WO2020099525A2

WO2020099525A2 - Oligonucleotides influencing the regulation of the fatty acid metabolism

Info

Publication number: WO2020099525A2
Application number: PCT/EP2019/081250
Authority: WO
Inventors: Stefan K. NILSSON; Gunilla Olivecrona; Frank Jaschinski; Anne Sadewasser; Sven MICHEL
Original assignee: Lipigon Pharmaceuticals AB
Current assignee: Lipigon Pharmaceuticals AB
Priority date: 2018-11-13
Filing date: 2019-11-13
Publication date: 2020-05-22
Anticipated expiration: 2021-05-13
Also published as: WO2020099525A3

Abstract

The present invention refers to a composition comprising an ANGPTL3 oligonucleotide and an ANGPTL4 oligonucleotide hybridizing with ANGPTL3 and ANGPTL4 mRNA, pre-mRNA and/or a combination thereof, and inhibiting the expression of ANGPTL3 and ANGPTL4. The composition is for example in use for treating a disorder such as cardiometabolic disease, obesity, diabetes such as type 2 diabetes, hypercholesterolemia, hypertriglyceridemia (HTG), dyslipidemia, pancreatitis, metabolic syndrome, familial chylomicronemia syndrome (PCS) and/or cancer.

Description

Oligonucleotides influencing the regulation of the fatty acid metabolism

The present invention refers to a composition comprising inhibitors of ANGPTL3 and/or ANGPTL4 such as antisense oligonucleotides and a pharmaceutical composition comprising such inhibitors as well as its use for treating a cardiovascular disease, obesity, diabetes type II, homozygote familial hypercholesterolemia (HoFH),

heterozygote familial hypercholesterolemia (HeFH) or dislipidemia.

Technical background

Disturbed plasma lipids are well-known risk factors in cardiometabolic disease.

Successful treatment for elevated LDL-cholesterol has been given since the mid-80’s and the following decades focus has broadened towards other lipid classes such as HDL- cholesterol and triglycerides. Epidemiological studies have revealed that increased plasma triacylglycerol (TG) and concomitant remnant cholesterol is an independent risk factor for coronary heart disease (Cullen, 2000). Furthermore, hypertriglyceridemia (HTG) is a hallmark of the metabolic syndrome (MS) and is often accompanied by 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 maintenance of plasma TG homeostasis is highly desirable.

Patients with severe hypertriglyceridemia can also develop pancreatitis (Athyros, 2002), particularly when TG levels exceed 1000-1500 mg/dl (Tsuang, 2009). As many as 40 different genes are now known to regulate plasma TG (Johansen, 2011), but only a few monogenetic disorders are known to markedly increase TG (Nordestgaard and Varbo, 2014). These comprises FCS which is described in more detail below.

Lipolysis is a key step in clearance of TG-rich lipoproteins that takes place on the luminal surface of capillaries of heart, skeletal muscle, and adipose tissues. LPL synthesized in muscle and adipocytes is translocated to capillary endothelial cells. Rare genetic defects in lipoprotein lipase (LPL) (Benlian, 1996), the main enzyme responsible for the hydrolysis of TG on lipoproteins, can lead to familial chylomicronemia syndrome (FCS) characterized by plasma TG levels well over >10 mmol. Homozygous defects in apolipoprotein C-II (apoC-II), a key protein activator of LPL, can also lead to a similar hypertriglyceridemic phenotype (Breckenridge, 1978). More recently, defects in

GPIHBP1, a protein that links LPL to the surface of endothelial cells (Beigneux, 2007), and mutations in apolipoprotein A-V (ApoA-V) (Ishihara, 2005) have also been described to cause hypertriglyceridemia in humans. Genetic defects in the lipase specific chaperone LMF1 has also been found to promote FCS. Taken together about 2-3:1 000 000 patients have FCS.

Not only LPL activating factors affect the LPL system; loss of function mutations in LPL negative regulators such as apoC3 and ANGPTL3, ANGPTL4 or ANGPTL8 has been shown to promote a favorable plasma lipid profile and a reduced risk for metabolic diseases. ANGPTL3 and ANGPTL4, respectively, are both regulators of different lipases and LPL in particular. Both proteins are unfolding chaperones that brakes up the dimeric catalytically active form of LPL into inactive monomers which is an irreversible event. The ANGPTLs are the only known factors to regulate LPL in this manner, compared to e.g. apoC3 which displaces LPL from lipid substrates. In addition

ANGPTL3 and ANGPTL4 affects hepatic lipase and endothelial lipase thus affecting not only the TG moiety of plasma lipids but also LDL-c and HDL-c. ANGPTL3 is mainly expressed by the liver and regulates postprandial plasma lipids in concert with co-factor ANGPTL8 whereas ANGPTL4 is a fasting induced factor expressed also by the liver but to a relevant extent by adipose tissue and skeletal muscle as well, i.e., ANGPTL4 is expressed ubiquitary. The expression of ANGPTL4 is regulated by different stimuli; it is for example induced in the liver by Peroxisome Proliferator- Activator Receptor (PPAR)ot, PPAR3 and the glucocorticoid receptor (GR), respectively. Animal models deficient for these ANGPTLs show increased LPL activity and decreased plasma lipids and mice with transgenic overexpression for the human variants show the opposite. The findings from animal studies are supported by human deficiency and loss of function mutations which for ANGPTL3 correlates with plasma TG levels and LDL-c and for ANGPTL4 with plasma TG levels and HDL-c. Both genes show a link to cardiometabolic diseases.

Thus, information to date provides new insights into the coordinate activities of LPL, GPIHBP1, ANGPTLs and apoA-V in plasma TG homeostasis. Among these factors, ANGPTL3s also regulates plasma cholesterol levels i.e. LDL-c, HDL-c and remnant-c intriguingly without being all dependent on the LDL-receptor which in most cases is non-functional in homozygote familial hypercholesterolemia (HoFH) and heterozygote familial hypercholesterolemia (HeFH). This provides an opportunity for an“all-purpose” plasma lipid drug while targeting ANGPTL3 and ANGPTL4.

Oligonucleotides often naturally accumulate in the liver, which is advantageous in the targeting of ANGPTL3, mainly expressed in the liver. ANGPTL3 regulates the activity of the lipoprotein lipase that plays an important role in the intake of free fatty acids into the liver. Dysregulation of lipoprotein lipase can lead to a lipid excess in the cells, which results for example in obesity, diabetes type II or cardiovascular diseases. ANGPTL3 Loss-of-Function mutations correlate with reduced plasma triglycerides and a reduced LDL cholesterol level. Further effects of ANGPTL3 Loss-of-Function mutations are increased activity of lipoprotein lipase and endothelial lipase, increased insulin sensitivity and reduced amounts of fatty acids in the serum.

ANGPTL4 knock-out mice show reduced triglyceride (TG) level based on increased degradation of very low-density lipoprotein (VLDL) and reduced VLDL production. The cholesterol level is influenced moderately. Food having high lipid level results in

ANGPTL4 knock-out mice which are treated with monoclonal antibodies to reduced viability due to lesions of lipogranuloma of the intestinal tissue, the effluent lymphatic system and/or the mesenteric lymph nodes (Desai et ah, 2007 PNAS). Humans being heterozygous for the ANGPTL4 variant E40K show significant lower plasma TG level when fasting. Also the high density lipoprotein (HDL) cholesterol levels were

significantly higher in E40K heterozygous humans. As the combination of high TG and low HDL cholesterol level leads to an increased risk to suffer from cardiovascular diseases, the reduction or inhibition of ANGPTL4 may reduce the risk. ANGPTL4 null alleles exist in humans, but a pathology comparable to ANGPTL4 knock-out mice has not been identified so far.

Oligonucleotides of the present invention inhibiting the expression of ANGPTL3 and/or ANGPTL4 reduce for example the plasma lipid level independent of LDL receptor functionality, which is relevant for example for use of the oligonucleotides in treating homozygote familial hypercholesterolemia (HoFH) or heterozygote familial

hypercholesterolemia (HeFH), where the LDL receptor is defect. So far no antisense oligonucleotides exist which are highly efficient in reduction and inhibition, respectively, of ANGPTL3 and ANGPTL4 expression and hybridize with ANGPTL3 and ANGPTL4 mRNA and/or pre-mRNA. Studies with siRNA to inhibit ANGPTL3 and ANGPTL4 expression showed that in vivo inhibition is only possible if siRNA is packed in suitable packaging material. Even if siRNA is packed the efficiency on the inhibition of mRNA expression can often not be improved.

A combination of oligonucleotides of the present invention is very successful in the inhibition of the expression of ANGPTL3 and ANGPTL4. The mode of action of an oligonucleotide differs from the mode of action of an antibody or small molecule, and oligonucleotides are highly advantageous regarding for example

(i) the penetration into tissues,

(ii) the blocking of multiple functions and activities, respectively, of a target,

(iii) the combination of oligonucleotides with each other or an antibody or a small molecule, and

(iv) the inhibition of intracellular effects which are not accessible or not specifically accessible for an antibody or inhibitable via a small molecule.

Summary of the invention

The present invention refers to a composition comprising or consisting of inhibitors of ANGPTL3 and ANGPTL4 such as oligonucleotides comprising or consisting of for example 12 to 22 nucleotides, 15 to 20 nucleotides, or 15, 16, 17, 18, 19 or 20 nucleotides, wherein at least one of the nucleotides is modified. The ANGPTL3 oligonucleotide hybridizes for example with a nucleic acid sequence of ANGPTL3 of SEQ ID NO.l (human; NM_014495.3), ANGPTL3 of SEQ ID NO. 2 (human;

GRCh38:l:62597487:62606159) and/or with a nucleic acid sequence of ANGPTL3 of SEQ ID NO.47 (mouse; NM_013913.4), wherein the oligonucleotide inhibits the expression of ANGPTL3. The ANGPTL4 oligonucleotide hybridizes for example with a nucleic acid sequence of ANGPTL4 of SEQ ID NO.98 (human; NM_139314), ANGPTL4 of SEQ ID NO. 99 (human; GROh38_19_8364151_8374373) ANGPTL4 of SEQ ID NO.155 (mouse; NM 020581.2) and/or SEQ ID NO.156 (mouse; GRCm38: 17:33773750:33781575), wherein the oligonucleotide inhibits the expression of ANGPTL4. Thus, the composition of the present invention comprises or consists of an ANGPTL3 oligonucleotide and an ANGPTL4 oligonucleotide hybridizing with ANGPTL3 and ANGPTL4 mRNA, pre- mRNA and/or a combination thereof, and inhibits the expression of ANGPTL3 and ANGPTL4. In addition, an ANGPTL3 oligonucleotide inhibits for example the expression of ANGPTL4 and/or an ANGPTL4 oligonucleotide inhibits for example the expression of ANGPTL3. Alternatively, the composition comprises

i) an ANGPTL3 modified oligonucleotide hybridizing with ANGPTL3 mRNA or pre-mRNA inhibiting the expression of ANGPTL3, and

ii) an ANGPTL4 modified oligonucleotide hybridizing with ANGPTL4 mRNA or pre-mRNA inhibiting the expression of ANGPTL4.

