Engineered mitochondrial vaccine, method for preparing same, and use thereof

Abstract

The present invention relates to the field of biopharmaceutical technology, and particularly, relates to an engineered mitochondrial vaccine against fibrosis, a method for preparing same, and use thereof. In order to develop more pharmaceuticals for preventing and treating fibrotic diseases, the present invention uses mitochondria as a fibrotic antigen delivery system to prepare an engineered mitochondrial vaccine targeting fibrosis-related antigens. In the present invention, WT1 was used as a model antigen to construct a cell line stably overexpressing WT1 on mitochondria, and transgenic mitochondria (Mito-WT1) therein were extracted for evaluating the efficacy thereof as a pulmonary fibrosis vaccine. Animal experiments have revealed that both the subcutaneous immunization and intranasal immunization with the Mito-WT1 vaccine exhibited therapeutic effects on the bleomycin-induced mouse pulmonary fibrosis model, and the intranasal immunization with the Mito-WT1 vaccine also exhibited prophylactic effects on mouse pulmonary fibrosis as well as good in vivo safety. It can be seen that the engineered mitochondrial vaccine of the present invention holds promise for inhibiting fibrosis.

Description

Engineered mitochondrial vaccines, their preparation methods and applications Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to engineered mitochondrial vaccines against fibrosis, their preparation methods, and applications. Background Technology

[0002] Fibrosis affects approximately one-quarter of the global population and is a common final pathological stage of many diseases. Pulmonary fibrosis, especially idiopathic pulmonary fibrosis (IPF), is the end-stage manifestation of interstitial lung disease, characterized by fibroblast proliferation and massive extracellular matrix deposition, ultimately leading to the destruction of lung tissue structure. The five-year survival rate for patients diagnosed with IPF is less than 30%, far lower than most types of cancer, and the median survival after an acute exacerbation is only 2.2 months. Currently, the FDA-approved IPF treatments—pirfenidone and nintedanib—while able to alleviate disease progression and slow the decline in lung function to some extent, cannot achieve a complete cure for IPF. Therefore, there is an urgent need to develop new treatment strategies to effectively prevent and reverse the progression of pulmonary fibrosis.

[0003] The occurrence and development of pulmonary fibrosis involve the interaction of multiple cell types, secreted factors, and complex signaling pathways. Precisely locating and effectively intervening in these key targets remains a major challenge in current research. In recent years, with in-depth exploration of the pathological mechanisms of pulmonary fibrosis, researchers have gradually revealed a variety of potential therapeutic targets. Among them, the abnormal activation of transcription factors is considered to play a key driving role in the early stages of pulmonary fibrosis. Specifically, in the subpleural mesenchymal cells, several transcription factors, such as Snail family zinc finger protein 1 (Snail1), Snail family zinc finger protein 2 (Snail2), kink family bHLH transcription factors, and immediate early genes c-Jun and c-Fos, have been found to be significantly activated. These transcription factors promote fibroblast proliferation and extracellular matrix deposition by regulating gene expression, thus driving the progression of fibrosis.

[0004] Vaccines can prevent disease or reverse pathological processes by activating the immune system. Mitochondria, as a vital energy metabolism and signal transduction center within cells, offer novel insights for vaccine development due to their unique biological characteristics. According to the endosymbiotic theory, mitochondria possess prokaryotic characteristics, being rich in cardiolipin, mitochondrial DNA (containing abundant CpG repeat sequences), and molecular patterns associated with mitochondrial damage. These components make mitochondria natural agonists of various Toll-like receptors, effectively activating the body's immune response. However, there are currently few reports on using mitochondria as a fibrosis antigen delivery system to prepare engineered mitochondrial vaccines targeting fibrosis-related antigens for the prevention and treatment of fibrosis. Summary of the Invention

[0005] In order to develop more drugs for the prevention and treatment of fibrotic diseases, this invention uses mitochondria as a fibrosis antigen delivery system to prepare engineered mitochondrial vaccines that target fibrosis-related antigens, thereby enhancing their immune activation capacity while ensuring their antigen specificity and safety.

[0006] To achieve the above-mentioned objectives, the technical solution adopted by this invention is as follows:

[0007] In a first aspect, the present invention provides an engineered mitochondrial vaccine containing transgenic mitochondria that express fibrosis-associated antigens in a directed manner.

[0008] The fibrosis includes at least one of pulmonary fibrosis, liver fibrosis, pancreatic fibrosis, kidney fibrosis, cardiac fibrosis, endometrial fibrosis, ocular fibrosis, splenic fibrosis, myelofibrosis, or skin fibrosis.

[0009] Preferably, the pulmonary fibrosis includes at least one of idiopathic pulmonary fibrosis (IPF), secondary pulmonary fibrosis, hereditary pulmonary fibrosis, or interstitial lung disease with pulmonary fibrosis characteristics.

[0010] The fibrosis-associated antigen is a pulmonary fibrosis-associated antigen, selected from at least one of nephroblastoma 1 (WT1), snail family zinc finger protein 1 (Snail1), snail family zinc finger protein 2 (Snail2), kink family bHLH transcription factor, immediate early gene c-Jun or c-Fos.

[0011] Preferably, the pulmonary fibrosis antigen is WT1.

[0012] More preferably, the nucleotide sequence of the WT1 is shown in SEQ ID NO.1.

[0013] The transgenic mitochondria are mitochondria expressing WT1, and the complete nucleotide sequence of its expression plasmid is shown in SEQ ID NO.2. Preferably, the target gene of the engineered mitochondrial vaccine is shown in SEQ ID NO.4.

[0014] The engineered mitochondrial vaccine uses mitochondria as a fibrosis antigen delivery system. This antigen delivery system is a mitochondrial-targeted fibrosis-related antigen expression platform established using plasmids with ornithine carbamoyltransferase (OTC) as the leader sequence and lentivirus transfection system.

[0015] Preferably, the leader sequence OTC is as shown in SEQ ID NO.3.

