Genetically engineered extracellular vesicle containing mitochondria and application thereof

Abstract

The invention discloses a gene engineering extracellular vesicle containing mitochondria and application of the gene engineering extracellular vesicle. The extracellular vesicle is prepared by introducing a target gene into a donor cell and then extracting; the target gene is selected from a CD38 gene, an IP3R gene or an RYR gene, and the donor cell is selected from a stem cell, a myocardial cell, a muscle cell, a nerve cell or an immune cell. Aiming at the technical problem of relatively low activity of transplanted mitochondria in the prior art, the invention designs that the mitochondria content of cells is increased through a genetic engineering means, and then the extracellular vesicles are taken as mitochondria carriers, so that the extracellular vesicles with high mitochondria content are obtained. The extracellular vesicles are used as a mitochondrial carrier to enhance the resistance of the extracellular vesicles to a high-calcium ion environment at a mitochondrial transplantation part, so that the treatment effect is enhanced.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a genetically engineered extracellular vesicle containing mitochondria and an application thereof. Background Art

[0002] Mitochondrial diseases are a group of primary or secondary metabolic disorders caused by mitochondrial dysfunction, which may affect almost all organs in the body and can occur at any age. They are the root cause of many diseases and there is currently no effective treatment. Mitochondrial transplantation is to regulate intracellular mitochondrial homeostasis by transferring functional mitochondria into cells with mitochondrial damage. In preclinical studies, mitochondrial transplantation therapy has been widely used in various diseases such as organ damage repair, tumor treatment, immune disorders, metabolic disorders, neurodegenerative diseases, and fibrosis. At present, there is an approved clinical therapy for mitochondrial transplantation therapy for the treatment of myocardial ischemia-reperfusion injury, which specifically uses mitochondria extracted from healthy tissues to perform intracardiac injections into the ischemic heart to restore the mitochondrial activity of myocardial cells and thus treat cellular oxidative damage; there are also some therapies in clinical research, such as Minovia using healthy mitochondria from the mother's cells of the child and transplanting them in vitro into the child's CD34+ hematopoietic stem cells to treat single large-scale mitochondrial DNA deletion syndrome (SLSMD). There are also some companies dedicated to improving mitochondrial activity, such as Luca science, which uses patented technology to improve mitochondrial activity by optimizing the mitochondrial extraction process.

[0003] Although there have been many mature studies on mitochondrial transplantation, the environment of mitochondrial transplantation contains too high calcium ion content, which will cause excessive opening of the mitochondrial permeability transition pore, resulting in a decrease in mitochondrial membrane potential and swelling of the mitochondrial membrane, leading to structural and functional damage, which seriously affects the activity of transplanted mitochondria and greatly reduces the therapeutic effect. Therefore, a modification method that can maintain mitochondrial activity in a high calcium environment is needed to ensure the activity of transplanted mitochondria. Summary of the invention

[0004] One of the purposes of the present invention is to provide an extracellular vesicle, which is prepared by extracting and introducing a target gene into a donor cell; The target gene is selected from CD38 gene, IP3R gene or RYR gene; The donor cells are selected from stem cells, cardiomyocytes, muscle cells, nerve cells or immune cells.

[0005] Furthermore, the target gene is introduced into the donor cells by transfection of a gene delivery vector.

[0006] In the present invention, the target gene can be in the form of mRNA, CRISPR-A, saRNA, plasmid, etc. In one embodiment of the present invention, a plasmid is used.

[0007] In the present invention, the gene delivery vector may be the one disclosed in patent CN 113082054 A, or may be a commercially available product, such as Lipofectamine™ 2000, Lipofectamine™ 3000, Lipofectamine™ StemTransfection Reagent, Lipofect5000 (Bio-Bio), ProteanFect (West Lake Coacervate), etc. In one embodiment of the present invention, CA3S2 disclosed in CN 113082054 A and commercially available Lipofectamine™ 3000 are used as gene delivery vectors.

