Antisense nucleic acids and their uses

Abstract

This antisense nucleic acid is for a target sequence at positions 96-330 or positions 400-530 in the base sequence represented by SEQ ID NO: 1, has a base sequence complementary to a sequence formed of 10 or more bases appearing consecutively in the target sequence, and targets intron-6 of TDP-43 mRNA.

Description

[Technical Field]

[0001] The present invention relates to an antisense nucleic acid and its use. Specifically, the present invention relates to an antisense nucleic acid, an enhancer of alternative splicing of intron 6 of TDP-43 mRNA, a pharmaceutical composition, a method for screening an enhancer of alternative splicing of intron 6 of TDP-43 mRNA, and a method for screening a candidate compound for preventing or treating TDP-43 proteinopathy. This application claims priority based on Japanese Patent Application No. 2020-198595, filed on November 30, 2020, the contents of which are incorporated herein by reference. [Background technology]

[0002] TDP-43 proteinopathy is a collective term for neurodegenerative diseases characterized by the accumulation of TDP-43 protein through aggregation. These disorders include frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS). There is no cure for FTLD, and treatment is primarily symptomatic for various complications. ALS is treated with edaravone, which scavenges free radicals, and riluzole, which reduces glutamate excitotoxicity. However, neither treatment is significantly effective, and the disease often leads to respiratory muscle paralysis and death within two to five years of onset. Phase III clinical trials of antisense oligonucleotides targeting SOD1 mRNA degradation are currently underway for ALS with SOD1 gene mutations. Molecular-specific therapies are being developed for autosomal dominant ALS. However, the causes of sporadic ALS and FTLD, which account for more than 90% of cases, remain unknown. While therapeutics targeting specific pathogenic molecules are needed, developing such therapeutics has proven extremely difficult.

[0003] TDP-43 protein is an RNA-binding protein that is primarily localized in the nucleus and is involved in various RNA metabolisms, including post-transcriptional regulation. TDP-43 proteinopathies are characterized by the loss of TDP-43 protein from the nucleus and its accumulation in the cytoplasm, which contributes to the pathology through both the loss of its nuclear function and the acquisition of cytoplasmic toxicity. For this reason, it is generally recognized that simply reducing or enhancing TDP-43 protein expression is not suitable for treatment.

[0004] The intrinsically disordered region (IDR) in the TDP-43 protein is the most important region determining the aggregation properties of the TDP-43 protein, and its coding region is located in the alternatively spliced ​​intron 6. Furthermore, the TDP-43 protein binds to the 3'UTR of its own pre-mRNA and induces alternative splicing of introns 6 and 7, thereby inducing nonsense-mediated mRNA decay (NMD) and autoregulating expression. The inventors have demonstrated that in motor neurons of ALS patients, where nuclear TDP-43 protein is reduced, the above-mentioned autoregulatory function of expression does not function, and TDP-43 mRNA retaining intron 6 increases (see, for example, Non-Patent Document 1). The inventors have also discovered an antisense oligonucleotide that specifically inhibits alternative splicing of intron 6, and have demonstrated that using this antisense oligonucleotide increases mRNA containing intron 6, resulting in insolubilization and fragmentation of TDP-43 protein in the mouse spinal cord and loss of motor neurons (see, for example, Non-Patent Document 2). Furthermore, in neurons derived from human iPS cells, inhibition of alternative splicing of intron 6 has been shown to reduce nuclear TDP-43 protein (see, for example, Non-Patent Document 2). These results, which demonstrate pathologies similar to ALS, suggest that attenuation of alternative splicing of intron 6 increases TDP-43 protein expression despite its accumulation in the cytoplasm, exacerbating TDP-43 proteinopathy, such as ALS pathology.

[0005] Meanwhile, many studies have investigated methods for regulating the expression of TDP-43 mRNA by RNA interference. For example, Patent Document 1 discloses antisense oligonucleotides for degrading and knocking down TDP-43 mRNA, and antisense oligonucleotides for targeting the binding region of the pre-mRNA of the TDP-43 protein itself to upregulate expression.

[0006] However, drugs that degrade TDP-43 mRNA cause loss of nuclear TDP-43 protein function, disrupting cellular function in both healthy and diseased cells. On the other hand, drugs that overexpress TDP-43 protein promote cytoplasmic aggregation and are cytotoxic. Therefore, drugs targeting the TDP-43 gene are concerned about adverse effects on both diseased and healthy cells, and these drugs are currently used in animal experiments as models to reproduce disease states. Therefore, there is a need for drugs that can modify their effects depending on the cellular state, improving the reduction of nuclear TDP-43 protein in diseased cells while alleviating cytoplasmic aggregation, while avoiding toxicity in healthy cells. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] International Publication No. 2019 / 013141 [Non-patent literature]

[0008] [Non-Patent Document 1] Koyama, A. et al., “Increased cytoplasmic TARDBP mRNA in affected spinal motor neurons in ALS caused by abnormal autoregulation of TDP-43.”, Nucleic Acids Res., Vol. 44, pp. 5820-5836, 2016. [Non-patent document 2] Sugai, A. et al., “Non-genetically modified models exhibit TARDBP mRNA increase due to perturbed TDP-43 autoregulation.” Neurobiol. Dis., Vol. 130, 104534, 2019. Summary of the Invention [Problem to be solved by the invention]

[0009] The present invention has been made in view of the above circumstances and provides a novel antisense nucleic acid that enhances alternative splicing of intron 6 of TDP-43 mRNA. The present invention also provides an enhancer of alternative splicing of intron 6 of TDP-43 mRNA and a pharmaceutical composition using the antisense nucleic acid. The present invention also provides a method for screening an enhancer of alternative splicing of intron 6 of TDP-43 mRNA and a method for screening a candidate compound for preventing or treating TDP-43 proteinopathy. [Means for solving the problem]

[0010] As a result of extensive research to achieve the above-mentioned object, the inventors focused on TDP-43 mRNA isoforms and discovered that the pathology of TDP-43 proteinopathy can be suppressed by adjusting the expression ratio of these isoforms using antisense nucleic acids that specifically enhance alternative splicing of intron 6, thereby completing the present invention.

[0011] That is, the present invention includes the following aspects. (1) The target sequence is from positions 96 to 330 or from positions 400 to 530 of the base sequence represented by SEQ ID NO: 1, An antisense nucleic acid that targets intron 6 of TDP-43 mRNA, comprising a base sequence complementary to a sequence consisting of 10 or more consecutive bases in the target sequence. (2) The antisense nucleic acid according to (1), which consists of a base sequence represented by any one of SEQ ID NOs: 2 to 7. (3) A selective splicing enhancer for intron 6 of TDP-43 mRNA, comprising the antisense nucleic acid according to (1) or (2) as an active ingredient. (4) A pharmaceutical composition containing the antisense nucleic acid according to (1) or (2) as an active ingredient, which is used for preventing or treating TDP-43 proteinopathy. (5) The pharmaceutical composition according to (4), wherein the TDP-43 proteinopathy is frontotemporal lobar degeneration or amyotrophic lateral sclerosis. (6) culturing cells expressing TDP-43 mRNA and in which CSE1L is knocked down in the presence of a test substance; and Quantifying splicing variants that do not contain intron 6 and splicing variants that contain intron 6 among TDP-43 mRNAs in the cells; Including, A method for screening for a selective splicing enhancer of intron 6 of TDP-43 mRNA, wherein a decrease in the expression level of a splicing variant containing intron 6 in the presence of the test substance compared to the absence of the test substance indicates that the test substance is a candidate for a selective splicing enhancer of intron 6 of TDP-43 mRNA. (7) culturing cells expressing TDP-43 mRNA and in which CSE1L is knocked down in the presence of a test substance; and Quantifying splicing variants that do not contain intron 6 and splicing variants that contain intron 6 among TDP-43 mRNAs in the cells; Including, A method for screening a candidate compound for the prevention or treatment of TDP-43 proteinopathy, wherein a decrease in the expression level of a splicing variant containing intron 6 in the presence of the test substance compared to the absence of the test substance indicates that the test substance is a candidate compound for the prevention or treatment of TDP-43 proteinopathy. [Effects of the Invention]

