MRNA (messenger ribonucleic acid) delivery system for in-situ generation of CAR-M (chimeric antigen receptor-M) by targeting M2 type macrophages and preparation method and application thereof

Abstract

The invention relates to an mRNA (messenger Ribonucleic Acid) delivery system for in-situ generation of CAR-M (Chimeric Antigen Receptor-Macrophage) by targeting M2 type macrophages as well The mRNA delivery system comprises a lipidosome and mRNA wrapped in the lipidosome; the liposome is prepared from the following raw materials: ionizable lipid, cholesterol, phosphatidylserine and dimyristoylglycerol polyethylene glycol; the mRNA encodes a chimeric antigen receptor of a fibroblast activation protein. The mRNA delivery system can specifically kill activated fibroblasts and reverse a fibrosis-promoting microenvironment, and can accurately play a role in relieving pulmonary fibrosis in vivo.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical preparations, and particularly relates to an mRNA delivery system for in-situ generation of CAR-M targeting M2 macrophages, and a preparation method and application thereof. Background Art

[0002] Lung macrophages are key regulators of pulmonary fibrosis. In response to tissue injury, the population of lung macrophages undergoes significant phenotypic, functional, and quantitative changes, playing an important role in the initiation, maintenance, and resolution stages of tissue repair. During this process, the number of M2-phenotype macrophages increases, and a large amount of cytokines and growth factors are released, including transforming growth factor-β1 (TGFβ1), platelet-derived growth factor α (PDGFα), vascular endothelial growth factor (VEGF), and Wnt gene (Wingless / Int-1), etc. Their function is to activate lung fibroblasts, resulting in the excessive production and secretion of extracellular matrix (ECM) proteins (including collagen, laminin, and fibronectin). Currently, drugs approved for the clinical treatment of pulmonary fibrosis, such as pirfenidone or nintedanib, although having certain efficacy in inhibiting fibroblast activation, do not target the macrophage population related to the disease process.

[0003] Considering the unique effector function of macrophages and their ability to penetrate tumors, it has been found that CARs gene-engineered macrophages show good antigen-specific phagocytosis and tumor clearance abilities. In the process of pulmonary fibrosis, lung macrophages play a key promoting role and have a numerical advantage. Therefore, CAR-M that can specifically eliminate activated fibroblasts is expected to show good anti-fibrotic effects. And according to previous experiments, after CARs gene engineering, the macrophage phenotype will transform from the M2 phenotype to the M1 phenotype, which can further inhibit the activation of fibroblasts.

[0004] Different from CAR-T, some natural characteristics of macrophages bring difficulties to the in-vitro construction of CAR-M. First, the phagocytic function of macrophages in innate immunity reduces the transfection ability of lentiviral vectors, so transfection easily fails or the transfection efficiency is very low. Second, macrophages have a relatively fast turnover rate, and there is a problem of difficult large-scale amplification in vitro. In addition, it is known that fibroblasts are mainly distributed in the lung interstitium, and it is difficult for CAR-M constructed in vitro and infused back to reach the lung interstitium. To achieve a better killing effect of CAR-M on activated fibroblasts, it is necessary to simultaneously achieve the expression of CARs in different lung macrophage subsets (AMs and IMs). Therefore, in-situ construction of CAR-M in vivo is crucial for the treatment of pulmonary fibrosis.

[0005] Therefore, the development of a delivery system that targets and mediates disease-related lung macrophage populations to achieve safe and efficient in vivo construction of CAR-M and specific treatment of pulmonary fibrosis has become one of the technical problems that need to be solved urgently. Summary of the Invention

[0006] Aiming at the deficiencies of the prior art, the purpose of the present invention is to provide an mRNA delivery system for in-situ generation of CAR-M targeting M2 macrophages, its preparation method and application. This mRNA delivery system can specifically kill activated fibroblasts and reverse the pro-fibrotic microenvironment, and can accurately play a role in alleviating pulmonary fibrosis in vivo.

[0007] To achieve the purpose of this invention, the following technical solutions are adopted:

[0008] In the first aspect, the present invention provides an mRNA delivery system for in-situ generation of CAR-M targeting M2 macrophages, and the mRNA delivery system includes liposomes and mRNA encapsulated in the liposomes.

[0009] The preparation raw materials of the liposomes include ionizable lipids, cholesterol, phosphatidylserine and dimyristoyl glycerol polyethylene glycol.

[0010] The mRNA encodes a chimeric antigen receptor of fibroblast activation protein.

