EP4687954A2 - Composition and method for treating or preventing tar dna-binding protein 43 related disease - Google Patents

Abstract

A composition for use in treatment or prevention of a TAR DNA-binding protein 43 related disease, comprising a promoting substance that increases an intracellular amount of a protein having ubiquitination activity.

Description

TITLE OF THE INVENTION

COMPOSITION AND METHOD FOR TREATING OR PREVENTING TAR DNA-BINDING PROTEIN 43 RELATED DISEASE

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001-01]

The present application is based upon and claims the benefit of priority to U.S. Applications No. 63/493, 462, filed March 31, 2023, and No. 63/597, 559, filed November 9, 2023. The entire contents of these applications are incorporated herein by reference.

REFERENCE TO A SEQUENCE LISTING

[0001-02]

In accordance with WIPO Standard ST.26 the specification makes reference to a Sequence Listing submitted electronically as a.xml file named “541677WO_ST26.xml.” The.xml file was generated on March 26, 2024 and is 49,152 bytes in size. The entire contents of the Sequence Listing are hereby incorporated by reference.

BACKGROUND OF THE INVENTION

Field of the Invention

[0001-03]

The present disclosure relates to a composition, a method for treating or preventing a TAR DNA- binding protein 43 related disease, and the like.

Description of Background Art

[0002] Patent Document 1 discloses a compound for improving splicing of messenger RNA (mRNA). The same document takes familial autonomic neuropathy as an example of a progressive neurodegenerative disease and discloses a treatment method for the disease.

[0003]

Non-Patent Document 1 focuses on Charcot-Marie-Tooth disease Type 2P and describes suppression of cell death associated with accumulation of misfolded protein. The same document discloses that LRSAM1 suppresses accumulation of misfolded luciferase and reduces cytotoxicity.

[0004]

Patent Document 1: US 2018/0118748 Al

[0005]

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

The entire contents of these publications are incorporated herein by reference.

SUMMARY OF THE INVENTION

[0006]

An embodiment of the present disclosure is a composition for use in treatment or prevention of a TAR DNA-binding protein 43 related disease, comprising a promoting substance that increases an intracellular amount of a protein having ubiquitination activity.

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

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

Further, an embodiment of the present disclosure is a method for suppressing cell damage, comprising suppressing occurrence or progression of cell damage in a neuron by bringing the neuron into contact with a promoting substance that increases an intracellular amount of a protein having ubiquitination activity.

Further, an embodiment of the present disclosure is a method for improving clearance of a protein, comprising improving clearance of a TAR DNA-binding protein 43 protein in a neuron by bringing the neuron into contact with a promoting substance that increases an intracellular amount of a protein having ubiquitination activity.

BRIEF DESCRIPTION OF THE DRAWINGS

A more complete appreciation of the invention and many of the attendant advantages thereof will be readily obtained as the same becomes better understood by reference to the following detailed description when considered in connection with the accompanying drawings, wherein:

[0007]

Fig. 1 is a graph showing degrees of cell damage in motor neurons derived from healthy individuals and motor neurons derived from ALS patients, using neurite length as an indicator.

Fig. 2 is a graph showing cytoplasmic/nuclear abundance ratios of TDP-43 protein in motor neurons derived from healthy individuals and motor neurons derived from ALS patients.

Fig. 3 is a graph showing an amount of phosphorylated TDP-43 protein present in cytoplasm in motor neurons derived from healthy individuals and motor neurons derived from ALS patients.

Fig. 4 is a graph showing an effect of presence or absence of a promoting substance on neurite length of motor neurons derived from healthy individuals.

Fig. 5 is a graph showing an effect of presence or absence of a promoting substance on neurite length of motor neurons derived from ALS patients. Fig. 6A is a graph showing cytoplasmic/nuclear abundance ratios of TDP-43 protein due to presence or absence of a promoting substance in motor neurons derived from healthy individuals.

Fig. 6B is a graph showing cytoplasmic/nuclear abundance ratios of TDP-43 protein due to presence or absence of a promoting substance in motor neurons derived from ALS patients.

Fig. 7A is a graph showing an amount of phosphorylated TDP-43 protein present in cytoplasm in motor neurons derived from healthy individuals.

Fig. 7B is a graph showing an amount of phosphorylated TDP-43 protein present in cytoplasm in motor neurons derived from ALS patients.

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

Fig. 9 is a graph showing an effect of presence or absence of a promoting substance on STMN2 splicing in motor neurons derived from healthy individuals.

Fig. 10 is a graph showing an effect of presence or absence of a promoting substance on STMN2 splicing in motor neurons derived from ALS patients.

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

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

Fig. 13 shows immunohistofluorescence staining images of TDP-43 protein (A), LRSAM1 (B), MAP2 (C), and DAPI (D) in brain (motor cortex) sections from a sporadic ALS patient. MAP2 is used to label neural cytoplasm, and DAPI is used to label nucleus. Arrows in Figs. 13 A and 13B indicate co-localization of TDP-43 and LRS AMI in cytoplasm. Figs. 14A and 14B show degrees of co-localization of TDP-43 and LRSAM1 in neural nuclei and cytoplasm, respectively, using Pearson's correlation coefficients. Fig. 14A shows distribution of the correlation coefficients and the cell counts in a histogram. Fig. 14B is a graph comparing average correlation coefficients of nucleus and cytoplasm.

DETAILED DESCRIPTION OF THE EMBODIMENTS

Embodiments will now be described with reference to the accompanying drawings, wherein like reference numerals designate corresponding or identical elements throughout the various drawings.

[0008]

In the following, the present disclosure is described based on embodiments thereof.

Contents disclosed in patent documents and non-patent documents cited in this specification are entirely incorporated herein by reference.

GenBank accession numbers and associated sequence information as well as other data available through databases are entirely incorporated herein by reference.

Sequence listing in electronic format submitted together with the present specification is entirely incorporated herein by reference.

Further, in mRNA base sequences disclosed in the sequence listing of the present application and the accession numbers described in the present specification, uracil (U) is expressed as thymine (T). However, an actual mRNA base sequence is applied with “T” read as “U.”

[0009]

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.

[0010]

TAR DNA-binding Protein of 43 kDa is an RNA-binding protein known as TDP-43 and is normally localized to nucleus. When an abnormal TDP-43 protein (such as a protein aggregate or a fragmented protein) is produced, it can contribute to development of various diseases. A TDP-43 related disease generally manifests symptoms such as motor dysfunction or cognitive impairment, which can greatly impact quality of life (QOL). However, there is no established effective treatment method that leads to a fundamental cure for a TDP-43 related disease.

[0011]

In one embodiment, the composition of the present disclosure is used for treating or preventing a TAR DNA-binding protein 43 (TDP-43) related disease. In other words, the composition of one embodiment is a composition for treatment or prevention of a TDP-43 related disease. 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.

[0012]

In the present specification, “treatment” includes, for example, suppression of progression, alleviation, mitigation, eradication, and the like of a disease, a disorder, or one or more symptoms related thereto.

In the present specification, “prevention” includes, for example, preventing or delaying onset of a disease, preventing or delaying recurrence of a disease, and the like. The composition of the present disclosure can be used for one or more purposes of treatment and prevention.

[0013]

In the following description, for convenience, unless otherwise specified, humans, as a type of mammal, or human cells will be described as examples to which the present disclosure is applied. However, as long as the effects of the present disclosure are achieved, it is also similarly applicable to other animal species or their cells besides humans, including rodents such as mice and rats, primates such as monkeys, and mammals such as rabbits, as well as orthologs of these animal species.

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

[0014]

In the following description, unless otherwise specified, “nucleic acid” means a polymer in which two or more nucleosides are linked together by internucleoside linkages, regardless of its length or backbone structure. Nucleic acid bases, sugars, and internucleoside linkages that form this polymer may each independently have a natural structure, or a non-natural structure in which a certain atom, functional group, or ring structure or the like has been added, substituted, or deleted from a natural structure.

Further, as long as the effects of the present disclosure are achieved, a nucleic acid in the present specification may also exist in a form of a pharmaceutically acceptable salt or ion. 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.

[0015]

TDP-43, an RNA-binding protein, is involved in regulation of transcription, splicing, and translation in cells. TDP-43 is a protein encoded by a TARDBP gene. A human wild-type TDP- 43 protein is a protein consisting of 414 amino acids in its full length and is a nuclear protein localized primarily to nucleus. As an example, an mRNA base sequence of a human wild-type TDP-43 is shown in SEQ ID NO: 1 (GenBank accession number: NM 007375.4). Further, an amino acid sequence of a human wild-type TDP-43 protein is shown in SEQ ID NO: 2 (GenBank accession number: NP_031401.1).

[0016]

In a TDP-43 related disease, production of an abnormal TDP-43 protein is considered to be one of causes. An abnormal protein typically becomes more likely to migrate into cytoplasm, form a protein aggregate, or have a change in amount and/or type of a post-translational modification such as phosphorylation. In this way, an abnormal TDP-43 protein often has a property that differs from that of a wild-type protein. Examples of abnormal TDP-43 proteins that can contribute to disease development and progression include, but are not limited to, an N-terminal fragment of a human wild-type TDP-43 protein (SEQ ID NO: 3; see Shenouda et al., Front Neurosci., 2022; 16: 868556), a C-terminal fragment protein consisting of amino acid residues 209 to 414 from the N-terminus of a human wild-type TDP-43 protein (SEQ ID NO: 4), a C-terminal fragment protein consisting of amino acid residues 90 to 414 from the N-terminus of a human wild-type TDP-43 protein (SEQ ID NO: 15), a C-terminal fragment protein consisting of amino acid residues 169 to 414 from the N- terminus of a human wild-type TDP-43 protein (SEQ ID NO: 16), a C-terminal fragment protein consisting of amino acid residues 174 to 414 from the N-terminus of a human wild-type TDP-43 protein (SEQ ID NO: 17), an amino acid mutant in a wild-type TDP-43 protein (see SEQ ID NO: 2), and the like. Examples of amino acid variants of TDP-43 protein include proteins with point mutations such as G294A, G298S, A315T, M337V, Q343R, A382T, and the like, or combinations thereof.

[0017]

TDP-43 related diseases in the present disclosure include diseases that may occur due to abnormal intracellular localization of TDP-43 protein. Examples of TDP-43 related diseases include one or more neuromuscular diseases selected from neurodegenerative diseases and muscle diseases (for example, diseases described in the 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 (Pans), 2022; 178 (3): 196-205., Walker et al., J Neurosci., 2014; 34 (19): 6448-58).

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

Examples of muscle diseases include inclusion body myositis.

By targeting these diseases, a TDP-43 related disease can be effectively treated or prevented by addressing specific pathological conditions such as abnormal intracellular localization or clearance abnormalities of TDP-43 protein. That is, in one embodiment, the composition described above can be a composition for improving clearance of TDP-43 protein. From a point of view of further enhancing effectiveness of disease treatment or prevention, the TDP-43 related disease is preferably a neurodegenerative disease selected from ALS, FTLD, and LATE, and is more preferable at least ALS.

[0018]

When the composition described above is used for treating or preventing a TDP-43 related disease, an applicable subject is preferably a subject who has developed or is likely to develop a TDP-43 related disease, and the subject may or may not have a specific mutation in a particular gene. That is, the composition described above can be used for a subject who does not have a mutation of a specific gene (for example, a normal homozygous subject). Further, the composition described above can also be used for a subject who has one or more mutations selected from a specific gene. When a subject has gene mutations, the gene mutations may each be independently a heterozygous mutation or a homozygous mutation. The subject is preferably a human subject (human living body) or a human-derived cell. Presence or absence of these mutations can be determined using a known method such as PCR.

