Angptl4 oligonucleotides affecting regulation of fatty acid metabolism

Abstract

To provide ANGPTL4 inhibitors.SOLUTION: ANGPTL4 inhibitors consisting of an oligonucleotide comprising 12-22 nucleotides, wherein at least one of the nucleotides is modified, wherein the oligonucleotide hybridizes to a nucleic sequence of ANGPTL4 (human) of a specific sequence, ANGPTL4 (human) of a specific sequence, ANGPTL4 (mouse) of a specific sequence and / or ANGPTL4 (mouse) of a specific sequence, and wherein the oligonucleotide inhibits the expression of ANGPTL4, and pharmaceutical compositions comprising such inhibitors and pharmaceutically acceptable carriers, excipients, diluents or combinations thereof are provided. ANGPTL4.SELECTED DRAWING: Figure 6-1

Description

[Technical Field]

[0001] The present invention relates to inhibitors of ANGPTL4, such as antisense oligonucleotides, and pharmaceutical compositions containing such inhibitors, and their use in the treatment of cardiovascular disease, obesity, type II diabetes, homozygous familial hypercholesterolemia (HoFH), heterozygous familial hypercholesterolemia (HeFH), or dyslipidemia.

[0002] Background of the Invention Disturbances in plasma lipids are well-known risk factors for cardiometabolic disease. Successful treatment of elevated LDL cholesterol began in the mid-1980s, and in subsequent decades, focus expanded to other lipid classes, such as HDL cholesterol and triglycerides. Epidemiological studies have demonstrated that elevated plasma triacylglycerol (TG) and associated remnant cholesterol are independent risk factors for coronary heart disease (Cullen, 2000). Furthermore, hypertriglyceridemia (HTG) is a hallmark of metabolic syndrome (MS) and is often associated with obesity and insulin resistance (Reaven, 1995). The increased risk of type 2 diabetes and cardiovascular disease (CVD) associated with metabolic syndrome and HTG suggests that maintaining plasma TG homeostasis is highly desirable.

[0003] Patients with severe hypertriglyceridemia may also develop pancreatitis (Athyros, 2002), especially if their triglyceride levels exceed 1000–1500 mg / dl (Tsuang, 2009). While 40 different genes are now known to regulate plasma triglycerides (Johansen, 2011), several monogenic disorders are known to significantly increase triglycerides (Nordestgaard and Varbo, 2014). These include FCS, which is discussed in more detail below.

[0004] Lipolysis is a key step in the clearance of triglyceride-rich lipoproteins (TG)-rich lipoproteins, occurring at the luminal surface of capillaries in the heart, skeletal muscle, and adipose tissue. LPL, synthesized in muscle and adipocytes, translocates to capillary endothelial cells. Rare genetic defects in lipoprotein lipase (LPL), the primary enzyme responsible for hydrolysis of TG on lipoproteins (Benlian, 1996), can cause familial hyperchylomicronemia syndrome (FCS), characterized by plasma TG levels well above 10 mmol / L. Homozygous defects in apolipoprotein C-II (apoC-II), the major protein activator of LPL, can also cause a similar hypertriglyceridemia phenotype (Breckenridge, 1978). Recently, defects in GPIHBP1, a protein that binds LPL to the surface of endothelial cells (Beigneux, 2007), and mutations in apolipoprotein AV (ApoA-V) (Ishihara, 2005), have also been described to cause hypertriglyceridemia in humans. Genetic defects in the lipase-specific chaperone LMF1 have also been found to promote FCS. Collectively, approximately 2–3 out of 1 million patients have FCS.

[0005] Not only do LPL activators affect the LPL system, but loss-of-function mutations in apoC3 and negative LPL regulators, such as ANGPTL4, ANGPTL3, or ANGPTL8, have been shown to promote desirable plasma lipid profiles and reduced risk of metabolic diseases. ANGPTL4 is a regulator of various lipases, particularly LPL. This protein is an unfolding chaperone that degrades the dimeric catalytically active form of LPL into an inactive monomer, an irreversible event. Compared to apoC3, which dissociates LPL from lipid substrates, ANGPTL4 is the only known factor that regulates LPL in this way. Additionally, ANGPTL4 influences hepatic lipase and endothelial lipase, thereby affecting not only the TG portion of plasma lipids but also LDL-c and HDL-c. ANGPTL4 is induced by fasting and is expressed not only in the liver but also, to an appropriate extent, by adipose tissue and skeletal muscle, making it ubiquitously expressed. ANGPTL4 expression is regulated by various stimuli, including peroxisome proliferator-activated receptor (PPAR)α, ​​PPARδ, and glucocorticoid receptor (GR) in the liver. Animal models lacking these ANGPTLs exhibit elevated LPL activity and reduced plasma lipids, whereas mice transgenic for human mutants exhibit the opposite results. These animal studies are supported by human deletion and loss-of-function mutations, which associate ANGPTL4 with plasma triglyceride levels and HDL-c. The ANGPTL4 gene has been linked to cardiometabolic disease.

[0006] Thus, previous information provides new insights into the coordinated activities of LPL, GPIHBP1, ANGPTL, and apoA-V in plasma TG homeostasis. Among these factors, ANGPTL4 interestingly also regulates plasma cholesterol levels, namely LDL-c, HDL-c, and remnant-c, without completely relying on the LDL receptor, which is often nonfunctional in homozygous familial hypercholesterolemia (HoFH) and heterozygous familial hypercholesterolemia (HeFH). This provides an opportunity for "universal" plasma lipid drugs by targeting ANGPTL4.

[0007] ANGPTL4 regulates the activity of lipoprotein lipase, which plays a key role in the uptake of free fatty acids into the liver. Dysregulation of lipoprotein lipase can lead to excess lipids in cells, which can lead to obesity, type II diabetes, or cardiovascular disease, for example.

[0008] ANGPTL4 knockout mice exhibit reduced triglyceride (TG) levels due to increased breakdown of very low-density lipoprotein (VLDL) and reduced VLDL production. Cholesterol levels are moderately affected. A high-lipid diet results in reduced viability in ANGPTL4 knockout mice treated with a monoclonal antibody due to lipogranulomatous lesions in the intestinal tissue, draining lymphatic system, and / or mesenteric lymph nodes (Desai et al., 2007 PNAS). Humans heterozygous for the ANGPTL4 variant E40K exhibit significantly lower plasma TG levels during fasting. High-density lipoprotein (HDL) cholesterol levels are also significantly higher in E40K heterozygotes. Because the combination of high TG levels and low HDL cholesterol levels increases the risk of developing cardiovascular disease, reducing or inhibiting ANGPTL4 may potentially reduce this risk. ANGPTL4 null alleles are found in humans, but no pathology equivalent to that seen in ANGPTL4 knockout mice has been identified to date.

[0009] The oligonucleotides of the present invention that inhibit the expression of ANGPTL4, for example, reduce plasma lipid levels independently of LDL receptor functionality, making them suitable for use in treating, for example, homozygous familial hypercholesterolemia (HoFH) or heterozygous familial hypercholesterolemia (HeFH), which are LDL receptor deficient.

[0010] ANGPTL4 is not only involved in the regulation of fatty acid metabolism but also in influenza infection. For example, ANGPTL4 is upregulated in influenza pneumonia via a STAT3-mediated mechanism and is a potential biomarker for respiratory infection and pneumonia (Li et al., Cell Reports 10, Feb. 2015).

[0011] Until now, there has been no antisense oligonucleotide that is highly effective in reducing and inhibiting ANGPTL4 expression, and hybridizes with ANGPTL4 mRNA and / or pre-mRNA.The study using siRNA that inhibits ANGPTL4 expression has shown that in vivo inhibition is only possible when siRNA is packaged in suitable packaging material.Even when siRNA is packaged, the efficiency of inhibiting mRNA expression often cannot be improved.

[0012] The oligonucleotides of the present invention have been highly successful in inhibiting the expression of ANGPTL4. The mode of action of oligonucleotides is different from that of antibodies or small molecules, and oligonucleotides can be used to inhibit the expression of ANGPTL4, e.g. (i) tissue penetration; (ii) blocking each of multiple target functions and activities; (iii) combinations between oligonucleotides or with antibodies or small molecules, and (iv) Inhibition of intracellular effects that are not accessible or specifically accessible to antibodies or that cannot be inhibited by small molecules It is highly advantageous in terms of

[0013] Summary of the Invention The present invention relates to an ANGPTL4 inhibitor comprising an oligonucleotide containing or consisting of, for example, 12 to 22 nucleotides, 15 to 20 nucleotides, or 15, 16, 17, 18, 19, or 20 nucleotides, at least one of which is modified. The ANGPTL4 oligonucleotide hybridizes to the nucleic acid sequence of, for example, ANGPTL4 (human; NM_139314) of SEQ ID NO: 1, ANGPTL4 (human; GRCh38_19_8364151_8374373) of SEQ ID NO: 2, ANGPTL4 (mouse; NM_020581.2) of SEQ ID NO: 58, and / or ANGPTL4 (mouse; GRCm38:17:33773750:33781575), and the oligonucleotide inhibits ANGPTL4 expression. The modified nucleotides are selected from the group consisting of bridged nucleic acids such as LNA, cET, ENA, 2'fluoro modified nucleotides, 2'O-methyl modified nucleotides, 2'O-methoxyethyl modified nucleotides and combinations thereof.

[0014] The ANGPTL4 oligonucleotides of the present invention include, for example, oligonucleotides of positions 1732 to 1759 of SEQ ID NO: 1 (e.g., A24044He, SEQ ID NO: 47; A24076He, SEQ ID NO: 47) and / or positions 234 to 261 (e.g., A24102He, SEQ ID NO: 177; A24103He, SEQ ID NO: 178) and / or positions 1264 to 1293 (e.g., A24110He, SEQ ID NO: 185; A24111He, SEQ ID NO: 186), and / or oligonucleotides of positions 1732 to 1759 of SEQ ID NO: 1 (e.g., A24044He, SEQ ID NO: 47; A24076He, SEQ ID NO: 47), and / or positions 234 to 261 (e.g., A24102He, SEQ ID NO: 177; A24103He, SEQ ID NO: 178), and / or positions 1264 to 1293 (e.g., A24110He, SEQ ID NO: 185; A24111He, SEQ ID NO: 186), and / or positions 1264 to 1293 of SEQ ID NO: 1 ... The inhibitor hybridizes to an active site selected from positions 2800 to 2872 (e.g., A24083Hi, SEQ ID NO: 158; A24085Hi, SEQ ID NO: 160; A24086Hi, SEQ ID NO: 161; A24087Hi, SEQ ID NO: 162) and / or positions 3415 to 3442 (e.g., A24089Hi, SEQ ID NO: 164) and / or positions 4968 to 4994 (e.g., A24097Hi, SEQ ID NO: 172) of sequence number 2, or a combination thereof. The inhibitor inhibits ANGPTL4 expression, for example, at nanomolar or micromolar concentrations.

