Oligonucleotide targeting amyloid precursor protein gene and use thereof

Abstract

An RNAi agent targeting an amyloid precursor protein, such as a double-stranded small interfering RNA (siRNA) agent. A method for inhibiting the expression of the APP gene by using the RNAi agent and a method for preventing and treating APP-related diseases, such as cerebral amyloid angiopathy (CAA) or Alzheimer's disease (AD), including early-onset familial Alzheimer's disease (EOFAD). The siRNA significantly inhibits the expression level of the APP gene and has a long-lasting drug effect.

Description

Oligonucleotides targeting amyloid precursor protein gene and uses thereof TECHNICAL FIELD

[0001] The present disclosure relates to an oligonucleotide, in particular to the inhibition of amyloid precursor protein (APP) gene expression and the treatment of related diseases caused by APP cleavage product beta amyloid. BACKGROUND

[0002] Amyloid precursor protein is a single transmembrane protein widely present in cells of tissues throughout the body, which produces beta-amyloid protein with toxic effect after protease cleavage. So far, people still know little about the true physiological function of APP, and many scholars believe that the senile plaques formed by the deposition of Aβ formed after APP cleavage is an important cause of the onset of Alzheimer's disease (AD).

[0003] It is generally believed that the factors that produce neurotoxicity in the process of AD mainly include Aβ, p-Tau, over-activated microglia, free radicals, etc. Among them, beta amyloid protein (Aβ) is a polypeptide composed of 39-43 amino acids, which is formed by the hydrolysis of APP. The latter has mainly two degradation pathways, most of which are hydrolyzed by α-secretase to generate sAPPα and C-terminal fragment α; a small part is cut by β-secretase and γ-secretase to generate Aβ (mainly Aβ40 and Aβ42) that can form amyloid aggregation. A large number of studies have shown that Aβ monomers and oligomers can produce neurotoxicity under different conditions in vivo and in vitro, affect synaptic plasticity by inhibiting the release of excitatory amino acids, induce Tau protein phosphorylation and immune cell activation of neurons, etc. to cause neuronal cell degeneration and necrosis. Therefore, the mainstream "Aβ theory" believes that Aβ is the initiating factor of AD. Tau protein is a microtubule binding protein that participates in the axonal transport and polarity establishment process of neurons. In the pathological process of AD, Tau protein is widely post-translationally modified, commonly known as phosphorylation modification, which reduces the binding of Tau protein to microtubules, affects the formation and stability of neuronal microtubules, and participates in neurodegenerative lesions.

[0004] AD is a neurodegenerative disease, and about 35 million people worldwide have been diagnosed with AD. It is common in people over 65 years old. In the context of an aging population, age-related pathologies are becoming more and more frequent, and some studies show that these data will triple by 2050. Some common clinical features of AD include cognitive impairment, memory loss, language impairment, emotional and behavioral changes, and orientation disorders in time and space, which hinder the patient's ability to perform normal daily activities.

[0005] The main pathological features of Alzheimer's disease are the presence of neurofibrillary tangles (NFTs), senile plaques (SP) and neuronal loss, which ultimately lead to brain atrophy (Figure 1). NFTs are composed of abnormal aggregates of Tau protein. Under normal conditions, Tau protein contributes to the cytoskeletal structure by interacting with tubulin to stabilize the microtubular network. However, it can undergo different post-translational modifications, such as cleavage or hyperphosphorylation. Although the causes of activation of these modifications remain elusive, there is evidence that this hyperphosphorylated form is prone to aggregation, leading to the formation of NFTs that accumulate inside the cell and are mainly located in the hippocampus. In addition, the dysfunction of Tau protein due to the collapse of the microtubular structure leads to cytoskeletal instability, loss of communication functions, resulting in Alzheimer's disease-mediated neurodegeneration. SP are composed of extracellular deposits of beta amyloid (Aβ) resulting from the degradation of APP, which are the cause of inflammation and neuronal death. APP, which is present in neurons, is a transmembrane protein that can be processed through two different pathways: the amyloidogenic pathway and the non-amyloidogenic pathway, both of which are mediated by secretases. The main reasons for the deposition of Aβ that generates neurotoxicity are the following three points: abnormal metabolism of Aβ, reduced levels of Aβ catabolism and Aβ transport imbalance. The neurotoxicity of Aβ involves complex molecular mechanisms, mainly including the promotion of the formation of free radicals, the disruption of intracellular Ca 2+ homeostasis, the reduction of the function of K + channels, the enhancement of the inflammatory response caused by proinflammatory cytokines, the impairment of small blood vessels causing ischemia and the activation of amyloid enzymes, the inhibition of the proliferation and differentiation of neural stem cells and the induction of apoptosis of neural stem cells. These toxic factors can also interact, causing a vicious cycle, especially the interaction between the neurotoxicity of Aβ and oxidative stress, which ultimately can lead to neuronal apoptosis and the occurrence of dementia.

[0006] Current treatment options for APP-related diseases and disorders are limited and largely ineffective. There is currently no therapy for genetic cerebral amyloid angiopathy (CAA), and attempts to treat sporadic AD and Early-onset familial Alzheimer's disease (EOFAD) have so far proven unsuccessful - for example, all trials using BACE1 (beta-secretase) inhibitors to treat sporadic AD have so far failed (Egan et al., The New England Journal of Medicine, 378: 1691-1703; Hung and Fu. Journal of Biomedical Science, 24:47). Meanwhile, many Abeta-directed immunotherapies are in various stages of development, and many plans for human gamma-secretase inhibitors have been halted due to toxicity (Selkoe and Hardy. EMBO Molecular Medicine, 8:595-608). Approved drug treatments for APP-related diseases or disorders to date are treatments for symptoms, not prevention or cure, and such treatments have limited efficacy, particularly when the APP-related disease or disorder is advanced in the affected individual. Thus, there is a need for treatment of individuals with APP-related diseases and disorders, including a particular need for treatment of individuals with genetic CAA and EOFAD. SUMMARY

[0007] The present disclosure aims to provide an inhibitor of APP expression that is effective, safe, and has a long-lasting effect.

[0008] The present disclosure provides an oligonucleotide or a pharmaceutically acceptable salt thereof, a conjugate thereof, or a composition thereof, and a method of inhibiting expression of an APP gene in a cell or a mammal using the oligonucleotide or a pharmaceutically acceptable salt thereof, a conjugate thereof, or a composition thereof, wherein the oligonucleotide targets an APP gene. Also provided herein are compositions and methods for treating pathological conditions and diseases in a mammal caused by deposition of the APP cleavage product, beta amyloid. The oligonucleotide is a double-stranded RNA (dsRNA) that directs sequence-specific degradation of mRNA through a process known as RNA interference (RNAi).

[0009] In one aspect, the present disclosure provides an oligonucleotide or a pharmaceutically acceptable salt thereof for inhibiting the expression of an APP gene, 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. 1-141 or a fragment thereof, or a modified sequence of the sequence or the fragment thereof, preferably having a sequence with 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. 144-284 or a fragment thereof, or a modified sequence of the sequence or the fragment thereof, preferably having a sequence with 65%, 70%, 75%, 80%, 85%, 90%, 95% or more sequence identity.

[0010] In another aspect, the present disclosure provides a conjugate or a pharmaceutically acceptable salt thereof for inhibiting the expression of an APP gene.

[0011] In another aspect, the present disclosure provides a composition comprising the oligonucleotide or the pharmaceutically acceptable salt thereof, or the conjugate or the pharmaceutically acceptable salt thereof, and optionally a pharmaceutically acceptable carrier.

[0012] In another aspect, the present disclosure provides use of the oligonucleotide or the pharmaceutically acceptable salt thereof, the conjugate or the pharmaceutically acceptable salt thereof, or the composition as described above in the manufacture of a medicament for treating or / and preventing an APP-related disorder.

[0013] In another aspect, the present disclosure provides a method of treating and / or preventing an APP-related disorder in a subject by administering to the subject a therapeutic agent (e.g., the oligonucleotide or the pharmaceutically acceptable salt thereof, or the conjugate or the pharmaceutically acceptable salt thereof, or the composition as described above, or a vector or a transgene encoding the oligonucleotide).

[0014] In another aspect, the present disclosure provides a method of treating and / or preventing an APP-related disorder in a subject by administering to the subject a therapeutic agent (e.g., the oligonucleotide or the pharmaceutically acceptable salt thereof, or the conjugate or the pharmaceutically acceptable salt thereof, or the composition as described above, or a vector or a transgene encoding the oligonucleotide).

[0015] Experiments demonstrate that the oligonucleotide of the present disclosure can effectively reduce the content of APP in vivo, and is an effective inhibitor of beta amyloid. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 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 upstream of autophagy signaling; COX-2: cyclooxygenase-2; NF-κΒ: nuclear factor kappa B; iNOS: inducible nitric oxide synthase.

[0017] Figure 2 shows an oligonucleotide solid-phase synthesis flowchart. DETAILED DESCRIPTION

[0018] In the present disclosure, the scientific and technical terms used herein have the meanings commonly understood by a person of ordinary skill in the art, unless otherwise indicated. Also, the terms related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, immunology, and laboratory operation procedures used herein are terms and procedures widely used in the corresponding fields. Meanwhile, for better understanding of the present disclosure, the definitions and explanations of the related terms are provided below.

[0019] 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 some embodiments, unless otherwise stated or otherwise clear from context, the term "approximately" or "about" refers to 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 stated reference value in either direction (greater than or less than), unless such number would exceed 100% of a possible value.

[0020] As used herein, the term "complementary" refers to a 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.

[0021] 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.

[0022] 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.

[0023] 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, a short siRNA, or a single-stranded siRNA. In some embodiments, a double-stranded oligonucleotide is an RNAi oligonucleotide.

[0024] 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 together base paired from a single nucleic acid strand that is folded (e.g., via a hairpin). 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.

[0025] 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., an APP 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.

[0026] As used herein, the terms "RNAi," "iRNA," "RNAi agent," "RNA interference agent" are used interchangeably herein to refer to an agent comprising the term 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 APP in a cell, e.g., a cell in an individual, e.g., a mammalian individual.

[0027] 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 the agents of the disclosure include all types of modifications disclosed herein or known in the art.

[0028] As used herein, "conjugate" refers to the linkage of two or more chemical moieties each having a specific function to one another 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 conjugate of the disclosure is sometimes also referred to simply as "conjugate". The siRNA conjugate should be understood in the context of the siRNA conjugate in general, the first siRNA conjugate or the second siRNA conjugate, or the siRNA sense strand conjugate or the siRNA antisense strand conjugate.

