MRNA (messenger ribonucleic acid) composition and application thereof in preparation of medicine for treating hepatic fibrosis

Abstract

The invention belongs to the technical field of preparation of medicines for treating hepatic fibrosis, and particularly relates to an mRNA composition and application thereof in preparation of medicines for treating hepatic fibrosis. According to the mRNA composition provided by the invention, the phagocytic rate of BMDM cells is remarkably improved, and the phagocytic rate induced by mRNA composition treatment can be more than ten times that of a WT group. After macrophages are transfected by the mRNA composition, hepatic fibrocytes (such as mouse hepatic stellate cell lines) can be effectively killed, and hepatic fibrosis can be effectively treated.

Description

Technical Field

[0001] The present invention belongs to the technical field of preparation of liver fibrosis drugs, and particularly relates to an mRNA composition and its application in the preparation of liver fibrosis therapeutic drugs. Background Art

[0002] Liver fibrosis is a progressive pathological process caused by chronic liver damage, with the core characteristics of abnormal activation and differentiation of hepatic stellate cells (HSCs) into myofibroblasts and excessive deposition of extracellular matrix (ECM). This process is accompanied by an imbalance in macrophage polarization (M1 / M2 phenotype imbalance) and abnormally high expression of fibrosis-related proteins (FAP, TGF-β, etc.), ultimately leading to liver failure or cancer. Existing clinical treatment options face a double dilemma: anti-inflammatory drugs (such as glucocorticoids) have the risk of systemic immunosuppression, while targeted signaling pathway inhibitors (such as TGF-β antagonists) have off-target toxicity and cannot reverse the established fibrotic network, highlighting the urgent need for precise regulation of specific cell subpopulations and targeted delivery systems.

[0003] In recent years, nano-delivery systems (such as lipid nanoparticles LNP) can increase liver enrichment through surface modification, but their targeting specificity for HSCs and fibrosis-related macrophages is insufficient, and they cannot achieve responsive drug release to the lesion microenvironment. Although genetically engineered cell therapies (such as CAR-T) have precise recognition capabilities, they are complex in cell preparation, uncontrollable in vivo survival, and have the potential risk of cytokine storms. It is worth noting that FAP, as a membrane surface marker specifically expressed on activated HSCs and myofibroblasts, is widely recognized as an ideal target, but its low abundance distribution characteristics in normal tissues place extremely high demands on the targeting accuracy of therapeutic vectors. Existing delivery systems are difficult to simultaneously meet the triple standards of target recognition, cell transfection efficiency, and biosafety.

[0004] The current technological gap focuses on the development of new therapeutic systems that combine intelligent targeting with synergistic effects of cell engineering. There is an urgent need to build integrated solutions that can direct transfection of key effector cells (such as macrophages), endow them with the ability to specifically recognize HSCs through engineering, while ensuring that the carrier system is biodegradable and immunogenicity is controllable. This requires breaking through the passive targeting limitations of traditional nanocarriers and innovatively combining cell membrane camouflage technology, controllable mRNA expression elements, and microenvironment-responsive release mechanisms to achieve: 1) precise delivery to fibrotic lesions; 2) reprogramming macrophages to transform into an anti-fibrotic phenotype; and 3) the multiple therapeutic effects of specifically eliminating activated HSCs. Summary of the Invention

[0005] Based on this, the present invention provides an mRNA composition of lipid nanoparticles encapsulating CAR structure mRNA, which can efficiently transfect macrophages. The transfected macrophages can accurately target liver fibroblasts to kill liver fibroblasts, thereby achieving the purpose of treating liver fibrosis.

[0006] In order to achieve the above-mentioned purpose, the present invention can adopt the following technical solutions:

[0007] On the one hand, the present invention provides an mRNA composition, including mRNA and lipid nanoparticles, wherein the lipid nanoparticles encapsulate the mRNA; the mRNA is a CAR structure mRNA, and the protein encoded by the CAR structure mRNA targets and recognizes fibroblast activation protein.

[0008] Preferably, in the above mRNA composition, the mRNA composition satisfies one or more of the following conditions:

[0009] (i) The amino acid sequence of the protein encoded by the CAR structural mRNA is shown in SEQ ID NO: 1;

[0010] (ii) Antibodies targeting macrophages in the liver are linked to the surface of lipid nanoparticles.

[0011] More preferably, in the above-mentioned CARmRNA composition, the antibody that targets and recognizes macrophages in the liver is a CD163 antibody.

[0012] Preferably, in the above mRNA composition, the lipid nanoparticles meet one or more of the following conditions:

[0013] (i) The lipid nanoparticles include ionizable lipids, auxiliary lipids, polyethylene glycol lipids and cholesterol, and the lipid nanoparticles have a core-shell structure;

[0014] (ii) The particle size of lipid nanoparticles was 118.2±7.8 nm, and the zeta potential was -4 mV to -5 mV.

[0015] More preferably, in the above mRNA composition,

[0016] Ionizable lipids are selected from one or more of C14-494, ALC-0315, C12-200, MC3, DLinDMA, DLinKC2DMA, ICE, HGT5000, HGT5001, OF-02, DODAC, DDAB, DMRIE, DOSPA, DOGS, DODAP, DOTMA, DLenDMA, CLinDMA, CpLinDMA, DMOBA, DOcarbDAP, DLinDAP, DLincarbDAP, DLinCDAP, DLin-K-XTC2-DMA, and HGT4003; and / or

[0017] The helper lipid is selected from one or more of DOPE, distearoylphosphatidylcholine, dioleoylphosphatidylcholine, dipalmitoylphosphatidylcholine, dioleoylphosphatidylglycerol, dipalmitoylphosphatidylglycerol, dioleoylphosphatidylethanolamine, palmitoyloleoylphosphatidylcholine, palmitoyloleoylphosphatidylethanolamine and dioleoylphosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate, dipalmitoylphosphatidylethanolamine, dimyristoylphosphatidylethanolamine, distearoylphosphatidylethanolamine, 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearoyl-2-oleoylphosphatidylethanolamine or 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine; and / or

[0018] The PEG lipid is selected from one or more of ALC-0159, DMG-PEG2000, DSPE-PEG2000, DPPE-PEG2000, DMPE-PEG2000, DOPE-PEG2000, DSPE-PEG1000, DPPE-PEG1000, DMPE-PEG1000, DOPE-PEG1000, DSPE-PEG550, DPPE-PEG550, DMPE-PEG550, PEG-cholesterol, PEG2000-ceramide, PEG1000-ceramide, PEG750-ceramide, and PEG550-ceramide.

