WO2024065651A1 - 一种向adam10基因敲除的hek293细胞外泌体中装载二聚体cd24的方法 - Google Patents

一种向adam10基因敲除的hek293细胞外泌体中装载二聚体cd24的方法 Download PDF

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WO2024065651A1
WO2024065651A1 PCT/CN2022/123202 CN2022123202W WO2024065651A1 WO 2024065651 A1 WO2024065651 A1 WO 2024065651A1 CN 2022123202 W CN2022123202 W CN 2022123202W WO 2024065651 A1 WO2024065651 A1 WO 2024065651A1
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exosomes
dimeric
loaded
apoe
adam10
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French (fr)
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王伊
杨佳蕾
董亚南
薛苗苗
李俊
王丹枫
周伟
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谛邈生物科技（新加坡）有限公司
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Priority to CN202280051569.3A priority Critical patent/CN118201601A/zh
Priority to PCT/CN2022/123202 priority patent/WO2024065651A1/zh
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  • the present invention relates to the technical field of exosome loading, and specifically, to a method of loading dimerized CD24 into exosomes to amplify the biological effect of CD24 and use it for treating severe inflammatory diseases, and to a technical field of loading ApoE and/or MyD88 inhibitor polypeptides into CD24-loaded exosomes to achieve multi-module synergistic effects and use it for treating severe inflammatory diseases.
  • CD24 is a highly sialylated protein molecule that is anchored to the cell membrane through glycosylated phosphatidylinositol (GPI). CD24 is expressed on a variety of cells and exerts immunosuppressive biological effects, such as inhibiting T cell activation, inducing neutrophil apoptosis, inhibiting B1 B cell maturation, inhibiting macrophage/monocyte inflammatory response, etc. Tumor cells express CD24 as a "Don't eat me" signal to achieve immune escape.
  • GPI glycosylated phosphatidylinositol
  • antagonizing tumor CD24 with monoclonal antibodies can indirectly activate tumor-associated macrophages to achieve anti-tumor effects; conversely, systemic or local administration of CD24 can effectively treat diseases caused by excessive immune responses, such as the use of CD24-Fc fusion protein to treat graft-versus-host disease (GVHD).
  • GVHD graft-versus-host disease
  • Extracellular vesicles are substances with vesicle-like structures secreted during cell growth. They can often carry intracellular proteins and nucleic acids during their formation, and carry extracellular proteins on the vesicle membrane or outside the cell like the cell membrane. There are many types of proteins on the extracellular vesicle membrane, so extracellular vesicles can bind to and be internalized by almost all cells to varying degrees.
  • Apolipoprotein E is a soluble protein secreted by the brain tissue and liver. It plays an important physiological role in energy metabolism homeostasis, innate immune regulation and other aspects. ApoE plays an important regulatory and improving role in severe inflammatory diseases such as acute brain injury (such as stroke, traumatic brain injury, etc.), chronic brain injury (such as Parkinson's disease, Alzheimer's disease, etc.), acute respiratory distress syndrome and sepsis.
  • MyD88 myeloid differentiation factor 88: It is an important hub molecule that connects the surface receptor signal of multiple immune cells with the downstream signal transduction in the cell. It mediates the intracellular signal transduction of important inflammatory receptors such as Toll-like receptors (TLR) and interleukin 1 receptor (IL1R), and downstream molecules include NF-kB.
  • TLR Toll-like receptors
  • IL1R interleukin 1 receptor
  • MyD88 is an important target for inhibiting innate immune response, but because it mainly mediates protein-protein interaction and lacks small molecule binding pockets in the protein structure, it has been impossible to achieve small molecule drug inhibition of MyD88 for decades. Loiarro (DOI: 10.1074/jbc.C400613200) et al. proved in 2005 that a series of peptide molecules can effectively inhibit the activity of MyD88, but due to the poor bioavailability and insufficient cell entry of such peptides, they are not druggable.
  • CD24 can be overexpressed in HEK293 cells, and exosomes (Exo-CD24) carrying CD24 in the cell culture supernatant can be collected and purified for the treatment of inflammation.
  • exosomes Exo-CD24
  • the efficacy of CD24 molecules loaded on exosomes by overexpression alone is not significantly superior to that of CD24-Fc fusion protein.
  • its production process is more complicated and costly than that of CD24-Fc fusion protein, and its drugability is limited.
  • the elimination half-life of MyD88 inhibitor polypeptide in vivo is very short, and it needs to penetrate the cell membrane to take effect, and its bioavailability is low.
  • the fusion peptide formed by MyD88 inhibitor polypeptide and membrane-penetrating peptide lacks targeting, and its widespread systemic immunosuppression poses a safety risk.
  • the full-length ApoE protein usually exists in the form of lipoprotein, and the efficacy of free ApoE protein or its simulated polypeptide is weak, and its in vivo stability is poor.
  • the present invention hopes to use extracellular vesicles/exosomes as carriers to load one, two or a combination of three of CD24, ApoE, and MyD88 inhibitor polypeptides to achieve the treatment of severe inflammatory response diseases.
  • dimeric CD24 and/or ApoE proteins are loaded into the exosomes of HEK293 cells with ADAM10 gene knockout (exosome protein loading technology and dimeric protein loading sequence are prior art, see patent: 202210549553.7), achieving a drug efficacy improvement of more than 1000 times compared with free CD24-Fc fusion protein and ApoE protein, and MyD88 inhibitor polypeptides are loaded into the above-mentioned exosomes (see patent: 202111263036.5 for polypeptide loading technology), thereby enhancing the inhibitory effect of exosomes on innate immune inflammatory response.
  • the present invention provides a method for loading dimer CD24 into ADAM10 gene knockout HEK293 cell exosomes, the specific steps comprising:
  • step (1) the ADAM10 gene knockout of HEK293 cells is achieved using a Crispr gene editing system or method.
  • step (2) the loading method includes:
  • the pCD24-Fc-Ig3(NPTN-Ig3)-TMD(NPTN-TMD)-ICD(EWI-F-ICD) plasmid was constructed and co-transfected with the Sleeping Beauty transposase expression vector pCMV-(CAT)T7-SB100X or the viral vector was infected into HEK293 cells and/or ADAM10 - HEK293 cells, and single clones were screened to obtain HEK293-CD24 or ADAM10 - HEK293-CD24 engineered stable cell lines.
  • step (2) after the engineered stably transfected cell line is expanded and cultured to a cell density of 5E+06/mL, the cell supernatant is collected by centrifugation at 5000 rpm for 30 min, and the engineered exosomes loaded with dimeric CD24 are purified from the cell supernatant.
  • step (3) the method for verifying the loading amount is: collecting cell proteins for Western blot experiment.
  • step (3) the functional verification method is: the role of dimeric CD24 exosomes in mortality and liver integrity in a mouse acute liver failure model.
  • step (2) dimeric ApoE protein is loaded into the exosomes.
  • the culture medium was replaced 16h and 24h after infection, and monoclonal clones were screened to obtain ADAM10 - HEK293-ApoE engineered stable cell line.
  • step (2) dimeric CD24 and dimeric ApoE proteins are loaded into the exosomes.
  • the constructed pApoE-Fc-Ig3(NPTN-Ig3)-TMD(NPTN-TMD)-ICD(EWI-F-ICD) plasmid and the constructed pCD24-Fc-Ig3(NPTN-Ig3)-TMD(NPTN-TMD)-ICD(EWI-F-ICD) plasmid were co-transfected or lentivirally infected into HEK293 cells to screen for the ADAM10 - HEK293-CD24-ApoE engineered stable cell line.
