Macrophage reprogrammed by mRNA (messenger ribonucleic acid) and anti-fibrosis application thereof

Abstract

The invention discloses an mRNA reprogrammed macrophage and an anti-fibrosis application thereof, and belongs to the technical field of biological medicines. The macrophage comprises an mRNA (messenger ribonucleic acid) encoding a BCL2 protein and optionally an mRNA encoding an IL10 protein and an mRNA encoding a BAMBI protein. According to the invention, the macrophages are engineered and modified by mRNA, the survival of the anti-inflammatory repair type macrophages is improved by introducing the mRNA encoding BCL2, and the anti-inflammatory ability and extracellular matrix degradation ability of the engineered macrophages in inflammatory fibrosis tissues are promoted.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and particularly relates to an mRNA reprogrammed macrophage and application thereof in anti-fibrosis. BACKGROUND

[0002] Fibrosis is a pathological process of tissue or organ damage repair, and its core feature is the excessive deposition of extracellular matrix (ECM) (such as collagen, fibronectin, and elastin) driven by fibroblasts and their activated form-myofibroblasts, which leads to the destruction of tissue structure and function. Various factors can lead to the occurrence and development of fibrosis, and chronic inflammation is one of the main causes of fibrosis. Inflammatory environment recruits inflammatory cells (such as macrophages and T cells) and promotes them to release pro-inflammatory factors (such as TGF-β, IL-1β, and TNF-α), stimulates fibroblast activation and promotes ECM production and deposition; TGF-β (transforming growth factor-β) and its downstream IL11 signaling pathway are key regulators of fibrosis, which can promote the transformation of fibroblasts into myofibroblasts; PDGF (platelet-derived growth factor) can promote fibroblast proliferation and migration; Wnt / β-catenin pathway is also involved in the occurrence and progression of fibrosis. In addition, the production and increase of reactive oxygen species (ROS) lead to tissue damage, cell aging and pro-fibrotic factors released by apoptosis, which also exacerbate the process of fibrosis. Fibrosis can involve multiple organs, such as lungs, liver, heart, and kidneys, and is a common end-stage pathological manifestation of many chronic diseases.

[0003] Pulmonary fibrosis occurs in the scar tissue produced during the repair process of damaged lung tissue. This thickened and stiff tissue makes the lungs work harder, and in clinical practice, patients mainly show symptoms such as dyspnea, dry cough, fatigue, and easy fatigue. Medical imaging tests show that a large amount of collagen fibers are deposited in the lungs of patients with pulmonary fibrosis, a large number of alveoli are lost, and the normal lung tissue structure is changed, and the patient's gas exchange function gradually loses, and eventually dies of respiratory failure. The factors of pulmonary fibrosis damage are various, including long-term exposure to certain toxins, certain medical conditions, radiotherapy, and certain drugs. However, in most cases, doctors cannot identify the root cause of the problem, so it is called idiopathic pulmonary fibrosis (IPF, idiopathic pulmonary fibrosis). IPF patients account for 30%-50% of all pulmonary fibrosis patients.

[0004] Clinically, anti-inflammatory, antioxidant and fibrosis-inhibiting treatment methods are mainly used, but these methods often have limited efficacy and certain side effects. Currently, the only treatment drugs are two types of pirfenidone and nintedanib, and non-drug treatment options include oxygen therapy, pulmonary rehabilitation therapy, and lung transplantation (MSD).

[0005] Cell therapy is an emerging therapeutic strategy that repairs damaged tissue, modulates immune responses, or inhibits pathological processes by transplanting or activating specific types of cells. Cell therapy may achieve long-term efficacy through paracrine effects and cell replacement. In the treatment of fibrosis, cell therapy has shown great potential, particularly in modulating inflammation, inhibiting fibrosis progression, and promoting tissue regeneration. Macrophages, as an important component of the immune system, can simultaneously modulate inflammation, inhibit fibrosis, and promote tissue repair. Macrophages have demonstrated anti-fibrotic potential in preclinical studies, and GMP-compliant autologous mononuclear cell-derived macrophage products have been used in clinical research to explore their safety and efficacy in patients with cirrhosis (MATCH, ISRCTN10368050; NCT06671275).

[0006] In recent years, strategies for modifying cell function based on RNA-regulated gene expression have shown great potential in the field of disease treatment. An RNA-modified macrophage has been validated for safety and efficacy in patients with end-stage liver disease (NCT06823713). Maintaining the anti-inflammatory repair phenotype of macrophages, improving the inflammatory environment of fibrotic tissues, and enhancing the ECM targeting and degradation capabilities of macrophages are important directions in the anti-fibrotic engineering of macrophages. For example, in patent WO2024068728A1, the ECM degradation ability of macrophages is enhanced by expressing MMP9 and MMP12; in patent WO2024074376A1, the anti-inflammatory and ECM degradation abilities of macrophages are enhanced by expressing MMP9 and IL-10; in patent CN112236445A, ECM-targeting proteins and proteases are introduced into macrophages to enhance their ECM targeting and degradation abilities; and patent CN115590880A discloses an engineered macrophage that can continuously express IL-10, sTGFR-Fc, or CD147 by means of lentivirus, which can be used for anti-fibrotic therapy.

