Compositions and methods for modulating APOE

Abstract

Aspects of this disclosure relate to compositions and methods for modulating the level, transcription, splicing, and / or translation of one or more RNA transcripts (e.g., mRNA transcripts) in cells or subjects. This disclosure is, in part, based on isolated nucleic acids that bind to mRNA transcripts of genes involved in neurodegenerative diseases and disorders, such as the apolipoprotein E gene (APOE), which encodes a protein involved in lipid homeostasis and amyloid plaque formation in the CNS. In some embodiments, the compositions of this disclosure are useful for treating neurodegenerative diseases or disorders such as Alzheimer's disease (AD) or Lewy body dementia.

Description

Technical Field

[0001] Related Applications This application claims the benefit under 35 U.S.C. 119(e) of U.S. Provisional Application No. 63 / 508,831, filed on June 16, 2023, U.S. Provisional Application No. 63 / 588,136, filed on October 5, 2023, and U.S. Provisional Application No. 63 / 556,391, filed on February 21, 2024, each of which is hereby incorporated by reference in its entirety. Reference to Electronic Sequence Listing

[0002] The contents of the electronic sequence listing (L090770040WO00-SEQ-KZM.xml; size: 57,014 bytes; and creation date: May 7, 2024) are hereby incorporated by reference in their entirety.

Background Art

[0003] Neurodegenerative disorders affect millions of people worldwide every year. The accumulation of amyloid plaques and neurofibrillary changes is associated with the development of neurodegenerative diseases such as Alzheimer's and Lewy body dementia. Some mutations on various proteins expressed in the brain appear to be important for the development of AD.

Summary of the Invention

[0004] Aspects of this disclosure relate to isolated nucleic acids that bind to mRNA transcripts of genes involved in certain diseases and disorders associated with amyloid plaque formation, such as genes encoding apolipoprotein E (APOE). In some embodiments, compositions of this disclosure are useful for treating neurodegenerative diseases or disorders, such as Alzheimer's disease, Lewy body dementia, Parkinson's disease and / or cognitive decline in Parkinson's disease, vascular dementia, neurodegenerative frontotemporal disorders, and amyloid-associated imaging abnormalities (ARIA). This disclosure is based in part on compositions and methods for modulating the level, transcription, splicing, and / or translation of one or more RNA transcripts (e.g., mRNA transcripts) in cells or subjects.

[0005] Accordingly, in several respects, the present disclosure provides an isolated nucleic acid comprising a nucleotide sequence that is at least 60% identical (e.g., 60-70%, 70-80%, 80-90%, 90-95%, 95-99%, or 100% identical) to any one of the nucleotide sequences defined in Sequence ID No. 1-52, which, upon binding to the mRNA transcript, reduces the level, transcription, splicing, and / or translation of the functional apolipoprotein E (ApoE) protein encoded by the mRNA transcript.

[0006] In some embodiments, the isolated nucleic acid contains RNA. In some embodiments, the isolated nucleic acid is an antisense oligonucleotide (ASO).

[0007] In some embodiments, the isolated nucleic acid contains or consists of nucleotides between 10 and 40. In some embodiments, the isolated nucleic acid contains or consists of nucleotides between 18 and 25.

[0008] In some embodiments, the isolated nucleic acid contains one or more chemical modifications. In some embodiments, the one or more chemical modifications include one or more nucleoside modifications and / or one or more sugar-phosphate backbone modifications. In some embodiments, the one or more nucleoside modifications include 2'-O-methyl (2'-OMe) modifications, 2'-O-methoxyethyl (2'-O-MOE) modifications, 2'-fluoro modifications, or loc nucleic acid (LNA) modifications. In some embodiments, the one or more sugar-phosphate backbone modifications include phosphorothioate backbone modifications. In some embodiments, the isolated nucleic acid is fully chemically modified (e.g., containing a fully modified sugar-phosphate backbone, where all nucleotides of the isolated nucleic acid are chemically modified).

[0009] In some embodiments, the isolated nucleic acid contains one or more deoxyribonucleotides. In some embodiments, the isolated nucleic acid is a gapmer.

[0010] In some embodiments, the complementary region is located on the untranslated region of the APOE mRNA transcript. In some embodiments, the untranslated region includes the 5'UTR, intron, or 3'UTR of the APOE mRNA transcript.

[0011] In some embodiments, the complementary region is located on the protein-coding region of the APOE mRNA transcript.

[0012] In some embodiments, the complementary region is located at the intron-exon boundary of the APOE mRNA transcript (for example, the complementary region straddles the intron-exon boundary so that the isolated nucleic acid hybridizes and binds to both introns and exons simultaneously).

[0013] In some embodiments, the complementary region includes at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 consecutive nucleotides from any one of the nucleotide sequences defined in SEQ ID NOs. 53-57.

[0014] In some embodiments, the nucleotide sequence includes a nucleic acid sequence defined by one of the nucleotide sequences defined in Table 1. In some embodiments, the nucleotide sequence includes a nucleic acid sequence defined by one of the nucleotide sequences defined in column A of Table 1. In some embodiments, the nucleotide sequence includes one or more chemical modifications (e.g., chemical modification patterns) defined in column C of Table 1. In some embodiments, the nucleotide sequence includes a nucleic acid sequence defined by one of the nucleotide sequences defined in column A of Table 1, and one or more chemical modifications (e.g., chemical modification patterns) defined in column C of Table 1. In some embodiments, the nucleic acid sequence from column A and one or more chemical modifications (e.g., chemical modification patterns) from column C are taken from the same row in Table 1.

[0015] In some respects, this disclosure provides methods for reducing the level, transcription, splicing, and / or translation of ApoE in cells or subjects, the methods comprising administering isolated nucleic acids described herein to subjects requiring it.

[0016] In some respects, this disclosure provides methods for reducing amyloid plaque formation in cells or subjects, the methods comprising administering isolated nucleic acids described herein to subjects requiring such reduction.

[0017] In some aspects, the cell is a neuron. In some aspects, the neuron is a presynaptic neuron.

[0018] In some embodiments, the subjects include one or more mutations in a gene associated with amyloid plaque formation. In some embodiments, the gene is APOE.

[0019] In some embodiments, the cells or subjects are human cells or subjects.

[0020] In some embodiments, the subject has or is suspected of having a neurodegenerative disease or disorder. In some embodiments, the disease or disorder is Alzheimer's disease. In some embodiments, the disease or disorder is dementia with Lewy bodies.

[0021] In some embodiments, the administration is systemic administration. In some embodiments, the systemic administration includes intravenous injection.

[0022] In some embodiments, the administration includes direct administration to the target tissue of the subject. In some embodiments, the direct administration includes direct injection into the central nervous system (CNS) of the subject. In some embodiments, the direct administration includes direct injection into the peripheral nervous system (PNS) of the subject. ·

[0023] In some embodiments, the administration includes placing the subject in the Trendelenburg position during administration.

[0024] In some embodiments, the subject does not contain a mutation in the APOE gene.

[0025] In some aspects, the present disclosure provides a method for preventing or treating a neurodegenerative disease or disorder in a subject that needs it, the method comprising administering to the subject an isolated nucleic acid described herein. [[ID= XXI]]

[0026] In some embodiments, the subject is human.

[0027] In some embodiments, the neurodegenerative disease or disorder is Alzheimer's disease. In some embodiments, the disease or disorder is dementia with Lewy bodies. In some embodiments, the administration includes direct administration to the target tissue of the subject. In some embodiments, the direct administration includes direct injection into the central nervous system (CNS) of the subject. In some embodiments, the direct administration includes direct injection into the peripheral nervous system (PNS) of the subject.

Brief Description of the Drawings

[0028] <U [Figure 1] Figure 1 shows a schematic diagram illustrating the regulation of RNA (e.g., mRNA, e.g., mature mRNA or premRNA) transcription, splicing, and / or translation by antisense oligonucleotides (ASOs). Composition "A" represents an ASO that binds to the 5' untranslated region (5'UTR) of RNA. Composition "B" represents an ASO that binds to an intron of RNA. Composition "C" represents an ASO that binds to the splice boundary (e.g., splice junction) between an exon and an intron of RNA. Composition "D" represents an ASO that binds to an exon of RNA (e.g., protein-coding region). Composition "E" represents a combination of ASOs that bind to the 3'UTR of RNA alone or with a trans-modulator. Composition "F" represents a "gapmer" ASO that binds to an exon of RNA (e.g., protein-coding region) and mediates RNaseH decay. Composition "G" represents a "gapmer" ASO that binds to the 3'UTR of RNA alone or with a trans-modulator and mediates RNaseH decay. In some embodiments, RNA-binding ASOs result in the translation of truncated proteins that have a dominant-negative effect on the wild-type full-length protein. [Figure 2]Figures 2A–2C show typical in vitro data for APOE RNA-targeting ASOs. Figure 2A shows APOE mRNA levels in HepG2 cells 48 hours after transfection with one of two different doses (5 nm or 20 nm; light and dark shading, respectively) of APOE ASO containing either a gapmer or a skipper, relative to a mock-transfected control (shown in black). Untargeted ASOs or APOE siRNAs were used as negative and positive controls, respectively. Figure 2B shows coordinates for APOE-targeting ASOs, representing the log2 scale changes in APOE mRNA levels relative to the mock control due to transfection at 20 nM and 5 nM concentrations on the X and Y axes as indicated. Figure 2C shows APOE mRNA levels in HepG2 cells 48 hours after transfection for 16 APOE ASOs pursued in multi-dose studies. Regarding Figures 2A and 2C: The bars indicate the mean expression in each condition; the error bars indicate the standard error; N = 2 biological replicates. [Figure 3]Figures 3A–3C show typical in vitro data for dose-dependent knockdown of APOE RNA. Figure 3A shows APOE mRNA levels in HepG2 cells 48 hours after transfection with APOE ASO (indicated in Figure 2C) as a function of eight different doses, and for each untargeted ASO-negative control or APOE siRNA-positive control, relative to mock-transfected control samples. Figure 3B shows the data presented in Figure 3A indicating the APOE mRNA expression patterns after transfection with the six ASOs shown. In Figures 3A–3B: Skippers are indicated by shaded bars labeled "APOE ASO" and indicated by arrows labeled "Skipper"; Gapmers are indicated by all other shaded bars labeled "APOE ASO"; Negative controls are shown in dark gray; Positive controls are shown in light gray. 0% knockdown and 50% knockdown are indicated by dashed lines (black and gray, respectively). Figure 3C shows the maximum inhibition (log2, Y-axis) plotted as a function of observed EC50 (X-axis). The dot plot shows the most potent ASO. "SEQ ID NO: 5" includes the nucleotide sequence, gapmer structure, and chemical modifications of SEQ ID NO: 5 as defined in columns A and C of row 6 of Table 1. "SEQ ID NO: 11" includes the nucleotide sequence, gapmer structure, and chemical modifications of SEQ ID NO: 11 as defined in columns A and C of row 12 of Table 1. "SEQ ID NO: 16" includes the nucleotide sequence, gapmer structure, and chemical modifications of SEQ ID NO: 16 as defined in columns A and C of row 17 of Table 1. "SEQ ID NO: 22" includes the nucleotide sequence, gapmer structure, and chemical modifications of SEQ ID NO: 22 as defined in columns A and C of row 23 of Table 1. "SEQ ID NO: 29" includes the nucleotide sequence, gapmer structure, and chemical modifications of SEQ ID NO: 29 as defined in columns A and C of row 30 of Table 1. "Sequence ID 50" includes the nucleotide sequence, gapmer structure, and chemical modifications of Sequence ID 50 as defined in columns A and C of row 51 in Table 1. [Figure 4]Figures 4A-4B show representative data for in vivo reduction of APOE mRNA levels in mouse brain. Figure 4A shows relative APOE mRNA levels in mouse target cortical tissue two weeks after single ICV injection of APOE ASO 1a at a base (artificial CSF) or at doses of 100ug or 200ug. Figure 4B shows relative APOE mRNA levels in mouse target hippocampal tissue two weeks after single ICV injection of APOE ASO 1 at a base (artificial CSF) or at doses of 100ug or 200ug. "APOE ASO 1" includes the nucleotide sequence, gapmer structure, and chemical modifications of Sequence ID No. 29, as defined in columns A and C of row 30 in Table 1. 0% knockdown and 50% knockdown are indicated by dashed lines (black and gray, respectively). [Figure 5] Figure 5 shows an unrestricted example of antisense oligonucleotide (ASO) design for targeting the 3' untranslated region (UTR) of an APOE mRNA transcript (e.g., one encoded by Ensembl ID NO: ENST00000252486). [Figure 6]Figures 6A–6J show the typical immunostimulatory effects of APOE ASO on human peripheral blood mononuclear cells (huPBMCs) harvested from healthy donors. huPBMCs were either untreated ("Mock" and "Culture Medium"), treated with a cytokine / chemokine response regulator (XD-01024, XD00366 transfection, poly(l:c) transfection, CL097, R837, TL8-506, ODN2395 transfection, ODN2395 gymnosis, ODN2216 transfection, ODN2216 gymnosis, ODN2006 transfection, or ODN2006 gymnosis), or treated for 24 hours with ASO at concentrations of 1 μM, 3 μM, or 10 μM (indicated on the x-axis). Then, cytokine / chemokine levels were analyzed using the MSD-U-Plex platform (indicated by the y-axis). "APOE ASO 1" includes the nucleotide sequence, gapmer structure, and chemical modifications of Sequence ID No. 29, defined in columns A and C of row 30 in Table 1. Plots show mean + / - standard error. Each dot represents an individual donor. N=4 donors (2 males and 2 females). Figure 6A shows the analysis of IFN-a2a levels. Figure 6B shows the analysis of IFN-b levels. Figure 6C shows the analysis of IL-1B levels. Figure 6D shows the analysis of IL-6 levels. Figure 6E shows the analysis of IL-10 levels. Figure 6F shows the analysis of IP-10 levels. Figure 6G shows the analysis of MCP-1 levels. Figure 6H shows the analysis of MIP-1a levels. Figure 6I shows the analysis of MIP-1b levels. Figure 6J shows the analysis of TNF-a levels. [Figure 7] Figure 7 shows an unspecified example of a study design in which non-human primate subjects were administered a series of four intrathecal injections of either a base (artificial CSF) or ASO at a dose of 80 mg (20 mg + 20 mg + 20 mg + 20 mg). Each intrathecal injection was administered 2 weeks apart (days 0, 14, 28, and 42). [Figure 8]Figure 8 shows ASO levels in dorsal root ganglia (DRG), hippocampus, lumbar spinal cord, motor cortex, prefrontal cortex, and temporal cortex samples. These were obtained from injected non-human primate subjects and evaluated by liquid chromatography-tandem mass spectrometry (LC-MS / MS). Non-human primate subjects received ASO at a dose of 80 mg (20 mg + 20 mg + 20 mg + 20 mg) via intrathecal injection, as illustrated in Figure 7. The indicated samples were obtained two weeks (day 56) after the last ASO injection. Each dot represents a sample obtained from a different non-human primate subject. N = 3 for each indicated sample group. Bars indicate the mean + / - mean standard error. “APOE ASO 1” includes the nucleotide sequence, gapmer structure, and chemical modifications of Sequence ID No. 29, defined in columns A and C of row 30 in Table 1. [Modes for carrying out the invention]

[0029] Aspects of this disclosure relate to compositions and methods for modulating the level, transcription, splicing, and / or translation of one or more RNA transcripts (e.g., mRNA transcripts) in cells or subjects. This disclosure is, in part, based on isolated nucleic acids that bind to mRNA transcripts of genes involved in amyloid plaque formation, such as genes encoding apolipoprotein E (APOE). In some embodiments, the compositions of this disclosure are useful for treating and / or preventing diseases or disorders associated with amyloid plaque formation. In some embodiments, the diseases or disorders are associated with neurodegeneration (e.g., Alzheimer's disease, Lewy body dementia, Parkinson's disease and / or cognitive decline in Parkinson's disease, vascular dementia, frontotemporal disorders associated with neurodegeneration, amyloid-associated imaging abnormalities (ARIA), etc.). In some embodiments, the compositions of this disclosure are useful for reducing amyloid plaques and neurofibrillary tangles in subjects where it is needed. Lipid homeostasis and amyloid plaque formation

[0030] Apolipoprotein E (APOE) is a lipid-binding protein involved in lipid metabolism in mammals. APOE is found in various lipoprotein particles, such as chylomicrons, chylomicron remnants, very low-density lipoprotein (VLDL), intermediate-density lipoprotein (IDL), and some high-density lipoproteins (HDL). These lipoprotein particles are found, for example, in plasma, interstitial fluid, and lymph, where they are involved in lipid transport throughout the body. Without being constrained by any particular theory, APOE interacts with lipids through its amphiphilic structure. In addition, APOE binds to various cellular receptors, which mediate the cellular uptake of lipoprotein particles such as the LDL receptor / LDLR, LDL receptor-related proteins LRP1, LRP2, and LRP8, and very low-density lipoprotein receptor / VLDLR. Thus, APOE is involved in the production, conversion, and clearance of lipoprotein particles in tissues / organs such as the liver, muscle, heart, and adipose tissue. In addition, APOE possesses heparin-binding activity, which allows it to bind to heparan sulfate proteoglycans on the cell surface. This property supports the capture of lipoproteins, including APOE, by cells and their uptake mediated by receptors.

[0031] APOE is expressed in various tissues and locations, including the liver, adrenal glands, testes, ovaries, skin, kidneys, spleen, adipose tissue, and macrophages. However, APOE is also expressed in the brain, by cells such as astrocytes and glial cells in the cerebral cortex, as well as neurons in the prefrontal cortex and hippocampus.

[0032] APOE is associated with a variety of diseases and disorders, such as cardiovascular diseases, hematopoietic cancers, and neurodegenerative diseases. One specific example of a disease associated with APOE is Alzheimer's disease (e.g., late-onset Alzheimer's disease). Alzheimer's is characterized by progressive dementia, loss of cognitive ability, and indicators such as intraneuronal neurofibrillary tangles, extracellular amyloid plaques, and deposition of fibrous amyloid proteins such as vascular amyloid deposits. APOE co-localizes with amyloid plaques containing amyloid-β (Aβ). Furthermore, APOE modulates amyloid plaque size and toxicity by promoting amyloidosis during the early stages of amyloid plaque formation and by impairing plaque clearance from the interstitial fluid of the brain. Accordingly, aspects of this disclosure relate to compositions for modulating the level, transcription, splicing, and / or translation of genes associated with amyloid plaque formation.

[0033] "Genes associated with amyloid plaque formation" refer to genes that encode gene products (e.g., mRNA, proteins, etc.) that are genetically, biochemically, or functionally involved in the amyloidogenesis processing of amyloid precursor protein (APP) in cells or objects. In some embodiments, genes associated with amyloid plaque formation are APOEs.

[0034] In some embodiments, genes associated with amyloid plaque formation encode mRNA that codes for the apolipoprotein E (APOE) protein. In humans, APOE is encoded by the APOE gene located on chromosome 19 (e.g., Ensembl ID NO: ENST00000252486.9, chromosome 19: 44,905,796-44,909,393 forward strand, location: 19q13.32). The APOE gene, including its uncoding region (UTR), contains 3,598 nucleotides that encode four exons and three introns. The human APOE gene may contain single nucleotide polymorphisms (SNPs) that result in variants such as ε2 (containing C112, C158) (also known as APOE2), ε3 (containing C112, R158) (also known as APOE3), and ε4 (containing R112, R158) (also known as APOE4). In some embodiments, APOE encodes a peptide defined by the NCBI reference sequences NP_000032.1, NP_001289617.1, NP_001289618.1, NP_001289619.1, or NP_001289620.1. In some embodiments, the APOE gene encodes mRNA containing a sequence defined by the NCBI reference sequences NM_014905.5, NM_001302688.2, NM_001302689.2, NM_001302690.2, or NM_001302691.2. In some embodiments, the mRNA encoded by the APOE gene contains one of the sequences defined below: NM_000041.4 NM_001302688.2 NM_001302689.2 NM_001302690.2 NM_001302691.2

[0035] A person skilled in the art will recognize that when referring to a gene sequence encoding mRNA, the mRNA sequence is identical to the described gene sequence, with the exception that each "T" is replaced by a "U".