[0016] Secondly, the present invention also provides a method for preparing the above-mentioned mitochondrial vaccine, comprising the following steps:

[0017] A recombinant plasmid containing fibrosis-associated antigen was constructed, packaged into lentivirus, and then cells were infected with the lentivirus system. Stable transgenic strains that stably expressed fibrosis-associated antigen in mitochondria were screened out. The mitochondria of the stable transgenic strains were then extracted to obtain transgenic mitochondria that directionally expressed fibrosis-associated antigen.

[0018] In the above method, the recombinant plasmid is prepared by synthesizing and linking OTC and template antigen using OTC as the mitochondrial localization peptide and fibrosis-associated antigen as the template antigen.

[0019] Preferably, the recombinant plasmid nucleotide sequence is as shown in SEQ ID NO.2; more preferably, the recombinant plasmid contains a nucleotide sequence consisting of OTC and template antigen, as shown in SEQ ID NO.4.

[0020] In the above method, the plasmid is selected from at least one of mammalian cell expression vectors, insect baculovirus expression vectors, Escherichia coli expression vectors, and yeast expression vectors.

[0021] Preferably, the plasmid is selected from any one of the vectors pLV-puro, pCDH-CMV, pCDH-EF1, pCDH-MSCV, and pCDH-RFP.

[0022] More preferably, the method for preparing the mitochondrial vaccine includes the following steps:

[0023] Using OTC as a mitochondrial localization peptide, OTC and the target gene WT1 were synthesized and ligated in the pLV-puro vector to construct a recombinant plasmid. The recombinant plasmid was then mixed with an helper plasmid and packaged into a lentiviral vector in HEK293T cells. The culture supernatant was collected, and the lentivirus was concentrated by ultracentrifugation. K562 cells were then infected with the lentivirus, and stable transfected cells were screened.

[0024] Thirdly, the present invention provides a pharmaceutical composition comprising the above-described engineered mitochondrial vaccine and pharmaceutically permissible excipients.

[0025] The excipients in the above-mentioned pharmaceutical composition are at least one of diluents, excipients, fillers, binders, wetting agents, disintegrants, absorption promoters, surfactants, protectants, adsorbent carriers, or lubricants.

[0026] Fourthly, the present invention also provides a combination drug containing the above-mentioned engineered mitochondrial vaccine and other drugs for the prevention and / or treatment of fibrosis.

[0027] The aforementioned engineered mitochondrial vaccines, pharmaceutical compositions, or combined pharmaceutical products are available in the form of injections, nasal drops, sprays, inhalers, or oral medications.

[0028] Preferably, the injection route is at least one of intramuscular injection, intravenous injection, subcutaneous injection, intradermal injection, intramyocardial injection, or intraperitoneal injection.

[0029] More preferably, the dosage form is an intramuscular or subcutaneous injection preparation or a nasal drop preparation.

[0030] Fifthly, the present invention provides the use of the above-described engineered mitochondrial vaccines, pharmaceutical compositions, or combinations thereof in the prevention and / or treatment of fibrotic diseases.

[0031] The fibrotic diseases mentioned include at least one of pulmonary fibrosis, liver fibrosis, pancreatic fibrosis, kidney fibrosis, cardiac fibrosis, endometrial fibrosis, ocular fibrosis, splenic fibrosis, myelofibrosis, or skin fibrosis.

[0032] Preferably, the pulmonary fibrosis includes at least one of idiopathic pulmonary fibrosis (IPF), secondary pulmonary fibrosis, hereditary pulmonary fibrosis, or interstitial lung disease with pulmonary fibrosis characteristics.

[0033] Beneficial Effects: This invention, for the first time, establishes a mitochondrial fibrosis-related antigen expression platform using OTC as a leader sequence plasmid and lentiviral transfection system. Furthermore, this invention discovers that WT1 antigen serves as a vaccine target for pulmonary fibrosis, and uses WT1 as a model antigen to construct a stable cell line overexpressing WT1 in mitochondria. Transgenic mitochondria (Mito-WT1) from these cells are extracted to evaluate their efficacy as a pulmonary fibrosis vaccine. Animal experiments show that the Mito-WT1 vaccine has therapeutic efficacy against bleomycin-induced pulmonary fibrosis in mice when administered subcutaneously or intranasally, and intranasal administration also has preventative efficacy against pulmonary fibrosis in mice, exhibiting good safety in vivo. Therefore, the engineered mitochondrial vaccine of this invention shows promise as a new hope for inhibiting pulmonary fibrosis. This innovative treatment method not only brings new hope to patients with IPF and other types of pulmonary fibrosis but also provides an important theoretical foundation and practical guidance for research in related fields. Attached Figure Description

[0034] Figure 1 shows the results of using the WT1 antigen as a potential target for a pulmonary fibrosis vaccine in Example 1. a is the immunohistochemical map of WT1 in normal human lung and IPF human lung, normal mouse lung tissue and fibrotic lung tissue, with scale bars at 30 μm (human) and 50 μm (mouse); b and c are the expression levels of the WT1 gene in normal and fibrotic lung tissues of humans and mice; d is the expression distribution of WT1 in different cell types of human and mouse lungs analyzed by single-cell transcriptome data.

[0035] Figure 2 shows the preparation and characterization results of Mito-WT1 in Example 2. a) shows the construction, lentiviral packaging, and transfection process of the Mito-WT1 plasmid, a mitochondrial-based pro-fibrosis antigen delivery system; b) shows a schematic diagram of the recombinant plasmid containing the mitochondrial localization signal peptide sequence and the WT1 sequence; c) shows transmission electron microscopy images of control mitochondria (Mito) and mitochondria expressing WT1 (Mito-WT1), with the scale bar representing 200 nm; d) shows the expression of WT1 protein in the mitochondria of the two cell types detected by Western blot, where VDAC is a multifunctional protein voltage-dependent anion channel 1, which can be used as a mitochondrial internal control, and Vinculin is a focal adhesion protein, which can be used as a whole-cell internal control; e) shows the co-localization of WT1 and mitochondria in the two cell types detected by immunofluorescence, with the scale bar representing 2 μm.

