Compositions and methods for decreasing 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 is filed on December 21, 2015, and is incorporated herein by reference in its entirety. This application claims the benefit of U.S. Provisional Patent Application No. 62 / 270,165.

[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. Tau protein interacts with tubulin to stabilize microtubules and The microtubule network is essential for the formation of the cytoskeleton. and maintenance of cell structure and morphology, and intracellular transport of vesicles, organelles, and macromolecules. They are involved in many important cellular processes, including providing a platform for transport. Tau binding to endothelial cells stabilizes microtubules, making it one of the most important cellular processes. It is a mediator.

[0004] At least six tau isoforms exist in the human brain, each ranging from 352 to 441 amino acids. Tau isoforms range in length from 1 to 20 amino acids. Derived from PT (microtubule-associated protein tau), MAPT transcripts are complex and regulated MAPT exons undergo alternative splicing to generate multiple mRNA species. 2 and 3 encode sequences of 29 and 58 amino acids, respectively, and thus Alternative splicing of tau 2 and / or 3 can result in 0N, 1N, or 2N tau and Zero, one, or two copies of a 29 amino acid acidic domain at the N-terminus, called Exon 10 of MAPT encodes the microtubule-binding domain, Thus, inclusion of exon 10 leads to the presence of an additional microtubule-binding domain. Since there are three microtubule-binding domains at the location of exon 10, tau isoforms containing exon 10 "4R tau" refers to a tau protein that has four repeats of the microtubule-binding domain. The tau isoform that does not contain exon 10 is called "3R tau" and This refers to tau proteins that have three repeats of the microtubule-binding domain. The tau isoform appears to bind more tightly to microtubules than the 3R tau isoform, This is because the 4R tau isoform contains one extra microtubule-binding domain. The ratio of 3R to 4R tau is developmentally regulated, with fetal tissues exclusively containing 3R tau. and adult human tissues express roughly equal levels of 3R tau and 4R tau .

[0005] Tau is a phosphoprotein, and the longest tau isoform contains approximately 85 phosphate ions. It has a carboxylable site (Ser, Thr, or Tyr) (Pedersen and Sigurdsson, T rends in Molecular Medicine 2015, 21 (6): 394). Phosphorylation is a process that inhibits normal tau protein. Tau is constantly phosphorylated during the cell cycle. Tau can only associate with microtubules in its dephosphorylated form. Thus, tau phosphorylation directly regulates the microtubule assembly-dissociation switch in neurons. Under pathological conditions, tau protein becomes hyperphosphorylated and acts as a tunica reductase inhibitor. This results in loss of purine binding and destabilization of microtubules, followed by the formation of pathogenic neurofibrillary tangles. This results in the aggregation and deposition of tau. Glu391, and Asp421) have also been identified in neurofibrillary tangles.

[0006] Summary of the Invention Antisense oligonucleotides targeting human microtubule-associated protein tau (MAPT) The present invention relates to antisense oligonucleotides, compositions containing the antisense oligonucleotides, and compositions comprising the antisense oligonucleotides. Using ance oligonucleotides to reduce tau mRNA and protein expression Methods are provided herein. The compositions and methods provided herein are directed to tau-related These compounds are useful in treating diseases such as

[0007] In one embodiment, the nucleic acid sequence is at least 70% identical to any of the nucleic acid sequences shown in Tables 2 to 17. For example, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) of sequence identity with the nucleic acid sequence. A oligonucleotide, wherein any C in the nucleic acid base sequence is a cytosine or a 5- methylcytosine, and at least one nucleotide of the oligonucleotide is Provided herein are oligonucleotides in which the end of the oligonucleotide has a 2'-modification. The nucleotide is an antisense oligonucleotide that targets human MAPT. The '-modifications are 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-dimethyl 2'-O-dimethylaminopropyl (2'-O-DMAP), 2'-O-dimethylaminoethyloxyethyl 2'-O-DMAEOE, and 2'-ON-methylacetamide (2'-O- In some embodiments, the 2'-modified .NMA. The modification is 2'-O-methoxyethyl (2'-O-MOE). wherein each C in any of the nucleobase sequences is a 5-methylcytosine.

[0008] In some embodiments, the antisense oligonucleotides provided herein The length of the antisense oligonucleotide is 12 to 30 nucleobases. Oligonucleotides are 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, Containing 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleobases In some embodiments, antisense oligonucleotides targeting MAPT can be used. The nucleotides are 12 to 25 nucleobases in length. For example, MAPT-targeting antibodies Sense oligonucleotides are 12, 13, 14, 15, 16, 17, 18, 19, 20 , 21, 22, 23, 24, or 25 nucleobases. In an embodiment, the antisense oligonucleotide targeting MAPT is 15 to 20 For example, antisense oligonucleotides targeting MAPT are The nucleic acid can comprise 15, 16, 17, 18, 19, or 20 nucleobases.

[0009] In some embodiments, the antisense oligonucleotides provided herein target MAPT are Such antisense oligonucleotides are steric blockers. Tide reduces tau mRNA and / or protein expression independently of RNAseH The internucleoside linkages of the steric blocking agents are phosphodiester or phosphorothioate. In some embodiments, the M Antisense oligonucleotides targeting APT are any of the sequences shown in Tables 2 to 8. or at least 70% (e.g., 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) of sequence identity wherein any one of the nucleic acid base sequences is a steric blocking agent comprising a nucleic acid base sequence C is either cytosine or 5-methylcytosine, and each nucleotide of the oligonucleotide The nucleotide has a 2'-modification. In some embodiments, the MAPT-targeting The antisense oligonucleotide has at least one of the sequences shown in Tables 2 to 8. In some embodiments, the MAP comprises a nucleic acid sequence having 80% sequence identity. The antisense oligonucleotide targeting T may be any of the sequences shown in Tables 2 to 8. In some embodiments, the nucleic acid sequence has at least 90% sequence identity with In the present invention, antisense oligonucleotides targeting MAPT are shown in Tables 2 to 8. In some embodiments, the MAPT targeting antigen comprises any one of the nucleic acid sequences. The antisense oligonucleotide has any one of the nucleic acid base sequences shown in Tables 2 to 8. In some embodiments, each C in any of the nucleobase sequences is a 5-methyl This is Toshin.

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

[0011] In some embodiments, antisense oligonucleotides targeting MAPT The oligonucleotide comprises a linker attached to the 3' end of the oligonucleotide through a phosphate bridge, A oligonucleotide has one of the following structures:

[0012] [ka]

[0013] In some embodiments, the antisense oligonucleotides provided herein target MAPT are The oligonucleotide is a gapmer, which has two nucleotides at the 5' and 3' ends. between the wing segments (also called the 5' wing and 3' wing, respectively) has a central gap segment of consecutive 2'-deoxyribonucleotides located at Such antisense oligonucleotides act by activating RNAse H. The gapmer nucleoside reduces the expression of tau mRNA and / or protein. The intersid linkages can be phosphorothioate or phosphodiester linkages. In some embodiments, the gapmer comprises at least 5 (e.g., 5, 6, 7, 8 , 9, 10, 11, 12) consecutive stretches of 2'-deoxyribonucleotides the 5' and 3' wing segments comprise one or more 2'-modified nucleotides. In some embodiments, such oligonucleotides include at least Each has seven (e.g., 7, 8, 9, 10, 11, or 12) consecutive 2'-deoxyribonucleic acids. In some embodiments, such an oligonucleotide comprises 1 0 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 ), and 2'-ON-methylacetamide (2'-O-NMA). In some embodiments, a gapmer can have a 5' wing and The 3' wing contains 2'-O-MOE modified nucleotides.

[0014] In some embodiments, the tau-targeting gapmer comprises a 20 nucleoside A 5-10-5 gapmer having a length of 10 Contiguous 2'-deoxynucleosides, flanked by 5' and 3' wings Each wing contains five nucleosides, each bearing a 2'-O-MOE modification.

[0015] In some embodiments, the antisense oligonucleotides provided herein target MAPT are The oligonucleotide has at least one sequence identical to any of the sequences shown in Tables 9 to 15 and 17. 70% (e.g. 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) of sequence identity wherein any C in the nucleic acid base sequence is a cytosine or or 5-methylcytosine, and at least one nucleotide of the oligonucleotide is In some embodiments, the nucleotide has a 2'-modification. The antisense oligonucleotide may be any of the sequences shown in Tables 9 to 15 and 17. In some embodiments, the nucleic acid sequence has at least 80% sequence identity. Therefore, antisense oligonucleotides targeting MAPT are listed in Tables 9 to 15 and 17. A nucleic acid sequence having at least 90% sequence identity with any of the nucleic acid sequences shown. In some embodiments, an antisense oligonucleotide targeting MAPT is The octides include any of the nucleic acid base sequences shown in Tables 9-15 and 17. In an embodiment, the antisense oligonucleotide targeting MAPT is selected from the group consisting of those listed in Tables 9-1. 5 and 17. The antisense oligonucleotides targeted to MAPT provided herein are listed in Table 9. A 5-10-5 group containing any of the nucleic acid base sequences shown in any of 15 and 17. a primer, wherein the first to fifth nucleotides are modified with 2'-O-MOE nucleotides 6 to 15 each contain a 2'-deoxynucleoside; The 16th to 20th nucleotides are each a 2'-O-MOE modified nucleotide. In some embodiments, any one of the nucleobase sequences may include a nucleotide. Each C is a 5-methylcytosine.

[0016] In some embodiments, antisense oligonucleotides targeting MAPT SEQ ID NOs: 208, 284, 285, 313, 329, 335, 366, 384, 38 6, 405, 473, and 474. In some embodiments, the antisense oligonucleotide targeting MAPT is , SEQ ID NO: 284 and at least 90% (e.g., 90%, 91%, 92%, 93%, 94 %, 95%, 96%, 97%, 98%, 99%, or 100%) of sequence identity In some embodiments, the antisense nucleic acid targeting MAPT comprises a nucleic acid sequence. The oligonucleotide comprises SEQ ID NO: 284. Antisense oligonucleotides targeting SEQ ID NO: 285 or 208 are at least 90% (e.g. 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97 %, 98%, 99%, or 100%) of sequence identity with the nucleic acid sequence. In some embodiments, the antisense oligonucleotide targeting MAPT is Contains column numbers 285 or 208.

[0017] In another embodiment, SEQ ID NO: 487-5 has 1, 2, or 3 mismatches. A nucleobase sequence that is complementary to at least 12 consecutive nucleobases of any one of wherein at least one nucleotide of the oligonucleotide comprises Provided herein are oligonucleotides in which the oligonucleotide has a 2'-modification. The oligonucleotides are antisense oligonucleotides that target MAPT. In some embodiments, such oligonucleotides include those of SEQ ID NOs: 487-506. A nucleobase sequence that is 100% complementary to at least 12 consecutive nucleobases of any one of the In some embodiments, such oligonucleotides include one or In some embodiments, such an oligonucleotide comprises a plurality of 5-methylcytosines. Leuotides have 2'-modifications. The 2'-modifications are 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- N-methylacetamide (2'-O-NMA). In an embodiment, the 2'-modification is 2'-O-methoxyethyl (2'-O-MOE). In some embodiments, such oligonucleotides include at least five (e.g., 5, 6, 7, 8, 9, 10, 11, 12) consecutive 2'-deoxyribonucleic acid residues In some embodiments, such oligonucleotides include at least At least seven (e.g., 7, 8, 9, 10, 11, 12) consecutive 2'-deoxyribonucleic acid residues In some embodiments, such oligonucleotides include , containing 10 consecutive 2'-deoxyribonucleotides.

[0018] In some embodiments, the antisense oligonucleotides provided herein target MAPT are The oligonucleotides increased the expression levels of tau mRNA or protein in vitro. , can be reduced by at least 30%.

[0019] In some embodiments, the antisense oligonucleotides provided herein target MAPT are The oligonucleotides reduce the expression levels of tau mRNA or protein in vivo. It can be reduced by at least 30%.

[0020] In another embodiment, any of the antisense oligonucleotides described herein. and a pharma- ceutically acceptable carrier.

[0021] In further embodiments, e.g., a subject suffering from or susceptible to a tau-related disease. Tau expression levels in a pancreatic subject are determined by administering to the subject an antisense oligonucleotide as described herein. The method of reducing the level of schizophrenia by administering a therapeutically effective amount of any of the oligonucleotides described herein. In some embodiments, such methods include administering a second agent to In some embodiments, the method includes administering to an elephant a compound that targets MAPT. Antisense oligonucleotides that target the target gene can be administered to a subject intrathecally, intracranially, intranasally, orally, or intravenously. In some embodiments, the subject may be administered intravenously or subcutaneously. is a human.

[0022] A subject in need thereof, e.g., a subject suffering from or suffering from a tau-related disease. The present invention provides a method for treating a tau-related disease in a subject susceptible to the use of the method described herein. Antisense oligonucleotides comprising the antisense sequences described herein are also provided. The present invention relates to an oligonucleotide or pharmaceutical composition for the treatment of tau-associated tau in a subject in need thereof. The present disclosure also includes uses for treating diseases associated with tau in a subject in need thereof. The use of an anti-cancer agent as described herein in the manufacture of a medicament for use in treating a disease associated with This also includes the use of sense oligonucleotides.

[0023] Tau-related diseases include Alzheimer's disease (AD), amyotrophic lateral sclerosis / Parkinson disease, and ALS-PDC, Argyrophilic grain dementia (AGD), British Amino cerebrovascular disease, cerebral amyloid angiopathy, chronic traumatic encephalopathy (CTE), corticobasal degeneration (CBD), Creutzfeldt-Jakob disease (CJD), Punch-Drunk Disease, Calcification Diffuse neurofibrillary tangles, Down syndrome, Dravet syndrome, epilepsy, frontotemporal dementia (F TD), frontotemporal with parkinsonism linked to chromosome 17 (FTDP-17) dementia, frontotemporal lobar degeneration, ganglioglioma, gangliocytoma, Gerstmann-Straussler - Scheinker's disease, Hallervorden-Spatz disease, Huntington's disease, inclusion body myositis, lead encephalopathy disease, Lytico-Bodig disease, meningioma hemangiomatosis, multiple system atrophy, myotonic dystrophy, Niemann-Pick disease type C (NP-C), non-Guamanian motor neuron disease with neurofibrillary tangles, Pick's disease (PiD), postencephalitic parkinsonism, prion protein, cerebral amyloidosis progressive subcortical gliosis, progressive supranuclear palsy (PSP), subacute sclerosing panencephalitis, neurological Tangle-only dementia, Neurofibrillary tangle-predominant dementia le-predominant dementia, multi-infarct dementia, ischemic stroke, or tuberous sclerosis You can choose from the following. [Brief description of the drawings]

[0024] [Figure 1-1] Figures 1A-1E show physical characterization of antisense oligonucleotides targeting MAPT. Figure 1A shows the structure of an antisense oligonucleotide (ASO) comprising SEQ ID NO:284 and having the formula C230H321N72O120P19S19 and a predicted molecular weight of 7212.3 Da. Figure 1B shows liquid chromatography mass spectrometry (LC-MS) data for an ASO comprising SEQ ID NO:284 and having a measured peak mass of 7214.3. Figure 1C shows a deconvoluted peak report of LC-MS for an ASO comprising SEQ ID NO:284. Figure 1D shows a deconvoluted peak report of LC-MS for an ASO comprising SEQ ID NO:285 and having a measured peak mass of 7232.5. Figure 1E shows a deconvoluted peak report of LC-MS for an ASO comprising SEQ ID NO:285. [Figure 1-2] (As stated above.) [Figure 1-3] (As stated above.) [Diagram 2]Figures 2A-2E show human tau mRNA and protein expression levels in representative hTau BAC transgenic mouse lines before and after antisense oligonucleotide treatment. Figure 2A shows representative RT-PCR results showing that all six human tau transcripts were found in the forebrain of hTau BAC transgenic mice (transgenic line 510, 2-month-old female mice). Exons 2, 3, and 10 are alternately spliced ​​to generate six tau isoforms: 2-3-10-; 2+3-10-; 2+3+10-; 2-3-10+; 2+3-10+; 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 exon 2 or exon 3; 1N represents a tau isoform with either exon 2 or exon 3; 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 ranging from 48 to 67 kD and amino acids ranging from 352 to 441. They differ in (1) the inclusion of zero, one or two inserts in the 29 amino acid N-terminal portion (0N, 1N, or 2N), or (2) the inclusion of three or four microtubule-binding domains (3R or 4R). Figure 2C is a representative immunohistochemistry image showing the distribution of normal axons of human tau stained with a human tau-specific antibody in the brain of an hTau BAC transgenic mouse. Figure 2D is a bar graph showing knockdown of tau mRNA in the cortex of an hTau BAC transgenic mouse 4 weeks after a single treatment with an antisense oligonucleotide comprising SEQ ID NO: 285. Figure 2E is a representative Western blot showing knockdown of tau protein in the hippocampus of an hTau BAC transgenic mouse 4 weeks after a single treatment with an antisense oligonucleotide comprising SEQ ID NO: 285. [Diagram 3] FIG. 3 is a series of in situ hybridization images showing widespread brain distribution of the antisense oligonucleotide of SEQ 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 SEQ ID NO:285. [Diagram 5] 5A and 5B are dot plots showing the time course of human tau mRNA (FIG. 5A) and protein (FIG. 5B) expression levels in hTau BAC transgenic mice following a single ICV injection of 200 μg of the antisense oligonucleotide of SEQ ID NO:285.

