Abstract:
The present invention relates to a non-human animal deficient in the N-terminal domain of the IL-33 gene. Also provided herein is the use of said non-human animal as an in vivo model of inflammatory diseases, especially with regard to screening methods for anti-inflammatory compounds, and methods for evaluating and optimising the pharmacological properties of a given anti-inflammatory compound.

Description:
CROSS REFERENCE TO RELATED APPLICATIONS 
       [0001]    This application is a continuation of International Application No. PCT/EP2011/068696 having an international filing date of Oct. 26, 2011, the entire contents of which are incorporated herein by reference, and which claims benefit under 35 U.S.C. §119 to European Patent Application No. 10189446.7 filed Oct. 29, 2010. 
     
    
     SEQUENCE LISTING 
       [0002]    The instant application contains a Sequence Listing submitted via EFS-Web and hereby incorporated by reference in its entirety. Said ASCII copy, created Apr. 17, 2013, is named P4670C1_Sequence_listing.txt, and is 28.132 bytes in size. 
       FIELD OF THE INVENTION 
       [0003]    The present invention relates to a non-human animal deficient in the N-terminal domain of the IL-33 gene. Also provided herein is the use of said non-human animal as an in vivo model of inflammatory diseases, especially with regard to screening methods for anti-inflammatory compounds, and methods for evaluating and optimising the pharmacological properties of a given anti-inflammatory compound. 
       BACKGROUND OF THE INVENTION 
       [0004]    The interleukin 33 (IL-33) cytokine is the newest member of the interleukin 1 (IL-1) family. Because of its nuclear localization, it was originally described as “Nuclear Factor from High Endothelial Venules”. IL-33 is primarily expressed by fibroblasts and epithelial, endothelial and airway smooth muscle cells. IL-33 is the ligand for the IL-1 receptor-related protein ST2. The ST2 receptor is expressed in almost all innate immune cells (mast cells, basophils, eosinophils, neutrophils, natural killer (NK) cells and macrophages) and in NK T and T helper (Th) 2 cells. The interaction of IL-33 with ST2 on targeted cells can trigger the expression and secretion of pro-inflammatory, Th1, Th2 and Th17 cytokines and expression of chemokines involved in Th1, Th2 and innate immune effector functions (published papers and Hicks et al. manuscript in preparation). IL-33 binds to its specific surface receptor through its pro-inflammatory cytokine domain. In addition, IL-33 also has a N-terminal domain that contains a typical DNA-binding helix-turn-helix motif. In its nuclear uncleaved form, IL-33 interacts with histones 2A and 2B in heterochromatin promoting chromatin compaction and functioning as a potential transcriptional repressor. There is strong support showing that IL-33 is similar to other chromatin-associated cytokines (IL-1α and HMGB1) that appears to exert a dual-function, regulating transcriptional repression in the nucleus and signaling via a classic receptor acting as a potent pro-inflammatory cytokine. Thus, it has been proposed that similarly to HMGB1, IL-33 may function as an ‘alarmin’ belonging to the larger family of damage-associated molecular pattern (DAMP) molecules. 
         [0005]    The IL-33/ST2 axis plays pivotal roles in the patho-physiology of human inflammatory diseases as confirmed by their high levels of expression in diseased tissues. Elevated levels of either IL-33 and/or its soluble receptor ST2 are observed in rheumatoid arthritis (RA), inflammatory bowel disease (IBD), psoriatic and ulcerative colitis, acute eosinophilic pneumonia, severe asthma, idiopathic pulmonary fibrosis, liver fibrotic diseases, atopic dermatitis, systemic sclerosis, autoimmune and trauma patients. Similar to its role in humans, the IL-33/ST2 axis has been shown to be critical in murine inflammatory models. IL-33 exacerbates collagen-induced arthritis (CIA), allergic conjunctivitis and experimental autoimmune encephalomyelitis (EAE), Interruption of IL-3/ST2 signaling with antibodies has been shown to be beneficial for the resolution of allergic airway inflammation and bleomycin-induced lung injury. It has also been shown recently that IL-33 is up-regulated in an IBD mouse model of chronic intestinal inflammation (Oboki et al, PNAS 2010 107 (43) 18581-18586). 
       SUMMARY OF THE INVENTION 
       [0006]    The non-human animal may be any non-human animal. Preferably, the non-human animal is a mammal, more preferably a rodent such as rat or a mouse, most preferably, the non-human animal is a mouse. In a preferred embodiment, said non-human animal is a mouse and the N-terminal deletion of the IL-33 gene comprises a deletion of the full DNA binding domain at the N-terminus of the IL-33 gene. Preferably, said N-terminal deletion of the IL-33 gene in the mouse comprises a deletion of amino acids 1-67 of the expression product of the IL-33 gene. 