The combination of an ANGPTL3 and ANGPTL4 oligonucleotide results for example in an increased inhibitory effect which higher than the inhibitory effect of each of these oligonucleotides separately. Hence, the composition comprising or consisting of an ANGPTL3 and ANGPTL4 oligonucleotide shows for example a synergistic effect.

The modified nucleotide is for example selected from the group consisting of a bridged nucleic acid such as LNA, cET, ENA, 2 'Fluoro modified nucleotide, 2O-Methyl modified nucleotide, 2’ O-Methoxyethyl modified nucleotide and a combination thereof.

The ANGPTL3 oligonucleotide of the present invention hybridizes for example with an active area selected from position 45-72 (e.g., A26004H, SEQ ID NO.6) or from position 1130-1170 (e.g., A26019H, SEQ ID NO.20; A26020H, SEQ ID NO.20; A26021H, SEQ ID NO. 21; A26022H, SEQ ID N0.22; A26023H, SEQ ID N0.23; A26024H, SEQ ID N0.24) of SEQ ID NO.l and/or from position 3060-3086 (e.g., A26033HΪ, SEQ ID NO.33) or from position 5768-5794 (e.g., A26036Hi, SEQ ID NO.36; A26037Hi, SEQ ID NO.36) of SEQ ID NO.2. The ANGPTL4 oligonucleotide of the present invention hybridizes for example with an active area selected from position 1732-1759 (e.g., A24044He, SEQ ID NO.143; A24076He, SEQ ID NO.143) and/or from position 234-261 (e.g. A24102He, SEQ ID NO. 276; A24103He, SEQ ID NO. 277) and/or from position 1264-1293 (e.g. A24110He, SEQ ID NO. 284; A24111He, SEQ ID NO. 285) of SEQ ID NO.98 and/or from position 2800- 2872 (e.g., A24083HΪ, SEQ ID NO. 257; A24085HΪ, SEQ ID NO. 259; A24086HΪ, SEQ ID NO. 260; A24087HΪ, SEQ ID NO. 261) and/or from position 3415-3442 (e.g. A24089HΪ, SEQ ID NO. 263) and/or from position 4968-4994 (e.g., A24097HΪ, SEQ ID NO. 271) of SEQ ID NO.99. Each of the ANGPTL3 oligonucleotide and the ANGPTL4 oligonucleotide inhibits the expression of ANGPTL3 and/or ANGPTL4 for example at a nanomolar or micromolar concentration. The composition of the present invention comprises or consists of for example one or more ANGPTL3 oligonucleotide(s) selected from SEQ ID NO. 3 to SEQ ID NO.45 and SEQ ID NO. 48 to SEQ ID NO. 97 and one or more ANGPTL4 oligonucleotide(s) selected from SEQ ID NO.100 to SEQ ID NO.154 , SEQ ID NO.157 to 249, SEQ ID NO. 252 to SEQ ID NO.298 and/or SEQ ID NO. 301 to SEQ ID NO.349, or a combination thereof.

The composition of the present invention comprises optionally a pharmaceutically acceptable carrier, excipient, dilutant or a combination thereof.

The composition is for example for use in a method of preventing and/or treating a disorder, where an ANGPTL3 and/or ANGPLT4 imbalance is involved. Such disorder is for example a cardiometabolic disease, obesity, diabetes such as type 2 diabetes, hypercholesterolemia, hypertriglyceridemia (HTG), dyslipidemia, pancreatitis, metabolic syndrome, familial chylomicronemia syndrome (FCS) and/or cancer. The

hypercholesterolemia is for example a homozygote familial hypercholesterolemia (HoFH) or heterozygote familial hypercholesterolemia (HeFH). The 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, gall bladder, pancreas, testicular, rectum, parathyroid, thyroid, adrenal, neural tissue, head and neck, colon, stomach, bronchi, kidneys, basal cell carcinoma, squamous cell carcinoma, metastatic skin carcinoma, osteo sarcoma, 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, hairy-cell tumor, adenoma, hyperplasia, medullary carcinoma, intestinal ganglioneuromas, Wilm's tumor, seminoma, ovarian tumor, leiomyomater tumor, cervical dysplasia, retinoblastoma, soft tissue sarcoma, malignant carcinoid, topical skin lesion, rhabdomyosarcoma, Kaposi's sarcoma, osteogenic sarcoma, malignant hypercalcemia, renal cell tumor, polycythermia vera, adenocarcinoma, anaplastic astrocytoma, glioblastoma multiforma, leukemia, or epidermoid carcinoma.

The composition of the present invention is for example suitable to be administered locally or systemically. It is suitable to be administered for example once or repeatedly.

All documents cited or referenced herein (“herein cited documents”), and all documents cited or referenced in herein cited documents, together with any manufacturer's instructions, descriptions, product specifications, and product sheets for any products mentioned herein or in any document incorporated by reference herein, are hereby incorporated herein by reference, and may be employed in the practice of the invention. More specifically, all referenced documents are incorporated by reference to the same extent as if each individual document was specifically and individually indicated to be incorporated by reference.

Description of the figures

Fig. 1 shows IC50 determination of nine selected human ANGPTL3-specific antisense oligonucleotides (A26004H (SEQ ID NO.6), A26019H (SEQ ID NO.20), A26020H (SEQ ID NO.20), A26022H (SEQ ID N0.22), A26023H (SEQ ID N0.23), A26024H (SEQ ID NO.24), A26033HΪ (SEQ ID NO.33), A26036HΪ (SEQ ID NO.36) and A26037HΪ (SEQ ID NO.36)). 15,000 primary human hepatocytes / well were seeded in 96-well plates and treated with different concentrations of the respective antisense oligonucleotides (1000 nM, 200 nM, 40 nM, 8 nM, 1.6 nM, 0.32 nM). Three days after treatment, cells were lysed and HPRT1 and ANGPTL3 mRNA expression were measured using the

QuantiGene RNA Singleplex assay. ANGPTL3-mRNA expression values were normalized to expression of the housekeeping gene HPRT1. Residual ANGPTL3-mRNA expression relative to mock-treated cells (set as 1). For graphic representation mock- treated cells were set as 0.16 nM. Triplicate wells, mean+/-SD. Data are represented as mean of triplicate wells +/-SD.

Fig. 2 depicts IC5₀ determination of selected human ANGPTL4-specific antisense oligonucleotides A24022HΪ (SEQ ID NO.121), A24023HΪ (SEQ ID NO.122), A24071HΪ (SEQ ID NO.152) and A24076He (SEQ ID NO.143). Human primary hepatocytes were treated with the respective ANGPTL4 antisense oligonucleotides at different

concentrations (5000 nM, 1000 nM, 200 nM, 40 nM, 8 nM, 1.6 nM) for three days and simultaneously treated with PPARy (1 mM). Three days after start of treatment, mRNA expression was analyzed using the QuantiGene Singleplex RNA assay. For graphic representation mock-treated cells were set as 0.32 nM. Data are represented as mean of triplicate wells +/-SD.

Fig. 3 shows IC50 determination of further 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 the respective ASO. Every 24 h, 70 mΐ of supernatant was replaced with fresh medium containing the respective ASOs at indicated concentrations. After three days, cells were lysed and HPRT1 and ANGPTL4 mRNA expression was measured using the QuantiGene RNA Singleplex assay. ANGPTL4-mRNA expression values were normalized to 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 represented as mean of triplicate wells +/-SD.

Fig. 4A - 4K shows an analysis of dose-dependent inhibition of human ANGPTL3- specific ASOs in combination with human ANGPTL4- specific ASOs in human

hepatocytes. 25,000 primary human hepatocytes / well were seeded in 96-well plates and treated with different concentrations (5000 nM, 1000 nM, 200 nM, 40 nM, 8 nM, 1.6 nM) of the respective ASO as well as 1 mM PPARy to induce ANGPTL4 expression. Equal amount of DMSO served as vehicle control. Every 24 hours, 70 mΐ of supernatant was replaced with fresh medium containing respective ASOs at indicated concentrations. After three days, cells were lysed and Hypoxanthine phosphoribosyltransferase 1 (HPRT1), ANGPTL4 and ANGPTL3 mRNA expression were measured using the QuantiGene RNA Singleplex assay. Fig. 4A-4E: ANGPTL4-mRNA expression values were normalized to expression of the housekeeping gene HPRT1. Residual ANGPTL4- mRNA expression relative to mock-treated cells (set as 1 (n=24), SD=0.23). Fig. 4F-4J: ANGPTL3-mRNA expression values were normalized to expression of the housekeeping gene HPRT1. Residual ANGPTL3-mRNA expression relative to mock-treated cells (set as 1 (n=24), SD=0.14). Fig. 4K: HPRTl-mRNA expression values were normalized to mock-treated cells (set as 100 (n=24), SD=11.5). Black lines indicate 0% (equivalent to residual mRNA level of 1.0 (Fig. 4A-4L) or 100 (Fig. 4M), respectively) knockdown efficacy. Data are represented as mean of triplicate wells +/-SD.

Fig. 5 shows IC50 determination of selected mouse ANGPTL3-specific antisense oligonucleotides (A26007M (SEQ ID NO.54), A26016M (SEQ ID NO.61), A26019M (SEQ ID NO.64), A26020M (SEQ ID NO.65), A26021M (SEQ ID NO.66), A26032M (SEQ ID NO.76), A26033M (SEQ ID NO.77), A26034M (SEQ ID N0.78), A26035M (SEQ ID NO.79), A26039M (SEQ ID NO.83) and A26047M (SEQ ID NO.90)). 15,000 primary human hepatocytes / well were seeded in 96-well plates and treated with different concentrations (5000 nM, 1000 nM, 200 nM, 40 nM, 8 nM, 1.6 nM) of the respective antisense oligonucleotides. Three days after start of treatment, cells were lysed and HPRT1 and ANGPTL3 mRNA expression were measured using the QuantiGene RNA Singleplex assay. ANGPTL3-mRNA expression values were normalized to expression of the housekeeping gene HPRT1. Residual ANGPTL3-mRNA expression relative to mock- treated cells (set as 1). Triplicate wells, mean+/-SD.

