Oligonucleotide targeting microtubule-associated protein tau gene and use thereof

Abstract

The present disclosure relates to an RNAi agent targeting MAPT. The RNAi agent significantly inhibits the expression level of the MAPT gene, and has a long-lasting efficacy.

Description

Oligonucleotides targeting the microtubule-associated protein tau gene and uses thereof TECHNICAL FIELD

[0001] The present disclosure relates to an oligonucleotide, in particular to the inhibition of the expression of the microtubule-associated protein tau (MAPT) gene and the treatment of related diseases caused by the cleavage product of MAPT, microtubule-associated protein tau. BACKGROUND

[0002] Tau protein has six isoforms, which are produced by alternative splicing of the microtubule-associated protein tau (MAPT) gene. The physiological function of tau protein is to stabilize the internal skeleton of brain neurons. Since tau is a microtubule-associated protein, microtubules facilitate neurons to maintain their structure and help cell division, which is also necessary for intercellular material transport.

[0003] Tau gene and tau isoforms. Microtubule organization, stability and polymerization are regulated by microtubule-associated proteins MAP1, MAP2 and tau. The MAPT gene is located on chromosome 17q21 and consists of 16 exons. Four types of repeat sequences are found in the MAPT gene: (a) short interspersed nuclear elements (sine), (b) long interspersed nuclear elements (LINEs), (c) DNA transposons, (d) transposons with long terminal repeats (LTRs). Among the total of 16 exons, exon 1 serves as a promoter, the whole or constituent exons include exons 1, 4, 5, 7, 9, 11, 12 and 13, and alternative spliced exons include exons 2, 3 and 10. Alternative splicing of exon 10 forms triple and quadruple repeat (3R and 4R) domains at the C-terminus, and dysregulation of exon 10 can lead to neurodegenerative diseases. Alternative splicing of exons 2 and 3 produces 0N, 1N or 2N inserts at the N-terminus. The MAPT gene encodes 6 tau isoforms containing 352-441 amino acids, which are expressed in neurons. The six possible tau isoforms are 3R0N, 3R1N, 3R2N, 4R0N, 4R1N and 4R2N. The structure of the isoforms is shown in Figure 1 (Rawat, P., et al. International Journal of Molecular Sciences, 2022. 23(21).).

[0004] Post-translational modifications (PTMs) and their mimics, nucleation motifs within tau paired helical filaments, disrupt microtubule interactions and the ability of tau to aggregate, thus promoting the pathological role of tau in the formation of oligomers. Tau protein undergoes multiple post-translational modifications and conformational changes in pathological states, which are protein hallmarks of tauopathies. About 35% of the amino acids in tau protein are susceptible to pre- or post-translational modifications, including serine, threonine, tyrosine, lysine, arginine, asparagine, histidine, and cysteine. Tau is a phosphorylated protein that contains 85 potential phosphorylation sites on serine (S), threonine (T), and tyrosine, respectively. In pathological states, tau phosphorylation increases, reducing its affinity for microtubules, leading to cytoskeletal instability, especially in neurons. Phosphorylation changes the electrostatic properties of the protein by adding a negatively charged, hydrophilic group, resulting in an overall hydrophilic protein. The phosphorylation process of tau protein is accomplished by a variety of kinases and phosphatases. Any imbalance in tau kinase and phosphatase activity is believed to lead to tau phosphorylation. There are more than 20 protein kinases that can phosphorylate tau protein.

[0005] In the brains of Alzheimer's disease (AD) patients, impaired glucose metabolism can lead to decreased tau protein O-GlcNAcylation, which in turn promotes hyperphosphorylation of tau protein, leading to neuronal death. Abnormal phosphorylation of tau protein can cause conformational changes, proteolytic cleavage, and aggregation. Abnormal phosphorylation reduces its affinity for microtubules and leads to loss of neurons and synapses in the brain.

[0006] Tau truncation plays a major role in tau aggregation and neurodegeneration. In the brains of AD patients, tau protein is truncated at different sites by multiple proteases, and proteolyzed tau protein fragments are prone to aggregation. Studies have shown that truncation of tau protein occurs at Asp41 (D421) and Glu391 sites, where truncation of tau protein makes tau protein more prone to aggregation. However, the truncation of tau under oligomeric conditions is not fully understood in AD brains. Carboxy-terminal truncation of tau is performed by caspase-3, and amyloid beta promotes caspase-3 activation in AD cases. Recent studies have shown that truncated tau at the C-terminus spreads to the brain and causes neuronal damage and death. Truncated tau protein also causes synaptic proteome dysregulation in presynaptic and postsynaptic compartments. Presynaptic truncated tau protein is associated with dysfunction of microtubule stability, which can be the cause of reduced synaptic vesicles, while postsynaptic truncated tau protein reduces the level of neurofilaments.

[0007] In addition, tau acetylation at lysine 280 is a pathological modification that can contribute to tau-mediated neurodegeneration; in AD brains, PHF are highly ubiquitinated, and ubiquitination can play a crucial role in the formation of tau filaments. Tau ubiquitination occurs on lysine residues, and there are seven lysine residues contained within tau; lysine methylation has been detected in extracted tau protein in both pathological and normal brains, and this process is carried out by a class of enzymes called methyltransferases; Tau SUMOylation at K340 reciprocally enhances its phosphorylation and inhibits ubiquitin-mediated Tau degradation.

[0008] In Alzheimer’s disease and other tauopathies, tau protein is abnormally phosphorylated, and this dysregulation of tau protein leads to loss of neurons and synapses in the brain. Tau phosphorylation in AD brains is characterized by at least a three-fold increase in phosphorylation. Three different types of tau protein pools are found in the brains of AD patients: (a) AD tau protein is most similar to normal tau protein and is not hyperphosphorylated, (b) AD phosphorylated tau (AD P-tau) is soluble, hyperphosphorylated tau, and (c) paired helical filament (PHF)-tau is insoluble and hyperphosphorylated. Tau accumulation leads to brain cell damage, but the exact process of Tau toxicity is not known. In AD, tau also exhibits loss of microtubule binding, which can be due to hyperphosphorylation at different sites that dissociate tau from microtubules and lead to disruption of intracellular traffic, resulting in death of neurons. Vigo Pelfrey et al. found increased tau protein in the cerebrospinal fluid, which can be due to neuronal death. Increasing evidence suggests that aggregated, hyperphosphorylated Tau can be a key driver of neurodegeneration in Alzheimer’s disease (as shown in Figure 2).

[0009] AD is a progressive neurodegenerative disease characterized by cognitive and functional decline leading to severe disability. The onset of pathology is marked by progression in neuroimaging and fluid biomarker measures (preclinical stage). Before subtle cognitive changes occur, it is referred to as mild cognitive impairment (MCI). The final progression to dementia occurs over a variable period of time characterized by cognitive and behavioral symptoms that impair an individual’s ability in daily life, symptoms typically occurring in patients 65 years of age and older, with less than 5% of all AD patients presenting symptoms before age 65. Historically, the diagnosis of AD has focused on clinical criteria, but increasing evidence suggests that cerebrospinal fluid (CSF) biomarkers, including amyloid-beta 42 (Ab42) and tau (total tau (t-tau) and phosphorylated tau (p-tau181)) and positron emission tomography (PET)-amyloid and PET-tau are reliable surrogates of neuropathological changes that can more powerfully characterize AD patients. For most AD patients, treatment remains limited to the multidisciplinary management of symptoms, including medications with disease-modifying effects. Recently, the U.S. Food and Drug Administration accelerated approval of aducanumab and lecanemab, providing the first treatments targeting the key disease mechanism of AD (amyloid plaque accumulation) for patients with MCI or mild AD. There are currently over 50 million people worldwide with dementia, primarily caused by Alzheimer’s disease, and this number is projected to double every 20 years, thus, additional disease-modifying treatments to prevent or slow the progression of this disease remain an important unmet need. SUMMARY

[0010] An object of the present disclosure is to provide an inhibitor of MAPT gene expression that is effective, safe, and has a long-lasting effect.

[0011] The present disclosure provides an oligonucleotide or a pharmaceutically acceptable salt thereof that inhibits MAPT expression and a method of using the oligonucleotide or the pharmaceutically acceptable salt thereof to inhibit MAPT gene expression in a cell or a mammal, wherein the oligonucleotide targets the MAPT gene. Also provided herein are compositions and methods for treating pathological conditions and diseases in a mammal caused by Tau phosphorylation. The oligonucleotide is a double-stranded RNA (dsRNA) that directs sequence-specific degradation of mRNA through a process known as RNA interference (RNAi).

[0012] In one aspect, the present disclosure provides an oligonucleotide or a pharmaceutically acceptable salt thereof for inhibiting MAPT expression, the oligonucleotide comprising a sense strand having a sequence with at least 60% or more sequence identity to a sequence set forth in any one of SEQ ID NO. 3-136 or a fragment thereof, or a modified sequence of the sequence or the fragment thereof, preferably a sequence with 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more sequence identity; and an antisense strand having a sequence with at least 60% or more sequence identity to a sequence set forth in any one of SEQ ID NO. 139-272 or a fragment thereof, or a modified sequence of the sequence or the fragment thereof, preferably a sequence with 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more sequence identity.

[0013] In another aspect, the present disclosure provides a conjugate or a pharmaceutically acceptable salt thereof for inhibiting MAPT gene expression.

[0014] In another aspect, the present disclosure provides a composition comprising the foregoing oligonucleotide or a pharmaceutically acceptable salt thereof, or the foregoing conjugate or a pharmaceutically acceptable salt thereof, and optionally a pharmaceutically acceptable carrier.

[0015] In another aspect, the present disclosure provides use of the foregoing oligonucleotide or a pharmaceutically acceptable salt thereof, conjugate or a pharmaceutically acceptable salt thereof, or composition in the manufacture of a medicament for treating and / or preventing a MAPT-related disorder.

[0016] In another aspect, the present disclosure provides a method of treating and / or preventing a MAPT-related disorder in a subject by administering to the subject a therapeutic agent (e.g., the foregoing oligonucleotide or a pharmaceutically acceptable salt thereof, or the foregoing conjugate or a pharmaceutically acceptable salt thereof, or the foregoing composition, or a vector or transgene encoding the oligonucleotide).

[0017] In another aspect, the present disclosure provides a method of treating and / or preventing a MAPT-related disorder in a subject by administering to the subject the foregoing oligonucleotide or a pharmaceutically acceptable salt thereof, conjugate or a pharmaceutically acceptable salt thereof, or composition in combination with other drugs and / or other therapeutic methods.

[0018] Experiments demonstrate that the oligonucleotide of the present disclosure can effectively reduce the content of MAPT in vivo, and is an effective inhibitor of beta tubulin associated protein Tau. BRIEF DESCRIPTION OF DRAWINGS

[0019] FIG. 1 shows the structure of the Tau gene.

[0020] Figure 2 shows the pathogenesis of Alzheimer's disease, wherein mTORCl: mammalian target of rapamycin complex 1; ULK1: unc-51 -like autophagy activating kinase 1; NOS: nitric oxide synthase; SOD: superoxide dismutase; catalase: catalase; NOS: nitric oxide synthase; Beclinl: a protein that regulates autophagy; mTORCl: mammalian target of rapamycin 1; ULK1: protein kinase of upstream signals of autophagy; COX-2: cyclooxygenase-2; NF-κΒ: nuclear factor kappa B; iNOS: inducible nitric oxide synthase.