[0007] Current methods for selecting targets in engineered macrophage modification still have certain limitations. Regarding maintaining / promoting the anti-inflammatory effects of macrophages, the focus is mainly on anti-inflammatory cytokines like IL-10; and regarding promoting the degradation of ECM in fibrotic tissue, this is mainly achieved through exogenous expression of matrix metalloproteinases such as MMP9.

[0008] Engineered macrophages are administered via intravenous infusion. Improving the survival of macrophages targeting fibrotic tissue and enhancing their anti-inflammatory and extracellular matrix (ECM) degradation capabilities within the fibrotic tissue microenvironment are key directions in the engineering of macrophages. Currently, no existing technology has been reported to simultaneously promote macrophage survival, reduce inflammation in fibrotic tissue, and enhance ECM degradation through single-gene modification.

[0009] BCL2 (B-cell lymphoma 2) is a core member of the anti-apoptotic protein family (BCL2 family), possessing functions such as inhibiting apoptosis, regulating autophagy, and participating in cellular metabolism. Currently, there are no studies or reports on the engineering of macrophages based on BCL2 expression. Summary of the Invention

[0010] To address the above technical problems, this invention utilizes mRNA-engineered macrophages. By introducing mRNA encoding BCL2, it improves the survival of anti-inflammatory and repair-promoting macrophages, thereby enhancing the anti-inflammatory capacity and extracellular matrix degradation ability of engineered macrophages in inflammatory fibrotic tissues. Specifically, The first aspect of the present invention provides engineered macrophages comprising introduced mRNA encoding the BCL2 protein.

[0011] In some embodiments, the BCL2 protein comprises the amino acid sequence shown in SEQ ID NO: 1 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 1.

[0012] In some embodiments, the mRNA encoding the BCL2 protein comprises the nucleotide sequence shown in any one of SEQ ID NO: 2-5 or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with any one of SEQ ID NO: 2-5.

[0013] In some embodiments, the mRNA encoding the BCL2 protein comprises the nucleotide sequence shown in any one of SEQ ID NO: 6-9 or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with any one of SEQ ID NO: 6-9.

[0014] In some embodiments, the engineered macrophages further comprise introduced mRNA encoding IL10 protein and / or BAMBI protein.

[0015] In some embodiments, the IL10 protein comprises the amino acid sequence shown in SEQ ID NO: 16 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 16.

[0016] In some embodiments, the BAMBI protein comprises the amino acid sequence shown in SEQ ID NO: 17 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 17.

[0017] In some embodiments, the mRNA encoding the IL10 protein comprises the nucleotide sequence shown in SEQ ID NO: 18 or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 18.

[0018] In some embodiments, the mRNA encoding the BAMBI protein comprises a nucleotide sequence as shown in SEQ ID NO: 19 or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 19.

[0019] In some embodiments, the mRNA encoding the IL10 protein comprises the nucleotide sequence shown in SEQ ID NO: 20 or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 20.

[0020] In some embodiments, the mRNA encoding the BAMBI protein comprises the nucleotide sequence shown in SEQ ID NO: 21 or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 21.

[0021] In some embodiments, the mRNA encoding the BCL2 protein is introduced into macrophages via nanolipid particle-nucleic acid complex or via electroporation.

[0022] A second aspect of the present invention provides a lipid nanoparticle-nucleic acid complex, the complex comprising mRNA encoding BCL2 protein and lipid nanoparticles (LNP); the mRNA comprising the nucleotide sequence shown in any one of SEQ ID NO: 2-5.

[0023] In some embodiments, the mRNA comprises a nucleotide sequence shown in any one of SEQ ID NO: 6-9 or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with any one of SEQ ID NO: 6-9.

[0024] In some embodiments, the nanolipid particles comprise ionizable cationic lipids, neutral cofactor phospholipids, sterols, and PEG lipids, wherein the sterols are a mixture of cholesterol and dexamethasone.

[0025] In some implementations, the molar ratio of cholesterol to dexamethasone in the cholesterol and dexamethasone mixture is 9:1.

[0026] A third aspect of the present invention provides a method for preparing engineered macrophages, comprising introducing the complex described in the second aspect of the present invention into macrophages.

[0027] The fourth aspect of the present invention provides a method for preparing engineered macrophages, comprising introducing mRNA encoding BCL2 protein, mRNA encoding IL10 and mRNA encoding BAMBI into macrophages.

[0028] In some embodiments, the BCL2 protein comprises the amino acid sequence shown in SEQ ID NO: 1 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 1.

[0029] In some embodiments, the IL10 protein comprises the amino acid sequence shown in SEQ ID NO: 16 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 16.

[0030] In some embodiments, the BAMBI protein comprises the amino acid sequence shown in SEQ ID NO: 17 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 17.

[0031] In some embodiments, the mRNA encoding the BCL2 protein comprises the nucleotide sequence shown in any one of SEQ ID NO: 2-5 or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with any one of SEQ ID NO: 2-5.

[0032] In some embodiments, the mRNA encoding the IL10 protein comprises the nucleotide sequence shown in SEQ ID NO: 18 or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with any one of SEQ ID NO: 18.

[0033] In some embodiments, the mRNA encoding the BAMBI protein comprises the nucleotide sequence shown in SEQ ID NO: 19 or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with any one of SEQ ID NO: 19.

[0034] In some embodiments, the mRNA encoding BCL2 protein, the mRNA encoding IL10, and the mRNA encoding BAMBI are simultaneously or sequentially introduced into macrophages.

[0035] The fifth aspect of the present invention provides engineered macrophages prepared according to the method described in the fourth aspect of the present invention.

