WO2016088797A1 - Nucleic acid reducing als-causing protein toxicity - Google Patents

Abstract

Provided are: a nucleic acid that reduces toxicity caused by abnormal accumulation of an RNA-binding protein in cells and is usable for preventing or treating a neurodegenerative disease; and a prophylactic or therapeutic agent for a neurodegenerative disease that comprises the nucleic acid. A repeat RNA that contains (UGGAA)_n [wherein n is an integer of 10-50); and a prophylactic or therapeutic agent for RBP proteinopathy which is a neurodegenerative disease induced by abnormal accumulation of an RNA-binding protein in cells, said agent comprising the repeat RNA as an active ingredient.

Description

Nucleic acids that reduce ALS-causing protein toxicity

The present invention relates to a nucleic acid that can be used for the prevention or treatment of neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS).

Amyotrophic lateral sclerosis (ALS) is an intractable neurological disease in which upper and lower motor neurons are selectively denatured and muscle strength of the whole body is reduced. The prevalence rate in Japan is 7 to 11 per 100,000 population, and the annual incidence rate is about 1 to 2.5 per 100,000 population. Symptoms are mainly muscular atrophy and muscle weakness, and limb and trunk dysfunction, gait disturbance, articulation disorder, dysphagia in the cranial nervous system appear, and finally respiratory disorder due to respiratory muscle paralysis To. The progression of the disease is relatively fast, and if you do not use a ventilator, you usually die in 2-5 years. As a therapeutic drug, a drug having an action of delaying the progression of ALS is used: Riluzole (trade name: Riltech (registered trademark)), but in reality the satisfactory therapeutic effect has not been obtained.

In recent years, it has become clear that RNA-binding proteins such as TDP-43 and FUS / TLS, which share a common function and structure, are deeply involved in the pathology (see Non-Patent Document 1). In particular, inclusion bodies positive for TDP-43 observed in degenerated neurons are pathological features common to not only sporadic amyotrophic lateral sclerosis but also most amyotrophic lateral sclerosis. That is, TDP-43 plays an important role in the onset and pathogenesis of amyotrophic lateral sclerosis. Currently, in ALS, in addition to the functions common to RNA-binding proteins and abnormal control of targets, research is being conducted from both the viewpoint of toxic effects due to the formation of aggregates of these proteins and the like.

The study of amyotrophic lateral sclerosis has greatly progressed with the discovery that TDP-43 accumulates in the cytoplasmic inclusions of degenerated neurons. For example, about 90% of TDP-43 is originally localized in the nucleus, but in degenerated neurons, almost all of them accumulate in cytoplasmic inclusions and disappear from the nucleus. It is that. Therefore, in the degenerated neurons, the physiological function of TDP-43 originally performed in the nucleus should be lost. For this reason, research on the pathological state has progressed from the viewpoint of acquiring toxicity that TDP-43 aggregates constituting the inclusion body exert toxicity and lead to cell death, and from the viewpoint of abnormal function of TDP-43. It has also been reported that FUS / TLS, which is physiologically present mainly in the nucleus, is excessively retained in the cytoplasm in nerve cells in ALS patients and exhibits abnormal localization (Non-patent Document 2). reference).

From previous studies, it has been clarified that transgenic mice overexpressing wild-type human TDP-43 and FUS / TLS exhibit amyotrophic lateral sclerosis-like symptoms due to degeneration of motor neurons. Similarly, transgenic mice expressing mutant TDP-43 identified in hereditary amyotrophic lateral sclerosis also exhibit amyotrophic lateral sclerosis-like symptoms. These findings suggest that certain neurons, such as motor neurons, are particularly vulnerable to an excess of TDP-43, FUS / TLS, regardless of the presence or absence of TDP-43, FUS / TLS mutations. . Excess TDP-43 and FUS / TLS are reported to exert toxicity through RNA binding ability by RNA recognition motif (RNA Recognition Motif; tifRRM) (see Non-Patent Documents 3 and 4).

The present invention relates to TDP-43 (TAR (transactivation responsive region) DNA-binding protein; transactivation response region DNA binding protein 43) and FUS / TLS (fused in sarcoma), which are proteins that cause neurodegenerative diseases such as ALS. The purpose of the present invention is to provide nucleic acids that can be used for the prevention or treatment of neurodegenerative diseases such as ALS by reducing abnormal accumulation in cells of RNA binding proteins such as / 結合 translated in liposarcoma) and excessive binding to RNA.

