An engineered macrophage, its preparation method and application in anti-fibrosis

Abstract

The present invention discloses an engineered macrophage, a preparation method thereof and an application in anti-fibrosis, belonging to the technical field of biomedicine. The macrophage contains a nucleic acid encoding a transmembrane fusion protein, the N-terminal of the transmembrane fusion protein is the extracellular domain and the seven-transmembrane domain of the CCR2 protein, and the C-terminal is composed of the intracellular domain of IL10RA and the intracellular domain of IL10RB. The engineered macrophage may further include one or more siRNAs selected from siRNAs targeting TIMP1, siRNAs targeting TIMP2 and siRNAs targeting TIMP3. It may further include a nucleic acid encoding a fusion protein of a leader secretion signal of IL12B and a catalytic activity domain of MMP.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technologies, and particularly relates to a function-enhanced macrophage obtained by RNA engineering transformation and its application in the treatment of fibrotic diseases. 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. 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 lung, liver, heart, kidney, etc., and is a common terminal pathological manifestation of many chronic diseases.

[0003] Pulmonary fibrosis occurs in the scar tissue generated during the process of lung tissue injury and repair. This thickened and stiff tissue makes it more difficult for the lung to work normally. Clinically, patients mainly show symptoms such as dyspnea, dry cough, fatigue, and easy tiredness. The factors causing pulmonary fibrosis are diverse, 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). IPF patients account for 30%-50% of all pulmonary fibrosis patients. Clinically, anti-inflammatory, antioxidant, and fibrosis-inhibiting treatment methods are mainly adopted, but these methods often have limited efficacy and certain side effects. The only existing treatment drugs are pirfenidone and nintedanib. Non-drug treatment options include oxygen therapy, pulmonary rehabilitation therapy, and lung transplantation, etc. (MSD).

[0004] 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 fibrosis process, and promoting tissue regeneration. As an important component of the immune system, macrophages can regulate inflammation, inhibit fibrosis, and promote tissue repair simultaneously. The key to engineering macrophages for anti-fibrotic treatment lies in how to regulate the functions of macrophages and enhance and maintain their anti-fibrotic 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 ability 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 ability of macrophages.

[0005] However, the selection of targets for engineering existing macrophages has limitations. Existing engineered macrophages restore the reduced ECM degradation ability by directly expressing MMPs proteins in response to the introduction of IL10. However, against the background of high expression of multiple MMPs proteins and their inhibitors TIMP1, TIMP2, and TIMP3 in macrophages themselves, the increased ECM degradation ability by directly overexpressing full-length MMP proteins is limited. In tissue homeostasis, inflammation and anti-inflammation maintain a balance. Existing engineered macrophages directly overexpress the anti-inflammatory cytokine IL10. This solution can maintain the anti-inflammatory phenotype of macrophages and may rapidly reverse the inflammatory microenvironment of fibrotic tissues. However, the potential risks brought about by the rapid disruption of the inflammatory and anti-inflammatory balance are unpredictable. On the other hand, existing macrophage engineering technologies mainly include virus infection, electroporation, and nanoparticle-based delivery. However, virus infection has the potential risk of insertion of virus-derived fragments, electroporation causes greater damage to cells, and lipid nanoparticles have an inflammation-inducing effect and are prone to M1 polarization of macrophages. Summary of the Invention

[0006] To address the above technical problems, engineered macrophages are provided that simultaneously achieve maintaining the anti-inflammatory phenotype of macrophages and enhancing the extracellular matrix degradation ability.

[0007] In the first aspect of the present invention, a transmembrane fusion protein is provided, with its N-terminus being the extracellular domain and the 7-transmembrane domain of the CCR2 protein, and its C-terminus being composed of the intracellular domain of IL10RA and the intracellular domain of IL10RB.

[0008] In the second aspect of the present invention, a nucleic acid is provided, which encodes the transmembrane fusion protein described in the first aspect. Among them, the nucleic acid is DNA or mRNA.

[0009] In the third aspect of the present invention, a nano-lipid particle-nucleic acid complex is provided, which comprises the nucleic acid described in the second aspect and nano-lipid particles (LNP).

[0010] In the fourth aspect of the present invention, engineered macrophages are provided, which contain the transmembrane fusion protein described in the first aspect of the present invention or the nucleic acid described in the second aspect.

[0011] In some embodiments, the engineered macrophages further comprise one or more siRNAs selected from siRNAs targeting TIMP1, siRNAs targeting TIMP2, and siRNAs targeting TIMP3;

[0012] In some embodiments, the engineered macrophages further contain a nucleic acid encoding a fusion protein comprising a signal peptide for guiding the secretion of IL12B and the catalytic active domain of MMP.

[0013] In the fifth aspect of the present invention, a method for preparing engineered macrophages is provided.

[0014] In the sixth aspect of the present invention, engineered macrophages obtained according to the preparation method described in the fifth aspect are provided.

[0015] In the seventh aspect of the present invention, the engineered macrophages described in the fourth or sixth aspect are used in the preparation of drugs for preventing and / or treating fibrotic diseases.

[0016] The advantages of the present invention over the prior art are as follows:

[0017] 1. The present invention screened anti-inflammatory LNPs and proved that the lipid formulation containing dexamethasone can reduce the expression of inflammation-related genes IL-1β and IL6 in macrophages, offsetting the inflammatory response induced by LNP.

[0018] 2. The present invention constructed novel engineered macrophages, achieving the purpose of maintaining the anti-inflammatory phenotype of macrophages and enhancing the extracellular matrix degradation ability at the same time.

[0019] 3. The chimeric protein introduced in the present invention can respond to the CCL2 signal in fibrotic tissues and be converted into an anti-inflammatory IL10 signal, achieving an anti-inflammatory effect based on the microenvironment signal of fibrotic tissues.

