Compositions and methods for reducing tau expression

Abstract

To provide compositions and methods for decreasing tau mRNA and protein expression.SOLUTION: Provided are: an oligonucleotide comprising a nucleobase sequence having 90% identity to any of the specific nucleobase sequences, where C in any of these nucleobase sequences is either cytosine or 5-methylcytosine and at least one nucleotide of the oligonucleotide has 2'-modification; a composition comprising the oligonucleotide and a pharmaceutically acceptable carrier; and a method for decreasing the tau expression level in a subject comprising administering to the subject a therapeutically effective amount of the oligonucleotide.SELECTED DRAWING: None

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the benefit of U.S. Provisional Patent Application No. 62 / 270,165, filed on Dec. 21, 2015, which is incorporated herein by reference in its entirety.

[0002] Technical Field The present invention provides compositions and methods for reducing tau mRNA and protein expression. These compositions and methods are useful for treating tau - related diseases.

[0003] Background Tau is a microtubule - associated protein that stabilizes microtubules and facilitates axonal transport. The tau protein interacts with tubulin to stabilize microtubules and promotes the assembly of tubulin into microtubules. The microtubule network is involved in many important cellular processes, including the formation of the cytoskeleton, the maintenance of cell structure and morphology, and the provision of a platform for the intracellular transport of vesicles, organelles, and macromolecules. The binding of tau to microtubules stabilizes the microtubules, so tau is one of the most important mediators in these cellular processes.

[0004] At least six tau isoforms exist in the human brain and are in the range of 352 - 441 amino acid residues in length. Tau isoforms are derived from a single gene, MAPT (microtubule - associated protein tau), located on chromosome 17. MAPT transcripts undergo complex, regulated alternative splicing to yield multiple mRNA species. Exons 2 and 3 of MAPT encode sequences of 29 - or 58 amino acids, respectively, and thus the exons ​​​​​​​​​​​​ Alternative splicings for Son 2 and / or 3 are 0N, 1N, or 2N tau, and These are referred to as zero, one, or two copies of the 29-amino acidic domain at the N-terminus. This results in inclusion. Exon 10 of MAPT encodes a microtubule-binding domain, Therefore, the inclusion of exon 10 leads to the presence of an additional microtubule-binding domain. Tau has other Because there are three microtubule-binding domains at that location, it contains the tau isoform with exon 10. "4R tau" refers to a tau protein that has four repeats of the microtubule-binding domain. It is called "3R tau". Tau isoforms that do not contain exon 10 are called "3R tau", and This refers to a tau protein that has three repeats of the microtubule-binding domain. 4R tau The isoform appears to bind more strongly to microtubules than the 3R isoform, and The reason is that the 4R tau isoform has one more microtubule-binding domain. The ratio of 3R tau to 4R tau is regulated during development, and fetal tissue is predominantly 3R tau. By expressing tau, adult human tissue expresses approximately equal levels of 3R tau and 4R tau. .

[0005] Tau is a phosphoprotein, and in its longest tau isoform, it has approximately 85 phosphate groups. It has a morphogenic site (Ser, Thr, or Tyr) (Pedersen and Sigurdsson, T rends in Molecular Medicine 2015, 21 (6): 394). Phosphorylation is normal tau protein. It has been reported in about half of these sites. Tau is constantly phosphorylated during the cell cycle. It is then dephosphorylated. Tau can only associate with microtubules in its dephosphorylated form. Therefore, tau phosphorylation directly switches microtubule association-dissociation within neurons. It acts as a tau protein. Under pathological conditions, tau protein is excessively phosphorylated, and tau This leads to loss of brin bonds and microtubule destabilization, followed by pathogenic neurofibrillary tangles. Aggregation and deposition of tau occurs. Protease-cleaved fragments of tau (Asp13, Glu391 and Asp421 have also been identified in neurofibrillary tangles.

[0006] Summary of the Invention Antisense oligonucleotides targeting tau (MAPT), a protein associated with human microtubules. Cleotide, composition comprising the antisense oligonucleotide, and these antisense Using lance oligonucleotides to reduce tau mRNA and protein expression Methods are provided herein. The compositions and methods provided herein relate to tau. It is useful in treating diseases.

[0007] In one embodiment, at least 70% of any of the nucleic acid base sequences shown in Tables 2-17 ( For example, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, Nucleic acid base sequences containing sequence identity of 97%, 98%, 99%, or 100% A ligonucleotide in which any of the nucleotide base sequences is cytosine or 5- It is either methylcytosine, and at least one nucleotypic of the oligonucleotide Oligonucleotides having a 2'-modification are provided herein. The nucleotide is an antisense oligonucleotide that targets human MAPT. 2 The 2'-modification can be selected from the group consisting of 2'-fluoro, 2'-deoxy-2'-fluoro, 2'-O-methyl, 2 '-O-methoxyethyl (2'-O-MOE), 2'-O-aminopropyl (2'-O- AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimeth ylaminopropyl (2'-O-DMAP), 2'-O-dimethylaminoethyloxyeth yl (2'-O-DMAEOE), and 2'-O-N-methylacetamide (2'-O- NMA). In some embodiments, the 2'-modification is 2'-O-methoxyethyl (2'-O-MOE). In some embodiments, [[ID=I2]]each C in any of the nucleobase sequences is 5-methylcytosine.

[0008] In some embodiments, the antisense oligonucleotides provided herein are 12 to 30 nucleobases in length. For example, an antisense oligonucleotide targeting MAPT can comprise 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleobases. In some embodiments, an antisense oligonucleotide targeting MAPT is 12 to 25 nucleobases in length. For example, an antisense oligonucleotide targeting MAPT can comprise 12, 13, 14, 15, 16, 17, 18, 19, 20 , 21, 22, 23, 24, or 25 nucleobases. In some embodiments, an antisense oligonucleotide targeting MAPT is 15 to 20 nucleobases in length. For example, an antisense oligonucleotide targeting MAPT can comprise 15, 16, 17, 18, 19, or 20 nucleobases. ' D can comprise 15, 16, 17, 18, 19, or 20 nucleobases.

[0009] In some embodiments, the antisense oligonucleotides targeting MAPT provided herein are steric blockers. Such antisense oligonucleotides reduce the expression of tau mRNA and / or protein independently of RNAseH. The internucleoside linkages of the steric blockers can be either phosphodiester or phosphorothioate linkages. In some embodiments, the antisense oligonucleotides targeting MAPT provided herein have at least 70% (e.g., 70%, 75%, 80%, 81%, 82%, 83%, <于 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, <于 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity with any of the sequences shown in Tables 2-8 and comprise a steric blocker having a nucleobase sequence, where C in any of the nucleobase sequences is either cytosine or 5-methylcytosine, and each nucleotide of the oligonucleotide has a 2'-modification. In some embodiments, the antisense oligonucleotides targeting MAPT have at least <于 80% sequence identity with any of the sequences shown in Tables 2-8. In some embodiments, the antisense oligonucleotides targeting MAPT have at least <于 90% sequence identity with any of the sequences shown in Tables 2-8. In some embodiments, the antisense oligonucleotides targeting MAPT have the sequences shown in Tables 2-8 <于 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, <于 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity and comprise a steric blocker having a nucleobase sequence, where C in any of the nucleobase sequences is either cytosine or 5-methylcytosine, and each nucleotide of the oligonucleotide has a 2'-modification. In some embodiments, the antisense oligonucleotides targeting MAPT have at least <于 80% sequence identity with any of the sequences shown in Tables 2-8. In some embodiments, the antisense oligonucleotides targeting MAPT have at least <于 90% sequence identity with any of the sequences shown in Tables 2-8. In some embodiments, the antisense oligonucleotides targeting MAPT have the sequences shown in Tables 2-8 <于 and comprise a nucleobase sequence having at least 80% sequence identity with any of the sequences shown in Tables 2-8. In some embodiments, the antisense oligonucleotides targeting MAPT have the sequences shown in Tables 2-8 <于 and comprise a nucleobase sequence having at least 90% sequence identity with any of the sequences shown in Tables 2-8. In some embodiments, the antisense oligonucleotides targeting MAPT have the sequences shown in Tables 2-8 <于 and comprise a nucleobase sequence having at least 80% sequence identity with any of the sequences shown in Tables 2-8. In some embodiments, the antisense oligonucleotides targeting MAPT have the sequences shown in Tables 2-8 <于 and comprise a nucleobase sequence having at least 90% sequence identity with any of the sequences shown in Tables 2-8. In some embodiments, the antisense oligonucleotides targeting MAPT have the sequences shown in Tables 2-8 <于 and comprise a nucleobase sequence having at least 90% sequence identity with any of the sequences shown in Tables 2-8. In some embodiments, the antisense oligonucleotides targeting MAPT have the sequences shown in Tables 2-8 <于 and comprise a nucleobase sequence having at least 80% sequence identity with any of the sequences shown in Tables 2-8. In some embodiments, the antisense oligonucleotides targeting MAPT have the sequences shown in Tables 2-8 <于 It contains any of the nucleic acid base sequences. In some embodiments, it targets MAPT. Nucleose oligonucleotides consist of one of the nucleic acid base sequences shown in Tables 2-8. In some embodiments, each C in any of the nucleic acid base sequences is 5-methylcysteine. It's Tosin.

[0010] In some embodiments, antisense oligonucleotides target MAPT Each nucleotide subunit has a 2'-O-MOE modification.

[0011] In some embodiments, antisense oligonucleotides target MAPT It contains a linker attached to the 3' end of an oligonucleotide via a phosphate crosslink, and A ligonucleotide has one of the following structures:

[0012] [ka]

[0013] In some embodiments, the anti-sensing agents that target MAPT as provided herein S-nucleotides are gapmers, and they have two at their 5' and 3' ends. Between the wing segments (also known as the 5' wing and the 3' wing, respectively) It has a central gap segment of consecutive 2'-deoxyribonucleotides located at [location]. Such antisense oligonucleotides activate RNAseH by The nucleo of the gapmer reduces the expression of tau mRNA and / or protein. Interside linkages can be phosphorothioate or phosphodiester linkages. In some embodiments, the gapmer has at least 5 (for example, 5, 6, 7, 8) A stretch of consecutive 2'-deoxyribonucleotides (9, 10, 11, 12) Including the 5' and 3' wing segments, one or more 2'-modified nuclei Contains an oligonucleotide. In some embodiments, such oligonucleotides are less Each of seven consecutive 2'-deoxyribonuclear compounds (for example, 7, 8, 9, 10, 11, 12) Contains a cleotide. In some embodiments, such an oligonucleotide is 1 It contains zero consecutive 2'-deoxyribonucleotides. The 2'-modification is 2'-fluoro , 2'-deoxy-2'-fluoro, 2'-O-methyl, 2'-O-methoxyethyl (2 '-O-MOE), 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethyl Aminoethyl(2'-O-DMAOE), 2'-O-dimethylaminopropyl(2'-O -DMAP), 2'-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE Select from the group consisting of ), and 2'-ON-methylacetamide (2'-O-NMA). It is possible. In some embodiments, the gapmer has a 5' wing and The 3' wing contains a nucleotide modified with 2'-O-MOE.

[0014] In some embodiments, a gapmer targeting tau is a 20-nucleoside The gapmer has a length of 5-10-5, and therefore the central gap segment is 10. It contains consecutive 2'-deoxynucleosides and is adjacent to the 5' wing and 3' wing. Each wing contains five nucleosides, each with a 2'-O-MOE modification.

[0015] In some embodiments, the anti-sensing agents that target MAPT as provided herein The solikinonucleotide is one of the sequences shown in Tables 9-15 and 17 and at least 70% (for example, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%) 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, Nucleic acid base sequences having sequence identity of 96%, 97%, 98%, 99%, or 100% It is a gapmer containing, where C in any of the nucleic acid base sequences is cytosine It is either 5-methylcytosine, and at least one of the alkyl groups The creotide has a 2'-modification. In some embodiments, MAPT is targeted. Antisense oligonucleotides are one of the sequences shown in Tables 9-15 and 17. It contains nucleic acid base sequences having at least 80% sequence identity. In some embodiments, The antisense oligonucleotides that target MAPT are listed in Tables 9-15 and 17. Nucleic acid sequences having at least 90% sequence identity with any of the shown nucleic acid sequences. Includes. In some embodiments, antisense oligonucleotides targeting MAPT Otide contains one of the nucleic acid base sequences shown in Tables 9-15 and 17. In the embodiment, antisense oligonucleotides that target MAPT are shown in Tables 9-1. It consists of one of the nucleic acid base sequences shown in 5 and 17. In some embodiments, The MAPT-targeting antisense oligonucleotides provided herein are shown in Table 9. 5-10-5 GA containing either of the nucleic acid base sequences shown in either ~15 or 17 This is a supmer, where the first to fifth nucleotides are modified with 2'-O-MOE. Each nucleotide contains a nucleoside, and the 6th to 15th nucleotides are 2'-deoxynucleo Each contains a side, and the 16th to 20th nucleotides are modified with 2'-O-MOE. Each contains a cleoside. In some embodiments, in any of the nucleic acid base sequences Each C is 5-methylcytosine.

[0016] In some embodiments, antisense oligonucleotides target MAPT These are sequence numbers 208, 284, 285, 313, 329, 335, 366, 384, and 38. It contains a nucleic acid base sequence selected from one of the following: 6, 405, 473, and 474. In some embodiments, antisense oligonucleotides targeting MAPT are , Array No. 284 and at least 90% (e.g., 90%, 91%, 92%, 93%, 94%) It has sequence identity of %, 95%, 96%, 97%, 98%, 99%, or 100%). Contains nucleic acid base sequences. In some embodiments, antisense targeting MAPT. The oligonucleotide includes SEQ ID NO: 284. In some embodiments, MAPT Antisense oligonucleotides that target this include SEQ ID NOs. 285 or 208, and a small number of others. All 90% (for example, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%) It contains nucleic acid base sequences with sequence identity of %, 98%, 99%, or 100%. In several embodiments, an antisense oligonucleotide targeting MAPT is distributed Includes column numbers 285 or 208.

[0017] In another embodiment, sequence numbers 487-5 have one, two, or three mismatches. A nucleic acid sequence that is complementary to at least 12 consecutive nucleic acid bases of any one of 06. An oligonucleotide comprising, at least one nucleo of the oligonucleotide These oligonucleotides having 2'-modifications are provided herein. Gonucleotides are antisense oligonucleotides that target MAPT. In some embodiments, such oligonucleotides are SEQ ID NOs: 487-506 Nucleic acid base pairings that are 100% complementary to any one of at least 12 consecutive nucleic acid bases Includes a column. In some embodiments, such oligonucleotides are one or Contains multiple 5-methylcytosine molecules. In some embodiments, such oligonucleotides The rheotide has a 2'-modification. This 2'-modification is 2'-fluoro, 2'-deoxy-2 '-Fluoro, 2'-O-methyl, 2'-O-methoxyethyl (2'-O-MOE), 2 '-O-aminopropyl(2'-O-AP), 2'-O-dimethylaminoethyl(2'- O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2'- O-dimethylaminoethyloxyethyl (2'-O-DMAEOE), and 2'-O- It can be selected from the group consisting of N-methylacetamide (2'-O-NMA). In the embodiment, the 2'-modification is 2'-O-methoxyethyl (2'-O-MOE) Yes. In some embodiments, such oligonucleotides are at least 5 (For example, 5, 6, 7, 8, 9, 10, 11, 12) consecutive 2'-deoxyribonu Contains a cleotide. In some embodiments, such oligonucleotides are small At least seven consecutive 2'-deoxyri It contains a bonucleotide. In some embodiments, such an oligonucleotide is It contains 10 consecutive 2'-deoxyribonucleotides.

[0018] In some embodiments, the anti-sensing agents that target MAPT as provided herein Soligonucleotides can be used in vitro to control the expression levels of tau mRNA or protein. It can be reduced by at least 30%.

[0019] In some embodiments, the anti-sensing agents that target MAPT as provided herein Soligonucleotides reduce the expression level of tau mRNA or protein in vivo. Even without it, it can be reduced by 30%.

[0020] In another embodiment, any of the antisense oligonucleotides described herein Compositions comprising, and a pharmaceutically acceptable carrier are provided herein.

[0021] In a further embodiment, for example, a person suffering from or prone to a tau-related disease. The tau expression level in the pancreatic subject is measured in the subject using the antisense method described herein. This specification describes a method of reducing by administering a therapeutically effective dose of any of the gonucleotides. Provided in the book. In some embodiments, such a method involves a second active substance against This may include administering it to elephants. In some embodiments, MAPT is targeted The antisense oligonucleotides used are administered intrathecally, intracranially, intranasally, orally, and intravenously. It can be administered internally or via a subcutaneous route. In some embodiments, the target It is a human being.

[0022] Those who need it, for example, those suffering from or suffering from tau-related diseases The following is described herein for use in treating tau-related diseases in susceptible subjects. Antisense oligonucleotides are also provided. Antisense oligonucleotides described herein A soligonucleotide or pharmaceutical composition related to tau in the target that requires it. This also includes use to treat diseases. This disclosure is for use in subjects that require it. Antimicrobial activity described herein in the manufacture of pharmaceuticals for use in the treatment of diseases related to This includes the use of sense oligonucleotides.

