Use of the lgmn overexpression plasmid in the preparation of a drug for treating sarcoidosis

Abstract

This invention discloses a lipid nanoparticle composition loaded with a DNA plasmid capable of overexpressing LGMN, its preparation method, and its applications, belonging to the field of biomedicine and drug delivery technology. The composition comprises a core nucleic acid construct encoding the LGMN gene and specific lipid nanoparticles encapsulating it. The composition significantly improves nucleic acid stability, cell delivery efficiency, and target gene expression levels, exhibiting excellent efficacy in the treatment of sarcoidosis. Furthermore, this composition can also be used in non-therapeutic fields such as in vitro cell engineering and recombinant protein production. The preparation method of this invention is simple and easy to scale up, providing an efficient and safe delivery tool for LGMN gene-based sarcoidosis treatments and related biotechnological applications.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of LGMN overexpression plasmid in the preparation of drugs for treating sarcoidosis. Background Technology

[0002] Sarcoidosis is a systemic granulomatous disease of unknown etiology, characterized by non-caseating necrotizing epithelioid granulomas. More than 90% of patients experience involvement of the lungs and intrathoracic lymph nodes, but vital organs such as the heart, central nervous system, skin, and eyes can also be affected. This multi-system involvement leads to complex and diverse clinical manifestations, posing a significant challenge to clinical diagnosis. Although approximately two-thirds of pulmonary sarcoidosis patients achieve clinical remission within several years, one-third experience a chronic progressive course, ultimately leading to irreversible fibrocystic remodeling of lung tissue. This pathological process is closely associated with a 12%-18% mortality rate within 5 years in sarcoidosis patients. Despite extensive research into alternative treatments for sarcoidosis, glucocorticoids remain the first-line treatment. However, some patients cannot tolerate the adverse effects of long-term glucocorticoid therapy, and some patients experience relapse during dose tapering. Therefore, a deeper understanding of the molecular mechanisms of granuloma formation is crucial to identifying new therapeutic targets for sarcoidosis.

[0003] Legumain (LGMN), also known as asparagine endopeptidase, is a member of the C13 family of cysteine ​​proteases that hydrolyzes the C-terminus of asparagine residues. Although LGMN is primarily located in late endosomes and lysosomes, participating in protein catabolism under physiological conditions, it can also be translocated from lysosomes to the cytoplasm, cell surface, or secreted by cells, participating in multiple biological processes. LGMN expression is upregulated in various pathological states, including cancer, neurodegenerative diseases, cardiovascular diseases, organ fibrosis, and inflammatory diseases. In tumor tissues, LGMN mainly originates from tumor-associated macrophages (TAMs). In aortic dissection and renal fibrosis, LGMN co-localizes with the M2 polarization marker CD206, and its expression level is significantly increased in interleukin (IL)-4-induced M2 macrophages. Previous studies have shown that LGMN can promote M2 polarization and drive macrophage transformation from M1 to M2, indicating that LGMN can regulate macrophage polarization direction. Based on the central role of macrophages in the formation of sarcoidosis granulomas and the functional state transitions mediated by macrophage polarization, this study hypothesizes that LGMN may be involved in the pathogenesis of sarcoidosis. Summary of the Invention

[0004] In view of this, in order to solve the above-mentioned technical problems currently faced in this field, the purpose of this invention is to provide a liposomal nanoparticle loaded with LGMN overexpression plasmid, providing a general and transferable new technical solution for the treatment of sarcoidosis.

[0005] To achieve the above objectives, the present invention adopts the following technical solution: A first aspect of the present invention provides a composition capable of overexpressing LGMN, the composition comprising a nucleic acid construct encoding LGMN and a lipid component capable of encapsulating the nucleic acid construct.

[0006] Furthermore, the nucleic acid construct is capable of including a nucleic acid molecule encoding the LGMN protein in a host cell.

[0007] Furthermore, the nucleic acid molecules include DNA molecules and RNA molecules.

[0008] Furthermore, the DNA molecules include genomic DNA, complementary DNA, linear DNA molecules, and circular DNA molecules.

[0009] Furthermore, the DNA molecule is a circular DNA molecule.

[0010] Furthermore, the circular DNA molecule includes plasmids, kinasemids, bacterial artificial chromosomes, and yeast artificial chromosomes.

[0011] In a specific embodiment of the present invention, the circular DNA molecule is a plasmid.

[0012] In this invention, the term "recombinant plasmid" refers to a circular recombinant DNA molecule formed by cutting a plasmid vector and a target DNA fragment with a restriction endonuclease and then ligating them in vitro. It refers both to the construction process and the final product, and is a core tool in genetic engineering for cloning and expressing foreign genes. A typical plasmid vector contains elements such as the origin of replication, a selective marker gene (e.g., an antibiotic resistance gene), and a multiple cloning site.

[0013] Furthermore, the lipid component comprises one or more of cationic lipids, neutral auxiliary lipids, and polyethylene glycol-modified lipids.

[0014] In this invention, the term "lipid nanoparticles" refers to closed vesicle-type or spherical nanoparticles composed of a lipid bilayer with an aqueous interior, typically with a particle size in the range of 1-1000 nm, particularly referring to liposomes at the nanoscale of 20-200 nm. As a drug delivery system, it can encapsulate hydrophilic or hydrophobic substances, mimicking cell membrane structures, and deliver its contents into the cell through fusion with the cell membrane or endocytosis. In this invention, this term is synonymous with "liposome nanoparticles," which are typically composed of cationic lipids, neutral accessory lipids (such as phospholipids and cholesterol), polyethylene glycol-modified lipids, etc.

[0015] Furthermore, the cationic lipids include ionizable cationic lipids, quaternary ammonium salt cationic lipids, and polyamine lipids.

[0016] Furthermore, the ionizable cationic lipids include SM-102, ALC-0315, Dlin-MC3-DMA, C12-200, and TT3.

[0017] Furthermore, the quaternary ammonium cationic lipids include, but are not limited to, (2,3-dioleoyl-propyl)-trimethylammonium chloride (DOTAP) and N-[1-(2,3-dilinoleoyloxy)propyl]-N,N,N-trimethylammonium (DOTMA).

[0018] Furthermore, the polyamine lipids include 2,3-dioleoxy-N-[2-(spermine-4-carbamoyl)ethyl]-N,N-dimethyl-1-propanediamine pentachloride (DOSPA), 3β-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol (DC-Chol), and cationic lipids based on macrocyclic polyamines (such as cyclen, cyclam) as hydrophilic heads.

[0019] In a specific embodiment of the present invention, the cationic lipid is SM102.

[0020] Furthermore, the neutral auxiliary lipids include phospholipids and cholesterol.

[0021] Furthermore, the phospholipids include phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phosphatidylserine (PS), phosphatidylinositol (PI), and sphingomyelin (SM).

[0022] Furthermore, the phospholipid is phosphatidylcholine (PC).