[0019] Preferably, in the above mRNA composition, the mRNA is mRNA modified with a CAR structure mRNA; the modification method includes one or more of the following:

[0020] (i) using m1Ψ-modified nucleosides instead of uridine;

[0021] (ii) Engineering of the 5'-end CleanCap structure;

[0022] (iii) Design of a 101-nucleotide homopolyadenylation tail.

[0023] Another aspect of the present invention provides a modified macrophage, which is obtained by transfecting a macrophage with the mRNA composition of the present invention.

[0024] In another aspect, the present invention provides a drug for treating liver fibrosis, comprising the mRNA composition of the present invention or the modified macrophage of the present invention.

[0025] In another aspect, the present invention further provides a use of the mRNA composition of the present invention or the modified macrophage of the present invention in the preparation of a drug for treating liver fibrosis.

[0026] Preferably, in the above application, liver fibrosis includes viral liver fibrosis, metabolic liver fibrosis, alcoholic liver fibrosis or drug / toxin-induced fibrosis.

[0027] The beneficial effects of the present invention include:

[0028] (1) The αCD163 / LNP-GFP conjugated with anti-CD163 antibodies provided in the present invention effectively delivered the GFP mRNA payload to target cells; in addition, after exposure to αCD163 / LNP-GFP, more than 80% of M cells expressed GFP, respectively, compared with less than 20% of M cells delivered GFP mRNA by LNPs not modified with anti-CD163 antibodies; in addition, free GFP mRNA not encapsulated in LNPs hardly expressed the target protein.

[0029] (2) The mRNA composition provided in the present invention significantly increases the phagocytic rate of BMDM cells, wherein the phagocytic rate induced by the mRNA composition treatment can be more than ten times that of the WT group.

[0030] (3) M cells transfected with the mRNA composition provided by the present invention significantly eliminated target cells expressing FAP in vitro.

[0031] (4) The mRNA composition provided in the present invention can effectively kill liver fibroblasts (such as mouse hepatic stellate cell line) after transfection into macrophages, and effectively treat liver fibrosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1A Schematic diagram of the modification process of FAPCAR encapsulated in LNP;

[0033] Figure 1B Transmission electron microscopy (TEM) size distribution of LNP-FAPCAR, counting more than 80 particles; the inset provides a closer view of LNP-FAPCAR (scale bar = 100 nm);

[0034] Figure 1CSchematic diagram of the structure of αCD163 / LNP-FAPCAR;

[0035] Figure 1D Transmission electron microscopy (TEM) size distribution of αCD163 / LNP-FAPCAR, counting more than 110 particles; the inset provides a closer view of αCD163 / LNP-FAPCAR (scale bar = 100 nm);

[0036] Figure 1E is the average zeta potential of LNP-FAPCAR and αCD163 / LNP-FAPCAR;

[0037] Figure 1F Statistical analysis of M cells (expressing CD163) transfected with mRNA, LNP-mRNA, or αCD163 / LNP-mRNA;

[0038] Figure 1G Representative confocal fluorescence images of M cells (expressing CD163) transfected with mRNA, LNP-mRNA, or αCD163 / LNP-mRNA. mRNA was labeled with Cy5 (red), and M expression was indicated by green fluorescence (scale bar = 10 μm);

[0039] Figure 1H This is a representative flow cytometric analysis of GFP in mouse M cells;

[0040] Figure 1I is the quantification of the amount of GFP positive staining in mouse M cells (%), across biological replicates (n=3);

[0041] Figure 1J Representative flow cytometric analysis of FAPCAR in mouse M cells;

[0042] Figure 1K Quantification of the amount of FAPCAR positive staining in murine M cells (%), across biological replicates (n = 3);

[0043] Figure 2A Screening for FAP by flow cytometry + JS-1 cell situation;

[0044] Figure 2B BMDM cells phagocytized FAP in different treatment groups + In the case of JS-1 cells, red fluorescence indicates specific phagocytosis and internalization of pHrodo markers, and green fluorescence indicates BMDM cells (scale bar = 10 μm);

[0045] Figure 2C Quantitative analysis of the phagocytic index of BMDM cells treated with different treatment groups;

[0046] Figure 2D pHrodo-labeled FAP + JS-1 cell situation;

[0047] Figure 2E To detect the phagocytic activity of BMDM cells after treatment in each treatment group by flow cytometry;

[0048] Figure 2F Statistical analysis of BMDM cell phagocytosis index in different treatment groups detected by flow cytometry;

[0049] Figure 2G The cell killing experiment is based on a flow chart;

[0050] Figure 2H The killing ability of phagocytic cells to target cells treated by different treatment groups;

[0051] Figure 2I Flow cytometry screening with FAP + Macrophage status in the JS-1 cell co-culture system;

[0052] Figure 2J Expression of CD80 and CD206 in macrophages screened for flow cytometric analysis;

[0053] Figure 2K is the relative expression of CD80, IL-1β, and IL-6 mRNA in macrophages;

[0054] Figure 2L is the relative expression of CD206, Arg1, and IL-10 mRNA in macrophages;

[0055] Figure 2M is the relative expression of MMP9, MMP12, and MMP13 mRNA in macrophages (n=3);

[0056] Figure 3A Schematic diagram of LNPs in vivo delivery experiment;

[0057] Figure 3B Bioluminescence analysis of CCL4-induced fibrosis mice after injection of LNP-Luc and αCD163 / LNP-Luc;

[0058] Figure 3C Immunofluorescence images of ZsGreen mRNA expression after injection of ZsGreen mRNA encapsulated in LNP (LNP-ZsGreen and αCD163 / LNP-ZsGreen);

