JP2016108249A - Therapeutic agent for spinocerebellar ataxia type 31 (sca31) - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a novel therapy based on the onset mechanism of spinocerebellar ataxia type 31 (SCA31).SOLUTION: The invention provides a therapeutic agent for spinocerebellar ataxia type 31 (SCA31) comprising RNA binding proteins selected from TDP-43 or FUS/TLS, or a fragment comprising these RNA binding regions.SELECTED DRAWING: None

Description

  The present invention relates to a therapeutic agent for spinocerebellar ataxia type 31 (SCA31), more specifically, the above treatment comprising a protein that specifically binds to an RNA repeat that abnormally aggregates in spinocerebellar ataxia type 31 (SCA31) It relates to the agent.

  Spinocerebellar degeneration is one of the intractable neurological diseases for which there is currently no cure, and it is said that there are about 30,000 affected people in Japan. When affected, the cerebellum and related brain functions are impaired, resulting in ataxia symptoms that often progress and become unable to walk in a few years.

It is known that spinocerebellar degeneration is hereditary and non-hereditary (spontaneous). Among hereditary ones, type 31 (SCA31) is one of the most common dominant hereditary spinocerebellar ataxias in Japan. The causative gene of SCA31 is a repeat sequence containing a TGAA 5-base repeat sequence of about 2.5 to 3.8 kbp present in the intron region common to the BEAN and TK2 genes located in the long arm of chromosome 16. Is abnormally expanded transcription (TGGAA) _n microsatellite repeats were in the genome, (UGGAA) repeat RNA containing _n that are aggregated accumulation has been reported by a group of the present inventors (Non-Patent Document intracellularly 1 and 2).

  Meanwhile, RNA binding proteins such as transactivation responsive region (TAR) DNA binding protein 43 (TAR DNA-binding protein 43, TDP-43) and fused in sarcoma / translated in liposarcoma (FUS / TLS) ( RBP) not only accumulates as ubiquitinated intracellular inclusions, but is also associated with the pathogenesis of familial amyotrophic lateral sclerosis (ALS) and frontotemporal lobe dementia (FTD) It has been reported (Non-Patent Document 3). TDP-43 has been reported to have high binding affinity for clusters of sequences rich in TG and UG (Non-patent Documents 4 and 5).

Sato, N. et al., The American Journal of Human Genetics 85, 544-557, November 13, 2009 Niimi, Y. et al., Neuropathology, 33, 600-611, 2013 Mackenzie, I.R. et al., The Lancet Neurology 9, 995-1007, 2010 Buratti, E. et al., The Journal of Biological Chemistry, 276, 36337-36343, 2001 Tollervey, J.R. et al., Nature Neuroscience 14, 452-458, 2011

  At present, there is no effective treatment for spinocerebellar ataxia type 31 (SCA31), and the development of fundamental therapies such as elucidation of the pathophysiology, treatment and prevention of neuromuscular diseases is expected. The object of the present invention is to elucidate the pathological condition of SCA31 and to provide a novel therapeutic agent that can be used for its prevention or treatment.

For the purpose of elucidating the pathology of SCA31 and developing a therapeutic method, the present inventors have created a SCA31 Drosophila model that expresses a repeat sequence of UGGAA related to SCA31. Hereinafter, in the present specification, (UGGAA) _n (n = 80-100), which is a repeat RNA having 80-100 UGGAA repeats prepared by the present inventors, is represented by (UGGAA) _exp and 22 repeats. (UGGAA) _n (n = 22) having (UGGAA) ₂₂ is described. In this in vivo model, it was demonstrated that expression of (UGGAA) _exp induced RNA accumulation / aggregation, resulting in neurodegeneration depending on repeat length and dose.

In examining the effects of various factors affecting the toxicity induced by the expression of (UGGAA) _exp , the present inventors surprisingly found that TDP-43, a protein involved in the pathology of ALS. And FUS / TLS were found to inhibit (UGGAA) _exp RNA misfolding and production of repeat-encoded pentapeptide repeat proteins and suppress (UGGAA) _exp- induced toxicity.

Co-expression of (UGGAA) _exp with TDP-43 or FUS / TLS dramatically suppresses compound eye degeneration of (UGGAA) _exp- expressing flies, and the suppression of these RNA binding regions (RRM: RNA recognition motif (RNA recognition motif) is required, and it was confirmed that protein fragments lacking other regions such as the C-terminus are more effective in suppressing RNA toxicity than full-length proteins. This inhibitory effect is based on the fact that these RNA-binding proteins function as RNA chaperones to suppress abnormal aggregation of (UGGAA) _exp RNA, and suppress RNA structural abnormalities that are thought to cause SCA31. It has been found that the toxicity can be greatly reduced, thereby exerting a therapeutic effect.

  Since the above proteins show the therapeutic effect of SCA31 against RNA toxicity due to abnormally extended UGGAA repeats, these proteins or fragments containing RNA binding regions thereof, or polynucleotides encoding these proteins or fragments are useful for SCA31 Gene therapy or peptide therapy.

That is, the present invention is as follows.
[1] Spinal cord containing an RNA binding protein selected from TDP-43 (transactivation responsive region DNA-binding protein 43) or FUS / TLS (fused in sarcoma / translated in liposarcoma), or a fragment containing these RNA binding regions Cerebellar ataxia type 31 (SCA31) treatment.
[2] TDP-43
(a) consisting of the amino acid sequence represented by SEQ ID NO: 1, or
(b) An amino acid sequence represented by SEQ ID NO: 1, consisting of an amino acid sequence in which one or several amino acids are deleted, substituted or added, and having binding activity to (UGGAA) _n (n = 80-100) The therapeutic agent according to [1] above, wherein
[3] FUS / TLS is
(a) consisting of the amino acid sequence represented by SEQ ID NO: 3, or
(b) The amino acid sequence shown in SEQ ID NO: 3 consisting of an amino acid sequence in which one or several amino acids are deleted, substituted or added, and having binding activity to (UGGAA) _n (n = 80-100) The therapeutic agent according to [1] above, wherein
[4] The fragment containing the RNA binding region of TDP-43 essentially contains an RRM1 region corresponding to amino acids 105 to 169 of SEQ ID NO: 1 and / or an RRM2 region corresponding to amino acids 193 to 257, The therapeutic agent according to [1] above.
[5] The therapeutic agent according to [1] above, wherein the fragment containing the RNA binding region of FUS / TLS essentially contains amino acids at positions 285 to 371 of SEQ ID NO: 3.

  The present invention revealed a protective role of TDP-43 and FUS / TLS as RNA chaperones in SCA31. Therefore, administration of these RNA-binding proteins and functional fragments thereof to patients can be used for treatment of SCA31 and reduction of symptoms.

