Construction method and application of a STING-related infantile-onset vasculitis model

Abstract

The present invention relates to a method for constructing a STING-related infantile-onset vasculitis model. The animal model obtained by editing the Sting1 gene in the present invention shows skin inflammation and lung disease similar to those of SAVI patients. The animal model can be naturally bred and a stable population can be established, which can meet the needs of large-scale preparation, and is conducive to further studying the pathogenic mechanism of SAVI in Sting1 mutant populations and testing the efficacy and safety of SAVI-related therapeutic or preventive drugs.

Description

Technical Field

[0001] The present invention belongs to the field of animal models, and specifically relates to a method for constructing a STING-related infantile-onset vasculitis disease model. Background Art

[0002] Sting1 (interferon response stimulator cGAMP interactor 1), also known as Tmem173, has 2',3'-cyclic GMP-AMP binding activity, cyclic di-GMP binding activity and ubiquitin ligase binding activity, and is located in various cellular components such as the perinuclear region of the cytoplasm, autophagosomes and peroxisomes. STING is involved in multiple life processes, including defense responses to other organisms, macroautophagy, upstream regulation of IFN-β responses and inflammatory responses, and positive regulation of transcription through RNA polymerase II.

[0003] Gain-of-function mutations in the STING gene are associated with STING-associated vasculopathy with onset in infancy (SAVI), in which patients develop extensive systemic inflammation due to increased levels of interferon β (IFN-β). Common clinical manifestations include chronic lung disease, finger necrosis, recurrent low-grade fever, and inflammatory skin lesions, most of which are related to vasculitis and tissue damage that relies on blood vessels for blood supply. A series of STING constitutively activating mutations have been found in SAVI patients. The three mutation sites that have received widespread attention are V147L (valine to leucine at position 147), N154S (asparagine to serine at position 154), and V155M (valine to methionine at position 155). The STING constitutively activating point mutation mouse model has been successfully used in the study of the pathogenesis of SAVI. For example, STING-p.V154M mice (corresponding to the human V155M mutation) develop clinical SAVI-like lung inflammation and pulmonary fibrosis, and STING-p.N153S mice (corresponding to the human N154S mutation) are reported to suffer from skin diseases. At the same time, the SAVI disease model has increased levels of interferon β (IFN-β), so it is also an effective model for JAK inhibitor screening. The above two types of mice cannot obtain homozygous mice due to poor breeding of heterozygous female mice, and heterozygous mice have severe inflammatory symptoms, short survival cycles, and limited window periods for efficacy evaluation, which limits the widespread application of STING-p.N153S and STING-p.V154M in drug development. On the other hand, although STING-p.N153S and STING-p.V154M mice partially simulate the disease phenotypes of SAVI patients, they cannot fully reflect the clinical manifestations of SAVI patients. Therefore, it is necessary to develop a SAVI disease model with good survival, more comprehensive phenotypes, and closer to clinical symptoms.

[0004] The traditional infantile-onset STING-associated vasculitis model is to directly mutate the corresponding SAVI site in mice. The activated STING protein is still endogenous to the mouse. The mouse STING protein has 68% sequence homology with the human STING protein, and there are structural differences. Therefore, drugs targeting human STING are very likely to fail to combine with mouse STING and exert ideal efficacy. At present, there are no reports on the construction method of SAVI disease model with a long survival period and close to clinical symptoms. Summary of the invention

[0005] The purpose of the present invention is to address the defects of existing models and provide a method for constructing and applying a STING-associated infantile-onset vasculitis (SAVI) model, thereby providing a favorable basis for in-depth research on the etiology and treatment of SAVI.

[0006] The first aspect of the present invention provides a method for constructing an animal model of STING-related infantile-onset vasculitis, the method comprising the following steps: replacing the Sting gene fragment in the genome of the target animal with a human hSTING1-p.V147L mutant gene fragment, the sequence of the human hSTING1-p.V147L mutant gene fragment being shown in SEQ ID NO:1.

[0007] Preferably, the target animal is a mouse, the mutant gene is a STING 147 valine to leucine mutation sequence, and the model mouse is named hSTING1-p.V147L mouse.

[0008] Preferably, the CRISPR / Cas9 gene editing technology is used to construct the animal model. Those skilled in the art will appreciate that the method for constructing the animal model of the present invention is not limited to using the third-generation gene editing technology CRISPR / Cas9, and other genome editing technologies can also achieve the same effect.

