Compositions and methods for regulating tau expression

Abstract

This specification provides MAPT antisense oligonucleotides that can modulate the expression of MAPT target nucleic acids. Methods of using them are also provided. In certain embodiments, a MAPT antisense oligonucleotide (MAPT ASO) is provided comprising a nucleic acid sequence comprising at least nine consecutive nucleobases of sequences selected from any of SEQ ID NOs: 4-9 and 19-37, and at least one modified internucleoside bond and / or at least one modified sugar.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims the benefits of U.S. Provisional Application No. 63 / 509,696, filed on 22 June 2023, which is incorporated herein by reference.

[0002] Sequence List This application includes a sequence listing submitted electronically in XML format, which is incorporated herein by reference in its entirety. A copy of the XML, created on 17 June 2024, is named DNL-038-02-WO_SeqListing.XMP and has a size of 328kb. [Background technology]

[0003] Alzheimer's disease is a progressive neurodegenerative disease, currently the seventh leading cause of death in the United States, and the most common cause of dementia in the elderly. A prominent pathological feature of Alzheimer's disease and other neurodegenerative diseases is the abnormal aggregation and inclusion formation of microtubule-associated protein tau. Mutations in the microtubule-associated protein tau (MAPT) gene, which encodes tau, are associated with tauopathies, which are related to neurodegenerative diseases. [Overview of the Initiative]

[0004] In certain embodiments, a MAPT antisense oligonucleotide (MAPT ASO) is provided, comprising a nucleic acid sequence containing at least nine consecutive nucleobases selected from any of the sequences 4-9 and 19-37, and at least one modified internucleoside bond and / or at least one modified sugar.

[0005] In certain embodiments, a MAPT antisense oligonucleotide (MAPT ASO) is provided, having a length of 17 to 19 nucleotides and comprising one nucleobase sequence from sequence numbers 4 to 9, 19 to 37, or 38 to 56, wherein the MAPT ASO comprises at least one modified internucleoside bond and / or at least one modified sugar. In certain embodiments, the MAPT ASO comprises a nucleobase sequence consisting of one of sequence numbers 4 to 9. In certain embodiments, the MAPT ASO according to claim 1 or 2 is a gapmer, wherein the gap segment consists of one nucleobase sequence from sequence numbers 10 to 15.

[0006] In certain embodiments, MAPT ASOs containing any one of the nucleobase sequences of SEQ ID NOs. 10-15 and SEQ ID NOs. 38-56 are provided, along with at least one modified nucleoside linkage and / or at least one modified sugar.

[0007] In certain embodiments, MAPT ASOs are provided that include a nucleic acid sequence described in any one of SEQ ID NOs: 4-9 and SEQ ID NOs: 19-37, having at least one modified nucleoside linkage and / or at least one modified sugar.

[0008] In certain embodiments, MAPT ASOs comprising any one of the nucleobase sequences of SEQ ID NOs. 10-15 and SEQ ID NOs. 38-56 have a length of 16-20 nucleotides and provide at least one modified nucleoside linkage and / or at least one modified sugar.

[0009] In certain embodiments, a MAPT ASO containing one of the nucleobase sequences from SEQ ID NOs. 10-15 and SEQ ID NOs. 38-56 has a length of 16 nucleotides and provides at least one modified nucleoside linkage and / or at least one modified sugar.

[0010] In certain embodiments, there is provided a MAPT ASO gapmer comprising a gap segment comprising any one of the nucleic acid base sequences of SEQ ID NOs: 10-15 and 38-56, and at least one modified internucleoside linkage and / or at least one modified sugar. In some embodiments, the MAPT ASO gapmer has a length of 16 nucleotides, with 5' and 3' wing segments each having a length of 3 nucleotides.

[0011] In certain embodiments, there is provided a MAPT ASO which is a gapmer having a gap segment consisting of any one of the nucleic acid bases of SEQ ID NOs: 10-15, and the MAPT ASO comprises at least one modified internucleoside linkage and at least one modified sugar. In certain embodiments, the MAPT ASO has a length of 15-25 linked nucleosides. In certain embodiments, the MAPT ASO has a length of 16-19 linked nucleosides.

[0012] In certain embodiments, there is provided a MAPT antisense oligonucleotide (MAPT ASO) which is a gapmer 16 nucleotides in length and comprises a gap segment consisting of any one of the nucleic acid base sequences of SEQ ID NOs: 38-56, and the MAPT ASO comprises at least one modified internucleoside linkage and at least one modified sugar. In certain embodiments, the MAPT ASO comprises a nucleic acid base sequence consisting of any one of the sequences of SEQ ID NOs: 19-37.

[0013] In certain embodiments, there is provided a MAPT ASO comprising at least 9 consecutive nucleic acid bases complementary to the sequence present at nucleic acid bases 66,750 to 66,786, 66,760 to 66,786, 66,750 to 66,776, 66,754 to 66,786, or 66,760 to 66,776 of SEQ ID NO: 1, the nucleic acid base sequence of the MAPT ASO having at least 90% complementarity to SEQ ID NO: 1, and further the MAPT ASO comprises at least one modified internucleoside linkage and / or at least one modified sugar.

[0014] In certain embodiments, provided is a MAPT ASO comprising at least 9 consecutive nucleic acid bases complementary to the sequence present at nucleic acid bases 78,431 to 78,468, 78,431 to 78,458, 78,441 to 78,468, or 78,441 to 78,458 of SEQ ID NO: 1, wherein the nucleic acid base sequence of the MAPT ASO has at least 90% complementarity to SEQ ID NO: 1, and further the MAPT ASO comprises at least one modified internucleoside bond and / or at least one modified sugar.

[0015] In certain embodiments, provided is a MAPT ASO comprising at least 9 consecutive nucleic acid bases complementary to the sequence present at nucleic acid bases 78,621 to 78,659, 78,621 to 78,649, 78,631 to 78,659, or 78,631 to 78,649 of SEQ ID NO: 1, wherein the nucleic acid base sequence of the MAPT ASO has at least 90% complementarity to SEQ ID NO: 1, and further the MAPT ASO comprises at least one modified internucleoside bond and / or at least one modified sugar.

[0016] In certain embodiments, provided is a MAPT ASO comprising at least 9 consecutive nucleic acid bases complementary to the sequence present at nucleic acid bases 78,818 to 78,855, 78,828 to 78,855, 78,818 to 78,845, or 78,828 to 78,845 of SEQ ID NO: 1, wherein the nucleic acid base sequence of the MAPT ASO has at least 90% complementarity to SEQ ID NO: 1, and further the MAPT ASO comprises at least one modified internucleoside bond and / or at least one modified sugar.

[0017] In certain embodiments, a MAPT ASO is provided that includes at least nine consecutive nucleic acid bases complementary to the sequences present in nucleic acid bases 78,431 to 78,659, 78,431 to 78,649, 78,441 to 78,659, or 78,441 to 78,649 of SEQ ID NO: 1, wherein the nucleic acid base sequence of the MAPT ASO has at least 90% complementarity to SEQ ID NO: 1, and further comprises at least one modified internucleoside bond and / or at least one modified sugar.

[0018] In certain embodiments, a MAPT ASO is provided that includes at least nine consecutive nucleic acid bases complementary to the sequence present at nucleic acid bases 78,641 to 78,855, 78,641 to 78,845, 78,631 to 78,855, or 78,631 of SEQ ID NO: 1, wherein the nucleic acid base sequence of the MAPT ASO has at least 90% complementarity to SEQ ID NO: 1, and further comprises at least one modified internucleoside bond and / or at least one modified sugar.

[0019] In certain embodiments, a MAPT ASO is provided that contains at least nine consecutive nucleic acid bases complementary to the sequences present in the nucleic acid bases 78,431 to 78,855, 78,441 to 78,855, 78,431 to 78,845, 78,441 to 78,845, 78,441 to 78,498, 78,517 to 78,678, or 78,697 to 78,845 of SEQ ID NO: 1, wherein the nucleic acid base sequence of the MAPT ASO has at least 90% complementarity to SEQ ID NO: 1, and further comprises at least one modified internucleoside bond and / or at least one modified sugar.

[0020] In certain embodiments, a MAPT ASO is provided that comprises at least nine consecutive nucleic acid bases complementary to the sequences of nucleic acid bases 90,205 to 90,243, 90,205 to 90,233, 90,215 to 90,243, 90,208 to 90,243, or 90,215 to 90,233, wherein the nucleic acid base sequence of the MAPT ASO has at least 90% complementarity to SEQ ID NO: 1, and further comprises at least one modified internucleoside bond and / or at least one modified sugar.

[0021] In some embodiments, MAPT ASO is bound to a target ligand or delivery medium to form a conjugate. The target ligand includes, but is not limited to, molecules that specifically bind to transferrin receptors (TfRs) or molecules expressed on the luminal surface of the blood-brain barrier. The molecule includes, but is not limited to, an anti-TfR antibody or its TfR-binding fragment, or an Fc polypeptide modified to bind to TfR (described in WO2023279099, incorporated herein by reference).

[0022] In certain embodiments, a pharmaceutical composition is provided comprising MAPT ASO as described herein, or a conjugate containing MAPT ASO as described herein, and a pharmaceutically acceptable carrier or diluent.

[0023] In certain embodiments, a method is provided for generating neurons with reduced tau expression, the method comprising delivering a MAPT ASO as described herein, or a conjugate containing a MAPT ASO as described herein, to the neurons, wherein the MAPT ASO reduces the expression level of the endogenous MAPT gene. The neurons may be, but are not limited to, brain cells, deep brain cells, or spinal cord cells.

[0024] In certain embodiments, a method is provided for modifying neurons to reduce tau expression, the method comprising delivering a MAPT ASO described herein, or a conjugate containing a MAPT ASO described herein, to the neurons, the MAPT ASO reducing the expression level of the endogenous MAPT gene. The neurons may be, but are not limited to, brain cells, deep brain cells, or spinal cord cells.

[0025] In certain embodiments, a method is provided for modifying neurons to reduce tau expression, the method comprising delivering a MAPT ASO described herein, or a conjugate containing a MAPT ASO described herein, to the neurons, the MAPT ASO specifically reducing the expression level of MAPT transcripts within the cells. The neurons may be, but are not limited to, brain cells, deep brain cells, or spinal cord cells.

[0026] In certain embodiments, a method is provided for delivering MAPT ASO to the CNS of a human subject requiring delivery of MAPT ASO to the CNS, the method comprising administering a pharmaceutical composition comprising MAPT ASO as described herein to the subject, wherein the MAPT ASO reduces the expression level of endogenous MAPT genes.

[0027] The delivery of the described MAPT ASO to neurons may be used to treat neurodegenerative disorders. The delivery of the described MAPT ASO to neurons may be used to treat tau-related neurodegenerative diseases. Neurodegenerative diseases may include, but are not limited to, Alzheimer's disease.

[0028] In certain embodiments, a method is provided for delivering MAPT ASO to cells of a human subject's CNS, the method comprising administering a pharmaceutical composition comprising MAPT ASO as described herein to the subject, the MAPT ASO being administered by intrathecal injection.

[0029] In certain embodiments, a method is provided for delivering MAPT ASO to cells of a human subject's CNS, comprising administering to the subject a pharmaceutical composition comprising MAPT ASO as described herein, wherein the MAPT ASO is conjugated to a molecule (e.g., a target ligand) or a delivery medium that facilitates the transport of MAPT ASO across the blood-brain barrier. MAPT ASO can be conjugated to any molecule or delivery medium known in the art to facilitate transport across the blood-brain barrier. Such molecules or delivery mediums include, but are not limited to, brain shuttles (e.g., those described in WO2018210898, WO2015101588, and WO2014033074, which are incorporated herein by reference, respectively).

