An engineered exosome for targeted intervention of mitochondrial gene expression and its construction method and application

Abstract

The present invention provides an engineered exosome for targeted intervention of mitochondrial gene expression and a preparation method and application thereof, and relates to the field of biomedical technology. The present invention combines exosomes with nucleic acid adsorption elements with mitochondrial targeting function to improve the stability of drug serum; the use of naturally derived exosomes greatly reduces drug toxicity and more effectively exerts therapeutic effects while ensuring safety. The mitochondrial gene intervention system constructed by the present invention is simple to prepare and can achieve precise delivery of mitochondria. As a carrier of gene therapy, it is of great significance.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to an engineered exosome for targeted intervention of mitochondrial gene expression, and a construction method and application thereof. Background Art

[0002] Mitochondria are double-membrane organelles that are ubiquitous in eukaryotic cells. They are the center of cell life activities and play an important role in cell energy metabolism and regulating programmed cell death. A large number of studies have shown that mitochondrial dysfunction is an important factor in the occurrence and development of a variety of diseases, which is closely related to abnormal mitochondrial gene expression.

[0003] Mitochondria have their own genome (mt-DNA), which replicates independently of the host genome, encodes 2 rRNAs, 22 tRNAs, and participates in the formation of 4 respiratory enzyme complexes. In addition, the mitochondrial genome can also encode a variety of non-coding RNAs, such as lncRNA, circRNA, sncRNA, and dsRNA, which can regulate mitochondrial function through RNA-RNA interactions, microRNA sponges, transcriptional regulators, protein translation templates, etc., thereby participating in various pathways such as cell death, proliferation, and differentiation. A large number of studies have shown that abnormal mitochondrial gene expression is the cause of mitochondrial dysfunction and further causes various diseases. For example, high expression of mc-COX2 leads to cell proliferation and promotes tumorigenesis. MitomiR-2392 reprograms cancer cells to enhance their drug resistance by regulating mitochondrial transcription in an AGO2-dependent manner. LIPCAR is closely related to the survival rate of patients with heart failure. Therefore, regulating mitochondrial gene expression may be an effective means to improve mitochondrial function and treat a variety of diseases.

[0004] Currently, there are two main strategies and methods for mitochondrial targeting: direct conjugation of mitochondrial targeting ligands to drugs, and drug delivery via nanocarriers with mitochondrial targeting function.

[0005] Direct conjugation of mitochondrial targeting ligands with drugs includes delocalized lipophilic cation guides (DLC) and mitochondrial targeting peptide fragments (MTS). Delocalized lipophilic cations mainly include triphenylphosphine (TPP), its derivatives such as octadecyltriphenylphosphine (STPP), triethylamine salts, dequalinium chloride (DQA), racemic-tetraphenylporphine, etc., which can carry small molecule drugs to target the inside of mitochondria under the drive of mitochondrial membrane potential, thereby exerting a role in regulating mitochondrial function. Mitochondrial targeting peptides are considered to be alternatives to DLC. The lipophilic residues (phenylalanine, tyrosine, isoleucine) and cationic residues (arginine, lysine) they carry are important reasons for their mitochondrial targeting.

[0006] In addition, drug delivery of nanocarriers with mitochondrial targeting function has also been widely studied. In this type of carrier, mitochondrial targeting is usually achieved by modifying molecules with mitochondrial targeting function. Taking the early desquinoxaline-based liposomes as an example, liposomes can obtain mitochondrial targeting through the delocalized positive charges present in them, thereby being able to deliver a series of molecules of different sizes, such as DNA and paclitaxel. In addition, polymer dendrimers combined with TPP+ are also used for mitochondrial targeted delivery. Their main advantage is that the surface functional groups can be precisely controlled, and the peripheral groups can be functionalized by different groups, making them multifunctional nanocarriers.

[0007] However, these targeted strategies all have some disadvantages. Most DLCs can produce mitochondrial toxicity at high concentrations, resulting in a significant decrease in ATP and a reduction in cell survival. Although MTS has been used to deliver a variety of cargoes (such as nucleic acids and proteins), its application is limited due to its large size, poor solubility and membrane permeability, and low serum stability. Targeted nanocarriers can ensure that drugs are efficiently enriched in the lesion site while reducing drug accumulation in normal tissues and organs, but their high immunogenicity and inability to cross biological barriers (such as the blood-brain barrier) greatly reduce their therapeutic effects. Summary of the invention

[0008] In view of this, the purpose of the present invention is to provide an engineered exosome for targeted intervention of mitochondrial gene expression and a construction method and application thereof, which can accurately target mitochondria and efficiently deliver RNA.