Fig. 6 depicts IC50 determination of selected mouse ANGPTL4-specific antisense oligonucleotides (A24018M (SEQ ID NO.174), A24019M (SEQ ID NO.175), A24020M (SEQ ID NO.176), A24021M (SEQ ID NO.177), A24047M (SEQ ID NO.203), A24054M (SEQ ID NO.210), A24065M (SEQ ID NO.176), A24070M (SEQ ID N0.224), A24072M (SEQ ID NO.226), A24082M (SEQ ID N0.235) and A24095MΪ (SEQ ID N0.248) which were selected for determination of half maximal inhibitory concentration (IC5₀) values. 4T1 cells were treated with these ANGPTL4 antisense oligonucleotides at different concentrations (5000 nM, 1000 nM, 200 nM, 40 nM, 8 nM, 1.6 nM) for three days. After three days, cell supernatant was replaced by fresh medium containing 5 mM of the respective ANGPTL4 antisense oligonucleotides and incubated for additional three days. Then, mRNA expression was analyzed using the QuantiGene Singleplex RNA assay. ANGPTL4-mRNA expression values were normalized to expression of the housekeeping gene HPRT1. Residual ANGPTL4-mRNA expression relative to mock-treated cells (set as 1). Triplicate wells, mean+/-SD.

Fig. 7 shows IC5₀ determination of further selected mouse Angptl4-specific ASOs. 15,000 primary mouse hepatocytes / well were seeded in 96-well flat bottom plates and treated with different concentrations (5000 nM, 1000 nM, 200 nM, 40 nM, 8 nM, 1.6 nM) of the respective ASO. After three days, cells were lysed and Hprtl and Angptl4 mRNA expression were measured using the QuantiGene RNA Singleplex assay. Angptl4-mRNA expression values were normalized to expression of the housekeeping gene Hprtl.

Residual Angptl4-mRNA expression relative to mock-treated cells (set as 1 (n=36), SD=0.17). Negl is displayed in black. Graph below shows Hprtl raw values relative to no oligo (set as 100 (n=36), SD=13.0). Data are represented as mean of triplicate wells +/- SD.

Fig. 8 depicts effects of ASO treatment on Hprtl mRNA levels of primary mouse hepatocytes upon testing of the in vitro compatibility of mouse ANGPTL3-specific and ANGPTL4-specific antisense oligonucleotides. 15,000 primary mouse hepatocytes (Lonza) were seeded in BioCoat Collagen I 96-well flat bottom plates in 100 mΐ Hepatocyte Plating Medium. Supernatant was removed 4-6 h after seeding and ASOs were added at indicated concentrations (5000 nM, 1000 nM, 200 nM, 40 nM, 8 nM, 1.6 nM) diluted in 100 mΐ Maintenance Medium. Cells were cultured for three days at 37°C. After three days, cells were lysed and Hypoxanthine phosphoribosyltransferase 1 (Hprtl) mRNA expression was measured using the QuantiGene RNA Singleplex assay. Hprtl- mRNA expression values were normalized to mock-treated cells (set as 1 (n=9), SD=0.29). Dotted line indicates 0% (equivalent to residual mRNA level of 1.0) decrease of Hprtl mRNA levels. Data are represented as mean of triplicate wells +/-SD.

Fig. 9 depicts an analysis of dose- dependent inhibition of mouse Angptl3-specific ASOs in combination with mouse Angptl4- specific ASOs in mouse hepatocytes. 15,000 primary mouse hepatocytes (Lonza) were seeded in BioCoat Collagen I 96-well flat bottom plates in 100 mΐ Hepatocyte Plating Medium. Supernatant was removed 4-6 h after seeding and ASOs were added at indicated concentrations (5000 nM, 1000 nM, 200 nM, 40 nM, 8 nM, 1.6 nM) diluted in 100 mΐ Maintenance Medium. Cells were cultured for three days at 37°C. After three days, cells were lysed and Hypoxanthine phosphoribosyltransferase 1 (Hprtl) and Angptl3 mRNA expression were measured using the QuantiGene RNA Singleplex assay. Angptl3-mRNA expression values were normalized to expression of the housekeeping gene Hprtl. Residual Angptl3-mRNA expression relative to mock-treated cells (set as 1 (n=9), SD=0.34). Dotted line indicates 0% (equivalent to residual mRNA level of 1.0) decrease of Angptl3 mRNA levels. Data are represented as mean of triplicate wells +/-SD.

Fig. 10 shows an analysis of dose-dependent inhibition of mouse Angptl4- specific ASOs in combination with mouse Angptl3-specific ASOs in mouse hepatocytes. 15,000 primary mouse hepatocytes (Lonza) were seeded in BioCoat Collagen I 96-well flat bottom plates in 100 mΐ Hepatocyte Plating Medium. Supernatant was removed 4-6 h after seeding and ASOs were added at indicated concentrations (5000 nM, 1000 nM, 200 nM, 40 nM, 8 nM, 1.6 nM) diluted in 100 mΐ Maintenance Medium. Cells were cultured for three days at 37°C. After three days, cells were lysed and Hypoxanthine phosphoribosyltransferase 1 (Hprtl) and Angptl4 mRNA expression were measured using the QuantiGene RNA Singleplex assay. Angptl4-mRNA expression values were normalized to expression of the housekeeping gene Hprtl. Residual Angptl4-mRNA expression relative to mock-treated cells (set as 1 (n=9), SD=0.28). Dotted line indicates 0% (equivalent to residual mRNA level of 1.0) decrease of Angptl4 mRNA levels. Data are represented as mean of triplicate wells +/-SD.

Fig. 11 shows effect of GalNAc conjugated specific ASO to Angptl3 (GalNAc-A26033M (SEQ ID NO.77)) and GalNAc conjugated specific ASO to Angptl4 (GalNAc-A24095Mi (SEQ ID NO.248)) on plasma TG in wild-type mice. C57BL/6J mice were subcutaneously injected with saline or GalNAc-Negl or GalNAc-A26033M or GalNAc-A24095Mi (1.25mg/kg BW, twice per week week) or a combination of GalNAc- A26033M and GalNAc- A24095MΪ (1.25mg/kg BW, twice per week of each of the ASOs) for 2 weeks. At day 14, the mice were fasted for 6 hrs (09.00 to 15.00) prior to blood sampling. Plasma lipid levels were measured by HPLC. **p<0.01 for comparisons made with Vehicle. Data are represented as mean of 8 mice/group +SD. T-test was used to determine p-values. **p<0.01 for comparisons made with Vehicle.

Fig. 12 depicts the effect of GalNAc conjugated Angptl3 and Angptl4- specific ASOs on the ANGPTL3 mRNA expression in the livers of wild-type mice measured by realtime qPCR. The GalNAc-Negl ASO was included as control. The different groups (n=8 male per group, age 10-12 weeks) were subcutaneously injected with the respective GalNAc conjugated oligonucleotides at a concentration of 1.25mg/kg BW/twice per week for two weeks. Data are represented as mean of 8 mice/group +SD. T-test was used to determine p values. *p<0.05, **p<0.01 for comparison made with Vehicle.

Fig. 13 depicts that GalNAc-A24095Mi and a combination of GalNAc-A26033M and GalNAc-A24095Mi significantly decreased the expression level of Angptl4 mRNA in the livers. Data are represented as mean of 8 mice/group +SD. T-test was used to determine p values. ***p<0.001 for comparisons made with Vehicle.

Fig. 14 shows the effects of the Angptl- specific ASOs in a disease model. The experiment has been performed according to the same protocol as described above for wild-type mice (Fig. 11-13) applied to APOA5-/- mice. Different groups of mice (n=6-7 male per group, age 22-23 weeks) were subcutaneously injected with the respective GalNAc conjugated oligonucleotides at a concentration of (1.25mg/kg BW twice per week) for two weeks. Total triglyceride levels in plasma were measured directly by a colorimetric assay. Data are represented as mean of 6-7 mice/group +SD. *p<0.05, ***p<0.001 for comparisons made with Vehicle Fig. 15 depicts the effects of GalNAc conjugated Angptl3 and Angptl4- specific ASOs on Angptl3 mRNA expression in the livers of APOA5-/- mice. Gene expression was measured by realtime qPCR and compared to the effects of the vehicle (Veh). Different groups (n=6-7 male mice in each group, age 20-22 weeks) were subcutaneously injected with the respective GalNAc conjugated oligonucleotides at a concentration of 1.25mg/kg BW/twice per week for two weeks. Data are represented as mean of 6-7 mice/group +SD. T-test was used to determine p values. *p<0.05, **p<0.01 for comparisons made with Vehicle.

Fig. 16 shows that the Angptl4 gene expression level in the livers of mice treated with GalNAc-A26095Mi and a combination of GalNAc-A26033M and GalNAc-A24095Mi is significantly lower than Vehicle, **p<0.01. Data are represented as mean of 6-7 mice/group +SD. T-test was used to determine p values. **p<0.01 for comparisons made with Vehicle.

Detailed description of the invention

The present invention provides successful inhibitor of ANGPTL3 expression, which is a human or mouse oligonucleotide hybridizing with mRNA and/or pre-mRNA sequences of ANGPTL3 and inhibits the expression and activity, respectively, of ANGPTL3. In addition, the present invention provides a successful inhibitor of ANGPTL4 expression, which is a human or mouse oligonucleotide hybridizing with mRNA and/or pre-mRNA sequences of ANGPTL4 and inhibits the expression and activity, respectively, of

ANGPTL4, which is combined with an ANGPTL3 inhibitor. Moreover, the ANGPTL3 oligonucleotide inhibits for example the expression of ANGPTL4 and/or the ANGPTL4 oligonucleotide inhibits for example the expression of ANGPTL3. A combination of these ANGPTL3 and ANGPTL4 inhibitors such as antisense oligonucleotides inhibit the expression of ANGPTL3 and ANGPTL4 mRNA and/or pre-mRNA. mRNA comprises only exons of the ANGPTL3 and/or ANGPTL4 encoding nucleic acid sequence, whereas pre- mRNA comprises exons and introns of the ANGPTL3 and/or ANGPTL4 encoding nucleic acid sequence. Thus, the oligonucleotides of the present invention represent an interesting and highly efficient tool for use in a method of preventing and/or treating disorders, where the ANGPTL3 and/or the ANGPTL4 expression and activity,

respectively, is increased. The combination of these ANGPTL3 and ANGPTL4 inhibitors such as antisense oligonucleotides includes conjugates, wherein two or more antisense oligonucleotides of the present invention such as an ANGPTL3 inhibitor and an

ANGPTL4 inhibitor are connected, e.g., via di-(or more) nucleotide or via a disulfide linker. The ANGPTL3-ANGPTL4 conjugate is linked at one or both free ends of the conjugate to e.g. N-acetylgalactosamine (GalNAc), in particular a multivalent GalNAc, e.g., triantennary GalNAc cluster. ANGPTL3-ANGPTL4 conjugates are optionally combined with a none conjugated ANGPTL3 oligonucleotide and/or -ANGPTL4

oligonucleotide of the present invention.