[0020] 4. During the process of the occurrence, development, and regression of fibrotic tissue, the balance between inflammation and anti-inflammation is constantly changing. The chimeric protein expressed by mRNA introduced in the present invention can bind to the inflammatory chemokines highly expressed in fibrotic tissue and be converted into an intracellular anti-inflammatory signal, achieving the effect of reversing and maintaining the anti-inflammatory state based on the inflammatory level in the fibrotic microenvironment. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Other features, objects, and advantages of the present invention will become more apparent by reading the following detailed description of non-limiting embodiments with reference to the accompanying drawings:

[0022] Figure 1 Preparation, phenotype detection, phagocytosis, and cell viability detection of macrophages. Figure 1 A in [reference] shows the cell morphology of macrophages differentiated from monocytes; Figure 1 B in [reference] shows the detection of macrophage surface marker proteins 25F9 and CD206 by flow cytometry, and the positive expression rates of the two marker proteins are statistically analyzed; Figure 1 C in [reference] shows the detection of macrophage viability and the phagocytosis efficiency of cells for fluorescently labeled particles by flow cytometry.

[0023] Figure 2 Showing agarose gel electrophoresis analysis of two types of mRNAs prepared by in vitro transcription and capping.

[0024] Figure 3 Showing the knockdown efficiency of siRNA verified in macrophages. Figure 3 A in [reference] shows that transcriptome sequencing analysis of different batches of macrophages shows high expression of TIMP1, TIMP2, and TIMP3 in macrophages; Figure 3 B in [reference] shows the knockdown efficiency of TIMP1 mRNA in macrophages electroporated with si#1, si#2, and si#3 after 2 days and 4 days by qPCR analysis; Figure 3 C in [reference] shows the knockdown efficiency of TIMP2 mRNA in macrophages electroporated with si#4, si#5, and si#6 after 2 days and 4 days by qPCR analysis; Figure 3 D in [reference] shows the knockdown efficiency of TIMP3 mRNA in macrophages electroporated with si#7, si#8, and si#9 after 2 days and 4 days by qPCR analysis.

[0025] Figure 4 Showing the screening of anti-inflammatory LNPs. Figure 4 A in [reference] shows the encapsulation efficiency of GFP mRNA analyzed by RiboGreen fluorescence detection method for different LNP lipid compositions; Figure 4B in [Figure] shows the expression of GFP mRNA delivered by LNP lipid formulations without dexamethasone (C#1-0 and C#2-0) and with dexamethasone (C#1-1 and C#2-1) in macrophages analyzed by flow cytometry; Figure 4 C in [Figure] shows the expression levels of inflammation-related genes IL-1β and IL6 in macrophages transfected with different lipid formulation LNP-GFP mRNA after 1 day analyzed by qPCR.

[0026] Figure 5 It shows that LNP-siRNA does not affect macrophage phenotype and cytokine secretion. Figure 5 A in [Figure] shows the expression levels of TIMP1, TIMP2, and TIMP3 proteins in macrophages transfected with LNP-RNA si#1 / 4 / 9 analyzed by Western-Blot after 2 days; Figure 5 B in [Figure] shows the expression percentages of macrophage surface markers 25F9 and CD206 analyzed by flow cytometry after 2 days of transfection with control siRNA (NC) and TIMP1 / 2 / 3 siRNA (si#1 / 4 / 9); Figure 5 C in [Figure] shows the viability of macrophages analyzed by flow cytometry after 2 days of transfection with control siRNA (NC) and TIMP1 / 2 / 3 siRNA (si#1 / 4 / 9); Figure 5 D in [Figure] shows the levels of cytokines IL6 and IL10 in the culture supernatant of macrophages detected by ELISA method after 2 days of transfection with control siRNA (NC) and TIMP1 / 2 / 3 siRNA (si#1 / 4 / 9).

[0027] Figure 6 It shows the ECM degradation ability of the culture supernatant of macrophages detected by microplate reader after 2 days of transfection with control siRNA (NC) and TIMP1 / 2 / 3 siRNA (si#1 / 4 / 9).

[0028] Figure 7 It shows that MMP9-CD mRNA promotes collagen degradation. Figure 7 A in [Figure] shows the extracellular expression levels of three kinds of MMP9-CD mRNA with different secretion signals detected by ELISA in macrophages; Figure 7 B in [Figure] shows the ECM degradation ability of the culture supernatant of 293T cells or macrophages transfected with MMP9-CD#3mRNA or control mRNA detected by microplate reader.

[0029] Figure 8 It shows that MMP9-CD mRNA does not affect phenotype and cytokine secretion. Figure 8 A in [Figure] shows the viability of macrophages transfected with MMP9-CD#3 mRNA or control macrophages analyzed by flow cytometry;Figure 8 B in it shows the percentage of the expression of the macrophage surface marker CD206 by flow cytometry analysis after transfection with MMP9-CD#3 mRNA or the control; Figure 8 C in it shows the mean fluorescence intensity of the expression of the macrophage surface markers CD40 and CD80 by flow cytometry analysis after transfection with MMP9-CD#3 mRNA or the control; Figure 8 D in it shows the mean fluorescence intensity of the expression of the macrophage surface markers CD163 and CD206 by flow cytometry analysis after transfection with MMP9-CD#3 mRNA or the control; Figure 8 E in it shows the levels of the cytokines IL6 and IL10 in the macrophage culture supernatant after 1 day of transfection with MMP9-CD#3 mRNA detected by the ELISA method.

[0030] Figure 9 Shows the expression and functional verification of CCR2-IL10RA-RB mRNA. Figure 9 A in it shows the expression of macrophages after LNP delivery of CCR2-10RA-RB mRNA detected by flow cytometry; Figure 9 B in it shows the induction of the downstream genes SOCS3 and IL1RA of the IL10 receptor by the CCL2 protein in macrophages with or without the expression of the CCR2-10RA-RB chimera analyzed by qPCR.