[0015] The present invention further relates to pharmaceutical compositions comprising an ANGPTL4 inhibitor of the present invention and a pharmaceutically acceptable carrier, excipient, diluent, or a combination thereof. The inhibitor and pharmaceutical composition are each used in methods for preventing and / or treating disorders associated with ANGPTL4 imbalance. Such disorders include, for example, cardiometabolic disease, obesity, diabetes, such as type 2 diabetes, hypercholesterolemia, hypertriglyceridemia (HTG), dyslipidemia, pancreatitis, metabolic syndrome, familial hyperchylomicronemia syndrome (FCS), influenza infection, and / or cancer. Hypercholesterolemia is, for example, homozygous familial hypercholesterolemia (HoFH) and heterozygous familial hypercholesterolemia (HeFH), and cancer is, for example, breast cancer, lung cancer, malignant melanoma, lymphoma, skin cancer, bone cancer, prostate cancer, liver cancer, brain cancer, cancer of the larynx, gallbladder, pancreas, testis, rectum, parathyroid gland, thyroid gland, adrenal gland, nervous tissue, head and neck, colon, stomach, bronchus, kidney, basal cell carcinoma, squamous cell carcinoma, metastatic skin cancer, osteosarcoma, Ewing's sarcoma, reticulum cell sarcoma, liposarcoma, myeloma, giant cell tumor, small cell lung tumor, islet cell tumor, primary brain tumor, meningioma, acute and chronic lymphocytic and granulocytic tumors, acute and chronic myeloid leukemia, trichocytoma, adenoma, hyperplasia, medullary carcinoma, intestinal ganglioneuroma, Wilms' tumor, seminoma, ovarian tumor, leiomyoma tumor), cervical dysplasia, retinoblastoma, soft tissue sarcoma, malignant carcinoid, localized skin lesion, rhabdomyosarcoma, Kaposi's sarcoma, osteogenic sarcoma, malignant hypercalcemia, renal cell tumor, polycythemia vera, adenocarcinoma, anaplastic astrocytoma, glioblastoma multiforme, leukemia, or epidermoid carcinoma.

[0016] The ANGPTL4 inhibitors or pharmaceutical compositions comprising an ANGPTL4 inhibitor of the present invention are administered locally or systemically.

[0017] All documents cited or referenced herein ("references herein"), and all documents cited or referenced in the references cited herein, together with any manufacturer's instructions, instructions, product standards, and product specifications for any product mentioned herein or in any document incorporated by reference, are hereby incorporated by reference and may be used in the practice of this invention. More specifically, all referenced documents are incorporated by reference to the same extent as if each individual document were specifically and individually indicated to be incorporated by reference. [Brief explanation of the drawings]

[0018] [Figure 1] Figure 1 shows the initial screening of 42 human ANGPTL4-specific antisense oligonucleotides. A control oligonucleotide (Neg1; SEQ ID NO: 57) with no sequence complementarity to any human or mouse mRNA was included as a control. Human epithelioid cervical carcinoma cells (HeLa) were treated with each oligonucleotide at a single concentration of 10 μM for 3 days. Three days after the start of treatment, cells were lysed, and ANGPTL4 and HPRT1 mRNA levels were determined using the QuantiGene Singleplex RNA assay. HPRT1 was used as a housekeeping gene for normalization of ANGPTL4 expression. Residual ANGPTL4-mRNA expression relative to mock-treated cells ("no oligo" set to 1). Triplicate wells, mean ± SD (Figure 1). [Figure 2] FIG. 10 shows another screen of human ANGPTL4-specific antisense oligonucleotides in SK-OV3 cells to investigate residual ANGPTL4 mRNA expression. [Figure 3] 1 shows additional human ANGPTL4-specific antisense oligonucleotides tested at a concentration of 10 μM in HeLa cells, with four of the tested antisense oligonucleotides showing greater than 50% knockdown of ANGPTL4 mRNA (corresponding to residual mRNA levels of less than 0.5). [Figure 4] FIG. 10 shows another screening of additional ANGPTL4-specific antisense oligonucleotides in SK-OV3 cells to investigate residual ANGPTL4 mRNA expression. [Figure 5] 1 shows HEK-Blue™ hTLR9 cells used to study human TLR9 stimulation by monitoring nuclear factor "kappa light-chain-enhancer" of activated B cells (NF-kB)-dependent activation of secreted embryonic alkaline phosphatase (SEAP) production in vitro. ANGPTL4 oligonucleotides A24022Hi (SEQ ID NO: 25), A24023Hi (SEQ ID NO: 26), A24071Hi (SEQ ID NO: 54), and A24076He (SEQ ID NO: 47), as well as controls Neg1 (SEQ ID NO: 57) and ODN2006 (5'-TCGTCGTTTTGTCGTTTTGTCGTT-3'PTO modified - Invivogen catalog no. tlrl-2006; SEQ ID NO: 153), were analyzed. [Figure 6] Figure 6 shows the human ANGPTL4-specific antisense oligonucleotides A24022Hi (SEQ ID NO: 25), A24023Hi (SEQ ID NO: 26), A24071Hi (SEQ ID NO: 54), and A24076He (SEQ ID NO: 47) with the most potent knockdown efficacy in HeLa and SK-OV3 cells, selected for determination of half-maximal inhibitory concentration (IC50) values. Primary hepatocytes were treated with different concentrations (5000 nM, 1000 nM, 200 nM, 40 nM, 8 nM, 1.6 nM) of each ANGPTL4 antisense oligonucleotide for 3 days, and simultaneously treated with PPARγ (1 μM). Three days after the start of treatment, mRNA expression was analyzed using the QuantiGene Singleplex RNA assay, as shown in Figure 6. For graphical representation, mock-treated cells were treated with 0.32 nM. [Figure 7]Figure 1 shows the first single-dose efficacy screening of additional ANGPTL4-specific antisense oligonucleotides (ASOs) in primary hepatocytes. 25,000 cells / well were seeded into 96-well collagen I-coated flat-bottom plates and treated with each ASO at a final concentration of 5 μM. To induce ANGPTL4 mRNA expression, cells were simultaneously treated with 1 μM PPARγ. As a vehicle control, cells were treated with the same volume of DMSO ("DMSO"). Every 24 hours, 70 μl of supernatant was replaced with fresh medium containing PPARγ and 5 μM of each ASO or DMSO. After 3 days, cells were lysed, and human HPRT1 and human ANGPTL4 mRNA expression was measured using the QuantiGene RNA Singleplex assay. ANGPTL4-mRNA expression values ​​were normalized to the expression of the housekeeping gene HPRT1. Residual ANGPTL4-mRNA expression is shown relative to mock-treated cells ("no oligo" (n=42) set as 1, SD=0.24). Solid and dotted lines indicate 70% and 0% knockdown efficacy (top graph) or 100% HPRT1 expression in mock-treated cells ("no oligo" (n=42) set to 100, SD=35.8, bottom graph). Data are presented as the mean ± SD of triplicate wells. Positive control ASO is indicated by an asterisk. [Figure 8]Figure 1 shows a second single-dose efficacy screen of additional ANGPTL4-specific ASOs in primary hepatocytes. 25,000 cells / well were seeded into 96-well collagen I-coated flat-bottom plates and treated with each ASO at a final concentration of 5 μM. To induce ANGPTL4 mRNA expression, cells were simultaneously treated with 1 μM PPARγ. As a vehicle control, cells were treated with the same amount of DMSO ("DMSO") or left untreated ("-DMSO"). Every 24 hours, 70 μl of supernatant was replaced with fresh medium containing PPARγ and 5 μM of each ASO, or DMSO or medium alone. After 3 days, cells were lysed, and human HPRT1 and human ANGPTL4 mRNA expression was measured using the QuantiGene RNA Singleplex assay. ANGPTL4 mRNA expression values ​​were normalized to the expression of the housekeeping gene HPRT1. Residual ANGPTL4-mRNA expression relative to mock-treated cells ("no oligo" (n=36) set to 1, SD=0.24) is shown. Solid and dotted lines indicate 70% and 0% knockdown efficacy, respectively (top graph) or 100% HPRT1 expression in mock-treated cells ("no oligo" (n=36) set to 100, SD=19.3, bottom graph). Data are presented as the mean ± SD of sextuplicate (A24076He and negative control oligonucleotide), ninuplicate (-DMSO) or triplicate (all other ASO) wells. Positive control ASOs are indicated by asterisks. [Figure 9]Figure 9. NF-kB activation in HEK-Blue hTLR9 SEAP reporter cells. HEK-Blue-hTLR9 cells were seeded in flat-bottom 96-well plates and treated with the indicated oligonucleotides for 24 hours. After incubation, cell supernatants were collected and incubated with QUANTI-Blue solution for 4 hours (Figures 9A, 9C, and 9D) or 3.5 hours (Figure 9B). SEAP activity was determined by measuring optical density (OD) at 620 nm. Data for cells treated with ANGPTL4-specific ASO and neg1 are shown as the mean ± SD of triplicates of OD units relative to the OD units of cells stimulated with 5000 nM ODN2006 (defined as 100%). Data for cells treated with ODN2006 are shown as the mean ± SD of sextuplicates (A), the mean ± SD of triplicates (D), or the mean ± SD of the mean of triplicates on each plate (B, C). [Figure 10] Figure 1 shows IC50 determination of selected ANGPTL4 ASOs. 25,000 primary human hepatocytes / well were seeded in 96-well plates and treated with different concentrations (5,000 nM, 1,000 nM, 200 nM, 40 nM, 8 nM, 1.6 nM) of each ASO. Every 24 hours, 70 μl of supernatant was replaced with fresh medium containing the indicated concentrations of each ASO. After 3 days, cells were lysed, and the mRNA expression of HPRT1 and ANGPTL4 was measured using the QuantiGene RNA Singleplex assay. ANGPTL4-mRNA expression values ​​were normalized to the expression of the housekeeping gene HPRT1. Residual ANGPTL4-mRNA expression relative to mock-treated cells (set as 1 (n=36), SD=0.26). Data are presented as the mean ± SD of triplicate wells. [Figure 11]Figure 1 shows the efficacy of selected ANGPTL4 ASOs on target gene expression after transfection of cynomolgus monkey hepatocytes. 25,000 primary cynomolgus monkey hepatocytes / well were seeded in a 96-well plate and transfected with different concentrations (2 nM, 0.2 nM) of each ASO. After 24 h of incubation at 37°C, cells were lysed, and the mRNA expression of HPRT1 and ANGPTL4 was measured using the QuantiGene RNA Singleplex assay. ANGPTL4-mRNA expression values ​​were normalized to the expression of the housekeeping gene HPRT1. Residual ANGPTL4-mRNA expression relative to mock-treated cells (set as 1 (n=12), SD=0.25) is shown. Data are presented as the mean ± SD of triplicate wells. [Figure 12] This figure shows mouse embryonic fibroblasts (3T3 cells) separately treated with the mouse ANGPTL4-specific antisense oligonucleotides shown in Figure 12 at a single concentration of 10 μM. Three days after the start of treatment, mRNA levels were determined by QuantiGene RNA Singleplex assay. Hprt1 was used as a housekeeping gene for normalization of ANGPTL4 expression. Eight ANGPTL4 antisense oligonucleotides, namely, A24047M (SEQ ID NO: 106), A24020M (SEQ ID NO: 79), A24017M (SEQ ID NO: 76), A24021M (SEQ ID NO: 80), A24049M (SEQ ID NO: 108), A24018M (SEQ ID NO: 77), A24041M (SEQ ID NO: 100), and A24010M (SEQ ID NO: 69), reduced normalized ANGPTL4 expression by more than 50%. [Figure 13]14 shows mouse renal carcinoma Renca cells separately treated with the ANGPTL4-specific antisense oligonucleotides shown in FIG. 13 at a single concentration of 10 μM. After 3 days, mRNA levels were determined by QuantiGene RNA Singleplex assay. Hprt1 was used as a housekeeping gene for normalization of ANGPTL4 expression. A24020M (SEQ ID NO: 79) and A24019M (SEQ ID NO: 78) resulted in more than 50% ANGPTL4 knockdown (corresponding to a residual mRNA level of less than 0.5). [Figure 14] Figure 14 shows nine previously tested mouse ANGPTL4-specific antisense oligonucleotides and 24 additional mouse ANGPTL4-specific antisense oligonucleotides tested in mouse breast cancer cell 4T1. Cells were treated with 5 μM of each ANGPTL4 antisense oligonucleotide shown in Figure 14 without using a transfection reagent. After 3 days, the cell supernatant was replaced with fresh medium containing 5 μM of each ANGPTL4 antisense oligonucleotide and incubated for an additional 3 days. Subsequently, mRNA levels were determined by QuantiGene RNA Singleplex assay. Gapdh was used as a housekeeping gene for normalization of ANGPTL4 expression. Twelve of the antisense oligonucleotides tested, namely, A24017M (SEQ ID NO: 76), A24070M (SEQ ID NO: 127), A24020M (SEQ ID NO: 79), A24019M (SEQ ID NO: 78), A24069M (SEQ ID NO: 126), A24021M (SEQ ID NO: 80), A24011M (SEQ ID NO: 70), A24073M (SEQ ID NO: 130), A24018M (SEQ ID NO: 77), A24055M (SEQ ID NO: 114), A24010M (SEQ ID NO: 69), and A24065M (SEQ ID NO: 79), exhibit greater than 80% knockdown of ANGPTL4 mRNA (corresponding to residual mRNA levels of less than 0.2). [Figure 15]Figure 15 shows 21 mouse ANGPTL4-specific antisense oligonucleotides tested in 4T1 cells by treating with 5 μM of each ANGPTL4 antisense oligonucleotide shown in Figure 15. After 3 days, the cell supernatant was replaced with fresh medium containing 5 μM of each ANGPTL4 antisense oligonucleotide and incubated for another 3 days. Then, mRNA levels were determined by QuantiGene RNA Singleplex assay. Hprt1 was used as a housekeeping gene for normalizing ANGPTL4 expression. A24047M (SEQ ID NO: 106), A24095Mi (SEQ ID NO: 151), A24093Mi (SEQ ID NO: 149), A24020M (SEQ ID NO: 79), A24090Mi (SEQ ID NO: 146) and A24082Mi (SEQ ID NO: 139) show more than 50% knockdown of ANGPTL4 mRNA (corresponding to a residual mRNA level of less than 0.5). [Figure 16] Figure 1 shows that A24018M (SEQ ID NO: 77), A24019M (SEQ ID NO: 78), A24020M (SEQ ID NO: 79), A24021M (SEQ ID NO: 80), A24047M (SEQ ID NO: 106), A24054M (SEQ ID NO: 113), A24065M (SEQ ID NO: 79), A24070M (SEQ ID NO: 127), A24072M (SEQ ID NO: 129), A24082M (SEQ ID NO: 139) and A24095Mi (SEQ ID NO: 151) have the most potent knockdown efficacy in 3T3, Renca and 4T1 cells, which were selected for determining half maximal inhibitory concentration (IC50) values. 4T1 cells were treated with these ANGPTL4 antisense oligonucleotides at different concentrations (5000nM, 1000nM, 200nM, 40nM, 8nM, 1.6nM) for 3 days. After 3 days, the cell supernatant was replaced with fresh medium containing 5 μM of each ANGPTL4 antisense oligonucleotide and incubated for an additional 3 days. mRNA expression was then analyzed using the QuantiGene Singleplex RNA assay. [Figure 17]Figure 1 shows the first single-dose efficacy screening of additional mouse Angptl4-specific ASOs in 4T1 cells. 2500 4T1 cells / well were seeded into 96-well flat-bottom plates and treated with each ASO at a final concentration of 5 μM. Three days after treatment, the cell supernatant was replaced with fresh medium containing the ASOs, and the cells were incubated at 37°C for an additional three days. Cells were then lysed, and mouse Hprtl and mouse Angptl4 mRNA expression was measured using the QuantiGene RNA Singleplex assay. Angptl4-mRNA expression values ​​were normalized to the expression of the housekeeping gene Hprtl. Residual Angptl4-mRNA expression is shown relative to mock-treated cells ("no oligo" (n=37) set to 1, SD=0.21). Solid and dotted lines indicate 75% and 0% knockdown efficacy (top graph) or 100% Hprt1 levels (bottom graph) ("no oligo" (n=37) set as 1, SD=0.17), respectively. Data are presented as the mean ± SD of triplicate wells. [Figure 18] Figure 1 shows a second single-dose screening of mouse Angptl4-specific ASOs in Renca cells. 2500 Renca cells / well were seeded in 96-well flat-bottom plates and treated with each ASO at a final concentration of 5 μM. Three days after treatment, the cell supernatant was replaced with fresh medium containing the ASOs, and the cells were incubated at 37°C for an additional three days. Cells were then lysed, and the mRNA expression of mouse Hprt1 and mouse Angptl4 was measured using the QuantiGene RNA Singleplex assay. Angptl4 mRNA expression values ​​were normalized to the expression of the housekeeping gene Hprt1. Residual Angptl4 mRNA expression is shown relative to mock-treated cells ("no oligo" (n = 37) set to 1, SD = 0.37). Solid and dotted lines indicate 75% and 0% knockdown efficacy (top graph) or 100% Hprt1 levels (bottom graph) ("no oligo" (n = 37) set to 1, SD = 0.36), respectively. Data are expressed as the mean ± SD of triplicate wells. [Figure 19]Figure 1 shows IC50 determination of selected Angptl4 ASOs. 15,000 primary mouse hepatocytes / well were seeded in 96-well flat-bottom plates and treated with each ASO at different concentrations (5,000 nM, 1,000 nM, 200 nM, 40 nM, 8 nM, and 1.6 nM). After 3 days, cells were lysed, and Hprtl and Angptl4 mRNA expression was measured using the QuantiGene RNA Singleplex assay. Angptl4-mRNA expression values ​​were normalized to the expression of the housekeeping gene Hprtl. Residual Angptl4-mRNA expression relative to mock-treated cells (set to 1 (n = 36), SD = 0.17). Negl is shown in black. The lower graph shows raw values ​​for Hprtl relative to no oligo (set to 100 (n = 36), SD = 13.0). Data are presented as the mean ± SD of triplicate wells.

