Oligonucleotides that modulate apolipoprotein E (APOE) expression and methods of use thereof

Abstract

The present disclosure relates to oligonucleotides, in particular antisense oligonucleotides (ASO), and pharmaceutically acceptable salts thereof, capable of hybridizing to and reducing expression of APOE pre-mRNA or mRNA. The ASO disclosed herein is capable of reducing the translation of APOE protein in mammals (e.g., humans). The disclosure further relates to methods of treating a disease or disorder in a subject in need thereof by administering an antisense oligonucleotide disclosed herein. In particular, the methods and ASOs described herein can be used to prevent and / or treat human diseases in which a reduction in the amount of APOE or its activity will be beneficial, including but not limited to neurodegenerative diseases such as Alzheimer's disease (AD), dementia with Lewy bodies (DLB), dementia with Parkinson's disease (PDD), and diseases generalized defined as tauopathy and synucleinopathy.

Description

[0001] Cross-reference to related applications

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 484,581, filed February 13, 2023, the entire contents of which are incorporated herein by reference.

[0003] References to sequence list XML

[0004] This application contains a sequence list that has been electronically submitted in XML format. The sequence list XML is incorporated herein by reference. The XML file, created on February 13, 2024, is named 767173_000017_SL.xml and is 403,882 bytes in size. Background Technology

[0005] Neurodegenerative diseases are caused by the gradual loss of neuronal structure or cellular function, followed by cell death in the neural tissue—a process known as neurodegeneration. Neurodegeneration can progress through different components or levels of neuronal circuits, leading to various clinical manifestations, such as Alzheimer's disease, Parkinson's disease, multiple system atrophy, amyotrophic lateral sclerosis (ALS), Huntington's disease, multiple sclerosis, prions, and other human diseases.

[0006] Alzheimer's disease (AD), as a non-limiting example, is the most common neurodegenerative disease and a leading cause of dementia in the elderly. Senile plaques composed of amyloid-β (Aβ) fibrils, neurofibrillary tangles composed of aggregated tau protein, and neuronal malnutrition and loss of neurons are characteristic pathologies of AD and constitute its diagnostic biomarker spectrum (see Jack CR, Jr. et al. (2018) Alzheimers Dement 14:535-562). The earliest pathological changes may occur 20 years or more before the onset of clinical symptoms in Alzheimer's disease (see Jack CR, Jr. et al., Alzheimer's Disease Neuroimaging I (2009) Brain 132:1355-1365; Braak H et al. (2011) J Neuropathol Exp Neurol 70:960-969; Bateman RJ et al. (2012) N Engl J Med 367:795-804; Gordon BA et al. (2018) Lancet Neurol 17:241-250; Barthelemy NR et al. (2020) Nat Med 26:398-407; Quiroz YT et al. (2020) Lancet Neurol 19:513-521), suggesting opportunities for early intervention and disease-modifying therapies.

[0007] AD susceptibility is influenced by common genetic variants, among which the APOE allele variant ε4 (APOE4) stands out due to its strong effect size and significant enrichment in the patient population. Compared to the risk-neutral ε3 / ε3 genotype, inheriting one or two ε4 alleles increases the risk of AD by 3-4 times or 9-15 times, respectively (see Farrer LA et al. (1997) JAMA 278:1349-1356; Genin E et al. (2011) Mol Psychiatry 16:903-907; Neu SC et al. (2017) JAMA Neurol 74:1178-1189). Furthermore, compared to non-carriers of ε4, inheriting one ε4 allele reduced the age of onset of Alzheimer's disease (AD) by 2–8 years, while inheriting two ε4 alleles reduced the age of onset by 5–16 years (see Corder EH et al. (1993) Science 261:921–923; Sando SB et al. (2008) BMC Neurol 8:9). A meta-analysis of 38 case-control pairs across multiple populations revealed a mean allele frequency of ε4 of 38% in patients with AD, compared to 14% in cognitively healthy individuals (Alzgene. APOE_2 / 3 / 4. http: / / www.alzgene.org / meta.asp?geneID=83, updated 2010).

[0008] Human APOE protein consists of a receptor-binding domain in the N-terminal domain and a lipid-binding domain in the C-terminal domain. Nascent APOE protein is secreted by cells and associates with the cell surface ATP-binding cassette transporters ABCA1 and ABCG1, which transfer cholesterol and phospholipids to nascent APOE to form lipoprotein particles (see Wahrle SE et al. (2004) J Biol Chem 279:40987-40993).

[0009] In healthy brains, APOE is primarily expressed in astrocytes, microglia, vascular parietal cells, and choroid plexus cells; however, under pathological or invasive conditions, APOE expression is significantly upregulated in myeloid cells, including microglia and macrophages (see Xu Q et al. (2006) J Neurosci 26:4985-4994; Yamazaki et al. (2019) Nat Rev Neurol 15(9):501-518). It plays a physiological and pathophysiological role in the transport of cholesterol and other lipids between different brain cell types via cell surface APOE receptors (see Hauser PS et al. (2011) Prog LipidRes 50:62-74; Yamazaki et al. (2019) Nat Rev Neurol 15(9):501-518).

[0010] Common APOE ε4 and ε3 allelic variants encode APOE4 and APOE3 protein isoforms, respectively. APOE4 differs from APOE3 at amino acid position 112 (APOE3: Cys112, APOE4: Arg112). Despite differences of only a single amino acid substitution, APOE4 has multiple effects on the pathogenesis of Alzheimer's disease (AD), see Yamazaki Y et al. (2019) Nat RevNeurol 15:501-518, which are generally described as toxic gain-of-function effects of APOE. The best-characterized of this toxic gain-of-function effect is APOE4's reduction of Aβ clearance and promotion of Aβ accumulation, which is associated with the clinical phenotype of accelerated Aβ plaque deposition in ε4 allelic carriers, with ε4 / ε4 homozygous carriers showing the fastest accelerated deposition. In both human and animal models, APOE4 is associated with impaired blood-brain barrier (BBB) ​​integrity (see Ishii M and Iadecola C (2020) Nature 581:31-32; Montagne A et al. (2020) Nature 581:71-76; Jackson RJ et al. (2022) Brain 145:3582-3593). Furthermore, the ε4 allele is also associated with higher pathological levels of TAR DNA-binding protein 43 (TDP-43) in the brains of individuals with AD (see Vossel KA et al. (2013) Neurocase 19:295-301; Josephs KA et al. (2017) Lancet Neurol 16:917-924; Yang HS et al. (2018) Lancet Neurol 17:773-781). In addition, the ε4 allele increases the risk of developing Lewy body dementia (see Tsuang D et al. (2013) JAMA Neurol 70:223-228; Bras J et al. (2014) Hum Mol Genet 23:6139-6146; Guerreiro R et al. (2018) Lancet Neurol 17:64-74) and Parkinson's disease dementia (PDD) (see Huang X et al. (2006) Arch Neurol 63:189-193; Irwin DJ et al. (2012) Ann Neurol 72:587-598; Tsuang D et al. (2013) JAMA Neurol 70:223-228; Tropea TF et al. (2018) Mov Disord 33:289-297).

[0011] Experimental studies have shown that APOE4 can exacerbate tau pathology and tau-mediated neurodegeneration in animal models. Furthermore, APOE4 has been shown to induce effects characterized by reduced APOE physiological and pathophysiological functions, including microglial responsiveness, neuroinflammation, energy metabolism, cholesterol and lipid transport and homeostasis, synaptic integrity and plasticity, glucose metabolism, and neurovascular integrity and function (see Liu CC et al. Nat Rev Neurol 9:106-118; Yamazaki Y et al. (2019) Nat Rev Neurol 15:501-518; Martens YA et al. (2022) Neuron 110:1304-1317). In addition, APOE4 also impairs the physiological functions of APOE, such as microglial reactivity, neuroinflammation, energy metabolism, cholesterol and lipid transport and homeostasis, synaptic integrity and plasticity, glucose metabolism, and neurovascular integrity and function (see Liu CC et al. Nat Rev Neurol 9:106-118; Yamazaki Y et al. (2019) Nat Rev Neurol 15:501-518; Martens YA et al. (2022) Neuron 110:1304-1317).

[0012] Available preclinical data demonstrate that certain APOE-targeting ASOs administered via intravenous infusion (icV) ​​in early stages of pathological development in AD model mice effectively reduce cerebral amyloid burden. Most importantly, this intervention protects AD mice from tau pathology, reduces neurite dystrophy, and lowers levels of neuronal damage markers such as neurofilament light chains (NfL), regardless of the timing of amyloid pathology or ASO administration relative to the onset of AD pathology. Further readings indicate that icV delivery of APOE ASOs protects against tau pathology and associated neurodegeneration, maintains synaptic density (reducing synaptic loss), and reduces the release of neuroinflammatory markers and pro-inflammatory cytokines in the brain without affecting lipid levels in the liver and plasma (see Huynh TV et al. (2017) Neuron 96:1013-1023e1014; Litvinchuk A. et al. (2021) Ann Neurol 89:952-966).

[0013] Recent data from biochemical, in vitro, and in vivo methods in rodent disease models suggest that certain monoclonal antibodies targeting non-lipomorphic aggregates of APOE (α-APOE antibodies) preferentially bind to APOE in amyloid plaques. These antibodies exhibit similar mechanisms of action and efficacy to monoclonal antibodies targeting Aβ (anti-Aβ antibodies), which also bind to Aβ aggregates and reduce Aβ aggregates via a microglia-mediated clearance mechanism (see Liao F et al. (2018) J Clin Invest 128:2144-2155). Furthermore, some α-APOE antibodies appear to specifically bind to dense-core plaques, while other anti-Aβ antibodies bind broadly to amyloid proteins, including those deposited in cerebral blood vessels, as seen in cerebral amyloid angiopathy (CAA). Mechanistically, α-APOE antibodies specifically targeting plaques initially increase glial cell activation, leading to sustained microglia recruitment and improved vascular function, while less specific anti-Aβ antibodies targeting vascular amyloid induce chronic astrocyte proliferation and persistent CAA pathology around the vessels (see Xiong M et al. (2021) Sci Transl Med 13, 581). The resulting neurovascular pathology and damage observed in animal models are consistent with amyloid-related imaging abnormalities (ARIA-E and ARIA-H pathology) observed in human subjects treated with anti-Aβ antibodies (see Sperling RA et al. (2011) Alzheimers Dement 7:367-385, 2011; Saloonay S et al. (2022) JAMA Neurol 79:13-21). Relevant datasets demonstrate that in AD mouse models, continuous treatment with α-APOE antibodies reduces tau seeding, spreading, and malnutrition in neurites in a manner consistent with a reduction in overall amyloid load in individuals (see Gratuze M, Jiang H, Wang C, Xiong M, Bao X, Holtzman DM (2022) Ann Neurol 91:847-852).It appears that while the efficacy of molecules targeting amyloid protein in reducing amyloid plaques is similar between those targeting amyloid protein and those targeting APOE, molecules targeting APOE may be superior because they mediate additional beneficial effects on vascular phenotype while also reducing tau seeding, spread, and pathology (see Liao F et al. (2018) J Clin Invest 128:2144-2155; Xiong M et al. (2021) SciTransl Med 13,581; Gratuze M et al. (2022) Ann Neurol 91:847-852). Unfortunately, antibody therapies (such as α-APOE antibodies) do not penetrate well into brain tissue due to their large molecular weight, short retention time in the brain after direct administration, and short duration of action, limiting their effects to extracellular processes. Therefore, alternative agents and methods are needed to reduce APOE expression. Summary of the Invention

[0014] As described herein, this disclosure relates to antisense oligonucleotides and methods of using them. Therefore, in one aspect, this disclosure provides antisense oligonucleotides or pharmaceutically acceptable salts thereof comprising at least 80% identical sequences to SEQ ID NO:5-11, 15, 16, 20-41, 43, 45, 47-50, 53, 55-57, 60-75, or 77-81.

[0015] In another aspect, this disclosure provides antisense oligonucleotides or pharmaceutically acceptable salts thereof comprising sequences selected from the following: SEQ ID NO: 5-11, 15, 16, 20-41, 43, 45, 47-50, 53, 55-57, 60-75, and 77-81. In some embodiments, the antisense oligonucleotide or pharmaceutically acceptable salt thereof has sequences selected from the following: SEQ ID NO: 7, 8, 10, 15, 32, 33, 35, 36, 37, 38, 39, 40, 41, 43, 45, 49, 56, 57, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 71, 71, 72, 73, 74, 77, 78, 79, 80, and 81.

[0016] In another aspect, this disclosure provides antisense oligonucleotides or pharmaceutically acceptable salts thereof, said antisense oligonucleotides or pharmaceutically acceptable salts comprising at least 80% identical sequences to SEQ ID NO: 84, 85, 88, 90, 91, 94, 96, 100, 103, 104, 108, 111, 113, 114, 115, 119, 120, 123, 124, 129, 130, 131, 133, 134, 135, 136, 138, 150, 151, 157, 163, 164, 168, 170, 175, 176, 180, 185, 210, 212, 216, 219, 228, or 229.

[0017] In another aspect, this disclosure provides antisense oligonucleotides or pharmaceutically acceptable salts thereof comprising sequences selected from the following: SEQ ID NO: 84, 85, 88, 90, 91, 94, 96, 100, 103, 104, 108, 111, 113, 114, 115, 119, 120, 123, 124, 129, 130, 131, 133, 134, 135, 136, 138, 150, 151, 157, 163, 164, 168, 170, 175, 176, 180, 185, 210, 212, 216, 219, 228, and 229. In some embodiments, the antisense oligonucleotide or a pharmaceutically acceptable salt thereof has a sequence selected from the following: SEQ ID NO: 127, 132, 147, 202 and 220.

[0018] In some embodiments, the antisense oligonucleotide or a pharmaceutically acceptable salt thereof comprises about 15 to about 25 nucleotides. In some embodiments, the antisense oligonucleotide or a pharmaceutically acceptable salt thereof comprises about 18 to about 22 nucleotides. In some embodiments, the antisense oligonucleotide or a pharmaceutically acceptable salt thereof comprises about 20 nucleotides. In some embodiments, the antisense oligonucleotide or a pharmaceutically acceptable salt thereof comprises about 18 nucleotides.

[0019] In some embodiments, the antisense oligonucleotide or a pharmaceutically acceptable salt thereof comprises about 12 to about 25 nucleobases. In some embodiments, the antisense oligonucleotide or a pharmaceutically acceptable salt thereof comprises about 14 to about 18 nucleobases. In some embodiments, the antisense oligonucleotide or a pharmaceutically acceptable salt thereof comprises about 16 nucleobases.

[0020] In some embodiments, the antisense oligonucleotide comprises one or more internucleotide linkers. The internucleotide linker may be selected from thiophosphates, dithiophosphates, methylphosphonates, methylthiophosphates, aminophosphates, diaminophosphates, thioaminophosphates, methanesulfonylaminophosphates, and combinations thereof. In some embodiments, the antisense oligonucleotide comprises one or more thiophosphate internucleotide links. In some embodiments, all internucleotide links are thiophosphate links.

[0021] In some embodiments, the antisense oligonucleotide comprises DNA. In some embodiments, the antisense oligonucleotide comprises RNA. In some embodiments, the antisense oligonucleotide comprises a protein and / or a polypeptide. In some embodiments, the antisense oligonucleotide comprises a small molecule.

