Patent Publication Number: US-2020283764-A1

Title: Oligonucleotide inhibiting the expression of chop

Description:
The present disclosure refers to an inhibitor of Chop expression consisting of an antisense oligonucleotide hybridizing with a nucleic acid sequence of C/EBP-homologous protein (Chop) mRNA and/or pre-mRNA, and to a pharmaceutical composition comprising such antisense oligonucleotide and a pharmaceutically acceptable carrier, excipient and/or dilutant. 
     BACKGROUND 
     C/EBP-homologous protein (Chop) is a member of the CCAAT/enhancer binding proteins (C/EBP) family of basic leucine zipper transcription factors, which is also known as growth arrest and DNA damage-inducible gene 153 (GADD153) (Back &amp; Kaufman,  Annu. Reu. Biochem,  2012, 81, 767-793). Chop consists of two functional domains, an N-terminal transcriptional activation domain and a C-terminal basic-leucine zipper (bZIP) domain (Li et al.,  Acta Biochim Biophys,  2014, 46 (8), 629-639). 
     While Chop is ubiquitously expressed at very low levels in the cytosol, its expression is induced in a wide variety of cells upon perturbations inducing endoplasmatic reticulum (ER) stress. This results in Chop accumulation in the nucleus (Ron &amp; Habener,  Genes Deu.,  1992, 6, 439-453). Factors inducing ER-stress are glucose deprivation, presumably by inhibiting N-linked protein glycosylation in the ER, amino acid starvation, tunicamycin, which blocks protein glycosylation, and dithiothreitol which disrupts disulfide bond formation. Accordingly, the human Chop promoter contains at least two stress response element motifs and one amino-acid-regulatory element motif for its transcriptional regulation. To achieve maximal induction of the CHOP promoter at ER stress, the interplay of three signaling pathways is required, which are the PKR-like endoplasmic reticulum kinase (PERK)/eIF2a signaling pathway, activating transcription factor 6a (ATF6) and Ire1/XBP-1 signaling pathway (Okada et al.,  Biochem. J,  2002, 366, 585-594). 
     Functionally, overexpression of Chop leads to cycle arrest and/or apoptosis. Chop downregulates the expression of anti-apoptotic protein factors such as Bcl-2 (Wang,  EMBO J.,  1998, 17(13), 3619-3630), and activates expression of genes encoding pro-apoptotic proteins such as Caspase-3, BAX, GADD34, DOCs and EORlalpha. Accordingly, induction of CHOP is involved in the development of various human diseases associated with severe ER stress and dysregulated apoptosis, such as neurodegenerative diseases, diabetes, ischemic diseases and acquired immune deficiency syndrome through cell loss (Li et al.,  Acta Biochim Biophys,  2014, 46 (8), 629-639). The ER-stress response has been demonstrated to be relevant in different diseases like diabetic nephropathy (DN), obesity, insulin resistance, type 2 diabetes mellitus and artheriosclerosis (Madhusudhan et al. 2015, Cnop et al. 2012, Back et al. 2012, Wang et al. 2012). Chop in particular has been shown to be involved in diabetic nephropathy (DN) and diabetes. More specifically, Chop knockout mice are protected from DN in a streptozotocin (STZ) induced diabetes model that reflects insulinopenic type 1 diabetes mellitus (Madhusudhan et al. 2015). Furthermore, in a model of type 2 diabetes (db/db mice) the genetic knockout of Chop results in improved glycemic control and expanded 6 cell mass by promoting 6 cell survival (Song et al. 2008). 
     Further, Chop plays an essential role in tumor derived immunosuppressive activity by inducing a state of T cell unresponsiveness toward tumor associated antigens (Gabrilouich &amp; Nagaraj,  Nat. Rev. Immunol.,  2009, 9(3), 162-174; Marigo et al.,  Immunity,  2010, 32, 790-802). Immune cells play an important role in the complex microenvironment of a tumor. On the one hand, there are immune effector cells like e.g. T cells or NK cells that are capable of recognizing immunogenic structures on the surface of tumor cells and thereby can attack tumor cells by the release of certain cytokines like e.g. interferon gamma (IFN-γ) or cytotoxic molecules like e.g. granzyme B. On the other hand, there are suppressive cells like e.g. regulatory T cells or myeloid-derived suppressor cells (MDSC), that produce cytokines like e.g. interleukin 10 (IL-10) or transforming growth factor beta (TGF-ß) and immunosuppressive enzymes like e.g. arginase. Those cytokines and enzymes have the capability to suppress the aforementioned immune effector cells. The expression of such suppressive factors is tightly regulated by transcription factors. In MDSC for example, the CCAAT/enhancer-binding protein-6 complex (C/EBPß) can activate the transcription of genes like IL-6 and arginase that contribute to the suppressive capacity of those cells (Corzo et al., 2009; Marigo et al., 2010; Sonda et al., 2013). C/EBPß contains a subdomain called C/EBPß liver-enriched inhibitory protein (LIP) that inhibits binding of the active C/EBPß complex (Hattori et al., 2003) to e.g. IL-6 and arginase promotors. C/EBP-homologous protein (Chop) binds to LIP and prevents its binding to the other domains of the C/EBPß complex, thereby allowing activation of the expression of e.g. IL-6 and arginase. Expression of Chop thereby contributes to the immunosuppressive activity of MDSC and is part of the endoplasmatic reticulum (ER)-stress response. The expression of Chop in MDSC has been shown to be induced by factors like e.g. reactive oxygen species (ROS) and peroxynitrite (PNT) in the tumor microenvironment (Thevenot et al. 2014). 
     As Chop is an intracellular factor with no enzymatic activity, inhibition of Chop by small molecules or antibodies are not or hardly suitable. Accordingly, an agent which is safe and effective in inhibiting the function of an intracellular mediator of immunosuppression like Chop is an important addition for the treatment of patients suffering from diseases or conditions affected for example by the activity of this factor. 
     So far no antisense oligonucleotide exists which is highly efficient in reduction and inhibition, respectively, of Chop expression and hybridizes with Chop mRNA and/or pre-mRNA. Studies with siRNA to inhibit Chop expression showed that in vivo inhibition is only possible if siRNA is packed in suitable packaging material. Even if siRNA is packed the efficiency on the inhibition of mRNA expression can often not be improved. 
     An oligonucleotide of the present invention is very successful in the inhibition of the expression of Chop. 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 tumor tissue in solid tumors,
 
(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 Chop 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 C/EBP-homologous protein (Chop) of SEQ ID NO.1 (human) and/or of SEQ ID NO.48 (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, 2′O-Methyl modified nucleotide or a combination thereof). In some embodiments, the oligonucleotide inhibits at least 50% of the Chop expression and in some embodiments the oligonucleotide inhibits the expression of Chop at a nanomolar concentration. 
     The present invention is further directed to a pharmaceutical composition comprising an antisense oligonucleotide of the present invention and optionally a pharmaceutically acceptable carrier, excipient, dilutant or a combination thereof. In some embodiments, this pharmaceutical composition additionally comprises a chemotherapeutic such as platinum or gemcitabine, another disease specific active agent such as insulin, angiotensin-converting enzyme inhibitor, angiotensin receptor blocker, another oligonucleotide, an antibody, a HERA fusion protein, a ligand trap, a Fab fragment, a nanobody, a BiTe and/or a small molecule which is for example effective in tumor treatment, treatment of diabetes (e.g., insulin resistance, type 2 diabetes mellitus) and its side effects (e.g., diabetic nephropathy), treatment of obesity, treatment of nephrological diseases, and/or treatment of artheriosclerosis. 
     In some embodiments, the oligonucleotide of the present invention is in combination with another oligonucleotide, an antibody, a HERA fusion protein, a ligand trap, a Fab fragment, a nanobody, a BiTe and/or a small molecule, either each of these compounds is separate or combined in a pharmaceutical composition, wherein the oligonucleotide, the antibody and/or the small molecule inhibits or stimulates an immune suppressive factor such as IDO1, IDO2, CTLA-4, PD-1, PD-L1, LAG-3, VISTA, A2AR, CD39, CD73, STAT3, TDO2, TIM-3, TIGIT, TGF-beta, BTLA, MICA, NKG2A, KIR, CD160, Chop, and/or Xbp1. In addition or alternatively, the oligonucleotide, the antibody and/or the small molecule inhibits or stimulates an immune stimulatory factor such as 4-1BB, Ox40, KIR, GITR, CD27 and/or 2B4. 
     Furthermore, the present invention relates to a method of preventing and/or treating a disorder, where a Chop imbalance is involved comprising administering the oligonucleotide or the pharmaceutical composition of the present invention. In some embodiments, the disorder is for example an autoimmune disorder, an immune disorder, diabetes, artheriosclerosis, a nephrological disorder and/or cancer. In some embodiments, the oligonucleotide 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&#39;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. 
    
    
     