[0009] Preferably, the construction method comprises the following steps:

[0010] (1) constructing a targeting vector expressing the humanized Sting1 gene for inserting the humanized Sting1 gene;

[0011] (2) Designing sgRNA targeting the translation start site and post-coding region of the mouse Sting1 gene, and obtaining the above sgRNA using in vitro transcription technology;

[0012] (3) co-injecting or co-electroporating the targeting vector constructed in step (1), the sgRNA obtained in step (2), and the Cas9 mRNA or Cas9 protein into the cytoplasm or nucleus of a fertilized egg of a background mouse, and transplanting the fertilized egg into a pseudo-pregnant mouse, performing genotyping on the pseudo-pregnant mouse offspring, and screening for positive F0 mice in which the correct human fragment was successfully inserted;

[0013] (4) The sexually mature positive F0 mice were bred with background mice to obtain F1 mice. After verification by PCR and sequencing, positive heterozygous F1 mice were obtained, and the Sting1 humanized mouse model that can be propagated was screened out.

[0014] Preferably, step (1) includes the following steps: according to the structure and function of human Sting1, a gene fragment of human Sting1 containing a valine to leucine mutation gene at position 147 is inserted into the translation start site of the mouse Sting1 transcript, the hSTING1-p.V147L mutant amino acid sequence expressed by the selected human hSTING1-p.V147L mutant gene fragment is shown in SEQ ID No.2, and the replaced mouse Sting1 gene amino acid sequence is shown in SEQ ID No.3.

[0015] Preferably, the step (1) includes the following steps: constructing a successful targeting vector sequence as shown in SEQ ID No.4.

[0016] More preferably, the gene sequence of sgRNA in step (2) is SEQ ID No.5 and SEQ ID No.7, or SEQ ID No.6 and SEQ ID No.8.

[0017] Further preferably, the gene sequence of sgRNA in step (2) is SEQ ID No.6 and SEQ ID No.8.

[0018] Preferably, the strain of mice providing fertilized eggs and pseudo-pregnant mice in step (3) is BALB / c.

[0019] Preferably, the 5' end identification primers used for the F0 mouse genotype identification in step (3) are shown as SEQ ID No.9 and SEQ ID No.10, and the 3' end identification primers are shown as SEQ ID No.11 and SEQ ID No.12.

[0020] Preferably, the PCR reaction system used for the genotype identification of F0 mice in step (3) is as follows:

[0021] Seg Reagent (VAZYME P112-03) Volume (μL) Specification 1 2x Taq Master Mix, Dye Plus 12.5 \ 2 <![CDATA[ddH 2 The]]> 9.5 \ 3 Primer A 1 10mM 4 Primer B 1 10mM 5 Template 1 100ng / μL .

[0022] Preferably, the PCR reaction conditions used for F0 mouse genotype identification in step (3) are as follows:

[0023]

[0024] Preferably, the animal model is consistent with the phenotype of clinical SAVI patients with Sting1 gene mutations, wherein the patients suffer from skin inflammation and lung disease.

[0025] A second aspect of the present invention provides the use of mice obtained by the above-mentioned construction method in studying the functions and mechanisms of action related to the STING gene.

[0026] Preferably, the use is for non-diagnostic and non-therapeutic purposes.

[0027] Preferably, the mouse is a BALB / c mouse.

[0028] A third aspect of the present invention provides the use of mice obtained by the above-mentioned construction method in screening drugs for treating diseases related to the STING gene.

[0029] Preferably, the disease associated with the STING gene is STING-associated vasculitis of infancy (SAVI).

[0030] Preferably, the use is for non-diagnostic and non-therapeutic purposes.

[0031] Preferably, the mouse is a BALB / c mouse.

[0032] The present invention provides the use of a humanized Sting1 mutant gene in preparing an anthropomorphic STING-related infantile-onset vasculitis animal model. Animals obtained by editing the Sting1 gene develop skin inflammation and lung disease similar to those of SAVI patients, can be naturally bred and a stable population can be established, which can meet the needs of large-scale preparation, and is conducive to further studying the pathogenic mechanism of SAVI in the Sting1 mutant population and testing the efficacy and safety of SAVI-related therapeutic or preventive drugs.