  • the engineered stable cell line was expanded and cultured to a cell density of 5E+06/mL
  • the cell supernatant was collected by centrifugation at 5000 rpm for 30 min, and the engineered loaded exosomes were purified from the cell supernatant.
  • the verification experiment is: detecting the immunosuppressive activity of different EVs and proteins in a PBMC inflammation model and/or establishing an acute liver failure model in mice, and verifying the liver protection effect of CD24 through the mortality rate.
  • step (2) while loading the dimeric CD24 into the exosomes, the MyD88 inhibitor polypeptide is also loaded.
  • the method is as follows: after the dimeric CD24 exosomes and the MyD88 inhibitor polypeptide are evenly mixed, the pH is adjusted to 8.0, and the free MyD88 inhibitor polypeptide is purified by Capto core700 to remove the free MyD88 inhibitor polypeptide, and the flow-through is collected to obtain CD24 exosomes loaded with the MyD88 inhibitor polypeptide.
  • the mixing ratio of dimeric CD24 exosomes and MyD88 inhibitor polypeptide was: 1E+12 exosome particles added with 1 mg MyD88 inhibitor polypeptide.
  • step (3) the verification method is: the PBMC inflammation model is used to detect the immunosuppressive activity of different EVs and proteins.
  • step (3) the verification experiment is: changes in mortality rate in a mouse sepsis model.
  • step (2) while loading the dimeric CD24 into the exosomes, the dimeric ApoE and MyD88 inhibitor polypeptide are also loaded.
  • the verification experiment is: an experiment to significantly reduce mortality and protect lung tissue integrity in a mouse acute respiratory distress syndrome model.
  • the present invention provides exosomes prepared by the above method.
  • the present invention provides a use of the above exosomes in preparing a drug.
  • the drug used is a drug for treating inflammation.
  • the present invention also provides a medicine, which comprises the exosomes of the present invention.
  • the present invention also provides a method of treatment, which comprises administering the medicament of the present invention.
  • the present invention loads dimeric CD24 and/or ApoE protein in ADAM10 knockout HEK293 cell exosomes, and further loads MyD88 inhibitor polypeptide in the above exosomes, thereby obtaining CD24-exosomes, ApoE-exosomes or CD24-ApoE-exosomes loaded with MyD88 inhibitor polypeptide with significantly improved efficacy.
  • the method and product provided by the present invention have the following advantages: (1) The efficacy of exosomes loaded with simple overexpression of CD24 is not significantly superior to that of CD24-Fc fusion protein.
  • the dimeric CD24 exosomes provided by the present invention only require a concentration of CD24-Fc fusion protein of one thousandth or even one ten-thousandth to achieve the same inflammation inhibition effect as the fusion protein.
  • a concentration of CD24-Fc fusion protein of one thousandth or even one ten-thousandth to achieve the same inflammation inhibition effect as the fusion protein.
  • the inflammation inhibition efficacy of ApoE loaded on exosomes is also significantly enhanced compared to free ApoE protein.
  • Exosomes loaded with dimeric CD24 and ApoE proteins simultaneously have achieved the technical effect of having both disease treatment and immune regulation functions.
  • exosome drugs are quickly cleared by innate immune cells in the body, after being loaded with MyD88 inhibitor peptides, even if the exosomes are phagocytosed and cleared by innate immune cells, they can still release MyD88 inhibitor peptides in their cells and continue to exert the effect of inhibiting inflammation, thereby significantly prolonging the duration of drug efficacy. This makes exosomes loaded with CD24/ApoE and MyD88 inhibitor peptides have better drugability than exosomes without peptides.
  • Figure 1 Western blot of the present invention verifies the background expression after ADAM10 knockout
  • FIG. 1 Expression of surface proteins of wild-type cell-derived exosomes of the present invention
  • FIG3 shows the expression of surface proteins of cell-derived exosomes after ADAM10 knockout of the present invention
  • FIG4 shows statistics of the inflammation inhibition activity of the free CD24-Fc fusion protein of the present invention, the exosomes loaded with monomeric CD24-EV and the exosomes loaded with dimeric CD24 in an in vitro PBMC cell inflammation model;
  • Figure 5 Statistics of mouse mortality after administration of CD24-EV in high-dose and low-dose groups of the present invention
  • FIG6 shows the protective effect of CD24EV of the present invention on liver tissue
  • FIG7 shows statistics of the inflammation inhibition activity of the free ApoE protein and the exosomes loaded with dimeric ApoE of the present invention in an in vitro PBMC cell inflammation model
  • Figure 8 Statistics of mouse mortality after administration of CD24EV and CD24-ApoE EV of the present invention
  • FIG9 shows statistics of the inflammation inhibition activity of free ApoE protein, exosomes loaded with dimeric CD24, and exosomes loaded with both dimeric CD24 and MyD88 inhibitor polypeptide in an in vitro PBMC cell inflammation model of the present invention
  • FIG10 shows the statistics of the mortality of mice after administration of the exosomes of the present invention simultaneously loaded with dimeric CD24, dimeric ApoE and MyD88 inhibitor polypeptide in a mouse acute respiratory distress syndrome model
  • Figure 11 Expression levels of cytokines in the blood of mice after administration of different groups of exosomes of the present invention.
  • FIG12 Statistics of mouse mortality after administration of the exosomes of the present invention to different administration groups.
  • Example 1 Knocking out ADAM10 on the surface of exosomes can significantly increase the loading amount of dimeric CD24 on the surface of exosomes
  • ADAM10 was knocked out in wild-type HEK293 cells to obtain ADAM10-KO cell line, which prevented the exosome surface membrane protein from being cleaved by ADAM10 and increased the protein loading amount on the exosome surface.
  • ADAM10 knockout Using the Crispr system and its specific methods, the ADAM10-gRNA plasmid was transfected into HEK293 cells. After transfection, the cells were plated and single clones were selected for sequencing to obtain a cell line with successful ADAM10 knockout. Western blot was used to further verify whether there was any residual background expression of ADAM10.
  • Plasmid design and construction VB220306-1137jmq (Yunzhou Biotechnology, Sleeping Beauty expression vector) was used as the vector to construct the pCD24-Fc-Ig3 (NPTN-Ig3)-TMD (NPTN-TMD)-ICD (EWI-F-ICD) plasmid.
  • the module design referenced the company's accepted patent (patent application number 202210549553.7).
  • the amino acid sequence is as follows:
  • the plasmid constructed in step (2) is co-transfected with the Sleeping Beauty (SB) transposase expression vector pCMV-(CAT)T7-SB100X into HEK293 cells or ADAM10-HEK29 cells, and single clones are screened to obtain HEK293-CD24 or ADAM10 - HEK293-CD24 engineered stable cell lines.
  • SB Sleeping Beauty
  • Loading capacity verification The exosomes purified from the supernatant of HEK293, ADAM10 - HEK293, HEK293-CD24, and ADAM10 - HEK293-CD24 cells were subjected to Western blot experiments using CD24 antibody, Fc antibody, and EGFP antibody to compare the loading capacity.
  • CD24-Fc-Ig3 NPTN-Ig3-TMD
  • NPTN-TMD NPTN-TMD
  • EWI-F-ICD The theoretical molecular weight of CD24-Fc-Ig3 (NPTN-Ig3)-TMD (NPTN-TMD)-ICD (EWI-F-ICD) is 60kD, and the molecular weight of the dimer is about 120kD.
  • Western blot shows the presence of dimer protein.