[0023] Further, the phosphatidylcholine (PC) includes one or more of distearylphosphatidylcholine (DSPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylcholine (DOPC), 1-palmitoyl-2-oleoylphosphatidylcholine (POPC), and hydrogenated soybean phosphatidylcholine (HSPC).

[0024] In a specific embodiment of the present invention, the phospholipid is DSPC.

[0025] Furthermore, the polyethylene glycol-modified lipid is a PEGylated phospholipid.

[0026] Furthermore, the PEGylated phospholipids include PEG-DMG, PEG-DSG, and PEG-DPPE.

[0027] Furthermore, the polyethylene glycol in the PEGylated phospholipid has a molecular weight of 1000~10000 Daltons.

[0028] In a specific embodiment of the present invention, the polyethylene glycol-modified lipid is PEG-DMG.

[0029] Furthermore, the polyethylene glycol in the PEG-DMG has a molecular weight of 2000 Daltons.

[0030] Furthermore, the molar ratio of SM102, cholesterol, DSPC, and PEG-DMG is (30-60):(20-50):(8-15):(1:10).

[0031] Furthermore, the molar ratio is 57.2:24.0:12.7:6.1.

[0032] A second aspect of the present invention provides a lipid nanoparticle overexpressing LGMN, wherein the lipid nanoparticle comprises a core of a nucleic acid construct encoding LGMN and an outer core of a lipid component as described in the first aspect of the present invention.

[0033] Furthermore, the nucleic acid construct encoding LGMN is loaded into the paper component to form the lipid nanoparticles.

[0034] Furthermore, the average hydrodynamic diameter of the lipid nanoparticles is 50-200 nm.

[0035] Preferably, the lipid nanoparticles have an average hydrodynamic diameter of 94.11 nm.

[0036] Preferably, the zeta potential of the lipid nanoparticles is -30 mV to +10 mV.

[0037] Preferably, the zeta potential of the lipid nanoparticles is -2.78 mV.

[0038] Furthermore, the lipid nanoparticles also carry fluorescently labeled molecules; Furthermore, the fluorescently labeled molecules include fluorescein, rhodamine, and cyanine dyes; Furthermore, the cyanine dye is DiR.

[0039] In this invention, the term "hydrodynamic diameter" refers to the diameter of an ideal sphere with the same translational diffusion coefficient as the test particle in solution, as measured by dynamic light scattering technology. It reflects the overall motion behavior of particles (including the solvent layer adsorbed on their surface) in the fluid and is a key parameter characterizing the apparent size of nanoparticles in solution. Its measurement is based on the Stokes-Einstein equation. The term "Zeta potential" refers to the potential at the interface (i.e., the sliding surface) where the counterion layer adsorbed on the surface of charged particles moves relative to the continuous phase fluid in a dispersion system. It is an important indicator characterizing the stability of colloidal dispersion systems. The higher the absolute value of the Zeta potential (typically > ±30 mV), the stronger the electrostatic repulsion between particles and the more stable the system; the lower the absolute value (closer to 0 mV), the easier it is for aggregation or flocculation to occur.

[0040] A third aspect of the present invention provides a medicament for the treatment and / or prevention of sarcoidosis, the medicament comprising one or more of the compositions provided in the first aspect of the present invention and the lipid nanoparticles provided in the second aspect.

[0041] Furthermore, the drug also contains pharmaceutically acceptable carriers and / or excipients.

[0042] Furthermore, the dosage form of the drug includes injections, inhalers, and topical administration.

[0043] Furthermore, the injectable includes an injection solution and a lyophilized powder injection; the inhalant includes a nebulized inhalation solution and a dry powder inhaler; and the topical medication includes a cream, a gel, and eye drops.

[0044] The pharmaceutically acceptable carriers described in this invention include, but are not limited to, nucleic acid delivery carriers (such as lipid nanoparticles, viral vectors, non-viral polymer carriers, etc.), protein / peptide delivery and modification carriers (such as fusion protein tags, chemically modified carriers, sustained-release microspheres, etc.), and carriers for the preparation of cell therapy products. The excipients include, but are not limited to, stabilizers, including sugars / polyols (such as sucrose, trehalose, mannitol, used as lyophilization protectants and stabilizers), surfactants (such as polysorbate 80, to prevent aggregation caused by interfacial adsorption), and certain amino acids (such as histidine, arginine); buffer systems, such as histidine buffer, Tris buffer, phosphate buffer, used to maintain a precise pH environment; and isotonic adjusters, such as sodium chloride, glycerol, used to adjust the osmotic pressure of the formulation.

[0045] Furthermore, the routes of administration of the drug include enteric administration and non-enteric administration.

[0046] Furthermore, the enteric route of administration includes oral administration and rectal administration; the non-enteric route of administration includes injection administration and topical application.

[0047] Furthermore, the route of administration is a non-enteric route.

[0048] Furthermore, the non-enteric route of administration includes injection and topical application.

[0049] Furthermore, the local medication includes airway delivery, stent delivery, and balloon delivery.

[0050] In a specific embodiment of the present invention, the route of administration is airway delivery.

[0051] In this invention, the term "enteral route of administration" refers to a method of drug delivery where the drug is absorbed into the systemic circulation through the digestive tract (including the stomach, small intestine, large intestine, and rectum). Its characteristic is that the drug must withstand the influence of gastrointestinal digestive juices, enzymes, and the first-pass effect. The term "non-enteral route of administration" refers to a method of drug delivery that bypasses the digestive tract, allowing the drug to directly enter the body's tissues, fluids, or bloodstream. This is the most common route of administration for macromolecular drugs (such as protein and nucleic acid drugs) and drugs easily destroyed by the gastrointestinal tract, to avoid the first-pass effect and degradation by digestive enzymes. The term "topical administration" refers to the direct application of a drug to the body surface or specific local body parts (such as skin, eyes, nasal cavity, oral cavity, vagina, etc.), aiming to ensure that the drug exerts its effect primarily in that local area, rather than producing a systemic effect. The term "airway delivery" refers to a method of drug delivery through the respiratory system to the lungs or upper respiratory tract. Specific forms include inhalation (such as via nebulizers, metered-dose inhalers, and dry powder inhalers) and intranasal administration. Drugs can be deposited in different parts of the respiratory tract to treat local diseases (such as asthma and chronic obstructive pulmonary disease) or to exert systemic effects through rapid absorption in the alveoli.

[0052] A fourth aspect of the present invention provides a drug delivery system comprising one or more of the composition provided in the first aspect of the present invention, lipid nanoparticles provided in the second aspect, and a drug provided in the third aspect.

[0053] Furthermore, the system also includes a drug delivery device.

[0054] Furthermore, the drug delivery device includes an oral delivery device, an injection delivery device, an inhalation delivery device, a local delivery device, and an implantable delivery device.

[0055] Furthermore, the inhalation drug delivery device includes a dry powder inhaler, a nebulizer, a pressure metering inhaler, and a nebulized inhaler.

