Application of OPA1-enriched exosome in preparation of medicine for improving mitochondrial dysfunction

Abstract

The invention discloses an application of an OPA1-enriched exosome in preparation of a medicine for improving mitochondrial dysfunction. The preparation method comprises the following steps: screening by adopting a phage display technology to obtain a CD9-targeted polypeptide P13, forming a fusion protein OPA1-P13 from the P13 and OPA1, constructing a recombinant plasmid for expressing the fusion protein, transfecting a cell for co-expression, and extracting the exosome, thereby obtaining the OPA1-enriched exosome OPA1-Exo. The OPA1-Exo is used for treating the mitochondrial dysfunction, and the result shows that the OPA1-Exo can activate antioxidant enzyme to reduce the ROS level in cells, promote an oxidative phosphorylation pathway to improve the ATP content of mitochondria, obviously improve a mitochondrial network and recover the mitochondrial dysfunction, so that the obvious mitochondrial function repair potential is shown. According to the invention, the OPA1 protein is successfully used for treating the mitochondrial dysfunction in the form of the exosome.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and more specifically, relates to the application of OPA1-enriched exosomes in the preparation of drugs for improving mitochondrial dysfunction. Background Art

[0002] When the body undergoes oxidative metabolism, the ETC in mitochondria is prone to a large amount of electron leakage, causing oxygen molecules to be reduced to form reactive oxygen species (ROS). Excessive ROS leads to mtDNA mutations, causing mitochondrial dysfunction, affecting mitochondrial homeostasis, and triggering various diseases.

[0003] Exosomes are tiny vesicles secreted by cells containing nucleic acids, proteins, and lipids. Exosomes have high targeting and the ability to penetrate biological barriers, low immunogenicity and high biocompatibility, multi-functional payloads and potential for combination therapy, can reduce side effects and enhance stability, and have natural sources and potential for large-scale production. Therefore, they can be used as drug delivery carriers for the treatment of various diseases.

[0004] OPA1 (Optic Atrophy 1) is an inner mitochondrial membrane fusion protein that participates in regulating the mitochondrial fusion process. Research shows that OPA1 plays a key role in regulating the fusion and fission processes of mitochondria, which is crucial for maintaining mitochondrial bioenergy production and cellular metabolic homeostasis. The morphological changes of mitochondria directly affect their function. The absence of OPA1 leads to mitochondrial fragmentation, thus affecting cell energy metabolism and viability. OPA1 can reduce oxidative stress damage in cells and improve mitochondrial dysfunction-related diseases. However, the OPA1 protein itself does not exist in exosomes, limiting its use in the form of exosomes for the treatment of mitochondrial dysfunction. Summary of the Invention

[0005] The purpose of the present invention is to overcome the above-mentioned defects and deficiencies existing in the prior art, and provide the application of OPA1-enriched exosomes in the preparation of drugs for improving mitochondrial dysfunction.

[0006] The second purpose of the present invention is to provide the application of OPA1-enriched exosomes in the preparation of drugs for treating related diseases caused by mitochondrial dysfunction.

[0007] The above objects of the present invention are achieved by the following technical solutions: The present invention first prepared OPA1 - enriched exosomes (OPA1 - Exo). First, the polypeptide P13 and OPA1 were formed into a fusion protein OPA1 - P13, then a recombinant plasmid expressing the fusion protein OPA1 - P13 was constructed and transfected into cell culture, and exosomes were extracted to obtain OPA1 - enriched exosomes OPA1 - Exo; that is, the coding sequence of polypeptide P13 was connected to the 3' end or 5' end of the OPA1 protein coding sequence to obtain the OPA1 - P13 fusion gene, then a recombinant plasmid expressing the OPA1 - P13 fusion gene was constructed, and then the recombinant plasmid was transfected into cells for co - expression, and exosomes were extracted to obtain OPA1 - enriched exosomes OPA1 - Exo; the amino acid sequence of the polypeptide P13 is shown in any one of SEQ ID No.1 - 4.

[0008] A peptide is a molecule formed by amino acids linked by peptide bonds, which can promote the interaction between proteins, thus forming a stable ternary complex or enhancing the existing protein - protein interaction.

[0009] CD9, also known as tetraspanin 9, belongs to the tetraspanin superfamily and is an important membrane protein. CD9 is one of the marker proteins of exosomes, naturally present on the exosome membrane, and is commonly used for the detection, separation and identification of exosomes, especially in the targeted application of engineered exosomes.

[0010] In the present invention, a phage library was incubated with CD9 protein to screen out the polypeptide P13 that targets and binds to CD9, and it was linked to the end of OPA1 protein through gene recombination technology to form a fusion protein OPA1 - P13 to promote the binding of OPA1 and CD9. Since CD9 is a transmembrane protein that already exists in exosomes, the polypeptide P13 can bind OPA1 protein to CD9, thus helping the OPA1 protein to achieve trans - organelle transport from the nucleus to exosomes, enabling it to be enriched in exosomes. Specifically, a recombinant plasmid expressing the fusion protein OPA1 - P13 was constructed (that is, the coding sequence of polypeptide P13 was linked to the 3' end or 5' end of the OPA1 protein coding sequence to obtain the OPA1 - P13 fusion gene, and then a recombinant plasmid expressing the OPA1 - P13 fusion gene was constructed), and transfected into cell culture; the polypeptide P13 can link OPA1 to CD9 in target exosomes, promoting the binding of OPA1 and CD9 to obtain OPA1 - enriched exosomes (OPA1 - Exo). When the amino acid sequences of OPA1 protein and polypeptide P13 are known, those skilled in the art can obtain the coding gene sequences of OPA1 protein and polypeptide P13 by conventional technical means and optimize them according to the host codon preference.

