Anti-inflammatory chimera, reprogramming macrophage, preparation method and application

Abstract

The invention provides an anti-inflammatory chimera, a reprogramming macrophage and a preparation method and application thereof, and belongs to the technical field of cell therapy. The anti-inflammatory chimera provided by the invention is a chimera based on conversion from an inflammatory signal to an anti-inflammatory signal, and can convert an inflammatory signal of a diseased region of a fibrosis disease into an IL-10 signal, so that the problem of an inflammatory environment of a fibrosis tissue is solved, and steady-state balance of the tissue is promoted. Moreover, the chimera is introduced into the recovery type macrophage, the recovery type phenotype stability of the recovery type macrophage is enhanced, and the purposes of improving the inflammatory environment of the diseased region of the fibrotic disease, reducing fibrotic cell activation, continuously degrading an extracellular matrix, swallowing dead cells and remodeling diseased tissues are achieved. In addition, the invention provides a preparation method of the reprogramming macrophage and a specific application of the reprogramming macrophage in fibrotic tissues.

Description

Technical Field

[0001] The present invention belongs to the technical field of cell therapy, and particularly relates to an anti-inflammatory chimera, reprogrammed macrophages, and a preparation method and application thereof. Background Art

[0002] Fibrosis can affect any organ. For a long time, it has been considered irreversible. However, preclinical models and clinical trials in various organ systems have shown that fibrosis is a highly dynamic process. Although significant progress has been made in our understanding of fibrosis pathology, there is still a translational gap between identifying anti-fibrotic targets and translating this knowledge into effective treatments for humans. So far, there is still no effective drug for treating fibrotic diseases.

[0003] The recovery of fibrotic diseases cannot be achieved by blocking a single target. Currently, most of the disclosed treatment technologies use chemical drugs for alleviation and improvement. For example, the fibrotic ameliorant composed of a zinc selenium complexing agent and an additive disclosed in CN107106602B, and the fibrotic inhibitor composed of a hydroquinone derivative disclosed in CN101559049B. However, these disclosed drugs cannot cure fibrosis to a certain extent, and there is a possibility of dependence and recurrence. CN118976114A discloses a lysosome-targeted chimera for PD-L1 / CD-47 dual-targeted protein degradation, which blocks and reverses fibrosis by inhibiting the activation and proliferation of HSCs and then reducing ECM synthesis. However, this technology only starts from the perspective of reducing ECM synthesis and cannot effectively solve the root cause of fibrosis.

[0004] Therefore, how to start from multiple targets, block the progression of fibrosis, reverse the fibrotic lesions of tissues, and promote the remodeling of normal tissues is a technical problem that needs to be solved urgently by those skilled in the art at present. Summary of the Invention

[0005] To solve the problems existing in the above-mentioned prior art, the present invention provides an anti-inflammatory chimera, reprogrammed macrophages, and a preparation method and application thereof. When tissue fibrosis progresses, there are a large number of inflammatory factors in the diseased tissue environment, which activate the pro-fibrotic cells in the diseased tissue, further increase the secretion of inflammatory factors, and at the same time secrete collagen to harden the tissue, change the physiological function of the tissue, and cause many complications of fibrotic diseases. Based on the characteristics of this disease environment, the present invention proposes a technical concept of converting the inflammatory factors in the diseased tissue into anti-inflammatory factors, so as to reduce and eliminate the inflammatory response, restore the steady-state environment of the tissue, and achieve the inducement of blocking the progression of fibrotic diseases. The specific content of the invention is as follows:

[0006] In a first aspect, the present invention provides an anti-inflammatory chimera. The anti-inflammatory chimera is a chimera based on the conversion of inflammatory signals into anti-inflammatory signals. The extracellular structure of the anti-inflammatory chimera is an inflammatory factor receptor, the intracellular structure is the intracellular signal of the anti-inflammatory factor receptor, and the transmembrane domain connecting the extracellular structure and the intracellular structure.

[0007] In some embodiments, the transmembrane domain is the transmembrane domain of a transmembrane protein, preferably the transmembrane region of an anti-inflammatory factor receptor or the transmembrane region of platelet-derived growth factor receptor β. Among them, when the transmembrane domain is the transmembrane region of the anti-inflammatory factor receptor, the intracellular signal is the intracellular region of the corresponding anti-inflammatory factor receptor. In addition, the CD28 transmembrane region can also be used in the present application.

[0008] In some embodiments, the inflammatory factor is one or more of TGF-β, IL-17, IL-4, IL-13, IL-11, IL6, IL33, IL-1, IL-12, IL-18, IFN-γ, and TNF-α. Among them, the TFG-b receptors TGF-βRA and / or TGF-βRB, NCBI gene IDs are 7406 and 7408 respectively. The IL-17 receptors IL-17RA and / or IL-17RB, NCBI gene IDs are 23765 and 55540 respectively. The IL-17 receptors IL-17RC and / or IL-17RD, NCBI gene IDs are 84818 and 54765 respectively. The IL-4 receptor IL-4R, NCBI gene ID is 3566. The IL-13 receptors IL-13RA1 and / or IL-13RA2, NCBI gene IDs are 3597 and 3598 respectively. The IL-6 receptor IL-6R, NCBI gene ID is 3570. The IL-11 receptor IL-11R, NCBI gene ID is 3590. The IL-33 receptor IL-33R, NCBI gene ID is 9173. The IL-1 receptor is IL-1RⅠ and / or IL-1RⅡ, NCBI gene IDs are 396481 and 7850 respectively. The IL-12 receptor is IL-12R, NCBI gene ID is 574199. The IL-18 receptor is IL-18R, NCBI gene ID is 16182. The IFN-γ receptor is IFN-γRα and / or IFN-γRβ, NCBI gene IDs are 3459 and 3460 respectively. The TNF-α receptor is TNFR1 and / or TNFR2, NCBI gene IDs are 21937 and 21938 respectively.

[0009] In some embodiments, the anti-inflammatory factor is IL-10. Among them, the IL-10 receptors are IL-10RA and / or IL-10RB, and the NCBI gene IDs are 3587 and 3588 respectively.

[0010] In some embodiments, the information carrier of the chimeric body is RNA or DNA.

[0011] In a second aspect, the present invention provides an anti-inflammatory cell. The anti-inflammatory cell contains the anti-inflammatory chimeric body described in the first aspect above.

[0012] In some embodiments, the cell is one of monocytes, T cells, B cells, NK cells, and macrophages.

[0013] In a third aspect, the present invention provides a method for preparing the anti-inflammatory cell described in the second aspect above. The anti-inflammatory chimeric body mRNA or anti-inflammatory chimeric body DNA is introduced into target cells.

[0014] In some embodiments, the method uses electroporation, lipid nanoparticles, or virus introduction.

[0015] In a fourth aspect, the present invention provides a cell vaccine. The cell vaccine contains the anti-inflammatory cell described in the second aspect above.

[0016] In a fifth aspect, the present invention provides an application of the anti-inflammatory chimeric body described in the first aspect above, or the anti-inflammatory cell described in the second aspect above, or the cell vaccine described in the fourth aspect above, in preventing or reducing tissue inflammation or treating fibrotic diseases.

[0017] In a sixth aspect, the present invention provides a reprogrammed macrophage. The information carrier of the anti-inflammatory chimeric body described in the first aspect above is introduced into the reprogrammed macrophage.

[0018] In some embodiments, the information carrier of the chimeric body is RNA or DNA.

[0019] In some embodiments, the reprogrammed macrophage also contains a introduced pro-regeneration gene.

[0020] In some embodiments, the pro-regeneration gene is a gene for promoting tissue regeneration.

[0021] In some embodiments, the factor corresponding to the pro-regeneration gene is HGF.

[0022] In some embodiments, a collagen targeting gene targeting the fibrotic lesion site is also introduced into the reprogrammed macrophage.

[0023] In some embodiments, the collagen targeting gene encodes a sequence of a collagen binding domain protein.

[0024] In some embodiments, the collagen-binding domain protein is derived from the von Willebrand factor-A3 domain.

[0025] In some embodiments, one or more of an anti-inflammatory gene, a phagocytosis-promoting gene, and an extracellular matrix degradation-promoting gene are further introduced into the reprogrammed macrophages. Among them, the information carrier of the gene is preferably DNA or RNA.

[0026] In a seventh aspect, the present invention provides a method for preparing the reprogrammed macrophages described in the sixth aspect above. The method includes: preparing the mRNA described in the sixth aspect above according to a sequence template and introducing it into the reparative macrophages.

[0027] In some embodiments, the method uses electroporation, lipid nanoparticles, or virus introduction.

