WO2019063791A1

WO2019063791A1 - Inhibitor inhibiting the expression of nfat5

Info

Publication number: WO2019063791A1
Application number: PCT/EP2018/076462
Authority: WO
Inventors: Frank Jaschinski; Ksenija SCHIRDUAN; Sven MICHEL
Original assignee: Secarna Pharmaceuticals Gmbh & Co. Kg
Priority date: 2017-09-28
Filing date: 2018-09-28
Publication date: 2019-04-04

Abstract

The present invention refers to an inhibitor consisting of oligonucleotides comprising to 25 nucleotides, wherein at least one of the nucleotides is modified, and the oligonucleotide hybridizes with a nucleic acid sequence of NFAT5 of SEQ ID NO.1, wherein the oligonucleotide inhibits at least 50 % of the NFAT5 expression. The invention is further directed to a pharmaceutical composition comprising such oligonucleotide.

Description

Inhibitor inhibiting the expression of NFAT5

The present disclosure refers to an inhibitor of NFAT5 expression consisting of an antisense oligonucleotide hybridizing with a nucleic acid sequence of Nuclear factor of activated T-cells 5 (NFAT5, NF-AT5, NFATL1, NFATZ, OREBP, TONEBP) and to a pharmaceutical composition comprising such antisense oligonucleotide and a

pharmaceutically acceptable carrier, excipient and/or dilutant. Further, the invention refers to the use of the inhibitor or the pharmaceutical composition comprising the inhibitor in a method of preventing and/or treating a chronic inflammatory disease.

Technical background

NFAT5 is a human gene that encodes a transcription factor that regulates the expression of genes involved in the osmotic stress. The product of this gene is a member of the nuclear factors of activated T cells (NFAT) family of transcription factors. Proteins belonging to this family play a central role in inducible gene transcription during the immune response. This protein regulates gene expression induced by osmotic stress in mammalian cells. Unlike monomeric members of this protein family, this protein exists as a homodimer and forms stable dimers with DNA elements. Multiple transcript variants encoding different isoforms have been found for this gene. Tissues that comprise the kidneys, skin, and eyes are often subjected to osmotic stresses. When the extracellular environment is hypertonic, cells lose water and consequently, shrink. To counteract this, cells increase their sodium uptake in order to lose less water. However, an increase in intracellular ionic concentration is harmful to the cell. Cells can alternatively synthesize enzymes and transporters that increase intracellular

concentration of organic osmolytes, which are less toxic than excess ions but which also aid in water retention. Under conditions of hyperosmolarity, NFAT5 is synthesized and accumulates in the nucleus. NFAT5 stimulates for example the transcription of genes for aldose reductase (AR), the sodium chloride-betaine cotransporter (SLC6A12) the so dium/myo- inositol cotransporter (SLC5A3), the taurine transporter (SLC6A6) and neuropathy target esterase which are involved in the production and uptake of organic osmolytes. Additionally, NFAT5 induces for example heat shock proteins, Hsp70, and osmotic stress proteins. NFAT5 is also implicated in cytokine production.

Kleinewietfeld et al, Nature 2013 and Wu et al, Nature 2013 showed that high salt promotes Thl7 development and worsens pathology of an autoimmune disease model, EAE (experimental encephalitis) providing a connection between high salt western diet and increased incidence of autoimmunity. Additionally, it has been shown that high salt in extracellular milieu mimics proinflammatory environment and leads to development of autoimmunity (Jantsch et al, Cell Metabolism 2015). As NFAT5 translates the high salt environment to the activation of immune cells, it is an ideal target for modulation of inflammatory diseases.

So far, no antisense oligonucleotide exists which is highly efficient in reduction and inhibition, respectively, of NFAT5 expression. An oligonucleotide of the present invention is very successful in the inhibition of the expression of NFAT5. The mode of action of an oligonucleotide differs from the mode of action of an antibody or small molecule, and oligonucleotides are highly advantageous regarding for example

(i) the penetration of tissues such as poorly vascularized tissues,

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

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

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

The present invention refers to an inhibitor of the expression of NFAT5 consisting of an antisense oligonucleotide comprising about 10 to 25 nucleotides, wherein at least one of the nucleotides is modified. The oligonucleotide hybridizes for example with a nucleic acid sequence of NFAT5 protein of SEQ ID NO.l (human). The modified nucleotide is for example selected from the group consisting of a bridged nucleic acid (e.g., LNA, cET, ENA, 2'Fluoro modified nucleotide, 2O-Methyl modified nucleotide, a 2"0-Methoxy modified nucleotide, a FANA or a combination thereof). In some embodiments, the oligonucleotide inhibits at least 50 % of the NFAT5 expression and in some embodiments the oligonucleotide inhibits the expression of NFAT5 at a nano- and/or micromolar concentration.