[0011] Furthermore, it also includes constructing a recombinant plasmid of the pro-vesicle secretion gene RAB31, and then co-transfecting the two recombinant plasmids, namely the recombinant plasmid expressing the OPA1-P13 fusion gene and the RAB31 recombinant plasmid promoting exosome secretion, into cells for co-expression, and extracting exosomes to obtain OPA1-enriched exosomes (OPA1-RAB31-Exo). RAB31 can further increase the yield of exosomes, and thus increase the content of OPA1 protein in exosomes, which can increase the yield of exosomes, thereby increasing the amount of OPA1 protein per milliliter of exosomes. The RAB31 protein is a small GTPase belonging to the RAS oncogene family (RAB subfamily), and plays a key role in intracellular membrane trafficking and signal regulation. As a member of the Rab family, RAB31 is activated by the GTP-bound state, recruits downstream effector proteins, and regulates vesicle formation, trafficking, and fusion with the target membrane. It is crucial for the functional integrity of the Golgi apparatus and the trans-Golgi network (TGN). When the amino acid sequence of the RAB31 protein is known, those skilled in the art can obtain the coding gene sequence of the RAB31 protein by using conventional technical means and optimize it according to the host codon preference.

[0012] The present invention uses the OPA1-enriched exosomes OPA1-Exo of the present invention to act on the 293T cell model with H 2 O 2 -induced oxidative damage, and detects the antioxidant activity index of cell mitochondria. The results show that the OPA1-enriched exosomes OPA1-Exo have an obvious improvement effect on mitochondrial dysfunction, indicating that the OPA1 protein successfully plays a role in the treatment of mitochondrial dysfunction in the form of exosome expression.

[0013] Specifically, the present invention adds the OPA1-enriched exosomes OPA1-Exo to the 293T cell model with H 2 O 2 -induced oxidative damage, evaluates its effect on cellular antioxidant stress, and measures indexes such as the activities of antioxidant enzymes SOD1, SOD2, CAT, and GSH-Px, the content of ROS, and the content of ATP in cells. The results show that OPA1-Exo reduces the ROS level in 293T cells by activating antioxidant enzymes, increases the ATP content in mitochondria by promoting the oxidative phosphorylation pathway, and improves the mitochondrial network structure, restoring mitochondrial function. It shows that OPA1-Exo has application value in improving mitochondrial dysfunction or treating related diseases caused by mitochondrial dysfunction.

[0014] Therefore, the present invention first provides the application of the above OPA1-enriched exosomes OPA1-Exo in the preparation of a drug for improving mitochondrial dysfunction.

[0015] The present invention also provides the use of the OPA1-enriched exosomes OPA1-Exo in the preparation of a drug for treating related diseases caused by mitochondrial dysfunction.

[0016] Preferably, the present invention also provides the use of the above OPA1-enriched exosomes OPA1-RAB31-Exo in the preparation of a drug for improving mitochondrial dysfunction or in the preparation of a drug for treating related diseases caused by mitochondrial dysfunction. Since RAB31 can further increase the amount of OPA1 protein in exosomes, compared with OPA1-Exo, OPA1-RAB31-Exo can further enhance the therapeutic effect on mitochondrial dysfunction.

[0017] Furthermore, the mitochondrial dysfunction is mitochondrial dysfunction caused by oxidative damage.

[0018] Furthermore, the drug clears ROS by upregulating the expression of antioxidant enzymes in the subject, promotes the oxidative phosphorylation pathway in the subject to increase the ATP content, improves the mitochondrial network structure in the subject and restores mitochondrial function, thereby achieving the improvement of mitochondrial dysfunction.

[0019] Furthermore, the subject is selected from mammals.

[0020] Furthermore, the mammals are selected from mice, cats, dogs, pigs, cows, horses, sheep, monkeys, and humans, etc.

[0021] Furthermore, the drug further comprises other pharmaceutically acceptable excipients.

[0022] Furthermore, the coding sequence of the polypeptide P13 is connected to the OPA1 protein coding sequence through a Flag tag, that is, the polypeptide P13 is connected to the OPA1 protein through a Flag tag.

[0023] Furthermore, the coding sequence of the polypeptide P13 is connected to the 3' end of the OPA1 protein coding sequence, that is, the polypeptide P13 is connected to the C-terminus of the OPA1 protein. The present invention constructs pcDNA3.1-OPA1-P13-C and pcDNA3.1-OPA1-P13-N recombinant plasmids, connects the polypeptide P13 to the C-terminus or N-terminus of the OPA1 protein through gene recombination technology to form an OPA1-polypeptide P13 fusion protein expression system, and after transfection of cells (engineered gene cells), the OPA1-enriched exosomes are successfully constructed. Through the analysis of the content of OPA1 in transfected cells (engineered gene cells), exosomes (engineered exosomes), and target cells (target cells) in the N+ and C+ groups, more co-localization of OPA1 and mitochondria can be seen in the C + group in target cells, indicating that connecting the polypeptide P13 at the C-terminus can more effectively promote the transport of OPA1 to mitochondria in target cells.

[0024] Furthermore, the amino acid sequence of the polypeptide P13 is shown in SEQ ID No.1: GKYCMATCCSMMA. Four polypeptide sequences capable of binding to the CD9 recombinant protein were screened from a 12-peptide phage peptide library in the present invention. After phage ELISA detection, the polypeptide with the sequence GKYCMATCCSMMA has a stronger binding force.

[0025] Furthermore, the template plasmid of the recombinant plasmid is pcDNA3.1, that is, pcDNA3.1-OPA1-P13 and pcDNA3.1-RAB31 recombinant plasmids are constructed.

[0026] Furthermore, the cell is 293T cell.

[0027] Furthermore, the extraction method of the exosomes is ultracentrifugation.