[0028] In an eighth aspect, the present invention provides a macrophage vaccine. The macrophage vaccine contains the reprogrammed macrophages described in the sixth aspect above.

[0029] In a ninth aspect, the present invention provides a cell population. The cell population contains any of the reprogrammed macrophages described in the sixth aspect above.

[0030] In a tenth aspect, the present invention provides an application of the reprogrammed macrophages described in the sixth aspect above, the macrophage vaccine described in the eighth aspect above, or the cell population described in the ninth aspect above in the prevention or treatment of fibrotic diseases.

[0031] In some embodiments, the fibrosis is one of liver fibrosis, kidney fibrosis, cardiac fibrosis, and pulmonary fibrosis.

[0032] The anti-inflammatory chimera provided by the present invention is a chimera that converts inflammatory signals to anti-inflammatory signals, which can convert the inflammatory signals in fibrotic lesion tissues into IL-10 signals, solve the inflammatory environment of fibrotic tissues, promote tissue homeostasis balance, inactivate pro-fibrotic cells, and slow down or block the progression of fibrosis. Moreover, the present invention introduces the chimera into macrophages to enhance the stability of the reparative phenotype of macrophages, achieve the improvement of the inflammatory environment at the lesion site of fibrotic diseases, promote the continuous degradation of the extracellular matrix by macrophages, phagocytose dead cells, and reshape the lesion tissues, and further reduce environmental inflammation.

[0033] In addition, the present invention provides a specific application of targeting fibrotic lesion tissues and pro-regenerative reprogrammed reparative macrophages in the treatment of fibrotic diseases. This cell has the following advantages:

[0034] 1. Under the GMP conditions established in the present invention, a large number of monocyte-derived macrophages can be stably produced to meet the needs of patients;

[0035] 2. The macrophages produced based on monocytes in the present invention have a robust restorative phenotype. Flow cytometry phenotypic analysis shows that the monocyte-derived macrophages disclosed in this patent have a robust phenotype, highly expressing 25F9 and CD206, with an average value > 90%; they do not secrete the M1 macrophage cytokine IL-6; they have a strong phagocytic ability towards E. coli biological particles, phosphatidylserine microspheres, and apoptotic cells; gene expression profile analysis shows the expression of matrix metalloproteinases such as MMP7, MMP9, and MMP19, and they can degrade collagen in vitro, indicating the ability to degrade the extracellular matrix. In an in vitro fibrosis model, they can effectively reduce the production of collagen and α-SMA.

[0036] 3. Based on the inflammatory signal to anti-inflammatory signal chimera, it can convert the inflammatory signal in fibrotic tissue into the downstream signal of IL-10, promote the stability of the restorative phenotype of macrophages, solve the inflammatory environment in fibrotic tissue, and promote the homeostasis balance of liver tissue.

[0037] 4. HGF can help the survival and proliferation of epithelial cells, endothelial cells, and hepatocytes, and promote the regeneration of the trachea, blood vessels, and liver tissue in fibrotic tissue. HGF can antagonize TGF-β, reduce the activation of HSCs, and reduce the accumulation of myofibroblasts.

[0038] 5. The collagen-binding domain protein can target and enrich macrophages in fibrotic lesion tissues, and better play the role of anti-inflammatory and pro-regenerative restorative macrophages.

[0039] 6. Experimental data in a mouse bleomycin-induced pulmonary fibrosis model and a CCL4-induced liver fibrosis model confirm that the anti-inflammatory chimera, collagen-binding domain, and pro-regenerative gene reprogrammed restorative macrophages provided by the present invention have good ability to improve fibrosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 Shows the characterization results of the restorative macrophages prepared in Example 1 of the present invention;

[0041] Figure 2 Shows the test results of the in vitro phagocytosis of the restorative macrophages prepared in the examples of the present invention;

[0042] Figure 3 Shows the test results of the extracellular matrix (EMC) degradation ability of the restorative macrophages prepared in the examples of the present invention;

[0043] Figure 4 Shows the test results of the detection of the in vitro anti-fibrosis ability of the restorative macrophages prepared in the examples of the present invention;

[0044] Figure 5Shows the detection results of genes downstream of IL-10 in the anti-inflammatory chimeric restored macrophages prepared in the embodiments of the present invention;

[0045] Figure 6 Shows the detection results of the anti-pulmonary fibrosis ability in vitro of the anti-inflammatory chimeric restored macrophages prepared in the embodiments of the present invention;

[0046] Figure 7 Shows the results of the detection of the anti-fibrosis ability in vitro of the anti-inflammatory chimeric restored macrophages prepared in the embodiments of the present invention;

[0047] Figure 8 Shows the detection results of the functions of the pro-regenerative restored macrophages prepared in the embodiments of the present invention;

[0048] Figure 9 Shows the detection results of the functions of the fibrosis tissue-targeting restored macrophages prepared in the embodiments of the present invention;

[0049] Figure 10 Shows the test results of the treatment of bleomycin-induced pulmonary fibrosis with the reprogrammed restored macrophages prepared in this embodiment;

[0050] Figure 11 Shows the test results of the treatment of bleomycin-induced liver fibrosis with the reprogrammed restored macrophages prepared in this embodiment. Detailed implementation manners

[0051] To make the above objects, features, and advantages of the present invention more obvious and understandable, the present invention will be further described in detail below with reference to the accompanying drawings and specific implementation manners. The following describes the embodiments of the present invention in detail. The embodiments are implemented on the premise of the technical solution of the present invention, and detailed implementation manners and specific operation processes are given, but the protection scope of the present invention is not limited to the following embodiments.

[0052] Various aspects of the present invention are described in detail in the following embodiments. The use of the embodiments is not intended to limit the present invention. Each embodiment can be applied to any aspect of the present invention. In this application, unless otherwise stated, the use of "or" means "and / or".

[0053] TGF-β is a key cytokine in fibrosis and the most intensively studied profibrotic factor to date. TGF-β is produced by infiltrating lymphocytes and monocytes, tissue-resident macrophages, and damaged hepatocytes. TGF-β effectively activates HSCs and induces the production of collagen, αSMA, and connective tissue growth factor in HSCs. IL-17, derived from TH17 cells and neutrophils, plays an important role in activating the TGF-β signaling pathway in HSCs, as it induces the upregulation of the TGF-β type II receptor and enhances the activation of downstream kinases. In addition, TGF-β can be deposited in the ECM and activate HSCs mediated by αV integrin. Interleukin-10 (IL-10) is an important anti-inflammatory cytokine that plays a central role in regulating and terminating inflammatory responses. IL-10 inhibits the synthesis of collagen I, suppresses Kupffer cell activation, and restricts the production of IL-17 by Th17, resulting in reduced activation of HSCs.

[0054] Hepatocyte growth factor (HGF) is a pleiotropic factor that induces cell motility, survival, proliferation, and morphogenesis. HGF has been shown to play a key role in tissue repair. In animal models of pulmonary fibrosis, the administration of HGF protein or the ectopic expression of HGF has been shown to induce normal tissue repair and prevent fibrosis. The inhibition of HGF-induced fibrotic remodeling may occur through multiple direct and indirect mechanisms, including inducing cell survival and proliferation of lung epithelial cells and endothelial cells, and reducing myofibroblast accumulation.

[0055] The present invention utilizes monocytes induced into restorative macrophages, which have high efficiency in degrading the extracellular matrix and strong phagocytic ability. On this basis, we further reprogram cells with an inflammatory signal converted into an anti-inflammatory signal chimera and a pro-regenerative mRNA. Among them, the conversion of the inflammatory signal into an anti-inflammatory signal chimera enables the restorative macrophages to release MMPs, efficiently degrade the fibrous tissue in the liver, phagocytize cell debris and apoptotic cells, and convert the surrounding inflammatory signals into IL-10 anti-inflammatory signals to inhibit the inflammatory signals; and then based on HGF mRNA, promote blood vessel and liver regeneration.

[0056] The mechanism proposed by the present invention is to capture inflammatory factors in the fibrotic environment and convert them into anti-inflammatory signals, promote the maintenance of the homeostasis of restorative macrophages, solve the fibrotic inflammatory environment, and promote tissue regeneration.

[0057] The specific content of the embodiments of the present invention is as follows:

[0058] In a first aspect, an embodiment of the present invention provides an anti-inflammatory chimera. The anti-inflammatory chimera is based on the conversion of an inflammatory signal into an anti-inflammatory signal chimera, and includes: an inflammatory factor receptor as the extracellular structure, an intracellular signal of an anti-inflammatory factor receptor as the intracellular structure, and a transmembrane domain connecting the extracellular structure and the intracellular structure.