The present invention is further directed to a pharmaceutical composition comprising an inhibitor of the present invention and optionally a pharmaceutically acceptable carrier, excipient, dilutant or a combination thereof. In some embodiments, this pharmaceutical composition additionally comprises an immunosuppressive agent such as a calcineurin inhibitor (cyclosporin A, tacrolimus), corticosteroids (prednison and prednisolon), azathioprine and mycophenolate mofetil, alkylating agents such as cyclophosphamide or chlorambucil, angiotensin-converting enzyme inhibitors (ACE -Inhibitors) such as captopril and ramipril, angiotensin receptor blockers (ARB) such as losartan and valsartan, fish-oil, another oligonucleotide, an antibody or a fragment thereof such as a Fab fragment, a HERA fusion protein, a ligand trap, a nanobody, a BiTe, a DARPin and or a small molecule which is for example effective in treatment of a disease selected from the group consisting of an autoimmune disorder, an immune disorder, an acute inflammatory disease, a chronic inflammatory disease such as chronic inflammatory disease of the kidney such as glomerulonephritis, infection-related glomerulonephritis, lupus nephritis, immunoglobulin A nephropathy, steroid- sensitive and resistant nephrotic syndrome, idiopathic membranous nephropathy, idiopathic membrano- proliferative glomerulonephritis, Henoch- Schonlein purpura nephritis, Pauci-immune focal and segmental necrotizing glomerulonephritis, Minimal-change disease, anti- glomerular basement membrane antibody glomerulonephritis, hypertension,

hypertension-related nephropathy, pulmonary hypertension, vasculitis, diabetes, diabetic nephropathy, retinopathy, neuropathy, diabetes vasculitis and a tumor such as a malignant or benign tumor.

In some embodiments, the oligonucleotide of the present invention is in combination with another oligonucleotide, an antibody and/or a small molecule, either each of these compounds is separate or combined in a pharmaceutical composition, wherein the oligonucleotide, the antibody or a fragment thereof such as a Fab fragment, a HERA fusion protein, a ligand trap, a nanobody, a BiTe, a DARPin and/or the small molecule inhibits or stimulates a RAAS factor such as ACE, Angiotensin receptor I and II, respectively, Renin, anti-diuretic hormone, vasopressin, bradykinin, kallidin, calcineurin, differentiation and signal transduction factors such as mTOR, CHOP, CTGF, VEGF, NF- kappa B, JAK1/2 and STAT1/3, a proinflammatory factor such as CCL2, CXCL2, Fraktalkine, IL18, TNF-alpha, IL1, IL6, IFN-gamma, IL12 and/or immune cell recruiting factor such as ICAM-1 and VCAM-1.

Furthermore, the present invention relates to the use of the inhibitor or the

pharmaceutical composition of the present invention in a method of preventing and/or treating a disorder, where an NFAT5 imbalance is involved. In some embodiments, the disorder is for example an autoimmune disorder, an immune disorder, an acute inflammatory disease, a chronic inflammatory disease such as chronic inflammatory disease of the kidney such as glomerulonephritis, infection-related glomerulonephritis, lupus nephritis, immunoglobulin A nephropathy, steroid- sensitive and resistant nephrotic syndrome, idiopathic membranous nephropathy, idiopathic

membranoproliferative glomerulonephritis, Henoch-Schonlein purpura nephritis, Pauci- immune focal and segmental necrotizing glomerulonephritis, Minimal-change disease, anti- glomerular basement membrane antibody glomerulonephritis, hypertension, hypertension-related nephropathy, pulmonary hypertension, vasculitis, diabetes, diabetic nephropathy, retinopathy, neuropathy, diabetes vasculitis and a tumor such as a malignant or benign tumor. In some embodiments, the inhibitor or the pharmaceutical composition of the present invention is for example administered locally or systemically. All documents cited or referenced herein ("herein cited documents"), and all documents cited or referenced in herein cited documents, together with any manufacturer's instructions, descriptions, product specifications, and product sheets for any products mentioned herein or in any document incorporated by reference herein, are hereby incorporated herein by reference, and may be employed in the practice of the invention. More specifically, all referenced documents are incorporated by reference to the same extent as if each individual document was specifically and individually indicated to be incorporated by reference.

Description of figures

Fig. 1 shows the mRNA sequence of human (h) NFAT5 (reference no. NM_138714.3).

Fig. 2 shows knock down efficacy of antisense oligonucleotides hybridizing with NFAT5 mRNA in human pancreas ductal adenocarcinoma cell line Panel. S6A01010HM is the negative control which is not complementary to any human or murine RNA. Cells were treated with oligonucleotides without any transfection reagent (gymnotic delivery) three days at a single concentration of 10 μΜ. After three days of treatment, cells were lysed and HPRTl and NFAT5 mRNA expression was measured using the QuantiGene RNA Singleplex assay. NFAT5 expression values were normalized to HPRTl. Mean relative expression of NFAT5 mRNA (triplicates and standard deviation) compared to untreated cells (no oligo, set as 1) is depicted in Fig. 2 for Panel cells.

Fig. 3 shows knock down efficacy of antisense oligonucleotides hybridizing with NFAT5 mRNA in murine renal adenocarcinoma cell line Renca. Cells were treated with oligonucleotides without any transfection reagent (gymnotic delivery) three days at a single concentration of 10 μΜ. After three days of treatment, cells were lysed and HPRTl and NFAT5 mRNA expression was measured using the QuantiGene RNA Singleplex assay. NFAT5 expression values were normalized to HPRTl. Mean relative expression of NFAT5 mRNA (triplicates and standard deviation) compared to untreated cells (no oligo, set as 1) is depicted in Fig. 3 for Renca cells.

Fig. 4 depicts correlation between knock-down efficacy of NFAT5- specific antisense oligonucleotides in human (Panel) and mouse (Renca) cell line. Mean relative expression of mouse (y axis) and human (x axis) NFAT5 mRNA (triplicates) compared to untreated cells (no oligo, set as 1) is depicted. Dotted line at 0.5 indicates 90 % knock-down efficacy.

