Preparation and anti-fibrosis application of engineered macrophage

Abstract

The invention discloses preparation and anti-fibrosis application of engineered macrophages, and belongs to the technical field of biological medicines. The engineered macrophages comprise a nucleic acid encoding a fusion protein comprising a VWF protein A3 domain and a PDGFR transmembrane domain. The engineered macrophages further include a nucleic acid encoding one or more of IL-10, IL-4, and IL-13.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to the preparation of engineered macrophages and their anti-fibrosis application. Background Art

[0002] Fibrosis is a pathological process of repair after tissue or organ damage. Its core feature is the excessive deposition of extracellular matrix (ECM) (such as collagen, fibronectin, elastin, etc.) driven by fibroblasts and their activated form, myofibroblasts, which leads to the destruction of tissue structure and function.

[0003] Various factors can lead to the development of fibrosis, and chronic inflammation is one of the main causes of fibrosis. The inflammatory environment recruits inflammatory cells (such as macrophages and T cells) and promotes their release of proinflammatory factors (such as TGF-β, IL-1β, TNF-α), stimulates fibroblast activation and promotes ECM production and deposition; TGF-β (transforming growth factor-β) and its downstream IL11 signaling pathway are key regulators of fibrosis, which can promote the transformation of fibroblasts into myofibroblasts; PDGF (platelet-derived growth factor) can promote fibroblast proliferation and migration; Wnt / β-catenin pathway is also involved in the occurrence and progression of fibrosis. In addition, the production and increase of reactive oxygen species (ROS) lead to tissue damage, cell senescence and apoptosis, which release profibrotic factors and can also aggravate the process of fibrosis. Fibrosis can affect multiple organs, such as the lungs, liver, heart, kidneys, etc., and is a common terminal pathological manifestation of many chronic diseases.

[0004] Pulmonary fibrosis occurs when scar tissue is produced during the damage and repair of lung tissue. This thickened and stiff tissue makes it more difficult for the lungs to work normally. Clinically, patients mainly show symptoms such as dyspnea, dry cough, fatigue, and easy fatigue. Medical imaging tests show that patients with pulmonary fibrosis have a large amount of collagen fiber deposition in the lungs, a large amount of alveolar loss, and changes in the structure of normal lung tissue. The patient's gas exchange function is gradually lost, and eventually he dies of respiratory failure. There are many factors that cause pulmonary fibrosis damage, including long-term exposure to certain toxins, certain medical conditions, radiotherapy, and certain drugs. However, in most cases, doctors cannot find the root cause of the problem, so it is called idiopathic pulmonary fibrosis (IPF). IPF patients account for 30%-50% of all patients with pulmonary fibrosis. Clinically, anti-inflammatory, antioxidant and anti-fibrosis treatment methods are mainly used, but these methods often have limited efficacy and have certain side effects. Currently, the only treatment drugs are pirfenidone and nindabuni. Non-drug treatment options include oxygen therapy, pulmonary rehabilitation and lung transplantation (MSD).

[0005] Cell therapy is an emerging therapeutic strategy that repairs damaged tissues, regulates immune responses, or inhibits pathological processes by transplanting or activating specific types of cells. Cell therapy may achieve long-term therapeutic effects through paracrine effects and cell replacement. In the treatment of fibrosis, cell therapy shows great potential, especially in regulating inflammation, inhibiting the process of fibrosis, and promoting tissue regeneration. Macrophages, as an important component of the immune system, can simultaneously regulate inflammation, inhibit fibrosis, and promote tissue repair.

[0006] In recent years, strategies based on RNA regulation of gene expression to transform cell functions have shown great potential in the field of disease treatment. The key to the transformation of engineered macrophages for anti-fibrosis treatment lies in how to regulate the function of macrophages and enhance and maintain their anti-fibrosis ability. Strategies such as maintaining the anti-inflammatory phenotype of macrophages, improving the inflammatory environment of fibrotic tissues, and enhancing the ECM targeting and degradation capabilities of macrophages are important directions for anti-fibrosis engineering of macrophages. For example, in patent WO2024068728A1, the ECM degradation ability of macrophages is enhanced by expressing MMP9 and MMP12 in macrophages; in patent WO2024074376A1, the anti-inflammatory and ECM degradation capabilities are enhanced by expressing MMP9 and IL10 in macrophages; in patent CN112236445A, ECM targeting proteins and proteases are introduced into macrophages to enhance the ECM targeting and degradation capabilities of macrophages.

[0007] Macrophage-based anti-fibrosis therapy mainly involves transplanting macrophages into patients through intravenous infusion. After entering the human body, macrophages circulate through the blood and eventually settle mainly in the liver. The residence time and residence amount of macrophages in organs such as the lungs, heart, and kidneys are very small. How to enhance the migration and colonization of macrophages into these organs is one direction of macrophage transformation. More and more studies have shown that vascular remodeling induced by pathological conditions is also an important manifestation of organ fibrosis. In fibrotic tissues and organs, ECM near remodeled blood vessels is often increased. Angiogenesis and remodeling are common in IPF lung tissues, and the site of occurrence is basically consistent with the site of fibrosis. Morphological studies have confirmed the existence of vascular remodeling in IPF, and have proved that the process of vascular remodeling is active in areas adjacent to fibroblast lesions, while the vascular density in fibroblast lesions is reduced. The pathological angiogenesis process is also included in the development of pulmonary fibrosis.