The ratio of the ANGPTL3 oligonucleotide and the ANGPTL4 oligonucleotide of the present invention is 1:1 or any other ratio such as 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9 1:10 or 10:1, 9:1. 8:1, 7:1, 6:1, 5:1, 4:1. 3:1 or 2:1. The ratio is for example selected depending on the level of ANGPTL3 and ANGPTL4 that has to be modified (i.e., increased or decreased) to bring an imbalance of ANGPTL3 and/or ANGPTL4 , e.g., in a patient, back to a normal level of a healthy cell, tissue or organism.

In the following, the elements of the present invention will be described in more detail. These elements are listed with specific embodiments, however, it should be understood that they may be combined in any manner and in any number to create additional embodiments. The variously described examples and embodiments should not be construed to limit the present invention to only the explicitly described embodiments. This description should be understood to support and encompass embodiments which combine the explicitly described embodiments with any number of the disclosed elements. Furthermore, any permutations and combinations of all described elements in this application should be considered disclosed by the description of the present application unless the context indicates otherwise.

Throughout this specification and the claims, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be

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. The terms "a" and "an" and "the" and similar reference used in the context of describing the invention (especially in the context of the claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by the context. Recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as",“for example”), provided herein is intended merely to better illustrate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.

An inhibitor which is an oligonucleotide of the present invention is for example an antisense oligonucleotide (ASO) consisting of or comprising 10 to 25 nucleotides, 12 to 22 nucleotides, 15 to 20 nucleotides or 16 to 18 nucleotides. The oligonucleotides for example consist of or comprise 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides.

The oligonucleotide of the present invention forms for example a gapmer consisting of or comprising a central block of at least 5 nucleotides, i.e., deoxynucleotides and/or ribonucleotides, which is flanked by for example naturally and/or artificially modified nucleotides such as deoxynucleotides and/or ribonucleotides.

The oligonucleotides of the present invention comprise at least one nucleotide which is modified. The modified nucleotide is for example a bridged nucleotide such as a locked nucleic acid (LNA, e.g., 2',4'-LNA), cET, ENA, a 2'Fluoro modified nucleotide, a 2Ό- 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 having the same or different modifications. In addition, the oligonucleotide of the present invention optionally comprises a modified phosphate backbone, wherein the phosphate is for example a phosphorothioate.

The oligonucleotide of the present invention comprises the one or more modified nucleotide at the 3'- and/or 5 end of the oligonucleotide and/or at any position within the oligonucleotide, wherein modified nucleotides follow in a row of for example 1, 2, 3, 4, 5, or 6 modified nucleotides, or a modified nucleotide is combined with one or more unmodified nucleotides. The following Tables 1 and 2 present examples of ANGPTL3 oligonucleotides comprising modified nucleotides for example LNA which are indicated by (+) and phosphorothioate (PTO) indicated by (*). The ANGPTL3 oligonucleotides consisting of or comprising the sequences of Table 1 (human) or Table 2 (mouse) may comprise any other modified nucleotide and/or any other combination of modified and unmodified nucleotides. ANGPTL3 oligonucleotides of Table 1 hybridize with mRNA and/or pre-m RNA of human ANGPTL3 and optionally with mRNA and/or pre-m RNA of mouse ANGPTL3, and/or mRNA and/or pre-m RNA of human and/or mouse ANGPTL4:

Table 1: List of antisense oligonucleotides hybridizing with human ANGPLT3 mRNA and/or pre-mRNA for example of SEQ ID NO. 1, 2, 47, 98, 99, 155 and/or SEQ ID NO. 156, preferably SEQ ID NO. 1 and/or 2; Negl, R01009 and R01019 are oligonucleotides representing negative controls which are not hybridizing with ANGPTL3 of SEQ ID NO. 1, 2, 47, 98, 99, 155 or SEQ ID NO. 156. Oligonucleotides primarily hybridizing with human ANGPLT3 mRNA are indicated by“H”, and oligonucleotides primarily hybridizing with human ANGPLT3 pre-mRNA are indicated by“Hi” as the

oligonucleotides hybridize with an intron.

Oligonucleotides of Table 2 hybridize with mRNA of mouse ANGPTL3 and optionally with mRNA and/or pre-m RNA of human ANGPTL3, and/or mRNA and/or pre-m RNA of human and/or mouse ANGPTL4:

Table 2: List of antisense oligonucleotides hybridizing with mouse ANGPLT3 mRNA for example of SEQ ID NO. 1, 2, 47, 98, 99, 155 and/or SEQ ID NO.156, preferably SEQ ID NO.47; Negl, R0109 and R01019 are oligonucleotides representing a negative control which are not hybridizing with ANGPTL3 of SEQ ID NO. 1, 2, 47, 47, 98, 99, 155 and/or SEQ ID NO.156 .

The oligonucleotides of the present invention hybridize for example with mRNA of human ANGPTL3 of SEQ ID NO. 1 and/or introns of the pre-mRNA of human ANGPTL3 of SEQ ID NO. 2. Such oligonucleotides are called ANGPTL3 antisense oligonucleotides. The oligonucleotides hybridize for example within a hybridizing active area which is one or more region(s) on the ANGPTL3 mRNA, e.g., of SEQ ID NO.l and/or the ANGPTL3 pre-mRNA, e.g., of SEQ ID NO.2, where hybridization with an oligonucleotide highly likely results in a potent knockdown of the ANGPTL3 expression. In the present invention surprisingly several hybridizing active areas were identified for example selected from hybridizing active areas for example selected from position 45-72 (e.g., A26004H, SEQ ID NO.6) or from position 1130-1170 (e.g., A26019H, SEQ ID NO.20; A26020H, SEQ ID NO.20; A26021H, SEQ ID NO. 21; A26022H, SEQ ID N0.22;

A26023H, SEQ ID NO.23; A26024H, SEQ ID NO.24) of SEQ ID NO. 1 and/or from position 3060-3086 (e.g., A26033HΪ, SEQ ID NO.33) or from position 5768-5794 (e.g., A26036HΪ, SEQ ID NO.36; A26037HΪ, SEQ ID NO.36) of SEQ ID NO.2. Hybridizing active areas on mouse SEQ ID NO.47 are for example from position 303-330 (e.g., A26007M, SEQ ID NO. 54) and/or from position 803-843 (e.g., A26016M, SEQ ID NO. 61; A26019M, SEQ ID NO. 64; A26020M, SEQ ID NO. 65; A26021M, SEQ ID NO. 66) and/or from position 1038-1134 (e.g., A26032M, SEQ ID NO. 76; A26033M, SEQ ID NO. 77; A26034M, SEQ ID NO. 78; A26035M, SEQ ID NO. 79; A26039M, SEQ ID NO. 83) and/or from position 1480-1507 (e.g., A26047M, SEQ ID NO. 90).

The following Tables 3, 4, 5 and 6 present examples of ANGPTL4 oligonucleotides comprising modified nucleotides for example LNA which are indicated by (+) and phosphorothioate (PTO) indicated by (*). The ANGPTL4 oligonucleotides consisting of or comprising the sequences of Table 3 and 4 (human) or Table 5 and 6 (mouse) may comprise any other modified nucleotide and/or any other combination of modified and unmodified nucleotides. ANGPTL4 oligonucleotides of Table 3 hybridize with mRNA and/or pre-m RNA of human ANGPTL4 and optionally with mRNA and/or pre-m RNA of mouse ANGPTL4, and/or mRNA and/or pre-m RNA of human and/or mouse ANGPTL3:

Table 3: List of antisense oligonucleotides hybridizing with human ANGPLT4 mRNA and/or pre-mRNA for example of SEQ ID NO. 1, 2, 47, 98, 99, 155 and/or SEQ ID NO. 156, preferably SEQ ID NO.l, 2, 98 and/or SEQ ID NO.99; Negl, R01009 and R01019 are oligonucleotides representing negative controls which are not hybridizing with ANGPTL4 of SEQ ID NO. 1, 2, 47, 98, 99, 155 or SEQ ID NO. 156.“He” means“human exonic region” and is an oligonucleotide primarily hybridizing with mRNA of human ANGPTL4 and“Hi” is an oligonucleotide primarily hybridizing with pre-mRNA.

ANGPTL4 oligonucleotides of Table 4 also hybridize with mRNA and/or pre-m RNA of human ANGPTL4 and optionally with mRNA and/or pre-m RNA of mouse ANGPTL4, and/or mRNA and/or pre-m RNA of human and/or mouse ANGPTL3:

Table 4: List of antisense oligonucleotides hybridizing with human ANGPLT4 mRNA and/or pre-mRNA for example of SEQ ID NO. 1, 2, 47, 98, 99, 155 and/or SEQ ID NO. 156, preferably SEQ ID NO.l, 2, 98 and/or SEQ ID NO.99; Negl, R01002, R01009, R01014 and R01019 are oligonucleotides representing negative controls which are not hybridizing with ANGPTL4 of SEQ ID NO. 1, 2, 98, 99, 155 or SEQ ID NO. 156.“He” means“human exonic region” and is an oligonucleotide primarily hybridizing with mRNA of human ANGPTL4, and“Hi” is an oligonucleotide hybridizing with introns of ANGPTL4 pre-mRNA.“HMe” indicates an oligonucleotide hybridizing with“human and mouse exonic region” of ANGPTL4.

Oligonucleotides of Table 5 hybridize particularly with mRNA and/or pre-m RNA of mouse ANGPTL4 and optionally with mRNA and/or pre-m RNA of human ANGPTL4, and/or mRNA and/or pre-m RNA of human and/or mouse ANGPTL3:

Table 5: List of antisense oligonucleotides hybridizing with mouse ANGPLT4 mRNA and/or pre-mRNA for example of SEQ ID NO. 1, 2, 47, 98, 99, 155 and/or SEQ ID NO. 156, preferably SEQ ID NO. 155 and/or SEQ ID NO. 156; Negl, R01009 and R01019 are oligonucleotides representing negative controls which are not hybridizing with ANGPTL4 of SEQ ID NO. 155 or SEQ ID NO. 156. Oligonucleotides primarily hybridizing with mouse ANGPLT4 mRNA are indicated by“M”, and oligonucleotides primarily hybridizing with mouse ANGPLT4 pre-mRNA are indicated by“Mi” as the oligonucleotides hybridize with an intron.