[0030] In certain embodiments, a method is provided for treating a tau-related neurodegenerative disease in a human subject requiring treatment for the disease, the method comprising administering to the human subject a MAPT ASO described herein, or a composition (e.g., a pharmaceutical composition) containing a MAPT ASO described herein.

[0031] In certain embodiments, a method for treating Alzheimer's disease is provided, which includes administering a MAPT ASO described herein, or a composition (e.g., a pharmaceutical composition) containing a MAPT ASO described herein, to a human subject in need thereof.

[0032] In certain embodiments, a method is provided for reducing the expression of MAPT messenger ribonucleic acid (mRNA) in a human subject that requires a reduction in MAPT messenger ribonucleic acid (mRNA) expression, the method comprising administering to the human subject a MAPT ASO described herein, or a composition (e.g., a pharmaceutical composition) containing a MAPT ASO described herein.

[0033] In certain embodiments, the MAPT ASO described herein, or a composition (e.g., a pharmaceutical composition) comprising the MAPT ASO described herein, is provided for use in the treatment of tau-related neurodegenerative diseases in human subjects requiring treatment of such diseases.

[0034] In certain embodiments, the MAPT ASO described herein, or a composition (e.g., a pharmaceutical composition) containing the MAPT ASO described herein, is provided for use in the treatment of Alzheimer's disease in human subjects requiring treatment for Alzheimer's disease.

[0035] In certain embodiments, the following are provided: a MAPT ASO described herein, or a composition (e.g., a pharmaceutical composition) containing the MAPT ASO described herein, for use in reducing the expression of MAPT mRNA in human subjects where it is necessary to reduce the expression of MAPT mRNA.

[0036] In certain embodiments, the use of MAPT ASO in the preparation of pharmaceuticals that reduce MAPT mRNA expression in human subjects requiring a reduction in MAPT mRNA expression is provided. [Brief explanation of the drawing]

[0037] [Figure 1] This study demonstrates in vivo knockdown of human tau in the cortex two weeks after ICV bolus administration.

[0038] [Figure 2-1] This study shows the effect of adding 5-methylcytosine (5meC) on the hepatotoxicity profile. [Figure 2-2] This study shows the effect of adding 5-methylcytosine (5meC) on the hepatotoxicity profile.

[0039] [Figure 3] This shows the ED50 curve of the brain.

[0040] [Figure 4] The ED50 curve in the spinal cord is shown.

[0041] [Figure 5-1] This shows the duration of knockdown in the brain, as well as the knockdown after 5 and 9 weeks. [Figure 5-2] This shows the duration of knockdown in the brain, as well as the knockdown after 5 and 9 weeks.

[0042] [Figure 6-1] This shows the duration of the knockdown effect in the spinal cord, as well as the knockdown after 5 and 9 weeks. [Figure 6-2] This shows the duration of the knockdown effect in the spinal cord, as well as the knockdown after 5 and 9 weeks.

[0043] [Figure 7A] The results of the nephrotoxicity assessment in rats are shown, specifically regarding serum urea nitrogen (BUN). [Figure 7B] The results of the nephrotoxicity assessment in rats are shown, specifically regarding creatinine.

[0044] [Figure 7C] The results of the urinary KIM-1:creatinine ratio in the evaluation of nephrotoxicity in rats are shown. [Modes for carrying out the invention]

[0045] Antisense oligonucleotides are single-stranded, small synthetic nucleic acid polymers used to regulate gene expression. They can target pre-mRNA, mRNA, or non-coding RNA to induce degradation, regulate splicing events, or interfere with protein translation. Described herein are antisense oligonucleotides that target MAPT RNA transcripts (e.g., MAPT mRNA such as pre-mRNA or mature mRNA) to reduce the expression level of MAPT genes.

[0046] The MAPT gene encodes the microtubule-associated protein tau (also referred to herein as tau), and its transcript undergoes complex and controlled alternative splicing to produce multiple mRNAs. MAPT transcripts exhibit different expression in the nervous system depending on the stage of neuronal maturation and neuronal cell type. Mutations in the MAPT gene have been associated with several neurodegenerative diseases, including Alzheimer's disease, Pick's disease, frontotemporal dementia, corticobasal degeneration, and progressive supranuclear palsy. In some embodiments, the human MAPT gene has the sequence identified by GenBank accession number NT_010783.15 (e.g., nucleotides 9,240,000 to 9,381,000 of the SEQ ID NO: 2). In some embodiments, the MAPT pre-mRNA has the sequence of SEQ ID NO: 2. Embodiments of the mRNA transcript are provided as SEQ ID NO: 3.

[0047] MAPT ASO as described herein can be used to treat tau-related disorders, such as Alzheimer's disease.

[0048] As used herein, the singular forms "a," "an," and "the" refer to multiple subjects unless their content clearly indicates otherwise.

[0049] When used herein, the terms “about” and “approximately” indicate that a reasonable deviation from the numerical value and values ​​known to those skilled in the art, e.g., ±20%, ±10%, or ±5%, is within the intended meaning of the enumerated value.

[0050] The term “antisense oligonucleotide (ASO)” refers to a single strand of a selected target polynucleotide sequence, for example, a DNA-like or RNA-like molecule (including, for example, modified internucleoside bonds, modified nucleic acid bases, and / or modified sugars, such as those described herein) that is complementary or partially complementary to mRNA. ASOs can modify or regulate gene expression by binding to a complementary target sequence, for example, by altering splicing (exon exclusion or exon inclusion); by employing RNase H resulting in target degradation; via translational inhibition; and via small RNA inhibition.

[0051] As used herein, “MAPT antisense oligonucleotide,” “MAPT ASO,” or “MAPT-targeted antisense oligonucleotide” refers to an ASO that can sequence-specifically bind (hybridize) to a MAPT-target nucleic acid (e.g., RNA transcript, MAPT mRNA (e.g., MAPT pre-mRNA or MAPT mature mRNA), or cDNA), resulting in reduced MAPT gene expression.

[0052] In this specification, "transferrin receptor" or "TfR" refers to transferrin receptor protein 1. Sequences of transferrin receptor protein 1 are known in several species (e.g., accession number NP_001121620.1 (or NP_001300894.1, NP_001300895.1, NP_003225.2) in humans, XP_003310238.1 in chimpanzees, NP_001244232.1 in rhesus macaques, NP_001003111.1 in dogs, NP_001193506.1 in cattle, NP_035768.1 in mice, NP_073203.1 in rats, and NP_990587.1 in chickens). Furthermore, the term "transferrin receptor" encompasses allelic variants of exemplary reference sequences (e.g., human sequences) encoded by the gene at the chromosomal locus of transferrin receptor protein 1. The full-length transferrin receptor protein contains a short N-terminal intracellular domain, a transmembrane domain, and a large extracellular domain. The extracellular domain is characterized by three domains: a protease-like domain, a helical domain, and an apical domain.

[0053] In relation to two or more polypeptide sequences, the term “identical” or “identity” percentage refers to two or more sequences or subsequences that, when compared and aligned to achieve the greatest match across a comparison window or specified region using a sequence comparison algorithm or by manual alignment and visual inspection, are identical across a specific region or have a specific percentage (%) of identical amino acid residues, e.g., at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% or more.

[0054] For polypeptide sequence comparison, typically, one amino acid sequence serves as a reference sequence compared to the candidate sequences. Alignment can be performed using various methods available to those skilled in the art, such as visual alignment or using publicly available software with known algorithms, to achieve maximum alignment. Such programs include the BLAST program, ALIGN, ALIGN-2 (Genentech, South San Francisco, Calif.), or Megalign (DNASTAR). The parameters used for alignment to obtain maximum alignment can be determined by those skilled in the art. For polypeptide sequence comparison for the purposes of this application, the standard protein BLAST of the BLASTP algorithm is used to align two protein sequences using default parameters.

[0055] The terms "corresponds to," "determined by reference to," or "numbered by reference to" used in relation to identifying a specific nucleotide residue in a nucleic acid sequence refer to the position of a residue in a particular reference sequence when the given nucleotide sequence is maximally aligned and compared to that reference sequence. The nucleic acid sequence aligned to the reference sequence does not need to be the same length as the reference sequence.

[0056] As used herein, the terms “nucleic acid” and “polynucleotide” refer to deoxyribonucleotides or ribonucleotides and their polymers in single-stranded or double-stranded forms, comprising monomers (nucleotides) containing a sugar moiety, a phosphate, and nucleic acid bases. Unless otherwise specified, these terms encompass both modified and unmodified nucleic acids.

[0057] As used herein, the term “nucleic acid base” refers to a nitrogen-containing compound that can bind to a sugar moiety to form a nucleoside, in other words, a component of a nucleotide. The ability of nucleic acid bases to form base pairs and stack with one another directly results in long helical structures such as ribonucleic acid (RNA) and deoxyribonucleic acid (DNA). Nucleic acid bases can be naturally occurring (i.e., adenine (A), cytosine (C), guanine (G), thymine (T), uracil (U)) or modified (e.g., 5-methylcytosine). Thus, a nucleic acid base moiety can be represented by a letter code corresponding to each nucleic acid base, namely A, T, G, C, or U, where each letter may include a modified nucleic acid base that is functionally equivalent (e.g., based on Watson-Crick type base-pairing ability).

[0058] As used herein, the term "nucleoside" refers to a compound comprising a nucleic acid base and a sugar moiety (e.g., deoxyribose or ribose, or modified variants thereof). The term nucleoside includes both modified and unmodified nucleosides.

[0059] As used herein, the term "nucleotide" refers to a compound comprising a nucleic acid base, a sugar moiety, and one or more phosphate groups. The term nucleotide includes both modified and unmodified nucleotides.

[0060] As used herein, the term “nucleoside bond” means a covalent bond between two nucleosides in an oligonucleotide. Nucleosides may be bonded via intrinsic bonds (i.e., phosphodiester (PO) bonds) or modified bonds.

[0061] The terms "chemical modification," "modification," or "modified" can refer to a chemical change in a compound compared to its naturally occurring counterpart. For example, nucleic acid bases, sugar moieties, or nucleoside bonds can be chemically modified.

[0062] The terms “nucleotide sequence,” “nucleic acid sequence,” “nucleic acid chain,” and “nucleic acid base sequence” refer to the sequence of bases (purines and / or pyrimidines, or their synthetic derivatives) in a DNA or RNA polymer, which may be single-stranded or double-stranded, and which may optionally be incorporated into a DNA or RNA polymer or a combination thereof, including synthetic, unnatural or modified nucleotides and / or skeletal modifications (e.g., modified oligomers).

[0063] The terms “oligo,” “oligonucleotide,” and “oligomer” may be used interchangeably and refer to such sequences of purines and / or pyrimidines. For example, oligonucleotides may include chemically modified or unmodified nucleic acid molecules (RNA or DNA) having a length of, for example, less than about 200 nucleotides (e.g., less than about 100 or 50 nucleotides). Oligonucleotides may include, for example, single-stranded DNA or RNA (e.g., ASO), double-stranded DNA or RNA (e.g., small interfering RNA (siRNA)), double-stranded DNA or RNA with hairpin loops, or DNA / RNA hybrids. In some embodiments, oligonucleotides have a length in the range of about 5 to about 60 nucleotides, or about 10 to about 50 nucleotides. In some embodiments, oligonucleotides have a length in the range of about 5 to about 30 nucleotides, or about 15 to about 30 nucleotides. In some embodiments, oligonucleotides have a length in the range of about 18 to about 24 nucleotides.

