Antisense nucleic acids and use of same

Abstract

Provided are antisense nucleic acids, a splicing promotor, and a pharmaceutical composition that, by inhibiting binding of a RNA-binding protein HNRNPA1 to an exitron more effectively than with conventional methods, can improve splicing abnormalities in an exitron of TDP-43, that can effectively suppress functional deterioration of intranuclear TDP-43 associated with abnormal accumulation of TDP-43, and that can be applied for treating neurodegenerative disorders. The present invention pertains to: antisense nucleic acids which promote splicing of TDP-43, which target a target sequence from position 136 to position 170 or from position 291 to position 339 in the base sequence represented by SEQ ID NO. 1, and which each have a base sequence complementary to a sequence formed of consecutive 10-30 bases in the target sequence; and a splicing promotor and a pharmaceutical composition containing the antisense nucleic acids.

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 that enhance alternative splicing of TDP-43 mRNA, and pharmaceutical compositions. This application claims priority to Japanese Patent Application No. 2024-107386, filed July 3, 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 by the present inventors in Patent Documents 3 and 4, methods for promoting splicing of TDP-43 excitrons have been shown to be 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 need for new strategies to directly and effectively regulate abnormal TDP-43 accumulation 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 restored in cells, the progression of these diseases could be delayed or even reversed. In other words, methods to promote the splicing of these TDP-43 excitrons are expected to be an innovative therapeutic approach. However, methods to promote this splicing more efficiently have not yet been fully explored. There is a strong need for the development of antisense nucleic acids that can more effectively regulate target regions that affect TDP-43 splicing than previously known methods.

[0011] The present invention has been made in view of the above circumstances, and its object is to provide an antisense nucleic acid, a splicing enhancer, and a pharmaceutical composition that can improve the abnormal splicing of the TDP-43 excitron by inhibiting the binding of the RNA-binding protein HNRNPA1 to the excitron more effectively than conventional methods, thereby effectively suppressing the functional decline of intranuclear TDP-43 associated with the abnormal accumulation of TDP-43, and that can be applied to the treatment of neurodegenerative diseases.

[0012] As a result of extensive research to achieve the above-mentioned objective, the inventors have searched for target sequences that more effectively inhibit the binding of HNRNPA1 to excitrons, identified several effective antisense nucleic acid sequences from the predicted target sequences, and verified their effectiveness, thereby discovering the present invention.

[0013] That is, the present invention includes the following aspects: (1) An antisense nucleic acid that promotes TDP-43 splicing, wherein the target sequence is from positions 136 to 170 or from positions 291 to 339 of the base sequence represented by SEQ ID NO: 1, and comprises a base sequence complementary to a sequence consisting of 10 to 30 consecutive bases in the target sequence. (2) The antisense nucleic acid according to (1), consisting of a base sequence represented by any of SEQ ID NOs: 2 to 4. (3) An agent for enhancing selective splicing of TDP-43 mRNA, comprising as an active ingredient the antisense nucleic acid according to (1) or (2). (4) A pharmaceutical composition used for preventing or treating TDP-43 proteinopathy, comprising as an active ingredient the antisense nucleic acid according to (1) or (2). (5) The pharmaceutical composition according to (4), 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 inhibit the binding of the RNA-binding protein HNRNPA1 to excitrons more effectively than conventional methods, thereby improving abnormal splicing of the TDP-43 excitron and effectively suppressing 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]

[0033] Figure 1 is a graph showing the inhibition of HNRNPA1 binding by each antisense nucleic acid.

[0034] Figure 2 is a photograph showing the results of RT-PCR indicating the splicing efficiency by each antisense nucleic acid of this example.

[0035] Figure 3 is a graph analyzing the relationship between the predicted binding value of RBPNet and the actual splicing promoting effect.

[0036] Figure 4 is a diagram showing verification of splicing rate in iPS cell-derived neurons.

[0037] Figure 5 is a graph showing the relative splicing efficiency when each antisense nucleic acid is introduced, with the control (Ctrl) set to 1.

