DITRA non-human animal models and their uses

Abstract

To provide non-human animal models of DITRA and uses thereof.SOLUTION: This disclosure relates to genetically modified rodent animals and rodent models of human diseases. More specifically, this disclosure relates to genetically modified rodents whose genome comprises a humanized Il1rl2 gene (coding for the IL1rl2 subunit of the IL-36R protein) and human IL-36 and ligand genes. The genetically modified rodents disclosed herein display enhanced skin and intestinal inflammation as a preclinical model of psoriasis and IBD, respectively, and serve as a rodent model of human DITRA disease.SELECTED DRAWING: None

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims the interests of U.S. Provisional Patent Application No. 62 / 698,459 filed July 16, 2018, and U.S. Provisional Patent Application No. 62 / 867,477 filed June 27, 2019, the entire contents of these provisional patent applications are incorporated herein by reference.

[0002] This disclosure relates to genetically modified rodents and rodent models of human diseases. More specifically, this disclosure relates to genetically modified rodents whose genomes include the humanized Il1rl2 gene (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 may serve as rodent models of human IL-36 receptor inhibitor deficiency (DITRA).

[0003] Reference to sequence listings A sequence listing file named 35950_10404US01_SequenceListing.txt, a 65KB ASCII text file created on July 9, 2019, and filed with the United States Patent and Trademark Office via EFS-Web, is incorporated herein by reference. [Background technology]

[0004] The interleukin (IL)-36 subfamily consists of three agonists (IL-36α, β, and γ, formerly known as IL1F6, IL1F8, and IL1F9, respectively), as well as one antagonist (IL36Ra) that, via signaling through the heterodimeric receptor (IL-36R), leads to the activation of nuclear factor-κB (NF-κB) and mitogen-activated protein kinase (MAPK). Similar to 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 demonstrated to be primarily expressed in squamous epithelial tissue, particularly in psoriatic skin lesions. The association between IL-36R and epithelial-mediated diseases was confirmed in humans based on loss-of-function mutations in IL-36Ra, leading to the diagnosis of generalized pustular psoriasis (abbreviated as "GPP"), which is now recognized as human IL-36 receptor inhibitor deficiency. d efficiency of the I nterleukin- 3 6 R a It is understood that it is a type of GPP called ntagonist (abbreviated as "DITRA"). It has been found that increased IL-36 expression has been reported in GPP, palmoplantar pustulosis (PPPP), inflammatory bowel disease (IBD), rheumatoid and psoriatic arthritis, asthma, chronic obstructive pulmonary disease, chronic kidney disease, and ichthyosis. [Overview of the Initiative]

[0005] This specification discloses some embodiments of genetically modified rodents comprising a humanized Il1rl2 gene encoding the Il1rl2 protein having an external domain substantially identical to that of human IL1RL2, as well as human Il1f6, Il1f8, and Il1f9 genes encoding human IL1F6, IL1F8, and IL1F9, respectively. Il1rl2 is a subunit of the heterodimeric IL-36R protein and binds to three agonists: IL1F6, IL1F8, and IL1F9 (also known as IL-36α, β, and γ, respectively). The rodents disclosed herein contain the humanized Il1rl2 gene and express three human agonist ligand genes, while retaining an endogenous IL-36Ra antagonist, the endogenous IL-36Ra having approximately 1 / 20th the potency in inhibiting human IL-36R signaling. Such quadruple humanized rodents (e.g., humanized Il1rl2 and human IL1F6, IL1F8, and IL1F9) are shown herein to exhibit symptoms of DITRA patients, resulting in enhanced IL-36R signaling due to mutations in IL-36Ra. Accordingly, genetically modified rodents, compositions and methods for producing such rodents, and methods for using such rodents as models for screening and developing therapeutic agents are provided herein.

[0006] In some embodiments, genetically modified rodents are disclosed herein, the genome comprising: (1) a humanized Il1rl2 gene at the endogenous rodent Il1rl2 locus that encodes a humanized Il1rl2 protein having an external domain substantially identical to that of the human IL1RL2 protein; (2) a human IL1F6 gene at the endogenous rodent Il1f6 locus; (3) a human IL1F8 gene at the endogenous rodent Il1f8 locus; and (4) a human IL1F9 gene at the endogenous rodent Il1f9 locus.

[0007] In some embodiments, the humanized Il1rl2 gene in rodents encodes a humanized Il1rl2 protein containing a transmembrane cytoplasmic sequence substantially identical to that of the endogenous rodent Il1rl2 protein. In some embodiments, the humanized Il1rl2 gene in rodents encodes a humanized Il1rl2 protein containing a signal peptide substantially identical to that of the endogenous rodent Il1rl2 protein.

[0008] In some embodiments, the humanized Il1rl2 gene in rodents encodes a humanized Il1rl2 protein containing an external domain substantially identical to that of the human IL1RL2 protein, and the human IL1RL2 protein contains the amino acid sequence described in Sequence ID No. 2.

[0009] In some embodiments, the humanized Il1rl2 gene in rodents encodes a humanized Il1rl2 protein including an external domain, the external domain containing amino acids 22-337 as described in Sequence ID No. 7.

[0010] In some embodiments, the humanized Il1rl2 gene in rodents encodes a humanized Il1rl2 protein containing the amino acid sequence described in Sequence ID No. 7.

[0011] In some embodiments, the humanized Il1rl2 gene in rodents is operably bound to the endogenous rodent Il1rl2 promoter at the endogenous rodent Il1rl2 locus.

[0012] In some embodiments, the humanized Il1rl2 gene in rodents is produced by substituting a genomic fragment of the endogenous rodent Il1rl2 gene at the endogenous rodent Il1rl2 locus with the nucleotide sequence of the human IL1RL2 gene.

[0013] In some embodiments, the nucleotide sequence of the human IL1RL2 gene is a genomic fragment of the human IL1RL2 gene that substantially encodes the external domain of the human IL1RL2 protein. In some embodiments, the genomic fragment of the human IL1RL2 gene includes exons 3–8 of the human IL1RL2 gene.

[0014] In some embodiments, the genomic sequence of the endogenous rodent Il1rl2 gene remaining after humanization substitution includes the 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 humanization substitution includes exons 1 and 2 of the endogenous rodent Il1rl2 gene.

[0015] In certain embodiments, the humanized Il1rl2 gene in rodents 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.

[0016] In some embodiments, the human IL1F6 gene in rodents disclosed herein replaces the endogenous rodent Il1f6 gene at the endogenous rodent Il1f6 locus.

[0017] In some embodiments, the human IL1F8 gene in rodents disclosed herein replaces the endogenous rodent Il1f8 gene at the endogenous rodent Il1f8 locus.

[0018] In some embodiments, the human IL1F9 gene in rodents disclosed herein replaces the endogenous rodent Il1f9 gene at the endogenous rodent Il1f9 locus.

[0019] In some embodiments, the rodents disclosed herein are homozygous for one or more of the four humanized genes and / or human genes.

[0020] In some embodiments, the rodents disclosed herein exhibit a shortened colon compared to wild-type rodents. In some embodiments, the rodents disclosed herein exhibit enhanced inflammation in an experimentally induced inflammation model compared to wild-type rodents. In some embodiments, the experimentally induced inflammation model is a skin inflammation model induced by imiquimod (IMQ). In some embodiments, the experimentally induced inflammation model is an intestinal inflammation model induced by dextran sulfate sodium (DSS) or oxazolone.

[0021] In some embodiments, the rodents disclosed herein are mice or rats.

[0022] Furthermore, isolated cells or tissues from the rodents described herein are disclosed herein.

[0023] In a further aspect of some embodiments, a method of generating a genetically modified rodent is disclosed herein, the method comprising modifying the genome of a rodent to comprise (1) a humanized Il1rl2 gene at the endogenous rodent Il1rl2 locus, the humanized Il1rl2 gene encoding a humanized Il1rl2 protein comprising an extracellular domain substantially identical to the extracellular domain of the human IL1RL2 protein; (2) the human IL1F6 gene at the endogenous rodent Il1f6 locus; (3) the human IL1F8 gene at the endogenous rodent Il1f8 locus; and (4) the human IL1F9 gene at the endogenous rodent Il1f9 locus, and generating a rodent comprising the modified genome.

[0024] In some embodiments, the rodent genome is modified by a process comprising: (i) creating a first rodent comprising a humanized Il1rl2 gene at the endogenous rodent Il1rl2 locus; (ii) creating 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) crossing the first rodent with the second rodent to obtain a modified rodent genome.

[0025] In some embodiments, a rodent comprising a humanized Il1rl2 gene at the endogenous rodent Il1rl2 locus is created by providing rodent embryonic stem (ES) cells, inserting the nucleotide sequence of the 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 rodent ES cells comprising the humanized Il1rl2 gene, and using the rodent ES cells comprising the humanized Il1rl2 gene to create a rodent.

[0026] In some embodiments, the nucleotide sequence of the inserted human IL1RL2 gene replaces a 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 external domain of the human IL1RL2 protein. In some embodiments, the genomic fragment of the human IL1RL2 gene includes exons 3-8 of the human IL1RL2 gene. In some embodiments, the genomic sequence of the endogenous rodent Il1rl2 gene remaining after substitution includes the 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 humanization substitution includes exons 1-2 of the endogenous rodent Il1rl2 gene. In some embodiments, the humanized Il1rl2 gene formed as a result of humanization substitution includes 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.

[0027] In some embodiments, rodents containing the human IL1F6 gene, human IL1F8 gene, and human IL1F9 gene are produced by providing rodent embryonic stem (ES) cells, inserting the human IL1F6 gene into the rodent Il1f6 locus of the rodent ES cells, the human IL1F8 gene into the rodent Il1f8 locus of the rodent ES cells, and the human IL1F9 gene into the rodent Il1f9 locus of the rodent ES cells, thereby obtaining rodent ES cells containing the human IL1F6 gene, human IL1F8 gene, and human IL1F9 gene, and producing rodents using rodent ES cells containing the human IL1F6 gene, human IL1F8 gene, and human IL1F9 gene.

[0028] In some embodiments, the human IL1F6 gene, the human IL1F8 gene, and the human IL1F9 gene are provided in a continuous nucleic acid molecule.

[0029] In some embodiments, the human IL1F6 gene replaces the endogenous rodent Il1f6 gene at the endogenous rodent Il1f6 locus.

[0030] In some embodiments, the human IL1F8 gene is replaced by the endogenous rodent Il1f8 gene at the endogenous rodent Il1f8 locus.

[0031] In some embodiments, the human IL1F9 gene replaces the endogenous rodent Il1f9 gene at the endogenous rodent Il1f9 locus.

[0032] In some embodiments, different aspects of certain embodiments, rodent embryonic stem (ES) cells are disclosed herein, comprising: (1) a humanized Il1rl2 gene at the endogenous rodent Il1rl2 locus encoding a humanized Il1rl2 protein, which includes an external domain substantially identical to that of the human IL1RL2 protein; (2) a human IL1F6 gene at the endogenous rodent Il1f6 locus; (3) a human IL1F8 gene at the endogenous rodent Il1f8 locus; and (4) a human IL1F9 gene at the endogenous rodent Il1f9 locus. In some embodiments, such ES cells can be produced by methods disclosed herein. In some embodiments, the use of such ES cells for producing rodents is also disclosed.

[0033] In yet another aspect of some embodiments, it is disclosed herein that nucleic acid constructs useful for modifying rodent genomes (e.g., rodent ES cells) to produce modified rodents may be targeted.

[0034] In some embodiments, a target nucleic acid construct is disclosed herein that includes the nucleotide sequence of the human IL1RL2 gene, adjacent to the 5' and 3' rodent nucleotide sequences, which can be homologously recombined and incorporated into the rodent Il1rl2 locus to form a humanized Il1rl2 gene, wherein the humanized Il1rl2 gene encodes a humanized Il1rl2 protein containing an external domain substantially identical to that of the human IL1RL2 protein.

[0035] In some embodiments, a targeted nucleic acid construct is disclosed herein, comprising a sequence of nucleic acid sequences including the human IL1F6 gene, the human IL1F8 gene, and the human IL1F9 gene, the sequence of nucleic acid sequences being adjacent to 5' and 3' rodent nucleotide sequences that can mediate homologous recombination and incorporation of the sequence of nucleic acid sequences into rodent loci containing the rodent Il1f6 gene, the rodent Il1f8 gene, and the rodent Il1f9 gene.

[0036] In further aspects of some embodiments, the use of rodents disclosed herein as a rodent model of human diseases associated with dysregulation of IL-36 signaling is disclosed herein. In non-limiting examples of embodiments disclosed herein, rodents can be used to study the pathophysiology and molecular basis of human diseases associated with dysregulation of IL-36 signaling (e.g., DITRA, but not limited to IL-36), or to screen, test, and develop therapeutic compounds useful for treating such diseases.

[0037] Furthermore, in further embodiments of some of the embodiments, a method for evaluating the therapeutic efficacy of a candidate compound for treating a disease associated with dysregulated IL-36 signaling is disclosed herein, which includes administering a drug to a rodent disclosed herein to induce inflammation, administering the candidate compound to the rodent, and determining whether the candidate compound inhibits and / or reduces the induced inflammation.

[0038] In some embodiments, the drug administered to induce inflammation is DSS or oxazolone, which induces intestinal inflammation in rodents. In some embodiments, the drug administered to induce inflammation is IMQ, which induces cutaneous inflammation in rodents.

