Non-human animal models of DITRA disease and their uses

Abstract

The present disclosure relates to genetically modified rodents and rodent models of human diseases. More specifically, the present disclosure relates to genetically modified rodents whose genomes contain humanized Il1rl2 genes (encoding the IL1rl2 subunit of IL-36R protein) and human IL-36, as well as ligand genes. The genetically modified rodents disclosed herein show enhanced skin and intestinal inflammation in preclinical models of psoriasis and IBD, respectively, and can serve as rodent models of human DITRA disease.

Description

[0001] This application is a divisional application of the Chinese patent application with application number 201980040953.1.

[0002] Cross-reference to related applications

[0003] This patent application claims the benefit of priority to U.S. Provisional Application No. 62 / 698,459, filed on July 16, 2018, and U.S. Provisional Application No. 62 / 867,477, filed on June 27, 2019, the entire contents of which are incorporated herein by reference. Technical Field

[0004] The present disclosure relates to genetically modified rodents and rodent models of human diseases. More specifically, the present disclosure relates to genetically modified rodents whose genomes contain humanized Il1rl2 genes (encoding the IL1rl2 subunit of IL-36R) and human IL-36α, β and γ ligand genes. The genetically modified rodents disclosed herein show enhanced skin and intestinal inflammation in preclinical models of psoriasis and inflammatory bowel disease (IBD), respectively, and can serve as rodent models of human interleukin-36R antagonist deficiency (DITRA) disease.

[0005] Sequence Listing Incorporated by Reference

[0006] The sequence listing is an ASCII text file named 35950_10404US01_SequenceListing.txt, with a size of 65KB, generated on July 9, 2019, which was submitted to the United States Patent and Trademark Office via EFS-Web and is incorporated herein by reference in its entirety. Background Art

[0007] The interleukin (IL)-36 subfamily consists of three agonists (IL-36α, β, and γ, previously named IL1F6, IL1F8, and IL1F9, respectively) and one antagonist (IL36Ra), which signal through a heterodimeric receptor (IL-36R), leading to activation of nuclear factor B (NFκB) and mitogen-activated protein kinase (MAPK). Similar to the classical IL-1 family members, IL-36 cytokines are involved in the initiation and regulation of immune responses. Members of the IL-36 family were first shown to be expressed primarily in squamous epithelial tissues and, in particular, in psoriatic skin lesions. The association between IL-36R and epithelial-mediated diseases has been demonstrated in humans based on loss of function mutations in IL-36Ra that result in the diagnosis of generalized pustular psoriasis (abbreviated "GPP"), which is currently understood to be a type of GPP, known as interleukin- 3 6R Deficiency in antagonist (abbreviated as "DITRA"). Elevated IL-36 expression has been reported in GPP, palmoplantar pustular psoriasis (PPPP), inflammatory bowel disease (IBD), rheumatoid and psoriatic arthritis, asthma, chronic obstructive pulmonary disease, chronic kidney disease, and ichthyosis. Summary of the invention

[0008] Disclosed herein are some embodiments of rodents that are genetically modified to contain a humanized Il1rl2 gene encoding an Il1rl2 protein having an extracellular domain substantially identical to that of human IL1RL2, and human Il1f6, Il1f8, and Il1f9 genes encoding human IL1F6, IL1F8, and IL1F9, respectively. Il1rl2 is a subunit of the heterodimeric IL-36R protein that binds three agonists: IL1F6, IL1F8, and IL1F9 (also referred to as IL-36α, β, and γ, respectively). The rodents disclosed herein, despite containing a humanized Il1rl2 gene and expressing three human agonist ligand genes, retain an endogenous IL-36Ra antagonist, wherein the endogenous IL-36Ra is about 20 times less potent in inhibiting human IL-36R signaling. Such quadruple humanized rodents (e.g., humanized 11112 and human IL1F6, IL1F8, and IL1F9) have been shown herein to display symptoms of DITRA patients in which mutations in IL-36Ra result in enhanced IL-36R signaling. Thus, provided herein are genetically modified rodents, compositions and methods for making such rodents, and methods for using such rodents as models for screening and developing therapeutic agents.

[0009] In one aspect of some embodiments, disclosed herein is a genetically modified rodent whose genome comprises (1) a humanized Il1rl2 gene at an endogenous rodent Il1rl2 locus, wherein the humanized Il1rl2 gene encodes a humanized Il1rl2 protein comprising an extracellular domain substantially identical to the extracellular domain of a human IL1RL2 protein; (2) a human IL1F6 gene at an endogenous rodent Il1f6 locus; (3) a human IL1F8 gene at an endogenous rodent Il1f8 locus; and (4) a human IL1F9 gene at an endogenous rodent Il1f9 locus.

[0010] In some embodiments, the humanized Il1rl2 gene in the rodent encodes a humanized Il1rl2 protein comprising a transmembrane cytoplasmic sequence that is substantially identical to that of the endogenous rodent Il1rl2 protein. In some embodiments, the humanized Il1rl2 gene in the rodent encodes a humanized Il1rl2 protein comprising a signal peptide that is substantially identical to that of the endogenous rodent Il1rl2 protein.

[0011] In some embodiments, the humanized Il1rl2 gene in the rodent encodes a humanized Il1rl2 protein comprising an extracellular domain substantially identical to the extracellular domain of a human IL1RL2 protein, wherein the human IL1RL2 protein comprises the amino acid sequence shown in SEQ ID NO:2.

[0012] In some embodiments, the humanized Il1rl2 gene in the rodent encodes a humanized Il1rl2 protein comprising an extracellular domain, wherein the extracellular domain comprises amino acids 22-337 of SEQ ID NO:7.

[0013] In some embodiments, the humanized Il1rl2 gene in the rodent encodes a humanized Il1rl2 protein comprising the amino acid sequence shown in SEQ ID NO:7.

[0014] In some embodiments, the humanized Il1rl2 gene in a rodent is operably linked to an endogenous rodent Il1rl2 promoter at the endogenous rodent Il1rl2 locus.

[0015] In some embodiments, a humanized IL1rl2 gene in a rodent is generated by replacing a genomic segment of an endogenous rodent IL1rl2 gene at the endogenous rodent IL1rl2 locus with a nucleotide sequence of a human IL1RL2 gene.

[0016] In some embodiments, the nucleotide sequence of the human IL1RL2 gene is a genomic fragment of the human IL1RL2 gene that substantially encodes the extracellular domain of the human IL1RL2 protein. In some embodiments, the genomic fragment of the human IL1RL2 gene comprises exons 3-8 of the human IL1RL2 gene.

[0017] In some embodiments, the genomic sequence of the endogenous rodent Il1rl2 gene remaining after the humanizing replacement comprises an exon downstream of exon 8 of the endogenous rodent Il1rl2 gene. In some embodiments, the genomic sequence of the endogenous rodent Il1rl2 gene remaining after the humanizing replacement comprises exons 1-2 of the endogenous rodent Il1rl2 gene.

[0018] In specific embodiments, the humanized Il1rl2 gene in a rodent disclosed herein comprises exons 1-2 of the endogenous rodent Il1rl2 gene, exons 3-8 of the human IL1RL2 gene, and the remaining exons downstream of exon 8 of the endogenous rodent Il1rl2 gene.

[0019] In some embodiments, the human IL1F6 gene in a rodent disclosed herein replaces an endogenous rodent Il1f6 gene at the endogenous rodent Il1f6 locus.

[0020] In some embodiments, the human IL1F8 gene in a rodent disclosed herein replaces an endogenous rodent Il1f8 gene at the endogenous rodent Il1f8 locus.

[0021] In some embodiments, the human IL1F9 gene in a rodent disclosed herein replaces an endogenous rodent Il1f9 gene at the endogenous rodent Il1f9 locus.

[0022] In some embodiments, the rodents disclosed herein are homozygous for one or more of the four humanized and / or human genes. In some embodiments, the rodents disclosed herein are homozygous for each of the four humanized and / or human genes.

[0023] In some embodiments, compared with wild-type rodents, rodents disclosed herein show shortened colon. In some embodiments, compared with wild-type rodents, rodents disclosed herein show enhanced inflammation in experimentally induced inflammatory models. In some embodiments, the experimentally induced inflammatory model is a skin inflammation model induced by imiquimod (IMQ). In some embodiments, the experimentally induced inflammatory model is an intestinal inflammation model induced by dextran sodium sulfate (DSS) or oxazolone.

[0024] In some embodiments, the rodent disclosed herein is a mouse or a rat.

[0025] Further disclosed herein are isolated cells or tissues from the rodents described herein.

[0026] In another aspect of some embodiments, disclosed herein is a method for preparing a genetically modified rodent, the method comprising modifying the rodent genome to comprise (1) a humanized Il1rl2 gene at an endogenous rodent Il1rl2 locus, wherein the humanized Il1rl2 gene encodes a humanized Il1rl2 protein comprising an extracellular domain substantially identical to the extracellular domain of a human IL1RL2 protein; (2) a human IL1F6 gene at an endogenous rodent Il1f6 locus; (3) a human IL1F8 gene at an endogenous rodent Il1f8 locus; and (4) a human IL1F9 gene at an endogenous rodent Il1f9 locus; and preparing a rodent comprising the modified genome.

[0027] In some embodiments, the rodent genome is modified by the following process, which includes: (i) preparing a first rodent comprising a humanized Il1rl2 gene at the endogenous rodent Il1rl2 locus; (ii) preparing a second rodent comprising a human IL1F6 gene at the endogenous rodent Il1f6 locus, a human IL1F8 gene at the endogenous rodent Il1f8 locus, and a human IL1F9 gene at the endogenous rodent Il1f9 locus; and (iii) hybridizing the first rodent with the second rodent to obtain a modified rodent genome.

[0028] In some embodiments, a rodent comprising a humanized Il1rl2 gene at an endogenous rodent Il1rl2 locus is prepared by providing rodent embryonic stem (ES) cells, inserting a nucleotide sequence of a human IL1RL2 gene into the rodent Il1rl2 locus of the rodent ES cells to form a humanized Il1rl2 gene at the rodent Il1rl2 locus, thereby obtaining a rodent ES cell comprising the humanized Il1rl2 gene, and preparing a rodent using the rodent ES cell comprising the humanized Il1rl2 gene.

[0029] In some embodiments, the nucleotide sequence of the inserted human IL1RL2 gene replaces the genomic fragment of the rodent Il1rl2 gene at the rodent Il1rl2 locus. In some embodiments, the nucleotide sequence of the human IL1RL2 gene is a genomic fragment of the human IL1RL2 gene that substantially encodes the extracellular domain of the human IL1RL2 protein. In some embodiments, the genomic fragment of the human IL1RL2 gene comprises exons 3-8 of the human IL1RL2 gene. In some embodiments, the genomic sequence of the endogenous rodent Il1rl2 gene remaining after the replacement comprises an exon downstream of exon 8 of the endogenous rodent Il1rl2 gene. In some embodiments, the genomic sequence of the endogenous rodent Il1rl2 gene remaining after the humanization replacement comprises exons 1-2 of the endogenous rodent Il1rl2 gene. In some embodiments, the humanized Il1rl2 gene as a result of the humanizing replacements comprises exons 1-2 of the endogenous rodent Il1rl2 gene, exons 3-8 of the human IL1RL2 gene, and the remaining exons downstream of exon 8 of the endogenous rodent Il1rl2 gene.

[0030] In some embodiments, a rodent comprising a human IL1F6 gene, a human IL1F8 gene, and a human IL1F9 gene is prepared by: providing rodent embryonic stem (ES) cells; inserting the human IL1F6 gene into the rodent Il1f6 locus of the rodent ES cells, inserting the human IL1F8 gene into the rodent Il1f8 locus of the rodent ES cells, and inserting the human IL1F9 gene into the rodent Il1f9 locus of the rodent ES cells, thereby obtaining rodent ES cells comprising a human IL1F6 gene, a human IL1F8 gene, and a human IL1F9 gene; and preparing a rodent using the rodent ES cells comprising a human IL1F6 gene, a human IL1F8 gene, and a human IL1F9 gene.

[0031] In some embodiments, the human IL1F6 gene, the human IL1F8 gene, and the human IL1F9 gene are provided in a contiguous nucleic acid molecule.

[0032] In some embodiments of the methods, the human IL1F6 gene replaces the endogenous rodent Il1f6 gene at the endogenous rodent Il1f6 locus.

[0033] In some embodiments of the methods, the human IL1F8 gene replaces the endogenous rodent Il1f8 gene at the endogenous rodent Il1f8 locus.

[0034] In some embodiments of the methods, the human IL1F9 gene replaces the endogenous rodent Il1f9 gene at the endogenous rodent Il1f9 locus.

[0035] In another aspect of some embodiments, disclosed herein is a rodent embryonic stem (ES) cell comprising (1) a humanized Il1rl2 gene at an endogenous rodent Il1rl2 locus, wherein the humanized Il1rl2 gene encodes a humanized Il1rl2 protein comprising an extracellular domain substantially identical to the extracellular domain of a human IL1RL2 protein; (2) a human IL1F6 gene at an endogenous rodent Il1f6 locus; (3) a human IL1F8 gene at an endogenous rodent Il1f8 locus; and (4) a human IL1F9 gene at an endogenous rodent Il1f9 locus. In some embodiments, such ES cells can be prepared by the methods disclosed herein. In some embodiments, the use of such ES cells to prepare rodents is also disclosed.

[0036] In yet another aspect of some embodiments, disclosed herein are targeting nucleic acid constructs that can be used to modify a rodent genome (eg, in a rodent ES cell) to produce a modified rodent.

[0037] In some embodiments, disclosed herein is a targeting nucleic acid construct comprising a nucleotide sequence of a human IL1RL2 gene flanked by 5' and 3' rodent nucleotide sequences capable of mediating homologous recombination and integration of the nucleotide sequence of the human IL1RL2 gene in the rodent Il1rl2 locus to form a humanized Il1rl2 gene, wherein the humanized Il1rl2 gene encodes a humanized Il1rl2 protein comprising an extracellular domain substantially identical to the extracellular domain of the human IL1RL2 protein.

[0038] In some embodiments, disclosed herein is a targeting nucleic acid construct comprising a contiguous nucleic acid sequence comprising: a human IL1F6 gene, a human IL1F8 gene, and a human IL1F9 gene, wherein the contiguous nucleic acid sequence is flanked by 5' and 3' rodent nucleotide sequences, and the 5' and 3' rodent nucleotide sequences are capable of mediating homologous recombination and integration of the contiguous nucleic acid sequence in a rodent locus comprising a rodent Il1f6 gene, a rodent Il1f8 gene, and a rodent Il1f9 gene.

[0039] In another aspect of some embodiments, disclosed herein is the use of the rodents disclosed herein as rodent models of human diseases associated with dysregulated IL-36 signaling. As non-limiting examples of the embodiments disclosed herein, the rodents can be used to study the pathology and molecular basis of human diseases associated with dysregulated IL-36 signaling (such as but not limited to DITRA), or to screen, test and develop therapeutic compounds that can be used to treat such diseases.

[0040] In yet another aspect of some embodiments, disclosed herein is a method of evaluating the therapeutic efficacy of a candidate compound for treating a disease associated with dysregulated IL-36 signaling, the method comprising administering an agent to a rodent disclosed herein to induce inflammation, administering a candidate compound to the rodent, and determining whether the candidate compound inhibits and / or reduces the induced inflammation.

[0041] In some embodiments, the agent administered to induce inflammation is DSS or oxazolone, which induces intestinal inflammation in rodents. In some embodiments, the agent administered to induce inflammation is IMQ, which induces skin inflammation in rodents.

