Novel APOE antisense oligonucleotide and use thereof

Abstract

The present invention relates to: an apolipoprotein E (APOE) antisense oligonucleotide; and a pharmaceutical composition for treating Alzheimer's disease comprising same. The antisense oligonucleotide of the present invention can reduce the expression of the APOE4 variant gene that contributes to increased risk and exacerbation of Alzheimer's disease, and can be used as an RNA therapeutic agent for diseases caused by abnormal levels of APOE proteins or APOE variant genome expression.

Description

Novel APOE antisense oligonucleotides and uses thereof

[0001] The present invention relates to an APOE (Apolipoprotein E) antisense oligonucleotide and a pharmaceutical composition comprising the same for preventing or treating Alzheimer's disease.

[0002] Therapeutics that inhibit RNA expression are broadly divided into antisense oligonucleotides (ASOs) and RNA interference (RNAi). Among these, antisense oligonucleotides are single-stranded nucleic acid polymers that possess specific sequence complementarity to target RNA and can regulate gene expression or influence mRNA splicing. When an ASO binds to a target site in pre-mRNA with a complementary sequence, an RNA-DNA heteroduplex is formed, activating ribonuclease H (RNase H), which hydrolyzes and cleaves the RNA strand, thereby inhibiting mRNA degradation and target protein expression. ASOs regulate gene and protein expression through various mechanisms, including not only mediating RNase H but also regulating splicing through the steric block of ribosomal subunits and targeting intron regions to interfere with RNA-binding protein interactions, thereby affecting protein translation (Annu. Rev. Med. 2019. 70: 307-21). Currently, gapmer ASOs are common and mediate RNase H activity, and have the advantage of being active in both the nucleus and the cytoplasm.

[0003] Since the FDA first approved the first antisense drug, VITRAVENE, in 1998 for the treatment of retinitis pigmentosa due to cytomegalovirus (CMV) infection in immunocompromised patients with HIV, and most recently in 2023, it granted accelerated approval to Biogen's QALSODY for adults with familial amyotrophic lateral sclerosis (ALS) who have a SOD1 mutation, indicating that antisense oligonucleotides have the potential to treat disease.

[0004] Meanwhile, apolipoprotein E (APOE) is one of the components of lipoprotein involved in cholesterol metabolism and lipid transport, and the APOE gene is composed of three common alleles, E2, E3, and E4. Among them, APOE3 is the normal protein, and the mutant type, APOE4, is known to be the strongest genetic risk factor associated with the development of late-onset or senile Alzheimer's disease that develops in people over 65 years of age (Neuron63(3), 287-303, 2009). Despite the recognition of this risk, the development of drugs targeting lipid metabolism and APOE4 is still insufficient, even in the clinical and preclinical stages. As a strategy to block the negative effects of the APOE4 gene, the development of a therapeutic agent that delivers the APOE2 gene, which has a protective effect on the development of Alzheimer's disease, using recombinant viral vector technology is currently underway. However, no treatment to reduce the risk gene APOE4 is currently in clinical trials, and few studies have confirmed the therapeutic effects of antisense compounds that suppress or inhibit APOE expression in human cells for Alzheimer's disease. Therefore, new treatment options that can effectively reduce APOE4 expression are urgently needed.

[0005] Against this backdrop, the present inventors studied an antisense oligonucleotide compound that targets the APOE transcript and induces a decrease in APOE protein expression, and as a result, confirmed that the expression level of APOE can be reduced in neurons, astrocytes, and brain organoids derived from APOE-expressing cell lines and human induced pluripotent stem cells expressing APOE4 homozygotes. In addition, it was confirmed that the amount of phosphorylated tau, known as a causative agent of Alzheimer's disease, was reduced by an antisense oligonucleotide targeting APOE in brain organoids generated from human induced pluripotent stem cells homozygous for APOE4, a causative gene that significantly increases the incidence of Alzheimer's disease. Therefore, it was suggested that the compound may be beneficial for diseases related to abnormal APOE expression and activity and Alzheimer's disease patients homozygous for APOE4.

[0006] Accordingly, one object of the present invention is to provide an antisense compound comprising a modified oligonucleotide consisting of 10 to 30 consecutively linked nucleotides that complementarily binds to a nucleic acid base sequence within a transcript of a gene encoding human Apolipoprotein E (APOE).

[0007] Another object of the present invention is to provide an antisense oligonucleotide having any one of SEQ ID NOs: 1 to 6, wherein at least one nucleoside among the nucleosides included in the antisense oligonucleotide is a 2'-O-methoxyethyl (2'-MOE) modified nucleoside, and all internucleoside linkages are phosphorothioate linkages.

