Exosome as well as preparation method, application and pharmaceutical composition thereof

Abstract

The invention provides an exosome, a preparation method and application thereof and a pharmaceutical composition, the exosome is derived from a genetically engineered mesenchymal stem cell, and the genetically engineered mesenchymal stem cell comprises an exogenous PD-L1 gene and an exogenous HGF gene. The exosome can improve functional damage caused by the ischemia condition of the tissue. Therefore, the exosome can be used for preparing the medicine for treating the ischemic condition of the tissue, and the medicine composition containing the exosome can be used for treating the ischemic condition of the tissue.

Description

Exosomes, preparation method thereof, use thereof and pharmaceutical composition Technical Field

[0001] The present invention relates to exosomes, their preparation method, their use and pharmaceutical compositions, and in particular to exosomes derived from genetically engineered mesenchymal stem cells, their preparation method, their use and pharmaceutical compositions. Background Art

[0002] Ischemia is a condition characterized by insufficient blood flow to tissues, leading to a lack of oxygen and nutrients. Ischemia is typically caused by vascular problems, but can also result from vasoconstriction, thrombosis, or embolism, leading to localized anemia. In addition to oxygen and nutrient deprivation, ischemia can also lead to the accumulation of metabolic products, which can damage tissues.

[0003] When ischemia occurs in the brain, it can cause ischemic stroke, the leading cause of death worldwide. Ischemic stroke results in a rapid loss of brain function due to an abnormal blood supply to the brain, and can easily lead to neurobehavioral impairment. Traditional stroke treatment relies primarily on medication to prevent subsequent strokes. While thrombolytics and intracranial artery thrombectomy are available in the acute phase of ischemic stroke, other drug treatments primarily aim to reduce the risk of subsequent strokes. However, these drugs have limited effectiveness in restoring damaged brain tissue and neurological function. More importantly, approximately 75% of survivors suffer from post-stroke complications, including paralysis, depression, speech disorders, and visual impairment. When ischemia occurs in the heart, it can cause ischemic heart disease, a leading cause of death worldwide. Many patients with heart failure secondary to acute myocardial ischemia are not suitable for invasive treatment and lack effective drug therapies.

[0004] Regenerative medicine has discovered that therapeutic cells, such as mesenchymal stem cells, possess multipotency and self-renewal abilities, making them promising treatments for ischemic heart disease and ischemic stroke. However, poor engraftment has been observed in ischemic heart disease treatments using these cells. This is likely due to the poor viability of the implanted therapeutic cells within the infarcted heart tissue, with only 1% of cells remaining viable after four days of implantation. Consequently, implanted therapeutic cells in the infarcted area exhibit little improvement in cardiac function. Therefore, there remains an urgent need for treatments that significantly improve tissue ischemia.

[0005] Summary of the Invention

[0006] In view of this, an object of the present invention is to provide exosomes derived from genetically engineered mesenchymal stem cells, which can significantly improve tissue ischemia and enhance nerve regeneration or reduce neuronal death, as well as reduce inflammatory responses. Exosomes are a cell-free product that is easier to produce and safer than mesenchymal stem cells and does not lead to unnecessary implantation.

[0007] One aspect of the present invention provides exosomes derived from a genetically engineered mesenchymal stem cell, wherein the genetically engineered mesenchymal stem cell comprises an exogenous PD-L1 gene and an exogenous HGF gene.

[0008] According to the aforementioned exosomes, the sequence of a peptide encoded by the exogenous PD-L1 gene may be shown as SEQ ID NO:4, and the sequence of a peptide encoded by the exogenous HGF gene may be shown as SEQ ID NO:5.

[0009] According to the aforementioned exosomes, the exogenous PD-L1 gene and the exogenous HGF gene can be linked to a sequence encoding a self-cleaving peptide.

[0010] According to the aforementioned exosomes, the exosomes may contain an overexpression of PD-L1 and an overexpression of HGF.

[0011] According to the aforementioned exosomes, when measured by flow cytometry, the expression level of PD-L1 on an exosome membrane of the exosomes can be increased relative to that of a control exosome, wherein the control exosomes are derived from a non-genetically engineered mesenchymal stem cell.

[0012] According to the aforementioned exosomes, when measured by ELISA, the HGF content of the exosomes may be increased relative to that of a control exosome, wherein the control exosome is derived from a non-genetically engineered mesenchymal stem cell.

[0013] According to the aforementioned exosomes, CXCR4 may be overexpressed on an exosome membrane of the exosomes.

[0014] According to the aforementioned exosomes, when measured by flow cytometry, the ratio of exosomes carrying a CXCR4 surface marker can be increased relative to a control exosome, wherein the control exosome is derived from a non-genetically engineered mesenchymal stem cell.

[0015] According to the aforementioned exosomes, a particle size of the exosomes may be between 30 nm and 200 nm.

[0016] According to the aforementioned exosomes, the particle size of the exosomes may be between 100 nm and 150 nm.

[0017] Another aspect of the present invention provides a method for producing exosomes, comprising constructing a genetically engineered mesenchymal stem cell, performing a culturing step, and performing an isolation step. During the construction of the genetically engineered mesenchymal stem cell, an exogenous HGF gene and an exogenous PD-L1 gene are transferred into the mesenchymal stem cell to produce the genetically engineered mesenchymal stem cell. In the culturing step, the genetically engineered mesenchymal stem cell is cultured in a culture medium to produce a conditioned medium. In the isolation step, exosomes are isolated from the conditioned medium.

[0018] According to the aforementioned method for preparing exosomes, in the culturing step, the genetically engineered mesenchymal stem cells may be cultured under a hypoxic condition.

[0019] According to the aforementioned method for preparing exosomes, the hypoxic condition may be an oxygen content below 3%.

[0020] According to the aforementioned method for preparing exosomes, the mesenchymal stem cells may be adipose-derived mesenchymal stem cells, umbilical cord-derived mesenchymal stem cells, or bone marrow-derived mesenchymal stem cells.

[0021] According to the aforementioned method for preparing exosomes, the mesenchymal stem cells may be adipose-derived mesenchymal stem cells or umbilical cord mesenchymal stem cells.

[0022] Another aspect of the present invention is to provide a pharmaceutical composition for treating an ischemic condition of a tissue, wherein the pharmaceutical composition comprises the aforementioned exosomes and a pharmaceutically acceptable carrier.

[0023] According to the aforementioned pharmaceutical composition, the administration route of the pharmaceutical composition is intravenous injection, intracarotid artery injection, intraarterial injection or a combination thereof.

[0024] Yet another aspect of the present invention is to provide a use of the aforementioned exosomes for preparing a medicament for treating an ischemic condition of a tissue.

[0025] According to the aforementioned use of exosomes, the tissue may be the brain.

[0026] According to the aforementioned use of exosomes, the ischemic condition may be ischemic stroke.

[0027] According to the aforementioned use of exosomes, the drug may be a drug that reduces the area of ​​brain damage caused by stroke.

[0028] Another aspect of the present invention is to provide a use of the aforementioned exosomes for preparing a drug for enhancing nerve regeneration or reducing neuronal death.

[0029] Another aspect of the present invention is to provide a use of the aforementioned exosomes for preparing a drug for reducing inflammatory response. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] To make the above and other objects, features, advantages and embodiments of the present invention more apparent, the following descriptions of the accompanying drawings are given:

[0031] FIG1 is a flow chart illustrating the steps of the method for preparing exosomes of the present invention;

[0032] Figure 2A is a micrograph of ADSCs, hTERT-ADSCs, and hTERT-ADSC-PD-L1-HGF differentiated into different tissue cells;

[0033] Figures 2B, 2C, and 2D are analysis results of PD-L1 expression and / or HGF content in hTERT-ADSCs and hTERT-ADSC-PD-L1-HGF;

[0034] FIG3A is a graph showing the analysis results of EXO-PD-L1-HGF production in hTERT-ADSC-PD-L1-HGF cultured under normoxic and hypoxic conditions;

[0035] FIG3B is a transmission electron micrograph of EXO and EXO-PD-L1-HGF;

[0036] FIG3C is a graph showing the nanoparticle tracking analysis results of EXO and EXO-PD-L1-HGF;

[0037] Figures 3D, 3E, 3F, 3G, 3H, and 3I are the analysis results of HGF content, PD-L1 expression, CD63 expression, CD9 expression, and / or CD81 expression in EXO and EXO-PD-L1-HGF;

[0038] FIG3J is a transmission electron micrograph of UMSC-EXO and UMSC-EXO-PD-L1-HGF;

[0039] FIG3K is a graph showing the nanoparticle tracking analysis results of UMSC-EXO and UMSC-EXO-PD-L1-HGF;

[0040] Figures 3L, 3M, and 3N are analysis results of HGF content, PD-L1 expression, CD63 expression, CD9 expression, and / or CD81 expression in UMSC-EXO and UMSC-EXO-PD-L1-HGF;

[0041] FIG4A is a graph showing the analysis results of DiO-EXO and DiO-EXO-PD-L1-HGF uptake by primary cortical cells (PCCs);

[0042] Figures 4B, 4C, 4D, and 4E are analysis results of TUNEL staining to detect that EXO-PD-L1-HGF reduces H2O2-induced apoptosis in PCCs and SH-SY5Y cells in vitro;

[0043] Figures 4F and 4G are the analysis results of the CCK-8 assay for the reduction of cell death induced by H2O2 in vitro by EXO-PD-L1-HGF;

[0044] Figures 4H, 4I, 4J, and 4K are analysis results showing that EXO-PD-L1-HGF reduces H2O2-induced apoptosis in PCCs and SH-SY5Y cells in vitro using Annexin V-FITC / 7-AAD double staining.

[0045] FIG4L is a graph showing the results of Western blot analysis of the expression of apoptosis-related proteins in H2O2-induced apoptosis of PCCs and SH-SY5Y cells after pretreatment with EXO or EXO-PD-L1-HGF;

[0046] FIG4M is a graph showing the analysis results of UMSC-EXO-PD-L1-HGF reducing H2O2-induced cell death in vitro using the CCK-8 assay;

[0047] FIG4N is a graph showing the results of Western blot analysis of the expression of apoptosis-related proteins in H2O2-induced apoptosis of PCCs after pretreatment with UMSC-EXO-PD-L1-HGF;

[0048] FIG5A is a graph showing the results of analyzing the inhibition of T cell proliferation by EXO-PD-L1-HGF using CFSE assay;

[0049] FIG5B is a graph showing the results of ELISA analysis of how EXO-PD-L1-HGF reduces interferon-γ expression;

[0050] FIG5C is a graph showing the analysis results of the percentage of regulatory T cells after treatment with EXO or EXO-PD-L1-HGF;

[0051] FIG5D is a graph showing the analysis results of stem cell marker expression in isolated neural progenitor cells (NPCs);

[0052] FIG5E is a graph showing the analysis results of EXO-PD-L1-HGF promoting NPCs proliferation detected by CCK-8 assay;

[0053] Figure 5F is a heat map of next-generation sequencing analysis of the control group, EXO group, and EXO-PD-L1-HGF group;

[0054] Figure 5G is a graph showing the results of Gene Set Enrichment Analysis (GSEA) for the control group, EXO group, and EXO-PD-L1-HGF group;

[0055] FIG6A is a graph showing the protein expression analysis results of phosphorylated STAT3 and phosphorylated FOXO3 in PCCs and NPCs of the PBS group, EXO group, and EXO-PD-L1-HGF group by Western blotting;

[0056] FIG6B is a graph showing the results of analyzing the protein expression of cytokines in cells after treatment with EXO or EXO-PD-L1-HGF using a human cytokine array;

[0057] FIG6C is a graph showing the results of ELISA analysis of cytokine protein expression in cells after treatment with EXO or EXO-PD-L1-HGF;

[0058] FIG6D is a graph showing the expression analysis results of apoptosis-related proteins in PCCs with attenuated FOXO3 expression after treatment with EXO-PD-L1-HGF by Western blotting;

[0059] Figures 6E and 6F are the results of flow cytometry analysis of cell apoptosis in different groups;

[0060] Figures 6G and 6H show the analysis results of the number of secondary neurospheres formed by NPCs in different groups;

[0061] Figures 6I and 6J show the results of size analysis of secondary neurospheres formed by NPCs from different groups;

[0062] FIG7A and FIG7B are graphs showing the fluorescence intensity analysis results after intravenous injection of DiD-EXO or DiD-EXO-PD-L1-HGF into ischemic stroke (middle cerebral artery occlusion, MCAO) mice;

[0063] FIG7C is an immunofluorescence staining result showing the co-localization of EXO-PD-L1-HGF and SDF-1α expressed in the injury site;

[0064] FIG7D is a graph showing the analysis results of CXCR4 expression on the exosome membranes of EXO and EXO-PD-L1-HGF detected by flow cytometry;

[0065] Figure 7E shows the expression of GFAP in the injured area by EXO and EXO-PD-L1-HGF + Cells and MAP2 + Immunofluorescence staining results of cell co-localization;

[0066] Figures 7F and 7G are immunofluorescence staining results showing that attenuating FOXO3 expression inhibits EXO-PD-L1-HGF and promotes the migration of nestin-GFP-expressing NPCs to the injury site;

[0067] Figures 7H, 7I, and 7J are analysis results of the proliferation of cells expressing nestin-GFP induced by EXO-PD-L1-HGF treatment;

[0068] Figures 8A, 8B, 8C, and 8D are analysis results showing the effects of EXO and EXO-PD-L1-HGF on the differentiation of NPCs.

