Oligonucleotides for modulating apolipoprotein E4 expression

Abstract

The present invention relates to antisense oligonucleotides capable of modulating the expression of apolipoprotein E4 (ApoE4) in target cells. The oligonucleotides can hybridize to APOE ε4 mRNA. The present invention further relates to oligonucleotide conjugates, pharmaceutical compositions, and methods for the treatment of, for example, Alzheimer's disease (AD), frontotemporal dementia (FTD), Pick's disease (PiD), progressive supranuclear palsy (PSP), movement disorders in Parkinson's disease (PD), dementia with Lewy bodies, dementia in Down's syndrome, and Niemann-Pick disease type C1.

Description

[Technical field]

[0001] The present invention relates to oligonucleotides (oligomers) that are complementary to APOEε4 nucleic acids (e.g., mRNA transcripts, etc.) and are useful for reducing expression of apolipoprotein E4 (ApoE4). Reduction of APOEε4 transcript and / or ApoE4 protein expression is beneficial for a variety of medical disorders, including, but not limited to, Alzheimer's disease (AD), frontotemporal dementia (FTD), Pick's disease (PiD), progressive supranuclear palsy (PSP), movement disorders such as Parkinson's disease (PD), dementia with Lewy bodies, dementia in Down's syndrome, and Niemann-Pick disease type C1. [Background technology]

[0002] Apolipoprotein E (ApoE) is a lipoprotein involved in binding lipids such as cholesterol and phospholipids for lipid transport. ApoE is mainly produced by hepatocytes and Kupffer cells in the liver, as well as in small amounts from the adrenal gland and adipose tissue. In the central nervous system, the main producers of ApoE are astrocytes and microglia in the healthy nervous system, while in disease states, microglia and neurons contribute more to ApoE production.

[0003] ApoE is encoded by the APOE gene (OMIM 107741), which is located on chromosome 19 and has two common single nucleotide polymorphisms (SNPs), rs429358 and rs7412. These result in three major isoforms of ApoE, ApoE2, ApoE3 and ApoE4, which differ by amino acids at positions 112 and 158 in the protein. In ApoE3, these are occupied by cysteine ​​and arginine, respectively, while in ApoE2 and ApoE4, both positions are occupied by cysteine ​​and arginine, respectively.

[0004] Amino acid differences affect the conformation of ApoE isoforms and their ability to bind different lipids and proteins. ApoE4, for example, has a higher tendency to bind heparin sulfate binding protein and very low density lipoprotein, and a lower interaction with the LDL receptor LRP1, resulting in reduced clearance of amyloid plaques. Preclinical studies have also shown that ApoE4 can accelerate blood-brain barrier (BBB) ​​breakdown, loss of cerebral blood flow, neuronal loss and behavioral deficits, independent of amyloid-β (Montagne et al., Nat. Aging 2021;1;506-20). It has further been revealed that the presence of ApoE4 in the P301S mouse model (a model of tauopathy) exacerbates already extensive tau-mediated neurodegeneration in a manner dependent on astroglial and microglial reactivity (Shi et al., Nature 2017;549(7673):523-527). Further studies showed that selective ablation of ApoE4 in astrocytes in the same mouse model attenuated tau-mediated neurodegeneration.

[0005] The most prevalent isoform of ApoE in the general population is ApoE3, whereas three percent (3%) of the world's population is homozygous for ApoE4, and 14% carry at least one copy of ApoE4. However, the proportion of AD patients with at least one copy of ApoE4 is 37%, which is higher than in the general population. ApoE4 is also associated with higher amyloid positivity and higher risk of developing late-onset AD in both normal and mild cognitive impairment patients.

[0006] ApoE4 has been implicated in other diseases and disorders besides AD. The presence of ApoE4 also reduces the age at which FTD-related neurodegeneration occurs. In PD, ApoE4 homozygous PD patients have a faster rate of cognitive decline compared to other ApoE genotypes, and PD patients have been found to develop dementia earlier in an ApoE4 copy number-dependent manner. ApoE4 alleles have been found to be over-represented in PiD, a cognitive disorder with tau protein-associated neuropathology. ApoE4 has also been reported to reduce the age at which dementia occurs in Down syndrome. In Niemann-Pick disease type C, disease severity is worsened when patients have the ApoE4 allele. ApoE4 allele frequency has also been shown to be higher in patients suffering from PSP with AD compared to patients with PSP alone.

[0007] There is a great need for robust and effective drugs for treating ApoE4-related diseases and disorders as well as other diseases and disorders.

[0008] Objective of the invention It is an object of the present invention to provide antisense oligonucleotides, including gapmer oligonucleotides, that target APOE ε4 nucleic acids, such as mRNA, and reduce ApoE4 expression in target cells in vivo and in vitro.

[0009] It is also an object to provide such antisense oligonucleotides that are capable of reducing ApoE4 expression more than ApoE3 expression.

[0010] It is also an object to provide such antisense oligonucleotides for use in methods of treating or preventing ApoE4-related diseases and disorders, including AD. Summary of the Invention

[0011] The present invention relates to oligonucleotides that target nucleic acids encoding ApoE4 and are thereby capable of modulating expression of ApoE4 in target cells. Oligonucleotides that target a segment corresponding to positions 516-556 of SEQ ID NO:1 are identified, specifically oligonucleotides whose target sequence includes a residue corresponding to residue 535 of SEQ ID NO:1, the site of the polymorphism that distinguishes APOE ε4 from APOE ε3.

[0012] Thus, the invention provides an oligonucleotide of 8 to 50, such as 10 to 30 nucleotides in length, comprising a contiguous nucleotide sequence of at least 10 nucleotides in length that has at least 80% complementarity to a target sequence of an APOE ε4 nucleic acid that comprises a residue corresponding to residue 535 of SEQ ID NO:1.

[0013] The oligonucleotide may be an antisense oligonucleotide, preferably with gapmer design.Preferably, the oligonucleotide can reduce the expression of ApoE4 by cleavage of target nucleic acid.Cleavage is preferably achieved through nuclease recruitment.Preferably, the oligonucleotide can reduce the expression of ApoE4 more than it reduces the expression of ApoE3.

[0014] In a further aspect, the present invention provides a pharmaceutical composition comprising an oligonucleotide of the invention and a pharma- ceutically acceptable diluent, carrier, salt and / or adjuvant.

[0015] In a further aspect, the present invention provides methods for modulating ApoE4 expression in a target cell by administering to said cell an effective amount of an oligonucleotide or composition of the present invention, including in vivo and in vitro methods.

[0016] In a further aspect, the present invention provides a method for treating or preventing a disease, disorder, or dysfunction associated with the in vivo activity or expression level of ApoE4, comprising administering a therapeutically or prophylactically effective amount of an oligonucleotide of the present invention to a subject suffering from or at risk of said disease, disorder, or dysfunction.

[0017] In certain embodiments, the oligonucleotides or compositions of the invention are used in the treatment or prevention of AD.

[0018] definition Oligonucleotides As used herein, the term "oligonucleotide" is defined as a molecule that contains two or more covalently linked nucleosides as generally understood by those skilled in the art. Such covalently linked nucleosides may also be referred to as nucleic acid molecules or oligomers. Oligonucleotides are usually made in the laboratory by solid-phase chemical synthesis followed by purification and isolation. When referring to the sequence of an oligonucleotide, the reference is to the sequence or order of the nucleobase moieties of the covalently linked nucleotides or nucleosides, or to modifications thereof. The oligonucleotides of the present invention are artificial, chemically synthesized, and typically purified or isolated. The oligonucleotides of the present invention may contain one or more modified nucleosides or nucleotides, such as, for example, 2' sugar modified nucleosides.

[0019] Antisense oligonucleotides As used herein, the term "antisense oligonucleotide" is defined as an oligonucleotide that can regulate the expression of a target gene by hybridizing to a target nucleic acid, particularly a continuous sequence on the target nucleic acid.Antisense oligonucleotides can be provided in single-stranded form, double-stranded form, essentially single-stranded form, or essentially double-stranded form.For example, antisense oligonucleotides that are provided as not essentially double-stranded and therefore not siRNA or shRNA are contemplated.Preferably, such antisense oligonucleotides are single-stranded.Antisense oligonucleotides that are provided in essentially double-stranded form, such as duplex form, are also contemplated.Single-stranded oligonucleotides can also form hairpin or intermolecular duplex structures (duplexes between two molecules of the same oligonucleotide), as long as the degree of intra- or inter-self-complementarity is greater than about 50% over the entire length of the oligonucleotide.

[0020] Contiguous nucleotide sequence The term "contiguous nucleotide sequence" refers to a region of an oligonucleotide that is complementary to a target nucleic acid or target sequence. This term is used interchangeably herein with the terms "contiguous nucleobase sequence" and "oligonucleotide motif sequence". In some embodiments, all nucleotides of an oligonucleotide constitute a contiguous nucleotide sequence. In some embodiments, an oligonucleotide comprises a contiguous nucleotide sequence, e.g., an FG-F' gapmer region, and may optionally comprise a nucleotide linker region that can be used to attach additional nucleotide(s), e.g., a functional group, to the contiguous nucleotide sequence. The nucleotide linker region may or may not be complementary to the target nucleic acid. It is understood that the contiguous nucleotide sequence of an oligonucleotide cannot be longer than the oligonucleotide itself, and that an oligonucleotide cannot be shorter than the contiguous nucleotide sequence.

[0021] nucleotide Nucleotides are the building blocks of oligonucleotides and polynucleotides, and for the purposes of the present invention, include both naturally occurring and non-naturally occurring nucleotides.Naturally, nucleotides, such as DNA nucleotides and RNA nucleotides, contain a ribose sugar moiety, a nucleic acid base moiety, and one or more phosphate groups (not present in nucleosides).Nucleosides and nucleotides can also be referred to interchangeably as "units" or "monomers".

[0022] Modified Nucleosides As used herein, the term "modified nucleoside" or "nucleoside modification" refers to a nucleoside that is modified compared to an equivalent DNA or RNA nucleoside by the introduction of one or more modifications in the sugar or (nucleic acid) base moieties. In a preferred embodiment, the modified nucleoside comprises a modified sugar moiety. The term modified nucleoside may also be used interchangeably with the terms "nucleoside analog" or modified "unit" or modified "monomer". Nucleosides with unmodified DNA or RNA sugar moieties are referred to herein as DNA or RNA nucleosides. Nucleosides with modifications in the base region of DNA or RNA nucleosides are still generally referred to as DNA or RNA if they are capable of Watson-Crick base pairing.

[0023] Modified Internucleoside Linkages The term "modified internucleoside linkage" is defined as a linkage other than a phosphodiester (PO) linkage that covalently links two nucleosides together, as generally understood by those skilled in the art. Thus, the oligonucleotide of the present invention may contain modified internucleoside linkages. In some embodiments, modified internucleoside linkages increase the nuclease resistance of the oligonucleotide compared to phosphodiester linkages. In naturally occurring oligonucleotides, the internucleoside linkages contain a phosphate group that creates a phosphodiester bond between adjacent nucleosides. Modified internucleoside linkages are particularly useful for stabilizing oligonucleotides for in vivo use, and may serve to protect against nuclease cleavage in regions of DNA or RNA nucleosides of the oligonucleotide of the present invention, such as in the gap region G of a gapmer oligonucleotide, and in regions F and F' of modified nucleosides.

[0024] In one embodiment, the oligonucleotide comprises one or more modified internucleoside linkages modified from natural phosphodiester, for example, such that the one or more modified internucleoside linkages are more resistant to nuclease attack. Nuclease resistance can be determined by incubating the oligonucleotide in serum or by using a nuclease resistance assay (e.g. snake venom phosphodiesterase (SVPD)), both of which are well known in the art. An internucleoside linkage that can enhance the nuclease resistance of an oligonucleotide is referred to as a nuclease-resistant internucleoside linkage. In some embodiments, at least 50% of the internucleoside linkages of the oligonucleotide or its consecutive nucleotide sequence are modified, for example at least 60%, for example at least 70%, for example at least 75%, for example at least 80%, or for example at least 90% of the internucleoside linkages of the oligonucleotide or its consecutive nucleotide sequence are nuclease-resistant internucleoside linkages. In some embodiments, all of the internucleoside linkages of the oligonucleotide or contiguous nucleotide sequence thereof are nuclease-resistant internucleoside linkages. It will be appreciated that in some embodiments, the nucleoside linking the oligonucleotide of the invention to a non-nucleotidic functional group, e.g., a conjugate, may be a phosphodiester.

[0025] The modified internucleoside linkage may be selected from the group including phosphorothioate, diphosphorothioate and boranophosphate. In some embodiments, the modified internucleoside linkage, such as phosphorothioate, diphosphorothioate or boranophosphate, is compatible with RNase H recruitment of the oligonucleotides of the present invention.

[0026] In some embodiments, the oligonucleotide comprises one or more neutral internucleoside linkages, in particular internucleoside linkages selected from phosphotriester, methylphosphonate, MMI, amide-3, formacetal, or thioformacetal.

[0027] Further internucleoside linkages are disclosed in WO 2009 / 124238, which is incorporated herein by reference. In one embodiment, the internucleoside linkage is selected from the linkers disclosed in WO 2007 / 031091, which is incorporated herein by reference. In particular, the internucleoside linkage is selected from the linkers disclosed in WO 2007 / 031091, which is incorporated herein by reference. In particular, the internucleoside linkage is selected from the linkers disclosed in WO 2007 / 031091, which is incorporated herein by reference. H )-O-, 0-PO(OCH3)-0-, -O-PO(NR H )-O-, -O-PO(OCH2CH2S-R)-O-, -O-PO(BH3)-O-, -O-PO(NHR H )-O-, -OP(O)2-NR H -, -NR H -P(O)2-O-, -NR H -CO-O-, -NR H -CO-NR H and / or the internucleoside linker may be selected from the following: -O-CO-O-, -O-CO-NR H -, -NR H -CO-CH2-, -O-CH2-CO-NR H -, -O-CH2-CH2-NR H -, -CO-NR H -CH2-, -CH2-NR H CO-, -O-CH2-CH2-S-, -S-CH2-CH2-O-, -S-CH2-CH2-S-, -CH2-SO2-CH2-, -CH2-CO-NR H -, -O-CH2-CH2-NR H -CO-, -CH-NCH-O-CH-, R H is selected from hydrogen and C1-4-alkyl.

[0028] Phosphorothioate Internucleoside Linkages One preferred modified internucleoside bond is phosphorothioate.Phosphorothioate internucleoside bond is particularly useful due to its nuclease resistance, favorable pharmacokinetics, and ease of manufacture.In some embodiments, at least 50% internucleoside bond of oligonucleotide or its consecutive nucleotide sequence is phosphorothioate, for example at least 60%, for example at least 70%, for example at least 80%, or for example at least 90% internucleoside bond of oligonucleotide or its consecutive nucleotide sequence is phosphorothioate.In some embodiments, all internucleoside bond of oligonucleotide or its consecutive nucleotide sequence is phosphorothioate.

[0029] Nuclease-resistant linkages such as phosphorothioate linkages are particularly useful in regions of oligonucleotides that can recruit nucleases when duplexed with a target nucleic acid, such as region G of a gapmer. However, phosphorothioate linkages may also be useful in non-nuclease recruiting and / or affinity enhancing regions, such as regions F and F' of a gapmer. Gapmer-type oligonucleotides may, in some embodiments, contain one or more phosphodiester linkages in regions F or F', or both regions F and F', and the internucleoside linkages of region G may be fully phosphorothioate.

[0030] Advantageously, all internucleoside linkages of the contiguous nucleotide sequence of the oligonucleotide are phosphorothioate linkages.

[0031] As disclosed in EP 2 742 135, it will be appreciated that antisense oligonucleotides may contain other internucleoside linkages (other than phosphodiester and phosphorothioate), such as alkylphosphonate / methylphosphonate internucleoside linkages, which according to EP 2 742 135 may be tolerated within the gap region of alternative DNA phosphorothioates.

[0032] Nucleic acid bases The term nucleobase includes the purine (e.g., adenine and guanine) and pyrimidine (e.g., uracil, thymine and cytosine) moieties present in nucleosides and nucleotides, which form hydrogen bonds during nucleic acid hybridization. In the context of the present invention, the term nucleobase also encompasses modified nucleobases that may differ from naturally occurring nucleobases but are functional during nucleic acid hybridization. In this context, "nucleobase" refers to both naturally occurring nucleobases such as adenine, guanine, cytosine, thymine, uracil, xanthine and hypoxanthine, as well as non-naturally occurring variants. Such variants are described, for example, in Hirao et al (2012) Accounts of Chemical Research vol 45, page 2055, Bergstrom (2009) Current Protocols in Nucleic Acid Chemistry Suppl.37 1.4.1, or PCT / EP2021 / 065266, which are incorporated herein by reference.

[0033] In some embodiments, the nucleobase moiety is modified by changing the purine or pyrimidine to a modified purine or pyrimidine, e.g., a substituted purine or substituted pyrimidine, e.g., a nucleobase selected from isocytosine, pseudoisocytosine, 5-methylcytosine, 5-thiozolo-cytosine, 5-propynyl-cytosine, 5-propynyl-uracil, 5-bromouracil 5-thiazolo-uracil, 2-thio-uracil, 2'-thio-thymine, inosine, diaminopurine, 6-aminopurine, 2-aminopurine, 2,6-diaminopurine, 7-deaza-8-azaguanine, and 2-chloro-6-aminopurine.

[0034] The nucleobase moiety may be represented by the letter code for each corresponding nucleobase, for example, A, T, G, C, or U, and each letter may optionally include modified nucleobases of equivalent function. For example, in the exemplified oligonucleotide, the nucleobase moiety is selected from A, T, G, C, and 5-methylcytosine. Optionally, in the LNA gapmer, 5-methylcytosine LNA nucleoside may be used.

[0035] Modified Oligonucleotides The term modified oligonucleotide refers to an oligonucleotide containing one or more sugar-modified nucleosides and / or modified internucleoside linkages. The term "chimeric" oligonucleotide is a term used in the literature to refer to oligonucleotides having modified nucleosides.

[0036] Complementarity The term "complementarity" refers to the ability of nucleosides / nucleotides to form Watson-Crick base pairs. Watson-Crick base pairs are guanine (G)-cytosine (C) and adenine (A)-thymine (T) / uracil (U). It will be understood that oligonucleotides may contain nucleosides with modified nucleobases, e.g., 5-methylcytosine is often used in place of cytosine, and thus the term complementarity encompasses Watson-Crick base pairing between unmodified and modified nucleobases (see, e.g., Hirao et al (2012) Accounts of Chemical Research vol 45, page 2055 and Bergstrom (2009) Current Protocols in Nucleic Acid Chemistry Suppl.37, 1.4.1).