[0023] Diseases associated with tau include Alzheimer's disease (AD), amyotrophic lateral sclerosis / parkinson's disease. ALS-PDC (Alopecia Syndrome-Dementia Complex), Argyrophilic Granule Dementia (AGD), British-type Ammonia Lloyd's vascular disease, cerebral amyloid angiopathy, chronic traumatic encephalopathy (CTE), corticobasal degeneration (CBD), Creutzfeldt-Jakob disease (CJD), punch-drunk syndrome, accompanied by calcification. Diffuse neurofibrillary tangles, Down syndrome, Dravet syndrome, epilepsy, frontotemporal dementia (F TD), frontotemporal Parkinson's syndrome linked to chromosome 17 (FTDP-17). Type 1 dementia, frontotemporal lobar degeneration, ganglioglioma, gangliocytoma, Gerstmann-Strouslä - Shainker's disease, Haller-Holden-Spats disease, Huntington's disease, inclusion body myositis, lead brain Diseases, Ritico-Bodig disease, meningiomatosis, multiple system atrophy, myotonic dystrophy, Niemann-Pick disease type C (NP-C), a non-Guanian motor neuron disorder with neurofibrillary tangles. Pick's disease (PiD), post-encephalitis parkinson's syndrome, prion protein brain amyloid blood vessels Disease, progressive subcortical gliosis, progressive supranuclear palsy (PSP), subacute sclerosing panencephalitis, neurological Tangle-only dementia, neurofibrillary tangle-dominant dementia (Tangle (L-predominant dementia), multiple infarct dementia, ischemic stroke, or tuberous sclerosis You can choose from these options. [Brief explanation of the drawing]

[0024] [Figure 1-1] Figures 1A–1E show the physical characterization of antisense oligonucleotides targeting MAPT. Figure 1A shows the structure of an antisense oligonucleotide (ASO) containing SEQ ID NO: 284, with formula C230H321N72O120P19S19 and expected molecular weight of 7212.3Da. Figure 1B shows liquid chromatography-mass spectrometry (LC-MS) data for the ASO containing SEQ ID NO: 284, with a measured peak mass of 7214.3. Figure 1C shows the LC-MS deconvolution peak report for the ASO containing SEQ ID NO: 284. Figure 1D shows the LC-MS data for the ASO containing SEQ ID NO: 285, with a measured peak mass of 7232.5. Figure 1E shows the LC-MS deconvolution peak report for the ASO containing SEQ ID NO: 285. [Figure 1-2] (As stated above.) [Figure 1-3] (As stated above.) [Figure 2]Figures 2A-2E show the expression levels of human tau mRNA and protein in representative hTau BAC transgenic mouse strains before and after antisense oligonucleotide treatment. Figure 2A is a representative RT-PCR result showing that all six human tau transcripts were found in the forebrain of hTau BAC transgenic mice (transgenic strain 510, 2-month-old female mice). Exons 2, 3, and 10 are alternately spliced ​​to produce six tau isoforms: 2-3-10-; 2+3-10-; 2+3+10-; 2-3-10+; 2+3-10+; and 2+3+10+. 4R represents a tau isoform with exon 10, 3R represents a tau isoform without exon 10; 0N represents a tau isoform without either exon 2 or exon 3; 1N represents a tau isoform with either exon 2 or exon 3; and 2N represents a tau isoform with both exon 2 and exon 3. Figure 2B is a representative Western blot showing six tau protein isoforms with molecular weights of 48–67 kD and amino acid ranges of 352–441. They differ in (1) the inclusion of zero, one, or two inserts in the 29-amino acid N-terminal region (0N, 1N, or 2N), or (2) the inclusion of three or four microtubule-binding domains (3R or 4R). Figure 2C is a representative immunohistochemical image showing the distribution of normal human tau axons stained with a human tau-specific antibody in the brain of an hTau BAC transgenic mouse. Figure 2D is a bar graph showing tau mRNA knockdown in the cortex of hTau BAC transgenic mice 4 weeks after single treatment with an antisense oligonucleotide containing SEQ ID NO: 285. Figure 2E is a representative Western blot showing tau protein knockdown in the hippocampus of hTau BAC transgenic mice 4 weeks after single treatment with an antisense oligonucleotide containing SEQ ID NO: 285. [Figure 3] Figure 3 shows a series of in situ hybridization images illustrating the broad brain distribution of the antisense oligonucleotide of Sequence ID No. 285 in hTau BAC transgenic mice. [Figure 4] Figures 4A and 4B are dot plots showing dose-dependent inhibition of human tau mRNA (Figure 4A) and protein (Figure 4B) expression in hTau BAC transgenic mice 4 or 12 weeks after a single ICV injection of 1, 10, 50, 200, or 400 μg of the antisense oligonucleotide of Sequence ID No. 285. [Figure 5] Figures 5A and 5B are dot plots showing the time course of human tau mRNA (Figure 5A) and protein (Figure 5B) expression levels in hTau BAC transgenic mice after a single ICV injection of 200 μg of the antisense oligonucleotide of Sequence ID No. 285.

[0025] Detailed description Antisense oligonucleotides targeting tau (MAPT), a protein associated with microtubules. Otide, compositions comprising the antisense oligonucleotide, and antisense thereof A method for reducing tau expression using oligonucleotides is provided herein. The compositions and methods provided in this specification are useful for treating diseases related to tau.

[0026] definition As used herein and in the claims, the singular forms "a," "an," and "the" are sentences. Unless otherwise clearly indicated by the pulse, it includes multiple references. For example, the term "a "Cell" contains multiple cells that include that mixture.

[0027] All numerical representations, such as pH, temperature, time, concentration, and molecular weight including ranges, are (+ This is an approximation that changes in increments of (0) or (-)0.1. It is not always explicitly stated. However, it should be understood that the term "approximately" precedes all numerical expressions. Although not necessarily stated explicitly, the reagents described herein are merely examples, and It should also be understood that equivalents of such reagents are known in the art. be.

[0028] The term "2'-modification" refers to the modification of the furanose ring of a nucleoside or nucleotide at the 2' position. This refers to the substitution of H or OH with another group.

[0029] The terms "2'-O-methoxyethyl", "2'-MOE", or used herein refer to the terms used herein. "2'-OCH2CH2-OCH3" is an O-methoxy compound at the 2' position of the furanose ring. This refers to chill modification. Sugars modified with 2'-O-methoxyethyl are modified sugars. "2'-MOE nucleoside / nucleotide" or "2'-O-methoxyethyl nucleo "Side / nucleotide" refers to a nucleoside / nucleotide containing a sugar moiety modified with 2'-MOE. This refers to Ochido.

[0030] "5-methylcytosine" refers to cytosine modified with a methyl group attached to the 5' position.

[0031] As used herein, the term "antisense oligonucleotide" refers to a target nucleic acid, e.g. For example, complementary to the target genome sequence, premRNA, or corresponding segment of the mRNA molecule. This refers to a single-stranded oligonucleotide having a specific nucleic acid base sequence. Several embodiments In this context, antisense oligonucleotides are 12 to 30 nucleic acid bases in length.

[0032] The term "complementarity" or "complementary" refers to the hydrogen bond between corresponding nucleic acid bases (e.g., Wa (by tson-Crick, Hoogsteen, or reverse Hoogsteen hydrogen bonds) The ability to form base pairs between the nucleic acid bases of the first nucleic acid chain and the nucleic acid bases of the second nucleic acid chain, which is mediated by the mediation of the base pair formation. This refers to the following: For example, in DNA, adenine (A) is complementary to thymine (T), and guanos Adenine (G) is complementary to cytosine (C). For example, in RNA, adenine (A) is complementary to cytosine (C). It is complementary to sil (U), and guanosine (G) is complementary to cytosine (C). In the application process, complementary nucleic acid bases can form base pairs with the nucleic acid bases of their target nucleic acid. It refers to the nucleic acid base of an antisense oligonucleotide. For example, antisense oligonucleotide A nucleic acid base at a certain position of a nucleotide corresponds to a nucleic acid base at a certain position of the target nucleic acid. If hydrogen bonding can be formed, then between the oligonucleotide and the target nucleic acid The positions of the hydrogen bonds are complementary in that nucleic acid base pair. Acids and bases can retain the ability to form pairs with their counterpart nucleic acid bases, therefore Therefore, nucleic acid base complementarity is indeed possible.

[0033] "Effective dose" refers to a quantity sufficient to produce a beneficial or desired result. For example, The therapeutic dose is the amount that achieves the desired therapeutic effect. This amount is used to predict the onset or symptoms of the disease. The effective preventive dose, which is the amount needed to prevent the disease, may be the same as or different from the effective preventive dose. The amount can be administered in one or more doses, applications, or prescriptions. Therapeutic compounds The "therapeutic effective dose" (i.e., effective dosage) of a substance depends on the selection of the therapeutic compound. The composition of the present invention is administered once or more times per day, or once every other day for a week. It can be administered once or multiple times. Those skilled in the art will know the severity of the disease or disability, and the previous Treatment of the subject, including general health and / or age and other pre-existing medical conditions, but these Factors, not limited to those mentioned above, that determine the dosage and amount of medication required to effectively treat the subject. It will be recognized that this may affect the timing. Furthermore, the therapeutic treatment described herein Treatment of a target using a therapeutically effective dose of the compound may include a single treatment or a series of treatments. Cut.

[0034] As used herein, the term "gapmer" refers to a continuous 2'-deoxyribonucle Chimeric antisense oligonucleotides containing a central gap segment composed of rheotide It refers to the fact that it can activate RNAseH, and at the 5' and 3' ends, respectively, One or more modified nucleotides that provide increased resistance to clease degradation It is adjacent to two wing segments, including the one mentioned above.

[0035] The term "hybridization" refers to the base pairing of complementary nucleic acid strands and the formation of double-strand structures. Hybridization refers to the formation of a hybrid between perfectly complementary or mismatched nucleic acid strands. This can occur between "substantially complementary" nucleic acid strands that contain non-essential regions. While not restricted, the most common mechanism of pair formation involves hydrogen bonding, which is complementary to the nucleic acid chain. Watson-Crick, Hoogsteen, or reverse Hoogst between nucleic acid bases It may also be a hydrogen bond of een. For example, adenine and thymine form hydrogen bonds. Throughout, they are complementary nucleic acid bases that form pairs. Hybridization is the process of changing strings This can occur under certain circumstances. The term "hybridization" as used herein refers to: Base pairing of complementary nucleic acid strands and at least under relatively low stringent conditions. This refers to the formation of a double-chain structure, for example, 2×SSC(0.3M sodium chloride, 0.03M sodium chloride). Hybridization of sodium enoate, 0.1% SDS at 37°C, and The subsequent washing in a 4×SSC, 0.1% SDS-containing solution can be performed at 37°C. Finally, it is washed in 1×SSC at 45°C.

[0036] The term "inhibit" or "suppress" refers to the reduction of the expression or activity of a target nucleic acid or protein. This refers to suppression or blockade, and does not necessarily indicate complete elimination of the target's expression or activity.

[0037] The term "internucleoside linkage" refers to the chemical bond between nucleosides.

[0038] The term "knockdown" or "knockdown of expression" refers to the use of reagents, such as antisense reagents. Reduced mRNA or protein expression of genes after oligonucleotide treatment This refers to the knockdown of expression, which occurs during transcription, mRNA splicing, or translation. obtain.

[0039] The term "mismatch" refers to a situation where a nucleic acid base in the first nucleic acid chain does not match the corresponding nucleic acid base in the second nucleic acid chain. This refers to a situation where something is not complementary to something else.

[0040] The term "nucleic acid base sequence" is defined independently of any sugar, linking group, and / or modification of the nucleic acid bases. This refers to the sequence of consecutive nucleic acid bases.

[0041] The term "oligonucleotide" refers to each of the modified or unmodified, linked molecules. Deoxyribonucleotides (DNA) and / or ribonucleotides (RNA) This refers to polymers. Unless otherwise specified, the term refers to polymers that have the property of binding in the same way as natural nucleic acids. Nucleic acids containing known analogs of natural nucleotides, and other than phosphodiester linkages. It includes nucleic acids that have alternative nucleoside linkages.

[0042] The term "phosphorothioate linkage" refers to a phosphodiester bond that connects one of the non-crosslinked oxygen atoms. This refers to nucleoside linkages modified by replacing one atom with a sulfur atom.

[0043] The term "sense strand" refers to the coding strand, the positive strand, of a double-stranded DNA molecule, or This refers to the non-template strand. The coding strand is where thymine (T) in DNA is replaced by uracil in RNA. It has the same sequence as the mRNA sequence, except that it is replaced by (U). An "integument strand" is the non-coding strand of a DNA molecule or a template for mRNA synthesis. This refers to a template chain that functions as such. Therefore, the sequence of antisense chains is the same as the sense chain. It is complementary to the mRNA sequence (where U is located at the T position in RNA).

[0044] As used herein, the term "steric blocker" refers to a target nucleic acid (e.g., a target genome). Activates RNAseH by hybridizing with a sequence (premRNA, or mRNA molecule). Anti-interference without interfering with the transcription, splicing, and / or translation of target nucleic acids This refers to sense oligonucleotides.

[0045] In this specification, the terms "targeted" or "targeted" refer to target nucleic acids, for example. For example, a target genome sequence, premRNA or mRNA molecule, or fragments thereof. Alternatively, it can specifically hybridize with variants, and transcribe and splice target nucleic acids. Antisense oligonucleotides that can modulate sings and / or translations. This refers to the design and selection of Ochido.

[0046] In this specification, the term "tau" (also known as "microtubule-related protein tau") is used to refer to "tau" (also known as "microtubule-related protein tau"). "is being", MAPT, MSTD;PPND;DDPAC;MAPTL;MTBT1;M TBT2;FTDP-17;PPP1R103) is encoded by the MAPT gene. This refers to proteins related to microtubules. The human MAPT gene is located at chromosome 17q2. It is mapped to 1.1, and the genome sequence of the human MAPT gene is available in GenBank. It can be found in NG_007398.1 (sequence number 304). MAPT IN Tron and exon sequences and branching points are in transcript: MAPT-203ENST0000 Based on the Ensembl genome database website, the determination was made using 0344290. It can be determined. In humans, based on complex alternating splicing, there are eight types of tauai. There are isoforms. The term "tau" refers collectively to all isoforms of tau. The longest human tau isoform protein and mRNA sequences are: Tau (MAPT), a protein associated with microtubules in Homo sapiens, transcript variant 6 mRNA(NM_001123066.3)

[0047] [ka]

[0048] [ka]

[0049] [ka] Homo sapiens microtubule-related protein tau isoform 6 (NP_00111) 6538.2)

[0050] [ka] That is the case. mRNA and protein sequences of other human tau isoforms can be found in GenBank. It can be found using the following accession number: Tau isoform 1: NM_016835.4 (mRNA) → NP_058519. 3 (protein); Tau isoform 2: NM_005910.5 (mRNA) → NP_005901. 2 (protein); Tau isoform 3: NM_016834.4 (mRNA) → NP_058518. 1 (protein); Tau isoform 4: NM_016841.4 (mRNA) → NP_058525. 1 (protein); Tau isoform 5: NM_001123067.3 (mRNA) → NP_0011 16539.1 (protein); Tau isoform 7: NM_001203251.1 (mRNA) → NP_0011 90180.1 (protein); Tau isoform 8: NM_001203252.1 (mRNA) → NP_0011 90181.1 (protein). The human tau protein used herein is one of the tau isoforms and its Along the entire length, at least approximately 70%, 71%, 72%, 73%, 74%, 75%, and 76%. %, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86 %, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96 This also includes proteins with sequence identity of %, 97%, 98%, 99%, or 100%. The sequences of tau proteins in mice, monkeys, and other animals are known in the art. Yes, they are.

[0051] The term "tau-related disease" refers to abnormal expression, secretion, phosphorylation, and cleavage of tau protein. This includes, but is not limited to, diseases associated with, and / or aggregation of tau. The diseases include Alzheimer's disease (AD), amyotrophic lateral sclerosis / Parkinsonian syndrome - dementia. Complex disease (ALS-PDC), argyrophilic grain dementia (AGD), British-type amyloid angiopathy, brain Amyloid angiopathy, chronic traumatic encephalopathy (CTE), corticobasal degeneration (CBD), clonal Itzfeldt-Jakob disease (CJD), punch-drunk syndrome, diffuse nerve root with calcification Blood vessel dysplasia, Down syndrome, Dravet syndrome, epilepsy, frontotemporal dementia (FTD), staining of cell number 17 Frontotemporal dementia with Parkinsonian syndrome linked to chromosome (FTDP-17), frontal Lobar degeneration, ganglioglioma, gangliocytoma, Gerstmann-Strössler-Scheinker Diseases, Haller-Holden-Spats disease, Huntington's disease, inclusion body myositis, lead encephalopathy, Ritico Bo Dig's disease, meningioma hemangiomatosis, multiple system atrophy, myotonic dystrophy, Niemann-Pick disease Pick's disease (PiD), a non-Guanian motor neuron disorder with neurofibrillary tangles, also known as NP-C. ), post-encephalitis parkinsonian syndrome, prion protein cerebral amyloid angiopathy, progressive subcortical disease Gliosis, progressive supranuclear palsy (PSP), subacute sclerosing panencephalitis, neurofibrillary tangle-dominant type Dementia (tangle-only dementia), neurofibrillary tangle-predominant dementia (tangle-predominant dementia) This includes dementia ementia, multiple infarct dementia, ischemic stroke, and tuberous sclerosis, but these include Not limited.

[0052] The term "homology" or "identity" refers to the relationship between two polymer molecules, for example, two nucleic acid molecules. Between, for example, two DNA molecules or two RNA molecules, or two polypeptides This refers to the identical arrangement of subunits between molecules. Subunit position in both molecules. However, if they are occupied by subunits of the same monomer; for example, two DNA molecules If each of the positions is occupied by adenine, then in that case they are in that position They are homologous or identical in position. Homologousity between two sequences means they match, i.e., they are identical. It is a direct function of the number of homologous positions. For example, half of the positions in two arrays (e.g. For example, if five of the lengths of the ten subunits in the polymer are homologous, then 2 Two sequences are 50% homologous, meaning that 90% of their positions (for example, 9 out of 10) coincide. If two sequences are homologous, then they are 90% homologous. The percentage of "sequence identity" is This is determined by comparing two optimally aligned sequences across a comparison window. It is possible, and in that case, there are two amino acid sequence fragments in the comparison window. The optimal alignment of the sequence is compared to the reference sequence (without additions or deletions), and the additions and deletions are considered. or may include deletions (e.g., gaps or overlaps). The percentage is the same Determine the number of positions in which the amino acid residue appears in both sequences, obtain the number of matching positions, and determine the number of matching positions. Divide the number of positions by the total number of positions in the comparison window, and set the result to 100. The result can be calculated by multiplying to obtain the percentage of sequence identity. The result is the percentage of identity of the target sequence with respect to the sequence in question.

[0053] The term "isolated" refers to something that has been altered or removed from its natural state. For example, nucleic acids or peptides that are naturally present in living animals are not "isolated". However, the same nucleic acid, which is partially or completely isolated from the material that coexists in its natural state, The peptide is “isolated.” Isolated nucleic acids or proteins are substantially purified. It can exist in a modified form, or in an environment that is not natural, such as a host cell. It can exist in such contexts.

[0054] The term "to treat" or "treatment" refers to both therapeutic procedures and preventive or preventive measures. The purpose is to prevent or slow down undesirable physiological changes or disorders. Therefore, for the purposes of the present invention, beneficial or desired clinical results are detectable or detectable. Whether impossible or not, the reduction of symptoms, the reduction of the disease's severity, and the stabilization of the disease (i.e., (No worsening of condition), delayed or slowed disease progression, relief or temporary reduction of symptoms, and remission. This includes, but is not limited to, solutions (whether incomplete or complete). "Treatment" is This can also mean a longer survival compared to the survival expected if no treatment is received. Cut.