[0008] The existing selection of targets for engineering macrophage transformation has certain limitations. In terms of enhancing the enrichment of macrophages to the ECM area, the targets selected by the existing transformation schemes are mainly integrins, lectin domain receptors, mannose family receptors and / or immunoglobulin-like receptors (patent CN112236445A).

[0009] VWF (von Willebrand factor) protein plays an important role in maintaining hemostasis. It promotes platelet adhesion to the site of vascular injury by forming a molecular bridge between the subendothelial collagen matrix and the platelet surface receptor complex GPIb-IX-V. It also acts as a molecular chaperone for coagulation factor VIII, delivering it to the site of injury, stabilizing its heterodimer structure and protecting it from being cleared from the plasma prematurely. There is no anti-fibrotic therapeutic strategy developed based on the collagen binding effect of VWF. Summary of the invention

[0010] In response to the above technical problems, the present invention constructs a fusion protein comprising a signal peptide, a VWF protein A3 domain (CBD) and a PDGFR transmembrane domain (TM), and utilizes LNP to encapsulate mRNA-LNP expressing the fusion protein or directly electrophoretically expresses a nucleic acid expressing the fusion protein, thereby achieving the expression of the fusion protein in macrophages and constructing anti-fibrotic engineered macrophages.

[0011] The first aspect of the present invention provides a fusion protein comprising a VWF protein A3 domain and a PDGFR transmembrane domain.

[0012] In some embodiments, the VWF protein A3 domain and the transmembrane domain of PDGFR are connected by a flexible linker.

[0013] In some embodiments, the fusion protein further comprises a signal peptide at the N-terminus; the signal peptide is derived from IL-2, IL-6, IL-11 or IL-12B.

[0014] The second aspect of the present invention provides a nucleic acid encoding the fusion protein of the first aspect of the present invention; wherein the nucleic acid is DNA or mRNA.

[0015] The third aspect of the present invention provides a nanolipid particle-nucleic acid complex, wherein the complex comprises the nucleic acid described in the first aspect of the present invention or the nanolipid particle (LNP) described in the second aspect, wherein the nucleic acid is mRNA.

[0016] In some embodiments, the nanolipid particle comprises an ionizable cationic lipid, a neutral auxiliary phospholipid, a sterol and a PEG lipid, wherein the sterol is a mixture of cholesterol and dexamethasone, wherein the molar ratio of cholesterol to dexamethasone in the mixture of cholesterol and dexamethasone is 9:1.

[0017] The fourth aspect of the present invention provides an engineered macrophage comprising the fusion protein of the first aspect of the present invention or the nucleic acid of the second aspect of the present invention.

[0018] In some embodiments, the engineered macrophage further comprises a nucleic acid encoding one or more of IL-10, IL-4, and IL-13.

[0019] The fifth aspect of the present invention provides a method for preparing engineered macrophages, comprising introducing the nucleic acid described in the second aspect of the present invention or the nanolipid particle-nucleic acid complex provided in the third aspect of the present invention into macrophages.

[0020] In some embodiments, the above method comprises: introducing the nucleic acid described in the second aspect of the present invention or the nanolipid particle-nucleic acid complex provided by the third aspect of the present invention into macrophages simultaneously or successively with LNPs coated with one or more nucleic acids selected from encoding one or more of IL-10, IL-4 and IL-13.

[0021] In some embodiments, when the nucleic acid is introduced, the introduction method is electroporation; when the complex is introduced, the introduction method is LNP delivery.

[0022] The sixth aspect of the present invention provides engineered macrophages prepared according to the method of the fourth aspect of the present invention.

[0023] The seventh aspect of the present invention provides use of the engineered macrophages according to the fourth aspect or the sixth aspect of the present invention in the preparation of a drug for preventing and / or treating fibrotic diseases.

[0024] The eighth aspect of the present invention provides use of the fusion protein according to the first aspect, the nucleic acid according to the second aspect, or the complex according to the third aspect of the present invention in the preparation of a drug for preventing and / or treating fibrotic diseases.

[0025] In some embodiments, the fibrotic disease includes pulmonary fibrosis, hepatic fibrosis, pneumoconiosis, cystic fibrosis, cirrhosis, myocardial fibrosis, renal interstitial fibrosis, scleroderma, keloid, pancreatic fibrosis, retroperitoneal fibrosis, systemic sclerosis and / or myelofibrosis. Wherein, the pulmonary fibrosis includes idiopathic pulmonary fibrosis and radiation-induced pulmonary fibrosis.

[0026] The advantages of the present invention over the prior art are: 1) For the first time, a fusion protein containing a signal peptide, VWF protein A3 domain (CBD) and PDGFR transmembrane domain (TM) was constructed, and the mRNA-LNP expressing the fusion protein was encapsulated in inflammation-suppressing LNP to achieve the expression of the fusion protein in macrophages.

[0027] 2) The VWF A3 domain has the ability to bind to collagen. After the VWF A3 domain is synthesized in macrophages, it is secreted outside the cells to exert its effects. Under the guidance of IL11-derived signals, the fusion protein (CBD-TM) can be expressed on the surface of the macrophage membrane. Macrophages transformed with CBD-TM mRNA have stronger ECM binding ability and can migrate more toward fibrotic tissues after being infused back into the body.