[0064] The terms “modified oligo”, “modified oligonucleotide”, or “modified oligomer” may be used interchangeably and refer to such sequences containing synthetic, unnatural, or modified bases, sugars, and / or skeletal modifications. References to oligonucleotides, nucleic acids, and polynucleotides herein include modified oligonucleotides, modified nucleic acids, and modified polynucleotides (i.e., oligonucleotides, nucleic acids, and polynucleotides having one or more modified bases, modified sugars, or internuclear bonds).

[0065] A "modified nucleotide" is a nucleotide other than a ribonucleotide (2'-hydroxyl nucleotide) or a deoxyribonucleotide (2'-H nucleotide). Modified nucleotides may include one or more modified nucleobases, modified ribose (sugar) moieties, or modified internuclear bonds to other nucleosides. Modified nucleotides include nucleotide mimes. Modified nucleosides include debasic nucleosides that lack a nucleic acid base, and nucleosides in which ribose is replaced by a non-sugar moiety (e.g., sugar substitutes, morpholinos, or those found in peptide nucleic acids).

[0066] A "2'-deoxynucleoside" is a nucleoside containing a 2'-deoxyribose sugar moiety. In naturally occurring DNA, 2'-deoxynucleosides contain ribose with a β-D-ribose structure.

[0067] A "2'-substituted nucleoside" or "2'-substituted nucleoside" or "2'-modified nucleoside" is a nucleoside in which the 2'-OH group of the ribose-type sugar moiety is substituted (for example, with a group other than hydrogen or hydroxyl). A 2'-substituted nucleoside contains at least one 2' substituent other than H or OH on the 2' carbon of the nucleoside ribose.

[0068] A "2′-MOE modified nucleoside" or "2′-MOE nucleoside" is a nucleoside in which the 2′-OH group of the ribose-type sugar moiety is substituted with 2′-OCH2CH2OCH3 (O-methoxyethyl).

[0069] A "2′-NMA modified nucleoside" or "2′-NMA nucleoside" is a nucleoside in which the 2′-OH group of the ribose-type sugar moiety is substituted with 2′-O-CH2-C(=O)-NH-CH3(ON-methylacetamide).

[0070] A "2′-OMe modified nucleoside" or "2′-OMe nucleoside" is a nucleoside in which the 2′-OH group of the ribose-type sugar moiety is substituted with a 2′-OCH3 group.

[0071] A "2′-F modified nucleoside" or "2′-F nucleoside" is a nucleoside that contains a 2′-fluoro substitution in place of the 2′-OH group in the ribose-type sugar moiety.

[0072] A "bicyclic nucleoside" (also called a bridged nucleoside) is a nucleoside containing a bicyclic sugar moiety. A "bicyclic sugar" or "bicyclic sugar moiety" is a modified sugar moiety containing two rings, where the second ring forms a bicyclic structure through a bridge connecting two atoms in the first ring. In some embodiments, the first ring of the bicyclic sugar moiety is a furanose moiety, for example, the ribose sugar moiety of a nucleoside.

[0073] A "non-bicyclic modified sugar moiety" is a modified sugar moiety that includes modifications, such as substituents, but does not form a bridge between two sugar atoms to form a second ring.

[0074] "Constrained ethyl," "cEt," "cEt-modified sugar moiety," or "cEt sugar moiety" is a β-D-ribose type bicyclic sugar moiety, where the second ring of the bicyclic sugar is formed by a bridge connecting the 4′-carbon and 2′-carbon of the β-D-ribose type sugar moiety, and the bridge has the structural formula 4′-CH(CH3)-O-2′, with the methyl group of the bridge being an S structure. "cEt-modified nucleoside" or "cEt nucleoside" is a nucleoside containing the cEt-modified sugar moiety.

[0075] "Locked nucleic acid," also known as "LNA nucleoside" or "LNA," is a bicyclic nucleoside having a 4′-CH2-O-2′ bridge between the 4′ and 2′ furanose ring atoms.

[0076] A "sugar surrogate" is the portion of a modified nucleoside that is not the ribose portion. Oligonucleotides containing one or more suitable sugar surrogates retain the ability to hybridize with complementary target nucleic acid bases or nucleic acid sequences.

[0077] "Nucleoside-to-nucleoside bond" refers to a covalent bond between adjacent nucleosides in an oligonucleotide. As used herein, "modified internuclear bond" refers to any internuclear bond other than phosphodiester internuclear bonds.

[0078] A "phosphorothioate internuclear bond" is a modified internuclear bond in which one of the non-bridged oxygen atoms in a phosphodiester internuclear bond is replaced with a sulfur atom.

[0079] "5-methylcytosine" contains cytosine modified with a methyl group attached to the 5th position. 5-methylcytosine is a modified nucleic acid base.

[0080] "Debasic nucleosides" include nucleosides that lack nucleic acid bases.

[0081] A "chirally enriched population" refers to a group of molecules with the same molecular formula in which the number or proportion of molecules containing a particular stereochemical configuration at a particular chiral center is greater than the number or proportion of molecules containing the same particular stereochemical configuration that would be predicted if that particular chiral center were sterically random. A chirally enriched population of molecules having multiple chiral centers in each molecule may contain one or more sterically random chiral centers. In some embodiments, the molecules are modified oligonucleotides.

[0082] A "stabilized phosphate group" refers to a 5'-chemical moiety that stabilizes the 5'-phosphate portion of the 5'-terminal nucleoside of an oligonucleotide compared to the stability of the unmodified 5'-phosphate group of an unmodified nucleoside under biological conditions. Examples of stabilized phosphate groups include, but are not limited to, 5'-vinyl phosphonate and 5'-cyclopropyl phosphonate.

[0083] The MAPT ASOs described herein may be synthesized using standard solid-phase or solution-phase synthesis techniques well known in the art. In certain embodiments, MAPT ASOs are synthesized using solid-phase phosphoramidite chemistry (U.S. Patent No. 6,773,885) by an automated synthesizer. Chemical synthesis of nucleic acids enables the production of modified ligatures, chimeric compositions, and various forms of nucleic acids having non-standard bases or modifying groups attached to selected locations along the entire length of the nucleic acid.

[0084] As used herein, the term “complementary” refers to the broad concept of complementary base pairing, which occurs between two nucleic acids aligned relative to each other at antisense positions. Nucleic acids are considered complementary at these positions if the nucleotide positions of both molecules are occupied by nucleotides that can normally base pair with each other. Thus, two nucleic acids are substantially complementary if at least about 50%, at least about 60%, or at least about 80% of the corresponding positions in each molecule are occupied by nucleotides that normally base pair with each other (e.g., A:T (A:U in the case of RNA) and G:C nucleotide pairs).

[0085] In the context of two or more nucleotide sequences, the term "percent complementarity" refers to two or more sequences or subsequences that, when compared and aligned for maximum match across a comparison window or specified region, are complementary, have complementary nucleotides, or possess a specified percentage of nucleotides, e.g., at least 60% identity, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% or more, when compared and aligned for maximum match across a comparison window or specified region, as measured using a sequence comparison algorithm or by manual alignment and visual inspection.

[0086] In the context of two or more nucleotide sequences, the term “identical” or “identity” percentage refers to two or more sequences or subsequences that, when compared and aligned for maximum match across a comparison window or specified region, are identical, the same, or have a specified percentage of nucleotides, e.g., at least 60% identity, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% or more, when measured using a sequence comparison algorithm or by manual alignment and visual inspection.

[0087] When determining the complementarity percentage or identity percentage, chemical modifications are ignored if the nucleic acid base retains its functional ability to form Watson-Crick base pairs (for example, 5-methylcytosine is considered identical to cytosine for the purpose of calculating % identity).

[0088] In oligonucleotide sequence comparison (e.g., to determine identity or complementarity), typically one nucleotide sequence serves as a reference sequence compared to the candidate sequence. Alignment can be performed using various methods available to those skilled in the art, such as visual alignment or using publicly available software with known algorithms, to achieve maximum alignment. Such programs include the BLAST program, ALIGN, ALIGN-2 (Genentech, South San Francisco, Calif.), or Megalign (DNASTAR). The parameters used for alignment to achieve maximum alignment can be determined by those skilled in the art.

[0089] As used herein, “hybridize” or “hybridization” means the pairing of complementary nucleotide sequences (e.g., between an antisense compound and its target nucleic acid, or between an antisense strand and a sense strand). As used herein, “specifically hybridize” means the ability of a reference nucleic acid to hybridize to another nucleic acid molecule with a higher affinity than it would hybridize to one nucleic acid molecule.

[0090] "Expression" refers to the transcription and / or translation of endogenous genes, heterologous genes or nucleic acid segments, or transgenes in a cell. For example, expression may refer to the transcription and stable accumulation of sense (mRNA) or functional RNA. Expression may also refer to the production of proteins.

[0091] The term "gene" refers to a nucleic acid (e.g., DNA or RNA) sequence containing the coding sequence necessary for the production of a polypeptide or its precursor.

[0092] The phrase "modulate the expression of a target gene or sequence" means a change in the expression of a target gene or sequence (e.g., increase or decrease) (e.g., via degradation or translation inhibition of the target). For example, this includes inhibiting, reducing, or decreasing the expression of a target gene or sequence. This also includes changes in alternative splicing that may result in a change in the absolute or relative amount of a particular splice variant.

[0093] The terms “subject,” “individual,” and “patient,” as used interchangeably herein, refer to mammals, including but not limited to humans, non-human primates, rodents (e.g., rats, mice, and guinea pigs), rabbits, cattle, pigs, horses, and other mammalian species. In some embodiments, the patient is a human.

[0094] Terms such as “treatment” and “treating” are used herein to generally mean obtaining a desired pharmacological and / or physiological effect. Terms such as “treating” and “treating” include methods or steps taken to reduce, improve or alleviate the number, severity, side effects, and / or frequency of at least one symptom or pathological consequence of a disease, disorder, or illness in a subject. Treating a disease, disorder, or illness may include improving at least one symptom of a particular disease, disorder, or illness, even if there is no impact on the underlying pathophysiology. “Treatment” or “treatment” may refer to any sign of success in treating or improving an injury, disease, or condition, and may include any objective or subjective parameters such as relief, remission, improved patient survival, increased survival time or survival rate, reduced symptoms, or increased patient tolerance to the injury, disease, or condition, slower rate of degeneration or decline, or improved physical or mental health. Treatment may aim for a cure in the sense of partial or complete cure of a disease, illness, symptom, or side effects resulting from a disease, disorder, or illness. Treatment can be preventive in the sense of preventing or partially preventing a disease, its symptoms, or condition. Prevention includes preventing the onset or recurrence of a disease in individuals who are susceptible to the disease but have not yet been diagnosed with it. Prevention also includes preventing the onset or recurrence of symptoms or pathological outcomes of a disease in individuals who are susceptible to the symptoms or pathological outcomes of the disease but have not yet been diagnosed with them. Treatment can also be preventive in the sense of delaying the onset of a disease or delaying the symptoms or condition of the disease. Delaying the onset of a disease or delaying the symptoms or pathological outcomes of a disease means delaying, hindering, slowing, postponing, stabilizing, suppressing, and / or postponing the onset of the disease or the onset of symptoms or pathological outcomes of the disease. This delay can be of a different duration depending on the disease history being treated and / or the individual being treated. Treatment can refer to treatment aimed solely at curing, treatment aimed solely at prevention, or treatment aimed at both curing and prevention.Those who require treatment (or are the subjects requiring treatment) may include individuals previously diagnosed with a disease, disability, or illness, or those identified as being at risk of developing a disease, disability, or illness. Furthermore, “treating” or “treatment” may refer to the regulation of target gene expression, such as gene knockdown or gene knockout. For example, the expression of a target gene or sequence is inhibited or reduced by, for example, at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100% compared to expression in a control. Treatment or improvement of symptoms may be based on objective or subjective parameters. The effect of treatment may be compared to individuals who have not received treatment or to a pool of individuals, or to the same patient at different point in time before or during treatment.

