Application of ENPP1 positive cells in prevention and / or treatment of arthritis

Abstract

The invention relates to application of ENPP1 positive cells in prevention and / or treatment of arthritis. Specifically, the invention provides an application of an inhibitor or an antagonist of the ENPP1 gene or the protein thereof, the inhibitor or the antagonist is used for preparing a composition or a preparation, and the composition or the preparation is used for (a) preventing and / or treating arthritis-related diseases; and / or (b) inhibiting synovial inflammation.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and more particularly, to use of ENPP1-positive cells in preventing and or treating arthritis. Background Art

[0002] Osteoarthritis (OA) is a degenerative disease with high morbidity and disability rates and a complex pathogenesis. Its primary manifestations are joint pain, limited mobility, and even functional loss, with a high disability rate in the late stages. OA is a pan-articular disease characterized by cartilage degeneration as its core and synovial inflammation as a key contributing factor. Epidemiological data indicate that OA affects over 300 million people worldwide. Although current treatments (such as medications or joint replacements) can alleviate symptoms, no FDA-approved medications are currently available to prevent or slow the progression of OA. Therefore, in-depth research into the pathogenesis of OA is urgently needed to develop novel therapeutics to ameliorate the disease.

[0003] With the rapid development of single-cell transcriptome sequencing technology, studies have defined seven functionally heterogeneous synovial fibroblast subsets during the progression of post-traumatic osteoarthritis (PTOA) and characterized their dynamic profiles at different time points. Another study, using single-cell technology, discovered the differentiation trajectory of the FAPα+THY1+ fibroblast subset and its important role in joint inflammation and damage. Researchers used single-cell technology to decipher the single-cell landscape of the infrapatellar fat pad in OA, revealing the mechanisms of fibrosis and inflammatory regulation in the infrapatellar fat pad and synovial tissue in OA. These studies suggest that synoviocytes exhibit functional heterogeneity and play diverse pathological roles in different diseases. Studies have shown that aging is a key driver of synovial degeneration in OA. However, the identification of synovial cell profiles and their dynamic changes in synovial tissue from humans and mice at different ages under aging conditions have been reported. In particular, the functional validation of key synovial pathological cells and the underlying molecular mechanisms remain unclear.

[0004] Synovial inflammation and cartilage degeneration are core pathological events in OA. However, the interplay between these two pathological changes, the sequence of these events, and their mechanisms of action remain unclear. Functional heterogeneity of synovial cells plays a crucial role in disease progression under pathological conditions such as trauma and inflammatory stimulation. However, the existence of a distinct subpopulation of synovial pathological cells in OA models and their communication with chondrocytes remain unclear. Summary of the Invention

[0005] In response to the above problems, the first purpose of the present invention is to clarify that the functional changes of ENPP1+ cell subpopulations induced by aging are the key driving factors promoting OA, thereby providing new insights into the pathogenesis of OA.

[0006] The second purpose of the present invention is to provide an important basis for the development of new drug targets that inhibit ENPP1+ cells from inducing cartilage aging and the occurrence and development of OA.

[0007] In a first aspect of the present invention, there is provided a use of ENPP1 as a biomarker or target in the preparation of products for diagnosis, monitoring, severity assessment, efficacy assessment, prognosis assessment, prevention and / or treatment of arthritis-related diseases.

[0008] In another preferred embodiment, the diagnosis, monitoring, severity assessment, efficacy assessment or prognosis assessment of arthritis-related diseases includes detecting the presence of ENPP1 or the expression level of its mRNA and / or protein, or the presence of ENPP1-positive cells.

[0009] In another preferred embodiment, the prevention and / or treatment of arthritis-related diseases comprises: knocking out or knocking down ENPP1, or eliminating ENPP1-positive cells.

[0010] In another preferred embodiment, the knockout or knockdown of ENPP1 comprises using the CRISPR / Cas system, tissue-specific knockout, or introduction of interfering RNA or microRNA that targets the knockout or knockdown of ENPP1.

[0011] The elimination of ENPP1-positive cells includes selectively eliminating ENPP1-positive cells by a bioablation method, wherein the bioablation method includes a gene knockout-mediated ablation method or an immune cell-mediated ablation method.

[0012] In another preferred embodiment, the ENPP1-positive cells include ENPP1-positive synovial fibroblasts and synovial immune cells.

[0013] In another preferred embodiment, the product includes a reagent for detecting the presence of ENPP1 or the expression level of its mRNA and / or protein, or the product includes a reagent for regulating the expression level of ENPP1 mRNA and / or protein.

[0014] In another preferred embodiment, the regulation includes up-regulation or down-regulation.

[0015] In a second aspect, the present invention provides a use of an inhibitor or antagonist of the ENPP1 gene or its protein for preparing a composition or formulation for (a) preventing and / or treating arthritis-related diseases; and / or (b) inhibiting synovial inflammation.

[0016] In another preferred embodiment, the composition or preparation is also used for (i) inhibiting the occurrence of cartilage degeneration; and / or (ii) inhibiting synovial lesions caused by aging.

[0017] In another preferred embodiment, the inhibitor refers to a substance that can reduce the activity and / or content of the ENPP1 gene or its protein in vivo or in vitro; the substance can be a synthetic or natural compound, protein, nucleotide, etc.

[0018] In another preferred embodiment, the ENPP1 inhibitor includes a substance that inhibits the expression of ENPP1.

[0019] In another preferred embodiment, the ENPP1 inhibitor includes a substance that inhibits ENPP1 protein.

[0020] In another preferred embodiment, the ENPP1 inhibitor includes an ENPP1 protein inhibitor and / or an ENPP1 gene inhibitor.