[0019]

Taking ALS, which is a type of TDP-43 related disease, as an example, gene mutations in humans include one or more gene mutations in a C9orf72 gene, a SOD1 gene, a TBK1 gene, a TARDBP gene, a FUS gene, a NEK1 gene, and the like, but are not limited to these. In one embodiment, the subject described above has, for example, a homozygous or heterozygous mutation in the TARDBP gene, but does not have a mutation in the FUS gene and/or the SOD1 gene.

Examples of the gene mutations in humans described above include one or more selected from: an abnormal elongation of a 6-base repeat sequence in intron 1 of the C9orf72 gene, a mutation in the SOD1 gene causing an A4V amino acid point mutation, a mutation in the TBK1 gene causing a T4A amino acid point mutation, a mutation in the TARDBP gene causing an A382T amino acid point mutation, a mutation in the FUS gene causing a P525L amino acid point mutation, a mutation in the NEK1 gene causing an R261H amino acid point mutation, and the like.

[0020] A base sequence of the TARDBP gene may differ between individuals. In the present embodiment, as long as it encodes a protein having an equivalent function to a wild-type TDP-43 protein, an mRNA base sequence of TDP-43 may a base sequence having 80% or higher, 85% or higher, 90% or higher, 95% or higher, 98% or higher, or 100% identity to the base sequence described in SEQ ID NO: 1. Examples of causes of differences in base sequences include, but are not limited to, degenerate codons and the like.

Further, as long as it encodes a protein having an equivalent function to a wild-type TDP-43 protein, an amino acid sequence of the TDP-43 protein may be an amino acid sequence having 80% or higher, 85% or higher, 90% or higher, 95% or higher, 98% or higher, or 100% identity to the amino acid sequence described in SEQ ID NO: 2.

Search for regions having sequence identity, complementarity or homology in the present specification, and calculation of percent complementarity, can be performed, for example, using commonly known software or methods such as the BLAST program or Genetyx software.

In the present specification, “complementary” or “complementarity” means ability to form a base pair between two nucleic acid bases. For example, adenine is complementary to thymidine or uracil, and cytosine is complementary to guanine.

[0021]

In one embodiment, the composition of the present disclosure includes a promoting substance. The promoting substance has one or more features that increase an amount and/or activity of a predetermined translation product in applicable targets including various cells such as cells, specimens, and living bodies. In the following explanation, this substance is also simply referred to as a “promoting substance.”

As one embodiment, the promoting substance directly or indirectly promotes gene expression from a predetermined nucleic acid. As a result, at least an intracellular amount of a predetermined translation product can be increased. In this specification, “gene expression” or “expression of a gene” includes not only the synthesis (generation) of a translation product based on information of a predetermined nucleic acid base sequence, but also synthesis (generation) of a transcription product. Specifically, the promoting substance according to one embodiment promotes, in a cell, at least one selected from production of a transcription product from a nucleic acid encoding a predetermined protein, production of a translation product, a function of a transcription product, and a function of a translation product. Specific examples of a nucleic acid to be promoted include polymers of deoxyribonucleotides such as endogenous DNA such as genomic DNA, exogenously introduced DNA, and synthetic DNA such as complementary DNA; polymers of ribonucleotides such as RNA; and the like.

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 the present disclosure may be a translation product itself to be increased. The promoting substances described above may each be used independently, or two or more of the promoting substances may be used in combination. Preferred embodiments of the promoting substance will be described later.

[0022]

Examples of transcription products preferably include any RNA synthesized using DNA as a template, regardless of presence or absence of RNA processing. Specific examples of RNA include polymers of various ribonucleotides such as mRNA precursors, mature mRNA, and noncoding RNAs such as microRNAs (miRNAs). In one embodiment, the RNA described above is preferably an mRNA precursor and/or a mature mRNA. By increasing an expression level of a transcription product such as mRNA, an amount of template of translation is increased.

Therefore, it can contribute to an increase in an expression level of a target protein.

Examples of translation products include proteins and the like generated through translation from any mRNA, regardless of presence or absence of post-translational modification.

These transcription products and translation products may independently be, for example, wildtype products transcribed and translated from predetermined DNA, or sequence variants such as various splicing variants and base substitution products (including SNPs), as well as non-wildtype proteins translated from these variants or products. The various types of DNA, transcription products, and translation products described above may be, as needed and independently, subjected to one or more commonly known extraction or purification steps to be isolated.

[0023]

It is preferable that a transcription product is capable of generating a wild-type protein or a protein having an equivalent function. Further, it is preferable that a translation product is a wild-type protein or a protein having an equivalent function to the wild-type protein. By generating these products, inherent functions of cells can be easily maintained or normalized, and occurrence or progression of cell damage can be easily further reduced. As a result, it can more effectively contribute to treatment or prevention of a disease.

In order to achieve the preferred modes described above, for example, as shown in examples to be described later, methods such as introducing an exogenous nucleic acid encoding a target protein or promoting splicing into a mature mRNA capable of translating a target protein can be used, but it is not limited to these methods.

In order to determine whether or not a generated translation product has an equivalent function to a wild-type, for example, enzyme activity measured using a commonly known method can be compared with activity of a wild-type translation product to make the determination.

[0024]

In the present specification, “promotion” means at least one of: an increase in abundance (for example, expression level) of a transcription product and/or a translation product from a predetermined gene in presence of any substance; and an increase in function (for example, activity) of a translation product. For example, in a case where an expression level of a transcription product or a translation product when a substance to be evaluated is brought into contact with or exposed to a predetermined applicable target is greater than an expression level of a transcription product or a translation product in absence of the substance to be evaluated, it can be determined that it has been promoted. In addition to or instead of this, that it has been promoted can also be determined by an increase in activity of a protein itself or an intracellular enzyme. As a criterion for determining whether or not it has been promoted, for example, it may be determined based on a magnitude of a measured value obtained using any measurement method, or it may be determined based on a magnitude of an arithmetic mean value, geometric mean value, or median value calculated from measured values or ratios thereof, or it may be determined by having a statistically significant difference. In a case where it is evaluated based on a ratio of measured values, for example, when a ratio (R2/R1) of a measured value (R2) in an experimental group to be determined to a measured value as a reference or a reference value (Rl) is, for example, 1.05 times or more, 1.10 times or more, 1.30 times or more, 1.50 times or more, or 2.00 times or more, it can be determined that it has been promoted.

[0025]

Examples of promotion of generation of a transcription product include one or more of: promotion of transcription from DNA to an mRNA precursor, inhibition of degradation of mRNA precursor, inhibition of degradation of mature mRNA, or control or activation of RNA processing that forms mature mRNA from mRNA precursor, and the like.

Examples of promotion of generation of a translation product include one or more of: promotion of translation from mature mRNA, inhibition of degradation of a protein generated through translation, and the like.

[0026]

For a measurement of an expression level of a transcription product, for example, various measurement methods such as PCR, microarray, and RNA sequencing can be used using measurement samples such as cultured cells, living bodies, or specimens collected from the living bodies. These measurement methods are preferably performed in a quantifiable manner. When necessary, processes to extract transcription products from the measurement samples described above may be performed, or processes to synthesize complementary DNAs (cDNAs) by reverse transcription reactions using transcription products as templates may be performed, and these products may be used for the measurements described above.

For a measurement of an expression level or activity of a translation product, for example, various measurement methods such as ELISA, western blot, flow cytometry, immunostaining, mass spectrometry, intracellular or in vivo accumulation of substrates labeled with fluorescent or radioactive substances, and in vitro activity measurement using a substrate can be used using measurement samples such as cultured cells, living bodies, or specimens collected from the living bodies. These measurement methods are preferably performed in a quantifiable manner. When necessary, processes to extract translation products from the measurement sample described above may be performed, and the extracts may be used for the measurement described above.

[0027]

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

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

As another embodiment, the promoting substance may be a substance that directly or indirectly promotes generation and/or activity of a translation product, which is a protein having ubiquitination activity. As a result, an amount of the translation product as a protein having ubiquitination activity can be increased.

[0028]

In order to increase a protein having ubiquitination activity in cells, for example, generation of a transcription product or translation product can be promoted. For example, the promoting substance described above may promote generation of a translation product by promoting translation from a transcription product encoding a protein having ubiquitination activity. Further, for example, the promoting substance described above may promote generation of a target translation product by increasing an expression level of a transcription product, controlling expression of other genes that control an expression level of the target translation product, and so on. Other means for achieving this include, for example, methods of introducing the protein itself having ubiquitination activity into cells. In either case, by these embodiments, for example, by increasing intracellular ubiquitination activity, a state of the cells can be easily improved.

[0029]

The proteins having ubiquitination activity described above include various ubiquitination- related proteins. Such proteins include, for example, ubiquitin ligases such as LRSAM1 (leucine rich repeat and sterile alpha motif containing 1), Gp78 (Glycoprotein 78), CHIP (C-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, ubiquitin conjugating enzymes, and the like. The above-described TDP-43 is excluded from proteins having ubiquitination activity.

Whether or not a generated protein or translation product has ubiquitination activity can be determined, for example, by an increase or decrease in ubiquitin amount or activity associated with presence or absence of exposure to a promoting substance, using methods such as ELISA, western blot, flow cytometry, immunostaining, and a TR-TUBE method to be described below. This determination can utilize, for example, the determination criterion described above.

[0030]

Human mRNA base sequences and protein amino acid sequences encoding the ubiquitin ligase described above are respectively shown below.

• LRSAM1: SEQ ID NO: 5 (mRNA base sequence; GenBank accession number

NM 001005373.4), and SEQ ID NO: 6 (amino acid sequence; GenBank accession number NP_001005373.1)

• Gp78: SEQ ID NO: 7 (mRNA base sequence; GenBank accession number NM_001144), and SEQ ID NO: 8 (amino acid sequence; GenBank accession number NP 001135.3)

• CHIP: SEQ ID NO: 9 (mRNA base sequence; GenBank accession number NM 005861.4), and SEQ ID NO: 10 (amino acid sequence; GenBank accession number NP 005852.2) • RNF19A: SEQ ID NO: 11 (mRNA base sequence; GenBank accession number NM_183419), and SEQ ID NO: 12 (amino acid sequence; GenBank accession number NP 056250.3)

• MGRN1 : SEQ ID NO: 13 (mRNA base sequence; GenBank accession number

NM 015246.4), and SEQ ID NO: 14 (amino acid sequence; GenBank accession number NP 056061.1)

[0031]

In one embodiment, the promoting substance is preferably a substance that at least increases an intracellular amount of the LRSAM1 protein as a protein having ubiquitination activity. In one embodiment, the promoting substance is preferably a substance that promotes generation of a transcription product and/or a translation product from a nucleic acid encoding the LRSAM1 protein, and more preferably a substance that promotes generation of at least a translation product. As a result, the promoting substance can at least promote generation of a protein having ubiquitination activity such as the LRSAM1 protein, and increase an intracellular amount of the LRS AMI protein. The LRS AMI protein described above is preferably a human LRS AMI protein, and also preferably a wild-type LRS AMI protein, and more preferably a wild-type human LRSAM1 protein.