[0095] As used herein, “relieve” means improving at least one symptom (for example, compared to the same symptom without treatment) using an effective dose of MAPT ASO and a conjugate containing it. In certain embodiments, relief is a reduction in the severity or frequency of a symptom, or a delay in the onset or progression of the severity or frequency of a symptom.

[0096] The terms “prevention” or “prevention” refer to the use of an effective dose of MAPT ASO and a conjugate containing it to reduce or prevent the onset or recurrence of a disease or its symptoms in subjects prone to developing or relapsing the disease.

[0097] The term "pharmaceutically acceptable additive" refers to a non-active medicinal ingredient (e.g., buffers, carriers, or preservatives) that is biologically or pharmacologically compatible for use in humans or animals.

[0098] As used herein, the “therapeutic dose” or “therapeutic effective dose” of a drug is the amount of drug that treats, reduces, alleviates, or reduces the severity of the symptoms of a disease in a subject. The “therapeutic dose” or “therapeutic effective dose” of a drug can improve patient survival, increase survival time or survival rate, reduce symptoms, make injury, disease or condition more tolerable, slow the rate of degeneration or decline, or improve the patient’s physical or mental well-being. The therapeutic effective dose may vary depending on factors such as the subject’s condition, age, sex, and weight, as well as the population of cells being administered.

[0099] "Dosage," "unit dose," or "administration" refers to physically separate units suitable for use in the subject, each unit containing a predetermined amount of active pharmaceutical ingredient and / or pharmaceutical composition.

[0100] The term "administration" refers to a method of delivering a drug, compound, or composition to a desired site of action. Such methods include, but are not limited to, topical delivery, parenteral delivery, intravenous delivery, intradermal delivery, intramuscular delivery, intrathecal delivery, colonic delivery, rectal delivery, or intraperitoneal delivery. In some embodiments, the proteins described herein are administered intravenously.

[0101] The terms “control” or “control value” refer to a reference value or baseline value. A suitable control can be determined by those skilled in the art. In some cases, the control value may be determined based on the baseline of the same subject or within the same experiment. For example, a measurement of MAPT gene expression measured before treatment with MAPT ASO or a conjugate or composition containing it described herein may serve as the control value for a measurement of MAPT levels after treatment in the same subject. In other cases, the control value may be determined against a control subject (e.g., a healthy control or a disease control) or against the mean in a population of control subjects (e.g., a population of 10, 20, 50, 100, 200, 500, 1000 or more control subjects). For example, a measurement of MAPT gene expression levels in a subject at baseline or after treatment may be compared to the value of a healthy control.

[0102] I. MAPT-targeted oligonucleotides This specification discloses MAPT antisense oligonucleotides (MAPT ASOs) complementary to the human MAPT gene. The MAPT ASOs described herein are at least 90% complementary to the sequence in the human MAPT gene. In some embodiments, the MAPT ASOs described herein are at least 90% complementary to the sequence contained in SEQ ID NO: 1. In some embodiments, the MAPT ASOs described herein contain, or consist of, a nucleic acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% complementary to the sequence contained in SEQ ID NO: 1. In some embodiments, the MAPT ASO described herein comprises or consists of a nucleic acid sequence having a length of 10 to 25 nucleotides and is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% complementary to the sequence contained in SEQ ID NO: 1. In some embodiments, the MAPT ASO described herein comprises or consists of a nucleic acid sequence having a length of 10 to 25 nucleotides and is at least 90% or at least 95% complementary to the sequence contained in SEQ ID NO: 1. In some embodiments, the MAPT ASO described herein comprises or consists of a nucleic acid sequence having a length of 10 to 25 nucleotides and is 100% complementary to the sequence contained in SEQ ID NO: 1.

[0103] For example, in some embodiments, the MAPT ASO disclosed herein comprises at least 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 consecutive nucleic acid bases, and is complementary to the sequences present at nucleic acid bases 66,750 to 66,786, 66,760 to 66,786, 66,750 to 66,776, 66,754 to 66,786, or 66,760 to 66,776 of SEQ ID NO: 1.

[0104] In some embodiments, the MAPT ASO disclosed herein comprises at least 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 consecutive nucleic acid bases, and is complementary to the sequences present at nucleo bases 78,431 to 78,468, 78,431 to 78,458, 78,441 to 78,468, or 78,441 to 78,458 of SEQ ID NO: 1.

[0105] In some embodiments, the MAPT ASO disclosed herein comprises at least 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 consecutive nucleic acid bases and is complementary to the sequences present at nucleic acid bases 78,621 to 78,659, 78,621 to 78,649, 78,631 to 78,659, or 78,631 to 78,649 of SEQ ID NO: 1.

[0106] In some embodiments, the MAPT ASO disclosed herein comprises at least 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 consecutive nucleic acid bases, and is complementary to the sequences present at nucleic acid bases 78,818 to 78,855, 78,828 to 78,855, 78,818 to 78,845, or 78,828 to 78,845 of SEQ ID NO: 1.

[0107] In some embodiments, the MAPT ASO disclosed herein comprises at least 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 consecutive nucleic acid bases, and is complementary to the sequences present at nucleic acid bases 78,431 to 78,659, 78,431 to 78,649, 78,441 to 78,659, or 78,441 to 78,649 of SEQ ID NO: 1.

[0108] In some embodiments, the MAPT ASO disclosed herein comprises at least 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 consecutive nucleic acid bases, and is complementary to the sequences present at nucleic acid bases 78,641 to 78,855, 78,641 to 78,845, 78,631 to 78,855, or 78,631 to 78,845 of SEQ ID NO: 1.

[0109] In some embodiments, the MAPT ASO disclosed herein comprises at least 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 consecutive nucleic acid bases and is complementary to the sequences present in the nucleic acid bases 78,431 to 78,855, 78,441 to 78,855, 78,431 to 78,845, 78,441 to 78,845, 78,441 to 78,498, 78,517 to 78,678, or 78,697 of SEQ ID NO: 1.

[0110] In some embodiments, the MAPT ASO disclosed herein comprises at least 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 consecutive nucleic acid bases, and is complementary to the sequences present in the nucleic acid bases 90,205 to 90,243, 90,205 to 90,233, 90,215 to 90,243, 90,208 to 90,243, or 90,215 to 90,233 of Sequence ID No. 1.

[0111] The length of the ASO is in the range of about 10 to 30 base pairs (bp), but may be longer or shorter. For example, in certain embodiments, the ASO is about 10 to about 60 nucleotides long (i.e., about 10 to about 60 linked nucleoside lengths), or 10 to about 50 nucleotides long, or about 10 to about 40 nucleotides long. In certain embodiments, the ASO is about 10 to 30 nucleotides long, or about 12 to 30 nucleotides long, or about 14 to about 30 nucleotides long, or about 15 to about 30 nucleotides long, or about 16 to about 30 nucleotides long, or about 17 to about 30 nucleotides long, or about 18 to about 30 nucleotides long, or about 18 to about 28 nucleotides long, or about 18 to about 26 nucleotides long, or about 18 to about 24 nucleotides long, or about 15 to about 25 nucleotides long, or about 16 to about 20 nucleotides long, or about 16 to about 19 nucleotides long, or about 17 to about 19 nucleotides long. In certain embodiments, the ASO is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides long. In certain embodiments, the ASO is 16, 17, 18, or 19 nucleotides long.

[0112] The length of MAPT ASO may vary, but in certain embodiments, the oligonucleotide is about 10 to about 60 nucleotides long, or about 10 to about 30 nucleotides long, or about 18 to about 30 nucleotides long, or about 15 to about 25 nucleotides long, or about 16 to about 20 nucleotides long. In some embodiments, MAPT ASO is about 16 to about 20 nucleotides long. In some embodiments, MAPT ASO is about 16 to about 19 nucleosides long. In some embodiments, MAPT ASO is about 17 to about 19 nucleosides long. In some embodiments, MAPT ASO is 16, 17, 18, 19, or 20 nucleotides long. In some embodiments, MAPT ASO is 16 nucleotides long. In some embodiments, MAPT ASO is 17 nucleotides long. In some embodiments, MAPT ASO is 18 nucleotides long. In some embodiments, MAPT ASO is 19 nucleotides long. In some embodiments, MAPT ASO is 20 nucleotides long.

[0113] In some embodiments, the MAPT ASO disclosed herein comprises or comprises a nucleic acid sequence comprising at least 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 consecutive nucleic acid bases and at least one modified internucleoside bond and / or at least one modified sugar, selected from any one of SEQ ID NOs. 4-9 and 19-37 (Table 1). In some embodiments, the MAPT ASO disclosed herein comprises a nucleic acid sequence selected from any one of SEQ ID NOs. 4-9 and 19-37, and further comprises at least one modified internucleoside bond and / or at least one modified sugar. In some embodiments, the MAPT ASO comprises a nucleic acid sequence described in any one of SEQ ID NOs. 4-9 and 19-37, and further comprises at least one modified internucleoside bond and / or at least one modified sugar. In some embodiments, the MAPT ASO disclosed herein targets an exon of the MAPT gene and comprises or consists of a nucleic acid sequence described in SEQ ID NO: 4 and SEQ ID NOs: 19-21, and further comprises at least one modified internucleoside linkage and / or at least one modified sugar. In some embodiments, the MAPT ASO disclosed herein targets an intron of the MAPT gene and comprises or consists of a nucleic acid sequence selected from SEQ ID NOs: 5-9 and SEQ ID NOs: 22-37, and further comprises at least one modified internucleoside linkage and / or at least one modified sugar. In some embodiments, the MAPT ASO disclosed herein comprises 15-25 linked nucleosides. In some embodiments, the MAPT ASO disclosed herein comprises 16-20 linked nucleosides. In some embodiments, the MAPT ASO disclosed herein comprises 16-19 linked nucleosides. In some embodiments, the MAPT ASO disclosed herein comprises 17 to 19 linked nucleosides.In some embodiments, the MAPT ASOs disclosed herein (e.g., 15-25 nucleotide length or 17-19 nucleotide length) are at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% complementary to SEQ ID NO: 1.