[0022] In some embodiments, the antisense oligonucleotide comprises one or more modified nucleosides. The modified nucleosides may be selected from 2'-modified nucleosides, 4'-modified nucleosides, bridging nucleosides, diaminophosphate morpholine, locked nucleic acids, ethylene-bridging nucleic acids, ethylene glycol nucleic acids, hexitol nucleic acids, cyclohexene nucleic acids, arabinose nucleic acids, peptide nucleic acids, threonose nucleic acids, tricyclic-2'-deoxynucleotides, 1'-deoxypentose, 1',2'-dideoxypentose, 2',3'-dideoxypentose, 2',3'-didehydro-2',3'-dideoxypentose, unlocked nucleic acids, and combinations thereof. The antisense oligonucleotide may comprise one or more 2'-nucleoside modifications. The antisense oligonucleotide may comprise at least eight 2'-nucleoside modifications. The antisense oligonucleotide may comprise at least ten 2'-nucleoside modifications.

[0023] In some embodiments, all nucleosides of the antisense oligonucleotide contain a 2'-nucleoside modification. The 2'-nucleoside modification may be selected from 2'-fluoro-nucleoside, 2'-O-methyl-nucleoside, 2'-O-methoxyethyl-nucleoside, 2'-O-benzyl-2'-deoxy-nucleoside, 2'-O-methyl-4-pyridyl-nucleoside (2'-O-CH2Py(4)), 2'-amino-nucleoside, and combinations thereof.

[0024] In some embodiments, the antisense oligonucleotide comprises one or more modified nucleobases. The modified nucleobase may be a methylated nucleobase. The methylated nucleobase may be selected from 5-methyluracil, N6-methyladenine, N4-methylcytosine, N7-methylguanine, 5-hydroxymethylcytosine, N3-methylcytosine, and combinations thereof. The modified nucleobase may also be an acetylated nucleobase. The acetylated nucleobase may be N4-acetylcytosine. The modified nucleobases can be selected from pseudouridine (Ψ), N1-methyl-pseudouridine (N1-methyl-Ψ), 2-thiouridine (s2U), 5-fluoro-2'-deoxyuridine (FUDR), 8-oxo-7,8-dihydroguanosine (8-oxoG), N-ethylpiperidine-7-EAA triazole modified adenine, N-ethylpiperidine-6-triazole modified adenine, 6-phenylpyrrolocytosine (PhpC), 2,4-difluorotoluyl-ribonucleoside (rF), N1(5-nitroindole)ribonucleoside, 5-methoxyuridine) and combinations thereof.

[0025] In some implementations, the antisense oligonucleotide or a pharmaceutically acceptable salt thereof further comprises a reverse nucleotide.

[0026] In some embodiments, the antisense oligonucleotide is a gapmer. The gapmer may comprise flanking RNA nucleotides at the 5', 3', or both ends. The range of flanking RNA nucleotides at the 5' and / or 3' ends can independently range from 1 nucleotide to approximately 9 nucleotides. The length of the gapmer can range from approximately 18 to approximately 22 nucleotides. The gapmer may be 18 nucleotides long. The gapmer may comprise ten central 2'-deoxynucleotides and four flanking RNA nucleotides at each of the 5' and 3' ends. The gapmer may be 20 nucleotides long. The gapmer may comprise ten central 2'-deoxynucleotides and / or five flanking ribonucleosides at each of the 5' and 3' ends.

[0027] In some implementations, the gapmer length ranges from about 14 to about 22 nucleotides. The range of flanking nucleotides at the 5' and / or 3' ends independently ranges from about 1 nucleotide to about 5 nucleotides. The gapmer may contain ten central 2'-deoxynucleotides and / or three flanking RNA nucleotides at each of the 5' and 3' ends. The gapmer length may be 16 nucleotides.

[0028] In some implementations, administration of antisense oligonucleotides to a subject reduces the expression of human APOE mRNA, pre-mRNA, protein, or a combination thereof. Human APOE pre-mRNA or mRNA may be reduced by about 10% or more. Human APOE pre-mRNA or mRNA may be reduced by about 30% or more. Human APOE pre-mRNA or mRNA may be reduced by about 50% or more. Human APOE mRNA may be reduced by about 25% or more. Human APOE mRNA may be reduced by about 50% or more. Human APOE mRNA may be reduced by about 70% or more. Human APOE protein may be reduced by about 25% or more. Human APOE protein may be reduced by about 50% or more. Human APOE protein may be reduced by about 70% or more.

[0029] In some embodiments, the antisense oligonucleotide can hybridize with human APOE mRNA. The human APOE mRNA may contain at least 80% of the same sequence as SEQ ID NO:1. The human APOE mRNA may encode a protein containing 285% of the same sequence as SEQ ID NO:8. APOE may be an isoform selected from APOE 2, APOE 3, APOE 4, APOE5f, APOE 5s, and APOE 7. Hybridization can be performed via Watson-Crick base pairing.

[0030] In some embodiments, the antisense oligonucleotide can hybridize with human APOE pre-mRNA. The human APOE pre-mRNA may contain at least 80% of the sequence identical to SEQ ID NO:230. The human APOE mRNA may encode a protein containing 85% of the sequence identical to SEQ ID NO:82. APOE may be an allotype selected from APOE 2, APOE 3, APOE 4, APOE 5f, APOE 5s, and APOE 7. Hybridization can be performed via Watson-Crick base pairing.

[0031] In another aspect, this disclosure relates to methods for treating a disease or disorder in a human subject in need, the methods comprising administering to the subject in need an antisense oligonucleotide as described herein or a pharmaceutically acceptable salt thereof.

[0032] In another aspect, this disclosure relates to methods for treating neurodegenerative diseases, said methods comprising administering to a subject in need an antisense oligonucleotide as described herein or a pharmaceutically acceptable salt thereof. Neurodegenerative diseases may be selected from, but are not limited to, Alzheimer's disease, amyotrophic lateral sclerosis, Huntington's disease, Lewy body dementia, multiple sclerosis, multiple system atrophy, Parkinson's disease, prions, and combinations thereof. Neurodegenerative diseases may include Alzheimer's disease. Neurodegenerative diseases may include tau protein diseases. Tau proteinopathy can be selected from, but is not limited to, primary age-related tau proteinopathy, chronic traumatic encephalopathy, progressive supranuclear palsy, corticobasal degeneration, chromosome 17-linked frontotemporal dementia with Parkinson's syndrome, vacuolet tau proteinopathy, lytico-bodig disease, ganglioglioma, gangliocytoma, meningeal angiomatosis, post-encephalitis Parkinson's syndrome, subacute sclerosing panencephalitis, lead poisoning encephalopathy, tuberous sclerosis, pantothenic kinase-related neurodegeneration, lipofuscinosis, behaviorally variant frontotemporal dementia (Pick's disease), argyrophilic granulation disease, and combinations thereof.

[0033] In another aspect, this disclosure relates to methods for treating acute or chronic injuries, the methods comprising administering to a subject in need an antisense oligonucleotide as described herein or a pharmaceutically acceptable salt thereof. Acute or chronic injuries may be selected from, but are not limited to, traumatic brain injury, spinal cord injury, chronic traumatic encephalopathy, chemotherapeutic brain, and combinations thereof.

[0034] In another aspect, this disclosure relates to methods for treating acute or chronic inflammatory conditions, the methods comprising administering to a subject in need an antisense oligonucleotide as described herein or a pharmaceutically acceptable salt thereof. Acute or chronic inflammatory conditions may be selected from, but are not limited to, stroke, multiple sclerosis, infectious diseases, immune responses to cancer development, and combinations thereof.

[0035] In another aspect, this disclosure relates to methods for treating vascular diseases or vascular pathological conditions, said methods comprising administering to a subject in need an antisense oligonucleotide as described herein or a pharmaceutically acceptable salt thereof. Vascular diseases or vascular pathological conditions may be selected from, but are not limited to, cerebral small vessel disease, blood-brain barrier leakage, atherosclerosis, cerebral amyloid angiopathy, and combinations thereof.

[0036] In another aspect, this disclosure relates to a method for treating lipid storage disorders, the method comprising administering to a subject in need an antisense oligonucleotide as described herein or a pharmaceutically acceptable salt thereof. Lipid storage disorders may be selected from, but are not limited to, tendon xanthomas, Fabry disease, Fabry disease, fucoside storage disorders, Gaucher disease, GM1 ganglioside storage disorders, GM2 ganglioside storage disorders AB variant, Krabby disease, metachromatic leukodystrophy, multiple sulfatase deficiencies, Niemann-Pick disease type A, Niemann-Pick disease type B, Niemann-Pick disease type C, Sankhov disease, Schindler disease, Ty-Sachs disease, Wollman disease, and combinations thereof.

[0037] In another aspect, this disclosure relates to the use of antisense oligonucleotides or pharmaceutically acceptable salts thereof, as described herein, in the manufacture of medicaments for the therapeutic treatment of diseases or disorders in human subjects in need.

[0038] In another aspect, this disclosure relates to the use of antisense oligonucleotides or pharmaceutically acceptable salts thereof, as described herein, for the treatment of diseases or disorders in human subjects in need.

[0039] In another aspect, this disclosure relates to the use of antisense oligonucleotides or pharmaceutically acceptable salts thereof, as described herein, in the manufacture of medicaments for the therapeutic treatment of neurodegenerative diseases in subjects in need.

[0040] In another aspect, this disclosure relates to the use of antisense oligonucleotides or pharmaceutically acceptable salts thereof, as described herein, for the treatment of neurodegenerative diseases in subjects of need. Neurodegenerative diseases may be selected from, but are not limited to, Alzheimer's disease, amyotrophic lateral sclerosis, Huntington's disease, Lewy body dementia, multiple sclerosis, multiple system atrophy, Parkinson's disease, prions, and combinations thereof. Neurodegenerative diseases may be tau protein diseases. Tau protein diseases can be selected from primary age-related tau protein diseases, chronic traumatic encephalopathy, progressive supranuclear palsy, corticobasal degeneration, chromosome 17-linked frontotemporal dementia with Parkinson's syndrome, vacuolet tau protein diseases, lytico-bodig disease, ganglioglioma, gangliocytoma, meningeal angiomatosis, post-encephalitis Parkinson's syndrome, subacute sclerosing panencephalitis, lead poisoning encephalopathy, tuberous sclerosis, pantothenic kinase-related neurodegeneration, lipofuscinosis, behaviorally variant frontotemporal dementia (Pick's disease), argyrophilic granulation disease, and combinations thereof.

[0041] In some embodiments, the oligonucleotide or a pharmaceutically acceptable salt thereof is administered topically to a subject in need. Topical administration may be intracranial, intracerebral, intramuscular, spinal, epidural, sacroiliac, or subdural injection.

[0042] In some implementations, oligonucleotides or pharmaceutically acceptable salts thereof are administered systemically to subjects in need. Attached Figure Description

[0043] Figures 1A-1C These are phase-contrast micrographs showing that some ASOs (e.g., S1038 (SEQ ID NO:24)) caused abnormal changes in cell morphology, while other ASOs resulted in HEP3B cells having morphology comparable to the untreated control. All cells were cultured for 48 hours with or without ASOs. Figure 1A These are microscopic images showing HEP3B cells that have not been treated with ASO (“untreated control”). Figure 1B These are microscopic images showing that HEP3B cells treated with ASO S1038 (12.5 nM (SEQ ID NO:24)) exhibit abnormal epithelial cell morphology (e.g., loss of confluence, dense condensed nuclei) compared to untreated HEP3B cells, indicating cytotoxicity and death. Figure 1C These are microscopic images showing that HEP3B cells treated with ASO S1094 (12.5 nM (SEQ ID NO: 80)) exhibit comparable morphology and confluence to untreated HEP3B cells. This indicates that ASO S1094 (SEQ ID NO: 80) is non-toxic to HEP3B cells. Detailed Implementation

[0044] Mutations and expression of apolipoprotein E (APOE) have been strongly associated with human diseases, particularly neurological disorders such as Alzheimer's disease (AD), Lewy body dementia, and Parkinson's disease. Susceptibility to AD is influenced by common genetic variants, among which the APOE allele variant ε4 (APOE4) stands out due to its large effect size and significant enrichment in the patient population. Inheriting one or both ε4 alleles increases the risk of AD by 3-4 times and 9-15 times, respectively, compared to the risk-neutral ε3 / ε3 genotype (see Farrer LA et al. (1997) JAMA 278:1349-1356; Genin E et al. (2011) Mol Psychiatry 16:903-907; Neu SC et al. (2017) JAMA Neurol 74:1178-1189). Therefore, APOE has been investigated as a potential target for treating neurological and other diseases where APOE may play a role in the disease pathology. Unfortunately, antibody therapies (such as α-APOE antibodies) cannot penetrate brain tissue well due to their large molecular weight, short retention time in the brain after direct administration, and short duration of action, and are limited to extracellular effects. Therefore, other agents and methods are needed to reduce APOE expression.

[0045] antisense oligonucleotides

[0046] This disclosure relates to antisense oligonucleotides and their pharmaceutically relevant salts that target APOE mRNA and / or pre-mRNA (e.g., hybridize with it, pair with it with its Watson-Crick bases, bind to it, or reduce its expression). In certain embodiments of this disclosure, the antisense oligonucleotide or its pharmaceutically acceptable salt comprises at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical sequence to at least one of SEQ ID NO: 5-11, 15, 16, 20-41, 43, 45, 47-50, 53, 55-57, 60-75, or 77-81. The antisense oligonucleotide or a pharmaceutically acceptable salt thereof may contain at least one of SEQ ID NO: 5-11, 15, 16, 20-41, 43, 45, 47-50, 53, 55-57, 60-75, or 77-81. In some embodiments, the antisense oligonucleotide or a pharmaceutically acceptable salt thereof contains at least one of SEQ ID NO: 7, 8, 10, 15, 32, 33, 35, 36, 37, 38, 39, 40, 41, 43, 45, 49, 56, 57, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 71, 71, 72, 73, 74, 77, 78, 79, 80, or 81.

[0047] In certain embodiments of this disclosure, the antisense oligonucleotide or a pharmaceutically acceptable salt thereof comprises at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical sequence to at least one of SEQ ID NO:3-81. In certain embodiments of this disclosure, the antisense oligonucleotide or a pharmaceutically acceptable salt thereof comprises at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical sequence to at least one of SEQ ID NO: 7, 8, 10, 15, 32, 33, 35, 36, 37, 38, 39, 40, 41, 43, 45, 49, 56, 57, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 71, 71, 72, 73, 74, 77, 78, 79, 80, or 81.

[0048] The target APOE oligonucleotide sequence can be a premRNA. PremRNA is the result of DNA transcription into premRNA. Post-transcriptional, premRNA undergoes processing steps such as adding a 5' cap, adding a multi-A tail, and removing introns to form mature mRNA. The target premRNA can be a premRNA encoding any APOE isoform, such as APOE 2, APOE 3, APOE 4, APOE 5f, APOE 5s, or APOE 7. For example, the target can be an APOE premRNA having the nucleic acid sequence according to SEQ ID NO:230. In some embodiments of this disclosure, the antisense oligonucleotide or a pharmaceutically acceptable salt thereof comprises the sequence of SEQ ID NO:230. IDNO: at least one of the following sequences is at least about 80%, 85%, 90%, 91%, 94%, 96%, 100%, 103%, 104%, 108, 111, 113, 114, 115, 119, 120, 123, 124, 129, 130, 131, 133, 134, 135, 136, 138, 150, 151, 157, 163, 164, 168, 170, 175, 176, 180, 185, 210, 212, 216, 219, 228 or 229, and is about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical. In certain embodiments of this disclosure, the antisense oligonucleotide or a pharmaceutically acceptable salt thereof comprises at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical sequence to at least one of SEQ ID NO: 84, 119, 120, 123, 124, 129, 133-136, 168, 185, 228, or 229. In certain embodiments of this disclosure, the antisense oligonucleotide or a pharmaceutically acceptable salt thereof comprises at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical sequence to at least one of SEQ ID NO: 84-229. In some embodiments of this disclosure, the antisense oligonucleotide or a pharmaceutically acceptable salt thereof comprises at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical sequence to at least one of SEQ ID NO: 127, 132, 147, 202, or 220.The antisense oligonucleotide or a pharmaceutically acceptable salt thereof may contain at least one of SEQ ID NO: 84, 85, 88, 90, 91, 94, 96, 100, 103, 104, 108, 111, 113, 114, 115, 119, 120, 123, 124, 129, 130, 131, 133, 134, 135, 136, 138, 150, 151, 157, 163, 164, 168, 170, 175, 176, 180, 185, 210, 212, 216, 219, 228 or 229. In some embodiments, the antisense oligonucleotide or a pharmaceutically acceptable salt thereof may comprise at least one of SEQ ID NO: 84, 119, 120, 123, 124, 129, 133-136, 168, 185, 228, or 229. In some embodiments, the antisense oligonucleotide or a pharmaceutically acceptable salt thereof comprises at least one of SEQ ID NO: 127, 132, 147, 202, or 220. In some embodiments, the antisense oligonucleotide or a pharmaceutically acceptable salt thereof comprises at least one of SEQ ID NO: 84-229.