[0033] The present invention has the following beneficial effects:

[0034] The construction method provided by the present invention can obtain an animal model of humanized hSTING1-p.V147L mutation. The present invention adopts gene editing technology, uses human V147L (valine to leucine) mutant gene to replace the corresponding fragment of mouse Sting1 gene, and the independently constructed SAVI mouse model expresses the same activated STING protein as the patient. The animal model has the following advantages: (1) The animal model of the present invention can show clinical disease characteristics similar to those of patients with STING-related infantile vasculitis. Compared with the existing models, the clinical disease characteristics are more comprehensive, so it can better simulate clinical manifestations. (2) The animal model of the present invention has good survival and a long time window for evaluating drug efficacy. (3) The STING mutation introduced in the animal model of the present invention is a human sequence, which can express pathogenic hSTING1-p.V147L mutant protein similar to that of patients, and is an ideal model for preclinical in vivo efficacy and safety evaluation of STING-related target drugs. (4) The present invention also provides the application of the STING-related infantile vasculitis disease model in the pathogenesis and mechanism research of SAVI or the screening of drugs for the prevention or treatment of SAVI. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 It is a schematic diagram of the overall gene editing strategy of the animal model of the present invention;

[0036] Figure 2 This is the electrophoresis result of hSTING1-p.V147L mouse identification;

[0037] Figure 3 This is a WB diagram showing the expression results of humanized hSTING1-p.V147L mutant protein in the SAVI model;

[0038] Figure 4 This is a diagram showing the results of Q-PCR demonstrating that the hSTING1-p.V147L mutation activates the interferon signaling pathway in the spleen of mice;

[0039] Figure 5 H&E staining pathological analysis of hSTING1-p.V147L mouse lungs;

[0040] Figure 6 H&E staining pathological analysis of the ear skin of hSTING1-p.V147L mice;

[0041] Figure 7 H&E staining of the tail skin of hSTING1-p.V147L mice for pathological analysis;

[0042] Figure 8 Masson staining pathological analysis of the ear skin of hSTING1-p.V147L mice;

[0043] Fig. 9 To detect the proportion of peripheral blood immune cell subsets in hSTING1-p.V147L mice;

[0044] Fig.10 To detect the proportion of peripheral blood immune cell subsets in hSTING1-p.V147L mice;

[0045] Fig.11 To detect the proportion of spleen immune cell subsets in hSTING1-p.V147L mice;

[0046] Fig.12 Detection of the proportion of spleen immune cell subsets in hSTING1-p.V147L mice. DETAILED DESCRIPTION

[0047] The present invention is further described by way of examples, but the present invention is not limited to the following examples.

[0048] Experimental Example 1: Establishment of a mouse model of STING-associated vasculitis with infantile onset

[0049] 1. Test methods

[0050] The animal model of the present invention uses CRISPR-Cas9 technology to replace the mouse Sting1 with the corresponding human V147L (valine to leucine) mutant gene fragment. The specific strategy is shown in Figure 1 , the implementation steps are as follows:

[0051] (1) Design of Sting site sgRNA

[0052] Determine the approximate region of sgRNA based on the Sting gene locus. Use sgRNA evaluation software to select several groups of sequences with the lowest off-target rate for the target fragment, identify the 5' end target site and the 3' end target site, and use the sequences in Table 1 as candidate sgRNAs. Synthesize and construct the sgRNA expression vector puc57-sgRNA.

[0053] Table 1 sgRNA sequences targeting Sting sites

[0054] gRNA name sequence Cutting efficiency Sting1-5S1 CCTCTGGGCCGTGGGATGGCTGG(SEQ ID NO.5) 92％ Sting1-5S2 GTGACCTCTGGGCCGTGGGATGG(SEQ ID NO.6) 86％ Sting1-3S1 GCTGTCCCATGCCTCAGATGAGG(SEQ ID NO.7) 83％ Sting1-3S2 CTCCGCACTGACCTCATCTGAGG(SEQ ID NO.8) 96％

[0055] sgRNA transcription preparation method: Using the PrimerStar or PrimerStar Max system, the correctly sequenced puc57-sgRNA plasmid (1:30 dilution) was used as a template for PCR, and the PCR product was purified to prepare the sgRNA transcription template. The sgRNA was transcribed using the T7-ShortScript in vitro transcription kit (AM1354).

[0056] (2) Construction of homologous DNA donor for targeting the human Sting1 mutant gene containing the valine to leucine mutation at position 147.

