less, and more preferably 50% or less. Methods for measuring RNA splicing abnormalities include, for example, using cultured cells, cells derived from ALS patients, etc., and measuring the expression ratio of STMN2CE and STMN2FL of the STMN2 gene (STMN2CE / STMN2FL). ) can be measured by a conventional method, for example, by absolute quantitative qPCR (see examples). In this method, cultured cells, cells derived from ALS patients, etc. are subjected to stress stimuli such as tunicamycin. You may give it. The degree to which promoting substances suppress RNA splicing abnormalities is, for example, the relationship between the promoting substance and the RNA When the process includes contact with various cells such as chlorofluorocarbons, the expression ratio of STMN2CE to STMN2FL is 90% or less, preferably 70% or less, and more preferably 50% or less, compared to the case where the process of contacting the promoting substance with the cells is not included.

[0075] In addition to the above description, this specification also discloses, as one embodiment, matters relating to the use of a substance in the manufacture of a composition for treating or preventing TDP-43-related diseases. The substance is, for example, the facilitator described above. The composition in this embodiment preferably contains the facilitator described above. This specification also discloses substances or compositions used to treat or prevent TDP-43-related diseases. These substances are the aforementioned promoting agents. The compositions in this embodiment preferably contain the aforementioned promoting agents.

[0076] Another embodiment in this disclosure relates to a method for screening substances that inhibit TDP-43 protein phosphorylation, or a method for screening substances that inhibit the extracellular translocation of TDP-43 protein. The proteins to which the above methods can be applied are, independently, wild-type TDP-43 protein, non-wild-type TDP-43 protein, or proteins derived from TDP-43. Specifically, these proteins include, for example, the abnormal TDP-43 proteins mentioned above, preferably TDP-43 protein or a fragment thereof, and more preferably non-wild-type TDP-43 protein. As a fragment of TDP-43 protein, the C-terminal fragment of TDP-43 protein is preferred. It can be done.

[0077] A screening method for TDP-43 protein phosphorylation inhibitors should include the steps of: contacting a test substance with TDP-43 protein; measuring the phosphorylation level of the TDP-43 protein that has been contacted with the test substance; comparing the measured phosphorylation level with the phosphorylation level measured for TDP-43 protein that has not been contacted with the test substance; and selecting a test substance that reduces the phosphorylation level of TDP-43 protein. The method of contacting the test substance with the TDP-43 protein is not particularly limited, and methods include direct or indirect contact between the test substance and the TDP-43 protein. Direct contact methods include, for example, using commercially available recombinant TDP-43 protein or recombinant TDP-43 protein produced by conventional methods. Indirect contact methods include, for example, administering the test substance to various cells such as neurons and exposing them to the TDP-43 protein, and are applicable in various environments such as in vitro, in vivo, and ex vivo. Examples of suitable cells include motor neurons differentiated from iPS cells derived from patients with TDP-43-related diseases, and are preferred. Examples include motor neurons differentiated from iPS cells derived from ALS patients, and more preferably motor neurons differentiated from iPS cells derived from ALS patients having a TDP-43 gene mutation. These cells may also be subjected to a step in which a stress-inducing agent such as tunicamycin is administered. The phosphorylation level of the TDP-43 protein can be measured by conventional methods, such as immunofluorescence staining (see Example 1) or Western blotting. By comparing the measured phosphorylation level with the TDP-43 protein phosphorylation level in a sample uncontacted with the test substance, it is possible to determine whether or not the test substance is a TDP-43 protein phosphorylation inhibitor. If the TDP-43 protein phosphorylation level in a sample contacted with the test substance is lower than the TDP-43 protein phosphorylation level in a sample uncontacted with the test substance, then the test substance can be determined to be a TDP-43 protein phosphorylation inhibitor and selected accordingly. For example, if the TDP-43 protein phosphorylation level in a sample contacted with the test substance is 70% or less, preferably 50% or less, and more preferably 25% or less, compared to the TDP-43 protein phosphorylation level in a sample uncontacted with the test substance, then the test substance can be determined to be a TDP-43 protein phosphorylation inhibitor.

[0078] A screening method for substances that inhibit the extracellular translocation of TDP-43 protein is acceptable as long as it includes the steps of: contacting a test substance with cells expressing TDP-43 protein; measuring the nuclear and / or cytoplasmic expression levels of TDP-43 protein in the cells that have been contacted with the test substance; comparing the measured nuclear and cytoplasmic expression levels of TDP-43 protein, and / or the ratio of cytoplasmic to nuclear TDP-43 protein expression levels (calculated by dividing the cytoplasmic expression level of TDP-43 by the nuclear expression level) with the nuclear and cytoplasmic expression levels of TDP-43 protein and / or the ratio of cytoplasmic to nuclear TDP-43 protein expression levels in cells that have not been contacted with the test substance; and selecting a test substance that increases the nuclear expression level of TDP-43 protein, or a test substance that decreases the cytoplasmic expression level and / or the ratio of cytoplasmic to nuclear TDP-43 protein expression levels. The method of bringing the test substance into contact with cells expressing the TDP-43 protein is not particularly limited; for example, the test substance may be administered to various cells such as neurons to expose them. It can be applied in various environments such as in vitro, in vivo, and ex vivo. Examples of cells include motor neurons differentiated from iPS cells derived from patients with TDP-43-related disease, and preferably Examples include motor neurons differentiated from iPS cells derived from ALS patients, and more preferably motor neurons differentiated from iPS cells derived from ALS patients having a TDP-43 gene mutation. These cells may also be subjected to a step in which a stress-inducing agent such as tunicamycin is administered. The expression levels of TDP-43 protein in the nucleus and / or cytoplasm can be measured by conventional methods, such as immunofluorescence staining (see Example 1) or Western blotting of nuclear and / or cytoplasmic proteins extracted from cells. By comparing the measured expression levels of TDP-43 protein in the nucleus and cytoplasm, and / or the ratio of TDP-43 protein expression levels in the cytoplasm and nucleus, with the expression levels of TDP-43 protein in the nucleus and cytoplasm, and / or the ratio of TDP-43 protein expression levels in the cytoplasm and nucleus, when not in contact with the test substance, it is possible to determine whether or not the test substance is an inhibitor of cell extranuclear translocation. If the nuclear expression level of TDP-43 protein increases, and the cytoplasmic expression level and / or the ratio of cytoplasmic to nuclear TDP-43 protein expression levels decreases, then the test substance can be determined to be a TDP-43 protein extranuclear translocation inhibitor and selected accordingly. For example, if the ratio of cytoplasmic to nuclear expression levels of TDP-43 protein after contact with the test substance is 80% or less, preferably 50% or less, and more preferably 25% or less, compared to when the test substance is not in contact with the protein, then the TDP-43 protein is determined to be an inhibitor. It is determined to be a substance that inhibits the extracellular translocation of plasmoplasmic cells.