[0025] Detailed Description Antisense oligonucleotides targeting microtubule-associated protein tau (MAPT) antisense oligonucleotides, compositions containing the antisense oligonucleotides, and antisense oligonucleotides thereof Methods for reducing tau expression using oligonucleotides are provided herein. The compositions and methods provided herein are useful for treating tau-related diseases.

[0026] definition As used in this specification and claims, the singular forms "a," "an," and "the" are used interchangeably. Unless the context clearly dictates otherwise, the term "a" includes plural references. "A cell" includes a plurality of cells, including mixtures thereof.

[0027] All numerical designations, e.g., pH, temperature, time, concentration, and molecular weight, including ranges, are given in the (+) range. ) or (-) 0.1 increments. Not always stated explicitly. However, it should be understood that all numerical designations are preceded by the term "about." Although not necessarily expressly stated, the reagents described herein are merely examples and are not intended to be limiting. It should also be understood that equivalents of such reagents are known in the art. be.

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

[0029] As used herein, "2'-O-methoxyethyl", "2'-MOE", or "2'-OCH2CH2-OCH3" refers to an O-methoxy group at the 2' position of the furanose ring. A 2'-O-methoxyethyl modified sugar is a modified sugar. 2'-MOE Nucleoside / Nucleotide" or "2'-O-Methoxyethyl Nucleoside / Nucleotide" "Nucleoside / Nucleotide" refers to a nucleoside / nucleotide that contains a sugar moiety modified with 2'-MOE. Refers to Ochido.

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

[0031] As used herein, the term "antisense oligonucleotide" refers to an oligonucleotide that binds to a target nucleic acid, e.g. For example, complementary to a corresponding segment of a target genomic sequence, pre-mRNA, or mRNA molecule. In some embodiments, the nucleic acid sequence of the nucleic acid molecule is a single stranded oligonucleotide having a nucleic acid base sequence that is In the present invention, the antisense oligonucleotides are 12 to 30 nucleobases in length.

[0032] The term "complementarity" or "complementary" refers to the formation of hydrogen bonds (e.g., tson-Crick, Hoogsteen or reverse Hoogsteen hydrogen bonds base pairing ability between a nucleobase of a first nucleic acid strand and a nucleobase of a second nucleic acid strand, mediated by For example, in DNA, adenine (A) is complementary to thymine (T) and guanosine (G) is complementary to thymine (T). For example, in RNA, adenine (A) is complementary to reverse transcription (Trimethylation). In some cases, cytosine (C) is complementary to selenium (U) and guanosine (G) is complementary to cytosine (C). In embodiments, a complementary nucleobase is capable of base pairing with a nucleobase of its target nucleic acid. For example, the antisense oligonucleotide The nucleic acid base at a certain position of the oligonucleotide is the same as the nucleic acid base at a certain position of the target nucleic acid. If hydrogen bonding is possible, then between the oligonucleotide and the target nucleic acid The hydrogen bond positions are complementary in the nucleic acid base pair. Acid-base pairs can be maintained with their nucleobase counterparts, and thus and may also be nucleobase complementary.

[0033] An "effective amount" refers to an amount sufficient to effect beneficial or desired results. A therapeutic amount is an amount that achieves the desired therapeutic effect. This amount prevents the onset of a disease or prevents disease symptoms. A prophylactically effective amount may be the same as or different from a prophylactically effective amount, which is the amount needed to prevent. The amount may be administered in one or more administrations, applications or dosages. The "therapeutically effective amount" of a compound (i.e., an effective dosage) depends on the choice of therapeutic compound. The compositions of the present invention may be administered one or more times per day to once per week, including once every other day. The dosage may be administered once or multiple times. Those skilled in the art will recognize the severity of the disease or disorder, prior the treatment of the condition, the general health and / or age of the subject and any other diseases present, Certain factors, including but not limited to, the dosage and It will be appreciated that the timing of administration of the therapeutic Treatment of a subject with a therapeutically effective amount of the compounds can include a single treatment or a series of treatments. Cut.

[0034] As used herein, the term "gapmer" refers to a sequence of consecutive 2'-deoxyribonucleic acid Chimeric antisense oligonucleotides containing a central gap segment consisting of nucleotides - Patents.com It refers to a nucleic acid that can activate RNAseH and binds nucleic acids at the 5' and 3' ends, respectively. One or more modified nucleotides that confer increased resistance to nuclease degradation. It is adjacent to two wing segments containing

[0035] The term "hybridization" refers to the formation of base pairs and a duplex structure between complementary nucleic acid strands. Hybridization can occur between perfectly complementary nucleic acid strands or between mismatched It may occur between "substantially complementary" nucleic acid strands that contain non-essential regions. Although not limited to this, the most common mechanism of pairing involves hydrogen bonding, which is the formation of complementary nucleic acid strands. Watson-Crick, Hoogsteen or reverse Hoogst between target nucleobases For example, adenine and thymine can form hydrogen bonds. Hybridization can occur at varying stringency. As used herein, hybridization can occur under a variety of circumstances. Base pairing of complementary nucleic acid strands under at least relatively low stringency conditions and This refers to the formation of a double-stranded structure, for example, in 2×SSC (0.3 M sodium chloride, 0.03 M sodium chloride, Hybridization at 37°C in 0.1% SDS and sodium ethyl acetate. Subsequent washes in 4x SSC, 0.1% SDS-containing solution at 37°C are sufficient for use. and finally washed in 1×SSC at 45° C.

[0036] The terms "inhibit" or "inhibition" refer to a decrease in the expression or activity of a target nucleic acid or protein. "Ablation" refers to reduction or blocking and does not necessarily indicate complete elimination of target 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 a reagent, e.g., an antisense Decreased mRNA or protein expression of genes following oligonucleotide treatment Knockdown of expression can occur during transcription, mRNA splicing, or translation. obtain.

[0039] The term "mismatch" refers to a mismatch between a nucleobase of a first nucleic acid strand and the corresponding nucleobase of a second nucleic acid strand. This refers to cases where the inequality is not complementary to the inequality.

[0040] The term "nucleobase sequence" refers to any sugar, linking group, and / or nucleobase modification. It refers to the order of consecutive nucleobases.

[0041] The term "oligonucleotide" refers to a linkage, each of which may be modified or unmodified. of deoxyribonucleotides (DNA) and / or ribonucleotides (RNA) Unless otherwise specified, the term refers to a polymer that has binding properties similar to those of natural nucleic acids. Nucleic acids containing known analogues of natural nucleotides, such as phosphodiester linkages, Nucleic acids having alternative internucleoside linkages are included.

[0042] The term "phosphorothioate linkage" refers to a phosphodiester bond that is linked to one of the non-bridging oxygen atoms. refers to an internucleoside linkage modified by replacing one with a sulfur atom.

[0043] The term "sense strand" refers to the coding strand, positive strand, or Refers to the non-template strand. The coding strand is where thymine (T) in DNA is replaced by uracil (U) in RNA. It has the same sequence as the mRNA sequence, except that (U) is replaced by "A The "antisense strand" is the non-coding strand of a DNA molecule or the template for the synthesis of mRNA. The sequence of the antisense strand is therefore the same as that of the sense strand and the template strand. and mRNA (U for T in the RNA).

[0044] As used herein, the term "steric blocker" refers to a steric blocker that acts to block a target nucleic acid (e.g., a target genomic sequence). hybridizes with a target nucleic acid (e.g., a nucleic acid sequence, pre-mRNA, or mRNA molecule) to activate RNAse H Antibodies that interfere with the transcription, splicing, and / or translation of a target nucleic acid without Refers to a sense oligonucleotide.

[0045] As used herein, "target" or "targeted" refers to the process of isolating a target nucleic acid, e.g. For example, a target genomic sequence, a pre-mRNA or mRNA molecule, or a fragment thereof. or a variant thereof and can inhibit transcription, splicing, or the like of a target nucleic acid. Antisense oligonucleotides capable of modulating transcription and / or translation This refers to the design and selection of the optode.

[0046] As used herein, "tau" (also known as "microtubule associated protein tau") ", MAPT, MSTD;PPND;DDPAC;MAPTL;MTBT1;M TBT2; FTDP-17; PPP1R103) are encoded by the gene MAPT , a protein associated with microtubules. The human MAPT gene is located at chromosome location 17q2 The human MAPT gene has been mapped to genomic sequence data in GenBank at The MAPT gene can be found in NG_007398.1 (SEQ ID NO: 304). Tron and exon sequences and branch points are shown in the transcript: MAPT-203ENST0000 0344290 based on the Ensembl genome database website In humans, eight tau genes are identified based on complex alternative splicing. The term "tau" refers collectively to all isoforms of tau. The protein and mRNA sequences of the longest human tau isoforms are: Homo sapiens microtubule-associated protein tau (MAPT), transcript variant 6 , mRNA (NM_001123066.3)

[0047] [ka]

[0048] [ka]

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

[0050] [ka] It is. The mRNA and protein sequences of other human tau isoforms are available in GenBank. The same can be found in US Pat. No. 6,333,493 under the following accession numbers: 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). As used herein, human tau protein refers to any of the tau isoforms and its At least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, %, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86 %, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96 %, 97%, 98%, 99% or 100% sequence identity to the The sequences of mouse, monkey, and other animal tau proteins are known in the art. There are.

[0051] The term "tau-related disease" refers to disorders characterized by abnormal tau protein expression, secretion, phosphorylation, or cleavage. , and / or aggregation-related diseases. Diseases include Alzheimer's disease (AD), amyotrophic lateral sclerosis / Parkinsonism-dementia Complex disease (ALS-PDC), argyrophilic grain dementia (AGD), British amyloid angiopathy, brain Amyloid angiopathy, chronic traumatic encephalopathy (CTE), corticobasal degeneration (CBD), CJD, Punch-Drunken Disease, Diffuse Neurofibromas with Calcification Fibromyalgia, Down's syndrome, Dravet syndrome, epilepsy, frontotemporal dementia (FTD), chromosome 17 Frontotemporal dementia with parkinsonism linked to chromosome (FTDP-17), frontotemporal Lobar degeneration, ganglioglioma, gangliocytoma, Gerstmann-Straussler-Scheinker disease, Hallervorden-Spatz disease, Huntington's disease, inclusion body myositis, lead encephalopathy, Lytico-Bonin disease Digg disease, meningioma hemangiomatosis, multiple system atrophy, myotonic dystrophy, Niemann-Pick disease Type C (NP-C), Non-Guamanian Motor Neuron Disease with Neurofibrillary Tangles, Pick's Disease (PiD) ), postencephalitic parkinsonism, prion protein cerebral amyloid angiopathy, progressive subcortical Gliosis, progressive supranuclear palsy (PSP), subacute sclerosing panencephalitis, neurofibrillary tangle predominant type Dementia (tangle only dementia), neurofibrillary tangle-predominant dementia (tangle-predominant dementia) ementia), multi-infarct dementia, ischemic stroke, and tuberous sclerosis. Not limited.

[0052] The terms "homology" or "identity" refer to the degree of similarity between two polymeric molecules, e.g., between two nucleic acid molecules. between, for example, two DNA molecules or two RNA molecules, or between two polypeptides Refers to the sequence identity of subunits between molecules. Subunit positions in both of two molecules are occupied by subunits of the same monomer; for example, two DNA molecules If a position in each of the is occupied by an adenine, then they are Homology between two sequences is a match, i.e., i.e., it is a direct function of the number of positions of homology. For example, half of the positions in the two sequences (e.g. For example, if 5 positions in the polymer (out of a length of 10 subunits) are homologous, then 2 The two sequences are 50% homologous, meaning that 90% of the positions (e.g., 9 out of 10) match. That is, if two sequences are homologous, they are 90% identical. The percentage of "sequence identity" is The best alignment is determined by comparing the two aligned sequences over a comparison window. In this case, the amino acid sequence fragments in the comparison window can be divided into two The sequence is compared to a reference sequence (not including additions or deletions) for optimal alignment of the sequence. The percentage may include identities or deletions (e.g., gaps or overlaps). Determine the number of positions where the amino acid residue of appears in both sequences, obtain the number of matching positions, and Divide the number of positions found by the total number of positions in the comparison window and multiply the result by 100. The percentage of sequence identity can be calculated by multiplying the results. The result is the percent identity of the subject sequence relative to the sequence in question.

[0053] The term "isolated" refers to altered or removed from the natural state. For example, a nucleic acid or peptide that is naturally present in a living animal is not "isolated." However, the same nucleic acid or nucleic acids partially or completely separated from the materials with which they coexist in their natural state A peptide is "isolated." An isolated nucleic acid or protein is substantially purified. The vector may exist in a modified form or in a non-native environment, such as a host cell. etc.

[0054] The terms "treat" or "treatment" refer to both therapeutic treatment and prophylactic or preventative measures. The purpose is to prevent or slow down undesirable physiological changes or disorders. For purposes of the present invention, a beneficial or desired clinical outcome is a detectable or detectable Whether or not this is possible, relief of symptoms, reduction in the extent of disease, or stabilization of disease (i.e., the absence of deterioration, delay or slowing of disease progression, relief or palliation of the condition, and remission. "Treatment" includes, but is not limited to, a solution (whether incomplete or complete). It can also mean prolonged survival compared with expected survival without treatment. Cut.

[0055] The term "subject" refers to an animal, human or non-human, to whom treatment according to the methods of the present invention is provided. Veterinary and non-veterinary applications are contemplated. The term refers to mammals, e.g., humans, other primates, pigs, rodents, e.g. mice and rats, rabbits, guinea pigs, hamsters Typical examples of such animals include, but are not limited to, cattle, horses, cats, dogs, sheep and goats. Suitable subjects include humans, livestock, and household pets, such as cats and dogs.

[0056] Unless otherwise specified, all technical and scientific terms used herein refer to the present invention. have the same meaning as commonly understood by one of ordinary skill in the art to which they pertain. Although methods and materials similar or equivalent to those described above can be used to practice the present invention. Suitable methods and materials are described below. All publications, patents, and other references mentioned herein are incorporated by reference in their entirety. Applications, patents, and other references are incorporated by reference in their entirety. The present specification, including definitions, is hereby incorporated by reference. In addition, materials, methods, and examples are provided herein. , for purposes of illustration only and are not intended to be limiting.

[0057] The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims. There will be.

[0058] Antisense oligonucleotides Antisense oligonucleotides (ASOs) reduce RNA, inhibit translation, and inhibit miRNAs. A inhibition, splicing modulation, and polyadenylation site selection. Antisense oligonucleotides are powerful and versatile agents that are being used in an increasing number of applications. The nucleotides are selected so that a sufficient number of nucleobases of the antisense oligonucleotide match the target nucleic acid. When it can form hydrogen bonds with corresponding nucleic acid bases, it binds to the target nucleic acid and promotes transcription and Therefore, the antisense oligonucleotides The acid-base sequence is a sequence of a target nucleic acid, e.g., a target genomic sequence, a pre-mRNA, or an mRNA molecule. The nucleic acid sequence is complementary to that of the nucleic acid sequence of son-Crick, Hoogsteen or reverse Hoogsteen hydrogen bonds) This occurs when a complementary nucleic acid base is formed between an antisense oligonucleotide and a target nucleic acid. Non-complementary nucleobases between the antisense oligonucleotide and the target nucleic acid result in the formation of an antisense oligonucleotide. The sense oligonucleotide still hybridizes specifically to the target nucleic acid. As far as possible, it is acceptable.