[0059] In some embodiments, the transmembrane domain is the transmembrane domain of a transmembrane protein, preferably the transmembrane region of an anti-inflammatory factor receptor or the transmembrane region of platelet-derived growth factor receptor β. Among them, when the transmembrane domain is the transmembrane region of an anti-inflammatory factor receptor, the intracellular signal is the intracellular region of the anti-inflammatory factor receptor. In addition, the CD28 transmembrane region can also be used in the present application.

[0060] In some embodiments, the inflammatory factors are one or more of TGF-β, IL-17, IL-4, IL-13, IL-11, IL6, IL33, IL-1, IL-2, IL-12, IL-18, IFN-γ, and TNF-α. Among them, the TGF-β receptors TGFBR1 and / or TGFBR2 have NCBI gene IDs of 7406 and 7408 respectively. The IL-17 receptors IL-17RA and / or IL-17RB have NCBI gene IDs of 23765 and 55540 respectively. The IL-17 receptors IL-17RC and / or IL-17RD have NCBI gene IDs of 84818 and 54765 respectively. The IL-4 receptor IL-4R has an NCBI gene ID of 3566. The IL-13 receptors IL-13RA1 and / or IL-13RA2 have NCBI gene IDs of 3597 and 3598 respectively. The IL-6 receptor IL-6R has an NCBI gene ID of 3570. The IL-11 receptor IL-11R has an NCBI gene ID of 3590. The IL-33 receptor IL-33R has an NCBI gene ID of 9173. The IL-1 receptors are IL-1RⅠ and / or IL-1RⅡ, and the NCBI gene IDs are 396481 and 7850 respectively. The IL-2 receptor is IL-2R, and the NCBI gene ID is 3559. The IL-12 receptor is IL-12R, and the NCBI gene ID is 574199. The IL-18 receptor is IL-18R, and the NCBI gene ID is 16182. The IFN-γ receptors are IFN-γRα and / or IFN-γRβ, and the NCBI gene IDs are 3459 and 3460 respectively. The TNF-α receptors are TNFR1 and / or TNFR2, and the NCBI gene IDs are 21937 and 21938 respectively.

[0061] In some embodiments, the anti-inflammatory factor is IL-10. Among them, the IL-10 receptors are IL-10RA and / or IL-10RB, and the NCBI gene IDs are 3587 and 3588 respectively.

[0062] In some embodiments, the information carrier of the chimeric body is RNA or DNA.

[0063] The anti-inflammatory chimeras provided by the present invention are introduced into macrophages and applied in the inflammatory environment of fibrotic diseases. After introduction, the reprogrammed macrophages express the chimeric protein, and the inflammatory factor receptors on the cell surface bind to the inflammatory factors in fibrotic diseases and convert this signal into an IL-10 signal, achieving the closure of inflammatory factors and blocking the inducement of fibrosis. The downstream feedback IL-10 signal, as an anti-inflammatory factor, further amplifies the anti-inflammatory effect, quiesces all inflammatory cells, reduces the release of inflammatory factors, and blocks the progression of fibrosis. At the same time, the IL-10 signal released by the chimeric protein can also promote the stability of the phenotype of restorative macrophages, maintain the ability of these cells to continuously clear necrotic cells and degrade the extracellular matrix, promote the restoration of the tissue homeostasis environment, and promote tissue repair.

[0064] In a second aspect, an embodiment of the present invention provides an anti-inflammatory cell. The anti-inflammatory cell contains the anti-inflammatory chimera described in the first aspect above.

[0065] In some embodiments, the cell is one of a monocyte, a T cell, a B cell, an NK cell, and a macrophage.

[0066] Since the anti-inflammatory chimera provided by the present invention is based on the intracellular IL-10 receptor and completes the conversion of extracellular inflammatory signals into IL-10 anti-inflammatory signals. Thus, in specific implementation, when the anti-inflammatory chimera is introduced into cells such as the above-mentioned monocytes, T cells, B cells, NK cells, and macrophages, the inflammatory signals can be converted into IL-10 in the inflammatory environment to achieve the purpose of anti-inflammation.

[0067] In a third aspect, an embodiment of the present invention provides a method for preparing the anti-inflammatory cell described in the second aspect above. The anti-inflammatory chimera mRNA or DNA is introduced into target cells.

[0068] In some embodiments, the method is introduced by any one of electroporation, lipid nanoparticles, or viruses.

[0069] In a fourth aspect, an embodiment of the present invention provides a cell vaccine. The cell vaccine contains the anti-inflammatory cell described in the second aspect above.

[0070] In a fifth aspect, an embodiment of the present invention provides an application of the anti-inflammatory chimera described in the first aspect above, the anti-inflammatory cell described in the second aspect above, or the cell vaccine described in the fourth aspect above in preventing or reducing tissue inflammation or treating fibrotic diseases.

[0071] In a sixth aspect, an embodiment of the present invention provides a reprogrammed macrophage. An information carrier of the anti-inflammatory chimera described in the first aspect above is introduced into the reprogrammed macrophage.

[0072] The technical concept of the embodiments of the present invention is as follows: Since restorative macrophages have the functions of anti-inflammation, phagocytosing necrotic cells, and degrading extracellular matrix, they have the ability to improve fibrosis. An anti-inflammatory chimera is introduced into pro-restorative macrophages and applied in the inflammatory environment of fibrotic diseases. The restorative macrophages express the chimeric protein, and the inflammatory factor receptors on the cell surface bind to the inflammatory factors in fibrotic diseases and convert this signal into an IL-10 signal, achieving the closure of inflammatory factors, blocking the inducement of fibrosis, and the downstream feedback IL-10 signal, as an anti-inflammatory factor, further amplifies the anti-inflammatory effect, quiesces all inflammatory cells, reduces the release of inflammatory factors, and blocks the progression of fibrosis; at the same time, the IL-10 signal released by the chimeric protein can also promote the phenotypic stability of pro-restorative macrophages, maintain the ability of these cells to continuously clear necrotic cells and degrade extracellular matrix, promote the restoration of tissue homeostasis environment, and promote tissue repair.

[0073] In the embodiments of the present invention, through condition optimization, a large number of monocyte-derived restorative macrophages can be stably produced. At the same time, through the collagen-binding domain, the restorative macrophages are promoted to target and enrich in fibrotic tissues, better exerting the anti-inflammatory and pro-regenerative effects. The enriched restorative macrophages further convert the inflammatory signals in fibrotic tissues into anti-inflammatory signals, enhancing the stability of the restorative phenotype of the restorative macrophages, improving the inflammatory environment of fibrotic tissues, continuously degrading extracellular matrix, and phagocytosing dead cells, and promoting the restoration and regeneration of liver tissue homeostasis.

[0074] In some embodiments, the information carrier of the chimera is RNA or DNA.

[0075] In some embodiments, the reprogrammed macrophages also contain introduced pro-regenerative genes.

[0076] In some embodiments, the pro-regenerative genes are genes for promoting angiogenesis and liver regeneration.

[0077] In some embodiments, the pro-regenerative gene encodes the sequence of HGF.

[0078] The pro-regenerative genes provided by the embodiments of the present invention, while eliminating the inflammatory response, enable macrophages to degrade extracellular matrix, phagocytose and clear necrotic cells, and restore the tissue homeostasis environment. When macrophages provide the conditions of space and environment for normal tissue remodeling, through regenerative factors, they promote angiogenesis and the regeneration of normal tissues, complete the remodeling of normal tissues, and reverse fibrotic lesion tissues.

[0079] In some embodiments, a collagen-targeting gene targeting fibrosis is also introduced into the reprogrammed macrophages.

[0080] In some embodiments, the collagen-targeting gene encodes a collagen-binding domain protein (CBD).

[0081] The function of the target gene (such as CBD encoded by the target mRNA) provided by the embodiments of the present invention is to promote the targeted enrichment of restorative macrophages at the fibrotic tissue lesion site.

[0082] In some embodiments, the collagen-binding domain protein is derived from the von Willebrand factor-A3 domain.

[0083] In some embodiments, one or more of anti-inflammatory genes, phagocytosis genes, and extracellular matrix degradation genes are also introduced into the reprogrammed macrophages. The information carriers of these genes are DNA or RNA.

[0084] In a seventh aspect, the embodiments of the present invention provide a method for preparing the reprogrammed macrophages described in the sixth aspect above. The method includes: preparing the mRNA described in the sixth aspect above according to a sequence template and introducing it into restorative macrophages.

[0085] In some embodiments, the method uses electroporation, lipid nanoparticles, or viral introduction.