Schematic representation of constructs for expression of SCA31 repeat (UGGAA) _exp and control repeats. The sequence of the repeat unit of Part 3 is shown in the sequence listing as SEQ ID NO: 14. Drosophila compound eyes expressing control repeat (A), (UGGAA) ₂₂ (B), (UGGAA) _exp (W) (C), (UGGAA) _exp (S) (D) using AD GMR-Gal4 system Are images observed with an optical microscope (left) and a scanning electron microscope (center and right). Control repeats (E), (UGGAA) ₂₂ (F), (UGGAA) _exp (W) (G), (UGGAA) _exp (S) on the GMR-Gal4 system using the EH LNA probe (red) and DAPI (blue) ) (H) shows the results of RNA FISH analysis of the compound eye primordium of Drosophila third instar larvae. Bar indicates 50 μm. In (G) and (H), RNA aggregation was observed in larvae that expressed (UGGAA) _exp (W) and (UGGAA) _exp (S). I shows compound eye degeneration in homozygous and heterozygous transgenic (UGGAA) _exp Drosophila. The results of RNA FISH analysis of the compound eye primordium of (UGGAA) _exp (W) -expressing Drosophila third instar larvae when pretreated with JRNaseA are shown. A shows the results of quantitative real-time PCR analysis of transgene expression in Drosophila. Mean ± S.E.M, * p <0.05, ** p <0.01, *** p <0.001, one-way ANOVA, n = 80 / genotype. B shows the results of quantification of RNA aggregates in larvae. (W): 2.1 ± 0.92%, (S): 12.4 ± 2.0%, mean ± S.D., ** p <0.01, Student t-test, n = 10 / genotype. The life analysis result using elav-GeneSwitch is shown. Drosophila expressing (UGGAA) _exp (S) (4) are shown to have a shorter lifespan than Drosophila expressing EGFP (1), control repeat (2), and UGGAA ₂₂ (3) (p <0.001, log-rank test, n = 100-120 animals / group). Changes in motor function with aging are shown. Drosophila expressing (UGGAA) _exp (S) (4) show more progressive motor dysfunction compared to Drosophila expressing EGFP (1), control repeat (2), and UGGAA ₂₂ (3) (Mean ± SD, n = 100-120, *** p <0.001, two-way ANOVA). (UGGAA) shows the results of an immunoblot showing the specific binding of TDP-43 to _exp RNA. We show that TDP-43 (green) and RNA aggregates (red) co-localize in Purkinje cells of human SCA31 patients by combining RNA FISH and immunostaining. Schematic diagrams of TDP-43 wild type (TDP-43 WT), N- and C-terminal deletion mutants (TDP-43 ΔN and TDP-43 ΔC), and RRM mutants are shown. Drosophila expressing TDP-43 or its fragments (N- and C-terminal deletion mutants) or mutants using GMR-Gal4, and Drosophila in which TDP-43 was knocked down by endogenous dTDP43 (TBPH) RNAi ( The optical microscope image of the compound eye of dTDP-43 KD) is shown. Drosophila expressing TDP-43 or a fragment or variant thereof together with (UGGAA) _exp (W) and (UGGAA) _exp (S) using GMR-Gal4, and TDP-43 by endogenous dTDP43 (TBPH) RNAi 2 shows an optical microscope image of a compound eye of Drosophila knocked down. Co-expression of TDP-43, or an N-terminal or C-terminal deletion fragment (ΔN or ΔC) thereof, dramatically reduces compound eye degeneration in Drosophila expressing (UGGAA) _exp . On the other hand, RRM mutants without RNA binding ability could not suppress (UGGAA) _exp- induced toxicity. Knockdown (KD) with dTDP-43 exacerbated compound eye degeneration. Quantitative results of (UGGAA) _exp transcript levels in Drosophila are shown as relative expression levels (mean ± SEM, ns (no significant difference), Student t-test, n = 80 / genotype). The RNA FISH analysis result of the compound eye primordium of (UGGAA) _exp (S) type | system | group 3rd instar larva coexpressing TDP-43 wild type (WT) or a RRM variant is shown. Bars indicate 50 μm and 10 μm (high magnification). RNA FISH analysis of the compound eye primordium of TDP-43 wild type (WT), RRM mutant, or TDP-43 knocked down by TDP-43 RNAi (UGGAA) _exp (W) Results are shown. Bars indicate 50 μm and 10 μm (high magnification). The quantification result of the RNA aggregate in each genotype is shown (mean ± S.E.M, ** p <0.01, one-way ANOVA, n = 10 / genotype). (UGGAA) shows the result of immunostaining of TDP-43 in the compound eye primordium of third-instar larvae expressing _exp (W) and TDP-43. TDP-43 colocalizes with the (UGGAA) _exp RNA transcript in the cell nucleus. Atomic Force Microscopy (AFM) image and aggregate size distribution of (UGGAA) _exp RNA solution showing reduced aggregation of (UGGAA) _exp in the presence of C-terminal deletion fragment (ΔC) of TDP-43 Indicates. Bar indicates 50 nm. ** p <0.01, 900 or more molecules were measured (mean ± SD). The distribution of single-stranded RNA (ssRNA), small aggregates (small) and large aggregates (large) after 10 minutes in the absence (−) and presence (+) of TDP-43ΔC is shown. (UGGAA) A schematic diagram of the pentapeptide repeat protein WNGME (SEQ ID NO: 12) encoded by _exp repeat RNA is shown. W: Trp, N: Asn, G: Gly, M: Met, E: Glu. The sequences of F1, F2, and F3 and the corresponding base sequences are shown in the sequence listing as SEQ ID NOs: 15 to 18, respectively. The result of immunostaining PPR protein with anti-WNGME antibodies (SGJ-1705 and SGJ-1706) in human brain tissue is shown. PPR protein is present as granules in the cell body and dendrites within Purkinje cells of SCA31 patients (arrows). Immunostaining with anti-WNGME antibody shows that no PPR protein is observed in the control repeats using the GMR-Gal4 system and the compound eye primordium of third-instar larvae expressing (UGGAA) ₂₂ . FIG. 5 shows PPR protein as shown by immunofluorescence analysis of compound eye primordium of (UGGAA) _exp (W) or (S) strain 3rd instar larvae co-expressing TDP-43, RRM mutant and dTDP-43 RNAi. Overexpression of TDP-43 reduced PPR protein production in both the (UGGAA) _exp (W) and (S) systems. Bars indicate 50 μm and 10 μm (high magnification). The quantification result of PPR protein in each genotype is shown (mean ± S.E.M, ** p <0.01, one-way ANOVA, n = 10 / genotype). The result of the immunoblotting of the PPR protein in the compound eye primate lysate using anti-WNGME antibody is shown. The quantification result from the immunoblot obtained in FIG. 7F is shown. Mean of 4 independent experiments ± S.E.M., * P <0.05, *** p <0.001 (Student t test). We show that overexpression of FUS / TLS reduces compound eye degeneration in Drosophila expressing (UGGAA) _exp (S). FUS / TLS reduces both RNA aggregation and PPR protein accumulation in the compound eye primordium of third-instar larvae. Results of quantitative real-time PCR analysis of transgene expression are shown (mean ± S.E.M, ns (no significant difference), one-way ANOVA, n = 80 / genotype). The quantitative results of RNA aggregates are shown (mean ± S.E.M, no significant difference, one-way ANOVA, n = 10 / genotype). The quantification result of PPR protein is shown (mean ± S.E.M, no significant difference, one-way ANOVA, n = 10 / genotype). Immunoblotting of PPR protein in lysate derived from compound eye primordia using anti-WNGME antibody and quantification results are shown (mean ± SEM of 4 independent experiments, * P <0.05, *** p <0.001 one-way ANOVA).