The present inventors used a Drosophila model having characteristics excellent in genetic analysis in order to examine the pathophysiology and therapeutic strategy of protein toxicity in an excessive state of TDP-43 and FUS / TLS in ALS.

Then, for TDP-43 and FUS / TLS, the genes expressing TDP-43 G298S mutation (familial ALS) and FUS / TLS (no mutation, sporadic ALS) were incorporated into Drosophila, and the toxicity of TDP-43 and FUS was examined. . Both TDP-43 and FUS / TLS overload caused compound eye degeneration in Drosophila. In TDP-43, protein aggregation of TDP-43 was also observed.

Therefore, it is a nucleic acid sequence derived from an abnormally extended UGGAA repeat sequence, which is present in the BEAN1 gene and causes spinocerebellar ataxia type 31 (SCA31), and is an RNA recognition site (RRM) for TDP-43 and FUS / TLS proteins When a repeat sequence (derived from the repeat RNA sequence of SCA31) containing nucleic acid sequence (UGGAA) ₂₂ that binds to the amino acid of SCA31 was designed and co-expressed in the above Drosophila, compound eye degeneration was suppressed. Furthermore, regarding TDP-43, It was confirmed that there was an inhibitory effect on protein aggregation.

As a result, it was found that (UGGAA) _n repeat RNA can be used as a nucleic acid drug for improving the symptoms of ALS by reducing the toxicity of TDP-43 and FUS / TLS, and the present invention has been completed.

That is, the present invention is as follows.
[1] Repeat RNA containing (UGGAA) _n (n is a natural number of 10 to 50).
[2] The repeat RNA of [1] containing (UGGAA) _n (n is a natural number of 15 to 25).
[3] The repeat RNA according to [1], including (UGGAA) ₂₂ .
[4] DNA encoding the repeat RNA of any one of [1] to [3].
[5] A pharmaceutical composition comprising the repeat RNA of any one of [1] to [3] or the DNA of [4] as an active ingredient.
[6] Prevention of RBP proteinopathy, which is a neurodegenerative disease caused by abnormal accumulation of RNA-binding protein in cells, containing the repeat RNA of any one of [1] to [3] or the DNA of [4] as an active ingredient Or therapeutic drugs.
[7] RBP proteinopathy, which is a neurodegenerative disease caused by abnormal accumulation of RNA-binding protein in cells, is a neurodegenerative disease caused by abnormal accumulation of TDP-43 or FUS / TLS in cells [6] Preventive or therapeutic drug.
[8] The preventive or therapeutic agent according to [6] or [7], wherein repeat RNA or DNA encoding the same suppresses abnormal accumulation of TDP-43 or FUS / TLS in a cell.
[9] The preventive or therapeutic agent according to [7] or [8], wherein the neurodegenerative disease is TDP-43 proteinopathy.
[10] Neurodegenerative diseases are sporadic amyotrophic lateral sclerosis (ALS) or familial amyotrophic lateral sclerosis, amyotrophic lateral sclerosis, frontotemporal lobar degeneration (FTLD: Frontotemporal lobar degeneration), Alzheimer's disease, Lewy body dementia, Huntington's disease, Parkinson's disease, addiction-granular dementia (Grain's disease), Perry syndrome, progressive supranuclear palsy, corticobasal degeneration and Pick's disease The prophylactic or therapeutic agent according to any one of [6] to [9] selected from the group consisting of
[11] The neurodegenerative disorder is sporadic amyotrophic lateral sclerosis (ALS) or amyotrophic lateral sclerosis, familial amyotrophic lateral sclerosis, or frontotemporal lobar degeneration (FTLD). : Frontotemporal lobar degeneration) [10]

This specification includes the disclosure of Japanese Patent Application No. 2014-244034, which is the basis of the priority of the present application.

Repeat RNA containing short (UGGAA) n derived from the abnormally extended UGGAA repeat sequence responsible for SCA31 is abnormal accumulation and abnormal aggregation in cells due to abnormal expression of RNA binding proteins such as TDP-43 and FUS / TLS. Cytotoxicity due to excessive binding to RNA can be reduced. Abnormal accumulation in cells due to abnormal expression of RNA-binding protein is caused by amyotrophic lateral sclerosis such as sporadic amyotrophic lateral sclerosis (ALS), familial amyotrophic lateral sclerosis, and frontal side. Causes neurodegenerative diseases such as frontotemporal lobar degeneration (FTLD). Therefore, repeat RNA containing (UGGAA) n can be used for prevention or treatment of neurodegenerative diseases caused by abnormal accumulation of RNA-binding proteins in cells.

