The present invention relates to an antagonist of the methyltransferase Setdb2 for use in treating an infection. Also provided herein are methods for treating, preventing or ameliorating infections comprising the administration of an antagonist of Setdb2 to a subject in need of such treatment. Herein preferred is the treatment of superinfections, in particular bacterial superinfections. The infection, in particular the bacterial superinfection, can be preceded by a viral infection.

Secondary bacterial pneumonia plays a predominant role in the morbidity of seasonal and pandemic influenza virus infection, thereby representing a significant clinical as well as socioeconomic challenge ^l ' ² ' ³ . Virus-induced immune responses are thought to be involved in the pathogenesis of bacterial superinfections. In general, upon initial pathogen recognition by receptors such as toll-like receptors (TLRs) ⁴ ' ⁵ , the subsequent downstream events lead to the induction of two major pathways, type-I interferon (IFN) and nuclear factor kappa B (NF-KB) signaling. The transcription of type I IFNs is regulated by the family of interferon regulatory factors (IRFs) ⁶ . Secreted IFNs bind to the ubiquitously expressed heteromeric receptor IFNa/β receptor 1 (Ifnarl) and Ifnar2, which results in the expression of a large number of interferon- stimulated genes (ISGs). Many ISGs encode effector proteins, which mediate the defense against viruses and other pathogens ' . The same triggering of TLRs can lead to the activation and nuclear translocation of NF-κΒ proteins, which in turn induce the expression of proinflammatory genes involved in antibacterial defense ⁵ ' ⁹ . Type-I IFN and NF-κΒ signaling are subjected to multiple layers of regulation, which are required to maintain a balance between effective pathogen clearance, the prevention of tissue damage and disease tolerance ^{10, n} ' ¹² ' ¹³ . This is of particular relevance in superinfections, where virus-induced host responses can lead to an increased susceptibility to bacterial infections through type I IFN-mediated interference with NF-κΒ signaling ^{15- ,6} . Maintaining a balance between effective pathogen defense and the prevention of excessive inflammation, autoimmunity, and immunopathology is the central task of immune regulation ¹⁰ ' ¹³ . Type I IFN and NF-κΒ signaling are two important pathways for this process and are subjected to multiple layers of crosstalk, many of which are still poorly understood.

In the prior art the therapy of infections often involves the use of drugs that target the pathogen directly. For example, fungal infections are treated by antifungal medication including macrocyclic polyenes and imidazole, thiazole and triazole derivates. The therapy of viral infections includes antiviral medication including entry inhibitors and inhibitors specific to viral enzymes such as reverse transcriptase, integrase and proteases. In case of protozoan infections, the therapy using antiprotozoal agents including metronidazole is recommended. In particular bacterial superinfections are treated by antibiotics such as penicillins, cephalosporins, chloramphenicol sulfonamides, trimethoprim-sulfamethoxazole, macrolides and quinolones.

However, often the pathogenic organisms develop a resistance to the conventionally used medicaments.

Thus, the technical problem underlying the present invention is the provision of means and methods for the therapy of infections, such as superinfections, and in particular bacterial superinfections.

The technical problem is solved by provision of the embodiments characterized in the claims.

The present invention relates to an antagonist of the methyltransferase Setdb2 for use in treating an infection. The present invention relates to a method for treating an infection comprising the administration of an antagonist of the methyltransferase Setdb2 to a subject in need of such a treatment.

In a preferred aspect, the present invention relates to an antagonist of the methyltransferase Setdb2 for use in treating a bacterial superinfection.

It is well known and accepted in the art that a bacterial superinfection is a distinct, specific type of a bacterial infection. The term "bacterial superinfection" as used herein can refer to a second bacterial infection superimposed on an earlier infection. In other words, the term "bacterial superinfection" can refer to a new bacterial infection occurring in a patient having an earlier or preexisting infection. The "earlier infection" or "pre-existing infection" can be a viral infection, bacterial infection, a fungal infection or a protozoan infection. Preferably, the "earlier infection" or "pre-existing infection" is a virus infection.

The present invention solves the above identified technical problem, as documented herein below and in the appended examples.

It was surprisingly shown that Setdb2 was the only methyltransferase induced/upregulated upon virus infection. In such a pathological setting (i.e. a setting characterized by activation/overexpression/upregulation of Setdb2) the downregulation of Setdb2 can exert beneficial therapeutic effects. In corresponding human and mouse cell culture experiments, the downregulation of Setdb2 resulted in enhanced expression/secretion of chemokine (C-X-C motif) ligand 1 (Cxcll) (and of its human ortholog chemokine (C-X-C motif) ligand 8 (CXCL8)). Cxcll /CXCL8 are chemoattractants for neutrophils. Because neutrophils are a key factor in the immune response it is believed that the enhanced Cxcll /CXCL8 expression or secretion triggered by downregulation of Setdb2 strengthens the immune response. Hence, the inhibition of Setdb2 is beneficial in a clinical setting that is characterized by Setdb2 activation/overexpression/upregulation, like viral infection, or an infection preceded by a viral infection, e.g. bacterial superinfection preceded by a viral infection.

As proof of principle, the knockdown of Setdb2 (Setdb2 genetrap mice (Setdb2 )) in an animal model of bacterial superinfection (Streptococcus pneumoniae) preceded by viral infection (influenza A virus) resulted indeed in an ameliorated pathogenesis compared to wild- type mice; see Example 1 and Fig. 5.

The mice were first infected intranasally with influenza virus (strain A/PR/8/34, or shortly PR8) and subsequently superinfected intranasally with Streptococcus pneumoniae (Sp). Thus, the mice used in Example 1 represent a model of bacterial superinfection, in particular bacterial superinfection of the lung.

Further, the mice showed signs of pneumonia and pulmonary edema as measured by lung wet weight two days after superinfection with Streptococcus pneumoniae (Sp). The pathology of pneumonia in this model includes increased size, weight, number of affected lobes, and hemorrhagic lesions of the lung. Thus, the mice are also a model for pneumonia, in particular bacterial pneumonia ¹⁶ . Consequently, it is shown herein that the loss of the methyltransferase Setdb2 in the in vivo mouse model for bacterial superinfection and pneumonia resulted in an ameliorating effect in pathogenesis.

Setdb2 genetrap mice (Setdb2 ) show a strong reduction of the Setdb2 protein compared to wild-type (WT) mice; see Fig. 2a. Therefore, the Setdb2 genetrap mice (Setdb2 ) reflect the activity of antagonists of Setdb2. The experiments show that the gross pathological appearance of the lungs (including size, weight, number of affected lobes, and hemorrhagic lesions) of Setdb2 genetrap mice (Setdb2 ) was significantly milder as compared to WT control mice upon superinfection of influenza virus-infected mice with Streptococcus pneumoniae (Sp); see Fig. 5e, f. The bacterial load (Streptococcus pneumoniae) was also significantly lower in

Setdb2 genetrap mice (Setdb2 ) as compared to WT mice upon superinfection; Fig. 5k. Histopathological analysis of lung sections confirmed the beneficial effect: Setdb2 mice showed reduced signs of pneumonia including bronchitis, endothelialitis and inflammatory infiltrates (Fig. 5g, h). Furthermore, Setdb2 mice at this advanced stage of bacterial superinfection showed decreased levels of mRNA and protein of the pro-inflammatory cytokine 116 (Fig. 5i, j). As a proof of principle, Setdb2 was knocked down by specific siRNAs in a human cell system, thus demonstrating the feasibility of the use of an antagonist of Setdb2 in a human model; see Example 2 and Fig. 18.

Thus, the in vivo experiments demonstrate that antagonists of Setdb2 are beneficial in the therapy of bacterial superinfections, in particular bacterial superinfections of the lung, and related diseases such as bacterial pneumonia. Therefore, the experimental data in the appended examples provide for a clear rationale to use antagonists of Setdb2 in the therapy of infections, in particular bacterial superinfections and related diseases.

It is shown herein that Setdb2 is specifically upregulated upon influenza virus infection; Fig lc. It is believed that the pronounced beneficial effect of Setdb2 inhibition in bacterial superinfection is due to the upregulation of Setdb2 induced by the preceding viral infection. Thus, it is believed that the provided therapy with Setdb2 antagonists is particularly advantageous in clinical settings that are characterized by or associated with increased expression of Setdb2 (or upregulation of Setdb2); see Example 1 and Fig. 13.

By contrast, it was shown that antagonizing Setdb2 is indeed not beneficial in a distinct therapeutic setting that does not involve upregulation of Setdb2. For example, Setdb2 genetrap mice (Setdb2 ^J " ⁱ ) and wild-type mice did, upon single intranasal infection of the lung with Streptococcus pneumoniae, not exhibit significant increased expression of Setdb2 or upregulation of Setdb2 (Fig. 13), differences of Cxcll, (Fig. 14d), of the number of neutrophils (Fig. 15e,f) and of the bacterial burden (Fig. 16b). Thus, the present invention demonstrates a specific and surprising effect in a therapeutic setting that involves upregulation of Setdb2, such as bacterial superinfection of the lung upon preceding viral infection.

Moreover, it is demonstrated herein that Setdb2 genetrap mice (Setdb2 ) exhibit increased neutrophil infiltration upon bacterial superinfection; see Fig. 5c, d. Neutrophils execute multiple roles including the regulation and resolution of inflammation and the elimination of bacterial pathogens ³³ ' ⁴¹ . Neutrophils migrate to inflammations sites and sites of bacterial infection following chemical signals, such as CXCL8. CXCL8 is the human ortholog of mouse Cxcll and plays a similar important antibacterial role as chemoattractant for neutrophils (Richmond A, Nature Reviews Immunology. 2002 Sep;2(9):664-74). Furthermore, it is shown herein that the reduced levels of Setdb2 in Setdbl mice lead to increased production and secretion of chemokine (C-X-C motif) ligand 1 (Cxcll); see Fig. 5b. Cxcll is a chemoattractant for neutrophils and is important for efficient pathogen clearance as well as implicated in immunopathologies . Furthermore, it is shown herein that the depletion of Setdb2 in human haploid cells resulted consistently in strong induction and secretion of CXCL8; see Example 3 and Fig. 18. Thus, the herein provided results in the mouse model are confirmed in a human model system. It is believed that the increased levels of Cxcll expression and subsequent increased neutrophil recruitment observed in Setdb2 mice and the increased expression of CXCL8, the human ortholog of Cxcll, in human Setdb2 depleted cells provides further evidence for a causal link of Setdb2 inhibition and the ameliorated pathogenesis of bacterial superinfection. Unexpectedly, it is shown herein that the protein lysine methyltransferase Setdb2 occupied the Cxcll promoter and mediated H3K9 tri-methylation, which is a repressive histone mark (Fig. 3). The increased H3K9 tri-methylation was absent at the Cxcll promoter in cells lacking

ΓΤ/ΓΤ

Setdb2 (Setdb2 cells). Thus, Setdb2 repressed the expression of the neutrophil attractant Cxcll and other NF-κΒ target genes. Without being bound by theory, it is believed that the inhibition of Setdb2 leads to reduced levels of the repressive histone mark, H3K9 tri-methyl at the Cxcll promoter, which consequently results in an increased Cxcll expression. This might, in turn, be useful to increase neutrophil infiltration and therefore be beneficial in the therapy of infections, in particular bacterial superinfections.

To summarize the above, the data presented herein provide a rationale for the therapy of infections/infectious diseases, in particular bacterial superinfections and related diseases, by inhibiting/antagonizing Setdb2.

Moreover, it is shown herein that the Cxcll was increased by Setdb2 knockdown in Setdb2 genetrap bone marrow-derived macrophages (BMDM) upon poly-IC treatment; see Example 4 and Fig. 20. Sinefungin and S-adenosyl-L-homocysteine (SAH), exemplary Setdb2 inhibitors, showed an effect on Cxcll levels in wild-type cells that is comparable to the effect of Setdb2 knockdown on Cxcll levels in Setdb2 genetrap BMDM. Thus, the present invention shows as a proof of principle that Setdb2 inhibitors indeed increase the level of Cxcll. Cxcll is a chemoattractant for neutrophils and its upregulation strengthens the immune response and contributes to an effective therapy of infections/infectious diseases. Further, it is shown herein that NFkB target genes were differently regulated by Setdb2 knockdown in Setdb2 genetrap mice; see Fig. 2b.

The model used in Example 4 reflects a virus infection (the BMDM cells were treated with the TLR3 ligand and synthetic dsRNA analog polykC) and thus a clinical setting characterized by Setdb2 activation/overexpression. The demonstrated upregulation/activation of Cxcll by the exemplary Setdb2 inhibitors Sinefungin and S-adenosyl-L-homocysteine (SAH) provide, as proof of principle, evidence that the inhibition of Setdb2 is indeed useful to exert a beneficial response in a setting that is characterized by increased Setdb2 expression/upregulation of Setdb2, such as a infection, like a viral infection or, in particular, a bacterial superinfection. The use of Sinefungin or S-adenosyl-L-homocysteine (SAH) in Setdb2 genetrap cells even showed a more than additive effect on Cxcll levels (compared to the effect observed in Setdb2 genetrap cells and Sinefungin in wild-type cells on Cxcll levels); see Fig. 20. Setdb2 genetrap cells are "knock-down" cells that show some residual levels/residual activity of Setdb2. Thus, the use of Sinefungin or S-adenosyl-L-homocysteine (SAH) in Setdb2 genetrap cells is believed to remove residual levels/residual activity of Setdb2 and thus to reflect a Setdb2 knockout. This in turn can reflect the use of a selective Setdb2 antagonist in accordance with the present invention. The model can also reflect the use of a combination therapy of an Setdb2 antagonist (e.g. a S-adenoxyl-L-methionine (SAM) analog, like Sinefungin or S-adenosyl-L- homocysteine (SAH)) and a different Setdb2 inhibitor (e.g. a selective Setdb2 inhibitor) in accordance with the present invention. Thus, this experiment shows that the use of a selective Setdb2 antagonist, or a combination therapy of an Setdb2 antagonist (e.g. a S-adenoxyl-L- methionine (SAM) analog, like Sinefungin or S-adenosyl-L-homocysteine (SAH)) and a different Setdb2 antagonist (e.g-. a selective Setdb2 antagonist), can provide excellent results in the therapy of infections as defined herein, such as viral infections, or, in particular, bacterial superinfections.

The present invention has, inter alia, the following advantages over conventional therapies of infections.

One advantage of the present invention is the fact that it strengthens the immune response against pathogens. A further advantage of the invention is that it can be used as a prophylactic treatment, in particular of high risk patients. A further advantage of the present invention is that it provides for a treatment option for long term treatment e.g., chronic infections. Furthermore, the present invention has less side effects compared to conventionally used therapies e.g., killing of commensals. As a further advantageous property, the provided therapy of the invention can be used independently of developed resistance of the pathogens to conventional medicaments.

In certain aspects, the present invention relates to the following items: An antagonist of the methyltransferase SET domain bifurcated 2 (Setdb2) for use in treating an infection. A method for treating an infection comprising the administration of an antagonist of the methyltransferase SET domain bifurcated 2 (Setdb2) to a subject in need of such a treatment. The antagonist of item 1 , or the method of item 2, wherein said infection is a bacterial, a protozoan, a fungal infection or a viral infection. The antagonist of item 1, or the method of item 2, wherein said infection is a bacterial infection. The antagonist of any one of items 1, 3 and 4, or the method of item 2 or 4, wherein said infection is a superinfection. The antagonist of any one of items 1 and 3 to 5 or the method of any one of items 2 to 5, wherein said bacterial infection is or comprises bacterial pneumonia. The antagonist of any one of items 1 and 3 to 6, or the method of any one of items 2 to 6, wherein said bacterial infection is a Streptococcus infection. The antagonist of item 7, or the method of item 7, wherein said Streptococcus infection is Streptococcus pneumoniae infection. The antagonist of any one of items 1 and 3 to 5, or the method of any one of items 2 to 5, wherein said infection is a bacterial infection selected from the group consisting of a Streptococcus infection, a Staphylococcus infection, a Haemophilus infection, a Mycobacterium infection, a Moraxella infection, a Pseudomonas infection, a Escherichia infection, a Yersinia infection, a Treponema infection, a Shigella infection, a Salmonella infection, a Rhodococcus infection, a Nocardia infection, a Campylobacter infection and a Clostridium infection. The antagonist of item 9 or the method of item 9,

wherein said Streptococcus infection is a Streptococcus pneumoniae or Streptococcus pyogenes infection;

wherein said Staphylococcus infection is a Staphylococcus aureus infection;

wherein said Haemophilus infection is a Haemophilus influenzae infection;

wherein said Mycobacterium infection is a Mycobacterium tuberculosis infection;

wherein said Moraxella infection is a Moraxella catarrhalis infection;

wherein said Pseudomonas infection is a Pseudomonas aeruginosa infection;

wherein said Escherichia infection is a Escherichia coli infection;

wherein said Yersinia infection is a Yersinia enterocolitica infection;

wherein said Treponema infection is a Treponema pallidum infection;

wherein said Shigella infection is a Shigella flexneri infection;

wherein said Salmonella infection is a Salmonella typhimurium infection;

wherein said Rhodococcus infection is a Rhodococcus equi infection;

wherein said Nocardia infection is a Nocardia asteroides infection;

wherein said Campylobacter infection is a Campylobacter jejuni infection; or wherein said Clostridium infection is a Clostridium difficile infection.

The antagonist of any one of items 1 and 3 to 5, or the method of any one of items 2 to 5, wherein said infection is a protozoan infection selected from the group consisting of a Toxoplasma infection, a Leishmania infection, an Isospora infection and a Plasmodium infection.

The antagonist of item 11 , or the method of item 11 ,

wherein said Toxoplasma infection is a Toxoplasma gondii infection;

wherein said Leishmania is a Leishmania infantum infection;

wherein said Isospora infection is a Isospora belli infection; or

wherein said Plasmodium infection is a Plasmodium falciparum infection.

The antagonist of any one of items 1 and 3 to 5, or the method of any one of items 2 to 5, wherein said infection is a fungal infection selected from the group consisting of a Candida infection, a Microsporidia infection, a Aspergillus infection, a Scedosporium infection, a Mucor infection, a Cryptococcus infection, a Coccidioides infection, a Histoplasma infection and a Pneumocystis infection. The antagonist of any one of items 1 and 3 to 13, or the method of any one of items 2 to 13, wherein said infection is preceded by a viral infection. The antagonist of item 14, or the method of item 14, wherein said viral infection is an influenza virus infection. The antagonist of item 14, or the method of item 14, wherein said viral infection is a viral infection selected from the group consisting of an orthomyxovirus infection, a herpesvirus infection, a hepadnavirus infection, a flavivirus infection, a lentivirus infection, a retrovirus infection, an arenavirus infection and a paramyxovirus infection. The antagonist of item 16, or the method of item 16,

wherein said orthomyxovirus infection is an influenza virus infection;

wherein said herpesvirus infection is a herpes simplex virus 1 (HSV-1), a

cytomegalovirus (CMV) or a Epstein-Barr virus (EBV) infection;

wherein said hepadnavirus infection is a hepatitis B virus (HBV) infection;

wherein said flavivirus is a hepatitis C virus (HCV) infection;

wherein said lentivirus infection is a human immunodeficiency virus (HIV) 1 or a human immunodeficiency virus (HIV) 2 infection;

wherein said retrovirus infection is a human T cell lymphotropic virus (HTLV) infection;

wherein said arenavirus infection is a lassa virus (LASV) or a lymphocytic

choriomeningitis virus (LCMV) infection; or

wherein said paramyxovirus infection is a measles virus infection.

The antagonist of item 15 or 17, or the method of item 15 or 17, wherein said influenza virus infection is an influenza A virus infection, influenza B virus infection or influenza C virus infection. The antagonist of any one of items 1 and 3 to 18, or the method of any one of items 2 to

18, wherein said methyltransferase SET domain bifurcated 2 (Setdb2) is human methyltransferase SET domain bifurcated 2 (Setdb2). The antagonist of any one of items 1 and 3 to 19, or the method of any one of items 2 to

19, wherein said methyltransferase SET domain bifurcated 2 (Setdb2) is selected from the group consisting of

a) a polypeptide comprising an amino acid encoded by a nucleic acid molecule having the nucleic acid sequence as depicted in SEQ ID NO: 1 or SEQ ID NO: 3;

b) a polypeptide having an amino acid sequence as depicted in SEQ ID NO:2 or SEQ ID NO:4;

c) a polypeptide encoded by a nucleic acid molecule encoding a peptide having an amino acid sequence as depicted in SEQ ID NO:2 or SEQ ID NO:4;

d) a polypeptide comprising an amino acid encoded by a nucleic acid molecule hybridizing under stringent conditions to the complementary strand of nucleic acid molecules as defined in (a) or (c);

e) a polypeptide having at least 65% identity to the polypeptide of any one of (a) to (d); and

f) a polypeptide comprising an amino acid encoded by a nucleic acid molecule being degenerated as a result of the genetic code to the nucleotide sequence of a nucleic acid molecule as defined in (a), (c) and (d). The antagonist of any one of items 1 and 3 to 20, or the method of any one of items 2 to

20, wherein said antagonist is selected from the group consisting of small molecule drugs, binding molecules, siRNA, shRNA, miRNA, dsRNA, stRNA and antisense molecules. The antagonist of item 21, or the method of item 21, wherein said small molecule drug is selected from the group consisting of

DZNep, Neplanocin A, CHEMBL61824, CHEMBL468927;

Sinefungin or S-adenosyl-L-homocysteine (SAH); BIX01294, UNC0321, UNC0638, NC0642, BRD4770, or UNC0224;

ΒΓΧ-01338, or Chaetocin;

Ell, UNC1999, EPZ-6438, EPZ005687, GSK126, or GSK343;

EPZ-5676, EPZ004777, or SGC0946;

14u;

17b, MethylGene and/orl7f; and

AZ505;

or pharmaceutically acceptable salts, solvates, and/or hydrates of said drug. The antagonist of item 21, or the method of item 21, wherein said binding molecule is selected from the group consisting of aptamers and intramers. The antagonist of item 21 or 23, or the method of item 21 or 23, wherein said binding molecule specifically binds to methyltransferase SET domain bifurcated 2 (Setdb2), particularly methyltransferase SET domain bifurcated 2 (Setdb2) as defined in item 20. The antagonist of item 21, or the method of item 21 , wherein said siRNA, shRNA, miRNA, dsRNA, stRNA, or antisense molecule targets a nucleic acid molecule having a sequence encoding methyltransferase SET domain bifurcated 2 (Setdb2). The antagonist of item 25, or the method of item 25, wherein said nucleic acid is selected from the group consisting of

a) a nucleic acid encoding a polypeptide comprising an amino acid sequence as depicted in SEQ ID NO:2 or SEQ ID NO:4;

b) a nucleic acid comprising a nucleotide sequence as depicted in SEQ ID NO: 1 or SEQ ID NO: 3;

c) a nucleic acid hybridizing under stringent conditions to the complementary strand of the nucleic acid as defined in (a) or (b);

d) a nucleic acid comprising a nucleotide sequence with at least 65 % identity to the nucleotide sequence of the nucleic acids of any one of (a) to (c); and

e) a nucleic acid comprising a nucleotide sequence which is degenerate as a result of the genetic code to the nucleotide sequence of a nucleic acid of any one of (a) to (d). 7. The antagonist of any one of items 21, 25 and 26, or the method of any one of items 21, 25 and 26, wherein one or both strands of said siRNA further comprises at least one base at the 5' end and/or at least one base at the 3' end. 8. The antagonist of any one of items 1 and 3 to 27, or the method of any one of items 2 to

27, wherein the antagonist is a selective antagonist of methyltransferase SET domain bifurcated 2 (Setdb2). 9. The antagonist of any one of items 1 and 3 to 28, or the method of any one of items 2 to

28, wherein said infection is an infection of the lung. 0. Method for assessing the activity of a candidate molecule suspected of being an antagonist of methyltransferase SET domain bifurcated 2 (Setdb2) comprising the steps of:

a) contacting a cell, tissue or a non-human animal comprising methyltransferase SET domain bifurcated 2 (Setdb2) with said candidate molecule;

b) detecting a decrease in activity of said methyltransferase SET domain bifurcated 2 (Setdb2); and

c) selecting a candidate molecule that decreases activity of said methyltransferase SET domain bifurcated 2 (Setdb2); wherein a decrease of the methyltransferase SET domain bifurcated 2 (Setdb2) activity is indicative for the capacity of the selected molecule to antagonise methyltransferase SET domain bifurcated 2 (Setdb2).

Setdb2 is a member of the SET-domain superfamily; see Dillon (2005), Genome Biol 6:227. All members of this superfamily share the conserved SET-domain, which transfers methyl residues from S-adenosyl-methionine to the amino group of target lysine. The terms "SET domain bifurcated 2", "methyltransferase SET domain bifurcated 2", "histone methyltransferase SET domain bifurcated 2", "Setdb2", "methyltransferase Setdb2", "histone methyltransferase Setdb2" and the like are used interchangeably herein. Setdb2 belongs to the SUV39 gene family, a sub-family of the SET-domain superfamily. The members of the SUV39 gene family share a Suvar 3-9/Enhancer-of-zeste/Trithorax (SET) domain that transfers methyl residues from S-adenosyl-methionine to the amino group of target lysines thereby catalyzing H3K9 methylation ²² ' ²³ . Setdbl, the closest related family member of Setdb2, is involved in pro-viral silencing, genomic stability and the onset of cancer ²⁴ ' ²⁵ . Furthermore, the SUV39 family members Suv39Hl , Ehmtl (alias: Glp) and Ehmt2 (alias: G9a) were shown to be involved in immunological processes such as the modulation of ISG expression, the NF-κΒ pathway and T cell differentiation ' ' . The prior art proposed that immune responses may be shaped by chromatin modifications ' ' ' ' . However, the prior art failed to disclose or propose that inhibiting/antagonizing Setdb2 might be useful in the therapy of infections, in particular bacterial infections. Hence the prior art implicated functional roles for Setdb2 only in embryonic development and cell division ' ' .

Two isoforms of human Setdb2 are known. Corresponding exemplary nucleic acid sequences and amino acid sequences of isoform a and b are shown in SEQ ID NOs. 1 and 2 (isoform a) and 3 and 4 (isoform b), respectively. One isoform of murine Setdb2 is known. A corresponding exemplary nucleic acid sequence and amino acid sequence are shown in SEQ ID NOs. 5 and 6; respectively.

Such nucleic acid sequences can be retrieved in public databases like NCBI using the following accession numbers:

NCBI :

Isoform a : NM_031915.2

Isoform b : NM_001160308

Murine Setdb2 : NM_001081024.1

Corresponding amino acid sequences can be retrieved in public database like NCBI using the following accession numbers:

NCBI :

Isoform a : NP 114121.2 Isoform b : NP 001 153780.1

Murine Setdb2 : NP_001074493.1

Amino acid sequences of SET domain bifurcated 2 (Setdb2) can also be obtained from Uniprot, e.g. for mouse Setdb2 under Uniprot accession number Q8C267 and for human Setdb2 under accession number Q96T68.

The term "Setdb2" as used herein refers primarily to a protein. Setdb2 as defined herein and to be used in accordance with the present invention is preferably human Setdb2.

In certain aspects, the Setdb2 as defined herein and to be used in accordance with the present invention can be selected from the group consisting of

a) a polypeptide comprising an amino acid encoded by a nucleic acid molecule having the nucleic acid sequence as depicted in SEQ ID NO: 1 or SEQ ID NO: 3;

b) a polypeptide having an amino acid sequence as depicted in SEQ ID NO:2 or SEQ ID NO:4;

c) a polypeptide encoded by a nucleic acid molecule encoding a peptide having an amino acid sequence as depicted in SEQ ID NO:2 or SEQ ID NO:4;

d) a polypeptide comprising an amino acid encoded by a nucleic acid molecule hybridizing under stringent conditions to the complementary strand of nucleic acid molecules as defined in (a) or (c);

e) a polypeptide having at least 65% identity to the polypeptide of any one of (a) to (d); and f) a polypeptide comprising an amino acid encoded by a nucleic acid molecule being degenerated as a result of the genetic code to the nucleotide sequence of a nucleic acid molecule as defined in (a), (c) and (d).

The Setdb2 can be isoform a of Setdb2. Accordingly, the Setdb2 as defined herein and to be used in accordance with the present invention can be selected from the group consisting of a) a polypeptide comprising an amino acid encoded by a nucleic acid molecule having the nucleic acid sequence as depicted in SEQ ID NO: 1 ;

b) a polypeptide having an amino acid sequence as depicted in SEQ ID NO:2;

c) a polypeptide encoded by a nucleic acid molecule encoding a peptide having an amino acid sequence as depicted in SEQ ID N0:2;

d) a polypeptide comprising an amino acid encoded by a nucleic acid molecule hybridizing under stringent conditions to the complementary strand of nucleic acid molecules as defined in (a) or (c);

e) a polypeptide having at least 65% identity to the polypeptide of any one of (a) to (d); and f) a polypeptide comprising an amino acid encoded by a nucleic acid molecule being degenerated as a result of the genetic code to the nucleotide sequence of a nucleic acid molecule as defined in (a), (c) and (d).

The Setdb2 can be isoform b of Setdb2. Accordingly, the Setdb2 as defined herein and to be used in accordance with the present invention can be selected from the group consisting of a) a polypeptide comprising an amino acid encoded by a nucleic acid molecule having the nucleic acid sequence as depicted in SEQ ID NO: 3;

b) a polypeptide having an amino acid sequence as depicted in SEQ ID NO:4;

c) a polypeptide encoded by a nucleic acid molecule encoding a peptide having an amino acid sequence as depicted in SEQ ID NO:4;

d) a polypeptide comprising an amino acid encoded by a nucleic acid molecule hybridizing under stringent conditions to the complementary strand of nucleic acid molecules as defined in (a) or (c);

e) a polypeptide having at least 65% identity to the polypeptide of any one of (a) to (d); and f) a polypeptide comprising an amino acid encoded by a nucleic acid molecule being degenerated as a result of the genetic code to the nucleotide sequence of a nucleic acid molecule as defined in (a), (c) and (d).

In a preferred aspect, the Setdb2 as defined herein and to be used in accordance with the present invention is

a) a polypeptide comprising an amino acid encoded by a nucleic acid molecule having the nucleic acid sequence as depicted in SEQ ID NO: 1 or SEQ ID NO: 3;

b) a polypeptide having an amino acid sequence as depicted in SEQ ID NO:2 or SEQ ID NO:4; or

c) a polypeptide having at least 95% identity to the polypeptide of (a) or (b). In a more preferred aspect, the Setdb2 as defined herein and to be used in accordance with the present invention is

a) a polypeptide comprising an amino acid encoded by a nucleic acid molecule having the nucleic acid sequence as depicted in SEQ ID NO: 1 or SEQ ID NO: 3;

b) a polypeptide having an amino acid sequence as depicted in SEQ ID NO:2 or SEQ ID NO:4; or

c) a polypeptide having at least 99% identity to the polypeptide of (a) or (b).

In a particularly preferred aspect, the Setdb2 as defined herein and to be used in accordance with the present invention is

a) a polypeptide comprising an amino acid encoded by a nucleic acid molecule having the nucleic acid sequence as depicted in SEQ ID NO: 1 or SEQ ID NO: 3; or

b) a polypeptide having an amino acid sequence as depicted in SEQ ID NO:2 or SEQ ID NO:4.

The use of (genetic) variants and fragments of the specific, exemplary, Setdb2 proteins and nucleic acid molecules as defined above (like those depicted in SEQ ID NOs: 1 to 6) in accordance with the present invention is envisaged herein. The following relates to such variants and fragments.

Setdb2 protein and related proteins/polypeptides (like variants, fragments, proteins/polypeptides having an identity of at least 65 % to the specific Setdb2 proteins provided and defined herein, and the like) have primarily the activity to methylate histone(s). However, it is also envisaged herein that Setdb2 protein and related proteins/polypeptides as defined herein can also have the activity to methylate non-histone proteins. Setdb2 protein and related proteins/polypeptides as defined herein can also have the activity, for example, to act as a scaffold or as a recruiting platform for interaction partners.

The nucleic acid sequence encoding for orthologous/homologous/identical (and thus related) sequences of the herein provided Setdb2 is at least 65% homologous/identical to the nucleic acid sequence as, inter alia, shown in SEQ ID NOs: 1, 3 and 5. More preferably, the nucleic acid sequence encoding orthologous/homologous/identical (and thus related) sequences of the herein provided Setdb2 is at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or 98% homologous/identical to the nucleic acid sequence as, inter alia, shown in SEQ ID NOs: 1, 3 and 5, wherein the higher values are preferred. Most preferably, the nucleic acid sequence encoding for orthologous/homologous/identical (and thus related) sequences of the herein provided Setdb2 is at least 99% homologous/identical to the nucleic acid sequence as, inter alia, shown in SEQ ID NOs: 1, 3 and 5. The above defined orthologous/homologous/identical sequences can also be encompassed in longer or shorter isoforms, spliced variants and fusion transcripts. The term "orthologous protein" or "orthologous gene" as used herein refers to proteins and genes, respectively, in different species that are similar to each other because they originated from a common ancestor.

Hybridization assays for the characterization of orthologs or other related sequences of known nucleic acid sequences are well known in the art; see e.g. Sambrook, Russell "Molecular Cloning, A Laboratory Manual", Cold Spring Harbor Laboratory, N.Y. (2001); Ausubel, "Current Protocols in Molecular Biology", Green Publishing Associates and Wiley Interscience, N.Y. (1989).

The term "hybridization" or "hybridizes" as used herein may relate to hybridizations under stringent or non-stringent conditions. If not further specified, the conditions are preferably non- stringent. Said hybridization conditions may be established according to conventional protocols described, e.g., in Sambrook (2001) loc. cit.; Ausubel (1989) loc. cit, or Higgins and Hames (Eds.) "Nucleic acid hybridization, a practical approach" IRL Press Oxford, Washington DC, (1985). The setting of conditions is well within the skill of the artisan and can be determined according to protocols described in the art. Thus, the detection of only specifically hybridizing sequences will usually require stringent hybridization and washing conditions such as, for example, the highly stringent hybridization conditions of 0.1 x saline sodium citrate buffer (SSC), 0.1% SDS at 65°C or 2 x SSC, 60°C, 0.1 % SDS. Low stringent hybridization conditions for the detection of homologous or not exactly complementary sequences may, for example, be set at 6 x SSC, 1% SDS at 65°C. As is well known, the length of the probe and the composition of the nucleic acid to be determined constitute further parameters of the hybridization conditions. In accordance with the present invention, the terms "homology" or "percent homology" or "identical" or "percent identity" or "percentage identity" or "sequence identity" in the context of two or more nucleic acid sequences refers to two or more sequences or subsequences that are the same, or that have a specified percentage of nucleotides that are the same (preferably at least 65% identity, more preferably at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or 98% identity, most preferably at least 99% identity), when compared and aligned for maximum correspondence over a window of comparison (preferably over the full length), or over a designated region as measured using a sequence comparison algorithm as known in the art, or by manual alignment and visual inspection. Sequences having, for example, 70% to 90% or greater sequence identity may be considered to be substantially identical. Such a definition also applies to the complement of a test sequence. Preferably the described identity exists over a region that is at least about 15 to 25 nucleotides in length, more preferably, over a region that is at least about 50 to 100 nucleotides in length and most preferably, over a region that is at least about 800 to 1200 nucleotides in length. Those having skill in the art will know how to determine percent identity between/among sequences using, for example, algorithms such as those based on CLUSTALW computer program (Thompson Nucl. Acids Res. 2 (1994), 4673-4680) or FASTDB (Brutlag Comp. App. Biosci. 6 (1990), 237-245), as known in the art.

Although the FASTDB algorithm typically does not consider internal non-matching deletions or additions in sequences, i.e., gaps, in its calculation, this can be corrected manually to avoid an overestimation of the % identity. CLUSTALW, however, does take sequence gaps into account in its identity calculations. Also available to those having skill in this art are the BLAST and BLAST 2.0 algorithms (Altschul, (1997) Nucl. Acids Res. 25:3389-3402; Altschul (1993) J. Mol. Evol. 36:290-300; Altschul (1990) J. Mol. Biol. 215:403-410). The BLASTN program for nucleic acid sequences uses as defaults a word length (W) of 11 , an expectation (E) of 10, M=5, N=4, and a comparison of both strands. The BLOSUM62 scoring matrix (Henikoff (1989) PNAS 89:10915) uses alignments (B) of 50, expectation (E) of 10, M=5, N=4, and a comparison of both strands.

In order to determine whether an nucleotide residue in a nucleic acid sequence corresponds to a certain position in the nucleotide sequence of e.g. SEQ ID NOs: 1, 3 and 5, respectively, the skilled person can use means and methods well-known in the art, e.g., alignments, either manually or by using computer programs such as those mentioned herein. For example, BLAST 2.0, which stands for Basic Local Alignment Search Tool BLAST (Altschul (1997), loc. cit; Altschul (1993), loc. cit; Altschul (1990), loc. cit), can be used to search for local sequence alignments. BLAST, as discussed above, produces alignments of nucleotide sequences to determine sequence similarity. Because of the local nature of the alignments, BLAST is especially useful in determining exact matches or in identifying similar sequences. The fundamental unit of BLAST algorithm output is the High-scoring Segment Pair (HSP). An HSP consists of two sequence fragments of arbitrary but equal lengths whose alignment is locally maximal and for which the alignment score meets or exceeds a threshold or cut-off score set by the user. The BLAST approach is to look for HSPs between a query sequence and a database sequence, to evaluate the statistical significance of any matches found, and to report only those matches which satisfy the user-selected threshold of significance. The parameter E establishes the statistically significant threshold for reporting database sequence matches. E is interpreted as the upper bound of the expected frequency of chance occurrence of an HSP (or set of HSPs) within the context of the entire database search. Any database sequence whose match satisfies E is reported in the program output.