[0029] 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.

[0030] As used herein, the term "naked sequence" refers to a nucleotide sequence that is unmodified.

[0031] 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.

[0032] The phrase "inhibiting expression of APP" is intended to refer to inhibiting expression of any APP gene, such as, for example, a mouse APP gene, a rat APP gene, a monkey APP gene, or a human APP gene, as well as variants or mutants of APP genes. Thus, in the context of genetically manipulating a cell, population of cells, or organism, the APP gene can be a wild-type APP gene, a mutant APP gene, or a transgenic APP gene.

[0033] "Inhibiting expression of an APP gene" includes inhibition of an APP gene at any level, e.g., at least partial inhibition of expression of an APP gene. Expression of an APP gene can be assessed based on the level or change in level of any variable associated with APP gene expression, e.g., APP mRNA level or APP protein level, or indirectly by inhibiting the mRNA level of a Gluc and APP fusion protein gene, which in turn reflects inhibition of the level of APP protein level by inhibiting the level of Gluc protein.

[0034] The term "lipophilic" or "lipophilic moiety" broadly refers to any compound or chemical moiety that has an affinity for lipids.

[0035] The term "pharmaceutically acceptable salt" refers to those salts of the free base or free acid that retain the biological effectiveness and properties of the free base or free acid, 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, and especially 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 alkaline metal or organic amine salts. Salts derived from inorganic bases include, but are not limited to, alkali metal salts (e.g., sodium salt, potassium salt, and lithium salt), ammonium salt, alkaline earth metal salts (e.g., calcium salt and magnesium salt). Salts derived from organic bases include, but are not limited to, salts of 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.

[0036] As used herein, the term "subject" is an animal, such as a mammal, including a primate (such as a human, non-human primate, e.g., monkey and 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.

[0037] As used herein, the term "treating" or "treatment" refers to a beneficial or desired result, such as a reduction in at least one sign or symptom of an APP-related disorder in a subject. Treatment also includes reducing one or more signs or symptoms associated with undesirable APP expression; lessening the extent of undesirable APP activation or stabilization; ameliorating or palliating undesirable APP activation or stabilization. Treatment also includes reducing one or more signs or symptoms associated with undesirable APP expression. "Treatment" can also mean prolonging survival as compared to the expected survival without treatment.

[0038] As used herein, the term "prevention" or "preventing" when used in reference to a disease or disorder, will benefit from a reduction in APP gene expression or APP protein production.

[0039] As used herein, the term "therapeutically effective amount" is intended to encompass an amount of an RNAi agent that is sufficient, when administered to a subject having an APP-related disorder, to affect treatment of the disease (e.g., by reducing, ameliorating, or maintaining an existing disease or one or more symptoms of the disease). The "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.

[0040] As used herein, the term "prophylactically effective amount" is intended to encompass an amount of an RNAi agent that is sufficient, when administered to a subject having an APP-related disorder, 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 that develops later. The "prophylactically effective amount" can vary depending on the RNAi agent, how the agent is administered, the extent of risk of the 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.

[0041] 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.

[0042] This level can be assessed in an individual cell or population of cells, including, for example, a sample from a subject. It can be appreciated that APP is a systemically expressed protein, not having tissue-specific expression, and is present in circulation.

[0043] Inhibition can be assessed by a decrease in the absolute or relative level of one or more variables associated with APP expression compared to a control level. The control level can be any type of control level used in the art, for example, 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 only.

[0044] In an aspect, the present disclosure provides an oligonucleotide or a pharmaceutically acceptable salt thereof for inhibiting the expression of APP, the oligonucleotide comprising a sense strand and an antisense strand, the 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. 1-141 or a fragment thereof, or a modified sequence of the sequence or the fragment thereof, preferably having a sequence of 65%, 70%, 75%, 80%, 85%, 90%, 95% or more sequence identity; the 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. 144-284 or a fragment thereof, or a modified sequence of the sequence or the fragment thereof, preferably having a sequence of 65%, 70%, 75%, 80%, 85%, 90%, 95% or more sequence identity.

[0045] In some embodiments, the sense strand and / or the antisense strand is 15-30, 17-25, or 19 to 25 nucleotides in length.

[0046] In some embodiments, each strand is independently 15-30, 17-25, or 19 to 25 nucleotides in length.

[0047] In some embodiments, the antisense strand is 19 to 23 nucleotides in length.

[0048] In some embodiments, the sense strand is 19 to 23 nucleotides in length.

[0049] 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.

[0050] In some embodiments, the antisense strand bears 1 or 2 overhangs.

[0051] In some embodiments, the sense strand bears 1 or 2 overhangs.

[0052] In some embodiments, the oligonucleotide comprises a 3'-overhang sequence of 1 or 2 nucleotides in length.

[0053] In some embodiments, the oligonucleotide comprises a 5'-overhang sequence of 1 or 2 nucleotides in length.

[0054] In some embodiments, the 3 '-overhang sequence is present on the antisense strand. In some embodiments, the overhang sequence is selected from the group consisting of: AA, AC, AG, AU, CA, CC, CG, CU, GA, GC, GG, GU, UA, UC, UG, and UU.

[0055] 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.

[0056] In some embodiments, the oligonucleotide comprises an antisense strand and a sense strand each ranging in length from 19 to 25 nucleotides.

[0057] In some embodiments, the oligonucleotide comprises an antisense strand and a sense strand each ranging in length from 19 to 23 nucleotides.

[0058] In some embodiments, the sense strand forms a duplex region with the antisense strand.

[0059] 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.

[0060] 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.

[0061] 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.

[0062] 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.

[0063] 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.

[0064] 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.

[0065] 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.

[0066] 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.

[0067] In some embodiments, the oligonucleotide or salt thereof can be in the form of a hydrate or solvate.

[0068] In some embodiments, the oligonucleotide comprises at least one modified nucleotide.

[0069] 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 mimic, a vinyl-phosphonate nucleotide, a thermally unstable nucleotide, a glycol modified nucleotide, a nucleotide comprising a 2' phosphate, and a 2-0-(N-methylacetamide) modified nucleotide; and combinations thereof.

[0070] 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.

[0071] 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.

[0072] In some embodiments, the oligonucleotide comprises at least one 2'-modified nucleotide.

[0073] In some embodiments, the 2'-modified nucleotide is selected from the group consisting of a 2'-alkoxy modified nucleotide, a 2'-substituted alkoxy modified nucleotide, a 2'-alkyl modified nucleotide, a 2'-substituted alkyl modified nucleotide, a 2'-fluoro modified nucleotide, a 2'-acylamino modified nucleotide, a 2'-deoxy 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'-amino modified nucleotide, a 2'-substituted amino modified nucleotide, a 2'-deoxynucleotide, a nucleotide comprising a 2' phosphate, and a 2'-O-(N-methylacetamide) modified nucleotide; 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.

[0074] In some embodiments, the 2'-modified nucleotide is selected from the group consisting of a 2'-alkoxy modified nucleotide, a 2'-substituted alkoxy modified nucleotide, a 2'-alkyl modified nucleotide, a 2'-substituted alkyl modified nucleotide, a 2'-amino modified nucleotide, a 2'-substituted amino modified nucleotide, a 2'-deoxynucleotide; and combinations thereof.

[0075] In some embodiments, the 2'-modification is selected from the group consisting of a 2'-methoxy, 2'-acetamido, 2'-aminoethyl, 2'-fluoro, 2'-O-methyl, and 2'-O-methoxyethyl modification; and combinations thereof.

[0076] In some embodiments, the 2'-modification is a 2'-methoxy modification.

[0077] In some embodiments, the 2'-modification is a 2'-acetamido modification.

[0078] In some embodiments, all of the nucleotides of the oligonucleotide are modified.

[0079] In some embodiments, the oligonucleotide comprises a modification at the 5' terminus comprising a 5'-phosphate analog or a 6-(3-(2-carboxyethyl)phenyl)purine (6-mCEPh-purine).

[0080] In some embodiments, the oligonucleotide has a 5'-phosphate analog modified nucleotide at the 5' terminal nucleotide.

[0081] In some embodiments, the 5'-phosphate analog modification is a 5'-(E)-vinyl phosphonate (5'-VP).

[0082] In some embodiments, the 5'-phosphate analog modification is a 5'-(E)-vinyl phosphonate (5'-VP).

[0083] In some embodiments, the 5'-phosphate analog modification is a 5'-(E)-vinyl phosphonate (5'-VP).

[0084] 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).

[0085] 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);

[0086] 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);

[0087] In some embodiments, the oligonucleotide comprises at least one modified internucleotide linkage.

[0088] In some embodiments, the at least one modified internucleotide linkage is a phosphorothioate linkage. The phosphorothioate internucleotide linkage modification can occur at any nucleotide on the sense strand, the antisense strand, or both strands at any position of the chain. For example, the internucleotide linkage modification can occur at every nucleotide on the sense strand or the antisense strand; every internucleotide linkage modification can occur in an alternating pattern on the sense strand or the antisense strand; or 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 have an 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 the 5’ end or the 3’ end.

[0089] In some embodiments, the foregoing sense strand comprises an unmodified oligonucleotide selected from any one of SEQ ID NO. 15, 25, 26, 27, 29, 31, 49, 50, 51, 53, 54, 55, 60, 62, 64, 98, 101, 102, 103, 105, 106, 107, 112, 113, 119, 123, 124, 125, 127, 128, 129, 130, 131, 132, or 133, or a modified oligonucleotide of any one of SEQ ID NO: 287-427; and the foregoing antisense strand comprises an unmodified oligonucleotide selected from any one of SEQ ID NO. 158, 168, 169, 170, 172, 174, 192, 193, 194, 196, 197, 198, 203, 205, 207, 241, 244, 245, 246, 248, 249, 250, 255, 256, 262, 266, 267, 268, 270, 271, 272, 273, 274, 275, or 276, or a modified oligonucleotide of any one of SEQ ID NO: 432-572.

[0090] In some embodiments, the foregoing sense strand comprises a modified oligonucleotide selected from any one of SEQ ID NO. 301, 311, 312, 313, 315, 317, 335, 336, 337, 339, 340, 341, 346, 348, 350, 384, 387, 388, 389, 391, 392, 393, 398, 399, 405, 409, 410, 411, 413, 414, 415, 416, 417, 418, or 419; and the foregoing antisense strand comprises a modified oligonucleotide selected from any one of SEQ ID NO. 446, 456, 457, 458, 460, 462, 480, 481, 482, 484, 485, 486, 491, 493, 495, 529, 532, 533, 534, 536, 537, 538, 543, 544, 550, 554, 555, 556, 558, 559, 560, 561, 562, 563, or 564.

