Antisense nucleic acid and use of same

Abstract

The present invention provides an antisense nucleic acid, a splicing promoter, and a pharmaceutical composition that improve TDP-43 exitron splicing anomalies, that can effectively suppress a decrease in function of TDP-43 in nuclei which occurs along with the accumulation of TDP-43 anomalies, and that can be applied to the treatment of neurodegenerative disease. Provided is an antisense nucleic acid for which a target sequence is position 760 onward in the base sequence represented by SEQ ID NO: 1, which includes a base sequence having complementarity with a sequence comprising not less than 10 consecutive bases in the target sequence, and which promotes TDP-43 splicing. Also provided are a splicing promoter and a pharmaceutical composition which comprise the same.

Description

Antisense nucleic acids and their uses

[0001] The present invention relates to antisense nucleic acids and uses thereof. Specifically, the present invention relates to antisense nucleic acids, agents for enhancing alternative splicing of TDP-43 mRNA, and pharmaceutical compositions. This application claims priority to Japanese Patent Application No. 2024-075776, filed May 8, 2024, the contents of which are incorporated herein by reference.

[0002] TDP-43 proteinopathy is a general term for neurodegenerative diseases in which TDP-43 protein aggregates and accumulates, including frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS). There is no cure for FTLD, and treatment is primarily symptomatic for various complications. For ALS, edaravone, which aims to eliminate free radicals, and riluzole, which aims to reduce glutamate neuroexcitotoxicity, are used as treatments; however, neither has significant efficacy, and patients often develop respiratory muscle paralysis or death within two to five years of onset. The causes of sporadic ALS and FTLD, which account for more than 90% of cases, are unknown, and therapeutic drugs targeting specific pathological molecules are needed, but developing such therapeutic drugs has proven extremely difficult.

[0003] For example, many studies have investigated methods for regulating the expression of TDP-43 mRNA by RNA interference. 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. Patent Document 2 discloses a technique for regulating specific splicing events by TDP-43.

[0004] However, these methods carry the risk of excessively suppressing the essential biological functions of TDP-43, and have not been able to be considered a fundamental solution to TDP-43 pathologies. Specifically, the TDP-43 protein is an RNA-binding protein that is primarily localized in the nucleus and is involved in various RNA metabolism processes, such as post-transcriptional regulation. TDP-43 proteinopathies are pathologically characterized by the loss of TDP-43 protein from the nucleus and its accumulation in the cytoplasm, and both the resulting loss of nuclear function and the resulting acquisition of toxicity in the cytoplasm are involved in the pathology. For this reason, it is generally recognized that methods that simply reduce or enhance TDP-43 protein expression are not suitable for treatment.

[0005] Meanwhile, the present inventors have delved deeper into the pathological mechanisms associated with TDP-43, focusing particularly on aberrant splicing of TDP-43, and have begun to develop a therapeutic strategy that fundamentally corrects its abnormal function. In Patent Document 3, the present inventors aimed to normalize aberrant splicing in specific intra-exon intron sequences called excitrons, which are associated with abnormal accumulation of TDP-43, and focused on the role of HNRNPA1, one of the major RNA-binding proteins (RBPs) involved in this process. As a result, they identified a novel antisense nucleic acid that specifically targets the HNRNPA1 binding region, and confirmed in a mouse model that this nucleic acid corrects the splicing pattern of TDP-43, suppresses TDP-43 accumulation, and consequently exhibits neuroprotective effects.

[0006] In Patent Document 4, the present inventors have confirmed in vitro and intracellular experiments that a short TDP-43 isoform (sTDP), produced by splicing of the TDP-43 excitron, inhibits TDP-43 aggregation. sTDP forms a heterodimer with TDP-43 via the N-terminal domain, alleviating the crowding of the prion-like domain involved in aggregation and inhibiting TDP-43 aggregation.

[0007] International Publication No. 2019 / 013141 International Publication No. 2022 / 018155 International Publication No. 2022 / 113799 International Publication No. 2023 / 204313

[0008] As shown in Patent Documents 3 and 4, the present inventors have demonstrated that a method for promoting splicing of TDP-43 excitrons is an innovative therapeutic approach. However, methods for promoting this splicing more efficiently have not yet been fully explored, and there is a strong demand for knowledge and technologies that can be applied to fundamental solutions to TDP-43 pathology and address significant unresolved issues in neurodegenerative diseases such as FTLD and ALS.

[0009] TDP-43 plays a central role in RNA metabolic processes, but its abnormal accumulation leads to neuronal dysfunction and death. Existing therapies are unable to effectively address TDP-43 abnormalities, and no direct interventions have been established to address its accumulation and functional impairment. Suppressing global TDP-43 expression through RNA interference targeting TDP-43 mRNA is undesirable, as it ignores the important biological functions of TDP-43. Conversely, approaches that promote TDP-43 overexpression have been shown to promote intracytoplasmic aggregate formation, resulting in increased cytotoxicity. Furthermore, therapies that target only one of the many RNA splicing pathways controlled by TDP-43 do not address the underlying TDP-43 pathology.

[0010] Given this background, there is a strong demand for new strategies to directly and effectively regulate the abnormal accumulation of TDP-43 in the treatment of TDP-43-related diseases. In particular, if the abnormal splicing pattern of TDP-43 could be more efficiently normalized and its normal function in cells could be restored, the progression of these diseases could be slowed or even reversed. To achieve this, it is essential to identify new target regions that affect TDP-43 splicing and to develop antisense nucleic acids to effectively regulate these regions.