[0039] In some embodiments, the candidate compound is administered to rodents before, during, or after administration of an anti-inflammatory agent. In some embodiments, the candidate compound may 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. In certain embodiments, the following items are provided, for example: (Item 1) A genetically modified rodent whose genome is (1) A humanized Il1rl2 gene located at the endogenous rodent Il1rl2 locus, which encodes a humanized Il1rl2 protein containing an external domain substantially identical to that of the human IL1RL2 protein, (2) The human IL1F6 gene at the endogenous rodent Il1f6 locus, (3) The human IL1F8 gene at the endogenous rodent Il1f8 locus, (4) A genetically modified rodent containing the human IL1F9 gene at the endogenous rodent Il1f9 locus. (Item 2) The genetically modified rodent described in item 1, wherein the humanized Il1rl2 protein contains a transmembrane cytoplasmic sequence substantially identical to the transmembrane cytoplasmic sequence of the endogenous rodent Il1rl2 protein. (Item 3) A genetically modified rodent according to item 1 or 2, wherein the humanized Il1rl2 protein contains a signal peptide substantially identical to the signal peptide of the endogenous rodent Il1rl2 protein. (Item 4) A genetically modified rodent according to any one of items 1 to 3, wherein the human IL1RL2 protein contains the amino acid sequence described in Sequence ID No. 2. (Item 5) A genetically modified rodent according to any one of items 1 to 3, wherein the external domain of the humanized Il1rl2 protein contains amino acids 22 to 337 as described in Sequence ID No. 7. (Item 6) A genetically modified rodent according to any one of items 1 to 3, wherein the humanized Il1rl2 protein contains the amino acid sequence described in Sequence ID No. 7. (Item 7) A genetically modified rodent according to any one of items 1 to 6, wherein the humanized Il1rl2 gene is operably bound to the endogenous rodent Il1rl2 promoter at the endogenous rodent Il1rl2 locus. (Item 8) A genetically modified rodent according to any one of items 1 to 7, wherein the humanized Il1rl2 gene is obtained by substituting the genomic fragment of the endogenous rodent Il1rl2 gene at the endogenous rodent Il1rl2 locus with the nucleotide sequence of the human IL1RL2 gene. (Item 9) The genetically modified rodent described in item 8, wherein the nucleotide sequence of the human IL1RL2 gene is a genomic fragment of the human IL1RL2 gene that substantially encodes the external domain of the human IL1RL2 protein. (Item 10) The genetically modified rodent described in item 9, wherein the genome fragment of the human IL1RL2 gene contains exons 3-8 of the human IL1RL2 gene. (Item 11) A genetically modified rodent according to any one of items 8 to 10, wherein the genome sequence of the endogenous rodent Il1rl2 gene remaining after the substitution includes the exon located downstream of exon 8 of the endogenous rodent Il1rl2 gene. (Item 12) A genetically modified rodent according to any one of items 8 to 11, wherein the genome sequence of the endogenous rodent Il1rl2 gene remaining after the substitution includes exons 1 to 2 of the endogenous rodent Il1rl2 gene. (Item 13) The genetically modified rodent described in item 8, wherein the humanized Il1rl2 gene includes exons 1-2 of the endogenous rodent Il1rl2 gene, exons 3-8 of the human IL1RL2 gene, and the remaining exon downstream of exon 8 of the endogenous rodent Il1rl2 gene. (Item 14) A genetically modified rodent according to any of items 1 to 13, wherein the human IL1F6 gene replaces the endogenous rodent Il1f6 gene at the endogenous rodent Il1f6 locus. (Item 15) A genetically modified rodent according to any of items 1 to 14, wherein the human IL1F8 gene replaces the endogenous rodent Il1f8 gene at the endogenous rodent Il1f8 locus. (Item 16) A genetically modified rodent according to any of items 1 to 15, wherein the human IL1F9 gene replaces the endogenous rodent Il1f9 gene at the endogenous rodent Il1f9 locus. (Item 17) A genetically modified rodent according to any of items 1 to 16, wherein the rodent is homozygous for each of the humanized Il1rl2 gene, human IL1F6 gene, human IL1F8 gene, and human IL1F9 gene. (Item 18) The aforementioned rodents are genetically modified rodents as described in item 20, which exhibit a shortened colon compared to wild-type rodents. (Item 19) A genetically modified rodent as described in any of items 1 to 18, wherein the rodent is a mouse or a rat. (Item 20) A method for creating genetically modified rodents, Rodent genomes, (1) A humanized Il1rl2 gene located at the endogenous rodent Il1rl2 locus, which encodes a humanized Il1rl2 protein containing an external domain substantially identical to that of the human IL1RL2 protein, (2) The human IL1F6 gene at the endogenous rodent Il1f6 locus, (3) The human IL1F8 gene at the endogenous rodent Il1f8 locus, (4) Modifying the human IL1F9 gene at the endogenous rodent Il1f9 locus to include, A method comprising creating a rodent containing the modified genome. (Item 21) The aforementioned rodent genome, (i) To produce a first rodent containing the humanized Il1rl2 gene at the endogenous rodent Il1rl2 locus, (ii) To create a second rodent containing the human IL1F6 gene at the endogenous rodent Il1f6 locus, the human IL1F8 gene at the endogenous rodent Il1f8 locus, and the human IL1F9 gene at the endogenous rodent Il1f9 locus, (iii) The method of item 20, modified by a process comprising: (iii) crossing the first rodent with the second rodent to obtain the modified rodent genome. (Item 22) The rodent containing the humanized Il1rl2 gene at the endogenous rodent Il1rl2 locus, To provide rodent embryonic stem (ES) cells, The nucleotide sequence of the human IL1RL2 gene is inserted into the rodent Il1rl2 locus of the rodent ES cells to form the humanized Il1rl2 gene at the rodent Il1rl2 locus, thereby obtaining rodent ES cells containing the humanized Il1rl2 gene. The method according to item 21, wherein a rodent is produced using the rodent ES cells containing the humanized Il1rl2 gene. (Item 23) The method according to item 22, wherein the nucleotide sequence of the human IL1RL2 gene replaces a genomic fragment of the rodent Il1rl2 gene at the rodent Il1rl2 locus. (Item 24) The method according to item 23, wherein the nucleotide sequence of the human IL1RL2 gene is a genomic fragment of the human IL1RL2 gene that substantially encodes the external domain of the human IL1RL2 protein. (Item 25) The method according to item 24, wherein the genomic fragment of the human IL1RL2 gene comprises exons 3 to 8 of the human IL1RL2 gene. (Item 26) The method according to any one of items 23 to 25, wherein the genome sequence of the endogenous rodent Il1rl2 gene remaining after the substitution includes the exon downstream of exon 8 of the endogenous rodent Il1rl2 gene. (Item 27) The method according to any one of items 23 to 26, wherein the genome sequence of the endogenous rodent Il1rl2 gene remaining after the substitution includes exons 1 to 2 of the endogenous rodent Il1rl2 gene. (Item 28) The method according to item 22, wherein the humanized Il1rl2 gene comprises exons 1-2 of the endogenous rodent Il1rl2 gene, exons 3-8 of the human IL1RL2 gene, and the remaining exon downstream of exon 8 of the endogenous rodent Il1rl2 gene. (Item 29) The second rodent, which includes the human IL1F6 gene, the human IL1F8 gene, and the human IL1F9 gene, To provide rodent embryonic stem (ES) cells, (i) The human IL1F6 gene is placed in the rodent Il1f6 locus of the rodent ES cell, (ii) The human IL1F8 gene is placed at the rodent Il1f8 locus of the rodent ES cell, and (iii) Inserting the human IL1F9 gene into the rodent Il1f9 locus of the rodent ES cell, This allows us to obtain rodent ES cells containing the human IL1F6 gene, the human IL1F8 gene, and the human IL1F9 gene, The method according to item 21, wherein a second rodent is produced using rodent ES cells containing the human IL1F6 gene, the human IL1F8 gene, and the human IL1F9 gene. (Item 30) The method according to item 29, wherein the human IL1F6 gene, the human IL1F8 gene, and the human IL1F9 gene are provided as a continuous nucleic acid molecule. (Item 31) The method according to any one of items 20 to 30, wherein the human IL1F6 gene replaces the endogenous rodent Il1f6 gene at the endogenous rodent Il1f6 locus. (Item 32) The method according to any one of items 20 to 31, wherein the human IL1F8 gene replaces the endogenous rodent Il1f8 gene at the endogenous rodent Il1f8 locus. (Item 33) The method according to any one of items 20 to 32, wherein the human IL1F9 gene replaces the endogenous rodent Il1f9 gene at the endogenous rodent Il1f9 locus. (Item 34) Isolated cells or tissues of any rodent described in any one of items 1 through 19. (Item 35) These are rodent embryonic stem (ES) cells, (1) A humanized Il1rl2 gene located at the endogenous rodent Il1rl2 locus, which encodes a humanized Il1rl2 protein containing an external domain substantially identical to that of the human IL1RL2 protein, (2) The human IL1F6 gene at the endogenous rodent Il1f6 locus, (3) The human IL1F8 gene at the endogenous rodent Il1f8 locus, (4) Rodent embryonic stem (ES) cells containing the human IL1F9 gene at the endogenous rodent Il1f9 locus. (Item 36) The use of rodent ES cells as described in item 35 in the creation of rodents. (Item 37) Use of any rodent described in item 1-19 as a rodent model for diseases associated with dysregulated IL-36 signaling. (Item 38) A method for evaluating the therapeutic efficacy of candidate compounds for treating diseases related to dysregulated IL-36 signaling, To provide a rodent as described in any one of items 1 to 19, Inducing inflammation by administering drugs to the aforementioned rodents, Administering the candidate compound to the rodents, and A method comprising determining whether the candidate compound inhibits induced inflammation. (Item 39) The method according to item 38, wherein the drug is DSS or oxazolone, and intestinal inflammation is induced in the rodent. (Item 40) The method according to item 38, wherein the drug is IMQ and skin inflammation is induced in the rodent. (Item 41) The method according to any one of items 38 to 40, wherein the candidate compound is administered to the rodent before, during, or after administration of the drug. (Item 42) The method according to any one of items 38 to 41, wherein the candidate compound is a small molecule compound, a nucleic acid inhibitor, or an antibody. (Item 43) The method according to item 42, wherein the candidate compound is an antibody. (Item 44) A target nucleic acid construct, A target nucleic acid construct comprising a nucleotide sequence of the human IL1RL2 gene, adjacent to 5' and 3' rodent nucleotide sequences, which can be incorporated into the rodent Il1rl2 locus via homologous recombination to form a humanized Il1rl2 gene, wherein the humanized Il1rl2 gene encodes a humanized Il1rl2 protein, the humanized Il1rl2 gene comprising an external domain substantially identical to that of the human IL1RL2 protein. (Item 45) A target nucleic acid construct, It includes a continuous nucleic acid sequence containing the human IL1F6 gene, the human IL1F8 gene, and the human IL1F9 gene, The continuous nucleic acid sequence is a target nucleic acid construct adjacent to 5' and 3' rodent nucleotide sequences, which can be incorporated into the rodent locus containing the rodent Il1f6 gene, the rodent Il1f8 gene, and the rodent Il1f9 gene via homologous recombination. [Brief explanation of the drawing]

[0040] The patent file includes at least one drawing produced in color. A copy of this patent, including the color drawing, will be provided by the Patent and Trademark Office upon request and payment of the required fees.

[0041] [Figure 1A] Figures 1A–1D illustrate exemplary strategies for the humanization of mouse Il1rl2. Figure 1A shows a non-scalar diagram of the genomic structures of the mouse Il1rl2 and human IL1RL2 genes. Exons are represented by thin bars positioned across the genomic sequence. The approximately 25,324 bp mouse genomic fragment to be deleted and the approximately 32,389 bp human genomic fragment to be inserted are shown. The locations of the probes used in the assay described in Example 1 are shown. Figure 1B, though not to exact scale, shows an exemplary modified BAC vector for humanization of the endogenous mouse Il1rl2 gene with junctional sequences at the bottom (sequences 26-28), achieving (i) the substitution of mouse Il1rl2 exons 3-8 and intercalating introns, and mouse intron 2 at 3'155 bp and mouse intron 8 at 5'642 bp with human IL1RL2 exons 3-8 and intercalating introns, as well as human intron 2 at 3'346 bp and human intron 8 at 5'1101 bp, and (ii) the insertion of the loxP-hUb1-em7-Neo-pA-mPrm1-Crei-loxP cassette (4996 bp) downstream of the insertion of the human genome fragment at intron 8. Figure 1C, though not to exact scale, shows the humanized Il1rl2 allele after the neomycin cassette has been deleted, along with junctional sequences at the bottom (sequences 26 and 29). Figure 1D shows 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). [Figure 1B] Same as above. [Figure 1C] Same as above. [Figure 1D] Same as above.

[0042] [Figure 2A]Figures 2A–2D illustrate exemplary strategies for replacing mouse Il1f8, Il1f9, and Il1f6 with human IL1F8, IL1F9, and IL1F6. Figure 2A, though not to exact scale, shows a diagram of the genomic composition of the mouse Il1f8, Il1f9, and Il1f6 genes and the human IL1F8, IL1F9, and IL1F6 genes. Exons are represented by thin bars positioned across the genomic sequence. The approximately 76,548 bp mouse genomic fragment to be deleted and the approximately 88,868 bp human genomic fragment to be inserted are shown. The locations of the probes used in the assay described in Example 1 are shown. Figure 2B, though not to exact scale, shows an exemplary modified BAC vector for substitution of the endogenous mouse Il1f8, Il1f9, and Il1f6 genes, along with the junctional sequences at the bottom (sequence numbers 30-32), achieving (i) substitution of the coding sequence and untranslated region (UTR), as well as the human sequences IL1F8, IL1F9, and IL1F6 corresponding to mouse Il1f8, Il1f9, and Il1f6, and (ii) insertion of the 'loxP-hUb1-em7-Hygro-pA-mPrm1-Crei-loxP cassette' (5,218 bp) downstream of the insertion of the human genome fragment. Figure 2C, though not to exact scale, shows the humanization locus after the neomycin cassette has been deleted, along with the junctional sequences at the bottom (sequence numbers 30 and 33). Figure 2D shows the sequence alignment 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. [Figure 2B] Same as above. [Figure 2C] Same as above. [Figure 2D-1] Same as above. [Figure 2D-2] Same as above.

[0043] [Figure 3A-C]Figures 3A-3E illustrate embodiments of the present invention and show 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 with topical application of IMQ for four consecutive days. On day 5, dorsal skin samples were collected for the following histopathological evaluation and qRT-PCR. (3A) Representative skin appearance of DITRA-like and WT mice on day 5 after topical treatment with Vaseline ("Vas", control) or IMQ-containing cream (Aldara) for four consecutive days. (3B) Representative hematoxylin and eosin ("H&E") staining of skin treated with Vaseline and IMQ from DITRA-like and WT mice. (3C) mRNA expression of pro-inflammatory molecules in the skin of DITRA and WT mice treated daily with Vaseline or IMQ-containing cream (n=10 in each group). After topical application of IMQ for four consecutive days, on day 5, dorsal skin samples were collected from Il1rl2 monohumanized mice ("1H"), DITRA-like mice, and wild-type mice for subsequent histopathological evaluation and qRT-PCR. The groups in each bar, from left to right, are: WT Vas, DITRA Vas, WT IMQ, and DITRA IMQ. (3D) Representative skin appearance of 1H, DITRA-like mice, and WT mice on day 5 after topical treatment with Vaseline (control) or IMQ-containing cream (Aldara) for four consecutive days. (3E) mRNA expression of pro-inflammatory molecules in the skin of 1H, DITRA, and WT mice treated daily with Vaseline or IMQ-containing cream (n=10 in each group). The groups in each bar, from left to right, are: WT Vas, 1H Vas, DITRA-like Vas, WT IMQ, 1H IMQ, and DITRA-like IMQ. [Figure 3D-E] Same as above.