[0059] ASOs can mediate targeting of proteins in either an RNaseH-dependent or RNaseH-independent manner. These proteins can be designed to reduce expression of the protein (Watts JK, et al., J Pathol. 2012 Jan 2012). (See Uary; 226(2): 365-379.) ASOs containing continuous stretches of DNA When hybridized with a target RNA, the DNA-RNA heteroduplex activates RNase H. It cleaves the double-stranded target RNA, leading to subsequent RNA fragmentation by cellular nucleases. ASOs promote the degradation of pre-mRNA fragments. By sterically blocking the translation of RNA into protein, it is possible to The expression of the target can also be decreased.

[0060] Antisense oligonucleotides targeting microtubule-associated protein tau (MAPT) In some embodiments, the agonists provided herein are Antisense oligonucleotides have 1, 2, 3, 4, or 5 mismatches. , a nucleic acid salt complementary to a segment of MAPT genomic DNA, pre-mRNA, or mRNA If perfect base pairing occurs (e.g., between A and T and between C and G), pairing), mismatches between the oligonucleotide and the corresponding target nucleic acid are counted. A mismatch occurs when the nucleobase of the first nucleic acid is This occurs when a nucleic acid is unable to pair with the corresponding nucleic acid base of a second nucleic acid. a position in a sequence having a nucleobase A and a corresponding position in a second sequence that cannot pair with A If it has a nucleobase (e.g., C or G), it constitutes a mismatch. If a position in a sequence has a nucleobase and the corresponding position in the other sequence does not have a nucleobase, Mismatches are counted even if they are to the sugar portion of a nucleotide or internucleoside linkage. The modification is not considered a mismatch. Thus, if one sequence contains a G and the second sequence contains a If the corresponding nucleobase contains a modified C (e.g., 5-methylcytosine), it is not counted. There will be no mismatches.

[0061] The antisense oligonucleotides provided herein relate to stretches of nucleic acid. segments of MAPT genomic DNA, pre-mRNA, or mRNA and segments At least 70%, 80%, 85%, 86%, 87%, 88%, 8 9%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 9 9%, or 100% complementary. Percent complementarity can be determined by routine methods known in the art, for example, using the BLAST protocol. Basic local alignment search tools or the PowerBLAST program (Altschul et al., J. Mol. Biol., 1990, 215, 4 03 410; Zhang and Madden, Genome Res., 1997, 7, 649 656). In some embodiments, the antisense oligonucleotides provided herein can be used to The nucleotides were synthesized by combining MAPT genomic DNA, pre-mRNA, or mRNA segments with 10 0% complementary (i.e., completely complementary) nucleobase sequences. As used herein, "fully complementary" or "100% complementary" refers to each nuclease of an antisense compound. It indicates that the acid base can form a precise base pair with the corresponding nucleobase of the target nucleic acid. For example, a 20 nucleobase antisense compound will have perfect complementarity to the antisense compound. As long as there is a corresponding 20 nucleobase portion of the target nucleic acid that is It is perfectly complementary to the target sequence.

[0062] In some embodiments, the antisense oligonucleotides provided herein 1, 2, or 3 mismatches in any of the sequences shown in Table 1. At least 12 consecutive nucleobases (e.g., 12, 13, 14, 15, 16, 17, or In some embodiments, the nucleobase sequence is complementary to In the present specification, the antisense oligonucleotides are selected from the group consisting of those shown in Table 1. At least 12 consecutive nucleic acid bases (e.g., 12, 13, 14, 15, 16, 17, or 18 consecutive nucleobases) Contains arrays.

[0063] Antisense compounds provided herein are directed to specific nucleotide sequences, SEQ ID NOs., or or a portion thereof. The antisense oligonucleotide used has the same nucleic acid base pairing ability as the is identical to the sequence disclosed herein. For example, uracil and thymidine are both Both of these pair with adenine, so they are substituted for thymine in the disclosed DNA sequence. RNA containing rasil would be considered identical to the DNA sequence presented herein. The antisense oligonucleotides described herein have non-identical bases to the antisense oligonucleotides provided herein. Antisense oligonucleotides and shortened and lengthened oligonucleotides Formats such as these are also contemplated. The non-identical bases may be adjacent to each other or may be anti-sequence. The antisense oligonucleotide may be dispersed throughout the antisense oligonucleotide. The percent sequence identity of a peptide is determined by the number of identical base pairings to the sequence to which it is being compared. The percentage of sequence identity can be calculated according to the number of bases that have the same sequence identity as the sequence of the original DNA. Routine methods known in the art, such as the BLAST program (basic loca l alignment search tools) or PowerBLAST Pro (Altschul et al., J. Mol. Biol., 1990, 215, 403-410; Zhang and Madden, Ge Nome Res., 1997, 7, 649 656); or using the GAP program (for Unix Wisconsin Sequence Analysis Package for Genetics, Version 8 ter Group, University Research Park, Mad ison (Wisconsin) based on the Smith and Waterman algorithm. (Adv. Appl. Math., 1981, 2, 482-489) to determine the initial setting. It is possible.

[0064] In some embodiments, any one of the nucleic acid sequences shown in any one of Tables 2 to 17. or at least 70% (e.g., 70%, 75%, 80%, 81%, 82%, 83%, 8 4%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 9 4%, 95%, 96%, 97%, 98%, 99%, or 100%) of sequence identity with An antisense oligonucleotide comprising a nucleic acid base sequence comprising any one of the nucleic acid base sequences. one C is either cytosine or 5-methylcytosine, At least one nucleotide of the antisense oligonucleotide has a 2'-modification. In some embodiments, a method for producing a medicament for use in ... At least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity Provided herein are antisense oligonucleotides comprising nucleobase sequences having the same identity as those described herein. In some embodiments, an antisense oligonucleotide targeting MAPT is The code includes any of the nucleic acid base sequences shown in any of Tables 2 to 17. In the form, the antisense oligonucleotide targeting MAPT is It consists of any of the nucleic acid base sequences shown in any of the above.

[0065] In some embodiments, the antisense oligonucleotides provided herein The length of the antisense oligonucleotide is 12 to 30 nucleobases. Oligonucleotides are 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, Containing 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleobases In some embodiments, antisense oligonucleotides targeting MAPT can be used. The nucleotides are 12 to 25 nucleobases in length. For example, MAPT-targeting antibodies Sense oligonucleotides are 12, 13, 14, 15, 16, 17, 18, 19, 20 , 21, 22, 23, 24, or 25 nucleobases. In an embodiment, the antisense oligonucleotide targeting MAPT is 15 to 20 For example, antisense oligonucleotides targeting MAPT are The base may contain 15, 16, 17, 18, 19, or 20 nucleobases. In some embodiments, the antisense oligonucleotide targeting MAPT is Contains 17 nucleobases. The length of the antisense oligonucleotide can be adjusted without eliminating the activity. and / or by increasing or decreasing the mismatch in the antisense oligonucleotide. It is possible to introduce multiple mismatches (e.g., 1, 2, 3, 4, or 5 mismatches). be.

[0066] Chemical modification of antisense oligonucleotides Oligonucleotides are linked together by internucleoside phosphodiester bonds. Each nucleotide is linked to a sugar moiety. A nucleic acid molecule that contains a nucleic acid base having a phosphate group covalently linked to the sugar moiety. The phosphodiester bonds link purines (guanine and / or adenine) nin) and / or pyrimidine bases (thymine and cytosine for DNA; and sugars linked to uracil and cytosine through glycosidic bonds Residues (either ribose for RNA or deoxyribose for DNA, collectively It is made from furanose.

[0067] The antisense oligonucleotides provided herein may comprise one or more modified It may contain nucleotide subunits and / or internucleoside linkages. Chemical modifications to oligonucleotides can be made at the internucleoside linkages, sugar moieties, nucleobases, Modifications include changes in the length, width, and / or backbone of the antisense oligonucleotide. improve the stability, efficacy, and / or reduce immunogenicity of the For example, the oligonucleotide may have a higher affinity for 5'-methyl-1'-propanediol compared to an unmodified oligonucleotide. , increased resistance to nucleases, enhanced binding affinity to nucleic acid targets, enhanced and / or modifying the cellular uptake and / or the cellular activity of the agonist. can be done.

[0068] In some embodiments, the antisense oligonucleotides provided herein includes naturally occurring phosphodiester and internucleoside linkages. can be used with other phosphorus-containing linkages, e.g., phosphorothioates, phosphotriesters, methylphosphonates, etc. such as phonate or phosphoramidate linkages, or may be modified to non-phosphorus-containing linkages. In some embodiments, the antisense oligonucleotides provided herein contains one or more modified internucleoside linkages. Therefore, the antisense oligonucleotides provided herein contain phosphorothioate linkages. In some embodiments, each nucleoside of the antisense oligonucleotide comprises The internucleoside linkages are phosphorothioate internucleoside linkages.

[0069] In some embodiments, the antisense oligonucleotides provided herein For example, antisense oligonucleotides may contain chemically modified sugar moieties. 2'-modifications in the lanose ring, bridging of non-geminal ring atoms to form bicyclic nucleic acids (BNAs) It may include bridges, replacement of oxygen atoms of the sugar ring with other atoms, or combinations thereof. In some embodiments, each nucleotide of the antisense oligonucleotide is , 2'-modified furanose ring. Exemplary 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), and 2'-ON-methylacetamide (2'-O-NMA). In this embodiment, each nucleotide of the antisense oligonucleotide has a 2'-O- Contains MOE modification.

[0070] In some embodiments, the antisense oligonucleotides provided herein indicates the replacement of a nucleotide at a given position with a modified form of the same nucleotide. For example, a nucleotide (A, G, C, or T) can be replaced by a corresponding hypoxamine. xanthine, 4-acetylcytosine, beta-D-galacrosil eosin, ibuprofen N6-Isopentenyladenine, 1-Methylguanine, 1-Methylinosine, 2 ,2-Dimethylguanine, 2-Methyladenine, 2-Methylguanine, 3-Methylcytosine 5-methylcytosine, N6-adenine, 7-methylguanine, beta-D-mannosine Luqueosin, 2-methylthio-N6-isopentenyladenine, wybutoxosin, It may be replaced by enosine, 2-thiocytosine, or 2,6-diaminopurine.

[0071] In some embodiments, the antisense oligonucleotides provided herein Chemically modified oligonucleotides that reduce the immunogenicity of oligonucleotides For example, oligonucleotides containing 5-methylcytosine or 2'-O-MOE modifications Retidoside has been shown to exhibit reduced 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 contain a 5-methyl group instead of a cytosine. In some embodiments, the antisense oligonucleotides provided herein include cytosine. The oligonucleotide comprises a 2'-O-MOE modification. The antisense oligonucleotides provided herein contain 5-methylcytosine and 2' Includes MOE modification.

[0072] In some embodiments, the antisense oligonucleotides provided herein target MAPT are The oligonucleotide comprises a C6 linker at the 3' end having the following structure:

[0073] [ka] The linker is attached to the 3' end of the oligonucleotide through a phosphate bridge, where R =PO2-O-oligonucleotides (for phosphodiester internucleoside linkages) or R=POS-O-oligonucleotide (for phosphorothioate internucleoside linkages) ) Such a 3'C6 linker blocks the attack of 3'-exonucleases. Therefore, the stability and duration of effect of antisense oligonucleotides can be improved. (For a similar strategy applied to siRNA, see Pan 2005 / 021749. (See FRET). In some cases, the 3' C6 linker is It may also facilitate the synthesis and / or purification of oligonucleotides. In this embodiment, the antisense oligonucleotide targeting MAPT has any of the following structures: It may have a structure.

[0074] [ka]

[0075] In some embodiments, the antisense oligonucleotides provided herein can be used with alternative backbones, e.g., morpholino, locked nucleic acid (LNA), unlocked nucleic acid (UNA), NA), threose nucleic acid (TNA), glycol nucleic acid (GNA), and / or peptide In some embodiments, the nucleic acid provided herein may include a polynucleotide sequence (PNA). The antisense oligonucleotides used are bicyclic oligonucleotides that contain a bridge connecting two carbon atoms of the sugar ring. For example, such BNAs can include nucleosides of the formula "Tethered" structures contain a 4'-CH(CH3)-O-2' bridge connecting the 4'-carbon and the 2'-carbon. In some embodiments, the aryl group may include aryloxyethyl (or "cEt"). The antisense oligonucleotides provided herein have a 4' position of the sugar unit of the nucleoside. and a locked nucleic acid (LNA) that includes a bridge connecting two carbon atoms between the 2' position. Such LNAs include α-L-methyleneoxy (4'-CH2-O-2') LNAs. 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. Pat. 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 No. 6,670,461; WO 98 / 39352 or any other LNA described in WO 99 / 14226. Other suitable LNAs include those described in Braasch et al., Chern. Biol. 8: 1-7, 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; or the LNAs described in Wahlestedt et al., PNAS 97: 5633-5638, 2000.

[0076] Steric Blocking Agents Antisense oligonucleotides bind to target nucleic acids, either DNA or RNA. proteins, transcription factors, splicing factors, ribosomes, and / or translation machinery can sterically block the access of the target nucleic acid to the target nucleic acid, thus preventing the activation of RNAse H. For example, such steric blocking agents can block the start codon of a target. It hybridizes to the surrounding sequences, blocking the branch point sequence of the intron and forming a splice site. Targeting intronic and / or exonic sequences or exon splicing By targeting regulatory sequences such as signaling enhancers, expression of the target protein can be reduced. Steric blocking agents can be used to reduce the amount of esters present in a given compound, which may be either predetermined or predicted. Different amplicons can be designed based on intron-exon boundaries and gene structure. A panel of antisense oligonucleotides can be generated to block the same site. BLAST analysis can be used to detect off-target hybridization for each ASO. This can be done to minimize

[0077] Steric blockers are an endogenous cellular surveillance pathway that recognizes and degrades aberrant mRNAs One such pathway is , nonsense-mediated mRNA decay (NMD), which modulates gene expression This prevents the production of potentially toxic proteins from the mRNA. Defects in NA processing lead to transcriptional regulation when a premature stop codon (PTC) is introduced. This leads to loss of function of the protein and disrupts the open reading frame. The PTC-containing mRNAs bind to the translating ribosome and exon junctions containing the essential NMD factor UPF1. It involves the exchange of information between components of the synthesis complex, and is involved in the endonuclease and exonuclease ASOs can be substrates for NMD, which degrades RNA through the activity of ribozymes. By directing target mRNAs into the NMD pathway, they can cause degradation of target mRNAs. This allows rational design of the sequence of the steric blocking agent by binding it to a specific coding region. exon, intron-exon junction, or other sequences required for proper pre-mRNA processing. The sequence was designed to be complementary to the exon skipping and frame shifting sequences. This can be achieved by introducing a PTC and / or a PTC.

[0078] In some embodiments, the antisense oligonucleotides provided herein target MAPT are The oligonucleotide may be a steric blocker, for example, a nucleic acid salt shown in any one of Tables 2 to 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% %, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) In some embodiments, the nucleic acid sequence is an oligonucleotide having a nucleobase sequence having sequence identity. In the present specification, the antisense oligonucleotides targeting MAPT are The steric blocker includes any one of the nucleic acid base sequences shown in Tables 2 to 8. In some embodiments, the antisense oligonucleotides targeted to MAPT provided herein The oligonucleotide is a nucleic acid having any one of the nucleic acid base sequences shown in Tables 2 to 8. As detailed in the Examples below, the steric blockers comprise target tau constructs. exons involved (e.g., exons 1, 4, 5, 7, 9, 11, 12, 13), MAPT Sequences surrounding the initiation codon, splice acceptors and donors, and splicing branch points , polypyrimidine track-related sequences, or splicing enhancers or inhibitors The target sequence was designed to target the initiation codon and exon 1. may block translation initiation, interfere with splicing and / or ASOs that induce sonskipping induce frameshifting and / or early downstream resulting in the introduction of a stop codon at the 2′ end of the tau gene, resulting in the reduction of MAPT mRNA and / or tau protein. It will lead down.