[0028] Compared with the prior art, the present invention has the following beneficial effects: The present invention provides the application of OPA1-enriched exosomes in the preparation of drugs for improving mitochondrial dysfunction. In the present invention, a phage library is incubated with a CD9 recombinant protein to screen out the targeting polypeptide P13. The polypeptide P13 and the OPA1 protein form a fusion protein OPA1-P13, and then a recombinant plasmid expressing the fusion protein OPA1-P13 is constructed and transfected into cells for culture. The polypeptide P13 can link to CD9 in OPA1-targeted exosomes, promote the binding of OPA1 and CD9, and help the OPA1 protein achieve transcellular transport and enrichment from the cell interior to exosomes. The OPA1-enriched exosomes OPA1-Exo are obtained by extracting the exosomes. When OPA1-Exo is used for the treatment of mitochondrial dysfunction, the results show that OPA1-Exo can activate antioxidant enzymes to reduce the ROS level in cells, promote the oxidative phosphorylation pathway to increase the mitochondrial ATP content, can significantly improve the mitochondrial network, and restore mitochondrial dysfunction, thus showing obvious potential for mitochondrial function repair. The present invention successfully realizes the use of the OPA1 protein in the form of exosomes for the treatment of mitochondrial dysfunction. Description of the Drawings

[0029] Figure 1 For screening CD9 affinity peptides using phage ELISA in Example 1. Among them, *: p<0.05, **: p<0.01.

[0030] Figure 2 Schematic diagrams of two modes of connection between P13 and the OPA1 terminus in Example 1.

[0031] Figure 3 For Co-IP detection of N + and C+ Protein binding of the recombinant protein to CD9.

[0032] Figure 4 For the ELISA determination of Flag and OPA1 contents in Example 1. Among them, Figure 4 A is the content of Flag in 293T-OPA1 transfected cells; B is the contents of Flag and OPA1 in exosomes secreted by 293T-OPA1 cells; C is the content of Flag in cells after co-culturing 293T with exosomes for 3 days. Among them, *: p < 0.05, ns: p > 0.05.

[0033] Figure 5 For the detection of engineered exosomes in Example 1. Among them, Figure 5 A is the microscopic structure images of Exo, OPA1-Exo and OPA1-RAB31-Exo under transmission electron microscopy (scale bar = 100 nm); B is the detection of the secretion levels of Exo, OPA1-Exo and OPA1-RAB31-Exo by BCA method, ns: p > 0.05; C is the detection of the particle size distribution of Exo, OPA1-Exo and OPA1-RAB31-Exo by nanoparticle tracking; D-E are the detection of exosome markers and OPA1 protein in Exo, OPA1-Exo and OPA1-RAB31-Exo by Western blot, and the quantitative analysis of OPA1 expression level based on the band gray value. Among them, #: p < 0.05 compared with the 293T group, *: p < 0.05 compared with the Exo group, **: p < 0.01 compared with the Exo group.

[0034] Figure 6 For the detection of antioxidant enzyme expression levels and ROS contents in Example 2. Among them, Figure 6 A is the detection of the activities of SOD1, SOD2, CAT and GSH-Px in normal 293T cells (Control group), oxidatively damaged 293T cells (H 2 O 2 group) and oxidatively damaged 293T cells with OPA1-RAB31-Exo added (H 2 O 2 -OPA1-RAB31-Exo group); B-C are the determination of cellular ROS by staining with CM-H2DCFDA probe and measurement by immunofluorescence and FACS (scale bar = 50 μm). Among them, *: P < 0.05, **: P < 0.01.

[0035] Figure 7 For the detection of ATP level in Example 2. Among them, **: P < 0.01.

[0036] Figure 8 It is a diagram for fluorescence detection of mitochondrial network structure (Mito-Tracker Green fluorescent probe) in Example 2. Among them, Figure 8 A in it is the mitochondrial structure of 293T cells induced by H 2 O 2 ; B is the mitochondrial structure of 293T cells after treatment with OPA1-Exo, scale bar = 50 μm.

[0037] Figure 9 This is a schematic diagram of the present invention. Detailed implementation manners

[0038] The present invention will be further described below in conjunction with the specification drawings and specific embodiments, but the embodiments do not impose any form of limitation on the present invention. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in the technical field.

[0039] Unless otherwise specified, the reagents and materials used in the following examples are all commercially available.

[0040] 1. Experimental materials Table 1 Cell sources Cell Characteristic description Source 293T Human embryonic kidney cells Merck KGaA <![CDATA[293T-OPA1 (N + )]]> Human embryonic kidney cells transfected with pcDNA3.1-OPA1-P13-N plasmid Constructed in the present invention <![CDATA[293T-OPA1 (C + )]]> Human embryonic kidney cells transfected with pcDNA3.1-OPA1-P13-C plasmid Constructed in the present invention 293T-OPA1-RAB31 Human embryonic kidney cells transfected with pcDNA3.1-OPA1-P13-C and pcDNA3.1-RAB31 plasmids Constructed in the present invention E.coli DH5α Host bacterium for plasmid construction Stored in the laboratory Table 2 Plasmid sources Plasmid Genetic marker and construction Source pcDNA3.1-OPA1-P13-N plasmid KpnI and XbaI restriction enzyme sites were designed at both ends of the P13-Flag-OPA1 sequence Wuhan Kingcare pcDNA3.1-OPA1-P13-C plasmid KpnI and XbaI restriction enzyme sites were designed at both ends of the OPA1-Flag-P13 sequence Wuhan Kingcare pcDNA3.1-RAB31 plasmid KpnI and XbaI restriction enzyme sites were designed at both ends of the RAB31 sequence Wuhan Kingcare DMEM powder, from Gibco; antioxidant enzyme activity assay kit, from Dojindo Molecular Technology Inc; fetal bovine serum FBS, from ThermoFisher; HEK293T cells are stored in the laboratory.

[0041] 2. Experimental methods 2.1 Cell culture (1) Preparation work in the cell room: If the cell room is being disinfected with ultraviolet light, turn it off and turn on the fluorescent lamp for illumination. Turn on the 37°C water bath, and put the DMEM medium and PBS for treating cells taken out from the 4°C chromatography cabinet into the water bath to heat; change into the special white coat and slippers for the cell room, wear a mask and hat, and spray the sleeves of the white coat with 75% ethanol and disinfect the hands. Disinfect the microscope stage with an alcohol cotton ball. After ultraviolet irradiation, start ventilation and fluorescent lamp illumination, and pull the glass baffle of the workbench to the appropriate position. Take out the DMEM medium and PBS that have returned to room temperature, disinfect them, and place them in the ultra-clean workbench for standby; take out the disinfected pipette tips needed from the 55°C oven, disinfect them with 75% ethanol, and place them on the left side of the ultra-clean workbench for standby. Turn on the special centrifuge for the cell room. Carefully take out the cells to be disposed of, place them on the disinfected microscope stage, and observe the cell growth status, whether there is contamination, etc. After preparation, start to process the cells.