[0021] In another preferred embodiment, the inhibiting of ENPP1 expression or activity refers to reducing the expression or activity of the ENPP1 gene or protein by ≥20%, preferably ≥50%, and more preferably ≥70%.

[0022] In another preferred embodiment, the ENPP1 inhibitor is selected from the group consisting of a small molecule compound, an antisense nucleic acid of the ENPP1 gene, an antibody against the protein encoded by the ENPP1 gene, or a combination thereof.

[0023] In another preferred embodiment, the protein comprises a full-length protein or a protein fragment.

[0024] In another preferred embodiment, the ENPP1 gene or its protein is derived from mammals, more preferably from rodents (such as mice, rats), primates and humans.

[0025] In another preferred embodiment, the ENPP1 protein further includes derivatives of the ENPP1 protein.

[0026] In another preferred embodiment, the derivatives of the ENPP1 protein include modified ENPP1 proteins, protein molecules whose amino acid sequences are homologous to natural ENPP1 proteins and have the activity of natural ENPP1 proteins, dimers or multimers of ENPP1 proteins, and fusion proteins containing the amino acid sequences of ENPP1 proteins.

[0027] In another preferred embodiment, the modified ENPP1 protein is a PEGylated ENPP1 protein.

[0028] In another preferred embodiment, the "protein molecule whose amino acid sequence is homologous to the natural ENPP1 protein and has the activity of the natural ENPP1 protein" refers to a protein molecule whose amino acid sequence has ≥85% homology, preferably ≥90% homology, more preferably ≥95% homology, and most preferably ≥98% homology with the ENPP1 protein; and has the activity of the natural ENPP1 protein.

[0029] In another preferred embodiment, the ENPP1 inhibitor is selected from the group consisting of a small molecule compound, an antisense nucleic acid of the ENPP1 gene, an antibody against the protein encoded by the ENPP1 gene, or a combination thereof.

[0030] In another preferred embodiment, the sequences of the forward primer and reverse primer targeting the ENPP1 mRNA are shown as SEQ ID NO: 1 and SEQ ID NO: 2, respectively.

[0031] In another preferred embodiment, the ENPP1 gene encodes ENPP1 protein.

[0032] In another preferred embodiment, the composition comprises a pharmaceutical composition.

[0033] In another preferred embodiment, the pharmaceutical composition contains (a) an inhibitor of ENPP1 gene or protein thereof; and (b) a pharmaceutically acceptable carrier.

[0034] In another preferred embodiment, the pharmaceutical composition is in liquid, solid, or semisolid form.

[0035] In another preferred embodiment, the dosage form of the pharmaceutical composition includes tablets, granules, capsules, oral solutions, or injections.

[0036] In another preferred embodiment, in the pharmaceutical composition, the component (a) accounts for 1-99 wt %, preferably 10-90 wt %, and more preferably 30-70 wt % of the total weight of the pharmaceutical composition.

[0037] In another preferred embodiment, the composition further comprises additional components for (a) preventing and / or treating arthritis-related diseases; and / or (b) inhibiting synovial inflammation.

[0038] In another preferred embodiment, the composition further comprises an additional component that inhibits cartilage degeneration.

[0039] In another preferred embodiment, the composition or preparation can be used alone or in combination for (a) preventing and / or treating arthritis-related diseases; and / or (b) inhibiting synovial inflammation.

[0040] In another preferred embodiment, the combined use includes: combined use with other drugs for (a) preventing and / or treating arthritis-related diseases; and / or (b) inhibiting synovial inflammation.

[0041] In another preferred embodiment, the other drugs for (a) preventing and / or treating arthritis-related diseases; and / or (b) inhibiting synovial inflammation are selected from the following groups: non-steroidal anti-inflammatory drugs, analgesics (non-anti-inflammatory), chondroprotective agents, intra-articular injection drugs, traditional Chinese medicines and external patches, or a combination thereof.

[0042] In another preferred embodiment, the nonsteroidal anti-inflammatory drug comprises celecoxib.

[0043] In another preferred embodiment, the bone protective agent comprises chondroitin sulfate.

[0044] In another preferred embodiment, the intra-articular injection drug includes sodium hyaluronate and glucocorticoid.

[0045] In another preferred embodiment, the arthritis-related disease is selected from the group consisting of osteoarthritis, rheumatoid arthritis, rheumatoid arthritis, infectious arthritis, gouty arthritis, or a combination thereof.

[0046] In a third aspect of the present invention, a pharmaceutical composition is provided, comprising:

[0047] (a1) a first active ingredient for preventing and / or treating arthritis-related diseases, the first active ingredient comprising: an inhibitor of the ENPP1 gene or its protein;

[0048] (a2) a second active ingredient for preventing and / or treating arthritis-related diseases, wherein the second active ingredient includes: other drugs for preventing and / or treating arthritis-related diseases; and

[0049] (b) a pharmaceutically acceptable carrier.

[0050] In another preferred embodiment, in the pharmaceutical composition, the component (a1) accounts for 1-99 wt %, preferably 10-90 wt %, and more preferably 30-70 wt % of the total weight of the pharmaceutical composition.

[0051] In another preferred embodiment, in the pharmaceutical composition, the component (a2) accounts for 1-99 wt %, preferably 10-90 wt %, and more preferably 30-70 wt % of the total weight of the pharmaceutical composition.

[0052] In another preferred embodiment, the weight ratio of the first active ingredient to the second active ingredient is 1:100 to 100:1, preferably 1:10 to 10:1.

[0053] In another preferred embodiment, the pharmaceutical composition further comprises an additional component that inhibits synovial inflammation.

[0054] In another preferred embodiment, the pharmaceutical composition further comprises an additional component that inhibits cartilage degeneration.