[0011] The present invention has been made in view of the above circumstances, and an object of the present invention is to provide an antisense nucleic acid, a splicing enhancer, and a pharmaceutical composition that can improve abnormal splicing of TDP-43 excitrons and effectively suppress the functional decline of intranuclear TDP-43 associated with abnormal accumulation of TDP-43, and that can be applied to the treatment of neurodegenerative diseases.

[0012] As a result of intensive research to achieve the above object, the present inventors identified an RNA-binding protein (RBP) that is deeply involved in the splicing of the TDP-43 excitron, and found that HNRNPC plays an important role in excitron splicing. Next, based on the distribution of HNRNPC binding motif sequences, the present inventors designed an antisense nucleic acid that inhibits excitron binding, and verified its effects, thereby discovering the present invention.

[0013] That is, the present invention includes the following aspects: (1) An antisense nucleic acid that promotes the splicing of TDP-43, wherein the target sequence is from position 760 onwards in the base sequence represented by SEQ ID NO: 1 and comprises 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), wherein the target sequence is from position 760 to position 985 in the base sequence represented by SEQ ID NO: 1. (3) The antisense nucleic acid according to (1), consisting of a base sequence represented by any of SEQ ID NOs: 2 to 4. (4) An agent for enhancing the selective splicing of TDP-43 mRNA, comprising as an active ingredient the antisense nucleic acid according to (1) or (2). (5) A pharmaceutical composition for use in the prevention or treatment of TDP-43 proteinopathy, comprising as an active ingredient the antisense nucleic acid according to (1) or (2). (6) The pharmaceutical composition according to (5), wherein the TDP-43 proteinopathy is frontotemporal lobar degeneration or amyotrophic lateral sclerosis.

[0014] The antisense nucleic acid, splicing enhancer, and pharmaceutical composition of the present invention can improve abnormal splicing of TDP-43 excitrons and effectively suppress the functional decline of intranuclear TDP-43 associated with abnormal accumulation of TDP-43, and can be applied to the treatment of neurodegenerative diseases.

[0015] 1 is a graph showing visualization of ENCODE RNA-Seq in this example. 2 is a diagram showing knockdown of HNRNPC in this example. 3 is another graph showing the analysis results of ENCODE RNA-Seq in this example. 4 is a graph showing inhibition of binding of HNRNPC by each antisense nucleic acid in this example. 5 is a photograph showing the investigation of splicing efficiency by each antisense nucleic acid in this example. 6 is a graph showing the splicing efficiency of each antisense nucleic acid targeting HNRNPC and HNRNPA1 in this example. 7 is a graph showing the splicing efficiency after CHX treatment of each antisense nucleic acid targeting HNRNPC and HNRNPA1 in this example. 8 is a photograph showing the selective splicing enhancing effect of antisense nucleic acids in CSE1L knockdown cells in this example. 9 is a photograph showing protein analysis after administration of antisense nucleic acids in CSE1L knockdown cells in this example. 10 is a graph showing the results of a test of functional recovery by antisense nucleic acids in this example. 1 is a graph showing the intracellular localization of TDP-43 due to CSE1L knockdown in SHS-SY5Y cells in this Example. 2 is a photograph of immunostaining showing the intracellular localization of TDP-43 in an iPS cell-derived neural model. 3 is a graph showing the intracellular localization, splicing rate, and expression level of various substances in an iPS cell-derived neural model.

[0016] The antisense nucleic acid, splicing enhancer, and pharmaceutical composition according to one embodiment of the present invention will be described in detail below.

[0017] <TDP-43 Protein> TDP-43 (TAR DNA-binding protein-43) protein is a protein encoded by the TARDBP gene in humans.

[0018] The amino acid sequence of the full-length human TDP-43 protein is disclosed in Genbank accession number NP_031401.1.

[0019] The nucleotide sequence of the full-length mRNA of human TDP-43 is disclosed in Genbank accession number NM_007375.4.

[0020] The present inventors have developed a novel antisense nucleic acid that promotes splicing of the region encoding the prion-like domain of TDP-43, thereby suppressing its abnormal accumulation and functional impairment. The prion-like domain of TDP-43 is located in an intron within a specific exon, called an excitron.

[0021] Here, the excitron (so-called exon 6) of human TDP-43, ie, the excitron sequence of TARDBP, is shown in SEQ ID NO:1.

[0022] We first began by identifying RNA-binding proteins (RBPs) critically involved in TDP-43 excitron splicing. This process included a thorough analysis of knockdown experimental data for 233 RNA-binding proteins provided by the ENCODE project. This analysis revealed that knockdown of specific RBPs significantly increased excitron splicing activity. Among these, HNRNPC, along with HNRNPA1, which we had previously focused on, was suggested to play an important role in excitron splicing. Through further experiments, we confirmed that reduction of HNRNPC promoted excitron splicing and revealed that this RNA-binding protein possesses multiple binding motifs on the 3' side of the excitron. This was supported by ECLIP data from the ENCODE project, demonstrating that HNRNPC indeed binds to this region. Based on these findings, we hypothesized that inhibiting HNRNPC binding to the excitron would promote splicing. To verify this hypothesis, antisense nucleic acids that inhibit the binding of HNRNPC were designed based on the distribution of binding motif sequences, and several effective antisense nucleic acid sequences were identified, leading to the completion of the present invention.