[0037] As used herein, the term "% complementary" refers to the percentage of nucleotides of a contiguous nucleotide sequence of a nucleic acid molecule (e.g., an oligonucleotide) that are complementary to a reference sequence (e.g., a target sequence or sequence motif) across the contiguous nucleotide sequence. The percentage of complementarity is calculated by counting the number of aligned nucleobases (from Watson-Crick base pairing) that are complementary between two sequences (when aligned with the target sequence 5'-3' and the oligonucleotide sequence from 3'-5'), dividing that number by the total number of nucleotides in the oligonucleotide, and multiplying by 100. In such a comparison, nucleobases / nucleotides that do not align (form base pairs) are referred to as mismatches. Insertions and deletions are not allowed in the calculation of the % complementarity of a contiguous nucleotide sequence. It will be understood that in determining complementarity, chemical modifications of nucleobases are disregarded so long as the functional ability of the nucleobase to form Watson-Crick base pairs is retained (e.g., 5-methylcytosine is considered identical to cytosine for purposes of calculating % identity).

[0038] The term "fully complementary" refers to 100% complementarity.

[0039] identity As used herein, the term "identity" refers to the proportion (expressed as a percentage) of nucleotides of a contiguous nucleotide sequence in a nucleic acid molecule (e.g., an oligonucleotide) that are identical to a reference sequence (e.g., a sequence motif) over the contiguous nucleotide sequence. Thus, the percentage of identity is calculated by counting the number of aligned identical (matching) nucleobases between two sequences (in the contiguous nucleotide sequence of the compound of the present invention and the reference sequence), dividing the number by the total number of nucleotides in the oligonucleotide, and multiplying by 100. Thus, the percentage of identity = (number of matches x 100) / length of the aligned region (e.g., contiguous nucleotide sequence). Insertions and deletions are not allowed in the calculation of the percentage identity of a contiguous nucleotide sequence. It will be understood that in determining identity, chemical modifications of nucleobases are disregarded as long as the functional ability of the nucleobase to form Watson Crick base pairs is maintained (e.g., 5-methylcytosine is considered to be identical to cytosine for the purpose of calculating identity %).

[0040] Hybridization As used herein, the term "hybridize" or "hybridize" should be understood as two nucleic acid strands (e.g., an oligonucleotide and a target nucleic acid) forming a duplex by forming hydrogen bonds between base pairs on opposing strands. The affinity of the binding between two nucleic acid strands is the strength of hybridization. This is determined by the melting temperature (T), defined as the temperature at which half of the oligonucleotide forms a duplex with the target nucleic acid. m ) is often explained by T m is not strictly proportional to affinity (Mergny and Lacroix, 2003, Oligonucleotides 13:515-537). The standard state Gibbs free energy, ΔG°, more accurately represents the binding affinity, ΔG°=-RTln(K d ) to calculate the dissociation constant (K d), where R is the gas constant and T is the absolute temperature. Thus, the very low ΔG° of the reaction between oligonucleotide and target nucleic acid reflects the strong hybridization between oligonucleotide and target nucleic acid. ΔG° is the energy associated with the reaction at an aqueous concentration of 1M, pH of 7, and temperature of 37°C. The hybridization of oligonucleotide to target nucleic acid is a spontaneous reaction, and in the case of spontaneous reaction, ΔG° is less than zero. ΔG° can be experimentally measured by using the isothermal titration calorimetry (ITC) method described, for example, in Hansen et al., 1965, Chem.Comm.36-38 and Holdgate et al., 2005, Drug Discov Today. Those skilled in the art will know that commercially available equipment is available for ΔG° measurement. ΔG° can also be numerically estimated by using the nearest neighbor model described by SantaLucia, 1998, Proc Natl Acad Sci USA. 95:1460-1465, using appropriately derived thermodynamic parameters described by Sugimoto et al., 1995, Biochemistry 34:11211-11216 and McTigue et al., 2004, Biochemistry 43:5388-5405. To ensure the possibility of modulating its intended nucleic acid target by hybridization, the oligonucleotides of the present invention hybridize to the target nucleic acid with an estimated ΔG° value of less than -10 kcal for oligonucleotides of 10-30 nucleotides in length. In some embodiments, the degree or strength of hybridization is measured by the standard state Gibbs free energy ΔG°. The oligonucleotides may hybridize to the target nucleic acid with estimated ΔG° values ​​in the range of less than −10 kcal, such as less than −15 kcal, such as less than −20 kcal, and such as less than −25 kcal for oligonucleotides 8 to 30 nucleotides in length.In some embodiments, the oligonucleotide hybridizes to the target nucleic acid with a calculated ΔG° value of -10 to -60 kcal, such as -12 to -40, such as -15 to -30 kcal, or -16 to -27 kcal, such as -18 to -25 kcal.

[0041] target nucleic acid According to the present invention, the target nucleic acid is the nucleic acid that codes mammalian ApoE4, and can be, for example, gene, RNA, mRNA, pre-mRNA, mature mRNA or cDNA sequence.Therefore, the target can be referred to as ApoE4 target nucleic acid or APOE ε4 target nucleic acid, and these terms can be used interchangeably.The oligonucleotide of the present invention can, for example, target APOE ε4 pre-mRNA or mRNA.

[0042] Suitably the target nucleic acid encodes an ApoE4 protein, in particular a mammalian ApoE4 protein, such as a human ApoE4 protein.

[0043] In some embodiments, the target nucleic acid comprises at least residues 522-548 of SEQ ID NO:1 and encodes a mammalian, eg, human, ApoE4 protein.

[0044] In some embodiments, the target nucleic acid comprises at least residues 516-556 of SEQ ID NO:1 and encodes a mammalian, eg, human, ApoE4 protein.

[0045] In some embodiments, the target nucleic acid is SEQ ID NO:1, or any naturally occurring variant thereof that encodes the ApoE4 protein of a mammal, such as a human. Thus, the target nucleic acid can be SEQ ID NO:1.

[0046] SEQ ID NO:2 is a human ApoE nucleic acid as set forth in NCBI Reference Sequence: NM_001302690.2 (Genbank) and encodes the human ApoE3 isoform. SEQ ID NO:2 differs from SEQ ID NO:1 at residue 535 due to the rs429358 single nucleotide polymorphism (SNP).

[0047] In some embodiments, the target nucleic acid comprises at least residues 522-548 of SEQ ID NO:2 having a t535c substitution and encodes a mammalian, eg, human, ApoE4 protein.

[0048] In some embodiments, the target nucleic acid comprises at least residues 516-556 of SEQ ID NO:2 having a t535c substitution and encodes a mammalian, eg, human, ApoE4 protein.

[0049] In some embodiments, the target nucleic acid is SEQ ID NO:2 with a t535c substitution, or any naturally occurring variant thereof that encodes a mammalian, e.g., human, ApoE4 protein. Thus, the target nucleic acid can be SEQ ID NO:2 with a t535c substitution.

[0050] In some embodiments, the target nucleic acid encodes the cynomolgus monkey ApoE4 protein. Suitably, the target nucleic acid encoding the cynomolgus monkey ApoE4 protein comprises the sequence shown in SEQ ID NO:3.

[0051] In some embodiments, the target nucleic acid comprises at least residues 450-490 of SEQ ID NO:3 and encodes a mammalian, eg, cynomolgus, ApoE4 protein.

[0052] In some embodiments, the target nucleic acid comprises at least residues 456-482 of SEQ ID NO:3 and encodes a mammalian, eg, cynomolgus, ApoE4 protein.

[0053] In some embodiments, the target nucleic acid is SEQ ID NO:3 or any naturally occurring variant thereof that encodes a mammalian ApoE4 protein, such as a cynomolgus monkey. Thus, the target nucleic acid can be SEQ ID NO:3.

[0054] For research or diagnostic uses of the oligonucleotides of the invention, the target nucleic acid can be a cDNA or a synthetic nucleic acid derived from DNA or RNA.

[0055] For in vivo or in vitro application, the oligonucleotide of the present invention can typically reduce the expression of ApoE4 protein in cells expressing APOE ε4 target nucleic acid. The contiguous sequence of nucleobases of the oligonucleotide of the present invention is typically complementary to APOE ε4 target nucleic acid, measured over the length of the oligonucleotide, optionally with one or two mismatches, and optionally with a nucleotide-based linker region that can link the oligonucleotide to any functional group, such as a conjugate, or other non-complementary terminal nucleotide (e.g., region D' or D").

[0056] SEQ ID NOs: 1-3 are presented as DNA sequences. It is understood that the target RNA sequence has uracil (U) bases in place of thymine (T) bases.

[0057] The target nucleic acid is advantageously a messenger RNA, such as a mature mRNA or a pre-mRNA.

[0058] In some embodiments, the oligonucleotides of the invention target SEQ ID NO:1.

[0059] In some embodiments, the oligonucleotide of the invention targets SEQ ID NO:2 having a t535c substitution.

[0060] In some embodiments, the oligonucleotide of the invention targets SEQ ID NO:3.

[0061] In some embodiments, the oligonucleotides of the invention target SEQ ID NO:1 and SEQ ID NO:3.

[0062] In some embodiments, oligonucleotides of the invention target SEQ ID NO:1, SEQ ID NO:2 with a t535c substitution, and SEQ ID NO:3. [Table 1]

[0063] Target sequence As used herein, the term "target sequence" refers to a sequence of nucleotides present in a target nucleic acid that contains a nucleobase sequence that is complementary to an oligonucleotide of the present invention. This region of the target nucleic acid may be interchangeably referred to as a target nucleotide sequence, a target sequence, or a target region.

[0064] In some embodiments, a target sequence consists of a region on a target nucleic acid having a nucleobase sequence that is complementary to a contiguous nucleotide sequence of an oligonucleotide of the invention. In some embodiments, a target sequence is longer than the complementary sequence of a single oligonucleotide, and may, for example, represent a preferred region of a target nucleic acid that may be targeted by several oligonucleotides of the invention.

[0065] Oligonucleotides of the invention comprise a contiguous nucleotide sequence that is complementary to or hybridizes to a target nucleic acid, such as a subsequence of a target nucleic acid, such as a target sequence described herein.

[0066] The oligonucleotide comprises a contiguous nucleotide sequence that is complementary to a target sequence present in a target nucleic acid molecule. The contiguous nucleotide sequence (and therefore the target sequence) comprises at least 8, such as at least 9, such as at least 10 contiguous nucleotides, such as 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 contiguous nucleotides, such as 12-25, such as 14-18 contiguous nucleotides.

[0067] Preferably, the target sequence is located in an RNA, such as a pre-mRNA, a mature mRNA, or both.

[0068] The target sequence contains a cytosine (c) residue at position 535 of SEQ ID NO:1, which corresponds to the site of the rs429358 SNP.

[0069] In some embodiments, the target sequence is located in a region within the segment defined by residues 516-556 of SEQ ID NO:1.

[0070] In some embodiments, the target sequence is located in a region within the segment defined by residues 522-548 of SEQ ID NO:1.

[0071] In some embodiments, the target sequence comprises a cytosine (c) at position 469 of SEQ ID NO:3.

[0072] In some embodiments, the target sequence is located in a region within the segment defined by residues 450-490 of SEQ ID NO:3.

[0073] In some embodiments, the target sequence is located in a region within the segment defined by residues 456-482 of SEQ ID NO:3.

[0074] In some embodiments, the target sequence is a sequence selected from those set forth in Table 2.

[0075] In some embodiments, the target sequence is selected from R_25, R_40, R_46, R_66 and R_91, e.g., R_25, R_40 and R_46.

[0076] In one embodiment, the target sequence is R_25, which corresponds to residues 522-535 of SEQ ID NO:1.

[0077] In one embodiment, the target sequence is R_40, which corresponds to residues 525-537 of SEQ ID NO:1.

[0078] In one embodiment, the target sequence is R_46, which corresponds to residues 526-538 of SEQ ID NO:1.

[0079] In one embodiment, the target sequence is R_66, which corresponds to residues 530-546 of SEQ ID NO:1.

[0080] In one embodiment, the target sequence is R_91, which corresponds to residues 535-548 of SEQ ID NO:1.

[0081] The target RNA sequence region should be understood to have uracil (U) bases in place of any thymine (T) bases. [Table 2] TIFF2024540271000003.tif255170

[0082] target cell As used herein, the term "target cell" refers to a cell expressing a target nucleic acid. In some embodiments, the target cell can be in vivo or in vitro. In some embodiments, the target cell is a mammalian cell, such as a rodent cell, such as a mouse cell or a rat cell, or a primate cell, such as a monkey cell (e.g., a cynomolgus monkey cell) or a human cell.

[0083] In preferred embodiments, the target cell expresses human ApoE4 mRNA, such as ApoE4 pre-mRNA, e.g., SEQ ID NO: 1, or ApoE4 mature mRNA. In some embodiments, the target cell expresses cynomolgus monkey ApoE4 mRNA, e.g., ApoE4 mature mRNA, e.g., SEQ ID NO: 3, and any polyA tail of ApoE4 mRNA is typically ignored for antisense oligonucleotide targeting.

[0084] Naturally occurring variants The term "naturally occurring variant" refers to a variant of the APOE ε4 gene or transcript that originates from the same locus as the target nucleic acid, but may differ, for example, due to the degeneracy of the genetic code resulting in multiple codons that code for the same amino acid, or alternative splicing of pre-mRNA, or the presence of polymorphisms, such as single nucleotide polymorphisms (SNPs) other than the rs429358 SNP (including silent SNPs), and allelic variants. Based on the presence of a sufficient complementary sequence to the oligonucleotide, the oligonucleotide of the present invention can thus target the target nucleic acid and its naturally occurring variants.

[0085] In some embodiments, the naturally occurring variant has at least 95%, such as at least 98% or at least 99% homology to a mammalian ApoE4 target nucleic acid, such as a target nucleic acid selected from the group consisting of SEQ ID NOs: 1 and 3. In some embodiments, the naturally occurring variant has at least 99% homology to the human APOE ε4 target nucleic acid of SEQ ID NO:1.

[0086] Regulation of expression As used herein, the term "modulation of expression" should be understood as a general term for the ability of an oligonucleotide to change the amount of ApoE4 protein or ApoE4 mRNA compared to the amount of ApoE4 protein or ApoE4 mRNA before administration of the oligonucleotide.Alternatively, the modulation of expression can be determined by referring to a control experiment.A control is generally understood to be an individual or target cell treated with a saline composition, or an individual or target cell treated with a non-targeting oligonucleotide (mock).

[0087] One type of modulation is the ability of an oligonucleotide to inhibit, decrease, reduce, suppress, eliminate, stop, block, prevent, decrease, reduce, avoid or terminate expression of ApoE4, for example, by degradation of ApoE4 mRNA or blocking transcription.

[0088] High-affinity modified nucleosides High affinity modified nucleosides are modified nucleotides which, when incorporated into an oligonucleotide, e.g., have a higher melting temperature (T m ) increases the affinity of the oligonucleotide for its complementary target. The high affinity modified nucleosides of the present invention preferably provide an increase in melting temperature of +0.5 to +12°C, more preferably +1.5 to +10°C, and most preferably +3 to +8°C per modified nucleoside. Numerous high affinity modified nucleosides are known in the art, including, for example, many 2'-substituted nucleosides and locked nucleic acids (LNAs) (see, for example, Freier & Altmann; Nucl. Acid Res., 1997, 25, 4429-4443 and Uhlmann; Curr. Opinion in Drug Development, 2000, 3(2), 293-213).

[0089] sugar modification Oligomers of the invention may contain one or more nucleosides which have modified sugar moieties, ie, sugar moieties which are modified compared to the ribose sugar moiety found in DNA and RNA.

[0090] A number of nucleosides with modifications in the ribose sugar moiety have been produced with the primary goal of improving certain properties of oligonucleotides, such as affinity and / or nuclease resistance.

[0091] Such modifications include those in which the ribose ring structure has been modified, for example by replacing it with a hexose ring (HNA) or bicyclic ring (typically having a biradical bridge between the C2 and C4 carbons of the ribose ring (Locked Nucleic Acid (LNA)), or an unlinked ribose ring that typically lacks a bond between the C2 and C3 carbons (e.g., Unlocked Nucleic Acid (UNA)). Other sugar-modified nucleosides include, for example, bicyclohexose nucleic acids (WO 2011 / 017521) or tricyclic nucleic acids (WO 2013 / 154798). Modified nucleosides also include nucleosides in which the sugar moiety has been replaced with a non-sugar moiety, for example in the case of peptide nucleic acids (PNAs) or morpholino nucleic acids.

[0092] Sugar modifications also include modifications made by changing the substituents on the ribose ring to groups other than hydrogen or to the 2'-OH group that occurs naturally in DNA and RNA nucleosides. Substituents can be introduced, for example, at the 2', 3', 4', or 5' positions.

[0093] 2' sugar modified nucleosides A 2' sugar modified nucleoside is a nucleoside that has a substituent other than H or -OH at the 2' position (2' substituted nucleoside) or that contains a 2' linked biradical that can form a bridge between the 2' carbon and a second carbon on the ribose ring, such as an LNA (2'-4' biradical bridged) nucleoside.

[0094] In fact, much attention has been paid to the development of 2' sugar-substituted nucleosides, and many 2' substituted nucleosides have been found to have beneficial properties when incorporated into oligonucleotides.For example, 2' modified sugars can provide oligonucleotides with enhanced binding affinity and / or increased nuclease resistance.Examples of 2' substituted modified nucleosides are 2'-O-alkyl-RNA nucleosides, 2'-O-methyl-RNA nucleosides, 2'-alkoxy-RNA nucleosides, 2'-O-methoxyethyl-RNA (MOE) nucleosides, 2'-amino-DNA nucleosides, 2'-fluoro-RNA nucleosides and 2'-F-ANA nucleosides. For further examples, see, for example, Freier & Altmann; Nucl. Acid Res., 1997, 25, 4429-4443 and Uhlmann; Curr. Opinion in Drug Development, 2000, 3(2), 293-213, and Deleavey and Damha, Chemistry and Biology 2012, 19, 937. Below are illustrative examples of some 2'-substituted modified nucleosides. [ka]

[0095] Locked Nucleoside (LNA) Nucleoside "LNA nucleosides" are 2' sugar modified nucleosides that contain a biradical (also called "2'-4' bridge") linking C2' and C4' of the ribose sugar ring of the nucleoside, which restricts or fixes the conformation of the ribose ring. These nucleosides are also called bridged nucleic acids or bicyclic nucleic acids (BNA) in the literature. Fixing the conformation of the ribose is associated with improved hybridization affinity (duplex stabilization) when LNA is incorporated into the oligonucleotide of a complementary RNA or DNA molecule. This can be routinely determined by measuring the melting temperature of the oligonucleotide / complementary duplex.

[0096] Non-limiting exemplary LNA nucleosides include those described in WO 99 / 014226, WO 00 / 66604, WO 98 / 039352, WO 2004 / 046160, WO 00 / 047599, WO 2007 / 134181, WO 2010 / 077578, WO 2010 / 036698, WO 2007 / 090071, WO 2009 / 006478, WO 2011 / 156202, WO 2008 / 154401, WO 2009 / 067647, WO 2008 / 150729, Morita et al. al., Bioorganic & Med. Chem. Lett. 12, 73-76, Seth et al. J. Org. Chem. 2010, Vol 75(5) pp. 1569-81, Mitsuoka et al., Nucleic Acids Research 2009, 37(4), 1225-1238, and Wan and Seth, J. Medical Chemistry 2016, 59, 9645-9667.