[0008] In the present invention, the donor cells are derived from mammals; the types of the donor cells are one or more of stem cells, cardiomyocytes, muscle cells, nerve cells, and immune cells; stem cells can be selected from one or more of mesenchymal stem cells, induced pluripotent stem cells, hematopoietic stem cells, and neural stem cells; the source of mesenchymal stem cells can be one or more of fat, bone marrow, umbilical cord blood, and dental pulp; immune cells can be activated M2 macrophages.

[0009] In a specific embodiment of the present invention, the target gene CD38 gene is constructed into a plasmid and self-assembled with a gene delivery vector to form nanoparticles as a gene transfection reagent, which is used to transfect rat adipose-derived mesenchymal stem cells to obtain cells overexpressing the target gene, and then extracellular vesicles are prepared.

[0010] In the present invention, in order to improve the targeting of the extracellular vesicles, they can be further modified, and the modification method can be targeting-responsive lipid insertion modification or cholesterol modification. The targeted-responsive lipid material used for the targeted-responsive lipid intercalation modification is connected by three parts: a hydrophobic tail, a pathological microenvironment responsive linker and a targeting group; the hydrophobic tail includes a hydrophobic segment and a linker segment polyethylene glycol, the hydrophobic segment is cholesterol or phospholipid, and the phospholipid is selected from one or more of 1,2-dimyristylglycerol, distearoylphosphatidylethanolamine, dioleoylphosphatidylethanolamine, 1,2-bis(diphenylphosphino)ethane, 1,2-tetradecanoylphosphatidylethanolamine, dimyristoylphosphatidylcholine, dipalmitoylphosphatidylcholine and dioleoylphosphatidylcholine; the pathological microenvironment responsive linker is selected from one or more of a pH responsive linker, a ROS responsive linker, a hypoxia responsive linker and an enzyme responsive linker; the targeting segment is selected from one or more of amylopectin, heparin precursor, alginate, mannan, hyaluronic acid and sialic acid.

[0011] The second object of the present invention is to provide a method for preparing the above-mentioned extracellular vesicles, comprising the following steps: Step 1, introducing the target gene into donor cells; Step 2, culturing the cells obtained in step 1, and then extracting the culture medium to obtain extracellular vesicles.

[0012] Furthermore, in step 1, the target gene is first mixed with the gene delivery vector, and the mixture is added to the culture medium for culturing the donor cells.

[0013] Furthermore, the gene delivery vector CA3S2 is mixed with a plasmid carrying the CD38 gene to obtain self-assembled nanoparticles, and then the nanoparticles are added to a serum-free medium for transfection of mesenchymal stem cells, and after the transfection, the medium containing the nanoparticles is replaced with a serum-free medium for continued culture. The mass ratio of the CD38 gene to the gene delivery vector is 1:10-1:100, preferably 1:20-50, and more preferably 1:30.

[0014] Furthermore, in step 2, the extracellular vesicles are extracted by differential centrifugation or tangential flow filtration.

[0015] Furthermore, the serum-free culture medium that continues to be cultured is collected and subjected to differential centrifugation or tangential flow filtration, and the resulting precipitate is the extracellular vesicles containing mitochondria.

[0016] The third object of the present invention is to provide the use of the above-mentioned extracellular vesicles in the preparation of therapeutic drugs for diseases related to mitochondrial dysfunction.

[0017] Further, the diseases related to mitochondrial dysfunction include primary mitochondrial diseases and secondary mitochondrial diseases. Primary mitochondrial diseases include: Leber hereditary optic neuropathy (LHON), mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes (MELAS) syndrome, myoclonic epilepsy with irregular red fibers (MERRF) syndrome, single large-scale mitochondrial DNA deletion (SLSMD) syndrome, progressive external ophthalmoplegia (PEO), Leigh syndrome (LS), Pearson syndrome (PS), Kearns-Sayre syndrome (KSS), neuropathic ataxia retinitis pigmentosa (NARP) syndrome one or more. Secondary mitochondrial diseases include: neurodegenerative diseases, fibrotic diseases, ischemic heart disease, metabolic diseases one or more.

[0018] A fourth object of the present invention is to provide a drug-loaded vesicle, using the above-mentioned extracellular vesicle as a drug carrier.