[0032]

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

The LRSAM1 protein exists mainly in cytoplasm and has ubiquitination activity for a target protein. The LRS AMI protein catalyzes addition of ubiquitin to a target protein (for example, an abnormal protein). As a result, an ubiquitinated target protein is promoted to be degraded by an ubiquitin-proteasome system in cells. As a result, it can contribute to clearance of an abnormal protein, suppression of cell damage caused by presence of an abnormal protein, and ultimately the treatment or prevention of a TDP-43 related disease.

[0033] In one embodiment, the promoting substance is also more preferably a substance that promotes generation of a transcription product capable of expressing a wild-type LRSAM1 protein or a protein having an equivalent function to the wild-type protein. In addition to this, or in place of this, the promoting substance is also more preferably a substance that promotes generation of a wild-type LRSAM1 protein or a protein having an equivalent function to the wild-type protein, as a translation product. When a promoting substance capable of expressing a wild-type LRSAM1 protein is used, an amino acid sequence of the protein is preferably a full-length wildtype sequence.

[0034]

By increasing expression levels of transcription products or translation products from nucleic acids (for example, genomic genes, exogenously introduced nucleic acids, mRNAs, and the like) encoding ubiquitination-related proteins such as LRS AMI , or by enhancing functions of these products, intracellular amounts and/or intracellular ubiquitination activities of proteins such as LRSAM1 can be increased. This can effectively contribute to maintaining or improving cellular functions. Specifically, by promoting induction of autophagy, or promoting degradation of an abnormal protein that contributes to onset or progression of a TDP-43 related disease through ubiquitination, intracellular clearance of the abnormal protein can be easily normalized. As a result, it can contribute to the treatment or prevention of a TDP-43 related disease. These functions can become particularly pronounced by expressing a wild-type protein or a protein having an equivalent function to the wild-type protein.

[0035]

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

Further, as long as it encodes a protein having an equivalent function to a wild-type LRSAM1 protein, an mRNA base sequence of LRS AMI may be a base sequence having 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99% or more, or 100% identity to a base sequence described in SEQ ID NO: 5. That is, for an mRNA base sequence of LRSAM1, it may be possible that a base is not deleted, substituted, or added, and, for example, it may be possible that 1 to 625 (or 1 to 468, 1 to 312, 1 to 156, 1 to 63, 1 to 31) bases are deleted, substituted, or added. Examples of such an mRNA base sequence include, but are not limited to, a degenerate codon, and the like.

Further, as long as it encodes a protein having an equivalent function to a wild-type LRSAM1 protein, an amino acid sequence of the LRSAM1 protein may be an amino acid sequence having 80% or higher, 85% or higher, 90% or higher, 95% or higher, 99% or higher, or 100% identity to the amino acid sequence described in SEQ ID NO: 6. That is, for the LRSAM1 protein, it may be possible that an amino acid is not deleted, substituted, or added, and, for example, it may be possible that 1 to 145 (or 1 to 108, 1 to 72, 1 to 36, 1 to 14, 1 to 7) amino acids are deleted, substituted, or added.

In order to determine whether or not a generated translation product has an equivalent function to a wild-type LRSAM1 protein, for example, ubiquitination activity or an amount of an ubiquitinated protein can be measured using a method such as the Trypsin Resistant Tandem Ubiquitin-binding Entity (TR-TUBE) method, and compared to activity or an amount of a wildtype translation product to make the determination. As a criterion for determining that it has an equivalent function, for example, a ratio to a wild-type result may be set to 70% or more, and preferably 80% or more.

[0036]

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

The expression enhancing nucleic acid means a nucleic acid for expressing a target gene, and is preferably an exogenously introduced nucleic acid. In one embodiment, the expressionenhancing nucleic acid is preferably a nucleic acid for promoting expression of a target gene. A target of gene expression promotion is, for example, an endogenous nucleic acid (for example, genomic DNA) encoding one or more of the proteins described above such as LRSAM1, Gp78, CHIP, RNF19A, and MGRN1, or an exogenously introduced nucleic acid. The promoting substances described above may each be used independently, or multiple types of the promoting substances may be used in combination.

By containing such a substance, for example, an amount of a translation product having ubiquitination activity can be easily controlled (for example, an increase in gene expression such as an increase in generation of a transcription product such as mRNA and/or a translation product such as a protein). As a result, intracellular clearance of an abnormal protein such as a TDP-43 mutant protein can be effectively easily normalized. This is advantageous in that, by taking a target translation product as the LRS AMI protein and, for example, taking a target nucleic acid for expression enhancement as a nucleic acid encoding the LRSAM1 protein, ubiquitination of an abnormal protein can be increased and intracellular clearance of the abnormal protein can be further improved.

[0037]

Examples of expression-enhancing nucleic acids include, but are not limited to, one or more nucleic acids selected from antisense, non-coding RNA, small activating RNA, and the like. These expression-enhancing nucleic acids have a function of enabling expression of a target gene in cells and/or promoting intracellular expression of the gene. As a result, an intracellular amount of a target translation product can be increased. These nucleic acids can be obtained or manufactured, for example, by screening using a commonly known method.

In one embodiment, an expression-enhancing nucleic acid preferably has fewer bases than a nucleic acid included in an expression vector to be described later. In one embodiment, an expression-enhancing nucleic acid has bases, for example, in a range of 5 to 300 bases per strand, and preferably, for example, 10 to 100 bases per strand.

[0038]

An antisense used as an expression-enhancing nucleic acid can take forms such as, for example, (a) a nucleic acid that controls RNA processing, (b) a nucleic acid partially or fully complementary to miRNA, and (c) a nucleic acid partially or fully complementary to Natural Antisense Transcript (NAT). These antisenses may bind complementarity to a full-length target nucleic acid, or may bind complementarity to a part of a sequence of a target nucleic acid.

[0039] The above form (a) binds complementarity to a splicing-related sequence in an mRNA precursor to promote RNA processing, such as splicing, to generate a desired mature mRNA (for example, mRNA capable of translating a full-length wild-type protein). This contributes to an increase in expression of a target gene (specifically, an increase in generation of a transcription product such as mRNA and/or a translation product such as a protein).

The above form (b) binds complementarity to miRNA and inhibits a function of miRNA. miRNA is generally a type of nucleic acid that binds complementarity to a 3'UTR region of mRNA. Therefore, miRNA works to suppress target gene expression by increasing degradability of mRNA or suppressing translation from mRNA. This form weakens or eliminates the function of miRNA by an antisense that binds complementarity to miRNA, thereby releasing the suppression of gene expression. As a result, generation of mRNA or translation from mRNA is promoted, thereby contributing to an increase in expression of a target gene (specifically, an increase in generation of a transcription product such as mRNA and/or a translation product such as a protein).

The above form (c) binds complementarity to NAT and inhibits a function of NAT. NAT is a type of nucleic acid that is generated in cells and binds complementarity to any region of a specific mRNA. In other words, NAT increases degradability of mRNA or suppresses translation from mRNA. This form weakens or eliminates the function of NAT by a mechanism similar to the above form (b), thereby releasing the suppression of gene expression. As a result, generation of mRNA or translation from mRNA is promoted, thereby contributing to an increase in expression of a target gene (specifically, an increase in generation of a transcription product such as mRNA and/or a translation product such as a protein).

[0040]

In addition to these, antisenses used as expression-enhancing nucleic acids include, for example, nucleic acids capable of binding complementarity to regions including sites such as a translation inhibitory element (HE) of mRNA, upstream ORF (uORF), premature termination codon (PTC), repetitive sequences of guanine or adenine, and the like.

These antisenses can contribute to suppressing mRNA degradation or promoting translation from mRNA, or can contribute to promoting generation of mRNA capable of generating a wildtype protein or a protein having an equivalent function to the wild-type protein. This contributes to an increase in expression of a target gene (specifically, an increase in generation of a transcription product such as mRNA and/or a translation product such as a protein).

These expression-enhancing nucleic acids contribute to improving generation of a target wildtype protein, which is preferable from a point of view of facilitating exertion of an inherent function of a wild-type protein and improving clearance of an abnormal protein.

[0041]

In addition, antisenses used as expression-enhancing nucleic acids include, for example, a nucleic acid that binds complementarily to a regulatory RNA that contributes to suppressing gene expression, a nucleic acid that binds complementarily to a recognition sequence of a RNA- binding protein that destabilizes mRNA, and the like. In this case, since a function of an RNA or protein that works to suppress expression is suppressed, it works to release the suppression of gene expression. As a result, generation of mRNA or translation from mRNA is promoted, thereby contributing to an increase in expression of a target gene (specifically, an increase in generation of a transcription product such as mRNA and/or a translation product such as a protein).

[0042]

The number of bases in an antisense used as an expression-enhancing nucleic acid is typically 10 to 30 bases per strand, and preferably 14 to 25 bases per strand.

An antisense used as an expression-enhancing nucleic acid, in any of the forms described above, has, as a percent complementarity, at least 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, or 100% complementarity to a region having homology (identity) with a complementary strand of a target nucleic acid base sequence. For example, in a case where a base sequence of an antisense is 20 bases, when a nucleic acid base sequence of the antisense and a nucleic acid base sequence in a complementary strand of a target nucleic acid base sequence are compared, it may be possible that a nucleic acid base is not deleted, substituted, or inserted, or, for example, it may be possible that 1 to 5 (or 1 to 4, 1 to 3, 1 to 2, 1) nucleic acid bases are deleted, substituted, or inserted. Such nucleic acids can be screened, designed and obtained using, for example, commonly known methods.

[0043] Examples of non-coding RNAs include nucleic acids such as regulatory RNAs and SINE element-containing translation upregulators (SINEUPs).

A regulatory RNA is a RNA that has a function of promoting transcription from a promoter incorporated in endogenous DNA such as genomic DNA or exogenously introduced nucleic acid, and promoting transcription from a target nucleic acid to mRNA. Along with this, generation of a target translation product (for example, the LRS AMI protein, and the like) can be promoted.

An SINEUP is an RNA that has a sequence of an SINE factor, and is a polyribonucleotide that has a domain promoting translation into a protein and a domain having a complementary sequence to mRNA. By using this, translation from mRNA is promoted (that is, it mainly contributes to an increase in generation of a translation product).

The number of bases of a non-coding RNA can be appropriately set according to a type or function thereof.

[0044]

A small activating RNA (saRNA) is a nucleic acid that is complementary to part or all of a promoter sequence in genomic DNA. As a result, translation from mRNA is promoted, thereby contributing to an increase in expression of a target gene (specifically, an increase in generation of a transcription product such as mRNA and/or a translation product such as a protein).

The number of bases of an saRNA is typically 10 to 30 bases per strand, and preferably 18 to 24 bases per strand. An saRNA is typically a single-stranded or double-stranded RNA.

[0045]

When it is formed to contain an expression-enhancing nucleic acid as a promoting substance, as long as the effects of the present disclosure are achieved, the expression-enhancing nucleic acid can be, for example, in a single-stranded or double-stranded form. Backbone structures of these nucleic acids may be, for example, deoxyribonucleotides, ribonucleotides, non-nucleotides containing bases such as pyrroles or piperidines, or combinations thereof.

As long as the effects of the present disclosure are achieved, a structural unit of a nucleic acid in an expression-enhancing nucleic acid may be of a natural type or a non-natural type. Nonnatural forms include, for example, nucleic acids in which sugars, bases, or atoms or molecules constituting internucleoside bonds in a nucleotide are substituted, modified, or deleted from a natural nucleotide.