       BRIEF DESCRIPTION OF DRAWINGS 
         FIG. 1  shows the mRNA sequence of human (h) Chop (SEQ ID No. 1; reference NM_001195053). 
         FIG. 2  depicts the distribution of hChop antisense oligonucleotide binding sites on the hChop mRNA of SEQ ID No. 1 as well as their modification(s) and length. hChop antisense oligonucleotides were aligned to the hChop mRNA sequence. The different grayscales indicate the different LNA modifications and symbols indicate the different length of the antisense oligonucleotides. 
         FIGS. 3A and 3B  depict hChop mRNA knockdown efficacy of hChop antisense oligonucleotides in human cancer cell lines EFO-21 (ovarian cystadenocarcinoma) in a first screening round ( FIG. 3A ) and SKOV-3 (ovarian adenocarcinoma) in a first screening round ( FIG. 3B ). EFO-21 and SKOV-3 cells were treated for 3 days with 5 μM of the respective antisense oligonucleotide without transfection reagent. As negative control, cells were treated with S6, an antisense oligonucleotide having the sequence TCTATCGTGATGTTTCT. Residual hChop mRNA expression relative to untreated cells is depicted. Expression values were normalized to expression of the housekeeping gene HPRT1. 
         FIGS. 4A and 4B  depict hChop mRNA knockdown efficacy of hChop antisense oligonucleotides in human cancer cell lines EFO-21 (ovarian cystadenocarcinoma) in a second screening round ( FIG. 4A ) and SKOV-3 (ovarian adenocarcinoma) in a second screening round ( FIG. 4B ). EFO-21 and SKOV-3 cells were treated for 3 days with 5 μM of the respective antisense oligonucleotide without transfection reagent. As negative control, cells were treated with neg1, an antisense oligonucleotide having the sequence CGTTTAGGCTATGTACTT (described in WO2014154843 A1). Residual hChop mRNA expression relative to untreated cells (EFO-21) or control antisense oligonucleotide treated cells (SKOV-3) is depicted. Expression values were normalized to expression of the housekeeping gene HPRT1. 
         FIG. 5  shows concentration-dependent hChop mRNA knockdown by selected hChop antisense oligonucleotides in EFO-21 cells which were A19001H (SEQ ID No.8), A19003H (SEQ ID No.4), A19018HM (SEQ ID No.2), A19020HM (SEQ ID No.3), A19027H (SEQ ID No.5), A19028H (SEQ ID No.6) and A19037H (SEQ ID No.7). EFO-21 cells were treated for 3 days with the indicated concentration of the respective antisense oligonucleotide. Residual hChop expression is depicted compared to untreated control cells. hChop mRNA expression values were normalized to expression of the housekeeping gene HPRT1. Concentration-dependent target knockdown was used for calculation of IC 50  values shown in Table 11. 
         FIG. 6  depicts knockdown of Chop mRNA in activated human CD8+ T cells in response to treatment with Chop antisense oligonucleotides. CD8+ T cells were activated with tetrameric CD2/CD3/CD28 antibody complexes for three days and treated with the Chop antisense oligonucleotide A19018HM (SEQ ID No. 2) or the negative control neg1 at a concentration of 5 μM. Residual hChop expression is depicted compared to untreated control cells. hChop mRNA expression values were normalized to expression of the housekeeping gene HPRT1. 
         FIGS. 7A and 7B  depict an efficacy screen of human/mouse cross reactive Chop antisense oligonucleotides in murine cancer cell lines Renca (mouse renal adenocarcinoma) in a first screening round (see  FIG. 7A ) and 4T1 (tumor of the mammary gland) in a first screening round, respectively, (see  FIG. 7B ). Cells were treated for 3 days with 5 μM of the respective antisense oligonucleotide without transfection reagent. As negative control, cells were treated with S6, an antisense oligonucleotide having the sequence TCTATCGTGATGTTTCT. Residual-Chop mRNA expression relative to untreated cells is depicted. Expression values were normalized to expression of the housekeeping gene HPRT1. 
         FIGS. 8A and 8B  depict an efficacy screen of human/mouse cross reactive Chop antisense oligonucleotides in murine cancer cell lines Renca (mouse renal adenocarcinoma) in a second screening round (see  FIG. 8A ) and 4T1 (tumor of the mammary gland) in a second screening round, respectively, (see  FIG. 8B ). Cells were treated for 3 days with 5 μM of the respective antisense oligonucleotide without transfection reagent. As negative control, cells were treated with neg1, an antisense oligonucleotide having the sequence CGTTTAGGCTATGTACTT (described in WO2014154843 A1). Residual-Chop mRNA expression relative to untreated cells is depicted. Expression values were normalized to expression of the housekeeping gene HPRT1. 
         FIG. 9  shows concentration-dependent Chop mRNA knockdown by selected-Chop antisense oligonucleotides in Renca cells which were A19018HM (SEQ ID No.2) and A19021HM (SEQ ID No.20). Renca cells were treated for 3 days with the indicated concentration of the respective antisense oligonucleotide. Residual-Chop expression is depicted compared to untreated control cells. Chop mRNA expression values were normalized to expression of the housekeeping gene HPRT1. Concentration-dependent target knockdown was used for calculation of IC 50  values shown in Table 16. 
         FIG. 10  depicts knockdown of Chop mRNA in murine myeloid derived suppressor cells (MDSC) using Chop antisense oligonucleotides of the present invention. Cells were treated for 3 days with 5 μM of A19018HM (SEQ ID No. 2) or the negative control neg1. Residual Chop mRNA expression relative to untreated cells is depicted. Expression values were normalized to expression of the housekeeping gene HPRT1. 
         FIG. 11  shows the introns of the pre-mRNA sequence of human (h) Chop (SEQ ID No. 48; reference ENST00000551116.5). 
         FIG. 12  depicts efficacy screening of intronic Chop antisense oligonucleotides in EFO-21 cells. In order to investigate the knockdown efficacy of the in silico designed intronic Chop antisense oligonucleotides, an efficacy screening round in the cancer cell line EFO-21 (human Ovarian Cystadenocarcinoma, DSMZ) was performed. Cells were treated with the respective antisense oligonucleotide at a concentration of 5 μM for three days without the addition of a transfection reagent. A19018HM (SEQ ID NO.2) was used as a reference Chop antisense oligonucleotide that binds to an exonic region of Chop RNA 
     
    
    
     DETAILED DESCRIPTION 
     The present invention provides for the first time human and murine oligonucleotides which hybridize with mRNA and/or pre-mRNA sequences of C/EBP-homologous protein (Chop) and inhibit the expression and activity, respectively, of Chop. mRNA comprises only exons and untranslated regions (UTRs) of the Chop encoding nucleic acid sequence, whereas pre-mRNA comprises exons, introns and UTRs of the Chop encoding nucleic acid sequence. Thus, the oligonucleotides of the present invention represent an interesting and highly efficient tool for use in a method of preventing and/or treating disorders, where the Chop expression and activity, respectively, is increased comprising administering the oligonucleotides of the present invention. 
     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 2′O-Methyl modified nucleotide 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 the phosphate is for example a phosphorothioate. 
     The oligonucleotide of the present invention comprises the one or more modified nucleotide at the 3′- and/or 5′-end of the oligonucleotide and/or at any position within the oligonucleotide, wherein modified nucleotides follow in a row of 1, 2, 3, 4, 5, or 6 modified nucleotides, or a modified nucleotide is combined with one or more unmodified nucleotides. The following Tables 1 and 2 present 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 or 2 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 Chop: 
     
       
         
           
               
             
               
                 TABLE 1 
               
             
            
               
                   
               
               
                 List of antisense oligonucleotides hybridizing with human Chop for example of 
               
               
                 SEQ ID No. 1; Neg1 and S6 are antisense oligonucleotides representing a negative control 
               
               
                 which is not hybridizing with Chop of SEQ ID No. 1. Some of these oligonucleotides do not 
               
               
                 only hybridize with human mRNA (H), but also with mouse and/or rat Chop mRNA (HM). 
               
            
           
           
               
               
               
               
            
               
                 Seq 
                   
                   
                   
               
               
                 ID 
                 Name 
                 Antisense Sequence 5′-3′ 
                 Antisense Sequence 5′-3′ with PTO (*) and LNA (+) 
               
               
                   
               
               
                  2 
                 A19018HM 
                 CTAGCTGTGCCACTTT 
                 +C*+T*+A*G*C*T*G*T*G*C*C*A*C*+T*+T*+T 
               
               
                   
               
               
                  3 
                 A19020HM 
                 TTTCCTGCTTGAGCCG 
                 +T*+T*+T*C*C*T*G*C*T*T*G*A*G*C*+C*+G 
               
               
                   
               
               
                  4 
                 A19003H 
                 ACATGATACGCTCAGT 
                 +A*+C*+A*T*G*A*T*A*C*G*C*T*C*+A*+G*+T 
               
               
                   
               
               
                  5 
                 A19027H 
                 CAGATTCACCATTCGGT 
                 +C*+A*+G*A*T*T*C*A*C*C*A*T*T*C*+G*+G*+T 
               
               
                   
               
               
                  6 
                 A19028H 
                 ATGCTTGGTGCAGATTC 
                 +A*+T*+G*C*T*T*G*G*T*G*C*A*G*A*+T*+T*+C 
               
               
                   
               
               
                  7 
                 A19037H 
                 TCTATATACAAGCTGA 
                 +T*+C*+T*A*T*A*T*A*C*A*A*G*C*+T*+G*+A 
               
               
                   
               
               
                  8 
                 A19001H 
                 TACGCTCAGTGCCTTAG 
                 +T*+A*+C*G*C*T*C*A*G*T*G*C*C*T*+T*+A*+G 
               
               
                   
               
               
                  9 
                 A19002H 
                 TGATACGCTCAGTGCCT 
                 +T*+G*+A*T*A*C*G*C*T*C*A*G*T*G*+C*+C*+T 
               
               
                   
               
               
                 10 
                 A19031HM 
                 GTTCATGCTTGGTGCAG 
                 +G*+T*+T*C*A*T*G*C*T*T*G*G*T*G*+C*+A*+G 
               
               
                   
               
               
                 11 
                 A19024HM 
                 TCAGGCGCTCGATTT 
                 +T*+C*+A*G*G*C*G*C*T*C*G*A*+T*+T*+T 
               
               
                   
               
               
                 12 
                 A19030HM 
                 TTCATGCTTGGTGCAG 
                 +T*+T*+C*A*T*G*C*T*T*G*G*T*G*+C*+A*+G 
               
               
                   
               
               
                 13 
                 A19017H 
                 CCTTCATGCGCTGCTTT 
                 +C*+C*+T*T*C*A*T*G*C*G*C*T*G*C*T*+T*+T 
               
               
                   
               
               
                 14 
                 A19022HM 
                 CTCGATTTCCTGCTTG 
                 +C*+T*+C*G*A*T*T*T*C*C*T*G*C*+T*+T*+G 
               
               
                   
               
               
                 15 
                 A19044HM 
                 GTTCATGCTTGGTGCA 
                 +G*+T*+T*C*A*T*G*C*T*T*G*G*T*+G*C*+A 
               
               
                   
               
               
                 16 
                 A19033H 
                 ACATCATTGGCACTAGT 
                 +A*+C*+A*T*C*A*T*T*G*G*C*A*C*T*+A*+G*+T 
               
               
                   
               
               
                 17 
                 A19015HM 
                 CCACTCTGTTTCCGTTT 
                 +C*+C*+A*C*T*C*T*G*T*T*T*C*C*G*+T*+T*+T 
               
               
                   
               
               
                 18 
                 A19004H 
                 AACATGATACGCTCAG 
                 +A*+A*+C*A*T*G*A*T*A*C*G*C*T*+C*+A*+G 
               
               
                   
               
               
                 19 
                 A19029H 
                 TCATGCTTGGTGCAGAT 
                 +T*+C*+A*T*G*C*T*T*G*G*T*G*C*A*+G*+A*+T 
               