[0069] FIG8E is a graph showing the analysis results of the rotarod test in MCAO mice treated with EXO-PD-L1-HGF;

[0070] FIG8F is a graph showing the analysis results of the balance beam test in MCAO mice treated with EXO-PD-L1-HGF;

[0071] Figures 8G and 8H are the analysis results of brain infarction in MCAO mice treated with EXO-PD-L1-HGF;

[0072] FIG8I is a graph showing the analysis results of the rotarod test in MCAO mice treated with UMSC-EXO-PD-L1-HGF;

[0073] FIG8J is a graph showing the analysis results of the balance beam test in MCAO mice treated with UMSC-EXO-PD-L1-HGF;

[0074] Figures 8K and 8L show the analysis results of brain infarction in MCAO mice treated with UMSC-EXO-PD-L1-HGF;

[0075] Figures 8M and 8N show the analysis results of TUNEL staining to detect cell apoptosis in brain tissues of MCAO mice treated with EXO-PD-L1-HGF;

[0076] Figure 9A shows the CD3 in the brain tissue of mice in different groups + Figure 2. T cell number analysis results.

[0077] Figures 9B, 9C, 9D, 9E, and 9F are analysis results showing that treatment with EXO or EXO-PD-L1-HGF significantly reduced the percentages of activated dendritic cells (DCs), cytotoxic T cells, natural killer (NK) cells, and M1 macrophages in the ischemic cerebral hemisphere;

[0078] Figures 9G, 9H, 9I, 9J, and 9K are analysis results showing that EXO or EXO-PD-L1-HGF treatment also reduces the percentages of activated DCs, cytotoxic T cells, NK cells, and M1 macrophages in the spleen;

[0079] Figures 9L, 9M, and 9N are graphs showing the analysis results of increased percentages of regulatory B cells, regulatory T cells, and M2 macrophages in the ischemic cerebral hemisphere after treatment with EXO or EXO-PD-L1-HGF; and

[0080] Figures 9O, 9P, and 9Q are graphs showing the analysis results of increased percentages of regulatory B cells, regulatory T cells, and M2 macrophages in the spleen after treatment with EXO or EXO-PD-L1-HGF.

[0081] Wherein, the reference numerals:

[0082] 100: Preparation method of exosomes

[0083] 110,120,130: Steps DETAILED DESCRIPTION

[0084] Unless otherwise defined, all scientific or technical terms used in this specification have the same meanings as those understood by those skilled in the art to which the present invention belongs. Those skilled in the art to which the present invention belongs can understand and use any methods and materials similar or equivalent to those described in this specification to practice the present invention.

[0085] Unless otherwise indicated, all numbers expressing quantities of ingredients, reaction conditions, and so forth used in the specification and claims of the present invention are to be understood as being modified in all instances by the term "about." Accordingly, unless indicated to the contrary, the numerical parameters set forth in the specification and claims of the present invention are approximations and may vary depending upon the requirements sought to be pursued by the present invention.

[0086] The term "a" or "an" used in this specification refers to one or more of the objects described. The term "and / or" refers to two or one of them.

[0087] The term "in vivo" as used herein generally refers to an experiment conducted in a living organism. The term "in vitro" generally refers to an experiment conducted outside a living organism, for example, in an artificial environment created outside a living organism.

[0088] In this specification, gene names are represented by italic letters, the symbol format of their RNA is the same as that of the gene, and the protein associated with the gene is represented by non-italic letters.

[0089] As used herein, "genetic engineering" refers to the manipulation of genes using genetic material to alter the number of copies and / or expression of genes in cells. Genetic material can be in the form of DNA or RNA. Genetic material can be transferred to cells via various methods, including viral transduction and non-viral transfection. Following genetic engineering, the expression of certain genes in cells can be permanently or temporarily altered.

[0090] "Exosomes," or "extracellular vesicles," as used herein, refer to vesicles secreted by cells with a particle size of approximately less than 500 nm, or between 30 nm and 200 nm. They are naturally present in various biological fluids, such as amniotic fluid, urine, and blood. Depending on the cell source, production pathway, and biological characteristics, exosomes vary in surface marker proteins, particle size, and composition of mRNA, long noncoding RNA (lncRNA), and microRNA (miRNA). Besides natural sources, exosomes can be isolated from various cell cultures (e.g., neurons, tumor cells, kidney cells, or stem cells), or from genetically engineered cells. The exosomes described herein are produced from genetically engineered mesenchymal stem cells. In some embodiments, the "exosomes" or "extracellular vesicles" described herein may have a particle size of 30 nm to 200 nm. In some embodiments, the particle size may be 100 nm to 150 nm.

[0091] The "mesenchymal stem cells" described in this specification may be derived from tissues such as bone marrow, umbilical cord blood, amniotic sac and amniotic fluid, placenta, skin, fat, muscle, vasculature, liver, pancreas or peripheral blood. The cell source may be xenogeneic, allogeneic or autologous. The "mesenchymal stem cells" described in this specification may refer to adipose mesenchymal stem cells, umbilical cord mesenchymal stem cells, bone marrow mesenchymal stem cells, dental pulp mesenchymal stem cells, amniotic fluid mesenchymal stem cells, amniotic membrane mesenchymal stem cells, induced mesenchymal stem cells (iMSC) differentiated from induced pluripotent stem cells (iPSCs) or immortalized mesenchymal stem cells (immortalized MSC). In some embodiments, the mesenchymal stem cells may be adipose mesenchymal stem cells. In some embodiments, the mesenchymal stem cells may be umbilical cord mesenchymal stem cells.

[0092] As used herein, an "expression vector" refers to a medium that carries exogenous genes into cells for expression without degradation. Expression vectors in the present invention may include plasmids, viral vectors, and artificial chromosomes. The exogenous genes described herein may be located in the same expression vector or in separate expression vectors.

[0093] The term "increased expression level" as used herein refers to an increase in the expression level of RNA or protein of a gene of interest in genetically engineered mesenchymal stem cells compared to the expression level of the corresponding gene in non-genetically engineered mesenchymal stem cells.

[0094] "Overexpression" as used herein refers to a significant increase in expression relative to normal conditions. For example, gene overexpression refers to a significant increase in the level of RNA transcribed relative to normal conditions. Increased RNA levels can lead to excessive accumulation of translated proteins, i.e., overexpression at the protein level.

[0095] In some embodiments, a "surface marker" refers to the presence or absence of a protein on the surface of a cell. In some embodiments, a "positive marker" refers to a surface marker that is present or expressed on the cell of interest and is expressed as + In some embodiments, a "negative marker" refers to a surface marker that is not present on the cell of interest and is represented by " - "express.

[0096] As used herein, "treatment" generally refers to obtaining a desired pharmacological and / or physiological effect. The effect may be preventative, i.e., complete or partial prevention of a disease, disorder, or its symptoms, and may be therapeutic, i.e., partial or complete cure of a disease, disorder, and / or symptoms attributable thereto. As used herein, "treatment" encompasses any treatment of a disease in a mammal (preferably a human), and includes inhibiting the progression of a disease, disorder, or its symptoms in a subject, or alleviating or ameliorating a disease, disorder, or its symptoms in a subject.

[0097] As used herein, "individual," "subject," and "patient" are used interchangeably and refer to any mammalian individual for whom diagnosis or treatment is desired.

[0098] "PD-L1," as used herein, refers to programmed death-ligand 1 (PD-L1), a 40 kDa type 1 transmembrane protein encoded by the CD274 gene. PD-L1 is also known as "CD274," "B7 homology 1," and "B7-H1." The PD-L1 receptor, programmed cell death protein-1 (PD-1), is present on activated T cells, B cells, and myeloid cells. Binding of PD-L1 to PD-1 inhibits T cell activation, reduces proliferation and cytotoxicity, and induces apoptosis. PD-L1 expressed on mesenchymal stem cells interacts with PD-1, providing inhibitory signals that regulate cell activation and proliferation. Consequently, in mouse stroke models, signaling through the PD-1 and PD-L1 pathways leads to T cell exhaustion, thereby suppressing the inflammatory cascade in the central nervous system. Previous studies have shown that mesenchymal stem cells inhibit the proliferation and function of effector T cells through direct contact with activated T cells and indirect secretion of soluble PD-L1. The PD-L1 described herein may refer to human PD-L1 or a functional variant thereof, such as a functional variant having a peptide sequence that is greater than 95%, greater than 90%, or greater than 80% similar to SEQ ID NO:4 and exhibits comparable activity to PD-L1. In some embodiments, the sequence of PD-L1 is SEQ ID NO:4.

[0099] "HGF," as used herein, refers to hepatocyte growth factor (HGF), a cytokine that significantly promotes tissue repair and organ regeneration after tissue and organ damage by binding to the c-Met tyrosine kinase receptor. Previous studies have found that HGF can induce functional improvement and promote peripheral nerve regeneration in rats with spinal cord injury (SCI) in a nerve crush model. The combination of HGF and PD-L1 may serve as an innovative genetic candidate to aid in neurogenesis and anti-inflammatory therapeutic intervention. The HGF described herein may refer to human HGF or a functional variant thereof, such as a functional variant having a peptide sequence that is greater than 95%, greater than 90%, or greater than 80% similar to SEQ ID NO:5 and exhibits similar activity to HGF. In some embodiments, the sequence of HGF is SEQ ID NO:5.

[0100] The nucleic acids for expressing PD-L1 and HGF can be polycistronic constructs, which allow for the expression of two exogenous genes (i.e., the PD-L1 gene and the HGF gene) under the control of a single promoter or two different promoters. Alternatively, the two exogenous genes can be expressed from the same promoter and encoded by the same protein sequence, separated by a self-cleavage peptide, or the two exogenous genes can be expressed from two separate gene vectors. In some embodiments, the exogenous genes (i.e., the PD-L1 gene and the HGF gene) are linked by a sequence encoding a self-cleavage peptide. The self-cleaving peptide encoded by the self-cleaving peptide sequence that can be used in the nucleic acid construct of the present invention can be the F2A peptide derived from foot-and-mouth disease virus (FMDV), the P2A peptide derived from porcine teschovirus (PTV), the T2A peptide derived from thosea asigna virus (TaV), and / or the E2A peptide derived from equine rhinitis A virus (ERAV). In some embodiments, the self-cleaving peptide encoding sequence is the T2A encoding sequence set forth in SEQ ID NO:3, which encodes the T2A peptide set forth in SEQ ID NO:6.

[0101] "CXCR4" as used herein refers to CXC motif chemokine receptor 4 (CXC motif chemokine receptor 4), a specific receptor for stromal cell-derived factor-1α (SDF-1α). CXCR4 is expressed in most tissues and organs in the body. It is a 352-amino acid G protein-coupled receptor (GPCR) with seven transmembrane domains. Its ligand, SDF-1α, has a strong chemotactic effect on lymphocytes.

[0102] In some embodiments, exosomes derived from genetically engineered mesenchymal stem cells exhibit significantly increased expression of PD-L1 on the exosome membrane relative to control exosomes derived from non-genetically engineered mesenchymal stem cells, as measured by flow cytometry or Western blot. When measured by flow cytometry, the number of exosomes with high PD-L1 expression increases by 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% or more. In some embodiments, the number of exosomes with high PD-L1 expression increases by approximately 50%. In some embodiments, the number of exosomes with high PD-L1 expression increases by approximately 90%. The proportion of exosomes with therapeutic efficacy that bear the PD-L1 surface marker may be greater than 95%, greater than 90%, greater than 80%, greater than 70%, greater than 60%, or greater than 50%. In some embodiments, the proportion of exosomes with therapeutic efficacy that bear the PD-L1 surface marker is greater than 90%.