[0049] Antisense oligonucleotides or pharmaceutically acceptable salts thereof contain about 15 to about 25 nucleotides, about 15 to about 24 nucleotides, about 15 to about 23 nucleotides, about 15 to about 22 nucleotides, about 15 to about 21 nucleotides, about 15 to about 20 nucleotides, about 15 to about 19 nucleotides, about 15 to about 18 nucleotides, about 18 to about 22 nucleotides, about 18 to about 21 nucleotides, about 18 to about 20 nucleotides, about 15 nucleotides, about 16 nucleotides, about 17 nucleotides, about 18 nucleotides, about 19 nucleotides, about 20 nucleotides, about 21 nucleotides, or about 22 nucleotides. In some embodiments, the antisense oligonucleotide or a pharmaceutically acceptable salt thereof may comprise about 12 to about 25 nucleotides, about 13 to about 25 nucleotides, about 14 to about 25 nucleotides, about 12 to about 24 nucleotides, about 12 to about 23 nucleotides, about 12 to about 22 nucleotides, about 12 to about 21 nucleotides, about 12 to about 20 nucleotides, about 12 to about 19 nucleotides, about 12 to about 18 nucleotides, about 12 to about 17 nucleotides, about 12 to about 16 nucleotides, about 12 About 15 nucleotides, about 12 to 14 nucleotides, about 12 to 13 nucleotides, about 14 to 24 nucleotides, about 14 to 23 nucleotides, about 14 to 22 nucleotides, about 14 to 21 nucleotides, about 14 to 20 nucleotides, about 14 to 19 nucleotides, about 14 to 18 nucleotides, about 14 to 17 nucleotides, about 14 to 16 nucleotides, about 14 to 15 nucleotides, about 12 nucleotides, about 13 nucleotides, or about 14 nucleotides. Antisense oligonucleotides or their pharmaceutically acceptable salts may contain about 20 nucleotides. Antisense oligonucleotides or their pharmaceutically acceptable salts may contain about 18 nucleotides. Antisense oligonucleotides or their pharmaceutically acceptable salts may contain about 16 nucleotides.

[0050] In some embodiments, the antisense oligonucleotide may include one or more internucleotide linking modifications. These internucleotide linking modifications may be thiophosphate, dithiophosphate, methylphosphonate, methylthiophosphate, aminophosphate, diaminophosphate, thioaminophosphate, methanesulfonylaminophosphate, or combinations thereof. The antisense oligonucleotide may include one or more thiophosphate internucleotide links. In some embodiments, all internucleotide links are thiophosphate links.

[0051] Antisense oligonucleotides may include DNA, RNA, proteins, and / or peptides. In some embodiments, antisense oligonucleotides include small molecules.

[0052] In some embodiments, the antisense oligonucleotide may contain one or more modified nucleosides. The modified nucleosides may be 2'-modified nucleosides, 4'-modified nucleosides, bridging nucleosides, diaminophosphate morpholine, locked nucleic acids, ethylene-bridging nucleic acids, ethylene glycol nucleic acids, hexitol nucleic acids, cyclohexene nucleic acids, arabinose nucleic acids, peptide nucleic acids, threonose nucleic acids, tricyclic-2'-deoxynucleotides, 1'-deoxypentose, 1',2'-dideoxypentose, 2',3'-dideoxypentose, 2',3'-didehydro-2',3'-dideoxypentose, unlocked nucleic acids, or combinations thereof. The antisense oligonucleotide may contain one or more 2'-nucleoside modifications. The antisense oligonucleotide may contain at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 2'-nucleoside modifications. The antisense oligonucleotide may contain at least eight 2'-nucleoside modifications. The antisense oligonucleotide may contain at least ten 2'-nucleoside modifications.

[0053] In some embodiments, all nucleosides of the antisense oligonucleotide contain a 2'-nucleoside modification. The 2'-nucleoside modification may be 2'-fluoro-nucleoside, 2'-O-methyl-nucleoside, 2'-O-methoxyethyl-nucleoside, 2'-O-benzyl-2'-deoxy-nucleoside, 2'-O-methyl-4-pyridyl-nucleoside (2'-O-CH2Py(4)), 2'-amino-nucleoside, or a combination thereof.

[0054] In some embodiments, the antisense oligonucleotide may contain one or more modified nucleosides (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 modified nucleosides). The modified nucleosides may be methylated nucleosides. Modified nucleosides may be 5-methyluracil, N6-methyladenine, N4-methylcytosine, N7-methylguanine, 5-hydroxymethylcytosine, N3-methylcytosine, or combinations thereof. Modified nucleosides may be acetylated nucleosides. Acetylated nucleosides may be N4-acetylcytosine. The modified nucleobases can be pseudouridine (Ψ), N1-methyl-pseudouridine (N1-methyl-Ψ), 2-thiouridine (s2U), 5-fluoro-2'-deoxyuridine (FUDR), 8-oxo-7,8-dihydroguanosine (8-oxoG), N-ethylpiperidine-7-EAA triazole modified adenine, N-ethylpiperidine-6-triazole modified adenine, 6-phenylpyrrolocytosine (PhpC), 2,4-difluorotoluyl-ribonucleoside (rF), N1(5-nitroindole)ribonucleoside, 5-methoxyuridine) or combinations thereof.

[0055] In some implementations, the antisense oligonucleotide or a pharmaceutically acceptable salt thereof further comprises a reverse nucleotide.

[0056] In some embodiments, the antisense oligonucleotide is a gapmer. The gapmer may comprise flanking RNA nucleotides at the 5', 3', or both ends. The range of flanking RNA nucleotides at the 5' and / or 3' ends can independently range from about 1 to about 10 nucleotides, about 1 to about 9 nucleotides, about 1 to about 8 nucleotides, about 1 to about 7 nucleotides, about 1 to about 6 nucleotides, about 1 to about 5 nucleotides, about 1 to about 4 nucleotides, or about 1 to about 3 nucleotides. The length of the gapmer can range from about 18 to about 22 nucleotides (e.g., about 18, 19, 20, 21, or 22 nucleotides). The gapmer may be 18 nucleotides long. The gapmer may comprise about ten central 2'-deoxynucleosides and about four flanking RNA nucleotides at each of the 5' and 3' ends. The gapmer may be 20 nucleotides long. The gapmer may comprise about ten central 2'-deoxynucleosides and / or about five flanking ribonucleosides at each of the 5' and 3' ends.

[0057] Gapmers can be of any length. The length of the gapmer nucleotide or nucleoside can be about 14 to about 22, about 15 to about 22, about 16 to about 22, about 17 to about 22, about 18 to about 22, about 19 to about 22, about 20 to about 22, about 21 to about 22, about 14 to about 21, about 14 to about 20, about 14 to about 19, about 14 to about 18, about 14 to about 17, about 14 to about 16, or about 14 to about 15. The length of the gapmer nucleotide or nucleoside can be about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, or about 22. A gapmer can contain about ten central 2'-deoxynucleosides and about three flanking RNA nucleotides at each of the 5' and 3' ends. A gapmer can contain about ten central 2'-deoxynucleosides and / or about three flanking RNA nucleotides at each of the 5' and 3' ends. In some embodiments, the gapmer is 16 nucleotides long. In some embodiments, the gapmer is 18 nucleotides long. In some embodiments, the gapmer length ranges from about 14 to about 22 nucleotides. In some embodiments, the range of flanking RNA nucleotides at the 5' and / or 3' ends independently ranges from about 1 nucleotide to about 5 nucleotides.

[0058] The gapmer can have any combination of the nucleotide length and number of flanking nucleotides (e.g., 3' flanking nucleotides, 5' flanking nucleotides, or both) as described herein. The flanking nucleotides (3' flanking nucleotides, 5' flanking nucleotides) can contain any number of modified nucleotides or nucleosides. Modified nucleosides or nucleotides can be modified in any way, such as any modification described herein (e.g., 2'-modification, locked nucleic acid modification). Modified nucleosides or nucleotides within the flanking region can be continuous. Modified nucleosides or nucleotides within the flanking region can be discontinuous (e.g., unmodified nucleotides may be present between two or more modified nucleotides in the flanking region). The gapmer can contain 14 nucleotides, and the 5' end and / or 3' end can independently contain about 1 to about 10 nucleotides, about 1 nucleotide to about 9 nucleotides, about 1 to about 8 nucleotides, about 1 to about 7 nucleotides, about 1 to about 6 nucleotides, about 1 to about 5 nucleotides, about 1 to about 4 nucleotides, about 1 to about 3 nucleotides, or about 1 to about 2 nucleotides. For example, a gapmer can be 1-12-1, 1-11-2, 2-11-1, 3-10-1, 1-10-3, 2-10-2, 3-9-2, 2-9-3, 1-9-4, 4-9-1, 5-8-1, 1-8-5, 4-8-2, 2-8-4, or 3-8-3, where the value XYZ indicates 5' flanking nucleotide-central nucleotide-3 flanking nucleotide. A gapmer can contain 15 nucleotides, and the 5' end and / or 3' end can independently contain about 1 to about 10 nucleotides, about 1 nucleotide to about 9 nucleotides, about 1 to about 8 nucleotides, about 1 to about 7 nucleotides, about 1 to about 6 nucleotides, about 1 to about 5 nucleotides, about 1 to about 4 nucleotides, about 1 to about 3 nucleotides, or about 1 to about 2 nucleotides. For example, a gapmer can be 1-13-1, 1-12-2, 2-12-1, 3-11-1, 1-11-3, 2-11-2, 3-10-2, 2-10-3, 1-10-4, 4-10-1, 5-9-1, 1-9-5, 4-9-2, 2-9-4, 3-9-3, 4-8-3, 3-8-4, 2-8-5, 5-8-2, 1-8-6, or 6-8-1. A gapmer can contain 16 nucleotides, and the 5' end and / or 3' end can independently contain about 1 to about 10 nucleotides, about 1 nucleotide to about 9 nucleotides, about 1 to about 8 nucleotides, about 1 to about 7 nucleotides, about 1 to about 6 nucleotides, about 1 to about 5 nucleotides, about 1 to about 4 nucleotides, about 1 to about 3 nucleotides, or about 1 to about 2 nucleotides.For example, gapmer can be 1-14-1, 1-13-2, 2-13-1, 3-12-1, 1-12-3, 2-12-2, 3-11-2, 2-11-3, 1-11-4, 4-11-1, 5-10-1, 1-10-5, 4-10-2, 2-10-4, 3-10-3, 4-9-3, 3-9-4, 2-9-5, 5-9-2, 1-9-6, 6-9-1, 7-8-1, 1-8-7, 6-8-2, 2-8-6, 5-8-3, 3-8-5, or 4-8-4. The gapmer may contain 17 nucleotides, and the 5' end and / or 3' end may independently contain about 1 to about 10 nucleotides, about 1 nucleotide to about 9 nucleotides, about 1 to about 8 nucleotides, about 1 to about 7 nucleotides, about 1 to about 6 nucleotides, about 1 to about 5 nucleotides, about 1 to about 4 nucleotides, about 1 to about 3 nucleotides, or about 1 to about 2 nucleotides. For example, gapmer can be 1-15-1, 1-14-2, 2-14-1, 3-13-1, 1-13-3, 2-13-2, 3-12-2, 2-12-3, 1-12-4, 4-12-1, 5-11-1, 1-11-5, 4-11-2, 2-11-4, 3-11-3, 4-10-3, 3-10-4, 2-10-5, 5-10-2, 1-10-6, 6-10-1, 7-9-1, 1-9-7, 6-9-2, 2-9-6, 5-9-3, 3-9-5, 4-9-4, 8-8-1, 1-8-8, 7-8-2, 2-8-7, 6-8-3, 3-8-6, 5-8-4, or 4-8-5. The gapmer may contain 18 nucleotides, and the 5' end and / or 3' end may independently contain about 1 to about 10 nucleotides, about 1 nucleotide to about 9 nucleotides, about 1 to about 8 nucleotides, about 1 to about 7 nucleotides, about 1 to about 6 nucleotides, about 1 to about 5 nucleotides, about 1 to about 4 nucleotides, about 1 to about 3 nucleotides, or about 1 to about 2 nucleotides.For example, gapmer can be 1-16-1, 1-15-2, 2-15-1, 3-14-1, 1-14-3, 2-14-2, 3-13-2, 2-13-3, 1-13-4, 4-13-1, 5-12-1, 1-12-5, 4-12-2, 2-12-4, 3-12-3, 4-11-3, 3-11-4, 2-11-5, 5-11-2, 1-11-6, 6-1 1-1, 7-10-1, 1-10-7, 6-10-2, 2-10-6, 5-10-3, 3-10-5, 4-10-4, 8-9-1, 1-9-8, 7-9-2, 2-9-7, 6-9-3, 3-9-6, 5-9-4, 4-9-5, 9-8-1, 1-8-9, 8-8-2, 2-8-8, 7-8-3, 3-8-7, 6-8-4, 4-8-6, or 5-8-5. The gapmer may contain 19 nucleotides, and the 5' end and / or 3' end may independently contain about 1 to about 10 nucleotides, about 1 nucleotide to about 9 nucleotides, about 1 to about 8 nucleotides, about 1 to about 7 nucleotides, about 1 to about 6 nucleotides, about 1 to about 5 nucleotides, about 1 to about 4 nucleotides, about 1 to about 3 nucleotides, or about 1 to about 2 nucleotides. For example, gapmer can be 1-17-1, 1-16-2, 2-16-1, 3-15-1, 1-15-3, 2-15-2, 3-14-2, 2-14-3, 1-14-4, 4-14-1, 5-13-1, 1-13-5, 4-13-2, 2-13-4, 3-13-3, 4-12-3, 3-12-4, 2-12-5, 5-12-2, 1-12-6, 6-12-1, 7-11-1, 1-11-7, 6-11-2, 2-11-6, 5-1 1-3, 3-11-5, 4-11-4, 8-10-1, 1-10-8, 7-10-2, 2-10-7, 6-10-3, 3-10-6, 5-10-4, 4-10-5, 9-9-1, 1-9-9, 8-9-2, 2-9-8, 7-9-3, 3-9-7, 6-9-4, 4-9-6, 5-9-5, 10-8-1, 1-8-10, 9-8-2, 2-8-9, 8-8-3, 3-8-8, 7-8-4, 4-8-7, 5-8-6 or 6-8-5. The gapmer may contain 20 nucleotides, and the 5' end and / or 3' end may independently contain about 1 to about 10 nucleotides, about 1 nucleotide to about 9 nucleotides, about 1 to about 8 nucleotides, about 1 to about 7 nucleotides, about 1 to about 6 nucleotides, about 1 to about 5 nucleotides, about 1 to about 4 nucleotides, about 1 to about 3 nucleotides, or about 1 to about 2 nucleotides.For example, gapmer can be 1-18-1, 1-17-2, 2-17-1, 3-16-1, 1-16-3, 2-16-2, 3-15-2, 2-15-3, 1-15-4, 4-15-1, 5-14-1, 1-14-5, 4-14-2, 2-14-4, 3-14-3, 4-13-3, 3-13-4, 2-13-5, 5-13-2, 1-13-6, 6-13-1, 7-12-1, 1-12-7, 6-12-2, 2-12-6, 5-12-3, 3-12-5, 4-12-4, 8-11-1, 1-11-8, 7-11-2. 2-11-7, 6-11-3, 3-11-6, 5-11-4, 4-11-5, 9-10-1, 1-10-9, 8-10-2, 2-10-8, 7-10-3, 3-10-7, 6-10-4, 4-10-6, 5-10-5, 10-9-1, 1-9-10, 9-9-2, 2-9-9, 8-9-3, 3-9-8, 7-9-4, 4-9-7, 5-9-6, 6-9-5, 11-8-1, 1-8-11, 10-8-2, 2-8-10, 9-8-3, 3-8-9, 8-8-4, 4-8-8, 7-8-5, 5-8-7 or 6-8-6. The gapmer may contain 21 nucleotides, and the 5' end and / or 3' end may independently contain about 1 to about 10 nucleotides, about 1 nucleotide to about 9 nucleotides, about 1 to about 8 nucleotides, about 1 to about 7 nucleotides, about 1 to about 6 nucleotides, about 1 to about 5 nucleotides, about 1 to about 4 nucleotides, about 1 to about 3 nucleotides, or about 1 to about 2 nucleotides.For example, gapmer can be 1-19-1, 1-18-2, 2-18-1, 3-17-1, 1-17-3, 2-17-2, 3-16-2, 2-16-3, 1-16-4, 4-16-1, 5-15-1, 1-15-5, 4-15-2, 2-15-4, 3-15-3, 4-14-3, 3-14-4, 2-14- 5, 5-14-2, 1-14-6, 6-14-1, 7-13-1, 1-13-7, 6-13-2, 2-13-6, 5-13-3, 3-13-5, 4-13-4, 8-12-1, 1-12-8, 7-12-2, 2-12-7, 6-12-3, 3-12-6, 5-12-4, 4-12-5, 9-11-1, 1- 11-9, 8-11-2, 2-11-8, 7-11-3, 3-11-7, 6-11-4, 4-11-6, 5-11-5, 10-10-1, 1-10-10, 9-10-2, 2-10-9, 8-10-3, 3-10-8, 7-10-4, 4-10-7, 5-10-6, 6-10-5, 11-9-1, 1-9- 11, 10-9-2, 2-9-10, 9-9-3, 3-9-9, 8-9-4, 4-9-8, 7-9-5, 5-9-7, 6-9-6, 12-8-1, 1-8-12, 11-8-2, 2-8-11, 10-8-3, 3-8-10, 4-8-9, 9-8-4, 8-8-5, 5-8-8, 7-8-6, or 6-8-7. The gapmer may contain 22 nucleotides, and the 5' end and / or 3' end may independently contain about 1 to about 10 nucleotides, about 1 nucleotide to about 9 nucleotides, about 1 to about 8 nucleotides, about 1 to about 7 nucleotides, about 1 to about 6 nucleotides, about 1 to about 5 nucleotides, about 1 to about 4 nucleotides, about 1 to about 3 nucleotides, or about 1 to about 2 nucleotides.For example, gapmer can be 1-20-1, 1-19-2, 2-19-1, 3-18-1, 1-18-3, 2-18-2, 3-17-2, 2-17-3, 1-17-4, 4-17-1, 5-16-1, 1-16-5, 4-16-2, 2-16-4, 3-16-3, 4-15-3, 3-15-4, 2-15-5, 5-15-2, 1-15-6, 6-15-1, 7- 14-1, 1-14-7, 6-14-2, 2-14-6, 5-14-3, 3-14-5, 4-14-4, 8-13-1, 1-13-8, 7-13-2, 2-13-7, 6-13-3, 3-13-6, 5-13-4, 4-13-5, 9-12-1, 1-12-9, 8-12-2, 2-12-8, 7-12-3, 3-12-7, 6-12-4, 4-12-6, 5- 12-5, 10-11-1, 1-11-10, 9-11-2, 2-11-9, 8-11-3, 3-11-8, 7-11-4, 4-11-7, 5-11-6, 6-11-5, 11-10-1, 1-10-11, 10-10-2, 2-10-10, 9-10-3, 3-10-9, 8-10-4, 4-10-8, 7-10-5, 5-10-7, 6-10-6, 12- 9-1, 1-9-12, 11-9-2, 2-9-11, 10-9-3, 3-9-10, 4-9-9, 9-9-4, 8-9-5, 5-9-8, 7-9-6, 6-9-7, 13-8-1, 1-8-13, 12-8-2, 2-8-12, 11-8-3, 3-8-11, 4-8-10, 10-8-4, 9-8-5, 5-8-9, 8-8-6, 6-8-8 or 7-8-7.

