Non-human animal models of DITRA and uses thereof

Abstract

The present disclosure relates to genetically modified rodents and rodent models of human diseases. More specifically, the present disclosure relates to the genome of a humanized Il1rl2 gene (encoding the IL1rl2 subunit of the IL-36R protein), as well as human IL-36 and ligand genes. The genetically modified rodents disclosed herein exhibit enhanced skin and intestinal inflammation as preclinical models of psoriasis and IBD, respectively, and serve as rodent models of human DITRA disease. [Selection diagram] None

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit 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 which are incorporated herein by reference.

[0002] The present disclosure relates to genetically modified rodents and rodent models of human disease.More specifically, the present disclosure relates to a genetically modified rodent whose genome comprises humanized Il1rl2 gene (encoding the IL1rl2 subunit of IL-36R) and human IL-36α, β, and γ ligand genes.The genetically modified rodent disclosed herein shows enhanced skin and intestinal inflammation in preclinical models of psoriasis and inflammatory bowel disease (IBD), respectively, and can serve as a rodent model of human IL-36 receptor inhibitor deficiency (DITRA).

[0003] INCORPORATION BY REFERENCE The sequence listing, a 65 KB ASCII text file entitled 35950_10404US01_SequenceListing.txt, created on July 9, 2019 and submitted to the U.S. 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, previously designated IL1F6, IL1F8, and IL1F9, respectively) and one antagonist (IL36Ra) that signal through a heterodimeric receptor (IL-36R) leading to activation of nuclear factor kappa B (NF-κB) and mitogen-activated protein kinases (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 predominantly expressed in squamous epithelial tissues, particularly in psoriatic skin lesions. The link between IL-36R and epithelial-mediated disease 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 known as human IL-36 receptor inhibitor deficiency (IL-36R). d eficiency of the I Interleukin- 3 6 R a It is understood to be a species of GPP called Diablos inflammatory antagonist (abbreviated as "DITRA"). 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. Summary of the Invention

[0005] Disclosed herein are some embodiments of rodents genetically modified to include a humanized Il1rl2 gene encoding an Il1rl2 protein having an ectodomain substantially identical to the human IL1RL2 ectodomain, and 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 a humanized Il1rl2 gene and express three human agonist ligand genes while retaining endogenous IL-36Ra antagonists, which are approximately 20-fold less potent 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 in which mutations in IL-36Ra result in enhanced IL-36R signaling. Accordingly, provided herein are genetically modified rodents, compositions and methods for making such rodents, and methods for using such rodents as models for screening and developing therapeutic agents.

[0006] In some embodiments, disclosed herein is a genetically modified rodent whose genome comprises: (1) a humanized Il1rl2 gene at the endogenous rodent Il1rl2 locus, encoding a humanized Il1rl2 protein comprising an ectodomain substantially identical to an ectodomain of a 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 the rodent encodes a humanized Il1rl2 protein that includes a transmembrane cytoplasmic sequence substantially identical to the transmembrane cytoplasmic sequence of an endogenous rodent Il1rl2 protein. In some embodiments, the humanized Il1rl2 gene in the rodent encodes a humanized Il1rl2 protein that includes a signal peptide substantially identical to the signal peptide of an endogenous rodent Il1rl2 protein.

[0008] In some embodiments, the humanized Il1rl2 gene in the rodent encodes a humanized Il1rl2 protein comprising an ectodomain substantially identical to an ectodomain of a human IL1RL2 protein, wherein the human IL1RL2 protein comprises the amino acid sequence set forth in SEQ ID NO:2.

[0009] In some embodiments, the humanized Il1rl2 gene in the rodent encodes a humanized Il1rl2 protein that includes an ectodomain, the ectodomain comprising amino acids 22-337 set forth in SEQ ID NO:7.

[0010] In some embodiments, the humanized Il1rl2 gene in a rodent encodes a humanized Il1rl2 protein comprising the amino acid sequence set forth in SEQ ID NO:7.

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

[0012] In some embodiments, a humanized Il1rl2 gene in a rodent is generated by replacing a genomic fragment of an endogenous rodent Il1rl2 gene at the endogenous rodent Il1rl2 locus with the nucleotide sequence of a 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 ectodomain of the human IL1RL2 protein. In some embodiments, the genomic fragment of the human IL1RL2 gene comprises exons 3-8 of the human IL1RL2 gene.

[0014] In some embodiments, the genomic sequence of the endogenous rodent Il1rl2 gene remaining after the humanization replacement comprises exons downstream of exon 8 of the endogenous rodent Il1rl2 gene. In some embodiments, the genomic sequence of the endogenous rodent Il1rl2 gene remaining after the humanization replacement comprises exons 1-2 of the endogenous rodent Il1rl2 gene.

[0015] In certain embodiments, the humanized Il1rl2 gene in a rodent disclosed herein comprises exons 1-2 of the endogenous rodent Il1rl2 gene, exons 3-8 of the human IL1RL2 gene, and the remaining exons downstream of exon 8 of the endogenous rodent Il1rl2 gene.

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

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

[0018] In some embodiments, the human IL1F9 gene in a rodent 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 and / or human genes. In some embodiments, the rodents disclosed herein are homozygous for each of the four humanized and / or human genes.

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

[0021] In some embodiments, the rodent disclosed herein is a mouse or a rat.

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

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

[0024] In some embodiments, the rodent genome is modified by a process comprising: (i) generating a first rodent that comprises a humanized Il1rl2 gene at the endogenous rodent Il1rl2 locus; (ii) generating a second rodent that comprises 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 produced by providing rodent embryonic stem (ES) cells, inserting a nucleotide sequence of a human IL1RL2 gene at 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 producing a rodent using the rodent ES cells comprising the humanized Il1rl2 gene.

[0026] In some embodiments, the nucleotide sequence of the human IL1RL2 gene that is inserted 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 ectodomain of the human IL1RL2 protein. In some embodiments, the genomic fragment of the human IL1RL2 gene comprises exons 3-8 of the human IL1RL2 gene. In some embodiments, the genomic sequence of the endogenous rodent Il1rl2 gene that remains after the replacement comprises an exon downstream of exon 8 of the endogenous rodent Il1rl2 gene. In some embodiments, the genomic sequence of the endogenous rodent Il1rl2 gene that remains after the humanization replacement comprises exons 1-2 of the endogenous rodent Il1rl2 gene. In some embodiments, the humanized Il1rl2 gene formed as a result of the humanizing replacement comprises exons 1 to 2 of the endogenous rodent Il1rl2 gene, exons 3 to 8 of the human IL1RL2 gene, and the remaining exons downstream of exon 8 of the endogenous rodent Il1rl2 gene.

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

[0028] In some embodiments, the human IL1F6, IL1F8, and IL1F9 genes are provided in a contiguous 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 replaces 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 another aspect of some embodiments, disclosed herein is a rodent embryonic stem (ES) cell, comprising: (1) a humanized Il1rl2 gene at endogenous rodent Il1rl2 locus, encoding a humanized Il1rl2 protein comprising an ectodomain substantially identical to that of human IL1RL2 protein; (2) a human IL1F6 gene at endogenous rodent Il1f6 locus; (3) a human IL1F8 gene at endogenous rodent Il1f8 locus; and (4) a human IL1F9 gene at endogenous rodent Il1f9 locus.In some embodiments, such ES cell can be produced by the method disclosed herein.In some embodiments, the use of such ES cell to produce rodent is also disclosed.

[0033] In yet another aspect of some embodiments, disclosed herein are targeting nucleic acid constructs useful for modifying a rodent genome (e.g., rodent ES cells) to generate modified rodents.

[0034] In some embodiments, disclosed herein is a target nucleic acid construct comprising a nucleotide sequence of a human IL1RL2 gene flanked by 5' and 3' rodent nucleotide sequences capable of homologous recombination and integration of the nucleotide sequence of the human IL1RL2 gene into a rodent Il1rl2 locus to form a humanized Il1rl2 gene, wherein the humanized Il1rl2 gene encodes a humanized Il1rl2 protein comprising an ectodomain that is substantially identical to the ectodomain of the human IL1RL2 protein.

[0035] In some embodiments, disclosed herein is a targeted nucleic acid construct comprising a contiguous nucleic acid sequence comprising a human IL1F6 gene, a human IL1F8 gene, and a human IL1F9 gene, wherein the contiguous nucleic acid sequence is flanked by 5' and 3' rodent nucleotide sequences capable of mediating homologous recombination and integration of the contiguous nucleic acid sequence into a rodent locus encompassing a rodent Il1f6 gene, a rodent Il1f8 gene, and a rodent Il1f9.

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

[0037] Moreover, in a further aspect of some embodiments, a method for evaluating the therapeutic efficacy of a candidate compound for treating a disease associated with deregulated IL-36 signaling is disclosed herein, comprising administering an agent disclosed herein to a rodent to induce inflammation, administering a candidate compound to the rodent, and determining whether the candidate compound inhibits and / or reduces the induced inflammation.