  • CD24 on the membrane surface is cleaved by ADAM10, and the CD24 antibody does not recognize the protein.
  • surface CD24 In exosomes derived from ADAM10-cells, surface CD24 is retained, and the loading amount of CD24 on the surface of exosomes is significantly increased.
  • Example 2 Exosomes loaded with dimeric CD24 showed immunosuppressive activity thousands of times stronger than free CD24-Fc fusion protein in an in vitro cell model
  • CD24 can play an immunosuppressive biological role, such as inhibiting T cell activation, inducing neutrophil apoptosis, inhibiting B1 B cell maturation, inhibiting macrophage/monocyte inflammatory response, etc., and detecting the inhibitory effect of CD24-EV on PBMC cell inflammatory response.
  • engineered exosomes loaded with dimeric CD24 can achieve local aggregation of CD24, amplify the biological effect, and thus exert stronger immunosuppressive activity than monomeric CD24-EV.
  • Plasmid design and construction Using VB220306-1137jmq (Yunzhou Biotechnology, Sleeping Beauty expression vector) as the vector, the pCD24-Ig3 (NPTN-Ig3)-TMD (NPTN-TMD)-ICD (EWI-F-ICD) plasmid was constructed.
  • the plasmid constructed in step (1) was co-transfected with the Sleeping Beauty transposase expression vector pCMV-(CAT)T7-SB100X into ADAM10 - HEK293 cells, and single clones were screened to obtain the ADAM10 - HEK293-CD24 engineered stable cell line.
  • Preparation of engineered monomeric CD24 exosomes After the engineered stable cell line was expanded and cultured to a cell density of 5E+06/mL, the cell supernatant was collected by centrifugation at 5000rpm for 30min. The engineered exosomes loaded with monomeric CD24 were purified from the cell supernatant and used for cell detection after confirming that the loading amount was normal.
  • PBMC inflammation model to detect the immunosuppressive activity of different EVs and proteins PBMC cells were used for the experiment. At the same time, 1ug/ml anti-human CD3 Antibody (coated) and 1ug/ml anti-human CD28 Antibody (free) were used to establish the inflammation model. Positive drug (dexamethasone), free CD24-Fc protein and different doses of engineered loaded dimer CD24EV and monomer CD24EV were treated for 72h to detect their inhibitory activity on PBMC inflammatory response.
  • exosomes loaded with monomeric CD24-EV and exosomes loaded with dimeric CD24 all showed good inflammation suppression activity in the in vitro PBMC cell inflammation model.
  • exosomes loaded with dimeric CD24 showed immunosuppressive activity thousands of times stronger than free CD24-Fc fusion protein and hundreds of times stronger than that loaded with monomeric CD24. And as the exosome dose increases, the immunosuppressive activity increases.
  • Example 3 Exosomes loaded with dimeric CD24 can effectively reduce the mortality rate of mice and maintain the integrity of liver tissue in a mouse acute liver failure model.
  • Engineered exosomes loaded with dimeric CD24 can achieve local aggregation of CD24, amplify the biological effects, establish a mouse model of acute liver failure, and verify the liver protective effect of CD24 through mortality and liver tissue section observations.
  • engineered dimeric CD24 exosomes The engineered stably transfected cell line (the same as the stably transfected cell line in Example 1) was expanded and cultured to a cell density of 5E+06/mL, and then the cell supernatant was collected by centrifugation at 5000 rpm for 30 min. The engineered exosomes loaded with dimeric CD24 were purified from the cell supernatant.
  • the drugs were administered three times within 24 hours, at 4 hours, 12 hours, and 20 hours after modeling, and the liver protection effect was detected by observing the mortality rate of mice.
  • the livers of mice in the CD24EV treatment group and the liver failure group were sliced to observe the damage of liver tissue.
  • Example 4 Exosomes loaded with dimeric ApoE showed stronger immunosuppressive efficacy than free ApoE protein in an in vitro cell model.
  • Plasmid design and construction The lentiviral vector pSLenti-CMV--PGK-PuroWPRE (Heyuan Bio) was used as the vector to construct the pApoE-Fc-Ig3 (NPTN-Ig3)-TMD (NPTN-TMD)-ICD (EWI-F-ICD) plasmid.
  • the module design referenced the company's accepted patent (patent application number 202210549553.7).
  • the amino acid sequence is as follows:
  • step (2) Obtaining a stable cell line:
  • the plasmid constructed in step (1) is packaged into a virus and used to infect ADAM10 - HEK293 cells at an MOI of 10.
  • the culture medium is replaced 16 h and 24 h after infection, and a single clone is screened to obtain an ADAM10 - HEK293-ApoE engineered stable cell line.
  • PBMC inflammation model to detect the immunosuppressive activity of different EVs and proteins PBMC cells were used for the experiment. At the same time, 1ug/ml anti-human CD3 Antibody (coated) and 1ug/ml anti-human CD28 Antibody (free) were used to establish the inflammation model. Positive drug (dexamethasone) and free ApoE protein and different doses of engineered loaded dimer ApoE-EV were treated for 72 hours to detect their inhibitory activity on PBMC inflammatory response.
  • Example 5 Exosomes simultaneously loaded with dimeric CD24 and dimeric ApoE significantly reduced the mortality rate of mice in a mouse acute liver failure model.
  • ADAM10 - HEK293 cell exosomes were loaded with dimeric CD24 and dimeric ApoE proteins at the same time to establish a mouse acute liver failure model, and the liver protective effect of CD24 was verified by the mortality rate.
  • Plasmid design and construction Using VB220306-1137jmq (Yunzhou Biotechnology, Sleeping Beauty expression vector) as the vector, the pCD24-Fc-Ig3(NPTN-Ig3)-TMD(NPTN-TMD)-ICD(EWI-F-ICD) plasmid and pApoE-Fc-Ig3(NPTN-Ig3)-TMD(NPTN-TMD)-ICD(EWI-F-ICD) plasmid were constructed respectively.
  • step (1) The two plasmids constructed in step (1) were combined with the Sleeping Beauty transposase expression vector pCMV
  • CATT7-SB100X were co-transfected into ADAM10 - HEK293 cells, and single clones were screened to obtain the ADAM10 - HEK293-CD24-ApoE engineered stable cell line.
  • CD24EV and CD24-ApoE EV groups showed a lower mortality effect than the positive drug, and the CD24-ApoE EV group was better at reducing the mortality of mice than the group loaded with CD24EV alone.
  • Example 6 Exosomes loaded with both dimeric CD24 and MyD88 inhibitor polypeptides significantly inhibited inflammatory responses in an in vitro cell model.
  • MyD88 inhibitor peptides into CD24-loaded exosomes can enhance the inhibitory effect of the exosomes on the innate immune inflammatory response. After the CD24-exosomes loaded with MyD88 inhibitor peptides are phagocytosed and cleared by immune cells, the MyD88 inhibitor peptides will be released inside the cells, continuing to exert the inhibitory effect on inflammation, effectively prolonging the duration of drug action.
  • PBMC inflammation model to detect the immunosuppressive activity of different EVs and proteins PBMC cells were used for the experiment. At the same time, 1ug/ml anti-human CD3 Antibody (coated) and 1ug/ml anti-human CD28 Antibody (free) were used to establish the inflammation model. Positive drug (dexamethasone), free CD24-Fc fusion protein, different doses of engineered loaded dimer CD24-EV and engineered loaded dimer CD24+MyD88 inhibitor peptide-EV were given for 72h and 120h treatment to detect their inhibitory activity on PBMC inflammatory response.