Fig. 5 shows concentration-dependent NFAT5 mRNA knockdown by the NFAT5 antisense oligonucleotide A18019HM (SEQ ID NO.4) and A18005HM (SEQ ID NO.2) in Panel cells and IC50 determination. Panel cells were treated for 3 days with the indicated concentration of the antisense oligonucleotides. NFAT5 mRNA expression values were normalized to expression of the housekeeping gene HPRTl. Normalized hNFAT5 mRNA expression relative to vehicle treated cells (set as 1) is depicted. IC50 for A18019HM is 498.3 nM and for A18005HM it is 723.8 nM. Detailed description

The present invention provides for the first time antisense oligonucleotides which hybridize with mRNA sequences of NFAT5 of different species such as human, mouse and rat, respectively, i.e., show cross-reactivity, and inhibit the expression and activity, respectively, of NFAT5. Thus, the oligonucleotides of the present invention represent an interesting and highly efficient tool for use in a method of preventing and/or treating disorders, where the NFAT5 expression and activity, respectively, is increased. The oligonucleotide of the present invention hybridizes for example with a nucleic acid sequence of NFAT5 of SEQ ID NO.l (human; for example reference no.NM_138714.3), wherein the oligonucleotide inhibits at least 50 % of the NFAT5 expression.

In the following, the elements of the present invention will be described in more detail.

These elements are listed with specific embodiments, however, it should be understood that they may be combined in any manner and in any number to create additional embodiments. The variously described examples and embodiments should not be construed to limit the present invention to only the explicitly described embodiments.

This description should be understood to support and encompass embodiments which combine the explicitly described embodiments with any number of the disclosed elements.

Furthermore, any permutations and combinations of all described elements in this application should be considered disclosed by the description of the present application unless the context indicates otherwise.

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

understood to imply the inclusion of a stated member, integer or step or group of members, integers or steps but not the exclusion of any other member, integer or step or group of members, integers or steps. The terms "a" and "an" and "the" and similar reference used in the context of describing the invention (especially in the context of the claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by the context. Recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as", "for example"), provided herein is intended merely to better illustrate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention. An oligonucleotide of the present invention is for example an antisense oligonucleotide (ASO) consisting of or comprising 10 to 25 nucleotides, 10 to 15 nucleotides, 15 to 20 nucleotides, 12 to 18 nucleotides, or 14 to 17 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 at least one nucleotide which is modified. The modified nucleotide is for example a bridged nucleotide such as a locked nucleic acid (LNA, e.g., 2',4'-LNA), cET, ENA, a 2"Fluoro modified nucleotide, a 2O-Methyl modified nucleotide, a 2O-Methoxy modified nucleotide, a FANA or a combination thereof. In some embodiments, the oligonucleotide of the present invention comprises nucleotides having the same or different modifications. In some embodiments the oligonucleotide of the present invention comprises a modified phosphate backbone, wherein one or more phosphate(s), e.g., all, is/are for example a phosphorothioate, a methylphosphonate or combinations thereof in the backbone.

A nucleotide forms the building block of an oligonucleotide, and is for example composed of a nucleobase (nitrogenous base, e.g., purine or pyrimidine), a five-carbon sugar (e.g., ribose, 2-deoxyribose, arabinose, xylose, lyxose, allose, altrose, glucose, mannose, gulose, idose, galactose, talose or stabilized modifications of those sugars), and one or more phosphate groups. Examples of modified phosphate groups are phosphorothioate or methylphosphonate. Each compound of the nucleotide is modifiable, and is naturally or non-naturally occurring. Examples of the latter are: locked nucleic acid (LNA), 2', 4' constrained ethyl nucleic acids (c-ET), 2'-0,4'-C-ethylene-bridged nucleic acid (ENA), polyalkylene oxide- (such as triethylene glycol (TEG)), 2'-fluoro-, 2'-deoxy-2'-fluoro-beta- D-arabinonucleic acid (FANA), 2'-0-methoxy- and 2'-0-methyl-modified nucleotides.

An "LNA" is a modified RNA nucleotide, wherein the ribose moiety is modified with an extra bridge connecting the 2' oxygen and 4' carbon (2'- 4' ribonucleoside). The bridge locks the ribose in the 3'-endo (North) conformation, which is often found in the A-form duplexes. LNA nucleosides and nucleotides, respectively, comprise for example the forms of thio-LNA, oxy-LNA, or amino-LNA, in alpha-D- or beta-L-configuration, and can be mixed or combined, respectively, with DNA or RNA residues in the oligonucleotide. A "bridged nucleic acid" is modified RNA nucleotide, sometimes also referred to as constrained or inaccessible RNA molecule, which may contain a five-membered, six- membered or even a seven-membered bridged structure with a "fixed" C3'-endo sugar puckering. The bridge is synthetically incorporated at the 2', 4'-position of the ribose to afford a 2', 4'-BNA monomer. Specific examples are "ENA" nucleotides, wherein the bridge is an ethylene bridge.

Oligonucleotides comprising modified nucleotides in specific regions having unmodified nucleotides in-between are entitled as "gapmers". The term "gapmer" for example refers to a chimeric antisense oligonucleotide that contains a central block of deoxynucleotide monomers sufficiently long to induce RNase H cleavage. The central block of a gapmer is for example flanked by blocks of 2'-0 modified ribonucleotides or other artificially modified ribonucleotide monomers such as bridged nucleic acids (BNAs) that for example protect the internal block from nuclease degradation. In many earlier studies modified DNA analogs were investigated for their stability in biological fluids.