Oligonucleotides of Table 6 also hybridize particularly with mRNA and/or pre-m RNA of mouse ANGPTL4 and optionally with mRNA and/or pre-m RNA of human ANGPTL4, and/or mRNA and/or pre-m RNA of human and/or mouse ANGPTL3:

Table 6: List of antisense oligonucleotides hybridizing with mouse ANGPLT4 mRNA and/or pre-mRNA for example of SEQ ID NO. 1, 2, 47, 98, 99, 155 and/or SEQ ID NO.156, preferably SEQ ID NO. 155 and/or SEQ ID NO.156; Negl, R01002, R01009, R01014 and R01019 are oligonucleotides representing negative controls which are not hybridizing with ANGPTL4 of SEQ ID NO.155 or SEQ ID NO.156. Oligonucleotides primarily hybridizing with mouse ANGPLT4 mRNA are indicated by“M”, and oligonucleotides primarily hybridizing with mouse ANGPLT4 pre-mRNA are indicated by“Mi” as the oligonucleotides hybridize with an intron.

Each oligonucleotide of the present invention can be combined with another

oligonucleotide of the present invention, in particular each ANGPLT3 oligonucleotide of the present invention can be combined individually with each ANGPLT4 oligonucleotide of the present invention.

The oligonucleotides hybridize for example within a hybridizing active area which is an area enriched for ASOs with high activity. The hybridizing active area is for example one or more region(s) on the ANGPTL4 mRNA, e.g., of SEQ ID NO.98 and/or the ANGPTL4 pre-mRNA, e.g., of SEQ ID NO.99, where hybridization with an oligonucleotide highly likely results in a potent knockdown of the ANGPTL4 expression. In the present invention surprisingly several hybridizing active areas were identified for example selected from hybridizing active areas for example selected from position 1732-1759 (e.g., A24044He, SEQ ID NO.143; A24076He, SEQ ID NO.143) of SEQ ID NO. 98 and/or from position 6603-6631 (e.g., A24022HΪ, SEQ ID NO.121; A24023HΪ, SEQ ID NO.122;

A24071HΪ, SEQ ID NO.152) of SEQ ID NO.99. Further hybridizing active areas are from position 234-261 (e.g., A24102He, SEQ ID N0.276; A24103He, SEQ ID N0.277) and/or from position 1264-1293 (e.g., A24110He, SEQ ID NO.284; A24111He, SEQ ID NO.285) of human SEQ ID NO. 98 and/or from position 2800-2872 (e.g., A24083HΪ, SEQ ID NO. 257; A24085HΪ, SEQ ID N0.259; A24086HΪ, SEQ ID NO.260; A24087HΪ, SEQ ID NO. 261) and/or from position 3415-3442 (e.g., A24089HΪ, SEQ ID NO.263) and/or from position 4968-4994 (e.g., A24097HΪ, SEQ ID NO. 271) of human SEQ ID NO.99.

Hybridizing active areas on mouse SEQ ID NO.155 or SEQ ID NO.156 are for example from position 137-163 (e.g., A24054M, SEQ ID NO. 210) and/or from position 215-299 (e.g., A24018M, SEQ ID NO.174; A24019M, SEQ ID NO.175; A24020M, SEQ ID NO.176; A24021M, SEQ ID NO.177; A24065M, SEQ ID NO.176) and/or from position 1343-1371 (e.g., A24042M, SEQ ID NO.198; A24109M, SEQ ID NO.313) and/or from position 1738- 1771 (e.g., A24047M, SEQ ID NO.203; A24070M, SEQ ID N0.224; A24072M, SEQ ID NO.226) of SEQ ID NO.155 and/or from position 1286-1314 (e.g., A24082MΪ, SEQ ID NO.235; A24125MΪ, SEQ ID NO.327) and/or from position 5485-5511 (e.g., A24095MΪ, SEQ ID NO.248; A24148MΪ, SEQ ID N0.248) of SEQ ID N0.156.

Each of the ANGPTL3 oligonucleotides for example of Tables 1 and 2, respectively, can be combined with one or more of the ANGPTL4 oligonucleotide(s) for example of Table 3, 4, 5, and/or 6. Alternatively, each of the ANGPTL4 oligonucleotides for example of Table 3, 4, 5, and/or 6 can be combined with one or more of the ANGPTL3 oligonucleotide(s) for example of Tables 1 and/or 2. An ANGPTL3 oligonucleotide and an ANGPTL4

oligonucleotide of the present invention form for example a conjugate. For example the ANGPTL3 oligonucleotide and an ANGPTL4 oligonucleotide are connected via a linker such as e.g., a di-(or more) -nucleotide or a disulfide linker. Furthermore, the ANGPTL3- ANGPTL4 conjugate is linked at one or both free ends of the conjugate to e.g. a polymer, in particular an amphipathic membrane active polyamine (e.g., described in

US2012/0157509) for example N-acetylgalactosamine (GalNAc).

A combination of oligonucleotides in the composition of the present invention or in a conjugate inhibits for example at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 93 %, 94%, 95%, 96%, 97%, 98%, 99% or 100% of ANGPTL3 and ANGPTL4 such as the, e.g., human or mouse, ANGPTL3 andANGPTL4 expression. The

combination of oligonucleotides of the present invention inhibits the expression of ANGPTL3 and the expression of ANGPTL4 at a nanomolar or micromolar concentration for example in a concentration range of 0.1 nM to 100 mM, 0.5 nM to 15 nM, 0,6 nM to 10 nM, 1 nM to 10 mM, 5 nM to 5 mM, 10 nM to 1 mM, 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 40 nM to 150 nM, or in 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 1, 10 or 100 mM.

Each of the ANGPTL3 oligonucleotide and the ANGPTL4 oligonucleotide of the present invention is for example used in a concentration range of 1 nM to 10 mM, 5 nM to 6.6 mM, 10 nM to 5 mM, 15 nM to 3 mM, 20 nM to 2.2 mM, 25 nM to 1 mM, 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 in 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 pM.

The ANGPTL4 and/or ANGPTL3 oligonucleotide of the present invention is for example administered once or repeatedly, e.g., every 12 h, every 24 h, every 48 h for some weeks, months or years, or it is administered every week, every two weeks, every three weeks or every months or every three or six months.

The composition of the present invention comprises an ANGPTL3 oligonucleotide and an ANGPTL4 oligonucleotide of the present invention and optionally a pharmaceutically acceptable carrier, excipient, dilutant or a combination thereof. Optionally, the

composition further comprises a chemotherapeutic, another disease specific active agent such as insulin, angiotensin-converting enzyme inhibitor, 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 which is for example effective in tumor treatment, treatment of diabetes and its side effects, treatment of a cardiovascular disease, obesity, diabetes type II, hypercholesterolemia such as homozygote familial hypercholesterolemia (HoFH), heterozygote familial hypercholesterolemia (HeFH) or dislipidemia.

The composition comprising or consisting of the ANGPTL3 oligonucleotide and the ANGPTL4 oligonucleotide of the present invention is for use in a method of preventing and/or treating a disorder for example a disorder where an ANGPTL3 imbalance and/or an ANGPTL4 imbalance is involved. Optionally, the use of the oligonucleotide or the pharmaceutical composition of the present invention in a method of preventing and/or treating a disorder is combined with radiotherapy. The radiotherapy may be further combined with a chemotherapy (e.g., platinum, gemcitabine) . The disorder is for example characterized by an ANGPTL3 and/or an ANGPTL4 imbalance, i.e., the ANGPTL3 level and/or the ANGPTL4 level is increased in comparison to the level in a normal, healthy cell, tissue, organ or subject. The ANGPTL3 level is for example increased by an increased ANGPTL3 expression and activity, respectively. The ANGPTL4 level is for example increased by an increased ANGPTL4 expression and activity, respectively. The ANGPTL3 level and/or the ANGPTL4 level is measured by any standard method such as immunohistochemistry, western blot, quantitative real time PCR or QuantiGene assay known to a person skilled in the art.

The composition comprising or consisting of the ANGPTL3 oligonucleotide and the ANGPTL4 oligonucleotide of the present invention is administered locally or

systemically for example orally, sublingually, nasally, subcutaneously, intravenously, intraperitoneally, intramuscularly, intratumoral, intrathecal, transdermal, and/or rectal. Alternatively or in combination ex vivo treated immune cells are administered. One or more ANGPTL3 antisense oligonucleotides of the present invention and one or more ANGPTL4 antisense oligonucleotides of the present invention are administered together. Optionally the combination of an ANGPTL3 and an ANGPTL4 antisense oligonucleotide of the present invention comprises additionally 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, a BiTe, a small molecule and/or a

chemotherapeutic (e.g., platinum, gemcitabine) and/or another disease specific agent such as insulin, angiotensin-converting enzyme inhibitor, and/or angiotensin receptor blocker.

The oligonucleotide not of the present invention, an antibody, a HERA fusion protein, a ligand trap, a Fab fragment, a nanobody, a BiTe, and/or the small molecule are effective in preventing and/or treating a tumor, diabetes such as diabetes type II and its side effects, a cardiovascular disease, obesity, hypercholesterolemia such as homozygote familial hypercholesterolemia (HoFH), heterozygote familial hypercholesterolemia (HeFH) or dislipidemia. The combination of an ANGPTL3 oligonucleotide and an

ANGPTL4 oligonucleotide of the present invention, or a pharmaceutical composition of the present invention is used for example in a method of preventing and/or treating a solid tumor or a hematologic tumor. Examples of cancers preventable and/or treatable by use of the oligonucleotide or pharmaceutical composition of the present invention are breast cancer, lung cancer, malignant melanoma, lymphoma, skin cancer, bone cancer, prostate cancer, liver cancer, brain cancer, cancer of the larynx, gall bladder, pancreas, testicular, rectum, parathyroid, thyroid, adrenal, neural tissue, head and neck, colon, stomach, bronchi, kidneys, basal cell carcinoma, squamous cell carcinoma, metastatic skin carcinoma, osteo sarcoma, 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, hairy-cell tumor, adenoma, hyperplasia, medullary carcinoma, intestinal ganglioneuromas, Wilm's tumor, seminoma, ovarian tumor, leiomyomater tumor, cervical dysplasia, retinoblastoma, soft tissue sarcoma, malignant carcinoid, topical skin lesion, rhabdomyosarcoma, Kaposi's sarcoma, osteogenic sarcoma, malignant hypercalcemia, renal cell tumor, polycythermia vera, adenocarcinoma, anaplastic astrocytoma, glioblastoma multiforma, leukemia, or epidermoid carcinoma.