[0012] The antisense nucleic acid of the above embodiment can enhance alternative splicing of intron 6 of TDP-43 mRNA. The alternative splicing enhancer of the above embodiment comprises the antisense nucleic acid and can enhance alternative splicing of intron 6 of TDP-43 mRNA. The pharmaceutical composition of the above embodiment comprises the antisense nucleic acid and can prevent or treat TDP-43 proteinopathy. The screening method of the above embodiment can screen for an alternative splicing enhancer of intron 6 of TDP-43 mRNA. The screening method of the above embodiment can screen for a candidate compound for the prevention or treatment of TDP-43 proteinopathy. [Brief explanation of the drawings]

[0013] [Figure 1] FIG. 1 is a schematic diagram showing the four isoforms of TDP-43 mRNA. [Figure 2] FIG. 1 illustrates the aggregation and functionality of TDP-43 protein. [Figure 3A] FIG. 1 is a schematic diagram showing the target positions of each morpholino antisense oligonucleotide (AS1, AS2.1, AS2, AS3, AS4, AS5, AS5.1, AS5.2, AS6, AS6.1, and AS7) within the sequence of TDP-43 mRNA in Example 1, as well as the binding positions of U2AF1 and HNRNPA1. [Figure 3B]1 shows an agarose gel electrophoresis image of PCR products obtained by reverse transcription PCR using RNA extracted from human HEK293T cells transfected with each morpholino antisense oligonucleotide in Example 1. [Figure 3C] 1 is a graph showing the ratio of the expression level of mRNA in which intron 6 is alternatively spliced ​​to the expression level of mRNA in which intron 6 is retained in human HEK293T cells transfected with each morpholino antisense oligonucleotide in Example 1. (In the graph, *: p<0.05, **: p<0.01, ***: p<0.001 (comparison with the control group, Dunnett's test)) [Figure 3D] 1 shows an agarose gel electrophoresis image of PCR products obtained by reverse transcription PCR using RNA extracted from human HEK293T cells transfected with each morpholino antisense oligonucleotide in Example 1 and treated with cycloheximide. [Figure 3E] 1 is a graph showing the ratio of the expression level of mRNA in which intron 6 is alternatively spliced ​​to the expression level of mRNA in which intron 6 is retained in human HEK293T cells transfected with each morpholino antisense oligonucleotide and treated with cycloheximide in Example 1. (In the graph, **: p<0.01, ***: p<0.001 (comparison with the control group, Dunnett's test)) [Figure 3F] 1 shows Western blotting of nuclear and cytoplasmic fractions of human HEK293T cells in which CSE1L was knocked down in Example 1. [Figure 3G] This is an agarose gel electrophoresis image of PCR products obtained by reverse transcription PCR using RNA extracted from human HEK293T cells that were treated with cycloheximide after CSE1L knockdown in Example 1 and then transfected with each morpholino antisense oligonucleotide. [Figure 3H]1 is a graph showing the ratio of the expression level of mRNA in which intron 6 is alternatively spliced ​​to the expression level of mRNA in which intron 6 is retained in human HEK293T cells that were transfected with each morpholino antisense oligonucleotide and treated with cycloheximide after knocking down CSE1L in Example 1. (In the graph, *: p<0.05, **: p<0.01, ***: p<0.001 (comparison of siCSE1L group with control group, Dunnett's test).) [Figure 3I] 1 is a graph showing the ratio of the expression level of mRNA in which only intron 6 is alternatively spliced ​​to the expression level of mRNA in which intron 6 and intron 7 are retained in human HEK293T cells that were transfected with each morpholino antisense oligonucleotide and treated with cycloheximide after knocking down CSE1L in Example 1. (In the graph, **: p<0.01, ***: p<0.001 (comparison of siCSE1L group with control group, Dunnett's test)) [Figure 4A] 1 shows an agarose gel electrophoresis image of PCR products obtained by reverse transcription PCR using RNA extracted from mouse neuroblastoma Neuro2a cells into which AS5 was introduced in Example 2. [Figure 4B] 1 is a graph showing the ratio of the expression level of mRNA retaining intron 6 to the expression level of total TDP-43 mRNA in mouse neuroblastoma Neuro2a cells transfected with AS5 in Example 2. (In the graph, ***: p<0.001 (two-tailed t-test)) [Figure 4C] 10 is a graph showing the ratio of the expression level of mRNA retaining intron 7 to the expression level of total TDP-43 mRNA in mouse neuroblastoma Neuro2a cells into which AS5 was introduced in Example 2. [Figure 4D] FIG. 10 shows the results of Western blotting analysis using a polyclonal antibody against the N-terminal side of the TDP-43 protein as an antigen in mouse neuroblastoma Neuro2a cells into which AS5 was introduced in Example 2. [Figure 5A]FIG. 1 shows the protocol for the test of intracerebroventricular administration of AS5 using newborn mice (C57BL / 6NJcl) in Example 3. [Figure 5B] 1 shows an agarose gel electrophoresis image of PCR products obtained by reverse transcription PCR using RNA extracted from various parts of the brain of a mouse to which AS5 was intracerebroventricularly administered in Example 3. [Figure 5C] 1 is a graph showing the expression level of mRNA retaining intron 6, determined by reverse transcription real-time PCR using GAPDH mRNA as a reference gene, in various regions of the mouse brain after intracerebroventricular administration of AS5 in Example 3. (In the graph, **: p<0.01, ***: p<0.001 (two-tailed t-test)) [Figure 5D] 1 is a graph showing the ratio of the expression level of mRNA retaining intron 6 to the expression level of total TDP-43 mRNA in each region of the mouse brain to which AS5 was intracerebroventricularly administered in Example 3. (In the graph, **: p<0.01, ***: p<0.001 (two-tailed t-test)) [Figure 5E] 1 is a graph showing the ratio of the expression level of mRNA retaining intron 7 to the expression level of total TDP-43 mRNA in each region of the mouse brain to which AS5 was intracerebroventricularly administered in Example 3. (In the graph, *: p<0.05 (two-tailed t-test)) [Figure 5F] FIG. 10 shows the results of Western blotting analysis using a polyclonal antibody against the C-terminal side of the TDP-43 protein as an antigen and an anti-GAPDH antibody as a control in various parts of the brain of a mouse to which AS5 was intracerebroventricularly administered in Example 3. [Figure 5G] 5B is a graph showing the ratio of the expression level of TDP-43 protein to the expression level of GAPDH quantified from the results of FIG. 5F (in the graph, **: p<0.01 (two-tailed t-test)). [Figure 5H] 10 is a graph showing the expression levels of Aif1 mRNA in the cerebrum, brainstem, and spinal cord after intracerebroventricular administration of AS5 in Example 3. [Figure 6A] FIG. 1 shows the protocol for the test of intrathecal administration of AS5 using adult mice (C57BL / 6NJcl) in Example 4. [Figure 6B] 1 shows an agarose gel electrophoresis image of PCR products obtained by reverse transcription PCR using RNA extracted from the cervical and lumbar spinal cord of mice administered AS5 intrathecally in Example 4. [Figure 6C] 1 is a graph showing the ratio of the expression level of mRNA in which intron 6 is retained to the expression level of mRNA in which intron 6 is alternatively spliced ​​in the cervical and lumbar vertebrae of mice administered AS5 intrathecally in Example 4. (In the graph, *: p<0.05, **: p<0.01 (two-tailed t-test)) [Figure 6D] 1 is a graph showing the body weights of male and female mice 8 weeks after intrathecal administration of AS5 in Example 4. [Figure 6E] 1 is a graph showing the grip strength of male and female mice 8 weeks after intrathecal administration of AS5 in Example 4. [Figure 6F] 10 is a graph showing the expression level of Aif1 mRNA in the lumbar spinal cord 8 weeks after intrathecal administration of AS5 in Example 4. [Figure 7A] FIG. 1 shows the protocol for the test of intracerebroventricular and intrathecal administration of AS5 using neonatal motor neuron-specific Rpt3 conditional knockout mice (Rpt3flox / flox; VAChT-Cre+ / -) in Example 5. [Figure 7B] 1 is a graph showing the change in body weight over time and the time to death in mice administered with AS5 in Example 5. [Figure 8A]Immunostaining images of spinal cord motor neurons of adult mice of the motor neuron-specific Rpt3 conditional knockout mouse (Rpt3flox / flox; VAChT-Cre+ / -) in Example 6 with anti-TDP-43 antibody, 4’,6-diamidino-2-phenylindole (DAPI), and anti-TUJ1 antibody. The scale bar indicates 40 μm. In the images, a to c represent the patterns observed in TDP-43 proteinopathies in human patients, respectively. Specifically, a is the staining image of a motor neuron in which nuclear TDP-43 has decreased and TDP-43 is diffusely mislocalized in the cytoplasm. b is the staining image of a motor neuron in which aggregates of TDP-43 are observed in the cytoplasm. c is the staining image of a motor neuron in which nuclear TDP-43 has almost disappeared. [Figure 8B] A diagram showing the protocol of the intrathecal administration test of AS5 using adult mice of the motor neuron-specific Rpt3 conditional knockout mouse (Rpt3flox / flox; VAChT-Cre+ / -) in Example 6. [Figure 8C] A graph showing the change in body weight over time in mice administered with AS5 in Example 6. [Figure 8D] A graph showing the change in grip strength over time in mice administered with AS5 in Example 6. [Figure 8E] A graph showing the relationship between body weight and grip strength in mice at 24 to 28 weeks of age in Example 6.