[0011] The mRNA delivery system involved in the present invention has a nanoscale size and can be inhaled into the body through the trachea. It can quickly home to the pulmonary fibrosis site. After lipid endocytosis mediated by the MerTK receptor, it targets the lung macrophage population, and the mRNA expression can induce a chimeric antigen receptor (CAR-M) that polarizes the macrophage phenotype. CAR-M specifically kills activated fibroblasts and inhibits the activation of unactivated fibroblasts, achieving specific reversal of the fibrotic pathological structure.

[0012] Among the preparation raw materials of the liposomes (LNP) involved in the present invention, phosphatidylserine represents the "eat me" signal on the surface of M2 macrophages, and LNP is phagocytosed by the lung macrophage population mediated by the MerTK receptor.

[0013] The present invention creatively discovers that ionizable lipids, cholesterol, phosphatidylserine and dimyristoyl glycerol polyethylene glycol are used together as the preparation raw materials of liposomes. Each component cooperates with each other and synergistically enhances the effect, and can be efficiently phagocytosed by the lung macrophage population, and has excellent delivery effect on mRNA.

[0014] Preferably, the ionizable lipid includes 1-octylnonyl 8-[(2-hydroxyethyl)[6-O-6-(undecyloxy)hexyl]amino]-octanoate.

[0015] Based on the synergistic effect of ionizable lipids, cholesterol, phosphatidylserine, and dimyristoyl glycerol polyethylene glycol, when the ionizable lipid includes 1-octylnonyl 8-[(2-hydroxyethyl)[6-O-6-(undecyloxy)hexyl]amino]-octanoate, the liposome can better exert the delivery effect on mRNA.

[0016] Preferably, the phosphatidylserine includes dioleoyl phosphatidylserine.

[0017] Preferably, the dimyristoyl glycerol polyethylene glycol includes 1,2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol.

[0018] Preferably, the molecular weight of polyethylene glycol in the dimyristoyl glycerol polyethylene glycol is 1000 - 5000 Da, and can be, for example, 1000 Da, 2000 Da, 3000 Da, 4000 Da, 5000 Da, etc.

[0019] Preferably, the molar ratio of the ionizable lipid, cholesterol, phosphatidylserine, and dimyristoyl glycerol polyethylene glycol is 50:(33.5 - 38.5):(10 - 14):(1.5 - 2.5), preferably 50:(35 - 36):(11 - 13):(1.5 - 2).

[0020] Among them, the specific point values in 35 - 36 can be selected as 35, 35.1, 35.2, 35.3, 35.4, 35.5, 35.6, 35.7, 35.8, 35.9, 36, etc.; the specific point values in 11 - 13 can be selected as 11, 11.2, 11.4, 11.6, 11.8, 12, 12.2, 12.4, 12.6, 12.8, 13, etc.; the specific point values in 1.5 - 2 can be selected as 1.5, 1.6, 1.7, 1.8, 1.9, 2, etc.

[0021] Preferably, the amino acid sequence encoded by the mRNA includes the sequence shown in SEQ ID NO:1.

[0022] The specific sequence of SEQ ID NO:1 is as follows: MALPVTALLLPLALLLHAARPQVQLKESGGLVQPGGSLKLSCAASGFTFSSYGMSWVRQTADKRLELVATTNNNGGVTYYPDSVKGRFTISRDNAKNTLYLQMSSLQSEDTAMYYCARYGYYAMDYWGQGISVTVSSGSTSGSGKPGSGEGSDVLMTQTPLWLPVSLGDQASISCRSSQSIVHSNGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTVKISRVEAEDLGVYYCFGGSHVPYTFGGGTKLEIKAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR。

[0023] The above-mentioned mRNA can encode a chimeric antigen receptor of fibroblast activation protein, enabling the mRNA delivery system to translate and express CAR-M containing the CD8α stalk and CD28 transmembrane domain, CD28, CD3ζ intracellular domain and anti-FAP scFv (single-chain variable fragment) domain in macrophages, thereby realizing the specific "transforming decay into wonder" transformation of M2 macrophages.

[0024] In a second aspect, the present invention provides a method for preparing an mRNA delivery system for in-situ generation of CAR-M targeting M2 macrophages as described in the first aspect, and the preparation method includes:

[0025] (1) Mix the raw materials for preparing liposomes to obtain liposomes;

[0026] (2) Mix and stir the mRNA dilution with the alcohol solution of the liposomes obtained in step (1) for self-assembly to obtain the mRNA delivery system.

[0027] Preferably, the temperature of the mixing in step (1) is 20-30°C and the time is 2-5 min.

[0028] Preferably, the rotation speed of the stirring in step (2) is 1200 - 1500 rpm, the temperature is 20 - 30 °C, and the time is 2 - 5 min.