[0008] Another object of the present invention is to provide a pharmaceutical composition for preventing or treating Alzheimer's disease comprising the antisense oligonucleotide.

[0009] Specifically, the following descriptions are provided. Meanwhile, each description and embodiment disclosed in the present invention can also be applied to each other description and embodiment. In other words, all combinations of the various elements disclosed in the present invention fall within the scope of the present invention. Furthermore, the scope of the present invention should not be considered limited by the specific descriptions described below.

[0010] One aspect of the present invention relates to an antisense oligonucleotide comprising at least one oligonucleotide selected from the group consisting of: an oligonucleotide of SEQ ID NO: 1 having 5'-GCATCCTGTGTGGAA-3'; an oligonucleotide of SEQ ID NO: 2 having 5'-GGACACTCACCTCAG-3'; an oligonucleotide of SEQ ID NO: 3 having 5'-GCCTGGCATCCTGTG-3'; an oligonucleotide of SEQ ID NO: 4 having 5'-CTCACCTCAGTTCCT-3'; an oligonucleotide of SEQ ID NO: 5 having 5'-CGCAGCCCACAGAACCTTCA-3'; and an oligonucleotide of SEQ ID NO: 6 having 5'-TGCAGGTCATCGGCATCGCG-3'.

[0011] The "oligonucleotide" of the present invention generally refers to a molecule comprising two or more covalently linked nucleosides, as defined by those skilled in the art. These covalently linked nucleosides may be referred to as nucleic acid molecules or oligomers. The oligonucleotide of the present invention may be chemically synthesized and may include one or more modified nucleosides or nucleotides.

[0012] The "antisense oligonucleotide" of the present invention may be defined as an oligonucleotide capable of regulating the expression of a target gene by hybridizing to a target nucleic acid, but is not limited thereto. The antisense oligonucleotide may be used interchangeably with an "antisense oligomer compound" or "ASO."

[0013] The antisense oligonucleotide of the present invention can complementarily bind to a nucleic acid base sequence within a transcript of a gene encoding human Apolipoprotein E (APOE), and can include a modified oligonucleotide composed of 10 to 30 consecutively linked nucleotides. In addition, the antisense oligonucleotide of the present invention can bind to an intron-exon or exon-intron boundary region of exon 3 of human APOE pre-mRNA.

[0014] The antisense oligonucleotides of the present invention can reduce APOE transcript expression and APOE protein expression in human cells expressing APOE (e.g., U373, THP-1 cells) and neurons, astrocytes or brain organoids derived from human induced pluripotent stem cells (iPSCs).

[0015] The antisense oligonucleotides of the present invention may comprise one or more modified nucleosides. The term "modified nucleoside" as used herein refers to a nucleoside that has been modified by the introduction of one or more modifications, compared to an equivalent DNA or RNA nucleoside. Specifically, the modified nucleosides of the present invention may comprise a modified sugar moiety and may be used interchangeably with "nucleoside analogs."

[0016] The above modified nucleoside may be, but is not limited to, 2'-O-alkyl-RNA, 2'-O-methyl RNA (2'-OMe), 2'-alkoxy-RNA, 2'-O-methoxyethyl-RNA (2'-MOE), 2'-amino-DNA, 2'-fluoro-RNA, 2'-fluoro-DNA, arabino nucleic acid (ANA), 2'-fluoro-ANA, a bicyclic nucleoside analogue (LNA, locked nucleic acid). In addition, the modified form may be, but is not limited to, a form in which a phosphorothioate bond is introduced, a phosphorodiamidate morpholino oligonucleotide (PMO), a peptide nucleic acid (PNA), a locked nucleic acid (LNA), and a form modified at the 2' position of a ribose sugar.

[0017] All nucleosides included in the antisense oligonucleotide of the present invention are 2'-O-methoxyethyl (2'-MOE) modified nucleosides, and all internucleoside linkages may be phosphorothioate linkages.

[0018] In addition, the antisense oligonucleotide of the present invention may be in the form of a gapmer. The gapmer has the design of NNNNNDDDDDDDDDDNNNNN, where N is a nucleoside variant and D is in the form of DNA, but the number of sequences may be modified. At this time, the nucleoside variant included in the antisense oligonucleotide may be selected from 2'-O-alkyl-RNA, 2'-O-methyl RNA (2'-OMe), 2'-alkoxy-RNA, 2'-O-methoxyethyl-RNA (2'-MOE), 2'-amino-DNA, 2'-fluoro-RNA, 2'-fluoro-DNA, arabino nucleic acid (ANA), 2'-fluoro-ANA, and a bicyclic nucleoside analog (LNA, locked nucleic acid). The antisense oligonucleotide of the present invention may be designed to degrade the target mRNA by RNase H, and the antisense oligonucleotide may be in the form of a gapmer, but is not limited thereto.