The oligonucleotide of the present invention comprises the one or more modified nucleotide at the 3'- and/or 5'- end of the oligonucleotide and/or at any position within the oligonucleotide, wherein modified nucleotides follow in a row of 1, 2, 3, 4, 5, or 6 modified nucleotides, or a modified nucleotide is combined with one or more unmodified nucleotides. For example the oligonucleotide of the present invention comprises at least one modified nucleotide, particularly at least one LNA, c-ET and/or ENA, at the 5'- and or 3'-end of the oligonucleotide; or the oligonucleotide comprises 1, 2, 3, or 4 LNAs or c-ETs or ENAs within the stretch of up to 5 nucleotides at the 5'-end, and 1, 2, 3, or 4 LNAs or c-ETs or ENAs within the stretch of up to 5 nucleotides at the 3 '-end; or the oligonucleotide comprises 1, 2, 3, or 4 LNAs, c-ETs, or ENAs within the stretch of 5 nucleotides at the 5'-end or 3'-end, and a polyalkylene oxide such as TEG within the stretch of 5 nucleotides at the 3'- or 5'-end.

The following Table 1 presents embodiments of oligonucleotides comprising modified nucleotides for example LNA which are indicated by (+) and phosphorothioate (PTO) indicated by (*). The oligonucleotides consisting of or comprising the sequences of Table 1 may comprise any other modified nucleotide and/or any other combination of modified and unmodified nucleotides. Oligonucleotides of Table 1 hybridize with mRNA of human and/or murine NFAT5: Seq Name Antisense Sequence 5'-3' Antisense Sequence 5'-3' with ΡΊΌ (*) and LNA (+) ID

2 A18005HM CATAGCCTTGCTGTCG +C*+A*+T*A*G*C*C*T*T*G*C*T*G*+T*+C*+G

3 A18020HM CCAATCTGGCACGAGT +C*+C*+A*A*T*C*T*G*G*C*A*C*G*+A*-K *+T

4 A18019HM CTTTGTTGTCCGTGGTA

5 A18023HM CAGACGCCACGTTGT +C*+A*+G*A*C*G*C*C*A*C*G*T*+T*+G*+T

6 A18011HM TTTCGTTTTCGTGATT

7 A18022HM ACGCCACGTTGTGGTA +A*+C*G*C*C*A*C*G*T*T*G*T*G*G*+T*+A

8 A18024HM ATCAGACGCCACGTTG +A*+T*+C*A*G*A*C*G*C*C*A*C*G*+T*+T*-K

9 A18010HM AGGACATCCGACTGT +A*-K *-K *A*C*A*T*C*C*G*A*C*+T*-K *+T

10 A18012HM AATCTCGTCGTTTGACC

11 A18027HM ACTGTTATCAGACGCC +A*+C*+T*G*T*T*A*T*C*A*G*A*C*+G*+C*+C

12 A18025HM TTATCAGACGCCACGTT +T*+T*+A*T*C*A*G*A*C*G*C*C*A*C*+G*+T*+T

13 A18026HM GTTATCAGACGCCAC +G*+T*+T*A*T*C*A*G*A*C*G*C*+C*+A*+C

14 A18004HM TTCAGAGAATCTCGCGA +T*+T*+C*A*G*A*G*A*A*T*C*T*C*G*+C*G*+A

15 A18001HM CATCGCAGCTCGACC +C*+A*+T*C*G*C*A*G*C*T*C*G*+A*+C*+C

16 A18002HM CTAGGTCCGCGCTGA +C*+T*+A*G*G*T*C*C*G*C*G*C*+T*-K *+A

17 A18003HM TCTAGGTCCGCGCTG +T*+C*+T*A*G*G*T*C*C*G*C*G*+C*+T*+G

18 A18006HM CCGACTGTTATCCAGC +C*+C*+G*A*C*T*G*T*T*A*T*C*C*+A*+G*+C

19 A18007HM CATCCGACTGTTATCC +C*+A*+T*C*C*G*A*C*T*G*T*T*A*+T*+C*+C

20 A18008HM CATCCGACTGTTATC +C*+A*+T*C*C*G*A*C*T*G*T*T*+A*+T*+C

21 A18009HM ACATCCGACTGTTAT +A*+C*+A*T*C*C*G*A*C*T*G*T*+T*+A*+T

22 A18013HM CACAATCTCGTCGTTT

23 A18014HM TCACAATCTCGTCGTT

24 A18015HM TCACAATCTCGTCGT +T*+C*+A*C*A*A*T*C*T*C*G*T*+C*+G*+T

25 A18016HM CGGCATCAAATATATCC +C*+G*G*C*A*T*C*A*A*A*T*A*T*+A*+T*+C*+C

26 A18017HM GTCGGCATCAAATATA +G*+T*+C*G*G*C*A*T*C*A*A*A*T*+A*+T*+A

27 A18018HM TGTCCGTGGTAAGCTGA +T*+G*+T*C*C*G*T*G*G*T*A*A*G*C*+T*-K *+A

28 A18021HM AACCAATCTGGCACGAG +A*A*+C*C*A*A*T*C*T*G*G*C*A*C*+G*+A*-K

29 S6A01010HM CATCGCAGCTCGACC +C*+A*+T*C*G*C*A*G*C*T*C*G*+A*+C*+C

Table 1: List of antisense oligonucleotides hybridizing for example with human and murine NFAT5 for example of SEQ ID No. 1. The annotation "HM" indicates that these antisense oligonucleotides target for example both human and mouse NFAT5. S6A01010HM is a negative control oligonucleotide that is not complementary to any human or murine RNA.

