Targeting exosome capable of delivering nucleic acid drugs and preparation method thereof

Abstract

The invention relates to the technical field of biological medicine, and provides a targeting exosome capable of delivering nucleic acid drugs and a preparation method of the targeting exosome. The exosome comprises a fusion polypeptide, the fusion polypeptide comprises a targeting protein molecule, an exosome transmembrane protein and a nucleic acid binding domain, and the exosome can load a nucleic acid molecule through binding of the nucleic acid binding domain. According to the invention, engineering modification is carried out on the exosome, so that nucleic acid molecules can be efficiently loaded into the exosome, and meanwhile, the exosome is endowed with the capability of targeting to specific target cells, tissues and organs.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a targeted exosome capable of delivering nucleic acid drugs and a preparation method thereof. Background Art

[0002] Nucleic acid drugs, also known as nucleotide drugs, are a general term for various oligoribonucleic acids (RNA) and oligodeoxyribonucleic acids (DNA) with different functions. This type of drug can directly act on the target site or target gene, exerting a therapeutic effect at the genetic level, especially showing good efficacy in gene therapy. However, nucleic acid drugs also face some challenges in clinical applications, such as poor stability in the body, easy degradation by nucleases, difficulty in crossing the body barrier, and weak targeting. In order to overcome these obstacles, researchers are constantly exploring the chemical structure modification of nucleic acid drugs and the optimization of delivery vectors to improve the stability and targeting of drugs, reduce immunogenicity, and thus achieve better therapeutic effects.

[0003] Exosomes are a type of nano-sized vesicles secreted by cells, with diameters usually between 30 and 150 nanometers. They carry a wealth of bioinformatic molecules, including proteins, nucleic acids (such as mRNA), and lipids, and can transmit information between cells. Exosomes have good biocompatibility and low immunogenicity, making them ideal carriers for delivering nucleic acid drugs.

[0004] The loading methods of nucleic acid molecules in exosomes are generally divided into two types: pre-loading method and post-loading method. Among them, the pre-loading method refers to the encapsulation of nucleic acid molecules into exosomes during the natural secretion of exosomes by cells. This can be achieved by changing the biosynthetic pathways in cells, such as regulating the mRNA synthesis and secretion process in cells through genetic engineering. The post-loading method is to load nucleic acid molecules into exosomes by physical or chemical methods after the secretion of exosomes. This usually requires the use of specific reagents or techniques to open the membrane structure of exosomes so that nucleic acid molecules can enter their interior.

[0005] Although the post-loading method of loading nucleic acid molecules into exosomes is feasible, it still has many limitations in terms of technology, efficiency, stability, biocompatibility, production standardization, cost and clinical application. The pre-loading method has the advantages of natural high efficiency, improved loading efficiency, reduced technical challenges, better biocompatibility and stability, and potential wide application. These advantages make the pre-loading method one of the current research hotspots in the field of exosome delivery of mRNA.

[0006] At present, there are few successful cases of preparing exosomes loaded with nucleic acid molecules through the pre-loading method. The relevant technology is not mature enough, and the efficiency of loading nucleic acid molecules into exosomes still has room for improvement; moreover, these exosomes loaded with nucleic acid molecules do not have the ability to target specific cells. Summary of the invention

[0007] The purpose of the present invention is to engineer exosomes to ensure that nucleic acid molecules are efficiently loaded into exosomes while giving exosomes the ability to target specific target cells, tissues, and organs; thus, the present invention provides engineered exosomes and a method for preparing targeted exosomes for delivering nucleic acid drugs.

[0008] In a first aspect of the present invention, there is provided an exosome capable of loading nucleic acid molecules and having targeting, comprising a fusion polypeptide, wherein the fusion polypeptide comprises a targeting protein molecule, an exosome transmembrane protein, and a nucleic acid binding domain, and the exosome can load nucleic acid molecules by binding to the nucleic acid binding domain.