[0028] 3) Based on the CBD-TM mRNA modification, the anti-inflammatory factors IL-10, IL-4 or IL-13 mRNA are further introduced, and the resulting fibrosis area-targeted anti-inflammatory macrophages have further enhanced anti-fibrosis effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Other features, objects and advantages of the present invention will become more apparent from the detailed description of non-limiting embodiments made with reference to the following drawings: Figure 1 . Construction of CBD-TM expression template and preparation of mRNA. A, Schematic diagram of fusion proteins with different signal peptides; B, Agarose gel electrophoresis analysis of the four prepared mRNAs.

[0030] Figure 2 . Comparison of different CBD-TM mRNA expressions and functional verification. A, Flow cytometry analysis of the expression percentage of CBD-TM fusion proteins with different signal peptides in macrophages; B, Flow cytometry analysis of the percentage of macrophages expressing CBD-TM fusion proteins with different signal peptides binding to collagen.

[0031] Figure 3 . CBD-TM LNP-mRNA does not affect macrophage phenotype and cytokine secretion. A, flow cytometry analysis of macrophage viability after 1 day of transfection with empty LNP control and CBD-TM (IL11 signal peptide) LNP-mRNA; B, flow cytometry analysis of the expression percentage of macrophage surface markers CD80 (M1) and CD206 (M2) after 1 day of transfection with empty LNP control and CBD-TM (IL11 signal peptide) LNP-mRNA; C, flow cytometry analysis of the levels of cytokines IL6 and IL10 in the supernatant of macrophage culture after 1 day of transfection with empty LNP control and CBD-TM (IL11 signal peptide) LNP-mRNA.

[0032] Figure 4. CBD-TM engineered macrophages have stronger anti-pulmonary fibrosis effects. A, qPCR analysis of the content of human gDNA in lung tissues of different treatment groups; B, hydroxyproline content in lung tissues of different treatment groups; C, qPCR analysis of Co1a1 and Col3a1 mRNA expression levels in lung tissues of different treatment groups; D and E, Masson staining results and statistical analysis results of lung tissues of different treatment groups.

[0033] Figure 5 . CBD-TM engineered macrophages have stronger anti-liver fibrosis effects. A, qPCR analysis of the expression levels of Mmp2 and Timp1 mRNA in liver tissues of different treatment groups; B, Sirius red staining results and statistical analysis results of liver tissue sections of different treatment groups; C, α-SMA staining results and statistical analysis results of liver tissue sections of different treatment groups.

[0034] Figure 6 . CBD-TM combined with IL-10 engineered macrophages has a stronger anti-pulmonary fibrosis effect. A, ELISA analysis of cytokine IL-10 levels in different macrophage culture supernatants; B, Hydroxyproline levels in lung tissues of different treatment groups; C, qPCR analysis of Col1a1 and Col3a1 mRNA expression levels in lung tissues of different treatment groups; D, Masson staining and statistical analysis results of lung tissues of different treatment groups.

[0035] Figure 7 . CBD-TM combined with IL-4 engineered macrophages has a stronger anti-pulmonary fibrosis effect. A, ELISA analysis of cytokine IL-4 levels in different macrophage culture supernatants; B, Hydroxyproline levels in lung tissues of different treatment groups; C, qPCR analysis of Timp1 and Col1a2 mRNA expression levels in lung tissues of different treatment groups; D, α-SMA staining and statistical analysis results of lung tissues of different treatment groups.

[0036] Figure 8 . CBD-TM combined with IL-13 engineered macrophages has a stronger anti-pulmonary fibrosis effect. A, ELISA analysis of cytokine IL-13 levels in different macrophage culture supernatants; B, Hydroxyproline levels in lung tissues of different treatment groups; C, qPCR analysis of Col1a1 and Col3a1 mRNA expression levels in lung tissues of different treatment groups; D, Masson staining and statistical analysis results of lung tissues of different treatment groups. DETAILED DESCRIPTION

[0037] In order to make the purpose, technical solution and advantages of the embodiment of the present invention clearer, the technical solution of the embodiment of the present invention will be clearly and completely described below in conjunction with the accompanying drawings of the embodiment of the present invention. Obviously, the described embodiment is a part of the embodiment of the present invention, not all of the embodiments. Based on the described embodiment of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0038] Unless otherwise defined, technical or scientific terms used herein shall have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs.

[0039] Example 1 Preparation of CBD-TM LNP-mRNA In this example, membrane-expressed collagen-targeted fusion protein mRNA with different guiding signals was designed and constructed. The N-terminus of the fusion protein is a signal peptide, and the A3 domain derived from VWF (SEQ ID NO: 1) is connected to the transmembrane domain derived from PDGFR (SEQ ID NO: 3) via a GS linker (SEQ ID NO: 2). Figure 1 A), the N-terminal signal peptides are from IL-2, IL-6, IL-11 and IL-12B. The signal peptide sequences of IL-2, IL-6, IL-11 and IL-12B are shown in SEQ ID NOs: 4-7, respectively. The full-length sequences of the fusion proteins of the signal peptide-VWF-A3 domain-GS linker-PDGFR transmembrane domain fused with the signal peptides of IL-2, IL-6, IL-11 and IL-12B at the N-terminus are shown in SEQ ID NOs: 8-11, respectively.