[0029] Among them, the specific point values in 20 - 30 °C can be selected as 20 °C, 22 °C, 24 °C, 26 °C, 28 °C, 30 °C, etc., the specific point values in 2 - 5 min can be selected as 2 min, 3 min, 4 min, 5 min, etc., and the specific point values in 1200 - 1500 rpm can be selected as 1200 rpm, 1250 rpm, 1300 rpm, 1350 rpm, 1400 rpm, 1450 rpm, 1500 rpm, etc.

[0030] Preferably, the alcohol solution includes absolute ethanol.

[0031] Preferably, the preparation method of the mRNA diluent includes: diluting mRNA in a citrate buffer solution.

[0032] In the third aspect, the present invention provides an application of the mRNA delivery system for in-situ generation of CAR-M targeting M2 macrophages as described in the first aspect in the preparation of anti-pulmonary fibrosis drugs or drugs for remodeling and reversing pulmonary fibrosis.

[0033] The numerical ranges described in the present invention not only include the above-listed point values, but also any point values between the above numerical ranges not listed. Due to space limitations and for the sake of simplicity, the present invention does not exhaustively list the specific point values included in the ranges.

[0034] Compared with the prior art, the present invention has the following beneficial effects:

[0035] The mRNA delivery system for in-situ generation of CAR-M targeting M2 macrophages involved in the present invention can quickly home to the pulmonary fibrosis site. After endocytosis of LNP mediated by the MerTK receptor, it targets the pulmonary macrophage population, and the mRNA expression can induce the CAR-M with macrophage phenotype polarization. CAR-M specifically kills activated fibroblasts and inhibits the activation of unactivated fibroblasts, realizing the specific reversal of the fibrotic pathological structure.

[0036] In the present invention, ionizable lipids, cholesterol, phosphatidylserine, and dimyristoyl glycerol polyethylene glycol are used together as the raw materials for preparing liposomes. Each component cooperates with each other and has a synergistic effect, and can be efficiently phagocytosed by the pulmonary macrophage population, and has an excellent delivery effect on mRNA. Description of the Drawings

[0037] Figure 1 It is a luciferase expression situation and quantitative analysis chart of the lungs of mice in Test Example 1.

[0038] Figure 2 It is a graph showing the results of in vitro macrophage targeting tests of different liposomes in Test Example 2.

[0039] Figure 3 It is a comparison graph of the expression of FAP-CAR in different test groups in Test Example 3.

[0040] Figure 4 It is a graph showing the endocytosis effect of CAR-M cells on L929 cells in Test Example 3.

[0041] Figure 5 It is a graph showing the differences in cell phenotypes and cytokine secretion levels in different test groups in Test Example 3, where A is the difference in the markers of macrophage M1 (CD86) and M2 (CD206) after the construction of CAR-M, and B is the difference in the expression of cytokine secretion levels.

[0042] Figure 6 It is a graph showing the imaging of L929 fibroblasts in different test groups in Test Example 3.

[0043] Figure 7 It is a graph showing the differences in the uptake of PS-LNP-mRNA2 by different immune cells in Test Example 4.

[0044] Figure 8 It is an immunofluorescence staining graph of the lung tissue of mice in Test Example 4.

[0045] Figure 9 It is a comparison graph of the lung distribution of adoptive transfer CAR-M and inhaled LNP-mRNA2 in Test Example 4.

[0046] Figure 10 It is a graph showing the H&E and Masson staining results of lung sections after treating a mouse model of pulmonary fibrosis in the early and late stages in Test Example 4.

[0047] Figure 11 It is a graph showing the H&E and Masson staining of lung sections after treating a silicosis model in different test groups in Test Example 5.

[0048] Figure 12 It is a graph showing the quantitative results of the HYP level after treating a mouse model of radiation-induced pulmonary fibrosis in different test groups in Test Example 5.

[0049] Figure 13 It is a graph showing the H&E staining results of the main organs of mice after treatment in different test groups in Test Example 6. Detailed implementation manners

[0050] The technical solution of the present invention will be further described below through specific implementation manners. Those skilled in the art should understand that the embodiments are only for helping to understand the present invention and should not be regarded as specific limitations on the present invention.

[0051] Preparation Example 1

[0052] This preparation example provides a liposome, which is prepared by the following method:

[0053] Take 1-octylnonyl 8-[(2-hydroxyethyl)[6-O-6-(undecyloxy)hexyl]amino]octanoate (SM-102), cholesterol (CHOL), dioleoyl phosphatidylserine (DOPS), 1,2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol 2000 (DMG-PEG2000) and mix them in a molar ratio of 50:36:12:2. Dissolve the above mixed lipid materials in absolute ethanol, and the final concentration is 10 mg / mL (named PS-LNP).