[0019] In view of the technical problem of low activity of transplanted mitochondria in the prior art, the present invention is designed to increase the mitochondrial content of cells by genetic engineering means, and then use extracellular vesicles as mitochondrial carriers to obtain extracellular vesicles with high mitochondrial content. The present invention uses extracellular vesicles as mitochondrial carriers to enhance its resistance to the high calcium ion environment at the mitochondrial transplantation site, thereby enhancing the therapeutic effect. On the one hand, extracellular vesicles have a complete lipid bilayer structure similar to the cell membrane, which can shield the external ion environment and protect mitochondrial activity as much as possible; on the other hand, extracellular vesicles also have good biocompatibility and cell membrane properties, which can give the mitochondria inside it specific targeting and promote endocytosis of target cells. Therefore, delivering mitochondria in the form of extracellular vesicles is a very effective engineering method.

[0020] Mesenchymal stem cells are in a state of mitochondrial surplus for a long time because their metabolism tends to be glycolytic and their own energy demand is not high. They tend to provide their own mitochondria to damaged cells in the body to promote their repair. However, their ability to deliver mitochondria is limited, and the mitochondria expelled may be damaged mitochondrial fragments, so they need to be transformed to further enhance the quantity and quality of their extracellular mitochondria. The CD38 gene is a transmembrane glycoprotein that is closely related to intracellular calcium ion signals, which is beneficial to promote endoplasmic reticulum-mitochondrial calcium transfer, enhance mitochondrial function and extracellular release. The present invention designs and uses the CD38 gene (Gene ID: 25668) to genetically engineer mesenchymal stem cells to obtain extracellular vesicles containing more mitochondria. Based on the previous research of the inventor's research group (CN113082054A), the carrier material CA3S2 used in the present invention has excellent gene delivery ability, and its transfection positive rate and fluorescence intensity are significantly higher than the commercially available carrier lipofectamine™3000 (Lipo3000).

[0021] The present invention uses a specific gene vector CA3S2 to overexpress CD38 in mesenchymal stem cells, and then uses serum-free culture medium to culture. The culture medium is subjected to differential centrifugation to obtain extracellular vesicles, that is, first centrifugation at a low speed to remove cells and cell debris, and then high speed is used to obtain extracellular vesicles containing mitochondria. Compared with the commercially available vector Lipo3000, the obtained extracellular vesicles were used for verification, proving that it contains more mitochondria with better quality, and the vesicle membrane has a protective effect on mitochondria. At the same time, the present invention also explores the possible mechanism by which CD38 promotes mitochondrial exudation, and provides a variety of genetic modification ideas. In addition, the present invention also explores the time parameters of cholesterol modification, which can promote the membrane fusion of vesicles and cells, laying the foundation for further improving cell uptake efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Screening results for gene to vector transfection ratio.

[0023] Figure 2 These are the results of the MTT toxicity experiment of CA3S2 vector.

[0024] Figure 3 DLS size analysis of mitochondrial vesicles in the CA3S2 group.

[0025] Figure 4 Particle size analysis for NTA nanoparticles.

[0026] Figure 5 The figure shows the WB results of vesicle and mitochondrial markers.

[0027] Figure 6TEM image of mitochondrial vesicles.

[0028] Figure 7 is the number of mitochondria in a single vesicle containing mitochondria.

[0029] Figure 8 SIM images of mitochondria-containing vesicles.

[0030] Fig. 9 Comparison of total fluorescence and mean fluorescence intensity of mitochondrial vesicles.

[0031] Fig.10 The results are for the fluorescence positive rate in mitochondrial vesicles.

[0032] Fig.11 The results are for the mitochondrial ROS content in mitochondrial vesicles.

[0033] Fig.12 The results of membrane integrity test of mitochondrial vesicles.

[0034] Fig.13 The results of the proteinase K digestion protection experiment of mitochondrial vesicles.

[0035] Fig.14 These are the results of calcium and hydrogen peroxide protection experiments on mitochondrial vesicles.

[0036] Fig.15 The results of the cellular uptake experiment of mitochondrial vesicles.

