MLKL-specific antisense oligonucleotides and their use in the prevention and / or treatment of a disorder caused by forms of cell death such as necroptosis

Abstract

The present invention refers to an oligonucleotide comprising 15 to 22 nucleotides, wherein at least one of the nucleotides is modified, and the oligonucleotide hybridizes with pre-mRNA of mixed lineage kinase domain like pseudokinase (MLKL) derived from SEQ ID NO.1289 (GRCh38.p 14: 16:74671855-74700960) or mRNA of MLKL derived from SEQ ID NO.1290 (RefSeq ID NM_152649.4). The invention is further directed to a pharmaceutical composition comprising such oligonucleotide. The pharmaceutical composition and the oligonucleotide are for use in a method of preventing and / or treating a hyperproliferative disorder, an inflammatory or autoimmune disorder, neurodegenerative disease, a neurological disorder, cardiovascular, metabolic disorder, renal disorder, liver disorder, lung disorder, skin disorder, ocular disorder, disorder of the gastro-intestinal tract, joint inflammation, organ malfunction after organ transplantation, fibrotic disorder and a combination thereof inflammatory disease, cancer, an ophthalmic disease, an autoimmune disorder and / or an immune disorder.

Description

[0001] MLKL-specific antisense oligonucleotides and their use in the prevention and / or treatment of a disorder caused by forms of cell death such as necroptosis

[0002] The present invention refers to oligonucleotides, in particular antisense oligonucleotides, comprising one or more modified nucleotides and hybridizing with mixed lineage kinase domain like pseudokinase (MLKL) such as MLKL pre-mRNA or mRNA. The oligonucleotides inhibit the expression of MLKL, MLKL mRNA and / or MLKL pre-mRNA and are used for preventing and / or treating diseases involving MLKL mediated cell death.

[0003] Technical Background

[0004] Mixed lineage kinase domain like pseudokinase (MLKL) is the central effector protein in necroptosis. In this form of inflammatory cell death, MLKL translocates to the cell membrane, oligomerizes which results in plasma membrane destabilization, leading to the release of so-called alarmins: These are cell components that are able to trigger local and systemic inflammatory reactions. Thus, necroptosis for example plays an important role in the immune defense against infections. However, the excessive activation of necroptotic processes itself is associated with numerous diseases. Furthermore, it can also lead to a vicious circle of chronic inflammatory reactions and is therefore associated with numerous inflammatory diseases.

[0005] MLKL is a key factor of the necroptosis pathway and its specific inhibition should result in a reduction of necroptosis and in turn break the vicious circle of chronic inflammatory reactions. However, MLKL is a difficult target for antibodies or small molecules to inhibit its undesired activities.

[0006] WO2021224505A1, WO2015172203A1, WO-2018157800-A1 disclose low-molecular active ingredients leading to the direct inhibition of MLK. None of these substances has been tested in clinical studies yet.

[0007] Martens S. et al. (Trends in Pharmacological Sciences, Vol. 41, Issue 3, p. 209-224) describe small molecule drugs acting upstream of MLKL, e.g. by inhibiting RIPK1 or RIPK3. Some of these drugs, especially RIPK1 inhibitors, are currently being tested in phase II trials for example ABBV-668 (AbbVie Inc); DNL-788, eclitasertib (Denali Therapeutics Inc); HNC-664 (Guangzhou Henovcom Bioscience Co Ltd); R-552 (Eli Lilly and Co); SIR-0365 (Sironax Ltd). Hence, until to date small molecule drugs have been developed that either specifically inhibit MLKL or intervene further upstream in the signaling pathway that leads to the activation of MLKL.

[0008] Low-molecular active substances, especially substances that aim to target proteinprotein interactions, are often non-specific and can therefore cause off-target effects. In addition, low-molecular active ingredients are often rapidly degraded in and excreted from the organism. This requires daily or even multiple daily applications of the active ingredient.

[0009] ASOs allow to avoid these problems and to inhibit the expression of MLKL specifically and permanently at low concentrations. Specific inhibition of MLKL compared to inhibitors that intervene further upstream in the necroptosis signaling pathway also reduce the risk of undesirable side effects.

[0010] So far only Rasheed A. et al. (Arteriosclerosis, Thrombosis and Vascular Biology, Vol. 40, No. 5) describe a mouse-specific ASO tested against MLKL for the treatment of atherosclerosis. However, they do not disclose human-specific ASOs that suppress human MLKL.

[0011] ASOs of the present invention are very successful in the inhibition of the expression and activity of MLKL, respectively. 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

[0012] (i) the blocking of multiple functions and activities, respectively, of a target,

[0013] (ii) the penetration of tumor tissue in solid tumors due to their small molecular size,

[0014] (iii) the combination of oligonucleotides with each other or an antibody or a small molecule, and

[0015] (iv) the inhibition of intracellular effects which are not accessible for an antibody or effects not inhibitable via a small molecule. ASOs of the present invention lead to the reliable and specific inhibition of MLKL expression. The substances inhibit the expression of MLKL in corresponding diseaserelevant cell types and minimize necroptotic cell death in these cells. This reduces undesired acute and chronic inflammatory reactions.

[0016] ASOs of the present invention are advantageous due to their specificity, high stability, strong target affinity, efficacy and potency. Moreover, the ASOs are advantageous due to their independence from delivery reagents to achieve target suppression in relevant cells.

[0017] Summary

[0018] The present invention refers to an oligonucleotide comprising 15 to 22 nucleotides, at least one nucleotide having a modification selected from the group consisting of a bridged nucleic acid such as LNA, ENA, TNA, cET, a 2'Fluoro modified nucleotide, a 2'O-Methyl modified nucleotide, a 2'O-Methoxyethyl modified nucleotide, a FANA, a bicyclo DNA, a tricyclo DNA and a combination thereof, and wherein the oligonucleotide hybridizes with pre-mRNA of mixed lineage kinase domain like pseudokinase (MLKL) derived from SEQ ID NO.1289 and / or with a transcript or an mRNA of MLKL derived from SEQ ID NO.1290. The modification of the nucleotide is preferably located at the 5'-end and / or 3'- end of the oligonucleotide.

[0019] The oligonucleotide of the present invention hybridizes outside a hybridizing active region or within a hybridizing active region. The hybridizing active region is for example a region of position 8401 to 8700, of position 17401 tol7700, of position 6301 to 6600, of position 9301 to 9600, of position 19801 to 20100, of position 1 to 300, of position 23701 to 24000, of position 23401 to 23700, of position 4501 to 4800, of position 3901 to 4200, of position 19201 to 19500, of position 301 to 600, of position 20101 to 20400, of position 11101 to 11400, of position 7501 to 7800, of position 3601 to 3900, of position 10801 to 11100, of position 3001 to 3300, of position 601 to 900, of position 901 to 1200, of position 1501 to 1800, of position 2401 to 2700, of position 3301 to 3600, of position 4201 to 4500, of position 4801 to 5100, of position 5701 to 6000, of position 6901 to 7200, of position 7201 to 7500, of position 8101 to 8400, of position 9001 to 9300, of position 9901 to 10200, of position 10201 to 10500, of position 10501 to 10800, of position 11401 to 11700, of position 11701 to 12000, of position 12301 to 12600, of position 12901 to 13200, of position 13201 to 13500, of position 13501 to 13800, of position 13801 to 14100, of position 15601 to 15900, of position 16501 to 16800, of position 17701 to 18000, of position 18301 to 18900, of position 18901 to 19200, of positionl9501 to 19800, of position 20401 to 20700, of position 20701 to 21000, of position 21001 to 21300, of position 21301 to 21600, of position 21901 to 22200, of position 22201 to 22500, of position 22501 to 22800, of position 23101 to 23400, of position 24001 to 24300, of position 24301 to 24600, of position 25201 to 25500, of position 25501 to 25800, of position 25801 to 26100, of position 26101 to 26400, of position 26401 to 26700, of position 26701 to 2700, of position 27301 to 27600, of position 27601 to 27900, of position 27901 to 28200, of position 28201 to 28500, of position 28501 to 28800, of position 28801 to 29100 or a combination thereof, preferably wherein the oligonucleotide hybridizes with a hot spot of position 8523 to 8549, of position 6338 to 6366, of position 3659 to 3696, of position 9518 to 9551, of position 19992 to 20020, of position 17596 to 17628, of position 17 to 46, of position 23838 to 23875 of SEQ ID NO.1289 or a combination thereof. The hybridizing active region is the region on the pre-mRNA derived from GRCh38.p 14: 16:74671855-74700960 (SEQ ID NO.1289).

[0020] The oligonucleotide of the present invention comprises for example SEQ ID NO: 117, SEQ ID NO:93, SEQ ID NO:128, SEQ ID NO:248, SEQ ID NO:116, SEQ ID NO:77, SEQ ID NO:260, SEQ ID NO:34, SEQ ID NO:45, SEQ ID NO: 134, SEQ ID NO: 136, SEQ ID NO:215, SEQ ID NO:41, SEQ ID NO:244, SEQ ID NO:140, SEQ ID NO:127, SEQ ID NO:219, SEQ ID NO:222, SEQ ID NO:162, SEQ ID NO:214, SEQ ID NO:106, SEQ ID NO:239, SEQ ID NO:247, SEQ ID NO:110, SEQ ID NO:43, SEQ ID NO:156 or a combination thereof or a combination thereof. In addition, the oligonucleotide of the present invention has for example 80 to 99 %, 85 to 98 %, 90 to 95 or 93 % sequence identity to any one of the oligonucleotides of these sequences for example any one of SEQ ID NO.l to SEQ ID NO.765 or any one of SEQ ID NO.1294 to SEQ ID NO.2581. Alternatively or in addition, the oligonucleotide of the present invention comprises for example at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 nucleotides of any one of these oligonucleotides for any one of SEQ ID NO.l to SEQ ID NO.765 or any one of SEQ ID NO.1294 to SEQ ID NO.2581, in particular for example any one of SEQ ID NO:117, SEQ ID NO:128, SEQ ID NO:248, SEQ ID NO:116, SEQ ID NO:77, SEQ ID NO:93, SEQ ID NO:260, SEQ ID NO:34, SEQ ID NO:45, SEQ ID NO:134, SEQ ID NO:136, SEQ ID NO:215, SEQ ID NO:41, SEQ ID NO:244, SEQ ID NO:140, SEQ ID NO:127, SEQ ID NO:219, SEQ ID NO:222, SEQ ID NO:162, SEQ ID NO:214, SEQ ID NO:106, SEQ ID NO:239, SEQ ID NO:247, SEQ ID NO:110, SEQ ID NO:43, or SEQ ID NO: 156.

[0021] The oligonucleotide is for example selected from the group consisting of +G*+G*A*T*A*A*G*G*T*G*G*T*A*T*G*+C*+A*+A*+G (SEQ ID NO: 117; A63331Hi), +T*+A*G*+G*T*A*A*A*T*C*T*T*A*C*G*+G*+T*+A (SEQ ID NO:93; A63294Hi),+T*A*+G*G*T*A*A*A*T*C*T*T*A*C*+G*+G*+T*+A (SEQ ID NO:93; A63297Hi), +G*+G*+A*T*A*A*G*G*T*G*G*T*A*T*G*C*+A*+A*+G (SEQ ID NO:117; A63117Hi), +G*+A*+A*A*G*G*T*T*T*G*G*C*G*C*T*+G*+C*+T (SEQ ID NO: 128; A63128Hi), +G*+T*+T*G*A*G*T*T*T*A*G*G*T*G*G*+T*+C*+C (SEQ ID NO:248; A63248Hi), +A*+A*+C*A*C*T*G*G*T*A*G*T*A*G*T*A*+T*+A*+G (SQ ID NO: 116; A63116Hi), +G*+A*+C*A*T*A*G*T*T*C*A*A*G*C*T*+C*+A*+G (SEQ ID NO:77; A63077Hi), +T*+A*+G*G*T*A*A*A*T*C*T*T*A*C*G*+G*+T*+A (SEQ ID NO:93; A63093Hi), +G*+T*+A*A*T*T*T*A*T*T*T*A*G*C*T*C*+C*+T*+G (SEQ ID NO:260;

[0022] A63260HMfa), +T*+C*+A*C*A*C*C*G*T*T*T*G*T*G*G*+A*+T*+G (SEQ ID NO:34; A63034H), +C*+C*+T*G*C*A*G*A*T*A*T*T*G*G*A*T*+C*+T*+A (SEQ ID NO:45 A63045Hmfa), +A*+G*+A*G*C*G*T*G*A*G*A*G*T*A*G*+T*+A*+G (SEQ ID NO: 134; A63134EH), +T*+T*+A*C*A*T*C*T*A*G*C*G*C*+C*+G*+T (SEQ ID NO: 136;

[0023] A63136Hi), +C*+T*+G*C*A*T*C*T*T*A*G*T*T*A*C*C*T*+C*+T*+T (SEQ ID NO:215; A63215EH), +G*+T*+T*C*A*T*G*G*C*T*G*T*G*G*T*+T*+A*+A (SEQ ID NO:41;

[0024] A63041H), +T*+T*+T*A*T*T*G*G*T*T*A*A*C*A*T*T*A*+T*+G*+G (SEQ ID NO:244; A63244EH), +A*+G*+A*C*C*T*G*C*T*A*A*G*T*T*G*+T*+A*+G (SEQ ID NO: 140; A63140Hi), +G*+G*+C*G*G*C*A*T*A*G*A*T*G*G*A*G*+A*+C*+T (SEQ ID NO: 127; A63127EH), +A*+G*+A*G*T*T*A*G*A*G*T*A*A*G*T*A*T*+T*+G*+G (SEQ ID NO:219; A63219Hi), +G*+T*+A*T*T*T*T*G*A*G*G*A*G*T*G*T*+G*+A*+T (SEQ ID NO:222; A63222Hi), +C*+A*+T*C*T*A*T*C*A*A*A*G*T*T*A*C*T*+G*+T*+C (SEQ ID NO:162; A63162HMfa), +C*+T*+G*C*T*A*G*A*A*G*G*T*A*T*C*+T*+T*+A (SEQ ID NO:214; A63214Hi),+c*+A*+T*A*A*T*A*C*T*T*G*G*C*T*T*G*+T*+C*+G (SEQ ID NO:106; A63106Hi), +G*+A*+T*T*T*G*T*A*T*C*A*A*G*A*A*G*+G*+T*+G (SEQ ID NO:239; A63239Hi), +C*+C*+T*A*T*T*A*T*G*C*A*C*A*T*C*T*+G*+A*+G (SEQ ID NO:247; A63247Hi), +A*+C*+T*A*G*G*A*C*C*A*T*G*C*G*Cmi*+T*+G (SEQ ID NO:110; A63110H), +A*+T*+T*G*C*T*G*A*A*C*T*T*T*T*C*T*+A*+T*+C (SEQ ID NO:43; A63043HMfa), +A*+C*+G*C*T*C*T*T*T*A*C*A*C*C*A*+G*+A*+A (SEQ ID NO:156, A63156Hi) and a combination thereof, wherein + indicates an LNA nucleotide and * indicates a phosphorothioate (PTO) linkage and / or a mesyl-modified phosphate linkage such as a mesyl-phosphoramidate between the nucleotides. In addition, the oligonucleotide of the present invention has for example 80 to 99 %, 85 to 98 %, 90 to 95 or 93 % sequence identity to any one of the oligonucleotides of these sequences.