[0044] [Figure 4]Figures 4A-4B. In some embodiments, DITRA-like mice exhibit a shortened colon in a steady state. (4A) Representative photographs of 3-month and 10-month-old DITRA-like mice and their IL-36R and WT littermates housed together. (4B) Quantified colonic length of 3-month-old and 10-month-old DITRA-like mice compared to their IL-36R KO and WT littermates housed together (n=6 in each group). Error bars indicate mean ± SD.

[0045] [Figure 5A-C] Figures 5A-5E. In some embodiments, DITRA-like mice exhibited impaired mucosal healing in DSS-induced chronic colitis. DITRA-like mice (n=16) and their co-contained WT littermates (n=14) were subjected to chronic DSS-induced colitis by administering 2.5% DSS for 7 days, followed by water for 11 days, then 1.5% DSS for 4 days, followed by water for 5 days, for a total of 27 days. Control mice (n=5) were administered normal water. (5A) Weight loss in DITRA-like mice and their WT co-contained littermates is calculated as the percentage difference between the original weight and the actual weight on any given day. (5B) Colon length in water and DSS-treated DITRA-like mice and WT mice. (5C) Hematoxylin and eosin (H&E) staining and pathological scores of the colons of water and DSS-treated DITRA-like and WT mice. (5D) Myeloperoxidase (MPO) activity in water and DSS-treated DITRA-like and WT mouse colon homogenates. (5E) Levels of pro-inflammatory cytokines in water and DSS-treated DITRA-like and WT mouse colon homogenates. Data are representative of at least three independent experiments. Error bars indicate mean ± SD. The groups for each bar, from left to right: WT / water, DITRA / water, WT / DSS, and DITRA / DSS. [Figure 5D-E] Same as above.

[0046] [Figure 6]Figures 6A-6B. In some embodiments, human IL-36R antagonistism using REGN anti-human IL-36R antibody improves IMQ-induced skin inflammation in DITRA-like mice when administered prophylactically. The dorsal skin of DITRA-like mice was shaved 3 days prior to the start of IMQ application and treated topically with Vaseline (control) or IMQ-containing cream (Aldara) for 4 consecutive days. PBS, αhIL-36R mAb, and hIgG4 isotype control were subcutaneously injected at 10 mg / mL on days -3 and 1 (n=9 for each treatment group). (6A) H&E tissue sections and pathological scores of dorsal skin isolated from DITRA-like mice on day 5. (6B) Levels of pro-inflammatory cytokines in skin homogenates of DITRA-like mice treated with daily IMQ application and injections of PBS, αIL-36R mAb, and isotype control on days -3 and 1 on day 5. Data are representative of three independent experiments. Error bars represent the mean ± SD. The groups for each bar, from left to right, are: PBS, αhIL-36R mAb, and hIgG4 isotype control.

[0047] [Figure 7]Figures 7A-7C. In some embodiments, therapeutic administration of anti-human IL-36R antibody improves chronic IMQ-induced skin inflammation in DITRA-like mice. The dorsal skin of DITRA-like mice was shaved 3 days prior to administration and treated topically twice with Vaseline (control) or IMQ-containing cream (Aldara) (5 consecutive days of application as the first dose, followed by 2 days of no application, and then 4 consecutive days of application as the second dose, for a total of 11 days of application). PBS, anti-human IL-36R antibody, and hIgG4 isotype control were subcutaneously injected at 10 mg / mL on days 5 and 9 (n=10 for each treatment group). (7A) Skin thickness was measured on day 12 in DITRA-like mice. Thickness is shown in μm. (7B) H&E tissue sections and pathological scores of dorsal skin from DITRA-like mice on day 12. (7C) Levels of pro-inflammatory cytokines were measured on day 12 in skin homogenates from DITRA-like mice. The data are representative of two independent experiments. Error bars indicate the mean ± SD. The groups for each bar, from left to right, are: phosphate-buffered saline (PBS), αhIL-36R mAb, and hIgG4 isotype control.

[0048] [Figure 8A-B]Figures 8A-8E. In some embodiments, therapeutic administration of anti-human IL-36R antibody, as well as IL-12p40 blockade, improves DSS-induced chronic inflammation in DITRA-like mice and rescues unhealable DSS-induced mucosal damage. DITRA-like mice (n=45) were subjected to chronic DSS-induced colitis by administering 3% DSS for 7 days, 2% DSS for 13 days, followed by water, for a total of 30 days. Control mice (n=5) were administered normal water. PBS (n=11), αIL-36R mAb (n=11), hIgG4 isotype control (n=6), αmIL-12p40 (BioxCell) (n=11), and rat IgG2a isotype control (n=6) were intraperitoneally injected at 10 mg / mL on days 7, 10, 14, 17, 21, and 24. (8A) Weight loss in DITRA-like mice is calculated as the percentage difference between the original weight and the actual weight on any given day. (8B) Fecal lipocalin-2 (Lcn2) levels in DITRA-like mice were measured at days 0, 7, 17, 23, and 30 throughout chronic colitis. (8C) Colon length in DITRA-like mice at day 30. (8D) Myeloperoxidase (MPO) activity in colon homogenate of DITRA-like mice at day 30. (8E) Pro-inflammatory cytokine levels in colon homogenate of DITRA-like mice at day 30. Data are representative of two independent experiments. Error bars indicate mean ± SD. The groups for each bar, from left to right: PBS / water, PBS / DSS, αhIL-36R / DSS, hIgG4 isotype control / DSS, αmIL-12p40 / DSS, and rat IgG2a isotype control / DSS. [Figure 8C-E] Same as above.

[0049] [Figure 9]Figures 9A-9C. Human IL-36R antagonistism 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 rectally on days 5, 6, and 7. PBS, anti-human IL-36R mAb, and hIgG4 isotype controls were intraperitoneally injected into the mice on days 2, 5, and 7 post-sensitization. (A) Body weight of PBS-, anti-human IL-36R mAb-, and hIgG4 control-treated DITRA-like mice during oxazolone administration. *p<0.05 from the PBS-treated group. (B) Colon length measured in DITRA-like mice on day 8. (C) Levels of pro-inflammatory cytokines in colon homogenates of oxazolone injected with PBS, anti-human IL-36R mAb, and hIgG4 isotype control, and vehicle-treated DITRA-like mice. *- Significant difference from the PBS-treated group, #- Significant difference from the isotype-treated group. Data are representative of two independent experiments with 5 mice in each group. Error bars represent mean ± SEM. *p<0.05, **p<0.005, ***p<0.0005, ****p<0.00001. [Modes for carrying out the invention]

[0050] Disclosed herein are genetically modified rodents (such as mice) that contain a humanized Il1rl2 gene (encoding a humanized IL1RL2 protein having an external domain substantially identical to that of the human IL1RL2 external domain) and a human gene encoding a human IL-36α ligand, while retaining the endogenous IL-36Ra antagonist, which exhibits a 1 / 20th lower potency 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. Therefore, the genetically modified rodents disclosed herein may serve as novel functional models of DITRA suitable for testing candidate therapeutic agents for treating DITRA and its associated disorders. Various embodiments are further described below.

[0051] Genetically modified rodents: Quadruple humanization In one aspect of certain embodiments, the Disclosure provides a rodent whose genome comprises a humanized Il1rl2 gene encoding the humanized IL1RL2 protein (a subunit of IL-36R), and human genes encoding human IL-36α, β, and γ ligands. Such a rodent is also referred to herein as a quadruple-humanized rodent (i.e., 4H or DITRA-like).

[0052] The term "humanized," when used in the context of nucleic acids or proteins, refers to rodent nucleic acids or proteins whose structure (i.e., nucleotide or amino acid sequence) has been altered in whole or in part to include structures found in the corresponding human nucleic acids or proteins. A rodent that contains a humanized gene or expresses a humanized protein is a "humanized" rodent.

[0053] In some embodiments, the rodents of this disclosure include, in non-limiting examples, mice, rats, and hamsters. In some embodiments, the rodents of this disclosure include, in non-limiting examples, mice and rats. In some embodiments, the rodents are selected from the superfamilies Muroidea. In some embodiments, the rodents of this disclosure are animals from families selected from the families Muridae (e.g., mouse-like hamsters), Cricetidae (e.g., hamsters, New World rats and mice, voles), Muridae (purebred mice and rats, gerbils, spiny mice, maned mice), Dolichonoides (tree mice, rock mice, white-tailed rats, Madagascar rats and mice), Glididae (e.g., spiny dormice), and Muridae (e.g., blind mice, bamboo mice, and highland mole mice). In some embodiments, the rodents of the Disclosure are selected from purebred mice or rats (Muridae), gerbils, spiny mice, and maned mice. In some embodiments, the mice of the Disclosure are derived from members of the Muridae family. Humanized IL-1RL2

[0054] IL-36R is a member of the interleukin-1 receptor family and is a heterodimer consisting of a receptor subunit named IL1RL2 (also known as IL-1Rrp2) and a co-receptor subunit, the interleukin-1 receptor accessory protein IL-1RAcP (the whole is referenced from Garlanda C et al., Immunity 39, 1003-1018 (2013); Towne JE et al., J. Biol. Chem. 279, 13677-13688 (2004). The receptor (IL-36R) recognizes and binds to three different agonists, IL-36α, IL-36β, and IL-36γ (also known as IL1F6, IL1F8, and IL1F9), which can induce the expression of inflammatory cytokines, as well as IL-36Ra, an antagonist that binds to IL-36R and reduces the expression of inflammatory cytokines.

[0055] IL1RL2 contains a signal peptide, an extracellular domain ("ECD" or "ectodomain"), a transmembrane domain, and an intracellular or cytoplasmic domain. See, for example, Figure 1D. Exemplary 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. [Table 1]

[0056] In some embodiments, the rodents disclosed herein include a humanized Il1rl2 gene in their genome, which includes the nucleotide sequence of the endogenous rodent Il1rl2 gene and the nucleotide sequence of the human IL1RL2 gene. As used herein, “nucleotide sequence of a gene” includes the genomic sequence, mRNA or cDNA sequence, in whole or in part. As a non-limiting example, the nucleotide sequence of the human IL1RL2 gene includes the genomic sequence, mRNA or cDNA sequence of all or part of the human IL1RL2 gene. The nucleotide sequences of the endogenous rodent Il1rl2 gene and the nucleotide sequence of the human IL1RL2 gene include the humanized Il1rl2 gene in the rodent genome having the Il1rl2 protein, i.e., the Il1rl2 protein structure (consisting of an external domain, a transmembrane domain and a cytoplasmic domain), and the Il1rl2 function (e.g., interleukin-1 receptor accessory protein). I nter l eukin- 1 r eceptor ac cessory p rotein (1L-1RacP) forms heterodimers and binds operably to each other in a protein-coding manner, such as a protein that performs the function of recognizing three ligands: IL-36α, IL-36β, and IL-36γ.

[0057] In some embodiments, the genetically modified rodent contains a humanized Il1rl2 gene in its genome, which encodes a humanized Il1rl2 protein, and this humanized Il1rl2 protein contains an external domain substantially identical to that of the human IL1RL2 protein. In some embodiments, the external domain substantially identical to that of the human IL1RL2 protein exhibits the same functionality (e.g., ligand-binding properties) as the external domain of the human IL1RL2 protein. In some embodiments, an external domain or polypeptide "substantially identical to that of the human IL1RL2 protein" means a polypeptide whose sequence is at least 95%, 98%, 99%, or 100% identical to that of the human IL1RL2 protein. In some embodiments, it means a polypeptide in which 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 or fewer amino acids differ from that of the human IL1RL2 protein. In some embodiments, a polypeptide is defined as having 5, 4, 3, 2, or 1 or fewer amino acids in the N or C-terminal portion of the external domain, which differs from the external domain of the human IL1RL2 protein only in the N or C-terminal portion, for example, by the addition, deletion, or substitution of amino acids. "N or C-terminal portion of the external domain" means 5 to 10 amino acids from the N or C-terminus of the external domain. In some embodiments, the human IL1RL2 protein has an amino acid sequence that is substantially identical (e.g., at least 95%, 98%, 99%, or 100% identical) to the amino acid sequence described in SEQ ID NO: 2. In certain embodiments, the human IL1RL2 protein includes the amino acid sequence described in SEQ ID NO: 2, and its external domain consists of amino acids 20 to 335 of SEQ ID NO: 2. In some embodiments, the humanized Il1rl2 gene encodes a humanized Il1rl2 protein that includes an external domain substantially identical to the external domain of the human IL1RL2 protein described in SEQ ID NO: 2, i.e., an external domain substantially identical to amino acids 20 to 335 of SEQ ID NO: 2. In some embodiments, the humanized Il1rl2 gene encodes a humanized Il1rl2 protein containing an external domain comprising amino acids 22-337 of sequence number 7.

[0058] In some embodiments, the humanized Il1rl2 gene encodes a humanized Il1rl2 protein containing a transmembrane cytoplasmic sequence (i.e., a sequence including both a transmembrane domain and a cytoplasmic domain) that is substantially identical to the transmembrane cytoplasmic sequence of the endogenous rodent Il1rl2 protein. In some embodiments, the transmembrane cytoplasmic sequence substantially identical to the transmembrane cytoplasmic sequence of the endogenous rodent Il1rl2 protein exhibits the same functionality (e.g., signal transduction and / or interaction with intracellular molecules) as the transmembrane cytoplasmic sequence of the endogenous rodent Il1rl2 protein. In some embodiments, a transmembrane cytoplasmic sequence or peptide "substantially identical to the transmembrane cytoplasmic sequence of the endogenous rodent Il1rl2 protein" means a polypeptide whose sequence is at least 95%, 98%, 99%, or 100% identical to the transmembrane cytoplasmic sequence of the endogenous rodent Il1rl2 protein. In some embodiments, a polypeptide is defined as having 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 or fewer amino acids that differ from the transmembrane cytoplasmic sequence of the endogenous rodent Il1rl2 protein. In some embodiments, a polypeptide is defined as having 5, 4, 3, 2, or 1 or fewer amino acids at the N or C terminus of the transmembrane cytoplasmic sequence, for example, due to the addition, deletion, or substitution of amino acids. "N or C-terminal portion of the transmembrane cytoplasmic sequence" means within 5 to 10 amino acids from the N terminus of the transmembrane domain, or within 5 to 10 amino acids from the C terminus of the cytoplasmic domain. In some embodiments, the humanized Il1rl2 protein contains a transmembrane cytoplasmic sequence of a mouse Il1rl2 protein, such as a mouse Il1rl2 protein that is substantially identical (at least 95%, 98%, 99%, or 100%) to SEQ ID NO: 4, or a transmembrane cytoplasmic sequence of a rat Il1rl2 protein, such as a rat Il1rl2 protein that is substantially identical (at least 95%, 98%, 99%, or 100%) to SEQ ID NO: 6.