[0036] The sixth aspect of the present invention provides the use of engineered macrophages according to the first or fifth aspect of the present invention in the preparation of medicaments for the prevention and / or treatment of fibrotic diseases.

[0037] In some implementations, the fibrotic diseases include idiopathic pulmonary fibrosis, radiation-induced pulmonary fibrosis, pneumoconiosis, cystic fibrosis, cirrhosis, myocardial fibrosis, renal interstitial fibrosis, scleroderma, keloids, pancreatic fibrosis, retroperitoneal fibrosis, systemic sclerosis, and / or myelofibrosis.

[0038] In the engineered macrophages provided by this invention, BCL2 participates in regulating macrophage survival, polarization, and function. Macrophages with high BCL2 expression have stronger survival ability in inflammatory environments; BCL2 regulates macrophage polarization by modulating macrophage OXPHOS (oxidative phosphorylation) and glycolysis. Anti-inflammatory and repair-promoting macrophages have higher levels of BCL2.

[0039] The advantages of this invention over the prior art are: 1. For the first time, engineered macrophages expressing BCL2 protein were constructed, which have the characteristics of improving the survival of anti-inflammatory and repair-type macrophages and promoting the anti-inflammatory ability and extracellular matrix degradation ability of engineered macrophages in inflammatory fibrotic tissues.

[0040] 2. By encapsulating and delivering BCL2 mRNA to macrophages using inflammation-suppressing lipid nanoparticles, BCL-2-engineered macrophages exhibit stronger anti-fibrotic effects.

[0041] 3. Based on the modification of BCL2 mRNA, the simultaneous introduction of mRNA expressing the anti-inflammatory cytokine IL10 and / or mRNA expressing the negative regulator of the TGF-β signaling pathway BAMBI can further enhance the anti-fibrotic effect of macrophages. Attached Figure Description

[0042] Other features, objects, and advantages of the present invention will become more apparent from the following detailed description of non-limiting embodiments with reference to the accompanying drawings: Figure 1 Demonstrates the preparation of BCL2 mRNA. Figure 1 A shows a schematic diagram of BCL2-encoding mRNAs with different degenerate sequences (BCL2-O0 mRNA CDS sequence is from NCBI database); Figure 1 B shows the four mRNAs prepared by agarose gel electrophoresis analysis.

[0043] Figure 2 This shows a comparison of the expression of different BCL2 mRNAs. Figure 2 A shows the percentage of BCL2 mRNA expression in macrophages with different expression levels as analyzed by flow cytometry; Figure 2 B shows flow cytometry analysis of the expression intensity of different BCL2 mRNAs in macrophages (mean fluorescence intensity of BCL2). Figure 2 C shows the expression of BCL2-O1 mRNA in macrophages using flow cytometry.

[0044] Figure 3 BCL2 mRNA was shown to promote macrophage survival. Figure 3 Figure A shows the flow cytometry analysis of macrophage viability 1 day after transfection with empty vector LNP control and BCL2-O1 LNP-mRNA; Figure 3 B shows the number of macrophages after 14 days of culture, as analyzed by cell counting of transfected empty vector LNP control and BCL2-O1 LNP-mRNA.

[0045] Figure 4 The results showed that BCL2 mRNA did not affect the macrophage ectotype. Figure 4 A shows the expression intensity of CD80 (M1 marker gene) in macrophages 2 days after transfection with empty LNP and BCL2-O1 LNP-mRNA by flow cytometry. Figure 4 B shows the expression intensity of CD206 (M2 marker gene) in macrophages 2 days after transfection with empty LNP and BCL2-O1 LNP-mRNA by flow cytometry. Figure 4 C shows the secretion of the inflammatory cytokine IL-6 by macrophages 2 days after transfection with empty LNP and BCL2-O1 LNP-mRNA using ELISA. Figure 4 D shows the secretion of the anti-inflammatory cytokine IL-10 by macrophages 2 days after transfection with empty vector LNP and BCL2-O1LNP-mRNA.

[0046] Figure 5 BCL2 was shown to reduce the expression of inflammatory factors in the HLF co-culture model. Figure 5 A shows the ELISA analysis of IL-1β secretion in macrophages transfected with empty vector LNP and BCL2-O1 LNP-mRNA after co-culturing with HLF (TGF-β-induced fibrosis) for 1 day; Figure 5 B shows the ELISA analysis of IL-6 secretion in macrophages transfected with empty vector LNP and BCL2-O1 LNP-mRNA after co-culturing with HLF (TGF-β-induced fibrosis) for 1 day; Figure 5 C shows the ELISA analysis of IL-8 secretion in macrophages transfected with empty vector LNP and BCL2-O1 LNP-mRNA after co-culturing with HLF (TGF-β-induced fibrosis) for 1 day; Figure 5 D-display ELISA analysis was performed on macrophages transfected with empty vector LNP and BCL2-O1 LNP-mRNA and co-cultured with HLF (TGF-β-induced fibrosis) for 1 day to determine TNF-α secretion.