[0057] A human Sting1 gene fragment donor containing a valine to leucine mutation at position 147 was constructed using SLIC connection, and the vector included: mouse Sting gene 5' homology arm sequence-hSTING1-p.V147L mutant gene-mouse Sting gene 3' homology arm sequence, wherein the nucleic acid sequence of the hSTING1-p.V147L mutant gene fragment is shown in SEQ ID NO.1 (the underline indicates the mutated p.V147L gene site), the amino acid sequence of the expressed hSTING1-p.V147L mutant protein is shown in SEQ ID NO.2, the replaced mouse Sting gene amino acid sequence is shown in SEQ ID NO.3, and the sequence of the successfully constructed targeting vector is shown in SEQ ID No.4 (italics indicate the junction of the mouse and human genes, and the underline indicates the inserted human STING gene fragment 28bp to 5471bp).

[0058] atgccccactccagcctgcatccatccatcccgtgtcccaggggtcacggggcccagaaggcagccttggttctgctgagtgcctgcctggtgaccctttgggggctaggagagccaccagagcacactctccggtacctggtgctccacctagcctccctgcagctgggactgctgttaaacggggtctgcagcctggctgaggagctgcgccacatccactccaggtgactcactgcagtacccagggacggggtatccaacgtgtgtcactcccttgatgcctagccctgcccctccttgaacctctctggctgagctgggctgggggctggggtctggggtctggctgtcactcacaggtaccggggcagctactggaggactgtgcgggcctgcctgggctgccccctccgccgtggggccctgttgctgctgtccatctatttctactactccctcccaaatgcggtcggcccgcccttcacttggatgcttgccctcctgggcctctcgcaggcactgaacatcctcctgggcctcaaggtatgacacagggggaggtagaagctctggccaagtggtggctgtggctggtgtgacctgccctgagctgagtactgggagtgggactggtttaaaggctggagtccatggagtagaacctataatgtcctggaacagtgggtttggcaatggcaaaagagggatcaagtcaggagcaggttgggaagccttggaggaggaggaggagttctctgggtgtccttgatggaggccccccagccacatcctgctgtccacagggcctggccccagctgagatctctgca c

[0059] MPHSSLHPSIPCPRGHGAQKAALVLLSACLVTLWGLGEPPEHTLRYLVLHLASLQLGLLLNGVCSLAE ELRHIHSRYRGSYWRTVRACLGCPLRRGALLLLSIYFYYSLPNAVGPPFTWmlALLGLSQALNILLGL KGLAPAEISALCEKGNFNVAHGLAWSYYIGYLRLILPELQARIRTYNQHYNNLLRGAVSQRLYILLPL DCGVPDNLSMADPNIRFLDKLPQQTGDHAGIKDRVYSNSIYELLENGQRAGTCVLEYATPLQTLFAMS QYSQAGFSREDRLEQAKLFCRTLEDILADAPESQNNCRLIAYQEPADDSSFSLSQEVLRHLRQEEKEE VTVGSLKTSAVPSTSTMSQEPELLISGMEKPLPLRTDFS(SEQ ID NO.2)

[0060] MPYSNLHPAIPRPRGHRSKYVALIFLVASLMILWVAKDPPNHTLKYLALHLASHELGLLLKNLCCLAE ELCHVQSRYQGSYWKAVRACLGCPIHCMAMILLSSYFYFLQNTADIYLSWMFGLLVLYKSLSMLLGLQ SLTPAEVSAVCEEKKLNVAHGLAWSYYIGYLRLILPGLQARIRMFNQLHNNMLSGAGSRRLYILFPLD CGVPDNLSVVDPNIRFRDMLPQQNIDRAGIKNRVYSNSVYEILENGQPAGVCILEYATPLQTLFAMSQ DAKAGFSREDRLEQAKLFCRTLEEILEDVPESRNNCRLIVYQEPTDGNSFSLSQEVLRHIRQEEKEEV TMNAPMTSVAPPPSVLSQEPRLLISGMDQPLPLRTDLI(SEQ ID NO.3)

[0061]

[0062]

[0063]

[0064]

[0065] (3) Establishment of hSTING1-p.V147L mouse model

[0066] The sgRNA obtained by in vitro transcription of the plasmid described in step 1 and the vector donor in step 2 were mixed with the Cas9 protein, injected into the fertilized eggs of 0.5-day-old background mice, and transplanted into the bodies of 0.5-day-old pseudo-pregnant female mice. After the mice were born, the targeted mice F0 were screened out by gene PCR and sequencing.