Mode for Carrying Out the Invention

[0014] Hereinafter, the antisense nucleic acid, the selective splicing enhancer of intron 6 of TDP-43 mRNA, the pharmaceutical composition, the screening method of the selective splicing enhancer of intron 6 of TDP-43 mRNA, and the screening method of the candidate compound for preventing or treating TDP-43 proteinopathy according to an embodiment of the present invention will be described in detail.

[0015] <TDP-43 protein> TDP-43 (TAR DNA-binding protein-43) is a protein encoded by the TARDBP gene in humans.

[0016] The full-length amino acid sequence of human TDP-43 protein is disclosed in Genbank accession number NP_031401.1.

[0017] The nucleotide sequence of the full-length mRNA of human TDP-43 is disclosed under Genbank accession number NM_007375.4.

[0018] The inventors have demonstrated that a combination of alternative splicing involving the 3'UTR of the TDP-43 gene pre-mRNA and the use of alternative polyadenylation sites results in the production of multiple isoforms (splice variants) of the TDP-43 gene mRNA. These splice variants can be broadly classified into: (i) variants in which neither intron 6 nor intron 7 of the TDP-43 gene is spliced ​​(hereinafter, sometimes referred to as "variant (i)"); (ii) variants in which only intron 6 of the TDP-43 gene is alternatively spliced ​​(hereinafter, sometimes referred to as "variant (ii)"); (iii) variants in which both intron 6 and intron 7 of the TDP-43 gene are alternatively spliced ​​(hereinafter, sometimes referred to as "variant (iii)"); and (iv) variants in which only intron 7 of the TDP-43 gene is alternatively spliced. These four splice variants are shown in Figure 1. The autoregulatory mechanism increases the production of variant (iii) or variant (iv) depending on the expression level of nuclear TDP-43 protein, but these are rapidly degraded due to their susceptibility to nonsense-mediated mRNA decay (NMD). Variant (i), which retains intron 6, encodes an IDR, and its increased expression induces aggregation and results in cytotoxicity.

[0019] Here, in cells with reduced nuclear TDP-43 protein, induction of alternative splicing of only intron 6 would reduce the IDR-encoding variant (i) and increase the proportion of variant (ii) retaining intron 7. Variant (ii) is not NMD-susceptible, resulting in the expression of TDP-43 proteins lacking the IDR. TDP-43 proteins form oligomers via the N-terminal domain, and their aggregation is known to be strongly dependent on the local concentration of the IDR at the C-terminus (see Figure 2(A)). Therefore, in cells with reduced nuclear TDP-43 protein, induction of alternative splicing of only intron 6 would suppress the expression of full-length TDP-43 protein and the production of IDR-less TDP-43 proteins, thereby reducing the local concentration of the IDR in the oligomers and suppressing TDP-43 protein aggregation (see Figure 2(C)). Suppression of aggregation leads to increased nuclear import of TDP-43 protein, resulting in the recovery of nuclear TDP-43 protein function. When the expression level of nuclear TDP-43 protein is sufficiently restored, alternative splicing of intron 7 is induced, reducing the expression ratio of variant (ii) and increasing the expression level of variant (iii). As a result, the production of IDR-deficient TDP-43 protein becomes negligible, and most of it is degraded via NMD. In addition, TDP-43 intron 6 contains a region where the U2AF protein, which is important for determining the 3' splicing site, binds. It has been reported that U2AF binding within the intron, together with HNRNPA1, suppresses proper splicing and is involved in intron retention. Based on these findings, as will be shown in the Examples below, the inventors designed an antisense nucleic acid that targets the binding regions of U2AF and HNRNPA1 and enhances the splicing of intron 6. Furthermore, by applying the above-mentioned autoregulatory mechanism of the TDP-43 protein, the inventors discovered that by inducing alternative splicing of only intron 6 using an antisense nucleic acid that targets intron 6 of the designed TDP-43 mRNA and changing the composition ratio of each isoform, it is possible to restore the function of the nuclear TDP-43 protein in pathological cells, suppress its aggregation in the cytoplasm, and maintain the expression of the full-length TDP-43 protein at a level that does not cause cytotoxicity in healthy cells, thereby completing the present invention.

[0020] <Antisense nucleic acid> The antisense nucleic acid of this embodiment targets intron 6 of TDP-43 mRNA, has as its target sequence the 96th to 330th or 400th to 530th bases of the nucleotide sequence represented by SEQ ID NO: 1, and contains a nucleotide sequence complementary to a sequence consisting of 10 or more consecutive nucleotides in the target sequence.