[0042] In some embodiments, the candidate compound is administered to the rodent before, during, or after administration of an agent that induces inflammation. In some embodiments, the candidate compound can be a small molecule compound, a nucleic acid inhibitor, or an antigen binding protein such as an antibody. In some embodiments, the candidate compound is an antibody that specifically binds to human IL-36R. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] This patent file contains at least one drawing executed in color. Copies of this patent with color drawing(s) will be provided by the Patent and Trademark Office upon request and payment of the necessary fee.

[0044] Figures 1A-1D An embodiment of an exemplary strategy for humanizing mouse Il1rl2 is shown. Figure 1A A diagram showing the genomic organization of the mouse IL1RL2 and human IL1RL2 genes is not shown to scale. The thin bars placed across the genomic sequence represent exons. The mouse genomic segment of approximately 25,324 base pairs (bp) to be deleted and the human genomic segment of approximately 32,389 bp to be inserted are indicated. The positions of the probes used in the assay described in Example 1 are indicated. Figure 1BShown not to scale is an exemplary modified BAC vector used to humanize the endogenous mouse Il1rl2 gene and the connecting sequence at the bottom (SEQ ID NOs: 26-28) to achieve (i) replacement of mouse Il1rl2 exons 3-8 and the middle intron, and 3'155bp mouse intron 2 and 5'642bp mouse intron 8 with human IL1RL2 exons 3-8 and the middle intron, and 3'346bp human intron 2 and 5'1101bp human intron 8, and (ii) insertion of a loxP-hUb1-em7-Neo-pA-mPrm1-Crei-loxP cassette (4996bp) downstream of the insertion of the human genomic fragment in intron 8. Figure 1C The humanized Il1rl2 allele after deletion of the neomycin cassette is shown not to scale, along with the junction sequence at the bottom (SEQ ID NOs: 26 and 29). Figure 1D Listed are the sequence alignments of mouse Il1rl2 protein (SEQ ID NO:4), human IL1RL2 protein (SEQ ID NO:2), and humanized Il1rl2 protein (SEQ ID NO:7).

[0045] Figures 2A-2D An embodiment of an exemplary strategy for replacing mouse Il1f8, Il1f9, and Il1f6 with human IL1F8, IL1F9, and IL1F6 is shown. Figure 2A A diagram of the genomic organization of the mouse Il1f8, Il1f9 and Il1f6 genes and the human IL1F8, IL1F9 and IL1F6 genes is not shown to scale. The thin bars placed across the genomic sequence represent exons. The mouse genomic fragment of about 76,548 bp to be deleted and the human genomic fragment of about 88,868 bp to be inserted are indicated. The positions of the probes used in the assay described in Example 1 are indicated. Figure 2B Shown not to scale is an exemplary modified BAC vector used to replace the endogenous mouse Il1f8, Il1f9 and Il1f6 genes and the connecting sequence at the bottom (SEQ ID NOs: 30-32) to achieve (i) replacement of the coding sequences and untranslated regions (UTRs) and intergenic sequences of mouse Il1f8, Il1f9 and Il1f6 with the corresponding human sequences IL1F8, IL1F9 and IL1F6; and (ii) insertion of a loxP-hUb1-em7-Hygro-pA-mPrm1-Crei-loxP box (5,218 bp) downstream of the insertion of the human genomic fragment. Figure 2C The humanized locus after the hygromycin cassette has been deleted, and the junction sequence at the bottom (SEQ ID NOs: 30 and 33) are shown not to scale. Figure 2DListed are the sequence alignments of mouse Il1f6 (SEQ ID NO: 11) and human IL1F6 (SEQ ID NO: 9) proteins; mouse Il1f8 (SEQ ID NO: 17) and human IL1F8 (SEQ ID NO: 15) proteins; and mouse Il1f9 (SEQ ID NO: 23) and human IL1F9 (SEQ ID NO: 21) proteins.

[0046] Figures 3A-3E illustrate embodiments of the present invention, showing enhanced IMQ-induced skin inflammation in humanized DITRA-like mice. DITRA-like (also abbreviated as "DITRA" in the figures herein) and wild-type (WT) mice were treated by topical application of IMQ for 4 consecutive days. On the 5th day, dorsal skin samples were collected for subsequent histopathological evaluation and qRT-PCR. (3A) On the 5th day, representative skin appearance of DITRA-like and WT mice treated topically with petrolatum ("Vas", control) or an IMQ-containing cream (Aldara) for 4 consecutive days. (3B) Representative hematoxylin and eosin ("H&E") staining of petrolatum and IMQ-treated skin from DITRA-like and WT mice. (3C) mRNA expression of proinflammatory molecules in the skin of DITRA and WT mice treated daily with petrolatum or an IMQ-containing cream (n=10 per group). After 4 consecutive days of topical application of IMQ, dorsal skin samples were collected from Il1rl2 single humanized mice ("1H"), DITRA-like mice, and wild-type mice on day 5 for subsequent histopathological evaluation and qRT-PCR. In each set of bars, from left to right are: WT Vas, DITRA Vas, WT IMQ, and DITRA IMQ. (3D) Representative skin appearance of 1H, DITRA-like, and WT mice treated topically with petrolatum (control) or an IMQ-containing cream (Aldara) for 4 consecutive days on day 5. (3E) mRNA expression of proinflammatory molecules in the skin of 1H, DITRA, and WT mice treated daily with petrolatum or an IMQ-containing cream (n=10 per group). In each set of bars, from left to right are: WT Vas, 1H Vas, DITRA-like Vas, WT IMQ, 1H IMQ, and DITRA-like IMQ.

[0047] Figures 4A-4B In some embodiments, DITRA-like mice exhibit shortened colons at steady state. (4A) Representative images of colons isolated from DITRA-like mice and their co-housed IL-36R KO and WT littermates at 3 and 10 months. (4B) Quantified colon lengths of DITRA-like mice at 3 and 10 months old compared to their co-housed IL-36R KO and WT littermates (n=6 per group). Error bars represent mean SD.

[0048] Figures 5A-5E. In some embodiments, DITRA-like mice exhibit defects in mucosal healing in DSS-induced chronic colitis. DITRA-like mice (n=16) and their co-housed WT littermates (n=14) were subjected to chronic DSS-induced colitis by administration of 2.5% DSS for 7 days, followed by water for 11 days and 1.5% DSS for 4 days, followed by water for 5 days, for a total of 27 days. Control mice (n=5) received regular water. (5A) Weight loss of DITRA-like mice and their WT co-housed littermates was calculated as the percentage difference between the original body weight and the actual body weight on any particular day. (5B) Colon length in water- and DSS-treated DITRA-like and WT mice. (5C) Hematoxylin and eosin (H&E) staining and pathological scoring of the colon of water- and DSS-treated DITRA-like and WT mice. (5D) Myeloperoxidase (MPO) activity in colon homogenates of water- and DSS-treated DITRA-like and WT mice. (5E) Levels of proinflammatory cytokines in colon homogenates of DITRA-like and WT mice treated with water and DSS. Data represent at least three independent experiments. Error bars represent mean SD. In each set of bars, from left to right: WT / water, DITRA / water, WT / DSS, and DITRA / DSS.

[0049] Figure 6A-6B In some embodiments, human IL-36R antagonism with REGN anti-human IL-36R antibodies improves IMQ-induced skin inflammation in DITRA-like mice when administered prophylactically. The skin on the back of DITRA-like mice was shaved 3 days before the start of IMQ application, and the skin was topically treated with petrolatum (control) or IMQ-containing cream (Aldara) for 4 consecutive days. PBS, hIL-36R mAb, and hIgG4 isotype control were injected subcutaneously at 10 mg / mL on days -3 and 1 (n=9 per treatment group). (6A) H&E histological sections and pathological scores of dorsal skin isolated from DITRA-like mice on day 5. (6B) Levels of proinflammatory cytokines in skin homogenates of DITRA-like mice treated with IMQ applied daily on days -3 and 1 and injected with PBS, IL-36R mAb, and isotype control on day 5. Data represent three independent experiments. Error bars represent mean SD. In each set of bars, from left to right: PBS, hIL-36R mAb, and hIgG4 isotype control.

[0050] Figures 7A-7C.In some embodiments, therapeutic administration of anti-human IL-36R antibodies improves chronic IMQ-induced skin inflammation in DITRA-like mice. The skin on the back of DITRA-like mice was shaved 3 days before the start of administration, and the skin was topically treated with petrolatum (control) or IMQ-containing cream (Aldara) for two rounds (5 consecutive days for the first round of application, followed by 2 days without application, and then 4 consecutive days for the second round, for a total of 11 days). PBS, anti-human IL-36R antibodies, and hIgG4 isotype controls were subcutaneously injected at 10 mg / mL on days 5 and 9 (n=10 for each treatment group). (7A) Skin thickness was measured in DITRA-like mice on day 12. Thickness is presented in μm. (7B) H&E histological sections and pathological scores of skin from DITRA-like mice on day 12. (7C) Levels of proinflammatory cytokines were measured in skin homogenates of DITRA-like mice on day 12. Data represent two independent experiments. Error bars represent mean SD. In each set of bars, from left to right are: phosphate buffered saline (PBS), hIL-36R mAb, and hIgG4 isotype control.

[0051] Figures 8A-8E. In some embodiments, similar to IL-12p40 blockade, therapeutic administration of anti-human IL-36R antibodies improves DSS-induced chronic inflammation in DITRA-like mice and rescues them from DSS-induced mucosal damage. DITRA-like mice (n = 45) were subjected to chronic DSS-induced colitis by administration of 3% DSS for 7 days and 2% DSS for 13 days, followed by water administration for a total of 30 days. Control mice (n = 5) received regular water. PBS (n = 11), IL-36R mAb (n = 11), hIgG4 isotype control (n = 6), mIL-12p40 (BioxCell) (n = 11), and rat IgG2a isotype control (n = 6) were injected intraperitoneally at 10 mg / mL on days 7, 10, 14, 17, 21, and 24. (8A) Weight loss in DITRA-like mice was calculated as the percentage difference between the original body weight and the actual body weight on any particular day. (8B) Fecal lipocalin-2 (Lcn2) levels in DITRA-like mice measured on days 0, 7, 17, 23, and 30 throughout chronic colitis. (8C) Colon length in DITRA-like mice on day 30. (8D) Myeloperoxidase (MPO) activity in colon homogenates of DITRA-like mice on day 30. (8E) Levels of proinflammatory cytokines in colon homogenates of DITRA-like mice on day 30. Data represent two independent experiments. Error bars represent mean SD. In each set of bars, from left to right are: PBS / water, PBS / DSS, hIL-36R / DSS, hIgG4 isotype control / DSS, mIL-12p40 / DSS, and rat IgG2a isotype control / DSS.

[0052] Figures 9A-9C.Human IL-36R antagonism improves oxazolone-induced colitis in DITRA-like mice. DITRA-like mice were pre-sensitized with a 3% oxazolone solution dissolved in 100% ethanol, and 1.5% oxazolone and vehicle (50% ethanol) were administered intrarectally on days 5, 6, and 7. On days 2, 5, and 7 after pre-sensitization, mice were injected intraperitoneally with PBS, anti-human IL-36R mAb, and hIgG4 isotype control. (A) Body weight of DITRA-like mice treated with PBS, anti-human IL-36R mAb, and hIgG4 control during oxazolone administration. *p<0.05 relative to the PBS-treated group. (B) Colon length measured in DITRA-like mice on day 8. (C) Levels of proinflammatory cytokines in colon homogenates of DITRA-like mice treated with oxazolone and vehicle injected with PBS, anti-human IL-36R mAb, and hIgG4 isotype control. *- represents significance relative to the PBS-treated group, #- represents significance relative to the isotype-treated group. Data represent two independent experiments with 5 mice per group. Error bars represent mean ± SEM. *p<0.05, **p<0.005, ***p<0.0005, ****p<0.00001. DETAILED DESCRIPTION

[0053] Disclosed herein are rodents (such as, but not limited to, mice) that are genetically modified to contain a humanized ll1rl2 gene (encoding a humanized IL1RL2 protein having an extracellular domain substantially identical to the extracellular domain of human IL1RL2) and human genes encoding human IL-36α, β, γ ligands, while retaining an endogenous IL-36Ra antagonist that is 20 times less potent in inhibiting human IL-36R signaling. The genetically modified rodents disclosed herein have been shown to exhibit symptoms of DITRA patients in which mutations in IL-36Ra result in enhanced IL-36R signaling. Thus, the genetically modified rodents disclosed herein can be used as a novel DITRA functional model suitable for testing candidate therapeutic agents for the treatment of DITRA and related disorders. Various embodiments are further described below.

[0054] Genetically modified rodents: quadruple humanization

[0055] In one aspect of some embodiments, the present disclosure provides a rodent comprising in its genome a humanized Il1rl2 gene encoding a humanized IL1RL2 protein (a subunit of IL-36R) and human genes encoding human IL-36α, β and γ ligands. Such rodents are also referred to herein as quadruple humanized rodents (i.e., 4H or DITRA-like).

[0056] When used in the context of nucleic acids or proteins, the term "humanized" includes rodent nucleic acids or proteins whose structure (i.e., nucleotide or amino acid sequence) has been modified in whole or in part to include the structure found in the corresponding human nucleic acid or protein. A rodent containing a humanized gene or expressing a humanized protein is a "humanized" rodent.

[0057] In some embodiments, rodents of the present disclosure include mice, rats and hamsters as non-limiting examples. In some embodiments, rodents of the present disclosure include mice and rats as non-limiting examples. In some embodiments, rodents are selected from the superfamily Muroidea. In some embodiments, rodents of the present disclosure are from a family selected from the following: Calomyscidae (e.g., hamsters of mouse-like species), Cricetidae (e.g., hamsters, New World rats and mice, voles), Muridae (Muridae) (true mice and rats, gerbils, spiny rats, crowned rats), Nesomyidae (climbing mice, rock mice, rats with tails, Madagascar rats and mice), Platacanthomyidae (e.g., dormouse) and Spalacidae (e.g., moles, bamboo rats and zokors). In some embodiments, the rodent of the present disclosure is selected from true mice or rats (family Muridae), gerbils, spiny mice, and crested mice. In some embodiments, the mouse of the present disclosure is from a member of the family Muridae.

[0058] IL-1RL2 humanization

[0059] IL-36R is a member of the interleukin 1 receptor family and is a heterodimer composed of a receptor subunit called IL1RL2 (also known as IL-1Rrp2) and a co-receptor subunit, the interleukin 1 receptor accessory protein IL-1RAcP (Garlanda C et al., Immunity 39, 1003–1018 (2013); Towne JE et al., J. Biol. Chem. 279, 13677–13688 (2004), which are incorporated by reference in their entirety). The receptor (IL-36R) can recognize and bind to three different agonists IL-36α, IL-36β, and IL-36γ (also known as IL1F6, IL1F8, and IL1F9) to induce the expression of inflammatory cytokines, and recognize and bind to the antagonist IL-36Ra, which binds to IL-36R and reduces the expression of inflammatory cytokines.

[0060] IL1RL2 contains a signal peptide, an extracellular domain ("ECD" or "ectodomain"), a transmembrane domain, and an intracellular or cytoplasmic domain. See, e.g. Figure 1DExemplary IL1RL2 sequences, including human, mouse, rat and humanized IL1RL2 nucleic acid and protein sequences, are disclosed in the Sequence Listing and summarized in the table below.