[0059] Figure 3D Immunofluorescence images of co-staining for hepatocyte markers (ALB), HSCs (Desmin), leukocytes (CD45), KCs (F4 / 80), and cholangiocytes (SOX9) (scale bar = 20 μm);

[0060] Figure 3E Schematic diagram of antibody-mediated targeted cellular uptake of LNPs mediated by surface receptor interactions;

[0061] Figure 3F is the change of CD163 level in liver (scale bar = 20 μm);

[0062] Figure 3G Fluorescence co-localization images of CD163 and ZsGreen in the liver of fibrotic mice (scale bar = 20 μm);

[0063] Figure 4A The body weights of mice in different groups;

[0064] Figure 4B Routine blood tests and biochemical evaluations of mice after different treatments;

[0065] Figure 4C H&E staining of the heart, liver, spleen, lung, and kidney of mice treated with oil and αCD163 / LNP-FAPCAR to evaluate pathological changes (bar = 20 μm);

[0066] Figure 5A Schematic diagram of the experimental design for in vivo experiments;

[0067] Figure 5B Immunofluorescence staining of FAPCAR (yellow) in liver tissue; F4 / 80 is highlighted + (green) macrophages (scale bar = 10 μm) and statistical analysis;

[0068] Figure 5C Statistical analysis of FAPCAR (yellow) in liver tissue;

[0069] Figure 5D Triple immunofluorescence staining of liver tissue sections using Desmin (red), F4 / 80 (green), and FAPCAR (yellow);

[0070] Figure 6A This is the quantitative status of hydroxyproline in mice;

[0071] Figure 6B is the serum ALT level of mice in different groups;

[0072] Figure 6CqPCR analysis of Col1a1, Col2a1, and Acta2 in liver tissues of fibrotic mice;

[0073] Figure 6D The liver tissues of fibrotic mice were stained with Sirius red, hematoxylin-eosin, and Masson's trichrome (scale bar = 50 μm) and immunohistochemically for Collagen III, TGF-β, MMP12, IL-6, and IL-10 (scale bar = 50 μm).

[0074] Figure 6E This is the quantitative analysis of Sirius red staining of liver tissue in fibrotic mice;

[0075] Figure 6F This is the quantitative analysis of Masson staining of liver tissue in fibrotic mice;

[0076] Figure 6G Quantitative results of CollagenIII immunohistochemical staining of liver tissue in fibrotic mice;

[0077] Figure 6H The quantitative results of TGF-β immunohistochemical staining in liver tissue of fibrotic mice;

[0078] Figure 6I The quantitative results of MMP12 immunohistochemical staining in liver tissue of fibrotic mice;

[0079] Figure 6J The quantitative results of IL-6 immunohistochemical staining in liver tissue of fibrotic mice;

[0080] Figure 6K The quantitative results of IL-10 immunohistochemical staining in liver tissue of fibrotic mice;

[0081] Figure 6L qPCR analysis of CollagenIII, TGF-β, MMP12, IL-6, and IL-10 in mouse liver;

[0082] In the figure, the differences between the groups were statistically evaluated by one-way analysis of variance, with significance indicated by *P<0.01 and non-significance indicated by nsP>0.05. DETAILED DESCRIPTION

[0083] The examples are provided to better illustrate the present invention, but are not intended to limit the present invention to the examples. Therefore, non-essential improvements and adjustments to the embodiments made by those skilled in the art based on the above-mentioned invention still fall within the scope of protection of the present invention.

[0084] The terms used herein are only used to describe specific embodiments and are not intended to limit the present disclosure. Unless the context has a significantly different meaning, expressions in the singular include expressions in the plural. As used herein, it should be understood that terms such as "include", "have", "comprise" and the like are intended to indicate the presence of features, numbers, operations, components, parts, elements, materials or combinations. The terms of the present invention are disclosed in the specification and are not intended to exclude the possibility that one or more other features, numbers, operations, components, parts, elements, materials or combinations thereof may exist or may be added. As used herein, " / " may be interpreted as "and" or "or", depending on the circumstances.

[0085] An embodiment of the present invention provides an mRNA composition, comprising mRNA and lipid nanoparticles, wherein the lipid nanoparticles encapsulate the mRNA; the mRNA is a CAR structure mRNA, and the protein encoded by the CAR structure mRNA targets and recognizes fibroblast activation protein.

[0086] It should be noted that in the present invention, the CAR structure mRNA is encapsulated using lipid nanoparticles and can then be targeted and delivered to macrophages. The CAR structure mRNA can be translated in situ in the macrophages and then target and recognize fibroblast activation protein, so that the macrophages can target and kill liver fibroblasts containing fibroblast activation protein.

[0087] In some specific examples, in the above mRNA composition, the mRNA composition satisfies one or more of the following conditions:

[0088] (i) The amino acid sequence of the protein encoded by the CAR structure mRNA is shown in SEQ ID NO: 1; specifically, the CAR structure of the present invention is well known in the art, and the amino acid sequence of the CAR structure of the present invention can be shown in SEQ ID NO: 1, and the nucleic acid sequence can be shown in SEQ ID NO: 2;

[0089] (ii) Antibodies that target and recognize macrophages in the liver are linked to the surface of the lipid nanoparticles; specifically, antibodies that target and recognize macrophages in the liver are linked to the surface of the lipid nanoparticles, thereby enabling recognition of macrophages and improving the efficiency of transfection of macrophages with the mRNA composition.

[0090] It should be noted that, either of the above two conditions can be met, but it is preferred that both conditions be met at the same time.

[0091] In some specific examples, in the above-mentioned CAR mRNA composition, the antibody targeting and recognizing macrophages in the liver is a CD163 antibody.

[0092] It should be noted that the antibodies targeting and recognizing macrophages in the liver in the present invention are well known in the art, such as CD163 antibodies.

[0093] In some specific examples, in the above mRNA composition, the lipid nanoparticles meet one or more of the following conditions:

[0094] (i) The lipid nanoparticles include ionizable lipids, auxiliary lipids, polyethylene glycol lipids and cholesterol, and the lipid nanoparticles have a core-shell structure;

[0095] (ii) The particle size of lipid nanoparticles was 118.2±7.8 nm, and the zeta potential was -4 mV to -5 mV.