  • Example 7 Exosomes loaded with both dimeric CD24 and MyD88 inhibitor polypeptides significantly reduced mortality in a mouse sepsis model.
  • MyD88 inhibitor peptides can inhibit inflammation and effectively prolong the duration of drug action.
  • CD24-exosomes loaded with the inhibitor peptides were used in a mouse sepsis model and could significantly reduce the mortality rate of mice.
  • Exosomes loaded with both dimeric CD24 and MyD88 inhibitor peptides can reduce the mortality rate of mice and the expression levels of inflammatory-related factors (IL6, IL10, IFN- ⁇ ) in the blood in a mouse sepsis model.
  • IL6 inflammatory-related factors
  • Example 8 Exosomes simultaneously loaded with dimeric CD24, dimeric ApoE and MyD88 inhibitor polypeptide significantly reduced mortality and protected lung tissue integrity in a mouse acute respiratory distress syndrome model.
  • MyD88 inhibitor peptides can inhibit inflammation and effectively prolong the duration of drug action.
  • CD24-exosomes, ApoE-exosomes or CD24-ApoE-exosomes loaded with the inhibitor peptides were used in the mouse acute respiratory distress syndrome model to reduce the mortality rate of mice and protect the integrity of lung tissue.
  • CD24-exosomes, ApoE-exosomes or CD24-ApoE-exosomes loaded with the inhibitor polypeptide are used in a mouse acute respiratory distress syndrome model to reduce the mortality rate of mice and protect the integrity of lung tissue.

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Abstract

本发明涉及一种向ADAM10基因敲除的HEK293细胞外泌体中装载二聚体CD24的方法，通过向ADAM10基因敲除的HEK293细胞外泌体中装载二聚体CD24和/或ApoE蛋白，实现相较于游离CD24-Fc融合蛋白以及ApoE蛋白超过1000倍的药效提升；同时向该外泌体中装载MyD88抑制剂多肽，提升了外泌体对固有免疫炎症反应的抑制效力。本申请得到的装载了上述抑制剂多肽的CD24-外泌体、ApoE-外泌体或CD24-ApoE-外泌体被免疫细胞吞噬清除后，会在该细胞内部释放MyD88抑制剂多肽，继续发挥炎症抑制效力，有效延长药物作用时间。

Description

一种向ADAM10基因敲除的HEK293细胞外泌体中装载二聚体CD24的方法 技术领域
本发明涉及外泌体的装载技术领域，具体地说，涉及一种向外泌体装载二聚化CD24实现CD24生物学效应的放大并用于治疗重症炎症性疾病，以及向装载有CD24的外泌体装载ApoE和/或MyD88抑制剂多肽实现多模块协同作用并用于治疗重症炎症性疾病的技术领域。
背景技术
CD24是一种高度唾液酸化的蛋白质分子，通过糖基化磷脂酰肌醇(GPI)锚定(anchored)于细胞膜上。CD24在多种细胞上表达，发挥免疫抑制类生物学作用，如抑制T细胞激活，诱导中性粒细胞凋亡，抑制B1型B细胞成熟，抑制巨噬细胞/单核细胞炎症反应等等。肿瘤细胞表达CD24作为“别吃我(Don’t eat me)”信号实现免疫逃逸。因此，在抗肿瘤领域，通过单克隆抗体拮抗肿瘤CD24可间接激活肿瘤相关巨噬细胞实现抗肿瘤作用；相反地，通过系统或局部给予CD24，可以有效治疗过度免疫反应造成的疾病，如使用CD24-Fc融合蛋白治疗移植物抗宿主病(GVHD)。
细胞外囊泡(Extracellular Vesicles，EVs)：是细胞生长过程中分泌的具有囊泡状结构的物质，往往在形成过程中可携带细胞内的蛋白、核酸，并且在囊泡膜上或胞外如细胞膜一样携带胞外蛋白。细胞外囊泡膜上蛋白种类非常多，因此细胞外囊泡几乎能与所有细胞不同程度的结合并内吞。
载脂蛋白E(Apolipoprotein E，ApoE)：是载脂蛋白E(Apolipoprotein E，ApoE)由脑组织和肝脏分泌的可溶性蛋白，在能量代谢稳态、固有免疫调节等多方面发挥重要生理作用；在急性脑损伤(如卒中、创伤性脑损伤等)、慢性脑损伤(如帕金森病、阿尔茨海默病等)，急性呼吸窘迫综合征和败血症等重症炎症性疾病中，ApoE均发挥重要的调节改善作用。
MyD88(髓样分化因子88)：是多种免疫细胞胞内连接膜表面受体信号与胞内下游信号转导的重要枢纽分子，它介导Toll样受体(TLR)和白介素1受体(IL1R)等重要炎症受体的胞内信号转导，下游分子包括NF-kB等。MyD88是抑制固有免疫反应的重要 靶点，但由于其主要介导蛋白质-蛋白质相互作用，蛋白结构中缺乏小分子结合口袋，数十年来一直无法实现对MyD88的小分子药物抑制。Loiarro(DOI：10.1074/jbc.C400613200)等人在2005年证明一系列多肽分子可有效抑制MyD88活性，但由于这类多肽生物利用度差，入胞能力不足，不具备成药性。
现有技术中，能够通过在HEK293细胞中过表达CD24，收集并纯化细胞培养上清中带有CD24的外泌体(Exo-CD24)，用于炎症治疗，然而仅通过过表达在外泌体上装载的CD24分子，其效力与CD24-Fc融合蛋白相比优势并不明显，反而因其生产工艺相较于CD24-Fc融合蛋白更复杂、成本更高，成药性有限，且MyD88抑制剂多肽体内消除半衰期很短，需要穿透细胞膜后才能起效，生物利用度低，MyD88抑制剂多肽与穿膜肽形成的融合肽，缺乏靶向性，其广泛的系统性免疫抑制存在安全性风险，此外，ApoE全长蛋白通常以脂蛋白形式存在，游离ApoE蛋白或其模拟多肽的效力较弱，且体内稳定性较差。
