Construction and application of spontaneous psoriasis and psoriatic arthritis animal models

Abstract

The present invention provides the construction and application of a spontaneous psoriasis and psoriatic arthritis animal model. The animal model is established by conditionally expressing IL23a in the animal's IL4i1-positive cells. The animal model mimics the pathogenesis of human psoriasis, exhibits stable and controllable conditions, exhibits typical disease symptoms, and is intuitive and easy to observe. The present invention provides an objective quantitative method for evaluating the proposed psoriasis-like animal model. This method can be strategically applied in future research to assess animal disease phenotypes, conduct in-depth studies on the pathogenesis of related diseases such as psoriasis and psoriatic arthritis, and provide a more scientific animal model for drug development.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology; more specifically, the present invention relates to the construction and application of a spontaneous psoriasis and psoriatic arthritis animal model. Background Art

[0002] Psoriasis (PsO) is a chronic, inflammatory autoimmune skin disease influenced by genetics and various environmental factors. Its pathogenesis primarily involves interactions between keratinocytes, immune cells, and other stromal cells of the skin tissue. The IL-23 / IL-17 axis, which regulates DCs and T cells, is key in driving the pathogenesis of psoriasis. Keratinocytes are activated by inflammatory factors such as TNF-α, IL-17, IL-21, and IL-22, producing antimicrobial peptides, cytokines, and chemokines, which promote the amplification of inflammatory signals. Numerous biologics targeting TNF-α or IL-17 have been developed and used to treat psoriasis. However, relapse after discontinuation of these biologics has prompted researchers to explore new treatment strategies.

[0003] Because most biologic agents used for psoriatic skin disease also reduce arthritis in psoriatic patients, it has been proposed that these joint and skin diseases share a common immunopathological mechanism. Recent genetic analyses have shown that psoriatic arthritis (PsA) and PsO share common susceptibility genes, including genes related to HLA class I, NF-κB, and IFN signaling. However, the underlying mechanisms of the "PsO to PsA transition" remain unclear. Therefore, the development of a mouse model that can reproduce the characteristics of psoriasis will facilitate the study of its pathological mechanisms and the development of therapeutic drugs.

[0004] Numerous psoriasis mouse models have been reported, including Imiquimod, K5-Tie2, and K14-AREG, but none reproduce the hallmarks of human psoriasis. Although the skin gene expression profiles associated with each mouse phenotype are statistically significant similar to those of human psoriasis, each model exhibits distinct similarities and differences with human psoriasis.

[0005] Therefore, there is an urgent need to develop new animal models of psoriasis with typical symptoms in this field to simulate the occurrence and development of the disease and provide a more effective approach for the development of pathological mechanisms and therapeutic drugs for psoriasis. Summary of the Invention

[0006] The purpose of the present invention is to provide a construction and application of a spontaneous psoriasis and psoriatic arthritis animal model.

[0007] In a first aspect of the present invention, a method for preparing a spontaneous psoriasis or psoriatic arthritis animal model is provided, comprising: conditionally expressing (overexpressing) IL23a in IL4i1-positive (IL4i1+) cells of the animal.

[0008] In one or more embodiments, the conditional expression is performed using a Cre / loxP recombination system; preferably, the method comprises: (1) providing animal 1, introducing a Cre recombinase expression element into the genome of its IL4i1-positive (IL4i1+) cells, which can be operably linked to the IL4i1-encoding nucleic acid in the genome; the Cre recombinase expression element comprises a Cre recombinase-encoding nucleic acid; (2) providing animal 2, introducing an expression cassette for conditional expression of IL23a into its genome, the expression cassette comprising: a promoter, Loxp-terminator-Loxp, and an IL23a expression element operably linked; the expression element comprises an IL23a-encoding nucleic acid; (3) mating the animals (1) and (2) to obtain offspring; wherein, when the Cre recombinase is expressed, it recognizes two LoxPs and cuts the terminator therebetween, so that the IL4i1-positive cells and their progeny cells continuously express Il23a.

[0009] In one or more embodiments, in (1), the Cre recombinase expression element further includes: a 2A peptide encoding nucleic acid located upstream of the Cre recombinase encoding nucleic acid, and a termination sequence is also set downstream of the Cre recombinase expression element located downstream of the Cre recombinase encoding nucleic acid.

[0010] In one or more embodiments, the Cre recombinase expression element is introduced into the stop codon position of IL4i1 in the animal genome.

[0011] In one or more embodiments, the Cre recombinase expression element is introduced into the genome of IL4i1-positive cells by setting homology arms on both sides.

[0012] In one or more embodiments, the 2A peptide encoding nucleic acid allows Cre recombinase encoding nucleic acid to be linked to IL4i1 encoding nucleic acid (IL4i1-2A-Cre-terminator sequence (eg, polyA)) for fusion expression.