Analogous computer techniques using BLAST (Altschul (1997), loc. cit.; Altschul (1993), loc. cit.; Altschul (1990), loc. cit.) are used to search for identical or related molecules in nucleotide databases such as GenBank or EMBL. This analysis is much faster than multiple membrane- based hybridizations. In addition, the sensitivity of the computer search can be modified to determine whether any particular match is categorized as exact or similar. The basis of the search is the product score, which is defined as:

% sequence identity x % maximum BLAST score

100 and it takes into account both the degree of similarity between two sequences and the length of the sequence match. For example, with a product score of 40, the match will be exact within a 1-2% eiTor; and at 70, the match will be exact. Similar molecules are usually identified by selecting those, which show product scores between 15 and 40, although lower scores may identify related molecules. Another example for a program capable of generating sequence alignments is the CLUSTALW computer program (Thompson (1994) Nucl. Acids Res. 2:4673- 4680) or FASTDB (Brutlag (1990) Comp. App. Biosci. 6:237-245), as known in the art.

The explanations and definitions given herein above in respect of "homology/identity of nucleic acid sequences" apply, mutatis mutandis, to "amino acid sequences" of members Setdb2, in particular an amino acid sequence as depicted in SEQ ID NO: 2 (Setdb2 isoform a), SEQ ID NO: 4 (Setdb2 isoform b) and SEQ ID NO: 6 (Setdb2 mouse).

In one embodiment, the polypeptide to be used in accordance with the present invention has at least 65 % homology/identity to a Setdb2 protein/polypeptide having the amino acid sequence as, for example, depicted in SEQ ID NOs: 2, 4 and 6. More preferably, the polypeptide has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or 98% homology/identity to a Setdb2 protein/polypeptide having the amino acid sequence as, for example, depicted in SEQ ID NOs: 2, 4 and 6, respectively, wherein the higher values are preferred. Most preferably, the polypeptide has at least 99% homology to a Setdb2 protein/polypeptide having the amino acid sequence as, for example, depicted in SEQ ID NO: 2, 4 and 6.

The terms "complement", "reverse complement" and "reverse sequence" referred to herein are described in the following example: For sequence 5'AGTGAAGT3', the complement is 3 CACTTCA5', the reverse complement is 3'ACTTCACT5' and the reverse sequence is 5 GAAGTGA3'.

The present invention provides antagonists of Setdb2 for the therapy of infections. These antagonists can be used as a medicament, i.e. the antagonists of Setdb2 provided and described herein are for use in medicine (e.g. for use in the therapy/treatment of a disease, in particular a disease associated with Setdb2 activation, like infections/infectious disease). The terms "medicament" and "pharmaceutical composition" are used interchangeably herein. Accordingly, definitions and explanations provided herein in relation to "pharmaceutical compositions", apply, mutatis mutandis, to the term "medicament". The terms "treatment", "treating" and the like are used herein to generally mean obtaining a desired pharmacological and/or physiological effect. The effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and/or may be therapeutic in terms of partially or completely curing a disease and/or adverse effect (e.g. a symptom) attributed to the disease, in particular an infectious disease. The term "treatment" as used herein covers any treatment of an infectious disease in a subject and includes: (a) preventing an infectious disease related in a subject, which may be predisposed to the infectious disease; (b) inhibiting the infectious disease, i.e. arresting its development; or (c) relieving the infectious disease, i.e. causing regression of the infectious disease.

"Treating an infection" as used herein can refer to (a) preventing the infection in a subject, which may be predisposed to the infection (e.g. preventing a bacterial infection in a subject that has an earlier or pre-existing virus infection); (b) inhibiting the infection, i.e. arresting its development (e.g. inhibiting the increase of the load of the pathogen in a subject, e.g. the viral load or bacterial load); or (c) relieving the infection, i.e. causing regression of the infection (e.g. reducing the load of the pathogen in a subject, e.g. reducing the viral load or bacterial load). Herein, a complete regression of the infection can refer to a complete reduction of the load of the pathogen in a subject, e.g. a complete reduction of the viral load or bacterial load. Preferably, no or essentially no residual pathogen can be detected in such a complete regression of the infection.

An "individual", "patient" or "subject" for the purposes of the present invention includes both humans and other animals, particularly mammals, and other organisms. Thus, the methods are applicable to both human therapy and veterinary applications. Preferably, the "individual", "patient" or "subject" is a mammal, and most preferably the "individual", "patient" or "subject" is human.

The following relates to "antagonist of Setdb2" provided and to be used in accordance with the present invention. The terms "antagonist of Setdb2" and "inhibitor of Setdb2" are used interchangeably herein. The terms "antagonist of Setdb2" or "inhibitor of Setdb2" means in context of the present invention a compound capable of fully or partially preventing or reducing the physiologic activity and/or expression level of Setdb2. The terms "antagonist" or "inhibitor" are used interchangeably herein. It is envisaged herein that the antagonist of Setdb2 is a selective antagonist of Setdb2.

In the context of the present invention said antagonist may, therefore, prevent, reduce, inhibit or inactivate the physiological activity of Setdb2 e.g. upon binding of said compound/substance (i.e. antagonist/inhibitor) to said Setdb2. As used herein, the term "antagonist" also encompasses competitive antagonists, (reversible) non-competitive antagonists or irreversible antagonist, as described, inter alia, in Mutschler, "Arzneimittelwirkungen" (1986), Wissenschaftliche Verlagsgesellschaft mbH, Stuttgart, Germany. Such an inhibition can be measured by determining substrate turnover.

An "antagonist" or "inhibitor" of Setdb2 may also be capable of preventing the function of Setdb2 by preventing/reducing the expression of the nucleic acid molecule encoding for said Setdb2. Thus, an antagonist/inhibitor of Setdb2 may lead to a decreased expression level of Setdb2 (e.g. decreased level of Setdb2 mRNA and/or of Setdb2 protein); this may be reflected in a decreased Setdb2 activity. The decreased activity and/or expression level can be measured/detected by known methods, which are also described herein.

An "antagonist/inhibitor of Setdb2" may, for example, interfere with transcription of (an) Setdb2 gene(s), processing (e.g. splicing, export from the nucleus and the like) of the gene product(s) (e.g. unspliced or partially spliced mRNA) and/or translation of the gene product (e.g. mature mRNA). The "antagonist/inhibitor of Setdb2" may also interfere with further modification (like glycosylation or phosphorylation) of the polypeptide/protein encoded by the Setdb2 gene(s) and thus completely or partially inhibit the activity of the Setdb2 protein(s) as described herein above. Furthermore, the "antagonist/inhibitor of a Setdb2" may interfere with interactions of the Setdb2 protein(s) with other proteins (thus, for example, interfering with the activity of complexes involving Setdb2 protein(s)) or, in general, with its synthesis, e.g. by interfering with upstream steps of Setdb2 expression or with signalling pathways in which the Setdb2 is involved. Depending on the mode of action, such antagonists may, for example, be denoted "sequestering antagonists" or "signaling antagonists".

In sum, the herein described Setdb2 antagonist/inhibitor will, accordingly, lead to a decrease or reduction of Setdb2 expression level and/or activity, and thereby reduce its contribution to the development, proliferation or progress of a disease associated with Setdb2 activation as defined herein, such as an infection, in particular a bacterial superinfection.

It is envisaged and preferred herein that the antagonist of Setdb2 targets, preferably specifically targets, the methyltransferase SET domain bifurcated 2 (Setdb2). The term "targeting" refers in this context to the binding to Setdb2 (and here in particular to the SET domain of Setdb2) and/or the inhibition of the activity of Setdb2, in particular the inhibition of the methyltransferase activity of Setdb2. Setdb2 can primarily have the activity to methylate histone(s). It is envisaged herein that Setdb2 can have the activity to methylate non-histone proteins. The inhibition of the activity of Setdb2 can also refer, for example, to the interference with/inhibition of the activity of Setdb2 to act as a scaffold or as a recruiting platform for interaction partners.

Preferably, the methyltransferase SET domain bifurcated 2 (Setdb2) is human methyltransferase SET domain bifurcated 2 (Setdb2) as defined above.

The antagonist(s) may be (a) small molecule drug(s), siRNA, shRNA, miRNA, dsRNA, small temporal RNA (stRNA), antisense molecules or (a) (small) binding molecule.

Antagonists to be used herein can be (a) small molecule drug(s). The terms "small molecule drug" and "small molecule compound" are used interchangeably herein. (A) small molecule drug(s) to be used herein as antagonist of Setdb2 can refer to an (organic) low molecular weight (<900 Daltons) compound. Small molecules can help to regulate a biological process and have usually a size in the order of 1(T ⁹ m. Antagonists to be used herein, like small molecules (drugs), can, for example, be identified by screening compound libraries, for example Enamine, Chembridge or Prestwick chemical libraries. For example, one or more of the following small molecule drug(s) can be used as Setdb2 inhibitors in accordance with the present invention:

Pan-methyltransferase inhibitors, such as DZNep, Neplanocin A, CHEMBL61824, CHEMBL468927, or Pan-methyltransferase inhibitors, such as an SAM analog like Sinefungin or S-adenosyl-L-homocysteine (SAH));

G9A-inhibitors, like BIX01294, UNC0321, UNC0638, NC0642, BRD4770, and/or U C0224;

Dual G9A/SUV39H1 inhibitors like BIX-01338, and/or Chaetocin;

EZH2 inhibitors like Ell, UNC1999, EPZ-6438, EPZ005687, GSK126, and/or GS 343;

DOT1L inhibitors like EPZ-5676, EPZ004777, and/or SGC0946;

PRMT3 inhibitors like 14u;

PRMT4 inhibitors like 17b, MethylGene and/orl7f; and/or

SMYD2 inhibitors like AZ505,

or pharmaceutically acceptable salts, solvates, and/or hydrates of the drug(s).

It is envisaged herein that Setdb2 antagonists/inhibitors can, in addition to Setdb2, also antagonize/inhibit another compound/other compounds, like G9A, SUV39H1, EZH2, DOT1 L, PRMT3, PRMT4 and/or SMYD2 (and optionally further compounds).

For example, small molecule drugs like BIX01294, UNC0321, UNC0638, NC0642, BRD4770, and/or UNC0224 are known to inhibit G9A. In accordance with the present invention they can be used as Setdb2 antagonists. The use of dual G9A/Setdb2 inhibitors/antagonists is envisaged in accordance with the present invention.

Small molecule drugs like BIX-01338, and/or Chaetocin are known to inhibit G9A and SUV39H1. In accordance with the present invention they can be used as Setdb2 antagonists. The use of trial G9A/SUV39H1/Setdb2 inhibitors/antagonists is envisaged in accordance with the present invention.

Small molecule drags like Ell, UNC1999, EPZ-6438, EPZ005687, GSK126, and/or GSK343 are known to inhibit EZH2. In accordance with the present invention they can be used as Setdb2 antagonists. The use of dual EZH2/Setdb2 inhibitors/antagonists is envisaged in accordance with the present invention. Small molecule drags like EPZ-5676, EPZ004777, and/or SGC0946 are known to inhibit DOTIL. In accordance with the present invention they can be used as Setdb2 antagonists. The use of dual DOTIL /Setdb2 inhibitors/antagonists is envisaged in accordance with the present invention.

Small molecule drugs like 14u are known to inhibit PRMT3. In accordance with the present invention they can be used as Setdb2 antagonists. The use of dual PRMT3/Setdb2 inhibitors/antagonists is envisaged in accordance with the present invention.

Small molecule drugs like 17b, Methyl Gene and/orl7f are known to inhibit PRMT4. In accordance with the present invention they can be used as Setdb2 antagonists. The use of dual PRMT4 /Setdb2 inhibitors/antagonists is envisaged in accordance with the present invention.

Small molecule drugs like AZ505 are known to inhibit SMYD2. In accordance with the present invention they can be used as Setdb2 antagonists. The use of dual SMYD2/Setdb2 inhibitors/ antagonists is envisaged in accordance with the present invention.

In certain aspects, the small molecule drug is one ore more of

A pan-methyltransferase inhibitor, such as DZNep, Neplanocin A, CHEMBL61824, CHEMBL468927;

an SAM analog like Sinefungin and/or S-adenosyl-L-homocysteine (SAH));

BIX01294, UNC0321, UNC0638, NC0642, BRD4770 and/or UNC0224;; and/or

Ell , UNC1999, EPZ-6438, EPZ005687, GSK126, and/or GS 343,

or pharmaceutically acceptable salts, solvates, and/or hydrates of the drugs.

In a preferred aspect, the small molecule drug is one or more of

a pan-methyltransferase inhibitor, such as DZNep, Neplanocin A, CHEMBL61824, CHEMBL468927;

a SAM analog like Sinefungin or S-adenosyl-L-homocysteine (SAH)); and/or

BIX01294, UNC0321, UNC0638, NC0642, BRD4770

or pharmaceutically acceptable salts, solvates, and/or hydrates of the drag. In a particularly preferred aspect, the small molecule drug is an SAM analog (like Sinefungin or S-adenosyl-L-homocysteine (SAH)) or phamiaceutically acceptable salts, solvates, and/or hydrates of the drug.

These exemplary small molecule drugs to be used herein as antagonists of Setdb2 are described below in detail:

D

Fujiwara T et al. J Bio Chem 2014

IUPAC Name: (lS,2R,5R)-5-(4-aminoimidazo[4,5-c]pyridin-l-yl)-3-

(hydroxymethyl)cyclopent-3-ene- 1 ,2-diol

Also known as: 3-Deazaneplanocin A; DZNep; 102052-95-9; (ls,2r,5r)-5-(4-amino-lh- imidazo[4,5-c]pyridin-l-yl)-3-(hydroxymethyl)cyclopent-3-ene -l ,2-diol

Molecular Formula: C ₁₂ Hi ₄ N ₄ 0 ₃

Molecular Weight: 262.26456

InChlKey: OMKH WTRU Y AGFG-IEBDPFPH S A-N

Vendor: Angene Chemical (SID 136397151 - External ID: AG-J-26404) Neplanocin A

Borchardt RT et al. J Biol Chem 1984

IUPAC Name: (1 S,2R,5R)-5-(6-aminopurin-9-yl)-3-(hydroxymethyl)cyclopent-3- ene- 1 ,2-diol Also known as: NPC-A; A-11079-B1B; CHEMBL8771 ; (ls,2r,5r)-5-(6-amino-9h-purin-9-yl)- 3-(hydroxymethyl)cyclopent-3-ene-l,2-diol; NSC 316458

Molecular Formula: CnHi ₃ N ₅ 0 ₃

Molecular Weight: 263.25262

InChIKey: XUGWUUDOWNZAGW-VDAHYXPESA-N

Vendor: Angene Chemical (SID 136514883 - External ID: AG-J-15319)

CHEMBL61824

Spannhoff A et al. Bioorg Med Cheni Lett 2007

Also known as: AGN-PC-046XPQ; GTPL7029; RM65; 2-(9H-xanthen-9-ylsulfanyl)-N-{2-[2- (9H-xanthen-9-ylsulfanyl)propanamido]ethyl}propanamide

IUPAC Name: 2-(9H-xanthen-9-ylsulfanyl)-N-[2-[2-(9H-xanthen-9- ylsulfanyl)propanoylamino]ethyl]propanamide

Molecular Formula: C ₃₄ H ₃₂ N ₂ 0 ₄ S ₂

Molecular Weight: 596.75888

InChlKey: KBXOGBYQKMAAIA-UHFFFAOYSA-N

Vendor: Angene Chemical (SID 181278197 - External ID: AGN-PC-046XPQ)

Huynh T et al. Biooorg Med Chem Lett 2009

IUPAC Name: (2S)-2-amino-N-[[3-[5-[5-(l,3-benzothiazol-7-yl)-l,3,4-oxadi azol-2-yl]-3-

(trifluoromethyl)pyrazol- 1 -yl]phenyl]methyl]propanamide

Also known as: BMS pyrazole inliibitor 7f; SureCN14565559; GTPL7030

Molecular Formula: C2 ₃ H ₁₈ F ₃ N ₇ 0 ₂ S

Molecular Weight: 513.49493

InChlKey: KODBDIFHMBDFOH-LBPRGKRZSA-N S-adenosyl-L-methionine (SAM) analoga can be used herein as antagonists of Setdb2. Herein preferred antagonists of Setdb2 are the SAM analoga Sinefungin and/or S- adenosylhomocysteine. Particularly preferred is Sinefungin.

Sinefungin

Also known as: Adenosylomithine, Compound 57926, Antibiotic A 9145, Sinefiingina,

Sinefungine, Sinefunginum

Molecular Formula: C15H23N705

Molecular Weight: 381.38702 g/mol

InChI Key: LMXOHSDXUQEUSF-YECHIGJVSA-N

IUPAC Name (2S,5S)-2,5-diamino-6-[(2R,3S,4R,5R)-5-(6-aminopurin-9-yl)-3 ,4- dihydroxyoxolan-2-yl]hexanoic acid

Vendor: Cayman Chemical #13829 S-adenosyl-L-homocysteine

Also known as: S-adenosylhomocysteine, S-adenosyl-L-homocysteine, AdoHcy, adenosylhomocysteine, Formycinylhomocysteine, Adenosyl-L-homocysteine

Molecular Formula: C14H20N6O5S

Molecular Weight: 384.4108 g mol

InChI Key: ZJUKTBDSGOFHSH-WFMPWKQPSA-N

lUPAC Name: (2S)-2-amino-4-[[(2S,3S,4R,5R)-5-(6-aminopurin-9-yl)-3,4-dih ydroxyoxolan-2- yl]methylsulfanyl]butanoic acid

Vendor: Cayman Chemical # 13603

Sinefungin is described in the prior art as an antifungal antibiotic and as a compound having antiparasitic activity (plasmodium, leishmania, trypanosome). Also some effects of Sinefungin on viruses (e.g. VSV, flavivirases) have been suggested. Yet, the use of Sinefungin in a therapy of infections associated with Setdb2 upregulation, like bacterial superinfections as defined herein, has not been disclosed.

Vaubourgeix (J Biol Chem (2009) 284, 19321-19330) is concerned with the design of potential drugs against tubercle bacilli. To this end, Vaubourgeix (loc. cit.) investigates the effect of Sinefungin, S-adenosyl-L-homocysteine and S-Adenosyl-N-decyl-aminoethyl on mycolic acid methyltransferases which are characteristic of tubercle bacilli. While Sinefungin and S- adenosyl-L-homocysteine are general inhibitors of S-Adenosylmethionine- (SAM) dependent methyltransferases, Vaubourgeix found that Sinefungin and S-adenosyl-L-homocysteine do not inhibit mycolic acid methyltransferases (paralogs of SAM-dependent methyltransferases) of tubercle bacilli and are therefore not useful in the treatment of tubercle bacilli infection. Vaubourgeix does therefore not envision that a Setdb2 antagonist might be beneficial in a setting wherein Setdb2 is upregulated, in particular in a bacterial superinfection of the lung (like Streptococcus pneumoniae superinfection) that may be preceded by a viral infection (like influenza virus A infection). Vaubourgeix teaches even away from the use of Setdb2 inhibitors like Sinefungin and S-adenosyl-L-homocysteine.

Yadav (BioMed Research International 2014, Article ID 156987) discloses that Sinefungin might inhibit Streptococcus pneumoniae biofilm growth. Yet, there is no proposal that a Setdb2 antagonist might be beneficial in a setting wherein Setdb2 is upregulated, in particular in a bacterial superinfection that may be preceded by a viral infection. Yadav investigates, inter alia, the effect of Sinefungin on pneumococcal infection of the ear without preceding viral infection. Yet, the authors of Yadav (loc. cit.) only draw the conclusion that Sinefungin might at most be used as a lead compound for developing antibiofilm agents. Hence, Yadav does not suggest that the data of that paper might provide a basis for the use of Sinefungin in the therapy of bacterial infections.

The appended examples of the present invention demonstrate that antagonizing Setdb2 for treating a single bacterial infection of the lung that is not associated with Setdb2 upregulation (e.g. without preceding viral infection) has, in contrast to a bacterial superinfection that is associated with Setdb2 upregulation (e.g. with preceding viral infection), no beneficial therapeutic effect; cf. Fig. 5 and 13. Thus, the result provided in the present application, namely that a Setdb2 antagonist provides excellent beneficial effects in a setting associated with Setdb2 upregulation, like in bacterial superinfection, in particular in bacterial superinfection of the lung, which may be preceded by viral infection (like influenza virus A), is surprising in view of Yadav. B

Kubicek S et al. Mol Cell 2007

IUP AC Name: N-( 1 -benzylpiperidin-4-yl)-6,7-dimethoxy-2-(4-methyl- 1 ,4-diazepan- 1 - yl)quinazolin-4-amine

Also known as: BIX-01294; BIX01294; CHEMBL569864; 935693-62-2

Molecular Formula: C ₂₈ H ₃ 8N ₆ 0 ₂

Molecular Weight: 490.64032

InChlKey: OSXFATOLZGZLSK-UHFFFAOYSA-N

Vendor: EMD Biosciences (SID 57571250 - External ID: 382190)

U

Liu F et al. J Med Chem 2010

IUP AC Name: 7-[2-[2-(dimethylamino)ethoxy]ethoxy]-6-methoxy-2-(4-methyl- 1 ,4-diazepan- 1 -yl)-N-(l -methylpiperidin-4-yl)quinazolin-4-amine

Also known as: UNC-0321; CHEMBL1214066; 1238673-32-9

Molecular Formula: C ₂₇ H ₄₅ N ₇ 0 ₃

Molecular Weight: 515.6913

InChlKey: AULLUGALUBVBDD-UHFFFAOYSA-N

Vendor: Angene Chemical (SID 188092173 - External ID: AGN-PC-080NC7)

U

Vedadi M et al. Nature Chem Biol 2011

IUP AC Name: 2-cyclohexyl-6-methoxy-N-( 1 -propan-2-ylpiperidin-4-yl)-7-(3 -pyrrolidin- 1 - ylpropoxy)quinazolin-4-amine

Also known as: UNC0638; UNC-0638; 1255580-76-7; UNII-26A103L2FO;

CHEMBL1231795

Molecular Formula: ¾οΗ ₄₇ Ν ₅ 0 ₂

Molecular Weight: 509.72648

InChlKey: QOECJCJVIMVJGX-UHFFFAOYSA-N

Vendor: Angene Chemical (SID 187406086 - External ID: AGN-PC-07NHBT)

Structural Genomics Consortium, Chemical Probes (SGC, 2013)

IUP AC Name: 2-(4,4-difluoropiperidin- 1 -yl)-6-methoxy-N-( 1 -propan-2-ylpiperidin-4-yl)-7-(3 - pyrrolidin- 1 -ylpropoxy)quinazolin-4-amine

Also known as: UNC0642; AGN-PC-09QG2O; GTPL7017; UNC 0642; IN2221 ; NCGC00189140-01 ; B-146019; 1481677-78-4

Molecular Formula: C ₂ 9H ₄₄ F ₂ N60 ₂

Molecular Weight: 546.695466

InChlKey: RNAMYOYQYRYFQY-UHFFFAOYSA-N

Vendor: Angene Chemical (SID 191713283 - External ID: AGN-PC-09QG2O)

B

Yuan Y et al. ACS Chem Biol 2012 IUPAC Name: methyl 2-benzamido-l -(3-phenylpropyl)benzimidazole-5-carboxylate

Also known as: GTPL7016; BRD 4770

Molecular Formula: C ₂₅ H ₂₃ N ₃ 0 ₃

Molecular Weight: 413.46842

InChl ey: UCGWYCMPZXDHNR-UHFFFAOYSA-N

Vendor: EMD Millipore (SID 170474678 - External ID: 382194)

U

Liu F et al. J Med Chem 2010

IUPAC Name: 7-[3-(dimethylamino)propoxy]-6-methoxy-2-(4-methyl- 1 ,4-diazepan- 1 -yl)-N-

( 1 -methylpiperidin-4-yl)quinazolin-4-amine

Also known as: CHEMBL576781; UNC-0224; 1197196-48-7

Molecular Formula: C ₂ 6H ₄₃ N ₇ 0 ₂

Molecular Weight: 485.66532

InChlKey: XIVUGRBSBIXXJE-UHFFFAOYSA-N

Vendor: Angene Chemical (SID 186512787 - External ID: AGN-PC-073VDJ)

Kubicek S et al. Mol Cell 2007

IUPAC Name: l-[2-[4-(4-memoxybenzoyl)oxyphenyl]ethyl]-2-[[4-

(trifluoromethyl)benzoyl]amino]benzimidazole-5-carboxylic acid;hydrate

Also known as: l-{2-[4-(4-Methoxybenzoyloxy)phenyl]ethyl}-2-(4- trifluoromethylbenzoylamino)- 1 H-benzoimidazole-5-carboxylic acid hydrate

Molecular Formula: C ₃₂ H ₂ F ₃ N ₃ 0 ₇

Molecular Weight: 621.55995

InChlKey: LLTPCGBKXOESTJ-UHFFFAOYSA-N

Vendor: Chembase.cn (SID 162248740 - External ID: 154602)

Chaetocin

Cherblanc FL et al. Nat Chem Biol 2013 Also known as: Chetocin; Chaetocin from Chaetomium minutum; 28097-03-2; BRN 5722505;

AC1L4PPK; AGN-PC-00BO57; C9492_SIGMA; MolPort-003-983-881

IUPAC Name: (3S,3'S,5aR,5'aR,10bR,10'bR,l laS.l l ^, aS)-2,2',3,3 ^, ,5a,5 ^, a,6,6'-Octahydro-3,3'- bis(hydroxymethyl)-2,2'-dimethyl-[10b,10'b(l 1H,1 l'H)-bi-3,l la-epidithio-1 laH- pyrazino[l',2': 1 ,5]pyrrolo[2,3-b]indole]- 1 , 1 ',4,4'-tetrone

Molecular Formula: C ₃ oH ₂₈ N ₆ 0 ₆ S ₄

Molecular Weight: 696.83992

InChlKey: PZPPOCZWRGNKIR-UHFFFAOYSA-N

Vendor: Angene Chemical (SID 172813026 - External ID: AGN-PC-00BO57)

E

Qi W et al. PNAS 2012

IUPAC Name: 6-cyano-N-[(4,6-dimethyl-2-oxo-lH-pyridin-3-yl)methyl]-l-pen tan-3-ylindole- 4-carboxamide

Also known as: EI 1 ; Ell ; KB-145943; 1418308-27-6; GTPL7013; CS-1654; HY-15573

Molecular Formula: C2 H ₂₆ N ₄ 0 ₂

Molecular Weight: 390.47814

InChlKey: PFHDWRIVDDIFRP-UHFFFAOYSA-N

Vendor: Chembo (SID 172821397 - External ID: KB-145943) UNC1999

onze KD et al. ACS Chem Biol 2013

IUPAC Name: N-[(6-methyl-2-oxo-4-propyl-lH-pyridin-3-yl)methyl]-l-propan -2-yl-6-[6-(4- propan-2-ylpiperazin- 1 -yl)pyiidin-3 -yl] indazole-4-carboxamide

Also known as: UNC 1999; 1431612-23-5; CS-1658; HY-15646; KB-145944;

S7165,1431612-23-5

Molecular Formula: C ₃₃ H ₄₃ N ₇ 0 ₂

Molecular Weight: 569.74022

InChlKey: DPJNKUOXBZSZAI-UHFFFAOYSA-N

Vendor: A Selleckchem (SID 172121846 - External ID: UNC1999)

E

Knutson SK et al. PNAS 2013 IUPAC Name: N-[(4,6-dimethyl-2-oxo-lH-pyridin-3-yl)methyl]-3-[ethyl(oxan -4-yl)amino]-2- methyl-5-[4-(morpholin-4-ylmethyl)phenyl]benzamide

Also known as: EPZ 6438; 1403254-99-8; E-7438; SureCNl 3276848; GTPL7011 ; CS-1758; HY-13803; EPZ-6438 (E7438); KB- 145940

Molecular Formula: C ₃₄ H ₄ N ₄ 0 ₄

Molecular Weight: 572.73756

InChlKey: NSQSAUGJQHDYNO-UHFFFAOYSA-N

Vendor: A Selleckchem (SID 171061163 - External ID: EPZ-6438 (E7438))

E

Knutson SK et al. Nature Chem Biol 2012

IUPAC Name: 1 -cyclopentyl-N-[(4,6-dimethyl-2-oxo- 1 H-pyridin-3-yl)methyl]-6-[4- (morpholin-4-ylmethyl)phenyl]indazole-4-carboxamide

Also known as: 1396772-26-1; EPZ 005687; SureCNl 2684069; CS-1215; HY-15555; KB-

145937; W-6045; S7004,EPZ 005687,1396772-26-1

Molecular Formula: C ₃₂ H ₃₇ N ₅ 0 ₃

Molecular Weight: 539.66788

InChlKey: ZOIBZSZLMJDVDQ-UHFFFAOYSA-N

Vendor: A Selleckchem (SID 164178278 - External ID: EPZ005687)

IUPAC Name: l-[(2S)-butan-2-yl]-N-[(4,6-dimethyl-2-oxo-lH-pyridin-3-yl)m ethyl]-3-methyl- 6-(6-piperazin- 1 -ylpyridin-3 -yl)indole-4-carboxamide

Mccabe MT et al. Nature 2012

Also known as: 1346574-57-9; GSK 126; GSK-126; SureCN12180401 ; GTPL7012; MolPort-

028-600-029; CS-1401 ; QC-9703; NCGC00347286-01

Molecular Formula: C ₃ iH ₃₈ N ₆ 0 ₂

Molecular Weight: 526.67242

InChlKey: FKSFKBQGSFSOSM-QFIPXVFZSA-N

Vendor: Chembo (SID 172821381 - External ID: KB-145928)

G

Amatangelo MD et al. Cell Cycle 2013 IUPAC Name: N-[(6-methyl-2-oxo-4-propyl-lH-pyridin-3-yl)methyl]-6-[2-(4- methylpiperazin- 1 -yl)pyridin-4-yl] - 1 -propan-2-ylindazole-4-carboxamide

Also known as: CHEMBL2204995; GSK 343; 1346704-33-3; SureCN 14716863; CS-1626; HY-13500; KB-145929

Molecular Formula: C ₃₁ H39 ₇ 0 ₂

Molecular Weight: 541.68706

InChlKey: ULNXAWLQFZMIHX-UHFFFAOYSA-N

Vendor: Chembo (SID 172821382 - External ID: KB-145929)

E

Daigle SR et al. Blood 2013

IUPAC Name: (2R,3R,4S,5R)-2-(6-aminopurin-9-yl)-5-[[[3-[2-(6-tert-butyl- lH-benzimidazol- 2-yl)ethyl]cyclobutyl]-propan-2-ylamino]methyl]oxolane-3,4-d iol

Also known as: CHEMBL3087499; EPZ 5676; 1380288-87-8; SureCN9106942;

SureCN9106946; U II-8V9YR09EF3; SureCN 10248515; 8V9YR09EF3

Molecular Formula: C3 ₀ H42NgO ₃

Molecular Weight: 562.70628

InChlKey: LXFOLMYKSYSZQS-XKHGBIBOSA-N

Vendor: A Selleckchem (SID 164178301 - External ID: EPZ-5676) E

Daigle SR et al. Cancer Cell 2011

IUPAC Name: l-[3-[[(2R,3S,4R,5R)-5-(4-aminopyrrolo[2,3-d]pyrimidin-7-yl) -3,4- dihydroxyoxolan-2-yl]methyl-propan-2-ylamino]propyl]-3-(4-te rt-butylphenyl)urea

Also known as: 1338466-77-5; CHEMBL2169919; EPZ-004777

Molecular Formula: C ₂ 8H ₄ iN ₇ 0 ₄

Molecular Weight: 539.66964

InChlKey: WXRGFPHDRFQODR-ICLZECGLSA-N

Vendor: A Selleckchem (SID 164178290 - External ID: EPZ004777)

SGC0946

Yu W et al. Nature Commun 2012

IUPAC Name: l-[3-[[(2R,3S,4R,5R)-5-(4-amino-6-bromopyrrolo[2,3-d]pyrimid in-7-yl)-3,4- dihydroxyoxolan-2-yl]methyl-propan-2-ylamino]propyl]-3-(4-te rt-butylphenyl)urea

Also known as: SGC 0946; S7079,SGC0946,

Molecular Formula: C ₂₈ H ₄ oBrN ₇ 0 ₄

Molecular Weight: 618.5657

InChI ey: CNTIACSNJOOHEX-HUBRGWSESA-N

Vendor: Selleckchem (SID 164178309 - External ID: SGC 0946)

14u

Reference: Liu F et al. J Med Chem 2013

MoL WL: 30536

IUPAC Name: l-Benzo[l ,2,3]thiadiazol-6-yl-3-(2-oxo-2-pyrrolidin-l-yl-ethyl)-urea

Chemical Name: PRMT3-IN-14u; l-Benzo[l ,2,3]thiadiazol-6-yl-3-(2-oxo-2-pyrrolidin-l-yl- ethyl)-urea

Molecular Weight: 305.36

Vendors: Glixx Laboratories (Cat No: GLXC-03626)

17b

Wan H et al. Bioorganic Med Chem Letters 2009 MethylGene

Allan M et al. Bioorg Med Chem Lett 2009

Also known as: compound 7a, CHEMBL475695

IUPAC name: 2-[5-[(2-aminopropanoylamino)methyl]thiophen-2-yl]-N-benzyl- l-heteroaryl-3-

(trifluoromethyl)- 1 H-pyrazole-5-carboxamide

Molecular formula: C20H20F3N5O2S

InChlKey: SZXSSTUGMFYLCV-LBPRGKRZSA-N

17

Wan H et al. Bioorg Med Chem Lett 2009, Sack JS et al. Biochem J 201 1

IUPAC Name: 2-[4-[2-(2,6-dimethoxyphenyl)-7-methyl-3H-benzimidazol-5-yl] piperidin-l-yl]-

N-methylethanamine

Also known as: AGN-PC-079OCP; GTPL7031 ; compound 17f (PMID: 19632837)

Molecular Formula: C ₂ 4H ₃ 2 ₄ 02 Molecular Weight: 408.53648

InChlKey: PFOQIHIIOLBCEQ-UHFFFAOYSA-N

Vendor: Angene Chemical (SID 186787438 - External ID: AGN-PC-079OCP)

Ferguson AD et al. Structure 2011

IUPAC Name: N-cyclohexyl-3-[2-(3,4-dichlorophenyl)ethylamino]-N-[2-[2-(5 -hydroxy-3-oxo- 4H- 1 ,4-benzoxazin-8-yl)ethylamino]ethyl]propanamide

Also known as: AZ505; AZ-505; CHEMBL2169920; 1035227-43-0; AGN-PC-04UPWA;

SureCN3598040; GTPL7021 ; CS-1735; HY-15226; KB-74794

Molecular Formula: C ₂₉ H ₃₈ C1 ₂ N ₄ 04

Molecular Weight: 577.54242

InChlKey: LIB VHXX HS ODII-UHFFF AO Y S A-N

Vendor: Angene Chemical (SID 182564262 - External ID: AGN-PC-04UPWA)

Antagonists of Setdb2 in general including the above exemplary small molecule drugs are believed to exert their antagonizing effect, inter alia, by interfering with/inhibiting the methyltransferase activity of Setdb2. The methyltransferase activity of methyltransferases that are members of the SET-domain protein superfamily (including the SUV39 family to which Setdb2 belongs) is believed to be mediated by the conserved SET-domain. Thus, antagonists of Setdb2 can exert their antagonizing effect by targeting the conserved SET-domain of Setdb2. The term "targeting" refers in this context to the binding to the SET-domain (e.g. by direct compound-protein interaction (like protein-protein-interaction (PPI)) in order to thereby interfere with/inhibit the methyltransferase activity of the SET-domain and/or the inhibition of the methyltransferase activity of the SET-domain by other means (e.g. by depleting the substrate S-adenosylmethionine of the methyltransferases) Therefore, the above exemplary small molecule drugs can be used as antagonists of Setdb2 in accordance with the present invention.

For example, antagonists of Setdb2 can be used herein, wherein said antagonists are competitive inhibitors/competitive antagonists of Setdb2. Such competitive inhibitors/antagonists of Setdb2 are, e.g. substrate analoga (like S-adenosylmethionine (SAM) analoga, such as Sinefungin and S-adenosyl-L-homocysteine. The terms "S- adenosylmethionine" and its abbreviated form "SAM" are used interchangeably herein. These competitive inhibitors/antagonists of Setdb2 bind to Setdb2 (in particular to the SET domain thereof). Thereby, they block the binding of the substrate S-adenosylmethionine (SAM) to Setdb2 (in particular to the conserved SET domain) and inhibit the methyltransferase activity of Setdb2. The use of exemplary inhibitors/antagonists Sinefungin and S-adenosyl-L- homocysteine in accordance with the present invention is demonstrated herein in Example 4 and Fig. 20.

