HK40109677A - Oligonucleotides for modulating apolipoprotein e4 expression - Google Patents

Description

Oligonucleotides for regulating apolipoprotein E4 expression

Technical Field

This invention relates to oligonucleotides (oligomers) that are complementary to APOEε4 nucleic acids (such as mRNA transcripts) and can be used to reduce the expression of apolipoprotein E4 (ApoE4). Reduction of APOEε4 transcript and/or ApoE4 protein expression is beneficial for a range of medical diseases, 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)), Lewy body dementia, Down syndrome dementia, and Niemann-Pick disease type C1.

Background Technology

Apolipoprotein E (ApoE) is a lipoprotein involved in the binding of lipids such as cholesterol and phospholipids for lipid transport. ApoE is primarily produced by hepatocytes and Kupffer cells in the liver, with smaller amounts produced by the adrenal glands and adipose tissue. In the central nervous system, the main producers of ApoE are astrocytes and microglia in healthy nerve systems, while in disease states, microglia and neurons contribute more to ApoE production.

ApoE is encoded by the APOE gene (OMIM 107741), located on chromosome 19, and carries two common single nucleotide polymorphisms (SNPs): rs429358 and rs7412. These result in three major isotypes of ApoE: ApoE2, ApoE3, and ApoE4, differing in amino acids at positions 112 and 158 in the protein. In ApoE3, these positions are occupied by cysteine and arginine, respectively, while in ApoE2 and ApoE4, these two positions are occupied by cysteine and arginine, respectively.

Amino acid differences affect the conformation of ApoE isoforms and their ability to bind to different lipids and proteins. For example, ApoE4 is more likely to bind to heparan sulfate-binding proteins and very low-density lipoproteins and has reduced interaction with the LDL receptor LRP1, resulting in reduced clearance of amyloid plaques. Preclinical studies have also shown that ApoE4 may accelerate blood-brain barrier (BBB) disruption, cerebral blood flow loss, neuronal loss, and behavioral deficits, independent of amyloid-β (Montagne et al., Nat. Aging 2021; 1; 506-20). It has been further demonstrated that the presence of ApoE4 in the P301S mouse model (a tau disease model) exacerbates the already widespread tau-mediated neurodegeneration in a way that depends on the responsiveness of astrocytes and microglia (Shi et al., Nature 2017; 549(7673): 523-527). Further research showed that selective removal of ApoE4 in astrocytes in the same mouse model alleviated tau-mediated neurodegeneration.

The most predominant ApoE isotype in the general population is ApoE3, while three percent (3%) of the world's population is homozygous for ApoE4, and 14% of the population carries at least one copy of ApoE4. However, the proportion of Alzheimer's disease (AD) patients carrying at least one copy of ApoE4 is higher than in the general population, at 37%. ApoE4 has also been associated with higher amyloid positivity in patients with normal and mild cognitive impairment and with an increased risk of developing late-onset AD.

ApoE4 has been associated with other diseases and conditions besides Alzheimer's disease (AD). The presence of ApoE4 also lowers the age of onset of FTD-related neurodegeneration. In Parkinson's disease (PD), ApoE4 homozygous PD patients have demonstrated 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. In PiD (a tau protein-related neuropathological dementia), an excess of the ApoE4 allele has been found. ApoE4 has also been reported to lower the age of onset of Down syndrome dementia. In Niemann-Pick disease type C, the severity of the disease is exacerbated if the patient carries the ApoE4 allele. The frequency of the ApoE4 allele has also been shown to be higher in patients with PSP and AD than in those with PSP alone.

There is a great need for robust and effective medications to treat these and other diseases and conditions associated with ApoE4.

Purpose of the invention

One object of the present invention is to provide antisense oligonucleotides, including nicked oligonucleotides, that target APOEε4 nucleic acids (such as mRNA) and reduce ApoE4 expression in target cells in vivo and in vitro.

Another objective is to provide antisense oligonucleotides that are more effective at reducing ApoE4 expression compared to ApoE3 expression.

Another objective is to provide such antisense oligonucleotides for use in methods of treating or preventing diseases and conditions (including AD) associated with ApoE4.

Summary of the Invention

This invention relates to oligonucleotides that target ApoE4-encoded nucleic acids and thereby regulate ApoE4 expression in target cells. Oligonucleotides targeting positions 516 to 556 of SEQ ID NO:1 have been identified, particularly oligonucleotides whose target sequence includes residue 535 of SEQ ID NO:1, which is a polymorphic site distinguishing APOEε4 from APOEε3.

Therefore, the present invention provides oligonucleotides of 8 to 50 nucleotides (such as 10 to 30 nucleotides) in length, comprising a continuous nucleotide sequence of at least 10 nucleotides in length, the continuous nucleotide sequence having at least 80% complementarity to a target sequence of APOEε4 nucleic acid, the target sequence including a residue corresponding to residue 535 in SEQ ID NO:1.

The oligonucleotide may be an antisense oligonucleotide, preferably with a gapmer design. Preferably, the oligonucleotide reduces ApoE4 expression by cleaving the target nucleic acid. Cleavage is preferably achieved through nuclease recruitment. Preferably, the oligonucleotide reduces ApoE4 expression more than it reduces ApoE3 expression.

In another aspect, the present invention provides pharmaceutical compositions comprising the oligonucleotides of the present invention, as well as pharmaceutical diluents, carriers, salts, and/or adjuvants.

In another aspect, the present invention provides a method for regulating ApoE4 expression in target cells, which is carried out by administering an effective amount of the antisense oligonucleotide or composition of the present invention to said cells. This method includes in vivo and in vitro methods.

In another aspect, the present invention provides a method for treating or preventing diseases, conditions, or functional disorders related to the in vivo activity of ApoE4, the method comprising: administering a therapeutically or preventively effective amount of the oligonucleotide of the present invention to a subject who has or is at risk of having the disease, condition, or functional disorder.

In certain aspects, the oligonucleotides or compositions of the present invention are used for the treatment or prevention of Alzheimer's disease (AD).

definition

Oligonucleotides

As used herein, the term "oligonucleotide" is defined, as commonly understood by those skilled in the art, as a molecule comprising two or more covalently linked nucleosides. Such covalently linked nucleosides may also be referred to as nucleic acid molecules or oligomers. Oligonucleotides are typically prepared in the laboratory by first synthesizing them in a solid-phase chemical process followed by purification and isolation. When referring to the sequence of an oligonucleotide, the meaning refers to the sequence or order of the nucleobase portion of the covalently linked nucleotide or nucleoside or its modifications. The oligonucleotides of the present invention are artificial and chemically synthesized, and are typically purified or isolated. The oligonucleotides of the present invention may comprise one or more modified nucleosides or nucleotides, such as 2'-sugar-modified nucleosides.

antisense oligonucleotides

As used herein, the term "antisense oligonucleotide" is defined as an oligonucleotide capable of regulating the expression of a target gene by hybridization with a target nucleic acid, particularly with a sequential sequence on the target nucleic acid. Antisense oligonucleotides can be provided in single-stranded, double-stranded, substantially single-stranded, or substantially double-stranded form. For example, it is contemplated that antisense oligonucleotides be provided as substantially non-double-stranded and therefore not siRNA or shRNA. Preferably, such antisense oligonucleotides are single-stranded. Antisense oligonucleotides provided in substantially double-stranded form (such as in a duplex form) are also considered. Single-stranded oligonucleotides can also form hairpin or intermolecular duplex structures (duplexes between two molecules of the same oligonucleotide), provided that their internal or mutual self-complementarity is greater than about 50% of the total length of the oligonucleotide.

Continuous nucleotide sequence

The term "adjacent nucleotide sequence" refers to a region of an oligonucleotide that is complementary to the target nucleic acid or target sequence. This term is used interchangeably herein with the terms "continuous nucleotide sequence" and "oligonucleotide motif sequence." In some embodiments, all nucleotides of the oligonucleotide constitute a continuous nucleotide sequence. In some embodiments, the oligonucleotide comprises a continuous nucleotide sequence, such as an F-G-F' nick polymer region, and may optionally comprise additional nucleotides, such as nucleotide linker regions for attaching functional groups to the continuous nucleotide sequence. The nucleotide linker region may be complementary to or not complementary to the target nucleic acid. It should be understood that the continuous nucleotide sequence of the oligonucleotide cannot be longer than the oligonucleotide itself, and the oligonucleotide cannot be shorter than the continuous nucleotide sequence.

Nucleotides

Nucleotides are the structural units of oligonucleotides and polynucleotides, and for the purposes of this invention, include both naturally occurring and non-naturally occurring nucleotides. In nature, nucleotides (such as DNA and RNA nucleotides) comprise a ribose moiety, a nucleobase moiety, and one or more phosphate ester groups (which are not present in nucleosides). Nucleosides and nucleotides may also be used interchangeably as “units” or “monomers”.

Modified nucleosides

As used herein, the term "modified nucleoside" or "nucleoside modification" refers to a nucleoside modified by introducing one or more modifications to a sugar moiety or (nucleo)base moiety compared to an equivalent DNA or RNA nucleoside. In a preferred embodiment, the modified nucleoside comprises a modified sugar moiety. The term modified nucleoside may also be used interchangeably herein with the terms "nucleoside analogue" or modified "unit" or modified "monomer". Nucleosides having unmodified DNA or RNA sugar moieties are referred to herein as DNA or RNA nucleosides. Nucleosides having modifications in the base regions of DNA or RNA nucleosides are generally still referred to as DNA or RNA if Watson Crick base pairing is permitted.

Modified internucleotide bonds

As commonly understood by those skilled in the art, the term "modified internucleotide bond" is defined as a bond other than a phosphodiester (PO) bond that covalently couples two nucleosides together. Therefore, the oligonucleotides of the present invention may contain modified internucleotide bonds. In some embodiments, modified internucleotide bonds increase the nuclease resistance of the oligonucleotide compared to phosphodiester bonds. For naturally occurring oligonucleotides, internucleotide bonds comprise phosphate ester groups that form phosphodiester bonds between adjacent nucleosides. Modified internucleotide bonds are particularly useful for stabilizing oligonucleotides for in vivo use and can protect DNA or RNA nucleoside regions (e.g., within the nick region G of a nicked oligonucleotide) and modified nucleoside regions (such as regions F and F') of the oligonucleotides of the present invention from nuclease cleavage.

In embodiments, the oligonucleotide comprises one or more nucleoside internucleotides modified from natural phosphodiester, such as one or more modified nucleoside internucleotides, which are, for example, 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. Nucleoside internucleotides that enhance the nuclease resistance of the oligonucleotide are called anti-nuclease nucleoside internucleotides. In some embodiments, at least 50% of the nucleoside internucleotides in the oligonucleotide or its continuous nucleotide sequence are modified; such as at least 60%, at least 70%, at least 75%, at least 80%, or at least 90% of the nucleoside internucleotides in the oligonucleotide or its continuous nucleotide sequence are anti-nuclease nucleoside internucleotides. In some embodiments, all the nucleoside internucleotides in the oligonucleotide or its continuous nucleotide sequence are anti-nuclease nucleoside internucleotides. It should be recognized that, in some embodiments, the nucleoside linking the oligonucleotide of the present invention to a non-nucleotide functional group such as a conjugate can be a phosphodiester.

The modified nucleoside internucleotides may be selected from the group consisting of phosphate thioates, diphosphorothioates, and boranophosphates. In some embodiments, the modified nucleoside internucleotides are compatible with the RNase H recruitment of the oligonucleotides of the present invention, such as phosphate thioates, diphosphorothioates, or boranophosphates.

In some embodiments, the oligonucleotide contains one or more neutral nucleoside bonds, particularly nucleoside bonds selected from phosphate triesters, methylphosphonates, MMI, amide-3, methyl acetals, or thiomethyl acetals.

Other nucleoside inter-bonds are disclosed in WO2009/124238 (incorporated herein by reference). In one embodiment, the nucleoside inter-bond is selected from the linker disclosed in WO2007/031091 (incorporated herein by reference). In particular, the nucleotide internucleotide bond can be selected from -OP(O) ₂ -O-, -OP(O,S)-O-, -OP(S) ₂ -O-, -SP(O) ₂ -O-, -SP(O,S)-O-, -SP(S) ₂ -O-, -OP(O) ₂ -S-, -OP(O,S)-S-, -SP(O) ₂ -S-, -O-PO(R ^H )-O-, O-PO(OCH ₃ )-O-, -O-PO(NR ^H )-O-, -O-PO(OCH ₂ CH ₂ SR)-O-, -O-PO(BH ₃ )-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 nucleotide inlet can be selected from the group consisting of: -O-CO-O-, -O-CO-NR ^H- , -NR ^H- CO-CH _2- , -O-CH ₂ -CO-NR ^H- , -O-CH ₂ -CH ₂ -NR ^H- , -CO-NR ^H -CH _2- , -CH ₂ -NR ^H CO-, -O-CH ₂ -CH ₂ -S-, -S-CH 2-CH ₂ -O-, -S-CH ₂ -CH _{2 -} S-, -CH ₂ -SO ₂ -CH _2- , -CH _2- CO-NR ^H- , -O-CH ₂ -CH ₂ -NR ^H -CO-, -CH ₂ -NCH ₃ -O-CH _2- , wherein ^RH is selected from hydrogen and C1-4-alkyl.

Nucleoside-to-thiophosphate bonds

A preferred modified nucleoside internucleotide bond is a thiophosphate. Thiophosphate nucleoside internucleotide bonds are particularly useful due to nuclease resistance, favorable pharmacokinetics, and ease of manufacture. In some embodiments, at least 50% of the nucleoside internucleotide bonds in the oligonucleotide or its adjacent nucleotide sequence are thiophosphates, such as at least 60%, at least 70%, at least 80%, or at least 90% of the nucleoside internucleotide bonds in the oligonucleotide or its adjacent nucleotide sequence are thiophosphates. In some embodiments, all nucleoside internucleotide bonds in the oligonucleotide or its continuous nucleotide sequence are thiophosphates.

Nuclease-resistant bonds, such as phosphate thioester bonds, are particularly useful in oligonucleotide regions that can recruit nucleases when forming a double strand with the target nucleic acid, such as region G of a spacer polymer. However, phosphate thioester bonds can also be used in non-nuclease-recruiting regions and/or affinity-enhancing regions, such as regions F and F' of a spacer polymer. In some embodiments, the spacer polymer oligonucleotide may contain one or more phosphodiester bonds in region F or F' or both regions F and F', wherein the internucleotide bonds in region G may be entirely phosphate thioesters.

Advantageously, all internucleotide bonds in the continuous nucleotide sequence of the oligonucleotide are phosphate thioester bonds.

It should be recognized that, as disclosed in EP2 742 135, antisense oligonucleotides may contain other nucleoside internucleotides (in addition to phosphodiester and thiophosphate), such as alkylphosphonate/methylphosphonate internucleotides, which, according to EP2742135, may be resistant, for example, to other DNA thiophosphate nick regions.

nucleobases

The term nucleobase includes 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 this invention, the term nucleobase also includes modified nucleobases that may differ from naturally occurring nucleobases but are functional during nucleic acid hybridization. In this context, "nucleobase" refers to naturally occurring nucleobases such as adenine, guanine, cytosine, thymine, uracil, xanthine, and hypoxanthine, as well as non-natural variants. Such variants are described, for example, in Hirao et al. (2012), Accounts of Chemical Research, Vol. 45, p. 2055, Bergstrom (2009) Current Protocols in Nucleic Acid Chemistry, Supplement 37 1.4.1, or by reference in PCT/EP2021/065266 incorporated herein by reference.

In some embodiments, the nucleobase moiety is modified by changing the purine or pyrimidine to a modified purine or pyrimidine, such as a substituted purine or substituted pyrimidine, such as a nucleobase selected from isocytosine, pseudoisocytosine, 5-methylcytosine, 5-thiazo-cytosine, 5-propynyl-cytosine, 5-propynyl-uracil, 5-bromouracil, 5-thiazo-uracil, 2-thiouracil, 2'-thio-thymidine, inosine, diaminopurine, 6-aminopurine, 2-aminopurine, 2,6-diaminopurine, 7-deaza-8-azanine, and 2-chloro-6-aminopurine.