因此，本发明是希望用细胞外囊泡/外泌体作为作为载体，装载CD24、ApoE、MyD88抑制剂多肽中的一种、两种或三种的组合，实现对重症炎症反应疾病的治疗。
发明内容
本发明中向ADAM10基因敲除的HEK293细胞外泌体中装载二聚体CD24和/或ApoE蛋白(外泌体蛋白装载技术以及二聚体蛋白装载序列为现有技术，参见专利：202210549553.7)，实现相较于游离CD24-Fc融合蛋白以及ApoE蛋白超过1000倍的药效提升，向上述外泌体中装载MyD88抑制剂多肽(多肽装载技术参见专利：202111263036.5)，提升外泌体对固有免疫炎症反应的抑制效力。
本发明提供一种向ADAM10基因敲除的HEK293细胞外泌体中装载二聚体CD24的方法，具体步骤包括：
(1)敲除HEK293细胞的ADAM10基因；
(2)向敲除ADAM10基因的HEK293细胞的外泌体中装载二聚体CD24和/或二聚体ApoE蛋白；
(3)装载量和/或功能验证。
进一步地，步骤(1)中，采用Crispr基因编辑系统或方法实现HEK293细胞的ADAM10基因敲除。
进一步地，步骤(2)中，装载的方法包括：
以VB220306-1137j mq为载体，构建pCD24-Fc-Ig3(NPTN-Ig3)-TMD(NPTN-TMD)-ICD(EWI-F-ICD)质粒，与睡美人转座酶表达载体pCMV-(CAT)T7-SB100X共同转染或病毒载体感染到HEK293细胞和/或ADAM10 -HEK293细胞，筛选单克隆得到HEK293-CD24或ADAM10 -HEK293-CD24工程化稳转细胞株。
进一步地，步骤(2)中，工程化稳转细胞株扩增培养至细胞密度为5E+06/mL后，5000rpm离心30min收集细胞上清，从细胞上清中纯化得到工程化装载二聚体CD24的外泌体。
进一步地，步骤(3)中，验证装载量的方法为：收集细胞蛋白进行Western blot实验。
进一步地，步骤(3)中，功能验证方法为：二聚体CD24外泌体在小鼠急性肝衰竭模型的死亡率以及肝完整度中所起作用。
进一步地，步骤(2)中，向外泌体中装载二聚体ApoE蛋白。
进一步地，向外泌体中装载二聚体ApoE时，以pSLenti-CMV--PGK-PuroWPRE为载体，构建pApoE-Fc-Ig3(NPTN-Ig3)-TMD(NPTN-TMD)-ICD(EWI-F-ICD)质粒，按MOI＝10感染ADAM10 -HEK293细胞，感染后16h和24h进行培养基更换，筛选单克隆得到ADAM10 -HEK293-ApoE工程化稳转细胞株。
进一步地，步骤(2)中，向外泌体中装载二聚体CD24和二聚体ApoE蛋白。
进一步地，向外泌体中装载二聚体CD24和二聚体ApoE时，将已构建的pApoE-Fc-Ig3(NPTN-Ig3)-TMD(NPTN-TMD)-ICD(EWI-F-ICD)质粒与已构建的pCD24-Fc-Ig3(NPTN-Ig3)-TMD(NPTN-TMD)-ICD(EWI-F-ICD)质粒共同经过转染或慢病毒感染HEK293细胞，筛选得到ADAM10 -HEK293-CD24-ApoE工程化稳转细胞株。
进一步地，工程化稳转细胞株扩增培养至细胞密度为5E+06/mL后，5000rpm离心30min收集细胞上清，从细胞上清中纯化得到工程化装载的外泌体。
进一步地，步骤(3)中，验证实验为：PBMC炎症模型检测不同EV及蛋白的免疫抑制活性和/或建立小鼠急性肝衰竭模型，通过死亡率情况验证CD24的肝保护效果。
进一步地，步骤(2)中，向外泌体中装载二聚体CD24的同时，也装载MyD88 抑制剂多肽。
进一步地，向外泌体中同时装载二聚体CD24和MyD88抑制剂多肽时，方法为：二聚体CD24外泌体与MyD88抑制剂多肽混匀后，调pH至8.0，用Capto core700纯化去除游离MyD88抑制剂多肽，收集流穿，得到装载了MyD88抑制剂多肽的CD24外泌体。
进一步地，二聚体CD24外泌体与MyD88抑制剂多肽混匀比例为：1E+12个外泌体颗粒加入1mg MyD88抑制剂多肽。
进一步地，步骤(3)中，验证方法为：PBMC炎症模型检测不同EV及蛋白的免疫抑制活性。
进一步地，步骤(3)中，验证实验为：小鼠脓毒血症模型中的死亡率变化。
进一步地，步骤(2)中，向外泌体中装载二聚体CD24的同时，也装载二聚体ApoE和MyD88抑制剂多肽。
进一步地，步骤(3)中，验证实验为：小鼠急性呼吸窘迫综合征模型中显著降低死亡率并保护肺组织完整性实验。
进一步地，本发明提供一种由上述方法制备得到的外泌体。
进一步地，本发明提供一种由上述外泌体在制备药物中的应用。
进一步地，所述应用的药物为炎症治疗的药物。
本发明还提供了药物，其包括本发明所述的外泌体。
本发明还提供了治疗方法，其包括给予本发明所述的药物。
有益效果：
本发明通过在ADAM10基因敲除的HEK293细胞外泌体中装载二聚体CD24和/或ApoE蛋白，并在上述外泌体中进一步地装载MyD88抑制剂多肽，得到了药效显著提升的装载MyD88抑制剂多肽的CD24-外泌体、ApoE-外泌体或CD24-ApoE-外泌体。本发明提供的方法以及产品具有一下优点：(1)单纯过表达CD24装载的外泌体，其效力相较于CD24-Fc融合蛋白优势不显著，本发明提供的的二聚体CD24外泌体仅需要CD24-Fc融合蛋白一千甚至一万分之一的浓度即可达到与融合蛋白相同的炎症抑制效果，体内药效研究也显示出二聚体CD24外泌体的起效剂量很低，这不仅降低了药物的单剂成本，也提升了药物安全性，也具有更好的成药性。(2)装载在 外泌体上的ApoE相较于游离ApoE蛋白的炎症抑制效力也有显著的增强。(3)同时装载二聚体CD24和ApoE蛋白的外泌体，达到了具有兼顾疾病治疗以及免疫调节双重功能的技术效果，其有望成为拥有免疫调节能力的药用外泌体的药物开发热点，具有巨大的市场应用潜力；(4)由于外泌体药物在体内会被固有免疫细胞快速清除，但装载了MyD88抑制剂多肽以后，即便外泌体被固有免疫细胞吞噬清除，它仍可在其胞内释放MyD88抑制剂多肽，继续发挥炎症抑制效果，从而显著延长药效持续时间，这使得同时装载CD24/ApoE和MyD88抑制剂多肽的外泌体与不含多肽的外泌体相比具有更好的成药性。
附图说明
图1：本发明的Western blot验证ADAM10敲除后本底表达情况；
图2：本发明的野生型细胞来源外泌体表面蛋白的表达情况；
图3：本发明的ADAM10敲除后细胞来源外泌体表面蛋白的表达情况；
图4：本发明的游离CD24-Fc融合蛋白，装载单体CD24-EV及装载二聚体CD24的外泌体在体外PBMC细胞炎症模型中的炎症抑制活力统计；
图5：本发明的高剂量和低剂量组CD24-EV给药后小鼠死亡率统计；
图6：本发明的CD24EV对肝组织的保护效果；
图7：本发明的游离ApoE蛋白及装载二聚体ApoE的外泌体在体外PBMC细胞炎症模型中的炎症抑制活力统计；
图8：本发明的CD24EV和CD24-ApoE EV给药后小鼠死亡率统计；
图9：本发明的游离ApoE蛋白，装载二聚体CD24的外泌体及同时装载二聚体CD24和MyD88抑制剂多肽的外泌体在体外PBMC细胞炎症模型中的炎症抑制活力统计；