[0055] The term "subject" refers to an animal, human, or non-human being to be treated by the method of the present invention. Veterinary and non-veterinary applications are intended. The term refers to mammals, e.g., humans, etc. Primates, pigs, rodents such as mice and rats, rabbits, guinea pigs, and hamsters This includes, but is not limited to, cattle, horses, cats, dogs, sheep, and goats. The subjects include humans, livestock, and domestic pets, such as cats and dogs.

[0056] Unless otherwise specified, all technical and scientific terms used herein are in accordance with the present invention. This has the same meaning as generally understood by those skilled in the art. Similar or equivalent methods and materials may be used to carry out the present invention. Appropriate methods and materials are described below. All publications and patents mentioned herein are subject to change without notice. Applications, patents, and other references are incorporated in their entirety by reference. In case of conflict, This specification, including its definitions, shall be governed by this provision. In addition, materials, methods, and examples This is for illustrative purposes only and is not intended to be limiting.

[0057] Details of one or more embodiments of the present invention are described in the accompanying drawings and the following description. Other features, purposes, and advantages of the present invention are evident from the description and drawings and the claims. It is likely.

[0058] Antisense oligonucleotides Antisense oligonucleotides (ASOs) reduce RNA levels, inhibit translation, and miRNs. This includes inhibition of A, splicing modulation, and selection of polyadenylation sites. It is a powerful and multi-functional active ingredient used in an increasing number of applications. Antisense oligo Nucleotides are formed when a sufficient number of nucleic acid bases in an antisense oligonucleotide are paired with the target nucleic acid. When it can form a hydrogen bond with the corresponding nucleic acid base, it binds to the target nucleic acid and mediated the transcription of the target nucleic acid. It modulates the translation. Therefore, the nucleus of antisense oligonucleotides. The acid-base sequence is used to target nucleic acids, such as target genome sequences, pre-mRNA, or mRNA molecules. It is complementary to the nucleic acid base sequence. Hybridization is a process involving hydrogen bonding (e.g., Wat (son-Crick, Hoogsteen, or reverse Hoogsteen hydrogen bond) This occurs when a complementary nucleic acid base of an oligonucleotide is formed between the target nucleic acid and the oligonucleotide. This is because the non-complementary nucleic acid base between the antisense oligonucleotide and the target nucleic acid is antisense. Sense oligonucleotides still do not hybridize specifically with target nucleic acids. It should be tolerated as much as possible.

[0059] ASO targets the target tongue in either an RNaseH-dependent or RNaseH-independent manner. It can be designed to reduce protein expression (Watts JK, et al., J Pathol. 2012 Jan) See uary; 226(2): 365-379). ASOs containing a continuous stretch of DNA When hybridized with target RNA, the DNA-RNA heteroduplex moves RNaseH It then cleaves the double-stranded target RNA, followed by RNA fracturing by cellular nucleases. It promotes the degradation of lagment. ASO processes premRNA and / or m By sterically blocking the translation of RNA into protein, independently of RNAseH, It is also possible to reduce the expression of the target.

[0060] Antisense oligonucleotides targeting tau (MAPT), a protein associated with microtubules. Otid is provided herein. In some embodiments, A is provided herein. An inthisense oligonucleotide has 1, 2, 3, 4, or 5 mismatches. , MAPT genomic DNA, premRNA, or nucleic acid salts complementary to mRNA segments It has a base sequence. If complete base pairing occurs (for example, between A and T and between C and G) Pair formation) Mismatches between oligonucleotides and their corresponding target nucleic acids can be counted. This is what happens. A mismatch occurs when, when the two sequences are perfectly aligned, the nucleic acid bases of the first nucleic acid are This occurs when the second nucleic acid cannot form a pair with the corresponding nucleic acid base. For example, the first The position in the sequence contains nucleic acid base A, and the corresponding position in the second sequence cannot form a pair with A. If it has a nucleic acid base (e.g., C or G), it constitutes a mismatch. When one position in a sequence contains a nucleic acid base, and the corresponding position in the other sequence does not contain a nucleic acid base. However, mismatches can be counted. For the sugar portion of a nucleotide or nucleoside linkage. Modifications are not considered mismatches. Therefore, if one sequence contains G and the second sequence contains G If the corresponding nucleic acid base contains a modified C (e.g., 5-methylcytosine), it is counted. There shouldn't be any mismatch.

[0061] Antisense oligonucleotides provided herein in relation to nucleic acid stretching This includes MAPT genomic DNA, premRNA, or mRNA segments, and segments Along its entire length, at least 70%, 80%, 85%, 86%, 87%, 88%, and 8 9%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 9 It is 9% or 100% complementary. The interaction between the antisense oligonucleotide and the target nucleic acid. The percentage of complementarity is calculated using common methods known in the art, such as BLAST Pro. Gram (basic local alignment search tools) Or the PowerBLAST program (Altschul et al., J. Mol. Biol., 1990, 215, 4 Determine using (03 410; Zhang and Madden, Genome Res., 1997, 7, 649 656). This is possible. In some embodiments, the antisense oligonucleotides provided herein Reotide is a MAPT genomic DNA, premRNA, or mRNA segment and 10 It has a 0% complementary (i.e., perfectly complementary) nucleic acid base sequence. The terms "completely complementary" or "100% complementary" are used to describe each nucleus of an antisense compound. This refers to the ability of an acid-base to form a precise base pair with the corresponding nucleic acid base of a target nucleic acid. For example, 20 nucleic acid base antisense compounds are perfectly complementary to the antisense compound. As long as there are 20 corresponding nucleic acid base portions of the target nucleic acid, the length of 400 nucleic acid bases is It is perfectly complementary to the target sequence.

[0062] In some embodiments, antisense oligonucleotides provided herein This refers to any of the sequences shown in Table 1 that have one, two, or three mismatches. At least 12 consecutive nucleic acid bases (for example, 12, 13, 14, 15, 16, 17, and It comprises a nucleic acid base sequence that is complementary to 18 consecutive nucleic acid bases. Several embodiments In this specification, the antisense oligonucleotides provided herein are shown in Table 1. At least 12 consecutive nucleic acid bases from any of the sequences (for example, 12, 13, 14, Nucleic acid bases that are 100% complementary to (15, 16, 17, or 18 consecutive nucleic acid bases) Includes arrays.

[0063] The antisense compounds provided herein are specific nucleotide sequences, sequence numbers, and Or they may have a percentage identity defined as a part of them. The antisense oligonucleotide used has the same nucleic acid base pairing ability. Therefore, it is identical to the sequence disclosed herein. For example, uracil and thymidine are both Since both form a pair with adenine, the position of thymidine in the disclosed DNA sequence is RNA containing racil is considered to be identical to the DNA sequence described above. The antisense oligonucleotides provided herein have non-identical bases. Antisense oligonucleotides and shortened and extended oligonucleotides Other forms are also considered. Non-identical bases may be adjacent to each other, or antisected. Antisense oligonucleotides can also be dispersed throughout the entire oligonucleotide. The percentage of sequence identity of a cydone is the same base pairing with respect to the sequence being compared. It can be calculated according to the number of bases that have it. The percentage of sequence identity is calculated according to the technical percentage Common methods known in the field, for example, the BLAST program (basic local) (alignment search tools) or PowerBLAST Pro Gram (Altschul et al., J. Mol. Biol., 1990, 215, 403-410; Zhang and Madden, Ge (nome Res., 1997, 7, 649 656) using; or gap program (Unix) Wisconsin sequence analysis package, version 8 Genetics Compu ter Group, University Research Park, Mad Ison (Wisconsin) by Smith and Waterman's algorithm Determine using the default settings provided in (Adv. Appl. Math., 1981, 2, 482-489). It is possible.

[0064] In some embodiments, any of the nucleic acid base sequences shown in Tables 2 to 17 And at least 70% (for example, 70%, 75%, 80%, 81%, 82%, 83%, 8 4%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 9 It has sequence identity of 4%, 95%, 96%, 97%, 98%, 99%, or 100%. An antisense oligonucleotide containing a nucleic acid base sequence, wherein any of the nucleic acid base sequences The C is either cytosine or 5-methylcytosine, and the oligonucleotide At least one nucleotide of which has a 2'-modification, antisense oligonucleotide A method is provided herein. In some embodiments, as shown in any of Tables 2 to 17 Any of the nucleic acid base sequences and at least 90% (for example, 90%, 91%, 92%) The same sequence of 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% Antisense oligonucleotides containing a unisexual nucleic acid base sequence are provided herein. In some embodiments, antisense oligonucleotides targeting MAPT are used. The sequence contains one of the nucleic acid base sequences shown in any of Tables 2-17. Morphologically, antisense oligonucleotides that target MAPT are shown in Tables 2-17. It consists of one of the nucleic acid base sequences shown in either of the following.

[0065] In some embodiments, antisense oligonucleotides provided herein It is 12 to 30 nucleic acid bases long. For example, antisense ointments targeting MAPT Ligonucleotides are 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, It must contain 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleic acid bases. This is possible. In some embodiments, antisense oligonucleotides targeting MAPT are used. Rheotides are 12 to 25 nucleic acid bases long. For example, anti-MAPT Sense oligonucleotides are 12, 13, 14, 15, 16, 17, 18, 19, 20 It can contain 21, 22, 23, 24, or 25 nucleic acid bases. In the application method, antisense oligonucleotides targeting MAPT are 15 to 2 0 is the length of nucleic acid bases. For example, antisense oligonucleotides targeting MAPT A molecule can contain 15, 16, 17, 18, 19, or 20 nucleic acid bases. In some embodiments, the antisense oligonucleotide targeting MAPT is It contains 17 nucleic acid bases. Without removing activity, the length of the antisense oligonucleotide Increasing or decreasing and / or mismatch in antisense oligonucleotides It is possible to introduce ti bases (for example, mismatches at 1, 2, 3, 4, or 5 positions). be.

[0066] Chemical modification of antisense oligonucleotides Oligonucleotides are linked together by phosphodiester bonds between nucleosides. It consists of repeating nucleotide units. Each nucleotide is linked to a sugar moiety. A nucleic acid base and a nucleotide containing a monophosphate group covalently linked to the sugar portion. It is composed of leosides. The phosphodiester bond is purine (guanine and / or adenoside). nin) and / or pyrimidine bases (thymine and cytosine for DNA; as well as For RNA, sugars (uracil and cytosine) are linked to it through glycoside bonding. Residue (either ribose for RNA or deoxyribose for DNA, collectively) It is made from furanose.

[0067] The antisense oligonucleotides provided herein are one or more modified It can contain nucleotide subunits and / or internucleoside links. Chemical modifications to oligonucleotides include nucleoside linkages, sugar moieties, and nucleic acid bases. , and / or changes in the skeleton. Modifications include antisense oligonucleotides. It can improve the stability and effectiveness of the drug, and / or reduce its immunogenicity. For example, when comparing an oligonucleotide with an unmodified oligonucleotide... Increased resistance to nucleases, enhanced binding affinity to nucleic acid targets, enhanced To modify cells to have increased uptake and / or inhibitory activity. It is possible.

[0068] In some embodiments, antisense oligonucleotides provided herein This includes naturally occurring phosphodiester and nucleoside linkages. This includes other phosphorus-containing links, such as phosphorothioates, phosphotriesters, and methylphospho. It can be modified with fonates, phosphoramidate links, or non-phosphorus-containing links. In some embodiments, antisense oligonucleotides provided herein It includes one or more modified nucleoside linkages. In some embodiments, The antisense oligonucleotides provided herein are phosphorothioate-linked. Includes. In some embodiments, each nucleocytan of the antisense oligonucleotide The internucleoside linkage is a nucleoside linkage mediated by phosphorothioates.

[0069] In some embodiments, antisense oligonucleotides provided herein It contains a chemically modified sugar moiety. For example, an antisense oligonucleotide is 2' modification in the lanose ring, the bridge of non-geminal ring atoms forming bicyclic nucleic acids (BNAs) This can include bridges, substitution of oxygen atoms in sugar rings with other atoms, or combinations thereof. In some embodiments, each nucleotide of an antisense oligonucleotide is It has a 2'-modified furanose ring. Typical 2'-modifications include 2'-fluoro, 2' -Deoxy-2'-fluoro, 2'-O-methyl, 2'-O-methoxyethyl(2'-O -MOE), 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylamino Ethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DM AP), 2'-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE), o It also contains 2'-ON-methylacetamide (2'-O-NMA). Several implementation forms In this state, each nucleotide of the antisense oligonucleotide has a 2'-O- in its sugar portion. It has MOE modification.

[0070] In some embodiments, antisense oligonucleotides provided herein This refers to the substitution of a nucleotide at a given position with a modified form of the same nucleotide. It can contain. For example, a nucleotide (A, G, C, or T) can contain the corresponding hypoxa Xanthine, 4-acetylcytosine, beta-D-galaclosil eosin, Nosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2 ,2-dimethylguanine,2-methyladenine,2-methylguanine,3-methylcytosine N, 5-methylcytosine, N6-adenine, 7-methylguanine, beta-D-mannosine Luqueosin, 2-methylthio-N6-isopentenyladenine, Weybutoxosin, C It can be replaced with enosine, 2-thiocytosine, or 2,6-diaminopurine.

[0071] In some embodiments, antisense oligonucleotides provided herein These are chemically modified oligonucleotides that reduce the immunogenicity of oligonucleotides. Includes, for example, oligonucleotides containing 5-methylcytosine or 2'-O-MOE modification. Reotide has been shown to reduce immune stimulation in mice (Henry S. et al.). al., J Pharmacol Exp Ther. 2000 Feb; 292(2):468-79). In some embodiments... The antisense oligonucleotides provided herein use 5-methyl instead of cytosine. Contains cytosine. In some embodiments, the antisense provided herein The oligonucleotide contains a 2'-O-MOE modification. In some embodiments, this The antisense oligonucleotides provided in the specification are 5-methylcytosine and 2' Includes MOE modification.

[0072] In some embodiments, the anti-sensing agents that target MAPT as provided herein The s-oligonucleotide contains a C6 linker with the following structure at its 3' end:

[0073] [ka] The linker is attached to the 3' end of the oligonucleotide via a phosphate crosslink, where R =PO2-O-oligonucleotide (due to phosphodiester nucleoside linkage) R=POS-O-oligonucleotide (due to inter-phosphorothioate nucleoside linkage) Such a 3'C6 linker blocks the attack of 3'-exonuclease. Therefore, the stability and duration of the effect of antisense oligonucleotides can be improved. Enhance (Regarding similar strategies applied to siRNA, see Pan 2005 / 021749) (See frets). In some cases, the 3'C6 linker is antisense original. It can also facilitate the synthesis and / or purification of gonucleotides. Several implementations In this state, an antisense oligonucleotide that targets MAPT can be any of the following structures It can have a structure.

[0074] [ka]

[0075] In some embodiments, antisense oligonucleotides provided herein This is an alternative skeleton, for example, morpholino, locked nucleic acid (LNA), unlocked nucleic acid (U NA), threose nucleic acid (TNA), glycol nucleic acid (GNA), and / or peptide It may include nucleic acid (PNA). In some embodiments, as provided herein Antisense oligonucleotides are bicyclic, containing a bridge connecting two carbon atoms in a sugar ring. It may include formula nucleosides (BNA). For example, such a BNA has a sugar moiety. "Bound" containing a 4'-CH(CH3)-O-2' bridge connecting 4'-carbon and 2'-carbon This may include "(cEt)" (or "cEt"). In some embodiments, this The antisense oligonucleotides provided in the specification are located at the 4' position of the sugar unit of the nucleoside. It contains locked nucleic acid (LNA) which includes a bridge connecting two carbon atoms between the 2' position. Yes, it is possible. Such an LNA is α-L-methyleneoxy(4'-CH2-O-2')LN A, β-D-methyleneoxy(4'-CH2-O-2')LNA, ethyleneoxy(4' -(CH2)2-O-2')LNA, aminooxy(4'-CH2-ON(R)-2' )LNA, oxyamino(4'-CH2-N(R)-O-2')LNA, or U.S. Patent Specification No. 7,053,207; Specification No. 6,268,490; No. 6,770,748 Specification No. 6,794,499; Specification No. 7,034,133; No. 6,52 Specification No. 5,191; Specification No. 7,696,345; Specification No. 7,569,575; Specification No. 7,314,923; Specification No. 7,217,805; No. 7,084,125 Specification No. 6,670,461; or Specification No. 6,670,461; International Publication No. 98 / 39352 pamphlet Let or any other LNAs listed in International Publication No. 99 / 14226 It may include other suitable LNAs, see Braasch et al., Chern. Biol. 8: l-7, 200 1; Elayadi et al., Curr. Opinion Invens. Drugs 2: 558-561, 2001; Frieden et al., Nucleic Acids Research, 21: 6365-6372, 2003; Koshkin et al., Tetrahedron, 54: 3 607-3630, 1998; Morita et al., Bioorganic Medicinal Chemistry, 11: 2211-2226, 20 03; Orum et al., Curr. Opinion Mol. Ther. 3: 239-243, 2001; Singh et al., Chem. Commun. 4: 455-456, 1998; Singh et al., J. Org. Chem., 63: 10035-10039, 1998; This includes LNAs as described in Wahlestedt et al., PNAS 97: 5633-5638, 2000.

[0076] Steric barrier Antisense oligonucleotides bind to target nucleic acids, specifically DNA or RNA. Proteins, transcription factors, splicing factors, ribosomes, and / or translation mechanisms It can sterically block the approach of RNAseH to the target nucleic acid, and therefore activate RNAseH. Without reducing the target expression, for example, such a steric blocker reduces the target start codon. Hybridizes with the surrounding sequence, blocking the branching point sequence of the intron, and creates a splice site. Targeting the intron and / or exon sequence, or exon splicing. By targeting regulatory sequences such as singly enhancers, the expression of the target protein can be reduced. It can be lowered. The steric barrier is determined in advance or predicted. It can be designed based on the nucleotide-exon boundary and gene structure, and different architectures A panel of thisense oligonucleotides can be generated to block the same area. BLAST analysis can determine off-target hybridization for each ASO. This can be done to minimize it.

[0077] Steric blockers are endogenous cellular surveillance pathways that recognize and degrade abnormal mRNA. By utilizing this, mRNA reduction can be achieved. One such pathway is Nonsense-mediated mRNA decay (NMD) modulates gene expression. This potentially prevents the production of toxic proteins from mRNA. A defect in NA processing occurs when premature stop codons (PTCs) are introduced. This causes a loss of protein function, destroying the open reading frame. PTC-containing mRNA is connected to the translation ribosome and the exon containing the essential NMD factor UPF1. This includes information exchange between the components of the composite complex, including endonucleases and exonucleas. ASO can be a substrate for NMD, which degrades RNA through the activity of the enzyme. By directing target mRNA towards the NMD pathway, a decrease in target mRNA levels is achieved. Therefore, it can be designed rationally. This is because the arrangement of steric barriers is determined by a specific code. xons, intron-exon junctions, or other necessary premRNA processing Designed to be complementary to the array, exon skipping and frame shifting This can be achieved by implementing and / or implementing PTC.