[0021] The antisense nucleic acid of this embodiment can induce selective splicing of only intron 6 of TDP-43 mRNA, and can suppress the production of an mRNA isoform (variant (i)) in which neither intron 6 nor intron 7 of the TDP-43 gene is spliced. This makes it possible to suppress aggregation of the TDP-43 protein in the cytoplasm and restore the function of the nuclear TDP-43 protein.

[0022] The antisense nucleic acid of this embodiment can be designed with reference to, for example, the full-length mRNA of human TDP-43 (Genbank accession number NM_007375.4). Specifically, the target sequence is the region of intron 6 in the full-length mRNA of human TDP-43 (SEQ ID NO: 1) other than the splicing site (the 5' alternative splicing site at positions 1, 65, and 74 of the base sequence represented by SEQ ID NO: 1, and the 3' alternative splicing site at position 1015), and the region to which U2AF1 and HNRNPA1 bind, i.e., the region from positions 96 to 330 or from positions 400 to 530 of the base sequence represented by SEQ ID NO: 1.

[0023] When targeting TDP-43 mRNA of a mammal other than human, the target sequence can be similarly designed by referring to known sequences.

[0024] The length of the antisense nucleic acid of this embodiment is 10 bases or more, preferably 10 to 50 bases, more preferably 15 to 35 bases, and even more preferably 20 to 30 bases.

[0025] The antisense nucleic acid of this embodiment may be composed of DNA, RNA, or a combination of DNA and RNA. Furthermore, the antisense nucleic acid of this embodiment is a nucleotide polymer in which nucleotides are linked by phosphodiester bonds, and may be a polymer of natural nucleotides, a polymer of natural nucleotides and non-natural nucleotides (an analog of a natural nucleotide, a nucleotide in which at least one of the base moiety, sugar moiety, and phosphate moiety is modified (e.g., a nucleotide having a phosphorothioate backbone or a monophosphorin ring)), or a polymer of non-natural nucleotides.

[0026] Specific examples of antisense nucleic acids include antisense nucleic acids consisting of a sequence containing the base sequence shown in any one of SEQ ID NOs: 2 to 7, and among these, antisense nucleic acids consisting of the base sequence shown in any one of SEQ ID NOs: 2 to 7 are preferred.

[0027] The antisense nucleic acid of this embodiment can be synthesized using known methods. Examples of the synthesis method include synthesis methods using genetic engineering techniques and chemical synthesis methods. Examples of the synthesis method using genetic engineering techniques include in vitro transcription synthesis methods, synthesis methods using vectors, and synthesis methods using PCR cassettes. Examples of chemical synthesis methods include the phosphoramidite method, the H-phosphonate method, etc. Chemical synthesis methods also include methods using commercially available automated nucleic acid synthesizers.

[0028] The antisense nucleic acid of this embodiment may be in the form of a vector that expresses the antisense nucleic acid. A vector that expresses an antisense nucleic acid can be prepared, for example, by inserting the base sequence of the target region into a commercially available vector.

[0029] Any vector can be used as long as it can express the antisense nucleic acid in target cells. The vector can include a promoter that controls expression of the antisense nucleic acid, wherein the sequence encoding the antisense nucleic acid is operably linked to the promoter.

[0030] The promoter is not particularly limited, and for example, a pol II promoter can be used. However, from the viewpoint of more accurate transcription of relatively short nucleic acids, a pol III promoter is preferred. Pol III promoters are not particularly limited, and examples include mouse and human U6-snRNA promoters, human H1-RNase P RNA promoters, and human valine-tRNA promoters. When using the U6 promoter, it is preferable that the 5' end of the antisense nucleic acid be "G" for transcription initiation. Therefore, it is preferable to design the sequence so that the 5' end of the antisense nucleic acid is "G," or to add "G" to the 5' end of the antisense nucleic acid.

[0031] In addition to the coding sequence and promoter of the antisense nucleic acid, the vector may optionally contain an enhancer, a poly(A) addition signal, a marker gene, a replication origin, a gene encoding a protein that binds to the replication origin and controls replication, and the like. The term "marker gene" refers to a gene that enables cell selection or sorting when introduced into cells. Specific examples of marker genes include drug resistance genes, fluorescent protein genes, luciferase genes, and chromogenic enzyme genes. These may be used alone or in combination. Specific examples of drug resistance genes include puromycin resistance genes, neomycin resistance genes, tetracycline resistance genes, kanamycin resistance genes, zeocin resistance genes, hygromycin resistance genes, and chloramphenicol resistance genes. Specific examples of fluorescent protein genes include green fluorescent protein (GFP) genes, yellow fluorescent protein (YFP) genes, and red fluorescent protein (RFP) genes. Specific examples of luciferase genes include luciferase genes. Specific examples of the chromogenic enzyme gene include the β-galactosidase gene, the β-glucuronidase gene, and the alkaline phosphatase gene.

[0032] The type of vector is not particularly limited, and any known expression vector can be used. Examples of expression vectors include plasmid vectors and viral vectors.

[0033] The plasmid vector is not particularly limited as long as it can be expressed in target cells. For example, in the case of animal cells, a plasmid vector commonly used for expression in animal cells can be used. Examples of plasmid vectors for expression in animal cells include, but are not limited to, pX459, pA1-11, pXT1, pRc / CMV, pRc / RSV, and pcDNAI / Neo.

[0034] Examples of viral vectors include retroviral (including lentiviral) vectors, adenoviral vectors, adeno-associated viral vectors, Sendai viral vectors, herpes viral vectors, vaccinia viral vectors, pox viral vectors, polio viral vectors, Silvis viral vectors, rhabdoviral vectors, paramyxoviral vectors, and orthomyxoviral vectors.

[0035] Among these, a plasmid vector is preferred as the expression vector.

[0036] <Alternative splicing enhancer> The selective splicing enhancer of this embodiment is an agent for enhancing selective splicing of intron 6 of TDP-43 mRNA, and contains the above-mentioned antisense nucleic acid as an active ingredient.

[0037] The selective splicing enhancer of this embodiment can effectively induce alternative splicing of intron 6 of TDP-43 mRNA.

[0038] For example, the selective splicing enhancer of this embodiment containing the antisense nucleic acid is administered to a subject having a TDP-43 gene. The administration method can be performed by contacting the subject with the antisense nucleic acid. The administration can be in vivo or in vitro.

[0039] The subjects of administration are not particularly limited, and examples include cells, tissues, or organs of mammals such as humans, monkeys, marmosets, mice, rats, guinea pigs, dogs, cats, rabbits, cows, horses, pigs, goats, and sheep.

[0040] The selective splicing enhancer of this embodiment may further contain a nucleic acid transfer reagent for the purpose of enhancing the efficiency of transfer of the antisense nucleic acid into target cells. Examples of nucleic acid introduction reagents include atelocollagen; liposomes; Lipofectamine (registered trademark), lipofectin, transfectam (dioctoadecylamidoglycylspermine; DOGS), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), didodecyldimethylammonium bromide (DDAB), DHDEAB (N,N-di-n-hexadecyl-N-methyl,N-(2-hydroxyethyl)ammonium bromide), polybrene, poly(ethyleneimine) (PEI), and other ionic lipids.