图10：本发明的同时装载二聚体CD24、二聚体ApoE和MyD88抑制剂多肽的外泌体在小鼠急性呼吸窘迫综合征模型中给药后小鼠死亡率统计；
图11：本发明的外泌体不同给药组给药后小鼠血液内细胞因子的表达水平；
图12：本发明的外泌体进行不同给药组给药后小鼠死亡率统计。
具体实施方式
下面将对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实 施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
实施例1：敲除外泌体表面的ADAM10可显著提升二聚体CD24在外泌体表面的装载量
野生型HEK293细胞上敲除ADAM10，得到ADAM10-KO细胞株，避免外泌体表面膜蛋白被ADAM10切掉，增加蛋白在外泌体表面的装载量。
1.方法：
(1)ADAM10敲除：利用Crispr系统以及其具体方法，向HEK293细胞中转染ADAM10-gRNA质粒，转染后进行铺板，挑单克隆细胞进行测序，得到成功敲除ADAM10的细胞株，Western blot进一步验证ADAM10是否有本底表达残留。
(2)质粒设计与构建：以VB220306-1137jmq(云舟生物，睡美人表达载体)为载体，构建pCD24-Fc-Ig3(NPTN-Ig3)-TMD(NPTN-TMD)-ICD(EWI-F-ICD)质粒，模块设计引用本公司已被受理专利(专利申请号202210549553.7)，氨基酸序列如下：
Seq1：
Figure PCTCN2022123202-appb-000001
(3)稳转细胞株获取：将步骤(2)中构建的质粒，与睡美人(Sleeping Beauty，SB)转座酶表达载体pCMV-(CAT)T7-SB100X共同转染到HEK293细胞或ADAM10- HEK29细胞，筛选单克隆得到HEK293-CD24或ADAM10 -HEK293-CD24工程化稳转细胞株。
(4)工程化二聚体CD24外泌体制备：工程化稳转细胞株扩增培养至细胞密度为5E+06/mL后，5000rpm离心30min收集细胞上清，从细胞上清中纯化得到工程化装载二聚体CD24的外泌体。
(5)装载量验证：将分别从HEK293、ADAM10 -HEK293、HEK293-CD24、ADAM10 -HEK293-CD24细胞上清中纯化得到的外泌体分别用CD24抗体、Fc抗体和EGFP抗体进行实验Western blot，比较装载量。
2.结果：
如图1-3所示。
3.结论：
ADAM10敲除后确认本底无ADAM10蛋白表达，
CD24-Fc-Ig3(NPTN-Ig3)-TMD(NPTN-TMD)-ICD(EWI-F-ICD)理论分子量60kD，二聚体分子量在120kD左右，Western blot显示有二聚体蛋白。野生型细胞来源的外泌体，膜表面CD24被ADAM10酶切，CD24抗体未识别到蛋白，ADAM10-细胞来源外泌体，表面CD24得到保留，CD24在外泌体表面的装载量得到显著提升。
实施例2：装载二聚体CD24的外泌体在体外细胞模型中显示出比游离CD24-Fc融合蛋白强数千倍的免疫抑制活力
利用CD24可发挥免疫抑制类生物学作用，如抑制T细胞激活，诱导中性粒细胞凋亡，抑制B1型B细胞成熟，抑制巨噬细胞/单核细胞炎症反应等特性，检测装载CD24-EV对PBMC细胞炎症反应的抑制效果。且工程化外泌体装载二聚体CD24，能够实现CD24的局部聚集，产生生物学效应的放大，从而发挥比单体CD24-EV更强的免疫抑制活性。
1.方法：
(1)装载二聚体CD24EV制备方法参见“实施例1”；
(2)装载单体CD24EV制备方法：
质粒设计与构建：以VB220306-1137jmq(云舟生物，睡美人表达载体)为载体，构建pCD24-Ig3(NPTN-Ig3)-TMD(NPTN-TMD)-ICD(EWI-F-ICD)质粒。
稳转细胞株获取：将步骤(1)中构建的质粒，与睡美人转座酶表达载体pCMV-(CAT)T7-SB100X共同转染到ADAM10 -HEK293细胞，筛选单克隆得到ADAM10 -HEK293-CD24工程化稳转细胞株。
工程化单体CD24外泌体制备：工程化稳转细胞株扩增培养至细胞密度为5E+06/mL后，5000rpm离心30min收集细胞上清，从细胞上清中纯化得到工程化装载单体CD24的外泌体，确认装载量正常后用于细胞检测。
PBMC炎症模型检测不同EV及蛋白的免疫抑制活性：利用PBMC细胞进行实验。同时利用1ug/ml anti-human CD3 Antibody(包被)及1ug/ml anti-human CD28 Antibody(游离)进行炎症模型造模，给予阳性药(地塞米松)，游离CD24-Fc蛋白和不同剂量的工程化装载二聚体CD24EV及单体CD24EV处理72h，检测其对PBMC炎症反应的抑制活性。
2.结果：
如表1以及图4所示。
Figure PCTCN2022123202-appb-000002
表1.不同处理组处理组PBMC上清中IFN-γ的浓度值
3.结论：
与模型组相比，游离CD24-Fc融合蛋白，装载单体CD24-EV及装载二聚体CD24的外泌体在体外PBMC细胞炎症模型中均显示出了较好的炎症抑制活性。尤其是装载二聚体CD24的外泌体，显示出了比游离CD24-Fc融合蛋白强数千倍的免疫抑制活力，比装载单体CD24的免疫抑制活力也要强几百倍。且随着外泌体剂量的增加，免疫抑制活力增强。
实施例3：装载二聚体CD24的外泌体在小鼠急性肝衰竭模型中可有效降低小鼠死亡率，维护肝组织完整性。
工程化外泌体装载二聚体CD24，能够实现CD24的局部聚集，产生生物学效应的放大，建立小鼠急性肝衰竭模型，通过死亡率和肝组织切片观测验证CD24的肝保护效果。
1.方法：
(1)工程化二聚体CD24外泌体制备：工程化稳转细胞株(与实施例1中稳转细胞株相同)扩增培养至细胞密度为5E+06/mL后，5000rpm离心30min收集细胞上清，从细胞上清中纯化得到工程化装载二聚体CD24的外泌体。
(2)肝保护效果验证：选择8周雄性Balb/c小鼠(北京维通利华)进行实验。按照5mL/kg量腹腔注射30％CCl 4(v/v)进行造模，注射CCl42h后给药，分组进行给阳性药(联苯双酯，Bifendate按照5mg/kg体重给药约100μg)和不同剂量的CD24EV(低剂量组每只鼠给药1E7个外泌体颗粒，高剂量组每只鼠给药1E9个外泌体颗粒，每1E9个外泌体装载1μg CD24蛋白)，24h内进行3次给药，时间点分别在造模后4h、12h、20h，通过观察小鼠死亡率检测肝保护效果。CD24EV治疗组和肝衰竭组小鼠肝脏进行组织切片，观察肝组织的损伤情况。
2.结果：
如图5-6所示。
3.结论：与模型组对比，高剂量和低剂量组CD24-EV均表现出死亡率降低的效果，尤其是高剂量组，比阳性药的死亡率更低。与肝衰竭组比较，CD24高剂量组对肝组织有明显的保护效果。
实施例4：装载二聚体ApoE的外泌体在体外细胞模型中显示出较游离ApoE蛋白更强的免疫抑制效力。
1.方法：
(1)质粒设计与构建：以慢病毒载体pSLenti-CMV--PGK-PuroWPRE(和元生物)为载体，构建pApoE-Fc-Ig3(NPTN-Ig3)-TMD(NPTN-TMD)-ICD(EWI-F-ICD)质粒，模块设计引用本公司已被受理专利(专利申请号202210549553.7)，氨基酸序列如下：