[0046]

In one embodiment, the promoting substance may be, for example, an expression vector formed capable of expressing a target gene. Specifically, the promoting substance may be an expression vector that contains a nucleic acid including a base sequence encoding a protein or substance that promotes expression, and is capable of expressing a target protein or substance in cells. With such an expression vector, for example, a target transcription product and/or translation product can be expressed in a subject and an amount of the translation product can be increased, in various environments such as in vivo, in vitro, or ex vivo. This can lead to beneficial effects such as maintaining or improving a cellular function and improving clearance of an abnormal protein.

[0047]

In the present specification, an “expression vector” means a vector that includes a nucleic acid including a promoter base sequence and a first base sequence operably linked to the promoter base sequence, and is capable of newly generating a nucleic acid or protein based on information of the first base sequence in cells or in a test tube. The first base sequence is formed, for example, capable of generating mRNA encoding a predetermined protein, or capable of generating the expression-enhancing nucleic acid described above. That is, the expressionenhancing nucleic acid itself described above is not included in the “expression vector.”

The nucleic acid forming the expression vector may further contain, for example, in its base sequence, a replication origin sequence for replicating in cells or a selection marker sequence such as a drug resistance gene. Examples of promoter base sequences include, but are not limited to, sequences capable of being expressed in mammalian cells such as SV40, and CMV, sequences capable of being expressed in E. coli such as trp, lacl, and lacZ, sequences capable of being expressed in test tubes such as SP6 and T7, and the like.

[0048] A type of the expression vector is not particularly restricted, and examples thereof include lipid particles in which a nucleic acid is encapsulated in a lipid membrane, plasmid vectors, or viral vectors in which a nucleic acid is encapsulated in a capsid, and the like.

Examples of the viral vectors include: DNA-containing viral vectors such as adenoviral vectors, and adeno-associated viral vectors; RNA-containing viral vectors such as retroviral vectors, and lentiviral vectors; and the like. Examples of structural units of a polynucleotide contained in a viral vector include a deoxyribonucleotide skeleton or a ribonucleotide skeleton, and the like. These skeletons are preferably each independently a natural type that does not contain a modified nucleic acid or an atomic substitution, and the like.

[0049]

In a case where it is structured such that a predetermined protein can be generated with the expression vector described above, as long as it has the intended function, the generated protein may include mutations such as deletions, substitutions, or additions of amino acids in its amino acid sequence, or may not include such mutations. Identity between a generated protein or an amino acid sequence in a protein, and the protein or an amino acid sequence in a wild-type protein corresponding to a protein can be, for example, 80% or more, 85% or more, 90% or more, 95% or more, or 100%.

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

[0050]

In one embodiment, the promoting substance can contain a substance that promotes a function of a translation product. One or more types of such promoting substances can be included in the composition. By including such promoting substances, it is possible to control to increase an expression level or activity of a protein that is decreased or may decrease due to a disease, and to effectively treat or prevent the disease. Examples of substances that promote the function of translation products include, but are not limited to, allosteric regulatory factors for proteins, substances that stabilize post-translational modifications, and the like.

[0051]

The promoting substances described above can each be independently synthesized or generated, and manufactured, for example, using a commonly known method in the technical field.

[0052]

Examples of application targets of the promoting substances include subjects such as living bodies of humans and non-human animals, and samples derived from these animals, preferably living humans or samples derived from humans. The application targets of the promoting substances may be healthy subjects (regardless of whether or not there is a risk of developing a disease), or subjects in which a disease has already developed (for example, human patients or disease model animals), or samples derived from these subjects. That is, the promoting substances can be used in various environments such as in vivo that includes or excludes humans, in vitro that includes humans, or ex vivo that includes humans. The applicable targets are more preferably human subjects, and even more preferably healthy individuals or human patients.

Examples of non-human animals include non-human mammals such as: rodents such as rats, mice, and guinea pigs; monkeys; pigs; dogs; and cats. In one embodiment, the non-human animals are land-dwelling mammals.

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

The promoting substances are applied to targets that include at least one or more cells. Such application targets may include, for example, isolated or pure cultured cells, samples containing cells, and subjects such as living bodies formed of cells.

[0053]

Examples of tissues include brain regions such as cerebrum, midbrain, diencephalon, pons, medulla oblongata, and cerebellum, spinal cord, stomach, pancreas, kidneys, liver, adrenal glands, skin, muscles such as skeletal muscles and smooth muscles, lungs, intestines such as large intestine and small intestine, heart, blood vessels, and the like. These are typically formed of aggregates of cells.

Cells may include differentiated cells that form tissues, precursor cells, or stem cells. Taking brain-derived cells as an example, brain-derived samples include various types of neural cells, such as neurons, glial cells (including astrocytes, microglia, and oligodendrocytes), neural precursor cells, neural stem cells, and the like. Among these, cells on which the promoting substances of the present disclosure act are preferably these neural cells. These cells may be used as aggregates of homogeneous cells obtained by isolation, pure culturing, or the like, or as aggregates of cells containing two or more different types of cells.

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

These samples can typically be collected from living or dead animals using commonly known methods such as biopsy or dissection. When necessary, an extraction step or a separation step, such as isolation, may be performed one or more times.

[0054]

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

[0055]

The promoting substance described above or a composition containing the substance can be in a solid or liquid state at 1 atm and 20 °C, depending on an intended usage. Unless otherwise specified, descriptions related to the state in the present specification refer to the state at 1 atm and 20 °C.

A liquid may be a solution containing a solvent or a dispersion containing a dispersion medium.

[0056]

When the promoting substance is used in a form of a composition, the composition may further contain a carrier, when necessary. These carriers are preferably pharmaceutically acceptable carriers from a point of view of reducing occurrence of an unintended effect on an application target. As the carriers, those used in the present technical field can be used without particular limitation. As the carriers, for example, excipients, disintegrants, disintegration aids, binders, lubricants, coating agents, colorants, diluents, vehicles, solvents, solubilizing agents, isotonic agents, pH adjusters, stabilizers, propellants, adhesives, and the like can be used. These carriers can each be used independently, or two or more of the carriers can be used in combination.

[0057]

As vehicles or diluents, for example, various liquids can be used, such as water, electrolytecontaining water such as saline, monovalent alcohols with 1 to 3 carbon atoms such as methanol, ethanol, and propanol, polyvalent alcohols such as glycerin, or culture media for cell culture. These liquids can each be used independently or two or more of the liquids can be used in combination, as the solvent or dispersion medium described above that can form a composition.

[0058]

The above is a description about the promoting substance and the composition. In the following, another embodiment is described. In the following description, content that differs from the embodiment described above will be mainly described, and for content that is not specifically described, the matters described in the present specification will be appropriately applied. The matters described in the present specification can each be applied independently or two or more of the matters can be applied in combination.

[0059]

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

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

In one embodiment, the method includes suppressing occurrence or progression of neural cell damage by administering the active ingredient to a subject (for example, various living bodies such as humans). As a result, a TDP-43 related disease can be more effectively treated or prevented.

[0060]

In the present specification, the “disorder” of cells includes one or more of the following cases: a case where cell morphology is normal but a function inherent in normal cell is reduced or lost; a case where cell morphology changes to a form different from the normal cell morphology; and a case where cell death such as apoptosis or necrosis occurs due to the above cases.

Examples of decreased cellular functions, in the case of neurons, include one or more of the following: reduction or loss of action potential generation, weakening of synapses or synaptic plasticity, decreased or lost energy metabolism, suppression of neurogenesis, and the like.

Examples of changes in cellular morphology, in the case of neurons, include one or more of the following: retraction of dendrites and axons, elongation suppression, reduction in spine structures, demyelination, swelling or shrinkage of a cell body, and the like.

[0061]

The promoting substance may be administered to a subject (for example, various living bodies such as humans) as it is, or may be administered to a subject in the form of the composition described above. When administering two or more promoting substances in combination, the promoting substances can be administered all at once, or the substances can be administered sequentially in any order.

[0062]

A method of administration to a subject can be suitably selected according to characteristics of a substance to be used. Examples of the administration methods include various in vivo administrations such as oral administration and parenteral administration. For oral administration, for example, solid dosage forms such as tablets, capsules, powders, fine granules, and granules may be used, and liquid dosage forms such as solutions, syrups, and suspensions can be used.

Examples of non-oral administration include intravenous, intramuscular, intraperitoneal, intrathecal, subcutaneous, or intradermal injections of liquids; injection or inhalation of solids or liquids into the gastrointestinal tract; and the like. These administrations may be a single rapid administration or multiple rapid administrations, or may be a continuous administration such as infusion.

[0063]

A dose to be administered to a subject can be suitably selected according to characteristics of a substance to be used and a therapeutically effective amount. When the subject is an adult human with a body weight of 60 kg, a daily dose of each active ingredient can be independently determined, for example, from 0.1 ng to 1000 mg per active ingredient. Further, when an active ingredient is a viral vector, the dose can be, for example, 1 x 10⁵ vector genome (vg)/kg to 1 x IO²⁰ vg/kg per 1 kg of human subject body weight.

The number of doses of an active ingredient per day can be suitably selected according to characteristics of a substance to be used and a therapeutically effective amount, and, for example, it may be administered once, or twice or more. Further, an administration method during a treatment period can be suitably selected according to characteristics of a substance to be used and a therapeutically effective amount. Regarding the administration methods described above, for example, administration can be carried out continuously every day, or it can be carried out intermittently by setting non-administration periods of one or more arbitrary days, months, or years.

[0064]

In the following, still another embodiment is described. In the following description, content that differs from the embodiments described above will be mainly described, and for content that is not specifically described, the matters described in the present specification will be appropriately applied. The matters and embodiments described in the present specification can each be applied independently or two or more of the matters and embodiments can be applied in combination.

[0065]

In one embodiment, the present disclosure relates to a method for suppressing cell damage. Further, in one embodiment, the present disclosure relates to a method for facilitating maintenance or improvement of a cellular function.

In one embodiment, the method includes a step of bringing a promoting substance into contact with a neuron, which is one of application targets. This can suppress occurrence or progression of cell damage in a neuron and maintain voluntary movement of skeletal muscles. As a result, a disease can be effectively treated or prevented.

The neuron as an application target is preferably a human neuron, and more preferably a human motor neuron.

Examples of normal cell functions in the case of neurons include one or more of the following: generation of action potentials, energy metabolism, and neurogenesis.

Measurement of cell damage can be performed, for example, as described in examples below, by measuring a neurite length when a stress stimulus is applied to neurons, preferably neurons derived from an ALS patient. An example of a stress stimulus is a tunicamycin treatment.

As a degree of suppression of cell damage by a promoting substance, a degree of cell damage when a step of bringing the promoting substance into contact with various cells such as neurons is included is preferably 90% or less, more preferably 70% or less, even more preferably 50% or less, compared to when the step of bringing the promoting substance into contact with the cells is not included.

[0066]

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

[0067]

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

Another embodiment of the present disclosure relates to a method for reducing or suppressing protein abnormal localization.

Another embodiment of the present disclosure relates to a method for regulating phosphorylation of a protein. More specifically, one embodiment of the present disclosure relates to a method for reducing or suppressing phosphorylation of a protein.

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

Another embodiment of the present disclosure relates to a method for suppressing protein accumulation in cytoplasm.

