Exosome-enriched polypeptide, genetically engineered exosome thereof, and preparation method and application thereof

Abstract

The present application provides a polypeptide highly enriched on the membrane of exosomes, and after the exogenous protein is expressed in fusion with the polypeptide, the exogenous protein can be highly enriched on the outer surface of the membrane of exosomes or in the inner cavity of the membrane, and can perform the biological function of the exogenous protein. The present application also provides a genetically engineered exosome comprising the polypeptide or the polypeptide fused with the exogenous protein, and a method for treating diseases using the genetically engineered exosome.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to an exosome-enriched polypeptide, genetically engineered exosomes loaded with exogenous targets using the polypeptide, a method for preparing the same, and applications of the exosomes loaded with exogenous targets. Background Technology

[0002] Exosomes (exo) are vesicle-like bodies secreted by cells, ranging in diameter from 30 nm to 150 nm. They possess a typical lipid bilayer membrane structure and are found in various biological fluids such as cell culture supernatants, plasma, serum, saliva, urine, and amniotic fluid. They play important roles in various physiological and pathological processes and have become a research hotspot in the biomedical field in recent years. Studies have shown that exosomes can maintain their biological functions even after being stored at -80°C for two years. Furthermore, due to their naturally derived cellular membrane structure, exosomes can easily cross the blood-brain barrier, suggesting a potential ability to treat neurological diseases.

[0003] In recent years, an increasing number of studies have shown that exosomes can participate in the body's immune regulation process as an important intercellular signaling system. Exosomes can directly fuse with the cell membrane of target cells to release mRNA and microRNA into the cytoplasm, or be taken up by target cells through endocytosis and recognize specific receptors on the cell surface to regulate immunity.

[0004] Bioengineering techniques can be used to improve the performance of exosomes. For example, gene engineering can be used to fuse the gene sequences of guide proteins or peptides with the gene sequences of selected exosome membrane proteins, and to modify the surface of exosomes through surface engineering. This can enhance the targeting of exosomes to organs or tissues or improve the ability of target cells to take up exosomes. High expression of certain functional nucleic acids or proteins on or inside the exosome membrane can effectively transport bioactive substances such as mRNA, microRNA, and proteins, thereby improving the therapeutic effect of exosomes on specific diseases. Summary of the Invention

[0005] This invention relates to a polypeptide that can accumulate on exosomes, which is homologous to prostaglandin F2 receptor negative regulator (PTGFRN). The polypeptide can be highly accumulated on the exosome membrane. After fusion expression of an exogenous protein with this polypeptide, the exogenous protein can be highly accumulated on the outer surface or inner lumen of the exosome membrane and perform its biological function.

[0006] The first aspect of this invention is to provide a polypeptide having 21 to 66 amino acids, and comprising at least the amino acid sequence shown in SEQ ID NO:19. SEQ ID NO:19 is shown below: LLIGVGLSTVIGLLSCLIGYC. The polypeptide can be highly enriched on the exosome membrane, forming a primary transmembrane structure on the exosome membrane, wherein the N-terminus is an extramembrane terminus and the C-terminus is an intramembrane terminus. By linking an exogenous protein to the N-terminus and / or C-terminus of the polypeptide through genetic engineering, the exogenous protein can be enriched and expressed on the extramembrane surface and / or intramembrane lumen of the exosome, thereby endowing the exosome with the biological functions derived from the exogenous protein.

[0007] In some embodiments of the invention, the polypeptide has an amino acid sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or about 100% identical to the amino acid sequence shown in SEQ ID NO:1. In one embodiment of the invention, the polypeptide has an amino acid sequence as shown in SEQ ID NO:1. SEQ ID NO:1 is shown below: SKPVFITVKMDVLNAFKYPLLIGVGLSTVIGLLSCLIGYCSSHWCCKKEVQETRRERRRLMSMEMD.

[0008] A second aspect of the present invention is to provide a fusion protein comprising the polypeptide described in the first aspect of the present invention and at least one exogenous protein.

[0009] In some embodiments of the present invention, the at least one exogenous protein is attached to the N-terminus of the polypeptide, and the exogenous protein is expressed on the extracellular surface of the exosome via the polypeptide. The at least one exogenous protein may be the same or different.

[0010] In some embodiments of the present invention, the at least one exogenous protein is linked to the C-terminus of the polypeptide, and the exogenous protein is expressed in the membrane of the exosome via the polypeptide. The at least one exogenous protein may be the same or different.

[0011] In some embodiments of the present invention, at least one first exogenous protein and at least one second exogenous protein are respectively linked to the N-terminus and C-terminus of the polypeptide. Via the polypeptide, the first exogenous protein linked to the N-terminus of the polypeptide is expressed on the extracellular surface of the exosome, and the second exogenous protein linked to the C-terminus of the polypeptide is expressed in the intracellular lumen of the exosome. The first exogenous protein and the second exogenous protein may be the same or different.