[0021] Figure 3 shows a flow chart of siRNA solid phase synthesis. DETAILED DESCRIPTION

[0022] In the present disclosure, unless otherwise indicated, the scientific and technical terms used herein have the meanings that would be generally understood by one of ordinary skill in the art. Also, the terms related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, immunology, and laboratory procedures steps used herein are terms and procedures commonly used in the corresponding fields. Also, for better understanding of the present disclosure, the definitions and explanations of the related terms are provided below.

[0023] As used herein, the term "about" or "approximately," as applied to one or more target values, refers to a value that is similar to a reference value. In certain embodiments, unless otherwise stated or otherwise clear from context, the term "approximately" or "about" means a range of values that fall within 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less of the recited reference value in either direction (greater than or less than), unless such number would exceed 100% of a possible value.

[0024] As used herein, the term "complementary" refers to the structural relationship between nucleotides (e.g., on opposite nucleic acids or on opposite regions of a single nucleic acid strand) that allows the nucleotides to form base pairs with one another. For example, a purine nucleotide of one nucleic acid that is complementary to a pyrimidine nucleotide of an opposite nucleic acid can base pair together by forming hydrogen bonds with one another. In some embodiments, complementary nucleotides can base pair in a Watson-Crick manner or in any other manner that allows for the formation of a stable duplex. In some embodiments, two nucleic acids can have nucleotide sequences that are complementary to one another so as to form a region of complementarity, as described herein.

[0025] As used herein, the term "strand" refers to a single continuous sequence of nucleotides linked together by internucleotide linkages (e.g., phosphodiester linkages, phosphorothioate linkages). In some embodiments, a strand has two free ends, e.g., a 5 '-end and a 3 '-end.

[0026] As used herein, the term "deoxyribonucleotide" refers to a nucleotide having a hydrogen at the 2' position of its pentose sugar as compared to a ribonucleotide. A modified deoxyribonucleotide is a deoxyribonucleotide having one or more modifications or substitutions (including modifications or substitutions in or of the sugar, phosphate group, or base or modifications or substitutions of the sugar, phosphate group, or base) other than at the 2' position.

[0027] As used herein, the term "oligonucleotide" refers to a short nucleic acid, e.g., a short nucleic acid less than 100 nucleotides in length. An oligonucleotide can comprise ribonucleotides, deoxyribonucleotides, and / or modified nucleotides, including, e.g., modified ribonucleotides. An oligonucleotide can be single-stranded or double-stranded. An oligonucleotide can or can not have duplex regions. As a non-limiting example, an oligonucleotide can be, but is not limited to, a small interfering RNA (siRNA), a microRNA (miRNA), a short hairpin RNA (shRNA), a Dicer substrate interfering RNA (dsiRNA), an antisense oligonucleotide, or a single-stranded siRNA. In some embodiments, a double-stranded oligonucleotide is an RNAi oligonucleotide.

[0028] As used herein, the term "double-stranded oligonucleotide" refers to an oligonucleotide that is substantially in duplex form. In some embodiments, one or more duplex regions of a double-stranded oligonucleotide form complementary base pairing between antiparallel sequences of nucleotides of covalently separate nucleic acid strands. In some embodiments, one or more duplex regions of a double-stranded oligonucleotide form complementary base pairing between antiparallel sequences of nucleotides of covalently linked nucleic acid strands. In some embodiments, one or more duplex regions of a double-stranded oligonucleotide form complementary base pairing of antiparallel sequences of nucleotides that are brought together, e.g., via a hairpin, from a single nucleic acid strand. In some embodiments, a double-stranded oligonucleotide comprises two covalently separate nucleic acid strands that are fully duplexed with each other. However, in some embodiments, a double-stranded oligonucleotide comprises two covalently separate nucleic acid strands that are partially duplexed, e.g., have overhangs at one or both ends. In some embodiments, a double-stranded oligonucleotide comprises antiparallel sequences of nucleotides that are partially complementary, and thus, can have one or more mismatches, which can include internal mismatches or terminal mismatches.

[0029] As used herein, the term "double-stranded RNA" or "dsRNA" refers to a complex of ribonucleic acid molecules having a duplex structure comprising two antiparallel and substantially complementary nucleic acid strands having "sense" and "antisense" orientation with respect to a target RNA (i.e., a MAPT gene). In some embodiments of the disclosure, double-stranded RNA (dsRNA) triggers the degradation of a target RNA (e.g., mRNA) through a post-transcriptional gene-silencing mechanism referred to herein as RNA interference or RNAi. Generally, most of the nucleotides of each strand of a dsRNA molecule are ribonucleotides, but as described in detail herein, each strand or both strands can also comprise one or more non-ribonucleotides, such as deoxyribonucleotides or modified nucleotides. In addition, as used herein, "RNAi" can comprise ribonucleotides having chemical modifications; RNAi can comprise substantial modifications at multiple nucleotides.

[0030] As used herein, the terms "RNAi," "iRNA," "RNAi agent," "RNA interference agent" are used interchangeably herein to refer to an agent comprising the RNA as defined herein and which mediates the targeted cleavage of an RNA transcript through the RNA-induced silencing complex (RISC) pathway. RNA interference (RNAi) is a process that directs the sequence-specific degradation of mRNA. RNAi modulation, e.g., inhibits the expression of MAPT in a cell, e.g., a cell in a subject, e.g., a mammalian subject.

[0031] As used herein, the term "modified nucleotide" refers to a nucleotide independently having a modified sugar moiety, a modified internucleotide linkage, or a modified nucleobase, or any combination thereof. Thus, the term "modified nucleotide" encompasses substitutions, additions, or deletions to the internucleosidic linkage, sugar moiety, or nucleobase, such as functional groups or atoms. Modifications suitable for use in the agents of the disclosure include all types of modifications disclosed herein or known in the art.

[0032] As used herein, "conjugation" refers to the linkage of two or more chemical moieties each having a specific function to each other in a covalent linkage; correspondingly, "conjugate" refers to a compound formed by the covalent linkage between the respective chemical moieties. Further, "siRNA conjugate" denotes a compound formed by the covalent linkage of one or more chemical moieties having a specific function to an siRNA. In the following, the siRNA conjugates of the disclosure are sometimes also referred to simply as "conjugates". The siRNA conjugate should be understood in the context of the total siRNA conjugate, the first siRNA conjugate or the second siRNA conjugate, or the siRNA sense strand conjugate or the siRNA antisense strand conjugate.

[0033] As used herein, the term "nucleotide overhang" refers to at least one unpaired nucleotide that overhangs from the duplex structure of a double-stranded RNAi. For example, a nucleotide overhang is present when the 3' end of one strand of a dsRNA extends beyond the 5' end of the other strand, or vice versa. A dsRNA can include an overhang of at least one nucleotide; alternatively, the overhang can include at least two nucleotides, at least three nucleotides, at least four nucleotides, at least five nucleotides, or more. The nucleotide overhang can include or consist of nucleotides / nucleoside analogs, including deoxynucleotides / nucleosides. The overhang can be on the sense strand, the antisense strand, or any combination thereof. Further, the nucleotides of the overhang can be present on the 5' end, the 3' end, or both ends of the antisense strand or the sense strand of a dsRNA.

[0034] As used herein, the term "naked sequence" refers to a nucleotide sequence that is unmodified.

[0035] As used herein, the term "inhibit" is used interchangeably with "knock down," "reduce," "silence," "down-regulate," "suppress," and other similar terms, and includes any degree of inhibitory effect.

[0036] The phrase "inhibit expression of MAPT" is intended to refer to inhibiting expression of any MAPT gene, such as, for example, a mouse MAPT gene, a rat MAPT gene, a monkey MAPT gene, or a human MAPT gene, as well as variants or mutants of a MAPT gene. Thus, in the context of genetically manipulating a cell, a population of cells, or an organism, the MAPT gene can be a wild-type MAPT gene, a mutant MAPT gene, or a transgenic MAPT gene.

[0037] "Inhibit expression of a MAPT gene" includes inhibition of a MAPT gene at any level, for example, at least partial inhibition of expression of a MAPT gene. Expression of a MAPT gene can be assessed based on the level or change in level of any variable associated with MAPT gene expression, for example, MAPT mRNA levels or MAPT protein levels, or indirectly by inhibiting the level of a Gluc and MAPT fusion protein gene, which in turn reflects inhibition of MAPT protein levels by inhibiting the level of a Gluc protein.

[0038] This level can be assessed in an individual cell or a population of cells, including, for example, a sample from a subject. It can be appreciated that MAPT is a systemically expressed protein, does not have tissue-specific expression, and is present in circulation.

[0039] Inhibition can be assessed by a decrease in the absolute or relative level of one or more variables associated with MAPT expression compared to a control level. The control level can be any type of control level used in the art, e.g., a pre-dose baseline level, or a level determined from a similar subject that has not been treated or treated with a control, such as, for example, a buffer control or a non-active agent control.

[0040] The term "lipophilic" or "lipophilic moiety" broadly refers to any compound or chemical moiety that has an affinity for lipids.

[0041] The term "pharmaceutically acceptable salt" refers to those salts which retain the biological effectiveness and properties of the free bases or free acids, which are not biologically or otherwise undesirable. These salts are prepared from inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, particularly hydrochloric acid, and organic acids such as acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, N-acetylcystein. In addition, these salts can be prepared as base salts with inorganic bases or organic bases. Salts derived from inorganic bases include, but are not limited to, alkali metal salts (such as sodium salt, potassium salt, and lithium salt), ammonium salt, alkaline earth metal salts (such as calcium salt and magnesium salt). Salts derived from organic bases include, but are not limited to, salts of a primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion-exchange resins such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, lysine, arginine, N-ethylpiperidine, piperidine, polyamine resins. The oligonucleotides of the present disclosure can also exist in a zwitterionic form. Particularly preferred pharmaceutically acceptable salts of the present disclosure are sodium, lithium, potassium and trialkylammonium salts.

[0042] As used herein, the term "subject" is an animal, such as a mammal, including a primate (such as a human, a non-human primate, e.g., a monkey and a chimpanzee), a non-primate (such as a cow, pig, horse, goat, rabbit, sheep, hamster, guinea pig, cat, dog, rat, or mouse), or a bird, that endogenously or heterologously expresses a target gene. In one embodiment, the subject is a human.

[0043] As used herein, the term "treating" or "treatment" refers to a beneficial or desired result, such as a reduction of at least one sign or symptom of a MAPT-associated disorder in a subject. Treatment also includes reducing one or more signs or symptoms associated with undesirable MAPT expression; lessening the extent of undesirable MAPT activation or stabilization; ameliorating or palliating undesirable MAPT activation or stabilization. Treatment also includes reducing one or more signs or symptoms associated with undesirable MAPT expression. "Treatment" can also mean prolonging survival as compared to expected survival without treatment.

[0044] As used herein, the term "prevention" or "preventing" when used in reference to a disease or disorder, will benefit from a reduction in MAPT gene expression or MAPT protein production.

[0045] As used herein, the term "therapeutically effective amount" is intended to include the amount of an RNAi agent that, when administered to a subject suffering from a MAPT-associated disorder, is sufficient to effect treatment of the disease (e.g., by reducing, ameliorating, or maintaining existing disease or one or more symptoms of the disease). A "therapeutically effective amount" can vary depending on the RNAi agent, how the agent is administered, the disease and its severity and history, the age, weight, family history, genetic makeup, type of prior or concomitant treatments, if any, and other individual characteristics of the subject to be treated.