[0056] A fifth aspect of the present invention provides a kit comprising one or more of the composition provided in the first aspect of the present invention, lipid nanoparticles provided in the second aspect, a drug provided in the third aspect, and a kit provided in the fourth aspect.

[0057] Furthermore, the kit also includes instructions.

[0058] The sixth aspect of the present invention provides any of the following methods: (1) A method for preparing any one of the compositions described in the first aspect of the present invention and the lipid nanoparticles described in the second aspect of the present invention, wherein the method comprises mixing an aqueous phase containing a nucleic acid construct encoding LGMN with a lipid phase containing a lipid component described in the first aspect of the present invention to form lipid nanoparticles loaded with the nucleic acid construct. (2) An in vitro detection method, characterized in that the method includes contacting target cells with lipid nanoparticles as described in the second aspect of the present invention and detecting them by fluorescence signals; (3) A method for increasing the expression level of gene LGMN in cells in vitro, characterized in that the method involves contacting one or more of the composition described in the first aspect of the present invention and the lipid nanoparticles described in the second aspect with target cells expressing gene LGMN in vitro. (4) A method for constructing engineered cells, characterized in that the method involves introducing one or more of the composition described in the first aspect of the present invention and the lipid nanoparticles described in the second aspect into target cells in vitro, and screening to obtain cells that overexpress the gene LGMN. (5) A method for producing LGMN protein in cells, characterized in that the method involves transfecting one or more of the composition of the first aspect of the present invention and the lipid nanoparticles of the second aspect into target cells to express and obtain the LGMN protein.

[0059] In this invention, the term "engineered cell" refers to a cell line that has unique traits or new functions with stable inheritance, obtained by modifying, altering, or recombining the genetic material of a host cell through genetic engineering techniques (such as gene transfection and gene editing) or cell fusion techniques. Examples include engineered CHO cells for producing recombinant proteins, or engineered T cells (such as CAR-T cells) for cell therapy. The term "transfection" refers to the process of artificially introducing exogenous nucleic acids (such as DNA and RNA) into eukaryotic cells through non-viral means (physical or chemical methods). Its purpose is to enable the exogenous nucleic acid to express or regulate gene function within the cell. Based on the duration of the exogenous nucleic acid's presence in the host cell, it can be divided into transient transfection (not integrated into the genome) and stable transfection (integrated into the genome). This term is distinct from "transformation," which is mainly used for prokaryotic cells, and virus-mediated "transduction."

[0060] The seventh aspect of the present invention provides for any of the following applications: (1) Use of any one of the compositions of the first aspect of the present invention, the lipid nanoparticles of the second aspect, the medicament of the third aspect, the drug delivery system of the fourth aspect, the kit of the fifth aspect, and the method of the sixth aspect in the preparation of a medicament for treating and / or preventing sarcoidosis; (2) The application of the lipid nanoparticles described in the second aspect of the present invention in the preparation of reagents for cell labeling or targeted imaging; (3) Use of a nucleic acid molecule encoding LGMN protein in the preparation of a drug for the treatment of sarcoidosis.

[0061] In this invention, the term "sarcoidosis" refers to a systemic inflammatory disease of unknown etiology, characterized by the formation of non-caseating granulomas mediated by an immune response in multiple organs or tissues. Sarcoidosis can affect the lungs, lymph nodes, skin, eyes, and other organs, and its development is related to abnormal immune responses and imbalances in inflammatory regulation. The preferred type of sarcoidosis involves primarily the lungs, but is not limited to this.

[0062] The advantages and beneficial effects of this invention are as follows: The composition provided by this invention can effectively encapsulate and protect LGMN overexpression plasmids, significantly improving their storage and in vivo stability, and possesses suitable particle size, potential, and high encapsulation efficiency, with well-defined and controllable physicochemical properties. Furthermore, it exhibits stronger target gene expression and therapeutic efficacy in both in vitro and in vivo sarcoidosis models. Functional experiments show that the encapsulated plasmid... Lgmn LNPs of pDNA P. acnesThe composition significantly reduces both induced and TDM-induced granulomatous inflammation and demonstrates good biocompatibility. The preparation process of the composition is simple, reproducible, easily scaled up for production, and adaptable to various clinical dosage forms. In summary, the liposome nanoparticles loaded with LGMN overexpression plasmid provided by this invention offer a novel, efficient, safe, and industrially promising strategy for the treatment and prevention of sarcoidosis. Attached Figure Description