[0103] In some embodiments, the HGF content of exosomes derived from genetically engineered mesenchymal stem cells is significantly increased relative to control exosomes derived from non-genetically engineered mesenchymal stem cells, as measured by ELISA or Western blot. When measured by ELISA, the HGF content of the exosomes is increased by about 25%, 50%, 75%, 100%, 150%, 200%, or more. In some embodiments, the HGF content of the exosomes is increased by about 200%. In some embodiments, the HGF content of the exosomes is increased by about 50%.

[0104] In some embodiments, the expression of CXCR4 on the exosome membrane of exosomes derived from genetically engineered mesenchymal stem cells is increased relative to control exosomes derived from non-genetically engineered mesenchymal stem cells, as measured by flow cytometry or Western blotting. When measured by flow cytometry, the proportion of exosomes bearing the CXCR4 surface marker is increased by 5% to 10%, 5% to 15%, 5% to 20%, 10% to 20%, 20% to 30%, 30% to 40%, 40% to 50%, 10% to 30%, 20% to 40%, 30% to 50%, or by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, or at least 50% relative to control exosomes. In some embodiments, the proportion of exosomes bearing the CXCR4 surface marker is increased by 5% to 10%, or at least 5%, relative to control exosomes. The proportion of exosomes with therapeutic effects carrying CXCR4 surface markers may be greater than 5%, greater than 10%, greater than 15%, greater than 20%, greater than 30%, or greater than 40%. In some embodiments, the proportion of exosomes with therapeutic effects carrying CXCR4 surface markers is greater than 5%.

[0105] Enhanced proliferation of endogenous neural progenitor cells (NPCs) within the neurogenic niche of the subventricular zone (SVZ) provides an important form of neural plasticity. During stroke induction, a large number of NPCs within the SVZ deviate from their initial migratory routes and migrate toward the peri-infarct region, suggesting a potential role for SVZ-derived NPCs in post-stroke recovery. Previous studies have shown that FOXO3 regulates the homeostasis of NPCs to maintain cell quiescence and prevent premature differentiation by inducing a self-renewal gene program. Hypoxia-ischemia-induced FOXO3 upregulation supports the hypothesis that specific therapeutic strategies to activate FOXO3 expression could promote NPC proliferation and enhance stroke recovery.

[0106] To enhance therapeutic efficacy, transferring exogenous PD-L1 and HGF genes into mesenchymal stem cells to generate genetically engineered mesenchymal stem cells and then harvesting exosomes derived from these engineered mesenchymal stem cells may be a better strategy for rescuing damaged glial tissue after stroke. Therefore, the present invention provides exosomes derived from genetically engineered mesenchymal stem cells, which overexpress PD-L1 and HGF, and overexpress CXCR4 on the exosome membrane. The disclosure herein demonstrates the therapeutic potential of the exosomes of the present invention in promoting neuroprotection, anti-inflammation, and neurogenesis after ischemic stroke using in vitro and animal studies. These studies demonstrate the ability of the exosomes of the present invention to enhance neuroregeneration or reduce neuronal death, and to induce enhanced neurological recovery and reduced infarct volume in an ischemic stroke mouse model. By exploring the molecular mechanisms of the STAT3-FOXO3 signaling axis, the exosomes of the present invention can serve as novel cell-free therapeutic products that can enhance neurological recovery after ischemic stroke.

[0107] The genetically engineered mesenchymal stem cells disclosed herein are modified to express PD-L1 and HGF. As used herein, the term "modified expression" in the present invention refers to the transfer of exogenous genes or gene fragments into mesenchymal stem cells so that they can express the exogenous genes or gene fragments. Preferably, this modification does not change the differentiation potential of the mesenchymal stem cells, nor does it change the immunoregulatory properties of the mesenchymal stem cells. On the other hand, this modification is preferably a stable modification, and the expression can be persistent or inducible. The genetically engineered mesenchymal stem cells according to the present invention are modified to express PD-L1 and HGF and still have multipotent differentiation potential, such as but not limited to adipogenesis, chondrogenesis, osteogenesis, and vascularization, and their multipotent differentiation potential is similar to that of non-genetically engineered mesenchymal stem cells.

[0108] The present invention also provides a method 100 for preparing exosomes. Referring to FIG. 1 , a flow chart of the method 100 for preparing exosomes of the present invention is shown, comprising step 110 , step 120 , and step 130 .

[0109] Step 110 constructs genetically engineered mesenchymal stem cells by transferring exogenous PD-L1 and HGF genes into them. Mesenchymal stem cells can be obtained from various sources, preferably adipose tissue, umbilical cord, or bone marrow. Depending on the source, mesenchymal stem cells can be adipose-derived stromal stem cells (ADSCs), umbilical cord mesenchymal stem cells (UMSCs), or bone marrow mesenchymal stem cells (BMSCs).

[0110] The term "transfer" as used herein may refer to the transfer of genetic material into cells by various means, including viral transduction and non-viral transfection. Transduction is the delivery of genetic material into cells by a virus, wherein the virus may be an integrating virus or a non-integrating virus. The integrating virus used in the present invention may be a lentivirus or a retrovirus, wherein the integrating virus allows its coding gene to be integrated into the transduced cell infected by the viral particles, and the non-integrating virus may be an adenovirus or a Sendai virus. Non-viral methods may also be used in the present invention, for example, by transfecting DNA material or RNA material into cells, wherein the DNA material may be in the form of a PiggyBac, a minicircle vector or an exon plasmid, and the RNA material may be in the form of an mRNA or a miRNA.

[0111] The genetically engineered mesenchymal stem cells comprise an expression vector containing an exogenous PD-L1 gene and an exogenous HGF gene. In addition to the sequences of the PD-L1 and HGF genes, the expression vector further comprises one or more control sequences to regulate the expression of the polynucleotides of the present invention. Depending on the expression vector used, manipulation of the isolated polynucleotide may be desirable or necessary prior to its insertion into the vector. Techniques for modifying polynucleotides and nucleic acid sequences using recombinant DNA methods are well known in the art. In some embodiments, control sequences specifically include promoters, leader sequences, polyadenylation sequences, propeptide sequences, signal peptide sequences, and transcription terminators. In some embodiments, an appropriate promoter is selected based on the host cell of choice. In some embodiments, the exogenous PD-L1 gene and the exogenous HGF gene are present on the same expression vector. In some embodiments, the exogenous PD-L1 gene and the exogenous HGF gene are present on different expression vectors.

[0112] Step 120 is a culturing step, in which the genetically engineered mesenchymal stem cells are cultured in a culture medium to obtain a conditioned medium. The conditioned medium is the supernatant of the cell culture. Furthermore, the genetically engineered mesenchymal stem cells can be cultured under hypoxic conditions. Hypoxic conditions can be adjusted depending on the cell culture process. Specifically, the hypoxic conditions can include an oxygen content of less than 10%, less than 5%, 1% to 5%, 2% to 10%, 2% to 8%, 2% to 5%, or 2% to 4%. In some embodiments, the genetically engineered mesenchymal stem cells of the present invention can be cultured under hypoxic conditions of 3% oxygen.

[0113] Step 130 is a separation step, in which exosomes are collected from the conditioned medium using a separation method. The separation method may include conventional exosome separation methods, such as ultracentrifugation, polymer precipitation, size exclusion chromatography, and tangential flow filtration (TFF).

[0114] The present invention also provides a pharmaceutical composition for treating tissue ischemia, wherein the pharmaceutical composition comprises the aforementioned exosomes and a pharmaceutically acceptable carrier.

[0115] The pharmaceutical compositions of the present invention include an effective amount of exosomes to treat tissue ischemia. The pharmaceutical compositions of the present invention may be in liquid or lyophilized form and may contain pharmaceutically acceptable excipients to stabilize the pharmaceutical compositions of the present invention. For administration to mammals, the pharmaceutical compositions of the present invention may contain exosomes suspended in a pharmaceutically acceptable carrier, such as buffered saline and other pharmaceutically acceptable saline solutions.

[0116] The route of administration of the pharmaceutical composition comprising the exosomes of the present invention depends on the tissue or organ in need of treatment. In some embodiments, for subjects with myocardial infarction, the route of administration of the exosomes may be intravenous, carotid, intraarterial, or a combination thereof. For subjects with stroke or acute myocardial infarction (AMI), the route of administration may be a combination of carotid and intravenous injections. Furthermore, the route of administration also includes administering the exosomes of the present invention to a subject in need of such treatment via an intra-arterial route combined with a venous route. Preferably, the intraarterial injection is via the carotid artery.

[0117] The present invention also provides a use of the aforementioned exosomes for preparing a medicament for treating ischemic conditions in tissues. "Ischemic conditions" refer to conditions caused by or associated with ischemic diseases, which are generally characterized by reduced blood flow to tissues or organs due to poor vascular conditions, such as vascular stenosis or aneurysm rupture. Myocardial infarction (MI), ischemic stroke, and critical limb ischemia are the three most common ischemic diseases. The tissues may be the brain and the heart. When the tissue is the brain, the ischemic condition may be an ischemic stroke, and when the tissue is the heart, the ischemic condition may be a myocardial infarction.

[0118] Ischemic diseases include, but are not limited to, ischemic heart disease, myocardial ischemia, stroke, myocardial infarction (MI), ischemic renal disease (IRD), and renal failure (e.g., acute renal failure). Preferably, MI is AMI. Administration of the present disclosure also includes reducing inflammation in ischemic tissue.

[0119] The present invention also provides a use of the aforementioned exosomes for preparing a drug for enhancing nerve regeneration or reducing neuronal death. In addition, the exosomes of the present invention can also be used to prepare a drug for reducing inflammatory response.

[0120] The exosomes of the present invention can be administered together with another active agent, for example, the exosomes and the other active agent can be administered simultaneously, separately or concurrently, or the exosomes and the other active agent can be administered periodically.

[0121] It will be appreciated that, if any prior art publication is referred to herein, this reference does not constitute an admission that the publication forms part of the common general knowledge in the technical field.

[0122] The present invention is further illustrated by the following specific experimental examples, which are intended to facilitate those with common knowledge in the technical field to which the present invention relates, so that they can fully utilize and practice the present invention without excessive interpretation. These experimental examples should not be construed as limiting the scope of the present invention, but are intended to illustrate how to implement the materials and methods of the present invention.

[0123] Part 1: Exosomes of the present invention

[0124] 1.1 Construction of the genetically engineered mesenchymal stem cells of the present invention

[0125] 1.1.1 Genetically engineered mesenchymal stem cells derived from adipose tissue

[0126] In this experimental example, the genetically engineered mesenchymal stem cells of the present invention were first constructed. The adipose tissue-derived mesenchymal stem cells used in the experiment were human telomerase reverse transcriptase immortalized adipose tissue-derived mesenchymal stem cells (hTERT-ADSCs) cell lines, which were purchased from ATCC (CRL-2266). The hTERT-ADSCs cell line analysis certificate (COA) provided by ATCC showed that the immortalized cells still had the characteristics of mesenchymal stem cells, including high expression of CD73, CD90 and CD105, and lacked expression of CD45, CD34, CD19, CD11b and HLA-DR. hTERT-ADSCs cells were cultured in MSC containing a supplement mixture. Culture medium (Biological Industries) and grown at 37°C in a humidified atmosphere of 5% CO2. In addition, hTERT-ADSCs were able to differentiate into osteoblasts, chondrocytes, and adipocytes in vitro.

[0127] In addition, adipose stem cells (ADSCs) were used as a control group in the experiment. Human adipose tissue was collected and approved by the Institutional Review Board (IRB) to remove Ca-free 2+ and Mg 2+ After washing three times with PBS (DPBS, Life Technology), adipose tissue was mechanically cut with scissors to separate the adipose tissue from the stem cells. The adipose tissue was then cut into pieces less than 0.5 cm. 3 The explants were treated with collagenase type 1 (Sigma-Aldrich) and then incubated in MSC culture medium containing a supplement mixture (Biological Industries) and antibiotics. The cells were cultured in a humidified atmosphere of 5% CO2 at 37°C for 5-7 days to allow ADSCs to grow outward from the explants. After 4-8 passages, the ADSCs became spindle-shaped and were analyzed by flow cytometry for specific surface molecules. TMCells were separated by enzyme selection (Gibco), washed with PBS, and incubated with corresponding antibodies conjugated with fluorescein isothiocyanate (FITC) or phycoerythrin (PE), including CD13, CD29, CD44, CD73, CD90, CD105, CD166, CD49b, CD1q, CD3, CD10, CD14, CD31, CD34, CD45, CD49d, CD56, CD117, HLA-ABC, and HLA-DR (BD Biosciences). Cells were then analyzed using a Becton Dickinson flow cytometer and FlowJo v.7.6 software.