[0031] Figure 10 Shows that CCR2-IL10RA-RB engineered macrophages resist M1 polarization. Flow cytometry analysis of the expression intensity of the marker proteins CD40 and CD80 in macrophages with or without the expression of the CCR2-IL10RA-RB chimera protein under M1 polarization conditions.

[0032] Figure 11 Shows that TIMPs siRNA combined with CCR2-IL10RA-RB mRNA enhances the anti-fibrotic effect of macrophages in vivo. Figure 11 A in it shows the hydroxyproline content in the lung tissues of different treatment groups; Figure 11 B in it shows the expression levels of Co1a1 and Col3a1 mRNA in the lung tissues of different treatment groups analyzed by qPCR; Figure 11 C in it shows the Masson staining results of the lung tissues of different treatment groups; Figure 11 D in it shows the statistical analysis results of the Masson staining of the lung tissues of different treatment groups; Figure 11 E in it shows the α-SMA staining results of the lung tissues of different treatment groups; Figure 11 F in it shows the statistical analysis results of the α-SMA staining of the lung tissues of different treatment groups.

[0033] Figure 12 It is shown that the combination of MMP9-CD mRNA and CCR2-IL10RA-RB mRNA enhances the anti-fibrotic effect of macrophages in vivo. Figure 12 Panel A in [reference] shows the hydroxyproline content in the lung tissues of different treatment groups; Figure 12 Panel B in [reference] shows the expression levels of Timp1 and Col3a1 mRNA in the lung tissues of different treatment groups analyzed by qPCR; Figure 12 Panel C in [reference] shows the Masson staining results of the lung tissues of different treatment groups; Figure 12 Panel D in [reference] shows the statistical analysis results of the Masson staining of the lung tissues of different treatment groups; Figure 12 Panel E in [reference] shows the α-SMA staining results of the lung tissues of different treatment groups; Figure 12 Panel F in [reference] shows the statistical analysis results of the α-SMA staining of the lung tissues of different treatment groups. Detailed implementation manners

[0034] To make the objectives, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Apparently, the described embodiments are some but not all of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the described embodiments of the present invention without creative efforts shall fall within the scope of protection of the present invention.

[0035] Unless otherwise defined, the technical terms or scientific terms used herein shall have the ordinary meanings as understood by those of ordinary skill in the art to which the present invention pertains.

[0036] The present invention provides nucleic acids encoding a transmembrane fusion protein, wherein the N-terminal of the transmembrane fusion protein is the extracellular domain and the seven-transmembrane domain of the CCR2 protein, and the C-terminal is composed of the intracellular domain of IL10RA and the intracellular domain of IL10RB.

[0037] Among them, the "extracellular domain and the seven-transmembrane domain of the CCR2 protein" is located at positions 1-309 of the CCR2 protein (ProteinID: NP_001116513.2). The extracellular domain formed by seven transmembrane spans can bind to the chemokine CCL2, promoting the chemotactic movement and migration of macrophages.

[0038] Among them, the "intracellular domain of IL10RA" is the intracellular domain of the IL10RA subunit (Protein ID: NP_001549.2), which is located at positions 257-578 of IL10RA and has the function of recruiting JAK1 and activating STAT3, activating anti-inflammatory and immunosuppressive signals.

[0039] Among them, the "IL10RB intracellular domain" is the intracellular domain of the IL10RB subunit (Protein ID: NP_000619.3), located at positions 243 - 325 of IL10RB, and has the function of forming a heterodimer with IL-10Rα and enhancing signal transduction.

[0040] In some embodiments, the engineered macrophages may further comprise one or more siRNAs selected from siRNAs targeting TIMP1, siRNAs targeting TIMP2, and siRNAs targeting TIMP3.

[0041] Among them, the "TIMP1", "TIMP2", and "TIMP3" are macrophage-derived MMPs activity inhibitory proteins, with Protein accession numbers NP_003245.1, NP_003245.1, and NP_000353.1 respectively; the siRNAs targeting the above genes can change the binding balance of endogenous TIMPs and MMPs proteins, release endogenous MMPs inhibited by TIMPs binding, thereby globally increasing the ECM degradation ability of macrophages.

[0042] In some embodiments, the engineered macrophages comprise a nucleic acid encoding a fusion protein of the leader secretion signal of IL12B and the catalytic active domain of MMP.

[0043] Among them, the "leader secretion signal of IL12B" is the leader secretion signal of the IL12B subunit (Protein ID: NP_002178.2), located at positions 1 - 22 of IL12B, and has the function of guiding IL12B into the secretory pathway and finally secreting it extracellularly through the Golgi apparatus.

[0044] Among them, the "MMP catalytic active domain" is the catalytic active domain of the MMP9 protein (Protein ID: NP_004985.2), located at positions 94 - 443 of MMP, and has the function of catalyzing extracellular matrix degradation.

[0045] Example 1 Macrophage Preparation

[0046] The source of macrophages can be autologous peripheral blood, which is induced from autologous peripheral blood mononuclear cells.

[0047] The preparation of macrophages can adopt methods known in the art. For example, PBMC is first prepared by Ficoll density gradient centrifugation, then the PBMC is processed to separate monocytes, and the separated monocytes are differentiated into macrophages.

[0048] The operation of preparing PBMC by Ficoll density gradient centrifugation is as follows: a certain volume of Ficoll is transferred to a centrifuge tube; whole blood is then covered on the Ficoll, and the centrifuge tube is centrifuged at room temperature at 200g for 45 minutes to obtain a red blood cell pellet, a Ficoll layer, a white layer containing PBMC, and a plasma layer; and the white layer containing PBMC is extracted.

[0049] PBMCs contain monocytes and lymphocytes, so PBMCs 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.