[0067] hSTING1-p.V147L-positive F0 mice were bred with Balb / cJGpt background mice to obtain F1 generation, and hSTING1-p.V147L gene was identified in the tails of F1 generation mice. The PCR reaction primers are shown in Table 2 below, and the PCR reaction system and reaction conditions are shown in Tables 3 and 4.

[0068] Table 2 Primers used to identify the correct insertion of hSTING1-p.V147L in F1

[0069]

[0070] Table 3 PCR reaction system

[0071] Seg Reagent (VAZYME P112-03) Volume (μL) Specification 1 2x Taq Master Mix, Dye Plus 12.5 \ 2 <![CDATA[ddH 2 The]]> 9.5 \ 3 Primer A 1 10mM 4 Primer B 1 10mM 5 Template 1 100ng / μL

[0072] Table 4 PCR reaction procedure

[0073]

[0074]

[0075] 2. Test results

[0076] The results of the PCR experiment on F1 mice are shown in Figure 2(WT is the background mouse Balb / cJGpt genomic DNA as template; N is blank control, no template control; P is positive control; TRANS2K PLUS II band: 8000bp\5000bp\3000bp\2000bp\1000bp\750bp\500bp\250bp\100bp). The 5' and 3' ends of the hSTING1-p.V147L gene of 33#, 34#, 36# and 40# mice were positive. The mouse clones identified as positive by PCR at both ends were sequenced for verification. The sequencing primer information is shown in Table 5. The clones with correct sequencing were identified as positive F1 mice.

[0077] Table 5 Sequencing primers

[0078]

[0079] The results showed that mice 33#, 34#, 36# and 40# were positive heterozygous mice with correct gene replacement. After a large number of F1 breeding, interbreeding was performed to obtain hSTING1-p.V147L homozygous mice.

[0080] Experimental Example 2: Western blot analysis of the expression of humanized hSTING1-p.V147L mutant protein in the SAVI model

[0081] 1. Test methods

[0082] (1) Total protein extraction: After euthanizing the mice, the thymus, lungs, and skin of the control and hSTING1-p.V147L mice were obtained, and an appropriate amount of pre-cooled RIPA protein lysis buffer (containing protease inhibitors) was added, homogenized, lysed on ice, centrifuged, and the supernatant was collected and stored in a -80°C refrigerator;

[0083] (2) Protein quantification: Protein quantification was performed using the BCA protein concentration assay kit;

[0084] (3) Gel running: prepare 10% separation gel and 5% stacking gel; Sample spotting: the total protein loading per well is 25 μg;

[0085] (4) Transfer: wet transfer;

[0086] (5) Blocking: 5% non-fat milkRT blocking;

[0087] (6) Primary antibody incubation: Incubate with primary antibody at 4°C overnight;

[0088] (7) Secondary antibody incubation: wash the membrane several times with TBST and incubate at room temperature;

[0089] (8) Development: Wash the membrane several times with TBST and expose with ECL.

[0090] 2. Test results

[0091] Western blot (WB) results are as follows Figure 3 As shown, KI / KI is a BALB / c-hSTING1-p.V147L homozygote, and WT / WT is a wild-type background mouse. The experimental results show that STING is expressed in the thymus, lungs, and skin of BALB / c-hSTING1-p.V147L mice and wild-type BALB / c mice.

[0092] Experimental Example 3: Analysis of Activation of Interferon Signaling Pathway in hSTING1-p.V147L Mice by Quantitative Real-time PCR (Q-PCR)

[0093] 1. Test methods

[0094] 1.1. Extraction of RNA

[0095] (1) After mice were euthanized, spleens of control and hSTING1-p.V147L mice were obtained, 10-50 mg of tissue was added to 500 μl of RNA-easy Isolation Reagent, and homogenized using a tissue homogenizer until the homogenate was transparent and free of particles;

[0096] (2) Add 2 / 5 volume of RNase-free H 2 O (use 200 μl for every 500 μl RNA-easy Isolation Reagent), cover the centrifuge tube tightly, mix by inverting, and let stand on ice for at least 5 minutes;

[0097] (3) Place the centrifuge tube in a refrigerated centrifuge and centrifuge at 12,000 g for 15 min at 4°C.