[0079] The TDP-43 protein phosphorylation inhibitors and TDP-43 protein extranuclear translocation inhibitors obtained by the screening method of one embodiment of this disclosure are useful as active ingredients for treating or preventing TDP-43-related diseases. In particular, they are useful for the prevention of ALS or the treatment of TDP-43-related diseases. It is useful as an effective ingredient for prevention.

[0080] Another embodiment in this disclosure is a composition comprising a substance (sometimes referred to as a binding promoter) that enhances the binding of the LRSAM1 protein to the TDP-43 protein for the treatment or prevention of TDP-43-related diseases. Binding promoters are substances that enhance the binding or interaction between the LRSAM1 protein and the TDP-43 protein or its fragments, such as PROTAC (Proteolysis targeting chimera) compounds. Examples include proteolytic chimeric molecules and MGD (Molecular glue degrader) compounds (molecular glue-type degrading compounds). Wild-type LRSAM1 protein is preferred. TDP-43 proteins can be independently wild-type TDP-43 protein, non-wild-type TDP-43 protein, or proteins derived from TDP-43. Specifically, these proteins include, for example, the abnormal TDP-43 proteins mentioned above, preferably TDP-43 protein or a fragment thereof, and more preferably non-wild-type TDP-43 protein. As a fragment of TDP-43 protein, the C-terminal fragment of TDP-43 protein is preferred.

[0081] PROTAC compounds are compounds that contain a TDP-43 protein target ligand, an LRSAM1 (E3 ubiquitin ligase) target ligand, and a linker. PROTAC compounds can be prepared as follows: TDP-43 protein target ligands and LRSAM1 (E3 ubiquitin ligase) target ligands are selected through screening of existing ligands, simulations based on X-ray crystal structure analysis, and screening using compound libraries. Leaning, biochemical evaluation (e.g., surface plasmon resonance (SPR) method, etc.) and / or The structure of proteins can be obtained using artificial intelligence programs such as Alphafold2, which perform protein structure prediction. Each obtained ligand can be linked with a linker using methods well known in this field to create a candidate PROTAC compound. Whether the candidate PROTAC compound binds to the TDP-43 protein and the LRSAM1 protein and enhances their interaction can be evaluated by attaching probes to each protein and competitively measuring the interaction. Specifically, this can be evaluated using methods such as fluorescence polarization (FP), time-resolved fluorescence resonance energy transfer (TR-FRET), and AlphaScreen / AlphaLISA technology. In this case, if a stronger interaction is observed in the presence of the candidate PROTAC compound compared to the absence of the candidate PROTAC compound, it can be identified as a candidate PROTAC compound. Furthermore, whether the candidate PROTAC compound degrades the TDP-43 protein can be evaluated using cells. For example, nerve cells, such as Neuro2a cells, can be used to evaluate TDP-43 The protein, or the TDP-43 protein fragment (the C-terminal fragment of the TDP-43 protein), is fluorescently labeled. The amount of TDP-43 protein can be quantified by introducing the plasmid into cells using a method well known in this field and measuring the fluorescence intensity. If a candidate PROTAC compound is added to these cells and the fluorescence intensity decreases, it can be determined that the compound has TDP-43 proteolytic activity and can be identified as a PROTAC compound. In this way, a protein PROTAC compound consisting of a TDP-43 protein target ligand, an LRSAM1 (E3 ubiquitin ligase) target ligand, and a linker can be obtained.

[0082] MGD compounds involve the interaction of TDP-43 protein and LRSAM1 (E3 ubiquitin ligase) protein. This compound enhances the interaction between the TDP-43 protein and the LRSAM1 protein by fitting into a pocket created during action. The MGD compound is prepared as follows: It is possible. TDP-43 protein, or a fragment thereof (the C-terminal fragment of the TDP-43 protein) and LRSAM1 protein Regarding the interaction between compounds, whether there is a pocket into which the compound can fit, X-ray crystal structure Structural analysis using structural analysis and nuclear magnetic resonance (NMR) spectroscopy and / or This can be explored using artificial intelligence programs such as Alphafold2, which perform protein structure prediction. This allows for the visualization and / or prediction of binding interfaces and pockets between the TDP-43 protein or its fragments and the LRSAM1 (E3 ubiquitin ligase) protein. MGD candidate compounds that fit into these pockets are then selected from a compound library using structure-based docking. The obtained MGD candidate compounds can be prepared by virtual screening via 3D or simulation, and / or compound design based on structural information from the pocket. Whether the TDP-43 protein or its fragments bind to and enhance the interaction between the TDP-43 protein and the LRSAM1 protein can be evaluated by quantifying the protein-protein interaction using time-resolved fluorescence resonance energy transfer (TR-FRET) or similar methods. At this time, if a stronger interaction is observed in the presence of the MGD candidate compound compared to the absence of the MGD candidate compound, it can be identified as an MGD compound. Furthermore, whether the MGD candidate compound has the effect of degrading the target protein can also be evaluated. Therefore, it can be evaluated using the same evaluation method as the PROTAC candidate compounds described above, and if it is determined to have TDP-43 proteolytic activity, it can be determined to be an MGD compound. In this way, we obtain MGD compounds that enhance the interaction between the target protein and LRSAM1. It is possible.

[0083] As one embodiment of the present disclosure, the binding promoter is the ubiquitin-proteasome system of the LRSAM1 protein, which binds to the TDP-43 protein or a TDP-43 protein fragment (the C of the TDP-43 protein). By promoting the degradation of terminal fragments, it is useful as an active ingredient for treating or preventing TDP-43-related diseases. In particular, for the prevention of ALS or for treating or preventing TDP-43-related diseases. It is useful as an active ingredient. Regarding the matters described above, unless otherwise specified, the provisions described in this specification shall apply as appropriate.

[0084] With regard to the embodiments described above, this specification further discloses the following embodiments.

[0085] <1> A composition containing a promoting substance. <2> The promoting substance is a substance having at least one, preferably two, more preferably three of the following (i) to (iii): <1> The composition described above. (i) Increase the intracellular amount of proteins with ubiquitination activity; (ii) Increase intracellular ubiquitination activity; and (iii) To promote the production of transcripts or translation products in cells. <3> The composition is used to treat or prevent TAR DNA-binding protein 43 (TDP-43) related diseases. <1> or <2> The composition described above. <4> The aforementioned TDP-43-related disease is one or more selected from neurodegenerative diseases and muscle diseases. <3> The composition described above. <5> The aforementioned neurodegenerative diseases include Parkinson's disease, amyotrophic lateral sclerosis (ALS), and spinal cord disease. Amyotrophic dysplasia (SMA), primary lateral sclerosis (PLS), multiple system atrophy (MSA), progressive supranuclear palsy The group consists of (PSP), Perry syndrome, Alexander disease, Alzheimer's disease, frontotemporal lobar degeneration (FTLD), and limbic-dominant senile TDP-43 encephalopathy (LATE). The aforementioned muscle disease is inclusion body myositis. <4> The composition described above. <6> The aforementioned neurodegenerative diseases include amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). ), and one selected from the group consisting of limbic system-dominant senile TDP-43 encephalopathy (LATE) or There are two or more types, Preferably, amyotrophic lateral sclerosis (ALS), the above <5> The composition described above.