[0041] <Pharmaceutical Composition> The pharmaceutical composition of this embodiment is used for the prevention or treatment of TDP-43 proteinopathy, and contains the above-mentioned antisense nucleic acid as an active ingredient.

[0042] As shown in the examples described below, the pharmaceutical composition of this embodiment can suppress the accumulation of TDP-43 protein in the cytoplasm and restore the function of intranuclear TDP-43 protein, and is effective in preventing or treating TDP-43 proteinopathy.

[0043] TDP-43 proteinopathy is a general term for neurodegenerative diseases in which the TDP-43 protein aggregates and accumulates, including frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS). FTLD and ALS include both genetically mutated (familial) and sporadic forms. Furthermore, accumulation of TDP-43 protein has been confirmed in addition to accumulation of tau protein, amyloid beta protein, huntingtin protein, etc. in neurodegenerative diseases such as Alzheimer's disease, dementia with Lewy bodies, Down syndrome, hippocampal sclerosis, familial British dementia, Perry syndrome, Parkinson's disease, polyglutamine diseases (e.g., Huntington's disease, spinocerebellar ataxia type 3, etc.), myopathy (e.g., sporadic inclusion body myositis, inclusion body myopathy, oculopharyngeal muscular dystrophy, distal myopathy, myofibrillar myopathy, etc.), corticobasal degeneration, progressive supranuclear palsy, and argyrophilic grain disease, and therefore the method can also be applied to these diseases. Among these TDP-43 proteinopathies, it is preferably used for treating or preventing FTLD or ALS.

[0044] The pharmaceutical composition of this embodiment may contain an effective amount of the antisense nucleic acid alone, or may be formulated in combination with a pharmaceutically acceptable carrier.

[0045] The antisense nucleic acid contained in the pharmaceutical composition of this embodiment may be in the form of a nucleic acid molecule, or may be in the form of a vector containing a nucleic acid encoding the antisense nucleic acid as described above, or may be a mixture of these forms.

[0046] Pharmaceutically acceptable carriers include, but are not limited to, excipients such as sucrose and starch; binders such as cellulose and methylcellulose; disintegrants such as starch and carboxymethylcellulose; lubricants such as magnesium stearate and aerosil; flavorings such as citric acid and menthol; preservatives such as sodium benzoate and sodium bisulfite; stabilizers such as citric acid and sodium citrate; suspending agents such as methylcellulose and polyvinylpyrrolide; dispersing agents such as surfactants; diluents such as water and saline; base waxes, etc.

[0047] The pharmaceutical composition of this embodiment may further comprise a nucleic acid transfer reagent to promote the transfer of the antisense nucleic acid into target cells. As the nucleic acid transfer reagent, the same reagents as those exemplified in the "alternative splicing enhancer" can be used.

[0048] The pharmaceutical composition of this embodiment may also be a pharmaceutical composition in which the antisense nucleic acid is encapsulated in a liposome. Liposomes are minute closed vesicles having an internal phase surrounded by one or more lipid bilayers, and can generally hold a water-soluble substance in the internal phase and a fat-soluble substance in the lipid bilayer. "Encapsulation" as used herein encompasses a state in which the antisense nucleic acid is held in the liposome internal phase and a state in which the antisense nucleic acid is held in the lipid bilayer.

[0049] The liposome may be a single-layer membrane or a multilayer membrane. The particle size of the liposome is, for example, 10 nm to 1000 nm, preferably 50 nm to 300 nm. In consideration of delivery to target cells or target tissues, the particle size is more preferably 50 nm to 200 nm, and even more preferably 50 nm to 100 nm.

[0050] Methods for encapsulating antisense nucleic acids into liposomes include, for example, the lipid film method (vortex method), reverse phase evaporation, surfactant removal method, freeze-thaw method, and remote loading method, but are not limited to these, and any known method can be appropriately selected.

[0051] The pharmaceutical composition of this embodiment can be administered orally or parenterally to mammals, but is preferably administered parenterally. As the mammal, the same mammals as those exemplified in the above "alternative splicing enhancer" can be used.

[0052] Examples of parenteral administration methods include subcutaneous injection, intramuscular injection, local injection, intraperitoneal administration, and intrathecal administration.

[0053] Preparations suitable for parenteral administration include aqueous and non-aqueous isotonic sterile injection solutions, which may further contain antioxidants, buffers, bacteriostats, isotonic agents, etc. Alternatively, aqueous and non-aqueous sterile suspensions may be used, which may further contain suspending agents, solubilizers, thickeners, stabilizers, preservatives, etc. These preparations can be enclosed in unit-dose or multiple-dose containers such as ampoules or vials. Alternatively, the active ingredient and a pharmaceutically acceptable carrier can be freeze-dried and stored in a state that only requires dissolving or suspending in an appropriate sterile vehicle immediately before use. Other preparations suitable for parenteral administration include sprays and the like.

[0054] The content of the antisense nucleic acid in the pharmaceutical composition of this embodiment is not particularly limited, but can be, for example, about 0.1% by mass or more and 100% by mass or less relative to the total mass of the pharmaceutical composition.

[0055] The dosage of the pharmaceutical composition of this embodiment varies depending on the purpose of administration, the administration method, the type and severity of the target disease, and the condition of the recipient (sex, age, body weight, etc.). For example, when administered systemically to an adult, the single dose of antisense nucleic acid can typically be 1 nmol / kg to 100 μmol / kg. Furthermore, when administered topically to an adult, the single dose can be 1 pmol / kg to 1 μmol / kg. This dosage can be administered once to 10 times. Furthermore, from the viewpoint of maintaining the high efficacy of the antisense nucleic acid, additional administration at regular intervals is preferable. The administration interval is not particularly limited, but examples include daily, every 3 days, weekly, every 2 weeks, monthly, every 3 months, and every 6 months.

[0056] The pharmaceutical composition of this embodiment can be used in combination with, for example, a therapeutic agent for a TDP-43 proteinopathy such as FTLD or ALS, for example, a therapeutic agent already on the market for these diseases. Examples of such therapeutic agents include neuroprotective agents (e.g., edaravone, etc.), glutamate inhibitors (e.g., riluzole, etc.), and neurotrophic factors (e.g., insulin-like growth factor-1, 5-HT1a receptor agonists (e.g., zaliproden, etc.)). These concomitant drugs can be formulated together with the pharmaceutical composition of this embodiment and administered as a single formulation, or they can be formulated separately from the pharmaceutical composition of this embodiment and administered simultaneously or at different times using the same or different administration method as the pharmaceutical composition of this embodiment. The dosage of these concomitant drugs may be the amount normally used when the drug is administered alone, or it may be reduced from the amount normally used.

[0057] <Treatment method> In one embodiment, the present invention provides a method for preventing or treating TDP-43 proteinopathy, comprising administering an effective amount of the antisense nucleic acid to a patient in need of treatment. Examples of the antisense nucleic acid include those described above. Examples of TDP-43 proteinopathy include those described above, with FTLD or ALS being preferred. In other words, the method for preventing or treating TDP-43 proteinopathy can also be considered a method for preventing or treating FTLD or ALS.

[0058] In one embodiment, the present invention provides the antisense nucleic acid for preventing or treating a TDP-43 proteinopathy. Examples of the antisense nucleic acid include those described above. Examples of TDP-43 proteinopathy include those described above, with FTLD or ALS being preferred.