[0025] According to the present invention, the oligonucleotide has 80 to 99 %, 85 to 98 %, 90 to 95 or 93 % sequence identity to any one of the oligonucleotides of SEQ ID NO.l - SEQ ID NO.1288.

[0026] The oligonucleotide of the present invention comprises for example at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 nucleotides of any one of the oligonucleotides of SEQ ID NO.l - SEQ ID NO.1288.

[0027] The oligonucleotide of the present invention inhibits the expression of MLKL, a MLKL mRNA, a MLKL pre-mRNA or a combination thereof at a nanomolar or micromolar concentration.

[0028] The present invention further refers to a pharmaceutical composition comprising an oligonucleotide of the present invention and a pharmaceutically acceptable carrier, excipient, dilutant, stimulant such as an adjuvant, or a combination thereof. The pharmaceutical composition further comprises for example a therapeutically active agent for example selected from the group consisting of another oligonucleotide, an antibody, a small molecule, a peptide, a lipid, a sugar and a combination thereof. The therapeutically active agent inhibits or stimulates a target which is for example selected from the group consisting of NLRP3, CD39, CD73, NRP1, FRA2, IL-1B, IL-1 receptor, IL- 1R accessory protein, IL- 18, IL- 18 receptor, ASC, NLRC4, AIM2, Caspase- 1, RIPK1, RIPK3, Gasdermin D, TLR4, Caspase-8, P2X7, NFKB, RORyt, TGF-6, IL-21, IL-17, IL- 22, IL-23, IL-6, TNF-u, CCR6, CCL20, STAT3, MMP-1, MMP-8, ACSL4, ADAMTS-5, STING, HMG-CoA, Myd-88, HMGB-1, ROS, TAK-1, Chop, FPR1, LIMCH1, caspase inhibitor and a combination thereof.

[0029] The pharmaceutical composition of the present invention and the oligonucleotide of the present invention, respectively, are for example for use in preventing and / or treating a disorder selected from the group consisting of a hyperproliferative disorder such as cancer, a disorder caused by forms of cell death such as necroptosis, an inflammatory or autoimmune disorder, neurodegenerative disease, a neurological disorder, cardiovascular disorder, metabolic disorder, renal disorder, liver disorder, lung disorder, skin disorder, ocular disorder, disorder of the gastro-intestinal tract, joint inflammation, organ transplantation, fibrotic disorder and a combination thereof. The preventable and / or treatable disorder is for example selected from the group consisting of Alzheimer’s disease, multiple sclerosis, autoimmune encephalitis, stroke, traumatic brain injury, atherosclerosis, nonalcoholic fatty liver disease, nonalcoholic steatohepatitis, hypertension, myocardial infarction, acute kidney injury, ischemia reperfusion injury, chronic kidney diseases, crystal-induced nephropathies, glomerulonephritis, silicosis, asthma, allergic airway inflammation, inflammatory bowel disease, colitis ulcerosa, osteoarthritis, dermatitis, rheumatoid arthritis, juvenile ideopathic arthritis, organ malfunction after transplantation of organs such as kidney, lung, liver and / or heart, fibrotic disorder of kidney, lung, liver and / or heart, hyperinflammation following influenza infection, graft-versus-host disease, interstitial cystitis, uveitis, sarcoidosis, sinusitis, peridontal disease, optic neuritis, myelodysplastic syndrome, gout, obesity-induced inflammation, insulin resistance, type 1 and type 2 diabetes, contact hypersensitivity and a combination thereof, and / or a cancer selected from the group consisting of 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, epidermoid carcinoma and a combination thereof. The pharmaceutical composition and / or the oligonucleotide is / are for example administrable locally or systemically.

[0030] The pharmaceutical composition of the present invention comprises for example an antiinflammatory therapeutic or the oligonucleotide of the present invention is administered in combination with an anti-inflammatory therapeutic. 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.

[0031] Description of figures

[0032] Fig. 1 shows a single concentration efficacy screen in human HeLa cells in a first screening round for human MLKL-specific antisense oligonucleotides (ASOs) after three days of treatment.

[0033] Fig. 2 shows a single concentration efficacy screen in human A549 cells in a first screening round for human MLKL-specific ASOs after three days treatment.

[0034] Fig. 3 depicts concentration-dependent MLKL mRNA knockdown by selected human MLKL-specific ASOs in human A549 cells after three days treatment.

[0035] Fig. 4 shows a single concentration efficacy screen in human HeLa cells in a second screening round for human MLKL-specific antisense oligonucleotides (ASOs) after three days treatment.

[0036] Fig. 5 shows a single concentration efficacy screen in human A549 cells in a second screening round for human MLKL-specific ASOs after three days treatment.

[0037] Fig. 6 depicts concentration- dependent MLKL mRNA knockdown by selected human MLKL-specific ASOs in human A549 cells after three days treatment.

[0038] Fig. 7 shows activity from a single concentration efficacy screen in human Hela cells of human MLKL-specific ASOs within the hybridizing active region of position 8401 to 8700 compared to activity of human MLKL-specific ASOs 6500bp upstream (position 8701- 15200) and downstream (position 1901-8400) of the respective hybridizing active region. Fig. 8 shows activity from a single concentration efficacy screen in human Hela cells of human MLKL-specific ASOs within the hybridizing active region of position 17401 to 17700 compared to activity of human MLKL-specific ASOs 6500bp upstream (position 17701-24200) and downstream (position 10901-17400) of the respective hybridizing active region.

[0039] Fig. 9 shows selected results from a single concentration efficacy screen in human Hela cells in a second screening round for human MLKL-specific ASOs with 50 to 95% sequence identity to SEQ ID NO: 117. SEQ ID NO: 117 is marked as star.

[0040] Detailed description

[0041] The present invention provides human-, monkey-, murine- and rat-specific oligonucleotides, in particular antisense oligonucleotides (ASOs), which hybridize with pre-mRNA I mRNA sequences of mixed lineage kinase domain like pseudokinase (MLKL) such as MLKL of human and / or mouse and / or monkey and / or rat and inhibit the expression and activity, respectively, of MLKL. MLKL plays a critical role for example in tumor necrosis factor (TNF)-induced necroptosis, a programmed cell death process, via interaction with receptor-interacting protein 3 (RIP3), which is a key signaling molecule in necroptosis pathway. Necroptosis has been implicated in the progression of disease in almost every physiological system and recent reports suggest a role for necroptosis in aging. MLKL represents an interesting and highly efficient target for use in a method of preventing and / or treating a disease where cell death is involved, e.g. an inflammatory diseases. ASOs of the present invention are used as single active agent for example in individual therapies. In addition or alternatively, these ASOs are used in combination with other therapeutics and therapies, e.g. anti-inflammatory therapeutics and therapies, respectively.

[0042] 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.

[0043] 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. The term “about” refers to a figure indicated and + / - 10 % of this figure.

[0044] Oligonucleotides of the present invention are for example antisense oligonucleotides consisting of or comprising 10 to 25 nucleotides, 15 to 22 nucleotides, 12 to 18 nucleotides, or 17 to 20 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 oligonucleotides of the present invention comprise for example at least one, at least two, at least three, at least four, at least five or at least six nucleotide(s) which is / are modified. The modified nucleotide is for example a bridged nucleotide such as a locked nucleic acid (LNA, e.g., 2',4'-LNA such as beta-D-LNA and / or alpha-L-LNA), cET, ENA, a threose nucleic acid (TNA), a 2'Fluoro modified nucleotide, a 2'O-Methyl, a 2’0- Methoxyethyl, bicyclo-DNA (e.g., 7', 5'-alpha-bicyclo-DNA), tricyclo-DNA modified nucleotide or a combination thereof. The oligonucleotide of the present invention comprises nucleotides having for example one or more, two or more, three or more or four or more of the same or different modifications. Further, the oligonucleotide of the present invention comprises optionally a modified phosphate backbone, wherein the phosphate is, for example, a phosphorothioate, methylphosphonate, mesyl - phosphoramidate (MsPA; e.g., WO 2021 / 030778), phosphoryl guanidine or a combination thereof. Optionally the cytosines of the oligonucleotide comprises one or more methyl group (s).

[0045] A mesyl-phosphoramidate for example is introduced as a stereorandom and / or stereoselective linkage in the gap and / or one or both wings of a gapmer oligonucleotide. Optionally all phosphate or phosphorothioate linkages of the oligonucleotide are replaced by mesyl-phosphoramidate, or all phosphate or phosphorothioate linkages of the gap or all phosphate or phosphorothioate linkages of one or both wings are replaced by mesyl- phosphoramidate. For example 1 to 5 phosphate or phosphorothioate linkages of the gap are replaced by mesyl-phosphoramidate and / or 3 to 5 phosphate or phosphorothioate linkages near the 5'-side of the gap are replaced by mesyl-phosphoramidate. The mesyl- phosphoramidate is for example introduced into the oligonucleotide by means of a Staudinger reaction, e.g., between methanesulfonyl azide (MsN3, 0.5M solution in 1:1 acetonitrile / toluene) and the trivalent phosphite intermediate produced upon phosphoramidite coupling (as described for example in Anderson B.A. et al., Nucleic Acid Research, 2021, Vol. 49, No. 16, 9026-9041).

[0046] Also a phosphorothioate introduces for example a stereorandom and / or a stereoselective linkage in the gap and / or one or both wings of a gapmer oligonucleotide.

[0047] According to the present invention, “gapmer” means an oligonucleotide having a central region comprising a plurality of nucleosides that support RNase H cleavage positioned between a 5’-region and a 3’-region. The positions of the central region refer to the order of the nucleosides of the central region and are counted starting from the 5’-end of the central region. Thus, the 5’-most nucleoside of the central region is at position 1 of the central region. The “central region” may be referred to as a “gap”, and the “5’-region” and “3’-region” may be referred to as “wings”.

[0048] The oligonucleotide of the present invention is for example an antisense oligonucleotide (ASO), e.g., DNA, siRNA, or miRNA. “Reducing” according to the present invention includes inhibiting an effect such as expression in different percentages and amounts, respectively.

[0049] “Hybridizing” according to the present invention is the interaction of an oligonucleotide of the present invention with mRNA and / or pre-mRNA for example coding for MLKL via Watson Crick-mediated base pairing under physiological conditions. The physiological conditions are for example the physiological conditions of a mammalian such as a human or animal, or a bird or a fish.

[0050] “Hot spot” according to the present invention is an area on the target mRNA and / or the target pre-mRNA where binding of ASOs of the present invention is most preferred and leads to highly efficient reduction of the expression of the target mRNA and / or pre- mRNA.

[0051] The present invention refers to the provision of an oligonucleotide such as an antisense oligonucleotide, mediating the limitation of available MLKL mRNA for protein expression. In order to limit protein expression, the oligonucleotide requires the presence of a complementary nucleic acid sequence representing a hybridization target, which allows the formation of heteroduplexes. The oligonucleotides of the present invention hybridize with the MLKL pre-mRNA derived from SEQ ID NO.1289

[0052] (GRCh38.p 14: 16:74671855-74700960) and / or mRNA of SEQ ID NO.1290 (RefSeq ID NM_152649.4). GRCh38.pl4:16:74671855-74700960 and ReflSeq ID NM_152649.4 specify the exact version of the sequences. The formation of a heteroduplex between the oligonucleotide and the target RNA leads to recruitment of enzymes such as RNaseH leading to degradation or inactivation of the target RNA or termination of transcription, and thus, limits the amount of available MLKL mRNA for protein expression.

[0053] The oligonucleotide of the present invention comprises one or more, two or more, three or more, or four or more modified nucleotide(s) for example at the 3'- and / or 5'- end of the oligonucleotide and / or at any position within the oligonucleotide. The modified nucleotides follow for example in a row of 1, 2, 3, 4, 5, or 6 modified nucleotides, or a modified nucleotide is combined with one or more, two or more, three or more, or four or more unmodified nucleotides. The modified nucleotides are for example in a sequence of 5 nucleotides at the 3'- and / or 5'- end of the oligonucleotide. This sequence needs not to start with the first nucleotide of the 3'- or 5'- end of the oligonucleotide. The following Table 1 presents embodiments of oligonucleotides comprising modified nucleotides for example LNA which are indicated by (+) and phosphorothioate (PTO) indicated by (*). Alternatively, in the oligonucleotides of Table 1 one or more PTO linkages are replaced for example by mesyl-phosphoramidate, methylphosphonate, mesyl-phosphoramidate, phosphoryl guanidine or a combination thereof. The oligonucleotides consisting of or comprising the sequences of Table 1 may comprise any other modified nucleotide and any other combination of modified and unmodified nucleotides. Oligonucleotides of Table 1 hybridize with pre-mRNA and / or mRNA of human, monkey, mouse and / or rat MLKL, preferably human MLKL: EE E17 Table 1: List of human MLKL-specific oligonucleotides, in particular ASOs. An “H” after the ASO ID indicates a human MLKL-specific sequence that binds to an exonic region of the pre-mRNA, a “HMfa” after the ASO ID indicates a human I monkey (e.g., macaca fascicularis aurea) MLKL- specific sequence that binds to an exonic region of the pre- mRNA and a “Hi” after the ASO ID indicates a human MLKL-specific sequence that binds to an intronic region of the pre-mRNA, and “HiMfa” after the ASO ID indicates a human I monkey (e.g., macaca fascicularis aurea) cross -re active MLKL sequence that binds to an intronic region of the pre-mRNA.