[0061] Table 1

[0062]

[0063]

[0064] In some embodiments, the rodent disclosed herein comprises in its genome a humanized Il1rl2 gene including the nucleotide sequence of an endogenous rodent Il1rl2 gene and the nucleotide sequence of a human IL1RL2 gene. As used herein, "a nucleotide sequence of a gene" includes all or part of a genomic sequence, mRNA or cDNA sequence of a gene. As a non-limiting example, the nucleotide sequence of a human IL1RL2 gene includes all or part of a genomic sequence, mRNA or cDNA sequence of a human IL1RL2 gene. The nucleotide sequence of the endogenous rodent Il1rl2 gene and the nucleotide sequence of the human IL1RL2 gene are operatively linked to each other, so that the humanized Il1rl2 gene in the rodent genome encodes an Il1rl2 protein, i.e., has an Il1rl2 protein structure (composed of an extracellular domain, a transmembrane domain and a cytoplasmic domain) and performs Il1rl2 functions (e.g., binding to IL-1 The receptor accessory protein (IL-1RacP) is a protein that forms heterodimers and recognizes three ligands: IL-36α, IL-36β, and IL-36γ).

[0065] In some embodiments, the genetically modified rodent contains a humanized Il1rl2 gene in its genome, wherein the humanized Il1rl2 gene encodes a humanized Il1rl2 protein containing an extracellular domain substantially identical to the extracellular domain of a human IL1RL2 protein. In some embodiments, the extracellular domain substantially identical to the extracellular domain of a human IL1RL2 protein exhibits the same functionality (e.g., ligand binding properties) as the extracellular domain of a human IL1RL2 protein. An extracellular domain or polypeptide that is "substantially identical to the extracellular domain of a human IL1RL2 protein" means, in some embodiments, a polypeptide that is at least 95%, 98%, 99% or 100% identical in sequence to the extracellular domain of a human IL1RL2 protein; in some embodiments, a polypeptide that differs from the extracellular domain of a human IL1RL2 protein by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acids; and in some embodiments, a polypeptide that differs from the extracellular domain of a human IL1RL2 protein only at the N or C terminal portion of the extracellular domain, for example, by having an addition, deletion or substitution of amino acids at the N or C terminal portion of the extracellular domain, but not more than 5, 4, 3, 2 or 1 amino acids. "N or C terminal portion of the extracellular domain" means within 5-10 amino acids of the N or C terminal of the extracellular domain. In some embodiments, the human IL1RL2 protein has an amino acid sequence substantially identical to the amino acid sequence shown in SEQ ID NO: 2 (e.g., at least 95%, 98%, 99% or 100% identical). In a specific embodiment, the human IL1RL2 protein comprises the amino acid sequence shown in SEQ ID NO: 2, and its extracellular domain consists of amino acids 20-335 of SEQ ID NO: 2. In some embodiments, the humanized Il1rl2 gene encodes a humanized Il1rl2 protein containing an extracellular domain substantially identical to the extracellular domain of the human IL1RL2 protein shown in SEQ ID NO: 2 (i.e., amino acids 20-335 of SEQ ID NO: 2). In some embodiments, the humanized Il1rl2 gene encodes a humanized Il1rl2 protein containing an extracellular domain comprising amino acids 22-337 of SEQ ID NO: 7.

[0066] In some embodiments, the humanized Il1rl2 gene encodes a humanized Il1rl2 protein containing a transmembrane cytoplasmic sequence that is substantially identical to the transmembrane cytoplasmic sequence of an endogenous rodent Il1rl2 protein (i.e., a sequence that includes both a transmembrane domain and a cytoplasmic domain). In some embodiments, the transmembrane cytoplasmic sequence that is substantially identical to the transmembrane cytoplasmic sequence of an endogenous rodent Il1rl2 protein exhibits the same functionality (e.g., signal transduction and / or interaction with intracellular molecules) as the transmembrane cytoplasmic sequence of an endogenous rodent Il1rl2 protein. A transmembrane cytoplasmic sequence or polypeptide that is "substantially identical to the transmembrane cytoplasmic sequence of an endogenous rodent Il1rl2 protein" means, in some embodiments, a polypeptide that is at least 95%, 98%, 99% or 100% identical to the transmembrane cytoplasmic sequence of an endogenous rodent Il1rl2 protein; in some embodiments, a polypeptide that differs from the transmembrane cytoplasmic sequence of an endogenous rodent Il1rl2 protein by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid; in some embodiments, a polypeptide that differs from the transmembrane cytoplasmic sequence of an endogenous rodent Il1rl2 protein only at the N or C terminus, such as by having an addition, deletion or substitution of amino acids in the N or C terminal portion of the transmembrane cytoplasmic sequence but not more than 5, 4, 3, 2 or 1 amino acid. "N or C terminal portion of the transmembrane cytoplasmic sequence" means within 5-10 amino acids from the N terminus of the transmembrane domain or within 5-10 amino acids from the C terminus of the cytoplasmic domain. In some embodiments, the humanized Il1rl2 protein contains a transmembrane cytoplasmic sequence that is substantially identical to a mouse Il1rl2 protein (e.g., a mouse Il1rl2 protein that is substantially identical (at least 95%, 98%, 99%, or 100% identical) to SEQ ID NO:4), or a transmembrane cytoplasmic sequence that is substantially identical (at least 95%, 98%, 99%, or 100% identical) to a rat Il1rl2 protein (e.g., a rat Il1rl2 protein that is substantially identical (at least 95%, 98%, 99%, or 100% identical) to SEQ ID NO:6).

[0067] In some embodiments, the humanized Il1rl2 gene encodes a humanized Il1rl2 protein containing a signal peptide that is substantially identical to the signal peptide of an endogenous rodent Il1rl2 protein. A signal peptide that is "substantially identical to the signal peptide of an endogenous rodent Il1rl2 protein" means, in some embodiments, a polypeptide that is at least 95%, 98%, 99% or 100% identical to the signal peptide of an endogenous rodent Il1rl2 protein; in some embodiments, a polypeptide that differs from the signal peptide of an endogenous rodent Il1rl2 protein by no more than 3, 2 or 1 amino acids; in some embodiments, a polypeptide that differs from the signal peptide of an endogenous rodent Il1rl2 protein only at the N or C terminus, such as by having an addition, deletion or substitution of amino acids in the N or C terminal portion of the signal peptide but not more than 3, 2 or 1 amino acids. "The N or C terminal portion of the signal peptide" means within 5 amino acids from the N or C terminus of the signal peptide. In some embodiments, the humanized Il1rl2 protein includes a signal peptide substantially identical to the signal peptide of a mouse Il1rl2 protein (e.g., a mouse Il1rl2 protein that is substantially identical (at least 95%, 98%, 99%, or 100% identical) to SEQ ID NO:4), or a signal peptide substantially identical to the signal peptide of a rat Il1rl2 protein (e.g., a rat Il1rl2 protein that is substantially identical (at least 95%, 98%, 99%, or 100% identical) to SEQ ID NO:6).

[0068] In some embodiments, the humanized Il1rl2 gene in the genome of the genetically modified rodent includes a nucleotide sequence of a human IL1RL2 gene (or "human IL1RL2 nucleotide sequence") and a nucleotide sequence of an endogenous rodent Il1rl2 gene (or "endogenous rodent Il1rl2 nucleotide sequence"), wherein the human IL1RL2 nucleotide sequence encodes a polypeptide substantially identical to the extracellular domain of a human IL1RL2 protein encoded by the human IL1RL2 gene. Such a human IL1RL2 nucleotide sequence is also referred to as substantially encoding the extracellular domain of a human IL1RL2 protein. In some embodiments, the human IL1RL2 nucleotide sequence is a genomic fragment of a human IL1RL2 gene. In some embodiments, the human IL1RL2 nucleotide sequence is a genomic fragment comprising exons 3-8 of a human IL1RL2 gene. In some embodiments, the human IL1RL2 nucleotide sequence is a genomic fragment comprising the 3' portion of intron 2, exons 3-8, and the 5' portion of intron 8 of a human IL1RL2 gene. In some embodiments, the human IL1RL2 nucleotide sequence is a cDNA sequence.

[0069] In some embodiments, the humanized Il1rl2 gene in the genome of the genetically modified rodent includes an endogenous rodent Il1rl2 nucleotide sequence and a human IL1RL2 nucleotide sequence, wherein the endogenous rodent Il1rl2 nucleotide sequence encodes a polypeptide substantially identical to the transmembrane cytoplasmic sequence of the endogenous rodent Il1rl2 protein. Such rodent Il1rl2 nucleotide sequences are also referred to as transmembrane cytoplasmic sequences that substantially encode endogenous rodent Il1rl2 proteins. In some embodiments, the endogenous rodent Il1rl2 nucleotide sequence present in the humanized Il1rl2 gene encodes the transmembrane cytoplasmic sequence of the endogenous rodent Il1rl2 protein. In some embodiments, the endogenous rodent Il1rl2 nucleotide sequence present in the humanized Il1rl2 gene includes the remaining exons downstream of exon 8 in the endogenous rodent Il1rl2 gene. In some embodiments, the endogenous rodent Il1rl2 nucleotide sequence present in the humanized Il1rl2 gene comprises the 3' portion of intron 8 and the remaining exons downstream of exon 8 in the endogenous rodent Il1rl2 gene.

[0070] In some embodiments, the humanized Il1rl2 gene in the genome of the genetically modified rodent includes an endogenous rodent Il1rl2 nucleotide sequence upstream (5') of the human IL1RL2 nucleotide sequence, wherein the endogenous rodent Il1rl2 nucleotide sequence encodes a polypeptide substantially identical to the signal peptide of the endogenous rodent Il1rl2 protein. Such a rodent Il1rl2 nucleotide sequence is also referred to as substantially encoding the signal peptide of the endogenous rodent Il1rl2 protein. In some embodiments, the endogenous rodent Il1rl2 nucleotide sequence encoding a polypeptide substantially identical to the signal peptide of the endogenous rodent Il1rl2 protein comprises exons 1-2 of the endogenous rodent Il1rl2 gene; and in some embodiments, the endogenous rodent Il1rl2 nucleotide sequence comprises the 5' portion of exons 1-2 and intron 2 of the endogenous rodent Il1rl2 gene.

[0071] In some embodiments, the humanized Il1rl2 gene is operably linked to endogenous rodent Il1rl2 regulatory sequences, for example one or more 5' transcriptional regulatory sequences such as a promoter and / or enhancer, such that expression of the humanized Il1rl2 gene is under the control of one or more rodent Il1rl2 5' regulatory sequences.

[0072] In some embodiments, the humanized Il1rl2 gene is located at an endogenous rodent Il1rl2 locus. In some embodiments, the humanized Il1rl2 gene is located at a locus other than the endogenous rodent Il1rl2 locus; for example, as a result of random integration. In some embodiments in which the humanized Il1rl2 gene is located at a locus other than the endogenous rodent Il1rl2 locus, the rodent is unable to express the rodent Il1rl2 protein, for example, as a result of inactivation (e.g., total or complete deletion) of the endogenous rodent Il1rl2 gene.

[0073] In some embodiments where the humanized Il1rl2 gene is located at an endogenous rodent Il1rl2 locus, the humanized Il1rl2 gene is generated by replacing the nucleotide sequence of the endogenous rodent Il1rl2 gene at the endogenous rodent Il1rl2 locus with the nucleotide sequence of the human IL1RL2 gene.

[0074] In some embodiments, the nucleotide sequence of the endogenous rodent Il1rl2 gene at the endogenous rodent Il1rl2 locus that is replaced is a genomic fragment of the endogenous rodent Il1rl2 gene that substantially encodes the extracellular domain of the rodent Il1rl2 protein. In some embodiments, the rodent genomic fragment that is replaced comprises exons 3-8 of the endogenous rodent Il1rl2 gene.

[0075] In some embodiments, the nucleotide sequence of the human IL1RL2 gene that replaces the genomic fragment of the rodent Il1rl2 gene at the endogenous rodent Il1rl2 locus is a cDNA sequence. In some embodiments, the human IL1RL2 nucleotide sequence that replaces the genomic fragment of the rodent Il1rl2 gene at the endogenous rodent Il1rl2 locus is a genomic fragment of the human IL1RL2 gene. In some embodiments, the genomic fragment of the human IL1RL2 gene that replaces the genomic fragment of the rodent Il1rl2 gene at the endogenous rodent Il1rl2 locus includes all or part of the exons of the human IL1RL2 gene that substantially encodes the extracellular domain of the human IL1RL2 protein. In some embodiments, the human genomic fragment comprises exons 3-8 of the human IL1RL2 gene.

[0076] In some embodiments, the genomic sequence of the endogenous rodent Il1rl2 gene that remains at the endogenous rodent Il1rl2 locus after the humanizing replacement and is operably linked to the inserted human IL1RL2 nucleotide sequence substantially encodes the transmembrane cytoplasmic sequence of the endogenous rodent Il1rl2 protein. In some embodiments, the genomic sequence of the endogenous rodent Il1rl2 gene that remains at the endogenous rodent Il1rl2 locus after the humanizing replacement includes exons downstream of exon 8 of the endogenous rodent Il1rl2 gene.

[0077] In some embodiments, the genomic sequence of the endogenous rodent Il1rl2 gene that remains at the endogenous rodent Il1rl2 locus after the humanizing replacement and is operably linked to the inserted human IL1RL2 nucleotide sequence substantially encodes the signal peptide of the endogenous rodent Il1rl2 protein. In some embodiments, the genomic sequence of the endogenous rodent Il1rl2 gene that remains at the endogenous rodent Il1rl2 locus after the humanizing replacement includes exons 1-2 of the endogenous rodent Il1rl2 gene.

[0078] In some embodiments, where the endogenous rodent Il1rl2 protein and the human IL1RL2 protein share common amino acids near the junction between the transmembrane domain and the extracellular domain, it may not be necessary to insert a human IL1RL2 nucleotide sequence that precisely encodes the extracellular domain of the human IL1RL2 protein. A slightly longer or shorter nucleotide sequence of the human IL1RL2 gene that substantially encodes the extracellular domain of the human IL1RL2 protein may be inserted, operatively linked to the genomic sequence of the endogenous rodent Il1rl2 gene that substantially encodes the transmembrane domain (and cytoplasmic domain) of the endogenous rodent Il1rl2 protein, such that the humanized Il1rl2 protein encoded by the resulting humanized Il1rl2 gene includes an extracellular domain identical to the extracellular domain of the human IL1RL2 protein and a transmembrane domain identical to the transmembrane domain of the endogenous rodent Il1rl2 protein.

[0079] In some embodiments, a genomic fragment comprising exons 3-8 of an endogenous rodent Il1rl2 gene at an endogenous rodent Il1rl2 locus has been replaced by a genomic fragment comprising exons 3-8 of a human IL1RL2 gene. As a result, a humanized Il1rl2 gene is formed at the endogenous rodent Il1rl2 locus and comprises exons 1-2 of an endogenous rodent Il1rl2 gene, exons 3-8 of a human IL1RL2 gene, and the remaining exons downstream of exon 8 of an endogenous rodent Il1rl2 gene.

[0080] In some embodiments, the rodents provided herein are heterozygous for the humanized Il1rl2 gene in their genome. In some embodiments, the rodents provided herein are homozygous for the humanized Il1rl2 gene in their genome.