[0019] At least one nucleoside among the nucleosides included in the antisense oligonucleotide of the present invention may be a 2'-O-methoxyethyl (2'-MOE) modified nucleoside, and all nucleoside linkages may be phosphorothioate linkages. In addition, at least one nucleoside at the 5'-terminus or at least one nucleoside at the 3'-terminus of the antisense oligonucleotide may be a modified nucleoside, and specifically, the first to n-th nucleosides at the 5'-terminus or the first to m-th nucleosides at the 3'-terminus of the antisense oligonucleotide may be a modified nucleoside. Here, n and m are each independently any integer selected from 1 to 10.

[0020] The antisense oligonucleotide of the present invention can target various regions within the pre-mRNA and mRNA of human Apolipoprotein E (APOE). Specifically, the antisense oligonucleotide of the present invention can have a sequence structure complementary to a sequence included in the human APOE gene, NCBI Reference Sequence: NC_000019.10 (SEQ ID NO: 7). The antisense oligonucleotide can target an intron-exon region (or an exon-intron region) where splicing occurs to reduce target gene and protein expression.

[0021] APOE (Apolipoprotein E) of the present invention is a fat-binding protein involved in fat metabolism in the body, and is known to be expressed in various organs, but especially in the liver and brain. APOE4, a mutation type of the APOE gene, is known to be the strongest genetic risk factor related to the development of late-stage Alzheimer's disease. In this regard, the antisense oligonucleotide of the present invention was confirmed to suppress the expression of the APOE4 mutation gene, suggesting that it can be used to treat diseases caused by the expression of the APOE mutation gene. In addition, a study was recently reported that cognitive function and brain function were affected when APOE4 was modulated between peripheral tissues rather than within the brain (Nat Neurosci. 2022 Aug;25(8):1020-1033). Therefore, the antisense oligonucleotide of the present invention has the advantage of being able to be delivered not only into the brain but also through peripheral tissues.

[0022] Another aspect of the present invention relates to a pharmaceutical composition for preventing or treating a disease caused by expression of an APOE mutant genome, comprising an antisense oligonucleotide.

[0023] Diseases caused by the expression of the above APOE mutant gene specifically include Alzheimer's disease, coronary artery disease such as atherosclerosis, or hypercholesterolemia, and are preferably Alzheimer's disease, but are not limited thereto.

[0024] The term "prevention" in the present invention refers to any action that suppresses or delays a disease by administering or treating a composition comprising the antisense oligonucleotide of the present invention as an active ingredient. The term "treatment" in the present invention refers to any action that improves or beneficially alters the symptoms of a disease by administering or treating a composition comprising the antisense oligonucleotide of the present invention as an active ingredient.

[0025] In one embodiment of the present invention, when the antisense oligonucleotide of the present invention was treated on a human induced pluripotent stem cell-derived brain organoid, it was confirmed that not only the protein expression of APOE but also the expression of phosphorylated tau, one of the causative proteins of Alzheimer's disease, was reduced, suggesting that the antisense oligonucleotide of the present invention can exhibit Alzheimer's disease therapeutic activity without using a separate carrier.

[0026] The pharmaceutical composition of the present invention may further comprise, in addition to the active ingredient, a pharmaceutically acceptable carrier, excipient or diluent commonly used in the manufacture of pharmaceutical compositions. Examples of the carrier, excipient or diluent include, but are not limited to, water, saline, ethanol, polyol, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinyl pyrrolidone, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil and suitable mixtures thereof. The pharmaceutical composition of the present invention may be in the form of a solution, gel, suspension, emulsion, cream, ointment, suppository, patch, pad, or spray, but is not limited thereto. When formulated as described above, the composition may be prepared using diluents or excipients, such as fillers, bulking agents, binders, wetting agents, disintegrants, and surfactants, commonly used in the art.

[0027] The present invention relates to an antisense oligonucleotide that regulates APOE expression. It suppresses the expression of the APOE4 variant gene, a risk gene that influences the onset and worsening of Alzheimer's disease, and thus can be used to treat other diseases, including Alzheimer's disease, caused by abnormal levels of APOE protein or expression of APOE variant genes. Furthermore, the antisense oligonucleotide of the present invention is expected to have therapeutic effects not only through intracerebral delivery but also through peripheral delivery.