[0059] In some embodiments, administration of antisense oligonucleotides to a subject reduces the expression of human APOE mRNA, pre-mRNA, protein, or a combination thereof. Human APOE mRNA may be reduced by approximately 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or more. In some embodiments, administration of antisense oligonucleotides to a subject reduces the expression of human APOE mRNA, pre-mRNA, protein, or a combination thereof. Human APOE pre-mRNA or mRNA may be reduced by approximately 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or more. Human APOE pre-mRNA can be reduced by approximately 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or more. Human APOE pre-mRNA or mRNA can be reduced by approximately 10% or more. Human APOE pre-mRNA or mRNA can be reduced by approximately 30% or more. Human APOE pre-mRNA or mRNA can be reduced by approximately 50% or more. Human APOE mRNA can be reduced by approximately 25% or more. Human APOE mRNA can be reduced by approximately 50% or more. Human APOE mRNA can be reduced by approximately 70% or more. Human APOE protein can be reduced by approximately 25% or more. Human APOE protein can be reduced by approximately 50% or more. Human APOE protein can be reduced by approximately 70% or more.

[0060] In some embodiments, the antisense oligonucleotide can hybridize with human APOE mRNA. The human APOE mRNA may contain at least 80%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical sequences to SEQ ID NO:1. The human APOE mRNA may encode a protein containing approximately 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical sequences to SEQ ID NO:82. APOE may be an allotype of APOE 2, APOE 3, APOE 4, APOE 5f, APOE 5s, and APOE 7. Hybridization can be performed via Watson-Crick base pairing.

[0061] In some embodiments, the antisense oligonucleotide may hybridize with human APOE pre-mRNA. The human APOE pre-mRNA may contain at least 80%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the sequence identical to SEQ ID NO:230. The human APOE pre-mRNA may encode a protein containing approximately 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the sequence identical to SEQ ID NO:82. APOE may be an allotype of APOE 2, APOE 3, APOE 4, APOE 5f, APOE 5s, or APOE 7. Hybridization can be achieved through Watson-Crick base pairing.

[0062] Antisense oligonucleotides can hybridize with APOE mRNA or APOE pre-mRNA containing a single nucleotide polymorphism (SNP). Any SNP can be present in the target APOE mRNA or APOE pre-mRNA (e.g., the mRNA or pre-mRNA ASO described herein hybridizes with), including but not limited to apoE3-R136S (“Christchurch” variant, apoE3-Ch), apoE3-V236E (apoE3-Jacksonville, apoE3-Jac), apoE4-R251G, or any combination thereof. In some embodiments, the APOE mRNA or APOE premRNA contains a single nucleotide polymorphism (SNP), and the SNP is selected from apoE3-R136S (“Christchurch” variant, apoE3-Ch), apoE3-V236E (apoE3-Jacksonville, apoE3-Jac), apoE4-R251G, or any combination thereof.

[0063] Treatment

[0064] This disclosure further relates to methods for treating diseases and disorders (e.g., human diseases and disorders, neurodegenerative diseases) using the oligonucleotides and pharmaceutically relevant salts disclosed herein. Oligonucleotides (such as those described herein) act in a manner different from that of antibodies or small molecules, and oligonucleotides offer significant advantages, for example, in the following ways:

[0065] (i) Due to their significantly smaller size and potential for direct delivery to the brain, they have superior permeability to tissues (such as brain tissue) compared to antibodies (e.g., Noguchi Y et al. (2017) MAbs 9:1210-1215; Pizzo ME et al. (2018) J Physiol 596:445-475); (ii) prolonged retention time and duration of action in the brain after direct administration; (iii) blocking multiple functions and activities of the target, respectively; (iv) combinations of oligonucleotides with each other or with antibodies or small molecules; and (v) inhibiting intracellular effects that antibodies cannot achieve or that cannot be inhibited by small molecules.

[0066] In summary, for Alzheimer's disease (AD) or neurodegenerative diseases in general, there is a clear need for effective treatments that target the underlying pathobiological mechanisms. For patients carrying APOEε4 and diagnosed with AD, DLB, PDD (see Li Y, Macyczko JR, Liu CC, Bu G (2022) Neurobiol Aging 115:20-28) or any other human patients or diseases associated with APOE dysregulation, counteracting the toxic gain-of-function effects of APOE variants may be a therapeutic approach. Specifically, therapeutic effects can be achieved by inhibiting the expression of mRNA encoding APOE. Therapeutic effects can also be achieved by inhibiting the expression of pre-mRNA encoding APOE.

[0067] This paper primarily describes two competitive technologies for the specific inhibition of mRNA expression: siRNA and antisense oligonucleotides (ASOs). siRNA targets mRNA in the cytoplasm. In contrast, RNase H-dependent ASOs target pre-mRNA in the nucleus. Therefore, intron regions inaccessible to siRNA can also be targeted by ASOs, resulting in a significantly larger target space for ASOs. Furthermore, due to their double-stranded nature, siRNAs themselves do not cross the cell membrane and require a delivery system to achieve their activity in vitro and in vivo. While efficient siRNA delivery systems exist for in vivo delivery of siRNA to hepatocytes, no system currently delivers siRNA to extrahepatic tissues with sufficient efficacy in vivo. In contrast, modified ASOs can enter multiple cell types in sufficient quantities in vitro without transfection or delivery methods, resulting in potent and sequence-specific target knockdown. In vivo, unconjugated naked ASOs achieved target knockdown in several different relevant tissues after systemic administration. Following direct administration, ASO achieved potent and durable target knockdown in the central nervous system (CNS), demonstrating its strong potential in treating CNS diseases such as neurodegenerative diseases (see Geary RS et al. (2015) Adv DrugDeliv Rev 87:46-51).

[0068] In some embodiments, this disclosure relates to a method of treating a disease or disorder in a human subject in need, the method comprising administering to the subject in need an antisense oligonucleotide as described herein or a pharmaceutically acceptable salt thereof.

[0069] In another aspect, this disclosure relates to methods for treating neurodegenerative diseases, said methods comprising administering to a subject in need an antisense oligonucleotide as described herein or a pharmaceutically acceptable salt thereof. Neurodegenerative diseases may be Alzheimer's disease, amyotrophic lateral sclerosis (ALS), Huntington's disease, Lewy body dementia, multiple sclerosis, multiple system atrophy, Parkinson's disease, prion diseases, or combinations thereof. Neurodegenerative diseases may include Alzheimer's disease. Neurodegenerative diseases may include tau protein diseases. Tau protein diseases can include primary age-related tau protein disease, chronic traumatic encephalopathy, progressive supranuclear palsy, corticobasal degeneration, chromosome 17-linked frontotemporal dementia with Parkinson's syndrome, vacuolet tau protein disease, lytico-bodig disease, ganglioglioma, gangliocytoma, meningeal angiomatosis, post-encephalitis Parkinson's syndrome, subacute sclerosing panencephalitis, lead poisoning encephalopathy, tuberous sclerosis, pantothenic kinase-related neurodegeneration, lipofuscinosis, behaviorally variant frontotemporal dementia (Pick's disease), argyrophilic granulation disease, or combinations thereof.

[0070] In another aspect, this disclosure relates to methods for treating acute or chronic injuries, said methods comprising administering to a subject in need an antisense oligonucleotide as described herein or a pharmaceutically acceptable salt thereof. Acute or chronic injuries can be any neurological injury, including but not limited to diffuse axonal injury, concussion, edema, hematoma, skull fracture, hemorrhage, hypoxic brain injury, hypoxic brain injury, carbon monoxide poisoning, cerebral aneurysm, stroke, hydrocephalus, traumatic brain injury, spinal cord injury, chronic traumatic encephalopathy, chemotherapy-induced brain, neuronal injury caused by tumors, neuronal injury caused by meningitis, encephalitis, and other inflammations of the body or nervous system, or combinations thereof.

[0071] In another aspect, this disclosure relates to a method for treating acute or chronic inflammatory conditions of the nervous system or peripheral regions of the body, the method comprising administering to a subject in need an antisense oligonucleotide as described herein or a pharmaceutically acceptable salt thereof. Acute or chronic inflammatory conditions can be autoimmune diseases, multiple sclerosis, infectious diseases, immune responses to cancer, tumors, or combinations thereof.

[0072] In another aspect, this disclosure relates to methods for treating vascular diseases or vascular pathological conditions, said methods comprising administering to a subject in need an antisense oligonucleotide as described herein or a pharmaceutically acceptable salt thereof. Vascular diseases or vascular pathological conditions can be any human vascular disease, including but not limited to cerebrovascular diseases, cerebral small vessel diseases, diseases caused by or affected by leakage of the blood-brain barrier, atherosclerosis, cerebral amyloid angiopathy, peripheral vascular diseases, peripheral artery diseases, carotid artery diseases, pulmonary embolism, abdominal aortic aneurysm, collagen vascular disease, chronic venous insufficiency, thrombosis, deep vein thrombosis, or combinations thereof.

[0073] In another aspect, this disclosure relates to a method for treating lipid storage disorders, the method comprising administering to a subject in need an antisense oligonucleotide as described herein or a pharmaceutically acceptable salt thereof. Lipid storage disorders can be tendon xanthomas, Fabry disease, Fabry disease, fucoside storage disorders, Gaucher disease, GM1 ganglioside storage disorders, GM2 ganglioside storage disorders AB variant, Krabby disease, metachromatic leukodystrophy, multiple sulfatase deficiencies, Niemann-Pick disease type A, Niemann-Pick disease type B, Niemann-Pick disease type C, Sankhov disease, Schindler disease, Ty-Sachs disease, Wollman disease, or combinations thereof.

[0074] In another aspect, this disclosure relates to the use of antisense oligonucleotides or pharmaceutically acceptable salts thereof, as described herein, in the manufacture of medicaments for the therapeutic treatment of diseases or disorders in human subjects in need.

[0075] In another aspect, this disclosure relates to the use of antisense oligonucleotides or pharmaceutically acceptable salts thereof, as described herein, for the treatment of diseases or disorders in human subjects in need.

[0076] In another aspect, this disclosure relates to the use of antisense oligonucleotides or pharmaceutically acceptable salts thereof, as described herein, in the manufacture of medicaments for the therapeutic treatment of neurodegenerative diseases in subjects in need.