[0036] Aspects of this disclosure relate to methods for inhibiting amyloid plaque formation in subjects with certain neurodegenerative diseases and disorders. In some embodiments, the subjects do not have any mutations in the APOE gene (for example, the subjects have wild-type APOE protein).

[0037] However, in some embodiments, the APOE gene (or mRNA encoded by the APOE gene) may contain one or more nucleotide substitutions, one or more nucleotide insertions, and / or one or more nucleotide deletions relative to the wild-type APOE gene (or mRNA encoded by the wild-type APOE gene) and may be referred to as a “mutant” APOE gene or APOE variant. The number of nucleotide substitutions in an APOE variant can vary. In some embodiments, an APOE variant may contain between 1 and 20, 5 and 10, 2 and 15, 10 and 30, or 20 and 100 nucleotide substitutions, insertions, and / or deletions relative to the wild-type APOE gene (or mRNA encoded by the wild-type APOE gene). In some embodiments, one or more nucleotide substitutions, one or more nucleotide insertions, and / or one or more nucleotide deletions result in amino acid substitutions on the protein encoded by the APOE variant. In some embodiments, one or more nucleotide substitutions, one or more nucleotide insertions, and / or one or more nucleotide deletions result in nonsense mutations (e.g., insertion of an immature stop codon) on mRNA encoded by the APOE variant.

[0038] In some embodiments, one or more nucleotide substitutions, one or more nucleotide insertions, and / or one or more nucleotide deletions result in a frameshift mutation of the APOE variant relative to the wild-type APOE gene. In some embodiments, one or more mutations present on the APOE variant result in the production of one or more splice variants of APOE mRNA. A “splice variant” can refer to mRNA resulting from one or more mutations in the DNA sequence occurring at the exon and intron boundaries (splice sites) of a gene. Splice site mutations generally disrupt RNA splicing, resulting in exon loss or intron inclusion, and a modulated protein-coding sequence (e.g., a “splice variant”).

[0039] Aspects of this disclosure relate to isolated nucleic acids, e.g., RNA processing modulators (e.g., ASOs), that bind to one or more target regions of mRNA encoded by genes associated with amyloid plaque formation. In some embodiments, the isolated nucleic acids bind to more splice variants of the APOE gene (e.g., human APOE splice variants, e.g., APOE2, APOE3, APOE4, etc.). In some embodiments, the isolated nucleic acids described herein bind to a region of an APOE splice variant (e.g., mRNA encoded by the APOE variant) selected from the untranslated region (UTR). In some embodiments, the UTR is the 5'UTR. In some embodiments, the UTR is the 3'UTR. In some embodiments, the isolated nucleic acids described herein bind to a region of the polyadenylated sequence of the APOE splice variant. In some embodiments, the isolated nucleic acids described herein bind to a region spanning the boundary on the 3'UTR of the APOE splice variant, where the 5' end of the polyadenylated sequence begins. In some embodiments, the UTR is an intron. In some embodiments, the isolated nucleic acid described herein binds to an intron-exon boundary of an APOE splice variant (e.g., mRNA encoded by the APOE variant). An intron-exon boundary refers to a contiguous nucleotide sequence that encloses adjacent intron and exon portions on the mRNA transcript. In some embodiments, the isolated nucleic acid (e.g., an antisense oligonucleotide) binds to mRNA expressed from a specific allele of APOE (e.g., binds to a target mRNA in an allele-specific manner). Isolated nucleic acids

[0040] In some aspects of this disclosure, nucleic acids are isolated nucleic acids. In some cases, nucleic acids are alternatively referred to as oligonucleotides. In some aspects, isolated nucleic acids include DNA (e.g., deoxyribonucleotides). In some aspects, isolated nucleic acids include RNA (e.g., ribonucleotides), such as isolated nucleic acids, which include a gapmer structure containing a region of deoxyribonucleotides flanked by a region of ribonucleotides. In some aspects, isolated nucleic acids include both DNA (e.g., deoxyribonucleotides) and RNA (e.g., ribonucleotides). Isolated nucleic acids can be single-stranded or double-stranded. In some aspects, isolated nucleic acids are RNA oligonucleotides. In some aspects, isolated nucleic acids are single-stranded RNA oligonucleotides (which may also be referred to as single-stranded RNA polynucleotides).

[0041] As used herein, the term "isolated" means artificially produced. The artificial production of isolated nucleic acids can be achieved, for example, through in vitro amplification via polymerase chain reaction (PCR), recombinant cloning, or chemosynthesis. Methods for synthesizing isolated nucleic acids, such as RNA, are known in the art and are described, for example, in Soukchareun et al. Preparation and characterization of antisense oligonucleotide-peptide hybrids containing viral fusion peptides. BioconjugHChem. 1995 Jan-Feb;6(1):43-53. doi:10.1021 / bc00031a004. PMID:7711103.

[0042] The length of isolated nucleic acids can vary. In some embodiments, isolated nucleic acids (e.g., RNA processing regulators described herein, e.g., ASOs described herein, which may include, but are not limited to, single-stranded RNA) have a length of 10, 15, 20, 25, 30, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or up to 120 nucleotides. In some embodiments, isolated nucleic acids have a length in the range of approximately 1–100, 2–30, 5–20, 10–40, or 20–80 nucleotides. In some embodiments, isolated nucleic acids have a length of 10 and 50 nucleotides. In some embodiments, the isolated nucleic acid contains nucleotides of length 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides. In some embodiments, the isolated nucleic acid has a length greater than 50 nucleotides (e.g., 60, 70, 80, 90, 100 nucleotides, etc.). In some embodiments, the isolated nucleic acid has a length of at most 200 nucleotides. In some embodiments, the isolated nucleic acid contains a nucleotide sequence encoding the full-length wild-type APOE protein.

[0043] In some embodiments, the isolated nucleic acids of the Disclosure (e.g., RNA processing regulators described herein) comprise an antisense oligonucleotide containing a sequence defined by any one of SEQ ID NOs: 1 to 52 (provided in column A of Table 1). In some embodiments, the isolated nucleic acids of the Disclosure comprise an antisense oligonucleotide containing at least 15 nucleotides (e.g., at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 nucleotides) of any one of the sequences defined by SEQ ID NOs: 1 to 52 (provided in column A of Table 1).

[0044] In some embodiments of the present invention, isolated nucleic acids are modified (e.g., one or more modifications, including chemical modifications such as those in column C of Table 1). Modified nucleic acids may refer to oligonucleotides that have been structurally modulated in a non-natural manner (e.g., in a manner that does not occur naturally). Nucleic acid modifications may be used to confer specific functional characteristics to nucleic acids relative to unmodified nucleic acids. In some embodiments, modifications of isolated nucleic acids facilitate the binding of the isolated nucleic acid to a target molecule or increase the stability of the isolated nucleic acid (e.g., make the isolated nucleic acid resistant to enzymatic degradation).

[0045] In some embodiments, one or more modifications are between 1 and 50 modifications, 2 and 20 modifications, 5 and 30 modifications, 10 and 40 modifications, or between 15 and 50 modifications. In some embodiments, the isolated nucleic acid contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 modifications. In some embodiments, the isolated nucleic acid contains more than 50 modifications (e.g., up to 60, 70, 80, 90, or 100 modifications). In some embodiments, isolated nucleic acids contain chemical modifications on each nucleotide and each sugar-phosphate backbone linkage. Such modified isolated nucleic acids may be referred to as “fully modified” isolated nucleic acids. In some embodiments, not all nucleotides of the isolated nucleic acid are modified.

[0046] Chemical modifications may include modifications of nucleic acid bases or nucleotides, and / or modifications of sugar-phosphate backbones (e.g., modifications of one or more sugar-phosphate backbone links).

[0047] In some embodiments, the isolated nucleic acids of this disclosure (e.g., RNA processing modulators described herein, e.g., ASOs described herein) include one or more chemical modifications listed in column C of Table 1.

[0048] In some embodiments, isolated nucleic acids (e.g., RNA processing regulators described herein, e.g., ASOs described herein) include one or more modifications to the 5' carbon atom (e.g., the 5'-carbon atom of a sugar) and / or one or more modifications to the 5-carbon of a nucleic acid base. Examples of modifications include, but are not limited to, 5-(2-amino)propyluridine, 5-bromouridine, 5-propyneuridine, 5-propenyluridine, 5-carboxymethylaminomethyl-2-thiouracil, and 5-carboxymethylaminomethyluracil. In other embodiments, nucleic acid modifications are targeted to the 6-carbon atom of the nucleic acid base. In some embodiments, isolated nucleic acids include one or more modifications to the 6-carbon atom (e.g., the 6-carbon atom of a nucleic acid base), e.g., 6-(2-amino)propyluridine. In some embodiments, isolated nucleic acids include one or more modifications to the 8-carbon atom (e.g., the 8-carbon atom of a nucleic acid base). Examples of 8-modifications include, but are not limited to, 8-bromoguanosine, 8-chloroguanosine, and 8-fluoroguanosine.

[0049] In some embodiments, isolated nucleic acids (e.g., RNA processing regulators described herein, e.g., ASOs described herein) include one or more modifications to the 2' carbon of a sugar group. Examples of modified sugar groups include, but are not limited to, D-ribose, 2'-O-alkyl (including 2'-O-methyl and 2'-O-ethyl), i.e., 2'-alkoxy, 2'-amino, 2'-S-alkyl, 2'-halo (including 2'-fluoro), 2'-2-O-methoxyethoxy, 2'-allyloxy (-OCH2CH=CH2), 2'-propargyl, 2'-propyl, ethynyl, ethenyl, propenyl, and cyano, and the like. In some embodiments, the modified sugar moiety includes a hexose and is incorporated onto the oligonucleotide as described (Augustyns, K., et al., Nucl.Acids.Res.18:4711 (1992)). Other examples of 2' modifications include, but are not limited to, the substitution of an OH group bonded by H, OR, R, F, Cl, Br, I, SH, SR, NH, NHR, NR, COOR, or OR, where R is a substituted or unsubstituted aliphatic group. Other 2' modifications are found in the art. As used herein, the term “aliphatic” encompasses both saturated and unsaturated, linear (i.e., unbranched), branched, acyclic, cyclic, or polycyclic aliphatic hydrocarbons, which may be optionally substituted by one or more functional groups. As will be understood by those skilled in the art, “aliphatic” as used herein is intended to encompass, but is not limited to, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, and cycloalkynyl moieties.

[0050] In some embodiments, modifications of isolated nucleic acids (e.g., RNA processing regulators described herein, e.g., ASOs described herein) include sugar-phosphate backbone modifications. One example of phosphate group modification is the substitution of an oxygen atom with a sulfur atom. In other embodiments, the backbone of the nucleic acid is modified. Examples of backbone modifications include, but are not limited to, phosphorothioates, boranophosphates, alkylphosphonate nucleic acids, peptide nucleic acids, and morpholinos. Morpholino backbones are described, for example, by Corey and Abrams Genome Biol. 2001; 2(5):reviews1015.1-reviews1015.3.

[0051] Other examples of modified bases include N4,N4-ethanocytosine, 7-deazaxanthosine, 7-deazaguanosine, 8-oxo-N6-methyladenine, 4-acetylcytosine, dihydrouracil, inosine, N6-isopentenyl-adenine, 1-methyladenine, 1-methylpseudracil, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-methyladenine, 7-methylguanine, 2-methylthio-N6-isopentenyladenine, pseudouracil, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 2-thiocytosine, and 2,6-diaminopurine. Other examples of nucleic acid modifications are described, for example, by Eckstein, Antisense Nucleic Acid Drug Dev.2000 Apr.10(2):117-21, Rusckowski et al. Antisense Nucleic Acid Drug Dev.2000 Oct.10(5):333-45, Stein, Antisense Nucleic Acid Drug Dev.2001 Oct.11(5):317-25, Vorobjev et al. Antisense Nucleic Acid Drug Dev.2001 Apr.11(2):77-85, Duffy, BMC Bio.2020 Sep.2(8):112, and US Patent No. US5684143.

[0052] Further modifications of isolated nucleic acids (e.g., ASOs) are described by Duffy et al. BMC Biology volume 18, Article number:112 (2020), the entire contents of which are incorporated herein by reference.

[0053] In some embodiments, the isolated nucleic acids of this disclosure (e.g., RNA processing modulators described herein, e.g., ASOs described herein) comprise a nucleic acid sequence from column A of Table 1 and one or more chemical modifications (or combinations of chemical modifications) from column C of Table 1, optionally where columns A and C are from the same row of Table 1. RNA processing modulators

[0054] Aspects of this disclosure relate to compositions (e.g., isolated nucleic acids, drugs, etc.) that modulate mRNA encoded by genes associated with amyloid plaque formation. In some embodiments, the gene associated with amyloid plaque formation is APOE (e.g., the human APOE gene). In some embodiments, the composition comprises an RNA processing modulator. As used herein, “RNA processing modulator” or “RPM” means a drug that binds to a target mRNA (e.g., mRNA encoded by a gene associated with amyloid plaque formation such as APOE, or a gene product such as a protein encoded by mRNA) and affects the transcription, level, splicing, and / or translation of the mRNA, thereby upregulating, downregulating, or otherwise altering its function or activity. The RNA processing modulator may be an isolated nucleic acid or ASO described herein. In some embodiments, the RNA processing modulator is an isolated nucleic acid that affects the transcription, level, splicing, and / or translation of a target mRNA (e.g., mRNA encoded by the APOE gene). In some embodiments, the RNA processing modulator is an antisense oligonucleotide (ASO) that affects the transcription, leveling, splicing, and / or translation of a target mRNA (e.g., mRNA encoded by the APOE gene). In some embodiments, the mRNA (e.g., target mRNA) is premRNA (e.g., RNA transcribed from a gene such as the APOE gene but not processed to remove introns, e.g., by splicing). In some embodiments, the mRNA is processed mature mRNA (e.g., mRNA transcribed from the APOE gene and having undergone processing).

[0055] In some embodiments, RNA processing modulators (e.g., ASOs as described herein) upregulate the transcription, level, splicing, and / or translation of a target mRNA. Upregulation of transcription, level, splicing, and / or translation may include binding to a regulatory region of the target mRNA (e.g., an untranslated region such as the 5'UTR or 3'UTR) and reducing unproductive splicing or translation initiation from an alternative start codon present on the target mRNA through steric blocking of, for example, an unproductive splicing site(s) or an alternative start codon (e.g., an “upstream alternative start codon” located on the 5'UTR of the target mRNA), or inducing an mRNA frameshift (e.g., a splice variant) resulting in the translation of a protein variant from the target mRNA lacking one or more inhibitory domains.

[0056] The amount of upregulation of transcription, level, splicing, and / or translation mediated by RNA processing modulators can vary. In some embodiments, RNA processing modulators increase the transcription, level, splicing, and / or translation of a target mRNA transcript by a value between 1x and 100x, 2x and 10x, 5x and 20x, 10x and 30x, 20x and 50x, or 25x and 100x, or any value in between (e.g., an increase relative to cells or subjects prior to RPM administration, or an increase relative to control cells or subjects). In some embodiments, RNA processing modulators increase the transcription, level, splicing, and / or translation of a target mRNA transcript by more than 100x, for example, at least 200x, 400x, 500x, or 1000x. In some embodiments, RNA processing modulators increase the transcription, level, splicing, and / or translation of a target mRNA transcript by up to 1000-fold. In some embodiments, upregulation of the level, transcription, splicing, and / or translation of a target mRNA is useful for increasing the expression of a desired (e.g., wild-type) allele encoding the target mRNA.

[0057] In some embodiments, RNA processing modulators (e.g., ASOs as described herein) downregulate the transcription, level, splicing, and / or translation of a target mRNA. Downregulation of transcription, level, splicing, and / or translation may include binding to a regulatory region of the target mRNA (e.g., an untranslated region such as the 5'UTR or 3'UTR) and blocking the transcription of the target mRNA, for example, through steric blocking of the transcription start site; binding to the mRNA and subsequently initiating RNAse H-mediated degradation (e.g., in the context of a “gapmer” RNA processing modulator); or causing an mRNA frameshift (e.g., a splice variant), resulting in the translation of a protein variant from the target mRNA having inactive or reduced function or activity (e.g., enzymatic activity, ability to interact with other proteins to form protein complexes, etc.). In some embodiments, the resulting protein variant is a dominant-negative protein variant. In some embodiments, downregulation of the level, transcription, splicing, and / or translation of target mRNA is useful for reducing the expression of undesirable (e.g., variant or disease-associated) alleles encoding the target mRNA.

[0058] The amount of downregulation of transcription, level, splicing, and / or translation mediated by RNA processing modulators can vary. In some embodiments, RNA processing modulators reduce the transcription, level, splicing, and / or translation of a target mRNA transcript by a value between 1x and 100x, 2x and 10x, 5x and 20x, 10x and 30x, 20x and 50x, or 25x and 100x, or any value in between. In some embodiments, RNA processing modulators reduce the transcription, level, splicing, and / or translation of a target mRNA transcript by more than 100x, for example, at least 200x, 400x, 500x, or 1000x. In some embodiments, RNA processing modulators reduce the transcription, level, splicing, and / or translation of a target mRNA transcript by at most 1000x.

[0059] RNA processing modulators (e.g., ASOs as described herein) can modulate the number and / or characteristics of splice variants of a target mRNA. In some embodiments, RNA processing modulators increase the number of different splice variants of the mRNA, or the ratio of different splice variants of the mRNA (relative to the native transcription or translation of the target mRNA). In some embodiments, RNA processing modulators decrease the number of different splice variants of the mRNA, or the ratio of different splice variants of the mRNA (relative to the native transcription or translation of the target mRNA). In some embodiments, contact of the target mRNA with an RNA processing modulator results in the transcription and / or translation of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more splice variants of the target mRNA. In some embodiments, contact of the target mRNA with an RNA processing modulator results in the transcription and / or translation of a single splice variant of the target mRNA.

[0060] The binding sites of RNA processing modulators can vary. In some embodiments, RNA processing modulators affect the splicing of target mRNA. For example, an RNA processing modulator may bind to target mRNA at a splice junction (e.g., a site spanning an intron-exon boundary) and mediate the skipping of one or more exons on the mRNA transcript. In some embodiments, the skipping of one or more exons on target mRNA results in the production of a shortened protein variant of the protein encoded by the target mRNA. In another example, an RNA processing modulator may bind to target mRNA at a splice junction and mediate alternative splicing, in which an intron is translated and a protein variant of the target gene is produced. In some embodiments, an RNA processing modulator binds to target mRNA at a site containing a coding sequence (e.g., a protein-coding sequence or exon).

[0061] In some embodiments, RNA processing modifiers (e.g., ASOs as described herein) include agents selected from the group consisting of nucleic acids, peptides (including polypeptides), and small molecules. Examples of small molecule RNA processing inhibitors include, but are not limited to, translation read-through inducing drugs (TRIDs), such as certain aminoglycosides, non-aminoglycoside antibiotics (e.g., negamycin), ataluren (PTC124), and anlexanox. Examples of peptides include, but are not limited to, activator proteins (e.g., transcription factors), suppressor proteins (e.g., inducible cAMP initial repressor (ICER), bZIP repressor, SP1 repressor, certain histone deacetylases, etc.), antibodies, etc. Examples of nucleic acids include, but are not limited to, suppressor tRNA, dsRNA, siRNA, microRNA (miRNA), artificial miRNA (ami-RNA), aptamers, and antisense oligonucleotides. In some embodiments, RNA processing modifiers include antisense oligonucleotides (ASOs).

[0062] In some embodiments, the isolated nucleic acids described herein are antisense nucleic acids, such as antisense oligonucleotides (ASOs). As used herein, “antisense nucleic acid” or “ASO” means a single-stranded nucleic acid that has sequence complementarity to a target sequence and is specifically hybridizable with a nucleic acid having the target sequence, for example, under stringent conditions. The antisense nucleic acid is specifically hybridizable when the binding of the antisense nucleic acid to the target nucleic acid is sufficient to produce complementary base pairing between the antisense nucleic acid and the target nucleic acid, and when there is a sufficient degree of complementarity to reduce or avoid nonspecific binding of the antisense nucleic acid to a non-target nucleic acid under conditions where specific binding is desired, such as physiological conditions in the case of an in vivo assay or therapeutic treatment, or under the conditions under which the assay is performed in the case of an in vitro assay. In some embodiments, ASOs are chemically synthesized. ASOs may be DNA polynucleotides, RNA polynucleotides, or DNA / RNA polynucleotides (e.g., ASOs containing a gapmer structure including a region of deoxyribonucleotides flanked by a region containing ribonucleotides).