[0086] <7> The composition is used to improve the clearance of the TDP-43 protein. <1> ~ <6> The composition described in any one of the following. <8> The composition is used to suppress cell damage. <1> ~ <7> A composition according to any one of the following. <9> The composition is used to suppress cell damage caused by the TDP-43 protein. <1> ~ <8> The composition described in any one of the following. <10> The composition is used to reduce or suppress abnormal localization of proteins. <1> ~ <9> A composition according to any one of the following. <11> The preceding composition is used to promote the degradation of proteins, <1> ~ <10> A composition according to any one of the following. <12> The composition is used to regulate the phosphorylation of proteins. <1> ~ <11> A composition according to any one of the following. <13> The protein is the full-length or fragment of the TDP-43 protein. <10> ~ <12> The composition described in any one of the following. <14> The early stage protein is the C-terminal fragment of the TDP-43 protein, as described above. <13> Note The composition of the listed items. <15> The composition suppresses abnormal splicing of RNA (preferably mRNA). The above <1> ~ <14> The composition described in any one of the following.

[0087] <16> The above applies to subjects or cells having mutations in the TARDBP gene. <1> ~ <15> A composition according to any one of the following. <17> The above applies to subjects or cells having heterozygous mutations in the TARDBP gene. <16> The composition described above. <18> The subject or cells are human subjects or human-derived cells. <16> or <17> The composition described above. <19> The aforementioned promoting substance comprises one or more selected from the group consisting of organic compounds, nucleic acids, proteins, lipids, and proteins. Preferably, the substance contains nucleic acids. <1> ~ <18> A composition according to any one of the following.

[0088] <20> The aforementioned promoting substance is a substance containing nucleic acids, The promoting substance preferably comprises one or more selected from the group consisting of expression-enhancing nucleic acids and expression vectors. <19> The composition described above. <21> The promoting substance includes the expression-enhancing nucleic acid, <20> The composition described above. <22> The expression-enhancing nucleic acids include antisense, non-coding RNA, and small molecule active The above is one or more selected from the group consisting of sexualized RNAs. <21> The composition described . <23> The antisense is one or more selected from (a) to (e) below. <22> The composition described above. (a) Nucleic acids that regulate RNA processing; (b) nucleic acids that are partially or fully complementary to the entire length or a portion of the miRNA; (c) Nucleic acids that are partially or fully complementary to the entire length or a portion of the Natural Antisense Transcript (NAT); (d) mRNA translation inhibitory element (TIE), upstream ORF (uORF), and immature In all or part of one or more regions selected from the group consisting of maturation stop codons (PTCs) Partially complementary or fully complementary nucleic acids; and (e) Partially complementary to the entire length or a portion of the regulatory RNA that contributes to gene expression repression. or fully complementary nucleic acids <24> The expression-enhancing nucleic acid has 5 to 300 base pairs per strand. Preferably, the number of bases per strand is 10 to 100, <21> ~ <23> either The composition described in (i).

[0089] <25> The promoting substance includes the expression vector, <20> The composition described above. <26> The aforementioned promoting substance is a substance that increases the intracellular amount of proteins having ubiquitination activity. The protein having ubiquitination activity is the LRSAM1 protein. <1> ~ <25> A composition according to any one of the following. <27> The protein having ubiquitination activity is the human LRSAM1 protein. <26> The composition described above. <28> The protein having ubiquitination activity is the wild-type LRSAM1 protein. <26> or <27> The composition described above. <29> The promoting substance is a substance that causes the production of transcripts and / or translation products from nucleic acids encoding proteins having ubiquitination activity. <1> ~ <28> A composition according to any one of the following. <30> The promoting substance is a substance that promotes the production of transcripts and / or translation products from nucleic acids encoding the full length of a wild-type protein having ubiquitination activity. <1> ~ <29> A composition according to any one of the following. <31> The aforementioned promoting substance is a substance that promotes the production of translation products. The aforementioned translation product is a protein having ubiquitination activity. The production of the aforementioned protein increases the intracellular amount of the protein and / or increases intracellular ubiquitination activity. <1> ~ <30> A composition according to any one of the following.

[0090] <32> The aforementioned promoting substance is a substance that increases the intracellular amount of a protein or translation product having ubiquitination activity. The aforementioned proteins or translation products are the group consisting of LRSAM1, Gp78, CHIP, RNF19A, and MGRN1. The above is one or more proteins selected from the above. <1> ~ <31> A composition according to any one of the following. <33> The protein having ubiquitination activity is the LRSAM1 protein. The aforementioned <29> ~ <32> A composition according to any one of the following. <34> The aforementioned promoting substance is a substance that promotes (i) and / or (ii) below: The aforementioned <1> ~ <32> A composition according to any one of the following. (i) Production of transcripts capable of expressing wild-type LRSAM1 protein or a protein having equivalent function; and (ii) Wild-type LRSAM1 protein or a protein having equivalent function to said protein Production of a certain transproduct <35> The aforementioned cells are nerve cells, Preferably a neuron, more preferably a human neuron, and even more preferably a human motor neuron, the <1> ~ <34> A composition according to any one of the following. <36> The above further comprises a pharmaceutically acceptable carrier. <1> ~ <35> A composition according to any one of the following.

[0091] <37> The pharmaceutical composition, <1> ~ <36> A composition according to any one of the following. <38> The above is used for subjects who have developed or are at risk of developing TDP-43-related disease. <1> ~ <37> A composition according to any one of the following. <39> in vivo (including or excluding humans), in vitro (including humans), or including humans. The above is used in either an ex vivo environment. <1> ~ <38> Any one of the following composition.

[0092] <a1>A method for treating or preventing a TDP-43-related disease, comprising the step of administering an effective amount of a promoting substance to a subject in need thereof. <a2>A method for improving TDP-43 protein clearance, comprising the step of administering an effective amount of a promoter to a subject in need thereof. <a3>A method for suppressing cell damage, comprising the step of administering an effective amount of a promoting substance to a subject in need of it.