[0077] In another aspect, this disclosure relates to the use of antisense oligonucleotides or pharmaceutically acceptable salts thereof, as described herein, for the treatment of neurodegenerative diseases in subjects of need. Neurodegenerative diseases may include Alzheimer's disease, amyotrophic lateral sclerosis (ALS), Huntington's disease, Lewy body dementia, multiple sclerosis, multiple system atrophy, Parkinson's disease, prions, or combinations thereof. Neurodegenerative diseases may include Alzheimer's disease. Neurodegenerative diseases may include tau protein diseases. Tau protein diseases can include primary age-related tau protein disease, chronic traumatic encephalopathy, progressive supranuclear palsy, corticobasal degeneration, chromosome 17-linked frontotemporal dementia with Parkinson's syndrome, vacuolet tau protein disease, lytico-bodig disease, ganglioglioma, gangliocytoma, meningeal angiomatosis, post-encephalitis Parkinson's syndrome, subacute sclerosing panencephalitis, lead poisoning encephalopathy, tuberous sclerosis, pantothenic kinase-related neurodegeneration, lipofuscinosis, behaviorally variant frontotemporal dementia (Pick's disease), argyrophilic granulation disease, or combinations thereof.

[0078] In some embodiments, the oligonucleotide or a pharmaceutically acceptable salt thereof is administered topically to a subject in need. Topical administration may be intracranial, intracerebral, intramuscular, spinal, epidural, sacroiliac, or subdural injection.

[0079] In some implementations, oligonucleotides or pharmaceutically acceptable salts thereof are administered systemically to subjects in need.

[0080] Example

[0081] The following examples illustrate non-limiting embodiments of the invention. The experiments were conducted on cells endogenously expressing APOE, i.e., cells that do not represent artificial systems containing transfected reporter constructs. Compared to endogenous systems that are closer to treatment-related in vivo systems, such artificial systems generally exhibited higher levels of inhibition and lower IC50 values.

[0082] In some embodiments, this disclosure provides antisense oligonucleotide compounds that target human APOE nucleic acid. In some embodiments, the human APOE nucleic acid is the APOE3 variant sequence shown in NCBI reference sequence NM_000041.4.

[0083]

[0084]

[0085] MKVEQAVETEPEPELRQQTEWQSGQRWELALGRFWDYLRWVQTLSEQVQEELLSSQVTQELRALMDETMKELKAYKSELEEQLTPVAEETRARLSKELQAAQARLGADMEDVCGRLVQYRGEVQAMLGQSTEELRVRLASHLRKLRKRLLRD ADDLQKRLAVYQAGAREGAERGLSAIRERLGPLVEQGRVRAATVGSLAGQPLQERAQAWGERLRARMEEMGSRTRDRLDEVKEQVAEVRAKLEEQAQQIRLQAEAFQARLKSWFEPLVEDMQRQWAGLVEKVQAAVGTSAAPVPSDNH(SEQ ID NO:82)

[0086] In some embodiments, this disclosure provides antisense oligonucleotide compounds that target human APOE pre-mRNA. An exemplary DNA sequence of APOE pre-mRNA is as follows:

[0087]

[0088]

[0089]

[0090]

[0091]

[0092]

[0093] In the APOE 3 pre-mRNA, exons are indicated by uppercase letters, while introns are not. The sequence contains a 5' untranslated region (5'UTR). The sequence was obtained from the Genome Explorer at the University of California, Santa Cruz Genome Institute, with the identifier: hg38 ENST00000252486.9.

[0094] This disclosure relates to oligonucleotides comprising about 18 to 20 nucleotides, at least one nucleotide having a modification selected from: bridging nucleic acids (such as LNA, ENA, cET), 2'-fluorinated nucleotides, 2O-methylated nucleotides, 2O-methoxylated nucleotides, 2'-fluoroarabinonucleotides (FANA), and combinations thereof, wherein the modification is located in a sequence of five nucleotides at the 5' end and / or 3' end of the oligonucleotide, and wherein the oligonucleotide hybridizes with the APOE transcript or mRNA of SEQ ID NO:1. Oligonucleotides, for example, hybridize outside or within the hybridization active regions at positions 39, 41, 42, 77, 84, 124, 128, 130, 131, 204, -206, 208-214, 226-228, 295-296, 298-299, 307-309, 311, 339, 341-348, 447, 541-542, 550, 573-576, 580, 589, 592, 809, 812, 901-905, 1000-1004, 1006-1014, 1016-1017, 1019-1020, 1122-1123, and 1145-1147.

[0095] This disclosure relates to oligonucleotides comprising about 14 to 18 nucleotides, at least one nucleotide having a modification selected from: bridging nucleic acids (e.g., LNA, ENA, cET, 2'-fluoro-modified nucleotides, 2O-methyl-modified nucleotides, 2O-methoxy-modified nucleotides, 2'-fluoroarabinonucleotides (FANA) and combinations thereof), wherein the modification is located in a sequence of 5 nucleotides at the 5' end and / or 3' end of the oligonucleotide, and wherein the oligonucleotide hybridizes with the APOE transcript or pre-mRNA of SEQ ID NO:230. Oligonucleotides, for example, at positions 45, 90, 127, 268, 269, 272, 273, 275, 276, 297, 298, 301, 319, 343, 344, 346, 347, 354, 380, 381, 384, 390, 391, 392, 394, 399, 400, 440, 444, 445, 446, 447, 448, 450, 451, 452, 453, 454, 456, 459, 462, 46 3, 464, 465, 485, 488, 489, 490, 492, 586, 769, 770, 788, 915, 916, 917, 919, 966, 967, 968, 969, 1005, 1006, 1007, 1008, 1009, 1036, 1037, 1327, 1328, 1329, 1330, 1331, 1333, 1334, 1335, 1336, 1386, 1424, 1425, 1 491, 1725, 1726, 1727, 1728, 1746, 1823, 1855, 1856, 1857, 1875, 1898, 1899, 1900, 1902, 1904, 1905, 1906, 1925, 1950, 1951, 1985, 2148, 2150, 2151, 2211, 2233, 2234, 2235, 2236, 2260, 2261, 2347, 2348, 2355 Hybridization outside or within the hybridization active region of 2356, 2357, 2358, 2359, 2360, 2362, 2363, 2364, 2699, 2700, 2704, 2743, 2775, 2777, 2778, 2779, 2780, 2781, 3337, 3441, 3442, 3443, 3444, 3445, 3447, 3448, 3449, 3453, 3455, 3582, or 3583.

[0096] Modified oligonucleotides complementary to APOE nucleic acids can be designed, and their effects on APOE mRNA can be tested in vitro. The modified oligonucleotides can be tested in a series of experiments with similar culture conditions.

[0097] The modified oligonucleotides in the table below can be uniformly modified oligonucleotides. The length of the oligonucleotide can be 21 nucleotides, and each nucleoside can have 2'-substitution or modification as described herein.

[0098] The modified oligonucleotides in Tables 1 and 2 below can be designed as gapmers. The gapmer can be 20 nucleotides long, with a central gap containing 10 2'-deoxynucleotides and flanking segments at both the 5' and 3' ends, each flanking segment containing five nucleotides (5-10-5 gapmers, except for ASO S1095 (SEQ ID NO:81), which is a 4-10-4 gapmer, Table 1). Each nucleotide in the 5' flanking segment and each nucleotide in the 3' flanking segment contains a 2'-modification. The 2'-modification is a 2'-methoxyethyl (e.g., 2'-MOE) modification.

[0099] In the implementation scheme, each nucleoside link is a thiophosphate nucleoside link.

[0100] Table 1. Target locations on ASO sequences and APOEs

[0101]

[0102]

[0103]

[0104]

[0105]

[0106]

[0107] Table 2. Selected ASOs

[0108]

[0109]

[0110] The modified oligonucleotides in Tables 3 and 4 below can be uniformly modified oligonucleotides. The length of the oligonucleotide can be 16 nucleotides, and each nucleoside can have a 2'-substitution or -modification as described herein.

[0111] The modified oligonucleotides in Tables 3 and 4 below can be designed as gapmers. The gapmer can be 16 nucleotides long, with a central gap containing 10 2'-deoxynucleotides and flanking segments at both the 5' and 3' ends, each flanking segment containing three nucleotides (3-10-3 gapmer). Each nucleotide in the 5' flanking segment and each nucleotide in the 3' flanking segment contains locked nucleic acid (LNA) modification.

[0112] In the implementation scheme, each nucleoside link is a thiophosphate nucleoside link.

[0113] Table 3. Target sites on ASO sequence and pre-APOE mRNA

[0114]

[0115]

[0116]

[0117]

[0118]

[0119]

[0120] Table 4. Selected ASOs targeting APOE pre-mRNA, with a >30% reduction in APOE mRNA compared to SEQ ID NO:83.

[0121]

[0122]

[0123] Table 5. Selected ASOs targeting APOE pre-mRNA, relative to HPRT1 gene-normalized APOE mRNA knockdown > 10％

[0124] ASO ID: SEQ ID NO: S1147 127 S1152 132 S1167 147 S1222 202 S1240 220

[0125] Example 1: Evaluation of the efficacy and toxicity of APOE-specific ASO knockdown in HEP3B cells

[0126] A detailed description of the methods used in this embodiment can be found in the Supplementary Experiments section below. In short, the knockdown efficacy of 79 APOE-specific ASOs in human HEP3B cells was evaluated in vitro. This was achieved using LIPOFECTAMINE. TMCells transfected with APOE (THERMO FISH ER SCIENTIFIC INC., 11668030) were treated with either APOE-specific ASO (12.5 nM) or a non-silencing control oligonucleotide (12.5 nM). Cells were lysed after 48 hours of treatment. APOE and HPRT1 mRNA levels were analyzed using quantitative real-time polymerase chain reaction (qPCR), and APOE expression values ​​were normalized relative to the endogenous housekeeping control gene HPRT1. Table 1 shows the knockdown results of APOE and HPRT1 in HEP3B cells as residual APOE or HPRT1 mRNA expression relative to the non-silencing control ASO (normalized to 1) used as a reference sample.

[0127] Treatment of HEP3B cells with ASOs S1043, S1042, S1044, S1041, and S1025 (SEQ ID NO: 11, 27-30) achieved >80% knockdown (expressed as residual APOE mRNA expression <0.2 compared to cells treated with non-targeted control ASOs) (see Table 1)). Notably, not all screened ASOs efficiently downregulated APOE mRNA. Some ASOs (e.g., ASOs S1017, S1027, S1033, S1018, and S1065 (SEQ ID NO: 3, 4, 13, 19, and 51)) downregulated target gene expression by approximately 30% compared to non-targeted control ASOs, indicating that our screening experiments identified target mRNA knockdown potency across 79 tested ASOs, ranging from approximately 30% to approximately 90% of the control reference value.

[0128] The altered HPRT1 housekeeping gene expression level in response to ASO treatment (i.e., transfection) was used as a substitute for identifying ASO that exhibited cytotoxicity under the experimental conditions employed. The use of HPRT1 readings as an approximation of cytotoxicity was validated by live-cell imaging of HEP3B cell cultures. Figure 1A , Figure 1B and Figure 1C The culture treated with ASO S1094 (SEQ ID NO:80) (see Table 1) without downregulating HPRT1 housekeeping gene expression showed that... Figure 1C It is non-toxic, as determined by images, its confluence and cell morphology are similar to those of cell cultures not treated with ASO. Figure 1A The results were comparable. In contrast, cell cultures treated with ASOs that downregulated HPRT1 housekeeping gene expression (e.g., ASO S1038 (SEQ ID NO:24)) (see Table 1) did not congregate, and many cells exhibited abnormal morphology and dense, condensed nuclei, indicating cytotoxicity and cell death. Figure 1BSince both ASO S1094 (SEQ ID NO:80) and S1038 (SEQ ID NO:24) have comparable efficiency in downregulating the target gene APOE (see Table 1, downregulation of approximately 70% to approximately 80%), the observed morphological changes and HPRT1 mRNA changes consistent with toxicity are not related to APOE downregulation.

[0129] Based on observations of HPRT1 housekeeping gene downregulation consistent with confluence and cell morphology (e.g., microscopic indicators of cytotoxicity), 79 screened molecules were further classified in a binary manner. Under the test conditions, ASOs that reduced HPRT1 mRNA in HEP3B cells by more than 20% were considered cytotoxic; conversely, ASOs that regulated HPRT1 mRNA by less than 20% were considered non-toxic. Table 1 summarizes the HPRT1 mRNA expression levels after transfection of HEP3B cells with ASOs. ASOS1024, S1088, S1047, S1077, and S1059 (SEQ ID NO: 10, 33, 45, 63, and 74) resulted in >70% robust inhibition of the APOE target (e.g., knockdown of APOE mRNA) without cytotoxic effects on HPRT1 housekeeping gene expression. In contrast, ASO S1043, S1042, S1044, S1041 and S1025 (SEQ ID NO: 11, 27, 28, 29 and 30) showed cytotoxic effects, resulting in a decrease in HPRT1 housekeeping gene expression in HEP3B cells ranging from about 25% to about 40%, although they downregulated APOE mRNA very effectively.

[0130] In summary, 37 ASOs were identified (see Table 2) that downregulated APOE target gene mRNA under experimental conditions without causing significant cytotoxicity (e.g., no change in confluence or morphology compared to the non-silencing control) (see Table 2). Figure 1A and Figure 1C Further assays can be used to demonstrate the potency and efficacy of these molecules in vitro and in vivo, as well as other characteristics.

[0131] Supplementary experimental section:

[0132] This section describes the specific techniques and reagents used in one or more of the above embodiments in detail.

[0133] Preparation of antisense oligonucleotides (ASO): ASO was synthesized by Integrated DNA Technologies (IDT) using its standard desalting process.

[0134] Cell Culture: HEP3B cells (ATCC HB-8064) were maintained at 37°C and 5% CO2 in growth medium containing EMEM (ATCC 30-2003) with 10% FBS (VWR 97068-085) and 1% w / w penicillin and 1% w / w streptomycin (Pen Strep) (THERMO FISHER SCIENTIFIC INC. 15140122). Cell dissociation was performed using 0.25% trypsin-EDTA (THERMO FISHER SCIENTIFIC INC. 25200-114). For ASO selection, HEP3B cells were seeded at 15,000 cells per well in 96-well plates in growth medium without Pen Strep.

[0135] ASO transfection using Lipofectamine 2000: Each ASO was prepared in ULTRAPURE TM The stock solution was prepared in 5 μM of DNase / RNase-free distilled water (THERMO FISHER INC., 10977023) and stored at -80°C until use. Transfection was performed in 96-well plates, with three wells transfected at a final ASO concentration of 12.5 nM each time, as a biological triplet. One day after cell seeding, the medium in each well was replaced with 90 μL of Pen Strep-free growth medium. For each ASO, ULTRAPURE was used. TM Pre-dilute the 5 μM stock solution (5 μM) to 1.25 μM with DNase-free / RNase-free distilled water. Use OPTI-MEM. TM (THERMO FISHER INC., 31985062) The pre-diluted ASO was further diluted to 0.25 μM. OPTI-MEM was used. TM LIPOFECTAMINE TM Pre-dilute 2000 (THERMO FISHER SCIENTIFIC INC., 11668030) to 6% (v / v) and incubate for 5 minutes. Add an equal volume of OPTI-MEM. TM 0.25 μM ASO and OPTI-MEM TM 6% (v / v) LIPOFECTAMINE TMMix 2000 (THERMO FISHER SCIENTIFIC INC., 11668030) (v / v) and incubate for 10 minutes. Add 10 μl of the mixture to each well, making a total volume of 100 μl per well. For the non-transfection control, all the above steps are the same, except for adding OPTI-MEM. TM The 0.25 μM ASO was replaced with a separate OPTI-MEM. TM By using The platform (SARTORIUS AG) performed live-cell imaging on each well at 0, 24, and 48 hours post-transfection to determine cell confluence as an alternative biomarker for monitoring cell health and potential toxicity of ASO treatment.