[0047] Figure 6 BCL2 showed that it reduced the expression of inflammatory factors in the LX2 co-culture model. Figure 6 A shows the secretion of IL-1β by ELISA analysis of macrophages transfected with empty vector LNP and BCL2-O1 LNP-mRNA and co-cultured with LX2 (TGF-β-induced fibrosis) for 1 day; Figure 6 B shows the ELISA analysis of IL-6 secretion in macrophages transfected with empty vector LNP and BCL2-O1 LNP-mRNA after co-culturing with LX2 (TGF-β-induced fibrosis) for 1 day; Figure 6 C shows the ELISA analysis of IL-8 secretion in macrophages transfected with empty LNP and BCL2-O1 LNP-mRNA after co-culturing with LX2 (TGF-β-induced fibrosis) for 1 day; Figure 6 D-display ELISA analysis was performed on macrophages transfected with empty LNP and BCL2-O1 LNP-mRNA and co-cultured with LX2 (TGF-β-induced fibrosis) for 1 day to determine TNF-α secretion.

[0048] Figure 7 The study showed that BCL2 enhances the in vitro anti-fibrotic effect of macrophages. Figure 7 A shows the secretion of TIMP1 protein by macrophages transfected with empty LNP and BCL2-O1 LNP-mRNA after 1 day of culture using ELISA. Figure 7B shows the expression intensity of Collagen I in HLF after macrophages transfected with empty LNP and BCL2-O1 LNP-mRNA were co-cultured with HLF (TGF-β-induced fibrosis) for 1 day using flow cytometry. Figure 7 C shows the expression intensity of Collagen I in LX2 cells after macrophages transfected with empty LNP and BCL2-O1LNP-mRNA were co-cultured with LX2 (TGF-β-induced fibrosis) for 1 day using flow cytometry. Figure 8 The study showed that BCL2 promotes the in vivo anti-pulmonary fibrosis effect of macrophages. Figure 8 A shows the detection of hydroxyproline (HYP) content in lung tissue of different treatment groups using acid hydrolysis (spectroscopy); Figure 8 B and Figure 8 C shows the expression levels of Col1a1 and Col3a1 mRNA in lung tissues of different treatment groups, as analyzed by qPCR. Figure 8 D shows the Masson staining results of lung tissue from different treatment groups; Figure 8 E shows the quantitative analysis results of Masson staining in lung tissues from different treatment groups.

[0049] Figure 9 The results showed that BCL2 combined with IL10 and BAMBI engineered macrophages had a stronger in vitro anti-pulmonary fibrosis effect. Figure 9 A shows the expression intensity of Collagen I in HLF after macrophages transfected with empty vector LNP, BCL2-O1 and mixRNA (BCL2-O1, IL10 and BAMBI) were co-cultured with HLF (TGF-β induced fibrosis) for 1 day by flow cytometry analysis. Figure 9 B shows flow cytometry analysis of the expression intensity of Collagen I in LX2 cells after macrophages transfected with empty vector LNP, BCL2-O1 and mixRNA (BCL2-O1, IL10 and BAMBI) were co-cultured with LX2 (TGF-β induced fibrosis) for 1 day. Figure 10 The results showed that BCL2 combined with IL10 and BAMBI engineered macrophages had a stronger in vivo anti-pulmonary fibrosis effect. Figure 10 A shows the content of hydroxyproline (HYP) in the lung tissue of model mice after treatment with macrophages, BCL2-engineered macrophages, and mixRNA-engineered macrophages (BCL2-O1, IL10, and BAMBI) using an acid hydrolysis method (spectrophotometry). Figure 10 B shows the expression level of Col1a1 mRNA in lung tissues of different treatment groups analyzed by qPCR; Figure 10C shows the expression level of Col3a1 mRNA in lung tissues of different treatment groups using qPCR analysis; Figure 10 D shows the Masson staining results of lung tissue from different treatment groups; Figure 10 E shows the quantitative analysis results of Masson staining in lung tissues from different treatment groups.

[0050] Figure 11 The results showed that BCL2 combined with IL10 and BAMBI engineered macrophages had a stronger in vivo anti-liver fibrosis effect. Figure 11 A shows the expression level of Col1a1 mRNA in liver tissue of mice with liver fibrosis after treatment with macrophages, whether or not the mRNA was engineered, using qPCR analysis. Figure 11 B shows the expression level of Col3a1 mRNA in liver tissue of mice with liver fibrosis after treatment with macrophages, regardless of whether the mRNA was engineered or not, using qPCR analysis. Figure 11 C shows the expression and quantitative analysis results of α-SMA in liver tissue of different treatment groups using immunohistochemical analysis. Figure 11 D shows the results of Sirius red staining analysis of collagen staining in liver tissue of different treatment groups and quantitative analysis. Detailed Implementation

[0051] 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. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. Based on the described embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0052] Unless otherwise defined, the technical or scientific terms used herein shall have the ordinary meaning as understood by one of ordinary skill in the art to which this invention pertains.

[0053] Example 1: BCL2 mRNA coding frame sequence optimization and LNP-mRNA preparation mRNAs with protein expression activity typically consist of a cap structure, a 5' UTR sequence (SEQ ID NO: 10), a protein coding frame sequence, a 3' UTR sequence (SEQ ID NO: 11), and a poly A sequence (SEQ ID NO: 12). Optimization of the mRNA coding frame (ORF) is a key step in improving mRNA stability, translation efficiency, and protein expression levels. In this embodiment, the BCL2 protein coding sequence (NM_000633.3) from the database was used as the original sequence (BCL-O0). Three open online mRNA sequence optimization software programs—GenSmart™ codon optimization (https: / / www.genscript.com.cn / tools / gensmart-codon-optimization?page=1), mRNArchitect (https: / / app.basefacility.org.au / ), and mRNAid (https: / / mrnaid.dichlab.org / )—were used to design BCL2-O1, BCL2-O2, and BCL2-O3 (…). Figure 1 A), Sequence information is shown in Table 3.