The nucleobase moiety may be represented by a letter code for each corresponding nucleobase, such as A, T, G, C, or U, wherein each letter may optionally include a modified nucleobase with equivalent function. For example, in an exemplary oligonucleotide, the nucleobase moiety is selected from A, T, G, C, and 5-methylcytosine. Optionally, for LNA spacer polymers, 5-methylcytosine LNA nucleosides may be used.

Modified oligonucleotides

The term "modified oligonucleotide" describes an oligonucleotide comprising one or more sugar-modified nucleosides and/or modified inter-nucleoside bonds. The term "chimeric" oligonucleotide is a term already used in the literature to describe oligonucleotides having modified nucleosides.

Complementarity

The term “complementarity” describes the ability of a nucleoside/nucleotide to pair with a Watson-Crick base. Watson-Crick base pairs are guanine (G)-cytosine (C) and adenine (A)-thymine (T)/uracil (U). It should be understood that oligonucleotides can contain nucleosides with modified nucleosides, for example, 5-methylcytosine is often used instead of cytosine, and therefore, the term complementarity covers Watson-Crick base pairing between unmodified and modified nucleosides (see, for example, Hirao et al. (2012) Accounts of Chemical Research, Vol. 45, p. 2055 and Bergstrom (2009) Current Protocols in Nucleic Acid Chemistry, Supplement 371.4.1).

As used herein, the term "complementarity percentage" refers (expressed as a percentage) the proportion of nucleotides in a continuous 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) spanning that continuous nucleotide sequence. The percentage of complementarity is calculated by counting the number of aligned nucleosides that are complementary (from Watson-Crick base pairing) between two sequences (when comparing oligonucleotide sequences to the target sequence at 5'-3' and 3'-5'), dividing that number by the total number of nucleotides in the oligonucleotide, and multiplying by 100. In this comparison, misaligned nucleosides/nucleotides (forming base pairs) are referred to as mismatches. Insertions and deletions are not permitted when calculating the percentage of complementarity for a continuous nucleotide sequence. It should be understood that chemical modifications of the nucleosides (e.g., 5'-methylcytosine is considered identical to cytosine used for the purpose of calculating the percentage of identity) are disregarded when determining complementarity, provided that the functional ability of the nucleosides forming Watson-Crick base pairs is preserved.

The term "perfect complementarity" refers to 100% complementarity.

identity

As used herein, the term "identity" refers to the percentage (expressed as a percentage) of consecutive nucleotide sequences in a nucleic acid molecule (e.g., an oligonucleotide) that are identical to a reference sequence (e.g., a sequence motif) spanning the consecutive nucleotide sequence. Therefore, the identity percentage is calculated by counting the number of identical (matching) aligned nucleobases in the two sequences (in the consecutive nucleotide sequence of the compound of the present invention and in the reference sequence), dividing that number by the total number of nucleotides in the oligonucleotide, and multiplying by 100. Thus, the identity percentage = (number of matches x 100) / length of the aligned region (e.g., consecutive nucleotide sequence). Insertions and deletions are not permitted when calculating the identity percentage of consecutive nucleotide sequences. It should be understood that, in determining identity, chemical modifications of the nucleobases are disregarded as long as the functional ability of the nucleobases forming Watson Crick base pairs is preserved (e.g., 5-methylcytosine is considered identical to cytosine when calculating the identity percentage).

Hybridization

As used herein, the term "hybridizing" should be understood as the formation of hydrogen bonds between base pairs on opposite strands of two nucleic acid chains (e.g., an oligonucleotide and a target nucleic acid), resulting in a double helix. The affinity between the two nucleic acid chains is the strength of the hybridization. It is usually described by the melting temperature (T_{_m} ), which is defined as the temperature at which half of the oligonucleotide and the target nucleic acid form a double helix. Under physiological conditions, T_{_m} is not exactly strictly proportional to the affinity (Mergny and Lacroix, 2003, Oligonucleotides 13:515-537). The Gibbs free energy ΔG° in standard conditions more accurately represents the binding affinity and is related to the dissociation constant (K_{_d} ) of the reaction by ΔG° = -RTln(K_{_d} ), where R is the gas constant and T is the absolute temperature. Therefore, a very low ΔG° in the reaction between the oligonucleotide and the target nucleic acid reflects a strong hybridization between the oligonucleotide and the target nucleic acid. ΔG° is the energy associated with a reaction at a water concentration of 1 M, pH 7, and temperature of 37 °C. Hybridization of oligonucleotides with target nucleic acids is a spontaneous reaction, and the ΔG° of a spontaneous reaction is less than zero. ΔG° can be measured experimentally, for example, by using isothermal titration calorimetry (ITC) methods as described 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 estimated numerically using the nearest neighbor model described in Santa Lucia, 1998, Proc Natl Acad Sci USA. 95:1460-1465, with appropriate use of the derived thermodynamic parameters described in Sugimoto et al., 1995, Biochemistry 34:11211-11216, and McTigue et al., 2004, Biochemistry 43:5388-5405. To enable the possibility of modulating their intended nucleic acid targets through hybridization, for oligonucleotides of 10-30 nucleotides in length, the oligonucleotides of the present invention hybridize with the target nucleic acid at an estimated ΔG° below -10 kcal. In some embodiments, the extent or intensity of hybridization is measured by the standard-state Gibbs free energy ΔG°. For oligonucleotides of 8-30 nucleotides in length, the oligonucleotides may hybridize with the target nucleic acid at an estimated ΔG° below -10 kcal, such as below -15 kcal, such as below -20 kcal, and such as below -25 kcal. In some embodiments, the oligonucleotides hybridize with the target nucleic acid at an estimated ΔG° of -10 kcal to -60 kcal, such as -12 kcal to -40 kcal, such as -15 kcal to -30 kcal, or -16 kcal to -27 kcal, such as -18 kcal to -25 kcal.

target nucleic acid

According to the present invention, the target nucleic acid is a nucleic acid encoding mammalian ApoE4, and can be, for example, a gene, RNA, mRNA and precursor mRNA, mature mRNA, or cDNA sequence. Therefore, the target can be referred to as ApoE4 target nucleic acid or APOEε4 target nucleic acid, and these terms are used interchangeably. The oligonucleotides of the present invention can, for example, target APOEε4 precursor mRNA or mRNA.

Suitable targets are nucleic acids that encode ApoE4 proteins, particularly mammalian ApoE4 proteins, such as human ApoE4 proteins.

In some implementations, the target nucleic acid contains at least residues 522 to 548 of SEQ ID NO:1 and encodes a mammalian (such as human) ApoE4 protein.

In some implementations, the target nucleic acid contains at least residues 516-556 of SEQ ID NO:1 and encodes a mammalian (such as human) ApoE4 protein.

In some embodiments, the target nucleic acid is SEQ ID NO:1 or any naturally occurring variant thereof that encodes a mammalian (such as human) ApoE4 protein. Therefore, the target nucleic acid can be SEQ ID NO:1.

SEQ ID NO:2 is the human ApoE nucleic acid shown in the NCBI reference sequence: NM_001302690.2 (gene bank), encoding the human ApoE3 isotype. Due to the rs429358 single nucleotide polymorphism (SNP), SEQ ID NO:2 differs from SEQ ID NO:1 at residue 535.

In some embodiments, the target nucleic acid contains at least residues 522 to 548 of SEQ ID NO:2 with a t535c substitution and encodes a mammalian (such as human) ApoE4 protein.

In some embodiments, the target nucleic acid contains at least residues 516-556 of SEQ ID NO:2 with a t535c substitution and encodes a mammalian (such as human) ApoE4 protein.

In some embodiments, the target nucleic acid is SEQ ID NO:2 with a t535c substitution, or any naturally occurring variant thereof encoding a mammalian (such as human) ApoE4 protein. Therefore, the target nucleic acid can be SEQ ID NO:2 with a t535c substitution.

In some embodiments, the target nucleic acid encodes the cynomolgus apoE4 protein. Suitable, the target nucleic acid encoding the cynomolgus apoE4 protein comprises the sequence shown in SEQ ID NO:3.

In some implementations, the target nucleic acid contains at least residues 450-490 of SEQ ID NO:3 and encodes a mammalian (such as a cynomolgus monkey) ApoE4 protein.

In some implementations, the target nucleic acid contains at least residues 456 to 482 of SEQ ID NO:3 and encodes a mammalian (such as a cynomolgus monkey) ApoE4 protein.

In some embodiments, the target nucleic acid is SEQ ID NO:3 or any naturally occurring variant of the mammalian (such as cynomolgus monkey) ApoE4 protein it encodes. Therefore, the target nucleic acid can be SEQ ID NO:3.

If the oligonucleotides of the present invention are used in research or diagnosis, the target nucleic acid may be cDNA or a synthetic nucleic acid derived from DNA or RNA.

For in vivo or in vitro applications, the oligonucleotides of the present invention are generally able to reduce the expression of ApoE4 protein in cells expressing APOEε4 target nucleic acids. The continuous nucleotide sequence of the oligonucleotides of the present invention is generally complementary to the APOEε4 target nucleic acid, as measured over the entire length of the oligonucleotide, optionally except for one or two mismatches, and optionally excluding nucleotide-based linker regions, such as conjugates or other non-complementary terminal nucleotides (e.g., regions D’ or D”), that could connect the oligonucleotide to optional functional groups.

SEQ ID NO 1-SEQ ID NO 3 are presented as DNA sequences. It should be understood that the target RNA sequences contain uracil (U) bases in place of thymine (T) bases.

The target nucleic acid is preferably a messenger RNA, such as mature mRNA or precursor mRNA.

In some embodiments, the oligonucleotide of the present invention targets SEQ ID NO:1.

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

In some embodiments, the oligonucleotide of the present invention targets SEQ ID NO:3.

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

In some embodiments, the oligonucleotides of the present invention target SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3 having t535c substitution.

Table 1. Examples of target nucleic acids

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 complementary to the oligonucleotide of the present invention. This region of the target nucleic acid may be interchangeably referred to as the target nucleotide sequence, target sequence, or target region.

In some embodiments, the target sequence comprises a region on the target nucleic acid having a nucleobase sequence complementary to the continuous nucleotide sequence of the oligonucleotide of the present invention. In some embodiments, the target sequence is longer than the complementary sequence of a single oligonucleotide and may, for example, represent a preferred region of the target nucleic acid that can be targeted by several oligonucleotides of the present invention.

The oligonucleotides of the present invention comprise a continuous nucleotide sequence that is complementary to or hybridizes with a target nucleic acid (such as a subsequence of the target nucleic acid, such as the target sequence described herein).

Oligonucleotides comprise a continuous nucleotide sequence complementary to a target sequence present in a target nucleic acid molecule. The continuous nucleotide sequence (and therefore the target sequence) comprises at least about 8 continuous nucleotides, such as at least about 9 continuous nucleotides, such as at least about 10 continuous 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 continuous nucleotides, such as 12-25 continuous nucleotides, such as 14-18 continuous nucleotides.

Preferably, the target sequence is located in RNA, such as precursor mRNA, mature mRNA, or both.

The target sequence contains the cytosine (c) residue at position 535 in SEQ ID NO:1, corresponding to the site of rs429358SNP.

In some implementations, the target sequence is located in the region defined by residues 516-556 of SEQ ID NO:1.

In some implementations, the target sequence is located in the region defined by residues 522 to 548 of SEQ ID NO:1.

In some implementations, the target sequence contains cytosine (c) at position 469 of SEQ ID NO:3.

In some implementations, the target sequence is located in the region defined by residues 450-490 of SEQ ID NO:3.

In some implementations, the target sequence is located in the region defined by residues 456 to 482 of SEQ ID NO:3.

In some implementations, the target sequence is a sequence selected from those sequences described in Table 2.

In some implementations, the target sequence is selected from R_25, R_40, R_46, R_66 and R_91, such as R_25, R_40 and R_46.

In some implementations, the target sequence is R_25, corresponding to residues 522 to 535 of SEQ ID NO:1.

In some implementations, the target sequence is R_40, corresponding to residues 525 to 537 of SEQ ID NO:1.

In some implementations, the target sequence is R_46, corresponding to residues 526 to 538 of SEQ ID NO:1.

In some implementations, the target sequence is R_66, corresponding to residues 530 to 546 of SEQ ID NO:1.

In some implementations, the target sequence is R_91, corresponding to residues 535 to 548 of SEQ ID NO:1.

It should be understood that the target RNA sequence region has uracil (U) bases that replace any thymine (T) bases.

Table 2. Examples of target sequences on SEQ ID NO:1

target cells

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

In a preferred embodiment, the target cells express human ApoE4 mRNA, such as ApoE4 precursor mRNA, for example, SEQ ID NO:1, or mature ApoE4 mRNA. In some embodiments, the target cells express cynomolgus monkey ApoE4 mRNA, such as mature ApoE4 mRNA, for example, SEQ ID NO:3, and any polyA tails of the ApoE4 mRNA are generally ignored for antisense oligonucleotide targeting.

Naturally occurring variants

The term "naturally occurring variant" refers to a variant of the APOEε4 gene or transcript that originates from the same genetic locus as the target nucleic acid, but may differ, for example, due to the degeneracy of the genetic code leading to diversity in codons encoding the same amino acid, or due to alternative splicing of the precursor 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 sufficiently complementary sequences to the oligonucleotides, the oligonucleotides of the present invention can therefore target the target nucleic acid and its naturally occurring variants.

In some embodiments, the naturally occurring variant has at least 95%, such as at least 98%, or at least 99%, homology with the mammalian ApoE4 target nucleic acid, such as the target nucleic acid selected from the group consisting of SEQ ID NO:1 and SEQ ID NO:3. In some embodiments, the naturally occurring variant has at least 99% homology with the human APOEε4 target nucleic acid of SEQ ID NO:1.

Regulation of expression

As used herein, the term "regulation of expression" should be understood as the collective ability of an oligonucleotide to alter the amount of ApoE4 protein or ApoE4 mRNA compared to the amount of ApoE4 protein or ApoE4 mRNA prior to administration. Alternatively, regulation of expression can be determined with reference to control experiments. As is generally known, controls are single or target cells treated with a saline composition or single or target cells treated with a non-targeting oligonucleotide (mimic).

One type of regulation is the ability of oligonucleotides to inhibit, downregulate, reduce, suppress, remove, stop, block, prevent, reduce, decrease, avoid, or terminate the expression of ApoE4, for example, by degrading ApoE4 mRNA or preventing transcription.

High affinity modified nucleosides

High-affinity modified nucleosides are modified nucleotides that, when incorporated into oligonucleotides, enhance the affinity of the oligonucleotides for their complementary targets, as measured, for example, by melting temperature (Tm ). The high-affinity modified nucleosides of the present invention preferably increase the melting temperature of each modified nucleoside by between +0.5°C and +12°C, more preferably between +1.5°C and +10°C, and most preferably between +3°C and +8°C. Many high-affinity modified nucleosides are known in the art and include, 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).

Sugar modification

Compared to the ribose moieties found in DNA and RNA, the oligomers of the present invention may contain one or more nucleosides with modified ribose moieties (i.e., modifications to the ribose moieties).

Many nucleosides modified with ribose moieties have been prepared, primarily with the aim of improving certain properties of oligonucleotides, such as affinity and/or nuclease resistance.

Such modifications include those in which the ribocyclic structure is modified, for example, by replacing the ribocyclic structure with a hexose ring (HNA) or a bicyclic ring, which typically has a bibasic bridge between the C2 and C4 carbons on the ribocyclic ring (locked nucleic acid or "LNA"), or an unlinked ribocyclic ring (e.g., unlocked nucleic acid or "UNA") typically lacking a bond between the C2 and C3 carbons. Other sugar-modified nucleosides include, for example, bicyclic hexosenes (WO2011/017521) or tricyclic nucleic acids (WO2013/154798). Modified nucleosides also include nucleosides in which the sugar portion is replaced by a non-sugar portion, such as in the case of peptide nucleic acids (PNA) or morpholino nucleic acids.

Sugar modification also includes modifications made by changing the substituents on the ribose ring to groups other than hydrogen or to the 2'-OH groups naturally present in DNA and RNA nucleosides. For example, substituents can be introduced at the 2', 3', 4', or 5' positions.