         [0007]    The non-human animal may be heterozygous or homozygous for the N-terminal IL-33 deletion. Preferably, the non-human animal is heterozygous for the N-terminal IL-33 deletion. 
         [0008]    The non-human animal deficient in the N-terminus of the IL-33 gene according to the present invention displays the typical characteristics of inflammatory diseases. For instance, a mouse according to the present invention has such characteristics as normal birth rate and growth until about 3-4 months of age, then hemorrhagic lesions in ears and repeatedly observed large coagula in the thoracic cavity, smaller size and general ill picture. On pathological examination these mice reveal multiorgan inflammation, including chronic, multifocal myocarditis; chronic, suppurative, strong ileitis with enlarged intestinal walls; chronic utreteritis with marked hydronephrosis and kidney atrophy, moderate multifocal and perivascular infiltrates in the lung, splenic hyperplasia with strong expansion of eosinophils and macrophages in immune organs, lung and intestine. 
         [0009]    Since the non-human animal according to the present invention exhibits an inflammatory phenotype, it is useful as an in vivo inflammation model. This non-human animal with an N-terminal IL-33 deletion can serve to practically prove the causal relationship between IL-33 dysfunctions and inflammatory disorders and allow the design of directed therapeutic strategies aimed to reduce or abolish the abnormal overproduction of IL-33 in patients. As massive production and secretion of IL-33 into the extracellular compartment is the cause of this severe inflammatory condition, this novel and unique non-human animal model can also be used to evaluate the in vivo efficacy and potency of IL-33 drug candidates if cross-reactivity with the non-human IL-33 or its receptor ST2 is given. Hence in a second object of the invention, said non-human test animal is used as an in vivo model of inflammatory diseases, especially with regard to screening for anti-inflammatory compounds. In one embodiment a method for screening for anti-inflammatory compounds is provided, comprising administering a candidate compound to a non-human animal with a N-terminal IL-33 deletion according to the present invention. In one embodiment, said method comprises a) providing the non-human animal with a N-terminal IL-33 deletion, b) administering to said non-human animal a candidate compound, c) comparing the inflammation symptoms of said non-human animal to those of a non-human animal with a N-terminal IL-33 deletion not administered said compound; wherein the compound that alleviates said inflammation symptoms is selected as an anti-inflammatory compound. 
         [0010]    Candidate compounds include, but are not limited to small molecules, (poly) peptides, (glyco) proteins, antibodies or antibody fragments, (poly) or (oligo) nucleotides, nucleosides, lipids, combinations thereof and modified derivatives thereof. 
         [0011]    Methods of administration of a candidate compound to be screened include, but are not limited to, oral administration and parenteral administration (e.g. intravenous administration, intraperitoneal administration and intranasal administration). In case of oral administration a candidate agent may be blended in a feed for administration. 
         [0012]    The candidate agent may be administered in combination with a pharmaceutically acceptable conventional excipient (such as carrier and diluent) or additives. Further, the candidate agent may be encapsulated in, or bound (or attached) to, liposomes (e.g. positively charged liposomes) or nano-particles and administered. 
         [0013]    Evaluation can be conducted for example using mitigation or recovery of inflammation symptoms, increase in body weight, recovery from hypertrophy of the ileon, or the like, as an indication, by observation with naked eye, measurement of body weight, histopathological observation (e.g. microscopic observation after tissue staining) and FACS analysis (see Examples below). 
         [0014]    Through use of the subject animals with a N-terminal IL-33 deletion or cells derived there from, one can identify ligands or substrates that bind to, modulate, antagonize or agonize cellular IL-33. Of particular interest are screening assays for anti-inflammatory compounds that have a low toxicity for human cells. A wide variety of assays may be used for this purpose, including in vivo studies, determination of the localization of drugs after administration, labeled in vitro protein-protein binding assays, protein-DNA binding assays, electrophoretic mobility shift assays, immunoassays for protein binding, and the like. Depending on the particular assay, whole animals may be used, or cells derived there from. Cells may be freshly isolated from an animal, or may be immortalized in culture. 
