Preparation of Engineered Macrophages and Their Application in Anti-Fibrosis

Abstract

The present invention discloses the preparation of engineered macrophages and their application in anti-fibrosis, belonging to the technical field of biomedicine. The engineered macrophages contain nucleic acids encoding a fusion protein, and the fusion protein contains the A3 domain of the VWF protein and the transmembrane domain of PDGFR. The engineered macrophages further include nucleic acids encoding one or more of IL-10, IL-4, and IL-13.

Description

Technical Field

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

[0002] Fibrosis is a pathological process of tissue or organ repair after injury. 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, resulting in the destruction of tissue structure and function.

[0003] Multiple factors can lead to the occurrence and development of fibrosis, and chronic inflammation is one of the main inducing factors of fibrosis. The inflammatory environment recruits inflammatory cells (such as macrophages, T cells) and promotes the release of pro-inflammatory factors (such as TGF-β, IL-1β, TNF-α), stimulates the activation of fibroblasts and promotes the production and deposition of ECM; TGF-β (transforming growth factor-β) and its downstream IL11 signaling pathway are key regulatory factors of fibrosis, which can promote the transformation of fibroblasts into myofibroblasts; PDGF (platelet-derived growth factor) can promote the proliferation and migration of fibroblasts; the Wnt / β-catenin pathway is also involved in the occurrence and progression of fibrosis. In addition, tissue damage caused by the generation and increase of reactive oxygen species (ROS), and profibrotic factors released by cellular senescence and apoptosis will also exacerbate the fibrosis process. 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 in the scar tissue generated during the process of lung tissue damage and repair. This thickened and stiff tissue makes it more difficult for the lungs to function normally. Clinically, patients mainly show symptoms such as dyspnea, dry cough, fatigue, and easy tiredness. Medical imaging examinations show that there are a large amount of collagen fiber depositions, a large number of alveolar losses, and changes in the normal lung tissue structure in the lungs of patients with pulmonary fibrosis. The gas exchange function of patients gradually loses, and they eventually die of respiratory failure. There are various factors causing pulmonary fibrosis damage, including long-term exposure to certain toxins, certain medical conditions, radiotherapy, and certain drugs, etc. But in most cases, doctors cannot identify the root cause of the problem, so it is called idiopathic pulmonary fibrosis (IPF). Patients with IPF account for 30%-50% of all patients with pulmonary fibrosis. Clinically, anti-inflammatory, antioxidant, and fibrosis-inhibiting treatment methods are mainly used, but these methods often have limited efficacy and certain side effects. The only current treatment drugs are pirfenidone and nintedanib. Non-drug treatment options include oxygen therapy, pulmonary rehabilitation therapy, and lung transplantation, etc. (MSD).

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

[0006] In recent years, strategies based on RNA to regulate gene expression and modify cell functions have shown great potential in the field of disease treatment. The key to engineering macrophages for anti-fibrotic treatment lies in how to regulate macrophage functions and enhance and maintain their anti-fibrotic capabilities. 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 engineering macrophages for anti-fibrotic treatment. 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 abilities of macrophages 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-fibrotic treatment mainly involves transplanting macrophages into patients via intravenous infusion. After entering the human body, macrophages eventually mainly colonize in the liver through blood circulation. 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 a direction for macrophage modification. More and more studies have shown that pathological state-induced blood vessel remodeling is also an important feature of organ fibrosis. In fibrotic tissues and organs, the ECM is often increased near the remodeled blood vessels. There is often angiogenesis and remodeling in the IPF lung tissue, and the occurrence site is basically the same as the fibrotic site. Morphological studies have confirmed the existence of blood vessel remodeling in IPF and proved that this process of blood vessel remodeling is active in the area adjacent to the fibroblast foci, while the blood vessel density in the fibroblast foci is reduced. The process of pathological angiogenesis is also included in the occurrence and development of pulmonary fibrosis.

[0008] The selection of existing engineering macrophage modification targets has certain limitations. In terms of enhancing the enrichment of macrophages in the ECM region, the targets selected by existing modification schemes are mainly integrins, Dictyostelium 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 heterodimeric structure and protecting it from premature clearance from the plasma. There has been no anti-fibrotic treatment strategy developed based on the collagen-binding effect of VWF. Summary of the Invention

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

[0011] In the first aspect of the present invention, there is provided a fusion protein, which comprises the A3 domain of VWF protein and the transmembrane domain of PDGFR.