               
                   
               
               
                 20 
                 A19021HM 
                 TCGATTTCCTGCTTG 
                 +T*+C*+G*A*T*T*T*C*C*T*G*C*+T*+T*+G 
               
               
                   
               
               
                 21 
                 A19035H 
                 TATACAAGCTGAGACC 
                 +T*+A*+T*A*C*A*A*G*C*T*G*A*G*+A*+C*+C 
               
               
                   
               
               
                 22 
                 A19034H 
                 GGTCACATCATTGGCAC 
                 +G*+G*+T*C*A*C*A*T*C*A*T*T*G*G*+C*+A*+C 
               
               
                   
               
               
                 23 
                 A19050HM 
                 TTTACCTCCAGCCTCCT 
                 +T*+T*+T*A*C*C*T*C*C*A*G*C*C*T*C*+C*+T 
               
               
                   
               
               
                 24 
                 A19032HM 
                 GTTCATGCTTGGTGCA 
                 +G*+T*+T*C*A*T*G*C*T*T*G*G*T*+G*+C*+A 
               
               
                   
               
               
                 15 
                 A19043HM 
                 GTTCATGCTTGGTGCA 
                 +G*+T*+T*C*A*T*G*C*T*T*G*G*T*G*+C*+A 
               
               
                   
               
               
                 25 
                 A19019H 
                 TTTCCTGCTTGAGCCGT 
                 +T*+T*T*C*C*T*G*C*T*T*G*A*G*C*+C*+G*+T 
               
               
                   
               
               
                 26 
                 T19010HM 
                 TCATACCAGGCTTCCAG 
                 +T*+C*+A*T*A*C*C*A*G*G*C*T*T*C*+C*+A*+G 
               
               
                   
               
               
                 27 
                 A19036H 
                 TATATACAAGCTGAGAC 
                 +T*+A*+T*A*T*A*C*A*A*G*C*T*G*A*+G*+A*+C 
               
               
                   
               
               
                 28 
                 A19009HM 
                 CATACCAGGCTTCCAGC 
                 +C*+A*+T*A*C*C*A*G*G*C*T*T*C*C*+A*+G*+C 
               
               
                   
               
               
                 29 
                 A19007H 
                 TGGCAAGCTGGTCTGAT 
                 +T*G*+G*C*A*A*G*C*T*G*G*T*C*T*+G*+A*+T 
               
               
                   
               
               
                 30 
                 A19048HM 
                 ATCCAGGCTGCTCTCTT 
                 +A*+T*+C*C*A*G*G*C*T*G*C*T*C*T*+C*+T*+T 
               
               
                   
               
               
                 31 
                 A19016H 
                 CATGCGCTGCTTTCCAG 
                 +C*+A*+T*G*C*G*C*T*G*C*T*T*T*C*+C*+A*+G 
               
               
                   
               
               
                 32 
                 A19014HM 
                 CACTCTGTTTCCGTTTC 
                 +C*+A*+C*T*C*T*G*T*T*T*C*C*G*T*+T*+T*+C 
               
               
                   
               
               
                 33 
                 A19023HM 
                 CAGGCGCTCGATTTC 
                 +C*+A*+G*G*C*G*C*T*C*G*A*T*+T*+T*+C 
               
               
                   
               
               
                 34 
                 A19013HM 
                 CTCTGACTGGAATCTGG 
                 +C*+T*+C*T*G*A*C*T*G*G*A*A*T*C*+T*+G*+G 
               
               
                   
               
               
                 35 
                 A19039HM 
                 GCTCTGTCGCTGCCACC 
                 +G*+C*+T*C*T*G*T*C*G*C*T*G*C*C*+A*C*+C 
               
               
                   
               
               
                 36 
                 A19042HM 
                 TCCTCATACCAGGCT 
                 +T*+C*C*T*C*A*T*A*C*C*A*G*G*+C*+T 
               
               
                   
               
               
                 36 
                 A19041HM 
                 TCCTCATACCAGGCT 
                 +T*+C*C*T*C*A*T*A*C*C*A*G*+G*+C*+T 
               
               
                   
               
               
                 35 
                 A19038HM 
                 GCTCTGTCGCTGCCACC 
                 +G*+C*+T*C*T*G*T*C*G*C*T*G*C*C*+A*+C*+C 
               
               
                   
               
               
                 37 
                 A19005HM 
                 GCTCTGTCGCTGCCACC 
                 +G*+C*+T*C*T*G*T*C*G*C*T*G*C*C*A*+C*+C 
               
               
                   
               
               
                 30 
                 A19049HM 
                 ATCCAGGCTGCTCTCTT 
                 +A*+T*C*C*A*G*G*C*T*G*C*T*C*T*C*+T*+T 
               
               
                   
               
               
                 38 
                 A19047HM 
                 CCAGGCTGCTCTCTTGT 
                 +C*+C*+A*G*G*C*T*G*C*T*C*T*C*T*T*+G*+T 
               
               
                   
               
               
                 39 
                 A19025HM 
                 TGGTCAGGCGCTCGA 
                 +T*+G*+G*T*C*A*G*G*C*G*C*T*+C*+G*+A 
               
               
                   
               
               
                 40 
                 A19026H 
                 CGAGTCGCCTCTACTTC 
                 +C*+G*A*G*T*C*G*C*C*T*C*T*A*C*+T*+T*+C 
               
               
                   
               
               
                 41 
                 A19011H 
                 GGTCCTCATACCAGGCT 
                 +G*+G*+T*C*C*T*C*A*T*A*C*C*A*G*+G*+C*+T 
               
               
                   
               
               
                 36 
                 A19040HM 
                 TCCTCATACCAGGCT 
                 +T*+C*+C*T*C*A*T*A*C*C*A*G*G*+C*+T 
               
               
                   
               
               
                 42 
                 A19045HM 
                 CAGGCTGCTCTCTTGT 
                 +C*+A*+G*G*C*T*G*C*T*C*T*C*T*+T*+G*+T 
               
               
                   
               
               
                 42 
                 A19046HM 
                 CAGGCTGCTCTCTTGT 
                 +C*+A*G*G*C*T*G*C*T*C*T*C*T*T*+G*+T 
               
               
                   
               
               
                 43 
                 A19008H 
                 TCTGCAGTTGGATCAGT 
                 +T*+C*+T*G*C*A*G*T*T*G*G*A*T*C*A*+G*+T 
               
               
                   
               
               
                 44 
                 A19012H 
                 GTGACCTCTGCTGGTT 
                 +G*T*+G*A*C*C*T*C*T*G*C*T*G*G*+T*+T 
               
               
                   
               
               
                 45 
                 A19006HM 
                 ACTCTCTCCTCAGGTTC 
                 +A*+C*T*C*T*C*T*C*C*T*C*A*G*G*+T*T*+C 
               
               
                   
               
               
                 46 
                 Neg1 
                   
                 +C*+G*+T*T*T*A*G*G*C*T*A*T*G*T*A*+C*+T*+T 
               
               
                   
               
               
                 47 
                 S6 
                   
                 +T*+C*+T*A*T*C*G*T*G*A*T*G*T*T*+T*+C*+T 
               
               
                   
               
            
           
         
       
     
     Oligonucleotides of Table 2 hybridize with intronic regions of the pre-mRNA of human Chop: 
     
       
         
           
               
             
               
                 TABLE 2 
               
             
            
               
                   
               
               
                 List of antisense oligonucleotides hybridizing with human Chop for example of 
               
               
                 SEQ ID No. 48; Neg1 is an antisense oligonucleotides representing a negative control 
               
               
                 which is not hybridizing with Chop of SEQ ID No. 48. Some of these oligonucleotides do 
               
               
                 not only hybridize with introns of human pre-mRNA (HI), but also with introns of mouse 
               
               
                 and/or rat Chop pre-mRNA (HM). 
               
            
           
           
               
               
               
               
            
               
                 Seq Id 
                   
                   
                   
               
               
                 No. 
                 Name 
                 Antisense sequence 5′-3′ 
                 Antisense sequence 5′-3′ with PTO and LNA (+) 
               
               
                   
               
               
                 42 
                 A19045HM 
                 CAGGCTGCTCTCTTGT 
                 +C*+A*+G*G*C*T*G*C*T*C*T*C*T*+T*+G*+T 
               
               
                   
               
               
                 42 
                 A19046HM 
                 CAGGCTGCTCTCTTGT 
                 +C*+A*G*G*C*T*G*C*T*C*T*C*T*T*+G*+T 
               
               
                   
               
               
                 38 
                 A19047HM 
                 CCAGGCTGCTCTCTTGT 
                 +C*+C*+A*G*G*C*T*G*C*T*C*T*C*T*T*+G*+T 
               
               
                   
               
               
                 30 
                 A19048HM 
                 ATCCAGGCTGCTCTCTT 
                 +A*+T*+C*C*A*G*G*C*T*G*C*T*C*T*+C*+T*+T 
               
               
                   
               
               
                 30 
                 A19049HM 
                 ATCCAGGCTGCTCTCTT 
                 +A*+T*C*C*A*G*G*C*T*G*C*T*C*T*C*+T*+T 
               
               
                   
               
               
                 23 
                 A19050HM 
                 TTTACCTCCAGCCTCCT 
                 +T*+T*+T*A*C*C*T*C*C*A*G*C*C*T*C*+C*+T 
               
               
                   
               
               
                 49 
                 A19051HI 
                 ATCCTAAAGAGCGGACG 
                 +A*+T*+C*C*T*A*A*A*G*A*G*C*G*G*+A*+C*+G 
               
               
                   
               
               
                 50 
                 A19052HI 
                 GTGAGGCCGATCCTAAA 
                 +G*+T*+G*A*G*G*C*C*G*A*T*C*C*T*+A*+A*+A 
               
               
                   
               
               
                 51 
                 A19053HI 
                 AGTGAGGCCGATCCTAA 
                 +A*+G*+T*G*A*G*G*C*C*G*A*T*C*C*+T*+A*+A 
               
               
                   
               
               
                 52 
                 A19054HI 
                 ATTCCTCTCTCGGACGG 
                 +A*+T*+T*C*C*T*C*T*C*T*C*G*G*A*C*+G*+G 
               
               
                   
               
               
                 53 
                 A19055HI 
                 GTCGTCCGAAGCAATAG 
                 +G*+T*+C*G*T*C*C*G*A*A*G*C*A*A*+T*+A*+G 
               
               
                   