[0079] Chemical modifications can be incorporated into steric blocking agents to improve their stability, efficacy, and / or Steric blockers can be used at each nucleotide position to improve cellular uptake. or at some selected position. For example, the 2' position of the sugar ring. -Incorporation of modifications (e.g. 2'-O-methoxyethyl, MOE), Locked Nucleic Acids (LNA) Inclusion of and / or backbone modifications (e.g., phosphorothioates) reduces nuclease degradation and / or increase the binding affinity of the antisense oligonucleotide. In addition to sugar and / or backbone modifications, steric blocking agents can be used to interact very well with DNA or RNA. Peptide nucleic acids (PNAs) are oligonucleotides that are made up of different oligomers. It is a mimetic whose nucleobases are linked by amide bonds. The amide backbone is uncharged. Therefore, binding is characterized by a high rate of association and high affinity (Bentin T, Biochem. mistry. 1996; 35:8863-8869; Smulevitch SV, Nat Biotech. 1996; 14:1700-1704. Phosphorodiamidate morpholino oligomers (PMOs or "morpholinos") PMOs, commonly referred to as "PNA analogs," are another uncharged DNA analog. Although they do not bind to complementary targets with the high affinity that characterizes mitosis, they are effectively expressed inside cells. (Summerton J, Antisense Nucleic Acid Drug Development . 1997; 7:187-195; see Corey DR, Genome Biol. 2001; 2:REVIEWS1015).

[0080] In some embodiments, the antisense oligonucleotides provided herein target MAPT are Oligonucleotides are steric blockers that contain 2'-modified nucleotides. The '-modifications are 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-dimethyl 2'-O-dimethylaminopropyl (2'-O-DMAP), 2'-O-dimethylaminoethyloxyethyl 2'-O-DMAEOE, and 2'-ON-methylacetamide (2'-O- In some embodiments, the present invention relates to a method for the preparation of a medicament for use in a medical device. The antisense oligonucleotides targeted to MAPT provided in It is a steric blocking agent with 2'-O-MOE modifications in the subunits.

[0081] In some embodiments, the antisense oligonucleotides provided herein target MAPT are Oligonucleotides are those that contain phosphodiester or phosphorothioate internucleoside bonds. It is a steric blocking agent having a linkage.

[0082] In some embodiments, the antisense oligonucleotides provided herein target MAPT are The oligonucleotides are steric blockers that contain backbone modifications that prevent the binding of RNase H. Such steric blocking agents include modified internucleoside linkages, e.g., methyl phosphatase. Sulfonate linkage, methylphosphonothioate linkage, phosphoromorpholidate linkage, phospho Some embodiments may include a piperazidate linkage or a phosphoramidite linkage. In the embodiment, every other internucleoside linkage is a 2' lower alkyl moiety (e.g., C 1-C4, linear or branched, saturated or unsaturated alkyl, e.g., methyl, ethyl, ethyl thenyl, propyl, 1-propenyl, 2-propenyl, and isopropyl) or It may also contain modified phosphate esters having combinations thereof. In an embodiment, the antisense oligonucleotides targeted to MAPT provided herein The otides may be one or more modified amines as described in U.S. Pat. No. 5,149,797. It is a steric blocking agent containing an internucleoside linkage.

[0083] In some embodiments, the MAPT-targeting steric blockade provided herein The agent comprises a C6 linker at the 3' end having the structure:

[0084] [ka] The structure is attached to the 3' end of an oligonucleotide through a phosphate bridge, wherein R=PO2-O-oligonucleotide (for phosphodiester internucleoside linkages) or R=POS-O-oligonucleotide (with phosphorothioate internucleoside linkages) Therefore, MAPT-targeting phosphodiester nucleosides The steric blocker having a linkage can have the following structure:

[0085] [ka] MAPT-targeting steric blocking agents with phosphorothioate internucleoside linkages can have the following structure:

[0086] [ka]

[0087] Gapmar Antisense oligonucleotides containing continuous stretches of DNA target the end of cells. The nuclease RNaseH is recruited to the target RNA:DNA heteroduplex, and the RNA:DNA A gapmer is a chimeric antisense molecule that can cleave the target RNA in the duplex. Chimeric antisense compounds are typically compounds that are resistant to nuclease degradation. increased tolerance, increased cellular uptake, increased binding affinity to target nucleic acids, and / or or at least one region modified to confer increased inhibitory activity, and The second region contains nucleotides that are chemically different from the nucleotides in the first region.

[0088] Gapmers are two gapmers consisting of modified nucleotides at the 5' and 3' ends. It consists of a stretch of consecutive 2'-deoxyribonucleotides located between the fragment segments. The gap segment is a central gap segment that is Serve as a substrate for seH cleavage, but have modified nucleotides The wing segments confer increased resistance to degradation by other nucleases. -Gap-wing segments can also be written as "XYZ", where "X" is "Y" represents the length of the gap, and "Z" represents the length of the 3' wing. "X" and "Z" represent the length of the saccharide. "X" and "Z" can include uniform, mutated, or alternating sugar moieties. It is also possible.

[0089] In some embodiments, the central gap segment of a gapmer comprises at least Five (e.g., 5, 6, 7, 8, 9, 10, 11, 12) consecutive 2'-deoxyribonucleic acid residues the 5' and 3' wing segments are each composed of one or more 2' -Contains modified nucleotides. Up to 4 consecutive 2'-deoxyribonucleotides It has been reported that chimeric oligonucleotides containing a stretch of See U.S. Patent No. 9,157,081. In one embodiment, the present invention provides an antisense oligonucleotide targeting MAPT. The tide is a sequence of at least seven (e.g., 7, 8, 9, 10, 11, or 12) consecutive 2'- In some embodiments, the present invention is a gapmer that includes deoxyribonucleotides. The antisense oligonucleotides targeted to MAPT provided herein comprise 10 A gapmer containing consecutive 2'-deoxyribonucleotides. The 2'-modification is '-Fluoro, 2'-deoxy-2'-fluoro, 2'-O-methyl, 2'-O-methoxy Diethyl (2'-O-MOE), 2'-O-aminopropyl (2'-O-AP), 2'- O-Dimethylaminoethyl (2'-O-DMAOE), 2'-O-Dimethylaminopropyl 2'-O-dimethylaminoethyloxyethyl (2'-O-DMAP) DMAEOE), and 2'-ON-methylacetamide (2'-O-NMA). The compound can be selected from the group consisting of:

[0090] In some embodiments, the MAPT targeting gapmer is a 20 nucleotide A 5-10-5 gapmer with a length of 10, where the central gap segment is 0 2'-deoxynucleosides, 5 nucleosides each having a 2'-modification and 5' and 3' wing segments each containing: 5-9-5 gapmer, 5-8-5 gapmer, 4-8-6 gapmer, 6- including, but not limited to, an 8-4 gapmer, or a 5-7-6 gapmer.

[0091] In some embodiments, the antisense oligonucleotides provided herein target MAPT are The oligonucleotide may be a gapmer, for example, any of those shown in Tables 9-15 and 17. or 0%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 9 0%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or or 100%) sequence identity. In some embodiments, the antisense oligonucleotides targeted to MAPT provided herein The oligonucleotide may be any of the nucleic acid base sequences shown in Tables 9 to 15 and 17. In some embodiments, the MA provided herein is a gapmer comprising: The antisense oligonucleotide targeting PT is any one of Tables 9 to 15 and 17. A gapmer having any of the nucleic acid base sequences shown in the following examples is described in detail below. Thus, gapmers are sequences surrounding the start codon, exon 1, or the MAPT transcript. In some embodiments, the 3′ untranslated region (UTR) of Thus, gapmers were designed to target the 3'UTR.

[0092] In some embodiments, the antisense oligonucleotides provided herein target MAPT are The oligonucleotide has any one of the nucleic acid base sequences shown in Tables 9 to 15 and 17. or a 5-10-5 gapmer comprising either , each of which contains a nucleoside modified with 2'-O-MOE, and the 6th to 15th nucleosides Nucleotides 16 to 20 each contain a 2'-deoxynucleoside. Each contains a nucleoside modified with -O-MOE.

[0093] MAPT genomic sequences targeted by antisense oligonucleotides In some embodiments, the antisense oligonucleotide is Specific regions of the sequence (GenBank accession number NG_007398.1; SEQ ID NO: 304) or a region of the corresponding tau mRNA or transcript (SEQ ID NO: 306). The intron and exon sequences and branch points of MAPT have been calculated using Ensemb Based on the genome database website, transcript: MAPT-203 ENST0 0000344290 was used to determine the exons and introns of the human MAPT gene. , and screening of intron / exon junctions identified MAPT genes or transcription factors Targeting certain regions in a gene with antisense oligonucleotides is a promising approach to targeting certain regions in a gene. was found to be more effective at reducing tau expression than targeting the For example, Table 1 shows that antisense oligonucleotides were used to Listed below are the sequences of some preferred regions that can be targeted by leucine.

[0094] In some embodiments, the antisense oligonucleotides provided herein Any one of SEQ ID NOs: 487 to 506 having 1, 2, or 3 mismatches. At least 12 consecutive nucleobases (e.g., 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 7, or 18 consecutive nucleobases) of a nucleic acid sequence that is complementary to In embodiments, the antisense oligonucleotides provided herein are At least 12 consecutive nucleic acid bases (e.g., 12, 13, 14, 15, 16, 17, or 18 consecutive nucleobases) It contains a certain nucleic acid base sequence.

[0095] [Table 1]

[0096] Antisense Oligonucleotide Conjugates Conjugation of the antisense oligonucleotide to another moiety can be used to Improve the activity, cellular uptake, and / or tissue distribution of oligonucleotides For example, the antisense oligonucleotide may be coupled to one or more diagnostic compounds, Reporter groups, cross-linkers, moieties that confer nuclease resistance, lipophilic molecules, cholesterol le, lipids, lectins, linkers, steroids, uvaol, hesigenin, diosgenin, Terpenes, triterpenes, sarsasapogenin, friedelin, epifriedelanol derivatives Solubilized lithocholic acid, vitamins, biotin, carbohydrates, dextran, dyes, pullulan, Chin, chitosan, synthetic carbohydrates, oligolactate 15-mer, natural polymers, low or medium Molecular weight polymers, inulin, cyclodextrin, hyaluronic acid, proteins, proteins Protein binding agents, integrin targeting molecules, polycations, peptides, polyamines, peptidomimetics Body, Transferrin, Coumarin, Phenazine, Folate, Phenanthridine, Ant Covalently linked to quinone, acridine, fluorescein, and / or rhodamine It is possible.

[0097] In some embodiments, the antisense oligonucleotides provided herein is attached to a linker molecule. The antisense oligonucleotides are linked to lipids or cholesterol. In some embodiments, the antisense oligonucleotide is delivered in a neutral liposome (NL) or lipid nanoparticles (LNPs). LNPs are self-assembling cationic lipids. Substrate-based systems, which include, for example, neutral lipids (liposome base); cationic Lipids (for oligonucleotide loading); cholesterol (for stabilizing liposomes) ); and PEG-lipids (for formulation stabilization, charge shielding, and enhanced circulation in the bloodstream) Neutral liposomes (NL) are particles based on non-cationic lipids. It is.

[0098] In some embodiments, the antisense oligonucleotides provided herein is linked to a fatty acid, e.g., an omega-3 fatty acid or an omega-6 fatty acid. The important omega-3 fatty acids are, for example, alpha-linolenic acid (ALA), docosahexaenoic acid (DH A), eicosapentaenoic acid (EPA), docosapentaenoic acid (DPA), eicosate Eicosapentaenoic acid (ETA), Eicosatrienoic acid (ETE), Eicosapentaenoic acid (EPA) , hexadecatrienoic acid (HTA), heneicosapentaenoic acid (HPA), stearyl These include tetracosapentaenoic acid (SDA), tetracosapentaenoic acid, and tetracosahexaenoic acid.

[0099] Testing for antisense oligonucleotide activity The activity of antisense oligonucleotides can be tested in vitro or in vivo. For in vitro testing, ASOs can be transfected into cultured cells. After a period of treatment, the ASO-treated cells The expression levels of MAPT (tau) in the cells were determined to compare the expression levels of MAPT in untreated control cells. (tau) expression levels can be compared.

[0100] The MAPT expression level can be measured by any suitable method, for example, by measuring the level of MAPT mRNA. The amount of MAPT mRNA produced by reverse transcription was measured by quantitative quantification of the The amount of tau protein can be determined by measuring the amount of tau protein. The method may be performed on a sample by sample basis or modified for high throughput analysis. This can be done.

[0101] The levels of MAPT mRNA were determined by hybridization specifically to a segment of the MAPT transcript. The corresponding probes can be used to detect and quantitate the protein, for example, by Northern blot analysis. The levels of MAPT mRNA were measured by polymerase chain reaction (PCR). It can also be detected and quantified using a pair of primers that recognize the PT transcript. The general procedure for CR is described in MacPherson et al., PCR: A Practical Approach, (IRL Pre ss at Oxford University Press (1991). However, each applied reaction The PCR conditions used for the amplification are determined empirically. Many parameters, e.g. Annealing temperature and time, weight gain time, Mg 2 + and / or ATP concentration, pH, and the relative concentrations of primers, templates, and / or deoxyribonucleotides. After amplification, the resulting DNA fragments are analyzed by agarose gel electrophoresis. Detection is by kinetic analysis followed by visualization using ethidium bromide staining and ultraviolet illumination. This can be done.

[0102] In some embodiments, the level of MAPT mRNA can be measured using any commercially available real-time Immuno-PCR systems are used to simultaneously incorporate a detectable dye or reporter during the amplification step. By incorporating the nucleotide sequence at specific times, the amplification of the target nucleic acid is monitored by quantitative real-time PCR. can be detected and quantified.

[0103] Alternatively, the labeling may be carried out on the original nucleic acid sample (e.g., mRNA, polyA, mRNA, cDNA, The label may be attached directly to the nucleic acid (e.g., nucleic acid fragment) or to the amplification product after amplification is complete. Means for attaching to nucleic acids are well known to those of skill in the art and include, for example, kinasing of the nucleic acid and subsequent Attachment of a nucleic acid linker (ligation) that joins the sample nucleic acid to a label (e.g., a fluorophore) nick translation or end labeling (e.g., using labeled RNA) (including

[0104] Detectable labels suitable for use in the present invention include spectroscopic, photochemical, biochemical, immunological, and This includes any composition detectable by epidemiological, electrical, optical or chemical means. A clear and useful label is a marker for staining with a labeled streptavidin conjugate. Biotin, magnetic beads (e.g., Dynabeads™), fluorescent dyes (e.g., Fluorescein, Texas Red, Rhodamine, Green Fluorescent Protein, etc.), radioactive labeling (e.g., 3H, 125I, 35S, 14C, or 32P), enzymes (e.g., horseradish peroxidase Biperoxidase, alkaline phosphatase and other enzymes commonly used in ELISA and colorimetric labels, such as colloidal gold or colored glass or plastic. Such materials include polymeric (e.g., polystyrene, polypropylene, latex, etc.) beads. Patents teaching the use of labels include U.S. Pat. Nos. 3,817,837; 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 specification No. 1.

[0105] Detection of labels is well known to those of skill in the art. Thus, for example, radioactive labels can be detected by a photographic filter. Fluorescent markers can be detected using a microscope or scintillation counter. The enzyme label can be detected using a photodetector to detect the emitted light. Providing a substrate to an enzyme and detecting the production of a reaction product by the action of the enzyme on the substrate. The label is detected by a fluorescent probe, and the colorimetric label is simply detected by visual detection of the colored label. For a detailed review of methods for detecting labeled hybridized nucleic acids, see Lab 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 reduces tau protein levels by a number of methods known in the art. It can also be assessed by measuring tauta using methods known in the art. Protein levels were assessed by Western blot analysis (immunoblotting), enzyme-linked immunosorbent assay (ELISA), and immunoassay. ELISA, immunohistochemistry, immunoassay, immunoprecipitation, immunofluorescence assay, immunoassay Immunocytochemistry, Fluorescence Activated Cell Sorting (FACS), Radioimmunoassay, Immunoradioassay, Rapid Liquid chromatography (HPLC), mass spectrometry, confocal microscopy, enzyme assays, can be quantified by surface plasmon resonance (SPR).

[0107] The in vivo activity of antisense oligonucleotides can be tested in animal models. Testing can be performed in normal animals or in animal models of experimental disease. The transsense oligonucleotides can be formulated in a pharma- ceutically acceptable diluent and can be used in a suitable After a period of treatment, tissue samples, e.g., brain tissue, cerebrospinal fluid, (CSF) and spinal cord were collected and tau expression levels were assayed using one of the methods described above. Histological analysis can be used to assess brain structure and / or the presence of neurofibrillary tangles. The present invention can be performed to assess the presence of a phenotypic change in treated animals, e.g. Improved cognition or mobility can also be monitored and assessed.