[0096] It should be noted that the lipid nanoparticles in the present invention are well known in the art, and preferably meet the above conditions.

[0097] In some specific examples, in the above mRNA composition,

[0098] Ionizable lipids are selected from one or more of C14-494, ALC-0315, C12-200, MC3, DLinDMA, DLinKC2DMA, ICE, HGT5000, HGT5001, OF-02, DODAC, DDAB, DMRIE, DOSPA, DOGS, DODAP, DOTMA, DLenDMA, CLinDMA, CpLinDMA, DMOBA, DOcarbDAP, DLinDAP, DLincarbDAP, DLinCDAP, DLin-K-XTC2-DMA, and HGT4003; and / or

[0099] The helper lipid is selected from one or more of DOPE, distearoylphosphatidylcholine, dioleoylphosphatidylcholine, dipalmitoylphosphatidylcholine, dioleoylphosphatidylglycerol, dipalmitoylphosphatidylglycerol, dioleoylphosphatidylethanolamine, palmitoyloleoylphosphatidylcholine, palmitoyloleoylphosphatidylethanolamine and dioleoylphosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate, dipalmitoylphosphatidylethanolamine, dimyristoylphosphatidylethanolamine, distearoylphosphatidylethanolamine, 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearoyl-2-oleoylphosphatidylethanolamine or 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine; and / or

[0100] The PEG lipid is selected from one or more of ALC-0159, DMG-PEG2000, DSPE-PEG2000, DPPE-PEG2000, DMPE-PEG2000, DOPE-PEG2000, DSPE-PEG1000, DPPE-PEG1000, DMPE-PEG1000, DOPE-PEG1000, DSPE-PEG550, DPPE-PEG550, DMPE-PEG550, PEG-cholesterol, PEG2000-ceramide, PEG1000-ceramide, PEG750-ceramide, and PEG550-ceramide.

[0101] In some specific examples, in the above mRNA composition, the mRNA is mRNA modified from CAR structure mRNA; the modification method includes one or more of the following:

[0102] (i) using m1Ψ-modified nucleosides instead of uridine;

[0103] (ii) Engineering of the 5'-end CleanCap structure;

[0104] (iii) Design of a 101-nucleotide homopolyadenylation tail.

[0105] It should be noted that the CAR structure mRNA in the present invention can be further modified to improve the expression efficiency of the CAR structure mRNA.

[0106] The present invention also provides a modified macrophage, which is obtained by transfecting the macrophage with the mRNA composition of the present invention.

[0107] It should be noted that after the mRNA composition of the present invention is transfected into macrophages, the mRNA composition can be expressed in situ in the macrophages, thereby achieving targeted killing of liver fibrosis cells.

[0108] An embodiment of the present invention further provides a drug for treating liver fibrosis, which comprises the mRNA composition of the present invention or the modified macrophages of the present invention.

[0109] It should be noted that the mRNA composition or modified macrophages of the present invention can kill liver fibroblasts, alleviate the symptoms of liver fibrosis, and achieve the purpose of treating liver fibrosis. In addition, the liver fibrosis treatment drugs of the present invention are generally injections; of course, other dosage forms can also be prepared according to clinical needs.

[0110] The embodiments of the present invention also provide a use of the mRNA composition of the present invention or the modified macrophages of the present invention in preparing a drug for treating liver fibrosis.

[0111] In some specific examples, in the above applications, liver fibrosis includes viral liver fibrosis, metabolic liver fibrosis, alcoholic liver fibrosis, or drug / toxin-induced fibrosis.

[0112] In order to better understand the present invention, the content of the present invention is further explained below with reference to specific examples, but the content of the present invention is not limited to the following examples.

[0113] In the following examples, the mouse hepatic stellate cell line (JS-1) was obtained from the Cell Bank of the Chinese Academy of Sciences; bone marrow cells were extracted from mouse femurs and induced to differentiate into bone marrow-derived macrophages (BMDMs), i.e., mouse macrophages (MΦs), in vitro; the cells were cultured in DMEM medium (containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (P / S)) at 37°C in a humidified atmosphere of 5% CO2; female BALB / c mice (6-8 weeks old) were purchased from Changsheng Bio (Liaoning, China) and maintained under specific pathogen-free conditions at the Laboratory Animal Center of Shengjing Hospital, China Medical University; all animal procedures were performed in accordance with the Guide for the Care and Use of Laboratory Animals of China Medical University (KT2022256) and approved by the Institutional Animal Care and Use Committee.

[0114] In the following examples, statistical analysis was performed using GraphPad Prism 7.0 (GraphPad Inc., La Jolla, CA, USA); continuous variables are presented as mean ± SD. Comparisons between groups were performed using one-way analysis of variance (ANOVA), and post hoc pairwise comparisons were performed using the Tukey test. For comparisons between multiple groups, ANOVA or the Mann-Whitney U test was used as appropriate. Statistical significance was set at a two-tailed p value < 0.05 for all analyses.

[0115] 1. Preparation and Characterization of CAR mRNA Complexes

[0116] (1) Preparation of lipid nanoparticles

[0117] C14-494, DOPE, cholesterol, and DSPE-PEG2000-Mal were dissolved in ethanol (final concentration 30% v / v) in a certain proportion (the molar ratio of C14-494, DOPE, cholesterol, and DSPE-PEG2000-Mal was 6:2:3:0.5) and mixed with acetate buffer (pH 4.0) by a microfluidic mixer (flow rate ratio 3:1) to form an LNP core-shell structure with a particle size of 69.4±6.3 nm.

[0118] (2) Lipid nanoparticle encapsulation of CAR mRNA

[0119] (1) Synthesis of FAP-CAR structure

[0120] In the following example, the FAP-CAR construct is composed of a scFv fragment from a mouse-specific FAP monoclonal antibody (clone 73.3) integrated with the mouse CD3ζ and CD28 cytoplasmic signaling domains. The sequence was codon-optimized for mammalian expression (DNA sequence shown in SEQ ID NO: 2) and cloned into an in vitro transcription (IVT) template plasmid containing a T7 promoter, 5' and 3' untranslated regions, and a poly(A) tail. Cloning was performed to prepare an endotoxin-free plasmid. The above cloning process was commissioned to Shanghai Gene Medical Technology Co., Ltd.