[0019] Detailed Description of the Invention The present invention provides a successful inhibitor of ANGPTL4 expression, which is a human or mouse oligonucleotide that hybridizes with the mRNA and / or pre-mRNA sequence of ANGPTL4 and inhibits the expression and activity of ANGPTL4, respectively.The mRNA contains only the exons of the nucleic acid sequence encoding ANGPTL4, and the pre-mRNA contains the exons and introns of the nucleic acid sequence encoding ANGPTL4.Therefore, the oligonucleotide of the present invention is an interesting and highly effective tool that can be used in methods for preventing and / or treating disorders in which ANGPTL4 expression and activity are increased, respectively.

[0020] The elements of the present invention are described in more detail below. While these elements are listed with specific embodiments, it should be understood that they can be combined in any manner and in any number to create additional embodiments. The various described examples and embodiments should not be construed as limiting the invention to only the explicitly described embodiments. The specification should be understood to support and encompass embodiments that combine the explicitly described embodiments with various disclosed elements. Furthermore, unless the context indicates otherwise, any permutation and combination of all elements described herein shall be deemed to be disclosed by the specification of this application.

[0021] Throughout this specification and the claims, unless the context requires otherwise, the word "comprise," and variations such as "comprises" and "comprising," are understood to imply the inclusion of a stated member, integer, or step, or group of members, integers, or steps, but not the exclusion of any other member, integer, or step, or group of members, integers, or steps. As used in the context of describing the invention (particularly in the context of the claims), the terms "a," "an," and "the," and similar referents, shall be construed to encompass both the singular and the plural, unless otherwise specified herein or clearly contradicted by context. The recitation of ranges of values ​​herein is merely intended to serve as a shorthand method of individually referring to each value falling within the range. Unless otherwise specified herein, each value is incorporated herein as if it were individually listed herein. All methods described herein can be performed in any suitable order unless otherwise specified herein or clearly contradicted by context. The use of any examples or exemplary language (e.g., "such as," "for example") presented herein is intended merely to better illustrate the invention and does not limit the scope of the invention as otherwise claimed. Nothing in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0022] The oligonucleotide inhibitors of the present invention are antisense oligonucleotides (ASOs) consisting of or containing, for example, 10 to 25 nucleotides, 12 to 22 nucleotides, 15 to 20 nucleotides, or 16 to 18 nucleotides. The oligonucleotides consist of or contain, for example, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides.

[0023] For example, the oligonucleotides of the invention form gapmers consisting of or including a central block of at least five nucleotides, i.e., deoxynucleotides and / or ribonucleotides, flanked by nucleotides, such as naturally and / or artificially modified deoxynucleotides and / or ribonucleotides.

[0024] The oligonucleotide of the present invention comprises at least one modified nucleotide. The modified nucleotide is, for example, a bridged nucleotide, such as locked nucleic acid (LNA, for example, 2',4'-LNA), cET, ENA, 2'fluoro-modified nucleotide, 2'O-methyl-modified nucleotide, 2'O-methoxyethyl-modified nucleotide, or a combination thereof. In some embodiments, the oligonucleotide of the present invention comprises one or more nucleotides with the same or different modifications. In addition, the oligonucleotide of the present invention optionally comprises a modified phosphate backbone, where the phosphate is, for example, phosphorothioate.