[0037] Fig.16 These are the results of the cell mitochondrial membrane potential experiment.

[0038] Fig.17 The results of the cell oxygen consumption rate test.

[0039] Fig.18 Schematic diagram and results of animal visual movement experiment.

[0040] Fig.19 These are the results of animal electroretinogram measurements.

[0041] Fig. 20 These are the results of WB measurement of animal eye tissue.

[0042] Fig.21 The quantitative results of CD38, IP3R and mitochondrial calcium content after transfection.

[0043] Fig. 22 Results of incubation time screening for cholesterol modification of mitochondria-containing vesicles.

[0044] Fig.23 The results are for the fluorescence positive rate in mitochondria-containing vesicles derived from cardiomyocytes. DETAILED DESCRIPTION

[0045] The preferred embodiments of the present invention will be described in detail below in conjunction with examples. It should be understood that the following examples are provided only for the purpose of illustration and are not intended to limit the scope of the present invention. Those skilled in the art may make various modifications and substitutions to the present invention without departing from the purpose and spirit of the present invention.

[0046] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0047] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0048] The gene vector CA3S2 used in the following examples can be synthesized according to the method in patent CN 113082054 A.

[0049] In the following examples, the CD38 plasmid was commissioned to Shanghai Jingnuo Biotechnology Co., Ltd. for synthesis and construction. The plasmid size is about 6000 bp, contains the CD38 functional gene, and carries ampicillin resistance (prokaryotic) and kanamycin resistance (eukaryotic). Example 1

[0050] 1. Screening of the ratio of carrier material to gene dosage In the preliminary experiment, the results of cell transfection using different ratios (w / w) of CA3S2 vector and gene were first examined to screen the optimal dosage ratio. 1μg of GFP plasmid was used to transfect DC2.4 cells in 24-well plates, and the mass ratio of vector to gene was adjusted. Four parameters of 30:1, 50:1, 80:1, and 100:1 were screened, and the gene transfection effect was analyzed by inverted fluorescence microscopy.

[0051] like Figure 1 As shown, when the mass ratio of the vector to the gene was 30:1, the GFP fluorescence intensity was the highest, so the parameter of 30:1 was subsequently selected for genetic engineering of stem cells.

[0052] Afterwards, the MTT toxicity test of CA3S2 carrier was conducted. Mesenchymal stem cells were plated in 96-well plates, 10k cells / well, and 12 hours later, serum-free DMEM medium was used to prepare different concentrations of CA3S2 material buffer, and the buffer was incubated with cells for 24 hours. The medium was changed, and serum-free DMEM medium containing 1 mg / mL MTT was added to culture for 4 hours. The medium was removed, and 100 μL DMSO was added to each well and shaken to fully dissolve the purple crystals. The absorbance was measured at 490 nm.

[0053] like Figure 2As shown, when the concentration was 20 μg / mL, the cell activity reached more than 90%, and the concentration was positively correlated with toxicity, so 30:1 was selected as the optimal transfection ratio for the experiment.

[0054] 2. Genetic engineering of mesenchymal stem cells The CA3S2 gene vector was dissolved in anhydrous DMSO at a concentration of 200 μg / μl as the mother solution, and the vector was diluted in DEPC water to form solution A at a concentration of 0.6 μg / μl; the CD38 plasmid was dissolved in DEPC water to form solution B at a concentration of 0.02 μg / μl, and the mass ratio of the vector to the gene was controlled to be 30:1. Solution B was vortexed and added dropwise to solution A to obtain self-assembled nanoparticles. Afterwards, the nanoparticles were dissolved in serum-free culture medium (Keygene, KGL1214-500) at a concentration of 2 μg / ml (calculated as gene) and used as gene transfection drugs for rat adipose-derived mesenchymal stem cell transfection. 0.5 ml of nanoparticles was used for every 100,000 cells transfected. After 4 hours, the drug liquid was replaced with serum-free culture medium. After 48 hours of culture, the next step of extracellular vesicle extraction was performed.