[0086] In an eighth aspect, the embodiments of the present invention provide a macrophage vaccine. The macrophage vaccine contains the reprogrammed macrophages described in the sixth aspect above.

[0087] In a ninth aspect, the embodiments of the present invention provide a cell population. The cell population contains any of the reprogrammed macrophages described in the sixth aspect above.

[0088] In a tenth aspect, the embodiments of the present invention provide the use of the reprogrammed macrophages described in the sixth aspect above, the macrophage vaccine described in the eighth aspect above, or the cell population described in the ninth aspect above in the prevention or treatment of fibrotic diseases.

[0089] In some embodiments, the fibrotic disease is one of liver fibrosis, kidney fibrosis, heart fibrosis, and lung fibrosis.

[0090] In order to enable those skilled in the art to better understand the anti-inflammatory chimeras and reprogrammed macrophages provided by the embodiments of the present invention, the following will be elaborated in detail through specific examples. Among them, the reagents, instruments, and other supplies used, if not otherwise specified, are all existing commercial products and can be directly purchased.

[0091] Example 1

[0092] Step 1.1 Preparation of restorative macrophages

[0093] Standard procedures for CMNC using apheresis systems involve aseptically collecting peripheral blood mononuclear cells, i.e., leukocyte concentrates, processing 2.5 times the blood volume, and collecting for 4 hours (4 - 5 hours are acceptable in actual operations). The isolation of monocytes is carried out in a closed system meeting GMP conditions, and is separated by a centrifugal countercurrent elutriation system or CD14 magnetic beads. Briefly, cell counting samples are taken from the leukocyte concentrate to determine the total white blood cell count, red blood cell volume, granulocyte and monocyte ratios, monocyte percentage, and absolute monocyte cell count, and the parameters are adjusted to meet the required selection criteria (≥1×10 9 monocytes, monocytes:granulocytes ≥ 3, volume ≤ 400 mL). At the end of the separation, the monocytes are washed in an EDTA / PBS buffer containing pharmaceutical-grade human albumin (HSA), counted, and then resuspended in TexMACS medium containing 25 ng / mL M-CSF cytokine at 0.5×10 6 monocytes / mL (parameters in actual operations are 10 - 500 ng / mL M-CSF cytokine and 0.5 - 5×10 6 monocytes / mL are both acceptable), perfused into a culture bag, and induced to macrophages for 7 days under conditions of 37°C, 5% CO2 concentration, and 75% humidity, with half-volume medium replacement every 2 days.

[0094] Flow cytometry combined with antibodies is used to analyze the surface markers of monocytes and macrophages. The purified monocytes or macrophages are incubated with FC blocking antibody for 5 minutes, then incubated with a 1:100 diluted antibody mixture at 4°C for 20 minutes, washed with flow cytometry wash buffer, and finally added with dead cell dye DRAQ7 at a 1:100 ratio. The cell surface marker antibodies are: CD45, CD14, CD206, and 25F9. Using forward and side scatter, DRAQ7 dead cell exclusion, gating is performed on monocytes and macrophages, and analysis is carried out using FlowJo software. Determine the percentages of 25F9 and CD206 in macrophages as the cell product release standard.

[0095] Step 1.2 Detection of phagocytic function of recovered macrophages

[0096] Detection of the phagocytic ability of monocytes and macrophages. Add 1 - 2 μL of pHrodo TM Red E.coli bioparticles to monocytes or macrophages and culture for 1 hour, then wash the cells, and quantitatively assess phagocytosis by flow cytometry of macrophage fluorescence microsphere uptake.

[0097] Detection of the ability of macrophages to phagocytose phosphatidylserine. Add FITC-labeled phosphatidylserine to macrophages and culture for 1 hour. Then wash the cells and use flow cytometry to quantitatively detect the uptake of fluorescent phosphatidylserine by macrophages to evaluate phagocytosis.

[0098] Detection of the ability of macrophages to phagocytose apoptotic cells. To detect the ability of macrophages to phagocytose apoptotic cells, zshikonin was used to induce apoptosis of K562 cells. After 20 hours, the cells were collected and counted. Then, according to the ratio of target cells (apoptotic K562 cells) to effector cells (macrophages) of 2:1, they were incubated together for 4 hours. Then, flow cytometry was used to analyze the percentage of macrophages that phagocytosed apoptotic cells.

[0099] Detection of non-M1 polarization of restored macrophages. Detection of IL-6, 1×10 6 Macrophages were cultured overnight, and the supernatant was collected. An ELISA kit was used to quantitatively detect the secretion of IL-6.

[0100] Analysis of the ability of restored macrophages to degrade the extracellular matrix (ECM). Transcriptome sequencing was used to analyze the expression of MMP genes in different batches of macrophage products, and a collagenase activity detection kit was used to analyze the collagen degradation ability of macrophages. Detection of MMP activity, 1×10 6 Macrophages were cultured overnight, and the supernatant was collected. Cell debris was removed by centrifugation. According to the instructions of the collagenase activity detection kit, the cell culture supernatant was reacted with the substrate to detect the ability of matrix metalloproteinases to degrade collagen.

[0101] Characterization results of the preparation of restored macrophages:

[0102] Figure 1 The characterization results of the restored macrophages prepared in Example 1 of the present invention are shown. Among them, Figure 1 A is the purity ratio of monocytes before separation, Figure 1 B is the purity ratio of monocytes after separation, Figure 1 C is the detection result of the viability of macrophages, Figure 1 D is the detection result of the induction rate of macrophages, Figure 1 E is the detection result of the number of cells produced by macrophages, Figure 1 F is the detection result of the marker 25F9, Figure 1 G is the detection result of the marker CD206, Figure 1 H is the detection result of the marker IL-6.

[0103] From Figure 1 the detection results, it can be seen that we can isolate monocytes with high purity. The average value before separation is 22.8%, and the range is 15.9 - 29.4 ( Figure 1A), the average value after separation is 78.5%, and the range is 65.1 - 98.5( Figure 1 B); the average viability of macrophages is 97.1%, and the range is 92.1 - 99.7%( Figure 1 C); the average induction rate is 70.7%, and the range is 54.1 - 84.5%( Figure 1 D); it is able to produce more than 10 8 macrophages, with an average of 6.7×10 8 cells, and the range is 4.7 - 9.9×10 8 cells( Figure 1 E); it has a high expression of the mature phenotype 25F9, with an average of 97.6%, and the range is 93.3 - 99.6%( Figure 1 F), and a high level of expression of the marker CD206 related to tissue repair and inflammation resolution, with an average of 96.1%, and the range is 90.6 - 99.6%( Figure 1 G), and does not express the M1-type inflammatory macrophage marker IL-6, with an average of 8.4 pg / ml, and the range is 2.9 - 14.5 pg / ml( Figure 1 H). From these test data, it can be seen that we have the ability to mass-produce restorative macrophage products.

[0104] Test results of the phagocytic function of restorative macrophages

[0105] During the liver injury repair process, a large number of apoptotic / necroptotic cells need to be cleared in a timely manner. The liver tissue-resident macrophages - Kupffer cells are the main executors in this process. However, in the livers with liver fibrosis / cirrhosis lesions, the number of Kupffer cells decreases, their activity reduces, and their ability to secrete anti-inflammatory factors is impaired.

[0106] Macrophages recognize the "find me" signal - phosphatidylserine (PtdSer) released by dead cells by expressing a series of receptors for apoptotic cells, and target and phagocytose apoptotic cells. After macrophages phagocytose apoptotic cells, they can induce the expression of MMPs, and then promote the degradation of the extracellular matrix (ECM) and liver fibrosis repair.

[0107] Figure 2 The test results of the in vitro phagocytosis of the restorative macrophages prepared in the embodiments of the present invention are shown. Among them, Figure 2 A is the test data of the restorative macrophages phagocytosing pHrodo TM Red E.coli microspheres, Figure 2 B is the test data of the restorative macrophages phagocytosing phosphatidylserine, Figure 2 C is the test data of the restorative macrophages phagocytosing apoptotic cells.

[0108] Figure 2 The results of the in vitro phagocytosis test shown in this embodiment indicate that macrophages have the ability to phagocytose Escherichia coli particles labeled with fluorescence, with an average percentage of 82.0% ( Figure 2 A); have the ability to phagocytose phosphatidylserine particles labeled with fluorescence, with an average percentage of 56.7% ( Figure 2 B); have the ability to phagocytose apoptotic cells labeled with fluorescent protein, with an average percentage of 40.7% ( Figure 2 C). These results confirm that the restored macrophages prepared in this embodiment have strong phagocytic ability.