[0093] <a4>A method for facilitating the maintenance or improvement of cellular function, comprising the step of administering an effective amount of a promoting substance to a subject in need thereof. <a5>A method for reducing or suppressing abnormal localization of a protein, comprising the step of administering an effective amount of a promoting substance to a subject in need thereof. <a6>A method for promoting protein degradation, comprising the step of administering an effective amount of a promoting substance to a subject in need thereof. <a7>A method for regulating protein phosphorylation, comprising the step of administering an effective amount of a promoter to a subject requiring it. <a8>RNA, comprising the step of administering an effective amount of the promoter to a subject requiring it. A method for suppressing splicing abnormalities (preferably mRNA).

[0094] <a9>The aforementioned promoting substance is administered to the subject to treat or prevent the disease by suppressing the onset or progression of nerve cell damage. <a1>Methods used. <a10>The protein is the full-length or fragment of the TDP-43 protein. <a5> ~ <a7>The method described in any one of the following ways. <a11>The protein is the C-terminal fragment of the TDP-43 protein. <a10> Methods used. <a12>The method described above is performed on subjects who have developed or are likely to develop TDP-43-related disease. <a1> ~ <a11>The method according to any one of the above. <a13>The aforementioned method is used for TDP-43-related diseases such as amyotrophic lateral sclerosis (ALS) and frontal lobe sclera. It consists of temporal lobar degeneration (FTLD) and limbic-dominant senile TDP-43 encephalopathy (LATE). The above procedure is performed on subjects selected from the group who have developed or are likely to develop one or more of the following conditions. <a1> ~ <a12>The method described in any one of the following ways. <a14>The subject is a human subject. <a1> ~ <a13>The method described in any one of the following ways. <a15>The promoting substance is a substance having at least one, preferably two, and more preferably three of the following (i) to (iii): <a1> ~ <a14>The method described in any one of the following ways. (i) Increase the intracellular amount of proteins with ubiquitination activity; (ii) Increase intracellular ubiquitination activity; and (iii) To promote the production of transcripts or translation products in cells. <a16>The method described above <1> ~ <39> The promoting substance is administered in the form of the composition described in any one of the above. <a1> ~ <a15>The method described in any one of the following ways.

[0095] <b1>Use of a promoter that increases the intracellular amount of ubiquitinating proteins in the manufacture of a composition for treating or preventing TDP-43-related diseases. <b2>The use of a promoter that increases the intracellular amount of ubiquitinated proteins in the production of a composition for increasing intracellular ubiquitination activity. <b3>The protein is the LRSAM1 protein. <b1>or <b2>Use as described above. <b4>The aforementioned promoting substance is a substance containing nucleic acids, The promoting substance preferably comprises one or more selected from expression-enhancing nucleic acids and expression vectors. <b1> ~ <b3>Use as described in any one of the following terms. <b5>The aforementioned cells are nerve cells, Preferably a neuron, more preferably a human neuron, and even more preferably a human motor neuron, the <b1> ~ <b4>Use as described in any one of the following terms. <b6>The composition, the <1> ~ <39> The composition is one of the compositions described above. <b1> ~ <b5>Use as described in any one of the following terms.

[0096] <c1>A method for suppressing cell damage, comprising bringing a promoting substance into contact with a neuron to suppress the occurrence or progression of cell damage in the neuron. <c2>A method for improving protein clearance, comprising bringing a promoting substance into contact with a neuron to improve the clearance of TDP-43 protein in the neuron. <c3>A method of bringing a promoting substance into contact with neurons to facilitate the maintenance or improvement of neuronal function. <c4>A method for reducing or suppressing the abnormal localization of proteins present within neurons by bringing a promoting substance into contact with the neurons. <c5>A method that involves bringing a promoting substance into contact with neurons to accelerate the breakdown of proteins present within the neurons. <c6>A method that involves bringing a promoting substance into contact with a neuron to regulate the phosphorylation of proteins present within the neuron. <c7>By bringing a promoting substance into contact with a neuron, RNA (preferred) in the neuron is stimulated. A method to suppress splicing abnormalities in mRNA.

[0097] <c8>The neuron is a motor neuron, preferably a human motor neuron. <c1> ~ <c7>The method described in any one of the following ways. <c9>The promoting substance is a substance having at least one, preferably two, and more preferably three of the following (i) to (iii): <c1> ~ <c8>The method described in any one of the following ways. (i) Increase the intracellular amount of proteins with ubiquitination activity; (ii) Increase intracellular ubiquitination activity; and (iii) To promote the production of transcripts or translation products in cells. <c10>The aforementioned promoting substance is a substance containing nucleic acids, The promoting substance preferably comprises one or more selected from the group consisting of expression-enhancing nucleic acids and expression vectors. <c1> ~ <c9>The method described in any one of the following ways. <c11>The method described above <1> ~ <39> Using the promoting substance in any one of the compositions described above, <c1> ~ <c10>The method described in any one of the following ways. [Examples]

[0098] The present disclosure will be further described below with reference to examples. However, the scope of the present disclosure is not limited to such examples. Unless otherwise specified, "%v / v" means "volume / volume%". Furthermore, in each figure, the group represented by "**" indicates that P<0.01 is statistically significant. In each figure, the group represented by "*" indicates that P<0.05 is statistically significant.

[0099] Example 1 Taking ALS as an example of a TDP-43-related disease, we will use a disease model to evaluate its cellular damage. It was built using the method below. First, human induced pluripotent stem (iPS) cells were induced from cells derived from human ALS patients and cells derived from healthy individuals using conventional methods. For the cells derived from ALS patients, those known to have a heterozygous mutation in the TARDBP gene indicating an ALS risk were used. Next, conventional... According to the law, motor neurons (hereinafter, motor neurons will simply be referred to as "neurons") are extracted from each iPS cell. They were each differentiated into (also known as ''). To preserve cells for long-term storage, neurons were sometimes frozen 2 to 7 days after differentiation, as needed. Nerves that have undergone this freezing process are also called "frozen neurons."

[0100] Next, using a 48-well culture plate (Corning, 3548), neurons were cultured under the following conditions. It was cultured. The culture plates were coated before cell seeding. Specifically, a 0.02% poly-L-ornithine solution (Sigma-Aldrich, P4957) was added to each well of the culture plate, and after standing in a 37°C 5% CO2 incubator for 2 hours, each well was washed and treated with 20 μg / mL laminin. The procedure was carried out using a solution (Thermo Fisher Scientific, 23017015) and by allowing it to stand for an additional 2 hours in a 37°C 5% CO2 incubator. After that, the frozen neurons were thawed by conventional methods. The thawed neurons were seeded at 50,000 cells / well and cultured in the following composition. The culture was performed in the presence of the solution.