It is a figure which shows suppression of the compound eye degeneration resulting from the overexpression of TDP-43 by the co-expression of (UGGAA) ₂₂ in Drosophila which overexpresses TDP-43. FIG. 3 is a graph showing reduction of cytoplasmic aggregation of TDP-43 by co-expression of (UGGAA) ₂₂ in Drosophila overexpressing TDP-43. FIG. 4 is a diagram showing suppression of compound eye degeneration caused by overexpression of FUS / TLS by co-expression of (UGGAA) ₂₂ in Drosophila overexpressing FUS / TLS. FIG. 4 shows suppression of compound eye degeneration caused by overexpression of TDP-43 by co-expression of (UGGAA) ₂₂ in Drosophila overexpressing TDP-43 full length or C-terminal fragment. FIG. 3 is a graph showing suppression of compound eye degeneration caused by overexpression of TDP-43 by co-expression of (UGGAA) ₂₂ in Drosophila overexpressing the entire TDP-43 length, by the thickness of the compound eye.

Hereinafter, the present invention will be described in detail.

The nucleic acids of the present invention are TDP-43 (TAR (transactivation responsive region) DNA-binding protein; transactivation response region DNA-binding protein 43) and FUS / TLS (fused in sarcoma / translocated in liposarcoma) ( It is a nucleic acid that suppresses toxicity, accumulation, and aggregation of RNA-binding proteins such as FUS). The nucleic acid is RNA, and RNA consisting of UGGAA, which is an abnormally elongated RNA that causes spinocerebellar ataxia type 31 (SCA31), or a repeat RNA of a derivative thereof. UGGAA derivatives are composed of 4 to 10 bases and contain 1 to 4 UG sequences, and the ratio of the number of bases in the UG sequence to the total number of bases is about 20 to 60%, preferably about 30 to 50%. Examples thereof include a nucleic acid comprising a sequence or a nucleic acid containing the sequence. The UG sequence may be continuous or discontinuous. However, the sequence (UG) n consisting only of the UG repeat sequence is excluded. That is, when the total number of bases is 4 to 6, the number of UGs is 1; when the total number of bases is 7 or 8, the number of UGs is 1 or 2. When the total number of bases is 9, the number of UGs When the total number of bases is 2, the number of UGs is 2 or 3.

The number of repetitions of the nucleic acid is 10 to 50 times, preferably 10 to 30 times, more preferably 15 to 25 times, still more preferably 20 to 25 times, and particularly preferably 22 times.

Examples of the nucleic acid of the present invention include a sequence in which the UGGAA sequence is repeated a plurality of times. In the present invention, the repeated sequence of n times is represented as (UGGAA) _n . Preferably, the sequence represented by (UGGAA) _{22 in} which the UGGAA sequence is repeated 22 times is mentioned. In the present invention, such RNA is also referred to as short repeat RNA.

Furthermore, the nucleic acid of the present invention includes DNA encoding the above short repeat RNA.

The nucleic acid of the present invention binds to an RNA binding protein. RNA binding proteins bind to RNA and have important RNA metabolic functions such as RNA splicing, miRNA synthesis, and RNA transport. RNA binding protein overexpression in the cytoplasm due to abnormal expression of RNA binding protein, abnormal accumulation in the cell, and binding to target RNA group affects RNA metabolism such as splicing abnormality and transport abnormality It is toxic to cells. It also exhibits toxicity by abnormal aggregation in the cytoplasm. When these RNA binding proteins are abnormally expressed in neurons, they exhibit toxicity to neurons and cause various neurodegenerative diseases. The nucleic acid of the present invention binds to an RNA recognition sequence (RRM: RNA-Recognition-Motif) (RNA-binding motif) of an RNA-binding protein that is abnormally expressed in the cytoplasm, and blocks the sequence so that an absorber, RNA-binding protein and RNA are blocked. It acts as a decoy that competes for the binding of RNA and reduces the toxicity of RNA-binding proteins.