The following Table 2 shows knock down effectiveness of the antisense oligonucleotides of Table 1 on NFAT5 expression for example in human Panel and murine Renca cells, respectively: Seq ID Name Position on IC50 in % Residual % Residual NFAT5 mRNA reporter NFAT5 mRNA in mRNA in murine system [nM] human Panel Renca cells

cells

2 A18005HM 746 10,00 1,80 5,83

3 A18020HM 1677 15,77 2,41 13,88

4 A18019HM 1172 9,16 3,24 18,52

5 A18023HM 7166 <1 4,19 58,89

6 A18011HM 1051 9,97 4,57 16,81

7 A18022HM 7162 <1 8,29 67,09

8 A18024HM 7167 <1 8,81 12,84

9 A18010HM 913 4,86 10,53 14,41

10 A18012HM 1085 13,52 11,16 31,57

11 A18027HM 7173 <1 12,41 8,93

12 A18025HM 7168 <1 15,73 19,26

13 A18026HM 7171 <1 18,42 11,39

14 A18004HM 403 14,42 40,41 67,20

15 A18001HM 325 5,32 ND ND

16 A18002HM 362 41,80 ND ND

17 A18003HM 363 52,89 ND ND

18 A18006HM 905 227,20 ND ND

19 A18007HM 908 11,91 ND ND

20 A18008HM 909 12,12 ND ND

21 A18009HM 910 3,79 ND ND

22 A18013HM 1089 25,29 ND ND

23 A18014HM 1090 26,63 ND ND

24 A18015HM 1091 90,72 ND ND

25 A18016HM 1118 70,58 ND ND

26 A18017HM 1121 23,00 ND ND

27 A18018HM 1166 7,23 ND ND

28 A18021HM 1678 <1 ND ND

29 S6A01010HM - - -

Table 2: Results of NFAT5 antisense oligonucleotides of Table 1 in inhibiting NFAT5 expression in human and murine cells; negative control is S6A01010HM antisense oligonucleotide.

The oligonucleotides of the present invention hybridize for example with mRNA of human and/or murine NFAT5 of SEQ ID No. 1 and 2, respectively. Such oligonucleotides are called NFAT5 antisense oligonucleotides.

In some embodiments, the oligonucleotide of the present invention inhibits for example at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of NFAT5 such as the, e.g., human, rat and/or murine, NFAT5 expression. Thus, the oligonucleotides of the present invention are for example nephritis- reverting oligonucleotides which inhibit and revert nephrosis, respectively, for example in a cell, tissue, organ, or a subject. The oligonucleotide of the present invention inhibits the expression of NFAT5 at a nanomolar or micromolar concentration for example in a concentration of 0.1, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900 or 950 nM, or 1, 1.5, 2.0, 2.5, 3.0, 10 or 100 μΜ.

In some embodiments, the oligonucleotide of the present invention is used in a

concentration of 1, 3, 5, 9, 10, 15, 27, 30, 40, 50, 75, 82, 100, 250, 300, 500, or 740 nM, or 1, 1.5, 2.2, 3, 5, 6.6 or 10 μΜ.

In some embodiments the present invention refers to a pharmaceutical composition comprising an oligonucleotide of the present invention and a pharmaceutically

acceptable carrier, excipient and/or dilutant. In some embodiments, the pharmaceutical composition further comprises an immunosuppressive agents such as calcineurin inhibitors (cyclosporin A and tacrolimus), corticosteroids (prednison and prednisolon), azathioprine and mycophenolate mofetil, alkylating agents such as cyclophosphamide or chlorambucil, angiotensin-converting enzyme inhibitors (ACE -Inhibitors) such as captopril and ramipril, angiotensin receptor blockers (ARB) such as losartan and valsartan, fish-oil, another oligonucleotide, an antibody or a fragment thereof such as a Fab fragment, a HERA fusion protein, a ligand trap, a nanobody, a BiTe and/or a small molecule, another oligonucleotide, an antibody or a fragment thereof such as a Fab fragment, a HERA fusion protein, a ligand trap, a nanobody, a BiTe and/or a small molecule which is for example effective in the prevention and/or treatment of

autoimmune disorder, an immune disorder, an acute inflammatory disease, a chronic inflammatory disease such as chronic inflammatory disease of the kidney such as glomerulonephritis, infection-related glomerulonephritis, lupus nephritis,

immunoglobulin A nephropathy, steroid- sensitive and resistant nephrotic syndrome, idiopathic membranous nephropathy, idiopathic membranoproliferative

glomerulonephritis, Henoch- Schonlein purpura nephritis, Pauci-immune focal and segmental necrotizing glomerulonephritis, Minimal-change disease, anti- glomerular basement membrane antibody glomerulonephritis, hypertension, hypertension-related nephropathy, pulmonary hypertension, vasculitis, diabetes, diabetic nephropathy, retinopathy, neuropathy, diabetes vasculitis and a tumor such as a malignant or benign tumor. The tumor is for example breast cancer, lung cancer, malignant melanoma, lymphoma, skin cancer, bone cancer, prostate cancer, liver cancer, brain cancer, cancer of the larynx, gall bladder, pancreas, testicular, rectum, parathyroid, thyroid, adrenal, neural tissue, head and neck, colon, stomach, bronchi, kidneys, basal cell carcinoma, squamous cell carcinoma, metastatic skin carcinoma, osteo sarcoma, Ewing's sarcoma, reticulum cell sarcoma, liposarcoma, myeloma, giant cell tumor, small-cell lung tumor, islet cell tumor, primary brain tumor, meningioma, acute and chronic lymphocytic and granulocytic tumors, acute and chronic myeloid leukemia, hairy-cell tumor, adenoma, hyperplasia, medullary carcinoma, intestinal ganglioneuromas, Wilm's tumor, seminoma, ovarian tumor, leiomyomater tumor, cervical dysplasia, retinoblastoma, soft tissue sarcoma, malignant carcinoid, topical skin lesion, rhabdomyosarcoma, Kaposi's sarcoma, osteogenic sarcoma, malignant hypercalcemia, renal cell tumor, polycythermia vera, adenocarcinoma, anaplastic astrocytoma, glioblastoma multiforma, leukemia, or epidermoid carcinoma.