[0121] (2) Preparation of FAP-CAR mRNA

[0122] FAP-CAR mRNA was produced using the MEGAScript T7 transcription kit, with UTP replaced by m1Ψ-5'-triphosphate and a 101-nucleotide poly(A) tail added. In addition, the in vitro transcribed FAP-CAR mRNA was co-transcriptionally modified with the CleanCap trinucleotide cap 1 analog and subsequently purified by cellulose. The purified FAP-CAR mRNA was analyzed by agarose gel electrophoresis and stored at -20°C.

[0123] (3) Lipid nanoparticle encapsulation of FAP-CAR mRNA

[0124] The FAP-CAR mRNA purified from cellulose was quickly mixed with the lipid nanoparticles prepared in (I) above, and the FAP-CAR mRNA purified from cellulose was encapsulated into lipid nanoparticles (LNPs) at an acidic pH to form LNP-FAP-CAR mRNA (also called LNP-mRNA) (see Figure 1A ).

[0125] The morphology of LNP-FAP-CAR mRNA was analyzed using transmission electron microscopy (TEM), and the zeta potential of LNP-FAP-CAR mRNA was tested using a Nicomp380 analyzer. Figure 1B As shown, LNP-FAP-CAR mRNA showed a spherical morphology with a uniform particle size of 69.4 ± 6.3 nm (the average particle size was determined by measuring the particle diameter in TEM images using a Hitachi imaging system) and a zeta potential of -4.95 mV.

[0126] (III) Lipid nanoparticle encapsulation of CAR mRNA-coupled antibodies

[0127] In order to improve the delivery of LNP-FAP-CAR mRNA to macrophages (MΦs), an antibody targeting CD163 was coupled to the surface of LNP-mRNA to prepare αCD163 / LNP-FAP-CAR mRNA (also known as αCD163 / LNP-mRNA) ( Figure 1C ); specifically as follows:

[0128] LNPs were prepared by conjugating purified anti-rat and anti-mouse CD163 antibodies and control antibodies using SATA-maleimide chemistry. Maleimide functional groups (DSPE-PEG-mal) were introduced into the LNPs via a post-insertion technique. The antibodies were functionalized with SATA (N-succinimidyl S-acetylthioacetate) to introduce sulfhydryl groups, enabling conjugation to the maleimide groups. After deprotection of SATA with 0.5 M hydroxylamine, unreacted components were removed using a G-25 Sephadex column. The active sulfhydryl groups on the antibodies were then covalently linked to the maleimide groups using thioether conjugation chemistry. The conjugated products were purified using an agarose CL-4B gel filtration column (MilliporeSigma). After ligand addition, all targeted and nontargeted LNP formulations were stored at 4°C until use.

[0129] The morphology of αCD163 / LNP-mRNA was analyzed using transmission electron microscopy (TEM). Figure 1D As shown, the results showed that the average particle size of αCD163 / LNP-mRNA was 118.2±7.8 nm.

[0130] In addition, the zeta potential of αCD163 / LNP-mRNA was tested using Nicomp380 analyzer, and the results were as follows: Figure 1E As shown, the results showed that the zeta potential of αCD163 / LNP-mRNA was -4.56 mV.

[0131] (III) Cellular uptake assessment

[0132] (1) Using a red fluorescent probe (Cy5) to label FAP-CAR mRNA to obtain Cy5-FAP-CAR mRNA (abbreviated as Cy5-mRNA);

[0133] (2) using lipid nanoparticles to encapsulate FAP-CAR mRNA according to the above-mentioned method, and then coupling the CD163-targeting antibody to the surface of LNP-Cy5-mRNA according to the above-mentioned method for encapsulating FAP-CAR mRNA with an antibody to obtain αCD163 / LNP-Cy5-mRNA;

[0134] (3) Bone marrow-derived macrophages (BMDMs) were cultured at 1×10 5 The cells were cultured in 24-well plates at a density of 100 cells / well and allowed to adhere overnight.

[0135] (4) BMDMs were incubated with Cy5-mRNA, LNP-Cy5-mRNA, and αCD163 / LNP-Cy5-mRNA mixtures (37°C, 5%, CO2) for four hours;

[0136] (5) After incubation, the cells were washed three times with PBS solution, stained with green fluorescent live cell dye, and observed using a confocal laser scanning microscope.

[0137] The results are as follows Figure 1F and Figure 1G As shown, the results showed that conjugation of αCD163 further increased the uptake of LNPs.

[0138] (IV) Cell transfection in vitro

[0139] (1) Green fluorescent protein (GFP) transfection test

[0140] 1) Green fluorescent protein mRNA (GFP mRNA) was encapsulated using lipid nanoparticles (LNPs) according to the above-mentioned method for encapsulating FAP-CAR mRNA with lipid nanoparticles to obtain LNP-GFP; in addition, an antibody targeting CD163 was coupled to the surface of LNP-GFP according to the above-mentioned method for encapsulating FAP-CAR mRNA with lipid nanoparticles to obtain αCD163 / LNP-GFP;

[0141] 2) BMDMs (M cells) in the logarithmic growth phase were plated at 1×10 4 The cells were seeded at a density of 100 cells / well in a 96-well plate and then divided into four groups: WT group, GFP group, LNP-GFP group, and αCD163 / LNP-GFP group. The GFP group was incubated with 100 ng of GFP, the LNP-GFP group was incubated with 100 LNP-GFP, and the αCD163 / LNP-GFP group was incubated with 100 ng of αCD163 / LNP-GFP. The incubation time was 48 h.

[0142] 3) After incubation, the transfection efficiency was tested by flow cytometry.

[0143] The results are shown in the figure Figure 1H and Figure 1IAs shown, the results showed that αCD163 / LNP-GFP coupled to anti-CD163 antibodies effectively delivered GFP mRNA payload to target cells; in addition, after exposure to αCD163 / LNP-GFP, more than 80% of M cells expressed GFP, respectively, compared with less than 20% of M cells delivered GFP mRNA by LNPs not modified with anti-CD163 antibodies; in addition, free GFP mRNA not encapsulated in LNPs hardly expressed the target protein.