[0042] (2)Cell culture conditions: 293T cells are cultured using DMEM medium. Among them, 50 mL of fetal bovine serum (FBS) that has been filtered through a filter and 5 mL of penicillin-streptomycin (double antibody) are added to every 445 mL of DMEM to prepare a complete medium. The cells are placed in a sterile cell culture incubator at a temperature of 37°C and containing 5% CO 2 . Regularly observe the cell growth status and decide whether to further process. After the cells grow to an appropriate density, they are passaged, plated, cryopreserved, etc. after being digested with sterile trypsin for subsequent experimental needs.

[0043] (3)Cell resuscitation: Take a 15 mL centrifuge tube and add 3 mL of complete medium for standby. Take out the cryopreserved cells from liquid nitrogen and heat them in a 37°C water bath. After thawing, disinfect the surface of the cryopreservation tube. Quickly transfer the thawed cell suspension to the prepared complete medium in a laminar flow hood; centrifuge at 800 rpm for five minutes. After centrifugation, discard the supernatant in the laminar flow hood and resuspend the cells with complete medium. Transfer them to a T25 cell culture flask at a ratio of 1:1, shake gently, and then culture in an incubator.

[0044] (4)Cell passage: Take out the cell culture flask and discard the old medium. Wash it once with PBS; add an appropriate amount of trypsin. After an appropriate time, observe under a microscope. If the cells gradually become independent spherical shapes at this time, immediately add complete medium with a volume four times that of trypsin to terminate digestion; gently pipette the digested cells down and transfer them to a 15 mL centrifuge tube. Centrifuge at 800 rpm for 5 min; take back the centrifuged cells, discard the supernatant in the centrifuge tube in a laminar flow hood, add an appropriate amount of complete medium to resuspend, and gently pipette evenly. Transfer them to a T25 cell culture flask at a ratio of 1:3 and continue to culture.

[0045] (5)Cell cryopreservation: Take out the cell culture flask and discard the old medium. Wash it once with PBS; add an appropriate amount of trypsin for digestion. After terminating digestion, gently pipette the cells and transfer them to a 15 mL centrifuge tube. Centrifuge at 800 rpm for 5 min; take back the centrifuged cells, discard the supernatant in the centrifuge tube in a laminar flow hood, add 1 mL of cell cryopreservation solution (prepared by mixing FBS: DMSO = 9:1) to resuspend, and gently pipette evenly. Then transfer them to a pre-prepared cryopreservation tube; finally, perform gradient cooling on the cells (4°C, 30 min; -20°C, 1.5 h; -80°C, 12 h) and then transfer them to liquid nitrogen for storage for later use.

[0046] Example 1 Preparation of OPA1-enriched exosomes I. Experimental method 1. Target peptide screening Target peptide screening was performed according to the operating instructions of the phage display peptide library kit (Tec Biotech, China). The library contained billions of fd-tet phage clones, and each clone displayed a random sequence of exogenous peptides at the N-terminus of the fd-tet phage coat protein. First, the phage library was incubated in a culture flask at 37 °C for 1 hour to remove phages that specifically bound to the flask. Then, after pretreatment to remove non-specifically bound phages, the remaining phage library was incubated with recombinant CD9 protein at room temperature for 1 hour. The CD9 protein was washed 10 times with bovine serum albumin (BSA) / Tween wash buffer to remove unbound phages. Phages bound to the CD9 protein were eluted for 10 minutes using a low pH elution buffer (0.1 N HCl, 1 mg / mL BSA, adjusted to pH 2.2 with glycine). The elution buffer was immediately neutralized with 1 M Tris-HCl (pH 8.8). The eluate from the first round of elution was concentrated using a Centricon 100 kDa ultrafiltration device (Merckmillipore, Germany). Then, the concentrated eluate was transferred into Escherichia coli medium and incubated with continuous shaking at 37 °C for 24 hours. Phage clones that internalized the CD9 protein were recovered using cell lysis buffer (2% sodium dodecyl sulfate, 10 mM Tris-HCl, 2 mM EDTA, pH 8.0). The cells were centrifuged at 130 g for 10 minutes, the supernatant was removed, and cell lysis buffer was added. Then the phages were amplified and further screened, similar to the above-described first round procedure. After the third round of screening, the eluted phages were titrated, and 40 clones were randomly selected for sequencing to determine the sequence of the CD9-binding peptide.

[0047] 2. Affinity verification The binding ability and specificity of the selected phages to CD9 were determined by enzyme-linked immunosorbent assay (ELISA). Prepare 150 μL of CD9 at a concentration of 100 μg / mL (dissolved in 0.1 M NaHCO 3 , pH 8.6) and coat it on a 96-well plate, then gently shake and incubate at 4 °C for 30 minutes, followed by overnight incubation at 4 °C. Subsequently, add 2×10 9 cfu of purified phages in blocking buffer and incubate at room temperature for 1 hour. Then, wash the plate three times with PBS containing 0.5% Tween 20 and then three more times with PBS. Thereafter, incubate the plate with alkaline phosphatase-conjugated anti-fd phage IgG (Abcam, MA, USA) at room temperature for 1 hour. After washing, add the substrate p-nitrophenyl phosphate to the wells and measure the absorbance at 405 nm using a plate reader.