[0013] In one or more embodiments, in (2), in the Loxp-terminator-Loxp, a resistance screening gene expression element (promoter-resistance screening gene; the promoter (such as PGK) is different from the promoter upstream of Loxp (such as CAG)) is also set upstream of the terminator.

[0014] In one or more embodiments, in (2), the IL23a expression element further comprises: a reporter gene connected to the IL23a encoding nucleic acid, and a termination sequence; preferably, the IL23a encoding nucleic acid and the reporter gene are connected via an IRES.

[0015] In one or more embodiments, in (2), the expression cassette for conditional expression of IL23a is introduced into the Rosa26 gene locus in its genome.

[0016] In one or more embodiments, the animal is a rodent.

[0017] In one or more embodiments, the rodents include: mice, rats.

[0018] In one or more embodiments, the method comprises:

[0019] Step 1: Construction of Il4i1-Cre mice;

[0020] Step 2: Construct the CAG-loxp-Neo-loxp-Il23a-IRES-EGFP sequence for site-directed knock-in of the Rosa26 gene to obtain Rosa-Il23a-EGFP mice;

[0021] Step 3: Il4i1-Cre heterozygous mice were mated with Rosa-Il23a-EGFP heterozygous mice;

[0022] Step 4: Obtain offspring Il4i1-Cre; Rosa-Il23a-EGFP mice (ear tissue testing can be performed after 10 weeks);

[0023] Step 4: Obtained offspring Il4i1-Cre; Rosa-Il23a-EGFP mice (micro-CT examination of mouse toe joints was performed after 10-14 weeks).

[0024] In another aspect of the present invention, there is provided the use of a spontaneous psoriasis or psoriatic arthritis animal model obtained by the method described above, for use as: an animal model for screening candidate drugs or therapeutic agents for alleviating or treating psoriasis or psoriatic arthritis; or as an animal model for studying psoriasis or psoriatic arthritis.

[0025] In one or more embodiments, the method or use is a non-diagnostic or therapeutic method or use, and is not intended to diagnose or treat a disease.

[0026] In another aspect of the present invention, a kit for preparing an animal model of spontaneous psoriasis or psoriatic arthritis is provided, comprising: a Cre recombinase expression element, comprising a Cre recombinase encoding nucleic acid; an expression cassette for conditionally expressing IL23a, comprising: a promoter, Loxp-terminator-Loxp, and an IL23a expression element operably linked; the expression element comprising an IL23a encoding nucleic acid.

[0027] In one or more embodiments, the Cre recombinase expression element further includes: a 2A peptide encoding nucleic acid located upstream of the Cre recombinase encoding nucleic acid, and a termination sequence is also provided downstream of the Cre recombinase expression element located downstream of the Cre recombinase encoding nucleic acid; preferably, homology arms are provided on both sides of the Cre recombinase expression element, so that the Cre recombinase expression element can be introduced into the genome of IL4i1-positive cells; preferably, it is introduced into the termination codon position of IL4i1 in the animal genome.

[0028] In one or more embodiments, in the Loxp-terminator-Loxp expression cassette for conditional expression of IL23a, a resistance selection gene expression element (promoter-resistance selection gene; the promoter (e.g., PGK) is different from the promoter upstream of Loxp (e.g., CAG)) is further provided upstream of the terminator.

[0029] In one or more embodiments, the IL23a expression element of the expression cassette for conditional expression of IL23a further comprises: a reporter gene connected to the IL23a encoding nucleic acid, and a termination sequence; preferably, the IL23a encoding nucleic acid and the reporter gene are connected via an IRES.

[0030] In another aspect of the present invention, a method for screening candidate drugs or therapeutic agents for relieving or treating psoriasis or psoriatic arthritis is provided, the method comprising: (1) preparing a spontaneous psoriasis or psoriatic arthritis animal model using any of the methods described above; (2) administering a candidate substance to the animal model of (1) to observe whether the candidate substance has an relieving or therapeutic effect on psoriasis or psoriatic arthritis; if it is observed that the psoriasis or psoriatic arthritis symptoms of the animal model are relieved, the candidate substance is a substance that relieves or treats psoriasis or psoriatic arthritis.

[0031] In one or more embodiments, the test further includes providing a control animal, wherein the control animal is not administered with the candidate substance.

[0032] Other aspects of the present invention will be apparent to those skilled in the art in view of the disclosure herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1Schematic diagram of the construction method of Il4i1-Cre mice, Il23a gene conditional overexpression mice and Il4i1-Cre;Rosa-Il23a-EGFP mice.

[0034] A shows the site-specific knock-in of the 2A-Cre-WPRE-polyA expression cassette at the stop codon of the Il4i1 gene. In this mouse, all cells expressing Il4i1 will simultaneously express the Cre recombinase.

[0035] B is a mouse model of conditional overexpression of the Il23a gene. Using the principle of homologous recombination and ES cell targeting, a CAG-Loxp-PGK-Neo-polyA-Loxp-Il23a-IRES-EGFP-polyA expression cassette was inserted into the Rosa26 gene locus.