[0091] In some embodiments, the foregoing oligonucleotide is selected from any one of the following combinations of sense and antisense strands:

[0092] (1) the sense strand comprises a sequence set forth in SEQ ID NO. 15, and the antisense strand comprises a sequence set forth in SEQ ID NO. 158;

[0093] (2) the sense strand comprises a sequence set forth in SEQ ID NO. 25, and the antisense strand comprises a sequence set forth in SEQ ID NO. 168;

[0094] (3) the sense strand comprises a sequence set forth in SEQ ID NO. 26, and the antisense strand comprises a sequence set forth in SEQ ID NO. 169;

[0095] (4) the sense strand comprises a sequence set forth in SEQ ID NO. 27, and the antisense strand comprises a sequence set forth in SEQ ID NO. 170;

[0096] (5) the sense strand comprises a sequence set forth in SEQ ID NO. 29, and the antisense strand comprises a sequence set forth in SEQ ID NO. 172;

[0097] (6) the sense strand comprises a sequence set forth in SEQ ID NO. 31, and the antisense strand comprises a sequence set forth in SEQ ID NO. 174;

[0098] (7) the sense strand comprises a sequence set forth in SEQ ID NO. 49, and the antisense strand comprises a sequence set forth in SEQ ID NO. 192;

[0099] (8) the sense strand comprises the sequence set forth in SEQ ID NO. 50, and the antisense strand comprises the sequence set forth in SEQ ID NO. 193;

[0100] (9) 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. 194;

[0101] (10) the sense strand comprises the sequence set forth in SEQ ID NO. 53, and the antisense strand comprises the sequence set forth in SEQ ID NO. 196;

[0102] (11) the sense strand comprises the sequence set forth in SEQ ID NO. 54, and the antisense strand comprises the sequence set forth in SEQ ID NO. 197;

[0103] (12) the sense strand comprises the sequence set forth in SEQ ID NO. 55, and the antisense strand comprises the sequence set forth in SEQ ID NO. 198;

[0104] (13) 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. 203;

[0105] (14) the sense strand comprises the sequence set forth in SEQ ID NO. 62, and the antisense strand comprises the sequence set forth in SEQ ID NO. 205;

[0106] (15) the sense strand comprises the sequence set forth in SEQ ID NO. 64, and the antisense strand comprises the sequence set forth in SEQ ID NO. 207;

[0107] (16) the sense strand comprises the sequence set forth in SEQ ID NO. 98, and the antisense strand comprises the sequence set forth in SEQ ID NO. 241;

[0108] (17) the sense strand comprises the sequence set forth in SEQ ID NO. 101, and the antisense strand comprises the sequence set forth in SEQ ID NO. 244;

[0109] (18) the sense strand comprises the sequence set forth in SEQ ID NO. 102, and the antisense strand comprises the sequence set forth in SEQ ID NO. 245;

[0110] (19) the sense strand comprises the sequence set forth in SEQ ID NO. 103, and the antisense strand comprises the sequence set forth in SEQ ID NO. 246;

[0111] (20) the sense strand comprises the sequence set forth in SEQ ID NO. 105, and the antisense strand comprises the sequence set forth in SEQ ID NO. 248;

[0112] (21) the sense strand comprises the sequence set forth in SEQ ID NO. 106, and the antisense strand comprises the sequence set forth in SEQ ID NO. 249;

[0113] (22) the sense strand comprises the sequence set forth in SEQ ID NO. 107, and the antisense strand comprises the sequence set forth in SEQ ID NO. 250;

[0114] (23) the sense strand comprises the sequence set forth in SEQ ID NO. 112, and the antisense strand comprises the sequence set forth in SEQ ID NO. 255;

[0115] (24) the sense strand comprises the sequence set forth in SEQ ID NO. 113, and the antisense strand comprises the sequence set forth in SEQ ID NO. 256;

[0116] (25) the sense strand comprises the sequence set forth in SEQ ID NO. 119, and the antisense strand comprises the sequence set forth in SEQ ID NO. 262;

[0117] (26) the sense strand comprises the sequence set forth in SEQ ID NO. 123, and the antisense strand comprises the sequence set forth in SEQ ID NO. 266;

[0118] (27) the sense strand comprises the sequence set forth in SEQ ID NO. 124, and the antisense strand comprises the sequence set forth in SEQ ID NO. 267;

[0119] (28) the sense strand comprises the sequence set forth in SEQ ID NO. 125, and the antisense strand comprises the sequence set forth in SEQ ID NO. 268;

[0120] (29) the sense strand comprises the sequence set forth in SEQ ID NO. 127, and the antisense strand comprises the sequence set forth in SEQ ID NO. 270;

[0121] (30) the sense strand comprises the sequence set forth in SEQ ID NO. 128, and the antisense strand comprises the sequence set forth in SEQ ID NO. 271;

[0122] (31) the sense strand comprises the sequence set forth in SEQ ID NO. 129, and the antisense strand comprises the sequence set forth in SEQ ID NO. 272;

[0123] (32) the sense strand comprises the sequence set forth in SEQ ID NO. 130, and the antisense strand comprises the sequence set forth in SEQ ID NO. 273;

[0124] (33) the sense strand comprises the sequence set forth in SEQ ID NO. 131, and the antisense strand comprises the sequence set forth in SEQ ID NO. 274;

[0125] (34) the sense strand comprises the sequence set forth in SEQ ID NO. 132, and the antisense strand comprises the sequence set forth in SEQ ID NO. 275; and

[0126] (35) the sense strand comprises the sequence set forth in SEQ ID NO. 133, and the antisense strand comprises the sequence set forth in SEQ ID NO. 276.

[0127] In some embodiments, the foregoing oligonucleotide is selected from any of the following combinations of sense strands and antisense strands:

[0128] (1) the sense strand comprises the sequence set forth in SEQ ID NO. 15, and the antisense strand comprises the sequence set forth in SEQ ID NO. 158;

[0129] (2) the sense strand comprises the sequence set forth in SEQ ID NO. 25, and the antisense strand comprises the sequence set forth in SEQ ID NO. 168;

[0130] (3) the sense strand comprises the sequence set forth in SEQ ID NO. 26, and the antisense strand comprises the sequence set forth in SEQ ID NO. 169;

[0131] (4) the sense strand comprises the sequence set forth in SEQ ID NO. 27, and the antisense strand comprises the sequence set forth in SEQ ID NO. 170;

[0132] (5) the sense strand comprises the sequence set forth in SEQ ID NO. 29, and the antisense strand comprises the sequence set forth in SEQ ID NO. 172;

[0133] (6) the sense strand comprises the sequence set forth in SEQ ID NO. 31, and the antisense strand comprises the sequence set forth in SEQ ID NO. 174;

[0134] (7) the sense strand comprises the sequence set forth in SEQ ID NO. 62, and the antisense strand comprises the sequence set forth in SEQ ID NO. 205;

[0135] (8) the sense strand comprises the sequence set forth in SEQ ID NO. 101, and the antisense strand comprises the sequence set forth in SEQ ID NO. 244;

[0136] (9) the sense strand comprises the sequence set forth in SEQ ID NO. 106, and the antisense strand comprises the sequence set forth in SEQ ID NO. 249;

[0137] (10) the sense strand comprises the sequence set forth in SEQ ID NO. 125, and the antisense strand comprises the sequence set forth in SEQ ID NO. 268;

[0138] (11) the sense strand comprises the sequence set forth in SEQ ID NO. 127, and the antisense strand comprises the sequence set forth in SEQ ID NO. 270;

[0139] (12) the sense strand comprises the sequence set forth in SEQ ID NO. 131, and the antisense strand comprises the sequence set forth in SEQ ID NO. 274;

[0140] (13) the sense strand comprises the sequence set forth in SEQ ID NO. 132, and the antisense strand comprises the sequence set forth in SEQ ID NO. 275; and

[0141] (14) the sense strand comprises the sequence set forth in SEQ ID NO. 133, and the antisense strand comprises the sequence set forth in SEQ ID NO. 276.

[0142] In some embodiments, the foregoing oligonucleotide is selected from any of the following sense strand and antisense strand combinations:

[0143] (1) the sense strand comprises the sequence set forth in SEQ ID NO. 301, and the antisense strand comprises the sequence set forth in SEQ ID NO. 446;

[0144] (2) 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. 456;

[0145] (3) 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. 457;

[0146] (4) the sense strand comprises the sequence set forth in SEQ ID NO. 313, and the antisense strand comprises the sequence set forth in SEQ ID NO. 458;

[0147] (5) the sense strand comprises the sequence set forth in SEQ ID NO. 315, and the antisense strand comprises the sequence set forth in SEQ ID NO. 460;

[0148] (6) the sense strand comprises the sequence set forth in SEQ ID NO. 317, and the antisense strand comprises the sequence set forth in SEQ ID NO. 462;

[0149] (7) the sense strand comprises the sequence set forth in SEQ ID NO. 335, and the antisense strand comprises the sequence set forth in SEQ ID NO. 480;

[0150] (8) the sense strand comprises the sequence set forth in SEQ ID NO. 336, and the antisense strand comprises the sequence set forth in SEQ ID NO. 481;

[0151] (9) 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. 482;

[0152] (10) the sense strand comprises the sequence set forth in SEQ ID NO. 339, and the antisense strand comprises the sequence set forth in SEQ ID NO. 484;

[0153] (11) the sense strand comprises the sequence set forth in SEQ ID NO. 340, and the antisense strand comprises the sequence set forth in SEQ ID NO. 485;

[0154] (12) the sense strand comprises the sequence set forth in SEQ ID NO. 341, and the antisense strand comprises the sequence set forth in SEQ ID NO. 486;

[0155] (13) the sense strand comprises the sequence set forth in SEQ ID NO. 346, and the antisense strand comprises the sequence set forth in SEQ ID NO. 491;

[0156] (14) the sense strand comprises the sequence set forth in SEQ ID NO. 348, and the antisense strand comprises the sequence set forth in SEQ ID NO. 493;

[0157] (15) the sense strand comprises the sequence set forth in SEQ ID NO. 350, and the antisense strand comprises the sequence set forth in SEQ ID NO. 495;

[0158] (16) the sense strand comprises the sequence set forth in SEQ ID NO. 384, and the antisense strand comprises the sequence set forth in SEQ ID NO. 529;

[0159] (17) the sense strand comprises the sequence set forth in SEQ ID NO. 387, and the antisense strand comprises the sequence set forth in SEQ ID NO. 532;