[0046] As used herein, the term "prophylactically effective amount" is intended to include the amount of an RNAi agent that, when administered to a subject suffering from a MAPT-associated disorder, is sufficient to prevent or ameliorate the disorder or one or more symptoms of the disorder. Ameliorating a disease includes slowing the progression of the disease or reducing the severity of the disease later developed. A "prophylactically effective amount" can vary depending on the RNAi agent, how the agent is administered, the extent of risk of disease, and the history, age, weight, family history, genetic makeup, type of prior or concomitant treatments, if any, and other individual characteristics of the patient to be treated.

[0047] As used herein, the term "pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, talc, magnesium stearate, or stearic acid), or solvent or encapsulation material, involved in carrying or transporting the subject compound from one organ, or portion of the body, to another. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the subject being treated. Such carriers are known in the art. Pharmaceutically acceptable carriers include those used for injection.

[0048] In one aspect, the present disclosure provides an oligonucleotide or a pharmaceutically acceptable salt thereof for inhibiting MAPT expression, the oligonucleotide comprising a sense strand having a sequence of at least 60% or more sequence identity to a sequence set forth in any one of SEQ ID NO. 3-136 or a fragment thereof, or a modified sequence of the sequence or the fragment thereof, preferably a sequence of 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more sequence identity; and an antisense strand having a sequence of at least 60% or more sequence identity to a sequence set forth in any one of SEQ ID NO. 139-272 or a fragment thereof, or a modified sequence of the sequence or the fragment thereof, preferably a sequence of 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more sequence identity.

[0049] In some embodiments, the sense strand and / or the antisense strand is 15-30, 17-25, or 19 to 25 nucleotides in length.

[0050] In some embodiments, each strand is independently 15-30, 17-25, or 19 to 25 nucleotides in length.

[0051] In some embodiments, the antisense strand is 19 to 23 nucleotides in length.

[0052] In some embodiments, the sense strand is 19 to 23 nucleotides in length.

[0053] In some embodiments, the oligonucleotide comprises a 5' and / or 3'-overhang sequence of one or more nucleotides in length, wherein the 5' and / or 3'-overhang sequence is present on the antisense strand and / or the sense strand. In one embodiment, the antisense strand of the oligonucleotide has an overhang of 1 to 10 nucleotides, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides, at the 3' end or the 5' end. In one embodiment, the sense strand of the dsRNA has an overhang of 1 to 10 nucleotides, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides, at the 3' end or the 5' end. In another embodiment, one or more of the nucleotides in the overhang is replaced by a nucleoside phosphorothioate.

[0054] In some embodiments, the antisense strand bears 1 or 2 overhangs.

[0055] In some embodiments, the sense strand bears 1 or 2 overhangs.

[0056] In some embodiments, the oligonucleotide comprises a 3'-overhang sequence of 1 or 2 nucleotides in length.

[0057] In some embodiments, the oligonucleotide comprises a 5'-overhang sequence of 1 or 2 nucleotides in length.

[0058] In some embodiments, the 3 '-overhang sequence is present on the sense strand; preferably, the overhang sequence is selected from the group consisting of: GG, GA, GC, UC, UG, UU, UA, CA, CC, CG, CU, AA, AG, AU, AC.

[0059] In some embodiments, the 3 '-overhang sequence is present on the antisense strand; preferably, the overhang sequence is selected from the group consisting of: UU, UC, UA, UG, GA, GG, GU, GC, TT, AG, AU, AA, AC, CA, CC, U; more preferably, the overhang sequence is UU.

[0060] In some embodiments, the 5 '-overhang sequence is present on the antisense strand. In some embodiments, the overhang sequence is selected from the group consisting of: A and G.

[0061] In some embodiments, the oligonucleotide comprises an antisense strand and a sense strand each in the range of 19 to 25 nucleotides in length.

[0062] In some embodiments, the oligonucleotide comprises an antisense strand and a sense strand each in the range of 19 to 23 nucleotides in length.

[0063] In some embodiments, the sense strand forms a duplex region with the antisense strand.

[0064] In some embodiments, the sense strand and the antisense strand are 19 / 21-paired, 21 / 21-paired, 21 / 23-paired, or 23 / 23-paired duplex structures, respectively.

[0065] In some embodiments, the oligonucleotide comprises a 5 '-overhang of 1 nucleotide in length and a 3 '-overhang sequence of 1 nucleotide in length, wherein the 5 '-overhang and the 3 '-overhang sequence are present on the antisense strand, and wherein the sense strand is 19 nucleotides in length and the antisense strand is 21 nucleotides in length, such that the sense strand and the antisense strand form a duplex of 19 nucleotides in length.

[0066] In some embodiments, the oligonucleotide comprises a 3 '-overhang sequence of 2 nucleotides in length, wherein the 3 '-overhang sequence is present on the antisense strand, and wherein the sense strand is 19 nucleotides in length and the antisense strand is 21 nucleotides in length, such that the sense strand and the antisense strand form a duplex of 19 nucleotides in length.

[0067] In some embodiments, the oligonucleotide comprises a 3 '-overhang sequence of 2 nucleotides in length, wherein the 3 '-overhang sequence is present on the antisense strand and the sense strand, and wherein the sense strand is 21 nucleotides in length and the antisense strand is 21 nucleotides in length, such that the sense strand and the antisense strand form a duplex of 19 nucleotides in length.

[0068] In some embodiments, the oligonucleotide comprises a 3 '-overhang sequence of 2 nucleotides in length, wherein the 3 '-overhang sequence is present on the antisense strand, and wherein the sense strand is 21 nucleotides in length and the antisense strand is 23 nucleotides in length, such that the sense strand and the antisense strand form a duplex of 21 nucleotides in length.

[0069] In some embodiments, the oligonucleotide comprises a 3 '-overhang sequence of 2 nucleotides in length, wherein the 3 '-overhang sequence is present on the antisense strand and the sense strand, and wherein the sense strand is 23 nucleotides in length and the antisense strand is 23 nucleotides in length, such that the sense strand and the antisense strand form a duplex of 21 nucleotides in length.

[0070] In some embodiments, the pharmaceutically acceptable salt of the oligonucleotide can be prepared by adding an inorganic or organic base to the free acid. Salts derived from inorganic bases include, but are not limited to, alkali metal salts (such as sodium salts, potassium salts, and lithium salts), ammonium salts, alkaline earth metal salts (such as calcium salts and magnesium salts). Salts derived from organic bases (e.g., organic amines) include, but are not limited to, salts formed with primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion-exchange resins, such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, lysine, arginine, N-ethylpiperidine, piperidine, polyamine resins.

[0071] In some embodiments, examples of the pharmaceutically acceptable salt of the oligonucleotide include, but are not limited to, ammonium salts, such as salts of tertiary alkylamine compounds (e.g., triethylamine salts), metal salts such as sodium salts, potassium salts, and magnesium salts, and the like.

[0072] In some embodiments, the oligonucleotide or salt thereof can be in the form of a hydrate or solvate.

[0073] In some embodiments, the oligonucleotide comprises at least one modified nucleotide.

[0074] In some embodiments, at least one of the modified nucleotides in the modified nucleotide is selected from the group consisting of: a deoxy nucleotide, a 3' terminal deoxythymidine (dT) nucleotide, a 2'-0-methyl modified nucleotide, a 2'-fluoro modified nucleotide, a 2'-deoxy modified nucleotide, a locked nucleotide, a 2'-5'-linked ribonucleotide (3'-RNA), an unlocked nucleotide, a conformationally restricted nucleotide, a constrained ethyl nucleotide, an abasic nucleotide, a 2'-amino modified nucleotide, a 2'-0-allyl modified nucleotide, a 2'-0-alkyl modified nucleotide, a 2'-hydroxyl modified nucleotide, a 2'-methoxyethyl modified nucleotide, a 2'-0-alkyl modified nucleotide, a morpholino nucleotide, a phosphoramidate, a nucleotide comprising a non-natural base, a tetrahydropyranyl modified nucleotide, a 1,5-anhydrohexitol modified nucleotide, a cyclohexenyl modified nucleotide, a nucleotide comprising a phosphorothioate group, a nucleotide comprising a methylphosphonate group, a nucleotide comprising a 5'-phosphate, a nucleotide comprising a 5'-phosphate analog, a vinyl-phosphonate nucleotide, a thermally unstable nucleotide, a glycol modified nucleotide, a nucleotide comprising a 2' phosphate, and a 2-O-(N-methylacetamide) modified nucleotide; and combinations thereof.

[0075] In some embodiments, at least one of the modified nucleotides in the modified nucleotide is selected from the group consisting of: a LNA modified nucleotide, a HNA modified nucleotide, a CeNA modified nucleotide, a 2'-methoxyethyl modified nucleotide, a 2'-0-alkyl modified nucleotide, a 2'-0-allyl modified nucleotide, a 2'-C-allyl modified nucleotide, a 2'-fluoro modified nucleotide, a 2'-deoxy modified nucleotide, a 2'-hydroxyl modified nucleotide, and a glycol modified nucleotide; and combinations thereof.

[0076] In some embodiments, at least one of the modified nucleotides in the modified nucleotide is selected from the group consisting of: a deoxy nucleotide, a 2'-0-methyl modified nucleotide, a 2'-fluoro modified nucleotide, a 2'-deoxy modified nucleotide, a glycol modified nucleotide (GNA), a nucleotide comprising a 2' phosphate, and a nucleotide comprising a phosphorothioate group; and combinations thereof.

[0077] In some embodiments, the oligonucleotide comprises at least one 2'-modified nucleotide.

[0078] In some embodiments, the 2’-modified nucleotides are selected from the group consisting of 2’-alkoxy modified nucleotides, 2’-substituted alkoxy modified nucleotides, 2’-alkyl modified nucleotides, 2’-substituted alkyl modified nucleotides, 2’-fluoro modified nucleotides, 2’-acylamido modified nucleotides, 2’-deoxy modified nucleotides, 2’-O-allyl modified nucleotides, 2’-O-alkyl modified nucleotides, 2’-hydroxyl modified nucleotides, 2’-methoxyethyl modified nucleotides, 2’-amino modified nucleotides, 2’-substituted amino modified nucleotides, 2’-deoxynucleotides, nucleotides including 2’ phosphate, and 2’-O-(N-methylacetamide) modified nucleotides; and combinations thereof. For example, C1-C3 alkoxy (e.g., methoxy); substituted alkoxy (e.g., C1-C3 alkoxy substituted C1-C3 alkoxy, e.g., methoxyethoxy); alkyl (e.g., C1-C3 alkyl, e.g., methyl), substituted alkyl (e.g., C1-C3 alkoxy substituted C1-C3 alkyl, e.g., methoxymethyl, methoxyethyl); amino (-NH2), substituted amino (e.g., C1-C3 alkyl mono or di-substituted amino, e.g., methylamino, ethylamino), but are not limited thereto.

[0079] In some embodiments, the 2’-modified nucleotides are selected from the group consisting of 2’-alkoxy modified nucleotides, 2’-substituted alkoxy modified nucleotides, 2’-alkyl modified nucleotides, 2’-substituted alkyl modified nucleotides, 2’-amino modified nucleotides, 2’-substituted amino modified nucleotides, 2’-deoxynucleotides; and combinations thereof.