[0009] Optionally, the nucleic acid molecule is a therapeutic agent or a preventive agent, including a nucleic acid drug; the exosome is loaded with the nucleic acid drug.

[0010] Optionally, the nucleic acid molecule includes one or more combinations of plasmid DNA, shRNA, siRNA, miRNA, mRNA, gRNA, pri-miRNA, pre-miRNA, circular RNA, piRNA, tRNA, rRNA, snRNA or lncRNA.

[0011] Optionally, the targeting protein molecule is an anti-CD3 single-chain antibody anti-CD3 scFv, whose amino acid sequence is shown in SEQ ID NO.1; the exosome transmembrane protein is CD63 truncated protein △33 CD63, whose amino acid sequence is shown in SEQ ID NO.2; and the nucleic acid binding domain is PUF, whose amino acid sequence is shown in SEQ ID NO.3.

[0012] Optionally, the targeting protein molecule comprises a combination of one or more of a targeting peptide, a monoclonal antibody, a bispecific antibody, a single-chain antibody or a bispecific T cell engager.

[0013] Optionally, the exosomal transmembrane protein includes CD63 truncated protein △33 CD63 or △103 CD63, whose amino acid sequences are shown in SEQ ID NO.2 and SEQ ID NO.4, respectively.

[0014] Optionally, the exosomal transmembrane proteins include CD9, CD63, CD81, FLOT1, FLOT2, NOTCH1, NOTCH2, TSPAN2, TSPAN8, LAMB1, LAMB2, PTGFRN, PDGFR, CD47, VSV-G, MARCKS, BASP1, TCRA, TCRB, TCRD, TCRG, Annexin, Cytokeratin, EGFR or TGFβ receptor and variants thereof.

[0015] In a second aspect of the present invention, the above-mentioned exosome fusion polypeptide expression vector is provided, comprising an exosome transmembrane protein signal peptide, a tag molecule, a targeting protein molecule, a connecting peptide, an exosome transmembrane protein and a nucleic acid binding domain.

[0016] Optionally, the amino acid sequence of the exosome transmembrane protein signal peptide is shown as SEQ ID NO.5, the amino acid sequence of the tag molecule is shown as SEQ ID NO.6, and the amino acid sequence of the connecting peptide is shown as SEQ ID NO.7.

[0017] In a third aspect, the present invention provides a method for preparing the above-mentioned exosomes, comprising: (1) transferring the constructed exosome fusion polypeptide expression vector into production cells to obtain engineered production cells; (2) detecting and enriching the engineered production cells; and (3) collecting and purifying from the screened production cells to obtain engineered exosomes.

[0018] In a fourth aspect of the present invention, a method for preparing targeted exosomes preloaded with nucleic acid drugs is provided, comprising the following steps: (1) designing and gene synthesizing a sequence capable of expressing the fusion polypeptide, constructing the sequence into a lentiviral expression vector with puromycin resistance, and obtaining a first recombinant expression vector; (2) Designing and synthesizing a nucleic acid molecule with a protein binding site, constructing it into a lentiviral expression vector with hygromycin resistance, and obtaining a second recombinant expression vector; (3) packaging the first recombinant expression vector obtained in step (1) and the second recombinant expression vector obtained in step (2) into lentivirus respectively to obtain a first lentivirus and a second lentivirus; (4) transfecting cells with the first lentivirus and the second lentivirus obtained in step (3) at the same time, screening the transfected cells with antibiotics, and obtaining a stable cell line; (5) Expanding and culturing the stable cell line obtained in step (4) and collecting exosomes; that is, obtaining the exosomes that are targeted and loaded with nucleic acid molecules or nucleic acid drugs.