[0036] Figure 3 shows the results of subcutaneous injection of Mito-WT1 vaccine to treat pulmonary fibrosis in mice in Example 3; where a is a flowchart of subcutaneous immunization with Mito-WT1 vaccine to treat pulmonary fibrosis in mice; b is the lung weight of mice; c is the H&E staining map (top) and Masson staining map (bottom) of mouse lung tissue, with the scale bar representing 50 μm; d is the Szapiel score; e is the Ashcroft score; f is the immunohistochemical map of Collagen 1 and α-SMA in mouse lung tissue, with the scale bar representing 50 μm; g is the immunohistochemical score of Collagen 1; h is the immunohistochemical score of α-SMA.

[0037] Figure 4 shows the results of pulmonary fibrosis in mice treated with Mito-WT1 vaccine via intranasal immunization in Example 4. In the figure, a is a flowchart of the pulmonary fibrosis treatment with Mito-WT1 vaccine via intranasal immunization; b is the lung weight of the mice; c is the H&E staining (top) and Masson staining (bottom) of mouse lung tissue, with the scale bar representing 50 μm; d is the Szapiel score; e is the Ashcroft score; f is the immunohistochemical map of Collagen 1 and α-SMA in mouse lung tissue; g is the immunohistochemical score of Collagen 1, with the scale bar representing 50 μm; h is the immunohistochemical score of α-SMA; j is the protein expression level of COLA1 and α-SMA in mouse lung tissue detected by Western blot, with VINCULIN as an internal control; km is the relative expression level of Col1α1, Fn1, and Acta2 genes in mouse lung tissue detected by real-time quantitative PCR (qRT-PCR).

[0038] Figure 5 shows the results of intranasal immunization against pulmonary fibrosis in mice using the Mito-WT1 vaccine in Example 5. In the figure, a is a flowchart of the intranasal immunization against pulmonary fibrosis in mice using the Mito-WT1 vaccine; b is the lung weight of the mice; c is the H&E staining (top) and Masson staining (bottom) images of mouse lung tissue, with the scale bar representing 50 μm; d is the Szapiel score; e is the Ashcroft score; f is the immunohistochemical image of Collagen 1 and α-SMA in mouse lung tissue; g is the immunohistochemical score of Collagen 1, with the scale bar representing 50 μm; h is the immunohistochemical score of α-SMA; j is the protein expression level of COLA1 and α-SMA in mouse lung tissue detected by Western blot, with VINCULIN as an internal control; and km is the relative expression level of Col1α1, Fn1, and Acta2 genes in mouse lung tissue detected by qRT-PCR.

[0039] Figure 6 shows the in vivo safety evaluation results of the Mito-WT1 vaccine in Example 6. In the figure, a represents the complete blood cell count of mice, including white blood cells, neutrophils, lymphocytes, monocytes, eosinophils, basophils, erythrocytes, hemoglobin, hematocrit, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH) content, mean corpuscular hemoglobin concentration (MCH), mean platelet volume (MPV), and platelet count; b represents the detection of serum biochemical indicators in mice, including total bilirubin, alanine aminotransferase (ALT), aspartate aminotransferase (AST), albumin, alkaline phosphatase (ALP), uric acid, creatinine, creatine kinase-MB isoenzyme, urea, and amylase; c represents the H&E staining of mouse organs (including heart, liver, spleen, lungs, kidneys, skin, small intestine, skeletal muscle, and nose). The scale bar represents 100 μm. Detailed Implementation

[0040] To make the technical problems, solutions, and beneficial effects of this application clearer, the following detailed description is provided in conjunction with the embodiments. Unless otherwise defined, all technical terms used herein have the same meaning as understood by one of ordinary skill in the art.

[0041] The nephroblastoma 1 (WT1) gene, located on human chromosome 11p13, encodes WT1, a dual-function transcription factor that inhibits tumor growth and activates oncogene transcription. WT1 is highly expressed in various solid tumors and leukemias, particularly acute myeloid leukemia (AML). As a key antigen in tumor immunotherapy, WT1 has demonstrated good safety and efficacy in multiple clinical trials. The applicant discovered high expression of WT1 in mesothelial and mesenchymal cells during the early stages of pulmonary fibrosis, suggesting its important role in the fibrotic process. Therefore, WT1 not only shows potential in cancer treatment but also represents an emerging target for the treatment of pulmonary fibrosis.

[0042] This invention establishes a mitochondrial-targeted pulmonary fibrosis-related antigen expression platform using OTC as a leader sequence plasmid and a lentiviral transfection system. Using WT1 as a model antigen, a stable WT1-overexpressing cell line was constructed, and transgenic mitochondria (Mito-WT1) were extracted to evaluate their efficacy as a pulmonary fibrosis vaccine. The results showed that the Mito-WT1 vaccine, administered subcutaneously and intranasally, had therapeutic efficacy against bleomycin-induced pulmonary fibrosis in a mouse model, and intranasal administration of the Mito-WT1 vaccine also demonstrated preventative efficacy against pulmonary fibrosis in mice, exhibiting good safety in vivo.

[0043] SEQ ID NO.1: Nucleotide sequence of pulmonary fibrosis antigen WT1

[0044] SEQ ID NO.2: Mito-WT1 nucleotide sequence expressing WT1

[0045] SEQ ID NO.2 is the nucleotide sequence of the entire expression plasmid, including the promoter, enhancer, OTC and WT1 nucleotide sequences, etc. The target gene sequence of the Mito-WT1 vaccine is SEQ ID NO.4OTC+WT1.