[0023] <Antisense Nucleic Acid> The antisense nucleic acid of this embodiment is an antisense nucleic acid that promotes splicing of TDP-43, and has a target sequence from position 760 onwards in the base sequence represented by SEQ ID NO: 1, and includes a base sequence that is complementary to a sequence consisting of 10 or more consecutive bases in the target sequence.

[0024] Here, "having complementarity with a specific base sequence" may refer to a complementary sequence.In addition, even if a part of antisense nucleic acid is not a base complementary to the target sequence, as long as antisense nucleic acid and target can be combined as a whole, it is considered to have complementarity.For example, taking into consideration the target, antisense nucleic acid and the structure after binding, some bases may be made non-complementary.As a guideline, "some bases" may refer to the number of bases (number of base pairs) of antisense nucleic acid that are not complementary to each other, such as approximately one-fourth or less, or approximately one-fifth or less.

[0025] According to the antisense nucleic acid of this embodiment, the novel antisense nucleic acid targets the HNRNPC-binding region within the excitron and inhibits the binding of HNRNPC, thereby improving the abnormal excitron splicing associated with the abnormal accumulation of TDP-43. Furthermore, correction of this abnormal splicing suppresses the decreased intranuclear function of TDP-43. This is demonstrated by the suppression of the decreased expression of the neuron-specific gene STMN2, which is reduced by TDP-43 dysfunction, providing a new opportunity for neuronal protection. The novel antisense nucleic acid of this embodiment can be applied to an innovative therapeutic approach to fundamentally suppress the abnormal expression and dysfunction of TDP-43 and slow the progression of ALS and FTLD.

[0026] 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), etc. Specifically, the target sequence is the region from position 760 onwards in the excitron sequence of human TDP-43 (SEQ ID NO: 1).

[0027] The antisense nucleic acid of this embodiment also preferably targets the HNRNPC binding region. Here, the HNRNPC binding region is from positions 760 to 956 in the excitron sequence of human TDP-43 shown in SEQ ID NO: 1. Furthermore, when the length of the antisense nucleic acid is considered to be approximately 30 bases, it is believed that binding can be significantly inhibited by a sequence including up to 30 bases starting from 956. In other words, it is also preferable that the antisense nucleic acid of this embodiment targets the base sequence from positions 760 to 985 in the base sequence shown in SEQ ID NO: 1.

[0028] When targeting TDP-43 mRNA of a mammal other than human, the target sequence can be similarly designed by referring to known sequences.

[0029] 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.

[0030] 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.

[0031] Specific examples of antisense nucleic acids include antisense nucleic acids comprising a sequence containing any of the nucleotide sequences represented by SEQ ID NOs: 2 to 4, with antisense nucleic acids comprising any of the nucleotide sequences represented by SEQ ID NOs: 2 to 4 being particularly preferred. These sequences were designed based on the distribution of HNRNPC binding motif sequences as antisense nucleic acids that inhibit the binding of HNRNPC. These sequences exhibit the activity of significantly increasing the splicing efficiency of the TDP-43 excitron.

[0032] The sequences of SEQ ID NOs: 2 to 4 (designated CS2, CS3, and CS4) are shown in Table 1. For CS4, the 11th base should be complementary to G in relation to the target sequence, but a mispair was introduced to prevent the formation of a quadruplex, and this position was designated C (indicated by a lowercase c in the table). The table also shows CS1, which targets a different site from CS2 to CS4 and is used in the Examples described below, and the Ctrl sequence used as a control.

[0033]

[0034] The antisense nucleic acid of this embodiment can be synthesized using known methods. Examples of synthesis methods include synthesis methods using genetic engineering techniques and chemical synthesis methods. Examples of synthesis methods 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 and the H-phosphonate method. Examples of chemical synthesis methods also include methods using commercially available automated nucleic acid synthesizers.

[0035] 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.

[0036] The vector may be any vector capable of expressing the antisense nucleic acid in target cells. The vector may contain a promoter that controls the expression of the antisense nucleic acid. In the vector, the sequence encoding the antisense nucleic acid is operably linked to the promoter.

[0037] 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 thereof 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.

[0038] 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 sorting or selection by introducing the marker gene 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.

[0039] The type of vector is not particularly limited, and any known expression vector can be used, including, for example, a plasmid vector and a viral vector.

[0040] 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.

[0041] 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.

[0042] Among these, a plasmid vector is preferred as the expression vector.

[0043] <Selective Splicing Enhancer> The selective splicing enhancer of this embodiment is an agent for enhancing selective splicing of TDP-43 mRNA, and contains the above-described antisense nucleic acid as an active ingredient.

[0044] The selective splicing enhancer of this embodiment can effectively induce selective splicing of the excitron sequence of TDP-43.

[0045] 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 can be carried out by contacting the subject with the antisense nucleic acid. The administration may be in vivo or in vitro.

[0046] 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.

[0047] The selective splicing enhancer of this embodiment may further comprise 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 transfer reagents include atelocollagen; liposomes; Lipofectamine®, lipofectin, transfectam (dioctadecylamidoglycylspermine; 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.

[0048] <Pharmaceutical Composition> The pharmaceutical composition of this embodiment is used for the prevention or treatment of TDP-43 proteinopathy, and contains the above-described antisense nucleic acid as an active ingredient.