[0009] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0010] The present invention provides an engineered exosome for targeted intervention of mitochondrial gene expression, wherein the engineered exosome comprises a mitochondrial targeting element, a nucleic acid adsorption element and a target RNA;

[0011] The mitochondrial targeting element and the nucleic acid adsorption element are electrotransferred into the engineered exosomes;

[0012] The engineered exosomes are loaded with the target RNA.

[0013] The present invention also provides a method for constructing the above-mentioned engineered exosomes, comprising the following steps: (1) selecting RNA having a function of regulating mitochondria as the target RNA, and constructing a related vector thereof;

[0014] (2) using the relevant vector obtained in step (1) to transfect exosome donor cells, collecting exosomes in the cell supernatant, and obtaining exosomes loaded with the target RNA;

[0015] (3) chemically synthesizing the mitochondrial targeting element and the nucleic acid adsorption element to obtain a loading material;

[0016] (4) electrotransferring the loading material into the exosomes loaded with the target RNA in step (2) to obtain the engineered exosomes;

[0017] There is no temporal sequence between step (1) and step (2) and step (3).

[0018] Preferably, the exosome donor cells in step (2) include HEK293T cells, or donor cells with targeting or therapeutic functions.

[0019] Preferably, the mitochondrial targeting element in step (3) includes TPP, and the nucleic acid adsorption element includes PDL 10 .

[0020] Preferably, for TPP and PDL 10 Chemical synthesis, including TPP, PDL 10 , EDC and DMAP were mixed and dissolved, reacted at 40°C for 8h, the product was loaded into a dialysis bag with a molecular weight cutoff of 1000Da, dialyzed in pure water, and the retained material was collected to obtain the loaded material TPP-PDL 10 .

[0021] Preferably, the loading in step (4) comprises loading the loading material into the exosomes by electroporation; during the electroporation, the voltage is set to 700 V, the current is set to 150 mF, the thickness of the electroporation cup is 4 mm, and the pulse electroporation is performed 5 times; and the precipitate is collected by centrifugation.

[0022] Preferably, the centrifugal force of the centrifugation is 12000g and the centrifugation time is 30min.

[0023] The present invention also provides the use of the engineered exosomes or the engineered exosomes constructed by the above-mentioned construction method in the preparation of gene drugs for improving serum stability.

[0024] The present invention also provides the use of the engineered exosomes or the engineered exosomes constructed by the above construction method in the preparation of gene drugs for treating sepsis;

[0025] The target gene expressed by the engineered exosomes is circRNA SCAR.

[0026] Beneficial effects: The present invention provides an engineered exosome for targeted intervention of mitochondrial gene expression, which fuses the exosome with a nucleic acid vector with mitochondrial targeting function to improve the stability of drug serum; the use of natural exosomes greatly reduces drug toxicity and more effectively exerts therapeutic effects while ensuring safety. In the embodiment of the present invention, TPP with mitochondrial targeting function is chemically coupled with the nucleic acid adsorption element PDL 10 Combined with TPP-PDL, it is loaded into exosomes by electroporation. While ensuring serum stability and safety, TPP-PDL 10 The high positive charge of TPP-PDL enables lysosomal escape, thereby effectively delivering the drug to the mitochondria to achieve the therapeutic purpose; specifically, 10 Rich in positive charge, exosomes are internalized by cells to form endosomes. When endosomes fuse with lysosomes, TPP-PDL 10 It can be released by exosomes and achieve lysosomal escape through the proton sponge effect, thereby effectively delivering the target RNA to mitochondria. The mitochondrial gene intervention system constructed by the present invention is simple to prepare and can achieve precise delivery to mitochondria. As a carrier of gene therapy, it is of great significance. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 Exos Mito TPP-PDL can be successfully 10 Delivery to macrophage mitochondria;

[0028] Figure 2 Exos Mito Available through TPP-PDL 10 Successfully delivered RNA into macrophage mitochondria;

[0029] Figure 3 Strategies for constructing engineered exosomes. DETAILED DESCRIPTION

[0030] The present invention provides an engineered exosome for targeted intervention of mitochondrial gene expression, wherein the engineered exosome comprises a mitochondrial targeting element, a nucleic acid adsorption element and a target RNA;

[0031] The mitochondrial targeting element and the nucleic acid adsorption element are loaded on the engineered exosomes;

[0032] The engineered exosomes are loaded with the target RNA.