[0054] The ASOs of the present invention hybridize, for example, with hybridizing active regions of the pre-mRNA / mRNA derived from SEQ ID NOs.1289 and / or 1290. Such regions comprise or consist of, for example, positions 1-300, positions 301-600, positions 601-900, positions 901-1200, positions 1501-1800, positions 2401-2700, positions 3001- 3300, positions 3301-3600, positions 3601-3900, positions 3901-4200, positions 4201- 4500, positions 4501-4800, positions 4801-5100, positions 5701-6000, positions 6301- 6600, positions 6901-7200, positions 7201-7500, positions 7501-7800, positions 8101- 8400, positions 8401-8700, positions 9001-9300, positions 9301-9600, positions 9901- 10200, positions 10201-10500, positions 10501-10800, positions 10801-11100, positions 11101-11400, positions 11401-11700, positions 11701-12000, positions 12301-12600, positions 12901-13200, positions 13201-13500, positions 13501-13800, positions 13801- 14100, positions 15601-15900, positions 16501-16800, positions 17401-17700, positions 17701-18000, positions 18301-18600, positions 18601-18900, positions 18901-19200, positions 19201-19500, positions 19501-19800, positions 19801-20100, positions 20101- 20400, positions 20401-20700, positions 20701-21000, positions 21001-21300, positions 21301-21600, positions 21901-22200, positions 22201-22500, positions 22501-22800, positions 23101-23400, positions 23401-23700, positions 23701-24000, positions 24001- 24300, positions 24301-24600, positions 25201-25500, positions 25201-25500, positions 25501-25800, positions 25801-26100, positions 26101-26400, positions 26401-26700, positions 26701-2700, positions 27301-27600, positions 27601-27900, positions 27901- 28200, positions 28201-28500, positions 28501-28800, or positions 28801-29100 for example of SEQ ID NO.1289. Following Table 2 shows these regions in more detail:

[0055] Table 2 shows some hybridizing active regions and oligonucleotides, in particular antisense oligonucleotides hybridizing in these regions. The ASOs of the present invention hybridize, for example, with hot spots on the pre- mRNA / mRNA derived from SEQ ID NOs.1289 and / or 1290. Such hot spots comprise or consist of, for example, positions 8523-8549, positions 6338-6366, positions 3659-3696, positions 9518-9551, positions 19992-20020, positions 17596-17628, positions 17-46, or positions23838-23875. Following Table 3 shows these hot spots in more detail:

[0056] Table 3 shows some hot spots and oligonucleotides, in particular antisense oligonucleotides hybridizing in these regions. The oligonucleotides of the present invention hybridize, for example, with pre-mRNA and / or mRNA of human MLKL derived from SEQ ID NO.1289 and / or SEQ ID NO.1290. Such oligonucleotides are called MLKL antisense oligonucleotides. Oligonucleotides of the present invention, which are for example antisense oligonucleotides, are shown in Table 1. The present invention further refers to oligonucleotides such as antisense oligonucleotides having about 80 % to 99 %, about 85 % to 98 %, about 90 % to 95 %, about 90 % to 99%, about 93% to about 99%, about 94% to about 99%, about 95% to about 99%, at least about 85 %, 86 %, 87 %, 88 %, 89 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, or 99 % sequence homology to an oligonucleotide of Table 1, e.g., of any one of SEQ ID NO.1 - SEQ ID NO.1288. Moreover, the present invention refers to oligonucleotides such as antisense oligonucleotides comprising or consisting of at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21 or at least 22 nucleotides of any one of Table 1, e.g., of any one of SEQ ID NO.1 - SEQ ID NO.1288.

[0057] Each nucleotide of the sequence can be modified, wherein ASOs of the present invention preferably comprise a core of 6 to 14, 8 to 12 or 10 unmodified nucleotides. ASOs of the present invention comprise, for example, one or more modified nucleotides, e.g., 1, 2, 3, 4 or 5 nucleotides at the 5'- and / or 3z-end of the oligonucleotide, i.e., on the 5'- and / or 3Z- side of the core. The 5'- and 3z-end are modified identically or differently. If the 5'- and 3z-ends are modified identically the nucleotides are modified at the same positions counted from the 5'- and 3'-end (in each case starting the counting with 1 from the end), respectively, having the same modification for example LNA-modification. If the 5'- and 3z-ends are modified differently the position of the modified nucleotide and / or the type of modification at the 5'- and 3'-ends differ; the type of nucleotide modification is the same (e.g., LNA) or different. Modified nucleotides such as LNA-modified nucleotides need not to be consecutive in a row, but may be separated by one or more 5'- and / or 3z-unmodified nucleotides. Preferably the backbone consists of phosphorothioate, alternatively or in addition of mesyl-phosphoramidate, methylphosphonate, mesyl-phosphoramidate, phosphoryl guanidine or a combination thereof. In the following table exemplary modification patterns at the 5'- and 3z-end of the ASOs of the present invention are described, wherein an unmodified nucleotide is indicated by “ and the number on either side of the “ refers to the number of modified nucleotides such as LNA-modified nucleotides in a row. The modified nucleotide(s) is / are at any position of the 5'- and / or 3Z- end of the ASO as shown for example in the following Table 4:

[0058] Specific positions of the modifications such as LNA modifications at the 5'- and 3 '-end of each ASO are described in the following Table 5:

[0059] In some embodiments, an oligonucleotide of the present invention reduces the amount of MLKL mRNA and / or the MLKL protein expression for example by about 30 % - 100 %, 35 % - 99 %, 40 % - 98 %, 45 % - 97 %, 50 % - 96 %, 55 % - 95 %, 60 % - 90 %, 65 % - 85 %, 70 % - 80 % or at least about 30 %, 35 %, 40 %, 45 %, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99% or 100%. The MLKL mRNA and / or protein expression is for example reduced in a cell, tissue, organ, or a subject.

[0060] The reduction of the amount of the MLKL mRNA and / or MLKL protein expression is for example determined by the comparison of the amount of the MLKL mRNA and / or MLKL protein expression in a sample treated with an oligonucleotide of the present invention and a corresponding untreated control. The untreated control is for example MLKL, MLKL mRNA, MLKL pre-mRNA expression, or a combination thereof in a subject prior to administration of an oligonucleotide of the present invention is administered or an untreated sample such as a cell, blood, urine, saliva etc.. The untreated sample is, for example, taken from a subject before an oligonucleotide of the present invention is administered.

[0061] The oligonucleotide of the present invention reduces the amount of MLKL mRNA and / or the expression of MLKL protein expression at a nanomolar or micromolar concentration for example at a concentration of about 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 jiM. The reduction of the MLKL expression results for example in the reduction of cell death, in particular necroptosis.

[0062] The oligonucleotide of the present invention is for example used in a concentration of about 1, 3, 5, 9, 10, 15, 27, 30, 40, 50, 75, 82, 100, 250, 300, 500, or 740 nM, or 1, 2.2, 3, 5, 6.6 or 10 pM.

[0063] The present invention also refers to a pharmaceutical composition comprising an oligonucleotide of the present invention and a pharmaceutically acceptable carrier, excipient and / or diluent. Optionally the pharmaceutical composition further comprises for example a chemotherapeutic, another oligonucleotide either from the present invention or different from the present invention, a protein, preferably an antagonistic protein such as a fusion protein, an antibody, an antibody fragment such as a FAB fragment, a lipid such as a cholesterol, a lipid monolayer or bilayer, a lipid nanoparticle, an exosome, a fatty acid, such as palmitic acid and / or a small molecule.

[0064] In some embodiments, the oligonucleotide or the pharmaceutical composition of the present invention is for use in a method of preventing and / or treating a disorder such as a disorder caused by forms of cell death such as necroptosis or an inflammatory disorder. In some embodiments, 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 and / or laser treatment. The radiotherapy may be further combined with a chemotherapy (e.g., platinum, gemcitabine).

[0065] The disorder is, for example, characterized by activity of MLKL, such as overactivity or a MLKL mRNA, MLKL pre-mRNA and / or protein imbalance, i.e., the MLKL mRNA and / or protein level is abnormal (e.g., increased) in comparison to the level in a normal, healthy cell, tissue, organ or subject, or MLKL activity is abnormal. The MLKL level is, for example, increased by an increased amount of MLKL mRNA and / or MLKL protein expression. Overactivity of MLKL function is, for example, caused by overactive upstream pathway components, by activating mutations or by unknown genetic, epigenetic or environmental mechanisms.

[0066] The disorder is for example selected from the group consisting of a hyperproliferative disorder such as cancer, a disorder caused by forms of cell death such as necroptosis, an inflammatory or autoimmune disorder, neurodegenerative disease, a neurological disorder, cardiovascular disorder, metabolic disorder, renal disorder, liver disorder, lung disorder, skin disorder, ocular disorder, disorder of the gastro-intestinal tract, joint inflammation, malfunction of a transplanted organa, fibrotic disorder and a combination thereof.

[0067] The disorder is for example selected from the group consisting of Alzheimer’s disease, multiple sclerosis, autoimmune encephalitis, stroke, traumatic brain injury, atherosclerosis, nonalcoholic fatty liver disease, nonalcoholic steatohepatitis, hypertension, myocardial infarction, acute kidney injury, ischemia reperfusion injury, chronic kidney diseases, crystal-induced nephropathies, glomerulonephritis, silicosis, asthma, allergic airway inflammation, inflammatory bowel disease, colitis ulcerosa, osteoarthritis, dermatitis, rheumatoid arthritis, juvenile ideopathic arthritis, organ malfunction after transplantation of an organ such as kidney, lung, liver and / or heart, fibrotic disorder of kidney, lung, liver and / or heart, hyperinflammation following influenza infection, graft-versus-host disease, interstitial cystitis, uveitis, sarcoidosis, sinusitis, peridontal disease, optic neuritis, myelodysplastic syndrome, gout, obesity-induced inflammation, insulin resistance, type 1 and type 2 diabetes, contact hypersensitivity and a combination thereof, and / or a cancer selected from the group consisting of 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, epidermoid carcinoma and a combination thereof. The MLKL mRNA and protein level, respectively, is measured for example by any standard method known to a person skilled in the art, such as immunohistochemistry, western blot, quantitative real time PCR or QuantiGene assay.

[0068] The oligonucleotide and the pharmaceutical composition comprising the oligonucleotide, respectively, of the present invention has an inhibitory effect on the MLKL expression for example for 1, 2, 3, 4, 5 or 6 days, 1, 2 or 3 weeks, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 months or 1 or 2 years. The treatment effect of the oligonucleotides of the present invention for example corresponds to the duration of the inhibitory effect.

[0069] An oligonucleotide or a pharmaceutical composition of the present invention is administered locally or systemically for example intravitreal, intracameral or subconjunctival, e.g., injection, topically via eye drops, orally, sublingually, nasally, subcutaneously, intravenously, intraperitoneally, intramuscularly, intratumorally, intrathecally, intraventricularily, transdermally, and / or rectally. Alternatively or in combination ex vivo treated immune cells are administered. The oligonucleotide is administered alone or in combination with another oligonucleotide of the present invention and optionally in combination with another compound such as another oligonucleotide, an antagonistic protein such as a fusion protein, an antibody, a small molecule and / or a chemotherapeutic (e.g., platinum, gemcitabine). Further, two or more oligonucleotides of the present invention are, for example, administered together, at the same time point, e.g., in a pharmaceutical composition or separately, or on staggered intervals.

[0070] One or more oligonucleotides of the present invention are for example administered together with another compound such as another oligonucleotide (i.e., not being part of the present invention), an antagonistic protein such as a fusion protein, an antibody, a small molecule and / or a chemotherapeutic, at the same time point for example in a pharmaceutical composition or separately, or on staggered intervals. The oligonucleotide inhibits for example the expression and activity, respectively, of a receptor such as a growth receptor and the other oligonucleotide (i.e., not being part of the present invention), an antagonistic protein such as a fusion protein, the antibody and / or small molecule inhibits (antagonist) the identical or a different growth receptor or it inhibits (antagonist) a signal transduction factor. The present invention further refers to a pharmaceutical composition comprising an oligonucleotide of the present invention and a pharmaceutically acceptable carrier, excipient, dilutant or a combination thereof. The pharmaceutical composition further comprises for example another active agent selected from the group consisting of an oligonucleotide, an antibody, a small molecule, a peptide, a lipid, a sugar and a combination thereof. The oligonucleotide and the other active agent inhibit for example the same target or a different target. The other active agent for example modulates, e.g., inhibits or stimulates the target selected from the group consisting of NLRP3, CD39, CD 73, NRP1, FRA2, IL- IB, IL-1 receptor, IL-1R accessory protein, IL- 18, IL- 18 receptor, ASC, NLRC4, AIM2, Caspase- 1, RIPK1, RIPK3, Gasdermin D, TLR4, Caspase-8, P2X7, NFKB, RORyt, TGF-B, IL-21, IL- 17, IL-22, IL-23, IL-6, TNF-u, CCR6, CCL20, STAT3, MMP-1, MMP-8, ACSL4, ADAMTS-5, STING, HMG-CoA, Myd-88, HMGB-1, ROS, TAK- 1, Chop, FPR1, LIMCH1, caspase inhibitor and a combination thereof.

[0071] A subject of the present invention is for example a mammalian (e.g., a human, monkey, dog, cat horse, cow, pig), bird, or fish.

[0072] Examples

[0073] The following examples illustrate different embodiments of the present invention, but the invention is not limited to these examples.

[0074] Example 1: Design of human MLKL antisense oligonucleotides

[0075] For ASOs with specificity for the human MLKL gene, the MLKL pre-mRNA sequence derived from the genomic region GRCh38.p 14:16:74671855-74700960 was used. An “H” after the ASO ID indicates a human MLKL-specific sequence that binds to an exonic region of the pre-mRNA, a “HMfa” after the ASO ID indicates a human I monkey (macaca fascicularis aured) cross -re active MLKL-specific sequence that binds to an exonic region of the pre-mRNA, a “Hi” indicates a human MLKL-specific sequence that binds to an intronic region of the pre-mRNA and “HiMfa” after the ASO ID indicates a human I monkey (macaca fascicularis aured) cross -re active MLKL sequence that binds to an intronic region of the pre-mRNA. 16, 17, 18, 19, and 20 mers were designed according to in house criteria, negl (SEQ ID NO:1291:+c*+G*+T*T*T*A*G*G*C*T*A*T*G*T*A*+C*+T*+T described in WO2014 / 154843 Al) was used as control oligonucleotide in all experiments (e.g., for Table 1).