Antagonists of Setdb2 can also exert their antagonizing effect, inter alia, by interfering witlVinhibiting the activity of Setdb2 to act as a scaffold or as a recruiting platform for interaction partners.

It is envisaged herein that compounds can be used as antagonists of Setdb2 that are known in the prior art as inhibitors of methyltransferases, such as S-adenosylmethionine (SAM)- dependent methyltransferases, and/or of members of the SET-domain protein superfamily. It is believed that these known compounds can be used herein, because they target the methyltransferase activity of Setdb2 and/or because they target the conserved SET-domain of Setdb2.

The "SET" domain of Setdb2 consists of a "pre-SET domain" and a "bifurcated SET Domain" (herein designated as "SET1" and "SET2", respectively). The SET domain of Setdb2 (or of related proteins) can be analyzed by appropriate computer programs, like world wide web at ncbi .nlm.rdh.gov/Stracture/cdd/wrpsb . cgi?

An exemplary nucleic acid sequence encoding a SET domain of Setdb2 can comprise the region encoding the "pre-SET domain" ranging from position 1639 to position 1983 of SEQ ID NO: 1, the region encoding SET1 ranging from position 2005 to position 2190 of SEQ ID NO: 1, and/or the region encoding SET2 ranging from position 2794 to position 2994 of SEQ ID NO: 1.

An exemplary amino acid sequence of a SET domain of Setdb2 can comprise the "pre-SET domain" having an amino acid sequence of from position 245 to 359 of SEQ ID NO: 2, SET1 having an amino acid sequence of from position 367 to 428 of SEQ ID NO: 2, and/or SET2 having an amino acid sequence of from position 630 to 696 of of SEQ ID NO: 2.

An exemplary nucleic acid sequence encoding a SET domain of Setdb2 can comprise the region encoding the "pre-SET domain" ranging from position 1039 to position 1383of SEQ ID NO: 3, the region encoding SET1 ranging from position 1405 to position 1590 of SEQ ID NO: 3, and/or the region encoding SET2 ranging from position 2194 to position 2394 of SEQ ID NO: 3.

An exemplary amino acid sequence of a SET domain of Setdb2 can comprise the "pre-SET domain" having an amino acid sequence of from position 233 to 347 of SEQ ID NO: 4, SET1 having an amino acid sequence of from position 355 to 416 of SEQ ID NO: 4, and/or SET2 having an amino acid sequence of from position 618 to 684 of SEQ ID NO: 4.

In certain aspects of the present invention an antagonist of Setdb2 is to be used, wherein said antagonist binds to/inhibits the activity of/targets methyltransferases. In certain aspects of the present invention an antagonist of Setdb2 is to be used, wherein said antagonist binds to/inhibits the activity of/targets SAM-dependent methyltransferases. In certain aspects of the present invention an antagonist of Setdb2 is to be used, wherein said antagonist binds to/inhibits the activity of/targets histone methyltransferases. In certain aspects of the present invention an antagonist of Setdb2 is to be used, wherein said antagonist binds to/inhibits the activity of/targets histone lysine methyltransferases. In certain aspects of the present invention an antagonist of Setdb2 is to be used, wherein said antagonist binds to/inhibits the activity of/targets SAM-dependent histone methyltransferases. In certain aspects of the present invention an antagonist of Setdb2 is to be used, wherein said antagonist binds to/inhibits the activity of/targets SAM-dependent histone lysine methyltransferases.

In certain aspects of the present invention an antagonist of Setdb2 is to be used, wherein said antagonist binds to/inhibits the activity of/targets SET domain-containing proteins. In certain aspects of the present invention an antagonist of Setdb2 is to be used, wherein said antagonist binds to/inhibits the activity of/targets the SET domain of SET domain containing proteins. In certain aspects of the present invention, the antagonist binds to/inhibits the activity of/targets SET domain containing proteins which are methyltransferases. In certain aspects of the present invention, the antagonist binds to/inhibits the activity of/targets SET domain containing proteins which are SAM-dependent methyltransferases. In certain aspects of the present invention, the antagonist binds to/inhibits the activity of/targets SET domain containing proteins which are histone methyltransferases. In certain aspects of the present invention the antagonist binds to/inhibits the activity of/targets SET domain containing proteins which are histone lysine methyltransferases. In certain aspects of the present invention the antagonist binds to SET domain containing proteins of the SUV39 family which are histone lysine methyltransferases. In certain aspects of the present invention, the antagonist binds to/inhibits the activity of/targets SET domain containing proteins which are SAM-dependent histone methyltransferases. In certain aspects of the present invention the antagonist binds to/inhibits the activity of/targets SET domain containing proteins which are SAM-dependent histone lysine methyltransferases. In certain aspects of the present invention the antagonist binds to SET domain containing proteins of the SUV39 family which are SAM-dependent histone lysine methyltransferases.

For example, compounds that broadly inhibit methyltransferases can be used herein as Setdb2 antagonists. Such broad-spectrum or pan-methyltransferase inhibitors are, for example, DZNep, Neplanocin A, CHEMBL61824, CHEMBL468927; or SAM analoga like Sinefungin or S- adenosyl-L-homocysteine (SAH). DOT1L (Gene ID: 84444, Uniprot: Q8TEK3), G9A (Gene ID: 10919, Uniprot: Q96KQ7 ), EZH2 (Gene ID: 2146, Uniprot: Q15910), SUV39H1 (Gene ID: 6839, Uniprot: 043463), PRMT3 (Gene ID: 10196, Uniprot: 060678) and PRMT4 (Gene ID: 10498, Uniprot: Q86X55) are SAM-dependent methyltransferases. DOT1L, G9A, SUV39H1, EZH2, PRMT3 and PRMT4 are SAM-dependent histone methyltransferases. DOT1L G9A, SUV39H1 and EZH2 are SAM-dependent histone lysine methyltransferases (KMTs). PRMT3 and PRMT4 are SAM- dependent histone arginine methyltransferases (PRMTs). G9A, SUV39H1 EZH2, PRMT3 and PRMT4 belong to the SET-domain protein superfamily. DOT1L belongs to the DOT1 family members of which methylate K79 in the globular region of histone H3 and which are structurally not related to SET-domain proteins.

Inhibitors of DOT1L, G9A, SUV39H1 , EZH2, PRMT3 and/or PRMT4 (like the small molecule drugs as defined herein above) can be used as antagonists of Setdb2 in accordance with the present invention.

For example, compounds inhibiting SAM-dependent histone lysine methyltransferases, which do not belong to the SET-domain protein family, like the DOT1 family (e.g. DOT1L), can be used herein as Setdb2 antagonists. Such exemplary inhibitors are EPZ-5676, EPZ004777, SGC0946.

Also compounds that inhibit members of the SET-domain protein superfamily, such as the SUV39-family, and preferably compounds that inhibit members that are closely related to Setdb2, can be used herein as Setdb2 antagonists.

PRMT3 or PRMT4 are SAM-dependent histone arginine methyltransferases and members of SET-domain protein superfamily. For example, compounds inhibiting PRMT3 or PRMT4 can be used herein as Setdb2 antagonists. Exemplary inhibitors are PRMT3 inhibitors like 14u; or PRMT4 inhibitors like 17b, MethylGene and/or 17f.

EZH2 is a SAM-dependent histone lysine methyltransferase and belongs to the EZ family of the SET-domain protein superfamily. For example, compounds inhibiting EZH2 can be used herein as Setdb2 antagonists. Exemplary inhibitors are Ell, U C1999, EPZ-6438, EPZ005687, GSK126, or GSK343.

In a preferred aspect of the present invention the antagonist to be used herein binds to SET domain containing proteins of the SUV39 family which are histone lysine methyltransferases and which are closely related to Setdb2, like G9A. -The phylogenetically closest-related family member of Setdb2 is Setdbl ; see Arrowsmith CH et al. Nat Rev Drug Discov 2012). G9a and SUV39H1 are members of the SUV39 family that are closely related to Setdb2. G9A and SUV39H1 are, like Setdb2, SAM-dependent histone lysine methyltransferases (KMT). Exemplary inhibitors of G9A that can be used herein as antagonists of Setdb2 are BEX01294, UNC0321, U C0638, NC0642, BRD4770 or UNC0224. Also the use of dual G9A/SUV39H1 inhibitors like ΒΓΧ-01338, or Chaetocin is envisaged herein.

SMYD2 is a SAM-dependent histone lysine methyltransferase and belongs to Smyd (SET and MYND domain containing protein) family of the SET-domain protein superfamily. Exemplary inhibitors of SMYD2, like AZ505, can be used in accordance with the present invention.

For example, an antagonist of Setdb2 can be a binding molecule(s), such as be (an) aptamer(s) and/or (an) intramer(s).

It is also envisaged in the present invention that peptides, particularly cyclic peptides can be used as antagonists of Setdb2. Cyclic peptides are polypeptide chains, wherein the amino termini and carboxyl termini, amino termini and side chain, carboxyl termini and side chain, or side chain and side chain are linked with a covalent bond that generates the ring. It is also envisaged herein that biological selection technology, such as phage display is used in order to select peptide ligands tethered to synthetic molecular structures. These peptide ligands show specificity to target Setdb2. In certain preferred aspects of the invention, monomeric monocyclic peptide inhibitors and dimeric bicyclic peptide inhibitors of Setdb2 are used.

Exemplary antagonists of Setdb2 provided and used herein are siRNA, shRNA, miRNA, dsR A, stRNA, or antisense molecule(s). These molecules target a nucleic acid molecule having a sequence encoding Setdb2. The nucleic acid molecule having a sequence encoding Setdb2 is especially mR A as defined herein. Exemplary nucleic acid molecules having a sequence encoding Setdb2 are shown in SEQ ID NOs: 1, 3 and 5. Exemplary mRNA molecules have a sequence corresponding to the sequences shown in SEQ ID NOs: 1, 3 and 5, respectively, with the exception that the thymidine (T) residue(s) of the sequences shown in SEQ ID NOs: 1 , 3 and 5, respectively, is/are replaced by (a) uracil (U) residue(s), if necessary. Also genome-editing techniques like TALEN, Zinc fingers and/or CrispR/Cas9 technique can be used to antagonize Setdb2.

The present invention relates to and provides in particular for the use of (an) siRNA(s) as antagonists of Setdb2, wherein said siRNA(s) specifically target the nucleic acid encoding the Setdb2 protein(s), whereby the nucleic is especially mRNA as defined herein.

Antagonist(s)/inhibitor(s) of Setdb2, which are nucleic acids, such as siRNAs, shRNAs, antisense molecules and the like can readily be prepared by known techniques using, for example, the following target sequences. For example, siRNAs and the like to be employed herein can comprise an RNA sequence corresponding to one of the target sequences further described below. The term "RNA sequence corresponding to" means in this context that the RNA sequence is (partially) identical to one of the target sequences below, if necessary with the exception that the thymidine (T) residues of the target sequence is replaced by a uracil (U) residue. It is understood that siRNAs usually comprise a first strand that is (partially) complementary to the target sequence and a second strand that is (partially) complementary to the first strand (and, hence, (partially) identical to the target sequence).

The siRNA can comprise a nucleic acid molecule comprising at least eight (or ten) contiguous bases. For example, the siRNA and the like can comprise at least eight (or ten) contiguous bases of an RNA sequence corresponding to one of the target sequences as defined herein. The siRNA and the like can comprise at least eight (or ten) contiguous bases of an RNA sequence corresponding to one of the target sequences as defined herein. For example, an antagonizing siRNA to be used herein can comprise at least eight (or ten) contiguous bases of an RNA sequence corresponding to one of the target sequences as shown in the sequence of SEQ ID NOs: 7 or 8. The term "RNA sequence corresponding to" means that the RNA sequence is identical to one of the target sequences as defined herein, if necessary with the exception that the thymidine (T) residues of the target sequence is replaced by a uracil (U) residue.

Up to 10% of the contiguous bases of the herein provided siRNAs can be non-complementary (to the target sequence). The siRNA can further comprise at least one base at the 5' end and/or at least one base at the 3' end of the first strand and/or of the second strand. The siRNA can comprise or consist of an RNA molecule having an RNA sequence corresponding to the target sequences as shown in SEQ ID NOs: 7 and/or an RNA molecule having an RNA sequence (partially) complementary to the sequence as shown in SEQ ID NOs: 7. The siRNA can comprise or consist of an RNA molecule having an RNA sequence corresponding to the target sequences as shown in SEQ ID NOs: 8 and/or an RNA molecule having an RNA sequence (partially) complementary to the sequence as shown in SEQ ID NOs: 8. Preferably, the siRNA consists of an RNA molecule having an RNA sequence corresponding to the target sequences as shown in SEQ ID NOs: 7 and an RNA molecule having an RNA sequence (partially) complementary to the sequence as shown in SEQ ID NOs: 7. Preferably, the siRNA consists of an RNA molecule having an RNA sequence corresponding to the target sequences as shown in SEQ ID NOs: 8 and an RNA molecule having an RNA sequence (partially) complementary to the sequence as shown in SEQ ID NOs: 8.

As shown in appended Example 2, the human embryonic kidney cell line Hek293T cell was used to deplete hSETDB2 by specific siRNAs (Qiagen) (Figure 17). Cells were transfected with a control siRNA, 2 different SETDB2-specific siRNAs (siRNA 1+2), a pool of both siRNAs (siRNA pool), or were left untreated as control (control siRNA). After 24h total RNA was extracted from transfected cells and real-time PCR was used to measure SETDB2 expression. Cells transfected with the siRNAs 1 and 2 as well as the siRNA pool showed strong reduction in SETDB2 expression. These experiments demonstrate the proof of principle that antagonist of Setdb2 can be used in accordance with the present invention in a human setting.

Accordingly, the use of these siRNA(s) alone or in combination is contemplated herein: siRNA 1 :

#SI00141393, Qiagen target sequence: CCGAGAGCATCTGAACTCTAA (SEQ ID NO: 7) siRNA 1 targets SETDB2 at nt position 2481-2501 of a nucleic acid molecule having a sequence encoding Setdb2 as shown in SEQ ID NO: 1. siRNA 1 targets SETDB2 at nt position 1881-1901 of a nucleic acid molecule having a sequence encoding Setdb2 as shown in SEQ ID NO: 3.

and/or siRNA 2:

#SI031 16904, Qiagen

target sequence: TCGGCCGCTTCCTTAATCATA (SEQ ID NO: 8)

siRNA2 targets SETDB2 at nt position 2843-2863 of a nucleic acid molecule having a sequence encoding Setdb2 as shown in SEQ ID NO: 1. siRNA2 targets SETDB2 at nt position 2243-2261 of a nucleic acid molecule having a sequence encoding Setdb2 as shown in SEQ ID NO: 3.

A preferred target sequence of the nucleic acids antagonists as defined above (e.g. siRNA, shRNA, miRNA, dsRNA, stRNA, or antisense molecule targets a nucleic acid molecule having a sequence encoding methyltransferase SET domain bifurcated 2 (Setdb2)) can be selected from the group consisting of

a) a nucleic acid encoding a polypeptide comprising an amino acid sequence as depicted in SEQ ID NO:2 or SEQ ID NO:4;

b) a nucleic acid comprising a nucleotide sequence as depicted in SEQ ID NO: 1 or SEQ ID NO: 3;

c) a nucleic acid hybridizing under stringent conditions to the complementary strand of the nucleic acid as defined in (a) or (b);

d) a nucleic acid comprising a nucleotide sequence with at least 65 % identity to the nucleotide sequence of the nucleic acids of any one of (a) to (c);

e) a nucleic acid comprising a nucleotide sequence which is degenerate as a result of the genetic code to the nucleotide sequence of a nucleic acid of any one of (a) to (d); and f) fragments of the nucleotide sequence of the nucleic acids of any one of (a) to (e). The target sequence to be used herein can be any of the can sequences encoding Setdb2 as defined above, and in particular fragments/portions of (or comprised in) the nucleotide sequence of the nucleic acids of any one of items (a) to (e) above. For example, fragments/portions of a length of 15 to 30, preferably 17 to 25, more preferably 18, 19, 20, 21, 22, 23 or 24 are contemplated herein as target sequence(s).

It is understood that the target Setdb2 molecule for siRNA(s) and other antisense compounds as explained and defined herien is an mRNA molecule encoding Setdb2. An exemplary target Setdb2 mRNA molecule has an RNA sequence corresponding to the sequence as shown in SEQ ID NO. 1 or 3 and related sequences as defined herein. The term "RNA sequence corresponding to" means that the RNA sequence is identical to one of the target sequences as defined herein, if necessary with the exception that the tymidine (T) residues of the target sequence is replaced by a uracil (U) residue.

Particular target sequences (especially for siRNA) are shown in SEQ ID NO. 7 or SEQ ID NO. 8. These target sequences correspond to nt position 2481-2501 of a nucleic acid molecule having a sequence encoding Setdb2 as shown in SEQ ID NO: 1 or nt position 1881-1901 of a nucleic acid molecule having a sequence encoding Setdb2 as shown in SEQ ID NO: 3; and to nt position 2843-2863 of a nucleic acid molecule having a sequence encoding Setdb2 as shown in SEQ ID NO: 1 or nt position 2243-2261 of a nucleic acid molecule having a sequence encoding Setdb2 as shown in SEQ ID NO: 3, respectively.

These sequences represent target sequence of the nucleic acids to be used as antagonists of Setdb2 as defined above (e.g. siRNA and the like, preferably siRNA).

The siRNAs described above can be used alone or in combination with each other as antagonists of Setdb2 in accordance with the present invention.

Usually a strand of an siRNA is of a length of 19 to 21 nt. In case of a complex of sense and antisense strand, each strand of the siRNA is of a length of 19 to 21 bp. For silencing it is crucial that bases from 2 to 8 (seed region) of the siRNA (i.e. in this context of the antisense strand) have perfect base pairing with the target sequence. The antagonist is preferably a selective antagonist of the methyltransferase SET domain bifurcated 2 (Setdb2). For example, the above provided and herein used siRNAs are selective inhibitors of Setdb2.

Selectivity expresses the biologic fact that at a given compound concentration enzymes (or proteins) are affected to different degrees. In the case of enzymes selective inhibition can be defined as preferred inhibition by a compound at a given concentration. Or in other words, an enzyme (or protein) is selectively inhibited over another enzyme (or protein) when there is a concentration, which results in inhibition of the first enzyme (or protein) whereas the second enzyme (or protein) is not, or not substantially, affected. To compare compound effects on different enzymes it is crucial to employ similar assay formats, such as the FRET assay, Plus assay, HMT assays, thermoshift assays, biological readouts (of reporter proteins/enzymes, such as Cxcll/CXCL8), or chemical proteomics.For example, commercially available test kits, like the commercial ELISA kit as used in the experiments herein can be employed (Mouse CXCL1/KC Quantikine ELISA Kit (MKC00B) from R&D Systems.

The inhibitors to be used herein are preferably specific for Setdb2, i.e. the compounds specifically inhibit Setdb2. In other words, the Setdb2 inhibitors/antagonists are preferably selective Setdb2 inhibitors/antagonists.

The term "selective Setdb2 antagonist(s)" as used herein refers to (a) Setdb2 antagonist(s) as defined herein (in particular (a) small molecule drug(s)) that inhibit(s) or display(s) antagonism towards Setdb2 without displaying substantial inhibition or antagonism towards another protein or enzyme, in particular another methyltransferase as defined herein above (e.g. another S- adenosylmethionine (SAM)-dependent methyltransferase (like DOT1L, G9A, EZH2, PRMT3 or PRMT4), another histone methyltransferase, another lysine methyltransferase, another SAM- dependent histone methyltransferase, another SAM-dependent histone lysine methyltransferases, another SET domain containing protein, another member of the SET- domain protein superfamily or another SET domain containing protein of the SUV39 family). Accordingly, an Setdb2 antagonist that is selective for Setdb2 exhibits an Setdb2 selectivity of greater than about 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 50-fold or greater than about 100-fold with respect to inhibition or antagonism of another protein or enzyme (in particular another methyltransferase as defined herein above, like another another S-adenosylmethionine (SAM)-dependent methyltransferase, e.g. DOT1L, G9A, EZH2, PRMT3 or PRMT4)).

For example, pan-methyltransferase inhibitors (i.e. compounds that broadly inhibit substantially any methyltransferase, like Sinefungin or S-adenosyl-L-homocysteine) are not considered herein as selective Setdb2 antagonists.

"Selectivity for Setdb2" can also be determined or defined by IC50 values. For example, the IC50 value of selective Setdb2inhibitors in relation to Setdb2 is low, preferably below 0.2 μΜ, more preferably, below 0.15 μΜ, 0.14 μΜ, 0.13 μΜ, 0.12 μΜ or even lower. More preferably, the IC50 value is below 0.1 μΜ, 0.095 μΜ, 0.090 μΜ, 0.085 μΜ, 0.080 μΜ, 0.075 μΜ, 0.070 μΜ, 0.065 μΜ, 0.060 μΜ, 0.055 μΜ, 0.050 μΜ, 0.045 μΜ, 0.040 μΜ , 0.035 μΜ, 0.030 μΜ, or even below 0.025 μΜ, wherein the lower values are preferred over the higher values. Even more preferably, the IC50 value is below 0.024 μΜ, 0.023 μΜ, 0.022 μΜ, 0.021 μΜ, 0.020 μΜ, 0.019 μΜ, 0.018 μΜ, 0.017 μΜ, 0.016 μΜ, 0.015 μΜ, 0.014 μΜ, 0.013 μΜ, 0.012 μΜ, or 0.011 μΜ. The IC50 value may even be lower, for example, below 0.010 μΜ, 0.009 μΜ, 0.008 μΜ, 0.007 μΜ, 0.006 μΜ, or 0.005 μΜ. Generally, the lower values are preferred herein over the higher values.

Selective Setdb2 inhibitors in accordance with the present invention can, in the alternative, or in addition to the IC50 value in relation to Setdb2, be defined by IC50 value in relation to another protein or enzyme (in particular another methyltransferase as defined herein above, like another another S-adenosylmethionine (SAM)-dependent methyltransferase, e.g. DOT1L, G9A, EZH2, PRMT3 or PRMT4)).

For example, the IC50 value of selective Setdb2 inhibitors in relation to another protein or enzyme (in particular another methyltransferase as defined herein above, like another another S-adenosylmethionine (SAM)-dependent methyltransferase, e.g. DOT1L, G9A, EZH2, PRMT3 or PRMT4)) is high, preferably higher than 0.001 μΜ, 0.002 μΜ, 0.003 μΜ, 0.004 μΜ, 0.005 μΜ, 0.006 μΜ, 0.007 μΜ, 0.008 μΜ, 0.009 μΜ, or 0.010 μΜ. More preferably, the IC50 value is higher than 0.011 μΜ, 0.012 μΜ, 0.013 μΜ, 0.014 μΜ, , 0.015 μΜ, 0.016 μΜ, 0.017 μΜ, 0.018 μΜ, 0.019 μΜ, 0.020 μΜ, 0.021 μΜ, 0.022 μΜ, 0.023 μΜ, or 0.024 μΜ. Even more preferably, the IC50 value is higher than 0.025 μΜ, 0.030 μΜ, 0.035 μΜ, 0.040 μΜ, 0.045 μΜ, 0.050 μΜ, 0.055 μΜ, 0.060 μΜ, 0.065 μΜ, 0.070 μΜ, 0.075 μΜ, 0.080 μΜ, 0.085 μΜ, 0.090 μΜ, 0.095 μΜ, 0.1 μΜ, or even higher, wherein the higher values are preferred over the lower values. Even more preferably, the IC50 value is higher than 0.12 μΜ, 0.13 μΜ, 0.14 μΜ, 0.15 μΜ, 0.2 μΜ or even higher.

Known IC50 values for G9a inhibitors in relation to G9a are e.g. as follows:

BIX01294: IC50 = 180nM

UNC0224: IC50 =43

UNC0638 IC50=81nM

UNC0321 IC50=6nM

U C0646: 1C50: 6 nM

U C0642: IC50=1 10 nM

UNC0123: IC50=230nM

UNC0558: IC50=64nM

SGC A-366 IC50=3nM

The above G9A inhibitors (i.e. one or more of ΒΓΧ01294, UNC0224, UNC0638, U C0321, UNC0646, UNC0642, UNC0123, UNC0558 and/or SGC A-366) can be used as Setdb2 antagonists in accordance with the present invention.

It is envisaged that IC50 value of selective Setdb2 inhibitors in relation to G9A are higher than IC50 values of such known G9A inhibitors, i.e. are higher than, for example, 3 nM, 6 nM, 43 nM, 64 nM, 81 nM, 1 10 nM, 180 nM or 230 nM in relation to G9A.

It is preferred herein that the ratio of IC50 values of selective Setdb2-inhibitors in relation to Setdb2 and IC50 values of another protein or enzyme (in particular another methyltransferase as defined herein above, like another another S-adenosylmethionine (SAM)-dependent methyltransferase, e.g. DOT1L, G9A, EZH2, PRMT3 or PRMT4)) in relation to Setdb2, preferably determined according to the same assay, is about 1 :10 or lower. A ratio of 1:10 or lower also indicates selectivity of the inhibitor for Setdb2. More preferred is a ratio of 1 :10, 1 :20, 1 :30, 1 :40, 1 :50, 1 :60, 1 :70, 1 :80, 1 :90 or 1 :100 or even lower.

Binding molecules are also envisaged herein as antagonists of Setdb2. It is envisaged herein that the binding molecule antagonizing Setdb2 specifically binds to Setdb2 as defined herein. It is envisaged herein that the aptamers/intramers can specifically target/bind to (functional) fragments or (functional) derivatives of the Setdb2 proteins as defined herein, for example also to polypeptides having at least 65% or more identity to herein provided Setdb2 protein(s). Accordingly, the present invention relates to the use of these aptamers/intramers in particular in the therapeutic methods of the present invention.

Inhibitors for use in accordance with the present invention are described and provided herein. Also the use of inhibitors yet to be generated or known compounds to be tested for their inhibiting activity is envisaged in context of the present invention.

Therefore, the present invention provides a method for assessing the activity of a candidate molecule suspected of being an antagonist of Setdb2 as defined and provided herein comprising the steps of:

a) contacting a cell, tissue or a non-human animal comprising methyltransferase SET domain bifurcated 2 (Setdb2) with said candidate molecule;

b) detecting a decrease in activity of said methyltransferase SET domain bifurcated 2 (Setdb2); and

c) selecting a candidate molecule that decreases activity of said methyltransferase SET domain bifurcated 2 (Setdb2).

A decrease of the methyltransferase SET domain bifurcated 2 (Setdb2) activity can indicate the capacity of the selected molecule to antagonize Setdb2.

The activity of Setdb2 can be reflected in e.g. methylation of histones and/or methylation of histone peptides in the presence of S-adenosyl-methionine (SAM), increase of histone marks,cytokine secretion (e.g. Cxcll/CXCL8) or methylation of non-histone proteins and/or methylation of non-histone peptides in the presence of S-adenosyl-methionine (SAM). Such assays are described in more detail further below.

Also a decrease in the (expression) level can indicate useful inhibitors of Setdb2. Accordingly, the term "activity" above can comprise and relate to the "expression level" and vice versa.

The present invention relates to a method for assessing the (expression) level of a candidate molecule suspected of being an antagonist of Setdb2 as defined and provided herein comprising the steps of:

a) contacting a cell, tissue or a non-human animal comprising methyltransferase SET domain bifurcated 2 (Setdb2) with said candidate molecule;

b) detecting a decrease in the (expression) level of said methyltransferase SET domain bifurcated 2 (Setdb2); and c) selecting a candidate molecule that decreases the (expression) level of said methyltransferase SET domain bifurcated 2 (Setdb2).

A decrease of the methyltransferase SET domain bifurcated 2 (Setdb2) (expression) level can indicate the capacity of the selected molecule to antagonize Setdb2.

All definitions and explanations provided herein above, inter alia, in relation to "Setdb2" (and related compounds), "antagonist", "activity" and the like, apply mutatis mutandis in the context of these methods for assessing the activity (or (expression) level) of a candidate molecule suspected of being an antagonist of Setdb2.

The Setdb2 can be any of the Setdb2 proteins/polypeptides as defined herein above or any of the nucleic acids (particularly mRNAs) as defined herein, which encode the Setdb2 proteins/polypeptides.

The following exemplary assays can be used in the determination that a candidate molecule is indeed an antagonist of Setdb2 to be used in accordance with the present invention: assays quantifying specific histone modifications after compound treatment by high throughput microscopy; assays comparing specific histone modifications and production of cytokines by cells either proficient or deficient of Setdb2 expression; and assays screening the activity of purified Setdb2 in the presence or absence of an inhibitor.

Such exemplary assays are described herein below in more detail:

Strategy 1 : Overexpression of Setdb2 in cell culture and quantification of specific histone modifications after compound treatment by high throughput microscopy:

Posttranslational modification of histones, including methylation and acetylation, can be quantified by immunofluorescence using antibodies that are specific for the respective histone mark. Overexpression of histone methyltransferases in cell culture leads to an increase of histone marks that are specific to the respective activity of the overexpressed enzyme. a) Setdb2-overexpression as readout for methyltransferase activity

The abundance of histone marks (e.g. H3K9mel , H3K9me2, H3K9me3, H3K4me3) in cell lines that overexpress human or mouse Setdb2 are quantified by immunofluorescence and compared to wild type cell lines. As a positive control, cell lines overexpressing methyltransferases of the SUV39 family with known activity (e.g. G9a, SUV39H1) are used. Cells are plated in pretreated 384 well plates (Corning, #3904) (5000 cells/well). After 48 h the cells are fixed for 20 min in 1% paraformaldehyde solution and than treated with 0.1% TritonX-100 for 1 h for permeabilization. Afterwards cells are blocked with 2% BSA-PBS-T over night. Thereafter, cells are stained for histone marks: 5 h incubation with primary antibodies (e.g. anti-H3K9me3, anti-H3K4me3) at room temperature. Cells are then washed three times for 10 minutes in PBS-T. Finally, cells are stained with DAPI and fluorescently labeled anti-species conjugates for 1 h at room temperature and washed again three times in PBS-T. Images can be acquired using a high-content microscope Operetta system (PerkinElmer, 20X objective). Nuclei are defined and signal insensitivities are quantified using the Harmony software. Number of nuclei versus relative signal intensity of the respective histone mark are displayed. Results from Setdb2 overexpressing cell lines are compared to the wild type situation. b) Screening for Setdb2-specific inhibitors in a overexpression system

The difference in histone modification between wild type cells and cells overexpressing Setdb2 (compare above) is used to screen for Setdb2-specific antagonists. Wild type as well as Setdb2 overexpressing cell lines are subjected to a 384 well plate based robotic platform. Firstly, a drug library of epigenetic compounds, including histone methyltransferase inhibitors, acetyltransferase inhibitors, and histone deacetylase inhibitors can be screened. In a second step, this system can be used to screen an unbiased library. ECHO acoustic transfer technology can be used to transfer compounds into 384 well tissue cultures. Different concentrations of compounds can also be tested. Quantification of histone marks can be performed as described in a). Histone marks in wild type and Setdb2-overexpressing cell lines are compared in order to identify molecules that show predominant effects specifically after overexpression of Setdb2, but not in wild type cells.

Strategy 2: High throughput comparison of specific histone modifications and cytokine production by cells being proficient or deficient in Setdb2 expression:

In contrast to strategy 1, wild type and Setdb2 knockout cells are used as a model system to screen for Setdb2-specific inhibitors. a) Identification of Setdbl antagonists by immunofluorescence based detection of histone mark alterations:

Wild type and Setdb2 knockout cell lines are plated into 384 well plates and then subjected to our robotic screening platform. Firstly, a drug library of epigenetic compounds can be screened, then, in a second step an unbiased screen can be performed. Treatment of cells can be done as described above. After treatment, cells are fixed and stained for histone marks as described above. This screen identifies compounds that turn the wild type phenotype into a situation observed in Setdb2 knockout cells. b) Identification of Setdb2 antagonists by alteration of cytokine secretion:

As we could show in this invention, Setdb2 is a negative regulator of Cxcll in primary mouse macrophages as well as in lungs of influenza infected mice. Reduced expression of Setdb2 leads to increased production and secretion of the neutrophil chemoattractant Cxcll . In order to study Cxcll secretion of wild type and Setdb2 knockout cells (e.g. primary macrophages), cells can be plated into 384 well plates and subjected to a robotic screening platform as described above. The secretion of Cxcll into the supernatant of the cell culture of treated cells is quantified as readout for Setdb2 activity. Compounds are screened, which increase, for example, the Cxcll expression in wild type cells. These compounds can be identified as Setdb2 antagonists and used in accordance with the invention as Setdb2 antagonists. If compounds are screened, which increase, for example, the Cxcll expression in wild type cells, but not in Setdb2 knockout cells, these compounds can be identified as selective Setdb2 antagonists and used in accordance with the invention as selective Setdb2 antagonists. For the detection of Cxcll ELISA kits (R&D systems) can be used with a robotics platform.

Strategy 3: Specific activity of purified Setdb2 can be tested in methyltransferases assays in the presence and absence of screening compounds:

Purified methyltransferases of the SUV39 family catalyze the methylation of histones and histone peptides in the presence of S-adenosyl-methionine (SAM). A similar assay can be performed with Setdb2:

Setdb2 is purified from different expression systems, including E. coli, insect cell culture and mammalian cell culture. In a biochemical assay, Setdb2 is incubated with the methyl group donor SAM and purified chromatin, purified histones, respectively histone tail peptides, in a suitable buffer allowing peptide methylation. Subsequently, methylated histones and histone tail peptides can be detected by SDS-PAGE and exposure of the dried gel to radiosensitive films (radioactive H ³ -SAM), by mass spectrometry, or western blot analysis using methyl- specific primary antibodies. Small molecules of a library are screened which interfere with Setdb2-specific methyltransferase activity.

The three approaches described above are used to identify antagonist of Setdb2. These candidate molecules can then be tested for the ability to inhibit Setdb2 in vitro and in vivo:

Primary murine macrophages from either wild type or Setdb2 knockout mice are incubated with candidate inhibitors or treated with DMSO as a control. Cells are then stimulated with toll-like receptor agonists, wherein the Cxcll production and secretion is quantified by realtime PCR and ELISA. Functional Setdb2 inhibitors should increase the Cxcll expression in wild type but not in Setdb2 knockout cells.

Cell lines that can be used herein in the screening assays are, inter alia, human cell lines, such as human embryonic kidney cell line HEK293, or human haploid cell lines, like human haploid KMB7 cells, or murine cell lines/cells, such as primary murine macrophages. The cell lines that can be used herein, like the exemplified cell lines above, can be either wild type or Setdb2 knockout/knockdown cells lines (or they can be derived from wild type or Setdb2 knockout/knockdown animals/animal models, like mice or rats).

Strategy 4: Use of Setdb2 inhibitors in animal models

In the mouse model of bacterial superinfecton used herein it is shown that expression of Setdb2 in mice led to susceptibility to bacterial superinfection in the situation of a primary influenza virus infection. Mice expressing reduced amounts of Setdb2 are less susceptible to bacterial superinfections and show reduced pathologies.

Therefore, candidate inhibitors of Setdb2 (e.g. candidate inhibitors fulfilling the criteria of the assays described above) can be used to treat wild-type mice in in vivo animal experiments in order to confirai that the candidate inhibitors/antagonists are indeed useful in the treatment of an infection, such as a viral infection or a bacterial superinfection. The wild-type mice to be used can, for example, be a model for an infection (e.g. a model for bacterial superinfection, like superinfection with Streptococcus pneumoniae upon infection with influenza virus (such as influenza virus A)). For example, wild-type mice can be first infected intranasally with influenza virus (strain A/PR/8/34, or shortly PR8) and subsequently superinfected intranasally with Streptococcus pneumoniae (Sp). These mice represent a model of bacterial superinfection, in particular bacterial superinfection of the lung.

An inhibitor/antagonist of Setdb2 significantly reduces the overall pathological symptoms of infected mice, e.g. pathology in the lungs of infected mice, like a reduced bacterial burden. Antibodies, in particular monoclonal antibodies, that specifically bind to Setdb2 as defined herein can be used in the herein provided screening assays in order to detect the expression level of Setdb2. For example, such antibodies can be used in techniques like global ChlP-seq, imaging/co-localisations, immunoprecipitation to find new interaction partners by mass-spec, and the like. Such antibodies are valuable research tools. Hence, the present application relates in certain aspects to an antibody specifically binding to Setdb2, in particular an antibody specifically binding to murine Setdb2 (e.g. as shown in SEQ ID NO: 6) or a fragment thereof. Preferably, the antibody is a monoclonal antibody.