2'-glycosides

A 2' sugar-modified nucleoside is a nucleoside that has a substituent other than H or -OH at the 2' position (2' substituted nucleosides) or contains a 2' linking dimer that can form a bridge between the 2' carbon and the second carbon in the ribose ring, such as LNA (2'-4' bimer bridged) nucleoside.

In fact, considerable effort has been devoted to developing 2'-sugar-substituted nucleosides, and many beneficial properties have been discovered when these 2'-substituted nucleosides are incorporated into oligonucleotides. For example, 2'-modified sugars can provide enhanced binding affinity to oligonucleotides and/or increased nuclease resistance. Examples of 2'-substituted modified nucleosides include 2'-O-alkyl-RNA, 2'-O-methyl-RNA, 2'-alkoxy-RNA, 2'-O-methoxyethyl-RNA (MOE), 2'-amino-DNA, 2'-fluoro-RNA, and 2'-F-ANA nucleosides. For further examples, see, for instance, 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 schematic diagrams of some 2'-substituted nucleosides.

Locked nucleoside (LNA nucleoside)

“LNA nucleotides” are 2’-sugar modified nucleotides containing a C2’ and C4’ bipolar junction (also called a “2’-4’ bridge”) that binds the ribosyl ring of the nucleotide, restricting or locking the conformation of the ribosyl ring. These nucleotides are also referred to in the literature as bridging nucleic acids or bicyclic nucleic acids (BNAs). When LNAs are incorporated into oligonucleotides of complementary RNA or DNA molecules, the locking of the ribosyl conformation is associated with increased hybridization affinity (double-strand stabilization). This can be routinely determined by measuring the melting temperature of the oligonucleotide/complementary double strand.

Non-limiting exemplary LNA nucleosides are disclosed in WO 99/014226, WO 00/66604, WO 98/039352, WO2004/046160, WO 00/047599, WO 2007/134181, WO 2010/077578, WO 2010/036698, WO2007/090071, WO 2009/006478, WO 2011/156202, WO 2008/154401, WO 2009/067647, WO 2008/150729, Morita et 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.

Other non-limiting exemplary LNA nucleosides are disclosed in Scheme 1.

Option 1:

Specific LNA nucleosides are β-D-oxy-LNA, 6'-methyl-β-D-oxy-LNA, such as (R) or (S)-6'-methyl-β-D-oxy-LNA (ScET) and ENA. A particularly advantageous LNA is β-D-oxy-LNA.

Definition of chemical groups

In this specification, the term "alkyl" refers, alone or in combination, to a straight-chain or branched alkyl group having 1 to 8 carbon atoms, particularly a straight-chain or branched alkyl group having 1 to 6 carbon atoms, and more particularly a straight-chain or branched alkyl group having 1 to 4 carbon atoms. Examples of straight-chain and branched _C1 - _C8 alkyl groups are methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, isopentyl, isohexyl, isoheptyl, and isoctyl, especially methyl, ethyl, propyl, butyl, and pentyl. Specific examples of alkyl groups are methyl, ethyl, and propyl.

The term "cycloalkyl" refers, alone or in combination, 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.

The term "alkoxy" alone or in combination represents a group of the formula alkyl-O-, wherein the term "alkyl" has the meaning previously given, such as methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, sec-butoxy, and tert-butoxy. Specific "alkoxy" groups are methoxy and ethoxy. Methoxyethoxy is a specific instance of "alkoxyalkoxy".

The term "oxygen group" refers to the -O- group, either alone or in combination.

The term "alkenyl" refers, alone or in combination, to a straight-chain or branched hydrocarbon residue comprising an olefinic bond and up to eight, preferably six, and particularly preferably four carbon atoms. Examples of alkenyl groups are vinyl, 1-propenyl, 2-propenyl, isopropenyl, 1-butenyl, 2-butenyl, 3-butenyl, and isobutenyl.

The term "alkynyl" alone or in combination refers to a straight-chain or branched hydrocarbon residue containing a triple bond and up to eight, preferably six, and particularly preferably four carbon atoms.

The term "halogen" or "halogenated" alone or in combination means fluorine, chlorine, bromine, or iodine, and particularly fluorine, chlorine, or bromine, and even more particularly fluorine. The term "halogenated" in combination with another group means that the group is substituted by at least one halogen, particularly by one to five halogens, especially one to four halogens, i.e., one, two, three, or four halogens.

The term "haloalkyl" refers, alone or in combination, to an alkyl group substituted with at least one halogen, particularly with one to five halogens, and especially with one to three halogens. Examples of haloalkyl groups include monofluoro-, difluoro-, or trifluoro-methyl, -ethyl, or -propyl, for example, 3,3,3-trifluoropropyl, 2-fluoroethyl, 2,2,2-trifluoroethyl, fluoromethyl, or trifluoromethyl. Fluoromethyl, difluoromethyl, and trifluoromethyl are specific "haloalkyl" groups.

The term "halocycloalkyl" refers, alone or in combination, to a cycloalkyl group as defined above that is substituted with at least one halogen, particularly with one to five halogens, especially with one to three halogens. Specific examples of "halocycloalkyl" are cyclopropyl halogens, especially fluorocyclopropyl, difluorocyclopropyl, and trifluorocyclopropyl.

The term "hydroxyl" (hydroxyl/hydroxy) refers to the -OH group, either alone or in combination.

The terms “hydrothioyl” and “thiol” refer, alone or in combination, to the -SH group.

The term "carbonyl" refers to the -C(O)- group, either alone or in combination.

The terms “carboxyl group” or “acidic group” refer, alone or in combination, to the -COOH group.

The term "amino" alone or in combination represents a primary amino group ( _-NH2 ), a secondary amino group (-NH-), or a tertiary amino group (-N-).

The term "alkylamino" alone or in combination refers to an amino group as defined above that is substituted by one or two alkyl groups as defined above.

The term "sulfonyl" alone or in combination represents the _-SO2 group.

The term "sulfinyl" alone or in combination represents the -SO- group.

The term "thio-" alone or in combination represents a -S- group.

The term "cyano" alone or in combination represents the -CN group.

The term "azido" alone or in combination refers to the -N ₃ group.

The term "nitro" alone or in combination refers to a _NO2 group.

The term "formyl" alone or in combination represents the -C(O)H group.

The term "carbamoyl" alone or in combination represents the -C(O) _NH2 group.

The term "urea group", alone or in combination, refers to the -NH-C(O)-NH ₂ group.

The term "aryl" refers, alone or in combination, to a monovalent aromatic carbocyclic monocyclic or bicyclic system comprising 6 to 10 carbon ring atoms, optionally substituted by 1 to 3 independent substituents selected from: halogen, hydroxyl, alkyl, alkenyl, alkoxy, alkoxyalkyl, alkenyloxy, carboxyl, alkoxycarbonyl, alkylcarbonyl, and formyl. Examples of aryl groups include phenyl and naphthyl, particularly phenyl.

The term "heteroaryl" alone or in combination refers to a monovalent aromatic heterocyclic monocyclic or bicyclic system having 5 to 12 ring atoms, comprising 1, 2, 3 or 4 heteroatoms selected from N, O and S, with the remaining ring atoms being carbon, optionally substituted by 1 to 3 substituents independently selected from halogen, hydroxyl, alkyl, alkenyl, alkoxy, alkoxyalkyl, alkenyloxy, carboxyl, alkoxycarbonyl, alkylcarbonyl and formyl. Examples of heteroaryl groups include pyrrole, furanyl, thiophene, imidazolyl, oxazolyl, thiazolyl, triazolyl, oxadiazolyl, thiadiazolyl, tetrazolyl, pyridyl, pyrazinyl, pyrazolyl, pyridazinyl, pyrimidinyl, triazinyl, azepinyl, diazepinyl, isoxazolyl, benzofuranyl, isothiazolyl, benzothiophene, indolyl, isoindolyl, isobenzofuranyl, benzimidazolyl, benzooxazolyl, benzoisooxazolyl, benzothiazolyl, benzooxadiazolyl, benzothiadiazolyl, benzotriazolyl, purinyl, quinolinyl, isoquinolinyl, quinazolinyl, quinoxolinyl, carbazole, or acridineyl.

The term "heterocyclic group," alone or in combination, refers to a monocyclic or bicyclic system of 4 to 12, particularly 4 to 9, ring atoms, which are monovalently saturated or partially unsaturated and contain 1, 2, 3 or 4 heteroatoms selected from N, O and S, with the remaining ring atoms being carbon, optionally substituted by 1 to 3 substituents independently selected from halogen, hydroxyl, alkyl, alkenyl, alkoxy, alkoxyalkyl, alkenyloxy, carboxyl, alkoxycarbonyl, alkylcarbonyl and formyl groups. Examples of monocyclic saturated heterocyclic groups are aziridine, pyrrolidinyl, tetrahydrofuranyl, tetrahydrothiophenyl, pyrazolidinyl, imidazodiphenyl, oxazolyl, isoxazolyl, tetrahydrothiazolyl, piperidinyl, tetrahydropiperanyl, tetrahydrothiopyranyl, piperazinyl, mofolinyl, thiomorpholinyl, 1,1-dioxo-thiomorpholin-4-yl, aziridine heptyl, diaziridine heptyl, homopiperidinyl, or oxaziridine heptyl. Examples of bicyclic saturated heterocyclic alkyl groups are 8-aza-bicyclo[3.2.1]octyl, quinoline, 8-oxo-3-aza-bicyclo[3.2.1]octyl, 9-aza-bicyclo[3.3.1]nonyl, 3-oxo-9-aza-bicyclo[3.3.1]nonyl, or 3-thio-9-aza-bicyclo[3.3.1]nonyl. Examples of partially unsaturated heterocyclic alkyl groups are dihydrofuranyl, imidazolinyl, dihydrooxazolyl, tetrahydropyridyl, or dihydropyranyl.

medicinal salt

The term "medicinal salt" refers to salts that retain the biological efficacy and properties of a free base or acid, and are not biologically or otherwise undesirable. These salts are formed from 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, and N-acetylcysteine. These salts can be prepared by adding an inorganic or organic base to a 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, salts of primary, secondary, and tertiary 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, and polyamine resins. Compounds of formula (I) may also exist in zwitterionic form. Particularly preferred pharmaceutical salts of compounds of formula (I) are salts of hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, and methanesulfonic acid.

Protecting group

The term "protecting group," used alone or in combination, refers to a group that selectively blocks a reactive site in a multifunctional compound, thereby allowing a chemical reaction to occur selectively at another unprotected reactive site. Protecting groups can be removed. Exemplary protecting groups are amino, carboxyl, or hydroxyl protecting groups.

nuclease-mediated degradation

Nuclease-mediated degradation means that an oligonucleotide can centrally influence the degradation of a sequence when it forms a double strand with a complementary nucleotide sequence.

In some embodiments, the oligonucleotides can function via nuclease-mediated degradation of the target nucleic acid, wherein the oligonucleotides of the present invention are capable of recruiting nucleases, particularly endonucleases, preferably ribonucleases (RNases), such as RNase H. Examples of oligonucleotide designs operating via a nuclease-mediated mechanism are oligonucleotides that typically comprise a region of at least 5 or 6 consecutive DNA nucleotides and have affinity-enhancing nucleotides flanking one or both sides, such as nick aggregates, head aggregates, and tail aggregates.

RNase H activity and recruitment

The RNase H activity of an antisense oligonucleotide refers to its ability to recruit RNase H when forming a double strand with a complementary RNA molecule. WO01/23613 provides an in vitro method for determining RNase H activity, which can be used to determine the ability to recruit RNase H. RNase H recruitment is generally considered possible if the initial rate of the oligonucleotide, when given a complementary target nucleic acid sequence, is at least 5%, such as at least 10% or more than 20%, of the initial rate of an oligonucleotide having the same base sequence as the oligonucleotide being tested but containing only DNA monomers in which all monomers are phosphate-thioester bonded, as measured using the method provided in Examples 91 to 95 of WO01/23613 (incorporated herein by reference). For the purpose of determining ribonuclease H activity, recombinant human ribonuclease H1 is available from Lubio Science GmbH, Lucerne, Switzerland.

Spacer

The antisense oligonucleotides or their sequential nucleotide sequences of the present invention can be spacer polymers, also known as spacer polymer oligonucleotides or spacer polymer designs. Antisense spacer polymers are generally used to inhibit target nucleic acids via RNase H-mediated degradation. The nicked polymer oligonucleotide comprises at least three distinct structural regions: a 5' flanking region in the "5 to 3" orientation, a nick, and a 3' flanking region F-G-F'. The "nick" region (G) contains a sequential DNA nucleotide that enables the oligonucleotide to recruit RNase H. The nick region is flanked by a 5' flanking region (F) containing one or more sugar-modified nucleosides (preferably high-affinity sugar-modified nucleosides), and a 3' flanking region (F') containing one or more sugar-modified nucleosides (preferably high-affinity sugar-modified nucleosides). One or more sugar-modified nucleosides in regions F and F' enhance the affinity of the oligonucleotide for the target nucleic acid (i.e., affinity-enhancing sugar-modified nucleosides). In some embodiments, one or more sugar-modified nucleosides in regions F and F' are 2' sugar-modified nucleosides, such as high-affinity 2' sugar modifications, such as those independently selected from LNA and 2'-MOE.

In the notched polymer design, the 5' and 3' terminal nucleotides of the notched region are DNA nucleotides, and are located near the sugar-modified nucleotides in the 5'(F) or 3'(F') region, respectively. The flanking region can be further defined as having at least one sugar-modified nucleotide at the end furthest from the notched region, i.e., at the 5' end of the 5' flanking region and the 3' end of the 3' flanking region.

The regions F-G-F’ form a continuous nucleotide sequence. The antisense oligonucleotides of the present invention, or their continuous nucleotide sequences, may contain spacer aggregate regions of the formula F-G-F’.

The overall length of the notched polymer design F-G-F' can be, for example, 12 to 32 nucleotides, such as 13 to 24 nucleotides, such as 14 to 22 nucleotides, such as 14 to 17 nucleotides, such as 16 to 18 nucleotides, such as 16 to 20 nucleotides.

For example, the notched oligonucleotide of the present invention can be represented by the following formula:

F _1-8 -G _5-16 -F' _1-8 , such as

F _1-8 -G _7-16 -F' _2-8, such as

F _3-8 -G _6-14 -F' _2-8

The condition is that the overall length of the notched polymer region F-G-F' is at least 10, such as at least 12, such as at least 14 nucleotides.

In aspects of the present invention, the antisense oligonucleotide or its continuous nucleotide sequence comprises or includes a nicked polymer of formula 5'-F-G-F'-3', wherein region F and region F' independently comprise or consist of 1-8 nucleotides, wherein 2-4 are modified with 2' sugars and define the 5' and 3' ends of regions F and F', and G is a region between 6 and 16 nucleotides capable of recruiting RNase H.

Regions F, G, and F' are further defined as follows and can be incorporated into the F-G-F' formula.

Notched aggregate - region G

The gap region G (gap region) of the gap polymer is the region that enables the oligonucleotide to recruit RNase H, such as human RNase H1, nucleosides (typically DNA nucleosides). RNase H is a cellular enzyme that recognizes the double strand between DNA and RNA and enzymatically cleaves the RNA molecule. A suitable spacer polymer may have a gap region (G) of at least 5 or 6 adjacent DNA nucleosides, such as 5-16 adjacent DNA nucleosides, such as 6-15 adjacent DNA nucleosides, such as 7-14 adjacent DNA nucleosides, such as 8-12 adjacent DNA nucleotides, such as a gap region of 8-12 adjacent DNA nucleotides. In some embodiments, the gap region G may consist of 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 adjacent DNA nucleosides. One or more cytosine (c) DNA residues in the gap region may be methylated in certain situations (e.g., when DNA c is followed by DNA g), and such 5'-methyl-cytosine residues may be annotated as ^meC or e instead of aC. 5'-substituted DNA nucleosides (such as 5'-methyl DNA nucleosides) have been reported for use in DNA gap regions (EP 2 742 136).

In some embodiments, the gap region G may consist of 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 consecutive phosphate-thioester-linked DNA nucleosides. In some embodiments, all internucleotide bonds in the gap are phosphate-thioester bonds.