[0036] C represents mice conditionally overexpressing the Il23a gene at the Rosa26 locus. When mated with Il4i1-Cre mice, Cre recombinase recognizes the two LoxP sequences at the Rosa26 locus and cleaves the intervening termination signal sequence (LoxP-PGK-Neo-polyA-LoxP). In the resulting offspring, the loxP-PGK-Neo-polyA-loxP expression cassette is knocked out, and the target gene Il23a is expressed in Il4i1+ cells under the drive of the CAG promoter.

[0037] Figure 2 Il4i1-Cre; Rosa-Il23a-EGFP (abbreviated as: Il4i1-Il23a OE ) Spontaneous psoriasis-like phenotype in mice;

[0038] A is Il4i1-Il23a OE Ear skin phenotype of heterozygous mice at 12 weeks of age;

[0039] B is Il4i1-Il23a OE H&E staining of histopathological sections of ear skin of mice and control group mice (abbreviated as: LSL-IL23a);

[0040] C is Il4i1-Il23a OE Keratinocyte abnormality markers in mice and control mice: Ki67, keratin 5 (K5), keratin 6 (K6); keratinocyte normal differentiation markers: 1 / 10 (K1 / 10);

[0041] D is flow cytometry analysis of Il4i1-Il23a OE Expression of IL-17A cells in the middle ear skin of mice and control mice;

[0042] E is RNA bulk-seq sequencing data analysis of Il4i1-Il23a OE The similarity of gene expression profiles between mice and the IMQ-induced psoriasis-like mouse model (the most widely used animal model of psoriasis to date).

[0043] Figure 3 Il4i1-Il23a OE Spontaneous psoriatic arthritis phenotype in mice

[0044] A is Il4i1-Il23a OE The mice developed a red and swollen phenotype in the paw pads;

[0045] B micro-CT image showing the proximal metacarpophalangeal joint of the mouse foot;

[0046] C micro-CT image of the mouse paw.

[0047] Figure 4 To validate the response of the established animal model to the drug using IL-17 antibody neutralization therapy. DETAILED DESCRIPTION

[0048] The inventors are committed to the establishment of an animal model of spontaneous psoriasis or psoriatic arthritis. In the early research work, multi-target and multi-faceted analysis was carried out, revealing targets suitable for targeting. By conditionally expressing (overexpressing) IL23a in the IL4i1-positive (IL4i1+) cells of the animal, an animal model capable of presenting symptoms of spontaneous psoriasis or psoriatic arthritis is provided. The animal model prepared by the method of the present invention simulates the pathogenesis of human psoriasis, has a stable and controllable state, typical disease symptoms, and is intuitive and easy to observe. The present invention provides an objective quantitative method for evaluating the proposed psoriasis-like animal model, which can be strategically used in future studies to evaluate animal disease phenotypes, conduct in-depth research on the pathogenesis of related diseases such as PsO and PsA, and provide a more scientific animal model for drug development.

[0049] As used in the present invention, "animal" refers to mammals; preferably, the "animal" includes rodents, such as mice and rats.

[0050] As used herein, "exogenous" refers to the relationship between two or more nucleic acid or protein sequences from different sources, or the relationship between a protein / gene and a host. For example, although the host itself may contain the corresponding gene or produce the corresponding protein, a synthetic / recombinant gene / protein is "exogenous" with respect to the host cell when introduced into the host cell through genetic engineering methods. The term "exogenous" includes "heterologous." A "heterologous" nucleic acid or protein is generally not present in the host cell itself.

[0051] As used in the present invention, the "psoriasis animal model" includes a "psoriasis-like animal model", wherein the meaning of "like" includes "simulation" or "replication".

[0052] As used herein, the term "psoriatic arthritis" includes "psoriatic-like arthritis".

[0053] The present invention provides a method for + A strategy for conditionally overexpressing Il23a in cells. The Il4i1-cre animal and construction strategy were first proposed. Combined with animals that incorporate the Il23a system (e.g., site-directed knock-in of the Rosa26 gene), spontaneous psoriasis and psoriatic arthritis animal models with phenotypes similar to human psoriasis were successfully constructed.

[0054] The present invention proposes an Il23a conditional overexpression system comprising: an Il4i1 gene-2A-Cre recombinase and a LoxP-Stop-LoxP-Il23a-EGFP site-directed knock-in gene.

[0055] In a preferred embodiment of the present invention, a system for constructing an animal with conditional overexpression of Il23a is provided, wherein the system comprises an Il4i1 gene, a 2A-Cre recombinase, and a CAG-loxp-Neo-loxp-Il23a-IRES-EGFP expression cassette system inserted into the Rosa26 gene locus. + An animal model of conditional Il23a overexpression in cells, referred to as an Il4i1-Cre; Rosa-Il23a-EGFP animal, is described. The Il4i1-Cre; Rosa-Il23a-EGFP animal spontaneously develops psoriasis and psoriatic arthritis phenotypes and is proposed for broad application in understanding the pathogenesis of related diseases and drug screening.