[0078] In some embodiments, the anti-sensing agents that target MAPT as provided herein The oligonucleotide is a steric barrier, for example, a nucleic acid salt shown in any of Tables 2-8. At least 70% (e.g., 70%, 75%, 80%, 81%, 82%) of any of the base sequences. %, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92% Distribution of %, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) This is an oligonucleotide containing a nucleic acid base sequence having serial identity. In some embodiments, In this specification, the MAPT-targeting antisense oligonucleotides provided herein are It is a steric barrier containing one of the nucleic acid base sequences shown in any of Tables 2-8. In some embodiments, antisense instruments targeting MAPT provided herein A ligonucleotide consists of one of the nucleic acid base sequences shown in any of Tables 2-8. It consists of a steric blocker. As detailed in the examples below, the steric blocker constitutes the target tau. Exons that do this (for example, exons 1, 4, 5, 7, 9, 11, 12, 13), MAPT Sequences surrounding the start codon, splice acceptors and donors, and splicing branch points. Polypyrimidine track-related sequences, or enhancers or inhibitors of splicing It was designed to target the sequence. Targeting the start codon and exon 1 is This may block the start of translation. It may interfere with splicing and / or excavation. ASO that induces Son skipping is a precursor to frame shifting and / or early downstream This results in the introduction of a stop codon, leading to a decrease in MAPT mRNA and / or tau protein levels. You will be leading those below you.

[0079] Chemical modifications can be incorporated into steric blockers to improve their stability, efficacy, and / or cellular uptake. Steric blockers can have chemical modifications at each nucleotide position or at some selected positions. For example, incorporation of 2'-modifications of the sugar ring (such as 2'-O-methoxyethyl, MOE), inclusion of locked nucleic acid (LNA) and / or backbone modifications (such as phosphorothioate) can reduce nuclease degradation and / or increase the binding affinity of antisense oligonucleotides. In addition to modifications of the sugar and / or backbone, steric blockers can be made from oligomers that are very different from DNA or RNA. Peptide nucleic acid (PNA) is an oligonucleotide mimic in which its nucleobases are linked by amide bonds. Since the amide backbone is uncharged, binding is characterized by a high rate of association and high affinity (see Bentin T, Biochemistry. 1996; 35:8863-8869; Smulevitch SV, Nat Biotech. 1996; 14:1700-1704). Phosphorodiamidate morpholino oligomers (PMO or commonly referred to as "morpholinos") are another type of uncharged DNA analog. PMO does not bind to complementary targets with the high affinity that characterizes PNA binding, but has been shown to be an effective agent inside cells (see Summerton J, Antisense Nucleic Acid Drug Dev. 1997; 7:187-195; Corey DR, Genome Biol. 2001; 2:REVIEWS1015). or can improve cellular uptake. Steric blockers can have chemical modifications at each nucleotide position or at some selected positions. For example, incorporation of 2'-modifications of the sugar ring (such as 2'-O-methoxyethyl, MOE), inclusion of locked nucleic acid (LNA) and / or backbone modifications (such as phosphorothioate) can reduce nuclease degradation and / or increase the binding affinity of antisense oligonucleotides. In addition to modifications of the sugar and / or backbone, steric blockers can be made from oligomers that are very different from DNA or RNA. Peptide nucleic acid (PNA) is an oligonucleotide mimic in which its nucleobases are linked by amide bonds. Since the amide backbone is uncharged, binding is characterized by a high rate of association and high affinity (see Bentin T, Biochemistry. 1996; 35:8863-8869; Smulevitch SV, Nat Biotech. 1996; 14:1700-1704). Phosphorodiamidate morpholino oligomers (PMO or commonly referred to as "morpholinos") are another type of uncharged DNA analog. PMO does not bind to complementary targets with the high affinity that characterizes PNA binding, but has been shown to be an effective agent inside cells (see Summerton J, Antisense Nucleic Acid Drug Dev. 1997; 7:187-195; Corey DR, Genome Biol. 2001; 2:REVIEWS1015). and / or increase the binding affinity of antisense oligonucleotides. In addition to modifications of the sugar and / or backbone, steric blockers can be made from oligomers that are very different from DNA or RNA. Peptide nucleic acid (PNA) is an oligonucleotide mimic in which its nucleobases are linked by amide bonds. Since the amide backbone is uncharged, binding is characterized by a high rate of association and high affinity (see Bentin T, Biochemistry. 1996; 35:8863-8869; Smulevitch SV, Nat Biotech. 1996; 14:1700-1704). Phosphorodiamidate morpholino oligomers (PMO or commonly referred to as "morpholinos") are another type of uncharged DNA analog. PMO does not bind to complementary targets with the high affinity that characterizes PNA binding, but has been shown to be an effective agent inside cells (see Summerton J, Antisense Nucleic Acid Drug Dev. 1997; 7:187-195; Corey DR, Genome Biol. 2001; 2:REVIEWS1015). In addition to modifications of the sugar and / or backbone, steric blockers can be made from oligomers that are very different from DNA or RNA. Peptide nucleic acid (PNA) is an oligonucleotide mimic in which its nucleobases are linked by amide bonds. Since the amide backbone is uncharged, binding is characterized by a high rate of association and high affinity (see Bentin T, Biochemistry. 1996; 35:8863-8869; Smulevitch SV, Nat Biotech. 1996; 14:1700-1704). Phosphorodiamidate morpholino oligomers (PMO or commonly referred to as "morpholinos") are another type of uncharged DNA analog. PMO does not bind to complementary targets with the high affinity that characterizes PNA binding, but has been shown to be an effective agent inside cells (see Summerton J, Antisense Nucleic Acid Drug Dev. 1997; 7:187-195; Corey DR, Genome Biol. 2001; 2:REVIEWS1015). Peptide nucleic acid (PNA) is an oligonucleotide mimic in which its nucleobases are linked by amide bonds. Since the amide backbone is uncharged, binding is characterized by a high rate of association and high affinity (see Bentin T, Biochemistry. 1996; 35:8863-8869; Smulevitch SV, Nat Biotech. 1996; 14:1700-1704). Phosphorodiamidate morpholino oligomers (PMO or commonly referred to as "morpholinos") are another type of uncharged DNA analog. PMO does not bind to complementary targets with the high affinity that characterizes PNA binding, but has been shown to be an effective agent inside cells (see Summerton J, Antisense Nucleic Acid Drug Dev. 1997; 7:187-195; Corey DR, Genome Biol. 2001; 2:REVIEWS1015). Peptide nucleic acid (PNA) is an oligonucleotide mimic in which its nucleobases are linked by amide bonds. Since the amide backbone is uncharged, binding is characterized by a high rate of association and high affinity (see Bentin T, Biochemistry. 1996; 35:8863-8869; Smulevitch SV, Nat Biotech. 1996; 14:1700-1704). Phosphorodiamidate morpholino oligomers (PMO or commonly referred to as "morpholinos") are another type of uncharged DNA analog. PMO does not bind to complementary targets with the high affinity that characterizes PNA binding, but has been shown to be an effective agent inside cells (see Summerton J, Antisense Nucleic Acid Drug Dev. 1997; 7:187-195; Corey DR, Genome Biol. 2001; 2:REVIEWS1015). Since the amide backbone is uncharged, binding is characterized by a high rate of association and high affinity (see Bentin T, Biochemistry. 1996; 35:8863-8869; Smulevitch SV, Nat Biotech. 1996; 14:1700-1704). Phosphorodiamidate morpholino oligomers (PMO or commonly referred to as "morpholinos") are another type of uncharged DNA analog. PMO does not bind to complementary targets with the high affinity that characterizes PNA binding, but has been shown to be an effective agent inside cells (see Summerton J, Antisense Nucleic Acid Drug Dev. 1997; 7:187-195; Corey DR, Genome Biol. 2001; 2:REVIEWS1015). Since the amide backbone is uncharged, binding is characterized by a high rate of association and high affinity (see Bentin T, Biochemistry. 1996; 35:8863-8869; Smulevitch SV, Nat Biotech. 1996; 14:1700-1704). Phosphorodiamidate morpholino oligomers (PMO or commonly referred to as "morpholinos") are another type of uncharged DNA analog. PMO does not bind to complementary targets with the high affinity that characterizes PNA binding, but has been shown to be an effective agent inside cells (see Summerton J, Antisense Nucleic Acid Drug Dev. 1997; 7:187-195; Corey DR, Genome Biol. 2001; 2:REVIEWS1015). Phosphorodiamidate morpholino oligomers (PMO or commonly referred to as "morpholinos") are another type of uncharged DNA analog. PMO does not bind to complementary targets with the high affinity that characterizes PNA binding, but has been shown to be an effective agent inside cells (see Summerton J, Antisense Nucleic Acid Drug Dev. 1997; 7:187-195; Corey DR, Genome Biol. 2001; 2:REVIEWS1015). Phosphorodiamidate morpholino oligomers (PMO or commonly referred to as "morpholinos") are another type of uncharged DNA analog. PMO does not bind to complementary targets with the high affinity that characterizes PNA binding, but has been shown to be an effective agent inside cells (see Summerton J, Antisense Nucleic Acid Drug Dev. 1997;​​​​​​​​​In some embodiments, the anti-sensing agents that target MAPT as provided herein S-oligonucleotides are steric barriers containing 2'-modified nucleotides. '- Modifications include 2'-fluoro, 2'-deoxy-2'-fluoro, 2'-O-methyl, 2 '-O-methoxyethyl (2'-O-MOE), 2'-O-aminopropyl (2'-O- AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimeth 2'-O-DMAP, 2'-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE), and 2'-ON-methylacetamide (2'-O- It can be selected from the group consisting of NMA). In some embodiments, as specified herein The MAPT-targeting antisense oligonucleotides provided are each nucleotide It is a steric barrier agent having a 2'-O-MOE modification in its subunit.

[0081] In some embodiments, the anti-sensing agents that target MAPT as provided herein S-oligonucleotides are phosphodiesters or phosphorothioates between nucleosides. It is a steric barrier agent with linkages.

[0082] In some embodiments, the anti-sensing agents that target MAPT as provided herein S-oligonucleotides are steric blockers containing skeletal modifications that prevent RNaseH binding. Such steric barriers are modified nucleoside links, for example, methyl hydroxylase. Phosphorate linkage, methylphosphonothioate linkage, phosphoromol holiday linkage, phospho This may include lopiperadiate or phosphoramidite linkages. Several implementations In some forms, every other linkage between nucleosides may contain a modified phosphate ester having a 2'-lower alkyl moiety (e.g., C 1-C4, straight or branched, saturated or unsaturated alkyl, such as methyl, ethyl, e thyl, propyl, 1-propenyl, 2-propenyl, and isopropyl, etc.) or combinations thereof. In some embodiments, the antisense oligonucleotides targeting MAPT provided herein are steric blockers that include one or more modified internucleoside linkages described in U.S. Patent No. 5,149,797.

[0083] ​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​ [ka]

[0087] Gapmar Antisense oligonucleotides containing a continuous stretch of DNA are found at the end of cells. The nuclease RNaseH is recruited to the target RNA:DNA heteroduplex, and RNA:DN It can cleave target RNA in the A double helix. Gapmers are chimeric antisense. It is a compound. Chimeric antisense compounds are typically increased against nuclease degradation. Significant resistance, increased cellular uptake, increased binding affinity to target nucleic acids, and / or at least one region modified to give increased inhibitory activity, and the first region It contains a second region with nucleotides that are chemically different from the nucleotides in the first region.

[0088] A gapmer is a pair of wins consisting of nucleotides modified at the 5' and 3' ends. From a stretch of consecutive 2'-deoxyribonucleotides located between segments It has a central gap segment. The gap segment contains endonuclease RNA. It serves as a substrate for seH cleavage, but on the other hand, it has modified nucleotides. The wing segment provides increased resistance to degradation by other nucleases. - The gap-wing segment can also be written as "XYZ", where "X" is 5' represents the length of the wing, "Y" represents the length of the gap, and "Z" represents the length of the 3' wing. Represents length. "X" and "Z" indicate the presence of uniform, mutated, or alternating sugar moieties. It's also possible.

[0089] In some embodiments, the central gap segment of the gapmer is at least Five consecutive 2'-deoxyri It consists of bonucleotides, and the 5' and 3' wing segments are one or more 2' - Contains modified nucleotides. Up to 4 consecutive 2'-deoxyribonucleotides. Chimeric oligonucleotides containing stretches of RNAseH have been reported not to activate RNAseH. It has been reported. See U.S. Patent No. 9,157,081. Several implementations In terms of form, the antisense oligonucleotides that target MAPT provided herein Chido has at least 7 consecutive 2'- This is a gapmer containing deoxyribonucleotides in some embodiments. The antisense oligonucleotides that target MAPT provided in the specification are 10 It is a gapmer containing consecutive 2'-deoxyribonucleotides. The 2'-modification is 2 '-Fluoro, 2'-deoxy-2'-fluoro, 2'-O-methyl, 2'-O-methoxy 2'-O-MOE, 2'-O-aminopropyl (2'-O-AP), 2'- O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2'-O-dimethylaminoethyloxyethyl(2'-O- DMAEOE) and 2'-ON-methylacetamide (2'-O-NMA) You can choose from the group.

[0090] In some embodiments, the gapmer targeting MAPT is 20 nucleos The length of the gap segment is 5-10-5, where the central gap segment is 1 Contains 0 2'-deoxynucleosides and 5 nucleosides, each with a 2'-modification. It is adjacent to the 5' and 3' wing segments, each containing the respective. Other suitable gap marks These are 5-9-5 gapmers, 5-8-5 gapmers, 4-8-6 gapmers, and 6- This includes, but is not limited to, 8-4 gapmers or 5-7-6 gapmers.

[0091] In some embodiments, the anti-sensing agents that target MAPT as provided herein Sulokkins are gapmers, for example, any of Tables 9-15 and 17. At least 70% (e.g., 70%, 75%, 8%) of any of the nucleic acid base sequences shown by ka 0%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 9 0%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, It is an oligonucleotide containing a nucleic acid base sequence that has 100% sequence identity. In some embodiments, antisense instruments targeting MAPT provided herein Ligonucleotides are any of the nucleic acid base sequences shown in Tables 9-15 and 17. It is a gapmer that includes or. In some embodiments, the MA provided herein Antisense oligonucleotides that target PT are any of those listed in Tables 9-15 and 17. It is a gapmer consisting of one of the nucleic acid base sequences shown. This will be described in detail in the examples below. Thus, a gapmer is a sequence around the start codon, exon 1, or MAPT transcript. It is designed to target the 3' untranslated region (UTR) of the molecule. In some embodiments, Therefore, the gapmar was designed to target the 3'UTR.

[0092] In some embodiments, the anti-sensing agents that target MAPT as provided herein Soligonucleotides are nucleic acid base sequences shown in any of Tables 9-15 and 17. It is a 5-10-5 gapmer containing either of the first to fifth nucleotides. Each contains a nucleoside modified with 2'-O-MOE, and the 6th to 15th nucleosides Each nucleotide contains a 2'-deoxynucleoside, and the 16th to 20th nucleotides are 2' Each contains a nucleoside modified with -O-MOE.

[0093] MAPT genome sequences targeted by antisense oligonucleotides In some embodiments, antisense oligonucleotides are used in MAPT genome distribution A specific area of ​​column (GenBank access number NG_007398.1; sequence number 304) Alternatively, it may be configured to target the region of the corresponding tau mRNA or transcript (SEQ ID NO: 306). The intron and exon sequences and branching points of MAPT are measured. Based on the genome database website, transcript: MAPT-203 ENST0 Determined using 0000344290. Exons and introns of the human MAPT gene. , and screening for intron / exon synchronicity, MAPT gene or transcription Targeting specific regions within a substance using antisense oligonucleotides is possible in other ways. It has been revealed that targeting this region is more effective in reducing tau expression than targeting the other region. For example, Table 1 shows that in the MAPT gene or transcript, antisense oligonucleotides The following is a list of sequences of several preferred regions that can be targeted by rheotide.

[0094] In some embodiments, antisense oligonucleotides provided herein This is one of the sequence numbers 487-506 that has 1, 2, or 3 mismatches. A sequence of at least 12 nucleic acid bases (for example, 12, 13, 14, 15, 16, 1) It contains a nucleic acid base sequence that is complementary to 7 or 18 consecutive nucleic acid bases. In embodiments, the antisense oligonucleotides provided herein are: At least 12 consecutive nucleic acid bases from any one of 487 to 506 (for example, 12, 13, 14, 15, 16, 17, or 18 consecutive nucleic acid bases) and 100% complementary It contains a certain nucleic acid base sequence.

[0095] [Table 1]

[0096] Antisense oligonucleotide conjugate The conjugation of an antisense oligonucleotide with another part is antisense This improves the activity, cellular uptake, and / or tissue distribution of oligonucleotides. Yes, it is possible. For example, antisense oligonucleotides are one or more diagnostic compounds. Reporter group, crosslinking agent, nuclease-resistance-constituting moiety, lipophilic molecule, cholesterol Lipids, lectins, linkers, steroids, ubaol, hesigenin, diosgenin, Terpenes, triterpenes, sarsasapogenins, friederin, epifriederanol inducer Modified lithocholic acid, vitamins, biotin, carbohydrates, dextran, dyes, pullulan, ki Chin, chitosan, synthetic carbohydrates, oligolactate 15-mer, natural polymers, low or moderate Molecular weight polymer, inulin, cyclodextrin, hyaluronic acid, protein, protein Particle binders, integrin target molecules, polycations, peptides, polyamines, peptide mimics Body, transferrin, coumarin, phenazine, folate, phenanthridine, ant Covalently linked with raquinone, acridine, fluorescein, and / or rhodamine. It is possible.

[0097] In some embodiments, antisense oligonucleotides provided herein It is attached to the linker molecule. In some embodiments, provided herein Antisense oligonucleotides are linked to lipids or cholesterol. In several embodiments, the antisense oligonucleotide is neutral liposome (NL) Alternatively, it is linked to lipid nanoparticles (LNPs). LNPs are self-assembling cationic lipids. It is a quality-based system, which includes, for example, neutral lipids (liposome base); cationic properties. Lipids (for oligonucleotide loading); cholesterol (for liposome stabilization) ); and PEG-lipids (stabilization of formulations, charge shielding and extended circulation in blood flow) (Therefore) it may include neutral liposomes (NL) are particles based on noncationic lipids. That is the case.