[0040] The mRNA was prepared by a one-tube multi-step method, that is, linearization, transcription and capping reactions were carried out in the same tube. After the reaction was completed, the mRNA was purified by magnetic beads. Agarose gel electrophoresis showed that the length of the prepared mRNA was close to the theoretical size, and the band was single and non-degraded ( Figure 1 B), the sequence information of mRNA is shown in Table 3. Then, the anti-inflammatory LNP lipid combination (Table 1) was used to prepare LNP-mRNA by microfluidic mixing, and the encapsulation efficiency, particle size, PDI and zeta charge of LNP-mRNA were tested as shown in Table 2.

[0041] The specific implementation plan is as follows: Plasmid construction: pUC57-Kan vector (Sangon Biotechnology, B522201-0100) was used as the backbone vector, which mainly includes the DNA replication initiation site, kanamycin resistance gene and its promoter. T7 promoter sequence (SEQ ID NO: 12), 5' UTR sequence (SEQ ID NO: 13), protein coding frame sequence, 3' UTR sequence (SEQ ID NO: 14), poly A sequence (SEQ ID NO: 15) and linearization restriction site were introduced through the multiple cloning site. The constructed vector was verified by sequencing, and the supercoiling ratio of the prepared plasmid should be greater than > 60%.

[0042] The 5' UTR sequence consists of the HBB (hemoglobin) mRNA 5UTR sequence (SEQ ID NO: 16), the IGG6 sequence (caatcaaac, SEQ ID NO: 17) and the Kozak sequence (gccacc, SEQ ID NO: 18). The 3' UTR sequence is the histone-derived 3UTR sequence, and the polyA length should be greater than 100.

[0043] Plasmid linearization: reaction volume 5 μL, restriction endonuclease (BspQI) concentration 1-10 U / μg plasmid, 50 ℃ enzyme digestion time 0.5-3 h; In vitro transcription: The total reaction volume was 10 μL, and the reaction system included 5 μL of restriction enzyme mixture, T7 transcriptase (Nearshore protein, GMP-E121-HC-U100) at a concentration of 100-500 U / μL, MgCl 2 The final concentration is 5-40 mM, the final concentration of Tris-HCl is 50 mM, the final concentration of NTP (UTP is replaced by m1ψ) is 1-5 mM, the concentration of pyrophosphatase (Near Shore Protein, GMP-M036-01A) is 0.02 U / μL, the concentration of RNase inhibitor (Near Shore Protein, GMP-E125-M001) is 1 U / μL, and the reaction is carried out at 37 °C for 0.5-12 h; Capping reaction: The total reaction volume was 100 μL, and the reaction system included 10 μL transcription reaction product, MgCl 2 The final concentration is 1-8 mM, the final concentration of Tris-HCl is 50 mM, the final concentration of SAM is 0.5 mM, the final concentration of GTP is 1-5 mM, the concentration of 2-O-methyltransferase (nearshore protein, GMP-M072-M001) is 50-250 U / μL, and the final concentration of vaccinia capping enzyme (nearshore protein, GMP-M062-M001) is 50-1000 U / μL. Incubate at 37 ℃ for 1-2 h.

[0044] mRNA purification: Use 0.5-1 μg of magnetic beads per μl of capped product for purification.

[0045] When preparing RNA, the above reaction system can be linearly amplified according to the requirements of mRNA.

[0046] LNP-mRNA encapsulation: In this embodiment, the molar percentage of ionizable cationic lipid: neutral auxiliary phospholipid: cholesterol and dexamethasone mixture: PEG-lipid is 40:12.6:46.2:1.2, and the total lipid mixture concentration is 12 mM (10-16mM), wherein the molar ratio of cholesterol to dexamethasone is 9:1 (Table 1). mRNA is diluted with sodium citrate buffer at pH 4.0 to a final RNA concentration of 110 ng / μ (50-150 ng / μ); during LNP preparation, the N / P (molar ratio of nitrogen content in the main lipid to phosphorus content in the nucleic acid) ratio is 6-10, preferably 6, and the total flow rate is 4-16 ml / min, preferably 12 ml / min.

[0047] The prepared LNP-mRNA is diluted 5-10 times (preferably 5 times) with sodium citrate (37.5 mM) at pH 4.0, then purified and concentrated to the original volume by a tangential flow filtration system, and then replaced with a freezing solution (pH 7.4-7.8) containing 10 mM Tris and 10% sucrose for 5-10 times the volume, and finally concentrated to 1 / 4 of the original volume; the purified LNP-mRNA is collected and aliquoted and frozen at -80°C; LNP-mRNA detection items include: detection of capping efficiency (>95%) and tailing distribution by mass spectrometry, detection of mRNA integrity (>90%) by capillary electrophoresis, detection of encapsulation efficiency (>90%) and mRNA content by RiboGreen staining, and detection of particle size (80-150 nm), PDI (<0.15) and surface potential (zeta potential, -2 to -10 mV) by nanoparticle size analyzer. As shown in Table 2, the prepared LNP-mRNA encapsulation efficiency is greater than 90%, the particle size is less than 150 nm, the PDI is less than 0.15, and the Zeta potential is between -10 mV and 0mV (Table 2).