[0055] In another preferred embodiment, the other drugs for preventing and / or treating arthritis-related diseases are selected from the following group: non-steroidal anti-inflammatory drugs, analgesics (non-anti-inflammatory), chondroprotective agents, intra-articular injection drugs, Chinese patent medicines and external patches, or a combination thereof.

[0056] In another preferred embodiment, the nonsteroidal anti-inflammatory drug comprises celecoxib.

[0057] In another preferred embodiment, the bone protective agent comprises chondroitin sulfate.

[0058] In another preferred embodiment, the intra-articular injection drug includes sodium hyaluronate and glucocorticoid.

[0059] In another preferred embodiment, the arthritis-related disease is selected from the group consisting of osteoarthritis, rheumatoid arthritis, rheumatoid arthritis, infectious arthritis, gouty arthritis, or a combination thereof.

[0060] In another preferred embodiment, the osteoarthritis includes: aging arthritis, lipid metabolism arthritis, and traumatic arthritis.

[0061] In another preferred embodiment, the pharmaceutical composition may contain a single compound or a mixture of multiple compounds.

[0062] In another preferred embodiment, the pharmaceutical composition is used to prepare a drug or preparation for treating or preventing arthritis-related diseases.

[0063] In another preferred embodiment, the pharmaceutical dosage form is an oral or parenteral dosage form.

[0064] In another preferred embodiment, the oral dosage form is a tablet, powder, granule or capsule, or an emulsion or syrup.

[0065] In another preferred embodiment, the non-oral dosage form is an injection or injection.

[0066] In another preferred embodiment, the total content of the active ingredient (a1) and the active ingredient (a2) is 1 to 99 wt %, more preferably 5 to 90 wt %, of the total weight of the composition.

[0067] In a fourth aspect of the present invention, a medicine kit is provided, comprising:

[0068] (i) a first container, and an active ingredient (a1) an inhibitor of ENPP1 gene or protein thereof, or a drug containing the active ingredient (a1) in the first container; and

[0069] (ii) a second container, and the active ingredient (a2) in the second container; other drugs for preventing and / or treating arthritis-related diseases, or drugs containing the active ingredient (a2).

[0070] In another preferred embodiment, the first container and the second container are the same or different containers.

[0071] In another preferred embodiment, the drug in the first container is a single-ingredient preparation containing an inhibitor of the ENPP1 gene or its protein.

[0072] In another preferred embodiment, the medicine in the second container is a single-ingredient preparation containing other medicines for preventing and / or treating arthritis-related diseases.

[0073] In another preferred embodiment, the dosage form of the drug is an oral dosage form or an injection dosage form.

[0074] In another preferred embodiment, the kit further comprises instructions for administering the active ingredient (a1) and the active ingredient (a2) in combination to (i) prevent and / or treat arthritis-related diseases; and / or (ii) inhibit synovial inflammation.

[0075] In another preferred embodiment, the dosage forms of the preparation containing the active ingredient (a1) ENPP1 gene or protein inhibitor or the preparation containing other drugs for preventing and / or treating arthritis-related diseases include capsules, tablets, suppositories, or intravenous injections.

[0076] In another preferred embodiment, the preparation containing the active ingredient (a1) an inhibitor of ENPP1 gene or protein thereof has a concentration of 0.0001-100 mg / kg body weight, preferably 0.1-50 mg / kg body weight, and more preferably 1-20 mg / kg body weight.

[0077] In a fifth aspect of the present invention, a method for inhibiting synovial inflammation is provided, comprising the steps of:

[0078] Synovial cells are cultured in the presence of an inhibitor of the ENPP1 gene or its protein, thereby inhibiting synovial inflammation.

[0079] In another preferred embodiment, the method is non-diagnostic and non-therapeutic.

[0080] In another preferred embodiment, the method is therapeutic.

[0081] In another preferred embodiment, the effective concentration of the ENPP1 inhibitor is 0.0001-100 mg / kg body weight, preferably 1-50 mg / kg body weight, and more preferably 5-20 mg / kg body weight.

[0082] In a sixth aspect of the present invention, a method for screening potential therapeutic agents for arthritis-related diseases is provided, comprising:

[0083] (a) in a test group, in a culture system, in the presence of a test compound, culturing cells expressing the ENPP1 gene for a period of time, and detecting the expression level E1 of the ENPP1 gene in the culture system of the test group;

[0084] and detecting the expression level E2 of the ENPP1 gene in the culture system of the control group in the absence of the test compound and under the same other conditions; and

[0085] (b) Compare E1 and E2. If E1 is significantly lower than E2, it indicates that the test compound is a potential therapeutic agent for arthritis.

[0086] In another preferred embodiment, the “significantly lower than” means E1 / E2≤1, preferably, ≤0.5, more preferably, ≤0.1.

[0087] In another preferred embodiment, the cells comprise synovial fibroblasts.

[0088] In another preferred embodiment, the method is non-diagnostic and non-therapeutic.

[0089] In another preferred embodiment, the method comprises the step (c): administering the potential therapeutic agent identified in step (a) to a mammal, thereby determining its effect on the arthritis-related disease in the mammal.

[0090] In another preferred embodiment, the mammal comprises a human or a non-human mammal.

[0091] In another preferred embodiment, the non-human mammals include rodents and primates, preferably, mice, rats, rabbits and monkeys.

[0092] In a seventh aspect of the present invention, a method for preventing and / or treating arthritis-related diseases is provided, comprising the steps of:

[0093] An inhibitor of the ENPP1 gene or its protein, the pharmaceutical composition according to the second aspect of the present invention, or the drug kit according to the third aspect of the present invention is administered to a subject in need.

[0094] In another preferred embodiment, said administering comprises oral administration.

[0095] In another preferred embodiment, the subject comprises a human or a non-human mammal.