[0054] Preparation Examples 2-9

[0055] Preparation Examples 2-9 each provide a liposome, the formulation and naming of which are shown in Table 1, and the preparation method refers to Preparation Example 1.

[0056] Table 1

[0057]

[0058]

[0059] Preparation Example 10

[0060] This preparation example provides a luciferase-labeled mRNA, which is purchased from Shenzhen Xinbosheng Biotechnology Co., Ltd., and the product number is FLuc mRNA (L-7602), named mRNA1.

[0061] Preparation Example 11

[0062] This preparation example provides an mRNA encoding a chimeric antigen receptor of fibroblast activation protein, which is synthesized by the following method:

[0063] (1) Obtain the FAP-CAR coding sequence containing the scFv fragment of the mouse-specific FAP monoclonal antibody and the cytoplasmic signal domains of CD3ζ and CD28, and link the FAP-CAR and the green fluorescent protein (EGFP) coding sequences together through the self-cleaving peptide P2A. The sequence of the prepared plasmid DNA is shown in SEQ ID NO:2.

[0064] (2) After the plasmid DNA with the sequence shown in SEQ ID NO:2 obtained in step (1) is expressed in mammalian cells and codon-optimized, the complete sequence is cloned into an in vitro transcription template plasmid carrying a T7 promoter, 5' and 3' UTR elements and a poly(A) tail.

[0065] (3) Linearize the plasmid DNA using a linearization kit digested with BspQI, purify the linearized plasmid using a DNA purification kit, and then transcribe mRNA in vitro using a T7 kit. Add 30 μL of DEPC water and 30 μL of lithium chloride to every 20 μL of the in vitro transcribed mRNA, place it at -20 °C for purification for 30 min, and then centrifuge at 12,000 rpm for 15 min. After washing twice with 70% ethanol, dilute it with DEPC water to a mRNA solution of 10 μg / μL.

[0066] (4) Perform capping treatment at the 5' end using a capping kit (NEB), then repeat the purification and washing steps of the above step (3), and finally resuspend it with DEPC water to 1 μg / μL to obtain the mRNA encoding the chimeric antigen receptor of fibroblast activation protein. All mRNAs are analyzed by agarose gel electrophoresis and stored at -80 °C. The amino acid sequence encoded by the prepared mRNA is shown in SEQ ID NO:1, and the mRNA is named mRNA2.

[0067] In addition, it should be noted that all mRNAs capable of expressing the amino acids shown in SEQ ID NO:1 can be used as the mRNAs of the mRNA delivery system described in the present invention, encoding the chimeric antigen receptor of fibroblast activation protein.

[0068] The specific sequence of SEQ ID NO:1 is: MALPVTALLLPLALLLHAARPQVQLKESGGLVQPGGSLKLSCAASGFTFSSYGMSWVRQTADKRLELVATTNNNGGVTYYPDSVKGRFTISRDNAKNTLYLQMSSLQSEDTAMYYCARYGYYAMDYWGQGISVTVSSGSTSGSGKPGSGEGSDVLMTQTPLWLPVSLGDQASISCRSSQSIVHSNGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTVKISRVEAEDLGVYYCFGGSHVPYTFGGGTKLEIKAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR。

[0069]

[0070] Comparative Preparation Example 1

[0071] Comparative Preparation Example 1 provides a commercially available liposome (purchased from AVT (Shanghai) Pharmaceutical Technology Co., Ltd., product number O02010), and its preparation raw materials include 1-octylnonyl 8-[(2-hydroxyethyl)[6-O-6-(undecyloxy)hexyl]amino]octanoate (SM-102), cholesterol (CHOL), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol 2000 (DMG-PEG2000), with a molar ratio of 50:38.5:10:1.5 and a final concentration of 10 mg / mL, named PC-LNP.

[0072] Example 1

[0073] This example provides a luciferase-modified LNP-mRNA, which is prepared by the following method:

[0074] (1) Take the luciferase-modified mRNA1 obtained in Preparation Example 10 and dilute it in 10 mM pH = 4 sodium citrate buffer to prepare an mRNA dilution of 0.16 mg / mL.

[0075] (2) According to the mass ratio of mRNA to LNP of 1:1 and the volume ratio of 1:3, add the PS-LNP obtained in Preparation Example 1 dropwise to the mRNA dilution, control the rotation speed at 1300 rpm, and the mixture is self-assembled to obtain LNP-mRNA.

[0076] (3) Dilute the LNP-mRNA with 20 times the volume of PBS, then transfer it to an ultrafiltration tube and ultrafilter at 3000 rpm and 4 °C for 20 min. After ultrafiltration and purification, collect the sample and store it in a 4 °C refrigerator. The purified LNP-mRNA is named PS-LNP-mRNA1.