[0136] Cell lysis and quantitative RT-PCR: 48 hours post-transfection, according to CELLS-TO-CT... TM 1-Step TAQMAN TM Lyse cells according to the instructions of the kit (THERMO FISHER SCIENTIFIC, INC. A25602). Take 50 μl of RNA lysate from each well and store at -80°C until RT-qPCR is performed. Perform a single-step RT-qPCR in a 384-well plate, with each reaction consisting of a 10 μl mixture containing 2.5 μl of TAQMAN. TM 1-step qRT-PCR mixture (THERMO FISHERSCIENTIFIC, INC.), 0.5 μl APOE TAQMAN TM Determination of FAM-MGB Hs00171168_m1 (THERMO FISHERSCIENTIFIC, INC. 4331182), 0.5 μl HPRT1TAQMAN TM VIC-MGB Hs02800695_m1 (THERMOFISHER SCIENTIFIC, INC. 4448489), 1 μl of RNA lysate, and 5.5 μl of UltraPure were measured. TMDistilled water free of DNase / RNase. The thermal cycling method was as follows: 50°C for 5 minutes, 95°C for 20 seconds, followed by 40 cycles: i) 95°C for 3 seconds, ii) 60°C for 30 seconds. Cycle threshold (Ct) values ​​were analyzed using the ΔΔCt method, with HRPT1 as the housekeeping gene and the non-silenced control ASO or untransfected control as reference samples. Differences between culture plates were controlled by including an untransfected control, a non-silenced control ASO, and an APOE knockdown positive control (S1087, SEQ ID NO:73) identified from pilot screening in each transfection protocol. Normalization between culture plates was performed using the "percentage of positive controls" method, as described in Malo N, Hanley JA, Cerquozzi S, Pelletier J, Nadon R (2006) Statistical practice in high-throughput screening data analysis. Nat Biotechnol 24:167-175.

[0137] Example 2: Evaluation of the efficacy and toxicity of APOE pre-mRNA-specific ASO in HEP3B cells for target knockdown.

[0138] A detailed description of the methods used in this embodiment can be found in the Supplementary Experiments section below. Unless otherwise stated, all other methods used in this embodiment are similar to those presented in Example 1 above. Briefly, the knockdown efficacy of 147 ASOs targeting APOE pre-mRNA in human HEP3B cells was evaluated in vitro. Cells were treated with ASOs targeting APOE pre-mRNA (5 μM) or the non-silencing control oligonucleotide S1250 (5 μM, SEQ ID NO: 83) via gymnotic delivery. Cells were lysed after 72 hours of treatment. APOE and HPRT1 mRNA levels were analyzed using quantitative real-time polymerase chain reaction (qPCR). The knockdown results of APOE and HPRT1 in HEP3B cells by ASOs targeting APOE pre-mRNA are shown in Table 3 as residual APOE or HPRT1 mRNA expression relative to the non-silencing control ASO (normalized to 1) used as a reference sample.

[0139] Using ASO S1104, S1105, S1108, S1110, S1111, S1114, S1116, S1120, S1123, S1124, S1128, S1131, S1133, S1134, S1135, S1139, S1140, S1143, S1144, S1149, S1150, S1151, S S1153, S1154, S1155, S1156, S1158, S1170, S1171, S1177, S1183, S1184, S1188, S1190, S1195, S1196, S1200, S1205, S1230, S1232, S1236, S1239, S1248 and S1249 (SEQ) Treatment of HEP3B cells with ID NO: 84, 85, 88, 90, 91, 94, 96, 100, 103, 104, 108, 111, 113, 114, 115, 119, 120, 123, 124, 129, 130, 131, 133, 134, 135, 136, 138, 150, 151, 157, 163, 164, 168, 170, 175, 176, 180, 185, 210, 212, 216, 219, 228, and 229 (e.g., ASO in Table 4) achieved a knockdown of >30% (expressed as <0.7 residual APOE mRNA expression compared to cells treated with the non-targeted control ASO) (see Table 3). >50% knockdown (indicated as <0.5% residual APOE mRNA expression compared to cells treated with non-targeted control ASO) was achieved by naked transfusion of HEP3B cells with ASO S1104, S1139, S1140, S1143, S1144, S1149, S1153, S1154, S1155, S1156, S1188, S1205, S1248 and S1249 (SEQ ID NO: 84, 119, 120, 123, 124, 129, 133, 134, 135, 136, 168, 185, 228 and 229) (see Table 3). Compared with cells treated with non-targeted control ASOs, some ASOs S1144, S1153, S1154, S1156, and S1188 (SEQ ID NO: 124, 133, 134, 136, and 168) resulted in a >60% reduction in HPRT1 mRNA. No visible cell death or morphological changes indicated any experimental or biological alteration in HPRT1 mRNA expression in the context of naked ASO delivery.

[0140] Supplementary experimental section:

[0141] This section describes the reagents, methods, and details regarding the specific techniques used in Example 2.

[0142] Treatment with LNA-modified ASO: Each ASO was prepared in ULTRAPURE TM The stock solution was prepared in 500 μM of DNase / RNase-free distilled water (THERMO FISHER INC., 10977023) and stored at -80°C until use. Treatments were performed in 96-well plates, with each treatment at a final ASO concentration of 5 μM in 3 wells, for a biological triplet. For each ASO, ULTRAPURE was used. TM Pre-dilute the 500 μM stock solution to 250 μM with DNase-free / RNase-free distilled water. One hour after cell plating, add 2 μl of pre-diluted ASO to each well. (The solution was then processed using INCUCY.) The platform (SARTORIUSAG) performed live-cell imaging on each well 72 hours after treatment to determine cell confluence as an alternative biomarker for monitoring cell health and potential toxicity of ASO treatment.

[0143] Cell lysis and quantitative RT-PCR: 72 hours after treatment (naked transfection), according to CELLS-TO-CT TM 1-Step TAQMAN TM Lyse cells according to the instructions of the kit (THERMO FISHER SCIENTIFIC, INC. A25602). Take 50 μl of RNA lysate from each well and store at -80°C until RT-qPCR is performed. Perform a single-step RT-qPCR in a 384-well plate, with each reaction consisting of a 10 μl mixture containing 2.5 μl of TAQMAN. TM 1-step qRT-PCR mixture (THERMO FISHER SCIENTIFIC, INC.), 0.5 μl APOE TAQMAN TM Determination of FAM-MGB Hs00171168_m1 (THERMO FISHER SCIENTIFIC, INC. 4331182), 0.5 μl HPRT1 TAQMAN TM VICMGBHs02800695_m1 (THERMO FISHER SCIENTIFIC, INC. 4448489), 1 μl of RNA lysate, and 5.5 μl of UltraPure were measured. TMDistilled water free of DNase / RNase. The thermal cycling method was as follows: 50°C for 5 minutes, 95°C for 20 seconds, followed by 40 cycles: i) 95°C for 3 seconds, ii) 60°C for 30 seconds. Cycle threshold (Ct) values ​​were analyzed using the ΔΔCt method, with HRPT1 as the housekeeping gene and the non-silenced control ASO or treatment control as the reference sample. Differences between culture plates were controlled by including a non-silenced control ASO (S1250 (SEQ ID NO: 83)) and an APOE knockdown assay control ASO (S1248 (SEQ ID NO: 228)) in each culture plate. Normalization between culture plates was performed using the "percentage of positive controls" method, as described in Malo N, Hanley JA, Cerquozzi S, Pelletier J, Nadon R (2006) Statistical practice in high-throughput screening data analysis. Nat Biotechnol 24:167-175.

[0144] definition

[0145] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. While any methods and materials similar to or equivalent to those described and used herein may be used in the practice or testing of this invention, preferred materials and methods are described herein. The following terms will be used in describing the invention and asserting its rights.

[0146] It will also be understood that the terminology used herein is for the purpose of describing particular implementations only and is not intended to be restrictive.

[0147] The articles “a” and “an” are used in this text to refer to one or more grammatical objects of the article (i.e., at least one / at least one). By way of example, “a component” means one component or more components.

[0148] As used herein, “about” when referring to measurable values ​​(such as quantity, duration of time, etc.) is intended to cover variations of 20% or 10%, more preferably 5%, even more preferably 1%, and still more preferably 0.1% from the specified value, because such variations are appropriate for carrying out the disclosed method.

[0149] The terms “effective amount” or “therapeutic effective amount” are used interchangeably herein and refer to the amount of a compound, formulation, material, or composition as described herein that effectively achieves a particular biological outcome or provides a therapeutic or preventive benefit.

[0150] "Encoding" refers to the inherent property of a specific nucleotide sequence in a polynucleotide (such as a gene, cDNA, or mRNA) that serves as a template for the synthesis of other polymers and macromolecules with defined nucleotide sequences (i.e., rRNA, tRNA, and mRNA) or defined amino acid sequences in biological processes, and the resulting biological properties. Therefore, if the transcription and translation of mRNA corresponding to a gene produces a protein in a cell or other biological system, then that gene encodes that protein. Both the coding strand (whose nucleotide sequence is identical to the mRNA sequence and is usually provided in the sequence listing) and the non-coding strand (which serves as a template for gene or cDNA transcription) can be referred to as encoding the protein or other product of that gene or cDNA.

[0151] Unless otherwise stated, "nucleotide sequences encoding amino acid sequences" includes all nucleotide sequences that are degenerate forms of each other and encode the same amino acid sequence. Phrases encoding protein or RNA nucleotide sequences may also include introns, provided that the protein-encoding nucleotide sequence may contain one or more introns in some form.

[0152] As used herein, the terms “peptide,” “polypeptide,” and “protein” are used interchangeably and refer to compounds containing amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and there is no limit to the maximum number of amino acids that can constitute a protein or peptide sequence. Polypeptides include any peptide or protein containing two or more amino acids linked together by peptide bonds. As used herein, the term refers both to short chains, which are commonly referred to in the art as, for example, peptides, oligopeptides, and oligomers; and to long chains, which are commonly referred to in the art as proteins, which come in many types. “Polypeptide” particularly includes, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, polypeptide variants, modified polypeptides, derivatives, analogs, and fusion proteins. Polypeptides include native peptides, recombinant peptides, synthetic peptides, or combinations thereof.

[0153] The term "subject" is intended to include any living organism (e.g., a mammal) capable of evoking an immune response. As used herein, "subject" or "patient" can be a human or a non-human mammal. Non-human mammals include, for example, livestock and pets such as sheep, cattle, pigs, dogs, cats, and rodents. Preferably, the subject is a human.

[0154] Scope: Throughout this disclosure, various aspects of the invention can be presented in the form of a scope. It should be understood that the scope description is merely for convenience and brevity and should not be construed as a rigid limitation on the scope of the invention. Therefore, the scope description should be considered to explicitly disclose all possible sub-scopes and individual numerical values ​​within that scope. For example, a scope description (such as 1 to 6) should be considered to explicitly disclose sub-scopes such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc.; and individual numbers within that scope, such as 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the width of the scope.

Claims

1. An antisense oligonucleotide or a pharmaceutically acceptable salt thereof, said antisense oligonucleotide or a pharmaceutically acceptable salt thereof comprising at least 80% identical sequence to SEQ ID NO:5-11, 15, 16, 20-41, 43, 45, 47-50, 53, 55-57, 60-75 or 77-81.

2. An antisense oligonucleotide or a pharmaceutically acceptable salt thereof, said antisense oligonucleotide or a pharmaceutically acceptable salt thereof comprising a sequence selected from the following: SEQ ID NO: 5-11, 15, 16, 20-41, 43, 45, 47-50, 53, 55-57, 60-75 and 77-81.

3. The antisense oligonucleotide of claim 1 or 2, or a pharmaceutically acceptable salt thereof, wherein the sequence is selected from SEQ ID NO: 7, 8, 10, 15, 32, 33, 35, 36, 37, 38, 39, 40, 41, 43, 45, 49, 56, 57, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 71, 71, 72, 73, 74, 77, 78, 79, 80, and 81.

4. The antisense oligonucleotide or a pharmaceutically acceptable salt thereof as described in any one of claims 1-3, wherein the antisense oligonucleotide or a pharmaceutically acceptable salt thereof comprises about 15 to about 25 nucleobases.

5. The antisense oligonucleotide or a pharmaceutically acceptable salt thereof as claimed in any one of claims 1-4, wherein the antisense oligonucleotide or a pharmaceutically acceptable salt thereof comprises about 18 to about 22 nucleobases.

6. The antisense oligonucleotide or a pharmaceutically acceptable salt thereof as described in any one of claims 1-5, wherein the antisense oligonucleotide comprises about 20 nucleobases.

7. The antisense oligonucleotide or a pharmaceutically acceptable salt thereof as described in any one of claims 1-5, wherein the antisense oligonucleotide comprises about 18 nucleobases.

8. The antisense oligonucleotide as described in any of the preceding claims, or a pharmaceutically acceptable salt thereof, wherein the antisense oligonucleotide comprises one or more nucleoside linkage modifications.

9. The antisense oligonucleotide of claim 8 or a pharmaceutically acceptable salt thereof, wherein the internucleotide linkage modification is selected from thiophosphates, dithiophosphates, methylphosphonates, methylthiophosphates, aminophosphates, diaminophosphates, thioaminophosphates, methanesulfonylaminophosphates, and combinations thereof.

10. The antisense oligonucleotide of claim 8 or a pharmaceutically acceptable salt thereof, wherein the antisense oligonucleotide comprises one or more phosphate thioester nucleosides linked together.

11. The antisense oligonucleotide of claim 8 or a pharmaceutically acceptable salt thereof, wherein all said nucleoside linkages are phosphate thioester linkages.

12. An antisense oligonucleotide or a pharmaceutically acceptable salt thereof as described in any of the preceding claims, wherein the antisense oligonucleotide comprises DNA.

13. The antisense oligonucleotide or a pharmaceutically acceptable salt thereof as described in any one of claims 1-11, wherein the antisense oligonucleotide comprises RNA.

14. The antisense oligonucleotide or a pharmaceutically acceptable salt thereof as claimed in any one of claims 1-11, wherein the antisense oligonucleotide comprises proteins and / or polypeptides.

15. The antisense oligonucleotide or a pharmaceutically acceptable salt thereof as claimed in any one of claims 1-11, wherein the antisense oligonucleotide comprises a small molecule.

16. The antisense oligonucleotide or a pharmaceutically acceptable salt thereof as described in any of the preceding claims, wherein the antisense oligonucleotide comprises one or more modified nucleosides.

17. The antisense oligonucleotide of claim 16, wherein the modified nucleoside is selected from 2'-modified nucleosides, 4'-modified nucleosides, bridging nucleosides, diaminophosphate morpholine, locked nucleic acids, ethylene-bridging nucleic acids, ethylene glycol nucleic acids, hexitol nucleic acids, cyclohexene nucleic acids, arabinose nucleic acids, peptide nucleic acids, threonose nucleic acids, tricyclic-2'-deoxynucleotides, 1'-deoxypentose, 1',2'-dideoxypentose, 2',3'-dideoxypentose, 2',3'-didehydro-2',3'-dideoxypentose, unlocked nucleic acids, and combinations thereof.

18. The antisense oligonucleotide or a pharmaceutically acceptable salt thereof as described in any one of claims 1-17, wherein the antisense oligonucleotide comprises one or more 2'-nucleoside modifications.

19. The antisense oligonucleotide of claim 18, wherein the antisense oligonucleotide comprises at least eight 2'-nucleoside modifications.

20. The antisense oligonucleotide of claim 18, wherein the antisense oligonucleotide comprises at least ten 2'-nucleoside modifications.