[0012] According to the present invention, the exogenous protein can be selected from any biologically active protein, including but not limited to: antibodies or fragments thereof, enzymes, ligands, receptors or fragments thereof, such as IL-10, NA-1, ATAD1, etc. In one embodiment of the present invention, the exogenous protein is IL-10, preferably, two IL-10 molecules are linked in series to form a dimer at the N-terminus of the polypeptide via a linker structure. In one embodiment of the present invention, the amino acid sequence of the linker is GSGGSGGSGGSGGSG, and the linker is used to link two IL-10 molecules in series to form a dimer. In one embodiment of the present invention, the amino acid sequence of IL-10 is as shown in SEQ ID NO. 5. In one embodiment of the present invention, the exogenous protein is NA-1 and ATAD1, preferably, three NA-1 molecules are linked in series at the N-terminus of the polypeptide, and ATAD1 with the signal peptide deleted is linked at the C-terminus of the polypeptide. In one embodiment of the present invention, the amino acid sequence of the three tandemly linked NA-1 molecules is as shown in SEQ ID NO. 11, and the amino acid sequence of ATAD1 with the signal peptide deleted is as shown in SEQ ID NO. 13.

[0013] According to the present invention, the exogenous protein can be directly linked to the polypeptide, or linked to the polypeptide via a linker. The linker can be a commonly used adapter polypeptide sequence in the art, including but not limited to GS linker sequences, DDK linker sequences, or combinations of these linker sequences. In one embodiment of the present invention, the amino acid sequence of the linker includes G4S, for example, G4S-G4S. In another embodiment of the present invention, the amino acid sequence of the linker is GGGGS. DYKDDDDK GGGGS.

[0014] The present invention also provides a nucleic acid that encodes the polypeptide described in the first aspect of the present invention or the fusion protein described in the second aspect of the present invention.

[0015] The present invention also provides an expression vector comprising the nucleic acid described above. In some embodiments, the vector is a plasmid vector or a viral vector. In some embodiments, the viral vector includes, but is not limited to, lentiviral expression vectors (e.g., pLVX-IRES-zsGreeen1, pLVX-IRES-puromycin, pLVX-P2A / T2A-zsGreeen1, pLVX-P2A / T2A-puromycin, etc.).

[0016] This invention also provides a host cell comprising the expression vector described above or its genome incorporating the nucleic acid described above. In some embodiments, the host cell is a eukaryotic cell. In some embodiments, the host cell is a mammalian cell. In some embodiments, the mammalian cell is a Chinese hamster ovary (CHO) cell. In some embodiments, the mammalian cell is a human cell. In some embodiments, the mammalian cell is a 293T cell. In some embodiments, the host cell is a normal somatic cell and / or stem cell, including but not limited to vascular endothelial cells, dendritic cells, and T cells (e.g., Foxp3). + Treg cells), neurons, oligodendrocytes, astrocytes, microglia, embryonic stem cells, adult stem cells (such as hematopoietic stem cells, neural stem cells, and mesenchymal stem cells derived from bone marrow, umbilical cord, fat, umbilical cord blood, amnion, (placental) chorionic villus, dental pulp, thymus, synovium, etc.), etc.

[0017] A third aspect of the present invention is to provide an engineered exosome comprising the polypeptide described in the first aspect of the present invention or the fusion protein described in the second aspect of the present invention.

[0018] According to the present invention, the engineered exosome comprises the polypeptide, the polypeptide forming a primary transmembrane structure on the membrane of the exosome, wherein the N-terminus is an extramembrane terminus and the C-terminus is an intramembrane terminus.

[0019] According to the present invention, the engineered exosome comprises the fusion protein, the fusion protein comprises the polypeptide, and at least one exogenous protein is further linked to the N-terminus and / or C-terminus of the polypeptide. The exogenous protein is directly or via a linker to the polypeptide, thereby expressing the exogenous protein on the extracellular surface and / or intracellular lumen of the exosome.

[0020] According to the present invention, the linker can be a commonly used adapter polypeptide sequence in the art, including but not limited to GS linker sequences, DDK linker sequences, or combinations of these linker sequences. In one embodiment of the present invention, the amino acid sequence of the linker contains G4S, for example, G4S-G4S. In another embodiment of the present invention, the amino acid sequence of the linker is GGGGS. DYKDDDDK GGGGS.

[0021] According to the present invention, the exogenous protein can be selected from any biologically active protein, including but not limited to: antibodies or fragments thereof, enzymes, ligands, receptors or fragments thereof, such as IL-10, NA-1, ATAD1, etc. In one embodiment of the present invention, the exogenous protein is IL-10, preferably, two IL-10s are tandemly linked by a linker structure to form a dimer. In one embodiment of the present invention, the exogenous protein is NA-1 and ATAD1.

[0022] In one embodiment of the present invention, the engineered exosome comprises the fusion protein described in the second aspect of the present invention, the fusion protein comprising the polypeptide and an exogenous protein, wherein the exogenous protein is IL-10; the polypeptide forms a primary transmembrane structure on the membrane of the exosome, wherein the N-terminus is an extramembrane terminus and the C-terminus is an intramembrane terminus, and the N-terminus of the polypeptide is linked to IL-10 via a linker. According to the present invention, the IL-10 may be one or more, and when there are two or more IL-10s, the IL-10s are also tandemly linked via linkers. In one embodiment of the present invention, the amino acid sequence of the linker is GSGGSGGSGGSGGSG, and this linker is used to tandemly link two IL-10s to form a dimer. In one embodiment of the present invention, the amino acid sequence of the linker connecting IL-10 and the polypeptide is G4S-G4S. In one embodiment of the present invention, the amino acid sequence of IL-10 is as shown in SEQ ID NO. 5.