[0048] Table 1 LNP formulation

[0049] Table 2 LNP-mRNA quality

[0050] Table 3 Sequence of CBD-TM mRNA

[0051] Example 2 Comparison of different CBD-TM mRNA expressions and functional verification Different CBD-TM LNP-mRNAs were used to transfect monocyte-derived macrophages, and then flow cytometry was used to detect the membrane expression of CBD-TM fusion proteins with different signal peptides ( Figure 2 A), and the ability of macrophages expressing CBD-TM with different signal peptides to bind to collagen ( Figure 2 B).

[0052] The source of macrophages can be autologous peripheral blood, specifically induced from autologous peripheral blood mononuclear cells. The preparation of macrophages can be carried out by methods known in the art. For example, PBMCs are first prepared by Ficoll density gradient centrifugation, and then the PBMCs are treated to separate mononuclear cells, and then the separated mononuclear cells are differentiated into macrophages.

[0053] Among them, the operation of preparing PBMC by Ficoll density gradient centrifugation can be: transferring a certain volume of Ficoll to a centrifuge tube; then covering the whole blood on the Ficoll, and centrifuging the centrifuge tube at room temperature according to the standard of 200g centrifugation for 45 minutes to obtain red blood cell sediment, Ficoll layer, white layer containing PBMC and plasma layer; extracting the white layer containing PBMC. PBMC contains monocytes and lymphocytes, so PBMC can be processed to separate monocytes. In this embodiment, monocytes are separated from lymphocytes by magnetic bead separation or centrifugal countercurrent elutriation to enrich monocytes.

[0054] Cultivation of macrophages: The monocytes isolated in this example (PBMCs can also be directly cultured) are cultured in a differentiation medium containing macrophage colony stimulating factor (M-CSF) to induce the culture to obtain macrophages. The culture conditions can be any suitable for culturing macrophages and are not particularly limited in this example. For example, the density of macrophages inoculated in the culture medium can be 5×10 5 cells / mL (can also be expressed as 5E+5 cells / mL in the specific experimental process). The culture temperature can be 37°C. The culture time can be appropriately set according to the growth state of the macrophages, for example, the culture time can be set to 5 to 8 days. The culture environment can be 5% CO 2 cultivation environment.

[0055] In this example, mononuclear cells were obtained by centrifugal countercurrent elutriation, and 1×10 6 Macrophages were obtained by culturing and differentiating at a cell concentration of 1×10 61 μg (mRNA mass) of CBD-TM LNP-mRNA was added to each macrophage, and the cells were collected after 6 h of culture, centrifuged at 300 g for 10 min, and replaced with fresh Texmacs medium for overnight culture. The expression of CBD-TM on the cell surface was detected by flow cytometry ( Figure 2 A) It was found that the constructed fusion protein can be successfully expressed on the surface of macrophages. The expression of CBD-TM mediated by different signal peptides is different, among which CBD-TM with IL-11-derived signal peptide fused to the N-terminus has a higher expression percentage. Macrophages were incubated with fluorescently labeled collagen, and then flow cytometry was used to detect that the expression of CBD-TM can significantly enhance the binding ability of macrophages to collagen ( Figure 2 B).

[0056] In this embodiment, the membrane-expressed collagen-targeted fusion protein mRNA prepared in Example 1 can also be directly introduced into macrophages by electroporation to obtain modified engineered macrophages. The conditions for electroporation can be: voltage of 250-300V, electroporation time of 8-12ms, and mRNA amount of 3-8μg / 106 cells. In actual operation, efficient and smooth electroporation can be achieved within the range of electroporation conditions.

[0057] Example 3 Phenotypic Analysis of CBD-TM mRNA-Engineering Macrophages According to the method of Example 2, CBD-TM fusion protein LNP-mRNA (SEQ ID NO: 25) with IL-11 signal peptide was transfected into engineered macrophages. After 1 day, cells in the experimental groups transfected with LNP empty control and mRNA were collected. Flow cytometry analysis showed that transfection with CBD-TM LNP-mRNA did not reduce the viability of macrophages ( Figure 3 A); Flow cytometry analysis of macrophage M1 polarization marker and M2 polarization marker CD206 ( Figure 3 B), the results showed that transfection with CBD-TM LNP-mRNA did not change the polarization state of macrophages; in addition, the inflammatory cytokine IL-6 and the anti-inflammatory cytokine IL-10 in the macrophage culture supernatant were analyzed by ELISA, and the results showed that the secretion levels of both did not change after transfection with CBD-TM LNP-mRNA ( Figure 3 C).

[0058] Example 4 CBD-TM engineered macrophages have stronger anti-pulmonary fibrosis effects Bleomycin-induced pulmonary fibrosis is currently the most widely used model of pulmonary fibrosis. After administration via bronchial aerosol, it can be concentrated in the lung tissue, leading to a large amount of oxidative stress, alveolar epithelial cell death, fibroblast proliferation, and finally immune cell infiltration. Chronic release of pro-inflammatory and pro-fibrotic molecules by immune cells and fibroblasts can lead to pneumonia and fibrosis. The bleomycin-induced pulmonary fibrosis model is often used to evaluate the anti-fibrotic effect of drugs in vivo.