[0109] Test results of the ability of restored macrophages to degrade extracellular matrix (EMC)

[0110] The extracellular matrix (ECM) is mainly secreted by myofibroblasts, which can lead to the loss of liver function and further exacerbate the fibrotic inflammatory response of the liver. ECM degradation is the most important aspect of fibrosis regression and requires the activation of MMP (matrix metalloproteinase), macrophage phagocytic activity, and the downregulation of MMP inhibitory molecules. MMP is the main matrix-degrading enzyme. During the repair process of liver fibrosis, MMPs contribute to the degradation of the extracellular matrix (ECM). At the same time, macrophages expressing MMP9 and TRAIL can induce the apoptosis of myofibroblasts, which helps macrophages phagocytose them.

[0111] Figure 3 The test results of the ability of the restored macrophages prepared in the embodiments of the present invention to degrade extracellular matrix (EMC) are shown. Among them, Figure 3 A is the data of the gene expression profile analysis of restored macrophages, Figure 3 B is the result of the in vitro collagen degradation test of restored macrophages.

[0112] Figure 3 The test results of the ability of the restored macrophages in this embodiment to degrade extracellular matrix (EMC) show that through the gene expression profile analysis of multiple batches of restored macrophages, macrophages have the ability to express a variety of matrix metalloproteinases ( Figure 3 A); at the same time, we confirmed through the in vitro collagen degradation test that the culture supernatant of macrophages has collagen degradation activity ( Figure 3 B).

[0113] Step 1.3 Evaluation of the in vitro fibrosis model of restored macrophages

[0114] In an in vitro fibrosis model, human hepatic stellate cell line LX2 (in vitro liver fibrosis model) was treated with 2.5 ng / ml TGF-β; primary pulmonary fibroblasts HLF from patients with idiopathic pulmonary fibrosis (IPF) (in vitro pulmonary fibrosis model) were treated; recovery macrophages were added to the two models respectively, and after 48 hours of treatment, the cells were collected, and the collagen content of LX2 and HLF cells was analyzed by immunofluorescence or flow cytometry.

[0115] Figure 4 The test results of detecting the anti-fibrotic ability of the recovery macrophages prepared in the examples of the present invention in vitro are shown. Among them, Figure 4 A is the fluorescence detection graph of the collagen content of HLF cells, Figure 4 B is the fluorescence detection result of the collagen content of HLF cells, Figure 4 C is the detection result of the percentage of collagen protein expression in HLF cells, Figure 4 D is the detection result of the average fluorescence intensity of collagen protein in HLF cells, Figure 4 E is the detection result of the percentage of collagen protein expression in LX2 cells, Figure 4 F is the detection result of the average fluorescence intensity of collagen protein in LX2 cells. Figure 4 In it, the "-" group in each chart refers to the corresponding fibrosis model control group without adding recovery macrophages.

[0116] Figure 4 The test results of detecting the anti-fibrotic ability of the recovery macrophages in vitro in this example show that: in the in vitro pulmonary fibrosis model, statistical analysis by collagen I immunofluorescence shows that the recovery macrophages reduce the collagen protein expression in 18.5% of HLF cells ( Figure 4 A / B). In the in vitro pulmonary and liver fibrosis models, the percentage of collagen protein expression and the average fluorescence intensity in HLF cells or LX2 cells were analyzed by flow cytometry. The results show that the recovery macrophages reduce the percentage of collagen protein expression in HLF cells or LX2 cells by 29.2% and 28.8% respectively; the average fluorescence intensity is reduced by 30.4% and 32.9% respectively ( Figure 4 C-F). From these data, it can be seen that the recovery macrophages prepared in this example have anti-fibrotic ability.

[0117] Example 2. Production of mRNA

[0118] Step 2.1 Production of chimeric mRNA for converting inflammatory signals into anti-inflammatory signals

[0119] The pro-fibrotic inflammatory factors selected in this example are TGF-β, IL-17, and IL-6. We designed the receptors of these factors as extracellular signals, with the transmembrane region being the transmembrane region of the IL-10 factor receptor and the intracellular signal being the intracellular region of the IL-10 factor receptor. The TGF-β receptor selected is TGF-βRA with NCBI gene ID 7406, the IL-17 receptor selected is IL-17RA with NCBI gene ID 23765, the IL-6 receptor selected is IL-6R with NCBI gene ID 3570, and the IL-10 receptor selected is IL-10RA with NCBI gene ID 3587.

[0120] In this example, the mRNA sequence of the produced TGF-βRA-IL-10RA chimera is shown in SEQ ID NO: 1, the amino acid sequence of the TGF-βRA-IL-10RA chimera is shown in SEQ ID NO: 2, and the ORF / CDS sequence of the TGF-βRA-IL-10RA chimera is shown in SEQ ID NO: 3.

[0121] In this example, the mRNA and sequence of the produced IL-17RA-IL-10RA chimera are shown in SEQ ID NO: 4, the amino acid sequence of the IL-17RA-IL-10RA chimera is shown in SEQ ID NO: 5, and the ORF / CDS sequence of the IL-17RA-IL-10RA chimera is shown in SEQ ID NO: 6.

[0122] In this example, the mRNA sequence of the produced IL-11RA / IL-6ST-IL-10RA chimera is shown in SEQ ID NO: 7, the amino acid sequence of the IL-11RA / IL-6ST-IL-10RA chimera is shown in SEQ ID NO: 8, and the ORF / CDS sequence of the IL-11RA / IL-6ST-IL-10RA chimera is shown in SEQ ID NO: 9.

[0123] Step 2.2 Production of HGF mRNA

[0124] HGF, with NCBI gene ID 3082. We produced HGF mRNA according to this nucleotide sequence.

[0125] In this example, the mRNA sequence of HGF is shown in SEQ ID NO: 10, the amino acid sequence of HGF is shown in SEQ ID NO: 11, and the ORF / CDS sequence of HGF is shown in SEQ ID NO: 12.

[0126] Step 2.3 Production of CBD mRNA

[0127] CBD, with NCBI gene ID 7450, is derived from the VWF A3 region. We produced the required CBD mRNA based on this nucleotide sequence.

[0128] In this example, the mRNA sequence of CBD is shown as SEQ ID NO: 13, the amino acid sequence of CBD is shown as SEQ ID NO: 14, and the ORF / CDS sequence of CBD is shown as SEQ ID NO: 15.

[0129] The specific operations of steps 2.1 - 2.3 above are similar, with the difference being the different mRNA templates; the specific similar operations are as follows:

[0130] One - tube multi - step method is used for mRNA preparation, that is, linearization, transcription, and capping reactions are carried out in the same tube, and after the reaction is completed, mRNA is purified by the magnetic bead method.

[0131] Plasmid construction: Using the pUC57 - Kan vector as the backbone vector, the backbone vector mainly includes a DNA replication origin, a kanamycin resistance gene and its promoter. The T7 promoter sequence, 5'UTR sequence, protein - coding frame sequence, 3'UTR sequence, poly A sequence, and linearization restriction site are introduced through the multiple cloning site. The constructed vector is verified by sequencing, and the proportion of supercoiled plasmid prepared should be greater than 60%.

[0132] Plasmid linearization: The reaction volume is 5 μL, the concentration of the restriction endonuclease (BspQI) is 1 - 10 U / μg plasmid, and the digestion time at 50 °C is 0.5 - 3 h;

[0133] In vitro transcription: The total reaction volume is 10 μL, the reaction system includes 5 μL of the digested mixture, the concentration of T7 transcriptase (Novoprotein, GMP - E121 - HC - U100) is 100 - 500 U / μL, the final concentration of MgCl2 is 5 - 40 mM, the final concentration of Tris - HCl is 50 mM, the final concentration of NTP (replacing UTP with m1ψ) is 1 - 5 mM, the concentration of pyrophosphatase (Novoprotein, GMP - M036 - 01A) is 0.02 U / μL, the concentration of RNase inhibitor (Novoprotein, GMP - E125 - M001) is 1 U / μL, and the reaction is carried out at 37 °C for 0.5 - 2 h;

[0134] Capping reaction: The total reaction volume is 100 μL. The reaction system includes 10 μL of the transcription reaction product, with the final concentration of MgCl2 being 1 - 8 mM, the final concentration of Tris-HCl being 50 mM, the final concentration of SAM being 0.5 mM, the final concentration of GTP being 1 - 5 mM, the concentration of 2-O-methyltransferase (Novoprotein, GMP-M072-M001) being 50 - 250 U / μL, and the final concentration of vaccinia capping enzyme (Novoprotein, GMP-M062-M001) being 50 - 1000 U / μL. React at 37 °C for 1 - 2 h.