[0097] Further non-limiting exemplary LNA nucleosides are disclosed in Scheme 1. Scheme 1: [ka]

[0098] Particular LNA nucleosides are beta-D-oxy-LNA, 6'-methyl-beta-D-oxy-LNA, such as (R) or (S)-6'-methyl-beta-D-oxy-LNA (ScET) and ENA. Particularly advantageous LNA is beta-D-oxy-LNA.

[0099] Chemical Group Definition As used herein, the term "alkyl", alone or in combination, refers to a straight or branched chain alkyl group having 1 to 8 carbon atoms, particularly a straight or branched chain alkyl group having 1 to 6 carbon atoms, more particularly a straight or branched chain alkyl group having 1 to 4 carbon atoms. Examples of straight and branched chain C1-C8 alkyl groups are methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, the isomeric pentyls, isomeric hexyls, isomeric heptyls, and isomeric octyls, particularly methyl, ethyl, propyl, butyl, and pentyl. Particular examples of alkyl are methyl, ethyl, and propyl.

[0100] The term "cycloalkyl", alone or in combination, refers to a cycloalkyl ring having 3 to 8 carbon atoms, particularly a cycloalkyl ring having 3 to 6 carbon atoms. Examples of cycloalkyl are cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl, more particularly cyclopropyl and cyclobutyl. A specific example of "cycloalkyl" is cyclopropyl.

[0101] The term "alkoxy", alone or in combination, denotes a radical of the formula alkyl-O-, giving the term "alkyl" the meaning given above, e.g. methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, sec.butoxy and tert.butoxy. Particular "alkoxy" are methoxy and ethoxy. Methoxyethoxy is a particular example of "alkoxyalkoxy".

[0102] The term "oxy", alone or in combination, signifies the group --O--.

[0103] The term "alkenyl", alone or in combination, denotes a straight-chain or branched hydrocarbon residue containing an olefinic bond and up to 8, preferably up to 6, particularly preferably up to 4 carbon atoms. Examples of alkenyl groups are ethenyl, 1-propenyl, 2-propenyl, isopropenyl, 1-butenyl, 2-butenyl, 3-butenyl and isobutenyl.

[0104] The term "alkynyl", alone or in combination, means a straight-chain or branched hydrocarbon residue containing a triple bond and up to 8, preferably up to 6, particularly preferably up to 4 carbon atoms.

[0105] The terms "halogen" or "halo", alone or in combination, mean fluorine, chlorine, bromine or iodine, particularly fluorine, chlorine or bromine, more particularly fluorine. The term "halo", in combination with another group, means substitution of said group with at least one halogen, particularly 1 to 5 halogens, particularly 1 to 4 halogens, i.e. 1, 2, 3 or 4 halogens.

[0106] The term "haloalkyl", alone or in combination, refers to an alkyl group substituted with at least one halogen, in particular substituted with 1 to 5 halogens, in particular substituted with 1 to 3 halogens. Examples of haloalkyl include monofluoro-, difluoro- or trifluoro-methyl, -ethyl or -propyl, such as 3,3,3-trifluoropropyl, 2-fluoroethyl, 2,2,2-trifluoroethyl, fluoromethyl or trifluoromethyl. Fluoromethyl, difluoromethyl and trifluoromethyl are specific "haloalkyl".

[0107] The term "halocycloalkyl", alone or in combination, refers to a cycloalkyl group as defined above substituted with at least one halogen, particularly substituted with 1 to 5 halogens, and especially substituted with 1 to 3 halogens. Particular examples of "halocycloalkyl" are halocyclopropyl, particularly fluorocyclopropyl, difluorocyclopropyl and trifluorocyclopropyl.

[0108] The terms "hydroxyl" and "hydroxy", alone or in combination, refer to an --OH group.

[0109] The terms "thiohydroxyl" and "thiohydroxy", alone or in combination, refer to a -SH group.

[0110] The term "carbonyl", alone or in combination, means the -C(O)- group.

[0111] The terms "carboxy" or "carboxyl", alone or in combination, refer to a -COOH group.

[0112] The term "amino," alone or in combination, means a primary amino group (-NH2), a secondary amino group (-NH-), or a tertiary amino group (-N-).

[0113] The term "alkylamino", alone or in combination, means an amino group as defined above substituted with one or two alkyl groups as defined above.

[0114] The term "sulfonyl", alone or in combination, means a -SO2 group.

[0115] The term "sulfinyl", alone or in combination, signifies the -SO- group.

[0116] The term "sulfanyl", alone or in combination, signifies the -S- group.

[0117] The term "cyano", alone or in combination, means the -CN group.

[0118] The term "azido", alone or in combination, means the -N3 group.

[0119] The term "nitro", alone or in combination, means a NO2 group.

[0120] The term "formyl", alone or in combination, means the group -C(O)H.

[0121] The term "carbamoyl", alone or in combination, means the group -C(O)NH.

[0122] The term "carbamide", alone or in combination, means the group -NH-C(O)-NH.

[0123] The term "aryl", alone or in combination, denotes a monovalent aromatic carbocyclic mono- or bicyclic ring system containing from 6 to 10 carbon ring atoms optionally substituted with from 1 to 3 substituents independently selected from halogen, hydroxyl, alkyl, alkenyl, alkynyl, alkoxy, alkoxyalkyl, alkenyloxy, carboxyl, alkoxycarbonyl, alkylcarbonyl, and formyl. Examples of aryl include phenyl and naphthyl, especially phenyl.

[0124] The term "heteroaryl", alone or in combination, denotes a monovalent aromatic heterocyclic mono- or bicyclic ring system of 5 to 12 ring atoms containing 1, 2, 3 or 4 heteroatoms selected from N, O and S, and the remaining ring atoms are carbon optionally substituted with 1 to 3 substituents independently selected from halogen, hydroxyl, alkyl, alkenyl, alkynyl, alkoxy, alkoxyalkyl, alkenyloxy, carboxyl, alkoxycarbonyl, alkylcarbonyl, and formyl. Examples of heteroaryl include pyrrolyl, furanyl, thienyl, imidazolyl, oxazolyl, thiazolyl, triazolyl, oxadiazolyl, thiadiazolyl, tetrazolyl, pyridinyl, pyrazinyl, pyrazolyl, pyridazinyl, pyrimidinyl, triazinyl, azepinyl, diazepinyl, isoxazolyl, benzofuranyl, isothiazolyl, benzothienyl, indolyl, isoindolyl, isobenzofuranyl, benzimidazolyl, benzoxazolyl, benzisoxazolyl, benzothiazolyl, benzisothiazolyl, benzoxadiazolyl, benzothiadiazolyl, benzotriazolyl, purinyl, quinolinyl, isoquinolinyl, quinazolinyl, quinoxalinyl, carbazolyl or acridinyl.

[0125] The term "heterocyclyl", alone or in combination, means a monovalent saturated or partially unsaturated monocyclic or bicyclic ring system of 4 to 12, especially 4 to 9, ring atoms containing 1, 2, 3, or 4 ring heteroatoms selected from N, O, and S, and the remaining ring atoms are carbon optionally substituted with 1 to 3 substituents independently selected from halogen, hydroxyl, alkyl, alkenyl, alkynyl, alkoxy, alkoxyalkyl, alkenyloxy, carboxyl, alkoxycarbonyl, alkylcarbonyl, and formyl. Examples of monocyclic saturated heterocyclyls are azetidinyl, pyrrolidinyl, tetrahydrofuranyl, tetrahydro-thienyl, pyrazolidinyl, imidazolidinyl, oxazolidinyl, isoxazolidinyl, thiazolidinyl, piperidinyl, tetrahydropyranyl, tetrahydrothiopyranyl, piperazinyl, morpholinyl, thiomorpholinyl, 1,1-dioxo-thiomorpholin-4-yl, azepanyl, diazepanyl, homopiperazinyl, or oxazepanyl. Examples of bicyclic saturated heterocycloalkyl are 8-aza-bicyclo[3.2.1]octyl, quinuclidinyl, 8-oxa-3-aza-bicyclo[3.2.1]octyl, 9-aza-bicyclo[3.3.1]nonyl, 3-oxa-9-aza-bicyclo[3.3.1]nonyl, or 3-thia-9-aza-bicyclo[3.3.1]nonyl. Examples of partially unsaturated heterocycloalkyl are dihydrofuryl, imidazolinyl, dihydro-oxazolyl, tetrahydro-pyridinyl, or dihydropyranyl.

[0126] Pharmaceutically acceptable salts The term "pharmaceutically acceptable salt" refers to a salt that retains the biological effectiveness and properties of the free base or free acid, without being biologically or otherwise undesirable. Salts are formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, especially hydrochloric acid, and organic acids such as acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, N-acetylcysteine, and the like. These salts can be prepared by adding an inorganic base or an organic base to the free acid. Salts derived from inorganic bases include, but are not limited to, sodium, potassium, lithium, ammonium, calcium, and magnesium salts. Salts derived from organic bases include, but are not limited to, primary, secondary and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, lysine, arginine, N-ethylpiperidine, piperidine, polyamine resin salts.The compound of formula (I) can also exist in the form of zwitterions.Particularly preferred pharmaceutically acceptable salts of the compound of formula (I) are the salts of hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid and methanesulfonic acid.

[0127] protecting group The term "protecting group", alone or in combination, means a group that selectively blocks a reactive site of a multifunctional compound so that a chemical reaction can be selectively carried out at an otherwise unprotected reactive site. The protecting group can be removed. Exemplary protecting groups are an amino protecting group, a carboxy protecting group, or a hydroxy protecting group.

[0128] Nuclease-mediated degradation Nuclease-mediated degradation refers to an oligonucleotide that, when duplexed with a complementary nucleotide sequence, is capable of mediating the degradation of such sequence.

[0129] In some embodiments, oligonucleotides may function through nuclease-mediated degradation of target nucleic acids, and the oligonucleotides of the invention are capable of recruiting nucleases, particularly endonucleases, preferably endonucleases such as RNase H, preferably endoribonucleases (RNases). Examples of oligonucleotide designs that act through a nuclease-mediated mechanism are oligonucleotides that typically contain a region of at least five or six contiguous DNA nucleosides and are flanked on one or both sides by affinity enhancing nucleosides, such as gapmers, headmers and tailmers.

[0130] RNase H activity and recruitment RNase H activity of an antisense oligonucleotide refers to its ability to recruit RNase H when in a duplex with a complementary RNA molecule. WO 01 / 23613 provides an in vitro method for determining RNase H activity, which can be used to determine the ability to recruit RNase H. Typically, an oligonucleotide is considered to be capable of recruiting RNase H if, when provided with a complementary target nucleic acid sequence, it has an initial rate measured in pmol / l / min that is at least 5%, e.g., at least 10% or more than 20% of the initial rate determined when using an oligonucleotide that has the same base sequence as the modified oligonucleotide being tested, but contains only DNA monomers with phosphorothioate linkages between all monomers in the oligonucleotide, and using the methodology provided by Examples 91-95 of WO 01 / 23613 (herein incorporated by reference). For use in determining RHase H activity, recombinant human RNase H1 is available from Lubio Science GmbH, Lucerne, Switzerland.

[0131] Gapmar The antisense oligonucleotide or its contiguous nucleotide sequence of the present invention may be a gapmer and may also be referred to as a gapmer oligonucleotide or gapmer design. Antisense gapmers are typically used to inhibit target nucleic acids via RNase H-mediated degradation. Gapmer-type oligonucleotides contain at least three distinct structural regions, 5'-flank, gap and 3'-flank, FG-F', in the 5'→3' direction. The "gap" region (G) contains a stretch of contiguous DNA nucleotides that allow the oligonucleotide to recruit RNase H. The gap region is flanked by a 5' flanking region (F) that contains one or more sugar-modified nucleosides, advantageously high affinity sugar-modified nucleosides, and a 3' flanking region (F') that contains one or more sugar-modified nucleosides, advantageously high affinity sugar-modified nucleosides. The one or more sugar-modified nucleosides in regions F and F' improve the affinity of the oligonucleotide for the target nucleic acid (i.e., it is an affinity-improving sugar-modified nucleoside). In some embodiments, one or more sugar modified nucleosides of regions F and F' are 2' sugar modified nucleosides, e.g., a high affinity 2' sugar modification, e.g., independently selected from LNA and 2'-MOE.

[0132] In a gapmer design, the 5' and 3' most nucleosides of the gap region are DNA nucleosides, positioned adjacent to sugar-modified nucleosides in the 5' (F) or 3' (F') regions, respectively. The flanks may be further defined by having at least one sugar-modified nucleoside at the end furthest from the gap region, i.e., at the 5' end of the 5' flank and the 3' end of the 3' flank.

[0133] The regions FG-F' form a contiguous nucleotide sequence.An antisense oligonucleotide of the invention, or a contiguous nucleotide sequence thereof, may comprise a gapmer region of the formula FG-F'.

[0134] The total length of the gapmer designed FG-F' may be, for example, 12 to 32 nucleosides, for example, 13 to 24, for example, 14 to 22 nucleosides, for example, 14 to 17, for example, 16 to 18 nucleosides, for example, 16 to 20 nucleotides. By way of example, a gapmer oligonucleotide of the invention can be represented by the formula: F 1-8 -G 5-16 -F' 1-8 ,for example F 1-8 -G 7-16 -F' 2-8、 for example F 3-8 -G 6-14 -F' 2-8

[0135] However, it is provided that the total length of the gapmer region FG-F' is at least 10, at least 12, for example at least 14 nucleotides in length.

[0136] In one embodiment of the invention, the antisense oligonucleotide or contiguous nucleotide sequence thereof consists of or comprises a gapmer of the formula 5'-FG-F'-3', in which regions F and F' independently comprise or consist of 1 to 8 nucleosides, 2 to 4 of which are 2' sugar modified, define the 5' and 3' ends of the F and F' regions, and G is a region of 6 to 16 nucleosides capable of recruiting RNase H.

[0137] Regions F, G, and F' are further defined below and can be combined into the formula FG-F'.

[0138] Gapmer region G The region G (gap region) of a gapmer is a region of nucleosides, typically DNA nucleosides, that enable the oligonucleotide to recruit RNase H, e.g. human RNase H1. RNase H is a cellular enzyme that recognizes duplexes between DNA and RNA and enzymatically cleaves the RNA molecule. Suitably, a gapmer may have a gap region (G) of at least 5 or 6 consecutive DNA nucleosides, such as 5-16 consecutive DNA nucleosides, such as 6-15 consecutive DNA nucleosides, such as 7-14 consecutive DNA nucleosides, such as 8-12 consecutive DNA nucleotides, such as 8-12 consecutive DNA nucleotides in length. The gap region G may in some embodiments consist of 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 consecutive DNA nucleosides. One or more cytosine (c) DNA in the gap region may optionally be methylated (e.g., when DNA c is followed by DNA g), and such 5'-methyl-cytosine residues are me It can be annotated as C or with e instead of c. 5' substituted DNA nucleosides, such as 5' methyl DNA nucleosides, have been reported for use in DNA gap regions (EP 2742136).

[0139] In some embodiments, the gap region G may consist of 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 consecutive phosphorothioate linked DNA nucleosides. In some embodiments, all internucleoside linkages within the gap are phosphorothioate linkages.

[0140] Although traditional gapmers have a DNA gap region, there are many examples of modified nucleosides that allow the recruitment of RNase H when used within the gap region. Modified nucleosides that have been reported to be able to recruit RNase H when included within the gap region include, for example, alpha-L-LNA, C4' alkylated DNA (as described in PCT / EP2009 / 050349 and Vester et al., Bioorg.Med.Chem.Lett.18(2008)2296-2300, both of which are incorporated herein by reference), arabinose-derived nucleosides such as ANA and 2'F-ANA (Mangos et al.2003 J.AM.CHEM.SOC.125,654-661), UNA (unlocked nucleic acid) (as described in Fluiter et al., Mol.Biosyst.,2009,10,1039, which is incorporated herein by reference). UNAs are typically unlocked nucleic acids in which the bond between C2 and C3 of the ribose has been removed to form an unlocked "sugar" residue. The modified nucleosides used in such gapmers may be nucleosides that adopt a 2'endo (DNA-like) structure when introduced into the gap region (i.e., modifications that allow RNase H recruitment). In some embodiments, the DNA gap region (G) described herein may optionally contain one to three sugar-modified nucleosides that adopt a 2'endo (DNA-like) structure when introduced into the gap region.

[0141] Area G - "Gap Breaker" On the other hand, there are many reports on the insertion of modified nucleosides that confer a 3'-end conformation to the gap region of a gapmer while retaining some RNaseH activity. Such gapmers with a gap region containing one or more 3'-end modified nucleosides are referred to as "gap breaker" or "gap-disrupting" gapmers, see, for example, WO2013 / 022984. Gap breaker oligonucleotides retain a sufficient region of DNA nucleosides within the gap region to allow RNaseH recruitment. The ability of gap breaker oligonucleotide designs to recruit RNaseH is typically sequence-specific or even compound-specific (see Rukov et al. 2015 Nucl.Acids Res.Vol.43 pp.8476-8487), and the literature discloses "gap breaker" oligonucleotides that recruit RNaseH, which in some cases provide more specific cleavage of target RNA. Modified nucleosides used within the gap region of a gap breaker oligonucleotide may be, for example, modified nucleosides that confer 3' end confirmation, such as 2'-O-methyl (OMe) or 2'-O-MOE (MOE) nucleosides, or beta-D LNA nucleosides (in which the bridge between the C2' and C4' of the ribose sugar ring of the nucleoside is in the beta conformation), such as beta-D-oxy LNA or ScET nucleosides.

[0142] Similar to the gapmers containing region G described above, the gap region of a gap breaker or gap disrupting gapmer has a DNA nucleoside at the 5' end of the gap (adjacent to the 3' nucleoside of region F) and a DNA nucleoside at the 3' end of the gap (adjacent to the 5' nucleoside of region F). Gapmers containing disruptive gaps typically retain a region of at least three or four contiguous DNA nucleosides at either the 5' or 3' end of the gap region. Exemplary designs of gap breaker oligonucleotides include: F 1-8 -[D 3-4-E1-D 3-4 ] - F' 1-8 F 1-8 -[D 1-4 -E1-D 3-4 ]-F' 1-8 F 1-8 -[D 3-4 -E1-D 1-4 ]-F' 1-8 The region G is the region of the square bracket [D n -E r -D m ], D is a contiguous sequence of DNA nucleosides, E is a modified nucleoside (gap breaker or gap disrupting nucleoside), and F and F' are flanking regions as defined herein, with the proviso that the total length of the gapmer region FG-F' is at least 12, e.g., 14, nucleotides in length.

[0143] In some embodiments, region G of the gap breaking gapmer comprises at least six DNA nucleosides, for example 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 DNA nucleosides. As noted above, the DNA nucleosides may be contiguous and, optionally, interspersed with one or more modified nucleosides, provided that the gap region G is capable of mediating RNase H recruitment.

[0144] Gapmer-flanking regions, F and F' Region F is positioned immediately adjacent to the 5' DNA nucleoside of region G. The 3'-most nucleoside of region F is a sugar-modified nucleoside, e.g., a high affinity sugar-modified nucleoside, e.g., a 2'-substituted nucleoside, e.g., an MOE nucleoside, or an LNA nucleoside.