[0114] In some embodiments, the MAPT ASO disclosed herein is 16 to 20 nucleotides long and includes the sequence described in any one of SEQ ID NOs. 10 to 15 and SEQ ID NOs. 38 to 56 (Table 2), and further includes at least one modified internucleoside bond and / or at least one modified sugar. In some embodiments, the MAPT ASO disclosed herein is 17 to 19 nucleotides long and includes the sequence described in any one of SEQ ID NOs. 10 to 15 and SEQ ID NOs. 38 to 56, and further includes at least one modified internucleoside bond and / or at least one modified sugar. In some embodiments, the MAPT ASO disclosed herein includes a gapmer-type ASO having a gap segment containing the sequence described in any one of SEQ ID NOs. 10 to 15 and SEQ ID NOs. 38 to 56. In some embodiments, the MAPT ASO disclosed herein includes a gapmer-type ASO having a gap segment consisting of the sequence described in any one of SEQ ID NOs. 10 to 15 and SEQ ID NOs. 38 to 56. In some embodiments, MAPT ASO includes sequences that are 100% identical to any of sequence numbers 10-15 and 38-56 (Table 2), and sequences that are at least 85%, at least 90%, at least 95%, or 100% identical to any of sequence numbers 4-9 and 19-37 (Table 1). [Table 1] [Table 2]

[0115] Biblical oligonucleotide modifications In certain embodiments, the MAPT ASOs described herein may include at least one nucleic acid modification, such as one selected from the group consisting of modified internucleoside bonds, modified nucleic acid bases, modified sugars, and combinations thereof (e.g., including at least one modified internucleoside bond and / or at least one modified sugar). Such modifications may be used to alter pharmacokinetics (extended half-life by improving nuclease resistance), pharmacodynamics (superior affinity to target RNA), or endocytotic uptake. However, many modifications prevent cleavage by RNase H, which is the desired mechanism of action for many ASOs. Therefore, certain RNase HASOs may be designed as chimeras, where different bases are mixtures of different chemicals, or as gapmers, where some modifications are located on the "wings" rather than the central base.

[0116] Therefore, the MAPT ASO described herein may include one or more nucleic acid modifications. In certain embodiments, the MAPT ASO includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or 40 or more modifications. In certain embodiments, the MAPT ASO described herein includes one or more nucleotide modifications (e.g., to nucleic acid bases or sugar moieties). In certain embodiments, 25% or more of the nucleotides present in the MAPT ASO are modified. In certain embodiments, 50% or more of the nucleotides present in the MAPT ASO are modified. In certain embodiments, 75% or more of the nucleotides present in the MAPT ASO are modified. In certain embodiments, 100% of the nucleotides present in the MAPT ASO are modified. Modified sugar portion, nucleoside / nucleotide

[0117] In certain embodiments, MAPT ASO comprises one or more nucleic acid base modifications. In certain embodiments, MAPT ASO comprises one or more modifications to a sugar moiety (e.g., a furanosyl with substituents at the 2', 3', 4', and / or 5' positions). In certain embodiments, the substituted sugar moiety comprises a bicyclic sugar moiety. In some embodiments, the bicyclic sugar moiety comprises a chemical bridge between the 4' and 2' positions of the sugar, each chemical bridge independently selected from 4'-CH(R)-O-2' and 4'-(CH2)2-O-2', wherein the formula, each R independently comprises H, C1 -Selected from C6 alkyl and C1-C6 alkoxy. In some embodiments, the bicyclic sugar portion includes a chemical bridge between the 4′ and 2′ positions of the sugar, where each chemical bridge is 4′-CH(R)-O-2′, and each R is independently H.

[0118] Modified nucleosides / nucleotides include, but are not limited to, 2'-O-methyl (2'OMe) residues, 2'O-methoxyethyl (MOE) residues, restricted nucleic acid residues (e.g., S-cEt, R-cEt, S-cMOE, and R-cMOE), peptide nucleic acid (PNA) residues, locked nucleic acid (LNA) residues, and 5-methylcytidine residues (methylated cytosine residues) (see also Scoles, et al., Neurol Genet Apr2019, 5(2)e323). In certain embodiments, MAPT ASO comprises one or more 2'-MOE residues. In certain embodiments, MAPT ASO comprises one or more OMe or F residues (e.g., 2'-F or 2'OMe). In certain embodiments, MAPT ASO comprises one or more restricted (e.g., S-cEt, R-cEt, S-cMOE, and R-cMOE) and / or LNA residues. Nucleic acids are considered "locked" if they have a methylene crosslink connection formed between the 2'-oxygen and the 4'-carbon of the ribose sugar molecule. In certain embodiments, MAPT ASO is morpholino (i.e., it contains certain modifications in the sugar moiety).

[0119] In certain embodiments, the MAPT ASO described herein comprises one or more LNA residues. In certain embodiments, the MAPT ASO described herein comprises one or more 5-methylcytidine residues. In certain embodiments, the MAPT ASO described herein comprises one or more LNA residues and one or more 5-methylcytidine residues. In some embodiments, all cytosines in the MAPT ASO are 5-methylcytidine. Inter-modified nucleoside bonding

[0120] In certain embodiments, MAPT ASO includes modifications to one or more nucleoside bonds (i.e., the native phosphodiester (PO) bond is modified). In certain embodiments, such modifications are made, for example, to reduce nuclease activity. Thus, in certain embodiments, MAPT ASO contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more modified nucleoside bonds. In certain embodiments, 25% or more of the modified nucleoside bonds are modified. In certain embodiments, 50% or more of the modified nucleoside bonds are modified. In certain embodiments, 75% or more of the modified nucleoside bonds are modified. In certain embodiments, 100% of the nucleoside bonds present in MAPT ASO are modified.

[0121] Skeletal modifications are well known in the art and include, but are not limited to, phosphorothioate (PS) bonds, chiral phosphorothioate bonds, phosphorodiamidate bonds, phosphorodithioate bonds, aminoalkyl phosphotriester bonds, phosphotriester bonds, thiophosphate bonds, phosphonate bonds, methylphosphonate bonds, alkylphosphonate bonds, 3'-alkylene phosphonate bonds, chiral phosphonate bonds, 3'-aminophosphoramide bonds, aminoalkylphosphoramide bonds, phosphinic acid bonds, thionoalkylphosphonate bonds, thionophosphoramide bonds, thionoalkyl-phosphotriester bonds, borano-phosphate bonds, morpholino bonds, and peptide nucleic acid (PNA) bonds. For example, in certain embodiments, one or more nucleoside bonds in MAPT ASO (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more) are substituted with phosphorothioate (PS) bonds. In certain embodiments, MAPT ASO contains a mixture of modified and unmodified bonds. Modification in one nucleoside bond may be independent of modification in another nucleoside bond. In certain embodiments, all nucleoside bonds in MAPT ASO are modified bonds. In certain embodiments, all nucleoside bonds in MAPT ASO are PS bonds. In some embodiments, all nucleoside bonds in LPA ASO are phosphorothioate bonds. In certain other embodiments, one or more nucleoside bonds in MAPT ASO (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25, or more) are replaced by phosphorodiamidate bonds. In certain embodiments, MAPT ASO is phosphorodiamidate morpholino (PMO).

[0122] In certain embodiments, the nucleoside bonds are sterically random with respect to the chiral centers (Rp and Sp). In certain embodiments, the arrangement of Rp and Sp in MAPT ASO is optimized for a specific configuration.

[0123] Antisense oligonucleotide motif In certain embodiments, nucleic acid modification by MAPT ASO is included in the pattern. In certain embodiments, MAPT ASO is a gapmer. The modification pattern of gapmer MAPT ASO generally has the formula 5'-X a -Y a -Z a -3', where X a and Z a are adjacent regions around the gap region Y a . In certain embodiments, the Y a region (gap region) is a continuous stretch of nucleotides (i.e., linked nucleosides), for example, a region of at least 6 DNA nucleotides that can mobilize an RNAse such as RNase H. In certain embodiments, the Ya region is at least 8 DNA nucleotides. In certain embodiments, the Y a region is from about 9 to about 15 DNA nucleotides. In certain embodiments, the Y a region is from about 11 to about 13 DNA nucleotides. In certain embodiments, the Y a region is 10, 11, 12, or 13 DNA nucleotides. In certain embodiments, the gapmer can bind to the target nucleic acid, at which point the RNAse is mobilized and then cleave the target nucleic acid. In certain embodiments, the Y a region is adjacent to both the 5' and 3' regions X a and Z a , and each contains 1 to 6 modified nucleotides in each of X a and Z a . In certain embodiments, the Y a region is adjacent to both the 5' and 3' regions X a and Z a , where X a and Z a contain modified nucleotides with modified sugars. In certain embodiments, each nucleotide in X a and Z aEach nucleotide in contains a modified nucleotide having sugar modification. Each modified nucleotide may, but is not limited to, a 2-MOE modified nucleotide, a bicyclic nucleotide, an LNA nucleotide, or a cET modified nucleotide. In certain embodiments, the modified nucleotides are located in the 5' and 3' regions of MAPT ASO, but certain modified nucleotides and / or modifying bonds may be located in the center (gap (i.e., Y) of the molecule). a They may or may not be present in the )) region. In certain embodiments, modified nucleotides are present in the 5' and 3' regions of MAPT ASO, and certain modified nucleotides are not present in the central region of the molecule (for example, LNA residues are not present in the central region, but the central region may contain modified bonds such as PS bonds). In certain embodiments, X a and Z a Each of them is independently about 3 to 6 nucleotides long. In certain embodiments, X a and Z a Each of these is independently 3, 4, or 5 nucleotides long. In certain embodiments, X a and Z a Each contains three modified nucleotides (e.g., modified sugars). In certain embodiments, X a and Z a Each contains three modified nucleotides, and each contains a modified sugar. In a particular embodiment, Xa 及び Za は Each comprises three modified nucleotides, each comprising a modified sugar and a modified nucleoside bond. In a particular embodiment, the three modified nucleotides are X a and Z a They are arranged in series in each of them.

[0124] In certain embodiments, MAPT ASO is a gapmer including LNA and PS modifications. For example, in certain embodiments, MAPT ASO is formula 5'-X a -Y a -Z a A gapmer having a -3' modification pattern, where X a and Z aThis is the gap region Y a Let X be the adjacent region around it. a and Z a Each contains three LNA-modified nucleotides (for example, three consecutive LNA-modified nucleotides), and the gap region Y a It contains PS bonds. In some embodiments, all nucleoside bonds in MAPT ASO contain PS bonds. In some embodiments, MAPT ASO contains a mixture of PS bonds and other modified nucleoside bonds. In certain embodiments, MAPT ASO further contains one or more 5-methylcytidine residues. In certain embodiments, gap region Y a It does not contain LNA residues.

[0125] In certain embodiments, MAPT ASO comprises the following configuration from 5′ to 3′: a 5′ wing segment having 1 to 6 nucleosides, each nucleoside of this 5′ wing segment containing a modified sugar; a gap segment having 8 to 15 nucleosides, each nucleoside of this gap segment containing a deoxynucleoside; and a 3′ wing segment having 1 to 6 nucleosides, each nucleoside of this 3′ wing segment containing a modified sugar. In some embodiments, all nucleoside bonds in MAPT ASO include modified nucleoside bonds. In some embodiments, all nucleoside bonds in MAPT ASO include PS bonds. In some embodiments, MAPT ASO contains a mixture of PS bonds and other modified nucleoside bonds. In some embodiments, all nucleosides within the 5′ and 3′ wing segments include 2-MOE modified nucleosides, bicyclic nucleosides, LNA nucleosides, or cET modified nucleosides.

[0126] In certain embodiments, MAPT ASO comprises the following configuration from 5′ to 3′: a 5′ wing segment having three nucleosides, each nucleoside of this 5′ wing segment containing a modified sugar; a gap segment having 11 to 13 nucleosides, each nucleoside of this gap segment containing a deoxynucleoside; and a 3′ wing segment having three nucleosides, each nucleoside of this 3′ wing segment containing a modified sugar. In some embodiments, all nucleoside bonds in MAPT ASO are modified nucleoside bonds. In some embodiments, all nucleoside bonds in MAPT ASO include PS bonds. In some embodiments, all nucleotides in the 5′ and 3′ wing segments include 2-MOE modified nucleotides, bicyclic nucleotides, LNA nucleotides, or cET modified nucleotides.