[0063] Complementarity refers to the ability of two nucleotides to form accurate pairs. For example, if a nucleotide at a certain position on an antisense nucleic acid can form a hydrogen bond with a nucleotide at a corresponding position on a target nucleic acid (e.g., target RNA), then the antisense nucleic acid and target nucleic acid are considered complementary to each other at that position. An antisense nucleic acid and target nucleic acid are complementary to each other when a sufficient number of corresponding positions on each molecule are occupied by nucleotides that can form hydrogen bonds with each other through their bases. Therefore, "complementary" is a term used to indicate a sufficient degree of complementarity or accurate pair formation between an antisense nucleic acid and a target nucleic acid that results in a stable and specific bond. However, it should be understood that 100% complementarity is not required. For example, in some embodiments, an antisense nucleic acid (e.g., an oligonucleotide) may be at least 80% complementary to a sequence of nucleotides of a target nucleic acid (e.g., a target nucleic acid comprising an mRNA sequence encoded by any one of sequence numbers 53-57) (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% complementary).

[0064] Sequence identity, which encompasses the determination of sequence complementarity for nucleic acid sequences, can be determined by sequence comparison and alignment algorithms known in the art. To determine the percentage identity of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (for example, gaps may be introduced on the first or second sequence for optimal alignment). Then, nucleotides at corresponding nucleotide positions are compared. The molecules are identical at a given position when the position on the first sequence is occupied by the same residue as the corresponding position on the second sequence. In some embodiments, the percentage identity between two sequences is a function of the number of identical positions shared by the sequences (e.g., % homology = # of identical positions / total # of positions × 100), and optionally, a penalty is imposed on the score for the number and / or length of gaps introduced.

[0065] In some embodiments, antisense oligonucleotides (e.g., ASOs as described herein) have lengths ranging from 5 to 40 nucleotides, 5 to 30 nucleotides, 10 to 30 nucleotides, 10 to 25 nucleotides, or 15 to 25 nucleotides. In some embodiments of this disclosure, antisense oligonucleotides include lengths of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 nucleotides.

[0066] In some embodiments, antisense nucleic acids (e.g., ASOs as described herein) contain complementary regions that are perfectly complementary to a portion of the target nucleic acid (e.g., 100% of the nucleotides of the ASO hybridize to nucleotides of the target RNA, such as the target mRNA (e.g., the mRNA sequence encoded by any one of SEQ ID NOs. 53–57)). However, it should be understood that in some embodiments, antisense nucleic acids contain less than 100% sequence complementarity with the target nucleic acid (e.g., 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the nucleotides of the ASO hybridize to nucleotides of the target RNA, such as the target mRNA (e.g., the mRNA sequence encoded by any one of SEQ ID NOs. 53–57)). In addition, to minimize the likelihood of off-target effects, antisense nucleic acids may be designed to ensure that they do not contain sequences (e.g., five or more consecutive nucleotides) that are complementary to off-target nucleic acids (e.g., mRNA not transcribed from the APOE gene).

[0067] In some embodiments, an antisense oligonucleotide (e.g., an ASO as described herein) includes a region complementary to mRNA encoded by (e.g., transcribed from) the APOE gene. In some embodiments, an antisense nucleic acid oligonucleotide includes a region complementary to mRNA encoded by a sequence defined in any one of SEQ ID NOs. 53-57. In some embodiments, the complementary region of the antisense nucleic acid hybridizes with at least six, e.g., at least seven, at least eight, at least nine, at least ten, at least fifteen, or more consecutive nucleotides of the target nucleic acid (e.g., mRNA encoded by a sequence defined in any one of SEQ ID NOs. 53-57). In some embodiments, an antisense oligonucleotide includes a region complementary to the 5'UTR, 3'UTR, intron sequence, exon sequence, splice donor sequence, splice acceptor sequence, or lariat branch point encoded by the human APOE gene. In some embodiments, an antisense oligonucleotide includes a region complementary to the 3'UTR encoded by the human APOE gene. In some embodiments, the antisense oligonucleotide contains a region complementary to an exon encoded by the human APOE gene, such as exon 4. In some embodiments, the oligonucleotide binds to mRNA expressed from a specific allele of APOE (e.g., binds to a target mRNA in an allele-specific manner).

[0068] In some embodiments, an antisense oligonucleotide (e.g., an ASO as described herein) includes a region complementary to mRNA encoded by (e.g., transcribed from) the APOE gene. In some embodiments, the antisense oligonucleotide includes a region complementary to a premRNA sequence encoded by, for example, the human APOE gene (e.g., Ensembl ID NO:ENST00000252486.9, chromosome 19:44,905,796-44,909,393 forward strand). In some embodiments, the complementary region of the antisense nucleic acid hybridizes with at least 6, e.g., at least 7, at least 8, at least 9, at least 10, at least 15, or more consecutive nucleotides of the target nucleic acid (e.g., premRNA encoded by Ensembl ID NO:ENST00000252486.9, chromosome 19:44,905,796-44,909,393 forward strand). In some embodiments, the antisense oligonucleotide comprises a region complementary to at least six, e.g., at least seven, at least eight, at least nine, at least ten, at least fifteen, or more consecutive nucleotides of an intron encoded by the forward strand of Ensembl ID NO:ENST00000252486.9, chromosome 19:44,905,796-44,909,393. Those skilled in the art will recognize that a premRNA transcript or the reverse strand of such nucleic acid encoding an mRNA transcript may also be targeted.

[0069] In some embodiments, an antisense oligonucleotide (e.g., an ASO as described herein) comprises an mRNA encoded by a sequence defined by any one of SEQ ID NOs: 53-57 and a complementary region which is complementary to 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 consecutive nucleotides. In some embodiments, an antisense oligonucleotide containing a region complementary to the mRNA transcript encoded by any one of SEQ ID NOs. 53-57 contains at least 60% sequence identity (e.g., 60-70%, 70-80%, 80-90%, 90-95%, or more than 95%) to the nucleic acid sequence defined by any one of SEQ ID NOs. 1-52 listed in column A of Table 1. In some embodiments, the antisense oligonucleotide contains a sequence of 10 or more consecutive nucleotides (e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, or more consecutive nucleotides) to any one of the sequences defined by SEQ ID NOs. 1-52 listed in column A of Table 1. In some embodiments, the antisense oligonucleotide contains a nucleic acid sequence defined by any one of SEQ ID NOs. 1-52 listed in column A of Table 1. In some embodiments, the antisense oligonucleotide comprises a nucleotide sequence having one or more mismatches (e.g., one or more bases that are not complementary to a nucleotide at a given position in the target mRNA) relative to the mRNA transcript encoded by the sequence defined in any one of SEQ ID NOs: 53-57. In some embodiments, the antisense oligonucleotide comprises a sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mismatches relative to the mRNA transcript encoded by the sequence defined in any one of SEQ ID NOs: 53-57.In some embodiments, an antisense oligonucleotide containing one or more mismatches relative to an mRNA transcript encoded by any one of sequence numbers 53-57 contains at least 60% sequence identity (e.g., 60-70%, 70-80%, 80-90%, 90-95%, or more than 95%) to a sequence of 10 or more consecutive nucleotides of any one of the sequences defined by sequence numbers 1-52 listed in column A of Table 1. In some embodiments, an antisense oligonucleotide having at least 60% sequence identity with respect to a sequence of 10 or more consecutive nucleotides (e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, or more consecutive nucleotides) of any one of the sequences defined by SEQ ID NOs: 1 to 52 is different at one or more nucleotide positions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more nucleotide positions, including substitutions, insertions, or deletions) relative to a sequence of 10 or more consecutive nucleotides of any one of the sequences defined by SEQ ID NOs: 1 to 52 listed in column A of Table 1. In some embodiments, an antisense oligonucleotide containing one or more mismatches relative to the mRNA transcript encoded by any one of SEQ ID NOs: 53-57 contains at least 60% sequence identity with respect to the nucleic acid sequence defined by any one of SEQ ID NOs: 1-52 (e.g., 60-70%, 70-80%, 80-90%, 90-95%, or more than 95% sequence identity). In some embodiments, an antisense oligonucleotide containing at least 60% sequence identity with respect to the nucleic acid sequence defined by any one of SEQ ID NOs: 1-52 differs relative to the nucleic acid sequence defined by any one of SEQ ID NOs: 1-52 at one or more nucleotide positions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more nucleotide positions, including substitutions, insertions, or deletions).

[0070] In some embodiments, the RNA processing regulator (e.g., antisense oligonucleotide) is provided in a homogeneous preparation in which, for example, at least 85%, at least 90%, at least 95%, or at least 99% of the RNA processing regulator (e.g., antisense oligonucleotide) is identical. In some embodiments, the homogeneous preparation is sterically pure (e.g., diastereomer). For example, in some embodiments, a homogeneous preparation of antisense oligonucleotide is provided in which at least 85%, at least 90%, at least 95%, or at least 99% of the oligonucleotide in the preparation is 10–25 nucleotides in length and includes a complementary region that is complementary to at least six consecutive nucleotides of an mRNA transcript encoded by an APOE gene (e.g., an APOE gene encoding an mRNA containing a nucleic acid sequence defined by any one of sequence numbers 53–57). In some embodiments, the RNA processing modifier (e.g., antisense oligonucleotide) is provided in a heterogeneous preparation comprising, for example, at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or more different RNA processing modifiers (e.g., antisense oligonucleotides, each targeting a different sequence of APOE mRNA transcript).

[0071] The RNA processing modulators (e.g., antisense oligonucleotides) of this disclosure can be modified to achieve one or more desired properties, such as improved cellular uptake, improved stability, reduced immunogenicity, improved titer, improved target hybridization, sensitivity to RNAse cleavage, etc. In some embodiments, the antisense nucleic acid is modified so that, when present in cells containing the APOE gene, it can hybridize with RNA transcribed from the APOE gene without inducing RNA cleavage by RNase. In some embodiments, the antisense nucleic acid is modified so that, when present in cells containing the APOE gene, it can hybridize with RNA transcribed from the APOE gene and induce RNA cleavage by RNase.

[0072] RNA processing regulators (e.g., antisense oligonucleotides; for example, nucleic acid sequences defined by any one of SEQ ID NOs. 1-52 listed in column A of Table 1) can be modified in the base portion, sugar portion, and / or phosphate backbone. Therefore, an RNA processing regulator (e.g., antisense oligonucleotide) may have one or more modified nucleotides (e.g., nucleotide analogs) and / or one or more backbone modifications (e.g., modified internucleotide linkages). An RNA processing regulator (e.g., antisense oligonucleotide) may have combinations of modified and unmodified nucleotides. An RNA processing regulator (e.g., antisense oligonucleotide) may also have combinations of modified and unmodified internucleotide linkages. An RNA processing regulator (e.g., antisense oligonucleotide) may, for example, include one or more chemical modifications (or combinations of chemical modifications) from column C of Table 1, in combination with any basic nucleotide sequence from column A of Table 1. In some embodiments, the RNA processing regulator comprises a nucleic acid sequence from column A of Table 1 and one or more chemical modifications (or combinations of chemical modifications) from column C of Table 1, optionally where columns A and C are from the same row of Table 1.

[0073] In some embodiments, one or more modifications are between 1 and 50 modifications, 2 and 20 modifications, 5 and 30 modifications, 10 and 40 modifications, or between 15 and 50 modifications. In some embodiments, RNA processing regulators (e.g., antisense oligonucleotides) include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 modifications. In some embodiments, the RNA processing regulator (e.g., antisense oligonucleotide) contains more than 50 modifications (e.g., 60, 70, 80, 90, 100 modifications, etc.). In some embodiments, the RNA processing regulator (e.g., antisense oligonucleotide) contains at most 100 modifications. In some embodiments, the RNA processing regulator (e.g., antisense oligonucleotide) contains chemical modifications on each nucleotide and each sugar-phosphate backbone linkage. Such modified RNA processing regulator (e.g., antisense oligonucleotide) may be referred to as a "fully modified" RNA processing regulator (e.g., antisense oligonucleotide). In some embodiments, a fully modified antisense oligonucleotide contains or consists of one of the nucleic acid sequences of SEQ ID NOs: 1 to 52. In some embodiments, not all nucleotides of the antisense oligonucleotide are modified.

[0074] RNA processing regulators (e.g., antisense oligonucleotides) may include ribonucleotides, deoxyribonucleotides, and combinations thereof (e.g., RNA processing regulators containing gapmer structures). Examples of modified nucleotides that can be used as antisense nucleic acids include, for example, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxymethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueusin, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, and 7-methylguanine. This includes 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueucine, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid(v), weybutoxosin, pseudouracil, queucine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methyl ester, uracil-5-oxyacetic acid(v), 5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl)uracil, and 2,6-diaminopurine.

[0075] In some embodiments, the modified nucleotide is a 2'-modified nucleotide. For example, 2'-modified nucleotides may be 2'-deoxy, 2'-fluoro, 2'-O-methyl, 2'-O-methoxyethyl, 2'-amino, and 2'-aminoalkoxy modified nucleotides. In some embodiments, the 2'-modified nucleotide includes a 2'-O-4'-C methylene bridge, such as a LOK nucleic acid (LNA) nucleotide. In some embodiments of the 2'-modified nucleotide, the 2'-hydroxyl group is linked to the 3' or 4' carbon atom of the sugar ring, thereby forming a bicyclic sugar moiety. In such embodiments, the linkage is a methylene(-CH2-) bridge between the 2' oxygen atom and the 3' or 4' carbon atom. n It can be a group, where n is 1 or 2. In some embodiments, the linkage involves cEt modification (e.g., -CH3 replacing a hydrogen on the methylene group of the bridge).

[0076] RNA processing modifiers (e.g., antisense oligonucleotides) may encompass combinations of LNA nucleotides and unmodified nucleotides. Antisense nucleic acids may encompass combinations of LNA and RNA nucleotides. Antisense nucleic acids may encompass combinations of LNA and DNA nucleotides. A more preferred oligonucleotide modification encompasses loc nucleic acid (LNA), in which a 2'-hydroxyl group is linked to the 3' or 4' carbon atom of a sugar ring, thereby forming a bicyclic sugar moiety.

[0077] The acids of RNA processing regulators (e.g., antisense oligonucleotides) may also include nucleotides with modified nucleic acid bases, such as nucleotides containing non-naturally occurring nucleic acid bases instead of naturally occurring ones. The bases may be modified to block the activity of, for example, adenosine deaminase. Examples of modified nucleic acid bases include, but are not limited to, uridine and / or cytidine modified at the 5-position, e.g., 5-(2-amino)propyluridine, 5-bromouridine; adenosine and / or guanosine modified at the 8-position, e.g., 8-bromoguanosine; deazanucleotides, e.g., 7-deazaadenosine; and O- and N-alkylated nucleotides, for example, N6-methyladenosine. It should be noted that the above modifications may be combined.

[0078] Within the antisense nucleic acids (e.g., antisense oligonucleotides) of this disclosure, as few as one and as many as all nucleotides may be modified. In some embodiments, a modified RNA processing modulator (e.g., an antisense oligonucleotide) will contain as few modified nucleotides as necessary to achieve a desired level of in vivo stability and / or bioaccessibility or other desired properties.

[0079] Certain antisense oligonucleotides may include nonionic DNA analogs, such as alkyl- and aryl-phosphonates (in which the charged non-crosslinked oxygen is replaced by an alkyl or aryl group), phosphodiesters, and alkylphosphotriesters in which the charged oxygen moiety is alkylated. Nucleic acids containing diols such as tetraethylene glycol or hexaethylene glycol at either or both ends have also been shown to be substantially resistant to nuclease degradation and may be used herein. In some embodiments, antisense nucleic acids may include at least one lipophilic-substituted nucleotide analog and / or pyrimidine-purine dinucleotide.

[0080] In some embodiments, RNA processing modifiers (e.g., antisense oligonucleotides) may have one or two accessible 5' ends. For example, modified oligonucleotides having two such 5' ends can be produced by attaching two oligonucleotides through a 3'-3' linkage to generate oligonucleotides having one or two accessible 5' ends. The 3'-3' linkage may be a phosphodiester, a phosphorothioate, or any other modified nucleoside crosslink. In addition, 3'-3' linked oligonucleotides where the linkage between the 3' terminal nucleosides is neither a phosphodiester nor a phosphorothioate nor any other modified crosslink can be prepared using additional spacers such as tri- or tetraethylene glycol phosphate moieties.

[0081] The phosphodiester nucleotide linkages of RNA processing regulators (e.g., antisense oligonucleotides) can be replaced by modified linkages. These modified linkages may be selected from, for example, phosphorothioates, phosphorodithioates, NR1R2-phosphoamidates, boranophosphates, α-hydroxybenzylphosphonates, phosphate-(C1-C21)-O-alkyl esters, phosphate-[(C6-C12)aryl-(C1-C21)-O-alkyl] esters, (C1-C8)alkylphosphonates and / or (C6-C12)arylphosphonate crosslinks, and (C7-C12)-α-hydroxymethyl-aryl. In some embodiments, triazole rings are used.

[0082] The phosphate backbone of RNA processing regulators (e.g., antisense oligonucleotides) can be modified to generate peptide nucleic acid molecules. As used herein, the term “peptide nucleic acid” or “PNA” refers to a nucleic acid mimic, e.g., a DNA mimic, in which the deoxyribose phosphate backbone is replaced by a pseudopeptide backbone, retaining only four native nucleic acid bases. The neutral backbone of PNA has been shown to allow specific hybridization to DNA and RNA under low ionic strength conditions. Synthesis of PNA oligomers can be carried out, for example, using standard solid-phase peptide synthesis protocols.

[0083] RNA processing regulators (e.g., antisense oligonucleotides) are also formulated as morpholino oligonucleotides. In this embodiment, the riboside portion of each subunit of the oligonucleotide reagent is converted to the morpholine portion. Morpholino can also be modified, for example, as peptide-conjugated morpholino.

[0084] Aspects of this disclosure relate to RNA processing regulators (e.g., antisense oligonucleotides) containing a "gapmer" structure. The "gapmer" is defined by the following formula X n1 -(Y) n2 -(X) n3The term "gapmer antisense oligonucleotide" refers to an antisense oligonucleotide containing (X) a ribonucleotide (e.g., an RNA base) and (Y) a deoxyribonucleotide (e.g., a DNA base), where n1, n2, and n3 are integers ranging from 1 to 50 (including all integers in between). In some embodiments, antisense oligonucleotides having a gapmer structure bind to (e.g., hybridize) a target mRNA (e.g., mRNA encoded by the APOE gene) and induce ribonuclease H1 (RNAseH1)-mediated degradation of the target mRNA. Gapmer antisense oligonucleotides are well known in the art and are described, for example, by Kasuya et al. Sci Rep. 2016; 6:30377.

[0085] The number of DNA bases on a gapmer can vary. In some embodiments, the gapmer contains between 1 and 10 DNA bases (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 DNA bases). In some embodiments, the gapmer contains between 2 and 6 DNA bases (e.g., 2, 3, 4, 5, or 6 DNA bases). The DNA bases of the gapmer antisense oligonucleotide may be located towards the 5' end of the ASO (e.g., within 1, 2, 3, 4, 5 nucleotides of the 5' terminal nucleotide of the ASO), towards the 3' end of the ASO (e.g., within 1, 2, 3, 4, 5 nucleotides of the 3' terminal nucleotide of the ASO), or in the central part of the ASO (e.g., having an equal number of RNA bases flanking the DNA bases).

[0086] In other embodiments, RNA processing modulators (e.g., antisense oligonucleotides) may be linked to functional groups such as peptides (e.g., for targeting host cell receptors in vivo) or agents that facilitate transport across the cell membrane or blood-brain barrier. For example, the oligonucleotide reagents of this disclosure may also be modified by chemical moieties (e.g., cholesterol) that improve the in vivo pharmacological properties of the RNA processing modulator. In some embodiments, the functional groups include peptides, small molecules, sugars, lipids, nucleic acids, or any combination thereof.