[0025] The oligonucleotides of the present invention comprise one or more modified nucleotides at the 3' and / or 5' end of the oligonucleotide, and / or at any position within the oligonucleotide, where the modified nucleotides are, for example, tandem with 1, 2, 3, 4, 5, or 6 modified nucleotides, or where the modified nucleotides are combined with one or more unmodified nucleotides. Tables 1 to 4 below list modified nucleotides, such as LNAs and ( * Examples of ANGPTL4 oligonucleotides containing phosphorothioates (PTOs) are provided below. ANGPTL4 oligonucleotides consisting of or containing the sequences of Table 1 or 2 (human) or Table 3 or 4 (mouse) may contain any other modified nucleotides and / or any other combination of modified and unmodified nucleotides. The ANGPTL4 oligonucleotides of Table 1 hybridize to human ANGPTL4 mRNA and / or pre-mRNA: [Table 1-1] [Table 1-2] [Table 1-3]

[0026] The ANGPTL4 oligonucleotides in Table 2 also hybridize to human ANGPTL4 mRNA and / or pre-mRNA: [Table 2-1] [Table 2-2] [Table 2-3]

[0027] The oligonucleotides in Table 3 specifically hybridize to mouse ANGPTL4 mRNA and / or pre-mRNA: [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4]

[0028] The oligonucleotides in Table 4 also hybridize specifically to mouse ANGPTL4 mRNA and / or pre-mRNA: [Table 4-1] [Table 4-2]

[0029] The oligonucleotide hybridizes at a hybridizing active area, e.g., a site enriched for highly active ASOs. The hybridizing active area is, for example, one or more regions on the ANGPTL4 mRNA, e.g., SEQ ID NO: 1, and / or the ANGPTL4 pre-mRNA, e.g., SEQ ID NO: 2, where hybridization with the oligonucleotide is likely to result in potent knockdown of ANGPTL4 expression. Surprisingly, the present invention has identified several hybridizing active sites selected from, for example, hybridizing active sites selected from positions 1732 to 1759 of SEQ ID NO: 1 (e.g., A24044He, SEQ ID NO: 47; A24076He, SEQ ID NO: 47) and / or positions 6603 to 6631 of SEQ ID NO: 2 (e.g., A24022Hi, SEQ ID NO: 25; A24023Hi, SEQ ID NO: 26; A24071Hi, SEQ ID NO: 54). Further hybridization active sites are at positions 234 to 261 of human SEQ ID NO: 1 (e.g., A24102He, SEQ ID NO: 178; A24103He, SEQ ID NO: 179) and / or positions 1264 to 1293 of human SEQ ID NO: 1 (e.g., A24110He, SEQ ID NO: 186; A24111He, SEQ ID NO: 187), and / or positions 2800 to 2872 of human SEQ ID NO: 2 (e.g., A24083Hi, SEQ ID NO: 159; A24085Hi, SEQ ID NO: 161; A24086Hi, SEQ ID NO: 162; A24087Hi, SEQ ID NO: 163) and / or positions 3415 to 3442 of human SEQ ID NO: 2 (e.g., A24089Hi, SEQ ID NO: 165) and / or positions 4968 to 4994 of human SEQ ID NO: 2 (e.g., A24097Hi, SEQ ID NO: 173).The hybridization active site on mouse SEQ ID NO: 58 or SEQ ID NO: 59 may be, for example, at positions 137 to 163 (e.g., A24054M, SEQ ID NO: 113) and / or positions 215 to 299 (e.g., A24018M, SEQ ID NO: 77; A24019M, SEQ ID NO: 78; A24020M, SEQ ID NO: 79; A24021M, SEQ ID NO: 80; A24065M, SEQ ID NO: 79) and / or positions 1343 to 1371 (e.g., A24042M, SEQ ID NO: 101; A24043M, SEQ ID NO: 102) of SEQ ID NO: 58. A24070M, SEQ ID NO: 127; A24072M, SEQ ID NO: 129), and / or positions 1286 to 1314 of SEQ ID NO: 59 (e.g., A24082Mi, SEQ ID NO: 139; A24125Mi, SEQ ID NO: 226) and / or positions 5485 to 5511 of SEQ ID NO: 59 (e.g., A24095Mi, SEQ ID NO: 151; A24148Mi, SEQ ID NO: 151).

[0030] For example, the oligonucleotides of the invention inhibit ANGPTL4, e.g., human or mouse ANGPTL4 expression, by at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%. The oligonucleotides of the present invention can inhibit ANGPTL4 expression at nanomolar or micromolar concentrations, for example, 0.1 nM to 100 μM, 0.5 nM to 15 nM, 0.6 nM to 10 nM, 1 nM to 10 μM, 5 nM to 5 μM, 10 nM to 1 μM, 15 nM to 950 nM, 20 nM to 900 nM, 25 nM to 850 nM, 30 nM to 800 nM, 35 nM to 750 nM, 40 nM to 700 nM, 45 nM to 650 nM, 50 nM to 500 nM, or Inhibition occurs at a concentration range of 40 nM to 150 nM, or at a concentration of 0.1, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900 or 950 nM, or at a concentration of 1, 10 or 100 μM.

[0031] The ANGPTL4 oligonucleotides of the present invention are used in a concentration range of, for example, 1 nM to 10 μM, 5 nM to 6.6 μM, 10 nM to 5 μM, 15 nM to 3 μM, 20 nM to 2.2 μM, 25 nM to 1 μM, 30 nM to 800 nM, 50 nM to 500 nM, 60 nM to 300 nM, 70 nM to 250 nM, 80 nM to 200 nM, 90 nM to 120 nM, or at a concentration of 1, 1.6, 3, 5, 8, 9, 10, 15, 20, 25, 27, 30, 40, 50, 75, 82, 100, 200, 250, 300, 500, or 740 nM, or 1, 2.2, 3, 5, 6.6, or 10 μM.

[0032] The ANGPTL4 oligonucleotides of the invention are administered, for example, once or repeatedly, e.g., once every 12 hours, once every 24 hours, once every 48 hours, or weekly, once every 2 weeks, once every 3 weeks, or monthly, or once every 3 months, or once every 6 months, for weeks, months, or years.

[0033] In some embodiments, the present invention relates to a pharmaceutical composition comprising an ANGPTL4 oligonucleotide of the present invention and a pharmaceutically acceptable carrier, excipient, and / or diluent. Optionally, the pharmaceutical composition further comprises a chemotherapeutic agent, another disease-specific active agent, such as insulin, an angiotensin-converting enzyme inhibitor, an angiotensin receptor blocker, another oligonucleotide not of the present invention, an antibody, a HERA fusion protein, a ligand trap, a Fab fragment, a nanobody, a BiTe, and / or a small molecule effective in, for example, tumor therapy, treatment of diabetes and its side effects, cardiovascular disease, obesity, type II diabetes, hypercholesterolemia such as homozygous familial hypercholesterolemia (HoFH), heterozygous familial hypercholesterolemia (HeFH), or dyslipidemia.

[0034] The ANGPTL4 oligonucleotide or pharmaceutical composition of the present invention is used in a method for preventing and / or treating disorders, such as disorders associated with ANGPTL4 imbalance.Optionally, the use of the oligonucleotide or pharmaceutical composition of the present invention in the method for preventing and / or treating disorders is combined with radiation therapy.Radiation therapy can further be combined with chemotherapy (e.g., platinum, gemcitabine).Disorders are characterized, for example, by ANGPTL4 imbalance, that is, ANGPTL4 level is elevated compared with the level in normal, healthy cells, tissues, organs or subjects.Angoploidy levels are elevated, for example, by the increase in ANGPTL4 expression and activity.Angoploidy levels are measured by any standard method known to those skilled in the art, such as immunohistochemistry, Western blot, quantitative real-time PCR or QuantiGene assay.

[0035] The ANGPTL4 oligonucleotide or pharmaceutical composition of the present invention can be administered locally or systemically, for example, orally, sublingually, nasally, subcutaneously, intravenously, intraperitoneally, intramuscularly, intratumorally, intrathecally, transdermally, and / or rectally. Alternatively, or in combination, ex vivo treated immune cells can be administered. The ANGPTL4 oligonucleotide can be administered alone or in combination with another ANGPTL4 antisense oligonucleotide of the present invention, optionally in combination with another compound such as another oligonucleotide not of the present invention, an antibody, a HERA fusion protein, a ligand trap, a Fab fragment, a nanobody, BiTe, a small molecule, and / or a chemotherapeutic drug (e.g., platinum, gemcitabine), and / or another disease-specific drug such as insulin, angiotensin-converting enzyme inhibitor, and / or angiotensin receptor blocker.

[0036] Oligonucleotides, antibodies, HERA fusion proteins, ligand traps, Fab fragments, nanobodies, BiTes, and / or small molecules not of the present invention are effective in preventing and / or treating tumors, influenza infection, diabetes such as type II diabetes and its side effects, cardiovascular disease, obesity, hypercholesterolemia such as homozygous familial hypercholesterolemia (HoFH) or heterozygous familial hypercholesterolemia (HeFH), or dyslipidemia. The ANGPTL4 oligonucleotides or pharmaceutical compositions of the present invention are used in methods for preventing and / or treating, for example, solid tumors or hematologic tumors. Examples of cancers that can be prevented and / or treated using the oligonucleotides or pharmaceutical compositions of the present invention include breast cancer, lung cancer, malignant melanoma, lymphoma, skin cancer, bone cancer, prostate cancer, liver cancer, brain cancer, cancer of the larynx, gallbladder, pancreas, testis, rectum, parathyroid gland, thyroid gland, adrenal gland, nervous tissue, head and neck, colon, stomach, bronchus, kidney, basal cell carcinoma, squamous cell carcinoma, metastatic skin cancer, osteosarcoma, Ewing's sarcoma, reticulum cell sarcoma, liposarcoma, myeloma, giant cell tumor, small cell lung tumor, islet cell tumor, primary brain tumor, Meningiomas, acute and chronic lymphocytic and granulocytic tumors, acute and chronic myeloid leukemia, pilocytoma, adenoma, hyperplasia, medullary carcinoma, intestinal ganglioneuroma, Wilms' tumor, seminoma, ovarian tumor, leiomyoma, cervical dysplasia, retinoblastoma, soft tissue sarcoma, malignant carcinoid, localized skin lesion, rhabdomyosarcoma, Kaposi's sarcoma, osteogenic sarcoma, malignant hypercalcemia, renal cell tumor, polycythemia vera, adenocarcinoma, anaplastic astrocytoma, glioblastoma multiforme, leukemia, or epidermoid carcinoma.

[0037] Further examples of diseases other than cancer that can be prevented and / or treated using the ANGPTL4 oligonucleotides or pharmaceutical compositions of the present invention include diabetes, such as type II diabetes, and its side effects, cardiovascular disease, obesity, hypercholesterolemia, such as homozygous familial hypercholesterolemia (HoFH) and heterozygous familial hypercholesterolemia (HeFH), or dyslipidemia.

[0038] In some examples, two or more ANGPTL4 oligonucleotides of the present invention are administered together at the same time, for example, in a pharmaceutical composition, or separately, or at staggered intervals.In some examples, two or more ANGPTL4 oligonucleotides of the present invention are administered together at the same time, for example, in a pharmaceutical composition, or separately, or at staggered intervals.In other examples, one or more oligonucleotides of the present invention are administered together with another compound, such as another oligonucleotide, antibody, HERA fusion protein, ligand trap, Fab fragment, nanobody, BiTe, small molecule and / or chemotherapeutic agent, that is not of the present invention, at the same time, for example, in a pharmaceutical composition, or separately, or at staggered intervals.