[0063] Figure 1 For package Lgmn LNP characterization of overexpression plasmids; a) containing Lgmn Transmission electron microscopy representation of LNPs in pDNA; scale bar = 100 nm. b) Encapsulation Lgmn c) Average particle size, zeta potential, and encapsulation efficiency of LNPs in pDNA; Lgmn The hydrodynamic diameter distribution of pDNA LNPs over 24 h (n = 3); d) The changes in the hydrodynamic diameter of prepared LNPs at different time points (0d, 1d, 3d, 6d) (n = 3); Figure 2 For package Lgmn In vivo biodistribution characteristics of LNPs overexpressing plasmids; a) DiR-labeled loading Lgmn In vivo fluorescence images of mouse LNPs containing pDNA at different time points (0d, 1d, 3d, 6d) after intratracheal instillation (n=4 per group); b) DiR-labeled inclusions Lgmn The in vitro fluorescence intensity of major organs in mice on day 3 after airway delivery of pDNA LNPs; Figure 3 For carrying Lgmn In vivo safety assessment of pDNA LNPs; a) Intratracheal injection of pDNA-containing LNPs into mice Lgmn Following LNPs of pDNA, H&E staining results of heart, liver, spleen, lung, and kidney (n = 3 per group); images acquired at 200x magnification, scale bar = 50 μm; b) delivery via airway of pDNA carrying empty vector plasmid (n = 6) or Lgmn After LNPs of pDNA (n = 7), P. acnes Serum biochemical parameters of model mice were analyzed; data are expressed as mean ± standard error, and unpaired t-tests were used for comparisons between groups; Figure 4 For airway delivery Lgmn Temporal changes in LGMN expression in lung tissue after pDNA LNPs; airway injection of pDNA-encapsulated pDNA. LgmnProtein expression of LGMN in mouse lung tissue at 0d, 1d, 3d, and 6d after LNPs of pDNA (n=4 per group); comparisons among multiple groups were performed using one-way ANOVA, followed by Tukey's multiple comparison test, *** p<0.001; Figure 5 For package Lgmn Prophylactic administration of pDNA LNPs reduces P. acnes Induced granulomatous inflammation; a) Encapsulation Lgmn Schematic diagram of the pDNA LNPs prophylactic treatment experimental protocol; b) In P. acnes Under the induction of airway delivery of empty vectors or Lgmn Histological analysis of granulomatous inflammation in mouse lung tissue after LNPs of pDNA (Lgmn-OE) (control group n = 3, Vector group n = 6, Lgmn -OE group n = 7), images were acquired at 200x magnification, scale bar = 50μm; c) in P. acnes Under the induction of [the drug], the blank control group (n = 3), the Vector group (n = 6), and [other groups] were [treated / treated]. Lgmn - IHC staining and quantitative analysis results of CD4, CD68, and Mac-2 in lung tissue sections of mice in the OE group (n = 7). Representative images were acquired at 200x magnification, scale bar = 50 μm; d) Blank control group (n = 3), Vector group (n = 4) or Lgmn Immunofluorescence co-staining results of p-S6 (green) and Mac-2 (red) in lung tissue sections of mice in the OE group (n = 4). Cell nuclei were stained blue by DAPI. Images were acquired at 400x magnification, scale bar = 20 μm; e) qRT-PCR detection of blank control group (n = 4), Vector group (n = 6) or Lgmn The mRNA expression levels of Cxcl9, Cxcl10, Tnf, and Ifng in lung tissue homogenates of mice in the OE group (n = 6) were measured. Data are expressed as mean ± standard error. Unpaired t-tests were used for comparisons between groups in Figure b, and one-way ANOVA was used for comparisons among multiple groups in Figures c, d, and e, followed by Tukey's multiple comparison test. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Figure 6 For package Lgmn Therapeutic administration of pDNA LNPs alleviates P. acnes Induced granulomatous inflammation; a) Representative image of H&E staining in mouse lung tissue on day 17 after P. acnes sensitization; images acquired at 200x magnification, scale bar = 50μm; b) Encapsulation LgmnSchematic diagram of the therapeutic dosing protocol for pDNA LNPs; c) Figure 3-7 b) Representative H&E staining images of lung tissue from each group of mice, scale bar = 50 μm; d) On day 21 after P. acnes sensitization, airway delivery of empty vectors (vector) (n = 5) or Lgmn pDNA ( Lgmn -OE)(n = 6) LNPs granuloma area statistics in mouse lung tissue; e) in P. acnes Under the guidance of [the vector group (n = 5) and] Lgmn Immunohistochemical staining and quantitative analysis of CD4 and Mac-2 in lung tissue sections of mice in the OE group (n = 6) were performed. Representative images were acquired at 200x magnification, with a scale bar of 50 μm. f) Western blot analysis of the Vector group and Lgmn - Protein expression levels of p-STAT1 (Ser727 and Tyr701 sites) and p-S6 in lung tissue of mice in the OE group (n = 6 per group); g) qRT-PCR detection of p-STAT1 (Ser727 and Tyr701 sites) and p-S6 in the Vector group (n = 5) and Lgmn -OE group (n = 6) mouse lung tissue homogenate Ccl4 , Cxcl9 , Cxcl10 , Tnf and Ifng mRNA expression levels; data are expressed as mean ± standard error, and unpaired t-tests were used for comparisons between groups, * p < 0.05, ** p < 0.01, *** p < 0.001; Figure 7 Polarization characteristics and mTORC1 / STAT1 activation levels in TDM-induced mouse lung granuloma tissue; a) Western blot analysis of iNOS and Arg-1 protein expression in lung tissues of mice in the emulsion control group (n = 5) and the TDM-induced pulmonary granuloma model group (n = 5); b) Western blot analysis of p-STAT1 (Ser727 and Tyr701 sites) and p-S6 protein expression levels in lung tissues of mice in the emulsion control group (n = 5) and the TDM-induced pulmonary granuloma model group (n = 5). Data are expressed as mean ± standard error. Unpaired t-tests were used for comparisons between groups. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Figure 8 For package Lgmn pDNA LNPs alleviate TDM-induced pulmonary granulomatous inflammation; a) Encapsulation Lgmna) Schematic diagram of the experimental protocol for reducing TDM-induced pulmonary granulomatous inflammation by pDNA LNPs; b) Schematic diagram of H&E staining of lung tissue in each group of mice shown in Figure a; c) Statistical graph of average granuloma area in lung tissue of each group of mice shown in Figure a; d) Analysis of IHC staining results of lung tissue in each group of mice shown in Figure a; e) Comparison of transcriptional levels of multiple pro-inflammatory factors in lung tissue of mice in the Lgmn-OE group and the Vector group; f) mRNA levels of M1 macrophage markers in lung tissue of mice in the Lgmn-OE group and the Vector group; g) protein expression levels of M1 macrophage markers in lung tissue of mice in the Lgmn-OE group and the Vector group; h) Statistical analysis of expression levels of p-STAT1 (Ser727 and Tyr701 sites) and p-S6 proteins in lung tissue of mice in the Lgmn-OE group and the Vector group. Detailed Implementation The present invention will be further described below with reference to embodiments. The following description is merely a preferred embodiment of the present invention and is not intended to limit the invention in any other way. Any person skilled in the art may make equivalent modifications to the disclosed technical content to create equivalent embodiments. Any simple modifications or equivalent changes made to the following embodiments based on the technical essence of the present invention without departing from the scope of the invention are all within the protection scope of the present invention.

[0064] Table 1 Experimental Materials

[0065] Table 2 Experimental Instrument Information

[0066] Example 1 Package Lgmn Overexpression plasmids ( Lgmn Construction and characterization of LNPs (pDNA) Preparation and characterization of LNPs 1) Lgmn Preparation of plasmid DNA loaded with LNPs Lgmn Sequence information was obtained from NCBI (ID: NM_011175.4), and the GV492 plasmid vector was purchased from Shanghai Jikai Gene Medical Technology Co., Ltd. Overexpression was constructed using conventional methods in the field. Lgmn The plasmid was prepared according to the method reported in the literature. LgmnThe pDNA LNPs (https: / / doi.org / 10.1038 / s41467-021-27684-9; https: / / doi.org / 10.1021 / ja301621z), SM102, cholesterol, DSPC, and PEG-DMG-2K were accurately weighed and dissolved in anhydrous ethanol at a molar ratio of 57.2:24.0:12.7:6.1 to prepare a lipid phase. Lgmn pDNA or vector was dissolved in PBS buffer to prepare an aqueous phase. Then, using the NanoGenerator™ Flex-M microfluidic system, the lipid phase and aqueous phase were mixed at a mass ratio of 1:3 to prepare LNPs via microfluidic technology. The constructed LNP suspension was then ultracentrifuged to remove residual ethanol and unencapsulated free pDNA, diluted to an appropriate volume with PBS buffer, and stored at 4°C for later use.

[0067] 2) Characterization of the physicochemical properties of LNPs The hydrodynamic diameter, polydispersity index (PDI), and zeta potential of the prepared LNPs were determined using dynamic light scattering and a Zetasizer Nano-ZS nanoparticle potentiometer. Following the manufacturer's instructions, the total pDNA and free pDNA content of the prepared LNPs were determined using a Quanti-iT PicoGreen dsDNA fluorescent dye assay kit to calculate the pDNA encapsulation efficiency of the LNPs. Furthermore, an appropriate amount of LNP suspension was negatively stained with 2% phosphotungstic acid, and the morphology and structure of the LNPs were observed under a transmission electron microscope.