[0048] 3. Synthesis and transfection of plasmids In this invention, the pcDNA3.1 plasmid was used as a template, and KpnI and XbaI restriction endonuclease sites were designed at both ends of the P13-Flag-OPA1 and OPA1-Flag-P13 sequences. Recombinant plasmids named pcDNA3.1-OPA1-P13-C (i.e., pcDNA3.1-OPA1-Flag-P13) and pcDNA3.1-OPA1-P13-N (i.e., pcDNA3.1-P13-Flag-OPA1) were constructed. 293T cells were evenly seeded in a 6-well plate and cultured until the cell density reached 30% - 50%. Take a 1.5 mL centrifuge tube, add 200 μL of serum-free medium and mix it with 3 μg of each of the above recombinant plasmids; take another centrifuge tube and mix 200 μL of serum-free medium with 6 μL of transfect-mate. Using the transfection method mediated by Lipofectamine 2000, react at room temperature for 5 minutes, then combine the contents of the two tubes and continue to react for 20 minutes. Add the transfection complex to the 6-well plate pre-laid with 2 mL of serum-free medium and shake well. Incubate in a cell culture incubator for 5 hours, then change to complete medium and continue to culture for 48 hours, and then collect the cells for subsequent experiments. The cells transfected with pcDNA3.1-OPA1-P13-N were named N + The cells transfected with pcDNA3.1-OPA1-P13-C were named C + (same as 293T-OPA1 in the following). At the same time, BstXⅠ and NotⅠ restriction enzyme sites were introduced at both ends of the RAB31 gene. After the plasmid pcDNA3.1 was digested with the corresponding restriction enzymes, it was ligated with the RAB31 gene fragment of interest to construct the pcDNA3.1-RAB31 recombinant plasmid; according to the above method, the pcDNA3.1-OPA1-P13-C recombinant plasmid and the pcDNA3.1-RAB31 recombinant plasmid were co-transfected into cells, and the cells were named 293T-OPA1-RAB31

[0049] 4. Co-immunoprecipitation (Co-IP) The co - immunoprecipitation technique was used to detect the fusion expression of OPA1 and CD9. The transfected cells were lysed, proteins were extracted, and incubated overnight at 4°C with the primary antibody (anti - Flag). Incubate for 4 hours at 4°C according to the guidelines provided by the manufacturer (Invitrogen, Carlsbad, CA, USA). After completing the immunoprecipitation step, the magnetic beads were washed three times with 1× phosphate - buffered saline (PBS). Subsequently, the proteins were eluted from the magnetic beads using 40 μL of elution buffer and applied to immunoblot analysis. Sample buffer containing 5% β - mercaptoethanol was added to the samples and heated at 55°C for 15 minutes, and then the binding with the target proteins was detected by Western blotting. The target proteins detected were OPA1 (diluted at a ratio of 1:1000, dissolved in TBST buffer), CD9 (diluted at a ratio of 1:1000, dissolved in TBST buffer), and Flag (diluted at a ratio of 1:1000, dissolved in TBST buffer).

[0050] 5. Exosome extraction After incubating 293T - OPA1 cells, 293T - OPA1 - RAB31 cells, and 293T cells in exosome - free medium for 48 hours, the culture supernatants were collected. First, the samples were centrifuged at 300 g for 10 minutes, then ultra - centrifuged at 2000 g for 10 minutes. Next, ultra - centrifugation was carried out at 10000 g for 30 minutes. The resulting cell pellets, membranes, and debris were discarded, and the supernatants were filtered through a 0.22 μm filter membrane from Merck Millipore. Finally, ultra - centrifugation was carried out at 120000 g for 90 minutes to isolate exosomes, and these exosomes were resuspended in PBS and washed at 120000 g for 90 minutes. The exosomes extracted from 293T were named Exo, the exosomes extracted from 293T - OPA1 were named OPA1 - Exo, and the exosomes extracted from 293T - OPA1 - RAB31 were named OPA1 - RAB31 - Exo.

[0051] 6. ELISA detection of mitochondrial internalization Take 293T cells (as target cells) and seed them in culture dishes, and culture them to an appropriate confluence (70% - 90%) to ensure good growth status and the ability to receive exosomes during co - culture. The exosomes extracted from the engineered gene cells in the N+ and C+ groups were respectively added to the 293T cell culture system, and co - cultured in a cell culture incubator. After the co - culture, the transfected cells (engineered gene cells in the N+ and C+ groups) and target cells (293T cells) were collected respectively. Mitochondrial protein extraction was performed on the transfected cells and target cells using a mitochondrial extraction kit. Then, proteins in the transfected cells, exosomes, and target cells were extracted respectively.

[0052] Operate according to the instructions of the ELISA kit using Flag and OPA1, and measure the contents of Flag and OPA1 proteins in each group of samples respectively. Use an enzyme-linked immunosorbent assay (ELISA) reader to read the absorbance values of the ELISA plate, and calculate the concentrations of Flag and OPA1 in each group of samples according to the standard curve.

[0053] 7. Western Blot (1) After one cell passage, Western Blot detection was performed, and a part of the protein lysate was extracted from the cells for Western Blot detection. First, wash the cells with pre-cooled PBS at 4 °C for 1 minute each time, for a total of 3 times, to ensure complete removal of residual liquid. Then, prepare the lysis buffer according to the ratio of 1 mL RIPA plus 10 μL PMSF (100 mM), shake well and place on ice. Subsequently, add 400 μL of the lysis buffer to each flask of cells and lyse on ice for 30 minutes, shaking the culture flask intermittently during this period to promote full reaction of the cells. After lysis, quickly scrape the cells to one side of the culture flask using a cell scraper on ice, and use a pipette to transfer the cell debris and lysis buffer to a 1.5 mL EP tube. Then, centrifuge these EP tubes at 8000 g for 10 minutes at 4 °C. After centrifugation, take the supernatant and store it at -20 °C for subsequent detection.

[0054] (2) Subsequently, the BCA protein concentration was measured. First, dissolve BSA in PBS to prepare a series of standard products with concentrations of 5, 2.5, 1, 0.5, 0.25, 0.125, 0.05, and 0.025 mg / mL. Then, take 20 μL of each concentration of the standard product and the total protein sample, add them to a 96-well plate, and set two replicates for each standard product and sample. Next, mix the solution A and solution B of the BCA kit at a volume ratio of 50:1 to form the working solution. After adding 200 μL of the working solution to each well, place the 96-well plate in an incubator at 37 °C for 30 minutes. Finally, use an ELISA reader to read the OD value at a wavelength of 562 nm, and draw a standard curve based on the OD values and concentrations of the standard products to obtain the protein concentration of the total protein sample.