  Hereinafter, the present invention will be described in detail.

  Neurodegenerative diseases such as spinocerebellar ataxia type 31 (SCA31) may develop in early life, but mainly after middle age. The progression of symptoms is often slow, and it is not easy to reproduce this condition in animal models.

In recent years, Drosophila has been used as a human disease model because of its unexpectedly high genetic similarity with mammals including humans, while having a short life cycle. The present inventors made a SCA31 Drosophila model expressing a UGAA repeat sequence (UGGAA) _exp related to SCA31 for the purpose of elucidating the pathology of SCA31 and developing a therapeutic method. Drosophila strains can be obtained from the University of Indiana at the Vienna Drosophila RNAi Center (VDRC) and the Bloomington Drosophila Stock Center (BDSC). RNAi Drosophila can be obtained similarly.

Transformed Drosophila expressing (UGGAA) _exp or (UGGAA) ₂₂ and Drosophila overexpressing TDP-43 or FUS / TLS were prepared by a conventional method using a pUAST vector incorporating a gene expressing them. A transformed line was created using the y, w line (BestGeen Inc.). Drosophila co-expressing (UGGAA) _exp or (UGGAA) ₂₂ , and TDP-43 or FUS / TLS or a fragment thereof was obtained by crossing between a Drosophila expressing one and a Drosophila expressing the other. Experiments using Drosophila were performed at 25 ° C. The phenotype of Drosophila eyes 1-2 days after emergence was evaluated using an SZX10 stereo microscope (Olympus) and a scanning electron microscope (Miniscope TM1000, Hitachi).

On the other hand, the present inventors mixed a nuclear extract derived from PC12 cells or mouse brain with (UGGAA) _exp repeat RNA synthesized by in vitro transcription and control repeat RNA (each labeled with biotin), and (UGGAA ) Proteins that specifically bind to _exp are obtained by RNA pull-down, and the band confirmed to be bound by gel electrophoresis is cut out and subjected to mass spectrometry, and by mass spectrometry (LC-MS) combined with liquid chromatography Identified.

The proteins identified by screening of (UGGAA) _exp binding protein by RNA pull-down assay were several RNA binding proteins including TDP-43 and FUS / TLS (Example 3).

The SCA31 Drosophila model expressing the repeat sequence (UGGAA) _exp produced compound eye degeneration depending on the dose of the repeat sequence (Examples 1 and 2). This Drosophila was crossed with Drosophila expressing the identified RNA binding proteins TDP-43 or FUS / TLS, which reduced compound eye degeneration and confirmed the inhibitory effect of (UGGAA) _exp- induced toxicity. (Example 4). It was revealed that this inhibitory effect was not due to a decrease in RNA expression, but due to suppression of abnormal aggregation of RNA both in vitro and in vivo (Examples 5 and 6). ).

  TDP-43 is an abbreviation for transactivation responsive region (TAR) DNA-binding protein 43 (TAR DNA-binding protein 43, TDP-43). TDP-43 and FUS / TLS (fused in sarcoma / translated in liposarcoma) have been identified as causative genes for familial ALS, and are also considered causative protein molecules in sporadic ALS. It is surprising that these proteins can be used to treat SCA31 because it has been reported that various neurodegenerative diseases such as ALS occur due to abnormal expression of TDP-43 and FUS / TLS. Met.

  Therefore, one embodiment of the present invention is a therapeutic agent for spinocerebellar ataxia type 31 (SCA31) comprising an RNA binding protein selected from TDP-43 and FUS / TLS, or a fragment containing these RNA binding regions.

  Examples of TDP-43 that can be suitably used in the present invention include TDP-43 derived from mammals and RNA-binding fragments thereof. For example, UniProtKB contains information such as sequences of TDP-43 derived from various mammals, including mutants, and those skilled in the art can easily obtain TDP-43, its fragments, and mutants. Can also be synthesized. Examples of the amino acid sequence of TDP-43 include human TDP-43 (Accession No. Q13148, SEQ ID NO: 1), mouse TDP-43 (Accession No. Q921F2, SEQ ID NO: 2) and the like. Drosophila is also known to express a similar protein and is referred to herein as dTDP-43.

  Human TDP-43 is a protein consisting of 414 amino acids, and its RNA binding region is RRM1 corresponding to amino acids 105 to 169 and RRM2 corresponding to amino acids 193 to 257 (FIG. 4A). The N-terminus of TDP-43 contains a region involved in nuclear translocation signals and splicing, while the C-terminus contains a region involved in binding to other proteins.

In the present invention, the above full length TDP-43 can be used. Therefore, one preferred embodiment of the present invention is that TDP-43 is
(a) consisting of the amino acid sequence represented by SEQ ID NO: 1, or
(b) An amino acid sequence represented by SEQ ID NO: 1, consisting of an amino acid sequence in which one or several amino acids are deleted, substituted or added, and having binding activity to (UGGAA) _n (n = 80-100) It is a therapeutic agent for spinocerebellar ataxia type 31 (SCA31).

In this specification, “several” means 5 or less, preferably 3 or less, more preferably 2 or less. Therefore, for example, “an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 1” means that the amino acid sequence represented by SEQ ID NO: 1 is 5 or less, preferably It shall mean an amino acid sequence in which no more than 3, more preferably no more than 2 amino acids have been deleted, substituted or added. As described above, the presence of several mutants has been reported in the protein according to the present invention such as TDP-43, and thus the amino acid sequence of human TDP-43 is limited to only one type. It is not possible. What is important in the present invention is “has binding activity to (UGGAA) _n (n = 80-100) ((UGGAA) _exp )”, thereby (UGGAA) _n (n = 80-100) ((UGGAA ) _exp ) Inhibiting RNA aggregation.