[0056] In cells expressing Il4i1, the Il4i1 gene drives the expression of Cre recombinase, which cuts the termination signal Stop before (upstream) the target gene Il23a-IRES-EGFP, thereby releasing the inhibition of the termination signal Stop on the expression of Il23a-IRES-EGFP, and then expressing Il23a and producing green fluorescent protein, thereby completing the overexpression of Il23a and cell labeling.

[0057] Preferably, the system may include a vector comprising a Cre recombinase encoding gene and LoxP-Stop-LoxP-Il23a-EGFP.

[0058] The Il4i1 gene is specifically expressed in a population of dendritic cells, and the sequence of the Il4i1 gene is shown in SEQ ID NO: 1. By homologous recombination, a 2A-Cre-WPRE-polyA expression cassette was site-directedly knocked into the Il4i1 gene stop codon site. A homologous recombination vector (donor vector) was constructed, comprising a 2.551 kb 5' homology arm, a 1.97 kb 2A-Cre-WPRE-polyA, and a 2.796 kb 3' homology arm. The sequence of the 5' homology arm is shown in SEQ ID NO: 2, the sequence of the 2A-Cre-WPRE-polyA is shown in SEQ ID NO: 3, and the sequence of the 3' homology arm is shown in SEQ ID NO: 4.

[0059] The Rosa26-(CAG-loxp-Neo-loxp-Il23a-IRES-EGFP) gene targeting vector comprises: a 1.085kb 5' homology arm, a 1.598kb CAG promoter, a 3.213kb loxp-PGK-Neo-polyA-loxp, a 2.74kb Il23a-IRES-EGFP coding region, a 4.322kb 3' homology arm, and a 1.472kb PGK-DTA-polyA negative selection marker gene element. The LoxP sequence is a DNA sequence that can be recognized by the Cre recombinase, which can cut out the DNA sequence between two LoxPs in the same direction. The Stop sequence is a DNA sequence that can prevent the expression of the gene behind it. Under normal circumstances, the expression of the target gene is inhibited due to the presence of the Stop sequence. When the Cre recombinase is present, the Stop sequence between the two LoxPs is cut out. Stop releases the inhibition of the target gene, and the target gene and reporter gene begin to express. The target gene is Il23a, and the reporter gene can be EGFP or a gene encoding another fluorescent protein, such as tdTomato or YFP. Preferably, the reporter gene in the plasmid is an EGFP element.

[0060] Wherein, the sequence of the 5' homology arm is shown in SEQ ID NO: 5;

[0061] Wherein, the sequence of the CAG promoter is shown in SEQ ID NO: 6;

[0062] Wherein, the sequence of loxp-PGK-Neo-polyA-loxp is shown in SEQ ID NO:7;

[0063] Wherein, the sequence of Il23a-IRES-EGFP-WPRE-PloyA is shown in SEQ ID NO: 8;

[0064] Wherein, the sequence of the 3' homology arm is shown in SEQ ID NO:9;

[0065] The sequence of the PGK-DTA-polyA is shown in SEQ ID NO: 10.

[0066] Preferably, the present invention proposes a conditional + A technical method for overexpressing Il23a in cells, wherein the construction method comprises the following steps:

[0067] (1) Inserting the 2A-Cre sequence after the Il4i1 gene to form Il4i1-2A-Cre, thereby achieving the expression of the Cre recombinase driven by Il4i1;

[0068] (2) site-directed insertion of a CAG-loxp-Neo-loxp-Il23a-IRES-EGFP-polyA expression cassette into the Rosa26 gene locus;

[0069] (3) By integrating Il4i1-2A-Cre with the LoxP target gene overexpression system (CAG-loxp-Neo-loxp-Il23a-IRES-EGFP-polyA), Il23a can be overexpressed in Il4i1-expressing cells.

[0070] Wherein, in the step (1), the 2A-Cre sequence can be constructed on a first vector and introduced into the cells expressing Il4i1.

[0071] Wherein, in the step (2), a LoxP target gene overexpression system can be constructed on a second vector and introduced into the cells expressing Il4i1.

[0072] Alternatively, in steps (1) to (2), the 2A-Cre sequence and the LoxP target gene overexpression system can be constructed on the same vector and introduced into the cells expressing Il4i1.