[0077] Examples 2-9

[0078] Examples 2-9 each provide a luciferase-modified LNP-mRNA, and the differences from Example 1 are only that the PS-LNP obtained in Preparation Example 1 is sequentially replaced with the LNPs prepared in Preparation Examples 2-9, and the remaining steps are the same as those in Example 1, and they are named LNP-1-mRNA1, LNP-2-mRNA1, LNP-3-mRNA1, LNP-5-mRNA1, LNP-6-mRNA1, LNP-7-mRNA1, LNP-8-mRNA1, LNP-9-mRNA1 respectively.

[0079] Example 10

[0080] This example provides a delivery system for encapsulating mRNA encoding a chimeric antigen receptor of fibroblast activation protein. The difference from Example 1 is only that the luciferase-modified mRNA1 obtained in Preparation Example 10 in step (1) is replaced with mRNA2 encoding a chimeric antigen receptor of fibroblast activation protein obtained in Preparation Example 11, and the remaining steps are the same as those in Example 1. The obtained LNP-mRNA is named PS-LNP-mRNA2.

[0081] Comparative Example 1

[0082] Comparative Example 1 provides a luciferase-modified LNP-mRNA. The difference from Example 1 is only that the PS-LNP obtained in Preparation Example 1 is replaced with PC-LNP prepared in Comparative Preparation Example 1, and the remaining steps are the same as those in Example 1, and it is named PC-LNP-mRNA1.

[0083] Comparative Example 2

[0084] Comparative Example 2 provides a delivery system for encapsulating mRNA encoding a chimeric antigen receptor of fibroblast activation protein. The difference from Example 10 is only that the PS-LNP obtained in Preparation Example 1 is replaced with PC-LNP prepared in Comparative Preparation Example 1, and the remaining steps are the same as those in Example 10, and it is named PC-LNP-mRNA2.

[0085] Test Example 1

[0086] This test example tests the transfection and expression effects of the luciferase-modified LNP-mRNA1 prepared in Examples 1-9 and Comparative Example 1.

[0087] Normal mice (C57BL / 6J, 6 weeks old, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.) were used as test models. Each mouse was delivered the luciferase-modified LNP-mRNA1 prepared in each example and comparative example to the lungs of the mouse by intratracheal inhalation at an mRNA dose of 5 μg. Through small animal fluorescence imaging technology, the luciferase expression in the lungs of the mouse was observed, and the expression level of luciferase was quantitatively analyzed. The results are as Figure 1 shown.

[0088] As can be seen from the above results, the luciferase-modified LNP-mRNA1 prepared in Example 1 has better expression effects in the lungs.

[0089] Test Example 2

[0090] This test example tests the in vitro macrophage targeting of LNP.

[0091] (1) In vitro test:

[0092] (1.1) Extract BMDMs cells from mouse bone marrow and divide them into three groups, which are induced into M0, M1, and M2 phenotypes respectively. The treatment methods are as follows:

[0093] ① M0 macrophages: No treatment is performed and they remain in an unpolarized state.

[0094] ② M1 macrophages: Treated with LPS (100 ng / mL) for 24 hours to induce the M1 phenotype.

[0095] ③ M2 macrophages: Treated with IL-4 (20 ng / mL) for 24 hours to induce the M2 phenotype.

[0096] (1.2) DiD fluorescence-label PS-LNP-mRNA1 prepared in Example 1 and PC-LNP-mRNA1 prepared in Comparative Example 1 respectively for subsequent observation.

[0097] (1.3) Seed M0, M1, and M2 macrophages in an eight-well plate, with 1×10 4 cells per well. According to the dose of 50 ng mRNA per well, add DiD-labeled PS-LNP-mRNA1 or PC-LNP-mRNA1 to macrophages of different phenotypes respectively. Observe the M2 targeting ability by confocal microscopy and flow cytometry at 1 h and 4 h after administration.

[0098] The results are as Figure 2 shown. It can be seen that there are significant differences in the uptake of PS-LNP-mRNA1 and PC-LNP-mRNA1 by M2 macrophages, while there are no significant differences in M0 and M1.

[0099] Test Example 3

[0100] This test example tests the in vitro construction efficiency and function of CAR-M of the delivery system LNP-mRNA2 encapsulating mRNA encoding chimeric antigen receptor of fibroblast activation protein.