[0039] The subject of the present invention is, for example, a mammal, a bird, or a fish.

[0040] Example The following examples illustrate various embodiments of the present invention, but the present invention is not limited to these examples. The following experiments were performed on cells that endogenously express ANGPTL4, i.e., the cells are not in an artificial system containing a transfected reporter construct. Such artificial systems generally produce higher inhibition and lower IC than endogenous systems, which more closely resemble therapeutically relevant in vivo systems. 50 Furthermore, no transfection agent was used in the following experiments, i.e., gymnotic delivery was performed. The transfection agent increased the activity of the oligonucleotide, which resulted in the IC 50It is known that transfection agents affect the values ​​(see, for example, Zhang et al., Gene Therapy, 2011, 18, 326-333; Stanton et al., Nucleic Acid Therapeutics, Vol. 22, No. 5, 2012). Because it is difficult or impossible to convert artificial systems using transfection agents into therapeutic approaches, and transfection agents have not been approved for oligonucleotides to date, the following experiments are performed without using transfection agents, except for the experiment in Example 11, which used a transfection reagent.

[0041] Example 1: First screening of human ANGPTL4-specific antisense oligonucleotides in HeLa cells Five thousand HeLa cells per well were seeded in a 96-well plate and treated with each antisense oligonucleotide shown in Figure 1 at a final concentration of 10 μM. To induce ANGPTL4 mRNA expression, the cells were simultaneously treated with 1 μM PPARδ (Sigma Aldrich, catalog no. SML1491). The PPARδ used in this example was based on a PPARδ stock solution (10 mM), prepared by dissolving 5 mg of PPARδ (molecular weight: 453.50) in 1.1 ml of DMSO. For a final concentration of 1 μM PPARδ, cells seeded in a 96-well plate were incubated with 100 μl of medium supplemented with 0.01 μl of PPARδ stock solution. As a negative control, cells were treated with the same volume of DMSO. Three days after the start of treatment, cells were lysed, and human HPRT1 and human ANGPTL4 mRNA expression was measured using the QuantiGene RNA Singleplex assay (Figure 1). The QuantiGene assay used in this example is based on branched DNA technology (bDNA), which relies on coordinate hybridization of target mRNA with a specific probe set (part of the QuantiGene Reagent System). The assay is performed according to the manufacturer's protocol (Thermo Fisher Scientific) and is used to determine RNA levels. It combines the QuantiGene Sample Processing Kit, which is used for cell lysis, with the QuantiGene Reagent System, which is used for hybridization, amplification, and detection of the RNA of interest. The QuantiGene Reagent System is based on an RNA-specific probe set designed to detect a specific RNA of interest.

[0042] ANGPTL4-mRNA expression values ​​were normalized to the expression of the housekeeping gene HPRT1. Residual ANGPTL4-mRNA expression is shown relative to mock-treated cells ("no oligo" set to 1). Solid and dotted lines indicate 70% and 50% or 0% knockdown efficacy, respectively. Data are presented as the mean ± SD of triplicate wells.

[0043] As shown in Figure 1, two antisense oligonucleotides (A24022Hi (SEQ ID NO: 25) and A24023Hi (SEQ ID NO: 26)) reduced normalized ANGPTL4 expression by more than 70% (corresponding to residual mRNA levels of less than 0.3). Furthermore, three antisense oligonucleotides (A24003He (SEQ ID NO: 5), A24042He (SEQ ID NO: 45), and A24005Hi (SEQ ID NO: 7)) showed knockdown efficacy ranging from 70% to 50%, while the control oligonucleotide (Neg1) did not reduce ANGPTL4 mRNA expression.

[0044] Example 2: Secondary screening of human ANGPTL4-specific antisense oligonucleotides in SK-OV3 cells 5000 SK-OV3 cells / well were seeded in a 96-well plate and treated with each ANGPTL4 antisense oligonucleotide at a final concentration of 10 μM. To induce ANGPTL4 mRNA expression, the cells were simultaneously treated with 1 μM PPARδ (Sigma Aldrich, Catalog No. SML1491; see Example 1 for the preparation of a 10 mM stock solution). For a final concentration of 1 μM PPARδ, the cells seeded in a 96-well plate were incubated with 100 μl of medium supplemented with 0.01 μl of PPARδ stock solution. As a negative control, the cells were treated with the same volume of DMSO.

[0045] Three days after the start of treatment, cells were lysed, and mRNA expression of human HPRT1 and human ANGPTL4 was measured using the QuantiGene RNA Singleplex assay. ANGPTL4-mRNA expression values ​​were normalized to the expression of the housekeeping gene HPRT1. Residual ANGPTL4-mRNA expression relative to mock-treated cells ("no oligo" set to 1) is shown in Figure 2. Solid and dotted lines indicate 60% and 0% knockdown efficacy, respectively. Data are presented as the mean ± SD of triplicate wells.

[0046] Repeated screening of ANGPTL4 antisense oligonucleotides in SK-OV3 cells yielded three ANGPTL4 antisense oligonucleotides (A24022Hi (sequence number 25), A24044He (sequence number 47), and A24023Hi (sequence number 26)) with knockdown efficiencies of over 60%.

[0047] Example 3: First single-dose efficacy screen of additional human ANGPTL4-specific antisense oligonucleotides in HeLa cells Five thousand HeLa cells / well were seeded in 96-well plates and treated with the most efficient ANGPTL4 antisense oligonucleotides (A24022Hi (SEQ ID NO: 25), A24023Hi (SEQ ID NO: 26), A24044He (SEQ ID NO: 47)) from the first round of screening in HeLa ( FIG. 1 ) and SK-OV3 ( FIG. 2 ) and additional ANGPTL4-specific antisense oligonucleotides (A24071Hi (SEQ ID NO: 54), A24076He (SEQ ID NO: 47), A24075He (SEQ ID NO: 5), A24073Hi (SEQ ID NO: 55), A24065Hi (SEQ ID NO: 51), A24067Hi (SEQ ID NO: 52), A24077He (SEQ ID NO: 47), A24074Hi (SEQ ID NO: 56)) at a final concentration of 10 μM. To induce ANGPTL4 mRNA expression, cells were simultaneously treated with 1 μM PPARδ (Sigma Aldrich, Catalog No. SML1491; see Example 1 for preparation of a 10 mM stock solution). For a final concentration of 1 μM PPARδ, cells seeded in a 96-well plate were incubated with 100 μl of medium supplemented with 0.01 μl of PPARδ stock solution. As a negative control, cells were treated with the same volume of DMSO.

[0048] Three days after the start of treatment, cells were lysed, and mRNA expression of human HPRT1 and human ANGPTL4 was measured using the QuantiGene RNA Singleplex assay. ANGPTL4-mRNA expression values ​​were normalized to the expression of the housekeeping gene HPRT1. Residual ANGPTL4-mRNA expression relative to mock-treated cells ("no oligo" set to 1) is shown in Figure 3. Solid and dotted lines indicate 50% and 0% knockdown efficacy, respectively. Data are presented as the mean ± SD of triplicate wells.

[0049] As shown in Figure 3, four of the ANGPTL4 antisense oligonucleotides tested (A24022Hi (sequence number 25), A24023Hi (sequence number 26), A24044He (sequence number 47), and A24071Hi (sequence number 54)) showed greater than 50% knockdown of ANGPTL4 mRNA (corresponding to residual mRNA levels of less than 0.5), and treatment with the Neg1 negative control oligonucleotide did not result in a reduction in ANGPTL4 mRNA levels.

[0050] Example 4: Second single-dose efficacy screen of additional human ANGPTL4-specific antisense oligonucleotides in SK-OV3 cells Five thousand SK-OV3 cells per well were seeded into a 96-well plate and treated with each ANGPTL4 antisense oligonucleotide (A24071Hi (SEQ ID NO: 54), A24022Hi (SEQ ID NO: 25), A24076He (SEQ ID NO: 47), A24044He (SEQ ID NO: 47), A24023Hi (SEQ ID NO: 26), A24077He (SEQ ID NO: 47), A24075He (SEQ ID NO: 5), A24073Hi (SEQ ID NO: 55)) at a final concentration of 10 μM. To induce ANGPTL4 mRNA expression, the cells were simultaneously treated with 1 μM PPARδ (Sigma Aldrich, Cat. No. SML1491; see Example 1 for the preparation of a 10 mM stock solution). For a final concentration of 1 μM PPARδ, cells seeded into a 96-well plate were incubated with 100 μl of medium supplemented with 0.01 μl of PPARδ stock solution. As a negative control, cells were treated with the same volume of DMSO.

[0051] Three days after the start of treatment, cells were lysed, and mRNA expression of human HPRT1 and human ANGPTL4 was measured using the QuantiGene RNA Singleplex assay. ANGPTL4-mRNA expression values ​​were normalized to the expression of the housekeeping gene HPRT1. Residual ANGPTL4-mRNA expression relative to mock-treated cells ("no oligo" set to 1) is shown in Figure 4. Solid and dotted lines indicate 50% and 0% knockdown efficacy, respectively. Data are presented as the mean ± SD of triplicate wells.

[0052] Repeated screening of the optimized ANGPTL4-specific antisense oligonucleotides in SK-OV3 cells yielded five ANGPTL4 antisense oligonucleotides (A24071Hi (sequence number 54), A24022Hi (sequence number 25), A24076He (sequence number 47), A24044He (sequence number 47), and A24023Hi (sequence number 26)) with knockdown efficiencies of greater than 50% (Figure 4).

[0053] Example 5: In vitro TLR9 assay of selected human ANGPTL4-specific antisense oligonucleotides Binding of immunostimulatory ligands, such as bacterial DNA or immunostimulatory oligonucleotides containing or not containing unmethylated CpG dinucleotides, leads to TLR activation. Because immune activation can lead to severe and potentially fatal pathologies of excessive cytokine release, there is an urgent need for preclinical testing systems that predict cytokine release in humans.

[0054] HEK-Blue-hTLR9 (Invivogen catalog no. hkb-htlr9) cells were seeded into flat-bottom 96-well plates and treated with ANGPTL4 oligonucleotides A24022Hi (SEQ ID NO: 25), A24023Hi (SEQ ID NO: 26), A24071Hi (SEQ ID NO: 54), and A24076He (SEQ ID NO: 47) for 24 hours. Cell supernatants were then collected and incubated with QUANTI-Blue solution (Invivogen catalog no. rep-qbs) for 4 hours. SEAP activity was determined by measuring optical density. The mean and standard deviation of OD units relative to the OD units (set at 100) of cells stimulated with 5000 nM ODN2006 are shown in Figure 5. Data are presented as the mean ± SD of triplicate wells.