[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] The present inventors searched for antisense nucleic acids with higher splicing activity that specifically target the binding region of HNRNPA1, an RNA-binding protein (RBP) deeply involved in the splicing of the TDP-43 excitron. To this end, the present inventors utilized a machine learning model called RBPNet, which uses the CLIP database, to find target sequences that effectively inhibit HNRNPA1 binding to the excitron. By examining the KL divergence calculated from binding predictions for the wild-type and for sequences within the excitron where 25 bases are consecutively substituted with an arbitrary base (N), sequences that effectively inhibit HNRNPA1 binding were predicted. From these predicted target sequences, multiple effective antisense nucleic acid sequences could be identified.

[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 positions 136 to 170 or from positions 291 to 339 of the base sequence represented by SEQ ID NO: 1, and contains a base sequence that is complementary to a sequence consisting of 10 to 30 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 HNRNPA1-binding region within excitrons and inhibits the binding of HNRNPA1, 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 cell protection. In addition, the novel antisense nucleic acid of this embodiment can promote splicing more efficiently than previously known antisense nucleic acids targeting the HNRNPA1-binding region. 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). Specifically, the target sequence is from positions 136 to 170 or from positions 291 to 339 of the excitron sequence of human TDP-43 (SEQ ID NO: 1). The antisense nucleic acid of this embodiment targets a site selected from the HNRNPA1 binding region.

[0027] When targeting TDP-43 mRNA of a mammal other than human, the target sequence can be similarly designed by referring to known sequences.

[0028] The length of the antisense nucleic acid of this embodiment is 10 to 30 bases, preferably 15 to 30 bases, more preferably 20 to 30 bases, even more preferably 22 to 28 bases, particularly preferably 24 to 26 bases, and more particularly preferably 25 bases.

[0029] 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.

[0030] Specific examples of antisense nucleic acids include antisense nucleic acids comprising a sequence containing a base sequence represented by any one of SEQ ID NOs: 2 to 4. In this embodiment, the sequence of SEQ ID NO: 2 will be referred to as AS3.1, the sequence of SEQ ID NO: 3 as AS5.3, and the sequence of SEQ ID NO: 3 as AS5.4. AS3.1 is complementary to 25 bases from positions 141 to 165 of the base sequence of SEQ ID NO: 1, AS5.3 is complementary to 25 bases from positions 296 to 320 of the base sequence of SEQ ID NO: 1, and AS5.4 is complementary to 25 bases from positions 304 to 328 of the base sequence of SEQ ID NO: 1. In other words, it is also preferable to use antisense nucleic acids that target sequences within 12 sequences, preferably within 10 sequences, and more preferably within 5 sequences of AS3.1, AS5.3, or AS5.4 of the base sequence of SEQ ID NO: 1 (for example, when selected from sequences with a high HNRNPA1 binding inhibitory effect (KL_Divergence > 0.1), the 5 sequences before and after AS3.1, from positions 136 to 170, or for sequences including partially overlapping AS5.3 and AS5.4, the 5 sequences before and 11 sequences after, from positions 291 to 339).

[0031] The antisense nucleic acid of this embodiment is preferably an antisense nucleic acid consisting of a base sequence represented by any one of SEQ ID NOS: 2 to 4. These sequences are designed as antisense nucleic acids of sequences predicted to effectively inhibit the binding of HNRNP1. 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 (AS3.1, AS5.3, AS5.4) are shown in Table 1. The table also shows the Ctrl antisense sequence (SEQ ID NO: 5) used as a control in the Examples described later.

[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 include 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, but Pol III promoters are preferred from the viewpoint of more accurate transcription of relatively short nucleic acids. 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.

[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] <Design of Antisense Nucleic Acid> (Search for Antisense Nucleic Acid Sequences) Based on the distribution of HNRNPA1 binding motif sequences, antisense nucleic acids that inhibit the binding of HNRNPA1 were designed. Based on the HNRNPA1 sequence, binding prediction for exon 6, which contains an excitron, was performed using RBPNet for each sequence of 25 base pairs of antisense nucleic acid, in an attempt to discover antisense nucleic acid sequences that can be predicted to inhibit HNRNPA1 binding particularly efficiently.

[0069] FIG. 1 is a graph showing the inhibition of HNRNPA1 binding by each antisense nucleic acid. Binding prediction of HNRNPA1 to the excitron was performed using RBPNet. 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 25-base sequence starting from each base position is substituted with an arbitrary base (N) and when not substituted (wild type). The larger the KL divergence, the more important that position is for HNRNPA1 binding, and the greater the predicted effect of binding inhibition by antisense nucleic acids.