[0059] After CBD-TM LNP-mRNA engineered macrophages were injected into the tail vein of mice, the reversal of pulmonary fibrosis in mice was achieved. 6 After 6 days of differentiation, macrophages were collected and prepared: unmodified macrophages (directly frozen) and CBD-TM LNP-mRNA (SEQ ID NO: 25) transfected macrophages (1×10 6 1.0 μg of mRNA was added to each macrophage. Cells were collected 6 h after transfection, centrifuged at 300 g for 10 min, and then 1×10 7 The concentration of 100 / mL was programmed to cool and freeze, and then stored in liquid nitrogen vapor phase for later use.

[0060] Six-week-old NOD-SCID mice were treated with 50 mg / kg bleomycin via pulmonary aerosol to establish a pulmonary fibrosis model. One week later, the above two groups of macrophages (1×10 6 cells / mouse), treated once a week for a total of three times. Two weeks after the last treatment, the mice were euthanized and the mouse lung tissue samples were collected. Genomic DNA (gDNA) was extracted from the lung tissue and qPCR analysis of human gDNA (primers are shown in Table 4). The results showed that there were more macrophages in the lung tissue of mice treated with macrophages expressing CBD-TM ( Figure 4 A); Lung tissue was taken to detect the level of hydroxyproline, one of the main components of collagen. It can be seen that compared with unmodified macrophages, CBD-TM-modified macrophages have lower hydroxyproline levels ( Figure 4 B); qPCR detection of fibrosis-related gene expression levels found that the expression levels of collagen-related genes Col1a1 and Col3a1 in lung tissues after treatment with CBD-TM-expressing macrophages were lower ( Figure 4 C); Masson staining and statistical analysis of lung tissue collagen showed that the extracellular matrix collagen content of lung tissue after treatment with macrophages expressing CBD-TM was lower ( Figure 4 D, 4E). Overall, CBD-TM mRNA-modified macrophages have better therapeutic effects on pulmonary fibrosis than unmodified macrophages.

[0061] Table 4 qPCR primers

[0062] Example 5 CBD-TM engineered macrophages have stronger anti-liver fibrosis effect The macrophages differentiated from monocytes were divided into 1×10 6 After 6 days of differentiation, macrophages were collected to prepare: unmodified macrophages (directly frozen) and macrophages transfected with CBD-TM LNP-mRNA (SEQ ID NO: 25) (1×10 6 1.0 μg of mRNA was added to each macrophage. Cells were collected 6 h after transfection, centrifuged at 300 g for 10 min, and then 1×10 7 The concentration of 100 / mL was programmed to cool and freeze, and then stored in liquid nitrogen vapor phase for later use.

[0063] CCl 4 Inducing liver fibrosis is a commonly used liver fibrosis model. In this embodiment, 4-5 week old mice were intraperitoneally injected with CCl twice a week. 4 (0.5 mL / kg), and after 6 weeks of modeling, macrophage therapy (1×10 6 cells / mouse), treated once a week for a total of three times. Two weeks after the last treatment, the mice were euthanized and liver tissue samples were collected. qPCR detection of fibrosis-related gene expression levels found that the expression levels of collagen-related genes Mmp2 and Timp1 in liver tissues treated with CBD-TM-expressing macrophages were lower ( Figure 5 A), primers are shown in Table 4 above; Sirius red staining and statistical analysis of liver tissue collagen showed that the extracellular matrix collagen content of liver tissue after treatment with macrophages expressing CBD-TM was lower ( Figure 5 B). The results of α-SMA staining and statistical analysis of liver tissue showed that after treatment with macrophages expressing CBD-TM, the expression area of ​​myofibroblast activation marker protein α-SMA was reduced more ( Figure 5 C). Overall, CBD-TM mRNA-modified macrophages have a better therapeutic effect on liver fibrosis than unmodified macrophages.

[0064] Example 6 CBD-TM combined with IL-10 engineered macrophages has a stronger anti-pulmonary fibrosis effect The macrophages differentiated from monocytes were divided into 1×10 6The cells were cultured at a concentration of 1×10 / mL. After 6 days of differentiation, the macrophages were collected and prepared as follows: unmodified macrophages, macrophages modified with CBD-TM LNP-mRNA, and macrophages modified with CBD-TM (SEQ ID NO: 25) and IL-10 LNP-mRNA (RNA sequence see Table 3, LNP formula see Table 1) (1×10 6 1.0 μg of mRNA was added to each macrophage. Cells were collected 6 h after transfection, centrifuged at 300 g for 10 min, and then 1×10 7 In addition, the culture supernatant of some cells was collected after overnight culture, and the expression of IL-10 was detected by ELISA. Compared with other groups, the secretion of IL-10 in the culture supernatant of macrophages expressing IL-10 mRNA (and CBD-TM) was significantly increased ( Figure 6 A).