21. The antisense oligonucleotide of claim 18 or a pharmaceutically acceptable salt thereof, wherein all nucleosides of the antisense oligonucleotide contain a 2'-nucleoside modification.

22. The antisense oligonucleotide of any one of claims 18-21 or a pharmaceutically acceptable salt thereof, wherein the 2'-nucleoside modification is selected from 2'-fluoro-nucleoside, 2'-O-methyl-nucleoside, 2'-O-methoxyethyl-nucleoside, 2'-O-benzyl-2'-deoxy-nucleoside, 2'-O-methyl-4-pyridyl-nucleoside (2'-O-CH2Py(4)), 2'-amino-nucleoside, and combinations thereof.

23. The antisense oligonucleotide or a pharmaceutically acceptable salt thereof as described in any of the preceding claims, wherein the antisense oligonucleotide comprises one or more modified nucleobases.

24. The antisense oligonucleotide of claim 23 or a pharmaceutically acceptable salt thereof, wherein the modified nucleobase is a methylated nucleobase.

25. The antisense oligonucleotide of claim 24 or a pharmaceutically acceptable salt thereof, wherein the methylated nucleobase is selected from 5-methyluracil, N6-methyladenine, N4-methylcytosine, N7-methylguanine, 5-hydroxymethylcytosine, N3-methylcytosine, and combinations thereof.

26. The antisense oligonucleotide of claim 23 or a pharmaceutically acceptable salt thereof, wherein the modified nucleobase is an acetylated nucleobase.

27. The antisense oligonucleotide of claim 26 or a pharmaceutically acceptable salt thereof, wherein the acetylated nucleobase is N4-acetylcytosine.

28. The antisense oligonucleotide of claim 23 or a pharmaceutically acceptable salt thereof, wherein the modified nucleobase is selected from pseudouridine (Ψ), N1-methyl-pseudouridine (N1-methyl-Ψ), 2-thiouridine (s2U), 5-fluoro-2'-deoxyuridine (FUDR), 8-oxo-7,8-dihydroguanosine (8-oxoG), N-ethylpiperidine-7-EAA triazole-modified adenine, N-ethylpiperidine-6-triazole-modified adenine, 6-phenylpyrrolocytosine (PhpC), 2,4-difluorotoluyl-ribonucleoside (rF), N1(5-nitroindole)ribonucleoside, 5-methoxyuridine), and combinations thereof.

29. The antisense oligonucleotide or a pharmaceutically acceptable salt thereof as claimed in any of the preceding claims, wherein the antisense oligonucleotide or a pharmaceutically acceptable salt thereof further comprises a reverse nucleotide.

30. An antisense oligonucleotide or a pharmaceutically acceptable salt as claimed in any of the preceding claims, wherein the antisense oligonucleotide is a gapmer.

31. The antisense oligonucleotide of claim 30, wherein the gapmer comprises flanking RNA nucleotides at the 5' end, 3' end, or both ends.

32. The antisense oligonucleotide of claim 31, wherein the range of the flanking RNA nucleotides at the 5' end and / or 3' end independently ranges from 1 nucleotide to about 9 nucleotides.

33. The antisense oligonucleotide of any one of claims 30-32, wherein the gapmer is in the range of about 18 to about 22 nucleotides.

34. The antisense oligonucleotide of any one of claims 30-32, wherein the gapmer is 18 nucleotides in length.

35. The antisense oligonucleotide of any one of claims 30-34, wherein the gapmer comprises ten central 2'-deoxynucleotides and four flanking RNA nucleotides at both the 5' and 3' ends.

36. The antisense oligonucleotide of any one of claims 30-33, wherein the gapmer is 20 nucleotides in length.

37. The antisense oligonucleotide of any one of claims 30-33 or 36, wherein the gapmer comprises ten central 2'-deoxynucleotides and / or five flanking ribonucleosides at each of the 5' and 3' ends.

38. The antisense oligonucleotide or a pharmaceutically acceptable salt thereof as described in any of the preceding claims, wherein administration of the antisense oligonucleotide to a subject reduces the expression of human APOE mRNA, pre-mRNA, protein, or a combination thereof.

39. The antisense oligonucleotide of claim 38 or a pharmaceutically acceptable salt thereof, wherein human APOE mRNA is reduced by about 25% or more relative to pre-treatment conditions.

40. The antisense oligonucleotide of claim 38 or a pharmaceutically acceptable salt thereof, wherein human APOE mRNA is reduced by about 50% or more relative to pre-treatment conditions.

41. The antisense oligonucleotide of claim 38 or a pharmaceutically acceptable salt thereof, wherein human APOE mRNA is reduced by about 70% or more relative to pre-treatment conditions.

42. The antisense oligonucleotide of claim 38 or a pharmaceutically acceptable salt thereof, wherein the human APOE protein is reduced by about 25% or more relative to the pre-treatment condition.

43. The antisense oligonucleotide of claim 38 or a pharmaceutically acceptable salt thereof, wherein the human APOE protein is reduced by about 50% or more relative to the pre-treatment condition.

44. The antisense oligonucleotide of claim 38 or a pharmaceutically acceptable salt thereof, wherein the human APOE protein is reduced by about 70% or more relative to the pre-treatment condition.

45. The antisense oligonucleotide or a pharmaceutically acceptable salt thereof as claimed in any of the preceding claims, wherein the antisense oligonucleotide is capable of hybridizing with human APOE mRNA.

46. ​​The antisense oligonucleotide of claim 45, wherein the human APOE mRNA comprises at least 80% of the same sequence as SEQ ID NO:

1.

47. The antisense oligonucleotide of claim 45, wherein the human APOE mRNA encodes a protein comprising 85% of the sequence identical to SEQ ID NO:

82.

48. The oligonucleotide of claim 45, wherein the APOE is selected from the following isoforms: APOE2, APOE3, APOE4, APOE5f, APOE5s and APOE7.

49. The oligonucleotide of claim 45, wherein the hybridization is performed by Watson-Crick base pairing.

50. A method for treating a disease or disorder in a human subject in need, the method comprising administering to the subject in need an antisense oligonucleotide as described in any of the preceding claims or a pharmaceutically acceptable salt thereof.

51. A method for treating a neurodegenerative disease, the method comprising administering to a subject in need an antisense oligonucleotide as described in any one of claims 1-49 or a pharmaceutically acceptable salt thereof.

52. The method of claim 51, wherein the neurodegenerative disease is selected from Alzheimer's disease, amyotrophic lateral sclerosis, Huntington's disease, Lewy body dementia, multiple sclerosis, multiple system atrophy, Parkinson's disease, prions, and combinations thereof.

53. The method of claim 51, wherein the neurodegenerative disease is Alzheimer's disease.

54. The method of claim 51, wherein the neurodegenerative disease is tau proteinosis.

55. The method of claim 54, wherein the tau disease is selected from primary age-related tau disease, chronic traumatic encephalopathy, progressive supranuclear palsy, corticobasal degeneration, chromosome 17-linked frontotemporal dementia with Parkinson's syndrome, vacuolet tau disease, lytico-Bodig disease, ganglioglioma, gangliocytoma, meningeal angiomatosis, post-encephalitis Parkinson's syndrome, subacute sclerosing panencephalitis, lead poisoning encephalopathy, tuberous sclerosis, pantothenic kinase-related neurodegeneration, lipofuscinosis, behaviorally variant frontotemporal dementia (Pick's disease), argyrophilic granuloma, and combinations thereof.

56. A method for treating acute or chronic injury, the method comprising administering to a subject in need an antisense oligonucleotide as described in any one of claims 1-49 or a pharmaceutically acceptable salt thereof.

57. The method of claim 56, wherein the acute or chronic injury is selected from diffuse axonal injury, concussion, edema, hematoma, skull fracture, hemorrhage, hypoxic brain injury, hypoxic brain injury, carbon monoxide poisoning, cerebral aneurysm, stroke, hydrocephalus, traumatic brain injury, spinal cord injury, chronic traumatic encephalopathy, chemotherapy-induced brain, neuronal injury caused by tumors, neuronal injury caused by meningitis, encephalitis and other inflammations of the body or nervous system, or combinations thereof.

58. A method for treating an acute or chronic inflammatory condition of the nervous system or peripheral regions of the body, the method comprising administering to a subject in need an antisense oligonucleotide as described in any one of claims 1-49 or a pharmaceutically acceptable salt thereof.

59. The method of claim 58, wherein the acute or chronic inflammatory condition is selected from autoimmune diseases, stroke, multiple sclerosis, infectious diseases, immune responses to cancer, tumors, or combinations thereof.

60. A method for treating vascular diseases or vascular pathological conditions, the method comprising administering to a subject in need an antisense oligonucleotide as described in any one of claims 1-49 or a pharmaceutically acceptable salt thereof.

61. The method of claim 60, wherein the vascular disease or the vascular pathological condition is selected from any human vascular disease, including but not limited to cerebrovascular disease, cerebral small vessel disease, diseases caused or affected by leakage of the blood-brain barrier, atherosclerosis, cerebral amyloid angiopathy, peripheral vascular disease, peripheral artery disease, carotid artery disease, pulmonary embolism, abdominal aortic aneurysm, collagen vascular disease, chronic venous insufficiency, thrombosis, deep vein thrombosis, cerebral small vessel disease, blood-brain barrier leakage, atherosclerosis, cerebral amyloid angiopathy, or combinations thereof.

62. A method for treating lipid storage disorder, the method comprising administering to a subject in need an antisense oligonucleotide as described in any one of claims 1-49 or a pharmaceutically acceptable salt thereof.

63. The method of claim 62, wherein the lipid storage disease is selected from tendon xanthomas, Fabry disease, Fabry disease, fucoside storage disease, Gaucher disease, GM1 ganglioside storage disease, GM2 ganglioside storage disease AB variant, Krabby disease, metachromatic leukodystrophy, multiple sulfatase deficiencies, Niemann-Pick disease type A, Niemann-Pick disease type B, Niemann-Pick disease type C, Sankhov disease, Schindler disease, Ty Sachs disease, Wollman disease, and combinations thereof.

64. The method of any one of claims 50-63, wherein the oligonucleotide or a pharmaceutically acceptable salt thereof is applied topically to a subject in need.

65. The method of claim 64, wherein the oligonucleotide or a pharmaceutically acceptable salt thereof is administered orally, sublingually, nasally, subcutaneously, intravenously, intraperitoneally, intramuscularly, intratumorally, intrathecally, transdermally, and / or rectally.

66. The method of claim 64, wherein the local application is an intracranial, intracerebral, intramuscular, spinal, epidural, sacroiliac, or subdural injection.

67. The method of any one of claims 50-63, wherein the oligonucleotide or a pharmaceutically acceptable salt thereof is administered systemically to a subject in need.

68. The method of any one of claims 50-67, wherein the oligonucleotide or a pharmaceutically acceptable salt thereof is administered in combination with one or more vaccines, antigens, antibodies, cytotoxic agents, chemotherapeutic agents (both conventional chemotherapy and modern targeted therapy), radiation, kinase inhibitors, allergens, antibiotics, agonists, antagonists, antisense oligonucleotides, ribozymes, RNAi molecules, siRNA molecules, miRNA molecules, aptamers, proteins, gene therapy vectors, DNA vaccines, adjuvants, co-stimulatory molecules, or combinations thereof.

69. Use of any antisense oligonucleotide or pharmaceutically acceptable salt thereof as described in any one of claims 1-49 in the manufacture of a medicament for the therapeutic treatment of a disease or disorder in a human subject in need.

70. Use of any antisense oligonucleotide or pharmaceutically acceptable salt thereof as described in any one of claims 1-49 for the treatment of a disease or disorder in a human subject in need.

71. Use of any antisense oligonucleotide or pharmaceutically acceptable salt thereof as described in any one of claims 1-49 in the manufacture of a medicament for the therapeutic treatment of a subject with a neurodegenerative disease.

72. Use of the antisense oligonucleotide or a pharmaceutically acceptable salt thereof as described in any one of claims 1-49 for the treatment of a neurodegenerative disease in a subject of need.

73. The use as described in claim 71 or 72, wherein the neurodegenerative disease is selected from Alzheimer's disease, amyotrophic lateral sclerosis, Huntington's disease, Lewy body dementia, multiple sclerosis, multiple system atrophy, Parkinson's disease, prions, and combinations thereof.

74. The use as described in claim 71 or 72, wherein the neurodegenerative disease is Alzheimer's disease.

75. The use as described in claim 71 or 72, wherein the neurodegenerative disease is tau proteinosis.

76. The use as described in claim 75, wherein the tau disease is selected from primary age-related tau disease, chronic traumatic encephalopathy, progressive supranuclear palsy, corticobasal degeneration, chromosome 17-linked frontotemporal dementia with Parkinson's syndrome, vacuolet tau disease, lytico-Bodig disease, ganglioglioma, gangliocytoma, meningeal angiomatosis, post-encephalitis Parkinson's syndrome, subacute sclerosing panencephalitis, lead poisoning encephalopathy, tuberous sclerosis, pantothenic kinase-related neurodegeneration, lipofuscinosis, behaviorally variant frontotemporal dementia (Pick's disease), argyrophilic granuloma, and combinations thereof.

77. An antisense oligonucleotide or a pharmaceutically acceptable salt thereof, said antisense oligonucleotide or a pharmaceutically acceptable salt thereof comprising at least 80% identical sequences to SEQ ID NO: 84, 85, 88, 90, 91, 94, 96, 100, 103, 104, 108, 111, 113, 114, 115, 119, 120, 123, 124, 129, 130, 131, 133, 134, 135, 136, 138, 150, 151, 157, 163, 164, 168, 170, 175, 176, 180, 185, 210, 212, 216, 219, 228 or 229.

78. An antisense oligonucleotide or a pharmaceutically acceptable salt thereof, said antisense oligonucleotide or a pharmaceutically acceptable salt thereof comprising a sequence selected from the following: SEQ ID NO: 84, 85, 88, 90, 91, 94, 96, 100, 103, 104, 108, 111, 113, 114, 115, 119, 120, 123, 124, 129, 130, 131, 133, 134, 135, 136, 138, 150, 151, 157, 163, 164, 168, 170, 175, 176, 180, 185, 210, 212, 216, 219, 228, and 229.

79. The antisense oligonucleotide of claim 77 or 78 or a pharmaceutically acceptable salt thereof, wherein the sequence is selected from SEQ ID NO: 127, 132, 147, 202 and 220.

80. The antisense oligonucleotide or a pharmaceutically acceptable salt thereof as claimed in any one of claims 77-79, wherein the antisense oligonucleotide or a pharmaceutically acceptable salt thereof comprises about 12 to about 25 nucleobases.

81. The antisense oligonucleotide or a pharmaceutically acceptable salt thereof as claimed in any one of claims 77-80, wherein the antisense oligonucleotide or a pharmaceutically acceptable salt thereof comprises about 14 to about 18 nucleobases.

82. The antisense oligonucleotide or a pharmaceutically acceptable salt thereof as described in any one of claims 77-81, wherein the antisense oligonucleotide comprises about 16 nucleobases.

83. The antisense oligonucleotide or a pharmaceutically acceptable salt thereof as described in any one of claims 77-81, wherein the antisense oligonucleotide comprises about 18 nucleobases.

84. The antisense oligonucleotide or a pharmaceutically acceptable salt thereof as described in any one of claims 77-83, wherein the antisense oligonucleotide comprises one or more internucleotide linkage modifications.

85. The antisense oligonucleotide of claim 84 or a pharmaceutically acceptable salt thereof, wherein the internucleotide linkage modification is selected from thiophosphates, dithiophosphates, methylphosphonates, methylthiophosphates, aminophosphates, diaminophosphates, thioaminophosphates, methanesulfonylaminophosphates, and combinations thereof.