[0049] 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.

[0050] TDP-43 proteinopathy is a general term for neurodegenerative diseases in which TDP-43 protein aggregates and accumulates, and examples thereof include frontotemporal lobar degeneration (FTLD), amyotrophic lateral sclerosis (ALS), etc. FTLD and ALS include both genetically mutated (familial) and sporadic forms. Furthermore, since accumulation of TDP-43 protein has been confirmed 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 degeneration type 3, etc.), myopathies (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, in addition to accumulation of tau protein, amyloid β protein, huntingtin protein, etc., the composition can also be applied to these diseases. Among these TDP-43 proteinopathies, the composition is preferably used for the treatment or prevention of FTLD or ALS.

[0051] 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.

[0052] 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.

[0053] Examples of 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 physiological saline; base waxes, etc.

[0054] 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.

[0055] 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 typically hold a water-soluble substance in the internal phase and a fat-soluble substance within 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 within the lipid bilayer.

[0056] The liposome may be a single-layer membrane or a multi-layer 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.

[0057] 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.

[0058] 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.

[0059] Examples of parenteral administration methods include subcutaneous injection, intramuscular injection, local injection, intraperitoneal administration, and intrathecal administration.

[0060] Preparations suitable for parenteral administration include aqueous and non-aqueous isotonic sterile injection solutions, which may further contain antioxidants, buffers, bacteriostats, isotonicity 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 packaged in unit-dose or multi-dose containers such as ampoules or vials. Alternatively, the active ingredient and a pharmaceutically acceptable carrier can be lyophilized and stored in a state that requires only dissolution or suspension in an appropriate sterile vehicle immediately before use. Other preparations suitable for parenteral administration include aerosols, etc.

[0061] 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.

[0062] 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 (gender, 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.

[0063] 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 neuroprotectants (e.g., edaravone, etc.), glutamate action 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 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 may be reduced from the amount normally used.

[0064] <Therapeutic 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 similar to those described above. Examples of TDP-43 proteinopathy include those similar to 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.

[0065] 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.

[0066] 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.

[0067] The present invention will be described below with reference to examples, but the present invention is not limited to the following examples.

[0068] [Test Example 1] <Sequence design of antisense nucleic acid> (Use of ENCODE shRNA data) The BAM file of the shRNA RNA-seq data from the ENCORE project (HepG2 cells, Genome annotation V29, Genome assembly GRCh38) was downloaded. Using McSplice (2.1.0) (Alqassem et al., 2021, https: / / github.com / canzarlab / McSplicer), an index was created as a reference annotation from GENCODE Release 39 (GRCh 38.p13). The utilization rate of the acceptor site in intron 6 (excitron) of TARDBP was calculated under 100 bootstrap conditions, and the median value was used as the splicing rate for analysis. The sum of the utilization rates of the different acceptor sites in intron 6 was calculated as 3' acceptor utilization rate + 5' acceptor utilization rate × (1 - 3' acceptor utilization rate), and similar sequential processing was performed to calculate the splicing rate for the entire intron 6. These splicing rates were compared with those of the respective control experiments, and Student's t-test was performed.

[0069] (Use of RBPmap) To confirm the presence of an HNRNPC binding motif within the excitron, RBPMap (Paz I, Kosti I, Ares M Jr, Cline M, Mandel-Gutfreund Y. (2014) RBPmap: a web server for mapping binding sites of RNA-binding proteins. Nucleic Acids Res., 2014. https: / / rbpmap.technion.ac.il / ) was used. The sequence from exon 6 to intron 7 of TARDBP was used as input, and analysis of the HNRNPC motif was performed under default conditions, and the Z-score was visualized.

[0070] (Use of ENCODE eCLIP) HNRNPC eCLIP data from the ENCODE project was visualized in IVG (Version 2.15.1) using ENCFF774JCA.

[0071] Figure 1 shows a graphical visualization of ENCODE RNA-Seq. Analysis of knockdown experimental data for 233 RNA-binding proteins provided by the ENCODE project revealed that knockdown of specific RBPs significantly increases excitron splicing activity. Among these, HNRNPA1 (shown by oval b in the figure), which we had already focused on, and HNRNPC (shown by oval a in the figure) were suggested to play important roles in excitron splicing.

[0072] [Test Example 2] <Verification of splicing rate by HNRNPC> (Knockdown of HNRNPC and Western blotting) HEK293T cells were subjected to knockdown of HNRNPC using Lipofectamine RNAiMAX (Invitrogen) and D siRNA (IDT). After 72 hours, protein extraction was performed using RIPA buffer (25 mM Tris-HCl, pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate and 0.1% sodium dodecyl sulfate) containing protease inhibitor cocktail (Sigma). After sonication, the samples were centrifuged at 12,000 x g for 10 minutes at 4°C, and the supernatant was collected. Western blotting was performed using anti-hnRNP C1 / C2 antibody (4F4) (SCB) as the primary antibody and HRP-labeled secondary antibody (Dako) as the secondary antibody. Total protein was detected using No-Stain Protein Labeling Reagent (Invitrogen).

[0073] (Construction of TARDBP minigene and RT-PCR) The region of TARDBP containing exon 5 through the excitron was inserted into the pCDNA3.1 / myc-His(-) A vector. A minigene-specific sequence was inserted into the 5' side of exon 5, excluding the Kozak sequence and translation initiation codon, and RT-PCR analysis was performed using the minigene-specific sequence. The minigene-specific sequences (forward primer: TARDBP minigene (F), reverse primer: TARDBP minigene (R)) are shown in Table 2.