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

[0039] In some embodiments, the candidate compound is administered to the rodent before, during, or after administration of an agent that induces inflammation. In some embodiments, the candidate compound 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 particular embodiments, for example, the following items are provided: (Item 1) 1. A genetically modified rodent, the genome of which comprises: (1) a humanized Il1rl2 gene at an endogenous rodent Il1rl2 locus, the humanized Il1rl2 gene encoding a humanized Il1rl2 protein comprising an ectodomain substantially identical to an ectodomain of a human IL1RL2 protein; (2) the human IL1F6 gene at the endogenous rodent Il1f6 locus; and (3) the human IL1F8 gene at the endogenous rodent Il1f8 locus; and (4) A genetically modified rodent comprising a human IL1F9 gene at the endogenous rodent Il1f9 locus. (Item 2) 2. The genetically modified rodent of item 1, wherein the humanized Il1rl2 protein comprises a transmembrane cytoplasmic sequence substantially identical to the transmembrane cytoplasmic sequence of the endogenous rodent Il1rl2 protein. (Item 3) 3. The genetically modified rodent of item 1 or 2, wherein the humanized Il1rl2 protein comprises a signal peptide substantially identical to the signal peptide of the endogenous rodent Il1rl2 protein. (Item 4) 4. The genetically modified rodent according to any one of items 1 to 3, wherein the human IL1RL2 protein comprises the amino acid sequence set forth in SEQ ID NO:2. (Item 5) 4. The genetically modified rodent according to any one of items 1 to 3, wherein the ectodomain of the humanized Il1rl2 protein comprises amino acids 22 to 337 as set forth in SEQ ID NO:7. (Item 6) 4. The genetically modified rodent according to any one of items 1 to 3, wherein the humanized Il1rl2 protein comprises the amino acid sequence set forth in SEQ ID NO:7. (Item 7) 7. The genetically modified rodent of any one of items 1 to 6, wherein the humanized Il1rl2 gene is operably linked to the endogenous rodent Il1rl2 promoter at the endogenous rodent Il1rl2 locus. (Item 8) 8. The genetically modified rodent of any one of items 1 to 7, wherein the humanized Il1rl2 gene results from replacing a genomic fragment of the endogenous rodent Il1rl2 gene at the endogenous rodent Il1rl2 locus with a nucleotide sequence of a human IL1RL2 gene. (Item 9) 9. The genetically modified rodent of item 8, wherein the nucleotide sequence of the human IL1RL2 gene is a genomic fragment of the human IL1RL2 gene that substantially encodes the ectodomain of the human IL1RL2 protein. (Item 10) 10. The genetically modified rodent according to item 9, wherein the genomic fragment of the human IL1RL2 gene comprises exons 3 to 8 of the human IL1RL2 gene. (Item 11) 11. The genetically modified rodent according to any one of items 8 to 10, wherein the genomic sequence of the endogenous rodent Il1rl2 gene remaining after the replacement comprises the exon downstream of exon 8 of the endogenous rodent Il1rl2 gene. (Item 12) 12. The genetically modified rodent of any one of items 8 to 11, wherein the genomic sequence of the endogenous rodent Il1rl2 gene remaining after the replacement comprises exons 1 to 2 of the endogenous rodent Il1rl2 gene. (Item 13) 9. The genetically modified rodent of item 8, wherein the humanized Il1rl2 gene comprises exons 1 to 2 of the endogenous rodent Il1rl2 gene, exons 3 to 8 of the human IL1RL2 gene, and the remaining exons downstream of exon 8 of the endogenous rodent Il1rl2 gene. (Item 14) 14. The 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) 15. The 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) 16. The 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) 17. The genetically modified rodent according to any of items 1 to 16, wherein the rodent is homozygous for each of the humanized Il1rl2 gene, the human IL1F6 gene, the human IL1F8 gene and the human IL1F9 gene. (Item 18) 21. The genetically modified rodent of item 20, wherein the rodent exhibits a shortened colon compared to a wild-type rodent. (Item 19) 19. The genetically modified rodent according to any one of items 1 to 18, wherein the rodent is a mouse or a rat. (Item 20) 1. A method for producing a genetically modified rodent, comprising: The rodent genome, (1) a humanized Il1rl2 gene at an endogenous rodent Il1rl2 locus, the humanized Il1rl2 gene encoding a humanized Il1rl2 protein comprising an ectodomain substantially identical to an ectodomain of a human IL1RL2 protein; (2) the human IL1F6 gene at the endogenous rodent Il1f6 locus; and (3) the human IL1F8 gene at the endogenous rodent Il1f8 locus; and (4) a human IL1F9 gene at the endogenous rodent Il1f9 locus; and generating a rodent comprising said modified genome. (Item 21) The rodent genome comprises: (i) generating a first rodent comprising the humanized Il1rl2 gene at the endogenous rodent Il1rl2 locus; (ii) generating a second rodent comprising 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) mating the first rodent with the second rodent to obtain the modified rodent genome. (Item 22) said rodent comprising said humanized Il1rl2 gene at said endogenous rodent Il1rl2 locus, Providing rodent embryonic stem (ES) cells; inserting a nucleotide sequence of a human IL1RL2 gene into the rodent Il1rl2 locus of the rodent ES cell to form the humanized Il1rl2 gene at the rodent Il1rl2 locus, thereby obtaining a rodent ES cell comprising the humanized Il1rl2 gene; and generating a rodent using the rodent ES cells containing the humanized Il1rl2 gene. (Item 23) 23. The method of claim 22, wherein the nucleotide sequence of the human IL1RL2 gene replaces the genomic fragment of the rodent Il1rl2 gene at the rodent Il1rl2 locus. (Item 24) 24. The method of claim 23, wherein the nucleotide sequence of the human IL1RL2 gene is a genomic fragment of the human IL1RL2 gene that substantially encodes the ectodomain of the human IL1RL2 protein. (Item 25) 25. The method of claim 24, wherein the genomic fragment of the human IL1RL2 gene comprises exons 3 to 8 of the human IL1RL2 gene. (Item 26) 26. The method of any one of items 23 to 25, wherein the genomic sequence of the endogenous rodent Il1rl2 gene remaining after the replacement comprises the exon downstream of exon 8 of the endogenous rodent Il1rl2 gene. (Item 27) 27. The method of any one of items 23 to 26, wherein the genomic sequence of the endogenous rodent Il1rl2 gene remaining after the replacement comprises exons 1 to 2 of the endogenous rodent Il1rl2 gene. (Item 28) 23. The method of claim 22, wherein the humanized Il1rl2 gene comprises exons 1 to 2 of the endogenous rodent Il1rl2 gene, exons 3 to 8 of the human IL1RL2 gene, and the remaining exons downstream of exon 8 of the endogenous rodent Il1rl2 gene. (Item 29) the second rodent comprising the human IL1F6 gene, the human IL1F8 gene, and the human IL1F9 gene; Providing rodent embryonic stem (ES) cells; (i) inserting the human IL1F6 gene into the rodent IL1F6 locus of the rodent ES cell; (ii) the human IL1F8 gene 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; and thereby obtaining rodent ES cells containing the human IL1F6 gene, the human IL1F8 gene, and the human IL1F9 gene; and generating the second rodent using the rodent ES cells containing the human IL1F6 gene, the human IL1F8 gene, and the human IL1F9 gene. (Item 30) 30. The method of claim 29, wherein the human IL1F6 gene, the human IL1F8 gene, and the human IL1F9 gene are provided in a contiguous nucleic acid molecule. (Item 31) 31. The method according to any of items 20 to 30, wherein the human IL1F6 gene replaces the endogenous rodent Il1f6 gene at the endogenous rodent Il1f6 locus. (Item 32) 32. The method according to any of items 20 to 31, wherein the human IL1F8 gene replaces the endogenous rodent Il1f8 gene at the endogenous rodent Il1f8 locus. (Item 33) 33. The method of any of items 20 to 32, wherein the human IL1F9 gene replaces the endogenous rodent Il1f9 gene at the endogenous rodent Il1f9 locus. (Item 34) 20. An isolated cell or tissue of the rodent according to any one of items 1 to 19. (Item 35) A rodent embryonic stem (ES) cell, (1) a humanized Il1rl2 gene at an endogenous rodent Il1rl2 locus, the humanized Il1rl2 gene encoding a humanized Il1rl2 protein comprising an ectodomain substantially identical to an ectodomain of a human IL1RL2 protein; (2) the human IL1F6 gene at the endogenous rodent Il1f6 locus; and (3) the human IL1F8 gene at the endogenous rodent Il1f8 locus; and (4) A rodent embryonic stem (ES) cell comprising a human IL1F9 gene at an endogenous rodent Il1f9 locus. (Item 36) 36. Use of the rodent ES cell of item 35 in generating a rodent. (Item 37) 20. Use of the rodent according to any one of items 1 to 19 as a rodent model for a disease associated with deregulated IL-36 signaling. (Item 38) 1. A method for evaluating the therapeutic efficacy of a candidate compound for treating a disease associated with deregulated IL-36 signaling, comprising: Providing a rodent according to any one of items 1 to 19; administering an agent to said rodent to induce inflammation; administering a candidate compound to said rodent; and determining whether the candidate compound inhibits the induced inflammation. (Item 39) 39. The method of claim 38, wherein the agent is DSS or oxazolone and intestinal inflammation is induced in the rodent. (Item 40) 39. The method of claim 38, wherein the agent is IMQ and skin inflammation is induced in the rodent. (Item 41) 41. The method of any one of items 38 to 40, wherein the candidate compound is administered to the rodent before, during, or after administration of the agent. (Item 42) 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) 43. The method of claim 42, wherein the candidate compound is an antibody. (Item 44) A target nucleic acid construct comprising: A target nucleic acid construct comprising a nucleotide sequence of a human IL1RL2 gene flanked by 5' and 3' rodent nucleotide sequences capable of mediating homologous recombination to integrate the nucleotide sequence of the human IL1RL2 gene into a rodent Il1rl2 locus to form a humanized Il1rl2 gene, said humanized Il1rl2 gene encoding a humanized Il1rl2 protein comprising an ectodomain substantially identical to the ectodomain of the human IL1RL2 protein. (Item 45) A target nucleic acid construct comprising: A contiguous nucleic acid sequence comprising the human IL1F6 gene, the human IL1F8 gene, and the human IL1F9 gene, A targeting nucleic acid construct, wherein the contiguous nucleic acid sequence is flanked by 5' and 3' rodent nucleotide sequences capable of mediating homologous recombination and integrating the contiguous nucleic acid sequence into the rodent locus including the rodent Il1f6 gene, the rodent Il1f8 gene, and the rodent Il1f9 gene. [Brief description of the drawings]

[0040] The file of this patent contains at least one drawing executed in color. Copies of this patent with color drawing(s) will be provided by the Patent and Trademark Office upon request and payment of the necessary fee.

[0041] [Figure 1A]1A-1D show an embodiment of an exemplary strategy for humanization of mouse Il1rl2. FIG. 1A shows a diagram, not to scale, of the genomic structure of the mouse Il1rl2 and human IL1RL2 genes. Exons are represented by thin bars distributed across the genomic sequence. The mouse genomic fragment of approximately 25,324 bp to be deleted and the human genomic fragment of approximately 32,389 bp to be inserted are shown. The positions of the probes used in the assay described in Example 1 are shown. FIG. 1B shows, not to scale, an exemplary modified BAC vector for humanization of the endogenous mouse Il1rl2 gene with junction sequences at the bottom (SEQ ID NOs: 26-28) to achieve (i) replacement of mouse Il1rl2 exons 3-8 and intervening intron, and 3' 155 bp mouse intron 2 and 5' 642 bp mouse intron 8 with human IL1RL2 exons 3-8 and intervening intron, and 3' 346 bp human intron 2 and 5' 1101 bp human intron 8, and (ii) insertion of a loxP-hUb1-em7-Neo-pA-mPrm1-Crei-loxP cassette (4996 bp) downstream of the insertion of the human genomic fragment in intron 8. FIG. 1C shows, not to scale, the humanized Il1rl2 allele after the neomycin cassette has been deleted with junction sequences at the bottom (SEQ ID NOs: 26 and 29). FIG. 1D depicts a sequence alignment 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 show an embodiment of an exemplary strategy for replacing mouse Il1f8, Il1f9, and Il1f6 with human IL1F8, IL1F9, and IL1F6. Figure 2A shows a diagram, not to scale, of the genomic organization of mouse Il1f8, Il1f9, and Il1f6 genes and human IL1F8, IL1F9, and IL1F6 genes. Exons are represented by thin bars distributed across the genomic sequence. The mouse genomic fragment of approximately 76,548 bp that is deleted and the human genomic fragment of approximately 88,868 bp that is inserted are shown. The positions of the probes used in the assay described in Example 1 are shown. Figure 2B, not to scale, shows an exemplary modified BAC vector for replacement of endogenous mouse Il1f8, Il1f9, and Il1f6 genes with the bottom junction sequences (SEQ ID NOs: 30-32) to achieve (i) replacement of coding sequences and untranslated regions (UTRs) and mouse Il1f8, Il1f9, and Il1f6 with the corresponding human sequences IL1F8, IL1F9, and IL1F6, and (ii) insertion of a human genomic fragment downstream of the loxP-hUb1-em7-Hygro-pA-mPrm1-Crei-loxP cassette (5,218 bp). Figure 2C, not to scale, shows the humanized locus after the neomycin cassette has been deleted with the bottom junction sequences (SEQ ID NOs: 30 and 33). Figure 2D shows a sequence alignment of mouse Il1f6 (sequence number 11) and human IL1F6 (sequence number 9) proteins, mouse Il1f8 (sequence number 17) and human IL1F8 (sequence number 15) proteins, and mouse Il1f9 (sequence number 23) and human IL1F9 (sequence number 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]3A-3E show an embodiment of the present invention, showing enhanced IMQ-induced skin inflammation in humanized DITRA-like mice. DITRA-like (also abbreviated as "DITRA" in figures herein) and wild-type (WT) mice were treated with topical application of IMQ for 4 consecutive days. On day 5, dorsal skin samples were taken for subsequent histopathological evaluation and qRT-PCR. (3A) Representative skin appearance of DITRA-like and WT mice on day 5, which were topically treated with Vaseline ("Vas", control) or IMQ-containing cream (Aldara) for 4 consecutive days. (3B) Representative hematoxylin and eosin ("H&E") staining of Vaseline- and IMQ-treated skin of DITRA-like and WT mice. (3C) mRNA expression of pro-inflammatory molecules in skin of DITRA and WT mice treated daily with Vaseline or IMQ-containing cream (n=10 per group). After 4 consecutive days of topical application of IMQ, on day 5, dorsal skin samples were taken from Il1rl2 single humanized mice ("1H"), DITRA-like mice, and wild-type mice for subsequent histopathological evaluation and qRT-PCR. The groups in each bar are, from left to right: WT Vas, DITRA Vas, WT IMQ, and DITRA IMQ. (3D) Representative skin appearance of 1H, DITRA-like, and WT mice on day 5, which were treated topically with Vaseline (control) or IMQ-containing cream (Aldara) for 4 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 per group). The groups in each bar are, from left to right: WT Vas, 1H Vas, DITRA-like Vas, WT IMQ, 1H IMQ, and DITRA-like IMQ. [Fig. 3D-E] Same as above.

[0044] [Figure 4]4A-4B. In some embodiments, DITRA-like mice exhibit shortened colons at steady state. (4A) Representative photographs of DITRA-like mice and their co-housed IL-36R and WT littermates at 3 and 10 months of age. (4B) Quantified colon length of DITRA-like mice at 3 and 10 months of age compared to their co-housed IL-36R KO and WT littermates (n=6 per group). Error bars indicate mean±SD.

[0045] [Figure 5A-C] Figures 5A-5E. In some embodiments, DITRA-like mice exhibit defects in mucosal healing in DSS-induced chronic colitis. DITRA-like mice (n=16) and their co-housed WT littermate controls (n=14) were subjected to chronic DSS-induced colitis by administration of 2.5% DSS for 7 days, followed by water for 11 days, 1.5% DSS for 4 days, and water for 5 days, for a total of 27 days. Control mice (n=5) were administered regular water. (5A) Weight loss in DITRA-like mice and their WT co-housed littermate controls is calculated as the percentage difference between the original weight and the actual weight on any particular day. (5B) Colon length in water and DSS-treated DITRA-like and WT mice. (5C) Hematoxylin and eosin (H&E) staining and pathology scores of colons in water and DSS-treated DITRA-like and WT mice. (5D) Myeloperoxidase (MPO) activity in colon homogenates of water- and DSS-treated DITRA-like and WT mice. (5E) Proinflammatory cytokine levels in colon homogenates of water- and DSS-treated DITRA-like and WT mice. Data are representative of at least three independent experiments. Error bars indicate mean ± SD. Groups in each bar are 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 antagonism using REGN anti-human IL-36R antibody ameliorates IMQ-induced skin inflammation in DITRA-like mice when administered prophylactically. The dorsal skin of DITRA-like mice was shaved 3 days before the start of IMQ application and topically treated with Vaseline (control) or IMQ-containing cream (Aldara) for 4 consecutive days. PBS, αhIL-36R mAb, and hIgG4 isotype control were injected subcutaneously at 10 mg / mL on days -3 and 1 (n=9 per treatment group). (6A) H&E histological sections and pathology scores of dorsal skin isolated from DITRA-like mice on day 5. (6B) Levels of proinflammatory cytokines in skin homogenates of DITRA-like mice on day 5 treated with daily application of IMQ and injections of PBS, αIL-36R mAb, and isotype control on days -3 and 1. Data show a representative of three independent experiments. Error bars indicate the mean ± SD. Groups in each bar are from left to right: 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 ameliorates chronic IMQ-induced skin inflammation in DITRA-like mice. The dorsal skin of DITRA-like mice was shaved 3 days before the start of treatment and topically treated twice with Vaseline (control) or IMQ-containing cream (Aldara) (first application for 5 consecutive days, then no application for 2 days, then second application for 4 consecutive days for a total of 11 days). PBS, anti-human IL-36R antibody, and hIgG4 isotype control were injected subcutaneously 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 pathology 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 of DITRA-like mice. Data are representative of two independent experiments. Error bars indicate mean ± SD. Groups in each bar are, from left to right: phosphate buffered saline (PBS), αhIL-36R mAb, and hIgG4 isotype control.