[0059] In some embodiments, the humanized Il1rl2 gene encodes a humanized Il1rl2 protein containing a signal peptide substantially identical to that of the endogenous rodent Il1rl2 protein. A signal peptide "substantially identical to that of the endogenous rodent Il1rl2 protein" means, in some embodiments, a polypeptide that is at least 95%, 98%, 99%, or 100% sequence-identical to the signal peptide of the endogenous rodent Il1rl2 protein. In some embodiments, this means a polypeptide that differs from the signal peptide of the endogenous rodent Il1rl2 protein by three, two, or one or fewer amino acids. In some embodiments, this means a polypeptide that differs from the signal peptide of the endogenous rodent Il1rl2 protein only at the N or C terminus, but only by three, two, or one amino acid in the N or C-terminal portion of the signal peptide, for example, through the addition, deletion, or substitution of amino acids. "N- or C-terminal portion of the signal peptide" means the five amino acids within the N- or C-terminus of the signal peptide. In some embodiments, the humanized Il1rl2 protein includes a signal peptide of the mouse Il1rl2 protein, such as the mouse Il1rl2 protein which is substantially identical (at least 95%, 98%, 99%, or 100%) to SEQ ID NO: 4, or a signal peptide of the rat Il1rl2 protein which is substantially identical (at least 95%, 98%, 99%, or 100%) to the rat Il1rl2 protein which is substantially identical (at least 95%, 98%, 99%, or 100%) to SEQ ID NO: 6.

[0060] In some embodiments, the humanized Il1rl2 gene in the genome of a genetically modified rodent comprises the nucleotide sequence of the human IL1RL2 gene (or "human IL1RL2 nucleotide sequence") and the nucleotide sequence of the endogenous rodent Il1rl2 gene (or "endogenous rodent Il1rl2 nucleotide sequence"), where the human IL1RL2 nucleotide sequence encodes a polypeptide substantially identical to the extradomain of the human IL1RL2 protein encoded by the human IL1RL2 gene. Such a human IL1RL2 nucleotide sequence is also referred to as substantially encoding the extradomain of the human IL1RL2 protein. In some embodiments, the human IL1RL2 nucleotide sequence is a genomic fragment of the human IL1RL2 gene. In some embodiments, the human IL1RL2 nucleotide sequence consists of exons 3-8 of the human IL1RL2 gene. In some embodiments, the human IL1RL2 nucleotide sequence consists of the 3' portion of intron 2, exons 3-8, and the 5' portion of intron 8 of the human IL1RL2 gene. In some embodiments, the human IL1RL2 nucleotide sequence is a cDNA sequence.

[0061] In some embodiments, the humanized Il1rl2 gene in the genome of a genetically modified rodent comprises an endogenous rodent Il1rl2 nucleotide sequence and a human Il1rl2 nucleotide sequence, the endogenous rodent Il1rl2 nucleotide sequence encoding a polypeptide substantially identical to the transmembrane cytoplasmic sequence of the endogenous rodent Il1rl2 protein. Such a rodent Il1rl2 nucleotide sequence is also referred to as substantially encoding 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 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 residual exon downstream of exon 8 of the endogenous rodent Il1rl2 gene. In some embodiments, the endogenous rodent Il1rl2 nucleotide sequence present in the humanized Il1rl2 gene includes the 3' portion of intron 8 and the residual exon downstream of exon 8 of the endogenous rodent Il1rl2 gene.

[0062] In some embodiments, the humanized Il1rl2 gene in the genome of a genetically modified rodent contains an endogenous rodent Il1rl2 nucleotide sequence upstream (5') of the human IL1RL2 nucleotide sequence, the endogenous rodent Il1rl2 nucleotide sequence encoding 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 exons 1-2 and the 5' portion of intron 2 of the endogenous rodent Il1rl2 gene.

[0063] In some embodiments, the humanized Il1rl2 gene is operablely linked to an endogenous rodent Il1rl2 regulatory sequence, such as a 5' transcriptional regulatory sequence including a promoter and / or enhancer, for example, so that the expression of the humanized Il1rl2 gene is under the control of the rodent Il1rl2 5' regulatory sequence.

[0064] In some embodiments, the humanized Il1rl2 gene is located at the 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 where 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., whole or partial deletion) of the endogenous rodent Il1rl2 gene.

[0065] In some embodiments where the humanized Il1rl2 gene is located at the endogenous rodent Il1rl2 locus, the humanized Il1rl2 gene arises as a result of the endogenous rodent Il1rl2 gene being substituted with the nucleotide sequence of the human IL1RL2 gene at the endogenous rodent Il1rl2 locus.

[0066] In some embodiments, the nucleotide sequence of the endogenous rodent Il1rl2 gene at the substituted endogenous rodent Il1rl2 locus is a genomic fragment of the endogenous rodent Il1rl2 gene that substantially encodes the external domain of the rodent Il1rl2 protein. In some embodiments, the substituted rodent genomic fragment includes exons 3-8 of the endogenous rodent Il1rl2 gene.

[0067] 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 encode the external domain of the human IL1RL2 protein. In some embodiments, the human genomic fragment includes exons 3-8 of the human IL1RL2 gene.

[0068] In some embodiments, the genomic sequence of the endogenous rodent Il1rl2 gene that remains at the endogenous rodent Il1rl2 locus after humanization substitution and operably binds 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 humanization substitution includes the exon downstream of exon 8 of the endogenous rodent Il1rl2 gene.

[0069] In some embodiments, the genomic sequence of the endogenous rodent Il1rl2 gene that remains at the endogenous rodent Il1rl2 locus after humanization substitution and operably binds 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 humanization substitution includes exons 1-2 of the endogenous rodent Il1rl2 gene.

[0070] In some embodiments, when the endogenous rodent Il1rl2 protein and the human IL1RL2 protein share common amino acids near the junction between the transmembrane and extramembrane domains, it may not be necessary to insert a human IL1RL2 nucleotide sequence that strictly codes for the extramembrane domain of the human IL1RL2 protein. It is possible to insert a slightly longer or shorter human IL1RL2 gene nucleotide sequence operably bound to the genomic sequence of the endogenous rodent Il1rl2 gene that substantially codes for the extramembrane domain of the human IL1RL2 protein and substantially codes for the transmembrane domain (and cytoplasmic domain) of the endogenous rodent Il1rl2 protein, thereby encoding a humanized Il1rl2 protein that includes an extramembrane domain identical to that of the human IL1RL2 protein and a transmembrane domain identical to that of the endogenous rodent Il1rl2 protein.

[0071] In some embodiments, the genomic fragment containing exons 3–8 of the endogenous rodent Il1rl2 gene at the endogenous rodent Il1rl2 locus is replaced with a genomic fragment containing exons 3–8 of the human IL1RL2 gene. As a result, the humanized Il1rl2 gene is formed at the endogenous rodent Il1rl2 locus and contains 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.

[0072] 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.

[0073] In some embodiments, the humanized Il1rl2 gene results in the expression of the humanized Il1rl2 protein encoded in rodents. In some embodiments, the humanized Il1rl2 protein is expressed in a pattern equivalent to, or substantially identical to, the corresponding rodent Il1rl2 protein in control rodents (e.g., rodents lacking the humanized Il1rl2 gene). In some embodiments, the humanized Il1rl2 protein is expressed at a level equivalent to, or substantially identical to, the corresponding rodent Il1rl2 protein in control rodents (e.g., rodents lacking the humanized Il1rl2 gene). In some embodiments, the humanized Il1rl2 protein is expressed and detected on the cell surface, for example, but not limited to, keratinocytes, monocytes, macrophages, neutrophils, bronchial and intestinal epithelial cells. In the context of comparing humanized genes or proteins of humanized rodents with endogenous rodent genes or proteins of control rodents, the term “comparable” means that the molecules or levels being compared may not be identical to one another, but are similar enough to allow comparison between them so that reasonable conclusions can be drawn based on the observed differences or similarities. In some embodiments, the term “substantially identical” includes levels being compared that do not differ from one another by more than 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% in terms of expression levels.

[0074] In some embodiments, the rodents disclosed herein are unable to express the rodent Il1rl2 protein as a result of, for example, inactivation (e.g., whole or partial deletion) or substitution (whole or partial) of the endogenous rodent Il1rl2 gene.

[0075] Humanization of IL-36 ligand The rodents disclosed herein include germline genes encoding human IL-36α, β, and γ ligands. [Table 2]

[0076] In some embodiments, the rodents disclosed herein have a genome containing the human IL-36α gene encoding the human IL-36α protein. The reference to the “human IL-36α gene” includes the human genomic DNA encoding the human IL-36α protein and the human IL-36α promoter. The human IL-36α protein may be the mature or precursor form of the human IL-36α protein. In some embodiments, the human IL-36α protein contains the amino acid sequence of SEQ ID NO: 9.

[0077] In some embodiments, the human IL-36α gene is located at the endogenous rodent Il-36α locus. In some embodiments, the human IL-36α gene is located at a locus other than the endogenous rodent Il-36α 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 Il-36α locus, the rodent is unable to express the rodent Il-36α protein, for example, as a result of inactivation (e.g., whole or partial deletion) of the endogenous rodent Il-36α gene.

[0078] In some embodiments, the human IL-36α gene is replaced by the endogenous rodent Il-36α gene at the endogenous rodent Il-36α locus.

[0079] 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.

[0080] In some embodiments, the human IL-36α gene results in the expression of the encoded human IL-36α protein in rodents, such as in serum, mucosal sites (e.g., skin, intestinal epithelium, lungs), and various types of cells in the immune system (e.g., monocytes, macrophages, T cells, dendritic cells). In some embodiments, the human IL-36α protein is expressed in a pattern equivalent to, or substantially identical to, the corresponding rodent IL-36α protein in control rodents (e.g., rodents lacking the human IL-36α gene). In some embodiments, the human IL-36α protein is expressed, for example, in serum, mucosal sites (e.g., skin, intestinal epithelium, lungs), and / or immune cells (e.g., monocytes, macrophages, T cells, dendritic cells) at levels equivalent to, or substantially identical to, the corresponding rodent IL-36α protein in control rodents (e.g., rodents lacking the human IL-36α gene).

[0081] In some embodiments, the rodents disclosed herein are unable to express the rodent Il-36α protein as a result of, for example, inactivation (e.g., whole or partial deletion) or substitution (whole or partial) of the endogenous rodent Il-36α gene. [Table 3]

[0082] In some embodiments, the rodents disclosed herein have a genome containing the human IL-36β gene encoding the human IL-36β protein. The reference to the “human IL-36β gene” includes the human genomic DNA encoding the human IL-36β protein and the human IL-36β promoter. The human IL-36β protein may be the mature or precursor form of the human IL-36β protein. In some embodiments, the human IL-36β protein contains the amino acid sequence of SEQ ID NO: 15.

[0083] In some embodiments, the human IL-36β gene is located at the endogenous rodent Il-36β locus. In some embodiments, the human IL-36β gene is located at a locus other than the endogenous rodent Il-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 Il-36β locus, the rodent is unable to express the rodent Il-36β protein, for example, as a result of inactivation (e.g., whole or partial deletion) of the endogenous rodent Il-36β gene.

[0084] In some embodiments, the human IL-36β gene replaces the endogenous rodent Il-36αβ gene at the endogenous rodent Il-36β locus.

[0085] 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.

[0086] In some embodiments, the human IL-36β gene results in the expression of the encoded human IL-36β protein in rodents, such as in serum, mucosal sites (e.g., skin, intestinal epithelium, lungs), and / or immune cells (e.g., monocytes, macrophages, T cells, dendritic cells). In some embodiments, the human IL-36β protein is expressed in a pattern equivalent to, or substantially identical to, the corresponding rodent IL-36β protein in control rodents (e.g., rodents lacking the human IL-36β gene). In some embodiments, the human IL-36β protein is expressed at levels equivalent to, or substantially identical to, the corresponding rodent IL-36β protein in control rodents (e.g., rodents lacking the human IL-36β gene), for example, in serum, mucosal sites (e.g., skin, intestinal epithelium, lungs), and various types of cells in the immune system (e.g., monocytes, macrophages, T cells, dendritic cells).

[0087] In some embodiments, the rodents disclosed herein are unable to express the rodent Il-36β protein as a result of, for example, inactivation (e.g., whole or partial deletion) or substitution (whole or partial) of the endogenous rodent Il-36β gene. [Table 4]

[0088] In some embodiments, the present invention provides a rodent whose genome contains a human IL-36γ gene encoding the human IL-36γ protein. The reference to the "human IL-36γ gene" includes human genomic DNA encoding the human IL-36γ protein and includes the human IL-36γ promoter. In some embodiments, the human IL-36γ protein may be a mature or precursor form of the human IL-36γ protein. In some embodiments, the human IL-36γ protein contains the amino acid sequence of SEQ ID NO: 21.

[0089] In some embodiments, the human IL-36γ gene is located at the endogenous rodent Il-36γ locus. In some embodiments, the human IL-36γ gene is located at a locus other than the endogenous rodent Il-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 Il-36γ locus, the rodent is unable to express the rodent Il-36γ protein, for example, as a result of inactivation (e.g., whole or partial deletion) of the endogenous rodent Il-36γ gene.

[0090] In some embodiments, the human IL-36γ gene is replaced by the endogenous rodent Il-36γ gene at the endogenous rodent Il-36γ locus.

[0091] 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.

[0092] In some embodiments, the human IL-36γ gene results in the expression of the encoded human IL-36γ protein (such as the same protein as the human IL-36γ protein) in rodents, including in serum, mucosal sites such as skin, intestinal epithelium, and lungs, and in various types of cells of the immune system (e.g., monocytes, macrophages, T cells, and dendritic cells). In some embodiments, the human IL-36γ protein is expressed in a pattern equivalent to, or substantially identical to, the corresponding rodent IL-36γ protein in control rodents (e.g., rodents lacking the human IL-36γ gene). In some embodiments, the human IL-36γ protein is expressed, for example, in serum, mucosal sites (e.g., skin, intestinal epithelium, lungs, etc.), and / or immune cells (e.g., monocytes, macrophages, T cells, dendritic cells) at levels equivalent to or substantially the same as the corresponding rodent IL-36γ protein in a control rodent (e.g., a rodent lacking the human IL-36γ gene).

[0093] In some embodiments, the rodents disclosed herein are unable to express the rodent Il-36γ protein as a result of, for example, inactivation (e.g., whole or partial deletion) or substitution (whole or partial) of the endogenous rodent Il-36γ gene.

[0094] In some embodiments, a rodent is provided whose genome comprises substitution of a contiguous nucleic acid at an endogenous locus with a contiguous genomic fragment containing coding sequences for three ligands, each substantially identical to human IL-36α, β, and γ, which are each coding sequences for all three IL-36 ligands. In some embodiments, the resulting locus comprises, from 5' to 3', (i) the human IL-36γ gene, (ii) the human IL-36γ gene, and (iii) the reverse strand of the human IL-36γ gene.