[0019] The present invention successfully constructs exosomes capable of targeted delivery of nucleic acid molecules; by engineering and modifying exosomes, while ensuring efficient loading of nucleic acid molecules into exosomes, exosomes are given the ability to target specific target cells, tissues, and organs. Through targeted delivery of nucleic acid molecules, precise treatment of specific cells or tissues can be achieved, and the bioavailability and targeting of drugs can be improved, thereby increasing the therapeutic effect. Compared with traditional systemic administration, targeted delivery can significantly reduce the accumulation of drugs in non-target tissues, reduce toxicity and side effects. The present invention also optimizes and improves exosome transmembrane proteins. By performing truncation analysis on exosome transmembrane proteins, the size of exosome transmembrane proteins is effectively shortened, and the loading efficiency of exosomes loaded with targeted proteins and nucleic acid molecules is improved. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 A schematic diagram of the engineered exosomes prepared by the present invention is shown.

[0021] Figure 2 The figure shows a comparison of the efficiency of loading target proteins with different exosome transmembrane proteins and their truncated proteins in the embodiments of the present invention.

[0022] Figure 3 The results of constructing Jurkat TCR / CD3- cells using CRISPR / Cas9 gene editing technology in an embodiment of the present invention are shown.

[0023] Figure 4 The targeting ability of exosomes loaded with CD19 CAR mRNA to target cells in the embodiments of the present invention is shown.

[0024] Figure 5 The figure shows the comparison of the in vitro tumor inhibition ability of CAR-T cells prepared by the engineered exosomes prepared by the present invention and lentiviral CAR-T in the examples of the present invention. DETAILED DESCRIPTION

[0025] The present invention provides a targeted exosome capable of delivering nucleic acid drugs, the exosome has targeting and can endogenously load nucleic acid molecules; the nucleic acid molecules are therapeutic agents or preventive agents, belonging to nucleic acid drugs; the exosome surface is fused with a targeted protein. Figure 1 As shown, the exosomes of the present invention contain at least one fusion polypeptide, which includes a targeting protein molecule, an exosome transmembrane protein, and a nucleic acid binding domain, and the exosomes can bind to one or more nucleic acid molecules through the nucleic acid binding domain to load the nucleic acid molecules. That is, the exosomes are loaded with nucleic acid drugs by a pre-loading method.

[0026] The nucleic acid molecule or nucleic acid drug can be a combination of any one or more of plasmid DNA, shRNA, siRNA, miRNA, mRNA, gRNA, pri-miRNA, pre-miRNA, circular RNA, piRNA, tRNA, rRNA, snRNA or lncRNA.

[0027] Targeted protein molecules can deliver engineered exosomes to target cells, target tissues and / or target organs. In some schemes, the targeted protein molecules can be in any form such as targeting peptides, monoclonal antibodies, bispecific antibodies, single chain antibody fragments (scFv), bispecific T cell engagers (BiTE), etc.

[0028] In some embodiments, the exosomal transmembrane protein is selected from CD9, CD63, CD81, FLOT1, FLOT2, NOTCH1, NOTCH2, TSPAN2, TSPAN8, LAMB1, LAMB2, PTGFRN, PDGFR, CD47, VSV-G, MARCKS, BASP1, TCRA, TCRB, TCRD, TCRG, Annexin, Cytokeratin, EGFR, TGFβ receptor and variants thereof.

[0029] In some embodiments, the exosome transmembrane protein is a truncated protein, including CD63 truncated protein △33 CD63 or △103 CD63, and their amino acid sequences are shown in SEQ ID NO. 2 and SEQ ID NO. 4, respectively. In the embodiment, by performing truncation analysis on the exosome transmembrane protein, the size of the exosome transmembrane protein is effectively shortened, thereby greatly improving the loading efficiency of the exosomes with targeted proteins and nucleic acid molecules.

[0030] In some more specific schemes, in the prepared exosomes, the targeting protein molecule is an anti-CD3 single-chain antibody anti-CD3 scFv, whose amino acid sequence is shown in SEQ ID NO.1; the exosomal transmembrane protein is a CD63 truncated protein △33 CD63, whose amino acid sequence is shown in SEQ ID NO.2; and the nucleic acid binding domain is PUF, whose amino acid sequence is shown in SEQ ID NO.3.