[0048] [Figure 8A-B]8A-8E. In some embodiments, therapeutic administration of anti-human IL-36R antibodies ameliorates DSS-induced chronic inflammation in DITRA-like mice, similar to IL-12p40 blockade, and rescues the inability to heal from DSS-induced mucosal injury. DITRA-like mice (n=45) were subjected to chronic DSS-induced colitis by administering 3% DSS for 7 days, 2% DSS for 13 days, and then water for a total of 30 days. Control mice (n=5) received regular 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 injected intraperitoneally 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 original weight and actual weight on any particular 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 homogenates of DITRA-like mice at day 30. (8E) Pro-inflammatory cytokine levels in colon homogenates of DITRA-like mice at day 30. Data are representative of two independent experiments. Error bars indicate mean ± SD. Groups in each bar are, 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 antagonism ameliorates oxazolone-induced colitis in DITRA-like mice. DITRA-like mice were pre-sensitized with 3% oxazolone solution in 100% ethanol and administered 1.5% oxazolone and vehicle (50% ethanol) intrarectally on days 5, 6, and 7. Mice were injected intraperitoneally with PBS, anti-human IL-36R mAb, and hIgG4 isotype control on days 2, 5, and 7 after pre-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 PBS-treated group. (B) Colon length measured in DITRA-like mice on day 8. (C) Proinflammatory cytokine levels in colon homogenates of oxazolone- and vehicle-treated DITRA-like mice injected with PBS, anti-human IL-36R mAb, and hIgG4 isotype control. *-significant difference from PBS-treated group, #-significant difference from isotype-treated group. Data are representative of two independent experiments with 5 mice per group. Error bars represent mean ± SEM. *p<0.05, **p<0.005, ***p<0.0005, ****p<0.00001. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0050] Disclosed herein is a genetically modified rodent (such as, but not limited to, a mouse) that contains a humanized Il1rl2 gene (encoding a humanized IL1RL2 protein having an ectodomain substantially identical to the human IL1RL2 ectodomain) and a human gene encoding a human IL-36α ligand, while retaining the IL-36Ra antagonist endogenous, which is 20 times less potent in inhibiting human IL-36R signaling. The genetically modified rodent disclosed herein has been shown to exhibit symptoms of DITRA patients, in which mutations in IL-36Ra result in enhanced IL-36R signaling. Thus, the genetically modified rodent disclosed herein can serve as a novel functional model 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 some embodiments, the disclosure provides a rodent comprising in its genome a humanized Il1rl2 gene encoding a humanized IL1RL2 protein (a subunit of IL-36R) and human genes encoding human IL-36 α, β, and γ ligands. Such rodents are also referred to herein as quadruple humanized rodents (i.e., 4H or DITRA-like).

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

[0053] In some embodiments, rodents of the present disclosure include, by way of non-limiting example, mice, rats, and hamsters. In some embodiments, rodents of the present disclosure include, by way of non-limiting example, mice and rats. In some embodiments, rodents are selected from the superfamily Murinoidea. In some embodiments, rodents of the present disclosure are animals from a family selected from the family Orycteridae (e.g., mouse-like hamsters), the family Cricetidae (e.g., hamsters, New World rats and mice, and voles), the family Muridae (pure mice and rats, gerbils, spiny mice, and maned mice), the family Tegretidae (tree mice, rock mice, white-tailed rats, Madagascar rats and mice), the family Euonymidae (e.g., spiny dormice), and the family Bucconidae (e.g., mole rats, bamboo mice, and plateau mole rats). In some embodiments, the rodent of the present disclosure is selected from a purebred mouse or rat (Muridae), a gerbil, a spiny mouse, and a maned mouse. In some embodiments, the mouse of the present disclosure is from a member of the Muridae family. IL-1RL2 humanization

[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 interleukin-1 receptor accessory protein IL-1RAcP (Garlanda C et al., Immunity 39, 1003-1018 (2013); Towne JE et al., J. Biol. Chem. 279, 13677-13688 (2004) which are incorporated by reference in their entirety). The receptor (IL-36R) can recognize and bind three different agonists, IL-36α, IL-36β, and IL-36γ (also known as IL1F6, IL1F8, and IL1F9), and 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, e.g., FIG. 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 rodent disclosed herein comprises in its genome a humanized Il1rl2 gene comprising the nucleotide sequence of an endogenous rodent Il1rl2 gene and the nucleotide sequence of a human IL1RL2 gene. As used herein, the "nucleotide sequence of a gene" includes the entire gene or a portion thereof, of a genomic sequence, an mRNA sequence or a cDNA sequence. As a non-limiting example, the nucleotide sequence of a human IL1RL2 gene includes the entire or a portion of a genomic sequence, an mRNA sequence or a cDNA sequence of a human IL1RL2 gene. The nucleotide sequence of an endogenous rodent Il1rl2 gene and the nucleotide sequence of a human IL1RL2 gene indicate that the humanized Il1rl2 gene in the rodent genome has an Il1rl2 protein, i.e., an Il1rl2 protein structure (consisting of an ectodomain, a transmembrane domain and a cytoplasmic domain), and an Il1rl2 function (e.g., an interleukin-1 receptor accessory protein (ILA)). I Inter l eukin- 1 r eceptor ac Accessory p The IL-36A and IL-36B proteins are operably linked to each other to encode proteins such as a protein that forms a heterodimer with IL-36A (protein 1L-1RacP) and recognizes 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, the humanized Il1rl2 gene encoding a humanized Il1rl2 protein, the humanized Il1rl2 protein containing an ectodomain substantially identical to the ectodomain of the human IL1RL2 protein. In some embodiments, the ectodomain substantially identical to the ectodomain of the human IL1RL2 protein exhibits the same functionality (e.g., ligand binding properties) as the ectodomain of the human IL1RL2 protein. An ectodomain or polypeptide "substantially identical to the ectodomain of the human IL1RL2 protein" refers, in some embodiments, to a polypeptide that is at least 95%, 98%, 99% or 100% identical in sequence to the ectodomain of the human IL1RL2 protein. In some embodiments, it refers to a polypeptide that differs from the ectodomain of the human IL1RL2 protein by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid. Also, in some embodiments, it refers to a polypeptide that differs from the ectodomain of the human IL1RL2 protein only at the N- or C-terminal portion of the ectodomain, e.g., by addition, deletion, or substitution of amino acids, but not more than 5, 4, 3, 2, 1 amino acid at the N- or C-terminal portion. "N- or C-terminal portion of the ectodomain" refers to 5-10 amino acids from the N- or C-terminus of the ectodomain. 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 set forth in SEQ ID NO:2. In certain embodiments, the human IL1RL2 protein comprises the amino acid sequence set forth in SEQ ID NO:2, and the ectodomain consists of amino acids 20-335 of SEQ ID NO:2. In some embodiments, the humanized Il1rl2 gene encodes a humanized Il1rl2 protein that comprises the ectodomain of the human IL1RL2 protein set forth in SEQ ID NO:2, i.e., an ectodomain that is substantially identical to amino acids 20-335 of SEQ ID NO:2. In some embodiments, the humanized Il1rl2 gene encodes a humanized Il1rl2 protein containing an ectodomain comprising amino acids 22-337 of SEQ ID NO:7.

[0058] In some embodiments, the humanized Il1rl2 gene encodes a humanized Il1rl2 protein that contains a transmembrane cytoplasmic sequence (i.e., a sequence that includes both a transmembrane domain and a cytoplasmic domain) that is substantially identical to the transmembrane cytoplasmic sequence of an endogenous rodent Il1rl2 protein. In some embodiments, the transmembrane cytoplasmic sequence that is substantially identical to the transmembrane cytoplasmic sequence of an endogenous rodent Il1rl2 protein exhibits the same functionality (e.g., signal transduction and / or interaction with intracellular molecules) as the transmembrane cytoplasmic sequence of an endogenous rodent Il1rl2 protein. A transmembrane cytoplasmic sequence or peptide that is "substantially identical to the transmembrane cytoplasmic sequence of an endogenous rodent Il1rl2 protein" refers, in some embodiments, to a polypeptide that is at least 95%, 98%, 99% or 100% identical in sequence to the transmembrane cytoplasmic sequence of an endogenous rodent Il1rl2 protein. In some embodiments, it refers to a polypeptide that differs from the transmembrane cytoplasmic sequence of an endogenous rodent Il1rl2 protein by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid. In some embodiments, it refers to a polypeptide that differs from the transmembrane cytoplasmic sequence of an endogenous rodent Il1rl2 protein only at the N- or C-terminus, e.g., by amino acid addition, deletion, or substitution, but by no more than 5, 4, 3, 2, or 1 amino acid at the N- or C-terminus of the transmembrane cytoplasmic sequence. By "N- or C-terminal portion of the transmembrane cytoplasmic sequence" it refers to within 5-10 amino acids from the N-terminus of the transmembrane domain, or within 5-10 amino acids from the C-terminus of the cytoplasmic domain. In some embodiments, the humanized Il1rl2 protein contains a transmembrane cytoplasmic sequence that is substantially identical to the transmembrane cytoplasmic sequence of a mouse Il1rl2 protein, such as, for example, a mouse Il1rl2 protein that is substantially identical (at least 95%, 98%, 99% or 100% identical) to SEQ ID NO:4, or a rat Il1rl2 protein, such as, for example, a rat Il1rl2 protein that is substantially identical (at least 95%, 98%, 99% or 100% identical) to SEQ ID NO:6.

[0059] In some embodiments, the humanized Il1rl2 gene encodes a humanized Il1rl2 protein that contains a signal peptide that is substantially identical to the signal peptide of an endogenous rodent Il1rl2 protein. A signal peptide that is "substantially identical to the signal peptide of an endogenous rodent Il1rl2 protein" refers in some embodiments to a polypeptide that is at least 95%, 98%, 99% or 100% identical in sequence to the signal peptide of an endogenous rodent Il1rl2 protein. In some embodiments, it refers to a polypeptide that differs from the signal peptide of an endogenous rodent Il1rl2 protein by 3, 2, or 1 amino acid or less. In some embodiments, it refers to a polypeptide that differs from the signal peptide of an endogenous rodent Il1rl2 protein only at the N- or C-terminus, for example, by addition, deletion or substitution of amino acids, but not more than 3, 2, or 1 amino acid at the N- or C-terminal portion of the signal peptide. "N- or C-terminal portion of the signal peptide" means within 5 amino acids from the N- or C-terminus of the signal peptide. In some embodiments, the humanized Il1rl2 protein comprises a signal peptide substantially identical to the signal peptide of a mouse Il1rl2 protein, such as, for example, a mouse Il1rl2 protein substantially identical (at least 95%, 98%, 99% or 100% identical) to SEQ ID NO: 4, or a rat Il1rl2 protein, such as, for example, a rat Il1rl2 protein substantially identical (at least 95%, 98%, 99% or 100% identical) to SEQ ID NO: 6.

[0060] In some embodiments, the humanized Il1RL2 gene in the genome of the genetically modified rodent comprises a nucleotide sequence of the human IL1RL2 gene (or "human IL1RL2 nucleotide sequence") and a nucleotide sequence of an endogenous rodent Il1rl2 gene (or "endogenous rodent Il1rl2 nucleotide sequence"), wherein the human IL1RL2 nucleotide sequence encodes a polypeptide substantially identical to an ectodomain of the human IL1RL2 protein encoded by the human IL1RL2 gene. Such a human IL1RL2 nucleotide sequence is also referred to as substantially encoding an ectodomain 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 is comprised of exons 3-8 of the human IL1RL2 gene. In some embodiments, the human IL1RL2 nucleotide sequence is comprised of a 3' portion of intron 2, exons 3-8, and a 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 genetically modified rodent comprises an endogenous rodent Il1rl2 nucleotide sequence and a human Il1rl2 nucleotide sequence, and the endogenous rodent Il1rl2 nucleotide sequence encodes a polypeptide that is substantially identical to the transmembrane cytoplasmic sequence of endogenous rodent Il1rl2 protein.Such a rodent Il1rl2 nucleotide sequence is also referred to as substantially encoding the transmembrane cytoplasmic sequence of 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 endogenous rodent Il1rl2 protein. In some embodiments, the endogenous rodent Il1rl2 nucleotide sequence present in the humanized Il1rl2 gene comprises the remaining exons 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 comprises the 3' portion of intron 8 and the remaining exons downstream of exon 8 of the endogenous rodent Il1rl2 gene.