[0135] When purifying mRNA, 0.5 - 1 μg of magnetic beads are used for purification per microliter of the capping product.

[0136] Example 3. Preparation and functional detection of anti-inflammatory chimeric reprogrammed macrophages

[0137] Take a part of the macrophages on the 7th day prepared in Step 1.1 and divide them into 4 groups. Use the LNP delivery system to introduce the 3 different chimeric mRNAs prepared in Step 2.1 (TGF-βRA-IL-10RA chimeric mRNA, IL-17RA-IL-10RA chimeric mRNA, and IL-11RA / IL-6R-IL-10RA chimeric mRNA) into the macrophages of the corresponding groups respectively. Let them rest overnight, collect the cells and freeze them to obtain 3 groups of reprogrammed macrophages into which different chimeric mRNAs are introduced and 1 group of unmodified macrophages into which no chimeric mRNA is introduced. Retain an appropriate amount of cells to detect the anti-inflammatory ability of the cells in the in vitro fibrosis model.

[0138] In the in vitro liver fibrosis model, treat the human hepatic stellate cell line LX2 (in vitro liver fibrosis model) with 2.5 ng / ml TGF-β, 2.5 ng / ml IL-17, and 2.5 ng / ml IL-6 cytokines, and then add TGF-βRA-IL-10RA chimeric reprogrammed macrophages, IL-17RA-IL-10RA chimeric reprogrammed macrophages, and IL-11RA / IL-6R-IL-10RA chimeric reprogrammed macrophages respectively. After 48 h of treatment, collect the cells, and analyze the collagen content of LX2 cells by flow cytometry. Use the LX2 cell line treated only with TGF-β, IL-17, and IL-6 cytokines as the control group.

[0139] In an in vitro fibrosis model, primary lung fibroblasts HLF from patients with idiopathic pulmonary fibrosis (IPF) (in vitro lung fibrosis model) were treated with 2.5 ng / ml TGF-β, 2.5 ng / ml IL-17, and 2.5 ng / ml IL-6, and then TGF-βRA-IL-10RA chimeric reprogrammed restorative macrophages, IL-17RA-IL-10RA chimeric reprogrammed restorative macrophages, and IL-11RA / IL-6R-IL-10RA chimeric reprogrammed restorative macrophages were added respectively. After 48 h of treatment, the cells were collected, and the collagen content of HLF cells was analyzed by immunofluorescence and flow cytometry. The HLF cell line treated only with TGF-β, IL-17, and IL-6 cytokines was used as the control group.

[0140] Detection results of genes downstream of anti-inflammatory chimeric restorative macrophage IL-10

[0141] Figure 5 The detection results of genes downstream of anti-inflammatory chimeric restorative macrophage IL-10 prepared in the examples of the present invention are shown. Among them, Figure 5 A shows the relative change results of the genes CCL18, CD163, and BCL3 downstream of IL-10 caused by the TGF-βRA-IL-10RA chimera respectively, Figure 5 B shows the relative change results of the genes CCL18, CD163, and BCL3 downstream of IL-10 caused by the IL-17RA-IL-10RA chimera respectively, Figure 5 C shows the relative change results of the genes CCL18, CD163, and BCL3 downstream of IL-10 caused by the IL-11RA / IL6ST-IL-10RA chimera respectively.

[0142] As Figure 5 shown by the detection results: The TGF-βRA-IL-10RA chimera increased the genes CCL18, CD163, and BCL3 downstream of IL-10 by 18.7, 15.0, and 12.8 times respectively ( Figure 5 A); The IL-17RA-IL-10RA chimera increased the genes CCL18, CD163, and BCL3 downstream of IL-10 by 20.7, 12.0, and 14.8 times respectively ( Figure 5 B); The IL-11RA / IL6ST-IL-10RA chimera increased the genes CCL18, CD163, and BCL3 downstream of IL-10 by 10.7, 19.0, and 8.4 times respectively ( Figure 5C). Among them, CCL18 and CD163 indicate that the chimera that converts inflammatory signals into anti-inflammatory signals can maintain more stable reparative macrophages in an inflammatory environment, and BCL3 indicates that the conversion of inflammatory signals into anti-inflammatory signals promotes reparative macrophages to reduce the production of inflammatory factors in an inflammatory environment. From the test results, it can be seen that the anti-inflammatory chimera reparative macrophages prepared in the embodiments of the present invention can effectively convert inflammatory factors into anti-inflammatory factors and have a high-fold anti-inflammatory effect.

[0143] Test results of the anti-fibrotic ability of anti-inflammatory chimera reparative macrophages in vitro

[0144] Figure 6 Shows the test results of the anti-pulmonary fibrosis ability of the anti-inflammatory chimera reparative macrophages prepared in the embodiments of the present invention in vitro. Among them, Figure 6 A is the fluorescence detection diagram of the collagen content of HLF cells, Figure 6 B is the fluorescence detection result of the collagen content of HLF cells. As Figure 6 shown, in the in vitro pulmonary fibrosis model, through immunofluorescence statistics of collagen I, it is shown that TGF-bRA-IL-10RA, IL-17RA-IL-10RA, and IL-11RA / IL6ST-IL-10RA chimera reparative macrophages reduce the collagen expression of HLF cells by 73.3%, 77.5%, and 68.3% respectively ( Figure 6 A / B).

[0145] Figure 7 Shows the test results of the anti-fibrotic ability detection of the anti-inflammatory chimera reparative macrophages prepared in the embodiments of the present invention in vitro. Among them, Figure 7 A is the detection result of the percentage of collagen expression of HLF cells corresponding to three anti-inflammatory chimera reparative macrophages, Figure 7 B is the detection result of the average fluorescence intensity of collagen of HLF cells corresponding to three anti-inflammatory chimera reparative macrophages, Figure 7 C is the detection result of the percentage of collagen expression of LX2 cells corresponding to three anti-inflammatory chimera reparative macrophages, Figure 7 D is the detection result of the average fluorescence intensity of collagen of LX2 cells corresponding to three anti-inflammatory chimera reparative macrophages.

[0146] As Figure 7As shown, in the in vitro lung and liver fibrosis models, the percentage of collagen expression and the mean fluorescence intensity in HLF cells or LX2 cells were analyzed by flow cytometry. The results showed that TGF-bRA-IL-10RA, IL-17RA-IL-10RA, and IL-11RA / IL6ST-IL-10RA chimeric reparative macrophages decreased the percentage of collagen expression in HLF cells by 74.3%, 64.5%, and 54.0% respectively, and decreased the mean fluorescence intensity of collagen in HLF cells by 75.9%, 73.0%, and 65.6%( Figure 7 A - B); TGF-bRA-IL-10RA, IL-17RA-IL-10RA, and IL-11RA / IL6ST-IL-10RA chimeric reparative macrophages decreased the percentage of collagen expression in LX2 cells by 79.5%, 70.8%, and 56.3% respectively; and decreased the mean fluorescence intensity of collagen in HLF cells by 81.7%, 76.3%, and 71.5%( Figure 7 C - D).

[0147] From the above Figure 6 and Figure 7 detection, it can be seen that the anti-inflammatory chimeric reparative macrophages prepared in the embodiments of the present invention have high in vitro anti-fibrotic ability for both liver fibrosis and lung fibrosis.

[0148] Example 4. Preparation and functional detection of pro-regenerative reparative macrophages

[0149] Take the macrophages on the 7th day prepared in part of step 1.1 and divide them into 2 groups. Use the electroporation system to introduce the HGF mRNA prepared in step 2.2 into the macrophages in one group, rest overnight, and collect the cells for cryopreservation. Retain an appropriate amount of cells to detect HGF secretion, promote epithelial cell growth, and the in vitro ability to resist TGF-β-induced fibrosis.

[0150] HGF detection, 1×10 6 Pro-regenerative reparative macrophages and un-reprogrammed macrophages were cultured overnight, and the supernatant was collected. The HGF secretion was quantitatively detected using an ELISA kit. For the detection of promoting epithelial cell growth, human lung epithelial cells A549 were co-cultured with the reprogrammed macrophages for 3 days, and the change in the number of cells was detected.

[0151] In the in vitro liver fibrosis model, human hepatic stellate cell line LX2 (in vitro liver fibrosis model) was treated with 2.5 ng / ml TGF-β. Then, pro-regenerative reparative macrophages were added and treated for 48 h. The cells were collected, and the collagen content in LX2 cells was analyzed by flow cytometry. The LX2 cell line treated only with the TGF-β cytokine was used as the control group.