[0070] Of the sequences shown in Figure 1, AS1 to AS5, AS5.1, AS5.2, and AS6 to AS6.1 are sequences found in the prior art. These sequences are shown in SEQ ID NOs: 6 to 15 in Table 2.

[0071]

[0072] In addition to these conventional sequences, the sequences AS3.1, AS5.3, and AS5.4 in the figure have a larger KL divergence than the conventionally known sequences, and are predicted to have a stronger binding inhibitory effect.

[0073] AS3.1 was a nucleotide sequence complementary to 25 bases from positions 141 to 165 of the nucleotide sequence of SEQ ID NO: 1, AS5.3 was a nucleotide sequence complementary to 25 bases from positions 296 to 320 of the nucleotide sequence of SEQ ID NO: 1, and AS5.4 was a nucleotide sequence complementary to 25 bases from positions 304 to 328 of the nucleotide sequence of SEQ ID NO: 1. The nucleotide sequences of AS3.1, AS5.3, and AS5.4 are shown in Table 1 above.

[0074] [Test Example 2] <Verification of splicing rate in HEK293 cells> The splicing rate was verified for AS3.1, AS5.3, and AS5.4, which were predicted to have a strong binding inhibitory effect. Each antisense nucleic acid was introduced into HEK293 cells, and the effect of HNRNPA1 on the splicing rate was examined.

[0075] 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 min, followed by 32 cycles of (94°C for 30 sec, 55°C for 30 sec, 72°C for 1 min), and 72°C for 5 min. 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 the TARDBP excitron (F) and (R) shown in SEQ ID NO: 16 and SEQ ID NO: 17 in Table 3.

[0077]

[0078] (Verification of splicing rate by antisense nucleic acids) The antisense nucleic acids AS3.1, AS5.3, AS5.4, and Ctlr nucleic acids were administered to HEK293T cells. The nucleic acids were added to the medium at a concentration of 10 μM and transfected into the human HEK293T cells using Endo-Porter (GeneTools). Cells transfected with a general-purpose control oligonucleotide were also prepared as a control. Forty-eight hours after transfection, RNA was extracted from each cell line using Nucleospin RNA II (Takara Bio Inc.), and the alternative splicing efficiency was examined.

[0079] The efficiency of alternative splicing can be confirmed from the intensity of molecular weight bands (not shown) in electrophoresis of extracted RNA. When TARDBP excitron (F) is used as the forward primer (F) and TARDBP excitron (R) is used as the reverse primer (R), if TARDBP is not spliced, a band (i) with a large molecular weight can be confirmed. If spliced, a band (ii) with a small molecular weight can be confirmed. The splicing efficiency can be confirmed from the ratio of the band signal intensities, (ii) / (i).

[0080] Furthermore, simultaneous splicing of the excitron and the 3' intron 7 increases susceptibility to nonsense-mediated mRNA decay, reducing the quantitative accuracy of the splicing band. Therefore, we analyzed the splicing rate after treatment with cycloheximide (CHX), which inhibits nonsense-mediated mRNA decay.

[0081] Figure 2 is a photograph showing the results of RT-PCR demonstrating the splicing efficiency of each antisense nucleic acid in this example. The (ii) / (i) ratio was calculated for each antisense nucleic acid administration example (control) and the control, and the ratio of each antisense nucleic acid to the control is shown. When antisense nucleic acids (AS3.1, AS5.3, AS5.4) predicted to efficiently inhibit NRNPA1 binding were introduced into HEK293T cells, the excitron (intron 6) splicing rate was significantly increased compared to the control nucleic acid (Ctrl). These results demonstrate that the antisense nucleic acids (AS3.1, AS5.3, AS5.4) actually increase the splicing rate in cells.

[0082] FIG. 3 is a graph analyzing the relationship between RBPNet's predicted binding value and actual splicing-promoting effect. A scatter plot of each antisense nucleic acid is shown, with the horizontal axis representing KL divergence calculated from the RBPNet's predicted binding value (target signal) and the vertical axis representing the actual splicing-promoting effect. In the figure, small circles represent each experimental value, and large circles represent the average value for each group. The antisense nucleic acids of this example (AS3.1, AS5.3, AS5.4) had a greater excitron (intron 6) splicing-promoting effect than previously disclosed antisense nucleic acids (AS1 to AS5, AS5.1, AS5.2, AS6 to AS6.1), and showed a positive correlation with KL divergence.