[0062] In some embodiments, the humanized Il1rl2 gene in the genome of the genetically modified rodent comprises 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 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 an 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 a 5' portion of intron 2 of the endogenous rodent Il1rl2 gene.

[0063] In some embodiments, the humanized Il1rl2 gene is operably linked to an endogenous rodent Il1rl2 regulatory sequence, e.g., a 5' transcriptional regulatory sequence, such as a promoter and / or enhancer, e.g., 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 at the endogenous rodent Il1rl2 locus. In some embodiments, the humanized Il1rl2 gene is at a locus other than the endogenous rodent Il1rl2 locus, for example, as a result of random integration. In some embodiments, the humanized Il1rl2 gene is at a locus other than the endogenous rodent Il1rl2 locus, for example, as a result of inactivation (e.g., total or partial deletion) of the endogenous rodent Il1rl2 gene, the rodent cannot express rodent Il1rl2 protein.

[0065] In some embodiments where the humanized Il1rl2 gene is at the endogenous rodent Il1rl2 locus, the humanized Il1rl2 gene results from replacement of the endogenous rodent Il1rl2 gene at the endogenous rodent Il1rl2 locus with the nucleotide sequence of the human IL1RL2 gene.

[0066] In some embodiments, the nucleotide sequence of the endogenous rodent Il1rl2 gene at the endogenous rodent Il1rl2 locus to be replaced is a genomic fragment of the endogenous rodent Il1rl2 gene that substantially encodes the ectodomain of the rodent Il1rl2 protein. In some embodiments, the rodent genomic fragment to be replaced comprises 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 comprises all or a portion of the exons of the human IL1RL2 gene that substantially encode the ectodomain of the human IL1RL2 protein. In some embodiments, the human genomic fragment comprises 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 replacement and is operably linked to the inserted human IL1RL2 nucleotide sequence substantially encodes the transmembrane cytoplasmic sequence of the endogenous rodent Il1rl2 protein. In some embodiments, the genomic sequence of the endogenous rodent Il1rl2 gene that remains at the endogenous rodent Il1rl2 locus after humanization replacement comprises an 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 the humanization replacement and is operably linked to the inserted human IL1RL2 nucleotide sequence substantially encodes the signal peptide of the endogenous rodent Il1rl2 protein. In some embodiments, the genomic sequence of the endogenous rodent Il1rl2 gene that remains at the endogenous rodent Il1rl2 locus after the humanization replacement comprises exons 1-2 of the endogenous rodent Il1rl2 gene.

[0070] In some embodiments, where the endogenous rodent Il1rl2 protein and the human IL1RL2 protein share common amino acids near the junction between the transmembrane and ectodomains, it may not be necessary to insert a human IL1RL2 nucleotide sequence that strictly codes for the ectodomain of the human IL1RL2 protein. It is possible to insert a slightly longer or shorter nucleotide sequence of the human IL1RL2 gene that substantially codes for the ectodomain of the human IL1RL2 protein and is operably linked to the genomic sequence of the endogenous rodent Il1rl2 gene that substantially codes for the transmembrane domain (and cytoplasmic domain) of the endogenous rodent Il1rl2 protein, such that the humanized Il1rl2 protein encoded by the resulting humanized Il1rl2 gene contains an ectodomain 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, a genomic fragment comprising exons 3-8 of the endogenous rodent Il1rl2 gene at the endogenous rodent Il1rl2 locus is replaced with a genomic fragment comprising exons 3-8 of the human IL1RL2 gene. As a result, a humanized Il1rl2 gene is formed at the endogenous rodent Il1rl2 locus, comprising 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 rodent provided herein is heterozygous for a humanized Il1rl2 gene in its genome. In some embodiments, the rodent provided herein is homozygous for a humanized Il1rl2 gene in its genome.

[0073] In some embodiments, the humanized Il1rl2 gene causes expression of the encoded humanized Il1rl2 protein in the rodent. In some embodiments, the humanized Il1rl2 protein is expressed in a pattern equivalent or substantially identical to the corresponding rodent Il1rl2 protein in a control rodent (e.g., a rodent that does not have a humanized Il1rl2 gene). In some embodiments, the humanized Il1rl2 protein is expressed at a level equivalent or substantially identical to the corresponding rodent Il1rl2 protein in a control rodent (e.g., a rodent that does not have a humanized Il1rl2 gene). In some embodiments, the humanized Il1rl2 protein is expressed and detected on the cell surface, such as, but not limited to, the cell surface of keratinocytes, monocytes, macrophages, neutrophils, bronchial and intestinal epithelial cells, among others. In the context of comparing a humanized gene or protein of a humanized rodent with an endogenous rodent gene or protein of a control rodent, the term "comparable" means that the molecules or levels being compared may not be identical to one another, but there is sufficient similarity to allow a comparison between them such that reasonable conclusions can be drawn based on the differences or similarities observed. In some embodiments, the term "substantially the same" includes compared levels 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, a rodent disclosed herein is unable to express a rodent Il1rl2 protein, e.g., as a result of inactivation (e.g., full or partial deletion) or replacement (full or partial) of the endogenous rodent Il1rl2 gene.

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

[0076] In some embodiments, the rodent disclosed herein has a genome comprising a human IL-36α gene encoding a human IL-36α protein. Reference to a "human IL-36α gene" includes human genomic DNA encoding a human IL-36α protein, including a human IL-36α promoter. The human IL-36α protein may be a mature or precursor form of the human IL-36α protein. In some embodiments, the human IL-36α protein comprises the amino acid sequence of SEQ ID NO:9.

[0077] In some embodiments, the human IL-36α gene is at the endogenous rodent Il-36α locus. In some embodiments, the human IL-36α gene is 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 at a locus other than the endogenous rodent Il-36α locus, the rodent cannot express rodent Il-36α protein, for example, as a result of inactivation (e.g., total or partial deletion) of the endogenous rodent Il-36α gene.

[0078] In some embodiments, the human IL-36α gene replaces 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 expression of the encoded human IL-36α protein in the rodent, such as in serum, at mucosal sites, e.g., skin, intestinal epithelium, lung, and in various types of cells of the immune system (e.g., monocytes, macrophages, T cells, dendritic cells). In some embodiments, the human IL-36α protein is expressed in a pattern comparable to or substantially identical to the corresponding rodent Il-36α protein in a control rodent (e.g., a rodent that does not have the human IL-36α gene). In some embodiments, the human IL-36α protein is expressed at a level comparable to or substantially the same as the corresponding rodent Il-36α protein in a control rodent (e.g., a rodent that does not have the human IL-36α gene), e.g., in serum, at mucosal sites (e.g., skin, intestinal epithelium, lung, etc.), and / or in immune cells (e.g., monocytes, macrophages, T cells, dendritic cells).

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

[0082] In some embodiments, the rodent disclosed herein has a genome comprising a human IL-36β gene encoding a human IL-36β protein. Reference to a "human IL-36β gene" includes human genomic DNA encoding a human IL-36β protein, including a human IL-36β promoter. The human IL-36β protein may be a mature or precursor form of the human IL-36β protein. In some embodiments, the human IL-36β protein comprises the amino acid sequence of SEQ ID NO: 15.

[0083] In some embodiments, the human IL-36β gene is at the endogenous rodent Il-36β locus. In some embodiments, the human IL-36β gene is at a locus other than the endogenous rodent Il-36β locus, for example, as a result of random integration. In some embodiments, the human IL-36β gene is at a locus other than the endogenous rodent Il-36β locus, the rodent cannot express rodent Il-36β protein, for example, as a result of inactivation (e.g., total 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 expression of the encoded human IL-36β protein in the rodent, such as in serum, at mucosal sites, e.g., skin, intestinal epithelium, lung, and / or in immune cells (e.g., monocytes, macrophages, T cells, dendritic cells). In some embodiments, the human IL-36β protein is expressed in a pattern comparable to or substantially identical to the corresponding rodent IL-36β protein in a control rodent (e.g., a rodent that does not have the human IL-36β gene). In some embodiments, the human IL-36β protein is expressed at levels comparable to or substantially the same as the corresponding rodent IL-36β protein in a control rodent (e.g., a rodent that does not have the human IL-36β gene), e.g., in serum, at mucosal sites, e.g., skin, intestinal epithelium, lung, and in various types of cells of the immune system (e.g., monocytes, macrophages, T cells, dendritic cells, etc.).

[0087] In some embodiments, a rodent disclosed herein is unable to express rodent Il-36β protein, e.g., as a result of inactivation (e.g., full or partial deletion) or replacement (full 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 a human IL-36γ protein. Reference to a "human IL-36γ gene" includes human genomic DNA encoding a human IL-36γ protein, including a human IL-36γ promoter. In some embodiments, the human IL-36γ protein can be a mature or precursor form of the human IL-36γ protein. In some embodiments, the human IL-36γ protein comprises the amino acid sequence of SEQ ID NO:21.

[0089] In some embodiments, the human IL-36γ gene is at the endogenous rodent Il-36γ locus. In some embodiments, the human IL-36γ gene is 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 at a locus other than the endogenous rodent Il-36γ locus, the rodent cannot express rodent Il-36γ protein, for example, as a result of inactivation (e.g., total or partial deletion) of the endogenous rodent Il-36γ gene.

[0090] In some embodiments, the human IL-36γ gene replaces 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 expression of the encoded human IL-36γ protein (e.g., a protein identical to the human IL-36γ protein) in the rodent, such as in serum, mucosal sites, e.g., skin, intestinal epithelium, lung, and in various types of cells of the immune system (e.g., monocytes, macrophages, T cells, dendritic cells). In some embodiments, the human IL-36γ protein is expressed in a pattern comparable to or substantially identical to the corresponding rodent IL-36γ protein in a control rodent (e.g., a rodent that does not have the human IL-36γ gene). In some embodiments, the human IL-36γ protein is expressed, e.g., in serum, mucosal sites (e.g., skin, intestinal epithelium, lung, etc.), and / or immune cells (e.g., monocytes, macrophages, T cells, dendritic cells) at levels comparable to or substantially the same as the corresponding rodent IL-36γ protein in a control rodent (e.g., a rodent that does not have the human IL-36γ gene).

[0093] In some embodiments, a rodent disclosed herein is unable to express rodent Il-36γ protein, e.g., as a result of inactivation (e.g., total or partial deletion) or replacement (total or partial) of an endogenous rodent Il-36γ gene.

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

[0095] Quadruple humanized rodent phenotypes The genetically modified rodents disclosed herein do not develop any spontaneous disease at steady state, but they exhibit shortened colons and increased expression of pro-inflammatory mediators in the skin compared to non-humanized age-matched control rodents at both steady state and disease states (e.g., after DSS or IMQ treatment). In some embodiments, the genetically modified rodents exhibit colon lengths that are at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% shorter than control rodents at steady state or disease states.

[0096] In some embodiments, the genetically modified rodent exhibits a colon length at steady state that is 10%-15% (±5%) shorter than a control rodent. In some embodiments, the genetically modified rodent exhibits a colon length at steady state that is 15%-20% (±5%) shorter than a control rodent. In some embodiments, the genetically modified rodent exhibits a colon length at steady state that is 10%-20% (±5%) shorter than a control rodent.

[0097] In other embodiments, the genetically modified rodent exhibits a colon length that is 20%-40% (±5%) shorter than control rodents in disease states (e.g., after DSS or oxazolone treatment). In other embodiments, the genetically modified rodent exhibits a colon length that is 30%-40% (±5%) shorter than control rodents in disease states (e.g., after DSS or oxazolone treatment). In other embodiments, the genetically modified rodent exhibits a colon length that is 20%-30% (±5%) shorter than control rodents in disease states (e.g., after DSS or oxazolone treatment). In other embodiments, the genetically modified rodent exhibits a colon length that is 25%-35% (±5%) shorter than control rodents in disease states (e.g., after DSS or oxazolone treatment).

[0098] The genetically modified rodents disclosed herein do not develop spontaneous disease at steady state, but have been shown to exhibit enhanced skin and intestinal inflammation in experimentally induced models of skin and intestinal inflammation (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 the rodent through at least 0.5%, at least 1%, at least 1.5%, at least 2.5% of drinking water. In some embodiments, DSS is administered to the rodent through no more than 10%, 9%, 8%, 7%, 6%, or 5% of drinking water. In some embodiments, the rodent is provided with drinking water containing 1.5% to 3% DSS. In some embodiments, the rodent is provided with drinking water containing 0.5% to 3% DSS. In some embodiments, the rodent is provided with drinking water containing 1% to 3% DSS. In some embodiments, the rodent is provided with drinking water containing 2% to 3% DSS. In some embodiments, the rodent is provided with drinking water containing 2.5% to 3% DSS. In some embodiments, the rodent is provided with drinking water containing 0.5% to 2.5% DSS. In some embodiments, the rodent is provided with drinking water containing 0.5% to 2% DSS. In some embodiments, the rodent is provided with drinking water containing 0.5% to 1.5% DSS. In some embodiments, the rodent is provided with drinking water containing 0.5% to 1% DSS. In some embodiments, the rodent is provided with drinking water containing 1% to 2.5% DSS. In some embodiments, the rodent is provided with drinking water containing 1.5% to 2% DSS.