[0080] In some embodiments, the 2’-modification is selected from the group consisting of 2’-methoxy, 2’-acetamido, 2’-aminoethyl, 2’-fluoro, 2’-O-methyl, and 2’-O-methoxyethyl modifications; and combinations thereof.

[0081] In some embodiments, the 2’-modification is a 2’-methoxy modification.

[0082] In some embodiments, the 2’-modification is a 2’-acetamido modification.

[0083] In some embodiments, all of the nucleotides of the oligonucleotide are modified.

[0084] In some embodiments, the oligonucleotide comprises a modification at the 5’ end comprising a 5’-phosphate analog or a 6-(3-(2-carboxyethyl)phenyl)purine (6-mCEPh-purine).

[0085] In some embodiments, the oligonucleotide has a 5’-phosphate analog modified nucleotide at the 5’ terminal nucleotide.

[0086] In some embodiments, the 5'-phosphate analog modification is a 5'-(E)-vinyl phosphonate (5'-VP).

[0087] In some embodiments, the 5'-phosphate analog modification is a 5'-(E)-vinyl phosphonate (5'-VP).

[0088] In some embodiments, the 5'-phosphate analog modification is a 5'-(E)-vinyl phosphonate (5'-VP).

[0089] In some embodiments, the modification is a modification selected from the group consisting of: 5'-phosphate analog modification, 2'-methoxy (CH3O-), 2'-fluoro (f), 2'-acetyl amino (CH3CO-NH-), and phosphorothioate (s).

[0090] In some embodiments, the oligonucleotide comprises a 6-(3-(2-carboxyethyl)phenyl) purine modified nucleotide at the 5' end. In some embodiments, the oligonucleotide comprises Formula M, which is a 2'-O-methyl-6-(3-(2-carboxyethyl)phenyl)-purine nucleotide of Formula (V);

[0091] In some embodiments, the oligonucleotide comprises at least one modified internucleotide linkage.

[0092] In some embodiments, the oligonucleotide comprises at least one modified internucleotide linkage.

[0093] In some embodiments, at least one modified internucleotide linkage is a phosphorothioate linkage. The phosphorothioate internucleotide linkage modification can occur at any nucleotide on either the sense strand, the antisense strand, or both strands at any position along the chain. For example, the internucleotide linkage modification can occur at every nucleotide on either the sense strand or the antisense strand; every internucleotide linkage modification can occur in an alternating pattern on either the sense strand or the antisense strand; or either the sense strand or the antisense strand can contain both internucleotide linkage modifications in an alternating pattern. The alternating pattern of internucleotide linkage modifications on the sense strand can be the same or different from the antisense strand, and the alternating pattern of internucleotide linkage modifications on the sense strand can be offset relative to the alternating pattern of internucleotide linkages on the antisense strand. In one embodiment, the double stranded RNAi agent comprises 6 to 8 phosphorothioate internucleotide linkages. In some embodiments, the antisense strand comprises two phosphorothioate internucleotide linkages at the 5’ end and two phosphorothioate internucleotide linkages at the 3’ end, and the sense strand comprises at least two phosphorothioate internucleotide linkages at either the 5’ end or the 3’ end.

[0094] In some embodiments of the disclosure, the sense strand comprises an unmodified oligonucleotide selected from any one of SEQ ID NO. 3, 4, 5, 6, 18, 32, 37, 38, 47, 51, 60, 63, 73, and 75, or a modified oligonucleotide of any one of SEQ ID NO: 277-410; and the antisense strand comprises an unmodified oligonucleotide selected from any one of SEQ ID NO. 139, 140, 141, 142, 154, 168, 173, 174, 183, 187, 196, 199, 209, and 211, or a modified oligonucleotide of any one of SEQ ID NO: 415-548.

[0095] In some embodiments of the disclosure, the sense strand comprises a modified oligonucleotide selected from any one of SEQ ID NO. 279, 280, 281, 282, 294, 308, 313, 314, 323, 327, 336, 339, 349, 350; and the antisense strand comprises a modified oligonucleotide selected from any one of SEQ ID NO. 417, 418, 419, 420, 432, 446, 451, 452, 461, 465, 474, 477, 487, 488.

[0096] In some embodiments of the disclosure, the oligonucleotide comprises any one of the following sense strand and antisense strand combinations:

[0097] (1) the sense strand comprises the sequence set forth in SEQ ID NO. 3, and the antisense strand comprises the sequence set forth in SEQ ID NO. 139;

[0098] (2) The sense chain contains the sequence shown in SEQ ID NO.4, and the antisense chain contains the sequence shown in SEQ ID NO.140;

[0099] (3) The sense chain contains the sequence shown in SEQ ID NO.5, and the antisense chain contains the sequence shown in SEQ ID NO.141;

[0100] (4) The sense chain contains the sequence shown in SEQ ID NO.6, and the antisense chain contains the sequence shown in SEQ ID NO.142;

[0101] (5) The sense chain contains the sequence shown in SEQ ID NO.18, and the antisense chain contains the sequence shown in SEQ ID NO.154;

[0102] (6) The sense chain contains the sequence shown in SEQ ID NO.32, and the antisense chain contains the sequence shown in SEQ ID NO.168;

[0103] (7) The positive chain contains the sequence shown in SEQ ID NO.37, and the negative chain contains the sequence shown in SEQ ID NO.173;

[0104] (8) The positive chain contains the sequence shown in SEQ ID NO.38, and the negative chain contains the sequence shown in SEQ ID NO.174;

[0105] (9) The positive chain contains the sequence shown in SEQ ID NO.47, and the negative chain contains the sequence shown in SEQ ID NO.183;

[0106] (10) The sense chain contains the sequence shown in SEQ ID NO.51, and the antisense chain contains the sequence shown in SEQ ID NO.187;

[0107] (11) The sense chain contains the sequence shown in SEQ ID NO.60, and the antisense chain contains the sequence shown in SEQ ID NO.196;

[0108] (12) The sense chain contains the sequence shown in SEQ ID NO.63, and the antisense chain contains the sequence shown in SEQ ID NO.199;

[0109] (13) The sense chain contains the sequence shown in SEQ ID NO. 73, and the antisense chain contains the sequence shown in SEQ ID NO. 209; and

[0110] (14) The positive chain contains the sequence shown in SEQ ID NO.75, and the negative chain contains the sequence shown in SEQ ID NO.211.

[0111] In some embodiments of this disclosure, the oligonucleotide comprises any of the following combinations of sense and antisense strands:

[0112] (1) The positive chain contains the sequence shown in SEQ ID NO.277, and the negative chain contains the sequence shown in SEQ ID NO.415;

[0113] (2) The sense chain contains the sequence shown in SEQ ID NO.278, and the antisense chain contains the sequence shown in SEQ ID NO.416;

[0114] (3) The sense chain contains the sequence shown in SEQ ID NO.279, and the antisense chain contains the sequence shown in SEQ ID NO.417;

[0115] (4) The sense chain contains the sequence shown in SEQ ID NO.280, and the antisense chain contains the sequence shown in SEQ ID NO.418;

[0116] (5) The sense chain contains the sequence shown in SEQ ID NO.292, and the antisense chain contains the sequence shown in SEQ ID NO.430;

[0117] (6) The sense chain contains the sequence shown in SEQ ID NO.306, and the antisense chain contains the sequence shown in SEQ ID NO.444;

[0118] (7) The positive chain contains the sequence shown in SEQ ID NO.311, and the negative chain contains the sequence shown in SEQ ID NO.449;

[0119] (8) The sense chain contains the sequence shown in SEQ ID NO.312, and the antisense chain contains the sequence shown in SEQ ID NO.450;

[0120] (9) The sense chain contains the sequence shown in SEQ ID NO.321, and the antisense chain contains the sequence shown in SEQ ID NO.459;

[0121] (10) The sense chain contains the sequence shown in SEQ ID NO.325, and the antisense chain contains the sequence shown in SEQ ID NO.463;

[0122] (11) The sense chain contains the sequence shown in SEQ ID NO.334, and the antisense chain contains the sequence shown in SEQ ID NO.472;

[0123] (12) The sense chain contains the sequence shown in SEQ ID NO.337, and the antisense chain contains the sequence shown in SEQ ID NO.475;

[0124] (13) The sense chain contains the sequence shown in SEQ ID NO. 347, and the antisense chain contains the sequence shown in SEQ ID NO. 485; and

[0125] (14) The positive chain contains the sequence shown in SEQ ID NO.349, and the negative chain contains the sequence shown in SEQ ID NO.487.

[0126] This disclosure also provides a conjugate or a pharmaceutically acceptable salt thereof for inhibiting MAPT expression, comprising: (i) the aforementioned oligonucleotide or a pharmaceutically acceptable salt thereof, and (ii) a targeting ligand, wherein at least one of the sense strand and antisense strand of the oligonucleotide is conjugated to the targeting ligand.

[0127] At least one nucleotide of an oligonucleotide is conjugated to one or more ligands to form an siRNA conjugate. The siRNA conjugate contains the aforementioned siRNA and a conjugating group attached to that siRNA. The term "oligonucleotide salt" refers to an oligonucleotide compound in salt form. Oligonucleotide salts include salts of oligonucleotide conjugated compounds and salts of unconjugated oligonucleotide compounds. Oligonucleotide salts are advantageously present in solid powder form.

[0128] Generally, the aforementioned conjugation group comprises at least one pharmaceutically acceptable targeting ligand and an optional linker, and the aforementioned siRNA, the aforementioned linker, and the ligand are sequentially linked. The targeting group can be a ligand commonly used in the field of siRNA drug delivery, such as the various ligands described in WO2009082607A2, the entire disclosure of which is incorporated herein by reference. In some embodiments, there are 2-4 aforementioned targeting ligands. The aforementioned siRNA molecule can be non-covalently or covalently conjugated to the aforementioned conjugation group, for example, it can be covalently conjugated to the aforementioned conjugation group. The conjugation site of the siRNA to the conjugation group can be at the 3' or 5' end of the sense or antisense strand of the siRNA, or it can be within the internal sequence of the siRNA. In some embodiments, the conjugation site of the siRNA to the conjugation group is at the 3' or 5' end of the sense strand of the siRNA. In some embodiments, the conjugation site of the siRNA to the conjugation group is at the 3' or 5' end of the antisense strand of the siRNA. In some preferred embodiments, the conjugation site of the aforementioned siRNA and the conjugating group is located at the 3' end of the siRNA's positive strand.

[0129] In some embodiments, the targeting ligand comprises a desialyl glycoprotein receptor ligand. In some embodiments, the desialyl glycoprotein receptor ligand comprises or is composed of one or more galactose derivatives. As used herein, the term "galactose derivative" includes galactose and lactose derivatives with an affinity for the desialyl glycoprotein receptor equal to or greater than that for galactose. Galactose derivatives include, but are not limited to, galactose, galactosamine, N-formylgalactosamine, N-acetylgalactosamine, N-propionyl-galactosamine, N-butyryl-galactosamine, and N-isobutyryl-galactosamine. Galactose derivatives and clusters of galactose derivatives that can be used to target the liver in vivo with oligonucleotides and other molecules are known in the art. Galactose derivatives have been used to target molecules to hepatocytes in vivo by binding to the desialyl glycoprotein receptor (ASGPr) expressed on the surface of hepatocytes. Binding of ASGPr ligands to ASGPr(s) facilitates cell-specific targeting of hepatocytes and the entry of endocytic molecules into hepatocytes. ASGPr ligands can be monomers (e.g., having a single galactose derivative) or polymers (e.g., having multiple galactose derivatives). Galactose derivatives or clusters of galactose derivatives can be linked to the 3' or 5' end of the siRNA using methods known in the art.