Although conventional cleavage aggregates contain DNA cleavage regions, there are numerous instances of modified nucleosides that, when used within the cleavage region, allow RNase H recruitment. Modified nucleosides reported to be able to recruit RNase H when included within the cleavage region include, for example, α-L-LNA, C4' alkylated DNA (as described in PCT/EP2009/050349 and Vester et al., Bioorg. Med. Chem. Lett. 18 (2008) 2296-2300 (both 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), and UNA (unlocked nucleic acid) (as described in Fluiter et al., Mol. Biosyst., 2009, 10, 1039 (which is incorporated herein by reference). UNA is a non-locked nucleic acid, typically formed by the removal of the C2-C3 bond of the ribose, resulting in an unlocked "sugar" residue. The nucleotide used for modification in such spacer aggregates can be a nucleotide with a 2' endoform (DNA-like) structure when introduced into the nick region (i.e., a modification that allows ribonuclease H recruitment). In some embodiments, the DNA nick region (G) described herein may optionally contain one to three sugar-modified nucleotides with a 2' endoform (DNA-like) structure when introduced into the nick region.

Region G - " Gap -breaker"

Alternatively, there are numerous reports of inserting modified nucleosides into the gap regions of spacer polymers, conferring a 3' endoform conformation, while retaining some ribonuclease H activity. Such spacer polymers with a gap region containing one or more 3' endoform modified nucleosides are called "gap-breakers" or "gap-disrupted" spacer polymers, see, for example, WO2013/022984. Gap-breaker oligonucleotides retain sufficient DNA nucleoside region within the gap region to allow recruitment of ribonuclease H. The ability of gap-breaker oligonucleotides to design recruitment of RNase H is often sequence-specific or even compound-specific—see Rukov et al. 2015 Nucl. Acids Res. Vol. 43 pp. 8476-8487, which discloses "gap-breaker" oligonucleotides that provide more specific target RNA cleavage activity for RNase H recruitment in some cases. The modified nucleosides used in the nick region of the nick interruptor oligonucleotide can be, for example, modified nucleosides that impart a 3' inner conformation, such as 2'-O-methyl (OMe) or 2'-O-MOE (MOE) nucleosides, or β-D-LNA nucleosides (the bridge between the C2' and C4' of the ribose ring of the nucleoside is the β conformation), such as β-D-oxyLNA or ScET nucleosides.

Similar to nicked aggregates containing region G, nicked disruptor nicked aggregates or nicked interruptor nicked aggregates have a DNA nucleoside at the 5' end of the nick (adjacent to the 3' nucleoside of region F) and a DNA nucleoside at the 3' end of the nick (adjacent to the 5' nucleoside of region F'). Nicked aggregates containing nicks that impede nicks generally retain a region with at least 3 or 4 consecutive DNA nucleosides at the 5' or 3' end of the nick region.

Exemplary designs of gap-breaking oligonucleotides include

F _1-8 -[D _3-4 -E ₁ -D _3-4 ] _- F' _1-8

F _1-8 -[D _1-4 -E ₁ -D _3-4 ]-F' _1-8

F _1-8 -[D _3-4 -E ₁ -D _1-4 ]-F' _1-8

In this context, region G is enclosed in brackets [D _n -E _r -D _m ], where D is a continuous sequence of DNA nucleosides, E is a modified nucleoside (a cleavage disruptor or a cleavage-breaking nucleoside), and F and F' are flanking regions as defined herein, provided that the overall length of the cleavage aggregate region FG-F' is at least 12 nucleotides, such as at least 14 nucleotides.

In some embodiments, the region G of the spacer aggregate that impedes the nick contains at least six DNA nucleotides, such as 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 DNA nucleotides. As described above, the DNA nucleotides may be adjacent or optionally dispersed with one or more modified nucleotides, provided that the nick region G is capable of mediating the recruitment of ribonuclease H.

Spacer aggregate-flank regions, F and F'

The 5' DNA nucleoside in region F is immediately adjacent to that in region G. The 3' terminal nucleoside in region F is a sugar-modified nucleoside, such as a high-affinity sugar-modified nucleoside, or a 2'-substituted nucleoside, such as MOE nucleoside or LNA nucleoside.

The 3' DNA nucleoside of region F' is immediately adjacent to region G. The 5' terminal nucleoside of region F' is a sugar-modified nucleoside, such as a high-affinity sugar-modified nucleoside, or a 2'-substituted nucleoside, such as MOE nucleoside or LNA nucleoside.

Region F is 1-8 consecutive nucleotides in length, such as 2-6, such as 3-4 consecutive nucleotides. Preferably, the 5' terminal nucleotide of region F is a sugar-modified nucleotide. In some embodiments, both 5' terminal nucleotides of region F are sugar-modified nucleotides. In some embodiments, the 5' terminal nucleotide of region F is an LNA nucleotide. In some embodiments, both 5' terminal nucleotides of region F are LNA nucleotides. In some embodiments, the two 5' terminal nucleotides of region F are 2'-substituted nucleotides, such as two 3' MOE nucleotides. In some embodiments, the 5' terminal nucleotide of region F is a 2'-substituted nucleotide, such as a MOE nucleotide.

The length of region F' is 1-8 consecutive nucleotides, such as 2-8 consecutive nucleotides, such as 3-6 consecutive nucleotides, such as 4-5 consecutive nucleotides. Advantageously, in embodiments, the 3' terminal nucleotide of region F' is a sugar-modified nucleotide. In some embodiments, both 3' terminal nucleotides of region F are sugar-modified nucleotides. In some embodiments, both 3' terminal nucleotides of region F are LNA nucleotides. In some embodiments, the 3' terminal nucleotide of region F is an LNA nucleotide. In some embodiments, both 3' terminal nucleotides of region F are 2'-substituted nucleotides, such as two 3' MOE nucleotides. In some embodiments, the 3' terminal nucleotide of region F is a 2'-substituted nucleotide, such as a MOE nucleotide.

It should be noted that when the length of region F or F' is one, it is preferably an LNA nucleoside.

In some embodiments, regions F and F’ independently consist of or contain a continuous sequence of a sugar-modified nucleoside. In some embodiments, the sugar-modified nucleoside 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, arabinonucleotide (ANA) units, and 2'-fluoro-ANA units.

In some embodiments, regions F and F′ independently contain both LNA and 2′-substituted modified nucleosides (hybrid wing design).

In some embodiments, regions F and F′ consist of only one type of sugar-modified nucleoside, such as only MOE, only β-D-oxyLNA, or only ScET. Such a design is also known as a uniformly flanked or uniformly spaced polymer design.

In some embodiments, all nucleosides in region F or F′, or F and F′, are LNA nucleosides, such as those independently selected from β-D-oxyLNA, ENA, or ScET nucleosides. In some embodiments, region F consists of 1 to 5, such as 2 to 4, such as 3 to 4, such as 1, 2, 3, 4, or 5 adjacent LNA nucleosides. In some embodiments, all nucleosides in regions F and F′ are β-D-oxyLNA nucleosides.

In some embodiments, all nucleotides in regions F or F', or F and F', are 2'-substituted nucleotides, such as OMe or MOE nucleotides. In some embodiments, region F consists of 1, 2, 3, 4, 5, 6, 7, or 8 adjacent OMe or MOE nucleotides. In some embodiments, only one flanking region may consist of 2'-substituted nucleotides, such as OMe or MOE nucleotides. In some embodiments, the 5'(F) flanking region consists of 2'-substituted nucleotides, such as OMe or MOE nucleotides, while the 3'(F') flanking region contains at least one LNA nucleotide, such as β-D-oxyLNA nucleotide or cET nucleotide. In some embodiments, the 3'(F') flanking region consists of 2'-substituted nucleotides, such as OMe or MOE nucleotides, while the 5'(F) flanking region contains at least one LNA nucleotide, such as β-D-oxyLNA nucleotide or cET nucleotide.

In some embodiments, all modified nucleosides in regions F and F' are LNA nucleosides, such as those independently selected from β-D-oxyLNA, ENA, or ScET nucleosides, wherein regions F or F' or F and F' may optionally contain DNA nucleosides (alternating flanking; see the definitions of these for more details).

In some embodiments, the 5' and 3' terminal nucleosides of regions F and F' are LNA nucleosides, such as β-D-oxyLNA nucleosides or ScET nucleosides.

In some embodiments, the internucleotide bond between region F and region G is a phosphate thioester internucleotide bond. In some embodiments, the internucleotide bond between region F' and region G is a phosphate thioester internucleotide bond. In some embodiments, the internucleotide bond between the nucleosides of region F or F', F and F' is a phosphate thioester internucleotide bond.

LNA spacer

LNA spacer polymers are spacer polymers in which one or both of regions F and F' contain or are composed of LNA nucleosides. β-D-oxy spacer polymers are spacer polymers in which one or both of regions F and F' contain or are composed of β-D-oxy LNA nucleosides.

In some embodiments, the LNA spacer aggregate has the following formula: [LNA] _1-5 - [Region G] - [LNA] _1-5 , where region G is as defined in the definition of spacer aggregate region G.

In one embodiment, the LNA notched polymer is of the formula [LNA] ₄ - [region G] _10-12 - [LNA] ₄ .

MOE notched polymer

MOE spacer polymers are spacer polymers in which regions F and F' are composed of MOE nucleotides. In some embodiments, the MOE spacer polymer is designed as [MOE] _1-8 - [region G] _5-16 - [MOE] _1-8 , such as [MOE] _2-7 - [region G] _6-14 - [MOE] _2-7 , such as [MOE] _3-6 - [region G] _8-12 - [MOE] _3-6 , wherein region G has the definition as described in the definition of spacer polymers. MOE spacer polymers with a 5-10-5 design (MOE-DNA-MOE) are widely used in the art.

Hybrid wing spacer

The hybrid wing spacer polymer is an LNA spacer polymer in which one or both of regions F and F' contain 2'-substituted nucleosides, such as 2'-substituted nucleosides 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, arabinonucleotide (ANA) units, and 2'-fluoro-ANA units, such as MOE nucleosides. In some embodiments, at least one of regions F and F', or both regions F and F', contains at least one LNA nucleoside, and the remaining nucleosides of regions F and F' are independently selected from the group consisting of MOE and LNA. In some embodiments, at least one of regions F or F', or both regions F and F', contains at least two LNA nucleosides, and the remaining nucleosides of regions F and F' are independently selected from the group consisting of MOE and LNA. In some embodiments of the hybrid wing, one or both of regions F and F' may further contain one or more DNA nucleosides.

The hybrid wing spacer design has been disclosed in WO2008/049085 and WO2012/109395, which are incorporated herein by reference.

Alternating flanking spacer

The flanking regions may contain both LNA and DNA nucleosides, and are referred to as “alternating flanking regions” because they contain alternating motifs of LNA-DNA-LNA nucleosides. Notched polymers including such alternating flanking regions are called “alternating flanking notched polymers.” An “alternating flanking notched polymer” is an LNA notched oligonucleotide in which at least one flanking region (F or F') contains DNA in addition to an LNA nucleoside. In some embodiments, at least one region of region F or region F', or both regions F and F', contains both an LNA nucleoside and a DNA nucleoside. In such embodiments, flanking regions F or F', or both F and F', contain at least three nucleosides, wherein the 5' and 3' terminal nucleosides of regions F and/or F' are LNA nucleosides.

Alternating flanking LNA spacer polymers are disclosed in WO2016/127002.

Alternating flanking regions may contain up to three consecutive DNA nucleotides, such as one to two, one, two, or three consecutive DNA nucleotides.

Alternating flanking can be annotated as a series of integers, representing many LNA nucleotides (L), followed by many DNA nucleotides (D), for example

[L] _1-3 -[D] _1-4 -[L] _1-3

[L] _1-2 -[D] _1-2 -[L] _1-2 -[D] _1-2 -[L] _1-2

In oligonucleotide design, these are typically 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') in oligonucleotides with alternating flanks can independently be 3 to 10 nucleotides, such as 4 to 8, 5 to 6, or 4, 5, 6, or 7 modified nucleotides. In some embodiments, only one flank in the nicked oligonucleotide is alternating, while the other flanks are composed of LNA nucleotides. It may be advantageous to have at least two LNA nucleotides at the 3' end of the 3' flank (F') to confer additional exonuclease resistance. In one embodiment, the flanks in this alternating flank nicked oligonucleotide have an overall length of 5 to 8 nucleotides, of which 3 to 5 are LNA nucleotides. Some examples of oligonucleotides with alternating flanks are:

[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

The condition is that the overall length of the nicked polymer is at least 12 nucleotides, such as at least 14 nucleotides.

Region D' or D” in oligonucleotides

In some embodiments, the oligonucleotides of the present invention may comprise or consist of the following: a continuous nucleotide sequence of an oligonucleotide complementary to a target nucleic acid, such as a nicked polymer F-G-F', and additional 5' and/or 3' nucleotides. The additional 5' and/or 3' nucleotides may or may not be fully complementary to the target nucleic acid. Such additional 5' and/or 3' nucleotides may be referred to herein as regions D' and D'".

For the purpose of conjugating a continuous nucleotide sequence (such as a spacer polymer) to a conjugate moiety or another functional group, the addition region D' or D'' can be used. When used to conjugate a continuous nucleotide sequence to a conjugate moiety, it can serve as a biolytic linker. Alternatively, it can be used to provide exonuclease protection or to facilitate synthesis or manufacturing.

Regions D’ and D'’ can be attached to the 5’ end of region F or the 3’ end of region F’, respectively, to generate the following formulas: D'-F-G-F', F-G-F'-D'’, or

The design is D'-F-G-F'-D''. In this case, F-G-F' is the spacer polymer portion of the oligonucleotide, while the region D' or D'' constitutes the individual portion of the oligonucleotide.

Regions D' or D” may independently contain or consist of one, two, three, four, or five 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 forms of these. Regions D' or D” can be used as nuclease-sensitive biolyzable linkers (see definition of linker). In some embodiments, additional 5' and/or 3' nucleotides are linked to phosphodiester bonds and are DNA or RNA. WO2014/076195 discloses nucleotide-based biolyzable linkers suitable for use as regions D' or D”, comprising, for example, phosphodiester-linked DNA dinucleotides. WO2015/113922 discloses the use of biolyzable linkers in polyoligonucleotide constructs, wherein they are used to link multiple antisense constructs (e.g., nicked aggregate regions) within a single oligonucleotide.

In one embodiment, the oligonucleotide of the present invention, in addition to the continuous nucleotide sequence constituting the spacer polymer, also includes regions D' and/or D'.

In some embodiments, the oligonucleotide of the present invention may 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

In some embodiments, the internucleotide bond between region D' and region F is a phosphodiester bond. In some embodiments, the internucleotide bond between region F' and region D' is a phosphodiester bond.

Conjugate

As used herein, the term “conjugate” refers to an oligonucleotide covalently linked to a nonnucleotide moiety (conjugate moiety or region C or third region).

The 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 its activity, cellular distribution, cellular uptake, or stability. In some embodiments, the conjugation moieties modify or enhance the pharmacokinetic properties of the oligonucleotide by improving its cellular distribution, bioavailability, metabolism, excretion, permeability, and/or cellular uptake. In particular, the conjugations can target the oligonucleotide to a specific organ, tissue, or cell type, thereby enhancing the oligonucleotide's effectiveness in that organ, tissue, or cell type. Simultaneously, the conjugations can be used to reduce the activity of the oligonucleotide in non-target cell types, tissues, or organs, for example, off-target activity or activity in non-target cell types, tissues, or organs.

Oligonucleotide conjugates and their synthesis are also reviewed by Manoharan in Antisense Drug Technology, Principles, Strategies, and Applications, edited by S.T. Crooke, Chapter 16, Marcel Dekker, Inc., 2001, and in Manoharan, Antisense and Nucleic Acid Drug Development, 2002, 12, 103, which are incorporated herein by reference in their entirety.

In one embodiment, the non-nucleotide portion (conjugated portion) is selected from the group consisting of carbohydrates (e.g., GalNAc), cell surface receptor ligands, pharmaceutical substances, hormones, lipophilic substances, polymers, proteins, peptides, toxins (e.g., bacterial toxins), vitamins, viral proteins (e.g., capsids), or combinations thereof.