[0074]

[0075] The HNRNPC knockdown HEK293T cells (siHNRNPC) were transfected with a plasmid containing the minigene using X-tremeGene HP DNA transfection reagent (Roche). After 24 hours, RNA was extracted using Nucleospin RNA (Takara Bio), and first-strand cDNA synthesis was performed using PrimeScript RT master mix (Takara Bio). PCR was performed using LA Taq polymerase (Takara Bio) at 94°C for 1 minute, 32 cycles of (94°C for 30 seconds, 55°C for 30 seconds, 72°C for 1 minute), and 72°C for 5 minutes. The densitometry of each band was quantified using ImageQuant TL analysis software, and the splicing rate of intron 6 was compared.

[0076] The primer sequences for RT-PCR used to analyze splicing of the TARDBP excitron were the sequences of TARDBP excitron (F) and (R) shown in Table 3.

[0077]

[0078] Figure 2 shows the knockdown of HNRNPC. (a) is a schematic diagram showing the location of the sequence of the TARDBP minigene transfected into cells. HEK293T cells in which HNRNPC was knocked down (siHNRNPC) were transfected with a minigene containing exon 5 to excitron (intron 6 in the figure) of the TARDBP gene encoding TDP-43. (b) shows the total protein derived from cells in which HNRNPC was knocked down (siHNRNPC, right lane) compared with the control (siCtrl, left lane), demonstrating that HNRNPC expression was knocked down in the knockdown cells. (c) shows the RNA expression level determined by RT-PCR for each cell type similar to (b). When the TARDBP excitron (F) primer (F) and the TARDBP excitron (R) primer (R) are used as the forward primer (F) and reverse primer (R), respectively, a band of large molecular weight (i) can be confirmed if TARDBP is not spliced. If spliced, a band of small molecular weight (ii) can be confirmed. In cells with HNRNPC knockdown, a strong band is observed at the site of splicing, indicating that a reduction in HNRNPC promotes excitron splicing. (d) shows a comparison of the expression levels of the intron band intensity. In (c), the intensity of the band containing the intron (i) and the band not containing the intron (ii) were measured, and the (ii / i) value is shown for siCtrl and siHNRNPC. Cells with HNRNPC knockdown contain more introns, indicating that splicing is promoted.

[0079] Figure 3 is another graph showing the results of ENCODE RNA-Seq analysis. (a) is a diagram of motif analysis of RNA-binding proteins using RBPmap. The binding sites of HNRNPA1 and HNRNPC to the excitron and intron 7 are shown by Z-score. In the figure, the sites marked "NRNPA1 rich" and "HNRNPC rich" indicate that the peaks at those sites are rich in HNRNPA1 and HNRNPC, respectively. HNRNPC has multiple binding motifs on the 3' side of the excitron. (b) shows a comparison with (a) in ECLIP data provided by the ENCODE project. The binding site was supported by both sets of data, demonstrating that HNRNPC indeed binds to this region.

[0080] Test Example 3 Design of Antisense Nucleic Acids (Sequence of Antisense Nucleic Acids) Based on the distribution of HNRNPC binding motif sequences, antisense nucleic acids that inhibit the binding were designed. The designed antisense nucleic acids were designated CS1 to CS4. The sequences of the antisense nucleic acids and controls are shown in Table 1 above. The binding sites of the antisense nucleic acids to excitrons are shown in Figure 4. A control (Ctrl) sequence that does not bind to excitrons was also designed (the site is not shown in Figure 4).

[0081] FIG. 4 is a graph showing the inhibition of HNRNPC binding by each antisense nucleic acid. Using RBPNet, binding prediction to the HNRNPC excitron was performed. The horizontal axis indicates the base position from exon 6, which contains the excitron. The vertical axis indicates the KL divergence calculated from the binding prediction value (target signal) by RBPNet when the 30-base sequence starting from each base position is substituted with an arbitrary base (N) and when it is not substituted (wild type). The larger the KL divergence, the more important that position is for HNRNPC binding, and the greater the binding inhibition effect by the antisense nucleic acid is predicted to be. From the results of the figure, the KL divergence is large at the sites to which CS1 to CS4 bind, and a strong binding inhibition effect could be predicted. In particular, the value is large for the site corresponding to 760 to 985 in SEQ ID NO: 1, which includes CS2 and CS3. It was also speculated that some sequences only inhibit a portion of the HNRNPC binding site, and also suppress binding at adjacent sites.

[0082] (Verification of splicing rate by antisense nucleic acids) The antisense nucleic acids CS1 to CS4 and Ctlr nucleic acid were administered to HEK293T cells. The nucleic acids were added to the medium at a concentration of 10 μM and transfected into human HEK293T cells using Endo-Porter (GeneTools). Cells transfected with a general-purpose control oligonucleotide were also prepared as a control. 48 hours after transfection, RNA was extracted from each cell line using Nucleospin RNA II (Takara Bio), and the alternative splicing efficiency was examined in the same manner as in Test Example 2.

[0083] 5 is a photographic representation of the splicing efficiency of each antisense nucleic acid. As shown in (a), administration of antisense nucleic acids (CS2, CS3, and CS4) targeting HNRNPC to HEK293T cells significantly increased the splicing rate compared to the control nucleic acid (Ctrl).