[0068] To verify the protective effect of LGMN in sarcoidosis-like granulomas and promote its clinical translation, this study constructed a membrane containing LGMN. Lgmn Overexpression plasmids ( Lgmn LNPs for pDNA were prepared. The encapsulation efficiency of the prepared LNPs for pDNA was 69.15%, and the zeta potential was -2.78 mV. Figure 1 a) Transmission electron microscopy revealed that it contained... Lgmn -pDNA LNPs are regular spherical in shape, with uniform particle size and an average diameter of approximately 100 nm. Figure 1 b). The results of dynamic light scattering analysis show that the substance carrying... Lgmn The hydrodynamic diameter distribution of -pDNA LNPs exhibits a normal distribution with an average diameter of 94.11 nm and a PDI of 0.216, indicating a relatively uniform particle size distribution. Figure 1c). To evaluate the storage stability of the prepared LNPs, the particle size changes were continuously monitored at different time points (0d, 1d, 3d, and 6d) after preparation. The results showed that the particle size of the LNPs did not increase significantly within 3 days after preparation, indicating that they have good stability in the short term. Figure 1 d).

[0069] Example 2: In vivo biodistribution and safety assessment of LNPs In vivo distribution experiment of LNPs To evaluate the tissue distribution of LNPs after intratracheal administration, DiR-labeled LNPs were prepared by diluting DiR dye to an appropriate concentration according to the manufacturer's instructions (https: / / doi.org / 10.7150 / thno.61085) and then injected into the airways of mice. Mice were anesthetized with isoflurane at 0, 1, 3, and 6 days post-injection, and in vivo fluorescence imaging was performed using a small animal in vivo imaging system to monitor the dynamic distribution of the DiR signal in vivo in real time. The imaging parameters were set to excitation wavelength 745 nm and emission wavelength 820 nm. To further clarify the specific distribution of LNPs in various organs, mice were sacrificed on day 3 post-LNP delivery, and major organs such as the heart, liver, spleen, lungs, and kidneys were rapidly isolated for in vitro fluorescence imaging. The tissue targeting and distribution characteristics of LNPs were evaluated by comparing the fluorescence signal intensity of each organ.

[0070] Biosafety assessment of LNPs 1) Histopathological assessment Take without P. acnes Healthy female C57BL / 6 WT mice were randomly divided into 4 groups, and each group of mice was given a single intratracheal injection of the encapsulated solution. Lgmn LNPs of pDNA (1 mg / kg) were administered to mice at 0, 1, 3, and 6 days post-drug administration. Hearts, livers, spleens, lungs, and kidneys were collected from these four groups of mice, fixed in 4% paraformaldehyde, embedded in paraffin, sectioned, and stained with H&E. Histopathological changes were observed under an optical microscope, and images were acquired using a 3DHISTECH digital slide scanner.

[0071] 2) Evaluation of liver and kidney function during treatment In the preventive intervention experiment, at P. acnes On day 14 after sensitization, the encapsulated material was delivered into the airway of mice. LgmnMice were treated with pDNA-based LNPs (1 mg / kg). Blood and urine samples were collected from mice on day 21 post-sensitization (day 7 post-drug administration). Blood samples were centrifuged after settling to separate serum, and liver function indicators (such as alanine aminotransferase and aspartate aminotransferase) and kidney function indicators (such as blood urea nitrogen and creatinine) were measured using an automated biochemical analyzer. Urine samples were used for routine urinalysis to comprehensively assess the potential impact of LNP treatment on liver and kidney function.

[0072] To clarify the tissue distribution characteristics of LNPs after intratracheal administration, this study used DiR fluorescent dye to label and encapsulate them. Lgmn -pDNA-labeled LNPs were dynamically monitored for DiR fluorescence signal using a small animal in vivo imaging system on days 0, 1, 3, and 6 after intratracheal injection. Results showed that the fluorescence signal of DiR-labeled LNPs was mainly concentrated in the lungs, and the signal intensity gradually decreased over time, indicating that LNPs could be effectively targeted and delivered to lung tissue. Figure 2 a). To further clarify the specific distribution of LNPs in various organs, mice were sacrificed on day 3 after LNP delivery, and their major organs (including heart, liver, spleen, lung, and kidney) were isolated for in vitro organ fluorescence imaging. The results were consistent with in vivo imaging, with fluorescence signals mainly distributed in the lungs, and no obvious fluorescence signals observed in other organs. Figure 2 b).

[0073] To assess the in vivo safety of the prepared LNPs, major organs (including heart, liver, spleen, lung, and kidney) of mice were collected on days 1, 3, and 6 after intratracheal injection of LNPs, and histopathological changes were observed by H&E staining. The results showed that the structures of the major organs were clear and normal at all time points, with no obvious pathological damage observed. Figure 3 a). In addition, this study collected data on the delivery of empty or... Lgmn LNPs overexpressing plasmids P. acnes Blood and urine samples from the model mice were analyzed for blood biochemistry and routine urinalysis. Results showed no statistically significant differences in any of the indicators between the two groups. Figure 3 b, Table 3). Data are expressed as the number of animals (percentage within a group). All p-values ​​were calculated using Fisher's exact test (Freeman-Halton extended test was used for contingency tables larger than 2×2). The above results indicate that the inclusion... Lgmn pDNA LNPs exhibit good biocompatibility and safety.

[0074] Table 3 Vector or Lgmn Urinary routine analysis parameters of OE group mice

[0075] Example 3: Prophylactic administration of encapsulation Lgmn LNPs in pDNA can alleviate P. acnes Induced granulomatous inflammation P. acnes Granuloma Model Construction 1) Inactivation P. acnes Suspension preparation: The inactivation used in this study P. acnes From (American Type Culture Collection, Manassas, VA, USA), the stock solution concentration was 30 mg / mL. Dilute with PBS as follows: P. acnes The suspension was diluted to low concentration (2 mg / mL) and high concentration (20 mg / mL).

[0076] 2) Establishment of a mouse lung granuloma model: ① Select wild-type female mice aged 6-8 weeks, weighing 18-22 g. Before modeling, check the general condition of the mice, including coat color, activity level, and food and water intake, to ensure that the mice used for modeling are in good health. ② Weigh the mice, disinfect the left lower abdomen with 75% ethanol, and administer an intraperitoneal injection of a 2 mg / mL suspension of *P. acnes* at a dose of 25 mg / kg to the mice to complete the first sensitization.