[0145] Region F' is positioned immediately adjacent to the 3' DNA nucleoside of region G. The 5'-most nucleoside of region F is a sugar-modified nucleoside, e.g., a high affinity sugar-modified nucleoside, e.g., a 2'-substituted nucleoside, e.g., an MOE nucleoside, or an LNA nucleoside.

[0146] Region F is 1 to 8 contiguous nucleotides in length, such as 2 to 6, such as 3 to 4 contiguous nucleotides in length. Advantageously, the 5'-most nucleoside of region F is a sugar-modified nucleoside. In some embodiments, the two 5'-most nucleosides of region F are sugar-modified nucleosides. In some embodiments, the two 5'-most nucleosides of region F are LNA nucleosides. In some embodiments, the two 5'-most nucleosides of region F are LNA nucleosides. In some embodiments, the two 5'-most nucleosides of region F are 2'-substituted nucleosides, such as two 3'MOE nucleosides. In some embodiments, the 5'-most nucleosides of region F are 2'-substituted nucleosides, such as MOE nucleosides.

[0147] Region F' is 1 to 8 contiguous nucleotides long, such as 2 to 8, such as 3 to 6, such as 4 to 5 contiguous nucleotides long. Advantageously, in embodiments, the 3'-most nucleoside of region F' is a sugar-modified nucleoside. In some embodiments, the two 3'-most nucleosides of region F' are sugar-modified nucleosides. In some embodiments, the two 3'-most nucleosides of region F' are LNA nucleosides. In some embodiments, the 3'-most nucleoside of region F' is an LNA nucleoside. In some embodiments, the two 3'-most nucleosides of region F' are 2'-substituted nucleosides, such as two 3'MOE nucleosides. In some embodiments, the 3'-most nucleoside of region F' is a 2'-substituted nucleoside, such as an MOE nucleoside.

[0148] It should be noted that when the length of region F or F' is 1, it is advantageously an LNA nucleoside.

[0149] In some embodiments, regions F and F' independently consist of or comprise a contiguous sequence of sugar-modified nucleosides. In some embodiments, the sugar-modified nucleosides of region F may be independently selected from 2'-O-alkyl-RNA units, 2'-O-methyl-RNA, 2'-amino-DNA units, 2'-fluoro-DNA units, 2'-alkoxy-RNA, MOE units, LNA units, arabinonucleic acid (ANA) units and 2'-fluoro-ANA units.

[0150] In some embodiments, regions F and F' independently comprise both LNA and 2'-substituted modified nucleosides (mixed wing design).

[0151] In some embodiments, regions F and F' consist of only one type of sugar modified nucleoside, for example only MOE, or only beta-D-oxy LNA, or only ScET. Such designs are also referred to as uniform flank or uniform gapmer designs.

[0152] In some embodiments, all nucleosides of regions F or F', or F and F', are LNA nucleosides, e.g., independently selected from beta-D-oxy LNA, ENA, or ScET nucleosides. In some embodiments, region F consists of 1 to 5, e.g., 2 to 4, e.g., 3 to 4, e.g., 1, 2, 3, 4, or 5 consecutive LNA nucleosides. In some embodiments, all nucleosides of regions F and F' are beta-D-oxy LNA nucleosides.

[0153] In some embodiments, all nucleosides of regions F or F', or F and F', are 2'-substituted nucleosides, such as OMe or MOE nucleosides. In some embodiments, region F consists of 1, 2, 3, 4, 5, 6, 7 or 8 consecutive OMe or MOE nucleosides. In some embodiments, only one of the flanking regions can consist of 2'-substituted nucleosides, such as OMe or MOE nucleosides. In some embodiments, it is the 5' (F) flanking region that consists of 2'-substituted nucleosides, such as OMe or MOE nucleosides, while the 3' (F') flanking region comprises at least one LNA nucleoside, such as a beta-D-oxy LNA nucleoside or a cET nucleoside. In some embodiments, it is the 3' (F') flanking region that consists of a 2' substituted nucleoside, such as an OMe or MOE nucleoside, while the 5' (F) flanking region comprises at least one LNA nucleoside, such as a beta-D-oxyLNA nucleoside or a cET nucleoside.

[0154] In some embodiments, all modified nucleosides in regions F and F' are LNA nucleosides, e.g., independently selected from beta-D-oxy LNA, ENA, or ScET nucleosides, while regions F or F', or F and F', may optionally include DNA nucleosides (alternating flanks, see these definitions for more details). In some embodiments, all modified nucleosides in regions F and F' are beta-D-oxy LNA nucleosides, while regions F or F', or F and F', may optionally include DNA nucleosides (alternating flanks, see these definitions for more details).

[0155] In some embodiments, the 5'-most and 3'-most nucleosides of regions F and F' are LNA nucleosides, such as beta-D-oxyLNA nucleosides or ScET nucleosides.

[0156] In some embodiments, the internucleoside linkage between region F and region G is a phosphorothioate internucleoside linkage. In some embodiments, the internucleoside linkage between region F' and region G is a phosphorothioate internucleoside linkage. In some embodiments, the internucleoside linkage between the nucleosides of regions F or F', F and F' is a phosphorothioate internucleoside linkage.

[0157] LNA gapmer An LNA gapmer is a gapmer which comprises or consists of LNA nucleosides in one or both of regions F and F'. A beta-D-oxy gapmer is a gapmer which comprises or consists of beta-D-oxy LNA nucleosides in one or both of regions F and F'.

[0158] In some embodiments, the LNA gapmer has the formula: [LNA] 1-5 -[Area G]-[LNA] 1-5 and region G is as defined in the definition of gapmer region G.

[0159] In one embodiment, the LNA gapmer has the formula [LNA]4-[region G] 10-12 -[LNA]4.

[0160] MOE Gapmar An MOE gapmer is a gapmer in which regions F and F' consist of MOE nucleosides. In some embodiments, an MOE gapmer has the design [MOE] 1-8 -[Area G] 5-16 -[MOE] 1-8 , e.g. [MOE] 2-7 -[Understood G] 6-14 -[MOE] 2-7 , e.g. [MOE] 3-6 -[Area G] 8-12 -[MOE] 3-6and region G is as defined in the gapmer definition. MOE gapmers with the 5-10-5 design (MOE-DNA-MOE) are widely used in the art.

[0161] Mixed Wing Gappar A mixed wing gapmer is an LNA gapmer in which one or both of regions F and F' comprise a 2'-substituted nucleoside, e.g., an MOE nucleoside, independently selected from the group consisting of 2'-O-alkyl-RNA units, 2'-O-methyl-RNA, 2'-amino-DNA units, 2'-fluoro-DNA units, 2'-alkoxy-RNA, MOE units, arabinonucleic acid (ANA) units, and 2'-fluoro-ANA units. In some embodiments in which at least one of regions F and F', or both of regions F and F' comprise at least one LNA nucleoside, the remaining nucleosides of regions F and F' are independently selected from the group consisting of MOE and LNA. In some embodiments in which at least one of regions F and F', or both of regions F and F' comprise at least two LNA nucleosides, the remaining nucleosides of regions F and F' are independently selected from the group consisting of MOE and LNA. In some mixed wing embodiments, one or both of regions F and F' may further comprise one or more DNA nucleosides.

[0162] Mixed wing gapmer designs are disclosed in WO 2008 / 049085 and WO 2012 / 109395, both of which are incorporated herein by reference.

[0163] Alternating Flank Gappar The flanking regions may comprise both LNA and DNA nucleosides and are referred to as "alternating flanks" because they comprise an alternating motif of LNA-DNA-LNA nucleosides. Gapmers comprising such alternating flanks are referred to as "alternating flank gapmers". An "alternating flank gapmer" is an LNA gapmer oligonucleotide where at least one of the flanks (F or F') comprises DNA in addition to the LNA nucleoside(s). In some embodiments, at least one of the regions F or F', or both regions F and F', comprise both LNA nucleosides and DNA nucleosides. In such embodiments, the flanking regions F or F', or both F and F', comprise at least three nucleosides and the 5' and 3' most nucleosides of the F and / or F' regions are LNA nucleosides.

[0164] Alternating flanking LNA gapmers are disclosed in WO 2016 / 127002.

[0165] The alternating flanking regions can include up to three consecutive DNA nucleosides, such as one or two, or one, or two, or three consecutive DNA nucleosides. The alternating flanks can be annotated, for example, as a series of integers representing the number of LNA nucleosides (L) followed by the number of DNA nucleosides (D), e.g., [L] 1-3 -[D] 1-4 -[L] 1-3 [L] 1-2 -[D] 1-2 -[L] 1-2 -[D] 1-2 -[L] 1-2 It is.

[0166] In oligonucleotide design, these are often represented as numbers such that 2-2-1 represents 5'[L]2-[D]2-[L]3' and 1-1-1-1-1 represents 5'[L]-[D]-[L]-[D]-[L]3'. The length of the flanks (regions F and F') of an oligonucleotide with alternating flanks can be independently 3 to 10 nucleosides, such as 4 to 8, such as 5 to 6 nucleosides, such as 4, 5, 6 or 7 modified nucleosides. In some embodiments, only one flank of a gapmer oligonucleotide is alternating, the other being composed of LNA nucleotides. It can be advantageous to have at least two LNA nucleosides at the 3' end of the 3' flank (F') to confer additional exonuclease resistance. In one embodiment, the flanks in an alternating flank gapmer have a total length of 5 to 8 nucleosides, of which 3 to 5 are LNA nucleosides. Some examples of oligonucleotides with alternating flanks are as follows: [L] 1-5 -[D] 1-4 -[L] 1-3 -[G] 5-16 -[L] 2-6 [L] 1-2 -[D] 2-3 -[L] 3-4 --[G] 5-7 -[L] 1-2 -[D] 2-3 -[L] 2-3 [L] 1-2 -[D] 1-2 -[L] 1-2 -[D] 1-2 -[L] 1-2 -[G] 5-16 -[L] 1-2 -[D] 1-3 -[L] 2-4 [L] 1-5 -[G] 5-16 -[L]-[D]-[L]-[D]-[L]2 [L]4-[G] 6-10 -[L]-[D]3-[L]2

[0167] However, the total length of the gapmer is at least 12, such as at least 14, nucleotides in length.

[0168] Region D' or D" in the oligonucleotide The oligonucleotides of the invention, in some embodiments, can comprise or consist of a contiguous nucleotide sequence of the oligonucleotide that is complementary to a target nucleic acid, such as a gapmer FG-F', as well as additional 5' and / or 3' nucleosides. The additional 5' and / or 3' nucleosides may or may not be fully complementary to the target nucleic acid. Such additional 5' and / or 3' nucleosides may be referred to herein as regions D' and D".

[0169] The addition of region D' or D" can be used for the purpose of linking a contiguous nucleotide sequence, such as a gapmer, to a conjugate moiety or another functional group. When used for linking, the contiguous nucleotide sequence with the conjugate moiety can serve as a biocleavable linker. Alternatively, it may be used to provide exonuclease protection or to facilitate synthesis or manufacturing. Regions D' and D" are linked to the 5' end of region F or the 3' end of region F', respectively, and have the following formula: D'-FG-F', FG-F'-D" or A D'-FG-F'-D" design can be generated, where FG-F' is the gapmer portion of the oligonucleotide and regions D' or D" constitute separate portions of the oligonucleotide.

[0170] Region D' or D" independently comprises or consists of 1, 2, 3, 4, or 5 additional nucleotides, which may or may not be complementary to the target nucleic acid. The nucleotides adjacent to the F or F' region are not sugar-modified nucleotides, such as DNA or RNA, or base-modified versions thereof. The D' or D" region may serve as a nuclease-sensitive biocleavable linker (see definition of linker). In some embodiments, the additional 5' and / or 3' terminal nucleotides are linked by phosphodiester bonds and are DNA or RNA. Nucleotide-based biocleavable linkers suitable for use as region D' or D" are disclosed in WO 2014 / 076195, including, by way of example, phosphodiester-linked DNA dinucleotides. The use of biocleavable linkers in polyoligonucleotide constructs is disclosed in WO 2015 / 113922, where they have been used to join multiple antisense constructs (e.g., gapmer regions) within a single oligonucleotide.

[0171] In one embodiment, the oligonucleotide of the invention comprises regions D' and / or D" in addition to the contiguous nucleotide sequence that constitutes the gapmer. In some embodiments, the oligonucleotide of the invention can be represented by the following formula: FG-F'; especially, F 2-8 -G 6-16 -F' 2-8 D'-FG-F', especially D' 2-3 -F 1-8 -G 6-16 -F' 2-8 FG-F'-D”, especially F 2-8 -G 6-16 -F' 2-8 -D” 1-3 D'-FG-F'-D", especially D' 1-3 -F 2-8 -G 6-16 -F' 2-8 -D”1-3

[0172] In some embodiments, the internucleoside linkage between region D' and region F is a phosphodiester linkage. In some embodiments, the internucleoside linkage between region F' and region D" is a phosphodiester linkage.

[0173] Conjugates As used herein, the term conjugate refers to an oligonucleotide covalently attached to a non-nucleotide moiety (the conjugate moiety or region C or a third region).

[0174] Conjugation of the oligonucleotide of the present invention to one or more non-nucleotide moieties can improve the pharmacology of the oligonucleotide, for example, by affecting the activity, cellular distribution, cellular uptake, or stability of the oligonucleotide.In some embodiments, the conjugate moiety regulates or improves the pharmacokinetic properties of the oligonucleotide by improving the cellular distribution, bioavailability, metabolism, excretion, permeability, and / or cellular uptake of the oligonucleotide.In particular, the conjugate can target the oligonucleotide to a specific organ, tissue, or cell type, thereby increasing the efficacy of the oligonucleotide in that organ, tissue, or cell type.At the same time, the conjugate can help to reduce the activity of the oligonucleotide in non-target cell types, tissues, or organs (e.g., off-target activity or activity in non-target cell types, tissues, or organs).

[0175] Oligonucleotide conjugates and their synthesis are also reported in comprehensive reviews by Manoharan in Antisense Drug Technology, Principles, Strategies, and Applications, STCrooke, ed., Ch. 16, Marcel Dekker, Inc., 2001, and Manoharan, Antisense and Nucleic Acid Drug Development, 2002, 12, 103, each of which is incorporated herein by reference in its entirety.

[0176] In one embodiment, the non-nucleotide moiety (conjugate moiety) is selected from the group consisting of a carbohydrate (e.g., GalNAc), a cell surface receptor ligand, a drug substance, a hormone, a lipophile, a polymer, a protein, a peptide, a toxin (e.g., a bacterial toxin), a vitamin, a viral protein (e.g., a capsid), or a combination thereof.

[0177] In some embodiments, the conjugate is an antibody or antibody fragment having specific affinity for the transferrin receptor, e.g., as disclosed in WO 2012 / 143379, which is incorporated herein by reference. In some embodiments, the non-nucleotide moiety is an antibody or antibody fragment, e.g., an antibody or antibody fragment that facilitates delivery across the blood-brain barrier, particularly an antibody or antibody fragment that targets the transferrin receptor.

[0178] Linker Bond or linker is a connection between two atoms that connects one chemical group or segment of interest to another chemical group or segment of interest through one or more covalent bonds.The conjugate moiety can be directly or through a linking moiety (e.g., linker or tether) to the oligonucleotide.The linker serves to covalently connect the third region, e.g., the conjugate moiety (region C), to the first region, e.g., the oligonucleotide or continuous nucleotide sequence (region A) that is complementary to the target nucleic acid.

[0179] In some embodiments of the invention, the conjugates or oligonucleotide conjugates of the invention may optionally comprise a linker region (second region or region B and / or region Y) located between the oligonucleotide or contiguous nucleotide sequence complementary to the target nucleic acid (region A or first region) and the conjugate moiety (region C or third region).

[0180] Region B refers to a biocleavable linker that comprises or consists of a physiologically labile bond that is cleavable under conditions that are normally or similar to those encountered in a mammalian body. Conditions under which a physiologically labile linker undergoes chemical transformation (e.g., cleavage) include chemical conditions such as pH, temperature, oxidative or reductive conditions, or drugs, as well as salt concentrations similar to those found or encountered in mammalian cells. Mammalian intracellular conditions also include the presence of enzymatic activities normally present in mammalian cells, such as proteolytic or hydrolytic enzymes or nucleases. In one embodiment, the biocleavable linker is susceptible to S1 nuclease cleavage. In a preferred embodiment, the nuclease-sensitive linker comprises between 1 and 10 nucleosides, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleosides, more preferably between 2 and 6 nucleosides, most preferably between 2 and 4 linked nucleosides, containing at least two consecutive phosphodiester bonds, such as at least three or four or five consecutive phosphodiester bonds. Preferably, the nucleosides are DNA or RNA. Phosphodiester-containing biocleavable linkers are described in more detail in WO 2014 / 076195, which is incorporated herein by reference.

[0181] Region Y refers to a linker that is not necessarily biocleavable but serves primarily to covalently attach the conjugate moiety (region C or third region) to the oligonucleotide (region A or first region). Region Y linkers may include chain structures or oligomers of repeating units such as ethylene glycol, amino acid units or aminoalkyl groups. The oligonucleotide conjugates of the invention may be constructed from the following region elements AC, ABC, ABYC, AYBC or AYC. In some embodiments, the linker (region Y) is an aminoalkyl, e.g., a C2-C36 aminoalkyl group, including a C6-C12 aminoalkyl group. In a preferred embodiment, the linker (region Y) is a C6 aminoalkyl group.

[0182] treatment As used herein, the term "treatment" refers to both the treatment of an existing disease (e.g., a disease or disorder referred to herein) or the prevention of disease, i.e., prophylaxis. Thus, it will be recognized that the treatments referred to herein may, in some embodiments, be prophylactic. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0183] The oligonucleotides of the present invention The present invention relates to oligonucleotides capable of modulating the expression of ApoE4, e.g., decreasing (downregulating) the expression of ApoE4. Modulation is achieved by hybridizing to a target nucleic acid encoding ApoE4. The target nucleic acid may be a mammalian APOE ε4 mRNA sequence, such as a sequence selected from the group consisting of SEQ ID NOs: 1 and 3.

[0184] The oligonucleotides are antisense oligonucleotides that target the APOE ε4 nucleic acid and result in a decrease in ApoE4 expression.

[0185] Advantageously, the oligonucleotide sequence is complementary to the target sequence of SEQ ID NO: 1 comprising residue 535 of SEQ ID NO: 1. Preferably, the nucleotide of the contiguous nucleotide sequence complementary to the nucleotide at position 535 of SEQ ID NO: 1 comprises a guanine (g) nucleobase or a modified nucleobase thereof that allows Watson-Crick base pairing with the cytosine (c) at position 535 of SEQ ID NO: 1.

[0186] Advantageously, antisense oligonucleotide can regulate the expression of target by reducing or downregulating it.Preferably, such regulation results in inhibition of at least 20% compared to the normal expression level of target, more preferably at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% compared to the normal expression level of target.It is also preferred that antisense oligonucleotide can reduce the expression level of target by at least 20% compared to the normal expression level of target, more preferably at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or 100% compared to the normal expression level of target.