[0081] In some embodiments, the humanized Il1rl2 gene results in expression of the encoded humanized Il1rl2 protein in a rodent. In some embodiments, the humanized Il1rl2 protein is expressed in a comparable or substantially identical pattern to the corresponding rodent Il1rl2 protein in a control rodent (e.g., a rodent without the humanized Il1rl2 gene). In some embodiments, the humanized Il1rl2 protein is expressed at a comparable or substantially identical level to the corresponding rodent Il1rl2 protein in a control rodent (e.g., a rodent without the humanized Il1rl2 gene). In some embodiments, the humanized Il1rl2 protein is expressed and detected on the surface of cells such as, but not limited to, keratinocytes, monocytes, macrophages, neutrophils, bronchial and intestinal epithelial cells. In the context of comparing a humanized gene or protein in a humanized rodent to an endogenous rodent gene or protein in a control rodent, the term "comparable" means that the molecules or levels being compared may be different from each other but are similar enough to allow comparison between the two, so that reasonable conclusions can be drawn based on the observed differences or similarities. In some embodiments, the term "substantially the same" when referring to expression levels includes that the levels being compared differ from each other by no more than 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1%.

[0082] In some embodiments, a rodent disclosed herein is unable to express a rodent Il1rl2 protein, for example as a result of inactivation (eg, deletion in whole or in part) or replacement (in whole or in part) of an endogenous rodent Il1rl2 gene.

[0083] IL-36 ligand humanization

[0084] The rodents disclosed herein also contain genes encoding human IL-36 α, β, and γ ligands in their germline.

[0085] Table 2

[0086]

[0087] In some embodiments, the rodent disclosed herein has a genome comprising a human IL-36α gene encoding a human IL-36α protein. Reference to a "human IL-36α gene" includes human genomic DNA encoding a human IL-36α protein and comprising a human IL-36α promoter. The human IL-36α protein may be a mature form or a precursor form of the human IL-36α protein. In some embodiments, the human IL-36α protein comprises the amino acid sequence of SEQ ID NO: 9.

[0088] In some embodiments, the human IL-36α gene is located at an endogenous rodent 11-36α locus. In some embodiments, the human IL-36α gene is located at a locus other than the endogenous rodent 11-36 locus; for example, as a result of random integration. In some embodiments where the human IL-36α gene is located at a locus other than the endogenous rodent 11-36α locus, the rodent is unable to express the rodent 11-36α protein, for example, as a result of inactivation (e.g., complete or partial deletion) of the endogenous rodent 11-36α gene.

[0089] In some embodiments, the human IL-36α gene replaces the endogenous rodent 11-36α gene at the endogenous rodent 11-36α locus.

[0090] In some embodiments, the rodents provided herein are heterozygous for the human IL-36α gene in their genome. In other embodiments, the rodents provided herein are homozygous for the human IL-36α gene in their genome.

[0091] In some embodiments, the human IL-36α gene causes the encoded human IL-36α protein to be expressed in rodents (e.g., in serum, at mucosal sites such as skin, intestinal epithelium, lungs, and in various types of immune system cells (e.g., monocytes, macrophages, T cells, dendritic cells). In some embodiments, the human IL-36α protein is expressed in a pattern comparable to or substantially the same as the corresponding rodent II-36α protein in a control rodent (e.g., a rodent without the human IL-36α gene). In some embodiments, the human IL-36α protein is expressed at levels comparable to or substantially the same as the corresponding rodent II-36α protein in a control rodent (e.g., a rodent without the human IL-36α gene), e.g., in serum, at mucosal sites (e.g., such as skin, intestinal epithelium, lungs), and / or in immune cells (e.g., monocytes, macrophages, T cells, dendritic cells).

[0092] In some embodiments, the rodents disclosed herein are unable to express rodent 11-36α protein, for example as a result of inactivation (eg, complete or partial deletion) or replacement (complete or partial) of an endogenous rodent 11-36α gene.

[0093] Table 3

[0094]

[0095] In some embodiments, the rodent disclosed herein has a genome comprising a human IL-36β gene encoding a human IL-36β protein. Reference to a "human IL-36β gene" includes human genomic DNA encoding a human IL-36β protein and comprising a human IL-36β promoter. The human IL-36β protein can be a mature form or a precursor form of the human IL-36β protein. In some embodiments, the human IL-36β protein comprises the amino acid sequence of SEQ ID NO: 15.

[0096] In some embodiments, the human IL-36β gene is located at an endogenous rodent 11-36β locus. In some embodiments, the human IL-36β gene is located at a locus other than the endogenous rodent 11-36β locus; for example, as a result of random integration. In some embodiments where the human IL-36β gene is located at a locus other than the endogenous rodent 11-36β locus, the rodent is unable to express the rodent 11-36β protein, for example, as a result of inactivation (e.g., complete or partial deletion) of the endogenous rodent 11-36β gene.

[0097] In some embodiments, the human IL-36β gene replaces the endogenous rodent 11-36β gene at the endogenous rodent 11-36β locus.

[0098] In some embodiments, the rodents provided herein are heterozygous for the human IL-36β gene in their genome. In other embodiments, the rodents provided herein are homozygous for the human IL-36β gene in their genome.

[0099] In some embodiments, the human IL-36β gene causes the encoded human IL-36β protein to be expressed in a rodent (e.g., in serum, at mucosal sites (e.g., such as skin, intestinal epithelium, lungs), and / or in immune cells (e.g., monocytes, macrophages, T cells, dendritic cells). In some embodiments, the human IL-36β protein is expressed in a pattern comparable to or substantially the same as the corresponding rodent II-36β protein in a control rodent (e.g., a rodent without the human IL-36β gene). In some embodiments, the human IL-36β protein is expressed at levels comparable to or substantially the same as the corresponding rodent II-36β protein in a control rodent (e.g., a rodent without the human IL-36β gene).

[0100] In some embodiments, the rodents disclosed herein are unable to express rodent 111-36β protein, for example as a result of inactivation (e.g., complete or partial deletion) or replacement (complete or partial) of an endogenous rodent 11-36β gene.

[0101] Table 4

[0102]

[0103]

[0104] In some embodiments, the present invention provides a rodent whose genome contains a human IL-36γ gene encoding a human IL-36γ protein. Reference to "human IL-36γ gene" includes human genomic DNA encoding a human IL-36γ protein and comprising a human IL-36 promoter. In some embodiments, the human IL-36 protein can be a mature form or a precursor form of the human IL-36γ protein. In some embodiments, the human IL-36 protein comprises the amino acid sequence of SEQ ID NO: 21.

[0105] In some embodiments, the human IL-36γ gene is located at an endogenous rodent 11-36γ locus. In some embodiments, the human IL-36γ gene is located at a locus other than the endogenous rodent 11-36γ locus; for example, as a result of random integration. In some embodiments where the human IL-36γ gene is located at a locus other than the endogenous rodent 11-36γ locus, the rodent is unable to express the rodent 11-36γ protein, for example, as a result of inactivation (e.g., complete or partial deletion) of the endogenous rodent 11-36γ gene.

[0106] In some embodiments, the human IL-36γ gene replaces the endogenous rodent 11-36γ gene at the endogenous rodent 11-36γ locus.

[0107] In some embodiments, the rodents provided herein are heterozygous for the human IL-36γ gene in their genome. In other embodiments, the rodents provided herein are homozygous for the human IL-36γ gene in their genome.

[0108] In some embodiments, the human IL-36γ gene causes the encoded human IL-36γ protein (e.g., a protein identical to the human IL-36γ protein) to be expressed in rodents (e.g., in serum, at mucosal sites such as the skin, intestinal epithelium, lungs, and in various types of immune system cells (e.g., monocytes, macrophages, T cells, dendritic cells). In some embodiments, the human IL-36γ protein is expressed in a pattern comparable to or substantially the same as the corresponding rodent II-36γ protein in a control rodent (e.g., a rodent without the human IL-36γ gene). In some embodiments, the human IL-36γ protein is expressed at levels comparable to or substantially the same as the corresponding rodent II-36γ protein in a control rodent (e.g., a rodent without the human IL-36γ gene), e.g., in serum, at mucosal sites (e.g., such as the skin, intestinal epithelium, lungs), and / or in immune cells (e.g., monocytes, macrophages, T cells, dendritic cells).

[0109] In some embodiments, the rodents disclosed herein are unable to express rodent 11-36γ protein, for example as a result of inactivation (e.g., complete or partial deletion) or replacement (complete or partial) of an endogenous rodent 11-36 gene.

[0110] In some embodiments, rodents are provided, wherein the locus comprises a continuous genomic segment covering all three Il-36 ligand coding sequences at the endogenous locus, and is replaced by a continuous nucleic acid comprising the coding sequences of the three ligands substantially identical to human IL-36α, β and γ, respectively. In some embodiments, the resulting locus comprises, from 5' to 3', (i) human IL-36β gene, (ii) human IL-36γ gene, and (iii) the antisense strand of human IL-36α gene.

[0111] Phenotype of quadruple humanized rodents

[0112] The genetically modified rodents disclosed herein do not develop any spontaneous diseases in a stable state; however, as compared to age-matched control rodents without humanization, these rodents do show shortened colon and increased expression of pro-inflammatory mediators in the skin in both a stable state and a disease state (e.g., after DSS or IMQ treatment). In some embodiments, the genetically modified rodents show a colon length that is at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or 50% shorter than that of control rodents in a stable state or a disease state.

[0113] In some embodiments, at steady state, the genetically modified rodents exhibit a colon length that is 10% to 15% (+ / - 5%) shorter than control rodents. In some embodiments, at steady state, the genetically modified rodents exhibit a colon length that is 15% to 20% (+ / - 5%) shorter than control rodents. In some embodiments, at steady state, the genetically modified rodents exhibit a colon length that is 10% to 20% (+ / - 5%) shorter than control rodents.

[0114] In other embodiments, in disease state (for example, after DSS or oxazolone treatment), genetically modified rodents show colon length 20% to 40% (+ / -5%) shorter than control rodents. In other embodiments, in disease state (for example, after DSS or oxazolone treatment), genetically modified rodents show colon length 30% to 40% (+ / -5%) shorter than control rodents. In other embodiments, in disease state (for example, after DSS or oxazolone treatment), genetically modified rodents show colon length 20% to 30% (+ / -5%) shorter than control rodents. In other embodiments, in disease state (for example, after DSS or oxazolone treatment), genetically modified rodents show colon length 25% to 35% (+ / -5%) shorter than control rodents.

[0115] Although the genetically modified rodents disclosed herein do not develop any spontaneous disease in the steady state, they have been shown to display enhanced skin and intestinal inflammation in experimentally induced models of skin and intestinal inflammation, such as preclinical models of psoriasis and IBD, respectively.

[0116] In some embodiments, DSS is used to induce chronic colitis. In some embodiments, DSS is administered to rodents by drinking at least 0.5%, at least 1%, at least 1.5%, at least 2.5% of water. In some embodiments, DSS is administered to rodents by drinking no more than 10%, 9%, 8%, 7%, 6% or 5% of water. In some embodiments, drinking water containing 1.5%-3% DSS is administered to rodents. In some embodiments, drinking water containing 0.5%-3% DSS is administered to rodents. In some embodiments, drinking water containing 1%-3% DSS is administered to rodents. In some embodiments, drinking water containing 2%-3% DSS is administered to rodents. In some embodiments, drinking water containing 2.5%-3% DSS is administered to rodents. In some embodiments, drinking water containing 0.5%-2.5% DSS is administered to rodents. In some embodiments, drinking water containing 0.5%-2% DSS is administered to rodents. In some embodiments, drinking water containing 0.5%-1.5% DSS is administered to rodents. In some embodiments, drinking water containing 0.5%-1% DSS is administered to rodents. In some embodiments, drinking water containing 1%-2.5% DSS is administered to rodents. In some embodiments, drinking water containing 1.5%-2% DSS is administered to rodents.

[0117] In some embodiments, the administration of DSS may be performed for a period of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more days, and may be continuous or interrupted by days without DSS administration. In some embodiments, rodents are provided with drinking water containing 2.5% DSS for 7 days, followed by drinking water containing 1.5% DSS for 5 days, followed by distilled water until 27-30 days before analysis. In some embodiments, rodents are provided with drinking water containing 3% DSS for 7 days, followed by drinking water containing 2% DSS for 13 days, followed by distilled water until 27-30 days before analysis. In some embodiments, DSS is not administered during the entire period. In some embodiments, rodents are provided with drinking water containing 2.5% DSS for 7 days, followed by distilled water for 11 days, followed by drinking water containing 1.5% DSS for 4 days, followed by distilled water for 5 days - a total of 27 days before analysis. In some embodiments, rodents are provided drinking water containing 3% DSS for 7 days, followed by water for 13 days, then drinking water containing 2% DSS for 4 days, followed by distilled water for 6 days until 30 days prior to analysis.

[0118] In some embodiments, oxazolone is used to induce colitis. In some embodiments, oxazolone is administered intrarectally to rodents to induce colitis. In some embodiments, oxazolone is administered intrarectally to rodents in three administrations to induce colitis. In some embodiments, oxazolone is administered topically to rodents for presensitization before intrarectal administration. In some embodiments, oxazolone is administered topically to rodents for presensitization by topical application of an oxazolone solution (e.g., a 3% oxazolone solution dissolved in 100% ethanol) on shaved skin, followed by three intrarectal administrations of an oxazolone solution (e.g., 1.0-2.0% oxazolone dissolved in 50% ethanol). In some embodiments, presensitization employs a 1.5%, 2.0%, 2.5%, 3.0%, 3.5%, or 4.0% oxazolone solution dissolved in 100% ethanol. In some embodiments, rectal administration adopts 1.0%, 1.1%, 1.2%, 1.3%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9% or 2.0% oxazolone solution dissolved in 50% ethanol.In some embodiments, rectal administration is carried out several days (such as 3, 4, 5, 6 or 7 days) after presensitization.In some embodiments, by topically applying 3.0% oxazolone solution dissolved in 100% ethanol to the skin, followed by three intrarectal administrations of 1.0-2.0% oxazolone solution dissolved in 50% ethanol on the 5th, 6th and 7th days, oxazolone is given to rodents.

[0119] In some embodiments, the extent of colitis is assessed by scoring the following features: inflammation (severity and extent), epithelial changes (erosions / ulcers), crypt changes (crypt loss, cryptitis / crypt abscesses, regeneration / hyperplasia, goblet cell loss), submucosal edema, and the percentage of tissue area with pathology relative to the total tissue area on the slide. A 0-4 scoring scale is used: 0-0-within the normal range, 1-minimal, 2-mild, 3-moderate, and 4-severe. An overall pathology score is calculated for each rodent by adding the scores for each histopathological feature. In some embodiments, colitis is assessed by measuring the level of lipocalin 2 (Lcn2) in fecal samples. In some embodiments, colitis is measured by measuring the level of myeloperoxidase (MPO) activity in colon homogenates. In some embodiments, colitis is assessed by measuring the level of inflammatory cytokines in colon homogenates.