[0076] Example 2: Target knockdown efficacy screen of human MLKL-specific ASOs

[0077] In order to investigate the knockdown efficacy of the in silico designed MLKL-specific ASOs, two efficacy screening rounds in human cell lines were performed. Therefore, cells were treated with the respective ASO at a concentration of 5 pM for three days without the addition of a transfection reagent. Cells were lysed after the three days treatment period, MLKL and HPRT1 mRNA expression was analyzed using the QuantiGene Singleplex assay (ThermoFisher) and the MLKL expression values were normalized to HPRT1 values and compared to mock-treated samples (set as 1). The results are shown in Fig. 1 and Fig. 2 as well as in Table 6 and 7. As depicted in Fig. 1 and Table 6, treatment of HeLa cells with 99 of the 260 tested ASOs (38%) resulted in a target inhibition of >50 %

[0078] (represented by a residual MLKL mRNA expression of <0.5 as compared to mock treated cells).

[0079] Table 6: List of the mean MLKL mRNA expression values in ASO-treated HeLa cells compared to mock treated cells. Expression values are normalized to HPRT1.

[0080] Knockdown efficacy of MLKL-specific ASOs was furthermore tested in A549 cells. As shown in Fig. 2 and Table 7, treatment with 113 of the 260 tested ASOs (43%) resulted in a target inhibition of >50 % (represented by a residual MLKL mRNA expression of <0.5 as compared to mock treated cells).

[0081] Table 7: List of the mean MLKL mRNA expression values in ASO-treated A549 cells compared to mock treated cells. Expression values are normalized to HPRT1. Example 3: Investigation of the concentration-dependent target knockdown by selected human MLKL-specific ASOs

[0082] The concentration- dependent knockdown of MLKL mRNA expression by MLKL-specific ASOs was investigated in A549 cells on mRNA level and the respective ICso values were calculated. Therefore, A549 cells were treated for three days with the respective ASO at the following concentrations: 5000 nM, 1667 nM, 556 nM, 185 nM, 62 nM, 21 nM and 7 nM. After the treatment period, cells were lysed, MLKL and HPRT1 mRNA expression was analyzed using the QuantiGene Singleplex assay (ThermoFisher) and the MLKL expression values were normalized to HPRT1 values and compared to mock-treated samples (set as 100%), i.e. the residual expression as compared to mock-treated samples was calculated (Fig. 3 as well as Table 8). % Inhibition was calculated by subtracting % residual expression from 100%, which corresponds to expression in mock-treated samples. A concentration- dependent knockdown of MLKL mRNA after treatment with all tested MLKL-specific ASOs in A549 was observed with ICso values between 222 nM (A63128FH) and 3802 nM (A63214Hi).

[0083] Table 8: Concentration- dependent inhibition of MLKL mRNA expression in A549 cells by selected MLKL-specific ASOs and respective ICso values. ND = Not determined.

[0084] Example 4: Further target knockdown efficacy screen of human MLKL-specific ASOs

[0085] In order to investigate the knockdown efficacy of the in silico designed MLKL-specific ASOs, two efficacy screening rounds in human cell lines were performed. Therefore, cells were treated with the respective ASO at a concentration of 5 uM for three days without the addition of a transfection reagent. Cells were lysed after the three days treatment period, MLKL and HPRT1 mRNA expression was analyzed using the QuantiGene Singleplex assay (ThermoFisher) and the MLKL expression values were normalized to HPRT1 values and compared to mock-treated samples (set as 1). The results are shown in Fig. 4 and Fig. 5 as well as in Table 9 and 10. As depicted in Fig. 4 and Table 9, treatment of HeLa cells with 224 of the 260 tested ASOs (86%) resulted in a target inhibition of >50 % (represented by a residual MLKL mRNA expression of <0.5 as compared to mock treated cells).

[0086] Table 9: List of the mean MLKL mRNA expression values in ASO-treated HeLa cells compared to mock treated cells. Expression values are normalized to HPRT1. Knockdown efficacy of MLKL-specific ASOs was furthermore tested in A549 cells. As shown in Fig. 5 and Table 10, treatment with 225 of the 260 tested ASOs (87%) resulted in a target inhibition of >50 % (represented by a residual MLKL mRNA expression of <0.5 as compared to mock treated cells).

[0087] Table 10: List of the mean MLKL mRNA expression values in ASO-treated A549 cells compared to mock treated cells. Expression values are normalized to HPRT1.

[0088] Example 5: Investigation of the concentration-dependent target knockdown by selected human MLKL-specific ASOs

[0089] The concentration- dependent knockdown of MLKL mRNA expression by MLKL-specific ASOs was investigated in A549 cells on mRNA level and the respective ICso values were calculated. Therefore, A549 cells were treated for three days with the respective ASO at the following concentrations: 5000 nM, 1667 nM, 556 nM, 185 nM, 62 nM, 21 nM and 7 nM. After the treatment period, cells were lysed, MLKL and HPRT1 mRNA expression was analyzed using the QuantiGene Singleplex assay (ThermoFisher) and the MLKL expression values were normalized to HPRT1 values and compared to mock-treated samples (set as 100%), i.e. the residual expression as compared to mock-treated samples was calculated (Fig. 6 as well as Table 11). % Inhibition was calculated by subtracting % residual expression from 100%, which corresponds to expression in mock-treated samples. A concentration- dependent knockdown of MLKL mRNA after treatment with all tested MLKL-specific ASOs in A549 was observed with ICso values between 86 nM (A63912Hi) and 2001 nM (A63295Hi).

[0090] Table 11: Concentration-dependent inhibition of MLKL mRNA expression in A549 cells by selected MLKL-specific ASOs and respective ICso values. ND = Not determined. Embodiments of the invention

[0091] 1. An oligonucleotide comprising 15 to 22 nucleotides, at least one nucleotide having a modification selected from the group consisting of a bridged nucleic acid such as LNA, ENA, TNA, cET, a 2'Fluoro modified nucleotide, a 2'0-Methyl modified nucleotide, a 2'0 -Methoxyethyl modified nucleotide, a FANA, a bicyclo DNA, a tricyclo DNA and a combination thereof, and wherein the oligonucleotide hybridizes with pre-mRNA of mixed lineage kinase domain like pseudokinase (MLKL) derived from SEQ ID NO.1289 and / or with a transcript or an mRNA of MLKL derived from SEQ ID NO.1290.

[0092] 2. The oligonucleotide of embodiment 1, wherein the nucleotide has a LNA modification.

[0093] 3. The oligonucleotide of embodiment 1 or 2, wherein the nucleotide has an ENA modification. 4. The oligonucleotide of any one of embodiments 1 to 3, wherein the nucleotide has a TNA modification.

[0094] 5. The oligonucleotide of any one of embodiments 1 to 4, wherein the nucleotide has a cET modification.

[0095] 6. The oligonucleotide of any one of embodiments 1 to 5, wherein the nucleotide is 2'Fluoro modified.

[0096] 7. The oligonucleotide of any one of embodiments 1 to 6, wherein the nucleotide is 2'0- Methyl modified.

[0097] 8. The oligonucleotide of any one of embodiments 1 to 7, wherein the nucleotide is 2'0- Methoxyethyl modified.

[0098] 9. The oligonucleotide of any one of embodiments 1 to 8, wherein the nucleotide has a FANA modification.

[0099] 10. The oligonucleotide of any one of embodiments 1 to 9, wherein the nucleotide has a FANA modification.

[0100] 11. The oligonucleotide of any one of embodiments 1 to 10, wherein the nucleotide has a bicyclo DNA, modification.

[0101] 12. The oligonucleotide of any one of embodiments 1 to 11, wherein the nucleotide has a tricyclo DNA, modification.

[0102] 13. The oligonucleotide of any one of embodiments 1 to 12, wherein the oligonucleotide hybridizes with pre-mRNA of mixed lineage kinase domain like pseudokinase (MEKE) derived from SEQ ID NO.1289.

[0103] 14. The oligonucleotide of any one of embodiments 1 to 13, wherein the oligonucleotide hybridizes with a transcript of MLKL derived from SEQ ID NO.1290. 15. The oligonucleotide of any one of embodiments 1 to 13, wherein the oligonucleotide hybridizes with a mRNA of MLKL derived from SEQ ID NO.1290.

[0104] 16. The oligonucleotide of any one of embodiments 1 to 15, wherein the modification is located at the 5'-end and / or 3'-end of the oligonucleotide.

[0105] 17. The oligonucleotide of any one of embodiments 1 to 16, wherein the modification is located at the 5'-end of the oligonucleotide.

[0106] 18. The oligonucleotide of any one of embodiments 1 to 17, wherein the modification is located at the 3'-end of the oligonucleotide.

[0107] 19. The oligonucleotide of any one of embodiments 1 to 18, wherein the oligonucleotide hybridizes outside a hybridizing active region or within a hybridizing active region of position 8401 to 8700, of position 17401 tol7700, of position 6301 to 6600, of position 9301 to 9600, of position 19801 to 20100, of position 1 to 300, of position 23701 to 24000, of position 23401 to 23700, of position 4501 to 4800, of position 3901 to 4200, of position 19201 to 19500, of position 301 to 600, of position 20101 to 20400, of position 11101 to 11400, of position 7501 to 7800, of position 3601 to 3900, of position 10801 to 11100, of position 3001 to 3300, of position 601 to 900, of position 901 to 1200, of position 1501 to 1800, of position 2401 to 2700, of position 3301 to 3600, of position 4201 to 4500, of position 4801 to 5100, of position 5701 to 6000, of position 6901 to 7200, of position 7201 to 7500, of position 8101 to 8400, of position 9001 to 9300, of position 9901 to 10200, of position 10201 to 10500, of position 10501 to 10800, of position 11401 to 11700, of position 11701 to 12000, of position 12301 to 12600, of position 12901 to 13200, of position 13201 to 13500, of position 13501 to 13800, of position 13801 to 14100, of position 15601 to 15900, of position 16501 to 16800, of position 17701 to 18000, of position 18301 to 18900, of position 18901 to 19200, of positionl9501 to 19800, of position 20401 to 20700, of position 20701 to 21000, of position 21001 to 21300, of position 21301 to 21600, of position 21901 to 22200, of position 22201 to 22500, of position 22501 to 22800, of position 23101 to 23400, of position 24001 to 24300, of position 24301 to 24600, of position 25201 to 25500, of position 25501 to 25800, of position 25801 to 26100, of position 26101 to 26400, of position 26401 to 26700, of position 26701 to 2700, of position 27301 to 27600, of position 27601 to 27900, of position 27901 to 28200, of position 28201 to 28500, of position 28501 to 28800, of position 28801 to 29100 or a combination thereof, preferably wherein the oligonucleotide hybridizes with a hot spot of position 8523 to 8549, of position 6338 to 6366, of position 3659 to 3696, of position 9518 to 9551, of position 19992 to 20020, of position 17596 to 17628, of position 17 to 46, of position 23838 to 23875 of SEQ ID NO.1289 or a combination thereof.

[0108] 20. The oligonucleotide of any one of embodiments 1 to 19, wherein the oligonucleotide hybridizes outside a hybridizing active region of of position 8401 to 8700, position 17401 tol7700, of position 6301 to 6600, of position 9301 to 9600, of position 19801 to 20100, of position 1 to 300, of position 23701 to 24000, of position 23401 to 23700, of position 4501 to 4800, of position 3901 to 4200, of position 19201 to 19500, of position 301 to 600, of position 20101 to 20400, of position 11101 to 11400, of position 7501 to 7800, of position 3601 to 3900, of position 10801 to 11100, of position 3001 to 3300, of position 601 to 900, of position 901 to 1200, of position 1501 to 1800, of position 2401 to 2700, of position 3301 to 3600, of position 4201 to 4500, of position 4801 to 5100, of position 5701 to 6000, of position 6901 to 7200, of position 7201 to 7500, of position 8101 to 8400, of position 9001 to 9300, of position 9901 to 10200, of position 10201 to 10500, of position 10501 to 10800, of position 11401 to 11700, of position 11701 to 12000, of position 12301 to 12600, of position 12901 to 13200, of position 13201 to 13500, of position 13501 to 13800, of position 13801 to 14100, of position 15601 to 15900, of position 16501 to 16800, of position 17701 to 18000, of position 18301 to 18900, of position 18901 to 19200, of positionl9501 to 19800, of position 20401 to 20700, of position 20701 to 21000, of position 21001 to 21300, of position 21301 to 21600, of position 21901 to 22200, of position 22201 to 22500, of position 22501 to 22800, of position 23101 to 23400, of position 24001 to 24300, of position 24301 to 24600, of position 25201 to 25500, of position 25501 to 25800, of position 25801 to 26100, of position 26101 to 26400, of position 26401 to 26700, of position 26701 to 2700, of position 27301 to 27600, of position 27601 to 27900, of position 27901 to 28200, of position 28201 to 28500, of position 28501 to 28800, of position 28801 to 29100 or a combination thereof, preferably wherein the oligonucleotide hybridizes with a hot spot of position 8523 to 8549, of position 6338 to 6366, of position 3659 to 3696, of position 9518 to 9551, of position 19992 to 20020, of position 17596 to 17628, of position 17 to 46, of position 23838 to 23875 of SEQ ID NO.1289 or a combination thereof. 21. The oligonucleotide of any one of embodiments 1 to 20, wherein the oligonucleotide hybridizes within a hybridizing active region of position 8401 to 8700, of position 17401 tol7700, of position 6301 to 6600, of position 9301 to 9600, of position 19801 to 20100, of position 1 to 300, of position 23701 to 24000, of position 23401 to 23700, of position 4501 to 4800, of position 3901 to 4200, of position 19201 to 19500, of position 301 to 600, of position 20101 to 20400, of position 11101 to 11400, of position 7501 to 7800, of position 3601 to 3900, of position 10801 to 11100, of position 3001 to 3300, of position 601 to 900, of position 901 to 1200, of position 1501 to 1800, of position 2401 to 2700, of position 3301 to 3600, of position 4201 to 4500, of position 4801 to 5100, of position 5701 to 6000, of position 6901 to 7200, of position 7201 to 7500, of position 8101 to 8400, of position 9001 to 9300, of position 9901 to 10200, of position 10201 to 10500, of position 10501 to 10800, of position 11401 to 11700, of position 11701 to 12000, of position 12301 to 12600, of position 12901 to 13200, of position 13201 to 13500, of position 13501 to 13800, of position 13801 to 14100, of position 15601 to 15900, of position 16501 to 16800, of position 17701 to 18000, of position 18301 to 18900, of position 18901 to 19200, of positionl9501 to 19800, of position 20401 to 20700, of position 20701 to 21000, of position 21001 to 21300, of position 21301 to 21600, of position 21901 to 22200, of position 22201 to 22500, of position 22501 to 22800, of position 23101 to 23400, of position 24001 to 24300, of position 24301 to 24600, of position 25201 to 25500, of position 25501 to 25800, of position 25801 to 26100, of position 26101 to 26400, of position 26401 to 26700, of position 26701 to 2700, of position 27301 to 27600, of position 27601 to 27900, of position 27901 to 28200, of position 28201 to 28500, of position 28501 to 28800, of position 28801 to 29100 or a combination thereof, preferably wherein the oligonucleotide hybridizes with a hot spot of position 8523 to 8549, of position 6338 to 6366, of position 3659 to 3696, of position 9518 to 9551, of position 19992 to 20020, of position 17596 to 17628, of position 17 to 46, of position 23838 to 23875 of SEQ ID

[0109] NO.1289 or a combination thereof.