[0023] In one embodiment of the present invention, the engineered exosome comprises the fusion protein described in the second aspect of the present invention. The fusion protein comprises the polypeptide and an exogenous protein, wherein the exogenous protein is NA-1 and ATAD1. The polypeptide forms a single transmembrane structure on the membrane of the exosome, wherein the N-terminus is an extramembrane terminal and the C-terminus is an intramembrane terminal. The N-terminus of the polypeptide is linked to NA-1 via a linker, and the C-terminus of the polypeptide is directly linked to ATAD1. In one embodiment of the present invention, the amino acid sequence of the linker is GGGGS. DYKDDDDK GGGGS. In one embodiment of the present invention, three NA-1 molecules are tandemly linked to the N-terminus of the polypeptide, and the ATAD1 of the deleted signal peptide is linked to the C-terminus of the polypeptide. In one embodiment of the present invention, the amino acid sequence of the three tandemly linked NA-1 molecules is shown in SEQ ID NO.11, and the amino acid sequence of the ATAD1 of the deleted signal peptide is shown in SEQ ID NO.13.

[0024] A fourth aspect of the present invention is to provide the use of the engineered exosomes in the preparation of pharmaceuticals.

[0025] In one embodiment of the present invention, the engineered exosome comprises the polypeptide described in the first aspect of the present invention, the polypeptide forming a primary transmembrane structure on the membrane of the exosome, wherein the N-terminus is an extramembrane terminus and the C-terminus is an intramembrane terminus, the N-terminus or C-terminus of the polypeptide is directly or through a linker to a foreign protein, the foreign protein being selected from any biologically active protein, including: antibodies or fragments thereof, enzymes, ligands, receptors or fragments thereof.

[0026] Preferably, the exogenous protein is IL-10, and the drug can be used to treat or improve inflammatory responses. The inflammatory responses include, but are not limited to, autoimmune inflammatory responses (e.g., ulcerative colitis, Crohn's disease, etc.) and inflammatory responses caused by pathogen infection (e.g., lung damage caused by pathogen infection). The treatment includes, but is not limited to, reducing GM-CSF, TNF-α, CCL2, CCL3, IL-2, IL-6, IL-1β, and / or CXCL10 in tissues or blood during an inflammatory response, especially reducing the levels of TNF-α, CCL2, and / or CCL3.

[0027] Preferably, the exogenous proteins are NA-1 and ATAD1, the N-terminus of the polypeptide is linked to NA-1 via a linker, and the C-terminus of the polypeptide is directly linked to ATAD1. The drug can be used for neurotrophic, neuroprotective, and / or therapeutic purposes for nerve injury.

[0028] A fifth aspect of the present invention is to provide a method for preparing the engineered exosomes, comprising:

[0029] 1) Construct an expression vector, wherein the expression vector contains a nucleotide sequence encoding the polypeptide or the fusion protein;

[0030] 2) Transfect the expression vector into host cells;

[0031] 3) Isolate and collect exosomes from transfected cells.

[0032] According to the present invention, the host cell may be a human or mammalian cell. The mammalian includes, but is not limited to, ungulates such as cattle, sheep, horses, and pigs; rodents such as rabbits, rats, mice, and guinea pigs; primates such as monkeys and apes; and animals such as cats and dogs.

[0033] The host cell can be a normal somatic cell and / or stem cell, including but not limited to vascular endothelial cells, dendritic cells, and T cells (e.g., Foxp3). + Treg cells), neurons, oligodendrocytes, astrocytes, microglia, embryonic stem cells, adult stem cells (such as hematopoietic stem cells, neural stem cells, and mesenchymal stem cells derived from bone marrow, umbilical cord, fat, umbilical cord blood, amnion, (placental) chorionic villus, dental pulp, thymus, synovium, etc.), etc.

[0034] Induced pluripotent stem cell (iPSC) technology can also be used to reprogram terminally differentiated somatic cells into pluripotent stem cells by introducing specific genes (such as Oct4, Sox2, Klf4, c-Myc, etc.). Exosomes derived from iPSCs can then be obtained using iPSCs as host cells. In this invention, iPSC exosomes are abbreviated as "iPSC-Exos". Using iPSC technology, proprietary induced pluripotent stem cells can be prepared from the patient's own somatic cells, and exosomes can then be obtained from these proprietary induced pluripotent stem cells for use by the patient, significantly reducing the possibility of immune rejection.

[0035] The host cells can also be modified using bioengineering techniques to overexpress one or more other proteins or RNAs, and / or underexpress another one or more other proteins or RNAs, thereby increasing or / and decreasing the protein content in exosomes derived from the modified cells to achieve better therapeutic effects or activity. Known bioengineering techniques can be used, for example, transfecting cells with a vector or mRNA carrying the target protein DNA to increase the expression level of the target protein in the cells. Alternatively, transfecting cells with a vector carrying siRNA or shRNA of the target gene to silence the expression of the target gene in the cells, thereby reducing the expression level of the corresponding protein.