An exemplary protocol (that is also exemplified in the appended experiments) for the generation of Setdb2-specific monoclonal antibody(ies) can be performed as follows:

A C-terminal 60 amino acid long sequence (amino acid position 541-600 of the amino acid sequence of murine Setdb2, e.g. as shown in SEQ ID NO: 6) can be fused into a hepatitis B carrier protein as immunogen. This region can be amplified by PC and inserted into a 6x histidine-tagged pB-His HBcAg_Linker plasmid ⁵⁶ . The fusion protein can be expressed in E. coli BL21 and purified on 1ml HisTrap HP columns (GE Healthcare) followed by a linear imidazole gradient on an A TA FPLC system (GE Healthcare). The fractions can be analyzed by SDS-Page and concentrated with Amicon Ultra 15-3K Centrifugal Filter Devices (Millipore). The immunization and generation of monoclonal B-cell hybridomas can be performed by challenging Setdb2 mice 3 times (every 2 weeks) with 50μg of purified fusion protein antigen mixed 1 :1 with adjuvant subcutaneously, before a final immunization intravenously with 50μg purified antigen (adjuvant-free). Mouse sera and clone pools can be tested by western blot against overexpressed and endogenous mouse Setdb2.

An exemplary monoclonal antibody provided herein is that of clone 7H7F11 which yielded the best signal-to-noise performance.

The present invention relates, inter alia, to the following aspects.

In certain aspects, the invention relates to an antagonist of the methyltransferase SET domain bifurcated 2 (Setdb2) for use in treating an infection.

In certain aspects, the invention relates to an antagonist of the methyltransferase SET domain bifurcated 2 (Setdb2) for use in treating a bacterial infection.

In certain aspects, the invention relates to an antagonist of the methyltransferase SET domain bifurcated 2 (Setdb2) for use in treating a viral infection.

In certain aspects, the invention relates to an antagonist of the methyltransferase SET domain bifurcated 2 (Setdb2) for use in treating a protozoan infection.

In certain aspects, the invention relates to an antagonist of the methyltransferase SET domain bifurcated 2 (Setdb2) for use in treating a fungal infection.

In certain aspects, the invention relates to an antagonist of the methyltransferase SET domain bifurcated 2 (Setdb2) for use in treating a superinfection.

In preferred aspects, the invention relates to an antagonist of the methyltransferase SET domain bifurcated 2 (Setdb2) for use in treating a bacterial superinfection.

In certain aspects, the invention relates to an antagonist of the methyltransferase SET domain bifurcated 2 (Setdb2) for use in treating a viral superinfection.

In certain aspects, the invention relates to an antagonist of the methyltransferase SET domain bifurcated 2 (Setdb2) for use in treating a protozoan superinfection.

In certain aspects, the invention relates to an antagonist of the methyltransferase SET domain bifurcated 2 (Setdb2) for use in treating a fungal superinfection.

In certain aspects, said bacterial infection is a Streptococcus infection.

In certain aspects, the invention relates to an antagonist of the methyltransferase SET domain bifurcated 2 (Setdb2) for use in treating a bacterial infection, wherein said bacterial infection is a Streptococcus infection.

In preferred aspects, the invention relates to an antagonist of the methyltransferase SET domain bifurcated 2 (Setdb2) for use in treating a bacterial superinfection, wherein said bacterial infection is a Streptococcus infection. In certain aspects, said Streptococcus infection is a Streptococcus pneumoniae infection.

In certain aspects, the invention relates to an antagonist of the methyltransferase SET domain bifurcated 2 (Setdb2) for use in treating a bacterial infection, wherein said bacterial infection is Streptococcus pneumoniae infection.

In preferred aspects, the invention relates to an antagonist of the methyltransferase SET domain bifurcated 2 (Setdb2) for use in treating a bacterial superinfection, wherein said bacterial superinfection is Streptococcus pneumoniae superinfection.

In preferred aspects, the infection is preceded by a viral infection.

In certain aspects, the invention relates to an antagonist of the methyltransferase SET domain bifurcated 2 (Setdb2) for use in treating an infection, wherein the infection is preceded by a viral infection.

In preferred aspects, the invention relates to an antagonist of the methyltransferase SET domain bifurcated 2 (Setdb2) for use in treating a bacterial infection, wherein the infection is preceded by a viral infection.

In certain aspects, the invention relates to an antagonist of the methyltransferase SET domain bifurcated 2 (Setdb2) for use in treating a viral infection, wherein the infection is preceded by a viral infection.

In certain aspects, the invention relates to an antagonist of the methyltransferase SET domain bifurcated 2 (Setdb2) for use in treating a protozoan infection, wherein the infection is preceded by a viral infection.

In certain aspects, the invention relates to an antagonist of the methyltransferase SET domain bifurcated 2 (Setdb2) for use in treating a fungal infection, wherein the infection is preceded by a viral infection.

In preferred aspects, a superinfection is preceded by a viral infection.

In certain aspects, the invention relates to an antagonist of the methyltransferase SET domain bifurcated 2 (Setdb2) for use in treating a superinfection, wherein the infection is preceded by a viral infection.

In preferred aspects, the invention relates to an antagonist of the methyltransferase SET domain bifurcated 2 (Setdb2) for use in treating a bacterial superinfection, wherein the infection is preceded by a viral infection.

In certain aspects, the invention relates to an antagonist of the methyltransferase SET domain bifurcated 2 (Setdb2) for use in treating a viral superinfection, wherein the infection is preceded by a viral infection.

In certain aspects, the invention relates to an antagonist of the methyltransferase SET domain bifurcated 2 (Setdb2) for use in treating a protozoan superinfection, wherein the infection is preceded by a viral infection.

In certain aspects, the invention relates to an antagonist of the methyltransferase SET domain bifurcated 2 (Setdb2) for use in treating a fungal superinfection, wherein the infection is preceded by a viral infection.

In certain aspects, the bacterial infection or superinfection is a Streptococcus infection that is preceded by a viral infection.

In certain aspects, the invention relates to an antagonist of the methyltransferase SET domain bifurcated 2 (Setdb2) for use in treating a bacterial infection, wherein said bacterial infection is a Streptococcus infection, wherein the infection is preceded by a viral infection.

In preferred aspects, the invention relates to an antagonist of the methyltransferase SET domain bifurcated 2 (Setdb2) for use in treating a bacterial superinfection, wherein said bacterial infection is a Streptococcus infection, wherein the infection is preceded by a viral infection. hi certain aspects, said Streptococcus infection is a Streptococcus pneumoniae infection that is preceded by a viral infection.

In certain aspects, the invention relates to an antagonist of the methyltransferase SET domain bifurcated 2 (Setdb2) for use in treating a Streptococcus pneumoniae infection, wherein the infection is preceded by a viral infection. In preferred aspects, a Streptococcus pneumoniae superinfection is preceded by a viral infection.

In preferred aspects, the invention relates to an antagonist of the methyltransferase SET domain bifurcated 2 (Setdb2) for use in treating a Streptococcus pneumoniae superinfection, wherein the infection is preceded by a viral infection.

In preferred aspects, the viral infection or viral superinfection is an influenza virus infection.

In preferred aspects, the invention relates to an antagonist of the methyltransferase SET domain bifurcated 2 (Setdb2) for use in treating a viral infection, wherein said viral infection is an influenza virus infection.

In certain aspects, the invention relates to an antagonist of the methyltransferase SET domain bifurcated 2 (Setdb2) for use in treating a viral superinfection, wherein said viral infection is an influenza virus infection.

In preferred aspects, the infection is preceded by an influenza virus infection.

In certain aspects, the invention relates to an antagonist of the methyltransferase SET domain bifurcated 2 (Setdb2) for use in treating an infection, wherein the infection is preceded by an influenza virus infection.

In preferred aspects, the invention relates to an antagonist of the methyltransferase SET domain bifurcated 2 (Setdb2) for use in treating a bacterial infection, wherein the infection is preceded by an influenza virus viral infection.

In certain aspects, the invention relates to an antagonist of the methyltransferase SET domain bifurcated 2 (Setdb2) for use in treating a viral infection, wherein the infection is preceded by an influenza virus viral infection.

In certain aspects, the invention relates to an antagonist of the methyltransferase SET domain bifurcated 2 (Setdb2) for use in treating a protozoan, wherein the infection is preceded by an influenza virus viral infection.

In certain aspects, the invention relates to an antagonist of the methyltransferase SET domain bifurcated 2 (Setdb2) for use in treating a fungal infection, wherein the infection is preceded by an influenza virus viral infection.

In preferred aspects, a superinfection is preceded by an influenza virus infection.

In certain aspects, the invention relates to an antagonist of the methyltransferase SET domain bifurcated 2 (Setdb2) for use in treating a superinfection, wherein the infection is preceded by an influenza virus infection.

h preferred aspects, the invention relates to an antagonist of the methyltransferase SET domain bifurcated 2 (Setdb2) for use in treating a bacterial superinfection, wherein the infection is preceded by an influenza virus infection.

In certain aspects, the invention relates to an antagonist of the methyltransferase SET domain bifurcated 2 (Setdb2) for use in treating a viral superinfection, wherein the infection is preceded by an influenza virus infection.

In certain aspects, the invention relates to an antagonist of the methyltransferase SET domain bifurcated 2 (Setdb2) for use in treating a protozoan superinfection, wherein the infection is preceded by an influenza virus infection.

In certain aspects, the invention relates to an antagonist of the methyltransferase SET domain bifurcated 2 (Setdb2) for use in treating a fungal superinfection, wherein the infection is preceded by an influenza virus infection.

In preferred aspects, the bacterial infection or superinfection is a Streptococcus infection that is preceded by an influenza virus infection.

In certain aspects, the invention relates to an antagonist of the methyltransferase SET domain bifurcated 2 (Setdb2) for use in treating a bacterial infection, wherein said bacterial infection is a Streptococcus infection, wherein the infection is preceded by an influenza virus infection. In preferred aspects, the invention relates to an antagonist of the methyltransferase SET domain bifurcated 2 (Setdb2) for use in treating a bacterial superinfection, wherein said bacterial infection is a Streptococcus infection, wherein the infection is preceded by an influenza virus infection.

In preferred aspects, said Streptococcus infection is a Streptococcus pneumoniae infection that is preceded by a viral infection.

In preferred aspects, the invention relates to an antagonist of the methyltransferase SET domain bifurcated 2 (Setdb2) for use in treating a Streptococcus pneumoniae infection, wherein the infection is preceded by an influenza virus infection.

In more preferred aspects, a Streptococcus pneumoniae superinfection is preceded by an influenza virus infection.

In more preferred aspects, the invention relates to an antagonist of the methyltransferase SET domain bifurcated 2 (Setdb2) for use in treating a Streptococcus pneumoniae superinfection, wherein the infection is preceded by an influenza virus infection.

In even more preferred aspects, the viral infection is an influenza virus A infection.

In even more preferred aspects, the invention relates to an antagonist of the methyltransferase SET domain bifurcated 2 (Setdb2) for use in treating a viral infection, wherein said viral infection is an influenza virus A infection.

In certain aspects, the invention relates to an antagonist of the methyltransferase SET domain bifurcated 2 (Setdb2) for use in treating a viral superinfection, wherein said viral infection is an influenza virus A infection.

In preferred aspects, the infection is preceded by an influenza virus A infection.

In certain aspects, the invention relates to an antagonist of the methyltransferase SET domain bifurcated 2 (Setdb2) for use in treating an infection, wherein the infection is preceded by an influenza virus A infection.

In preferred aspects, the invention relates to an antagonist of the methyltransferase SET domain bifurcated 2 (Setdb2) for use in treating a bacterial infection, wherein the infection is preceded by an influenza virus A infection.

In certain aspects, the invention relates to an antagonist of the methyltransferase SET domain bifurcated 2 (Setdb2) for use in treating a viral infection, wherein the infection is preceded by an influenza virus viral infection.

In certain aspects, the invention relates to an antagonist of the methyltransferase SET domain bifurcated 2 (Setdb2) for use in treating a protozoan infection, wherein the infection is preceded by an influenza virus A infection.

In certain aspects, the invention relates to an antagonist of the methyltransferase SET domain bifurcated 2 (Setdb2) for use in treating a fungal infection, wherein the infection is preceded by an influenza virus A infection.

In preferred aspects, a superinfection is preceded by an influenza virus A infection.

In certain aspects, the invention relates to an antagonist of the methyltransferase SET domain bifurcated 2 (Setdb2) for use in treating a superinfection, wherein the infection is preceded by an influenza virus A infection.

In preferred aspects, the invention relates to an antagonist of the methyltransferase SET domain bifurcated 2 (Setdb2) for use in treating a bacterial superinfection, wherein the infection is preceded by an influenza virus A infection.

In certain aspects, the invention relates to an antagonist of the methyltransferase SET domain bifurcated 2 (Setdb2) for use in treating a viral superinfection, wherein the infection is preceded by an influenza virus A infection.

In certain aspects, the invention relates to an antagonist of the methyltransferase SET domain bifurcated 2 (Setdb2) for use in treating a protozoan superinfection, wherein the infection is preceded by an influenza virus A infection.

In certain aspects, the invention relates to an antagonist of the methyltransferase SET domain bifurcated 2 (Setdb2) for use in treating a fungal superinfection, wherein the infection is preceded by an influenza virus A infection.

In more preferred aspects, the bacterial infection or superinfection is a Streptococcus infection that is preceded by an influenza virus A infection.

In more preferred aspects, the invention relates to an antagonist of the methyltransferase SET domain bifurcated 2 (Setdb2) for use in treating a bacterial infection, wherein said bacterial infection is a Streptococcus infection, wherein the infection is preceded by an influenza virus A infection.

In even more preferred aspects, the invention relates to an antagonist of the methyltransferase SET domain bifurcated 2 (Setdb2) for use in treating a bacterial superinfection, wherein said bacterial infection is a Streptococcus infection, wherein the infection is preceded by an influenza virus A infection.

In preferred aspects, said Streptococcus infection is a Streptococcus pneumoniae infection that is preceded by an influenza virus A infection.

In preferred aspects, the invention relates to an antagonist of the methyltransferase SET domain bifurcated 2 (Setdb2) for use in treating a Streptococcus pneumoniae infection, wherein the infection is preceded by an influenza virus A infection.

In particularly preferred aspects, a Streptococcus pneumoniae superinfection is preceded by an influenza virus A infection.

In particularly preferred aspects, the invention relates to an antagonist of the methyltransferase SET domain bifurcated 2 (Setdb2) for use in treating a Streptococcus pneumoniae superinfection, wherein the infection is preceded by an influenza virus A infection.

In preferred aspects, the infection is an infection of the lung.

In certain aspects, the invention relates to an antagonist of the methyltransferase SET domain bifurcated 2 (Setdb2) for use in treating an infection of the lung.

In certain aspects, the invention relates to an antagonist of the methyltransferase SET domain bifurcated 2 (Setdb2) for use in treating a bacterial infection of the lung.

In certain aspects, the invention relates to an antagonist of the methyltransferase SET domain bifurcated 2 (Setdb2) for use in treating a viral infection of the lung.

In certain aspects, the invention relates to an antagonist of the methyltransferase SET domain bifurcated 2 (Setdb2) for use in treating a protozoan infection of the lung.

In certain aspects, the invention relates to an antagonist of the methyltransferase SET domain bifurcated 2 (Setdb2) for use in treating a fungal infection of the lung.

In preferred aspects, the invention relates to an antagonist of the methyltransferase SET domain bifurcated 2 (Setdb2) for use in treating a superinfection of the lung.

In certain aspects, the invention relates to an antagonist of the methyltransferase SET domain bifurcated 2 (Setdb2) for use in treating a bacterial superinfection of the lung.

In certain aspects, the invention relates to an antagonist of the methyltransferase SET domain bifurcated 2 (Setdb2) for use in treating a viral superinfection of the lung.

In certain aspects, the invention relates to an antagonist of the methyltransferase SET domain bifurcated 2 (Setdb2) for use in treating a protozoan superinfection of the lung.

In certain aspects, the invention relates to an antagonist of the methyltransferase SET domain bifurcated 2 (Setdb2) for use in treating a fungal superinfection of the lung.

In preferred aspects, said bacterial infection or superinfection is a Streptococcus infection or superinfection of the lung.

In certain aspects, the invention relates to an antagonist of the methyltransferase SET domain bifurcated 2 (Setdb2) for use in treating a bacterial infection, wherein said bacterial infection is a Streptococcus infection of the lung.

In preferred aspects, the invention relates to an antagonist of the methyltransferase SET domain bifurcated 2 (Setdb2) for use in treating a bacterial superinfection, wherein said bacterial infection is a Streptococcus infection of the lung.

In more preferred aspects, said Streptococcus infection or superinfection is a Streptococcus pneumoniae infection or superinfection of the lung.

In certain aspects, the invention relates to an antagonist of the methyltransferase SET domain bifurcated 2 (Setdb2) for use in treating a bacterial infection, wherein said bacterial infection is Streptococcus pneumoniae infection of the lung.

In more preferred aspects, the invention relates to an antagonist of the methyltransferase SET domain bifurcated 2 (Setdb2) for use in treating a bacterial superinfection, wherein said bacterial superinfection is Streptococcus pneumoniae superinfection of the lung.

In preferred aspects, the infection of the lung is preceded by a viral infection of the lung.

In certain aspects, the invention relates to an antagonist of the methyltransferase SET domain bifurcated 2 (Setdb2) for use in treating an infection of the lung, wherein the infection is preceded by a viral infection of the lung.

In preferred aspects, the invention relates to an antagonist of the methyltransferase SET domain bifurcated 2 (Setdb2) for use in treating a bacterial infection of the lung, wherein the infection is preceded by a viral infection of the lung.

In certain aspects, the invention relates to an antagonist of the methyltransferase SET domain bifurcated 2 (Setdb2) for use in treating a viral infection of the lung, wherein the infection is preceded by a viral infection of the lung.

In certain aspects, the invention relates to an antagonist of the methyltransferase SET domain bifurcated 2 (Setdb2) for use in treating a protozoan infection of the lung, wherein the infection is preceded by a viral infection of the lung.

In certain aspects, the invention relates to an antagonist of the methyltransferase SET domain bifurcated 2 (Setdb2) for use in treating a fungal infection of the lung, wherein the infection is preceded by a viral infection of the lung.

In certain aspects, the infection is preceded by a viral infection of the lung.

In certain aspects, the invention relates to an antagonist of the methyltransferase SET domain bifurcated 2 (Setdb2) for use in treating an infection, wherein the infection is preceded by a viral infection of the lung.

In certain aspects, the invention relates to an antagonist of the methyltransferase SET domain bifurcated 2 (Setdb2) for use in treating a bacterial infection, wherein the infection is preceded by a viral infection of the lung.

In certain aspects, the invention relates to an antagonist of the methyltransferase SET domain bifurcated 2 (Setdb2) for use in treating a viral infection, wherein the infection is preceded by a viral infection of the lung.

In certain aspects, the invention relates to an antagonist of the methyltransferase SET domain bifurcated 2 (Setdb2) for use in treating a protozoan infection, wherein the infection is preceded by a viral infection of the lung.

In certain aspects, the invention relates to an antagonist of the methyltransferase SET domain bifurcated 2 (Setdb2) for use in treating a fungal infection, wherein the infection is preceded by a viral infection of the lung.

In preferred aspects, a superinfection of the lung is preceded by a viral infection of the lung.

In certain aspects, the invention relates to an antagonist of the methyltransferase SET domain bifurcated 2 (Setdb2) for use in treating a superinfection of the lung, wherein the infection is preceded by a viral infection of the lung.

In certain aspects, the invention relates to an antagonist of the methyltransferase SET domain bifurcated 2 (Setdb2) for use in treating a bacterial superinfection of the lung, wherein the infection is preceded by a viral infection of the lung.

In certain aspects, the invention relates to an antagonist of the methyltransferase SET domain bifurcated 2 (Setdb2) for use in treating a viral superinfection of the lung, wherein the infection is preceded by a viral infection of the lung.

In certain aspects, the invention relates to an antagonist of the methyltransferase SET domain bifurcated 2 (Setdb2) for use in treating a protozoan superinfection of the lung, wherein the infection is preceded by a viral infection of the lung.

In certain aspects, the invention relates to an antagonist of the methyltransferase SET domain bifurcated 2 (Setdb2) for use in treating a fungal superinfection of the lung, wherein the infection is preceded by a viral infection of the lung.

In certain aspects, a superinfection is preceded by a viral infection of the lung.

In certain aspects, the invention relates to an antagonist of the methyltransferase SET domain bifurcated 2 (Setdb2) for use in treating a superinfection, wherein the infection is preceded by a viral infection of the lung.

In certain aspects, the invention relates to an antagonist of the methyltransferase SET domain bifurcated 2 (Setdb2) for use in treating a bacterial superinfection, wherein the infection is preceded by a viral infection of the lung. In certain aspects, the invention relates to an antagonist of the methyltransferase SET domain bifurcated 2 (Setdb2) for use in treating a viral superinfection, wherein the infection is preceded by a viral infection of the lung.

In certain aspects, the invention relates to an antagonist of the methyltransferase SET domain bifurcated 2 (Setdb2) for use in treating a protozoan superinfection, wherein the infection is preceded by a viral infection of the lung.

In certain aspects, the invention relates to an antagonist of the methyltransferase SET domain bifurcated 2 (Setdb2) for use in treating a fungal superinfection, wherein the infection is preceded by a viral infection of the lung.

In preferred aspects, the bacterial infection or superinfection of the lung is a Streptococcus infection of the lung that is preceded by a viral infection of the lung.

In certain aspects, the invention relates to an antagonist of the methyltransferase SET domain bifurcated 2 (Setdb2) for use in treating a bacterial infection of the lung, wherein said bacterial infection is a Streptococcus infection, wherein the infection is preceded by a viral infection of the lung.

In preferred aspects, the invention relates to an antagonist of the methyltransferase SET domain bifurcated 2 (Setdb2) for use in treating a bacterial superinfection of the lung, wherein said bacterial infection is a Streptococcus infection, wherein the infection is preceded by a viral infection of the lung.

In certain aspects, the bacterial infection or superinfection is a Streptococcus infection that is preceded by a viral infection of the lung.

In certain aspects, the invention relates to an antagonist of the methyltransferase SET domain bifurcated 2 (Setdb2) for use in treating a bacterial infection, wherein said bacterial infection is a Streptococcus infection, wherein the infection is preceded by a viral infection of the lung. In certain aspects, the invention relates to an antagonist of the methyltransferase SET domain bifurcated 2 (Setdb2) for use in treating a bacterial superinfection, wherein said bacterial infection is a Streptococcus infection, wherein the infection is preceded by a viral infection of the lung. In preferred aspects, said Streptococcus infection is a Streptococcus pneumoniae infection of the lung that is preceded by a viral infection of the lung.

In preferred aspects, the invention relates to an antagonist of the methyltransferase SET domain bifurcated 2 (Setdb2) for use in treating a Streptococcus pneumoniae infection of the lung, wherein the infection is preceded by a viral infection of the lung.

In certain aspects, said Streptococcus infection is a Streptococcus pneumoniae infection that is preceded by a viral infection of the lung.

In certain aspects, the invention relates to an antagonist of the methyltransferase SET domain bifurcated 2 (Setdb2) for use in treating a Streptococcus pneumoniae infection, wherein the infection is preceded by a viral infection of the lung.

In more preferred aspects, a Streptococcus pneumoniae superinfection of the lung is preceded by a viral infection of the lung.

In more preferred aspects, the invention relates to an antagonist of the methyltransferase SET domain bifurcated 2 (Setdb2) for use in treating a Streptococcus pneumoniae superinfection of the lung, wherein the infection is preceded by a viral infection of the lung.

In certain aspects, a Streptococcus pneumoniae superinfection is preceded by a viral infection of the lung.

In certain aspects, the invention relates to an antagonist of the methyltransferase SET domain bifurcated 2 (Setdb2) for use in treating a Streptococcus pneumoniae superinfection, wherein the infection is preceded by a viral infection of the lung.

In preferred aspects, said viral infection or viral superinfection is an influenza virus infection of the lung. In certain aspects, the invention relates to an antagonist of the methyltransferase SET domain bifurcated 2 (Setdb2) for use in treating a viral infection, wherein said viral infection is an influenza virus infection of the lung.

In certain aspects, the invention relates to an antagonist of the methyltransferase SET domain bifurcated 2 (Setdb2) for use in treating a viral superinfection, wherein said viral infection is an influenza virus infection of the lung.

In certain aspects, the infection of the lung is preceded by an influenza virus infection of the lung.

In certain aspects, the invention relates to an antagonist of the methyltransferase SET domain bifurcated 2 (Setdb2) for use in treating an infection of the lung, wherein the infection is preceded by an influenza virus infection of the lung.

In preferred aspects, the invention relates to an antagonist of the methyltransferase SET domain bifurcated 2 (Setdb2) for use in treating a bacterial infection of the lung, wherein the infection is preceded by an influenza virus viral infection of the lung.

In certain aspects, the invention relates to an antagonist of the methyltransferase SET domain bifurcated 2 (Setdb2) for use in treating a viral infection of the lung, wherein the infection is preceded by an influenza virus viral infection of the lung.

In certain aspects, the invention relates to an antagonist of the methyltransferase SET domain bifurcated 2 (Setdb2) for use in treating a protozoan infection of the lung , wherein the infection is preceded by an influenza virus viral infection of the lung.

In certain aspects, the invention relates to an antagonist of the methyltransferase SET domain bifurcated 2 (Setdb2) for use in treating a fungal infection of the lung, wherein the infection is preceded by an influenza virus viral infection of the lung.

In certain aspects, the infection is preceded by an influenza virus infection of the lung.

In certain aspects, the invention relates to an antagonist of the methyltransferase SET domain bifurcated 2 (Setdb2) for use in treating an infection, wherein the infection is preceded by an influenza virus infection of the lung.

In preferred aspects, the invention relates to an antagonist of the methyltransferase SET domain bifurcated 2 (Setdb2) for use in treating a bacterial infection, wherein the infection is preceded by an influenza virus viral infection of the lung.

In certain aspects, the invention relates to an antagonist of the methyltransferase SET domain bifurcated 2 (Setdb2) for use in treating a viral infection, wherein the infection is preceded by an influenza virus viral infection of the lung.

In certain aspects, the invention relates to an antagonist of the methyltransferase SET domain bifurcated 2 (Setdb2) for use in treating a protozoan infection, wherein the infection is preceded by an influenza virus viral infection of the lung.

In certain aspects, the invention relates to an antagonist of the methyltransferase SET domain bifurcated 2 (Setdb2) for use in treating a fungal infection, wherein the infection is preceded by an influenza virus viral infection of the lung.

In preferred aspects, a superinfection of the lung is preceded by a viral infection of the lung.

In certain aspects, the invention relates to an antagonist of the methyltransferase SET domain bifurcated 2 (Setdb2) for use in treating a superinfection of the lung, wherein the infection is preceded by an influenza virus infection of the lung.

In preferred aspects, the invention relates to an antagonist of the methyltransferase SET domain bifurcated 2 (Setdb2) for use in treating a bacterial superinfection of the lung, wherein the infection is preceded by an influenza virus infection of the lung.

In certain aspects, the invention relates to an antagonist of the methyltransferase SET domain bifurcated 2 (Setdb2) for use in treating a viral superinfection of the lung, wherein the infection is preceded by an influenza virus infection of the lung.

In certain aspects, the invention relates to an antagonist of the methyltransferase SET domain bifurcated 2 (Setdb2) for use in treating a protozoan superinfection of the lung, wherein the infection is preceded by an influenza virus infection of the lung.

In certain aspects, the invention relates to an antagonist of the methyltransferase SET domain bifurcated 2 (Setdb2) for use in treating a fungal superinfection of the lung, wherein the infection is preceded by an influenza virus infection of the lung.

In certain aspects, a superinfection is preceded by a viral infection of the lung. In certain aspects, the invention relates to an antagonist of the methyltransferase SET domain bifurcated 2 (Setdb2) for use in treating a superinfection, wherein the infection is preceded by an influenza virus infection of the lung.

In certain aspects, the invention relates to an antagonist of the methyltransferase SET domain bifurcated 2 (Setdb2) for use in treating a bacterial superinfection, wherein the infection is preceded by an influenza virus infection of the lung.

In certain aspects, the invention relates to an antagonist of the methyltransferase SET domain bifurcated 2 (Setdb2) for use in treating a viral superinfection, wherein the infection is preceded by an influenza virus infection of the lung.

In certain aspects, the invention relates to an antagonist of the methyltransferase SET domain bifurcated 2 (Setdb2) for use in treating a protozoan superinfection, wherein the infection is preceded by an influenza virus infection of the lung.

In certain aspects, the invention relates to an antagonist of the methyltransferase SET domain bifurcated 2 (Setdb2) for use in treating a fungal superinfection, wherein the infection is preceded by an influenza virus infection of the lung.

In preferred aspects, the bacterial infection or superinfection of the lung is a Streptococcus infection of the lung that is preceded by an influenza virus infection of the lung.

In certain aspects, the invention relates to an antagonist of the methyltransferase SET domain bifurcated 2 (Setdb2) for use in treating a bacterial infection of the lung, wherein said bacterial infection is a Streptococcus infection, wherein the infection is preceded by an influenza virus infection of the lung.

In preferred aspects, the invention relates to an antagonist of the methyltransferase SET domain bifurcated 2 (Setdb2) for use in treating a bacterial superinfection of the lung, wherein said bacterial infection is a Streptococcus infection, wherein the infection is preceded by an influenza virus infection of the lung.

In preferred aspects, the bacterial infection or superinfection is a Streptococcus infection that is preceded by an influenza virus infection of the lung.

In certain aspects, the invention relates to an antagonist of the methyltransferase SET domain bifurcated 2 (Setdb2) for use in treating a bacterial infection, wherein said bacterial infection is a Streptococcus infection, wherein the infection is preceded by an influenza virus infection of the lung.

In certain aspects, the invention relates to an antagonist of the methyltransferase SET domain bifurcated 2 (Setdb2) for use in treating a bacterial superinfection, wherein said bacterial infection is a Streptococcus infection, wherein the infection is preceded by an influenza virus infection of the lung.

In preferred aspects, said Streptococcus infection is a Streptococcus pneumoniae infection of the lung that is preceded by a viral infection of the lung.

In preferred aspects, the invention relates to an antagonist of the methyltransferase SET domain bifurcated 2 (Setdb2) for use in treating a Streptococcus pneumoniae infection of the lung, wherein the infection is preceded by an influenza virus infection of the lung.

In certain aspects, said Streptococcus infection is a Streptococcus pneumoniae infection that is preceded by a viral infection of the lung.

In certain aspects, the invention relates to an antagonist of the methyltransferase SET domain bifurcated 2 (Setdb2) for use in treating a Streptococcus pneumoniae infection, wherein the infection is preceded by an influenza virus infection of the lung.

In preferred aspects, a Streptococcus pneumoniae superinfection of the lung is preceded by an influenza virus infection of the lung.

In preferred aspects, the invention relates to an antagonist of the methyltransferase SET domain bifurcated 2 (Setdb2) for use in treating a Streptococcus pneumoniae superinfection of the lung, wherein the infection is preceded by an influenza virus infection of the lung.

In certain aspects, a Streptococcus pneumoniae superinfection is preceded by an influenza virus infection of the lung. In certain aspects, the invention relates to an antagonist of the methyltransferase SET domain bifurcated 2 (Setdb2) for use in treating a Streptococcus pneumoniae superinfection, wherein the infection is preceded by an influenza virus infection of the lung.

In preferred aspects, said viral infection is an influenza virus A infection of the lung.

In certain aspects, the invention relates to an antagonist of the methyltransferase SET domain bifurcated 2 (Setdb2) for use in treating a viral infection, wherein said viral infection is an influenza virus A infection of the lung.

In certain aspects, the invention relates to an antagonist of the methyltransferase SET domain bifurcated 2 (Setdb2) for use in treating a viral superinfection, wherein said viral infection is an influenza virus A infection of the lung.

In preferred aspects, the infection of the lung is preceded by an influenza virus A infection of the lung.

In certain aspects, the invention relates to an antagonist of the methyltransferase SET domain bifurcated 2 (Setdb2) for use in treating an infection of the lung, wherein the infection is preceded by an influenza virus A infection of the lung.

In preferred aspects, the invention relates to an antagonist of the methyltransferase SET domain bifurcated 2 (Setdb2) for use in treating a bacterial infection of the lung, wherein the infection is preceded by an influenza virus A infection of the lung.

In certain aspects, the invention relates to an antagonist of the methyltransferase SET domain bifurcated 2 (Setdb2) for use in treating a viral infection of the lung, wherein the infection is preceded by an influenza virus A infection of the lung.

In certain aspects, the invention relates to an antagonist of the methyltransferase SET domain bifurcated 2 (Setdb2) for use in treating a protozoan infection of the lung, wherein the infection is preceded by an influenza virus A infection of the lung.

In certain aspects, the invention relates to an antagonist of the methyltransferase SET domain bifurcated 2 (Setdb2) for use in treating a fungal infection of the lung, wherein the infection is preceded by an influenza virus A infection of the lung. In preferred aspects, the infection is preceded by an influenza virus A infection of the lung.

In certain aspects, the invention relates to an antagonist of the methyltransferase SET domain bifurcated 2 (Setdb2) for use in treating an infection, wherein the infection is preceded by an influenza virus A infection of the lung.

In preferred aspects, the invention relates to an antagonist of the methyltransferase SET domain bifurcated 2 (Setdb2) for use in treating a bacterial infection, wherein the infection is preceded by an influenza virus A infection of the lung.

In certain aspects, the invention relates to an antagonist of the methyltransferase SET domain bifurcated 2 (Setdb2) for use in treating a viral infection, wherein the infection is preceded by an influenza virus A infection of the lung.

In certain aspects, the invention relates to an antagonist of the methyltransferase SET domain bifurcated 2 (Setdb2) for use in treating a protozoan infection, wherein the infection is preceded by an influenza virus A infection of the lung.

In certain aspects, the invention relates to an antagonist of the methyltransferase SET domain bifurcated 2 (Setdb2) for use in treating a fungal infection, wherein the infection is preceded by an influenza virus A infection of the lung.

In preferred aspects, a superinfection of the lung is preceded by an influenza virus A infection of the lung.

In certain aspects, the invention relates to an antagonist of the methyltransferase SET domain bifurcated 2 (Setdb2) for use in treating a superinfection of the lung, wherein the infection is preceded by an influenza virus A infection of the lung.

In preferred aspects, the invention relates to an antagonist of the methyltransferase SET domain bifurcated 2 (Setdb2) for use in treating a bacterial superinfection of the lung, wherein the infection is preceded by an influenza virus A infection of the lung.

In certain aspects, the invention relates to an antagonist of the methyltransferase SET domain bifurcated 2 (Setdb2) for use in treating a viral superinfection of the lung, wherein the infection is preceded by an influenza virus A infection of the lung.

In certain aspects, the invention relates to an antagonist of the methyltransferase SET domain bifurcated 2 (Setdb2) for use in treating a protozoan superinfection of the lung, wherein the infection is preceded by an influenza virus A infection of the lung.

In certain aspects, the invention relates to an antagonist of the methyltransferase SET domain bifurcated 2 (Setdb2) for use in treating a fungal superinfection of the lung, wherein the infection is preceded by an influenza virus A infection of the lung.

In certain aspects, a superinfection is preceded by an influenza virus A infection of the lung.

In certain aspects, the invention relates to an antagonist of the methyltransferase SET domain bifurcated 2 (Setdb2) for use in treating a superinfection, wherein the infection is preceded by an influenza virus A infection of the lung.

In certain aspects, the invention relates to an antagonist of the methyltransferase SET domain bifurcated 2 (Setdb2) for use in treating a bacterial superinfection, wherein the infection is preceded by an influenza virus A infection of the lung.

In certain aspects, the invention relates to an antagonist of the methyltransferase SET domain bifurcated 2 (Setdb2) for use in treating a viral superinfection, wherein the infection is preceded by an influenza virus A infection of the lung.

In certain aspects, the invention relates to an antagonist of the methyltransferase SET domain bifurcated 2 (Setdb2) for use in treating a protozoan superinfection, wherein the infection is preceded by an influenza virus A infection of the lung.

In certain aspects, the invention relates to an antagonist of the methyltransferase SET domain bifurcated 2 (Setdb2) for use in treating a fungal superinfection, wherein the infection is preceded by an influenza virus A infection of the lung.

In preferred aspects, the bacterial infection or superinfection of the lung is a Streptococcus infection of the lung that is preceded by an influenza virus A infection of the lung.

In certain aspects, the invention relates to an antagonist of the methyltransferase SET domain bifurcated 2 (Setdb2) for use in treating a bacterial infection of the lung, wherein said bacterial infection is a Streptococcus infection, wherein the infection is preceded by an influenza virus A infection of the lung.

In preferred aspects, the invention relates to an antagonist of the methyltransferase SET domain bifurcated 2 (Setdb2) for use in treating a bacterial superinfection of the lung, wherein said bacterial infection is a Streptococcus infection, wherein the infection is preceded by an influenza virus A infection of the lung.

In preferred aspects, the bacterial infection or superinfection is a Streptococcus infection that is preceded by an influenza virus A infection of the lung.

In certain aspects, the invention relates to an antagonist of the methyltransferase SET domain bifurcated 2 (Setdb2) for use in treating a bacterial infection, wherein said bacterial infection is a Streptococcus infection, wherein the infection is preceded by an influenza virus A infection of the lung.

In preferred aspects, the invention relates to an antagonist of the methyltransferase SET domain bifurcated 2 (Setdb2) for use in treating a bacterial superinfection, wherein said bacterial infection is a Streptococcus infection, wherein the infection is preceded by an influenza virus A infection of the lung.