[0096] In another preferred embodiment, the non-human mammals include rodents and primates, preferably mice, rats, rabbits, monkeys.

[0097] In another preferred embodiment, the ENPP1 inhibitor is administered at a dosage of 0.0001-100 mg / kg body weight, preferably 1-50 mg / kg body weight, and most preferably 5-20 mg / kg body weight.

[0098] In another preferred embodiment, the ENPP1 inhibitor is administered 1-150 times per month, preferably once a day.

[0099] In another preferred embodiment, the ENPP1 inhibitor is administered for 5-100 days, preferably 10-50 days, and most preferably 14-42 days.

[0100] In an eighth aspect of the present invention, a method for constructing an animal model for specific ablation of ENPP1-positive cells is provided, comprising the following steps:

[0101] ENPP1-positive cells are selectively eliminated by a biological ablation method, thereby obtaining the animal model of specific ablation of ENPP1-positive cells; wherein the biological ablation method includes a gene knockout-mediated ablation method or an immune cell-mediated ablation method.

[0102] In another preferred embodiment, the ENPP1-positive cells include ENPP1-positive synovial fibroblasts or synovial immune cells.

[0103] In another preferred embodiment, the gene knockout-mediated ablation method includes: constructing an ENPP1 gene knockout animal model and / or an animal model for eliminating ENPP1-positive cells based on CRISPR / Cas9 technology.

[0104] In another preferred embodiment, the method for constructing the ENPP1 gene knockout animal model comprises the following steps:

[0105] 1) Design of gRNA targeting mouse ENPP1 sequence:

[0106] 2) Purifying the pair of gRNAs from step 1) and co-injecting them with cas9 mRNA into mouse embryos to obtain F0 generation mice;

[0107] 3) Identify the ENPP1 genotype and select positive F0 generation mice;

[0108] 4) Positive F0 mice are crossed with wild-type mice to obtain F1 mice, and the F1 heterozygous mice are self-pollinated to obtain homozygous offspring, thereby constructing an animal model for ENPP1 gene knockout. This invention utilizes CRISPR / Cas9 technology to achieve site-specific knockout of the ENPP1 gene, offering the advantages of simple operation and high ENPP1 gene knockout efficiency.

[0109] In another preferred embodiment, the method for constructing an animal model for eliminating ENPP1-positive cells comprises the following steps:

[0110] 1) Design of gRNA targeting mouse ENPP1 sequence:

[0111] 2) Purifying the pair of gRNAs from step 1) and co-injecting them with cas9 mRNA into mouse embryos to obtain F0 generation mice;

[0112] 3) Identify the ENPP1 genotype and select positive F0 generation mice;

[0113] 4) Positive F0 generation mice were crossed with wild-type mice to obtain F1 generation mice, and the F1 generation heterozygous mice were self-pollinated to obtain homozygous offspring, thereby constructing an animal model for the elimination of ENPP1-positive cells.

[0114] In another preferred embodiment, the gene knockout-mediated ablation method further comprises: inducing the Caspase 9 system, causing ENPP1-positive cells to express Caspase 9 protein through genetic engineering methods, and injecting a chemical inducer (CID) to trigger apoptosis.

[0115] In another preferred embodiment, the immune cell-mediated ablation method includes: CAR-T cell therapy, CAR-NK cell therapy or CAR-M cell therapy.

[0116] In another preferred embodiment, the CAR-T cell therapy comprises: using a viral vector (such as a lentivirus) to introduce a chimeric antigen receptor (CAR) gene into T cells, causing them to express a receptor capable of recognizing a target cell antigen (such as Enpp1);

[0117] The CAR-NK cell therapy or CAR-M cell therapy includes: utilizing natural killer cells (NK) and macrophages (M) to transform and target the elimination of pathogenic ENPP1-positive cells.

[0118] In a ninth aspect, the present invention provides use of an animal model obtained by the construction method described in the eighth aspect of the present invention in screening drugs for preventing and / or treating arthritis-related diseases.

[0119] In another preferred embodiment, the arthritis-related disease is selected from the group consisting of osteoarthritis, rheumatoid arthritis, infectious arthritis, gouty arthritis, or a combination thereof.

[0120] In another preferred embodiment, the osteoarthritis includes: aging arthritis, lipid metabolism arthritis, and traumatic arthritis.

[0121] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features described in detail below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be listed here one by one.

[0122] Compared with the prior art, the present invention has the following technical effects:

[0123] (1) The present invention discovered a new group of pathogenic cells that appear during synovial aging. This group of cells increases with age, and the proportion of these cells gradually increases during the pathogenesis of OA. This could serve as a new intervention target for the treatment of OA.

[0124] (2) The present invention discloses for the first time the application of synovial pathogenic cells in the diagnosis, evaluation or prognosis of OA. By detecting the number level of synovial pathogenic cells, the important marker cells of synovial aging in the young, middle-aged and elderly groups can be distinguished.

[0125] (3) The present invention discloses for the first time the use of synovial pathogenic cell inhibitors in the preparation of drugs for preventing and / or treating OA. By inhibiting the formation of synovial pathogenic cells, the effect of preventing and / or treating OA drugs can be achieved.

[0126] (4) The present invention also provides transgenic ablation cells targeting synovial pathogenic cells, which can achieve the effect of preventing and / or treating OA drugs by targeting and ablating the expression of synovial pathogenic cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0127] Figure 1 The HE staining results show that in the aging process of human joints, pathological changes in the synovium occur earlier than cartilage degeneration. AC are the pathological changes in the synovium and cartilage degeneration of the young group (Young, 18-28 years old), the middle-aged group (Middle, 35-50 years old), and the elderly group (Old, over 65 years old), respectively. The green arrow indicates inflammatory infiltration, the blue arrow indicates fibrosis, and the red arrow indicates ossification.