[0108] Oligonucleotide synthesis and characterization The single stranded oligonucleotide can be polymerized by 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; SL Beaucage and RP Iyer, Tetrahedron, 1992, 48, 2223) They can be synthesized using H-phosphonate, phosphortriester chemistry, or enzymatic synthesis. Automated synthesizers available commercially, such as BioAutomation (I rving, TX), or Applied Biosystems (Foste A synthesizer from the company AB (Richard City, Calif.) can be used. In this embodiment, the single-stranded oligonucleotide can be prepared by the method described in, for example, Nucleic Acid Chemistry, Beauca ge, SL et al. (Edrs.), Current Pr of John Wiley & Sons, Inc., New York, NY, USA. The phosphatase was generated using standard solid-phase phosphoramidite chemistry as described in protocols. The holothioate linkage was prepared using phenylacetyl disulfide or DDTT (((dimethylacetamide). (amino-methylidene)amino)-3H-1,2,4-dithiazoline-3-thione and other sulfur compounds Modified amidites can be introduced using similar techniques and commercially available modified amidites. and controlled-pore glass (CPG) products, e.g. For example, amidines modified with biotin, fluorescein, acridine, or psoralen may be used. The peptides and / or CPGs are coupled to modified oligonucleotides or fluorescently labeled, Used to synthesize conjugated oligonucleotides with thiocyanates or other This is well known.

[0109] Control of the quality of the starting materials and products from each synthesis step is essential to minimize impurities in the final product. However, it is extremely important to minimize the synthesis level per coupling. Given the number of steps and couplings, the presence of impurities is inevitable. The method is used to eliminate undesirable impurities from the final oligonucleotide product. A commonly used purification technique for single-stranded oligonucleotides is reverse phase isopropanol. on-pair high performance liquid chromatography (RP-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 spectrophotometrically by wavelength.

[0111] Therapeutic Uses and Methods Tau expression levels in a subject, e.g., a human, can be determined by administering an antisense oligonucleotide to the subject, such as an antisense oligonucleotide, as described herein. and a method for reducing the level of schizophrenia by administering a therapeutically effective amount of any one of the oligonucleotides. In some embodiments, an antisense oligonucleotide is provided. The drug is administered to the subject via intrathecal, intracranial, intranasal, intravenous, oral, or subcutaneous routes. In some embodiments, such methods include administering to a patient suffering from a tau-related disease. The method further includes identifying and selecting a subject who has or is susceptible to the disease.

[0112] The antisense oligonucleotides or pharmaceutical compositions thereof provided herein are It can be used to treat or prevent a tau-related disease in a subject. In some embodiments, the present invention provides an antisense oligonucleotide comprising the antisense oligonucleotides described herein. or a pharmaceutical composition thereof for the treatment or prevention of a tau-related disease in a patient. In a further embodiment, the present invention provides a method for treating tau in a patient. In the manufacture of a medicament for use in the treatment or prevention of a related disease, The present invention provides uses of antisense oligonucleotides.

[0113] Tau-related diseases include Alzheimer's disease (AD), amyotrophic lateral sclerosis / Parkinson disease, and ALS-PDC, Argyrophilic grain dementia (AGD), British Amino cerebrovascular disease, cerebral amyloid angiopathy, chronic traumatic encephalopathy (CTE), corticobasal degeneration (CBD), Creutzfeldt-Jakob disease (CJD), Punch-Drunk Disease, Calcification Diffuse neurofibrillary tangles, Down syndrome, Dravet syndrome, epilepsy, frontotemporal dementia (F TD), frontotemporal with parkinsonism linked to chromosome 17 (FTDP-17) dementia, frontotemporal lobar degeneration, ganglioglioma, gangliocytoma, Gerstmann-Straussler - Scheinker's disease, Hallervorden-Spatz disease, Huntington's disease, inclusion body myositis, lead encephalopathy disease, Lytico-Bodig disease, meningioma hemangiomatosis, multiple system atrophy, myotonic dystrophy, Niemann-Pick disease type C (NP-C), non-Guamanian motor neuron disease with neurofibrillary tangles, Pick's disease (PiD), postencephalitic parkinsonism, prion protein, cerebral amyloidosis progressive subcortical gliosis, progressive supranuclear palsy (PSP), subacute sclerosing panencephalitis, neurological Tangle-only dementia, Neurofibrillary tangle-predominant dementia le-predominant dementia, multi-infarct dementia, ischemic stroke, and tuberous sclerosis Including, but not limited to:

[0114] Combination Therapy The various oligonucleotides described above may be used in combination with other therapeutic partners. Thus, the methods of treating tau-related diseases described herein can further include administering a second agent to a subject in need of treatment. For example, antisense oligonucleotides targeting microtubule-associated protein tau (MAPT) have been The oligonucleotide is an antibody that specifically binds to tau protein and / or an amyloid vector. Agents that target Aβ or beta-secretase (BACE) In some embodiments, the antibody may be used in combination with an antibody that binds to the inhibitor. Therefore, antisense oligonucleotides targeting MAPT specifically bind to tau protein. In some embodiments, the antibody is used in combination with an antibody that binds to MAPT. The antisense oligonucleotides are used in combination with a BACE inhibitor.

[0115] The term "combination" refers to a fixed combination in one unit dosage form or to a combination of the compounds of the present invention and A combination partner (e.g., another drug, a "therapeutic agent," or a "co-agent," as described below) (also called agonists) may act independently, simultaneously, or in combination with other combination partners that exert a cooperative, e.g., synergistic, effect. Refers to any combination administration that may be administered separately within a time interval that allows The individual components may be packaged in a kit or packaged separately. One or both of the formulations (e.g., powder or liquid) may be reconstituted or diluted to the desired dose prior to administration. As used herein, the terms "co-administration" or "administration in combination" and the like refer to a selection and administering the combination partners to a single subject (e.g., a patient) in need thereof. It is meant to encompass all of the above, provided that the active substances are administered by the same route of administration or simultaneously. As used herein, the term "treatment regimen" is intended to include treatment regimens that are not necessarily "Pharmaceutical combination" means a product resulting from the mixing or combining of two or more therapeutic agents; The term "fixed combination" includes both fixed and non-fixed combinations of agents. " refers to a combination partner that is a therapeutic agent, e.g., an oligonucleotide of the invention, and a combination partner that is a The term "unfixed" refers to a single entity or dosage form that is administered simultaneously to a patient. A "combination" is a combination in which a therapeutic agent, e.g., an oligonucleotide of the invention and a combination partner are Both are identified to the patient as separate entities, either simultaneously, in parallel, or sequentially. and such administration means that the above-mentioned The latter provides therapeutically effective levels of two compounds. This also applies to the administration of the above therapeutic agents.

[0116] As used herein, the term "pharmaceutical combination" refers to a combination fixed in one unit dosage form. or two or more therapeutic agents are administered independently, simultaneously or separately within a time interval. In particular, these time intervals may be used when the combination partners have a cooperative, e.g. synergistic, effect. A kit of parts for administering a combination or combination that can be used to This refers to one of the following:

[0117] The term "combination therapy" refers to a combination of two or more therapeutic agents for treating a therapeutic condition or disorder described in this disclosure. Such administration refers to the administration of one or more therapeutic agents in a substantially simultaneous manner, e.g., effectively This involves co-administration of these therapeutic agents in a single capsule with a fixed ratio of ingredients. Alternatively, such administration may be in multiple or separate containers for each active ingredient (e.g. This includes co-administration in various dosage forms (tablets, capsules, powders, and liquids). The liquid formulation can be reconstituted or diluted to the desired dose prior to administration. Such administration may be in a sequential manner, either at about the same time or at different times. In either case, the treatment regimen includes the use of any of the types of therapeutic agents described herein. The combination of drugs may provide a beneficial effect in treating a given condition or disorder.

[0118] Sample preparation Tissue samples are probed with antisense oligonucleotides using any method known in the art. The nucleic acid sequence can be obtained from the subject to be treated with the nucleic acid, for example, by biopsy or surgery. For example, a sample containing cerebrospinal fluid is collected by inserting a thin needle attached to a syringe into the spinal canal at the lumbar region. Once inserted, a vacuum is 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 are used to obtain this type of procedure. It can be used to guide the order.

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

[0120] Pharmaceutical Compositions, Dosages, and Administration Compositions, such as one or more of the antisense oligonucleotides provided herein, Also provided herein are pharmaceutical compositions that contain the tide. Pharmaceutical compositions typically contain pharma- ceuticals that are pharma- ceutical acceptable. As used herein, the term "pharmacologically acceptable carrier" includes a pharma- ceutically acceptable carrier. The present invention provides a pharmaceutical composition that includes physiological salines, solvents, dispersion media, coatings, antibacterial agents, and antifungal agents that are compatible with pharmaceutical administration. Antifungal agents, isotonic and absorption delaying agents, etc. Pharmaceutical compositions typically contain the active ingredient(s) for their intended purpose. It is formulated to be compatible with the route of administration. Examples of routes of administration include intrathecal, intracranial, intranasal, This includes intravenous, intravenous, oral, or subcutaneous administration.

[0121] In some embodiments, the antisense oligonucleotides described herein can be conjugated to an antibody capable of crossing the blood-brain barrier (e.g., Binds to the transferrin receptor, insulin, leptin, or insulin-like growth factor 1 (Antibody that targets the inflammatory bowel disease) 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, minington: The Science and Practice of Pharmacy. 21st ed., 2005; and Drugs and e Pharmaceutical Sciences: a Series of Textbooks and Monographs (Dekker, NY). See, e.g., Leeds, for parenteral, intradermal, intrathecal, or subcutaneous administration. The solutions or suspensions used may contain the following components: a sterile diluent, for example, water for injection; Saline solution, fixed oil, polyethylene glycol, glycerin, propylene glycol alcohol or other synthetic solvents; antibacterial agents, such as benzyl alcohol or methylparaben; Ben etc.; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates, etc.; and isotonicity adjusting agents, e.g., sodium chloride or dextrose, etc. The pH can be adjusted using an acid or base such as hydrochloric acid or sodium hydroxide. Parenteral preparations may be packaged in ampoules, disposable syringes or glass or It can be packaged in plastic multi-dose vials.

[0123] Pharmaceutical compositions suitable for injectable use may be prepared as sterile aqueous solutions (where water soluble) or dispersions. and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL ( (BASF, Parsippany, NJ) or phosphate-buffered saline. In all cases, the composition must be sterile and easily disposable. The composition should be stable under the conditions of manufacture and storage. and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier may be, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, recall, and liquid polyethylene glycol, etc.), and suitable mixtures thereof. The appropriate fluidity may be, for example, a solvent or dispersion medium such as lecithin. By using coatings, by maintaining the required particle size in the case of dispersions, and Prevention of microbial action can be achieved by the use of various antibacterial agents and and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, This can be achieved by the use of isotonicity agents such as merosal. Often, the composition will contain isotonicity agents such as , sugars, polyalcohols such as mannitol, sorbitol, sodium chloride, etc. It would be preferable to include a medicament for injectable composition that has a long absorption time. Time-delaying agents, such as aluminum monostearate and gelatin, may be included. It can be brought about by.

[0124] Sterile solutions for injection may be prepared by mixing the active compound in the required amount in an appropriate solvent such as one or more of the above listed solvents. The composition may be prepared by incorporating the above-mentioned components in a combination, followed by sterilization by filtration, if necessary. This can be done.

[0125] Generally, the dispersion will consist of a basic dispersion medium and any other components required from those listed above. The injection is prepared by incorporating the active compound into a sterile vehicle containing the ingredient. In the case of sterile powders for preparation of bacterial solutions, the preferred methods of preparation are vacuum drying and freeze drying. These methods include adding any additional desired ingredients to the active ingredient, and This produces a powder from the sterile filtered solution.

[0126] Oral compositions generally include an inert diluent or an edible carrier. For this purpose, the active compound may be incorporated with excipients and formulated into tablets, troches, or capsules. Oral compositions may be used in the form of capsules, e.g., gelatin capsules. For use in pharmaceutical compositions, the compositions may also be formulated using a fluid carrier. and / or adjunct ingredients may be included as part of the composition. The lozenges and the like can contain any of the following ingredients, or compounds of a similar nature: a binder, e.g., microcrystalline cellulose, Crystalline cellulose, gum tragacanth or gelatin; excipients, e.g., starch or lactose; disintegrants, such as alginic acid, Primogel, or tow sorghum starch, etc.; lubricants, such as magnesium stearate or Stero glidants, such as colloidal silicon dioxide; sweeteners, such as sucrose; or saccharin; or flavoring agents, e.g., peppermint, methyl salicylate , or orange flavoring, etc.

[0127] For administration by inhalation, the compounds are administered in a suitable propellant, e.g., a gas such as carbon dioxide. The aerosol is sprayed from a pressurized container or dispenser or nebulizer containing the Such a method can be delivered in the form of a capsule. Systemic administration of the therapeutic compounds described herein includes: It may also be by transmucosal or transdermal means. For transmucosal or transdermal administration, the barrier must be penetrated. A penetrant appropriate for permeating the blood is used in the formulation. Such penetrants are well known in the art. For example, for transmucosal administration, detergents, bile salts, and fluorescein are commonly known. Transmucosal administration is accomplished by use of nasal sprays or suppositories. For transdermal administration, the active compound can be administered in any suitable manner as generally known in the art. It may be formulated into an ointment, salve, gel, or cream.

[0128] In one embodiment, the therapeutic compound is adapted to inhibit rapid elimination from the body. The use of carriers that protect the subject, including implants and microencapsulated delivery systems In addition, the drug is prepared as a controlled release formulation.

[0129] The pharmaceutical compositions may be included in a container, pack, or dispenser with instructions for administration. can be included together.

[0130] In a non-limiting example, a pharmaceutical composition containing at least one pharmaceutical agent is provided in a liquid form (e.g., as solid components (e.g., powders or biodegradable biocompatible polymers) Polymers (e.g., cationic biodegradable biocompatible polymers) or in the form of gels In some embodiments, the composition is formulated as a component (e.g., a biodegradable, biocompatible polymer). In the method, at least one composition containing at least one pharmaceutical agent is an alginate gel ( e.g., sodium alginate), cellulose-based gels (e.g., carboxymethyl cellulose cellulose or carboxyethylcellulose), or chitosan-based gels (e.g., chitosan The composition is formulated as a gel selected from the group consisting of glycerophosphates, glycerophosphates, and the like. Additional examples of drug eluting polymers that may be used to formulate any of the pharmaceutical compositions include: Carrageenan, carboxymethylcellulose, hydroxypropylcellulose, polyvinyl Dextran combined with polyacrylic acid, dextran combined with polyacrylic acid Orchid, polygalacturonic acid, galacturonic polysaccharide, polylactic acid, polyglycolic acid, tamarin Do gum, xantham gum, cellulose gum, guar gum (carboxymethyl guar), pe Cutin, polyacrylic acid, polymethacrylic acid, N-isopropyl polyacrylamide, poly Polyoxyethylene, polyoxypropylene, pluronic acid, polylactic acid, cyclodextrin , cycloamylose, resilin, polybutadiene, N-(2-hydroxypropyl)meth Acrylamide (HPMA) copolymer, maleic anhydride-alkyl vinyl ether, poly Depsipeptide, polyhydroxybutyrate, polycaprolactone, polydioxanone, Polyethylene glycol, polyorganophosphorazene, polyorthoester, polyvinyl Pyrrolidone, polylactic-co-glycolic acid (PLGA), polyanhydride, polysilane These include, but are not limited to, poly(N-vinylcaprolactam), poly(N-vinylcaprolactam), and gellan. stomach.

[0131] In some embodiments, delivery of antisense oligonucleotides to a target tissue comprises , cationic liposomes, cyclodextrins, porphyrin derivatives, branched dendrimers -, Polyethylenimine Polymers, Nanoparticles and Microspheres (Dass CR. J Pharm Pharmacal 2002; 54(1):3-27) .

[0132] An "effective amount" is an amount sufficient to effect beneficial or desired results. A therapeutic amount is an amount that achieves the desired therapeutic effect. This amount prevents the onset of a disease or prevents disease symptoms. It may be the same as or different from a prophylactically effective amount, which is the amount needed to prevent. The amount may be administered in one or more administrations, applications or dosages. The therapeutically effective amount (i.e., effective dosage) of the compound will depend on the choice of therapeutic compound. The composition may be administered in multiple doses, including once or multiple times per day to once per week or once every other day. Those skilled in the art will recognize that a therapeutic approach can be used based on the severity of the disease or disorder, previous treatments, the general health of the subject, and other factors. Certain factors, including but not limited to, the patient's health and / or age, and other existing diseases, may play a role. It has been found that factors such as the amount and timing of the drug required to effectively treat a subject can affect Moreover, it will be appreciated that the therapeutically effective amounts of the therapeutic compounds described herein may be used Treatment of a subject can include a single treatment or a series of treatments.