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. The disorder is for example characterized by an NFAT5 imbalance, i.e., the NFAT5 level is increased in comparison to the level in a normal, healthy cell, tissue, organ or subject. The NFAT5 level is for example increased by an increased NFAT5 such as NFAT5 expression and activity, respectively. The NFAT5 level can be measured by any standard method such as immunohistochemistry, western blot, quantitative real time PCR or QuantiGene assay known to a person skilled in the art. An oligonucleotide or a pharmaceutical composition of the present invention is administered locally or systemically for example orally, sublingually, nasally,

inhalationally, subcutaneously, intravenously, intraperitoneally, intramuscularly, intratumoral, intrathecal, transdermal, and/or rectal. Alternatively or in combination ex vivo treated immune cells are administered. The oligonucleotide is administered alone or in combination with another antisense oligonucleotide of the present invention and optionally in combination with another compound such as another oligonucleotide, an antibody or a fragment thereof such as a Fab fragment, a HERA fusion protein, a ligand trap, a nanobody, a BiTe, DARPin, a small molecule and/or another disease specific agent calcineurin inhibitors (cyclosporin A and tacrolimus), corticosteroids (predsnison and prednisolon), azathioprine and mycophenolate mofetil, alkylating agents such as cyclophosphamide or chlorambucil, angiotensin-converting enzyme inhibitors (ACE- Inhibitors) such as captopril and ramipril, angiotensin receptor blockers (ARB) such as losartan and valsartan and/or fish-oil. In some embodiments, the other oligonucleotide (i.e., not being part of the present invention), the antibody or a fragment thereof such as a Fab fragment, a HERA fusion protein, a ligand trap, a nanobody, a BiTe, DARPin and or the small molecule are effective in preventing and/or treating an autoimmune disorder, an immune disorder, diabetes, artheriosclerosis, a nephrological disorder and/or cancer. An oligonucleotide or a pharmaceutical composition of the present invention is used for example in a method of preventing and/or treating a kidney disease. Examples of kidney disease preventable and/or treatable by use of the oligonucleotide or pharmaceutical composition of the present invention are glomerulonephritis, infection- related glomerulonephritis, lupus nephritis, immunoglobulin A nephropathy, steroid- sensitive and resistant nephrotic syndrome, idiopathic membranous nephropathy, idiopathic membranoproliferative glomerulonephritis, Henoch-Schonlein purpura nephritis, Pauci-immune focal and segmental necrotizing glomerulonephritis, Minimal- change disease and anti-glomerular basement membrane antibody glomerulonephritis

In some embodiments two or more oligonucleotides of the present invention are administered together, at the same time point for example in a pharmaceutical composition or separately, or on staggered intervals. In other embodiments, one or more oligonucleotides of the present invention are administered together with another compound such as another oligonucleotide (i.e., not being part of the present invention), 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. In some embodiments of these combinations the oligonucleotide (i.e., not being part of the present invention), the antibody or a fragment thereof such as a Fab fragment, a HERA fusion protein, a ligand trap, a nanobody, a BiTe, DARPin and/or small molecule inhibits (antagonist) or stimulates (agonist) a RAAS factor such as ACE, Angiotensin receptor I and II, respectively, Renin, anti- diuretic hormone, vasopressin, bradykinin, kallidin, calcineurin differentiation and signal transduction factors such as mTOR, TGF-beta, CHOP, CTGF, VEGF, NF-kappa B. JAK1/2 and STAT1/3, proinflammatory factor such as CCL2, CXCL2, Fraktalkine, IL18, TNF-alpha, IL1, IL6, IFN-gamma, IL12 and/or immune cell recruiting factor such as ICAM-1 and VCAM- 1. An antibody in combination with the oligonucleotide or the pharmaceutical composition of the present invention is for example an anti-TNF-alpha antibody, an anti-TNF- alpha antibody, or a bispecific antibody. A small molecule in combination with the

oligonucleotide or the pharmaceutical composition of the present invention are for example cyclosporine A, ramipril or valsartan.

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

Examples

The following examples illustrate different embodiments of the present invention, but the invention is not limited to these examples. The following experiments are performed on cells endogenously expressing NFAT5, i.e., the cells do not represent an artificial system comprising transfected reporter constructs. Such artificial systems generally show a higher degree of inhibition and lower IC50 values than endogenous systems which are closer to therapeutically relevant in vivo systems. Further, in the following

experiments no transfecting agent is used, i.e., gymnotic delivery is performed.