[0187] In some embodiments, the antisense oligonucleotide can reduce the expression level of ApoE4 mRNA by at least 60% or 70% in vitro after applying 25 μM oligonucleotide to neuroblastoma cells that comprise at least one copy of APOE ε4 in their genome, compared to the normal expression level of ApoE4 mRNA.In some embodiments, the antisense oligonucleotide can reduce the expression level of ApoE4 mRNA by at least 50% or 60% in vitro after applying 5 μM oligonucleotide to neuroblastoma cells that comprise at least one copy of APOE ε4 in their genome, compared to the normal expression level of ApoE4 mRNA.Suitably, the examples provide an assay that can be used to measure ApoE4 RNA inhibition (Example 1).

[0188] Target regulation is caused by hybridization between the continuous nucleotide sequence of the oligonucleotide and the target nucleic acid.In some embodiments, the oligonucleotide comprises a mismatch between the oligonucleotide and the target nucleic acid.Despite the mismatch, hybridization to the target nucleic acid may still be sufficient to show the desired regulation of ApoE4 expression.The decrease in binding affinity resulting from the mismatch can be advantageously compensated by increasing the number of nucleotides in the oligonucleotide and / or the number of modified nucleosides, such as 2' sugar modified nucleosides, including LNA, present in the oligonucleotide sequence, which can increase the binding affinity to the target.

[0189] Advantageously, antisense oligonucleotides can reduce ApoE4 expression more than they can reduce ApoE3 expression. In some embodiments, antisense oligonucleotides can reduce ApoE4 expression in target cells containing both ApoE4 and ApoE3 nucleic acids, such that the ratio of the percentage of remaining ApoE3 nucleic acid (when compared to a control) to the percentage of remaining ApoE4 nucleic acid (when compared to a control) is greater than 1, preferably at least 1.5, more preferably at least 2, at least 2.5, at least 3, at least 3.5, or at least 4 (normal expression level of ApoE3 nucleic acid).

[0190] In some embodiments, the antisense oligonucleotide is capable of reducing the expression level of ApoE4 mRNA in target cells containing both ApoE4 mRNA and ApoE3 mRNA such that the ratio of the percentage of remaining ApoE3 mRNA (compared to a control) to the percentage of remaining ApoE4 mRNA (compared to a control) following application of 25 μM oligonucleotide to neuroblastoma cells having a heterozygous genotype for APOE ε3 and APOE ε4 is greater than 1, preferably at least 1.5, more preferably at least 2, at least 2.5, at least 3, at least 3.5 or at least 4.

[0191] In particular, antisense oligonucleotides may be capable of reducing the expression of ApoE4 in target cells, including by inhibiting the expression of ApoE4 in the target cells. (a) an mRNA encoding a human ApoE4 protein encoded by SEQ ID NO: 1, the mRNA comprising a segment corresponding to positions 516 to 556 of SEQ ID NO: 1; and (b) an mRNA encoding a human ApoE3 protein encoded by SEQ ID NO: 2, the mRNA comprising a segment corresponding to positions 516 to 556 of SEQ ID NO: 2; Antisense oligonucleotides reduce the levels of mRNA encoding human ApoE4, resulting in (i) the percentage of remaining ApoE3 mRNA levels compared to controls; and (ii) the ratio between the percentage of remaining ApoE4 mRNA levels compared to the control is greater than 1, such as at least 1.5, such as at least 2, such as at least 2.5, such as at least 3, such as at least 3.5, such as at least 4.

[0192] Suitable controls reflecting the normal expression level of ApoE4 nucleic acid and / or ApoE3 nucleic acid include the expression level of ApoE3 mRNA and ApoE4 mRNA in the absence of antisense oligonucleotide, for example, target cells contacted with vehicle (e.g., PBS or culture medium) only, or with a control oligonucleotide that is known not to target the target sequence as defined herein and is preferably known not to have any off-target activity (i.e., does not have any relevant effect on the expression of the cell / organism genome). Reference (control) values ​​may also be known from the literature. Suitable target cells include human KELLY neuroblastoma cells with heterozygous APOE ε3 / ε4 genotype (Schaffer et al., Genes Nutr. 2014 Jan;9(1)). Example 1 provides an assay that can be used to measure the relative inhibition of ApoE3 and ApoE4 mRNA inhibition using human KELLY neuroblastoma cells.

[0193] One aspect of the present invention relates to an antisense oligonucleotide comprising a contiguous nucleotide sequence of 10 nucleotides in length having at least 80% complementarity to SEQ ID NO: 1. The antisense oligonucleotide may also, or alternatively, have at least 80% complementarity to SEQ ID NO: 3. In some embodiments, the antisense oligonucleotide comprises a contiguous nucleotide sequence of at least 10 nucleotides in length having at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% complementarity to SEQ ID NO: 1. In some embodiments the antisense oligonucleotide also, or alternatively, comprises a contiguous nucleotide sequence of at least 10 nucleotides in length having at least 80%, such as at least 81%, for example at least 82%, such as at least 83%, for example at least 84%, such as at least 85%, for example at least 86%, such as at least 87%, for example at least 88%, such as at least 89%, for example at least 90%, such as at least 91%, for example at least 92%, such as at least 93%, for example at least 94%, such as at least 95%, for example at least 96%, such as at least 97%, for example at least 98%, for example at least 99%, such as 100% complementarity to SEQ ID NO:3.

[0194] In some embodiments, the oligonucleotide comprises a contiguous sequence of 10-30 nucleotides in length that is at least 80%, such as at least 81%, for example at least 82%, such as at least 83%, for example at least 84%, such as at least 85%, for example at least 86%, such as at least 87%, for example at least 88%, such as at least 89%, for example at least 90%, such as at least 91%, for example at least 92%, such as at least 93%, for example at least 94%, such as at least 95%, for example at least 96%, such as at least 97%, for example at least 98%, for example at least 99%, such as 100% complementary to a region of a target nucleic acid or to a target sequence.

[0195] It is advantageous if the oligonucleotide of the invention, or its contiguous nucleotide sequence, is fully complementary (100% complementary) to a region of a target nucleic acid or to a target sequence.

[0196] In some embodiments, the oligonucleotide or its contiguous nucleotide sequence may contain 0 to 3 mismatches compared to the target sequence to which it is complementary, selected arbitrarily from 1 mismatch, 2 mismatches, and 3 mismatches. For example, the oligonucleotide or its contiguous nucleotide sequence may contain 1 or 2 mismatches between the oligonucleotide or its contiguous nucleotide sequence and the target sequence in the target nucleic acid. The mismatch is not at the nucleotide at position 535 of SEQ ID NO: 1. Thus, the nucleotide of the contiguous nucleotide sequence complementary to the nucleotide at position 535 of SEQ ID NO: 1 contains a guanine (g) nucleobase.

[0197] In some embodiments, the oligonucleotide comprises a contiguous nucleotide sequence of 8 to 50 nucleotides in length that is at least 80% complementary, such as at least 90% complementary, such as completely (or 100%) complementary to a target sequence within positions 516 to 556 of SEQ ID NO:1, such as within positions 522 to 548 of SEQ ID NO:1.

[0198] In some embodiments, the oligonucleotide comprises a contiguous nucleotide sequence of 10 to 30 nucleotides in length that is at least 80% complementary, such as at least 90% complementary, such as completely (or 100%) complementary to a target sequence within positions 516 to 556 of SEQ ID NO:1, such as within positions 522 to 548 of SEQ ID NO:1.

[0199] In some embodiments, the oligonucleotide sequence is 100% complementary to the corresponding target sequence present in SEQ ID NO:1.

[0200] Antisense oligonucleotides are also advantageous if they are complementary to a target sequence selected from one of the regions listed in Table 2. In some embodiments, the contiguous nucleotide sequence of the antisense oligonucleotide is at least 80% complementary or at least 90% complementary, e.g., fully complementary, to a target sequence selected from R_4 to R_96. In some embodiments, the oligonucleotide sequence is 100% complementary to a target sequence selected from R_25, R_40, R_46, R_66 and R_91 (Table 2).

[0201] The oligonucleotide may comprise or consist of 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 or 50 nucleotides in length. The oligonucleotide may be, for example, 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 or 50 nucleotides in length. In some embodiments, the oligonucleotide comprises or consists of 8-50, such as 8-40, such as 10-40, such as 10-35, such as 10-30, such as 11-25, such as 12-22, such as 14-20 or 14-18 consecutive nucleotides in length. In one embodiment, the oligonucleotide comprises or consists of 16-22 nucleotides in length. In a preferred embodiment, the oligonucleotide comprises or consists of a length of 16 to 20 nucleotides.

[0202] In some embodiments, the oligonucleotide or its contiguous nucleotide sequence comprises or consists of 22 or less nucleotides, such as 20 or less nucleotides, such as 16, 17, 18, 19, or 20 nucleotides. Any range provided herein should be understood to include the end points of the range. Thus, when an oligonucleotide is described as comprising 10 to 30 nucleotides, both 10 nucleotides and 30 nucleotides are included.

[0203] In some embodiments, the contiguous nucleotide sequence comprises or consists of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 contiguous nucleotides in length. In preferred embodiments, the oligonucleotide comprises or consists of 16, 17, 18, 19 or 20 nucleotides in length.

[0204] Preferably, the contiguous nucleotide sequence is 100% complementary to the target nucleic acid. [Table 3] TIFF2024540271000007.tif255170 TIFF2024540271000008.tif116170

[0205] In some embodiments, the oligonucleotide or contiguous nucleotide sequence comprises or consists of a sequence selected from those listed in Table 3.

[0206] In some embodiments, the antisense oligonucleotide or contiguous nucleotide sequence comprises or consists of a length of 10-30 nucleotides that is at least 90% identical, preferably 100% identical, to a sequence selected from the group consisting of SEQ ID NOs: 4-96 (see motif sequences listed in Table 3).

[0207] In some embodiments, the antisense oligonucleotide or contiguous nucleotide sequence comprises or consists of a length of 10 to 30 nucleotides having at least 90% identity, preferably 100% identity, to a sequence selected from the group consisting of SEQ ID NOs: 25, 40, 46, 66 and 91.

[0208] In some embodiments, the antisense oligonucleotide or contiguous nucleotide sequence comprises or consists of a length of 10 to 30 nucleotides having at least 90% identity, preferably 100% identity, to a sequence selected from the group consisting of SEQ ID NOs: 25, 40, and 46.

[0209] In some embodiments, the antisense oligonucleotide or contiguous nucleotide sequence comprises or consists of a length of 10-30 nucleotides that is at least 90% identical, preferably 100% identical, to the sequence of SEQ ID NO:25.

[0210] In some embodiments, the antisense oligonucleotide or contiguous nucleotide sequence comprises or consists of a length of 10-30 nucleotides that is at least 90% identical, preferably 100% identical, to the sequence of SEQ ID NO:40.

[0211] In some embodiments, the antisense oligonucleotide or contiguous nucleotide sequence comprises or consists of a length of 10 to 30 nucleotides that is at least 90% identical, preferably 100% identical, to the sequence of SEQ ID NO:46.

[0212] In some embodiments, the antisense oligonucleotide or contiguous nucleotide sequence comprises or consists of a length of 10-30 nucleotides that is at least 90% identical, preferably 100% identical, to the sequence of SEQ ID NO:66.

[0213] In some embodiments, the antisense oligonucleotide or contiguous nucleotide sequence comprises or consists of a length of 10 to 30 nucleotides that is at least 90% identical, preferably 100% identical, to the sequence of SEQ ID NO:91.

[0214] In some embodiments, the contiguous nucleotide sequence comprises a sequence selected from SEQ ID NOs: 4-96.

[0215] In some embodiments, the contiguous nucleotide sequence consists of a sequence selected from SEQ ID NOs: 4-96.

[0216] In some embodiments, the antisense oligonucleotide comprises a sequence selected from SEQ ID NOs: 4-96.

[0217] In some embodiments, the antisense oligonucleotide consists of a sequence selected from SEQ ID NOs: 4-96.

[0218] In some embodiments, the contiguous nucleotide sequence comprises or consists of the nucleotide sequence set forth in SEQ ID NO:25.

[0219] In some embodiments, the contiguous nucleotide sequence comprises or consists of the nucleotide sequence set forth in SEQ ID NO:40.

[0220] In some embodiments, the contiguous nucleotide sequence comprises or consists of the nucleotide sequence set forth in SEQ ID NO:46.

[0221] In some embodiments, the contiguous nucleotide sequence comprises or consists of the nucleotide sequence set forth in SEQ ID NO:66.

[0222] In some embodiments, the contiguous nucleotide sequence comprises or consists of the nucleotide sequence set forth in SEQ ID NO:91.

[0223] In some embodiments, the antisense oligonucleotide sequence comprises or consists of the nucleotide sequence set forth in SEQ ID NO:25.

[0224] In some embodiments, the antisense oligonucleotide sequence comprises or consists of the nucleotide sequence set forth in SEQ ID NO:40.

[0225] In some embodiments, the antisense oligonucleotide sequence comprises or consists of the nucleotide sequence set forth in SEQ ID NO:46.

[0226] In some embodiments, the antisense oligonucleotide sequence comprises or consists of the nucleotide sequence set forth in SEQ ID NO:66.

[0227] In some embodiments, the antisense oligonucleotide sequence comprises or consists of the nucleotide sequence set forth in SEQ ID NO:91.

[0228] It is understood that the consecutive nucleobase sequence (motif sequence) can be modified, for example, to increase nuclease resistance and / or binding affinity to the target nucleic acid.

[0229] The pattern in which modified nucleosides (such as high affinity modified nucleosides) are incorporated into an oligonucleotide sequence is commonly referred to as the oligonucleotide design.

[0230] Advantageously, the oligonucleotide sequence does not contain RNA nucleosides, because it reduces nuclease resistance.Furthermore, as described elsewhere herein, the antisense oligonucleotide can advantageously contain one or more modified nucleosides or nucleotides, such as 2' sugar modified nucleosides.Furthermore, it is advantageous that the unmodified nucleoside is a DNA nucleoside.Therefore, the oligonucleotide of the present invention can be designed using modified nucleosides and DNA nucleosides.Preferably, high affinity modified nucleosides are used.

[0231] In one embodiment, the oligonucleotide comprises at least one modified nucleoside, such as at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, or at least 16 modified nucleosides. In one embodiment, the oligonucleotide comprises 1-10 modified nucleosides, such as 2-9 modified nucleosides, such as 3-8 modified nucleosides, such as 4-7 modified nucleosides, such as 6 or 7 modified nucleosides. Suitable modifications are described in the "Definitions" sections of "Modified Nucleosides", "High Affinity Modified Nucleosides", "Sugar Modifications", "2' Sugar Modifications", and Locked Nucleic Acid (LNA).

[0232] In one embodiment, the oligonucleotide comprises one or more sugar-modified nucleosides, such as 2' sugar-modified nucleosides.Preferably, the oligonucleotide of the present invention comprises one or more 2' sugar-modified nucleosides independently selected from the group consisting of 2'-O-alkyl-RNA, 2'-O-methyl-RNA, 2'-alkoxy-RNA, 2'-O-methoxyethyl-RNA, 2'-amino-DNA, 2'-fluoro-DNA, arabino nucleic acid (ANA), 2'-fluoro-ANA, and LNA nucleoside.It is advantageous if one or more of the modified nucleoside(s) is locked nucleic acid (LNA).

[0233] In further embodiments, the oligonucleotide comprises at least one modified internucleoside bond. Suitable internucleoside modifications are described in the "Definitions" section under "Modified Internucleoside Bonds". It is advantageous if at least 75%, such as 80%, such as all internucleoside bonds in a contiguous nucleotide sequence are phosphorothioate or boranophosphate internucleoside bonds. In some embodiments, all internucleoside bonds in a contiguous sequence of the oligonucleotide are phosphorothioate bonds.

[0234] In some embodiments, the oligonucleotide of the present invention comprises at least one LNA nucleoside, such as 1, 2, 3, 4, 5, 6, 7, or 8 LNA nucleosides, such as 2-6 LNA nucleosides, such as 3-7 LNA nucleosides, 4-8 LNA nucleosides, or 3, 4, 5, 6, 7, or 8 LNA nucleosides. In some embodiments, at least 75% of the modified nucleosides in the oligonucleotide are LNA nucleosides, such as 80%, such as 85%, such as 90% of the modified nucleosides are LNA nucleosides, in particular beta-D-oxyLNA or ScET. In yet further embodiments, all of the modified nucleosides in the oligonucleotide are LNA nucleosides. In further embodiments, the oligonucleotide may comprise both beta-D-oxy-LNA and one or more of the following LNA nucleosides: thio-LNA in either beta-D or alpha-L configuration, amino-LNA, oxy-LNA, ScET, and / or ENA, or a combination thereof. In further embodiments, all LNA cytosine units are 5-methyl-cytosine. For nuclease stability of an oligonucleotide or a contiguous nucleotide sequence, it is advantageous to have at least one LNA nucleoside at the 5' end and at least two LNA nucleosides at the 3' end of the nucleotide sequence.

[0235] In one embodiment of the invention, the oligonucleotide of the invention is capable of recruiting RNase H.

[0236] In the present invention, advantageous structural designs are those gapmer designs described in the "Definitions" section, e.g., "Gapmer", "LNA Gapmer", "MOE Gapmer", and "Mixed Wing Gapmer", "Alternating Flank Gapmer". Gapmer designs include gapmers with uniform flanks, mixed wing flanks, alternating flanks, and gap breaker designs.

[0237] Advantageously, the oligonucleotide is a gapmer of FG-F' design, in particular a gapmer of the formula 5'-FG-F'-3', where regions F and F' independently comprise 1 to 8 nucleosides, 2 to 5 of which are 2' sugar modified and define the 5' and 3' ends of the F and F' regions, and G is a region of 6 to 16 nucleosides capable of recruiting RNase H, e.g. a region comprising 6 to 16 DNA nucleosides.

[0238] In some embodiments, both flanks have 2' sugar modified nucleosides at the 5' and 3' termini.

[0239] In some embodiments the gapmer is an LNA gapmer.

[0240] In some embodiments of the invention, the LNA gapmer is selected from the following uniform flank designs: 2-13-2, 2-8-3, 3-8-2, 2-9-3, 3-9-2, 2-8-4, 2-9-2.

[0241] In some embodiments, the LNA gapmer has an alternating flank design. In the alternating flank design, at least one of the flanks (F or F') contains one or more DNA nucleosides in addition to an LNA nucleoside. The flanking regions F or F', or both F and F', then contain at least three nucleosides, and the 5' and 3' most nucleosides of the F and / or F' regions are LNA nucleosides. A gapmer with an alternating flank design with F having five nucleosides that is LNA-LNA-DNA-DNA-LNA, a gap region with six DNA nucleosides, and F' having two LNA nucleosides can therefore be represented as 2-2-1-6-2, since each "-" (dash) represents a shift between LNA / DNA nucleosides, the number of nucleosides, and the most number of DNA nucleosides in the gap region G.