[0083] [Test Example 3] <Verification of splicing rate in iPS cell-derived neurons> To examine the effect of antisense nucleic acids on iPS cell-derived neurons (ReproNeuro) (ReproCell, RCDN001N), the cells were seeded onto a 24-well plate and cultured according to the manufacturer's instructions. 14 days after seeding, the Ctrl nucleic acid, a conventionally known AS5 antisense nucleic acid, and the antisense nucleic acid AS5.4 and Ctrl nucleic acids of the present embodiment were administered using the method described above. After an additional 3 days of culture, the splicing rate was verified in the same manner as in Test Example 2.

[0084] Figure 4 shows the verification of splicing rates in iPS cell-derived neurons. (a) shows the band intensities of gel electrophoresis indicating RNA expression levels by RT-PCR. The left side of the figure is a gel photograph, and the right side is a schematic diagram of splicing. As shown in the schematic diagram, the large molecular weight (i) indicates the unspliced ​​band, and the small molecular weight (ii) indicates the spliced ​​band. The splicing rate can be determined by comparing the signal intensities of these bands. (b) The intensities of the intron-containing band (i) and the intron-free band (ii) were measured, and the (ii / i) values ​​are shown for Ctrl, AS5, and AS5.4. Splicing was significantly promoted in cells treated with AS5 and AS5.4 compared to Ctrl, but AS5.4 was particularly efficient compared to AS5. These results demonstrate that the antisense nucleic acid of this embodiment has a higher splicing efficiency in iPS cell-derived neurons than conventionally known antisense nucleic acids.

[0085] [Test Example 4] <Further optimization of antisense nucleic acids> Based on the results of Test Example 2 and the correlation between KL divergence and splicing-promoting effect shown in Figure 3, further optimization of antisense nucleic acids was attempted around the target region of AS5.4, which was particularly promising. AS5.5 (SEQ ID NO: 18) and AS5.6 (SEQ ID NO: 19), shown in Table 4 below, were newly designed as antisense nucleic acids targeting sequences adjacent to or partially overlapping the target region of AS5.4.

[0086]

[0087] To verify the effects of these novel antisense nucleic acids (AS5.5, AS5.6), AS5.4 was used as a comparison and Ctrl nucleic acid was used as a negative control. Using the same method as in Test Example 2, each antisense nucleic acid was introduced into HEK293T cells, and RNA was extracted from the cells after CHX treatment, followed by RT-PCR to calculate the excitron splicing rate (excitron exclusion / inclusion ratio). The results were subjected to one-way analysis of variance (ANOVA) and multiple comparison testing using the Tukey method.

[0088] Figure 5 is a graph showing the relative splicing efficiency when each antisense nucleic acid was introduced, with the control (Ctrl) set to 1. In the figure, bars indicate mean values, error bars indicate standard errors, and asterisks () indicate statistical significance (*, p < 0.05; **, p < 0.01; ***, p < 0.001). As shown in Figure 5, AS5.5 and AS5.6 promoted splicing extremely strongly compared to the control (Ctrl). Furthermore, AS5.5 and AS5.6 had significantly higher splicing-promoting effects than AS5.4, which had already been confirmed to be highly effective. These results demonstrate that AS5.5 and AS5.6 have significant splicing-promoting activity exceeding that of conventional techniques and are highly promising candidates for therapeutic agents for TDP-43 proteinopathy.

[0089] The antisense nucleic acid, splicing enhancer, and pharmaceutical composition of the present invention can inhibit the binding of the RNA-binding protein HNRNPA1 to excitrons more effectively than conventional methods, thereby improving abnormal splicing of the TDP-43 excitron and effectively suppressing 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 positions 136 to 170 or from positions 291 to 339 of the base sequence represented by SEQ ID NO: 1 and contains a base sequence that is complementary to a sequence consisting of 10 to 30 consecutive bases in the target sequence.

2. 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.

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

4. 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.

5. The pharmaceutical composition according to claim 4, wherein the TDP-43 proteinopathy is frontotemporal lobar degeneration or amyotrophic lateral sclerosis.

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