[0101] The culture medium used was a mixture with the following composition. ·DMEM / F12 (Thermo Fisher Scientific, 21331-020): 50%v / v ·Neurobasal Medium (Thermo Fisher Scientific, 21103-049): 50%v / v ·Glutamax Supplement (Thermo Fisher Scientifc, 35050061): 1% v / v ·Penicillin-Streptomycin (10000 units / mL) (Thermo Fisher Scietific, 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

[0102] The culture medium, containing a solution of the stress-inducing agent Dimethyl sulfoxide (DMSO) (final concentration: tunicamycin 0.1 μg / mL, DMSO 0.01% v / v), was applied to neurons after 7 days of culture, and the occurrence of cell damage was evaluated over time. Separately, a group without the stress-inducing agent (a group with the same concentration of DMSO added) was also prepared. Cell damage is assessed using IncuCyte S3 (Sartorius) according to the attached protocol. Ibcell imaging was performed to determine the neurite length per unit area (unit: mm / mm). 2 ) over time The data was analyzed and used as an evaluation index. A smaller neurite length value indicates that cell damage has occurred.

[0103] Cell damage assessment (1) Regarding neurite length in each experimental group, the results at the start of stress-inducing agent treatment (0hr) are as follows: The results calculated based on the ratio are shown in Figure 1. A smaller value on the vertical axis indicates that the neurite length decreases over time, and that cell damage is occurring. The experimental groups in Figure 1 are as follows: • Healthy-DMSO: A group using neurons derived from healthy individuals and without stress-inducing agents. • Healthy-Tunicamycin: A group containing stress-inducing agents, using neurons derived from healthy individuals. • ALS-DMSO: A group using neurons derived from ALS patients, without stress-inducing agents. • ALS-Tunicamycin: A group of cells containing stress-inducing agents, using neurons derived from ALS patients.

[0104] Neurons derived from ALS patients show a significant reduction in neurite length compared to neurons derived from healthy individuals. A small number was observed. In addition, in both neurons derived from healthy individuals and neurons derived from ALS patients... When tunicamycin treatment is performed, the neurite length in neurons derived from ALS patients is increased compared to untreated neurons. The neurite length became significantly smaller than that of neurons derived from healthy individuals, indicating cellular damage. In contrast, no significant decrease in neurite length was observed in neurons derived from healthy individuals (Figure 1).

[0105] Localization assessment of TDP-43 (1) The intracellular localization of TDP-43 protein was evaluated by immunofluorescence staining of neurons. Cellular neurons were washed with Phosphate-Buffered Saline (PBS) and fixed with 4% Paraformaldehyde (PFA). Blocking was performed with PBS containing 5% Fetal Bovine Serum (FBS) and 0.1% Triton-X. Next, add a primary antibody dilution containing anti-TDP-43 antibody and anti-β-III tubulin antibody, and incubate overnight at 4°C. The cells were incubated. Anti-β-III tubulin antibody was used to visualize the cell bodies of neurons. The following day, after washing with PBS, a secondary antibody dilution conjugated with Alexa dye was added, and the cells were incubated at room temperature for 1 hour. After further nuclear staining, the cells were washed with PBS and imaged using a fluorescence microscope. The acquired images were analyzed using Matlab (Mathworks), and the fluorescence intensity of TDP-43 in the nuclear and cytoplasmic regions was calculated, respectively.

[0106] The results of the intracellular localization of the TDP-43 protein are shown in Figure 2. The experimental groups in Figure 2 are as follows: • Healthy-DMSO: A group using neurons derived from healthy individuals and without stress-inducing agents. • Healthy-Tunicamycin: A group containing stress-inducing agents, using neurons derived from healthy individuals. • ALS-DMSO: A group using neurons derived from ALS patients, without stress-inducing agents. • ALS-Tunicamycin: A group of cells containing stress-inducing agents, using neurons derived from ALS patients.

[0107] The intracellular localization of TDP-43 protein, as shown in Figure 2, is expressed as the ratio of the fluorescence intensity based on cytoplasmic TDP-43 to that based on nuclear TDP-43 (in Figure 2, "cytoplasmic / nuclear ratio"). A higher value of this ratio indicates a higher proportion of TDP-43 protein localization in the cytoplasm, suggesting localization abnormalities. Two-way ANOVA revealed that the neurons originated from ALS patients and that the treatment with stress-inducing drugs was effective. This interaction significantly promotes the abnormal localization of TDP-43 protein in the cytoplasm. This abnormal localization is similar to the phenomenon observed in the brain tissue of ALS patients. Ta.

[0108] Evaluation of the degree of phosphorylation of TDP-43 (1) The degree of TDP-43 protein phosphorylation was evaluated by immunofluorescence staining of neurons. In addition to the primary antibody used in the immunofluorescence staining described above, anti-phosphorylated TDP-43 (S409) antibody was used instead of the anti-TDP-43 antibody in the same immunofluorescence staining procedure. Otherwise, the procedure described above was followed. The fluorescence intensity was calculated in the same manner as described above. Phosphorylation of the TDP-43 protein is thought to indicate the following intracellular states. Specifically, it has been reported that TDP-43 that is insoluble in the cytoplasm is phosphorylated. Insolubilization of TDP-43 in the cytoplasm leads to inhibition of the proteolytic pathway and mitochondrial toxicity. In addition, insoluble TDP-43 can inhibit the normal nuclear translocation of the TDP-43 protein and promote TDP-43 dysfunction in the nucleus. In other words, a high degree of phosphorylation of TDP-43 protein means that the normal function of the TDP-43 protein is inhibited, and the cell is more susceptible to cytotoxicity.

[0109] Abundance of phosphorylated TDP-43 protein (pTDP-43) in the cytoplasm (arithmetic mean of fluorescence intensity) The values ​​are shown in Figure 3. The experimental groups in Figure 3 are all the same as the experimental groups shown in Figure 2. As shown in Figure 3, neurons derived from ALS patients have phosphorylated TDP-43 in the cytoplasm. Expression was significantly higher in neurons derived from healthy individuals than in neurons derived from healthy individuals. Furthermore, in the presence of stress inducers, TDP-43 phosphorylation significantly increased in neurons derived from ALS patients. This suggests that the normal function of TDP-43 is impaired. Based on the above, this model is considered to be an experimental model that can appropriately reflect the pathology of ALS patients. It was done.

[0110] Example 2 Using the experimental model described above, the effect of the promoter on neuronal cell damage was evaluated. The target for gene expression promotion was LRSAM1. LRSAM1 is one of the proteins with intracellular ubiquitination activity. The promoter was the wild-type human LRSAM1 protein (SEQ ID NO: 6). A lentiviral vector containing the nucleic acid encoding (see CDS base sequence in SEQ ID NO: 5) was prepared by a conventional method. This promoter is configured to promote the production of LRSAM1 transcripts and / or translation products in cells, thereby increasing the amount of LRSAM1 protein in cells. Separately, a lentiviral vector was constructed that does not contain nucleic acid encoding the wild-type protein of LRSAM1. This lentiviral vector does not fall under the category of a promoter as defined herein, and even when applied to cells, it does not increase the intracellular amount of ubiquitinating proteins.