[0242] Using the same expression, in some embodiments of the invention, the LNA gapmer is selected from the following alternating flank designs: 2-1-1-1-1-6-2, 2-7-1-1-2, 2-1-1-6-3, 2-1-2-6-2, 2-8-1-1-2, 2-7-1-1-3, 2-7-1-2-2, 2-2-1-6-2 and 2-1-1-7-2.

[0243] Tables 4 and 5 list the preferred designs for each motif sequence.

[0244] In all cases, the FG-F' design may further comprise regions D' and / or D" as described in the "Definitions" section of "Region D' or D" in an Oligonucleotide." In some embodiments, oligonucleotides of the invention have one, two, or three phosphodiester-linked nucleoside units, e.g., DNA units, at the 5' or 3' end of the gapmer region.

[0245] In some embodiments, the oligonucleotides of the invention contain, in addition to the phosphorodithioate bond(s), both phosphorothioate internucleoside bond(s) and at least one phosphodiester bond, such as 2, 3, or 4 phosphodiester bonds. In gapmer-type oligonucleotides, phosphodiester bonds, if present, are not necessarily positioned between consecutive DNA nucleosides within the gap region G.

[0246] Advantageously, all internucleoside bonds of the consecutive nucleotide sequence of the oligonucleotide are phosphorothioate or all internucleoside bonds of the oligonucleotide are phosphorothioate. Nuclease-resistant bonds such as phosphorothioate bonds are particularly useful in oligonucleotide regions that can recruit nucleases when forming a duplex with a target nucleic acid, such as region G of a gapmer. However, phosphorothioate bonds can also be useful in non-nuclease recruiting regions and / or affinity enhancing regions, such as regions F and F' of a gapmer. Gapmer oligonucleotides may, in some embodiments, contain one or more phosphodiester bonds in regions F or F', or both regions F and F', and all internucleoside bonds of region G can be phosphorothioate.

[0247] In some embodiments, the oligonucleotide is an oligonucleotide compound having compound number 91_1. In certain embodiments, the oligonucleotide is an oligonucleotide compound having compound number 66_1. In certain embodiments, the oligonucleotide is an oligonucleotide compound having compound number 46_1. In certain embodiments, the oligonucleotide is an oligonucleotide compound having compound number 46_2. In certain embodiments, the oligonucleotide is an oligonucleotide compound having compound number 46_3. In certain embodiments, the oligonucleotide is an oligonucleotide compound having compound number 46_4. In certain embodiments, the oligonucleotide is an oligonucleotide compound having compound number 46_5. In certain embodiments, the oligonucleotide is an oligonucleotide compound having compound number 25_1. In certain embodiments, the oligonucleotide is an oligonucleotide compound having compound number 25_2. In certain embodiments, the oligonucleotide is an oligonucleotide compound having compound number 25_3. In certain embodiments, the oligonucleotide is an oligonucleotide compound having compound number 25_4. In certain embodiments, the oligonucleotide is an oligonucleotide compound having compound number 25_5. In certain embodiments, the oligonucleotide is an oligonucleotide compound having compound number 25_6. In certain embodiments, the oligonucleotide is an oligonucleotide compound having compound number 40_1. In certain embodiments, the oligonucleotide is an oligonucleotide compound having compound number 40_2. In certain embodiments, the oligonucleotide is an oligonucleotide compound having compound number 40_3.

[0248] Particularly advantageous antisense oligonucleotides in the context of the present invention are ACcAgGcggccgCG (SEQ ID NO: 91; Compound No. 91_1) CAggcggccgcgcacGT (SEQ ID NO: 66; Compound No. 66_1) CGcgcacgtCcTC (SEQ ID NO: 46; Compound No. 46_1) CGcgcacgtcCTC (SEQ ID NO: 46; Compound No. 46_2) CGcGcacgtcCTC (SEQ ID NO: 46; Compound No. 46_3) CGCgcacgtccTC (SEQ ID NO: 46; compound number 46_4) CGcGCacgtccTC (SEQ ID NO: 46; Compound No. 46_5) GCacgtcctccATG (SEQ ID NO: 25; Compound No. 25_1) GCAgtcctccaTG (SEQ ID NO: 25; Compound No. 25_2) GCacgtcctcCaTG (SEQ ID NO: 25; Compound No. 25_3) GCacgtcctCcATG (SEQ ID NO: 25; Compound No. 25_4) GCacgtcctCcaTG (SEQ ID NO: 25; Compound No. 25_5) GCacgtcctcCATG (SEQ ID NO: 25; Compound No. 25_6) GCgcacgtcctCC (SEQ ID NO: 40; Compound No. 40_1) GCgcAcgtcctCC (SEQ ID NO: 40; compound number 40_2) GCgCacgtcctCC (SEQ ID NO: 40; compound number 40_3) wherein the capital letters are beta-D-oxy LNA nucleosides, the lower case letters are DNA nucleosides, all LNA C's are 5-methylcytosine, and all internucleoside linkages are phosphorothioate internucleoside linkages.

[0249] Further particular advantageous oligonucleotides in the context of the present invention are oligonucleotide compounds selected from the group consisting of the compounds defined in Table 4 by HELM annotation, i.e. the structure of each compound is described by the Hierarchical Editing Language for Macromolecules (HELM) (for details see Zhang et al., Chem. Inf. Model. 2012, 52, 10, 2796-2806 or J. Chem. Inf. Model. 2017, 57, 6, 1233-1239) using the following HELM annotation key: [LR](G) is beta-D-oxy-LNA guanine nucleoside; [LR](T) is beta-D-oxy-LNA thymine nucleoside; [LR](A) is beta-D-oxy-LNA adenine nucleoside; [LR] ([5meC]) is beta-D-oxy-LNA 5-methylcytosine nucleoside; [dR](G) is a DNA guanine nucleoside, [dR](T) is a DNA thymine nucleoside, [dR](A) is a DNA adenine nucleoside, [dR]([C]) is a DNA cytosine nucleoside, [sP] is a phosphorothioate internucleoside linkage (sterically undefined).

[0250] Further information and open source tools for HELM can be found at the internet addresses www.pistoiaalliance.org / helm-tools / and www.pistoiaalliance.org / membership / about / . In particular, in Table 4, the designations "RNA1" and "$$$$V2.0" represent information useful for computer analysis of the HELM sequence and should not be construed as limiting the oligonucleotide sequence defined between the brackets (i.e., between "{" and "}"). [Table 4] TIFF2024540271000010.tif207170

[0251] Manufacturing method In a further aspect, the present invention provides a method for producing the oligonucleotide of the present invention, comprising reacting nucleotide units to thereby form covalently linked consecutive nucleotide units of the oligonucleotide. Preferably, the method uses phosphoramidite chemistry (see, for example, Caruthers et al, 1987, Methods in Enzymology vol. 154, pages 287-313). In a further embodiment, the method further comprises reacting the consecutive nucleotide sequence with a conjugating moiety (ligand) to covalently link the conjugate moiety to the oligonucleotide. In a further aspect, a method for producing the composition of the present invention is provided, comprising mixing the oligonucleotide of the present invention or the conjugated oligonucleotide with a pharma- ceutically acceptable diluent, solvent, carrier, salt, and / or adjuvant.

[0252] Pharmaceutical salts The compound according to the present invention may exist in the form of its pharmaceutically acceptable salt. The term "pharmaceutically acceptable salt" refers to a conventional acid or base addition salt that retains the biological effectiveness and properties of the compound of the present invention and is formed from suitable non-toxic organic or inorganic acids or organic or inorganic bases. Acid addition salts include, for example, those derived from inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, sulfamic acid, phosphoric acid and nitric acid, and those derived from organic acids such as p-toluenesulfonic acid, salicylic acid, methanesulfonic acid, oxalic acid, succinic acid, citric acid, malic acid, lactic acid, fumaric acid, and the like. Base addition salts include those derived from ammonium, potassium, sodium and quaternary ammonium hydroxides, such as tetramethylammonium hydroxide. Chemical modification of pharmaceutical compounds into salts is a technique well known to pharmaceutical scientists to obtain improved physical and chemical stability, hygroscopicity, flowability and solubility of the compound. This is described, for example, in Bastin, Organic Process Research & Development 2000, 4, 427-435 or in Ansel, In: Pharmaceutical Dosage Forms and Drug Delivery Systems, 6th ed. (1995), pp. 196 and 1456-1457. For example, the pharma- ceutically acceptable salt of the compounds provided herein can be a sodium salt.

[0253] In a further aspect, the present invention provides a pharma- ceutically acceptable salt of the antisense oligonucleotide or conjugate thereof. In a preferred embodiment, the pharma- ceutically acceptable salt is a sodium or potassium salt.

[0254] Pharmaceutical Compositions In a further aspect, the present invention provides a pharmaceutical composition comprising any of the aforementioned oligonucleotides and / or oligonucleotide conjugates or salts thereof, and a pharma- ceutically acceptable diluent, carrier, salt and / or adjuvant. Pharmaceutically acceptable diluents include phosphate buffered saline (PBS) and pharma- ceutically acceptable salts include, but are not limited to, sodium and potassium salts. In some embodiments, the pharma- ceutical acceptable diluent is sterile phosphate buffered saline. In some embodiments, the oligonucleotide is used in the pharma- ceutical acceptable diluent at a concentration of 50-300 μM solution.

[0255] Formulations suitable for use in the present invention can be found in Remington's Pharmaceutical Sciences, Mack Publishing Company, Philadelphia, Pa., 17th ed., 1985. For a brief review of methods of drug delivery, see, for example, Langer (Science 249:1527-1533, 1990). WO 2007 / 031091 provides further suitable and preferred examples of pharma-ceutically acceptable diluents, carriers and adjuvants (herein incorporated by reference). Suitable dosages, formulations, administration routes, compositions, dosage forms, combinations with other therapeutic agents, prodrug formulations are also provided in WO 2007 / 031091.

[0256] The oligonucleotide or oligonucleotide conjugate of the present invention can be mixed with pharmaceutically acceptable active or inactive substances for the preparation of pharmaceutical compositions or formulations.The composition and method for the preparation of pharmaceutical compositions depend on many criteria, including but not limited to, the route of administration, the extent of disease, or the dose to be administered.

[0257] These compositions may be sterilized by conventional sterilization techniques or sterile filtered. The resulting aqueous solutions may be packaged for immediate use or lyophilized, the lyophilized preparation being combined with a sterile aqueous carrier prior to administration. The pH of the preparation will typically be 3-11, more preferably 5-9 or 6-8, most preferably 7-8, e.g., 7-7.5. The resulting solid form compositions may be packaged in a plurality of single-dose units, each containing a fixed amount of the agent or agents, such as a sealed package of tablets or capsules. The solid form compositions may also be packaged in flexible volume containers, such as squeezable tubes designed for topically applicable creams or ointments.

[0258] In some embodiments, the oligonucleotide or oligonucleotide conjugate of the invention is a prodrug. Particularly with respect to oligonucleotide conjugates, when the prodrug is delivered to the site of action, e.g., a target cell, the conjugate moiety is cleaved from the oligonucleotide.

[0259] Purpose The oligonucleotides of the invention can be utilized, for example, as research reagents for diagnostic, therapeutic and prophylactic methods.

[0260] In research, such oligonucleotides can be used to specifically regulate the synthesis of ApoE4 protein in cells (e.g., in vitro cell cultures) and experimental animals, thereby facilitating the functional analysis of the target or the evaluation of its usefulness as a target for therapeutic intervention.Typically, target regulation is achieved by degrading or inhibiting the mRNA that produces the protein, thereby preventing protein formation, or by degrading or inhibiting the modulator of the gene or mRNA that produces the protein.

[0261] For research or diagnostic uses of the oligonucleotides of the invention, the target nucleic acid can be a cDNA or a synthetic nucleic acid derived from DNA or RNA.

[0262] The present invention provides an in vivo or in vitro method for modulating ApoE4 expression in a target cell expressing ApoE4, comprising administering to said cell an effective amount of an oligonucleotide of the invention.

[0263] In some embodiments, the target cells are mammalian cells, particularly human cells. The target cells may be in vitro cell cultures or in vivo cells that form part of mammalian tissue. In a preferred embodiment, the target cells are present in the brain or central nervous system. In particular, cells of the cerebral cortex, medulla / pons, midbrain, frontal cortex, brainstem, cerebellum, and spinal cord may be relevant target regions. For the treatment of AD, targeted reduction of the cerebral cortex, medulla / pons, and midbrain regions of the brain region is advantageous. For the treatment of PSP, such as PSP with AD-like pathology, targeted reduction of the brain regions medulla / pons and midbrain is advantageous. In particular, microglia, neurons, nerve cells, astrocytes, axons, and basal ganglia are or contain relevant cell types.

[0264] In diagnostics, oligonucleotides can be used to detect and quantitate ApoE4 expression in cells and tissues by Northern blotting, in situ hybridization or similar techniques.

[0265] For purposes of therapy, the oligonucleotides may be administered to an animal or human suspected of having a disease or disorder that can be treated by modulating the expression of ApoE4.

[0266] For therapeutic purposes, oligonucleotides may also be administered to animals or humans at risk of developing a disease or disorder that can be treated by modulating the expression of ApoE4.

[0267] For therapeutic purposes, oligonucleotides may also be administered to animals or humans diagnosed with a disease or disorder that can be treated by modulating the expression of ApoE4.

[0268] In particular, the present invention provides a method of treating or preventing a disease or disorder comprising administering a therapeutically or prophylactically effective amount of an oligonucleotide, oligonucleotide conjugate or pharmaceutical composition of the invention to a subject suffering from, at risk of, or susceptible to the disease or disorder.

[0269] The present invention also relates to an oligonucleotide, a composition or a conjugate as defined herein for use as a medicament.

[0270] The oligonucleotides, oligonucleotide conjugates or pharmaceutical compositions according to the invention are typically administered in effective amounts.

[0271] The present invention also provides the use of an oligonucleotide or oligonucleotide conjugate of the invention as described for the manufacture of a medicament for treating or preventing a disease or disorder referred to herein, or for a method of treating or preventing a disorder referred to herein.

[0272] The diseases or disorders as referred to herein are associated with the expression of ApoE4. The therapeutic applications of the present invention are preferably used for the treatment or prevention of diseases or disorders caused by abnormal levels and / or activity of ApoE4.

[0273] Advantageously, the therapeutic applications of the present invention are used to treat subjects suffering from or at risk of such diseases or disorders who have at least one copy of the APOE ε4 gene in their genome.

[0274] In some embodiments, a subject suffering from or at risk for a disease or disorder carries a copy of the APOE ε4 gene in their genome.

[0275] In some embodiments, a subject suffering from or at risk for a disease or disorder carries a copy of the APOE ε3 gene in their genome.

[0276] In some embodiments, the subject suffering from or at risk for the disease has the heterozygous APO ε3 / ε4 genotype.

[0277] In some embodiments, the subject suffering from or at risk for the disease has the heterozygous APO ε2 / ε4 genotype.

[0278] In some embodiments, the subject suffering from or at risk for the disease has the homozygous genotype APO ε4 / ε4.

[0279] In some embodiments, the subject suffering from or at risk for the disease is homozygous for the APO ε4 gene.

[0280] In some embodiments, the treatment is administered to a subject at risk for, suspected of having, or diagnosed with a neurological disorder selected from the group consisting of neurodegenerative diseases including Alzheimer's Disease (AD), Frontotemporal Dementia (FTD), Pick's Disease (PiD), Progressive Supranuclear Palsy (PSP), movement disorders such as Parkinson's Disease (PD), dementia with Lewy bodies, dementia in Down's Syndrome, and Niemann-Pick Disease Type C1, etc.

[0281] In one embodiment, the invention relates to an oligonucleotide, an oligonucleotide conjugate or a pharmaceutical composition for use in the treatment of a disease or disorder selected from AD, FTD, PiD, PSP, movement disorders such as PD, dementia with Lewy bodies, dementia in Down's syndrome, and Niemann-Pick disease type C1.

[0282] In certain embodiments, the disease or disorder is AD. AD can be, for example, late-onset AD (over 65 years old). In some embodiments, AD can be AD without dominant AD mutation. AD can be early-onset AD. AD can also be AD or AD-type pathology in patients with progressive supranuclear palsy (PSP).

[0283] In certain embodiments, the disease or disorder is frontotemporal dementia (FTD).

[0284] In certain embodiments, the disease or disorder is Pick's Disease (PiD).

[0285] In certain embodiments, the disease or disorder is progressive supranuclear palsy (PSP).

[0286] In certain embodiments, the disease or disorder is a movement disorder. For example, the movement disorder can be Parkinson's disease (PD).

[0287] In certain embodiments, the disease or disorder is dementia with Lewy bodies.

[0288] In certain embodiments, the disease or disorder is dementia in a subject with Down's syndrome.

[0289] In certain embodiments, the disease or disorder is Niemann-Pick disease type C1.

[0290] In certain embodiments, the disease or disorder is dementia. Optionally, the dementia is dementia in or related to any one or more of AD, FTD, PiD, PSP, PD, dementia with Lewy bodies, dementia in subjects with Down's syndrome, or Niemann-Pick disease type C1.

[0291] Administration The oligonucleotide or pharmaceutical composition of the present invention can be administered parenterally (intravenous, subcutaneous, intramuscular, intracerebral, intraventricular, intraocular, or intrathecal administration, etc.).

[0292] In some embodiments, administration is via intrathecal administration.

[0293] Advantageously, eg for the treatment of neurological disorders, the oligonucleotides or pharmaceutical compositions of the invention are administered intrathecally or intracranially, eg via intracerebral or intraventricular administration.

[0294] The invention also provides the use of an oligonucleotide or conjugate thereof, such as a pharmaceutical salt or composition of the invention, for the manufacture of a medicament in a dosage form for subcutaneous administration.

[0295] The invention also provides the use of an oligonucleotide of the invention or a conjugate thereof, such as a pharmaceutical salt or composition of the invention, for the manufacture of a medicament in a dosage form for intrathecal administration.

[0296] The invention also provides the use of an oligonucleotide or oligonucleotide conjugate of the invention as described for the manufacture of a medicament in a dosage form for intrathecal administration.

[0297] Combination therapy In some embodiments, the oligonucleotide, oligonucleotide conjugate or pharmaceutical composition of the invention is used in combination treatment with another therapeutic agent, which may be, for example, a standard of care for the above-mentioned disease or disorder.