[0054] The pUC57-Kan vector was used as the backbone vector, which mainly included the DNA replication initiation site, the kanamycin resistance gene, and its promoter. The T7 promoter sequence (SEQ ID NO: 22), 5' UTR sequence (SEQ ID NO: 10), protein coding frame sequence, 3' UTR sequence (SEQ ID NO: 11), poly A sequence (SEQ ID NO: 12), and linearized restriction enzyme sites were introduced through the multiple cloning site. The constructed vector was verified by sequencing, and the supercoiled proportion of the prepared plasmid should be greater than 60%. The 5' UTR sequence consists of the HBB (hemoglobin) mRNA 5UTR sequence (SEQ ID NO: 13), the IGG6 sequence (CAATCAAAC), and the Kozak sequence (GCCACC). The 3' UTR sequence is a stem-loop structure sequence derived from histones, and the polyA length should be greater than 100.

[0055] In this embodiment, a multi-step method is used for mRNA preparation, that is, linearization, transcription, and capping reactions are performed in the same tube. After the reaction is completed, the mRNA is purified by magnetic bead method. Figure 1 B). Subsequently, an anti-inflammatory LNP lipid combination (Table 1) was used to prepare LNP-mRNA via microfluidic mixing. The encapsulation efficiency, particle size, PDI, and zeta charge of the LNP-mRNA are shown in Table 2. The specific implementation scheme is as follows: Plasmid linearization: reaction volume 5 μL, restriction endonuclease (BspQI) concentration 1-10 U / μg plasmid, digestion time 0.5-3 h at 50 ℃; In vitro transcription: The total reaction volume was 10 μL. The reaction system included 5 μL of enzyme digestion mixture, T7 transcriptase (nearshore protein, GMP-E122-HC-U100) concentration of 100-500 U / μL, MgCl2 final concentration of 5-40 mM, Tris-HCl final concentration of 50 mM, NTP (replacing UTP with m1ψ) final concentration of 1-5 mM, pyrophosphatase (nearshore protein, GMP-M036-01A) concentration of 0.02 U / μL, and RNase inhibitor (nearshore protein, GMP-E125-M001) concentration of 1 U / μL. The reaction was carried out at 37 ℃ for 0.5-12 h. Capping reaction: The total reaction volume was 100 μL. The reaction system included 10 μL of transcription reaction product, MgCl2 final concentration of 1-8 mM, Tris-HCl final concentration of 50 mM, SAM final concentration of 0.5 mM, GTP final concentration of 1-5 mM, 2-oxomethyltransferase (nearshore protein, GMP-M072-M001) concentration of 50-250 U / μL, and vaccinia capping enzyme (nearshore protein, GMP-M062-M001) final concentration of 50-1000 U / μL. The reaction was carried out at 37 ℃ for 1-2 h.

[0056] mRNA purification: Use 0.5-1 μg of magnetic beads for purification per microliter of capped product.

[0057] During RNA preparation, the above reaction system can be linearly scaled up according to the requirements of mRNA.

[0058] LNP-mRNA encapsulation:

[0059] In this embodiment, the molar percentage of ionizable cationic lipids:neutral cofactor phospholipids:cholesterol:dexamethasone mixture:PEG-lipids was 44.6:8:41.58:4.62:1.2, and the total lipid mixture concentration was 8 mM (6-16 mM), wherein the molar ratio of cholesterol to dexamethasone was 9:1 (Table 1). mRNA was diluted with sodium citrate buffer at pH 4.0 to a final RNA concentration of 101 ng / μ (50-150 ng / μ). During LNP preparation, the N / P ratio (molar ratio of nitrogen in the main lipids to phosphorus in the nucleic acids) was 4-10, preferably 4, and the total flow rate was 4-16 ml / min, preferably 10 ml / min.

[0060] 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 using a tangential flow filtration system. The medium was then replaced 5-10 times with 10 mM Tris (pH 7.4-7.8), and finally concentrated to 1 / 4 of the original volume. The purified LNP-mRNA was collected, added to sucrose solution to a final concentration of 6%, aliquoted, and stored at -80 °C. The LNP-mRNA detection methods included: mass spectrometry to detect capping efficiency (>95%) and tailing distribution; capillary electrophoresis to detect mRNA integrity (>90%); RiboGreen staining to detect encapsulation efficiency (>90%) and mRNA content; and nanoparticle size analysis to detect particle size (80-150 nm), PDI (<0.15), and surface potential (zeta potential, -2 to -10 mV). In this example, the prepared LNP-mRNAs had encapsulation efficiencies greater than 90%, particle sizes less than 150 nm, PDI less than 0.15, and zeta potentials between -10 mV and 0 mV (Table 2).

[0061] Table 1. Lipid composition of anti-inflammatory LNPs

[0062] Table 2. Results of BCL2 LNP-mRNA detection

[0063] Table 3 BCL2 related sequence information

[0064] Example 2: Comparison of expression of different BCL2 mRNAs In this embodiment, macrophages derived from monocyte differentiation were transfected with different BCL2 LNP-mRNAs. The percentage of BCL2 protein expression was then detected by flow cytometry, and the expression intensity of different mRNAs was analyzed. Figure 2 ).