[0087] The sequences and chemical modifications of representative RNA processing modulators (e.g., antisense oligonucleotides) that target APOE (e.g., mRNA encoded by the APOE gene, such as pre-mRNA or mature mRNA) are shown in columns A and C of Table 1, respectively.

[0088] Table 1: Representative RPMs that target APOE [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5]

[0089] In some embodiments, an antisense oligonucleotide (e.g., an ASO as described herein) contains or consists of one, two, three, four, five, six, seven, eight, nine, ten, one, two, three, four, five, six, seven, eight, nine, ten, one, one, one, two, one, three, three, four, five, one, six, seven, eight, nine, one, one, two, three In some embodiments, an antisense oligonucleotide comprises or consists of one, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 consecutive nucleotides from any one of the nucleotide sequences defined by SEQ ID NOs. 1 to 52 listed in column A of Table 1, and the corresponding chemical modification patterns of the SEQ ID NOs.

[0090] In some embodiments, an antisense oligonucleotide (e.g., an ASO as described herein) contains or consists of one, two, three, four, five, six, seven, eight, nine, ten, one, two, three, four, five, six, seven, eight, nine, ten, one, two, one, three, four, five, six, five, five, six In some embodiments, an antisense oligonucleotide comprises or consists of one, two, three, four, five, six, seven, eight, nine, ten,

[0091] In some embodiments, an antisense oligonucleotide (e.g., an ASO as described herein) contains or consists of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 consecutive nucleotides in any one of the nucleotide sequences defined by SEQ ID NOs. 13, 14, 15, 17, 17, 17, and 46 listed in column A of Table 1. In some embodiments, an antisense oligonucleotide contains one or more of the chemical modifications described herein. In some embodiments, an antisense oligonucleotide contains the chemical modification patterns in column C of Table 1 corresponding to any one of the nucleotide sequences defined by SEQ ID NOs. 1 to 52 listed in column A of Table 1. In some embodiments, an antisense oligonucleotide comprises or consists of one, two, three, four, five, six, seven, eight, nine, ten, one, two, three, four, five, six, seven, eight, nine, ten, one, two, three, seven, eight, nine, six

[0092] In some embodiments, the RNA processing regulator (e.g., antisense oligonucleotide) comprises at least 18 consecutive nucleotides from any one of the nucleic acid sequences defined in SEQ ID NOs: 1-52 (e.g., comprising or consisting of 18 nucleotides, 19 nucleotides, or 20 nucleotides) (see column A of Table 1). In some embodiments, the RNA processing regulator comprises 18 consecutive nucleotides from any one of the nucleic acid sequences defined in column A of Table 1, labeled "18mer" in column C of the same row in Table 1. In some embodiments, the RNA processing modulator comprises 18 consecutive nucleotides from any one of the nucleic acid sequences defined in column A of Table 1, labeled "18mer" in column C of the same row of Table 1, and includes one additional nucleotide (either at the 5' or 3' end) or two additional nucleotides (both at the 5' end, both at the 3' end, or one at the 5' end and the other at the 3' end), which are complementary to the target sequence on APOE mRNA that hybridize to 18 consecutive nucleotides of the nucleic acid sequence selected from column A of Table 1. In some embodiments, the RNA processing modulator comprises 19 consecutive nucleotides from any one of the nucleic acid sequences defined in column A of Table 1, labeled "20mer" in column C of the same row of Table 1, and includes one additional nucleotide at either the 5' or 3' end, which are complementary to the target sequence on APOE mRNA that hybridize to 20 consecutive nucleotides of the nucleic acid sequence selected from column A of Table 1. In some embodiments, the RNA processing regulator comprises or consists of 20 consecutive nucleotides from any one of the nucleic acid sequences defined in column A of Table 1, which are labeled "20mer" in column C of the same row in Table 1.In some embodiments, the RNA processing modulator comprises 20 consecutive nucleotides from any one of the nucleic acid sequences defined in column A of Table 1, labeled "20mer" in column C of the same row in Table 1, and comprises one or more additional nucleotides at either the 5' end, the 3' end, or both the 5' and 3' ends, which are complementary to the target sequence on the APOE mRNA that hybridizes to the 20 consecutive nucleotides of the nucleic acid sequence selected from column A of Table 1. In some embodiments, the RNA processing modulator comprising at least 18 consecutive nucleotides from any one of the nucleic acid sequences defined in SEQ ID NOs: 1-52 (e.g., an ASO comprising or consisting of 18, 19, or 20 consecutive nucleotides from any one of the nucleic acid sequences shown in column A of Table 1) comprises one or more chemical modifications defined in any one of the rows in column C of Table 1. In some embodiments, an RNA processing modulator containing at least 18 consecutive nucleotides from any one of the nucleic acid sequences defined by SEQ ID NOs: 1-52 (e.g., an ASO containing or consisting of 18, 19, or 20 consecutive nucleotides from any one of the nucleic acid sequences shown in column A of Table 1) includes a pattern of chemical modification defined in any one of the rows in column C of Table 1. In some embodiments, the at least 18 consecutive nucleotides contained in the RNA processing modulator are defined in any one of the nucleotide sequences defined by SEQ ID NOs: 2-3, 5-6, 8-18, 20-25, 27, 29-31, 34-42, 44, 46, 48, and 50-52. In some embodiments, the at least 18 consecutive nucleotides contained in the RNA processing modulator are defined in the nucleic acid sequence of SEQ ID NO: 5. In some embodiments, the at least 18 consecutive nucleotides contained in the RNA processing modulator are defined in the nucleic acid sequence of SEQ ID NO: 11. In some embodiments, the RNA processing regulator comprises at least 18 consecutive nucleotides defined by the nucleic acid sequence of Sequence ID No. 16.In some embodiments, the RNA processing regulator consists of at least 18 consecutive nucleotides defined by the nucleic acid sequence of SEQ ID NO: 22. In some embodiments, the RNA processing regulator consists of at least 18 consecutive nucleotides defined by the nucleic acid sequence of SEQ ID NO: 29. In some embodiments, the RNA processing regulator consists of at least 18 consecutive nucleotides defined by the nucleic acid sequence of SEQ ID NO: 50. In some embodiments, an RNA processing modulator comprising at least 18 consecutive nucleotides of any one nucleic acid sequence defined in Sequence ID No. 1 to 52 reduces the levels of APOE mRNA (e.g., mature mRNA or premRNA) and / or APOE protein by 50% or more (e.g., 50-60%, 60-70%, 70-80%, 80-90%, 90-95%, or 95-100%) in cells or one or more tissues in a subject (e.g., cerebrospinal fluid, plasma, and / or brain tissue) when the RNA processing modulator or a composition thereof is administered to the subject in an effective amount. In some embodiments, an RNA processing modulator comprising at least 18 consecutive nucleotides of any one nucleic acid sequence defined in SEQ ID NOs. 5, 11, 16, 22, 29, and 50 reduces the levels of APOE mRNA (e.g., mature mRNA or premRNA) and / or APOE protein by 50% or more (e.g., 50-60%, 60-70%, 70-80%, 80-90%, 90-95%, or 95-100%) in cells or one or more tissues in a subject (e.g., cerebrospinal fluid, plasma, and / or brain tissue) when the RNA processing modulator or a composition thereof is administered to the subject in an effective amount.

[0093] In some embodiments, the RNA processing regulator contains or consists of 18 consecutive nucleotides from any one of the nucleic acid sequences defined in SEQ ID NOs: 1-52, 19 consecutive nucleotides from any one of them, or 20 consecutive nucleotides from any one of them (see column A of Table 1), where one or more positions containing a "T" residue are replaced with a "U" residue. In some embodiments, an RNA processing regulator comprising at least 18 consecutive nucleotides from any one nucleic acid sequence defined in Sequence ID No. 1 to 52 (e.g., an ASO comprising or consisting of 18 nucleotides, 19 nucleotides, or 20 consecutive nucleotides from any one nucleic acid sequence shown in column A of Table 1), wherein one or more positions containing "T" residues are substituted with "U" residues, and the RNA processing regulator comprises one or more chemical modifications defined in any one row of column C of Table 1. In some embodiments, one or more positions on an RNA processing regulator containing a "U" residue include uracil nitrogen bases or chemically modified uracil nitrogen bases as described herein, and deoxyribose sugars or chemically modified deoxyribose sugars as described herein.In some embodiments, at least 18 consecutive nucleotides in the RNA processing regulator are defined by the nucleic acid sequence of SEQ ID NO: 5, where one or more positions on SEQ ID NO: 5 containing a "T" residue (e.g., each position on SEQ ID NO: 5 containing a "T" residue) are replaced with a "U" residue. In some embodiments, at least 18 consecutive nucleotides in the RNA processing regulator are defined by the nucleic acid sequence of SEQ ID NO: 11, where one or more positions on SEQ ID NO: 11 containing a "T" residue (e.g., each position on SEQ ID NO: 11 containing a "T" residue) are replaced with a "U" residue. In some embodiments, at least 18 consecutive nucleotides in the RNA processing regulator are defined by the nucleic acid sequence of SEQ ID NO: 16, where one or more positions on SEQ ID NO: 16 containing a "T" residue (e.g., each position on SEQ ID NO: 16 containing a "T" residue) are replaced with a "U" residue. In some embodiments, the RNA processing regulator consists of at least 18 consecutive nucleotides defined by the nucleic acid sequence of Sequence ID No. 22, where one or more positions on Sequence ID No. 22 containing a "T" residue (e.g., each position on Sequence ID No. 22 containing a "T" residue) are replaced with a "U" residue. In some embodiments, the RNA processing regulator consists of at least 18 consecutive nucleotides defined by the nucleic acid sequence of Sequence ID No. 29, where one or more positions on Sequence ID No. 29 containing a "T" residue (e.g., each position on Sequence ID No. 29 containing a "T" residue) are replaced with a "U" residue. In some embodiments, the RNA processing regulator consists of at least 18 consecutive nucleotides defined by the nucleic acid sequence of Sequence ID No. 50, where one or more positions on Sequence ID No. 50 containing a "T" residue (e.g., each position on Sequence ID No. 50 containing a "T" residue) are replaced with a "U" residue.

[0094] In some embodiments, the RNA processing regulator (e.g., antisense oligonucleotide) comprises a skipper structure, where each nucleotide position comprises a ribose sugar, which is linked to a nitrogenase base (e.g., thymine (T), cytosine (C), guanine (G), adenine (A), or uracil (U)), the ribose sugar comprises a 2'-O-methoxyethyl (-OCH2CH2OCH3(2'MOE)) modification, and each nucleotide position is linked by phosphorothioate or phosphodiester linkage. In some embodiments, each nucleotide position is linked by phosphorothioate linkage. In some embodiments, the RNA processing regulator (e.g., antisense oligonucleotide) comprising the skipper structure comprises or consists of 18 consecutive nucleotides from any one of the nucleic acid sequences defined by SEQ ID NOs: 1-52 (see column A in Table 1). In some embodiments, an RNA processing regulator (e.g., an antisense oligonucleotide) containing a skipper structure contains or consists of 18 consecutive nucleotides from any one of the nucleic acid sequences defined by SEQ ID NOs: 1-52 (see column A of Table 1), where one or more positions containing thymine (T) on the nucleic acid sequence are replaced by uracil (U). In some embodiments, an RNA processing regulator (e.g., an antisense oligonucleotide) containing a skipper structure contains or consists of 18 consecutive nucleotides from any one of the nucleic acid sequences defined by SEQ ID NOs: 3, 5, 7, 11, 16, 22, 29, 31, 34, 43, or 48-50. In some embodiments, an RNA processing regulator (e.g., an antisense oligonucleotide) containing a skipper structure contains or consists of 18 consecutive nucleotides from the nucleic acid sequence of SEQ ID NO: 5. In some embodiments, an RNA processing regulator (e.g., an antisense oligonucleotide) containing a skipper structure contains or consists of 18 consecutive nucleotides from the nucleic acid sequence of SEQ ID NO: 11. In some embodiments, the RNA processing regulator (e.g., antisense oligonucleotide) containing a skipper structure comprises or consists of 18 consecutive nucleotides of the nucleic acid sequence of SEQ ID NO: 16.In some embodiments, an RNA processing regulator (e.g., an antisense oligonucleotide) containing a skipper structure contains or comprises 18 consecutive nucleotides of the nucleic acid sequence of SEQ ID NO: 22. In some embodiments, an RNA processing regulator (e.g., an antisense oligonucleotide) containing a skipper structure contains or comprises 18 consecutive nucleotides of the nucleic acid sequence of SEQ ID NO: 29. In some embodiments, an RNA processing regulator (e.g., an antisense oligonucleotide) containing a skipper structure contains or comprises 18 consecutive nucleotides of the nucleic acid sequence of SEQ ID NO: 50. In some embodiments, RNA processing modulators comprising a skipper structure reduce the levels of APOE mRNA (e.g., mature mRNA or premRNA) and / or APOE protein in cells or one or more tissues in a subject (e.g., cerebrospinal fluid, plasma, and / or brain tissue) by 50% or more (e.g., 50-60%, 60-70%, 70-80%, 80-90%, 90-95%, or 95-100%) when the RNA processing modulator or a composition thereof is administered to the subject in an effective amount.

[0095] In some embodiments, the RNA processing regulator (e.g., antisense oligonucleotide) comprises a gapmer structure in which a region of 10 deoxyribonucleotides is flanked by regions each containing 4 ribonucleotides (thus resulting in a total of 8 ribonucleotides and 10 deoxyribonucleotides). In some embodiments, the 1, 2, 3, or 4 ribonucleotides in each region flanking the region of 10 deoxyribonucleotides contain a 2'-O-methoxyethyl (-OCH2CH2OCH3(2'MOE)) modification. In some embodiments, the 1, 2, 3, or 4 ribose sugars contained in each region flanking the region of 10 deoxyribonucleotides are linked by phosphorothioate or phosphodiester linkages. In some embodiments, 1 to 10 deoxyribose sugars (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 deoxyribose sugars) contained within a region of 10 deoxyribonucleotides are linked by phosphorothioate linkages. In some embodiments, one or more ribose sugars (e.g., 1, 2, 3, or 4 ribose sugars) contained within each region flanking the region of 10 deoxyribonucleotides are linked by phosphorothioate linkages. In some embodiments, 16 of the 18 positions are linked by phosphorothioate linkages. In some embodiments, 16 of the 18 positions are linked by phosphorothioate linkages, where the second position is linked to the third position (relative to the 5' end) by a phosphodiester linkage, and the 16th position is linked to the 17th position (relative to the 5' end) by a phosphodiester linkage. In some embodiments, RNA processing regulators containing a gapmer structure (e.g., antisense oligonucleotides) contain or consist of 18 consecutive nucleotides from any one of the nucleic acid sequences defined by SEQ ID NOs: 1-52 (see column A in Table 1).In some embodiments, an RNA processing regulator (e.g., an antisense oligonucleotide) containing a gapmer structure contains or consists of 18 consecutive nucleotides of the nucleic acid sequence of SEQ ID NO: 5. In some embodiments, an RNA processing regulator (e.g., an antisense oligonucleotide) containing a gapmer structure contains or consists of 18 consecutive nucleotides of the nucleic acid sequence of SEQ ID NO: 11. In some embodiments, an RNA processing regulator (e.g., an antisense oligonucleotide) containing a gapmer structure contains or consists of 18 consecutive nucleotides of the nucleic acid sequence of SEQ ID NO: 16. In some embodiments, an RNA processing regulator (e.g., an antisense oligonucleotide) containing a gapmer structure contains or consists of 18 consecutive nucleotides of the nucleic acid sequence of SEQ ID NO: 22. In some embodiments, an RNA processing regulator (e.g., an antisense oligonucleotide) containing a gapmer structure contains or consists of 18 consecutive nucleotides of the nucleic acid sequence of SEQ ID NO: 29. In some embodiments, an RNA processing regulator (e.g., an antisense oligonucleotide) containing a gapmer structure contains or consists of 18 consecutive nucleotides of the nucleic acid sequence of SEQ ID NO: 50. In some embodiments, RNA processing modulators comprising a gapmer structure reduce the levels of APOE mRNA (e.g., mature mRNA or premRNA) and / or APOE protein in cells or one or more tissues in a subject (e.g., cerebrospinal fluid, plasma, and / or brain tissue) by 50% or more (e.g., 50-60%, 60-70%, 70-80%, 80-90%, 90-95%, or 95-100%) when the RNA processing modulator or a composition thereof is administered to the subject in an effective amount.

[0096] In some embodiments, the RNA processing regulator (e.g., antisense oligonucleotide) comprises a gapmer structure in which a region of 10 deoxyribonucleotides is flanked by regions each containing 5 ribonucleotides (thus resulting in a total of 10 ribonucleotides and 10 deoxyribonucleotides). In some embodiments, the 1, 2, 3, 4, or 5 ribonucleotides in each region flanking the region of 10 deoxyribonucleotides comprises a 2'-O-methoxyethyl (-OCH2CH2OCH3(2'MOE)) modification. In some embodiments, the 1, 2, 3, 4, or 5 ribose sugars contained in each region flanking the region of 10 deoxyribonucleotides are linked by phosphorothioate or phosphodiester linkages. In some embodiments, 1 to 10 deoxyribose sugars (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 deoxyribose sugars) within a region of 10 deoxyribonucleotides are linked by phosphorothioate linkages. In some embodiments, one or more ribose sugars (e.g., 1, 2, 3, 4, or 5 ribose sugars) within each region flanking the region of 10 deoxyribonucleotides are linked by phosphorothioate linkages. In some embodiments, 16 of the 20 positions are linked by phosphorothioate linkages. In some embodiments, 16 of the 20 positions are linked by phosphorothioate linkages, where the second and third positions (relative to the 5' end), the third and fourth positions (relative to the 5' end), the 17th and 18th positions (relative to the 5' end), and the 18th and 19th positions (relative to the 5' end) are each linked by phosphodiester linkages. In some embodiments, 18 of the 20 positions are linked by phosphorothioate linkages.In some embodiments, 18 of the 20 positions are linked by phosphorothioate linkages, where the second and third positions (relative to the 5' end) are linked by phosphodiester linkages, and the 17th and 18th positions (relative to the 5' end) are linked by phosphodiester linkages. In some embodiments, the RNA processing regulator (e.g., antisense oligonucleotide) containing the gapmer structure contains or consists of 20 consecutive nucleotides from any one of the nucleic acid sequences defined by SEQ ID NOs: 1 to 52 (see column A in Table 1). In some embodiments, the RNA processing regulator (e.g., antisense oligonucleotide) containing the gapmer structure contains or consists of 20 consecutive nucleotides from the nucleic acid sequence of SEQ ID NO: 16. In some embodiments, the RNA processing regulator (e.g., antisense oligonucleotide) containing the gapmer structure contains or consists of 20 consecutive nucleotides from the nucleic acid sequence of SEQ ID NO: 22. In some embodiments, an RNA processing modulator (e.g., an antisense oligonucleotide) containing a gapmer structure contains or comprises 20 consecutive nucleotides of the nucleic acid sequence of SEQ ID NO: 29. In some embodiments, an RNA processing modulator (e.g., an antisense oligonucleotide) containing a gapmer structure contains or comprises 20 consecutive nucleotides of the nucleic acid sequence of SEQ ID NO: 50. In some embodiments, an RNA processing modulator containing a gapmer structure reduces the levels of APOE mRNA (e.g., mature mRNA or premRNA) and / or APOE protein by 50% or more (e.g., 50-60%, 60-70%, 70-80%, 80-90%, 90-95%, or 95-100%) in cells or one or more tissues in a subject (e.g., cerebrospinal fluid, plasma, and / or brain tissue) when the RNA processing modulator or a composition thereof is administered to the subject in an effective amount.