[0127] In some embodiments, the MAPT ASOs provided herein include or consist of modified nucleic acid sequences having at least about 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the modified sequences shown in Table 7. In certain embodiments, the MAPT ASO includes the modified nucleic acid sequences shown in Table 7. In certain embodiments, the MAPT ASO includes a nucleic acid base sequence consisting of the modified nucleic acid sequences shown in Table 7. In certain embodiments, the MAPT ASO includes a nucleic acid base sequence consisting of the modified nucleic acid sequences shown in Table 7, and the MAPT ASO is conjugated to a target ligand or delivery vehicle (e.g., a vehicle that facilitates delivery across the blood-brain barrier). The MAPT ASOs in Table 7 are gapmers having wing segments with LNA-modified nucleosides. Similar MAPT ASO gapmers can be constructed that have wing segments containing, but not limited to, 2'-MOE nucleotides and / or cEt nucleosides, as well as other ribose-modified nucleosides.

[0128] In some embodiments, the modified oligonucleotides (MAPT ASOs) provided herein are those conforming to one of the following chemical structures: [ka] [ka] [ka] [ka] [ka] [ka] , or its salt.

[0129] ASO end clipping In certain embodiments, MAPT ASO targeted to tau nucleic acid may be shortened or cleaved. In some embodiments, MAPT ASO may be shortened or cleaved by endonuclease activity. For example, a single subunit (e.g., a nucleotide or a portion thereof) may be deleted from the 5' end (5' cleavage) or, instead, deleted from the 3' end (3' cleavage). A shortened or cleaved antisense compound targeting tau nucleic acid may have two or more subunits removed from the 5' end of MAPT ASO, or two or more subunits removed from the 3' end.

[0130] II. MAPT ASO Conjugate Any of the described MAPT ASOs can be conjugated to a targeting group or delivery vehicle. MAPT ASOs can be directly or indirectly conjugated to a targeting group or delivery vehicle. MAPT ASOs can be conjugated to a targeting group or delivery vehicle, or to any nucleotide in the ASO, at the 5' end and the 3' end of the ASO. The targeting group or delivery vehicle can be conjugated to the sugar moiety, base moiety, nucleoside bond, or terminal (5' or 3') of the MAPT ASO. Any targeting group or delivery vehicle known in the art can be conjugated to a MAPT ASO to facilitate or improve the delivery of the MAPT ASO to target cells or tissues of interest. Targeting groups include, but are not limited to, molecules that specifically bind to transferrin receptors (TfRs) or molecules expressed on the luminal surface of the blood-brain barrier. The molecules may be, but are not limited to, anti-TfR antibodies or their TfR-binding fragments, or Fc polypeptides modified to bind to TfR. The delivery vehicle may be, but is not limited to, a molecule, brain shuttle, protein, liposome, lipoplex, or lipid nanoparticle.

[0131] In some embodiments, MAPT ASO is linked to a targeting group or delivery vehicle via a linker. Any linker known in the art that is suitable for conjugating MAPT ASO to another molecule can be used.

[0132] III. How to use MAPT ASO can be used for a variety of purposes, including therapeutic indications.

[0133] In some embodiments, methods for suppressing MAPT gene expression in a subject are described, which include administering an effective amount of the MAPT ASO, conjugate, or composition described herein to the subject. In some embodiments, MAPT ASO, conjugate, or composition thereof is provided for use in reducing MAPT gene expression in cells of a subject. In certain embodiments, administration of the described MAPT ASO (or a conjugate or composition containing the MAPT ASO) to cells or a subject reduces MAPT gene expression in the cells or subject. The expression can be reduced by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% or more compared to the expression in a control (e.g., cells or a subject not administered with the MAPT ASO, conjugate, or composition described herein) or the MAPT expression level in cells or a subject before administration of the MAPT ASO.

[0134] In certain embodiments, methods are provided for reducing MAPT mRNA expression in subjects where it is necessary to reduce MAPT mRNA expression, the methods comprising administering to the subject a MAPT ASO, conjugate, or composition described herein.

[0135] Certain embodiments also provide a method for reducing tau expression in a subject that requires a reduction in tau expression, the method comprising administering to the subject a MAPT ASO, conjugate, or composition described herein.

[0136] In certain embodiments, the MAPT ASO described herein may be used to reduce MAPT expression in brain cells. Brain cells may be, but are not limited to, neurons, deep brain cells, hippocampal cells, or entorhinal cortex cells. Accordingly, in certain embodiments, a method is provided for generating neuronal cells with reduced tau expression, the method comprising delivering a MAPT ASO, conjugate, or composition described herein to neuronal cells, wherein the MAPT ASO reduces the expression level of the MAPT gene (e.g., endogenous MAPT gene). In certain embodiments, a method is provided for generating neuronal cells with reduced tau expression, the method comprising delivering a MAPT ASO, conjugate, or composition described herein to neuronal cells, wherein the MAPT ASO reduces the expression level of the MAPT gene (e.g., endogenous MAPT gene). In certain embodiments, the MAPT ASO binds to MAPT transcripts and reduces the expression level of MAPT transcripts in cells. In certain embodiments, the MAPT ASO binds to MAPT transcripts and recruits RNase H to degrade the transcripts.

[0137] In certain embodiments, the MAPT ASO described herein may be used to reduce MAPT expression in the spinal cord or cells of the spinal cord.

[0138] In certain embodiments, the MAPT ASO described herein is delivered to target cells in the brain, such as neurons, deep brain cells, hippocampal cells, or entorhinal cortex cells.

[0139] In certain embodiments, a method is provided for delivering MAPT ASO to the CNS of a subject that requires delivery of MAPT ASO to the CNS, the method comprising administering MAPT ASO, a conjugate, or a composition described herein to the subject, wherein the MAPT ASO reduces the expression level of a MAPT gene (e.g., an endogenous MAPT gene).

[0140] In certain embodiments, MAPT ASO reduces the expression level of the endogenous MAPT gene or the level of MAPT transcript by about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 95% compared to the level of MAPT transcript without administration of MAPT ASO. In certain embodiments, the expression of the endogenous MAPT gene or the level of MAPT transcript is reduced by at least about 50% compared to the expression level before administration of MAPT ASO. In certain embodiments, the expression of the endogenous MAPT gene or the level of MAPT transcript is reduced by at least about 70% compared to the expression level before administration of MAPT ASO.

[0141] The MAPT ASOs, conjugates, and compositions described herein (e.g., pharmaceutical compositions) may be used to treat, prevent, or improve tau-related diseases, disorders, and conditions. Accordingly, in some embodiments, methods for treating, preventing, or improving tau-related diseases, disorders, and conditions in subjects requiring treatment, prevention, or improvement are provided herein. In certain embodiments, tau-related diseases are tau-related neurodegenerative disorders. In certain embodiments, tau-related diseases include tauopathies, Alzheimer's disease, frontotemporal dementia (FTD), FTDP-17, progressive supranuclear palsy (PSP), chronic traumatic encephalopathy (CTE), corticobasal degeneration (CBD), epilepsy, and Dravet syndrome.

[0142] Accordingly, in certain embodiments, methods are provided for treating subjects in need of treatment for tau-related neurodegenerative diseases, the methods comprising administering to the subject a MAPT ASO, conjugate, or composition described herein.

[0143] In certain embodiments, the tau-related neurodegenerative disease is Alzheimer's disease. Therefore, in certain embodiments, a method is provided for treating Alzheimer's disease, the method comprising administering one of the MAPT ASOs, conjugates, or compositions described herein to a subject in need. The subject may have been diagnosed with Alzheimer's disease, been diagnosed with one or more symptoms of Alzheimer's disease, or be at risk of developing one or more symptoms of Alzheimer's disease or related thereto.

[0144] In certain embodiments, the subject is a human subject.

[0145] The MAPT ASO, MAPT ASO conjugate, or compositions containing MAPT ASO or conjugate described herein are administered to the subject in a therapeutically effective dose. However, the dose may vary depending on several factors, including the selected route of administration, the composition formulation, the patient's response, the severity of the condition, the subject's body weight, and the prescribing physician's judgment. The dose may be increased or decreased over time as needed for the individual patient.

[0146] In various embodiments, MAPT ASO, MAPT ASO conjugate, or compositions comprising MAPT ASO or conjugate as described herein are administered parenterally. In some embodiments, MAPT ASO, conjugate, or compositions are administered intravenously. Intravenous administration may be carried out, for example, by drip infusion over about 10 to about 30 minutes, or over at least 1, 2, or 3 hours. In some embodiments, MAPT ASO or conjugate is administered as an intravenous bolus. A combination of infusion and bolus administration may be used.

[0147] In some parenteral administration embodiments, MAPT ASO, conjugate, or composition is administered intraperitoneally, subcutaneously, intradermally, or intramuscularly. In some embodiments, MAPT ASO, conjugate, or composition is administered intradermally or intramuscularly. In some embodiments, MAPT ASO, conjugate, or composition is administered intrathecally, such as by epidural administration, or intraventricularly.

[0148] In other embodiments, the MAPT ASOs, conjugates, or compositions described herein may be administered orally, pulmonaryly, intranasally, intraocularly, or topically. Pulmonary administration may also be performed, for example, by using an inhaler or nebulizer, and by using a formulation with an aerosolizing agent.

[0149] IV. Pharmaceutical compositions and kits In another embodiment, pharmaceutical compositions and kits comprising MAPT ASO or conjugate as described herein are provided.

[0150] Pharmaceutical composition Guidelines for preparing formulations for the purposes of use described herein can be found in various handbooks on pharmaceutical formulations and preparations known to those skilled in the art.

[0151] In some embodiments, the pharmaceutical composition comprises MAPT ASO or MAPT ASO conjugate as described herein, and further comprises one or more pharmaceutically acceptable carriers, diluents and / or excipients.

[0152] As used herein, a pharmaceutically acceptable carrier comprises any solvent, dispersion medium, or coating agent that is physiologically compatible and preferably does not interfere with or otherwise inhibit the activity of the active substance. A variety of pharmaceutically acceptable excipients are well known. In some embodiments, the carrier is suitable for intravenous, intrathecal, lateral ventricular, intramuscular, oral, intraperitoneal, transdermal, topical, or subcutaneous administration. A pharmaceutically acceptable carrier may contain, for example, one or more physiologically acceptable compounds that stabilize the composition of MAPT ASO or conjugate, or increase or decrease its absorption. Examples of physiologically acceptable compounds include carbohydrates such as glucose, sucrose, or dextran; antioxidants such as ascorbic acid or glutathione; chelating agents; low molecular weight proteins; compositions that reduce the clearance or hydrolysis of the active substance; or excipients or other stabilizers and / or buffers. Other pharmaceutically acceptable carriers and their formulations are also available in the art.

[0153] The pharmaceutical compositions described herein may be prepared in ways known to those skilled in the art, for example, by conventional mixing, dissolution, granulation, sugar-coated tablet production, emulsification, encapsulation, encapsulation, or freeze-drying processes. The following methods and excipients are merely illustrative and not limiting.

[0154] For oral administration, the MAPT ASO or conjugate described herein can be formulated by combining it with pharmaceutically acceptable carriers well known in the art. The compounds can be formulated with such carriers as tablets, pills, sugar-coated tablets, capsules, emulsions, lipophilic and hydrophilic suspensions, liquids, gels, syrups, slurries, and the like for oral administration by the patient being treated. Pharmaceutical preparations for oral use can be obtained by mixing the MAPT ASO or conjugate with a solid excipient, optionally grinding the resulting mixture, processing the granular mixture, and, if desired, adding appropriate excipients to obtain tablets or sugar-coated tablet cores. Suitable excipients include fillers such as sugars containing lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as corn starch, wheat starch, rice starch, potato starch, gelatin, tragacanth gum, methylcellulose, hydroxypropyl methylcellulose, sodium carboxymethylcellulose, and / or polyvinylpyrrolidone. Disintegrants such as cross-linked polyvinylpyrrolidone, agar, or alginic acid or salts thereof such as sodium alginate may be added as needed.