As used herein, "binding activity to _{(UGGAA) n (n = 80-100} ) ", attached in vitro and / or in vivo and _{(UGGAA) n (n = 80-100} ) , the absence Below, it is intended to mean an activity capable of inhibiting the aggregation of (UGGAA) _n (n = 80-100) RNA having a tendency to aggregate. According to the findings of the present inventors, toxicity in Drosophila is not induced with a repeat length of about (UGGAA) ₂₂ . On the other hand, it has been reported that RNA with a repeat length of about 200 times exists in Purkinje cells of SCA31 patients, and the binding activity to (UGGAA) _n (n = 80-100) has therapeutic effects in vivo It is assumed that it is necessary and sufficient for this purpose.

As described above, the therapeutic agent of the present invention is capable of inhibiting (UGGAA) _n (n = 80-100) RNA aggregation in vitro and / or in vivo. The degree of inhibition is, for example, the amount of RNA aggregates in a tissue sample from a patient or a sample obtained from a laboratory animal such as Drosophila, or an in vitro sample, or the RNA aggregation region is 50% or more compared to the control, 60 %, 70%, 80%, or 90% or more. Alternatively, the therapeutic agent of the present invention, when described from another aspect, produces a protein encoded by (UGGAA) _n (n = 80-100) by 50% or more, 60% or more, 70% compared to the control. % Or more, 80% or more, or 90% or more.

  According to the knowledge of the present inventors, it became clear that the RNA binding region is essential for the purpose of the present invention, and its N-terminal and C-terminal are not necessary for the function of the present invention. (Example 4). The C-terminus of TDP-43 is believed to have a prion domain that is itself associated with toxicity, and making it a TDP-43 fragment that does not contain such a domain reduces the potential risk of developing ALS. It can be avoided and a safer and more effective therapeutic effect can be obtained. Furthermore, smaller fragments than full-length proteins can be more effective when prepared as pharmaceuticals and when administered and delivered to a target site compared to larger protein molecules.

Therefore, the fragment that can be used in the present invention may be a fragment containing the RNA binding region (RRM) of TDP-43 protein. Therefore, in another preferred embodiment of the present invention, the fragment containing the RNA binding region of TDP-43 is an RRM1 region corresponding to amino acids 105 to 169 of SEQ ID NO: 1 and / or amino acids 193 to 257. It is a therapeutic agent for spinocerebellar ataxia type 31 (SCA31), which essentially contains the RRM2 region corresponding to. The effect of the present invention can be obtained when the binding activity to (UGGAA) _n (n = 80-100) ((UGGAA) _exp ) is lost as a result of mutation such as amino acid substitution in the RNA binding region. Can not. Therefore, it is preferable that no amino acid deletion, substitution or addition exists in the RNA binding region of TDP-43. On the other hand, the region other than the RNA binding region can be examined in consideration of various factors including the effect as a therapeutic agent and the stability when considering the preparation and administration.

  FUS / TLS (fused in sarcoma / translated in liposarcoma, also known as hnRNP P2) is not only a disease-related RBP, but also has a standard RNA recognition motif (RRM) and a putative prion domain Shares functional and structural similarities with TDP-43 and functions in transcriptional regulation and RNA processing in neurons. The present inventors have found that FUS / TLS also has the same aggregation inhibitory effect on SCA31-derived RNA repeats as TDP-43 (Example 8).

  Examples of FUS / TLS that can be suitably used in the present invention include FUS / TLS derived from mammals and RNA-binding fragments thereof. For example, information such as FUS / TLS sequences derived from various mammals is registered in UniProtKB, including mutants. Examples of amino acid sequences include human FUS / TLS (Accession No. P35637, SEQ ID NO: 3). Mouse FUS / TLS (Accession No. P56959, SEQ ID NO: 4) and the like. Human FUS / TLS is a protein consisting of 526 amino acids, and its RNA binding region corresponds to amino acids at positions 285 to 371.

Therefore, a preferred aspect of the present invention is that FUS / TLS is
(a) consisting of the amino acid sequence represented by SEQ ID NO: 3, or
(b) The amino acid sequence shown in SEQ ID NO: 3 consisting of an amino acid sequence in which one or several amino acids are deleted, substituted or added, and having binding activity to (UGGAA) _n (n = 80-100) It is a therapeutic agent for spinocerebellar ataxia type 31 (SCA31).

Another preferred embodiment of the present invention provides treatment for spinocerebellar ataxia type 31 (SCA31), wherein the fragment containing the RNA binding region of FUS / TLS essentially comprises amino acids at positions 285 to 371 of SEQ ID NO: 3. It is an agent. The effect of the present invention can be obtained when the binding activity to (UGGAA) _n (n = 80-100) ((UGGAA) _exp ) is lost as a result of mutation such as amino acid substitution in the RNA binding region. Can not. Therefore, it is preferable that no amino acid deletion, substitution or addition exists in the RNA binding region of FUS / TLS. On the other hand, the region other than the RNA binding region can be examined in consideration of various factors including the effect as a therapeutic agent and the stability when considering the preparation and administration.

It has been shown that disease-causing microsatellite repeats can be abnormally translated into proteins without an ATG-initiated open reading frame (Zu, T. et al., Proc. Natl. Acad. Sci. USA, 108, 260-265, 2011). Non-ATG (RAN) translated proteins associated with this repeat have been reported in a number of diseases including SCA8, DM1, FXTAS, and C9ORF72 ALS / FTD (Cleary, JD and Ranum, LP, Current Opinion in Genetics & Development 26C, 6-15, 2014). Unlike other non-coding repeat elongation disorders, the ATG-start codon is included in the TGGAA repeat sequence in SCA31. However, the present inventors have hypothesized that intron TGGAA repeats can be abnormally translated into pentapeptide repeat (PPR) proteins by the RAN translation mechanism, and have studied to verify this hypothesis. As a result, it was confirmed that the PPR protein encoded by the TGGAA repeat was present in the brains of SCA31 patients, and that this PPR protein was also synthesized in Drosophila expressing (UGGAA) _exp . Then, it was confirmed that a protein such as TDP-43 according to the present invention can also act suppressively on the protein production encoded by the (UGGAA) _exp repeat (Example 7).