[0120] In some embodiments, the genetically modified rodents exhibit increased intestinal pathology scores, e.g., an increase of at least 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400% or more, as compared to wild-type control rodents that have undergone the same DSS administration. In some embodiments, the genetically modified rodents exhibit increased intestinal pathology scores, e.g., an increase of 50%-400%, 50%-300%, 50%-200%, 50%-100%, 100%-400%, 100%-300%, 100-200%, or 200%-400%, as compared to wild-type control rodents that have undergone the same DSS administration. In some embodiments, the genetically modified rodents exhibit increased myeloperoxidase ("MPO") levels in colon homogenates, e.g., an increase of at least 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400% or more, compared to wild-type control rodents that have undergone the same DSS administration. In some embodiments, the genetically modified rodents exhibit increased MPO levels in colon homogenates, e.g., an increase of 50%-400%, 50%-300%, 50%-200%, 50%-100%, 100%-400%, 100%-300%, 100-200%, or 200%-400%, compared to wild-type control rodents that have undergone the same DSS administration. In some embodiments, the genetically modified rodents exhibit increased fecal Lcn levels, e.g., an increase of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400% or more, as compared to wild-type control rodents that have undergone the same DSS administration. In some embodiments, the genetically modified rodents exhibit increased mRNA expression and / or protein levels of one or more proinflammatory mediators (e.g., KC-GRO, IL-6, IL-1β, TNFα, IL-21, IL-12p40, IL-17f, IL-17a, and IL-17c) in colon homogenate, e.g., by at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900% or more, as compared to wild-type control rodents that have undergone the same DSS administration.In some embodiments, the genetically modified rodents exhibit increased mRNA expression and / or protein levels of one or more proinflammatory mediators in colon homogenates, such as 20%-900%, 20%-800%, 20%-700%, 20%-600%, 20%-500%, 20%-400%, 20%-300%, 20%-200% or 20%-100%. In some embodiments, the genetically modified rodents exhibit increased mRNA expression and / or protein levels of one or more proinflammatory mediators in colon homogenates, such as 30%-900%, 30%-800%, 30%-700%, 30%-600%, 30%-500%, 30%-400%, 30%-300%, 30%-200% or 30%-100%. In some embodiments, the genetically modified rodents exhibit increased mRNA expression and / or protein levels of one or more proinflammatory mediators in colon homogenates, such as 40%-900%, 40%-800%, 40%-700%, 40%-600%, 40%-500%, 40%-400%, 40%-300%, 40%-200% or 40%-100%. In some embodiments, the genetically modified rodents exhibit increased mRNA expression and / or protein levels of one or more proinflammatory mediators in colon homogenates, such as 50%-900%, 50%-800%, 50%-700%, 50%-600%, 50%-500%, 50%-400%, 50%-300%, 50%-200% or 50%-100%.

[0121] In some embodiments, IMQ is topically applied to the skin of rodents to induce skin inflammation. In some embodiments, IMQ is provided in a carrier suitable for topical application, and the carrier is, for example, a cream, a gel, including commercially available IMQ creams (for example, such as those from Aldara). In some embodiments, in order to induce skin inflammation, IMQ is applied to rodent skin every day with a daily dose of 1 to 5mg, 2 to 4mg or 3-3.5mg, for a period of 2, 3, 4, 5, 6, 7, 8, 9, 10 days or longer. In some embodiments, topical application every day with a daily dose of about 3.125mg lasts for 4 days and is suitable for inducing acute skin inflammation, and topical application every day with a daily dose of about 3.125mg lasts for 9 days and is suitable for inducing chronic skin inflammation. In some embodiments, before analysis or determination, IMQ is topically applied multiple rounds (for example, 2, 3 or 4 rounds), wherein the IMQ of 4-5 days in a row is applied, and then the IMQ of 2 days in each round is applied. In specific embodiments, IMQ is topically administered for two rounds prior to analysis, with 5 consecutive days of IMQ administration followed by 2 days of no IMQ administration in the first round and then 4 consecutive days of IMQ administration in the second round (see, e.g., FIGS. 7A-7C ). In some embodiments, the severity of inflammation can be assessed by: (i) using an adapted version of the clinical Psoriasis Area and Severity Index based on measuring erythema, desquamation, and thickening of the skin; (ii) performing histopathological analysis of skin tissue, e.g., to assess the presence of parakeratosis, orthokeratosis, Munro microabscesses, acanthosis, epidermal ulcers, inflammation of the dermis and subcutaneous tissue, vascular congestion of the dermis and subcutaneous tissue, follicular hyperkeratosis, and epithelial hyperplasia, and determining an overall pathology score; (iii) measuring mRNA expression and / or protein levels of proinflammatory mediators in skin homogenates, including, e.g., mRNA expression and / or protein levels of Cxcl1, IL-17f, IL-17a, IL-23a, S100A8, and Defb4; and (iv) combinations of (i)-(iii).

[0122] In some embodiments, the genetically modified rodents show an increased skin pathology score, such as an increase of at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200% or more, as compared to wild-type control rodents that have undergone the same IMQ administration. In some embodiments, the genetically modified rodents exhibit an increase in mRNA expression and / or protein levels of one or more proinflammatory mediators (such as, but not limited to, Cxcl1, IL-17f, IL-17a, IL-23a, S100A8, and Defb4), such as an increase of at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300% or more, as compared to wild-type control rodents that have undergone the same IMQ administration. In some embodiments, the genetically modified rodent at steady state exhibits increased mRNA expression and / or protein levels of one or more proinflammatory mediators (such as, but not limited to, Cxcl1, IL-17f, IL-17a, IL-23a, S100A8, and Defb4) in skin homogenate as compared to a control wild-type rodent at steady state, e.g., an increase of at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300% or more.

[0123] For IMQ-induced skin inflammation, single Il1rl2 humanized mice exhibited a phenotype similar to wild-type rodents as reflected by skin histopathology and RNA expression of proinflammatory molecules in the skin (see Figures 3D and 3E). On the other hand, after IMQ administration, DITRA-like mice (i.e., quadruple humanized mice containing humanized Il1rl2 and human IL1F6, IL1F8, and IL1F9) developed increased skin inflammation compared to WT and 1H mice (see Figures 3D and 3E).

[0124] Genetically modified rodent tissues and cells

[0125] In some embodiments, disclosed herein are cells or tissues from the separation of rodents as described herein. In some embodiments, cells are selected from dendritic cells, lymphocytes (e.g., B cells or T cells), macrophages and monocytes. In some embodiments, tissues are selected from fat, bladder, brain, breast, bone marrow, eyes, heart, intestines, kidneys, liver, lungs, lymph nodes, muscles, pancreas, plasma, serum, skin, spleen, stomach, thymus, testis, ovum and combinations thereof. In some embodiments, the cells or tissues separated include quadruple humanization features as described herein in their genome.

[0126] Compositions and methods for making quadruple humanized rodents

[0127] Additional aspects of the present disclosure relate to methods for preparing the above-described genetically modified rodents, as well as nucleic acid vectors and rodent embryonic stem cells suitable for use in preparing genetically modified rodents.

[0128] Quadruple humanized rodents, i.e., rodents comprising humanized Il1rl2 and human IL1F6, IL1F8, and IL1F9 genes, can be produced by preparing a single Il1rl2 humanized rodent strain, and a triple human rodent strain of Il1f6, Il1f8, and Il1f9, and then breeding the single humanized and triple human strains together to obtain quadruple humanized rodents. As used herein with respect to rodents, the terms "breeding" or "crossing" refer to the mating of rodents to produce offspring. Those skilled in the art will appreciate that more than one cross may be required to achieve homozygosity.

[0129] In some embodiments, disclosed herein is a targeting vector (or nucleic acid construct) comprising a human nucleotide sequence that is expected to be integrated into a rodent locus. In some embodiments, the human nucleotide sequence may be a human IL1RL2 gene nucleotide sequence encoding an extracellular domain substantially identical to the human IL1RL2 extracellular domain, for example, a nucleotide sequence comprising exons 3-8 of the human IL1RL2 gene. In some embodiments, the human nucleotide sequence may be a nucleotide sequence covering the complementary strand of the coding sequence of human IL-36α, the coding sequence of human Il-36β, and the coding sequence of human IL-36γ. The targeting vector also includes 5' and 3' rodent sequences, also referred to as homology arms, that mediate homologous recombination and integration of human nucleotide sequences in target rodent loci (e.g., Il1rl2 loci, or loci where rodent Il1f6, Il1f8, and Il1f9 genes are located), and that are located on the flanks of the human nucleotide sequence to be integrated. In some embodiments, the 5' and 3' flanking rodent sequences are nucleotide sequences that flank the corresponding rodent nucleotide sequences of the target rodent locus that will be replaced by human nucleotide sequences. For example, in embodiments where a nucleotide sequence encoding a rodent extracellular domain (e.g., exons 3-8 of a rodent Il1rl2 gene) is replaced with a sequence encoding a human extracellular domain (e.g., exons 3-8 of a human IL1RL2 gene), the 5' flanking sequence may include exons 1-2 of the rodent Il1rl2 gene, and the 3' flanking sequence may include the remaining exons downstream of exon 8 of the rodent Il1rl2 gene. In some embodiments where the rodent nucleotide sequences encoding all three IL-36 ligands are to be replaced by human nucleotide sequences, the 5' flanking sequence may include a rodent nucleotide sequence upstream of the coding sequence of the Il1f6 gene, and the 3' flanking sequence may include a rodent nucleotide sequence upstream of the coding sequence of the Il1f9 gene.

[0130] In some embodiments, the targeting vector comprises a selectable marker gene. In some embodiments, the targeting vector comprises one or more site-specific recombination sites. In some embodiments, the targeting vector comprises a selectable marker gene flanked by site-specific recombination sites such that the selectable marker gene can be deleted due to recombination between sites.

[0131] In an exemplary embodiment, bacterial homologous recombination and Bacterial artificial chromosome (BAC) clones carrying rodent genomic fragments are modified using techniques (see, e.g., US6,586,251 and Valenzuela et al. (2003) Nature Biotech. 21(6): 652-659; the document is incorporated herein by reference in its entirety). As a result, the rodent genomic sequence is deleted from the original BAC clone, and the human nucleotide sequence is inserted, thereby generating a modified BAC clone carrying a human nucleotide sequence flanked by 5' and 3' rodent homology arms. In some embodiments, the human nucleotide sequence can be a cDNA sequence or human genomic DNA encoding (i) all or part of human IL1RL2 (e.g., the extracellular domain of human IL1RL2 protein) or (ii) all three of human IL1F6, IL1F8 and IL1F9. Once the modified BAC clone is linearized, it can be introduced into rodent embryonic stem (ES) cells.

[0132] In some embodiments, the invention provides the use of a targeting vector as described herein for preparing modified rodent embryonic stem (ES) cells. For example, the targeting vector can be introduced into rodent ES cells by, for example, electroporation. Mouse ES cells and rat ES cells have been described in the art. See, for example, US 7,576,259, US 7,659,442, US 7,294,754, and US 2008-0078000 A1 (all of which are incorporated herein by reference in their entirety), which describe mouse ES cells and methods for preparing genetically modified mice. Method; US 2014 / 0235933A1 (Regeneron Pharmaceuticals, Inc.), US 2014 / 0310828 A1 (Regeneron Pharmaceuticals, Inc.), Tong et al. (2010) Nature 467:211-215, and Tong et al. (2011) Nat Protoc.6(6):doi:10.1038 / nprot.2011.338 (all of which are incorporated herein by reference in their entirety), which describe rat ES cells and methods for preparing genetically modified rats, which can be used to prepare modified rodent embryos, which can in turn be used to prepare rodents.

[0133] In some embodiments, ES cells can be selected that have human nucleotide sequences integrated into the genome. In some embodiments, ES cells are selected based on the loss of rodent alleles and / or the gain of human alleles. In some embodiments, the selected ES cells are then used as donor ES cells to be used in the transplantation of rodents by using Method (see, for example, US 7,576,259, US 7,659,442, US 7,294,754 and US 2008-0078000 A1, all of which are incorporated by reference in their entirety) or in US 2014 / 0235933 A1 and US 2014 / 0310828 A1 (all of which are incorporated by reference in their entirety) The method described in is injected into the pre-morula embryo (e.g., 8-cell stage embryo). In some embodiments, the embryo comprising the donor ES cell is incubated until the blastocyst stage and then implanted into a surrogate mother to produce a F0 rodent derived entirely from the donor ES cell. The rodent pups carrying human nucleotide sequences can be identified by using the loss of rodent alleles and / or the acquisition of human allele determinations, by genotyping the DNA separated from the tail clip.

[0134] In some embodiments, rodents heterozygous for a humanized gene can be crossed to produce homozygous rodents.

[0135] A quadruple humanized rodent approach

[0136] The rodents disclosed herein provide a useful in vivo system and source of biological material for identifying and testing compounds useful for treating diseases or disorders associated with dysregulated IL-36 signaling.

[0137] "Disease associated with dysregulated IL-36 signaling" means a disease in which abnormal IL-36 signaling is manifested, which may directly or indirectly cause the disease or exacerbate the symptoms of the disease. Non-limiting examples of diseases associated with dysregulated IL-36 signaling include generalized pustular psoriasis (GPP or DITRA) (Marrakchi S. et al., NEngl J. Med. 365:620-628 (2011) and Onoufriadis A. et al., Am J. Hum Genet 89:432-437 (2011), which are incorporated herein by reference in their entireties), palmoplantar pustular psoriasis (PPPP) (Bissonnette R. et al., PLoS One 11:e0155215 (2016), which are incorporated herein by reference in their entireties), inflammatory bowel disease (IBD) (Medina-Contreras et al., J Immunol 196:34-38 (2016); Nishida A. et al., Inflamm Bowel Dis 22:303-314 (2016) and Russell SE et al., Mucosal Immunol. 9: 1193-1204 (2016), which is incorporated herein by reference in its entirety), rheumatoid arthritis and psoriatic arthritis (Frey S. et al., Ann Rheum Dis 72: 1569-1574 (2013), which is incorporated herein by reference in its entirety), asthma, chronic obstructive pulmonary disease (Chen H. et al., J. Proteomics 75: 2835-2843 (2012), which is incorporated herein by reference in its entirety), chronic kidney disease (Shaik Y. et al., Int J Immunopathol Pharmacol 26: 27-36 (2013), which is incorporated herein by reference in its entirety), and ichthyosis (Paller AS et al., J Allergy Clin Immunol 139: 152-165 (2017), which is incorporated herein by reference in its entirety).

[0138] In some embodiments, compounds that can be evaluated using the disclosed rodents include candidate inhibitors of IL-36 signaling, such as, but not limited to, small molecule inhibitors, nucleic acid-based inhibitors (e.g., siRNA, ribozymes, antisense constructs, etc.), antigen binding proteins (e.g., antibodies or antigen-binding fragments thereof), or blocking peptides / peptide inhibitors.

[0139] In some embodiments, the candidate inhibitor is an antibody or an antigen-binding fragment thereof. Both monoclonal and polyclonal antibodies are suitable for testing in rodents disclosed herein. In some embodiments, the antibody specifically binds to human IL-36R protein. In some embodiments, the antibody specifically binds to the IL1RL2 subunit of human IL-36R protein.

[0140] Candidate compounds can be evaluated by inducing inflammation (e.g., IMQ-induced skin inflammation or DSS-induced intestinal inflammation) in rodents disclosed herein and determining whether the candidate compound can treat or inhibit the induced inflammation. The terms "treat" or "inhibit" include improving the severity of the induced inflammation and symptoms or a combination thereof, slowing the progression of the induced inflammation and symptoms or a combination thereof, eliminating the induced inflammation and symptoms or a combination thereof, delaying or preventing the onset of the induced inflammation and symptoms or a combination thereof.

[0141] In some embodiments, before, simultaneously or after the administration of the medicament inducing inflammation, the candidate compound is administered to the rodent. The candidate compound can be administered via various desired routes of administration (including parenteral and non-parenteral routes of administration). Parenteral routes include, for example, intravenous, intraarterial, intraportal, intramuscular, subcutaneous, intraperitoneal, intraspinal, intrathecal, intraventricular, intracranial, intrapleural or other injection routes. Non-parenteral routes include, for example, oral, nasal, transdermal, pulmonary, rectal, cheek, vagina, eye. Administration can also be carried out by continuous infusion, topical application, sustained release from implants (gels, films, etc.) and / or intravenous injection.