[0065] Six-week-old NOD-SCID mice were treated with 50 mg / kg bleomycin via pulmonary aerosol to establish a pulmonary fibrosis model. After one week, they were randomly divided into three groups: untreated control group, CBD-TM-modified macrophages, and CBD-TM and IL-10 LNP-mRNA-modified macrophages. Three groups of macrophages (1×10 6 The mice were treated once a week for three times. Two weeks after the last treatment, the mice were euthanized and lung tissue samples were collected. The lung tissue was taken to detect the level of hydroxyproline, one of the main components of collagen. The results showed that compared with macrophages expressing CBD-TM, the hydroxyproline level in the lung tissue of macrophages expressing both CBD-TM and IL-10 LNP-mRNA was lower after treatment ( Figure 6 B); qPCR detection of fibrosis-related gene expression levels found that after treatment with macrophages expressing both CBD-TM and IL-10 LNP-mRNA, the expression levels of collagen-related genes Col1a1 and Col3a1 in lung tissue were lower ( Figure 6 C), primers are shown in Table 4 above; Masson staining and statistical analysis of lung tissue collagen showed that after treatment with macrophages expressing both CBD-TM and IL-10 LNP-mRNA, the extracellular matrix collagen content of lung tissue was lower ( Figure 6 D). Overall, macrophages transformed with both CBD-TM and IL-10 LNP-mRNA have a better therapeutic effect on pulmonary fibrosis than macrophages transformed with CBD-TM alone.

[0066] Example 7 CBD-TM combined with IL-4 engineered macrophages has a stronger anti-pulmonary fibrosis effect In the embodiment, the macrophages differentiated from monocytes were divided into 1×10 6 The cells were cultured at a concentration of 1×10 cells / mL. After 6 days of differentiation, the macrophages were collected and prepared as follows: unmodified macrophages, macrophages modified with CBD-TM LNP-mRNA, and macrophages modified with CBD-TM (SEQ ID NO: 25) and IL-4 LNP-mRNA (RNA sequence see Table 3, LNP formula see Table 1) (1×10 6 1.0 μg of mRNA was added to each macrophage. Cells were collected 6 h after transfection, centrifuged at 300 g for 10 min, and then 1×10 7 In addition, the culture supernatant of some cells was collected after overnight culture, and the expression of IL-4 was detected by ELISA. Compared with other groups, the secretion of IL-4 in the culture supernatant of macrophages expressing IL-4 mRNA (and CBD-TM) was significantly increased ( Figure 7 A).

[0067] Six-week-old NOD-SCID mice were treated with 50 mg / kg bleomycin via pulmonary aerosol to establish a pulmonary fibrosis model. After one week, they were randomly divided into three groups: untreated control group, CBD-TM-modified macrophages, and CBD-TM and IL-4 LNP-mRNA-modified macrophages. Three groups of macrophages (1×10 6 The mice were treated once a week for three times. Two weeks after the last treatment, the mice were euthanized and lung tissue samples were collected. The lung tissue was taken to detect the level of hydroxyproline, one of the main components of collagen. The results showed that compared with macrophages expressing CBD-TM, the hydroxyproline level in the lung tissue of macrophages expressing both CBD-TM and IL-4LNP-mRNA was lower after treatment ( Figure 7 B); qPCR detection of fibrosis-related gene expression levels found that after treatment with macrophages expressing both CBD-TM and IL-4 LNP-mRNA, the expression levels of collagen-related genes Timp1 and Col1a2 in lung tissue were lower ( Figure 7 C), primers are shown in Table 4 above; the results of lung tissue α-SMA staining and statistical analysis showed that after treatment with macrophages expressing both CBD-TM and IL-4 LNP-mRNA, the expression area of ​​myofibroblast activation marker protein α-SMA was less ( Figure 7D). Overall, macrophages transformed with both CBD-TM and IL-4 LNP-mRNA have a better therapeutic effect on pulmonary fibrosis than macrophages transformed with CBD-TM alone.

[0068] Example 8 CBD-TM combined with IL-13 engineered macrophages has a stronger anti-pulmonary fibrosis effect In the embodiment, the macrophages differentiated from monocytes were divided into 1×10 6 The cells were cultured at a concentration of 1×10 cells / mL. After 6 days of differentiation, the macrophages were collected and prepared as follows: unmodified macrophages, macrophages modified with CBD-TM LNP-mRNA, and macrophages modified with CBD-TM (SEQ ID NO: 25) and IL-13 LNP-mRNA (RNA sequence see Table 3, LNP formula see Table 1) (1×10 6 1.0 μg of mRNA was added to each macrophage. Cells were collected 6 h after transfection, centrifuged at 300 g for 10 min, and then 1×10 7 The cells were cryopreserved at a concentration of 100 / mL and then stored in liquid nitrogen vapor phase for future use. In addition, the culture supernatant was collected after overnight culture of some cells. The expression of IL-13 was detected by ELISA. Compared with other groups, the secretion of IL-13 in the culture supernatant of macrophages expressing IL-13 mRNA (and CBD-TM) was significantly increased ( Figure 8 A).

[0069] Six-week-old NOD-SCID mice were treated with 50 mg / kg bleomycin via pulmonary aerosol to establish a pulmonary fibrosis model. After one week, they were randomly divided into three groups: untreated control group, CBD-TM-modified macrophages, and CBD-TM and IL-13 LNP-mRNA-modified macrophages. Three groups of macrophages (1×10 6 The mice were treated once a week for three times. Two weeks after the last treatment, the mice were euthanized and lung tissue samples were collected. The lung tissue was taken to detect the level of hydroxyproline, one of the main components of collagen. The results showed that compared with macrophages expressing CBD-TM, the hydroxyproline level in the lung tissue of macrophages expressing both CBD-TM and IL-13 LNP-mRNA was lower after treatment ( Figure 8 B); qPCR detection of fibrosis-related gene expression levels found that after treatment with macrophages expressing both CBD-TM and IL-13 LNP-mRNA, the expression levels of collagen-related genes Col1a1 and Col3a1 in lung tissue were lower ( Figure 8C), primers are shown in Table 4 above; Masson staining and statistical analysis of lung tissue collagen showed that after treatment with macrophages expressing both CBD-TM and IL-13 LNP-mRNA, the extracellular matrix collagen content of lung tissue was lower ( Figure 8 D). Overall, macrophages transformed with both CBD-TM and IL-13 LNP-mRNA have a better therapeutic effect on pulmonary fibrosis than macrophages transformed with CBD-TM alone.