In one embodiment, the methods described above each include a step of bringing a promoting substance into contact with a neuron, which is one of application targets.

Proteins as application targets of the methods described above are each independently a TDP-43 protein, a protein derived from TDP-43, or the like. Specifically, these proteins are each, for example, an abnormal TDP-43 protein described above, preferably a TDP-43 protein or a fragment thereof, and more preferably a non-wild-type TDP-43 protein. A preferred example of a fragment of the TDP-43 protein is a C-terminal fragment of the TDP-43 protein, which is known to be generated when the full-length TDP-43 protein is cleaved by caspase 3 or the like. Examples of an amino acid sequence of the C-terminal fragment include sequences shown in SEQ ID NO: 4, SEQ ID NO: 15, SEQ ID NO: 16, and SEQ ID NO: 17. Among these, the protein shown in SEQ ID NO: 15 is about 35 kDa (which may be referred to as CTF35), and the protein shown in SEQ ID NO: 16 or SEQ ID NO: 17 is about 25 kDa (which may be referred to as CTF25). The proteins may each be a full-length amino acid sequence or a peptide fragment having any residue. Abnormal localization of abnormal proteins and formation of aggregates can be suppressed by suppressing translocation of the TDP-43 protein out of a cell nucleus, suppressing accumulation of the TDP-43 protein in cytoplasm, suppressing abnormal post- translational modification of the TDP-43 protein, or promoting degradation of an abnormal TDP- 43 protein. Further, clearance of an abnormal protein can be normally maintained. As a result, maintenance or improvement of a cellular function can be facilitated, and consequently, a TDP- 43 related disease can be treated or prevented.

[0068]

Further, another embodiment of the present disclosure relates to a method for suppressing abnormal RNA splicing. Examples of targets to be suppressed in the present method include an STMN2 (Stathmin-2) gene described in the examples to be described later, or a transcription product (for example, an mRNA precursor) from that gene.

[0069]

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

An example of a method for measuring abnormal protein localization is a method of measuring a ratio between expression levels of the TDP-43 protein in the cytoplasm and in the nucleus, using cultured cells, cells derived from an ALS patient, or the like, as described in a screening method for TDP-43 protein nuclear export inhibitors to be described below. In this method, a stress stimulus such as tunicamycin may be applied to cultured cells, cells derived from an ALS patient, or the like.

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

An example of a method for measuring protein phosphorylation is a method of measuring a phosphorylation level of, for example, TDP-43 protein using cultured cells, cells derived from an ALS patient, or the like, using a conventional method, for example, immunofluorescence staining or Western blot or the like, as described in the examples to be described later. In this method, a stress stimulus such as tunicamycin may be applied to cultured cells, cells derived from an ALS patient, or the like.

As a degree of reduction or suppression in protein phosphorylation by a promoting substance, for example, when a step of bringing the promoting substance into contact with various types of cells such as neurons is included, the phosphorylation level of the TDP-43 protein is 90% or less, preferably 70% or less, and more preferably 50% or less, compared to that when the step of bringing the promoting substance into contact with the cells is not included.

An example of a method for measuring protein degradation is a method of measuring an expression level of a C-terminal fragment of the TDP-43 protein using cultured cells, cells derived from an ALS patient, or the like, using a conventional method, for example, using Western blot or the like. In this method, a stress stimulus such as tunicamycin may be applied to cultured cells, cells derived from an ALS patient, or the like.

As a degree of suppression in protein degradation by a promoting substance, for example, when a step of bringing the promoting substance into contact with various types of cells such as neurons is included, the expression level of the C-terminal fragment of the TDP-43 protein is 90% or less, preferably 70% or less, and more preferably 50% or less, compared to that when the step of bringing the promoting substance into contact with the cells is not included. An example of a method for measuring abnormal RNA splicing is a method of measuring a ratio (STMN2CE/STMN2FL) between expression levels of STMN2CE and STMN2FL for the STMN2 genes using cultured cells, cells derived from an ALS patient, or the like, using a conventional method, for example, using absolute quantitative qPCR (see Examples). In this method, a stress stimulus such as tunicamycin may be applied to cultured cells, cells derived from an ALS patient, or the like.

As a degree of suppression in abnormal RNA splicing by a promoting substance, for example, when a step of bringing the promoting substance into contact with various types of cells such as neurons is included, the ratio between the expression levels of STMN2CE and STMN2FL is 90% or less, preferably 70% or less, and more preferably 50% or less, compared to that when the step of bringing the promoting substance into contact with the cells is not included.

[0070]

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

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

[0071]

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

[0072]

The screening method for a TDP-43 protein phosphorylation inhibitor may include: a step of bringing a test substance into contact with a TDP-43 protein; a step of measuring a phosphorylation level of the TDP-43 protein brought into contact with the test substance; a step of comparing the measured phosphorylation level with a phosphorylation level measured for a TDP-43 protein not brought into contact with the test substance; and a step of selecting a test substance that reduces the phosphorylation level of the TDP-43 protein.

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

The phosphorylation level of a TDP-43 protein can be measured using a conventional method, such as immunofluorescence staining (see Example 1) or Western blot, or the like.

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

[0073]

A screening method for a TDP-43 protein nuclear export inhibitor may include: a step of bringing a test substance into contact with cells expressing a TDP-43 protein; a step of measuring expression levels of the TDP-43 protein in nucleus and/or cytoplasm of the cells exposed to the test substance; a step of comparing the measured expression levels of the TDP-43 protein in the nucleus and in the cytoplasm, and/or a ratio between the expression levels of the TDP-43 protein in the cytoplasm and in the nucleus (calculated by dividing the expression level in the cytoplasm by the expression level in the nucleus), with expression levels of the TDP-43 protein in nucleus and in cytoplasm, and/or a ratio between the expression levels of the TDP-43 protein in the cytoplasm and in the nucleus in cells not exposed to the test substance; and a step of selecting a test substance that increases the expression level of the TDP-43 protein in the nucleus, or reduces the expression level of the TDP-43 protein in the cytoplasm and/or the ratio between the expression levels of the TDP-43 protein in the cytoplasm and in the nucleus.

A method for bringing a test substance into contact with cells expressing a TDP-43 protein is not particularly limited, and an example thereof is a method in which a test substance is administered to various types of cells such as neurons to expose the cells, and which is applicable in various environments such as in vitro, in vivo, and ex vivo. Examples of the cells include motor neurons differentiated from iPS cells derived from a patient with a TDP-43 related disease, preferably motor neurons differentiated from iPS cells derived from an ALS patient, more preferably motor neurons differentiated from iPS cells derived from an ALS patient with a TDP-43 gene mutation. Further, these cells may also include a step of administering a stress inducer such as tunicamycin.

The expression levels of a TDP-43 protein in the nucleus and/or in cytoplasm can be measured using a conventional method, for example, by immunofluorescence staining (see Example 1) or by Western blot of nuclear and/or cytoplasmic proteins extracted from cells. By comparing the measured expression levels of the TDP-43 protein in the nucleus and in the cytoplasm, and/or the ratio between the expression levels in the cytoplasm and in the nucleus, with the expression levels of the TDP-43 protein in the nucleus and in the cytoplasm, and/or the ratio between the expression levels in the cytoplasm and in the nucleus not exposed to the test substance, it can be determined whether or not the test substance is a nuclear export inhibitor. By comparing with the expression levels of the TDP-43 protein in the nucleus and in the cytoplasm, and/or the ratio between the expression levels of the TDP-43 protein in the cytoplasm and in the nucleus when not exposed to the test substance, when the expression level of the TDP- 43 protein in the nucleus increases, and the expression level in the cytoplasm and/or the ratio between the expression levels of the TDP-43 protein in the cytoplasm and in the nucleus decrease when exposed to the test substance, it can be determined that the test substance is a TDP-43 protein nuclear export inhibitor, and the test substance may be selected. For example, when the ratio between the expression levels of the TDP-43 protein in the cytoplasm and in the nucleus when exposure to the test substance is 80% or less, preferably 50% or less, more preferably 25% or less, compared to when not exposed to the test substance, the test substance can be determined to be a TDP-43 protein nuclear export inhibitor.

[0074]

The TDP-43 protein phosphorylation inhibitor and the TDP-43 protein nuclear export inhibitor obtained according the screening methods of one embodiment of the present disclosure are useful as active ingredients for treating or preventing a TDP-43 related disease. In particular, these inhibitors are useful as active ingredients for preventing ALS or for treating or preventing a TDP-43 related disease.

[0075]

Another embodiment of the present disclosure is a composition comprising a substance (which may be referred to as a binding promoting substance) that enhances binding between an LRS AMI protein and a TDP-43 protein for treating or preventing a TDP-43 related disease.

A binding promoting substance is a substance that enhances binding or interaction between an LRSAM1 protein and a TDP-43 protein or between their fragments, and examples thereof include a PROTAC (Proteolysis Targeting Chimera) compound or an MGD (Molecular Glue Degrader) compound. A preferred example of an LRS AMI protein is of a wild-type. TDP-43 proteins are each independently a wild-type TDP-43 protein, a non-wild-type TDP-43 protein, a protein derived from TDP-43, or the like. Specifically, these proteins are each, for example, an abnormal TDP-43 protein described above, preferably a TDP-43 protein or a fragment thereof, and more preferably a non-wild-type TDP-43 protein. A preferred example of a fragment of the TDP-43 protein is a C-terminal fragment of the TDP-43 protein.

[0076]

A PROTAC compound is a compound that contains a TDP-43 protein target ligand, an LRSAM1 (E3 ubiquitin ligase) target ligand, and a linker.

A PROTAC compound can be prepared as follows. A TDP-43 protein target ligand and an LRSAM1 (E3 ubiquitin ligase) target ligand can be obtained by screening existing ligands, simulation based on X-ray crystallography, screening using compound libraries, biochemical evaluation (such as surface plasmon resonance (SPR) method, or the like), and/or the use of an artificial intelligence program such as Alphafold2 that performs protein structure prediction. The obtained ligands can be linked with a linker using a method commonly known in the art to prepare a compound that can be used as a PROTAC candidate compound. Whether a PROTAC candidate compound binds to a TDP-43 protein and an LRSAM1 protein and enhances interaction between them can be evaluated by binding a probe to each of the TDP-43 protein and LRSAM1 protein and competitively measuring the interaction. Specifically, for example, the evaluation can be performed using methods such as fluorescence polarization (FP), time-resolved fluorescence resonance energy transfer (TR-FRET), the AlphaScreen/ AlphaLISA technology, and the like. In this case, when a strong interaction is observed in the presence of a PROTAC candidate compound compared to that in the absence of the PROTAC candidate compound, the compound can be determined to be a PROTAC compound. Further, whether or not a PROTAC candidate compound has the ability to degrade a TDP-43 protein can be evaluated using cells. An amount of a TDP-43 protein can be quantified by introducing a plasmid in which the TDP-43 protein or a fragment of a TDP-43 protein (a C-terminal fragment of a TDP-43 protein) is fluorescently labeled into nerve cells, such as Neuro2a cells, using a method commonly known in the art and measuring a fluorescence intensity. A PROTAC candidate compound is added to the cells, and when the fluorescence intensity decreases, it can be determined that the compound has the ability to degrade the TDP-43 protein and is therefore 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.