Further examples of diseases preventable and/or treatable by use of an ANGPTL3 oligonucleotide in combination with an ANGPTL4 oligonucleotide or the pharmaceutical composition of the present invention other than cancer are for example diabetes such as diabetes type II and its side effects, a cardiovascular disease, obesity,

hypercholesterolemia such as homozygote familial hypercholesterolemia (HoFH), heterozygote familial hypercholesterolemia (HeFH) or dislipidemia.

The ANGPTL3 and the ANGPTL4 oligonucleotide of the present invention are administered together, at the same time point for example in a pharmaceutical composition or separately, or on staggered intervals. In other examples, one or more oligonucleotides of the present invention are administered together 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, a BiTe, a small molecule and/or a chemotherapeutic, at the same time point for example in a

pharmaceutical composition or separately, or on staggered intervals.

A subject of the present invention is for example a mammalian, a bird or a fish. Examples

The following examples illustrate different embodiments of the present invention, but the invention is not limited to these examples. The following experiments are performed on cells endogenously expressing ANGPTL3 and/or ANGPTL4, i.e., the cells do not represent an artificial system comprising transfected reporter constructs. Such artificial systems generally show a higher degree of inhibition and lower IC50 values than endogenous systems which are closer to therapeutically relevant in vivo systems.

Further, in the following experiments no transfecting agent is used, i.e., gymnotic delivery is performed. Transfecting agents are known to increase the activity of an oligonucleotide which influences the IC50 value (see for example Zhang et al., Gene Therapy, 2011, 18, 326-333; Stanton et al., Nucleic Acid Therapeutics, Vol. 22, NO. 5, 2012). As artificial systems using a transfecting agent are hard or impossible to translate into therapeutic approaches and no transfection formulation has been approved so far for oligonucleotides, the following experiments are performed without any transfecting agent.

Example 1: IC50 determination of selected human ANGPTL3- specific antisense oligonucleotides

15,000 primary human hepatocytes / well were seeded in 96-well plates and treated with different concentrations (1000 nM, 200 nM, 40 nM, 8 nM, 1.6 nM, 0.32 nM) of nine antisense oligonucleotides (A26004H (SEQ ID NO.6), A26019H (SEQ ID NO.20), A26020H (SEQ ID NO.20), A26022H (SEQ ID N0.22), A26023H (SEQ ID N0.23), A26024H (SEQ ID N0.24), A26033HΪ (SEQ ID N0.33), A26036HΪ (SEQ ID N0.36) and A26037HΪ (SEQ ID NO.36)). Three days after start of treatment, cells were lysed and HPRT1 and ANGPTL3 mRNA expression was measured using the QuantiGene RNA Singleplex assay. The QuantiGene Assay in the examples is built upon the branched DNA technology (bDNA), which relies on cooperative hybridization between a target mRNA and a specific probe set (part of QuantiGene Reagent System). The assay was performed according to manufacturer’s protocol and was used for determination of RNA levels. It combines the QuantiGene Sample Processing Kit that is used for cell lysis and the QuantiGene Reagent System that is used for hybridization, amplification and detection of RNA of interest. The QuantiGene Reagent System is based on an RNA- specific probe set, designed to detect a particular RNA of interest. ANGPTL3-mRNA expression values were normalized to expression of the housekeeping gene HPRT1. Residual ANGPTL3-mRNA expression relative to mock-treated cells (set as 1). For graphic representation mock-treated cells were set as 0.16 nM. Data are represented as mean of triplicate wells +/-SD.

Fig. 1 and Table 5 show that the selected human ANGPTL3-specific antisense oligonucleotides inhibit ANGPTL3 mRNA expression dose- dependency with IC50 values in the nanomolar range.

Table 5: IC50 values and R squares of selected human ANGPTL3-specific antisense oligonucleotides determined in primary human hepatocytes.

Example 2: IC50 determination of selected human ANGPTL4- specific antisense oligonucleotides

30,000 primary human hepatocytes / well were seeded in 96-well plates and treated with different ANGPTL4 antisense oligonucleotides in different concentrations of 5000 nM, 1000 nM, 200 nM, 40 nM, 8 nM and 1.6 nM: A24022HΪ (SEQ ID N0.121), A24023HΪ (SEQ ID NO.122), A24071Hi (SEQ ID NO.152) and A24076He (SEQ ID NO.143). To induce ANGPTL4 mRNA expression, cells were simultaneously treated with 1 mM peroxisome proliferator-activator receptor g (PPARy) (Sigma Aldrich, cat. no. R2408). PPARy stock solution (10 mM) was prepared by dissolving 10 mg PPARy (Molecular weight: 357.43) in 2.8 ml DMSO. For final concentration of 1 mM PPARy, cells seeded in 96-well plates were incubated with 100 mΐ medium supplemented with 0.01 mΐ PPARy stock solution. As a negative control, cells were treated with equal volume of DMSO. Every 24 h, 70 mΐ of supernatant was replaced with fresh medium containing 1 mM PPARy as well as the respective ANGPTL4 antisense oligonucleotides at indicated concentrations. Three days after start of treatment, cells were lysed and HPRT1 and ANGPTL4 mRNA expression was measured using the QuantiGene RNA Singleplex assay. ANGPTL4-mRNA expression values were normalized to expression of the housekeeping gene HPRT1. Residual ANGPTL4-mRNA expression relative to mock- treated cells (set as 1). For graphic representation mock-treated cells were set as 0.32 nM. Data are represented as mean of triplicate wells +/-SD.

Fig. 2 and Table 6 demonstrate that the selected ANGPTL4-specific antisense oligonucleotides inhibit ANGPTL4 mRNA expression dose- dependency with IC50 values in the nanomolar range.

Table 6: IC50 values and R squares of selected human ANGPTL4-specific antisense oligonucleotides determined in primary human hepatocytes.

Example 3: IC50 determination of selected human ANGPTL4- specific antisense oligonucleotides (ASOs)

Based on knockdown efficiency in primary hepatocytes, ASOs with most potent knockdown efficacy and no induction of caspase3/7 upon transfection were selected for determination of IC50 values. As a positive control ANGPTL4-specific ASO A24076He (SEQ ID NO.143) with verified knockdown efficiency was used.

Primary human hepatocytes (Primacyt) were treated with ANGPTL4-specific ASOs or negative control oligonucleotides Negl, R01009 and R01019 at different concentrations for three days. Simultaneously, cells were treated with PPARy (1 mM) (Sigma Aldrich, cat. no. R2408; for preparation of 10 mM stock solution see example 2) to induce ANGPTL4 expression. For final concentration of 1 mM PPARy, cells seeded in 96-well plates were incubated with 100 mΐ medium supplemented with 0.01 mΐ PPARy stock solution. As a negative control, cells were treated with equal volume of DMSO. After three days, mRNA expression was analyzed using the QuantiGene Singleplex RNA assay.

Table 7 and Fig. 3 demonstrate that the eight ANGPTL4-specific ASOs (A24083HΪ (SEQ ID NO.257), A24085HΪ (SEQ ID N0.259), A24087HΪ (SEQ ID N0.261), A24089HΪ (SEQ ID NO.263), A24097HΪ (SEQ ID N0.271), A24103He (SEQ ID N0.277), A24110He (SEQ ID NO.284) and A24111He (SEQ ID NO.285)) and the positive control A24076He (SEQ ID NO.143) inhibit ANGPTL4 mRNA expression dose-dependently with IC5₀ values in the nanomolar range.

Following Table 7 shows IC5₀ values and R squares of selected human ANGPTL4- specific antisense oligonucleotides determined in primary human hepatocytes. *, R square below 0.85.

Example 4: In vitro compatibility of human ANGPTL3-specific and ANGPTL4- specific oligonucleotides in human hepatocytes

Human ANGPTL3- specific antisense-oligonucleotides A26004H (SEQ ID NO.6) and A26022H (SEQ ID NO.22) with verified knockdown efficiency in human cell lines and primary hepatocytes were tested for compatibility with human ANGPTL4-specific antisense oligonucleotide (ASO) A24076H (SEQ ID NO.143) in primary human hepatocytes (Fig. 4). Previously, efficacy of ASO A24076H (SEQ ID NO.143) was verified in human cell lines and primary hepatocytes.

For efficacy testing in human hepatocytes, 25,000 cells were seeded in Collagen Coated 96well plates (Primacyt) using the Plating and Thawing Kit. After incubation for 6 hours at 37°C, supernatant was replaced by 100 mΐ Human Hepatocyte Maintenance Medium supplemented with ASOs at indicated concentrations (5000 nM, 1000 nM, 200 nM, 40 nM, 8 nM, 1.6 nM) each. Simultaneously, cells were treated with PPARy (Sigma Aldrich, cat. no. R2408; for preparation of 10 mM stock solution see example 2) at a final concentration of 1 mM to induce ANGPTL4 expression. For final concentration of 1 mM PPARy, cells seeded in 96-well plates were incubated with 100 mΐ Maintenance Medium supplemented with 0.01 mΐ PPARy stock solution As a vehicle control, cells were treated with equal volume of DMSO. Every 24 hours, 70 mΐ of supernatant was replaced with fresh medium containing respective ASOs at indicated concentrations (5000 nM, 1000 nM, 200 nM, 40 nM, 8 nM, 1.6 nM). Cells were cultured for three days at 37°C and 5% C0₂.

Human primary hepatocytes were treated with the respective oligonucleotides at indicated concentrations at a 1:1 ratio (if combined) for three days without the use of a transfection reagent. Three control oligonucleotides (R01009 (SEQ ID NO.250), R01019 (SEQ ID NO.251), Negl (SEQ ID NO.46)) with different lengths (16, 17 and 18 nucleotides, respectively) that do not have sequence complementarity to any human or mouse RNA were included as negative controls (Fig. 4B - 4C, 41 - 4K).

As shown in Fig. 4A, treatment with human ANGPTL4-specific ASO A24076H (SEQ ID NO.143) alone or in combination with ANGPTL3-specific ASO A26022H (SEQ ID NO.22) led to dose-dependent reduction of ANGPTL4 mRNA expression in primary human hepatocytes up to 50% (equivalent to residual ANGPTL4-mRNA expression of 0.5), while combination of ASO A24076H (SEQ ID NO.143) with ANGPTL3-specific ASO A26004H (SEQ ID NO.6) slightly decreased efficacy of ASO A24076H (SEQ ID NO.143) (residual ANGPTL4-mRNA expression of 0.73). Treatment with ANGPTL3-specific ASOs A26004H (SEQ ID NO.6) or A26022H (SEQ ID NO.22) alone did not affect ANGPTL4 mRNA expression levels (Fig. 4D - 4E). Control oligonucleotides R01009 (SEQ ID NO.250), R01019 (SEQ ID NO.251) and Negl (SEQ ID NO.46) did also not diminish ANGPTL4 mRNA expression (Fig. 4B - 4C).