[0055] As shown in Figure 5, none of the tested ANGPTL4-specific antisense oligonucleotides induced TLR9 activation. In contrast, the positive control CpG oligonucleotide ODN2006 (5'-TCGTCGTTTTGTCGTTTTGTCGTT-3'PTO modified - Invivogen catalog number tlrl-2006; SEQ ID NO: 153) clearly stimulated NFκB activation (Figure 5).

[0056] Example 6: IC of selected human ANGPTL4-specific antisense oligonucleotides 50 decision 30,000 primary human hepatocytes / well were seeded in a 96-well plate and treated with various ANGPTL4 antisense oligonucleotides: A24022Hi (SEQ ID NO: 25), A24023Hi (SEQ ID NO: 26), A24071Hi (SEQ ID NO: 54) and A24076He (SEQ ID NO: 47) at various concentrations of 5000nM, 1000nM, 200nM, 40nM, 8nM and 1.6nM. To induce ANGPTL4 mRNA expression, cells were simultaneously treated with 1 μM PPARγ (Sigma Aldrich, Cat. No. R2408). PPARγ stock solution (10mM) was prepared by dissolving 10mg of PPARγ (molecular weight: 357.43) in 2.8ml of DMSO. For a final concentration of 1 μM PPARγ, cells seeded in 96-well plates were incubated with 100 μl of medium supplemented with 0.01 μl of PPARγ stock solution. As a negative control, cells were treated with the same volume of DMSO. Every 24 h, 70 μl of the supernatant was replaced with fresh medium containing 1 μM PPARγ and the indicated concentrations of the respective ANGPTL4 antisense oligonucleotides. Three days after the start of treatment, cells were lysed, and the mRNA expression of HPRT1 and ANGPTL4 was measured using the QuantiGene RNA Singleplex assay. ANGPTL4 mRNA expression values ​​were normalized to the expression of the housekeeping gene HPRT1. Residual ANGPTL4 mRNA expression relative to mock-treated cells (set at 1). For graphical presentation, mock-treated cells were set at 0.32 nM. Data are presented as the mean ± SD of triplicate wells.

[0057] Figure 6 and Table 5 show that the selected ANGPTL4-specific antisense oligonucleotides suppressed ANGPTL4 mRNA expression with IC in the nanomolar range. 50 It is demonstrated that the inhibition is dose-dependent at high values.

[0058] [Table 5]

[0059] Example 7: First single-dose screening of additional human ANGPTL4-specific antisense oligonucleotides (ASOs) in primary hepatocytes A total of 49 additional ANGPTL4-specific antisense oligonucleotides were designed. Based on two initial rounds of screening in human cell lines (data not shown), 17 promising ASOs were selected for a first round of screening in primary human hepatocytes (Figure 7). Three control oligonucleotides (R01002, R01014, and Neg1) of different lengths (16, 17, and 18 nucleotides, respectively) that had no sequence complementarity to any human or mouse mRNA were included as negative controls, and three ANGPTL4-specific oligonucleotides with confirmed knockdown efficiency (A24022Hi (SEQ ID NO: 25), A24071Hi (SEQ ID NO: 54), and A24076He (SEQ ID NO: 47)) were used as positive controls. Human primary hepatocytes (Lonza) were treated with each oligonucleotide at a single concentration of 5 μM for 3 days without the use of transfection reagent. To induce ANGPTL4 mRNA expression, cells were simultaneously treated with 1 μM PPARγ (Sigma Aldrich, Catalog No. R2408; see Example 6 for preparation of a 10 mM stock solution). For a final concentration of 1 μM PPARγ, cells seeded in a 96-well plate were incubated with 100 μl of medium supplemented with 0.01 μl of PPARγ stock solution. As a negative control, cells were treated with the same volume of DMSO.

[0060] Three days after the start of treatment, cells were lysed, and mRNA levels were determined by QuantiGene RNA Singleplex assay. Hypoxanthine phosphoribosyltransferase 1 (HPRT1) was used as a housekeeping gene for normalization of ANGPTL4 expression. As shown in Figure 7, PPARγ stimulation resulted in an approximately 50% increase in ANGPTL4 levels compared to DMSO-treated cells (no oligo).

[0061] Treatment with eight ANGPTL4-specific ASOs (A24096Hi (SEQ ID NO: 172), A24103He (SEQ ID NO: 179), A24091Hi (SEQ ID NO: 167), A24123He (SEQ ID NO: 197), A24083Hi (SEQ ID NO: 159), A24087Hi (SEQ ID NO: 163), A24102He (SEQ ID NO: 178), A24116He (SEQ ID NO: 192)) and positive control ASOs (A24022Hi (SEQ ID NO: 25), A24071Hi (SEQ ID NO: 54), A24076He (SEQ ID NO: 47)) reduced ANGPTL4 expression by more than 70% (corresponding to a residual ANGPTL4-mRNA expression of less than 0.3). Control oligonucleotides (Neg1, R01002, R01014) did not reduce ANGPTL4 mRNA expression.

[0062] Example 8: Second single-dose screening of additional human ANGPTL4-specific antisense oligonucleotides (ASOs) in primary hepatocytes The remaining newly designed ANGPTL4-specific ASOs were tested in primary hepatocytes under the experimental conditions of Example 7 (FIG. 8). These ASOs were well tolerated in subsequent in vitro tests (data not shown). One ANGPTL4-specific oligonucleotide with confirmed knockdown efficiency (A24076He (SEQ ID NO: 47)) was used as a positive control, and three control oligonucleotides (R01009, R01019, Neg1) of different lengths (16, 17, and 18 nucleotides, respectively) that did not have sequence complementarity to any human or mouse mRNA were included as negative controls.

[0063] Treatment with 10 ANGPTL4-specific ASOs (A24083Hi (SEQ ID NO: 159), A24089Hi (SEQ ID NO: 165), A24117He (SEQ ID NO: 193), A24124He (SEQ ID NO: 46), A24103He (SEQ ID NO: 179), A24097Hi (SEQ ID NO: 173), A24110He (SEQ ID NO: 186), A24121He (SEQ ID NO: 195), A24086Hi (SEQ ID NO: 162), A24085Hi (SEQ ID NO: 161)) reduced ANGPTL4 expression by more than 70% (corresponding to a residual ANGPTL4-mRNA expression of less than 0.3), whereas incubation with the positive control ASO A24076H reduced ANGPTL4 mRNA expression by approximately 63% (Figure 8). Control oligonucleotides (Neg1, R01009, R01019) only slightly reduced ANGPTL4 mRNA expression (6–20%).

[0064] Example 9: Activation of human Toll-like receptor 9 (hTLR9) in response to human angiopoietin-like protein 4 (ANGPTL4)-specific LNA-modified antisense oligonucleotides The potential of human ANGPTL4-specific LNA-modified antisense oligonucleotides A24076H (SEQ ID NO: 47), A24083Hi (SEQ ID NO: 159), A24085Hi (SEQ ID NO: 161), A24086Hi (SEQ ID NO: 162), A24087Hi (SEQ ID NO: 163), A24089Hi (SEQ ID NO: 165), A24096Hi (SEQ ID NO: 172), A24102He (SEQ ID NO: 178), A24103He (SEQ ID NO: 179), A24110He (SEQ ID NO: 186), A24111He (SEQ ID NO: 187), A24113He (SEQ ID NO: 189), A24116He (SEQ ID NO: 192), and A24123He (SEQ ID NO: 197) to activate TLR9 was tested. Experiments were performed once (A24076H, A24096H, A24102He, A24113He, A24116He, and A24123He) or twice (all other ASOs) in HEK-Blue-hTLR9 cells (Invivogen catalog number hkb-htlr9), a TLR9 reporter cell line, under the experimental conditions described in Example 5. As shown in Figures 9A, 9B, 9C, and 9D, no dose-dependent activation of NF-κB was observed after treatment of cells with different concentrations of each human ANGPTL4 ASO. In contrast, NF-κB was activated in a dose-dependent manner after treatment with the positive control ODN2006 (5'-TCGTCGTTTTGTCGTTTTGTCGTT-3'PTO modified - Invivogen catalog number tlrl-2006; SEQ ID NO: 153).

[0065] Example 10: IC of selected human ANGPTL4-specific antisense oligonucleotides (ASOs) 50 decision Based on knockdown efficiency in primary hepatocytes (Figures 7 and 8), the ASO with the most potent knockdown efficacy and showing no induction of caspase 3 / 7 after transfection (data not shown) was selected as IC 50 As a positive control, ANGPTL4-specific ASO A24076He (SEQ ID NO: 47), whose knockdown efficiency was confirmed, was used.

[0066] Primary human hepatocytes (Primacyt) were treated with different concentrations of ANGPTL4-specific ASO or negative control oligonucleotides Neg1, R01009, and R01019 for 3 days. At the same time, cells were treated with PPARγ (1 μM) (Sigma Aldrich, Cat. No. R2408; see Example 6 for the preparation of a 10 mM stock solution) to induce ANGPTL4 expression. For a final concentration of 1 μM PPARγ, cells seeded in a 96-well plate were incubated with 100 μl of medium supplemented with 0.01 μl of PPARγ stock solution. As a negative control, cells were treated with the same volume of DMSO. After 3 days, mRNA expression was analyzed using the QuantiGene Singleplex RNA assay.

[0067] Table 6 and Figure 10 show that eight ANGPTL4-specific ASOs (A24083Hi (SEQ ID NO: 159), A24085Hi (SEQ ID NO: 161), A24087Hi (SEQ ID NO: 163), A24089Hi (SEQ ID NO: 165), A24097Hi (SEQ ID NO: 173), A24103He (SEQ ID NO: 179), A24110He (SEQ ID NO: 186), and A24111He (SEQ ID NO: 187)) and the positive control A24076He (SEQ ID NO: 47) suppressed ANGPTL4 mRNA expression with IC50 in the nanomolar range. 50 Treatment with ASO A24086Hi (SEQ ID NO: 162) did not result in a dose-dependent decrease in ANGPTL4 mRNA expression (R 2 =0.5). Therefore, the data were not included.

[0068] Table 6 below lists the IC of select human ANGPTL4-specific antisense oligonucleotides determined in primary human hepatocytes. 50 value and R 2 Shows. * is R 2 is less than 0.85.

[0069] [Table 6]

[0070] Example 11: In vitro efficacy of human ANGPTL4-specific oligonucleotides in cynomolgus monkey hepatocytes The human ANGPTL4-specific antisense oligonucleotide A24076H (SEQ ID NO: 47), which is tolerated in vivo, and three additional human ANGPTL4-specific ASOs (Table 7) with only one mismatch to the cynomolgus monkey ANGPTL4 sequence were tested in primary cynomolgus monkey hepatocytes (Figure 11). All tested ASOs were shown to have confirmed knockdown efficiency in human cell lines and primary hepatocytes. Two control oligonucleotides (R01009, R01019) of different lengths (16 and 17 nucleotides, respectively) that do not have sequence complementarity to any human or mouse RNA were included as negative controls.