[0144] (2) FAP-CAR mRNA transfection test

[0145] The transfection efficiency of FAPCAR, LNP-FAPCAR and αCD163 / LNP-FAPCAR was tested according to the green fluorescent protein (GFP) transfection test method described above. The results are shown in Figure 5. Figure 1J and Figure 1K As shown, the transfection results were consistent with those of green fluorescent protein (GFP) transfection.

[0146] 2. In vitro killing test

[0147] (1) Cell pretreatment

[0148] (1) FAP-positive cell screening

[0149] JS-1 cells (mouse hepatic stellate cells) were stimulated with 5 ng / ml TGF-β1 (Peprotech, USA) for 48 hours and then screened for FAP by flow cytometry. + JS-1 cells (FAP-positive cells) (see Figure 2a).

[0150] (2) Macrophage preconditioning

[0151] BMDM were seeded in 12-well plates (density: 1×10 5 cells / well), cultured at 37°C, 5% CO2 until semi-adherent, and then divided into four groups, with three routine experiments set up in each group; the groups were αCD163 / LNP-Luc group, LNP-FAPCAR group, αCD163 / LNP-FAPCAR group and WT group (blank culture medium); LNP-Luc, LNP-FAPCAR and αCD163 / LNP-FAPCAR group were transfected into BMDM according to the above-mentioned cell in vitro transfection method, respectively.

[0152] (II) In vitro phagocytosis assay

[0153] (1) FAP-positive cell labeling and conditioning

[0154] FAP-positive JS-1 cells were washed twice with PBS (centrifugation conditions: 300 × g, 5 minutes, room temperature), and target cells were labeled with pHrodo dye according to the instructions (protected from light). They were washed twice with pre-cooled PBS (centrifugation conditions were the same as above) to remove unbound dye. FAP-positive cells were then treated with opsonin (anti-FAP antibody) and incubated at 37°C for 1 hour to enhance macrophage recognition.

[0155] (2) Co-culture of FAP-positive cells with macrophages

[0156] Add labeled pHrodo+ target cells (JS-1) to the transfected macrophage wells (target cell:effector cell = 5:1); pre-cool the 12-well plate in a 4°C refrigerator for 15 minutes; centrifuge at 150 × g for 5 minutes at 4°C to promote adhesion of target cells to macrophages; discard the supernatant, replace with preheated complete culture medium, and incubate in a 37°C, 5% CO2 incubator.

[0157] (3) In vitro phagocytosis test

[0158] The effects of various treatments on the phagocytosis of FAP-positive cells by BMDM cells were examined using confocal microscopy and flow cytometry. Figure 2B 、 Figure 2C 、 Figure 2D 、 Figure 2E and Figure 2F As shown, the results showed that αCD163 / LNP-FAPCAR and LNP-FAPCAR treatment significantly increased the phagocytic rate of BMDM cells, among which the phagocytic rate induced by αCD163 / LNP-FAPCAR treatment was more than ten times that of the WT group and about three times that of the LNP-FAPCAR group; in contrast, the αCD163 / LNP-Luc group did not show an increase in phagocytic efficiency, indicating that FAPCAR expression is essential for enhancing M cell-mediated phagocytosis of FAP-positive cells.

[0159] (3) In vitro killing effect test

[0160] (1) Co-culturing the FAP-positive cells screened above with the transfected macrophages (M cells) overnight to obtain mixed cells;

[0161] (2) The mixed cells were washed with PBS solution and then lysed using Promega E151A kit. After lysis, luciferase luminescence was measured using PerkinElmer VictorX3 plate reader (Waltham, MA) (see Figure 2G); a decrease in luciferase signal indicated that functional FAP-CAR-M cells successfully eliminated FAP-expressing JS-1 target cells; the killing efficiency was calculated according to the following formula: 100–((test RLU / average RLU of the control group without M cells)×100), where RLU represents relative luminescence unit.

[0162] Cell killing effect Figure 2H As shown, the results showed that M cells transfected with αCD163 / LNP-FAPCAR significantly eliminated FAP-expressing target cells in vitro.

[0163] (IV) M cell polarization test

[0164] (1) Co-culturing the FAP-positive cells screened above with the transfected macrophages (M cells) overnight to obtain mixed cells;

[0165] (2) Using flow cytometry to screen M cells in mixed cells, the results are as follows Figure 2I As shown;

[0166] (3) Flow cytometry and qPCR were used to detect the polarization of M cells. The results showed that M cells (especially those treated with αCD163 / LNP) significantly promoted the expression of M1 marker CD80 ( Figure 2J ), and the transcriptional activity of IL-1β and IL-6 ( Figure 2K ); Meanwhile, the αCD163 / LNP-FAPCAR group reduced the expression of M2 marker CD206 ( Figure 2J ) and the anti-inflammatory gene transcriptional activity of CD206, ARG1 and IL-10 ( Figure 2L ); In addition, the αCD163 / LNP-FAPCAR group was able to induce high MMP12 expression ( Figure 2M ).

[0167] 3. In vivo testing

[0168] In the following experiments, for blood biochemistry analysis, blood samples (200 μL) were collected from mice receiving various treatments, and serum was prepared and stored at −80°C. Liver function was assessed by measuring bilirubin and alanine aminotransferase (ALT) levels. In addition, plasma concentrations of various cytokines and chemokines were quantified using a multiplex protein array kit according to the manufacturer's protocol (BioRad Laboratories, Hercules, CA).

[0169] In the following experiments, immunohistochemistry (IHC) and immunofluorescence (IF) detection methods were performed according to the following steps: liver tissue was fixed with 4% formaldehyde, embedded in paraffin, and cut into 4-μm-thick sections for immunohistochemical analysis; after dewaxing, hematoxylin staining was performed using an appropriate kit; the sections were hydrated and incubated in hematoxylin solution for one hour, followed by six washes with acetic acid solution and absolute alcohol; then dehydrated and mounted; immunofluorescence staining of target proteins was performed according to standardized protocols; and quantitative analysis of immunofluorescence and immunohistochemical staining was performed in a blinded manner using ImageJ software.