[0097] In some embodiments, the antisense oligonucleotide (e.g., ASO as described herein) includes or consists of a region complementary to the 3'UTR of APOE RNA (see, for example, Figure 5). In some embodiments, the antisense oligonucleotide includes or consists of a region having complementarity to the 3'UTR found 1 to 4000 nucleotides downstream of the last exon of APOE RNA. In some embodiments, the antisense oligonucleotide includes regions 1 to 10, 10 to 20, 20 to 30, 30 to 40, 40 to 50, 50 to 60, 60 to 70, 70 to 80, 80 to 90, 90 to 100, 100 to 200, 200 to 300, 300 to 400, 400 to 500, 500 to 600, 600 to 700, 700 to 800, 800 to 900 of the last exon of APOE RNA. 900-1,000, 1,000-1,100, 1,100-1,200, 1,200-1,300, 1,300-1,400, 1,400-1,500, 1,500-1,600, 1,600-1,700, 1,700-1,800, 1,800-1,900, 1,900-2,000, 2,000 It contains or consists of a region that is complementary to the 3'UTR found downstream of ~2,100, 2,100~2,200, 2,200~2,300, 2,300~2,400, 2,400~2,500, 2,500~2,600, 2,600~2,700, 2,700~2,800, 2,800~2,900, 2,900~3,000, 3,000~3,100, 3,100~3,200, 3,200~3,300, 3,300~3,400, 3,400~3,500, 3,500~3,600, 3,600~3,700, 3,700~3,800, or 3,900~4,000 nucleotides. In some embodiments, the complementary region contains or consists of 1 to 20 nucleotides in length. In some embodiments, the complementary region contains or consists of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides in length. In some embodiments, the antisense oligonucleotide contains one or more of the chemical modifications described herein.In some embodiments, the antisense oligonucleotide contains the chemical modification pattern in column C of Table 1, corresponding to any one of the nucleotide sequences defined by SEQ ID NOs. 1-52 in column A of Table 1. In some embodiments, the antisense oligonucleotide contains or consists of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 consecutive nucleotides in any one of the nucleotide sequences defined by SEQ ID NOs. 3, 5, 7, 11, 16, 29, 31, 34, 43, and 48-50 in column A of Table 1. In some embodiments, an antisense oligonucleotide comprises or consists of 1 to 20 consecutive nucleotides from any one of the nucleotide sequences defined by SEQ ID NOs: 3, 5, 7, 11, 16, 29, 31, 34, 43, and 48-50 (see column A of Table 1), and a chemical modification pattern corresponding to any one of SEQ ID NOs: 1-52 as defined in column C of Table 1. In some embodiments, an antisense oligonucleotide comprises or consists of 1 to 20 consecutive nucleotides from any one of the nucleotide sequences defined by SEQ ID NOs: 3, 5, 7, 11, 16, 29, 31, 34, 43, or 48-50 (see column A of Table 1), and a chemical modification pattern corresponding to the SEQ ID NOs as defined in the same row of column C of Table 1. In some embodiments, the antisense oligonucleotide comprises 18 to 20 consecutive nucleotides from any one nucleotide sequence defined by SEQ ID NOs: 3, 5, 7, 11, 16, 29, 31, 34, 43, or 48-50 (see column A in Table 1), and the following modification patterns: complete PS; 2'MOE. In some embodiments, the antisense oligonucleotide comprises 18 consecutive nucleotides from any one nucleotide sequence defined by SEQ ID NOs: 3, 5, 7, 11, 16, 29, 31, 34, 43, or 48-50 (see column A in Table 1), and the following modification patterns: complete PS; 2'MOE; 4-10-4, PO after the second base from the 5' end; PO after the third base from the 3' end.In some embodiments, an antisense oligonucleotide consists of 20 consecutive nucleotides from any one of the nucleotide sequences defined by SEQ ID NOs: 3, 5, 7, 11, 16, 29, 31, 34, 43, or 48-50 (see column A in Table 1), and includes the following modification patterns: complete PS; 2' MOE; 5-10-5, PO after the second base from the 5' end. In any of the aforementioned embodiments: a position containing a "T" residue contains a thymine (T) nitrogen base bonded to the 1' carbon of either ribose or deoxyribose; isolated nucleic acids containing a gapmer structure are indicated by a structure denoted as "(X)-(Y)-(X)", where "(X)" refers to a region flanking a region called "(Y)", containing a number of nucleotide positions of "X" having ribose and a number of nucleotide positions of "Y" having deoxyribose; isolated nucleic acids containing a skipper structure are indicated by the absence of the notation describing "(X)-(Y)-(X)", where each nucleotide position contains ribose; "complete PS" refers to a nucleic acid containing a phosphodiester group (PO) at the 3' carbon of ribose. Unless otherwise indicated by the rheotide position, it means that each nucleotide position is linked by an internucleotide linkage containing a phosphorothioate modification; "2'MOE" refers to the 2' carbon of ribose bonded to the oxygen atom bonded to the methoxyethyl group; "#mer" refers to the number of the nucleotide position; "PO" refers to a nucleotide position where the 3' carbon of ribose contains a phosphodiester bond, which links the nucleotide position to an immediately adjacent nucleotide position in the 3' direction, where a nucleotide position containing a PO group at the 3' carbon of ribose (e.g., the second or third position from the 5' or 3' end) is indicated by the location of the PO group, which is said to be either "to" or "after".

[0098] In some embodiments, an antisense oligonucleotide (e.g., an ASO as described herein) contains or comprises a region complementary to the polyadenylated sequence of APOE RNA. In some embodiments, the complementary region contains or comprises 1 to 20 nucleotides in length. In some embodiments, the antisense oligonucleotide contains or comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides in length. In some embodiments, the antisense oligonucleotide contains one or more of the chemical modifications described herein. In some embodiments, the antisense oligonucleotide containing the complementary region contains the chemical modification pattern in column C of Table 1, corresponding to any one of the nucleotide sequences defined by SEQ ID NOs. 1 to 52 in column A of Table 1. In some embodiments, the complementary region contains or consists of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 consecutive nucleotides as defined by the nucleotide sequence of SEQ ID NO: 5 or SEQ ID NO: 34 listed in column A of Table 1. In some embodiments, the antisense oligonucleotide contains or consists of 1 to 20 consecutive nucleotides of the nucleotide sequence defined by SEQ ID NO: 5 or SEQ ID NO: 34 (see column A of Table 1), and one of the chemical modification patterns of SEQ ID NOs: 1 to 52 as defined in column C of Table 1. In some embodiments, the antisense oligonucleotide contains or consists of 1 to 20 consecutive nucleotides of the nucleotide sequence defined by SEQ ID NO: 5 or SEQ ID NO: 34 (see column A of Table 1), and the corresponding chemical modification pattern of the SEQ ID NO as defined in the same row of column C of Table 1. In some embodiments, the antisense oligonucleotide comprises 18 to 20 consecutive nucleotides of the nucleotide sequence defined in SEQ ID NO: 5 or SEQ ID NO: 34 (see column A of Table 1), and the following modification pattern: complete PS; 2'MOE.In some embodiments, the antisense oligonucleotide comprises 18 consecutive nucleotides of the nucleotide sequence defined in SEQ ID NO: 5 or SEQ ID NO: 34 (see column A in Table 1), and the following modification pattern: complete PS; 2' MOE; 4-10-4, PO after the second base from the 5' end; PO after the third base from the 3' end. In some embodiments, the antisense oligonucleotide comprises 20 consecutive nucleotides of the nucleotide sequence defined in SEQ ID NO: 5 or SEQ ID NO: 34 (see column A in Table 1), and the following modification pattern: complete PS; 2' MOE; 5-10-5, PO after the second base from the 5' end. In any of the aforementioned embodiments: a position containing a "T" residue contains a thymine (T) nitrogen base bonded to the 1' carbon of either ribose or deoxyribose; isolated nucleic acids containing a gapmer structure are indicated by a structure denoted as "(X)-(Y)-(X)", where "(X)" refers to a region flanking a region called "(Y)", containing a number of nucleotide positions of "X" having ribose and a number of nucleotide positions of "Y" having deoxyribose; isolated nucleic acids containing a skipper structure are indicated by the absence of the notation describing "(X)-(Y)-(X)", where each nucleotide position contains ribose; "complete PS" refers to a nucleic acid containing a phosphodiester group (PO) at the 3' carbon of ribose. Unless otherwise indicated by the rheotide position, it means that each nucleotide position is linked by an internucleotide linkage containing a phosphorothioate modification; "2'MOE" refers to the 2' carbon of ribose bonded to the oxygen atom bonded to the methoxyethyl group; "#mer" refers to the number of the nucleotide position; "PO" refers to a nucleotide position where the 3' carbon of ribose contains a phosphodiester bond, which links the nucleotide position to an immediately adjacent nucleotide position in the 3' direction, where a nucleotide position containing a PO group at the 3' carbon of ribose (e.g., the second or third position from the 5' or 3' end) is indicated by the location of the PO group, which is said to be either "to" or "after".

[0099] In some embodiments, an antisense oligonucleotide (e.g., an ASO as described herein) comprises or consists of a region complementary to a sequence that straddles the boundary on the 3'UTR of the APOE RNA in which the polyadenylated sequence begins. In some embodiments, the complementary region comprises or consists of 1 to 20 nucleotides in length. In some embodiments, the complementary region comprises or consists of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides in length. In some embodiments, the complementary region comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides found on the 3'UTR that is not on the polyadenylated sequence. In some embodiments, the complementary region comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides found on the polyadenylated sequence. In some embodiments, the antisense oligonucleotide comprises one or more of the chemical modifications described herein. In some embodiments, the antisense oligonucleotide comprises a chemical modification pattern defined in column C of Table 1, corresponding to any one of the nucleotide sequences defined by SEQ ID NOs. 1 to 52, listed in column A of Table 1. In some embodiments, the complementary region comprises or consists of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 consecutive nucleotides (see column A of Table 1) of any one of the nucleotide sequences defined by SEQ ID NOs. 1 to 52, and the corresponding chemical modification pattern of the SEQ ID NOs. In some embodiments, the antisense oligonucleotide comprises 18 to 20 consecutive nucleotides (see column A in Table 1) from any one of the nucleotide sequences defined by SEQ ID NOs: 3, 5, 7, 11, 16, 29, 31, 34, 43, or 48-50, and the following modification pattern: complete PS; 2'MOE.In some embodiments, the antisense oligonucleotide comprises 18 consecutive nucleotides from any one nucleotide sequence defined by SEQ ID NOs: 3, 5, 7, 11, 16, 29, 31, 34, 43, or 48-50 (see column A in Table 1), and includes the following modification pattern: complete PS; 2' MOE; 4-10-4, PO after the second base from the 5' end; PO after the third base from the 3' end. In some embodiments, the antisense oligonucleotide comprises 20 consecutive nucleotides from any one nucleotide sequence defined by SEQ ID NOs: 3, 5, 7, 11, 16, 29, 31, 34, 43, or 48-50 (see column A in Table 1), and includes the following modification pattern: complete PS; 2' MOE; 5-10-5, PO after the second base from the 5' end. In any of the aforementioned embodiments: a position containing a "T" residue contains a thymine (T) nitrogen base bonded to the 1' carbon of either ribose or deoxyribose; isolated nucleic acids containing a gapmer structure are indicated by a structure denoted as "(X)-(Y)-(X)", where "(X)" refers to a region flanking a region called "(Y)", containing a number of nucleotide positions of "X" having ribose and a number of nucleotide positions of "Y" having deoxyribose; isolated nucleic acids containing a skipper structure are indicated by the absence of the notation describing "(X)-(Y)-(X)", where each nucleotide position contains ribose; "complete PS" refers to a nucleic acid containing a phosphodiester group (PO) at the 3' carbon of ribose. Unless otherwise indicated by the rheotide position, it means that each nucleotide position is linked by an internucleotide linkage containing a phosphorothioate modification; "2'MOE" refers to the 2' carbon of ribose bonded to the oxygen atom bonded to the methoxyethyl group; "#mer" refers to the number of the nucleotide position; "PO" refers to a nucleotide position where the 3' carbon of ribose contains a phosphodiester bond, which links the nucleotide position to an immediately adjacent nucleotide position in the 3' direction, where a nucleotide position containing a PO group at the 3' carbon of ribose (e.g., the second or third position from the 5' or 3' end) is indicated by the location of the PO group, which is said to be either "to" or "after".