In some embodiments, the conjugate is an antibody or antibody fragment having a specific affinity for the transferrin receptor, such as that disclosed in WO 2012/143379, which is incorporated herein by reference. In some embodiments, the nonnucleotide portion is an antibody or antibody fragment, such as an antibody or antibody fragment that facilitates the delivery of medicament across the brain-vascular barrier, particularly an antibody or antibody fragment targeting the transferrin receptor.

connector

A linker or tie is a connection between two atoms that links one target chemical group or segment to another via one or more covalent bonds. The conjugate portion can be directly attached to an oligonucleotide or via a linker portion (e.g., a linker or tie). A linker can covalently attach a third region, such as the conjugate portion (region C), to a first region, such as an oligonucleotide or a continuous nucleotide sequence (region A) complementary to the target nucleic acid.

In some embodiments of the present invention, the conjugates or oligonucleotide conjugates of the present invention may optionally include a linker region (second region or region B and/or region Y) located between an oligonucleotide or continuous nucleotide sequence complementary to the target nucleic acid (region A or first region) and a conjugate portion (region C or third region).

Region B refers to a biocleavable linker containing physiologically unstable bonds or composed of such bonds, which are cleavable under conditions commonly encountered in mammals or similar conditions. Conditions under which a physiologically unstable linker undergoes chemical transformation (e.g., cleavage) include chemical conditions such as pH, temperature, oxidative or reducing conditions or reagents, and salt concentrations encountered in mammalian cells or similar salt concentrations. Intracellular mammalian conditions also include enzymatic activities commonly present in mammalian cells, such as enzymatic activities from proteolytic enzymes or hydrolases or nucleases. In one embodiment, the biocleavable linker is sensitive to S1 nuclease cleavage. In a preferred embodiment, the nuclease-susceptible linker comprises 1 to 10 nucleosides, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleosides, more preferably 2 to 6 nucleosides, and most preferably 2 to 4 linked nucleosides containing at least two consecutive phosphodiester bonds, such as at least 3, 4, or 5 consecutive phosphodiester bonds. Preferably, the nucleoside is DNA or RNA. Biodegradable linkers containing phosphate diesters are described in more detail in WO 2014/076195 (incorporated herein by reference).

Region Y refers to a linker that is not necessarily biodegradable but is primarily used to covalently link the conjugate portion (Region C or the third region) to an oligonucleotide (Region A or the first region). The Region Y linker may comprise oligomers of chain structures or repeating units such as ethylene glycol, amino acid units, or aminoalkyl groups. The oligonucleotide conjugates of the present invention can be constructed from the following region elements: A-C, A-B-C, A-B-Y-C, A-Y-B-C, or A-Y-C. In some embodiments, the linker (Region Y) is an aminoalkyl group, such as a C2-C36 aminoalkyl group, which includes, for example, C6 to C12 aminoalkyl groups. In a preferred embodiment, the linker (Region Y) is a C6 aminoalkyl group.

treat

As used herein, the term "treatment" refers to the treatment of an existing disease (e.g., a disease, symptom, or functional impairment as understood herein) or the prevention of a disease, i.e., prevention. Therefore, it will be appreciated that in some embodiments, the treatment referred to herein may be preventative.

Detailed Implementation

The oligonucleotides of the present invention

This invention relates to oligonucleotides capable of regulating ApoE4 expression (such as reducing (downregulating) ApoE4 expression). Regulation is achieved by hybridization with a target nucleic acid encoding ApoE4. The target nucleic acid can be a mammalian APOEε4 mRNA sequence, such as a sequence selected from the group consisting of SEQ ID NO:1 and SEQ ID NO:3.

The oligonucleotide is an antisense oligonucleotide that targets the APOEε4 nucleic acid, thereby reducing ApoE4 expression.

Advantageously, the oligonucleotide sequence is complementary to the target sequence in SEQ ID NO:1, which contains residue 535 of SEQ ID NO:1. Preferably, the nucleotide complementary to the nucleotide at position 535 of SEQ ID NO:1 in the continuous nucleotide sequence contains a guanine (g) nucleobase or a modified nucleobase that allows pairing with a Watson-Crick base at position 535 of SEQ ID NO:1.

Advantageously, antisense oligonucleotides can regulate target expression by reducing or downregulating target expression. Preferably, this regulation produces at least 20% inhibition of expression compared to the normal expression level of the 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% inhibition compared to the normal expression level of the target. Also preferably, the antisense oligonucleotide can reduce the target expression level by at least 20% compared to the normal expression level of the target; more preferably, the inhibition is 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 level of the target.

In some embodiments, after application of 25 μM oligonucleotides to neuroblastoma cells containing at least one copy of APOEε4 in their genome, the antisense oligonucleotides are able to reduce the expression level of ApoE4 mRNA by at least 60% or 70% in vitro compared to the normal expression level of ApoE4 mRNA. In some embodiments, after application of 5 μM oligonucleotides to neuroblastoma cells containing at least one copy of APOEε4 in their genome, the antisense oligonucleotides are able to reduce the expression level of ApoE4 mRNA by at least 50% or 60% in vitro compared to the normal expression level of ApoE4 mRNA. Suitablely, examples provide an assay that can be used to measure ApoE4 RNA inhibition (Example 1).

Targeted regulation is triggered by hybridization between the adjacent nucleotide sequence of the oligonucleotide and the target nucleic acid. In some embodiments, the oligonucleotide contains a mismatch between the oligonucleotide and the target nucleic acid. Despite the mismatch, hybridization with the target nucleic acid can still be sufficient to exhibit the desired regulation of ApoE4 expression. The reduced binding affinity caused by the mismatch can preferably be compensated by an increase in the number of nucleotides in the oligonucleotide and/or an increase in the number of modified nucleosides capable of increasing the binding affinity to the target, such as 2' sugar-modified nucleosides present in the oligonucleotide sequence, including LNA.

Advantageously, the antisense oligonucleotide is able to reduce ApoE4 expression more than it reduces ApoE3 expression. In some embodiments, the antisense oligonucleotide is able to reduce ApoE4 expression such that, in target cells containing both ApoE4 and ApoE3 nucleic acids, the ratio of the percentage of remaining ApoE3 nucleic acid (as compared to a control) to the percentage of remaining ApoE4 nucleic acid (as 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 (the normal expression level of ApoE3 nucleic acid).

In some embodiments, the antisense oligonucleotide can reduce the expression level of ApoE4 mRNA such that, in target cells containing both ApoE4 mRNA and ApoE3 mRNA, after applying 25 μM oligonucleotide to neuroblastoma cells with heterozygous APOEε3 and APOEε4 genotypes, the ratio of the percentage of remaining ApoE3 mRNA (as compared to the control) to the percentage of remaining ApoE4 mRNA (as compared to the 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.

Specifically, antisense oligonucleotides may be able to reduce ApoE4 expression, thereby making it more effective in the presence of...

(a) mRNA encoding the human ApoE4 protein encoded by SEQ ID NO:1 and containing the region corresponding to positions 516 to 556 of SEQ ID NO:1 and

(b) Target cells containing mRNA encoding the human ApoE3 protein encoded by SEQ ID NO:2 and comprising the region corresponding to positions 516 to 556 of SEQ ID NO:2,

This antisense oligonucleotide reduces the mRNA level encoding human ApoE4, making...

(i) The percentage of remaining ApoE3 mRNA levels compared to the control.

(ii) The ratio of the percentage of remaining ApoE4 mRNA levels compared to the control.

Higher 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.

Suitable controls reflecting normal expression levels of ApoE4 and/or ApoE3 nucleic acids include the expression levels of ApoE3 mRNA and ApoE4 mRNA in the absence of antisense oligonucleotides, such as target cells exposed only to a medium (e.g., PBS or culture medium) or target cells exposed to control oligonucleotides known not to target the target sequences defined in this specification and preferably known not to have any off-target activity (i.e., no relevant effect on the expression of the cell/organism genome). Reference (control) values are also known from the literature. Suitable target cells include human KELLY neuroblastoma cells with heterozygous APOE ε3/ε4 genotypes (Schaffer et al., Genes Nutr., January 2014; 9(1)). Example 1 provides an assay that can be used to measure the relative inhibition of ApoE3 and ApoE4 mRNA using human KELLY neuroblastoma cells.

This invention relates to antisense oligonucleotides comprising a continuous nucleotide sequence of at least 10 nucleotides having at least 80% complementarity with SEQ ID NO:1. The antisense oligonucleotide may also, or alternatively, have at least 80% complementarity with SEQ ID NO:3. In some embodiments, the antisense oligonucleotide comprises a continuous nucleotide sequence of at least 10 nucleotides having at least 80% complementarity with SEQ ID NO:1 (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 100%). In some embodiments, the antisense oligonucleotide also comprises, or alternatively comprises, a continuous nucleotide sequence of at least 10 nucleotides in length, which is complementary to SEQ ID NO:3 by at least 80% (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 100%).

In some embodiments, the oligonucleotide comprises a continuous nucleotide sequence of at least 10 to 30 nucleotides in length, which is complementary to the region of the target nucleic acid or to the target sequence at least 80% (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 100%).

It is advantageous if the oligonucleotide or its continuous nucleotide sequence of the present invention is completely complementary (100% complementary) to the region of the target nucleic acid or to the target sequence.

In some embodiments, the oligonucleotide or its sequential nucleotide sequence may contain zero to three mismatches compared to its complementary target sequence, optionally selected from one, two, and three mismatches. For example, the oligonucleotide or its sequential nucleotide sequence may contain one or two mismatches between the oligonucleotide or its sequential nucleotide sequence and the target sequence in the target nucleic acid. The mismatch is not in the nucleotide at position 535 of SEQ ID NO:1. Therefore, the nucleotide of the sequential nucleotide sequence complementary to the nucleotide at position 535 of SEQ ID NO:1 contains guanine (g) nucleobases.

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

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

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

It is also advantageous if the antisense oligonucleotide is complementary to a target sequence selected from one of the regions listed in Table 2. In some embodiments, the consecutive nucleotide sequences of the antisense oligonucleotide are at least 80% or at least 90% complementary to the selected target sequences R_4 to R_96, such as being completely complementary. In some embodiments, the oligonucleotide sequence is 100% complementary to the target sequences selected from R_25, R_40, R_46, R_66, and R_91 (Table 2).

Oligonucleotides can contain or be composed 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, 3, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, or 50 nucleotides. Oligonucleotides can be composed of, 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, 3, 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 to 50 nucleotides in length, such as 8 to 40, 10 to 40, 10 to 35, 11 to 25, 12 to 22, 14 to 20, or 14 to 18 consecutive nucleotides. In one embodiment, the oligonucleotide comprises or consists of 16 to 22 nucleotides in length. In a preferred embodiment, the oligonucleotide comprises or consists of 16 to 20 nucleotides in length.

In some embodiments, the oligonucleotide or its adjacent nucleotide sequence comprises or consists of 22 or fewer nucleotides, such as 20 or fewer nucleotides, such as 16, 17, 18, 19, or 20 nucleotides. It should be understood that any range given herein includes the endpoints of the range. Accordingly, if an oligonucleotide is described herein as comprising from 10 to 30 nucleotides, then both 10 and 30 nucleotides are included.

In some embodiments, the continuous 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 consecutive nucleotides in length. In a preferred embodiment, the oligonucleotide comprises or consists of 16, 17, 18, 19, or 20 nucleotides in length.

Preferably, the continuous nucleotide sequence is 100% complementary to the target nucleic acid.

Table 3. Examples of motif sequences for oligonucleotides or continuous nucleotide sequences

In some implementations, the oligonucleotide or continuous nucleotide sequence comprises or consists of sequences selected from those listed in Table 3.

In some embodiments, the antisense oligonucleotide or continuous nucleotide sequence comprises or consists of 10 to 30 nucleotides in length and has at least 90% identity, preferably 100% identity, with a sequence selected from the group consisting of SEQ ID NO:4 to SEQ ID NO:96 (see motif sequences listed in Table 3).

In some embodiments, the antisense oligonucleotide or continuous nucleotide sequence comprises or consists of 10 to 30 nucleotides in length and has at least 90% identity, preferably at least 100% identity, with a sequence selected from the group consisting of SEQ ID NO:25, SEQ ID NO:40, SEQ ID NO:46, SEQ ID NO:66 and SEQ ID NO:91.

In some embodiments, the antisense oligonucleotide or continuous nucleotide sequence comprises or consists of 10 to 30 nucleotides in length and has at least 90% identity with a sequence selected from the group consisting of SEQ ID NO:25, SEQ ID NO:40 and SEQ ID NO:46, preferably at least 100% identity.

In some embodiments, the antisense oligonucleotide or continuous nucleotide sequence comprises or consists of 10 to 30 nucleotides in length and has at least 90% identity with the sequence of SEQ ID NO:25, preferably at least 100% identity.

In some embodiments, the antisense oligonucleotide or continuous nucleotide sequence comprises or consists of 10 to 30 nucleotides in length and has at least 90% identity with the sequence of SEQ ID NO:40, preferably at least 100% identity.

In some embodiments, the antisense oligonucleotide or continuous nucleotide sequence comprises or consists of 10 to 30 nucleotides in length and has at least 90% identity with the sequence of SEQ ID NO:46, preferably at least 100% identity.

In some embodiments, the antisense oligonucleotide or continuous nucleotide sequence comprises or consists of 10 to 30 nucleotides in length and has at least 90% identity with the sequence of SEQ ID NO:66, preferably at least 100% identity.

In some embodiments, the antisense oligonucleotide or continuous nucleotide sequence comprises or consists of 10 to 30 nucleotides in length and has at least 90% identity with the sequence of SEQ ID NO:91, preferably at least 100% identity.

In some embodiments, the continuous nucleotide sequence comprises a sequence selected from SEQ ID NO:4 to SEQ ID NO:96.

In some embodiments, the continuous nucleotide sequence consists of sequences selected from SEQ ID NO:4 to SEQ ID NO:96.

In some implementations, the antisense oligonucleotide comprises a sequence selected from SEQ ID NO:4 to SEQ ID NO:96.

In some implementations, the antisense oligonucleotide consists of sequences selected from SEQ ID NO:4 to SEQ ID NO:96.

In some embodiments, the continuous nucleotide sequence comprises or consists of a nucleotide sequence as shown in SEQ ID NO:25.

In some embodiments, the continuous nucleotide sequence comprises or consists of a nucleotide sequence as shown in SEQ ID NO:40.

In some embodiments, the continuous nucleotide sequence comprises or consists of a nucleotide sequence as shown in SEQ ID NO:46.

In some embodiments, the continuous nucleotide sequence comprises or consists of a nucleotide sequence as shown in SEQ ID NO:66.

In some embodiments, the continuous nucleotide sequence comprises or consists of a nucleotide sequence as shown in SEQ ID NO:91.

In some implementations, the antisense oligonucleotide sequence comprises or consists of a nucleotide sequence as shown in SEQ ID NO:25.

In some implementations, the antisense oligonucleotide sequence comprises or consists of a nucleotide sequence as shown in SEQ ID NO:40.

In some implementations, the antisense oligonucleotide sequence comprises or consists of a nucleotide sequence as shown in SEQ ID NO:46.

In some embodiments, the antisense oligonucleotide sequence comprises or consists of a nucleotide sequence as shown in SEQ ID NO:66.

In some embodiments, the antisense oligonucleotide sequence comprises or consists of a nucleotide sequence as shown in SEQ ID NO:91.

It should be understood that continuous nucleobase sequences (motif sequences) can be modified, for example, to increase nuclease resistance and/or binding affinity to target nucleic acids.

The pattern of incorporating modified nucleosides (such as high-affinity modified nucleosides) into oligonucleotide sequences is generally referred to as oligonucleotide design.

Advantageously, the oligonucleotide sequence does not contain RNA nucleosides, as this would reduce nuclease resistance. Additionally, as described elsewhere herein, the antisense oligonucleotide may advantageously contain one or more modified nucleosides or nucleotides, such as 2'-sugar-modified nucleosides. Furthermore, it is preferred that the unmodified nucleosides are DNA nucleosides. Therefore, the oligonucleotides of the present invention can be engineered to contain both modified nucleosides and DNA nucleosides. Preferably, high-affinity modified nucleosides are used.