[0059] In one embodiment, the present invention provides use of the antisense nucleic acid for producing a pharmaceutical composition for use in preventing or treating a TDP-43 proteinopathy. Examples of the antisense nucleic acid include those described above. Examples of the TDP-43 proteinopathy include those described above, with FTLD or ALS being preferred.

[0060] <Screening method> The screening method of this embodiment is a method for screening for a candidate compound for preventing or treating TDP-43 proteinopathy, and includes the following steps: Culturing cells expressing TDP-43 mRNA in the presence of a test substance; A step of quantifying splicing variants that do not contain intron 6 and splicing variants that include intron 6 among TDP-43 mRNAs in the cells.

[0061] If the expression level of the splicing variant containing intron 6 decreases in the presence of the test substance compared to the absence of the test substance, or if the expression level of the splicing variant not containing intron 6 increases relative to the expression level of the splicing variant containing intron 6, this indicates that the test substance is a candidate compound for the prevention or treatment of TDP-43 proteinopathy.

[0062] As described above, a substance that reduces the expression level of a splicing variant containing intron 6, or a substance that increases the expression level of a splicing variant that does not contain intron 6 relative to the expression level of a splicing variant that contains intron 6, can be said to be a candidate compound for preventing or treating TDP-43 proteinopathy. Therefore, the screening method of this embodiment makes it possible to screen for candidate compounds for preventing or treating TDP-43 proteinopathy.

[0063] The screening method of this embodiment can also be said to be a method for screening for an agent that enhances alternative splicing of intron 6 of TDP-43 mRNA. In this case, if the expression level of the splicing variant containing intron 6 decreases in the presence of the test substance compared to the absence of the test substance, or if the expression level of the splicing variant not containing intron 6 increases relative to the expression level of the splicing variant containing intron 6, this indicates that the test substance is a candidate for an enhancer of selective splicing of intron 6 of TDP-43 mRNA.

[0064] The test substance is not particularly limited, and examples thereof include natural compound libraries, synthetic compound libraries, existing drug libraries, metabolite libraries, and the like.

[0065] Cells that express TDP-43 mRNA include, but are not limited to, human HEK293T cells, mouse neuroblastoma Neuro2a cells, and induced pluripotent stem cell-derived neurons.

[0066] The screening method of this embodiment also includes screening using cells treated with cycloheximide, which inhibits degradation of NMD-sensitive mRNA, for the purpose of improving analytical accuracy. It also includes screening using cells that mimic TDP-43 proteinopathy accompanied by reduced alternative splicing of intron 6 by adding a factor that induces mislocalization of TDP-43, such as by knocking down CSE1L.

[0067] The expression levels of splicing variants of TDP-43 mRNA that do not contain intron 6 and splicing variants that contain intron 6 can be quantified by, for example, RNA-Seq, quantitative RT-PCR, etc. Primer sets for detecting splicing variants of TDP-43 mRNA that do not contain intron 6 and splicing variants that contain intron 6 can be appropriately designed from known sequences. Examples of such primer sets include a combination of a forward primer consisting of the nucleotide sequence represented by SEQ ID NO: 8, a first reverse primer consisting of the nucleotide sequence represented by SEQ ID NO: 9, and a second reverse primer consisting of the nucleotide sequence represented by SEQ ID NO: 10. [Example]

[0068] The present invention will be described below with reference to examples, but the present invention is not limited to the following examples.

[0069] [Example 1] Antisense nucleic acids enhance alternative splicing of intron 6 in human cells We designed six morpholino antisense oligonucleotides (AS2, AS3, AS4, AS5, AS5.1, AS5.2, and AS6.1) targeting the U2AF1 and HNRNPA1 binding peaks obtained from the public ENCODE eCLIP experiment data (ENCFF811WVR and ENCFF080DPL) shown in Figure 3A, and two (AS2.1 and AS6) that deviated from these binding peaks. One (AS1) targeted a canonical splice site, and one (AS7) targeted a sequence within intron 7. Using the GeneTools algorithm, we designed 25-base sequences from target regions of 35 to 50 bases each. Figure 3A shows a schematic diagram of the target locations of AS1, AS2.1, AS2, AS3, AS4, AS5, AS5.1, AS5.2, AS6, AS6.1, and AS7 within the TDP-43 mRNA sequence. The base sequences of these antisense nucleic acids are shown in Table 1 below.

[0070] [Table 1]

[0071] These morpholino antisense oligonucleotides were then added to the medium at a concentration of 10 μM and transfected into human HEK293T cells using Endo-Porter (GeneTools). Control cells were also prepared by transfecting a generic control oligo. Forty-eight hours after transfection, RNA was extracted from each cell line using Nucleospin RNA II (Takara Bio), and the efficiency of alternative intron 6 splicing was examined by reverse transcription-PCR. The primer sequences used for PCR are listed in Table 2. Figure 3B shows the results of agarose gel electrophoresis of the PCR products, and Figure 3C shows a graph depicting the ratio of the expression level of mRNA with alternatively spliced ​​intron 6 to the expression level of mRNA with retained intron 6 in each cell line.

[0072] [Table 2]

[0073] As shown in Figures 3B and 3C, five types of morpholino antisense oligonucleotides (AS2, AS4, AS5, AS5.1, AS5.2, and AS6.1) increased splicing and reduced the expression level of mRNA retaining intron 6.

[0074] To improve the quantitation of spliced ​​mRNA, cells were treated with cycloheximide for 6 hours before RNA extraction. The results are shown in Figure 3D and Figure 3E. Five morpholino antisense oligonucleotides (AS2, AS4, AS5, AS5.1, AS5.2, and AS6.1) enhanced intron 6 splicing and reduced the expression of mRNA containing intron 6.

[0075] Next, we investigated the selective splicing-enhancing effect of morpholino antisense oligonucleotides in a condition mimicking TDP-43 proteinopathy, in which nuclear TDP-43 is reduced and cytoplasmic TDP-43 accumulates. Specifically, CSE1L, which is involved in the nuclear translocation of TDP-43, was knocked down in HEK293T cells using RNA interference (On-TARGETplus siRNA, Dharmacon) and Lipofectamine RNAiMAX (Invitrogen). After 48 hours, nuclear and cytoplasmic proteins were extracted using NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Scientific). Anti-LaminB1 antibody (MBL, PM064) was used as a loading control for the nuclear fraction, and anti-GAPDH antibody (MBL, M171-3) was used as a loading control for the cytoplasmic fraction. CSE1L expression was confirmed using an anti-CSE1L antibody (Abcam, ab151546), and TDP-43 expression in each fraction was analyzed by Western blotting using an anti-TDP-43 antibody (Proteintech, 12892-1-AP). The results are shown in Figure 3F.

[0076] As shown in Figure 3F, reduction of CSE1L resulted in a decrease in TDP-43 in the nucleus and an increase in TDP-43 in the cytoplasm.

[0077] Next, 18 hours after transfection with the CSE1L siRNA, five morpholino antisense oligonucleotides (AS2, AS4, AS5, AS5.1, and AS5.2) were transfected and treated with cycloheximide (100 μg / mL in the medium for 6 hours). Splicing of intron 6 and intron 7 was analyzed by reverse transcription-PCR as described above. The primer sequences used for PCR are listed in Table 2. Figure 3G shows the results of agarose gel electrophoresis of the PCR products. Figure 3H shows a graph of the ratio of the expression level of mRNA with alternatively spliced ​​intron 6 (ii) to the expression level of mRNA with alternatively spliced ​​intron 6 (i) in each cell line. Figure 3I shows the ratio of the expression level of mRNA with alternatively spliced ​​intron 6 only (v) to the expression level of mRNA with alternatively spliced ​​introns 6 and 7 (iii) in each cell line.