[0128] The PiggyBac transposon system was used to construct the genetically engineered mesenchymal stem cells of the present invention. The human PD-L1 (NM_014143) marker ORF clone (Cat. No. RC213071, OriGene Technologies) was used as a template for PCR amplification of PD-L1 cDNA. The resulting PD-L1 cDNA sequence is shown in SEQ ID NO:1. The human HGF (NM_000601) marker lentiviral ORF clone (Cat. No. RC215593L3, OriGene Technologies) was used as a template for PCR amplification of HGF cDNA. The resulting HGF cDNA sequence is shown in SEQ ID NO:2. A bicistronic expression construct was constructed using specific restriction enzyme linkers and PCR amplification to produce the PD-L1-T2A-HGF fragment (sequence shown in SEQ ID NO:7), with the T2A encoding sequence shown in SEQ ID NO:3. The PD-L1-T2A-HGF fragment was then sub-replicated with NotI into the PB-CMV-MCS-EF1α-Puro PiggyBac cDNA clone and expression vector (Cat. No. PB510B-1, System Biosciences) to construct the PB510B-PD-L1-HGF expression construct (hereinafter referred to as PB510B-PD-L1-HGF). pPB-CMV-MCS-EF1α-Puro contains a multiple cloning site (MCS) driven by human EF1α, PiggyBac terminal repeats (PB-TRs), core insulators (CIs), and a puromycin selection marker.

[0129] To obtain stable genetically engineered mesenchymal stem cells, Amaxa Nucleofector TMPB510B-PD-L1-HGF and PiggyBac transposase (System Bioscience) were co-transfected into hTERT-ADSCs using a 2b device (Lonza), and stable adipose tissue-derived genetically engineered mesenchymal stem cells (hereinafter referred to as hTERT-ADSC-PD-L1-HGF) were selected by puromycin.

[0130] First, to verify that genetic manipulation would not affect the stemness of genetically engineered mesenchymal stem cells, ADSCs, hTERT-ADSCs, and hTERT-ADSC-PD-L1-HGF were subjected to in vitro differentiation assays including adipogenesis, chondrogenesis, and osteogenesis.

[0131] In the in vitro adipogenic differentiation assay, confluent monolayer cultures of ADSCs, hTERT-ADSCs, and hTERT-ADSC-PD-L1-HGF were grown in adipogenic medium consisting of StemPro Adipogenic Differentiation Basal Medium (A10410-01, Gibco), supplements (A10065-01, Gibco), 15% rabbit serum, and 100 mg / mL penicillin-streptomycin (Sigma-Aldrich). Cells in culture medium served as negative controls. Adipogenesis medium was changed three times a week until 7-14 days. To assess adipogenesis, cells in each group were stained with 0.3% Oil Red staining solution (Sigma-Aldrich) at room temperature for 30 minutes and protected from light to mark intracellular lipid accumulation.

[0132] In the in vitro differentiation assay for chondrogenesis, high density (2 × 10 6 ADSCs, hTERT-ADSCs, and hTERT-ADSC-PD-L1-HGF were induced to differentiate using a 100 μL / 100 μL cell suspension culture system. The cells were then washed with chondrogenic medium consisting of osteocyte / chondrocyte differentiation basal medium (A10069-01, Gibco), supplements (A10064-01), and 100 mg / mL penicillin-streptomycin (Sigma-Aldrich). The chondrogenic medium was changed twice weekly for 14-21 days. Chondrogenesis in pellet cultures was confirmed histologically using Alcian blue staining for sulfated proteoglycans (Sigma-Aldrich) at room temperature overnight in the dark.

[0133] In the in vitro differentiation assay for osteogenesis, confluent monolayer cultures of ADSCs, hTERT-ADSCs, and hTERT-ADSC-PD-L1-HGF were grown in osteogenic medium consisting of osteocyte / chondrocyte differentiation basal medium (A10069-01, Gibco), supplements (A10066-01), and 100 mg / mL penicillin-streptomycin (Sigma-Aldrich). Cells in culture medium served as a negative control. Osteogenesis medium was replaced three times a week until day 21-28. Bone formation and calcium mineralization were determined using 2% Alizarin Red S staining solution (Sigma-Aldrich) at room temperature in the dark for 45 minutes.

[0134] Please refer to Figure 2A, which is a micrograph of ADSCs, hTERT-ADSCs, and hTERT-ADSC-PD-L1-HGF differentiated into different tissue cells. The results in Figure 2A show that the differentiation potential of hTERT-ADSC-PD-L1-HGF into adipocytes, chondrocytes, and osteoblasts is the same as that of the parental hTERT-ADSCs and primary ADSCs.

[0135] To verify the successful electroporation of PB510B-PD-L1-HGF into hTERT-ADSCs, PD-L1 expression and / or HGF content in hTERT-ADSCs and hTERT-ADSC-PD-L1-HGF were analyzed by Western blotting, enzyme-linked immunosorbent assay (ELISA), and flow cytometry. The primary antibodies used were PD-L1 (OriGene), HGF (Millipore), and actin (Millipore), with actin serving as an internal control. Figures 2B to 2D show the analysis results of PD-L1 expression and / or HGF content in hTERT-ADSCs and hTERT-ADSC-PD-L1-HGF. Figure 2B shows the Western blotting analysis results, Figure 2C shows the ELISA analysis results, and Figure 2D shows the flow cytometry analysis results. The results of Figure 2B show that PD-L1 and HGF are significantly overexpressed in hTERT-ADSC-PD-L1-HGF compared to hTERT-ADSCs. The results of Figure 2C show that the HGF content in hTERT-ADSC-PD-L1-HGF is approximately 1.5 times higher than that in hTERT-ADSCs. The results of Figure 2D show that PD-L1 expression on the cell surface of hTERT-ADSC-PD-L1-HGF is also upregulated (approximately 85%) compared to hTERT-ADSCs.

[0136] 1.1.2 Genetically Engineered Umbilical Cord-Derived Mesenchymal Stem Cells

[0137] In this study, umbilical cord-derived mesenchymal stem cells were used to construct the genetically engineered mesenchymal stem cells of the present invention. Human umbilical cord tissue was collected and approved by the Human Research Ethics Committee (IRB). 2+ and Mg 2+ After washing three times with DPBS (Life Technology), the umbilical artery, vein, and adventitial vessels were separated from Wharton's jelly (WJ) by cutting with scissors in the midline direction. The interstitial tissue of Wharton's jelly was then cut into pieces less than 0.5 cm 3 The explants were treated with collagenase type 1 (Sigma-Aldrich) and incubated at 37°C in 5% CO2 for 3 hours. Supplement blend (Sartorius), 5% UltraGRO TM -Advanced-PURE cell culture supplement (AventaCell Biomedical) and antibiotics for MSCs The cells were cultured in a 5% CO2 atmosphere at 37°C for 5-7 days to allow umbilical cord mesenchymal stem cells (UMSCs) to grow outward from the explants. The morphology of UMSCs changed to a uniform spindle shape after 4-8 generations of culture, and specific surface molecules of UMSCs were analyzed by flow cytometry. TM Cells were detached with selection enzyme (Gibco), washed with PBS, and incubated with corresponding antibodies conjugated with FITC or PE, including CD13, CD29, CD44, CD73, CD90, CD105, CD166, CD49b, CD1d, CD3, CD10, CD14, CD31, CD34, CD45, CD49d, CD56, CD117, HLA-ABC, and HLA-DR (BD Biosciences). Cells were analyzed using a Becton Dickinson flow cytometer and FlowJo v.7.6 software.

[0138] To obtain stable genetically engineered mesenchymal stem cells, Amaxa Nucleofector TM The PB510B-PD-L1-HGF described in the previous paragraph was co-transfected with PiggyBac transposase (System Bioscience) into UMSCs using a 2b device (Lonza), and stable umbilical cord-derived genetically engineered mesenchymal stem cells (hereinafter referred to as UMSC-PD-L1-HGF) were selected by puromycin.

[0139] 1.2 Preparation and Isolation of Exosomes Derived from Genetically Engineered Mesenchymal Stem Cells

[0140] To determine the optimal conditions for exosome production, hTERT-ADSC-PD-L1-HGF was cultured under normoxic conditions (20% O₂) or hypoxic conditions (3% O₂) for 72 hours. Please refer to Figure 3A for the analysis of EXO-PD-L1-HGF production when hTERT-ADSC-PD-L1-HGF was cultured under normoxic and hypoxic conditions. The results in Figure 3A show that exosome production (EXO-PD-L1-HGF) was higher under hypoxic conditions (3% O₂) than under normoxic conditions. Therefore, all subsequent experiments used hypoxic conditions to produce the exosomes of the present invention.

[0141] Furthermore, the exosomes of the present invention (hereinafter referred to as EXO-PD-L1-HGF) were prepared using hTERT-ADSCs, and the control exosomes (hereinafter referred to as EXO) were prepared using hTERT-ADSCs. hTERT-ADSCs and hTERT-ADSC-PD-L1-HGF were respectively cultured in a conventional culture medium (MSC culture medium containing penicillin-streptomycin). Culture medium) at 75cm 2 Cultured in tissue culture flasks. To prepare EXO and EXO-PD-L1-HGF, the regular culture medium of hTERT-ADSCs and hTERT-ADSC-PD-L1-HGF was replaced with fresh DMEM medium without serum, and cultured under hypoxic conditions (3% O2) for 72 hours to obtain conditioned medium. The conditioned medium was then collected, first centrifuged at 500×g for 10 minutes, and then further centrifuged at 3,000×g for 20 minutes to eliminate cells and debris, and then separated by polymer precipitation or tangential flow filtration. In the polymer precipitation method, the centrifugal supernatant was centrifuged at 5,000×g for 60 minutes at 4°C using an Amicon ultra-15 centrifugal filter 3K device (Millipore), and the centrifugal supernatant was filtered using a 0.22 μm pore size filter to obtain a concentrated supernatant. The concentrated supernatant was mixed with a total exosome isolation reagent (Invitrogen) and incubated overnight at 4°C. The supernatant was then centrifuged at 10,000 × g for 1 hour at 4°C to recover EXO and EXO-PD-L1-HGF. The supernatant was then filtered through a 300 kDa MWCO hollow fiber filter and concentrated 10-fold. The final product was sterile filtered through a 0.22 μm pore size filter to recover EXO and EXO-PD-L1-HGF. The resulting exosomes can be used fresh or resuspended in sterile 1× PBS or RIPA buffer and stored at -80°C. If stored at -80°C, mix the exosomes with a freeze-drying protective agent, freeze them at -80°C under atmospheric pressure for 6-10 hours, and then vacuum dry them at -50°C for 21 hours. The resulting freeze-dried exosomes can be stored at -80°C.

[0142] The morphology and particle size of EXO and EXO-PD-L1-HGF were analyzed using transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA). Please refer to Figures 3B and 3C. Figure 3B shows TEM images of EXO and EXO-PD-L1-HGF, and Figure 3C shows the NTA results of EXO and EXO-PD-L1-HGF. The results in Figure 3B show that the morphology of EXO and EXO-PD-L1-HGF is uniform, round membrane vesicles, while the results in Figure 3C show that the particle size distribution of EXO and EXO-PD-L1-HGF ranges from 30 nm to 200 nm, with a predominant distribution between 100 nm and 150 nm.

[0143] EXO and EXO-PD-L1-HGF were then analyzed for HGF, PD-L1, CD63, CD9, and / or CD81 expression using Western blotting, ELISA, and flow cytometry. The primary antibodies used were PD-L1 (OriGene), HGF (Millipore), CD63 (Invitrogen), CD9 (ABclonal), and CD81 (ABclonal), respectively.

[0144] Please refer to Figures 3D to 3I , which show the analysis results of HGF content, PD-L1 expression, CD63 expression, CD9 expression, and / or CD81 expression in EXO and EXO-PD-L1-HGF. Figure 3D shows the analysis results by Western blot, Figure 3E shows the analysis results by ELISA, and Figures 3F to 3I show the flow cytometry analysis results for PD-L1, CD63, CD9, and CD81, respectively. The results in Figure 3D show that both EXO and EXO-PD-L1-HGF express the exosomal markers CD9, CD63, and CD81, confirming that they are exosomes. Furthermore, the PD-L1 expression and HGF content in EXO-PD-L1-HGF are higher than those in EXO. The results in Figure 3E show that the HGF content in EXO-PD-L1-HGF is significantly higher than that in EXO, consistent with the results in Figure 3D . The results in Figures 3F to 3I show that the expression levels of PD-L1, CD9, CD63, and CD81 in EXO-PD-L1-HGF were significantly higher than those in EXO, which is consistent with the results in Figure 3D.