[0101] (1) Test grouping and intervention methods:

[0102] ① Blank group: No treatment is performed on M2 BMDMs;

[0103] ② PS-LNP-mRNA2 group: Transfect PS-LNP-mRNA2 obtained in Example 10 into M2 BMDMs according to the mRNA dose of 1 μg per well and culture for 24 h;

[0104] ③PC-LNP-mRNA2 group: Transfect the PC-LNP-mRNA2 obtained in Comparative Example 2 into M2-type BMDMs at an mRNA dose of 1 μg per well and culture for 24 h;

[0105] ④PS-LNP group: Transfect the PS-LNP obtained in Preparation Example 1 into M2-type BMDMs at the same LNP dose as the PS-LNP-mRNA2 group and culture for 24 h.

[0106] (2) Index test:

[0107] (2.1) In vitro construction efficiency test of CAR-M:

[0108] Detect the construction efficiency of CAR-M cells in the blank group, PS-LNP-mRNA2 group and PC-LNP-mRNA2 group by flow cytometry. The results are as Figure 3 shown. It can be seen that the expression of FAR-CAR of PS-LNP-mRNA2 is 1.6 times that of PC-LNP-mRNA2, which proves the successful in vitro construction of macrophages expressing FAP-CAR (CAR-M). At the same time, this further proves the successful construction of the mRNA encoding the chimeric antigen receptor of fibroblast activation protein in Preparation Example 11.

[0109] (2.2) Killing ability test:

[0110] Activate L929 cells with TGF-β1 to make them highly express FAP, and stably transfect L929 cells with luciferase for subsequent observation.

[0111] Co-culture the constructed CAR-M cells in the PS-LNP-mRNA2 group with activated L929 cells (fibroblasts) for 90 min. During this period, observe the endocytosis of CAR-M cells on L929 cells by laser confocal microscopy. The results are as Figure 4 shown, and it can be observed that CAR-M cells gradually endocytose L929 cells.

[0112] (2.3) Changes in cell phenotype and cytokine secretion level:

[0113] Analyze the cell phenotypes after different administration methods in each test group by flow cytometry, and test the cytokine secretion levels after different administration methods in each test group by PCR. The results are as Figure 5 shown, where A is the change difference in the markers of macrophages M1 (CD86) and M2 (CD206) after CAR-M construction, and B is the expression levels of TNF-α1, IL-6, TGF-β1, and IL-10, indicating that macrophages mainly transform into M1, and at the same time, the secretion of profibrotic cytokines is down-regulated.

[0114] (2.4) Test the proliferation and migration ability of fibroblasts:

[0115] The supernatant of BMDMs in each test group was used to culture L929 fibroblasts. A complete medium was prepared by mixing 20% supernatant + 69% DMEM + 10% serum + 1% double antibody. The fibroblasts were observed under a laser confocal microscope. The results are as Figure 6 shown. It can be seen that the PS-LNP-mRNA2 group significantly inhibited the activation ability of fibroblasts, and the FAP level was significantly reduced. PS-LNP-mRNA2 has a certain inhibitory effect on the proliferation and migration ability of fibroblasts.

[0116] Test Example 4

[0117] This test example tests the in-situ CAR-M construction in vivo and the anti-fibrotic effect of the delivery system LNP-mRNA2 encapsulating mRNA encoding a chimeric antigen receptor for fibroblast activation protein.

[0118] (1) Establish a mouse model of pulmonary fibrosis:

[0119] Bleomycin (BLM) was injected into the trachea once at a dose of 30 μg / mouse. An early mouse fibrosis model was obtained after 7 days, and a late mouse fibrosis model was obtained after 14 days.

[0120] (2) Experimental grouping and intervention methods:

[0121] ① Blank group: Both the early mouse model and the late mouse model were not given any additional treatment.

[0122] ② PS-LNP-mRNA2 group: For both the early mouse model and the late mouse model, the PS-LNP-mRNA2 obtained in Example 10 was delivered to the lungs of mice by intratracheal inhalation at a dose of 5 μg of mRNA2 per mouse. The drug was administered once every 3 days for a total of 3 times.

[0123] ③ PC-LNP-mRNA2 group: For both the early mouse model and the late mouse model, the PC-LNP-mRNA2 obtained in Comparative Example 2 was delivered to the lungs of mice by intratracheal inhalation at a dose of 5 μg of mRNA2 per mouse. The drug was administered once every 3 days for a total of 3 times.

[0124] ④ PS-LNP group: For both the early mouse model and the late mouse model, the PS-LNP prepared in Preparation Example 1 was delivered to the lungs of mice by intratracheal inhalation at a dose of 5 μg of mRNA per mouse. The drug was administered once every 3 days for a total of 3 times.