In one embodiment, the oligonucleotide comprises at least one modified nucleoside, such as at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen, at least fourteen, at least fifteen, or at least sixteen modified nucleosides. In another embodiment, the oligonucleotide comprises one to ten modified nucleosides, such as two to nine modified nucleosides, three to eight modified nucleosides, four to seven modified nucleosides, or six or seven modified nucleosides. Suitable modifications are described under “Modified Nucleosides,” “High-Affinity Modified Nucleosides,” “Sugar Modifications,” “2' Sugar Modifications,” and “Locked Nucleic Acids (LNAs)” in the “Definitions” section.

In one embodiment, the oligonucleotide comprises one or more sugar-modified nucleosides, such as 2' sugar-modified nucleosides. Preferably, the oligonucleotides of the present invention comprise one or more 2' sugar-modified nucleosides, independently selected from 2'-O-alkyl-RNA, 2'-O-methyl-RNA, 2'-alkoxy-RNA, 2'-O-methoxyethyl-RNA, 2'-amino-DNA, 2'-fluoro-DNA, arabinonucleotide (ANA), 2'-fluoro-ANA, and LNA nucleosides. It is preferred if one or more modified nucleosides are locked nucleic acids (LNAs).

In another embodiment, the oligonucleotide comprises at least one modified internucleotide bond. Suitable internucleotide modifications are described under “Modified Internucleotide Bonds” in the “Definitions” section. It is advantageous if at least 75%, such as 80%, or such as all, of the internucleotide bonds within the adjacent nucleotide sequence are phosphate thioester or borane phosphate internucleotide bonds. In some embodiments, all internucleotide bonds in the continuous sequence of the oligonucleotide are phosphate thioester bonds.

In some embodiments, the oligonucleotides of the present invention comprise at least one LNA nucleoside, such as 1, 2, 3, 4, 5, 6, 7, or 8 LNA nucleosides, such as 2 to 6 LNA nucleosides, such as 3 to 7 LNA nucleosides, 4 to 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%, 85%, or 90% of the modified nucleosides are LNA nucleosides, particularly β-D-oxy-LNA or ScET. In yet another embodiment, all the modified nucleosides in the oligonucleotide are LNA nucleosides. In another embodiment, the oligonucleotide may simultaneously comprise β-D-oxy-LNA and one or more of the following LNA nucleosides: thio-LNA, amino-LNA, oxy-LNA, ScET, and/or ENA, or be in the β-D configuration or the α-L configuration or a combination thereof. In another embodiment, all LNA cytosine units are 5-methylcytosine. For the nuclease stability of oligonucleotides or continuous nucleotide sequences, it is preferred to have at least one LNA nucleotide at the 5' end of the nucleotide sequence and at least two LNA nucleotides at the 3' end of the nucleotide sequence.

In one embodiment of the present invention, the oligonucleotide of the present invention is capable of recruiting RNase H.

In this invention, advantageous structural designs are notched aggregate designs as described in the "Definitions" section, such as "notched aggregate," "LNA notched aggregate," "MOE notched aggregate," and "hybrid wing notched aggregate," and "alternating wing notched aggregate." Notched aggregate designs include notched aggregates with uniform wing, hybrid wing wing, alternating wing, and notched interruptor designs.

It is advantageous if the oligonucleotide is a nicked polymer with an F-G-F' design, particularly a nicked polymer of the formula 5'-F-G-F'-3', wherein region F and region F' independently contain 1-8 nucleotides, wherein 2-5 nucleotides are modified with 2' sugars and define the 5' and 3' ends of the F and F' regions, and G is a region containing 6 to 16 nucleotides capable of recruiting RNase H, such as a region containing 6-16 DNA nucleotides.

In some implementations, both flanks have 2' sugar-modified nucleosides at both the 5' and 3' ends.

In some embodiments, the notched polymer is an LNA notched polymer.

In some embodiments of the invention, the LNA notched polymer 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, and 2-9-2.

In some embodiments, the LNA nick polymer has an alternating flanking design. In an alternating flanking design, at least one flanking region (F or F') contains one or more DNA nucleotides in addition to LNA nucleotides. Flanking region F or flanking region F', or both F and F', contains at least three nucleotides, and the 5' terminal and 3' terminal nucleotides of regions F and/or F' are LNA nucleotides. Each "-" (dash) represents a transfer between LNA/DNA nucleotides, the number represents the number of nucleotides, and the highest number represents the DNA nucleotide in the nick region G. Therefore, a nick polymer with an alternating flanking design (where F has 5 nucleotides, which is LNA-LNA-DNA-DNA-LNA, the nick region has 6 DNA nucleotides, and F' has 2 LNA nucleotides) can be represented as 2-2-1-6-2.

Using the same designation, in some embodiments of the invention, the LNA notched polymer is selected from the following alternating flanking 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.

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

In all cases, the F-G-F' design may further include regions D' and/or D'", as described under "Regions D' or D' in oligonucleotides" in the "Definitions" section. In some embodiments, the oligonucleotides of the present invention have one, two, or three phosphodiester-linked nucleoside units, such as DNA units, at the 5' or 3' end of the nicked polymer region.

In some embodiments, in addition to dithiophosphate bonds, the oligonucleotides of the present invention comprise both a thiophosphate nucleoside internucleotide bond and at least one phosphodiester bond, such as two, three, or four phosphodiester bonds. In spacer oligonucleotides, the phosphodiester bonds (when present) are suitably not located between consecutive DNA nucleosides in the gap region G.

Advantageously, all internucleotide bonds in the continuous nucleotide sequence of the oligonucleotide are phosphate thioesters, or all internucleotide bonds in the oligonucleotide are phosphate thioester bonds. Nuclease-resistant bonds, such as phosphate thioester bonds, are particularly useful in oligonucleotide regions capable of recruiting nucleases when forming a double strand with the target nucleic acid, such as region G of the spacer polymer. However, phosphate thioester bonds can also be used in non-nuclease-recruiting regions and/or affinity-enhancing regions, such as regions F and F' of the spacer polymer. In some embodiments, the spacer polymer oligonucleotide may contain one or more phosphodiester bonds in region F or F', or simultaneously in regions F and F', wherein all internucleotide bonds in region G may be phosphate thioesters.

In some embodiments, the oligonucleotide is an oligonucleotide compound having CMP ID NO: 91_1. In some embodiments, the oligonucleotide is an oligonucleotide compound having CMP ID NO: 66_1. In some embodiments, the oligonucleotide is an oligonucleotide compound having CMP ID NO: 46_1. In some embodiments, the oligonucleotide is an oligonucleotide compound having CMP ID NO: 46_2. In some embodiments, the oligonucleotide is an oligonucleotide compound having CMP ID NO: 46_3. In some embodiments, the oligonucleotide is an oligonucleotide compound having CMP ID NO: 46_4. In some embodiments, the oligonucleotide is an oligonucleotide compound having CMP ID NO: 46_5. In some embodiments, the oligonucleotide is an oligonucleotide compound having CMP ID NO: 25_1. In some embodiments, the oligonucleotide is an oligonucleotide compound having CMP ID NO: 25_2. In some embodiments, the oligonucleotide is an oligonucleotide compound having CMP ID NO: 25_3. In some embodiments, the oligonucleotide is an oligonucleotide compound having CMP ID NO: 25_4. In some embodiments, the oligonucleotide is an oligonucleotide compound having CMP ID NO: 25_5. In some embodiments, the oligonucleotide is an oligonucleotide compound having CMP ID NO: 25_6. In some embodiments, the oligonucleotide is an oligonucleotide compound having CMP ID NO: 40_1. In some embodiments, the oligonucleotide is an oligonucleotide compound having CMP ID NO: 40_2. In some embodiments, the oligonucleotide is an oligonucleotide compound having CMP ID NO: 40_3.

The antisense oligonucleotide that is particularly advantageous according to the present invention is an oligonucleotide compound selected from the group consisting of:

ACcAgGcggccgCG(SEQ ID NO:91; CMP ID NO:91_1)

CAggcggccgcgcacGT(SEQ ID NO:66; CMP ID NO:66_1)

CGcgcacgtCcTC (SEQ ID NO:46; CMP ID NO:46_1)

CGcgcacgtcCTC (SEQ ID NO:46; CMP ID NO:46_2)

CGcGcacgtcCTC (SEQ ID NO:46; CMP ID NO:46_3)

CGCgcacgtccTC (SEQ ID NO:46; CMP ID NO:46_4)

CGcGCacgtccTC (SEQ ID NO:46; CMP ID NO:46_5)

GCacgtcctccATG(SEQ ID NO:25; CMP ID NO:25_1)

GCAcgtcctccaTG(SEQ ID NO:25; CMP ID NO:25_2)

GCacgtcctcCaTG(SEQ ID NO:25; CMP ID NO:25_3)

GCacgtcctCcATG(SEQ ID NO:25; CMP ID NO:25_4)

GCacgtcctCcaTG(SEQ ID NO:25; CMP ID NO:25_5)

GCacgtcctcCATG(SEQ ID NO:25; CMP ID NO:25_6)

GCgcacgtcctCC(SEQ ID NO:40; CMP ID NO:40_1)

GCgcAcgtcctCC(SEQ ID NO:40; CMP ID NO:40_2)

GCgCacgtcctCC(SEQ ID NO:40; CMP ID NO:40_3)

The uppercase letters represent β-D-oxyLNA nucleosides, the lowercase letters represent DNA nucleosides, all LNA Cs are 5-methylcytosine, and all internucleotide bonds are thiophosphate internucleotide bonds.

In the context of this invention, another particularly advantageous oligonucleotide is an oligonucleotide compound selected from the group of compounds defined in section 4 by HELM annotation. That is, the structure of each compound is described using 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 keys:

[LR](G) is a β-D-oxy-LNA guanine nucleoside.

[LR](T) is a β-D-oxy-LNA thymidine.

[LR](A) is a β-D-oxy-LNA adenine nucleoside.

[LR]([5meC]) is a β-D-oxy-LNA 5-methylcytosine nucleoside.

[dR](G) is a DNA guanine nucleoside.

[dR](T) is a DNA thymidine nucleoside.

[dR](A) is a DNA adenine nucleoside.

[dR]([C]) is a DNA cytosine nucleoside.

[sP] is an inter-nucleoside bond of thiophosphate (stereotype not defined).

Further information on HELM and open-source tools can be found at www.pistoiaalliance.org/helm-tools/ and www.pistoiaalliance.org/membership/about/. Specifically, in Table 4, the names “RNA1” and “$$$$V2.0” represent information useful for computerized analysis of HELM sequences and should not be considered as restrictions on the oligonucleotide sequences defined between the brackets (i.e., between “{” and “}”).

Table 4. Compounds defined by HELM annotations.

Manufacturing method

In another aspect, the present invention provides a method for manufacturing the oligonucleotide of the present invention, the method comprising reacting nucleotide units to form covalently linked sequential nucleotide units contained in the oligonucleotide. Preferably, the method uses a phosphoramidite chemistry method (see, for example, Caruthers et al., 1987, Methods in Enzymology, Vol. 154, pp. 287-313). In another embodiment, the method further comprises reacting the sequential nucleotide sequence with a conjugate moiety (ligand) to covalently link the conjugate moiety to the oligonucleotide. In another aspect, a method for manufacturing the compositions of the present invention is provided, the method comprising mixing the oligonucleotide or conjugated oligonucleotide of the present invention with a pharmaceutically acceptable diluent, solvent, carrier, salt, and/or adjuvant.

medicinal salt

The compounds according to the invention can exist in the form of their pharmaceutical salts. The term "pharmaceutical salt" refers to a conventional acid addition salt or base addition salt formed from a suitable non-toxic organic or inorganic acid or organic or inorganic base, while retaining the bioavailability and properties of the compounds of the invention. Acid addition salts include, for example, salts derived from inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, aminosulfonic acid, phosphoric acid, and nitric acid, and salts 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, etc. Base addition salts include salts derived from ammonium, potassium, sodium, and quaternary ammonium hydroxides (such as, for example, tetramethylammonium hydroxide). The chemical modification of pharmaceutical compounds into salts to obtain improved physical and chemical stability, hygroscopicity, flowability, and solubility of the compounds is a well-known technique among medicinal chemists. For example, Bastin describes this in *Organic Process Research & Development*, 2000, Vol. 4, pp. 427-435, or Ansel describes it in *Pharmaceutical Dosage Forms and Drug Delivery Systems, 6th ed. (1995)*, pp. 196 and 1456-1457. For example, the pharmaceutical salt of the compounds provided herein may be a sodium salt.

In another aspect, the present invention provides pharmaceutically acceptable salts of antisense oligonucleotides or conjugates thereof. In a preferred embodiment, the pharmaceutically acceptable salt is a sodium or potassium salt.

Pharmaceutical Composition

In another aspect, the present invention provides pharmaceutical compositions comprising any of the aforementioned oligonucleotides and/or oligonucleotide conjugates or salts thereof, as well as pharmaceutical diluents, carriers, salts, and/or adjuvants. Pharmaceutical diluents include phosphate-buffered saline (PBS), and pharmaceutical salts include, but are not limited to, sodium and potassium salts. In some embodiments, the pharmaceutical diluent is sterile phosphate-buffered saline. In some embodiments, the oligonucleotides are used in the pharmaceutical diluent at a concentration of 50-300 μM solution.

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

The oligonucleotides or oligonucleotide conjugates of the present invention can be mixed with pharmaceutically active or inert substances to prepare pharmaceutical compositions or formulations. The composition and formulation method of the pharmaceutical composition depend on many criteria, including but not limited to route of administration, disease severity, or dosage.

These compositions can be sterilized using conventional sterilization techniques or by aseptic filtration. The resulting aqueous solution can be used directly after packaging or lyophilized, with the lyophilized formulation mixed with a sterile aqueous carrier prior to application. The pH of the formulation is typically between 3 and 11, more preferably between 5 and 9 or between 6 and 8, and most preferably between 7 and 8, such as 7 to 7.5. The resulting compositions in solid form can be packaged in multiple single-dose units, each unit containing a fixed amount of one or more of the aforementioned reagents, such as in sealed packages of tablets or capsules. The compositions in solid form can also be flexibly quantified in containers, such as in squeeze tubes designed for topical application of creams or ointments.

In some embodiments, the oligonucleotide or oligonucleotide conjugate of the present invention is a prodrug. In particular, for the oligonucleotide conjugate, once the prodrug is delivered to the site of action (e.g., a target cell), the conjugate portion is cleaved from the oligonucleotide.

application

The oligonucleotides of the present invention can be used as research reagents, for example, for diagnosis, treatment and prevention.

In research, such oligonucleotides can be used to specifically regulate the synthesis of the ApoE4 protein in cells (e.g., in vitro cell cultures) and laboratory animals, thereby facilitating functional analysis of the target or evaluating its applicability as a therapeutic intervention target. Typically, target regulation is achieved by preventing protein formation through degradation or inhibition of the mRNA that produces the protein, or by degradation or inhibition of the gene or mRNA that produces the protein.

If the oligonucleotides of the present invention are used in research or diagnosis, the target nucleic acid may be cDNA or a synthetic nucleic acid derived from DNA or RNA.

The present invention provides an in vivo or in vitro method for regulating ApoE4 expression in target cells expressing ApoE4, comprising administering an effective amount of the oligonucleotide of the present invention to said cells.

In some embodiments, the target cells are mammalian cells, particularly human cells. Target cells can be in vitro cell cultures or in vivo cells that form part of mammalian tissue. In preferred embodiments, the target cells are present in the brain or central nervous system. Cells in the cerebral cortex, medulla oblongata/pons, midbrain, frontal cortex, brainstem, cerebellum, and spinal cord are particularly relevant target regions. For the treatment of AD, reduction of targets in the cerebral cortex, medulla oblongata/pons, and midbrain is advantageous. For the treatment of PSP (such as PSP with AD-type pathology), reduction of targets in the medulla oblongata/pons and midbrain is advantageous. In particular, microglia, neurons, nerve cells, astrocytes, axons, and basal ganglia are or contain relevant cell types.

In diagnostics, oligonucleotides can be used to detect and quantify ApoE4 expression in cells and tissues using techniques such as Northern blotting, in situ hybridization, or similar methods.