[0084] Furthermore, simultaneous splicing of the excitron and the 3' intron 7 increases susceptibility to nonsense-mediated mRNA decay, resulting in reduced quantitative detection of splicing bands. Therefore, we analyzed the splicing rate after treatment with cycloheximide (CHX), which inhibits nonsense-mediated mRNA decay. The results are shown in Figure 1(b). Antisense nucleic acids (CS2, CS3, and CS4) targeting the HNRNPC binding region significantly increased the splicing rate compared to the control nucleic acid (Ctrl).

[0085] We also transfected iPS cell-derived neurons with an antisense nucleic acid (CS3) targeting the HNRNPC binding domain. As shown in (c), administration of CS3 significantly increased the splicing rate compared with the control nucleic acid (Ctrl).

[0086] Next, splicing efficiency was compared, including comparison with antisense nucleic acids targeting HNRNPA1 previously reported by the present inventors. AS1-7, AS2.1, AS5.1, AS5.2, AS6.1, and AS6.2 described in Patent Document 3 were used as antisense nucleic acids targeting HNRNPA1. The sequences of these are shown in Table 4.

[0087]

[0088] 6 is a graph showing the splicing efficiency of each antisense nucleic acid targeting HNRNPC and HNRNPA1. The graph shows the band quantification and results of RT-PCR for each antisense nucleic acid administered in the same manner as above. It was suggested that the antisense nucleic acids targeting HNRNPC (CS2, CS3, CS4) have a stronger splicing-promoting effect than the antisense nucleic acids targeting the HNRNPA1 binding region (AS2, AS3, AS4, AS5, AS2.1, AS5.1, AS5.2, AS6.1).

[0089] 7 is a graph showing the splicing efficiency after CHX treatment of antisense nucleic acids targeting HNRNPC and HNRNPA1. Analysis after CHX treatment also showed that the effects of antisense nucleic acids targeting the HNRNPC binding region (CS2, CS3, CS4) were stronger than those of antisense nucleic acids targeting the HNRNPA1 binding region (AS2, AS3, AS4, AS5, AS2.1, AS5.1, AS5.2, AS6.1).

[0090] [Test Example 4] <CSE1L knockdown experiment in HEK293 cells> The selective splicing enhancing effect of each antisense nucleic acid was examined under conditions mimicking TDP-43 proteinopathy, in which intranuclear TDP-43 is reduced and cytoplasmic TDP-43 accumulates.

[0091] Specifically, in HEK293T cells, CSE1L, which is involved in the nuclear translocation of TDP-43, was knocked down by RNA interference (On-TARGETplus siRNA, manufactured by Dharmacon) using Lipofectamine RNAiMAX (manufactured by Invitrogen).

[0092] These knockdown cells were administered CHK and antisense nucleic acids, and RNA expression levels were examined, as in Test Examples 2 and 3. Figure 8 is a photograph showing the selective splicing enhancement effect of antisense nucleic acids in CSE1L knockdown cells. (a) shows the RNA expression level, and (b) shows a comparison of expression levels. In HEK293T cells, knockdown of CSE1L (siCSE1L) reduced the excitron (intron 6) splicing rate, but antisense nucleic acid (C3) targeting the HNRNPC binding region improved this.

[0093] Test Example 5 CSE1L Knockdown Experiment in SHS-SY5Y Cells (Protein Expression Analysis by CSE1L Knockdown in SH-SY5Y Cells) Human neuroblastoma-derived SH-SY5Y cells were cultured in DMEM / F-12 medium containing 10% FBS and GlutaMAX™. For CSE1L knockdown, Lipofectamine RNAiMAX reagent was used for transfection, and ON-TARGETplus Human CSE1L siRNA-SMARTpool or ON-TARGETplus Non-targeting Control siRNA as a control were transfected at a final concentration of 20 nM.

[0094] After 48 hours, proteins from the nuclear and cytoplasmic fractions were extracted using NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Scientific). Anti-Lamin A / C 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. Expression of CSE1L was confirmed using anti-CSE1L antibody (Abcam, ab151546), and TDP-43 expression in each fraction was analyzed by Western blotting using anti-TDP-43 antibody (Proteintech, 12892-1-AP).

[0095] Figure 9 is a photograph showing protein analysis of CSE1L knockdown cells after administration of antisense nucleic acid. (a) shows a Western blot image. (b) shows the expression ratio based on the band intensity ratio of nuclear TDP-43 / Lamin C. Here, the nuclear membrane protein Lamin A / C was used as an endogenous control for nuclear proteins. As shown in the figure, knocking down CSE1L (siCSE1L) in neuroblastoma (SH-SY5Y cells) reduced nuclear TDP-43.

[0096] Test Example 6: Test for Functional Recovery by Antisense Nucleic Acid in SHS-SY5Y Cells. SH-SY5Y cells were subjected to CSE1L knockdown using the same procedure as in Test Example 5, and functional recovery by antisense nucleic acid (ASO, antisense oligo) was examined. 24 hours after CSE1L knockdown, antisense nucleic acid was introduced at a final concentration of 10 μM and the cells were cultured for an additional 48 hours. Standard control morpholino oligo was used as the control nucleic acid, and CS3 was used in the experimental group. Endo-Porter PEG reagent was used to introduce antisense nucleic acid at a final concentration of 6 μM. Total RNA was extracted using the NucleoSpin RNA II kit. Reverse transcription was performed using Prime Script RT Master Mix to prepare cDNA. Real-time PCR was performed using SYBR Green Premix Ex Taq II with a Thermal Cycler Dice Real Time System III (TaKaRa).