[0100] In some embodiments, the isolated nucleic acid (e.g., an RNA processing regulator such as an antisense oligonucleotide) includes the nucleic acid sequence defined in Sequence ID No. 1 and the following modification pattern: complete PS; 2' MOE; 5-10-5, PO after the second base from the 5' end; PO after the third base from the 3' end; 20 mer. In some embodiments, the isolated nucleic acid (e.g., an RNA processing regulator such as an antisense oligonucleotide) includes the nucleic acid sequence defined in Sequence ID No. 2 and the following modification pattern: complete PS; 2' MOE; 4-10-4; PO after the second base from the 5' end, PO after the third position from the 3' end; 18 mer. In some embodiments, the isolated nucleic acid (e.g., an RNA processing regulator such as an antisense oligonucleotide) includes the nucleic acid sequence defined in Sequence ID No. 3 and the following modification pattern: complete PS; 2' MOE; 4-10-4, PO after the second base from the 5' end; PO after the third base from the 3' end; 18 mer. In some embodiments, the isolated nucleic acid (e.g., an RNA processing regulator such as an antisense oligonucleotide) includes the nucleic acid sequence defined in Sequence ID No. 4 and the following modification pattern: complete PS; 2' MOE; 5-10-5, PO after the second base from the 5' end; PO after the third base from the 3' end; 20 mer. In some embodiments, the isolated nucleic acid (e.g., an RNA processing regulator such as an antisense oligonucleotide) includes the nucleic acid sequence defined in Sequence ID No. 5 and the following modification pattern: complete PS; 2' MOE; 4-10-4, PO after the second base from the 5' end; PO after the third base from the 3' end; 18 mer. In some embodiments, the isolated nucleic acid (e.g., an RNA processing regulator such as an antisense oligonucleotide) includes the nucleic acid sequence defined in Sequence ID No. 6 and the following modification pattern: complete PS; 2' MOE; 4-10-4, PO after the second base from the 5' end; PO after the third base from the 3' end; 18 mer. In some embodiments, the isolated nucleic acid (e.g., an RNA processing regulator such as an antisense oligonucleotide) comprises the nucleic acid sequence defined in Sequence ID No. 7 and the following modification pattern: complete PS; 2' MOE; 20 mer.In some embodiments, the isolated nucleic acid (e.g., an RNA processing regulator such as an antisense oligonucleotide) includes the nucleic acid sequence defined in SEQ ID NO: 8 and the following modification pattern: complete PS; 2' MOE; 4-10-4, PO after the second base from the 5' end; PO after the third base from the 3' end; 18mer. In some embodiments, the isolated nucleic acid (e.g., an RNA processing regulator such as an antisense oligonucleotide) includes the nucleic acid sequence defined in SEQ ID NO: 9 and the following modification pattern: complete PS; 2' MOE; 18mer. In some embodiments, the isolated nucleic acid (e.g., an RNA processing regulator such as an antisense oligonucleotide) includes the nucleic acid sequence defined in SEQ ID NO: 10 and the following modification pattern: complete PS; 2' MOE; 18mer. In some embodiments, the isolated nucleic acid (e.g., an RNA processing regulator such as an antisense oligonucleotide) includes the nucleic acid sequence defined in SEQ ID NO: 11 and the following modification pattern: complete PS; 2' MOE; 4-10-4, PO after the second base from the 5' end; PO after the third base from the 3' end; 18mer. In some embodiments, the isolated nucleic acid (e.g., an RNA processing regulator such as an antisense oligonucleotide) comprises the nucleic acid sequence defined in SEQ ID NO: 12 and the following modification pattern: complete PS; 2' MOE; 18 mer. In some embodiments, the isolated nucleic acid (e.g., an RNA processing regulator such as an antisense oligonucleotide) comprises the nucleic acid sequence defined in SEQ ID NO: 13 and the following modification pattern: complete PS; 2' MOE; 5-10-5; PO after the second base from the 5' end, PO after the third position from the 3' end; 20 mer. In some embodiments, the isolated nucleic acid (e.g., an RNA processing regulator such as an antisense oligonucleotide) comprises the nucleic acid sequence defined in SEQ ID NO: 14 and the following modification pattern: complete PS; 2' MOE; 5-10-5, PO after the second base from the 5' end; PO after the third base from the 3' end; 20 mer.In some embodiments, the isolated nucleic acid (e.g., an RNA processing regulator such as an antisense oligonucleotide) includes the nucleic acid sequence defined in Sequence ID No. 15 and the following modification pattern: complete PS; 2' MOE; 5-10-5, PO after the second base from the 5' end; PO after the third base from the 3' end; 20mer. In some embodiments, the isolated nucleic acid (e.g., an RNA processing regulator such as an antisense oligonucleotide) includes the nucleic acid sequence defined in Sequence ID No. 16 and the following modification pattern: complete PS; 2' MOE; 5-10-5, PO after the second base from the 5' end; PO after the third base from the 3' end; 20mer. In some embodiments, the isolated nucleic acid (e.g., an RNA processing regulator such as an antisense oligonucleotide) includes the nucleic acid sequence defined in Sequence ID No. 17 and the following modification pattern: complete PS; 2' MOE; 5-10-5, PO after the second base from the 5' end; PO after the third base from the 3' end; 20mer. In some embodiments, the isolated nucleic acid (e.g., an RNA processing regulator such as an antisense oligonucleotide) includes the nucleic acid sequence defined in SEQ ID NO: 18 and the following modification pattern: complete PS; 2' MOE; 18 mer. In some embodiments, the isolated nucleic acid (e.g., an RNA processing regulator such as an antisense oligonucleotide) includes the nucleic acid sequence defined in SEQ ID NO: 19 and the following modification pattern: complete PS; 2' MOE; 5-10-5, PO after the second base from the 5' end; PO after the third base from the 3' end; 20 mer. In some embodiments, the isolated nucleic acid (e.g., an RNA processing regulator such as an antisense oligonucleotide) includes the nucleic acid sequence defined in SEQ ID NO: 20 and the following modification pattern: complete PS; 2' MOE; 5-10-5, PO after the second base from the 5' end; PO after the third base from the 3' end; 20 mer. In some embodiments, isolated nucleic acids (e.g., RNA processing modifiers such as antisense oligonucleotides) contain the nucleic acid sequence defined in Sequence ID No. 21 and the following modification pattern: complete PS; 2' MOE; 4-10-4, PO after the second base from the 5' end; PO after the third base from the 3' end; 18mer.In some embodiments, the isolated nucleic acid (e.g., an RNA processing regulator such as an antisense oligonucleotide) includes the nucleic acid sequence defined in SEQ ID NO: 22 and the following modification pattern: complete PS; 2' MOE; 5-10-5, PO after the second base from the 5' end; PO after the third base from the 3' end; 20 mer. In some embodiments, the isolated nucleic acid (e.g., an RNA processing regulator such as an antisense oligonucleotide) includes the nucleic acid sequence defined in SEQ ID NO: 23 and the following modification pattern: complete PS; 2' MOE; 4-10-4; PO after the second base from the 5' end, PO after the third position from the 3' end; 18 mer. In some embodiments, the isolated nucleic acid (e.g., an RNA processing regulator such as an antisense oligonucleotide) includes the nucleic acid sequence defined in SEQ ID NO: 24 and the following modification pattern: complete PS; 2' MOE; 5-10-5; PO after the second base from the 5' end, PO after the third position from the 3' end; 20 mer. In some embodiments, the isolated nucleic acid (e.g., an RNA processing regulator such as an antisense oligonucleotide) includes the nucleic acid sequence defined in SEQ ID NO: 25 and the following modification pattern: complete PS; 2' MOE; 4-10-4, PO after the second base from the 5' end; PO after the third base from the 3' end; 18mer. In some embodiments, the isolated nucleic acid (e.g., an RNA processing regulator such as an antisense oligonucleotide) includes the nucleic acid sequence defined in SEQ ID NO: 26 and the following modification pattern: complete PS; 2' MOE; 5-10-5, PO after the second base from the 5' end; PO after the third base from the 3' end; 20mer. In some embodiments, the isolated nucleic acid (e.g., an RNA processing regulator such as an antisense oligonucleotide) includes the nucleic acid sequence defined in SEQ ID NO: 27 and the following modification pattern: complete PS; 2' MOE; 4-10-4, PO after the second base from the 5' end; PO after the third base from the 3' end; 18mer.In some embodiments, the isolated nucleic acid (e.g., an RNA processing regulator such as an antisense oligonucleotide) includes the nucleic acid sequence defined in SEQ ID NO: 28 and the following modification pattern: complete PS; 2' MOE; 5-10-5, PO after the second base from the 5' end; PO after the third base from the 3' end; 20 mer. In some embodiments, the isolated nucleic acid (e.g., an RNA processing regulator such as an antisense oligonucleotide) includes the nucleic acid sequence defined in SEQ ID NO: 29 and the following modification pattern: complete PS; 2' MOE; 5-10-5; PO after the second base from the 5' end, PO after the third position from the 3' end; 20 mer. In some embodiments, the isolated nucleic acid (e.g., an RNA processing regulator such as an antisense oligonucleotide) includes the nucleic acid sequence defined in SEQ ID NO: 30 and the following modification pattern: complete PS; 2' MOE; 4-10-4, PO after the second base from the 5' end; PO after the third base from the 3' end; 18 mer. In some embodiments, the isolated nucleic acid (e.g., an RNA processing regulator such as an antisense oligonucleotide) includes the nucleic acid sequence defined in SEQ ID NO: 31 and the following modification pattern: complete PS; 2' MOE; 4-10-4, PO after the second base from the 5' end; PO after the third base from the 3' end; 18 mer. In some embodiments, the isolated nucleic acid (e.g., an RNA processing regulator such as an antisense oligonucleotide) includes the nucleic acid sequence defined in SEQ ID NO: 32 and the following modification pattern: complete PS; 2' MOE; 5-10-5; PO after the second base from the 5' end, PO after the third position from the 3' end; 20 mer. In some embodiments, the isolated nucleic acid (e.g., an RNA processing regulator such as an antisense oligonucleotide) includes the nucleic acid sequence defined in SEQ ID NO: 33 and the following modification pattern: complete PS; 2' MOE; 5-10-5, PO after the second base from the 5' end; PO after the third base from the 3' end; 20 mer.In some embodiments, the isolated nucleic acid (e.g., an RNA processing regulator such as an antisense oligonucleotide) includes the nucleic acid sequence defined in SEQ ID NO: 34 and the following modification pattern: complete PS; 2' MOE; 4-10-4; PO after the second position from the 5' end, PO after the third position from the 3' end; 18mer. In some embodiments, the isolated nucleic acid (e.g., an RNA processing regulator such as an antisense oligonucleotide) includes the nucleic acid sequence defined in SEQ ID NO: 35 and the following modification pattern: complete PS; 2' MOE; 5-10-5; PO after the second position from the 5' end, PO after the third position from the 3' end; 20mer. In some embodiments, the isolated nucleic acid (e.g., an RNA processing regulator such as an antisense oligonucleotide) includes the nucleic acid sequence defined in SEQ ID NO: 36 and the following modification pattern: complete PS; 2' MOE; 18mer. In some embodiments, the isolated nucleic acid (e.g., an RNA processing regulator such as an antisense oligonucleotide) includes the nucleic acid sequence defined in Sequence ID No. 37 and the following modification pattern: complete PS; 2' MOE; 5-10-5; PO after the second base from the 5' end, PO after the third position from the 3' end; 20mer. In some embodiments, the isolated nucleic acid (e.g., an RNA processing regulator such as an antisense oligonucleotide) includes the nucleic acid sequence defined in Sequence ID No. 38 and the following modification pattern: complete PS; 2' MOE; 5-10-5, PO after the second base from the 5' end; PO after the third base from the 3' end; 20mer. In some embodiments, the isolated nucleic acid (e.g., an RNA processing regulator such as an antisense oligonucleotide) includes the nucleic acid sequence defined in Sequence ID No. 39 and the following modification pattern: complete PS; 2' MOE; 5-10-5; PO after the second base from the 5' end, PO after the third position from the 3' end; 20mer. In some embodiments, isolated nucleic acids (e.g., RNA processing modifiers such as antisense oligonucleotides) include the nucleic acid sequence defined in Sequence ID No. 40 and the following modification pattern: complete PS; 2' MOE; 4-10-4, PO after the second base from the 5' end; PO after the third base from the 3' end; 18mer.In some embodiments, the isolated nucleic acid (e.g., an RNA processing regulator such as an antisense oligonucleotide) has the nucleic acid sequence defined in SEQ ID NO: 41 and the following modifications. The pattern includes: complete PS; 2' MOE; 4-10-4, PO after the second base from the 5' end; PO after the third base from the 3' end; 18mer. In some embodiments, the isolated nucleic acid (e.g., an RNA processing regulator such as an antisense oligonucleotide) includes the nucleic acid sequence defined in SEQ ID NO: 42 and the following modification pattern: complete PS; 2' MOE; 18mer. In some embodiments, the isolated nucleic acid (e.g., an RNA processing regulator such as an antisense oligonucleotide) includes the nucleic acid sequence defined in SEQ ID NO: 43 and the following modification pattern: complete PS; 2' MOE; 5-10-5, PO after the second base from the 5' end; PO after the third base from the 3' end; 20mer. In some embodiments, the isolated nucleic acid (e.g., an RNA processing regulator such as an antisense oligonucleotide) includes the nucleic acid sequence defined in SEQ ID NO: 44 and the following modification pattern: complete PS; 2' MOE; 18mer. In some embodiments, the isolated nucleic acid (e.g., an RNA processing regulator such as an antisense oligonucleotide) includes the nucleic acid sequence defined in SEQ ID NO: 45 and the following modification pattern: complete PS; 2' MOE; 5-10-5, PO after the second base from the 5' end; PO after the third base from the 3' end; 20 mer. In some embodiments, the isolated nucleic acid (e.g., an RNA processing regulator such as an antisense oligonucleotide) includes the nucleic acid sequence defined in SEQ ID NO: 46 and the following modification pattern: complete PS; 2' MOE; 5-10-5, PO after the second base from the 5' end; PO after the third base from the 3' end; 20 mer. In some embodiments, the isolated nucleic acid (e.g., an RNA processing regulator such as an antisense oligonucleotide) includes the nucleic acid sequence defined in SEQ ID NO: 47 and the following modification pattern: complete PS; 2' MOE; 18 mer. In some embodiments, isolated nucleic acids (e.g., RNA processing modifiers such as antisense oligonucleotides) contain the nucleic acid sequence defined in Sequence ID No. 48 and the following modification pattern: complete PS; 2' MOE; 4-10-4, PO after the second base from the 5' end; PO after the third base from the 3' end; 18mer.In some embodiments, the isolated nucleic acid (e.g., an RNA processing regulator such as an antisense oligonucleotide) includes the nucleic acid sequence defined in Sequence ID No. 49 and the following modification pattern: complete PS; 2' MOE; 5-10-5, PO after the second base from the 5' end; PO after the third base from the 3' end; 20 mer. In some embodiments, the isolated nucleic acid (e.g., an RNA processing regulator such as an antisense oligonucleotide) includes the nucleic acid sequence defined in Sequence ID No. 50 and the following modification pattern: complete PS; 2' MOE; 5-10-5, PO after the second base from the 5' end; PO after the third base from the 3' end; 20 mer. In some embodiments, the isolated nucleic acid (e.g., an RNA processing regulator such as an antisense oligonucleotide) includes the nucleic acid sequence defined in Sequence ID No. 51 and the following modification pattern: complete PS; 2' MOE; 4-10-4, PO after the second base from the 5' end; PO after the third base from the 3' end; 18 mer. In some embodiments, isolated nucleic acids (e.g., RNA processing modifiers such as antisense oligonucleotides) contain the nucleic acid sequence defined in Sequence ID No. 52 and the following modification pattern: complete PS; 2' MOE; 4-10-4, PO after the second base from the 5' end; PO after the third base from the 3' end; 18mer.In any of the aforementioned embodiments: a position containing a "T" residue contains a thymine (T) nitrogen base bonded to the 1' carbon of either ribose or deoxyribose; isolated nucleic acids containing a gapmer structure are indicated by a structure denoted as "(X)-(Y)-(X)", where "(X)" refers to a region flanking a region called "(Y)", containing a number of nucleotide positions of "X" having ribose and a number of nucleotide positions of "Y" having deoxyribose; isolated nucleic acids containing a skipper structure are indicated by the absence of the notation describing "(X)-(Y)-(X)", where each nucleotide position contains ribose; "complete PS" refers to a nucleic acid containing a phosphodiester group (PO) at the 3' carbon of ribose. Unless otherwise indicated by the rheotide position, it means that each nucleotide position is linked by an internucleotide linkage containing a phosphorothioate modification; "2'MOE" refers to the 2' carbon of ribose bonded to the oxygen atom bonded to the methoxyethyl group; "#mer" refers to the number of the nucleotide position; "PO" refers to a nucleotide position where the 3' carbon of ribose contains a phosphodiester bond, which links the nucleotide position to an immediately adjacent nucleotide position in the 3' direction, where a nucleotide position containing a PO group at the 3' carbon of ribose (e.g., the second or third position from the 5' or 3' end) is indicated by the location of the PO group, which is said to be either "to" or "after". Pharmaceutical composition

[0101] In some aspects of this disclosure, RNA processing modulators (e.g., antisense oligonucleotides) are formulated into compositions for therapeutic purposes. In some aspects, the compositions are designed to enhance the therapeutic effect of the RNA processing modulator. For example, by increasing biocompatibility, targeting the RNA processing modulator to a desired site in vivo, reducing the clearance of isolated nucleic acids (e.g., antisense oligonucleotides) in vivo, increasing the stability of isolated nucleic acids (e.g., antisense oligonucleotides) in vivo, increasing the uptake of isolated nucleic acids (e.g., antisense oligonucleotides) in target cells, or amplifying the intended effect of isolated nucleic acids (e.g., antisense oligonucleotides) in vivo. Such effects may also be enhanced in vitro or ex vivo.

[0102] In some embodiments, RNA processing modulophores (e.g., antisense oligonucleotides) are provided in combination with pharmaceutically acceptable carriers. “pharmaceutically acceptable carrier” means a pharmaceutically acceptable material, composition, or carrier, such as a liquid or solid filler, stabilizer, dispersant, suspending agent, diluent, excipient, thickener, solvent, or encapsulating material, that is involved in carrying or transporting a useful compound within the present invention within or to a patient, so that it can perform its intended function. Additional components that may be incorporated into pharmaceutical compositions used in the implementation of the present invention are known in the art and are described, for example, in Remington's Pharmaceutical Sciences (Genaro, Ed., Mack Publishing Co., 1985, Easton, PA), which is incorporated herein by reference. Methods and medical uses

[0103] Aspects of this disclosure relate to methods for modulating the transcription, translation, function, and / or activity of genes associated with amyloid plaque formation in cells or subjects. In some embodiments, the method involves administering to cells or subjects a composition comprising one or more RNA processing modulators described herein (e.g., 1, 2, 3, 4, 5, or more RNA processing modulators, e.g., 1, 2, 3, 4, 5, or more antisense oligonucleotides). In some embodiments, one or more RNA processing modulators described herein (e.g., 1, 2, 3, 4, 5, or more RNA processing modulators, e.g., 1, 2, 3, 4, 5, or more antisense oligonucleotides) in a composition for use as a pharmaceutically acceptable agent. In some embodiments, the use of one or more RNA processing modulators described herein (e.g., 1, 2, 3, 4, 5, or more RNA processing modulators, e.g., 1, 2, 3, 4, 5, or more antisense oligonucleotides) in a composition for use in the manufacture of a pharmaceutically acceptable agent for the treatment of a disease described herein. In some embodiments, administration of a composition (e.g., an RNA processing modulator) results in modulation (e.g., reduction) of APOE levels, modulation (e.g., reduction) of one or more markers of neurodegeneration in cells or subjects, and / or modulation (e.g., reduction) of one or more biological products associated with amyloid plaque formation in subjects. Cells may be in vivo, ex vivo, or in vitro.

[0104] For example, in some embodiments, administration of RNA processing modulators (e.g., antisense oligonucleotides) that target APOE mRNA results in inhibition of amyloid plaque formation in cells or subjects. In some embodiments, administration of RNA processing modulators (e.g., antisense oligonucleotides) that target APOE mRNA results in a reduction in the production of one or more amyloid-forming markers in cells or subjects. This disclosure is based in part on the recognition that contacting cells or subjects with RNA processing modulators that reduce the transcription, translation, function, or activity of APOE proteins results in a reduction in one or more amyloid-forming markers in the subjects.

[0105] Neurodegenerative diseases, including Alzheimer's disease, are characterized by indicators such as progressive dementia and loss of cognitive ability. Such changes may manifest in the subject as, for example, memory loss, forgetfulness, increased anxiety, dysphoria or euphoria, apathy, disinhibition, and / or agitation. Not limited to neurodegenerative diseases, neurodegenerative diseases include Lewy body dementia, early-onset Alzheimer's disease, late-onset Alzheimer's disease, sporadic late-onset Alzheimer's disease, APOE4-positive Alzheimer's disease, familial Alzheimer's disease, frontotemporal disorders associated with neurodegeneration, Parkinson's disease and / or cognitive decline in Parkinson's disease, vascular dementia, and amyloid-related imaging abnormalities (ARIA).

[0106] The molecular pathology of Alzheimer's disease is associated with altered amyloidogenesis in cells of the central nervous system (CNS). Such alterations include, without limitation, abnormal endosomal trafficking, increased amyloid plaque levels, and / or increased intracellular fibrillary tangles containing hyperphosphorylated tau protein. Mechanisms of amyloid plaque biosynthesis involve the dysregulated processing of amyloid precursor protein (APP). For example, the β-secretase and γ-secretase-dependent release of APP from endosomes allows for cleavage and subsequent processing of APP into a form that can bind to other APP molecules, thereby forming plaques inside the cell. Several proteins are involved in the amyloidogenesis of APP. For example, increased levels and / or activity of amyloid-β peptide species such as Aβ38, Aβ40, and Aβ42 are markers of amyloidogenesis. In addition, various proteins involved in endosomal trafficking, such as SORL1, VPS26, and VPS35, are understood to produce soluble APPα, which is non-amyloidogenic.

[0107] APOE mutations are associated with cellular mechanisms of neurodegeneration. For example, APOE has been shown to be involved in the deposition of fibrous amyloid proteins as intraneuronal neurofibrillary tangles, extracellular amyloid plaques, and vascular amyloid deposits. APOE has also been found to co-localize with amyloid plaques containing Aβ protein. Furthermore, APOE is associated with regulating amyloid plaque size and toxicity by promoting amyloidosis during the early stages of Aβ plaque formation and by impairing plaque clearance from the interstitial fluid.

[0108] Accordingly, in several aspects, this disclosure provides methods for reducing one or more amyloid-forming markers in cells or subjects, the methods comprising administering isolated nucleic acids described herein to subjects requiring such reduction. In some embodiments, the isolated nucleic acids comprise antisense oligonucleotides comprising sequences defined by any one of SEQ ID NOs: 1 to 52 (provided in column A of Table 1, optionally comprising one or more modifications in column C of Table 1, optionally, where the sequences in column A and the chemistry in column C are provided in the same rows of Table 1). In some embodiments, the isolated nucleic acids (e.g., antisense oligonucleotides) are administered as monotherapy. In some embodiments, the isolated nucleic acids (e.g., antisense oligonucleotides) are administered as a component of combination therapy with one or more additional therapeutic agents (e.g., one or more selective serotonin reuptake inhibitors (SSRIs), other antidepressants, or antipsychotics).

[0109] In general, it is desirable to inhibit amyloid (or tau) plaque formation (e.g., by reducing APOE levels, transcription, splicing, and / or translation) in certain subjects (e.g., subjects with certain neurodegenerative diseases or disorders, such as Alzheimer's disease or Lewy body dementia, Parkinson's disease and / or cognitive decline in Parkinson's disease, vascular dementia, neurodegenerative frontotemporal disorders, amyloid-associated imaging abnormalities (ARIA), etc.). However, in some embodiments, this disclosure provides methods for increasing APOE function and / or activity in cells or subjects (e.g., by increasing APOE levels, transcription, splicing, and / or translation), and it should be understood that the methods include administering isolated nucleic acids described herein to subjects requiring them. In some embodiments, the isolated nucleic acid comprises or consists of an antisense oligonucleotide containing a sequence defined by any one of SEQ ID NOs: 1 to 52 (provided in column A of Table 1, optionally including one or more modifications in column C of Table 1, optionally where the sequences in column A and the chemistry in column C are provided in the same row of Table 1).

[0110] In some embodiments, isolated nucleic acids (e.g., RNA processing modulators described herein, e.g., ASOs described herein) bind to mRNA expressed from a specific allele of APOE (e.g., bind to target mRNA in an allele-specific manner).

[0111] In some aspects, the RNA processing modulators (e.g., antisense oligonucleotides) described herein are useful for treating diseases or disorders associated with amyloid plaque formation. Therefore, in some aspects, RNA processing modulators (e.g., antisense oligonucleotides) described herein are provided herein for use in methods of treating and / or preventing diseases or disorders associated with amyloid plaque formation. Diseases or disorders associated with amyloid plaque formation refer to diseases or disorders in which a subject (e.g., a patient) has 1) enlarged intracellular fibrillary tangles and / or amyloid plaques containing hyperphosphorylated tau protein, and / or 2) one or more mutations in one or more genes associated with amyloid plaque formation, and / or 3) one or more mutations in one or more genes involved in pathways for the degradation, synthesis, and / or trafficking of APP. In some embodiments, the amyloid-β peptide species include Aβ38, Aβ40, and / or Aβ42. In some embodiments, amyloid plaque formation involves modulated levels and / or activity of SORL1, VPS26, and / or VPS35. In some embodiments, the disease is Alzheimer's disease or Lewy body dementia. Treatment and / or prevention may include reducing the level, transcription, splicing, and / or translation of ApoE in cells or subjects, and / or reducing amyloid plaque formation.

[0112] In some embodiments, markers of amyloid plaque formation include modulated levels and / or activity of APP and / or regulators of APP processing. In some embodiments, modulated APP processing during amyloid plaque formation includes increased levels and / or activity of amyloid-β peptide species. In some embodiments, the amyloid-β peptide species include Aβ38, Aβ40, and / or Aβ42. In some embodiments, modulated APP processing during amyloid plaque formation includes modulated levels and / or activity of SORL1, VPS26, and / or VPS35. In some embodiments, intracellular fibrillary tangles and / or amyloid plaque formation, including hyperphosphorylated tau protein, are revealed by assaying single-photon emission computed tomography or positron emission tomography.

[0113] Methods for measuring protein levels and / or activity in cells or subjects are known in the art. In some embodiments, the levels of Aβ38, Aβ40, Aβ42, soluble APPα, and / or amyloid plaques produced by cells (e.g., cells in a subject) are determined by measuring the concentration of soluble APPα and / or amyloid plaques in a sample (e.g., a biological sample obtained from the subject, e.g., a blood sample, serum sample, cerebrospinal fluid (CSF) sample, etc.).

[0114] Methods for measuring cholesterol levels in a subject are known in the art. In some embodiments, cholesterol levels in a subject are determined by measuring the concentration of cholesterol or cholesterol-containing lipoprotein particles in a sample (e.g., a biological sample obtained from the subject, such as a blood sample, serum sample, or cerebrospinal fluid (CSF) sample).

[0115] In some embodiments, the subject has one or more mutations in the APOE gene. In some embodiments, the subject with one or more mutations in the APOE gene has (or is at risk of developing) a neurodegenerative disease or disorder. Methods for detecting mutations in the subject's gene are known in the art and include, for example, DNA sequencing, RNA sequencing, and microarray analysis.

[0116] In some embodiments, subjects with a disease or disorder associated with amyloid plaque formation have one or more mutations in one or more other genes involved in neurodegeneration. Examples of other genes involved in neurodegeneration include those encoding amyloid precursor protein (APP), SORL1, VPS26, VPS35, and others.