Seq2：
Figure PCTCN2022123202-appb-000003
(2)稳转细胞株获取：将步骤(1)中构建的质粒包装病毒，按MOI＝10感染ADAM10 -HEK293细胞，感染后16h和24h进行培养基更换，筛选单克隆得到ADAM10 -HEK293-ApoE工程化稳转细胞株。
(3)二聚体ApoE外泌体制备：工程化稳转细胞株扩增培养至细胞密度为5E+06/mL后，5000rpm离心30min收集细胞上清，从细胞上清中纯化得到工程化装载二聚体ApoE的外泌体。
PBMC炎症模型检测不同EV及蛋白的免疫抑制活性：利用PBMC细胞进行实验。同时利用1ug/ml anti-human CD3 Antibody(包被)及1ug/ml anti-human CD28 Antibody(游离)进行炎症模型造模，给予阳性药(地塞米松)及游离ApoE蛋白和不同剂量的工程化装载二聚体ApoE-EV处理72h，检测其对PBMC炎症反应的抑制活性。
2.结果：
如表2以及图7所示。
Figure PCTCN2022123202-appb-000004
表2.不同处理组处理组PBMC上清中IFN-γ的浓度值
3.结论：与模型组相比，游离ApoE蛋白及装载二聚体ApoE的外泌体在体外PBMC细胞炎症模型中均显示出了较好的炎症抑制活性。尤其是装载二聚体ApoE的外泌体，显示出了比游离ApoE蛋白强超过一千倍的免疫抑制活力。且随着外泌体剂量的增加，免疫抑制活力增强。
实施例5：同时装载二聚体CD24和二聚体ApoE的外泌体在小鼠急性肝衰竭模型中显著降低小鼠死亡率。
ADAM10 -HEK293细胞外泌体中同时装载二聚体CD24和二聚体ApoE蛋白，建立小鼠急性肝衰竭模型，通过死亡率情况验证CD24的肝保护效果。
1.方法：
(1)质粒设计与构建：以VB220306-1137jmq(云舟生物，睡美人表达载体)为载体，分别构建pCD24-Fc-Ig3(NPTN-Ig3)-TMD(NPTN-TMD)-ICD(EWI-F-ICD)质粒和pApoE-Fc-Ig3(NPTN-Ig3)-TMD(NPTN-TMD)-ICD(EWI-F-ICD)质粒。
(2)稳转细胞株获取：将步骤(1)中构建的两种质粒，与睡美人转座酶表达载体pCMV
(CAT)T7-SB100X一起共同转染到ADAM10 -HEK293细胞，筛选单克隆得到ADAM10 -HEK293-CD24-ApoE工程化稳转细胞株。
(3)CD24-ApoE外泌体制备：工程化稳转细胞株扩增培养至细胞密度为5E+06/mL后，5000rpm离心30min收集细胞上清，从细胞上清中纯化得到工程化装载二聚体CD24-ApoE的外泌体。
肝保护效果验证：选择8周雄性Balb/c小鼠(北京维通利华)进行实验。按照5mL/kg量腹腔注射30％CCl4(v/v)进行造模，注射CCl42h后给药，分组进行给阳性药(联苯双酯，Bifendate，按照5mg/kg体重给药约100μg)和CD24EV、ApoE EV、CD24-ApoE EV(每组各1E9个外泌体，装载蛋白1μg)，24h内进行3次给药，时间点分别在造模后4h、12h、20h，通过观察小鼠死亡率检测肝保护效果。
2.结果：
如图8所示。
3.结论：CD24EV和CD24-ApoE EV组表现出比阳性药死亡率更低的效果，且CD24-ApoE EV组降低小鼠死亡率效果优于只装载CD24EV组。
实施例6：同时装载二聚体CD24和MyD88抑制剂多肽的外泌体在体外细胞模型中显著抑制炎症反应。
向装载CD24的外泌体中装载MyD88抑制剂多肽，可提升该外泌体对固有免疫炎症反应的抑制效力。且装载了MyD88抑制剂多肽的CD24-外泌体被免疫细胞吞噬清除后，会在该细胞内部释放MyD88抑制剂多肽，继续发挥炎症抑制效力，有效延长药物作用时间。
1.方法：
(1)二聚体CD24外泌体制备：质粒构建、细胞株筛选和外泌体制备过程与实施例1一致。
(2)MyD88抑制剂多肽装载：二聚体CD24外泌体与MyD88抑制剂多肽混匀(1E+12个外泌体颗粒加入1mg MyD88)后，将溶液pH用Na 2CO3调节至8.0(或9.0、10.0)，然后用Capto core700纯化去除游离MyD88抑制剂多肽，收集流穿，即为装载了MyD88抑制剂多肽的CD24外泌体，ELISA检测MyD88抑制剂多肽装载量(多肽装载技术参见专利：202111263036.5)。
(3)PBMC炎症模型检测不同EV及蛋白的免疫抑制活性：利用PBMC细胞进行实验。同时利用1ug/ml anti-human CD3 Antibody(包被)及1ug/ml anti-human CD28  Antibody(游离)进行炎症模型造模，给予阳性药(地塞米松)，游离CD24-Fc融合蛋白，不同剂量的工程化装载二聚体CD24-EV及工程化装载二聚体CD24+MyD88抑制剂多肽-EV处理72h及120h，检测其对PBMC炎症反应的抑制活性。
2.结果：
如表3-4以及图9所示。
Figure PCTCN2022123202-appb-000005
表3.二聚体CD24外泌体装载MyD88抑制剂多肽结果
Figure PCTCN2022123202-appb-000006
表4.不同处理组PBMC上清中IFN-γ的浓度值
3.结论：与模型组相比，游离ApoE蛋白，装载二聚体CD24的外泌体及同时装载二聚体CD24和MyD88抑制剂多肽的外泌体在体外PBMC细胞炎症模型中均显示出了较好的炎症抑制活性。且同时装载二聚体CD24和MyD88抑制剂多肽的外泌体的作用时间更长。
实施例7：同时装载二聚体CD24和MyD88抑制剂多肽的外泌体在小鼠脓毒血症模型中显著降低死亡率。
MyD88抑制剂多肽能够发挥炎症抑制效力，有效延长药物作用时间。装载了该抑制剂多肽的CD24-外泌体用于小鼠脓毒血症模型，能够显著降低小鼠死亡率。
1.方法：
(1)二聚体CD24外泌体制备：质粒构建、细胞株筛选和外泌体制备过程与实施例1一致。
(2)MyD88抑制剂多肽装载：与实施例6一致。
(3)死亡率及血内炎症因子降低效果验证：选择8周雄性C57BL6小鼠进行实验。腹腔注射10mg/kg LPS进行造模、尾静脉给予不同剂量的CD24-MyD88抑制剂多肽-EV(每只鼠1E8-1E10个外泌体颗粒，1E9个外泌体装载1μg蛋白)。24小时内监测并记录不同时间点各组实验动物的死亡率。24h处死后采血检测细胞因子(IL6，IL10，IFN-γ)的水平。
2.结果：
如表5-7以及图10-11所示。
Figure PCTCN2022123202-appb-000007
表5.不同给药组小鼠血液内细胞因子IL6的浓度值
Figure PCTCN2022123202-appb-000008
表6.不同给药组小鼠血液内细胞因子IL10的浓度值
Figure PCTCN2022123202-appb-000009
表7.不同给药组小鼠血液内细胞因子IFN-γ的浓度值
3.结论：同时装载二聚体CD24和MyD88抑制剂多肽的外泌体在小鼠脓毒血症模型中可降低小鼠死亡率及血液内炎症相关因子(IL6，IL10，IFN-γ)的表达水平。
实施例8：同时装载二聚体CD24、二聚体ApoE和MyD88抑制剂多肽的外泌体在小鼠急性呼吸窘迫综合征模型中显著降低死亡率并保护肺组织完整性。