[0097] Primers for detecting total STMN2 expression were used to analyze STMN2 expression levels. The primers used for STMN2 expression level analysis are shown in Table 5 (forward primer: STMN2(F), reverse primer: STMN2(R)). The endogenous reference gene RPLP1 for quantitative PCR was used as an endogenous control. The RPLP1 primer used was HA067802 (Takara). To examine excitron splicing efficiency, real-time PCR was performed using three primer sets to specifically detect two splicing variants and one unspliced ​​product. The primers used are shown in Table 6. Specifically, the forward primers (splicing 1(F) and splicing 2(F)) and the common reverse primer (splicing(R)) used to detect the two splicing variants are shown, as well as the primers (forward primer: unspliced(F), reverse primer: unspliced(R)) used to detect the unspliced ​​product. The influence of these expression levels on the splicing efficiency of intron 6 was compared. Statistical analysis was performed using R. After confirming equal variance among the three groups with a Bartlett test, one-way analysis of variance and multiple comparison tests using the Tukey method were performed. A significance level of less than 5% was considered significant.

[0098]

[0099]

[0100] Figure 10 is a graph showing the results of a functional recovery test using antisense nucleic acids. (a) shows the spliced / unspliced ​​mRNA ratio, and (b) shows the STMN2 / RPLP1 mRNA ratio. In SH-SY5Y cells with reduced CSE1L expression (siCSE1L), real-time quantitative PCR showed reduced excitron (intron 6) splicing, but this was improved by antisense nucleic acid (C3) targeting the HNRNPC binding region.

[0101] Test Example 7: Intracellular localization of TDP-43 following CSE1L knockdown in SHS-SY5Y cells. Human neuroblastoma-derived SH-SY5Y cells were seeded onto a μ-Slide 8-well ibiTreat (Ibidi) plate coated with Matrigel Matrix in DMEM / F12 medium containing 10% fetal bovine serum (FBS) and GlutaMAX. ON-TARGETplus Human CSE1L siRNA - SMARTpool was used for CSE1L knockdown. ON-TARGETplus Non-targeting Control siRNA was used as a control. siRNA was transfected at a final concentration of 20 nM. Lipofectamine RNAiMAX reagent was used for transfection. 24 hours later, standard control morpholino oligos or TDP-43-specific antisense nucleic acids (CS3) were introduced at a final concentration of 10 μM using Endo-Porter PEG reagent. At the time of antisense nucleic acid transfection, differentiation medium was switched to DMEM / F12 medium supplemented with 1% FBS, 10 μM retinoic acid, and 50 ng / mL BDNF. 48 hours after antisense nucleic acid administration, cells were fixed with 4% paraformaldehyde, washed with PBS, and then treated with 0.1% Triton X-100 for 5 minutes to permeabilize the cell membrane. After blocking the samples with 5% bovine serum albumin (BSA), anti-TDP-43 antibody (12892-1-AP) and anti-TUJ1 antibody (ab7751) were added as primary antibodies and incubated overnight at 4°C. After washing with PBS, the samples were incubated with Alexa Fluor 568-labeled goat anti-rabbit IgG and Alexa Fluor 488-labeled goat anti-mouse IgG as secondary antibodies for 1 hour at room temperature in the dark. Ibidi Mounting Medium with DAPI was used for mounting. Fluorescence images were captured using a BIOREVO BZ-9000 (KEYENCE) with a 40x objective. The obtained images were analyzed using ImageJ software, and the TDP-43 intensity in the nucleus and cytoplasm of each cell was quantified.200 cells were randomly selected from each group, and the ratio of nuclear / cytoplasmic TDP-43 brightness was calculated, followed by ANOVA and multiple comparison test by Tukey's method.

[0102] Figure 11 is a graph showing the intracellular localization of TDP-43 following CSE1L knockdown in SHS-SY5Y cells. (a) shows the localization of TDP-43 in SH-SY5Y cells subjected to CSE1L knockdown using siRNA and treatment with antisense nucleic acid (CS3 or control). Scale bar: 20 μm. The spectrum represents the fluorescence intensity of TDP-43, and the black line indicates the outline of the nucleus. (b) is a graph showing the nuclear to cytoplasmic expression ratio of TDP-43. 200 cells were analyzed for each condition, and the fluorescence intensity ratio of TDP-43 in the nucleus and cytoplasm was calculated.

[0103] [Test Example 8] <TDP-43 pathology in an iPS cell-derived neural model and functional recovery by antisense nucleic acid> In order to verify the therapeutic effect of TARDBP excitron splicing targeted therapy in a pathology-related model of TDP-43 proteinopathy, an iPS cell-derived neural model was prepared in which CSE1L, which is essential for the nuclear transport of TDP-43 and has been reported to be reduced in the frontal lobe of FTLD-TDP patients, was knocked down.