[0130] In some embodiments, the pharmaceutically acceptable targeting ligand in the aforementioned siRNA conjugate can be galactose or N-acetylgalactosamine (GalNAc), wherein the galactose or N-acetylgalactosamine molecule can be monovalent, divalent, trivalent, or tetravalent. It should be understood that the monovalent, divalent, trivalent, and tetravalent meanings refer to the molar ratio of siRNA molecules to galactose or N-acetylgalactosamine molecules in the siRNA conjugate being 1:1, 1:2, 1:3, or 1:4, respectively, after the siRNA molecule forms a conjugate with a conjugate group containing galactose or N-acetylgalactosamine as a targeting ligand. In some embodiments, the pharmaceutically acceptable targeting ligand is N-acetylgalactosamine. In some embodiments, when the siRNA of the present invention is conjugated with a conjugate group containing N-acetylgalactosamine, the N-acetylgalactosamine molecule is trivalent or tetravalent. In some embodiments, when the siRNA of the present invention is conjugated with a conjugating group containing N-acetylgalactosamine, the N-acetylgalactosamine molecule is trivalent.

[0131] In some embodiments of this disclosure, the targeting ligand comprises an N-acetylgalactosamine (GalNAc) moiety.

[0132] In some embodiments of this disclosure, the GalNac portion is a monovalent GalNAc portion, a divalent GalNAc portion, a trivalent GalNAc portion, or a tetravalent GalNAc portion.

[0133] The target ligand is L96.

[0134] This disclosure also provides a conjugate or a pharmaceutically acceptable salt thereof for inhibiting MAPT expression, comprising: (i) an oligonucleotide or a pharmaceutically acceptable salt thereof, and (ii) a lipophilic moiety, wherein at least one of the sense and antisense strands of the oligonucleotide is conjugated to one or more lipophilic moieties, optionally, the lipophilic moieties being conjugated via a linker or a vector.

[0135] In some implementations, the lipophilic moiety is conjugated at the 2'-position of a nucleotide or modified nucleotide within the sense or antisense strand. Internal positions include all positions except the two terminal positions at each end of at least one strand of the oligonucleotide. For example, the lipophilic moiety is conjugated at position 6 of the sense strand, meaning the lipophilic moiety is located at position 6 of the sense strand.

[0136] In some embodiments, the lipophilic moiety comprises one to four 2'-O-alkyl modifications. Optionally, the 2'-O-alkyl modified nucleotide is a 2'-C16-modified nucleotide. Optionally, the RNAi agent comprises a single 2'-O-C16-modified nucleotide. Optionally, the single 2'-C16-modified nucleotide is located at the 6th nucleotide position at the 5' end of the positive strand.

[0137] In some embodiments of this disclosure, another modification of the RNAi agent chemically links one or more ligands, portions or conjugates that enhance RNAi activity, cellular distribution or cellular uptake to the RNA. Such moieties include, but are not limited to, lipid moieties, such as cholesterol moieties (Letsinger et al., (1989) Proc. Natl. Acid. Sci. USA, 86: 6553-6556), bile acids (Manohara et al., (1994) Biorg. Med. Chem. Let., 4: 1053-1060), thioethers, such as beryl-S-triphenylmethylthiol (Manoharan et al., (1992) Ann. NY Acad. Sci., 660: 306-309; Manoharan et al., (1993) Biorg. Med. Chem. Let., 3: 2765-2770), sulfur cholesterol (Oberhauser et al., (1992) Nucl. Acids Res., 20: 533-538), and fatty acid chains, such as dodecanediol or undecyl residues (Saison-Behmoaras et al., (1991) EMBO J, 10:1111-1118; Kabanov et al., (1990) FEBS Lett, 259:327-330; Svinarchuk et al., (1993) Biochimie, 75:49-54), phospholipids, such as di-hexadecyl-racemic-glycerol or triethyl-ammonium 1,2-di-O-hexadecyl-racemic-glycerol-3-phosphate (Manoharan et al., (1995) Tetrahedron Lett., 36:3651-3654; Shea et al., (1990) Nucl. Acids Res., 18:3777-3783), polyamines or polyethylene glycol chains (Manoharan et al., (1995) Nucleosides & Nucleotides, 14:969-973), or adamantane acetic acid (Manoharan et al., (1995) Tetrahedron Lett., 36:3651-3654), palmitic moiety (Mishra et al., (1995) Biochim. Biophys. Acta, 1264:229-237), or octadecylamine or hexylamino-carbonyl-oxocholesterol moiety (Crooke et al., (1996) J. Pharmacol. Exp. Ther., 277:923-937).

[0138] In some embodiments, the lipophilic moiety is an aliphatic, cyclic (e.g., alicyclic) or polycyclic (e.g., polyalicyclic) compound, such as a steroid (e.g., a sterol) or a straight-chain or branched aliphatic hydrocarbon. The lipophilic moiety may typically comprise a hydrocarbon chain, which may be cyclic or acyclic. The hydrocarbon chain may contain various substituents and / or one or more heteroatoms, such as oxygen or nitrogen atoms. Such lipophilic aliphatic moietyes include, but are not limited to, saturated or unsaturated C4-C... 30 Hydrocarbons (e.g., C6-C) 22 Hydrocarbons), saturated or unsaturated fatty acids, waxes (e.g., monohydric alcohol esters of fatty acids and fatty diamides), terpenes (e.g., C464 ... 10 Terpenes, C 15 Sesquiterpenes, C 20 Diterpenes, C 30 Triterpenes and C 40 Tetraterpenes and other polycyclic hydrocarbons. For example, the lipophilic moiety can contain C4 to C5. 30 Hydrocarbon chains (e.g., C4 to C5) 30 Alkyl or alkenyl). In some embodiments, the lipophilic moiety comprises saturated or unsaturated C6 to C6 groups. 18 Hydrocarbon chains (e.g., straight-chain C6 to C15) 22 (alkyl or alkenyl). In one embodiment, the lipophilic moiety comprises saturated or unsaturated C. 16 Hydrocarbon chains (e.g., straight-chain C) 16 Alkyl or alkenyl). The lipid moiety is 2'-O-alkyl, consisting of 10-30 hydrocarbon chains, including single and / or branched chains.

[0139] In another embodiment, the lipophilic portion comprises lipids, cholesterol, retinoic acid, cholic acid, adamantaneacetic acid, 1-pyrenebutyric acid, dihydrotestosterone, 1,3-bis-O-(hexadecyl)glycerol, geranyloxyhexanol, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecanyl, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, ibuprofen, naproxen, dimethoxytriphenylmethyl, or phenoxazine.

[0140] The lipophilic moiety is bound to the double-stranded RNAi agent via a linker, which may contain ether, thioether, urea, carbonate, amine, amide, maleimide-thioether, disulfide, phosphate diester, sulfonamide bond, click reaction (e.g., triazole from azide-alkynyl cycloaddition), or carbamate.

[0141] In some embodiments of this disclosure, the ligand that enhances RNAi activity, cell distribution, or cellular uptake comprises C 12 Alkyl, C 16 Alkyl, C 18 Alkyl, C 22Alkyl or branched lipids such as DDA (cationic dimethyl dioctadecyl ammonium) and TDB (trehalose 6,6,9-disorbate).

[0142] For example, the straight-chain lipophilic portion (C16) attached to a position on the chain has the following structure:

[0143] Base is a nucleotide base or a nucleotide base analogue. In some implementations, Base is selected from the group consisting of adenine, guanine, cytosine, thymine, and uracil.

[0144] For example, 2'-O-hexadecyluridine has the following structure:

[0145] This disclosure also provides a conjugate or a pharmaceutically acceptable salt thereof for inhibiting MAPT expression, wherein the lipid conjugate has the following structure:

[0146] A is a modified double-stranded oligonucleotide or a modified single-stranded oligonucleotide, wherein the modified double-stranded oligonucleotide or the modified single-stranded oligonucleotide is conjugated to a lipid-containing moiety at the 3' end of one strand of the modified double-stranded oligonucleotide or the 3' end of the modified single-stranded nucleic acid.

[0147] X1 is:

[0148] L1 is -(CH2)n-, -(CH2) n L2(CH2) n - or key;

[0149] L2 is -C(=O)NH-, -C(=O)O-, -OC(=O)O-, -NHC(=O)O-, -NHC(=O)NH-, -C(=S)NH-, -C(=O)S-, -NH-, O (oxygen) or S (sulfur).

[0150] Each m is an integer from 10 to 18, and each n is an integer from 1 to 6.

[0151] For example, DTX-1

[0152] This disclosure also provides a conjugate or a pharmaceutically acceptable salt thereof for inhibiting MAPT expression, comprising: (i) an oligonucleotide or a pharmaceutically acceptable salt thereof, and (ii) a targeting ligand and a lipophilic moiety, wherein at least one of the sense strand and antisense strand of the oligonucleotide is conjugated to the targeting ligand, and at least one of the sense strand and antisense strand of the oligonucleotide is conjugated to one or more lipophilic moieties.

[0153] In some embodiments of the methods disclosed herein, MAPT gene expression is suppressed by at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, or below the assay detection level. In a preferred embodiment, MAPT expression is suppressed by at least 70%. It should also be understood that it may be desirable to suppress MAPT expression in certain tissues (e.g., the brain) without significantly suppressing expression in other tissues. In a preferred embodiment, expression levels are determined in suitable species-matched cell lines using the assay method provided in Example 2 with siRNA concentrations of 50 nM, 10 nM, 1 nM, and 0.1 nM.

[0154] In some implementations, inhibition of in vivo expression is determined by knocking down the human group in rodents expressing human genes, for example, in AAV-infected mice expressing the human target gene (i.e., MAPT), for example, by confirming the inhibitory effect on the human gene when administered as a single dose, for example, by the point of minimum MAPT expression after a subcutaneous injection of 3 mg / kg. Such systems are useful when the nucleic acid sequences of the human gene and the model animal gene are sufficiently similar that the human iRNA provides effective knockdown of the model animal gene.

[0155] Inhibition of MAPT gene expression can be represented by a reduction in the amount of mRNA expressed in a cell line (such cells may be present, for example, in a sample derived from a subject), in which the MAPT gene has been transcribed and treated (e.g., by contacting one or more cells with the iRNA of this disclosure, or by administering the iRNA of this disclosure to a subject in which cells are present or were previously present), such that MAPT gene expression is inhibited compared to a substantially identical cell line that has not been treated (control cells not treated with iRNA or not treated with iRNA targeting the target gene). In a preferred embodiment, inhibition is assessed in a species-matched cell line using a 10 nM siRNA concentration as described in Example 2, expressed as 2^-ΔΔCT of the mRNA expression level in the treated cells relative to the mRNA level in the control cells, using the following formula, where a housekeeping gene (e.g., GAPDH) is used as an internal control for normalization:

[0156] △CT=CT MAPT -CT GAPDH

[0157] △△CT=△CT 处理细胞 -△CT 对照细胞

[0158] mRNA level = 2^-△△CT

[0159] In other embodiments, inhibition of MAPT gene expression can be assessed based on a decrease in parameters associated with MAPT gene expression function, such as the level of MAPT protein in the blood or serum of a subject. MAPT gene silencing can be determined in any cells expressing MAPT, whether endogenous or heterologous from the expression construct, and by any assay known in the art.