[0055] For lipofectamine™3000 (Lipo3000), follow the instructions of the reagent and transfect mesenchymal stem cells with the same dosage (calculated in genes). The rest of the operations are the same as CA3S2.

[0056] The same volume of serum-free medium was used in the transfection process, and the obtained mesenchymal stem cells were used as a control (Control).

[0057] 3. Extraction and purification of extracellular vesicles The serum-free culture medium after engineering transformation of mesenchymal stem cells was collected, and first centrifuged at 600g for 5 minutes to remove suspended cells in the culture medium; then centrifuged at 2500g for 10 minutes to remove cell debris; finally centrifuged at 18000g for 30 minutes, and the precipitate obtained was the extracellular vesicles containing mitochondria.

[0058] The extracellular vesicles of each group prepared above were characterized and measured below.

[0059] 1. Characterization of the physical properties of extracellular vesicles The obtained precipitate was added to PBS and resuspended, and DLS particle size measurement and zeta potential measurement were performed at a protein concentration of 0.2 mg / ml; NTA measurement was performed at a protein concentration of about 1 mg / ml to obtain particle size distribution data.

[0060] like Figure 3As shown, the average particle size of the extracellular vesicles using CA3S2 carrier is 520.52 ± 11.12nm; the control group is 683.77 ± 22.26nm, and the Lipo3000 group is 580.25 ± 5.50nm. Since the size of mitochondria is mostly 1-2μm, and the size of fission mitochondria is smaller, 500μm-1μm, it can be seen from the vesicle particle size that vesicles of this size have the objective conditions to wrap mitochondria. Zeta potential is -1.37 ± 0.13mV (Control), -1.35 ± 0.06mV (Lipo3000), -1.76± 0.27mV (CA3S2).

[0061] like Figure 4 As shown, the number of extracellular vesicles increased after transfection.

[0062] 2. Protein extraction and component identification of extracellular vesicles The obtained precipitate was added to lysis buffer for lysis, vortexed in an ice bath for 30 seconds, waited for 4 minutes, and cycled 5 times for protein extraction. The protein was quantified using the BCA method. The protein components were semi-quantitatively analyzed by Western blot, including the internal reference protein β-actin, the extracellular vesicle marker CD81, and the mitochondrial markers COXⅠ, COXⅣ, and MT-ND4.

[0063] like Figure 5 As shown, the protein component contained extracellular vesicle marker CD81, as well as mitochondrial proteins COXⅠ, COXⅣ and mtND4; and the content was CA3S2 group>Lipo3000 group>Control group.

[0064] 3. Imaging and analysis of extracellular vesicles The obtained precipitate was added to the fixative, and then TEM pre-treatment and photography were performed, and the obtained image results were statistically analyzed for the number of mitochondria. Figure 6 and Figure 7 As shown, the number of mitochondria contained in the transfected vesicles is more distributed at higher values.

[0065] The obtained precipitate was added to a PBS solution containing DIO (5μM) and Mitotracker Red (400nM) dyes, and stained at 37°C for 30 minutes; then centrifuged at 18000g for 30 minutes, resuspended in PBS and centrifuged at 18000g for another 30 minutes. The final precipitate was resuspended in PBS at a concentration of 1mg / ml, and the solution was spread into a confocal dish and covered with a coverslip in a fixed position. Use a SIM microscope to take pictures to obtain images of the outer green circle and the inner red contents, which are the mitochondria wrapped in vesicles. Figure 8As shown, the vesicle membrane is intact and the contents contain mitochondria.

[0066] 4. Analysis of Mitochondrial Quantity Before genetic engineering of mesenchymal stem cells, the cells were stained with Mitotracker Green, i.e., the dye was diluted in serum-free medium at a concentration of 400nM, and stained at 37°C for 30min, followed by the steps of "genetic engineering" and "extraction and purification of extracellular vesicles". The mitochondria in the obtained extracellular vesicles will contain green fluorescence. The precipitate obtained by the above purification method was resuspended in PBS, and then analyzed by flow cytometry to obtain the data of positive rate and average fluorescence intensity; or after protein quantification, the same amount of protein was plated into a microplate for fluorescence intensity detection.