[0160] (18) the sense strand comprises the sequence set forth in SEQ ID NO. 388, and the antisense strand comprises the sequence set forth in SEQ ID NO. 533;

[0161] (19) the sense strand comprises the sequence set forth in SEQ ID NO. 389, and the antisense strand comprises the sequence set forth in SEQ ID NO. 534;

[0162] (20) the sense strand comprises the sequence set forth in SEQ ID NO. 391, and the antisense strand comprises the sequence set forth in SEQ ID NO. 536;

[0163] (21) the sense strand comprises the sequence set forth in SEQ ID NO. 392, and the antisense strand comprises the sequence set forth in SEQ ID NO. 537;

[0164] (22) the sense strand comprises the sequence set forth in SEQ ID NO. 393, and the antisense strand comprises the sequence set forth in SEQ ID NO. 538;

[0165] (23) the sense strand comprises the sequence set forth in SEQ ID NO. 398, and the antisense strand comprises the sequence set forth in SEQ ID NO. 543;

[0166] (24) the sense strand comprises the sequence set forth in SEQ ID NO. 399, and the antisense strand comprises the sequence set forth in SEQ ID NO. 544;

[0167] (25) the sense strand comprises the sequence set forth in SEQ ID NO. 405, and the antisense strand comprises the sequence set forth in SEQ ID NO. 550;

[0168] (26) the sense strand comprises the sequence set forth in SEQ ID NO. 409, and the antisense strand comprises the sequence set forth in SEQ ID NO. 554;

[0169] (27) the sense strand comprises the sequence set forth in SEQ ID NO. 410, and the antisense strand comprises the sequence set forth in SEQ ID NO. 555;

[0170] (28) the sense strand comprises the sequence set forth in SEQ ID NO. 411, and the antisense strand comprises the sequence set forth in SEQ ID NO. 556;

[0171] (29) the sense strand comprises the sequence set forth in SEQ ID NO. 413, and the antisense strand comprises the sequence set forth in SEQ ID NO. 558;

[0172] (30) the sense strand comprises the sequence set forth in SEQ ID NO. 414, and the antisense strand comprises the sequence set forth in SEQ ID NO. 559;

[0173] (31) the sense strand comprises the sequence set forth in SEQ ID NO. 415, and the antisense strand comprises the sequence set forth in SEQ ID NO. 560;

[0174] (32) the sense strand comprises the sequence set forth in SEQ ID NO. 416, and the antisense strand comprises the sequence set forth in SEQ ID NO. 561;

[0175] (33) the sense strand comprises the sequence set forth in SEQ ID NO. 417, and the antisense strand comprises the sequence set forth in SEQ ID NO. 562;

[0176] (34) the sense strand comprises the sequence set forth in SEQ ID NO. 418, and the antisense strand comprises the sequence set forth in SEQ ID NO. 563; and

[0177] (35) the sense strand comprises the sequence set forth in SEQ ID NO. 419, and the antisense strand comprises the sequence set forth in SEQ ID NO. 564.

[0178] In some embodiments, the foregoing oligonucleotide is selected from any of the following combinations of sense strands and antisense strands:

[0179] (1) the sense strand comprises the sequence set forth in SEQ ID NO. 301, and the antisense strand comprises the sequence set forth in SEQ ID NO. 446;

[0180] (2) 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. 456;

[0181] (3) 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. 457;

[0182] (4) the sense strand comprises the sequence set forth in SEQ ID NO. 313, and the antisense strand comprises the sequence set forth in SEQ ID NO. 458;

[0183] (5) the sense strand comprises the sequence set forth in SEQ ID NO. 315, and the antisense strand comprises the sequence set forth in SEQ ID NO. 460;

[0184] (6) the sense strand comprises the sequence set forth in SEQ ID NO. 317, and the antisense strand comprises the sequence set forth in SEQ ID NO. 462;

[0185] (7) the sense strand comprises the sequence set forth in SEQ ID NO. 348, and the antisense strand comprises the sequence set forth in SEQ ID NO. 493;

[0186] (8) the sense strand comprises the sequence set forth in SEQ ID NO. 387, and the antisense strand comprises the sequence set forth in SEQ ID NO. 532;

[0187] (9) the sense strand comprises the sequence set forth in SEQ ID NO. 392, and the antisense strand comprises the sequence set forth in SEQ ID NO. 537;

[0188] (10) the sense strand comprises the sequence set forth in SEQ ID NO. 411, and the antisense strand comprises the sequence set forth in SEQ ID NO. 556;

[0189] (11) the sense strand comprises the sequence set forth in SEQ ID NO. 413, and the antisense strand comprises the sequence set forth in SEQ ID NO. 558;

[0190] (12) the sense strand comprises the sequence set forth in SEQ ID NO. 417, and the antisense strand comprises the sequence set forth in SEQ ID NO. 562;

[0191] (13) the sense strand comprises the sequence set forth in SEQ ID NO. 418, and the antisense strand comprises the sequence set forth in SEQ ID NO. 563; and

[0192] (14) the sense strand comprises the sequence set forth in SEQ ID NO. 419, and the antisense strand comprises the sequence set forth in SEQ ID NO. 564.

[0193] The present disclosure also provides a conjugate or a pharmaceutically acceptable salt thereof for inhibiting expression of APP, comprising: (i) the foregoing oligonucleotide or a pharmaceutically acceptable salt thereof, and (ii) a targeting ligand, wherein at least one of the sense strand and the antisense strand of the oligonucleotide is conjugated to the targeting ligand.

[0194] In some embodiments, the targeting ligand is conjugated at the 3' end or the 5' end of the sense strand. In some embodiments, the targeting ligand is conjugated at the 3' end of the sense strand.

[0195] In some embodiments, the foregoing targeting ligand comprises an N-acetylgalactosamine (GalNAc) moiety. In some embodiments, the foregoing GalNac moiety is a monovalent GalNAc moiety, a bivalent GalNAc moiety, a trivalent GalNAc moiety, or a tetravalent GalNAc moiety. In some embodiments, the foregoing targeting ligand is L96.

[0196] The present disclosure also provides a conjugate or a pharmaceutically acceptable salt thereof for inhibiting expression of APP, comprising: (i) the foregoing oligonucleotide or a pharmaceutically acceptable salt thereof, and (ii) a lipophilic moiety, wherein at least one of the sense strand and the antisense strand of the oligonucleotide is conjugated to one or more lipophilic moieties, optionally, the lipophilic moiety is conjugated through a linker or a carrier.

[0197] In some embodiments, the foregoing lipophilic moiety is conjugated at the 2'-position of a nucleotide or modified nucleotide within the sense or antisense strand. An internal position includes all positions except the terminal two positions from each end of at least one strand of the foregoing oligonucleotide. For example, a lipophilic moiety is conjugated to position 6 of the sense strand, i.e., the lipophilic moiety is at the 6th position from the 5' end of the sense strand.

[0198] In some embodiments, the foregoing lipophilic moiety comprises between one and four 2'-O-alkyl modifications. Optionally, the 2'-O-alkyl modified nucleotide is a 2'-C16-modified nucleotide. Optionally, the RNAi agent includes a single 2'-O-C16 modified nucleotide. Optionally, the single 2'-C16 modified nucleotide is at the 6th nucleobase position from the 5' end on the sense strand.

[0199] In some embodiments, another modification of the RNA of the RNAi agent chemically links one or more ligands, moieties, or conjugates that enhance the RNAi activity, cellular distribution, or cellular uptake of the RNA. Such moieties include, but are not limited to, lipid moieties such as a cholesterol moiety (Letsinger et al. (1989) Proc. Natl. Acad. Sci. USA, 86:6553-6556), a cholic acid (Manoharan et al. (1994) Biorg. Med. Chem. Let., 4:1053-1060), a thioether, for example, a green fluorescent protein- S-tritylthiol (Manoharan et al. (1992) Ann. N.Y. Acad. Sci., 660:306-309; Manoharan et al. (1993) Biorg. Med. Chem. Let., 3:2765-2770), a sulfur containing cholesterol (Oberhauser et al. (1992) Nucl. Acids Res., 20:533-538), a fatty chain, for example, a dodecandiol or undecyl residue (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), a phospholipid, for example, di-hexadecyl-rac- glycerol or triethyl- ammonium 1,2-di-O-hexadecyl-rac-glycero-3-Hphosphocholine (Manoharan et al. (1995) Tetrahedron Lett., 36:3651-3654; Shea et al. (1990) Nucl. Acids Res., 18:3777-3783), a polyamine or a polyethylene glycol chain (Manoharan et al. (1995) Nucleosides & Nucleotides, 14:969-973), or adamantane acetic acid (Manoharan et al. (1995) Tetrahedron Lett., 36:3651-3654), a palmityl moiety (Mishra et al. (1995) Biochim. Biophys. Acta, 1264:229-237), or an octadecylamine or hexylamino-carbonyloxy cholesterol moiety (Crooke et al. (1996) J. Pharmacol. Exp. Ther., 277:923-937).

[0200] In some embodiments, the lipophilic moiety is aliphatic, cyclic, e.g., alicyclic, or polycyclic, e.g., polya!icyclic, such as a steroid (e.g., a sterol) or a straight or branched aliphatic hydrocarbon. The lipophilic moiety can generally comprise a hydrocarbon chain, which can be cyclic or acyclic. The hydrocarbon chain can comprise various substituents and / or one or more heteroatoms, such as oxygen or nitrogen atoms. Such lipophilic aliphatic moieties 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., monoalcohol esters of fatty acids and fatty diamides), terpenes (e.g., C 10 terpenes, C 15 sesquiterpenes, C 20 diterpenes, C 30 triterpenes, and C 40 tetra terpenes) and other polya!icyclic hydrocarbons. For example, the lipophilic moiety can comprise a C4to C 30 hydrocarbon chain (e.g., a C4to C 30 alkyl or alkenyl group). In some embodiments, the lipophilic moiety comprises a saturated or unsaturated C6to C 18 hydrocarbon chain (e.g., a straight chain C6to C 22 alkyl or alkenyl group). In one embodiment, the lipophilic moiety comprises a saturated or unsaturated C 16 hydrocarbon chain (e.g., a straight chain C 16 alkyl or alkenyl group). The lipid moiety is a 2'-O-alkyl, including single and or branched 10-30 hydrocarbon chain compositions.