[0036] The expression vector can be a commonly used expression vector in the art, including but not limited to lentiviral expression vectors (such as pLVX-IRES-zsGreeen1, pLVX-IRES-puromycin, pLVX-P2A / T2A-zsGreeen1, pLVX-P2A / T2A-puromycin, etc.).

[0037] Exosomes can be isolated and prepared from cell culture medium by in vitro culture of transfected cells and secreted by the cells themselves. The culture of each cell type can be performed using methods and culture media known in the art. For example, human skin fibroblast culture medium can be DMEM + 15% FBS + NEAA, umbilical cord mesenchymal stem cell culture medium can be DMEM + 10% FBS, and neuronal cell culture medium can be B27 + Neurobasal + GlutaMAX.

[0038] To increase the yield of exosomes in cell cultures, the content of chemical substances in the culture medium can be adjusted according to the characteristics of the cultured cells. For example, essential chemical substances for cell growth can be retained in the culture medium, while non-essential chemical substances or their content can be removed or reduced, putting the cells in a "starved" state to increase the yield of exosomes in the culture. For example, in the case of culturing stem cells to obtain stem cell exosomes, L-ascorbic acid or its salts, selenium or its salts, insulin, etc., can be added to the basic stem cell culture medium, such as DMEM, F12, or DMEM / F12, to induce stem cells to secrete large amounts of exosomes. For example, the exosome secretion inducer described in Chinese patent application CN112920991A is incorporated herein by reference in its entirety.

[0039] In some embodiments of the present invention, the cells are human mesenchymal stem cells, ESCs (embryonic stem cells), or iPSCs. The method for preparing exosomes from these cells can be as follows: L-ascorbic acid or its salt, selenium or its salt, and insulin are added to the basal culture medium of the corresponding cells to adjust the pH of the medium to 7.0-7.5, obtaining a cell culture medium capable of producing a high yield of exosomes. Human mesenchymal stem cells, ESCs (embryonic stem cells), or iPSCs are cultured using this cell culture medium, and the cell culture supernatant is obtained from which the exosomes of the corresponding cells are isolated. The operation of obtaining the cell culture supernatant can be repeated at appropriate intervals before the end of the culture, for example, obtaining the cell culture supernatant daily during the culture period. Commonly used pH adjusters in the art can be used to adjust the pH of the culture medium, including but not limited to carbonates or bicarbonates, such as Na₂CO₃, K₂CO₃, NaHCO₃, KHCO₃, etc. In one embodiment of the present invention, L-ascorbic acid or its salt at a concentration of 30-100 mg / L, selenium or its salt at a concentration of 5-50 μg / L, and insulin at a concentration of 10-30 mg / L are added to the basal culture medium, and the pH of the culture medium is adjusted to 7.0-7.5. In one embodiment of the present invention, the basal culture medium is DMEM. In another embodiment of the present invention, the basal culture medium is DMEM / F12.

[0040] In one specific embodiment of the present invention, the cells are iPSCs (e.g., iPSCs induced from human peripheral blood cells), using DMEM / F12 as the basal medium. L-ascorbic acid or its salt (e.g., magnesium L-ascorbic acid diphosphate) at a concentration of 50-75 mg / L, selenium or its salt (e.g., sodium selenium) at a concentration of 10-20 μg / L, and insulin at a concentration of 15-25 mg / L are added to the basal medium. The pH is adjusted to 7.0-7.5 to obtain a cell culture medium capable of producing high yields of exosomes. The iPSCs are cultured in this cell culture medium. From 15-30 hours after the start of culture until the end of culture, the cell culture supernatant is collected every 20-25 hours, from which exosomes are isolated.

[0041] A sixth aspect of the present invention is to provide a pharmaceutical composition comprising the engineered exosomes described in the third aspect of the present invention.

[0042] According to the present invention, the pharmaceutical composition further comprises pharmaceutically acceptable excipients. The pharmaceutical composition can be in various formulations known in the art, such as liquid formulations (including but not limited to suspensions), solid formulations (including but not limited to tablets, powder for injection, etc.), and semi-solid formulations (including but not limited to ointments). In one embodiment of the present invention, the pharmaceutical composition is a suspension containing the engineered exosomes and a buffer solution. The buffer solution can be a commonly used buffer solution in the art, including but not limited to PBS buffer, DPBS buffer, Tris buffer, Hepes buffer, sodium phosphate, sodium citrate, sodium succinate, histidine (or histidine-HCl), sodium malate, sodium carbonate, etc. In one embodiment of the present invention, the pharmaceutical composition is a lyophilized formulation containing the engineered exosomes and a cryoprotectant. The cryoprotectant can be a commonly used cryoprotectant in the art, including but not limited to mannitol, sucrose, trehalose, lactose, glycerol, dextran, etc. For lyophilized formulations, water for injection or a buffer solution can be added before use to reconstitute the suspension.