[0073] The vector can be any vector used for gene targeting. Specifically, the construction method includes:

[0074] (1) The steps for constructing the Il4i1-Cre; Rosa-Il23a-EGFP animal conditional overexpression system are as follows:

[0075] A 2A-Cre fragment was inserted after the Il4i1 stop codon in the animal. In this animal, all cells expressing Il4i1 will also express Cre recombinase. This animal was then crossed with a Rosa26-(CAG-loxp-Neo-loxp-Il23a-IRES-EGFP) animal to create an Il4i1-Cre;Rosa-Il23a-EGFP animal. In this Il4i1-Cre;Rosa-Il23-EGFP animal, Cre recombinase recognizes the two LoxP sequences at the Rosa26 locus and cleaves the intervening stop signal sequence (LoxP-Stop-LoxP). Once the stop signal sequence is cleaved, the cell and its progeny will continue to express Il23a and EGFP, producing green fluorescence that can be detected by flow cytometry and fluorescence microscopy.

[0076] (2) Construction of Il4i1-Cre; Rosa-Il23a-EGFP animal conditional overexpression system to produce spontaneous psoriasis-like and psoriatic arthritis phenotypes

[0077] In mice, it was observed that the ear skin of Il4i1-Cre; Rosa-Il23a-EGFP heterozygous animals was slightly thickened at 8 weeks of age. After 8-10 weeks, scaly patches began to appear on the ears, paws, and tail skin of the animals, and the skin thickened significantly. After 10-12 weeks of age, swelling of the toes (dactylitis) and paw tissue was observed in Il4i1-Cre; Rosa-Il23a-EGFP heterozygous animals. MicroCT scans of the animal paws were performed, and bone destruction of the metacarpophalangeal joints was observed.

[0078] The animal models constructed by the present invention can be used for screening and testing specific drugs. In drug screening, candidate drugs or therapeutic agents are substances known to have certain pharmacological activities or substances being tested for potential pharmacological activities, including but not limited to nucleic acids, proteins, carbohydrates, chemically synthesized small or large molecules, cells, etc. The candidate drugs or therapeutic agents can be administered orally, intravenously, intraperitoneally, subcutaneously, through the spinal canal, or directly into the brain.

[0079] The animal model constructed by the present invention can be used as a powerful tool for scientific research and new drug evaluation.

[0080] Those skilled in the art understand that obtaining animal models that exhibit typical disease symptoms is difficult due to factors such as the complexity of genetics, the multiple signaling pathways that affect diseases, and the body's own compensatory or repair mechanisms. The present invention, through optimized design, overcomes these technical challenges.

[0081] In terms of disease mechanism research, the animal model constructed by the present invention, which can stably present spontaneous psoriasis or psoriatic arthritis, can be used to conduct research on disease mechanisms, explore the key factors that lead to the transition from normal inhibition to spontaneous psoriasis or psoriatic arthritis, and explore the intermediate mechanisms that prevent or delay the progression of these diseases. The model system of the present invention helps to better understand spontaneous psoriasis or psoriatic arthritis and is used to explore / identify candidate drugs / therapeutics that can prevent, delay, or reverse the disease process.

[0082] In terms of preclinical drug in vivo testing, the animal model constructed by the present invention that can stably present spontaneous psoriasis or psoriatic arthritis is expected to be used for preclinical drug metabolism, toxicity and efficacy testing. The animal model is physiologically close to the human body and supports long-term sampling, detection and tracking, which is conducive to promoting new drug research and development. In the present invention, the type of candidate drug used for drug testing is not particularly limited and can be obtained from a variety of sources, including synthetic or natural compound libraries. For example, there are many ways to randomly and directed synthesize a variety of organic compounds and biomolecules, including expressing random oligonucleotides and oligopeptides; alternatively, natural compound libraries in the form of bacterial, fungal, plant and animal extracts can be obtained or easily produced. In addition, libraries and compounds produced by natural or synthetic methods can be easily modified by conventional chemical, physical and biochemical methods and can be used to produce combinatorial libraries. Known pharmacological agents can be subjected to directed or random chemical modifications (such as acylation, alkylation, esterification, amidation, etc.) to produce structural analogs.

[0083] The method for preparing an animal model is simple and controllable, resulting in a stable animal model that effectively mimics the defective phenotype of human psoriasis or psoriatic arthritis. The resulting disease symptoms are highly typical, and phenotypic changes are easily observed. The animal model provides a new avenue for studying the pathogenic mechanisms of spontaneous psoriasis or psoriatic arthritis, as well as for drug screening and clinical treatment.

[0084] Based on the method of the present invention, the present invention also provides a kit for preparing an animal model of spontaneous psoriasis or psoriatic arthritis, wherein the kit comprises: a Cre recombinase expression element, which includes a Cre recombinase encoding nucleic acid; an expression cassette for conditionally expressing IL23a, which includes an operably linked promoter, Loxp-terminator-Loxp, and an IL23a expression element; the expression element includes an IL23a encoding nucleic acid.

[0085] The kit may also include instructions for use describing the method for preparing the animal model of the present invention, so as to facilitate use by those skilled in the art.

[0086] After obtaining the animal model of the present invention, it can be used to screen for substances of interest that can (or potentially can) alleviate or treat spontaneous psoriasis or psoriatic arthritis. After screening, truly useful drugs can be found from the substances of interest.