As shown in Fig. 4F - 4G, treatment with human ANGPTL3- specific ASOs A26004H (SEQ ID NO.6) or A26022H (SEQ ID NO.22) led to dose-dependent reduction of ANGPTL3 mRNA expression in primary human hepatocytes up to approx. 90% (equivalent to residual ANGPTL3-mRNA expression of 0.1). Combination with ANGPTL4-specific ASO A24076H (SEQ ID NO.143) did not affect efficacy of A26022H (SEQ ID NO.22) (Fig. 5G), while efficacy of ASO A26004H (SEQ ID NO.6) was slightly reduced at lower concentrations (40 nM, 8 nM, 1.6 nM) (Fig. 4F). However, half maximal inhibitory concentration (IC50) values were similar (A26004H (SEQ ID NO.6). 66.3 nM (linear regression) vs. A26004H (SEQ ID NO.6) xA24076H (SEQ ID NO.143): 36.9 nM (nonlinear fit)) (Fig. 4F). Negative control oligonucleotides Negl (SEQ ID NO.46), R01009 (SEQ ID NO.250) and R01019 (SEQ ID NO.251) (Fig. 41 - 4J) as well as ANGPTL4-specific ASO A24076H (SEQ ID NO.143) (Fig. 4H) did not affect ANGPTL3 mRNA expression.

Treatment with none of the oligonucleotides led to substantial decrease of HPRT1 mRNA levels (Fig. 4K).

Example 5: IC50 determination of selected mouse ANGPTL3-specific antisense oligonucleotides

11 selected mouse ANGPTL3-specific antisense oligonucleotides, (A26019M (SEQ ID NO.64), A26016M (SEQ ID NO.61), A26020M (SEQ ID N0.65), A26007M (SEQ ID

NO.54), A26021M (SEQ ID NO.66), A26032M (SEQ ID N0.76), A26034M (SEQ ID

NO.78), A26033M (SEQ ID NO.77), A26039M (SEQ ID N0.83), A26035M (SEQ ID

NO.79), A26047M(SEQ ID NO.90)) selected due to high knockdown efficacy in primary mouse hepatocytes were selected for determination of half maximal inhibitory

concentration (IC50) values. Primary mouse hepatocytes were treated with the respective antisense oligonucleotides at different concentrations (5000 nM, 1000 nM, 200 nM, 40 nM, 8 nM, 1.6 nM) for three days and mRNA expression was analyzed using the QuantiGene Singleplex RNA assay. Hprtl was used as a housekeeping gene for normalization of ANGPTL3 expression. Residual ANGPTL3-mRNA expression relative to mock-treated cells (“no oligo” set as 1) is shown. Data are represented as mean of triplicate wells +/-SD.

Fig. 5 and Table 8 demonstrate that the selected ANGPTL3-specific antisense oligonucleotides inhibit ANGPTL3 mRNA expression dose- dependency with IC50 values in the nanomolar range. Table 8 in the following shows IC50 values and R square of selected ANGPTL3- specific antisense oligonucleotides determined in mouse primary hepatocytes:

Table 8: IC50 values and R squares of selected mouse ANGPTL3- specific antisense oligonucleotides determined in primary hepatocytes.

Example 6: IC50 determination of selected mouse ANGPTL4-specific antisense oligonucleotides

2,500 4T1 cells / well were seeded in 96-well plates and treated with the respective ANGPTL4 antisense oligonucleotides (ASO) A24018M (SEQ ID NO.174), A24019M (SEQ ID NO.175), A24020M (SEQ ID NO.176), A24021M (SEQ ID NO.177), A24047M (SEQ ID NO.203), A24054M (SEQ ID NO.210), A24065M (SEQ ID NO.176), A24070M (SEQ ID NO.224), A24072M (SEQ ID N0.226), A24082MΪ (SEQ ID N0.235) and A24095MΪ (SEQ ID NO.248) at different concentrations of 5000 nM, 1000 nM, 200 nM, 40 nM, 8 nM and 1.6 nM. Three days after start of treatment, cell supernatant was replaced by fresh medium w/ ASO and cells were incubated for additional 3 d. Then, cells were lysed and mouse Hprtl and mouse ANGPTL4 mRNA expression was measured using the

QuantiGene RNA Singleplex assay. ANGPTL4-mRNA expression values were normalized to expression of the housekeeping gene Hprtl. Residual ANGPTL4-mRNA expression relative to mock-treated cells (“no oligo” control set as 1) is shown. Data are represented as mean of triplicate wells +/-SD.

Fig. 6 and Table 9 demonstrate that the selected ANGPTL4-specific antisense oligonucleotides inhibit ANGPTL4 mRNA expression dose- dependency with IC50 values in the nanomolar range.

Table 9: IC50 values and R squares of selected mouse ANGPTL4-specific antisense oligonucleotides determined in 4T1 cells.

Example 7: IC50 determination of further selected mouse ANGPTL4-specific antisense oligonucleotides

Nine ASOs (A24103M (SEQ ID NO.307), A24110M (SEQ ID NO.314), A24122HMe (SEQ ID NO.294), A24120MΪ (SEQ ID N0.322), A24125MΪ (SEQ ID NO.327), A24139MΪ (SEQ ID NO.341), A24143MΪ (SEQ ID N0.345), A24146MΪ (SEQ ID NO.348), A24148MΪ (SEQ ID NO.248)) with most potent knockdown efficacy Renca and 4T1 cells (data not shown) were selected for determination of half maximal inhibitory concentration (IC50) values.

Primary mouse hepatocytes were treated with the respective ASO at different concentrations for three days. After three days, mRNA expression was analyzed using the QuantiGene Singleplex RNA assay. Fig. 7 and Table 10 demonstrate that the selected Angptl4- specific ASOs inhibit Angptl4 mRNA expression dose-dependently with IC50 values in the nanomolar range.

ASOs with increased potential to induce caspase3/7 (data not shown) led to dose- dependent decrease of Hprtl levels (Fig. 7, graph below).

Table 10 shows IC50 values and R squares of selected Angptl4- specific ASOs determined in primary mouse hepatocytes. *, R square below 0.85

Example 8: In vitro compatibility of mouse ANGPTL3-specific and ANGPTL4-specific oligonucleotides in mouse hepatocytes

Mouse Angptl3-specific antisense-oligonucleotides A26007M (SEQ ID NO.54) and A26033M (SEQ ID NO.77) with verified knockdown efficiency in mouse cell lines and primary mouse hepatocytes were tested for compatibility with mouse Angptl4- specific ASOs A24047M (SEQ ID NO.203), A24072M (SEQ ID N0.226) and A24095MΪ (SEQ ID NO.248) in primary mouse hepatocytes. Previously, efficacy of ASOs A24047M (SEQ ID NO.203), A24072M (SEQ ID NO.226) and A24095MΪ (SEQ ID NO.248) was verified in mouse cell lines.

Primary mouse hepatocytes were treated with the respective oligonucleotides at indicated concentrations at a 1:1 ratio (if combined) for three days without the use of a transfection reagent. Negative control oligonucleotide Negl (SEQ ID NO.46) that does not have sequence complementarity to any human or mouse RNA was included as negative control.

As shown in Fig. 8, treatment with none of the oligonucleotides led to substantial decrease of Hprtl mRNA levels. Mixture of ASOs A26033M (SEQ ID NO.77) x A24072M (SEQ ID NO.226) and A26033M (SEQ ID N0.77) x A24095MΪ (SEQ ID N0.248) led to decreased Hprtl levels at highest concentration (5 mM per ASOs) by about 40% (equivalent to residual mRNA expression of 0.6). Treatment with mouse Angptl3-specific ASOs A26007M (SEQ ID NO.54) or A26033M (SEQ ID NO.77) led to dose-dependent reduction of Angptl3 mRNA expression in primary mouse hepatocytes up to 99% (equivalent to residual Angptl3-mRNA expression of 0.01) (Fig. 9).

Combination with Angptl4- specific ASO A24047M (SEQ ID NO.203), A24072M (SEQ ID NO.226) or A24095MΪ (SEQ ID NO.248) did not affect efficacy of mouse Angptl 3 -specific ASO A26007M (SEQ ID NO.54) (Fig. 9A, 9C and 9E), while efficacy of ASO A26033M (SEQ ID NO.77) was slightly reduced if combined with Angptl4- specific ASO A24047M (SEQ ID NO.203) or A24072M (SEQ ID NO.226) (Fig. 9B and 9D). Combination of ASO A26033M (SEQ ID NO.77) with ASO A24095MΪ (SEQ ID NO.248) did not decrease the ability of A26033M (SEQ ID NO.77) to knock-down Angptl3 mRNA expression (Fig. 9F). Negative control oligonucleotide Negl as well as Angptl4-specific ASOs A24047M (SEQ ID NO.203), A24072M (SEQ ID N0.226) or A24095MΪ (SEQ ID N0.248) did not affect Angptl3 mRNA expression at low concentrations (1.6-200 nM) (Fig. 9G). At 1 mM and 5 mM oligonucleotide concentration an unspecific reduction of about 50% (equivalent to residual Angptl3-mRNA expression of 0.5) of Angptl3 mRNA expression was detected (Fig. 9G). However, treatment with Angptl3-specific ASOs led to strongly decreased Angptl3 mRNA levels (Fig. 9A - 9F).

As shown in Fig. 10, treatment with mouse Angptl4- specific ASOs A24047M (SEQ ID NO.203), A24072M (SEQ ID N0.226) and A24095MΪ (SEQ ID N0.248) alone or in combination with Angptl3-specific ASOs A26007M (SEQ ID NO.54) or A26033M (SEQ ID NO.77) led to dose- dependent reduction of Angptl4 mRNA expression in primary mouse hepatocytes up to 80% (equivalent to residual ANGPTL4-mRNA expression of 0.2). Combination of Angptl4- specific ASOs with Angptl3-specific ASOs had either no effect on Angptl4 ASO efficacy (Fig. IOC, 10E - 10F) or even slightly increased the capability of Angptl4-specific ASOs to reduce Angptl4 mRNA expression (Fig. 10A - 10B, 10D). Treatment with Angptl3- specific ASOs A26007M (SEQ ID NO.54) or A26033M (SEQ ID NO.77) alone did not affect Angptl4 mRNA expression levels (Fig. 10G). Control oligonucleotide Negl (SEQ ID NO.46) did also not diminish Angptl4 mRNA expression (Fig. 10G). Example 9: Testing triglyceride levels in plasma of wild- type mice treated with GalNAc conjugated Angptl3 and Angptl4- specific ASOs in wild-type mice

C67BL/6 mice were divided into six groups. Each of the treatment groups received subcutaneous injections (SC) of the following compounds: 1) GalNAc conjugated A26033M (SEQ ID NO.77), 2) GalNAc conjugated A24095MΪ (SEQ ID NO.248), 3) a mix of GalNAc conjugated A26033M and GalNAc conjugated A24095MΪ, 4) GalNAc conjugated Neg 1. The vehicle (or control) group received subcutaneous injections of saline solution (NaCl 0,9% w/v).