[0142] In some embodiments, suitable control rodents may include, for example, humanized rodents that have not undergone induced inflammation; humanized rodents that have undergone induced inflammation without the use of any compound or with the use of a control compound that is not expected to have any therapeutic efficacy (e.g., an isotype control antibody); and humanized rodents that have undergone induced inflammation and have been treated with a compound known to have a therapeutic effect.

[0143] To evaluate the efficacy of a candidate compound on skin inflammation, the compound can be administered to a rodent before, during, or after IMQ treatment. In a specific embodiment, the candidate compound is administered subcutaneously at or near the skin area where IMQ is administered. If the compound inhibits skin inflammation compared to a control rodent to which the compound is not administered, the compound is considered effective. For example, if the total pathology score is reduced by 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more (e.g., reduced by 20%-90%, 20%-80%, 20%-70%, 20%-60%, 20%-50%, 20%-40%, 30%-90%, 30%-80%, 30%-70%, 30%-60%, 30%-50% or 30%-40%); or if one or more A compound is considered effective if the concentration of proinflammatory mediators is reduced by 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more (e.g., a reduction of 20%-90%, 20%-80%, 20%-70%, 20%-60%, 20%-50%, 20%-40%, 30%-90%, 30%-80%, 30%-70%, 30%-60%, 30%-50%, or 30%-40%).

[0144] In order to evaluate the efficacy of candidate compounds on colon inflammation, compounds can be administered to rodents before, during or after DSS or oxazolone treatment. In some embodiments, candidate compounds are administered intraperitoneally several days (e.g., 5, 6, 7, 8 or 9 days) after the start of DSS treatment. In some embodiments, candidate compounds are administered intraperitoneally multiple times during oxazolone treatment; for example, 2-3 days after topical administration of oxazolone, when oxazolone is administered intrarectally at one or more times (e.g., when oxazolone is administered intrarectally for the first time, and when oxazolone is administered intrarectally for the third time). If the compound suppresses colitis compared to a control DITRA rodent not administered with the compound, the compound is considered to be effective.For example, if the total pathology score is reduced by 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more (e.g., by 20%-90%, 20%-80%, 20%-70%, 20%-60%, 20%-50%, 20%-40%, 30%-90%, 30%-80%, 30%-70%, 30%-60%, 30%-50%, or 30%-40%); if the concentration of one or more pro-inflammatory mediators is reduced by 10%, 15%, 20%, 30%, 40%, 50%, 60% if fecal Lcn2 is reduced by 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more (e.g., a decrease of 20%-90%, 20%-80%, 20%-70%, 20%-60%, 20%-50%, 20%-40%, 30%-90%, 30%-80%, 30%-70%, 30%-60%, 30%-50% or 30%-40%); if fecal Lcn2 is reduced by 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more (e.g., a decrease of 20%-90%, 20%-80%, 20%-70%, if the MPO activity in the colon homogenate is reduced by 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more (e.g., by 20%-90%, 20%-80%, 20%-70%, 20%-60%, 20%-50%, 20%-40%, 30%-90%, 30%-80%, 30%-70%, 30%-60%, 30%-50% or 30%-40%); if the MPO activity in the colon homogenate is reduced by 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more (e.g., by 20%-90%, 20%-80%, 20%-70%, 20%-60%, 20%-50%, 20%-40%, 30%-90%, 30%-80%, 30%-40%) The compound is considered effective if the colon length increases by 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more (e.g., decreases by 20%-90%, 20%-80%, 20%-70%, 20%-60%, 20%-50%, 20%-40%, 30%-90%, 30%-80%, 30%-70%, 30%-60%, 30%-50%, or 30%-40%); or a combination thereof.

[0145] The present description is further illustrated by the following examples, which should not be construed as limiting in any way. The contents of all cited documents (including references, issued patents, and published patent applications cited throughout this application) are hereby expressly incorporated by reference in their entirety.

[0146] Example 1. Generation of a quadruple humanized mouse strain

[0147] use Two genetically engineered mouse strains were created using the technology (Poueymirou et al., Nat Biotechnol. 2007 Jan; 25(1):91-9; Valenzuela et al., Nat Biotechnol 2003 Jun; 21:652-59; the documents are incorporated herein by reference in their entirety): the Il1rl2 single humanized mouse strain and the Il1f6, Il1f8, Il1f9 triple humanized mouse strain. A "quadruple humanized" strain was generated by mating the single humanized and triple humanized strains together.

[0148] Generation of a single humanized Il1rl2 mouse line

[0149] A "single humanized" strain was generated in which the portion of the mouse Il1rl2 gene encoding the extracellular domain of mouse Il1rl2 (interleukin 1 receptor-like 2 protein) was replaced by a fragment of the human IL1RL2 gene encoding the corresponding extracellular domain of human IL1RL2 (Il1rl2 / IL1RL2 exons 3-8 with an inserted intron and parts of the flanking introns, respectively) ( Figure 1A The resulting humanized gene encodes a chimeric receptor that maintains the mouse intracellular signaling specificity while making the extracellular domain human and capable of binding the human ligands IL1F6, IL1F8, and IL1F9, also known as IL36A, B, and G, respectively. Homozygous humanized Il1rl2 mice are referred to as Il1rl2 hu / hu .

[0150] More specifically, a mouse bacterial artificial chromosome (BAC) clone RP23-235L22 containing the mouse Il1rl2 gene was used and modified as follows to provide a targeting vector. A DNA fragment was generated to include a 5' mouse homologous nucleotide sequence, approximately 32,389 bp of human IL1RL2 genomic DNA (3'346 bp containing intron 2, exons 3-8 with all inserted introns, and 5'1101 bp of intron 8), approximately 4,996 bp of a self-deleting neomycin cassette, and a 3' mouse homologous sequence ( Figure 1B ). This DNA fragment was used to modify the BAC clone RP23-235L22 by homologous recombination in bacterial cells. As a result, the mouse Il1rl2 genomic fragment encoding the extracellular domain in the BAC clone (about 25,324 bp, including 3'155 bp of mouse intron 2, exons 3 to 8 with all inserted introns, and 5'642 bp of mouse intron 8) was replaced by human IL1RL2 genomic DNA, followed by a self-deleted neomycin cassette ( Figure 1B). The resulting modified BAC clone includes, from 5' to 3', (i) a 5' mouse homology arm containing approximately 112.2 kb of mouse genomic DNA, mouse Il1rl2 exons 1-2, and the 5' portion of intron 2; (ii) a human IL1RL2 genomic fragment including the 3' portion of intron 2, exons 3 to 8, and the 5' portion of intron 8; (iii) an approximately 4,996 bp self-deleting neomycin cassette, followed by (iv) a 31.3 kb 3' mouse homology arm containing the remaining mouse Il1rl2 exons downstream of exon 8, all inserted introns, and the 3'UTR. See Figure 1B .exist Figure 1B The connection sequence is also listed at the bottom of the .

[0151] As described above, a modified BAC clone containing the humanized Il1rl2 gene was used as a targeting vector to electroporate mouse F1H4 embryonic stem cells (50% C57BL / 6NTac / 50% 129S6 / SvEvTac) to create modified ES cells containing the humanized Il1rl2 gene. Forward-targeted ES cells containing the humanized Il1rl2 gene were identified by detecting the presence of human IL1RL2 sequences and confirming the loss and / or retention of mouse Il1rl2 sequences (Valenzuela et al., supra). The primers and probes used to confirm humanization as described above are listed in Table 5. Once a correctly targeted ES cell clone is selected, the neomycin selection cassette can be excised. Figure 1C The humanized Il1rl2 locus after deletion of the cassette is depicted in FIG. , and the junction sequence is also shown in FIG. Figure 1C middle.

[0152] Table 5

[0153]

[0154]

[0155] Selected ES cell clones were microinjected into 8-cell embryos from Charles River Laboratories SwissWebster albino mice, generating F0 embryos that were 100% derived from targeted cells. (Poueymirou et al. 2007, supra). Mice carrying the humanized locus were reconfirmed and identified by genotyping of DNA isolated from tail snips using allele assays that detect the presence of human gene sequences (Valenzuela et al., supra). Animals homozygous for the humanized locus were prepared by crossing heterozygous animals.

[0156] exist Figure 1DAn alignment of the resulting amino acid sequences of the humanized / chimeric Il1rl2 receptor (SEQ ID NO:7), mouse Il1rl2 protein (SEQ ID NO:4), and human IL1RL2 protein (SEQ ID NO:2) is provided in .

[0157] Generation of triple humanized mouse strains

[0158] A "triple humanized" strain was generated in which the entire coding sequence of each of the mouse Il1f6, Il1f8, and Il1f9 genes was replaced with the entire coding sequence of the human IL1F6, IL1F8, and IL1F9 genes, respectively. This strategy resulted in humanized genes encoding human ligands that were able to bind to the human extracellular domain of the chimeric Il1rl2 receptor. The homozygous humanized Il1f6, Il1f8, Il1f9 mouse was referred to as the Il1f6 hu / hu ,Il1f8 hu / hu ,Il1f9 hu / hu .

[0159] More specifically, a mouse bacterial artificial chromosome (BAC) clone RP23-90G23 containing mouse Il1f8, Il1f9 and Il1f6 genes and intergenic sequences was used and modified as follows to provide a targeting vector. A DNA fragment was generated to include a 5' mouse homologous nucleotide sequence, approximately 88,868 bp of human genomic DNA (containing the promoter, untranslated region, and coding sequences for human IL1F8, IL1F9 and IL1F6), approximately 5,218 bp of a self-deleting hygromycin cassette, and a 3' mouse homologous sequence ( Figure 2B This DNA fragment was used to modify the BAC clone RP23-90G23 by homologous recombination in bacterial cells. As a result, the mouse genomic fragment of about 76,548 bp in the BAC clone was replaced by human genomic DNA, followed by a self-deletion cassette ( Figure 2A-2B ). The resulting modified BAC clone includes, from 5' to 3', (i) a 5' mouse homology arm containing about 5.7 kb of mouse genomic DNA, (ii) an approximately 88,868 bp human genomic fragment (containing a promoter, untranslated region, and coding sequences of human IL1F8, IL1F9, and IL1F6), (iii) a self-deleting hygromycin cassette of about 5,218 bp, and (iv) about 29.7 kb of 3' mouse homology sequence. See Figure 2B .exist Figure 2B The connection sequence is also listed at the bottom of the .

[0160] As described above, modified BAC clones containing human IL1F8, IL1F9, and IL1F6 gene sequences are used as targeting vectors to electroporate mouse F1H4 embryonic stem cells (50% C57BL / 6NTac / 50% 129S6 / SvEvTac) to create modified ES cells containing human IL1F8, IL1F9, and IL1F6 gene sequences. Forward-targeted ES cells containing human IL1F8, IL1F9, and IL1F6 gene sequences are identified by assays to detect the presence of human sequences and confirm the loss and / or retention of mouse sequences (Valenzuela et al., supra). The primers and probes used to confirm humanization as described above are listed in Table 6. Once a correctly targeted ES cell clone is selected, the hygromycin selection cassette can be excised. Figure 2C The humanized locus after deletion of the cassette is depicted in and the junction sequence is also shown in Figure 2C middle.

[0161] Table 6

[0162]

[0163] Selected ES cell clones were microinjected into 8-cell embryos from Charles River Laboratories SwissWebster albino mice, generating F0 embryos that were 100% derived from targeted cells. (Poueymirou et al. 2007, supra). Mice carrying the humanized locus were reconfirmed and identified by genotyping of DNA isolated from tail snips using allele assays that detect the presence of human gene sequences (Valenzuela et al., supra). Animals homozygous for the humanized locus were prepared by crossing heterozygous animals.

[0164] Generation of triple humanized mouse strains

[0165] A "quadruple humanized" line was generated by mating the single and triple humanized lines together and breeding homozygous for both loci on a 100% C57BL / 6NTac background. The homozygous quadruple humanized line is called Il1rl2 hu / hu Il1f6 hu / hu Il1f8 hu / hu Il1f9 hu / hu , or "DITRA-like" mice. The Neo and hygro cassettes were deleted in the F0 germline by self-deletion technology.

[0166] Example 2. Characterization of DITRA mice

[0167] Materials and methods

[0168] Acute and chronic IMQ-induced skin inflammation induction and antibody treatment in DITRA-like mice - To induce skin inflammation, the backs of 8-10 week old humanized DITRA-like female mice were shaved using a mouse hair trimmer (Oster, MiniMax, catalog #78049-100) and the skin was depilated with 0.5 g Veet depilatory gel three days before the application of IMQ cream. A 62.5 mg daily topical dose of commercially available IMQ cream (5%) (Aldara, GM Health Care Limited, NDC 99207-206-12, batch number QJ044A) or petrolatum (CVS Pharmacy, NDC 59779-902-88) was applied to the shaved back skin of the mice for four consecutive days for chronic disease and nine consecutive days for chronic disease induction. The 62.5 mg daily topical dose of Aldara converted to a 3.125 mg daily dose of active compound. In acute IMQ-induced skin inflammation, anti-human IL-36R antibody was administered subcutaneously into the back skin at 10 mg / kg 3 days before and 1 day after the start of IMQ administration. The control group received PBS and 10 mg / kg of hIgG4 isotype control injections. In chronic IMQ-induced skin inflammation, the same anti-human IL-36R antibody was therapeutically administered subcutaneously into the back skin at 10 mg / kg on days 4 and 8 after the start of IMQ administration. Two or three days after treatment, the back skin of the mice began to show signs of erythema, desquamation, and thickening. The severity of inflammation was measured daily using a modified version of the Clinical Psoriasis Area and Severity Index. Erythema, desquamation, and thickening were scored independently on a scale of 0–4: 0, none; 1, mild; 2, moderate; 3, significant; and 4, very significant (van der Fits et al., J Immunol 2009, 182: 5836–5845, which is incorporated herein by reference in its entirety). Skin thickness was measured using a caliper (Kaefer) on day 4 of acute IMQ-induced skin inflammation and on day 11 of chronic IMQ-induced skin inflammation.

[0169] Histopathology-6 mm diameter skin tissue from the back of the mouse was fixed in 10% formalin buffer, and 4-5 μm paraffin-embedded sections were stained with hematoxylin and eosin. The skin sections were blindly evaluated for the presence of parakeratosis, orthokeratosis, Munro microabscesses, acanthosis, epidermal ulcers, inflammation of the dermis and subcutaneous tissue, vascular congestion of the dermis and subcutaneous tissue, follicular hyperkeratosis, and epithelial hyperplasia. A 0-4 scoring scale was used: 0-within the normal range, 1-minimal degree, 2-mild, 3-moderate, and 4-severe (van der Fits et al., J Immunol 2009, 182: 5836-5845, which is incorporated herein by reference in its entirety). The total pathology score for each mouse was calculated by adding the scores of the individual histopathological features. Data analysis was performed using GraphPad Prism software.