[0110] 22. The oligonucleotide of embodiment 20 or 21, wherein the hybridizing active region is of position 8401 to 8700.

[0111] 23. The oligonucleotide of any one of embodiments 20 to 22, wherein the hybridizing active region is of position 17401 tol7700. 24. The oligonucleotide of any one of embodiments 20 to 23, wherein the hybridizing active region is of position 6301 to 6600.

[0112] 25. The oligonucleotide of any one of embodiments 20 to 24, wherein the hybridizing active region is of position 9301 to 9600.

[0113] 26. The oligonucleotide of any one of embodiments 20 to 25, wherein the hybridizing active region is of position 19801 to 20100.

[0114] 27. The oligonucleotide of any one of embodiments 20 to 26, wherein the hybridizing active region is of position 1 to 300.

[0115] 28. The oligonucleotide of any one of embodiments 20 to 27, wherein the hybridizing active region is of position 23701 to 24000.

[0116] 29. The oligonucleotide of any one of embodiments 20 to 28, wherein the hybridizing active region is of position 23401 to 23700.

[0117] 30. The oligonucleotide of any one of embodiments 20 to 29, wherein the hybridizing active region is of position 4501 to 4800.

[0118] 31. The oligonucleotide of any one of embodiments 20 to 30, wherein the hybridizing active region is of position 3901 to 4200.

[0119] 32. The oligonucleotide of any one of embodiments 20 to 31, wherein the hybridizing active region is of position 19201 to 19500.

[0120] 33. The oligonucleotide of any one of embodiments 20 to 32, wherein the hybridizing active region is of position 301 to 600.

[0121] 34. The oligonucleotide of any one of embodiments 20 to 33, wherein the hybridizing active region is of position 20101 to 20400.

[0122] 35. The oligonucleotide of any one of embodiments 20 to 34, wherein the hybridizing active region is of position 11101 to 11400. 36. The oligonucleotide of any one of embodiments 20 to 35, wherein the hybridizing active region is of position 7501 to 7800.

[0123] 37. The oligonucleotide of any one of embodiments 20 to 36, wherein the hybridizing active region is of position 3601 to 3900.

[0124] 38. The oligonucleotide of any one of embodiments 20 to 37, wherein the hybridizing active region is of position 10801 to 11100.

[0125] 39. The oligonucleotide of any one of embodiments 20 to 38, wherein the hybridizing active region is of position 3001 to 3300.

[0126] 40. The oligonucleotide of any one of embodiments 20 to 39, wherein the hybridizing active region is of position 601 to 900.

[0127] 41. The oligonucleotide of any one of embodiments 20 to 40, wherein the hybridizing active region is of position 901 to 1200.

[0128] 42. The oligonucleotide of any one of embodiments 20 to 41, wherein the hybridizing active region is of position 1501 to 1800.

[0129] 43. The oligonucleotide of any one of embodiments 20 to 42, wherein the hybridizing active region is of position 2401 to 2700.

[0130] 44. The oligonucleotide of any one of embodiments 20 to 43, wherein the hybridizing active region is of position 3301 to 3600.

[0131] 45. The oligonucleotide of any one of embodiments 20 to 44, wherein the hybridizing active region is of position 4201 to 4500.

[0132] 46. The oligonucleotide of any one of embodiments 20 to 45, wherein the hybridizing active region is of position 4801 to 5100.

[0133] 47. The oligonucleotide of any one of embodiments 20 to 46, wherein the hybridizing active region is of position 5701 to 6000. 48. The oligonucleotide of any one of embodiments 20 to 47, wherein the hybridizing active region is of position 6901 to 7200.

[0134] 49. The oligonucleotide of any one of embodiments 20 to 48, wherein the hybridizing active region is of position 7201 to 7500.

[0135] 50. The oligonucleotide of any one of embodiments 20 to 49, wherein the hybridizing active region is of position 8101 to 8400.

[0136] 51. The oligonucleotide of any one of embodiments 20 to 50, wherein the hybridizing active region is of position 9001 to 9300.

[0137] 52. The oligonucleotide of any one of embodiments 20 to 51, wherein the hybridizing active region is of position 9901 to 10200.

[0138] 53. The oligonucleotide of any one of embodiments 20 to 52, wherein the hybridizing active region is of position 10201 to 10500.

[0139] 54. The oligonucleotide of any one of embodiments 20 to 53, wherein the hybridizing active region is of position 10501 to 10800.

[0140] 55. The oligonucleotide of any one of embodiments 20 to 54, wherein the hybridizing active region is of position 11401 to 11700.

[0141] 56. The oligonucleotide of any one of embodiments 20 to 55, wherein the hybridizing active region is of position 11701 to 12000.

[0142] 57. The oligonucleotide of any one of embodiments 20 to 56, wherein the hybridizing active region is of position 12301 to 12600.

[0143] 58. The oligonucleotide of any one of embodiments 20 to 57, wherein the hybridizing active region is of position 12901 to 13200.

[0144] 59. The oligonucleotide of any one of embodiments 20 to 58, wherein the hybridizing active region is of position 13201 to 13500. 60. The oligonucleotide of any one of embodiments 20 to 59, wherein the hybridizing active region is of position 13501 to 1380.

[0145] 61. The oligonucleotide of any one of embodiments 20 to 60, wherein the hybridizing active region is of position 13801 to 14100.

[0146] 62. The oligonucleotide of any one of embodiments 20 to 61, wherein the hybridizing active region is of position 15601 to 15900.

[0147] 63. The oligonucleotide of any one of embodiments 20 to 62, wherein the hybridizing active region is of position 16501 to 16800.

[0148] 64. The oligonucleotide of any one of embodiments 20 to 63, wherein the hybridizing active region is of position 17701 to 18000.

[0149] 65. The oligonucleotide of any one of embodiments 20 to 64, wherein the hybridizing active region is of position 18301 to 18900.

[0150] 66. The oligonucleotide of any one of embodiments 20 to 65, wherein the hybridizing active region is of position 18901 to 19200.

[0151] 67. The oligonucleotide of any one of embodiments 20 to 66, wherein the hybridizing active region is of positionl9501 to 19800.

[0152] 68. The oligonucleotide of any one of embodiments 20 to 67, wherein the hybridizing active region is of position 20401 to 20700.

[0153] 69. The oligonucleotide of any one of embodiments 20 to 68, wherein the hybridizing active region is of position 20701 to 21000.

[0154] 70. The oligonucleotide of any one of embodiments 20 to 69, wherein the hybridizing active region is of position 21001 to 21300.

[0155] 71. The oligonucleotide of any one of embodiments 20 to 70, wherein the hybridizing active region is of position 21301 to 21600. 72. The oligonucleotide of any one of embodiments 20 to 71, wherein the hybridizing active region is of position 21901 to 22200.

[0156] 73. The oligonucleotide of any one of embodiments 20 to 72, wherein the hybridizing active region is of position 22201 to 22500.

[0157] 74. The oligonucleotide of any one of embodiments 20 to 73, wherein the hybridizing active region is of position 22501 to 22800.

[0158] 75. The oligonucleotide of any one of embodiments 20 to 74, wherein the hybridizing active region is of position 23101 to 23400.

[0159] 76. The oligonucleotide of any one of embodiments 20 to 75, wherein the hybridizing active region is of position 24001 to 24300.

[0160] 77. The oligonucleotide of any one of embodiments 20 to 76, wherein the hybridizing active region is of position 24301 to 24600.

[0161] 78. The oligonucleotide of any one of embodiments 20 to 77, wherein the hybridizing active region is of position 25201 to 25500.

[0162] 79. The oligonucleotide of any one of embodiments 20 to 78, wherein the hybridizing active region is of position 25501 to 25800.

[0163] 80. The oligonucleotide of any one of embodiments 20 to 79, wherein the hybridizing active region is of position 25801 to 26100.

[0164] 81. The oligonucleotide of any one of embodiments 20 to 80, wherein the hybridizing active region is of position 26101 to 26400.

[0165] 82. The oligonucleotide of any one of embodiments 20 to 81, wherein the hybridizing active region is of position 26401 to 26700.

[0166] 83. The oligonucleotide of any one of embodiments 20 to 82, wherein the hybridizing active region is of position 26701 to 2700. 84. The oligonucleotide of any one of embodiments 20 to 83, wherein the hybridizing active region is of position 27301 to 27600.

[0167] 85. The oligonucleotide of any one of embodiments 20 to 84, wherein the hybridizing active region is of position 27601 to 27900.

[0168] 86. The oligonucleotide of any one of embodiments 20 to 85, wherein the hybridizing active region is of position 27901 to 28200.

[0169] 87. The oligonucleotide of any one of embodiments 20 to 86, wherein the hybridizing active region is of position 28201 to 28500.

[0170] 88. The oligonucleotide of any one of embodiments 20 to 87, wherein the hybridizing active region is of position 28501 to 28800.

[0171] 89. The oligonucleotide of any one of embodiments 20 to 88, wherein the hybridizing active region is of position 28801 to 29100.

[0172] 90. The oligonucleotide of any one of embodiments 20 to 89, wherein the oligonucleotide hybridizes with a hot spot of position 8523 to 8549.

[0173] 91. The oligonucleotide of any one of embodiments 20 to 90, wherein the oligonucleotide hybridizes with a hot spot of position 6338 to 6366.

[0174] 92. The oligonucleotide of any one of embodiments 20 to 91, wherein the oligonucleotide hybridizes with a hot spot of position 3659 to 3696.

[0175] 93. The oligonucleotide of any one of embodiments 20 to 92, wherein the oligonucleotide hybridizes with a hot spot of position 9518 to 9551.

[0176] 94. The oligonucleotide of any one of embodiments 20 to 93, wherein the oligonucleotide hybridizes with a hot spot of position 19992 to 20020.

[0177] 95. The oligonucleotide of any one of embodiments 20 to 94, wherein the oligonucleotide hybridizes with a hot spot of position 17596 to 17628. 96. The oligonucleotide of any one of embodiments 20 to 95, wherein the oligonucleotide hybridizes with a hot spot of position 17 to 46.

[0178] 97. The oligonucleotide of any one of embodiments 20 to 96, wherein the oligonucleotide hybridizes with a hot spot of position 23838 to 23875.

[0179] 98. The oligonucleotide of any one of embodiments 1 to 97, wherein the oligonucleotide comprises SEQ ID NO:93.

[0180] 99. The oligonucleotide of any one of embodiments 1 to 98, wherein the oligonucleotide comprises SEQ ID NO: 117.

[0181] 100. The oligonucleotide of any one of embodiments 1 to 99, wherein the oligonucleotide comprises SEQ ID NO: 128.

[0182] 101. The oligonucleotide of any one of embodiments 1 to 100, wherein the oligonucleotide comprises SEQ ID NO:248,

[0183] 102. The oligonucleotide of any one of embodiments 1 to 101, wherein the oligonucleotide comprises SEQ ID NO: 116.

[0184] 103. The oligonucleotide of any one of embodiments 1 to 102, wherein the oligonucleotide comprises SEQ ID NO:77.

[0185] 104. The oligonucleotide of any one of embodiments 1 to 103, wherein the oligonucleotide comprises SEQ ID NO:260.

[0186] 105. The oligonucleotide of any one of embodiments 1 to 104, wherein the oligonucleotide comprises SEQ ID NO:34.

[0187] 106. The oligonucleotide of any one of embodiments 1 to 105, wherein the oligonucleotide comprises SEQ ID NO:45.

[0188] 107. The oligonucleotide of any one of embodiments 1 to 106, wherein the oligonucleotide comprises SEQ ID NO: 134. 108. The oligonucleotide of any one of embodiments 1 to 107, wherein the oligonucleotide comprises SEQ ID NO: 136.

[0189] 109. The oligonucleotide of any one of embodiments 1 to 108, wherein the oligonucleotide comprises SEQ ID NO:215.

[0190] 110. The oligonucleotide of any one of embodiments 1 to 109, wherein the oligonucleotide comprises SEQ ID NO:41.

[0191] 111. The oligonucleotide of any one of embodiments 1 to 110, wherein the oligonucleotide comprises SEQ ID NO:244.

[0192] 112. The oligonucleotide of any one of embodiments 1 to 111, wherein the oligonucleotide comprises SEQ ID NO: 140.

[0193] 113. The oligonucleotide of any one of embodiments 1 to 112, wherein the oligonucleotide comprises SEQ ID NO: 127.

[0194] 114. The oligonucleotide of any one of embodiments 1 to 113, wherein the oligonucleotide comprises SEQ ID NO:219.

[0195] 115. The oligonucleotide of any one of embodiments 1 to 114, wherein the oligonucleotide comprises SEQ ID NO:222.

[0196] 116. The oligonucleotide of any one of embodiments 1 to 115, wherein the oligonucleotide comprises SEQ ID NO: 162.

[0197] 117. The oligonucleotide of any one of embodiments 1 to 116, wherein the oligonucleotide comprises SEQ ID NO:214.

[0198] 118. The oligonucleotide of any one of embodiments 1 to 117, wherein the oligonucleotide comprises SEQ ID NO: 106.

[0199] 119. The oligonucleotide of any one of embodiments 1 to 118, wherein the oligonucleotide comprises SEQ ID NO:239. 120. The oligonucleotide of any one of embodiments 1 to 119, wherein the oligonucleotide comprises SEQ ID NO:247.

[0200] 121. The oligonucleotide of any one of embodiments 1 to 120, wherein the oligonucleotide comprises SEQ ID NO: 110,.

[0201] 122. The oligonucleotide of any one of embodiments 1 to 121, wherein the oligonucleotide comprises SEQ ID NO:43.

[0202] 123. The oligonucleotide of any one of embodiments 1 to 122, wherein the oligonucleotide comprises SEQ ID NO: 156.