[0046] SEQ ID NO.3: OTC leader sequence (NCBI Reference Sequence: NM_000531.6)

[0047] SEQ ID NO.4: OTC+WT1 full-length sequence (NCBI Reference Sequence: NM_000378.6)

[0048] The following specific embodiments will be provided to explain the solution of the present invention. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0049] The main materials used in the following embodiments are as follows:

[0050] Bleomycin (HY-108345) was purchased from MedChemExpress. The mitochondrial isolation kit (MITOIS02) was purchased from Sigma-Aldrich. Antibodies rabbit polyclonal anti-Voltage-dependent anion channel (VDAC) (#4866), rabbit monoclonal anti-Wilms' Tumor 1 (WT1) (#83535), and HRP-conjugated anti-rabbit IgG (7074) were all purchased from Cell Signaling Technology. Antibodies rabbit polyclonal anti-α-smooth muscle actin (α-SMA) (14395-1-A) were purchased from Wuhan Sanying Biotechnology Co., Ltd. Antibodies rabbit polyclonal anti-Collagen I alpha 1 (501352) were purchased from Chengdu Zhengneng Biotechnology Co., Ltd. The fluorescent secondary antibody GOAT anti-rabbit AF647 (A27040) was purchased from Invitrogen.

[0051] K562 cells and K562 WT1 Cells were cultured in Gibco 1640 medium containing 10% fetal bovine serum (FBS), 100 U / mL penicillin, and 100 mg / mL streptomycin. Human embryonic kidney 293T (HEK293T) cells were cultured in DMEM medium containing 4.5 g / L D-glucose, 10% FBS, 100 U / mL penicillin, and 100 mg / mL streptomycin. Male C57BL / 6 mice (weighing 26–28 g) were purchased from Vital River Pharmaceuticals (Beijing, China). Experiments were conducted one week after mouse acclimatization. Mice were housed in the SPF-grade animal facility of the State Key Laboratory of Biotherapy, Sichuan University. All experimental protocols were reviewed and approved by the Experimental Animal Management Committee of Sichuan University (Chengdu, Sichuan).

[0052] The main detection methods used in the following embodiments are as follows:

[0053] 1. Real-time quantitative PCR (qRT-PCR) detection

[0054] Total RNA was extracted from tissues and cells using a total RNA isolation kit (FOREGENE, RE-03113) and analyzed using RT Easy. TMcDNA synthesis was performed using a reverse transcription kit (FOREGENE, RT-01031 / 01032). Real-time quantitative PCR was performed using a Bio-Rad CFX96 Touch system. TM qRT-PCR detection was performed. The PCR reaction system included front primers, back primers, and... Green Supermix (Bio-Rad, iTaq Universal SYBR Green), cDNA, and ddH2O were used. 18SRNA, present in all eukaryotic cells, was selected as an internal control gene. Primer sequences used for qRT-PCR are shown in Table 1.

[0055] Table 1. Primer sequences

[0056] 2. Histopathology, hematoxylin and eosin (H&E) staining, and Masson staining

[0057] (1) Sampling, dehydration, embedding, and sectioning

[0058] After fresh tissue sampling, each tissue and organ is soaked in 4% paraformaldehyde for at least 2 days. The tissue is then removed from the fixative and trimmed using a scalpel in a fume hood. The trimmed tissue and corresponding labels are placed in a dehydration box. After washing, the tissue undergoes gradient dehydration in the following order: 70%, 80%, 90%, 95%, and 100% alcohol (anhydrous ethanol), with each stage lasting 30 minutes. The paraffin-soaked tissue is then embedded in a Leica paraffin embedding machine and cooled on a Leica automated tissue embedding machine's cold stage. After the wax solidifies, the wax block is removed from the embedding frame and trimmed. The trimmed wax block is then sectioned on a paraffin microtome to a thickness of 3 μm. The sections are floated on 40°C warm water in a slide spreader to flatten the tissue. The tissue is then retrieved using a glass slide and placed in a 60°C oven to bake. After the water has dried and the wax has melted, the sections are removed and stored at room temperature or 4°C for later use.

[0059] (2) Dewaxing and hydration

[0060] First, place the slides orderly on a copper slide holder and bake them in a slide warmer at 65-70℃ for 30 minutes to dissolve the wax layer. Then, dewax them three times with xylene, 10 minutes each time. Next, hydrate them in a gradient of alcohol: anhydrous ethanol, 95%, 85%, and 75%, with each hydration stage lasting 5 minutes. After hydration, transfer the slides to a histochemistry chamber and wash them twice with ddH2O on a shaker, 5 minutes each time. Immerse the slides in the histochemistry chamber containing 200 mL of 1× antigen retrieval solution, transfer them to an autoclave, and cook for 10-12 minutes after the autoclave reaches boiling point. Remove the histochemistry chamber and allow it to cool naturally to room temperature. Wash twice with 1×PBS on a shaker, 5 minutes each time.

[0061] (3) H&E staining

[0062] After the paraffin sections have been processed according to the above steps, they are stained using an H&E staining kit (Beyotime, C0105S). Generally, hematoxylin staining is performed for 5-10 minutes, followed by rinsing with tap water to remove excess stain, and then washing once with ddH2O. Eosin staining is performed for 3-30 seconds. These times can be adjusted according to the staining results and requirements. After drying at room temperature, the slides are mounted with neutral resin and scanned for analysis using a pathological slide scanner.

[0063] (4) Masson staining

[0064] After paraffin sections were processed using standard methods, they were stained with Masson's trichrome staining reagent (Beyotime, C0189M). First, the sections were stained with Weigert iron hematoxylin staining solution for 5-10 minutes. After staining, the sections were rinsed with tap water to remove excess staining and then washed once with distilled water. Next, the sections were stained with acid fuchsin staining solution for 1-3 minutes and then rinsed again with water. Following this, phosphotungstic acid-sapphire blue staining was performed, typically for 5-10 minutes, which could be adjusted depending on the tissue type and staining desired effect. After staining, the sections were dehydrated until clean and transparent, then mounted with neutral resin and scanned for analysis using a pathological slide scanner.