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

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

[0014] In the second aspect of the present invention, there is provided a nucleic acid encoding the fusion protein of the first aspect of the present invention; wherein, the nucleic acid is DNA or mRNA.

[0015] In the third aspect of the present invention, there is provided a nano-lipid particle-nucleic acid complex, which comprises the nucleic acid of the first aspect of the present invention or the nano-lipid particle (LNP) of the second aspect, wherein the nucleic acid is mRNA.

[0016] In some embodiments, the nano-lipid 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. The molar ratio of cholesterol to dexamethasone in the mixture of cholesterol and dexamethasone is 9:1.

[0017] In the fourth aspect of the present invention, there is provided an engineered macrophage containing the fusion protein of the first aspect of the present invention or the nucleic acid of the second aspect.

[0018] In some embodiments, the engineered macrophages further comprise 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 nano-lipid 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 nano-lipid particle-nucleic acid complex provided in the third aspect of the present invention, and one or more LNPs coated with one or more nucleic acids encoding one or more of IL-10, IL-4, and IL-13 into macrophages simultaneously or successively.

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

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

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

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

[0025] In some embodiments, the fibrotic diseases include pulmonary fibrosis, liver fibrosis, silicosis, cystic fibrosis, liver cirrhosis, myocardial fibrosis, renal interstitial fibrosis, scleroderma, keloid, pancreatic fibrosis, retroperitoneal fibrosis, systemic sclerosis, and / or myelofibrosis. Among them, the pulmonary fibrosis includes idiopathic pulmonary fibrosis and radiation-induced pulmonary fibrosis.

[0026] The advantages of the present invention over the prior art are as follows:

[0027] 1) For the first time, a fusion protein containing a signal peptide, the VWF protein A3 domain (CBD), and the PDGFR transmembrane domain (TM) was constructed, and the expression of this fusion protein in macrophages was achieved by encapsulating the mRNA-LNP expressing the fusion protein with inflammation-inhibiting LNPs.

[0028] 2) The VWF A3 domain has the ability to bind collagen. After being synthesized in macrophages, the VWF A3 domain is secreted extracellularly to exert its function. Under the action of the guiding signal derived from IL11, the fusion protein (CBD-TM) can be expressed on the surface of macrophage membranes. Macrophages modified with CBD-TM mRNA have a stronger ability to bind to the ECM and can migrate more to fibrotic tissues after being infused back into the body.

[0029] 3) On the basis of the modification with CBD-TM mRNA, anti-inflammatory factors IL-10, IL-4 or IL-13 mRNA are further introduced. The fibrotic region-targeted anti-inflammatory macrophages obtained have a further enhanced anti-fibrotic effect. Brief Description of the Drawings

[0030] By reading the detailed description of the non-limiting embodiments with reference to the following drawings, other features, objectives and advantages of the present invention will become more prominent:

[0031] 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 4 kinds of mRNAs prepared.

[0032] Figure 2 . Comparison of different CBD-TM mRNA expressions and function 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.

[0033] Figure 3 . CBD-TM LNP-mRNA does not affect macrophage phenotype and cytokine secretion. A, Flow cytometry analysis of the viability of macrophages transfected with empty LNP control and CBD-TM (IL11 signal peptide) LNP-mRNA for 1 day; B, Flow cytometry analysis of the expression percentages of macrophage surface markers CD80 (M1) and CD206 (M2) after transfection with empty LNP control and CBD-TM (IL11 signal peptide) LNP-mRNA for 1 day; C, Flow cytometry analysis of the levels of cytokines IL6 and IL10 in the culture supernatant of macrophages after transfection with empty LNP control and CBD-TM (IL11 signal peptide) LNP-mRNA for 1 day.

[0034] Figure 4. CBD-TM engineered macrophages have a stronger anti-pulmonary fibrosis effect. 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 the expression levels of Col1a1 and Col3a1 mRNA in lung tissues of different treatment groups; D and E, Masson staining results and statistical analysis results of lung tissues in different treatment groups.

[0035] Figure 5 . CBD-TM engineered macrophages have a stronger anti-hepatic fibrosis effect. 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 in different treatment groups; C, α-SMA staining results and statistical analysis results of liver tissue sections in different treatment groups.