In preferred aspects, said Streptococcus infection is a Streptococcus pneumoniae infection of the lung that is preceded by a influenza virus A infection of the lung.

In preferred aspects, the invention relates to an antagonist of the methyltransferase SET domain bifurcated 2 (Setdb2) for use in treating a Streptococcus pneumoniae infection of the lung, wherein the infection is preceded by an influenza virus A infection of the lung.

In preferred aspects, said Streptococcus infection is a Streptococcus pneumoniae infection of the lung that is preceded by a influenza virus A infection of the lung.

In preferred aspects, the invention relates to an antagonist of the methyltransferase SET domain bifurcated 2 (Setdb2) for use in treating a Streptococcus pneumoniae infection of the lung, wherein the infection is preceded by an influenza virus A infection of the lung.

In preferred aspects, said Streptococcus infection is a Streptococcus pneumoniae infection that is preceded by a influenza virus A infection of the lung. In preferred aspects, the invention relates to an antagonist of the methyltransferase SET domain bifurcated 2 (Setdb2) for use in treating a Streptococcus pneumoniae infection, wherein the infection is preceded by an influenza virus A infection of the lung.

In particularly preferred aspects, a Streptococcus pneumoniae superinfection of the lung is preceded by an influenza virus A infection of the lung.

In particularly preferred aspects, the invention relates to an antagonist of the methyltransferase SET domain bifurcated 2 (Setdb2) for use in treating a Streptococcus pneumoniae superinfection of the lung, wherein the infection is preceded by an influenza virus A infection of the lung.

In preferred aspects, a Streptococcus pneumoniae superinfection is preceded by an influenza virus A infection of the lung.

In particularly preferred aspects, the invention relates to an antagonist of the methyltransferase SET domain bifurcated 2 (Setdb2) for use in treating a Streptococcus pneumoniae superinfection, wherein the infection is preceded by an influenza virus A infection of the lung.

The antagonist of the methyltransferase SET domain bifurcated 2 (Setdb2) for use in treating an infection or the method for treating an infection comprising the administration of an antagonist of Setdb2. The antagonist of Setdb2 can be of use to treat infections, infectious disease or superinfections. A superinfection is generally defined as a second infection superimposed on a previous one.

The bacterial infection or superinfection to be treated in accordance with the present invention can, for example, be a Streptococcus infection (e.g. Streptococcus pneumoniae or Streptococcus pyogenes, wheien Streptococcus pneumoniae is preferred), a Staphylococcus infection (e.g. Staphylococcus aureus), a Haemophilus infection (e.g. Haemophilus influenza), a Mycobacterium infection (e.g. Mycobacterium tuberculosis), a Moraxella infection (e.g. Moraxella catarrhalis), a Pseudomonas infection (e.g. Pseudomonas aeruginosa), a Escherichia infection (e.g. Escherichia coli), a Yersinia infection (e.g. Yersinia enterocolitica), a Treponema infection (e.g. Treponema pallidum), a Shigella infection (e.g. Shigella flexneri), a Salmonella infection (e.g. Salmonella typhimurium), a Rhodococcus infection (e.g. Rhodococcus equi), a Nocardia infection (e.g. Nocardia asteroides), a Campylobacter infection (e.g. Campylobacter jejuni) and a Clostridium infection (e.g. Clostridium difficile).

In certain aspects the present invention relates to an antagonist of the methyltransferase SET domain bifurcated 2 (Setdb2) for use in treating a bacterial infection or bacterial superinfection, for example a Streptococcus infection (e.g. Streptococcus pneumoniae or Streptococcus pyogenes), a Staphylococcus infection (e.g. Staphylococcus aureus), a Haemophilus infection (e.g. Haemophilus influenza), a Mycobacterium infection (e.g. Mycobacterium tuberculosis), a Moraxella infection (e.g. Moraxella catarrhalis), a Pseudomonas infection (e.g. Pseudomonas aeruginosa), a Escherichia infection (e.g. Escherichia coli), a Yersinia infection (e.g. Yersinia enterocolitica), a Treponema infection (e.g. Treponema pallidum), a Shigella infection (e.g. Shigella flexneri), a Salmonella infection (e.g. Salmonella typhimurium), a Rhodococcus infection (e.g. Rhodococcus equi), a Nocardia infection (e.g. Nocardia asteroides), a Campylobacter infection (e.g. Campylobacter jejuni) and/or a Clostridium infection (e.g. Clostridium difficile).

The protozoan infection or superinfection to be treated in accordance with the present invention can, for example, be a Toxoplasma infection (e.g. Toxoplasma gondii), a Leishmania infection (e.g. Leishmania infantum), an Isospora infection (e.g. Isospora belli) and/or a Plasmodium infection (e.g. Plasmodium falciparum).

In certain aspects the present invention relates to an antagonist of the methyltransferase SET domain bifurcated 2 (Setdb2) for use in treating a protozoan infection, for example a Toxoplasma infection (e.g. Toxoplasma gondii), a Leishmania infection (e.g. Leishmania infantum), an Isospora infection (e.g. Isospora belli) and a Plasmodium infection (e.g. Plasmodium falciparum).

The fungal infection or superinfection to be treated in accordance with the present invention can, for example, be a Candida infection, a Microsporidia infection, a Aspergillus infection, a Scedosporium infection, a Mucor infection, a Cryptococcus infection, a Coccidioides infection, a Histoplasma infection and a Pneumocystis infection.

In certain aspects the present invention relates to an antagonist of the methyltransferase SET domain bifurcated 2 (Setdb2) for use in treating a fungal infection, for example a Candida infection, a Microsporidia infection, a Aspergillus infection, a Scedosporium infection, a Mucor infection, a Cryptococcus infection, a Coccidioides infection, a Histoplasma infection and/or a Pneumocystis infection.

The infection or superinfection to be treated in accordance with the present invention can be preceded by a viral infection, for example, an orthomyxovirus infection (e.g. influenza virus), a herpesvirus infection (e.g. herpes simplex virus 1 (HSV-1), a cytomegalovirus (CMV) or a Epstein-Barr virus (EBV)), a hepadnavirus infection (e.g. hepatitis B virus (HBV)), a flavivirus infection (e.g. hepatitis C virus (HCV)), a lentivirus infection (e.g. human immunodeficiency virus (HIV) 1 or a human immunodeficiency virus (HIV) 2), a retrovirus infection (e.g. human T cell lymphotropic virus (HTLV)), an arenavirus infection (e.g. lassa virus (LASV) or a lymphocytic choriomeningitis virus (LCMV)) and a paramyxovirus infection (e.g. measles virus). Preferably, said viral infection is an influenza virus infection, particularly preferred an influenza virus A infection.

In certain aspects the present invention relates to an antagonist of the methyltransferase SET domain bifurcated 2 (Setdb2) for use in treating an infection, wherein said infection is preceded by a viral infection. In a preferred embodiment of the invention is said viral infection an influenza virus infection, particularly preferred an influenza virus A infection.

The viral infection or superinfection to be treated in accordance with the present invention can, for example, be an orthomyxovirus infection (e.g. influenza virus), a herpesvirus infection (e.g. herpes simplex virus 1 (HSV-1), a cytomegalovirus (CMV) or a Epstein-Barr virus (EBV)), a hepadnavirus infection (e.g. hepatitis B virus (HBV)), a flavivirus infection (e.g. hepatitis C virus (HCV)), a lentivirus infection (e.g. human immunodeficiency virus (HIV) 1 or a human immunodeficiency virus (HIV) 2), a retrovirus infection (e.g. human T cell lymphotropic virus (HTLV)), an arenavirus infection (e.g. lassa virus (LASV) or a lymphocytic choriomeningitis virus (LCMV)) and a paramyxovirus infection (e.g. measles virus). Preferably, said viral infection an influenza virus infection, particularly preferred an influenza virus A infection.

In certain aspects the present invention relates to an antagonist of the methyltransferase SET domain bifurcated 2 (Setdb2) for use in treating a viral infection, for example an orthomyxovirus infection (e.g. influenza virus), a herpesvirus infection (e.g. herpes simplex virus 1 (HSV-1), a cytomegalovirus (CMV) or a Epstein-Barr virus (EBV)), a hepadnavirus infection (e.g. hepatitis B virus (HBV)), a flavivirus infection (e.g. hepatitis C virus (HCV)), a lentivirus infection (e.g. human immunodeficiency virus (HIV) 1 or a human immunodeficiency virus (HIV) 2), a retrovirus infection (e.g. human T cell lymphotropic virus (HTLV)), an arenavirus infection (e.g. lassa virus (LASV) or a lymphocytic choriomeningitis virus (LCMV)) and a paramyxovirus infection (e.g. measles virus).

In certain aspects the present invention relates to an antagonist of the methyltransferase SET domain bifurcated 2 (Setdb2) for use in treating an orthomyxovirus infection, wherein said orthomyxovirus infection is an influenza A virus infection, influenza B virus infection or influenza C virus infection, preferably an influenza A virus infection,.

The Setdb2 knockout mouse model showed ameliorated pathogenesis upon superinfection of influenza virus-infected mice with Streptococcus pneumoniae. Thus, in a preferred embodiment of the invention the antagonist of the methyltransferase SET domain bifurcated 2 (Setdb2) can be used in treating a superinfection preceded by an influenza virus infection. It is envisaged herein that said superinfection is a bacterial infection, wherein said bacterial infection is a Streptococcus infection.

One advantage of the present invention is the fact that it strengthens the immune response against pathogens. A further advantage of the invention is that it is a prophylactic treatment of high risk patients. A further advantage of the present invention is that it is a better option for long term treatment e.g., chronic infections. Furthermore, the present invention has less side effects compared to conventional used therapies e.g., killing of commensals. As a further advantageous property, the invention is independent of developed resistance of the pathogens. It is envisaged herein that the antagonist of Setdb2 can share characteristics of medicaments (like antibiotics) that are conventionally used in the treatment of infections. Yet, the antagonist of Setdb2 to be used herein exerts its therapeutic effect primarily (and preferably solely) by strengthening the immune response.

It is envisaged herein that an antagonist of Setdb2 to be used in accordance with the present invention is not cytotoxic/non-toxic.It is envisaged herein that an antagonist of Setdb2 to be used in accordance with the present invention is not an anti-biofilm agent.lt is envisaged herein that an antagonist of Setdb2 to be used in accordance with the present invention is not bacteristatic.lt is envisaged herein that an antagonist of Setdb2 to be used in accordance with the present invention is not bactericidal.

The antagonist for use in treating an infection in accordance with the present invention can be used to strengthen the immune response against pathogens.The infection/infectious disease to be treated in accordance with the present invention can be characterized by/associated with activation/overexpression/upregulation of Setdb2 as defined herein. It is believed that the herein provided therapy with Setdb2 antagonists is particularly advantageous in clinical settings/pathological conditions that are characterized by or associated with increased expression of Setdb2 (or upregulation of Setdb2); see Example 1 and Fig. 13. Such clinical settings (e.g. pathological conditions, like the infections/infectious diseases as defined herein) characterized by/associated with an increased expression of Setdb2 or upregulation of Setdb2 can be determined, e.g. by measuring the (expression) level or activity of Setdb2 in a sample, for example, a sample from a patient suffering or suspected of suffering from an infection (as defined herein, e.g. the exemplary infections explained and defined herein), and comparing the measured (expression) level or activity of Setdb2 with a control (e.g. control values, such as (expression) level or activity of Setdb2 in a sample from a healthy person). The (increased) production or presence of interferons (like Interferon-alpha, Interferon-beta, Interferon-gamma, and/or, optionally Interferon-lambda) in a sample from a patient suffering or suspected of suffering from an infection as defined herein can also be indicative of a clinical setting/pathological conditions (like infections/infectious diseases, like virus infections or bactierla infections, as defined herein) that are characterized by or associated with increased expression of Setdb2. It is envisaged herein that an antagonist of the present invention can be used in a prophylactic treatment e.g. of high risk patients. It is envisaged herein that an antagonist of the present invention can be used as a long term treatment in e.g., chronic infections. It is envisaged and preferred herein that an antagonist of the present invention can be used in the early phase of an infection, such as the early phase of a viral infection or the early phase of a bacterial superinfection as defined herein.

In certain aspects the present invention an antagonist of the methyltransferase SET domain bifurcated 2 (Setdb2) is for use in treating an infection, wherein said treatment has less side effects (compared to conventional therapy, like conventional antibiotics) e.g., the treatment is associated with/leads to a reduced killing of commensals.

In certain aspects the present invention relates to an antagonist of the methyltransferase SET domain bifurcated 2 (Setdb2) for use use in treating an infection, wherein said treatment is independent of developed resistance of the pathogens (to conventional therapy, like conventional antibiotics).

The term "infection" can refer to the invasion of a host organism's (e.g. subject's/patient's) body/body tissues/body organs by (a) disease-causing agent(s), their multiplication, and the reaction of the host organism's (e.g. subject's/patient's) body/body tissues/body organs to these disease-causing agent(s) and, optionally, the reaction of the host organism's (e.g. subject's/patient's) body/body tissues/body organs to the toxin(s) that the disease-causing agent(s) produce. Disease-causing agents (such as bacteria, viruses, and parasites) are not normally present within the body of a host (like a subject/patient). Infections are normally caused by disease-causing agents. Non-limiting examples of disease-causing agents are infectious agents such as viruses, viroids, and prions; microorganisms such as bacteria; protozoans, such as Plasmodium or Trypanosoma species;nematodes such as roundworms and pinworms; arthropods such as ticks, mites, fleas, and lice; fungi such as ringwomi; and other macroparasites such as tapeworms. Herein, the term "infection" can refer to a bacterial infection, a viral infection, a protozoan infection or a fungal infection.

An infection may cause no symptoms and be subclinical, or it may cause symptoms and be clinically apparent. Thus, the tern 'infection" as used herein can refer to symptoms of the infection (e.g. in case the disease-causing agent is no longer detectable) and/or to the presence of the disease-causing agent in the subject/patient. Accordingly, a patient/subject to be treated in accordance with the invention that has/suffers from an infection (or superinfection as defined herein) has either symptoms that are characteristic of the infection (or superinfection) or does not have symptoms characteristic of the infection (or superinfection). Subjects/patients without symptoms may be in the recovery phase or they may be in an early phase of an infection. Whether a subject/patient suffers from/has an infection (or, in other words, whether an infection occurs in the patient/subject) can be determined by routine tests in accordance with clinical practice. For example, tests for the presence of antigen characteristic of the disease- causing agent (like ELISA tests) can be performed, for example, to confirm whether a disease- causing agent is present in a subject/patient. Such tests can be used to identify patients/subject that show no symptoms (or no symptoms characteristic of an infection).

An infection may remain localized, or it may spread through the blood or lymphatic vessels to become systemic (bodywide). Microorganisms that live naturally in the body are normally not considered infections. For example, bacteria that normally live within the mouth and intestine are normally not considered to be infections.

The definition of an "infection" and the corresponding explanations provided herien apply, generally, mutatis mutandis, to a "superinfection" to be treated in accordance with the present invention.

Yet, it is well known and accepted in the art that a superinfection is a distinct, specific type of an infection.

The term "superinfection" as used herein can be defined as a second infection or a new infection superimposed on an earlier infection or a pre-existing infection. In other words, the term "superinfection" can refer to a new/second infection occurring in a patient or subject having/suffering from an earlier infection or a pre-existing infection, such as bacterial superinfection in viral respiratory disease or a superinfection of a chronic hepatitis B carrier with hepatitis D virus. Patients suffering from an infection, in particular a superinfection, like a bacterial superinfection, or being at risk to suffer from an infection, in particular a superinfection, like a bacterial superinfection, can have a suppressed immune system. For example, cancer patients (e.g. undergoing chemo- and/or radiotherapy), or HIV patients have a suppressed immune system. The therapy of such patients (like patients with a suppressed immune system) with Setdb2 antagonists in accordance with the present invention is contemplated herein. Such patients can especially benefit from the herein provided therapy because one advantage of the provided therapy is, inter alia, its stimulation of the immune system e.g. by increasing the infiltration of neutrophils.

The new/second infection can especially be caused by a different disease-causing agent (such as a microbial agent) that is resistant to the treatment used against the first infection. The "superinfection", "second infection" or "new infection" and the like can be a bacterial superinfection, a viral superinfection, a fungal superinfection or a protozoan superinfection. Preferably, the "superinfection" is a "bacterial superinfection". The terms "superinfection", "second infection" or "new infection" and other terms used in the art in this context are used interchangeably herein. The "earlier infection" or "pre-existing infection" can be a bacterial infection, a viral infection, a fungal infection or a protozoan infection. Preferably, the "earlier infection'V'pre-existing infection" is a virus infection. The terms "earlier infection" or "preexisting infection" and other terms used in the art in this context are used interchangeably herein.

The terms "earlier infection" or "pre-existing infection" as used herein can refer to an infection occurring in a patient prior to a superinfection/second infection/new infection. In certain aspects, the earlier/pre-existing infection has started prior to the superinfection/second infection/new infection and both the earlier/pre-existing infection and the superinfection/second infection/new infection then occur simultaneously in the subject/patient. In certain aspects, the earlier/pre-existing infection has started prior to the superinfection/second infection/new infection and the earlier/pre-existing infection e.g. in its active phase (like replicative phase of the disease-causing agent) no longer occurs/has ceased in the subject/patient at the time the superinfection/second infection/new infection occurs in the subject/patient. In the latter situation, it is contemplated herein that the subject/patient can still have symptoms of the earlier/pre-existing infection, although the disease-causing agent(s) is/are no longer detectable in the subject/patient. It is contemplated herein that the term "infection" (or "superinfection") can refer to one or multiple infections or superinfections. For example, one, two or more (earlier/pre-existing) infections (or superinfections) can occur simultaneously in a subject/patient.

In one situation, a subject/patient may, for example, suffer from one (earlier/pre-existing) infection, such as a viral infection (like influenza virus A). The (earlier/pre-existing) infection may be followed by one superinfection (like a bacterial superinfection, such as Streptococcus pneumoniae infection). As explained herein, an infection can refer to symptoms of the infection (e.g. in case the disease-causing agent is no longer detectable) and/or to the presence of the disease-causing agent in the subject/patient.

In another situation a subject/patient may, for example, suffer from two (earlier/pre-existing) infections, such as viral infections, e.g. one infection caused by one disease-causing agent (e.g. a first virus) and another infection caused by a different disease-causing agent (e.g. a second virus that differs from the first virus, like another viral strain). The two or more (earlier/preexisting) infections may be followed by one, two or more superinfections that can occur simultaneously in the subject/patient. For example, bacterial pneunomia can involve simultaneous infection with Streptococcus pneumonia, Haemophilus influenza, Klebsiella pneumonia and/or Staphylococcus aureus.

The term "simultaneous" as used herein can refer to a complete or partial overlap of the infections (or superinfections) occuring in the subject/patient. As explained herein, an infection can refer to symptoms of the infection (e.g. the disease-causing agent is no longer detectable) and/or to the presence of the disease-causing agent in the subject/patient.

It is contemplated herein that a subject/patient may, for example, suffer from one (earlier/preexisting) infection, such as a viral infection (like influenza virus A).The (earlier/pre-existing) infection may be followed by one, two or more superinfections that can occur simultaneously in the subject/patient. The term "simultaneous" can refer to a complete or partial overlap of the infections (or superinfections) occuring in the subject/patient. As explained herein, an infection can refer to symptoms of the infection (e.g. the disease-causing agent is no longer detectable) and/or to the presence of the disease-causing agent in the subject/patient. The treatment of infectious diseases, such as pneumonia (in particular bacterial pneumonia), that result from or are associated with an infection, by antagonists of Setdb2 is also contemplated and encompassed in the present invention. In certain aspects, the present invention relates to an antagonist of the methyltransferase SET domain bifurcated 2 (Setdb2) for use in treating an infectious disease, preferably bacterial pneumonia, in particular bacterial pneumonia associated with or characterized by Streptococcus pneumoniae infection. Preferably, said infectious disease is preceded by a viral infection, preferably influenza virus infection, and most preferably influenza virus A infection.

Infectious diseases, also known as transmissible or communicable diseases, comprise clinically evident illness (i.e., characteristic medical signs and/or symptoms of disease) resulting from the infection, presence and growth of pathogenic biological agents in an individual host organism. The treatment of an "infection" in accordance with the present invention can encompass the treatment of an associated infectious disease or an infectious diseases resulting from the infection in particular in cases when characteristic medical signs and/or symptoms of the disease are manifest. By treating the underlying infection in accordance with the present invention, the resulting/associated infectious disease is likewise treated. Also envisaged is the therapy of a patient/subject suffering from an infection by antagonists of Setdb2, if the patient does not (e.g. not yet) suffer from the associated infectious disease, for example, because the characteristic medical signs and/or symptoms of the disease are not (e.g. not yet) manifest. Also envisaged is the therapy of a patient/subject suffering from an infection by antagonists of Setdb2, if the patient does no longer suffer from the associated infectious disease, for example, because the characteristic medical signs and/or symptoms of the disease have already disappeared, decreased or ameliorated e.g. because the infectious disease was treated by conventional therapy.

In a preferred aspect, the therapy of bacterial pneumonia is contemplated herein. In other words, the treatment of an infection, in particular a bacterial superinfection (such as Streptococcus pneumoniae superinfection), is or comprises the treatment of bacterial pneumonia. Bacterial pneumonia can be (or be caused by) an infection (preferably a superinfection) with one or more of Streptococcus pneumoniae, Haemophilus influenza, Klebsiella pneumonia, and/or Staphylococcus aureus. Preferably, bacterial pneumonia is (or is cause by) an infection (preferably a superinfection) with Streptococcus pneumoniae.

Bacterial pneumonia is (or is caused by) an infection in one or both lungs. The bacteria cause the lung's air sacs (alveoli) to become inflamed and engorged with pus, fluid, and cellular debris. This often impairs the body's ability to exchange oxygen and carbon dioxide. If a patient/subject has/suffers from bacterial pneumonia he/she might experience breathlessness or pain as he/she struggles to take in oxygen. Bacterial pneumonia can be mild or serious, even leading to respiratory failure or death. How a subject/patient will be affected depends on the potency of the bacterial agent and his/her age, health, and immune status. Early treatment of the infection with the Setdb2 antagonists may significantly reduce the danger of acute respiratory distress.

Bacterial pneumonia is classified based on where you acquire it— outside or inside a hospital. This is generally known as community-acquired pneumonia (CAP) and hospital-acquired pneumonia (HAP), respectively. The therapy of community-acquired pneumonia (CAP) is preferred herein. An infection that occurs in a healthcare setting (like and hospital-acquired pneumonia (HAP)) is usually more serious because the patient/ subject is already sick.

Community-acquired pneumonia (CAP) refers to an infection that is the result of exposure to pathogens outside of a healthcare setting. This is the most common type. A subject/patient may be infected by respiratory droplets in coughs or sneezes or by skin-to-skin contact.

The bacteria that commonly cause bacterial pneumonia, and in particular CAP, include:

• Streptococcus pneumoniae: This is the leading cause of bacterial pneumonia. This bacterium lives in the noses and throats of healthy people. It can enter lungs through inhalation, or it can travel to the bloodstream from a wound or infection site within the body.

• Haemophilus influenzae: This bacterium may live in the upper respiratory tract and does not cause harm or illness until opportunity strikes, such as after a viral infection or when immune function is impaired. It is the second most common cause of bacterial pneumonia.

• Klebsiella pneumoniae:It is found in the mouth, skin, and digestive tract. This bacterium is more prone to infect those with weakened immunity. • Staphylococcus aureus: Infection from this bacterium occurs more frequently among intravenous drug abusers, patients with chronic illness, or young children with immature immune systems. Approximately 1 in 4 healthy individuals carries the staph germ. It usually lives on skin or within the pharynx or intestine. About 2 in 100 individuals carry an antibiotic-resistant strain of the bacterium known as methicillin-resistant Staphylococcus aureus (MRSA). MRSA occurs more commonly in medical settings but is becoming increasingly common within the general community. It is spread by the sharing of personal items or through contact sports such as rugby or wrestling.

A subject-patient usually gets hospital-acquired pneumonia (HAP) within two to three days of exposure to germs in an inpatient or outpatient healthcare facility. This type of pneumonia is often more resistant to antibiotics and more difficult to treat than CAP. Examples of the germs that give rise to HAP include MRSA (methicillin resistant staphylococcus aureus) and Pseudomonas aeruginosa.

Those who are at higher risk for developing bacterial pneumonia include infants and children, adults over age 65, individuals who are ill or have impaired immunity, long-term users of immunosuppressant drugs, chronic obstructive pulmonary disease (COPD) patients who use inhaled corticosteroids for lengthy periods or smokers. The most common symptoms of bacterial pneumonia are cough with yellow, green, or blood-tinged mucus, chest pain that worsens when coughing or breathing, sudden onset of chills, fever of 102 degrees Fahrenheit or above (corresponding to about 38.9 °C or above) or above (lower than 102 degrees Fahrenheit/38.9 °C in older persons), headache, muscle pain, breathlessness or rapid breathing, lethargy, moist, pale skin, confusion (especially among the elderly), or loss of appetite.

To diagnose bacterial pneumonia, one can listen for abnormal chest sounds that indicate heavy mucus secretion and/or possible obstruction of the airways, take a blood sample to get a white blood cell count (a high count usually indicates infection), take blood and/or mucus samples to identify the infection-causing pathogen, or order chest X-rays to confirm the presence and extent of infection. Therapy of bacterial pneumonia involves in accordance with the present invention the use of one or more antagonists of Setdb2. The therapy may, in addition to Setdb2 antagonists, involve conventional therapeutic approaches, like (an) antibiotic that fights the specific bacterium causing the infection, a cough medicine to calm the cough and to help expectorate sputum and/or fever medication to reduce temperature.

The inhibitor/antagonist of Setdb2 can be used herein as a single agent (i.e., in form of a monotherapy) or in form of a combination therapy, for example, in combination with other antagonist(s) of Setdb2 and/or in combination with conventional therapies like antibacterial treatment (e.g., antibiotics such as penicillins, cephalosporins, chloramphenicol sulfonamides, trimethoprim-sulfamethoxazole, macrolides and quinolones), antifungal treatment (e.g., macrocyclic polyenes, imidazole, thiazole and triazole derivates), antiprotozoal treatment (e.g., metronidazole) and/or antiviral treatment (e.g., entry inhibitors and inhibitors specific to viral enzymes such as reverse transcriptase, integrase and proteases).

The combination therapy can, for example, comprise the use of an Setdb2 antagonist (e.g. a competitive substrate analog of a methyltransferase, like a S-adenoxyl-L-methionine (SAM) analog, such as Sinefungin or S-adenosyl-L-homocysteine (SAH)) and of a different Setdb2 inhibitor (e.g. a selective Setdb2 inhibitor) in accordance with the present invention. For example, the present invention relates in certain aspects, to an inhibitor/antagonist of Setdb2 (e.g. a competitive substrate analog of a methyltransferase, like a S-adenoxyl-L-methionine (SAM) analog, such as Sinefungin or S-adenosyl-L-homocysteine (SAH)) for use in the treatment of an infection (e.g. a viral infection, such as influenza virus infection (like influenza virus A infection) or a bacterial superinfection e.g. of Streptococcus pneumoniae as defined herein) combined with e.g. a different Setdb2 inhibitor (e.g. a selective Setdb2 inhibitor) in accordance with the present invention.

It is contemplated herein that the infection/infectious disease can affect various organs/organ systems, tissues or cells of an organism. For example, the following organs/organ systems can be affected:

• Cardiovascular system: pumping and channeling blood to and from the body and lungs with heart, blood and blood vessels. • Digestive system: digestion and processing food with salivary glands, esophagus, stomach, liver, gallbladder, pancreas, intestines, colon, rectum and anus.

• Endocrine system: communication within the body using hormones made by endocrine glands such as the hypothalamus, pituitary gland, pineal body or pineal gland, thyroid, parathyroids and adrenals, i.e., adrenal glands.

• Excretory system: kidneys, ureters, bladder and urethra involved in fluid balance, electrolyte balance and excretion of urine.

• Immune System: structures involved in the transfer of lymph between tissues and the blood stream, the lymph and the nodes and vessels that transport it including the Immune system: defending against disease-causing agents with leukocytes, tonsils, adenoids, thymus and spleen.

• Integumentary system: skin, hair and nails.

• Muscular system: movement with muscles.

• Nervous system: collecting, transferring and processing information with brain, spinal cord and nerves.

• Reproductive system: the sex organs, such as ovaries, fallopian tubes, uterus, vagina, mammary glands, testes, vas deferens, seminal vesicles, prostate and penis.

• Respiratory system: the organs used for breathing, the pharynx, larynx, trachea, bronchi, lungs and diaphragm.

• Skeletal system: structural support and protection with bones, cartilage, ligaments and tendons.

It is preferred herein that the respiratory system (including the organs used for breathing, the pharynx, larynx, trachea, bronchi, lungs and diaphragm) is affected by the infection(s)/infectious disease(s) to be treated in accordance with the present invention. In other words, the infection(s)/infectious disease(s) to be treated herein is preferably an infection(s)/infectious disease(s) of the respiratory system, particularly preferably of the lung. Pneumonia (in particular bacterial pneumonia) is the preferred infectious disease(s) of the respiratory system, particularly preferably of the lung, that is to be treated in accordance with the present invention. The antagonist of the methyltransferase SET domain bifurcated 2 (Setdb2) can be used herein for the treatment of an infection, in particular a bacterial superinfection. Thus, the antagonist of Setdb2 can be comprised in or formulated as a pharmaceutical composition.

The pharmaceutical composition will be formulated and dosed in a fashion consistent with good medical practice, taking into account the clinical condition of the individual patient, the site of delivery of the pharmaceutical composition, the method of administration, the scheduling of administration, and other factors known to practitioners. The "effective amount" of the pharmaceutical composition for purposes herein is thus determined by such considerations.

The skilled person knows that the effective amount of pharmaceutical composition administered to an individual will, inter alia, depend on the nature of the compound.

For example, if said inhibitor is a small molecule, the total (pharmaceutically) effective amount of the inhibitor in the pharmaceutical composition administered orally per dose will be in the range of about 50 mg inhibitor per day to 1000 mg inhibitor per day of patient, although, as noted above, this will be subject to therapeutic discretion. More preferably, this dose is at least 50 mg inhibitor per day, and most preferably for humans between about 50mg and 600 mg inhibitor per day. For example, an inhibitor may be administered at a dose of 15 mg kg body weigth per day. If given continuously, the inhibitor is typically administered at a dose rate of about 50 mg per day to about 600 mg per day. An intravenous bag solution may also be employed. The length of treatment needed to observe changes and the interval following treatment for responses to occur appears to vary depending on the desired effect. The particular amounts may be determined by conventional tests which are well known to the person skilled in the art. The length of treatment needed to observe changes and the interval following treatment for responses to occur appears to vary depending on the desired effect. The particular amounts may be determined by conventional tests which are well known to the person skilled in the art. The administration of the herein provided compositions may, inter alia, comprise an administration twice daily, every day, every other day, every third day, every forth day, every fifth day, once a week, once every second week, once every third week, once every month, etc.

For example, if said compound is a (polypeptide or protein the total pharmaceutically effective amount of pharmaceutical composition administered parenterally per dose will be in the range of about 1 μg protein /kg/day to 15 mg protein /kg/day of patient body weight, although, as noted above, this will be subject to therapeutic discretion. More preferably, this dose is at least 0.01 mg protein /kg/day, and most preferably for humans between about 0.01 and 1 mg protein /kg/day. If given continuously, the pharmaceutical composition is typically administered at a dose rate of about 1 μg/kg/hour to about 50 either by 1-4 injections per day or by continuous subcutaneous infusions, for example, using a mini-pump. An intravenous bag solution may also be employed. The length of treatment needed to observe changes and the interval following treatment for responses to occur appears to vary depending on the desired effect. The particular amounts may be determined by conventional tests which are well known to the person skilled in the art.

Pharmaceutical compositions of the invention may be administered orally, rectally, parenterally, intracisternally, intravaginally, intraperitoneally, topically (as by powders, ointments, drops or transdemial patch), bucally, intratrachially, intranasally or as an oral or nasal spray.

Pharmaceutical compositions of the invention preferably comprise a pharmaceutically acceptable carrier. By "pharmaceutically acceptable carrier" is meant a non-toxic solid, semisolid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. The term "parenteral" as used herein refers to modes of administration which include intravenous, intramuscular, intraperitoneal, intratracheal, intranasal, intrastemal, subcutaneous and intraarticular injection and infusion.

The pharmaceutical composition is also suitably administered by sustained release systems. Suitable examples of sustained-release compositions include semi-permeable polymer matrices in the form of shaped articles, e.g., films, or mirocapsules. Sustained-release matrices include polylactides (U.S. Pat. No. 3,773,919, EP 58,481), copolymers of L-glutamic acid and gamma- ethyl-L-glutamate (Sidman, U. et al, Biopolymers 22:547-556 (1983)), poly (2-hydroxyethyl methacrylate) (R. Langer et al, J. Biomed. Mater. Res. 15:167-277 (1981), and R. Langer, Chem. Tech. 12:98-105 (1982)), ethylene vinyl acetate (R. Langer et al., Id.) or poly-D-(-)-3- hydroxybutyric acid (EP 133,988). Sustained release pharmaceutical composition also include liposomally entrapped compound. Liposomes containing the pharmaceutical composition are prepared by methods known per se: DE 3,218,121 ; Epstein et al, Proc. Natl. Acad. Sci. (USA) 82:3688-3692 (1985); Hwang et al., Proc. Natl. Acad. Sci. (USA) 77:4030-4034 (1980); EP 52,322; EP 36,676; EP 88,046; EP 143,949; EP 142,641 ; Japanese Pat. Appl. 83-118008; U.S. Pat. Nos. 4,485,045 and 4,544,545; and EP 102,324. Ordinarily, the liposomes are of the small (about 200-800 Angstroms) unilamellar type in which the lipid content is greater than about 30 mol. percent cholesterol, the selected proportion being adjusted for the optimal therapy.

For parenteral administration, the pharmaceutical composition is formulated generally by mixing it at the desired degree of purity, in a unit dosage injectable form (solution, suspension, or emulsion), with a pharmaceutically acceptable carrier, i.e., one that is non-toxic to recipients at the dosages and concentrations employed and is compatible with other ingredients of the formulation.

Generally, the formulations are prepared by contacting the components of the pharmaceutical composition uniformly and intimately with liquid carriers or finely divided solid carriers or both. Then, if necessary, the product is shaped into the desired formulation. Preferably the carrier is a parenteral carrier, more preferably a solution that is isotonic with the blood of the recipient. Examples of such carrier vehicles include water, saline, Ringer's solution, and dextrose solution. Non aqueous vehicles such as fixed oils and ethyl oleate are also useful herein, as well as liposomes. The carrier suitably contains minor amounts of additives such as substances that enhance isotonicity and chemical stability. Such materials are non-toxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, succinate, acetic acid, and other organic acids or their salts; antioxidants such as ascorbic acid; low molecular weight (less than about ten residues) (polypeptides, e.g., polyarginine or tripeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids, such as glycine, glutamic acid, aspartic acid, or arginine; monosaccharides, disaccharides, and other carbohydrates including cellulose or its derivatives, glucose, manose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; counterions such as sodium; and/or nonionic surfactants such as polysorbates, poloxamers, or PEG.

The components of the pharmaceutical composition to be used for therapeutic administration must be sterile. Sterility is readily accomplished by filtration through sterile filtration membranes (e.g., 0.2 micron membranes). Therapeutic components of the pharmaceutical composition generally are placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.

The components of the pharmaceutical composition ordinarily will be stored in unit or multi- dose containers, for example, sealed ampoules or vials, as an aqueous solution or as a lyophilized formulation for reconstitution. As an example of a lyophilized formulation, 10-ml vials are filled with 5 ml of sterile-filtered 1% (w/v) aqueous solution, and the resulting mixture is lyophilized. The infusion solution is prepared by reconstituting the lyophilized compound(s) using bacteriostatic Water-for-Injection.

As used herein, the terms "comprising" and "including" or grammatical variants thereof are to be taken as specifying the stated features, integers, steps or components but do not preclude the addition of one or more additional features, integers, steps, components or groups thereof. This term encompasses the terms "consisting of and "consisting essentially of."

Thus, the terms "comprising"/"including"/"having" mean that any further component (or likewise features, integers, steps and the like) can be present.

The term "consisting of means that no further component (or likewise features, integers, steps and the like) can be present.

The term "consisting essentially of or grammatical variants thereof when used herein are to be taken as specifying the stated features, integers, steps or components but do not preclude the addition of one or more additional features, integers, steps, components or groups thereof but W

105

only if the additional features, integers, steps, components or groups thereof do not materially alter the basic and novel characteristics of the claimed composition, device or method.

Thus, the term "consisting essentially of means that specific further components (or likewise features, integers, steps and the like) can be present, namely those not materially affecting the essential characteristics of the composition, device or method. In other words, the term "consisting essentially of (which can be interchangeably used herein with the term "comprising substantially"), allows the presence of other components in the composition, device or method in addition to the mandatory components (or likewise features, integers, steps and the like), provided that the essential characteristics of the device or method are not materially affected by the presence of other components.

The term "method" refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, biological and biophysical arts.

As used herein the term "about" refers to ± 10%.