[0128] Figure 2HE staining results show that pathological changes in the synovium precede cartilage degeneration during joint aging in mice. (a) and (b) show pathological changes in the synovium and cartilage degeneration in young (3-month-old), middle (9-month-old), and old (18-month-old) mice, respectively. In synovial tissue, green arrows indicate inflammatory infiltration, and blue arrows indicate hyperplasia; in cartilage tissue, yellow arrows indicate cartilage matrix degradation. (d) shows the synovial inflammation score; (e) shows the cartilage thickness measurement results. n = 5, scale bar, 200 μm. *P < 0.05, ***P < 0.001.

[0129] Figure 3 Figures show the results of single-cell analysis in mice. (a) shows the design of single-cell analyses of synovial tissue from mice of different ages (3, 9, and 18 months) (n = 5). (b) shows the division of synovial tissue into nine subpopulations based on cell-specific genes. (c) shows the dynamic cell ratios in synovial tissue, with Y-Sy, M-Sy, and O-Sy representing synovial tissue from mice of different ages (3, 9, and 18 months), respectively. (d) shows the most significant changes in the interactions between fibroblast clusters (SSF and SIF) and other cells. This suggests that synovial fibroblasts are the cell subpopulation with the most significant functional changes in OA.

[0130] Figure 4 Figures show the correlation between ENPP1+ cells and the early stages of OA. (a) shows the most significant dynamic changes in the C3 subpopulation in mice of different ages (3M_Sy: 3-month-old young synovium, 9M_Sy: 9-month-old middle synovium, and 18M_Sy: 18-month-old old synovium). Numbers 0-8 represent C0-C8 cell subpopulations, respectively. (b) shows the perturbation scores of different cell subpopulations during aging. (c) shows the specific high expression of ENPP1 and PRG4 in the C3 subpopulation. (d) show the multiple immunofluorescence analysis of ENPP1+PRG4+ cell subpopulations and their statistical results, demonstrating a significant increase in the ENPP1+PRG4+ cell subpopulation with aging. (T, M, and S represent tibia, meniscus, and synovium, respectively; n = 5; scale bars, 75 and 10 μm; **P < 0.01, ***P < 0.001).

[0131] Figure 5 The results of the in vivo animal experiments of the present invention confirm that ENPP1+ cells promote the pathological progression of arthritis (OA). (a) shows the results of flow cytometry sorting and identification of ENPP1+ cells in the synovial tissue of aged mice. (b) shows the schematic diagram of the experimental design flow for the mouse model of cell transfusion: a DMM OA mouse model (n=4) was established. Two weeks after modeling, ENPP1+ cells (5x10 3Cells were injected into the right limb (right limb) or PBS for 8 weeks. c shows the results of SO staining analysis of joint tissue. Yellow arrows indicate cartilage matrix degradation and wear; scale bar is 200 μm. d shows the results of OARSI scoring of joint tissue. (e) and (f) show the results of experiments related to ENPP1+ cell-induced hyperalgesia in mice. *P < 0.05, **P < 0.01, ***P < 0.001. DETAILED DESCRIPTION

[0132] To make the purpose, technical solutions, beneficial effects, and significant improvements of the embodiments of the present invention more clear, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, all the embodiments described are only some embodiments of the present invention, not all embodiments; based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0133] the term

[0134] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0135] As used herein, the term “including” or “comprising” encompasses “comprising,” “consisting mainly of,” “consisting essentially of,” and “consisting of;” “consisting mainly of,” “consisting essentially of,” and “consisting of” are subordinate concepts of “containing,” “having,” or “including.”

[0136] ENPP1 (Ectonucleotide Pyrophosphatase / Phosphodiesterase 1)

[0137] ENPP1, short for ectonucleotide pyrophosphatase / phosphodiesterase 1, is a cell-surface enzyme involved in regulating nucleotide metabolism and mineralization. Fibroblasts (ENPP1) are typically transformed from quiescent fibroblasts under chronic inflammatory or pathological conditions (similar to activated fibroblasts or myofibroblasts). They highly express proinflammatory cytokines (such as IL-6 and IL-8), chemokines, and matrix metalloproteinases (MMPs), recruiting immune cells and maintaining an inflammatory microenvironment. They promote fibrosis: Excessive secretion of ECM leads to tissue sclerosis (e.g., pulmonary fibrosis).

[0138] Experimental methods:

[0139] 1. Inclusion and exclusion criteria for volunteers of different age groups

[0140] This study involved volunteers undergoing thigh resections due to accidental trauma. Discarded synovial tissue from the joints was collected. All volunteers were informed and provided signed informed consent before specimen collection. This study protocol has been approved by the Ethics Review Committee of the Yangzhi Rehabilitation Hospital Affiliated to Tongji University (No. SBKT-2025-021).

[0141] 1.1 Inclusion criteria for the study subjects:

[0142] ① Volunteers who had accidental trauma within 24 hours and needed to undergo thigh resection;

[0143] ② Unilateral or bilateral thigh injuries with intact joints were divided into three age groups: young group (18-28 years old), middle-aged group (35-50 years old) and elderly group (over 65 years old);

[0144] ③ No history of any drug injection into the joint cavity.

[0145] 1.2 Exclusion criteria for study subjects:

[0146] ① Patients with bone and cartilage metabolic diseases;

[0147] ② Patients with autoimmune arthritis, including rheumatoid arthritis or combined gouty arthritis;

[0148] ③ Patients with bone tumor diseases;

[0149] ④ Those who have received drug or surgical treatment in the past 6 months.