[0098] (4) Carefully remove the centrifuge tube from the centrifuge. The homogenate is now separated into an upper aqueous phase (containing RNA) and a dark lower precipitate (containing impurities such as protein, DNA, and polysaccharides). Carefully pipette the supernatant and transfer it to another new centrifuge tube.

[0099] (5) Add an equal volume of isopropanol to the supernatant, mix thoroughly by inverting the tube, and let stand on ice for at least 10 min.

[0100] (6) Centrifuge at 12000g, 4°C for 10 minutes. Usually a white precipitate will be visible. Carefully discard the supernatant.

[0101] (7) Add 500 μL of 75% ethanol, gently tap the bottom of the tube to resuspend the precipitate, and invert it several times;

[0102] (8) Centrifuge at 12000g for 3-5 minutes at 4°C and discard the supernatant;

[0103] (9) Repeat steps (6) and (7) and discard the supernatant;

[0104] (10) Dry on ice and add an appropriate amount of RNase-free ddH 2 O dissolves and precipitates;

[0105] (11) After measuring the RNA concentration using an ultra-micro spectrophotometer, 1 μl of RNA was reverse transcribed into cDNA (according to PrimeScript TM RT reagent Kit with gDNA Eraser recommended system).

[0106] 1.2 Real-time fluorescence quantitative PCR

[0107] cDNA was added with ddH 2 O was diluted to 10 ng / μl for qPCR, and the amplification primer sequences were as follows:

[0108]

[0109] The RT-qPCR reaction system is as follows:

[0110]

[0111] The RT-qPCR reaction procedure is as follows:

[0112] Reagents Volume (μl) Specification 2X SYBR Mix 5 \ Primer 0.2 10μM Primer 0.2 10μM Rox 0.2 \ <![CDATA[Template+ddH 2 The]]> 4.4 ~10ng / μl

[0113] 2. Test results

[0114] The results of real-time fluorescence quantitative PCR (Q-PCR) are as follows Figure 4 As shown, the expression levels of interferon-induced genes Isg15, Isg10, and Ifit2 in the spleen of hSTING1-p.V147L (V147L) mice were significantly higher than those in background mice (WT), indicating that the hSTING1-p.V147L mutant protein overactivated the interferon signaling pathway.

[0115] Example 4: Pathological analysis of lung and skin staining in hSTING1-p.V147L mice

[0116] 1. Test methods

[0117] 1.1. Tissue paraffin embedding and sectioning

[0118] After euthanasia, the lungs, ear skin, and tail skin were obtained from 8-week-old control background mice (WT-8W), 8-week-old and 15-week-old hSTING-p.V147L mice (V147L-8W, V147L-15W). After the tissue was fixed with 4% PFA overnight, it was placed in an automatic dehydrator and dehydrated according to the set process. The embedding operation was performed using an embedding machine, and the slicing operation was performed using a paraffin slicer. The slice thickness was 3μm, and the slides were placed in a 37°C oven for drying.

[0119] 1.2 H&E staining

[0120] After the paraffin slides were dewaxed and covered with gradient alcohol, they were immersed in hematoxylin staining solution for several minutes, rinsed with running water, differentiated and turned blue, dehydrated with gradient ethanol, immersed in eosin staining for several minutes, dehydrated with gradient ethanol, transparent, and sealed with neutral gum.

[0121] 1.3 Masson staining

[0122] The corresponding staining was performed according to the instructions of the kit (modified Masson trichrome staining solution, Senbega BP-DL02250ml).

[0123] 2. Test results

[0124] The results of H&E staining of lung tissue are shown in Figure 5 (Alveolar collapse (blue arrow), inflammatory cell infiltration (green arrow); first row: Bar = 200 μm; second row: Bar = 50 μm). The lung tissue structure of wild-type BALB / c mice was normal, with no obvious pathological changes. Local alveolar collapse appeared in the lung tissue of 8-week-old BALB / c-hSTING1-p.V147L homozygous mice; inflammatory cell infiltration appeared in the lung tissue of 15-week-old BALB / c-hSTING1-p.V147L homozygous mice.