         [0015]    In another embodiment of the invention, said in vivo model is used for evaluation of the pharmacological effects such as in vivo efficacy and potency of IL-33 drug candidates. Therefore in another embodiment of the invention a method for evaluation of the pharmacological effects of a IL-33 drug candidate is provided, said method comprising administering said IL-33 drug candidate to a non-human animal with a N-terminal IL-33 deletion according to the present invention. 
         [0016]    The present invention also relates to descendants of the non-human animals with an N-terminal IL-33 deletion as provided by the invention, obtained by breeding with the same or with another genotype. Preferably, the descendant is obtained by breeding with the same genotype. Said descendant comprises an N-terminal IL-33 deletion as the non-human animal with an N-terminal IL-33 deletion described above. A further object of the invention is the use of said descendants as an in vivo model of inflammatory diseases. In one embodiment, said descendants are used as an in vivo model for screening of anti-inflammatory compounds. In another embodiment, said descendants are used as an in vivo model for evaluation of the pharmacological effects of an anti-inflammatory compound. 
         [0017]    Furthermore, the present invention relates to a cell line or primary cell culture derived from a non-human animal with an N-terminal IL-33 deletion or its descendants as described above. 
         [0018]    In addition, the present invention also provides a tissue or an organ explant or culture thereof, derived from a non-human animal with an N-terminal IL-33 deletion or its descendants as described above. 
         [0019]    The present invention also provides a tissue or cell extract derived a non-human animal with an N-terminal IL-33 deletion or its descendants as described above. 
         [0020]    In another embodiment of the invention, above-mentioned cell line or primary cell culture, tissue or an organ explant or culture thereof, tissue or cell extract derived from a non-human animal with an N-terminal IL-33 deletion or its descendants is used as a model of inflammatory diseases. In one embodiment, said model of inflammatory diseases is used for screening of anti-inflammatory compounds, in another embodiment, said model of inflammatory diseases is used for or evaluation of the pharmacological effects of an anti-inflammatory compound, as outlined above. 
       DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS 
     Definitions 
       [0021]    Methods for producing a non-human animal with a deletion in a gene, such as the N-terminus of the IL-33 gene, are well known in the art. Suitable methods are described i.e. in Hogan B et al: Manipulating the mouse embryo, A laboratory manual, 2nd Edition (1994), Cold Spring Harbor Laboratory Press. 
         [0022]    The term “IL-33 gene” as used herein relates in particular to the interleukin 33 gene also known as DVS27; NF-HEV; NFEHEV; C9orf26; DKFZp586H0523; RP11-575C20.2; IL33, IL-1F11, 9230117N10Rik, RGD1311155 and the like. Said gene comprises a N-terminal domain with a putative DNA-binding helix-turn-helix motif and a C-terminal cytokine domain. The IL33 gene is conserved in humans, chimpanzee, dog, cow, mouse, and rat. By way of an example the DNA sequence of the mouse IL-33 gene is depicted in SEQ ID. 1. The term “expression product of the IL-33 gene” refers to the translated protein of the IL-33 gene, i.e. the IL-33 protein. 
         [0023]    “N-terminal IL-33 deletion” as used herein means that all or part of the N-terminus of the IL-33 gene is modified (for example substituted, deleted, added and/or inserted) or disrupted, whereby the expression product of the IL-33 gene lacks the whole or parts of the N-terminal domain and/or does not exhibit the function of the N-terminal domain. The N-terminal domain as used herein comprises a putative helix-turn-helix motif which is responsible for DNA binding and nucleon localization of IL-33. In a preferred embodiment the non-human animal is a mouse and amino acids 1-67 of the mouse expression product of the IL-33 gene (IL-33 protein) are modified or disrupted. 