[0150] 2. Preparation of Synovial Tissue from Humans and Mice of Different Ages

[0151] Synovial tissue was collected from six humans and divided into three age groups: young (Y; 18-28 years old), middle-aged (M; 35-50 years old), and elderly (O; 65 years and older). Synovial tissue was also collected from mice of different ages and divided into young (Y; 3 months old), middle-aged (M; 9 months old), and elderly (O; 18 months old). Synovial tissue was obtained from mouse joints, paraffin-embedded, and histologically analyzed using hematoxylin and eosin (HE) and sulfur dioxide (SO) staining to assess synovial degeneration. The animal husbandry and experimental protocols for this invention were reviewed by the Tongji University Laboratory Animal Ethics Committee, with animal ethics approval number TJAA11225104, and adhered to animal protection, animal welfare, and ethical principles, as well as relevant national regulations on laboratory animal welfare ethics.

[0152] 3. Construction and Sequencing of Synovial Tissue Senescent Cells from Different Age Groups of Mice

[0153] 10x Genomics was used to investigate the dynamics of synoviocyte aging at the single-cell level. Synovial tissue was collected from 3-, 9-, and 18-month-old mice (n=5 pooled samples per group). The tissue was enzymatically digested into single cells using a collagenase complex solution (type I and II collagenase, neutral protease, elastase, hyaluronic phytase, and DNAse). Excess red blood cells were removed using an erythrocyte lysis buffer, and single-cell suspensions were collected for synovial cell library construction and sequencing.

[0154] 4. Single-cell transcriptional data analysis reveals dynamic maps of various cells and identifies ENPP1+ cell subpopulations and their characteristics

[0155] Single-cell raw data were processed using CellRanger software and analyzed using Seurat software, excluding cells expressing high levels of mitochondrial genes (>10%). The harmony algorithm was used for multi-sample integration, and cell type annotation and subpopulation clustering were performed based on currently known cell markers. For example, SSF fibroblast clusters highly express Thy1, SIF fibroblast clusters highly express Prg4, endothelial cells highly express Cdh5 and VWF, pericytes highly express Mcam and Pdgfrb, monocytes and macrophages highly express Csf1r and CD14, and neutrophils highly express S100a8, S100a9, and Cd177.

[0156] A second-level cluster analysis was performed based on the definition of synovial marker subpopulations. The FindAllMarkers and FindMarkers functions were used to identify subpopulation-specifically expressed genes and perform enrichment analysis. Synovial cell subpopulations were annotated, and the changes in the number and proportion of each cell subpopulation across different age groups were analyzed.

[0157] Further cluster analysis of synovial fibroblast subpopulations was performed to screen the key synovial pathological cell subpopulation (ENPP1+ cell) in the joint degeneration process; the changes in the cell composition ratio of this subpopulation in different age groups were analyzed to clarify the molecular characteristics of the ENPP1+ cell subpopulation and its distribution in the synovial tissue.

[0158] The forward primer sequence targeting ENPP1 is 5'-GTCGTCAGTGGTCCTGTGTT -3' (SEQ ID NO: 1), and the reverse primer sequence targeting ENPP1 is 5'-TGCAAAGGCGTCTGAGATGT -3' (SEQ ID NO: 2).

[0159] Monocle2 and Velocyto algorithms were used to predict cell differentiation trajectories and analyze RNA rates for synovial cell subsets, respectively. Key cell subsets were predicted to which cell subsets they could be traced. Markers specifically expressed by these cell subsets were then screened for subsequent lineage tracing experiments. Differential expression analysis, combined with GO, KEGG, and GSEA methods, identified pathways that played a crucial role in the differentiation process of the target subpopulations from which these key cell subsets were derived.

[0160] 5. Verification of ENPP1+ cells and differential gene changes in human and mouse synovial tissue samples

[0161] GO, GSEA enrichment analysis, and intercellular communication analysis were used to investigate the functions and biological changes of individual cell subpopulations at different time points, including the interaction between ENPP1+ cells and chondrocytes, receptor-ligand pairs, and highly abundant secreted factors. Multiple immunofluorescence staining was used to co-stain specific proteins expressed in ENPP1+ cells and their upstream source cells to preliminarily verify the localization of ENPP1+ cells during joint degeneration. Flow cytometry was used to determine the proportions of various cell subpopulations in human and mouse synovial tissue, and multiple immunofluorescence staining was used to identify marker gene expression levels in ENPP1+ cells.

[0162] 6. Establishment of the OA Model with Medial Collateral Ligament Transecting (DMM)

[0163] We used a mouse model of DMM to investigate the effects of pathogenic cells on articular cartilage degeneration. Eight-week-old male C57BL / 6 mice were anesthetized with an intraperitoneal injection of 1.25% avertin (0.2 ml / 10 g). The right leg joint was then disinfected with 75% alcohol. A scalpel was used to incise the skin medially to expose the joint cavity. The medial collateral ligament was then severed, and the joint cavity and muscle and skin were sutured. In the sham-operated (Sham) group, only the joint cavity was exposed and sutured. Subsequent experiments were performed 8 weeks later.

[0164] 7. ENPP1+ cells were infused back into DMM mice to verify their ability to promote the pathological progression of OA

[0165] ENPP1+ cells were isolated from the synovial tissue of 18-month-old aged mice by flow cytometry, and a young (3-month-old) mouse model was established. Two weeks after modeling, ENPP1+ cells (5x10 3 Six weeks after modeling, the mechanical and thermal pain in the joints of the mice were detected, as well as the OARSI score, synovitis score, SO and HE staining. Immunofluorescence was used to detect changes in the expression of cartilage matrix degrading metalloproteinase (MMP13) protein in cartilage tissue to further clarify whether ENPP1+ cells can promote the pathological process of OA.

[0166] The present invention will be further described below in conjunction with 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. The experimental methods in the following examples, for which specific conditions are not specified, are generally performed under conventional conditions such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are calculated by weight. The experimental materials and reagents involved in the present invention can be obtained from commercial sources unless otherwise specified.