[0036] Figure 6 . CBD-TM combined with IL-10 engineered macrophages have a stronger anti-pulmonary fibrosis effect. A, ELISA analysis of the cytokine IL-10 level in the culture supernatant of different macrophages; B, hydroxyproline levels in lung tissues of different treatment groups; C, qPCR analysis of the expression levels of Col1a1 and Col3a1 mRNA in lung tissues of different treatment groups; D, Masson staining and statistical analysis results of lung tissues in different treatment groups.

[0037] Figure 7 . CBD-TM combined with IL-4 engineered macrophages have a stronger anti-pulmonary fibrosis effect. A, ELISA analysis of the cytokine IL-4 level in the culture supernatant of different macrophages; B, hydroxyproline levels in lung tissues of different treatment groups; C, qPCR analysis of the expression levels of Timp1 and Col1a2 mRNA in lung tissues of different treatment groups; D, α-SMA staining and statistical analysis results of lung tissues in different treatment groups.

[0038] Figure 8 . CBD-TM combined with IL-13 engineered macrophages have a stronger anti-pulmonary fibrosis effect. A, ELISA analysis of the cytokine IL-13 level in the culture supernatant of different macrophages; B, hydroxyproline levels in lung tissues of different treatment groups; C, qPCR analysis of the expression levels of Col1a1 and Col3a1 mRNA in lung tissues of different treatment groups; D, Masson staining and statistical analysis results of lung tissues in different treatment groups. Detailed implementation methods

[0039] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the following will clearly and completely describe the technical solutions of the embodiments of the present invention in conjunction with the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the described embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the scope of protection of the present invention.

[0040] Unless otherwise defined, the technical terms or scientific terms used herein shall have the ordinary meaning as understood by those of ordinary skill in the field to which the present invention pertains.

[0041] Example 1 Preparation of CBD-TM LNP-mRNA

[0042] In this example, membrane-expressed collagen-targeted fusion protein mRNAs with different guiding signals were 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) through a GS linker (SEQ ID NO: 2) ( Figure 1 A). The N-terminal signal peptides are respectively 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 signal peptide-VWF-A3 domain-GS linker-PDGFR transmembrane domain with IL-2, IL-6, IL-11, and IL-12B signal peptides fused at the N-terminus are shown in SEQ ID NOs: 8-11 respectively.

[0043] The one-tube multi-step method was used for mRNA preparation, that is, linearization, transcription, and capping reaction were carried out in the same tube. After the reaction was completed, mRNA was purified by the magnetic bead method. Agarose gel electrophoresis showed that the length of the prepared mRNA was close to the theoretical size, and the band was single without degradation ( Figure 1 B), and the sequence information of the mRNA is shown in Table 3. Subsequently, an anti-inflammatory LNP lipid combination (Table 1) was used to prepare LNP-mRNA by microfluidic mixing. The detection results of the encapsulation efficiency, particle size, PDI, and zeta charge of LNP-mRNA are shown in Table 2.

[0044] The specific implementation scheme is as follows:

[0045] Plasmid construction: The pUC57-Kan vector (Sangon Biotech, B522201-0100) was used as the backbone vector, which mainly includes a DNA replication origin, a kanamycin resistance gene and its promoter. The 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 sites were introduced through the multiple cloning site. The constructed vector was verified by sequencing, and the proportion of supercoiled plasmid prepared should be greater than 60%.

[0046] The 5' UTR sequence consists of the HBB (hemoglobin) mRNA 5' UTR 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 3' UTR sequence derived from histone, and the length of polyA should be greater than 100.

[0047] Plasmid linearization: The reaction volume was 5 μL, the concentration of the restriction enzyme (BspQI) was 1-10 U / μg plasmid, and the digestion time at 50 °C was 0.5-3 h;

[0048] In vitro transcription: The total reaction volume was 10 μL. The reaction system included 5 μL of the digested mixture, the concentration of T7 transcriptase (Novoprotein, GMP-E121-HC-U100) was 100-500 U / μL, the final concentration of MgCl2 was 5-40 mM, the final concentration of Tris-HCl was 50 mM, the final concentration of NTP (using m1ψ to replace UTP) was 1-5 mM, the concentration of pyrophosphatase (Novoprotein, GMP-M036-01A) was 0.02 U / μL, the concentration of RNase inhibitor (Novoprotein, GMP-E125-M001) was 1 U / μL, and the reaction was carried out at 37 °C for 0.5-12 h;

[0049] Capping reaction: The total reaction volume was 100 μL. The reaction system included 10 μL of the transcription reaction product, the final concentration of MgCl2 was 1-8 mM, the final concentration of Tris-HCl was 50 mM, the final concentration of SAM was 0.5 mM, the final concentration of GTP was 1-5 mM, the concentration of 2-O-methyltransferase (Novoprotein, GMP-M072-M001) was 50-250 U / μL, the final concentration of vaccinia capping enzyme (Novoprotein, GMP-M062-M001) was 50-1000 U / μL, and the reaction was carried out at 37 °C for 1-2 h.