[0111] In the experimental model described above, on day 3 of culture, each lentiviral vector is prepared to achieve a predetermined multiplicity of infection (MOI) (e.g., MOI 0.01 or 0.03) The mixture was added to Ron's culture plates, and the culture was continued. On day 7 of culture, a stress inducer (or DMSO alone) was added to the final concentration described above, and the mixture was cultured for a maximum of 96 hours in the presence of the stress inducer.

[0112] Cell damage assessment (2) Figures 4 and 5 show the results of evaluating the occurrence of neuronal cell damage by morphological analysis of cells using the same method as in Example 1. Figure 4 shows the results using neurons from healthy individuals, and Figure 5 shows the results using neurons from ALS patients. As shown in these figures, in neurons derived from ALS patients, the group exposed to the stimulant (Figure 5) Reference: In the MOI 0.03 group, the occurrence or progression of cytotoxicity was significantly suppressed. Also, in healthy individuals... No significant difference in LRSAM1 protein expression was observed in the derived neurons (see Figure 4). However, the inventors confirmed that LRSAM1 expression levels increased in the neurons of the experimental group shown in Figure 5.

[0113] Localization assessment of TDP-43 (2) The cytoplasmic / nuclear ratio of TDP-43 with and without the promoting substance was calculated using the same method as in Example 1, and the results are shown in Figures 6A and 6B. The experimental groups shown in Figures 6A and 6B are as follows. In Figures 6A and 6B, the value of the Null-DMSO group in each neuron is set to 100%. The results for each group were expressed as a percentage. • Null-DMSO: Group that does not contain stress inducers or stimulants. • Null-Tunicamycin: A group containing stress inducers but no stress promoters. • LRSAM1-DMSO: A group that does not contain stress-inducing agents but contains stress-promoting substances. • LRSAM1-Tunicamycin: A group containing stress inducers and stimulants.

[0114] As shown in Figures 6A and 6B, promoting the production of LRSAM1 protein as a translation product and increasing intracellular ubiquitination activity reduced the cytoplasm / nucleus ratio in both healthy human-derived neurons and ALS patient-derived neurons. The degree of decline was more pronounced in neurons derived from ALS patients than in neurons derived from healthy individuals. Therefore, by using promoting substances, the abnormal localization and clearance of TDP-43 can be improved, allowing cells such as motor neurons to perform their inherent functions.

[0115] Evaluation of the degree of phosphorylation of TDP-43 (2) Results regarding the amount of pTDP-43 in the cytoplasm of neurons derived from healthy individuals, with and without the presence of a promoting substance. Figure 7A shows the results for neurons derived from ALS patients, and Figure 7B shows the results for neurons derived from ALS patients. The experimental groups in Figures 7A and 7B are the same as the experimental groups shown in Figures 6A and 6B. In this case, the value of the Null-DMSO group in each neuron is taken as 100%, and each neuron is expressed as a percentage of that value. The results for each group are shown. As shown in these figures, the expression of phosphorylated TDP-43, which was increased by tunicamycin treatment, was suppressed by the promoting agent. As mentioned above, phosphorylation of TDP-43 can lead to inhibition of the proteolytic system, the development of mitochondrial toxicity, and TDP-43 dysfunction in the nucleus. Therefore, the regulation of phosphorylation by the promoting agent, which suppresses the expression of phosphorylated TDP-43, leads to the suppression of the aforementioned abnormalities. As a result, the promoting agent can improve the function of cells such as motor neurons so that they can perform their inherent functions.

[0116] Evaluation of improved splicing regulation by promoting substances Focusing on STMN2, one of the genes whose splicing is regulated by TDP-43, we evaluated the amount of STMN2 transcript produced using the following method. Specifically, RNA was extracted from cultured neurons using the Quick-RNA micro kit (Zymoresearch). After measuring the RNA concentration, a certain amount of RNA was reverse transcribed using SuperScript IV VILO Master Mix (ThermoFisher Scientific) to synthesize cDNA. A calibration curve was created using nucleic acids that were identical to the amplified sequence by qPCR and whose copy number was known, and absolute quantitative qPCR was performed. By absolute quantitative qPCR, the wild-type STMN2 was identified. The transcript production levels of full-length mRNA (hereinafter also referred to as STMN2FL, including in the figures) and mRNA containing the cryptic exon of STMN2 (hereinafter also referred to as STMN2CE, including in the figures) were measured. Cryptic exons are sequences that are originally located in the intron region but are unintentionally included as exons, and are caused by splicing errors. (Example: STMN2FL generation) A high ratio of STMN2CE production to total TDP-43 output (STMN2CE / STMN2FL) indicates a relatively high production of non-wild-type transcripts, making splicing abnormalities more likely. In other words, a high STMN2CE / STMN2FL ratio suggests a reduction or loss of normal TDP-43 function. It means that it is doing so.

[0117] The results are shown in Figure 8. The experimental groups shown in Figure 8 represent the following: • Healthy-Null-DMSO: A group using neurons derived from healthy individuals, without stress inducers or stimulants. • Healthy-Null-Tunicamycin: Contains a stress-inducing agent derived from neurons of healthy individuals. and the group that does not contain promoting substances. • Healthy-LRSAM1-DMSO: A group using neurons derived from healthy individuals, containing no stress inducers but containing stress-promoting substances. • Healthy-LRSAM1-Tunicamycin: Using neurons derived from healthy individuals, stress inducers and Group containing promoting substances • ALS-Null-DMSO: A group using neurons derived from ALS patients, without containing stress inducers or stimulants. · ALS -Null- Tunicamin: A group of drugs using neurons derived from ALS patients that contain stress-inducing agents but no stress-promoting substances. • ALS-LRSAM1-DMSO: Uses neurons derived from ALS patients, does not contain stress inducers, and promotes Group containing advanced substances · ALS-LRSAM1-Tunicamycin: A group of substances containing stress inducers and stimulants, derived from neurons of ALS patients.

[0118] As shown in Figure 8, neurons derived from ALS patients had a significantly higher STMN2CE / STMN2FL ratio than neurons derived from healthy individuals (comparison of healthy-Null-DMSO and ALS-Null-DMSO in Figure 8). This indicates that splicing abnormalities are more likely to occur in TDP-43-related diseases such as ALS, and that the normal function of TDP-43 is reduced or lost. In neurons derived from ALS patients, the STMN2CE / STMN2FL ratio was significantly higher in the tunicamycin-treated group (comparison of ALS-Null-DMSO and ALS-Null-tunicamycin in Figure 10). This indicates that tunicamycin treatment exacerbates splicing abnormalities. Furthermore, tunicamycin In the Kamycin-treated group, when LRSAM1 was expressed with a promoting substance, the STMN2CE / STMN2FL ratio was superior. The ratio decreased to the lowest level (comparison of ALS-Null-tunicamycin and ALS-LRSAM1-tunicamycin in Figure 10). On the other hand, in neurons derived from healthy individuals, no significant changes in the STMN2CE / STMN2FL ratio were observed in each group. It was not possible (Figure 9). From the above, it appears that in neurons derived from ALS patients, the normal function of TDP-43 is promoted by the substance. Therefore, it was determined that he was recovering.