[0298] array SEQ ID NO:1 - NM_001302690.2 with rs429358 (APOE4, mRNA) 1 ggatggggag ataagagaag accaggaggg agttaaatag ggaatgggtt gggggcggct 61 tggtaaatgt gctgggatta ggctgttgca gataatgcaa caaggcttgg aaggctaacc 121 tgggactggc caatcacagg caggagatg aaggttctgt gggctgcgtt gctggtcaca 181 ttcctggcag gatgccaggc caaggtggag caagcggtgg agacagagcc ggagcccgag 241 ctgcgccagc agaccgagtg gcagagcggc cagcgctggg aactggcact gggtcgcttt 301 tgggattacc tgcgctgggt gcagacactg tctgagcagg tgcaggagga gctgctcagc 361 tcccaggtca cccaggaact gagggcgctg atggacgaga ccatgaagga gttgaaggcc 421 tacaaatcgg aactggagga acaactgacc ccggtggcgg aggagacgcg ggcacggctg 481 tccaaggagc tgcaggcggc gcaggcccgg ctgggcgcgg acatggagg cgtgcgcggc 541 cgcctggtgc agtaccgcgg cgaggtgcag gccatgctcg gccagagcac cgaggagctg 601 cgggtgcgcc tcgcctccca cctgcgcaag ctgcgtaagc ggctcctccg cgatgccgat 661 gacctgcaga agcgcctggc agtgtaccag gccggggccc gcgagggcgc cgagcgcggc 721 ctcagcgcca tccgcgagcg cctggggccc ctggtggaac agggccgcgt gcggggccgcc 781 actgtgggct ccctggccgg ccagccgcta caggagcggg cccaggcctg gggcgagcgg 841 ctgcgcgcgc ggatggagga gatgggcagc cggacccgcg accgcctgga cgaggtgaag 901 gagcaggtgg cggaggtgcg cgccaagctg gaggagcagg cccagcagat acgcctgcag 961 gccgaggcct tccaggcccg cctcaagagc tggttcgagc ccctggtgga agacatgcag 1021 cgccagtggg ccgggctggt ggagaaggtg caggctgccg tgggcaccag cgccgcccct 1081 gtgcccagcg acaatcactg aacgccgaag cctgcagcca tgcgacccca cgccaccccg 1141 tgcctcctgc ctccgcgcag cctgcagcgg gagaccctgt ccccgcccca gccgtcctcc 1201 tggggtggac cctagtttaa taaagattca ccaagtttca cgca Accession No. 2 - NM_001302690.2 (APOE3, mRNA) 1 ggatggggag ataagagaag accaggaggg agttaaatag ggaatgggtt gggggcggct 61 tggtaaatgt gctgggatta ggctgttgca gataatgcaa caaggcttgg aaggctaacc 121 tgggactggc caatcacagg caggaagatg aaggttctgt gggctgcgtt gctggtcaca 181 ttcctggcag gatgccaggc caaggtggag caagcggtgg agacagagcc ggagcccgag 241 ctgcgccagc agaccgagtg gcagagcggc cagcgctggg aactggcact gggtcgcttt 301 tgggattacc tgcgctgggt gcagacactg tctgagcagg tgcaggagga gctgctcagc 361 tcccaggtca cccaggaact gagggcgctg atggacgaga ccatgaagga gttgaaggcc 421 tacaaatcgg aactggagga acaactgacc ccggtggcgg aggagacgcg ggcacggctg 481 tccaaggagc tgcaggcggc gcaggcccgg ctgggcgcgg acatggagga cgtgtgcggc 541 cgcctggtgc agtaccgcgg cgaggtgcag gccatgctcg gccagagcac cgaggagctg 601 cgggtgcgcc tcgcctccca cctgcgcaag ctgcgtaagc ggctcctccg cgatgccgat 661 gacctgcaga agcgcctggc agtgtaccag gccggggccc gcgagggcgc cgagcgcggc 721 ctcagcgcca tccgcgagcg cctggggccc ctggtggaac agggccgcgt gcggggccgcc 781 actgtgggct ccctggccgg ccagccgcta caggagcggg cccaggcctg gggcgagcgg 841 ctgcgcgcgc ggatggagga gatgggcagc cggacccgcg accgcctgga cgaggtgag 901 gagcaggtgg cggaggtgcg cgccaagctg gagcagg cccagcagat acgcctgcag 961 gccgaggcct tccaggcccg cctcaagagc tggttcgagc ccctggtgga agacatgcag 1021 cgccagtggg ccgggctggt ggagaaggtg caggctgccg tgggcaccag cgccgcccct 1081 gtgcccagcg acaatcactg aacgccgaag cctgcagcca tgcgacccca cgccaccccg 1141 tgcctcctgc ctccgcgcag cctgcagcgg gagaccctgt ccccgcccca gccgtcctcc 1201 tggggtggac cctagtttaa taaagattca ccaagtttca cgca Accession No. 3 - XM__005589554.2 (Macaca fascicularis apolipoprotein E (APOE), mRNA) 1 atgagctcag gcgcctctag aaagtgtagc tgggacctcg ggaagccctg gcctccagac 61 tggccaatca caggcaggaa gatgaaggtt ctgtgggctg cgttgctggt cacattcctg 121 gcaggatgcc aggccaaggt ggagcaaccg gtggagccag agacggaacc cgagcttcgc 181 cagcaggctg aggggcagag cggccagccc tgggagctgg cactgggtcg cttttgggat 241 tacctgcgct gggtgcagac actgtctgag caggtgcagg aggagctgct cagcccccag 301 gtcacccagg aactgacgac gctgatggat gagaccatga aggagttgaa ggcctacaaa 361 tcggaactgg aggaacagct gagcccggtg gcggaggaga cgcgggcacg gctgtccaag 421 gagctgcagg cggcgcaggc ccggctgggt gccgacatgg aggacgtgcg cagccgcctg 481 gtgcagtacc gcagcgaggt gcaggccatg ctgggccaga gtaccgagga gctgcgggcg 541 cgcctcgcct cccacctgcg caagctgcgc aagcggctcc tccgcgatgc tgatgacctg 601 cagaagcgcc tggcagtgta tcaggccggg gcccgcgagg gcgccgagcg cggggtcagc 661 gccatccgcg agcgcctggg acccctggtg gagcagggcc gcgtgcgggc cgccactgtg 721 ggctccctgg ccagccagcc gcttcaggag cgggcccagg ccttgggtga gcggcttcgc 781 gcacggatgg aggagatggg cagccggacc cgcgaccgcc tggacgaggt gaaggagcag 841 gtggcggagg tgcgcgccaa gctggaggaa caggcccagc agataagcct gcaggccgag 901 gccttccagg cccgcctcaa gagctggttc gagcccctcg tggaagatat gcagcgccag 961 tgggctgggc tggtggagaa ggtgcaggct gccgtgggcg ccagcaccgc ccctgtgccc 1021 agcgacaatc actgaacgcc caggcctaca gccatgcgac ccgactccac cccatgcctc 1081 ctctctccgc tcagcctgca gcgggagacc ctgtccccgc cccagccgtc ctccaggggt 1141 gggccctagt ttaataaaga ttcgccaagt ttcaccgca

[0299] EMBODIMENTS OF THE PRESENT DISCLOSURE An antisense oligonucleotide having a length of 1.8 to 50 nucleotides, for example, 10 to 30 nucleotides, comprising a contiguous nucleotide sequence of at least 10 nucleotides that is at least 80% complementary to a target sequence within positions 516 to 556 of an apolipoprotein (Apo) E4-encoding nucleic acid shown as SEQ ID NO:1, wherein the target sequence comprises position 535 of SEQ ID NO:1.

[0300] 2. The antisense oligonucleotide according to item 1, wherein the contiguous nucleotide sequence has 0 to 3 mismatches compared to the target sequence, optionally selected from 1 mismatch, 2 mismatches, and 3 mismatches, provided that the nucleotide of the contiguous nucleotide sequence complementary to the nucleotide at position 535 of SEQ ID NO:1 comprises a guanine (g) nucleobase.

[0301] 3. The antisense oligonucleotide of any one of the preceding items, wherein the contiguous nucleotide sequence is at least 90% complementary to a target sequence within positions 516 to 556 of SEQ ID NO:1.

[0302] 4. The antisense oligonucleotide of any one of the preceding items, wherein the contiguous nucleotide sequence is 100% complementary to a target sequence within positions 516 to 556 of SEQ ID NO:1.

[0303] 5. The antisense oligonucleotide of any one of the preceding items, wherein the contiguous nucleotide sequence is complementary to a target sequence within positions 522 to 548 of SEQ ID NO:1.

[0304] 6. The antisense oligonucleotide of any one of the preceding items, wherein the contiguous nucleotide sequence is complementary to a target sequence selected from R_4 to R_96 in Table 2.

[0305] 7. The antisense oligonucleotide of any one of the preceding items, wherein the contiguous nucleotide sequence is complementary to a target sequence selected from residues 522-535 (R_25), residues 525-537 (R_40), residues 526-538 (R_46), residues 530-546 (R_66) and residues 535-548 (R_91) of SEQ ID NO:1.

[0306] 8. The antisense oligonucleotide according to any one of the preceding items, wherein the contiguous nucleotide sequence comprises or consists of a nucleotide sequence selected from the group consisting of SEQ ID NOs: 4 to 96.

[0307] 9. The antisense oligonucleotide of any one of the preceding items, wherein the contiguous nucleotide sequence comprises or consists of a nucleotide sequence selected from the group consisting of SEQ ID NOs: 25, 40, 46, 66 and 91.

[0308] 10. The antisense oligonucleotide of any one of the preceding items, wherein the contiguous nucleotide sequence comprises or consists of SEQ ID NO:25.

[0309] 11. The antisense oligonucleotide of any one of the preceding items, wherein the contiguous nucleotide sequence comprises or consists of SEQ ID NO:40.

[0310] 12. The antisense oligonucleotide of any one of the preceding items, wherein the contiguous nucleotide sequence comprises or consists of SEQ ID NO:46.

[0311] 13. The antisense oligonucleotide of any one of the preceding items, wherein the contiguous nucleotide sequence comprises or consists of SEQ ID NO:66.

[0312] 14. The antisense oligonucleotide of any one of the preceding items, wherein the contiguous nucleotide sequence comprises or consists of SEQ ID NO:91.

[0313] 15. The antisense oligonucleotide of any one of the preceding items, wherein the antisense oligonucleotide is capable of reducing expression of apolipoprotein (Apo) E4 in a mammal, such as a human.

[0314] 16. The antisense oligonucleotide according to any one of the preceding items, wherein the antisense oligonucleotide is capable of reducing the expression level of mRNA encoding apolipoprotein (Apo) E4 of a mammal, such as a human, in a target cell by at least 20%, such as at least 30%, for example at least 40%, such as at least 50%, for example at least 60%, such as at least 70%, for example at least 80%, for example at least 90%, compared to the normal expression level of the target.

[0315] 17. The antisense oligonucleotide can reduce the expression of ApoE4, resulting in: (a) an mRNA encoding a human ApoE4 protein encoded by SEQ ID NO: 1, the mRNA comprising a segment corresponding to positions 516 to 556 of SEQ ID NO: 1; and (b) in a target cell containing an mRNA encoding a human ApoE3 protein encoded by SEQ ID NO: 2, the mRNA containing a segment corresponding to positions 516 to 556 of SEQ ID NO: 2, The antisense oligonucleotide reduces the level of mRNA encoding human ApoE4, thereby (i) the percentage of remaining ApoE3 mRNA levels compared to controls; and (ii) the ratio between the percentage of remaining ApoE4 mRNA levels compared to the control is greater than 1, such as at least 1.5, such as at least 2, such as at least 2.5, such as at least 3, such as at least 3.5, such as at least 4, and optionally said control is the level of mRNA in a control target cell in the absence of said antisense oligonucleotide.

[0316] 18. The antisense oligonucleotide of any one of the preceding items, wherein the target sequence is located within an RNA.

[0317] 19. The antisense oligonucleotide according to item 18, wherein the RNA is mRNA.

[0318] 20. The antisense oligonucleotide according to item 19, wherein the mRNA is a mature mRNA.

[0319] 21. The antisense oligonucleotide of any one of the preceding items, comprising or consisting of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides in length.

[0320] 22. The antisense oligonucleotide according to any one of the preceding items, comprising or consisting of a length of 14 to 30 nucleotides.

[0321] 23. The antisense oligonucleotide according to any one of the preceding items, comprising or consisting of a length of 16 to 24 nucleotides.

[0322] 24. The antisense oligonucleotide according to any one of the preceding items, wherein the contiguous nucleotide sequence has a length of 14 to 22 nucleotides.

[0323] 25. The antisense oligonucleotide of any one of the preceding items, wherein the contiguous nucleotide sequence has a length of at least 16 nucleotides, such as 16, 17, 18, 19, 20, 21 or 22 nucleotides.

[0324] 26. The antisense oligonucleotide of any one of the preceding items, which is single-stranded.

[0325] 27. The antisense oligonucleotide of any one of the preceding items, wherein the contiguous nucleotide sequence comprises one or more modified nucleosides, such as 2' sugar modified nucleosides or unlocked nucleic acid (UNA) nucleosides.

[0326] 28. The antisense oligonucleotide of any one of the preceding items, wherein the contiguous nucleotide sequence comprises one or more 2' sugar modified nucleosides.

[0327] 29. The antisense oligonucleotide of claim 28, wherein the one or more 2' sugar modified nucleosides are independently selected from the group consisting of 2'-O-alkyl-RNA, 2'-O-methyl-RNA, 2'-alkoxy-RNA, 2'-O-methoxyethyl-RNA, 2'-amino-DNA, 2'-fluoro-DNA, arabinonucleic acid (ANA), 2'-fluoro-ANA, and locked nucleic acid (LNA) nucleosides.

[0328] 30. The antisense oligonucleotide of paragraph 29, wherein the one or more 2' sugar modified nucleosides comprises at least one LNA nucleoside.

[0329] 31. The antisense oligonucleotide of paragraph 30, wherein the at least one LNA nucleoside is independently selected from the group consisting of oxy-LNA, amino-LNA, thio-LNA, cET and ENA LNA nucleosides.

[0330] 32. The antisense oligonucleotide of paragraph 31, wherein the modified LNA nucleoside is oxy-LNA with a 2'-4' bridge -O-CH2-.

[0331] 33. The antisense oligonucleotide according to item 32, wherein the oxy-LNA is beta-D-oxy-LNA.

[0332] 34. The antisense oligonucleotide according to any one of items 29 to 33, wherein the contiguous nucleotide sequence comprises 4 to 8 LNA nucleosides.

[0333] 35. The antisense oligonucleotide of any one of the preceding items, comprising at least one modified internucleoside linkage.

[0334] 36. The antisense oligonucleotide of any one of the preceding items, comprising nuclease-resistant modified internucleoside linkages.

[0335] 37. The antisense oligonucleotide of any one of the preceding items, wherein at least 50% of the internucleoside linkages in the contiguous nucleotide sequence are phosphorothioate internucleoside linkages.

[0336] 38. The antisense oligonucleotide of any one of the preceding items, wherein at least 80% of the internucleoside linkages in the contiguous nucleotide sequence are phosphorothioate internucleoside linkages.

[0337] 39. The antisense oligonucleotide of any one of the preceding items, wherein the antisense oligonucleotide is capable of recruiting RNase H.

[0338] 40. The antisense oligonucleotide according to any one of the preceding items, wherein the oligonucleotide or the contiguous nucleotide sequence thereof is a gapmer.

[0339] 41. The antisense oligonucleotide according to any one of the preceding items, wherein the oligonucleotide or its contiguous nucleotide sequence is a gapmer of the formula 5'-FG-F'-3', wherein each of regions F and F' independently comprises or consists of 1 to 8 nucleosides, of which 2 to 5 are 2' sugar modified nucleosides, and region G is a region of between 6 and 16 nucleosides capable of recruiting RNase H.

[0340] 42. The antisense oligonucleotide according to item 41, wherein the region G is a region containing 6 to 16 DNA nucleosides.

[0341] 43. The antisense oligonucleotide of any one of paragraphs 41 and 42, wherein 2' sugar modified nucleosides define the 5' and 3' ends of the F and F' regions.

[0342] 44. The antisense oligonucleotide according to any one of paragraphs 41 to 43, wherein the 2' sugar modified nucleoside is according to any one of paragraphs 29 to 33.

[0343] 45. (a) the F region is 2 to 8 nucleotides in length and consists of 2 to 5 identical LNA nucleosides and 0 to 4 DNA nucleosides; (b) the F' region is 2 to 6 nucleotides in length and consists of 2 to 4 identical LNA nucleosides and 0 to 2 DNA nucleosides; (c) The antisense oligonucleotide according to any one of items 41 to 44, wherein region G is 6 to 14 DNA nucleotides.

[0344] 46. ​​The antisense oligonucleotide according to any one of claims 1 to 45, wherein the antisense oligonucleotide is or comprises a compound selected from the group consisting of: ACcAgGcggccgCG (SEQ ID NO: 91; Compound No. 91_1) CAggcggccgcgcacGT (SEQ ID NO: 66; Compound No. 66_1) CGcgcacgtCcTC (SEQ ID NO: 46; Compound No. 46_1) CGcgcacgtcCTC (SEQ ID NO: 46; Compound No. 46_2) CGcGcacgtcCTC (SEQ ID NO: 46; Compound No. 46_3) CGCgcacgtccTC (SEQ ID NO: 46; compound number 46_4) CGcGCacgtccTC (SEQ ID NO: 46; Compound No. 46_5) GCacgtcctccATG (SEQ ID NO: 25; Compound No. 25_1) GCAgtcctccaTG (SEQ ID NO: 25; Compound No. 25_2) GCacgtcctcCaTG (SEQ ID NO: 25; Compound No. 25_3) GCacgtcctCcATG (SEQ ID NO: 25; Compound No. 25_4) GCacgtcctCcaTG (SEQ ID NO: 25; Compound No. 25_5) GCacgtcctcCATG (SEQ ID NO: 25; Compound No. 25_6) GCgcacgtcctCC (SEQ ID NO: 40; Compound No. 40_1) GCgcAcgtcctCC (SEQ ID NO: 40; compound number 40_2) GCgCacgtcctCC (SEQ ID NO: 40; compound number 40_3) (wherein capital letters indicate beta-D-oxy LNA nucleosides, lower case letters indicate DNA nucleosides, capital C indicates 5-methylcytosine beta-D-oxy LNA nucleosides, and all internucleoside linkages are phosphorothioate internucleoside linkages).

[0345] 47. The antisense oligonucleotide according to item 46, which is AChAgGcggccgCG (sequence number 91; compound number 91_1).

[0346] 48. The antisense oligonucleotide according to item 46, which is CAggcggccgcgcacGT (sequence number 66; compound number 66_1).

[0347] 49. The antisense oligonucleotide according to item 46, which is CGcgcacgtCcTC (sequence number 46; compound number 46_1).

[0348] 50. The antisense oligonucleotide according to item 46, which is CGcgcacgtcCTC (sequence number 46; compound number 46_2).

[0349] 51. The antisense oligonucleotide according to item 46, which is CGcGcacgtcCTC (sequence number 46; compound number 46_3).

[0350] 52. The antisense oligonucleotide according to item 46, which is CGCgcacgtccTC (sequence number 46; compound number 46_4).

[0351] 53. The antisense oligonucleotide according to item 46, which is CGcGCacgtccTC (sequence number 46; compound number 46_5).

[0352] 54. The antisense oligonucleotide according to item 46, which is GCacgtcctccATG (sequence number 25; compound number 25_1).

[0353] 55. The antisense oligonucleotide according to item 46, which is GCAgtcctccaTG (sequence number 25; compound number 25_2).

[0354] 56. The antisense oligonucleotide according to item 46, which is GCacgtcctcCaTG (sequence number 25; compound number 25_3).