[0155] As disclosed above, the MAPT ASO or conjugate described herein can be formulated for parenteral administration, for example, by bolus injection or continuous infusion. For injection, the MAPT ASO or conjugate can be formulated into a preparation by dissolving, suspending or emulsifying it in an aqueous or non-aqueous solvent, such as a vegetable or other similar oil, synthetic fatty acid glycerides, esters of higher fatty acids or propylene glycol, and, if necessary, with conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifiers, stabilizers and preservatives. In some embodiments, the MAPT ASO or conjugate can be formulated in an aqueous solution, preferably a physiologically compatible buffer such as Hanks' solution, Ringer's solution and physiological saline buffer. The formulation for injection can be provided in unit dosage forms (e.g., ampoules or multi-dose containers) with added preservatives. The composition can take the form of a suspension, solution or emulsion in an oily or aqueous medium, and may contain formulation agents such as suspending agents, stabilizers and / or dispersants.

[0156] Pharmaceutical compositions intended for in vivo administration are typically sterilized. Sterilization can be achieved by methods known in the art, such as heat sterilization, steam sterilization, sterile filtration, or irradiation.

[0157] The dosages and desired drug concentrations of the pharmaceutical compositions described herein may vary depending on the specific intended use.

[0158] kit In some embodiments, a kit comprising the MAPT ASO or MAPT ASO conjugate described herein is provided. In some embodiments, the kit is intended for use in reducing the expression of the MAPT gene.

[0159] The MAPT ASO (including MAPT ASO conjugate) and MAPT ASO-containing pharmaceutical compositions described herein may be packaged in or contained in kits, containers, packs, or dispensers. MAPT ASO and MAPT ASO-containing pharmaceutical compositions may be packaged in pre-filled syringes or vials. Any of the MAPT ASO or MAPT ASO-containing pharmaceutical compositions described herein may be formulated or packaged in single-dose or multi-dose form. Any of the MAPT ASO or MAPT ASO-containing pharmaceutical compositions described herein may be formulated for repeated use.

[0160] In some embodiments, the kit further includes instructions for use, which include instructions for carrying out the methods described herein (i.e., protocols) (e.g., instructions for using the kit to administer a composition across the blood-brain barrier). Instructional materials typically consist of written or printed materials, but are not limited thereto. Any medium capable of storing such instructions and communicating them to end users is contemplated herein. Such media include, but are not limited to, electronic storage media (e.g., magnetic disks, magnetic tapes, magnetic cartridges, magnetic chips), optical media (e.g., CD-ROMs), and the like. Such media may include addresses to internet sites providing such instructional materials. [Examples]

[0161] The present invention will be described in more detail through specific examples. The following examples are provided for illustrative purposes only and are not intended to limit the invention in any way. Those skilled in the art will readily recognize various non-essential parameters that can be changed or modified to produce essentially the same results. Efforts have been made to ensure accuracy with respect to the numerical values ​​used (e.g., quantity, temperature, etc.), but some experimental errors and deviations may exist. Unless otherwise specified, the implementation of this disclosure will utilize conventional methods of protein chemistry, biochemistry, recombinant DNA technology and pharmacology within the scope of the art. Such techniques are well described in the literature.

[0162] Example 1: In vitro screening of ASO for MAPT gene The selection of ASO sequences specific to particular target sequences is based on the analysis of the selected target sequences and the evaluation of several factors, including their in vitro and in vivo effects, as well as their hepatotoxicity profiles.

[0163] Initial MAPT ASO screening Initial identification of 393 MAPT ASO sequences that are 100% complementary to MAPT precursor mRNA (Ensembl ENST0000034429, SEQ ID NO: 2) and / or MAPT mRNA (RefSeq NM_001123066, SEQ ID NO: 3) was performed based on computational predictions regarding target binding, specificity, efficacy, and safety.

[0164] The knockdown effect of MAPT-specific ASOs in human MiaPaCa-2 and HDLM2 cells was investigated. Cells were treated with MAPT-specific ASOs or control oligonucleotides at a concentration of 5 μM without the use of transfection reagents. After 3 days of treatment, the cells were lysed. MAPT and HPRT1 mRNA expression was analyzed using the QuantiGene Singleplex assay (ThermoFisher). MAPT expression levels were normalized relative to HPRT1 levels, and the degree of knockdown was determined compared to mock-treated cells.

[0165] 2. Concentration screening Fifty-five MAPT-specific ASOs that demonstrated a knockdown effect of 99.9% or more (at 5 μM) in both test cell lines were tested using two-concentration screening (100 nM, 1000 nM) with MiaPaCa-2 cells. After 3 days of treatment, the cells were lysed. MAPT and HPRT1 mRNA expression was analyzed using the QuantiGene Singleplex assay (ThermoFisher). MAPT expression levels were normalized by HPRT1 levels, and the degree of knockdown was measured compared to mock-treated cells. Twenty-two ASOs were analyzed for TLR9 activation ability and concentration-response relationship (IC1). 50 It was selected for the investigation of the measurement of [something].

[0166] Investigation of TLR9 activation ability Twenty-two types of ASOs were tested for their TLR9-dependent pro-inflammatory activity. Reporter cell lines (HEK-Blue-hTLR9 cells, Invivogen) were treated with MAPT-specific ASOs, positive controls, and negative controls at a concentration of 5 μM without the use of transfection reagents. After 20 hours, Quanti-Blue® solution (Invivogen) was added to the cells. Optical density was measured after 2 hours to assess the TLR9 activating ability of the tested ASOs.

[0167] In vitro IC50 measurement Concentration-response relationships and IC50 values ​​were investigated in MiaPaCa-2 cells using 22 selected MAPT-specific ASOs. Cells were treated with the following concentrations of MAPT-specific ASOs without transfection reagents: 5000, 1667, 556, 185, 62, 21, and 7 nM. After 3 days of treatment, the cells were lysed. MAPT and HPRT1 mRNA expression was analyzed using the QuantiGene Singleplex assay (Thermo Fisher). MAPT expression values ​​were normalized relative to HPRT1 values, and the degree of knockdown was determined compared to mock-treated cells. The results are shown in Table 3. [Table 3]

[0168] Fifteen sequences were selected based on their efficacy and activity in vitro (Table 3 above), and subsequent in vivo safety and activity analyses. The selected 15 sequences were ASO2, ASO3, ASO5, ASO6, ASO7, ASO8, ASO9, ASO10, ASO12, ASO13, ASO16, ASO17, ASO18, ASO21, and ASO22.

[0169] Example 2: In vivo knockdown screening The 15 selected ASOs listed above were synthesized according to the procedure described below for in vivo administration. The ASOs were diluted in sterile saline and administered to h-tau mice (m-tau - / -) in single doses of 50 μg or 100 μg per mouse in a total volume of 10 μL via intracerebroventricular injection (ICV). The 50 μg or 100 μg dose was determined based on the in vivo tolerability of each ASO. ASO sequences that induced acute neurotoxicity by ICV bolus administration were administered at a 50 μg dose.

[0170] Two weeks after administration, tissue (brain and spinal cord) was collected, and terminal blood was obtained. MAPT expression was measured in the brain and spinal cord as follows: A portion of the frontal lobe and cervical spinal cord were homogenized together in trizole using a bead homogenizer, and bulk RNA was separated. Briefly, the homogenized tissue was incubated with chloroform for 3-5 minutes, followed by centrifugation and phase separation. The aqueous phase was then incubated with isopropanol for 10 minutes to precipitate the RNA, followed by washing with 75% ethanol and resuspending in nuclease-free water. MAPT expression was then measured by qPCR using the Express One-Step Superscript Kit and normalized to the expression of the housekeeping gene Gapdh. A group of glial activation markers (Aif1, Gfap, Tlr9, Itgax) were quantified by qPCR using the Express One-Step Superscript Kit to identify whether any of the ASOs were causing the chronic neuroinflammatory response.

[0171] Figure 1 shows the human tau knockdown induced by each ASO (excluding ASO9, ASO12, and ASO22). Mice administered with ASO9, ASO12, and ASO22 exhibited acute neurotoxicity and were sacrificed. All ASOs were shown to achieve at least 50% knockdown in vivo. ASO10 and ASO7 showed the highest levels of human tau knockdown at 100 μg (88% and 82%, respectively). ASO6 and ASO13 showed the highest levels of human tau knockdown at 50 μg (76% and 70%, respectively). ASO10 was excluded from further analysis due to extreme glial activation (i.e., significant upregulation of the entire glial activation marker group) that persisted for two weeks after administration.

[0172] Example 3: In vivo hepatotoxicity screening The liver safety of each ASO was evaluated. Alanine aminotransferase (ALT), aspartate aminotransferase (AST), and lactate dehydrogenase (LDH) are enzymes that are abundant in the liver. When liver cells are damaged, they release these enzymes into the bloodstream. Therefore, serum levels of ALT, AST, and LDH were measured to determine whether ASOs induce liver damage.

[0173] The ASOs shown in Table 4 below were diluted with sterile saline and administered subcutaneously to wild-type mice at a dose of 20 mpk per day for 5 consecutive days. The body weight of the mice was measured daily for 12 days, and blood samples were collected on days 5, 8, and 12. After coagulation of the blood samples, serum was prepared by centrifugation. Subsequently, the levels of ALT, AST, and LDH in the serum samples were measured. A summary of the results is shown in Table 4 below. [Table 4]

[0174] Using a combination of survival data, weight change data, and liver enzyme data, nine ASOs (ASO2, ASO6, ASO7, ASO8, ASO9, ASO12, ASO13, ASO18, and ASO22) were selected for follow-up activity studies.

[0175] Furthermore, to determine whether internal cytosine methylation, i.e., methylation of cytosines within the gap region of ASO, affects the hepatic safety profile of ASO, internal cytosines in ASO6 were replaced with 5-methylcytosine (see Table 7; sequence number 5, all cytosines modified with 5-methylcytosine). Hepatic safety was analyzed using the same protocol described above. The results of this hepatotoxicity test are shown in Figure 2. The addition of 5-methylcytosine showed a slightly beneficial effect on the hepatic safety profile of ASO6, meaning that the slight elevation in liver enzyme levels was improved by cytosine methylation.

[0176] Example 4: In vivo ED50 screening The nine ASOs selected above (based on the absence of chronic neuroinflammation and significant hepatotoxicity) were diluted with sterile saline and administered by ICV injection to h tau (m tau - / -) mice at multiple doses of 5–50 μg, as shown in Table 5. All ASOs were administered at a final volume of 10 μL. Subsequently, ASO12 was excluded from the study due to paroxysmal acute neurotoxicity and delayed recovery time after ICV injection. ASO9 and ASO22 were not tested in the initial high-dose screening due to acute neurotoxicity related to the route of administration. [Table 5]

[0177] Tissue samples were collected two weeks after administration, and bulk RNA was isolated according to the protocol described above. MAPT expression in the brain and spinal cord was then measured using qPCR for MAPT and Gapdh. MAPT knockdown in the cortex and spinal cord is shown in Figures 3 and 4, respectively. ASO2, ASO6, ASO7, ASO9, ASO13, and ASO22 were selected for further study (see Table 7 for corresponding modified sequences).