               
               
                 54 
                 A19056HI 
                 CCGTCGTCCGAAGCAAT 
                 +C*+C*+G*T*C*G*T*C*C*G*A*A*G*C*+A*+A*+T 
               
               
                   
               
               
                 55 
                 A19057HI 
                 GCCGTCGTCCGAAGCAA 
                 +G*+C*+C*G*T*C*G*T*C*C*G*A*A*G*+C*+A*+A 
               
               
                   
               
               
                 56 
                 A19058HI 
                 TAACGATGAGGCCTGAA 
                 +T*A*+A*C*G*A*T*G*A*G*G*C*C*T*+G*A*+A 
               
               
                   
               
               
                 57 
                 A19059HI 
                 GGACAGGCCTAACGATG 
                 +G*+G*+A*C*A*G*G*C*C*T*A*A*C*G*+A*+T*+G 
               
               
                   
               
               
                 58 
                 A19060HI 
                 GACAGGCCTAACGATG 
                 +G*+A*+C*A*G*G*C*C*T*A*A*C*G*+A*+T*+G 
               
               
                   
               
               
                 59 
                 A19061HI 
                 AACGGCCTGAGCGATGG 
                 +A*+A*+C*G*G*C*C*T*G*A*G*C*G*A*+T*+G*+G 
               
               
                   
               
               
                 60 
                 A19062HI 
                 GGAAACGGCCTGAGCGA 
                 +G*+G*+A*A*A*C*G*G*C*C*T*G*A*G*+C*+G*+A 
               
               
                   
               
               
                 61 
                 A19063HI 
                 GAAGCGGCGGAAACGGC 
                 +G*+A*+A*G*C*G*G*C*G*G*A*A*A*C*+G*+G*+C 
               
               
                   
               
               
                 62 
                 A19064HI 
                 AGAGACCTCTACGGCAA 
                 +A*+G*A*G*A*C*C*T*C*T*A*C*G*G*C*+A*+A 
               
               
                   
               
               
                 63 
                 A19065HI 
                 GGCCTCCTACACTTAAG 
                 +G*G*+C*C*T*C*C*T*A*C*A*C*T*T*A*+A*+G 
               
               
                   
               
               
                 64 
                 A19066HI 
                 TGACCACCTGATGTAAC 
                 +T*+G*+A*C*C*A*C*C*T*G*A*T*G*T*+A*+A*+C 
               
               
                   
               
               
                 65 
                 A19067HI 
                 TAAGGTAATTCCTGACC 
                 +T*+A*+A*G*G*T*A*A*T*T*C*C*T*G*+A*+C*+C 
               
               
                   
               
               
                 66 
                 A19068HI 
                 CCTGCAATCCAAAGTAC 
                 +C*+C*+T*G*C*A*A*T*C*C*A*A*A*G*+T*+A*+C 
               
               
                   
               
               
                 67 
                 A19069HI 
                 TTGTGATCCATCGCTTC 
                 +T*+T*+G*T*G*A*T*C*C*A*T*C*G*C*+T*+T*+C 
               
               
                   
               
               
                 68 
                 A19070HI 
                 CCTTGTGATCCATCGCT 
                 +C*+C*+T*T*G*T*G*A*T*C*C*A*T*C*+G*+C*+T 
               
               
                   
               
               
                 69 
                 A19071HI 
                 TACTGTCCTGCCGTTTA 
                 +T*+A*+C*T*G*T*C*C*T*G*C*C*G*T*+T*+T*+A 
               
               
                   
               
               
                 70 
                 A19072HI 
                 AAGAGTCACGCTAGAGA 
                 +A*+A*+G*A*G*T*C*A*C*G*C*T*A*G*+A*+G*+A 
               
               
                   
               
               
                 46 
                 neg1 
                   
                 +C*+G*+T*T*T*A*G*G*C*T*A*T*G*T*A*+C*+T*+T 
               
               
                   
               
            
           
         
       
     
     The oligonucleotides of the present invention hybridize for example with mRNA of human Chop of SEQ ID No. 1 and/or introns of the pre-mRNA of human Chop of SEQ ID No. 48. Such oligonucleotides are called Chop antisense oligonucleotides. In some embodiments, the oligonucleotides hybridize within a hybridizing active area which is one or more region(s) on the Chop mRNA, e.g., of SEQ ID No.1 and/or the Chop pre-mRNA, e.g., of SEQ ID No.48, where hybridization with an oligonucleotide highly likely results in a potent knockdown of the Chop expression. In the present invention surprisingly several hybridizing active areas were identified for example selected from hybridizing active areas for example selected from position 0 or 1 to 60, position 695 to 755, position 725 to 785, position 800 to 860, and/or position 970 to 1030 (including the terminal figures of the ranges) of Chop mRNA for example of SEQ ID No. 1. Examples of antisense oligonucleotides hybridizing within the above mentioned positions of Chop mRNA for example of SEQ ID No. 1 are shown in the following Tables 3 to 7 and examples of antisense oligonucleotides hybridizing within the above mentioned positions of Chop pre-mRNA for example of SEQ ID No. 48 are shown in the following Tables 8 to 21: 
     
       
         
           
               
             
               
                 TABLE 3 
               
             
            
               
                   
               
               
                 Nucleotide position 0 to 60 of Chop mRNA of SEQ ID No. 1 
               
            
           
           
               
               
               
            
               
                   
                   
                 SEQ ID 
               
               
                   
                 Binding site on hChop mRNA 
                 No./ASO 
               
               
                   
                 (Position of the first nucleotide) 
                 Name 
               
               
                   
                   
               
               
                   
                 19 
                 8/A19001H 
               
               
                   
                 22 
                 9/A19002H 
               
               
                   
                 26 
                 4/A19003H 
               
               
                   
                 27 
                 18/A19004H  
               
               
                   
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 4 
               
             
            
               
                   
               
               
                 Nucleotide position 695 to 755 of Chop mRNA of SEQ ID No. 1 
               
            
           
           
               
               
               
            
               
                   
                   
                 SEQ ID 
               
               
                   
                 Binding site on hChop mRNA 
                 No./ASO 
               
               
                   
                 (Position of the first nucleotide) 
                 65Name 
               
               
                   
                   
               
               
                   
                 725 
                 2/A19018HM 
               
               
                   
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 5 
               
             
            
               
                   
               
               
                 Nucleotide position 725 to 785 of Chop mRNA of SEQ ID No. 1 
               
            
           
           
               
               
               
            
               
                   
                   
                 SEQ ID 
               
               
                   
                 Binding site on hChop mRNA 
                 No./ASO 
               
               
                   
                 (Position of the first nucleotide) 
                 Name 
               
               
                   
                   
               
               
                   
                 754 
                 25/A19019H  
               
               
                   
                 755 
                  3/A19020HM 
               
               
                   
                 760 
                 20/A19021HM 
               
               
                   
                 760 
                 14/A19022HM 
               
               
                   
                 767 
                 33/A19023HM 
               
               
                   
                 768 
                 11/A19024HM 
               
               
                   
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 6 
               
             
            
               
                   
               
               
                 Nucleotide position 800 to860 of Chop mRNA of SEQ ID No. 1 
               
            
           
           
               
               
               
            
               
                   
                   
                 SEQ ID 
               
               
                   
                 Binding site on hChop mRNA 
                 No. 
               
               
                   
                 (Position of the first nucleotide) 
                 Name 
               
               
                   
                   
               
               
                   
                 819 
                 5/A19027H  
               
               
                   
                 829 
                 6/A19028H  
               
               
                   
                 831 
                 19/A19029H  
               
               
                   
                 833 
                 12/A19030HM 
               
               
                   
                 833 
                 10/A19031HM 
               
               
                   
                 834 
                 24/A19032HM 
               
               
                   
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 7 
               
             
            
               
                   
               
               
                 Nucleotide position 970 to 1030 of Chop mRNA of SEQ ID No. 1 
               
            
           
           
               
               
               
            
               
                   
                   
                 SEQ ID 
               
               
                   
                 Binding site on hChop mRNA 
                 No./ASO 
               
               
                   
                 (Position of the first nucleotide) 
                 Name 
               
               
                   
                   
               
               
                   
                 992 
                 21/A19035H 
               
               
                   
                 993 
                 27/A19036H 
               
               
                   
                 996 
                  7/A19037H 
               
               
                   
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 8 
               
             
            
               
                   
               
               
                 Nucleotide position 29 to 89 of Chop pre-mRNA of SEQ ID No. 48 
               
            
           
           
               
               
               
            
               
                   
                   
                 SEQ ID 
               
               
                   
                 Binding site on hChop pre-mRNA 
                 No./ASO 
               
               
                   
                 (Position of the first nucleotide) 
                 Name 
               
               
                   
                   
               
               
                   
                 54 
                 49/A19051HI 
               
               
                   
                 63 
                 50/A19052HI 
               
               
                   
                 64 
                 51/A19053HI 
               
               
                   
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 9 
               
             
            
               
                   
               
               
                 Nucleotide position 90 to 132 of Chop pre-mRNA of SEQ ID No. 48 
               
            
           
           
               
               
            
               
                   
                 SEQ ID 
               
               
                 Binding site on hChop pre-mRNA 
                 No./ASO 
               
               
                 (Position of the first nucleotide) 
                 Name 
               
               
                   
               
               
                 102 
                 52/A19054HI 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 10 
               
             
            
               
                   
               
               
                 Nucleotide position 130 to 190 of Chop pre-mRNA of SEQ ID  
               
               
                 No. 48 (e.g., intron 1) 
               
            
           
           
               
               
               
            
               
                   
                   
                 SEQ ID 
               
               
                   
                 Binding site on hChop pre-mRNA 
                 No./ASO 
               
               
                   
                 (Position of the first nucleotide) 
                 Name 
               
               
                   
                   
               
               
                   
                 158 
                 53/A19055HI 
               
               
                   
                 160 
                 54/A19056HI 
               
               
                   
                 161 
                 55/A19057HI 
               
               
                   
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 11 
               
             
            
               
                   
               
               
                 Nucleotide position 256 to 316 of Chop pre-mRNA of SEQ ID  
               
               
                 No. 48 (e.g., intron 1) 
               
            
           
           
               
               
               
            
               
                   
                   
                 SEQ ID 
               
               
                   
                 Binding site on hChop pre-mRNA 
                 No./ASO 
               
               
                   
                 (Position of the first nucleotide) 
                 Name 
               
               
                   