[0028] Figure 1 is a schematic diagram of the human APOE gene and the antisense oligonucleotide (ASO) of the present invention targeting it.

[0029] Figure 2 shows data confirming a decrease in APOE protein expression through ASO in U373 cells.

[0030] Figure 3 shows data confirming a decrease in APOE mRNA expression through ASO in THP-1 cells.

[0031] Figures 4a to c are data confirming a decrease in APOE protein expression through ASO in THP-1 cells.

[0032] Figure 5 shows data confirming a decrease in APOE mRNA expression through ASO without the use of a transporter in THP-1 cells.

[0033] Figure 6 shows data confirming a decrease in phosphorylated tau protein along with a decrease in APOE mRNA and protein expression through ASO in human induced pluripotent stem cell (iPSC)-derived neural cells.

[0034] Figure 7 is data confirming a decrease in APOE protein expression and a decrease in the amount of APOE protein secreted out of cells through ASO in human induced pluripotent stem cell-derived astrocytes.

[0035] Figure 8 shows data confirming a decrease in APOE protein expression and phosphorylated tau protein through ASO in human induced pluripotent stem cell-derived brain organoids.

[0036] Hereinafter, the present invention will be described in detail based on examples. However, the following examples are intended only to illustrate the present invention and are not intended to limit the spirit or scope of the present invention.

[0037] Experimental Example 1. Building ASO

[0038] The antisense oligonucleotides (SEQ ID NOs: 1 to 4) described in the present invention are designed to target various regions within human APOE pre-mRNA and mRNA. Specifically, they target intron-exon regions (or exon-intron regions) where splicing occurs to reduce target gene and protein expression, thereby altering splicing outcomes by masking splicing signals and interfering with spliceosome recognition.

[0039] The ASO of SEQ ID NO: 1 was designed to target the 3' splice site of Exon3 (intron 10 mer + exon 5 mer), the ASO of SEQ ID NO: 2 was designed to target the 5' splice site of Exon3 (intron 10 mer + exon 5 mer), the ASO of SEQ ID NO: 3 was designed to target the 3' splice site of Exon3 (intron 5 mer + exon 10 mer), and the ASO of SEQ ID NO: 4 was designed to target the 5' splice site of Exon3 (intron 5 mer + exon 10 mer). SEQ ID NOs: 5 and 6 are Gapmer type ASOs that degrade target mRNA by RNase H. Therefore, the ASO of SEQ ID NO: 5 was designed to have a target sequence in Exon2 (5-10-5 type), and the ASO of SEQ ID NO: 6 was designed to have a target sequence in Exon4 (5-10-5 type). The above designed ASO was synthesized by Integrated DNA technologies.

[0040]

[0041] More specifically, the ASOs of SEQ ID NOs: 1 to 4 have all nucleosides modified with 2'-O-methoxyethyl-RNA (2'-MOE), and all nucleosides are linked by phosphorothioate bonds. In addition, the gapmer ASOs of SEQ ID NOs: 5 and 6 have five nucleosides at both the 5' and 3' ends modified with 2'-O-methoxyethyl-RNA (2'-MOE), and all nucleosides are linked by phosphorothioate bonds.

[0042] Experimental Example 2: Cell Culture and ASO Treatment

[0043] Human monocytic THP-1 cells and human astrocytoma U373 cells were cultured in DMEM medium containing 10% FBS and 0.1 mg / ml penicillin and streptomycin (P / S) at 37°C in a humidified 5% CO2 atmosphere. For the experiment, 200,000 cells per well of a 12-well plate were cultured in 1 ml of medium. After approximately 24 hours of incubation, the cells were mixed with lipofecatmine 2000 in opti-mem medium to a final concentration of ASO and transfected for 24 hours.

[0044] To differentiate neural progenitor cells (NPCs) derived from human induced pluripotent stem cells (iPSCs) into neurons, they were cultured in a Matrigel-coated environment in Neurobasal-A medium containing B-27 supplement and penicillin and streptomycin (P / S), with the growth factors BDNF and NT3. After 14 days of culture to generate mature neurons, ASO was treated for 3 days.