[0152] In an in vitro pulmonary fibrosis model, primary pulmonary fibroblasts HLF (in vitro pulmonary fibrosis model) from patients with idiopathic pulmonary fibrosis (IPF) were treated with 2.5 ng / ml TGF-β, and pro-regenerative and restorative macrophages were added. After 48 hours of treatment, the cells were collected, and the collagen content of HLF cells was analyzed by flow cytometry. The HLF cell line treated only with TGF-β cytokine was used as a control group.

[0153] Detection results of the function of pro-regenerative and restorative macrophages

[0154] Figure 8 The detection results of the function of the pro-regenerative and restorative macrophages prepared in the examples of the present invention are shown. Among them, Figure 8 A shows the detection results of HGF secreted by pro-regenerative and restorative macrophages, Figure 8 B shows the results of the effect of pro-regenerative and restorative macrophages on the proliferation of A549 cells, Figure 8 C shows the effect of pro-regenerative and restorative macrophages on the percentage of collagen expression in HLF cells, Figure 8 D shows the effect of pro-regenerative and restorative macrophages on the mean fluorescence intensity of collagen in HLF cells, Figure 8 E shows the effect of pro-regenerative and restorative macrophages on the percentage of collagen expression in LX2 cells, Figure 8 F shows the effect of pro-regenerative and restorative macrophages on the mean fluorescence intensity of collagen in LX2 cells.

[0155] As Figure 8 A and Figure 8 B show that after introducing HGF mRNA into pro-regenerative and restorative macrophages, the secretion of HGF by macrophages can be enhanced, with an average value of 217.5 ng / mL and a range of 164.6 - 280.2 ng / mL ( Figure 8 A). Moreover, it also promotes the proliferation of bleomycin-treated A549 cells, with an average increase of 2.7 times and a range of 2.1 - 4.2 times ( Figure 8 B). This indicates that the pro-regenerative and restorative macrophages prepared in this example all have a high pro-regenerative ability in the pulmonary fibrosis model.

[0156] As Figure 8 C - F show that HGF-restorative macrophages reduced the percentage of collagen expression in HLF cells by 49.5% ( Figure 8 C), and reduced the mean fluorescence intensity by 54.1% respectively ( Figure 8 D); HGF-restorative macrophages reduced the percentage of collagen expression in LX2 cells by 54.3% respectively ( Figure 8 E); and reduced the mean fluorescence intensity of collagen in LX2 cells by 49.9% respectively ( Figure 8F). These data results indicate that the regenerative recovery-promoting macrophages prepared in this example have a high regenerative capacity in the liver fibrosis model.

[0157] Example 5. Preparation and functional detection of fibrosis-targeted recovery macrophages

[0158] Take a part of the macrophages on the 7th day prepared in Step 1.1 and divide them into 2 groups. Use the electroporation system to introduce the CBD mRNA prepared in Step 2.3 into the macrophages of one group, rest overnight, collect the cells and freeze them. Reserve an appropriate amount of cells to detect the CBD-collagen binding ability.

[0159] Detection of CBD-collagen binding ability, 1×10 6 For reprogrammed macrophages and unmodified macrophages, incubate with FITC collagen I at 5 μg / mL, and use unincubated macrophages as a control. Analyze the percentage of FITC collagen I by flow cytometry.

[0160] Functional detection of fibrosis-targeted tissue recovery macrophages

[0161] Figure 9 The results of the functional detection of fibrosis-targeted tissue recovery macrophages prepared in the examples of the present invention are shown. Among them, Figure 9 A is the flow analysis chart of macrophage collagen binding, Figure 9 B is the analysis result of the macrophage collagen binding percentage.

[0162] As Figure 9 shown in A and 9B, the average value of the macrophage collagen binding percentage is 26.0, and the range is 21.3 - 30.1% ( Figure 9 A - B); the average value of the fibrosis-targeted tissue recovery macrophage collagen binding percentage is 95.9 ng / mL, and the range is 91.2 - 99.8% ( Figure 9 A - B). From these data, it can be seen that after introducing the collagen domain CBD mRNA into the fibrosis-targeted tissue recovery macrophages, the macrophage collagen binding can be enhanced.

[0163] Example 6. Preparation of fibrosis-targeted, anti-inflammatory chimeric and regenerative reprogramming recovery macrophages and detection of their in vivo anti-fibrosis effect

[0164] Take the recovered macrophages on the 7th day prepared in part of step 1.1 and divide them into 4 groups. Use the LNP delivery system to introduce the TGF-βRA-IL-10RA chimeric mRNA, IL-17RA-IL-10RA chimeric mRNA, IL-11RA / IL-6R-IL-10RA chimeric mRNA prepared in step 2.1 and HGF mRNA, CBD mRNA into the macrophages of 3 of the groups respectively. Let them rest overnight, collect the cells and freeze them to obtain 3 groups of reprogrammed macrophages (respectively: TGF-βRA-IL-10RA+HGF+CBD reprogrammed group, IL-17RA-IL-10RA+HGF+CBD reprogrammed group, IL-11RA / IL-6R-IL-10RA+HGF+CBD reprogrammed group) and 1 group of recovered macrophages without any mRNA introduced. Retain an appropriate amount of cells to detect the anti-inflammatory ability of the cells in the in vitro fibrosis model.

[0165] Using the carbon tetrachloride (CCL4) mouse fibrosis model, inject CCL4 intraperitoneally into NOG mice twice a week. At the 6th week, intravenously transfuse TGF-bR-IL-10R mRNA+HGF mRNA+CBD mRNA reprogrammed macrophages, IL-17R-IL-10R mRNA+HGF mRNA+CBD mRNA reprogrammed macrophages, IL-6R-IL-10R mRNA+HGF mRNA+CBD mRNA reprogrammed macrophages into the bodies of 3 groups of mice. In addition, intravenously transfuse the recovered macrophages prepared in step 1.1 without modification into another group of mice to obtain the recovered macrophage group, and a control group without transfusing macrophages. 3×10 6 cells / mouse, and then continuously inject CCL4 until the 10th week. Anesthetize and sacrifice the mice, collect the liver tissues, and perform histochemical staining with Sirius red and α-SMA to evaluate the regression of liver tissue fibrosis.

[0166] Using the bleomycin mouse fibrosis model, instill bleomycin into NOG mice through the trachea. At the 2nd week, intravenously transfuse TGF-bR-IL-10R mRNA+HGF mRNA+CBD mRNA reprogrammed macrophages, IL-17R-IL-10RmRNA+HGF mRNA+CBD mRNA reprogrammed macrophages, IL-6R-IL-10RmRNA+HGF mRNA+CBD mRNA reprogrammed macrophages into the bodies of 3 groups of mice. Similarly, intravenously transfuse the recovered macrophages prepared in step 1.1 without modification into another group of mice to obtain the recovered macrophage group, and a control group without transfusing macrophages. 3×10 6 cells / mouse. At the 6th week, anesthetize and sacrifice the mice, collect the lung tissues, and perform histochemical staining with Sirius red and α-SMA to evaluate the regression of lung tissue fibrosis.

[0167] Detection Results of Anti-Fibrotic Effects of Targeted Fibrosis, Anti-Inflammatory Chimeras, and Pro-Regenerative Reprogrammed Macrophages In Vivo

[0168] Figure 10 The test results of treating bleomycin-induced pulmonary fibrosis with the reprogrammed reparative macrophages prepared in this example are shown. Figure 10 A is a schematic diagram of treating bleomycin-induced pulmonary fibrosis model NOG mice with reprogrammed reparative macrophages;

[0169] Figure 10 B shows the changes in COL1A1 gene after treatment with different macrophages; Figure 10 C shows the changes in TIMP-1 gene after treatment with different macrophages; Figure 10 D shows the changes in Sirius red staining after treatment with different macrophages; Figure 10 E shows the changes in α-SMA after treatment with different macrophages.

[0170] As Figure 10 Shown in A, the anti-fibrotic effects of reprogrammed reparative macrophages were evaluated by detecting the changes in the genes of type I collagen alpha 1 chain (COL1A1) and tissue inhibitor of metalloproteinase 1 (TIMP-1) by qPCR, and Sirius red and α-SMA immunohistochemical staining. Type I collagen is the main component of the extracellular matrix (ECM). A decrease in TIMP-1 levels can increase MMP activity and promote ECM degradation. Sirius red staining can be used to distinguish type I, II, III, and IV collagens, and can directly observe and analyze the degree of collagen fibrosis; α-smooth muscle actin (α-SMA) is a marker of fibrotic cell activation. It has been proven that α-SMA expression increases with the severity of fibrosis and can indicate the severity of different fibrotic diseases.