                   
               
               
                   
                 282 
                 56/A19058HI 
               
               
                   
                 291 
                 57/A19059HI 
               
               
                   
                 291 
                 58/A19060HI 
               
               
                   
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 12 
               
             
            
               
                   
               
               
                 Nucleotide position 331 to 391 of Chop pre-mRNA of SEQ ID  
               
               
                 No. 48 (e.g., intron 1) 
               
            
           
           
               
               
               
            
               
                   
                   
                 SEQ ID 
               
               
                   
                 Binding site on hChop pre-mRNA 
                 No./ASO 
               
               
                   
                 (Position of the first nucleotide) 
                 Name 
               
               
                   
                   
               
               
                   
                 356 
                 59/A19061HI 
               
               
                   
                 359 
                 60/A19062HI 
               
               
                   
                 367 
                 61/A19063HI 
               
               
                   
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 13 
               
             
            
               
                   
               
               
                 Nucleotide position 534 to 576 of Chop pre-mRNA of SEQ ID  
               
               
                 No. 48 (e.g., intron 1) 
               
            
           
           
               
               
               
            
               
                   
                   
                 SEQ ID 
               
               
                   
                 Binding site on hChop pre-mRNA 
                 No./ASO 
               
               
                   
                 (Position of the first nucleotide) 
                 Name 
               
               
                   
                   
               
               
                   
                 564 
                 62/A19064HI 
               
               
                   
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 14 
               
             
            
               
                   
               
               
                 Nucleotide position 577 to 600 of Chop pre-mRNA of SEQ ID  
               
               
                 No. 48 (e.g., intron 1) 
               
            
           
           
               
               
               
            
               
                   
                   
                 SEQ ID 
               
               
                   
                 Binding site on hChop pre-mRNA 
                 No./ASO 
               
               
                   
                 (Position of the first nucleotide) 
                 Name 
               
               
                   
                   
               
               
                   
                 592 
                 42/A19045HM 
               
               
                   
                 592 
                 42/A19046HM 
               
               
                   
                 592 
                 38/A19047HM 
               
               
                   
                 594 
                 30/A19048HM 
               
               
                   
                 594 
                 30/A19049HM 
               
               
                   
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 15 
               
             
            
               
                   
               
               
                 Nucleotide position 601 to 643 of Chop pre-mRNA of SEQ ID  
               
               
                 No. 48 (e.g., intron 1) 
               
            
           
           
               
               
               
            
               
                   
                   
                 SEQ ID 
               
               
                   
                 Binding site on hChop pre-mRNA 
                 No./ASO 
               
               
                   
                 (Position of the first nucleotide) 
                 Name 
               
               
                   
                   
               
               
                   
                 613 
                 63/A19065HI 
               
               
                   
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 16 
               
             
            
               
                   
               
               
                 Nucleotide position 1108 to 1168 of Chop  
               
               
                 pre-mRNA of SEQ ID No. 48 (e.g., intron 1) 
               
            
           
           
               
               
               
            
               
                   
                 Binding site on hChop 
                 SEQ ID 
               
               
                   
                 pre-mRNA 
                 No./ASO 
               
               
                   
                 (Position of the first nucleotide) 
                 Name 
               
               
                   
                   
               
               
                   
                 1132 
                 64/A19066HI 
               
               
                   
                 1144 
                 65/A19067HI 
               
               
                   
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 17 
               
             
            
               
                   
               
               
                 Nucleotide position 1739 to 1799 of Chop 
               
               
                 pre-mRNA of SEQ ID No. 48 (e.g., intron 1) 
               
            
           
           
               
               
               
            
               
                   
                 Binding site on hChop 
                 SEQ ID 
               
               
                   
                 pre-mRNA 
                 No./ASO 
               
               
                   
                 (Position of the first nucleotide) 
                 Name 
               
               
                   
                   
               
               
                   
                 1769 
                 66/A19068HI 
               
               
                   
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 18 
               
             
            
               
                   
               
               
                 Nucleotide position 2241 to 2301 of Chop 
               
               
                 pre-mRNA of SEQ ID No. 48 (e.g., intron 1) 
               
            
           
           
               
               
               
            
               
                   
                 Binding site on hChop 
                 SEQ ID 
               
               
                   
                 pre-mRNA 
                 No./ASO 
               
               
                   
                 (Position of the first nucleotide) 
                 Name 
               
               
                   
                   
               
            
           
           
               
               
               
            
               
                   
                 2271 
                 23/A19050HM 
               
               
                   
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 19 
               
             
            
               
                   
               
               
                 Nucleotide position 2312 to 2372 of Chop  
               
               
                 pre-mRNA of SEQ ID No. 48 (e.g., intron 1) 
               
            
           
           
               
               
               
            
               
                   
                 Binding site on hChop 
                 SEQ ID 
               
               
                   
                 pre-mRNA 
                 No./ASO 
               
               
                   
                 (Position of the first nucleotide) 
                 Name 
               
               
                   
                   
               
               
                   
                 2341 
                 67/A19069HI 
               
               
                   
                 2343 
                 68/A19070HI 
               
               
                   
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 20 
               
             
            
               
                   
               
               
                 Nucleotide position 17 to 47 of Chop  
               
               
                 pre-mRNA of SEQ ID No. 48 (e.g., intron 2) 
               
            
           
           
               
               
               
            
               
                   
                 Binding site on hChop 
                 SEQ ID 
               
               
                   
                 pre-mRNA 
                 No./ASO 
               
               
                   
                 (Position of the first nucleotide) 
                 Name 
               
               
                   
                   
               
            
           
           
               
               
               
            
               
                   
                 32 
                 69/A19071HI 
               
               
                   
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 21 
               
             
            
               
                   
               
               
                 Nucleotide position 61 to 91 of Chop  
               
               
                 pre-mRNA of SEQ ID No. 48 (e.g., intron 2) 
               
            
           
           
               
               
               
            
               
                   
                 Binding site on hChop 
                 SEQ ID 
               
               
                   
                 pre-mRNA 
                 No./ASO 
               
               
                   
                 (Position of the first nucleotide) 
                 Name 
               
               
                   
                   
               
               
                   
                 76 
                 70/A19072HI 
               
               
                   
                   
               
            
           
         
       
     
     In Tables 3 to 21 “ASO” is the abbreviation for “antisense oligonucleotide” and the sequences and LNA patterns of the ASOs are specified in Tables 1 and 2, respectively. 
     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 Chop such as the, e.g., human, rat or murine, Chop expression. Thus, the oligonucleotides of the present invention are for example immunosuppression-reverting oligonucleotides which inhibit and revert immunosuppression, respectively, for example in a cell, tissue, organ, or a subject. The oligonucleotide of the present invention inhibits the expression of Chop 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, 10 or 100 μM. 
     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, 2.2, 3, 5, 6.6 or 10 μM. 
     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 a chemotherapeutic, another disease specific active agent such as insulin, angiotensin-converting enzyme inhibitor, angiotensin receptor blocker, another oligonucleotide, an antibody, a HERA fusion protein, a ligand trap, a Fab fragment, a nanobody, a BiTe and/or a small molecule which is for example effective in tumor treatment, treatment of diabetes and its side effects, treatment of obesity, treatment of nephrological diseases, and/or treatment of artheriosclerosis. 
     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 comprising administering the oligonucleotide or the pharmaceutical composition to a subject. In some embodiments, the method of preventing and/or treating a disorder is combined with radiotherapy. The radiotherapy may be further combined with a chemotherapy (e.g., platinum, gemcitabine). The disorder is for example characterized by an Chop imbalance, i.e., the Chop level is increased in comparison to the level in a normal, healthy cell, tissue, organ or subject. The Chop level is for example increased by an increased Chop expression and activity, respectively. The Chop 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, 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, a HERA fusion protein, a ligand trap, a Fab fragment, a nanobody, a BiTe, a small molecule and/or a chemotherapeutic (e.g., platinum, gemcitabine) and/or another disease specific agent such as insulin, angiotension-converting enzyme inhibitor, and/or angiotensin receptor blocker. In some embodiments, the other oligonucleotide (i.e., not being part of the present invention), the antibody, a HERA fusion protein, a ligand trap, a Fab fragment, a nanobody, a BiTe, and/or the small molecule are effective in preventing and/or treating 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 solid tumor or a hematologic tumor comprising administering the oligonucleotide or the pharmaceutical composition of the present invention to a subject. Examples of cancers preventable and/or treatable by use of the oligonucleotide or pharmaceutical composition of the present invention are breast cancer, lung cancer, malignant melanoma, lymphoma, skin cancer, bone cancer, prostate cancer, liver cancer, brain cancer, cancer of the larynx, gall bladder, pancreas, testicular, rectum, parathyroid, thyroid, adrenal, neural tissue, head and neck, colon, stomach, bronchi, kidneys, basal cell carcinoma, squamous cell carcinoma, metastatic skin carcinoma, osteo sarcoma, Ewing&#39;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&#39;s tumor, seminoma, ovarian tumor, leiomyomater tumor, cervical dysplasia, retinoblastoma, soft tissue sarcoma, malignant carcinoid, topical skin lesion, rhabdomyosarcoma, Kaposi&#39;s sarcoma, osteogenic sarcoma, malignant hypercalcemia, renal cell tumor, polycythermia vera, adenocarcinoma, anaplastic astrocytoma, glioblastoma multiforma, leukemia, or epidermoid carcinoma. 
     Further examples of diseases preventable and/or treatable by use of the oligonucleotide or pharmaceutical composition of the present invention other than cancer are for example diabetes, insulin resistance, type 2 diabetes mellitus, diabetic nephropathy, obesity and artheriosclerosis. 
     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 HERA fusion protein, a ligand trap, a Fab fragment, a nanobody, a BiTe, a small molecule and/or a chemotherapeutic, at the same time point for example in a pharmaceutical composition or separately, or on staggered intervals. In some embodiments of these combinations, the antisense oligonucleotide inhibits the expression and activity, respectively, of an immune suppressive factor and the other oligonucleotide (i.e., not being part of the present invention), the antibody, a HERA fusion protein, a ligand trap, a Fab fragment, a nanobody, a BiTe and/or small molecule inhibits (antagonist) or stimulates (agonist) the same and/or another immune suppressive factor and/or an immune stimulatory factor. The immune suppressive factor is for example selected from the group consisting of IDOL IDO2, CTLA-4, PD-1, PD-L1, LAG-3, VISTA, A2AR, CD39, CD73, STAT3, TDO2, TIM-3, TIGIT, TGF-beta, BTLA, MICA, NKG2A, KIR, CD160, Chop, Xbp1 and a combination thereof. The immune stimulatory factor is for example selected from the group consisting of 4-1BB, Ox40, KIR, GITR, CD27, 2B4 and a combination thereof. 
     The immune suppressive factor is a factor whose expression and/or activity is for example increased in a cell, tissue, organ or subject. The immune stimulatory factor is a factor whose level is increased or decreased in a cell, tissue, organ or subject depending on the cell, tissue, organ or subject and its individual conditions. 
     An antibody in combination with the oligonucleotide or the pharmaceutical composition of the present invention is for example an anti-PD-1 antibody, an anti-PD-L1 antibody, or a bispecific antibody. A small molecule in combination with the oligonucleotide or the pharmaceutical composition of the present invention are for example NLG919, Indoximod, or Epacadostat. 
     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 Chop, 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 IC 50  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 IC 50  value (see for example Zhang et al., Gene Therapy, 2011, 18, 326-333; Stanton et al., Nucleic Acid Therapeutics, Vol. 22, No. 5, 2012). As artificial systems using a transfecting agent are hard or impossible to translate into therapeutic approaches and no transfection formulation has been approved so far for oligonucleotides, the following experiments are performed without any transfecting agent. 
     Example 1: Efficacy Screen of Chop Antisense Oligonucleotides in Human Cancer Cell Lines (First Screening Round) 
     In order to investigate the knockdown efficacy of the in silico designed Chop antisense oligonucleotides of Table 1, two efficacy screening rounds were performed in the cancer cell lines EFO-21 (human Ovarian Cystadenocarcinoma, DSMZ) and SKOV-3 (human Ovary Adenocarcinoma, ATCC). Therefore, cells were treated with the respective antisense oligonucleotide at a concentration of 5 μM for three days without the addition of a transfection reagent. Cells were lyzed after the three days treatment period, Chop and HPRT1 mRNA expression was analyzed using the QuantiGene Singleplex assay (ThermoFisher) and the Chop expression values were normalized to HPRT1 values. The results for the first screening round of antisense oligonucleotide are shown in  FIGS. 3A and 3B  as well as in the following Tables 22 and 23, wherein Chop expression has been calculated. As depicted in  FIG. 3A ) and Table 22, treatment of EFO-21 cells with the antisense oligonucleotides A19001H (SEQ ID NO.8), A19002H (SEQ ID NO.9), A19015HM (SEQ ID NO.17), A19022HM (SEQ ID NO.14), A19033H (SEQ ID NO.16), A19034H (SEQ ID NO.22), A19035H (SEQ ID NO.21) and A19044HM (SEQ ID NO.15) resulted in a residual Chop mRNA expression of &lt;0.3 compared to untreated cells. The control antisense oligonucleotide S6 showed only a minimal effect on the Chop mRNA expression in this experiment. Selected antisense oligonucleotides were furthermore screened in SKOV-3 cells with regard to their Chop knockdown efficacy. As shown in  FIG. 3B ) and Table 23, treatment with the antisense oligonucleotides A19001H, A19002H, A19022HM, A19033H, A19034H, A19039HM (SEQ ID NO.35) and A19044HM resulted in a residual Chop mRNA expression of &lt;0.5, whereas the control antisense oligonucleotide S6 had no effect. 
     