         [0024]    Such non-human animal is depicted herein as a non-human animal with a N-terminal IL-33 deletion, or a non-human animal deficient in the N-terminus of the IL-33 gene or a N-terminal IL-33 gene knock-out non-human animal and the like. In one special preferred embodiment said modification of the N-terminus of the IL-33 gene is achieved through knock-in of a DsRed cassette, thus deleting the N-terminal part of the IL-33 gene. Said non-human animal is accordingly also referred to as DsRed-IL33/COOH non-human animal. The term “wild type” as used herein refers to a non-human animal having a full-length IL-33 gene. 
         [0025]    “Non-human animal” as described herein refers to any animal that is not a human. Preferably, the non-human animal is a mammal, more preferably a rodent such as rat or a mouse, most preferably, the non-human animal is a mouse. 
         [0026]    The non-human animal with a N-terminal IL-33 deletion as described above can be used as a model for treatment of inflammatory diseases. It allows to investigate the effect of an potentially non-inflammatory compound on the inflammatory disease in a non-human in vivo model. Since the transgenic animal is immunotolerant for the transgenic human maB11 antibody, the effect of chronic treatment with a therapeutic antibody such as for example Mab 11 can be determined. Also, the effect on extracellular IL-33 levels and the process and kinetics of an inflammatory disease can be followed. 
         [0027]    The term “inflammatory disease” as used herein relates to any impairment of health or a condition of abnormal functioning characterized by inflammation. In particular, the term “inflammatory disease” as used herein relates to diseases connected to an increased level of IL-33, for example, but not limited to inflammatory bowel disease, rheumatoid arthritis, urticuria, atherosclerotic vascular disease, psoriatic colitis, ulcerative colitis, acute eosinophilic pneumonia, severe asthma, idiopathic pulmonary fibrosis, liver fibrotic diseases, atopic dermatitis, systemic sclerosis, autoimmune diseases and the like. 
         [0028]    “Anti-inflammatory compounds” as used herein means any molecule with the capability of reducing inflammatory responses, in detail, affecting the biological action of IL-33. As such, “Anti-inflammatory compound” includes, but is not limited to small molecules, (poly) peptides, (glyco) proteins, antibodies or antibody fragments, (poly) or (oligo) nucleotides, nucleosides, lipids, combinations thereof and modified derivatives thereof “Anti-inflammatory compound” also includes molecules that mediate RNA interference such as shRNA, microRNA, siRNA, antisense oligonucleotides, spiegelmers, LNA or PNA oligomers, or combinations thereof. Putative anti-inflammatory compounds as used herein are also depicted as “IL-33 drug candidates” or “candidate compounds”. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0029]      FIG. 1   a : Schematic drawing of the targeted mutation of IL-33. The DsRed cassette was inserted at the Start codon and fused with IL-33 at its amino acid position 68. 
           [0030]      FIG. 1   b : Targeting strategy. RFP: red fluorescent protein; tm1: targeted mutation 1; wt: wild-type; Marker: marker X (Roche); kb: 1 kilo base pairs; EcoRI: Restriction endonuclease site; NeoR. Neomycin selection cassette; triangles: lox sites 
           [0031]      FIG. 2 : Phenotype of heterozygous DsRedIL-33/COOH knock-in mice. Growth retardation, erected fur. 
           [0032]      FIG. 3 : Phenotype of heterozygous DsRedIL-33/COOH knock-in mice. Organ morphology. 
           [0033]      FIG. 4 : Phenotype of heterozygous DsRedIL-33/COOH knock-in mice. Organ morphology compared to wild-type mouse. SI: small intestine; He: heart; Th: thymus; Ki: kidney; Lu: lung; Sp: spleen; Li: liver Co*: blood coagulum in thoracic cavity 
           [0034]      FIG. 5 : Phenotype of heterozygous DsRedIL-33/COOH knock-in mice. Ileon hypertrophy. Intestine preparation opened longitudinally. 
           [0035]      FIG. 6 : Phenotype of heterozygous DsRedIL-33/COOH knock-in mice (DsRedIL-33/COOH-KI) via FACS analysis. Cell suspensions of the indicated organs were stained with antibodies specific for defined surface marker: CD45(labeled APC-Cy7), total leukocytes; CD11b (labeled allophycocyanin, APC), macrophages; SiglecF (labeled phycoerythrin PE), eosinophils; Gr1 (labeled PE-Cy7), granulocytes; F4/80 (labeled Alexa 488), monocytes. Stained cells were acquired using a FACS Canto I device (BD Corp.) and analysed with FlowJo software. Y-axis: SiglecF, X-Axis: F4/80. Figures show the superposed stainings of different cell populations, DsRedIL-33/COOH knock-in mice have an increase in eosinophils as determined by high expression of SiglecF and F4/80 (see marked populations, “Eos”=Eosinophils) 
           [0036]      FIG. 7 : Phenotype of heterozygous DsRedIL-33/COOH knock-in mice. Serum IL-33 levels. 
       