[0050] Cultivation of macrophages: The monocytes isolated in this example (PBMCs can also be directly cultured) are cultured in TexMACS differentiation medium containing macrophage colony stimulating factor (M-CSF) to induce culture to obtain macrophages. The culture conditions can be any suitable for culturing macrophages, and there is no particular limitation in this example. For example, the density of macrophages inoculated in the culture medium can be 5×10 5 cells / mL. The culture temperature may be 37°C. The culture time may be appropriately set according to the growth state of the macrophages, for example, the culture time may be set to 5 to 8 days. The culture environment may be a 5% CO2 culture environment.

[0051] In this example, monocytes were obtained by centrifugal countercurrent elutriation. The differentiated macrophages showed a relatively uniform spherical morphology. Each macrophage had a small protrusion ( Figure 1 A). Flow cytometry was used to detect the surface markers of differentiated macrophages 25F9 and CD206. Statistical analysis showed that the positive rates were greater than 90% ( Figure 1 B). Flow cytometry detection of cell viability showed that the viability of macrophages prepared in different batches was greater than 90% ( Figure 1 C). Macrophages were co-incubated with fluorescently labeled bio-microsphere particles, and then flow cytometry was used to detect that macrophages had a strong phagocytic ability ( Figure 1 C).

[0052] Example 2 RNA preparation

[0053] The one-tube multi-step method was used for mRNA preparation, that is, linearization, transcription and capping reactions were carried out in the same tube, and mRNA was purified by magnetic beads after the reaction was completed.

[0054] Plasmid construction: The pUC57-Kan vector (Sangon Biotech, B522201-0100) was used as the backbone vector, which mainly includes the DNA replication origin, kanamycin resistance gene and its promoter. The T7 promoter sequence (SEQ ID NO: 22), 5’ UTR sequence (SEQ ID NO: 23), protein coding frame sequence, 3’ UTR sequence (SEQ ID NO: 27), poly A sequence (SEQ ID NO: 28) 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%.

[0055] Plasmid linearization: The reaction volume was 5 μL, the concentration of the restriction endonuclease (BspQI) was 1 - 10 U / μg plasmid, and the digestion time at 50 °C was 0.5 - 3 h;

[0056] 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 (replacing UTP with m1ψ) 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;

[0057] 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, and the final concentration of vaccinia capping enzyme (Novoprotein, GMP-M062-M001) was 50 - 1000 U / μL. The reaction was carried out at 37 °C for 1 - 2 h.

[0058] mRNA purification: Each microliter of the capped product was purified using 0.5 - 1 μg of magnetic beads.

[0059] When preparing RNA, the above reaction system can be linearly amplified according to the demand for mRNA. The protein coding frames of MMP9-CD mRNA (9CD) that enhances the ECM degradation ability and mRNA CCR2-IL10RA-RB (R2-AB) that maintains the anti-inflammatory phenotype of macrophages have been sequence-optimized (as shown in Table 4 and Table 5 below). The 5' UTR sequence consists of the HBB (hemoglobin) mRNA 5' UTR sequence (SEQ ID NO: 24), the IGG6 sequence (SEQ ID NO: 25), and the Kozak sequence (SEQ ID NO: 26, caatcaaac). The 3' sequence is the 3' UTR sequence derived from histone, and the polyA length should be greater than 100.

[0060] In this example, based on the above scheme, these two types of mRNA were prepared. 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 2 ).

[0061] Example 3 Preparation and Verification of siRNA

[0062] In this example, siRNAs targeting TIMP1, TIMP2, and TIMP3 proteins (the gene sequences of TIMP1, TIMP2, and TIMP3 are shown in SEQ ID NO: 19 - 21 respectively) were prepared by in vitro transcription. The siRNA is formed by annealing the sense strand and the antisense strand to form a double-stranded structure. The transcription templates for the sense strand and the antisense strand of the siRNA are formed by annealing two complementary polyoligonucleotides, and the formed transcription template carries a T7 promoter sequence. The main steps for preparing the siRNA are as follows:

[0063] Template preparation: Two complementary polyoligonucleotides are annealed to form a double-stranded DNA transcription template. The annealing system contains 1×NEB buffer 2, and the concentration of the two polyoligonucleotides is 4.5 μM (1 - 10 μM); the annealing conditions of the PCR instrument are 95°C for 5 min; 65°C for 2 min; 55°C for 2 min; 40°C for 2 min; 22°C for 2 min.

[0064] In vitro transcription: The total reaction volume was 20 μL. In the reaction system, the transcription templates of the sense and antisense strand siRNAs were each 0.5 μM (0.1 - 2.0 μM), 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 (replacing UTP with m1ψ and / or A with m6A) 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 4 - 12 h;

[0065] Nuclease digestion: After transcription was completed, DNase I with a final concentration of 0.2 - 2.0 U / μL and RNase T1 with a final concentration of 0.1 - 1.0 U / μL were added to the transcription system, and the reaction was carried out at 37 °C for 0.5 - 3.0 h;

[0066] siRNA purification: The siRNA products obtained by nuclease digestion were purified using the RNeasy kit from QIAGEN.

[0067] TIMP family proteins are inhibitors of extracellular matrix metalloproteinases. Sequencing of the macrophage transcriptome in the early stage of the study found that macrophages highly expressed TIMP1, TIMP2, and TIMP3 and lowly expressed TIMP4 ( Figure 3 A); In this example, siRNAs targeting TIMP1 (si#1, si#2, and si#3), TIMP2 (si#4, si#5, and si#6), and TIMP3 (si#7, si#8, and si#9) mRNAs were prepared (Table 1). After electroporating macrophages, the expression levels of TIMP1, TIMP2, and TIMP3 mRNAs were detected by qPCR at 2 days and 4 days respectively. The results showed that si#1, si#4, and si#9 could efficiently reduce the expression of TIMP1 ( Figure 3 B), TIMP2 ( Figure 3 C), and TIMP3 ( Figure 3 D) protein mRNAs.