[0125] The results of H&E staining of ear skin tissue are shown in Figure 6(Herma abscess (orange arrow), epidermal thickening (yellow arrow), inflammatory cell infiltration (blue arrow), plasma cells (black arrow), fibroblasts (white arrow), fibrocytes (pink arrow); first row: Bar = 200 μm; second row: Bar = 50 μm). The ear skin tissue structure of wild-type BALB / c mice is normal, with no obvious pathological changes. In the ear skin tissue of 8-week-old BALB / c-hSTING1-p.V147L homozygous mice, local stratum corneum abscesses are formed, the stratum corneum is incompletely keratinized, and inflammatory cells are infiltrated; the epidermis is slightly thickened, and the number of spinous cell layers increases; some collagen fibers in the dermis are degenerated and necrotic, the collagen fiber structure disappears, and the necrotic area is accompanied by a small amount of inflammatory cell infiltration and a small amount of fibrous tissue hyperplasia. In the ear skin tissue of 15-week-old BALB / c-hSTING1-p.V147L homozygous mice, local stratum corneum parakeratosis was observed; the epidermis was significantly thickened; the fibrous tissue in the dermis was slightly proliferated, and a small amount of inflammatory cells infiltrated.

[0126] The results of H&E staining of tail skin tissue are shown in Figure 7 (abscess (yellow arrow), edema (light blue arrow), inflammatory cell infiltration (dark green arrow), epidermal thickening (orange arrow), parakeratosis (black arrow), fibroblasts (light green arrow), fibrocytes (dark blue arrow), collagen fiber necrosis (pink arrow), scab (white arrow); first row: Bar=200μm; second row: Bar=50μm). The wild-type BALB / c tail skin tissue structure is normal, without obvious pathological changes. The tail skin of BALB / c-hSTING1-p.V147L homozygous 8-week-old mice has parakeratosis and local abscess formation; the dermis is thickened, and the number of spinous cell layers increases; the collagen fibers in the dermis are necrotic, and the hair follicles and sebaceous glands are reduced; the dermis and subcutaneous tissue are infiltrated with inflammatory cells and more fibrous tissue hyperplasia; the subcutaneous tissue has local edema, and the muscle fibers in the muscular layer are loosely arranged. The tail skin tissue of 15-week-old BALB / c-hSTING1-p.V147L homozygous mice showed extensive necrosis. The entire layer of skin tissue in the necrotic area was necrotic with structural loss, and a large number of neutrophils infiltrated, accompanied by exudation of strong eosinophilic serous substances, forming large abscesses and thick scabs. The dermis at the edge of the necrotic area was edematous, the epidermis was necrotic and thickened, and a small amount of new epidermis was formed. The collagen fibers in the dermis were necrotic, the hair follicles and sebaceous glands were significantly reduced, and there was a large number of neutrophils infiltrating and fibrous tissue hyperplasia.

[0127] Masson staining results of ear skin tissue are shown in Figure 8(Collagen fibers (green arrows); first row: Bar=500μm; second row: Bar=100μm). The ear skin tissue structure of wild-type BALB / c mice was normal, with no obvious pathological changes, and a small amount of collagen fibers could be seen. The collagen fibers in the ear skin tissue of homozygous BALB / c-hSTING1-p.V147L mice aged 8 and 15 weeks were slightly increased.

[0128] Experimental Example 5: Detection of immune cell subset ratios in BALB / c-hSTING1-p.V147L mice by flow cytometry

[0129] 1. Experimental methods (flow cytometry sample preparation and staining)

[0130] Peripheral blood or spleen of BALB / c-hSTING1-p.V147 homozygous mice and BALB / c background mice were collected. The spleen was cut to the required experimental weight, ground and digested. Blood was collected from the heart into EP tubes containing anticoagulants. The extracellular proteins of tissue cells were stained with mouse T\B\NK\DC\macrophages\monocytes\neutrophils\eosinophils\basophils surface antibodies. After washing the cells with PBS, flow cytometry was used to detect the proportions of immune cells such as T (CD4+, CD8+), B, NK, DC, monocytes, neutrophils, eosinophils, basophils, and macrophages.

[0131] 2. Test results

[0132] 2.1. Detection of peripheral blood immune cell subsets in BALB / c and BALB / c-hSTING1-p.V147L

[0133] like Fig. 9 and 10 As shown, the percentages of immune cells such as T cells, NK cells, dendritic cells, eosinophils, and macrophages in the peripheral blood of BALB / c-hSTING1-p.V147L mice were similar to those of BALB / c, indicating that hSTING1-p.V147L replacing mSTING1 did not change the development, differentiation, and distribution of these cells in the peripheral blood. The percentage of B cells decreased, while the percentage of neutrophils increased, which is similar to other existing SAVI model studies.