[0043] According to the present invention, the content of the engineered exosomes in the pharmaceutical composition can be 100–50,000 μg, for example 200–40,000 μg, 350–35,000 μg. When the pharmaceutical composition is a suspension, the concentration of the engineered exosomes can be 0.01–20 μg / mL, for example 0.01–10 μg / mL, 0.1–5 μg / mL, etc.

[0044] According to the present invention, the engineered exosomes or pharmaceutical compositions containing the engineered exosomes can be administered using various methods of administration known in the art, such as oral, nasal, subcutaneous, topical, or intravenous administration.

[0045] A seventh aspect of the present invention is to provide a method of treating a disease by administering an effective amount of the engineered exosomes described in the third aspect of the present invention, or the pharmaceutical composition described in the sixth aspect of the present invention, to a patient in need of treatment.

[0046] In one embodiment of the invention, the engineered exosome comprises the fusion protein described in the second aspect of the invention. The fusion protein comprises the polypeptide and an exogenous protein. The polypeptide forms a primary transmembrane structure on the membrane of the exosome, wherein the N-terminus is extramembrane and the C-terminus is intramembrane. The exogenous protein is IL-10. The N-terminus of the polypeptide is linked to IL-10 via a linker. The disease is an inflammatory response. The inflammatory response includes, but is not limited to, autoimmune inflammatory responses (e.g., ulcerative colitis, Crohn's disease, etc.), and inflammatory responses caused by pathogen infection (e.g., lung damage caused by pathogen infection). The treatments described include, but are not limited to, reducing GM-CSF, TNF-α, CCL2, CCL3, IL-2, IL-6, IL-1β, and / or CXCL10 in tissues or blood during an inflammatory response, particularly reducing the levels of TNF-α, CCL2, and / or CCL3.

[0047] In one embodiment of the invention, the engineered exosome expresses the fusion protein described in the second aspect of the invention on its membrane. The fusion protein comprises the polypeptide and an exogenous protein. The polypeptide forms a single transmembrane structure on the exosome membrane, wherein the N-terminus is extramembrane and the C-terminus is intramembrane. The exogenous protein is NA-1 and ATAD1. The N-terminus of the polypeptide is linked to NA-1 via a linker, and the C-terminus of the polypeptide is linked to ATAD1. The disease is nerve damage.

[0048] The polypeptides of the present invention can accumulate on the exosome membrane and can carry various exogenous proteins of different lengths, thereby accumulating them on the outer surface or inner lumen of the exosome membrane.

[0049] In this invention, various exosome isolation methods in the art can be used to isolate exosomes from cell culture medium.

[0050] Exosomes can be separated and concentrated by differential ultracentrifugation, which precipitates different impurity components in a sample under varying centrifugal forces. Relatively pure exosomes can be obtained at speeds ranging from 100,000 × g to 200,000 × g, making it the most commonly used method for exosome separation and concentration. This method can be further improved by combining ultrafiltration with 0.22 μm or 0.45 μm pore size membranes to enhance product purity and reduce exosome aggregation.

[0051] Exosomes, as a subgroup of extracellular vesicles, have diameters ranging from 30 to 150 nm. They can also be separated based on particle size using methods such as ultrafiltration, size exclusion chromatography, hydrostatic filtration dialysis, and asymmetric field flow separation.

[0052] Commercial exosome extraction kits such as Exo-spin can also be used. TM ExoQuick TM Invitrogen TM We use products to separate exosomes.

[0053] Extracted exosomes can be suspended in buffer solutions, such as phosphate buffer, and frozen at -80°C to 4°C. Alternatively, they can be lyophilized with the addition of cryoprotectants, such as trehalose.

[0054] Terminology definition:

[0055] "And / or" will be considered as a specific disclosure of each of the two specified features or components having or not having the other. Therefore, the term "and / or" as used in phrases such as "A and / or B" is intended to include "A and B", "A or B", "A" (alone), and "B" (alone). Similarly, the term "and / or" as used in phrases such as "A, B, and / or C" is intended to cover each of the following: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0056] "Comprising" and "including" have the same meaning and are intended to be open and allow, but do not require, the inclusion of additional elements or steps. When the terms "comprising" or "including" are used herein, the terms "consisting of" and / or "substantially consisting of" are also included and disclosed.

[0057] Homology: As used herein, the term "homology" refers to the overall correlation between polymer molecules, for example, between nucleic acid molecules (e.g., DNA and / or RNA molecules) and / or between polypeptide molecules. Generally, the term "homology" implies an evolutionary relationship between two molecules. Therefore, two homologous molecules will share a common evolutionary ancestor. In the context of this disclosure, the term homology includes both identity and similarity.

[0058] In some embodiments, polymer molecules are considered “homological” if at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the monomers in the molecule are identical (completely identical monomers) or similar (conservative substitutions). The term “homological” necessarily refers to a comparison between at least two sequences (polynucleotide or polypeptide sequences).