[0033] In the present invention, the mitochondrial targeting element preferably includes TPP (2-(carboxyethyl)triphenylphosphonium chloride); the nucleic acid adsorption element preferably includes PDL 10 (polylysine). The present invention preferably combines the above-mentioned TPP and PDL 10 Connect to form TPP-PDL 10 , are loaded on the exosomes together. The TPP-PDL of the present invention 10 Rich in positive charge, exosomes are internalized by cells to form endosomes. When endosomes fuse with lysosomes, TPP-PDL 10 It can be released by exosomes and achieve lysosomal escape through the proton sponge effect, thereby effectively delivering the target RNA to the mitochondria.

[0034] The present invention also provides a method for constructing the above-mentioned engineered exosomes, such as Figure 3 As shown, the steps include: (1) selecting RNA with mitochondrial regulation function as target RNA, and constructing its related vector;

[0035] (2) using the relevant vector obtained in step (1) to transfect exosome donor cells, collecting exosomes in the cell supernatant, and obtaining exosomes loaded with the target RNA;

[0036] (3) chemically synthesizing the mitochondrial targeting element and the nucleic acid adsorption element to obtain a loading material;

[0037] (4) loading the loading material onto the exosomes loaded with the target RNA in step (2) to obtain the engineered exosomes;

[0038] There is no temporal sequence between step (1) and step (2) and step (3).

[0039] The present invention selects RNA that regulates mitochondrial function as the target RNA and constructs its related vector. The present invention does not specifically limit the category of the target RNA. CircRNA SCAR is used as an example in the embodiment, but it cannot be identified as the entire protection scope of the present invention.

[0040] After obtaining the relevant vector, the present invention uses the obtained relevant vector to transfect exosome donor cells, collects the exosomes in the cell supernatant, and obtains exosomes loaded with the target RNA. The present invention does not specifically limit the type of exosome donor cells, preferably including HEK293T cells, or donor cells with targeting or therapeutic functions, such as: when treating inflammatory diseases, anti-inflammatory macrophages can be selected as donors. HEK293T cells are preferably used for illustration in the embodiment, but they cannot be identified as the entire protection scope of the present invention. In the embodiment of the present invention, HEK293T cells are preferably cultured with DMEM+10% FBS cell culture medium. The cells are inoculated into a 6-well plate one day before transfection and cultured in an incubator at 37°C. When the cell density reaches 80%, transfection is started. The present invention transfects the circRNA SCAR expression vector into HEK293T cells, preferably including: a. changing the medium of the HEK293T cells laid the day before, and adding 2 ml of serum-containing culture medium to each well; b. adding 3 μg of the circRNA SCAR expression vector to 200 μL PBS; c. taking 7.2 μL HighGene and adding it to the DNA solution, shaking it thoroughly, and standing it at room temperature for 15 minutes; d. culturing the above mixed solution with HEK293T cells.

[0041] In the present invention, the serum-free medium is preferably replaced 6 hours after the above co-culture, and the cell culture supernatant is collected after 48 hours. In the present invention, the collected cell supernatant is preferably centrifuged at 2000g for 10 minutes to remove dead cells and at 10000g for 30 minutes to remove cell debris; the cell culture supernatant treated as above is ultracentrifuged at 100000g for 70 minutes, and the resulting precipitate is the exosomes rich in target RNA.

[0042] The present invention chemically synthesizes the mitochondrial targeting element and the nucleic acid carrier element to obtain a loading material. In the embodiment, TPP and PDL 10 Chemical synthesis is described as an example, preferably comprising TPP, PDL 10 , EDC and DMAP were mixed and dissolved, reacted at 40°C for 8h, the product was loaded into a dialysis bag with a molecular weight cutoff of 1000Da, dialyzed in pure water, and the retained material was collected to obtain the loaded material TPP-PDL 10 More preferably, 100 mg PDL 10Dissolved in 1 mL of dimethyl sulfoxide (DMSO), TPP (1.2 mol), 1-ethyl-(3-dimethylaminopropyl) carbodiimide (EDC) (2.0 mol) and 4-dimethylaminopyridine (DMAP) (0.1 mol) were added and completely dissolved. After the dissolution, the present invention was placed at 40°C for reaction for 8 hours, the product was placed in a dialysis bag (molecular weight cutoff 1000Da), dialyzed in pure water for 24 hours, the retained material was collected, and freeze-dried to obtain TPP-PDL 10 .