[0145] In addition, the exosomes of the present invention (hereinafter referred to as UMSC-EXO-PD-L1-HGF) were prepared using UMSC-PD-L1-HGF, and the control exosomes (hereinafter referred to as UMSC-EXO) were prepared using UMSC. UMSCs and UMSC-PD-L1-HGF were cultured in conventional culture medium (MSC culture medium containing penicillin-streptomycin). Culture medium) at 75cm 2 To prepare UMSC-EXO and UMSC-EXO-PD-L1-HGF, the conventional culture medium of UMSCs and UMSC-PD-L1-HGF was replaced with fresh serum-free DMEM medium and cultured under hypoxic conditions (3% O2) for 72 hours to obtain conditioned medium. The conditioned medium was then collected and centrifuged at 500 × g for 10 minutes and then at 3,000 × g for 20 minutes to eliminate cells and debris. Separation steps were then performed using polymer precipitation or tangential flow filtration to obtain UMSC-EXO and UMSC-EXO-PD-L1-HGF.

[0146] The morphology and particle size of UMSC-EXO and UMSC-EXO-PD-L1-HGF were analyzed experimentally by TEM and NTA. Please refer to Figure 3J and Figure 3K, which are TEM photos and NTA results of UMSC-EXO and UMSC-EXO-PD-L1-HGF, respectively. The results of Figure 3J show that the morphology of UMSC-EXO and UMSC-EXO-PD-L1-HGF is uniform round membrane vesicles, while the results of Figure 3K show that the particle size distribution of UMSC-EXO and UMSC-EXO-PD-L1-HGF is between 30nm and 200nm, mainly distributed between 100nm and 150nm. The number of UMSC-EXO in Figure 3K is approximately 9.3×10 11 The number of particles / mL of UMSC-EXO-PD-L1-HGF is approximately 1.5×10 12 particles / mL.

[0147] Western blotting, ELISA, and flow cytometry were used to analyze HGF content, PD-L1 expression, CD63 expression, CD9 expression, and / or CD81 expression in UMSC-EXO and UMSC-EXO-PD-L1-HGF. The primary antibodies used were PD-L1 (OriGene), HGF (Millipore), CD63 (Invitrogen), CD9 (ABclonal), and CD81 (ABclonal), respectively.

[0148] Please refer to Figures 3L to 3N , which show the analysis results of HGF content, PD-L1 expression, CD63 expression, CD9 expression, and / or CD81 expression in UMC-EXO and UMC-EXO-PD-L1-HGF. Figure 3L shows the results of Western blot analysis, Figure 3M shows the results of ELISA analysis of HGF content, and Figure 3N shows the results of flow cytometry analysis of PD-L1 expression. The results in Figure 3L show that both UMC-EXO-PD-L1-HGF and UMC-EXO express the exosomal markers CD9, CD63, and CD81, confirming that they are exosomes. Furthermore, the PD-L1 expression and HGF content in UMC-EXO-PD-L1-HGF are higher than those in UMC-EXO. The results in Figure 3M show that the HGF content in UMC-EXO-PD-L1-HGF is significantly higher than that in UMC-EXO, consistent with the results in Figure 3L . The results in Figure 3N show that the expression level of PD-L1 in UMSC-EXO-PD-L1-HGF is significantly higher than that in UMSC-EXO, which is consistent with the results in Figure 3L.

[0149] In experiments, flow cytometry can also be used to quantify multiple production batches of EXO-PD-L1-HGF or UMSC-EXO-PD-L1-HGF, measure and quantify the expression of PD-L1 surface markers, and define the proportion of therapeutic exosomes that should possess the PD-L1 surface marker.

[0150] Experimentally, ELISA analysis can be used to quantify multiple production batches of EXO-PD-L1-HGF or UMSC-EXO-PD-L1-HGF to measure and quantify the HGF content in exosomes, defining the HGF content of exosomes that are therapeutically effective.

[0151] Part II: Regulatory Mechanism of Exosomes of the Present Invention

[0152] 2.1 The exosomes of the present invention significantly attenuate hydrogen peroxide (H2O2)-induced neuronal apoptosis in vitro

[0153] To investigate whether EXO-PD-L1-HGF attenuates H2O2-induced cell death, the cells tested in the experiment were primary cortical cells (PCCs) and human SH-SY5Y cells (hereinafter referred to as SH-SY5Y). PCCs were prepared from C57BL / 6JNarl mice on embryonic day 18 (E18), and the isolated cerebral cortex was collected, separated by mechanical grinding, and suspended in growth medium (composed of Neurobasal medium, 2% B-27 supplement, 0.5mM L-glutamine, and 25μM glutamine). Cells were counted using the trypan blue exclusion method and then counted at 3×10 4 Cells were plated at a density of 10 cells / 24 well (cortex) in poly-d-lysine-coated 24-well or 6-well plates (Nunc) or 2-3 × 10 6 Cells / 6 wells. The culture was maintained in a humidified incubator at 37°C and 5% CO2. After 3 days in vitro (DIV 3), half of the growth medium was removed and replaced with maintenance medium (composed of Neurobasal medium, 2% B-27 supplement, and 0.5 mM L-glutamine). The maintenance medium was replaced in the same manner every 3-4 days. More than 95% of the cells in the culture produced by the above method were neurons. SH-SY5Y cells were cultured in DMEM / F12 (1:1, v:v) medium (Gibco) containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin and grown at 37°C in a humidified atmosphere of 5% CO2.

[0154] The experiment first examined exosome uptake by PCCs in vitro using immunofluorescence staining. EXO and EXO-PD-L1-HGF were labeled with 1 mg / mL DiO green dye (Invitrogen) at room temperature for 1 hour and reprecipitated overnight at 4°C using a total exosome isolation reagent. The exosomes were then centrifuged at 10,000 × g for 60 minutes at 4°C, and the fluorescently labeled exosomes (hereinafter referred to as DiO-EXO and DiO-EXO-PD-L1-HGF) were resuspended in sterile 1× PBS. Cell nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI). After incubating DiO-EXO and DiO-EXO-PD-L1-HGF-labeled cells with DiD (Invitrogen) at 37°C for 1 hour, images of exosome uptake were captured using a Leica SP2 conjugate spectral microscope. Please refer to Figure 4A, which shows the analysis results of PCCs uptake of DiO-EXO and DiO-EXO-PD-L1-HGF. The results in Figure 4A show that the white dots of DiO-EXO and DiO-EXO-PD-L1-HGF are internalized into MAP2 + in the cytoplasm of PCCs.

[0155] When the H2O2-induced cell death assay was performed, PCCs or SH-SY5Y cells were cultured at a cell density of 3 × 10 5 cells / 24-well plate or 1×10 5 Cells were seeded in 24-well plates and cultured overnight. At 24 hours, cells were treated with different concentrations of H2O2 (0 μM, 100 μM, 200 μM, 300 μM, 400 μM, and 500 μM) at 37°C and 5% CO2 for 3 hours. Cell viability was then determined by the CCK-8 method to determine the optimal concentration and duration of H2O2 treatment to induce toxic effects on PCCs and SH-SY5Y.

[0156] The experimental results found that H2O2 treatment significantly induced cell death in PCCs and SH-SY5Y in a concentration-dependent manner, and the optimal treatment dose was 300 μM (data not shown). Therefore, subsequent experiments pretreated PCCs or SH-SY5Y with 20 μg of EXO or EXO-PD-L1-HGF for 24 hours, and then treated PCCs or SH-SY5Y with 300 μM H2O2 for 3 hours. Cell viability was then determined using the CCK-8 method to investigate whether the exosomes of the present invention could exert a neuroprotective effect against H2O2-induced cell death, and TUNEL staining was used to detect cell apoptosis.

[0157] Please refer to Figures 4B to 4L. Figures 4B and 4C are the analysis results of EXO-PD-L1-HGF reducing H2O2-induced PCCs apoptosis in vitro by TUNEL staining, wherein Figure 4C is the statistical result of Figure 4B; Figures 4D and 4E are the analysis results of EXO-PD-L1-HGF reducing H2O2-induced SH-SY5Y apoptosis in vitro by TUNEL staining, wherein Figure 4E is the statistical result of Figure 4D; Figures 4F and 4G are the analysis results of EXO-PD-L1-HGF reducing H2O2-induced PCCs and SH-SY5Y cell death in vitro by CCK-8 assay; Figures 4H and 4I are the analysis results of Annexin V assay. Figure 4I is a statistical result diagram of Figure 4H ; Figures 4J and 4K are analysis results of the Annexin V-FITC / 7-AAD double staining method for detecting EXO-PD-L1-HGF to reduce H2O2-induced SH-SY5Y cell apoptosis in vitro, wherein Figure 4K is a statistical result diagram of Figure 4J ; Figure 4L is a Western blot analysis of the expression of apoptosis-related proteins in H2O2-induced PCCs and SH-SY5Y cell apoptosis after pretreatment with EXO or EXO-PD-L1-HGF. In Figures 4B to 4L , the control group represents the untreated group, PBS represents the group pretreated with PBS followed by H₂O₂, EXO represents the group pretreated with EXO followed by H₂O₂, and EXO-PD-L1-HGF represents the group pretreated with EXO-PD-L1-HGF followed by H₂O₂. Data in the above statistical figures are expressed as mean ± SD, with * indicating p < 0.05, ** indicating p < 0.01, and *** indicating p < 0.001.

[0158] The results of Figures 4B to 4E show that, in both PCCs and SH-SY5Y, pretreatment with EXO-PD-L1-HGF overnight significantly reduced the rate of H2O2-induced cell apoptosis compared with the EXO and PBS groups (white dots indicate TUNEL-positive cells). The CCK-8 assay results in Figures 4F and 4G show that pretreatment with 20 μg of EXO-PD-L1-HGF further attenuated H2O2-induced cell death compared with the EXO and PBS groups, with the cell viability of PCCs increasing to 80% and that of SH-SY5Y increasing to 85%. The results of Figures 4H to 4K show that pretreatment with EXO-PD-L1-HGF significantly reduced cell apoptosis after exposure to H2O2 compared with the EXO and PBS groups. In addition, the results of Figure 4L showed that compared with the EXO group and PBS group, after 24 hours of pretreatment with EXO-PD-L1-HGF, the expression levels of pro-apoptotic proteins Bax and Cleaved Caspase-3 in PCCs and SH-SY5Y were significantly reduced, and the expression level of anti-apoptotic protein Bcl-2 was increased to the same level as the observation control group without adding H2O2.

[0159] In addition, this study also tested whether UMSC-EXO-PD-L1-HGF could also attenuate H2O2-induced cell death. 5 Cells were seeded in 24-well plates and cultured overnight. At 24 hours, the cells were treated with different concentrations of H2O2 (0 μM, 100 μM, 200 μM, 300 μM, 400 μM, and 500 μM) for 3 hours at 37°C and 5% CO2, and then the cell viability was determined by the CCK-8 method. The test results showed that the optimal treatment dose was 200 μM (data not shown), so a H2O2 treatment dose of 200 μM was selected for subsequent experiments. In order to study whether the exosomes of the present invention can exert a neuroprotective effect under H2O2-induced cell death, PCCs were pretreated with 20 μg of UMSC-EXO or UMSC-EXO-PD-L1-HGF for 24 hours, and then treated with 200 μM H2O2 for 3 hours. Cell viability was determined by the CCK-8 method, and cell apoptosis was detected by TUNEL staining.

[0160] Please refer to Figures 4M and 4N. Figure 4M shows the CCK-8 assay to assess the effect of UMSC-EXO-PD-L1-HGF on H2O2-induced cell death in vitro. Data are presented as mean ± SD, with * indicating p < 0.05 and ** indicating p < 0.01. Figure 4N shows the Western blot analysis of the expression of apoptosis-related proteins in H2O2-induced apoptosis in PCCs following pretreatment with UMSC-EXO-PD-L1-HGF.