[0068] Table 1 siRNA information

[0069] Note: Uracil (U) in Table 1 was replaced by m1ψ.

[0070] Example 4 Screening of anti-inflammatory LNP formulations

[0071] Both ionizable cationic lipids and PEG lipids in the LNP delivery system have an immune-mediated effect, which is not conducive to the maintenance of the anti-inflammatory phenotype of macrophages. Dexamethasone is a long-acting glucocorticoid with an anti-inflammatory effect. In this example, when preparing LNP, a certain concentration of dexamethasone was added to offset the inflammatory response induced by LNP.

[0072] In this example, mRNA or siRNA was diluted with sodium citrate buffer at pH 4.0 to a final RNA concentration of 110 ng / μL (50 - 150 ng / μL).

[0073] A lipid mixture (ionizable cationic lipid: neutral helper phospholipid: cholesterol and dexamethasone mixture: PEG lipid) was prepared with a total lipid mixture concentration of 12 mM (10 - 16 mM), and the molar ratio of the four components was 35 - 53∶5 - 14∶39 - 52∶1 - 5. When preparing LNP, the N / P (molar ratio of nitrogen content in the main lipid to phosphorus content in nucleic acid) ratio was 6 - 10, preferably 6, and the total flow rate was 4 - 16 ml / min, preferably 12 ml / min.

[0074] The prepared LNP-mRNA was 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 exchanged with a freezing solution containing 10 mM Tris and 10% sucrose (pH 7.4 - 7.8) at 5 - 10 times the volume, and finally concentrated to 1 / 4 of the original volume; the purified LNP-mRNA was collected and aliquoted and stored at -80 °C.

[0075] 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 efficiency (>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 sizer.

[0076] In this example, the ionizable cationic lipid selected was SM-102, and the molar percentages of ionizable cationic lipid∶neutral auxiliary phospholipid∶cholesterol and dexamethasone mixture∶PEG lipid were 50∶10∶38.5∶1.5 or 40∶12.6∶46.2∶1.2 respectively; the molar ratios of cholesterol and dexamethasone mixture were 10∶0, 9∶1, 8∶2, 5∶5, 2∶8, 1∶9, and 0∶10 (Table 2). Encapsulation efficiency detection showed that among the two LNP lipid formulations (50∶10∶38.5∶1.5 or 40∶12.6∶46.2∶1.2), only when the molar ratio of cholesterol and dexamethasone mixture was 9∶1 (C#1-1, C#2-1), the encapsulation efficiency of LNP-mRNA was close to that of the LNP formulation without dexamethasone (C#1-0, C#2-0) ( Figure 4 A). Flow cytometry detection 1 day after transfection of LNP-mRNA (GFP) into macrophages showed that after adding dexamethasone (C#1-1, C#2-1), the transfection efficiency of LNP-mRNA was not weakened ( Figure 4 B), and the qPCR analysis results showed that the lipid formulations C#1-1 and C#2-1 containing dexamethasone could reduce the expression of inflammation-related genes IL-1β and IL6 ( Figure 4 C).

[0077] In this example, LNP-RNA was prepared with the molar percentages of ionizable cationic lipid∶neutral auxiliary phospholipid∶cholesterol and dexamethasone mixture∶EG lipid being 40∶12.6∶46.2∶1.2, where the molar ratio of cholesterol and dexamethasone was 9∶1. The encapsulation efficiencies of the 5 prepared LNP-RNAs were all greater than 90%, the particle size was less than 150 nm, the PDI was less than 0.15, and the Zeta potential was between -10 mV and -0 mV (Table 3).

[0078] Table 2 LNP formulations

[0079]

[0080] Table 3 Detection results of anti-inflammatory LNP-RNA

[0081]

[0082] Example 5 Inhibiting TIMP1 / 2 / 3 proteins and increasing the ability of macrophages to degrade ECM

[0083] In this example, macrophages were engineered by co-transfecting with LNP-siRNA (si#1, si#4, and si#9). In the example, the differentiated macrophages were cultured at a cell concentration of 1×10 6 cells / mL, and every 1×10 6Add 3 kinds of LNP-siRNA with a final concentration of 25 μM respectively to macrophages,

[0084] After culturing for 6 hours, collect the cells, centrifuge at 300 g for 10 min, and replace with fresh Texmacs medium for culturing for 48 hours. After collecting the cell pellet, it can be detected by Western blotting that the proteins of three kinds of TIMP1, TIMP2 and TIMP3 can be effectively reduced by siRNA ( Figure 5 A); It can be seen by antibody staining and flow cytometry that the macrophage surface markers 25F9 and CD206 are detected. Compared with the control, the expression percentage of macrophage surface marker proteins has not changed after transfection with LNP-siRNA ( Figure 5 B); The results of flow cytometry analysis show that the cell viability is not affected by siRNA either ( Figure 5 C); By collecting the macrophage culture supernatant and detecting by ELISA, it is found that transfection with LNP-siRNA (si#1 / 4 / 9) does not change the expression of inflammatory factor IL6 and anti-inflammatory factor IL10 secreted by macrophages ( Figure 5 D).

[0085] Collect the macrophage culture supernatant in the examples and detect it using a collagenase activity detection kit (Abcam, ab234624). It is found that compared with the control (NC), transfection with LNP-siRNA (si#1 / 4 / 9) can increase the ability of macrophages to degrade the extracellular matrix ( Figure 6 ).

[0086] Example 6 MMP9-CD mRNA increases the ability of macrophages to degrade ECM

[0087] In this example, macrophages were engineered with MMP9-CD mRNA. Three kinds of MMP9-CD expressing mRNAs were constructed in the examples. The N-termini of MMP9-CD#1, MMP9-CD#2 and MMP9-CD#3 were respectively fused with the secretion signals from the IL2, IL6 and IL12B genes. The sequence information involved in the construction of MMP9-CD is shown in Table 4.