[0055] (3) Next, prepare the SDS-PAGE gel, including a 15% separating gel and a 5% stacking gel. First, pour the separating gel into the gap between the glass plates to a position 1.5 cm from the upper edge, and add an appropriate amount of 75% ethanol on the upper layer. After the separating gel solidifies, pour out the upper-layer ethanol, pour in the stacking gel, then insert the comb and let it dry naturally.

[0056] (4)Before electrophoresis, the total protein sample was heated in a 95°C water bath for 5 minutes and mixed with protein loading buffer. Then, electrophoresis buffer was poured into the electrophoresis tank, and 10 μL of protein marker was added to the lanes on both sides, and 15 μL of the sample was added to each lane. During electrophoresis, first, a voltage of 90 V was used for 30 minutes in the stacking gel stage, and then a voltage of 160 V was used for electrophoresis in the separating gel stage until the bromophenol blue ran to the bottom of the gel.

[0057] (5)After electrophoresis, transfer was performed. First, filter paper of appropriate size and a 0.22 μm PVDF membrane were cut, and the PVDF membrane was activated with methanol for 1 minute. Then, the transfer cassette was assembled in the order of "sponge - filter paper - gel - PVDF membrane - filter paper - sponge", and it was ensured that there were no bubbles in it. After assembly, the transfer cassette was inserted into the transfer tank, and transfer buffer was poured in. Under ice bath conditions, a constant current of 200 mA was used for 60 minutes of transfer operation.

[0058] (6)After transfer, antibody incubation was carried out. First, the membrane was washed with TBST solution for 5 minutes, and blocked with 5% skim milk powder (prepared with PBS solution) at room temperature for 1 hour. Subsequently, the membrane was washed 3 times with TBST solution, 5 minutes each time. Then, the primary antibody diluted with primary antibody diluent was added and incubated overnight at 4°C. The next day, the membrane was washed 3 times with TBST solution, 5 minutes each time, then the secondary antibody diluted with secondary antibody diluent was added and incubated on a shaker at room temperature for 1 hour. Finally, the membrane was washed again 3 times with TBST solution, 5 minutes each time.

[0059] (7)Finally, luminescence detection was performed. After adding ECL luminescence solution and incubating for 3 minutes, exposure imaging was carried out. Finally, the gray values of the internal reference gene and the target gene were measured using ImageJ software, thus completing the entire Western Blot detection process.

[0060] According to the recommendations of the "Minimum Information for Studies of Extracellular Vesicles (MISEV2018)", in this invention, Western Blot was used to detect exosome positive markers OPA1, CD9, and ALIX, and the corresponding antibodies used were diluted at ratios of 1:1000, 1:500, and 1:1000 respectively.

[0061] 8. Nanoparticle Tracking Analysis Nanoparticle tracking analysis (NTA) was performed using a ZetaView PMX 110 (Particle Metrix) and its software (ZetaView 8.02.28). The exosomes were diluted in particle-free PBS and placed in the sample chamber. Subsequently, their size and concentration were measured at a wavelength of 405 nm, and finally, the particle size of the exosomes was quantitatively analyzed and recorded.

[0062] 9. Transmission Electron Microscopy 10 μL of the exosome sample was dropped onto a copper grid and incubated at room temperature for 10 minutes. Then, negative staining was performed with 2% uranyl acetate and air-dried at the edge of the filter paper. The prepared sample was imaged using an H-7650 transmission electron microscope (Hitachi) at 80 kV. The detection method for mitochondria was the same as described above.

[0063] 10. Statistical Analysis Statistical analysis of continuous variables was expressed as "mean ± standard deviation". Statistical analysis was performed using GraphPad Prism 10.0. Independent sample t-tests or one-way ANOVA were used to analyze the statistics between groups. A p value less than 0.05 was considered statistically significant.

[0064] II. Experimental Results 1. Four polypeptide sequences that could bind to the CD9 recombinant protein were screened out from a 12-peptide phage peptide library. These polypeptide sequences and binding frequencies are shown in Table 3. After phage ELISA detection, the polypeptide with the sequence GKYCMATCCSMMA had stronger binding ability, and the results are as Figure 1 shown. This polypeptide sequence was named P13.

[0065] Table 3 Polypeptide sequences screened by phage and the binding frequency of polypeptides to CD9 Polypeptide sequence Polypeptide binding frequency GKYCMATCCSMMA (SEQ ID No.1) 25 AGMTCMASCCKYM (SEQ ID No.2) 19 GKMTCYCMASCMG (SEQ ID No.3) 16 GCASMKYCCMMTS (SEQ ID No.4) 10 2. After confirming that P13 could bind to CD9, the coding sequences of the P13 sequence, Flag tag, and OPA1 protein were ligated into a recombinant plasmid, and the OPA1-P13 recombinant protein was expressed in 293T cells to enable targeted binding of this protein to CD9. Given that previous studies have not reported whether the ligation of polypeptides to OPA1 affects its protein structure, it was planned to ligate Flag and P13 from the end of the protein sequence, but it was uncertain whether to ligate them to the N-terminus or C-terminus of OPA1. Therefore, these two ligation methods were studied. When P13 was ligated to the N-terminus of OPA1, it was named the N + group, and when ligated to the C-terminus, it was named the C + group, as Figure 2 shown.

[0066] 3. After constructing the above recombinant plasmid and transfecting 293T cells, the protein-protein interactions in the cells were detected by Co-IP. The results are as follows: Figure 3 As shown, it can be seen that there is no Flag band in the untransfected cells (293T group), and the content of OPA1 is significantly less than that in the N + and C + groups. The reason is that after plasmid transfection, OPA1-P13 with Flag was overexpressed. After separation with Flag magnetic beads, it was found that the 122 kDa OPA1-CD9 complex appeared in both the N + and C + groups, indicating that P13 can bind to CD9 regardless of which end of OPA1 it is connected to, which clarifies the role of P13.