Therefore, the nucleic acid of the present invention can be used for the prevention or treatment of diseases caused by abnormal accumulation in cells due to abnormal expression of RNA-binding proteins or abnormal aggregation in the cytoplasm. Such a disease is called RBP (RNA binding protein) proteinopathy. Examples of RNA binding proteins that cause RBP proteinopathy and that can be bound by the nucleic acids of the present invention include TDP-43 (TAR DNA-binding protein), FUS / TLS (fused in sarcoma / translocated in liposarcoma), etc. The nucleic acid of the present invention can be used for the prevention or treatment of diseases caused by abnormal expression of these RNA-binding proteins, particularly neurodegenerative diseases. RBP proteinopathy includes multisystem proteinopathy (MSP) in which TDP-43 and the like are abnormally accumulated in bones, muscles, and nerves.

The nucleic acid of the present invention can be preferably used for prevention or treatment of neurodegenerative diseases caused by abnormal expression of TDP-43 and FUS / TLS. TDP-43 and FUS / TLS are proteins belonging to the hnRNP family, and belong to a group of RNA chaperones that bind to RNA and have important RNA metabolic functions such as RNA splicing, miRNA synthesis, and RNA transport. TDP-43 and FUS / TLS have an RNA recognition sequence (RNA binding domain (RPM)) and a glycine-rich region, and the nucleic acid of the present invention binds to the RNA recognition sequence. It can be overexpressed when mutations occur in TDP-43 and FUS / TLS. For example, TDP-43 occurs mainly in the glycine-rich region, and the protein aggregation of TDP-43 is enhanced by the mutation. When TDP-43 and FUS / TLS are overexpressed, they are toxic to cells. In addition, overexpression of TDP-43 aggregates in the cytoplasm and exhibits toxicity. When cells are neurons, nerves degenerate, causing neurodegenerative diseases. When the nucleic acid of the present invention binds to TDP-43 and FUS / TLS, for example, accumulation and aggregation of TDP-43 in cells can be suppressed, and cytotoxicity of TDP-43 and FUS / TLS can be reduced.

Examples of diseases caused by overexpression due to abnormal expression of TDP-43 or FUS / TLS include diseases caused by excessive expression of TDP-43 and abnormal accumulation in cells. As such diseases, diseases caused by abnormal aggregation and accumulation in the cytoplasm and formation of intracellular inclusions, for example, neurodegenerative diseases caused by abnormal aggregation and accumulation of TDP-43 in the cytoplasm of neurons Is mentioned. Here, the neurodegenerative disease refers to a progressive disease in which nerve cells in the central nervous system gradually degenerate due to accumulation of abnormal protein aggregates, resulting in malfunction or cell death. A disease caused by abnormal accumulation of TDP-43 or abnormal aggregation is particularly referred to as TDP-43 proteinopathy among RBP proteinopathy. The above MSP proteinopathy is also one of the TDP-43 proteinopathy. Specific examples of RBP proteinopathy include sporadic amyotrophic lateral sclerosis (ALS), amyotrophic lateral sclerosis such as familial amyotrophic lateral sclerosis, frontotemporal lobar degeneration (FTLD) : Frontotemporal lobar degeneration), Alzheimer's disease, dementia with Lewy bodies, Huntington's disease, Parkinson's disease, granulopathy of the taste buds (Grain disease), Perry syndrome, progressive supranuclear paralysis, basal ganglia degeneration, pick Diseases and the like. It can also be used for the prevention or treatment of neurodegenerative diseases associated with dementia. Among these, it is useful for the prevention or treatment of amyotrophic lateral sclerosis and frontotemporal lobar degeneration.

In patients with sporadic amyotrophic lateral sclerosis (ALS) and some familial amyotrophic lateral sclerosis, a C-terminal fragment of TDP-43 with a molecular weight of 25-35 kDa was observed in the brain tissue. Known to be involved. This C-terminal fragment has aggregation properties like full-length TDP-43, and the C-terminal fragment coaggregates with full-length TDP-43 and exhibits more toxicity. The nucleic acid of the present invention exerts a therapeutic effect against sporadic amyotrophic lateral sclerosis (ALS) by suppressing aggregation and toxicity of C-terminal fragment in addition to full-length TDP-43 in brain tissue. .

The nucleic acid of the present invention can be used for the prevention or treatment of the above-mentioned neurodegenerative diseases. Moreover, the nucleic acid of the present invention can suppress the progression of the above-mentioned neurodegenerative diseases and prevent recurrence. In the present invention, suppression of progression of neurodegenerative diseases and prevention of recurrence are also included in prevention or treatment of neurodegenerative diseases. Furthermore, the nucleic acid of the present invention can be used for the suppression or inhibition of abnormal intracellular accumulation and cytoplasmic aggregation of RNA binding proteins such as TDP-43 and FUS / TLS.