[0078] As shown in Fig. 3G (top) and Fig. 3H, in cells transfected with the control morpholino antisense oligo, knockdown of CSE1L attenuated intron 6 splicing. In contrast, the reduction in intron 6 splicing was suppressed by four types of morpholino antisense oligos (AS2, AS5, AS5.1, and AS5.2). Furthermore, as shown in the lower panel of Figure 3G and Figure 3I, three types of morpholino antisense oligos (AS2, AS5.1, and AS5.2) suppressed the increase in mRNAs retaining introns 6 and 7, and increased the proportion of mRNAs in which only intron 6 was spliced.

[0079] [Example 2] Antisense nucleic acids enhance alternative splicing of intron 6 in mouse cells Of the four morpholino antisense oligonucleotides confirmed to enhance alternative splicing of intron 6 in human HEK293T cells in Example 1, one morpholino antisense oligonucleotide (AS5) with a target sequence identical to that of mouse TDP-43 mRNA was added to the medium at a concentration of 10 μM and transfected into mouse neuroblastoma-derived Neuro2a cells using Endo-Porter (GeneTools). Cells without transfection of the morpholino antisense oligonucleotide were also prepared as controls. Forty-eight hours after transfection, RNA was extracted from each cell line using Nucleospin RNA II (Takara Bio Inc.), and the efficiency of alternative splicing of intron 6 was examined by reverse transcription-PCR. The sequences of the primers used in reverse transcription-PCR are listed in Table 3. The results of agarose gel electrophoresis of the PCR products are shown in Figure 4A. Figure 4B shows a graph showing the ratio of the expression level of mRNA retaining intron 6 to the total expression level of TDP-43 mRNA in each cell, using droplet digital PCR. Figure 4C shows a graph showing the ratio of the expression level of mRNA retaining intron 7 to the total expression level of TDP-43 mRNA in each cell. The primer sequences used in droplet digital PCR are shown in Table 4.

[0080] [Table 3]

[0081] [Table 4]

[0082] Furthermore, protein was extracted from each cell line using RIPA buffer, and TDP-43 protein expression was confirmed by Western blotting using a polyclonal antibody (Proteintech, 10782-2-AP) against the N-terminus of the TDP-43 protein. The results are shown in Figure 4D.

[0083] As shown in Figure 4A, similar to the results in human HEK293T cells, increased alternative splicing of intron 6 was confirmed. As shown in Figure 4B, the percentage of mRNAs retaining the alternative intron 6 among all isoforms of TDP-43 mRNA was reduced to an average of 57%. On the other hand, as shown in Figure 4C, mRNAs retaining intron 7 remained unchanged. These findings confirmed that the action of AS5 is a splicing modification effect specific to intron 6. Furthermore, as shown in Figure 4D, AS5-transfected Neuro2a cells produced a TDP-43 protein (approximately 32 kDa) lacking the IDR generated by splicing out intron 6.

[0084] [Example 3] (Enhancing alternative splicing of intron 6 in the mouse central nervous system) A glass capillary was inserted into the ventricle of newborn mice (C57BL / 6NJcl) under hypothermic anesthesia and transmitted light, and AS5 (4 mM, 2 μL) as designed in Example 1 was administered (see Figure 5A). A control group was also administered PBS. One week later, RNA was extracted from the cerebrum, brainstem, and spinal cord of the AS5- or PBS-administered side using Nucleospin RNA II (Takara Bio Inc.), and the efficiency of alternative splicing of intron 6 was examined by reverse transcription-PCR. The primer sequences used for PCR are listed in Table 2. Figure 5B shows the results of agarose gel electrophoresis of PCR products in various regions of the mouse central nervous system, and Figure 5C shows the results of real-time quantitative PCR, indicating the expression levels of mRNA containing intron 6, using GAPDH as a reference gene. Figure 5D shows a graph showing the ratio of the expression level of mRNA retaining intron 6 to the expression level of total TDP-43 mRNA, as determined by droplet digital PCR, and Figure 5E shows a graph showing the ratio of the expression level of mRNA retaining intron 7 to the expression level of total TDP-43 mRNA. The primer sequences used to detect GAPDH cDNA in real-time quantitative PCR are shown in Table 5. Other primer sequences used are as shown in Tables 3 and 4 above.

[0085] [Table 5]

[0086] Furthermore, protein was extracted from mouse spinal cord using RIPA buffer, and TDP-43 protein expression was confirmed by Western blotting using a polyclonal antibody (Proteintech, 12892-1-AP) against the C-terminus of TDP-43 protein as an antigen and an anti-GAPDH antibody (MBL, M171-3) as a control. Figure 5F shows the results of Western blotting, and Figure 5G shows a graph of TDP-43 protein expression levels, quantified from the Western blotting results and corrected for GAPDH expression levels. Figure 5H shows the results of real-time quantitative PCR to detect Aif1 mRNA expression, which reflects immunoreactive inflammation, in various central nervous system regions, using GAPDH as the reference gene. The primer sequences used to detect Aif1 cDNA in real-time quantitative PCR are listed in Table 6. The primer sequences used to detect GAPDH cDNA are as listed in Table 5 above.

[0087] [Table 6]

[0088] As shown in Figure 5B, increased alternative splicing of intron 6 was confirmed. Furthermore, a decrease in mRNAs retaining intron 6 was confirmed (see Figure 5C). At this time, the proportion of mRNAs retaining intron 6 decreased (see Figure 5D), but alternative splicing of intron 7 did not decrease (see Figure 5E). Furthermore, as shown in Figures 5F and 5G, full-length TDP-43 protein was reduced to approximately 70% in the spinal cord. At this time, there was no difference in Aif1 mRNA expression, which reflects immunoreactive inflammation.

[0089] [Example 4] (Effect of intron 6 alternative splicing enhancement and toxicity evaluation in adult mouse spinal cord) Eight-week-old adult mice (C57BL / 6NJcl) were intrathecally administered AS5 (4 mM, 15 μL) via lumbar puncture (Figure 6A). A control group was also prepared without AS5 administration. Eight weeks after administration, RNA was extracted from the neck and lumbar vertebrae of the mice using Nucleospin RNA II (Takara Bio Inc.), and the efficiency of alternative splicing of intron 6 was examined by reverse transcription-PCR. The primer sequences used for PCR are listed in Table 3. Figure 6B shows the results of agarose gel electrophoresis of the PCR products, and Figure 6C shows a graph showing the ratio of the expression level of mRNA with intron 6 retained to the expression level of mRNA with alternatively spliced ​​intron 6 at each site. Furthermore, the body weight and grip strength of each male and female mouse used in the administration test were measured, and the results are shown in Figure 6D (body weight) and Figure 6E (grip strength).

[0090] Furthermore, we analyzed the expression level of Aif1 mRNA, which reflects immunoreactive inflammation, using reverse transcription real-time PCR in lumbar spinal cord RNA. GAPDH mRNA was used as a reference gene. The results are shown in Figure 6F.