[0065] 3. Immunohistochemistry

[0066] Immunohistochemical staining was performed according to the Histonstain-SP kit instructions (ZSGB-BIO, SPN-9002), with minor adjustments. The specific steps were as follows: Paraffin sections were dewaxed and rehydrated, followed by antigen retrieval. Subsequently, the sections were permeabilized with 0.2% Triton X-100 (Sigma Aldrich, 9036-19-5) and non-specific binding sites were blocked with 10% goat serum (Solarbio, SL038). 100 μL of primary antibody was added, and the sections were incubated at 37°C for 1 hour. After incubation, the sections were washed three times with PBS for 5 minutes each time. Next, 100 μL of biotin-labeled goat anti-rabbit IgG was added, and the sections were incubated at room temperature for 10-15 minutes. After washing three more times with PBS, 100 μL of horseradish peroxidase (HRP)-labeled streptavidin working solution was added, and the sections were incubated at room temperature for 10-15 minutes. The colorimetric reaction was performed using freshly prepared diaminobenzidine (DAB) (5-8 minutes at room temperature), followed by counterstaining the cell nuclei with hematoxylin for 30 seconds and rinsing with tap water for 5 minutes. After drying, the sections were mounted with neutral resin (Solarbio, G8590). The primary antibodies used in this experiment included: WT1 (Servicebio, GB11382, 1:300), α-SMA (Servicebio, GB111364, 1:1000), and Col1α1 (Servicebio, GB11022-3, 1:1000). All images were acquired using a PANNORAMIC digital slide scanner (3DHISTECH, PANNORAMIC MIDI II).

[0067] 4. Western blot

[0068] Western blot analysis was performed according to the established experimental protocol. For cell samples, after discarding the supernatant, the cell pellet was lysed using RIPA buffer (Beyotime, P0013B) with the addition of a phosphatase inhibitor (MedChemExpress, HY-K0022) and a protease inhibitor (MedChemExpress, HY-K0010). The lysate was sonicated and then boiled at 98°C for 10 minutes to denature the proteins. For tissue samples, 100 mg of lung tissue was homogenized in 1 mL of RIPA buffer containing 10 μL of phosphatase inhibitor and 10 μL of protease inhibitor, and further homogenized using a high-speed tissue homogenizer (Servicebio, KZ-II). The homogenized tissue lysate was centrifuged at 13,000 rpm for 10 minutes, and the supernatant was collected. Subsequently, the tissue lysate was mixed with SDS-PAGE sample loading buffer (Beyotime, P0015L), boiled for 10 minutes, and stored at -80°C.

[0069] Protein electrophoresis and membrane transfer were performed according to standard protocols. Protein electrophoresis was performed using 7.5% SDS-PAGE, and membrane transfer was performed at 370 mA for 2 hours. The PVDF membrane was then collected. The PVDF membrane was blocked with 5% skim milk at room temperature for 1 hour. After blocking, the PVDF membrane was incubated with primary antibody, washed with 1×TBST, and then incubated with secondary antibody. The primary antibodies used included: rabbit polyclonal anti-Voltage-dependent anion channel (VDAC) (Cell Signaling Technology, #4866, 1:1000), rabbit monoclonal anti-Wilms' Tumor 1 (WT1) (Cell Signaling Technology, #83535, 1:1000), mouse monoclonal anti-Vinculin (Sigma-Aldrich, V9264, 1:1000), rabbit polyclonal anti-α-smooth muscle actin (α-SMA) (Proteintech, 14395-1-AP, 1:2000), rabbit polyclonal anti-Fibronectin (Proteintech, 15613-1-AP, 1:2000), and rabbit polyclonal anti-Collagen I alpha 1 (Zenbio, 501352, 1:1000). VDAC and Vinculin were used as internal control proteins. Secondary antibodies were HRP-labeled anti-rabbit IgG (Cell Signaling Technology, 7074, 1:5000) and HRP-labeled anti-mouse IgG (Cell Signaling Technology, 7076, 1:5000). Chemiluminescence was performed using Clarity Western ECL Substrate (Bio-Rad, 1705061). Image data were acquired using a Western Blot imaging system (e-BLOT).

[0070] 5. Immunofluorescence

[0071] The co-localization of WT1 protein with cellular mitochondria was detected by immunofluorescence assay, specifically for K562 and K562 mitochondria. WT1Cells were first gently washed 2-3 times with 1×PBS to remove residual serum from the culture medium. They were then resuspended in RPMI 1640 medium containing 50 μM Mitotracker Red CMXRos (Invitrogen, M7512) preheated to 37°C. Cells were incubated at 37°C in a cell culture incubator with 5% CO2 for 30 minutes in the dark, followed by centrifugation and washing with preheated physiological saline (37°C). Cell smears were prepared. Cells were washed with physiological saline at 37°C. K562 cells and K562 cells were then smeared separately using a cell smearer (Thermo Scientific, Cytospin 4). WT1 Cells (5×10) 5 Cells were evenly and gently deposited onto a glass slide and centrifuged at 500g for 5 minutes. Cells were fixed and perforated using a commercial fixation and perforation kit (BD Biosciences, #554715). After washing, blocking, and incubation with primary antibody (Rabbit anti-human WT1), Goat anti-rabbit IgG-FITC secondary antibody was added, followed by DAPI staining. The slides were then mounted with an anti-fluorescence quencher. Finally, the co-localization of mitochondria and WT1 in K562WT1 cells was observed under a Zeiss 880 confocal microscope.

[0072] Example 1. WT1 antigen is a potential target for pulmonary fibrosis vaccines.