[0045] To differentiate neural progenitor cells derived from human induced pluripotent stem cells into astrocytes, Y-27632 was added to the culture medium consisting of DMEM / F12, GlutaMAX (1:100), MEM-NEAA (Non-Essential Amino Acids) (1:100), B27 minus Vitamin A (1:50), N2 Supplement (1:100), recombinant human EGF (10 ng / mL), and recombinant human basic FGF (10 ng / mL) and cultured. After 24 hours, the culture medium was replaced with astrocyte culture medium, and this day was set as Day 0. The astrocyte culture medium is a Sciencell Cat#1801 product, consisting of AM Basal Medium + P / S (1:100) + FBS (1:50). Thereafter, all culture medium was removed every 2-3 days and replaced with fresh culture medium. 15,000 cells / cm in astrocyte culture medium using Accutase solution every 5-6 days 2 Subculture was performed at a cell density of 100 μl. On day 30, cells that had fully matured into astrocytes were collected and used for experiments. ASO was mixed with lipofectamine 2000 and transfected into astrocytes in opti-mem medium for 24 hours.

[0046] Experimental Example 3. Production of Human Brain Organoids

[0047] To generate brain organoids, human induced pluripotent stem cells homozygous for APOE4 were cultured in AggreWell 800 24-well plates in EB (Embryo body) formation medium containing ROCK inhibitor Y-27632 on Day 0. The following day, the EB formation medium was replaced with Y-27632-free EB formation medium, and from Day 2, the medium was replaced daily for 3 days with DMEM / F-12 (containing GlutaMAX) supplemented with 20% KnockOut Serum Replacement (20%), 1% MEM Non-Essential Amino Acids Solution, 0.1 mM 2-mercaptoethanol, antibiotic P / S (100 U / ml), and SMAD inhibitors 10 μM dorsomorphin and 10 μM SB-431542 to induce neural induction. On day 6, individual embryo-like bodies were transferred one by one to ultra-low attachment 96-well plates and cultured in Neurobasal-A neural media containing B-27 supplement, P / S, GlutaMAX, and 0.5% (v / v) Matrigel Basement Membrane Matrix, supplemented with EGF (20 ng / ml) and bFGF (20 ng / ml), and replaced daily until day 24 to generate organoids. From day 25 to day 42, the culture was replaced every other day with neural media containing BDNF and NT-3 (each 20 ng / ml), and thereafter, the culture was maintained by replacing the medium with basic neural media without Matrigel and growth factors once every four days. For ASO treatment, organoids cultured until day 86 were used.

[0048] Experimental Example 4. RNA Extraction and Quantitative Real-Time Reverse Transcription PCR

[0049] After incubation, the medium was removed, washed with PBS, and RNA was extracted using an RNA extraction kit (RNeasy Mini Kit, Qiagen) according to the manufacturer's instructions. The extracted RNA was quantified and equal amounts of RNA were extracted using MaximeTM cDNA was synthesized using RT PreMix (iNtRON Biotechnology), and quantitative real-time reverse transcription PCR was performed using the KAPA SYBR Fast qPCR kit. The obtained results were calculated as a percentage by comparing them with the negative control group.

[0050] Experimental Example 5. Western Blot Analysis

[0051] The medium was removed, washed with PBS, and cells were harvested with RIPA buffer. After sonication, the cells were disrupted and centrifuged at 4°C and 13,000 rpm for 15 minutes to obtain the supernatant. 10 μg of protein was calculated by BCA quantitation and loaded onto a 4-12% Bis-Tris polyacrylamide precast gel (NuPAGE gel) for separation. Next, the proteins were transferred from the gel to a PVDF membrane, and then placed in a 5% skim milk solution and reacted at room temperature for 1 hour to prevent nonspecific protein binding. Primary antibodies (APOE, AT8, pT181, PSD-95, actin) were added and reacted at 4°C for more than 16 hours. After the primary antibody reaction, the cells were washed 5 times with 1x PBST and reacted with HRP-conjugated secondary antibodies at room temperature for 1 hour. The signal was amplified with ECL solution and analyzed by Amersham. TM The images were developed using the Imager600 device, and the obtained protein band images were analyzed and quantified using MultiGauge software.

[0052] To analyze APOE protein secreted outside the cells by Western blot, the conditioned medium of astrocytes was mixed with methanol / chloroform to precipitate the proteins present in the medium and used as a sample.

[0053] Example 1. Confirmation of decreased APOE protein expression following ASO treatment in U373 cells.

[0054] When human astrocytoma cells, U373 cells, were transfected with ASOs of sequence numbers 1 to 4 at a concentration of 50 nM or 100 nM using lipofectamine 2000 for 24 hours, it was confirmed that the expression of APOE protein was reduced compared to untreated cells (see Fig. 2).