[0065] In this embodiment, the macrophages can be derived from autologous peripheral blood, specifically from autologous peripheral blood mononuclear cells induced from them. Macrophages can be prepared using methods known in the art. For example, PBMCs can be prepared first using Ficoll density gradient centrifugation, then the PBMCs can be processed to isolate mononuclear cells, and finally, the isolated mononuclear cells can be differentiated into macrophages.

[0066] The procedure for preparing PBMCs using the Ficoll density gradient centrifugation method can be as follows: a certain volume of Ficoll is transferred to a centrifuge tube; then whole blood is placed on top of the Ficoll, and the centrifuge tube is centrifuged at room temperature at 200g for 45 minutes to obtain red blood cell precipitate, Ficoll layer, white layer containing PBMCs, and plasma layer; the white layer containing PBMCs is then extracted.

[0067] PBMCs contain monocytes and lymphocytes, therefore, PBMCs can be processed to isolate monocytes. In this embodiment, monocytes and lymphocytes are separated by magnetic bead separation or countercurrent centrifugation and washing, thereby enriching the monocytes.

[0068] Macrophage culture: In this embodiment, the isolated monocytes (or PBMCs can be cultured directly) are induced to obtain macrophages by culturing in differentiation medium containing macrophage colony-stimulating factor (M-CSF, concentration of 10 ng / mL to 100 ng / mL). Culture conditions suitable for macrophage culture are acceptable, and there are no particular limitations in this embodiment. For example, the density of macrophages seeded in the culture medium can be 5 × 10⁶ cells / mL. 5 Cells / mL (can also be expressed as 5E+05 cells / mL in specific experiments). The culture temperature can be 37℃. The culture time can be appropriately set according to the growth status of the macrophages, for example, 5 to 8 days. The culture environment can be a 5% CO2 environment.

[0069] In this embodiment, mononuclear cells were obtained by countercurrent centrifugation and washing, at a ratio of 1×10⁻⁶. 6 Macrophages were obtained by culturing and differentiating cells at a concentration of 10 cells / mL. The specific transfection procedure was as follows: macrophages were collected by centrifugation at 300g for 10 min, the supernatant was removed, and the macrophage pellet was resuspended in fresh culture medium (TexMACS medium) until the final cell concentration was 1×10⁻⁶ cells / mL. 6 / mL, gently mix by pipetting and slowly add different LNP-mRNAs, 1×10⁶ per mL. 6 Add 1 μg (mRNA mass) of BCL2 LNP-mRNA to the cells, then immediately gently pipette the cells to mix them, and then incubate them at 37°C and 5% CO2 for 6 h before collecting the cells.

[0070] Example 3: Effects of BCL2 mRNA on macrophages Following the method in Example 2, macrophages were transfected with empty vector LNP and BCL2-O1 LNP-mRNA. Cells were collected one day later, and flow cytometry analysis revealed that macrophages transfected with empty vector LNP and BCL2-O1 LNP-mRNA exhibited similar cell viability.Figure 3 A). To further investigate the long-term effects of transient BCL2 expression on transfected macrophages, we cultured transfected macrophages for 14 consecutive days. Count analysis revealed that macrophages transfected with BCL2-mRNA had a higher survival rate compared to those transfected with empty LNP. Figure 3 B).

[0071] In addition, the effect of BCL2 protein overexpression on macrophage phenotype was investigated. Two days after transfection of macrophages with empty vector LNP and BCL2-O1 LNP-mRNA, flow cytometry analysis was performed on the macrophage M1 polarization marker gene CD80 (…). Figure 4 A) and M2 polarization marker gene CD206 ( Figure 4 The expression intensity of BCL2 was measured, indicating that BCL2 expression did not affect the expression of CD80 and CD206. ELISA analysis of the culture supernatant from transfected macrophages revealed that BCL2 expression did not alter the expression of the inflammatory cytokine IL-6 (B). Figure 4 C) and anti-inflammatory cytokine IL-10 ( Figure 4 The level of expression of D).

[0072] Example 4: In vitro antifibrotic effect of BCL2 mRNA-engineered macrophages TGF-β-induced fibroblast fibrosis is an important model for studying in vitro anti-fibrotic effects. In an in vitro co-culture model of macrophages and fibrosis-induced fibroblasts, macrophages can inhibit fibroblast fibrosis through multiple pathways, including suppressing the secretion of inflammatory factors and promoting the degradation of the extracellular matrix. HLF cells are immortalized human lung fibroblasts, and LX2 cells are immortalized human hepatic stellate cells; TGF-β can induce fibrotic responses in both.

[0073] HLF and LX2 cells cultured adherently were induced for 48 h with TGF-β at a final concentration of 5 ng / mL, followed by co-culture for 1 day with macrophages transfected with LNP-mRNA (macrophage to fibroblast ratio of 5:1). The culture supernatant of the co-culture system was collected, and ELISA was used to detect inflammatory cytokines. The results showed that, compared with macrophages transfected with empty LNP vector, macrophages overexpressing BCL2-O1 mRNA, after co-culturing with HLF or LX2 cells, exhibited significantly lower levels of the inflammatory cytokines IL-1β and TNF-α in the culture supernatant. Figure 5 A, 5D, 6A, 6D), the differences in inflammatory factors IL-6 and IL-8 were small ( Figure 5 B, 5C, 6B, 6C).