[0050] mRNA Purification: The capped product is purified using 0.5 - 1 μg of magnetic beads per microliter.

[0051] When preparing RNA, the above reaction system can be linearly amplified according to the demand for mRNA.

[0052] LNP-mRNA Encapsulation: In this example, 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 - 16 mM), where the molar ratio of cholesterol to dexamethasone is 9 ∶ 1 (Table 1). The mRNA is diluted with sodium citrate buffer at pH 4.0 to a final RNA concentration of 110 ng / μ (50 - 150 ng / μ); when preparing LNP, the N / P (molar ratio of nitrogen content in the main lipid to phosphorus content in nucleic acid) ratio is 6 - 10, preferably 6, and the total flow rate is 4 - 16 ml / min, preferably 12 ml / min.

[0053] 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 through a tangential flow filtration system, and then the volume is exchanged 5 - 10 times with a freezing solution containing 10 mM Tris and 10% sucrose (pH 7.4 - 7.8), and finally concentrated to 1 / 4 of the original volume; the purified LNP-mRNA is collected and aliquoted and stored frozen at -80 °C;

[0054] The detection items of LNP-mRNA include: detecting the capping efficiency (>95%) and poly(A) tail distribution by mass spectrometry, detecting the mRNA integrity (>90%) by capillary electrophoresis, detecting the encapsulation rate (>90%) and mRNA content by RiboGreen staining, and detecting the particle size (80 - 150 nm), PDI (<0.15), and surface potential (zeta potential, -2 to -10 mV) using a nanoparticle size analyzer. As shown in Table 2, the encapsulation rate of the prepared LNP-mRNA 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 0 mV (Table 2).

[0055] Table 1 LNP Formulation

[0056]

[0057] Table 2 LNP-mRNA Quality

[0058]

[0059] Table 3 Sequences of CBD-TM mRNA

[0060]

[0061] Example 2 Comparison of Different CBD-TM mRNA Expressions and Function Verification

[0062] Macrophages differentiated from monocytes were transfected with different CBD-TM LNP-mRNAs, and then the membrane expression of CBD-TM fusion proteins with different signal peptides was detected by flow cytometry ( Figure 2 A), and the binding ability of macrophages expressing different signal peptides of CBD-TM to collagen ( Figure 2 B).

[0063] The source of macrophages can be autologous peripheral blood, specifically induced from autologous peripheral blood monocytes. The preparation of macrophages can adopt methods known in the art. For example, PBMC is first prepared by Ficoll density gradient centrifugation, then PBMC is processed to separate monocytes, and the separated monocytes are differentiated into macrophages.

[0064] Among them, the operation of preparing PBMC by Ficoll density gradient centrifugation can be: transferring a certain volume of Ficoll into a centrifuge tube; then covering whole blood on Ficoll, and centrifuging the centrifuge tube at room temperature according to the standard of centrifuging at 200g for 45 minutes to obtain a red blood cell pellet, a Ficoll layer, a white layer containing PBMC, and a plasma layer; extracting the white layer containing PBMC. PBMC contains monocytes and lymphocytes, so PBMC can be processed to separate monocytes. In this example, monocytes were separated from lymphocytes by magnetic bead separation or countercurrent elutriation centrifugation to enrich monocytes.

[0065] Cultivation of macrophages: The monocytes separated in this example (or PBMC can also be directly cultured) were cultured in a differentiation medium containing macrophage colony-stimulating factor (M-CSF) for induction culture to obtain macrophages. The culture conditions can be any suitable for culturing macrophages, and there is no special limitation in this example. For example, the density of inoculating macrophages in the medium can be 5×10 5 cells / mL (which 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 macrophages. For example, the culture time can be set to 5 days to 8 days. The culture environment can be a 5% CO2 culture environment.