[0067] 4. To clarify the content of OPA1 in the transfected cells (engineered gene cells), exosomes (engineered exosomes), and target cells (target cells) in the N + and C + groups, exosomes were extracted from the cells of the N + and C + groups respectively. The exosomes were co-cultured with 293T cells, and the mitochondria of the transfected cells and target cells were extracted. The ELISA results of Flag and OPA1 are as follows: Figure 4 As shown: In the transfected cells, there was no significant difference in the total content of Flag between the two groups. The Flag in the mitochondria of the C + group was slightly higher than that in the N + group, but there was no significant statistical difference (A in Figure 4 ). In the exosomes, there was also no statistical difference in the content of Flag and OPA1 (B in Figure 4 ). However, in the target cells, the Flag in the mitochondria of the C + group was significantly more than that in the N + group, indicating that a large amount of OPA1-P13 molecules in the exosomes entered the mitochondria (C in Figure 4 ). The above results suggest that connecting P13 at the C-terminus may promote the transport of OPA1 to the mitochondria in the target cells. Therefore, theoretically, the C + group is more conducive to mitochondrial targeted therapy.

[0068] 5. In the subsequent experiments, the recombinant plasmid pcDNA3.1-OPA1-P13-C with P13 connected to the C-terminus will be used. Under the transmission electron microscope, the exosomes showed a vesicle-like structure, and a double-membrane structure could be seen in some fields of view, as shown in A in Figure 5 . The level of exosome secretion by the cells was detected by the BCA method. The results are as follows: Figure 5As shown in B of [reference], in a T25 culture flask with a cell density of 80%, 39.55 ± 7.25 μg of Exo can be isolated from every 1 mL of culture medium, while the yield of OPA1-Exo is 42.87 ± 6.97 μg / mL, and the yield of OPA1-RAB31-Exo is 40.77 ± 6.5 μg / mL. There is no significant statistical difference among the three. The results of nanoparticle tracking detection are as shown in Figure 5 C of [reference]. The particle size ranges of Exo, OPA1-Exo, and OPA1-RAB31-Exo are all in the interval of 40 - 150 nm, and there is no significant difference in the particle size concentration among the three. By detecting the markers contained in exosomes through Western blot, the results are as shown in Figure 5 D of [reference]. In 293T, 293T-OPA1, and 293T-OPA1-RAB31 cells, compared with Exo, OPA1-Exo, and OPA1-RAB31-Exo, the expression levels of CD9 and ALIX in cells are lower, while the expression level of GAPDH is higher. This is because at the unit concentration, exosomes contain more relevant markers, while exosomes basically do not contain housekeeping proteins. Affected by plasmid overexpression, the OPA1 protein in 293T-OPA1 and 293T-OPA1-RAB31 cells is 40.34% and 39.22% higher than that in 293T cells respectively. In exosomes, the OPA1 content in OPA1-Exo exosomes is 11 times that of Exo, and the OPA1 content in OPA1-RAB31-Exo exosomes is further increased compared with OPA1-Exo exosomes, which is 16.1 times that of Exo ( Figure 5 E of [reference]). This result indicates that the engineered exosomes enriched with OPA1 were successfully constructed based on the polypeptide P13 strategy.

[0069] Example 2 Improvement of H by OPA1-enriched exosomes 2 O 2 In vitro study on inducing mitochondrial dysfunction in 293T cells I. Experimental methods 1. Establishment of cell model 293T cells were cultured in DMEM (GIBCO BRL) medium containing 10% heat-inactivated fetal bovine serum (FBS) and 1% antibiotic / antifungal solution (GIBCO BRL). 293T cells were exposed to 500 μM H 2 O 2 in FBS-free DMEM for 30 min to induce oxidative stress, and then the protected groups were treated with 1×10 7 cfu / ml suspended in FBS-free DMEM. The cells were seeded at 2.0×10 6 cells / 100 mm 2Inoculate the culture dish with the density, and add OPA1-Exo for culture.

[0070] The cell experiments were divided into 3 groups, namely: blank group (Control), H 2 O 2 group, H 2 O 2 group with OPA1-Exo added (H 2 O 2 -OPA1-Exo).

[0071] 2. Detection of antioxidant enzymes and ROS The activities of SOD1, SOD2, CAT and GSH-Px were determined according to the kit instructions (Dojindo Molecular Technology Inc.). The intracellular production of ROS was quantified by using the cell-permeable oxidation-sensitive fluorescent probe CM-H2DCFDA (Molecular Probes). Treat the 293T cells co-cultured with OPA1-Exo with up to 500 μM of H 2 O 2 for 16 hours. Seed the 293T cells at a density of 2.5×10 5 cells per well in a six-well plate containing complete medium, and pre-treat with DMEM containing 1 μM CM-H2DCFDA at 37°C for 30 minutes. Harvest the cells, wash them twice with DMEM, resuspend them in DMEM, and analyze them on a FACSCalibur flow cytometer (BD Biosciences). Culture the cells in a normal medium in an 11 cm 2 culture dish (BD Biosciences), and treat them with 1 μM CM-H2DCFDA for 30 minutes. Wash the cells 3 times in the normal medium, and then analyze them at 37°C using a Leica laser scanning confocal microscope with an excitation wavelength of 488 nm and an emission wavelength of 500–540 nm.

[0072] 3. ATP level detection Operate according to the instructions of the ATP kit. First, aspirate the culture medium, add 200 μL of lysis solution to each well of the 6-well plate, repeatedly pipette and lyse, then centrifuge at 12000 g for 5 minutes at 4 °C, take the supernatant for subsequent determination. Then thaw the reagents to be used on ice, and dilute the ATP standard solution with ATP detection lysis solution to 0.01, 0.03, 0.1, 0.3, 1, 3, and 10 μM. Add 100 μL of ATP detection working solution to the light-shielded 96-well plate, and let it stand at room temperature for 3 - 5 min to consume all the background ATP, thereby reducing the background. Add 20 μL of the test solution or standard solution to the well, quickly mix, select the luminometer function of the full-wavelength multifunctional microplate reader to detect the chemiluminescence value, draw a standard curve, and calculate the ATP content of each group of cells according to the luminescence value.