[0074] Flow cytometry analysis of the extracellular matrix component Collagen I was used to analyze the fibrosis-inducing effect of TGF-β on HLF and LX2 in the co-culture system. The results showed that BCL2-O1 mRNA-engineered macrophages could reduce TGF-β-induced Collagen I protein levels. Figure 7 B, 7C).

[0075] In summary, BCL2-O1 mRNA-engineered macrophages express lower levels of the matrix metalloproteinase inhibitor TIMP1 protein (… Figure 7 A) and inhibiting the expression of inflammatory factors in a fibrotic environment ( Figure 5 , 6 This will enable a stronger anti-fibrotic effect.

[0076] Example 5: In vivo antifibrotic effect of BCL2 mRNA-engineered macrophages Bleomycin-induced pulmonary fibrosis is currently the most widely recognized and used model of pulmonary fibrosis. After administration via bronchial nebulization, it concentrates in lung tissue, leading to significant oxidative stress, alveolar epithelial cell death, fibroblast proliferation, and ultimately, immune cell infiltration. Chronic release of pro-inflammatory and pro-fibrotic molecules from immune cells and fibroblasts results in pneumonia and fibrosis. Bleomycin-induced pulmonary fibrosis models are frequently used to evaluate the in vivo anti-fibrotic effects of drugs.

[0077] In this embodiment, macrophages engineered with empty LNP or BCL2-O1 mRNA were injected via the tail vein of mice to treat pulmonary fibrosis. In this embodiment, macrophages differentiated from mononuclear sources were used at a ratio of 1×102 6 Macrophages were cultured at a concentration of 10 cells / mL, and after 6 days of differentiation, macrophages were collected to prepare macrophages transfected with empty vector LNP and BCL2-O1 mRNA (1×10⁶ cells / mL). 6 1.0 μg of mRNA was added to each macrophage. Cells were collected 6 h after transfection, centrifuged at 300 g for 10 min, and then divided into 1×10⁻⁶ mRNA cells. 7 After being programmed to freeze at a concentration of 1 / mL, the sample is stored in liquid nitrogen gas phase for later use.

[0078] Six-week-old NOD-SCID mice were given 75 mg / Kg of bleomycin via pulmonary nebulization to establish a pulmonary fibrosis model. One week later, macrophages from the two groups mentioned above (2×10⁶ cells / kg) were injected via the tail vein. 6Mice were treated with BCL2-O1 mRNA-modified macrophages (1 cell / mouse), once a week for a total of three treatments. One week after the last treatment, the mice were euthanized, and lung tissue samples were collected. The lung tissue was analyzed to detect the level of hydroxyproline, a major component of collagen. It was observed that macrophages modified with BCL2-O1 mRNA had lower hydroxyproline levels compared to macrophages transfected with empty LNP. Figure 8 A); qPCR detection of fibrosis-related gene expression levels revealed that lung tissue treated with macrophages expressing BCL2-O1 mRNA showed lower expression levels of collagen-related genes Col1a1 and Col3a1. Figure 8 B, 8C); Masson staining and statistical analysis of lung tissue collagen showed that lung tissue treated with macrophages expressing BCL2-O1 mRNA had less extracellular matrix collagen content (B, 8C). Figure 8 (D, 8E). Overall, macrophages modified with BCL2-O1 mRNA showed better therapeutic effects on pulmonary fibrosis compared to unmodified macrophages.

[0079] Example 6: Antifibrotic effect of BCL2 combined with IL10 and BAMBI engineered macrophages IL10 is an important anti-inflammatory cytokine that stabilizes the anti-inflammatory phenotype of macrophages and inhibits inflammatory responses in a fibrotic environment. BAMBI is a pseudoreceptor for TGF-β, lacking an intracellular serine / threonine kinase domain. It can competitively bind to the TGF-β receptor, inhibiting downstream signaling pathways, reducing excessive extracellular matrix deposition, and thus alleviating tissue fibrosis.

[0080] To further enhance the antifibrotic effect of BCL2 mRNA-engineered macrophages, IL10 and BAMBI mRNA were introduced into the BCL2 mRNA matrix. The antifibrotic effect of macrophages co-expressing BCL2-O1, IL10, and BAMBI mRNA (mixRNA) was evaluated using in vitro and in vivo models. The sequence information of IL10 and BAMBI mRNA is shown in Table 4 below.

[0081] Empty vector LNP, BCL2-O1 mRNA and mixRNA (each 1×10) were prepared using the same method as in the previous embodiments. 6 One macrophage was added to macrophages transfected with 1.0 μg each of BCL2-O1, IL10, and BAMBI mRNA. The specific transfection procedure was the same as that in Example 2 above, and will not be repeated here.

[0082] After co-culturing engineered macrophages with TGF-β-induced HLF or LX2 cells, flow cytometry analysis of collagen I expression levels in HLF and LX2 cells revealed that macrophages co-expressing BCL2-O1, IL10, and BAMBI mRNA (mixRNA) exhibited stronger inhibitory effects on collagen I protein compared to macrophages transfected solely with BCL2-O1 mRNA. Figure 9 ).

[0083] Using the same research methods as the aforementioned mouse model of pulmonary fibrosis, the results confirmed that, compared with BCL2-O1 mRNA expression alone, macrophages engineered with mixRNA had a stronger in vivo anti-pulmonary fibrosis effect. Figure 10 ).