[0095] Phenotype of quadruple humanized rodents The genetically modified rodents disclosed herein do not develop any spontaneous diseases in a steady state, but these rodents exhibit a shortened colon and increased expression of pro-inflammatory mediators in the skin, both in a steady state and in a disease state (e.g., after DSS or IMQ treatment), compared to unhumanized, age-matched control rodents. In some embodiments, the genetically modified rodents exhibit a colon length that is at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% shorter than that of control rodents, either in a steady state or in a disease state.

[0096] In some embodiments, genetically modified rodents exhibit a colon length 10% to 15% (±5%) shorter than that of control rodents in a steady state. In some embodiments, genetically modified rodents exhibit a colon length 15% to 20% (±5%) shorter than that of control rodents in a steady state. In some embodiments, genetically modified rodents exhibit a colon length 10% to 20% (±5%) shorter than that of control rodents in a steady state.

[0097] In other embodiments, genetically modified rodents exhibit a colon length 20% to 40% (±5%) shorter than control rodents in a diseased state (e.g., after DSS or oxazolone treatment). In other embodiments, genetically modified rodents exhibit a colon length 30% to 40% (±5%) shorter than control rodents in a diseased state (e.g., after DSS or oxazolone treatment). In other embodiments, genetically modified rodents exhibit a colon length 20% to 30% (±5%) shorter than control rodents in a diseased state (e.g., after DSS or oxazolone treatment). In other embodiments, genetically modified rodents exhibit a colon length 25% to 35% (±5%) shorter than control rodents in a diseased state (e.g., after DSS or oxazolone treatment).

[0098] The genetically modified rodents disclosed herein do not develop spontaneous diseases under steady-state conditions, but have been shown to exhibit enhanced skin inflammation and intestinal inflammation in experimentally induced skin inflammation and intestinal inflammation models (e.g., preclinical models of psoriasis and IBD, respectively).

[0099] In some embodiments, DSS is used to induce chronic colitis. In some embodiments, DSS is administered to rodents through drinking water containing at least 0.5%, at least 1%, at least 1.5%, or at least 2.5%. In some embodiments, DSS is administered to rodents through drinking water containing 10%, 9%, 8%, 7%, 6%, or 5% or less. In some embodiments, drinking water containing 1.5% to 3% DSS is given to rodents. In some embodiments, drinking water containing 0.5% to 3% DSS is given to rodents. In some embodiments, drinking water containing 1% to 3% DSS is given to rodents. In some embodiments, drinking water containing 2% to 3% DSS is given to rodents. In some embodiments, drinking water containing 2.5% to 3% DSS is given to rodents. In some embodiments, drinking water containing 0.5% to 2.5% DSS is given to rodents. In some embodiments, rodents are given drinking water containing 0.5% to 2% DSS. In some embodiments, rodents are given drinking water containing 0.5% to 1.5% DSS. In some embodiments, rodents are given drinking water containing 0.5% to 1% DSS. In some embodiments, rodents are given drinking water containing 1% to 2.5% DSS. In some embodiments, rodents are given drinking water containing 1.5% to 2% DSS.

[0100] In some embodiments, DSS administration can be carried out over a period of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 days, or longer, and may be continuous or intermittent with no DSS administration. In some embodiments, rodents are given drinking water containing 2.5% DSS for 7 days, then 1.5% DSS for 5 days, then distilled water until 27-30 days before analysis. In some embodiments, rodents are given 3% In some embodiments, rodents are given drinking water containing DSS for 7 days, followed by 2% DSS for 13 days, and then distilled water until 27-30 days before analysis. In some embodiments, DSS is not given for the entire period. In some embodiments, rodents are given drinking water containing 2.5% DSS for 7 days, followed by distilled water for 11 days, followed by 1.5% DSS for 4 days, followed by distilled water for 5 days, for a total of 27 days before analysis. In some embodiments, rodents are given drinking water containing 3% DSS for 7 days, followed by water for 13 days, followed by 2% DSS for 4 days, followed by distilled water for 6 days, until 30 days before analysis.

[0101] In some embodiments, oxazolone is used to induce colitis. In some embodiments, oxazolone is administered rectally to rodents to induce colitis. In some embodiments, oxazolone is administered rectally to rodents three times to induce colitis. In some embodiments, oxazolone is applied topically to rodents for pre-sensitization before rectal administration. In some embodiments, oxazolone is administered to rodents for pre-sensitization by topically applying an oxazolone solution (e.g., a 3% oxazolone solution dissolved in 100% ethanol) onto shaved skin, followed by three rectal administrations of an oxazolone solution (e.g., 1.0-2.0% oxazolone dissolved in 50% ethanol). In some embodiments, pre-sensitization uses 1.5%, 2.0%, 2.5%, 3.0%, 3.5%, or 4.0% oxazolone solutions dissolved in 100% ethanol. In some embodiments, rectal administration is performed using 1.0%, 1.1%, 1.2%, 1.3%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, or 2.0% solutions of oxazolone dissolved in 50% ethanol. In some embodiments, rectal administration is performed several days later, such as 3, 4, 5, 6, or 7 days after pre-sensitization. In some embodiments, oxazolone is administered to rodents by topical application of an oxazolone solution (e.g., a 3.0% oxazolone solution dissolved in 100% ethanol) onto shaved skin, followed by three rectal administrations of a 1.0-2.0% oxazolone solution dissolved in 50% ethanol on days 5, 6, and 7.

[0102] In some embodiments, the severity of colitis is assessed by scoring the following features: inflammation (severity and degree), epithelial changes (erosions / ulcers), crypt changes (crypt defects, cryptitis / crypt abscesses, regeneration / hyperplasia, goblet cell defects), submucosal edema, and the percentage of tissue area with pathology relative to the total tissue area on a slide. A scoring scale of 0 to 4 is used: 0-0 - within normal range, 1 - minimal, 2 - mild, 3 - moderate, 4 - severe. A total pathology score is calculated for each rodent by adding the individual histopathological feature scores. In some embodiments, colitis is assessed by measuring the level of lipocalin-2 (Lcn2) in a fecal sample. In some embodiments, colitis is measured by measuring the level of myeloperoxidase (MPO) activity in a colon homogenate. In some embodiments, colitis is assessed by measuring the level of inflammatory cytokines in a colon homogenate.

[0103] In some embodiments, genetically modified rodents exhibit an increase in intestinal pathology scores of, for example, at least 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, or 400% compared to wild-type control rodents administered the same DSS. In some embodiments, genetically modified rodents exhibit an increase in intestinal pathology scores of, for example, 50%-400%, 50%-300%, 50%-200%, 50%-100%, 100%-400%, 100%-300%, 100%-200%, or 200%-400% compared to wild-type control rodents administered the same DSS. In some embodiments, genetically modified rodents exhibit increased myeloperoxidase ("MPO") levels in colon homogenate by, for example, at least 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, or 400% compared to wild-type control rodents administered the same DSS. In some embodiments, genetically modified rodents exhibit increased MPO levels in colon homogenate by, for example, 50%–400%, 50%–300%, 50%–200%, 50%–100%, 100%–400%, 100%–300%, 100%–200%, or 200%–400% compared to wild-type control rodents administered the same DSS. In some embodiments, genetically modified rodents exhibit increased levels of fecal Lcn, for example, by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, or 400% compared to wild-type control rodents administered the same DSS. In some embodiments, genetically modified rodents exhibit increased mRNA expression and / or protein levels of one or more pro-inflammatory mediators (e.g., KC-GRO, IL-6, IL-1β, TNFα, IL-21, IL-12p40, IL-17f, IL-17a, and IL-17c) in colon homogenates, for example, by at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, and 900% or more, compared to wild-type control rodents administered the same DSS.In some embodiments, the genetically modified rodents exhibit increased mRNA expression and / or protein levels of one or more pro-inflammatory mediators in colon homogenates, for example, 20%-900%, 20%-800%, 20%-700%, 20%-600%, 20%-500%, 20%-400%, 20%-300%, 20%-200%, or 20%-100%. In some embodiments, genetically modified rodents exhibit increased mRNA expression and / or protein levels of one or more pro-inflammatory mediators in colon homogenates, for example, 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 pro-inflammatory mediators in colon homogenates, for example, 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 pro-inflammatory mediators in colon homogenates, for example, 50%-900%, 50%-800%, 50%-700%, 50%-600%, 50%-500%, 50%-400%, 50%-300%, 50%-200%, or 50%-100%.

[0104] In some embodiments, IMQ is applied topically to the skin of rodents to induce skin inflammation. In some embodiments, IMQ is provided in a carrier suitable for topical application, such as a cream, gel, or a commercially available IMQ cream (e.g., from Aldara). In some embodiments, to induce skin inflammation, IMQ is applied daily to the skin of rodents for 2, 3, 4, 5, 6, 7, 8, 9, or 10 days or more, in a daily dose of 1-5 mg, 2-4 mg, or 3-3.5 mg. In some embodiments, daily topical application at a daily dose of approximately 3.125 mg for 4 days is suitable for inducing acute skin inflammation, and daily topical application at a daily dose of approximately 3.125 mg for 9 days is suitable for inducing chronic skin inflammation. In some embodiments, IMQ is applied topically multiple times (e.g., two, three, or four times) by applying IMQ for four to five consecutive days before the analysis or assay is performed, followed by two days of IMQ non-application for each subsequent application. In certain embodiments, IMQ is applied topically twice: five consecutive days before the analysis, followed by two days of IMQ non-application for the first application, and then four consecutive days of IMQ application for the second application (see Figures 7A-7C). In some embodiments, the severity of inflammation may be assessed by (i) using an adapted version of the Clinical Psoriasis Area and Severity Index (PASI) based on the measurement of erythema, desquamation and skin thickening; (ii) histopathological analysis of skin tissue to assess, for example, parakeratosis, normal keratosis, Munro microabscess, acanthosis, skin ulceration, inflammation of the dermis and subcutaneous tissue, vascular congestion in the dermis and subcutaneous tissue, follicular keratosis and epithelial hyperplasia, and to determine the total pathology score; (iii) measuring the mRNA expression and / or protein levels of pro-inflammatory mediators in skin homogenates, for example, including mRNA expression and / or protein levels of Cxcl1, IL-17f, IL-17a, IL-23a, S100A8 and Defb4; and (iv) by a combination of (i) to (iii).

[0105] In some embodiments, genetically modified rodents exhibit increased skin pathology scores, for example, by at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or 200% compared to wild-type control rodents receiving the same IMQ dose. In some embodiments, genetically modified rodents exhibit increased mRNA expression and / or protein levels of one or more pro-inflammatory mediators (e.g., Cxcl1, IL-17f, IL-17a, IL-23a, S100A8, and Defb4) in skin homogenates, for example, by at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, or 300% compared to wild-type control rodents receiving the same IMQ dose. In some embodiments, genetically modified rodents exhibit increased mRNA expression and / or protein levels of one or more pro-inflammatory mediators (e.g., Cxcl1, IL-17f, IL-17a, IL-23a, S100A8, and Defb4) in skin homogenates in a steady state compared to a steady-state wild-type control rodent, for example, by at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, or 300% or more.

[0106] Regarding IMQ-induced skin inflammation, one Il1rl2 humanized mouse exhibited a phenotype similar to wild-type rodents, as reflected in the histopathology and RNA expression of pro-inflammatory molecules in the skin (Figures 3D and 3E). On the other hand, DITRA-like mice (i.e., quadruple-humanized mice containing humanized Il1rl2 and human IL1F6, IL1F8, and IL1F9) showed increased skin inflammation after IMQ application compared to both WT and 1H mice (Figures 3D and 3E).

[0107] Tissues and cells of genetically modified rodents In some embodiments, isolated cells or tissues from rodents described herein are disclosed 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, mammary gland, bone marrow, eye, heart, intestine, kidney, liver, lung, lymph node, muscle, pancreas, plasma, serum, skin, spleen, stomach, thymus, testis, oocyte, and combinations thereof. In some embodiments, isolated cells or tissues have a genome containing the quadruple humanization features described herein.

[0108] Composition and method for producing quadruple humanized rodents Further aspects of this disclosure are aimed at methods for producing the genetically modified rodents described above, as well as nucleic acid vectors and rodent embryonic stem cells suitable for use in the production of genetically modified rodents.

[0109] Quadruple humanized rodents, i.e., rodents containing the humanized Il1rl2 and human IL1F6, IL1F8, and IL1F9 genes, can be produced by creating a single Il1rl2 humanized rodent strain and a triple humanized rodent strain of Il1f6, Il1f8, and Il1f9, and then breeding the single humanized rodent strain and the triple humanized rodent strain to obtain quadruple humanized rodents. The terms “breeding” or “crossing,” as used herein in relation to rodents, refer to mating rodents to produce offspring. Those skilled in the art will understand that multiple crosses may be necessary to achieve homozygosity.

[0110] In some embodiments, target vectors containing human nucleotide sequences that are desirable to be incorporated into rodent loci are disclosed herein. In some embodiments, the human nucleotide sequence may be a nucleotide sequence of the human IL1RL2 gene encoding an external domain substantially identical to that of the human IL1RL2 external domain, for example, a nucleotide sequence containing exons 3-8 of the human IL1RL2 gene. In some embodiments, the human nucleotide sequence may be a nucleotide sequence encompassing the complementary strands of the coding sequence for human IL-36α, human IL-36β, and human IL-36γ. The target vector also includes 5' and 3' rodent sequences adjacent to the human nucleotide sequence to be incorporated, also known as homologous arms, which mediate homologous recombination and the incorporation of the human nucleotide sequence into a target rodent locus (e.g., the Il1rl2 locus, or the locus where the rodent Il1f6, Il1f8, and Il1f9 genes are located). In some embodiments, the 5' and 3' flanking rodent sequences are nucleotide sequences adjacent to the corresponding rodent nucleotide sequence at the target rodent locus that is substituted by the human nucleotide sequence. For example, in embodiments where the nucleotide sequence encoding the rodent external domain (e.g., exons 3-8 of the rodent Il1rl2 gene) is substituted with the sequence encoding the human external domain (e.g., exons 3-8 of the 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 substituted with human nucleotide sequences, the 5' flanking sequence may include the rodent nucleotide sequence upstream of the coding sequence of the Il1f6 gene, and the 3' flanking sequence may include the rodent nucleotide sequence upstream of the coding sequence of the Il1f9 gene.

[0111] In some embodiments, the targeting vector includes a selection marker gene. In some embodiments, the targeting vector includes one or more site-specific recombination sites. In some embodiments, the targeting vector includes a selection marker gene adjacent to a site-specific recombination site, and as a result, the selection marker gene may be deleted as a result of recombination between sites.