[0134] 2.2. Detection of the proportion of spleen immune cell subsets in BALB / c and BALB / c-hSTING1-p.V147L

[0135] like Fig.11 and 12As shown, the percentages of immune cells such as T cells, NK cells, dendritic cells, monocytes, eosinophils, and macrophages in the spleen of BALB / c-hSTING1-p.V147L mice were similar to those of BALB / c, indicating that hSTING1-p.V147L did not change the development, differentiation, and distribution of these cells in the spleen after replacing mSTING1. The percentage of B cells decreased, while the percentage of neutrophils increased, which is similar to other existing SAVI model studies.

[0136] Experimental Example 6: Statistics of the incidence of skin lesions in BALB / c-hSTING1-p.V147L

[0137] 1. Test methods

[0138] In the longitudinal study of the incidence of the existing population, the incidence of skin lesions in all mice of the BALB / c-hSTING1-p.V147L existing population was evaluated weekly for a total of 12 weeks of observation and statistics, and the weekly statistical results were summarized. During the initial statistics, the number of mice of different ages was distributed differently. Small-age mice grew into large-age mice during the long-term statistical process. Each mouse was counted once in each age group, and newborn small-age mice reaching 6 weeks of age were also included in the statistics.

[0139] 2. Test results

[0140] Table 6 Proportion of skin lesions in BALB / c-hSTING1-p.V147L mice

[0141]

[0142]

[0143] The first three columns in Table 6 indicate the percentage of mice with the phenotype at the corresponding age, and the last column indicates the total number of mice evaluated at the corresponding age. As shown in the experimental results in Table 6, 6-week-old hSTING1-p.V147L mice began to develop tail and ear skin inflammation, with a high incidence, and did not heal spontaneously.

[0144] Although specific embodiments of the present invention have been described, it will be appreciated by those skilled in the art that various changes and modifications may be made to the present invention without departing from the scope or spirit of the present invention. Therefore, the present invention is intended to cover all such changes and modifications that fall within the scope of the appended claims and their equivalents.

Claims

1. A method for constructing an animal model of STING-related infantile-onset vasculitis, characterized in that: The method comprises the following steps: replacing the Sting gene fragment in the genome of the target animal with a human hSTING1-p .V147L mutant gene fragment, the sequence of which is shown in SEQ ID NO: 1; the construction method comprises the following steps: (1) constructing a targeting vector expressing the humanized Sting1 gene for inserting the humanized Sting1 gene; (2) Designing sgRNA targeting the translation start site and post-coding region of the mouse Sting1 gene, and obtaining the above sgRNA using in vitro transcription technology; (3) co-injecting or co-electroporating the targeting vector constructed in step (1), the sgRNA obtained in step (2), and the Cas9 mRNA or Cas9 protein into the cytoplasm or nucleus of a fertilized egg of a background mouse, and transplanting the fertilized egg into a pseudo-pregnant mouse, performing genotyping on the pseudo-pregnant mouse offspring, and screening for positive F0 mice in which the correct human fragment was successfully inserted; (4) The sexually mature positive F0 mice were bred with background mice to obtain F1 mice. After verification by PCR and sequencing, positive heterozygous F1 mice were obtained, and the Sting1 humanized mouse model that can be propagated was screened; The step (1) includes the following steps: constructing a successful targeting vector sequence as shown in SEQ ID No. 4; The gene sequences of sgRNA in step (2) are SEQ ID No. 6 and SEQ ID No.

8.

2. The construction method according to claim 1, characterized in that: The target animal is a mouse, and the mutant gene is a STING 147 valine to leucine mutation sequence.

3. The construction method according to claim 1, characterized in that: The step (1) includes the following steps: according to the structure and function of human Sting1, a gene fragment of human Sting1 containing a valine to leucine mutation gene at position 147 is inserted into the translation start site of the mouse Sting1 transcript, the hSTING1-p .V147L mutant amino acid sequence expressed by the selected human hSTING1-p .V147L mutant gene fragment is shown in SEQ ID No. 2, and the replaced mouse Sting1 gene amino acid sequence is shown in SEQ ID No.

3.

4. The construction method according to any one of claims 1 to 3, characterized in that: The animal model is consistent with the phenotype of clinical SAVI patients with Sting1 gene mutations, who suffer from skin inflammation and lung disease.

5. Use of the mouse obtained by the construction method according to any one of claims 1 to 3 in screening drugs for treating STING-related infantile-onset vasculitis.

Images