[0160] Inhibition of MAPT protein expression can be demonstrated by a decrease in the level of MAPT protein or secreted luciferase expressed in cells or cell populations, or in a subject sample (e.g., protein levels in a blood sample from the subject). As described above, to assess mRNA inhibition, inhibition of protein expression levels in treated cells or cell populations can similarly be expressed as a percentage of protein levels in control cells or cell populations, or as a change in protein levels in a subject sample (e.g., blood or serum from it), expressed as a percentage of MAPT or Gluc expression in the treated sample (e.g., blood or serum from it) relative to MAPT or Gluc expression in control cells, using the following formula.

[0161] mRNA inhibition percentage = (protein expression level) 处理细胞 - Protein expression level 对照细胞 Protein expression level 对照细胞 *100%

[0162] Control cells, cell populations, or subject samples that can be used to assess inhibition of MAPT gene expression include cells, cell populations, or subject samples that have not yet been exposed to the RNAi agent of this disclosure. For example, control cells, cell lines, or subject samples may be derived from individual subjects (e.g., human or animal subjects) before treating subjects or appropriately matched cohorts with the RNAi agent.

[0163] In some embodiments of the methods disclosed herein, RNAi is administered to a subject to deliver RNAi to a specific site within the subject. Inhibition of MAPT expression can be assessed by measuring the level or changes in MAPT mRNA or MAPT protein or fusion secreted luciferase in fluid or tissue samples from a specific site in the subject (e.g., the brain or blood).

[0164] This disclosure also provides a method for using the RNAi of this disclosure or a composition containing the RNAi of this disclosure to inhibit MAPT expression, thereby preventing or treating MAPT-related conditions, such as cerebral amyloid angiopathy (CAA) or Alzheimer's disease (AD), including early-onset familial Alzheimer's disease (EOFAD), dementia, etc.

[0165] Cells suitable for treatment using the methods of this disclosure can be any cell expressing the MAPT gene, such as hepatocytes, nerve cells, gallbladder cells, heart cells, or kidney cells, but preferably nerve cells. Cells suitable for use in the methods of this disclosure can be mammalian cells, such as primate cells (e.g., human cells, including human cells in chimeric nonhuman animals, or nonhuman primate cells, such as monkey cells or chimpanzee cells) or non-primate cells. In some embodiments, the cells are human cells, such as human nerve cells. In the methods of this disclosure, MAPT expression in cells is inhibited by at least 30%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, or below a measured detection level.

[0166] The in vivo methods of this disclosure may include administering a composition comprising RNAi to a subject, wherein the RNAi comprises a nucleotide sequence complementary to at least a portion of the RNA transcript of the MAPT gene of the mammal to which the RNAi agent is administered. The composition may be administered in any manner known in the art, including but not limited to oral, intraperitoneal, or parenteral routes, including intracranial (e.g., intraventricular, intraparenchymal, and intrathecal), intravenous, intramuscular, subcutaneous, transdermal, airway (aerosol), nasal, rectal, and local (including oral and sublingual) administration. In some embodiments, the composition is administered by intravenous infusion or injection. In some embodiments, the composition is administered subcutaneously. In some embodiments, the composition is administered by intrathecal injection.

[0167] In one aspect, this disclosure also provides a method for inhibiting MAPT gene expression in mammals. The method comprises administering an oligonucleotide or a pharmaceutically acceptable salt thereof, or a combination thereof, to a mammal. The oligonucleotide is a double-stranded RNA (dsRNA) that targets the MAPT gene in mammalian cells and sustains the mammal for a sufficient time to allow for degradation of the mRNA transcript of the MAPT gene, thereby inhibiting the expression of the MAPT protein in the cells. The reduction in gene expression can be assessed by any method known in the art and by means of methods such as qRT-PCR described herein, for example, as in Example 2. The reduction in protein product can be assessed by any method known in the art (e.g., ELISA). In other embodiments, a blood sample is used as a subject sample to monitor the reduction in MAPT protein expression.

[0168] This disclosure also provides methods of treatment in subjects who require it, such as those diagnosed with MAPT-related conditions, cerebral amyloid angiopathy (CAA), or Alzheimer's disease (AD), including early-onset familial Alzheimer's disease (EOFAD), dementia, etc.

[0169] In one implementation, MAPT-related diseases are cerebral amyloid angiopathy (CAA) or Alzheimer's disease (AD), including early-onset familial Alzheimer's disease (EOFAD), dementia, etc.

[0170] The RNAi disclosed herein can be administered as “free RNAi.” Free RNAi is administered in the absence of a pharmaceutical composition. Naked RNAi can be administered in a suitable buffer solution. The buffer solution may contain acetate, citrate, lactate, tartrate, carbonate, or phosphate, or any combination thereof. In one embodiment, the buffer solution is phosphate-buffered saline (PBS). The pH and osmotic pressure of the buffer solution containing RNAi can be adjusted to suit its administration to the subject.

[0171] The administration of RNAi according to the method of this disclosure can lead to the prevention or treatment of MAPT-related conditions, such as cerebral amyloid angiopathy (CAA) or Alzheimer's disease (AD), including early-onset familial Alzheimer's disease (EOFAD), dementia, etc. A therapeutic dose of RNAi can be administered to the subject, such as from about 0.01 mg / kg to about 200 mg / kg. Preferably, it is from 1 mg / kg to about 50 mg / kg. RNAi is preferably administered subcutaneously, i.e., by intrathecal injection. One or more injections can be used to deliver the desired dose of RNAi to the subject. Injections can be repeated over a period of time.

[0172] It can be administered periodically. In some embodiments, treatment can be administered at a lower frequency after the initial treatment regimen. Repeated dosing regimens may include periodic administration of therapeutic doses of RNAi, such as once a month to once a year. In some embodiments, RNAi is administered approximately once a month to approximately every three months, or approximately every three months to approximately every six months, or even once a year.

[0173] This disclosure further provides RNAi agents or pharmaceutical compositions thereof in combination with other drugs and / or other treatments (e.g., known drugs and / or known treatments, such as those currently used to treat these conditions) to treat subjects who would benefit from reduced and / or suppressed MAPT gene expression, such as subjects with MAPT-related diseases. Other therapeutic agents and treatments suitable for treating subjects who would benefit from reduced MAPT expression (e.g., subjects with MAPT-related diseases) include tacolin, donepezil, rivastigmine, galantamine, memantine and memantine-donepezil combination preparations, cholinesterase inhibitors donepezil, huperzine A, galantamine hydrobromide, rivastigmine bitartrate, etc.

[0174] Example

[0175] Example 1. Preparation of ligands and siRNA

[0176] Unless otherwise specified in this article, such reagents can be obtained from any molecular biology reagent supplier, and their quality / purity standards are applicable to molecular biology.

[0177] Abbreviations for nucleotide monomers used in nucleic acid sequence representation. It should be understood that when a nucleotide contains a 2'-fluorine modification, the fluorine replaces the hydroxyl group at that position in the parent nucleotide (i.e., it is a 2'-deoxy-2'-fluorine nucleotide).

[0178] Table A. Abbreviations for nucleotide monomers used in nucleic acid sequence representation

[0179] Preparation of ligands

[0180] L96 was prepared according to the method described in patent CN104717982B.

[0181] C16 was purchased from Chengdu Pioneer Pharmaceuticals Co., Ltd.

[0182] The preparation of DTX was carried out in accordance with the method described in patent application CN113166191A.

[0183] Preparation of oligonucleotides

[0184] (1) Preparation of siRNA

[0185] First, a computer-based algorithm was used to generate candidate oligonucleotide sequences complementary to human MAPT mRNA (NM_016841.4, Table 1). Some of these sequences were also complementary to or had no more than two mismatches with cynomolgus monkey MAPT mRNA (XM_005584540.2, Table 1). Some were designed as double-stranded siRNAs with 19 / 21 pairings on the sense and antisense strands, with the antisense strand having two drooping ends complementary to the mRNA sequence. In some cases, the drooping ends of the antisense strand were non-complementary UU. Some sequences were designed as double-stranded siRNAs with 21 / 23 pairings on the sense and antisense strands, with the antisense strand having two drooping ends complementary to the mRNA sequence. Some sequences were designed as double-stranded siRNAs with 21 / 21 and 23 / 23 pairings. In some complementary pairing sequences, the first base at the 5' end of the antisense strand (the last base at the 3' end of the sense strand) was replaced with a base that did not match the MAPT mRNA.

[0186] Table 1. MAPT mRNA sequences of humans and cynomolgus monkeys

[0187] The siRNA sequence was synthesized separately on a solid support via the sense strand (SS) and antisense strand (AS), and was obtained after deprotection, cleavage, purification, annealing, purification and lyophilization.

[0188] Solid-phase synthesis (Figure 3): The sense and antisense strands were synthesized separately on a solid support using an automated oligonucleotide synthesizer, employing phosphoramide technology. The synthesizer used was, for example, the AKTA Oligopilot (Cytiva) or Dr. Oligo 192XLc (Kunshan Berlik Precision Instruments Co., Ltd.). Solid-phase synthesis began at the 3' end of the sequence, with monomers sequentially coupled into the sequence. Each coupling of a phosphoramide monomer involved four chemical steps: 1) unblocking or deprotection (de-hydroxyl protecting group); 2) coupling; 3) oxidation; and 4) end-capping. All phosphoramidite monomers, reagents, and purification consumables used were commercially available. For example, various phosphoramidite monomers (such as 5'-O-(4,4'-Dimethoxytrityl)-2'-O-methyl-Uridine-3'-CE-Phosphoramidite) were purchased from Shanghai Zhaowei Technology Development Co., Ltd., and reaction reagents (such as 40wt% methylamine aqueous solution and 28wt% ammonium hydroxide aqueous solution) were purchased from Sigma-Aldrich LLC. The siRNA synthesis and purification methods used in this paper are as described in US20130178612A1 and US2015100197A1; the synthesis methods for VPUm and APU structural sequences are as described in J.Med.Chem.2018,61,734-744.

[0189] (2) Preparation of double-stranded RNA reagent

[0190] (a) The synthesis of the chain of justice

[0191] Solid-phase phosphoramide synthesis is a mature method for synthesizing oligonucleotides. A computer-controlled synthesizer is used, and the reaction takes place in a stainless steel column. The positive chain synthesis begins with a solid support loaded with a targeting ligand (e.g., L96), or directly with the solid support. Different starting materials, reagents, and solvents are injected sequentially from sequence 3' to 5' using the solid-phase synthesizer, linking phosphoramide nucleoside monomers one by one. The reaction process involves four cyclic steps: DMT protection removal, condensation, oxidation or thiolation, and end-capping. One nucleotide unit is linked in each cycle, yielding an oligonucleotide sequence of 19 or 21 nucleotides. After synthesis, the protecting group (2-cyanoethyl) is removed on the solid-phase column, and the synthesized sequence is cleaved from the solid support via ammonolysis. The sequence is filtered, the filter cake is washed with ethanol, and the filtrate and washings are collected and concentrated to obtain the crude positive chain. The crude product is purified by chromatography (SOURCE 15Q) and lyophilized to obtain the target product, the positive chain. In the synthesizer, siRNA positive strand conjugates were synthesized starting with a solid support loaded with a targeting ligand (e.g., L96); siRNA was synthesized directly starting with a solid support.