    
    
     EXAMPLES 
       [0037]    The present invention will now be described in more detail by working examples, provided that the examples should not be interpreted as those limiting the scope of the present invention. 
       Example 1 
     Generation of a Targeting Vector 
       [0038]    The targeting vector (SEQ ID. NO. 2) was generated using recombineering technology as supplied by Gene Bridges GmbH, Heidelberg. It contains the following elements: 
         [0000]    
       
         
               
               
               
             
           
               
                   
                   
               
             
             
               
                   
                   1-70 
                 IL-33 exon 1b 
               
               
                   
                  71-3871 
                 intron 
               
               
                   
                 3872-3882 
                 IL-33 exon 2 
               
               
                   
                 3883-4557 
                 dsRed monomer CDS 
               
               
                   
                 4558-4579  
                 IL-33 exon 3 
               
               
                   
                 4580-4590  
                 intronic sequence 
               
               
                   
                 4591-4626  
                 lox site 
               
               
                   
                 4625-6040  
                 neomycin selection cassette 
               
               
                   
                 6041-6074  
                 lox site 
               
               
                   
                 6075-6571  
                 intronic sequence 
               
               
                   
                 6572-6682  
                 IL-33 exon 4 
               
               
                   
                 6683-7285  
                 intron 
               
               
                   
                 7286-7603 
                 pBluescript SKII+ 
               
               
                   
                 7604-8463  
                 diphteria toxin a selection cassette 
               
               
                   
                 8464-10697 
                 pBluescript SKII+ 
               
               
                   
                   
               
             
          
         
       
     
         [0039]    The targeting vector was used for homologous recombination in BALB/c ES cells. Positive clones were identified using PCR screening strategies (sequences of oligonucleotides see below). Following electroporation of an Cre expressing plasmid site specific recombination leads to the removal of the neomycin selection cassette in vitro. After blastocyst injection of ES cell clones chimeric animals were breed and DNA preparations of biopsies of the F1 and F2 generation were used to confirm the identity of the targeted mutation as well as genotyping using PCR (PCR AB, CD, EF and EFG in  FIG. 1   b ). 
         [0000]    
       
         
               
               
             
           
               
                   
                 Oligonucletides 
               
               
                   
                 Oligonucletide A: 
               
               
                   
                 (SEQ ID. NO. 3) 
               
               
                   
                 TAG AAA GAG CCC AGT GTT AAG C 
               
               
                   
                   
               
               
                   
                 Oligonucletide B: 
               
               
                   
                 (SEQ ID. NO. 4) 
               
               
                   
                 GGC TTG CCC TCG CCC TCG 
               
               
                   
                   
               
               
                   
                 Oligonucletide C: 
               
               
                   
                 (SEQ ID. NO. 5) 
               
               
                   
                 CAC CTG CGA CTT CAA GAC C 
               
               
                   
                   
               
               
                   
                 Oligonucletide D: 
               
               
                   
                 (SEQ ID. NO. 6) 
               
               
                   