[0098] In some embodiments, antisense oligonucleotides provided herein It is linked to fatty acids, such as omega-3 fatty acids or omega-6 fatty acids. Examples of omega-3 fatty acids include alpha-linolenic acid (ALA) and docosahexaenoic acid (DH). A) Eicosapentaenoic acid (EPA), docosapentaenoic acid (DPA), eicosapentaenoic acid Eicosapentaenoic acid (ETA), eicosatrienoic acid (ETE), eicosapentaenoic acid (EPA) Hexadecatrienoic acid (HTA), Heneicosapentaenoic acid (HPA), Stearyl Contains tetracosapentaenoic acid (SDA), tetracosapentaenoic acid, and tetracosahexaenoic acid.

[0099] Testing of antisense oligonucleotide activity The activity of antisense oligonucleotides should be tested in vitro or in vivo. This can be done. To test in vitro, ASO must be transfected into cultured cells. It can be introduced by electroporation or by injection. After the treatment period, the ASO-treated tissue Determine the expression level of MAPT (tau) in cells and compare it to MAPT in untreated control cells. It can be compared with the expression level of (tau).

[0100] MAPT expression levels can be determined by any suitable method, for example, by the level of MAPT mRNA. By quantifying the amount of MAPT mRNA, the amount of cDNA produced by reverse transcription of MAPT mRNA can be measured. This can be determined by determining the amount of tau protein. The law requires that the analysis be performed on the sample according to the sample standards or modified for high-capacity analysis. It is possible.

[0101] MAPT mRNA levels are specifically hybridized in segments of MAPT transcripts. The probe can be used to detect and quantify, for example, by Northern blot analysis. Yes, it is possible. The level of MAPT mRNA can be determined by polymerase chain reaction (PCR). PT can also be detected and quantified using a pair of primers that recognize PT transcripts. The general procedure for CR is described in MacPherson et al., PCR: A Practical Approach, (IRL Pre This is taught in ss at Oxford University Press (1991). However, each application reaction The PCR conditions used are determined empirically. Many parameters, for example, Kneeling temperature and time, increase time, Mg 2 + and / or ATP concentration, pH, if The relative concentrations of primer, template, and / or deoxyribonucleotides are This affects the success of the reaction. After amplification, the resulting DNA fragments are subjected to agarose gel electrophoresis. Following motion detection, the substance is visualized using ethidium bromide staining and ultraviolet illumination. It is possible.

[0102] In some embodiments, the level of MAPT mRNA is compared to any commercially available real-time Using an immunoPCR system, detectable dyes or reporters are added during the amplification step. By incorporating it at times, quantitative real-time PCR can be used to monitor the amplification of target nucleic acids. It can be detected and quantified.

[0103] Alternatively, the label can be applied to the original nucleic acid sample (e.g., mRNA, poly(A), mRNA, cDNA, The label may be attached directly to the product (otherwise) or to the amplified product after the amplification is complete. Methods for attaching nucleic acids are well known to those skilled in the art, for example, kinase manipulation of nucleic acids and subsequent The attachment of a nucleic acid linker (ligase) to the sample nucleic acid to the label (e.g., a fluorophore) Nick translation or terminal labeling (for example, using labeled RNA) (including)

[0104] Suitable detectable labels for use in this invention include spectroscopic, photochemical, biochemical, and immunological labels. This includes any composition detectable by epidemiological, electrical, optical, or chemical means. A clear and useful label is for staining using labeled streptavidin conjugates. Biotin, magnetic beads (e.g., Dynabeads®), fluorescent dyes (e.g., Fluorescein, Texas Red, Rhodamine, Green Fluorescent Protein, etc., radioactive labeling (For example, 3H, 125I, 35S, 14C, or 32P), enzymes (for example, horseradish) Biperoxidase, alkaline phosphatase, and other commonly used in ELISAs (and other similar materials), and colorimetric labels, such as colloidal gold or colored glass or plastic. Includes beads made of materials such as polystyrene, polypropylene, latex, etc. A patent teaching the use of the mark is U.S. Patent No. 3,817,837; No. 3,850, Specification No. 752; Specification No. 3,939,350; Specification No. 3,996,345; No. 4 ,277,437; 4,275,149; and 4,366,24 Includes Item 1.

[0105] The detection of labels is well known to those skilled in the art. Therefore, for example, radioactive labels are detected in photographic film. It can be detected using a meter or scintillation counter, and the fluorescent marker is It can be detected using a photodetector that detects light emission. Enzyme labeling is typically, To provide a substrate to an enzyme and detect the production of a reaction product resulting from the enzyme's action on the substrate. Nucleic acid is detected by, and colorimetric labeling simply detects the colored label visually. For a detailed review of methods for detecting identified, labeled, and hybridized nucleic acids, see Labo ratory Techniques in Biochemistry and Molecular Biology, Vol. 24: Hybridization See *with Nucleic Acid Probes*, P. Tijssen, ed. Elsevier, NY (1993).

[0106] The activity of antisense oligonucleotides is related to tau protein levels in this technology. It can also be evaluated by measuring using known methods. For example, tauta Protein levels are determined by Western blot analysis (immunoblotting) and enzyme-linked immunoassay. Immunoassay (ELISA), immunohistochemistry, immunoassay, immunoprecipitation, immunofluorescence assay, immuno Epidemiological cytochemistry, fluorescence-activated cell sorting (FACS), radioimmunoassay, immunoradioassay, high-speed Liquid chromatography (HPLC), mass spectrometry, confocal microscopy, enzyme assays, and This can be quantified by surface plasmon resonance (SPR).

[0107] The in vivo activity of antisense oligonucleotides is tested in animal models. It is also possible. The test can be conducted in normal animals or experimental disease animal models. Chisen oligonucleotides can be formulated with pharmaceutically acceptable diluents, and It is delivered through the appropriate route of administration. After the treatment period, tissue samples, such as brain tissue and cerebrospinal fluid, are administered. (CSF), collect spinal cord and measure tau expression levels using one of the methods described above It can be measured by histological analysis of the brain structure and / or the presence of neurofibrillary tangles. This can be done to evaluate the present. Changes in the phenotype of treated animals, for example, Improved cognitive or motor skills can also be monitored and evaluated.

[0108] Synthesis and characterization of oligonucleotides Single-stranded oligonucleotides can be polymerized using any nucleic acid polymerization method known in the art, e.g. For example, the phosphoramidite method (SL Beaucage and RP Iyer, Tetrahedron, 1993, 49, 6123; Solid phase bonding using SL Beaucage and RP Iyer, Tetrahedron, 1992, 48, 2223) It is synthesized using chemical, H-phosphonate, phosphortryster, or enzymatic synthesis. This can be done using an automated, commercially available synthesizer, such as BioAutomation(I rving, Texas, or Applied Biosystems (Foste A synthesizer in r City, California can be used. Several implementations In this state, single-stranded oligonucleotides are, for example, Nucleic Acid Chemistry, Beauca ge, SL et al. (Edrs.), Current Pr of John Wiley & Sons, Inc., New York, NY, USA. It is produced using standard solid-phase phosphoramidite chemistry as described in otocols. Holothioate linkage is phenylacetyl disulfide or DDTT(((dimethyl Sulfur (mino-methylidene)amino)-3H-1,2,4-dithiazoline-3-thion) It can be introduced using chemical reagents. Similar techniques and commercially available modified amidites and controlled-pore glass (CPG) products, e.g. For example, Amidai modified with biotin, fluorescein, acridine, or psoralen. The nitrate and / or CPG are modified oligonucleotides, or fluorescently labeled nitrates. Used to synthesize otin or other conjugate oligonucleotides. This is common knowledge.

[0109] Controlling the quality of the starting materials and the products from each synthesis step involves controlling impurities in the final product. This is extremely important for minimizing the level. However, the synthesis per coupling Considering the number of steps and couplings, the presence of impurities is inevitable. The method is used to remove undesirable impurities from the final oligonucleotide product. It is possible. A common purification technique used for single-stranded oligonucleotides is reverse-phase ionization. ON-IP-HPLC, capillary gel electrophoresis (C GE), anion exchange HPLC (AX-HPLC), and size exclusion chromatography —(SEC) included.

[0110] After purification, the oligonucleotides can be analyzed by mass spectrometry at 260 nm. It is quantified spectroscopically by wavelength.

[0111] Use and methods of treatment For example, the tau expression level in humans is used in relation to the antiseptic described herein. A method of reducing by administering a therapeutically effective dose of any of the lance oligonucleotides. This is provided herein. In some embodiments, antisense oligonucleotides The drug is administered to the target via intrathecal, intracranial, intranasal, intravenous, oral, or subcutaneous routes. To obtain. In some embodiments, such a method is used to treat tau-related diseases. This further includes identifying and selecting individuals that are susceptible to the disease or are prone to it.

[0112] The antisense oligonucleotides or their pharmaceutical compositions provided herein are It can be used to treat or prevent tau-related diseases in the subject. In several embodiments, the present invention relates to the antisense oligonucleotide described herein. Otide or its pharmaceutical compositions are used to treat or prevent tau-related diseases in patients. Provided for use in a patient. In a further embodiment, the present invention provides for use in a patient. In the manufacture of pharmaceuticals for use in the treatment or prevention of related diseases, as described herein This provides the use of antisense oligonucleotides.

[0113] Diseases associated with tau include Alzheimer's disease (AD), amyotrophic lateral sclerosis / parkinson's disease. ALS-PDC (Alopecia Syndrome-Dementia Complex), Argyrophilic Granule Dementia (AGD), British-type Ammonia Lloyd's vascular disease, cerebral amyloid angiopathy, chronic traumatic encephalopathy (CTE), corticobasal degeneration (CBD), Creutzfeldt-Jakob disease (CJD), punch-drunk syndrome, accompanied by calcification. Diffuse neurofibrillary tangles, Down syndrome, Dravet syndrome, epilepsy, frontotemporal dementia (F TD), frontotemporal Parkinson's syndrome linked to chromosome 17 (FTDP-17). Type 1 dementia, frontotemporal lobar degeneration, ganglioglioma, gangliocytoma, Gerstmann-Strouslä - Shainker's disease, Haller-Holden-Spats disease, Huntington's disease, inclusion body myositis, lead brain Diseases, Ritico-Bodig disease, meningiomatosis, multiple system atrophy, myotonic dystrophy, Niemann-Pick disease type C (NP-C), a non-Guanian motor neuron disorder with neurofibrillary tangles. Pick's disease (PiD), post-encephalitis parkinson's syndrome, prion protein brain amyloid blood vessels Disease, progressive subcortical gliosis, progressive supranuclear palsy (PSP), subacute sclerosing panencephalitis, neurological Tangle-only dementia, neurofibrillary tangle-dominant dementia (Tangle (1-predominant dementia), multiple infarct dementia, ischemic stroke, and tuberous sclerosis This includes, but is not limited to, these items.

[0114] Combination therapy The various oligonucleotides listed above are used in combination with other therapeutic partners. Therefore, the method for treating tau-related diseases described herein This may further include administering a second active substance to a subject in need of treatment. For example, antisense antisense targeting the microtubule-related protein tau (MAPT) Gonucleotides are antibodies and / or amyloid proteins that specifically bind to tau protein. A substance that targets Aβ, for example, Aβ or beta-secretase (BACE). ) It can be used in combination with an antibody that binds to an inhibitor. Therefore, antisense oligonucleotides that target MAPT are specific to tau protein. It is used in combination with an antibody that binds to MAPT. In some embodiments, it targets MAPT. Antisense oligonucleotides are used in combination with BACE inhibitors.

[0115] The term "combination" refers to a combination fixed in one unit dosage form, or the compounds of the present invention and Combination partners (for example, other drugs described below, also known as "therapeutic agents" or "co-acting substances") (as it is called), independently, simultaneously, or in combination, partners collaborate, for example, to produce synergistic effects. This refers to any combination of administrations that may be administered separately within a time interval that allows for the indication to be shown. A single component may be packaged in the kit or packaged separately. Component One or both (for example, powder or liquid) are reconstituted or diluted to the desired dose before administration. It may be interpreted as follows. The terms "co-administration" or "combination administration" used herein are selective. The combination of partners is administered to a single target (e.g., a patient) that needs it. This means that the active substance must be administered via the same route or simultaneously. It is intended to include treatment plans that do not necessarily have [specific characteristics]. The term " as used herein "Pharmaceutical combination" refers to a product resulting from the mixing or combination of two or more therapeutic agents. This includes both fixed and unfixed combinations of agents. The term "fixed combination" is a general term. "The therapeutic agent, for example, the oligonucleotide and combination partner of the present invention, is both This also means a single entity or dosage form that is administered to the patient simultaneously. The "combination" refers to a therapeutic agent, for example, the oligonucleotide and combination partner of the present invention. Both are identified in the patient as separate entities, either simultaneously, in parallel, or sequentially. This means that it is administered without time restrictions, and such administration is performed in the patient's body. It provides therapeutically effective levels of two compounds. The latter is used in cocktail therapy, for example, with three or more compounds. This also applies to the administration of the above-mentioned therapeutic agents.

[0116] As used herein, the term "pharmaceutical combination" refers to a combination fixed in a single unit dosage form. One or more therapeutic agents are administered independently, simultaneously, or separately within a time interval. These time intervals, in particular, allow for collaborative, synergistic effects between partners. Kit of parts for non-fixed combination or combination administration that makes it possible to demonstrate It refers to one of the following:

[0117] The term “combination therapy” refers to two methods used to treat the therapeutic conditions or disorders described in this disclosure. This refers to the administration of more than one type of therapeutic agent. Such administration is carried out in a substantially simultaneous manner, for example, effectively This involves co-administration of these therapeutic agents in a single capsule with a fixed ratio of components. Alternatively, such administration may be in multiple or separate containers for each active ingredient (for example). This includes co-administration in the form of tablets, capsules, powders, and liquids. Alternatively, liquid preparations can be reconstituted or diluted to the desired dose before administration. In addition... Such administrations are administered sequentially, either at approximately the same time or at different times. This also includes the use of different types of therapeutic agents. In all cases, the treatment plan is as described herein. This will provide beneficial effects from drug combinations in the treatment of a condition or disorder.

[0118] Sample preparation Tissue samples are subjected to antisense oligonucleotide analysis using any method known in the art. It can be obtained from subjects treated with creotides, for example, by biopsy or surgery. For example, a sample containing cerebrospinal fluid is injected into the spinal canal in the lumbar region using a thin needle attached to a syringe. Once inserted, a vacuum can be created, and cerebrospinal fluid can be drawn through the needle and collected in a syringe. It can be obtained by lumbar puncture. CT imaging, ultrasound, or endoscopy can also be used for this type of procedure. It can be used to derive order.

[0119] The sample can also be flash-frozen and stored at -80°C for later use. It is fixed with a fixative such as formaldehyde, paraformaldehyde, or acetic acid / ethanol. It can also be determined. RNA or protein can be fresh, frozen, or fixed. It can also be extracted from the material for analysis.

[0120] Pharmaceutical composition, dosage, and administration Compositions, for example, one or more antisense oligonucleotides provided herein. Pharmaceutical compositions containing tides are also provided herein. Pharmaceutical compositions are typically pharmaceutically acceptable. Includes a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" as used herein. These include saline solutions, solvents, dispersions, coatings, antibacterial agents, and antimicrobial agents suitable for pharmaceutical administration. The pharmaceutical composition typically includes antifungal agents, isotonic agents, and absorption retarders. The drug is formulated to be compatible with the route of administration. Examples of routes of administration include intrathecal, intracranial, and nasal. This includes internal, intravenous, oral, or subcutaneous administration.

[0121] In some embodiments, the antisense oligonucleotides described herein It can be conjugated with antibodies that can cross the blood-brain barrier (for example, It binds to transferrin receptors, insulin, leptin, or insulin-like growth factor 1. Antibodies that can be delivered intravenously (Evers et al., Advanced Drug Delivery Reviews 8) 7 (2015): 90-103).

[0122] Methods for formulating suitable pharmaceutical compositions are known in the art, for example, Re mington: The Science and Practice of Pharmacy. 21st ed., 2005; and Drugs and the e Pharmaceutical Sciences: a Series of Textbooks and Monographs (Dekker, NY). Please refer to Leeds's book, for example, for parenteral, intradermal, intrathecal, or subcutaneous application. The solution or suspension used consists of the following components: sterile diluent, e.g., water for injection. Physiological saline solution, non-volatile oil, polyethylene glycol, glycerin, propylene glycol Alcohol or other synthetic solvents; antibacterial agents, e.g., benzyl alcohol or methyl parabens. Benefits, etc.; antioxidants, such as ascorbic acid or sodium bisulfite; chelates Agents, such as ethylenediaminetetraacetic acid; buffering agents, such as acetates, citrates or Phosphates, etc.; and isotonic modifiers, such as sodium chloride or dextrose. It may include. pH can be determined using an acid or base such as hydrochloric acid or sodium hydroxide. It can be adjusted. Parenteral preparations can be in ampoules, disposable syringes or glass or It can be enclosed in multiple dose vials made of plastic.

[0123] Suitable pharmaceutical compositions for use by injection are sterile aqueous solutions (if water-soluble) or dispersions. This may include sterile powders for the immediate preparation of liquids and sterile solutions or dispersions for injection. Suitable carriers for intravenous administration include physiological saline, bacteriostatic water, and Cremophor EL( (Trademark) (BASF, Parsippany, New Jersey) or Phosphate-buffered Physiology Contains saline solution (PBS). In all cases, the composition must be sterile and easy to use. It should be fluid enough to be injected. The composition should be stable under manufacturing and storage conditions. It should be present and must be protected against contamination by microorganisms such as bacteria and fungi. The carrier is, for example, water, ethanol, polyol (e.g., glycerol, propylene Contains (such as recall and liquid polyethylene glycol) and suitable mixtures thereof. It can be a solvent or dispersion medium. Suitable fluidity is, for example, lecithin. By using coating, the particle size required in the case of a dispersion is maintained, and It can be maintained by the use of surfactants. Prevention of microbial activity can be achieved with various antimicrobial agents. and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, This can be achieved with merosal, etc. In many cases, the composition contains an isotonic agent, for example , sugars, polyalcohols, such as mannitol, sorbitol, sodium chloride, etc. It would be preferable to include. Long-term absorption of the injectable composition is due to the absorption of the composition. The substance containing the delaying agent, for example, aluminum monostearate and gelatin. It can be brought about by...

[0124] A sterile solution for injection contains the required amount of active compound in a suitable solvent, one or more of the compounds listed above. The mixture is prepared by incorporating the specified combination of components and, if necessary, subsequently sterilizing by filtration. It is possible.