[0104] iPS cell-derived neurons (ReproNeuro, ReproCell, RCDN001N) were coated onto μ-Slide 8-well ibiTreat slides (ibidi) according to the manufacturer's protocol and cultured in 200 μL of dedicated medium. Thirteen days after seeding, CSE1L knockdown was performed using a recombinant lentivirus expressing shRNA. Specifically, lentivirus containing shRNA targeting hCSE1L (LVS(VB230809-1203tuz)-K1, pLV[shRNA]-Puro-U6>hCSE1L[shRNA#1]) was added to 150 μL of medium at an MOI of 5. As a control, a lentivirus containing a scrambled shRNA (LVS(VB010000-9460fht)-K1, pLV[shRNA]-Puro-U6>Scramble[shRNA#1]) was used at the same MOI. Antisense nucleic acids (CS3 or control) were administered on the day after viral transduction (day 14).

[0105] For immunocytochemical staining, 6 days after administration of the antisense nucleic acid (day 20), cells were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, and blocked with 5% BSA. Primary antibodies included anti-TDP-43 antibody (Proteintech, 12892-1-AP, 1:200 dilution) and anti-TUJ1 antibody (Abcam, ab7751, 1:400 dilution), and the cells were incubated overnight at 4°C. Secondary antibodies included Alexa Fluor 568-labeled goat anti-rabbit IgG and Alexa Fluor 488-labeled goat anti-mouse IgG (both diluted 1:400), and the cells were incubated for 1 hour at room temperature. The sections were mounted using DAPI-containing mounting medium (ibidi, ib50011) and photographed using a BIOREVO BZ-9000 microscope (KEYENCE). Nuclear and cytoplasmic regions were defined based on DAPI and TUJ1 signals using ImageJ software, and TDP-43 fluorescence intensity was measured in more than 400 neurons per condition. For statistical analysis, 400 neurons were randomly selected from each condition and analyzed using R. The primer sequences targeting POLDIP3 ver1 and ver2 and their respective targets are shown in Table 7, SEQ ID NOs: 29 to 31.

[0106]

[0107] Figure 12 shows immunostaining images showing the intracellular localization of TDP-43 in an iPS cell-derived neural model. The images show TUJ1 (neuron marker), Hoechst (nuclear stain), TDP-43, and the TDP-43 fluorescence intensity spectrum for the control (shCtrl+Ctrl), CSE1L knockdown model (shCSE1L+Ctrl), and the group treated with antisense nucleic acid after CSE1L knockdown (shCSE1L+CS3). It was observed that CSE1L knockdown reduced nuclear TDP-43, and that its localization was partially restored by CS3 treatment.

[0108] Figure 13 is a graph showing the results of quantitative analysis of various parameters in the iPS cell-derived neural model. (a) shows the nuclear / cytoplasmic fluorescence intensity ratio of TDP-43. This ratio was significantly reduced by CSE1L knockdown (shCSE1L + Ctrl) (p<0.001) and significantly restored by CS3 treatment (shCSE1L + CS3) (p<0.001), indicating that CS3 improves the nuclear localization of TDP-43. (b) shows the splicing efficiency of excitron (intron 6). The ratio of spliced / unspliced ​​mRNA measured by real-time PCR is displayed on a log2 scale. The primers listed in Table 6 were used for quantitative PCR. Splicing efficiency was reduced by CSE1L knockdown but significantly restored by CS3 treatment (p<0.001). (c) shows the STMN2 expression level on a log2 scale. Quantitative PCR was performed using STMN2(F) and STMN2(R) primers, with RPLP1 used as an endogenous control. CSE1L knockdown significantly reduced STMN2 expression (p=0.007), which was restored by CS3 treatment (p=0.004). (d) shows aberrant splicing of POLDIP3 (ver2 / ver1 ratio). POLDIP3 is considered one of the most sensitive indicators of TDP-43 dysfunction. CSE1L knockdown increased aberrant splicing (p<0.001), which was significantly suppressed by CS3 treatment (p<0.001). These results indicate that even under conditions in which TDP-43 nuclear transport is impaired, promotion of excitron splicing by an antisense nucleic acid (CS3) that inhibits HNRNPC binding suppresses the decline in TDP-43 nuclear function and interrupts the pathological spiral (a vicious cycle of TDP-43 aggregation and loss of nuclear function).

[0109] The antisense nucleic acid, splicing enhancer, and pharmaceutical composition of the present invention can improve abnormal splicing of TDP-43 excitrons and effectively suppress the functional decline of intranuclear TDP-43 associated with abnormal accumulation of TDP-43, and can be applied to the treatment of neurodegenerative diseases.

Claims

1. An antisense nucleic acid that promotes the splicing of TDP-43, which targets the base sequence from position 760 onwards in the base sequence represented by SEQ ID NO: 1 and contains a base sequence that is complementary to a sequence consisting of 10 or more consecutive bases in the target sequence.

2. The antisense nucleic acid according to claim 1, which targets the 760th to 985th positions of the base sequence represented by SEQ ID NO:

1.

3. The antisense nucleic acid according to claim 1, which consists of a base sequence represented by any one of SEQ ID NOs: 2 to 4.

4. A selective splicing enhancer for TDP-43 mRNA, comprising the antisense nucleic acid according to claim 1 or 2 as an active ingredient.

5. A pharmaceutical composition containing the antisense nucleic acid according to claim 1 or 2 as an active ingredient, which is used for the prevention or treatment of TDP-43 proteinopathy.

6. The pharmaceutical composition according to claim 5, wherein the TDP-43 proteinopathy is frontotemporal lobar degeneration or amyotrophic lateral sclerosis.

Images