[0133] Dosage, toxicity and therapeutic efficacy of therapeutic compounds are determined in cell cultures or experimental animals. For example, LD50 (the dose lethal to 50% of the population) and ED50 (the The therapeutically effective dose can be determined by standard pharmaceutical procedures. The dose ratio between toxic and therapeutic effects is the therapeutic index and it is expressed as the ratio LD50 / ED50. Compounds that exhibit high therapeutic indices are preferred. Compounds that exhibit toxic side effects are Sometimes, other agents are used, but they should be used in a way that minimizes the chance of damaging uninfected cells. This allows for targeting of such compounds to the site of affected tissue to reduce side effects. Care should be taken to design the delivery system.

[0134] for human use using data obtained from cell culture assays and animal studies Dosage ranges of such compounds can be formulated with little to no toxicity. Dosages should preferably be within a range of circulating concentrations that include the ED50, but not necessarily all of the above. It may vary within this range depending on the dosage form used and the route of administration utilized. For any compound used in the method, a therapeutically effective dose is first determined by cell culture assays. The dose can be estimated based on the IC50 (i.e., the maximum dose at which symptoms are observed) determined in cell culture. Achieve a circulating plasma concentration range that includes the concentration of the test compound that achieves half the inhibition of Such information can be used in animal models to develop therapeutic options that are useful in humans. This can be used to determine the dosage more accurately. It can be measured by high performance liquid chromatography.

[0135] In some embodiments, the antisense oligonucleotides described herein For administration, sterile water, saline (e.g., phosphate buffered saline), or intracerebral In some embodiments, the antibodies described herein are dissolved in spinal fluid (CSF). The antisense oligonucleotides are delivered intrathecally, e.g., into the L3 or L4 disc space. It is given by bolus injection or by infusion using an intrathecal pump.

[0136] In some embodiments, about 0.001 to 1000 mg (e.g., about 0.1 to 80 0mg, approx. 1-600mg, approx. 10-500mg, approx. 50-450mg, approx. 80-300 mg, about 100-200 mg) of the antisense oligonucleotide described herein. is administered to a subject in need thereof.

[0137] kit One or more of the antisense oligonucleotides described above and instructions for use. Also provided is a kit comprising the method for diagnosing or treating a tau-related disease. The kits provided herein can include any of the methods described herein. The kits provided herein can be used in accordance with the methods described herein. The appropriate use of the kit is known and the kit may be used for such purposes. The kits provided herein are used to return samples, e.g., to a laboratory, for analysis. A mailing container (e.g., a postage-paid envelope or mailing pack) that can be used to The kit may include one or more containers for the samples, Alternatively, the sample can be in a standard blood collection vial. A number of informed consent forms, study request forms, and methods described herein were used Such kits may include instructions on how to use the kit. Methods for use are also included herein. The sample container may be labeled with, for example, a barcode to identify the subject who provided the sample. It can be coded in

[0138] Those skilled in the art will recognize many methods and materials similar or equivalent to those described herein. Those skilled in the art will recognize and be able to use them in the practice of the present invention. It is in no way limited to the methods and materials described in the document. EXAMPLES

[0139] The invention is further described in the following examples, which are provided to illustrate the invention as defined in the claims. Does not limit the scope of clarity.

[0140] [Example 1] General Materials and Methods Synthesis and purification of antisense oligonucleotides The modified antisense oligonucleotides described in this invention are similar to standard phosphoramidites. Mermade 192 Syn for in vitro use using midite chemistry thesizer (BioAutomation) and Merm for in vivo purposes The phosphoramidites were prepared using an ade12 kit (BioAutomation). Dissolved in nitrile at 0.15M (0.08M in Mermade 192); The coupling was performed with a 0.5 M solution of 5-ethylthiotetrazole in acetonitrile (Mer This is done by activating phosphoramidites with 0.25M fluoride (192). Coupling times were typically between 3 and 4 minutes. Sulfurization was carried out using phenylacetyl disulfide. The oxidation was carried out using a 0.2 M solution of phenylalanine in pyridine for 5 min. 2M iodine solution (20%) / water (9.5%) / tetrahydrofuran (70.5%) Capping was performed using standard capping reagents. The growing chain of oxodides was detrimented with 3% dichloroacetic acid in toluene for the next coupling. After completion of the sequence, the support-bound compound was cleaved and cooled to 100° C. The resulting crude solution was immediately purified by HPLC (Akta HPLC). The purified fraction was analyzed by mass spectrometry. The fractions were quantified by UV according to their extinction coefficients in nM. and freeze-dried.

[0141] In vitro testing of antisense oligonucleotides Human cell lines, such as Huh7 cells, Hela cells, and SH-SY5Y cells, The amyloid plaques were expressed in various cell lines, including, but not limited to, COS1 and COS1 green monkey cell lines. Antisense oligonucleotides were tested in vitro. Cells were purchased from commercial suppliers (e.g., American Type Culture Collection (ATCC), M The cultures were obtained from Biochem., Inc. (Anassas, VA) and cultured according to the manufacturer's instructions.

[0142] Antisense oligonucleotides were purchased from Sigma-Aldrich, Inc., Madison, WI, USA. Human embryonic stem cells obtained from WiCell Research Institute, Inc. We also tested human embryonic stem cell (hESC)-derived human neurons. hESCs were transfected with neurogenin-2 (N gn2), forced expression of a neuronal lineage-specific transcription factor induces functional neuronal cell Constitutive expression of rtTA and tetO promoter-driven Using lentiviral delivery for tetracycline-inducible expression of exogenous proteins The Ngn2 construct was delivered to hESCs by the recombinant human ES cell line, Sigma-Aldrich. al Council Institute of Medicine of theN The Guidelines established by the National Academies for human Embryonic Stem Cell Research NAS guidelines and the Office for Human Subjects Protection Research Protections, Department of Heal Department of Federal and Human Services ("DHHS") regulations (4 C.F.R. This standard complies with Part 1Q of Volume 5.

[0143] Antisense oligonucleotides were administered to cultured cells until the cells were approximately 60-80% complete. When fluency is reached, either transfection or nucleofection can be performed. For transfection, antisense oligonucleotides were used. The transfection reagent was OptiFect™ Transfection Reagent (catalog available from Life Tech) The desired antisense oligonucleotide is mixed with the desired nucleotide sequence (DNA fragment number 12579-017) in an appropriate cell culture medium. Oligonucleotide concentration and 2 to 100 nM antisense oligonucleotide OptiFect™ concentrations in the range of 12 μg / mL were achieved. Nucleofection To achieve this, antisense oligonucleotides were injected into neuroblastoma SH-SY5Y cells. Amaxa Nucleofector-II device (Lonza, Walkersv To test the efficacy of ASOs, Preliminary experiments showed high viability and Select Nucleofector Solution SF based on its transfection efficiency and On the day of nucleofection, 60-80% confluent cultures were trypsinized. hESC-derived human neurons were cultured in one or two wells. The mice were treated with 10 μM antisense oligonucleotide in the medium. Incorporation was achieved by uptake.

[0144] Cells were harvested 24 to 72 hours after antisense oligonucleotide treatment and immediately analyzed for tau expression. The mRNA or protein may be isolated and analyzed using methods known in the art and described herein. In general, when treatments were performed in multiple replicates, Data are expressed as the average of repeated treatments. The concentration of antisense oligonucleotides varied depending on the cell line. Methods for determining the concentration of antisense oligonucleotides are well known in the art. The oligonucleotides are typically transfected using OptiFect. at concentrations ranging from 1 nM to 1,000 nM; and using nucleofection When transfected with ribosomal RNA, concentrations ranging from 25 nM to 20,000 nM were used. Ta.

[0145] Quantification of MAPT (tau) mRNA levels was performed using the ViiA7 real-time PCR system. (Life Technologies) according to the manufacturer's instructions. This was accomplished by quantitative real-time PCR. The NA is subjected to reverse transcription to produce complementary DNA (cDNA), which is then used for real-time PCR. The reverse transcription and real-time PCR reactions were carried out using the 100% ... , sequentially in the same sample well. A kit (Qiagen Cat. No. 216513) was used to lyse the cells in the wells. was used to reverse transcribe mRNA directly into cDNA without purifying RNA from cultured cells. The cDNA was then used to analyze tau expression using quantitative real-time PCR. The levels of tau mRNA, as determined by real-time PCR, were compared to those of cells with expression levels of tau. The expression level of a housekeeping gene that is constant, e.g., human glyceraldehyde 3- phosphate dehydrogenase (GAPDH), human TATA-box binding protein (TB P), or human hypoxanthine-guanine phosphoribosyltransferase (HPR T1) was used for standardization.

[0146] QuantiTect Multiplex RT-PCR Kit (Qiagen The duplex RT-PC was connected according to the protocol described in the Catalog No. 204643. TaqMan gene expression assays were run in real time using duplex RT-PCR reactions. Human MAPT (Life Tech Assa yID number Hs00902194_m1: FAM-MGB), human GAPDH (Life Tech Assay ID number Hs02758991_g1:VIC-MGB), human T BP (LifeTech Catalog No. 4326322E) or human HPRT1 (Li Uses a Taqman probe specific for feTech Catalog No. 4333768T The samples were cycled according to the recommended cycling conditions for duplex RT-PCR. The results were analyzed using a ViiA7 real-time PCR system (Life Technologies). All data were controlled for the amount of cDNA input and tau mRNA levels were internalized. Tau and control genes were normalized to the levels of the causative reference gene. Amplification was performed in the same reaction with PCR efficiency to allow relative quantification by the ΔΔCT method. is expressed as the percentage of remaining tau mRNA relative to PBS-treated control cells .

[0147] In vivo testing of antisense oligonucleotides Antisense oligonucleotides against MAPT were administered to mice in the cerebrospinal fluid (CSF) of mice. The compound was tested in vivo by delivery via intracranial cavity (ICV) administration into the SF. Mice were anesthetized with 5% isoflurane and then incubated with isoflurane-containing bromine in oxygen / nitrous oxide. The rectal temperature was maintained throughout the surgical procedure by using a constant temperature heating blanket and The temperature was maintained at 36.9 ± 1.0 °C using a rectal probe and a cerebrospinal fluid (PEF). The skin on the head was shaved and disinfected with povidone-iodine solution (Betadine). Before incision, mice were injected with buprenorphine (Temgesic, 0.03 mg / kg, 1 The mice were then given 100 ml / kg subcutaneous injection. The skull was exposed for injection. A 10 μl Hamilton syringe and a micropump (Ha All animals were injected with a 28G needle equipped with a varard apparatus. In the right lateral ventricle, the following coordinates: AP=+0.5 mm; ML=+1.0 mm; DV=-2.5 The flow rate was 1 μl / min, and the needle was withdrawn after infusion. The skin was closed and the mice were allowed to recover in single cages. The rats were then returned to their home cage. An additional dose of buprenorphine (Temgesic, 0.03 mM) was administered. 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. General condition of the animals and wounds Recovery was monitored and body weights were measured daily. At the end point, animals were treated with sodium pentobarbital. in tar (60 mg / kg Mebunat, OrionPharma, Finland). Deeply anesthetized mice were then subjected to cisternal puncture to collect CSF (3-5 µL per mouse). The mice were then cardiac punctured to collect blood samples. Approximately 0.4-0.5 mL of blood was collected. Collect 100 µL of each sample into a 500 µL plastic lavender K2EDTA anticoagulant tube and discard. The plasma was aliquoted by centrifugation at 0,000 g for 10 min at 4°C. Mice were then decapitated and the brains were The mice were harvested and dissected into different brain regions, including the cortex, hippocampus, and cerebellum. was also collected.

[0149] [Example 2] Suppression of human tau expression in Huh7 cells by 18-mer 2'-O-MOE steric blocker Inhibition The steric blocker of the antisense oligonucleotide is present in all isoforms. Intron-exon junctions of constitutive exons in human tau, an invariant exon The steric blockade of the antisense oligonucleotide was designed to target the It hybridizes to the branch point sequence of an intron, directly targets the splice site, and By flanking the exon sequence and exon splicing enhancer site, Steric blockers targeting MAPT were designed to induce cytoskipping. It acts on all nucleosides by steric hindrance, inhibiting RNase H or RISC. The inactive 2'-O-(2-methoxyethyl) (2'-O-MOE) ribose sugar modification It was originally designed as a length of 18 nucleosides, with the internucleoside linkages at the corners. The entire sequence is phosphodiester-linked. The study was carried out on the above 18-mer ASO of uniformly modified 2'-MOE with a tert-butyl backbone. To avoid off-target hybridization, BLAST analysis was performed on each molecule. A polynucleotide sequence was performed.

[0150] A 18-mer 2'-O-MOE steric blocker targeting tau was synthesized in vitro and in human Their activity in inhibiting tau mRNA was tested. Huh7 cells were cultured at 1 well per well. Plate at a density of 10,000 cells per well and infuse with OptiFect reagent (LifeTec ch, Catalog No. 12579-017) at 25 nM of antisense oligonucleotide. After a 48-hour treatment period, cells were transfected with Fastlane ce ll multiplex kit (Qiagen, Cat. No. 216513) The levels of tau mRNA were measured using human MAPT (L ifeTech Assay ID number Hs00902194_m1:FAM-MGB) and human TBP (TATA-box binding protein) endogenous control (LifeTech, Calif.). A Taqman probe specific for the 4326322E gene was used to identify duplexes. Quantitative real-time PCR was performed using a 3x RT-PCR reaction. All data are shown in cD To control for the amount of NA input, the levels of tau mRNA were measured using the expression level of the endogenous reference gene TBP. The tau and TBP control genes were normalized to the same PCR level with similar high PCR efficiency. Amplification was performed in the same reaction to allow relative quantification by the ΔΔCT method. The results are expressed as the percentage of remaining tau mRNA relative to control cells. The steric blocking activity of these 18-mer 2'-O-MOEs in cells is shown.

[0151] [Table 2-1]

[0152] [Table 2-2]

[0153] [Table 2-3]

[0154] [Example 3] Steric blocker of 18-mer 2'-O-MOE inhibits human TATA in SH-SY5Y cells Inhibition of expression In Example 2, a steric blocker that significantly reduced tau expression was selected and used to treat human neuroblastoma S The test was performed on H-SY5Y cells. Cultured SH-SY5Y cells were incubated with 1,000 nM of the selected The cells were nucleofected with the selected antisense oligonucleotide. The treatment period was approximately 24 hours. After the interval, a Fastlane cell multiplex kit (Qiagen, Cat. cDNA was prepared directly from cultured cells using the log number 216513. Tau mRNA The levels of The human MAPT (LifeTech Assay ID number Hs00902194_ m1:FAM-MGB) and human GAPDH (LifeTech Assay ID number H s02758991_g1: VIC-MGB) specific Taqman probe was used. All data were controlled for the amount of cDNA input and tau mRNA levels were compared with those of endogenous The tau and GAPDH control genes were normalized to the levels of the gender reference gene GAPDH. Amplification in the same reaction with similar high PCR efficiency allows relative quantification by the ΔΔCT method Results are expressed as the percentage of remaining tau mRNA relative to PBS-treated control cells. Table 3 shows the 2'-O-MOE structure of selected 18-mers in SH-SY5Y cells. It shows the activity of a somatic blocking agent.

[0155] [Table 3]

[0156] Steric blockers that showed significant in vitro inhibition of tau mRNA were tested at different doses. Cultured SH-SY5Y cells were treated with 0.125 nM, 0.25 nM, 0.5 nM, 1, One selected concentration of 000nM, 2,000nM, 4,000nM and 8,000nM After a treatment period of approximately 24 hours, cDNA was prepared directly and tau mRNA levels were analyzed by quantitative real-time immunoassay as described above. The 50% inhibitory concentration (IC50) was determined by constructing a dose-response curve. To investigate the effect of different concentrations of antisense oligonucleotides on lowering tau mRNA, The concentration required to inhibit half of the maximum biological response of a compound was determined by The potency of the antisense oligonucleotide was evaluated by calculating IC50 values. Table 4 shows the 2'-O-MOE structure of selected 18-mers. The IC50 values ​​of the somatic blocking agents are shown.