                 ACG ATT CCT TAG TGA TGG GGC 
               
               
                   
                   
               
               
                   
                 Oligonucletide E: 
               
               
                   
                 (SEQ ID. NO. 7) 
               
               
                   
                 GTT GCT TCT GAT GAC TTC AGG 
               
               
                   
                   
               
               
                   
                 Oligonucletide F: 
               
               
                   
                 (SEQ ID. NO. 8) 
               
               
                   
                 GCA ATA GCC CTT GCC AAG GC 
               
               
                   
                   
               
               
                   
                 Oligonucletide G: 
               
               
                   
                 (SEQ ID. NO. 9) 
               
               
                   
                 TGC TGT TCC AGC CTC TGT TGG 
               
             
          
         
       
     
       Example 2 
     Generation of N-Terminal IL-33 Knockout Mouse 
       [0040]    Two genetically modified knock-in mouse models in a Balb/c background were generated. The first mutant targets the N-terminal intracellular transcriptional factor-like activity by in-frame replacing it with the fluorochrome DsRd-monomer (DsRed-IL33/COOH) keeping the functional cytokine domain complete ( FIG. 1 ). The second mutant replaces the pro-inflammatory cytokine domain by in-frame knocking-in the fluorochrome DsRed keeping the DNA binding domain intact (NH2/L33-DsRed). 
       Example 3 
     Characterization of N-Terminal IL-33 Knockout Mouse 
       [0041]    Unexpectedly, the genetic engineered heterozygous mutant mice missing the IL-33 N-terminal domain but maintaining an active the proinflammatory cytokine activity in the cytoplasm (DsRed-IL33/COOH) die at around of 4 months of age. Apparently normal at birth, they become progressively sick and finally moribund between 4 and 5 months later with an estimated phenotype penetration of around 60%. At the age of 3-4 months, these mutants start displaying bloody lesions in ears and enlarged bellies. Upon necropsy, a massive splenomegaly, hypertrophy of the ilion suggestive of intestinal inflammation, the presence of large coagula in the thoracic cavity and kidney atrophy are repeatedly observed ( FIGS. 2-5 ). Closer examination of different organs in FACS analysis showed a strong eosinophilia in lungs, spleen, lymph nodes, Payer&#39;s Patches and peripheral blood ( FIG. 6 ). Serum IL-33 levels are elevated in DsRed-IL33/COOH knockout mice ( FIG. 7 ). 
       Discussion 
       [0042]    We have recently found that intranasal administration of IL-33 evoked profound lung inflammation with multinucleated giant cells of macrophage origin in the interstitium (Hicks et al. manuscript in preparation). In the same manner, IL-33 also evoked a bone marrow hyperplasia with big cluster of myeloid/granulocytic cells as shown by histopathology (Hicks et al. manuscript in preparation). In addition, high levels of soluble IL-33 are found in the plasma of peripheral blood of these mice, strongly suggesting that the immunopathological effects seen in our DsRed-IL33/COOH mutant animals is the result of the continuous presence of the fully active DsRed-IL33/COOH cytokine in the cytoplasm and its possible release into the extracellular compartment, thus behaving as a potent endogenous DAMP signal as it has been hypothesized. 
         [0043]    This “alarmin”-like effect of knocking out the N-terminal domain of IL-33 is thus similar to human immune-pathological conditions (whose diagnosis remains unknown) when a potent danger signal is released after tissue damage, necrosis or autoimmunity. As the occurrence of severe multi-organ inflammation in patients with homozygous mutations or deletions of gene encoding IL-1RA and its blockade with mAbs have demonstrated the central role of IL-1α and IL-β in a number of auto-inflammatory diseases (Weber at al, Science signaling, 3, 2010). Possibly genetic variations of IL-33 could result in over-expression and/or secretion of this possible “danger” molecule and it may contribute to the pathogenesis of auto-inflammatory or allergic diseases. Precisely, it has recently been shown a positive association between polymorphisms in the IL-33 gene and higher IL-33 levels with susceptibility of Japanese cedar pollinosis (Sakashita et al. Clin. Exp. Allergy December 2008).