       
         
           
               
             
               
                 TABLE 22 
               
             
            
               
                   
               
               
                 List of the mean Chop mRNA expression values in 
               
               
                 antisense oligonucleotide (ASO)-treated (first screening 
               
               
                 round) EFO-21 cells compared to untreated cells. 
               
               
                 Expression values are normalized to HPRT1. 
               
            
           
           
               
               
               
            
               
                   
                   
                 Residual Chop expression 
               
               
                   
                   
                 (compared to untreated cells) 
               
               
                   
                   
               
               
                   
                 A19001H 
                 0.22 
               
               
                   
                 A19002H 
                 0.24 
               
               
                   
                 A19022HM 
                 0.26 
               
               
                   
                 A19044HM 
                 0.26 
               
               
                   
                 A19033H 
                 0.26 
               
               
                   
                 A19015HM 
                 0.27 
               
               
                   
                 A19035H 
                 0.28 
               
               
                   
                 A19034H 
                 0.28 
               
               
                   
                 A19032HM 
                 0.30 
               
               
                   
                 A19043HM 
                 0.30 
               
               
                   
                 A19010HM 
                 0.31 
               
               
                   
                 A19036H 
                 0.33 
               
               
                   
                 A19009HM 
                 0.33 
               
               
                   
                 A19016H 
                 0.42 
               
               
                   
                 A19013HM 
                 0.45 
               
               
                   
                 A19039HM 
                 0.51 
               
               
                   
                 A19042HM 
                 0.53 
               
               
                   
                 A19041HM 
                 0.55 
               
               
                   
                 A19038HM 
                 0.55 
               
               
                   
                 A19005HM 
                 0.56 
               
               
                   
                 A19011H 
                 0.63 
               
               
                   
                 A19040HM 
                 0.64 
               
               
                   
                 Control ASO (S6) 
                 0.87 
               
               
                   
                 Untreated cells 
                 1.00 
               
               
                   
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 23 
               
             
            
               
                   
               
               
                 List of the mean Chop mRNA expression values 
               
               
                 in antisense oligonucleotide-treated (first screening  
               
               
                 round) SKOV-3 cells compared to untreated  
               
               
                 cells. Expression values are normalized to HPRT1. 
               
            
           
           
               
               
               
            
               
                   
                   
                 Residual Chop expression 
               
               
                   
                 ASO 
                 (compared to untreated cells) 
               
               
                   
                   
               
               
                   
                 A19001H 
                 0.32 
               
               
                   
                 A19002H 
                 0.35 
               
               
                   
                 A19044HM 
                 0.39 
               
               
                   
                 A19022HM 
                 0.39 
               
               
                   
                 A19039HM 
                 0.39 
               
               
                   
                 A19034H 
                 0.44 
               
               
                   
                 A19033H 
                 0.45 
               
               
                   
                 A19035H 
                 0.51 
               
               
                   
                 A19015HM 
                 0.51 
               
               
                   
                 A19043HM 
                 0.58 
               
               
                   
                 A19036H 
                 0.66 
               
               
                   
                 A19032HM 
                 0.66 
               
               
                   
                 A19010HM 
                 0.73 
               
               
                   
                 A19013HM 
                 0.91 
               
               
                   
                 A19009HM 
                 0.93 
               
               
                   
                 A19016H 
                 0.96 
               
               
                   
                 Untreated cells 
                 1.00 
               
               
                   
                 Control ASO (S6) 
                 1.16 
               
               
                   
                   
               
            
           
         
       
     
     Example 2: Efficacy Screen of Chop Antisense Oligonucleotides in Human Cancer Cell Lines (Second Screening Round) 
     The results of the second screening round are shown in  FIGS. 4A and 4B  as well as in Tables 24 and 25. As depicted in  FIG. 4A ) and Table 24, treatment of EFO-21 cells with the antisense oligonucleotides A19001H (SEQ ID NO.8), A19018HM (SEQ ID NO.2), A19020HM (SEQ ID NO.3), A19027H (SEQ ID NO.5), A19028H (SEQ ID NO.6) and A19037H(SEQ ID NO.7) resulted in a residual Chop mRNA expression of &lt;0.3 compared to untreated cells. The control antisense oligonucleotide neg1 showed a moderate negative effect on the Chop mRNA expression in this particular experiment which has not been observed in further experiments. Selected antisense oligonucleotides were furthermore screened in SKOV-3 cells with regard to their Chop knockdown efficacy. As shown in  FIG. 4B ) and Table 25, treatment with the antisense oligonucleotides A19001H (SEQ ID NO.8), A19003H (SEQ ID NO.4), A19018HM (SEQ ID NO.2), A19027H (SEQ ID NO.5), A19029H (SEQ ID NO.19) and A19037H (SEQ ID NO.7) resulted in a residual Chop mRNA expression of &lt;0.5 compared to control antisense oligonucleotide (neg1) treated cells. 
     
       
         
           
               
             
               
                 TABLE 24 
               
             
            
               
                   
               
               
                 List of the mean Chop mRNA expression values  
               
               
                 in antisense oligonucleotide-treated (second 
               
               
                 screening round) EFO-21 cells compared to untreated 
               
               
                 cells. Expression values are normalized to HPRT1. 
               
            
           
           
               
               
               
            
               
                   
                   
                 Residual Chop expression 
               
               
                   
                 ASO 
                 (compared to untreated cells) 
               
               
                   
                   
               
               
                   
                 A19037H 
                 0.19 
               
               
                   
                 A19027H 
                 0.20 
               
               
                   
                 A19028H 
                 0.21 
               
               
                   
                 A19020HM 
                 0.29 
               
               
                   
                 A19018HM 
                 0.30 
               
               
                   
                 A19001H 
                 0.30 
               
               
                   
                 A19003H 
                 0.31 
               
               
                   
                 A19031HM 
                 0.33 
               
               
                   
                 A19024HM 
                 0.34 
               
               
                   
                 A19030HM 
                 0.34 
               
               
                   
                 A19017H 
                 0.35 
               
               
                   
                 A19004H 
                 0.37 
               
               
                   
                 A19029H 
                 0.37 
               
               
                   
                 A19021HM 
                 0.37 
               
               
                   
                 A19050HM 
                 0.40 
               
               
                   
                 A19019H 
                 0.41 
               
               
                   
                 A19007H 
                 0.53 
               
               
                   
                 A19048HM 
                 0.56 
               
               
                   
                 A19014HM 
                 0.57 
               
               
                   
                 A19023HM 
                 0.57 
               
               
                   
                 Control ASO (neg1) 
                 0.58 
               
               
                   
                 A19049HM 
                 0.78 
               
               
                   
                 A19047HM 
                 0.82 
               
               
                   
                 A19025HM 
                 0.83 
               
               
                   
                 A19026H 
                 0.84 
               
               
                   
                 A19045HM 
                 0.88 
               
               
                   
                 A19046HM 
                 0.98 
               
               
                   
                 A19008H 
                 1.00 
               
               
                   
                 Untreated cells 
                 1.00 
               
               
                   
                 A19012H 
                 1.06 
               
               
                   
                 A19006HM 
                 1.07 
               
               
                   
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 25 
               
             
            
               
                   
               
               
                 List of the mean Chop mRNA expression values  
               
               
                 in antisense oligonucleotide-treated (second  
               
               
                 screening round) SKOV-3 cells compared to control 
               
               
                 antisense oligonucleotide (neg1) treated cells. 
               