[0077]

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

The interaction between a TDP-43 protein, or a fragment thereof (a C-terminal fragment of the TDP-43 protein) and an LRS AMI protein can be explored to see whether or not there is a pocket into which the compound can fit, using three-dimensional structure analysis using X-ray crystallography or nuclear magnetic resonance (NMR) spectroscopy and/or an artificial intelligence program such as Alphafold2 that performs protein structure prediction. This allows visualization and/or prediction of a binding interface or pocket between the TDP-43 protein or a fragment thereof and the LRS AMI (E3 ubiquitin ligase) protein. An MGD candidate compound that can fit into the pocket can be generated by virtual screening from a compound library via structure-based docking or simulation, and/or by compound designing based on structural information of the pocket. Whether or not an obtained MGD candidate compound binds to and enhances the interaction between the TDP-43 protein or a fragment thereof and the LRSAM1 protein when they interact can be evaluated by quantitatively measuring the protein-protein interaction using a method such as time-resolved fluorescence resonance energy transfer (TR- LRET). In this case, when a stronger interaction is observed in the presence of an MGD candidate compound compared to that when the MGD candidate compound is absent, it can be determined that the compound is an MGD compound. Lurther, whether or not an MGD candidate compound has the ability to degrade a target protein can be evaluated using the same evaluation method as the PROTAC candidate compound described above, and when a compound is determined to have the ability to degrade the TDP-43 protein, it can be determined to be an MGD compound. In this way, an MGD compound that enhances the interaction between a target protein and LRSAM1 can be obtained.

[0078] As one embodiment of the present disclosure, a binding promoting substance is useful as an active ingredient for treating or preventing a TDP-43 -related disease by promoting the degradation of the TDP-43 protein or a fragment of the TDP-43 protein (such as a C-terminal fragment of the TDP-43 protein) through the ubiquitin-proteasome system of the LRSAM1 protein. In particular, the binding promoting substance is useful as an active ingredient for preventing ALS or for treating or preventing a TDP-43 related disease.

Regarding the matters described above, for content that is not particularly described, the matters described in the present specification shall be appropriately applied.

[0079]

Regarding the embodiments described above, the present specification further discloses the following embodiments.

[0080]

<1> A composition comprising a promoting substance.

<2> The composition according to <1>, wherein the promoting substance is a substance having at least one, preferably two, more preferably three of the following (i) to (iii):

(i) increasing an intracellular amount of a protein having ubiquitination activity;

(ii) increasing intracellular ubiquitination activity; and

(iii) promoting generation of a transcription product or a translation product in the cells.

<3> The composition according to <1> or <2> above being used for treating or preventing a TAR DNA-binding protein 43 (TDP-43) related disease.

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

<5> The composition of <4> above, wherein the neurodegenerative disease is selected from a group consisting of Parkinson's disease, amyotrophic lateral sclerosis (ALS), spinal muscular atrophy (SMA), primary lateral sclerosis (PLS), multiple system atrophy (MSA), progressive supranuclear palsy (PSP), Perry syndrome, Alexander disease, Alzheimer's disease, frontotemporal lobar degeneration (FTLD), and limbic-predominant age-related TDP-43 encephalopathy (LATE), and the muscle disease is inclusion body myositis. <6> The composition according to <5> above, wherein the neurodegenerative disease is one or more selected from a group consisting of amyotrophic lateral sclerosis (ALS), frontotemporal lobar degeneration (FTLD), and limbic-predominant age-related TDP-43 encephalopathy (LATE), preferably amyotrophic lateral sclerosis (ALS).

[0081]

<7> The composition according to any one of <1> to <6> above, being used for improving clearance of TDP-43 protein.

<8> The composition according to any one of <1> to <7> above, being used for suppressing cell damage.

<9> The composition according to any one of <1> to <8> above, being used for suppressing cell damage caused by TDP-43 protein.

<10> The composition according to any one of <1> to <9> above, being used for reducing or suppressing abnormal localization of a protein.

<11> The composition according to any one of <1> to <10> above, being used for promoting degradation of a protein.

<12> The composition according to any one of <1> to <11> above, being used for regulating phosphorylation of a protein.

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

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

<15> The composition according to any one of <1> to <14> above, being used for suppressing abnormal splicing of RNA (preferably mRNA).

[0082]

<16> The composition according to any one of <1> to <15> above, being applied to a subject or cell having a mutation in a TARDBP gene. <17> The composition according to <16> above, being applied to a subject or cell having a heterozygous mutation in the TARDBP gene.

<18> The composition according to <16> or <17> above, wherein the subject or cell is a human subject or human- derived cell.

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

[0083]

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

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

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

<23 > The composition according to <22> above, wherein the antisense is one or more selected from the following (a) to (e):

(a) a nucleic acid that controls RNA processing;

(b) a nucleic acid that is partially or fully complementary to a full length or a partial region of miRNA;

(c) a nucleic acid that is partially or fully complementary to a full length or a partial region of Natural Antisense Transcript (NAT);

(d) a nucleic acid that is partially or fully complementary to all or a part of one or more regions selected from a group consisting of a translation inhibitory element (TIE) of mRNA, an upstream ORF (uORF), and a premature termination codon (PTC); and

(e) a nucleic acid that is partially or fully complementary to the full-length or a partial region of a regulatory RNA that contributes to gene expression suppression. <24> The composition according to any one of <21 > to <23> above, wherein the expressionenhancing nucleic acid has 5 to 300 bases per strand, and preferably 10 to 100 bases per strand.

[0084]

<25> The composition according to <20> above, wherein the promoting substance includes an expression vector.

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

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

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

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

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

<31> The composition according to any one of <1> to <30> above, wherein the promoting substance is a substance that promotes generation of a translation product, the translation product is a protein having ubiquitination activity, and by promoting the generation of the protein, an intracellular amount of the protein is increased, and/or intracellular ubiquitination activity is increased.

[0085]

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

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

<34> The composition according to any one of <1> to <32> above, wherein the promoting substance is a substance that promotes the following (i) and/or (ii):

(i) generation of a transcription product capable of expressing a wild-type LRSAM1 protein or a protein having an equivalent function to the wild-type LRSAM1 protein; and

(ii) generation of a translation product that is a wild-type LRS AMI protein or a protein having an equivalent function to the wild-type LRS AMI protein.

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

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

[0086]

<37> The composition according to any one of <1> to <36> above, being a pharmaceutical composition.

<38> The composition according to any one of <1> to <37> above, being used for a subject who has developed or is likely to develop a TDP-43 related disease.

<39> The composition according to any one of <1> to <38> above, being used in any of the following environments: in vivo including or excluding humans; in vitro including humans; or ex vivo including humans.

[0087]

<A1> A method for treating or preventing a TDP-43 related disease, comprising a 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 a step of administering an effective amount of a promoting substance to a subject in need thereof.

<A3> A method for suppressing cell damage, comprising a step of administering an effective amount of a promoting substance to a subject in need thereof.

[0088]

<A4> A method for facilitating maintenance or improvement of a cellular function, comprising a 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 a step of administering an effective amount of a promoting substance to a subject in need thereof.

<A6> A method for promoting degradation of a protein, comprising a step of administering an effective amount of a promoting substance to a subject in need thereof.

<A7> A method for regulating phosphorylation of a protein, comprising a step of administering an effective amount of a promoting substance to a subject in need thereof.

<A8> A method for suppressing abnormal splicing of RNA (preferably mRNA), comprising a step of administering an effective amount of a promoting substance to a subject in need thereof.

[0089]

<A9> The method according to <A1>, wherein the disease is treated or prevented by suppressing occurrence or progression of neural cell damage by administering the promoting substance to the subject.

<A10> The method according to any one of <A5> to <A7> above, wherein the protein is a full- length TDP-43 protein or a fragment thereof.

<A11> The method according to <A10> above, wherein the protein is a C-terminal fragment of TDP-43 protein.

<A12> The method according to any one of <A1> to <A11> above, being used for a subject who has developed or is likely to develop a TDP-43 related disease.

<A13> The method according to any one of <A1> to <A12> above, being used for a subject who has developed or is likely to develop one or more diseases selected from a group consisting of amyotrophic lateral sclerosis (ALS), frontotemporal lobar degeneration (FTLD), and limbic- predominant age-related TDP-43 encephalopathy (LATE) as TDP-43 related diseases.

<A14> The method according to any one of <A1> to <A13> above, wherein the subject is a human subject.

<A15> The method according to any one of <A1> to <A14> above, wherein the promoting substance is a substance having at least one, preferably two, more preferably three of the following (i) to (iii):

(i) increasing an intracellular amount of a protein having ubiquitination activity;

(ii) increasing intracellular ubiquitination activity; and

(iii) promoting generation of a transcription product or a translation product in the cells.

<A16> The method according to any one of <A1> to <A15> above, wherein the promoting substance is administered in the form of the composition according to any one of <1> to <39> above.

[0090]

<B1> Use of a promoting substance that increases an intracellular amount of a protein having ubiquitination activity in manufacture of a composition for treating or preventing a TDP-43 related disease.

<B2> Use of a promoting substance that increases an intracellular amount of a protein having ubiquitination activity in manufacture of a composition for increasing intracellular ubiquitination activity.

<B3> The use according to <B1> or <B2> above, wherein the protein is an LRSAM1 protein.

<B4> The use according to any one of <B1> to <B3> above, wherein the promoting substance is a substance containing a nucleic acid, and the promoting substance preferably contains one or more selected from an expression-enhancing nucleic acid and an expression vector.

<B5> The use according to any one of <B1> to <B4> above, wherein the cell is a neural cell, preferably a neuron, more preferably a human neuron, even more preferably a human motor neuron. <B6> The use according to any one of <B1> to <B5> above, wherein the composition is the composition according to any one of <1> to <39> above.

[0091]

<C1> A method for suppressing cell damage, comprising suppressing occurrence or progression of cell damage in a neuron by bringing a promoting substance into contact with the neuron.

<C2> A method for improving clearance of a protein, comprising improving clearance of a TDP- 43 protein in a neuron by bringing a promoting substance into contact with the neuron.

<C3> A method for facilitating maintenance or improvement of a function of a neuron by bringing a promoting substance into contact with the neuron.

<C4> A method for reducing or suppressing abnormal localization of a protein in a neuron by bringing a promoting substance into contact with the neuron.

<C5> A method of promoting degradation of a protein in a neuron by bringing a promoting substance into contact with the neuron.

<C6> A method of regulating phosphorylation of a protein in a neuron by bringing a promoting substance into contact with the neuron.

<C7> A method for suppressing abnormal splicing of RNA (preferably mRNA) in a neuron by bringing a promoting substance into contact with the neuron.

[0092]

<C8> The method according to any one of <C1> to <C7> above, wherein the neuron is a motor neuron, preferably a human motor neuron.

<C9> The method according to any one of <C1> to <C8> above, wherein the promoting substance is a substance having at least one, preferably two, and more preferably three of the following (i) to (iii):

(i) increasing an intracellular amount of a protein having ubiquitination activity;

(ii) increasing intracellular ubiquitination activity; and

(iii) promoting generation of a transcription product or a translation product in the cells. <C10> The method according to any one of <C1> to <C9> above, wherein the promoting substance is a substance containing a nucleic acid, and the promoting substance preferably contains one or more selected from a group consisting of an expression-enhancing nucleic acid and an expression vector.

<C11> The method according to any one of <C1> to <C10> above, wherein the promoting substance is used in the form of the composition according to any one of <1> to <39> above.