86. The antisense oligonucleotide of claim 84 or a pharmaceutically acceptable salt thereof, wherein the antisense oligonucleotide comprises one or more phosphate thioester nucleosides linked together.

87. The antisense oligonucleotide of claim 84 or a pharmaceutically acceptable salt thereof, wherein all said nucleoside linkages are phosphate thioester linkages.

88. The antisense oligonucleotide or a pharmaceutically acceptable salt thereof as claimed in any one of claims 77-87, wherein the antisense oligonucleotide comprises DNA.

89. The antisense oligonucleotide or a pharmaceutically acceptable salt thereof as claimed in any one of claims 77-87, wherein the antisense oligonucleotide comprises RNA.

90. The antisense oligonucleotide or a pharmaceutically acceptable salt thereof as described in any one of claims 77-87, wherein the antisense oligonucleotide comprises proteins and / or polypeptides.

91. The antisense oligonucleotide or a pharmaceutically acceptable salt thereof as claimed in any one of claims 77-87, wherein the antisense oligonucleotide comprises a small molecule.

92. The antisense oligonucleotide or a pharmaceutically acceptable salt thereof as described in any one of claims 77-91, wherein the antisense oligonucleotide comprises one or more modified nucleosides.

93. The antisense oligonucleotide of claim 92, wherein the modified nucleoside is selected from 2'-modified nucleoside, 4'-modified nucleoside, bridging nucleoside, diaminophosphate morpholine, locked nucleic acid, ethylene-bridging nucleic acid, ethylene glycol nucleic acid, hexitol nucleic acid, cyclohexene nucleic acid, arabinose nucleic acid, peptide nucleic acid, threonine nucleic acid, tricyclic-2'-deoxynucleotide, 1'-deoxypentose, 1',2'-dideoxypentose, 2',3'-dideoxypentose, 2',3'-didehydro-2',3'-dideoxypentose, unlocked nucleic acid, and combinations thereof.

94. The antisense oligonucleotide or a pharmaceutically acceptable salt thereof as described in any one of claims 77-93, wherein the antisense oligonucleotide comprises one or more 2'-nucleoside modifications.

95. The antisense oligonucleotide of claim 94, wherein the antisense oligonucleotide comprises at least eight 2'-nucleoside modifications.

96. The antisense oligonucleotide of claim 94, wherein the antisense oligonucleotide comprises at least ten 2'-nucleoside modifications.

97. The antisense oligonucleotide of claim 94 or a pharmaceutically acceptable salt thereof, wherein all nucleosides of the antisense oligonucleotide contain a 2'-nucleoside modification.

98. The antisense oligonucleotide of any one of claims 94-97 or a pharmaceutically acceptable salt thereof, wherein the 2'-nucleoside modification is selected from 2'-fluoro-nucleoside, 2'-O-methyl-nucleoside, 2'-O-methoxyethyl-nucleoside, 2'-O-benzyl-2'-deoxy-nucleoside, 2'-O-methyl-4-pyridyl-nucleoside (2'-O-CH2Py(4)), 2'-amino-nucleoside, and combinations thereof.

99. The antisense oligonucleotide or a pharmaceutically acceptable salt thereof as described in any one of claims 77-98, wherein the antisense oligonucleotide comprises one or more modified nucleobases.

100. The antisense oligonucleotide of claim 99 or a pharmaceutically acceptable salt thereof, wherein the modified nucleobase is a methylated nucleobase.

101. The antisense oligonucleotide of claim 100 or a pharmaceutically acceptable salt thereof, wherein the methylated nucleobase is selected from 5-methyluracil, N6-methyladenine, N4-methylcytosine, N7-methylguanine, 5-hydroxymethylcytosine, N3-methylcytosine, and combinations thereof.

102. The antisense oligonucleotide of claim 99 or a pharmaceutically acceptable salt thereof, wherein the modified nucleobase is an acetylated nucleobase.

103. The antisense oligonucleotide of claim 102 or a pharmaceutically acceptable salt thereof, wherein the acetylated nucleobase is N4-acetylcytosine.

104. The antisense oligonucleotide of claim 99 or a pharmaceutically acceptable salt thereof, wherein the modified nucleobase is selected from pseudouridine (Ψ), N1-methyl-pseudouridine (N1-methyl-Ψ), 2-thiouridine (s2U), 5-fluoro-2'-deoxyuridine (FUDR), 8-oxo-7,8-dihydroguanosine (8-oxoG), N-ethylpiperidine-7-EAA triazole-modified adenine, N-ethylpiperidine-6-triazole-modified adenine, 6-phenylpyrrolocytosine (PhpC), 2,4-difluorotoluyl-ribonucleoside (rF), N1(5-nitroindole)ribonucleoside, 5-methoxyuridine), and combinations thereof.

105. The antisense oligonucleotide or a pharmaceutically acceptable salt thereof as claimed in any one of claims 77-104, wherein the antisense oligonucleotide or a pharmaceutically acceptable salt thereof further comprises an inverse nucleotide.

106. The antisense oligonucleotide or pharmaceutically acceptable salt of any one of claims 77-105, wherein the antisense oligonucleotide is a gapmer.

107. The antisense oligonucleotide of claim 106, wherein the gapmer comprises flanking RNA nucleotides at the 5' end, 3' end, or both ends.

108. The antisense oligonucleotide of claim 107, wherein the range of the flanking RNA nucleotides at the 5' end and / or 3' end independently ranges from about 1 nucleotide to about 5 nucleotides.

109. The antisense oligonucleotide of any one of claims 106-108, wherein the gapmer is in the range of about 14 to about 22 nucleotides.

110. The antisense oligonucleotide of any one of claims 106-108, wherein the gapmer is 18 nucleotides in length.

111. The antisense oligonucleotide of any one of claims 106-110, wherein the gapmer comprises ten central 2'-deoxynucleotides and three flanking RNA nucleotides at both the 5' and 3' ends.

112. The antisense oligonucleotide of any one of claims 106-109, wherein the gapmer is 16 nucleotides in length.

113. The antisense oligonucleotide of any one of claims 106-109 or 112, wherein the gapmer comprises ten central 2'-deoxyribonucleosides and / or three flanking ribonucleosides at each of the 5' and 3' ends.

114. The antisense oligonucleotide or a pharmaceutically acceptable salt thereof as described in any one of claims 77-113, wherein administration of said antisense oligonucleotide to a subject reduces the expression of human APOE mRNA, pre-mRNA, protein, or a combination thereof.

115. The antisense oligonucleotide of claim 114 or a pharmaceutically acceptable salt thereof, wherein the human APOE pre-mRNA or mRNA is reduced by about 10% or more relative to the pre-treatment condition.

116. The antisense oligonucleotide of claim 114 or a pharmaceutically acceptable salt thereof, wherein the human APOE pre-mRNA or mRNA is reduced by about 30% or more relative to the pre-treatment condition.

117. The antisense oligonucleotide of claim 114 or a pharmaceutically acceptable salt thereof, wherein the human APOE pre-mRNA or mRNA is reduced by about 50% or more relative to the pre-treatment condition.

118. The antisense oligonucleotide of claim 114 or a pharmaceutically acceptable salt thereof, wherein the human APOE protein is reduced by about 25% or more relative to the pre-treatment condition.

119. The antisense oligonucleotide of claim 114 or a pharmaceutically acceptable salt thereof, wherein the human APOE protein is reduced by about 50% or more relative to the pre-treatment condition.

120. The antisense oligonucleotide of claim 114 or a pharmaceutically acceptable salt thereof, wherein the human APOE protein is reduced by about 70% or more relative to the pre-treatment condition.

121. The antisense oligonucleotide or a pharmaceutically acceptable salt thereof as described in any one of claims 77-120, wherein the antisense oligonucleotide is capable of hybridizing with human APOE pre-mRNA.

122. The antisense oligonucleotide of claim 121, wherein the human APOE premRNA comprises at least 80% of the same sequence as SEQ ID NO:

230.

123. The antisense oligonucleotide of claim 121, wherein the human APOE premRNA encodes a protein comprising 85% of the sequence identical to SEQ ID NO:

82.

124. The oligonucleotide of claim 121, wherein the APOE is selected from the following isoforms: APOE2, APOE3, APOE4, APOE5f, APOE5s and APOE7.

125. The oligonucleotide of claim 121, wherein the hybridization is performed by Watson-Crick base pairing.

126. A method of treating a disease or disorder in a human subject in need, the method comprising administering to the subject in need an antisense oligonucleotide as described in any one of claims 77-125 or a pharmaceutically acceptable salt thereof.

127. A method for treating a neurodegenerative disease, the method comprising administering to a subject in need an antisense oligonucleotide as described in any one of claims 77-125 or a pharmaceutically acceptable salt thereof.

128. The method of claim 127, wherein the neurodegenerative disease is selected from Alzheimer's disease, amyotrophic lateral sclerosis, Huntington's disease, Lewy body dementia, multiple sclerosis, multiple system atrophy, Parkinson's disease, prions, and combinations thereof.

129. The method of claim 127, wherein the neurodegenerative disease is Alzheimer's disease.

130. The method of claim 127, wherein the neurodegenerative disease is tau proteinosis.

131. The method of claim 130, wherein the tau disease is selected from primary age-related tau disease, chronic traumatic encephalopathy, progressive supranuclear palsy, corticobasal degeneration, chromosome 17-linked frontotemporal dementia with Parkinson's syndrome, vacuolet tau disease, lytico-Bodig disease, ganglioglioma, gangliocytoma, meningeal angiomatosis, post-encephalitis Parkinson's syndrome, subacute sclerosing panencephalitis, lead poisoning encephalopathy, tuberous sclerosis, pantothenic kinase-related neurodegeneration, lipofuscinosis, behaviorally variant frontotemporal dementia (Pick's disease), argyrophilic granuloma, and combinations thereof.

132. A method for treating acute or chronic injury, the method comprising administering to a subject in need an antisense oligonucleotide as described in any one of claims 77-125 or a pharmaceutically acceptable salt thereof.

133. The method of claim 132, wherein the acute or chronic injury is selected from diffuse axonal injury, concussion, edema, hematoma, skull fracture, hemorrhage, hypoxic brain injury, hypoxic brain injury, carbon monoxide poisoning, cerebral aneurysm, stroke, hydrocephalus, traumatic brain injury, spinal cord injury, chronic traumatic encephalopathy, chemotherapy-induced brain, neuronal injury caused by tumors, neuronal injury caused by meningitis, encephalitis and other inflammations of the body or nervous system, and combinations thereof.

134. A method for treating an acute or chronic inflammatory condition of the nervous system or peripheral regions of the body, the method comprising administering to a subject in need an antisense oligonucleotide or a pharmaceutically acceptable salt thereof as described in any one of claims 77-125.

135. The method of claim 134, wherein the acute or chronic inflammatory condition is selected from autoimmune diseases, stroke, multiple sclerosis, infectious diseases, immune responses to cancer, tumors, and combinations thereof.

136. A method for treating vascular diseases or vascular pathological conditions, the method comprising administering to a subject in need an antisense oligonucleotide as described in any one of claims 77-125 or a pharmaceutically acceptable salt thereof.

137. The method of claim 136, wherein the vascular disease or the vascular pathological condition is selected from any human vascular disease, including but not limited to cerebrovascular disease, cerebral small vessel disease, diseases caused or affected by leakage of the blood-brain barrier, atherosclerosis, cerebral amyloid angiopathy, peripheral vascular disease, peripheral artery disease, carotid artery disease, pulmonary embolism, abdominal aortic aneurysm, collagen vascular disease, chronic venous insufficiency, thrombosis, deep vein thrombosis, cerebral small vessel disease, blood-brain barrier leakage, atherosclerosis, cerebral amyloid angiopathy, and combinations thereof.

138. A method for treating lipid storage disorder, the method comprising administering to a subject in need an antisense oligonucleotide as described in any one of claims 77-125 or a pharmaceutically acceptable salt thereof.

139. The method of claim 138, wherein the lipid storage disease is selected from tendon xanthomas, Fabry disease, Fabry disease, fucoside storage disease, Gaucher disease, GM1 ganglioside storage disease, GM2 ganglioside storage disease AB variant, Krabby disease, metachromatic leukodystrophy, multiple sulfatase deficiencies, Niemann-Pick disease type A, Niemann-Pick disease type B, Niemann-Pick disease type C, Sankhov disease, Schindler disease, Ty-Sachs disease, Wollman disease, and combinations thereof.

140. The method of any one of claims 126-139, wherein the oligonucleotide or a pharmaceutically acceptable salt thereof is applied topically to a subject in need.

141. The method of claim 140, wherein the oligonucleotide or a pharmaceutically acceptable salt thereof is administered orally, sublingually, nasally, subcutaneously, intravenously, intraperitoneally, intramuscularly, intratumorally, intrathecally, transdermally, and / or rectally.

142. The method of claim 140, wherein the local application is an intracranial, intracerebral, intramuscular, spinal, epidural, sacroiliac, or subdural injection.

143. The method of any one of claims 126-139, wherein the oligonucleotide or a pharmaceutically acceptable salt thereof is administered systemically to a subject in need.

144. The method of any one of claims 126-143, wherein the oligonucleotide or a pharmaceutically acceptable salt thereof is administered in combination with one or more vaccines, antigens, antibodies, cytotoxic agents, chemotherapeutic agents (both conventional chemotherapy and modern targeted therapy), radiation, kinase inhibitors, allergens, antibiotics, agonists, antagonists, antisense oligonucleotides, ribozymes, RNAi molecules, siRNA molecules, miRNA molecules, aptamers, proteins, gene therapy vectors, DNA vaccines, adjuvants, co-stimulatory molecules, or combinations thereof.

145. Use of any antisense oligonucleotide or pharmaceutically acceptable salt thereof as described in any one of claims 77-125 in the manufacture of a medicament for the therapeutic treatment of a disease or disorder in a human subject in need.

146. The use of any antisense oligonucleotide or pharmaceutically acceptable salt thereof as described in any one of claims 77-125 for the treatment of a disease or disorder in a human subject in need.

147. Use of any antisense oligonucleotide or a pharmaceutically acceptable salt thereof as described in any one of claims 77-125 in the manufacture of a medicament for the therapeutic treatment of a subject with a neurodegenerative disease.

148. Use of any antisense oligonucleotide or pharmaceutically acceptable salt thereof as described in any one of claims 77-125 for the treatment of a neurodegenerative disease in a subject of need.

149. The use as described in claim 147 or 148, wherein the neurodegenerative disease is selected from Alzheimer's disease, amyotrophic lateral sclerosis, Huntington's disease, Lewy body dementia, multiple sclerosis, multiple system atrophy, Parkinson's disease, prions, and combinations thereof.

150. The use as described in claim 147 or 148, wherein the neurodegenerative disease is Alzheimer's disease.

151. The use as described in claim 147 or 148, wherein the neurodegenerative disease is tau proteinosis.

152. The use as described in claim 151, wherein the tau disease is selected from primary age-related tau disease, chronic traumatic encephalopathy, progressive supranuclear palsy, corticobasal degeneration, chromosome 17-linked frontotemporal dementia with Parkinson's syndrome, vacuolet tau disease, lytico-Bodig disease, ganglioglioma, gangliocytoma, meningeal angiomatosis, post-encephalitis Parkinson's syndrome, subacute sclerosing panencephalitis, lead poisoning encephalopathy, tuberous sclerosis, pantothenic kinase-related neurodegeneration, lipofuscinosis, behaviorally variant frontotemporal dementia (Pick's disease), argyrophilic granuloma, and combinations thereof.

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