[0125] Generally, dispersions consist of a basic dispersion medium and other components required from those listed above. It is prepared by incorporating the active compound into a sterile vehicle containing [a specific substance]. For sterile powders used in the preparation of bacterial solutions, preferred methods of preparation include vacuum drying and freeze-drying. These methods involve adding any additional desired ingredients to the active ingredient, and also before them. This process produces a powder from a sterile, filtered solution.

[0126] Oral compositions generally contain an inert diluent or an edible carrier. Purpose of oral therapeutic administration. Therefore, the active compound is incorporated together with excipients to form tablets, lozenges, or capsules. It can be used in the form of a liquid preparation, for example, a gelatin capsule. The oral composition is used as a mouthwash. It can also be prepared using a fluid carrier for use. A pharmaceutically compatible binder. , and / or auxiliary materials may be included as part of the composition. Tablets, pills, capsules Lozenges and the like may contain any of the following ingredients or compounds with similar properties: binders, for example, fine Crystalline cellulose, tragacanth gum, or gelatin, etc.; excipients, e.g., starch. Or lactose, etc.; disintegrants, such as alginate, Primogel, or tou Sorghum starch, etc.; lubricants, for example, magnesium stearate or Stero te, etc.; flow promoters, e.g., colloidal silicon dioxide; sweeteners, e.g., sucrose - or saccharin, etc.; or flavoring agents, e.g., peppermint, methyl salicylate. Alternatively, it may contain orange flavoring or other similar ingredients.

[0127] For administration by inhalation, the compound is administered with a suitable propellant, such as a gas like carbon dioxide. From the pressurized container, dispenser, or nebulizer containing the aerosol spray It can be delivered in the form of a ray. Such a method is permitted under U.S. Article 6,468,798 This includes those described in the specification. Systemic administration of the therapeutic compounds described herein is It can also be administered via mucosal or percutaneous means. For mucosal or percutaneous administration, the barrier must be cleared. A suitable penetrating agent is used in the formulation to facilitate this process. Such penetrating agents are available in the art. It is commonly known that, for example, for transmucosal administration, detergents, bile salts, and Contains sisic acid derivatives. Transmucosal administration is performed using nasal spray or suppositories. It is possible. For transdermal administration, the active compound is known in the art. It is formulated into ointments, plasters, gels, or creams.

[0128] In one embodiment, the therapeutic compound is rapidly eliminated from the body. Includes embedded and microencapsulated delivery systems using a protective carrier. The excretion is then prepared as a controlled-release formulation, etc.

[0129] The pharmaceutical composition is packaged in a container, pack, or dispenser with instructions for administration and They can be included together.

[0130] In a non-limiting example, a pharmaceutical composition containing at least one pharmaceutical agent is a liquid (e.g., For example, as a thermosetting liquid agent, a solid component (e.g., powder or biodegradable biocompatible powder) As a polymer (e.g., a cationic biodegradable biocompatible polymer) or as part of a gel composition. It is formulated as an element (e.g., a biodegradable biocompatible polymer). Several embodiments In this, at least one composition containing at least one pharmaceutical agent is an alginate gel ( For example, sodium alginate, cellulose gels (for example, carboxymethylcellulose) (Cells or carboxyethylcellulose), or chitosan-based gels (e.g., chitosan gels) Formulated as a gel selected from the group of lyserophosphates. Additional examples of drug-eluting polymers that can be used to formulate any pharmaceutical composition include: Carrageenan, carboxymethylcellulose, hydroxypropylcellulose, polyvinyl Dextran combined with alcohol, dextrin combined with polyacrylic acid Orchid, polygalacturonic acid, galacturone polysaccharide, polylactic acid, polyglycolic acid, tamarin Dogum, xanthan gum, cellulose gum, guar gum (carboxymethyl guar), pe Kuchin, polyacrylic acid, polymethacrylic acid, N-isopropyl polyacrylamide, Polyoxyethylene, polyoxypropylene, puluronic acid, polylactic acid, cyclodextrin cycloamylose, resilience, polybutadiene, N-(2-hydroxypropyl)meth Crylamide (HPMA) copolymer, maleic anhydride-alkyl vinyl ether, poly Depsipeptide, polyhydroxybutyrate, polycaprolactone, polydioxanone, Polyethylene glycol, polyorganophosphorazene, polyorthoester, polyvinyl Pyrrolidone, polylactic acid-co-glycolic acid (PLGA), polyanhydride, polysilamine This includes, but is not limited to, mine, poly-N-vinylcaprolactam, and Guerlain. stomach.

[0131] In some embodiments, delivery of antisense oligonucleotides to target tissue is , cationic liposomes, cyclodextrins, porphyrin derivatives, branched-chain dendrims - Polyethyleneimine polymer, nanoparticles and microspheres (Dass CR. J Pharm Pharmacal It can be enhanced by carrier-mediated delivery, including but not limited to those mentioned in 2002; 54(1):3-27). .

[0132] An "effective dose" is a sufficient amount to produce a beneficial or desired result. For example, The therapeutic dose is the amount that achieves the desired therapeutic effect. This amount is used to predict the onset or symptoms of the disease. The effective preventive dose, which is the amount needed to prevent infection, may be the same as or different from the effective preventive dose. The amount can be administered in one or more doses, applications, or dosages. Therapeutic compounds The therapeutically effective dose of a substance (i.e., the effective dosage) depends on the selection of the therapeutic compound. The medication is administered multiple times a day, including once or more times a day, or once a week or every other day. A person skilled in the art can determine the severity of the disease or disability, previous treatments, and the general health of the subject. Certain conditions include, but are not limited to, health and / or age, and other pre-existing medical conditions. The cause can influence the dosage and timing of medication needed to effectively treat the target. They will recognize. Furthermore, using the therapeutically effective amount of the therapeutic compounds described herein The treatment may include a single procedure or a series of procedures.

[0133] The dosage, toxicity, and therapeutic efficacy of therapeutic compounds in cell cultures or experimental animals For example, LD50 (a lethal dose in 50% of the population) and ED50 (a lethal dose in 50% of the population) This can be determined by standard pharmaceutical procedures for determining a therapeutically effective dose. The dose-to-toxicity ratio is an indicator of treatment, and it is expressed as the LD50 / ED50 ratio. Compounds that show high therapeutic indicators are preferred. Compounds that show toxic side effects Although materials may be used, the possibility of damaging uninfected cells is minimized. Therefore, in order to reduce side effects, such compounds are directed towards the affected tissue. Care should be taken when designing the delivery system.

[0134] Using data obtained from cell culture assays and animal studies, for use in humans. The dosage range can be formulated. The dosage of such compounds has almost no toxicity. It is preferable that there is no effusion at all, and that the circulating concentration is within the range including ED50. The dosage used is This range may vary depending on the dosage form and the route of administration used. For any compound used in the method, the therapeutically effective dose is first determined by a cell culture assay. It can be estimated. The dose is determined by the IC50 (i.e., the peak of symptoms) determined in cell culture. Achieve a concentration range in circulating plasma that includes the concentration of the test compound that achieves half the inhibition. Thus, formulations can be developed using animal models. Such information is useful in humans. It can be used to determine the dose more accurately. The level in plasma is, for example, high It can be measured by fast liquid chromatography.

[0135] In some embodiments, the antisense oligonucleotides described herein For administration, use sterile water, saline solution (e.g., phosphate-buffered saline), or brain It is dissolved in cerebrospinal fluid (CSF). In some embodiments, as described herein Inthisense oligonucleotides enter the spinal cavity, for example, in the L3 or L4 intervertebral disc space. It is administered by bolus injection or by intravenous infusion using an intrathecal pump.

[0136] In some embodiments, approximately 0.001 to 1000 mg (for example, approximately 0.1 to 80 mg) 0mg, approximately 1-600mg, approximately 10-500mg, approximately 50-450mg, approximately 80-300mg (mg, approximately 100-200 mg) of antisense oligonucleotides as described herein However, it is administered to those who need it.

[0137] kit One or more antisense oligonucleotides and instructions for use as described above A kit containing the product is also provided. Instructions for use are for the diagnosis or treatment of tau-related diseases. Instructions may be included. The kits provided herein are any of the kits described herein. It can be used according to the method. Those skilled in the art will know about the kit provided herein. Knowing the appropriate use of this, the kit can be used for such purposes. The kit provided in the specification is for use in returning samples for analysis, for example, to a laboratory. Includes a mailing container (e.g., a postage-paid envelope or mailing pack) It is also possible that the kit may include one or more containers for the sample. Alternatively, the sample can be in a standard blood collection vial. The kit can be one or multiple A number of informed consent forms, test request forms, and in the manner described herein. The kit may include instructions on how to use it. Methods for use are also included herein. One or more forms (e.g., for requesting an examination) Paper) and a container for storing the sample, for example, a barcode for identifying the subject from which the sample was provided. It can be coded using a code.

[0138] Those skilled in the art will recognize many methods and materials similar to or equivalent to those described herein. It will be possible to recognize and use them in the implementation of the present invention. In fact, the present invention will be described in this specification. The methods and materials described in this book are by no means limited to those specified. [Examples]

[0139] The present invention will be further described by the following embodiments, which are the same as those described in the claims. The scope of brightness is not limited.

[0140] [Example 1] General materials and methods Synthesis and Purification of Antisense Oligonucleotides The modified antisense oligonucleotides described in this invention are standard phosphors Mermade 192 Syn for in vitro use using Midite Chemical Thesizer (BioAutomation) and Merm for in vivo purposes Prepared using ade12 (BioAutomation). Phosphoramidite was converted to acetone. Dissolve in nitrile at a concentration of 0.15 M (0.08 M for Mermade 192); Plugging is performed using a 0.5 M solution of 5-ethylthiotetrazole in acetonitrile (Mer In made 192, the phosphoramidite was activated by (0.25M) The coupling time was typically between 3 and 4 minutes. Sulfidation was performed using phenylacetyl disulfide. The oxidation was performed by using a 0.2 M solution of Fied for 5 minutes. Oxidation was performed using 0.02 in pyridine. M iodine solution (20%) / water (9.5%) / tetrahydrofuran (70.5%) The procedure was performed for several minutes. Capping was performed using standard capping reagents. Oligonucleotides The growing chain of othide is detritated with 3% dichloroacetic acid in toluene for the next coupling. Chilled. After the sequence was complete, the compound bound to the support was cleaved, and liquid ammonium hydroxide was used. The solution was deprotected at 65°C for 2 hours using a spectrometry device. The resulting crude solution was immediately subjected to HPLC (Akta E). It was purified using an Xplorer. The purified fraction was analyzed by mass spectrometry, yielding 260. They were quantified by UV light according to their absorption coefficients in nM. The extracted fractions were desalted. It was freeze-dried.

[0141] In vitro testing of antisense oligonucleotides Human cell lines, such as Huh7 cells, HeLa cells, and SH-SY5Y cells, This includes, but is not limited to, various cell lines including and COS1 green monkey cell lines, and Inthisense oligonucleotides were tested in vitro. Cells were supplied by a commercially available company (e.g., American Type Culture Collection (ATCC), M Obtained from Anasas (Virginia) and cultured according to the supplier's instructions.

[0142] Antisense oligonucleotides are located in Madison, Wisconsin, USA. Human embryonic stem cells obtained from WiCell Research Institute, Inc. The experiment was also conducted on human neurons derived from endothelial cells (hESCs). hESCs were treated with neurogenin-2(N gn2), by forcibly expressing transcription factors specific to the neuronal lineage, functional neuronal cells Converted to cells. Driven by the constitutive expression of rtTA and the tetO promoter. Lentiviral delivery is used for tetracycline-inducible expression of exogenous proteins. By doing so, the Ngn2 construct was delivered to the hESC. The sample was sent to the Nation al Council Institute of Medicine of theN Guidelines established by the National Academies for human Embryonic Stem Cell Research NAS Guidelines) and the Office for Human Protection Research Protections, Department of Heal Regulations concerning the throne and Human Services ("DHHS") (Code of Federal Regulations, Section 4) It conforms to Part 1 of Chapter 5.

[0143] Antisense oligonucleotides are added to cultured cells when the cells have grown to approximately 60-80% capacity. When the influence is reached, the transfection or nucleofection It was introduced by one of the following. For transfection, an antisense oligonucleotide was used. OptiFect® Transfection Reagent (Life Tech Catalog) Mix with (G number 12579-017) in a suitable cell culture medium and use the desired antisense Oligonucleotide concentration and 2 per 100 nM antisense oligonucleotide The OptiFect® concentration reached a range of 12 μg / mL. Nucleofection For this purpose, antisense oligonucleotides are introduced into neuroblastoma SH-SY5Y cells. Amaxa Nucleofector-II device (Lonza, Walkersv) It was introduced using ille (Maryland). To test the effectiveness of ASO, Nuku Rheofection was performed in a 96-well plate. After preliminary experiments, high viability and Based on efficient transfection, select Nucleofector solution SF. On the day of nucleofection, the 60-80% confluent culture was treated with trypsin. The cells were processed and plated in each well. hESC-derived human neurons were then plated in one well. The treatment was performed by adding 10 μM antisense oligonucleotide to the culture medium. It was imported via import.

[0144] Cells were collected 24-72 hours after antisense oligonucleotide treatment and immediately treated with tau Isolating mRNA or protein, as known in the art and described herein. Measurements were taken using the methods described. Generally, if the procedure is performed multiple times, Data are expressed as the average over repeated treatments. Antisense oligonucleotides used. The concentration of thiocyanate varied depending on the cell line. The optimal antisense thiocyanate for a specific cell line was determined. Methods for determining the concentration of nucleotides are well known in the art. Ligonucleotides are typically transfected using OptiFect. For the combination, use concentrations ranging from 1 nM to 1,000 nM; and use nucleofection. When transfected, use at concentrations ranging from 25 nM to 20,000 nM. Ta.

[0145] Quantification of MAPT (tau) mRNA levels was performed using the ViiA7 real-time PCR system. (Life Technologies) Quantitatively use according to the manufacturer's instructions. The procedure was performed by real-time PCR. Prior to real-time PCR, isolated R NA is subjected to a reverse transcription reaction to produce complementary DNA (cDNA), which is then used in the next real It was used as a substrate for time-based PCR amplification. Reverse transcription and real-time PCR reactions were performed. The procedure was performed sequentially in the same sample well. Fastlane cell multiplex Use the kit (Qiagen catalog number 216513) to lyse the cells in the wells. This method was used to directly reverse transcribe mRNA into cDNA from cultured cells without purifying the RNA. Next, the cDNA was used to analyze tau expression using quantitative real-time PCR. The level of tau mRNA, determined by real-time PCR, was used to determine the expression level in cells. The expression level of housekeeping genes remains constant, for example, human glyceraldehyde 3- Phosphate dehydrogenase (GAPDH), human TATA-box binding protein (TB) P), or human hypoxanthine-guanine phosphoribosyltransferase (HPR Standardization was performed using T1, etc.

[0146] QuantiTect Multiplex RT-PCR Kit (Qiagen) Duplex RT-PC Using a duplex RT-PCR (R) reaction, the TaqMan gene expression assay was performed in real time. This was performed for quantitative RT-PCR. Human MAPT (Life Tech Assa) yID number Hs00902194_m1:FAM-MGB), Human GAPDH (Life Tech AssayID number Hs02758991_g1:VIC-MGB), Human T BP (LifeTech catalog number 4326322E) or human HPRT1 (Li Use a Taqman probe specific to feTech catalog number 4333768T. The sample was then processed according to the recommended cycling conditions for duplex RT-PCR. This was achieved using the ViiA7 real-time PCR system (Life Technologies). We controlled all data relative to the amount of cDNA input to control the level of tau mRNA. The levels were standardized against the causal reference gene. Tau and control genes were standardized to similar high levels. Amplification was performed using the same reaction with PCR efficiency, enabling relative quantification by the ΔΔCT method. Results This is expressed as the percentage of residual tau mRNA compared to control cells treated with PBS. .

[0147] In vivo testing of antisense oligonucleotides Antisense oligonucleotides against MAPT were used to target ASO in mouse cerebrospinal fluid (C The drug was tested in vivo by delivery via intracranial cavity (ICV) administration during SF (Sclerosis). Mice were anesthetized with 5% isoflurane, and then the isoflurane content in oxygen / nitrous oxide was measured. The rate was reduced to 1.5-2% and maintained through surgical procedures. Rectal temperature was controlled using a constant-temperature heated blanket. The temperature was maintained at 36.9 ± 1.0°C using a rectal probe. Anesthetized mice were placed in a stereotactic device. The scalp was shaved and disinfected with povidone-iodine solution (Betadine). Before incision, mice were given buprenorphine (Temgesic, 0.03 mg / kg, 1 (ml / kg, administered subcutaneously). Then, an incision was made to determine the brain coordinates for the injection. The skull was exposed. A 10 μl Hamilton syringe and micropump (Ha For all animals, use a 28G hypodermic needle with rvard Apparatus. In the right ventricle, the following coordinates are observed: AP=+0.5mm;ML=+1.0mm;DV=-2.5 It was injected into mm (total volume 2 μl). The flow rate was 1 μl / min, and the injection needle was withdrawn after infusion. They were left in place for one minute before being removed. After closing the skin, the mice were allowed to recover in a single cage. Returned to home cage. Additional dose of buprenorphine (Temgesic, 0.03mg) was administered. The drug (g / kg, 1 ml / kg, subcutaneous injection) was administered twice daily for the first 48 hours.

[0148] The animals were monitored daily by laboratory / animal technicians. The animals' general condition and any injuries were monitored. Recovery was monitored, and weight was measured daily. At the end of the treatment, the animals were given sodium pentobarbital. Tar (60 mg / kg Mebunat, OrionPharma, Finland) The mice were deeply anesthetized. CSF (3-5 μL per mouse) was collected by puncturing the cisterna magna of the mouse. Then, the mice were paracentesis and blood samples were collected. Approximately 0.4-0.5 mL of blood was collected. The samples were collected in a 500 μL plastic lavender K2 EDTA anticoagulant tube, and 2 The plasma was divided equally after centrifuging at 4°C for 10 minutes at 000g. Then the mice were decapitated and the brains were extracted. The samples were collected and segmented into different brain regions such as the cortex, hippocampus, and cerebellum. In addition, the spinal cord was also included. I also collected them.