The effects of TDP-43 and FUS / TLS on RNA aggregation were determined by C9ORF72 ALS / FTD (Donnelly, CJ et al., Neuron, 80, 415-428, 2013) or DM1 / DM2 (Querido, E. et al., Journal of Cell Science, 124, 1703-1714, 2011), which is different from other RBPs such as ADARB2 or MBNL 1 reported to promote RNA aggregation. The protective role that TDP-43 and FUS / TLS suppress its target RNA aggregation is similar to the role of molecular chaperones that suppress protein aggregation in neurodegenerative diseases. However, molecular chaperones such as HSP70 or HSP40 reduce the phenotype of Drosophila expressing CGG repeats associated with FXTAS (Jin, P. et al., Neuron, 39, 739-747, 2003), but the present invention They have confirmed that these molecular chaperones cannot reduce the degenerative phenotype of Drosophila expressing (UGGAA) _exp (data not shown).

The TDP-43 and FUS / TLS proteins and fragments thereof that can be used as the therapeutic agent of the present invention may be in the form of a pharmaceutically acceptable salt for the purpose of improving the stability as a preparation. In addition, as long as it does not impair the effect as a therapeutic agent, specifically (UGGAA) _exp binding activity, and does not cause harmful effects such as toxicity to patients, derivatives that are usually acceptable in the art You can also TDP-43 and FUS / TLS proteins and fragments thereof, including these salts and derivatives, may be used alone or in combination.

  In the present invention, the above-mentioned polynucleotides encoding TDP-43 and FUS / TLS can also be administered as a nucleic acid drug. The polynucleotide may be DNA or RNA, and the design of an expression system such as a vector suitable for human expression, and the delivery system for effective delivery to the affected area, specifically the cerebellum, is based on the common general technical knowledge in the field. Can be appropriately performed.

  Accordingly, another embodiment of the present invention is a therapeutic agent for spinocerebellar ataxia type 31 (SCA31) comprising a nucleotide sequence encoding TDP-43 or FUS / TLS, or a fragment containing these RNA binding regions.

  The therapeutic agent of the present invention can be used alone or in combination with a commonly used pharmaceutical carrier as a pharmaceutical composition, such as intrathecal, intravenous, intramuscular, subcutaneous and intraperitoneal injection or non-enteral route. It can be administered by the oral route as well as the oral route, the parenteral route is preferred, and intrathecal injection is more preferred. Examples of the dosage form include injections, tablets, capsules, vulcanizing agents, syrups, emulsions, sprays and the like. 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 therapeutic agent of the present invention, which is an active ingredient, 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, 0.0001-1 mg is divided into once to several times a day for parenteral administration. Can be administered.

  The present invention will be specifically described by the following examples, but the present invention is not limited to these examples.

[Example 1]
Demonstration of RNA aggregation and toxicity induction by expression of (UGGAA) _exp in Drosophila
To characterize the toxicity of (UGGAA) _exp that may be involved in Purkinje cell degeneration in SCA31 in vivo, we used the UAS / GAL4 system to identify disease-specific (UGGAA) _exp Expression constructs were made to create transgenic or Drosophila lines that express repeats or control repeats consisting of the UAGAA and UAAAAUAGAA (SEQ ID NO: 5) complex found in the normal population (Figure 1-1).

SCA31 or control repeat insert DNA was amplified by PCR from genomic DNA. For complete repeat amplification, primers were designed to amplify the transcript region of the pUAST vector (forward primer: 5'-TCTACGGAGCGACAATTCAA-3 '(SEQ ID NO: 6), reverse primer: 5'-TGCTCCCATTCATGAG- 3 '(SEQ ID NO: 7)). (UGGAA) For PCR amplification of the _exp region (FIG. 1-1 Part 1), forward primer: 5′-AGGGAATTGGGAATTCGTTAACA-3 ′ (SEQ ID NO: 8) and reverse primer: 5′-TCCATTCCATTCTATTCTATTCCCAT-3 ′ (SEQ ID NO: 8) 9) was used. The approximate size of the repeat amplified by PCR was confirmed by gel electrophoresis. To identify the exact repeat length, PCR products were separated by electrophoresis on a 1% agarose gel, cloned into the PCRII TA vector (Invitrogen), and the repeat length was determined by DNA sequencing. The PCR product obtained contained _exp (UGGAA) containing approximately 80-100 TGGAA repeats and 22 (UGGAA) ₂₂ containing ₂₂ TGGAA.

  The PCR product was digested with HaeIII, purified and cloned into the pBluescript KS (+) vector (Stratagene). The Drosophila expression construct was made by inserting a DNA fragment in pBluescript KS (+) between the NotI and XhoI cloning sites of the Drosophila transformation vector pUAST. These constructs contain a TAG- or TAA-translation stop codon immediately upstream of the repeat sequence. The transformed Drosophila was prepared by a standard method using the y, w strain (Bestgene Inc.).

  Unless otherwise stated, all experiments were performed at 25 ° C. The phenotype of Drosophila eyes 1-2 days after the emergence was evaluated with an SZX10 stereo microscope (Olympus) and a scanning electron microscope (Miniscope TM1000, Hitachi).

For the transgene in the Drosophila genome, confirmed by TGGAA-specific PCR, several Drosophila strains with approximately 80-100 TGGAA repeats ((UGGAA) _exp strong expression system (S), weak expression system ( W), and the most strongly expressed system (L)) and 22 (UGGAA) ₂₂ systems with ₂₂ TGGAA repeats were established. To evaluate the effect of (UGGAA) _exp length, (UGGAA) _exp , (UGGAA) ₂₂ or control repeats were expressed in Drosophila compound eyes.

  For quantitative real-time PCR, total RNA of 20 third-instar larvae was extracted using RNeasy kit (Quiagen), and cDNA was obtained using PrimeScript ™ RT Master Mix (Takara Bio). Prior to PCR, the cDNA was treated with DNase I (Invitrogen) to remove genomic DNA. Real-time PCR was performed three times using LightCycler 480 (Roche), and data was performed using the ΔΔCt method or the standard curve method (Rp49 level). Primers used for insertion of SCA31 repeats or control repeats are as follows. Forward primer: 5′-AGGGAATTGGGAATTCGTTAACA-3 ′ (SEQ ID NO: 8), reverse primer: 5′-CTGGAGTGCAGTGACGTGATCT-3 ′ (SEQ ID NO: 10).

As a result, the expression of (UGGAA) ₂₂ and the control repeat did not significantly affect the eye morphology (Figures 1-2A and B), but the (UGGAA) _exp strong expression (S) system had an eye size of It became smaller and caused collapse of the eye morphology with individual abnormalities, resulting in a slightly disturbed phenotype in the weak expression (W) system (FIGS. 1-2C and D). (UGGAA) In the system in which _exp was most strongly expressed, lethality was induced at the early developmental stage (L system) (FIGS. 1-3A). RT-PCR using primers that amplify the entire transcript showed that the transgene was transcribed in all systems. Homozygous transgenic (UGGAA) _exp Drosophila showed more severe compound eye degeneration compared to heterozygous transgenic (UGGAA) _exp Drosophila (Figure 1-2I). Therefore, (UGGAA) _exp is shown to act in a dose-dependent manner.