Examples

[0093]

In the following, the present disclosure is further described in detail based on examples. However, the scope of the present disclosure is not limited to such examples. Unless otherwise specified, “%v/v” means “volume/volume%.” Further, in the figures, groups indicated with “**” show statistical significance at P < 0.01. In the figures, groups indicated with show statistical significance at P < 0.05.

[0094]

Example 1

Taking ALS as an example of a TDP-43 related disease, a pathological model for evaluating cell damage was constructed using the following method.

First, human induced pluripotent stem (iPS) cells were induced from cells derived from an ALS patient and cells derived from a healthy individual, respectively, using a conventional method. The cells derived from the ALS patient were used that are known to have an ALS risk mutation as a heterozygous mutation in the TARDBP gene. Next, each iPS cell was differentiated into a motor neuron (hereinafter, a motor neuron is also simply referred to as a “neuron”) using a conventional method.

For long-term preservation of the cells, when necessary, the neurons were subjected to a freezing treatment 2 to 7 days after the differentiation treatment. Neurons that have been subjected to the freezing treatment are also referred to as “frozen neurons.”

[0095] Subsequently, neurons were cultured using a 48-well culture plate (Corning, 3548) under the conditions shown below.

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

[0096]

The culture solution 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 ? (Elixirgen): 0.05% v/v

[0097]

A treatment was performed in which the culture solution dissolved with a Dimethyl sulfoxide (DMSO) solution (final concentration: Tunicamycin 0.1 pg/mL, DMSO 0.01% v/v) as a stress inducer was brought into contact with the neurons after 7 days of culture, and occurrence of cell damage was evaluated over time. Separately, a group containing no stress inducer (a group to which the same concentration of DMSO was added) was also prepared. To evaluate cell damage, live cell imaging was performed according to the attached protocol using an IncuCyte S3 (Sartorius), and neurite length per unit area (unit: mm/mm²) was analyzed over time, which was used as an evaluation indicator. A smaller value of the neurite length indicates that cell damage has occurred.

[0098]

Cell damage evaluation (1)

The results for the neurite length in each experimental group, calculated as a ratio based on the results at the time of stress inducer treatment initiation (0 hr), are shown in Fig. 1. The smaller the value on the vertical axis, the more the neurite length decreases over time, indicating that cell damage has occurred. The experimental groups in Fig. 1 are as follows.

• Healthy-DMSO: Group using neurons derived from a healthy individual, containing no stress inducer

• Healthy-tunicamycin: Group using neurons derived from a healthy individual, containing a stress inducer

• ALS-DMSO: Group using neurons derived from an ALS patient, containing no stress inducer

• ALS-tunicamycin: Group using neurons derived from an ALS patient, containing a stress inducer

[0099]

The neurons derived from an ALS patient showed a significant decrease in neurite length compared to neurons derived from a healthy individual. Further, when neurons derived from a healthy individual and neurons derived from an ALS patient were treated with tunicamycin, the neurite length in neurons derived from an ALS patient became significantly shorter than that in untreated neurons, indicating the occurrence of cell damage. In contrast, no significant decrease in neurite length was observed in neurons derived from a healthy individual (Fig. 1).

[0100]

TDP-43 localization evaluation (1) Intracellular localization of TDP-43 protein was evaluated by immunofluorescent staining of neurons. The cultured neurons were washed with phosphate buffered saline (PBS) and fixed with 4% paraformaldehyde (PF A). After blocking with PBS containing 5% Fetal Bovine Serum (FBS) and 0.1% Triton-X, a primary antibody dilution containing anti-TDP-43 antibody and anti-P-III tubulin antibody was added, and incubated overnight at 4 °C. The anti-P-III tubulin antibody was used to visualize cell bodies of neurons. The next day, after washing with PBS, a secondary antibody dilution conjugated with Alexa dye was added and incubated for 1 hour at room temperature. After further nuclear staining, washing with PBS, and imaging under a fluorescence microscope were performed. The acquired images were analyzed using Matlab (Mathworks) to calculate fluorescence intensities of TDP-43 in a nuclear region and a cytoplasmic region, respectively.

[0101]

The results of intracellular localization of TDP-43 protein are shown in Fig. 2 The experimental groups in Fig. 2 are as follows.

• Healthy-DMSO: Group using neurons derived from a healthy individual, containing no stress inducer

• Healthy-tunicamycin: Group using neurons derived from a healthy individual, containing a stress inducer

• ALS-DMSO: Group using neurons derived from an ALS patient, containing no stress inducer

• ALS-tunicamycin: Group using neurons derived from an ALS patient, containing a stress inducer

[0102]

The intracellular localization of TDP-43 protein shown in Fig. 2 is expressed as a ratio of an intensity based on TDP-43 in cytoplasm to a TDP-43 fluorescence intensity in nucleus (“cytoplasm/nucleus ratio” in Fig. 2). The higher the value of this ratio, the higher the proportion of TDP-43 protein localized in the cytoplasm, meaning that abnormal localization has occurred. A two-way analysis of variance showed that the fact that the neurons are derived from an ALS patient and the treatment with a stress inducer interact to significantly promote the abnormal localization of TDP-43 protein in the cytoplasm. This abnormal localization was a phenomenon similar to the phenomenon observed in brain tissues of ALS patients.

[0103]

Evaluation of the degree of phosphorylation of TDP-43 (1)

The degree of phosphorylation of the TDP-43 protein was evaluated by immunofluorescent staining on neurons. In addition to the primary antibody used in the immunofluorescent staining described above, in the procedure of the immunofluorescent staining described above, an antiphosphorylated TDP-43 (S409) antibody was used instead of the anti-TDP-43 antibody. Other than that, the fluorescence intensity was calculated in the same way as in the procedure described above.

The phosphorylation of the TDP-43 protein is thought to indicate the following intracellular state. Specifically, it has been reported that TDP-43 insolubilized in the cytoplasm is phosphorylated. The insolubilization of TDP-43 in the cytoplasm can cause inhibition of protein degradation systems, mitochondrial toxicity, and the like. In addition, insolubilized TDP-43 can inhibit normal nuclear translocation of the TDP-43 protein and promote TDP-43 dysfunction in the nucleus. That is, a high degree of phosphorylated TDP-43 protein means a state where a normal function of the TDP-43 protein is inhibited and cell damage is likely to occur.

[0104]

Fig. 3 shows the abundance (arithmetic mean value of the fluorescence intensity) of the phosphorylated TDP-43 protein (p TDP-43) in the cytoplasm. The experimental groups in Fig. 3 are the same as those shown in Fig. 2.

As shown in Fig. 3, neurons derived from an ALS patient had a significantly higher expression of phosphorylated TDP-43 in the cytoplasm compared to neurons derived from a healthy individual. Further, in the presence of a stress inducer, phosphorylation of TDP-43 was significantly increased in neurons derived from an ALS patient. From this, it was inferred that a normal function of TDP-43 was inhibited. Based on the above, the present model is considered an appropriate experimental model capable of reflecting a pathological condition of an ALS patient.

[0105]

Example 2

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

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

[0106]

In the above experimental model, on the third day of culture, the lentiviral vectors described above were added to culture plates of neurons at a predetermined multiplicity of infection (MOI) (for example, MOI 0.01 or 0.03), and the culture was continued. Then, on the seventh day of culture, a stress inducer (or DMSO alone) was added to achieve the final concentration described above, and the culture was further continued for up to 96 hours in the presence of the stress inducer.

[0107]

Cell damage evaluation (2)

Results of evaluating occurrence of cell damage in neurons by cell morphological analysis, using the same method as in Example 1, are shown in Figs. 4 and 5. Fig. 4 shows the results using neurons derived from a healthy individual, and Fig. 5 shows the results using neurons derived from an ALS patient. As shown in these figures, in neurons derived from an ALS patient, occurrence or progression of cell damage was significantly suppressed in the group exposed to the promoting substance (see Fig. 5; the MOI 0.03 group). Further, in neurons derived from a healthy individual, no significant difference was observed depending on the expression of LRSAM1 protein (see Fig. 4). In the experimental groups shown in Fig. 5, it was confirmed by the inventors that the expression level of LRSAM1 in the neurons had increased.

[0108]

TDP-43 localization evaluation (2)

The results of calculating the cytoplasmic/nuclear ratio of TDP-43 in the presence or absence of a promoting substance, using the same method as in Example 1, are shown in Figs. 6A and 6B. The experimental groups shown in Figs. 6A and 6B are as follows. In Figs. 6A and 6B, the value of the Null-DMSO group in each neuron was set to 100%, and the result for each group was expressed as a percentage of that value.

• Null-DMSO: Group containing no stress inducer and no promoting substance

• Null-tunicamycin: Group containing a stress inducer but no promoting substance

• LRSAM1-DMSO: Group containing no stress inducer but a promoting substance

• LRSAMl-Tunicamycin: Group containing both a stress inducer and a promoting substance [0109]

As shown in Figs. 6A and 6B, by promoting generation of the LRSAM1 protein as a translation product and thereby increasing the intracellular ubiquitination activity, the cytoplasmic/nuclear ratio decreased in both neurons derived from a healthy individual and neurons derived from an ALS patient. And, the degree of decrease in the cytoplasmic/nuclear ratio was more pronounced in neurons derived from an ALS patient compared to neurons derived from a healthy individual. Therefore, by using the promoting substance, abnormal localization and clearance of TDP-43 can be improved, allowing an improvement to be made such that cells such as motor neurons can exert their inherent functions.

[0110] Evaluation of the degree of phosphorylation of TDP-43 (2)

Regarding the abundance of cytoplasmic pTDP-43, the results for neurons derived from a healthy individual in the presence or absence of a promoting substance are shown in Fig. 7A, and the results for neurons derived from an ALS patient are shown in Fig. 7B. The experimental groups in Figs. 7A and 7B are the same as the experimental groups shown in Figs. 6A and 6B. In Figs. 7A and 7B, the value of the Null-DMSO group in each neuron was set to 100%, and the result for each group was expressed as a percentage of that value.

As shown in these figures, the expression of the phosphorylated TDP-43, which was increased by the tunicamycin treatment, was suppressed by the promoting substance. As described above, the phosphorylation of TDP-43 can lead to inhibition of proteolytic systems and mitochondrial toxicity, and can also lead to TDP-43 dysfunction in the nucleus. Therefore, the regulation of phosphorylation by the promoting substance to suppress the expression of phosphorylated TDP- 43 leads to the suppression of the abnormalities described above. As a result, the promoting substance can make an improvement such that cells such as motor neurons can exert their inherent functions.

[01 H]

Evaluation of splicing regulation improvement by promoting substance

Focusing on STMN2, which is one of the genes of which splicing is regulated by TDP-43, a generation amount of a transcription product of STMN2 was evaluated using the following method. Specifically, RNA extraction from cultured neurons was performed using a Quick-RNA Micro Kit (Zymo Research). After measuring a concentration of RNA, a certain amount of RNA was reverse transcribed using SuperScript IV VILO Master Mix (ThermoFisher Scientific) to synthesize cDNA. Absolute quantitative qPCR was performed by generating a calibration curve using a nucleic acid that is identical to a sequence amplified by qPCR and with a known copy number. By the absolute quantitative qPCR, a transcription product generation amount of a full- length wild-type mRNA of STMN2 (hereafter referred to as STMN2FL, including in the figures) and a transcription product generation amount of mRNA containing a cryptic exon of STMN2 (hereafter referred to as STMN2CE, including in the figures) were measured. A cryptic exon refers to a sequence that is originally located in an intron region but is unintentionally included as an exon, which occurs due to abnormal splicing. A high ratio of the STMN2CE generation amount to the STMN2FL generation amount (STMN2CE / STMN2FL) means that the generation amount of the non-wild-type transcription product is relatively high, and abnormal splicing is more likely to occur. That is, a high STMN2CE/STMN2FL ratio means that the normal function of TDP-43 is weakened or lost.