[0161] The results of Figure 4M show that compared with the UMSC-EXO group and the PBS group, pretreatment with 20 μg of UMSC-EXO-PD-L1-HGF can further attenuate H2O2-induced cell death, among which the cell survival rate of PCCs can be increased to 80%. The results of Figure 4N show that compared with the UMSC-EXO group and the PBS group, after 24 hours of pretreatment with UMSC-EXO-PD-L1-HGF, the expression levels of pro-apoptotic proteins Bax and Cleaved Caspase-3 in PCCs were significantly reduced, and the expression level of anti-apoptotic protein Bcl-2 was significantly increased. The above results show that the exosomes of the present invention have neuroprotective ability against H2O2-induced cytotoxicity.

[0162] 2.2 Exosomes of the present invention can inhibit cytotoxic T cells and promote the activation of regulatory T cells

[0163] To verify whether EXO-PD-L1-HGF affects T cell proliferation in vitro, proliferating T cells stimulated with CD3 / CD28 antibodies were stained using a CFSE cell proliferation kit (Invitrogen). The cells were then treated with either EXO or EXO-PD-L1-HGF for 3 days, and the fluorescence intensity of the CFSE-labeled cells was measured by flow cytometry. Figure 5A shows the results of the CFSE assay analyzing the inhibition of T cell proliferation by EXO-PD-L1-HGF. The unstimulated group represents the untreated group, the CD3 / CD28Ab group represents the group stimulated with CD3 / CD28 antibodies alone (hereinafter referred to as the vehicle control group), the CD3 / CD28Ab+EXO group represents the group stimulated with CD3 / CD28 antibodies and then treated with EXO (hereinafter referred to as the EXO group), and the CD3 / CD28Ab+EXO-PD-L1-HGF group represents the group stimulated with CD3 / CD28 antibodies and then treated with EXO-PD-L1-HGF (hereinafter referred to as the EXO-PD-L1-HGF group). The results of Figure 5A showed that the percentage of T cell proliferation in the EXO-PD-L1-HGF group was significantly decreased compared with the other groups.

[0164] In order to evaluate the activation of T cells, the expression of pro-inflammatory cytokine interferon-γ (IFN-γ) in each group was detected by ELISA. Please refer to Figure 5B, which shows the result of ELISA analysis of EXO-PD-L1-HGF to reduce IFN-γ expression. The results of Figure 5B show that the expression of IFN-γ in T cells in the EXO-PD-L1-HGF group was significantly reduced. In addition, the expression of regulatory T cells (CD8 + CD122+ IL-10 + ). Referring to Figure 5C , the analysis results for the percentage of regulatory T cells after treatment with EXO or EXO-PD-L1-HGF show that the number of regulatory T cells was also upregulated in the EXO-PD-L1-HGF-treated group. These results demonstrate that the administration of the exosomes of the present invention can exert an immunosuppressive effect.

[0165] 2.3 The exosomes of the present invention can promote the proliferation of primary NPCs in vitro

[0166] To validate the potential of EXO-PD-L1-HGF to stimulate NPC proliferation, NPCs were isolated from the cerebral cortex of embryonic day 17 (E17) C57BL / 6JNarl mice by aseptically dissociating and dissociating the collected cerebral cortex. Neurosphere cultures were then prepared in Neurobasal medium (Gibco BRL) supplemented with 2% B-27 (Gibco BRL), 0.5 mM L-glutamine (PAN-Biotech), and 100 U / mL penicillin / 0.1 mg / L streptomycin (Gibco BRL). To maintain and expand the neurosphere cultures, 20 ng / mL bFGF (Gibco BRL) and 20 ng / mL epidermal growth factor (EGF) (Gibco BRL) were further added to the Neurobasal medium. Neurosphere cultures were maintained in a humidified incubator at 37°C and 5% CO2. To establish clonal propagation lines and low-density cultures, primary neurospheres grown at high density were dissociated into single-cell suspensions and plated at 1 × 10 5 Cells were reseeded at a density of 10 cells / mL in 6-well plates, and newly formed secondary neurospheres were counted after 7 days. The isolated NPCs were analyzed for stem cell marker expression, such as nestin and Sox2, by immunofluorescence staining. Figure 5D shows the analysis of stem cell marker expression in isolated NPCs. The results in Figure 5D demonstrate that nestin and Sox2 protein expression was observed in all NPCs isolated using the above method.

[0167] Furthermore, NPCs were treated with PBS, EXO, and EXO-PD-L1-HGF for 72 hours, and cell viability was measured using the CCK-8 assay. Figure 5E shows the CCK-8 assay results for the effect of EXO-PD-L1-HGF on NPC proliferation. The results in Figure 5E show that compared to the PBS and EXO groups, the EXO-PD-L1-HGF group exhibited significantly higher NPC proliferation. These results demonstrate that the exosomes of the present invention can significantly enhance NPC proliferation.

[0168] 2.4 The exosomes of the present invention can positively regulate the STAT3 / FOXO3 signaling pathway involved in NPCs-induced neural regeneration

[0169] In this experimental example, we further validated the potential mechanism by which the exosomes of the present invention may contribute to the neurotrophic effects of NPCs. NPCs were treated with EXO and EXO-PD-L1-HGF for 24 hours. Total RNA was extracted using the Quick-RNA miniPrep kit (Zymo). RNA-seq-based next-generation sequencing (NGS) (hereinafter referred to as RNA-seq) was used to analyze total mRNA transcript expression in the NPCs of the control, EXO (EXO) and EXO-PD-L1-HGF groups (EXO-PD-L1-HGF) groups. RNA-seq results were provided as BAM files and converted to FASTQ format using Picard. FASTQ files were then aligned to the hg19 human reference genome using Top Hat 2.0.14. Expression values ​​were calculated using FeatureCounts v1.4.6-p2, and differential expression analysis was determined using DESeq2. Genes downregulated by RNA-seq were defined as counts > 100, fold-rank > 2, and p < 0.05. Go analysis was performed using the Gene Ontology Consortium website.

[0170] Please refer to Figure 5F, which shows a heat map of RNA-seq analysis of the control, EXO, and EXO-PD-L1-HGF groups. The results in Figure 5F show that the EXO-PD-L1-HGF group significantly upregulated multiple growth factors, chemokines, apoptosis genes, and transcription factors, including hepatocyte growth factor receptor (HGFR), vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), stromal cell-derived factor-1 (SDF-1), CXCR4, signal transducer and activator of transcription 3 (STAT3), and FOXO3 (fold change [FC] ≥ 2). Fold change [FC] ≥ 2 and p-value < 0.05 were used to further identify differentially expressed genes between the EXO-PD-L1-HGF group and the control group, and approximately 613 upregulated genes were confirmed (data not shown).

[0171] Gene Set Enrichment Analysis (GSEA) was also performed to identify biological pathways significantly altered after EXO-PD-L1-HGF treatment (p-value < 0.05). Please refer to Figure 5G for the GSEA results for the control, EXO, and EXO-PD-L1-HGF groups. The results in Figure 5G show that in the EXO-PD-L1-HGF group, multiple biological pathways related to the JAK-STAT pathway, FOXO pathway, chemokine pathway, stem cell proliferation, and stem cell population maintenance were significantly upregulated.

[0172] Furthermore, NPCs and PCCs were treated with PBS, EXO, and EXO-PD-L1-HGF for 5 hours, respectively. Cell lysates and total proteins were collected using 1× RIPA lysis buffer (Sigma-Aldrich), and Western blotting was used to detect the protein expression of phosphorylated STAT3 (hereinafter referred to as p-STAT3) and phosphorylated FOXO3 (hereinafter referred to as p-FOXO3) in NPCs and PCCs in the PBS group, EXO group, and EXO-PD-L1-HGF group. The primary antibodies used were p-STAT3 (cell signaling), STAT3 (cell signaling), p-FOXO3 (cell signaling), FOXO3 (cell signaling), and actin (Millipore), among which actin was used as an internal control. Please refer to Figure 6A, which shows the results of Western blot analysis of the protein expression of p-STAT3 and p-FOXO3 in PCCs and NPCs in the PBS group, EXO group, and EXO-PD-L1-HGF group. The results show that EXO-PD-L1-HGF treatment significantly increased the protein expression of p-STAT3 and p-FOXO3 in both PCCs and NPCs.

[0173] To systematically investigate the regulatory molecules encapsulated in the exosomes of the present invention, this experimental example further analyzed the important substances contained in EXO and EXO-PD-L1-HGF using a human cytokine array. Since mesenchymal stem cells can exert immune regulatory effects through paracrine secretion (e.g., IL-10 and TGFβ), conditioned medium from PCCs treated with EXO or EXO-PD-L1-HGF was collected, and exosomal proteins were extracted using a lysis buffer supplemented with a protease and phosphatase inhibitor cocktail. The exosomal proteins were then analyzed using a human cytokine array (R&D Systems). Please refer to Figure 6B, which shows the results of human cytokine microarray analysis of cytokine protein expression in PCCs after treatment with EXO or EXO-PD-L1-HGF. The results show that the expression levels of six cytokines in the EXO-PD-L1-HGF group were significantly higher than those in the EXO group. They are SDF-1α (circle 1), VEGF (circle 2), brain-derived neurotrophic factor (BDNF; circle 3), fibroblast growth factor (FGF; circle 4), HGF (circle 5), and leukemia inhibitory factor (LIF; circle 6). ELISA was then used to examine the expression of cytokines in the EXO and EXO-PD-L1-HGF groups. Figure 6C shows the ELISA analysis of cytokine protein expression in PCCs after treatment with either EXO or EXO-PD-L1-HGF. The results in Figure 6C confirm that the EXO-PD-L1-HGF group expressed significantly higher levels of SDF-1α, VEGF, BDNF, FGF, HGF, and LIF than the EXO group. These results suggest that the exosomes of the present invention may have the potential to inhibit harmful damage and rescue neural injury.

[0174] To further confirm the role of FOXO3 in neuroprotection and regeneration, FOXO3 shRNA (purchased from the RNAi Core Center, hereafter referred to as sh-FOXO3) and scrambled GFP shRNA (purchased from the RNAi Core Center, hereafter referred to as sh-control) were cloned into the expression vector pLKO1. The constructed plasmids and the lentiviral packaging constructs pCMV-ΔR8.91 and pMD.G were co-transfected into HEK293T cells using Lipofectamine 3000 transfection reagent (Invitrogen). After 24–48 hours, lentiviral particles containing sh-FOXO3 (LV-sh-FOXO3) and sh-control (LV-sh-control) were collected using a 0.45 mm filter and concentrated by centrifugation at 4,000 × g for 15 minutes. The concentrated virus was then centrifuged a second time at 1,000 × g for 2 minutes at room temperature. The concentrated virus can be used fresh or stored at -80°C.

[0175] In the experiment, PCCs were infected with either LV-sh-FOXO3 or LV-sh-control cells, then pretreated with EXO and EXO-PD-L1-HGF, respectively, and then treated with 300 μM H₂O₂. The expression of apoptosis-related proteins in each group of PCCs was analyzed by Western blotting and Annexin V-FITC / 7-AAD double staining. Please refer to Figure 6D for Western blotting analysis of apoptosis-related protein expression in PCCs treated with the exosomes of the present invention, which showed knockdown of FOXO3 expression. The results showed that knockdown of FOXO3 expression reduced the increased expression of p-FOXO3 and Bcl-2 proteins after treatment with EXO-PD-L1-HGF. Please refer to Figures 6E and 6F for flow cytometry analysis of apoptosis in different groups. Figure 6F shows the statistical results of Figure 6E. Data are presented as mean ± SD, with *** indicating p < 0.001. The results of Figures 6E and 6F showed that the attenuated expression of FOXO3 significantly reduced the effect of EXO-PD-L1-HGF on attenuating H2O2-induced PCCs neuronal apoptosis.

[0176] To verify the potential of EXO-PD-L1-HGF to regulate NPC proliferation through FOXO3 gene, FOXO3-NL mice (genotype FOXO3 + / + , hereinafter referred to as FOXO3-NL) and FOXO3 knockout mice (genotype FOXO3 - / - , the following is FOXO3 - / -NPCs were isolated from the cerebral cortex of embryos at day 17 (E17) of embryonic development (denoted by [element of the missing text]), and the self-renewal capacity of the isolated NPCs was further analyzed, including analysis of the number of secondary neurospheres formed by each NPC and measurement of the size of the secondary neurospheres formed. Please refer to Figures 6G to 6J. Figures 6G and 6H show the analysis results of the number of secondary neurospheres formed by NPCs in different groups, and Figure 6H shows the statistical results of Figure 6G. Figures 6I and 6J show the analysis results of the size of secondary neurospheres formed by NPCs in different groups, and Figure 6J shows the statistical results of Figure 6I. Data in Figures 6H and 6J are expressed as mean ± SD, where * indicates p < 0.05, ** indicates p < 0.01, and *** indicates p < 0.001. The results showed that the elimination of FOXO3 expression in NPCs inhibited the proliferation of NPCs promoted by EXO-PD-L1-HGF as measured by the number and size of secondary neurospheres.