[0073] To evaluate the potential of the WT1 antigen as a vaccine target for pulmonary fibrosis, we performed immunohistochemistry on normal human lungs, IPF human lungs, normal mouse lung tissues, and bleomycin-induced pulmonary fibrosis mouse lung tissues. We found that the WT1 antigen was not expressed or expressed with limited expression in normal human and mouse lungs (Fig. 1a), but was highly expressed in IPF human lungs and fibrotic mouse lung tissues (Fig. 1a). Real-time quantitative PCR (qRT-PCR) was used to detect the relative expression of the WT1 gene in 4 normal human lungs and 5 IPF human lungs. We found that the expression level of the WT1 gene was significantly increased in IPF human lungs (Fig. 1b), and the qRT-PCR results in mouse lung tissues were consistent with those in human lungs. The WT1 gene expression level in fibrotic mouse lung tissues was significantly increased (Fig. 1c). Furthermore, we used the human single-cell transcriptome database (GSE136831: containing 26 normal human lungs and 32 IPF human lungs) to analyze which cell group in fibrotic lungs was mainly highly expressed. We found that in the lungs of IPF patients, WT1 was mainly expressed in mesenchymal cells. The mouse single-cell transcriptome sequencing results were consistent with those in humans, showing that the WT1 gene was mainly enriched in the mesenchymal cells of the lungs of mice with bleomycin-induced pulmonary fibrosis (Figure 1d). Based on these results, we found that the WT1 antigen was significantly overexpressed in the fibrotic lungs of both humans and mice, suggesting that the WT1 antigen could serve as a target for developing vaccines against pulmonary fibrosis.

[0074] Example 2. Preparation and Characterization of Mito-WT1 Vaccine

[0075] We obtained K562 cells that stably target and express WT1 by constructing recombinant plasmids, packaging lentiviruses, infecting human chronic myeloid leukemia cells (K562) with lentiviruses, and screening with puromycin. WT1 Wild-type K562 cells and K562 cells were isolated using a mitochondrial isolation kit (Sigma-Aldrich, MITOIS02). WT1 Mitochondria of the cells (Figure 2a). First, we constructed the recombinant plasmid and packaged it with lentivirus: specifically, using OTC as the mitochondrial targeting peptide, OTC and the target gene WT1 were synthesized and ligated in the open reading frame region of the pLV-puro vector (OTC+WT1), successfully constructing the pLV-EF1a-OTC-WT1-pGK-Puro(pLV-WT1) recombinant plasmid (Figure 2b). The pLV-WT1 plasmid contains the puromycin resistance gene and can be used later for screening stable tumor cell lines after transfection. Subsequently, the plasmid vector was transformed and expanded. The plasmid DNA was extracted in large quantities using an endotoxin-free plasmid large-scale extraction kit (TIANGEN, #DP117). Lentiviral vectors are an effective tool for introducing foreign genes. We directionally transferred the foreign WT1 gene into the mitochondria of tumor cells through lentiviral transfection. We mixed the pLV-WT1 recombinant plasmid with helper plasmids (pSPAX2 and pMD2.G) in a volume ratio of 5:3:2, totaling 10 μg, and mass-packaged the lentiviral vector into HEK293T cells. Six to eight hours after transfection, we carefully aspirated the cell culture medium and added 10 mL of fresh cell culture medium for further culture. We collected the viral supernatant and concentrated the lentivirus using ultracentrifugation at 4°C, 35,000 rpm, and 90 minutes. Then, we infected K562 cells with the lentivirus and screened for stable transfected cells. We co-cultured K562 cells in logarithmic growth phase with different volumes of concentrated virus, added polybrene to increase transfection efficiency, and incubated at 37°C for 8 hours, replacing the medium with fresh 1640 double-sided medium. After 3-4 days of continued infection, we observed the cell status and used a puromycin gradient from low to high concentrations to select stable strains, with the highest selection concentration of puromycin being 9 μg / mL.

[0076] Next, we isolated and identified mitochondria overexpressing WT1 (Mito-WT1). The specific steps are as follows: Mito mitochondria were extracted from wild-type K562 cells and K562 cells using the Sigma-Aldrich Mitochondrial Isolation Kit (MITOIS02). WT1Mitochondria of cells. First, cells in the logarithmic growth phase were collected and counted. Cells were washed twice with sterile, pre-chilled PBS and centrifuged at 600g for 5 minutes at 4°C to collect the cell pellet. The cell pellet was resuspended in diluted 1×Extraction Buffer A (EBA) (Sigma-Aldrich, #E2778) containing protease inhibitors (1-2.5 mL: 2-5×10⁻⁵). 7 Cells were incubated on ice for 10-15 minutes. The cell suspension was repeatedly aspirated with a syringe to disrupt the cell structure until microscopic examination showed that more than 50% of the cells were broken. The cells were centrifuged at 1000g for 10 minutes at 4°C, and the supernatant was carefully collected. The supernatant was then centrifuged again for 10 minutes (4°C, 3000g) to collect the mitochondrial precipitate. This was obtained from K562 cells and K562... WT1 Control mitochondria (Mito) and mitochondria expressing WT1 (Mito-WT1) were obtained from the cells.

[0077] Transmission electron microscopy results showed that Mito and Mito-WT1 were essentially identical in morphology and structure, suggesting that engineered modification of mitochondria does not affect mitochondrial structure (Figure 2c). Mitochondria resuspended in RIPA were lysed using a probe (5 seconds sonication, 5-second interval, 30% power), and SDS-PAGE protein loading buffer (5X) was added to prepare protein samples. We detected WT1 protein expression in mitochondria using Western blot and found K562... WT1 The expression of WT1 protein in mitochondria was significantly higher than that in wild-type K562 cells. While the expression levels of the mitochondrial reference protein VDAC were high and similar in both groups, the expression level of the whole-cell reference protein Vinculin was lower, indicating higher purity of the extracted mitochondria and fewer cytoplasmic impurities (Figure 2d). The co-localization of WT1 protein with mitochondria was detected by immunofluorescence assay, as described in Method 5 above. We analyzed the expression location of WT1 protein using immunofluorescence, as shown in Figure 2e. Although WT1 was expressed in wild-type K562 cells, it did not co-localize with mitochondria. Conversely, in K562 cells… WT1 In the cells, WT1 protein expression was enhanced, and the fluorescence distribution of the WT1 antibody was consistent with that of the mitochondrial tracer, indicating that the WT1 protein was co-localized with mitochondria in K562 cells. Based on these results, we successfully constructed a K562 cell line that targets mitochondrial expression of WT1 and built a Mito-WT1 pulmonary fibrosis vaccine production platform.