[0077] ③ On day 14 after sensitization, mice were anesthetized by intraperitoneal injection of tribromoethanol. After the mice had completely lost their righting reflex, their weight was checked again. Based on a dose of 50 mg / kg, the required volume of 20 mg / mL *P. acnes* suspension was calculated and pipetted into sterile EP tubes for later use. Based on the calculated volume, a 1 mL syringe connected to a lacrimal duct needle was used to draw... P. acnes Suspension. The fully anesthetized mice were fixed in a prone position on a mouse restraint frame. The mouse's tongue was gently pulled out and fixed to one side using forceps. A mouse laryngoscope was used with a tongue depressor to hold down the base of the tongue, fully exposing the glottis. Under the guidance of a laryngoscope light source, a lacrimal duct needle was inserted into the trachea along the glottis, and the suspension was slowly injected. P. acnes Suspension.

[0078] 3) Control group treatment: Control group mice were intraperitoneally injected with an equal volume of sterile PBS buffer in the same manner, instead of... P. acnes Sensitization treatment: On day 14 after intraperitoneal injection, an equal volume of sterile PBS buffer was injected via the airway using the same method as the model group.

[0079] 4) Sample collection: Injection into the peritoneum P. acnesOn day 21 after suspension or PBS buffer (i.e., day 7 after airway provocation), mice were deeply anesthetized. After the mice completely lost their righting reflex, they were fixed in a supine position on the operating table, and then cardiac perfusion was performed using physiological saline. Lung tissue was collected from the mice. The left lung was fixed in 4% paraformaldehyde, and each lobe of the right lung was flash-frozen in liquid nitrogen and stored at -80 °C for later use.

[0080] Preventive intervention experiment: On day 14 after sensitization, i.e., 50 mg / kg was administered. P. acnes On the same day that the suspension was used for airway provocation, it was delivered via the airway. Lgmn The study aimed to evaluate the preventive effect of pDNA LNPs (1 mg / kg) against granuloma formation. All mice were given the first intraperitoneal injection. P. acnes They were then sacrificed on the 21st day, and their lung tissue was collected for subsequent experiments.

[0081] RT-PCR: The RT-PCR experimental procedure was performed according to the standard procedures in this field. The relevant primer sequences are shown in Table 4.

[0082] Table 4 qPCR primer sequences

[0083] Tissue paraffin embedding: Mouse heart, liver, spleen, lung, and kidney tissues were collected and fixed with 4% paraformaldehyde. After fixation, the tissues were removed from the 4% paraformaldehyde solution and gently rinsed three times (5 min each time) in PBS buffer to remove residual fixative. The tissues were then transferred to embedding frames for later use. The tissues were then sequentially immersed in a gradient of alcohols for dehydration: 70% ethanol for 2 h → 85% ethanol for 1 h → 95% ethanol I for 1 h → 95% ethanol II for 1 h → anhydrous ethanol I for 30 min → anhydrous ethanol II for 30 min. During dehydration, the tissues were ensured to be completely submerged. After dehydration, the tissues were transferred to xylene for 15 min. A water bath was preheated to melt paraffin wax. The tissues were then sequentially immersed in paraffin I and paraffin II for 30 min each, ensuring thorough paraffin penetration. A constant temperature was maintained during the paraffin embedding process to prevent solidification. The embedding machine was preheated to 65°C. Place the paraffin-impregnated tissue evenly at the center of the bottom of the embedding mold according to the desired cut surface. Quickly drip molten paraffin to cover the tissue, cover with the embedding cassette, and add more paraffin until completely submerged. Smoothly transfer the mold to a freezing stage. After the paraffin has completely solidified, remove the embedded block and store it at room temperature for later use. Freeze the paraffin-embedded block at -20°C to increase tissue hardness, and then perform continuous sectioning on a microtome with a section thickness of 3-5 μm.

[0084] H&E staining: The prepared paraffin sections were dewaxed by sequentially passing them through xylene, anhydrous ethanol, and a series of ethanol solutions for rehydration. The sections were then placed in deionized water for later use. Hematoxylin staining: The dewaxed and rehydrated sections were immersed in hematoxylin staining solution and stained at room temperature for 2 minutes. After removal, they were rinsed with a slow stream of water for several minutes to remove excess staining solution. Differentiation and blueing: The sections from the previous step were immersed in 1% hydrochloric acid-ethanol solution for differentiation for 10-15 seconds. After differentiation, the sections were rinsed with a slow stream of running tap water and blued for 5 minutes. Eosin staining: The blued sections were immersed in eosin staining solution and stained at room temperature for 1 minute. After removal, they were soaked in anhydrous ethanol I and anhydrous ethanol II for 2 minutes each to dehydrate the sections. Xylene permeation and mounting: The dehydrated sections were soaked in xylene I and xylene II for 5 minutes each. After min, the slide is removed, and an appropriate amount of neutral resin is added to the tissue in the fume hood. A coverslip is carefully placed on top to avoid air bubbles. For image acquisition, after the neutral resin has solidified, a full-field scan is performed using a 3DHISTECH digital slide scanner to acquire images of the H&E stained slide.

[0085] IHC staining: Performed according to standard procedures in this field; relevant primary antibody information is shown in Table 5.

[0086] Table 5 Information on IHC staining primary antibodies

[0087] Immunofluorescence staining: Performed according to standard procedures in this field; relevant primary antibody information is shown in Table 6.

[0088] Table 6 Information on primary antibody used for immunofluorescence staining

[0089] Western Blot: Performed according to standard procedures in the field; relevant antibody information is shown in Table 7.

[0090] Table 7 Information on primary antibody used in Western Blot experiments.

[0091] To clarify the time-series changes in LGMN protein expression after LNP administration, lung tissue was collected at different time points following intratracheal injection of LNPs, and LGMN expression levels were detected by Western blot. Results showed that LGMN protein expression significantly increased in lung tissue on day 3 post-administration, returning to baseline levels by day 6. Figure 4 This result indicates that LNPs can achieve transient high expression of LGMN in lung tissue, providing a time window for subsequent efficacy evaluation.

[0092] To further clarify its efficacy, this study first evaluated the inclusion factor. Lgmn LNPs of pDNA P. acnes The prophylactic therapeutic effect of induced sarcoidosis-like granulomas. Experimental design as follows: Figure 5 As shown in a, P. acnes On day 14 after intraperitoneal injection, a vector containing control or [other drugs] was administered via airway. Lgmn LNPs (1 mg / kg) of pDNA (Lgmn-OE). Histopathological analysis showed that, compared with the Vector control group, delivery of LNPs carrying pDNA (Lgmn-OE) was significantly more effective. Lgmn After LNPs of pDNA, P. acnes The induced pulmonary granuloma area was significantly reduced. Figure 5 b). Immunostaining revealed that CD4 cells, the key effector cells in the lung tissue of the treated mice, were... + Decreased infiltration of T cells and macrophages suggests that the inflammatory response is suppressed. Figure 5 c). Simultaneously, the proportion of p-S6 positive macrophages was significantly reduced, indicating that the activation of the mTORC1 signaling pathway within macrophages was inhibited. Figure 5 d). Furthermore, qRT-PCR results showed that... Lgmn The mRNA expression levels of pro-inflammatory cytokines (including Cxcl9, Cxcl10, Tnf, and Ifng) in the lung tissue of mice in the -OE group were significantly downregulated, further confirming the effectiveness of LNP delivery. Lgmn The anti-inflammatory effect of pDNA ( Figure 5 e).