[0059] Identity: As used herein, the term “identity” refers to the overall monomer conservation between polymer molecules, such as between polynucleotide molecules (e.g., DNA molecules and / or RNA molecules) and / or between polypeptide molecules. For example, the percentage identity of two polynucleotide sequences can be calculated by aligning them for optimal comparison purposes (e.g., vacancies can be introduced in one or both of the first and second nucleic acid sequences for optimal alignment, and dissimilar sequences can be omitted for comparison purposes). In some embodiments, the length of the sequence aligned for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% of the length of the reference sequence. Nucleotides at corresponding nucleotide positions are then compared. When a position in the first sequence is occupied by the same nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percentage identity between two sequences is a function of the number of common positions shared by the sequences, taking into account the number of vacancies that need to be introduced to achieve optimal alignment of the two sequences and the length of each vacancy. Sequence comparison and determination of the percentage identity between two sequences can be accomplished using mathematical algorithms. When comparing DNA and RNA, thymine (T) and uracil (U) can be considered equivalent.

[0060] Suitable software programs are available from various sources and can be used for the alignment of both protein and nucleotide sequences. Examples include Bl2seq, Needle, Stretcher, Water, or Matcher. Sequence alignment can be performed using methods known in the art, such as MAFFT, Clustal (ClustalW, ClustalX, or Clustal Omega), MUSCLE, etc.

[0061] "Pharmaceutically acceptable excipients" means any component other than exosomes described herein that has substantially non-toxic and non-inflammatory properties in patients, including but not limited to any and all solvents, dispersion media or other liquid carriers, dispersing or suspending agents, diluents, isotonic agents, preservatives, colorants, sweeteners or flavorings, stabilizers, antioxidants, antimicrobial or antifungal agents, molar osmolarity regulators, pH regulators, buffers, chelating agents, cryoprotectants, and / or fillers, as appropriate for the desired specific dosage form. Various excipients used to formulate pharmaceutical compositions and techniques used to prepare compositions are known in the art. Exemplary antimicrobial or antifungal agents include, but are not limited to, benzalkonium chloride, benzyl chloride, methylparaben, ethylparaben, propylparaben, butylparaben, benzoic acid, hydroxybenzoic acid, potassium or sodium benzoate, potassium or sodium sorbate, sodium propionate, sorbic acid, and combinations thereof. Exemplary preservatives include, but are not limited to, vitamin A, vitamin C, vitamin E, beta-carotene, citric acid, ascorbic acid, and combinations thereof. Exemplary buffer solutions for controlling pH may include, but are not limited to, sodium phosphate, sodium citrate, sodium succinate, histidine (or histidine-HCl), sodium malate, sodium carbonate, and / or combinations thereof. Exemplary cryoprotectants include, but are not limited to, mannitol, sucrose, trehalose, lactose, glycerol, dextrose, and combinations thereof. Attached Figure Description

[0062] Figure 1 : Schematic diagram of the connection between the exosome enrichment domain and EGFP, where A is a schematic diagram of the connection between the exosome enrichment polypeptide of the present invention (hereinafter referred to as "MH-EP1", SEQ ID NO.1) and EGFP, i.e., EGFP-MH-EP1; B is a schematic diagram of the connection between the prior art truncated PTGFRN (PTGFRN-454, SEQ ID NO.17) and EGFP, i.e., EGFP-PTGFRN-454.

[0063] Figure 2 Images show fluorescence micrographs of EGFP expression in 293T cells. A shows a fluorescence micrograph of EGFP without MH-EP1 linkage in 293T cells; B shows a fluorescence micrograph of EGFP-MH-EP1 expression in 293T cells. The scale bar is 10 μm.

[0064] Figure 3Characterization analysis results of exosomes, where A is the particle size distribution of exosomes containing EGFP-MH-EP1 as determined by nanoflow cytometry; B is the EGFP expression rate of exosomes containing EGFP-MH-EP1 as determined by nanoflow cytometry; C is the particle size distribution of exosomes containing EGFP-PTGFRN-454 as determined by nanoflow cytometry; and D is the EGFP expression rate of exosomes containing EGFP-PTGFRN-454 as determined by nanoflow cytometry.

[0065] Figure 4 : Confocal micrograph of 293T cells after co-incubation with exosomes containing EGFP-MH-EP1 for 24 h.

[0066] Figure 5 : Schematic diagram of the connection between MH-EP1 and IL-10, i.e. 2×IL10-MH-EP1.

[0067] Figure 6 Immunofluorescence image of 2×IL10-MH-EP1 expression in 293T cells. Scale bar in the image is 10 μm.

[0068] Figure 7 Characterization analysis results of exosomes containing 2×IL10-MH-EP1, where A is the particle size distribution of exosomes containing 2×IL10-MH-EP1 as determined by nanoflow cytometry; B is the expression rate of IL-10 in exosomes containing 2×IL10-MH-EP1 as determined by nanoflow cytometry; C is the expression rate of CD63, a surface marker of exosomes containing 2×IL10-MH-EP1, as determined by nanoflow cytometry; D is the detection results of common proteins and surface-specific proteins in exosomes containing 2×IL10-MH-EP1, where WCL represents cell lysis buffer and Exo represents exosome lysis buffer.

[0069] Figure 8 : Schematic diagram of the modeling and drug administration procedure for the LPS-induced acute lung injury disease model.

[0070] Figure 9 Statistical results of leukocytes (A), neutrophils (B), and macrophages (C) in bronchoalveolar lavage fluid after administration of exosomes containing 2×IL10-MH-EP1 in an LPS-induced acute lung injury disease model.