[0067] like Fig. 9 and Fig.10 As shown, the average and total fluorescence intensity, as well as the positive rate of the engineered extracellular vesicles were significantly increased; and the content was CA3S2 group>Lipo3000 group>Control group.

[0068] 5. Mitochondrial Quality Analysis Extracellular vesicles containing green fluorescent mitochondria were obtained according to the method in "Analysis of Mitochondrial Quantity". The vesicles were stained with mitoSOX (500nM, 37°C, 30min), then centrifuged at 18000g for 30min, resuspended in PBS and centrifuged again at 18000g for 30min. The final precipitate was resuspended in PBS at a concentration of 0.5mg / ml and analyzed using a flow cytometer. Using green fluorescence as a correction parameter, the fluorescence intensity of mitoSOX was compared, such as Fig.11 As shown, the mitoSOX fluorescence of the genetically engineered vesicles was weaker, indicating that the mitochondrial ROS in them was less and the quality was higher, and the mitochondrial quality was CA3S2 group>Lipo3000 group>Control group.

[0069] 6. Analysis of vesicle membrane integrity The obtained precipitate was stained with calcein-AM at a concentration of 2 μM, and then a portion of it was freeze-thawed for different times, and a portion was permeabilized with Triton. The positive rate was tested using a flow cytometer, such as Fig.12 As shown in the figure, the fluorescence positivity rate of vesicles that have been frozen and thawed three times is not significantly different from that of vesicles that have not been frozen and thawed (FT refers to the number of freeze-thaw cycles), that is, the vesicle membrane remains intact, reflecting the preservation stability of extracellular vesicles.

[0070] 7. Analysis of vesicle membrane protection ability The obtained precipitate was digested with proteinase K and compared with the undigested one. That is, 200 μg / mL proteinase K was used for digestion on ice for 15 min, and then 5 mM PMSF was used to terminate the digestion for 5 min. The protein expression was analyzed according to the "Protein extraction and component identification of extracellular vesicles". Fig.13 As shown, CD81 located on the vesicle membrane was destroyed by proteinase K, while the internal mitochondrial proteins remained unchanged, demonstrating the protective effect of the vesicle membrane on the internal mitochondria.

[0071] According to the method in "Mitochondrial Quantity Analysis", the stem cells were stained and genetically engineered, that is, the stem cells were stained with Mitotracker Green in advance, and then transfected with CD38 plasmid. After 24 hours, the vesicles in the culture medium and the mitochondria in the cells were extracted, and hydrogen peroxide and calcium chloride solutions of different concentrations were prepared. The vesicles and free mitochondria were placed in the prepared solutions for 24 hours, and then the fluorescence intensity was detected in a microplate. Fig.14 As shown, in high concentrations of hydrogen peroxide and calcium chloride, the percentage decrease in fluorescence of extracellular vesicles was less than that of free mitochondria, proving that vesicles can protect mitochondrial activity in harsh transplantation environments.

[0072] 8. Cellular efficacy Cell uptake: Before genetically engineering mesenchymal stem cells, the cells were stained with Mitotracker Red, that is, the dye was diluted in serum-free medium at a concentration of 400nM, and stained at 37°C for 30 minutes. After that, "genetic engineering" and "extracellular vesicle extraction and purification" were performed, and the mitochondria in the obtained extracellular vesicles will contain red fluorescence. The precipitate obtained by the above purification method was administered to cells at a dose of 40μg / ml, that is, the precipitated protein was quantified and resuspended in DMEM medium containing 10% serum, and then compared with GM10742 cells (immortalized B cells of human LHON patients with the same mitochondrial gene defect). After 24 hours, the cell positivity rate and the average fluorescence intensity data were obtained by flow cytometry. Fig.15 As shown, in terms of fluorescence intensity and positive rate, the order of GM10742 cells was CA3S2 group>Lipo3000 group>Control group, and the positive rate of CA3S2 group was more than 6 times that of Control group, indicating that CA3S2 group enabled GM10742 cells to obtain more exogenous mitochondria.