[0201] In another embodiment, the lipophilic moiety comprises a lipid, cholesterol, retinoic acid, cholic acid, adamantane acetic acid, 1-pyrene butyric acid, dihydrotestosterone, 1,3-bis-0(hexadecyl)glycerol, geranyloxyhexanol, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl, palmitic acid, myristic acid, 03-(oleoyl)lithocholic acid, 03-(oleoyl)cholenic acid, ibuprofen, naproxen, dimethoxybenzophenone, or phenoxazine.

[0202] The lipophilic moiety is conjugated to the double stranded RNAi agent via a linker comprising an ether, a thioether, a urea, a carbonate, an amine, an amide, a maleimide-thioether, a disulfide, a phosphodiester, a sulfonamide linkage, a click reaction (e.g., a triazole from an azide-alkyne cycloaddition), or a carbamate.

[0203] In some embodiments, the foregoing ligand that enhances RNAi activity, cellular distribution, or cellular uptake comprises a C 12 alkyl group, a C 16 alkyl group, a C 18 alkyl group, a C 22alkyl, or branched lipids such as DDA (cationic dimethyl dioctadecyl ammonium), TDB (trehalose 6,6,9-ditridecanoate), and the like.

[0204] For example, a linear lipophilic moiety (C16) conjugated to one position on the chain has the following structure:

[0205] Base is a nucleotide base or a nucleotide base analog. In some embodiments, Base is selected from the group consisting of adenine, guanine, cytosine, thymine, and uracil.

[0206] For example, 2'-0-hexadecyluridine has the following structure:

[0207] The present disclosure also provides a conjugate or a pharmaceutically acceptable salt thereof for inhibiting expression of APP, comprising: (i) a modified oligonucleotide having the following structure:

[0208] 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 the 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;

[0209] X1is:

[0210] L1is -(CH2)n-, -(CH2) n L2(CH2) n - or a bond;

[0211] L2is -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),

[0212] wherein each m is independently an integer from 10 to 18, and wherein each n is independently an integer from 1 to 6.

[0213] For example, DTX-1

[0214] The present disclosure also provides a conjugate or a pharmaceutically acceptable salt thereof for inhibiting expression of APP, comprising: (i) a modified oligonucleotide or a pharmaceutically acceptable salt thereof as described above, and (ii) a targeting ligand and 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 at least one of the sense strand and the antisense strand of the oligonucleotide is conjugated to one or more lipophilic moieties.

[0215] In some embodiments of the disclosure, expression of the APP gene is inhibited by at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, or to below the level of detection of the assay. In preferred embodiments, expression of APP is inhibited by at least 70%. It will also be appreciated that it can be desirable to inhibit expression of APP in certain tissues (e.g., the brain) without significantly inhibiting expression in other tissues. In preferred embodiments, expression levels are determined in a suitably species-matched cell line using the assay method provided in Example 2 using 50 nM, 10 nM, 1 nM, and 0.1 nM siRNA concentrations.

[0216] In certain embodiments, inhibition of expression in vivo is determined by knocking down the human gene in a rodent expressing the human gene, for example, a mouse infected with an AAV expressing the human target gene (i.e., APP), for example, the effect of inhibition of the human gene is confirmed by the nadir of APP expression after, for example, a single dose administration, for example, a 3 mg / kg subcutaneous injection. Such systems are useful when the nucleic acid sequences of the human gene and the model animal gene are sufficiently close that human RNAi provides effective knockdown of the model animal gene. RNA expression in the liver is determined using the PCR method provided in Example 2.

[0217] Inhibition of APP gene expression can be represented by a decrease in the amount of mRNA expressed by a cell line (such cells can be present in, for example, a sample derived from a subject), the APP gene is transcribed in the cell or cell population and it is treated (e.g., by contacting one or more cells with an iRNA of the disclosure, or by administering an RNAi of the disclosure to a subject in which the cells are present or were once present), such that expression of the APP gene is inhibited as compared to a cell line that is substantially identical to the cell line but is not treated (control cells that are not treated with RNAi or not treated with RNAi targeting the gene of interest). In preferred embodiments, inhibition is assessed using 10 nM siRNA concentration in a species-matched cell line using the method provided in Example 2, using the following formula, representing the amount of mRNA expression in the treated cells relative to the mRNA level in the control cells: CT APP - CT GAPDH CT 处理细胞 - CT 对照细胞 mRNA level = 2^-△△CT

[0218] In other embodiments, inhibition of APP gene expression can be assessed according to a decrease in a parameter that is functionally related to APP gene expression, e.g., APP protein levels in blood or serum from a subject. APP gene silencing can be determined in any cell expressing APP, whether endogenous or heterologous from an expression construct, and by any assay known in the art.

[0219] Inhibition of APP protein expression can be manifested by a decrease in the level of APP protein expressed by a cell or cell population or in a subject sample, e.g., protein levels in a blood sample from a 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 a change in protein levels in a subject sample, e.g., blood or serum therefrom, using the following formula, expressing the amount of APP expression or Gluc expression in the treated sample, e.g., blood or serum therefrom, as a percentage of the amount of APP expression or Gluc expression in control cells.

[0220] Percentage inhibition of mRNA = (amount of protein expression 处理细胞 - amount of protein expression 对照细胞 ) / amount of protein expression 对照细胞 * 100%

[0221] Control cells, cell populations, or subject samples that can be used to assess inhibition of APP gene expression include cells, cell populations, or subject samples that have not been contacted with an RNAi agent of the disclosure. For example, control cells, cell lines, or subject samples can be from an individual subject (e.g., a human or animal subject) prior to treatment of the subject or appropriately matched population control with an RNAi agent.

[0222] In some embodiments of the methods of the disclosure, the RNAi is administered to a subject such that the RNAi is delivered to a particular site within the subject. Inhibition of APP expression can be assessed by measuring the level or change in APP mRNA or APP protein or fusion secreted luciferase in a sample of fluid or tissue from a particular site in the subject, e.g., in the brain or in the blood.

[0223] The disclosure also provides methods of using the RNAi of the disclosure or compositions comprising the RNAi of the disclosure to inhibit APP expression, thereby preventing or treating an APP-related disorder, e.g., cerebral amyloid angiopathy (CAA) or Alzheimer’s disease (AD), including early onset familial Alzheimer’s disease (EOFAD), dementia, etc.

[0224] Cells suitable for treatment using the methods of the disclosure can be any cell that expresses an APP gene, for example, a liver cell, a neural cell, a gall bladder cell, a heart cell, or a kidney cell, but preferably a neural cell. Cells suitable for use in the methods of the disclosure can be mammalian cells, for example, primate cells (such as human cells, including human cells in chimeric non-human animals, or non-human primate cells, for example, monkey cells or chimpanzee cells) or non-primate cells. In certain embodiments, the cells are human cells, for example, human neural cells. In the methods of the disclosure, expression of APP in the cells is inhibited by at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, or below the level of detection of the assay.

[0225] The in vivo methods of the disclosure can comprise administering to a subject a composition comprising RNAi, wherein the RNAi comprises a nucleotide sequence that is complementary to at least a portion of an RNA transcript of an APP gene of the mammal to which the RNAi agent is administered. The composition can be administered by any means 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 topical (including buccal and sublingual) administration. In certain embodiments, the composition is administered by intravenous infusion or injection. In certain embodiments, the composition is administered subcutaneously. In certain embodiments, the composition is administered intrathecally.

[0226] In one aspect, the disclosure also provides methods for inhibiting expression of an APP gene in a mammal. The method comprises administering to the mammal an oligonucleotide or a pharmaceutically acceptable salt thereof or a composition thereof. The oligonucleotide is a double-stranded RNA (dsRNA) that targets an APP gene in a mammalian cell and is maintained in the mammal for a sufficient time to obtain degradation of an mRNA transcript of the APP gene, thereby inhibiting expression of an APP protein in the cell. Reduction in gene expression can be assessed by any method known in the art and by methods, for example, qRT-PCR as described herein, for example, in Example 2. Reduction in protein product can be assessed by any method known in the art (e.g., ELISA). In other embodiments, blood samples are taken as a sample from the subject for monitoring reduction in APP protein expression.

[0227] The disclosure also provides methods of treatment in a subject in need thereof, for example, a subject diagnosed with an APP-related disorder, cerebral amyloid angiopathy (CAA) or Alzheimer’s disease (AD), including early-onset familial Alzheimer’s disease (EOFAD), dementia, and the like.

[0228] In one embodiment, the APP-related disease is cerebral amyloid angiopathy (CAA) or Alzheimer's disease (AD), including early onset familial Alzheimer's disease (EOFAD), dementia, and the like.

[0229] The RNAi of the disclosure can be administered as "free RNAi." Free RNAi is administered without a pharmaceutical composition. The naked RNAi can be in a suitable buffered solution. The buffered solution can comprise acetate, citrate, lactate, tartrate, carbonate, or phosphate, or any combination thereof. In one embodiment, the buffered solution is phosphate buffered saline (PBS). The pH and osmolarity of the buffered solution containing the RNAi can be adjusted to be suitable for administration to a subject.

[0230] Administration of the RNAi according to the methods of the disclosure can result in the prevention or treatment of an APP-related disorder, e.g., cerebral amyloid angiopathy (CAA) or Alzheimer's disease (AD), including early onset familial Alzheimer's disease (EOFAD), dementia, and the like. A therapeutic amount of the RNAi can be administered to a subject, such as about 0.01 mg / kg to about 200 mg / kg. Preferably, 1 mg / kg to about 50 mg / kg. The RNAi is preferably administered subcutaneously, i.e., by intrathecal injection. One or more injections can be used to deliver the desired dose of the RNAi to the subject. Injections can be repeated over a period of time.

[0231] Administration can be repeated on a regular basis. In certain embodiments, after an initial treatment regimen, treatment can be performed at a lower frequency. Repeated dosing regimens can include the periodic administration of a therapeutic amount of the RNAi, such as once a month to once a year. In certain embodiments, the RNAi is administered about once a month to about once every three months, or about once every three months to about once every six months, or even once a year.

[0232] The disclosure further provides that the RNAi agent or pharmaceutical composition thereof be used in combination therapy with other drugs and / or other treatment methods (e.g., known drugs and / or known treatment methods, such as, for example, those currently used to treat these disorders) for subjects that would benefit from a reduction and / or inhibition of APP gene expression, e.g., subjects having an APP-related disease. For example, other therapeutic agents and treatment methods suitable for treating subjects that would benefit from a reduction in APP expression, e.g., subjects having an APP-related disease, include tacrine, donepezil, rivastigmine, galantamine, memantine and memantine donepezil combination formulation, cholinesterase inhibitor donepezil, huperzine A, galantamine hydrobromide, rivastigmine tartrate, and the like.