[0066] In this example, monocytes were obtained by countercurrent elutriation centrifugation, at a ratio of 1×10 6Macrophages were obtained by culturing and differentiating at a cell concentration of 6 per mL. 1 μg (mRNA mass) of CBD-TM LNP-mRNA was added to every 1×10 Figure 2 macrophages, and the cells were collected after 6 h of culture. After centrifugation at 300 g for 10 min, the cells were replaced with fresh Texmacs medium and cultured overnight. 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 could be successfully expressed on the surface of macrophages, and there were differences in the expression of CBD-TM mediated by different signal peptides. Among them, CBD-TM with the signal peptide derived from IL-11 fused at the N-terminus had a higher expression percentage. Macrophages were incubated with fluorescently labeled collagen, and then it was detected by flow cytometry that the binding ability of macrophages to collagen was significantly enhanced after the expression of CBD-TM (

[0067] B).

[0068] Example 3 Phenotype analysis of CBD-TM mRNA-engineered macrophages

[0069] According to the method of Example 2, macrophages were engineered by transfecting CBD-TM fusion protein LNP-mRNA (SEQ ID NO: 25) with the IL-11 signal peptide. One day later, the experimental group cells 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 was used to analyze the M1-type polarization marker and M2-type polarization marker CD206 of macrophages ( 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 anti-inflammatory cytokine IL-10 in the culture supernatant of macrophages were analyzed by ELISA. The results showed that the secretion levels of both did not change after transfection with CBD-TM LNP-mRNA ( Figure 3 C).

[0070] Example 4 CBD-TM-engineered macrophages have a stronger anti-pulmonary fibrosis effect

[0071] The bleomycin-induced pulmonary fibrosis lesion is currently the most widely recognized and used pulmonary fibrosis model. After transbronchial aerosol administration, it can concentrate in lung tissue, leading to a large amount of oxidative stress, alveolar epithelial cell death, fibroblast proliferation, and finally immune cell infiltration. The chronic release of pro-inflammatory and profibrotic molecules by immune cells and fibroblasts can cause pneumonia and fibrosis. The bleomycin-induced pulmonary fibrosis model is commonly used to evaluate the in vivo antifibrotic effects of drugs.

[0072] After engineering macrophages with CBD-TM LNP-mRNA, they were injected into the tail vein of mice to achieve the reversal treatment of pulmonary fibrosis in mice. Macrophages differentiated from monocytes were cultured at a cell concentration of 1×10 6 cells / mL. After 6 days of differentiation, macrophages were collected and prepared: unmodified macrophages (directly cryopreserved) and macrophages transfected with CBD-TM LNP-mRNA (SEQ ID NO: 25) (1.0 μg of mRNA was added to every 1×10 6 macrophages). Cells were collected 6 h after transfection. After centrifugation at 300 g for 10 min, they were cryopreserved by programmed cooling at a concentration of 1×10 7 cells / mL and stored in the gas phase of liquid nitrogen for later use.

[0073] One week after constructing a pulmonary fibrosis model by treating 6-week-old NOD-SCID mice with 50 mg / Kg of bleomycin via lung aerosolization, the above two groups of macrophages (1×10 6 cells / mouse) were injected into the tail vein. The treatment was given once a week for a total of three times. Two weeks after the last treatment, the mice were euthanized and lung tissue samples were collected. Genomic DNA (gDNA) of the lung tissue was extracted and qPCR was performed to analyze human gDNA (primers are shown in Table 4). The results showed that there were more macrophages remaining in the lung tissue of mice treated with macrophages expressing CBD-TM ( Figure 4 A); The level of hydroxyproline, one of the main components of collagen, was detected in the lung tissue. It was found that the macrophages modified with CBD-TM had a lower hydroxyproline level compared to unmodified macrophages ( Figure 4 B); qPCR was used to detect the expression levels of fibrosis-related genes. It was found that the expression levels of collagen-related genes Col1a1 and Col3a1 were lower in the lung tissue treated with macrophages expressing CBD-TM ( Figure 4 C); Masson staining and statistical analysis of lung tissue collagen showed that the extracellular matrix collagen content was less in the lung tissue treated with macrophages expressing CBD-TM ( Figure 4 D, 4E). Overall, macrophages modified with CBD-TM mRNA had a better effect on treating pulmonary fibrosis compared to unmodified macrophages.