[0055] Example 2. Confirmation of decreased APOE mRNA expression following ASO treatment in THP-1 cells.

[0056] After transfecting THP-1 cells, which are human monocyte cells, with gapmer ASOs of sequence numbers 5 and 6 at concentrations of 10 nM, 25 nM, and 50 nM using lipofectmaine 2000 for 24 hours, APOE mRNA expression was analyzed. It was confirmed that APOE mRNA expression was significantly reduced by treatment with gapmer ASOs of sequence numbers 5 or 6 (see Fig. 3).

[0057] Example 3. Confirmation of decreased APOE protein expression following ASO treatment in THP-1 cells.

[0058] When human monocyte THP-1 cells were treated with gapmer ASOs of sequence numbers 5 and 6 at concentrations of 10 nM, 25 nM, and 50 nM, and then APOE protein expression was analyzed, it was confirmed that APOE protein expression decreased depending on the concentration of gapmer ASO treatment of sequence numbers 5 or 6 (see Figures 4a to c).

[0059] Example 4. Confirmation of decreased APOE mRNA expression by ASO treatment alone without the use of a delivery vehicle in THP-1 cells.

[0060] After treating human monocyte THP-1 cells with ASO (Gapmer #1) of sequence number 5 for 24 hours without the use of a carrier, we analyzed APOE mRNA expression. As a result, we confirmed that APOE mRNA expression decreased depending on the treatment concentration (see Fig. 5). This indicates that APOE ASO can enter cells and suppress gene expression without the aid of a carrier.

[0061] Example 5. Confirmation of decreased APOE mRNA and protein expression through ASO treatment in human induced pluripotent stem cell (iPSC)-derived neural cells.

[0062] When differentiated neural cells from human induced pluripotent stem cells homozygous for the APOE4 gene were treated with ASO (Gapmer #1) of sequence number 5 at a concentration of 5 μM for 3 days without using a carrier, it was confirmed that the amount of APOE mRNA and protein decreased (see Fig. 6). In addition, when APOE expression was reduced by ASO, the amount of phosphorylated tau protein, a major pathogenic factor of Alzheimer's disease, was observed to decrease when confirmed with AT8 antibody, and conversely, the amount of PSD-95, a synaptic protein, was confirmed to increase.

[0063] Example 6. Confirmation of decreased APOE protein expression through ASO treatment in human induced pluripotent stem cell (iPSC)-derived astrocytes.

[0064] Human induced pluripotent stem cell-differentiated APOE3 homozygous-expressing astrocytes and APOE4 homozygous-expressing astrocytes were transfected with 100 nM of ASO (Gapmer #1) of sequence number 5 mixed with lipofectamine 2000 for 24 hours. The changes in APOE expression in astrocytes and the amount of APOE secreted from astrocytes were confirmed by Western blotting. The results of Western blotting confirmed that the gapmer ASO of sequence number 5 decreased APOE protein expression in both astrocytes expressing APOE3 homozygous and APOE4 homozygous, respectively (see Fig. 7). In addition, the amount of APOE protein released into the cells was also measured by Western blotting, and the result confirmed that the gapmer ASO of sequence number 5 also decreased the amount of APOE protein released into the cells (see Fig. 7).

[0065] Example 7. Confirmation of decreased APOE protein expression and phosphorylated tau protein through ASO treatment in human induced pluripotent stem cell (iPSC)-derived brain organoids.

[0066] To confirm its potential as a treatment for Alzheimer's disease, human brain organoids were used. Brain organoids derived from human induced pluripotent stem cells homozygous for the APOE4 gene were treated with ASO (Gapmer #1) of sequence number 5 at a concentration of 10 μM twice every three days without a vehicle, and a decrease in APOE protein expression was confirmed (see Figure 8). Furthermore, under conditions where APOE expression was reduced, the amount of phosphorylated tau, one of the causative proteins of Alzheimer's disease, was confirmed to be reduced in the group treated with ASO (Gapmer #1) of sequence number 5 (see Figure 8).

[0067] While specific aspects of the present invention have been described in detail above, it will be apparent to those skilled in the art that these specific descriptions merely represent preferred embodiments and are not intended to limit the scope of the present invention. Therefore, the substantial scope of the present invention is defined by the appended claims and their equivalents.