[0167] Example 1: Synovial lesions occur earlier than cartilage degeneration and can promote the development of OA

[0168] Based on the above experimental method, the results of this example show that low-grade inflammation of the synovium precedes cartilage degeneration in OA patients. The inventors found that the changes in joint structure of volunteers of different age groups who underwent traumatic amputation surgery were different from those of young patients with synovial tissue ( Figure 1 a) Compared with the elderly, cartilage degeneration (characterized by cartilage wear and matrix degradation) Figure 1 c) Before the onset of synovial tissue in middle-aged patients, the synovial tissue has already shown pathological features such as low-grade inflammation and hyperplasia ( Figure 1 b) This phenomenon was also verified in OA mouse models of different age groups ( Figure 2 a-2e).

[0169] These results suggest that pathological changes in the synovium already occur in middle age, potentially contributing to the development of OA. Therefore, identifying and intervening in potential targeted interventions targeting aging-induced synovial lesions is a crucial breakthrough in addressing the clinical challenge of OA.

[0170] Example 2: Synovial fibroblasts are the cell subpopulation with the most significant functional changes in OA

[0171] Based on the above experimental methods and the results of Example 1, this example further fully analyzes the identification of senescent cell atlas in mouse synovial tissue and its dynamic changes in different aging states. Synovial tissues of wild mice aged 3, 9, and 18 months were collected (n=5 per group) ( Figure 3 a) Perform single-cell sequencing analysis.

[0172] The results showed that based on cell markers, mouse synovial cells were divided into 9 different cell clusters, including two different synovial tissue fibroblasts (SSF and SIF), endothelial cells, pericytes, Schwann cells, monocytes-macrophages, neutrophils, lymphoid cells ( Figure 3b) Among them, SSF fibroblast clusters express Thy1; SIF fibroblast clusters express Prg4; endothelial cells express Cdh5 and VWF; pericytes express Mcam and Pdgfrb; Schwann cells express Mpz and Sox10; monocytes and macrophages express Csf1r and CD14; and neutrophils express S100a8, S100a9, and Cd177.

[0173] The dynamic proportions of cells in the synovial tissue of mice at different ages showed that the dynamic proportions of synovial fibroblast clusters (SSF and SIF) in the synovial tissue of mice changed most significantly ( Figure 3 cd). These results suggest that synovial fibroblasts are the cell subpopulation with the most significant functional changes in OA.

[0174] Example 3: ENPP1+ cells play an important role in the early progression of OA

[0175] Whether pathological synovial cell subpopulations contribute to the progression of OA during joint aging remains unexplored. Therefore, based on the aforementioned experimental methods, this example used single-cell sequencing technology to analyze synovial cells from mice of different ages to investigate whether pathological synovial cell subpopulations contribute to the progression of OA during joint aging.

[0176] The results showed that from the C0-C8 subgroups, the C3 subgroup was almost not expressed in young synovial tissue, but increased in the middle-aged stage and significantly increased in the elderly stage (( Figure 4 a). Further analysis revealed that the C3 subgroup had the highest perturbation score ( Figure 4 b). Bioinformatics analysis combined with multiple immunofluorescence results revealed that the C3 subpopulation highly expressed ENPP1 and PRG4 ( Figure 4 c), and the C3 subpopulation that specifically and highly expresses ENPP1 and PRG4 increases significantly with aging ( Figure 4 These results suggest that ENPP1+ cells may play an important role in the early progression of OA.

[0177] Example 4: ENPP1+ cells were infused into DMM mice to verify their ability to promote the pathological progression of OA

[0178] To investigate whether ENPP1+ cell subpopulations can promote the pathological process of OA, this example used flow cytometry to sort and identify ENPP1+ cells in the synovial tissue of aged mice ( Figure 5 a) Meanwhile, a DMM-induced OA mouse model was established, and ENPP1+ cells (5x10 3 cells, right limb) or PBS, modeling for 6 weeks ( Figure 5 b).

[0179] SO staining and OARSI scoring results showed that ENPP1+ cells promoted the degradation and wear of cartilage matrix in OA ( Figure 5 cd). In addition, the results of mechanical and thermal pain experiments in mice showed that ENPP1+ cells induced hyperalgesia in mice ( Figure 5 These results provide an important foundation for revealing that ENPP1+ cells are the key driving factor in the development of OA.

[0180] Example 5: ENPP1 inhibitors can treat arthritis (OA)

[0181] This example uses conventional methods to test the protective effect of ENPP1 inhibitors on chondrocytes in arthritis. The results show that ENPP1 inhibitors can protect chondrocytes from senescence and apoptosis in arthritis and inhibit the expression of ENPP1 and other inflammatory factors in cells.

[0182] Example 6: Elimination of ENPP1-positive cells can treat arthritis (OA)

[0183] This example constructs an animal model for specific ablation of ENPP1-positive cells. The specific construction method mainly includes the following steps:

[0184] The animal model of specific ablation of ENPP1-positive cells is obtained by selectively eliminating ENPP1-positive cells (such as ENPP1-positive synovial fibroblasts) through a bioablation method; wherein the bioablation method includes a gene knockout-mediated ablation method or an immune cell-mediated ablation method.

[0185] 1. Knockout-mediated ablation methods include: constructing ENPP1 gene knockout animal models and / or animal models that eliminate ENPP1-positive cells based on CRISPR / Cas9 technology; including the following steps:

[0186] 1) Design of gRNA targeting mouse ENPP1 sequence:

[0187] 2) Purifying the pair of gRNAs from step 1) and co-injecting them with cas9 mRNA into mouse embryos to obtain F0 generation mice;

[0188] 3) Identify the ENPP1 genotype and select positive F0 generation mice;

[0189] 4) Positive F0 generation mice are crossed with wild-type mice to obtain F1 generation mice, and the F1 generation heterozygous mice are self-pollinated to obtain homozygous offspring, thereby constructing an ENPP1 gene knockout animal model and / or an animal model for eliminating ENPP1-positive cells.