[0112] In exemplary embodiments, a bacterial artificial chromosome (BAC) clone carrying a rodent genome fragment may be modified using bacterial homologous recombination and VELOCIGENE® technology (see, for example, U.S. Patent No. 6,586,251 and Valenzuela et al. (2003) Nature Biotech. 21(6):652-659, which are incorporated herein by reference in their entirety). As a result, the rodent genome sequence is deleted from the original BAC clone, and a human nucleotide sequence is inserted, resulting in a modified BAC clone carrying the human nucleotide sequence adjacent to the 5' and 3' rodent homologous arms. In some embodiments, the human nucleotide sequence may be a cDNA sequence or (i) all or part of human IL1RL2 (e.g., the external domain of the human IL1RL2 protein), or (ii) human genomic DNA encoding all three of human IL1F6, IL1F8, and IL1F9. Modified BAC clones, once linearized, can be introduced into rodent embryonic stem (ES) cells.

[0113] In some embodiments, the present invention provides a method for using the targeted vectors described herein to produce modified rodent embryonic stem (ES) cells. For example, the targeted vectors can be introduced into rodent ES cells, for example, by electroporation. Mouse ES cells and rat ES cells are both described in the Art. For example, US7,576,259, US7,659,442, US7,294,754, and US2008-0078000A1 (all of which are incorporated herein by reference) describe the VELOCIMOUSE® method for producing mouse ES cells and genetically modified mice; US2014 / 0235933A1 (Regeneron Pharmaceuticals, Inc.), US2014 / 0310828A1 (Regeneron Pharmaceuticals, Inc.), Tong et al. (2010) Nature 467:211-215 and Tong et al. (2011) Nat See Protoc.6(6):doi:10.1038 / nprot.2011.338 (the entire document is incorporated herein by reference).

[0114] In some embodiments, ES cells having human nucleotide sequences incorporated into their genome can be selected. In some embodiments, ES cells are selected based on rodent allele loss and / or human allele acquisition assays. In some embodiments, the selected ES cells are then used as donor ES cells for injection into premorlature embryos (e.g., 8-cell stage embryos) using the VELOCIMOUSE® method (see, for example, US7,576,259, US7,659,442, US7,294,754, and US2008-0078000A1, both of which are invoked by reference) or the methods described in US2014 / 0235933A1 and US2014 / 0310828A1, both of which are invoked by reference. In some embodiments, the embryos containing the donor ES cells are incubated to the blastocyst stage and then transplanted into a surrogate mother to produce F0 rodents that are entirely derived from the donor ES cells. Rodent offspring possessing human nucleotide sequences can be identified by genotyping of DNA isolated from tail fragments using assays for loss of rodent alleles and / or acquisition of human alleles.

[0115] In some embodiments, homozygous rodents can be produced by crossing them with rodents that are heterozygous for the humanization gene.

[0116] Method using quadruple humanized rodents The rodents disclosed herein provide a useful in vivo system and source of biomaterials for identifying and testing compounds useful for treating diseases or conditions associated with dysregulation of IL-36 signaling.

[0117] "Diseases associated with dysregulated IL-36 signaling" refers to diseases in which abnormalities in IL-36 signaling manifest, and which may directly or indirectly cause or worsen the symptoms of such diseases. Non-exclusive examples of diseases associated with dysregulated IL-36 signaling include generalized pustular psoriasis (GPP or DITRA) (the entirety of which is incorporated herein by reference, Marrakchi S. et al., N Engl J. Med. 365:620-628 (2011) and Onoufriadis A. et al., Am J. Hum Genet 89:432-437 (2011)), palmoplantar pustulosis (PPPP) (the entirety of which is incorporated herein by reference, Bissonnette R. et al., PLoS One 11:e0155215 (2016)), and inflammatory bowel disease (IBD) (the entirety of which is incorporated herein by reference, 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), rheumatoid arthritis and psoriatic arthritis (the whole of which is incorporated herein by reference, Frey S. et al., Ann Rheum Dis 72:1569-1574 (2013)), asthma, chronic obstructive pulmonary disease (the whole of which is incorporated herein by reference, Chen H. et al., J. Proteomics 75:2835-2843 (2012)), chronic kidney disease (the whole of which is incorporated herein by reference, Shaik Y. et al., Int J Immunopathol Pharmacol 26:27-36(2013)), and ichthyosis (the entire text is incorporated herein by reference, Paler AS et al.) This includes al., J Allergy Clin Immunol 139:152-165 (2017).

[0118] In some embodiments, compounds that may be evaluated using the disclosed rodents include, for example, but are not limited to, small molecule inhibitors, nucleoside inhibitors (e.g., siNRAs, ribozymes, antisense constructs, etc.), antigen-binding proteins (e.g., antibodies or their antigen-binding fragments), or candidate inhibitors of IL-36 signaling, such as blocking peptides / peptide inhibitors.

[0119] In some embodiments, the candidate inhibitor is an antibody or an antibody-conjugated fragment thereof. Both monoclonal and polyclonal antibodies are suitable for testing in the rodents disclosed herein. In some embodiments, the antibody specifically binds to the human IL-36R protein. In some embodiments, the antibody specifically binds to the IL1RL2 subunit of the human IL-36R protein.

[0120] Candidate compounds can be evaluated by inducing inflammation in rodents disclosed herein, for example, IMQ-induced dermatitis or DSS-induced enteritis, and determining whether the candidate compound can treat or inhibit the induced inflammation. The terms “treat” or “inhibit” include improvement of severity, mitigation of progression, elimination, delay or prevention of the onset of induced inflammation and symptoms, or a combination thereof.

[0121] In some embodiments, rodents are administered the candidate compound before, during, or after administration of an anti-inflammatory agent. The candidate compound can be administered via any desired route of administration, including parenteral and non-parenteral routes. Parenteral routes include, for example, intravenous, intra-arterial, intra-portal, intramuscular, subcutaneous, intraperitoneal, intrathecal, subarachnoid, lateral ventricle, intracranial, intrathoracic, or other routes of infusion. Non-injectable routes include, for example, oral, nasal, percutaneous, pulmonary, rectal, oral cavity, vaginal, and ocular. Administration may also be by continuous infusion, local administration, sustained release from an implant (gel, membrane, etc.), and / or intravenous injection.

[0122] In some embodiments, suitable control rodents may include, for example, humanized rodents not subjected to induced inflammation, humanized rodents subjected to induced inflammation without a compound or control compound not expected to have therapeutic efficacy (e.g., an isotype control antibody), and humanized rodents subjected to induced inflammation and compounds known to have therapeutic efficacy.

[0123] To evaluate the efficacy of candidate compounds against skin inflammation, the compounds may be administered to rodents before, during, or after IMQ treatment. In certain embodiments, the candidate compounds are administered subcutaneously in or near the skin area where IMQ is applied. A compound is considered effective if it inhibits skin inflammation compared to control rodents that do not receive the compound. For example, a compound is considered effective if the total pathology score decreases by 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more (e.g., 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 one or more of these factors. A reduction in the concentration of pro-inflammatory mediators of 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% or more (for example, 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%) is considered effective.

[0124] To evaluate the efficacy of candidate compounds against colitis, the compounds may be administered to rodents before, during, or after oxazolone treatment. In some embodiments, the candidate compounds are administered intraperitoneally for several days after the initiation of DSS treatment (e.g., 5, 6, 7, 8, or 9 days). In some embodiments, the candidate compounds are administered intraperitoneally multiple times during oxazolone treatment, for example, 2-3 days after topical application of oxazolone, and with one or more rectal administrations of oxazolone (e.g., with the first rectal administration of oxazolone and with the third rectal administration of oxazolone). The compounds are considered effective if they inhibit colitis compared to control DITRA rodents that do not receive the compound.For example, when the total pathology score decreases by 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% or more (e.g., 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 when the concentration of one or more pro-inflammatory mediators decreases by 15%, 20%, 30%, 40%, 50%, or 60%. When Lcn2 in feces decreases by 70%, 80%, 90% or more (for example, 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%); when Lcn2 in feces decreases by 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more (for example, 20%~90%, 20%~80%, 20%~70%, 20%~6%) When MPO activity in colon homogenate decreases by 0%, 20%~50%, 20%~40%, 30%~90%, 30%~80%, 30%~70%, 30%~60%, 30%~50%, or 30%~40%; when MPO activity in colon homogenate decreases by 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% or more (for example, 20%~90%, 20%~80%, 20%~70%, 20%~60%, 20%~50%, 20%~40%, 30%~90%, 30%~80%, 30%~70%) It is considered effective when there is a 30%-60%, 30%-50%, or 30%-40% decrease in colon length; when colon length is extended by 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% or more (for example, 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% shortening); or a combination thereof.

[0125] This specification is further illustrated by the following examples, which should not be construed as limiting. All cited references (including references to documents, published patents, and published patent applications cited throughout this application) are expressly incorporated herein by reference in their entirety. [Examples]

[0126] Example 1. Preparation of a quadruple humanized mouse line Two genetically modified mouse lines—the Il1rl2 single-humanized mouse line and the Il1f6, Il1f8, and Il1f9 triple-humanized mouse lines—were created using VelociGene® technology (the entirety of which is incorporated herein by reference; Poueymirou et al., Nat Biotechnol. 2007 Jan;25(1):91-9; Valenzuela et al., Nat Biotechnol 2003 Jun;21:652-59). A "quadriple-humanized" line was created by crossing the single-humanized and triple-humanized lines. Creation of a single humanized Il1rl2 mouse strain

[0127] The "single-humanized" line was created by substituting a portion of the mouse Il1rl2 gene (interleukin-1 receptor-like protein 2), which encodes the extracellular domain of mouse Il1rl2, with a fragment of the human IL1RL2 gene (Il1rl2 / IL1RL2 exons 3-8, each containing an intercellular intron and an adjacent intron) that encodes the corresponding extracellular domain of human IL1RL2 (Figure 1A). The resulting humanized gene encodes a chimeric receptor that maintains the intracellular signaling specificity of mice, while humanizing the extracellular domain to enable binding to IL1F6, IL1F8, and IL1F9 (also known as IL36A, B, and G), respectively. Homozygous humanized Il1rl2 mice are Il1rl2 hu / hu It is called that.

[0128] More specifically, we used the mouse bacterial artificial chromosome (BAC) clone RP23-235L22 containing the mouse Il1rl2 gene and modified it as follows to provide a target vector. The DNA fragment was constructed to contain a 5' mouse homologous nucleotide sequence, approximately 32,389 bp of human IL1RL2 genomic DNA (containing 3'346 bp of intron 2, exons 3-8 with all intervening introns, and 5'1101 bp of intron 8), approximately 4,996 bp of auto-deletion neomycin cassette, and a 3' mouse homologous sequence (Figure 1B). Using this DNA fragment, we modified the BAC clone RP23-235L22 via homologous recombination in bacterial cells. As a result, a mouse Il1rl2 genome fragment encoding the external domain in the BAC clone (approximately 25,324 bp, including 3' 155 bp of mouse intron 2, exons 3-8 with all intervening introns, and 5' 642 bp of mouse intron 8) was subsequently replaced with human IL1RL2 genomic DNA, followed by a self-deletion neomycin cassette (Figure 1B). The resulting modified BAC clone contained, from 5' to 3', (i) a 5' mouse homologous arm containing approximately 112.2 kb of mouse genomic DNA, including the 5' portion of mouse Il1rl2 exons 1-2 and intron 2; (ii) a human IL1RL2 genomic fragment containing the 3' portion of intron 2, exons 3-8, and the 5' portion of intron 8; (iii) a self-deleting neomycin cassette of approximately 4,996 bp, followed by (iv) a 31.3 kb 3' mouse homologous arm containing the remaining mouse Il1rl2 exon downstream of exon 8, all intervening introns, and the 3' UTR. See Figure 1B. The junction sequence is also shown at the bottom of Figure 1B.

[0129] Using the modified BAC clone containing the humanized Il1rl2 gene described above as a target vector, mouse F1H4 embryonic stem cells were electroporated (50% C57BL / 6NTac / 50% 129S6 / SvEvTac) to generate modified ES cells containing the humanized Il1rl2 gene. Clearly targeted ES cells containing the humanized Il1rl2 gene were identified by an assay detecting the presence of the human IL1RL2 sequence (Valenzuela et al., see above), confirming the loss and / or retention of the mouse Il1rl2 sequence. The primers and probes used to confirm the humanization described above are shown in Figure 5. Once correctly targeted ES cell clones are selected, the neomycin selection cassette can be removed. The humanized Il1rl2 locus after cassette removal is shown in Figure 1C, and the junction sequence is also shown in Figure 1C. [Table 5]

[0130] Selected ES cell clones were microinjected into 8-cell embryos derived from albino mice at Charles River Laboratories Swiss Webster to obtain 100% target cell-derived F0 VelociMice® (Poueymirou). (See et al. 2007, above). Mice possessing the humanization locus were re-confirmed and identified by genotyping of DNA isolated from tail fragments using an improved allele assay (Valenzuela et al., above) that detects the presence of human gene sequences. Homozygous animals for the humanization locus were generated by mating them with heterozygous animals.

[0131] The resulting amino acid sequence alignments 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) are provided in Figure 1D.

[0132] Creation of a triple-humanized mouse strain We created "triple-humanized" lines in which the complete coding sequences of the mouse Il1f6, Il1f8, and Il1f9 genes were replaced with the complete coding sequences of the human IL1F6, IL1F8, and IL1F9 genes, respectively. This strategy resulted in humanized genes encoding human ligands that can bind to the human extracellular domain of the chimeric Il1rl2 receptor. Homozygous humanized Il1f6, Il1f8, and Il1f9 mice were found to be Il1f6 hu / hu ,Il1f8 hu / hu ,Il1f9 hu / hu It is called that.

[0133] More specifically, the mouse bacterial artificial chromosome (BAC) clone RP23-90G23 containing the mouse Il1f8, Il1f9, and Il1f6 genes, along with intergenetic sequences, were modified as follows to provide a target vector. The DNA fragment was constructed to contain 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-deletion hygromycin cassette, and a 3' mouse homologous sequence (Figure 2B). Using this DNA fragment, the BAC clone RP23-90G23 was modified via homologous recombination in bacterial cells. As a result, approximately 76,548 bp of the genomic fragment in the BAC clone was replaced with human genomic DNA, followed by the self-deletion cassette (Figures 2A-2B). The resulting modified BAC clone contained, in the 5' to 3' direction, (i) a 5' mouse homologous arm containing approximately 5.7 kb of mouse genomic DNA, (ii) a human genome fragment of approximately 88,868 bp (containing promoters, untranslated regions, and coding sequences for human IL1F8, IL1F9, and IL1F6), (iii) an auto-deleted hygromycin cassette of approximately 5,218 bp, and a 3' mouse homologous sequence of approximately 29.7 bp. See Figure 2B. The junction sequence is also shown at the bottom of Figure 2B.