[0170] In the following experiments, the reverse transcription and quantitative polymerase chain reaction (qPCR) detection procedures were as follows: total RNA was isolated from cells or tissues using TRIzol reagent (Qiagen) according to the manufacturer's instructions; complementary DNA (cDNA) synthesis was performed using TaqMan reverse transcription reagents (ThermoFisher); quantitative PCR was performed using TaqMan (Invitrogen) or SYBR Green (ThermoFisher) gene expression detection protocols on a 96-well ABI Step OnePlus real-time PCR system (ThermoFisher); all samples were analyzed in triplicate; gene expression levels were normalized to GAPDH as a reference housekeeping gene to determine relative expression.

[0171] In the following example, the steps for constructing CCl4-induced fibrosis mice include: diluting CCl4 in corn oil (mass concentration of 40%) to obtain an injection solution; subcutaneously injecting mice (C57BL / 6J mice, male, about 5 weeks old, weighing 22-26 g) with 3 mL / kg of the injection solution; injecting twice a week for 12 consecutive weeks to obtain CCl4-induced fibrosis mice.

[0172] (1) In vivo bioluminescence assay

[0173] The following bioluminescence test process is schematically shown for reference Figure 3A The process shown is as follows:

[0174] (1) αCD163 / LNP (LNP was first prepared according to the method in Example 1, and then an antibody targeting CD163 was coupled to the surface of LNP) and LNP (prepared according to the method in Example 1) were linked to luciferase (Luc) mRNA and green fluorescent protein (ZsGreen) mRNA to obtain LNP-Luc and αCD163 / LNP-Luc, as well as LNP-ZsGreen and αCD163 / LNP-ZsGreen.

[0175] (2) CCl4-induced fibrosis mice were intravenously injected with LNP-Luc and αCD163 / LNP-Luc (40 μg of mRNA), respectively. The whole-body luciferase fluorescence signal was monitored using an IVIS imaging system at 0, 0.5 h, 2 h, 8 h, 24 h, and 72 h after injection, and the data were analyzed using LivingImage software. The results are shown in Figure 2. Figure 3B As shown, the results showed that the bioluminescence intensity of the αCD163 / LNP-Luc group and the LNP-Luc group reached a peak 8 hours after injection, and obvious signal attenuation was observed at 24 hours; the bioluminescence of the αCD163 / LNP group was mainly concentrated in the abdomen of the mice, and the fluorescence signal gradually weakened. In addition, the ability of LNP to deliver specific targets to cells was enhanced by antibody coupling, which enabled it to interact with the corresponding receptors and subsequently undergo endocytosis.

[0176] (3) CCl4-induced fibrosis mice were intravenously injected with LNP-ZsGreen and αCD163 / LNP-ZsGreen respectively; ZsGreen fluorescence was detected on the 15th day after injection to evaluate the expression of different LNPs in the liver. Figure 3C As shown, the results showed that the liver fluorescence intensity of mice injected with αCD163 / LNP-ZsGreen was significantly higher than that of the LNP-ZsGreen group 14 days after injection, indicating that the CD163 antibody-conjugated LNPs exhibited excellent accumulation and persistence in the liver.

[0177] (4) ZsGreen-encoded LNP injection demonstrated the specific expression of ZsGreen protein in macrophages of fibrotic mice in co-localization studies, while other hepatocytes showed no expression ( Figure 3D ). Antibody-mediated targeted cellular uptake of LNPs was subsequently observed ( Figure 3E Compared with the LNP-ZsGreen group, a significant decrease in CD163 levels was observed in the αCD163 / LNP-ZsGreen group ( Figure 3F In contrast to the LNP-ZsGreen group, cells expressing ZsGreen were mainly distinct from CD163+ cells, indirectly validating the effect of the conjugated antibody on targeted mRNA delivery ( Figure 3G ).

[0178] The above data indicate that the combination of CD163 antibody and LNP-encapsulated mRNA technology can specifically and effectively target macrophages in the liver of fibrotic mice.

[0179] (2) In vivo safety assessment

[0180] (1) Mouse modeling and medication

[0181] CCl4-induced fibrosis mice were constructed according to the construction steps of the above-mentioned CCl4-induced fibrosis mice; the CCl4-induced fibrosis mice were intravenously injected with the above-prepared αCD163 / LNP-FAPCAR and an equal amount of corn oil.

[0182] (2) Safety assessment test

[0183] The body weight changes of CCl4-injected mice after injection of αCD163 / LNP-FAPCAR and corn oil were monitored. Figure 4A As shown; CCl4-injected mice were subjected to hematological and biochemical tests on the 15th day after injection of αCD163 / LNP-FAPCAR and corn oil. The results are shown in the figure Figure 4B In addition, 15 days after injection, the main organs such as heart, liver, spleen, lung and kidney were subjected to histopathological staining and analysis, and the results were shown in Figure 4C Analysis of the above results showed that the body weight, hematological and biochemical parameters and major organs of mice injected with αCD163 / LNP-FAPCAR had no significant changes compared with the oil group.

[0184] (3) In vivo liver function improvement test

[0185] (1) Mouse modeling and medication

[0186] The present invention also investigated the potential of αCD163 / LNP-FAPCAR to inhibit CCl4-induced liver fibrosis in a mouse model (refer to Figure 5A ), specifically as follows: CCl4-induced fibrosis mice were constructed according to the construction steps of the above-mentioned CCl4-induced fibrosis mice, and CCl4 was replaced with corn oil (oil) to construct a control mouse model (normal mice); mice injected with CCl4 were intravenously injected with the above-prepared αCD163 / LNP-FAPCAR and an equal amount of PBS on the 41st day after CCl4 injection; mice injected with corn oil were intravenously injected with the above-prepared αCD163 / LNP-FAPCAR on the 41st day after corn oil injection;

[0187] (2) Detection of relevant cell indicators

[0188] On the 48th day after the mice were injected with CCl4 and oil, the indicators of mice in each group were detected.