[0355] 57. The antisense oligonucleotide according to item 46, which is GCacgtcctCcATG (sequence number 25; compound number 25_4).

[0356] 58. The antisense oligonucleotide according to item 46, which is GCacgtcctCcaTG (sequence number 25; compound number 25_5).

[0357] 59. The antisense oligonucleotide according to item 46, which is GCacgtcctcCATG (sequence number 25; compound number 25_6).

[0358] 60. The antisense oligonucleotide according to item 46, which is GCgcacgtcctCC (sequence number 40; compound number 40_1).

[0359] 61. The antisense oligonucleotide according to item 46, which is GCgcAcgtcctCC (sequence number 40; compound number 40_2).

[0360] 62. The antisense oligonucleotide according to item 46, which is GCgCacgtcctCC (sequence number 40; compound number 40_3).

[0361] 63. The antisense oligonucleotide according to any one of clauses 1 to 46, wherein one of five, for example one of four, for example one of the first, third and fourth most proximal nucleotides to the 5' end of said antisense oligonucleotide, or a contiguous nucleotide sequence thereof, is complementary to position 535 of SEQ ID NO: 1 and comprises a guanine nucleobase.

[0362] 64. The antisense oligonucleotide according to any one of clauses 1 to 46, wherein one of the six, for example one of the first and sixth most proximal nucleotides to the 3' end of the antisense oligonucleotide, or a contiguous nucleotide sequence thereof, is complementary to position 535 of SEQ ID NO: 1 and comprises a guanine nucleobase.

[0363] 65. The antisense oligonucleotide of any one of clauses 1-46, wherein one of the nucleotides of region G is complementary to position 535 of SEQ ID NO:1 and comprises a guanine nucleobase.

[0364] 66. The antisense oligonucleotide of any one of clauses 1-46, wherein one of the nucleotides of region F is complementary to position 535 of SEQ ID NO:1 and comprises a guanine nucleobase.

[0365] 67. The antisense oligonucleotide according to any one of clauses 1 to 46, wherein one of the nucleotides of region F' is complementary to position 535 of SEQ ID NO:1 and comprises a guanine nucleobase.

[0366] 68. A conjugate comprising an antisense oligonucleotide according to any one of the preceding items and at least one conjugate moiety covalently attached to said oligonucleotide.

[0367] 69. The conjugate of claim 68, wherein the conjugate moiety is selected from a carbohydrate, a cell surface receptor ligand, a drug substance, a hormone, a lipophilic substance, a polymer, a protein, a peptide, a toxin, a vitamin, a viral protein, or a combination thereof.

[0368] 70. The conjugate of paragraph 69, wherein the conjugate moiety facilitates delivery across the blood-brain barrier.

[0369] 71. The conjugate of paragraph 70, wherein the conjugate moiety is an antibody or antibody fragment that targets the transferrin receptor.

[0370] 72. The conjugate of any one of clauses 68 to 70, comprising a linker disposed between the antisense oligonucleotide and the conjugate moiety.

[0371] 73. The conjugate according to paragraph 72, wherein the linker is a physiologically labile linker.

[0372] 74. A pharma- ceutically acceptable salt of the antisense oligonucleotide according to any one of clauses 1 to 67 or the conjugate according to any one of clauses 68 to 73.

[0373] 75. A pharmaceutical composition comprising an antisense oligonucleotide according to any one of clauses 1 to 67, and / or a conjugate according to any one of clauses 68 to 73, and / or a pharma- ceutically acceptable salt according to clause 74, and a pharma- ceutically acceptable diluent, solvent, carrier, salt and / or adjuvant.

[0374] 76. A method for producing an antisense oligonucleotide according to any one of paragraphs 1 to 67, comprising reacting nucleotide units, thereby forming covalently linked consecutive nucleotide units contained in said oligonucleotide.

[0375] 77. The method of claim 76, further comprising reacting the contiguous nucleotide sequence with a non-nucleotide conjugation moiety.

[0376] 78. A method for producing a pharmaceutical composition according to paragraph 75, comprising mixing the antisense oligonucleotide with a pharma- ceutically acceptable diluent, carrier, salt and / or adjuvant.

[0377] 79. A method for regulating ApoE4 expression in a target cell expressing ApoE4, the method comprising administering to the cell an effective amount of an antisense oligonucleotide according to any one of clauses 1 to 67, a conjugate according to any one of clauses 68 to 73, or a pharmaceutical composition according to clause 74.

[0378] 80. The method according to paragraph 79, which is an in vivo method or an in vitro method.

[0379] 81. A method for treating or preventing a disease, comprising administering a therapeutically or prophylactically effective amount of an antisense oligonucleotide according to any one of clauses 1 to 67, a conjugate according to any one of clauses 68 to 73, a pharma- ceutically acceptable salt according to clause 74, or a pharmaceutical composition according to clause 75 to a subject suffering from or at risk of the disease.

[0380] 82. The antisense oligonucleotide according to any one of clauses 1 to 67, the conjugate according to any one of clauses 68 to 73, the pharma- ceutically acceptable salt according to clause 74, or the pharmaceutical composition according to clause 75, for use as a pharmaceutical.

[0381] 83. The antisense oligonucleotide according to any one of clauses 1 to 67, the conjugate according to any one of clauses 68 to 73, the pharma- ceutically acceptable salt according to clause 74, or the pharmaceutical composition according to clause 75, for use in a method for treating or preventing a disease.

[0382] 84. Use of the antisense oligonucleotide according to any one of clauses 1 to 67, the conjugate according to any one of clauses 68 to 73, the pharma- ceutically acceptable salt according to clause 74, or the pharmaceutical composition according to clause 75 for the preparation of a medicament for a method for treating or preventing a disease.

[0383] 85. The method according to any one of clauses 79 to 81, the antisense oligonucleotide, conjugate, salt or pharmaceutical composition for use according to any one of clauses 82 and 83, or the use according to clause 84, wherein the disease is associated with the in vivo activity of ApoE4.

[0384] 86. The method of use, the antisense oligonucleotide, conjugate, salt or pharmaceutical composition for use, or the use according to clause 85, wherein the in vivo activity of ApoE4 is reduced by at least 30%, such as at least 40%, for example at least 50%, such as at least 60%, for example at least 70%, for example at least 80%, for example at least 90% compared to a control, optionally wherein the control is the in vivo activity of ApoE4 before administering the antisense oligonucleotide, conjugate or pharmaceutical composition.

[0385] 87. The method of any one of clauses 79 to 81 and 85 to 86, the antisense oligonucleotide, conjugate, salt or pharmaceutical composition for use of any one of clauses 82 and 83, or the use of clause 84, wherein the disease is optionally associated with the expression level of ApoE4 in a biological sample from the subject.

[0386] 88. The method, antisense oligonucleotide, conjugate, salt or pharmaceutical composition according to clause 87, or use, wherein the expression level of ApoE4 is reduced by at least 30%, such as at least 40%, for example at least 50%, such as at least 60%, for example at least 70%, for example at least 80%, for example at least 90% compared to a control, optionally wherein the control is the expression level of ApoE4 before administering the antisense oligonucleotide, conjugate or pharmaceutical composition.

[0387] 89. The method, antisense oligonucleotide, conjugate, salt for use or pharmaceutical composition, or use according to any one of paragraphs 79 to 88, wherein the subject suffering from or at risk of the disease has at least one copy of the APOE ε4 gene in the genome.

[0388] 90. The method, antisense oligonucleotide, conjugate, salt or pharmaceutical composition for use, or use according to paragraph 89, wherein the subject suffering from or at risk of the disease is a subject of the APOE ε3 / ε4 genotype.

[0389] 91. The method, antisense oligonucleotide, conjugate, salt or pharmaceutical composition for use, or use according to paragraph 89, wherein the subject suffering from or at risk of the disease is a subject of the APOE ε4 / ε4 genotype.

[0390] 92. The method, antisense oligonucleotide, conjugate, salt or pharmaceutical composition for use, or use according to any one of paragraphs 79 to 91, wherein the disease is a dementia-related disease.

[0391] 93. The antisense oligonucleotide, conjugate, salt or pharmaceutical composition for use, or the use according to any one of paragraphs 79 to 92, wherein the disease is selected from Alzheimer's disease (AD), frontotemporal dementia (FTD), Pick's disease (PiD), progressive supranuclear palsy (PSP), movement disorders such as Parkinson's disease (PD), dementia with Lewy bodies, dementia in Down's syndrome, and Niemann-Pick disease type C1, etc.

[0392] 94. The method, antisense oligonucleotide, conjugate, salt or pharmaceutical composition for use, or use according to any one of paragraphs 79 to 93, wherein the disease is AD.

[0393] 95. The method, antisense oligonucleotide, conjugate, salt or pharmaceutical composition for use, or use according to any one of paragraphs 79 to 94, wherein the subject suffering from or at risk of a disease is a mammalian subject, such as a human. EXAMPLES

[0394] material and method Oligonucleotide motif sequences and oligonucleotide compounds [Table 5]

[0395] A motif sequence represents a contiguous sequence of nucleobases present in an oligonucleotide.

[0396] The design refers to gapmer design FG-F' containing alternating flanks as described elsewhere herein.

[0397] In the oligonucleotide compounds, capital letters indicate beta-D-oxy LNA nucleosides, lower case letters indicate DNA nucleosides, capital C indicates 5-methylcytosine beta-D-oxy LNA nucleosides, and all internucleoside linkages are phosphorothioate internucleoside linkages. See Table 4 for a description of these compounds according to HELM annotation.

[0398] Oligonucleotide synthesis Oligonucleotide synthesis is generally known in the art. Below are applicable protocols. The oligonucleotide compounds described herein may be produced by methods that differ slightly in terms of the equipment, support and concentration used.

[0399] Oligonucleotides are synthesized on Unylinker universal solid support (Org. Process Res. Dev. 2008, 12, 3, 399-410) using the phosphoramidite method on a 1 μmol scale MermMade 192 oligonucleotide synthesizer. At the end of the synthesis, oligonucleotides are cleaved from the solid support with aqueous ammonia at 60 °C for 5-16 h. Oligonucleotides are purified by reversed-phase HPLC (RP-HPLC) or solid-phase extraction, characterized by UPLC, and molecular weights are further confirmed by ESI-MS.

[0400] Oligonucleotide extension: Coupling of 5'DMTr protected nucleoside β-cyanoethyl-phosphoramidites including DNA-A(Bz), DNA-G(iBu), DNA-C(Bz), DNA-T, LNA-5-methyl-C(Bz), LNA-A(Bz), LNA-G(dmf), LNA-T, 2'OMe-A(Bz), 2'OMe(U), 2'OMe(T), 2'OMe-C(Ac), 2'OMe-G(iBu), 2'OMe-G(dmf) is carried out by using 0.1 M of 5'-O-DMT protected phosphorumidites in acetonitrile and a solution of DCI (4,5-dicyanoimidazole) in acetonitrile (0.25 M) as activating agent.

[0401] Purification by RP-HPLC: The crude compound is purified by preparative RP-HPLC on a Phenomenex Jupiter C18 10μ 150x10mm column. 0.1M ammonium acetate pH 8 and acetonitrile are used as buffers at a flow rate of 5mL / min. The collected fractions are lyophilized to give the purified compound, typically as a white solid. Abbreviation: DCI: 4,5-dicyanoimidazole DCM: dichloromethane DMF: Dimethylformamidine DMT: 4,4'-dimethoxytrityl THF: tetrahydrofuran Bz: Benzoyl Ibu: Isobutyryl RP-HPLC: Reversed-phase high-performance liquid chromatography

[0402] Example 1: 15 Oligonucleotides Screened for Effects on APOE3 and APOE4 Expression Levels Human KELLY neuroblastoma cells (ACC 355, DSMZ) were seeded in 96-well plates at 30000 cells / well in 190ul of standard cell culture medium (RPMI-1640 Sigma R2405, 10% FBS, 25μg / ml penicillin-streptomycin) the day before treatment. KELLY cells were selected based on their heterozygous genotype for APOE3 and APOE4 (Schaffer et al., Genes Nutr. 2014 Jan;9(1)). On the day of treatment, oligonucleotides diluted in PBS (Gibco#14190-094) were added to obtain gymnotic uptake at final concentrations in medium of 5μM and 25μM, respectively, in a total volume of 200μl per well.

[0403] Cells were incubated for 5 days at 37 °C, 5% CO2, and 98% humidity in an active evaporative incubator. On day 5, cell medium was removed from the culture wells by pipetting, 125 μL of RLT buffer (Qiagen) was added, and RNA was extracted by using Qiagen's RNeasy 96 kit and protocol. cDNA synthesis was performed using 4 μL of input RNA and was performed using the IScript Advanced cDNA Synthesis Kit for RT-qPCR (Bio-Rad), and 2.5 μL was used as input for digital droplet PCR using ddPCR supermix for probes (no dUTP) (Bio-Rad) according to the manufacturer's protocol. The following assays were used: APOE3 / 4: TaqMan® SNP Genotyping Assay, rs429358 (Assay ID C___3084793_20, Thermo Fischer) HPRT1: HPRT1 (ddPCR GEX CY5.5 Assay 12005587 from Biorad)

[0404] APOE3 and APOE4 mRNA concentrations were quantified relative to the housekeeping gene HPRT1 using QuantaSoft Software (Bio-Rad).

[0405] Expression levels were then normalized to the average of the untreated PBS controls for APOE3 and APOE4, respectively. The results are shown in Table 6, including the ratio of APOE3 to APOE4 (higher ratios indicate APOE4 selectivity). [Table 6]

[0406] Various modifications and variations of the described modes of carrying out the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with certain preferred embodiments, the claimed invention should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes of carrying out the invention that are obvious to those skilled in the relevant arts are intended to be within the scope of the following embodiments.

Claims

1. 1. An antisense oligonucleotide of 8 to 50 nucleotides in length, for example 10 to 30 nucleotides in length, comprising a contiguous nucleotide sequence of at least 10 nucleotides in length that is at least 80% complementary to a target sequence within positions 516 to 556 of an apolipoprotein (Apo) E4-encoding nucleic acid set forth as SEQ ID NO:1, wherein said target sequence comprises position 535 of SEQ ID NO:

1.

2. 2. The antisense oligonucleotide of claim 1, wherein the contiguous nucleotide sequence has 0 to 3 mismatches compared to the target sequence, optionally selected from 1 mismatch, 2 mismatches, and 3 mismatches, provided that the nucleotide of the contiguous nucleotide sequence complementary to nucleotide 535 of SEQ ID NO: 1 comprises a guanine (g) nucleobase.

3. The antisense oligonucleotide of claim 1, wherein the contiguous nucleotide sequence is at least 90% complementary to a target sequence within positions 516 to 556 of SEQ ID NO:

1.

4. The antisense oligonucleotide of claim 1, wherein the contiguous nucleotide sequence is 100% complementary to a target sequence within positions 516 to 556 of SEQ ID NO:

1.

5. The antisense oligonucleotide of claim 1, wherein the contiguous nucleotide sequence is complementary to a target sequence within positions 522 to 548 of SEQ ID NO:

1.

6. 2. The antisense oligonucleotide of claim 1, wherein the contiguous nucleotide sequence is complementary to a target sequence selected from residues 522-535 (R_25), residues 525-537 (R_40), residues 526-538 (R_46), residues 530-546 (R_66), and residues 535-548 (R_91) of SEQ ID NO:

1.

7. 2. The antisense oligonucleotide of claim 1, wherein the contiguous nucleotide sequence comprises or consists of a nucleotide sequence selected from the group consisting of SEQ ID NOs: 4 to 96.

8. 2. The antisense oligonucleotide of claim 1, wherein the contiguous nucleotide sequence comprises or consists of a nucleotide sequence selected from the group consisting of SEQ ID NOs: 25, 40, 46, 66 and 91.

9. The antisense oligonucleotide of claim 1, wherein the antisense oligonucleotide is capable of reducing the expression of apolipoprotein (Apo) E4 in a mammal, such as a human.

10. 2. The antisense oligonucleotide of claim 1, wherein the antisense oligonucleotide is or comprises a compound selected from the group consisting of: ACcAgGcggccgCG (SEQ ID NO: 91; Compound No. 91_1) CAggcggccgcgcacGT (SEQ ID NO: 66; Compound No. 66_1) CGcgcacgtCcTC (SEQ ID NO: 46; Compound No. 46_1) CGcgcacgtcCTC (SEQ ID NO: 46; Compound No. 46_2) CGcGcacgtcCTC (SEQ ID NO: 46; Compound No. 46_3) CGCgcacgtccTC (SEQ ID NO: 46; Compound No. 46_4) CGcGCacgtccTC (SEQ ID NO: 46; Compound No. 46_5) GCacgtcctccATG (SEQ ID NO: 25; Compound No. 25_1) GCAcgtcctccaTG (SEQ ID NO: 25; Compound No. 25_2) GCacgtcctcCaTG (SEQ ID NO: 25; Compound No. 25_3) GCacgtcctCcATG (SEQ ID NO: 25; Compound No. 25_4) GCacgtcctCcaTG (SEQ ID NO: 25; Compound No. 25_5) GCacgtcctcCATG (SEQ ID NO: 25; Compound No. 25_6) GCgcacgtcctCC (SEQ ID NO: 40; Compound No. 40_1) GCgcAcgtcctCC (SEQ ID NO: 40; Compound No. 40_2) GCgCacgtcctCC (SEQ ID NO: 40; Compound No. 40_3) (wherein capital letters indicate beta-D-oxy LNA nucleosides, lower case letters indicate DNA nucleosides, capital C indicates 5-methylcytosine beta-D-oxy LNA nucleosides, and all internucleoside linkages are phosphorothioate internucleoside linkages).

11. A conjugate comprising the antisense oligonucleotide of claim 1 and at least one conjugate moiety covalently attached to the oligonucleotide.

12. A pharmaceutically acceptable salt of the antisense oligonucleotide of claim 1 or the conjugate of claim 11.

13. A pharmaceutical composition comprising an antisense oligonucleotide according to claim 1, and / or a conjugate according to claim 11, and / or a pharmaceutically acceptable salt of the antisense oligonucleotide according to claim 1 or the conjugate according to claim 11, and a pharmaceutically acceptable diluent, solvent, carrier, salt and / or adjuvant.

14. A pharmaceutical composition for use in a method for treating or preventing a disease, comprising an antisense oligonucleotide described in claim 1, a conjugate described in claim 11, or a pharmaceutically acceptable salt of the antisense oligonucleotide described in claim 1 or the conjugate described in claim 11.

15. The pharmaceutical composition of claim 14, wherein the disease is associated with the in vivo activity of ApoE4.

16. The pharmaceutical composition according to claim 14, wherein the disease is a dementia-related disease.

17. 15. The pharmaceutical composition of claim 14, wherein the disease is selected from Alzheimer's disease (AD), frontotemporal dementia (FTD), Pick's disease (PiD), progressive supranuclear palsy (PSP), movement disorders such as Parkinson's disease (PD), dementia with Lewy bodies, dementia in Down's syndrome, and Niemann-Pick disease type C1.

18. The pharmaceutical composition of claim 17, wherein the disease is AD.