[0134] Using modified BAC clones containing the aforementioned human IL1F8, IL1F9, and IL1F6 gene sequences as target vectors, mouse F1H4 embryonic stem cells were electroporated (50% C57BL / 6NTac / 50% 129S6 / SvEvTac) to generate modified ES cells containing the human IL1F8, IL1F9, and IL1F6 gene sequences. Clearly targeted ES cells containing the human IL1F8, IL1F9, and IL1F6 gene sequences were identified by an assay detecting the presence of human sequences (Valenzuela et al., see above), confirming the loss and / or retention of mouse sequences. The primers and probes used to confirm the aforementioned humanization are shown in Figure 6. Once correctly targeted ES cell clones are selected, the hydromycin selection cassette can be removed. The humanization locus after cassette removal is shown in Figure 2C, and the junction sequence is also shown in Figure 2C. [Table 6]

[0135] Selected ES cell clones were microinjected into 8-cell embryos derived from albino mice at Charles River Laboratories Swiss Webster to obtain 100% target cell-derived F0 VelociMice® (Poueymirou). (See et al. 2007, above). Mice possessing the humanization locus were re-confirmed and identified by genotyping of DNA isolated from tail fragments using an improved allele assay (Valenzuela et al., above) that detects the presence of human gene sequences. Homozygous animals for the humanization locus were generated by mating them with heterozygous animals. Creation of a triple-humanized mouse strain

[0136] The "triple-humanized" line was created by crossing a single-humanized line, which was bred homozygously for both loci in a 100% C57BL / 6NTac background, with a triple-humanized line. The homozygous triple-humanized line was Il1rl2hu / hu Il1f6 hu / hu Il1f8 hu / hu Il1f9 hu / hu These mice are also referred to as "DITRA-like" mice. In the F0 germline, the Neo cassette and hygro cassette were removed by self-deletion technology.

[0137] Example 2. Characterization of DITRA mice material and method Acute and Chronic IMQ-Induced Dermatoinflammation Induction and Antibody Therapy in DITRA-like Mice - To induce dermatoinflammation, 8-10 week old humanized DITRA-like female mice were treated with mouse hair trimmers (Oster, MiniMax, Cat#) three days prior to the application of IMQ cream. The dorsal hair was shaved using 78049-100), and the skin was depilated with 0.5 g of Veet hair removal gel. A daily topical dose of 62.5 g of commercially available IMQ cream (5%) (Aldara, GM Health Care Limited, NDC 99207-206-12, lot # QJ044A) or Vaseline (CVS Pharmacy, NDC 59779-902-88) was applied to the shaved dorsal skin of mice for four consecutive days for acute disease induction and for nine days for chronic disease induction. The daily topical dose of Aldara 62.5 mg is equivalent to the daily dose of the active compound 3.125 mg. In acute IMQ-induced dermatitis, anti-human IL-36R antibody was subcutaneously administered to the dorsal skin at a dose of 10 mg / kg three days before and one day after the start of IMQ application. The control group received PBS and a 10 mg / kg hIgG4 isotype control injection. In acute IMQ-induced dermatitis, the same anti-human IL-36R antibody was therapeutically administered subcutaneously to the dorsal skin at a dose of 10 mg / kg four days before and eight days after the initiation of IMQ treatment. Two or three days after treatment, the dorsal skin of mice showed 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 (PASI). Erythema, desquamation, and thickening were independently scored on a scale of 0 to 4: 0, none, 1, mild, 2, moderate, 3, severe, and 4, very severe (the entire score is incorporated herein by reference, van der Fits et al., J Immunol 2009, 182:5836-5845). Skin thickness was measured using a caliper (Kaefer) on day 4 of acute IMQ-induced dermatitis and on day 11 of chronic IMQ-induced dermatitis.

[0138] Histopathology – 6 mm diameter skin tissue sections from the dorsal side of mice were fixed in 10% buffered formalin, and 4–5 μm paraffin-embedded sections were stained with hematoxylin and eosin. Skin sections were blindly evaluated for the presence of parakeratosis, normal keratosis, Munro microabscesses, acanthosis, cutaneous ulceration, inflammation of the dermis and subcutaneous tissue, vascular congestion of the dermis and subcutaneous tissue, keratosis pilaris, and epithelial hyperplasia. A scoring scale of 0–4 was used: 0-normal, 1-minimal, 2-mild, 3-moderate, and 4-severe (the entire scale is incorporated herein by reference, van der Fits et al., J Immunol 2009, 182:5836–5845). A total pathology score was calculated for each mouse by adding the individual histopathological feature scores. Data analysis was performed using GraphPad Prism® software.

[0139] Measurement of cytokines in skin homogenates - Full-thickness skin tissue with a diameter of 6 mm was collected from the back of mice and placed in 15 mL test tubes containing T-per buffer (Thermo Scientific, Cat#378510, lot#RF236217), 1× Halt Protease Inhibitor Cocktail (Thermo Scientific, Cat#87786, lot#QG221763), and 5 M EDTA solution (Thermo Scientific, Cat3 78429). The skin tissue was disrupted using a Polytron (PT10-35 GT-D, Cat#9158158) at 28000 rpm for 1 minute and placed on ice. The prepared skin homogenates were 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 the Bradford protein assay using a protein assay dye (BioRad, Cat# 500-0006, Lot# 210008149) to quantify the total protein content. Cytokine concentrations in skin homogenates were measured using the Proinflammatory Panel 1 (mouse) multiplex immunoassay kit (MesoScale Discovery, Cat# K15048D) according to the manufacturer's instructions. Briefly, 50 μL / well of calibrator and sample (diluted with Diluent 41) were added to a plate pre-coated with capture antibody and incubated at room temperature with shaking at 700 rpm for 2 hours. The plate was then washed three times with 1×PBS containing 0.05% (w / v) Tween-20, followed by the addition of 25 μL of detection antibody solution diluted with Diluent 45. After incubation at room temperature for 2 hours with shaking, the plates were washed three times, and 150 μL of 2× Read Buffer was added to each well. Electrochemiluminescence was immediately read using an MSD Spector® instrument. Data analysis was performed using GraphPad Prism® software. Cytokine levels were normalized against total protein content.

[0140] Induction of a DSS-induced model of chronic colitis and antibody treatment in DITRA-like mice - To induce DSS-mediated colitis, female DITRA-like mice aged 12–20 weeks with an average body weight of over 23g were given 1.5–3% DSS (Sigma-Aldrich Cat# 87786, lot# PJ203966B) in drinking water for 7 days, followed by 11–13 days of distilled water. A second cycle of DSS treatment (4 days), followed by water (5–6 days), was administered until day 27–30. The control group was given distilled water throughout the study period. Anti-human IL-36R antibody and mIL-12p40 (Bioxcell Cat# BE0051, clone C17.8) antibody were administered intraperitoneally at a dose of 10 mg / kg once a week starting from day 7. The control group was administered PBS and 10 mg / kg of the respective hIgG4 and ratIgG2a (Bioxcell Cat#BE0089, clone 2A3) isotype control injections. Mice were weighed daily and monitored for clinical signs of colitis (e.g., stool consistency and blood in stool). Mice were euthanized on days 27–30, and colon length was measured. To assess DSS colitis, the following features were scored: inflammation (severity and degree), epithelial changes (erosions / ulcers), crypt changes (crypt defects, cryptitis / crypt abscesses, regeneration / hyperplasia, goblet cell defects), submucosal edema, and the percentage of tissue area with pathology relative to the total tissue area on a slide. A scoring scale of 0–4 was used: 0–0 - within normal range, 1 - minimal, 2 - mild, 3 - moderate, 4 - severe. A total pathology score was calculated for each mouse by adding the scores of the individual histopathological features.

[0141] Measurement of Lcn-2 in fecal samples - To monitor intestinal inflammation through the test, feces from individual DITRA-like mice were collected weekly into 2 mL deep well plates and stored at -80 °C. At the end of the test, feces collected on different days were homogenized. Briefly, fecal samples were reconstituted with 1 mL of PBS containing 0.1% Tween-20, 1× Halt Protease Inhibitor Cocktail (Thermo Scientific, Cat# 87786, lot# QG221763), and 5 M EDTA solution (Thermo Scientific, Cat# 78429). After adding two tungsten 3 mm carbide beads to the well (Qiagen, Cat# 69997), the plate was placed on a shaker at the highest speed overnight at 4 °C. The homogeneous fecal suspension was centrifuged at 1200 rpm for 10 minutes at 4 °C, and the supernatant was collected into a 96-well plate. Fecal lipocalin-2 (Lcn2) levels were measured using the Mouse Duoset Lipocalin-2 / NGAL ELISA kit (R&D Systems, Cat# DY1857, lot# P116359) according to the manufacturer's instructions. Data analysis was performed using GraphPad Prism (trademark) software.

[0142] Measurement of myeloperoxidase (MPO) activity in colon homogenates - Fragments of the distal part of the colon were placed into 2 mL microcentrifuge tubes containing two tungsten 3 mm carbide beads (Qiagen, Cat# 69997) with T-per buffer (Thermo Scientific, Cat# 378510, lot# RF236217), 1× Halt Protease Inhibitor Cocktail (Thermo Scientific, Cat# 87786, lot# QG221763) and 5 M EDTA solution (Thermo Scientific, Cat# 78429). Using a Qiagen Tissue Lyser II, for 27.5 s -1Colon tissue was disrupted at a frequency of 10 minutes. The tubes were centrifuged at 1500 rpm for 8 minutes at 4°C, and the supernatant was collected in a 96-well plate. Colon homogenates were subjected to the Bradford protein assay using a protein assay dye (BioRad, Cat# 500-0006, Lot# 210008149) to quantify the total protein content. Myeloperoxidase (MPO) activity in colon homogenates was analyzed using a mouse MPO ELISA kit (HHycult Biotech, Cat# HK210-02, Lot# 210008149). Measurements were taken using 21022KO617-Y) according to the manufacturer's instructions. Data analysis was performed using GraphPad Prism® software. MPO levels were normalized relative to the total protein content.

[0143] Cytokine Measurement in Colon Homogenates - Cytokine concentrations in colon homogenates were measured using the Proinflammatory Panel 1 (mouse) multiplex immunoassay kit (MesoScale Discovery, Cat# K15048D) according to the manufacturer's instructions. Briefly, 50 μL / well of calibrator and sample (diluted with Diluent 41) were added to a plate pre-coated with capture antibody and incubated at room temperature for 2 hours with shaking at 700 rpm. The plate was then washed three times with 1×PBS containing 0.05% (w / v) Tween-20, followed by the addition of 25 μL of detection antibody solution diluted with Diluent 45. After incubation at room temperature for 2 hours with shaking, the plate was washed three times, and 150 μL of 2× Read Buffer was added to each well. Electrochemiluminescence was immediately read using an MSD Spector® instrument. Data analysis was performed using GraphPad. The analysis was performed using Prism™ software. Cytokine levels were normalized relative to total protein content.

[0144] Statistical analysis - Tukey's multiple comparison post-hoc test (*p<0.05, **p<0.005, ***p<0.0005, ****p<0.0001) was used to determine intragroup statistical significance using a one-way ANOVA method (the same method was used for both *-statistical significance from the PBS treatment group and #-statistical significance from the corresponding isotype treatment group).

[0145] result Humanized DITRA-like mice exhibit enhanced skin inflammation in an IMQ-induced model of psoriasis.

[0146] To investigate the role of dysregulated IL-36R signaling similar to that seen in GPP or DITRA ("Interleukin 36 Receptor Antagonist Deficiency") patients, mice were constructed to possess humanized IL-36R (e.g., Il1rl2) and human IL-36α,β,γ ligands, but lacking IL-36Ra antagonists, as described in Example 1. The resulting hIL-36R / hIL-36α,β,γ mice were designated as DITRA-like mice due to a 1 / 20 reduction in the affinity of mouse IL36Ra to human IL-36R, resulting in enhanced IL-36R signaling similar to that seen in DITRA patients (for a more detailed study of DITRA patients, see, for example, Marrakchi et al., N Engl J Med 2011, 365:620-628, the entire work of which is incorporated herein by reference).

[0147] Under no-load conditions, DITRA-like mice did not develop any spontaneous disease. In contrast, in a preclinical model of IMQ-induced psoriasis-like dermatitis that closely resembled human psoriatic lesions in terms of phenotypic and histological features (for a more detailed discussion of the characteristics of psoriasis-like dermatitis, see van der Fits et al., J Immunol 2009, 182:5836-5845; Swindell et al., PLoS One 2011, 6:e18266, which are incorporated herein by reference in their entirety), DITRA-like mice exhibited enhanced skin inflammation compared to their wild littermates (Figures 3A-3B). Briefly, IMQ was applied daily to shaved dorsal skin of DITRA-like and wild-type mice for four consecutive days. On day 5, skin was collected for subsequent histopathological evaluation and protein and RNA isolation. IMQ-treated DITRA-like mice developed more severe psoriatic lesions, including desquamation, erythema, and skin thickening, compared to wild-caught littermates (Figure 3A). Consistent with the clinical features, histopathological evaluation of the skin revealed more enhanced acanthosis, parakeratosis, and disruption of keratinocyte differentiation in DITRA-like mice (Figure 3B). IMQ-treated DITRA-like mice also exhibited a significant increase in the number of Munro abscesses or pustules, similar to GPP patients. Furthermore, IMQ application resulted in increased levels of disregulated pro-inflammatory molecules in the skin of DITRA-like mice compared to wild-caught littermates (e.g., pro-inflammatory molecules including IL-17a, IL-17f, IL-23a, S100A8, and Defb4) in psoriasis (Figure 3C). Furthermore, while 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 to Vaseline-treated DITRA-like mice and Vaseline-treated wild-type mice, the protein levels of IL-36α and IL-36β cytokines were detected at twice the concentration in the inflamed skin of DITRA-like mice compared to the inflamed skin of wild-type mice.

[0148] Humanized DITRA-like mice exhibit impaired mucosal healing in a DSS-induced chronic colitis model. Recent studies in a subset of patients have suggested the potential contribution of dysregulated IL-36 axis expression and its role in intestinal inflammation in IBD. IL-36α and IL-36β expression have been shown to be elevated in the inflamed mucosa of ulcerative colitis patients (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 preclinical models, IL-36R deficiency protects against DSS-induced and oxazolone-induced colitis (see 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). At steady state, DITRA-like mice did not develop spontaneous ileitis / colitis but exhibited a significantly shortened colon at young age (3 and 10 months) (Figures 4A and 4B). Furthermore, compared to steady-state and wild-type mice, DITRA-like mice showed elevated levels of IL-36 cytokines, as well as a trend toward elevated levels of IL-17F and IL-17A, and decreased levels of IL-21 in the colon.