For treatment, oligonucleotides can be administered to animals or humans suspected of having the disease or condition, and can be used to treat it by regulating the expression of ApoE4.

For treatment, oligonucleotides can also be applied to animals or humans at risk of developing diseases or conditions, and can be used to treat them by regulating the expression of ApoE4.

For treatment, oligonucleotides can also be applied to animals or humans diagnosed with diseases or conditions, and can be used to treat the disease by regulating the expression of ApoE4.

In particular, the present invention provides a method for treating or preventing a disease or condition, the method comprising: administering a therapeutically or preventively effective amount of the oligonucleotide, oligonucleotide conjugate, or pharmaceutical composition of the present invention to a subject who has, is at risk of having, or is susceptible to the disease or condition.

The present invention also relates to oligonucleotides, compositions or conjugates that can be used as pharmaceuticals as defined herein.

The oligonucleotides, oligonucleotide conjugates, or pharmaceutical compositions according to the present invention are typically administered in an effective amount.

The present invention also provides the use of the oligonucleotides or oligonucleotide conjugates of the present invention as described herein in the manufacture of medicaments for the treatment or prevention of the diseases or conditions mentioned herein, or provides methods for the treatment or prevention of the conditions mentioned herein.

As mentioned herein, diseases or conditions are associated with the expression of ApoE4. The therapeutic application of this invention is preferably for treating or preventing diseases or conditions caused by abnormal levels and/or activity of ApoE4.

Advantageously, the therapeutic application of the present invention is used to treat subjects who have or are at risk of having such disease or condition, who carry at least one copy of the APOEε4 gene in their genome.

In some implementations, subjects who have or are at risk of having a disease or condition carry a copy of the APOEε4 gene in their genome.

In some implementations, subjects who have or are at risk of having a disease or condition carry a copy of the APOEε3 gene in their genome.

In some implementations, subjects who have or are at risk of having the disease have the heterozygous genotype APOε3/ε4.

In some implementations, subjects who have or are at risk of having the disease have the heterozygous genotype APOε2/ε4.

In some implementations, subjects who have or are at risk of having the disease have the homozygous genotype APOε4/ε4.

In some implementations, subjects who have or are at risk of having the disease are homozygous for the APOε4 gene.

In some implementations, treatment is given to subjects who are at risk of having, suspected of having, or have been diagnosed with a neurological disorder, such as those selected from a group consisting of neurodegenerative diseases (including Alzheimer's disease (AD), frontotemporal dementia (FTD), Pick's disease (PiD), progressive supranuclear palsy (PSP), and movement disorders such as Parkinson's disease (PD), Lewy body dementia, Down syndrome dementia, and Niemann-Pick disease type C1).

In one embodiment, the present invention relates to oligonucleotides, oligonucleotide conjugates, or pharmaceutical compositions for use in treating diseases or conditions selected from AD, FTD, PiD, PSP, movement disorders such as PD, Lewy body dementia, Down syndrome dementia, and Niemann-Pick disease type C1.

In some implementations, the disease or condition is Alzheimer's disease (AD). AD can be, for example, late-onset AD (over 65 years of age). In some implementations, AD can be AD without a dominant AD mutation. AD can be early-onset AD. In patients with progressive supranuclear palsy (PSP), AD can also be AD or AD-type pathology.

In some implementations, the disease or condition is frontotemporal dementia (FTD).

In some implementations, the disease or condition is Pick's disease (PiD).

In some implementations, the disease or condition is progressive supranuclear palsy (PSP).

In some implementations, the disease or condition is a movement disorder. For example, a movement disorder could be Parkinson's disease (PD).

In some implementations, the disease or symptom is Lewy body dementia.

In some implementations, the disease or symptom is dementia in subjects with Down syndrome.

In some implementations, the disease or condition is Niemann-Pick disease type C1.

In some implementations, the disease or condition is dementia. Optionally, dementia is dementia or dementia associated with any or more of AD, FTD, PiD, PSP, PD, Lewy body dementia, dementia in subjects with Down syndrome, or Niemann-Pick disease type C1.

application

The oligonucleotides or pharmaceutical compositions of the present invention can be administered via parenteral routes (such as intravenous injection, subcutaneous, intramuscular, intracerebral, intraventricular, intraocular, or intrathecal administration).

In some embodiments, the administration is performed intrathecally.

Advantageously, for example, in the treatment of neurological disorders, the oligonucleotide or pharmaceutical composition of the present invention is administered intrathecally or intracranially, for example, via intracerebral or intraventricular administration.

The present invention also provides the use of the oligonucleotides or conjugates thereof, such as pharmaceutical salts or compositions, in the manufacture of pharmaceuticals, wherein the pharmaceuticals are dosage forms for subcutaneous administration.

The present invention also provides the use of the oligonucleotides of the present invention, or conjugates thereof, such as pharmaceutical salts or compositions of the present invention, in the manufacture of pharmaceuticals, wherein the pharmaceuticals are dosage forms for intrathecal administration.

The present invention also provides the use of the oligonucleotides or oligonucleotide conjugates of the present invention as described herein in the manufacture of pharmaceuticals, wherein the pharmaceuticals are dosage forms for intrathecal administration.

Combination therapy

In some embodiments, the oligonucleotides, oligonucleotide conjugates, or pharmaceutical compositions of the present invention are used to treat in combination with another therapeutic agent. The therapeutic agent may be, for example, a standard of care for the aforementioned disease or ailment.

sequence

SEQ ID NO:1-NM_001302690.2 (APOE4, mRNA) containing rs429358

SEQ ID NO:2-NM_001302690.2(APOE3,mRNA)

SEQ ID NO:3-XM__005589554.2 (cynomolgus monkey apolipoprotein E (APOE), mRNA)

Embodiments of the present invention

1. An antisense oligonucleotide of 8 to 50 nucleotides in length, such as 10 to 30 nucleotides in length, comprising a continuous nucleotide sequence of at least 10 nucleotides in length, the continuous nucleotide sequence being at least 80% complementary to a target sequence within positions 516 to 556 of the nucleic acid encoded by apolipoprotein (Apo) E4 shown in SEQ ID NO:1, wherein the target sequence comprises position 535 of SEQ ID NO:1.

2. The antisense oligonucleotide according to Project 1, wherein the continuous nucleotide sequence has zero to three mismatches compared to the target sequence, optionally selected from one mismatch, two mismatches, and three mismatches, and the precursor is a nucleotide of the continuous nucleotide sequence complementary to the nucleotide at position 535 of SEQ ID NO:1 containing a guanine (g) nucleobase.

3. The antisense oligonucleotide according to any one of the preceding items, wherein the continuous nucleotide sequence is at least 90% complementary to the target sequence at positions 516 to 556 of SEQ ID NO:1.

4. The antisense oligonucleotide according to any one of the preceding items, wherein the continuous nucleotide sequence is 100% complementary to the target sequence at positions 516 to 556 of SEQ ID NO:1.

5. The antisense oligonucleotide according to any one of the preceding items, wherein the continuous nucleotide sequence is complementary to the target sequence at positions 522 to 548 of SEQ ID NO:1.

6. The antisense oligonucleotide according to any one of the preceding items, wherein the continuous nucleotide sequence is complementary to the target sequences selected from R_4 to R_96 in Table 2.

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

8. The antisense oligonucleotide according to any one of the preceding items, wherein the continuous nucleotide sequence comprises or consists of a nucleotide sequence selected from or composed of the group consisting of SEQ ID NO:4 to SEQ ID NO:96.

9. The antisense oligonucleotide according to any one of the preceding items, wherein the continuous nucleotide sequence comprises or consists of a nucleotide sequence selected from or composed of the group consisting of SEQ ID NO:25, SEQ ID NO:40, SEQ ID NO:46, SEQ ID NO:66 and SEQ ID NO:91.

10. The antisense oligonucleotide according to any one of the preceding items, wherein the continuous nucleotide sequence comprises or consists of SEQ ID NO:25.

11. The antisense oligonucleotide according to any one of the preceding items, wherein the continuous nucleotide sequence comprises or consists of SEQ ID NO:40.

12. The antisense oligonucleotide according to any one of the preceding items, wherein the continuous nucleotide sequence comprises or consists of SEQ ID NO:46.

13. The antisense oligonucleotide according to any one of the preceding items, wherein the continuous nucleotide sequence comprises or consists of SEQ ID NO:66.

14. The antisense oligonucleotide according to any one of the preceding items, wherein the continuous nucleotide sequence comprises or consists of SEQ ID NO:91.

15. The antisense oligonucleotide according to any one of the preceding items, wherein the antisense oligonucleotide is capable of reducing the expression of mammalian (such as human) apolipoprotein (Apo) E4.

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 mammalian (e.g., human) apolipoprotein (Apo) E4 in the target cell by at least 20%, such as at least 30%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, compared with the normal expression level of the target cell.

17. The antisense oligonucleotide according to any one of the preceding items, wherein the antisense oligonucleotide is capable of reducing the expression of ApoE4, such that in the presence of...

(a) mRNA encoding the human ApoE4 protein encoded by SEQ ID NO:1 and containing the region corresponding to positions 516 to 556 of SEQ ID NO:1 and

(b) Target cells containing mRNA encoding the human ApoE3 protein encoded by SEQ ID NO:2 and comprising the region corresponding to positions 516 to 556 of SEQ ID NO:2,

This antisense oligonucleotide reduces the mRNA level encoding human ApoE4, making...

(i) The percentage of remaining ApoE3 mRNA levels compared to the control.

(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, optionally, wherein the control is the level of mRNA in the control target cells in the absence of antisense oligonucleotides.

18. The antisense oligonucleotide according to any one of the preceding items, wherein the target sequence is located in RNA.

19. The antisense oligonucleotide as described in Item 18, wherein the RNA is mRNA.

20. The antisense oligonucleotide as described in Item 19, wherein the mRNA is a mature mRNA.

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

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

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

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

25. The antisense oligonucleotide according to any one of the preceding items, wherein the continuous nucleotide sequence has a length of at least 16 nucleotides (such as 16, 17, 18, 19, 20, 21 or 22 nucleotides).

26. The antisense oligonucleotide according to any one of the preceding items is single-stranded.

27. The antisense oligonucleotide according to any one of the preceding items, wherein the continuous nucleotide sequence comprises one or more modified nucleosides, such as 2' sugar-modified nucleosides or unlocked nucleic acid (UNA) nucleosides.

28. The antisense oligonucleotide according to any one of the preceding items, wherein the continuous nucleotide sequence comprises one or more 2' sugar-modified nucleosides.

29. The antisense oligonucleotide according to item 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, arabinose (ANA), 2'-fluoro-ANA and LNA nucleosides.

30. The antisense oligonucleotide according to item 29, wherein the one or more 2' sugar-modified nucleosides comprise at least one LNA nucleoside.

31. The nucleic acid molecule according to item 30, wherein the at least one LNA nucleotide is selected from the group consisting of oxy-LNA, amino-LNA, thio-LNA, cET and ENA LNA nucleotides.

32. The antisense oligonucleotide according to item 31, wherein the modified LNA nucleotide is an oxy-LNA with a 2'-4' bridge -O-CH ₂ -.

33. The antisense oligonucleotide according to item 32, wherein the oxy-LNA is β-D-oxy-LNA.

34. The antisense oligonucleotide according to any one of items 29 to 33, wherein the continuous nucleotide sequence comprises 4 to 8 LNA nucleotides.

35. The antisense oligonucleotide according to any one of the preceding items, comprising at least one modified nucleoside internucleotide bond.

36. An antisense oligonucleotide according to any of the preceding items, which contains a nuclease-resistant modified nucleoside bond.

37. The antisense oligonucleotide according to any one of the preceding items, wherein at least 50% of the internucleotide bonds in the continuous nucleotide sequence are phosphate thioester internucleotide bonds.

38. The antisense oligonucleotide according to any one of the preceding items, wherein at least 80% of the internucleotide bonds in the continuous nucleotide sequence are phosphate thioester internucleotide bonds.

39. The antisense oligonucleotide according to any one of the preceding items, wherein the antisense oligonucleotide is capable of recruiting RNase H.

40. The antisense oligonucleotide according to any one of the preceding items, wherein the oligonucleotide or its continuous nucleotide sequence is a nicked polymer.

41. The antisense oligonucleotide according to any one of the preceding items, wherein the oligonucleotide or its sequential nucleotide sequence is a nicked polymer of formula 5'-F-G-F'-3', wherein each of regions F and F' independently contains or consists of 1-8 nucleotides, wherein 2 to 5 nucleotides are 2' sugar-modified nucleotides, and region G is a region between 6 and 16 nucleotides that is capable of recruiting RNase H.

42. The antisense oligonucleotide according to item 41, wherein region G is a region containing 6 to 16 DNA nucleotides.

43. An antisense oligonucleotide according to any one of items 41 and 42, wherein the 2' sugar-modified nucleoside defines the F region and the 5' and 3' ends of the F' region.

44. An antisense oligonucleotide according to any one of items 41 to 43, wherein the 2' sugar-modified nucleoside is according to any one of items 29 to 33.

45. An antisense oligonucleotide according to any one of items 41 to 44, wherein

(a) The F region is between 2 and 8 nucleotides in length and consists of 2 to 5 identical LNA nucleotides and 0 to 4 DNA nucleotides; and

(b) The F' region is between 2 and 6 nucleotides in length and consists of 2 to 4 identical LNA nucleotides and 0 to 2 DNA nucleotides; and

(c) Region G is between 6 and 14 DNA nucleotides.

46. The antisense oligonucleotide according to any one of items 1 to 45, wherein the antisense oligonucleotide is or comprises a compound selected from the group consisting of:

ACcAgGcggccgCG(SEQ ID NO:91; CMP ID NO:91_1)

CAggcggccgcgcacG(SEQ ID NO:66; CMP ID NO:66_1)T

CGcgcacgtCcTC (SEQ ID NO:46; CMP ID NO:46_1)

CGcgcacgtcCTC (SEQ ID NO:46; CMP ID NO:46_2)

CGcGcacgtcCTC (SEQ ID NO:46; CMP ID NO:46_3)

CGCgcacgtccTC (SEQ ID NO:46; CMP ID NO:46_4)

CGcGCacgtccTC (SEQ ID NO:46; CMP ID NO:46_5)

GCacgtcctccATG (SEQ ID NO:25; CMP ID NO:25_1)

GCAcgtcctccaTG (SEQ ID NO:25; CMP ID NO:25_2)

GCacgtcctcCaTG (SEQ ID NO:25; CMP ID NO:25_3)

GCacgtcctCcATG (SEQ ID NO:25; CMP ID NO:25_4)

GCacgtcctCcaTG (SEQ ID NO:25; CMP ID NO:25_5)

GCacgtcctcCATG (SEQ ID NO:25; CMP ID NO:25_6)

GCgcacgtcctCC (SEQ ID NO:40; CMP ID NO:40_1)

GCgcAcgtcctCC (SEQ ID NO:40; CMP ID NO:40_2)

GCgCacgtcctCC (SEQ ID NO:40; CMP ID NO:40_3)

The uppercase letters represent β-D-oxyLNA nucleosides, the lowercase letters represent DNA nucleosides, the uppercase C represents 5-methylcytosine β-D-oxyLNA nucleosides, and all nucleoside bonds are thiophosphate nucleoside bonds.