[0068] SEQ ID NO: 1: APOE antisense oligonucleotide

[0069] gcatcctgtg tggaa

[0070] SEQ ID NO: 2: APOE antisense oligonucleotide

[0071] ggacactcac ctcag

[0072] SEQ ID NO: 3: APOE antisense oligonucleotide

[0073] gcctggcatc ctgtg

[0074] SEQ ID NO: 4: APOE antisense oligonucleotide

[0075] ctcacctcag ttcct

[0076] SEQ ID NO: 5: APOE antisense oligonucleotide

[0077] cgcagcccac agaaccttca

[0078] SEQ ID NO: 6: APOE antisense oligonucleotide

[0079] tgcaggtcat cggcatcgcg

[0080] Sequence number 7: Human APOE gene

[0081] ctactcagcc ccagcggagg tgaaggacgt ccttccccag gagccggtga gaagcgcagt

[0082] cgggggcacg gggatgagct caggggcctc tagaagagc tgggaccctg ggaacccctg

[0083] gcctccaggt agtctcagga gagctactcg gggtcgggct tggggagg aggagcgggg

[0084] gtgaggcaag cagcagggga ctggacctgg gaagggctgg gcagcagaga cgacccgacc

[0085] cgctagaagg tggggtgggg agagcagctg gactgggatg taagccatag caggactcca

[0086] cgagttgtca ctatcattta tcgagcacct actggtgtc cccagtgtcc tcagatctcc

[0087] aactgggg agccaggggc agcgacacgg tagctagccg tcgattggag aactttaaaa

[0088] tgaggactga attagctcat aaatggaaca cggcgcttaa ctgtgaggtt ggagcttaga

[0089] atgtgaaggg agaatgagga atgcgagact gggactgaga tggaaccggc ggtggggagg

[0090] gggtgggggg atggaatttg aaccccggga gaggagatg gattttcta tggaggccga

[0091] cctggggatg gggagatag agagaccag gagggagtta atagggaat gggttgggg

[0092] cggcttgta aatgtgctgg gattaggctg tgcagataa tgcacaagg cttggaggc

[0093] taacctgggg tgaggccggg ttggggccgg gctgggggtg ggaggagtcc tcactggcgg

[0094] ttgattgaca gtttctcctt ccccagactg gccaatcaca ggcaggaaga tgaaggttct

[0095] gtgggctgcg ttgctggtca cattcctggc aggtatgggg gcggggcttg ctcggttccc

[0096] cccgctcctc cccctctcat cctcacctca acctcctggc cccattcagg cagaccctgg

[0097] gccccctctt ctgaggcttc tgtgctgctt cctggctctg aacagcgatt tgacgctctc

[0098] tgggcctcgg tttcccccat ccttgagata ggagttagaa gttgttttgt tgttgttgtt

[0099] tgttgttgtt gttttgtttt tttgagatga agtctcgctc tgtcgcccag gctggagtgc

[0100] agtggcggga tctcggctca ctgcaagctc cgcctcccag gtccacgcca ttctcctgcc

[0101] tcagcctccc aagtagctgg gactacaggc acatgccacc acacccgact aacttttttg

[0102] tattttcagt agagacgggg tttcaccatg ttggccaggc tggtctggaa ctcctgacct

[0103] caggtgatct gcccgtttcg atctcccaaa gtgctgggat tacaggcgtg agccaccgca

[0104] cctggctggg agttagaggt ttctaatgca ttgcaggcag atagtgaata ccagacacgg

[0105] ggcagctgtg atctttattc tccatcaccc ccacacagcc ctgcctgggg cacacaagga

[0106] cactcaatac atgcttttcc gctgggcgcg gtggctcacc cctgtaatcc cagcactttg

[0107] ggaggccaag gtgggaggat cacttgagcc caggagttca acaccagcct gggcaacata

[0108] gtgagaccct gtctctacta aaaatacaaa aattagccag gcatggtgcc acacacctgt

[0109] gctctcagct actcaggagg ctgaggcagg aggatcgctt gagcccagaa ggtcaaggtt

[0110] gcagtgaacc atgttcaggc cgctgcactc cagcctgggt gacagagcaa gaccctgttt

[0111] ataaatacat aatgctttcc aagtgattaa accgactccc ccctcaccct gcccaccatg

[0112] gctccaaaga agcatttgtg gagcaccttc tgtgtgcccc taggtactag atgcctggac

[0113] ggggtcagaa ggaccctgac ccaccttgaa cttgttccac acaggatgcc aggccaaggt

[0114] ggagcaagcg gtggagacag agccggagcc cgagctgcgc cagcagaccg agtggcagag

[0115] cggccagcgc tgggaactgg cactgggtcg cttttgggat tacctgcgct gggtgcagac

[0116] actgtctgag caggtgcagg aggagctgct cagctcccag gtcacccagg aactgaggtg

[0117] agtgtcccca tcctggccct tgaccctcct ggtgggcggc tatacctccc caggtccagg

[0118] tttcattctg cccctgtcgc taagtcttgg ggggcctggg tctctgctgg ttctagcttc