[0100] In some embodiments, administration of DSS can be for 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more days, even if continuous, with breaks in between where no DSS is administered. 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 prior to analysis. In some embodiments, rodents are given drinking water containing 3% DSS for 7 days, then 2% DSS for 13 days, then distilled water until 27-30 days prior to 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, then distilled water for 11 days, then 1.5% DSS for 4 days, then distilled water for 5 days, for a total of 27 days prior to analysis. In some embodiments, rodents are given drinking water containing 3% DSS for 7 days, then water for 13 days, then 2% DSS for 4 days, then distilled water for 6 days, until 30 days prior to analysis.

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

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

[0103] In some embodiments, the genetically modified rodent exhibits, for example, at least a 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400% or more increase in intestinal pathology score compared to a wild-type control rodent receiving the same DSS administration. In some embodiments, the genetically modified rodent exhibits, for example, a 50%-400%, 50%-300%, 50%-200%, 50%-100%, 100%-400%, 100%-300%, 100%-200%, or 200%-400% increase in intestinal pathology score compared to a wild-type control rodent receiving the same DSS administration. In some embodiments, the genetically modified rodent exhibits an increase in myeloperoxidase ("MPO") levels in colon homogenates, e.g., at least 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400% or more, compared to a wild-type control rodent receiving the same DSS administration. In some embodiments, the genetically modified rodent exhibits an increase in MPO levels in colon homogenates, e.g., 50%-400%, 50%-300%, 50%-200%, 50%-100%, 100%-400%, 100%-300%, 100%-200%, or 200%-400%, compared to a wild-type control rodent receiving the same DSS administration. In some embodiments, the genetically modified rodents exhibit increased levels of fecal Lcn, e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400% or more, compared to wild-type control rodents receiving the same DSS administration. In some embodiments, the genetically modified rodents exhibit increased mRNA expression and / or increased 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, e.g., by at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900% or more, compared to wild-type control rodents receiving the same DSS administration.In some embodiments, the genetically modified rodent exhibits increased mRNA expression and / or protein levels of one or more pro-inflammatory mediators in colon homogenates, e.g., by 20%-900%, 20%-800%, 20%-700%, 20%-600%, 20%-500%, 20%-400%, 20%-300%, 20%-200%, or 20%-100%. In some embodiments, the genetically modified rodent exhibits increased mRNA expression and / or protein levels of one or more pro-inflammatory mediators in colon homogenates, e.g., by 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 rodent exhibits increased mRNA expression and / or protein levels of one or more pro-inflammatory mediators in colon homogenates, e.g., by 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 rodent exhibits increased mRNA expression and / or protein levels of one or more pro-inflammatory mediators in colon homogenates, e.g., by 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 the rodent to induce skin inflammation. In some embodiments, IMQ is provided in a carrier suitable for topical application, such as a cream, gel, a commercially available IMQ cream (e.g., from Aldara), and the like. In some embodiments, IMQ is applied daily to the skin of the rodent at a daily dose of 1-5 mg, 2-4 mg, or 3-3.5 mg for 2, 3, 4, 5, 6, 7, 8, 9, 10 or more days to induce skin inflammation. In some embodiments, daily topical application with a daily dose of about 3.125 mg for 4 days is suitable for inducing acute skin inflammation, and daily topical application with a daily dose of about 3.125 mg for 9 days is suitable for inducing chronic skin inflammation. In some embodiments, IMQ is applied topically multiple times (e.g., 2, 3, or 4 times) before the analysis or assay is performed, with IMQ applied for 4-5 consecutive days, followed by 2 days without IMQ each time. In certain embodiments, IMQ is applied topically twice, with IMQ applied for 5 consecutive days, followed by 2 days without IMQ for the first time, followed by 4 consecutive days with IMQ applied for the second time (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 measurements of erythema, scaling and skin thickening; (ii) histopathological analysis of skin tissue to evaluate, for example, parakeratosis, normal keratosis, Munro's microabscesses, 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 a total pathology score; (iii) measuring the mRNA expression and / or protein levels of pro-inflammatory mediators in skin homogenates, including, for example, the mRNA expression and / or protein levels of Cxcl1, IL-17f, IL-17a, IL-23a, S100A8 and Defb4, among others; and (iv) a combination of (i)-(iii).

[0105] In some embodiments, the genetically modified rodent exhibits an increase in skin pathology score, e.g., at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200% or more, compared to a wild-type control rodent receiving the same IMQ administration. In some embodiments, the genetically modified rodent exhibits an increase in mRNA expression and / or protein levels of one or more pro-inflammatory mediators (e.g., but not limited to, Cxcl1, IL-17f, IL-17a, IL-23a, S100A8, and Defb4) in skin homogenates, e.g., at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300% or more, compared to a wild-type control rodent receiving the same IMQ administration. In some embodiments, the genetically modified rodent exhibits increased mRNA expression and / or protein levels of one or more pro-inflammatory mediators (e.g., but not limited to, Cxcl1, IL-17f, IL-17a, IL-23a, S100A8 and Defb4) in skin homogenates at steady state compared to wild-type control rodents at steady state, e.g., at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300% or more.

[0106] With regard to IMQ-induced skin inflammation, single Il1rl2 humanized mice exhibited a phenotype similar to wild-type rodents, as reflected by histopathology and RNA expression of pro-inflammatory molecules in the skin (Figures 3D and 3E), whereas 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] Genetically modified rodent tissues and cells In some embodiments, disclosed herein is an isolated cell or tissue from a rodent as described herein. In some embodiments, the cell is selected from a dendritic cell, a lymphocyte (e.g., a B cell or a T cell), a macrophage, and a monocyte. In some embodiments, the tissue is selected from adipose, bladder, brain, breast, bone marrow, eye, heart, intestine, kidney, liver, lung, lymph node, muscle, pancreas, plasma, serum, skin, spleen, stomach, thymus, testis, egg, and combinations thereof. In some embodiments, the isolated cell or tissue comprises in its genome the quadruple humanization feature as described herein.

[0108] Compositions and methods for generating quadruple-humanized rodents Further aspects of the present disclosure are directed to methods for producing the genetically modified rodents described above, as well as nucleic acid vectors and rodent embryonic stem cells suitable for use in producing the genetically modified rodents.

[0109] Quadruple humanized rodents, i.e., rodents containing humanized Il1rl2 and human IL1F6, IL1F8, and IL1F9 genes, can be made by creating a single Il1rl2 humanized rodent line and a triple human rodent line of Il1f6, Il1f8, and Il1f9, and then breeding the single humanized rodent line with the triple humanized rodent line to obtain a quadruple humanized rodent. The term "breeding" or "crossing" as used herein in relation to rodents refers to mating rodents to produce offspring. Those skilled in the art will understand that multiple matings may be required to achieve homozygosity.

[0110] In some embodiments, a targeting vector is disclosed herein that includes a human nucleotide sequence that is desired to be integrated into a rodent locus. In some embodiments, the human nucleotide sequence can be a nucleotide sequence of the human IL1RL2 gene that encodes an ectodomain that is substantially identical to the ectodomain of human IL1RL2, for example, a nucleotide sequence that includes exons 3-8 of the human IL1RL2 gene. In some embodiments, the human nucleotide sequence can be a nucleotide sequence that includes the coding sequence of human IL-36α, the coding sequence of human Il-36β, and the complementary strand of the coding sequence of human IL-36γ. The targeting vector also includes 5′ and 3′ rodent sequences, also known as homology arms, that flank the human nucleotide sequence to be integrated, which mediate homologous recombination and integration of the human nucleotide sequence into the 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 to be replaced by the human nucleotide sequence. For example, in an embodiment in which a nucleotide sequence encoding a rodent ectodomain (e.g., exons 3-8 of the rodent Il1rl2 gene) is replaced with a sequence encoding a human ectodomain (e.g., exons 3-8 of the human IL1RL2 gene), the 5' flanking sequence can include exons 1-2 of the rodent Il1rl2 gene, and the 3' flanking sequence can include the remaining exons downstream of exon 8 of the rodent Il1rl2 gene. In some embodiments in which rodent nucleotide sequences encoding all three IL-36 ligands are replaced with human nucleotide sequences, the 5' flanking sequence can include rodent nucleotide sequences upstream of the coding sequence of the Il1f6 gene, and the 3' flanking sequence can include rodent nucleotide sequences upstream of the coding sequence of the Il1f9 gene.

[0111] In some embodiments, the targeting vector comprises a selectable marker gene. In some embodiments, the targeting vector comprises one or more site-specific recombination sites. In some embodiments, the targeting vector comprises a selectable marker gene adjacent to the site-specific recombination sites, such that the selectable marker gene can be deleted as a result of recombination between the sites.

[0112] In an exemplary embodiment, a bacterial artificial chromosome (BAC) clone carrying a rodent genomic fragment can be modified using bacterial homologous recombination and VELOCIGENE® technology (see, e.g., U.S. Pat. No. 6,586,251 and Valenzuela et al. (2003) Nature Biotech. 21(6):652-659, which are incorporated by reference in their entireties). As a result, the rodent genomic sequence is deleted from the original BAC clone and the human nucleotide sequence is inserted, resulting in a modified BAC clone carrying the human nucleotide sequence flanked by 5' and 3' rodent homology arms. In some embodiments, the human nucleotide sequence can be a cDNA sequence or human genomic DNA encoding (i) all or a portion of human IL1RL2 (e.g., the ectodomain of the human IL1RL2 protein), or (ii) all three of human IL1F6, IL1F8, and IL1F9. Once linearized, the modified BAC clone can be introduced into rodent embryonic stem (ES) cells.

[0113] In some embodiments, the invention provides for the use of the targeting vectors described herein to generate modified rodent embryonic stem (ES) cells. For example, the targeting vectors can be introduced into rodent ES cells, for example, by electroporation. Both mouse ES cells and rat ES cells have been described in the art. For example, US 7,576,259, US 7,659,442, US 7,294,754, and US 2008-0078000 A1, all of which are incorporated herein by reference, which describe the VELOCIMOUSE® method of generating mouse ES cells and genetically modified mice; US 2014 / 0235933 A1 (Regeneron Pharmaceuticals, Inc.), US 2014 / 0310828 A1 (Regeneron Pharmaceuticals, Inc.), which describe methods for generating rat ES cells and genetically modified rats, which can be used to generate modified rodent embryos, which can be used to generate rodent animals; Tong et al. (2010) Nature 467:211-215 and Tong et al. (2011) Nature 467:211-215, which describe methods for generating rat ES cells and genetically modified rats, which can be used to generate modified rodent embryos, which can be used to generate rodent animals; See Protoc. 6(6):doi:10.1038 / nprot.2011.338, which is incorporated by reference in its entirety.

[0114] In some embodiments, ES cells with human nucleotide sequences integrated into the genome can be selected. In some embodiments, ES cells are selected based on rodent allele loss and / or human allele gain assays. In some embodiments, the selected ES cells are then used as donor ES cells to be injected into pre-morula embryos (e.g., 8-cell embryos) by using the VELOCIMOUSE® method (see, e.g., US7,576,259, US7,659,442, US7,294,754, and US2008-0078000A1, which are incorporated by reference in their entirety), or the methods described in US2014 / 0235933A1 and US2014 / 0310828A1, both of which are incorporated by reference in their entirety. In some embodiments, the embryos containing donor ES cells are incubated until the blastocyst stage and then implanted into surrogate mothers to produce F0 rodents entirely derived from donor ES cells. Rodent pups carrying the human nucleotide sequence can be identified by genotyping DNA isolated from tail fragments using assays for loss of rodent alleles and / or gain of human alleles.

[0115] In some embodiments, rodents heterozygous for the humanized gene can be bred to produce homozygous rodents.

[0116] Quadruple-humanized rodent method 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 deregulated IL-36 signaling.

[0117] By "disease associated with deregulated IL-36 signaling" is meant a disease in which abnormalities in IL-36 signaling are manifested and may directly or indirectly cause or exacerbate the symptoms of the disease. Non-limiting examples of diseases associated with deregulated IL-36 signaling include generalized pustular psoriasis (GPP or DITRA) (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), which are incorporated by reference in their entireties), palmoplantar pustulosis (PPPP) (Bissonnette R. et al., PLoS One 11:e0155215 (2016), which are incorporated by reference in their entireties), inflammatory bowel disease (IBD) (Medina-Contreras et al., J Immunol 196:34-38 (2016; Nishida A. et al., Inflamm Bowel Dis 22:303-314 (2016) and Russell SE et al., Mucosal Immunol. 9:1193-1204 (2016)), rheumatoid arthritis and psoriatic arthritis (Frey S. et al., Ann Rheum Dis 72:1569-1574 (2013), which is incorporated by reference in its entirety), asthma, chronic obstructive pulmonary disease (Chen H. et al., J. Proteomics 75:2835-2843 (2012), which is incorporated by reference in its entirety), chronic kidney disease (Shaik Y. et al., Int J Immunopathol Pharmacol 26:27-36 (2013), which is incorporated by reference in its entirety), and ichthyosis (Paller AS et al., J Allergy Clin Immunol 26:27-36 (2013), which is incorporated by reference in its entirety). 139:152-165(2017)).