[0177] Part III: Use of the exosomes and pharmaceutical compositions of the present invention in treating tissue ischemia

[0178] Based on the aforementioned knowledge that the exosomes of the present invention have the ability to protect neurons against H2O2-induced cytotoxicity, inhibit cytotoxic T cells and promote the activation of regulatory T cells, promote the proliferation of NPCs, and positively regulate the STAT3 / FOXO3 signaling pathway involved in neuroprotection and neuroregeneration, this section of the experimental examples evaluated the efficacy of the exosomes of the present invention in treating tissue ischemia in mice with ischemic stroke. Two neurobehavioral measurements were used to assess neurological behavior in mice before and after stroke to evaluate neurological recovery.

[0179] An ischemic stroke mouse model was established using an ischemia–reperfusion model to simulate the symptoms of transient focal cerebral ischemia in mice. Adult male C57BL / 6 mice weighing 25–30 g underwent bivascular ligation. All surgical procedures, animal experimental protocols, and methods were performed in accordance with institutional guidelines and approved by the Institutional Animal and Clinical Research Committee. Mice were anesthetized with chloral hydrate (0.4 g / kg, intraperitoneal injection), and the right middle cerebral artery (MCA) and bilateral common carotid arteries (CCAs) were ligated. The CCAs were occluded with non-invasive artery clamps. Using a surgical microscope, a 2 × 2 mm craniotomy was made at the fusion site of the zygomatic bone and squamosal bone. The right middle cerebral artery was ligated with a 10-0 nylon suture. Cortical blood flow was continuously measured in anesthetized animals using a laser Doppler flowmeter (PF-5010, Periflux Systems). A 1 mm diameter burr hole was made in the right frontoparietal region to facilitate the placement of the photodetector. The probe (0.45 mm diameter) was stereotaxically placed in the cortex (1.3 mm posterior, 2.8 mm lateral to the fontanelle, and 1.0 mm subdural). After 120 minutes of ischemia, the sutures on the MCA and the arterial clamps on the CCAs were removed to allow reperfusion to obtain ischemic stroke (middle cerebral artery occlusion, MCAO) mice (hereinafter referred to as MCAO mice). After 30 minutes of reperfusion, EXO or EXO-PD-L1-HGF (200 μg dissolved in 100 μL of PBS) or vehicle (100 μL of PBS) was injected into the right femoral vein of the experimental mice via a 26-gauge syringe. Core body temperature was monitored using a thermistor probe and maintained at 37°C with a heating pad during anesthesia. After recovery from anesthesia, the body temperature of the MCAO mice was maintained at 37°C with a heating lamp.

[0180] 3.1 Exosomes of the present invention significantly migrate into the ischemic brain

[0181] To examine the biodistribution and migration of EXO and EXO-PD-L1-HGF in vivo, the lipophilic dye DiD (Invitrogen) was used to label the surface of EXO and EXO-PD-L1-HGF. 50 μg of exosomes (EXO or EXO-PD-L1-HGF) were incubated with DiD at 37°C for 1 hour. After staining, free dye was removed by gel filtration using an exosome spin column. MCAO mice were randomly divided into groups and injected intravenously with fluorescently labeled exosomes (DiD-EXO or DiD-EXO-PD-L1-HGF) at a dose of 1 mg / kg per mouse. Analyses were performed 4, 24, and 72 hours after injection. Mice were anesthetized with 2% isoflurane in oxygen, and fluorescence images (excitation wavelength: 745 nm, emission wavelength: 830 nm) were acquired using an IVIS in vivo imaging system with Living Image 3.0 software (Xenogen Corp.). Mice were sacrificed 72 hours later. Relative quantification of fluorescence signals was performed using the ROI tool.

[0182] Please refer to Figures 7A and 7B for fluorescence intensity analysis results after intravenous injection of DiD-EXO or DiD-EXO-PD-L1-HGF into MCAO mice. Figure 7B is a statistical graph of Figure 7A. Data are presented as mean ± SD, where *** indicates p < 0.001. The results in Figures 7A and 7B show that the fluorescence signals of DiD-EXO and DiD-EXO-PD-L1-HGF could be detected by the IVIS in vivo imaging system 4 hours after injection. The fluorescence intensity observed at the stroke site increased significantly with detection time. Compared with the EXO and PBS groups, the fluorescence signal in the stroke brain of the EXO-PD-L1-HGF group was significantly enhanced in a time-dependent manner.

[0183] In the experiment, the brain tissue of MCAO mice was stained with immunofluorescence to detect the ischemic injury site. Please refer to Figure 7C for the immunofluorescence staining results of the co-localization of the exosomes of the present invention with SDF-1α expressed in the injury site. The results of Figure 7C show that in the EXO-PD-L1-HGF group, SDF-1α co-localized with DiD-EXO-PD-L1-HGF + The number of cells in the EXO group was higher than that in the EXO group, indicating that the ability of EXO-PD-L1-HGF to home to the ischemic injury site may be related to the SDF-1α expressed in the injury site after stroke.

[0184] Previous studies have shown that SDF-1 / CXCR4 plays an important role in guiding mesenchymal stem cells to home to sites of injury. To verify whether EXO-PD-L1-HGF indeed enhances homing by increasing CXCR4 expression, the experiment further analyzed the expression of CXCR4 on the exosome membranes of EXO and EXO-PD-L1-HGF using flow cytometry. Please refer to Figure 7D for the analysis of CXCR4 expression on the exosome membranes of EXO and EXO-PD-L1-HGF using flow cytometry. The results show that the expression of CXCR4 on the exosome membranes of EXO-PD-L1-HGF is significantly increased compared to EXO.

[0185] In addition, please refer to Figure 7E, which shows the expression of GFAP in the injured area with EXO and EXO-PD-L1-HGF. + Cells and MAP2 + The results of immunofluorescence staining of cell co-localization. By immunofluorescence staining, it can be found that 72 hours after intravenous injection of DiD-EXO-PD-L1-HGF into MCAO mice, the fluorescence signal of DiD-EXO-PD-L1-HGF is colocalized with GFAP in the stroke brain. + or MAP2 + The above results show that the exosomes of the present invention implanted into MCAO mice can home to the ischemic injury site, accumulate in the ischemic brain, and then be internalized into glial cells to exert their regenerative effects.

[0186] The experiment can also be further analyzed by flow cytometry to measure the expression of CXCR4 on the exosome membrane of UMSC-EXO and UMSC-EXO-PD-L1-HGF, and to measure and quantify the differences in exosomes with CXCR4 surface markers between the UMSC-EXO-PD-L1-HGF group and the UMSC group.

[0187] Flow cytometry can also be used to quantify multiple production batches of EXO-PD-L1-HGF or UMSC-EXO-PD-L1-HGF, measure and quantify exosomes with the CXCR4 surface marker, and define the proportion of therapeutic exosomes that have the CXCR4 surface marker.

[0188] 3.2 Intravenous injection of the exosomes of the present invention enhances endogenous nestin-GFP + Cell-induced neurogenesis in the peri-infarct area

[0189] To further understand the endogenous nestin-GFP after each treatment + To investigate the regenerative potential of cells, we used nestin-GFP gene transgenic mice to construct MCAO mice (hereinafter referred to as GFP mice) to study the effect of nestin-GFP gene transgenic mice on the regenerative potential of cells after stroke.+ Cell migration and proliferation to the sensorimotor cortex. In the experiment, GFP mice were divided into five groups. Four of the groups were infected with LV-sh-FOXO3 or LV-sh-control GFP mice 7 days after stroke, and then 100-200μg of EXO or EXO-PD-L1-HGF was administered to each GFP mouse (approximately 25g) for treatment. Another group was injected with PBS as a control. The experiment also included a sham group (sham) of mice, and immunofluorescence staining was used for analysis. The sham group of mice underwent the same surgical procedures as the GFP mice, but the left anterior descending coronary artery of the mice was not occluded in the sham group.

[0190] Please refer to Figure 7F and Figure 7G, which show the immunofluorescence staining results of the inhibition of EXO-PD-L1-HGF to promote the migration of nestin-GFP-expressing NPCs to the injury site by reducing FOXO3 expression. The results showed that in the group without reducing FOXO3 expression, compared with the EXO group and the control group, the nestin-GFP-expressing NPCs in the EXO-PD-L1-HGF group migrated to the lateral and ventral peri-infarct areas. + Conversely, attenuating FOXO3 expression inhibited the expression of nestin-GFP promoted by EXO-PD-L1-HGF in the infarcted area. + of cell migration.

[0191] In addition, bromodeoxyuridine (BrdU, Sigma-Aldrich) and Ki-67 (Abcam) were used as cell proliferation markers to observe the expression of nestin-GFP in the brain of GFP mice after treatment with EXO and EXO-PD-L1-HGF. + Cell proliferation. Please refer to Figures 7H, 7I and 7J for the analysis results of the proliferation of cells expressing nestin-GFP induced by EXO-PD-L1-HGF. Figures 7I and 7J are the statistical results of Figure 7H. The data are expressed as mean ± SD, where * indicates p < 0.05 and *** indicates p < 0.001. The results of Figures 7H to 7J show that the application of EXO-PD-L1-HGF significantly induced GFP + BrdU + and GFP + Ki-67 + Cell proliferation.

[0192] Previous studies have shown that ischemic stroke can stimulate the proliferation of endogenous NPCs and their ectopic migration to the infarct area, and that the proliferating NPCs can functionally integrate with adult nerves to enhance stroke recovery. In order to explore whether the exosomes of the present invention promote the proliferation of endogenous NPCs to induce glial differentiation and then induce adult neurogenesis to achieve functional recovery, the experiment was conducted by immunohistochemistry to detect glial markers and proliferation markers in nestin and nestin-GFP. + Colocalization examination of populations.

[0193] Please refer to Figures 8A, 8B, 8C and 8D for the analysis results of the effects of EXO and EXO-PD-L1-HGF on the differentiation of NPCs. Figures 8B, 8C and 8D are the statistical results of Figure 8A. The data are presented as mean ± SD, where *** indicates p < 0.001. The results of Figures 8A to 8D show that, in addition to oligodendrocytes (GFP) expressing GFP in GFP mice after stroke, + NG2 + ) and stellate cells (GFP + GFAP + Nestin-GFP + Nestin-GFP can also be observed outside the cell + Importantly, at 1 week after stroke, more GFP was observed in the ventral peri-infarct area of ​​the EXO-PD-L1-HGF group compared with the EXO and control groups. + DCX + , GFP + NG2 + and GFP + GFAP + cell.

[0194] 3.3 FOXO3 regulates the activation of the exosomes of the present invention to enhance functional recovery and reduce infarct volume after stroke

[0195] In order to verify the neuroregenerative ability of EXO-PD-L1-HGF in MCAO mice through the FOXO3 signaling pathway, neurobehavioral measurements were performed on different groups of MCAO mice. The MCAO mice were sacrificed on the 28th day after treatment, and the infarct volume of MCAO mice was measured by triphenyltetrazolium chloride (TTC) staining. The infarct volume of MCAO mice in different groups was measured. The experimental groups included sham operation group, PBS group, EXO group (genotype FOXO3 + / + ), EXO-PD-L1-HGF group (genotype FOXO3 + / + ), LV-sh-FOXO3+EXO group (genotype FOXO3 - / - ) and LV-sh-FOXO3+EXO-PD-L1-HGF group (genotype FOXO3 - / - ).

[0196] Neurobehavioral measurements were performed on MCAO mice 5 days before the cerebral ischemia / reperfusion model and on days 0, 7, 14, 21, and 28 after exosome (EXO or EXO-PD-L1-HGF) treatment. Neurobehavioral measurements included a rotarod test and a balance beam test. The scores of the baseline test were recorded to normalize the scores after cerebral ischemia. (1) The rotarod test used an automatic rotating rod treadmill, which consisted of a 5-cm diameter roller that was appropriately processed to provide grip and a power source for rotating the roller. Five circular partitions divided the rod into compartments of equal size (each compartment was 5 cm long), allowing four MCAO mice to be on the treadmill at the same time. The time each MCAO mouse spent on the rotating rod (20-25 rpm) was measured by a timer. When the MCAO mouse fell from the treadmill to the plate below or stayed on the rod for up to 3 minutes, the timer would detect and record it. (2) The balance beam is a 160 cm long, 2.5 cm diameter pole. A plastic platform (7 cm × 4 cm) is placed at one end of the pole as a starting point, and a black plastic box (15 cm × 15 cm × 8 cm) is placed at the other end of the pole as a nest to encourage MCAO mice to cross the beam. The apparatus is suspended 90 cm above the mat and 50 cm from the wall to protect the animals from falling. The balance beam test records the time it takes for MCAO mice to cross the beam.