47. The antisense oligonucleotide according to item 46 is ACcAgGcggccgCG (SEQ ID NO: 91; CMPID NO: 91_1).

48. The antisense oligonucleotide according to item 46 is CAggcggccgcgcacGT (SEQ ID NO: 66; CMP ID NO: 66_1).

49. The antisense oligonucleotide according to item 46 is CGcgcacgtCcTC (SEQ ID NO:46; CMP ID NO:46_1).

50. The antisense oligonucleotide according to item 46 is CGcgcacgtcCTC (SEQ ID NO:46; CMP ID NO:46_2).

51. The antisense oligonucleotide according to item 46 is CGcGcacgtcCTC (SEQ ID NO:46; CMP ID NO:46_3).

52. The antisense oligonucleotide according to item 46 is CGCgcacgtccTC (SEQ ID NO:46; CMP ID NO:46_4).

53. The antisense oligonucleotide according to item 46 is CGcGCacgtccTC (SEQ ID NO:46; CMP ID NO:46_5).

54. The antisense oligonucleotide according to item 46 is GCacgtcctccATG (SEQ ID NO:25; CMPID NO:25_1).

55. The antisense oligonucleotide according to item 46 is GCAcgtcctccaTG (SEQ ID NO:25; CMPID NO:25_2).

56. The antisense oligonucleotide according to item 46 is GCacgtcctcCaTG (SEQ ID NO:25; CMPID NO:25_3).

57. The antisense oligonucleotide according to item 46 is GCacgtcctCcATG (SEQ ID NO:25; CMPID NO:25_4).

58. The antisense oligonucleotide according to item 46 is GCacgtcctCcaTG (SEQ ID NO:25; CMPID NO:25_5).

59. The antisense oligonucleotide according to item 46 is GCacgtcctcCATG (SEQ ID NO:25; CMPID NO:25_6).

60. The antisense oligonucleotide according to item 46 is GCgcacgtcctCC (SEQ ID NO:40; CMP ID NO:40_1).

61. The antisense oligonucleotide described in item 46 is GCgcAcgtcctCC (SEQ ID NO: 40; CMP ID NO: 40_2).

62. The antisense oligonucleotide according to item 46 is GCgCacgtcctCC (SEQ ID NO:40; CMP ID NO:40_3).

63. The antisense oligonucleotide according to any one of items 1 to 46, wherein one of the five nucleotides of the antisense oligonucleotide or its continuous nucleotide sequence (such as one of the four nucleotides, such as one of the terminal 5' nucleotides of the 1st, 3rd and 4th) is complementary to position 535 in SEQ ID NO:1 and contains a guanine nucleobase.

64. The antisense oligonucleotide according to any one of items 1 to 46, wherein one of the six nucleotides of the antisense oligonucleotide or its sequential nucleotide sequence (such as one of the terminal 3' nucleotides of the 1st and 6th sequences) is complementary to position 535 in SEQ ID NO:1 and contains a guanine nucleobase.

65. The antisense oligonucleotide according to any one of items 1 to 46, wherein one nucleotide of region G is complementary to position 535 in SEQ ID NO:1 and contains a guanine nucleobase.

66. The antisense oligonucleotide according to any one of items 1 to 46, wherein one nucleotide of region F is complementary to position 535 in SEQ ID NO:1 and contains a guanine nucleobase.

67. The antisense oligonucleotide according to any one of items 1 to 46, wherein one nucleotide in the F' region is complementary to position 535 in SEQ ID NO:1 and contains a guanine nucleobase.

68. A conjugate comprising an antisense oligonucleotide according to any one of the preceding items and at least one conjugate moiety covalently linked to the oligonucleotide.

69. The conjugate compound according to item 68, wherein the conjugate portion is selected from carbohydrates, cell surface receptor ligands, active pharmaceutical ingredients, hormones, lipophilic substances, polymers, proteins, peptides, toxins, vitamins, viral proteins, or combinations thereof.

70. The conjugate according to item 69, wherein the conjugate portion facilitates delivery across the blood-brain barrier.

71. The conjugate according to item 70, wherein the conjugate portion is an antibody or antibody fragment targeting the transferrin receptor.

72. The conjugate according to any one of items 68 to 70, comprising a linker located between the antisense oligonucleotide and the conjugate moiety.

73. The conjugate according to item 72, wherein the conjugate is a physiologically unstable conjugate.

74. A pharmaceutical salt of an oligonucleotide or a conjugate according to any one of items 1 to 67 or any one of items 68 to 73.

75. A pharmaceutical composition comprising an antisense oligonucleotide according to any one of items 1 to 67 and/or a conjugate according to any one of items 68 to 73 and/or a pharmaceutical salt according to item 74; and a pharmaceutical diluent, solvent, carrier, salt and/or adjuvant.

76. A method for manufacturing an antisense oligonucleotide according to any one of items 1 to 67, the method comprising: reacting nucleotide units to form covalently linked continuous nucleotide units contained in the oligonucleotide.

77. The method according to item 76 further comprises: reacting a continuous nucleotide sequence with a non-nucleotide conjugated portion.

78. A method for manufacturing a pharmaceutical composition according to item 75, the method comprising: mixing the oligonucleotide with a pharmaceutical diluent, a carrier, a salt and/or an adjuvant.

79. A method for regulating ApoE4 expression in target cells expressing ApoE4, the method comprising: administering to the cells a therapeutically effective amount of an antisense oligonucleotide according to any one of items 1 to 67, a conjugate according to any one of items 68 to 73, or a pharmaceutical composition according to item 74.

80. The method described in Item 79 is an in vivo method or an in vitro method.

81. A method for treating or preventing a disease, the method comprising: administering to a subject who has or is at risk of having the disease a therapeutically or preventively effective amount of an antisense nucleotide according to any one of items 1 to 67, a conjugate according to any one of items 68 to 73, a pharmaceutical salt according to item 74, or a pharmaceutical composition according to item 75.

82. An antisense oligonucleotide according to any one of items 1 to 67, a conjugate according to any one of items 68 to 73, a pharmaceutical salt according to item 74, or a pharmaceutical composition according to item 75, for use as a medicament.

83. An antisense oligonucleotide according to any one of items 1 to 67, a conjugate according to any one of items 68 to 73, a pharmaceutical salt according to item 74, or a pharmaceutical composition according to item 75, for use in a method of treating or preventing a disease.

84. Use of any antisense oligonucleotide according to any one of items 1 to 67, any conjugate according to any one of items 68 to 73, any pharmaceutical salt according to item 74, or any pharmaceutical composition according to item 75 in the preparation of a medicament for the treatment or prevention of a disease.

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

86. The method, antisense oligonucleotide, conjugate, salt, or pharmaceutical composition for use, or application according to item 85, wherein the in vivo activity of ApoE4 is reduced by at least 30%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, compared with a control, optionally wherein the control is the in vivo activity of ApoE4 prior to administration of the antisense oligonucleotide, conjugate, or pharmaceutical composition.

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

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

89. The method, antisense oligonucleotide, conjugate, salt, or pharmaceutical composition, or use according to any one of items 79 to 88, wherein the subject who has or is at risk of having the disease carries at least one copy of the APOEε4 gene in his/her genome.

90. The method, antisense oligonucleotide, conjugate, salt, or pharmaceutical composition, or use according to item 89, wherein the subject who has or is at risk of having the disease has the APOE ε3/ε4 genotype.

91. The method, antisense oligonucleotide, conjugate, salt, or pharmaceutical composition, or use according to item 89, wherein the subject who has or is at risk of having the disease has the APOE ε4/ε4 genotype.

92. The method, antisense oligonucleotide, conjugate, salt or pharmaceutical composition, or use according to any one of items 79 to 91, wherein the disease is a disease related to dementia.

93. The method, antisense oligonucleotide, conjugate, salt, or pharmaceutical composition, or use according to any one of items 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), Lewy body dementia, Down syndrome dementia, and Niemann-Pick disease type C1.

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

95. The method, antisense oligonucleotide, conjugate, salt or pharmaceutical composition, or use according to any one of items 79 to 94, wherein the subject who has or is at risk of having the disease is a mammal, such as a human subject.

Example

Materials and methods

Oligonucleotide motif sequences and oligonucleotide compounds

Table 5: Compound Table

A list of oligonucleotide motif sequences (represented by SEQ ID NO), their design, and specific oligonucleotide compounds designed based on the motif sequences (represented by CMP ID NO).

Motif sequences represent a continuous sequence of nucleobases present in an oligonucleotide.

The design refers to a notched polymer design, F-G-F', which includes alternating flanks as described elsewhere in this document.

In oligonucleotide compounds, uppercase letters represent β-D-oxyLNA nucleosides, lowercase letters represent DNA nucleosides, uppercase C represents 5-methylcytosine β-D-oxyLNA nucleosides, and all internucleotide bonds are phosphate thioester internucleotide bonds. See Table 4 for descriptions of these compounds in HELM_annotations.

Oligonucleotide synthesis

Oligonucleotide synthesis is well known in the art. The following are feasible methods. The oligonucleotide compounds described herein can already be produced by slightly modified methods, in terms of equipment, carriers, and concentrations used.

Oligonucleotides were synthesized on a MermMade 192 oligonucleotide synthesizer using the phosphoramide method on a Unilinker universal solid support (Org. Process Res. Dev. 2008, 12, 3, 399-410) at a scale of 1 μmol. At the end of the synthesis, the oligonucleotides were cleaved from the solid support using ammonia at 60 °C for 5–16 hours. The oligonucleotides were purified by reversed-phase HPLC (RP-HPLC) or solid-phase extraction, characterized by UPLC, and their molecular weight was further confirmed by ESI-MS.

Oligonucleotide extension:

The coupling of 5'-DMTr-protected nucleoside β-cyanoethyl phosphorimides (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)) was carried out using a 0.1 M solution of 5'-O-DMT-protected amidite in acetonitrile and DCI (4,5-dicyanimidazolium) in acetonitrile (0.25 M) as an activator.

Purified by RP-HPLC:

The crude compound was purified by preparative RP-HPLC on a Phenomenex Jupiter C18 10μm 150x10mm column. 0.1M ammonium acetate (pH 8) and acetonitrile were used as buffer at a flow rate of 5 mL/min. The collected fractions were lyophilized to give the purified compound, typically as a white solid.

abbreviation:

DCI: 4,5-Dicyanoimidazole

DCM: Dichloromethane

DMF: Dimethylformamide

DMT: 4,4'-Dimethoxytriphenylmethyl

THF: Tetrahydrofuran

Bz: Benzoyl group

Ibu: Isobutyryl

RP-HPLC: Reversed-phase high-performance liquid chromatography

Example 1: Screening of fifteen oligonucleotides based on their effects on APOE3 and APOE4 expression levels

One day prior to treatment, human KELLY neuroblastoma cells (ACC 355, DSMZ) were seeded in 96-well plates at 30,000 cells per well in 190 μL of standard cell culture medium (RPMI-1640 Sigma R2405, 10% FBS, 25 μg/ml penicillin-streptomycin). KELLY cells were selected based on their heterozygous APOE3 and APOE4 genotypes (Schaffer et al., Genes Nutr. Jan 2014; 9(1)). On the day of treatment, oligonucleotides diluted in PBS (Gibco#14190-094) were added to obtain naked uptake at final concentrations of 5 μM and 25 μM in the culture medium, with a total volume of 200 μL per well.

Cells were cultured for 5 days in an active evaporation incubator at 37°C, 5% _CO2 , and 98% humidity. On day 5, the cell culture medium was removed from the wells by pipetting, and RNA was extracted by adding 125 μL of RLT buffer (Qiagen) and using the RNeasy 96 kit and protocol from Qiagen. cDNA synthesis was performed using 4 μL of input RNA and 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 as a probe (dUTP-free) (Bio-Rad), according to the manufacturer's protocol. The following assays were performed:

APOE3/4: SNP genotyping detection, rs429358 (test ID C__3084793_20, Thermo Fischer)

HPRT1: HPRT1 (ddPCR GEX CY5.5 assay 12005587, from Biorad)

The concentrations of APOE3 and APOE4 mRNA relative to the housekeeping gene HPRT1 were quantified using QuantaSoft software (Bio-Rad).

The expression levels were then normalized to the mean of the untreated PBS controls for APOE3 and APOE4, respectively. The results are shown in Table 6, which also includes the ratio of APOE3 to APOE4 (a high ratio indicates APOE4 selectivity).

Table 6: Data Table

The residual expression levels of APOE3 and APOE4 and the ratio of APOE3 to APOE4 are shown separately (a high ratio indicates APOE4 selectivity).

Various modifications and variations of the described aspects of 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 conjunction with specific preferred embodiments, the claimed invention should not be unduly limited to these specific embodiments. Indeed, it will be apparent to those skilled in the art that various modifications to the manner in which the invention is practiced are intended to fall within the scope of the following embodiments.

Claims (18)

1. An antisense oligonucleotide of 8 to 50 nucleotides in length, such as an antisense oligonucleotide of 10 to 30 nucleotides in length, said antisense oligonucleotide comprising a continuous nucleotide sequence of at least 10 nucleotides in length, said continuous nucleotide sequence being at least 80% complementary to a target sequence within positions 516 to 556 of the nucleic acid encoded by apolipoprotein (Apo) E4 shown in SEQ ID NO:1, said target sequence comprising position 535 of SEQ ID NO:1. 2. The antisense oligonucleotide of claim 1, wherein the continuous nucleotide sequence has zero to three mismatches compared to the target sequence, optionally selected from one mismatch, two mismatches, and three mismatches, provided that the nucleotide complementary to the nucleotide at position 535 of SEQ ID NO:1 in the continuous nucleotide sequence contains a guanine (g) nucleobase. 3. The antisense oligonucleotide according to any one of the preceding claims, wherein the continuous nucleotide sequence is at least 90% complementary to the target sequence at positions 516 to 556 of SEQ ID NO:1. 4. The antisense oligonucleotide according to any one of the preceding claims, wherein the continuous nucleotide sequence is 100% complementary to the target sequence at positions 516 to 556 of SEQ ID NO:1. 5. The antisense oligonucleotide according to any one of the preceding claims, wherein the continuous nucleotide sequence is complementary to the target sequence at positions 522 to 548 of SEQ ID NO:1. 6. The antisense oligonucleotide according to any one of the preceding claims, wherein the continuous nucleotide sequence is complementary to the target sequence selected from residues 522 to 535 (R_25), residues 525 to 537 (R_40), residues 526 to 538 (R_46), residues 530 to 546 (R_66) and residues 535 to 548 (R_91) of SEQ ID NO:1. 7. The antisense oligonucleotide according to any one of the preceding claims, wherein the sequential nucleotide sequence comprises or consists of a nucleotide sequence selected from or composed of the group consisting of SEQ ID NO:4 to SEQ ID NO:96. 8. The antisense oligonucleotide according to any one of the preceding claims, wherein the continuous nucleotide sequence comprises or consists of a nucleotide sequence selected from or composed of the group consisting of SEQ ID NO:25, SEQ ID NO:40, SEQ ID NO:46, SEQ ID NO:66 and SEQ ID NO:91. 9. The antisense oligonucleotide according to any one of the preceding claims, wherein the antisense oligonucleotide is capable of reducing the expression of mammalian apolipoprotein (Apo) E4, such as human apolipoprotein (Apo) E4. 10. The antisense oligonucleotide according to any one of the preceding claims, wherein the antisense oligonucleotide is or comprises a compound selected from the group consisting of: The uppercase letters represent β-D-oxyLNA nucleosides, the lowercase letters represent DNA nucleosides, the uppercase C represents 5-methylcytosine β-D-oxyLNA nucleosides, and all nucleoside bonds are thiophosphate nucleoside bonds. 11. A conjugate comprising an antisense oligonucleotide according to any one of the preceding claims and at least one conjugate moiety covalently linked to the oligonucleotide. 12. A pharmaceutical salt of an antisense oligonucleotide according to any one of claims 1 to 10 or a conjugate according to claim 11. 13. A pharmaceutical composition comprising an antisense oligonucleotide according to any one of claims 1 to 10 and/or a conjugate according to claim 11 and/or a pharmaceutical salt according to claim 12; and a pharmaceutical diluent, solvent, carrier, salt and/or adjuvant. 14. The antisense oligonucleotide according to any one of claims 1 to 10, the conjugate according to claim 11, the pharmaceutical salt according to claim 12, or the pharmaceutical composition according to claim 13, for use in methods of treating or preventing disease. 15. The antisense oligonucleotide, conjugate, salt, or pharmaceutical composition for use according to claim 14, wherein the disease is associated with the in vivo activity of ApoE4. 16. The antisense oligonucleotide, conjugate, salt, or pharmaceutical composition for use according to claim 14 or 15, wherein the disease is a disease associated with dementia. 17. The antisense oligonucleotide, conjugate, salt, or pharmaceutical composition for use according to any one of claims 14 to 16, 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), Lewy body dementia, Down syndrome dementia, and Niemann-Pick disease type C1. 18. The antisense oligonucleotide, conjugate, or pharmaceutical composition for use according to any one of claims 14 to 17, wherein the disease is AD.