[0031] In addition, the present invention also relates to a method for preparing the above-mentioned engineered exosomes. The method comprises at least the following steps: (1) transferring the constructed exosome fusion polypeptide expression vector into production cells; (2) detecting and enriching the engineered production cells; (3) collecting and purifying the engineered exosomes from the screened production cells; (4) identifying and detecting the purified exosomes.

[0032] The constructed exosome fusion polypeptide expression vector includes at least one exosome transmembrane protein signal peptide, at least one tag molecule, at least one targeting protein molecule, at least one connecting peptide, at least one exosome transmembrane protein and at least one nucleic acid binding domain. In some more specific schemes, for the signal peptide or tag molecule in the recombinant expression vector, the amino acid sequence of the exosome transmembrane protein signal peptide is shown in SEQ ID NO.5, the amino acid sequence of the tag molecule is shown in SEQ ID NO.6, and the amino acid sequence of the connecting peptide is shown in SEQ ID NO.7.

[0033] Some schemes of the present invention are achieved by genetically engineering exosome-producing cells, such as 293F, mesenchymal stem cells (MSC), induced pluripotent stem cells (iPSC), neural stem cells, etc., through genetic engineering technology. The engineered production cells can efficiently and massively produce and release the above-mentioned exosomes.

[0034] The method for preparing the targeted exosomes preloaded with nucleic acid drugs by engineering modified exosomes, i.e., preloaded with nucleic acid drugs, of the present invention, in some specific embodiments, comprises the following steps: (1) Designing and gene synthesizing a sequence capable of expressing the fusion polypeptide, constructing the sequence into a lentiviral expression vector with puromycin resistance to obtain a first recombinant expression vector; (2) Designing and synthesizing a nucleic acid molecule with a protein binding site, constructing it into a hygromycin-resistant lentiviral expression vector to obtain a second recombinant expression vector; (3) packaging the first recombinant expression vector obtained in step (1) and the second recombinant expression vector obtained in step (2) into lentivirus respectively to obtain a first lentivirus and a second lentivirus; (4) transfecting cells with the first lentivirus and the second lentivirus obtained in step (3) at the same time, screening the transfected cells with antibiotics, and obtaining a stable cell line; (5) Expanding the stable cell line obtained in step (4) and collecting exosomes; that is, obtaining exosomes that are targeted and loaded with nucleic acid drugs.

[0035] The present invention can achieve precise treatment of specific cells or tissues through targeted delivery of nucleic acid molecules, improve the bioavailability and targeting of drugs, and thus increase the therapeutic effect. Compared with traditional systemic administration, targeted delivery can significantly reduce the accumulation of drugs in non-target tissues, reduce toxicity and side effects. It has the potential to treat a variety of non-liver targeted diseases, including rare diseases, neurodegenerative diseases, cardiovascular diseases, autoimmune diseases, etc., and by delivering mRNA with anti-tumor activity (such as encoding CAR mRNA), it can achieve specific killing of tumor cells or enhance the anti-tumor response of the immune system.

[0036] The following provides specific embodiments, which are combined with the accompanying drawings and specific experimental operations to further illustrate the solutions of the present invention.

[0037] The experimental operation method involved in the specific embodiment of the present invention is as follows: 1. Construction of gene synthesis vector The splicing designed expression vector open reading frame (ORF) was obtained by gene synthesis and subcloned into the target vector pLenti-EF1a. That is, the target gene and plasmid vector were digested with the corresponding restriction endonucleases to produce the same sticky ends, and the target gene and the linearized plasmid vector were connected using T4 ligase to form a recombinant plasmid. The recombinant plasmid was transformed into Escherichia coli competent cells for screening and amplification. The screened monoclonal strain was subjected to plasmid extraction according to the supplier's (Qiagen) operating instructions (EndoFree Plasmid Maxi Kit).