[0190] This method induces the Caspase 9 system, uses genetic engineering methods to make ENPP1-positive cells express Caspase 9 protein, and injects a chemical inducer (CID) to trigger apoptosis. Based on CRISPR / Cas9 technology, it achieves site-specific knockout of the ENPP1 gene, which has the advantages of simple operation and high efficiency of ENPP1 gene knockout.

[0191] 2. Immune cell-mediated ablation methods include: CAR-T cell therapy, CAR-NK cell therapy or CAR-M cell therapy. Among them,

[0192] 2.1 CAR-T cell therapy involves using viral vectors (such as lentivirus) to introduce chimeric antigen receptor (CAR) genes into T cells, causing them to express receptors that can recognize target cell antigens (such as Enpp1);

[0193] 2.2 CAR-NK cell therapy or CAR-M cell therapy involves the use of natural killer (NK) cells and macrophages (M) to modify and target the clearance of pathogenic ENPP1-positive cells.

[0194] Based on the above-constructed animal model, relevant tests were performed.

[0195] The results showed that animals that eliminated ENPP1-positive cells were less sensitive to pain, delayed the degradation and wear of cartilage matrix, and inhibited the pathological process of arthritis (OA), indicating that eliminating ENPP1-positive cells can treat arthritis.

[0196] The above describes the preferred embodiments of the present invention. It should be understood that the present invention is not limited to the above-mentioned specific embodiments, and the devices and structures that are not described in detail should be understood to be implemented in a common manner in the art; any technician familiar with the art can use the above-mentioned disclosed methods and technical contents to make many possible changes and modifications to the technical solutions of the present invention without departing from the scope of the technical solutions of the present invention, or modify them into equivalent embodiments of equivalent changes, which does not affect the essential content of the present invention. Therefore, any simple modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention that do not depart from the content of the technical solutions of the present invention are still within the scope of protection of the technical solutions of the present invention.

Claims

1. Use of ENPP1 as a biomarker or target in the preparation of products for diagnosis, monitoring, severity assessment, efficacy assessment, prognosis assessment, prevention and / or treatment of arthritis-related diseases, characterized in that: The diagnosis, monitoring, severity assessment, efficacy assessment or prognostic assessment of arthritis-related diseases includes detecting the presence of ENPP1 or the expression level of its mRNA and / or protein, or the presence of ENPP1-positive cells; The prevention and / or treatment of arthritis-related diseases includes: knocking out or knocking down ENPP1, or eliminating ENPP1-positive cells.

2. The use according to claim 1, characterized in that The knockout or knockdown of ENPP1 includes using the CRISPR / Cas system, tissue-specific knockout, and introduction of interfering RNA or microRNA that targets the knockout or knockdown of ENPP1; The elimination of ENPP1-positive cells includes selectively eliminating ENPP1-positive cells by a biological ablation method, wherein the biological ablation method includes a gene knockout-mediated ablation method or an immune cell-mediated ablation method.

3. Use of an inhibitor or antagonist of the ENPP1 gene or its protein for preparing a composition or preparation for (a) preventing and / or treating arthritis-related diseases; and / or (b) inhibiting synovial inflammation.

4. The use according to claim 3, characterized in that The composition or preparation is also used for (i) inhibiting the occurrence of cartilage degeneration; and / or (ii) inhibiting synovial lesions caused by aging.

5. A pharmaceutical composition, characterized in that include: (a1) a first active ingredient for preventing and / or treating arthritis-related diseases, the first active ingredient comprising: an inhibitor of the ENPP1 gene or its protein; (a2) a second active ingredient for preventing and / or treating arthritis-related diseases, wherein the second active ingredient includes: other drugs for preventing and / or treating arthritis-related diseases; and (b) a pharmaceutically acceptable carrier.

6. The pharmaceutical composition according to claim 5, characterized in that The arthritis-related disease is selected from the group consisting of osteoarthritis, rheumatoid arthritis, rheumatoid arthritis, infectious arthritis, gouty arthritis, or a combination thereof.

7. A medicine box, characterized in that: include: (i) a first container, and an active ingredient (a1) an inhibitor of ENPP1 gene or protein thereof, or a drug containing the active ingredient (a1) in the first container; and (ii) a second container, and the active ingredient (a2) in the second container; other drugs for preventing and / or treating arthritis-related diseases, or drugs containing the active ingredient (a2).

8. A method for screening potential therapeutic agents for arthritis-related diseases, characterized in that: include: (a) in a test group, in a culture system, in the presence of a test compound, culturing cells expressing the ENPP1 gene for a period of time, and detecting the expression level E1 of the ENPP1 gene in the culture system of the test group; and detecting the expression level E2 of the ENPP1 gene in the culture system of the control group in the absence of the test compound and under the same other conditions; and (b) comparing E1 and E2; if E1 is significantly lower than E2, it indicates that the test compound is a potential therapeutic agent for arthritis; The methods described are non-diagnostic and non-therapeutic.

9. A method for constructing an animal model for specific ablation of ENPP1-positive cells, characterized in that: The steps include: Selectively eliminating ENPP1-positive cells by a bioablation method, thereby obtaining the animal model of specific ablation of ENPP1-positive cells; The bioablation method includes a gene knockout-mediated ablation method or an immune cell-mediated ablation method.

10. Use of the animal model obtained by the construction method according to claim 9 in screening drugs for preventing and / or treating arthritis-related diseases.

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