[0088] Table 4 MMP9-CD related sequence information

[0089]

[0090] The differentiated macrophages were cultured at a cell concentration of 1×10 6 cells / mL, and every 1×10 6Add 1 μg (mRNA mass) of LNP-mRNA to each macrophage, collect the cells after culturing for 6 h, centrifuge at 300 g for 10 min and replace with fresh Texmacs medium for culturing. Take the engineered macrophages cultured overnight, centrifuge and collect the culture supernatant. ELISA detection of the expression of MMP9-CD protein in the supernatant found that the secretion signal derived from IL12B can mediate a higher expression level of MMP9-CD#3 in macrophages ( Figure 7 A). After transfection of 293T cells or macrophages with MMP9-CD#3 LNP-mRNA, the ECM degradation ability of their culture supernatant can be enhanced ( Figure 7 B). Figure 7 In it, the difference between the control group (Con group) and the experimental group is only that no LNP-mRNA is transfected.

[0091] In the example, 1 day after transfection of macrophages with MMP9-CD#3 LNP-mRNA, it was detected that the transfection of MMP9-CD#3 did not reduce the viability of macrophages ( Figure 8 A) and the expression of surface marker CD206 ( Figure 8 B); analysis of macrophage M1-type polarization markers CD40 and CD80 by flow cytometry ( Figure 8 C), M2-type polarization markers CD163 and CD206 ( Figure 8 D), and the results showed that transfection with MMP9-CD#3 LNP-mRNA did not affect the polarization phenotype of macrophages. In addition, ELISA detection results showed that there were no significant changes in the inflammatory factor IL6 and anti-inflammatory factor IL10 in the culture supernatant of transfected macrophages ( Figure 8 E). Figure 8 In it, the difference between the control group (Con group) and the experimental group is only that no LNP-mRNA is transfected.

[0092] Example 7 CCR2-IL10RA-RB mRNA-engineered macrophages have a stable anti-inflammatory phenotype

[0093] In this example, macrophages were engineered with CCR2-IL10RA-RB mRNA. CCR2-IL10RA-RB mRNA expresses a transmembrane chimeric protein. The extracellular domain has the ability to bind to CCL2, which is highly expressed in fibrotic tissues. The intracellular domains of IL10RA and IL10RB are connected by a GS linker and serve as the intracellular domain of the chimeric protein. On the one hand, CCR2-IL10RA-RB can bind to CCL2 and promote the downstream signals of IL10 intracellularly. On the other hand, it can consume CCL2 in fibrotic tissues and reduce the recruitment of inflammatory cells to the fibrotic tissue area. The sequence information involved in the construction of CCR2-IL10RA-RB is shown in Table 5:

[0094] Table 5 Sequence information involved in CCR2-IL10RA-RB

[0095]

[0096] In this example, macrophages differentiated from monocytes were cultured at a cell concentration of 1×10 6 cells / mL. 0.5 μg (mRNA mass) of CCR2-IL10RA-RB LNP-mRNA was added to every 1×10 6 macrophages. After culturing for 6 hours, the cells were collected. After centrifugation at 300 g for 10 min, they were replaced with fresh Texmacs medium and cultured for 24 hours. Flow cytometry analysis of the macrophages after collecting the cells showed that the macrophages transfected with CCR2-IL10RA-RB LNP-mRNA had stronger surface signals of CCR2 ( Figure 9 A). Different concentrations of CCL2 protein were added to the macrophages transfected with CCR2-IL10RA-RB mRNA. After 24 hours, the cells were collected and qPCR was used to analyze the mRNA expression levels of the downstream genes SOCS3 and IL1RA of the IL10 signaling pathway. The results showed that compared with the control, the CCR2-IL10RA-RB chimeric protein could respond to the CCL2 protein signal and induce the expression of the downstream genes SOCS3 and IL1RA of the IL10 receptor intracellularly ( Figure 9 B). Figure 9 In the control group (Con group), the only difference from the experimental group was that no LNP-mRNA was transfected.

[0097] To further verify whether macrophages expressing CCR2-IL10RA-RB have the ability to resist M1 polarization, we added IFN-γ at a final concentration of 50 ng / ml and LPS at 10 ng / mL to the culture media of macrophages and macrophages expressing CCR2-IL10RA-RB to induce M1 polarization for 48 h,

[0098] Flow cytometry analysis revealed that IFN-γ and LPS could increase the expression of M1-type marker genes CD40 and CD80. After expressing CCR2-IL10RA-RB, adding 10 ng / mL of CCL2 simultaneously during M1-type polarization could weaken the increase in CD40 and CD80 ( Figure 10 ), that is, macrophages expressing the CCR2-IL10RA-RB chimeric protein could resist M1-type polarization. Figure 10 In [the experiment], the only difference between the control group (Con group) and the experimental group was that no LNP-mRNA was transfected.

[0099] Example 8 TIMPs siRNA combined with CCR2-IL10RA-RB mRNA-engineered macrophages have stronger in vivo anti-fibrotic effects

[0100] Bleomycin is currently the most widely recognized and used pulmonary fibrosis-inducing drug. After being administered by bronchial atomization, it can concentrate 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 cause pneumonia and fibrosis. The bleomycin-induced pulmonary fibrosis model is often used to evaluate the in vivo anti-fibrotic effects of drugs.

[0101] In this example, siRNAs (si#1 / 4 / 9) targeting TIMP1, TIMP2, and TIMP3 proteins and chimeric expression protein CCR2-IL10RA-RB mRNA (SEQ ID NO: 58) were simultaneously delivered into macrophages via LNP. After engineering the macrophages, they were injected via the tail vein of mice for treatment to reverse pulmonary fibrosis in mice.