[0149] [Example 2] Human tau expression in Huh7 cells mediated by 18-mer 2'-O-MOE steric blockers Inhibition The steric barrier agents of antisense oligonucleotides are present in all isoforms. The intron-exon junction of the constitutive exon in hi-tau, which is an invariant exon. It was designed to target. The steric blocker of antisense oligonucleotides is By hybridizing to the branching point sequence of introns, the splice site is directly targeted, and the intron And by sandwiching the exon sequence and the exon splicing enhancer region, Designed to induce xon skipping, MAPT-targeted steric blockers In all nucleosides, sterically hindrance acts to target RNaseH or RISC. Inactive 2'-O-(2-methoxyethyl)(2'-O-MOE) ribose sugar modification It was initially designed to have a length of 18 nucleosides. The nucleoside connections are at the corners. It is phosphodiester coupled from corner to corner. Unbiased screening, phosphodiester The procedure was carried out on ASOs of the above 18-mer having a tel skeleton, which are uniformly modified 2'-MOEs. To avoid off-target hybridization, BLAST analysis was performed on each mole. The study was conducted on the horino oligonucleotide sequence.

[0150] A steric blocker of an 18-mer 2'-O-MOE targeting tau was used in vitro in humans. Their activity in inhibiting tau mRNA was tested. Huh7 cells per well Plating is performed at a density of 10,000 cells, and OptiFect reagent (LifeTe Using ch (catalog number 12579-017), 25nM antisense oligonucleotides Transfected with creotide. After a 48-hour treatment period, Fastlane ce Using the ll multiplex kit (Qiagen, catalog number 216513) Then, cDNA was directly prepared from cultured cells. The level of tau mRNA was compared to human MAPT(L). ifeTech AssayID number Hs00902194_m1:FAM-MGB) and human TBP (TATA-box binding protein) endogenous control (LifeTech, Ca Using a Taqman probe specific to catalog number 4326322E, duplex The results were measured by quantitative real-time PCR using the RT-PCR reaction. All data were collected in cD It controls the level of tau mRNA in accordance with the amount of NA input, and the endogenous reference gene TBP The levels were standardized. Tau and TBP control genes were standardized to have similar high PCR efficiency. The reaction was amplified, enabling relative quantification by the ΔΔCT method. The results were treated with PBS. Table 2 shows the percentage of residual tau mRNA compared to control cells. These 18-mers in the cell exhibit steric blocking activity against 2'-O-MOE.

[0151] [Table 2-1]

[0152] [Table 2-2]

[0153] [Table 2-3]

[0154] [Example 3] Steric blocking of 18-mer 2'-O-MOE in SH-SY5Y cells Inhibition of expression In Example 2, a steric blocker that significantly reduced tau expression was selected, and human neuroblastoma S The test was performed using H-SY5Y cells. Cultured SH-SY5Y cells were selected at 1,000 nM. Nucleofect was performed with selected antisense oligonucleotides. Treatment period was approximately 24 hours. After the interval, Fastlane cell multiplex kit (Qiagen, Cata cDNA was directly prepared from cultured cells using log number 216513. Tau mRNA The level is measured using a duplex RT-PCR reaction via quantitative real-time PCR. Measured using human MAPT (LifeTechAssayID number Hs00902194_ m1:FAM-MGB) and human GAPDH (LifeTechAssayID number H Using a Taqman probe specific to s02758991_g1:VIC-MGB) All data was controlled according to the amount of cDNA input, and the level of tau mRNA was controlled to match the endogenous The levels were standardized to the sex reference gene GAPDH. The tau and GAPDH control genes were used. Amplification using the same reaction with similarly high PCR efficiency enables relative quantification by the ΔΔCT method. The results were expressed as the percentage of residual tau mRNA compared to control cells treated with PBS. Table 3 shows the 2'-O-MOE levels of selected 18-mers in SH-SY5Y cells. It exhibits somatic blocking activity.

[0155] [Table 3]

[0156] We tested steric blockers that showed significant in vitro inhibition of tau mRNA at different doses. Cultured SH-SY5Y cells were diluted to 0.125nM, 0.25nM, 0.5nM, 1, One of the following selected values: 000nM, 2,000nM, 4,000nM, and 8,000nM Nucleofect was performed with antisense oligonucleotides. After a treatment period of approximately 24 hours, By directly preparing cDNA, the level of tau mRNA was quantitatively measured as described above. The 50% inhibitory concentration (IC50) was measured by time-PCR. The dose-response curve was constructed using this method. We investigated the effects of different concentrations of antisense oligonucleotides on reducing tau mRNA. This was determined by the concentration required to inhibit half of the compound's maximum biological response. By determining the IC50 value, the potency of the antisense oligonucleotide is calculated. It can be used as a measurement. Table 4 shows the 2'-O-MOE ratio of the selected 18-mers. The IC50 values ​​of the systemic blocking agents are shown.

[0157] [Table 4]

[0158] [Example 4] Human SH-SY5Y cells mediated by 12-25 mer 2'-O-MOE steric blockers Inhibition of tau expression The steric blockers that significantly reduced tau expression in Examples 2 and 3 ranged from 12 to 25 nuclei. They were selected and prepared to have different osidic lengths. 2' of these 12-25mers -O-MOE steric blockers were tested in SH-SY5Y cells. Cells are nucleofected with 2,000 nM selected antisense oligonucleotides. After a treatment period of approximately 24 hours, the cDNA was prepared directly as described above, and then tau The mRNA levels were measured as described above. Table 5 shows the levels of 12-25 mers. This demonstrates the activity of the steric blocker '-O-MOE' in SH-SY5Y cells.

[0159] [Table 5-1]

[0160] [Table 5-2]

[0161] Selected 12-25 mers 2'-OM having phosphodiester nucleoside linkages The IC50 values ​​of the OE steric barrier were determined as described above and are shown in Table 6.

[0162] Many 12-25 mers (2'-O-MO) with phosphorothioate nucleoside linkages E. Steric barriers were synthesized, and the IC50 values ​​of some of these steric barriers are shown in Table 7. did.

[0163] [Table 6]

[0164] [Table 7]

[0165] [Example 5] Human tau production in SH-SY5Y cells induced by the 17-mer 2'-O-MOE steric blocker Current inhibition A 2'-MOE steric barrier agent of the length of 17 nucleosides is used in human tau. Designed to target Son. These 17-mer 2'-O-MOE steric blockers The test was performed using SH-SY5Y cells. Cultured SH-SY5Y cells were selected at 2,000 nM. Transfected with an antisense oligonucleotide. Treatment period of approximately 24 hours. Afterward, cDNA was directly prepared and the tau mRNA levels were measured as described above. Table 8 shows the effects of a 17-mer 2'-O-MOE steric blocker on SH-SY5Y cells. It shows activity.

[0166] [Table 8-1]

[0167] [Table 8-2]

[0168] [Example 6] Inhibition of human tau expression in Huh7 cells by a 5-10-5 gapmer Antisense oligonucleotide sequences, the shortest tau isoforms, and the transcript barrier. 4 is designed to be complementary to mRNA (GenBank: NM_016841.4). We calculated the results. To avoid off-target hybridization, we performed BLAST analysis on each original sample. The procedure was performed on gonucleotide sequences. Newly designed and modified chimeric antisense was used. Oligonucleotides are 5-10-5 gapmers with a length of 20 nucleosides. Designed so that the central gap segment contains 10 2'-deoxynucleosides Each of the 5'-directions contains five nucleosides modified with 2'-O-MOE ribose sugar. It is adjacent to the wing segment in the 3' direction. Nucleo throughout each gapmer Interside junctions are phosphorothioate (P=S) junctions.

[0169] A gapmer targeting tau was used to inhibit human tau mRNA expression in vitro. The experiment was conducted. Huh7 cells were plated at a density of 10,000 cells per well. Using OptiFect reagent (LifeTech, catalog number 12579-017) The cells were transfected with a 25 nM antisense oligonucleotide. Treatment period: 48 hours. After a certain period, cDNA was extracted from cultured cells using Fastlane cell multiplex. Prepared directly using a kit (Qiagen, catalog number 216513). Tau mR The level of NA is determined by quantitative real-time PCR using a duplex RT-PCR reaction. Human MAPT (LifeTech, AssayID number Hs00902194_m1:F) AM-MGB) and human TBP (TATA-box binding protein) endogenous control (Li Use a Taqman probe specific to feTech (catalog number 4326322E). The measurements were taken by controlling all data to match the amount of cDNA input and the level of tau mRNA. The tau and TBP control genes were standardized to the level of the endogenous reference gene TBP. It can be amplified using the same reaction with similarly high PCR efficiency, enabling relative quantification by the ΔΔCT method. The result was the percentage of residual tau mRNA compared to control cells treated with PBS. The data was displayed as shown. Table 9 shows the activity of the gapmer in Huh7 cells.

[0170] [Table 9-1]

[0171] [Table 9-2]

[0172] [Example 7] Inhibition of human tau expression by the 5-10-5 gapmer in SH-SY5Y cells In Example 6, a gapmer that significantly reduced tau mRNA expression was selected, and S The test was performed using H-SY5Y cells. Cultured H-SY5Y cells were treated with 2,000 nM antimicrobial agents. Nucleofect was performed with sense oligonucleotides. After a treatment period of approximately 24 hours, the culture cells were... From the cell, Fastlane cell multiplex kit (Qiagen catalog) cDNA was directly prepared using (G-number 216513). The level of tau mRNA was measured. Quantitative real-time PCR is used to measure the duplex RT-PCR reaction, and MAPT (LifeTech AssayID number Hs00902194_m1:FA) M-MGB) and human GAPDH (LifeTech AssayID number Hs027) A Taqman probe specific to 58991_g1 (VIC-MGB) was used. The data is controlled according to the amount of cDNA input, and the level of tau mRNA is used as an endogenous reference. The levels were standardized to the GAPDH gene level. Tau and GAPDH control genes were standardized to similar high levels. By amplifying the sample using the same reaction with high PCR efficiency, relative quantification by the ΔΔCT method was made possible. The results are expressed as the percentage of residual tau mRNA compared to control cells treated with PBS. Table 10 shows the activity of selected 5-10-5 gapmers in SH-SY5Y cells. This shows the IC50 of selected 5-10-5 gapmers having 5-methylcytosine. The values ​​were determined using SH-SY5Y cells as described above and are shown in Table 11.

[0173] [Table 10]

[0174] [Table 11]

[0175] [Example 8] Characterization of antisense oligonucleotides targeting MAPT Antisense oligonucleotides targeting MAPT, Thermo Scien We use a Tific high-performance liquid chromatography-mass spectrometry (LC-MS) instrument. This was used to characterize the antisense oligonucleotide (ASO) Confirm the mass and gather information about the purity of the sample and the identification of the main components present. Used to provide. For example, antisense oligonucleotides containing sequence number 284. The structure shown in Figure 1A and the formula C230H321N72O120P19S19 are It has. Therefore, the expected molecular weight for ASO containing SEQ ID NO: 284 is approximately 721. 2.3Da. Figure 1B shows the measurement of ASO including sequence number 284 by LC-MS. The determined peak mass is 7214.3 Da. Figure 1C shows sequence number 284. The LC-MS deconvolution peaks for ASO, including this compound, are reported.

[0176] Antisense oligonucleotides containing sequence number 285 are C230N69O124P It has the formula 19S19H318, and its estimated molecular weight is 7231.11Da. (Figure 1D) For ASO containing sequence number 285, the peak mass measured by LC-MS was 72. This indicates 32.5. Figure 1E shows LC-M for ASO including sequence number 285. The deconvolution peak of S is reported.

[0177] [Example 9] In vivo trials of gapmers targeting MAPT Development of human tau (hTau) transgenic mice A BAC vector (pBACe3.6) containing the human tau gene MAPT was developed by Life. Obtained from technologies' human genome library. All MAPT genes. Three vectors predicted to contain the regulatory region were screened for the human genome. DNA is subjected to standard PCR to identify the protrusions that span across each exon and intron of the human tau gene. Used to test for the presence of rimers and regulatory regions and to determine the sequence of clones. By comparing it with human DNA, one clone (RP11669E14) was found to possess tau genes. It was shown to be complete for all parts of the child. This BAC vector is hTau B Used to generate AC transgenic mice. Purified DNA was used in C57BL / It was injected into 6 mouse fertilized embryos. Tail DNA from the progenitor mouse offspring was digested with restriction enzymes. Then, the exon-specific probe was hybridized with the digested human DNA. They were used as a control to test the integrity of the introduced gene. The offspring mice of the positive progenitor were multiplied. They were propagated. Several hTau BAC transgenic lines were generated, and one line was human M This strain showed expression of APT mRNA and protein (Figures 2A-2C). This strain is related to the human brain. All six human brain transcripts and protein isoforms found in [the study] were expressed (Figure 2). A~2C). Heterozygous hTau BAC transgenic mice have one copy With the transgene carried, the levels of RNA and human tau expression are similar to those of endogenous mouse tau expression. They were roughly equivalent.

[0178] In vivo knockdown of human tau using antisense oligonucleotides Selected antisense oligonucleotides were tested in vivo. Five groups of hTau Choose from 1, 10, 50, 200, or 400 μg of BAC transgenic mice. The antisense oligonucleotides are administered by intraventricular (ICV) bolus injection. Either treated or left untreated as a control group of mice. All procedures were performed using isofluoran The procedure was performed under anesthesia, in accordance with IACUC regulations. For the ICV bolus injection, antiseptic was used. The lance oligonucleotide was introduced into the right ventricular region of hTau BAC transgenic mice. Injected. 2 or 4 microliters containing 100 μg / μl oligonucleotide. The PBS solution was injected immediately after oligonucleotide administration, and at 1 hour, 4 hours, and 24 hours. Tissue was collected after 2 weeks, 4 weeks, 12 weeks, or 24 weeks. R was collected from the hippocampus or cortex. NA was extracted, and human tau mRNA expression was examined using real-time PCR analysis. The levels of human tau mRNA were measured as described above. The results were compared to the untreated control group. Inhibition rate of human tau mRNA expression standardized to GAPDH levels compared to Us (percentage) The calculation was performed as follows: Proteins were extracted from the hippocampus or cortex and subjected to ELISA. The total protein expression level was investigated and standardized to match the total protein level. Ta.

[0179] In vivo 5-10-5 gapmers containing SEQ ID NO: 284 or SEQ ID NO: 285 The activity was tested using the method described above. As shown in Table 12, both annealing agents were tested. Antisense oligonucleotides after a single ICV injection of antisense oligonucleotides At two weeks, it significantly inhibited the expression of human tau mRNA in the cortex and hippocampus. Sequence number For gapmers including no. 285, knockdown of human tau mRNA is effective in the cortex and The percentage was approximately 65% ​​in both the hippocampus and other parts of the brain. For gapmers including sequence number 284, Hitota U mRNA knockdown was approximately 42% in both the cortex and hippocampus. ASO treatment Regarding tau protein levels two weeks later, gapmers including SEQ ID NO: 285 showed that the skin In terms of quality, it knocks down approximately 50% of tau protein expression; including sequence number 284 Pumper knocked down approximately 36% of tau protein expression in the cortex. They observed a significant decrease in tau protein levels two weeks after ASO treatment in the hippocampus. It wasn't there.

[0180] [Table 12]

[0181] Tau mRNA and protein levels were measured 4 weeks after a single ICV injection of gapmer. It was also tested. Antisense oligonucleotides containing SEQ ID NO: 285 were found to be effective in the brain. The expression of totau mRNA (Figure 2D) and protein (Figure 2E) was significantly inhibited. Sequence number For gapmers including no. 285, knockdown of human tau mRNA is effective in the cortex and Approximately 60% were observed in both the hippocampus and other parts of the hippocampus (Figure 2D and data not shown). Western blot analysis The gapmer containing sequence number 285 increased human tau protein levels four weeks after treatment. This showed that approximately 50% of the cells were knocked down in the hippocampus (Figure 2E).

[0182] Antisense oligonucleotides in the brains of hTau BAC transgenic mice To detect the brain distribution of cydoplasm, in situ hybridization experiments were performed in duplicate. Digoxigenin (DIG)-labeled locked nucleic acid (LNA®, Exiqon) The procedure was performed using a probe. Two complementary antisense oligonucleotides were used. The heavy-DIG LNA probe was hybridized overnight. Next, the probe was subjected to sheep ash. DIG alkaline phosphatase conjugate antibody (Roche Diagnostics) s, catalog number 11093274910) and Nitroblue Tetrazolium and Alka Conjugation of 5-bromo-4-chloro-3-indolyl phosphate, a substrate of riphosphatase Detection was performed using a colorimetric reaction with jugate (BCIP). Sequence ID No. 2 in a representative experiment. Figure 3 shows the brain distribution of 85 antisense oligonucleotides, which is the distribution of ASOs. It shows initial diffusion from the ventricles to the mouse brain parenchyma, and the distribution signal changes between 24 hours and 2 weeks. It does not transform. (Figure 3). Antisense oligonucleotides remain stable in the brain even after 4 weeks. Yes (data not shown).

[0183] hTau BAC transfusion by antisense oligonucleotide of sequence number 285 Human tau mRNA (Figure 4A) and protein (Figure 4B) in genic mice Dose-dependent inhibition of expression was observed (Figures 4A and 4B).

[0184] After a single ICV injection of 200 μg of the antisense oligonucleotide of SEQ ID NO: 285 Human tau mRNA in hTau BAC transgenic mice (Figure 5A) and The time course of protein expression levels (Figure 5B) is shown using the antisense oligonucleotide sequence number 285. Cleotide demonstrated sustained inhibition of tau mRNA and protein expression for up to 12 weeks. (Figures 5A and 5B).

[0185] [Example 10] Additional 5-1 methylcytosine in Huh7 and SH-SY5Y cells Inhibition of human tau expression by 0-5 gapmer An additional gapmer sequence containing 5-methylcytosine targeting tau is described above. Regarding the inhibition of human tau mRNA expression in Huh7 and SH-SY5Y cells, The study was performed in vitro. The results showed that residual tau mR was higher compared to control cells treated with PBS. Expressed as a percentage of NA. Table 13 shows the results in Huh7 and SH-SY5Y cells. This shows the activity of additional screened sequences of the 5-10-5 gapmer.

[0186] [Table 13-1]

[0187] [Table 13-2]

[0188] [Table 13-3]

[0189] [Table 13-4]

[0190] Select the gapmers in Table 13 that significantly reduced tau mRNA expression, and then perform SH-SY Tested in 5Y cells. Selected 5-methylcytosine-containing 5-10-5 gapmers. The IC50 values ​​were determined in SH-SY5Y cells as described above and are shown in Table 14.

[0191] [Table 14]

[0192] [Example 11] Antisense oligonucleotides containing 5-methylcytosine can be used to treat monkey and human thromboembolism. Inhibition of expression We selected several gapmers that significantly reduced tau mRNA expression, and then used COS1 The study was conducted using green monkey cells. The results showed that residual tau m³ was higher compared to control cells treated with PBS. Expressed as a percentage of RNA. Table 15 shows the selected 5-10- RNA in COS1 cells. It shows the activity of the 5-gapmer.