[0125] (3) Test the anti-fibrotic effect:

[0126] (3.1) In vivo targeting test of LNP-mRNA2:

[0127] Take the lung tissues of the late-stage mice in the PS-LNP-mRNA2 group, isolate the T cells, NK cells, macrophages (M2), monocytes, and dendritic cells in them, and analyze the internalization efficiency of PS-LNP-mRNA2 in different immune cells by flow cytometry to evaluate the uptake differences of PS-LNP-mRNA2 by various immune cells. The results are as Figure 7 shown. It can be seen that the mean fluorescence intensity of M2 macrophages is 10.7 times that of monocytes and 1.8 times that of dendritic cells, indicating that PS-LNP has a good targeting effect on M2 macrophages.

[0128] (3.2) Verification of in situ CAR-M construction in the lungs with LNP-mRNA2:

[0129] (3.2.1) Take the lung tissues of the late-stage mice in the PS-LNP-mRNA2 group, perform immunofluorescence staining on the lung tissues of the mice, observe the expression of CARs on the surface of macrophages and their co-localization with FAP, and verify the construction and function of CAR-M. The results are as Figure 8 shown. It can be seen that co-localization of F4 / 80 and FAP-CAR appears on the surface of macrophages in the PS-LNP-mRNA2 administration group, proving the successful construction of CARs on the surface of macrophages. In addition, compared with the control group, the FAP level in the tissues of the PS-LNP-mRNA2 administration group decreases, proving the effectiveness of FAP CAR-M.

[0130] (3.2.2) To highlight the therapeutic advantages of in situ CAR-M construction, extract alveolar macrophages from the bronchoalveolar lavage fluid of mice. After in vitro transfection with DiD-labeled PS-LNP-mRNA2 for 24 hours, CAR-M is labeled with Dil and re-infused into the mice through the trachea for comparison. The results are as Figure 9 shown. After re-infusion, CAR-M is mainly distributed near the airways and blood vessels, while in situ construction of CAR-M by inhaling LNP-mRNA2 in vivo has deeper interstitial infiltration.

[0131] (3.3) Test of the degree of pulmonary fibrosis reversal:

[0132] After the mice in each experimental group have completed drug administration, evaluate whether pulmonary fibrosis reversal has been achieved through Masson staining and H&E staining pathological sections. The results are as Figure 10 shown. It can be seen that the early-stage mouse fibrosis model has basically achieved pathological reversal of pulmonary fibrosis, that is, early intervention can play a preventive effect; while after PS-LNP-mRNA administration in the late-stage mouse fibrosis model, the progression of pulmonary fibrosis is significantly inhibited, that is, late intervention can significantly inhibit the progression of pulmonary fibrosis.

[0133] Test Example 5

[0134] In this test example, the anti-fibrotic effect of the delivery system LNP-mRNA2, which encapsulates the mRNA encoding the chimeric antigen receptor of fibroblast activation protein, was evaluated in other models.

[0135] (1) Construction of mouse pulmonary fibrosis model:

[0136] Silicosis mouse model: Male C57BL / 6J mice at 7 weeks of age were selected for the construction of the silicosis model. First, a SiO2 suspension (300 mg / mL) was prepared and instilled into the trachea at a dose of 40 μL / 20 g. After 2 weeks, a silicosis mouse model was obtained.

[0137] Radiation-induced pulmonary fibrosis mouse model: Mice were irradiated with a single dose of 17 Gy. After 3.5 months, a radiation-induced pulmonary fibrosis mouse model was obtained.

[0138] (2) Experimental grouping and intervention methods:

[0139] ① Blank group: The mouse pulmonary fibrosis model was not treated additionally.

[0140] ② PS-LNP-mRNA2 group: According to the mRNA dose of 5 μg per mouse, the PS-LNP-mRNA2 obtained in Example 10 was delivered to the lungs of mice in different models by intratracheal inhalation, once every 3 days, for a total of 3 times.

[0141] ③ PC-LNP-mRNA2 group: According to the mRNA dose of 5 μg per mouse, the PC-LNP-mRNA2 obtained in Comparative Example 2 was delivered to the lungs of mice in different models by intratracheal inhalation, once every 3 days, for a total of 3 times.

[0142] ④ PS-LNP group: According to the mRNA dose of 5 μg per mouse, the PS-LNP prepared in Preparation Example 1 was delivered to the lungs of mice in different models by intratracheal inhalation, once every 3 days, for a total of 3 times.

[0143] (3) Testing the alleviating effect of LNP-mRNA2 on pulmonary fibrosis in silicosis mice:

[0144] After the mice in each experimental group were administered as described above, the H&E and Masson staining results of the lung sections after treatment in different groups were evaluated. The H&E and Masson staining results of the lung sections are as Figure 11 shown. The intervention with PS-LNP-mRNA2 significantly reduced the number and size of nodules in silicosis.