[0203] 124. The oligonucleotide of any one of embodiments 1 to 123, wherein the oligonucleotide is selected from the group consisting of +G*+G*A*T*A*A*G*G*T*G*G*T*A*T*G*+C*+A*+A*+G (SEQ ID NO: 117; A63331Hi), +T*+A*G*+G*T*A*A*A*T*C*T*T*A*C*G*+G*+T*+A (SEQ ID NO:93; A63294Hi),+T*A*+G*G*T*A*A*A*T*C*T*T*A*C*+G*+G*+T*+A (SEQ ID NO:93; A63297Hi), +G*+G*+A*T*A*A*G*G*T*G*G*T*A*T*G*C*+A*+A*+G (SEQ ID NO:117; A63117Hi), +G*+A*+A*A*G*G*T*T*T*G*G*C*G*C*T*+G*+C*+T (SEQ ID NO: 128; A63128Hi), +G*+T*+T*G*A*G*T*T*T*A*G*G*T*G*G*+T*+C*+C (SEQ ID NO:248; A63248Hi), +A*+A*+C*A*C*T*G*G*T*A*G*T*A*G*T*A*+T*+A*+G (SQ ID NO: 116; A63116Hi), +G*+A*+C*A*T*A*G*T*T*C*A*A*G*C*T*+C*+A*+G (SEQ ID NO:77; A63077Hi), +T*+A*+G*G*T*A*A*A*T*C*T*T*A*C*G*+G*+T*+A (SEQ ID NO:93; A63093Hi), +G*+T*+A*A*T*T*T*A*T*T*T*A*G*C*T*C*+C*+T*+G (SEQ ID NO:260;

[0204] A63260HMfa), +T*+C*+A*C*A*C*C*G*T*T*T*G*T*G*G*+A*+T*+G (SEQ ID NO:34; A63034H), +C*+C*+T*G*C*A*G*A*T*A*T*T*G*G*A*T*+C*+T*+A (SEQ ID NO:45 A63045Hmfa), +A*+G*+A*G*C*G*T*G*A*G*A*G*T*A*G*+T*+A*+G (SEQ ID NO: 134; A63134Hi), +T*+T*+A*C*A*T*C*T*A*G*C*G*C*+C*+G*+T (SEQ ID NO: 136;

[0205] A63136Hi), +C*+T*+G*C*A*T*C*T*T*A*G*T*T*A*C*C*T*+C*+T*+T (SEQ ID NO:215; A63215Hi), +G*+T*+T*C*A*T*G*G*C*T*G*T*G*G*T*+T*+A*+A (SEQ ID NO:41; A63041H), +T*+T*+T*A*T*T*G*G*T*T*A*A*C*A*T*T*A*+T*+G*+G (SEQ ID NO:244; A63244Hi), +A*+G*+A*C*C*T*G*C*T*A*A*G*T*T*G*+T*+A*+G (SEQ ID NO: 140; A63140Hi), +G*+G*+C*G*G*C*A*T*A*G*A*T*G*G*A*G*+A*+C*+T (SEQ ID NO: 127; A63127Hi), +A*+G*+A*G*T*T*A*G*A*G*T*A*A*G*T*A*T*+T*+G*+G (SEQ ID NO:219; A63219Hi), +G*+T*+A*T*T*T*T*G*A*G*G*A*G*T*G*T*+G*+A*+T (SEQ ID NO:222; A63222Hi), +C*+A*+T*C*T*A*T*C*A*A*A*G*T*T*A*C*T*+G*+T*+C (SEQ ID NO:162; A63162HMfa), +C*+T*+G*C*T*A*G*A*A*G*G*T*A*T*C*+T*+T*+A (SEQ ID NO:214; A63214IH), +C*+A*+T*A*A*T*A*C*T*T*G*G*C*T*T*G*+T*+C*+G (SEQ ID NO:106; A63106IH), +G*+A*+T*T*T*G*T*A*T*C*A*A*G*A*A*G*+G*+T*+G (SEQ ID NO:239; A63239IH), +C*+C*+T*A*T*T*A*T*G*C*A*C*A*T*C*T*+G*+A*+G (SEQ ID NO:247; A63247H1), +A*+C*+T*A*G*G*A*C*C*A*T*G*C*G*Cmi*+T*+G (SEQ ID NO:110; A63110H), +A*+T*+T*G*C*T*G*A*A*C*T*T*T*T*C*T*+A*+T*+C (SEQ ID NO:43; A63043HMfa), +A*+C*+G*C*T*C*T*T*T*A*C*A*C*C*A*+G*+A*+A (SEQ ID NO:156, A63156EH) and a combination thereof, wherein + indicates a modifies nucleotide and * indicates a modified phosphate linkage the nucleotides.

[0206] 125. The oligonucleotide of any one of embodiments 1 to 124, wherein the oligonucleotide is selected from the group consisting of +G*+G*A*T*A*A*G*G*T*G*G*T*A*T*G*+C*+A*+A*+G (SEQ ID NO: 117; A63331Hi), +T*+A*G*+G*T*A*A*A*T*C*T*T*A*C*G*+G*+T*+A (SEQ ID NO:93; A63294Hi),+T*A*+G*G*T*A*A*A*T*C*T*T*A*C*+G*+G*+T*+A (SEQ ID NO:93; A63297Hi), +G*+G*+A*T*A*A*G*G*T*G*G*T*A*T*G*C*+A*+A*+G (SEQ ID NO:117; A63117Hi), +G*+A*+A*A*G*G*T*T*T*G*G*C*G*C*T*+G*+C*+T (SEQ ID NO: 128; A63128Hi), +G*+T*+T*G*A*G*T*T*T*A*G*G*T*G*G*+T*+C*+C (SEQ ID NO:248; A63248Hi), +A*+A*+C*A*C*T*G*G*T*A*G*T*A*G*T*A*+T*+A*+G (SQ ID NO: 116; A63116Hi), +G*+A*+C*A*T*A*G*T*T*C*A*A*G*C*T*+C*+A*+G (SEQ ID NO:77; A63077Hi), +T*+A*+G*G*T*A*A*A*T*C*T*T*A*C*G*+G*+T*+A (SEQ ID NO:93; A63093Hi), +G*+T*+A*A*T*T*T*A*T*T*T*A*G*C*T*C*+C*+T*+G (SEQ ID NO:260;

[0207] A63260HMfa), +T*+C*+A*C*A*C*C*G*T*T*T*G*T*G*G*+A*+T*+G (SEQ ID NO:34; A63034H), +C*+C*+T*G*C*A*G*A*T*A*T*T*G*G*A*T*+C*+T*+A (SEQ ID NO:45 A63045Hmfa), +A*+G*+A*G*C*G*T*G*A*G*A*G*T*A*G*+T*+A*+G (SEQ ID NO: 134; A63134Hi), +T*+T*+A*C*A*T*C*T*A*G*C*G*C*+C*+G*+T (SEQ ID NO: 136;

[0208] A63136Hi), +C*+T*+G*C*A*T*C*T*T*A*G*T*T*A*C*C*T*+C*+T*+T (SEQ ID NO:215; A63215Hi), +G*+T*+T*C*A*T*G*G*C*T*G*T*G*G*T*+T*+A*+A (SEQ ID NO:41; A63041H), +T*+T*+T*A*T*T*G*G*T*T*A*A*C*A*T*T*A*+T*+G*+G (SEQ ID NO:244; A63244Hi), +A*+G*+A*C*C*T*G*C*T*A*A*G*T*T*G*+T*+A*+G (SEQ ID NO: 140; A63140Hi), +G*+G*+C*G*G*C*A*T*A*G*A*T*G*G*A*G*+A*+C*+T (SEQ ID NO: 127; A63127Hi), +A*+G*+A*G*T*T*A*G*A*G*T*A*A*G*T*A*T*+T*+G*+G (SEQ ID NO:219; A63219Hi), +G*+T*+A*T*T*T*T*G*A*G*G*A*G*T*G*T*+G*+A*+T (SEQ ID NO:222; A63222Hi), +C*+A*+T*C*T*A*T*C*A*A*A*G*T*T*A*C*T*+G*+T*+C (SEQ ID NO:162; A63162HMfa), +C*+T*+G*C*T*A*G*A*A*G*G*T*A*T*C*+T*+T*+A (SEQ ID NO:214; A63214H1), +C*+A*+T*A*A*T*A*C*T*T*G*G*C*T*T*G*+T*+C*+G (SEQ ID NO:106; A63106IH), +G*+A*+T*T*T*G*T*A*T*C*A*A*G*A*A*G*+G*+T*+G (SEQ ID NO:239; A63239IH), +C*+C*+T*A*T*T*A*T*G*C*A*C*A*T*C*T*+G*+A*+G (SEQ ID NO:247; A63247H1), +A*+C*+T*A*G*G*A*C*C*A*T*G*C*G*Cmi*+T*+G (SEQ ID NO:110; A63110H), +A*+T*+T*G*C*T*G*A*A*C*T*T*T*T*C*T*+A*+T*+C (SEQ ID NO:43; A63043HMfa), +A*+C*+G*C*T*C*T*T*T*A*C*A*C*C*A*+G*+A*+A (SEQ ID NO:156, A63156EH) and a combination thereof, wherein + indicates an LNA nucleotide and * indicates a phosphorothioate (PTO) linkage.

[0209] 126. The oligonucleotide of any one of embodiments 1 to 125, wherein the oligonucleotide is selected from the group consisting of +G*+G*A*T*A*A*G*G*T*G*G*T*A*T*G*+C*+A*+A*+G (SEQ ID NO: 117; A63331Hi), +T*+A*G*+G*T*A*A*A*T*C*T*T*A*C*G*+G*+T*+A (SEQ ID NO:93; A63294Hi),+T*A*+G*G*T*A*A*A*T*C*T*T*A*C*+G*+G*+T*+A (SEQ ID NO:93; A63297Hi), +G*+G*+A*T*A*A*G*G*T*G*G*T*A*T*G*C*+A*+A*+G (SEQ ID NO:117; A63117Hi), +G*+A*+A*A*G*G*T*T*T*G*G*C*G*C*T*+G*+C*+T (SEQ ID NO: 128; A63128Hi), +G*+T*+T*G*A*G*T*T*T*A*G*G*T*G*G*+T*+C*+C (SEQ ID NO:248; A63248Hi), +A*+A*+C*A*C*T*G*G*T*A*G*T*A*G*T*A*+T*+A*+G (SQ ID NO: 116; A63116Hi), +G*+A*+C*A*T*A*G*T*T*C*A*A*G*C*T*+C*+A*+G (SEQ ID NO:77; A63077Hi), +T*+A*+G*G*T*A*A*A*T*C*T*T*A*C*G*+G*+T*+A (SEQ ID NO:93; A63093Hi), +G*+T*+A*A*T*T*T*A*T*T*T*A*G*C*T*C*+C*+T*+G (SEQ ID NO:260;

[0210] A63260HMfa), +T*+C*+A*C*A*C*C*G*T*T*T*G*T*G*G*+A*+T*+G (SEQ ID NO:34; A63034H), +C*+C*+T*G*C*A*G*A*T*A*T*T*G*G*A*T*+C*+T*+A (SEQ ID NO:45 A63045Hmfa), +A*+G*+A*G*C*G*T*G*A*G*A*G*T*A*G*+T*+A*+G (SEQ ID NO: 134; A63134Hi), +T*+T*+A*C*A*T*C*T*A*G*C*G*C*+C*+G*+T (SEQ ID NO: 136;

[0211] A63136Hi), +C*+T*+G*C*A*T*C*T*T*A*G*T*T*A*C*C*T*+C*+T*+T (SEQ ID NO:215; A63215Hi), +G*+T*+T*C*A*T*G*G*C*T*G*T*G*G*T*+T*+A*+A (SEQ ID NO:41; A63041H), +T*+T*+T*A*T*T*G*G*T*T*A*A*C*A*T*T*A*+T*+G*+G (SEQ ID NO:244; A63244Hi), +A*+G*+A*C*C*T*G*C*T*A*A*G*T*T*G*+T*+A*+G (SEQ ID NO: 140; A63140Hi), +G*+G*+C*G*G*C*A*T*A*G*A*T*G*G*A*G*+A*+C*+T (SEQ ID NO: 127; A63127Hi), +A*+G*+A*G*T*T*A*G*A*G*T*A*A*G*T*A*T*+T*+G*+G (SEQ ID NO:219; A63219Hi), +G*+T*+A*T*T*T*T*G*A*G*G*A*G*T*G*T*+G*+A*+T (SEQ ID NO:222; A63222Hi), +C*+A*+T*C*T*A*T*C*A*A*A*G*T*T*A*C*T*+G*+T*+C (SEQ ID NO:162; A63162HMfa), +C*+T*+G*C*T*A*G*A*A*G*G*T*A*T*C*+T*+T*+A (SEQ ID NO:214; A63214Hi), +c*+A*+T*A*A*T*A*C*T*T*G*G*C*T*T*G*+T*+C*+G (SEQ ID

[0212] NO:106; A63106H1), +G*+A*+T*T*T*G*T*A*T*C*A*A*G*A*A*G*+G*+T*+G (SEQ ID

[0213] NO:239; A63239IH), +C*+C*+T*A*T*T*A*T*G*C*A*C*A*T*C*T*+G*+A*+G (SEQ ID

[0214] NO:247; A63247H1), +A*+C*+T*A*G*G*A*C*C*A*T*G*C*G*Cmi*+T*+G (SEQ ID

[0215] NO:110; A63110H), +A*+T*+T*G*C*T*G*A*A*C*T*T*T*T*C*T*+A*+T*+C (SEQ ID

[0216] NO:43; A63043HMfa), +A*+C*+G*C*T*C*T*T*T*A*C*A*C*C*A*+G*+A*+A (SEQ ID

[0217] NO:156, A63156EH) and a combination thereof, wherein + indicates an LNA nucleotide and * indicates a mesyl-modified phosphate linkage between the nucleotides.

[0218] 127. The oligonucleotide of any one of embodiments 1 to 126, wherein the mesyl-modified phosphate linkage is a mesyl-phosphoramidate between the nucleotides.

[0219] 128. The oligonucleotide of any one of embodiments 1 to 127, wherein the oligonucleotide is +G*+G*A*T*A*A*G*G*T*G*G*T*A*T*G*+C*+A*+A*+G (SEQ ID NO: 117;

[0220] A63331Hi).

[0221] 129. The oligonucleotide of any one of embodiments 1 to 128, wherein the oligonucleotide is +T*+A*G*+G*T*A*A*A*T*C*T*T*A*C*G*+G*+T*+A (SEQ ID NO:93; A63294Hi).

[0222] 130. The oligonucleotide of any one of embodiments 1 to 129, wherein the oligonucleotide is +T*A*+G*G*T*A*A*A*T*C*T*T*A*C*+G*+G*+T*+A (SEQ ID NO:93; A63297Hi).