[0171] Figure 10 The experimental results of B - E showed that after treatment with reparative macrophages, TGF-βRA-IL-10RA anti-inflammatory chimera-targeted pro-regenerative reparative macrophages, IL-17RA-IL-10RA anti-inflammatory chimera-targeted pro-regenerative reparative macrophages, and IL-11RA / IL6ST-IL-10RA anti-inflammatory chimera-targeted pro-regenerative reparative macrophages, the decreases in COL1A1 gene in the lungs of mice were 41.9%, 74.9%, 69.3%, and 68.5% respectively ( Figure 10 B); the decreases in TIMP-1 gene were 44.6%, 74.4%, 65.1%, and 71.5% respectively ( Figure 10 C); the decreases in α-SMA were 44.3%, 68.3%, 70.0%, and 61.8% respectively ( Figure 10D); The decreases in Sirius red staining were 51.0%, 81.3%, 81.7% and 72.1% respectively ( Figure 10 E). These data results indicate that the reprogrammed regenerative macrophages prepared in this example have stronger effects in improving pulmonary fibrosis, stimulating lung regeneration and improving function than regenerative macrophages.

[0172] Figure 11 The test results of treating liver fibrosis with the reprogrammed regenerative macrophages prepared in this example are shown. Figure 11 A is a schematic diagram of treating CCL4-induced liver fibrosis model NOG mice with reprogrammed regenerative macrophages; Figure 11 B shows the change results of COL1A1 gene after treatment with different macrophages; Figure 11 C shows the change results of TIMP-1 gene after treatment with different macrophages; Figure 11 D shows the change results of α-SMA after treatment with different macrophages; Figure 11 E shows the change results of Sirius red staining after treatment with different macrophages

[0173] As Figure 11 shown in A, the changes in type I collagen alpha 1 chain (COL1A1) and tissue inhibitor of metalloproteinase 1 (TIMP-1) genes were detected by qPCR, and Sirius red and α-SMA immunohistochemical staining were used to evaluate the anti-fibrotic effects of reprogrammed regenerative macrophages. Type I collagen is the main component of the extracellular matrix (ECM). A decrease in TIMP-1 level can increase MMP activity and promote ECM degradation.

[0174] Figure 11 The experimental results of B-E show that after treatment with regenerative macrophages, TGF-βRA-IL-10RA anti-inflammatory chimeric targeted regenerative macrophages, IL-17RA-IL-10RA anti-inflammatory chimeric targeted regenerative macrophages and IL-11RA / IL6ST-IL-10RA anti-inflammatory chimeric targeted regenerative macrophages respectively, in the mouse liver, the decreases in COL1A1 gene were 38.7%, 59.4%, 55.8% and 52.8% respectively ( Figure 11 B); the decreases in TIMP-1 gene were 21.2%, 68.6%, 62.4% and 53.2% respectively ( Figure 11 C); the decreases in α-SMA were 29.1%, 70.3%, 65.0% and 58.5% respectively ( Figure 11 D); the decreases in Sirius red staining were 40.1%, 86.3%, 75.7% and 76.9% respectively ( Figure 11 E), indicating that reprogrammed regenerative macrophages have stronger effects in improving liver fibrosis, stimulating liver regeneration and improving function than regenerative macrophages.

[0175] For the method embodiments, for the sake of simplicity of description, they are all expressed as a series of combinations of actions. However, those skilled in the art should be aware that the present invention is not limited by the described order of actions, because according to the present invention, certain steps can be performed in other orders or simultaneously. Secondly, those skilled in the art should also be aware that the embodiments described in the specification are all preferred embodiments, and the actions and components involved are not necessarily essential to the present invention.

[0176] The anti-inflammatory chimeras, reprogrammed macrophages, and their preparation methods and applications provided by the present invention have been introduced in detail above. Specific examples are used herein to elaborate on the principles and implementation manners of the present invention. The description of the above embodiments is only used to help understand the method and its core idea of the present invention; at the same time, for those of ordinary skill in the art, according to the idea of the present invention, there will be changes in the specific implementation manners and application scopes. In summary, the content of this specification should not be construed as a limitation to the present invention.

Claims

1. An anti-inflammatory chimera, characterized in that The anti-inflammatory chimera is based on the conversion of inflammatory signals into anti-inflammatory signal chimeras; the extracellular structure of the anti-inflammatory chimera is an inflammatory factor receptor, and the intracellular structure is an anti-inflammatory factor receptor intracellular signal.

2. The anti-inflammatory chimera according to claim 1, characterized in that The inflammatory factor is one or more of TGF-β, IL-17, IL-4, IL-13, IL-11, IL-6, IL-33, IL-1, IL-12, IL-18, IFN-γ and TNF-α.

3. The anti-inflammatory chimera according to claim 1, characterized in that The transmembrane domain of the anti-inflammatory chimera is the transmembrane domain of a transmembrane protein, preferably the transmembrane region of an anti-inflammatory factor receptor or the transmembrane region of a platelet-derived growth factor receptor β.

4. The anti-inflammatory chimera according to any one of claims 1 to 3, characterized in that The anti-inflammatory factor is IL-10.

5. The anti-inflammatory chimera according to any one of claims 1 to 4, characterized in that The information carrier of the chimera is RNA or DNA.

6. An anti-inflammatory cell, characterized in that The anti-inflammatory cells contain the anti-inflammatory chimera according to any one of claims 1-5.

7. The anti-inflammatory cell according to claim 6, characterized in that The cell is one of monocytes, T cells, B cells, NK cells and macrophages.

8. A method for preparing anti-inflammatory cells as claimed in claim 6 or 7, characterized in that: The anti-inflammatory chimeric mRNA or anti-inflammatory chimeric DNA is introduced into the target cells.

9. The preparation method according to claim 8, characterized in that: The method is to introduce the drug by any one of electroporation, lipid nanoparticles or viruses.

10. A cell vaccine, characterized in that: The cell vaccine comprises the anti-inflammatory cells according to claim 6 or 7.

11. Use of the anti-inflammatory chimera according to any one of claims 1 to 5, the anti-inflammatory cells according to claim 6 or 7, or the cell vaccine according to claim 10 in preventing or reducing tissue inflammation or treating fibrotic diseases.

12. A reprogrammed macrophage, characterized in that: The reprogrammed macrophages are introduced with an information carrier of the anti-inflammatory chimera according to any one of claims 1 to 5.

13. The reprogrammed macrophage according to claim 12, characterized in that The information carrier of the chimera is DNA or RNA.

14. The reprogrammed macrophage according to claim 12, characterized in that The reprogrammed macrophages also contained pro-regeneration genes.

15. The reprogrammed macrophage according to claim 14, characterized in that The pro-regeneration gene is a gene that promotes tissue regeneration.

16. The reprogrammed macrophage according to claim 15, characterized in that The pro-regeneration gene encodes the sequence of HGF.

17. The reprogrammed macrophage according to any one of claims 12 to 16, characterized in that: A collagen targeting gene targeting fibrotic lesions is also introduced into the reprogrammed macrophages.

18. The reprogrammed macrophage according to claim 17, characterized in that The collagen targeting gene encodes the sequence of a collagen binding domain protein.

19. The reprogrammed macrophage according to claim 18, characterized in that The collagen binding domain protein is derived from the von Willebrand factor-A3 domain.

20. The reprogrammed macrophage according to any one of claims 12 to 19, characterized in that One or more of anti-inflammatory genes, pro-phagocytosis genes and pro-degradation extracellular matrix genes are also introduced into the reprogrammed macrophages; wherein the information carrier of the gene is preferably DNA or RNA.

21. A method for preparing reprogrammed macrophages according to any one of claims 12 to 20, characterized in that: The method comprises: preparing the gene according to any one of claims 12 to 20 according to a sequence template, and introducing the gene into a recovery macrophage.

22. The method according to claim 21, characterized in that The method uses any one of electroporation, lipid nanoparticles or viruses to introduce the drug.

23. A macrophage vaccine, characterized in that: The macrophage vaccine comprises the reprogrammed macrophage according to any one of claims 12-20.

24. A cell population, characterized in that The cell population comprises the reprogrammed macrophages according to any one of claims 12-20.

25. Use of the reprogrammed macrophage according to any one of claims 12 to 20, the macrophage vaccine according to claim 23, or the cell population according to claim 24 in preventing or treating tissue fibrosis diseases.

26. The use according to claim 25, characterized in that The fibrotic disease is one of liver fibrosis, lung fibrosis, cardiac fibrosis and kidney fibrosis.

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