[0157] [Table 4]

[0158] [Example 4] Human 12-25 mer 2'-O-MOE steric blockers in SH-SY5Y cells Inhibition of tau expression The steric blockers that significantly reduced tau expression in Examples 2 and 3 were 12 to 25 nucleotides. The 2' amino acid sequence of these 12- to 25-mers was selected and constructed to have different lengths. The -O-MOE steric blocker was tested in SH-SY5Y cells. Nucleofect the cells with 2,000 nM of the selected antisense oligonucleotide. After a treatment period of approximately 24 hours, cDNA was prepared directly as described above to identify tau The mRNA levels were measured as described above. Table 5 shows the results of the 25-mer 12-mer 1 shows the activity of the '-O-MOE steric blocker in SH-SY5Y cells.

[0159] [Table 5-1]

[0160] [Table 5-2]

[0161] Selected 12- to 25-mer 2'-OMs with phosphodiester internucleoside linkages The IC50 values ​​of the OE steric blockers were determined as described above and are shown in Table 6.

[0162] Many 12- to 25-mer 2'-O-MO complexes with phosphorothioate internucleoside linkages Steric blockers were synthesized and the IC50 values ​​of some of these steric blockers are shown in Table 7. did.

[0163] [Table 6]

[0164] [Table 7]

[0165] [Example 5] Expression of human tau by 17-mer 2'-O-MOE steric blocker in SH-SY5Y cells Current obstruction A 17-nucleoside long 2'-MOE steric blocker was used to characterize the structural exon in human tau. These 17-mer 2'-O-MOE steric blockers were designed to target the 2'-O-MOE 17-mer esters. The test was performed on SH-SY5Y cells. SH-SY5Y cells were cultured with 2,000 nM of selection The cells were transfected with the antisense oligonucleotides prescribed in Example 1. Treatment period was approximately 24 hours. Afterwards, cDNA was directly prepared and tau mRNA levels were measured as described above. Table 8 shows the effect of the 17-mer 2'-O-MOE steric blocker on SH-SY5Y cells. Shows activity.

[0166] [Table 8-1]

[0167] [Table 8-2]

[0168] [Example 6] Inhibition of human tau expression by 5-10-5 gapmer in Huh7 cells Antisense oligonucleotide sequences were then applied to the shortest tau isoform, the barrier to transcription, and 4, designed to be complementary to mRNA (GenBank: NM_016841.4) BLAST analysis was performed on each origin to avoid off-target hybridization. The newly designed and modified chimeric antisense The oligonucleotides are arranged as 5-10-5 gapmers, 20 nucleosides in length. was designed, in which the central gap segment contained 10 2'-deoxynucleosides. , each of which contains five nucleosides modified with 2'-O-MOE ribose sugars in the 5' and 6' directions. The gapmer is flanked by 3′ wing segments and 4′ wing segments. The interside linkages are phosphorothioate (P=S) linkages.

[0169] Tau-targeting gapmers were assayed in vitro for inhibition of human tau mRNA expression. 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 25 nM of antisense oligonucleotides for 48 hours. After a period of time, cDNA was extracted from the cultured cells and then purified using Fastlane cell multiplex. Directly prepared using a kit (Qiagen, Cat. No. 216513). The levels of NA were analyzed by quantitative real-time PCR in duplex RT-PCR reactions. Human MAPT (LifeTech, Assay ID number Hs00902194_m1:F AM-MGB) and human TBP (TATA-box binding protein) endogenous control (Li feTech, Cat. No. 4326322E) All data were controlled for the amount of cDNA input and tau mRNA levels were measured. The levels were normalized to the levels of the endogenous reference gene TBP. , amplified in the same reaction with similar high PCR efficiency, allowing relative quantification by the ΔΔCT method The results were as follows: percent remaining tau mRNA versus PBS-treated control cells Table 9 shows the activity of gapmers in Huh7 cells.

[0170] [Table 9-1]

[0171] [Table 9-2]

[0172] [Example 7] Inhibition of human tau expression by 5-10-5 gapmer in SH-SY5Y cells Gapmers that significantly reduced tau mRNA expression in Example 6 were selected and used as S H-SY5Y cells were cultured with 2,000 nM of anti-SH-SY5Y cells. After a treatment period of approximately 24 hours, the cultured cells were nucleofected with From the cells, Fastlane cell multiplex kit (Qiagen catalog) cDNA was prepared directly using the ELISA kit (catalog number 216513). Tau mRNA levels were measured using Quantitative real-time PCR was performed using duplex RT-PCR reactions to measure histamine release. MAPT (LifeTech Assay ID number Hs00902194_m1:FA M-MGB) and human GAPDH (LifeTech Assay ID number Hs027 A Taqman probe specific for 58991_g1: VIC-MGB was used. Data were controlled for the amount of cDNA input and tau mRNA levels were compared to the endogenous reference Normalized to the level of the gene GAPDH. Tau and GAPDH control genes were similarly elevated. Both were amplified in the same reaction with high PCR efficiency, allowing relative quantification by the ΔΔCT method. Results are expressed as the percentage of remaining tau mRNA relative to PBS-treated control cells. Table 10 shows the activity of selected 5-10-5 gapmers in SH-SY5Y cells. The IC50 values ​​of selected 5-10-5 gapmers with 5-methylcytosine are shown. Values ​​were determined in 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 were synthesized by Thermo Scientific Use a high performance liquid chromatography-mass spectrometry (LC-MS) instrument from This method was used to predict the activity of antisense oligonucleotides (ASOs). The masses obtained are then checked to provide information about the purity of the sample and the identity of the major components present. For example, an antisense oligonucleotide comprising SEQ ID NO: 284 was used to provide The structure shown in Figure 1A and the formula C230H321N72O120P19S19 are Thus, the predicted molecular weight for an ASO containing SEQ ID NO:284 is approximately 721. FIG. 1B shows the LC-MS results for the ASO containing SEQ ID NO:284. The peak mass determined to be 7214.3 Da is shown in FIG. 1C. The LC-MS deconvoluted peaks for the ASO containing this compound are reported.

[0176] The antisense oligonucleotide containing SEQ ID NO: 285 is C230N69O124P It has the formula 19S19H318 and an estimated molecular weight of 7231.11 Da. The peak mass measured by LC-MS for the ASO containing SEQ ID NO:285 was 72 FIG. 1E shows the LC-M for ASO containing SEQ ID NO:285. Shown is a report of the deconvoluted peaks of S.

[0177] [Example 9] In vivo testing of MAPT-targeting gapmers Generation of human tau (hTau) transgenic mice The BAC vector (pBACe3.6) containing the human tau gene MAPT was cloned into Life Sciences. The entire MAPT gene was obtained from the human genome library of the University of California, Berkeley. Three vectors predicted to contain all of the regulatory regions were screened. The DNA was subjected to standard PCR to identify fragments spanning each exon and intron of the human tau gene. Used to sequence clones, testing for the presence of primers and regulatory regions. Comparison with human DNA identified one clone (RP11669E14) as belonging to the tau gene. All parts of the gene were shown to be intact. Purified DNA was then transferred to C57BL / The tail DNA from the founder mice was digested with restriction enzymes and then injected into fertilized embryos of 6 mice. and hybridizing it with exon-specific probes and similarly digested human DNA. These were used as controls to test the integrity of the transgene. Several hTau BAC transgenic lines were generated, one of which was a human M The expression of APT mRNA and protein was shown to be consistent with that of human brain. We expressed all six human brain transcripts and protein isoforms found in (Figure 2 A-2C). Heterozygous hTau BAC transgenic mice contain one copy of Carrying the transgene, the levels of RNA and human tau expression were nearly equal to endogenous mouse tau expression. The results were almost equal.

[0178] In vivo knockdown of human tau by antisense oligonucleotides Selected antisense oligonucleotides were tested in vivo. Five groups of hTau BAC transgenic mice were injected with 1, 10, 50, 200, or 400 μg of the selected The antisense oligonucleotides were administered by intracerebroventricular (ICV) bolus injection. A group of mice were either treated with isofluorane or left untreated as a control. The procedure was performed under anesthesia and in accordance with IACUC regulations. The nucleotide oligonucleotide was then delivered to the right lateral ventricle of hTau BAC transgenic mice. Two or four microliters containing 100 μg / μl of oligonucleotide were injected. The mice were injected with 100 µg of PBS solution immediately, 1 hour, 4 hours, and 24 hours after oligonucleotide administration. Tissues were collected after 2, 4, 12 or 24 weeks. NAs were extracted and examined for human tau mRNA expression by real-time PCR analysis. Human tau mRNA levels were measured as described above. Results were compared with control untreated mice. Percent inhibition of human tau mRNA expression normalized to GAPDH levels compared to mice Proteins were extracted from the hippocampus or cortex and assayed for histochemical staining by ELISA. Totau protein expression levels were examined and normalized to the levels of total protein. Ta.

[0179] In vivo analysis of 5-10-5 gapmers containing SEQ ID NO:284 or SEQ ID NO:285 The activity of both anti-cancer drugs in the treatment of osteoporosis was tested using the methods described above. As shown in Table 12, Antisense oligonucleotides were administered intravenously after a single ICV injection of antisense oligonucleotides. At 2 weeks, it significantly inhibited the expression of human tau mRNA in the cortex and hippocampus. For gapmers containing No. 285, knockdown of human tau mRNA was observed in the cortex and The results for the gapmer containing SEQ ID NO:284 were approximately 65% ​​in both the human and hippocampal tissues. Knockdown of the mRNA was approximately 42% in both the cortex and hippocampus. For tau protein levels after 2 weeks of treatment, a gapmer containing SEQ ID NO:285 knocks down approximately 50% of tau protein expression in the glial cell; The co-receptor knocked down approximately 36% of tau protein expression in the cortex. observed a significant reduction in tau protein levels after 2 weeks of ASO treatment in the hippocampus. There wasn't.

[0180] [Table 12]

[0181] Tau mRNA and protein levels were measured 4 weeks after a single ICV injection of gapmers. The antisense oligonucleotide containing SEQ ID NO: 285 was also tested in the brain. The expression of totau mRNA (FIG. 2D) and protein (FIG. 2E) was significantly inhibited. For gapmers containing No. 285, knockdown of human tau mRNA was observed in the cortex and The difference was approximately 60% in both the hippocampus and the hippocampus (Fig. 2D and data not shown). 2011, pp. 1111-1115, 2011. The results show that a gapmer comprising SEQ ID NO: 285 increases human tau protein levels at 4 weeks post-treatment. showed approximately 50% knockdown in the hippocampus (Figure 2E).

[0182] Antisense oligonucleotide synthesis in the brains of hTau BAC transgenic mice To detect the brain distribution of tides, in situ hybridization experiments were performed in duplicate. Digoxigenin (DIG)-labeled Locked Nucleic Acid (LNA™, Exiqon) The probe was complementary to the antisense oligonucleotide of the target. The heavy-DIG LNA probe was hybridized overnight. The probe was then hybridized with sheep anti- DIG alkaline phosphatase conjugated antibody (Roche Diagnostics s, Catalog No. 11093274910) and Nitroblue Tetrazolium and Alkaline Conjugates with 5-bromo-4-chloro-3-indolyl phosphate, the substrate for phosphatase Detection was performed using the colorimetric reaction of BCIP. The intracerebral distribution of 85 antisense oligonucleotides is shown in Figure 3, which indicates that ASO It shows initial diffusion from the ventricles to the mouse brain parenchyma, and the distribution signal does not change between 24 hours and 2 weeks. (Figure 3). Antisense oligonucleotides were stable in the brain even after 4 weeks. (data not shown).

[0183] hTau BAC transfection with antisense oligonucleotide of SEQ ID NO: 285 Human tau mRNA (Figure 4A) and protein (Figure 4B) in transgenic mice A dose-dependent inhibition of expression was observed (Figures 4A and 4B).

[0184] Following a single ICV injection of 200 μg of antisense oligonucleotide of SEQ ID NO:285 Human tau mRNA in hTau BAC transgenic mice (Figure 5A) and The time course of protein (Figure 5B) expression levels was measured using the antisense oligonucleotide of SEQ ID NO: 285. The study showed that nucleotides inhibited tau mRNA and protein expression for up to 12 weeks. (Figures 5A and 5B).

[0185] [Example 10] Additional 5-1 with 5-methylcytosine in Huh7 and SH-SY5Y cells Inhibition of human tau expression by 0-5 gapmers Additional gapmer sequences with 5-methylcytosine that target tau are described above. Inhibition of human tau mRNA expression in Huh7 and SH-SY5Y cells The results showed that the residual tau mRNA levels were significantly lower in PBS-treated control cells than in PBS-treated control cells. The results are expressed as a percentage of NA. Table 13 shows the results in Huh7 and SH-SY5Y cells. 1 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] Gapmers that significantly reduced tau mRNA expression in Table 13 were selected and used to identify SH-SY Tested in 5Y cells. 5-10-5 gapmer with 5-methylcytosine selected The IC50 values ​​of 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 inhibit the transcription of tyrosine in monkeys and humans Inhibition of expression Several gapmers that significantly reduced tau mRNA expression were selected and analyzed using COS1 The results showed that the residual tau m was significantly lower in green monkey cells than in PBS-treated control cells. The results are expressed as a percentage of RNA. Table 15 shows the results of the selected 5-10- 5 shows the activity of gapmers.

[0193] [Table 15]

[0194] We selected several antisense oligonucleotides that significantly reduced tau mRNA expression. We selected human embryonic stem cell (hESC)-derived neurons for testing. Results showed that PBS-treated The results are expressed as the percentage of remaining tau mRNA compared to control cells. 1 shows the activity of selected antisense oligonucleotides in the IL-1 gene.

[0195] [Table 16]

[0196] [Example 12] In vivo testing of MAPT-targeting gapmers The in vivo activity of selected 5-10-5 gapmers was evaluated as described in Example 9. As shown in Table 17, several antisense oligonucleotides were tested using the methods described above. Reotide significantly inhibited human tau mRNA and protein expression in the cortex and hippocampus. Harmful.

[0197] [Table 17]

[0198] Unless otherwise defined, technical and scientific terms used herein are intended to be understood as meaning those terms to which this disclosure pertains. "term" has the same meaning as commonly understood by a person skilled in the art.

[0199] Unless otherwise noted, all methods, steps, techniques and methods not specifically described are The procedures and operations can be carried out in a manner that is clear and known to those skilled in the art. The standard handbooks and general background art mentioned in this specification and the examples therein Reference is again made to the further references cited. Each of the references cited is incorporated by reference in its entirety.

[0200] The claims of the present invention are non-limiting and are provided below.

[0201] Although certain embodiments and claims have been disclosed in detail herein, this is for illustrative purposes only. are made by way of example only and are not intended to be limiting unless otherwise specified in the appended claims or any corresponding future claims. No limitations are intended as to the scope of the subject matter of the applicable claims. Various substitutions, modifications, and variations are possible without departing from the spirit and scope of the present disclosure as defined herein. It is contemplated by the inventors that modifications and variations may be made to the present disclosure. The selection of nucleic acid starting materials, clones, or library types can be carried out as described herein. It is believed that this would be a routine matter for one of ordinary skill in the art having knowledge of the above aspects. and modifications are contemplated to be within the scope of the following claims. Those of ordinary skill in the art will appreciate that such modifications and variations will be apparent to those skilled in the art using no more than routine experimentation. Without wishing to use any particular technique, it is recognized that there are many equivalents to the specific embodiments of the invention described herein. Such equivalents are defined in the following claims. The scope of the claims in the corresponding later-filed application is intended to be encompassed by the present application. Rewriting of the claims may be based on limitations imposed by patent laws in various countries and may affect the release of the claimed subject matter. should not be construed as abandonment. The following aspects may be included. [1] At least 90% sequence identity with any of the nucleic acid sequences shown in Tables 2 to 17 any one of said nucleobase sequences having the formula: wherein C is either cytosine or 5-methylcytosine, An oligonucleotide, wherein at least one nucleotide of the oligonucleotide has a 2'-modification. [2] A nucleic acid having at least 90% sequence identity with any of the sequences shown in Tables 2 to 8. a nucleic acid base sequence, wherein any C in said nucleic acid base sequence is cytosine or 5-methyl cytosine, and each nucleotide of the oligonucleotide has a 2'-modification. The oligonucleotide according to the above item [1]. [3] A nucleic acid having at least 90% sequence identity with any of the sequences shown in Tables 2 to 8. The oligonucleotide according to [2] above, comprising an acid-base sequence. [4] At least 95% sequence identity with any of the nucleic acid sequences shown in Tables 2 to 8. The oligonucleotide according to [2] above, comprising a nucleic acid base sequence having the following structure: [5] The oligonucleotide according to [2] above, which contains any one of the nucleic acid base sequences shown in Tables 2 to 8. nucleotide. [6] The oligonucleotide according to [2] above, which has any one of the nucleic acid base sequences shown in Tables 2 to 8. Gonucleotide. [7] The internucleoside linkages of the oligonucleotide are phosphodiester or phospho The oligonucleotide according to any one of the above [2] to [6], which is either a thiol linkage or a thiol linkage. Cleotide. [8] A linker attached to the 3' end of the oligonucleotide through a phosphate bridge. , having the following structure: [ka] The oligonucleotide according to any one of [2] to [7] above, having any one of the following: [9] Reduces expression of tau mRNA or protein independent of RNAseH; An oligonucleotide according to any one of [2] to [8] above.