[0223] 131. The oligonucleotide of any one of embodiments 1 to 130, wherein the oligonucleotide is +G*+G*+A*T*A*A*G*G*T*G*G*T*A*T*G*C*+A*+A*+G (SEQ ID NO: 117;

[0224] A63117Hi).

[0225] 132. The oligonucleotide of any one of embodiments 1 to 131, wherein the oligonucleotide is +G*+A*+A*A*G*G*T*T*T*G*G*C*G*C*T*+G*+C*+T (SEQ ID NO: 128; A63128Hi).

[0226] 133. The oligonucleotide of any one of embodiments 1 to 132, wherein the oligonucleotide is +G*+T*+T*G*A*G*T*T*T*A*G*G*T*G*G*+T*+C*+C (SEQ ID NO:248; A63248Hi).

[0227] 134. The oligonucleotide of any one of embodiments 1 to 133, wherein the oligonucleotide is +A*+A*+C*A*C*T*G*G*T*A*G*T*A*G*T*A*+T*+A*+G (SQ ID NO: 116; A63116Hi). 135. The oligonucleotide of any one of embodiments 1 to 134, wherein the oligonucleotide is +G*+A*+C*A*T*A*G*T*T*C*A*A*G*C*T*+C*+A*+G (SEQ ID NO:77; A63077Hi).

[0228] 136. The oligonucleotide of any one of embodiments 1 to 135, wherein the oligonucleotide is +T*+A*+G*G*T*A*A*A*T*C*T*T*A*C*G*+G*+T*+A (SEQ ID NO:93; A63093Hi).

[0229] 137. The oligonucleotide of any one of embodiments 1 to 136, wherein the oligonucleotide is +G*+T*+A*A*T*T*T*A*T*T*T*A*G*C*T*C*+C*+T*+G (SEQ ID NO:260;

[0230] A63260HMfa).

[0231] 138. The oligonucleotide of any one of embodiments 1 to 137, wherein the oligonucleotide is +T*+C*+A*C*A*C*C*G*T*T*T*G*T*G*G*+A*+T*+G (SEQ ID NO:34; A63034H).

[0232] 139. The oligonucleotide of any one of embodiments 1 to 138, wherein the oligonucleotide is +C*+C*+T*G*C*A*G*A*T*A*T*T*G*G*A*T*+C*+T*+A (SEQ ID NO:45 A63045Hmfa).

[0233] 140. The oligonucleotide of any one of embodiments 1 to 139, wherein the oligonucleotide is +A*+G*+A*G*C*G*T*G*A*G*A*G*T*A*G*+T*+A*+G (SEQ ID NO: 134; A63134Hi).

[0234] 141. The oligonucleotide of any one of embodiments 1 to 140, wherein the oligonucleotide is +T*+T*+A*C*A*T*C*T*A*G*C*G*C*+C*+G*+T (SEQ ID NO: 136; A63136Hi).

[0235] 142. The oligonucleotide of any one of embodiments 1 to 141, wherein the oligonucleotide is +c*+T*+G*C*A*T*C*T*T*A*G*T*T*A*C*C*T*+C*+T*+T (SEQ ID NO:215;

[0236] A63215Hi).

[0237] 143. The oligonucleotide of any one of embodiments 1 to 142, wherein the oligonucleotide is +G*+T*+T*C*A*T*G*G*C*T*G*T*G*G*T*+T*+A*+A (SEQ ID NO:41; A63041H).

[0238] 144. The oligonucleotide of any one of embodiments 1 to 143, wherein the oligonucleotide is +T*+T*+T*A*T*T*G*G*T*T*A*A*C*A*T*T*A*+T*+G*+G (SEQ ID NO:244;

[0239] A63244Hi).

[0240] 145. The oligonucleotide of any one of embodiments 1 to 144, wherein the oligonucleotide is +A*+G*+A*C*C*T*G*C*T*A*A*G*T*T*G*+T*+A*+G (SEQ ID NO: 140; A63140Hi). 146. The oligonucleotide of any one of embodiments 1 to 145, wherein the oligonucleotide is +G*+G*+C*G*G*C*A*T*A*G*A*T*G*G*A*G*+A*+C*+T (SEQ ID NO: 127;

[0241] A63127Hi).

[0242] 147. The oligonucleotide of any one of embodiments 1 to 146, wherein the oligonucleotide is +A*+G*+A*G*T*T*A*G*A*G*T*A*A*G*T*A*T*+T*+G*+G (SEQ ID NO:219;

[0243] A63219Hi).

[0244] 148. The oligonucleotide of any one of embodiments 1 to 147, wherein the oligonucleotide is +G*+T*+A*T*T*T*T*G*A*G*G*A*G*T*G*T*+G*+A*+T (SEQ ID NO:222; A63222Hi).

[0245] 149. The oligonucleotide of any one of embodiments 1 to 148, wherein the oligonucleotide is +C*+A*+T*C*T*A*T*C*A*A*A*G*T*T*A*C*T*+G*+T*+C (SEQ ID NO: 162;

[0246] A63162HMfa).

[0247] 150. The oligonucleotide of any one of embodiments 1 to 149, wherein the oligonucleotide is +C*+T*+G*C*T*A*G*A*A*G*G*T*A*T*C*+T*+T*+A (SEQ ID NO:214; A63214Hi).

[0248] 151. The oligonucleotide of any one of embodiments 1 to 150, wherein the oligonucleotide is +c*+A*+T*A*A*T*A*C*T*T*G*G*C*T*T*G*+T*+C*+G (SEQ ID NO: 106; A63106Hi).

[0249] 152. The oligonucleotide of any one of embodiments 1 to 151, wherein the oligonucleotide is +G*+A*+T*T*T*G*T*A*T*C*A*A*G*A*A*G*+G*+T*+G (SEQ ID NO:239;

[0250] A63239Hi).

[0251] 153. The oligonucleotide of any one of embodiments 1 to 152, wherein the oligonucleotide is +C*+C*+T*A*T*T*A*T*G*C*A*C*A*T*C*T*+G*+A*+G (SEQ ID NO:247; A63247Hi).

[0252] 154. The oligonucleotide of any one of embodiments 1 to 153, wherein the oligonucleotide is +A*+C*+T*A*G*G*A*C*C*A*T*G*C*G*C*+T*+T*+G (SEQ ID NO:110; A63110H).

[0253] 155. The oligonucleotide of any one of embodiments 1 to 154, wherein the oligonucleotide is +A*+T*+T*G*C*T*G*A*A*C*T*T*T*T*C*T*+A*+T*+C (SEQ ID NO:43;

[0254] A63043HMfa). 156. The oligonucleotide of any one of embodiments 1 to 155, wherein the oligonucleotide is +A*+C*+G*C*T*C*T*T*T*A*C*A*C*C*A*+G*+A*+A (SEQ ID NO: 156, A63156Hi).

[0255] 157. The oligonucleotide of any one of embodiments 1 or 156, wherein the oligonucleotide has 80 to 99 %, 85 to 98 %, 90 to 95 or 93 % sequence identity to any one of the oligonucleotides of SEQ ID NO.l - SEQ ID NO.1288.

[0256] 158. The oligonucleotide of any one of embodiments 1 to 157, wherein said oligonucleotide comprises at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 nucleotides of any one of the oligonucleotides of SEQ ID NO.l - SEQ ID NO.1288.

[0257] 159. The oligonucleotide of any one of embodiments 1 to 158, wherein the oligonucleotide inhibits the expression of MLKL, a MLKL mRNA, a MLKL pre-mRNA or a combination thereof at a nanomolar or micromolar concentration.

[0258] 160. A pharmaceutical composition comprising an oligonucleotide of any one of embodiments 1 to 159 and a pharmaceutically acceptable carrier, excipient, dilutant, stimulant such as an adjuvant, or a combination thereof.

[0259] 161. The pharmaceutical composition of embodiment 160, further comprising a therapeutically active agent.

[0260] 162. The pharmaceutical composition of embodiment 160 or 161, wherein the therapeutically active agent is selected from the group consisting of another oligonucleotide, an antibody, a small molecule, a peptide, a lipid, a sugar and a combination thereof.

[0261] 163. The pharmaceutical composition of any one of embodiments 160 to 162, wherein the therapeutically active agent is another oligonucleotide.

[0262] 164. The pharmaceutical composition of any one of embodiments 160 to 163, wherein the therapeutically active agent is an antibody. 165. The pharmaceutical composition of any one of embodiments 160 to 164, wherein the therapeutically active agent is a small molecule.

[0263] 166. The pharmaceutical composition of any one of embodiments 160 to 165, wherein the therapeutically active agent is a peptide.

[0264] 167. The pharmaceutical composition of any one of embodiments 160 to 166, wherein the therapeutically active agent is a lipid.

[0265] 168. The pharmaceutical composition of any one of embodiments 160 to 167, wherein the therapeutically active agent is a sugar.

[0266] 169. The pharmaceutical composition of any one of embodiments 160 to 168, wherein the therapeutically active agent inhibits or stimulates a target selected from the group consisting of NLRP3, CD39, CD73, NRP1, FRA2, IL- IB, IL-1 receptor, IL-1R accessory protein, IL- 18, IL- 18 receptor, ASC, NLRC4, AIM2, Caspase- 1, RIPK1, RIPK3, Gasdermin D, TLR4, Caspase-8, P2X7, NFKB, RORyt, TGF-B, IL-21, IL- 17, IL-22, IL-23, IL-6, TNF- u, CCR6, CCL20, STAT3, MMP-1, MMP-8, ACSL4, ADAMTS-5, STING, HMG-CoA, Myd- 88, HMGB-1, ROS, TAK-1, Chop, FPR1, LIMCH1, caspase inhibitor and a combination thereof.

[0267] 170 The pharmaceutical composition of any one of embodiments 160 to 169, or the oligonucleotide of any one of embodiments 1 to 159 for use in preventing and / or treating a disorder selected from the group consisting of a hyperproliferative disorder such as cancer, a disorder caused by forms of cell death such as necroptosis, an inflammatory or autoimmune disorder, neurodegenerative disease, a neurological disorder, cardiovascular disorder, metabolic disorder, renal disorder, liver disorder, lung disorder, skin disorder, ocular disorder, disorder of the gastro-intestinal tract, joint inflammation, organ transplantation, fibrotic disorder and a combination thereof.

[0268] 171. The pharmaceutical composition of any one of embodiments 160 to 170, or the oligonucleotide of any one of embodiments 1 to 159 for use in treating a hyperproliferative disorder. 172. The pharmaceutical composition of any one of embodiments 160 to 171, or the oligonucleotide of any one of embodiments 1 to 159 for use in treating an inflammatory or autoimmune disorder.

[0269] 173. The pharmaceutical composition of any one of embodiments 160 to 172, or the oligonucleotide of any one of embodiments 1 to 159 for use in treating a neurodegenerative disease.

[0270] 174. The pharmaceutical composition of any one of embodiments 160 to 173, or the oligonucleotide of any one of embodiments 1 to 159 for use in treating a neurological disorder.

[0271] 175. The pharmaceutical composition of any one of embodiments 160 to 174, or the oligonucleotide of any one of embodiments 1 to 159 for use in treating a cardiovascular disorder.

[0272] 176. The pharmaceutical composition of any one of embodiments 160 to 175, or the oligonucleotide of any one of embodiments 1 to 159 for use in treating metabolic disorder.

[0273] 177. The pharmaceutical composition of any one of embodiments 160 to 176, or the oligonucleotide of any one of embodiments 1 to 159 for use in treating renal disorder.

[0274] 178. The pharmaceutical composition of any one of embodiments 160 to 177, or the oligonucleotide of any one of embodiments 1 to 159 for use in treating liver disorder.

[0275] 179. The pharmaceutical composition of any one of embodiments 160 to 178, or the oligonucleotide of any one of embodiments 1 to 159 for use in treating lung disorder.

[0276] 180. The pharmaceutical composition of any one of embodiments 160 to 179, or the oligonucleotide of any one of embodiments 1 to 159 for use in treating skin disorder.

[0277] 181. The pharmaceutical composition of any one of embodiments 160 to 180, or the oligonucleotide of any one of embodiments 1 to 159 for use in treating ocular disorder. 182. The pharmaceutical composition of any one of embodiments 160 to 181, or the oligonucleotide of any one of embodiments 1 to 159 for use in treating disorder of the gastro-intestinal tract.

[0278] 183. The pharmaceutical composition of any one of embodiments 160 to 182, or the oligonucleotide of any one of embodiments 1 to 159 for use in treating joint inflammation.

[0279] 184. The pharmaceutical composition of any one of embodiments 160 to 183, or the oligonucleotide of any one of embodiments 1 to 159 for use in treating organ transplantation.

[0280] 185. The pharmaceutical composition of any one of embodiments 160 to 184, or the oligonucleotide of any one of embodiments 1 to 159 for use in treating fibrotic disorder.

[0281] 186. The pharmaceutical composition of any one of embodiments 160 to 185, or the oligonucleotide of any one of embodiments 1 to 159 for use in treating cancer.

[0282] 187. The pharmaceutical composition of any one of embodiments 160 to 186, or the oligonucleotide of any one of embodiments 1 to 159 for use in treating necroptosis.

[0283] 188. The pharmaceutical composition or the oligonucleotide for use according to any one of embodiments 170 to 187, wherein the disorder selected from the group consisting of Alzheimer’s disease, multiple sclerosis, autoimmune encephalitis, stroke, traumatic brain injury, atherosclerosis, nonalcoholic fatty liver disease, nonalcoholic steatohepatitis, hypertension, myocardial infarction, acute kidney injury, ischemia reperfusion injury, chronic kidney diseases, crystal-induced nephropathies, glomerulonephritis, silicosis, asthma, allergic airway inflammation, inflammatory bowel disease, colitis ulcerosa, osteoarthritis, dermatitis, rheumatoid arthritis, juvenile ideopathic arthritis, organ malfunction after transplantation of organs such as kidney, lung, liver and / or heart, fibrotic disorder of kidney, lung, liver and / or heart, hyperinflammation following influenza infection, graft-versus-host disease, interstitial cystitis, uveitis, sarcoidosis, sinusitis, peridontal disease, optic neuritis, myelodysplastic syndrome, gout, obesity-induced inflammation, insulin resistance, type 1 and type 2 diabetes, contact hypersensitivity and a combination thereof, and / or a cancer selected from the group consisting of 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, epidermoid carcinoma and a combination thereof.

[0284] 189. The pharmaceutical composition or the oligonucleotide for use according to any one of embodiments 170 to 188, wherein the oligonucleotide or the composition is administered locally or systemically.