[0043] After obtaining the exosomes (or exosomes rich in target RNA) and the loading material loaded with the target RNA and the loading material, the loading material is loaded onto the exosomes loaded with the target RNA to obtain the engineered exosomes. The present invention preferably performs the loading by electroporation, and more preferably includes setting the voltage to 700V, the current to 150mF, the thickness of the electroporation cup to 4mm, and pulse electroporation for 5 times; centrifugation to collect the precipitate. After the pulse electroporation, the present invention preferably also includes rapidly transferring to ice for incubation for 30min, and then centrifuging again, the centrifugal force of the centrifugation is preferably 12000g, and the centrifugation time is preferably 30min. After the above centrifugation, the present invention preferably also includes washing with cold PBS to remove free peptides, and then continuing to centrifuge at 12000g for 30min, and the resulting precipitate is an exosome that can target mitochondrial genes.

[0044] The present invention also provides the use of the engineered exosomes or the engineered exosomes constructed by the above-mentioned construction method in the preparation of gene drugs for improving serum stability.

[0045] The present invention combines exosomes with a nucleic acid vector having a mitochondrial targeting function to improve the serum stability of the drug.

[0046] The present invention also provides the use of the engineered exosomes or the engineered exosomes constructed by the above construction method in the preparation of gene drugs for treating sepsis;

[0047] The target gene loaded by the engineered exosomes is circRNA SCAR.

[0048] The present invention chemically couples TPP with mitochondrial targeting function with nucleic acid carrier PDL 10 Combined with TPP-PDL, it is loaded into exosomes by electroporation. While ensuring serum stability and safety, TPP-PDL 10 The high positive charge of the exosome enables lysosomal escape, thereby effectively delivering the drug to the mitochondria to achieve the therapeutic purpose. After the above modification, an engineered exosome that targets mitochondrial gene expression can be produced to achieve precise and efficient delivery of the target RNA.

[0049] The following is a detailed description of an engineered exosome for targeted intervention of mitochondrial gene expression provided by the present invention, as well as its construction method and application, in conjunction with the embodiments. However, they should not be construed as limiting the scope of protection of the present invention.

[0050] Example 1

[0051] 1. Construction and production of engineered exosomes that can precisely target and intervene in mitochondrial genes

[0052] 1. Construct a plasmid expressing the target RNA (the target RNA used in the present invention is circRNA SCAR).

[0053] The circRNA SCAR overexpression plasmid was constructed by Geneseed Biotechnology Company, and the process is as follows:

[0054] (1) EcoRⅠ and BamHⅠ were selected as restriction sites;

[0055] (2) Adding cyclization-mediating sequences at both ends of the circRNA SCAR sequence to promote the circularization of circRNA SCAR;

[0056] (3) After enzyme digestion, ligation and other steps, the sequence was successfully inserted into the PLC5-Cir vector.

[0057] 2. Produce exosomes enriched with target RNA.

[0058] (1) HEK293T cells were cultured in DMEM+10% FBS cell culture medium. The cells were seeded into 6-well plates one day before transfection and cultured in a 37°C incubator. When the cell density reached 80%, transfection was started.

[0059] (2) Transfect the circRNA SCAR expression vector into HEK293T cells. The specific steps are as follows: a. Replace the medium of the HEK293T cells that were plated the day before, and add 2 ml of serum-containing culture medium to each well; b. Add 3 μg of the circRNA SCAR expression vector to 200 μL PBS; c. Take 7.2 μL HighGene and add it to the DNA solution, shake it thoroughly, and let it stand at room temperature for 15 minutes; d. Culture the above mixture with HEK293T cells.

[0060] (3) After 6 h, the medium was changed to serum-free medium and the cell culture supernatant was collected after 48 h.

[0061] (4) The collected cell supernatant was centrifuged at 2000 g for 10 min to remove dead cells and at 10000 g for 30 min to remove cell debris.