To investigate how (UGGAA) _exp exhibits neurotoxicity in the eye, we followed early events in the development of the retina that expressed (UGGAA) _exp . Since photoreceptor differentiation occurs in the compound eye primordium of third-instar larvae, the transgene was induced only to the differentiating photoreceptors by the GMR-GAL4 system.

(UGGAA) To determine whether _exp- derived RNA aggregates are present in the Drosophila compound eye primordium, we used a DIG-conjugated locked nucleic acid (LNA) probe to produce a yellow-white RNA fluorescence in situ hybridization (RNA FISH) was performed on weak and strong expression systems of (yw), (UGGAA) ₂₂ , and (UGGAA) _exp (FIG. 1-2 EH). RNA FISH to detect sense strand aggregation (red) was combined with nuclear DNA staining with DAPI (blue). As a result, RNA aggregation was not detected in the control repeat or (UGGAA) ₂₂ expressing system (E and F), but in the (UGGAA) _exp (W) and (S) compound eye discs (UGGAA) _exp RNA aggregation (arrowheads) was observed, similar to the pathology of human SCA31 (FIGS. 1-2G and H). RNA aggregation appears to be present in both the nucleus (arrow) and the cytoplasm, but the tissue degeneration is so severe that RNA aggregation observed to be present in the cytoplasm is also present. It may have existed in the nucleus originally. Importantly, the amount of RNA aggregation correlated with the expression level of (UGGAA) _exp and the severity of compound eye degeneration (FIGS. 1-3). In addition, in the (S) system, which is a strong expression system of (UGGAA) _exp , it was shown that the amount of RNA aggregates in the compound eye primordia increased more than the difference in the expression level (Fig. 1-3B). . Aggregation decreases when pretreated with RNase and is apparently due to RNA (FIG. 1-2J).

The above results demonstrated that (UGGAA) _exp exerts toxicity through the formation of abnormally aggregated structures, depending on dose and length.

[Example 2]
Demonstration of Toxicity Induction by Expression of (UGGAA) _{exp in} Adult Drosophila Next, we will examine the elav-GAL4 system to investigate the effect of (UGGAA) _exp expression in the Drosophila central nervous system (CNS) Was used. Since the persistent expression of (UGGAA) _{exp in} the CNS using elav-GAL4 killed the pupa, in this example we used an inducible Gene-Switch expression system, and in the CNS of Drosophila after emergence (UGGAA) _exp Expression was induced.

  Life analysis and climbing tests were performed with some modifications to the methods described in Feany, M.B. and Bender, W.W., Nature, 404, 394-398, 2000. Specifically, 20 Drosophila were placed in a plastic vial, and the state of climbing was observed after the vial was tapped and dropped on the bottom of the vial. When 15 seconds had elapsed, Drosophila that reached the top of the vial (> 10 cm) were counted. More than 20 tests were performed at each time point, and more than 100 Drosophila were tested for each group.

Analysis of neurotoxicity of (UGGAA) _exp by life analysis using elav-GeneSwitch shows that (UGGAA) _exp (S) Drosophila has a shorter life span than Drosophila expressing EGFP, control repeats, and (UGGAA) ₂₂ The controls survived for 80-90 days, whereas the average survival of (UGGAA) _exp (S) Drosophila was 34 days (p <0.001, log-rank test, n = 100-120 animals / group) (FIG. 2A).

Furthermore, in the climbing test, Drosophila expressing (UGGAA) _exp (S) showed more progressive motor dysfunction, and only 34.4 ± 11.7% of Drosophila could climb on day 28. In contrast, 80.2 ± 6.9% of Drosophila expressing EGFP, 79.1 ± 8,1% of Drosophila expressing control repeats, and 72.1 ± 9.2% of Drosophila expressing (UGGAA) ₂₂ We were able to climb to the top (mean ± sd, n = 100-120, *** p <0.001, two-way ANOVA) (FIG. 2B).

These data indicate that Drosophila expressing (UGGAA) _exp reproduce the neurological and pathological properties of human SCA31.

[Example 3]
Interaction between TDP-43 and UGGAA RNA in vitro and in vivo
Potential (UGGAA) _exp binding partners were screened by in vitro RNA pull-down assay. Specifically, biotinylated RNA containing (UGGAA) _exp or control repeats was synthesized by in vitro transcription using T7 RNA polymerase (Roche), and a nuclear fraction of PC12 cells or mouse brain (NE-PER extraction reagent ( The cells were lysed and fractionated using Pierce) and incubated. It was then pulled down using streptavidin-coated magnetic beads (Dynabeads M-280 Streptavidin (Invitrogen)) and the captured protein- (UGGAA) _exp complexes were separated by SDS-PAGE and stained with Coomassie blue. The band of interest was excised from the gel and the protein was identified by LC-MS analysis.

Preparation of human brain tissue (patient 1: 80-year-old male, patient 2: 74-year-old male) and detection of repeat transcripts in Purkinje cells by FISH were performed using a digoxigenin (DIG) -conjugated locked nucleic acid (LNA) probe ( 5 nM, Exiqon), (TTCCA) ₅ or (TTTTATTCTA) _2.5 (SEQ ID NO: 11). Operations such as fixation of a human brain section on a slide and washing were performed by general techniques. For the staining, an anti-TBP-43 antibody (1/100 dilution, Abcam AB41972) was used as the primary antibody, and a goat anti-rabbit antibody (1/500, Fisher) conjugated to Alexa-Fluor 488 was used as the secondary antibody.

  As a result, several RNA-binding proteins and nuclear paraspeckle components that function in RNA transport, splicing, and editing have been identified. The inventors focused on TDP-43, one of the most abundant proteins among the identified proteins, and analyzed in detail the role of SCA31 in the pathology.

The results of Western blot analysis confirmed that TDP-43 interacts specifically with (UGGAA) _exp in vitro (FIG. 3A). Furthermore, RNA FISH and RBP immunofluorescence analysis in human SCA31 Purkinje cells showed that TDP-43 co-localizes with (UGGAA) _exp RNA lesions, and TDP-43 is also (UGGAA) _exp in vivo. (Fig. 3B). These results strongly suggested that TDP-43 plays a role in the development of SCA31.