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

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

[0120] Candidate compounds can be evaluated by inducing inflammation, such as, for example, IMQ-induced skin inflammation or DSS-induced intestinal inflammation, in rodents as disclosed herein and determining whether the candidate compound can treat or inhibit the induced inflammation. The terms "treat" or "inhibit" include ameliorating the severity, alleviating the progression, eliminating, delaying or preventing the onset of induced inflammation and symptoms, or a combination thereof.

[0121] In some embodiments, the rodent is administered a candidate compound before, during, or after administration of an agent that induces inflammation. The candidate compound can be administered via any desired route of administration, including parenteral and non-parenteral routes of administration. Parenteral routes include, for example, intravenous, intraarterial, intraportal, intramuscular, subcutaneous, intraperitoneal, intrathecal, intraventricular, intracranial, intrathoracic, or other routes of injection. Non-injection routes include, for example, oral, nasal, transdermal, pulmonary, rectal, buccal, vaginal, and ocular. Administration may also be by continuous infusion, topical administration, sustained release from implants (gels, membranes, etc.), and / or intravenous injection.

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

[0123] To evaluate the efficacy of a candidate compound against skin inflammation, the compound may be administered to the rodent before, during, or after IMQ treatment. In certain embodiments, the candidate compound is administered subcutaneously at or near the skin area where IMQ is applied. A compound is considered effective if it inhibits skin inflammation compared to a control rodent not receiving the compound. For example, a compound may be effective if it inhibits skin inflammation by 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more (e.g., a 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% reduction); or one or more A treatment is considered effective if it reduces concentrations of pro-inflammatory mediators by 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more (e.g., a 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% reduction).

[0124] To evaluate the effectiveness of a candidate compound against colon inflammation, the compound may be administered to rodents before, during, or after oxazolone treatment. In some embodiments, the candidate compound is administered intraperitoneally for several days (e.g., 5, 6, 7, 8, or 9 days) after the initiation of DSS treatment. In some embodiments, the candidate compound is administered intraperitoneally multiple times during oxazolone treatment, for example, 2-3 days after topical application of oxazolone, at one or more rectal doses of oxazolone (e.g., at the first rectal dose of oxazolone and at the third rectal dose of oxazolone). A compound is considered effective if it inhibits colitis compared to control DITRA rodents not receiving the compound.For example, a compound may be effective when the total pathology score is reduced by 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more (e.g., by 20%-90%, 20%-80%, 20%-70%, 20%-60%, 20%-50%, 20%-40%, 30%-90%, 30%-80%, 30%-70%, 30%-60%, 30%-50%, or 30%-40%); or the concentration of one or more pro-inflammatory mediators is reduced by 15%, 20%, 30%, 40%, 50%, 60%, or more , 70%, 80%, 90% or more decreased (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% decreased); when fecal Lcn2 is decreased by 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more (e.g., 20%-90%, 20%-80%, 20%-70%, 20%-60%, 30%-50%, or 30%-40% decreased); when MPO activity in colon homogenates is decreased by 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more (e.g., 20%-90%, 20%-80%, 20%-70%, 20%-40%, 30%-90%, 30%-80%, 3 ...80%, 30%-40%, 30%-40%, 30%-50%, 30%-50%, 30%-60%, 30%-80%, 30%-70%, 20%-50%, 20%-60%, 30%-80%, 30%-80%, 30%-90%, 30%-50%, 20%-50%, 20%-60%, 30%-80%, 30%-90%, 30 a 30%-60%, 30%-50%, or 30%-40% reduction); a 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more increase in colon length (e.g., a 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 any combination thereof.

[0125] The present specification is further illustrated by the following examples, which should not be construed as limiting. All cited references, including literature references, issued patents, and published patent applications cited throughout this application, are expressly incorporated herein by reference in their entirety. EXAMPLES

[0126] Example 1. Generation of quadruple humanized mouse strains Two genetically engineered mouse strains were generated using VelociGene® technology (Poueymirou et al., Nat Biotechnol. 2007 Jan;25(1):91-9; Valenzuela et al., Nat Biotechnol 2003 Jun;21:652-59, which are incorporated by reference in their entireties). A "quadruple humanized" strain was generated by crossing the single and triple humanized strains. Generation of a single humanized Il1rl2 mouse line

[0127] The "single humanized" line was created by replacing a portion of the mouse Il1rl2 gene (interleukin-1 receptor-like 2 protein) encoding the extracellular domain of mouse Il1rl2 with a fragment of the human IL1RL2 gene (Il1rl2 / IL1RL2 exons 3-8, with portions of the intervening and flanking introns, respectively) encoding the corresponding extracellular domain of human IL1RL2 (Figure 1A). The resulting humanized gene encodes a chimeric receptor that maintains mouse intracellular signaling specificity while humanizing the extracellular domain, allowing it to bind IL1F6, IL1F8, and IL1F9 (also called IL36A, B, and G), respectively. Homozygous humanized Il1rl2 mice express Il1rl2 hu / hu It is called.

[0128] More specifically, mouse bacterial artificial chromosome (BAC) clone RP23-235L22 containing the mouse Il1rl2 gene was used and modified to provide a targeting vector as follows: A DNA fragment was generated containing the 5' mouse homologous nucleotide sequence, approximately 32,389 bp of human IL1RL2 genomic DNA (containing the 3' 346 bp of intron 2, exons 3-8 with all intervening introns, and the 5' 1101 bp of intron 8), approximately 4,996 bp of the auto-deleted neomycin cassette, and the 3' mouse homologous sequence (Figure 1B). This DNA fragment was used to modify the BAC clone RP23-235L22 via homologous recombination in bacterial cells. As a result, the mouse Il1rl2 genomic fragment encoding the ectodomain in the BAC clone (approximately 25,324 bp, including the 3′ 155 bp of mouse intron 2, exons 3 to 8 with all intervening introns, and the 5′ 642 bp of mouse intron 8) was replaced by human IL1RL2 genomic DNA, followed by a self-deleting neomycin cassette ( Fig. 1B ). The resulting modified BAC clone contained, from 5' to 3', (i) a 5' mouse homology arm containing approximately 112.2 kb of mouse genomic DNA, mouse Il1rl2 exons 1-2 and the 5' portion of intron 2; (ii) a human IL1RL2 genomic fragment containing the 3' portion of intron 2, exons 3-8, and the 5' portion of intron 8; (iii) an approximately 4,996 bp self-deleting neomycin cassette, followed by (iv) a 31.3 kb 3' mouse homology arm containing the remaining mouse Il1rl2 exons downstream of exon 8, all intervening introns and the 3'UTR. See Figure 1B. The junction sequences are also listed at the bottom of Figure 1B.

[0129] The modified BAC clone containing the humanized Il1rl2 gene described above was used as a targeting vector to electroporate mouse F1H4 embryonic stem cells (50% C57BL / 6NTac / 50%129S6 / SvEvTac) to generate modified ES cells containing the humanized Il1rl2 gene. Specifically targeted ES cells containing the humanized Il1rl2 gene were identified by an assay that detects the presence of the human IL1RL2 sequence (Valenzuela et al., supra), confirming the loss and / or retention of the mouse Il1rl2 sequence. The primers and probes used to confirm the humanization described above are listed in FIG. 5. Once a correctly targeted ES cell clone is selected, the neomycin selection cassette can be deleted. The humanized Il1rl2 locus after cassette deletion is shown in FIG. 1C, and the junction sequence is also shown in FIG. 1C. [Table 5]

[0130] Selected ES cell clones were microinjected into 8-cell embryos derived from Charles River Laboratories Swiss Webster albino mice to obtain F0 VelociMice®, which were 100% derived from the target cells (Poueymirou et al. 2007, see above). Mice carrying the humanized locus were again confirmed and identified by genotyping DNA isolated from tail fragments using a modified method of allele assay (Valenzuela et al., see above) that detects the presence of human gene sequences. Animals homozygous for the humanized locus were generated by mating heterozygous animals.

[0131] An alignment of the resulting amino acid sequences of the humanized / chimeric Il1rl2 receptor (SEQ ID NO:7), the mouse Il1rl2 protein (SEQ ID NO:4), and the human IL1RL2 protein (SEQ ID NO:2) is provided in FIG. 1D.

[0132] Generation of triple-humanized mouse strains A "triple humanized" line was generated in which the complete coding sequence of each of the mouse Il1f6, Il1f8, and Il1f9 genes was replaced with the complete coding sequence of the human IL1F6, IL1F8, and IL1F9 genes, respectively. This strategy resulted in humanized genes encoding human ligands capable of binding to the human extracellular domain of the chimeric Il1rl2 receptor. Homozygous humanized Il1f6, Il1f8, Il1f9 mice were generated using the Il1f6 hu / hu , Il1f8 hu / hu , Il1f9 hu / hu It is called.

[0133] More specifically, mouse bacterial artificial chromosome (BAC) clone RP23-90G23 containing mouse Il1f8, Il1f9, and Il1f6 genes and intergenic sequences were used to modify and provide a targeting vector as follows. A DNA fragment was generated to contain a 5' mouse homologous nucleotide sequence, approximately 88,868 bp of human genomic DNA (containing the promoter, untranslated regions, and coding sequences of human IL1F8, IL1F9, and IL1F6), an approximately 5,218 bp self-deleting hygromycin cassette, and a 3' mouse homologous sequence (Figure 2B). This DNA fragment was used to modify BAC clone RP23-90G23 via homologous recombination in bacterial cells. As a result, an approximately 76,548 bp genomic fragment in the BAC clone was replaced with human genomic DNA followed by the self-deleting cassette (Figures 2A-2B). The resulting modified BAC clone contained, from 5' to 3', (i) a 5' mouse homology arm containing approximately 5.7 kb of mouse genomic DNA, (ii) an approximately 88,868 bp human genomic fragment (containing the promoter, untranslated regions, and coding sequences for human IL1F8, IL1F9, and IL1F6), (iii) an approximately 5,218 bp self-deleting hygromycin cassette, and approximately 29.7 bp of 3' mouse homology sequence. See Figure 2B. The junction sequences are also set forth at the bottom of Figure 2B.

[0134] The modified BAC clones containing the human IL1F8, IL1F9, and IL1F6 gene sequences described above were used as targeting vectors to electroporate mouse F1H4 embryonic stem cells (50% C57BL / 6NTac / 50%129S6 / SvEvTac) to generate modified ES cells containing human IL1F8, IL1F9, and IL1F6 gene sequences. Specifically targeted ES cells containing human IL1F8, IL1F9, and IL1F6 gene sequences were identified by an assay that detects the presence of the human sequences (Valenzuela et al., supra) to confirm the loss and / or retention of the mouse sequences. The primers and probes used to confirm the humanization described above are listed in FIG. 6. Once a correctly targeted ES cell clone is selected, the hydromycin selection cassette can be deleted. The humanized locus after cassette deletion is shown in FIG. 2C, and the junction sequences are also shown in FIG. 2C. [Table 6]

[0135] Selected ES cell clones were microinjected into 8-cell embryos derived from Charles River Laboratories Swiss Webster albino mice to obtain F0 VelociMice®, which were 100% derived from the target cells (Poueymirou et al. 2007, see above). Mice carrying the humanized locus were again confirmed and identified by genotyping DNA isolated from tail fragments using a modified method of allele assay (Valenzuela et al., see above) that detects the presence of human gene sequences. Animals homozygous for the humanized locus were generated by mating heterozygous animals. Generation of triple-humanized mouse strains

[0136] The "triple humanized" strain was generated by crossing a single humanized strain with a triple humanized strain that had been bred homozygous for both loci on a 100% C57BL / 6NTac background. The homozygous triple humanized strain is Il1rl2 hu / hu Il1f6hu / hu Il1f8 hu / hu Il1f9 hu / hu or "DITRA-like" mice. The Neo and hygro cassettes were deleted in the F0 germline by self-deletion technique.

[0137] Example 2. Characterization of DITRA mice material and method Acute and chronic IMQ-induced skin inflammation induction and antibody treatment in DITRA-like mice - To induce skin inflammation, 8-10 week old humanized DITRA-like female mice were shaved on the back using a mouse hair trimmer (Oster, MiniMax, Cat# 78049-100) and the skin was depilated with 0.5 g of Veet hair removal gel three days prior to IMQ cream application. A daily topical dose of 62.5 g of commercial 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 the mice for four consecutive days for acute disease induction and nine days for chronic disease induction. A daily topical dose of 62.5 mg of Aldara translates to a daily dose of 3.125 mg of active compound. For acute IMQ-induced skin inflammation, anti-human IL-36R antibodies were administered subcutaneously to the dorsal skin at 10 mg / kg 3 days before and 1 day after the start of IMQ application. Control groups received PBS and 10 mg / kg hIgG4 isotype control injections. For acute IMQ-induced skin inflammation, the same anti-human IL-36R antibodies were administered therapeutically subcutaneously to the dorsal skin at 10 mg / kg 4 days before and 8 days after the start of IMQ application. Two or three days after treatment, the dorsal skin of the mice showed signs of erythema, scaling, and thickening. The severity of inflammation was measured daily using an adapted version of the clinical Psoriasis Area and Severity Index (PASI). Erythema, scaling, and thickening were scored independently on a scale of 0 to 4: 0, none; 1, mild; 2, moderate; 3, severe; and 4, extremely severe (van der Fits et al., J Immunol 2009, 182:5836-5845, incorporated herein by reference in its entirety). Skin thickness was measured using calipers (Kaefer) on day 4 of acute IMQ-induced skin inflammation and day 11 of chronic IMQ-induced skin inflammation.