[0073] 4. Fluorescence detection of mitochondrial network structure (Mito-Tracker Green fluorescent probe) Prepare a 1 mM stock working solution of Mito-Tracker Green solution using anhydrous DMSO and store it in the dark at -20 °C for later use; Transfer cells in good growth state to a culture dish at a density of 1×10 5 , routinely culture each group of cells. After 24 h of culture, remove the culture medium, add the pre-warmed Mito-Tracker Green staining working solution at 37 °C, and incubate for 2 h; After the incubation, replace the staining solution with fresh culture medium and observe the mitochondrial network structure under a laser confocal microscope.

[0074] 5. Statistical analysis Continuous variables in statistical analysis are expressed as "mean ± standard deviation". Statistical analysis is performed using GraphPad Prism 10.0. Independent sample t-tests or one-way ANOVA are used to analyze the statistics between groups. A p value less than 0.05 indicates a statistically significant difference.

[0075] II. Experimental results 1. OPA1-Exo activates antioxidant enzymes and reduces the ROS level in 293T cells To study the effect of OPA1-Exo on the mitochondrial antioxidant activity of mammalian cells. First, treat 293T cells with hydrogen peroxide to establish an oxidative damage cell model, and then add OPA1-Exo and H 2 O 2 to the 293T cell culture as the experimental group and the control group respectively. The results are as Figure 6 shown. It was found that in the experimental group compared with the control group, the antioxidant enzyme SOD1 was restored, and the expression levels of SOD2 and CAT were significantly increased ( Figure 6In A), CM-H2DCFDA probe staining was used to detect cellular ROS, and the quantitative analysis graphs all showed that the ROS in the experimental group was significantly reduced ( Figure 6 as shown in B-C). Therefore, our data indicate that OPA1-Exo can scavenge intracellular ROS by upregulating the expression of antioxidant enzymes, and its action pathway is as Figure 9 shown.

[0076] 2. OPA1-Exo promotes the oxidative phosphorylation pathway to increase ATP content Due to the impairment of the oxidative phosphorylation pathway in cells with mitochondrial dysfunction, the anaerobic glycolysis pathway in cells is enhanced, and the ATP content decreases accordingly. Therefore, we investigated whether OPA1-Exo affects the oxidative phosphorylation pathway to increase ATP content. For this purpose, OPA1-Exo was transfected into 293T cells. An ATP assay kit was used to measure the ATP level in the cells. The results are as Figure 7 shown. After co-culture with OPA1-Exo, the ATP level in the cells increased significantly, indicating that OPA1-Exo strengthened the oxidative phosphorylation pathway in the cells, thereby increasing the ATP content.

[0077] 3. OPA1-Exo improves the mitochondrial network structure Mitochondrial dysfunction can lead to the destruction of the mitochondrial network structure, and the mitochondrial morphology usually changes from slender tubular structures to fragmented spherical structures. After Mito-Tracker Green fluorescence staining of cells in each group, as shown in A of Figure 8 , the mitochondria of M293T cells induced by H 2 O 2 were fragmented and the structure was incomplete; while when OPA1-Exo was transfected into 293T cells, it was found that the mitochondria were interconnected and presented a network shape, as shown in B of Figure 8 , indicating that OPA1-Exo can significantly improve the mitochondrial network and the mitochondrial dysfunction is restored. Further, OPA1-RAB31-Exo was used to conduct in vitro studies on mitochondrial dysfunction according to the above method, and the results showed that OPA1-RAB31-Exo can better improve the mitochondrial dysfunction induced by H 2 O 2 compared with OPA1-Exo. The present invention successfully realizes the use of OPA1 protein in the form of exosomes for the treatment of mitochondrial dysfunction.

Claims

1. Application of OPA1-enriched exosomes in the preparation of a drug for improving mitochondrial dysfunction, characterized in that: The preparation method of the OPA1-enriched exosomes is to connect the coding sequence of the polypeptide P13 to the 3' end or the 5' end of the OPA1 protein coding sequence to obtain the OPA1-P13 fusion gene, then construct a recombinant plasmid expressing the OPA1-P13 fusion gene, and then transfect the recombinant plasmid into cells for co-expression, and extract exosomes to obtain; the amino acid sequence of the polypeptide P13 is shown in any one of SEQ ID No. 1 to 4.

2. Use of OPA1-enriched exosomes in the preparation of a drug for treating diseases related to mitochondrial dysfunction, characterized in that: The preparation method of the OPA1-enriched exosomes is to connect the coding sequence of the polypeptide P13 to the 3' end or 5' end of the OPA1 protein coding sequence to obtain the OPA1-P13 fusion gene, then construct a recombinant plasmid expressing the OPA1-P13 fusion gene, and then transfect the recombinant plasmid into cells for co-expression, and extract exosomes to obtain; the amino acid sequence of the polypeptide P13 is shown in any one of SEQ ID No. 1 to 4.

3. The use according to claim 1 or 2, characterized in that: The preparation method of the OPA1-enriched exosomes also includes constructing a RAB31 recombinant plasmid, and then co-transfecting the recombinant plasmid expressing the OPA1-P13 fusion gene and the RAB31 recombinant plasmid into cells for co-expression, and extracting exosomes to obtain OPA1-enriched exosomes.

4. The use according to claim 1 or 2, characterized in that: The amino acid sequence of the polypeptide P13 is GKYCMATCCSMMA.

5. The use according to claim 1 or 2, characterized in that: The coding sequence of the polypeptide P13 is connected to the 3' end of the coding sequence of the OPA1 protein.

6. The use according to claim 1 or 2, characterized in that: The coding sequence of the polypeptide P13 is connected to the coding sequence of the OPA1 protein via a Flag tag.

7. The use according to claim 1 or 2, characterized in that: The template plasmid of the recombinant plasmid is pcDNA3.

1.

8. The use according to claim 1 or 2, characterized in that: The drug removes ROS by upregulating the expression of antioxidant enzymes in the subject, promotes the oxidative phosphorylation pathway in the subject to increase ATP content, improves the subject's mitochondrial network structure and restores mitochondrial function, thereby improving mitochondrial dysfunction.

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