The total triglyceride (TG) level of the plasma samples in the experiment was quantified by HPLC. A control oligonucleotide that does not have sequence complementarity to any human or mouse mRNA (Negl) was included as control. The different groups of mice (n=8 males in each group, age 10-12 weeks) were subcutaneously injected with the respective GalNAc conjugated oligonucleotides at a concentration of 1.25mg/kg BW/twice per week for two weeks.

In wild-type mice, the basal TG levels are very low and therefore the free glycerol levels that are normally present in plasma causes an unacceptable background that interferes with the determination of the TG levels by the GPO-PAP kit described above. Therefore, the plasma samples from these mice have to be separated by size exclusion chromatography (SEC) on a Superose 6 3.2/300 column (GE Healthcare Life Science). For this 10 mΐ of plasma is applied to the column which is then run in Elution Buffer A (NaCl 150mM, TRIS lOmM, Azid 0.02%, PG 7.4) at a rate of 4 ml/min. The TG concentration in the eluate is continuously determined by GPO-PAP reagent as previously described and glycerol elutes from the column well separated after all lipoprotein classes. The amount of TG in the lipoprotein fractions is combined and the total TG content of the plasma samples can then be calculated. For this analysis only single samples of plasma is run. As shown in Fig. 11, GalNAc-A24095Mi (SEQ ID NO. 248) and a combination of GalNAc-A26033M (SEQ ID NO. 77) and GalNAc-A24095Mi (SEQ ID NO. 248) showed statistically significant decrease of plasma TG level compared to Vehicle. Example 10: Testing expression levels of Angptl3 and Angptl4 in the livers of wild type mice treated with GalNAc conjugated Angptl3 and Angptl4- specific ASOs

The effect of GalNAc conjugated Angptl3 (GalNAc-A26033M) and GalNAc conjugated Angptl4 (GalNAc-A24095Mi) specific ASOs on Angptl3 gene expression was tested in the livers of wild-type mice. C57BL/6J mice (n=8 male per group, age 10-12 weeks) were subcutaneously injected with saline or GalNAc-Negl or GalNAc-A26033M (SEQ ID NO.77) or GalNAc-A24095Mi (SEQ ID NO.248; 1.25mg/kg BW twice per week) or a combination of GalNAc- A26033M and GalNAc- A24095MΪ (1.25mg/kg BW twice per week of each ASOs) for 2 weeks. At day 14, the mice were fasted for 6 hrs (09.00 to 15.00) prior cervical dislocation and tissue collection. Data are presented as expression level compared to Vehicle. The Angptl3 expression level in the livers of mice treated with GalNAc-A26033M and a combination of GalNAc-A26033M and GalNAc-A24095Mi is lower than Vehicle group.

The results of this experiment have shown that, the combination of GalNAc-A26033M and GalNAc-A24095Mi has significantly stronger effect on lowering Angptl3 gene expression level in the liver. *P<0.05, **p<0.01. Results are shown in Fig. 12.

Further it is shown in Fig. 13 that GalNAc-A24095Mi and a combination of GalNAc - A26033M and GalNAc-A24095Mi significantly decreased the expression level of Angptl4 gene in the livers***p<0.001.

Example 11: Testing triglyceride levels in plasma of APOA5-/- mice treated with

GalNAc conjugated Angptl3 and Angptl4- specific ASOs in an APOA5 disease mouse model

Effects of GalNAc conjugated Angptl3 (GalNAc-A26033M; SEQ ID NO.77) or GalNAc conjugated Angptl4 (GalNAc-A24095Mi; SEQ ID NO.248) specific ASOs on plasma TG levels in APOA5-/- mice. APOA5-/- FVB mice (n=6-7 male per group, age 20-22 weeks) were subcutaneously injected with saline or GalNAc-A26033M or GalNAc-A24095Mi (1.25mg/kg BW twice per week) or a combination of GalNAc- A26033M and GalNAc - A24095MΪ (1.25mg/kg BW twice per week of each ASOs) for 2 weeks. At day 14, the mice were fasted for 6 hrs (09.00 to 15.00) prior to blood sampling. Plasma lipid levels were measured directly by a colorimetric assay. The results of this experiment show that the plasma TG level in the mice treated with GalNAc-A26033M and a combination of GalNAc-A26033M and GalNAc-A24095Mi is significantly lower than Vehicle. Treatment with the combination of GalNAc-A26033M and GalNAc-A24095Mi has an increased effect on plasma TG compared to single GalNAc-A26033M and GalNAc-A24095Mi treatment respectively (the difference is not statistically significant)*P<0.05,*** P<0.001. for comparisons made with Vehicle. The results are shown in Fig. 14.

Example 12: Testing the expression levels of Angptl3 and Angptl4 in the livers of APOA5-/- mice treated with GalNAc conjugated Angptl3 and Angptl4- specific ASOs

Testing the effect of GalNAc conjugated Angptl3 specific (GalNAc-A26033M (SEQ ID NO.77)) and GalNAc conjugated Angptl4 specific (GalNAc-A24095Mi (SEQ ID NO.248)) ASOs on Angptl3 gene expression in the livers of APOA5-/- mice. APOA5-/- FVB mice (n=6-7 male per group, age 20-22 weeks) were subcutaneously injected with saline or GalNAc-A26033M or GalNAc-A24095Mi (1.25mg/kg BW twice per week) or a combination of GalNAc- A26033M and GalNAc- A24095MΪ (1.25mg/kg BW twice per week of each ASOs) for 2 weeks. At day 14, the mice were fasted for 6 hrs (09.00 to 15.00) before blood sampling followed by cervical dislocation and tissue collection.

Data are presented as expression level compared to Vehicle. The expression level of Angptl3 gene is significantly lower in the liver of mice treated with GalNAc-A26033M and a combination of GalNAc-A26033M and GalNAc-A24095Mi compared to Vehicle, *P<0.05, **p<0.01. Results are shown in Fig. 15.

Fig. 16 further shows the Angptl4 gene expression level in the livers of mice treated with GalNAc-A26095Mi and a combination of GalNAc-A26033M and GalNAc-A24095Mi is significantly lower than Vehicle, **p<0.01.

Claims

1. Composition comprising an ANGPTL3 oligonucleotide and an ANGPTL4

oligonucleotide hybridizing with ANGPTL3 and ANGPTL4 mRNA, pre-mRNA and/or a combination thereof, and inhibiting the expression of ANGPTL3 and ANGPTL4.

2. Composition according to claim 1, wherein a hybridizing active area of ANGPTL3 is selected from position 45 to 72 and/or 1130 to 1170 of SEQ ID NO. 1 and/or from position 3060 to 3086 and/or from position 5768 to 5794 of SEQ ID NO.2 and a hybridizing active area of ANGPTL4 is selected from positionl732 to 1759 of SEQ ID NO.98 and/or from position 234-261 and/or from position 1264-1293 of SEQ ID NO.98 and/or from position 2800-2872 and/or from position 3415-3442 and/or from position 4968-4994 of SEQ ID NO.99.

3. Composition according to claim 1 or 2, wherein the modified nucleotide is selected from the group consisting of a bridged nucleic acid such as LNA, cET, ENA, 2'Fluoro modified nucleotide, 2O-Methyl modified nucleotide, 2’ O-Methoxyethyl modified nucleotide and a combination thereof.

4. Composition according to any one of claims 1 to 3, wherein one or more ANGPTL3 oligonucleotide(s) is/are selected from SEQ ID NO. 3 to SEQ ID NO.45 and SEQ ID NO. 48 to SEQ ID NO. 97 and one or more ANGPTL4 oligonucleotide(s) is/are selected from SEQ ID NO.100 to SEQ ID NO.154, SEQ ID NO.157 to 249, SEQ ID NO.252 to 298 and SEQ ID NO.301 to 349 or a combination thereof.

5. Composition according to any one of claims 1 to 4, wherein the inhibitor inhibits the expression of ANGPTL3 and/or ANGPTL4 at a nanomolar or micromolar concentration.

6. Composition according to any one of claims 1 to 5 further comprising a

pharmaceutically acceptable carrier, excipient, dilutant or a combination thereof.

7. Composition according to any one of claims 1 to 6 for use in a method of preventing and/or treating a disorder, where an ANGPTL3 and/or ANGPLT4 imbalance is involved.

8. Composition for use according to claim 7, wherein the disorder is a cardiometabolic disease, obesity, diabetes such as type 2 diabetes, hypercholesterolemia,

hypertriglyceridemia (HTG), dyslipidemia, pancreatitis, metabolic syndrome, familial chylomicronemia syndrome (FCS) and/or cancer.

9. Composition for use according to claim 7 or 8, wherein the hypercholesterolemia is homozygote familial hypercholesterolemia (HoFH) or heterozygote familial

hypercholesterolemia (HeFH).

10. Composition for use according to any one of claims 7 to 9, wherein the cancer is breast cancer, lung cancer, malignant melanoma, lymphoma, skin cancer, bone cancer, prostate cancer, liver cancer, brain cancer, cancer of the larynx, gall bladder, pancreas, testicular, rectum, parathyroid, thyroid, adrenal, neural tissue, head and neck, colon, stomach, bronchi, kidneys, basal cell carcinoma, squamous cell carcinoma, metastatic skin carcinoma, osteo sarcoma, 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, hairy-cell tumor, adenoma, hyperplasia, medullary carcinoma, intestinal ganglioneuromas, Wilm's tumor, seminoma, ovarian tumor, leiomyomater tumor, cervical dysplasia, retinoblastoma, soft tissue sarcoma, malignant carcinoid, topical skin lesion, rhabdomyosarcoma, Kaposi's sarcoma, osteogenic sarcoma, malignant hypercalcemia, renal cell tumor, polycythermia vera, adenocarcinoma, anaplastic astrocytoma, glioblastoma multiforma, leukemia, or epidermoid carcinoma.

11. Composition for use according to any one of claims 7 to 10, wherein the composition is suitable to be administered locally or systemically.

12. Composition for use according to any one of claims 7 to 11, wherein the composition is suitable to be administered once or repeatedly.