The present invention is further described by reference to the following non-limiting figures and examples. Unless otherwise indicated, established methods of recombinant gene technology were used as described, for example, in Sambrook, Russell "Molecular Cloning, A Laboratory Manual", Cold Spring Harbor Laboratory, N.Y. (2001) ) which is incorporated herein by reference in its entirety.

The Figures show:

Figure 1: Setdb2 is induced upon influenza virus infection and TLR stimulation in an Ifnarl dependent manner, (a) WT mice were intranasally infected with influenza virus or mock treated. 18 hours later lung sections were stained using an H IN 1 -specific antibody. H1N1 infected areas are indicated by arrowheads. Scale bar, 250μηι. (b) Gene ontology enrichment analysis of up-regulated genes in lungs of influenza virus-infected mice (n=6 per group from two pooled experiments), (c) Expression profile of protein lysine methyltransferases (PKMTs) in lungs upon influenza virus infection compared to untreated controls (n=6). Heatmap illustrations of (left) log2-fold change compared to uninfected mice respectively (right) Robust Multi-array Average (RMA) values are shown, (d) WT, lfnarl ^' ' ^' , Irp ^~ ' ^~ and Statl ^' ^ mice were intranasally infected with influenza virus (PR8) or mock treated (control). 18 hours later lung tissue was collected and expression of Setdb2 was determined by real-time PCR. (e-g) BMDMs from Ifna ^1' and WT mice were either left untreated (control), (e) infected with influenza virus (PR8, MOI 10), (f) stimulated with IFNp, IFNy or IFN , or (g) stimulated with the indicated TLR agonists polyI:C, PAM3 and LPS. Setdb2 mRNA expression was quantified by real-time PCR 8 hours after stimulation. Biological triplicates of one out of two similar independent experiments are shown. For protein detection, Setdb2 expression was analyzed (e) 8 hours respectively (f, g) 24 hours after stimulation by western blot using the Setdb2-specific mAb clone 7H7F11. Antibodies specific for the IFN-stimulated protein Zbpl and actin served as controls for induction and loading, respectively, (e, f) Western blots show results of one out of two representative experiments, (d-g) Statistical significance was calculated by unpaired t-test.

Figure 2: Setdb2 ^GT/GT macrophages show increased expression of a subset of NF-KB target genes including Cxcll. (a) Spleen and lung tissue as well as bone marrow-derived macrophages (BMDMs) from naive Setdb ¹ ™ ¹ or WT mice were analyzed for the expression of Setdb2 by western blot using the Setdb2-specific mAb clone 7H7F11. Actin served as loading control, (b) BMDMs were treated with polyI:C for 0, 2 and 8 hours and expression profiling was performed by RNAseq using biological triplicates for each condition. Heatmap illustrations for (left) differential gene expression between WT and Setdbl BMDMs of all protein-coding genes, respectively (right) the expression profiles at 2 and 8 hours compared to untreated BMDMs of the respective genotype are shown. NF-κΒ target genes are indicated as grey boxes._BMDMs from either WT or Setdbl mice were either left untreated (control), (c, d) stimulated with the indicated TLR agonists and with IFN or (e, f) infected with influenza virus (PR8, MOI 10). (c, e) Cxcll mRNA expression was quantified by real-time PCR after 2 hours of stimulation, (d, f) Cxcll secretion was quantified by ELISA after 8 hours of stimulation. Biological triplicates of one out of two similar independent experiments are shown, (c-f) Statistical significance was calculated by unpaired t-test. W

107

Figure 3: Setdb2 binds to the Cxcll promoter region and associates with H3K9 tri- methylation. (a-c) WT or Setdb2 ^GT/GT BMDMs were stimulated with polyl.C for 2 hours or left untreated (control) before cells were prepared for chromatin immunoprecipitation (ChIP). Specific enrichment of indicated promoter elements was quantified by real-time PCR. Data are depicted as normalized recovery to the highest response in WT of two independent experiments (a, b), and one experiment (c). Empty beads were used as control (Mock), (a) ChIP for endogenous Setdb2 using the Setdb2-specific mAb clone 7H7F11. (b) ChIP for H3K9me3. (c) ChIP for H3K9ac. Statistical significance was calculated by unpaired t-test.

Figure 4: Setdb2 ^G1[IGT mice show exacerbated lung inflammation in a model of LPS- induced neutrophilia. WT and Setdb2 mice were intranasally challenged with LPS for 4 hours or mock treated (control). Graphs show pooled results of three independent experiments (n=10-14 mice per group), (a) Total lung RNA from challenged and control mice was extracted and analyzed for the expression of Cxcll by real-time PCR. (b) Bronchoalveolar lavage (BAL) was performed and supernatants were analyzed for levels of Cxcll by ELISA. (c-e), Total BAL fluid cells as well as neutrophils and macrophages were quantified. Cell numbers of individual experiments were normalized to the respective means of LPS-stimulated WT mice (relative number = cell number/mean T+LPs)- Statistical analysis was performed by unpaired t-test.

Figure 5: Setdb2 mediates influenza virus-induced susceptibility to superinfection with

Streptococcus pneumoniae. WT and Setdb2 mice were either left untreated (control), infected with a sublethal dose of influenza virus (PR8) or superinfected with Streptococcus pneumoniae (Sp) 5 days after PR8 infection, (a) Cxcl 1 RNA was quantitified by real-time PCR from lung tissue 5 days after influenza virus infection, (b-d) WT and Setdb2 mice were harvested 16 hours after Sp superinfection. Superinfected mice received ~2xl0 ⁴ CFU of Sp. (b) Results of Cxcll ELISA from BAL and enumeration of neutrophils from (c) lung tissue and (d) BAL are shown, (e-k) 2 days after superinfection with ~2xl 0 ³ CFU of Sp, WT and Setdb2 ^GTm mice were sacrificed and (e) images of lungs were taken (size bar, 1cm). Lungs were aligned according to the degree of gross pathology, (f) The lung wet weight was determined from the right lobes and normalized to the body weight on day zero. A dotted line depicts the average lung weight to body weight ratio of three uninfected lungs, (g) H/E histological staining of representative sections of the left lung lobes of WT and Setdb2 mice are shown. Scale bar of low magnification is 200μπι, high magnification 50μηι. (h) Histopathological scoring of lung sections (see Methods), (i) Expression of 116 in lung tissue was determined by real-time PCR. (j) Levels of 116 were detected by ELISA. (k) Bacterial burden was determined as CFU in tissue homogenates of the right lung lobes. Scatter blots represent individual mice from (a, e-k) one or (b-d) two pooled experiments, (f, k) show one out of two similar experiments. Statistical significance was calculated by unpaired t-test. *P 0.05, **P 0.01 , ***P 0.001 and ****p 0.0001.

Figure 6: PAM3 induction of Setdb2 and Zbpl is TLR2 dependent. WT and Tlrl ^{1 ~} Cd36 ^' ' ^~ BMDMs were treated with PAM3 (two different batches from Invivogen), LPS or left untreated (control), (a) Setdb2 or (b) Zbpl mRNA expression was quantified by real-time PCR 8 hours after stimulation. Statistical significance was calculated by unpaired t-test.

Figure 7: Expression kinetics of PKMTs in bone marrow-derived macrophages (BMDMs) upon stimulation with polyI:C. WT BMDMs were stimulated with polyLC and gene expression levels were determined at the indicated time points by microarray. The top three up- regulated genes are highlighted. The data is derived from systemsimmunology.org.

Figure 8: Generation of Setdbl mice, (a) Schematic of recombinant Setdb2 genetrap targeting strategy, (b) Southern blot of transfected ES cells, (c) Genotyping PCR of Setdb2 genetrap mice.

Figure 9: No difference in ΙκΒα degradation between polyI:C stimulated WT and

Setdb2 ^CTIGJ BMDMs. WT or Setdb2 ^GVm BMDMs were stimulated with polyLC for indicated times points and ΙκΒ expression/degradation was analyzed by western blot using the antibody sc-371. Western blot against Actin served as control for loading. One out of two similar experiments is shown.

Figure 10: Blockade of IFNARl leads to increased Cxcll expression upon polyLC stimulation. WT and Setdb2 ^GllGX BMDMs were treated with 2(^g/ml IFNARl -specific antibody (clone MAR 1-5 A3, anti-Ifnar) or with isotype control followed by polyLC stimulation, (a) Setdbl and (b) Cxcll expression were quantified by real-time PCR after 2 hours of stimulation. One out of two similar experiments is shown. Statistical significance was calculated by unpaired t-test.

Figure 11: Time kinetics of Cxcll and Setdb2 expression in BMDMs upon stimulation with polyI:C. WT BMDMs were stimulated with polyLC and Cxcll and Setdb2 mRNA expression was determined by microarray at the indicated time points. The data was derived from systemsimmunology.org.

Figure 12: Setdbl alveolar macrophages express increased levels of Cxcll upon infection with influenza virus. Alveolar macrophages from BAL of naive WT and Setdbl mice were seeded on 96-well tissue culture plates and subsequently infected with influenza virus (PR8) (MOI 100). 12 hours after stimulation, (a) Cxcll mRNA expression was quantified by real-time PCR and (b) Cxcll protein was quantified by ELISA. Results from one out of two similar experiments are shown. Statistical significance was calculated by unpaired t- test.

Figure 13: Comparison of Setdb2 induction in the lungs of mice infected with either Streptococcus pneumoniae or influenza virus.

WT as well as Setdbl mice were either infected with SP, PR8, or left untreated as control (Ctrl) and lungs were harvested at indicated time points. Total RNA was extracted from lungs and real-time PCR was used to measure Setdbl expression. Interestingly, SP infection did not induce Setdbl expression at any of the time points investigated. In contrast, Setdb2 was strongly induced 5 days (5d) after influenza virus infection, the time point used to perform superinfection experiments (see previous figures). Setdb2 expression is displayed as fold induction compared to uninfected mice. Scatter blot indicate individual mice pooled from different experiments. Statistical analysis was performed by unpaired t-test. **** p < 0.0001.

Figure 14: Cytokine profiling of mice infected with either influenza virus, Sp or superinfected with both pathogens. WT and Setdbl mice were either left untreated (control), infected with Streptococcus pneumoniae (Sp) (~2xl 0 ⁴ CFU), infected with a sublethal dose of influenza virus (PR8), or superinfected with Sp (~2xl 0 ⁴ CFU) (PR8+5p) on day 5 after PR8 infection. BAL was harvested 16 hours after Sp infection respectively 5 days and 16 hours for the groups infected with PR8 or PR8+,¾?. Levels of (a) Cxcl2, (b) 116, (c) 1110 and (d) Cxcll were determined by ELISA. (a-c) Pooled data of two experiments is shown. No statistically significant differences between WT and Setdb2 groups of the respective infections were detected by unpaired t-test.

Figure 15: Cellular lung profiling of WT and Setdb2 ^GTIGT mice. WT and Set<¾2 ^GT/GT mice were either (a-b) left untreated, (c-d) infected with influenza virus for 5 days and 16 hours, (e- f) infected with Sp for 16 hours, or (g-h) superinfected with Sp for 16 hours on day 5 after influenza virus infection (compare Fig. 14 legend for respective infectious doses), (a, c, e, g) Representative FACS plots with gating strategies for neutrophils (Neutr), monocytes/macrophages/dendritic cells (Mac/DC), alveolar macrophages (AM), NK cells (N ), T cells and B cells are shown. Scatter plots represent total cell numbers per lung from individual mice, (b, d, f, h) Scatter plots represent relative percentages of live CD45 ⁺ cells in BAL from individual mice, (i) Representative backgating plot of the population of AMs from CD45 ⁺ live lung cells, (a-d, f-h) Pooled data of two or more experiments are shown. Statistical significance was calculated by unpaired t-test.

Figure 16: No differences in pathogen loads in Setdb2 ^GJIGT compared to WT mice upon single infection with either influenza virus or Streptococcus pneumoniae, (a) WT and

Setdb2 mice were infected by a sublethal dose of influenza virus (PR8). Five days later, mice were sacrificed and total RNA was prepared from the right lung lobes. Viral loads were quantified by real-time PCR for the M gene, (b) WT and Setdb2 mice were infected with ~2xl0 ³ respectively 4xl0 ⁴ CFU of Sp. Two days later mice were sacrificed and bacterial burden was analyzed by colony formation assay on blood agar plates. Lysates from total lung tissue were analyzed. No statistically significant differences between WT and Setdb2 groups of the respective infections were detected by unpaired t-test.

Figure 17: Working model: The role of Setdb2 in mediating virus-induced susceptibility to bacterial superinfection. The illustration highlights three important stages of superinfection, comparing the situation in a WT and a Setdb2-deficient lung, (a) Primary viral infection. Viral particles enter the respiratory tract and are recognized by intracellular pattern recognition receptors (PRR). This induces the activation of type-I interferon (IFN) signaling and, subsequently, the expression of IFN-stimulated genes (ISGs) including Setdb2. In addition, activation of NF-κΒ signaling initiates the expression of genes such as the neutrophil chemoattractant Cxcll . (b) Bacterial superinfection. Bacteria enter the lung and are recognized by cell surface PRRs. The dashed arrows indicate possible cross-activation of IFNs and NF-KB by different PRRs. In the WT lung, high Setdb2 expression leads to a virus-induced and Setdb2-mediated repression of Cxcll and, as consequence, to reduced neutrophil infiltration. In the Setdb2-deficient lung this repression does not occur resulting in increased Cxcll expression and neutrophil infiltration, (c) Late stage pathology after superinfection. In WT lungs, the repression of Cxcll leads to uncontrolled bacterial growth, formation of edema, accumulation of inflammatory cells, and tissue damage (depicted in red color and increased lung size). This extent of severe lung pathology is reduced in the Setdb2-deficient lung.

Figure 18: Knockdown of SETDB2 in human cells

Knockdown of SETDB2 by siRNA in the human embryonic kidney cell line HEK293

Figure 19: CXCL8 expression is induced by the knockdown of Setdb2 in human cells

Characterization human haploid KMB7 cells in the presence and absence of SETDB2. a) Detection of SETDB2 expression by real-time PCR. b) Time kinetics of CXCL8 expression as detected by real-time PCR. c) Time kinetics of CXCL8 expression as detected by ELISA. Statistical analysis was performed by unpaired t-test (with or without Welch's correction), p- values were indicated as follows: * p<0.05, ** <0.01.

Figure 20: Setdb2 inhibitors induce CXCL1 expression

Effect of Setdb2 inhibitors on the secretion of Cxcll BMDMs after polyLC stimulation. The Examples illustrate the invention. Example 1: A Setdb2-knockdown animal model provides evidence that an antagonist of Setdb2 can be used in the therapy of infections

Material and Methods Mice.

C57BL/6J mice (WT) were obtained from The Jackson Laboratory and IfnarF^ ⁴⁸ , Irf7 ^{~ 49} , Statr ⁵⁰ and TlrZ^CdS^- ⁵¹ ' ⁵² mice were on a C57BL/6J background. To generate Setdb2 ^GT/GT mice, the targeting vector pEKF106 was made by recombineering using Lambda RED system in E. coli strain EL350 ⁵³ . A genomic PAC library (i.e. from the 129/SvevTACfBr genetic background, RPCI mouse PAC library 21 ; MRC Geneservice) was screened for the full-length Setdbl gene using specific cDNA probes. A positive PAC clone, RP21-498J23, was used for recovery of flanking genomic sequences. We generated a 2.3kb genomic fragment introducing an EcoRV restriction site and loxP -Neo using plasmid pLMJ237, for insertion downstream of exon 3. Neo was removed by arabinose induction of Cre recombinase. Similarly, a 2.9kb genomic fragment harboring the frt-loxP flanked gene trap cassette and a heterologous BamHI restriction site was generated by recombineering using pLTM330. The gene trap cassette contains a pGK/EM7 dual promoter, a strong splice acceptor site from the engrailed 2 gene, and Neo reporter gene, followed by a strong polyadenylation signal (Fig. 8A). Notl linearized targeting vector D A was transfected into v6.4 embryonic stem (ES) cells, a hybrid of the C57BL/6J x 129/SvJae lineages. Transfected ES cells were selected for neomycin resistance with G418 and positive clones were tested by Southern blot analysis of EcoRV-digested DNA (Fig. 8B). Positive ES cell clones were microinjected into blastulae and transferred to pseudopregnant female mice, following standard methods. Mice were genotyped for the presence of the gene trap cassette by Southern blot hybridization (not shown) and by PCR amplification of genomic DNA using primers 5 '-AATGGGCCATATTAGTAGAAGC-3 ' and 5 ' -G ATCTTGCTC AA AGGTC ACCA-3 ' (Fig. 8C). The WT allele amplicon was a 422 bp PCR product, while the knocked-in gene trap amplicon was a 516 bp PCR product. All

GT/GT

Setdb2 mice used in this study were backcrossed for >10 generations onto a C57BL/6J background. All mice were kept under specific pathogen-free conditions at the Institute of Molecular Biotechnology (1MB A) of the Austrian Academy of Sciences, the Medical University of Vienna, National Cancer institute, the Ohio State University, and/or the Institute for Systems Biology, Seattle. For all experiments sex- and age-matched mice were used. The animal protocols were approved by the Institutional Animal Care and Use Committees of the National Cancer Institute-Frederick, the Ohio State University, the Institute of Systems Biology in Seattle respectively by the ethical committee of the Medical University of Vienna and the Austrian Federal Ministry of Science and Research.

Bone marrow-derived macrophages.

Bone marrow-derived macrophages (BMDMs) were isolated from C57BL/6J, Ifnarl ^'1' and Setdb2 mice and cultured in RPMI medium containing 10% FBS, penicillin-streptomycin- glutamine (Life Tech #10378-016) and 50ng/ml recombinant mouse macrophage colony stimulating factor (eBioscience #34-8983-85) ⁵⁴ . BMDMs were stimulated with PAM3CS 4 (PAM3) (500ng/ml, Invivogen tlrl-pms), polyFC (6μ Ίη1, Invivogen tlrl-pic), LPS (20ng/ml, Salmonella enterica serotype Minnesota, Sigma #L4641), mouse IFN (1000 IU/ml, PBL Interferon Source #12400-1), mouse IFNy (lOOng ml, Peptrotech #315-05) or IFN 2 (lOOng/ml, Biomedica 4635-ML-025). For in vitro infections with influenza virus A/PR/8/34, cells were grown in OPTI-MEM medium (Life Tech, #31985-070) containing 4% BSA (Sigma, #A7979), lx MEM vitamins solution (Life Tech, #11120-037) and ^g/ml TPC trypsin (Sigma, #T8802). To block Ifnarl, we pre-treated BMDMs with 20 g/ml of IFNAR1 -specific antibody (clone MAR 1-5 A3, BioXCell #BE0241) ⁵⁵ or with mouse IgGl isotype control (clone MOPC-21, BioXCell #BE0083) 18 hours prior to polyI:C stimulation and added fresh antibody upon stimulation.

Microarray analysis.

Total RNA was extracted with Trizol (Life Technologies) and RNA quality was detemiined by Bioanalyzer (Agilent). For the experiments shown in Fig. la-c, RNA was processed for hybridization to GeneChip Mouse Exon 1.0 ST arrays according to the manufacturer's instructions (Affymetrix). Exon-level signal values were generated by Affymetrix Power Tools. The Institute of Systems Biology's GenePattern Exon pipeline was used to generate transcript- level expression values. PERL scripts were used to combine and annotate these data. For the experiments shown in Fig. 7 , publicly available array expression data derived from polyI:C stimulated BMDMs was downloaded from the website http://www.systemsimmunology.org.

RNA sequencing.

For the experiments in Fig. 2b, we performed expression analysis by RNAseq. Briefly, BMDMs from each genotype were prepared and stimulated with polyI:C for the indicated time points. RNA was extracted by QIAzol lysis reagent (Qiagen) and the libraries were prepared with the Truseq RNA sample preparation kit v2 according to the manufacturer's instructions (Illumina). Quality control analysis was performed by Experion DNA Analysis chip (Biorad) and Qubit Fluorometric Quantitation (Life Technologies). The samples were multiplexed with 9 samples per lane and run on a 50bp single-end flow cell in a Hiseq2000 sequencer (Illumina). RNA-Seq reads were aligned to the mouse genome assembly GRCm38 (UCSC mmlO) with the TopHat splice junction mapper (version 2.0.12) utilizing the mouse gene and transcript annotation from Ensembl version 70 as reference transcriptome. The TopHat max-multihits option were set to 100, while the length (-L) of the seed substrings of the underlying Bowtie 2 aligner (version 2. 2.3) were reduced from 20 to 15. Programs from the Cufflinks package (version 2._2.1) were used to assemble transcripts, merge transcript assemblies of replicates and samples before finally testing for differential expression with the Cuffdiff program. The default false discovery rate (FDR) of 0.05 was left unchanged. Cuffdiff comparisons were post- processed, and quality assessment plots were drawn with the Bioconductor package cummeRbund (version 2.6.1).

Bioinformatics analyses.

Gene ontology enrichment analyses were done by DAVID Bioinformatics Resources 6.7 and the Molecular Signatures Database of Gene Set Enrichment Analysis (GSEA). Enrichment analysis of transcription factor binding targets was performed by GSEA using transcription factor binding sites as defined in TRANSFAC version 7.4. The computed Robust Multi-array Average (RMA) values were subjected to Significance Analysis of Microanalysis (SAM) using Microarray expression Viewer v4.9. A two-class paired test with default parameters was used and calculated the Delta and a cutoff of 3 to compute the significantly regulated genes. Heatmaps were plotted in R. The motif scanning for transcription factor binding sites was performed +/- 3kb up- and downstream of the transcriptional start site of Setdb2 as described previously ³⁰ . The raw data from our array and RNAseq data are deposited at ArrayExpress (http://www.ebi.ac.uk/arrayexpress/) with accession numbers E-MTAB-2845 and_E-MTAB- 2263.

A list of NF-KB target genes was compiled using resources from the website of The Gilmore Lab, Boston University (http://www.bu.edu/nf-kb/gene-resources/target-genes/), from the website of the Institut de Biologie de Lille et LIFL (http://bioinfo.lifl.fr/NF- KB/#haut%20de%20page) as well as from recent literature. The resulting total list of 373 NF- KB target genes was used to calculate the hypergeometric distribution (assuming a total number of 24561 coding genes, source: MGI informatics.jax.org) of overlapping genes within the protein-coding genes that were significantly upregulated > 1.5-fold at ≥ one time point (p<0.001).

Real-time PCR.

For the measurement of gene expression by real-time PCR, total RNA was isolated using QIAzol lysis reagent (Qiagen) and reverse-transcribed with the First Strand cDNA Synthesis Kit (Fermentas). Subsequently gene expression was analyzed using Taqman Fast Universal PCR Mastermix and Taqman Gene Expression assays (Setdb2: Mm01318748_ml, Cxcll: Mm00433859_ml, 116: Mm00446190_ml) (Life Technologies) as well as an assay for the M gene of influenza virus A PR/8/34 using the oligonucleotides F 5"- CATGGAATGGCTAAAGACAAGACC- '3 (SEQ ID NO: 9), R 5'- CCATTAAGGGCATTTTGGACA-3 ^x (SEQ ID NO: 10) and taqman probe 5 ^X -FAM- TTTGTGTTCACGCTCACCGTGCCCA-BHQ 1 - '3 (SEQ ID NO: 1 1). Real-time PCR experiments were run on a 7900HT Real-Time PCR system or a StepOnePlus Real-Time PCR system (Life Technologies). Expression data was normalized to the housekeeping gene Eeflal (encoding eukaryotic translation elongation factor 1 ocl) ⁵⁴ . Fold-induction was calculated by comparison to the untreated WT control group.

Generation of a Setdb2-specific monoclonal antibody.

A C-terminal 60 amino acid long sequence (amino acid position 541-600) was fused into a hepatitis B carrier protein as immunogen. This region was amplified by PCR and inserted into a 6x histidine-tagged pB-His HBcAg_Linker plasmid ⁵⁶ . The fusion protein was expressed in E. coli BL21 and purified on 1ml HisTrap HP columns (GE Healthcare) followed by a linear imidazole gradient on an AKTA FPLC system (GE Healthcare). The fractions were analyzed by SDS-Page and concentrated with Amicon Ultra 15-3K Centrifugal Filter Devices (Millipore). The immunization and generation of monoclonal B-cell hybridomas was performed by challenging Setdbl mice 3 times (every 2 weeks) with 5(^g of purified fusion protein antigen mixed 1 : 1 with adjuvant subcutaneously, before a final immunization intravenously with 5(^g purified antigen (adjuvant-free). Mouse sera and clone pools were tested by western blot against overexpressed and endogenous mouse Setdb2. Clone 7H7F11 yielded the best signal-to-noise performance.

Western blot

Protein concentration of cell lysates and organs were determined with a Coomassie Protein Assay kit (Thermo Scientific). Proteins were analyzed by SDS-Page using NuPAGE® Novex 4-12% Bis-Tris Gels (Life Technologies), Westran® Clear signal PVDF membranes (Whatman) and the following antibodies: anti-Zbpl (kindly provided by the Superti-Furga laboratory), anti-Setdb2 (clone 7H7F11, described in this study), anti-ΙκΒ (Santa Cruz Biotechnology sc-371 clone C-21, respectively, Cell Signaling n.4814 clone L35A5) and anti- actin (Sigma #A2066). The protein size was determined with the PageRuler™ Prestained Protein Ladder (Thermo Scientific). Detection was done with Pierce ECL Western blotting substrate (Thermo Scientific) or Amersham ECL select Western blotting detection reagent (GE Healthcare Life Sciences). Gels were visualized with the chemiluminescent gel documentation system F-ChemiBIS 3.2 (DNR Bio-Imaging Systems).

ELISA.

Protein concentrations were determined using the Mouse CXCLl/KC Quantikine ELISA Kit (#MKC00B) or the Mouse CXCLl/KC DuoSet (#DY453), the Mouse CXCL2/MIP-2 DuoSet (#DY452), the Mouse IL-6 DuoSet (#DY406) or the Mouse BD OptEIA IL-10 kit (BD Biosciences, #555252). The ELISAs were performed according to the manufacturer's instructions. Chromatin immunoprecipitation analysis.

BMDMs derived from WT or Setdb2 ^GVGT mice were either treated with polyI:C for 2h or left untreated. The same number of cells was subsequently harvested, fixed with 1% formaldehyde for lOmin at RT, and lysed in 1% SDS buffer. Chromatin was sheared to an average size of 300bp using S2X Focused-ultrasonicator (Covaris), and the amount was adjusted using ND- 1000 spectrophotometer (NanoDrop) measurement to match WT and Setdb2 ^GT/GT samples before histone mark ChlPs. The prepared chromatin of WT or WT and Setdb2 samples were subjected to ChIP with anti-Setdb2 clone 7H7F11 (see above), anti-H3 9me3 (Abeam Ab8898) or anti-H3K9acetyl (Millipore #07-352) respectively following a procedure as described previously ⁵⁷ that was modified by the use of magnetic Dynabeads Protein-G beads (Life Technologies). For mock ChIP controls, empty beads were used. For mock controls in Setdb2 ChIP, the medium used for culturing the hybridoma 7H7F11 was added instead. The ChIP efficiency was controlled by quantitative real-time PCR analysis using the primers for: Cxcll promoter region, F 5'-CCTCTTCACATGCCTCCCTG-3' (SEQ ID NO: 12) and R 5'- CGGGGATGGAAGCTTGTCTT-3 ' (SEQ ID NO. 13); Cxcll exon 1 region, F 5'- GTTCCAGCACTCCAGACTCC-3' (SEQ ID NO: 14) and R 5'- AGTGGCGAGACCTACCTGT-3 ' (SEQ ID NO: 15); Actb promoter region, F 5'- CCTCTGGGTGTGGATGTCAC-3 ' (SEQ ID NO: 16) and R 5'- TGTCCATTCAATCC AGGCCC-3 ' (SEQ ID NO: 17).

LPS-induced pulmonary neutrophilia model.

Mice were anesthetized with ketamine/xylazine and intranasally administered with 0.4 μg of LPS (E. coli serotype 01 1 1 :B4, Sigma #L4391) as described previously ⁵⁸ . Four hours later, mice were sacrificed and bronchoalveolar lavage (BAL) was taken by washing the lungs 3 times with PBS in a total volume of 1 ml. The total number of cells in the BAL was enumerated with an improved Neubauer hemocytometer. Cytocentrifuged preparations (Cytospin-4, Shandon Instruments) were stained with Kwik-Diff Stains (Thermo Fisher Scientific) and the percentage of inflammatory cells was determined by morphological examination of at least 300 cells per sample. Infection models.

For the lung analyses in Fig. 1, mice were anesthetized with ketamine/xylazine and intranasally infected with 15μ1 or 50μ1 of PBS containing ~10 ⁵ plaque forming units (PFU) of influenza virus A/PR/8/34 (PR8) (originally obtained from Charles River Laboratories). In all other experiments mice were infected intranasally with a sublethal dose of PR8 (~10 ² PFU) or the indicated dose of Streptococcus pneumoniae (Sp) strain ATCC 6303. Mice for the 16h samples in Fig. 5 were harvested in the time span corresponding to 14-16h after superinfection. Bacterial titers were determined from lung homogenates by plating 10- fold serial dilutions on blood agar plates ⁵⁹ . The lung wet weights were determined with a Pioneer precision balance (Ohaus).

Flow cytometric analysis.

Lung tissue was harvested as indicated and single cell suspension were prepared using a metal mesh. Absolute cell numbers were counted with a Neubauer chamber. Single-cell suspensions of the lungs respectively collected BAL cells were incubated with CD16/CD32 Fc block (BioLegend, 101310) to inhibit unspecific antibody binding. For flow cytometry, cells were stained with the following antibodies: anti-B220/CD45R (eBioscience, 45-0452), anti-CD3e (BioLegend, 100320), anti-CDl lb (BioLegend, 101206), anti-Ly-6G (eBioscience, 17-9668), anti-CD45 (BioLegend, 103137), anti-CDl lc (Biolegend, 117333) and anti-SiglecF (BD, 562681). To exclude dead cells from the analysis, the samples were labeled with the Fixable Viability Dye eFluor 780 (eBioscience, 65-0865).

Histology.

Lung tissue was fixed with either 4% paraformaldehyde or 10% formalin and embedded in paraffin. Immunohistochemistry was performed on 3-4 μη thick sections. Endogenous peroxidase was neutralized (PBS/3% H202) and unspecific binding blocked (PBS/10% FCS). Sections were then incubated with goat-anti influenza antibody (Serotec, Product Code 5315- 0064) overnight at 4°C. Bound primary antibody was visualized by a biotin technique with 3,3' diaminobenzidine as chromogen (haemalaun counterstaining of nuclei).

In Fig. 5h, histology scores were obtained by a trained pathologist, blinded for groups, from lung sections stained with hematoxylin and eosin ⁵⁹ . The severity of inflammation and pneumonia was evaluated based on the presence of interstitial inflammation, alveolar inflammation, pleuritis, bronchitis and endothelitis with 0 representing absent, 1 mild, 2 moderate, and 3 severe. Additionally, 1 point was added for the presence of pneumonia, edema or thrombi formation, and 0.5 point for every infiltrate covering 10% of the lung area. The sum of all parameters indicates the total histology score.

Statistical Analysis.

Results are indicated as line graph, bar graph or scatter plot with the mean +/- standard error of the mean as indicated. Statistical differences between experimental groups were determined with either paired or unpaired t-test as detailed in the figure legends. Significant p- values were indicated as follows: * p<0.05, ** p<0.01, *** p≤0.001, **** p≤0.0001. Graphs and statistical tests were done with GraphPad Prism version 5 and 6.

Results

Influenza virus infection induces Setdbl expression

To identify novel regulatory immune mechanisms that are involved in virus-induced susceptibility to bacterial superinfection, we infected wild type (WT) mice with influenza virus and collected lung tissue at 18 hours after infection. At this early time point, the distribution of viral antigen was limited to a small percentage of epithelial cells (Fig. la). We next performed a global expression profiling of lung tissue from infected and uninfected WT mice (Fig. lb-c). We identified more than 200 virus-induced genes with many of them being known ISGs ⁸ . A gene ontology analysis highlighted the enrichment of genes involved in IFN-mediated immune responses (Fig. lb). This was confirmed by the enrichment of transcription factor binding targets for Irfl (p-value 0 e°), Irf8 (0 e°), Irf7 (1.11 e ¹⁶ ) and Irf2 (2.43 e ^"11 ) (Methods). Next, we analyzed the expression of the mouse orthologs of the previously annotated PKMTs ²⁹ . This revealed Setdb2 as the only statistically significant PKMT that was induced upon influenza virus infection (Fig. lc).

Type-I interferon signaling drives Setdb2 expression

We identified several putative IRF binding sites by motif scanning of the Setdb2 gene

30

(Methods) . To study whether the induction of Setdb2 depended on IFN signaling, we infected WT as well as Ifnarl^ ^' , IrfT' ^~ and StatV^ mice, each of them lacking key molecules required for IFN signaling, with influenza virus. We collected lung tissue at 18 hours after infection and detected increased expression of Setdb2 in infected WT lungs (Fig. Id). In contrast, the induction of Setdb2 was strongly reduced in Ifnarl ^' ' ^' lungs, indicating that type I IFN signaling is essential for Setdb2 up-regulation in vivo. A reduction of Setdb2 expression was also observed in lrp ^~ ' ^' and Stall ^' ' ^' mice (Fig. Id). Similarly, infection of primary mouse bone marrow-derived macrophages (BMDMs) with influenza virus resulted in an Ifnarl - dependent up-regulation of Setdb2 transcription (Fig. le). To investigate its protein levels, we raised a monoclonal antibody (mAb) against a c-terminal region of mouse Setdb2 (clone 7H7F11, Methods). Consistent with RNA expression, we detected increased Setdb2 protein levels after infection of BMDMs in an Ifnarl -dependent manner (Fig. le). Detection of the known IFN-stimulated protein Zbpl (alias Dai) served as a control ³¹ . Stimulation of BMDMs with IFN (type I IFN), IFNy (type II IFN) or IFN (type III IFN) revealed that Setdb2 and Zbpl are induced by type I IFN as well as by type II IFN (Fig. If). The induction of Setdb2 by type II IFN was partially dependent on Ifnarl, indicating a secondary requirement for endogenous type I IFN ³² .

Next, we elucidated the mRNA and protein expression profiles of Setdb2 after stimulation of BMDMs with the TLR2 agonist PAM3, the synthetic viral RNA analog and TLR3 agonist polyLC and the TLR4 agonist LPS ⁵ . Treatment of WT BMDMs resulted in up-regulation of Setdb2 on the RNA and protein level (Fig. lg). Again, this induction was entirely dependent on type I IFN signaling, as Setdb2 was not induced in Ifnarl ^{' '} BMDMs. The induction of Setdb2 and Zbpl upon PAM3 stimulation was TLR2-dependent (Fig. 6). In addition to Setdb2, stimulation of WT BMDMs with polyLC led to an induction of other PKMTs such as Setdlb and Prdml (alias: Blimpl), a well-studied regulator of B- and T-cells ²¹ , that were however not altered upon influenza virus infection in vivo (Fig. 7, Fig. lc). Elevated expression of Setdb2 mRNA in WT BMDMs persisted for at least 24 hours (Fig. 7).

Setdbl modulates expression ofNF-κΒ target genes

(~~"~r

We generated Setdb2 genetrap mice (Setdb2 ) to investigate the biological function of Setdb2 (Fig. 8 and Methods). Western blot analysis of spleen, lung and BMDMs showed a strong reduction of the Setdb2 protein level in the hypomorphic Setdb2 mice compared to WT controls (Fig. 2a). Next, we compared the transcriptomes of polyLC-stimulated WT and GT/GX

Setdb2 BMDMs by RNAseq. This experiment revealed a significant enrichment of known NF- B target genes among the up-regulated transcripts in Setdbl BMDMs (p-value = 1.35xl0 ^"8 , Fig. 2b, and Methods). Interestingly, these NF- Β target genes represented a subset of genes including Cxcll, 1112b, SlOOaS/9, Lcnl, Cxcl2, Chi3ll and Ltf, each of which has been implicated in antibacterial defense. Other established NF-κΒ regulated genes such as 116, however, were not affected. Upstream activation of the NF-κΒ cascade as measured by ΙκΒ degradation showed no difference between stimulated WT and Setdb2 ^GVGT BMDMs (Fig. 9), suggesting that Setdb2 acts downstream of ΙκΒα-mediated NF-κΒ activation.

One of the genes found with particularly high levels of expression in polyLC stimulated Setdb2T ^Jl BMDMs was Cxcll. It encodes a key chemoattractant for neutrophils, a type of leukocyte shown to be critically involved in bacterial clearance in superinfection ¹⁴ ' ¹⁵ ' ¹⁶ . Thus, we decided to focus on the effects of Setdb2 -mediated regulation of Cxcll. Indeed, stimulation with different TLR agonists resulted in the induction of significantly more Cxcll transcripts in Setdb2" ^{Ji jl} BMDMs as compared to WT controls (Fig. 2c). This finding was confirmed at the level of secreted protein (Fig. 2d). To investigate the effects of type I IFN signaling on Cxcll expression, we stimulated BMDMs with polyI:C in the presence of Ifnarl -blocking antibodies. This led to diminished levels of Setdb2, which correlated inversely with the expression of Cxcll (Fig. 10). These differences were seen not only in WT but also in Setdb2 ^GT/GT BMDMs, which may be due to the residual levels of Setdb2 in the Setdb2 BMDMs and/or other IFN-I driven Setdb2-independent pathway. Finally and in agreement with the previous results, infection with influenza virus led to increased Cxcll expression in Setdb2 compared to WT BMDMs (Fig. 2e, f).