[0285] 190. The pharmaceutical composition or the oligonucleotide for use according to any one of embodiments 170 to 189, wherein the oligonucleotide or the composition is administered locally.

[0286] 191. The pharmaceutical composition or the oligonucleotide for use according to any one of embodiments 170 to 189, wherein the oligonucleotide or the composition is administered systemically.

[0287] 192. The pharmaceutical composition or the oligonucleotide for use according to any one of embodiments 170 to 191, wherein the pharmaceutical composition comprises an antiinflammatory therapeutic.

[0288] 193. The pharmaceutical composition or the oligonucleotide for use according to any one of embodiments 170 to 191, wherein the oligonucleotide is administered in combination with an anti-inflammatory therapeutic. 194. The oligonucleotide of any one of embodiments 1 to 169 or the oligonucleotide for use according to any one of embodiments 170 to 193, wherein the oligonucleotide is an antisense oligonucleotide. 195. The oligonucleotide of any one of embodiments 1 to 169 or the oligonucleotide for use according to any one of embodiments 170 to 194, wherein the modified nucleotide is located in position 1 to 5 of the 5'-end and / or 3'-end of the oligonucleotide.

[0289] 196. The oligonucleotide of any one of embodiments 1 to 169 or the oligonucleotide for use according to any one of embodiments 170 to 195, wherein the modified nucleotide is located in position 1 to 5 of the 5 '-end of the oligonucleotide.

[0290] 197. The oligonucleotide of any one of embodiments 1 to 169 or the oligonucleotide for use according to any one of embodiments 170 to 196, wherein the modified nucleotide is located in position 1 to 5 of the 3 '-end of the oligonucleotide.

Claims

Claims1. An oligonucleotide comprising 15 to 22 nucleotides, at least one nucleotide having a modification selected from the group consisting of a bridged nucleic acid such as LNA, ENA, TNA, cET, a 2'Fluoro modified nucleotide, a 2'0-Methyl modified nucleotide, a 2'0 -Methoxyethyl modified nucleotide, a FANA, a bicyclo DNA, a tricyclo DNA and a combination thereof, and wherein the oligonucleotide hybridizes with pre-mRNA of mixed lineage kinase domain like pseudokinase (MLKL) derived from SEQ ID NO.1289 and / or with a transcript or an mRNA of MLKL derived from SEQ ID NO.1290.

2. The oligonucleotide of claim 1, wherein the modification is located at the 5'-end and / or 3'-end of the oligonucleotide, optionally wherein the modified nucleotide is located at position 1 to 5 of the 5 '-end and / or 3 '-end of the oligonucleotide.

3. The oligonucleotide according to claim 1 or 2, wherein the oligonucleotide hybridizes outside a hybridizing active region or within a hybridizing active region of position 8401 to 8700, of position 17401 tol7700, of position 6301 to 6600, of position 9301 to 9600, of position 19801 to 20100, of position 1 to 300, of position 23701 to 24000, of position 23401 to 23700, of position 4501 to 4800, of position 3901 to 4200, of position 19201 to 19500, of position 301 to 600, of position 20101 to 20400, of position 11101 to 11400, of position 7501 to 7800, of position 3601 to 3900, of position 10801 to 11100, of position 3001 to 3300, of position 601 to 900, of position 901 to 1200, of position 1501 to 1800, of position 2401 to 2700, of position 3301 to 3600, of position 4201 to 4500, of position 4801 to 5100, of position 5701 to 6000, of position 6901 to 7200, of position 7201 to 7500, of position 8101 to 8400, of position 9001 to 9300, of position 9901 to 10200, of position 10201 to 10500, of position 10501 to 10800, of position 11401 to 11700, of position 11701 to 12000, of position 12301 to 12600, of position 12901 to 13200, of position 13201 to 13500, of position 13501 to 13800, of position 13801 to 14100, of position 15601 to 15900, of position 16501 to 16800, of position 17701 to 18000, of position 18301 to 18900, of position 18901 to 19200, of positionl9501 to 19800, of position 20401 to 20700, of position 20701 to 21000, of position 21001 to 21300, of position 21301 to 21600, of position 21901 to 22200, of position 22201 to 22500, of position 22501 to 22800, of position 23101 to 23400, of position 24001 to 24300, of position 24301 to 24600, of position 25201 to 25500, of position 25501 to 25800, of position 25801 to 26100, of position 26101 to 26400, of position 26401 to 26700, of position 26701 to 2700, of position27301 to 27600, of position 27601 to 27900, of position 27901 to 28200, of position 28201 to 28500, of position 28501 to 28800, of position 28801 to 29100 or a combination thereof, preferably wherein the oligonucleotide hybridizes with a hot spot of position 8523 to 8549, of position 6338 to 6366, of position 3659 to 3696, of position 9518 to 9551, of position 19992 to 20020, of position 17596 to 17628, of position 17 to 46, of position 23838 to 23875 of SEQ ID NO.1289 or a combination thereof.

4. The oligonucleotide according to any one of claims 1 to 3, wherein the oligonucleotide comprises SEQ ID NO: 117, SEQ ID NO:93, SEQ ID NO: 128, SEQ ID NO:248, SEQ ID NO:116, SEQ ID NO:77, SEQ ID NO:260, SEQ ID NO:34, SEQ ID NO:45, SEQ ID NO:134, SEQ ID NO:136, SEQ ID NO:215, SEQ ID NO:41, SEQ ID NO:244, SEQ IDNO:140, SEQ ID NO:127, SEQ ID NO:219, SEQ ID NO:222, SEQ ID NO:162, SEQ IDNO:214, SEQ ID NO:106, SEQ ID NO:239, SEQ ID NO:247, SEQ ID NO:110, SEQ IDNO:43, SEQ ID NO: 156 or a combination thereof or a combination thereof.

5. The oligonucleotide according to any one of claims 1 to 4, wherein the oligonucleotide is selected from the group consisting of +G*+G*A*T*A*A*G*G*T*G*G*T*A*T*G*+C*+A*+A*+G (SEQ ID NO: 117; A63331Hi), +T*+A*G*+G*T*A*A*A*T*C*T*T*A*C*G*+G*+T*+A (SEQ ID NO:93; A63294Hi),+T*A*+G*G*T*A*A*A*T*C*T*T*A*C*+G*+G*+T*+A (SEQ ID NO:93; A63297Hi), +G*+G*+A*T*A*A*G*G*T*G*G*T*A*T*G*C*+A*+A*+G (SEQ ID NO:117; A63117Hi), +G*+A*+A*A*G*G*T*T*T*G*G*C*G*C*T*+G*+C*+T (SEQ ID NO: 128; A63128Hi), +G*+T*+T*G*A*G*T*T*T*A*G*G*T*G*G*+T*+C*+C (SEQ ID NO:248; A63248Hi), +A*+A*+C*A*C*T*G*G*T*A*G*T*A*G*T*A*+T*+A*+G (SEQ ID NO:116; A63116Hi), +G*+A*+C*A*T*A*G*T*T*C*A*A*G*C*T*+C*+A*+G (SEQ ID NO:77; A63077Hi), +T*+A*+G*G*T*A*A*A*T*C*T*T*A*C*G*+G*+T*+A (SEQ ID NO:93; A63093Hi), +G*+T*+A*A*T*T*T*A*T*T*T*A*G*C*T*C*+C*+T*+G (SEQ ID NO:260;A63260HMfa), +T*+C*+A*C*A*C*C*G*T*T*T*G*T*G*G*+A*+T*+G (SEQ ID NO:34; A63034H), +C*+C*+T*G*C*A*G*A*T*A*T*T*G*G*A*T*+C*+T*+A (SEQ ID NO:45 A63045Hmfa), +A*+G*+A*G*C*G*T*G*A*G*A*G*T*A*G*+T*+A*+G (SEQ ID NO: 134; A63134Hi), +T*+T*+A*C*A*T*C*T*A*G*C*G*C*+C*+G*+T (SEQ ID NO: 136;A63136Hi), +C*+T*+G*C*A*T*C*T*T*A*G*T*T*A*C*C*T*+C*+T*+T (SEQ ID NO:215; A63215Hi), +G*+T*+T*C*A*T*G*G*C*T*G*T*G*G*T*+T*+A*+A (SEQ ID NO:41; A63041H), +T*+T*+T*A*T*T*G*G*T*T*A*A*C*A*T*T*A*+T*+G*+G (SEQ ID NO:244; A63244Hi), +A*+G*+A*C*C*T*G*C*T*A*A*G*T*T*G*+T*+A*+G (SEQ ID NO: 140;A63140Hi), +G*+G*+C*G*G*C*A*T*A*G*A*T*G*G*A*G*+A*+C*+T (SEQ ID NO: 127; A63127Hi), +A*+G*+A*G*T*T*A*G*A*G*T*A*A*G*T*A*T*+T*+G*+G (SEQ ID NO:219; A63219H1),+G*+T*+A*T*T*T*T*G*A*G*G*A*G*T*G*T*+G*+A*+T (SEQ ID NO:222; A63222H1), +C*+A*+T*C*T*A*T*C*A*A*A*G*T*T*A*C*T*+G*+T*+C (SEQ ID NO:162; A63162HMfa), +C*+T*+G*C*T*A*G*A*A*G*G*T*A*T*C*+T*+T*+A (SEQ ID NO:214; A63214H1), +C*+A*+T*A*A*T*A*C*T*T*G*G*C*T*T*G*+T*+C*+G (SEQ ID NO:106; A63106H1), +G*+A*+T*T*T*G*T*A*T*C*A*A*G*A*A*G*+G*+T*+G (SEQ ID NO:239; A63239H1), +C*+C*+T*A*T*T*A*T*G*C*A*C*A*T*C*T*+G*+A*+G (SEQ ID NO:247; A63247H1), +A*+C*+T*A*G*G*A*C*C*A*T*G*C*G*Cmi*+T*+G (SEQ ID NO:110; A63110H), +A*+T*+T*G*C*T*G*A*A*C*T*T*T*T*C*T*+A*+T*+C (SEQ ID NO:43; A63043HMfa), +A*+C*+G*C*T*C*T*T*T*A*C*A*C*C*A*+G*+A*+A (SEQ ID NO:156, A63156EH) and a combination thereof, wherein + indicates an LNA nucleotide and * indicates a phosphorothioate (PTO) linkage and / or a mesyl-modified phosphate linkage such as a mesyl-phosphoramidate between the nucleotides.

6. The oligonucleotide according to any one of claims 1 or 5, wherein the oligonucleotide has 80 to 99 %, 85 to 98 %, 90 to 95 or 93 % sequence identity to any one of the oligonucleotides of SEQ ID NO.1 - SEQ ID NO.765 or SEQ ID NO.1294 to SEQ ID NO.2581.

7. The oligonucleotide according to any one of claims 1 to 6, wherein said oligonucleotide comprises at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 nucleotides of any one of the oligonucleotides of SEQ ID NO.1 - SEQ ID NO.765 or SEQ ID NO.1294 to SEQ ID NO.2581.

8. The oligonucleotide according to any one of claims 1 to 7, wherein the oligonucleotide inhibits the expression of MLKL, a MLKL mRNA, a MLKL pre-mRNA or a combination thereof at a nanomolar or micromolar concentration.

9. A pharmaceutical composition comprising an oligonucleotide according to any one of claims 1 to 8 and a pharmaceutically acceptable carrier, excipient, dilutant, stimulant such as an adjuvant, or a combination thereof.

10. The pharmaceutical composition according to claim 9, further comprising a therapeutically active agent for example selected from the group consisting of another oligonucleotide, an antibody, a small molecule, a peptide, a lipid, a sugar and a combination thereof.

11. The pharmaceutical composition of claim 9 or 10, wherein the therapeutically active agent inhibits or stimulates a target selected from the group consisting of NLRP3, CD39, CD 73, NRP1, FRA2, IL- IB, IL-1 receptor, IL-1R accessory protein, IL- 18, IL- 18 receptor, ASC, NLRC4, AIM2, Caspase- 1, RIPK1, RIPK3, Gasdermin D, TLR4, Caspase-8, P2X7, NFKB, RORyt, TGF-B, IL-21, IL- 17, IL-22, IL-23, IL-6, TNF-u, CCR6, CCL20, STAT3, MMP-1, MMP-8, ACSL4, ADAMTS-5, STING, HMG-CoA, Myd-88, HMGB-1, ROS, TAK- 1, Chop, FPR1, LIMCH1, caspase inhibitor and a combination thereof.

12. The pharmaceutical composition of any one of claims 9 to 11, or the oligonucleotide according to any one of claims 1 to 8 for use in preventing and / or treating a disorder selected from the group consisting of a hyperproliferative disorder such as cancer, a disorder caused by forms of cell death such as necroptosis, an inflammatory or autoimmune disorder, neurodegenerative disease, a neurological disorder, cardiovascular disorder, metabolic disorder, renal disorder, liver disorder, lung disorder, skin disorder, ocular disorder, disorder of the gastro-intestinal tract, joint inflammation, organ transplantation, fibrotic disorder and a combination thereof.

13. The pharmaceutical composition or the oligonucleotide for use according to claim 12, wherein the disorder selected from the group consisting of Alzheimer’s disease, multiple sclerosis, autoimmune encephalitis, stroke, traumatic brain injury, atherosclerosis, nonalcoholic fatty liver disease, nonalcoholic steatohepatitis, hypertension, myocardial infarction, acute kidney injury, ischemia reperfusion injury, chronic kidney diseases, crystal-induced nephropathies, glomerulonephritis, silicosis, asthma, allergic airway inflammation, inflammatory bowel disease, colitis ulcerosa, osteoarthritis, dermatitis, rheumatoid arthritis, juvenile ideopathic arthritis, organ malfunction after transplantation of organs such as kidney, lung, liver and / or heart, fibrotic disorder of kidney, lung, liver and / or heart, hyperinflammation following influenza infection, graft - versus-host disease, interstitial cystitis, uveitis, sarcoidosis, sinusitis, peridontal disease, optic neuritis, myelodysplastic syndrome, gout, obesity-induced inflammation, insulin resistance, type 1 and type 2 diabetes, contact hypersensitivity and a combination thereof,and / or a cancer selected from the group consisting of 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, epidermoid carcinoma and a combination thereof.

14. The pharmaceutical composition or the oligonucleotide for use according to claim 12 or 13, wherein the oligonucleotide or the composition is administrable locally or systemically.

15. The pharmaceutical composition or the oligonucleotide for use according to any one of claims 12 to 14, wherein the pharmaceutical composition comprises an anti-inflammatory therapeutic or the oligonucleotide is administered in combination with an antiinflammatory therapeutic.

Images