[0084] The progression and elimination of fibrosis in different organs share similar mechanisms. Using a CCl4-induced liver fibrosis treatment model as an example, this study further explores the therapeutic effects of engineered macrophages on fibrosis in other organs besides pulmonary fibrosis.

[0085] In the example, 4-5 week old mice were intraperitoneally injected with CCl4 (0.5 mL / kg) twice a week for 6 consecutive weeks to model the growth of macrophages. Then, they were treated with different macrophages prepared above (4 × 10⁻⁶) via tail vein injection. 6 Mice were treated with a single cell per mouse, once a week for a total of three weeks. One week after the last treatment, mice were euthanized, and liver tissue samples were collected. qPCR analysis of fibrosis-related gene expression levels revealed that, compared to unmodified macrophages, liver tissue treated with mixRNA-engineered macrophages showed lower expression levels of collagen-related genes col1a1 and col3a1. Figure 11 A, 11B); α-SMA staining and statistical analysis of liver tissue sections showed that after treatment with macrophages expressing mixRNA, the expression area of ​​the myofibroblast activation marker protein α-SMA was reduced more significantly. Figure 11 C). Sirius red staining and statistical analysis of liver tissue collagen showed that the extracellular matrix collagen content in liver tissue treated with macrophages expressing mixRNA was lower. Figure 11 D). Overall, macrophages engineered with BCL2-O1, IL10, and BAMBI mRNA showed better therapeutic effects on liver fibrosis compared to unengineered macrophages.

[0086] Table 4 Sequence information of IL10 and BAMBI mRNA

[0087] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. It will be apparent to those skilled in the art that the invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from its spirit or essential characteristics. Therefore, the embodiments should be considered illustrative and non-limiting in all respects, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, all variations falling within the meaning and scope of equivalents of the claims are intended to be included within the scope of the invention. No reference numerals in the claims should be construed as limiting the scope of the claims.

[0088] Furthermore, it should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This description is merely for clarity. Those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. Engineered macrophages containing introduced mRNA encoding the BCL2 protein.

2. The engineered macrophage according to claim 1, wherein the BCL2 protein comprises the amino acid sequence shown in SEQ ID NO:

1.

3. The engineered macrophage according to claim 1 or 2, wherein the mRNA encoding the BCL2 protein comprises the nucleotide sequence shown in any one of SEQ ID NO: 2-5.

4. The engineered macrophage according to claim 3, wherein the mRNA encoding the BCL2 protein comprises the nucleotide sequence shown in any one of SEQ ID NO: 6-9.

5. The engineered macrophage of claim 1, further comprising introduced mRNA encoding IL10 protein and / or encoding BAMBI protein.

6. The engineered macrophage according to claim 5, wherein the IL10 protein comprises the amino acid sequence shown in SEQ ID NO: 16, and / or the BAMBI protein comprises the amino acid sequence shown in SEQ ID NO:

17.

7. The engineered macrophage according to claim 6, wherein the mRNA encoding the IL10 protein comprises the nucleotide sequence shown in SEQ ID NO: 18, and / or the mRNA encoding the BAMBI protein comprises the nucleotide sequence shown in SEQ ID NO:

19.

8. The engineered macrophage according to claim 7, wherein the mRNA encoding the IL10 protein comprises the nucleotide sequence shown in SEQ ID NO: 20, and / or the mRNA encoding the BAMBI protein comprises the nucleotide sequence shown in SEQ ID NO:

21.

9. The engineered macrophages according to claim 1 or 2, characterized in that, The mRNA encoding the BCL2 protein is introduced into macrophages via either a nanolipid particle-nucleic acid complex or via electroporation.

10. A method for preparing engineered macrophages, characterized in that, This includes introducing mRNA encoding BCL2 protein, and / or mRNA encoding IL-10, and / or mRNA encoding BAMBI into macrophages.

11. The method of claim 10, wherein the BCL2 protein comprises the amino acid sequence shown in SEQ ID NO: 1, and / or the IL10 protein comprises the amino acid sequence shown in SEQ ID NO: 16, and / or the BAMBI protein comprises the amino acid sequence shown in SEQ ID NO:

17.

12. The method according to claim 10 or 11, wherein the mRNA encoding the BCL2 protein comprises the nucleotide sequence shown in any one of SEQ ID NO: 2-5, and / or the mRNA encoding the IL10 protein comprises the nucleotide sequence shown in SEQ ID NO: 18, and / or the mRNA encoding the BAMBI protein comprises the nucleotide sequence shown in SEQ ID NO:

19.

13. The method according to claim 11 or 12, wherein the mRNA encoding BCL2 protein, the mRNA encoding IL-10, and the mRNA encoding BAMBI are simultaneously or sequentially introduced into macrophages.

14. Engineered macrophages prepared by the method according to any one of claims 10-13.

15. The use of engineered macrophages according to any one of claims 1-9 or 14 in the preparation of medicaments for the prevention and / or treatment of fibrotic diseases.

16. The application according to claim 15, wherein the fibrotic disease includes idiopathic pulmonary fibrosis, radiation-induced pulmonary fibrosis, pneumoconiosis, cystic fibrosis, cirrhosis, myocardial fibrosis, renal interstitial fibrosis, scleroderma, keloids, pancreatic fibrosis, retroperitoneal fibrosis, systemic sclerosis, and / or myelofibrosis.

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