[0189] The results showed that both liver fibrosis and normal mice injected with LNPs showed FAPCAR-positive macrophages, mainly F4 / 80 + and FAPCAR + (His tag), indicating that FAPCAR mRNA transduction was successful; in contrast, the control group cells did not have FAPCAR co-staining ( Figure 5B and Figure 5C ).

[0190] In addition, in the experimental group of mice injected with CD5 / LNP-FAPCAR, FAPCAR-M cells were observed in the area rich in HSC cells (Desmin + ) accumulation, which was not observed in the control group. In contrast, mice that received standard LNP injections showed FAPCAR-M cells, but not in Desmin + There is no accumulation in the area ( Figure 5D ).

[0191] (3) Test of related protein expression

[0192] In order to explore whether αCD163 / LNP-FAPCAR can improve liver function in vivo, the present invention evaluated the effect of hydroxyproline ( Figure 6A ) and ALT( Figure 6B ) content and the levels of fibrosis-related genes were analyzed by qPCR ( Figure 6C ) to detect the extent of liver damage (48h after medication).

[0193] The results showed that the expression levels of the above targets were higher in the CCL4 group and were suppressed by αCD163 / LNP-FAPCAR treatment. At the same time, it was observed that the mice treated with αCD163 / LNP-FAPCAR showed significant differences in expression levels by histology, hematoxylin-eosin staining, and Masson trichrome staining ( Figure 6D 、 Figure 6E and Figure 6F ) and fibrosis markers collagen III and TGF-β ( Figure 6D 、 Figure 6G Figure 6H ) can significantly reduce liver fibrosis.

[0194] In addition, it was found that the expression of MMP12 and IL-6 was lower in the CCL4 group, but higher in the αCD163 / LNP-FAPCAR treatment group ( Figure 6D 、 Figure 6I and Figure 6J ).

[0195] In addition, IL-10 was significantly inhibited by αCD163 / LNP-FAPCAR ( Figure 6D and Figure 6K ), the gene expression levels of collagen III, TGF-β, MMP12, IL-6, and IL-10 were the same as the protein levels ( Figure 6L ).

[0196] The above data showed that αCD163 / LNP-FAPCAR effectively alleviated liver injury and fibrosis in mice.

[0197] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not limiting. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the purpose and scope of the technical solutions of the present invention, which should all be covered by the scope of the claims of the present invention.

Claims

1. An mRNA composition, characterized in that It includes mRNA and lipid nanoparticles, the lipid nanoparticles encapsulate the mRNA; the mRNA is a CAR structure mRNA, and the protein encoded by the CAR structure mRNA targets and recognizes fibroblast activation protein.

2. The mRNA composition according to claim 1, characterized in that The mRNA composition meets one or more of the following conditions: (i) The amino acid sequence of the protein encoded by the CAR structural mRNA is shown in SEQ ID NO: 1; (ii) Antibodies targeting macrophages in the liver are linked to the surface of lipid nanoparticles.

3. The CARmRNA complex according to claim 2, characterized in that The antibody that targets and recognizes macrophages in the liver is the CD163 antibody.

4. The mRNA composition according to claim 2 or 3, characterized in that The lipid nanoparticles meet one or more of the following conditions: (i) The lipid nanoparticles include ionizable lipids, auxiliary lipids, polyethylene glycol lipids and cholesterol, and the lipid nanoparticles have a core-shell structure; (ii) The particle size of lipid nanoparticles was 118.2±7.8 nm, and the zeta potential was -4 mV to -5 mV.

5. The mRNA composition according to claim 4, characterized in that Ionizable lipids are selected from one or more of C14-494, ALC-0315, C12-200, MC3, DLinDMA, DLinKC2DMA, ICE, HGT5000, HGT5001, OF-02, DODAC, DDAB, DMRIE, DOSPA, DOGS, DODAP, DOTMA, DLenDMA, CLinDMA, CpLinDMA, DMOBA, DOcarbDAP, DLinDAP, DLincarbDAP, DLinCDAP, DLin-K-XTC2-DMA, and HGT4003; and / or The helper lipid is selected from one or more of DOPE, distearoylphosphatidylcholine, dioleoylphosphatidylcholine, dipalmitoylphosphatidylcholine, dioleoylphosphatidylglycerol, dipalmitoylphosphatidylglycerol, dioleoylphosphatidylethanolamine, palmitoyloleoylphosphatidylcholine, palmitoyloleoylphosphatidylethanolamine and dioleoylphosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate, dipalmitoylphosphatidylethanolamine, dimyristoylphosphatidylethanolamine, distearoylphosphatidylethanolamine, 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearoyl-2-oleoylphosphatidylethanolamine or 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine; and / or The PEG lipid is selected from one or more of ALC-0159, DMG-PEG2000, DSPE-PEG2000, DPPE-PEG2000, DMPE-PEG2000, DOPE-PEG2000, DSPE-PEG1000, DPPE-PEG1000, DMPE-PEG1000, DOPE-PEG1000, DSPE-PEG550, DPPE-PEG550, DMPE-PEG550, PEG-cholesterol, PEG2000-ceramide, PEG1000-ceramide, PEG750-ceramide, and PEG550-ceramide.

6. The mRNA composition according to claim 1, 2, 3 or 5, characterized in that The mRNA is modified from the CAR structure mRNA; the modification method includes one or more of the following: (i) using m1Ψ-modified nucleosides instead of uridine; (ii) Engineering of the 5'-end CleanCap structure; (iii) Design of a 101-nucleotide homopolyadenylation tail.

7. Modified macrophage, characterized in that Obtained by transfecting macrophages with the mRNA composition according to any one of claims 1 to 6.

8. A drug for treating liver fibrosis, characterized in that: The method comprises the mRNA composition according to any one of claims 1 to 6 or the modified macrophage according to claim 7.

9. Use of the mRNA composition according to any one of claims 1 to 6 or the modified macrophage according to claim 7 in the preparation of a drug for treating liver fibrosis.

10. The use according to claim 9, characterized in that Liver fibrosis includes viral fibrosis, metabolic fibrosis, alcoholic fibrosis, or drug / toxin-induced fibrosis.

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