[0233] Examples

[0234] Example 1. Preparation of Targeting Ligands and siRNAs

[0235] In cases where the source of reagents is not specifically given herein, such reagents can be obtained from any molecular biology reagent supplier following the quality / purity standards appropriate for molecular biology.

[0236] Abbreviations used for nucleotide monomers in nucleic acid sequence representation. It is understood that when a nucleotide contains a 2'-fluoro modification, then the fluorine replaces the hydroxyl group at that position in the parent nucleotide (i.e., it is a 2'-deoxy-2'-fluoro nucleotide).

[0237] Table A. Abbreviations for nucleotide monomers used in nucleic acid sequence representation

[0238] Preparation of targeting ligands and lipophilic moieties

[0239] Preparation of L96 was performed according to the method described in patent CN104717982B.

[0240] C16 was purchased from Chengdu Lead Pharmaceutical Development Co., Ltd.

[0241] Preparation of DTX was performed according to the method described in patent application CN113166191A.

[0242] Preparation of oligonucleotides

[0243] (1) Preparation of siRNA

[0244] First, candidate oligonucleotide sequences complementary to human APP mRNA (NM_201414.3, Table 1) were generated using computer-based algorithms, some of which were also complementary to cynomolgus monkey APP mRNA (XM_005548883.3, Table 1) or no more than 2 mismatches. Some of them were designed as double-stranded siRNA with 19 / 21 pairing of sense and antisense strands respectively, and the antisense strand had two overhanging ends complementary to the mRNA sequence, in some cases the overhanging ends of the antisense strand were non-complementary UU; some of the sequences were designed as double-stranded siRNA with 21 / 23 pairing of sense and antisense strands respectively, and the antisense strand had two overhanging ends complementary to the mRNA sequence; some of the sequences were designed as double-stranded siRNA with 21 / 21, 23 / 23 pairing. The 1st base at the 5' end of the antisense strand of some of the complementary pairing sequences (the last 1st base at the 3' end of the sense strand) was replaced with a base that did not match the APP mRNA.

[0245] Table 1 Human, cynomolgus monkey APP mRNA sequences

[0246] The siRNA sequences were synthesized separately on a solid support via the sense strand (SS) and the antisense strand (AS), and obtained after deprotection, cleavage, purification, annealing, purification, and lyophilization.

[0247] Solid-phase synthesis (Figure 2): The sense strand and the antisense strand were synthesized separately on a solid support using an oligonucleotide synthesizer using the phosphoramidite technique. The synthesizer was AKTA Oligopilot (Cytiva), Dr. Oligo 192XLc (Kunshan Boli Ke Precision Instrument Co., Ltd.). The solid-phase synthesis started from the 3' end of the sequence, and the monomers were coupled into the sequence in sequence order. Each coupling of a phosphoramidite monomer included four chemical steps: 1) deblocking or deprotection (removing the hydroxyl protecting group); 2) coupling; 3) oxidation; and 4) capping. The phosphoramidite monomers, reagents, and purification consumables used were commercialized and circulated reagents and consumables, such as various phosphoramidite monomers (such as 5'-O-(4,4'-Dimethoxytrityl)-2'-O-methyl-Uridine-3'-CE-Phosphoramidite) purchased from Shanghai Zhiwei Technology Development Co., Ltd., and reaction reagents (such as 40wt% methylamine aqueous solution, 28wt% ammonium hydroxide aqueous solution, etc.) purchased from Sigma-Aldrich LLC. The siRNA synthesis and purification methods used in this paper are described in US20130178612A1, US2015100197A1, etc.; the synthesis method of the sequence containing VPUm and APU is described in J. Med. Chem. 2018, 61, 734-744.

[0248] (2) Preparation of double-stranded RNA reagent

[0249] (a) Synthesis of the sense strand

[0250] Solid phase phosphoramidite method is a mature oligonucleotide synthesis method. Using computer-controlled synthesizer, the reaction is carried out in a stainless steel synthesis column. In the synthesis of the sense strand, the solid phase support loaded with targeting ligand (such as L96) is used as the starting material, or the solid phase support is directly used as the starting material. Different raw materials, reagents and solvents are injected into different pipelines in the order of 3' to 5' sequence under the control of the solid phase synthesizer, and the phosphoramidite nucleotide monomers are connected one by one. The reaction process includes four steps of DMT protecting group removal reaction, condensation reaction, oxidation or thio reaction, and capping reaction, and one nucleotide unit is connected each time to obtain an oligonucleotide sequence of 19 or 21 nucleotide units. After the synthesis is completed, the protecting group (2-cyanoethyl) is removed on the solid phase synthesis column, and the synthesized sequence is then cut from the solid phase support through an ammonolysis reaction. The filtrate and the washing liquid are collected after the filter cake is washed with ethanol. The crude product of the sense strand is obtained by concentration. The crude product is purified by chromatography (SOURCE 15Q) and freeze-dried to obtain the target product of the sense strand. In the synthesizer, the siRNA sense strand conjugate is synthesized by using the solid phase support loaded with the targeting ligand (such as L96) as the starting material; and the siRNA is synthesized by directly using the solid phase support as the starting material.

[0251] (b) Synthesis of antisense strand

[0252] The synthesis of the antisense strand is similar to that of the sense strand. Different raw materials, reagents and solvents are injected into different pipelines in the order of 3' to 5' sequence under the control of the solid phase synthesizer, and the phosphoramidite nucleotide monomers are connected one by one. The reaction process includes four steps of DMT protecting group removal reaction, condensation reaction, oxidation or thio reaction, and capping reaction, and one nucleotide unit is connected each time to obtain an oligonucleotide sequence of 21 or 23 nucleotide units. After the synthesis is completed, the protecting group (2-cyanoethyl) is removed on the solid phase synthesis column, and the synthesized sequence is then cut from the solid phase support through an ammonolysis reaction. The filtrate and the washing liquid are collected after the filter cake is washed with ethanol. The crude product of the sense strand is obtained by concentration. The crude product is purified by chromatography (SOURCE 15Q) and freeze-dried to obtain the target product of the sense strand. In the synthesizer, the siRNA sense strand conjugate is synthesized by using the solid phase support loaded with the targeting ligand (such as L96) as the starting material; and the siRNA is synthesized by directly using the solid phase support as the starting material.

[0253] (c) Preparation of double-stranded siRNA

[0254] The AS strand and the SS strand are dissolved in injection water respectively, mixed in a certain proportion (1.01:1.0-1.2:1.0), incubated at 30-50°C for 30-90 min, and cooled to room temperature. After freeze-drying, the double-stranded siRNA product is obtained.

[0255] According to the same method, the double-stranded siRNA reagents in Tables 2, 3 and 4 below are prepared.

[0256] In Tables 2, 3 and 4, “G”, “C”, “A”, “U” and “T” generally represent nucleotides with guanine, cytosine, adenine, uracil and thymine as the base, respectively.

[0257] Modification: m represents 2'-methoxy; f represents 2'-deoxy-2'-fluoro; s represents phosphorothioate; VPUm is 2'-methoxy modified uridine; M formula is 2'-O-methyl-6-(3-(2-carboxyethyl)phenyl)-purine nucleotide; L96 is N-[tris(GalNAc-alkyl)amidodecanoyl]-4-hydroxyprolinol (Hyp-(GalNAc-alkyl)3).

[0258] Table 2 Oligonucleotide naked sequence

[0259] Table 3 Oligonucleotide modified sequence

[0260] Example 2. In vitro activity screening of APP-siRNA

[0261] (1) Cell culture and transfection:

[0262] Take human neuroblastoma (also known as Be2C cells) (Tianbai (Shanghai) Biotechnology Co., Ltd., item number BE(2)-C cells identification), place in a 37°C, 5% CO2 incubator, use DMEM, high glucose (Thermo, item number 11965-092), add 10% FBS (GIBCO, 12483020), 1% penicillin-streptomycin (GIBCO, 15140-122) for culture, when the cell confluence reaches 90%, digest by trypsin-EDTA (Thermo, 25200-072), count with a counting instrument (Countstar, IC1000), 190 μl of cell suspension per well, inoculate in a 96-well plate, the inoculation number of Be2C cells is: 1*10 4 cells / well, wait until the next day to adhere for transfection.

[0263] Transfection was performed using Lipofectamine™ RNAiMAX (thermofisher, 13778150), 2.2 μl (2 μM) diluted compound, 19.1 μl Opti-MEM (thermofisher, 1105821), 0.7 μl RNAiMAX were mixed to form transfection complex, incubated for 5 minutes, then the transfection complex was added to the cells (two technical replicates per complex), 10 μl per well, the final concentration of siRNA was 10 nM, and incubated at 37℃, 5% CO2 incubator for 24 hours.

[0264] (2) RNA extraction and detection

[0265] (i) One day after transfection, the culture medium was removed; PBS was added for washing; 200 μl lysis buffer was added to each well, and incubated at room temperature for 15 minutes, then blown evenly. RNA was extracted using a 96-channel automatic nucleic acid extractor (Hanwei Technology, HW-96 series). The RNA concentration was determined for subsequent experiments. The extracted RNA can be used immediately for subsequent experiments, or stored at -80℃ for later use.

[0266] (ii) Using The cDNA was synthesized using the IIQ RT SuperMix for qPCR (+gDNA wiper) reverse transcription kit (Novozyme, R223-01):

[0267] The mixture was prepared in an RNase-free centrifuge tube: 4 μl 4×gDNA wiper Mix, 1 μg template RNA, and RNase-free ddH2O to 16 μl to remove genomic DNA, and gently mixed with a pipette. Then 4 μl 5×HiScript II qRT SuperMix II was added directly to the reaction tube and gently mixed with a pipette, and placed in a PCR instrument (Bio-Rad C1000 Touch PCR instrument) at 50℃ for 15 min; 85℃ for 5 sec, 4℃. The product can be used immediately for qPCR reaction, or stored at -20℃ and used within six months. Long-term storage requires aliquoting and storage at -80℃. The cDNA should avoid repeated freezing and thawing.

[0268] (iii) qPCR quantification using ChamQ SYBR qPCR Master Mix (Novozyme, Q311-02):

[0269] The mixture was configured with 10 μΐ 2x ChamQ SYBR qPCR Master Mix, 0.5 μΐ Forword primer (Ribobio), 0.5 μΐ Reverse primer (Ribobio), 1 μΐ Template cDNA, 8 μΐ ddH2O to 20 μΐ system, 3 repeats for each sample, and the 96-well plate was placed into a qPCR instrument (Quantstudio 5) to execute the program: pre-denaturation, 95°C, 30 sec; amplification, 95°C, 10 sec, 60°C, 30 sec, 40 cycles; melting curve, 95°C, 15 sec, 60°C, 60 sec, 95°C, 15 sec.