[0074] Table 4 qPCR primers

[0075]

[0076] Example 5 CBD-TM engineered macrophages have stronger anti-hepatic fibrosis effect

[0077] Macrophages differentiated from monocytes were cultured at a cell concentration of 1×10 6 cells / mL. After 6 days of differentiation, macrophages were collected to prepare: unmodified macrophages (directly cryopreserved) and macrophages transfected with CBD-TM LNP-mRNA (SEQ ID NO: 25) (1.0 μg of mRNA was added per 1×10 6 macrophages). Cells were collected 6 h after transfection. After centrifugation at 300 g for 10 min, they were cryopreserved by programmed cooling at a concentration of 1×10 7 cells / mL and stored in the gas phase of liquid nitrogen for later use.

[0078] CCl4-induced liver fibrosis lesion is a commonly used liver fibrosis model. In this example, 4-5-week-old mice were intraperitoneally injected with CCl4 (0.5 mL / kg) twice a week for 6 consecutive weeks. After modeling, macrophage treatment was administered via tail vein injection (1×10 6 cells / mouse), 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 was used to detect the expression levels of fibrosis-related genes. It was found that the expression levels of collagen-related genes Mmp2 and Timp1 in the liver tissue after treatment with macrophages expressing CBD-TM were lower ( Figure 5 A), and the primers are shown in Table 4 above; Sirius red staining and statistical analysis of liver tissue collagen showed that the extracellular matrix collagen content in the liver tissue after treatment with macrophages expressing CBD-TM was less ( 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 the myofibroblast activation marker protein α-SMA decreased more ( Figure 5 C). Overall, macrophages modified with CBD-TM mRNA have a better therapeutic effect on liver fibrosis than unmodified macrophages.

[0079] Example 6 CBD-TM combined with IL-10 engineered macrophages have stronger anti-pulmonary fibrosis effect

[0080] Macrophages differentiated from monocytes were cultured at a concentration of 1×10 6Cells were cultured at a cell concentration of 6 cells / mL. After 6 days of differentiation, macrophages were collected and prepared: unmodified macrophages, macrophages modified with CBD-TM LNP-mRNA, and macrophages co-modified with CBD-TM (SEQ ID NO: 25) and IL-10 LNP-mRNA (the RNA sequence is shown in Table 3 and the LNP formulation is shown in Table 1) (1.0 μg of mRNA was added to every 7 cells). Cells were collected 6 h after transfection. After centrifugation at 300 g for 10 min, they were cryopreserved by programmed cooling at a concentration of Figure 6 cells / mL and stored in the gas phase of liquid nitrogen for later use. In addition, after overnight culture of a part of the cells, the culture supernatant was collected. ELISA detection of IL-10 expression showed that, compared with other groups, the secretion of IL-10 in the culture supernatant of macrophages expressing IL-10 mRNA (simultaneously expressing CBD-TM) was significantly increased (

[0081] A). 6 One week after constructing a pulmonary fibrosis model by treating 6-week-old NOD-SCID mice with 50 mg / Kg of bleomycin by pulmonary atomization, the mice were randomly divided into 3 groups: untreated control group, treatment with macrophages modified with CBD-TM, and treatment with macrophages co-modified with CBD-TM and IL-10 LNP-mRNA. The three groups of macrophages ( Figure 6 cells / mouse) were injected via the tail vein once a week for a total of three times. Two weeks after the last treatment, the mice were euthanized and lung tissue samples were collected. The level of hydroxyproline, one of the main components of collagen, was detected in the lung tissue. The results showed that the level of hydroxyproline in the lung tissue after treatment with macrophages simultaneously expressing CBD-TM and IL-10 LNP-mRNA was lower than that after treatment with macrophages expressing CBD-TM ( Figure 6 B); qPCR detection of the expression levels of fibrosis-related genes found that after treatment with macrophages simultaneously expressing CBD-TM and IL-10 LNP-mRNA, the expression levels of collagen-related genes Col1a1 and Col3a1 in the lung tissue were lower ( Figure 6 C), and the primers are shown in Table 4 above; Masson staining and statistical analysis of lung tissue collagen showed that after treatment with macrophages simultaneously expressing CBD-TM and IL-10 LNP-mRNA, the content of extracellular matrix collagen in the lung tissue was less (

[0082] D). Overall, macrophages co-modified with CBD-TM and IL-10 LNP-mRNA have a better therapeutic effect on pulmonary fibrosis than macrophages modified with CBD-TM alone.Example 7: CBD-TM combined with IL-4 engineered macrophages have stronger anti-pulmonary fibrosis effects