[0117] Accordingly, in several aspects, this disclosure provides methods for treating diseases or disorders associated with amyloid plaque formation, the methods comprising administering isolated nucleic acids described herein to a subject requiring them. RNA processing modulators (e.g., antisense oligonucleotides) described herein are also provided for use in methods for treating diseases or disorders associated with amyloid plaque formation. The methods may comprise administering isolated nucleic acids described herein to a subject requiring them. In some embodiments, the isolated nucleic acids comprise antisense oligonucleotides comprising or consisting of a sequence defined in any one of SEQ ID NOs: 1 to 52 (provided in column A of Table 1, optionally comprising one or more modifications in column C of Table 1, optionally where the sequence in column A and the chemistry in column C are provided in the same row of Table 1). In some embodiments, the disease is Alzheimer's disease or Lewy body dementia.

[0118] As used herein, “to treat” or “to treat” means to prevent or delay the onset of a disease, to reduce or prevent the occurrence of disease-related symptoms, to reduce the severity of a disease, and / or to prevent the worsening of disease-related symptoms. Accordingly, in several respects, this disclosure provides methods for treating subjects having or suspected to have a disease caused by amyloid plaque formation. Treatment of subjects involves administering compositions described herein (e.g., RNA processing modulators such as antisense oligonucleotides) to the subjects.

[0119] As used herein, the term “to treat” means the application or administration of a composition (e.g., RNA processing modulators such as antisense oligonucleotides described herein) to a subject having a disease or disorder associated with amyloid plaque formation for the purpose of curing, resolving, mitigating, alleviating, modulating, treating, improving, refining, or influencing a disorder, symptom of a disease, or predisposition to a disease.

[0120] Mitigating amyloid plaque formation-related disease encompasses preventing or delaying the onset or progression of the disease, or reducing the severity of the disease. Mitigating a disease does not necessarily require a curative outcome. As used herein, “delaying” the onset of a disease (e.g., amyloid plaque formation-related disease) means inhibiting, interfering with, slowing, delaying, stabilizing, and / or postponing the progression of the disease. This delay can be of varying lengths depending on the disease history and / or the individual being treated. A method for “delaying” the onset of a disease or mitigating the onset of a disease is a method that, when compared to not using the method, reduces the probability of developing one or more symptoms of the disease and / or reduces the severity of symptoms within a given time frame. Such comparisons are typically based on clinical trials using a sufficient number of subjects to yield statistically significant results.

[0121] The “onset” or “progression” of a disease means the initial signs and / or subsequent progression of the disease. The onset of a disease may be detectable and assessed using standard clinical techniques well known in the art. However, onset also refers to progression that may be undetectable. For the purposes of this disclosure, onset or progression refers to the biological process of symptoms. “Onset” includes occurrence, recurrence, and manifestation. As used herein, “onset” or “progression” of a disease is related to amyloid plaque formation.

[0122] The subjects may be humans, mice, rats, pigs, dogs, cats, or non-human primates. In some embodiments, the subjects have or are suspected of having a disease or disorder associated with amyloid plaque formation. In some embodiments, subjects having a disease or disorder associated with amyloid plaque formation include at least one APOE allele having a mutation. In some embodiments, the APOE allele having a mutation (e.g., a mutation associated with amyloid plaque formation, e.g., APOE loss-of-function mutation, or other mutations that cause abnormal APOE function or activity, e.g., mutations associated with Alzheimer's disease or Lewy body dementia) includes frameshift mutations, splice site mutations, missense mutations, shortened mutations, or nonsense mutations. The subjects may have two APOE alleles with the same mutation (homozygous state) or two APOE alleles with different mutations (compound heterozygous state).

[0123] The optimal process for administering or delivering the RNA processing modulators (e.g., antisense oligonucleotides) of this disclosure may vary depending on the desired outcome and / or the target to be treated. As used herein, “administration” means bringing cells into contact with the processing modulator, which may be done in vitro or in vivo. The compositions provided herein (e.g., pharmaceutical compositions) may be administered by several routes, including but not limited to oral administration, intravenous administration (e.g., systemic intravenous injection / administration), administration to the brain and / or spinal cord, intracerebral injection, intracerebroventricular injection, intracerebral (ICV) injection, intracisional injection, intraparenchymal injection, intrathecal injection, and any combination thereof. In some embodiments, administration includes administration to the cerebrospinal fluid and / or direct administration to the affected site (e.g., target tissue, e.g., central nervous system (CNS) tissue or peripheral nervous system (PNS) tissue).

[0124] Generally, the most appropriate route of administration will depend on various factors, including the properties of the drug (e.g., its stability in the gastrointestinal environment) and / or the condition of the subject (e.g., whether the subject can tolerate oral administration, injection, etc.). In some embodiments, the administration (e.g., by injection) of the compound or pharmaceutical composition is carried out to a patient in the Trendelenburg position. In some embodiments, the composition is administered to the subject through only one route of administration. In some embodiments, multiple routes of administration may be utilized (e.g., sequentially or simultaneously) for the administration of the composition to the subject.

[0125] In some embodiments, it may be desirable to deliver the RNA processing modulators of this disclosure (e.g., antisense oligonucleotides) to the target CNS. By “CNS”, we mean all cells and tissues of the brain and spinal cord of vertebrates. Thus, the term includes, but is not limited to, neurons, glial cells, astrocytes, cerebrospinal fluid (CSF), interstitial spaces, bone, cartilage, and similar tissues. The RNA processing modulators (e.g., antisense oligonucleotides) of this disclosure may be delivered directly to the CNS or brain, e.g., into the ventricular region, and by injection into the striatum (e.g., the caudate nucleus or putamen of the striatum), spinal cord, neuromuscular junctions, or cerebellar lobules, by needles, catheters, or related devices, using neurosurgical techniques known in the art, such as stereotactic injection (see, e.g., Stein et al., J Virol 73:3424-3429, 1999; Davidson et al., PNAS 97:3428-3432, 2000; Davidson et al., Nat. Genet. 3:219-223, 1993; and Alisky and Davidson, Hum. Gene Ther. 11:2315-2329, 2000). In some embodiments, the RNA processing modulators of this disclosure (e.g., antisense oligonucleotides) are administered by intravenous injection.

[0126] In some embodiments, the RNA processing modulators (e.g., antisense oligonucleotides) of the Disclosure are administered by intracerebral injection. In some embodiments, the RNA processing modulators (e.g., antisense oligonucleotides) of the Disclosure are administered by intraventricular (ICV) injection. In some embodiments, the RNA processing modulators (e.g., antisense oligonucleotides) of the Disclosure are administered by intrathecal injection. In some embodiments, the RNA processing modulators (e.g., antisense oligonucleotides) of the Disclosure are administered by intrastriatal injection. In some embodiments, the RNA processing modulators (e.g., antisense oligonucleotides) of the Disclosure are delivered by intracranial injection. In some embodiments, the RNA processing modulators (e.g., antisense oligonucleotides) of the Disclosure are delivered by cisterna magna injection. In some embodiments, the RNA processing modulators (e.g., antisense oligonucleotides) of the Disclosure are delivered by lateral ventricle injection. Those skilled in the art will also recognize that the aforementioned routes of administration may be combined in a single subject (e.g., a subject may be administered the RNA processing modulators (e.g., antisense oligonucleotides) of the Disclosure using two or more combinations of the aforementioned techniques).

[0127] In some embodiments, an effective dose (e.g., an amount sufficient to increase the transcription, translation, function, or activity of the target mRNA) is administered to the subject. In some embodiments, the effective dose of the RNA processing modulator (e.g., antisense oligonucleotide) is sufficient to increase the transcription, translation, function, and / or activity of the target mRNA (e.g., a desired variant, variant, and / or allele). In some embodiments, the effective dose of the RNA processing modulator (e.g., antisense oligonucleotide) is sufficient to decrease the transcription, translation, function, or activity of the target mRNA (e.g., an unwanted variant, variant, and / or allele). The effective dose will depend primarily on factors such as the species, age, weight, health, and tissue to be targeted of the subject. Therefore, it may vary between animals and tissues. In some embodiments, the effective dose may be a combination of an effective dose, frequency, and duration for administration.

[0128] In some embodiments, the effective dose (e.g., an amount sufficient to increase the transcription, translation, function, or activity of the target mRNA, or an amount sufficient to decrease the transcription, translation, function, or activity of the target mRNA) is 1 ng to 500 mg. In some embodiments, the effective dose (e.g., an amount sufficient to increase the transcription, translation, function, or activity of the target mRNA, or an amount sufficient to decrease the transcription, translation, function, or activity of the target mRNA) is 1 to 1000 ng. In some embodiments, the effective dose (e.g., an amount sufficient to increase the transcription, translation, function, or activity of the target mRNA, or an amount sufficient to decrease the transcription, translation, function, or activity of the target mRNA) is 1 to 10, 10 to 50, 50 to 100, 100 to 200, 200 to 300, 300 to 500, 500 to 750, or 750 to 1000 ng. In some embodiments, the effective dose (e.g., an amount sufficient to increase the transcription, translation, function, or activity of the target mRNA, or an amount sufficient to decrease the transcription, translation, function, or activity of the target mRNA) is 0.1 μg to 100.0 μg. In some embodiments, the effective dose (e.g., an amount sufficient to increase the transcription, translation, function, or activity of the target mRNA, or an amount sufficient to decrease the transcription, translation, function, or activity of the target mRNA) is 0.1 to 1.0, 1.0 to 5.0, 5.0 to 20.0, 20.0 to 50.0, or 50.0 to 100.0 μg. In some embodiments, the effective dose (e.g., an amount sufficient to increase the transcription, translation, function, or activity of the target mRNA, or an amount sufficient to decrease the transcription, translation, function, or activity of the target mRNA) is 1 μg to 1000 μg. In some embodiments, the effective dose (for example, a sufficient amount to increase the transcription, translation, function, or activity of the target mRNA, or a sufficient amount to decrease the transcription, translation, function, or activity of the target mRNA) is 100-250, 250-500, 500-750, or 750-1000 μg.In some embodiments, the effective dose (for example, an amount sufficient to increase the transcription, translation, function, or activity of the target mRNA, or an amount sufficient to decrease the transcription, translation, function, or activity of the target mRNA) is 0.1–1.0, 1.0–20.0, 20.0–50.0, 50.0–200.0, or 200.0–500.0 mg.

[0129] During the course of treatment, the administration of the composition may be modulated or adjusted as appropriate. For example, the expression of proteins encoded by nucleic acids targeted by isolated nucleic acids of the pharmaceutical composition may be monitored to inform the method of use of the composition. Expression information may be obtained, for example, by measuring changes in the levels of the protein or RNA product of the target nucleic acid. Alternatively, sequencing analysis of the target nucleic acid may be used to determine whether the expression change involves a structural or sequence modification of the protein or RNA product of the target nucleic acid sequence.

[0130] The amount of composition will vary depending on several factors, including, but not limited to, the clinical characteristics of the subject (e.g., disease severity, rate of disease progression, physical characteristics, etc.) and the mode of administration. Therefore, in some cases, the composition may be administered to a single subject once or more times. In some cases, the composition may be administered to the same subject at different times during the treatment process through different modes or routes. [Examples]

[0131] Example 1: RNA processing modulators (RPMs) This example describes the use of RNA processing modulators (RPMs) to regulate the translation of one or more mRNA transcripts in a cell or subject. RPMs function by binding to a target-specific mRNA sequence and modulating (e.g., upregulating or downregulating) the translation of the mRNA sequence into proteins.

[0132] In some embodiments, RPM is an antisense oligonucleotide (ASO). Antisense oligonucleotides (ASOs) are typically in the range of about 10 to 30 nucleotides in length and may contain a non-natural sugar-phosphate backbone (e.g., a phosphorodiamidate morpholino backbone, a phosphorothioate backbone, etc.) and / or one or more modified sugar moieties (e.g., a 2'-O-methoxyethylribose (2'-O-MOE) modification, etc.).

[0133] In some embodiments, RPMs (e.g., ASOs as described herein) target structural elements of an mRNA transcript, such as the untranslated region (UTR), to regulate the expression of a target (e.g., a target gene encoding the mRNA transcript) by increasing or decreasing the transcription and / or translation of the protein encoded by the mRNA transcript (alternatively, this is referred to as regulating expression upward or downward). In another example, RPMs (e.g., ASOs as described herein) may regulate the expression of a target upward or downward by targeting regulatory regions of the UTR region (and thus interfering with protein binding, such as ribosomal protein binding). Alternatively, R A protein modifier (e.g., an ASO as described herein) can target splice sites (e.g., splice acceptor sites or splice donor sites on the UTR region, or one or more nucleotide positions thereof) to regulate the expression of the target upward or downward (thus generating a novel protein variant). Additional structural elements that can be targeted by a protein modifier (e.g., an ASO) include, but are not limited to, intron regulatory sites, exon regulatory sites, exon-intron boundaries, antisense binding sites on target mRNA transcripts, long non-coding RNA (LncRNA) binding sites on target genes, and retained exons of classical mRNA.

[0134] Figure 1 shows an unspecified number of ASOs that target various structural elements of mRNA. Composition "A" represents an ASO that binds to the 5' untranslated region (5'UTR) of RNA. Composition "B" represents an ASO that binds to an intron of RNA. Composition "C" represents an ASO that binds to the splice boundary (e.g., splice junction) between an exon and an intron of RNA. Composition "D" represents an ASO that binds to an exon of RNA (e.g., protein-coding region). Composition "E" represents a combination of ASOs that bind to the 3'UTR of RNA alone or with a trans-modulator. Composition "F" represents a "gapmer" ASO that binds to an exon of RNA (e.g., protein-coding region) and mediates RNaseH decay. Composition "G" represents a "gapmer" ASO that binds to the 3'UTR of RNA alone or with a trans-modulator and mediates RNaseH decay. In some embodiments, the RNA-binding ASO results in the translation of a truncated protein with a dominant-negative effect against the wild-type full-length protein. Example 2: Lipid homeostasis regulators and neurodegenerative diseases

[0135] This example describes diseases and disorders associated with amyloid plaque formation, specifically those associated with neurodegeneration. Neurodegenerative diseases, including Alzheimer's disease, are characterized by indicators such as progressive dementia and loss of cognitive ability. Such changes may manifest in the subject as, for example, memory loss, forgetfulness, increased anxiety, dysphoria or euphoria, apathy, disinhibition, and / or agitation. Not limited examples of neurodegenerative diseases include Lewy body dementia, early-onset Alzheimer's disease, late-onset Alzheimer's disease, sporadic late-onset Alzheimer's disease, APOE4-positive Alzheimer's disease, familial Alzheimer's disease, frontotemporal disorders associated with neurodegeneration, Parkinson's disease and / or cognitive decline in Parkinson's disease, vascular dementia, and amyloid-associated imaging abnormalities (ARIA).

[0136] The molecular pathology of Alzheimer's disease is associated with altered amyloidogenesis in cells of the central nervous system (CNS). Such alterations include, without limitation, abnormal endosomal trafficking, increased amyloid plaque levels, and / or increased intracellular fibrillary tangles containing hyperphosphorylated tau protein. Mechanisms of amyloid plaque biosynthesis involve the dysregulated processing of amyloid precursor protein (APP). For example, the β-secretase and γ-secretase-dependent release of APP from endosomes allows for cleavage and subsequent processing of APP into a form that can bind to other APP molecules, thereby forming plaques inside the cell. Several proteins are involved in the amyloidogenesis of APP. For example, increased levels and / or activity of amyloid-β peptide species such as Aβ38, Aβ40, and Aβ42 are markers of amyloidogenesis. In addition, various proteins involved in endosomal trafficking, such as SORL1, VPS26, and VPS35, are understood to produce soluble APPα, which is non-amyloidogenic.

[0137] Accordingly, in some embodiments, markers of amyloid plaque formation include modulated levels and / or activity of APP and / or modulators of APP processing. In some embodiments, modulated APP processing during amyloid plaque formation includes increased levels and / or activity of amyloid β peptide species. In some embodiments, the amyloid β peptide species include Aβ38, Aβ40, and / or Aβ42. In some embodiments, modulated APP processing during amyloid plaque formation includes modulated levels and / or activity of SORL1, VPS26, and / or VPS35. In some embodiments, intracellular fibrillary tangles and / or amyloid plaque formation, including hyperphosphorylated tau protein, are revealed by assaying single-photon emission computed tomography or positron emission tomography.

[0138] APOE mutations are associated with cellular mechanisms of neurodegeneration. For example, APOE has been shown to be involved in the deposition of fibrous amyloid proteins as intraneuronal neurofibrillary tangles, extracellular amyloid plaques, and vascular amyloid deposits. APOE has also been found to co-localize with amyloid plaques containing Aβ protein. Furthermore, APOE is associated with regulating amyloid plaque size and toxicity by promoting amyloidosis during the early stages of Aβ plaque formation and by impairing plaque clearance from the interstitial fluid. Example 3: ASO targeting APOE

[0139] This example describes the design of an RPM (e.g., ASO) that targets human APOE. In the context of neurodegenerative diseases, it is desirable to reduce the protein levels of apolipoprotein E (APOE) (e.g., by reducing APOE mRNA levels, transcription, splicing, and / or translation, or by reducing the activity of the APOE protein). In some embodiments, the ASO is designed to target a region of APOE mRNA that would result in reduced APOE levels, transcription, splicing, and / or translation, reduced APOE mRNA levels, and / or reduced APOE protein activity.

[0140] Table 1 describes embodiments of antisense oligonucleotides (ASOs) that target APOE mRNA. In some embodiments, the ASO comprises one or more chemical modifications and / or a non-natural sugar-phosphate backbone (e.g., a phosphorothioate backbone). In some embodiments, the ASO has a "gapmer" structure. Example 4: In vitro screening of ASO

[0141] Cell lines (e.g., HepG2 human hepatocellular carcinoma cells) were cultured and maintained in appropriate media (e.g., Dulbecco's modified Eagle medium containing 10% fetal bovine serum). Where appropriate, several approaches were used to generate in vitro models for evaluating apolipoprotein E (APOE) function. For example, cell lines could be engineered to stably express APOE. Where appropriate, cells were selected based on APOE expression.

[0142] Screening for ASOs that target APOE RNA (Table 1) was performed in a 96-well plate format, seeding approximately 20,000 cells per well, and treating with ASOs at different concentrations of 5 nM and 20 nM using the RNAiMAX Lipofectamine protocol. Each concentration was transfected in two independent wells for two biological replicates. Two different ASO chemistrys were assayed for APOE RNA targeting. In addition to mock transfected wells treated with PBS or water, untargeted ASO sequences with matched chemistry and length were used as negative controls. Cells were incubated in a cell culture incubator at 37°C for 48 hours before isolating total RNA for gene expression measurement. Total RNA was isolated and evaluated using the TaqMan® Fast Advanced Cells-to-CT® kit (ThermoFisher A35378) according to the manufacturer's instructions. The qPCR reaction was multiplexed with probes targeting APOE and the housekeeping gene hypoxanthine guanine phosphoribosyltransferase 1 (HPRT1) as an internal control. To quantify APOE gene regulation, the generated cycle threshold (Ct) values ​​for both APOE and HPRT1 were used.

[0143] APOE gene expression levels were analyzed using the ΔΔCt method. For each sample, the APOE gene expression level provided as the cycle threshold (Ct) value was normalized relative to the housekeeping gene HPRT1 (2-(APOE Ct - HPRT1 Ct)). Then, APOE expression relative to the control was calculated for each sample based on the mean value of the untransfected control wells treated with water in each plate, and expressed as a percentage ((sample / control mean) * 100). The resulting values ​​for all treatment groups are shown in Figure 2A.

[0144] Sixteen ASOs resulted in a greater than 50% reduction in APOE RNA expression at a 5 nM dose. The effects of 16 of the most potent ASOs from either the 5 nM or 20 nM doses are shown in Figures 2A-2B.

[0145] Sixteen (16) potent antisense ASOs with two different chemistrys targeting APOE were selected from two concentration screenings for further testing in multiconcentration-response analyses and tested in eight dose-response analyses.