[0062] (5) The cell culture supernatant treated as above is ultracentrifuged at 100,000 g for 70 min, and the resulting precipitate is exosomes rich in target RNA.

[0063] 3.TPP-PDL 10 Synthesis

[0064] (1) 100 mg PDL 10 Dissolve in 1 mL of dimethyl sulfoxide, add TPP (1.2 mol), EDC (2.0 mol) and 4-dimethylaminopyridine (0.1 mol) to completely dissolve.

[0065] (2) The reaction was carried out at 40°C for 8 h, the product was placed in a dialysis bag (molecular weight cutoff 1000 Da), dialyzed in pure water for 24 h, the retained material was collected, and freeze-dried to obtain TPP-PDL 10 .

[0066] 4.TPP-PDL 10 of loading.

[0067] (1) Mix 400 μL of PBS with exosomes with a protein content of 100 μg and add TPP-PDL 10 , making the final concentration 1μM, and using Gene pulser Xcell system for electroporation, setting the parameters voltage 700V, current 150mF, electroporation cup thickness 4mm, pulse electroporation 5 times. After successful electroporation, quickly transfer to ice and incubate for 30min.

[0068] (2) Exosomes were collected by centrifugation at 12000 g for 30 min, washed with cold PBS to remove free peptides, and then centrifuged at 12000 g for 30 min. The resulting precipitate is the exosome that can target mitochondrial genes.

[0069] 2. Verification of exosome loading efficiency and delivery efficiency.

[0070] 1. Verification of exosome loading efficiency.

[0071] qRT-PCR was used to observe the content of target RNA in exosomes. Total RNA of exosomes was extracted using TRIzol (Invitrogen, USA). The target RNA was reverse transcribed using Transcriptor First-strand cDNA Synthesis Kit according to the manufacturer's instructions. FastStart Essential DNA Green Master (Indianapolis, IN, USA) was used for 20 μL qPCR reaction. Relative gene expression was normalized to β-actin, and quantitative comparison was performed using the -ΔCt method. The results showed that the expression of circRNA SCAR in exosomes produced by HEK293T cells after transfection was significantly increased.

[0072] 2. Verification of the delivery efficiency of exosomes.

[0073] To evaluate the targeting of exosomes, FITC-labeled TPP-PDL 10 The targeting of FAM-labeled RNA to exosomes was observed. The specific method is as follows:

[0074] (1) Collect exosomes rich in target RNA and electroporate FITC-labeled TPP-PDL 10 Loaded into exosomes, the specific method is the same as above. RAW264.7 cells were plated in a confocal culture dish one day in advance, and after adherence, they were loaded with FITC-labeled TPP-PDL. 10 After 24 hours, 100 nM MitoTracker TM Mitochondria were stained with ELISA Red for 30 min and then washed three times with PBS. Cell nuclei were stained with Hoechst (Invitrogen, USA). At the end of the incubation, cells were washed with PBS and imaged under a confocal microscope. Figure 1 Display, TPP-PDL 10 Can be successfully delivered to macrophage mitochondria.

[0075] In order to further observe whether exosomes can successfully deliver the target RNA to mitochondria, FAM-labeled RNA was transfected into HEK293T cells to visualize the target RNA carried by the exosomes. The rest of the steps are the same as above. RAW264.7 cells were plated in a confocal culture dish one day in advance, and after adherence, they were co-incubated with exosomes loaded with FAM-labeled RNA. After 24 hours, mitochondria were stained with 100nM MitoTrackerTM Red for 30 minutes, and then washed three times with PBS. The cell nucleus was stained with Hoechst (Invitrogen, USA). At the end of the incubation, the cells were rinsed with PBS and imaged under a confocal microscope. The results are shown in Figure 2. Figure 2 showed that RNA could be successfully delivered to macrophage mitochondria.

[0076] The above experiments prove that the present invention has successfully constructed an exosome strategy that can target the delivery of circular RNA to intervene in mitochondrial function.

[0077] Example 2

[0078] The system was validated using circRNA SCAR as the target gene and septic mice as the disease model.

[0079] The method for constructing plasmid is the same as above.