[0038] 2. Lentiviral packaging and cell infection The cell line used for lentiviral packaging is 293T cells. The cells were digested and inoculated into T75 cell culture flasks one day before the experiment to ensure that the cell confluence reached more than 80% on the second day. The third-generation lentiviral packaging system used for lentiviral packaging includes lentiviral vectors with target genes, pMDLg / pRRE, pRSV-Rev, and pMD2.G, and the plasmid dosage ratio is 4:2:1:1. Mix the mixed plasmid and transfection reagent PEI in the transfection buffer Opti-MEM at a ratio of 1:3, incubate at room temperature for 15 minutes, and then add it dropwise to the transfected cells and mix gently. Collect the viral supernatant 48 hours after transfection and concentrate and aliquot for use.

[0039] The exosome production cells used in the present invention are Expi293F cells (Thermofisher, A14527). The cells were counted on the day of cell infection, and the number of cells was calculated based on 5×10 5 / ml cell concentration was inoculated in a 125ml triangular shake flask with a volume of 30ml. Concentrated lentivirus was added to the cells, and polybrene was added at a final concentration of 4ug / ml. Cultured at 37℃, 120RPM, 8% CO2 for 48 hours.

[0040] Prepare fresh Expi293™ expression medium containing 0.5ug / ml puromycin and 50ug / ml hygromycin B, and change the medium by centrifugation every 2-3 days until the control cells are completely killed.

[0041] 3. Exosome Preparation and Purification Resuscitate the established stable cell line and expand the cells under appropriate culture conditions. For small-volume production (less than 1.2 liters), 293F cells are cultured in shake flasks at 37°C, 8.0% CO2 concentration, 80% humidity, and 120 rpm. The working volume (i.e., the volume of liquid actually used for cell culture) is between 200 ml and 1200 ml. After the culture is terminated, the cells are removed by centrifugation at 3000g for 20 minutes at room temperature, and the supernatant after the cells are removed is centrifuged at 16000g for 30 minutes to remove large cell debris. The clarified culture medium is supplemented with 1 mM MgCl2 and benzonase (20 U / mL, Millipore) and incubated at 37°C for 3 hours or overnight at room temperature to digest the nucleic acids outside the exosomes.

[0042] The clarified and digested sample is loaded onto the filter membrane of the TFF device. By applying pressure, the liquid in the sample passes through the filter membrane in a tangential flow, while larger molecules (including exosomes) are retained on the filter membrane. At the same time, new buffer is continuously added for dialysis to remove small molecule impurities in the sample. When the desired concentration multiple is reached, the concentrated liquid on the filter membrane is collected. The SEC column is balanced with the corresponding buffer to ensure that the column is in the best working condition, and the sample concentrated and dialyzed by TFF is loaded onto the SEC column. Buffer is used for elution. Depending on the size of the molecule, the exosomes will flow out of the column before other impurities. The exosome portion of the effluent is collected.

[0043] 4. Fluorescence labeling and nanoflow cytometry of exosome samples Take 50 ul of exosomes and dilute them to 150 ul. Take 30 ul of diluted exosomes and add 20 ul of fluorescent-labeled antibodies respectively. Mix well and incubate at 37°C for 30 min in the dark. Add 1 ml of pre-cooled DPBS, select an ultraspeed rotor, and ultracentrifuge at 4°C, 110,000 × g for 70 min. Carefully remove the supernatant, add 1 ml of pre-cooled DPBS, select an ultraspeed rotor, and ultracentrifuge at 4°C, 110,000 × g for 70 min. Carefully remove the supernatant, resuspend with 50 ul of pre-cooled DPBS, and then load the sample for exosome nanoflow detection.

[0044] Example 1: Detection of the expression of anti-CD3 scFv loaded with different exosome transmembrane proteins in exosomes.

[0045] In order to evaluate the ability of different transmembrane proteins and protein truncations to load target proteins into exosomes, several exosomes fused with anti-CD3 scFv were first constructed, including: aCD3-PTGFRN, aCD3-Lamp2b, aCD3-CD63, aCD3-△33 CD63, and aCD3-△103 CD63.