[0146] Cells plated in 96-well plates were transfected using the RNAiMAX Lipofectamine protocol. Each ASO was transfected at eight concentrations (40 nM, 20 nM, 10 nM, 5 nM, 2.5 nM, 1.25 nM, 0.625 nM, and 0.3125 nM), and each concentration was transfected in three independent wells for three biological replicates. Mock transfected wells and untargeted ASOs were used as negative controls. After a 48-hour incubation period, transfected cells were assayed for gene expression. mRNA levels were assessed by quantitative reverse transcription polymerase chain reaction (RT-qPCR). Cells were assayed using the TaqMan® Fast Advanced Cells-to-CT® kit (ThermoFisher A35378) according to the vendor's protocol. The qPCR reaction was multiplexed with probes targeting APOE and the housekeeping HPRT1 gene as an internal control. The generated cycle threshold (Ct) values ​​for both APOE and HPRT1 were used for analysis.

[0147] APOE gene expression levels were analyzed using the ΔΔCt method. For each sample, the APOE gene expression level, provided as the cycle threshold (Ct) value, was normalized relative to the housekeeping gene HPRT1 (2-(APOE Ct - HPRT1 Ct)). Then, relative APOE expression to the control was calculated for each sample based on the mean value of the untransfected control wells treated with water in each plate and expressed as a percentage ((sample / control mean) * 100). Sixteen ASOs tested exhibited concentration-dependent APOE mRNA knockdown (Figure 3A). Of these, six ASOs showed EC50 values ​​below 20 nM (Figures 3B-3C), and one ASO showed an EC50 below 5 nM in HepG2 cells (Figure 3C, square in the lower left corner of the plot).

[0148] The effects of these ASOs on the regulation of pathways associated with neurodegeneration can be further measured. Treatment of cells with ASOs (e.g., cells containing Alzheimer's disease-related mutations such as APOE mutations) allows for profiling of amyloid plaque levels using methods well known in this field (e.g., Mavrogiorgou et al. Psychiatria Danubina Vol 23, No.4:334-339 (2011) and Lamy et al. Neuropathology and Applied Neurobiology Vol.15, Issue 6:563-578). As described in (1989), in short, silver staining is an established approach for visualizing amyloid plaques using stains that include, but are not limited to, Gallyas, Bielschowsky, or Campbell. Alternatively, immunological detection of amyloid plaques includes, but is not limited to, the use of antibodies against the amyloid plaque protein Aβ4 or amyloid-specific Congo red staining. To further characterize ASO-dependent changes in APOE function, cytotoxicity is measured to understand the physiological impact of changes in APOE transcript levels. Cell viability is measured by generating viability curves through manual counting of trypan blue stains of cells after ASO treatment. Alternatively, propidium iodide staining of cells, followed by flow cytometry analysis, is used to measure cell death. Example 5: In vivo ASO method

[0149] Rodent models of Alzheimer's disease (e.g., APPPS1-21 mice, including overexpression of mutant APP and mutant PSEN1 genes, as well as knock-in of human APOE alleles) may be used. Animals are maintained in a consistent light-dark cycle and allowed to acclimate for at least 5 days prior to the experiment. Regular feeding is performed each day at a consistent time, frequency, and amount. ASO targeting APOE is administered to the animals by infusion. When multiple ASO infusions are performed, the ASO administrations are made simultaneously on each day to minimize metabolic changes due to circadian rhythms. The ASO infusion is delivered either directly to the affected area or into the cerebrospinal fluid (CSF). To aid in the distribution of ASO into the animal's tissues (e.g., CNS tissue), the animals may be placed in the Trendelenburg position during and after infusion. The ASO is solubilized in a suitable buffer and sterilized prior to infusion. After infusion, the animals are maintained for a predetermined period prior to analysis. In some cases, animals are fed a diet containing radioactive lipids (e.g., cholesterol) to determine the degree of lipid homeostasis. To analyze the effects of ASO treatment, animals are anesthetized and tissue is harvested. Harvested tissue samples are flash-frozen in a suitable extraction buffer. Blood samples are isolated when appropriate and mixed with a buffer for preservation purposes. Harvested tissue samples are frozen-sectioned and used for immunohistochemical analysis. Tissue samples are used to measure soluble APPα and / or amyloid plaque levels. Example 6: In vivo knockdown of APOE mRNA in mice

[0150] This example describes in vivo knockdown of APOE mRNA using the ASO described herein. Briefly, it describes knock-in of human APOE4 in 6-week-old mice (B6(SJL)-Apoe) in which mouse ApoE exons 2, 3, and 4 are replaced by human exons 2, 3, 4, and 3'UTR elements. tm1.1(APOE*4)AdiujA single ICV injection of APOE ASO was administered to the brain (J), and mRNA levels were quantified in brain tissue (samples harvested from left cortex 1 and left hippocampus) two weeks after ICV injection. Frozen tissue was lysis and homogenized in RLT buffer using beads (MP Biomedical) before RNA extraction using the RNeasy Mini kit (Qiagen) at a QIAcube station (Qiagen). RNA concentration was assessed using a nanodrop spectrometer (ThermoFisher), and integrity was assessed using a 2100 Bioanalyzer LabChip (Agilent). 500 ng of RNA was reverse transcribed using SuperScript IV VILO Master Mix ezDNase (Invitrogen). qPCR was performed using TaqMan Fast Advance Master Mix (Invitrogen) on a Quantstudio thermocycler (Applied Biosystems) by two independent Taqman (VIC) assays for APOE (TF Hs00171168_m1, TF Hs03037354_mH). PGK1, PPIA, and GAPDH levels were measured using a Taqman (FAM) assay (Mm00435617_m1, TF Mm99999915_g1, Thermofischer) for normalization using the ΔΔCt method.

[0151] Figures 4A–4B show representative data for in vivo reduction of APOE mRNA levels in the brain. Figure 4A shows relative APOE mRNA levels in the cortical tissue of mouse subjects two weeks after a single ICV injection of APOE ASO 1 or a base (synthetic CSF) at doses of 100 ug or 200 ug. Figure 4B shows relative APOE mRNA levels in the hippocampal tissue of mouse subjects two weeks after a single ICV injection of APOE ASO 1 or a base (synthetic CSF) at doses of 100 ug or 200 ug. "APOE ASO 1" includes the nucleotide sequence, gapmer structure, and chemical modifications of SEQ ID NO: 29, as defined in columns A and C of row 30 in Table 1. Values ​​were averaged between two technical replicates and shown as a percentage of the base control group. Statistical analysis was performed using a linear model. The treatment group was compared to the aCSF group, adjusted for RNA isolation batches (*: p<0.05, **: p<0.01, ***: p<0.001). 0% knockdown and 50% knockdown are indicated by dashed lines (black and gray, respectively). N = 3 and 2 for animals injected with the basil and APOE ASO1, respectively.

[0152] In single-dose ICV injection studies, some animals showed minor acute physiological symptoms after administration of the base or APOE ASO 1, which resolved within approximately 1-2 hours. No significant effects on life were observed in the treated animals. Furthermore, single ICV injection of APOE ASO 1 resulted in 25-40% APOE mRNA knockdown in both the cortex and hippocampus (Figures 4A-4B). Example 7: Immunostimulatory effect of ASO in vitro

[0153] This example describes the analysis of the immunostimulatory effects of APOE ASO in human peripheral blood mononuclear cells (huPBMCs).

[0154] All ASOs were prepared using in vivo quality grade materials. HuPBMCs were harvested from healthy donors and were either left untreated, treated with cytokine / chemokine response regulators, or treated for 24 hours with APOE ASO 1 (including the nucleotide sequence, gapmer structure, and chemical modifications of SEQ ID NO: 29 as defined in columns A and C of column 30 in Table 1) at concentrations of 1 μM, 3 μM, or 10 μM.

[0155] Cytokine / chemokine response regulatory agents included: cholesterol-conjugated ApoB with TLR7 / 8 agonist effect. siRNA, XD-01024; CL097, a water-soluble derivative of the imidazoquinoline compound R848, which is a TLR7 / 8 ligand; imiquimod (R837), an immune response modifier that has potent antiviral activity, also induces cytokine production, and activates TLR7; ODN2216, a 20-mer oligonucleotide containing unmethylated CpG and exhibiting TLR9 agonist activity; ODN2006, with preference for TLR9, containing one or more CpGs, class B; ODN2395, with TLR9, a CpG containing a palindromic motif, class C; TL8-506, a benzoazepine compound exhibiting TLR8 agonist activity; LMW poly(l:c), exhibiting TLR3 agonist activity; and XD-00366, a 25-mer double-stranded unmodified blunt-end LacZ RNA double-stranded RNA exhibiting TLR7 / 8 agonist activity. After treatment with huPBMCs, levels of IFN-a2a, IFN-b, IL-1B, IL-6, IL-10, IP-10, MCP-1, MIP-1a, MIP-1b, and TNF-a were measured using the MSD-U-Plex platform. Representative data from these analyses are shown in Figures 6A–6J. Negative control cells showed minimal or no detectable increase in chemokine / cytokine levels after treatment. Treatment with TLR agonist-positive controls resulted in increased chemokine / cytokine levels, as expected. No immunogenic response to APOE ASO was detected when compared relatively to cell samples treated under negative or positive control conditions (Figures 6A–6J). Example 8: In vivo ASO administration to non-human primates

[0156] This example describes the in vivo administration and in vivo pharmacokinetic (ASO level) analysis of APOE ASO in cynomolgus monkeys (Macaca fascicularis), also known as "non-human primate subjects." Briefly, three male non-human primate subjects received a series of four intrathecal ("IT") injections of either ASO ("APOE ASO 1," including the nucleotide sequence, gapmer structure, and chemical modifications of SEQ ID NO: 29, defined in columns A and C of row 30 in Table 1) or a base (artificial CSF) at a dose of 80 mg (20 mg + 20 mg + 20 mg + 20 mg). For all non-human primate subjects, each round of IT injections was spaced two weeks apart (days 0, 14, 28, and 42). Cerebrospinal fluid and tissue samples from the brain (frontal cortex, sensory cortex, and hippocampus), lumbar spinal cord, dorsal root ganglia, kidneys, liver, spleen, heart, stomach, and gonadal tissues were collected two weeks after the last IT injection (Figure 7). Liquid chromatography-tandem mass spectrometry (LC-MS / MS) was used to measure the pharmacokinetics (ASO levels) of ASO in tissue samples obtained from injected non-human primates. The ASO concentration in the tissue samples was quantified to determine the ASO level as a result of the IT injection (Figure 8). Equal parts

[0157] While several aspects of the present invention are described and illustrated herein, those skilled in the art will readily conceive of various other means and / or structures to perform the functions described herein and / or to obtain one or more of the results and / or advantages. Each such variation and / or modification is considered to fall within the scope of the aspects of the present invention described herein. More generally, those skilled in the art will readily understand that all parameters, dimensions, materials, and configurations described herein are illustrative, and that the actual parameters, dimensions, materials, and / or configurations will depend on the specific application (one or more) in which the teachings of the present invention are used. Those skilled in the art will recognize many equivalents of the specific aspects of the invention described herein, or at best can verify them using conventional experimental work. Thus, it should be understood that the aforementioned aspects are presented only as examples, and that within the scope of the appended claims and their equivalents, aspects of the invention may be carried out in ways different from those specifically described and claimed. The aspects of the invention of this disclosure cover each individual feature, system, article, material, kit, and / or method described herein. In addition, any combination of two or more such features, systems, articles, materials, kits, and / or methods is included within the scope of the invention of this disclosure, provided that such features, systems, articles, materials, kits, and / or methods are not mutually inconsistent.

[0158] All definitions defined and used herein should be understood to take precedence over dictionary definitions, definitions in documents incorporated by reference, and / or the ordinary meanings of the defined terms.

[0159] All references, patents, and patent applications disclosed herein are incorporated by reference with respect to the subject matter they refer to. In some cases, this may encompass the entire document.

[0160] In this application, the indefinite articles "a" and "an" as used herein and in the claims should be understood to mean "at least one" unless explicitly indicated otherwise.

[0161] In this application, the phrase “and / or” as used herein and in the claims should be understood to mean “either or both” of the elements thus connected, i.e., elements that exist conjunct in some cases and disjunct in others. Multiple elements enumerated by “and / or” should be interpreted similarly; that is, “one or more” of the elements thus connected. Other elements other than those specifically identified by the “and / or” clause may exist, whether related to or unrelated to those specifically identified elements. Thus, as an unlimited example, when used in conjunction with open-ended phrases such as “including,” “in one embodiment” could mean only A (optionally including elements other than B); in another embodiment only B (optionally including elements other than A); and in yet another embodiment both A and B (optionally including other elements); and so on.

[0162] As used herein and in the claims, “or” should be understood to have the same meaning as “and / or” as defined above. For example, when separating items in a list, “or” or “and / or” is interpreted as inclusive, that is, inclusion of several or at least one of the elements of the list, but also including more than one and optionally additional unlisted items. Only terms that explicitly indicate the opposite, such as “only one of” or “exactly one of” or “consisting of” as used in a claim, would refer to the inclusion of several or exactly one of the elements of the list. In general, as used herein, the term “or” is interpreted as indicating an exclusive substitution (i.e., “one or the other, but not both”) only when preceded by terms of exclusivity, such as “either,” “one of,” “only one of” or “exactly one of”. “Essentially consisting of” has its usual meaning as used in the field of patent law when used in a claim.

[0163] In this application, the phrase “at least one” as used herein and in the claims, referring to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily encompassing at least one of each specifically enumerated element in the list of elements, nor excluding any combination of elements in the list of elements. This definition also allows for the presence of elements other than those specifically identified in the list of elements referred to by the phrase “at least one,” whether related to or unrelated to those specifically identified elements. Therefore, as an unrestricted example, “at least one of A and B” (or equivalently “at least one of A or B,” or equivalently “at least one of A and / or B”) could mean, in one embodiment, at least one A that includes any more than one element and B is absent (and optionally includes elements other than B); in another embodiment, at least one B that includes any more than one element and A is absent (and optionally includes elements other than A); in yet another embodiment, at least one A that includes any more than one element, and at least one B that includes any more than one element (and optionally includes other elements); and so on.

[0164] Unless explicitly indicated otherwise, it should also be understood that in any method claimed in this application that includes more than one step or action, the order of the steps or actions of the method is not necessarily limited to the order in which the steps or actions of the method are described.

[0165] In the claims and the above specification, all transitional phrases such as “include,” “encompass,” “carry,” “have,” “contain,” “related to,” “have,” “composed of,” and similar phrases should be understood to be open-ended, meaning they include but are not limited. As specified in Section 2111.03 of the U.S. Patent and Trademark Office's Patent Examination Procedure Manual, only the transitional phrases “consist of” and “essentially consist of” are closed or semi-closed transitional phrases, respectively. It should be understood that aspects described in this document using an open-ended transitional phrase (e.g., “include”) are also intended in alternative aspects to be features “consist of” and “essentially consist of” as described by the open-ended transitional phrase. For example, where this disclosure describes “a composition comprising A and B,” this disclosure also intends the alternative aspects “a composition comprising A and B” and “a composition essentially consisting of A and B.”

Claims

1. An isolated nucleic acid comprising a region complementary to a human APOE mRNA transcript, a nucleotide sequence at least 60% identical to one of the nucleotide sequences defined by any one of sequence numbers 1–52, and which, upon binding to the mRNA transcript, reduces the transcription, splicing, and / or translation of the functional apolipoprotein E (APOE) protein encoded by the mRNA transcript.

2. The isolated nucleic acid according to claim 1, wherein the isolated nucleic acid comprises RNA.

3. The isolated nucleic acid according to claim 1 or 2, wherein the isolated nucleic acid is an antisense oligonucleotide.

4. An isolated nucleic acid according to any one of claims 1 to 3, comprising or consisting of nucleotides between 10 and 40.

5. The isolated nucleic acid according to claim 4, wherein the isolated nucleic acid comprises or consists of nucleotides between 18 and 25.

6. The isolated nucleic acid according to any one of claims 1 to 5, wherein the isolated nucleic acid comprises one or more chemical modifications.

7. The isolated nucleic acid according to claim 6, wherein one or more chemical modifications comprise one or more nucleoside modifications and / or one or more sugar-phosphate backbone modifications.

8. The isolated nucleic acid according to claim 7, wherein one or more nucleoside modifications include a 2'-O-methyl (2'-OMe) modification, a 2'-O-methoxyethyl (2'-O-MOE) modification, a 2'-O-fluoro modification, or a loc nucleic acid (LNA) modification.

9. The isolated nucleic acid according to claim 7 or 8, wherein one or more sugar-phosphate backbone modifications include a phosphorothioate backbone modification.

10. The isolated nucleic acid according to any one of claims 1 to 9, wherein the isolated nucleic acid is completely chemically modified.

11. The isolated nucleic acid according to any one of claims 1 to 10, wherein the isolated nucleic acid comprises one or more deoxyribonucleotides, and optionally, wherein the isolated nucleic acid is a gapmer.

12. The isolated nucleic acid according to any one of claims 1 to 11, wherein the complementary region is located on the untranslated region (UTR) of the APOE mRNA transcript.

13. The isolated nucleic acid according to claim 12, wherein the untranslated region comprises the 5'UTR, intron, or 3'UTR of the APOE mRNA transcript.

14. The isolated nucleic acid according to claim 12 or 13, wherein the untranslated region comprises the 3'UTR of the APOE mRNA transcript.

15. The isolated nucleic acid according to claim 12, wherein the complementary region is located on a sequence that spans the boundary on the 3'UTR of the APOE mRNA transcript, where the 5' end of the polyadenylated sequence begins.

16. The isolated nucleic acid according to claim 12, wherein the complementary region is located on the polyadenylated sequence of the APOE mRNA transcript.

17. The isolated nucleic acid according to any one of claims 1 to 11, wherein the complementary region is located on the protein-coding region of the APOE mRNA transcript.

18. The isolated nucleic acid according to any one of claims 1 to 11, wherein the complementary region is located on the intron-exon boundary of the APOE mRNA transcript.

19. An isolated nucleic acid according to any one of claims 1 to 18, wherein the complementary region comprises at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 consecutive nucleotides of any one of the nucleotide sequences defined in SEQ ID NOs. 53 to 57.

20. An isolated nucleic acid according to any one of claims 1 to 19, comprising a nucleotide sequence defined by one of the nucleotide sequences defined in Table 1.

21. A composition comprising an isolated nucleic acid according to any one of claims 1 to 20 and a pharmaceutically acceptable excipient.

22. A method for inhibiting amyloid plaque formation in cells or subjects, comprising administering an isolated nucleic acid according to any one of claims 1 to 20 or a composition according to claim 21 to cells or subjects requiring it.

23. The method according to claim 22, wherein the cell is a neuron cell.

24. The method according to claim 22 or 23, wherein the subject comprises one or more mutations in a gene associated with a neurodegenerative disease or disorder, and optionally, wherein the gene is APOE.

25. The method according to any one of claims 22 to 24, wherein the cell is a human cell, and optionally, therein the cell is in the subject.

26. The method according to any one of claims 22 to 25, wherein the subject is a human subject.

27. The method according to any one of claims 22 to 26, wherein the subject has or is suspected to have a neurodegenerative disease or disorder.

28. The method according to claim 27, wherein the disease or disorder is Alzheimer's disease or Lewy body dementia.

29. The method according to any one of claims 22 to 28, wherein the administration is systemic, and optionally, the systemic administration includes intravenous injection.

30. The method according to any one of claims 22 to 28, wherein the administration includes direct administration to a target tissue, optionally wherein the direct administration includes direct injection into the central nervous system (CNS) or direct injection into the peripheral nervous system.

31. The method according to claim 30, wherein the administration includes placing the subject in the Trendelenburg position during the administration.

32. The method according to any one of claims 22 to 31, characterized in that the subject does not have a mutation in APOE.

33. The method according to any one of claims 22 to 31, wherein the subject comprises one or more mutations in a gene associated with amyloid plaque formation, and optionally, wherein the gene is APOE.

34. A method for preventing or treating a neurodegenerative disease or disorder in a subject requiring the use of the same, comprising administering to a subject requiring the use of the same an isolated nucleic acid according to any one of claims 1 to 20 or a composition according to claim 21.

35. The method according to claim 34, wherein the subject is a human.

36. The method according to claim 34 or 35, wherein the disease or disorder is Alzheimer's disease or Lewy body dementia.

37. The method according to any one of claims 34 to 36, wherein the administration includes direct administration to a target tissue, optionally wherein the direct administration includes direct injection into the central nervous system (CNS) or direct injection into the peripheral nervous system (PNS).

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