[0119] Example 3 The effect of promoting substances on the degradation of the C-terminal fragment of the TDP-43 protein was evaluated. C-terminal fragments of protein with a magnitude of approximately 35 kDa and approximately 25 kDa are known. These will be referred to as CTF35 and CTF25. Similar to the experimental model used in Example 2, on day 3 of culturing each neuron, each lentiviral vector was added to the culture plate to achieve a predetermined MOI (e.g., MOI 0.03), and then cultured. This was continued. Then, on day 7 of culture, the stress inducer (or DMSO alone) was administered to the predetermined final concentration. (For example, add to a concentration of 0.1 μg / mL) and continue for up to 96 hours in the presence of a stress inducer. The cells were cultured. Tunicamycin was used as a stress inducer. Culture using an 8M Urea, 50mM Tris-HCl solution containing a 1x Proteinase / Phosphatase inhibitor. The cells were then lysed on ice to obtain a cell lysate. The protein concentration in the cell lysate was measured, and after denaturation, the C-terminal fragment protein of the TDP-43 protein was quantified using the Wes (SimpleProtein) fully automated capillary electrophoresis immunoassay system. In fully automated capillary electrophoresis immunoassay, similar to Western blotting, the amount of the target protein can be quantified by analyzing the detected bands.

[0120] The evaluation results for the C-terminal fragment proteins CTF35 and CTF25 of the TDP-43 protein are shown in Figures 11 and 12. The experimental groups shown in Figures 11 and 12 represent the following: • Healthy-Null-DMSO: A group using neurons derived from healthy individuals, without stress inducers or stimulants. • Healthy-Null-Tunicamycin: Contains a stress-inducing agent derived from neurons of healthy individuals. and the group that does not contain promoting substances. • Healthy-LRSAM1-DMSO: A group using neurons derived from healthy individuals, containing no stress inducers but containing stress-promoting substances. • Healthy-LRSAM1-Tunicamycin: Using neurons derived from healthy individuals, stress inducers and Group containing promoting substances • ALS-Null-DMSO: A group using neurons derived from ALS patients, without containing stress inducers or stimulants. · ALS -Null- Tunicamin: A group of drugs using neurons derived from ALS patients that contain stress-inducing agents but no stress-promoting substances. • ALS-LRSAM1-DMSO: Uses neurons derived from ALS patients, does not contain stress inducers, and promotes Group containing advanced substances · ALS-LRSAM1-Tunicamycin: A group of substances containing stress inducers and stimulants, derived from neurons of ALS patients.

[0121] Using the software Compass for SW included with Wes, the total lengths of TDP-43, CTF35, and CTF25 can be measured. The intensity of the enzyme was quantified. Subsequently, the quantitative values ​​of each protein—full-length TDP-43, CTF35, and CTF25—were divided by the sum of the TDP-43 quantitative values, and the results are shown in Figures 11 and 12. As a result, the enzyme activity of ALS patients was reduced. In the acquired neurons, the amounts of CTF35 and CTF25 were significantly higher than in neurons derived from healthy individuals (comparison of healthy-Null-DMSO and ALS-Null-DMSO in Figures 11 and 12). This suggests that in TDP-43-related diseases such as ALS, the amount of protein in the C-terminal fragment of the TDP-43 protein is increased. This was shown. Furthermore, in neurons derived from ALS patients, treating them with stress-inducing agents... This showed a significant increase in the protein content of the C-terminal fragment of the TDP-43 protein (Figure 11). (12. Comparison of ALS-Null-DMSO and ALS-Null-tunicamycin). In neurons derived from ALS patients treated with stress inducers, when LRSAM1 was expressed by a stimulant, CTF35 and CTF25 were lower compared to the ALS-Null-tunicamycin group. From the above, the stimulant It was shown that the quality induces the degradation of the C-terminal fragment proteins CTF35 and CTF25 of the TDP-43 protein.

[0122] Colocalization of LRSAM1 protein and TDP-43 protein using brain tissue sections from sporadic ALS patients. The degree of the disease was evaluated by immunohistochemistry. Tissue samples were obtained through TARGET ALS MULTICENTER POSTMORTEM TISSUE CORE. Paraffin sections of motor cortical tissue were deparaffinized and dehydrated using conventional methods. The cell membranes were then permeated with PBS containing 0.5% Triton-X, and bleeding was performed using Blocking One (Nacalai). Locking was performed. Subsequently, the above-mentioned anti-TDP-43 antibody and LRSAM1 antibody (Sigma), anti-MAP2 antibody were administered. Abcam was used as the primary antibody, diluted with Signal Enhancer A (Nacalai), and added to the sample. The sample was incubated overnight at 4°C. Anti-MAP2 antibody was used to visualize nerve cells. The sample was washed with PBS the following day. Next, a secondary antibody diluent conjugated with Alexa dye and DAPI solution (Invitrogen) for nuclear staining were added, and the mixture was incubated at room temperature for 1 hour. After washing with PBS, the mounting solution was applied. The samples were mounted on coverslips using [a specific method]. The samples were imaged with a fluorescence microscope, and the images were analyzed using Matlab (Mathworks). The analysis method involved surrounding the nucleus and cytoplasm within the image. Furthermore, the signals of TDP-43 and LRSAM1 in the nucleus or cytoplasm of a single cell were calculated, and the degree of co-localization of these signals was calculated as Pearson's correlation coefficient. A higher value indicates a higher degree of co-localization, allowing for a comparison of the degree of localization of TDP-43 and LRSAM1 in the nucleus and cytoplasm of nerve cells.

[0123] Figure 13 shows the results of the intracellular localization of TDP-43 protein and LRSAM1 in the brains of ALS patients. The images in Figure 13 are fluorescence-stained images of TDP-43 (A), LRSAM1 (B), MAP2 (C) representing the neuronal cytoplasm, and DAPI (D) representing the nucleus, respectively. The dashed lines in Figures 13A and B indicate the positions of cell nuclei, derived from Figure 13D. Figure 13A shows that TDP-43 leaks outside the cell nucleus in neurons in the brains of sporadic ALS patients, as previously reported. Furthermore, comparing Figures 13A and 13B, co-localization of TDP-43 and LRSAM1 in the cytoplasm was observed, as indicated by the arrows. Brain samples from six patients were stained and imaged using the method described above, and the cell nuclei were extracted from the acquired images. The cytoplasm was defined, and the correlation coefficient was calculated as described above. Figure 14(A) shows the distribution of the correlation coefficient. This is a histogram. When comparing the correlation coefficients of TDP-43 and LRSAM1 in the nucleus and cytoplasm, it was found that there were many cells showing a higher correlation in the cytoplasm than in the nucleus (Figure 14(A)). Also, when calculating the average correlation coefficients of the nucleus and cytoplasm, the correlation coefficient in the cytoplasm was statistically significantly higher (Figure 14(B)). That is, TDP-43 outside the nucleus shows a high proportion of co-localization with LRSAM1. This was shown.