[Example 4]
Inhibition of (UGGAA) _exp- induced toxicity by TDP-43
To investigate the role of TDP-43 in (UGGAA) _exp- induced toxicity, we crossed (UGGAA) _exp- expressing Drosophila with human wild-type TDP-43-expressing Drosophila. A schematic of the construct used for the expression of TDP-43 is shown in FIG. 4A.

  Transgenic Drosophila expressing human TDP-43 were generated using standard techniques using the pUAST vector. The pUAST-TDP-43ΔN and pUAST-TDP-43ΔC constructs were made by deleting the regions encoding amino acids 1-89 and 276-414, respectively. The TDP-43 RRM mutant construct was generated by mutating phenylalanine residues 147, 149, 194, 229 and 231 to leucine using the QuikChange Multi Site-Directed Mutagenesis kit (Stratagene).

As a result, in line with the results of previous studies suggesting TDP-43 toxicity in the Drosophila model of ALS / FTD, the expression of human wild-type TDP-43 (TDP-43 WT) resulted in slight ocular degeneration. (FIG. 4B). However, in Drosophila expressing (UGGAA) _exp , co-expression of TDP-43 WT dramatically suppressed compound eye degeneration (FIG. 4C). On the other hand, RNA interference (RNAi) -mediated knockdown of endogenous Drosophila TDP-43 (dTDP-43) (Lin, MJ et al., PloS one 6, e20371, 2011) is (UGGAA) _exp (W) The system significantly increased compound eye degeneration, and the (UGGAA) _exp (S) system induced a lethal phenotype (FIG. 4C). These data indicate that TDP-43 plays an important role in the toxicity of (UGGAA) _exp in vivo.

(UGGAA) To determine which part of TDP-43 is required to suppress _exp- induced toxicity, we lack the N-terminal (ΔN) or C-terminal (ΔC) region, Inability to interact with other proteins for splicing control and microRNA (miRNA) biosynthesis / silencing (Buratti, E. et al., The Journal of Biological Chemistry 280, 37572-37584, 2005; Ling, SC et al., Neuron 79, 416-438, 2013), or a TDP-43 variant with 5 amino acid substitutions in the RNA recognition motif (TDP-43 RRM variant) and reduced ability to bind to its RNA target. (Buratti, E. and Baralle, FE, The Journal of Biological Chemistry 276, 36337-36343, 2001; Elden, AC et al., Nature 466, 1069-1075, 2010) were produced. 4A).

Since some Drosophila expressing only the TDP-43 fragment lacking the N-terminal (ΔN) and C-terminal (ΔC) also showed mild compound eye degeneration (FIG. 4B), the present inventors A line with a relatively weak phenotype was selected and crossed with (UGGAA) _exp expressing Drosophila.

As a result, surprisingly, co-expression of either of the two TDP-43 deletion mutants significantly reduced the (UGGAA) _exp- expressing Drosophila phenotype, but mutated the RRM region. The modified TDP-43 RRM mutant could not reduce the denaturation (FIG. 4C). These results indicated that direct interaction between TDP-43 and (UGGAA) _exp may have a role in reducing (UGGAA) _exp- induced toxicity.

[Example 5]
(UGGAA) Function of TDP-43 as an ATP-independent RNA chaperone for _exp RNA folding
In order to analyze the mechanism by which TDP-43 suppresses (UGGAA) _exp- induced toxicity through its RNA binding ability, we have (UGGAA) in Drosophila co-expressing (UGGAA) _exp and TDP-43. The expression level of _exp transcript and RNA aggregation were examined.

As a result, co-expression of TDP-43 WT did not significantly affect the expression level of (UGGAA) _exp transcript (FIG. 5A), but RNA aggregation was caused by (UGGAA) _exp -TDP-43 interaction. It was greatly suppressed (FIGS. 5B-5D). In the (UGGAA) _exp (W) system, the amount of RNA aggregates decreased from 2.08% to 0.37% per area of the compound eye primordium by co-expression of TDP-43, indicating about 82% aggregation inhibitory activity. In addition, in the (UGGAA) _exp (S) system, the amount of RNA aggregates decreased from 12.9% to 4.3%, indicating that it has about 67% aggregation inhibitory activity (FIG. 5D).

On the other hand, co-expression of the TDP-43 RRM mutant failed to suppress RNA aggregation in Drosophila expressing (UGGAA) _exp (FIGS. 5B-5D). In addition, fluorescent immunostaining of RNA FISH and RBP in the (UGGAA) _exp (W) compound eye disc revealed that TDP-43 binds to (UGGAA) _exp RNA transcripts in the nucleus. (FIG. 5E). Furthermore, consistent with compound eye morphology deterioration, knockdown of dTDP-43 dramatically increased RNA aggregation (FIGS. 5C-5D). These results indicate that TDP-43 does not promote degradation of (UGGAA) _exp transcripts, but eliminates RNA aggregation through RNA-TDP-43 interactions in vivo, thereby toxicating RNA. Was shown to neutralize.

[Example 6]
from the data of the interaction Example 5 of the TDP-43 in in vitro and (UGGAA) _exp, formation of non-functional conformation in in vivo of TDP-43 is (UGGAA) _exp interact directly with abnormal RNA It was suggested that this could be suppressed. The inventors then tested whether TDP-43 directly inhibits (UGGAA) _exp aggregation in vitro using an atomic force microscope (High-speed AFM, NanoExplorer, RIBM).

Immediately after heat denaturation in solution, most of the (UGGAA) _exp RNA transcribed in vitro using T7 RNA polymerase is large RNA aggregates (height> 1.5 nm, width> 40 nm, asterisks, 3.2 ± 1.9%) than as single-stranded RNA (ssRNA, height <0.9nm, arrow, 19.5 ± 2.6%) or small aggregates (height> 1.5nm, width> 15nm, <40nm, arrowhead, 77.3 ± 3.4%), but after incubation for 10 minutes at room temperature in RNA-only conditions, the proportion of large aggregates increased significantly (38.9 ± 9.5%, ** p <0.01). However, incubation with purified recombinant TDP-43ΔC (Wang, IF et al., Nature Communications 3, 766, 2012), which lost its own tendency to aggregate, resulted in (UGGAA) _exp RNA being maintained as a single strand. (20.6 ± 5.0%, ** p <0.01), the formation of large RNA aggregates (6.1 ± 3.1%, ** p <0.01) was suppressed as compared with the RNA-only conditions (FIGS. 6A-6B).

Combined with the results of Example 5, it was shown that TDP-43 can suppress the aggregation of the target RNA (UGGAA) _exp by directly binding, and as a result, the toxicity of (UGGAA) _exp can be eliminated. . This process does not require adenosine triphosphate (ATP) or other proteins, unlike DEAD-box helicase DDX6, which unwinds CUG repeat duplex in an ATP-dependent manner, and does not require TDP-43 in RNA quality control. It reveals new functions.