[0193] [Table 15]

[0194] We selected several antisense oligonucleotides that significantly reduced tau mRNA expression. Selected and tested with human embryonic stem cell (hESC)-derived neurons. The results were obtained using PBS-treated neurons. This is expressed as the percentage of residual tau mRNA compared to the control cells. Table 16 shows human tau mRNA. This shows the activity of selected antisense oligonucleotides in ¹¹.

[0195] [Table 16]

[0196] [Example 12] In vivo trials of gapmers targeting MAPT The in vivo activity of the selected 5-10-5 gapmer is described in Example 9. The following methods were used for testing. As shown in Table 17, several antisense oligonucleotides Rheotide significantly inhibits human tau mRNA and protein expression in the cortex and hippocampus. He caused harm.

[0197] [Table 17]

[0198] Unless otherwise specified, the technical and scientific terms used herein belong to the same entity as those used in this disclosure. It has the same meaning as it is usually understood by experts familiar with the field.

[0199] Unless otherwise noted, all methods, steps, and techniques not specifically described in detail are not included. The operation itself can be carried out in a manner that is obvious and known to those skilled in the art, and the operation This has been done. The standard manuals and general background art mentioned herein and therein, as examples. Further references cited are mentioned again. Unless otherwise noted, all references cited herein are... Each of the listed references is incorporated in its entirety by reference.

[0200] The claims of the present invention are not limiting and are provided below.

[0201] Certain embodiments and claims have been disclosed in detail herein, but this is for illustrative purposes only. This is done for illustrative purposes only and is not limited to the scope of the attached claims or any corresponding future claims. No limitation is intended with respect to the scope of the subject matter of the claims to which the application applies. In particular, the claims Without departing from the spirit and scope of this disclosure as defined above, various substitutions, modifications, and It is intended by the inventors that modifications may be made to this disclosure. The selection of nucleic acid starting materials, or library-type nucleic acids, and clones is as described herein. This is considered commonplace for those skilled in the art who have knowledge of the embodiments. Other embodiments, advantages And modifications are also considered to be within the scope of the following claims. A person skilled in the art will be able to perform more than routine experiments. Without using [a specific method], one can recognize many equivalent things of the particular aspects of the present invention described herein. or it may be possible to verify such equivalent things. It is intended to be included in the scope of claims in the corresponding application filed later. The rewriting of the claim may be subject to restrictions under the patent laws of various countries, and the subject matter of the claim may be subject to restrictions. This should not be interpreted as abandonment. The following embodiments may be included: [1] At least 90% sequence identity with any of the nucleic acid base sequences shown in Tables 2-17 An oligonucleotide comprising a nucleic acid base sequence having any of the nucleic acid base sequences In the above, C is either cytosine or 5-methylcytosine, and the oligonucleotide An oligonucleotide in which at least one nucleotide of the nucleotide has a 2'-modification. [2] A nucleus having at least 90% sequence identity with any of the sequences shown in Tables 2-8 The acid-base sequence is included, and the C in any of the nucleic acid base sequences is cytosine or 5-methyl It is either cytosine, and each nucleotide of the oligonucleotide has a 2'-modification. The oligonucleotide described in [1] above. [3] A nucleus having at least 90% sequence identity with any of the sequences shown in Tables 2-8 The oligonucleotide described in [2] above, comprising an acid-base sequence. [4] At least 95% sequence identity with any of the nucleic acid base sequences shown in Tables 2-8 The oligonucleotide described in [2] above, comprising a nucleic acid base sequence. [5] The oligos described in [2] above, which include any of the nucleic acid base sequences shown in Tables 2 to 8. nucleotide. [6] The original [2] described above, consisting of any of the nucleic acid base sequences shown in Tables 2 to 8. Gonucleotide. [7] The nucleoside linkage of the oligonucleotide is a phosphodiester or phosphodiester The oligonucleus described in any of the above [2] to [6] is either rhozioate linked. Cleotide. [8] The linker is attached to the 3' end of the oligonucleotide via a phosphate crosslink. The following structure: [ka] An oligonucleotide according to any one of the above [2] to [7], having any of the following: [9] Independent of RNAseH, it reduces the expression of tau mRNA or protein. The oligonucleotide described in any of the above [2] to [8].

[10] At least 90% of the sequences are identical to any of the sequences shown in Tables 9-15 and 17. It comprises a nucleic acid base sequence of uniformity, wherein C in any of the nucleic acid base sequences is cytosine or 5-methylcytosine, and at least one of the alkyl groups The oligonucleotide described in [1] above, wherein the nucleotide has a 2'-modification.

[11] At least 90% of the sequences are identical to any of the sequences shown in Tables 9-15 and 17. The oligonucleotide described above

[10] , comprising a unisexual nucleic acid base sequence.

[12] At least 95% of any of the nucleic acid sequences shown in Tables 9-15 and 17 The oligonucleotide described in

[10] above, comprising a nucleic acid base sequence having sequence identity.

[13] Oligonucleotides as described in

[0010] above, comprising any of the nucleic acid base sequences shown in Tables 9-15 and 17.

[14] Oligonucleotides as described in

[0010] above, comprising any of the nucleic acid base sequences shown in Tables 9-15 and 17.

[15] The nucleoside linkage of the oligonucleotide is a phosphorothioate linkage. or, an oligonucleotide as described in any of

[10] to

[14] above.

[16] The above

[10] containing at least five consecutive 2'-deoxynucleosides An oligonucleotide as described in any of

[15] .

[17] The above

[10] containing at least seven consecutive 2'-deoxynucleosides Oligonucleotides as described in any of the 15 from to 15.

[18] An oligonucleotide according to any one of

[10] to

[0015] above, comprising 10 consecutive 2'-deoxynucleosides.

[19] Activating RNAseH reduces tau mRNA or protein expression. The oligonucleotide described in any of the above

[10] to

[18] , which reduces the amount of oligonucleotides.

[20] The above, wherein each C in any of the nucleic acid base sequences is 5-methylcytosine. An oligonucleotide as described in any of [1] to

[19] .

[21] At least 12 consecutive nucleic acid salts of any one of sequence numbers 487-506 Oligonucleotides comprising a nucleic acid base sequence complementary to the group, the oligonucleotide An oligonucleotide in which at least one nucleotide of the nucleotide has a 2'-modification.

[22] At least 12 consecutive nucleic acid salts of any one of sequence numbers 487-506 The oligonucleotide sequence described in

[21] above, comprising a nucleic acid base sequence that is 100% complementary to the base. Do.

[23] The oligonucleotides described in

[21] above, comprising one or more 5-methylcytosines. Cleotide.

[24] The oligonucleotide according to

[0021] above, wherein each nucleotide of the oligonucleotide has a 2'-modification.

[25] The above

[21] containing at least five consecutive 2'-deoxynucleosides The oligonucleotides listed above.

[26] The above

[21] containing at least seven consecutive 2'-deoxynucleosides The oligonucleotides listed above.

[27] The same as described in

[21] above, comprising 10 consecutive 2'-deoxynucleosides. Oligonucleotides.

[28] A nucleic acid containing 12 to 30 nucleic acid bases, as described in any of [1] to

[27] above. Oligonucleotides.

[29] A nucleic acid containing 12 to 25 nucleic acid bases, as described in any of [1] to

[27] above. Oligonucleotides.

[30] A nucleic acid containing 15 to 20 nucleic acid bases, as described in any of [1] to

[27] above. Oligonucleotides.

[31] The 2'-modification is 2'-fluoro, 2'-deoxy-2'-fluoro, 2' -O-methyl, 2'-O-methoxyethyl (2'-O-MOE), 2'-O-aminopro Pill (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2'-O-dimethylamino Ethyloxyethyl (2'-O-DMAEOE) and 2'-ON-methylacetate Select from the group consisting of mid(2'-O-NMA), any of the above [1] to

[30] Oligonucleotides as described above.

[32] The above 2'-modification is 2'-O-methoxyethyl (2'-O-MOE), An oligonucleotide as described in any of [1] to

[30] .

[33] In vitro, tau mRNA or protein expression levels were reduced by at least 30% The oligonucleotides described in any of the above [1] to

[32] can be reduced. Do.

[34] In vivo, tau mRNA or protein expression levels were reduced by at least 30%. The oligonucleotide described in any of the above [1] to

[32] can be reduced. .

[35] Oligonucleotides and pharmaceuticals described in any of the above [1] to

[34] A composition containing a carrier that is permissible.

[36] Therapeutic effective amount of oligonucleotide described in any of [1] to

[34] above A method for reducing tau expression levels in a subject, comprising administering the substance to the subject.

[37] The subject is suffering from or is susceptible to a tau-related disease, as described above. The method described in

[36] .

[38] Diseases associated with tau include Alzheimer's disease (AD) and amyotrophic lateral sclerosis. Parkinson's syndrome-dementia complex disease (ALS-PDC), argyrophilic grain dementia (AGD) British-type amyloid angiopathy, cerebral amyloid angiopathy, chronic traumatic encephalopathy (CTE), cerebral cortex Basal ganglia degeneration (CBD), Creutzfeldt-Jakob disease (CJD), punch-drunk syndrome, Diffuse neurofibrillary tangles with calcification, Down syndrome, Dravet syndrome, epilepsy, frontotemporal syndrome Type 1 dementia (FTD), Parkinson's syndrome linked to chromosome 17 (FTDP-17) Associated frontotemporal dementia, frontotemporal lobar degeneration, ganglioglioma, gangliocytoma, Gerstmann's disease Stroisler-Scheinker disease, Haller-Holden-Spats disease, Huntington's disease, inclusion Body myositis, lead encephalopathy, Ritico Bodig disease, meningiomatosis, multiple system atrophy, myotonic dysplasia Trophy, Niemann-Pick disease type C (NP-C), non-Guanian with neurofibrillary tangles Motor neurological disorders, Pick's disease (PiD), post-encephalitis-related Parkinsonian syndrome, prion protein brain a Myloid angiopathy, progressive subcortical gliosis, progressive supranuclear palsy (PSP), subacute sclerosing Panencephalitis, neurofibrillary tangle-dominant dementia (tangle-only dementia), neurofibrillary tangle-dominant Dementia (Tangle-predominant dementia), multiple infarct dementia, ischemic stroke, or The method described above

[37] , selected from articular sclerosis.

[39] The oligonucleotide is administered to the target intrathecal, intracranial, intranasal, oral, or intravenous. The method according to

[36] above, administered intravascularly or via a subcutaneous route.

[40] The same as described in

[36] , further comprising administering a second active substance to the subject. The method.

[41] The method according to any one of the above

[36] to

[40] , wherein the subject is a human.

[42] The above [for treating tau-related diseases in subjects that require it] Use of an oligonucleotide as described in any of [1] to

[34] .

[43] Medical use for the treatment of tau-related diseases in persons who need it In the manufacture of a drug, use of an oligonucleotide described in any of the above [1] to

[34] For.

[44] The oligonucleotides are nucleic acid base sequences shown in Tables 9-15 and 17. Nucleosy cells containing any of the above, in which the first to fifth nucleotides are modified with 2'-O-MOE Each contains a nucleotide, and the 6th to 15th nucleotides each contain a 2'-deoxynucleoside. , nucleosides in which the 16th to 20th nucleotides are modified with 2'-O-MOE, respectively Including the oligonucleotides described in

[10] above.

[45] Sequence IDs 208, 284, 285, 313, 329, 335, 366, 384 A nucleic acid base sequence selected from one of 386, 405, 473, and 474. Contains oligonucleotides.

[46] Nucleosides in which the first to fifth nucleotides are modified with 2'-O-MOE Each contains, and the 6th to 15th nucleotides each contain a 2'-deoxynucleoside, Each of the 16th to 20th nucleotides contains a nucleoside modified with 2'-O-MOE. , the oligonucleotides described in

[45] above.

Claims

1. A pharmaceutical composition, below: 5'-G*G*T*T*G*A m CAT m CGT m CTG m C* m C*T*G*T*-3' (SEQ ID NO: 285) Includes the nucleotide sequence, Each nucleotide marked with an asterisk (*) contains a 2'-O-methoxyethyl modification at the 2' position of the furanose ring, each nucleotide without an asterisk (*) contains a 2'-deoxynucleoside, mC contains 5-methylcytosine, and all internucleoside links are phosphorothioates, forming an antisense oligonucleotide. A pharmaceutically acceptable carrier, The aforementioned pharmaceutical composition is a pharmaceutical composition intended for intrathecal administration.

2. A pharmaceutical composition, below: 5'-G*G*T*T*G*A m CAT m CGT m CTG m C* m C*T*G*T*-3' (SEQ ID NO: 285) It consists of the nucleotide sequence, Each nucleotide marked with an asterisk (*) contains a 2'-O-methoxyethyl modification at the 2' position of the furanose ring, each nucleotide without an asterisk (*) contains a 2'-deoxynucleoside, mC contains 5-methylcytosine, and all internucleoside links are phosphorothioates, forming an antisense oligonucleotide. A pharmaceutically acceptable carrier, The aforementioned pharmaceutical composition is a pharmaceutical composition intended for intrathecal administration.

3. The pharmaceutical composition according to Claim 1, The aforementioned pharmaceutical composition is a pharmaceutical composition for treating diseases related to tau.

4. The pharmaceutical composition according to Claim 1, The aforementioned pharmaceutical composition is used for Alzheimer's disease (AD), amyotrophic lateral sclerosis / Parkinson's syndrome-dementia complex (ALS-PDC), argyrophilic grain dementia (AGD), British-type amyloid angiopathy, cerebral amyloid angiopathy, chronic traumatic encephalopathy (CTE), corticobasal degeneration (CBD), Creutzfeldt-Jakob disease (CJD), punch-drunk syndrome, diffuse neurofibrillary tangles with calcification, Down syndrome, Dravet syndrome, epilepsy, frontotemporal dementia (FTD), frontotemporal dementia with Parkinson's syndrome linked to chromosome 17 (FTDP-17), and frontotemporal lobe A pharmaceutical composition for the treatment of degenerative diseases, ganglioglioma, gangliocytoma, Gerstmann-Streussler-Scheinker disease, Haller-Holden-Spats disease, Huntington's disease, inclusion body myositis, lead encephalopathy, Ritico-Bodig disease, meningioma hemangioma, multiple system atrophy, myotonic dystrophy, Niemann-Pick disease type C (NP-C), non-Guamanian motor neuropathy with neurofibrillary tangles, Pick's disease (PiD), post-encephalitis-related parkinsonian syndrome, prion protein cerebral amyloid angiopathy, progressive subcortical gliosis, progressive supranuclear palsy (PSP), subacute sclerosing panencephalitis, tangle-only dementia, tangle-predominant dementia, polyinfarct dementia, ischemic stroke, or tuberous sclerosis.

5. The pharmaceutical composition according to Claim 1, The aforementioned pharmaceutical composition is for the treatment of progressive supranuclear palsy (PSP).

6. The pharmaceutical composition according to Claim 1, The aforementioned pharmaceutical composition is a pharmaceutical composition for treating Alzheimer's disease.

7. The pharmaceutical composition according to Claim 1, The aforementioned pharmaceutical composition is for the treatment of frontotemporal dementia (FTD).

8. The pharmaceutical composition according to Claim 1, The aforementioned pharmaceutical composition is for the treatment of corticobasal degeneration (CBD).

9. The pharmaceutical composition according to claim 2, The aforementioned pharmaceutical composition is a pharmaceutical composition for treating diseases related to tau.

10. The pharmaceutical composition according to claim 2, The aforementioned pharmaceutical composition is used for Alzheimer's disease (AD), amyotrophic lateral sclerosis / Parkinson's syndrome-dementia complex (ALS-PDC), argyrophilic grain dementia (AGD), British-type amyloid angiopathy, cerebral amyloid angiopathy, chronic traumatic encephalopathy (CTE), corticobasal degeneration (CBD), Creutzfeldt-Jakob disease (CJD), punch-drunk syndrome, diffuse neurofibrillary tangles with calcification, Down syndrome, Dravet syndrome, epilepsy, frontotemporal dementia (FTD), frontotemporal dementia with Parkinson's syndrome linked to chromosome 17 (FTDP-17), and frontotemporal lobe A pharmaceutical composition for the treatment of degenerative diseases, ganglioglioma, gangliocytoma, Gerstmann-Streussler-Scheinker disease, Haller-Holden-Spats disease, Huntington's disease, inclusion body myositis, lead encephalopathy, Ritico-Bodig disease, meningioma hemangioma, multiple system atrophy, myotonic dystrophy, Niemann-Pick disease type C (NP-C), non-Guamanian motor neuropathy with neurofibrillary tangles, Pick's disease (PiD), post-encephalitis-related parkinsonian syndrome, prion protein cerebral amyloid angiopathy, progressive subcortical gliosis, progressive supranuclear palsy (PSP), subacute sclerosing panencephalitis, tangle-only dementia, tangle-predominant dementia, polyinfarct dementia, ischemic stroke, or tuberous sclerosis.

11. The pharmaceutical composition according to claim 2, The aforementioned pharmaceutical composition is for the treatment of progressive supranuclear palsy (PSP).

12. The pharmaceutical composition according to claim 2, The aforementioned pharmaceutical composition is a pharmaceutical composition for treating Alzheimer's disease.

13. The pharmaceutical composition according to claim 2, The aforementioned pharmaceutical composition is for the treatment of frontotemporal dementia (FTD).

14. The pharmaceutical composition according to claim 2, A pharmaceutical composition for the treatment of corticobasal degeneration (CBD).

15. The following: 5'-G*G*T*T*G*A m CAT m CGT m CTG m C* m C*T*G*T*-3' (SEQ ID NO: 285) Includes the nucleotide sequence, A cell containing antisense oligonucleotides, where each nucleotide marked with an asterisk (*) to the right of the nucleotide contains a 2'-O-methoxyethyl modification at the 2' position of the furanose ring, each nucleotide without an asterisk to the right of the nucleotide contains a 2'-deoxynucleoside, mC contains 5-methylcytosine, and all internucleoside links are phosphorothioates.

16. The following: 5'-G*G*T*T*G*A m CAT m CGT m CTG m C* m C*T*G*T*-3' (SEQ ID NO: 285) It consists of the nucleotide sequence, A cell containing antisense oligonucleotides, where each nucleotide marked with an asterisk (*) to the right of the nucleotide contains a 2'-O-methoxyethyl modification at the 2' position of the furanose ring, each nucleotide without an asterisk to the right of the nucleotide contains a 2'-deoxynucleoside, mC contains 5-methylcytosine, and all internucleoside links are phosphorothioates.

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