[0071] Table 7 lists human ANGPTL4-specific ASOs with demonstrated knockdown efficiency in human cells that do not induce caspase 3 / 7 in vitro. Cross-reactivity and number of mismatches with the cynomolgus monkey (Mfa, macaque fascicularis) ANGPTL4 sequence, as well as activity in primary cynomolgus monkey hepatocytes in vitro (Figure 11) are shown: [Table 7]

[0072] Primary cynomolgus monkey hepatocytes (Primacyt) were transfected with different concentrations of ANGPTL4-specific ASO or negative control oligonucleotides R01009 and R01019 for 3 days. At the same time, cells were treated with PPARδ (1 μM) (Sigma Aldrich, Cat. No. R2408; see Example 6 for the preparation of a 10 mM stock solution) to induce ANGPTL4 expression. For a final concentration of 1 μM PPARγ, cells seeded in a 96-well plate were incubated with 100 μl of medium supplemented with 0.01 μl of PPARγ stock solution. As a negative control, cells were treated with the same volume of DMSO. After 3 days, mRNA expression was analyzed using the QuantiGene Singleplex RNA assay.

[0073] As shown in Figure 11, treatment with PPARδ induced approximately 8-fold ANGPTL4 expression (Figure 11, DMSO control). Treatment with the ANGPTL4-specific ASO A24076He (SEQ ID NO: 47), which is completely cross-reactive with the cynomolgus monkey ANGPTL4 sequence, resulted in a knockdown of ANGPTL4 by more than 70% (corresponding to a residual mRNA expression of 0.3). In addition, human ANGPTL4-specific oligonucleotides (A24089Hi (SEQ ID NO: 165), A24110He (SEQ ID NO: 186), and A24111He (SEQ ID NO: 187)) with one mismatch with the cynomolgus monkey sequence reduced cynomolgus monkey ANGPTL4 mRNA by up to 51% (corresponding to a residual ANGPTL4-mRNA expression of 0.49). The negative control oligonucleotide R01009 did not reduce Angptl4 expression in primary cynomolgus monkey hepatocytes, whereas the control oligonucleotide R01019 only slightly reduced ANGPTL4 mRNA expression by approximately 25% when used at a concentration of 2 nM (corresponding to a residual ANGPTL4-mRNA expression of 0.75).

[0074] Conclusions based on Example 11: In summary, our in vitro experiments identified a highly potent human ANGPTL4-specific ASO suitable for testing ASO-based ANGPTL4-targeted therapeutics in cynomolgus monkeys. Such drugs could be used, for example, for the systemic treatment of patients with dyslipidemia to reduce ANGPTL4-mediated inhibition of lipoprotein lipase L and prevent cellular lipid overload, obesity, type II diabetes, and cardiovascular disease.

[0075] Example 12: First single-dose screening of mouse ANGPTL4-specific antisense oligonucleotides in 3T3 cells 4500 3T3 cells / well were seeded into 96-well plates and treated with the ANGPTL4 antisense oligonucleotides shown in Figure 12 at a final concentration of 10 μM. Cells were lysed, and the mRNA expression of mouse Hprt1 and mouse ANGPTL4 was measured using the QuantiGene RNA Singleplex assay. ANGPTL4-mRNA expression values ​​were normalized to the expression of the housekeeping gene Hprt1. Residual ANGPLT4-mRNA expression relative to mock-treated cells ("no oligo" set to 100) is shown in Figure 12. Solid and dotted lines indicate 50% and 0% knockdown efficacy, respectively. Data are presented as the mean ± SD of triplicate wells.

[0076] As shown in Figure 12, eight ANGPTL4 antisense oligonucleotides, namely, A24047M (sequence number 106), A24020M (sequence number 79), A24017M (sequence number 76), A24021M (sequence number 80), A24049M (sequence number 108), A24018M (sequence number 77), A24041M (sequence number 100), and A24010M (sequence number 69), reduced normalized ANGPTL4 expression by more than 50%, while the control oligonucleotide (Neg1) did not reduce ANGPTL4 mRNA expression (Figure 12).

[0077] Example 13: Second single-dose efficacy screen of mouse ANGPTL4-specific antisense oligonucleotides in Renca cells Five thousand Renca cells per well were seeded in a 96-well plate and treated with the ANGPTL4 antisense oligonucleotides shown in Figure 13 at a final concentration of 10 μM. Three days after the start of treatment, cells were lysed, and the mRNA expression of mouse Hprt1 and mouse ANGPTL4 was measured using the QuantiGene RNA Singleplex assay. ANGPTL4-mRNA expression values ​​were normalized to the expression of the housekeeping gene Hprt1. Residual ANGPTL4-mRNA expression relative to mock-treated cells ("no oligo" set to 1) is shown. Solid and dotted lines indicate 50% and 0% knockdown efficacy, respectively. Data are presented as the mean ± SD of triplicate wells.

[0078] As shown in Figure 13, treatment with two ANGPTL4-specific antisense oligonucleotides, namely, A24020M (sequence number 79) and A24019M (sequence number 78), resulted in greater than 50% ANGPTL4 knockdown (corresponding to residual mRNA levels of less than 0.5) (Figure 13).

[0079] Example 14: Testing of additional mouse ANGPTL4-specific antisense oligonucleotides in 4T1 cells 2500 4T1 cells / well were seeded in 96-well plates and treated with the ANGPTL4 antisense oligonucleotides (ASOs) shown in Figure 14 at a final concentration of 5 μM. After 3 days, the cell supernatant was replaced with fresh medium containing ASOs. Six days after the start of treatment, cells were lysed, and the mRNA expression of mouse Gapdh and mouse ANGPTL4 was measured using the QuantiGene RNA Singleplex assay. ANGPTL4-mRNA expression values ​​were normalized to the expression of the housekeeping gene Gapdh. Residual ANGPTL4-mRNA expression relative to Neg1-treated cells (set as 1) is shown. Solid and dotted lines indicate 80% and 0% knockdown efficacy, respectively. Data are presented as the mean ± SD of triplicate wells.

[0080] As shown in Figure 14, 12 of the antisense oligonucleotides tested, namely, A24017M (SEQ ID NO: 76), A24070M (SEQ ID NO: 127), A24020M (SEQ ID NO: 79), A24019M (SEQ ID NO: 78), A24069M (SEQ ID NO: 126), A24021M (SEQ ID NO: 80), A24011M (SEQ ID NO: 70), A24073M (SEQ ID NO: 130), A24018M (SEQ ID NO: 77), A24055M (SEQ ID NO: 114), A24010M (SEQ ID NO: 69), and A24065M (SEQ ID NO: 79), showed greater than 80% knockdown of ANGPTL4 mRNA (corresponding to residual mRNA levels of less than 0.2).

[0081] Example 15: Single-dose efficacy screening of intron-targeted murine ANGPTL4-specific antisense oligonucleotides in 4T1 cells 2500 4T1 cells / well were seeded in a 96-well plate and treated with the ANGPTL4 antisense oligonucleotides (ASOs) shown in Figure 15 at a final concentration of 5 μM. Three days after the start of treatment, the cell supernatant was replaced with fresh medium containing ASOs, and the cells were incubated for an additional three days. Cells were then lysed, and the mRNA expression of mouse Hprt1 and mouse ANGPTL4 was measured using the QuantiGene RNA Singleplex assay. ANGPTL4-mRNA expression values ​​were normalized to the expression of the housekeeping gene Hprt1. Residual ANGPTL4-mRNA expression relative to mock-treated cells (the "no oligo" control, set to 1) is shown. Solid and dotted lines indicate 50% and 0% knockdown efficacy, respectively. Data are presented as the mean ± SD of triplicate wells.

[0082] As shown in Figure 15, six of the ANGPTL4 antisense oligonucleotides tested, namely, A24047M (sequence number 106), A24095Mi (sequence number 151), A24093Mi (sequence number 149), A24020M (sequence number 79), A24090Mi (sequence number 146) and A24082Mi (sequence number 139), showed greater than 50% knockdown of ANGPTL4 mRNA (corresponding to residual mRNA levels of less than 0.5).

[0083] Example 16: IC of selected mouse ANGPTL4-specific antisense oligonucleotides 50 decision 2500 4T1 cells / well were seeded in a 96-well plate and treated with each of the ANGPTL4 antisense oligonucleotides (ASOs) A24018M (SEQ ID NO: 77), A24019M (SEQ ID NO: 78), A24020M (SEQ ID NO: 79), A24021M (SEQ ID NO: 80), A24047M (SEQ ID NO: 106), A24054M (SEQ ID NO: 113), A24065M (SEQ ID NO: 79), A24070M (SEQ ID NO: 127), A24072M (SEQ ID NO: 129), A24082M (SEQ ID NO: 139), and A24095Mi (SEQ ID NO: 151) at various concentrations of 5000 nM, 1000 nM, 200 nM, 40 nM, 8 nM, and 1.6 nM. Three days after the start of treatment, the cell supernatant was replaced with fresh medium containing ASO, and the cells were incubated for an additional three days. Cells were then lysed, and the mRNA expression of mouse Hprt1 and mouse ANGPTL4 was measured using the QuantiGene RNA Singleplex assay. ANGPTL4-mRNA expression values ​​were normalized to the expression of the housekeeping gene Hprt1. Residual ANGPTL4-mRNA expression is shown relative to mock-treated cells (the "no oligo" control, set at 1). Data are presented as the mean ± SD of triplicate wells.

[0084] Figure 16 and Table 8 show that selected ANGPTL4-specific antisense oligonucleotides suppressed ANGPTL4 mRNA expression with IC in the nanomolar range. 50 It is demonstrated that the inhibition is dose-dependent at high values.

[0085] [Table 8]

[0086] Example 17: First single-dose screening of additional murine Angptl4-specific ASOs in 4T1 cells Antisense oligonucleotides specific to mouse Angptl4 were designed. In the initial screening, 53 ASOs targeting Angptl4 mRNA were tested. Three control oligonucleotides (R01002, R01014, and Neg1) of different lengths (16, 17, and 18 nucleotides, respectively) with no sequence complementarity to any human or mouse mRNA were included as negative controls. Three ANGPTL4-specific oligonucleotides with confirmed knockdown efficiency (A24047M (SEQ ID NO: 106), A24072M (SEQ ID NO: 129), and A24095Mi (SEQ ID NO: 151)) were used as positive controls. Mouse breast cancer cells (4T1 cells) were treated with each oligonucleotide at a single concentration of 5 μM. After 3 days, the cell supernatant was replaced with fresh medium containing 5 μM of each ASO and incubated for an additional 3 days. Cells were then lysed, and mRNA levels were determined by QuantiGene RNA Singleplex assay. Hprt1 was used as a housekeeping gene for normalization of Angptl4 expression.