[0046] The amino acid sequence of the targeted protein molecule anti-CD3 scFv is shown in SEQ ID NO. 1. The amino acid sequences of different transmembrane proteins, CD63, PTGFRN, and Lamp2b are shown in SEQ ID NO. 8-10, respectively. The amino acid sequences of △33 CD63 and △103 CD63 are shown in SEQ ID NO. 2 and SEQ ID NO. 4, respectively.

[0047] The particle concentration was measured using NTA (Nanoparticle tracking analysis), and the proportion of anti-CD3 scFv-positive exosomes in the exosome samples was detected using nanoflow cytometry. Figure 2 As shown, among the exosomes of different transmembrane proteins, the aCD3-△33 CD63 sample had the highest positive rate of anti-CD3 scFv in nanoflow cytometry detection, indicating that the CD63 truncated protein △33 CD63 had a higher loading efficiency compared with other transmembrane proteins.

[0048] Example 2: Detection of exosomes loaded with CD19 CAR mRNA for targeted delivery of mRNA to target cells for expression.

[0049] In order to further verify the targeting ability of engineered modified exosomes loaded with nucleic acid molecule mRNA to target cells and whether the delivered mRNA can be normally expressed in target cells, engineered modified exosomes (aCD3-mCD19 exo) with anti-CD3 scFv loaded on the surface and CD19 CAR mRNA mounted in the exosome cavity were constructed.

[0050] The specific construction steps of aCD3-mCD19 exo are the preparation methods of targeted exosomes preloaded with nucleic acid drugs as mentioned above. In this embodiment, CD19 CAR mRNA is the nucleic acid drug loaded by the exosomes. In the exosome aCD3-mCD19 exo, the targeting protein molecule is the anti-CD3 scFv described in Example 1, the transmembrane protein is the △33 CD63 described in Example 1, and the nucleic acid binding domain is PUF, and its amino acid sequence is shown in SEQ ID NO.3.

[0051] At the same time, the TRAC gene in the Jurkat genome was knocked out by CRISPR / Cas9 gene editing technology (TRAC sgRNA sequence is attached) to construct Jurkat TCR / CD3- cells ( Figure 3 ). Wild-type Jurkat cells and Jurkat TCR / CD3- cells were used as target cells, and equal amounts of aCD3-mCD19 exo were added. The results showed that the engineered exosomes had a higher delivery capacity for wild-type CD3+ Jurkat cells, and the delivery efficiency increased with increasing doses ( Figure 4 ).

[0052] TRAC sgRNA (SEQ ID NO.11): mU*mC*mU*rCrUrCrArGrCrUrGrGrUrArCrArCrGrGrCrGrUrUrUrUrArGrArGrCrUrArGrArArUrArGrCrArArGrUrUrArArArU rArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrArArCrUrUrGrArArArArGrUrGrGrCrArCrCrGrArGrUrCrGrGrUrGrCrU*mU*mU*mU Example 3: Comparison of in vitro functions of CAR-T cells prepared by engineered exosomes and traditional lentiviral CAR-T.

[0053] In order to verify whether the CAR-T cells prepared by engineered exosomes (i.e., the exosomes aCD3-mCD19 exo prepared in Example 2) have the same in vitro tumor suppression function as lentivirus CAR-T, human peripheral blood mononuclear cells (PBMCs) were infected with engineered exosomes and lentivirus to prepare CAR-T cells ( Figure 5 ), in vitro killing experiments and ELISA IFN-γ release detection were performed using K562-CD19 cells as target cells. The results showed that CAR-T cells prepared by engineered exosomes (EXO CD19 CAR) and lentiviral CAR-T (Lenti CD19 CAR) had similar in vitro killing abilities, but the IFN-γ release level was significantly lower than that of the lentiviral experimental group, showing the characteristics of low cytokine release, indicating potential good safety.