[0178] Example 5: Measurement of ASO half-life in vivo Five types of ASOs (ASO2, ASO6, ASO7, ASO13, and ASO22) were diluted in sterile saline and administered to h-tau (m-tau- / -) mice via ICV injection at a moderate dose of 25 μg per 10 μL total volume. These ASOs were selected based on their low hepatotoxicity and high knockdown activity, with ASO2 being an exception due to its cross-reactivity to mice, although its activity was not as high. Tissue samples were collected at 5 and 9 weeks post-administration to confirm the duration of tau knockdown. ASO9 was administered to h-tau (m-tau- / -) mice, but tissue samples were collected only at 5 weeks. After isolating bulk RNA according to the protocol described above, MAPT expression in the brain and spinal cord was measured using qPCR for MAPT and Gapdh.

[0179] The results for the duration of knockdown effects in the brain and spinal cord are shown in Figures 5 and 6, and Table 6, respectively. MAPT knockdown was shown to last for several weeks after ICV injection, with ASO6 maintaining potent tau knockdown for the longest period. [Table 6]

[0180] Example 6: Evaluation of nephrotoxicity in rats in vivo Rats are considered the most sensitive species for detecting ASO-mediated nephrotoxicity and are recommended for use in risk assessment because they can over-predict susceptibility to nephrotoxicity in humans. The purpose of this study was to determine whether repeated administration of five ASOs (ASO2, ASO6, ASO9, ASO13, and ASO22) had any effect on renal function and morphology (gross and histological evaluation) in a two-week rat study.

[0181] Male Wistar Harnver rats (7-8 weeks old) were divided into seven groups of five animals each. Each group received subcutaneous administration of 40 mg / kg of ASO (control ASO A, ASO2, ASO6, ASO9, ASO13, or ASO22) or physiological saline (vehicle control) on days 1 and 8, and the animals were euthanized on day 15. Body weight was measured on days 1 and 8 before administration, and again on days 5, 10, and 15. Clinical observations were performed on days 1 and 8 after administration. Blood samples were collected on days 5, 10, and 15 to measure blood urea nitrogen (BUN) and creatinine as indicators of renal function. On day 15, the kidney was collected for microscopic evaluation, and urine was collected to measure the KIM-1:creatinine ratio as an indicator of renal impairment.

[0182] All ASOs showed no changes in clinical signs or body weight throughout the treatment period and were well tolerated in Wistar Hannover rats. In all treatment groups, serum BUN and creatinine levels (Figure 7A-B) were within the previously observed normal range and comparable to the vehicle control group. On day 15, the change in the urinary KIM-1 / creatinine ratio was below the critical limit and was considered to be within the range of change commonly observed in rats under similar test conditions (Figure 7C). One animal in test group ASO9 had a KIM-1 / creatinine ratio above the normal range for Wistar Hannover rats (KIM-1 / creatinine ratio: approximately 0.1-2). However, based on the fact that no similar changes were observed in other rats in the same group, the association of this finding was considered questionable.

[0183] In all ASO-treated groups, minimal to mild degeneration of the proximal tubular epithelium in the kidney was observed, associated with minimal regeneration and mild to minimal interstitial, perivascular, and / or periglomerular mononuclear cell infiltration. These subtle changes were deemed non-harmful based on their minimal to mild severity and the absence of functional changes in clinical signs, body weight, and serum and urinary renal biomarkers (BUN, creatinine, KIM-1). In conclusion, this study did not detect any evidence of adverse renal function or histopathological effects in Wistar Han rats administered two doses of 40 mg / kg with one of the six selected ASOs (control ASO A, ASO2, ASO6, ASO9, ASO13, or ASO22). [Table 7-1] [Table 7-2] [Table 8]

Claims

1. A MAPT antisense oligonucleotide (MAPT ASO) having a length of 17 to 19 nucleotides and comprising one nucleic acid base sequence of sequence numbers 4 to 9, 19 to 37, and 38 to 56, wherein the MAPT ASO comprises at least one modified internucleoside bond and / or at least one modified sugar.

2. The MAPT ASO according to claim 1, wherein the MAPT ASO comprises a nucleic acid base sequence consisting of any one of sequence numbers 4 to 9.

3. The MAPT ASO according to claim 1 or 2, wherein the MAPT ASO is a gapmer having a gap segment consisting of any one nucleic acid base sequence of sequence numbers 10 to 15.

4. A MAPT antisense oligonucleotide (MAPT ASO), wherein the MAPT ASO is a gapmer having a gap segment consisting of any one nucleic acid base sequence of SEQ ID NOs: 10-15, and the MAPT ASO comprises at least one modified nucleoside bond and at least one modified sugar.

5. The MAPT ASO according to claim 4, wherein the MAPT ASO has a nucleoside length of 15 to 25 linked nucleosides.

6. The MAPT ASO according to claim 5, wherein the MAPT ASO has a length of 16 to 19 nucleotides.

7. A MAPT antisense oligonucleotide (MAPT ASO), wherein the MAPT ASO is a 16-nucleotide gapmer and comprises a gap segment consisting of one nucleic acid base sequence from SEQ ID NOs. 38 to 56, and the MAPT ASO comprises at least one modified internucleoside bond and at least one modified sugar.

8. The MAPT ASO according to claim 7, wherein the MAPT ASO comprises a nucleic acid base sequence consisting of any one of sequence numbers 19 to 37.

9. The MAPT ASO according to any one of claims 1 to 8, wherein the at least one modified nucleoside bond is a phosphorothioate nucleoside bond.

10. The MAPT ASO according to any one of claims 1 to 9, wherein the MAPT ASO contains at least one modified sugar including a bicyclic sugar.

11. The MAPT ASO according to claim 10, wherein the MAPT ASO contains 2 to 6 modified sugars, each containing a bicyclic sugar.

12. Each bicyclic sugar contains a chemical crosslink between the 4' and 2' positions of the sugar, and each chemical crosslink is 4'-CH(R)-O-2' and 4'-(CH ２ ) ２ Independently selected from the group consisting of -O-2', where each R is H, C １ ~C ６ Alkyl and C １ ~C ６ A MAPT ASO according to claim 11, which is independently selected from alkoxys.

13. The MAPT ASO according to claim 12, wherein each bicyclic sugar contains a chemical bridge between the 4' and 2' positions of the sugar, and each chemical bridge is 4'-CH(R)-O-2', and each R is independently H.

14. MAPT ASO according to any one of claims 1 to 13, further comprising at least one modified nucleic acid base.

15. The compound according to claim 14, wherein the modified nucleic acid base comprises 5-methylcytosine.

16. The aforementioned MAPT ASO changes from 5' to 3'. A 5' wing segment comprising three nucleosides, wherein each nucleoside of the 5' wing segment comprises a modified sugar, The gap segment contains 10 to 13 nucleosides, and each nucleoside in the gap segment is a deoxynucleoside. A MAPT ASO according to any one of claims 1 to 15, comprising a 3'-wing segment comprising three nucleosides, wherein each nucleoside of the 3'-wing segment comprises a modified sugar.

17. (a) Seventeen consecutive nucleic acid bases complementary to nucleic acid bases 66,760-66,776 of SEQ ID NO: 1, (b) 18 consecutive nucleic acid bases complementary to nucleic acid bases 78,441-78,458 of Sequence ID No. 1, (c) 19 consecutive nucleic acid bases complementary to nucleic acid bases 78,631-78,649 of Sequence ID No. 1, (d) 18 consecutive nucleic acid bases complementary to nucleic acid bases 78,828-78,845 of Sequence ID No. 1, or (e) A MAPT ASO consisting of 19 consecutive nucleic acid bases complementary to nucleic acid bases 90,215 to 90,233, The MAPT ASO comprising at least one modified nucleoside linkage and / or at least one modified sugar.

18. MAPT ASO follows the following chemical structure: 【Chemistry 11】 or its salt.

19. MAPT ASO follows the following chemical structure: 【Chemistry 12】 or its salt.

20. MAPT ASO follows the following chemical structure: 【Chemistry 13】 or its salt.

21. MAPT ASO follows the following chemical structure: 【Chemistry 14】 or its salt.

22. MAPT ASO follows the following chemical structure: 【Chemistry 15】 or its salt.

23. MAPT ASO follows the following chemical structure: 【Chemistry 16】 or its salt.

24. The MAPT ASO according to any one of claims 1 to 23, wherein the MAPT ASO is conjugated to a target ligand or a delivery vehicle.

25. The MAPT ASO according to claim 24, wherein the target ligand specifically binds to a transferrin receptor (TfR) or a molecule expressed on the luminal surface of the blood-brain barrier.

26. A pharmaceutical composition comprising MAPT ASO according to any one of claims 1 to 25, and a pharmaceutically acceptable carrier or excipient.

27. A method for generating nerve cells with reduced tau expression, wherein the method comprises delivering a MAPT ASO according to any one of claims 1 to 25 to the nerve cells, the MAPT ASO reducing the expression level of the endogenous MAPT gene.

28. A method for modifying nerve cells to reduce tau expression, wherein the method comprises delivering a MAPT ASO according to any one of claims 1 to 25 to the nerve cells, the MAPT ASO reducing the expression level of an endogenous MAPT gene.

29. A method for modifying nerve cells to reduce tau expression, wherein the method comprises delivering a MAPT ASO according to any one of claims 1 to 25 to the nerve cells, the MAPT ASO specifically reduces the expression level of a MAPT transcript within the cells.

30. A method for reducing tau expression in target spinal cord cells, comprising administering MAPT ASO according to any one of claims 1 to 25 or the pharmaceutical composition according to claim 26 by intrathecal administration.

31. A method for reducing the expression of tau in a target, comprising administering to the target MAPT ASO according to any one of claims 1 to 25 or the pharmaceutical composition according to claim 26.

32. The method according to claim 31, wherein tau expression is reduced in the target CNS.

33. The method according to claim 31, wherein the MAPT ASO is administered to the subject by intrathecal administration.

34. The method according to any one of claims 27 to 33, wherein the MAPT ASO reduces the expression level of the endogenous MAPT gene or reduces the level of the MAPT transcript by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95% compared to the level when the MAPT ASO is not administered.

35. The method according to any one of claims 31 to 34, wherein the MAPT ASO is administered intravenously to the subject.

36. A method for treating tau-related neurodegenerative disease in human subjects who require treatment for tau-related neurodegenerative disease. The method comprises administering to a human subject the MAPT ASO described in any one of claims 1 to 25 or the pharmaceutical composition described in claim 26.

37. The method according to claim 36, wherein the tau-related neurodegenerative syndrome is Alzheimer's disease.

38. A method for treating Alzheimer's disease, the method comprising administering the pharmaceutical composition described in claim 26 to a human subject in need thereof.

39. A method for reducing the expression of MAPT messenger ribonucleic acid (mRNA) in a human subject in which it is necessary to reduce the expression of MAPT messenger ribonucleic acid (mRNA), the method comprising administering to the human subject MAPT ASO according to any one of claims 1 to 25 or the pharmaceutical composition according to claim 26.

40. The pharmaceutical composition according to claim 26, which is used for delivery to a human CNS requiring MAPT ASO, wherein the MAPT ASO reduces the expression level of the endogenous MAPT gene.

41. The pharmaceutical composition according to claim 26, for use in human subjects requiring treatment for tau-related neurodegenerative disorders.

42. The pharmaceutical composition according to claim 41, wherein the tau-related neurodegenerative disease is Alzheimer's disease.

43. The pharmaceutical composition according to claim 26, for use in reducing MAPT mRNA expression in human subjects where it is necessary to reduce MAPT mRNA expression.

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