Setdbl binds to the Cxcll promoter and mediates H3K9 tri-methylation

Methyltransferases of the SUV39 family preferentially methylate the histone substrate H3K9 ²² ' . Setdb2 was shown to catalyze the repressive mark H3K9me3 ' . We, therefore, hypothesized that Setdb2 would inhibit Cxcll expression by introducing repressive marks in the Cxcll promoter region. To test if Setdb2 was able to bind to the Cxcll promoter, we used the Setdb2-specific mAb clone 7H7F11 to perform chromatin immunoprecipitation (ChIP) experiments. BMDMs were treated with polyI:C for two hours and Setdb2-specific enrichment of genomic DNA was quantified by real-time PCR. Compared to sequences of the Actb promoter, we found a significant enrichment of the promoter region and exon 1 of Cxcll in Setdb2 ChlPs (Fig. 3a), indicating that Setdb2 binds to the Cxcll promoter.

Next, we addressed whether Setdb2 binding correlated with altered chromatin modifications. To test this, we performed H3K9me3 ChIP experiments in the presence or absence of polyLC stimulation in either WT or Setdb2 ^GT/GT BMDMs. Under unstimulated conditions, Setdb2 ^GVGT cells displayed less H3K9 tri-methylation compared to WT controls (Fig. 3b). This repressive mark was significantly increased at the Cxcll promoter upon polyLC stimulation of WT cellSi which is consistent with the rapid induction but only transient expression of this gene (Fig. 11). This increase of H3K9me3 was absent in Setdb2 cells. The promoter of the actively transcribed gene Actb had low levels of H3K9 tri-methylation in both WT and Setdb2 ^GVGT BMDMs. Finally, we analyzed the presence of the activation mark H3K9ac and, as expected, found enrichment at the promoter of Actb (Fig. 3c). Together, these data suggested that Setdb2 mediated the repression of Cxcll expression at the chromatin level.

Setdbl deficiency results in exacerbated lung inflammation

Experiments with Setdb2 BMDMs revealed an increased expression of Cxcll, which as chemoattractant for neutrophils is important for efficient pathogen clearance as well as implicated in immunopathologies . To corroborate our in vitro findings, we challenged WT and Setdb2 mice using a model of LPS-induced pulmonary neutrophilia. Four hours after an intranasal application of LPS, bronchoalveolar lavage fluid (BAL) was obtained to determine potential changes in Cxcll secretion and cell infiltration. In addition, total RNA was extracted from lung tissue to analyze differential gene expression. This demonstrated increased levels of Cxcll mRNA expression (Fig. 4a) and protein secretion (Fig. 4b) in Setdb2 ^GllGT mice. The Cxcll increase was accompanied by elevated total cell infiltration (Fig. 4c) and neutrophilia in the airways (Fig. 4d). Macrophage numbers were similar in WT and Setdb2 mice (Fig. 4e). We conclude that Setdb2 mice show increased lung infiltration of neutrophils in the early phase of inflammation. W

123

Setdb2 mediates influenza virus-induced susceptibility to superinfection by Streptococcus pneumoniae

Importantly, the rapid recruitment of neutrophils by the early cytokine Cxcll is crucial for the prevention of excessive lung inflammation as well as for the clearance of Streptococcus pneumoniae (Sp), the most common bacterial agent found in influenza virus-induced superinfection ' ' ' . Therefore, we sought to investigate the response of Setdbl mice in a superinfection model of Sp after primary influenza virus infection.

Upon intranasal infection with influenza virus, Setdb2 mice induced more Cxcll compared to WT controls (Fig. 5a, b). In the lung, Cxcll is secreted by multiple cell types including alveolar macrophages ³⁵ . Indeed, influenza virus infection of alveolar macrophages taken ex vivo from BAL of Setdb2 mice expressed more Cxcll compared to WT controls (Fig. 12), implicating this cell population as one source of Cxcll in our infection model in vivo. The levels of other inflammatory mediators such as Cxcl2, 116 and 1110 were similar in BAL of Setdb2 ^GVGT and WT mice (Fig. 14a-c). Despite increased levels of Cxcll in Setdb2 ^GVGT mice upon influenza virus infection, both mouse strains showed comparable neutrophil infiltration in the lung at this stage of infection (Fig. 5c, d). A comprehensive profiling of other cell populations in the lung tissue and BAL, including monocytes/macrophages/dendritic cells, alveolar macrophages, NK cells, T cells and B cells revealed no differences in uninfected as well as in influenza virus infected Setdb2 ^Jl " ^Ji and WT mice (Fig. 15a-d). The viral loads were comparable in both mouse strains (Fig. 16a).

In single infection with Sp, Setdb2 and WT mice exhibited no significant differences in the induction of Cxcll (Fig. 14d), the numbers of neutrophils (Fig. 15e, f) and the bacterial burden (Fig. 16b).

Superinfection of influenza virus-infected mice with Sp led to a further increase of Cxcll (Fig. 5b). This was accompanied by increased infiltration of neutrophils in lung tissue and in BAL of Setdb2^ ¹ but not WT mice (Fig. 5c, d, Fig. 15g, h). Two days after superinfection with Sp, mice showed severe signs of pneumonia (Fig. 5e) and pulmonary edema as measured by lung wet weight (Fig. 5f). Interestingly, the gross pathological appearance including size, weight, number of affected lobes, and hemorrhagic lesions of Setdb2 lungs was milder as compared to WT controls (Fig. 5e, f). Histopathological analysis of lung sections confirmed and extended the macroscopic findings, showing reduced signs of pneumonia including bronchitis, endothelialitis and inflammatory infiltrates (Fig. 5g, h). In line with the ameliorated pathological findings in Setdb2 mice at this advanced stage of bacterial superinfection, we found decreased levels of mRNA and protein of the pro-inflammatory cytokine 116 (Fig. 5i, j) and a reduced bacterial burden compared to WT control (Fig. 5k).

In order to confirm that Setdb2 is indeed upregulated upon viral infection and, hence, that its targeting is beneficial in the therapy of e.g. bacterial superinfections, we compared Setdb2 induction in the lungs of mice infected with either Streptococcus pneumoniae or influenza virus. Setdbl mice express decreased amounts of Setdb2 and are less sensitive to bacterial superinfection after primary influenza virus infection, compared to wild-type WT mice. This is in contrast to the situation after infection with Streptococcus pneumoniae (SP) only, where no difference in bacterial clearance and overall pathology can be observed between the two genotypes. To elucidate this difference on the molecular level, we measured the induction of Setdb2 after SP and influenza virus (PR8) infection. We show that influenza virus, but not SP infection was able to induce Setdb2 expression. This highlights the crucial importance of Setdb2 induction in the context of infections, such as viral infections or, in particular, bacterial superinfections.

WT as well as Setdb2 mice were either infected with SP, PR8, or left untreated as control (Ctrl) and lungs were harvested at indicated time points. Total RNA was extracted from lungs and real-time PCR was used to measure Setdb2 expression. Interestingly, SP infection did not induce Setdbl expression at any of the time points investigated, see Fig. 13. In contrast, Setdb2 was strongly induced 5 days (5d) after influenza virus infection, the time point used to perform superinfection experiments (see e.g. Fig. 5). Setdb2 expression is displayed as fold inducion compared to uninfected mice. Scatter blot indicate individual mice pooled from different experiments. Statistical analysis was performed by unpaired t-test. **** p < 0.0001. The Setdbl genetrap mice show a residual expression of about 20 % of that of the wild-type mice. Nevertheless Setdbl mice can show an induction of Setdb2 upon influenza virus infection relative to non-infected Setdbl genetrap mice. This induction of Setdb2 in Setdbl mice can be due to the presence of the wild-type promoter of the Setdb2 gene in W 201

125

GT/GT

Setdb2 mice. The expression of Setdb2 can be increased by stimuli of the promoter like interferons, TLR ligands etc. In sum, the Setdb2 mice represent a model for antagonists of Setdb2. They can also be used as a model for a clinical setting characterized by upregulation of Setdb2, e.g. upon influenza infection, for example, relative to non-infected Setdb2 mice.

Taken together, we conclude that influenza virus-induced Setdb2 expression had a detrimental effect on the early recruitment of neutrophils, the subsequently delayed pathogen clearance and the impaired tissue integrity during bacterial superinfection.

The data above provide thus a scientific rationale for the use of Setdb2 antagonists in a clinical setting that is characterized by Setdb2 induction/upregulation, e.g. upon viral infection. Because Setdb2 is upregulated in such a setting, it can serve as a target for Setdb2 antagonists. The antagonists in turn induce, by downregulation of Setdb2, the upregulation/secretion/activation/enhanced infilitration of chemoattractans for neutrophils, like Cxcll/CXCL8. The enhanced/stimulated immune response (involving the upregulation etc. of neutrophils) has a beneficial therapeutic effect on the infection/infectious disease.

Maintaining a balance between effective pathogen defense and the prevention of excessive inflammation, autoimmunity, and immunopathology is the central task of immune regulation ¹⁰ '

13

. Type I IFN and NF-κΒ signaling are two important pathways for this process and are subjected to multiple layers of crosstalk, many of which are still poorly understood.

We identified Setdb2 as a crucial part of the IFN-mediated immune response that provides a hitherto unknown layer of regulatory crosstalk between the type I IFN and NF-κΒ signaling pathways. Our finding of Setdb2 being an interferon-stimulated gene (ISG) itself points towards an important role in the innate immune response, which may contribute to the prevention of excessive immune-mediated pathology in particular infections and/or inflammatory conditions. This evolutionary strategy, however, may turn into a double-edged sword during bacterial superinfection, by causing impaired bacterial clearance and severe tissue damage (Fig. 17). Previously, type I IFN signaling was shown to have a detrimental role in the pathogenesis of virus-induced susceptibility to bacterial superinfection ¹⁴ ' ^{15, 16} . Accordingly, Setdb2 may be responsible and mediate at least parts of this important type I IFN-dependent mechanism.

Chromatin modifiers can be recruited to their targets through specific interactions with transcriptional regulators and/or chromatin-associated factors. We hypothesize that a similar mechanism facilitates the specific recruitment of Setdb2 to its target gene promoters to introduce repressive H3 9me3 chromatin marks. Yet, we cannot exclude the possibility that Setdb2 may recruit other methyltransferases or transcription factors. Likewise, the function of Setdb2 may be determined by mutually non-exclusive cellular and immunological parameters (e.g. cell type, inflammatory state, pathogen type) as well as by the complex epigenetic context of multivalent chromatin modifications ³⁸ . Independent of direct histone methylation and in analogy to other PKMTs, Setdb2 may also methylate non-histone protein targets ³⁶ ' ³⁹ ' ⁰ .

This data shows that reduced levels of Setdb2 lead to increased production and secretion of Cxcll . Our data suggest that the increased levels of Cxcll expression and neutrophil recruitment observed in Setdb2 mice may be causally involved in the ameliorated pathogenesis of bacterial superinfection. Neutrophils execute multiple roles including the regulation and resolution of inflammation and the elimination of bacterial pathogens ³³ ' ⁴¹ . In this study we observed increased Cxcll expression in the lungs of influenza virus infected Setdbr ¹ '"' compared to WT mice. This alteration in chemokine production and secretion was not sufficient to augment the virus-induced neutrophil response. The increased Cxcll expression in Setdb2 mice manifested phenotypically only upon superinfection as elevated numbers of neutrophils in the lungs. This could be due to altered time kinetics of the host response and/or the involvement of additional signals provided by the bacterial superinfection. Together, our data suggest that the influenza virus-induced expression of Setdb2 reduced the antibacterial response in WT mice, leading to aggravated lung pathology.

These findings provide evidence for a regulatory function of Setdb2 in Cxcll expression, the recruitment of neutrophils, and the pathological outcome of superinfections. However, we cannot exclude that other Setdb2-modulated genes may contribute to improved bacterial clearance and reduced tissue inflammation in Setdb2 mice. Our RNAseq experiment performed with polyLC stimulation identified several other upregulated antibacterial genes in Setdb2" ^{l Ji} BMDMs. These genes included the chemotactic proteins S100a8/9 , Marco, a macrophage scavenger receptor that is implicated in the phagocytosis and clearance of bacteria in the lung ⁴³ ' ⁴⁴ , as well as Chi311, which was demonstrated to promote both improved clearance of Sp and disease tolerance ⁴⁵ . Yet, their expression profiling by real-time PCR revealed no differences between superinfected WT and Setdb2 ^GJ/GT mice under the experimental conditions of this study (data not shown). Finally, Setdb2 may not only regulate antibacterial responses but may as well modulate genes involved in disease tolerance in superinfection ⁴⁶ .

In summary, our study assigns Setdb2 an important regulatory role in the IFN-mediated immune response and in the pathogenesis of virus-induced susceptibility to bacterial superinfection. Several inhibitors for PKMTs are currently in clinical trials ⁴¹ . Thus, Setdb2 is an attractive therapeutic target for the treatment of superinfections and other inflammatory conditions.

Example 2: Characterization of S£TZ)i?2-knockdown in human cell lines

To study the role of human SETDB2 on immune responses, we used two different approaches to interfere with its expression:

The human embryonic kidney cell line Hek293T cell was used to deplete hSETDB2 by specific siRNAs (Qiagen) (Figure 17). Cells were transfected with a control siRNA, 2 different SETDB2-specific siRNAs (siRNA 1+2), a pool of both siRNAs (siRNA pool), or were left untreated as control (control siRNA). siRNA 1 :

#SI00141393, Qiagen

Target sequence: CCGAGAGCATCTGAACTCTAA (SEQ ID NO: 7)

siRNA 1 targets SETDB2 at nt position 2481-2501 of a nucleic acid molecule having a sequence encoding Setdb2 as shown in SEQ ID NO: 1. siRNA 1 targets SETDB2 at nt position W 201

128

1881-1901 of a nucleic acid molecule having a sequence encoding Setdb2 as shown in SEQ ID NO: 3.

siRNA 2:

#SI031 16904, Qiagen

Target sequence: TCGGCCGCTTCCTTAATCATA (SEQ ID NO: 8) siRNA2 targets SETDB2 at nt position 2843-2863of a nucleic acid molecule having a sequence encoding Setdb2 asshown in SEQ ID NO: 1. siRNA2 targets SETDB2 at nt position 2243-2261 of a nucleic acid molecule having a sequence encoding Setdb2 asshown in SEQ ID NO: 3.

After 24h total RNA was extracted from transfected cells and real-time PCR was used to measure SETDB2 expression. Cells transfected with the siRNAs 1 and 2 as well as the siRNA pool showed strong reduction in SETDB2 expression.

Example 3: Induction of CXCL8 in human SET.Di?2-knockdown cell lines

As a second strategy of interfering with Setdb2 expression, human haploid KBM7 cells were targeted with a retroviral gene trap cassette and clones with insertions in Setdb2 were selected. Wild type (WT) and hSETDB2 genetrap (GT) cells were harvested and total RNA was prepared. Measurements by real-time PCR revealed that SETDB2 GT cells show a reduction in SETDB2 expression as compared to WT cells (Figure 18a).

Functional consequences of SETDB2 depletion in human cells

In the murine system we could identify the neutrophil chemoattractant Cxcll as one critical Setdb2-regulated antibacterial gene. To validate this finding in the human system, we studied CXCL8 expression and secretion in a human cell line lacking SETDB2. CXCL8 is the human ortholog of mouse Cxcll and plays a similar important antibacterial role as chemoattractant for neutrophils {Richmond A, Nature Reviews Immunology 2002). WT as well as SETDB2 GT human haploid KBM7 cells (as characterized in Figure 18a) were stimulated with the TLR3 agonist polyLC or left untreated as control. After indicated time points, total RNA was extracted and real-time PCR was used to measure CXCL8 expression. KBM7 GT cells that have reduced SETDB2 expression showed a stronger induction of CXCL8 as compared to WT cells, resembling the phenotype observed in the murine system (Figure 18b).

WT as well as SETDB2 GT human haploid KBM7 cells (as characterized in Figure 18a) were stimulated with the TLR3 agonist polyI:C or left untreated as control. After indicated time points, cell supernatant was harvested and analyzed for secreted CXCL8 protein by ELISA. Similar to the real-time analysis in (Figure 18b) KBM7 GT cells secreted an increased amount of CXCL8 as compared to WT cells (Figure 18c). Again, these results are in line with our observations in the murine system.

Example 4: Treatment of Bone marrow-derived macrophages (BMDMs) with Sinefungin and SAH increases Cxcll expression after TLR stimulation

Setdb2 belongs to the SUV39 gene family of histone methyltransferases. All family members share a Suvar 3-9/Enhancer-of-zeste/Trithorax (SET) domain that transfers methyl residues from S-adenosyl-methionine (SAM) to the amino group of target lysines thereby catalyzing H3K9 methylation. After we identified Setdb2 as a repressor of Cxcll expression, we aimed to mimic the activating effects seen in Setdb2 knockdown cell, using specific inhibitors of Setdb2 that can interfere with target lysine methylation.

Bone marrow-derived macrophages (BMDM) from either wild type (WT) or Setdb2 ^GT/GT mice were stimulated with the toll-like receptor 3 agonist polyI:C or left untreated. In addition to polyLC cells were either treated with PBS, the organic solvent DMSO (inhibitors dissolved in DMSO) or two different methyltransferase inhibitors; Sinefungin, a SAM analogue or S- adenosyl-L-homocysteine SAH, a SAM-derived product that is usually generated after methylation (20μΜ each). Treatment with inhibitors or controls (PBS, DMSO) was performed 1 hours prior to stimulation with polyI:C. 8 hours after polyLC stimulation the supernatant was harvested and Cxcll protein secretion was quantified by ELISA. The results are shown in Fig. 20. BMDMs that were treated with PBS or DMSO only did not show detectable Cxcll expression. BMDMs that were stimulated with polyI:C and PBS/DMSO secreted Cxcll . Again cells derived from Setdb2 mice produced more Cxcll compared to WT controls. Interestingly, both the treatment of BMDMs with Sinefungin and SAH significantly increased the production of Cxcll in both genotypes. From these results we conclude that the inhibition of Setdb2 can mimic the effects seen in a situation with low Setdb2 expression. Statistical analysis was performed by unpaired t-test. p-values were indicated as follows: * p<0.05, ** <0.01.

The present invention refers to the following nucleotide and amino acid sequences:

The sequences provided herein are available in the NCBI database and can be retrieved from world wide web at ncbi.nlm.nih.gov/sites/entrez?db=gene; Theses sequences also relate to annotated and modified sequences. The present invention also provides techniques and methods wherein homologous sequences, and variants and fusion transcripts/fusion genes of the concise sequences provided herein are used. Preferably, such "variants" are genetic variants or fusion transcripts/fusion genes.

SEQ ID NO: 1

Nucleotide sequence encoding Homo sapiens methyltransferase SET domain bifurcated 2 (Setdb2), isoform a (cDNA); NM__031915.2. A corresponding mRNA molecule encoding Homo sapiens Setdb2, isoform a, has a nucleotide sequence that corresponds to the sequence shown in SEQ ID NO: 1, with the exception that the thymidine (T) residue(s) of the sequence shown in SEQ ID NO: 1, is/are replaced by (a) uracil (U) residue(s). The target sequence of siRNA 1 (SEQ ID NO: 7) is underlined. The target sequence of siRNA 2 (SEQ ID NO: 8) is underlined and in bold letters. The coding region ranges from position 907 (start codon) to position 3066 (stop codon).

The "SET" domain of Setdb2 consists of a "pre-SET domain" and a "bifurcated SET Domain" (herein designated as "SET1" and "SET2", respectively). The region encoding the "pre-SET domain" ranges from position 1639 to position 1983. The region encoding SET1 ranges from position 2005 to position 2190. The region encoding SET2 ranges from position 2794 to position 2994.

ATCCCCGGTAGAGGCAGGGCGGGACTGTTGTGGTTGAGATGAAGGCTAGTAAATGGT GAAGTA CTTCCCGGCCAGAGGGCACCTGCGCTCGGGAGGTTTGGGCGGCTTGGCGTCGGAGGAGAG CCC CACCCGCGGAGGAACCCAGCCTTGCCAACGGAGCTGGCGGAGCTCACTCCTCAGGTCAGG CGG

GCGGCGTAGAAAACGCAGCGGAGCCAGGTGAAACCAAGGCACCGCCGTGGCTGGCCC CCGACA

GTTCCTCTAGCCGGGAGGTTGGAGGAGCTGAAAACGCCGCGGAGCCCTCGGCCGCCC GAGCAG

GGGCTGGACCCCAGCCCTTGCAGCCTCCCTTCTCCTGGCACCCAAGTGCAGTCCTGG CTGCAG

AAGGGGCCGCGGGCGCACTGAGTTTCCAACCTCCATTTCAGCCTGTCTGTCTCAGGG TGCAGC

CTTAATGAGAGGTGATTCCTAAGCTGCTGGGAACCTGAGGTTGTCAAAGGGGCGGCA GGAAAT

GGACAGCAGTATAAAACCCAGAAGCAGAACTTGAAGGTTAAACCACTAGCCCATTTC ACAGAA

TGTTTCATCCATTTGTGGACCAAAAGATGGAGTTGGTTTTTATTTTTAAAAAGATAA TGTTAA

TGATCTGATACCACTACAAATATTTACGTGAGAAGATTCATGGACTTGTCTTTTGGT TGGACT

GTCACTCATTTCTGAAAGTTTCTTCAGCCACAATTTCTATTTGAAAATTCAAGTATC AAAGGA

TACCAGGTTTAGAATGGTATAATGATGTATTTTGTCTGAGGACTGCAAATTTTATAG AGACCA

CAGTTGGATTCCAGTGATATTCTGCAATCAAAGTGATTTGATAAACCTAATTTTGAA GCATTT

TATATTTATAAGCGACATCAAAAGATGGGAGAAAAAAATGGCGATGCAAAAACTTTC TGGATG

GAGCTAGAAGATGATGGAAAAGTGGACTTCATTTTTGAACAAGTACAAAATGTGCTG CAGTCA

CTGAAACAAAAGATCAAAGATGGGTCTGCCACCAATAAAGAATACATCCAAGCAATG ATTCTA

GTGAATGAAGCAACTATAATTAACAGTTCAACATCAATAAAGGGAGCATCACAGAAA GAAGTG

AATGCCCAAAGCAGTGATCCTATGCCTGTGACTCAGAAGGAACAGGAAAACAAATCC AATGCA

TTTCCCTCTACATCATGTGAAAACTCCTTTCCAGAAGACTGTACATTTCTAACAACA GAAAAT

AAGGAAATTCTCTCTCTTGAAGATAAAGTTGTAGACTTTAGAGAAAAAGACTCATCT TCGAAT

TTATCTTACCAAAGTCATGACTGCTCTGGTGCTTGTCTGATGAAAATGCCACTGAAC TTGAAG

GGAGAAAACCCTCTGCAGCTGCCAATCAAATGTCACTTCCAAAGACGACATGCAAAG ACAAAC

TCTCATTCTTCAGCACTCCACGTGAGTTATAAAACCCCTTGTGGAAGGAGTCTACGA AACGTG

GAGGAAGTTTTTCGTTACCTGCTTGAGACAGAGTGTAACTTTTTATTTACAGATAAC TTTTCT

TTCAATACCTATGTTCAGTTGGCTCGGAATTACCCAAAGCAAAAAGAAGTTGTTTCT GATGTG

GATATTAGCAATGGAGTGGAATCAGTGCCCATTTCTTTCTGTAATGAAATTGACAGT AGAAAG

CTCCCACAGTTTAAGTACAGAAAGACTGTGTGGCCTCGAGCATATAATCTAACCAAC TTTTCC

AGCATGTTTACTGATTCCTGTGACTGCTCTGAGGGCTGCATAGACATAACAAAATGT GCATGT

CTTCAACTGACAGCAAGGAATGCCAAAACTTCCCCCTTGTCAAGTGACAAAATAACC ACTGGA

TATAAATATAAAAGACTACAGAGACAGATTCCTACTGGCATTTATGAATGCAGCCTT TTGTGC

AAATGTAAT CGACAATTGTGTCAAAACCGAGTTGTCCAACATGGTCCTCAAGTGAGGTTACAG

GTGTTCAAAACTGAGCAGAAGGGATGGGGTGTACGCTGTCTAGATGACATTGACAGA GGGACA

TTTGTTTGCATTTATTCAGGAAGATTACTAAGCAGAGCTAACACTGAAAAATCTTAT GGTATT

GAT GAAAAC GGGAGAGAT GAGAAT AC T AT GAAAAAT AT AT T T T CAAAAAAGAGGAAAT TAGAA

GTTGCATGTTCAGATTGTGAAGTTGAAGTTCTCCCATTAGGATTGGAAACACATCCT AGAACT

GCTAAAACTGAGAAATGTCCACCAAAGTTCAGTAATAATCCCAAGGAGCTTACTGTG GAAACG AAAT AT GAT AAT AT T T CAAGAAT T C AA A C ATT C AGT T AT AGAGA CCTGAAT C CAAGACA

GCCATTTTTCAACACAATGGGAAAAAAATGGAATTTGTTTCCTCGGAGTCTGTCACT CCAGAA

GATAATGATGGATTTAAACCACCCCGAGAGCATCTGAACTCTAAAACCAAGGGAGCA CAAAAG

GAC T CAAGTT CAAAC CATGTTGATGAGTTT GAAGAT AAT C T GCT GA T GAAT C AGAT GT G AT A

GATATAACTAAATATAGAGAAGAAACTCCACCAAGGAGCAGATGTAACCAGGCGACC ACATTG

GATAATCAGAATATTAAAAAGGCAATTGAGGTTCAAATTCAGAAACCCCAAGAGGGA CGATCT

ACAGCATGTCAAAGACAGCAGGTATTTTGTGATGAAGAGTTGCTAAGTGAAACCAAG AATACT

TCATCTGATTCTCTAACAAAGTTCAATAAAGGGAATGTGTTTTTATTGGATGCCACA AAAGAA

GGAAATGTCGGCCGCTTCCTTAATCATAGTTGTTGCCCAAATCTCTTGGTACAGAAT GTTTTT

GTAGAAACACACAACAGGAATTTTCCATTGGTGGCATTCTTCACCAACAGGTATGTG AAAGCA

AGAACAGAGCTAACATGGGATTATGGCTATGAAGCTGGGACTGTGCCTGAGAAGGAA ATCTTC

TGCCAATGTGGGGTTAATAAATGTAGAAAAAAAATATTATAAATATGTAACTAACGC CTGTTT

GTGAAATTAGCTTATCAGGCTGAAATTAAAGCCATGCAAAAGAAGGTCTAGGTCCAT CAAGGA

AATTCCCCTCCGTTTTCCTTTGTCATGGGGTTTATGTTTTATTTCAGATTTTATTTG TGTGAC

TTAGAAATTCCAGGAACACAATTAGGATATTTTCATACACATAGGGTATCTTGTTCA CTGCTG

TGCTACTTTACATGAGTAGGATGGAAGTGTATATTTTATATGAAATACCACTGTACA ATTTAT

AATTTATTTACAAATTATATATTAAGAGAAACAAATGTCATAACAGAACTCAGCTGT TTCTAA

TTGCTTTTGTGACTGTTACCTTTTAGTTCATGCCCCCCCAAAGAGCTAAATTTCACA TTTTTA

CCTACAAAATTGATTTTTAATTCCTGGCAAATAATTTACCATTATGAGCTACAAGGT GGGCAA

CAGCGCCTGAGGATCTAATTTTATGCATATTACTCCCAAGTATTTTAACACTTGTTG GAGAAG

CAATATCTGGATCGATAAAACACTGTCCCATCAACCATTTGAGTGGGGAGAGGGAGA AGCTCT

TCTGTAAGTAAGATTCTGGCAAGCTCTTTGAAATGAGTCTTCTTTCCCACAGATTTT CTCTAC

TCTTTCTATACAAACAGATAGGAGAAGAGGGAATAGAAACCTGGAGGAACTTGAATA TTTTTG

TTCT AGAT AGAGAT ACAGTTACTGAAAAGGAAACCTAGAAAGTAGTCACACGTTGCTTATTT A

GGCCAGAAGTAATTGTACTGGGCAAAAATTTCACTTAAAAAACACAAGAAGTCCAGG TATGGT

GGCTCAGACCTGTAATCCCAGCACTTTGAGAGGCCGAGGCAGGTGGATTACTTGAGC CTAGGG

GTTCAAGACCAGCTTGGGCAACATGTCAAAACCCTGTCTCTACAAAAAATACAAAAA TTAGCC

TGGCATGATGGCATGTGCCCGTAGTCTCAGCTACTCAGGAGTGAGGTGGGAGGATCA TTTGAG

CTCAGAAGGTCAAGGCTGCAATGAGACATAATTTCACCATAGTACTTCCAGCCTGGG CAATAG

AGCAAGACTCTCTCTCAAAAAAAACAGCACACACACACACACACGAAAACAATTCTG AACTAT

GAAATCTGAAACAGCCCCTTGGTATCTCCTGGGCATGATTTGCAAATCTTTTTTTTT TACAGA

AAAAAGGCAAAGAGTAAGCACTTTGCCATAGGTTACTTGGCCGTGATCATCTATCTA GTGGAA

AAGGGGACTGGGAAGCCCAAGCAGACTGGGAAACCAGACAGCTAGGAAAAGGAGCAA AACATA

GCCCAGCAACCTACAGATGAAGAAAGTTGAGAAATCCATTTATTCACCATAGAGACG CAGGAA

TTTCAGGCAATGCACTAAAATGAAATGGGGGAAAAAAGCTTGATCAGTATGGGAACC ATTTTT GTGCAAAAGGGAATATTATGGATCAGCCAGTATTTCTTTGAGCTCTGCCTGTGGAGTCCA TTT

GACCTTTAGAAATATGAGGTATTCTGTCAGTTTTATCTTCTTGGAGAAATTTCTCCT AAAATC

TTGATTTGCTTTAGTCTGGACTGGTTCATAGCCATCATCTTCCATCAGTACCCCAGA GATTCA

CTTTGTCTCTTATGTGGGATCTGTTTCCAGTTAGATGCCATTATTTTCCTTTTCCTT GGTTTA

CTCTTCCACATATTGGTAAAGCTCTTCCAATAGCTTTTGGAAAGGAAAAATGAAAAG TAAATG

TTTTGAATCTCTGTGTGTTTGACAATGTCTTTATTTTACCCTTATACCTGATTGCTG TTTTGG

TTGGCAAGGTATAGGATTCTTTAGTGGTCTCCATGCCCAGTTTTGAAGACATCTGCT AGCTTT

CAGTGCTGTTGCTGTGGAGTCTGAAAATCTGTCTTCTGGCTTCCAGGGTGACTACTG GAAATT

GAATGCCATTCTGTTCCTTCTCTTTTGCATATATAATCCATTTTTATCTCTCTTGAA GCTTAT

AGGTTTATCTTTGTCTCAATGTTCTGTCCCTGTTAAGAGTCCATTTTCATCCTTTGT ACTAGG

TGCCTGGTGGGATCATTCCGTCTGAAACTAATGATTTCCCATCTCTTCACTGTTTCT GGAATT

CCTGTTTTCCAGATGTTAGACCTCCAGAATTTGATCTCTAATTTTCCTATCTTTTCT CTTAAC

TTTCAGCTCTGTCTTCTTGCTAGGACCTTTTCCTAGGAGCATTTCTCAATTTAATCT TCCAGT

TCATCTGTTGCATTTTATTTTTCTAGTCTCATATTGTCTCATATTTTTAATTTCTAA GAGCTC

CCCTTCTCCGAATATTCTTTTTTTTTAATAGCATCCTATTTTGGCTCATGGTTGCAG TATTTT

ATCTCCTTGAAGATGTTTGTGTGTTTATGTATGTATATGCACACACGTATACATACA CATACA

GGCATGCATCTCTGTATTCTTTCGGCATAATCTGTGTCCTCCAGGGTTTGTTTCTTT GTTTCC

CCTGTATGTTTGTTTTGGTCGTTCACATTATAGGCTTTCCTCAGAGTTAATGGTCTT GGTAGT

CTACTCATATTTAAGTGTGGAACACCAAAAAGCTTACTATAAGCTGAGAGTGTGGTA AAGGGC

TCTTTGTTTTACTATGACCTACCTGAGCTATCTTGCTGGGGAACACCCTAATGTCAG TCTCTT

TATAAAGGGCCTTTCATTTTGGCCTGGCAAGAAATACTCTTTCATCCTCCTGCATGG AGGGCA

AAAAAAAATTTAAAAATTGGCTGCTAGGGTCTGTCTGCTCACTTCCCTGTTTTGCAG ACCCCA

CACTCTTCTGCAATTCATTTCATAGTTGTCAAGACTATACAAATTGTCCTTTTTAAT GTTCTC

TCTTCTGCTATCCCTAGTTGGCAGTCTTCCTCTTTACAACCTGCTGAAAGTGGAAGA CCTCCA

GTTTTCCTTTAATTCCTCAGCAAACCACCAACTATTATATGTCTTTTTTCCAGAACA ACTTAT

TTTTTAACTATAATTATATGCATTTATGTTAGATTCACTGAAAACCTCATCTTGTAT GGTGCT

CTGTACCCTATGGGTGCTAAATAAAGGCTTGCTACTGGCAACTGGAAAAAAAAAAAA AAAAA

SEQ ID NO:2

Amino acid sequence of Homo sapiens methyltransferase SET domain bifurcated 2 (Setdb2), isoform a; UniProtKB: Q96T68; NP_114121.2

The "SET" domain of Setdb2 consists of a "pre-SET domain" and a "bifurcated SET Domain" (herein designated as "SETl" and "SET2", respectively). The "pre-SET domain" has an amino acid sequence of from position 245 to 359. SETl has an amino acid sequence of from position 367 to 428. SET2 has an amino acid sequence of from position 630 to 696.

MGEKNGDAKTFWMELEDDGKVDFI FEQVQNVLQSLKQKIKDGSATNKEYIQAMILVNEATI IN S STS IKGASQKEVNAQSSDPMPVTQKEQENKSNAFPSTSCEN SFPEDCTFLTTENKEILSLED KVVDFREKDSSSNLSYQSHDCSGACLMKMPLNLKGENPLQLPIKCHFQRRHAKTNSHSSA LHV SYKTPCGRSLRNVEEVFRYLLETECNFLFTDNFS FNTYVQLARNYPKQKEVVSDVDI SNGVES VPI SFCNEI DSRKLPQFKYRKTVWPRAYNLTNFSSMFTDSCDCSEGCI DITKCACLQLTARNA KTS PLS S DKITTGYKYKRLQRQI PTGIYECSLLCKCNRQLCQNRVVQHGPQVRLQVFKTEQKG GVRCLDDI DRGTFVCIYSGRLLSRANTEKSYGI DENGRDENTMKNI FSKKRKLEVACS DCEV EVLPLGLETHPRTAKTEKCPPKFSNN PKELTVETKYDN I SRIQYHSVIRDPESKTAI FQHNGK KMEFVS SESVT PEDNDGFKPPREHLNSKTKGAQKDS SSNHVDEFEDNLLIESDVI DITKYREE T PPRSRCNQATTLDNQNIKKAI EVQIQKPQEGRSTACQRQQVFCDEELLSETKNTSSDSLTKF NKGNVFLLDATKEGNVGRFLNHSCCPNLLVQNVFVETHNRNFPLVAFFTNRYVKARTELT WDY GYEAG VPEKE I FCQCGVNKCRKKIL

SEQ ID NO: 3

Nucleotide sequence encoding Homo sapiens methyltransferase SET domain bifurcated 2 (Setdb2), isoform b (cDNA); NM_001160308. A corresponding mRNA molecule encoding Homo sapiens Setdb2, isoform b, has a nucleotide sequence that corresponds to the sequence shown in SEQ ID NO: 3, with the exception that the tymidine (T) residue(s) of the sequence shown in SEQ ID NO: 3, is/are replaced by (a) uracil (U) residue(s). The target sequence of siRNA 1 (SEQ ID NO: 7) is underlined. The target sequence of siRNA 2 (SEQ ID NO: 8) is underlined and in bold letters. The coding region ranges from position 343 (start codon) to position 2466 (stop codon).

The "SET" domain of Setdb2 consists of a "pre-SET domain" and a "bifurcated SET Domain" (herein designated as "SETl" and "SET2", respectively). The region encoding the "pre-SET domain" ranges from position 1039 to position 1383. The region encoding SETl ranges from position 1405 to position 1590. The region encoding SET2 ranges from position 2194 to position 2394.