[0197] Please refer to Figures 8E and 8F. Figure 8E shows the analysis results of the rotarod test in MCAO mice treated with EXO-PD-L1-HGF, and Figure 8F shows the analysis results of the balance beam test in MCAO mice treated with EXO-PD-L1-HGF. The results in Figures 8E and 8F show that compared with the other groups, post-stroke neurological function in the EXO-PD-L1-HGF group was significantly improved 28 days after ischemic stroke, as measured by neurobehavioral measurements of the rotarod test and the balance beam test. In contrast, the EXO-PD-L1-HGF-induced neurological function improvement was significantly inhibited in the LV-sh-FOXO3 + EXO group and the LV-sh-FOXO3 + EXO-PD-L1-HGF group.

[0198] Please refer to Figures 8G and 8H for the analysis of brain infarction in MCAO mice treated with EXO-PD-L1-HGF. Figure 8H is the statistical result of Figure 8G. The data are expressed as mean ± SD, where * indicates p < 0.05, ** indicates p < 0.01, and *** indicates p < 0.001. The TTC staining results in Figures 8G and 8H show that in the FOXO3 genotype, + / + In the FOXO3-NL group, the ischemic hemisphere infarct volume in the EXO-PD-L1-HGF group was smaller than that in the EXO group and the control group, but the FOXO3 genotype - / - The group inhibited the EXO-PD-L1-HGF-induced infarct volume reduction.

[0199] The experiment also tested whether the administration of UMSC-EXO-PD-L1-HGF could also improve functional recovery and reduce infarct volume after stroke in MCAO mice. After the MCAO mice were established, they were divided into three groups. In two groups, each MCAO mouse (approximately 25g) was administered 100-200μg of UMSC-EXO or UMSC-EXO-PD-L1-HGF, and the other group was injected with PBS as a control group. MCAO mice underwent neurobehavioral measurements using the rotarod test and balance beam test 5 days before undergoing the cerebral ischemia / reperfusion model and on days 0, 7, 14, 21, and 28 after exosome (UMSC-EXO or UMSC-EXO-PD-L1-HGF) treatment. The MCAO mice were sacrificed on day 28 after treatment, and the infarct volume of the MCAO mice was measured by TTC staining.

[0200] Please refer to Figures 8I and 8J. Figure 8I shows the analysis results of the rotarod test in MCAO mice treated with UMSC-EXO-PD-L1-HGF, and Figure 8J shows the analysis results of the balance beam test in MCAO mice treated with UMSC-EXO-PD-L1-HGF. The results show that neurobehavioral measurements performed through the rotarod test and the balance beam test show that post-stroke neurological function in the UMSC-EXO-PD-L1-HGF group can be significantly improved 28 days after ischemic stroke compared with other groups. These results show that the exosomes of the present invention can significantly improve the neurobehavioral function of MCAO mice and reduce the infarct volume in MCAO mice.

[0201] Please also refer to Figures 8K and 8L for analysis of brain infarction in MCAO mice treated with UMSC-EXO-PD-L1-HGF. Figure 8L shows the statistical results of Figure 8K. Data are presented as mean ± SD, with * indicating p < 0.05. TTC staining results show that the infarct volume in the ischemic hemisphere in the UMSC-EXO-PD-L1-HGF group was smaller than that in the UMSC-EXO and control groups. These results demonstrate that the exosomes of the present invention can modulate neuroplasticity in the brain after stroke.

[0202] Furthermore, TUNEL staining was used to examine apoptosis in brain tissue of MCAO mice treated with EXO-PD-L1-HGF. Figures 8M and 8N show the TUNEL staining analysis results, with Figure 8N showing the statistical results of Figure 8M. Data are presented as mean ± SD, with * indicating p < 0.05. The results showed that the number of TUNEL-positive cells in the ischemic hemisphere was significantly reduced in the EXO-PD-L1-HGF group, while treatment with LV-sh-FOXO3 abolished the EXO-PD-L1-HGF-induced decrease in TUNEL-positive cells in the ischemic hemisphere. These results suggest that FOXO3 activation triggers EXO-PD-L1-HGF-mediated neuroplasticity in the stroke-affected brain.

[0203] 3.4 Exosomes of the present invention can control immune regulation

[0204] To demonstrate that the exosomes of the present invention can control immune regulation, flow cytometry was used to examine the percentage of white blood cell populations in the brain and spleen of MCAO mice treated with EXO and EXO-PD-L1-HGF, or sham-operated mice.

[0205] Please refer to Figure 9A, which shows the CD3 + The results of the T cell analysis showed that compared with the sham operation group, the ischemic cerebral hemisphere of MCAO mice treated with EXO-PD-L1-HGF contained CD3 + The total number of viable white blood cells, including T cells, increased significantly, while there was no significant difference in the total number of cells among the control group, EXO group, and EXO-PD-L1-HGF group in both brain hemispheres.

[0206] 9B to 9Q, FIG9B, FIG9C, FIG9D, FIG9E and FIG9F show that after treatment with EXO or EXO-PD-L1-HGF, the activation of dendritic cells (DCs), cytotoxic T cells, and natural killer (NK) cells in the ischemic hemisphere was significantly reduced. Figures 9G, 9H, 9I, 9J and 9K are graphs showing that EXO or EXO-PD-L1-HGF treatment also reduces the percentages of activated DCs, cytotoxic T cells, NK cells and M1 macrophages in the spleen; Figures 9L, 9M and 9N are graphs showing that EXO or EXO-PD-L1-HGF treatment increases the percentages of regulatory B cells, regulatory T cells and M2 macrophages in the ischemic hemisphere; Figures 9O, 9P and 9Q are graphs showing that EXO or EXO-PD-L1-HGF treatment increases the percentages of regulatory B cells, regulatory T cells and M2 macrophages in the spleen.

[0207] The results of Figures 9B to 9K showed that compared with the EXO group and the control group in the ischemic cerebral hemisphere and spleen, intravenous injection of EXO-PD-L1-HGF into MCAO mice significantly reduced the expression of activated DC cells (CD11c + CD80 + and CD11c + CD86 + ), cytotoxic T cells (CD3 + CD8 + IFN-γ + ), NK cells (CD3 - NK1.1 + ), M1 macrophages (CD11b + CD80 + The results of Figures 9L to 9Q show that compared with the EXO group and the control group in the ischemic cerebral hemisphere and spleen, intravenous injection of EXO-PD-L1-HGF into MCAO mice significantly increased the number of regulatory B cells (CD19 + IL-10 + ), regulatory T cells (CD8 + CD122 + IL-10 + ) and M2 macrophages (CD11b + CD206 + f / 4 / 80 + The above results show that the exosomes of the present invention reduce pro-inflammatory responses by regulating anti-inflammatory leukocytes in ischemic brain and spleen.

[0208] In summary, the exosomes of the present invention can enter target cells through endocytosis, significantly reduce the cytotoxicity caused by H2O2, and increase the expression of anti-apoptotic proteins in neurons by inducing some cytokines. The exosomes of the present invention can inhibit T cell proliferation and reduce IFN-γ + cells to reduce inflammatory damage. The exosomes of the present invention promote neurogenesis and angiogenesis induced by endogenous progenitor cells through the STAT3-FOXO3 signaling axis, play an important role in cell survival and neuroprotection, and can reduce infarct volume and enhance neurological recovery in an ischemic stroke mouse model. In addition, the exosomes of the present invention can provide an anti-inflammatory microenvironment to protect glial cells in the ischemic brain by increasing the number of regulatory B cells and regulatory T cells and reducing the number of DCs, NK cells, and cytotoxic T cells. Therefore, the exosomes of the present invention can be used as a new drug delivery strategy and may regulate the ecological niche that induces neuroplasticity and regeneration in stroke brains. The data in the specification prove that the exosomes and pharmaceutical compositions comprising the same proposed by the present invention are not only safe and effective, but also enable the exosomes to play an important role in rescuing brain ischemic damage.

[0209] Although the present invention has been disclosed above in terms of embodiments, they are not intended to limit the present invention. Anyone skilled in the art may make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention shall be determined by the scope defined by the appended claims.

Claims

1. An exosome, It is characterized in that Derived from a genetically engineered mesenchymal stem cell, wherein the genetically engineered mesenchymal stem cell comprises an exogenous PD-L1 gene and an exogenous HGF gene.

2. The exosome according to claim 1, It is characterized in that The sequence of a peptide encoded by the exogenous PD-L1 gene is shown in SEQ ID NO:4, and the sequence of a peptide encoded by the exogenous HGF gene is shown in SEQ ID NO:

5.

3. The exosome according to claim 1, It is characterized in that The exogenous PD-L1 gene is connected to the exogenous HGF gene via a coding self-cleaving peptide sequence.

4. The exosome according to claim 1, It is characterized in that The exosomes contain an overexpression of PD-L1 and an overexpression of HGF.

5. The exosome according to claim 4, It is characterized in that When measured by flow cytometry, the expression level of PD-L1 on an exosome membrane of the exosome is increased relative to that of a control exosome, wherein the control exosome is derived from a non-genetically engineered mesenchymal stem cell.

6. The exosome according to claim 4, It is characterized in that When measured by ELISA, the exosomes have an increased HGF content relative to a control exosome derived from a non-genetically engineered mesenchymal stem cell.

7. The exosome according to claim 1, It is characterized in that An exosome membrane of the exosome has an overexpression of CXCR4.

8. The exosome according to claim 7, It is characterized in that As measured by flow cytometry, the proportion of the exosomes carrying a CXCR4 surface marker was increased relative to a control exosome derived from a non-genetically engineered mesenchymal stem cell.

9. The exosome according to claim 1, It is characterized in that The particle size of the exosome is between 30nm and 200nm.

10. The exosome according to claim 9, It is characterized in that The particle size of the exosome is between 100nm and 150nm.

11. A method for preparing exosomes, It is characterized in that Include: Constructing a genetically engineered mesenchymal stem cell comprises transferring an exogenous HGF gene and an exogenous PD-L1 gene into the mesenchymal stem cell to obtain the genetically engineered mesenchymal stem cell; performing a culturing step, culturing the genetically engineered mesenchymal stem cells in a culture medium to obtain a conditioned medium; and The step of separating is to collect an exosome from the conditioned medium in a separation manner.

12. The method for preparing exosomes according to claim 11, It is characterized in that In the culturing step, the genetically engineered mesenchymal stem cells are cultured under a hypoxic condition.

13. The method for preparing exosomes according to claim 12, It is characterized in that The hypoxic condition is an oxygen content of less than 3%.

14. The method for preparing exosomes according to claim 11, It is characterized in that The mesenchymal stem cells are adipose mesenchymal stem cells, umbilical cord mesenchymal stem cells or bone marrow mesenchymal stem cells.

15. The method for preparing exosomes according to claim 11, It is characterized in that The mesenchymal stem cells are adipose-derived mesenchymal stem cells or umbilical cord mesenchymal stem cells.

16. A pharmaceutical composition for treating an ischemic condition of a tissue, It is characterized in that Include: The exosomes according to claim 1: and A pharmaceutically acceptable carrier.

17. The pharmaceutical composition according to claim 16, It is characterized in that The pharmaceutical composition is administered by intravenous injection, intracarotid injection, intraarterial injection or a combination thereof.

18. A use of the exosomes according to claim 1, It is characterized in that It is used to prepare a drug for treating an ischemic condition of a tissue.

19. The use of exosomes according to claim 18, It is characterized in that The tissue is the brain.

20. The use of exosomes according to claim 19, It is characterized in that The ischemic condition is an ischemic stroke.

21. The use of exosomes according to claim 20, It is characterized in that This drug is a medicine that reduces the area of ​​the brain damaged by a stroke.

22. A use of the exosomes according to claim 1, It is characterized in that It is used to prepare drugs for enhancing nerve regeneration or reducing neuron death.

23. A use of the exosomes according to claim 1, It is characterized in that It is used to prepare drugs for reducing inflammatory response.

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