The method of administering the preventive or therapeutic agent for the neurodegenerative disease of the nucleic acid of the present invention includes parenteral routes such as intrathecal fluid, intravenous, intramuscular, subcutaneous and intraperitoneal injection or parenteral route, and oral route. The parenteral route is preferred, but intrathecal injection is more preferred. Examples of the form include injections, tablets, capsules, vulcanizing agents, syrups, emulsions, sprays and the like. The nucleic acid of the present invention is used in combination with a commonly used pharmaceutical carrier. Carriers include, but are not limited to, physiological saline, phosphate buffered saline, glucose solution and buffered saline. Additives such as sugars such as xylose, trehalose and fructose; sugar alcohols such as mannitol, xylitol and sorbitol; buffer solutions such as phosphate buffer, citrate buffer and glutamate buffer; surfactants such as fatty acid esters Can be used as When blended with a carrier, the content of the nucleic acid may be appropriately selected depending on the preparation, and varies depending on the preparation, but is preferably 0.001 to 1% by weight. The dosage is determined by the patient's age, sex, weight, symptoms, therapeutic purpose, etc. Generally, for parenteral administration, 0.0001 to 1 mg is divided into once to several times a day for parenteral administration. Can be administered. This dose can be appropriately increased or decreased depending on the type of disease, patient age, weight, symptoms and the like.

The present invention will be specifically described by the following examples, but the present invention is not limited to these examples.
Example 1 Effect of Non-Toxic Short Repeat RNA ((UGGAA) ₂₂ ) on Mutant TDP-43 Toxicity Clarifies whether the interaction between RNA and TDP-43 affects TDP-43 toxicity in vivo Therefore, the effect of non-toxic short repeat RNA ((UGGAA) ₂₂ ) on the toxicity of mutant TDP-43 was evaluated.

Drosophila strains were obtained from the University of Indiana at the Vienna Drosophila RNAi Center (VDRC) and the Bloomington Drosophila Stock Center (BDSC). Drosophila overexpressing human TDP-43 or FUS / TLS, and transformed Drosophila expressing (UGGAA) ₂₂ derived from the repeat sequence of SCA31 were prepared by a conventional method using a pUAST vector (GAL4-UAS system). A transformed line was created using a yellow, white line (BestGeen Inc.). GMR-GAL4 driver is used for compound eye expression. Experiments using Drosophila were performed at 25 ° C. The phenotype of 1 to 2 day-old Drosophila eyes was evaluated using a binocular stereomicroscope SZX10 (Olympus) and a scanning electron microscope (Miniscope TM1000, Hitachi).

For TDP-43 and FUS / TLS, a gene expressing TDP-43 G298S mutation (with TDP-43 G298S mutation) (familial ALS) and FUS / TLS (no mutation, sporadic ALS) is incorporated into Drosophila It is. Drosophila incorporating a gene that expresses the TDP-43 G298S mutation (familial ALS) or FUS / TLS (no mutation, sporadic ALS) is an ALS model Drosophila. Furthermore, a nucleic acid sequence derived from the abnormally extended UGGAA repeat sequence that causes spinocerebellar ataxia type 31 (SCA31), which binds to the amino acids of TDP-43 and FUS / TLS protein RNA recognition sequence (RRM) A repeat sequence (derived from the repeat RNA sequence of SCA31) containing the sequence (UGGAA) ₂₂ was designed and incorporated into Drosophila. TDP-43 G298S mutation (familial ALS), FUS / TLS (no mutation, sporadic ALS) expressing Drosophila and a repeat sequence containing (UGGAA) ₂₂ (derived from SCA31 repeat RNA sequence) Drosophila were crossed to obtain Drosophila that co-express both genes.