[0087] Therefore, the present invention also provides a method for screening potential substances, which comprises: (1) preparing an animal model of spontaneous psoriasis or psoriatic arthritis using the method or kit described above; (2) administering a candidate substance to the animal model of (1), and observing whether the candidate substance has an alleviating or therapeutic effect on spontaneous psoriasis or psoriatic arthritis; if it is observed that the symptoms of spontaneous psoriasis or psoriatic arthritis in the animal model are alleviated, the candidate substance is a substance that alleviates or treats spontaneous psoriasis or psoriatic arthritis.

[0088] In a preferred embodiment of the present invention, during screening, a control group may be set up to more easily observe changes in the symptoms of spontaneous psoriasis or psoriatic arthritis. The control group may be the animal model to which the candidate substance is not added.

[0089] As a preferred embodiment of the present invention, the method further comprises: conducting further cell experiments and / or animal experiments and / or clinical experiments on the obtained potential substances to further select and determine truly useful substances.

[0090] In another aspect, the present invention also provides potential substances of interest obtained using the screening method. These initially screened substances can constitute a screening library, from which safe, relatively ideal substances that are truly effective in alleviating or treating spontaneous psoriasis or psoriatic arthritis can be ultimately screened.

[0091] The beneficial effect of the present invention is that the Il23a of the present invention is in a group of Il4i1 + The overexpression system in cells can induce spontaneous psoriasis and psoriatic arthritis, which is beneficial to the study of their pathogenesis and recurrence mechanisms and drug development.

[0092] The present invention will be further described below with reference to specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. Experimental procedures in the following examples, where specific conditions are not specified, were generally performed under conventional conditions, such as those described in J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd Edition, Science Press, or according to the conditions recommended by the manufacturer.

[0093] Example 1: The present invention is Il4i1-Il23a OE Construction of mice

[0094] like Figure 1As shown, the construction method includes the following steps:

[0095] 1. Construction of Il4i1-Cre mice

[0096] The DNA sequence of Il4i1 is shown in SEQ ID NO: 1. A homologous recombination vector was constructed, comprising a 5' homology arm (5'am) (SEQ ID NO: 2), 2A-Cre-WPRE-polyA (SEQ ID NO: 3), and a 3' homology arm (3'am) (SEQ ID NO: 4).

[0097] Using CRISPR / Cas9 technology, a 2A-Cre-WPRE-polyA expression cassette was inserted into the Il4i1 gene stop codon site by homologous recombination, located after exon 9 and before the 3'UTR ( Figure 1 A) Cas9 mRNA, gRNA, and homologous recombination vectors were microinjected into C57BL / 6J fertilized eggs to obtain F0 mice. Positive F0 mice were identified and mated with C57BL / 6J mice to obtain F1 Il4i1-Cre-positive mice.

[0098] At the same time, a Rosa26 site-directed knock-in mouse of the IL-23a gene was constructed, and an ES cell targeting vector was constructed, which contained a 5' homology arm (SEQ ID NO: 5), a CAG promoter (SEQ ID NO: 6), loxp-PGK-Neo-polyA-loxp (SEQ ID NO: 7), Il23a-IRES-EGFP-WPRE-PloyA (SEQ ID NO: 8), a 3' homology arm (SEQ ID NO: 9), and a PGK-DTA-polyA negative selection marker (SEQ ID NO: 10) ( Figure 1 B) The vector was linearized and electroporated into ES cells derived from C57BL / 6J×129 / Sv mice. After selection with G418 and Ganc, positive clones demonstrating correct homologous recombination in resistant ES cells were expanded and injected into blastocysts of C57BL / 6J mice to generate chimeric Rosa-Il23a-EGFP mice.

[0099] 2. Il4i1-Il23a OE Preparation of mice

[0100] Il4i1-Cre mice were crossed with Rosa-Il23a-EGFP mice to obtain Il4i1-Il23a OE Mouse ( Figure 1C) In cells expressing Il4i1, Cre recombinase is expressed simultaneously with Il4i1. Cre recombinase recognizes the Stop sequence at the Rosa26 site and removes it, causing Il23a to begin expressing. Therefore, Il4i1 + All cells overexpress Il23a and have green fluorescence.

[0101] Example 2, Il4i1-Il23a OE Detection of spontaneous psoriasis-like phenotype and indicators in mice

[0102] 1. Il4i1-Il23a OE Heterozygous mice

[0103] Observation of Il4i1-Il23a OE Growth and disease progression of heterozygous mice.

[0104] Eight weeks after birth, the skin of the mouse's ears was slightly thickened. After 10-12 weeks, scaly patches began to appear on the hairless areas of the mouse's ears, and the skin became significantly thicker. Figure 2 A).

[0105] 2. Ear skin tissue analysis

[0106] Il4i1-Il23a was collected at 12 weeks of age OE The ear skin tissues of heterozygous mice and control mice (LSL-Il23a) were pathologically sectioned and stained with H&E.