[0080] A severe sepsis model was established by cecal ligation and puncture (CLP). Before all operations, mice were anesthetized by intraperitoneal injection of sodium pentobarbital (1%). After anesthesia, the peritoneum was opened aseptically, 75% of the cecum was ligated with a 4.0 silk suture end, and punctured once with a 21-gauge needle. A small amount of feces was squeezed out from the perforation site to ensure the patency of the perforation. The cecum was returned to the abdominal cavity and the abdominal incision was sutured with 4.0 silk. After CLP, fluid resuscitation was performed immediately with preheated saline (50 ml / kg subcutaneously). Except for the cecal ligation and puncture, the other steps of the sham group were the same.

[0081] C57 / BL6N mice (male, 6-8 weeks) were randomly divided into 4 groups for tail vein injection, including Sham group, CLP group, CLP+Exos group SCAR Group, CLP+Exos Mito-SCAR Group.

[0082] The Sham group and CLP group received no treatment.

[0083] CLP+Exos SCAR The mice underwent cecal ligation and puncture (CLP) to simulate sepsis. After obvious symptoms appeared, the extracted exosomes loaded with circRNA SCAR were injected into the mice at a dose of 4 μg / g, once every 6 hours, for a total of 4 injections.

[0084] CLP+Exos Mito-SCAR The mice were subjected to cecal ligation and puncture (CLP) to simulate sepsis. After obvious symptoms appeared, the extracted TPP-PDL 10 The exosomes expressing circRNA SCAR were injected into mice at a dose of 4 μg / g, once every 6 h, for a total of 4 injections.

[0085] Afterwards, the organs (heart, liver, spleen, lung, kidney and intestine) of each group of mice were stained with HE to observe the pathological changes of each organ under different treatments.

[0086] The results showed that after loading with TPP-PDL 10 After treatment with exosomes of circRNA SCAR, the inflammation and pathological changes of various organs in septic mice were significantly higher than those in the CLP group and CLP+Exos group. SCAR group was significantly reduced.

[0087] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. An engineered exosome for targeted intervention of mitochondrial gene expression, characterized in that: The engineered exosomes carry a mitochondrial targeting element, a nucleic acid adsorption element and a target RNA; the mitochondrial targeting element includes TPP, and the nucleic acid adsorption element includes PDL 10 ; The mitochondrial targeting element and the nucleic acid adsorption element are electrotransferred into the engineered exosomes; The engineered exosomes are loaded with the target RNA, and the target RNA is circRNA SCAR; the donor cells of the engineered exosomes include HEK293T cells.

2. The method for constructing engineered exosomes according to claim 1, characterized in that: The following steps are involved: (1) Selecting RNA that regulates mitochondrial function as the target RNA and constructing its related vector; (2) using the relevant vector obtained in step (1) to transfect exosome donor cells, collecting exosomes in the cell supernatant, and obtaining exosomes loaded with the target RNA; (3) chemically synthesizing the mitochondrial targeting element and the nucleic acid adsorption element to obtain a loading material; the mitochondrial targeting element includes TPP, and the nucleic acid adsorption element includes PDL 10 ; (4) loading the loading material into the exosomes loaded with the target RNA in step (2) by electroporation to obtain the engineered exosomes; There is no temporal sequence between step (1) and step (2) and step (3).

3. The construction method according to claim 2, characterized in that: On TPP and PDL 10 Chemical synthesis, including TPP, PDL 10 , EDC and DMAP were mixed and dissolved, reacted at 40°C for 8h, the product was loaded into a dialysis bag with a molecular weight cutoff of 1000Da, dialyzed in pure water, and the retained material was collected to obtain the loaded material TPP-PDL 10 .

4. The construction method according to claim 2, characterized in that: The loading in step (4) includes loading the loading material into the exosomes by electroporation; the voltage during the electroporation is set to 700 V, the current is 150 mF, the thickness of the electroporation cup is 4 mm, and the pulse electroporation is performed 5 times; Collect the precipitate by centrifugation.

5. The construction method according to claim 4, characterized in that: The centrifugal force of the centrifugation is 12000g, and the centrifugal time is 30 minutes.

6. Use of the engineered exosomes according to claim 1 or the engineered exosomes constructed by the construction method according to any one of claims 2 to 5 in the preparation of a gene drug for improving serum stability.

7. Use of the engineered exosomes according to claim 1 or the engineered exosomes constructed by the construction method according to any one of claims 2 to 5 in the preparation of gene drugs for treating sepsis; The target gene expressed by the engineered exosomes is circRNA SCAR.

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