FIG. 1 shows a compound eye state when mutant TDP-43 G298S is expressed and a compound eye state when mutant TDP-43 G298S and (UGGAA) ₂₂ are co-expressed. The lower panel of FIG. 1 shows the compound eye of Drosophila (TDP-43 G298S / (UGGAA) ₂₂ ) co-expressing mutant TDP-43 G298S (TDP-43 G298S) and (UGGAA) ₂₂ , and the upper panel (UGGAA). ) Shows compound eyes of Drosophila (TDP-43 G298S / +) not co-expressing ₂₂ . FIG. 1A shows a compound eye stereomicrograph, and FIGS. 1B and C show scanning electron micrographs (FIG. 1C is an enlarged photo). FIG. 1D shows stained images of DAPI (47,6-diamidino-2-phenylindole) and fluorescently labeled anti-TDP-43. The red-purple portion in FIGS. 1D and E shows the portion stained with DAPI, and the entire compound eye is stained red-purple in both the upper and lower photographs. FIG. 1E is an enlarged photograph of the framed part in FIG. 1D, and the green part of the arrow indicates the part stained with the anti-TDP-43 antibody, and only the part of the upper panel, the part stained green is observed. There is no green-stained portion in the lower panel photo, indicating that TDP-43 aggregation has disappeared.

As shown in FIG. 1, Drosophila expressing mutant TDP-43 G298S produced retinal degeneration along with pigment loss and eye disturbance, and compound eye degeneration was observed. TDP-43 protein aggregation was also observed. On the other hand, co-expression of (UGGAA) ₂₂ alleviated this denaturation. Further, FIG. 2 shows the degree of aggregation of TDP-43. FIG. 2 shows the ratio of TDP-43 G298S and TDP-43 G298S / (UGGAA) ₂₂ aggregated cells in the Drosophila cytoplasm. (UGGAA) Coexpression of ₂₂ and TDP-43 reduces intracytoplasmic aggregation of mutant TDP-43 in the compound eye primordium of Drosophila eyes compared to Drosophila expressing mutant TDP-43 alone (P <0.01, TDP-43 inclusion volume decreased from 15.1 ± 1.42% to 1.8 ± 0.20%). Involvement of TDP-43 in multiple pathways of RNA metabolism via RNA binding activity, and that TDP-43 RRM mutant Drosophila did not show neurodegeneration (Voigt et al., 2010, PloS one 5, e12247) In view of this, the interaction between RNA and TDP-43 may be crucial in the toxicity of TDP-43.

FIG. 3 shows the compound eye state when FUS / TLS is expressed and the compound eye state when (UGGAA) ₂₂ is co-expressed. The lower panel of FIG. 3 shows compound eyes of Drosophila (FUS / (UGGAA) ₂₂ ) co-expressed with FUS / TLS and (UGGAA) ₂₂ , and the upper panel shows Drosophila not co-expressed with (UGGAA) ₂₂ (FUS / +). Showing compound eyes. FIG. 3A shows a compound eye stereomicrograph, and FIGS. 3B and C show scanning electron micrographs (FIG. 3C is an enlarged photo). As shown in FIG. 3, compound eye degeneration was observed in Drosophila not co-expressing (UGGAA) ₂₂ , but compound eye degeneration was not observed in Drosophila co-expressing (UGGAA) ₂₂ . This result indicates that co-expression of FUS / TLS and (UGGAA) ₂₂ suppressed the toxicity of wild-type FUS / TLS up-regulation.

Combined with these results, our findings indicate that the protective effect of non-toxic (UGGAA) ₂₂ acts like an absorber or decoy and interacts with the target RNA, which is a toxic mediator, RRM-based interactions. The possibility is raised that it may be a targeted block of action.

As noted above, non-toxic short repeat RNA itself plays a protective role against RNA binding protein (RBP) -induced toxicity associated with ALS (FIGS. 1-3). Non-toxic short repeat RNAs likely act as absorbers or decoys and interact with RNA binding proteins to suppress binding of RNA binding proteins to other important intracellular RNAs.
Example 2 Effect of non-toxic short repeat RNA ((UGGAA) ₂₂ ) on C-terminal fragment toxicity of TDP-43 In addition to full-length TDP-43 in the brain of ALS patients, C-terminal fragment cleaved near the center of the molecule Is known to stand out. Like the full-length TDP-43, the fragmented C-terminal TDP-43 also has an aggregating property and is expected to exhibit cytotoxicity by coaggregating with the full-length TDP-43 in the cell. Therefore, it was confirmed whether non-toxic short repeat RNA (UGGAA RNA) also suppresses the toxicity of the C-terminal fragment of TDP-43.

In the same manner as in Example 1, a sporadic ALS model Drosophila was prepared by incorporating a gene expressing the full-length wild-type TDP-43 or a gene expressing the C-terminal fragment (CTF) of TDP-43 into the Drosophila strain. Then, the effect of UGGAA short repeat ((UGGAA) ₂₂ ) was verified in the wild-type TDP-43 full length and C-terminal fragment (CTF) expression model. The C-terminal fragment is a full-length TDP-43 fragment with a molecular weight of 25-35 kDa.