[0170] Measurement of cytokines in skin homogenate - Full thickness skin tissue with a diameter of 6 mm was taken from the back of the mouse and placed in a 15 mL tube containing T-per buffer (Thermo Scientific, catalog number 378510, batch number RF236217) and 1x Halt protease inhibitor mixture (Thermo Scientific, catalog number 87786, batch number QG221763) and 5M EDTA solution (ThermoScientifi, catalog number 3 78429). The skin tissue was disrupted at 28000 rpm for 1 minute using a Polytron (PT10-35 GT-D, catalog number 9158158) and placed on ice. The resulting skin homogenate was centrifuged at 1500 rpm for 8 minutes at 4°C, and the supernatant was collected in a 96-well plate. Skin homogenates were subjected to Bradford protein assay using protein assay dye (BioRad, catalog number 500-0006, batch number 210008149) to quantify total protein content. Cytokine concentrations in skin homogenates were measured using the Proinflammatory Panel 1 (mouse) multiplex immunoassay kit (MesoScale Discovery, catalog number K15048D) according to the manufacturer's instructions. Briefly, 50 L / well of calibrants and samples (diluted in diluent 41) were added to a plate pre-coated with capture antibodies and incubated for 2 hours at room temperature while shaking at 700 rpm. The plate was then washed 3 times with 1xPBS containing 0.05% (w / v) Tween-20, followed by addition of 25 μL of detection antibody solution diluted in diluent 45. After incubation for 2 hours at room temperature while shaking, the plate was washed 3 times and 150 μL of 2x reading buffer was added to each well. Immediately in MSD Electrochemiluminescence was read on the instrument. Data were analyzed using GraphPad Prism software. Cytokine levels were normalized to total protein content.

[0171] Induction of DSS-induced chronic colitis model and antibody treatment in DITRA-like mice - To induce chronic DSS-mediated colitis, female DITRA-like mice aged 12-20 weeks with an average body weight of more than 23 g were given 1.5-3% DSS (Sigma-Aldrich catalog number 87786, batch number PJ203966B) in drinking water for 7 days, followed by distilled water for 11-13 days. A second cycle of DSS treatment (lasting 4 days) was performed, followed by water (5-6 days) until days 27-30. During the study, the control group received distilled water. Starting from day 7, anti-human IL-36R and mIL-12p40 (Bioxcell catalog number BE0051, clone C17.8) antibodies were administered intraperitoneally at 10 mg / kg every two weeks. The control group received 10 mg / kg of PBS and the corresponding hIgG4 and rat IgG2a (Bioxcell catalog number BE0089, clone 2A3) isotype control injections. Mice were weighed daily and monitored for clinical signs of colitis (e.g., stool consistency and fecal blood). On days 27-30, mice were euthanized and colon length was measured. To evaluate DSS colitis, the following features were scored: inflammation (severity and extent), epithelial changes (erosions / ulcers), crypt changes (crypt loss, cryptitis / crypt abscesses, regeneration / hyperplasia, goblet cell loss), submucosal edema, and the percentage of tissue area with pathology relative to the total tissue area on the slide. A 0-4 scoring scale was used: 0-0-within the normal range, 1-minimal extent, 2-mild, 3-moderate, and 4-severe. The total pathology score for each mouse was calculated by adding the scores for each histopathological feature.

[0172] Measurement of Lcn-2 in fecal samples-In order to monitor intestinal inflammation throughout the study, feces from individual DITRA-like mice were collected into 2mL deep well plates every week and stored at -80°C. After the study was completed, the feces collected on different days were homogenized. In brief, fecal samples were reconstructed with 1mL PBS containing 0.1% Tween-20, 1x Halt protease inhibitor mixture (Thermo Scientific, catalog number 87786, batch number QG221763) and 5M EDTA solution (Thermo Scientifi, catalog number 3 78429). After adding 2 3mm tungsten carbide beads (Qiagen, catalog number 69997) to the wells, the plate was placed overnight at 4°C on a shaker at maximum speed. Homogeneous fecal suspension was centrifuged at 1200rpm for 10 minutes at 4°C, and the supernatant was collected in a 96-well plate. The levels of lipocalin 2 (Lcn2) in feces were measured using the Mouse Duoset Lipocalin 2 / NGAL ELISA Kit (R&D Systems, Cat. No. DY1857, Lot No. P116359) according to the manufacturer's instructions. Data analysis was performed using GraphPad Prism software.

[0173] Measurement of myeloperoxidase (MPO) activity in colon homogenate - Several pieces of distal colon were placed in 2 mL microcentrifuge tubes containing 2 3 mm tungsten carbide beads (Qiagen, catalog number 69997) and T-per buffer (Thermo Scientific, catalog number 378510, lot number RF236217), 1x Halt protease inhibitor cocktail (Thermo Scientific, catalog number 87786, lot number QG221763) and 5M EDTA solution (Thermo Scientifi, catalog number 78429). The samples were lysed using a Qiagen Tissue Lyser II at 27.5 s. -1 The colon tissue was destroyed at a frequency of 10 minutes. The tube was centrifuged at 1500rpm for 8 minutes at 4°C, and the supernatant was collected in a 96-well plate. Bradford protein assay was performed on colon homogenate using protein assay dye (BioRad, catalog number 500-0006, batch number 210008149) to quantify total protein content. Myeloperoxidase (MPO) activity in colon homogenate was measured using mouse MPO ELISA kit (Hycult Biotech, catalog number HK210-02, batch number 21022KO617-Y) according to the manufacturer's instructions. Data analysis was performed using GraphPad Prism software. MPO levels were normalized relative to total protein content.

[0174] Measurement of cytokines in colon homogenates - Cytokine concentrations in skin homogenates were measured using the Proinflammatory Panel 1 (mouse) multiplex immunoassay kit (MesoScale Discovery, catalog number K15048D) according to the manufacturer's instructions. Briefly, 50 L / well of calibrators and samples (diluted in diluent 41) were added to a plate pre-coated with capture antibody and incubated for 2 hours at room temperature while shaking at 700 rpm. The plate was then washed 3 times with 1xPBS containing 0.05% (w / v) Tween-20, followed by the addition of 25 μL of detection antibody solution diluted in diluent 45. After incubation for 2 hours at room temperature while shaking, the plate was washed 3 times and 150 μL of 2x reading buffer was added to each well. Immediately incubate at MSD Electrochemiluminescence was read on the instrument. Data were analyzed using GraphPad Prism software. Cytokine levels were normalized to total protein content.

[0175] Statistical analysis - Statistical significance within groups was determined by one-way ANOVA with Tukey's multiple comparison post-test (*p<0.05, **p<0.005, ***p<0.0005, ****p<0.0001) (for * - statistical significance with PBS-treated group and # - statistical significance with corresponding isotype-treated group).

[0176] result

[0177] Humanized DITRA-like mice exhibit enhanced skin inflammation in the IMQ-induced psoriasis model

[0178] To interrogate the role of dysregulated IL-36R signaling similar to that seen in GPP or DITRA (for "deficiency of interleukin 36 receptor antagonist") patients, mice with humanized IL-36R (e.g., Il1rl2) and human IL-36α, β, γ ligands but not an IL-36Ra antagonist were generated as described in Example 1. The resulting hIL-36R / hIL-36α, β, γ mice are referred to as DITRA-like mice because the 20-fold reduction in affinity of mouse IL-36Ra for human IL-36R results in enhanced IL-36R signaling similar to that seen in DITRA patients (for a more detailed discussion of DITRA patients, see, e.g., Marrakchi et al., N Engl J Med 2011, 365:620-628, which is incorporated herein by reference in its entirety).

[0179] In the unchallenged state, DITRA-like mice did not develop any spontaneous disease. In contrast, in the preclinical model of psoriatic dermatitis induced by IMQ, which is very similar to human psoriasis lesions in terms of phenotype and histological characteristics (for a more detailed discussion of the characteristics of psoriatic dermatitis, see van der Fits et al., J Immunol 2009, 182: 5836–5845; Swindell et al., PLoS One 2011, 6: e18266, all of which are incorporated herein by reference as a whole), DITRA-like mice showed enhanced skin inflammation (Fig. 3A-Fig. 3B) compared with their WT littermates. In brief, IMQ was applied to the shaved back skin of DITRA-like and WT mice every day for four consecutive days. On the 5th day, the skin was harvested for subsequent histopathological evaluation, protein and RNA separation. Compared with WT littermates, the DITRA-like mice treated with IMQ developed more serious psoriasis lesions, such as peeling, erythema and skin thickening (Fig. 3A). Consistent with clinical features, histopathological evaluation of skin revealed more enhanced acanthosis, parakeratosis and disturbed keratinocyte differentiation (Fig. 3B) in DITRA-like mice. The DITRA-like mice treated with IMQ also showed significantly increased Munro abscesses or pustule numbers similar to those of GPP patients. In addition, IMQ used proinflammatory molecules (proinflammatory molecules include, for example, IL-17a, IL-17f, IL-23a, S100A8, Defb4) levels that were dysregulated in psoriasis in DITRA-like mice skin compared with WT littermates (Fig. 3C). Furthermore, the expression of IL-36α, IL-36β, and IL-36γ mRNA was equally increased in IMQ-treated DITRA-like mice and IMQ-treated wild-type mice compared with petroleum jelly-treated DITRA-like mice and petroleum jelly-treated wild-type mice, respectively; however, 2-fold higher concentrations of IL-36α and IL-36β cytokine protein levels were detected in the inflamed skin of DITRA-like mice compared with the levels in the inflamed skin of wild-type mice.

[0180] Humanized DITRA-like mice show defects in mucosal healing in a DSS-induced chronic colitis model - In recent years, multiple studies on patient subgroups have demonstrated the dysregulated expression of the IL-36 axis in IBD and its possible contribution to intestinal inflammation. It was shown that the expression of IL-36α and IL-36β was elevated in the inflamed mucosa of patients with ulcerative colitis (see Medina-Contreras et al., J Immunol 2016, 196: 34-38; Nishida et al., Inflamm Bowel Dis 2016, 22: 303-314, and Russell, Mucosal Immunol 2016, 9: 1193-1204, all of which are incorporated herein by reference in their entirety). In preclinical models, IL-36R deficiency prevents DSS- and oxazolone-induced colitis (Medina-Contreras et al., J Immunol 2016, 196: 34-38; Harusato et al., Mucosal Immunol 2017, 10: 1455-1467, all of which are incorporated herein by reference in their entirety). In the steady state, DITRA-like mice did not develop spontaneous ileitis / colitis, but showed significantly shortened colons in both young and old ages (3 months and 10 months old, respectively) (Figures 4A and 4B). In addition, in the steady state and compared with wild-type mice, DITRA-like mice showed elevated levels of IL-36 cytokines in the colon, as well as increased levels of IL-17F and IL-17A and a trend toward decreased levels of IL-21.

[0181] To evaluate the role of the IL-36 axis in intestinal inflammation, a chemically induced intestinal injury model was used by oral administration of DSS, which damages the colonic epithelium (Okayasu et al., Gastroenterology 1990, 98: 694-702, incorporated herein by reference in its entirety) and induces an effective inflammatory response (Rakoff-Nahoum et al., Cell 2004, 118: 229-241, incorporated herein by reference in its entirety), thereby exhibiting the main features of IBD (particularly ulcerative colitis). DITRA-like mice and their co-housed WT littermates were subjected to a DSS-induced chronic colitis regimen by administering 2.5% DSS for 7 days, followed by 11 days of water (first cycle) and 1.5% DSS for 4 days, followed by 5 days of water (second cycle). In the acute phase of the disease, DITRA-like mice developed intestinal inflammation similar to co-housed WT littermates (Figure 5A). Interestingly, during the repair phase of the disease, DITRA-like mice showed an inability to recover from DSS-induced mucosal damage, as reflected in sustained weight loss (Figure 5A), a significant reduction in colon length (Figure 5B), more severe pathological scores (associated with more severe ulcers, extensive epithelial erosions and neutrophil infiltration) (Figure 5C), a significant increase in fecal lipocalin 2 (Lcn2) levels and myeloperoxidase activity (Figure 5D), upregulation of proinflammatory cytokines (such as KC-GRO, TNF-α and IL-6), and a decrease in IL-22 levels in the colon (Figure 5E). In addition, DITRA-like mice exhibited a mortality rate of approximately 62.5% (data not shown). In contrast, WT mice did recover during the repair phase of colitis and had a mortality rate of 25%. Intestinal epithelial integrity was also studied by oral enema of DITRA-like mice and wild-type mice with fluorescein isothiocyanate (FITC)-dextran. On day 14 of DSS treatment, a significant increase in FITC-dextran levels was observed in the serum of DITRA-like mice, indicating that the IL-36 pathway is involved in regulating intestinal permeability during intestinal injury. Thus, enhanced IL-36 signaling in DITRA-like mice resulted in exacerbated intestinal inflammation and defects in mucosal repair, suggesting a role for IL-36 in regulating mechanisms involved in intestinal tissue remodeling.

[0182] Anti-human IL-36R monoclonal antibodies inhibit acute skin inflammation in DITRA-like mice when administered prophylactically - To examine the role of IL-36R in skin inflammation, anti-human IL-36R monoclonal antibodies were tested in an IMQ-induced psoriatic dermatitis model. IMQ was applied to the shaved back skin of DITRA-like mice every day for four consecutive days. Anti-human IL-36R monoclonal antibodies were administered at 10 mg / kg 3 days before (-3d) and one day after (d1) the start of IMQ administration. The control group received 10 mg / kg of PBS and hIgG4 isotype control injections. On day 5, the skin was harvested for subsequent histopathological evaluation and protein separation. Compared with the PBS and isotype control treatment groups, anti-human IL-36R monoclonal antibodies significantly reduced the total pathological scores induced by IMQ, including parakeratosis and Munro microabscesses (Figure 6A). Human IL-36R blockade also resulted in a 66-93% reduction in KC-GRO, IL-6, and TNFα (Figure 6B). Importantly, anti-human IL-36R antibody treatment significantly reduced the levels of proinflammatory cytokines that are dysregulated in psoriasis (e.g., IL-12p40, IL-17f, and IL-17a) (Figure 6B), indicating that the interactions between these cytokine pathways are tightly regulated. The observed efficacy of anti-human IL-36R monoclonal antibody in eliminating acute IMQ-induced skin inflammation was dose-dependent, with the 10 mg / kg dose resulting in greater inhibition of skin inflammation compared to the 1 mg / kg dose, as determined by pathological scores, skin thickness, and proinflammatory cytokine levels in skin homogenates (data not shown).

[0183] Anti-human IL-36R monoclonal antibodies inhibit chronic skin inflammation at therapeutic doses - To further examine the therapeutic efficacy of human IL-36R antagonism in vivo, the same anti-human IL-36R monoclonal antibodies were tested in a chronic IMQ-induced skin inflammation model. Over a two-week duration, IMQ was applied to the shaved back skin of DITRA-like mice for nine consecutive days in two rounds separated by two days without treatment. Anti-human IL-36R monoclonal antibodies were administered subcutaneously on days 5 and 9 after IMQ administration began at a dose of 10 mg / kg. The control group received 10 mg / kg of PBS and hIgG4 isotype control injections. On day 12, skin thickness was measured and tissue was harvested for subsequent histopathological evaluation and protein separation. Similar to acute IMQ-induced inflammation, prolonged IMQ administration resulted in upregulation of proinflammatory mediators in the skin of DITRA-like mice (data not shown). Anti-human IL-36R monoclonal antibodies showed significant efficacy in reducing IMQ-induced skin thickness and pathological lesion scores in DITRA-like mice (Figures 7A and 7B, respectively). In addition, administration of anti-human IL-36R monoclonal antibodies resulted in significant inhibition of IMQ-induced pro-inflammatory cytokine production in the skin of DITRA-like mice (Figure 7C).