[0091] The enhanced effect of intron 6 alternative splicing was maintained for up to 8 weeks after administration (Fig. 6B and 6C). Furthermore, there were no differences in body weight (Fig. 6D) or grip strength (Fig. 6E) compared with the PBS-administered group, and there was no difference in Aif mRNA expression, which reflects immune-reactive inflammation (Fig. 6F). These results suggest that AS5 exerts the expected splicing-enhancing effect in the central nervous system of adult mice without causing tissue toxicity.

[0092] [Example 5] (Survival extension effect in a mouse model of TDP-43 proteinopathy) Motor neuron-specific Rpt3 conditional knockout mice (Rpt3) show aggregation of TDP-43 protein due to impaired proteasome function. flox / flox ;VAChT-Cre + / -Three newborn mice were administered AS5 (4 mM, 2 μL) intracerebroventricularly, followed by intrathecal administration of AS5 (4 mM, 15 μL) via lumbar puncture at 18 and 30 weeks of age (Figure 7A). A control group was also prepared without AS5 administration. Figure 7B is a graph showing the time course of body weight change and time to death in AS5-administered mice.

[0093] As shown in Figure 7B, compared with six mice not administered AS5, three mice administered AS5 showed reduced weight loss and prolonged survival from week 30 onwards (median survival time: 405.5 days (non-AS5 administration group) vs. 558.0 days (AS5 administration group), P value 0.011, Logrank test).

[0094] [Example 6] (Improvement of motor function in a mouse model after TDP-43 proteinopathy) The pathological abnormalities observed in TDP-43 proteinopathy appear in adult mice (Rpt3 flox / flox ;VAChT-Cre + / - We investigated the effect of AS5 on the development of rhesus monkeys. In this model mouse, Rpt3 expression in spinal motor neurons is lost at 6 weeks of age, and TDP-43 protein disappears from the nucleus and aggregates in the cytoplasm at 8 weeks of age (Figure 8A).

[0095] Therefore, we compared Rpt3 mice at 8 and 24 weeks of age. flox / flox ;VAChT-Cre + / - Mice were administered AS5 (4 mM, 15 μL) intrathecally by lumbar puncture (see FIG. 8B). A control group was also prepared that underwent lumbar puncture alone (sham surgery group). The body weight and grip strength of the mice subjected to the administration test were measured over time. The results are shown in Figure 8C (body weight) and Figure 8D (grip strength). The relationship between body weight and grip strength in 24- to 28-week-old mice is shown in Figure 8E.

[0096] Up to 40 weeks of age, the AS5-treated group showed improved weight gain compared to the sham-operated group (two-way analysis of variance; p 0.001; AS5-treated group: 12, sham-operated group: 16) (Fig. 8C). Regarding motor function, grip strength improved in the AS5-treated group at 24 weeks of age (two-tailed t-test; p 0.001; AS5-treated group: 15, sham-operated group: 18) and 28 weeks of age (two-tailed t-test; p 0.012; AS5-treated group: 12, sham-operated group: 16) (Fig. 8D). Grip strength at 24 and 28 weeks of age was significantly increased in the AS5-treated group, even after adjustment for body weight (analysis of covariance; p 0.0001) (Fig. 8E).

[0097] These results suggest that antisense oligonucleotides that enhance alternative splicing of intron 6 of TDP-43 mRNA may be effective in treating diseases associated with TDP-43 proteinopathy. [Industrial Applicability]

[0098] The antisense nucleic acid of this embodiment can enhance alternative splicing of intron 6 of TDP-43 mRNA. The selective splicing enhancer of this embodiment comprises the antisense nucleic acid and can enhance alternative splicing of intron 6 of TDP-43 mRNA. The pharmaceutical composition of this embodiment comprises the antisense nucleic acid and can prevent or treat TDP-43 proteinopathy. The screening method of this embodiment can screen for an agent that enhances alternative splicing of intron 6 of TDP-43 mRNA. The screening method of this embodiment can screen for a candidate compound for the prevention or treatment of TDP-43 proteinopathy.

Claims

1. a base sequence complementary to a sequence consisting of 15 to 25 consecutive bases selected from (i) positions 96 to 140, (ii) positions 160 to 184, (iii) positions 217 to 298, or (iv) positions 408 to 432 of the base sequence represented by SEQ ID NO: 1; An antisense nucleic acid that targets intron 6 of TDP-43 pre-mRNA, the antisense nucleic acid having a length of 30 bases or less.

2. An antisense nucleic acid described in claim 1, comprising a base sequence complementary to a sequence consisting of 15 to 25 consecutive bases at positions 96 to 120, 116 to 140, 160 to 184, 217 to 240, 230 to 254, 274 to 298, or 408 to 432 of the base sequence represented by SEQ ID NO:

1.

3. The antisense nucleic acid according to claim 1, comprising a base sequence represented by any one of SEQ ID NOs: 2 to 7 and 13.

4. An antisense nucleic acid described in any one of claims 1 to 3, wherein the antisense nucleic acid is a morpholino antisense oligonucleotide.

5. 4. An agent for enhancing the selective splicing of intron 6 of TDP-43 pre-mRNA, comprising the antisense nucleic acid according to claim 1 as an active ingredient.

6. A pharmaceutical composition containing the antisense nucleic acid according to any one of claims 1 to 3 as an active ingredient, which is used for preventing or treating TDP-43 proteinopathy.

7. The pharmaceutical composition according to claim 6, wherein the TDP-43 proteinopathy is frontotemporal lobar degeneration or amyotrophic lateral sclerosis.

8. Culturing cells expressing TDP-43 mRNA in the presence of a test substance; Quantifying the splicing variants that do not contain intron 6 and the splicing variants that contain intron 6 among the TDP-43 mRNA in the cells; Including, A method for screening for an agent that enhances selective splicing of intron 6 of TDP-43 mRNA, wherein a decrease in the expression level of a splicing variant containing intron 6 in the presence of the test substance compared to the absence of the test substance indicates that the test substance is a candidate agent for enhancing selective splicing of intron 6 of TDP-43 mRNA.

9. Culturing cells expressing TDP-43 mRNA in the presence of a test substance; Quantifying the splicing variants that do not contain intron 6 and the splicing variants that contain intron 6 among the TDP-43 mRNA in the cells; Including, A method for screening a candidate compound for the prevention or treatment of TDP-43 proteinopathy, wherein a decrease in the expression level of a splicing variant containing intron 6 in the presence of the test substance compared to the absence of the test substance indicates that the test substance is a candidate compound for the prevention or treatment of TDP-43 proteinopathy.

10. A method for enhancing selective splicing of intron 6 of TDP-43 pre-mRNA using an antisense nucleic acid comprising a base sequence complementary to a sequence consisting of 15 to 25 consecutive bases within (i) positions 96 to 140, (ii) positions 160 to 184, (iii) positions 217 to 298, or (iv) positions 408 to 432 of the base sequence represented by SEQ ID NO:

1.

11. A pharmaceutical composition for use in the prevention or treatment of TDP-43 proteinopathy, comprising as an active ingredient an antisense nucleic acid targeting intron 6 of TDP-43 pre-mRNA, the antisense nucleic acid comprising a base sequence complementary to a sequence consisting of 15 to 25 consecutive bases selected from (i) positions 96 to 140, (ii) positions 160 to 184, (iii) positions 217 to 298, or (iv) positions 408 to 432 of the base sequence represented by SEQ ID NO: 1.

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