[0138] Histopathology - Skin tissues 6 mm in diameter from the back of the mice were fixed in 10% buffered formalin, and 4-5 μm paraffin-embedded sections were stained with hematoxylin and eosin. Skin sections were blindly assessed for the presence of parakeratosis, normal keratosis, Munro microabscesses, acanthosis, skin ulceration, inflammation in the dermis and subcutaneous tissue, vascular congestion in the dermis and subcutaneous tissue, keratosis pilaris, and epidermal hyperplasia. A scoring scale of 0-4 was used: 0-within normal range, 1-minimal, 2-mild, 3-moderate, and 4-severe (van der Fits et al., J Immunol 2009,182:5836-5845, incorporated herein by reference in its entirety). 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 tissues with a diameter of 6 mm were collected from the back of mice and placed in 15 mL test tubes containing T-per buffer (Thermo Scientific, Cat#378510, Lot#RF236217), 1x Halt Protease Inhibitor Cocktail (Thermo Scientific, Cat#87786, Lot#QG221763) and 5M EDTA solution (Thermo Scientific, Cat3 78429). Skin tissues were disrupted using a Polytron (PT10-35 GT-D, Cat#9158158) at 28000 rpm for 1 min and placed on ice. The generated skin homogenates were centrifuged at 1500 rpm for 8 min at 4°C and the supernatants were collected into 96-well plates. Skin homogenates were subjected to Bradford protein assay using protein assay dye dye (BioRad, Cat# 500-0006, Lot# 210008149) to quantify total protein content. Cytokine concentrations in skin homogenates were measured using Proinflammatory Panel 1 (mouse) multiplex immunoassay kit (MesoScale Discovery, Cat# K15048D) according to the manufacturer's instructions. Briefly, 50 μL / well of calibrators and samples (diluted in Diluent 41) were added to the capture antibody pre-coated plates and incubated at room temperature with shaking at 700 rpm for 2 hours. The plates were 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 in Diluent 45. After 2 hours of incubation at room temperature with shaking, the plates were washed three times and 150 μL of 2× Read Buffer was added to each well. Electrochemiluminescence was immediately read on an MSD Spector® instrument. Data analysis was performed using GraphPad Prism™ software. Cytokine levels were normalized to total protein content.

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

[0141] Measurement of Lcn-2 in fecal samples - To monitor intestinal inflammation throughout the study, feces from individual DITRA-like mice were collected weekly in 2 mL deep well plates and stored at -80°C. At the end of the study, feces collected on different days were homogenized. Briefly, fecal samples were reconstituted with 1 mL of PBS containing 0.1% Tween-20, 1x Halt Protease Inhibitor Cocktail (Thermo Scientific, Cat# 87786, Lot# QG221763), and 5M EDTA solution (Thermo Scientific, Cat3 78429). After adding two tungsten 3 mm carbide beads to the wells (Qiagen, Cat# 69997), the plate was placed on a shaker at maximum speed overnight at 4°C. The homogenous fecal suspension was centrifuged at 1200 rpm for 10 minutes at 4°C, and the supernatant was collected in 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™ software.

[0142] Measurement of myeloperoxidase (MPO) activity in colon homogenates - Distal colon fragments were taken into a 2 mL microcentrifuge tube containing two tungsten 3 mm carbide beads (Qiagen, Cat# 69997) containing T-per buffer (Thermo Scientific, Cat# 378510, Lot# RF236217), 1× Halt Protease Inhibitor Cocktail (Thermo Scientific, Cat# 87786, Lot# QG221763) and 5M EDTA solution (Thermo Scientific, Cat# 78429). The homogenates were lysed for 27.5 s using a Qiagen Tissue Lyser II. -1Colon tissue was disrupted for 10 min at a frequency of 100 rpm. The tubes were centrifuged at 1500 rpm for 8 min at 4° C., and the supernatant was collected in a 96-well plate. Colon homogenates were subjected to Bradford protein assay using protein assay dye (BioRad, Cat# 500-0006, Lot# 210008149) to quantify total protein content. Myeloperoxidase (MPO) activity in colon homogenates was measured using Mouse MPO ELISA Kit (HHycult Biotech, Cat# HK210-02, Lot# 21022KO617-Y) according to the manufacturer's instructions. Data analysis was performed using GraphPad Prism™ software. MPO levels were normalized to total protein content.

[0143] Measurement of cytokines 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 calibrators and samples (diluted in Diluent 41) were added to the capture antibody pre-coated plates and incubated at room temperature with shaking at 700 rpm for 2 hours. The plates were 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 in Diluent 45. After 2 hours of incubation at room temperature with shaking, the plates were washed three times and 150 μL of 2× Read Buffer was added to each well. Electrochemiluminescence was read immediately on an MSD Spector® instrument. Data analysis was performed using GraphPad Prism™ software. Cytokine levels were normalized to total protein content.

[0144] Statistical analysis - Statistical significance within groups was determined by one-way ANOVA using Tukey's multiple comparison post-hoc test (*p<0.05, **p<0.005, ***p<0.0005, ****p<0.0001) (same for both *-statistically significant difference from PBS-treated group, and #-statistically significant difference from corresponding isotype-treated group).

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

[0146] To investigate the role of deregulated IL-36R signaling similar to GPP or DITRA ("Interleukin 36 Receptor Antagonist Deficiency") patients, mice were generated with humanized IL-36R (e.g., Il1rl2) and human IL-36α, β, γ ligands, but no IL-36Ra antagonist, as described in Example 1. The resulting hIL-36R / hIL-36α, β, γ mice were termed DITRA-like mice due to a 20-fold reduction in the affinity of mouse IL36Ra for human IL-36R, resulting in enhanced IL-36R signaling similar to DITRA patients (for a more detailed discussion of DITRA patients, see, e.g., Marrakchi et al., N Engl J Med 2011,365:620-628, which is incorporated herein by reference in its entirety).

[0147] In the unloaded condition, DITRA-like mice did not develop any spontaneous disease. In contrast, in a preclinical model of IMQ-induced psoriasiform dermatitis, which closely resembles human psoriatic lesions in terms of phenotypic and histological features (for a more detailed discussion of the properties of psoriasiform 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 WT littermates (Figures 3A-3B). Briefly, IMQ was applied daily for four consecutive days to the shaved dorsal skin of DITRA-like and WT mice. On the fifth day, the skin was harvested for subsequent histopathological evaluation, protein and RNA isolation. IMQ-treated DITRA-like mice developed more severe psoriatic lesions, such as desquamation, erythema, and skin thickening, compared with WT littermates (Figure 3A). Consistent with the clinical characteristics, histopathological evaluation of the skin revealed more enhanced acanthosis, parakeratosis, and disturbance of keratinocyte differentiation in DITRA-like mice (Figure 3B). IMQ-treated DITRA-like mice also presented with a marked increase in the number of Munro abscesses or pustules, similar to GPP patients. Furthermore, application of IMQ led to an increase in the levels of pro-inflammatory molecules that were deregulated in psoriasis (e.g., pro-inflammatory molecules including IL-17a, IL-17f, IL-23a, S100A8, Defb4) in the skin of DITRA-like mice compared with WT littermates (Figure 3C). Furthermore, IL-36α, IL-36β, and IL-36γ mRNA expression was equally increased in IMQ-treated DITRA-like and IMQ-treated wild-type mice compared with Vaseline-treated DITRA-like and Vaseline-treated wild-type mice, whereas protein levels of IL-36α and IL-36β cytokines were detected at two-fold higher concentrations in the inflamed skin of DITRA-like mice compared with the levels in the inflamed skin of wild-type mice.

[0148] Humanized DITRA-like mice exhibit defects in mucosal healing in a DSS-induced chronic colitis model - Recently, several studies in a subset of patients have demonstrated deregulated expression of the IL-36 axis in IBD and its possible contribution to intestinal inflammation. Expression of IL-36α and IL-36β has been shown to be elevated in the inflamed mucosa of patients with ulcerative colitis (see Medina-Contreras et al., J Immunol 2016,196:34-38; Nishida et al., Inflamm Bowel Dis 2016,22:303-314, and Russell, Mucosal Immunol 2016,9:1193-1204, all of which are incorporated herein by reference). In 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, which are incorporated by reference in their entirety). At steady state, DITRA-like mice did not develop spontaneous ileitis / colitis, but exhibited significantly shortened colons at young ages (3 and 10 months of age) (Figures 4A and 4B). In addition, compared to steady state and wild-type mice, DITRA-like mice showed elevated levels of IL-36 cytokines, as well as trends toward elevated levels of IL-17F and IL-17A, and reduced 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 (Okayasu et al., Gastroenterology 1990, 98:694-702, herein incorporated by reference in its entirety) and triggers a strong inflammatory response (Rakoff-Nahoum et al., Cell 2004, 118:229-241), which represents a major feature of IBD, especially in ulcerative colitis. DITRA-like mice and their co-housed WT littermates were subjected to a DSS-induced chronic colitis regimen with 2.5% DSS for 7 days, followed by water (first dose) for 11 days and 1.5% DSS for 4 days, followed by water (second dose) for 5 days. During the acute phase of the disease, DITRA-like mice developed intestinal inflammation similar to their co-housed WT littermates (Figure 5A). Interestingly, during the recovery phase of the disease, DITRA-like mice exhibited persistent weight loss (Figure 5A), significant shortening of colon length (Figure 5B), more severe pathology scores (associated with more severe ulceration, extensive epithelial erosion and neutrophil infiltration) (Figure 5C), a significant elevation of 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 an inability to recover from DSS-induced mucosal injury reflected by reduced IL-22 levels in the colon (Figure 5E). Moreover, DITRA-like mice exhibited a mortality rate of approximately 62.5% (data not shown). In contrast, WT mice recovered during the recovery phase of colitis with a mortality rate of 25%. Intestinal epithelial integrity was also investigated by oral gavage of DITRA-like and wild-type mice with fluorescein isothiocyanate (FITC)-dextran.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 the control of intestinal permeability during intestinal injury.Thus, enhanced IL-36 signaling in DITRA-like mice leads to exacerbated intestinal inflammation and defective mucosal repair, suggesting a role for IL-36 in controlling mechanisms involved in intestinal tissue remodeling.

[0150] Anti-human IL-36R monoclonal antibodies inhibit 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 antibodies were tested in an IMQ-induced model of psoriasis-like dermatitis. IMQ was applied daily for four consecutive days to the shaved dorsal skin of DITRA-like mice. Anti-human IL-36R monoclonal antibodies were administered at 10 mg / kg 3 days before (-3d) and 1 day after (d1) IMQ application was initiated. Control groups received PBS and hIgG4 isotype control injections at 10 mg / kg. On day 5, skin was harvested for later histopathological evaluation and protein isolation. Anti-human IL-36R monoclonal antibodies significantly reduced IMQ-induced total pathology scores, including parakeratosis and Munro microabscesses, compared to the PBS- and isotype control-treated groups (Figure 6A). Moreover, 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 pro-inflammatory cytokines deregulated in psoriasis (e.g., IL-12p40, IL-17f, and IL-17a) (Figure 6B), suggesting a tightly regulated interplay 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 the 10 mg / kg dose resulting in a more potent inhibition of skin inflammation as determined based on skin homogenate pathology scores, skin thickness, and pro-inflammatory cytokine levels compared to the 1 mg / kg dose (data not shown).

[0151] Anti-human IL-36R monoclonal antibodies inhibit chronic skin inflammation upon therapeutic administration - To further explore the therapeutic efficacy of human IL-36R antagonism in vivo, the same anti-human IL-36R monoclonal antibodies were tested in a chronic IMQ-induced skin inflammation model. Over a two-week period, IMQ was applied to the shaved dorsal skin of DITRA-like mice twice over nine days, with two days of no treatment in between. Anti-human IL-36R monoclonal antibodies were administered subcutaneously at 10 mg / kg on days 5 and 9 after the start of IMQ application. Control groups received PBS and hIgG4 isotype control injections at 10 mg / kg. On day 12, skin thickness was measured and tissues were harvested for subsequent histopathological evaluation and protein isolation. Similar to acute IMQ-induced inflammation, chronic IMQ application led to upregulation of proinflammatory mediators in the skin of DITRA-like mice (data not shown). Anti-human IL-36R monoclonal antibodies showed significant efficacy in reducing IMQ-induced skin thickness and pathological lesion scores in DITRA-like mice (Figures 7A and 7B, respectively). 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 ameliorating acute and chronic IMQ-induced skin inflammation in vivo.