               
                 Expression values are normalized to HPRT1. 
               
            
           
           
               
               
               
            
               
                   
                   
                 Residual Chop expression 
               
               
                   
                   
                 (compared to  
               
               
                   
                 ASO 
                 neg1-treated cells) 
               
               
                   
                   
               
               
                   
                 A19003H 
                 0.39 
               
               
                   
                 A19027H 
                 0.45 
               
               
                   
                 A19029H 
                 0.45 
               
               
                   
                 A19018HM 
                 0.48 
               
               
                   
                 A19037H 
                 0.48 
               
               
                   
                 A19001H 
                 0.50 
               
               
                   
                 A19020HM 
                 0.52 
               
               
                   
                 A19004H 
                 0.52 
               
               
                   
                 A19028H 
                 0.53 
               
               
                   
                 A19021HM 
                 0.56 
               
               
                   
                 A19017H 
                 0.67 
               
               
                   
                 A19031HM 
                 0.69 
               
               
                   
                 Control ASO (neg1) 
                 1.01 
               
               
                   
                   
               
            
           
         
       
     
     Example 3: Determination of IC 50  Values of Selected Chop Antisense Oligonucleotide in EFO-21 Cells 
     The dose-dependent knockdown of Chop mRNA expression by Chop antisense oligonucleotides in EFO-21 cells was investigated and the respective IC 50  values were calculated. Therefore, EFO-21 cells were treated for three days with the respective antisense oligonucleotide at the following concentrations: 6 μM, 2 μM, 600 nM, 200 nM, 60 nM, 20 nM, 6 nM, 2 nM. After the treatment period, cells were lyzed, Chop and HPRT1 mRNA expression was analyzed using the QuantiGene Singleplex assay (ThermoFisher) and the Chop expression values were normalized to HPRT1 values. A dose-dependent knockdown of Chop mRNA with all tested Chop antisense oligonucleotides was observed ( FIG. 5 ) with IC 50  values below 500 nM (Table 26): 
     
       
         
           
               
             
               
                 TABLE 26 
               
             
            
               
                   
               
               
                 Dose-dependent inhibition of Chop mRNA expression in EFO-21 cells by 
               
               
                 selected Chop antisense oligonucleotides and respective IC 50  values. 
               
            
           
           
               
               
               
            
               
                   
                 IC 50   
                 Inhibition (%) 
               
            
           
           
               
               
               
               
               
               
               
               
               
               
            
               
                 ASO 
                 (nM) 
                 6 μM 
                 2 μM 
                 600 nM 
                 200 nM 
                 60 nM 
                 20 nM 
                 6 nM 
                 2 nM 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
               
            
               
                 A19001H 
                 338.5 
                 80.8 
                 76.0 
                 58.3 
                 28.5 
                 4.3 
                 5.1 
                 4.0 
                 0.0 
               
               
                 A19003H 
                 252.8 
                 81.7 
                 74.7 
                 61.7 
                 41.0 
                 6.2 
                 13.6 
                 0.0 
                 0.0 
               
               
                 A19018HM 
                 227.8 
                 72.7 
                 67.0 
                 54.3 
                 35.5 
                 14.8 
                 7.2 
                 0.0 
                 0.0 
               
               
                 A19020HM 
                 241.8 
                 70.5 
                 53.6 
                 43.8 
                 22.9 
                 11.1 
                 0.0 
                 0.0 
                 0.0 
               
               
                 A19027H 
                 256.6 
                 80.8 
                 71.2 
                 57.0 
                 22.7 
                 20.9 
                 0.0 
                 0.0 
                 0.0 
               
               
                 A19028H 
                 263 
                 78.1 
                 76.6 
                 55.0 
                 36.2 
                 10.6 
                 0.0 
                 2.2 
                 0.0 
               
               
                 A19037H 
                 311.9 
                 81.2 
                 68.5 
                 51.1 
                 29.5 
                 0.0 
                 0.0 
                 0.0 
                 0.0 
               
               
                   
               
            
           
         
       
     
     Example 4: Knockdown of Chop mRNA in Activated Human CD8+ T Cells after Treatment with Chop Antisense Oligonucleotides 
     To test the activity of Chop antisense oligonucleotides in activated immune cells (CD8+ T cells), the antisense oligonucleotide A19018HM (SEQ ID NO.2) was selected. T cells were activated for three days using tetrameric CD2/CD3/CD28 antibody complexes. During the activation period, cells were treated with the control antisense oligonucleotide neg1 or the Chop-specific antisense oligonucleotide A19018HM at a concentration of 5 μM. After the treatment period, cells were lyzed, Chop and HPRT1 mRNA expression was analyzed using the QuantiGene Singleplex assay (ThermoFisher) and the Chop expression values were normalized to HPRT1 values. As shown in  FIG. 6 , treatment of T cells with the Chop-specific antisense oligonucleotide A19018HM resulted in a residual Chop mRNA expression of 0.45 compared to untreated cells whereas treatment with the control antisense oligonucleotide had no effect. 
     Example 5: Efficacy Screen of Human/Mouse Cross-Reactive Chop Antisense Oligonucleotides in Murine Cancer Cell Lines (First Screening Round) 
     In order to investigate the knockdown efficacy of human/mouse cross-reactive Chop antisense oligonucleotides (Table 1), two efficacy screening rounds were performed in the cancer cell lines Renca (mouse renal adenocarcinoma, ATCC) and 4T1 (tumor of the mammary gland, ATCC). Therefore, cells were treated with the respective antisense oligonucleotide at a concentration of 5 μM for three days without the addition of a transfection reagent. Cells were lyzed after the three days treatment period, Chop and HPRT1 mRNA expression was analyzed using the QuantiGene Singleplex assay (ThermoFisher) and the Chop expression values were normalized to HPRT1 values. The results of the first screening round are shown in  FIGS. 7A and 7B  as well as in Tables 27 and 28. As shown in  FIG. 7A ) and Table 27, treatment of Renca cells with the antisense oligonucleotides A19015HM (SEQ ID NO.17), A19022HM (SEQ ID NO.14), A19032HM (SEQ ID NO.24), A19042HM (SEQ ID NO.36), A19043HM (SEQ ID NO.15) and A19044HM (SEQ ID NO.15) resulted in a residual Chop mRNA expression of &lt;0.5 compared to untreated cells. Selected antisense oligonucleotides were furthermore screened in 4T1 cells with regard to their Chop knockdown efficacy. As shown in  FIG. 7B ) and Table 28, treatment with the antisense oligonucleotides A19015HM and A19022HM resulted in a residual Chop mRNA expression of &lt;0.5 compared to untreated cells. The control antisense oligonucleotide S6 showed only a moderate effect in both tested cell lines. 
     
       
         
           
               
             
               
                 TABLE 27 
               
             
            
               
                   
               
               
                 List of the mean Chop mRNA expression values  
               
               
                 in antisense oligonucleotide-treated (first  
               
               
                 screening round) Renca cells compared to untreated 
               
               
                 cells. Expression values are normalized to HPRT1. 
               
            
           
           
               
               
               
            
               
                   
                   
                 Residual Chop expression 
               
               
                   
                 ASO 
                 (compared to untreated cells) 
               
               
                   
                   
               
               
                   
                 A19022HM 
                 0.26 
               
               
                   
                 A19044HM 
                 0.37 
               
               
                   
                 A19015HM 
                 0.40 
               
               
                   
                 A19032HM 
                 0.41 
               
               
                   
                 A19042HM 
                 0.46 
               
               
                   
                 A19043HM 
                 0.49 
               
               
                   
                 A19009HM 
                 0.51 
               
               
                   
                 A19041HM 
                 0.52 
               
               
                   
                 A19010HM 
                 0.56 
               
               
                   
                 A19039HM 
                 0.58 
               
               
                   
                 A19040HM 
                 0.59 
               
               
                   
                 A19038HM 
                 0.59 
               
               
                   
                 A19005HM 
                 0.66 
               
               
                   
                 Control ASO (S6) 
                 0.74 
               
               
                   
                 A19013HM 
                 0.85 
               
               
                   
                 Untreated cells 
                 1.01 
               
               
                   
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 28 
               
             
            
               
                   
               
               
                 List of the mean Chop mRNA expression values 
               
               
                 in antisense oligonucleotide-treated (first 
               
               
                 screening round) 4T1 cells compared to untreated  
               
               
                 cells. Expression values are normalized to HPRT1. 
               
            
           
           
               
               
               
            
               
                   
                   
                 Residual Chop expression 
               
               
                   
                 ASO 
                 (compared to untreated cells) 
               
               
                   
                   
               
               
                   
                 A19015HM 
                 0.22 
               
               
                   
                 A19022HM 
                 0.28 
               
               
                   
                 A19032HM 
                 0.58 
               
               
                   
                 A19011H 
                 0.67 
               
               
                   
                 A19044HM 
                 0.67 
               
               
                   
                 Control ASO (S6) 
                 0.78 
               
               
                   
                 A19042HM 
                 0.79 
               
               
                   
                 Untreated cells 
                 1.00 
               
               
                   
                 A19043HM 
                 1.13 
               
               
                   
                   
               
            
           
         
       
     
     Example 6: Efficacy Screen of Human/Mouse Cross-Reactive Chop Antisense Oligonucleotides in Murine Cancer Cell Lines (Second Screening Round) 
     The results of the second screening round are shown in  FIGS. 8A and 8B  as well as in Tables 29 and 30. As shown in  FIG. 8A ) and Table 29, treatment of Renca cells with the antisense oligonucleotides A19014HM (SEQ ID NO.32), A19018HM (SEQ ID NO.2), A19020HM (SEQ ID NO.3), A19021HM (SEQ ID NO.20), A19022HM (SEQ ID NO.14), A19024HM (SEQ ID NO.11), A19025HM (SEQ ID NO.39) and A19031HM (SEQ ID NO.10) resulted in a residual Chop mRNA expression of &lt;0.3 compared to untreated cells. Selected antisense oligonucleotides were furthermore screened in 4T1 cells with regard to their Chop knockdown efficacy. As shown in  FIG. 8B ) and Table 30, treatment with the antisense oligonucleotides A19014HM, A19020HM, A19021HM, A19022HM, A19023HM (SEQ ID NO.33), A19024HM, A19045HM (SEQ ID NO.42), A19047HM (SEQ ID NO.38) and A19048HM (SEQ ID NO.30) resulted in a residual Chop mRNA expression of &lt;0.3 compared to untreated cells. The control antisense oligonucleotide neg1 showed no effect in both tested cell lines. 
     