[0093] Example 4: Therapeutic administration of LNPs loaded with Lgmn pDNA can alleviate... P. acnes Induced granulomatous inflammation To evaluate the therapeutic potential of LGMN overexpression for established granulomas, this study first identified... P. acnes The timeframe for induced pulmonary granuloma formation. H&E staining results indicated that... P. acnes Day 17 after sensitization (i.e. P. acnes On the 3rd day after airway provocation, typical granulomatous lesions had formed in the lung tissue of mice. Figure 6 a). Based on this, this study was conducted in P. acnes On day 17 after sensitization, an empty vector or... Lgmn LNPs of pDNA Figure 6(b) To evaluate its therapeutic effect on existing granulomas. Therapeutic intervention experiment: On day 14 after sensitization, challenge was performed with 75 mg / kg of P. acnes suspension. After granuloma formation (i.e., day 17 after sensitization), LNPs loaded with Lgmn pDNA (1 mg / kg) were injected via the airway to evaluate its therapeutic effect on existing granulomas.

[0094] Histopathological analysis showed that even after administration following granuloma formation, the drug contained... Lgmn The pDNA LNPs still showed significant therapeutic effects. Compared with the Vector control group, the area of ​​granulomas in the lung tissue of mice in the treatment group was significantly reduced. Figure 6 c). IHC staining results showed that the proportion of CD4-positive T cells and Mac-2-positive macrophages in the lung tissue of mice in the treatment group was significantly decreased. Figure 6 d). qRT-PCR results showed that LNPs were delivered Lgmn Following pDNA delivery, the mRNA expression levels of multiple pro-inflammatory cytokine-related genes (including Ccl4, Cxcl9, Cxcl10, Tnf, and Ifng) in mouse lung tissue were significantly reduced, further confirming the anti-granulomatous inflammatory effect of Lgmn overexpression plasmid delivered via LNP. Figure 6 e). This study also examined the activation levels of downstream signaling pathways of LGMN. Western blot results showed that... Lgmn LNPs treatment with pDNA significantly downregulated p-S6 protein levels in lung tissue and inhibited STAT1 phosphorylation at Ser727 and Tyr701 sites. Figure 6 f) indicates that mTORC1 / STAT1 activation is inhibited. These results suggest that even after granuloma formation, administration via the airway carrying [a specific drug / product] is effective. Lgmn pDNA's LNPs can still effectively alleviate P. acnes Induced sarcoidosis-like granulomatous inflammation.

[0095] Example 5: Application of LNPs for Delivery Lgmn pDNA significantly reduced TDM-induced pulmonary granulomatous inflammation. Construction and treatment of a mouse pulmonary granuloma model induced by 6,6'-trehalose-6,6'-dimycolate (TDM): 1) Preparation of TDM emulsion (100 μg / mL): 100 μg TDM, 2 μL Tween-80, 32 μL incomplete Freund's adjuvant, 966 μL PBS; 2) Construction of the TDM-induced mouse granuloma model: 6-8 week old SPF-grade wild-type healthy female mice were selected. Before the experiment, the mice were observed to ensure they were in good condition. After weighing and recording the weight of the mice, they were properly fixed in the fixture of a mouse tail vein injection device with a built-in light source. The mouse tail was repeatedly wiped with an alcohol swab to stimulate tail vein dilation and congestion, while also disinfecting the area. After the tail vein was clearly visible, TDM emulsion was precisely drawn using an insulin syringe at a dose of 1.25 μg TDM / g body weight. The needle was inserted into the tail vein at an angle almost parallel to the mouse tail. After observing blood return and confirming that the needle tip was in the blood vessel, the TDM emulsion was slowly injected. After the injection, a sterile cotton ball was used to press and stop the bleeding while preventing extravasation of the drug. 3) Control group mice: Control group mice were injected with the same tail vein injection method as the experimental group, but with the same volume of blank emulsion without TDM, to ensure that the experimental conditions were consistent except for TDM induction. 4) To evaluate the therapeutic effect of overexpressing LGMN, WT mice were given a load via airway on the day of TDM tail vein injection (day 0). Lgmn LNPs of pDNA (1 mg / kg). Mice were sacrificed on day 7 after TDM injection, and lung tissue was collected to assess the degree of granulomatous inflammation.

[0096] The above experiments have confirmed that airway delivery of the package... Lgmn LNPs of pDNA can be found in P. acnes It exerts a therapeutic effect in a sarcoidosis-like granuloma model. Given the lack of a universally accepted animal model of sarcoidosis, to verify the universality of the anti-granulomatous effect of LGMN, this study further constructed a TDM-induced mouse lung granuloma model to evaluate the role of LGMN overexpression in this model.

[0097] This study first analyzed the polarization phenotype of macrophages in pulmonary granuloma tissues of mice after tail vein injection of TDM. Western blot results showed that, compared with... P. acnes Similar to the control group, compared with the control group, the protein expression of both the M1 macrophage marker iNOS and the M2 macrophage marker Arg-1 in the lung tissue of TDM group mice was significantly upregulated, suggesting that the co-activation of M1 and M2 polarization also exists in TDM-induced granulomatous inflammation. Figure 7a). This study further examined the activation level of downstream signaling pathways of LGMN and found that the protein content of p-S6 and p-STAT1 (Ser727 and Tyr701 sites) in mouse lung tissue was also significantly increased after TDM induction. Figure 7 b).

[0098] To investigate the role of LGMN overexpression in TDM-induced granulomatous inflammation, this study constructed a vector containing... Lgmn pDNA or empty vector plasmids containing LNPs were delivered via airway on the same day as tail vein injection of TDM. The experimental design was as follows: Figure 8 As shown in figure a. Histopathological analysis showed that, compared with the Vector group, Lgmn The number of granulomas in the lung tissue of mice in the -OE group was significantly reduced. Figure 8 b-8c). IHC staining results showed that... Lgmn The infiltration of CD4+ T cells and Mac-2+ macrophages in the lung tissue of mice in the OE group was significantly reduced compared with the control group. Figure 8 d). In addition, Lgmn The transcriptional levels of multiple pro-inflammatory factors in the lung tissue of mice in the -OE group were significantly downregulated compared with those in the Vector group, including Ccl4 , Cxcl9 , Cxcl10 , Tnf , Ifng and Il1b ( Figure 8 e). These results indicate that delivery via LNPs... Lgmn pDNA can effectively reduce TDM-induced granuloma burden.