[0192] (b) Synthesis of antisense chains

[0193] Similar to the sense strand synthesis, the antisense strand is synthesized using a solid-phase synthesizer. Different starting materials, reagents, and solvents are injected sequentially from the 3' to 5' ends of the sequence through different tubing, linking phosphoramidine nucleoside monomers one by one. The reaction process involves four cyclic steps: DMT protection removal, condensation, oxidation or thiolation, and end-capping. One nucleotide unit is linked in each cycle, yielding an oligonucleotide sequence of 21 or 23 nucleotides. After synthesis, the protecting group (2-cyanoethyl) is removed on a solid-phase column, and the synthesized sequence is cleaved from the solid support via ammonolysis. The sequence is filtered, the filter cake is washed with ethanol, and the filtrate and washings are collected and concentrated to obtain the crude antisense strand. The crude product is purified by chromatography (SOURCE 15Q), ultrafiltered, and lyophilized to obtain the target product, antisense siRNA.

[0194] (c) Preparation of double-stranded siRNA

[0195] The AS and SS strands were dissolved separately in injection water and mixed in a defined ratio (1.01:1.0-1.2:1.0). The mixture was incubated at 30-50°C for 30-90 minutes and then cooled to room temperature. The double-stranded siRNA product was obtained by freeze-drying.

[0196] The double-stranded siRNA reagents listed in Tables 2, 3, and 4 below were prepared using the same method.

[0197] In Tables 2, 3, and 4, “G”, “C”, “A”, “U”, and “T” usually represent nucleotides with guanine, cytosine, adenine, uracil, and thymine as bases, respectively.

[0198] Modifications: m represents 2'-methoxy; f represents 2'-deoxy-2'-fluorine; s represents thiophosphate; VPUm is 2'-methoxy modified uridine; L96 is N-[tris(GalNAc-alkyl)amidodecyl]-4-hydroxyprolyl (Hyp-(GalNAc-alkyl)3).

[0199] Some oligonucleotides may contain GNA (glucan-diol nucleic acid) modifications. In some oligonucleotides, individual ribonucleic acid units are replaced with (Tgn), which is the S-isomer of thymidine-diol nucleic acid (GNA) shown in formula (I). In some oligonucleotides, individual ribonucleic acid units are replaced with (Cgn), which is the S-isomer of cytidine-diol nucleic acid (GNA) shown in formula (II). In some oligonucleotides, individual ribonucleic acid units are replaced with (Agn), which is the S-isomer of adenosine-diol nucleic acid (GNA) shown in formula (III). In some oligonucleotides, individual ribonucleic acid units are replaced with (Ggn), which is the S-isomer of guanosine-diol nucleic acid (GNA) shown in formula (IV). The structural formulas of Tgn, Cgn, Agn, and Ggn are as follows:

[0200] Table 2. Naked Oligonucleotide Sequences

[0201] Table 3 Oligonucleotide Modification Sequences

[0202] Example 2. In vitro activity screening of MAPT-siRNA

[0203] (1) Cell culture and transfection:

[0204] Human neuroblastoma cells (also known as Be2C cells) (Tongpai (Shanghai) Biotechnology Co., Ltd., catalog number BE(2)-C cells) were collected and placed in a 37℃, 5% CO2 incubator. They were cultured using DMEM, high glucose (Thermo, catalog number 11965-092), with the addition of 10% FBS (GIBCO, 12483020) and 1% penicillin-streptomycin (GIBCO, 15140-122). Once the cell confluence reached 90%, the cells were digested with trypsin-EDTA (Thermo, 25200-072), counted using a Countstar (IC1000), and seeded with 190 μl of cell suspension per well in 96-well plates. The seeding number of Be2C cells was 1*102. 4 Cells / wells will adhere to the culture vessel the following day for transfection.

[0205] Transfection was performed using Lipofectamine™ RNAiMAX (thermofisher, 13778150). A transfection complex was prepared by mixing 2.2 μl (2 μM) of the diluted compound, 19.1 μl of Opti-MEM (thermofisher, 1105821), and 0.7 μl of RNAiMAX. After incubation for 5 minutes, the transfection complex was added to the cells (two replicates per complex), 10 μl per well, with a final siRNA concentration of 10 nM. The cells were incubated at 37°C in a 5% CO2 incubator for 24 hours.

[0206] (2) RNA extraction and detection

[0207] (i) One day after transfection, remove the culture medium; wash with PBS; add 200 μl of lysis buffer to each well, incubate at room temperature for 15 minutes, and then rehydrate. RNA was extracted using a 96-throughput automated nucleic acid extractor (Hanwei Technology, HW-96 series). RNA concentration was measured for subsequent experiments. The extracted RNA can be used immediately for subsequent experiments or stored at -80℃ for later use.

[0208] (ii) Use IIQ RT SuperMix for qPCR (+gDNA wiper) reverse transcription kit (Novizan, R223-01) for cDNA synthesis:

[0209] Prepare a mixture in an RNase-free centrifuge tube: 4 μl 4×g DNA wiper Mix, 1 μg template RNA, and RNase-free ddH2O to a final volume of 16 μl to remove genomic DNA. Gently pipette to mix and incubate at 42°C for 2 min. Then, directly add 4 μl 5×HiScript II qRT SuperMix II to the reaction tube and gently pipette to mix. Incubate in a PCR instrument (Bio-Rayer C1000 Touch PCR instrument) at 50°C for 15 min, then at 85°C for 5 sec, and finally at 4°C. The product can be used immediately for qPCR reactions or stored at -20°C and used within six months. For long-term storage, aliquot and store at -80°C. Avoid repeated freeze-thaw cycles for cDNA.

[0210] (iii) Quantitative PCR was performed using ChamQ SYBR qPCR Master Mix (Novazia, Q311-02):

[0211] Prepare a 20 μl mixture by adding 10 μl 2×ChamQ SYBR qPCR Master Mix, 0.5 μl Forward primer (Ruiboxingke), 0.5 μl Reverse primer (Ruiboxingke), 1 μl Template cDNA, and 8 μl ddH2O. Each sample was replicated in triplicate. The 96-well plate was placed in a qPCR instrument (Quantstudio 5), and the following program was executed: pre-denaturation, 95℃, 30 sec; amplification, 95℃, 10 sec, 60℃, 30 sec, 40 cycles; melting curve, 95℃, 15 sec, 60℃, 60 sec, 95℃, 15 sec.

[0212] (3) Data statistical analysis:

[0213] Export the data to Excel format using CT. MAPT -CT GAPDH The control group was normalized. To calculate the fold change in relative silencing efficiency, the data were analyzed using the ΔΔCT method. The mean and standard deviation of the three parallel replicates were calculated.

[0214] The results of the two screenings of Be2C cells are shown in Tables 5-6.

[0215] As shown in Table 4, at a dosage of 10 nM, the MAPT mRNA inhibition rate of 42 siRNAs reached or approached 30% or higher. AL0255005, AL0255006, AL0255007, AL0255008, AL0255020, AL0255034, AL0255039, AL0255040, AL0255049, AL0255053, AL0255062, AL0255065, AL0255075, and AL0255077 showed the best screening results, with inhibition rates exceeding 50% at 10 nM. AL0255005, AL0255006, and AL0255007 achieved the highest inhibition rate, reaching up to 70%.

[0216] Table 4 shows the MAPT siRNA sequence knockdown level in Be2C cells.

Claims

1. An oligonucleotide or a pharmaceutically acceptable salt thereof for use in inhibiting MAPT expression, the oligonucleotide comprising a sense strand and an antisense strand, the sense strand having a sequence with at least 60% or more sequence identity to a sequence set forth in any one of SEQ ID NO. 3-136 or a fragment thereof, or a modified sequence of the sequence or the fragment thereof, preferably having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more sequence identity; the antisense strand having a sequence with at least 60% or more sequence identity to a sequence set forth in any one of SEQ ID NO. 139-272 or a fragment thereof, or a modified sequence of the sequence or the fragment thereof, preferably having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more sequence identity.

2. The oligonucleotide or pharmaceutically acceptable salt thereof of claim 1, wherein, the pharmaceutically acceptable salt of the oligonucleotide is selected from the group consisting of a carboxylate salt, an alkali metal salt, an ammonium salt, an alkaline earth metal salt, a salt with an organic base, and other pharmaceutically acceptable salts; preferably, the salt is an alkali metal salt, more preferably a sodium salt or a potassium salt; preferably, the salt is an alkaline earth metal salt, more preferably a magnesium salt or a calcium salt; preferably, the salt is an ammonium salt.