[0054] The present invention has been described in detail above in conjunction with specific implementation methods and exemplary examples, but these descriptions cannot be understood as limiting the present invention. Those skilled in the art will appreciate that, without departing from the spirit and scope of the present invention, various equivalent substitutions, modifications or improvements may be made to the technical solution of the present invention and its implementation methods, all of which fall within the scope of the present invention.

Claims

1. An exosome comprising a fusion polypeptide, characterized in that: The fusion polypeptide includes a targeting protein molecule, an exosome transmembrane protein and a nucleic acid binding domain, and the exosome can be loaded with nucleic acid molecules through binding with the nucleic acid binding domain.

2. The exosome according to claim 1, characterized in that The nucleic acid molecule is a therapeutic agent or a preventive agent, including a nucleic acid drug; the exosome is loaded with the nucleic acid drug.

3. The exosome according to claim 1, characterized in that The nucleic acid molecule includes one or more combinations of plasmid DNA, shRNA, siRNA, miRNA, mRNA, gRNA, pri-miRNA, pre-miRNA, circular RNA, piRNA, tRNA, rRNA, snRNA or lncRNA.

4. The exosome according to claim 1, characterized in that The targeting protein molecule is an anti-CD3 single-chain antibody anti-CD3 scFv, whose amino acid sequence is shown in SEQ ID NO.1; the exosome transmembrane protein is a CD63 truncated protein △33 CD63, whose amino acid sequence is shown in SEQ ID NO.2; and the nucleic acid binding domain is PUF, whose amino acid sequence is shown in SEQ ID NO.

3.

5. The exosome according to claim 1, characterized in that The targeting protein molecule includes a combination of one or more of a targeting peptide, a monoclonal antibody, a bispecific antibody, a single-chain antibody or a bispecific T cell engager.

6. The exosome according to claim 1, characterized in that The exosome transmembrane protein includes CD63 truncated protein △33 CD63 or △103 CD63, and the amino acid sequences thereof are shown in SEQ ID NO.2 and SEQ ID NO.4, respectively.

7. The exosome according to claim 1, characterized in that The exosomal transmembrane proteins include CD9, CD63, CD81, FLOT1, FLOT2, NOTCH1, NOTCH2, TSPAN2, TSPAN8, LAMB1, LAMB2, PTGFRN, PDGFR, CD47, VSV-G, MARCKS, BASP1, TCRA, TCRB, TCRD, TCRG, Annexin, Cytokeratin, EGFR or TGFβ receptor and variants thereof.

8. The exosome fusion polypeptide expression vector according to any one of claims 1 to 7, characterized in that: The fusion polypeptide expression vector comprises an exosome transmembrane protein signal peptide, a tag molecule, a targeting protein molecule, a connecting peptide, an exosome transmembrane protein and a nucleic acid binding domain.

9. The fusion polypeptide expression vector according to claim 8, characterized in that: The amino acid sequence of the exosome transmembrane protein signal peptide is shown in SEQ ID NO.5, the amino acid sequence of the tag molecule is shown in SEQ ID NO.6, and the amino acid sequence of the connecting peptide is shown in SEQ ID NO.

7.

10. The method for preparing exosomes according to any one of claims 1 to 7, characterized in that: include: (1) Designing and gene synthesizing a sequence capable of expressing the fusion polypeptide, constructing the sequence into a lentiviral expression vector with puromycin resistance to obtain a first recombinant expression vector; (2) Designing and synthesizing a nucleic acid molecule with a protein binding site, constructing it into a lentiviral expression vector with hygromycin resistance, and obtaining a second recombinant expression vector; (3) packaging the first recombinant expression vector obtained in step (1) and the second recombinant expression vector obtained in step (2) into lentivirus respectively to obtain a first lentivirus and a second lentivirus; (4) transfecting cells with the first lentivirus and the second lentivirus obtained in step (3) at the same time, screening the transfected cells with antibiotics, and obtaining a stable cell line; (5) Expanding the stable cell line obtained in step (4) and collecting exosomes.

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