[0102] 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 to prepare: unmodified macrophages (directly cryopreserved), macrophages transfected with LNP-siRNA (si#1 / 4 / 9) alone (the final concentrations of the 3 siRNAs were 25 μM respectively), macrophages transfected with CCR2-IL10RA-RB mRNA alone (1.0 μg of mRNA was added to every 1×10 6 macrophages), and macrophages co-transfected with LNP-siRNA (si#1 / 4 / 9) and CCR2-IL10RA-RB mRNA. After 6 h of transfection, the cells were collected, centrifuged at 300 g for 10 min, and then 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.

[0103] After 6-week-old NOD-SCID mice were treated with 50 mg / Kg bleomycin by pulmonary atomization to construct a pulmonary fibrosis model, 1 week later, the engineered macrophages of the above different groups were injected via the tail vein (1×10 6 cells / mouse), once a week for a total of three times. Three weeks after treatment, the mice were euthanized, and lung tissues were taken to detect the level of hydroxyproline, one of the main components of collagen. It was found that compared with unmodified macrophages and macrophages modified with siRNA (si#1 / 4 / 9) or CCR2-IL10RA-RB mRNA (R2-AB) alone, the treatment group of macrophages co-modified with si#1 / 4 / 9 and R2-AB had lower hydroxyproline levels ( Figure 11 A); 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 in the lung tissues after treatment with macrophages co-modified with siRNA (si#1 / 4 / 9) and CCR2-IL10RA-RB mRNA (R2-AB) were lower ( Figure 11 B); Masson staining and statistical analysis of lung tissue collagen showed that after treating the pulmonary fibrosis model with macrophages co-modified with siRNA (si#1 / 4 / 9) and CCR2-IL10RA-RB mRNA (R2-AB), the extracellular matrix collagen content was less ( Figure 11 C, D); At the same time, staining of the myofibroblast marker protein α-SMA in lung tissues confirmed that the co-modified macrophages had a better therapeutic effect on pulmonary fibrosis compared with unmodified or singly modified macrophages ( Figure 11 E, F).

[0104] Example 9 MMP9-CD mRNA combined with CCR2-IL10RA-RB mRNA engineered macrophages have stronger in vivo anti-fibrotic effects

[0105] In this example, MMP9-CD#3 mRNA (SEQ ID NO: 39) and chimeric expressed protein CCR2-IL10RA-RB mRNA (SEQ ID NO: 48) were simultaneously delivered into macrophages via LNP. After engineering the macrophages, they were injected via the tail vein of mice for treatment to achieve the reversal of pulmonary fibrosis in mice.

[0106] 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 (directly cryopreserved), macrophages transfected with MMP9-CD#3 mRNA (9CD) alone (per 1×10 6mRNA (1.0 μg) was added to each macrophage, and macrophages were transfected with CCR2-IL10RA-RB mRNA (R2-AB) alone (1.0 μg of mRNA was added to each 1×10 6 macrophages), and macrophages were co-transfected with MMP9-CD#3 mRNA and CCR2-IL10RA-RB mRNA. 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.

[0107] One week after establishing a pulmonary fibrosis model by treating 6-week-old NOD-SCID mice with 50 mg / kg bleomycin via pulmonary atomization, the engineered macrophages of the above different groups were injected via the tail vein (1×10 6 cells / mouse), once a week for a total of three times. Three weeks after treatment, the mice were euthanized, and lung tissues were taken to detect the level of hydroxyproline, one of the main components of collagen. It was found that compared with macrophages without modification and macrophages modified with MMP9-CD#3 mRNA (9CD) or CCR2-IL10RA-RB mRNA (R2-AB) alone, the treatment group of macrophages co-modified with 9CD and R2-AB had a lower hydroxyproline level ( Figure 12 A); qPCR was used to detect the expression levels of fibrosis-related genes. It was found that the expression levels of the collagen-related genes Timp1 and Col3a1 in the lung tissues after treatment with macrophages co-modified with MMP9-CD#3 mRNA (9CD) and CCR2-IL10RA-RB mRNA (R2-AB) were lower ( Figure 12 B); Masson staining of lung tissue collagen and statistical analysis showed that after treating the pulmonary fibrosis model with macrophages co-modified with MMP9-CD#3 mRNA (9CD) and CCR2-IL10RA-RB mRNA (R2-AB), the extracellular matrix collagen content was less ( Figure 12 C, D); at the same time, staining of the myofibroblast marker protein α-SMA in lung tissue confirmed that the co-modified macrophages had a better effect on treating pulmonary fibrosis than unmodified or singly modified macrophages ( Figure 12 E, F). The primers involved in qPCR are shown in Table 6.

[0108] Table 6 qPCR primers

[0109]

Claims

1. A transmembrane fusion protein, characterized in that, The N-terminus of the transmembrane fusion protein is the extracellular domain of the CCR2 protein and its seven transmembrane domains, and the C-terminus is successively composed of the intracellular domain of IL10RA and the intracellular domain of IL10RB; the extracellular domain of the CCR2 protein and its seven transmembrane domains are composed of the amino acid sequence shown in SEQ ID NO: 40, the intracellular domain of IL10RA is composed of the amino acid sequence shown in SEQ ID NO: 41, and the intracellular domain of IL10RB is composed of the amino acid sequence shown in SEQ ID NO:

42.

2. The transmembrane fusion protein according to claim 1, wherein the transmembrane fusion protein is composed of the amino acid sequence shown in SEQ ID NO:

43.

3. A nucleic acid, characterized in that, The nucleic acid encodes the transmembrane fusion protein according to any one of claims 1-2; wherein, the nucleic acid is DNA or mRNA.