[0184] Taken together, these data demonstrate the preventive and therapeutic efficacy of anti-human IL-36R antibodies in ameliorating acute and chronic IMQ-induced skin inflammation in vivo.

[0185] Anti-human IL-36R monoclonal antibody improves DSS-induced chronic colitis in DITRA-like mice at therapeutic doses-Having demonstrated defects in mucosal healing due to enhanced IL-36 signaling in DITRA-like mice, it was further investigated whether IL-36 blockade could rescue the observed phenotype. DITRA-like mice were subjected to chronic DSS-induced colitis by administering 3% DSS for 7 days, followed by 13 days of water for two cycles. Starting from day 7, the same anti-human IL-36R monoclonal antibody and anti-mIL-12p40 monoclonal antibody (an approved mouse replacement for Ustekinumab for the treatment of Crohn's disease) were administered at 10 mg / kg every two weeks. The control group received PBS and 10 mg / kg of the corresponding hIgG4 and rat IgG2a isotype control injections. Compared with PBS and isotype control treatments in DITRA-like mice, treatment with anti-human IL-36R monoclonal antibodies rescued DITRA-like mice from the inability to recover from DSS-induced mucosal damage and reduced the severity of the disease (Figure 8A). To monitor intestinal inflammation at different stages of the disease, feces of individual mice were collected weekly to measure fecal lipocalin 2 (Lcn2) protein, a non-invasive biomarker of inflammation in intestinal injury (procedures are described, e.g., in Thorsvik et al., J Gastroenterol Hepatol 2017, 32: 128-135, which is incorporated herein by reference in its entirety). As shown in FIG8B , groups treated with PBS, hIgG4, and rat IgG2a showed significant upregulation of fecal Lcn2 levels at days 17, 23, and 30 compared to water alone. Administration of anti-human IL-36R monoclonal antibodies surprisingly resulted in significant reductions in Lcn2 levels compared to PBS and isotype treated groups. Sustained reductions in fecal Lcn2 levels were observed in anti-human IL-36 antibody treated groups at days 17, 23, and 30 ( FIG8B ).

[0186] Further results included that hIL-36R blockade with anti-human IL-36R monoclonal antibody resulted in increased colon length (Fig. 8C) and decreased myeloperoxidase (MPO) activity (Fig. 8D) and 61-95% reduction in proinflammatory cytokines (Fig. 8E) in the colons of DSS-treated DITRA-like mice. Anti-human IL-36R monoclonal antibody showed comparable efficacy to IL-12p40 blockade in improving DSS-induced chronic colitis, as reflected by similar reductions in fecal Lcn2, MPO activity, and levels of proinflammatory cytokines in the colons of DITRA-like mice (Fig. 8A-8E).

[0187] IL-36R Blockade in Oxazolone-Induced Colitis - The efficacy of IL-36R blockade was tested in oxazolone-induced colitis, another preclinical model of IBD that is histologically similar to human ulcerative colitis (Heller et al., Immunity 17, 629-638 (2002), which is incorporated by reference in its entirety). Prophylactic administration of anti-human IL-36R antibodies in DITRA-like mice significantly reduced the severity of oxazolone-induced disease compared to PBS and isotype control treatment groups, as reflected by mean weight loss and mean colon length (Figures 9A-9B). In addition, IL-36R antagonism resulted in a significant reduction in the levels of IL-4 and TNF-α in the colon of DITRA-like mice treated with oxazolone (Figure 9C).

[0188] Various publications, including patents, patent applications, published patent applications, accession numbers, technical articles, and scholarly articles, are cited throughout the specification. Each of these cited publications is incorporated herein by reference in its entirety for all purposes.

Claims

1. A genetically modified mouse genome comprising: (1) a humanized Il1rl2 gene at the endogenous mouse Il1rl2 locus, wherein the humanized Il1rl2 gene encodes a humanized Il1rl2 protein comprising the extracellular domain of the human IL1RL2 protein and the transmembrane-cytoplasmic sequence of the endogenous mouse Il1rl2 protein; (2) human IL1F6 gene at the endogenous mouse Il1f6 locus; (3) human IL1F8 gene at the endogenous mouse Il1f8 locus; and (4) Human IL1F9 gene at the endogenous mouse Il1f9 locus.

2. The genetically modified mouse genome of claim 1, wherein the humanized Il1rl2 protein comprises a signal peptide of the endogenous mouse Il1rl2 protein.

3. The genetically modified mouse genome according to claim 1, wherein the human IL1RL2 protein comprises the amino acid sequence shown in SEQ ID NO:

2.

4. The genetically modified mouse genome of claim 1, wherein the extracellular domain of the humanized Il1rl2 protein comprises amino acids 22-337 of SEQ ID NO:

7.

5. The genetically modified mouse genome of claim 1, wherein the humanized Il1rl2 gene is operably linked to the endogenous mouse Il1rl2 promoter at the endogenous mouse Il1rl2 locus.

6. The genetically modified mouse genome of claim 1, wherein the humanized Il1rl2 gene is generated by replacing a genomic fragment of the endogenous mouse Il1rl2 gene at the endogenous mouse Il1rl2 locus with a nucleotide sequence of a human IL1RL2 gene. 7 . The genetically modified mouse genome according to claim 6 , wherein the nucleotide sequence of the human IL1RL2 gene is a genomic fragment of the human IL1RL2 gene encoding the extracellular domain of the human IL1RL2 protein.

8. The genetically modified mouse genome of claim 7, wherein the genomic fragment of the human IL1RL2 gene comprises exons 3-8 of the human IL1RL2 gene.

9. The genetically modified mouse genome of claim 6, wherein the genomic sequence of the endogenous mouse Il1rl2 gene remaining after the replacement comprises an exon downstream of exon 8 of the endogenous mouse Il1rl2 gene.

10. The genetically modified mouse genome of claim 6, wherein the genomic sequence of the endogenous mouse Il1rl2 gene remaining after the replacement comprises exons 1-2 of the endogenous mouse Il1rl2 gene.

11. The genetically modified mouse genome of claim 1, wherein the human IL1F6 gene replaces the endogenous mouse Il1f6 gene at the endogenous mouse Il1f6 locus.

12. The genetically modified mouse genome of claim 1, wherein the human IL1F8 gene replaces the endogenous mouse Il1f8 gene at the endogenous mouse Il1f8 locus.

13. The genetically modified mouse genome of claim 1, wherein the human IL1F9 gene replaces the endogenous mouse Il1f9 gene at the endogenous mouse Il1f9 locus.

14. The genetically modified mouse genome of claim 1, wherein the mouse genome is homozygous for each of the humanized Il1rl2 gene, human IL1F6 gene, human IL1F8 gene, and human IL1F9 gene.

15. A method for preparing a genetically modified mouse, the method comprising Modifying the mouse genome to contain (1) a humanized Il1rl2 gene at the endogenous mouse Il1rl2 locus, wherein the humanized Il1rl2 gene encodes a humanized Il1rl2 protein comprising the extracellular domain of the human IL1RL2 protein and the transmembrane-cytoplasmic sequence of the endogenous mouse Il1rl2 protein; (2) human IL1F6 gene at the endogenous mouse Il1f6 locus; (3) human IL1F8 gene at the endogenous mouse Il1f8 locus; and (4) human IL1F9 gene at the endogenous mouse Il1f9 locus; and A mouse comprising the modified genome is prepared.

16. The method of claim 15, wherein the mouse genome is modified by the following process: the process comprising (i) preparing a first mouse comprising the humanized Il1rl2 gene at the endogenous mouse Il1rl2 locus; (ii) preparing a second mouse comprising the human IL1F6 gene at the endogenous mouse Il1f6 locus, the human IL1F8 gene at the endogenous mouse Il1f8 locus, and the human IL1F9 gene at the endogenous mouse Il1f9 locus; and (iii) hybridizing the first mouse with the second mouse to obtain a modified mouse genome.

17. The method of claim 16, wherein the first mouse comprising the humanized Il1rl2 gene at the endogenous mouse Il1rl2 locus is prepared by: Provide mouse embryonic stem (ES) cells, Inserting the nucleotide sequence of the human IL1RL2 gene into the endogenous mouse Il1rl2 locus of the mouse ES cell to form the humanized Il1rl2 gene at the endogenous mouse Il1rl2 locus, thereby obtaining a mouse ES cell comprising the humanized Il1rl2 gene, and The first mouse is prepared using the mouse ES cells containing the humanized Il1rl2 gene.

18. The method of claim 17, wherein the nucleotide sequence of the human IL1RL2 gene replaces the genomic fragment of the mouse Il1rl2 gene at the endogenous mouse Il1rl2 locus.

19. The method according to claim 18, wherein the nucleotide sequence of the human IL1RL2 gene is a genomic fragment of the human IL1RL2 gene encoding the extracellular domain of the human IL1RL2 protein.

20. The method of claim 19, wherein the genomic fragment of the human IL1RL2 gene comprises exons 3-8 of the human IL1RL2 gene.

21. The method of claim 18, wherein the genomic sequence of the endogenous mouse Il1rl2 gene remaining after the replacement comprises an exon downstream of exon 8 of the endogenous mouse Il1rl2 gene.

22. The method of claim 18, wherein the genomic sequence of the endogenous mouse Il1rl2 gene remaining after the replacement comprises exons 1-2 of the endogenous mouse Il1rl2 gene.

23. The method of claim 17, wherein the humanized Il1rl2 gene comprises exons 1-2 of the endogenous mouse Il1rl2 gene, exons 3-8 of the human IL1RL2 gene, and the remaining exons downstream of exon 8 of the endogenous mouse Il1rl2 gene.

24. The method according to claim 16, wherein the second mouse comprising the human IL1F6 gene, the human IL1F8 gene and the human IL1F9 gene is prepared by: Provide mouse embryonic stem (ES) cells, insert: (i) inserting the human IL1F6 gene into the mouse Il1f6 locus of the mouse ES cell, (ii) inserting the human IL1F8 gene into the mouse Il1f8 locus of the mouse ES cell, and (iii) inserting the human IL1F9 gene into the mouse Il1f9 locus of the mouse ES cell, Thus, mouse ES cells containing the human IL1F6 gene, the human IL1F8 gene and the human IL1F9 gene are obtained, and The second mouse is prepared using the mouse ES cell containing the human IL1F6 gene, the human IL1F8 gene, and the human IL1F9 gene.

25. The method according to claim 24, wherein the human IL1F6 gene, the human IL1F8 gene and the human IL1F9 gene are provided in a continuous nucleic acid molecule.

26. The method of claim 15, wherein the human IL1F6 gene replaces the endogenous mouse Il1f6 gene at the endogenous mouse Il1f6 locus.

27. The method of claim 15, wherein the human IL1F8 gene replaces the endogenous mouse Il1f8 gene at the endogenous mouse Il1f8 locus.

28. The method of claim 15, wherein the human IL1F9 gene replaces the endogenous mouse Il1f9 gene at the endogenous mouse Il1f9 locus.

29. An isolated mouse cell or tissue comprising the mouse genome of any one of claims 1-14, wherein the mouse cell or tissue does not include an animal species.

30. A method for preparing a genetically modified mouse embryonic stem (ES) cell, the method comprising modifying the genome of the mouse ES cell so that the modified genome comprises (1) a humanized Il1rl2 gene at the endogenous mouse Il1rl2 locus, wherein the humanized Il1rl2 gene encodes a humanized Il1rl2 protein comprising the extracellular domain of the human IL1RL2 protein and the transmembrane-cytoplasmic sequence of the endogenous mouse Il1rl2 protein; (2) human IL1F6 gene at the endogenous mouse Il1f6 locus; (3) human IL1F8 gene at the endogenous mouse Il1f8 locus; and (4) Human IL1F9 gene at the endogenous mouse Il1f9 locus.

31. Use of a genetically modified mouse ES cell in preparing a mouse, wherein the genetically modified ES cell comprises: (1) a humanized Il1rl2 gene at the endogenous mouse Il1rl2 locus, wherein the humanized Il1rl2 gene encodes a humanized Il1rl2 protein comprising the extracellular domain of the human IL1RL2 protein and the transmembrane-cytoplasmic sequence of the endogenous mouse Il1rl2 protein; (2) human IL1F6 gene at the endogenous mouse Il1f6 locus; (3) human IL1F8 gene at the endogenous mouse Il1f8 locus; and (4) Human IL1F9 gene at the endogenous mouse Il1f9 locus.

32. Use of a mouse comprising the mouse genome of any one of claims 1-14 as a mouse model for a disease associated with dysregulated IL-36 signaling.

33. A method for evaluating the therapeutic efficacy of a candidate compound for treating a disease associated with dysregulated IL-36 signaling, the method comprising: Providing a mouse comprising the mouse genome according to any one of claims 1-14, administering an agent to the mice to induce inflammation, administering the candidate compound to the mouse, and It is determined whether the candidate compound inhibits inflammation.

34. The method of claim 33, wherein the agent is DSS or oxazolone and intestinal inflammation is induced in the mouse.

35. The method of claim 33, wherein the agent is IMQ and skin inflammation is induced in the mouse.

36. The method of claim 33, wherein the candidate compound is administered to the mouse before, during, or after administration of the agent.

37. The method of claim 33, wherein the candidate compound is a small molecule compound, a nucleic acid inhibitor, or an antibody.

38. The method of claim 37, wherein the candidate compound is an antibody.

39. A targeting nucleic acid construct comprising A nucleotide sequence of a human IL1RL2 gene flanked by 5' and 3' mouse nucleotide sequences, wherein the 5' and 3' mouse nucleotide sequences are capable of mediating homologous recombination and integration of the nucleotide sequence of the human IL1RL2 gene in the mouse Il1rl2 locus to form a humanized Il1rl2 gene, wherein the humanized Il1rl2 gene encodes a humanized Il1rl2 protein comprising the extracellular domain of the human IL1RL2 protein and the transmembrane-cytoplasmic sequence of the mouse Il1rl2 protein.

40. A targeting nucleic acid construct comprising A continuous nucleic acid sequence comprising a human IL1F6 gene, a human IL1F8 gene and a human IL1F9 gene, The continuous nucleic acid sequence is flanked by 5' and 3' mouse nucleotide sequences, which are capable of mediating homologous recombination and integration of the continuous nucleic acid sequence in a mouse locus comprising the mouse Il1f6 gene, the mouse Il1f8 gene and the mouse Il1f9 gene.

41. A method of determining whether a compound is capable of inhibiting inflammation, the method comprising: (a) inducing intestinal inflammation in a mouse having the genetically modified mouse genome of claim 1 by administering DSS or oxazolone; (b) administering the compound to the mouse; (c) evaluating the mouse, comprising measuring colon length in the mouse; and (d) if the colon length in the mouse is increased compared to a control mouse comprising the same genetic modification that was not administered the compound, then the compound is determined to inhibit inflammation.

42. The method of claim 41, wherein the evaluation further comprises one or more of the following: Determine the pathological score of the intestine, The levels of myeloperoxidase (MPO) activity in colon homogenates were measured. Measuring the levels of inflammatory cytokines in colon homogenates, and The levels of lipocalin 2 (Lcn2) were measured in stool samples.

43. The method of claim 41, wherein the humanized Il1rl2 protein comprises the amino acid sequence shown in SEQ ID NO:

7.

44. The method of claim 41, wherein the humanized Il1rl2 gene comprises exons 1-2 of the endogenous mouse Il1rl2 gene, exons 3-8 of the human IL1RL2 gene, and exons 9-11 of the endogenous mouse Il1rl2 gene.

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