[0270] (3) Data statistical analysis:

[0271] The data was exported to EXCEL format, and CT APP -CT GAPDH The control group was normalized, and in order to calculate the fold change of the relative silencing efficiency, the data was analyzed using the △△CT method, and the average value and standard deviation of the obtained three parallel repeated data were calculated.

[0272] The screening results of Be2C cells in two times are shown in Tables 5-6. The data in each table is obtained from a single test, and since the batches of cells are different, the silencing efficiency of the target gene will be different. In some tests, due to the state of the cells, low concentrations may occur, and the error between the replicate wells of biological repeats is large, so reference should be made to other concentrations and the data with smaller error at the same concentration.

[0273] As can be seen from Table 4, when the dosage is 10 nM, the inhibition rate of APP mRNA of 42 siRNAs can reach or approach 40% or more. In order to further evaluate the more preferred sequence, 42 sequences with better inhibition rate were selected for double-concentration screening at 50 nM and 10 nM, and the detection results are shown in Table 5. The results show that the double-concentration screening results of AL0235015, AL0235025, AL0235026, AL0235027, AL0235029, AL0235031, AL0235062, AL0235101, AL0235106, AL0235107, AL0235112, AL0235113, AL0235123, AL0235125, AL0235127, AL0235131, AL0235132 and AL0235133 are better, the inhibition rate at 10 nM can reach more than 60% or approach 60%, and the highest can be greater than 80%.

[0274] Table 4 APP siRNA sequence knockdown level in Be2C cells

[0275] Table 5 APP siRNA sequence knockdown levels in Be2C cells

Claims

1. An oligonucleotide or a pharmaceutically acceptable salt thereof for use in inhibiting the expression of APP, 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. 1-141 or a fragment thereof, or a modified sequence of the sequence or the fragment thereof, preferably having 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. 144-284 or a fragment thereof, or a modified sequence of the sequence or the fragment thereof, preferably having 65%, 70%, 75%, 80%, 85%, 90%, 95% or more sequence identity.

2. The oligonucleotide or pharmaceutically acceptable salt thereof of claim 1, wherein, the oligonucleotide or the pharmaceutically acceptable salt thereof 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 thereof; 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 mimic, 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 LNA modified nucleotide, a HNA modified nucleotide, a CeNA modified nucleotide, a 2'-methoxyethyl modified nucleotide, a 2'-O-alkyl modified nucleotide, a 2'-O-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; Preferably, the at least one modified nucleotide 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, a nucleotide comprising a 2' phosphate, and a nucleotide comprising a 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 a 2'-alkoxy modified nucleotide, a 2'-substituted alkoxy modified nucleotide, a 2'-alkyl modified nucleotide, a 2'-substituted alkyl modified nucleotide, a 2'-fluoro modified nucleotide, a 2'-acylamino modified nucleotide, a 2'-deoxy 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'-amino modified nucleotide, a 2'-substituted amino modified nucleotide, a 2'-deoxy nucleotide, a nucleotide comprising a 2' phosphate, and a 2'-0-(N-methylacetamide) modified nucleotide; and combinations thereof; Preferably, the 2'-modification is selected from the group consisting of a 2'-methoxy, a 2'-acetamino, a 2'-aminoethyl, a 2'-fluoro, a 2'-0-methyl, and a 2'-0-methoxyethyl modification; and combinations thereof; Preferably, the oligonucleotide comprises a modification at the 5' terminus, the modification comprising a 5'-phosphate analog or a 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'-0-methoxyethyl; preferably, the nucleotide comprising a 5'-phosphate analogue modification has the structure shown in Formula (II); more preferably, the nucleotide comprising a 5'-phosphate analogue modification 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. 15, 25, 26, 27, 29, 31, 49, 50, 51, 53, 54, 55, 60, 62, 64, 98, 101, 102, 103, 105, 106, 107, 112, 113, 119, 123, 124, 125, 127, 128, 129, 130, 131, 132, or 133, or a modified oligonucleotide of any one of SEQ ID NO: 287-427; the antisense strand comprises an unmodified oligonucleotide selected from any one of SEQ ID NO. 158, 168, 169, 170, 172, 174, 192, 193, 194, 196, 197, 198, 203, 205, 207, 241, 244, 245, 246, 248, 249, 250, 255, 256, 262, 266, 267, 268, 270, 271, 272, 273, 274, 275, or 276, or a modified oligonucleotide of any one of SEQ ID NO: 432-572; Preferably, the sense strand comprises a modified oligonucleotide selected from any of SEQ ID NO. 301, 311, 312, 313, 315, 317, 335, 336, 337, 339, 340, 341, 346, 348, 350, 384, 387, 388, 389, 391, 392, 393, 398, 399, 405, 409, 410, 411, 413, 414, 415, 416, 417, 418, or 419; and the antisense strand comprises a modified oligonucleotide selected from any of SEQ ID NO. 446, 456, 457, 458, 460, 462, 480, 481, 482, 484, 485, 486, 491, 493, 495, 529, 532, 533, 534, 536, 537, 538, 543, 544, 550, 554, 555, 556, 558, 559, 560, 561, 562, 563, or 564.

6. The oligonucleotide or a pharmaceutically acceptable salt thereof of any one of claims 1-5, wherein, The oligonucleotide comprises any of the following sense and antisense strand combinations: (1) the sense strand comprises the sequence set forth in SEQ ID NO. 15, and the antisense strand comprises the sequence set forth in SEQ ID NO. 158; (2) the sense strand comprises the sequence set forth in SEQ ID NO. 25, and the antisense strand comprises the sequence set forth in SEQ ID NO. 168; (3) the sense strand comprises the sequence set forth in SEQ ID NO. 26, and the antisense strand comprises the sequence set forth in SEQ ID NO. 169; (4) the sense strand comprises the sequence set forth in SEQ ID NO. 27, and the antisense strand comprises the sequence set forth in SEQ ID NO. 170; (5) the sense strand comprises the sequence set forth in SEQ ID NO. 29, and the antisense strand comprises the sequence set forth in SEQ ID NO. 172; (6) the sense strand comprises the sequence set forth in SEQ ID NO. 31, and the antisense strand comprises the sequence set forth in SEQ ID NO. 174; (7) the sense strand comprises the sequence set forth in SEQ ID NO. 49, and the antisense strand comprises the sequence set forth in SEQ ID NO. 192; (8) the sense strand comprises the sequence set forth in SEQ ID NO. 50, and the antisense strand comprises the sequence set forth in SEQ ID NO. 193; (9) 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. 194; (10) the sense strand comprises the sequence set forth in SEQ ID NO. 53, and the antisense strand comprises the sequence set forth in SEQ ID NO. 196; (11) the sense strand comprises the sequence set forth in SEQ ID NO. 54, and the antisense strand comprises the sequence set forth in SEQ ID NO. 197; (12) the sense strand comprises the sequence set forth in SEQ ID NO. 55, and the antisense strand comprises the sequence set forth in SEQ ID NO. 198; (13) 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. 203; (14) the sense strand comprises the sequence set forth in SEQ ID NO. 62, and the antisense strand comprises the sequence set forth in SEQ ID NO. 205; (15) the sense strand comprises the sequence set forth in SEQ ID NO. 64, and the antisense strand comprises the sequence set forth in SEQ ID NO. 207; (16) the sense strand comprises the sequence set forth in SEQ ID NO. 98, and the antisense strand comprises the sequence set forth in SEQ ID NO. 241; (17) the sense strand comprises the sequence set forth in SEQ ID NO. 101, and the antisense strand comprises the sequence set forth in SEQ ID NO. 244; (18) the sense strand comprises the sequence set forth in SEQ ID NO. 102, and the antisense strand comprises the sequence set forth in SEQ ID NO. 245; (19) the sense strand comprises the sequence set forth in SEQ ID NO. 103, and the antisense strand comprises the sequence set forth in SEQ ID NO. 246; (20) the sense strand comprises the sequence set forth in SEQ ID NO. 105, and the antisense strand comprises the sequence set forth in SEQ ID NO. 248; (21) the sense strand comprises the sequence set forth in SEQ ID NO. 106, and the antisense strand comprises the sequence set forth in SEQ ID NO. 249; (22) the sense strand comprises the sequence set forth in SEQ ID NO. 107, and the antisense strand comprises the sequence set forth in SEQ ID NO. 250; (23) the sense strand comprises the sequence set forth in SEQ ID NO. 112, and the antisense strand comprises the sequence set forth in SEQ ID NO. 255; (24) the sense strand comprises the sequence set forth in SEQ ID NO. 113, and the antisense strand comprises the sequence set forth in SEQ ID NO. 256; (25) the sense strand comprises the sequence set forth in SEQ ID NO. 119, and the antisense strand comprises the sequence set forth in SEQ ID NO. 262; (26) the sense strand comprises the sequence set forth in SEQ ID NO. 123, and the antisense strand comprises the sequence set forth in SEQ ID NO. 266; (27) the sense strand comprises the sequence set forth in SEQ ID NO. 124, and the antisense strand comprises the sequence set forth in SEQ ID NO. 267; (28) the sense strand comprises the sequence set forth in SEQ ID NO. 125, and the antisense strand comprises the sequence set forth in SEQ ID NO. 268; (29) the sense strand comprises a sequence set forth in SEQ ID NO. 127, and the antisense strand comprises a sequence set forth in SEQ ID NO. 270; (30) the sense strand comprises a sequence set forth in SEQ ID NO. 128, and the antisense strand comprises a sequence set forth in SEQ ID NO. 271 ; (31) the sense strand comprises a sequence set forth in SEQ ID NO. 129, and the antisense strand comprises a sequence set forth in SEQ ID NO. 272; (32) the sense strand comprises a sequence set forth in SEQ ID NO. 130, and the antisense strand comprises a sequence set forth in SEQ ID NO. 273; (33) the sense strand comprises a sequence set forth in SEQ ID NO. 131, and the antisense strand comprises a sequence set forth in SEQ ID NO. 274; (34) the sense strand comprises a sequence set forth in SEQ ID NO. 132, and the antisense strand comprises a sequence set forth in SEQ ID NO. 275; and (35) the sense strand comprises a sequence set forth in SEQ ID NO. 133, and the antisense strand comprises a sequence set forth in SEQ ID NO. 276; Preferably, the oligonucleotide comprises any one of a sense strand and an antisense strand combination selected from: (1) the sense strand comprises a sequence set forth in SEQ ID NO. 15, and the antisense strand comprises a sequence set forth in SEQ ID NO. 158; (2) the sense strand comprises a sequence set forth in SEQ ID NO. 25, and the antisense strand comprises a sequence set forth in SEQ ID NO. 168; (3) the sense strand comprises a sequence set forth in SEQ ID NO. 26, and the antisense strand comprises a sequence set forth in SEQ ID NO. 169; (4) the sense strand comprises a sequence set forth in SEQ ID NO. 27, and the antisense strand comprises a sequence set forth in SEQ ID NO. 170; (5) the sense strand comprises a sequence set forth in SEQ ID NO. 29, and the antisense strand comprises a sequence set forth in SEQ ID NO. 172; (6) the sense strand comprises a sequence set forth in SEQ ID NO. 31, and the antisense strand comprises a sequence set forth in SEQ ID NO. 174; (7) the sense strand comprises a sequence set forth in SEQ ID NO. 62, and the antisense strand comprises a sequence set forth in SEQ ID NO. 205; (8) the sense strand comprises a sequence set forth in SEQ ID NO. 101, and the antisense strand comprises a sequence set forth in SEQ ID NO. 244; (9) the sense strand comprises a sequence set forth in SEQ ID NO. 106, and the antisense strand comprises a sequence set forth in SEQ ID NO. 249; (10) the sense strand comprises a sequence set forth in SEQ ID NO. 125, and the antisense strand comprises a sequence set forth in SEQ ID NO. 268; (11) the sense strand comprises a sequence set forth in SEQ ID NO. 127, and the antisense strand comprises a sequence set forth in SEQ ID NO. 270; (12) the sense strand comprises a sequence set forth in SEQ ID NO. 131, and the antisense strand comprises a sequence set forth in SEQ ID NO. 274; (13) the sense strand comprises a sequence set forth in SEQ ID NO. 132, and the antisense strand comprises a sequence set forth in SEQ ID NO. 275; and (14) the sense strand comprises a sequence set forth in SEQ ID NO. 133, and the antisense strand comprises a sequence set forth in SEQ ID NO.