[0149] To evaluate the role of the IL-36 axis in intestinal inflammation, we used a chemically induced intestinal injury model by oral administration of DSS, which damages the colonic epithelium (the entirety of which is incorporated herein by reference, Okaasu et al., Gastroenterology 1990, 98:694-702, herein incorporated by reference in its entirety) and triggers a potent inflammatory response (Rakoff-Nahoum et al., Cell 2004, 118:229-241), particularly in ulcerative colitis, which exhibits the major features of IBD. DITRA-like mice and their co-enclosed wild littermates were subjected to a DSS-induced chronic colitis regimen consisting of 7 days of 2.5% DSS followed by 11 days of water (first dose) and 4 days of 1.5% DSS followed by 5 days of water (second dose). During the acute phase of the disease, DITRA-like mice developed enteritis similar to that of co-carried wild-type littermates (Figure 5A). Interestingly, during the recovery phase of the disease, DITRA-like mice showed irreversible damage from DSS-induced mucosal injury, reflected in persistent weight loss (Figure 5A), significant shortening of colon length (Figure 5B), a more severe pathology score (associated with more severe ulceration, extensive epithelial erosion, and neutrophil infiltration) (Figure 5C), significant increases in fecal lipocalin-2 (Lcn2) levels and myeloperoxidase activity (Figure 5D), upregulation of pro-inflammatory cytokines such as KC-GRO, TNF-α, and IL-6, and decreased IL-22 levels in the colon (Figure 5E). Furthermore, DITRA-like mice exhibited a mortality rate of approximately 62.5% (data not shown). In contrast, wild-type mice recovered during the recovery phase of colitis, with a mortality rate of 25%. Furthermore, the integrity of the intestinal epithelium was investigated by orally administering fluorescein isothiocyanate (FITC)-dextran to DITRA-like mice and wild-type mice. A significant increase in FITC-dextran levels was observed in the serum of DITRA-like mice on day 14 of DSS treatment, suggesting that the IL-36 pathway is involved in regulating intestinal permeability during intestinal injury.Therefore, the enhancement of IL-36 signaling in DITRA-like mice leads to exacerbation of intestinal inflammation and defects in mucosal repair, suggesting a role of IL-36 in regulating mechanisms involved in intestinal tissue remodeling.

[0150] Anti-human IL-36R monoclonal antibody inhibits acute skin inflammation in DITRA-like mice when administered prophylactically. To investigate the role of IL-36R in skin inflammation, anti-human IL-36R monoclonal antibody was tested in an IMQ-induced model of psoriasis-like dermatitis. IMQ was applied daily to shaved dorsal skin of DITRA-like mice for four consecutive days. Anti-human IL-36R monoclonal antibody was administered at 10 mg / kg three days before (-3d) and one day after (d1) the initiation of IMQ application. The control group received PBS and hIgG4 isotype control injections at 10 mg / kg. On day 5, skin was collected for subsequent histopathological evaluation and protein isolation. Anti-human IL-36R monoclonal antibody significantly reduced the IMQ-induced total pathology score, including parakeratosis and Munro microabscesses, compared to the PBS-treated and isotype control-treated groups (Figure 6A). Furthermore, human IL-36R blockade 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 disregulated pro-inflammatory cytokines in psoriasis (e.g., IL-12p40, IL-17f, and IL-17a) (Figure 6B), suggesting tightly controlled interactions between these cytokine pathways. The observed efficacy of anti-human IL-36R monoclonal antibodies in suppressing acute IMQ-induced skin inflammation was dose-dependent, with a dose of 10 mg / kg resulting in a more potent inhibition of skin inflammation, determined based on skin homogenate pathology scores, skin thickness, and pro-inflammatory cytokine levels, compared to a dose of 1 mg / kg (data not shown).

[0151] Anti-human IL-36R monoclonal antibodies inhibit chronic skin inflammation with therapeutic administration. To further investigate the therapeutic efficacy of human IL-36R antagonistism in vivo, the same anti-human IL-36R monoclonal antibodies were tested in a chronic IMQ-induced skin inflammation model. For two weeks, IMQ was applied to the shaved dorsal skin of DITRA-like mice in two nine-day increments, with a two-day untreated period in between. Anti-human IL-36R monoclonal antibodies were subcutaneously administered at 10 mg / kg on days 5 and 9 after the start of IMQ application. The control group received PBS and hIgG4 isotype control injections at 10 mg / kg. On day 12, skin thickness was measured and tissue was collected for subsequent histopathological evaluation and protein isolation. Similar to acute IMQ-induced inflammation, long-term IMQ application resulted in upregulation of pro-inflammatory mediators in the skin of DITRA-like mice (data not shown). Anti-human IL-36R monoclonal antibodies demonstrated significant efficacy in reducing IMQ-induced skin thickness and pathological lesion scores in DITRA-like mice (Figures 7A and 7B, respectively). Furthermore, administration of anti-human IL-36R monoclonal antibodies resulted in significant inhibition of IMQ-induced production of pro-inflammatory cytokines in the skin of DITRA-like mice (Figure 7C).

[0152] Overall, the data demonstrated the prophylactic and therapeutic efficacy of anti-human IL-36R antibodies in improving acute and chronic IMQ-induced skin inflammation in vivo.

[0153] Anti-human IL-36R monoclonal antibodies improve DSS-induced chronic colitis in DITRA-like mice when administered therapeutically. Further investigation was conducted to determine if they rescue phenotypes in DITRA-like mice that exhibited mucosal healing defects as a result of enhanced IL-36 signaling and in which IL-36 blockade was observed. DITRA-like mice were subjected to chronic DSS-induced colitis by administering 3% DSS for 7 days, followed by water for 13 days, in two cycles. The same anti-human IL-36R monoclonal antibody and anti-mIL-12p40 monoclonal antibody (a mouse surrogate of ustekinumab, approved for the treatment of Crohn's disease) were administered every other week at 10 mg / kg starting on day 7. The control group received PBS and corresponding hIgG4 and rat IgG2a isotype control injections at 10 mg / kg. Treatment with an anti-human IL-36R monoclonal antibody rescued DITRA-like mice from DSS-induced mucosal injury and reduced disease severity in DITRA-like mice compared to PBS and isotype control treatments (Figure 8A). To monitor enteritis at different stages of the disease, individual mouse feces were collected weekly, and fecal lipocalin-2 (Lcn2) protein, a non-invasive biomarker of inflammation in enteritis, was measured (e.g., Thorsvik et al., J Gastroenterol Hepatol 2017, 32:128-135, the entire text of which is incorporated herein by reference). As shown in Figure 8B, the PBS-treated, hIgG4-treated, and rat IgG2a-treated groups showed significant upregulation of fecal Lcn2 levels at days 17, 23, and 30 compared to the water-only group. Surprisingly, administration of anti-human IL-36R monoclonal antibody resulted in a significant reduction in Lcn2 levels compared to the PBS-treated and isotype-treated groups. Sustained reductions in fecal Lcn2 levels were observed in the anti-human IL-36 antibody-treated group on days 17, 23, and 30 (Figure 8B).

[0154] Furthermore, hIL-36R blockade using an anti-human IL-36R monoclonal antibody resulted in colon lengthening (Figure 8C), decreased myeloperoxidase (MPO) activity (Figure 8D), and a 61-95% reduction in pro-inflammatory cytokines (Figure 8E) in the colon of DSS-treated DITRA-like mice. Anti-human IL-36R monoclonal showed comparable efficacy to IL-12p40 blockade in improving DSS-induced chronic colitis, and similarly reflected reductions in fecal Lcn2, MPO activity, and pro-inflammatory cytokines in the colon of DITRA-like mice (Figures 8A-8E).

[0155] The efficacy of IL-36R blockade in oxazolone-induced colitis was tested in oxazolone-induced colitis, another preclinical model of IBD histologically similar to human ulcerative colitis (the whole study is cited by reference, Heller et al., Immunity 17, 629-638 (2002)). Prophylactic administration of anti-human IL-36R antibody significantly reduced the severity of oxazolone-induced disease in DITRA-like mice compared to PBS and isotype control treatment groups, as reflected in a decrease in mean body weight and mean colon length (Figure 9A-9B). Furthermore, IL-36R antagonistism resulted in a significant decrease in IL-4 and TNF-α levels in the colon of oxazolone-treated DITRA-like mice (Figure 9C).

[0156] Various publications, including patents, patent applications, published patent applications, accession numbers, technical papers, and academic papers, are referenced herein. Each of these referenced publications is incorporated herein by reference in whole for all purposes.

Claims

1. A rodent embryo containing genetically modified rodent embryonic stem (ES) cells, wherein the rodent ES cells are (1) A humanized Il1rl2 gene located at the endogenous rodent Il1rl2 locus, wherein the humanized Il1rl2 gene encodes a humanized Il1rl2 protein having an external domain having an amino acid sequence that is at least 95% identical to the external domain of the human IL1RL2 protein, and the human IL1RL2 protein contains the amino acid sequence described in Sequence ID No. 2; (2) The human IL1F6 gene located at the endogenous rodent IL1f6 locus; (3) The human IL1F8 gene located at the endogenous rodent Il1f8 locus; and (4) The human IL1F9 gene located at the endogenous rodent IL1f9 locus. A rodent embryo comprising, wherein the rodent is a mouse or a rat, and the rodent embryo produces a genetically modified rodent that exhibits disregulated IL-36 signaling.

2. The rodent embryo according to claim 1, wherein the humanized Il1rl2 protein includes a transmembrane cytoplasmic sequence that is at least 95% identical to the transmembrane cytoplasmic sequence of the endogenous rodent Il1rl2 protein.

3. The rodent embryo according to claim 1, wherein the humanized Il1rl2 protein comprises the amino acid sequence described in Sequence ID No.

7.

4. A rodent embryo according to any one of claims 1 to 3, wherein the humanized Il1rl2 gene is operably bound to the endogenous rodent Il1rl2 promoter at the endogenous rodent Il1rl2 locus.

5. A rodent embryo according to any one of claims 1 to 4, wherein the humanized Il1rl2 gene is produced by the substitution of the nucleotide sequence of the human IL1RL2 gene in a genomic fragment of the endogenous rodent Il1rl2 gene at the endogenous rodent Il1rl2 locus.

6. The rodent embryo according to claim 5, wherein the nucleotide sequence of the human IL1RL2 gene includes exons 3 to 8 of the human IL1RL2 gene.

7. The rodent embryo according to claim 6, wherein the humanized Il1rl2 gene includes exons 1-2 of the endogenous rodent Il1rl2 gene, exons 3-8 of the human IL1RL2 gene, and an exon downstream of exon 8 of the endogenous rodent Il1rl2 gene.

8. A rodent embryo according to any one of claims 1 to 7, wherein the human IL1F6 gene replaces the endogenous rodent Il1f6 gene at the endogenous rodent Il1f6 locus, the human IL1F8 gene replaces the endogenous rodent Il1f8 gene at the endogenous rodent Il1f8 locus, and the human IL1F9 gene replaces the endogenous rodent Il1f9 gene at the endogenous rodent Il1f9 locus.

9. The rodent embryo according to any one of claims 1 to 8, wherein the rodent is homozygous for each of the humanized Il1rl2 gene, human IL1F6 gene, human IL1F8 gene, and human IL1F9 gene.

10. A method for determining whether a compound can inhibit inflammation, wherein the method is a) Inducing enteritis in genetically modified rodents by administering DSS or oxazolone, wherein the genetically modified rodents have the following genome: (1) A humanized Il1rl2 gene located at the endogenous rodent Il1rl2 locus, wherein the humanized Il1rl2 gene encodes a humanized Il1rl2 protein having an external domain having an amino acid sequence that is at least 95% identical to the external domain of the human IL1RL2 protein, and the human IL1RL2 protein contains the amino acid sequence described in Sequence ID No. 2; (2) The human IL1F6 gene located at the endogenous rodent IL1f6 locus; (3) The human IL1F8 gene located at the endogenous rodent Il1f8 locus; and (4) The human IL1F9 gene located at the endogenous rodent IL1f9 locus; The genetically modified rodent is a mouse or a rat; the genetically modified rodent exhibits disregulated IL-36 signaling; b) Administering the compound to the rodents; c) Evaluating the rodents, including measuring the length of the colon in the rodents; d) If the colon length of the rodent increases compared to a control rodent containing the same genetic modification but without the compound, it is determined that the compound inhibits inflammation. Methods that include...

11. Further evaluation of the above Determining the pathological score of the aforementioned intestine, Measuring the level of myeloperoxidase (MPO) activity in colon homogenates, Measuring the levels of inflammatory cytokines in colon homogenates, To measure the level of lipocalin-2 (Lcn2) in fecal samples. The method according to claim 10, comprising one or more of the above.

12. The method according to claim 10 or 11, wherein the humanized Il1rl2 protein comprises a transmembrane cytoplasmic sequence that is at least 95% identical to the transmembrane cytoplasmic sequence of the endogenous rodent Il1rl2 protein.

13. The method according to claim 12, wherein the humanized Il1rl2 protein comprises the amino acid sequence described in SEQ ID NO:

7.

14. The method according to claim 12 or 13, wherein the humanized Il1rl2 gene is operably bound to the endogenous rodent Il1rl2 promoter at the endogenous rodent Il1rl2 locus.

15. The method according to any one of claims 12 to 14, wherein the humanized Il1rl2 gene is produced by the substitution of the nucleotide sequence of the human IL1RL2 gene in a genomic fragment of the endogenous rodent Il1rl2 gene at the endogenous rodent Il1rl2 locus.

16. The method according to claim 15, wherein the nucleotide sequence of the human IL1RL2 gene comprises exons 3 to 8 of the human IL1RL2 gene.

17. The method according to claim 16, wherein the humanized Il1rl2 gene includes exons 1-2 of the endogenous rodent Il1rl2 gene, exons 3-8 of the human IL1RL2 gene, and an exon downstream of exon 8 of the endogenous rodent Il1rl2 gene.

18. The method according to any one of claims 12 to 17, wherein the human IL1F6 gene replaces the endogenous rodent Il1f6 gene at the endogenous rodent Il1f6 locus, the human IL1F8 gene replaces the endogenous rodent Il1f8 gene at the endogenous rodent Il1f8 locus, and the human IL1F9 gene replaces the endogenous rodent Il1f9 gene at the endogenous rodent Il1f9 locus.

19. The method according to any one of claims 12 to 18, wherein the rodent is homozygous for each of the humanized Il1rl2 gene, human IL1F6 gene, human IL1F8 gene, and human IL1F9 gene.

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