[0087] As shown in Figure 17, 15 ASOs (A24146Mi (SEQ ID NO: 247), A24047M (SEQ ID NO: 106), A24126Mi (SEQ ID NO: 227), A24120Mi (SEQ ID NO: 221), A24104M (SEQ ID NO: 207), A24108M (SEQ ID NO: 211), A24110M (SEQ ID NO: 213), A24139Mi (SEQ ID NO: 240), A24103M (SEQ ID NO: 206), A24112M (SEQ ID NO: 217)) were used. 107), A24122HMe (SEQ ID NO: 196), A24113M (SEQ ID NO: 108), A24125Mi (SEQ ID NO: 226), A24099M (SEQ ID NO: 202), A24095Mi (SEQ ID NO: 151)) reduced Angptl4 expression by more than 75% (corresponding to a residual Angptl4-mRNA expression of less than 0.25), while control oligonucleotides (Neg1, R01002, R01014) reduced Angptl4 mRNA expression by less than 50% (corresponding to a residual Angptl4-mRNA expression of more than 0.5) (Figure 17).

[0088] Example 18: Second single-dose screening of mouse Angptl4-specific ASOs in Renca cells For further confirmation in a different cell line, mouse Renca cells were treated with the same procedure as in Example 17. As a result, 19 Angptl4-specific ASOs (A24143Mi (SEQ ID NO: 244), A24047M (SEQ ID NO: 106), A24095Mi (SEQ ID NO: 151), A24125Mi (SEQ ID NO: 226), A24110M (SEQ ID NO: 213), A24148Mi (SEQ ID NO: 151), A24120Mi (SEQ ID NO: 221), A24104M (SEQ ID NO: 207), A24109M (SEQ ID NO: 212), A24139Mi (SEQ ID NO: 240), A24103M (SEQ ID NO: 213)) were detected. 06), A24122HMe (SEQ ID NO: 196), A24131Mi (SEQ ID NO: 232), A24138Mi (SEQ ID NO: 239), A24123Mi (SEQ ID NO: 224), A24146Mi (SEQ ID NO: 247), A24117Mi (SEQ ID NO: 218), A24130Mi (SEQ ID NO: 231), A24144Mi (SEQ ID NO: 245)) resulted in greater than 75% Angptl4 knockdown (corresponding to residual Angptl4-mRNA expression of less than 0.25) (Figure 18).

[0089] Example 19: IC of selected mouse ANGPTL4-specific antisense oligonucleotides 50 decision Based on the knockdown efficiency results in 4T1 and Renca cells (Figures 17 and 18), the nine ASOs with the most potent knockdown efficacy in Renca and 4T1 cells (A24103M (SEQ ID NO: 206), A24110M (SEQ ID NO: 213), A24122HMe (SEQ ID NO: 196), A24120Mi (SEQ ID NO: 221), A24125Mi (SEQ ID NO: 226), A24139Mi (SEQ ID NO: 240), A24143Mi (SEQ ID NO: 244), A24146Mi (SEQ ID NO: 247), and A24148Mi (SEQ ID NO: 151)) were selected based on their half inhibitory concentrations (IC 50 ) values ​​were selected for determination.

[0090] Primary mouse hepatocytes were treated with different concentrations of each ASO for 3 days. After 3 days, mRNA expression was analyzed using the QuantiGene Singleplex RNA assay. Figure 19 and Table 9 show that the selected Angptl4-specific ASOs suppressed Angptl4 mRNA expression with IC values ​​in the nanomolar range. 50 It is demonstrated that the inhibition is dose-dependent at high values.

[0091] Table 9 shows the IC of select Angptl4-specific ASOs determined in primary human hepatocytes. 50 Indicates the value. * is R 2 <0.85; ASOs with increased potential to induce caspase 3 / 7, resulting in a dose-dependent decrease in Hprt1 levels: [Table 9]

[0092] Conclusions based on Examples 7-11 and 17-19: A series of 47 additional ASOs with specificity for human ANGPTL4 were tested to select several ASOs that strongly reduced human ANGPTL4 expression at the mRNA level in primary hepatocytes. Treatment with 16 of the tested ASOs demonstrated greater than 70% knockdown of ANGPTL4 mRNA in primary human hepatocytes, thereby identifying the most potent candidates as IC 50 Values ​​were in the low nanomolar range.

[0093] To conduct in vivo experiments in a mouse model, ASOs with specificity for mouse Angptl4 were designed, and successful candidate ASOs were selected for in vivo studies to strongly knockdown mouse Angptl4 expression in vitro.

[0094] In summary, a comprehensive series of in vitro experiments identified a highly potent human ANGPTL4-specific ASO suitable for the development of ASO-based ANGPTL4-targeting therapeutics.

Claims

1. An ANGPTL4 inhibitor comprising an oligonucleotide containing 12 to 22 nucleotides, wherein at least one of the nucleotides is modified, the oligonucleotide hybridizes with the nucleic acid sequence of ANGPTL4 (human) within positions 234 to 261 of SEQ ID NO: 1, and / or within positions 2800 to 2872, 3415 to 3442, 4968 to 4994, or 6603 to 6631 of SEQ ID NO: 2, the oligonucleotide inhibits the expression of ANGPTL4, and the oligonucleotide contains a sequence selected from the group consisting of SEQ ID NO: 159, SEQ ID NO: 161, SEQ ID NO: 162, SEQ ID NO: 163, SEQ ID NO: 165, SEQ ID NO: 173, SEQ ID NO: 178, SEQ ID NO: 179, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 54, and combinations thereof.

2. The inhibitor according to claim 1, wherein the oligonucleotide consists of 12 to 22 nucleotides.

3. The inhibitor according to claim 1 or 2, wherein the modified nucleotide is selected from the group consisting of cross-linked nucleic acids such as LNA, cET, ENA, 2'-fluoro-modified nucleotide, 2'O-methyl-modified nucleotide, 2'O-methoxyethyl-modified nucleotide, and combinations thereof.

4. The oligonucleotide is +T ＊ +C ＊ +G ＊ A ＊ G ＊ A ＊ T ＊ G ＊ A ＊ A ＊ C ＊ G ＊ G ＊ +A ＊ +G ＊ +A (SEQ ID NO: 159), +A ＊ +A ＊ +C ＊ T ＊ T ＊ A ＊ G ＊ A ＊ G ＊ A ＊ A ＊ C ＊ C ＊ G ＊ +C ＊ +G ＊ +A (SEQ ID NO: 161), +T ＊ +T ＊ +A[[ID=6%]] ＊ G ＊ A ＊ G ＊ A ＊ A ＊ C ＊ C ＊ G ＊ C ＊ G ＊ +A ＊ +G ＊ +T (SEQ ID NO: 163), +T ＊ +C ＊ +G ＊ A ＊ A ＊ A ＊ T ＊ G ＊ A ＊ G ＊ T ＊ C ＊ T ＊ G ＊ +C ＊ +A ＊ +C (SEQ ID NO: 165), +C ＊ +T ＊ +T ＊ A ＊ A ＊ C ＊ A ＊ G ＊ T ＊ G ＊ G ＊ A ＊ T ＊ G ＊ +A ＊ +C ＊ +C (allocation number 173), +T ＊ +A ＊ +G ＊ C ＊ A ＊ C ＊ G ＊ G ＊ C ＊ G ＊ G ＊ T ＊ G ＊ +G ＊ +C ＊ +G (allocation number 179), +G ＊ +T ＊ +G ＊ T ＊ T ＊ G ＊ T ＊ A ＊ A ＊ C ＊ C ＊ T ＊ C ＊ T ＊ +T ＊ +G ＊ +T (array number 25), +C ＊ +C ＊ +G ＊ T ＊ G ＊ T ＊ T ＊ G ＊ T ＊ A ＊ A ＊ C ＊ C ＊ T ＊ +C ＊ +T ＊ +T (array number 26), +C ＊ +G ＊ +T ＊ G ＊ T ＊ T ＊ G ＊ T ＊ A ＊ A ＊ C ＊ C ＊ T ＊ C ＊ +T ＊ +T ＊ +G (SEQ ID NO: 54), +G * +C * +C * G * T * G * A * A * C * T * T * A * G * A * +G * +A * +A (SEQ ID NO: 162), +T * +A * +G * C * A * C * G * G * C * G * G * T * G * G * +C * +G * +G (SEQ ID NO: 178) and combinations thereof are selected from the group, where + indicates an LNA nucleotide. ＊ The inhibitor according to any one of claims 1 to 3, wherein is a phosphorothioate (PTO) bond between nucleotides.

5. The inhibitor according to any one of claims 1 to 4, wherein the inhibitor inhibits the expression of ANGPTL4 at a nanomolar or micromolar concentration.

6. A pharmaceutical composition comprising the inhibitor according to any one of claims 1 to 5 and a pharmaceutically acceptable carrier, excipient, diluent, or combination thereof.

7. An inhibitor according to any one of claims 1 to 5 or a pharmaceutical composition according to claim 6, for use in a method of preventing and / or treating disorders involving ANGPTL4 imbalance.

8. The inhibitor or pharmaceutical composition for use according to claim 7, wherein the disorder is a cardiovascular metabolic disease, obesity, diabetes such as type 2 diabetes, hypercholesterolemia, hypertriglyceridemia (HTG), dyslipidemia, pancreatitis, metabolic syndrome, familial hyperchylomicronemia syndrome (FCS), influenza infection and / or cancer.

9. The inhibitor or pharmaceutical composition for use according to claim 7 or 8, wherein the hypercholesterolemia is homozygous familial hypercholesterolemia (HoFH) or heterozygous familial hypercholesterolemia (HeFH).

10. The aforementioned cancers include breast cancer, lung cancer, malignant melanoma, lymphoma, skin cancer, bone cancer, prostate cancer, liver cancer, brain cancer, larynx, gallbladder, pancreas, testis, rectum, parathyroid gland, thyroid gland, adrenal gland, nerve tissue, head and neck, colon, stomach, bronchi, kidney cancer, basal cell carcinoma, squamous cell carcinoma, metastatic skin cancer, osteosarcoma, Ewing's sarcoma, reticulum sarcoma, liposarcoma, myeloma, giant cell tumor, small cell lung tumor, islet cell tumor, primary brain tumor, meningioma, acute and chronic lymphocytic and granulocytic tumors, and acute and chronic myeloid leukoma. An inhibitor or pharmaceutical composition for use according to any one of claims 7 to 9, wherein the patient is a hematological disease, pilosarcoma, adenoma, hyperplasia, medullary carcinoma, enteric ganglioneuroma, Wilms' tumor, seminomas, ovarian tumor, leiomyoma, cervical dysplasia, retinoblastoma, soft tissue sarcoma, malignant carcinoid, focal skin lesion, rhabdomyosarcoma, Kaposi's sarcoma, osteogenic sarcoma, malignant hypercalcemia, renal cell tumor, polycythemia vera, adenocarcinoma, anaplastic astrocytoma, glioblastoma multiforme, leukemia, or epidermoid carcinoma.

11. The inhibitor or pharmaceutical composition for use according to any one of claims 7 to 10, wherein the inhibitor or composition is suitable for local or systemic administration.

12. An inhibitor or pharmaceutical composition for use according to any one of claims 7 to 11, wherein the composition is suitable for single or repeated administration.

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