[0071] Figure 10 Statistical results of serum immune factor concentrations in animals after administration of exosomes containing 2×IL10-MH-EP1 in an LPS-induced acute lung injury model.

[0072] Figure 11: Schematic diagram of the connection between MH-EP1 and NA-1 and ATAD1, i.e. 3×NA-1-MH-EP1-ATAD1.

[0073] Figure 12 Characterization analysis results of exosomes containing 3×NA-1-MH-EP1-ATAD1, where A is the particle size distribution of exosomes containing 3×NA-1-MH-EP1-ATAD1 as determined by nanoflow cytometry; B is the expression rate of Flag tag in exosomes containing 3×NA-1-MH-EP1-ATAD1 as determined by nanoflow cytometry; C is the expression rate of CD63, a surface marker of exosomes containing 3×NA-1-MH-EP1-ATAD1, as determined by nanoflow cytometry; and D is the detection of Flag tag protein, where WCL represents cell lysis buffer and Exo represents exosome lysis buffer. Detailed Implementation

[0074] The present invention will be further described below with reference to embodiments. It should be noted that the embodiments are not intended to limit the scope of protection of the present invention, and those skilled in the art will understand that any improvements and variations made based on the present invention are within the scope of protection of the present invention.

[0075] All commonly used reagents used in the following examples are commercially available. The biological experiments performed are routine biological experiments in the field and can be conducted according to the instructions in the relevant lab manual or kit.

[0076] The experimental materials and methods in the following embodiments:

[0077] 1. Laboratory animals

[0078] All experimental procedures were performed in accordance with the Regulations on the Management of Laboratory Animals of the Ministry of Health of the People's Republic of China (Document No. 55, 2001). Eight-week-old male mice C57BL6 / J were purchased from Vital River.

[0079] 2. Lentiviral

[0080] Lentiviral vectors: pLVX-IRES-zsGreeen1 and / or pLVX-IRES-puromycin are used for cloning and linking exogenous load targets, and lentiviral packaging plasmids are psPAX2 and pCMV-VSVG.

[0081] 3. Experimental Methods

[0082] (1) Design and construction of the carrier

[0083] The codons for the linker, MH-EP1, and the foreign protein were optimized and the gene was synthesized. The synthesized gene fragment was cloned into the pLVX-IRES-zsGreeen1 or pLVX-IRES-puromycin lentiviral expression vector to obtain a lentiviral expression vector expressing the MH-EP1 fusion foreign protein.

[0084] (2) Packaging and purification of lentiviruses

[0085] HEK293T cells were cultured to the logarithmic growth phase, digested, centrifuged, and collected. The cells were resuspended and counted, and seeded at 5 × 10⁶ cells / year. 6 293T cells were cultured overnight in 100mm cell culture dishes at 37°C with 5% CO2 until they reached 70%-80% confluence, at which point they were ready for transfection. Two hours before transfection, the cell culture medium was replaced with antibiotic-free medium. The lentiviral expression vector expressing the MH-EP1 fusion exogenous protein, pVSVG, and psPAX2 plasmid were mixed in a 3:1:2 ratio, vortexed for 1-2 seconds, and incubated at room temperature for 15 minutes. The mixture was then slowly added dropwise to the prepared 293T cells, gently mixed, and incubated at 37°C with 5% CO2. After 8 hours, the medium was discarded, and 10ml of fresh medium was added with sodium butyrate to a final concentration of 10mM. 48 hours after the medium change, the cell supernatant was collected, centrifuged at 1000rpm for 10 minutes at 4°C to remove cell debris, digested with 0.1% DNase for 5 minutes, and filtered through a 0.45μm filter. Schedule an ultracentrifuge, centrifuge at 25,000 rpm and 4°C for 2 hours, resuspend the virus pellet overnight in 0.1M PBS, aliquot the virus solution into 0.6ml EP tubes, and store at -80°C for later use.

[0086] (3) Enrichment and purification of exosomes

[0087] Exosomes were extracted using ultrafiltration, specifically as follows: The cells were centrifuged at 300g for 20 min at 4°C to remove cell debris, and the supernatant was filtered through a 0.22 μM filter. The supernatant was then filtered through a 100kD ultrafiltration tube at 4°C, centrifuged at 3000g for 5-10 min, and repeated 4-5 times. The resulting concentrate was added to PBS at a 1:10 volume ratio, filtered through a 100kD ultrafiltration tube, centrifuged at 3000g for 5-10 min, and this step was repeated three times to obtain the exosomes.

[0088] (4) Nanoflow cytometry detection

[0089] The procedure for using a Fuliu-Nanoflow cytometer (U30, Xiamen Fuliu) was employed to detect the particle size and number of exosomes. The instrument was calibrated using 250nm particle size standards of known concentrations, and the number of particles was recorded at a specific time (120 seconds) and pressure (1.0 kPa) to establish a concentration coefficient ratio between the detected particles and the standards. A series of standards with known particle sizes were loaded, and a standard working curve was established comparing scattered light intensity with particle size. Samples were then loaded, and the number of particles was recorded at 120 seconds and 1.0 kPa. The particle concentration was obtained through the concentration coefficient ratio between the particles and the standards, and the particle diameter was obtained through the standard working curve comparing scattered light intensity with particle size. FITC and AF647 fluorescence signals were detected using 525 / 40 and 670 / 30 filters.