Transfecting agents are known to increase the activity of an oligonucleotide which influences the IC50 value (see for example Zhang et al., Gene Therapy, 2011, 18, 326-333; Stanton et al., Nucleic Acid Therapeutics, Vol. 22, No. 5, 2012). As artificial systems using a transfecting agent are hardly or impossible to translate into therapeutic approaches and no transfection formulation has been approved so far for oligonucleotides, the following experiments are performed without any transfecting agent. Example 1: Single dose efficacy screens of NFAT5 antisense oligonucleotides in Panel cells

10000 Panel cells / well were seeded in 96-well flat bottom plates and treated with the respective antisense oligonucleotide of Table 1 (black bars), or negative control (grey bar) at a final concentration of 10 μΜ or left untreated (white bar). After three days, cells were lysed and human HPRT1 (hHPRTl) and human NFAT5 (hNFAT5) mRNA expression were measured using the QuantiGene RNA Singleplex assay. hNFAT5 expression values were normalized to hHPRTl. Mean relative expression of hNFAT5 mRNA (triplicates and standard deviation) compared to untreated cells (no oligo, set as 1) is depicted in Fig. 2. Dotted lines at 0.5 and 0.1 indicate 50 and 90 % knock-down efficacy.

Example 2: Single dose efficacy screens of NFAT5 ASOs in Renca cells

15000 Renca cells / well were seeded in 96-well flat bottom plates and treated with the respective antisense oligonucleotide of Table 1 (black bars) at a final concentration of 10 μΜ or left untreated (white bar). After three days, cells were lysed and mouse HPRT1 (mHPRTl) and mouse NFAT5 (mNFAT5) mRNA expression values were measured using the QuantiGene RNA Singleplex assay. mNFAT5 expression values were normalized to mHPRTl. Mean relative expression of mNFAT5 mRNA (triplicates and standard deviation) compared to untreated cells (no oligo, set as 1) is depicted in Fig. 3. Dotted lines at 0.5 and 0.1 indicate 50 and 90% knock-down efficacy. Example 3: Dose dependency of effects and IC50 determination for NFAT5- specific ASOs A18019HM and A18005HM

Panel cells were treated with different concentrations of the respective antisense oligonucleotide for three days. After three days, cells were lysed and hHPRTl and hNFAT5 mRNA expression was measured using the QuantiGene RNA Singleplex assay. hNFAT5 expression values were normalized to hHPRTl. Normalized hNFAT5 mRNA expression relative to untreated cells (set as 1) is depicted in Fig. 5. IC50 for A18019HM (SEQ ID NO.4) is 498.3 nM and for A18005HM (SEQ ID NO.2) is 723.8 nM.

Claims

1. NFAT5 inhibitor consisting of an antisense oligonucleotide comprising 12 to 20 nucleotides, wherein at least one of the nucleotides is modified, and the oligonucleotide hybridizes with a nucleic acid sequence of NFAt5 of SEQ ID NO.l, wherein the

oligonucleotide inhibits at least 50 % of the PRRX1 expression.

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

3. The inhibitor of claim 1 or 2 hybridizing with NFAT5 of SEQ ID. NO.l comprising a sequence selected from the group consisting of SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11, SEQ ID NO.12, SEQ ID NO.13, SEQ ID N0.14, SEQ ID NO.15, SEQ ID NO.16, SEQ ID NO.17, SEQ ID NO.18, SEQ ID NO.19, SEQ ID NO.20, SEQ ID N0.21, SEQ ID N0.22, SEQ ID N0.23, SEQ ID N0.24, SEQ ID N0.25, SEQ ID N0.26, SEQ ID N0.27, SEQ ID N0.28 and a combination thereof.