[0153] Anti-human IL-36R monoclonal antibody, upon therapeutic administration, ameliorates DSS-induced chronic colitis in DITRA-like mice - DITRA-like mice exhibited defects in mucosal healing as a result of enhanced IL-36 signaling, and we further investigated whether IL-36 blockade would rescue the observed phenotype. DITRA-like mice were subjected to chronic DSS-induced colitis by administering two cycles of 3% DSS for 7 days followed by water for 13 days. The same anti-human IL-36R monoclonal antibody and anti-mIL-12p40 monoclonal antibody (a mouse surrogate for ustekinumab approved for the treatment of Crohn's disease) were administered at 10 mg / kg every other week starting on day 7. Control groups received PBS and the corresponding hIgG4 and rat IgG2a isotype control injections at 10 mg / kg. Treatment with anti-human IL-36R monoclonal antibody rescued DITRA-like mice from DSS-induced mucosal injury and reduced disease severity compared to PBS and isotype control treatment in DITRA-like mice (Figure 8A). To monitor intestinal inflammation at different stages of disease, feces from individual mice were collected weekly and fecal lipocalin-2 (Lcn2) protein, a non-invasive biomarker of inflammation in intestinal injury, was measured (e.g., Thorsvik et al., J Gastroenterol Hepatol 2017,32:128-135, incorporated herein by reference in its entirety). As shown in Figure 8B, the PBS-treated, hIgG4-treated, and rat IgG2a-treated groups exhibited significant upregulation of fecal Lcn2 levels on days 17, 23, and 30 compared to the water-only group. Surprisingly, administration of anti-human IL-36R monoclonal antibody resulted in a significant decrease in Lcn2 levels compared to the PBS- and isotype-treated groups. A sustained decrease in fecal Lcn2 levels was observed in the anti-human IL-36 antibody-treated group on days 17, 23, and 30 (Figure 8B).

[0154] Furthermore, hIL-36R blockade with anti-human IL-36R monoclonal antibodies resulted in increased colon length (Figure 8C) and reduced 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 monoclonals were as effective as IL-12p40 blockade in ameliorating DSS-induced chronic colitis, as reflected by reduced fecal Lcn2, MPO activity, and pro-inflammatory cytokines in the colon of DITRA-like mice (Figures 8A-8E).

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

[0156] Various publications, including patents, patent applications, published patent applications, accession numbers, technical articles, and journal articles, are cited herein. Each of these cited publications is incorporated by reference in its entirety and for all purposes.

Claims

1. 1. A genetically modified rodent, the genome of which comprises: (1) a humanized Il1rl2 gene at an endogenous rodent Il1rl2 locus, the humanized Il1rl2 gene encoding a humanized Il1rl2 protein comprising an ectodomain having at least 95% sequence identity with an ectodomain of a human IL1RL2 protein, the ectodomain of the human IL1RL2 protein comprising amino acids 20-335 of SEQ ID NO:2; (2) the human IL1F6 gene at the endogenous rodent Il1f6 locus; and (3) the human IL1F8 gene at the endogenous rodent Il1f8 locus; and (4) a human IL1F9 gene at an endogenous rodent Il1f9 locus; A genetically modified rodent, wherein said genetically modified rodent exhibits deregulated IL-36 signaling.

2. The genetically modified rodent of claim 1 , wherein the humanized Il1rl2 protein comprises a transmembrane cytoplasmic sequence that is at least 95% identical to the transmembrane cytoplasmic sequence of an endogenous rodent Il1rl2 protein.

3. 3. The genetically modified rodent of claim 1 or 2, wherein the humanized Il1rl2 protein comprises a signal peptide having at least 95% sequence identity to the signal peptide of an endogenous rodent Il1rl2 protein.

4. The genetically modified rodent of any one of claims 1 to 3, wherein the human IL1RL2 protein comprises the amino acid sequence set forth in SEQ ID NO:

2.

5. The genetically modified rodent of any one of claims 1 to 3, wherein the ectodomain of the humanized Il1rl2 protein comprises amino acids 22 to 337 of SEQ ID NO:

7.

6. The genetically modified rodent of any one of claims 1 to 3, wherein the humanized Il1rl2 protein comprises the amino acid sequence set forth in SEQ ID NO:

7.

7. 7. The genetically modified rodent of claim 1, wherein the humanized Il1rl2 gene is operably linked to an endogenous rodent Il1rl2 promoter at the endogenous rodent Il1rl2 locus.

8. 8. The genetically modified rodent of claim 1, wherein the humanized Il1rl2 gene results from replacing a genomic fragment of an endogenous rodent Il1rl2 gene at the endogenous rodent Il1rl2 locus with the nucleotide sequence of a human IL1RL2 gene.

9. The genetically modified rodent of claim 8, wherein the nucleotide sequence of the human IL1RL2 gene is a genomic fragment of the human IL1RL2 gene encoding a polypeptide having at least 95% sequence identity to the ectodomain of the human IL1RL2 protein.

10. The genetically modified rodent of claim 9, wherein the genomic fragment of the human IL1RL2 gene comprises exons 3-8 of the human IL1RL2 gene.

11. 11. The genetically modified rodent of claim 8, wherein exons downstream of exon 8 of the endogenous rodent Il1rl2 gene remain after the replacement.

12. 12. The genetically modified rodent of claim 8, wherein exons 1-2 of the endogenous rodent Il1rl2 gene remain after the replacement.

13. 9. The genetically modified rodent of claim 8, wherein the humanized Il1rl2 gene comprises exons 1-2 of the endogenous rodent Il1rl2 gene, exons 3-8 of the human IL1RL2 gene, and exons downstream of exon 8 of the endogenous rodent Il1rl2 gene.

14. A genetically modified rodent according to any one of claims 1 to 13, wherein the human IL1F6 gene replaces the endogenous rodent Il1f6 gene at the endogenous rodent Il1f6 locus.

15. 15. The genetically modified rodent of any one of claims 1 to 14, wherein the human IL1F8 gene replaces the endogenous rodent Il1f8 gene at the endogenous rodent Il1f8 locus.

16. 16. The genetically modified rodent of any one of claims 1 to 15, wherein the human IL1F9 gene replaces the endogenous rodent Il1f9 gene at the endogenous rodent Il1f9 locus.

17. 17. The genetically modified rodent of any one of claims 1 to 16, wherein the rodent is homozygous for each of the humanized Il1rl2 gene, the human IL1F6 gene, the human IL1F8 gene and the human IL1F9 gene.

18. The genetically modified rodent of claim 17 , wherein the rodent exhibits a shortened colon compared to a wild-type rodent.

19. A genetically modified rodent according to any one of claims 1 to 18, wherein the rodent is a mouse or a rat.

20. 1. A method for producing a genetically modified rodent, comprising: The rodent genome, (1) a humanized Il1rl2 gene at an endogenous rodent Il1rl2 locus, the humanized Il1rl2 gene encoding a humanized Il1rl2 protein comprising an ectodomain having at least 95% sequence identity with an ectodomain of a human IL1RL2 protein, the ectodomain of the human IL1RL2 protein comprising amino acids 20-335 of SEQ ID NO:2; (2) the human IL1F6 gene at the endogenous rodent Il1f6 locus; and (3) the human IL1F8 gene at the endogenous rodent Il1f8 locus; and (4) a human IL1F9 gene at the endogenous rodent Il1f9 locus; and generating a rodent comprising said modified genome; The genetically modified rodent exhibits deregulated IL-36 signaling.

21. The rodent genome comprises: (i) generating a first rodent comprising the humanized Il1rl2 gene at the endogenous rodent Il1rl2 locus; (ii) generating a second rodent comprising 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) mating the first rodent with the second rodent.

22. said rodent comprising said humanized Il1rl2 gene at said endogenous rodent Il1rl2 locus, Providing rodent embryonic stem (ES) cells; inserting a nucleotide sequence of a human IL1RL2 gene into the rodent Il1rl2 locus of the rodent ES cell to form the humanized Il1rl2 gene at the rodent Il1rl2 locus, thereby obtaining a rodent ES cell comprising the humanized Il1rl2 gene; and generating a rodent using the rodent ES cells containing the humanized Il1rl2 gene.

23. 23. The method of claim 22, wherein the nucleotide sequence of the human IL1RL2 gene replaces a genomic fragment of a rodent Il1rl2 gene at the endogenous rodent Il1rl2 locus.

24. 24. The method of claim 23, wherein the nucleotide sequence of the human IL1RL2 gene is a genomic fragment of the human IL1RL2 gene encoding a polypeptide having at least 95% sequence identity to the ectodomain of the human IL1RL2 protein.

25. The method of claim 24, wherein the genomic fragment of the human IL1RL2 gene comprises exons 3 to 8 of the human IL1RL2 gene.

26. 26. The method of any one of claims 23 to 25, wherein exons downstream of exon 8 of the endogenous rodent Il1rl2 gene remain after the replacement.

27. 27. The method of any one of claims 23 to 26, wherein exons 1-2 of the endogenous rodent Il1rl2 gene remain following the replacement.

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

29. the second rodent comprising the human IL1F6 gene, the human IL1F8 gene, and the human IL1F9 gene; Providing rodent embryonic stem (ES) cells; (i) inserting the human IL1F6 gene into the rodent IL1F6 locus of the rodent ES cell; (ii) the human IL1F8 gene 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; and Thereby, obtaining rodent ES cells containing the human IL1F6 gene, the human IL1F8 gene, and the human IL1F9 gene; and generating the second rodent using the rodent ES cells containing the human IL1F6 gene, the human IL1F8 gene, and the human IL1F9 gene.

30. The method of claim 29, wherein the human IL1F6 gene, the human IL1F8 gene, and the human IL1F9 gene are provided in a contiguous nucleic acid molecule.

31. The method of any of claims 20 to 30, wherein the human IL1F6 gene replaces the endogenous rodent Il1f6 gene at the endogenous rodent Il1f6 locus.

32. The method of any of claims 20 to 31, wherein the human IL1F8 gene replaces the endogenous rodent Il1f8 gene at the endogenous rodent Il1f8 locus.

33. The method of any of claims 20 to 32, wherein the human IL1F9 gene replaces the endogenous rodent Il1f9 gene at the endogenous rodent Il1f9 locus.

34. 20. An isolated cell or tissue of a rodent according to any one of claims 1 to 19.

35. 1. A rodent embryonic stem (ES) cell, comprising: (1) a humanized Il1rl2 gene at an endogenous rodent Il1rl2 locus, the humanized Il1rl2 gene encoding a humanized Il1rl2 protein comprising an ectodomain having at least 95% sequence identity with an ectodomain of a human IL1RL2 protein, the ectodomain of the human IL1RL2 protein comprising amino acids 20-335 of SEQ ID NO:2; (2) the human IL1F6 gene at the endogenous rodent Il1f6 locus; and (3) the human IL1F8 gene at the endogenous rodent Il1f8 locus; and (4) A rodent embryonic stem (ES) cell comprising a human IL1F9 gene at an endogenous rodent Il1f9 locus.

36. 36. Use of the rodent ES cell of claim 35 in generating a rodent.

37. 20. Use of a rodent according to any one of claims 1 to 19 as a rodent model for a disease associated with deregulated IL-36 signalling.

38. 1. A method for evaluating the therapeutic efficacy of a candidate compound for treating a disease associated with deregulated IL-36 signaling, comprising: Providing a rodent according to any one of claims 1 to 19, administering an agent to said rodent to induce inflammation; administering a candidate compound to said rodent; and determining whether the candidate compound inhibits the induced inflammation.

39. 39. The method of claim 38, wherein the agent is DSS or oxazolone and intestinal inflammation is induced in the rodent.

40. 39. The method of claim 38, wherein the agent is IMQ and skin inflammation is induced in the rodent.

41. The method of any one of claims 38 to 40, wherein the candidate compound is administered to the rodent before, during or after administration of the agent.

42. The method of any one of claims 38 to 41, wherein the candidate compound is a small molecule compound, a nucleic acid inhibitor, or an antibody.

43. 43. The method of claim 42, wherein the candidate compound is an antibody.

44. 36. A combination of a first targeting nucleic acid construct and a second targeting nucleic acid construct for generating a genetically modified rodent according to claim 1 or a rodent embryonic stem (ES) cell according to claim 35, comprising: the first targeting nucleic acid construct comprises a nucleotide sequence of a human IL1RL2 gene flanked by 5' and 3' rodent nucleotide sequences capable of mediating homologous recombination to integrate the nucleotide sequence of the human IL1RL2 gene into the rodent Il1rl2 locus to form a humanized Il1rl2 gene, the humanized Il1rl2 gene encoding a humanized Il1rl2 protein comprising an ectodomain having at least 95% sequence identity to an ectodomain of a human IL1RL2 protein, the ectodomain of the human IL1RL2 protein comprising amino acids 20-335 of SEQ ID NO:2; and A combination wherein the second targeting nucleic acid construct comprises a contiguous nucleic acid sequence comprising a human IL1F6 gene, a human IL1F8 gene, and a human IL1F9 gene, said contiguous nucleic acid sequence flanked by 5' and 3' rodent nucleotide sequences capable of mediating homologous recombination and integrating said contiguous nucleic acid sequence into a rodent locus comprising a rodent Il1f6 gene, a rodent Il1f8 gene, and a rodent Il1f9 gene.

Images