GAATGTTTCATCCATTTGTGGACCAAAAGATGGAGTTGGTTTTTATTTTTAAAAAGA TAATGT TAATGATCTGATACCACTACAAATATTTACGTGAGAAGATTCATGGACTTGTCTTTTGGT TGG ACTGTCACTCATTTCTGAAAGTTTCTTCAGCCACAATTTCTATTTGAAAATTCAAGTATC AAA

GGATACCAGGTTTAGAATGGTATAATGATGTATTTTGTCTGAGGACTGCAAATTTTA TAGAGA

CCACAGTTGGATTCCAGTGATATTCTGCAATCAAAGTGATTTGATAAACCTAATTTT GAAGCA

TTTTATATTTATAAGCGACATCAAAAGATGGGAGAAAAAAATGGCGATGCAAAAACT TTCTGG

ATGGAGCTAGAAGATGATGGAAAAGTGGACTTCATTTTTGAACAAGTACAAAATGTG CTGCAG

TCACTGAAACAAAAGATCAAAGATGGGTCTGCCACCAATAAAGAATACATCCAAGCA ATGATT

CTAGTGAATGAAGCAACTATAATTAACAGTTCAACATCAATAAAGGATCCTATGCCT GTGACT

CAGAAGGAACAGGAAAACAAATCCAATGCATTTCCCTCTACATCATGTGAAAACTCC TTTCCA

GAAGACTGTACATTTCTAACAACAGAAAATAAGGAAATTCTCTCTCTTGAAGATAAA GTTGTA

GACTTTAGAGAAAAAGACTCATCTTCGAATTTATCTTACCAAAGTCATGACTGCTCT GGTGCT

TGTCTGATGAAAATGCCACTGAACTTGAAGGGAGAAAACCCTCTGCAGCTGCCAATC AAATGT

CACTTCCAAAGACGACATGCAAAGACAAACTCTCATTCTTCAGCACTCCACGTGAGT TATAAA

ACCCCTTGTGGAAGGAGTCTACGAAACGTGGAGGAAGTTTTTCGTTACCTGCTTGAG ACAGAG

TGTAACTTTTTATTTACAGATAACTTTTCTTTCAATACCTATGTTCAGTTGGCTCGG AATTAC

CCAAAGCAAAAAGAAGTTGTTTCTGATGTGGATATTAGCAATGGAGTGGAATCAGTG CCCATT

TCTTTCTGTAATGAAATTGACAGTAGAAAGCTCCCACAGTTTAAGTACAGAAAGACT GTGTGG

CCTCGAGCATATAATCTAACCAACTTTTCCAGCATGTTTACTGATTCCTGTGACTGC TCTGAG

GGCTGCATAGACATAACAAAATGTGCATGTCTTCAACTGACAGCAAGGAATGCCAAA ACTTCC

CCCTTGTCAAGTGACAAAATAACCACTGGATATAAATATAAAAGACTACAGAGACAG ATTCCT

ACTGGCATTTATGAATGCAGCCTTTTGTGCAAATGTAATCGACAATTGTGTCAAAAC CGAGTT

GTCCAACATGGTCCTCAAGTGAGGTTACAGGTGTTCAAAACTGAGCAGAAGGGATGG GGTGTA

CGCTGTCTAGATGACATTGACAGAGGGACATTTGTTTGCATTTATTCAGGAAGATTA CTAAGC

AGAGCTAACACTGAAAAATCTTATGGTATTGATGAAAACGGGAGAGATGAGAATACT ATGAAA

AATATATTTTCAAAAAAGAGGAAATTAGAAGTTGCATGTTCAGATTGTGAAGTTGAA GTTCTC

CCATTAGGATTGGAAACACATCCTAGAACTGCTAAAACTGAGAAATGTCCACCAAAG TTCAGT

AATAATCCCAAGGAGCTTACTGTGGAAACGAAATATGATAATATTTCAAGAATTCAA TATCAT

TCAGTTATTAGAGATCCTGAATCCAAGACAGCCATTTTTCAACACAATGGGAAAAAA ATGGAA

TTTGTTTCCTCGGAGTCTGTCACTCCAGAAGATAATGATGGATTTAAACCACCCCGA GAGCAT

CTGAACTCTAAAACCAAGGGAGCACAAAAGGACTCAAGTTCAAACCATGTTGATGAG TTTGAA

GATAATCTGCTGATTGAATCAGATGTGATAGATATAACTAAATATAGAGAAGAAACT CCACCA

AGGAGCAGATGTAACCAGGCGACCACATTGGATAATCAGAATATTAAAAAGGCAATT GAGGTT

CAAATTCAGAAACCCCAAGAGGGACGATCTACAGCATGTCAAAGACAGCAGGTATTT TGTGAT

GAAGAGTTGCTAAGTGAAACCAAGAATACTTCATCTGATTCTCTAACAAAGTTCAAT AAAGGG

AATGTGTTTTTATTGGATGCCACAAAAGAAGGAAATGTCGGCCGCTTCCTTAATCAT AGTTGT

TGCCCAAATCTCTTGGTACAGAATGTTTTTGTAGAAACACACAACAGGAATTTTCCA TTGGTG GCATTCTTCACCAACAGGTATGTGAAAGCAAGAACAGAGCTAACATGGGATTATGGCTAT GAA

GCTGGGACTGTGCCTGAGAAGGAAATCTTCTGCCAATGTGGGGTTAATAAATGTAGA AAAAAA

ATATTATAAATATGTAACTAACGCCTGTTTGTGAAATTAGCTTATCAGGCTGAAATT AAAGCC

ATGCAAAAGAAGGTCTAGGTCCATCAAGGAAATTCCCCTCCGTTTTCCTTTGTCATG GGGTTT

AT GTT T TAT T T C AGAT T T TAT T T GT GT GAC T TAGAAAT C C AGGAAC AC AAT T AGGA A T T T

CATACACATAGGGTATCTTGTTCACTGCTGTGCTACTTTACATGAGTAGGATGGAAG TGTATA

TTTTATATGAAATACCACTGTACAATTTATAATTTATTTACAAATTATATATTAAGA GAAACA

AATGTCATAACAGAACTCAGCTGTTTCTAATTGCTTTTGTGACTGTTACCTTTTAGT TCATGC

CCCCCCAAAGAGCTAAATTTCACATTTTTACCTACAAAATTGATTTTTAATTCCTGG CAAATA

ATTTACCATTATGAGCTACAAGGTGGGCAACAGCGCCTGAGGATCTAATTTTATGCA TATTAC

TCCCAAGTATTTTAACACTTGTTGGAGAAGCAATATCTGGATCGATAAAACACTGTC CCATCA

ACCATTTGAGTGGGGAGAGGGAGAAGCTCTTCTGTAAGTAAGATTCTGGCAAGCTCT TTGAAA

TGAGTCTTCTTTCCCACAGATTTTCTCTACTCTTTCTATACAAACAGATAGGAGAAG AGGGAA

T AGAAAC CT GGAGGAAC T T GAAT T T T T T GT T CT GAT AGAG AT ACAGT AC T GAAAAGGAAA

CCTAGAAAGTAGTCACACGTTGCTTATTTAGGCCAGAAGTAATTGTACTGGGCAAAA ATTTCA

CTTAAAAAACACAAGAAGTCCAGGTATGGTGGCTCAGACCTGTAATCCCAGCACTTT GAGAGG

CCGAGGCAGGTGGATTACTTGAGCCTAGGGGTTCAAGACCAGCTTGGGCAACATGTC AAAACC

CTGTCTCTACAAAAAATACAAAAATTAGCCTGGCATGATGGCATGTGCCCGTAGTCT CAGCTA

CTCAGGAGTGAGGTGGGAGGATCATTTGAGCTCAGAAGGTCAAGGCTGCAATGAGAC ATAATT

TCACCATAGTACTTCCAGCCTGGGCAATAGAGCAAGACTCTCTCTCAAAAAAAACAG CACACA

CACACACACACGAAAACAATTCTGAACTATGAAATCTGAAACAGCCCCTTGGTATCT CCTGGG

CATGATTTGCAAATCTTTTTTTTTTACAGAAAAAAGGCAAAGAGTAAGCACTTTGCC ATAGGT

TACTTGGCCGTGATCATCTATCTAGTGGAAAAGGGGACTGGGAAGCCCAAGCAGACT GGGAAA

CCAGACAGCTAGGAAAAGGAGCAAAACATAGCCCAGCAACCTACAGATGAAGAAAGT TGAGAA

ATCCATTTATTCACCATAGAGACGCAGGAATTTCAGGCAATGCACTAAAATGAAATG GGGGAA

AAAAGCTTGATCAGTATGGGAACCATTTTTGTGCAAAAGGGAATATTATGGATCAGC CAGTAT

TTCTTTGAGCTCTGCCTGTGGAGTCCATTTGACCTTTAGAAATATGAGGTATTCTGT CAGTTT

TATCTTCTTGGAGAAATTTCTCCTAAAATCTTGATTTGCTTTAGTCTGGACTGGTTC ATAGCC

ATCATCTTCCATCAGTACCCCAGAGATTCACTTTGTCTCTTATGTGGGATCTGTTTC CAGTTA

GATGCCATTATTTTCCTTTTCCTTGGTTTACTCTTCCACATATTGGTAAAGCTCTTC CAATAG

CTTTTGGAAAGGAAAAATGAAAAGTAAATGTTTTGAATCTCTGTGTGTTTGACAATG TCTTTA

TTTTACCCTTATACCTGATTGCTGTTTTGGTTGGCAAGGTATAGGATTCTTTAGTGG TCTCCA

TGCCCAGTTTTGAAGACATCTGCTAGCTTTCAGTGCTGTTGCTGTGGAGTCTGAAAA TCTGTC

TTCTGGCTTCCAGGGTGACTACTGGAAATTGAATGCCATTCTGTTCCTTCTCTTTTG CATATA

TAATCCATTTTTATCTCTCTTGAAGCTTATAGGTTTATCTTTGTCTCAATGTTCTGT CCCTGT TAAGAGTCCATTTTCATCCTTTGTACTAGGTGCCTGGTGGGATCATTCCGTCTGAAACTA ATG

ATTTCCCATCTCTTCACTGTTTCTGGAATTCCTGTTTTCCAGATGTTAGACCTCCAG AATTTG

ATCTCTAATTTTCCTATCTTTTCTCTTAACTTTCAGCTCTGTCTTCTTGCTAGGACC TTTTCC

TAGGAGCATTTCTCAATTTAATCTTCCAGTTCATCTGTTGCATTTTATTTTTCTAGT CTCATA

TTGTCTCATATTTTTAATTTCTAAGAGCTCCCCTTCTCCGAATATTCTTTTTTTTTA ATAGCA

TCCTATTTTGGCTCATGGTTGCAGTATTTTATCTCCTTGAAGATGTTTGTGTGTTTA TGTATG

TATATGCACACACGTATACATACACATACAGGCATGCATCTCTGTATTCTTTCGGCA TAATCT

GTGTCCTCCAGGGTTTGTTTCTTTGTTTCCCCTGTATGTTTGTTTTGGTCGTTCACA TTATAG

GCTTTCCTCAGAGTTAATGGTCTTGGTAGTCTACTCATATTTAAGTGTGGAACACCA AAAAGC

TTACTATAAGCTGAGAGTGTGGTAAAGGGCTCTTTGTTTTACTATGACCTACCTGAG CTATCT

TGCTGGGGAACACCCTAATGTCAGTCTCTTTATAAAGGGCCTTTCATTTTGGCCTGG CAAGAA

ATACTCTTTCATCCTCCTGCATGGAGGGCAAAAAAAAATTTAAAAATTGGCTGCTAG GGTCTG

TCTGCTCACTTCCCTGTTTTGCAGACCCCACACTCTTCTGCAATTCATTTCATAGTT GTCAAG

ACTATACAAATTGTCCTTTTTAATGTTCTCTCTTCTGCTATCCCTAGTTGGCAGTCT TCCTCT

TTACAACCTGCTGAAAGTGGAAGACCTCCAGTTTTCCTTTAATTCCTCAGCAAACCA CCAACT

ATTATATGTCTTTTTTCCAGAACAACTTATTTTTTAACTATAATTATATGCATTTAT GTTAGA

TTCACTGAAAACCTCATCTTGTATGGTGCTCTGTACCCTATGGGTGCTAAATAAAGG CTTGCT

AC GGCAACTGGAAAAAAAAAAAAAAAAA

SEQ ID NO:4

Amino acid sequence of Homo sapiens methyltransferase SET domain bifurcated 2 (Setdb2), isoform b; UniProtKB: Q96T68; NP_001153780.1

The "SET" domain of Setdb2 consists of a "pre-SET domain" and a "bifurcated SET Domain" (herein designated as "SETl" and "SET2", respectively). The "pre-SET domain" has an amino acid sequence of from position 233 to 347. SETl has an amino acid sequence of from position 355 to 416. SET2 has an amino acid sequence of from position 618 to 684.

MGEKNGDAKTF MELEDDGKVDFIFEQVQNVLQSLKQKIKDGSATNKEYIQAMILVNEATIIN SSTSIKDPMPVTQKEQENKSNAFPSTSCENSFPEDCTFLTTENKEILSLEDKVVDFREKD SSS NLSYQSHDCSGACLMKMPLNLKGENPLQLPIKCHFQRRHAKTNSHSSALHVSYKTPCGRS LRN VEEVFRYLLETECNFLFTDNFSFNTYVQLARNYPKQKEVVSDVDISNGVESVPISFCNEI DSR KLPQFKYRKTVWPRAYNLTNFSSMFTDSCDCSEGCIDITKCACLQLTARNAKTSPLSSDK ITT GYKYKRLQRQIPTGIYECSLLCKCNRQLCQNRVVQHGPQVRLQVFKTEQKGWGVRCLDDI DRG TFVCIYSGRLLSRANTEKSYGIDENGRDENTMKNIFSKKRKLEVACSDCEVEVLPLGLET HPR TAKTEKCPPKFSNNPKELTVETKYDNISRIQYHSVIRDPESKTAIFQHNGKKMEFVSSES VTP EDNDGFKPPREHLNSKTKGAQKDSSSNHVDEFEDNLLIESDVIDITKYREETPPRSRCNQ ATT LDNQNIKKAIEVQIQKPQEGRSTACQRQQVFCDEELLSETKNTSSDSLTKFNKGNVFLLD ATK EGNVGRFLNHSCCPNLLVQNVFVETHNRNFPLVAFFTNRYVKARTELTWDYGYEAGTVPE KEI FCQCGVNKCRKKIL

SEQ ID NO: 5

Nucleotide sequence encoding murine methyltransferase SET domain bifurcated 2 (Setdb2), mouse (cDNA); NM_001081024.1. A corresponding mRNA molecule encoding murine Setdb2 has a nucleotide sequence that corresponds to the sequence shown in SEQ ID NO: 5, with the exception that the thymidine (T) residue(s) of the sequence shown in SEQ ID NO: 5, is/are replaced by (a) uracil (U) residue(s).

GCGGGGCCGCGGAAACTGTGTGGAGCTCCCTTGGGGCTGATGTCTCCCCCGCTGAGG TGAAGC

CGCCTCGGGACTGCAGACTCTGGTGCGGACAGTGCAAGGCGCGCGGGGTGCCAGGCC GCCTCA

ACCCGCTGTCTCAGTAGCGGGGGTGGCCGGGGCTCTCAGTCGCGTTTCCCCCACCGT CTTGCG

GCGCTCACTTTCGTGGCTGGTGATTTCTGAGTAATTTGGCTATCATGGGCTGTAGAC AGCGGA

GCACAGACAAGATCTTCAGGACACTGGTCCATTCCACAGAGTGTTTCATGCATTTGT GGACCA

AAACATGGGATGAACTTTAATTTTAAAAAGACATTGTCAGTAGTCCTGATCCGGCTT TCAACC

TGGCCTTGAAGAAGATGCTGTGCCTGTTTTTCTTGGACTAGTAGTCATTCTGAAAGT CTATTC

AAGCACAGTTTCTATTTGCAAATTCAAATACTAAAGACTATTAGGAAGTGGCCTAAC GAGATA

CTCTCTTTGGGGACTACAGATTTCATAGAGACTGCAGTTGGAGCCCATTTAGTTTTG ATTAAC

TTCATTTTGAAGAATTTTATATAAGCAACACCAAAAGATGGAAGAAAAAAATGGTGA TGCAAA

GACTTTCTGGATGGAGCTACAAGATGATGGTAAAGTTGACTTAATGTTTGAGAAAAC ACAAAA

TGTCCTACATTCACTGAAACAGAAGATAAAGGATGGGTCTGCCACAAATGGAGACTA TGTCCA

AGCAATGAATCTAGTAAATGAAGCCACTCTGAGTAACACGCAAACACTGGAAAAGGG TATGTT

CATTACTTATTCCAATCCTGAAGTGAATACTCATCGTAGCAATCATACACCTGTGAC TCAGAG

TGAACAGGAAAACAAATCAAGTGCGGTTCCCTCTGCATCATGTGACAACTCCTGTCC TAAGGG

CTGTACTATCCCATCTCCAGGAAAAAAAGTATTCCTCCCTGTGAAGAATAAAGCTGA CAATTT

AGTGAAAAAGGAAGCCCCACTGCATATATCTTTCCATCGCCATATCTGCTCCAGGAC TTGTCT

AATGGAAACCCCACTGTCCTTGAAGGGAGAAAACCCCCTGCAGCTACCAATCAGATG TCACTT

CCAAAGACGACATGCAAAGACAAACTCTCATTCTTCTGCCCTCCATGTGAATTATAA AACGCC

CTGTGGACGGAATCTACGAAACATGGAGGAAGTTTTCCATTACCTGCTTGAAACAGA GTGTAA

CTTTTTATTCACAGACAACTTCTCTTTCAATACTTATGTCCAGTTGACTCGGAATCA CCCAAA

GCAAAATGAAGTTGTTTCTGACGTGGATATTAGTAATGGAGTGGAATCAGTGTCGAT TCCTTT CTGTAATGAAATTGACAACAGTAAACTTCCACGGTTTAAGTATAGAAATACAGTATGGCC CCG

AATATATCATCTGAACTTTTCCAACATGTTTTCTGATTCATGTGACTGTTCTGAGGG CTGCAT

AGACATAAAAAAATGTGCATGTCTTCAGTTGACAGCAAAGAATGCCAAGGCATGTCC CTTGTC

ATCGGATGGAGAATGTGCTGGATATAAATACAAAAGACTGCAGAGGCTCATACCTAC TGGCAT

TTATGAATGCAACCTGCTCTGCAAGTGTAACAAACAGATGTGTCAAAACCGAGTTAT CCAGCA

TGGTGTCCGGGTGAGGCTACAGGTGTTCAAAAGTGAGAAGAAGGGCTGGGGAGTACG CTGCCT

GGATGACATTGACAAAGGGACATTTGTGTGCATTTATTCAGGAAGGTTACTGCGCAG AGCCAC

TCCTGAGAAAACTAATATAGGTGAAAATGGAAGAGAGCAACAGCACATTGTGAAAAA TTCATT

TTCCAAAAAGAGGAAACTAGAAGTTGTGTGTTCAGATTGTGACGCACACTGTGACAG TCCTAA

GGCTGAGGACTGCCCTCCCAAGCTTAGTGGTGATCTCAAAGAGCCCGCTGTGGAAAT GAACCA

TAGAAATATTTCAAGAACTCAGCATCATTCAGTCATTAGAAGAACTAAATCCAAGAC AACTGT

TTTTCATTACAGTGAGAAAAACATGGGATTTGTTTGCTCAGATTCTGCCGCCCCAGA AGATAA

GAATGGATTTAAACCAGCTCAAGAACACGTGAACTCTGAAGCTAGGAGAGCTCACGA GGACTT

AAGTTCAAACCCAGCTGGAGATTCTGAAGACACACAGCTGACTGAATCAGACGTGAT AGATAT

AACTGCAAGTAGAGAAGACTCTGCCCCAGCATACAGGTGTAAGCACGCAACCATAGT GGATCG

TAAGGACACTAAACAGGTGCTTGAAGTCCCAGGAAAGAAGTCCCAAGAGGAAGAGCC TGCAGC

CTCTCAAAGCCAGCAGGCCTTGTGTGATGAGGAGCTGCCAAGTGAGAGGACAAAGAT TCCATC

TGCTTCCCTGATGCAGCTCAGTAAGGAGAGTCTGTTTCTATTGGATGCTTCAAAAGA AGGAAA

TGTGGGCCGTTTCCTCAATCATAGTTGCTGTCCAAATCTCTGGGTGCAGAATGTTTT TGTAGA

AACACATGACAGGAATTTCCCATTGGTGGCCTTCTTCACCAACAGGTATGTGAAAGC AAGGAC

AGAACTAACGTGGGATTATGGTTATGAAGCTGGGGCCACGCCTGCAAAGGAAATCCT CTGCCA

ATGTGGGTTTAATAAGTGTCGGAAAAAATTAATATAACTTTATATCTGATATCCATC CGTGAA

CTGAGCTGCTCAATGTATAACAATATCAGTTGTCAAAGAGATTATGGTTAATACTTC TGTTTC

CTTTGTTCAGGGTATTGATGTTTTATGTCTAATTTTGTATCTTAGGTTAGAAGCAAA ATAAGG

GCACTCATCAAGTATCACAGTGTTATTACAGTTAATTAACTTTACATGAGTAGAATA TAAATA

ATATATTTTATGTACATATCATTGTATAATTTGTAACTTTTGTATTTTATATATTAA TATAAA

ATTATACTAG

SEQ ID NO:6

Amino acid sequence of murine methyltransferase SET domain bifurcated 2 (Setdb2) mouse; Uniprot B: Q8C267; NP_001074493.1

MEEKNGDAKTFWMELQDDGKVDLMFEKTQNVLHSLKQKIKDGSATNGDYVQAMNLVN EATLSN TQTLEKGMFITYSNPEVNTHRSNHTPVTQSEQENKSSAVPSASCDNSCPKGCTIPSPGKK VFL PVKNKADNLVKKEAPLHISFHRHICSRTCLMETPLSLKGENPLQLPIRCHFQRRHAKTNS HSS ALHVNYKTPCGRNLRNMEEVFHYLLETECNFLFTDNFSFNTYVQLTRNHPKQNEVVSDVD ISN GVESVSIPFCNEIDNSKLPRFKYRNTVWPRIYHLNFSNMFSDSCDCSEGCIDIKKCACLQ LTA KNAKACPLSSDGECAGYKYKRLQRLIPTGIYECNLLCKCNKQMCQNRVIQHGVRVRLQVF KSE KKGWGVRCLDDIDKGTFVCIYSGRLLRRATPEKTNIGENGREQQHIVKNSFSKKRKLEVV CSD CDAHCDSPKAEDCPP LSGDLKEPAVEMNHRNISRTQHHSVIRRT SKTTVFHYSEKNMGFVC SDSAAPEDKNGFKPAQEHVNSEARRAHEDLSSNPAGDSEDTQLTESDVIDITASREDSAP AYR CKHATIVDRKDTKQVLEVPGKKSQEEEPAASQSQQALCDEELPSERTKIPSASL QLSKESLF LLDASKEGNVGRFLNHSCCPNLWVQNVFVETHDRNFPLVAFFTNRYVKARTELTWDYGYE AGA TPAKEILCQCGFNKCRKKLI

SEQ ID NO:7

Nucleotide sequence of the target sequence of siRNA 1 targeting nucleotide sequence encoding Homo sapiens methyltransferase SET domain bifurcated 2 (Setdb2) siRNA 1 :

#SI00141393, Qiagen

Target sequence: CCGAGAGCATCTGAACTCTAA siRNA 1 targets SETDB2 at nt position 2481-2501 of a nucleic acid molecule having a sequence encoding Setdb2 as shown in SEQ ID NO: 1. siRNA 1 targets SETDB2 at nt position 1881-1901 of a nucleic acid molecule having a sequence encoding Setdb2 as shown in SEQ ID NO: 3.

SEQ ID NO:8

Nucleotide sequence of the target sequence of siRNA 2 targeting nucleotide sequence encoding Homo sapiens methyltransferase SET domain bifurcated 2 (Setdb2) siRNA 2:

#SI03116904, Qiagen

Target sequence: TCGGCCGCTTCCTTAATCATA siRNA2 targets SETDB2 at nt position 2843-2863of a nucleic acid molecule having a sequence encoding Setdb2 asshown in SEQ ID NO: 1. siRNA2 targets SETDB2 at nt position 2243-2261 of a nucleic acid molecule having a sequence encoding Setdb2 as shown in SEQ ID NO: 3.

References:

1. Morens DM, Taubenberger J , Fauci AS. Predominant role of bacterial pneumonia as a cause of death in pandemic influenza: implications for pandemic influenza preparedness. The Journal of infectious diseases 2008, 198(7): 962-970.

2. Molinari NA, Ortega-Sanchez IR, Messonnier ML, Thompson WW, Wortley PM, Weintraub E, et al. The annual impact of seasonal influenza in the US: measuring disease burden and costs. Vaccine 2007, 25(27): 5086-5096.

3. van der Sluijs KF, van der Poll T, Lutter R, Juffermans NP, Schultz MJ. Bench-to- bedside review: bacterial pneumonia with influenza - pathogenesis and clinical implications. Critical care 2010, 14(2): 219.

4. Aderem A, Ulevitch RJ. Toll-like receptors in the induction of the innate immune response. Nature 2000, 406(6797): 782-787.

5. Kawai T, Akira S. The role of pattern-recognition receptors in innate immunity: update on Toll-like receptors. Nat Immunol 2010, 11(5): 373-384.

6. Taniguchi T, Ogasawara K, Takaoka A, Tanaka N. IRF family of transcription factors as regulators of host defense. Annu Rev Immunol 2001, 19: 623-655.

7. Sadler AJ, Williams BR. Interferon-inducible antiviral effectors. Nat Rev Immunol 2008, 8(7): 559-568.

8. Schoggins JW, Macduff DA, Imanaka N, Gainey MD, Shrestha B, Eitson JL, et al. Pan- viral specificity of IFN-induced genes reveals new roles for cGAS in innate immunity. Nature 2013.

9. Oeckinghaus A, Ghosh S. The NF-kappaB family of transcription factors and its regulation. Cold Spring Harbor perspectives in biology 2009, 1(4): a000034.

10. Medzhitov R, Schneider DS, Soares MP. Disease tolerance as a defense strategy.

Science 2012, 335(6071): 936-941. Liew FY, Xu D, Brint EK, O'Neill LA. Negative regulation of toll-like receptor- mediated immune responses. Nat Rev Immunol 2005, 5(6): 446-458.

Litvak V, Ratushny AV, Lampano AE, Schmitz F, Huang AC, Raman A, et al. A FOX03-IRF7 gene regulatory circuit limits inflammatory sequelae of antiviral responses. Nature 2012, 490(7420): 421-425.

Rouse BT, Sehrawat S. Immunity and immunopathology to viruses: what decides the outcome? Nat Rev Immunol 2010, 10(7): 514-526.

Navarini AA, Recher M, Lang KS, Georgiev P, Meury S, Bergthaler A, et al. Increased susceptibility to bacterial superinfection as a consequence of innate antiviral responses. Proc Natl Acad Sci USA 2006, 103(42): 15535-15539.

Nakamura S, Davis KM, Weiser JN. Synergistic stimulation of type I interferons during influenza virus coinfection promotes Streptococcus pneumoniae colonization in mice. J Clin Invest 2011, 121(9): 3657-3665.

Shahangian A, Chow EK, Tian X, Kang JR, Ghaffari A, Liu SY, et al. Type I IFNs mediate development of postinfluenza bacterial pneumonia in mice. J Clin Invest 2009, 119(7): 1910-1920.

Foster SL, Hargreaves DC, Medzhitov R. Gene-specific control of inflammation by TLR-induced chromatin modifications. Nature 2007, 447(7147): 972-978.

Fang TC, Schaefer U, Mecklenbrauker I, Stienen A, Dewell S, Chen MS, et al. Histone H3 lysine 9 di-methylation as an epigenetic signature of the interferon response. J Exp Med 2012, 209(4): 661-669.

Satoh T, Takeuchi O, Vandenbon A, Yasuda K, Tanaka Y, Kumagai Y, et al. The Jmjd3-Irf4 axis regulates M2 macrophage polarization and host responses against helminth infection. Nat Imm unol 2010, 11(10): 936-944.

Allan RS, Zueva E, Cammas F, Schreiber HA, Masson V, Belz GT, et al. An epigenetic silencing pathway controlling T helper 2 cell lineage commitment. Nature 2012, 487(7406): 249-253.

Nutt SL, Fairfax KA, Kallies A. BLIMP 1 guides the fate of effector B and T cells. Nat Rev Immunol 2007, 7(12): 923-927.

Dillon SC, Zhang X, Trievel RC, Cheng X. The SET-domain protein superfamily: protein lysine methyltransferases. Genome Biol 2005, 6(8): 227. Black JC, Van Rechem C, Whetstine JR. Histone lysine methylation dynamics: establishment, regulation, and biological impact. Mol Cell 2012, 48(4): 491-507.

Matsui T, Leung D, Miyashita H, Maksakova IA, Miyachi H, Kimura H, et al. Proviral silencing in embryonic stem cells requires the histone methyltransferase ESET. Nature 2010, 464(7290): 927-931.

Ceol CJ, Houvras Y, Jane-Valbuena J, Bilodeau S, Orlando DA, Battisti V, et al. The histone methyltransferase SETDB1 is recurrently amplified in melanoma and accelerates its onset. Nature 2011 , 471(7339): 513-517.

Xu PF, Zhu KY, Jin Y, Chen Y, Sun XJ, Deng M, et al. Setdb2 restricts dorsal organizer territory and regulates left-right asymmetry through suppressing fgf8 activity. Proc Natl Acad Sci USA 2010, 107(6): 2521-2526.

Falandry C, Fourel G, Galy V, Ristriani T, Horard B, Bensimon E, et al. CLLD8/KMT1F is a lysine methyltransferase that is important for chromosome segregation. J Biol Chem 2010, 285(26): 20234-20241.

Hogarth CA, Mitchell D, Evanoff R, Small C, Griswold M. Identification and expression of potential regulators of the mammalian mitotic-to-meiotic transition. Biol Reprod 201 1, 84(1): 34-42.

Richon VM, Johnston D, Sneeringer CJ, Jin L, Majer CR, Elliston K, et al. Chemogenetic analysis of human protein methyltransferases. Chem Biol Drug Des 2011, 78(2): 199-210.

Litvak V, Ramsey SA, Rust AG, Zak DE, Kennedy KA, Lampano AE, et al Function of C/EBPdelta in a regulatory circuit that discriminates between transient and persistent TLR4-induced signals. Nat Immunol 2009, 10(4): 437-443.

Takaoka A, Wang Z, Choi MK, Yanai H, Negishi H, Ban T, et al. DAI (DLM- 1 /ZBP 1 ) is a cytosolic DNA sensor and an activator of innate immune response. Nature 2007, 448(7152): 501-505.

Takaoka A, Mitani Y, Suemori H, Sato M, Yokochi T, Noguchi S, et al. Cross talk between interferon-gamma and -alpha/beta signaling components in caveolar membrane domains. Science 2000, 288(5475): 2357-2360.

Mantovani A, Cassatella MA, Costantini C, Jaillon S. Neutrophils in the activation and regulation of innate and adaptive immunity. Nat Rev Immunol 2011 , 11(8): 519-531. McNamee LA, Harmsen AG. Both influenza-induced neutrophil dysfunction and neutrophil-independent mechanisms contribute to increased susceptibility to a secondary Streptococcus pneumoniae infection. Infect Immun 2006, 74(12): 6707-6721. Cai S, Batra S, Lira SA, Kolls JK, Jeyaseelan S. CXCL1 regulates pulmonary host defense to Klebsiella Infection via CXCL2, CXCL5, NF-kappaB, and MAPKs. J Immunol 2010, 185(10): 6214-6225.

Levy D, Kuo AJ, Chang Y, Schaefer U, Kitson C, Cheung P, et al. Lysine methylation of the NF-kappaB subunit RelA by SETD6 couples activity of the histone methyltransferase GLP at chromatin to tonic repression of NF-kappaB signaling. Nat Immunol 2011, 12(1): 29-36.

Zhang Y, Leaves NI, Anderson GG, Ponting CP, Broxholme J, Holt R, et al. Positional cloning of a quantitative trait locus on chromosome 13ql4 that influences immunoglobulin E levels and asthma. Nat Genet 2003, 34(2): 181-186.

Ruthenburg AJ, Li H, Patel DJ, Allis CD. Multivalent engagement of chromatin modifications by linked binding modules. Nat Rev Mol Cell Biol 2007, 8(12): 983-994. Yang XD, Huang B, Li M, Lamb A, Kelleher NL, Chen LF. Negative regulation of NF- kappaB action by Set9-mediated lysine methylation of the RelA subunit. Embo J 2009, 28(8): 1055-1066.

Marazzi I, Ho JS, Kim J, Manicassamy B, Dewell S, Albrecht RA, et al. Suppression of the antiviral response by an influenza histone mimic. Nature 2012, 483(7390): 428-433. Nauseef WM, Borregaard N. Neutrophils at work. Nat Immunol 2014, 15(7): 602-611. Raquil MA, Anceriz N, Rouleau P, Tessier PA. Blockade of antimicrobial proteins S100A8 and S100A9 inhibits phagocyte migration to the alveoli in streptococcal pneumonia. J Immunol 2008, 180(5): 3366-3374.

Arredouani M, Yang Z, Ning Y, Qin G, Soininen R, Tryggvason et al. The scavenger receptor MARCO is required for lung defense against pneumococcal pneumonia and inhaled particles. J Exp Med 2004, 200(2): 267-272.

Sun K, Metzger DW. Inhibition of pulmonary antibacterial defense by interferon- gamma during recovery from influenza infection. Nat Med 2008, 14(5): 558-564.

Dela Cruz CS, Liu W, He CH, Jacoby A, Gornitzky A, Ma B, et al. Chitinase 3 -like- 1 promotes Streptococcus pneumoniae killing and augments host tolerance to lung antibacterial responses. Cell host & microbe 2012, 12(1): 34-46. Jamieson AM, Pasman L, Yu S, Gamradt P, Homer RJ, Decker T, et al. Role of tissue protection in lethal respiratory viral-bacterial coinfection. Science 2013, 340(6137): 1230-1234.

Helin K, Dhanak D. Chromatin proteins and modifications as drug targets. Nature 2013, 502(7472): 480-488.

Muller U, Steinhoff U, Reis LF, Hemmi S, Pavlovic J, Zinkernagel RM, et al. Functional role of type I and type II interferons in antiviral defense. Science 1994, 264(5167): 1918-1921.

Honda K, Yanai H, Negishi H, Asagiri M, Sato M, Mizutani T, et al. IRF-7 is the master regulator of type-I interferon-dependent immune responses. Nature 2005, 434(7034): 772-777.

Durbin JE, Hackenmiller R, Simon MC, Levy DE. Targeted disruption of the mouse Statl gene results in compromised innate immunity to viral disease. Cell 1996, 84(3): 443-450.

Takeuchi O, Hoshino K, Kawai T, Sanjo H, Takada H, Ogawa T, et al. Differential roles of TLR2 and TLR4 in recognition of gram-negative and gram-positive bacterial cell wall components. Immunity 1999, 11(4): 443-451.

Febbraio M, Podrez EA, Smith JD, Hajjar DP, Hazen SL, Hoff HF, et al. Targeted disruption of the class B scavenger receptor CD36 protects against atherosclerotic lesion development in mice. J Clin Invest 2000, 105(8): 1049-1056.

Lee EC, Yu D, Martinez de Velasco J, Tessarollo L, Swing DA, Court DL, et al. A highly efficient Escherichia coli-based chromosome engineering system adapted for recombinogenic targeting and subcloning of BAC DNA. Genomics 2001 , 73(1): 56-65. Gilchrist M, Thorsson V, Li B, Rust AG, Korb M, Roach JC, et al. Systems biology approaches identify ATF3 as a negative regulator of Toll-like receptor 4. Nature 2006, 441(7090): 173-178.

Sheehan KC, Lai KS, Dunn GP, Bruce AT, Diamond MS, Heutel JD, et al. Blocking monoclonal antibodies specific for mouse IFN-alpha/beta receptor subunit 1 (IFNAR-1) from mice immunized by in vivo hydrodynamic transfection. J Interferon Cytokine Res 2006, 26(11): 804-819. 6. Kratz PA, Bottcher B, Nassal M. Native display of complete foreign protein domains on the surface of hepatitis B virus capsids. Proc Natl Acad Sci U S A 1999, 96(5): 1915- 1920.

57. Schebesta A, McManus S, Salvagiotto G, Delogu A, Busslinger GA, Busslinger M.

Transcription factor Pax5 activates the chromatin of key genes involved in B cell signaling, adhesion, migration, and immune function. Immunity 2007, 27(1): 49-63.

58. Szarka RJ, Wang N, Gordon L, Nation PN, Smith RH. A murine model of pulmonary damage induced by lipopolysaccharide via intranasal instillation. Journal of immunological methods 1997, 202(1): 49-57.

59. Warszawska JM, Gawish R, Sharif O, Sigel S, Doninger B, Lakovits K, et al. Lipocalin 2 deactivates macrophages and worsens pneumococcal pneumonia outcomes. J Clin Invest 2013.

All references cited herein are fully incorporated by reference. Having now fully described the invention, it will be understood by a person skilled in the art that the invention may be practiced within a wide and equivalent range of conditions, parameters and the like, without affecting the spirit or scope of the invention or any embodiment thereof.