[0107] The results showed that the skin tissue had epidermal hyperplasia (spinous layer), loss of granular layer, hyperkeratosis and parakeratosis of the epidermis, accumulation of microabscesses on the surface of the thickened epidermis, and a large number of cell infiltrations in the dermis ( Figure 2 B).

[0108] 3. Analysis of markers related to abnormal keratinocyte proliferation

[0109] After 12 weeks of birth, Il4i1-Il23a was analyzed OE The markers related to abnormal proliferation of keratinocytes in heterozygous mice are: Ki67, keratin 5 (K5), keratin 6 (K6); and the marker related to normal differentiation of keratinocytes is: 1 / 10 (K1 / 10).

[0110] The results showed that compared with the control group, the expression of Ki67, keratin 5 (K5), and keratin 6 (K6), markers related to abnormal proliferation of keratinocytes, increased; while the expression of 1 / 10 (K1 / 10), a marker related to normal differentiation of keratinocytes, decreased ( Figure 2 C).

[0111] 4. Cell Type Analysis

[0112] In traditional psoriasis-like mouse models, the T cells infiltrating the lesion area are mainly γδT cells, which is very different from the immune phenotype dominated by αβT cells in the lesions of psoriasis patients.

[0113] The inventors used Il4i1-Il23a OE Ear skin from mice and a control group was placed in a 5mg / ml Dispase II solution and incubated at 37°C for 1-2 hours to separate the epidermis. The separated dermal tissue was minced in 3ml of a solution containing 2mg / ml Collagenase P and 1mg / ml DNase I and incubated at 37°C for 45 minutes. The digested tissue was gently blown open to form a single-cell suspension. The separated epidermis was then placed in 15ml of a 37°C preheated 0.05% Typsin solution and digested in a 37°C water bath for 15 minutes to separate single cells. Cell types were analyzed.

[0114] The results showed that Il4i1-Il23a OE IL-17A in mouse skin lesions + T cells are mainly αβT cells ( Figure 2 D). This suggests that this mouse model is closer to human psoriasis than traditional psoriasis-like mouse models and can be used as an animal model for human psoriasis.

[0115] 5. Bulk-RNA Sequencing Analysis

[0116] IMQ-induced psoriasis-like mice are the most widely used animal model of psoriasis so far and were used as a control. OE Bulk-RNA sequencing was performed on mice and the control group and compared with bulk-RNA data from ear skin tissue of IMQ-induced psoriasis-like mice (obtained from no. GSE27628).

[0117] Sequencing data confirmed that Il4i1-Il23a OE The genes of mice induced by IMQ-induced psoriasis-like mice are significantly closer to those of human psoriasis at the gene transcription level ( Figure 2 E).

[0118] Example 3, Il4i1-Il23a OE Mice develop psoriatic arthritis-like symptoms

[0119] Il4i1-Il23a OE When mice were 12-16 weeks old, the inventors observed redness and swelling in the soles of the mice ( Figure 3 A) The hind paw tissue of mice was fixed with 4% paraformaldehyde and the metacarpophalangeal joints were observed by Micro-CT scanning. Il4i1-Il23aOE Some of the mice's proximal toe joints showed wear and tear, showing typical psoriatic arthritis-like symptoms ( Figure 3 B, C).

[0120] Example 4. Application of animal model: IL-17 antibody neutralization therapy

[0121] 100 μg of mouse IL-17A antibody (BioxCell cat. BE0173) or isotype control (BioxCell cat. BE0083) were intraperitoneally injected to treat Il4i1-Il23a cells at 6 weeks of age. OE Mice, once a week for 4 weeks.

[0122] The results showed that IL-17A antibody could significantly inhibit the expression of Il4i1-Il23a OE Increased skin thickness in mice ( Figure 4 ). Therefore, the Il4i1-Il23a OE Mouse models can be used to screen inhibitory drugs such as IL-17.

[0123] In summary, the Il4i1-Il23a provided by the present invention OE The mouse and traditional psoriasis and psoriatic arthritis mouse models are highly similar to the phenotype and mechanism of human psoriasis, which is more conducive to in-depth research on the pathogenesis and drug screening of psoriasis, psoriatic arthritis, psoriatic enteritis and related diseases.

[0124] The above-described embodiments merely represent several implementation methods of the present invention, and their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the present invention. It should be noted that a person skilled in the art may make several modifications and improvements without departing from the concept of the present invention, and these modifications and improvements fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention shall be based on the appended claims. At the same time, all documents mentioned in this application are cited as references in this application, just as if each document was cited as a reference individually.