Claims

1. A fusion protein, characterized in that The fusion protein comprises the VWF protein A3 domain and the PDGFR transmembrane domain. 2 . The fusion protein according to claim 1 , wherein the VWF protein A3 domain and the transmembrane domain of PDGFR are connected by a flexible linker. The fusion protein according to claim 1 , further comprising a signal peptide at the N-terminus. The fusion protein according to claim 3 , wherein the signal peptide is derived from IL-2, IL-6, IL-11 or IL-12B. 5 . The fusion protein according to claim 1 , wherein the A3 domain of the VWF protein comprises the amino acid sequence shown in SEQ ID NO: 1, and the PDGFR transmembrane domain comprises the amino acid sequence shown in SEQ ID NO:

3. The fusion protein according to claim 5 , comprising the amino acid sequence shown in any one of SEQ ID NOs: 8-11.

7. A nucleic acid, characterized in that The nucleic acid encodes the fusion protein according to any one of claims 1 to 6; wherein the nucleic acid is DNA or mRNA. The nucleic acid according to claim 7 , comprising the nucleotide sequence shown in any one of SEQ ID NOs: 19-22. 9 . The nucleic acid according to claim 8 , comprising the nucleotide sequence shown in any one of SEQ ID NOs: 23-26.

10. A nanolipid particle-nucleic acid complex, characterized in that: The complex comprises the nucleic acid according to any one of claims 7 to 9 and a lipid nanoparticle (LNP), wherein the nucleic acid is mRNA.

11. The composite according to claim 10, wherein The nano lipid particle comprises ionizable cationic lipid, neutral auxiliary phospholipid, sterol and PEG lipid, wherein the sterol is a mixture of cholesterol and dexamethasone.

12. The composite according to claim 11, wherein The molar ratio of cholesterol to dexamethasone in the cholesterol and dexamethasone mixture is 9:

1.

13. An engineered macrophage, characterized in that The macrophage contains the fusion protein according to any one of claims 1 to 6 or the nucleic acid according to any one of claims 7 to 9.

14. The macrophage of claim 13, further comprising a nucleic acid encoding one or more of IL-10, IL-4 and IL-13. 15 . The macrophage according to claim 14 , wherein the IL-10, IL-4 and IL-13 comprise the amino acid sequences shown in SEQ ID NOs: 27-29, respectively.

16. The macrophage according to claim 15, wherein the nucleic acids encoding IL-10, IL-4 and IL-13 comprise the polynucleotide sequences shown in SEQ ID NOs: 30-32, respectively.

17. A method for preparing engineered macrophages, characterized in that: The method comprises introducing the nucleic acid according to any one of claims 7 to 9 or the complex according to any one of claims 10 to 12 into macrophages.

18. The method according to claim 17, comprising introducing the nucleic acid according to any one of claims 7 to 9 or the nanolipid particle-nucleic acid complex according to any one of claims 10 to 12 into macrophages simultaneously or sequentially with LNPs coated with one or more nucleic acids selected from one or more of encoding IL-10, IL-4 and IL-13.

19. The method according to claim 17 or 18, wherein when the nucleic acid is introduced, the introduction is carried out by electroporation; and when the complex is introduced, the introduction is carried out by LNP delivery.

20. The method according to claim 19, wherein the IL-10, IL-4 and IL-13 comprise the amino acid sequences shown in SEQ ID NOs: 27-29, respectively.

21. The method according to claim 20, wherein the nucleic acids encoding IL-10, IL-4 and IL-13 comprise the polynucleotide sequences shown in SEQ ID NOs: 30-32, respectively.

22. The engineered macrophage prepared according to the method according to any one of claims 17 to 21.

23. Use of the engineered macrophage according to any one of claims 13 to 16 or claim 22 in the preparation of a drug for preventing and / or treating fibrotic diseases.

24. Use of the fusion protein according to any one of claims 1 to 6, the nucleic acid according to any one of claims 7 to 9, or the complex according to any one of claims 10 to 12 in the preparation of a drug for preventing and / or treating fibrotic diseases.

25. The use according to claim 23 or 24, wherein the fibrotic disease comprises pulmonary fibrosis, liver fibrosis, pneumoconiosis, cystic fibrosis, cirrhosis, myocardial fibrosis, renal interstitial fibrosis, scleroderma, keloid, pancreatic fibrosis, retroperitoneal fibrosis, systemic sclerosis and / or myelofibrosis.

26. The use according to claim 25, wherein the pulmonary fibrosis comprises idiopathic pulmonary fibrosis and radiation-induced pulmonary fibrosis.

Images