[0073] Analysis of mitochondrial membrane potential: GM10742 cells were plated in 24-well plates at 10w / well, and mesenchymal stem cells were genetically engineered. Vesicles were extracted after 48 hours. After protein quantification, they were resuspended in DMEM medium containing 10% serum and administered at a dose of 6μg / well. JC-1 staining was performed after 48 hours. JC-1 staining was performed according to the standard procedure of the kit (Biyuntian, C2006), and then confocal microscopy was used to photograph the cells. ImageJ software was used to perform fluorescence quantitative analysis of the cells. The ratio of red fluorescence (dye aggregates) to green fluorescence (dye monomers) is the relative value of mitochondrial membrane potential. Fig.16 As shown, the mitochondrial membrane potential increased after administration, and the treatment effect was CA3S2>Lipo3000>Control group.

[0074] Cellular oxygen consumption rate detection: Using the same administration method as mitochondrial membrane potential, the GM10742 cells after administration were plated at a density of 80k / well in a cell culture plate supporting a cell energy metabolism instrument (Agilent, SeahorseXFe96), and the detection was performed according to the instrument standard operating procedures. Fig.17 As shown, the cell oxygen consumption rate curve increased, and both the maximum oxygen consumption rate and respiratory potential increased, indicating the recovery of cell mitochondrial function.

[0075] 9. Animal efficacy Animal model: C57BL / 6J mice with in situ mutation of mitochondrial genes were used for the experiment. The animals were purchased from Aurora Bioscience.

[0076] Animal experiment process: The experimental groups were WT, PBS, IDE, and CA3S2. IDE is idebenone, which is the only clinically approved drug for the treatment of LHON. The WT group did not undergo any operation, the PBS group received an intraocular injection of 2 μl PBS solution, and the CA3S2 group received an intraocular injection of 1 μg mitochondrial vesicles. The mice began the first intraocular administration at 12 weeks of age (day 0), and the second administration at 13 weeks of age (day 7). The IDE group received daily idebenone gavage (60 mg / kg). All experimental mice in all groups underwent optokinetic and electroretinographic experiments at 15 weeks of age (day 21) and were killed to collect eyeball samples for western blot analysis.

[0077] Visual motion test: The mouse is placed on a circular platform with a radius of 6 cm, and surrounded by a black and white cardboard background. After the mouse adapts for 5 minutes, the cardboard background begins to rotate, and the number of times the mouse's head rotates following the background within 2 minutes is recorded, once in the clockwise and counterclockwise directions, with a 30-second rest period in between. The visual motion test reflects the mouse's vision status through animal behavior, such as Fig.18As shown, both the IDE group and the CA3S2 group were therapeutic, and the number of head rotations in the CA3S2 group was greater, indicating that the efficacy was better than that of clinically approved positive drugs.

[0078] Electroretinogram (ERG) experiment: Mice were placed in a dark room overnight to adapt to the dark, then anesthetized and placed on a platform. Ring electrodes were placed on the cornea, and needle electrodes were inserted into the skin and tail for subsequent measurements. Dark-adapted 0.01 ERG detection (flash intensity = 10 cd·s / cm2) was performed using the Espion Visual Electrophysiology System (Diagnosys LLC, USA). The ERG experiment measures the electrophysiological response of the optic nerve, reflecting the ability of retinal cells to respond to light, such as Fig.19 As shown, the CA3S2 group had a significant therapeutic effect, while the IDE group had no significant therapeutic effect.

[0079] Western blot detection: After the animals were killed, the eyeballs were used to extract proteins according to the standard procedure in the instruction manual (Keygen Biotechnology, KGB5303-100). Western blot detection was performed using the steps mentioned above, such as Fig. 20 As shown, the MT-ND4 and COX Ⅳ contents in both IDE and CA3S2 groups were increased, and the CA3S2 group was superior to the IDE group.