[0125] Sequence information

[0126] Il4i1 DNA (SEQ ID NO:1; 4523bp)

[0127]

[0128] 5’am(SEQ ID NO:2;2551bp)

[0129]

[0130] 2A-Cre-WPRE-polyA(SEQ ID NO:3;1970bp)

[0131]

[0132] 3’am(SEQ ID NO:4;2796bp)

[0133]

[0134] 5’am(SEQ ID NO:5;1085bp)

[0135]

[0136] CAG promoter(SEQ ID NO:6;1598bp)

[0137]

[0138] loxp-PGK-Neo-polyA-loxp(SEQ ID NO:7;3213bp)

[0139]

[0140] Il23a-IRES-EGFP-WPRE-PloyA(SEQ ID NO:8;2740bp)

[0141]

[0142] 3’am(SEQ ID NO:9;4322bp)

[0143]

[0144] PGK-DTA-polyA(SEQ ID NO:10;1472bp)

[0145]

Claims

1. A method for preparing an animal model of spontaneous psoriasis or psoriatic arthritis, comprising: Conditional expression of IL23a in IL4i1-positive cells of animals; The conditional expression is performed using a Cre / loxP recombination system; the method comprises: (1) Providing an animal, introducing a Cre recombinase expression element into the genome of its IL4i1-positive cells, which can be operably linked to the IL4i1-encoding nucleic acid in the genome; the Cre recombinase expression element comprises a Cre recombinase-encoding nucleic acid; the Cre recombinase expression element further comprises: a 2A peptide-encoding nucleic acid located upstream of the Cre recombinase-encoding nucleic acid, and a termination sequence is further provided downstream of the Cre recombinase expression element located downstream of the Cre recombinase-encoding nucleic acid; the Cre recombinase expression element is introduced into the stop codon position of IL4i1 in the animal genome; and the Cre recombinase expression element is introduced into the genome of the IL4i1-positive cells by providing homology arms on both sides; (2) providing an animal, into whose genome an expression cassette for conditional expression of IL23a is introduced, wherein the expression cassette comprises: a promoter, a Loxp-terminator-Loxp, and an IL23a expression element operably linked; the expression element comprises an IL23a encoding nucleic acid; (3) The animals of (1) and (2) are mated to obtain offspring; wherein, when the Cre recombinase is expressed, it recognizes the two LoxPs and cuts the terminator between them, so that the IL4i1-positive cells and their progeny cells continue to express Il23a.

2. The method according to claim 1, wherein (2) In the Loxp-terminator-Loxp, a resistance screening gene expression element is further provided upstream of the terminator; (2), the IL23a expression element further comprises: a reporter gene connected to the IL23a encoding nucleic acid, and a termination sequence; the IL23a encoding nucleic acid and the reporter gene are connected via an IRES.

3. The method according to claim 1, wherein (2) In its genome Rosa26 The expression cassette for conditional expression of IL23a was introduced into the gene locus.

4. The method according to any one of claims 1 to 3, characterized in that: The animal is a rodent.

5. Use of the spontaneous psoriasis or psoriatic arthritis animal model obtained by the method according to any one of claims 1 to 4 for: As an animal model for screening candidate drugs or therapeutic agents for alleviating or treating psoriasis or psoriatic arthritis; or As an animal model for studying psoriasis or psoriatic arthritis.

6. A kit for preparing an animal model of spontaneous psoriasis or psoriatic arthritis, comprising: Cre recombinase expression element, which includes a Cre recombinase encoding nucleic acid; An expression cassette for conditionally expressing IL23a, comprising: a promoter, Loxp-terminator-Loxp, and an IL23a expression element operably linked; the expression element comprises an IL23a encoding nucleic acid; The Cre recombinase expression element further comprises: a 2A peptide encoding nucleic acid located upstream of the Cre recombinase encoding nucleic acid, and a termination sequence is further provided downstream of the Cre recombinase expression element located downstream of the Cre recombinase encoding nucleic acid; homology arms are provided on both sides of the Cre recombinase expression element, so that the Cre recombinase expression element can be introduced into the genome of IL4i1-positive cells; and introduced into the stop codon position of IL4i1 in the animal genome; In the Loxp-terminator-Loxp of the expression cassette for conditional expression of IL23a, a resistance screening gene expression element is further provided upstream of the terminator; The IL23a expression element of the expression cassette for conditional expression of IL23a further comprises: a reporter gene connected to the IL23a encoding nucleic acid, and a termination sequence; the IL23a encoding nucleic acid and the reporter gene are connected via an IRES.

7. A method for screening candidate drugs or therapeutic agents for alleviating or treating psoriasis or psoriatic arthritis, the method comprising: (1) Preparing a spontaneous psoriasis or psoriatic arthritis animal model using the method described in any one of claims 1 to 4; (2) administering the candidate substance to the animal model of (1) to observe whether the candidate substance has an alleviating or therapeutic effect on psoriasis or psoriatic arthritis; If alleviation of psoriasis or psoriatic arthritis symptoms is observed in an animal model, the candidate substance is a substance that alleviates or treats psoriasis or psoriatic arthritis.

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