FIG. 4 shows the state of the compound eye (FIG. 4A) when expressing full-length TDP-43 and the compound eye (FIG. 4B) when expressing the C-terminal fragment of TDP-43. The lower panel of FIG. 4 shows a stereoscopic micrograph of a Drosophila compound eye co-expressed with full-length TDP-43 or C-terminal fragment and (UGGAA) _22, and the upper panel of FIG. 4 shows a Drosophila not co-expressing (UGGAA) ₂₂ A stereomicrograph of a compound eye is shown. As shown in FIG. 4, in the Drosophila which do not co-express (UGGAA) _22, although the compound eye of degeneration was observed in the Drosophila coexpressed (UGGAA) _22, compound eye degeneration was observed. This result indicates that compound eye degeneration due to toxicity of wild-type TDP-43 and CTF was reduced by UGGAA RNA. In addition, since the wild type was difficult to understand only by the compound eye appearance, a section was prepared, and the compound eye tissue was stained with hematoxylin and eosin. The results are shown in FIG. FIG. 5A is a section of a Drosophila compound eye in which a highly sensitive green fluorescent protein (EGFP, molecular weight 27 kDa) having no toxicity is expressed, and is an example in which a normal retina thickness is maintained. FIG. 5B shows a compound eye section of Drosophila expressing the full-length wild-type TDP-43, and FIG. 5C shows a compound eye section of Drosophila co-expressing the wild-type TDP-43 full-length and UGGAA) ₂₂ . As shown in FIG. 5, the thickness of the compound eye (Retina) collapsed by denaturation was improved by the expression of UGGAA RNA.

The results of this example show that UGGAA ₂₂ is effective in treating sporadic ALS, where aggregation of wild type TDP-43 and C-terminal fragments is observed in brain tissue.

The nucleic acid of the present invention can be used as a preventive or therapeutic agent for neurodegenerative diseases caused by abnormal expression of RNA-binding proteins.

All publications, patents and patent applications cited in this specification are incorporated herein by reference in their entirety.

Claims (11)

1. (UGGAA) Repeat RNA containing _n (n is a natural number from 10 to 50).
2. The repeat RNA according to claim 1, comprising (UGGAA) _n (n is a natural number of 15 to 25).
3. The repeat RNA according to claim 1, comprising (UGGAA) ₂₂ .
4. DNA encoding the repeat RNA according to any one of claims 1 to 3.
5. A pharmaceutical composition comprising the repeat RNA according to any one of claims 1 to 3 or the DNA according to claim 4 as an active ingredient.
6. Prevention of RBP proteinopathy, which is a neurodegenerative disease caused by abnormal accumulation of RNA-binding protein in cells, comprising the repeat RNA according to any one of claims 1 to 3 or the DNA according to claim 4 as an active ingredient Or therapeutic drugs.
7. The prevention according to claim 6, wherein the RBP proteinopathy that is a neurodegenerative disease caused by abnormal accumulation of RNA-binding protein in cells is a neurodegenerative disease caused by abnormal accumulation of TDP-43 or FUS / TLS in cells. Or therapeutic drugs.
8. The preventive or therapeutic agent according to claim 6 or 7, wherein the repeat RNA or the DNA encoding the same suppresses abnormal accumulation of TDP-43 or FUS / TLS in a cell.
9. The prophylactic or therapeutic agent according to claim 7 or 8, wherein the neurodegenerative disease is TDP-43 proteinopathy.
10. Neurodegenerative disease is sporadic amyotrophic lateral sclerosis (ALS) or familial amyotrophic lateral sclerosis, amyotrophic lateral sclerosis, frontotemporal lobar degeneration (FTLD) ), Alzheimer's disease, Lewy body dementia, Huntington's disease, Parkinson's disease, addiction-granular dementia (Grain's disease), Perry syndrome, progressive supranuclear palsy, corticobasal degeneration, and Pick's disease The prophylactic or therapeutic agent according to any one of claims 6 to 9, selected from:
11. The neurodegenerative disease is sporadic amyotrophic lateral sclerosis (ALS) or amyotrophic lateral sclerosis, which is familial amyotrophic lateral sclerosis, or frontotemporal lobar degeneration (FTLD). The prophylactic or therapeutic agent according to claim 10, which is degeneration).

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