4. The inhibitor of any one of claims 1 to 3, wherein the oligonucleotide is selected from the group consisting of

+C*+A*+T*A*G*C*C*T*T*G*C*T*G*+T*+C*+G (SEQ ID NO.2),

+C*+C*+A*A*T*C*T*G*G*C*A*C*G*+A*+G*+T (SEQ ID NO.3),

+C*+T*+T*T*G*T*T*G*T*C*C*G*T*G*+G*+T*+A (SEQ ID NO.4),

+C*+A*+G*A*C*G*C*C*A*C*G*T*+T*+G*+T (SEQ ID NO.5),

+T*+T*+T*C*G*T*T*T*T*C*G*T*+G*+A*+T*+T (SEQ ID NO .6) ,

+A*+C*G*C*C*A*C*G*T*T*G*T*G*G*+T*+A (SEQ ID NO.7),

+A*+T*+C*A*G*A*C*G*C*C*A*C*G*+T*+T*+G (SEQ ID NO.8),

+A*+G*+G*A*C*A*T*C*C*G*A*C*+T*+G*+T (SEQ ID NO.9),

+A*+A*+T*C*T*C*G*T*C*G*T*T*T*G*+A*+C*+C (SEQ ID NO.10),

+A*+C*+T*G*T*T*A*T*C*A*G*A*C*+G*+C*+C (SEQ ID NO.11),

_+T*_+T*_+A*_T*_C*_A*_G*_A*_{TO ID} NO.12),

+G*+T*+T*A*T*C*A*G*A*C*G*C*+C*+A*+C (SEQ ID NO.13),

+T*+T*+C*A*G*A*G*A*A*T*C*T*C*G*+C*G*+A (SEQ ID NO.14),

+C*+A*+T*C*G*C G*C*T*C*G*+A*+C*+C (SEQ ID NO.15),

+C*+T*+A*G*G^*C*C*G*C*G*C*+T*+G*+A (SEQ ID NO.16),

+T*+C*+T*A*G*G*T*C*C*G*C*G*+C*+T*+G (SEQ ID NO.17),

+C*+C*+G*A*C*T*G*T*T*A*T*C*C*+A*+G*+C (SEQ ID NO.18), +C*+A*+T*C*C*G*A*C*T*G*T*T*A*+T*+C*+C (SEQ ID NO.19),

+C*+A*+T*C*C*G*A*C*T*G*T*T*+A*+T*+C (SEQ ID NO.20),

+A*+C*+A*T*C*C*G*A*C*T*G*T*+T*+A*+T (SEQ ID N0.21),

+C*+A*+C*A*A*T*C*T*C*G*T*C*G*+T*+T*+T (SEQ ID N0.22),

+T*+C*+A*C*A*A*T*C*T^irC*G*T*C*+G*+T*+T (SEQ ID NO .23) ,

+T*+C*+A*C*A*A*T*C*T*C*G*T*+C*+G*+T (SEQ ID N0.24),

+C*+G*G*C*A*T*C*A*A*A*T*A*T*+A*+T*+C*+C (SEQ ID N0.25),

+G*+T*+C*G*G*C*A*T*C*A*A*A*T*+A*+T*+A (SEQ ID N0.26),

+T*+G*+T*C*C*G*T*G*G*T*A*A*G*C*+T*+G*+A (SEQ ID NO.27),

+A*A*+C*C*A*A*T*C*T*G*G*C*A*C*+G*+A*+G (SEQ ID N0.28),

and a combination thereof, wherein + indicates an LNA nucleotide and * indicates a phosphorothioate (PTO) linkage between the nucleotides.

5. The inhibitor of any one of claims 1 to 4, wherein the inhibitor inhibits the expression of NFAT5 at a nanomolar concentration.

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

7. The pharmaceutical composition of claim 6, further comprising an immunosuppressive agent such as cyclosporin A and tacrolimus, a corticosteroid (prednison and prednisolon), azathioprine and mycophenolate mofetil, alkylating agent such as cyclophosphamide or chlorambucil, angiotensin-converting enzyme inhibitors (ACE -Inhibitors) such as captopril and ramipril, angiotensin receptor blockers (ARB) such as losartan and valsartan and/or fish-oil, another oligonucleotide, an antibody or a fragment thereof such as a Fab fragment, a HERA fusion protein, a ligand trap, a nanobody, a BiTe, a DARPin and/or a small molecule.

8. The pharmaceutical composition of claim 7, wherein the other oligonucleotide, the antibody and/or the small molecule inhibits or stimulates a RAAS factor such as ACE, Angiotensin receptor I and II, Renin, anti-diuretic hormone, vasopressin, bradykinin, kallidin, calcineurin, differentiation and signal transduction factors such as mTOR, TGF-beta, CHOP, CTGF, VEGF, NF-kappa B. JAK1/2 and STAT1/3, proinflammatory factor such as CCL2, CXCL2, Fraktalkine, IL18, TNF-alpha, ILl, IL6, IFN-gamma, IL12 and/or immune cell recruiting factor such as ICAM-1 and VCAM-1.

9. The Inhibitor of any one of claims 1 to 5 or the pharmaceutical composition of any one of claims 6 to 7 for use in a method of preventing and/or treating a disorder, where an NFAT5 imbalance is involved.

10. The inhibitor or the pharmaceutical composition for use according to claim 9, wherein the disorder is an autoimmune disorder, an immune disorder, an acute inflammatory disease, a chronic inflammatory disease such as chronic inflammatory disease of the kidney or a diabetic vasculitis.

11. The inhibitor or the pharmaceutical composition for use according to claim 10, wherein the disorder is an autoimmune disorder, an immune disorder, an acute inflammatory disease, a chronic inflammatory disease such as chronic inflammatory disease of the kidney such as glomerulonephritis, infection-related glomerulonephritis, lupus nephritis, immunoglobulin A nephropathy, steroid- sensitive and resistant nephrotic syndrome, idiopathic membranous nephropathy, idiopathic

membranoproliferative glomerulonephritis, Henoch-Schonlein purpura nephritis, Pauci- immune focal and segmental necrotizing glomerulonephritis, Minimal-change disease, anti- glomerular basement membrane antibody glomerulonephritis, hypertension, hypertension-related nephropathy, pulmonary hypertension, vasculitis, diabetes, diabetic nephropathy, retinopathy, neuropathy, diabetes vasculitis and a tumor such as a malignant or benign tumor.

12. The inhibitor or the pharmaceutical composition for use according to claim 11, wherein the cancer is breast cancer, lung cancer, malignant melanoma, lymphoma, skin cancer, bone cancer, prostate cancer, liver cancer, brain cancer, cancer of the larynx, gall bladder, pancreas, testicular, rectum, parathyroid, thyroid, adrenal, neural tissue, head and neck, colon, stomach, bronchi, kidneys, basal cell carcinoma, squamous cell carcinoma, metastatic skin carcinoma, osteo sarcoma, Ewing's sarcoma, reticulum cell sarcoma, liposarcoma, myeloma, giant cell tumor, small-cell lung tumor, islet cell tumor, primary brain tumor, meningioma, acute and chronic lymphocytic and granulocytic tumors, acute and chronic myeloid leukemia, hairy-cell tumor, adenoma, hyperplasia, medullary carcinoma, intestinal ganglioneuromas, Wilm's tumor, seminoma, ovarian tumor, leiomyomater tumor, cervical dysplasia, retinoblastoma, soft tissue sarcoma, malignant carcinoid, topical skin lesion, rhabdomyosarcoma, Kaposi's sarcoma, osteogenic sarcoma, malignant hypercalcemia, renal cell tumor, polycythermia vera, adenocarcinoma, anaplastic astrocytoma, glioblastoma multiforma, leukemia, or epidermoid carcinoma.

13. The inhibitor or the pharmaceutical composition for use according to claim 10 or 11, wherein the inhibitor or the composition is suitable to be administered locally or systemically.