[0080] 10. Study on the mechanism of CD38 pathway Determination of CD38 protein expression: Mesenchymal stem cells were plated into 24-well plates at a density of 5w / well. 12 hours after plating, the cells were genetically engineered according to the transfection method of "genetic engineering". 12 hours after gene transfection, the cells were digested and incubated with PE-Anti-rat-CD38 antibody (Biolegend, 250505) at 37°C for 30 minutes, with an antibody dilution ratio of 1:100. Fluorescence detection was then performed using a flow cytometer. The experimental results showed that the expression of CD38 protein in both the Lipo3000 group and the CA3S2 group was increased, and the CA3S2 group was superior to the Lipo3000 group.

[0081] IP3R protein expression determination: The same steps as CD38 protein determination, 12 hours after gene transfection, cells were collected and lysed to extract protein. WB detection was performed using the aforementioned steps. The experimental results showed that the IP3R protein expression in both the Lipo3000 group and the CA3S2 group was increased, and the CA3S2 group was superior to the Lipo3000 group.

[0082] Mitochondrial calcium content determination: The steps for determining CD38 protein were the same. Rhod2-AM (Abcam, ab142780) was used for staining at 37°C for 30 minutes 12 hours after gene transfection, and the dye concentration was 2μM. Fluorescence detection was then performed using a flow cytometer. The experimental results showed that the mitochondrial calcium content of both the Lipo3000 group and the CA3S2 group increased, and the CA3S2 group was higher than the Lipo3000 group. Example 2

[0083] Cholesterol-modified mitochondrial-containing vesicles PBS solution was used to prepare a 0.02 mg / mL cholesterol buffer, and the obtained vesicles were placed in the cholesterol buffer and incubated at 4°C for 0.5 h, 24 h, and 48 h, respectively. The vesicles were then collected by centrifugation and the total lipid and cholesterol contents were detected.

[0084] like Fig. 22 As shown, there is no significant difference between the incubation time of 0.5 h and 24 h and 48 h. The incubation time of 0.5 h has made the cholesterol modification close to saturation, and a shorter time can preserve better mitochondrial activity. Therefore, 0.5 h can be selected as the incubation time for cholesterol modification. Example 3

[0085] Cardiomyocyte transfection AC16 cells were used to perform the experiment according to the transfection, staining and extraction methods in "Analysis of Mitochondrial Quantity", and the fluorescence positive rate of mitochondrial vesicles was determined by flow cytometry.

[0086] like Fig.23 As shown, after CD38 transfection, the fluorescence positivity rate of vesicles increased significantly, indicating the effectiveness of CD38 gene engineering in cardiomyocytes.

Claims

1. An extracellular vesicle, characterized in that It is prepared by introducing the target gene into donor cells and then extracting it; The target gene is selected from CD38 gene, IP3R gene or RYR gene; The donor cells are selected from stem cells, cardiomyocytes, muscle cells, nerve cells or immune cells.

2. The extracellular vesicle according to claim 1, characterized in that The target gene is introduced into the donor cells through gene delivery vector transfection.

3. The extracellular vesicle according to claim 1, characterized in that The stem cells are selected from mesenchymal stem cells, induced pluripotent stem cells, hematopoietic stem cells or neural stem cells.

4. The extracellular vesicle according to claim 1, characterized in that The extracellular vesicles are also decorated with lipid materials.

5. The method for preparing extracellular vesicles according to claim 1, characterized in that: The following steps are involved: Step 1, introducing the target gene into donor cells; Step 2, culturing the cells obtained in step 1, and then extracting the culture medium to obtain the extracellular vesicles.

6. The preparation method according to claim 5, characterized in that: In step 1, the target gene is first mixed with the gene delivery vector, and the mixture is added to the culture medium for culturing the donor cells.

7. The preparation method according to claim 5, characterized in that: In step 2, the extracellular vesicles are extracted by differential centrifugation or tangential flow filtration.

8. Use of the extracellular vesicles according to claim 1 in the preparation of therapeutic drugs for diseases related to mitochondrial dysfunction.

9. The use according to claim 9, characterized in that: The mitochondrial dysfunction-related disease is a primary mitochondrial disease or a secondary mitochondrial disease.

10. A drug-loaded vesicle, characterized in that: The extracellular vesicles according to claim 1 are used as drug carriers.

Images