[0112]

The results are shown in Fig. 8. The experimental groups shown in Fig. 8 represent the following contents.

• Healthy-Null-DMSO: Group using neurons derived from a healthy individual, containing no stress inducer and no promoting substance.

• Healthy-Null-Tunicamycin: Group using neurons derived from a healthy individual, containing a stress inducer but no promoting substance.

• Healthy-LRSAMl-DMSO: Group using neurons derived from a healthy individual, containing no stress inducer but a promoting substance.

• Healthy-LRSAMl-Tunicamycin: Group using neurons derived from a healthy individual, containing a stress inducer and a promoting substance.

• ALS-Null-DMSO: Group using neurons derived from an ALS patient, containing no stress inducer and no promoting substance.

• ALS-Null-Tunicamycin: Group using neurons derived from an ALS patient, containing a stress inducer but no promoting substance.

• ALS-LRSAM1-DMSO: Group using neurons derived from an ALS patient, containing no stress inducer but a promoting substance.

• ALS-LRSAMl-Tunicamycin: Group using neurons derived from an ALS patient, containing a stress inducer and a promoting substance.

[0113] As shown in Fig. 8, neurons derived from an ALS patient had a significantly higher STMN2CE/STMN2FL ratio compared to neurons derived from a healthy individual (comparison between Healthy-Null-DMSO and ALS-Null-DMSO in Fig. 8). This indicates that in a TDP-43 related disease such as ALS, abnormal splicing is more likely to occur, and the normal function of TDP-43 is weakened or lost.

In neurons derived from an ALS patient, the tunicamycin-treated groups had significantly higher STMN2CE/STMN2FL ratios (comparison between ALS-Null-DMSO and ALS-Null- Tunicamycin in Fig. 10). This shows that the tunicamycin treatment exacerbates abnormal splicing. Further, in the tunicamycin-treated groups, expressing LRSAM1 with the promoting substance significantly lowered the STMN2CE/STMN2FL ratio (comparison between ALS- Null-Tunicamycin and ALS-LRS AMI -Tunicamycin in Fig. 10). On the other hand, in neurons derived from a healthy individual, no significant changes in the STMN2CE/STMN2FL ratio were observed among the groups (Fig. 9).

From the above, it was found that the normal function of TDP-43 was restored by the promoting substance in neurons derived from an ALS patient.

[0114]

Example 3

The effect of a promoting substance on the degradation of C-terminal fragments of the TDP-43 protein was evaluated. C-terminal fragments of the TDP-43 protein of about 35 kDa and about 25 kDa are known. Hereinafter, they will be referred to as CTF35 and CTF25, respectively.

Similar to the experimental model used in Example 2, on the third day of culture of neurons, lentiviral vectors were added to culture plates to achieve a predetermined MOI (for example, MOI 0.03), and the culture was continued. On the seventh day of culture, a stress inducer (or DMSO alone) was added to achieve a predetermined final concentration (for example, 0.1 pg/mL), and the culture was continued for up to 96 hours in the presence of a stress inducer. As the stress inducer, tunicamycin was used.

The cultured cells were lysed on ice using an 8M urea, 50mM Tris-HCl solution containing lx Proteinase/Phosphatase inhibitor to obtain a cell lysate. A protein concentration in the cell lysate was measured, and after denaturation, quantification of the C-terminal fragment of the TDP-43 protein was performed using a fully automated capillary electrophoresis immunoassay system Wes (SimpleProtein). In the automated capillary electrophoresis immunoassay, similar to the western blot method, an amount of a target protein can be quantified by analyzing a detected band.

[0115]

The evaluation results for the C-terminal fragment proteins CTF35 and CTF25 of the TDP-43 protein are shown in Figs. 11 and 12. The experimental groups shown in Figs. 11 and 12 represent the following contents.

• Healthy-Null-DMSO: Group using neurons derived from a healthy individual, containing no stress inducer and no promoting substance.

• Healthy-Null-Tunicamycin: Group using neurons derived from a healthy individual, containing a stress inducer but no promoting substance.

• Healthy-LRSAMl-DMSO: Group using neurons derived from a healthy individual, containing no stress inducer but a promoting substance.

• Healthy-LRSAMl-Tunicamycin: Group using neurons derived from a healthy individual, containing a stress inducer and a promoting substance.

• ALS-Null-DMSO: Group using neurons derived from an ALS patient, containing no stress inducer and no promoting substance.

• ALS-Null-Tunicamycin: Group using neurons derived from an ALS patient, containing a stress inducer but no promoting substance.

• ALS-LRSAM1-DMSO: Group using neurons derived from an ALS patient, containing no stress inducer but a promoting substance.

• ALS-LRSAMl-Tunicamycin: Group using neurons derived from an ALS patient, containing a stress inducer and a promoting substance.

[0116] The Compass for SW software included with the Wes was used to quantify band intensities of full-length TDP-43, CTF35, and CTF25. After that, the quantitative values of full-length TDP- 43, CTF35, and CTF25 proteins were divided by a sum of the TDP-43 quantitative values, and the results are shown in Figs. 11 and 12. As a result, in neurons derived from an ALS patient, the amounts of CTF35 and CTF25 were significantly higher compared to neurons derived from a healthy individual (comparison between Healthy-Null-DMSO and ALS-Null-DMSO in Figs. 11 and 12). This indicates that the protein amount of the C-terminal fragment of theTDP-43 protein is increased in a TDP-43 related disease such as ALS. Further, it indicates that in neurons derived from an ALS patient, treating with a stress inducer significantly increased the protein amounts of the C-terminal fragments of the TDP-43 protein (comparison between ALS-Null- DMSO and ALS-Null-Tunicamycin in Figs. 11 and 12). In neurons derived from an ALS patient treated with a stress inducer, expressing LRSAM1 with a promoting substance resulted in lower values for CTF35 and CTF25 compared to the ALS-Null-Tunicamycin group. From the above, it was shown that the promoting substance has induced the degradation of the C-terminal fragment proteins CTF35 and CTF25 of the TDP-43 protein.

[0117]

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

From paraffin sections of a brain motor cortex tissue, deparaffinization and dehydration were performed using conventional methods. After that, cell membranes were permeabilized with PBS containing 0.5% Triton-X, and blocking was performed using Blocking One (Nacalai). Next, the above-described anti-TDP-43 antibody, and LRSAM1 antibody (Sigma), as well as the anti-MAP2 antibody (Abeam), were added as primary antibodies after being diluted with Signal Enhancer A (Nacalai), and incubated overnight at 4 °C. The anti-MAP2 antibody was used to visualize neurons. The next day, after washing with PBS, a secondary antibody dilution conjugated with Alexa dye and a DAPI solution for nuclear staining (Invitrogen) were added and incubated at room temperature for 1 hour. After washing with PBS, the samples were sealed with a cover glass using a mounting solution. The samples were imaged using a fluorescence microscope, and the captured images were analyzed using Matlab (Mathworks). As an analysis method, after enclosing nucleus and cytoplasm in an image, signals of TDP-43 and LRSAM1 in the nucleus or cytoplasm per single cell were calculated, and a degree of colocalization of these signals was calculated as Pearson's correlation coefficient. The higher this value, the higher the degree of colocalization, and the degrees of localization of TDP-43 and LRS AMI in the nucleus and cytoplasm of the neurons can be compared.

[0118]

The results of intracellular localization of TDP-43 protein and LRS AMI in the brain of an ALS patient are shown in Fig. 13. The images in Fig. 13 are respectively fluorescent staining images of TDP-43 (A), LRS AMI (B), MAP2 indicating neural cytoplasm (C), and DAPI indicating nuclei (D). The dashed lines in Fig. 13A and 13B indicate a position of the cell nucleus created from Fig. 13D. Fig. 13A shows that TDP-43 leaks out of the cell nucleus in neurons in the brain of a sporadic ALS patient, as previously reported. Further, comparing Fig. 13A and Fig. 13B, colocalization of TDP-43 and LRS AMI 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 nucleus and cytoplasm were defined from the acquired images, and the correlation coefficients were calculated as described above. Fig. 14 (A) is a histogram showing the distribution of the correlation coefficients. Comparing the correlation coefficients between TDP- 43 and LRS AMI in the nucleus and cytoplasm, it was found that more cells showed a higher correlation in the cytoplasm than in the nucleus (Fig. 14(A)). Further, when an average correlation coefficient of the nucleus and cytoplasm was calculated, the correlation coefficient in the cytoplasm was statistically significantly higher (Fig. 14(B)). In other words, it was shown that extranuclear TDP-43 colocalized with LRSAM1 at a high rate.

[0119]

The above results show that a promoting substance targeting a predetermined target (for example, a target that contributes to an increase in ubiquitination activity such as LRS AMI) can contribute to maintenance or improvement of normal cell function. As a result, a promoting substance can contribute to advantageous effects such as suppression of cell damage, improvement of intracellular TDP-43 clearance, appropriate control of post-translational modifications such as phosphorylation, and treatment or prevention of TDP-43 related diseases. Further, it is presumed that a technology that increases an intracellular amount of a protein having ubiquitination activity such as LRSAM1 and thereby increases intracellular ubiquitination activity, can improve the function and localization of TDP-43 in a normal direction. Therefore, the technology can be a suitable method for treating or preventing TDP-43 -related diseases. These matters are new findings that have become clear by completing the present disclosure, and are useful, for example, for protecting neural cells such as motor neurons.

Further, from the results of the present example, it will be clear to those skilled in the art that the same effect can be obtained even when the expression-enhancing nucleic acid according to the present embodiment is used as a promoting substance targeting a predetermined target (for example, LRSAM1).

[0120]

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

[0121]

Further, the ALS patient-derived neurons used in the present example have a heterozygous mutation in the TARDBP gene that encodes the TDP-43 protein. Therefore, roughly, two types of proteins can be generated in the neurons: normal TDP-43 proteins (wild-type TDP-43 proteins) and mutant TDP-43 proteins. As in the present embodiment, by using a promoting substance targeting a predetermined target (for example, LRSAM1), the abundance ratio of the normal TDP-43 protein in the cells can be increased. Further, the inherent functions of TDP-43 can be enhanced, which can contribute to the improvement of normal cellular functions. In particular, since a mutant TDP-43 protein tends to accumulate in the cytoplasm, the mutant TDP- 43 protein is more susceptible to degradation by the LRSAM1 protein present in the cytoplasm, while the normal TDP-43 protein present in the nucleus is able to exert its inherent function in the cell as it is.

[0122]

In Patent Document 1 and Non-Patent Document 1, there is no discussion related to the TDP-43 protein and TDP-43 related diseases.

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

[0123]

According to the present embodiment, maintenance or improvement of a cell function can be facilitated. This is useful in the pharmaceutical field.

Obviously, numerous modifications and variations of the present invention are possible in light of the above teachings. It is therefore to be understood that within the scope of the appended claims, the invention may be practiced otherwise than as specifically described herein.

Claims

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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