4. The nucleic acid according to claim 3, wherein the nucleic acid encoding the extracellular domain of the CCR2 protein and its seven transmembrane domains comprises the nucleotide sequence shown in SEQ ID NO: 44 or its degenerate sequence, and / or the nucleic acid encoding the intracellular domain of IL10RA comprises the nucleotide sequence shown in SEQ ID NO: 45 or its degenerate sequence, and / or the nucleic acid encoding the intracellular domain of IL10RB comprises the nucleotide sequence shown in SEQ ID NO: 46 or its degenerate sequence.

5. The nucleic acid according to claim 3 or 4, which comprises the nucleotide sequence shown in SEQ ID NO: 47 or its degenerate sequence.

6. The nucleic acid according to claim 5, which contains the nucleotide sequence shown in SEQ ID NO:

48.

7. A nano-lipid particle-nucleic acid complex, characterized in that, The complex comprises the nucleic acid according to any one of claims 3-6 and a nano-lipid particle (LNP), wherein the nucleic acid is mRNA.

8. The composite according to claim 7, wherein, The nano-lipid particle contains an ionizable cationic lipid, a neutral auxiliary phospholipid, a sterol and a PEG lipid, wherein the sterol is a mixture of cholesterol and dexamethasone.

9. The composite according to claim 8, wherein, The molar ratio of cholesterol to dexamethasone in the cholesterol and dexamethasone mixture is 9:

1.

10. Engineered macrophages, characterized in that, The macrophage contains the transmembrane fusion protein according to any one of claims 1-2 or the nucleic acid according to any one of claims 3-6.

11. The macrophage according to claim 10, which further comprises one or more siRNAs selected from siRNA targeting TIMP1, siRNA targeting TIMP2 and siRNA targeting TIMP3.

12. The macrophage according to claim 11, wherein the siRNA targeting TIMP1 comprises the sense sequence of SEQ ID NO: 1 and the antisense sequence of SEQ ID NO: 2; and / or the siRNA targeting TIMP2 comprises the sense sequence of SEQ ID NO: 7 and the antisense sequence of SEQ ID NO: 8; and / or the siRNA targeting TIMP3 comprises the sense sequence of SEQ ID NO: 17 and the antisense sequence of SEQ ID NO:

18.

13. The macrophage according to any one of claims 11-12, further comprising a nucleic acid encoding a fusion protein of a leader secretion signal of IL12B and a MMP catalytic activity domain.

14. The macrophage according to claim 13, wherein the leader secretion signal of IL12B comprises the amino acid sequence shown in SEQ ID NO: 31, and the MMP catalytic activity domain comprises the amino acid sequence shown in SEQ ID NO:

32.

15. The macrophage according to claim 14, wherein the fusion protein comprises the amino acid sequence shown in SEQ ID NO:

33.

16. The macrophage according to claim 14, wherein the nucleic acid encoding the leader secretion signal of IL12B comprises the nucleotide sequence shown in SEQ ID NO: 36 or its degenerate sequence, and / or the nucleic acid encoding the MMP catalytic activity domain comprises the nucleotide sequence shown in SEQ ID NO: 37 or its degenerate sequence.

17. The macrophage according to claim 16, wherein the nucleic acid encoding the fusion protein comprises the nucleotide sequence shown in SEQ ID NO:

38.

18. A method for preparing engineered macrophages, characterized in that, It includes introducing the nucleic acid according to any one of claims 3-6 or the complex according to any one of claims 7-9 into macrophages.

19. The method according to claim 18, which includes introducing the nano-lipid particle-nucleic acid complex according to any one of claims 7-9 and an LNP coated with one or more of siRNAs targeting TIMP1, siRNAs targeting TIMP2, and siRNAs targeting TIMP3 into macrophages simultaneously or successively.

20. The method according to claim 19, wherein the siRNA targeting TIMP1 comprises the sense sequence of SEQ ID NO: 1 and the antisense sequence of SEQ ID NO: 2; and / or the siRNA targeting TIMP2 comprises the sense sequence of SEQ ID NO: 7 and the antisense sequence of SEQ ID NO: 8; and / or the siRNA targeting TIMP3 comprises the sense sequence of SEQ ID NO: 17 and the antisense sequence of SEQ ID NO:

18.

21. The method according to claim 18, which includes introducing the nano-lipid particle-nucleic acid complex according to any one of claims 7-9 and an LNP coated with a nucleic acid encoding a fusion protein of a leader secretion signal of IL12B and a MMP catalytic activity domain into macrophages simultaneously or successively, wherein the leader secretion signal of IL12B comprises the amino acid sequence shown in SEQ ID NO: 31, and the MMP catalytic activity domain comprises the amino acid sequence shown in SEQ ID NO:

32.

22. The method according to claim 21, wherein the fusion protein comprises the amino acid sequence shown in SEQ ID NO:

33.

23. The method according to claim 21, wherein the nucleic acid encoding the leader secretion signal of IL12B comprises the nucleotide sequence shown in SEQ ID NO: 36 or its degenerate sequence, and / or the nucleic acid encoding the MMP catalytic domain comprises the nucleotide sequence shown in SEQ ID NO: 37 or its degenerate sequence.

24. The method according to claim 23, wherein the nucleic acid encoding the fusion protein comprises the nucleotide sequence shown in SEQ ID NO: 38 or its degenerate sequence.

25. The method according to claim 24, wherein the encoding nucleic acid is mRNA, which comprises the nucleotide sequence shown in SEQ ID NO:

39.

26. Engineered macrophages obtained by the method according to any one of claims 18-25.

27. Use of the engineered macrophages according to any one of claims 10-17 or claim 26 in the preparation of a medicament for treating fibrotic diseases.

28. The use according to claim 27, wherein the fibrotic diseases include idiopathic pulmonary fibrosis, radiation-induced pulmonary fibrosis, pneumoconiosis, cystic fibrosis, liver cirrhosis, myocardial fibrosis, renal interstitial fibrosis, scleroderma, keloid, pancreatic fibrosis, retroperitoneal fibrosis, systemic sclerosis and / or myelofibrosis.

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