[0119] ctcttcccat ttctgactcc tggctttagc tctctggaat tctctctctc agctttgtct

[0120] ctctctcttc ccttctgact cagtctctca cactcgtcct ggctctgtct ctgtccttcc

[0121] ctagctcttt tatatagaga cagagagatg gggtctcact gtgttgccca ggctggtctt

[0122] gaacttctgg gctcaagcga tcctcccgcc tcggcctccc aaagtgctgg gattagaggc

[0123] atgagccacc ttgcccggcc tcctagctcc ttcttcgtct ctgcctctgc cctctgcatc

[0124] tgctctctgc atctgtctct gtctccttct ctcggcctct gccccgttcc ttctctccct

[0125] cttgggtctc tctggctcat ccccatctcg cccgccccat cccagccctt ctccccgcct

[0126] cccactgtgc gacaccctcc cgccctctcg gccgcagggc gctgatggac gagaccatga

[0127] aggagttgaa ggcctacaaa tcggaactgg aggaacaact gaccccggtg gcggaggaga

[0128] cgcgggcacg gctgtccaag gagctgcagg cggcgcaggc ccggctgggc gcggacatgg

[0129] aggacgtgtg cggccgcctg gtgcagtacc gcggcgaggt gcaggccatg ctcggccaga

[0130] gcaccgagga gctgcgggtg cgcctcgcct cccacctgcg caagctgcgt aagcggctcc

[0131] tccgcgatgc cgatgacctg cagaagcgcc tggcagtgta ccaggccggg gcccgcgagg

[0132] gcgccgagcg cggcctcagc gccatccgcg agcgcctggg gcccctggtg gaacagggcc

[0133] gcgtgcgggc cgccactgtg ggctccctgg ccggccagcc gctacaggag cgggcccagg

[0134] cctggggcga gcggctgcgc gcgcggatgg aggagatggg cagccggacc cgcgaccgcc

[0135] tggacgaggt gaaggagcag gtggcggagg tgcgcgccaa gctggaggag caggcccagc

[0136] agatacgcct gcaggccgag gccttccagg cccgcctcaa gagctggttc gagcccctgg

[0137] tggaagacat gcagcgccag tgggccgggc tggtggagaa ggtgcaggct gccgtgggca

[0138] ccagcgccgc ccctgtgccc agcgacaatc actgaacgcc gaagcctgca gccatgcgac

[0139] cccacgccac cccgtgcctc ctgcctccgc gcagcctgca gcgggagacc ctgtccccgc

[0140] cccagccgtc ctcctggggt ggaccctagt ttaataaaga ttcaccaagt ttcacgca

Claims

1. An antisense oligonucleotide having any one of the sequence numbers 1 to 4, An antisense oligonucleotide wherein all nucleosides contained in the above antisense oligonucleotide are 2'-O-methoxyethyl (2'-MOE) modified nucleosides and all internucleoside linkages are phosphorothioate linkages.

2. An antisense oligonucleotide having sequence number 5 or 6, An antisense oligonucleotide, wherein at least one nucleoside among the nucleosides included in the above antisense oligonucleotide is a 2'-O-methoxyethyl (2'-MOE) modified nucleoside, and all internucleoside linkages are phosphorothioate linkages.

3. In paragraph 2, An antisense oligonucleotide, wherein at least one nucleoside at the 5'-terminus or at least one nucleoside at the 3'-terminus of the antisense oligonucleotide is a modified nucleoside.

4. In paragraph 3, An antisense oligonucleotide wherein the first to nth nucleosides from the 5'-end or the first to mth nucleosides from the 3'-end of the antisense oligonucleotide are modified nucleosides (wherein n and m are each independently an integer of 1 to 10).

5. In paragraph 3, An antisense oligonucleotide, wherein the first to fifth nucleosides from the 5'-terminus or the first to fifth nucleosides from the 3'-terminus of the antisense oligonucleotide are modified nucleosides.

6. In paragraph 1 or 2, The above antisense oligonucleotide is an antisense oligonucleotide that can reduce the expression of APOE (Apolipoprotein E).

7. A pharmaceutical composition for preventing or treating Alzheimer's disease, comprising the antisense oligonucleotide of claim 1 or 2.

Images