3. The oligonucleotide or pharmaceutically acceptable salt thereof of claim 1 or 2, wherein, the oligonucleotide comprises at least one modified nucleotide; preferably, the at least one modified nucleotide in the modified nucleotide is selected from the group consisting of a deoxynucleotide, a 3' terminal deoxythymidine (dT) nucleotide, a 2'-O-methyl modified nucleotide, a 2'-fluoro modified nucleotide, a 2'-deoxy modified nucleotide, a locked nucleotide, a 2'-5'-linked ribonucleotide (3'-RNA), an unlocked nucleotide, a conformationally restricted nucleotide, a constrained ethyl nucleotide, an abasic nucleotide, a 2'-amino modified nucleotide, a 2'-O-allyl modified nucleotide, a 2'-O-alkyl modified nucleotide, a 2'-hydroxyl modified nucleotide, a 2'-methoxyethyl modified nucleotide, a 2'-O-alkyl modified nucleotide, a morpholino nucleotide, a phosphoramidate, a nucleotide comprising a non-natural base, a tetrahydropyran modified nucleotide, a 1,5-anhydrohexitol modified nucleotide, a cyclohexenyl modified nucleotide, a nucleotide comprising a phosphorothioate group, a nucleotide comprising a methylphosphonate group, a nucleotide comprising a 5'-phosphate, a nucleotide comprising a 5'-phosphate analog, a vinyl-phosphonate nucleotide, a thermally unstable nucleotide, a glycol modified nucleotide, a nucleotide comprising a 2' phosphate, and a 2-O-(N-methylacetamide) modified nucleotide; and combinations thereof; preferably, the at least one modified nucleotide in the modified nucleotide is selected from the group consisting of a deoxynucleotide, a 3' terminal deoxythymidine (dT) nucleotide, a 2'-O-methyl modified nucleotide, a 2'-fluoro modified nucleotide, a 2'-deoxy modified nucleotide, a locked nucleotide, a 2'-5'-linked ribonucleotide (3'-RNA), an unlocked nucleotide, a conformationally restricted nucleotide, a constrained ethyl nucleotide, an abasic nucleotide, a 2'-amino modified nucleotide, a 2'-O-allyl modified nucleotide, a 2'-O-alkyl modified nucleotide, a 2'-hydroxyl modified nucleotide, a 2'-methoxyethyl modified nucleotide, a 2'-O-alkyl modified nucleotide, a morpholino nucleotide, a phosphoramidate, a nucleotide comprising a non-natural base, a tetrahydropyran modified nucleotide, a 1,5-anhydrohexitol modified nucleotide, a cyclohexenyl modified nucleotide, a nucleotide comprising a phosphorothioate group, a nucleotide comprising a methylphosphonate group, a nucleotide comprising a 5'-phosphate, a nucleotide comprising a 5'-phosphate analog, a vinyl-phosphonate nucleotide, a thermally unstable nucleotide, a glycol modified nucleotide, a nucleotide comprising a 2' phosphate, and a 2-O-(N-methylacetamide) modified nucleotide; and combinations thereof; Preferably, the at least one modified nucleotide in the modified nucleotide is selected from the group consisting of: LNA modified nucleotide, HNA modified nucleotide, CeNA modified nucleotide, 2'-methoxyethyl modified nucleotide, 2'-O-alkyl modified nucleotide, 2'-O-allyl modified nucleotide, 2'-C-allyl modified nucleotide, 2'-fluoro modified nucleotide, 2'-deoxy modified nucleotide, 2'-hydroxyl modified nucleotide, and glycol modified nucleotide; and combinations thereof; Preferably, the at least one modified nucleotide in the modified nucleotide is selected from the group consisting of: deoxy nucleotide, 2'-O-methyl modified nucleotide, 2'-fluoro modified nucleotide, 2'-deoxy modified nucleotide, glycol modified nucleotide, nucleotide comprising 2' phosphate, and nucleotide comprising phosphorothioate group; and combinations thereof; Preferably, the oligonucleotide comprises at least one 2'-modified nucleotide; Preferably, the 2'-modified nucleotide is selected from the group consisting of: 2'-alkoxy modified nucleotide, 2'-substituted alkoxy modified nucleotide, 2'-alkyl modified nucleotide, 2'-substituted alkyl modified nucleotide, 2'-fluoro modified nucleotide, 2'-acylamino modified nucleotide, 2'-deoxy modified nucleotide, 2'-O-allyl modified nucleotide, 2'-O-alkyl modified nucleotide, 2'-hydroxyl modified nucleotide, 2'-methoxyethyl modified nucleotide, 2'-amino modified nucleotide, 2'-substituted amino modified nucleotide, 2'-deoxy nucleotide, nucleotide comprising 2' phosphate, and 2'-O-(N-methylacetamide) modified nucleotide; and combinations thereof; Preferably, the 2'-modification is selected from the group consisting of: 2'-methoxy, 2'-acetamido, 2'-aminoethyl, 2'-fluoro, 2'-O-methyl, and 2'-O-methoxyethyl modification; and combinations thereof; Preferably, the oligonucleotide comprises a modification at the 5' end, the modification comprising a 5'-phosphate analog or 6-(3-(2-carboxyethyl)phenyl)purine (6-mCEPh-purine); Preferably, the 5'-phosphate analogue modification is a 5'-(E)-vinylphosphonate (5'-VP); preferably, the nucleotide comprising a 5'-(E)-vinylphosphonate modification at the 5' terminus has the structure shown in Formula (I); wherein Base represents a natural or modified base; preferably Base is selected from A, G, C, and U; R is selected from H, fluoro, 2'-methoxy, 2'-acetamido, 2'-aminoethyl, and 2'-O-methoxyethyl; preferably, the 5'-phosphate analogue modified nucleotide has the structure shown in Formula (II); more preferably, the 5'-phosphonate analogue modified nucleotide is an APU of Formula (III) or a VPUm of Formula (IV); Preferably, the oligonucleotide comprises a 6-(3-(2-carboxyethyl)phenyl) purine modified nucleotide at the 5' terminus; preferably, the oligonucleotide comprises Formula M, which is a 2'-0-methyl-6-(3-(2-carboxyethyl)phenyl)-purine nucleotide as shown in Formula (V); 4. The oligonucleotide or pharmaceutically acceptable salt thereof of any one of claims 1-3, wherein, the oligonucleotide comprises at least one modified internucleotide linkage; Preferably, the at least one modified internucleotide linkage is a phosphorothioate linkage.

5. The oligonucleotide or a pharmaceutically acceptable salt thereof according to any one of claims 1-4, wherein, the sense strand comprises an unmodified oligonucleotide selected from any one of SEQ ID NO. 3, 4, 5, 6, 18, 32, 37, 38, 47, 51, 60, 63, 73, and 75, or a modified oligonucleotide of any one of SEQ ID NO: 277-410; the antisense strand comprises an unmodified oligonucleotide selected from any one of SEQ ID NO. 139, 140, 141, 142, 154, 168, 173, 174, 183, 187, 196, 199, 209, and 211, or a modified oligonucleotide of any one of SEQ ID NO: 415-548; Preferably, the sense strand comprises a modified oligonucleotide selected from any one of SEQ ID NO. 277, 278, 279, 280, 292, 306, 311, 312, 321, 325, 334, 337, 347, and 349; and the antisense strand comprises a modified oligonucleotide selected from any one of SEQ ID NO. 415, 416, 417, 418, 430, 444, 449, 450, 459, 463, 472, 475, 485, and 487.

6. The oligonucleotide or a pharmaceutically acceptable salt thereof of any one of claims 1-3, wherein, The oligonucleotide comprises any one of the following sense strand and antisense strand combinations: (1) the sense strand comprises the sequence set forth in SEQ ID NO. 3, and the antisense strand comprises the sequence set forth in SEQ ID NO. 139; (2) the sense strand comprises the sequence set forth in SEQ ID NO. 4, and the antisense strand comprises the sequence set forth in SEQ ID NO. 140; (3) the sense strand comprises the sequence set forth in SEQ ID NO. 5, and the antisense strand comprises the sequence set forth in SEQ ID NO. 141; (4) the sense strand comprises the sequence set forth in SEQ ID NO. 6, and the antisense strand comprises the sequence set forth in SEQ ID NO. 142; (5) the sense strand comprises the sequence set forth in SEQ ID NO. 18, and the antisense strand comprises the sequence set forth in SEQ ID NO. 154; (6) the sense strand comprises the sequence set forth in SEQ ID NO. 32, and the antisense strand comprises the sequence set forth in SEQ ID NO. 168; (7) the sense strand comprises the sequence set forth in SEQ ID NO. 37, and the antisense strand comprises the sequence set forth in SEQ ID NO. 173; (8) the sense strand comprises the sequence set forth in SEQ ID NO. 38, and the antisense strand comprises the sequence set forth in SEQ ID NO. 174; (9) the sense strand comprises the sequence set forth in SEQ ID NO. 47, and the antisense strand comprises the sequence set forth in SEQ ID NO. 183; (10) the sense strand comprises the sequence set forth in SEQ ID NO. 51, and the antisense strand comprises the sequence set forth in SEQ ID NO. 187; (11) the sense strand comprises the sequence set forth in SEQ ID NO. 60, and the antisense strand comprises the sequence set forth in SEQ ID NO. 196; (12) the sense strand comprises the sequence set forth in SEQ ID NO. 63, and the antisense strand comprises the sequence set forth in SEQ ID NO. 199; (13) the sense strand comprises the sequence set forth in SEQ ID NO. 73, and the antisense strand comprises the sequence set forth in SEQ ID NO. 209; and (14) the sense strand comprises the sequence set forth in SEQ ID NO. 75, and the antisense strand comprises the sequence set forth in SEQ ID NO.

211.

7. The oligonucleotide or pharmaceutically acceptable salt thereof of any one of claims 1-6, wherein, The oligonucleotide comprises any one selected from the following combinations of sense strand and antisense strand: (1) the sense strand comprises the sequence set forth in SEQ ID NO. 277, and the antisense strand comprises the sequence set forth in SEQ ID NO. 415; (2) the sense strand comprises the sequence set forth in SEQ ID NO. 278, and the antisense strand comprises the sequence set forth in SEQ ID NO. 416; (3) the sense strand comprises the sequence set forth in SEQ ID NO. 279, and the antisense strand comprises the sequence set forth in SEQ ID NO. 417; (4) the sense strand comprises the sequence set forth in SEQ ID NO. 280, and the antisense strand comprises the sequence set forth in SEQ ID NO. 418; (5) the sense strand comprises the sequence set forth in SEQ ID NO. 292, and the antisense strand comprises the sequence set forth in SEQ ID NO. 430; (6) the sense strand comprises the sequence set forth in SEQ ID NO. 306, and the antisense strand comprises the sequence set forth in SEQ ID NO. 444; (7) the sense strand comprises the sequence set forth in SEQ ID NO. 311, and the antisense strand comprises the sequence set forth in SEQ ID NO. 449; (8) the sense strand comprises the sequence set forth in SEQ ID NO. 312, and the antisense strand comprises the sequence set forth in SEQ ID NO. 450; (9) the sense strand comprises the sequence set forth in SEQ ID NO. 321, and the antisense strand comprises the sequence set forth in SEQ ID NO. 459; (10) the sense strand comprises the sequence set forth in SEQ ID NO. 325, and the antisense strand comprises the sequence set forth in SEQ ID NO. 463; (11) the sense strand comprises the sequence set forth in SEQ ID NO. 334, and the antisense strand comprises the sequence set forth in SEQ ID NO. 472; (12) the sense strand comprises the sequence set forth in SEQ ID NO. 337, and the antisense strand comprises the sequence set forth in SEQ ID NO. 475; (13) the sense strand comprises the sequence set forth in SEQ ID NO. 347, and the antisense strand comprises the sequence set forth in SEQ ID NO. 485; and (14) the sense strand comprises the sequence set forth in SEQ ID NO. 349, and the antisense strand comprises the sequence set forth in SEQ ID NO.

487.

8. A conjugate or a pharmaceutically acceptable salt thereof for use in inhibiting MAPT expression, comprising: (i) the oligonucleotide of any one of claims 1-7 or a pharmaceutically acceptable salt thereof, and (ii) a targeting ligand and / or a lipophilic moiety, wherein at least one of the sense strand and the antisense strand of the oligonucleotide is conjugated to the targeting ligand and / or at least one of the sense strand and the antisense strand of the oligonucleotide is conjugated to one or more of the lipophilic moieties; Preferably, the targeting ligand comprises an N-acetylgalactosamine (GalNAc) moiety; preferably, the GalNac moiety is a monovalent GalNAc moiety, a bivalent GalNAc moiety, a trivalent GalNAc moiety or a tetravalent GalNAc moiety; more preferably, the targeting ligand is L96; Preferably, the targeting ligand is conjugated to the 3' end or the 5' end of the sense strand; more preferably, the targeting ligand is conjugated to the 3' end of the sense strand; Preferably, the lipophilic moiety is an aliphatic, cycloaliphatic or polycycloaliphatic compound; preferably, the lipophilic moiety contains a saturated or unsaturated C16 hydrocarbon chain; Preferably, the lipophilic moiety comprises a lipid moiety; Preferably, the lipophilic moiety is located at position 6 of the sense strand.

9. A composition comprising the oligonucleotide or a pharmaceutically acceptable salt thereof of any one of claims 1 to 7, or the conjugate or a pharmaceutically acceptable salt thereof of claim 8, and optionally a pharmaceutically acceptable carrier; Preferably, the dosage form of the composition is an oral dosage, an intravenous injection, a subcutaneous injection, an intramuscular injection or an intrathecal injection; preferably an intrathecal injection; Preferably, the composition further comprises other drugs for treating and / or preventing MAPT-related disorders.

10. Use of the oligonucleotide or a pharmaceutically acceptable salt thereof of any one of claims 1 to 7, the conjugate or a pharmaceutically acceptable salt thereof of claim 8, or the composition of claim 9, for the manufacture of a medicament for treating and / or preventing MAPT-related disorders. Preferably, the MAPT-related disorders are selected from cerebral amyloid angiopathy (CAA) or Alzheimer's disease (AD), including early-onset familial Alzheimer's disease (EOFAD), dementia, etc.

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