[10] At least 90% sequence identity with any of the sequences shown in Tables 9-15 and 17 wherein any C in said nucleic acid base sequence is a cytosine or or 5-methylcytosine, and at least one of said oligonucleotides The oligonucleotide according to [1] above, wherein the nucleotide has a 2'-modification.

[11] At least 90% sequence identity with any of the sequences shown in Tables 9-15 and 17 The oligonucleotide according to

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

[12] A nucleic acid sequence that is at least 95% identical to any of the nucleic acid sequences shown in Tables 9 to 15 and 17. The oligonucleotide according to

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

[13] The oligonucleotide described in

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

[14] The oligonucleotide according to the above

[0010] , which consists of any one of the nucleic acid base sequences shown in Tables 9 to 15 and 17.

[15] The internucleoside linkage of the oligonucleotide is a phosphorothioate linkage. The oligonucleotide according to any one of

[10] to

[14] above.

[16] The above

[10] containing at least five consecutive 2'-deoxynucleosides. An oligonucleotide according to any one of claims 1 to 15.

[17] The above

[10] , which contains at least seven consecutive 2'-deoxynucleosides. 16. An oligonucleotide according to any one of claims 1 to 15.

[18] An oligonucleotide described in any one of

[10] to

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

[19] Reduces tau mRNA or protein expression by activating RNAseH. The oligonucleotide according to any one of

[10] to

[18] above, which reduces

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

[19] .

[21] At least 12 consecutive nucleoside salts of any one of SEQ ID NOs: 487 to 506 an oligonucleotide comprising a nucleobase sequence complementary to a base, said oligonucleotide An oligonucleotide, wherein at least one nucleotide of the oligonucleotide has a 2'-modification.

[22] At least 12 consecutive nucleoside salts of any one of SEQ ID NOs: 487 to 506 The oligonucleotide according to

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

[23] The oligonucleotide according to

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

[24] The oligonucleotide described in

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

[25] The above

[21] , which contains at least five consecutive 2'-deoxynucleosides. The oligonucleotide according to claim 1,

[26] The above

[21] , which contains at least seven consecutive 2'-deoxynucleosides. The oligonucleotide according to claim 1,

[27] The nucleic acid according to

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

[28] Any of the above [1] to

[27] , containing 12 to 30 nucleobases. of oligonucleotides.

[29] Any of the above [1] to

[27] containing 12 to 25 nucleobases. of oligonucleotides.

[30] Any of the above [1] to

[27] containing 15 to 20 nucleobases. of oligonucleotides.

[31] The 2'-modification is 2'-fluoro, 2'-deoxy-2'-fluoro, 2' -O-Methyl, 2'-O-Methoxyethyl (2'-O-MOE), 2'-O-Aminopropanol Pyr (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 Any of the above [1] to

[30] is selected from the group consisting of 2'-O-NMA and 2'-O-NMA. The oligonucleotide according to any one of claims 1 to 4,

[32] The above, wherein the 2'-modification is 2'-O-methoxyethyl (2'-O-MOE). An oligonucleotide according to any one of [1] to

[30] .

[33] In vitro, tau mRNA or protein expression levels were increased by at least 30% The oligonucleotide according to any one of the above [1] to

[32] can reduce Do.

[34] Reduce tau mRNA or protein expression levels by at least 30% in vivo The oligonucleotide according to any one of the above [1] to

[32] , .

[35] The oligonucleotide according to any one of [1] to

[34] above and a pharmaceutical A composition comprising a carrier acceptable to.

[36] A therapeutically effective amount of the oligonucleotide according to any one of [1] to

[34] above. 23. A method for reducing tau expression levels in a subject comprising administering to the subject.

[37] The method of any one of the above, wherein the subject is suffering from or prone to suffer from a tau-related disease. The method described in

[36] .

[38] The tau-related disease is Alzheimer's disease (AD), amyotrophic lateral sclerosis (AMSC), or / Parkinsonism-Dementia Complex (ALS-PDC), Argyrophilic Grain Dementia (AGD) , British amyloid angiopathy, cerebral amyloid angiopathy, chronic traumatic encephalopathy (CTE), cerebral cortex Basal ganglionic degeneration (CBD), Creutzfeldt-Jakob disease (CJD), Punch-drunk syndrome, Diffuse neurofibrillary tangles with calcification, Down syndrome, Dravet syndrome, epilepsy, frontotemporal Forehead-to-head dementia (FTD), Parkinson's syndrome linked to chromosome 17 (FTDP-17) Frontotemporal dementia, frontotemporal lobar degeneration, ganglioglioma, gangliocytoma, Gerstmann- Straussler-Scheinker disease, Hallervorden-Spatz disease, Huntington's disease, inclusion Myositis, lead encephalopathy, Ritiko-Bodig disease, meningioma hemangiomatosis, multiple system atrophy, myotonic dysplasia Trophies, Niemann-Pick disease type C (NP-C), non-Guamanian with neurofibrillary tangles Motor neuron disease, Pick's disease (PiD), postencephalitic parkinsonism, prion protein brain amyloidosis Myloid angiopathy, progressive subcortical gliosis, progressive supranuclear palsy (PSP), subacute sclerosis Panencephalitis, tangle only dementia, neurofibrillary tangle predominant Tangle-predominant dementia, multi-infarct dementia, ischemic stroke, or stroke The method according to

[37] above, wherein the disease is selected from segmental sclerosis.

[39] The oligonucleotide is administered to the subject intrathecally, intracranially, intranasally, orally, intravenously, or intravenously. The method according to

[36] above, wherein the compound is administered intravenously or subcutaneously.

[40] The method of

[36] above, further comprising administering a second agent to the subject. How to.

[41] The method according to any one of

[36] to

[40] above, wherein the subject is a human.

[42] The above-mentioned [ Use of an oligonucleotide according to any one of claims 1 to 34.

[43] A pharmaceutical composition for use in treating a tau-related disease in a subject in need thereof. Use of the oligonucleotide according to any one of the above [1] to

[34] in the manufacture of a drug. For.

[44] The oligonucleotide has any of the nucleic acid base sequences shown in Tables 9 to 15 and 17. A nucleoside containing either one of the above, and the first to fifth nucleotides are modified with 2'-O-MOE. nucleotides 6 to 15 each contain a 2'-deoxynucleoside; , and the 16th to 20th nucleotides are 2'-O-MOE modified nucleosides, respectively. The oligonucleotide according to

[10] above,

[45] SEQ ID NOs: 208, 284, 285, 313, 329, 335, 366, 384 , 386, 405, 473, and 474. An oligonucleotide comprising:

[46] The first to fifth nucleotides are modified with 2'-O-MOE. each of the 6th to 15th nucleotides comprises a 2'-deoxynucleoside; Nucleotides 16 through 20 each contain a nucleoside modified with 2'-O-MOE. , the oligonucleotide described in

[45] above.

Claims

1. Having at least 90% sequence identity with any of the nucleic acid sequences shown in Tables 2 to 17 an oligonucleotide comprising a nucleobase sequence comprising any one of said nucleobase sequences, C is either cytosine or 5-methylcytosine, An oligonucleotide, wherein at least one nucleotide has a 2'-modification.

2. A nucleobase having at least 90% sequence identity with any of the sequences shown in Tables 2 to 8. wherein any C in said nucleic acid base sequence is cytosine or 5-methylcytosine. or each nucleotide of the oligonucleotide has a 2'-modification; The oligonucleotide of claim 1.

3. A nucleobase having at least 90% sequence identity with any of the sequences shown in Tables 2 to 8. The oligonucleotide of claim 2 comprising the sequence

4. Has at least 95% sequence identity with any of the nucleic acid sequences shown in Tables 2 to 8 The oligonucleotide of claim 2 comprising a nucleobase sequence.

5. The oligonucleotide of claim 2, comprising any one of the nucleic acid sequences shown in Tables 2 to 8. Chid.

6. The oligonucleotide according to claim 2, which consists of any one of the nucleic acid base sequences shown in Tables 2 to 8. Punch.

7. The internucleoside linkages of the oligonucleotide are phosphodiester or phosphorothioate.

7. The oligonucleotide of claim 2, wherein the linkage is either an ate linkage or an ester linkage.

8. a linker attached to the 3' end of said oligonucleotide through a phosphate bridge, The structure below: 【Chemistry 1】 The oligonucleotide according to any one of claims 2 to 7, having any one of the following structures:

9. The present invention relates to a method for the treatment of tau, comprising the steps of:

9. An oligonucleotide according to any one of 2 to 8.

10. A sequence having at least 90% sequence identity with any of the sequences shown in Tables 9 to 15 and 17. wherein C in any one of said nucleic acid base sequences is cytosine or 5- methylcytosine, and at least one nucleoside of the oligonucleotide is The oligonucleotide of claim 1, wherein the tide has a 2'-modification.

11. A sequence having at least 90% sequence identity with any of the sequences shown in Tables 9 to 15 and 17. The oligonucleotide of claim 10, comprising a nucleobase sequence which

12. A nucleic acid sequence having at least 95% sequence identity with any of the nucleic acid sequences shown in Tables 9 to 15 and 17. The oligonucleotide of claim 10, comprising a nucleic acid base sequence having identity.

13. The nucleic acid sequence according to claim 10, comprising any of the nucleic acid sequences shown in Tables 9 to 15 and 17. Oligonucleotides.

14. The nucleic acid sequence according to claim 10, which consists of any one of the nucleic acid sequences shown in Tables 9 to 15 and 17. of oligonucleotides.

15. The internucleoside linkages of said oligonucleotides are phosphorothioate linkages. Item 15. The oligonucleotide according to any one of items 10 to 14.

16. 16. The nucleic acid according to claim 10, comprising at least five consecutive 2'-deoxynucleosides. Any of the oligonucleotides described above.

17. 16. The nucleic acid according to claim 10, comprising at least 7 consecutive 2'-deoxynucleosides. Any of the oligonucleotides described above.

18. Any one of claims 10 to 15, comprising 10 consecutive 2'-deoxynucleosides. The oligonucleotide according to claim 1,

19. Reducing tau mRNA or protein expression by activating RNAse H 19. An oligonucleotide according to any one of claims 10 to 18.

20. 1 to 5, wherein each C in any of said nucleic acid base sequences is 5-methylcytosine.

20. The oligonucleotide according to any one of 19.

21. Complementary to at least 12 consecutive nucleobases of any one of SEQ ID NOs:487-506 an oligonucleotide comprising a nucleic acid base sequence targeting said nucleic acid; An oligonucleotide in which at least one nucleotide has a 2'-modification.

22. At least 12 contiguous nucleobases of any one of SEQ ID NOs: 487 to 506 and 10 22. The oligonucleotide of claim 21, comprising a nucleobase sequence that is 0% complementary.

23. The oligonucleotide of claim 21, comprising one or more 5-methylcytosines. 。

24. 22. The oligonucleotide of claim 21, wherein each nucleotide of the oligonucleotide has a 2'-modification. Oligonucleotides.

25. The oligonucleotide of claim 21, comprising at least five consecutive 2'-deoxynucleosides. Oligonucleotides.

26. The oligonucleotide of claim 21, comprising at least seven consecutive 2'-deoxynucleosides. Oligonucleotides.

27. The oligonucleotide of claim 21, comprising 10 consecutive 2'-deoxynucleosides. Leotide.

28. 28. The oligonucleotide of any one of claims 1 to 27, comprising 12 to 30 nucleobases. Punch.

29. 28. The oligonucleotide of any one of claims 1 to 27, comprising from 12 to 25 nucleobases. Punch.

30. 28. The oligonucleotide of any one of claims 1 to 27, comprising 15 to 20 nucleobases. Punch.

31. The 2'-modifications are 2'-fluoro, 2'-deoxy-2'-fluoro, 2'-O-methyl ethyl, 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-dimethylaminoethyl 2'-O-DMAEOE, and 2'-O-N-methylacetamide (2 31. The oligonucleotide according to claim 1, selected from the group consisting of: Gonucleotide.

32. 1 or 2, wherein the 2'-modification is 2'-O-methoxyethyl (2'-O-MOE).

31. The oligonucleotide according to any one of claims 2 to 30.

33. Reduce tau mRNA or protein expression levels by at least 30% in vitro 33. The oligonucleotide of any one of claims 1 to 32.

34. Reduce tau mRNA or protein expression levels by at least 30% in vivo 33. The oligonucleotide of any one of claims 1 to 32.

35. 35. The oligonucleotide of claim 1 and a pharma- ceutically acceptable carrier. A composition comprising a body.

36. Administering a therapeutically effective amount of an oligonucleotide according to any one of claims 1 to 34 to a subject. A method for reducing tau expression levels in a subject comprising:

37. 37. The method of claim 36, wherein the subject is suffering from or susceptible to a tau-related disease. The method described.

38. The tau-related disease is Alzheimer's disease (AD), amyotrophic lateral sclerosis / parkinsonism, Nson syndrome-dementia complex disease (ALS-PDC), argyrophilic granular dementia (AGD), British type Amyloid angiopathy, cerebral amyloid angiopathy, chronic traumatic encephalopathy (CTE), corticobasal ganglia degeneration CBD, Creutzfeldt-Jakob disease (CJD), punch-drunk syndrome, calcification Diffuse neurofibrillary tangles, Down syndrome, Dravet syndrome, epilepsy, frontotemporal dementia (FTD), frontal temporal lobe disorder with parkinsonism linked to chromosome 17 (FTDP-17) Temporal dementia, Frontotemporal lobar degeneration, Ganglioglioma, Gangliocytoma, Gerstmann-Streu Thuller-Scheinker disease, Hallervorden-Spatz disease, Huntington's disease, inclusion body myositis, Lead encephalopathy, Lytcho-Bodig disease, meningioma hemangiomatosis, multiple system atrophy, myotonic dystrophy - Niemann-Pick disease type C (NP-C), non-Guamanian motor neuron disease with neurofibrillary tangles disease, Pick's disease (PiD), postencephalitic parkinsonism, prion protein cerebral amyloidosis Vasculopathy, progressive subcortical gliosis, progressive supranuclear palsy (PSP), subacute sclerosing panencephalitis, Tangle only dementia, tangle only dementia Tangle-predominant dementia, multi-infarct dementia, ischemic stroke, or tuberous sclerosis 38. The method of claim 37, wherein the disease is selected from the group consisting of rheumatoid arthritis, ... and rheumatoid arthritis.

39. The oligonucleotide is administered to the subject intrathecally, intracranially, intranasally, orally, intravenously, or 37. The method of claim 36, wherein the administration is via intravenous or subcutaneous route.

40. 37. The method of claim 36, further comprising administering a second agent to the subject.

41. 41. The method of any of claims 36 to 40, wherein the subject is a human.

42. A method according to any one of claims 1 to 3 for treating a tau-related disease in a subject in need thereof.

5. Use of the oligonucleotide according to any one of claims 4 to 4.

43. Manufacture of a medicament for use in treating a tau-related disease in a subject in need thereof 35. Use of an oligonucleotide according to any one of claims 1 to 34 in

44. The oligonucleotide has any of the nucleobase sequences shown in Tables 9 to 15 and 17. Each of the first to fifth nucleotides is a nucleoside modified with 2'-O-MOE. the sixth to fifteenth nucleotides each contain a 2'-deoxynucleoside, and the sixteenth nucleotides 1 to 20 each contain a nucleoside modified with 2'-O-MOE, The oligonucleotide according to claim 10.

45. SEQ ID NOs: 208, 284, 285, 313, 329, 335, 366, 384, 386 405, 473, and 474. Gonucleotide.

46. the first to fifth nucleotides each contain a nucleoside modified with 2'-O-MOE; , the 6th to 15th nucleotides each contain a 2'-deoxynucleoside, and the 16th to wherein the 20th nucleotide each comprises a nucleoside modified with 2'-O-MOE.

45. The oligonucleotide according to claim 45.

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