MyD88抑制剂多肽能够发挥炎症抑制效力，有效延长药物作用时间。装载了该抑制剂多肽的CD24-外泌体、ApoE-外泌体或CD24-ApoE-外泌体用于小鼠急性呼吸窘迫综合征模型，能够降低小鼠死亡率，保护肺组织完整性。
1.方法：
(1)CD24-ApoE外泌体制备：质粒构建、细胞株筛选和外泌体制备过程与实施例5一致。
(2)MyD88抑制剂多肽装载：CD24-ApoE外泌体与MyD88抑制剂多肽混匀(1E+12个外泌体颗粒加入1mg MyD88)后，将溶液pH用NaCO3调节至8.0(或9.0、10.0)，然后用Capto core700纯化去除游离MyD88，收集流穿，即为装载了MyD88抑制剂多肽的CD24-ApoE外泌体，ELISA检测MyD88装载量。
(3)肺组织保护效果验证：雄性C57BL/6小鼠(8-10周)用异氟醚麻醉，腹腔注射5mg/kg体重的LPS进行造模、注射LPS后24h尾静脉注射给予不同类型的CD24EV， 各1E9个外泌体颗粒(装载1μg蛋白)，通过死亡率检测肝保护效果。肺组织切片观察肺组织完整性。将Evans标记的5％白蛋白生理盐水以每kg体重4ml灌入小鼠右肺下叶内，30min计算肺泡液体清除率(Alveolar fuid clearance，AFC)。
2.结果：
如表8以及图12所示。
Figure PCTCN2022123202-appb-000010
表8.CD24-ApoE外泌体装载MyD88抑制剂多肽结果
3.结论：
装载了该抑制剂多肽的CD24-外泌体、ApoE-外泌体或CD24-ApoE-外泌体用于小鼠急性呼吸窘迫综合征模型，能够降低小鼠死亡率，保护肺组织完整性。
本发明可用其他的不违背本发明的精神或主要特征的具体形式来概述。因此，无论从哪一点来看，本发明的上述实施方案都只能认为是对本发明的说明而不能限制本发明，权利要求书指出了本发明的范围，而上述的说明并未指出本发明的范围，因此，在与本发明的权利要求书相当的含义和范围内的任何改变，都应认为是包括在本发明的权利要求书的范围。

Claims (24)

  1. 一种向ADAM10基因敲除的HEK293细胞外泌体中装载二聚体CD24的方法，其特征在于，具体步骤包括：
    (1)敲除HEK293细胞的ADAM10基因；
    (2)向敲除ADAM10基因的HEK293细胞的外泌体中装载二聚体CD24和/或二聚体ApoE蛋白；
    (3)装载量和/或功能验证。
  2. 如权利要求1所述的方法，其特征在于，步骤(1)中，采用Crispr基因编辑系统或方法实现HEK293细胞的ADAM10基因敲除。
  3. 如权利要求1或2所述的方法，其特征在于，步骤(2)中，装载的方法包括：以VB220306-1137jmq为载体，构建pCD24-Fc-Ig3(NPTN-Ig3)-TMD(NPTN-TMD)-ICD(EWI-F-ICD)质粒，与睡美人转座酶表达载体pCMV-(CAT)T7-SB100X共同转染或病毒载体感染到HEK293细胞和/或ADAM10 -HEK293细胞，筛选单克隆得到HEK293-CD24或ADAM10 -HEK293-CD24工程化稳转细胞株。
  4. 如权利要求1所述的方法，其特征在于，步骤(2)中，工程化稳转细胞株扩增培养至细胞密度为5E+06/mL后，5000rpm离心30min收集细胞上清，从细胞上清中纯化得到工程化装载二聚体CD24的外泌体。
  5. 如权利要求1所述的方法，其特征在于，步骤(3)中，验证装载量的方法为：收集细胞蛋白进行Western blot实验。
  6. 如权利要求1所述的方法，其特征在于，步骤(3)中，功能验证方法为：二聚体CD24外泌体在小鼠急性肝衰竭模型的死亡率以及肝完整度中所起作用。
  7. 如权利要求1所述的方法，其特征在于，步骤(2)中，向外泌体中装载二聚体ApoE蛋白。
  8. 如权利要求7所述的方法，其特征在于，向外泌体中装载二聚体ApoE时，以慢病毒质粒载体pSLenti-CMV--PGK-PuroWPRE为基础，构建慢病毒表达载体pApoE-Fc-Ig3(NPTN-Ig3)-TMD(NPTN-TMD)-ICD(EWI-F-ICD)质粒并包装病毒，按MOI＝10感染ADAM10 -HEK293细胞，感染后16h和24h进行培养基更换，筛选单 克隆得到ADAM10 -HEK293-ApoE工程化稳转细胞株。
  9. 如权利要求1所述的方法，其特征在于，步骤(2)中，向外泌体中装载二聚体CD24和二聚体ApoE蛋白。
  10. 如权利要求7所述的方法，其特征在于，向外泌体中装载二聚体CD24和二聚体ApoE时，将已构建的pApoE-Fc-Ig3(NPTN-Ig3)-TMD(NPTN-TMD)-ICD(EWI-F-ICD)质粒与已构建的pCD24-Fc-Ig3(NPTN-Ig3)-TMD(NPTN-TMD)-ICD(EWI-F-ICD)质粒共同经过转染或慢病毒感染HEK293细胞，筛选得到ADAM10 -HEK293-CD24-ApoE工程化稳转细胞株。
  11. 如权利要求7-10所述任一项的方法，工程化稳转细胞株扩增培养至细胞密度为5E+06/mL后，5000rpm离心30min收集细胞上清，从细胞上清中纯化得到工程化装载的外泌体。
  12. 如权利要求11所述的方法，其特征在于，步骤(3)中，验证实验为：PBMC炎症模型检测不同EV及蛋白的免疫抑制活性和/或建立小鼠急性肝衰竭模型，通过死亡率情况验证CD24EV的肝保护效果。
  13. 如权利要求1所述的方法，其特征在于，步骤(2)中，向外泌体中装载二聚体CD24的同时，也装载MyD88抑制剂多肽。
  14. 如权利要求13所述的方法，其特征在于，向外泌体中同时装载二聚体CD24和MyD88抑制剂多肽时，方法为：二聚体CD24外泌体与MyD88抑制剂多肽混匀后，调pH至8.0，用Capto core700纯化去除游离MyD88抑制剂多肽，收集流穿，得到装载了MyD88抑制剂多肽的CD24外泌体。
  15. 如权利要求14所述的方法，其特征在于，二聚体CD24外泌体与MyD88抑制剂多肽混匀比例为：1E+12个外泌体颗粒加入1mg MyD88抑制剂多肽。
  16. 如权利要求13所述的方法，其特征在于，步骤(3)中，验证方法为：PBMC炎症模型检测不同EV及蛋白的免疫抑制活性。
  17. 如权利要求13所述的方法，其特征在于，步骤(3)中，验证实验为：小鼠脓毒血症模型中的死亡率变化。
  18. 如权利要求1所述的方法，其特征在于，步骤(2)中，向外泌体中装载二聚体CD24的同时，也装载二聚体ApoE和MyD88抑制剂多肽。
  19. 如权利要求18所述的方法，其特征在于，步骤(3)中，验证实验为：小鼠急性呼吸窘迫综合征模型中显著降低死亡率并保护肺组织完整性实验。
  20. 一种由权利要求1-19所述方法制备得到的外泌体。
  21. 一种由权利要求20的外泌体在制备药物中的应用。
  22. 如权利要求21所述的应用，其特征在于，所述药物为炎症治疗的药物。
  23. 药物，其包括权利要求20所述的外泌体。
  24. 治疗方法，其包括给予权利要求23所述的药物。
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