[Example 7]
Based on the hypothesis that the intron TGGAA repeat suppressed by TDP-43 of the repeat-encoded pentapeptide repeat protein production can be abnormally translated into a pentapeptide repeat (PPR) protein by the RAN translation mechanism, as shown in FIG. In all frames, translation of the TGGAA repeat yields one PPR protein, poly- (Trp-Asn-Gly-Met-Glu) (SEQ ID NO: 12). The present inventors prepared polyclonal antibodies (anti-WNGME SGJ1705 and SGJ1706) against the [H] CMEWNGMEWMGMEWNG [OH] (SEQ ID NO: 13) peptide by a conventional method. The specificity of affinity-purified PPR antibody was confirmed by processing cerebellar tissue sections containing recombinant pentapeptide. The amount of PPR protein encapsulated was reduced, and the affinity-purified PPR antibody was produced in bacteria as a GST fusion. Replacement poly- (WNGME) peptide was detected.

  An immunohistochemical analysis was then performed on cerebellar tissue sections to assess whether PPR protein was present in the brains of SCA31 patients. In tissue sections of SCA31 patients, poly-WNGME-specific antibodies detected Purkinje cell bodies and punctate granules in the dendrites (black arrows) that were not seen in controls, and PPR protein was detected in SCA31 patients. It was suggested to exist in the brain (FIG. 7B).

Next, it was examined whether poly-WNGME peptide is also expressed in Drosophila expressing (UGGAA) _exp . Immunostaining with anti-WNGME-antibody showed no evidence of PPR protein production in control repeats or Drosophila lines expressing (UGGAA) ₂₂ , but (UGGAA) _exp (W) and (S) lines It was revealed that PPR protein was synthesized in the compound eye primordium of both third-instar larvae (FIGS. 7C and 7D, arrows).

(UGGAA) Given the remarkable effect of TDP-43 as an RNA chaperone on the suppression of _exp- induced toxicity, the present inventors examined whether TDP-43 also affects PPR protein production in Drosophila. As a result, up-regulation of TDP-43 significantly reduced PPR protein production and RNA aggregation in both (UGGAA) _exp (W) and (S) systems (** P <0.01, PPR protein levels are compound eyes) Decreased from 2.5 ± 0.38 and 4.1 ± 0.48% per primordial to 0.1 ± 0.02 and 0.9 ± 0.23%, respectively) (FIGS. 7D and 7E). However, RNA accumulation and co-expression of TDP-43 RRM mutants that cannot suppress (UGGAA) _exp- induced toxicity also suppressed PPR protein synthesis in (UGGAA) _exp- expressing Drosophila (PPR protein levels per compound eye primordium) Reduced to 0.9 ± 0.15 and 1.0 ± 0.38%, respectively (* p <0.05, ** p <0.01). Furthermore, silencing of dTDP-43 did not statistically significantly increase PPR protein synthesis in the (UGGAA) _exp (W) system (3.3 ± 0.68% per compound eye primordium).

These data suggest that the amount of PPR protein does not necessarily correlate with the degree and amount of RNA aggregation in Drosophila expressing (UGGAA) _exp , suggesting that PPR protein is not translated from RNA in this Drosophila model It was done.

On the other hand, Western blot analysis of Drosophila lysates expressing (UGGAA) _exp reveals molecular species with immunoreactivity with WNGME, and TDP-43 is involved in the inhibition of PPR production in vivo (Relative PPR protein levels decreased from 1.0 and 6.7 ± 1.36% per compound eye disc to 0.1 ± 0.05 and 1.3 ± 0.33%, respectively. * P <0.05, *** p <0.001) (FIG. 7F and 7G).

[Example 8]
Inhibitory effect of (UGGAA) _exp- induced toxicity by FUS / TLS The effect of FUS / TLS on (UGGAA) _exp toxicity in Drosophila was evaluated. Up-regulation of human FUS / TLS dramatically reduced compound eye degeneration in the (UGGAA) _exp (S) system, similar to TDP-43 (FIG. 8A). Furthermore, FUS / TLS, like TDP-43, suppressed RNA aggregation and production of PPR protein encoding repeat RNA in third-instar larvae expressing (UGGAA) _exp . Co-expression of FUS / TLS with (UGGAA) _exp did not affect the expression level of (UGGAA) _exp transcripts (Figure 8B), but their expression was RNA aggregation (** p <0.01, compound eye Statistically reduced from 12.4 ± 0.76 to 4.1 ± 0.26% per group, about 67% aggregation inhibitory activity) and PPR protein (** p <0.001, reduced from 4.1 ± 0.48 to 1.3 ± 0.16% per compound eye primordium) There was a significant decrease (FIGS. 8C and 8D). Immunoblot analysis also revealed that FUS / TLS inhibited PPR protein synthesis (*** p <0.001, relative PPR protein levels from 1.0 to 0.5 ± 0.05% per compound eye primordium) (Reduction) (FIG. 8E).

  According to the present invention, it is possible to provide a new therapeutic agent based on the onset mechanism of spinocerebellar ataxia type 31 (SCA31) for which there has been no effective treatment.

Claims (5)

  Spinocerebellar ataxia containing RNA binding protein selected from TDP-43 (transactivation responsive region DNA-binding protein 43) or FUS / TLS (fused in sarcoma / translated in liposarcoma), or a fragment containing these RNA binding regions Type 31 (SCA31) treatment. TDP-43
(a) consisting of the amino acid sequence represented by SEQ ID NO: 1, or
(b) An amino acid sequence represented by SEQ ID NO: 1, consisting of an amino acid sequence in which one or several amino acids are deleted, substituted or added, and having binding activity to (UGGAA) _n (n = 80-100) The therapeutic agent according to claim 1, wherein FUS / TLS
(a) consisting of the amino acid sequence represented by SEQ ID NO: 3, or
(b) The amino acid sequence shown in SEQ ID NO: 3 consisting of an amino acid sequence in which one or several amino acids are deleted, substituted or added, and having binding activity to (UGGAA) _n (n = 80-100) The therapeutic agent according to claim 1, wherein   The fragment containing the RNA binding region of TDP-43 essentially comprises an RRM1 region corresponding to amino acids 105 to 169 of SEQ ID NO: 1 and / or an RRM2 region corresponding to amino acids 193 to 257. The therapeutic agent described.   The therapeutic agent according to claim 1, wherein the fragment containing the RNA binding region of FUS / TLS essentially comprises amino acids at positions 285 to 371 of SEQ ID NO: 3.

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