[0124] From the above results, a promoting substance for a predetermined target (for example, a target contributing to an increase in ubiquitination activity such as LRSAM1) may contribute to the maintenance or improvement of normal cell functions. As a result, the promoting substance may contribute to advantageous effects such as suppression of cell damage, improvement of clearance of intracellular TDP-43, appropriate control of post-translational modifications such as phosphorylation, treatment or prevention of TDP-43-related diseases, etc. Also, it is speculated that a technique for increasing the intracellular amount of a protein having ubiquitination activity such as LRSAM1 and increasing the intracellular ubiquitination activity can improve the function and localization of TDP-43 in a normal direction. Therefore, the said technique may become a suitable treatment method or prevention method for TDP-43-related diseases. These matters are new findings clarified by the completion of the present disclosure and are useful, for example, for protecting nervous system cells such as motor neurons. Also, it will be obvious to those skilled in the art that even when the expression-enhancing nucleic acid according to the present embodiment is used as a promoting substance for a predetermined target (for example, LRSAM1), an equivalent effect can be obtained.

[0125] Focusing on ALS, which is a kind of TDP-43-related disease, its pathological image is known to be mainly composed of TDP-43 (Ling SC et al., Neuron. 2013;79(3):416-38.). Also, TDP-43 and FUS are known. It is also known that the pathologies related to these are mutually exclusive (Guo L et al., Cold Spring Harb Perspect Med. 2017;7(9):a024554.). Therefore, abnormal localization of TDP-43 in cells Using promoting substances to reduce the amount of TDP-43 protein or to increase the clearance of abnormal TDP-43 protein can reduce cell damage such as motor neurons, which is advantageous for the treatment or prevention of TDP-43-related diseases. Furthermore, since ALS has been reported to be primarily pathologically caused by TDP-43, TDP-43 protein By increasing the intracellular clearance of proteins, the amount of pathologically active proteins can be reduced. As a result, those skilled in the art will know that the promoting substance is particularly useful in the treatment or prevention of ALS. That should be obvious.

[0126] Furthermore, the ALS patient-derived neurons used in this embodiment have heterozygous mutations in the TARDBP gene, which encodes the TDP-43 protein. Therefore, within these neurons, there are broadly two types: Two types of TDP-43 protein can be produced: normal TDP-43 protein (wild-type TDP-43 protein) and mutant TDP-43 protein. As in this embodiment, by using a promoter for a predetermined target (e.g., LRSAM1), the proportion of normal TDP-43 protein in the cell can be increased. Furthermore, the functions inherent to TDP-43 can be enhanced, contributing to the improvement of normal cellular function. In particular, mutant TDP-43 protein tends to accumulate in the cytoplasm, so it is more easily degraded by LRSAM1 protein present in the cytoplasm, while normal TDP-43 protein present in the nucleus can continue to exert its inherent functions within the cell.

[0127] Neither Patent Document 1 nor Non-Patent Document 1 mentioned above discusses anything related to the TDP-43 protein or diseases associated with it. Non-patent document 1 describes the effects of stress-inducing agent treatment on cell death. However, the cells used in non-patent document 1 are non-neuronal cells, which have different properties from the nervous cells used in this embodiment. This is supported, for example, by the fact that the intracellular localization of the TDP-43 variant differs between non-neuronal and nervous cells (Shenouda M et al., Front Neurosci. 2022;16:868556). Therefore, the results of this embodiment using nervous cells such as motor neurons represent one of the new findings revealed by the completion of this disclosure, suggesting new therapeutic or preventive effects against nervous system diseases.

[0128] According to this embodiment, it is possible to easily maintain or improve cell function. This is useful in the pharmaceutical field.

[0129] In light of the above implications, the present invention is subject to numerous modifications and variations. Therefore, it should be understood that, within the scope of the appended claims, the invention is not limited to the forms specifically described herein. < / c1> < / c1> < / c1> < / c1> < / b1> < / b1> < / b1> < / a1> < / a1> < / a1> < / a1> < / a1> < / a5>

Claims

1. A composition for use in the treatment or prevention of TAR DNA-binding protein 43-related diseases, comprising a substance that promotes the increase of intracellular amounts of proteins having ubiquitination activity.

2. A composition for improving the clearance of TAR DNA-binding protein 43, comprising a substance that promotes the increase of intracellular amounts of proteins with ubiquitination activity.

3. The composition according to claim 1 or 2, for use in subjects who have developed or are at risk of developing 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 limbic system-dominant senile TDP-43 encephalopathy.

5. The aforementioned promoting substance is a protein having ubiquitination activity, and is a component of the LRSAM1 protein. The composition according to claim 1 or 2, which is a substance that increases intracellular volume.

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

7. The aforementioned promoting substance promotes the production of translation products from nucleic acids encoding the LRSAM1 protein. The combination according to claim 1 or 2, which is a substance that increases the intracellular amount of LRSAM1 protein. Finished product.

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 nucleic acid.

10. The composition according to claim 9, wherein the promoting substance comprises one or more selected from expression-enhancing nucleic acids and expression vectors.

11. The composition according to claim 1 or 2, wherein the cells are nerve cells.

12. The process includes administering an effective amount of a promoting agent to a subject requiring it, A method for treating or preventing TAR DNA-binding protein 43-related disease, wherein the promoting substance is a substance that increases the intracellular amount of a protein having ubiquitination activity.

13. The method according to claim 12, wherein the promoting substance is administered to the subject to treat or prevent the disease by suppressing the occurrence or progression of nerve cell damage.

14. Use of a promoter that increases the intracellular amount of ubiquitinating proteins in the manufacture of a composition for treating or preventing TAR DNA-binding protein 43-related diseases.

15. A method for suppressing cell damage, comprising bringing a substance that increases the intracellular amount of a protein having ubiquitination activity into contact with a neuron, thereby suppressing the occurrence or progression of cell damage in the neuron.

16. A method for improving protein clearance, comprising bringing a neuron into contact with a substance that increases the intracellular amount of a protein having ubiquitination activity, thereby improving the clearance of TAR DNA-binding protein 43 in the neuron.

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