[0083] In the example, macrophages differentiated from monocytes were cultured at a cell concentration of 1×10 6 cells / mL. After 6 days of differentiation, macrophages were collected and prepared: unmodified macrophages, macrophages modified with CBD-TM LNP-mRNA, and macrophages co-modified with CBD-TM (SEQ ID NO: 25) and IL-4 LNP-mRNA (the RNA sequence is shown in Table 3, and the LNP formulation is shown in Table 1) (1.0 μg of mRNA was added per 1×10 6 macrophages). Cells were collected 6 h after transfection. After centrifugation at 300 g for 10 min, they were cryopreserved by programmed cooling at a concentration of 1×10 7 cells / mL and stored in the gas phase of liquid nitrogen for later use. In addition, after overnight culture of a part of the cells, the culture supernatant was collected. ELISA detection of IL-4 expression showed that, compared with other groups, the secretion of IL-4 in the culture supernatant of macrophages expressing IL-4 mRNA (simultaneously expressing CBD-TM) was significantly increased ( Figure 7 A).

[0084] One week after a pulmonary fibrosis model was constructed by treating 6-week-old NOD-SCID mice with 50 mg / Kg of bleomycin via pulmonary atomization, the mice were randomly divided into 3 groups: untreated control group, macrophages modified with CBD-TM for treatment, and macrophages co-modified with CBD-TM and IL-4 LNP-mRNA. The three groups of macrophages (1×10 6 cells / mouse) were injected via the tail vein once a week for a total of three times. Two weeks after the last treatment, the mice were euthanized and lung tissue samples were collected. The level of hydroxyproline, one of the main components of collagen, was detected in the lung tissue. The results showed that the hydroxyproline level in the lung tissue after treatment with macrophages simultaneously expressing CBD-TM and IL-4 LNP-mRNA was lower than that after treatment with macrophages expressing CBD-TM ( Figure 7 B); qPCR detection of the expression levels of fibrosis-related genes found that after treatment with macrophages simultaneously expressing CBD-TM and IL-4 LNP-mRNA, the expression levels of the collagen-related genes Timp1 and Col1a2 in the lung tissue were lower ( Figure 7 C), and the primers are shown in Table 4 above; the results of α-SMA staining and statistical analysis of the lung tissue showed that after treatment with macrophages simultaneously expressing CBD-TM and IL-4 LNP-mRNA, the expression area of the myofibroblast activation marker protein α-SMA was smaller ( Figure 7D). Overall, macrophages co-engineered with CBD-TM and IL-4 LNP-mRNA have better therapeutic effects on pulmonary fibrosis than macrophages engineered with CBD-TM alone.

[0085] Example 8 CBD-TM combined with IL-13 engineered macrophages have stronger anti-pulmonary fibrosis effects

[0086] In the example, macrophages differentiated from monocytes were cultured at a cell concentration of 1×10 6 cells / mL. After 6 days of differentiation, macrophages were collected and prepared: unengineered macrophages, macrophages engineered with CBD-TM LNP-mRNA, and macrophages co-engineered with CBD-TM (SEQ ID NO: 25) and IL-13 LNP-mRNA (RNA sequence is shown in Table 3, LNP formulation is shown in Table 1) (1.0 μg of mRNA was added per 1×10 6 macrophages). Cells were collected 6 h after transfection. After centrifugation at 300 g for 10 min, they were cryopreserved by programmed cooling at a concentration of 1×10 7 cells / mL and stored in the gas phase of liquid nitrogen for later use. In addition, after overnight culture of a part of the cells, the culture supernatant was collected. ELISA detection of IL-13 expression showed that, compared with other groups, the secretion of IL-13 in the culture supernatant of macrophages expressing IL-13 mRNA (simultaneously expressing CBD-TM) was significantly increased ( Figure 8 A).