[0145] (4) To test the regulatory effect of LNP-mRNA2 on radiation-induced pulmonary fibrosis in mice:

[0146] After the mice in each experimental group were administered drugs in the above manner, an Elisa kit was used to test the content of hydroxyproline (HYP), a pulmonary fibrosis marker, in the mice after treatment in different groups. The results are as Figure 12 shown. After the intervention of PS-LNP-mRNA2, the level of HYP was significantly decreased. It is indicated that PS-LNP-mRNA2 also has a significant alleviating effect on the degree of pulmonary fibrosis in radiation-induced pulmonary fibrosis mice.

[0147] Test Example 6

[0148] This test example evaluates the safety of the mice in each experimental group of Test Example 4.

[0149] (1) Test method:

[0150] After the administration of drugs to the mice in the PS-LNP-mRNA2 group, PC-LNP-mRNA2 group, and PS-LNP group in Test Example 4 was completed, their main organs were taken, and the pathological damage of the main organs was evaluated by H&E staining; meanwhile, normal mice injected with 100 μL of PBS were used as controls. The results are as Figure 13 shown. The results indicate that compared with normal mice, there was no significant damage to the organs of each mouse treated with drugs, indicating that the LNP-mRNA used to produce CAR-M in vivo has good safety.

[0151] The applicant declares that the present invention uses the above embodiments to illustrate the technical solutions of the present invention, but the present invention is not limited to the above embodiments, that is, it does not mean that the present invention must rely on the above embodiments to be implemented. Those skilled in the art should understand that any improvement to the present invention, the equivalent replacement of each raw material of the products of the present invention, the addition of auxiliary components, and the selection of specific methods, etc., all fall within the protection scope and the disclosure scope of the present invention.

[0152] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept scope of the present invention, various simple modifications can be made to the technical solutions of the present invention, and these simple modifications all fall within the protection scope of the present invention.

[0153] In addition, it should be noted that in the above specific embodiments, the various specific technical features described can be combined in any appropriate manner without conflict. To avoid unnecessary repetition, the present invention will not separately describe various possible combination methods.

Claims

1. An mRNA delivery system for in-situ generation of CAR-M targeting M2 macrophages, characterized in that, The mRNA delivery system includes liposomes and mRNA encapsulated in the liposomes; The raw materials for preparing the liposomes include ionizable lipids, cholesterol, phosphatidylserine, and dimyristoyl glycerol polyethylene glycol; The mRNA encodes a chimeric antigen receptor of fibroblast activation protein.

2. The mRNA delivery system according to claim 1, wherein The ionizable lipid includes 1-octyl nonyl 8-[(2-hydroxyethyl)[6-O-6-(undecyloxy)hexyl]amino]-octanoate.

3. The mRNA delivery system according to claim 1 or 2, characterized in that, The phosphatidylserine includes dioleoyl phosphatidylserine; Preferably, the dimyristoyl glycerol polyethylene glycol includes 1,2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol; Preferably, the molecular weight of polyethylene glycol in the dimyristoyl glycerol polyethylene glycol is 1000-5000 Da.

4. The mRNA delivery system according to any one of claims 1-3, characterized in that, The molar ratio of the ionizable lipid, cholesterol, phosphatidylserine, and dimyristoyl glycerol polyethylene glycol is 50:(33.5-38.5):(10-14):(1.5-2.5), preferably 50:(35-36):(11-13):(1.5-2).

5. The mRNA delivery system according to any one of claims 1-4, characterized in that, The amino acid sequence encoded by the mRNA includes the sequence shown in SEQ ID NO:

1.

6. A method for preparing an mRNA delivery system for in-situ generation of CAR-M targeting M2 macrophages as described in any one of claims 1-5, characterized in that, The preparation method includes: (1) Mix the raw materials for preparing the liposomes to obtain liposomes; (2) Mix and stir the mRNA diluent with the alcohol solution of the liposomes obtained in step (1), and self-assemble to obtain the mRNA delivery system.

7. The preparation method according to claim 6, characterized in that, The temperature of the mixing in step (1) is 20-30 °C, and the time is 2-5 min.

8. The preparation method according to claim 6 or 7, characterized in that The rotation speed of the stirring in step (2) is 1200-1500 rpm, the temperature is 20-30 °C, and the time is 2-5 min.

9. The preparation method according to any one of claims 6-8, characterized in that, The alcohol solution includes absolute ethanol; Preferably, the preparation method of the mRNA diluent includes: diluting the mRNA in a citrate buffer solution.

10. Use of an mRNA delivery system for in-situ generation of CAR-M targeting M2 macrophages as described in any one of claims 1-5 in the preparation of an anti-pulmonary fibrosis drug or a drug for remodeling and reversing pulmonary fibrosis.

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