       
         
           
               
             
               
                 TABLE 29 
               
             
            
               
                   
               
               
                 List of the mean Chop mRNA expression values  
               
               
                 in antisense oligonucleotide-treated (second  
               
               
                 screening round) Renca cells compared to untreated  
               
               
                 cells. Expression values are normalized to HPRT1. 
               
            
           
           
               
               
               
            
               
                   
                   
                 Residual Chop expression 
               
               
                   
                 ASO 
                 (compared to untreated cells) 
               
               
                   
                   
               
               
                   
                 A19018HM 
                 0.10 
               
               
                   
                 A19021HM 
                 0.12 
               
               
                   
                 A19022HM 
                 0.21 
               
               
                   
                 A19031HM 
                 0.22 
               
               
                   
                 A19024HM 
                 0.24 
               
               
                   
                 A19014HM 
                 0.25 
               
               
                   
                 A19020HM 
                 0.25 
               
               
                   
                 A19025HM 
                 0.27 
               
               
                   
                 A19023HM 
                 0.34 
               
               
                   
                 A19047HM 
                 0.35 
               
               
                   
                 A19048HM 
                 0.36 
               
               
                   
                 A19046HM 
                 0.38 
               
               
                   
                 A19045HM 
                 0.38 
               
               
                   
                 A19030HM 
                 0.43 
               
               
                   
                 A19049HM 
                 0.56 
               
               
                   
                 A19050HM 
                 0.63 
               
               
                   
                 A19006HM 
                 0.64 
               
               
                   
                 Control ASO (neg1) 
                 0.94 
               
               
                   
                 Untreated cells 
                 1.00 
               
               
                   
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 30 
               
             
            
               
                   
               
               
                 List of the mean Chop mRNA expression values  
               
               
                 in antisense oligonucleotide-treated (second 
               
               
                 screening round) 4T1 cells compared to untreated  
               
               
                 cells. Expression values are normalized to HPRT1. 
               
            
           
           
               
               
               
            
               
                   
                   
                 Residual Chop expression 
               
               
                   
                 ASO 
                 (compared to untreated cells) 
               
               
                   
                   
               
               
                   
                 A19021HM 
                 0.16 
               
               
                   
                 A19048HM 
                 0.19 
               
               
                   
                 A19022HM 
                 0.22 
               
               
                   
                 A19023HM 
                 0.24 
               
               
                   
                 A19024HM 
                 0.25 
               
               
                   
                 A19045HM 
                 0.26 
               
               
                   
                 A19047HM 
                 0.27 
               
               
                   
                 A19014HM 
                 0.27 
               
               
                   
                 A19020HM 
                 0.28 
               
               
                   
                 A19046HM 
                 0.31 
               
               
                   
                 A19049HM 
                 0.33 
               
               
                   
                 A19018HM 
                 0.37 
               
               
                   
                 A19050HM 
                 0.70 
               
               
                   
                 A19030HM 
                 0.78 
               
               
                   
                 A19006HM 
                 0.84 
               
               
                   
                 A19031HM 
                 1.00 
               
               
                   
                 Untreated cells 
                 1.01 
               
               
                   
                 Control ASO (neg1) 
                 1.14 
               
               
                   
                 A19025HM 
                 1.14 
               
               
                   
                   
               
            
           
         
       
     
     Example 7: Determination of IC 50  Values of Selected Human/Mouse Cross-Reactive Chop Antisense Oligonucleotides in Renca Cells 
     The dose-dependent knockdown of Chop mRNA expression by Chop antisense oligonucleotides in Renca cells was investigated and the respective IC 50  values were calculated. Therefore, Renca cells were treated for three days with the respective antisense oligonucleotide at the following concentrations: 6 μM, 2 μM, 600 nM, 200 nM, 60 nM, 20 nM, 6 nM, 2 nM. After the treatment period, cells were lyzed, Chop and HPRT1 mRNA expression was analyzed using the QuantiGene Singleplex assay (ThermoFisher) and the Chop expression values were normalized to HPRT1 values. A dose-dependent knockdown of Chop mRNA with the two tested Chop antisense oligonucleotides A19018HM and A19021HM ( FIG. 9 ) with IC 50  values below 500 nM (Table 31) was observed. 
     
       
         
           
               
             
               
                 TABLE 31 
               
             
            
               
                   
               
               
                 Dose-dependent inhibition of Chop mRNA expression in Renca cells by 
               
               
                 selected Chop antisense oligonucleotides and respective IC 50  values 
               
            
           
           
               
               
               
            
               
                   
                 IC 50   
                 Inhibition (%) 
               
            
           
           
               
               
               
               
               
               
               
               
               
               
            
               
                 ASO 
                 (nM) 
                 6 μM 
                 2 μM 
                 600 nM 
                 200 nM 
                 60 nM 
                 20 nM 
                 6 nM 
                 2 nM 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
               
            
               
                 A19018HM 
                 439.4 
                 86.5 
                 67.7 
                 37.7 
                 9.8 
                 0 
                 0 
                 0 
                 0 
               
               
                 A19021HM 
                 406.3 
                 71.4 
                 53.0 
                 25.9 
                 6.2 
                 0 
                 0 
                 0 
                 0 
               
               
                   
               
            
           
         
       
     
     Example 8: Knockdown of Chop mRNA in Murine Myeloid Derived Suppressor Cells with Chop Antisense Oligonucleotides 
     Myeloid-derived suppressor cells (MDSC) are one of the major immune cell subset that contribute to the suppressive tumor-microenvironment. Chop seems to play an important role in the regulation of the suppressive capacity of MDSC, as genetic knockout of Chop in those cells has been shown to revert them into immune stimulatory cells in murine model systems (Thevenot et al. 2014). Therefore, it was tested if Chop antisense oligonucleotides have the capacity to knock down Chop in in vitro generated bone marrow-derived MDSC. Murine bone marrow was isolated, cells were plated in tissue culture treated 96-well plates and differentiation to MDSC was induced by addition of granulocyte macrophage colony-stimulating factor (GM-CSF) and macrophage colony-stimulating factor (M-CSF). Cells were additionally treated with the Control antisense oligonucleotide neg1 or the Chop specific antisense oligonucleotide A19018HM at a concentration of 5 μM. Three days later, cells were treated with the N-linked glycosylation inhibitor Tunicamycin in order to mimic stress factors that are present in the tumor-microenvironment as reactive oxygen species (ROS) and nitrogen species (RNS). Tunicamycin as well as ROS and RNS lead to induction of the endoplasmatic reticulum (ER)-stress response including upregulation of Chop. As shown in  FIG. 10 , treatment of Tunicamycin-treated MDSC with the Chop-specific antisense oligonucleotide A19018HM resulted in a residual Chop mRNA expression of 0.19 compared to cells that had been treated with Tunicamycin but no antisense oligonucleotide (=Untreated cells). Importantly, the Chop mRNA expression in Chop antisense oligonucleotide-treated cells was comparable to cells that had not been treated with Tunicamycin (expression compared to untreated cells: 0.2). Treatment of cells with the control antisense oligonucleotide neg1 had only a moderate negative effect on Chop mRNA expression. 
     Example 9: Efficacy Screen of Chop Antisense Oligonucleotides in Human Cancer Cell Line 
     In order to investigate the knockdown efficacy of the in silico designed intronic Chop antisense oligonucleotides, an efficacy screening round in the cancer cell line EFO-21 (human Ovarian Cystadenocarcinoma, DSMZ) was performed. Cells were treated with the respective antisense oligonucleotide at a concentration of 5 μM for three days without the addition of a transfection reagent. Cells were lyzed after the three days treatment period, Chop and HPRT1 mRNA expression was analyzed using the QuantiGene Singleplex assay (ThermoFisher) and the Chop expression values were normalized to HPRT1 values. As depicted in  FIG. 12  and Table 32, treatment of EFO-21 cells with the antisense oligonucleotides A19072HI, A19071HI, A19066HI, A19054H1 and A19062H1 resulted in a residual Chop RNA expression of &lt;0.6. The control oligonucleotide neg1 showed no effect on Chop RNA expression. A19018HM was used as a reference Chop antisense oligonucleotide that binds to an exonic region of Chop RNA and resulted in a residual Chop RNA expression of 0.55 in this experiment. 
     
       
         
           
               
             
               
                 TABLE 32 
               
             
            
               
                   
               
               
                 Residual Chop expression in 
               
               
                 comparison to untreated cells. 
               
            
           
           
               
               
               
            
               
                   
                   
                 Residual Chop expression 
               
               
                   
                 ASO 
                 (compared to untreated cells) 
               
               
                   
                   
               
               
                   
                 A19072HI 
                 0.39 
               
               
                   
                 A19071HI 
                 0.44 
               
               
                   
                 A19018HM 
                 0.55 
               
               
                   
                 (reference) 
                   
               
               
                   
                 A19066HI 
                 0.57 
               
               
                   
                 A19054HI 
                 0.57 
               
               
                   
                 A19062HI 
                 0.59 
               
               
                   
                 A19070HI 
                 0.63 
               
               
                   
                 A19063HI 
                 0.65 
               
               
                   
                 A19067HI 
                 0.71 
               
               
                   
                 A19064HI 
                 0.72 
               
               
                   
                 A19053HI 
                 0.78 
               
               
                   
                 A19055HI 
                 0.81 
               
               
                   
                 A19056HI 
                 0.81 
               
               
                   
                 A19051HI 
                 0.82 
               
               
                   
                 A19052HI 
                 0.83 
               
               
                   
                 A19069HI 
                 0.84 
               
               
                   
                 A19059HI 
                 0.85 
               
               
                   
                 A19061HI 
                 0.86 
               
               
                   
                 A19060HI 
                 0.87 
               
               
                   
                 A19065HI 
                 0.89 
               
               
                   
                 A19058HI 
                 0.94 
               
               
                   
                 A19057HI 
                 1.08 
               
               
                   
                 A19068HI 
                 1.20 
               
               
                   
                 neg1 
                 1.00