[0087] One week after constructing a pulmonary fibrosis model by treating 6-week-old NOD-SCID mice with 50 mg / Kg of bleomycin by pulmonary atomization, the mice were randomly divided into 3 groups: untreated control group, treatment with macrophages engineered with CBD-TM, and treatment with macrophages co-engineered with CBD-TM and IL-13 LNP-mRNA. The three groups of macrophages (1×10 6 cells / mouse) were injected via the tail vein once a week for a total of three times. Two weeks after the last treatment, the mice were euthanized and lung tissue samples were collected. The level of hydroxyproline, one of the main components of collagen, was detected in the lung tissue. The results showed that the level of hydroxyproline in the lung tissue after treatment with macrophages simultaneously expressing CBD-TM and IL-13 LNP-mRNA was lower than that in the lung tissue treated with macrophages expressing CBD-TM ( Figure 8 B); qPCR detection of the expression levels of fibrosis-related genes found that after treatment with macrophages simultaneously expressing CBD-TM and IL-13 LNP-mRNA, the expression levels of collagen-related genes Col1a1 and Col3a1 in the 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 co-expressing CBD-TM and IL-13 LNP-mRNA, the extracellular matrix collagen content in the lung tissue was less ( Figure 8 D). Overall, macrophages co-engineered with CBD-TM and IL-13 LNP-mRNA have a better therapeutic effect on pulmonary fibrosis than macrophages engineered with CBD-TM alone.

Claims

1. A fusion protein, characterized in that, The fusion protein comprises a VWF protein A3 domain and a PDGFR transmembrane domain connected in sequence; wherein the VWF protein A3 domain is composed of the amino acid sequence shown in SEQ ID NO: 1, and the PDGFR transmembrane domain is composed of the amino acid sequence shown in SEQ ID NO: 3; the VWF protein A3 domain and the transmembrane domain of PDGFR are connected by a flexible linker.

2. The fusion protein according to claim 1, which further comprises a signal peptide at the N-terminus.

3. The fusion protein according to claim 2, wherein the signal peptide is derived from IL-2, IL-6, IL-11 or IL-12B.

4. The fusion protein according to any one of claims 1-3, which comprises the amino acid sequence shown in any one of SEQ ID NO: 8-11.

5. A nucleic acid, characterized in that, The nucleic acid encodes the fusion protein according to any one of claims 1-4; wherein the nucleic acid is DNA or mRNA.

6. The nucleic acid according to claim 5, which comprises the nucleotide sequence shown in any one of SEQ ID NO: 19-22.

7. The nucleic acid according to claim 6, which comprises the nucleotide sequence shown in any one of SEQ ID NO: 23-26.

8. A nano-lipid particle-nucleic acid complex, characterized in that, The complex comprises the nucleic acid according to any one of claims 5-7 and a nano-lipid particle (LNP), wherein the nucleic acid is mRNA.

9. The composite according to claim 8, wherein, The nano-lipid 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.

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

1.

11. Engineered macrophages, characterized in that, The macrophage contains the fusion protein according to any one of claims 1-4 or the nucleic acid according to any one of claims 5-7.

12. The macrophage according to claim 11, which further comprises a nucleic acid encoding one or more of IL-10, IL-4 and IL-13.

13. The macrophage according to claim 12, wherein IL-10, IL-4 and IL-13 are the amino acid sequences shown in SEQ ID NO: 27-29 respectively.

14. The macrophage according to claim 13, wherein the nucleic acids encoding IL-10, IL-4 and IL-13 are the polynucleotide sequences shown in SEQ ID NO: 30-32 respectively.

15. A method for preparing engineered macrophages, characterized in that, It includes introducing the nucleic acid according to any one of claims 5-7 or the complex according to any one of claims 8-10 into macrophages.

16. The method according to claim 15, which includes introducing the nucleic acid according to any one of claims 5-7 or the nano-lipid particle-nucleic acid complex according to any one of claims 8-10, and LNP coated with one or more of the nucleic acids encoding one or more of IL-10, IL-4 and IL-13 into macrophages simultaneously or successively.

17. The method according to claim 15 or 16, when introducing the nucleic acid, the introducing method is electroporation; when introducing the complex, the introducing method is LNP delivery.

18. The method according to claim 17, wherein the IL-10, IL-4, and IL-13 are amino acid sequences shown in SEQ ID NO: 27-29, respectively.

19. The method according to claim 18, wherein the nucleic acids encoding IL-10, IL-4, and IL-13 are polynucleotide sequences shown in SEQ ID NO: 30-32, respectively.

20. An engineered macrophage obtained by the method according to any one of claims 15-19.

21. Use of the engineered macrophage according to any one of claims 11-14 or claim 20 in the preparation of a medicament for treating fibrotic diseases; the fibrotic diseases are pulmonary fibrosis or liver fibrosis.

22. Use of the fusion protein according to any one of claims 1-4, the nucleic acid according to any one of claims 5-7, or the complex according to any one of claims 8-10 in the preparation of a medicament for treating fibrotic diseases; the fibrotic diseases are pulmonary fibrosis or liver fibrosis.

Images