[0099] This study further examined the effects of encapsulation. Lgmn The degree of M1 polarization in macrophages and the activation level of downstream signaling pathways of LGMN were observed in mouse lung tissue after treatment with pDNA-containing LNPs. The results showed that pDNA-containing LNPs delivered via the airway... Lgmn LNPs of pDNA significantly reduced the mRNA and protein expression levels of M1 macrophage markers in mouse lung tissue after TDM injection. Figure 8 f-8g). Meanwhile, compared to the Vector group, Lgmn In the lung tissue of mice in the -OE group, the expression of p-STAT1 (Ser727 and Tyr701 sites) and p-S6 proteins, which were elevated by TDM, was significantly reduced. Figure 8 h). These results are consistent with P. acnes The results observed in the model are consistent.

[0100] In summary, in a TDM-induced mouse lung granulation model, LGMN inhibits the activation of the mTORC1 / STAT1 signaling pathway, thereby suppressing macrophage M-type polarization and ultimately alleviating granulomatous inflammation. These results further confirm the universality of LGMN's anti-granulomatous effect in different models. They also validate... Lgmn The feasibility and efficacy of pDNA-loaded LNPs inhalation administration suggest that LGMN supplementation or targeting LGMN overexpression may be a potential treatment strategy to alleviate granulomatous inflammation in sarcoidosis.

[0101] The above description of the embodiments is only for understanding the method and core ideas of the present invention. It should be noted that those skilled in the art can make various improvements and modifications to the present invention without departing from the principles of the invention, and these improvements and modifications will also fall within the protection scope of the claims of the present invention.

Claims

1. A composition for overexpressing LGMN, characterized in that, The composition comprises a nucleic acid construct encoding LGMN and a lipid component capable of encapsulating the nucleic acid construct.

2. The composition according to claim 1, characterized in that, The nucleic acid construct includes a nucleic acid molecule encoding the LGMN protein; Preferably, the nucleic acid molecule includes DNA molecules and RNA molecules; Preferably, the DNA molecule is a circular DNA molecule; Preferably, the circular DNA molecule is a plasmid.

3. The composition according to claim 1, characterized in that, The lipid component includes one or more of cationic lipids, neutral auxiliary lipids, and polyethylene glycol-modified lipids; Preferably, the cationic lipids include ionizable cationic lipids, quaternary ammonium salt cationic lipids, and polyamine lipids; Preferably, the cationic lipid is an ionizable cationic lipid; Preferably, the ionizable cationic lipids include SM-102, ALC-0315, Dlin-MC3-DMA, C12-200, and TT3; Preferably, the cationic lipid is SM102; Preferably, the neutral auxiliary lipids include phospholipids and cholesterol; Preferably, the phospholipid is DSPC; Preferably, the polyethylene glycol-modified lipid is a PEGylated phospholipid; Preferably, the PEGylated phospholipids include PEG-DMG, PEG-DSG, and PEG-DPPE; Preferably, the molecular weight of the polyethylene glycol in the PEGylated phospholipid is 1000~10000 Daltons; Preferably, the polyethylene glycol-modified lipid is PEG-DMG; Preferably, the polyethylene glycol in the PEG-DMG has a molecular weight of 2000 Daltons.

4. A lipid nanoparticle overexpressing LGMN, characterized in that, The lipid nanoparticles comprise a core of a nucleic acid construct encoding LGMN and an outer core of a lipid component from the compositions of claims 1-3; Preferably, the nucleic acid construct encoding LGMN is loaded within the paper component to form the lipid nanoparticles; Preferably, the average hydrodynamic diameter of the lipid nanoparticles is 50-200 nm; Preferably, the average hydrodynamic diameter of the lipid nanoparticles is 94.11 nm; Preferably, the zeta potential of the lipid nanoparticles is -30 mV to +10 mV; Preferably, the zeta potential of the lipid nanoparticles is -2.78 mV.

5. The lipid nanoparticles according to claims 3-4, characterized in that, The lipid nanoparticles also contain fluorescently labeled molecules; Preferably, the fluorescent labeling molecule includes fluorescein, rhodamine, and anthocyanin dyes; Preferably, the cyanine dye is DiR.

6. A medicine for the treatment and / or prevention of sarcoidosis, characterized in that, The drug comprises one or more of the compositions of claims 1-3 and the lipid nanoparticles of claims 4-5; Preferably, the drug further comprises a pharmaceutically acceptable carrier and / or excipients; Preferably, the dosage form of the drug includes injection, inhalation, and topical administration. Preferably, the route of administration of the drug includes enteral administration and non-enteric administration; Preferably, the route of administration is a non-enteric route; Preferably, the non-enteric route of administration includes injection and topical application; Preferably, the local medication includes airway delivery, stent delivery, and balloon delivery. Preferably, the route of administration is airway delivery.

7. A drug delivery system, characterized in that, The system comprises one or more of the compositions of claims 1-3, the lipid nanoparticles of claims 4-5, and the drug of claim 6; Preferably, the system further includes a drug delivery device; Preferably, the drug delivery device includes an oral delivery device, an injection delivery device, an inhalation delivery device, a local delivery device, and an implantable delivery device; Preferably, the inhalation drug delivery device includes a dry powder inhaler, a nebulizer, a pressure metering inhaler, and a nebulizer.

8. A reagent kit, characterized in that, The kit comprises one or more of the compositions of claims 1-3, the lipid nanoparticles of claims 4-5, the drug of claim 6, and the drug delivery system of claim 7; Preferably, the kit also includes instructions.

9. Any of the following methods: (1) A method for preparing any one of the compositions according to claims 1-3 and the lipid nanoparticles according to claims 4-5, characterized in that... The method involves mixing an aqueous phase containing a nucleic acid construct encoding LGMN with a lipid phase containing the lipid components described in claims 1-3 to form lipid nanoparticles loaded with the nucleic acid construct. (2) An in vitro detection method, characterized in that the method includes contacting the lipid nanoparticles of claim 5 with target cells and detecting them by fluorescence signal; (3) A method for increasing the expression level of gene LGMN in cells in vitro, characterized in that the method involves contacting one or more of the compositions of claims 1-3 and the lipid nanoparticles of claims 4-5 with target cells in vitro. (4) A method for constructing engineered cells, characterized in that the method involves introducing one or more of the compositions of claims 1-3 and the lipid nanoparticles of claims 4-5 into target cells in vitro, and screening to obtain cells overexpressing LGMN; (5) A method for producing LGMN protein in cells, characterized in that the method involves transfecting one or more of the compositions of claims 1-3 and the lipid nanoparticles of claims 4-5 into target cells to express and obtain the LGMN protein.

10. Any of the following applications: (1) Use of any one of the compositions of claims 1-3, the lipid nanoparticles of claims 4-5, the medicament of claim 6, the drug delivery system of claim 7, the kit of claim 8, or the method of claim 9 in the preparation of a medicament for the treatment and / or prevention of sarcoidosis; (2) The use of the lipid nanoparticles of claim 5 in the preparation of reagents for cell labeling or targeted imaging; (3) Use of a nucleic acid molecule encoding LGMN protein in the preparation of a drug for the treatment of sarcoidosis.

Images