276.

7. The oligonucleotide or a pharmaceutically acceptable salt thereof of any one of claims 1-6, wherein, the oligonucleotide comprises any one of a sense strand and an antisense strand combination selected from the following: (1) the sense strand comprises a sequence set forth in SEQ ID NO. 301, and the antisense strand comprises a sequence set forth in SEQ ID NO. 446; (2) the sense strand comprises a sequence set forth in SEQ ID NO. 311, and the antisense strand comprises a sequence set forth in SEQ ID NO. 456; (3) the sense strand comprises a sequence set forth in SEQ ID NO. 312, and the antisense strand comprises a sequence set forth in SEQ ID NO. 457; (4) the sense strand comprises a sequence set forth in SEQ ID NO. 313, and the antisense strand comprises a sequence set forth in SEQ ID NO. 458; (5) the sense strand comprises a sequence set forth in SEQ ID NO. 315, and the antisense strand comprises a sequence set forth in SEQ ID NO. 460; (6) the sense strand comprises a sequence set forth in SEQ ID NO. 317, and the antisense strand comprises a sequence set forth in SEQ ID NO. 462; (7) the sense strand comprises a sequence set forth in SEQ ID NO. 335, and the antisense strand comprises a sequence set forth in SEQ ID NO. 480; (8) the sense strand comprises a sequence set forth in SEQ ID NO. 336, and the antisense strand comprises a sequence set forth in SEQ ID NO. 481; (9) the sense strand comprises a sequence set forth in SEQ ID NO. 337, and the antisense strand comprises a sequence set forth in SEQ ID NO. 482; (10) the sense strand comprises a sequence set forth in SEQ ID NO. 339, and the antisense strand comprises a sequence set forth in SEQ ID NO. 484; (11) the sense strand comprises a sequence set forth in SEQ ID NO. 340, and the antisense strand comprises a sequence set forth in SEQ ID NO. 485; (12) the sense strand comprises a sequence set forth in SEQ ID NO. 341, and the antisense strand comprises a sequence set forth in SEQ ID NO. 486; (13) the sense strand comprises a sequence set forth in SEQ ID NO. 346, and the antisense strand comprises a sequence set forth in SEQ ID NO. 491; (14) the sense strand comprises the sequence set forth in SEQ ID NO. 348, and the antisense strand comprises the sequence set forth in SEQ ID NO. 493; (15) the sense strand comprises the sequence set forth in SEQ ID NO. 350, and the antisense strand comprises the sequence set forth in SEQ ID NO. 495; (16) the sense strand comprises the sequence set forth in SEQ ID NO. 384, and the antisense strand comprises the sequence set forth in SEQ ID NO. 529; (17) the sense strand comprises the sequence set forth in SEQ ID NO. 387, and the antisense strand comprises the sequence set forth in SEQ ID NO. 532; (18) the sense strand comprises the sequence set forth in SEQ ID NO. 388, and the antisense strand comprises the sequence set forth in SEQ ID NO. 533; (19) the sense strand comprises the sequence set forth in SEQ ID NO. 389, and the antisense strand comprises the sequence set forth in SEQ ID NO. 534; (20) the sense strand comprises the sequence set forth in SEQ ID NO. 391, and the antisense strand comprises the sequence set forth in SEQ ID NO. 536; (21) the sense strand comprises the sequence set forth in SEQ ID NO. 392, and the antisense strand comprises the sequence set forth in SEQ ID NO. 537; (22) the sense strand comprises the sequence set forth in SEQ ID NO. 393, and the antisense strand comprises the sequence set forth in SEQ ID NO. 538; (23) the sense strand comprises the sequence set forth in SEQ ID NO. 398, and the antisense strand comprises the sequence set forth in SEQ ID NO. 543; (24) the sense strand comprises the sequence set forth in SEQ ID NO. 399, and the antisense strand comprises the sequence set forth in SEQ ID NO. 544; (25) the sense strand comprises the sequence set forth in SEQ ID NO. 405, and the antisense strand comprises the sequence set forth in SEQ ID NO. 550; (26) the sense strand comprises the sequence set forth in SEQ ID NO. 409, and the antisense strand comprises the sequence set forth in SEQ ID NO. 554; (27) the sense strand comprises the sequence set forth in SEQ ID NO. 410, and the antisense strand comprises the sequence set forth in SEQ ID NO. 555; (28) the sense strand comprises the sequence set forth in SEQ ID NO. 411, and the antisense strand comprises the sequence set forth in SEQ ID NO. 556; (29) the sense strand comprises the sequence set forth in SEQ ID NO. 413, and the antisense strand comprises the sequence set forth in SEQ ID NO. 558; (30) the sense strand comprises the sequence set forth in SEQ ID NO. 414, and the antisense strand comprises the sequence set forth in SEQ ID NO. 559; (31) the sense strand comprises a sequence set forth in SEQ ID NO. 415, and the antisense strand comprises a sequence set forth in SEQ ID NO. 560; (32) the sense strand comprises a sequence set forth in SEQ ID NO. 416, and the antisense strand comprises a sequence set forth in SEQ ID NO. 561; (33) the sense strand comprises a sequence set forth in SEQ ID NO. 417, and the antisense strand comprises a sequence set forth in SEQ ID NO. 562; (34) the sense strand comprises a sequence set forth in SEQ ID NO. 418, and the antisense strand comprises a sequence set forth in SEQ ID NO. 563; and (35) the sense strand comprises a sequence set forth in SEQ ID NO. 419, and the antisense strand comprises a sequence set forth in SEQ ID NO. 564; Preferably, the oligonucleotide comprises any one of a sense strand and an antisense strand combination selected from the following: (1) the sense strand comprises a sequence set forth in SEQ ID NO. 301, and the antisense strand comprises a sequence set forth in SEQ ID NO. 446; (2) the sense strand comprises a sequence set forth in SEQ ID NO. 311, and the antisense strand comprises a sequence set forth in SEQ ID NO. 456; (3) the sense strand comprises a sequence set forth in SEQ ID NO. 312, and the antisense strand comprises a sequence set forth in SEQ ID NO. 457; (4) the sense strand comprises a sequence set forth in SEQ ID NO. 313, and the antisense strand comprises a sequence set forth in SEQ ID NO. 458; (5) the sense strand comprises a sequence set forth in SEQ ID NO. 315, and the antisense strand comprises a sequence set forth in SEQ ID NO. 460; (6) the sense strand comprises a sequence set forth in SEQ ID NO. 317, and the antisense strand comprises a sequence set forth in SEQ ID NO. 462; (7) the sense strand comprises a sequence set forth in SEQ ID NO. 348, and the antisense strand comprises a sequence set forth in SEQ ID NO. 493; (8) the sense strand comprises a sequence set forth in SEQ ID NO. 387, and the antisense strand comprises a sequence set forth in SEQ ID NO. 532; (9) the sense strand comprises a sequence set forth in SEQ ID NO. 392, and the antisense strand comprises a sequence set forth in SEQ ID NO. 537; (10) the sense strand comprises a sequence set forth in SEQ ID NO. 411, and the antisense strand comprises a sequence set forth in SEQ ID NO. 556; (11) the sense strand comprises a sequence set forth in SEQ ID NO. 413, and the antisense strand comprises a sequence set forth in SEQ ID NO. 558; (12) the sense strand comprises a sequence set forth in SEQ ID NO. 417, and the antisense strand comprises a sequence set forth in SEQ ID NO. 562; (13) the sense strand comprises a sequence as set forth in SEQ ID NO. 418, and the antisense strand comprises a sequence as set forth in SEQ ID NO. 563; and (14) the sense strand comprises a sequence as set forth in SEQ ID NO. 419, and the antisense strand comprises a sequence as set forth in SEQ ID NO.

564.

8. A conjugate or a pharmaceutically acceptable salt thereof for use in inhibiting the expression of APP, 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, alicyclic or polycyclic 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 of any one of claims 1 to 7 or a pharmaceutically acceptable salt thereof, or the conjugate of claim 8 or a pharmaceutically acceptable salt thereof, 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 or / and preventing APP-related disorders.

10. Use of the oligonucleotide of any one of claims 1 to 7 or a pharmaceutically acceptable salt thereof, the conjugate of claim 8 or a pharmaceutically acceptable salt thereof, or the composition of claim 9 in the manufacture of a medicament for treating or / and preventing APP-related disorders. The APP-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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