[0078] Example 3. Subcutaneous immunization with Mito-WT1 vaccine demonstrates therapeutic efficacy for pulmonary fibrosis in mice.

[0079] To evaluate the efficacy of subcutaneous immunization with the Mito-WT1 vaccine in treating pulmonary fibrosis in mice, as shown in Figure 3a, mice were subcutaneously injected with 100 μL of different drugs (saline, 50 μg Mito, 5 μg WT1 protein, and 50 μg Mito-WT1) on days 0, 7, and 14, respectively. A mouse model of pulmonary fibrosis was established on day 1 by sublingual instillation of 50 μL bleomycin (3 mg / kg). Lung tissue was collected on day 28 after modeling to observe the therapeutic efficacy of the vaccine. We found that although subcutaneous immunization with Mito-WT1 did not significantly reduce lung weight in fibrotic mice (Figure 3b), pathological section results showed significant relief of lung inflammation in the Mito-WT1 group, a decreased Szapiel score indicating a reduction in damaged and fibrotic alveolitis areas (Figure 3c-H&E, 3d), reduced lung collagen deposition, and a decreased Ashcroft score indicating a reduction in fibrotic areas (Figure 3c-Masson, 3e). Type I collagen (Collagen 1) and α-smooth muscle actin (α-SMA) are markers of pulmonary fibrosis, and their expression increases in fibrotic lungs. Furthermore, immunohistochemistry showed that subcutaneous immunization with Mito-WT1 reduced the deposition and expression of Collagen 1 and α-SMA (Figure 3f-h). Based on these results, subcutaneous immunization with the Mito-WT1 vaccine is an effective treatment for pulmonary fibrosis in a bleomycin-induced mouse model.

[0080] Example 4. Mito-WT1 vaccine intranasal immunization has therapeutic efficacy for pulmonary fibrosis in mice.

[0081] To evaluate the efficacy of intranasal immunization with Mito-WT1 vaccine in treating pulmonary fibrosis in mice, as shown in Figure 4a, mice were intranasally immunized with 25 μL of saline, 50 μg Mito, and 50 μg Mito-WT1 on days 0, 7, and 14, respectively. A mouse model of pulmonary fibrosis was established on day 1 by retrolingual injection of 50 μL bleomycin (3 mg / kg). Lung tissue was collected 28 days after modeling to observe the therapeutic efficacy of intranasal immunization with Mito-WT1 vaccine. Therapeutic intranasal immunization with Mito-WT1 significantly reduced lung weight in fibrotic mice (Figure 4b). H&E and Masson staining results showed that therapeutic intranasal immunization with Mito-WT1 significantly alleviated lung inflammation and collagen deposition in fibrotic mice (Figures 4c-e). Immunohistochemical results showed that intranasal immunization with Mito-WT1 significantly reduced the expression of pulmonary fibrosis markers Collagen 1 and α-SMA (Figures 4f-h). Western blot results showed that intranasal immunization with Mito-WT1 significantly reduced the protein expression levels of COL1A1 and α-SMA (Figure 4j). Furthermore, fibronectin (Fn) is also a biomarker of pulmonary fibrosis, and qRT-PCR results showed that intranasal immunization with Mito-WT1 significantly reduced the relative expression levels of genes involved in pulmonary fibrosis, including Col1α1, Fn1, and Acta2 (Figure 4k-m). Based on these results, intranasal immunization with the Mito-WT1 vaccine was also effective in treating pulmonary fibrosis in a bleomycin-induced mouse model.

[0082] Example 5. Mito-WT1 vaccine nasal immunization in mice has the effect of preventing pulmonary fibrosis.

[0083] To evaluate the ability of intranasal immunization with Mito-WT1 vaccine to prevent pulmonary fibrosis in mice, as shown in Figure 5a, mice were intranasally immunized with 25 μL of saline (50 μg Mito, 50 μg Mito, and 50 μg Mito-WT1) on days 0, 14, and 21, respectively. A mouse model of pulmonary fibrosis was established on day 22 by retrolingual injection of 50 μL bleomycin (3 mg / kg). Lung tissue was collected on day 50 to observe the prophylactic efficacy of intranasal immunization with Mito-WT1 vaccine. Prophylactic intranasal immunization with Mito-WT1 significantly reduced lung weight in fibrotic mice (Figure 5b). H&E staining and Masson staining results showed that prophylactic intranasal immunization with Mito-WT1 significantly alleviated lung inflammation and collagen deposition in fibrotic mice (Figures 5c-e). Immunohistochemical results showed that intranasal immunization with Mito-WT1 significantly reduced the expression of pulmonary fibrosis markers Collagen 1 and α-SMA (Figures 5f-h). Western blot results showed that intranasal immunization with Mito-WT1 significantly reduced the protein expression levels of COL1A1 and α-SMA (Figure 5j). qRT-PCR results showed that intranasal immunization with Mito-WT1 significantly reduced the relative expression levels of Col1α1, Fn1, and Acta2 genes (Figure 5k-m). Based on these results, intranasal immunization with Mito-WT1 vaccine has the efficacy of preventing pulmonary fibrosis in a bleomycin-induced mouse model of pulmonary fibrosis.

[0084] Example 6. Safety evaluation of Mito-WT1 in mice

[0085] To evaluate the safety of the Mito-WT1 vaccine, mice were immunized intranasally three times with different vaccines (saline, 50 μg Mito, and 50 μg Mito-WT1) according to the prophylactic vaccine immunization protocol. One week after the third immunization, fresh anticoagulated whole blood and serum were collected. Complete blood cell counts and serum biochemical tests were performed. There were no statistically significant differences among the control group, the Mito group, and the Mito-WT1 group (Figures 6a and 6b). H&E staining of pathological sections showed no obvious pathological changes in the heart, liver, spleen, lungs, kidneys, skin, small intestine, skeletal muscle, and nasal mucosa of the immunized mice (Figure 6c).

Claims

1. An engineered mitochondrial vaccine, characterized in that: Transgenic mitochondria containing targeted expression of fibrosis-associated antigens.

2. The engineered mitochondrial vaccine of claim 1, wherein: The fibrosis includes at least one of pulmonary fibrosis, liver fibrosis, pancreatic fibrosis, kidney fibrosis, cardiac fibrosis, endometrial fibrosis, ocular fibrosis, splenic fibrosis, myelofibrosis, or skin fibrosis. Preferably, the pulmonary fibrosis includes at least one of idiopathic pulmonary fibrosis, secondary pulmonary fibrosis, hereditary pulmonary fibrosis, or interstitial lung disease with pulmonary fibrosis characteristics.

3. The engineered mitochondrial vaccine of claim 1 or 2, wherein: The fibrosis-associated antigen is a pulmonary fibrosis-associated antigen, which is selected from at least one of nephroblastoma 1, snail family zinc finger protein 1, snail family zinc finger protein 2, kink family bHLH transcription factor, immediate early gene c-Jun or c-Fos; Preferably, the pulmonary fibrosis antigen is WT1; More preferably, the nucleotide sequence of the WT1 is shown in SEQ ID NO.

1.

4. The engineered mitochondrial vaccine of any one of claims 1-3, wherein: The transgenic mitochondria are mitochondria expressing WT1, and the entire expression plasmid nucleotide sequence is shown in SEQ ID NO.2; preferably, the target gene of the engineered mitochondrial vaccine is shown in SEQ ID NO.

4.

5. The engineered mitochondrial vaccine of any one of claims 1-4, wherein: The engineered mitochondrial vaccine uses mitochondria as a fibrosis antigen delivery system. This antigen delivery system is a mitochondrial-targeted fibrosis-related antigen expression platform established using a plasmid with ornithine carbamoyltransferase as the leader sequence and a lentivirus transfection system. Preferably, the leader sequence is as shown in SEQ ID NO.

3.

6. The method for preparing the mitochondrial vaccine according to any one of claims 1 to 5, characterized in that: Includes the following steps: A recombinant plasmid containing fibrosis-associated antigen was constructed, packaged into lentivirus, and then cells were infected with the lentivirus system. Stable transgenic strains that stably expressed fibrosis-associated antigen in mitochondria were screened out. The mitochondria of the stable transgenic strains were then extracted to obtain transgenic mitochondria that directionally expressed fibrosis-associated antigen.

7. The method of claim 6, wherein: The recombinant plasmid was prepared by synthesizing and linking OTC and template antigen using OTC as the mitochondrial localization peptide and fibrosis-associated antigen as the template antigen. Preferably, the recombinant plasmid nucleotide sequence is as shown in SEQ ID NO.2; more preferably, the recombinant plasmid contains a nucleotide sequence consisting of OTC and template antigen, as shown in SEQ ID NO.

4.

8. The production method according to claim 6 or 7, characterized in that: The plasmid is selected from at least one of mammalian cell expression vectors, insect baculovirus expression vectors, Escherichia coli expression vectors, and yeast expression vectors; Preferably, the plasmid is selected from any one of the vectors pLV-puro, pCDH-CMV, pCDH-EF1, pCDH-MSCV, and pCDH-RFP.

9. The method of any one of claims 6 to 8, wherein: The method for preparing the mitochondrial vaccine includes the following steps: Using OTC as a mitochondrial localization peptide, OTC and the target gene WT1 were synthesized and ligated in the pLV-puro vector to construct a recombinant plasmid. The recombinant plasmid was then mixed with an helper plasmid and packaged into a lentiviral vector in HEK293T cells. The culture supernatant was collected, and the lentivirus was concentrated by ultracentrifugation. K562 cells were then infected with the lentivirus, and stable transfected cells were screened.

10. A pharmaceutical composition, characterized by: It includes the engineered mitochondrial vaccine as described in any one of claims 1 to 5, and pharmaceutically permissible excipients.

11. The pharmaceutical composition of claim 10, wherein: The excipients are at least one of diluents, excipients, fillers, binders, wetting agents, disintegrants, absorption promoters, surfactants, protectants, adsorbent carriers, or lubricants.

12. A combination drug, characterized in that: Contains engineered mitochondrial vaccines as described in any one of claims 1 to 5 and other drugs for the prevention and / or treatment of fibrosis.

13. The engineered mitochondrial vaccine according to any one of claims 1 to 5, the pharmaceutical composition according to claim 10 or 11, or the combination drug according to claim 12 is in the form of an injection, nasal drops, spray, inhaler, or oral preparation; Preferably, the injection route is at least one of intramuscular injection, intravenous injection, subcutaneous injection, intradermal injection, intramyocardial injection, or intraperitoneal injection. More preferably, the dosage form is an intramuscular or subcutaneous injection preparation or a nasal drop preparation.

14. The use of the engineered mitochondrial vaccine according to any one of claims 1 to 5, the pharmaceutical composition according to claim 10 or 11, or the combination drug according to claim 12 in the prevention and / or treatment of fibrotic diseases.

15. The use according to claim 14, characterized in that: The fibrotic diseases include at least one of pulmonary fibrosis, liver fibrosis, pancreatic fibrosis, kidney fibrosis, cardiac fibrosis, endometrial fibrosis, ocular fibrosis, splenic fibrosis, myelofibrosis, or skin fibrosis. Preferably, the pulmonary fibrosis includes at least one of idiopathic pulmonary fibrosis, secondary pulmonary fibrosis, hereditary pulmonary fibrosis, or interstitial lung disease with pulmonary fibrosis characteristics.

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