[0090] (5) Western blot of proteins

[0091] Extraction of total cellular protein: Discard the culture medium from cells in 6-well plates and wash twice with PBS. Then add 100 μL of RIPA (containing PMSF) and place on ice to fully lyse the cells. Collect the cell lysate in a 1.5 mL centrifuge tube and centrifuge at 12,000 rpm for 10 min at 4°C. The supernatant is the extracted total cellular protein and should be stored at -80°C.

[0092] Protein sample preparation: The protein concentration was determined using a BCA protein assay kit. Based on the measured protein concentration, the required total protein sample volume for 100 μg was calculated. The protein sample and 5× loading buffer were added to an EP tube to achieve a final concentration of 1×, and the volume was brought to the nearest whole number (RIPA). After mixing, the sample was centrifuged and boiled for 5 min to denature the protein. The sample was then stored at 4°C or -80°C.

[0093] Separating and stacking gels for protein electrophoresis were prepared using a rapid gel preparation kit. Proteins were transferred to NC membranes using the classic SDS-PAGE electrophoresis-electrotransfer method. After blocking the NC membranes with skim milk powder, they were incubated overnight with primary antibody. The bands on the NC membranes were then imaged, quantitatively analyzed, and qualitatively analyzed using ECL chemiluminescence solution under an electrochemiluminescence analyzer.

[0094] (6) Immunofluorescence detection of intracellular localization and expression of specific targets

[0095] 293T cells were seeded at 10,000 cells / well in 8-well glass slides, and 300 μL of 10% FBS / DMEM complete medium was added. The cells were incubated at 37°C and 5% CO2 for 24 h. The cell culture medium was removed, and the cells were washed once with PBS. Then, 4% PFA was added to each well for fixation and incubation at room temperature for 45 min. The cells were washed once with pre-cooled PBS and permeabilized with 0.5% PBS-Triton x100 for 20 min. The cells were washed twice with PBS for 5 min each time. The cells were blocked with 1% BSA at room temperature for 1 h. Primary antibody diluted with 1% BSA was added, and hybridization was performed at room temperature for 2 h. The cells were washed three times with PBS for 5 min each time. Secondary fluorescent antibody diluted with 1% BSA was added, and hybridization was performed at room temperature for 1 h. The cells were washed three times with PBS for 5 min each time. The expression of fluorescent proteins was detected under a confocal microscope.

[0096] A design scheme for single-target enrichment of exosomes involves sequentially linking exogenous proteins, such as EGFP or IL-10, to a linker and MH-EP1, thereby enriching EGFP or IL-10 on the outer surface of the exosome membrane. A schematic diagram of this structure is shown below. Figure 1 and Figure 5 As shown.

[0097] The design scheme for dual-target enrichment of exosomes involves binding exogenous proteins to the N-terminus and C-terminus of MH-EP1, respectively. This results in the enrichment of the first exogenous protein linked to the N-terminus on the outer surface of the exosome membrane, while the second exogenous protein linked to the C-terminus is enriched in the inner lumen of the exosome membrane.

[0098] The amino acid sequence of MH-EP1:

[0099]

[0100] The encoding DNA sequence of MH-EP1 is as follows:

[0101]

[0102]

[0103] Example 1

[0104] Experiments were conducted using EGFP as the exogenous protein.

[0105] The amino acid sequence of the linker is: GGGGSGGGGS (SEQ ID NO.3), and the DNA sequence of the linker is:

[0106]

[0107] 1) Design of expression vector sequence:

[0108] The linker and MH-EP1 amino acids were codon-optimized, followed by gene synthesis. The synthesized nucleotide sequence was cloned into the pLVX-IRES-puromycin lentiviral expression vector. The EGFP gene was then ligated into the vector via a multiple cloning site to obtain a lentiviral expression vector expressing the EGFP-linker-MH-EP1 fusion protein. A schematic diagram of the fusion protein is shown below. Figure 1 As shown in A in the diagram.

[0109] The amino acid sequence of PTGFRN-454, a known truncated form of PTGFRN in the prior art, is as follows:

[0110]

[0111] Its DNA sequence is:

[0112]

[0113] As a positive control, a lentiviral expression vector expressing the EGFP-linker-PTGFRN-454 fusion protein was constructed using the same method described above. A schematic diagram of the structure of the fusion protein is shown below. Figure 1 As shown in B in the diagram.

[0114] 2) Identification of expression plasmids:

[0115] The lentiviral expression vector constructed in step 1) was sequenced and identified. The correctly sequenced plasmid was transformed into high-fidelity Stbl3 competent cells, and single clones were selected for amplification culture. Endotoxin was removed from the plasmid. The extracted lentiviral expression plasmid was transfected into 293T cells in 6-well plates. EGFP expression was observed under a fluorescence microscope 24 hours after transfection.

[0116] Under a fluorescence microscope, EGFP signaling was observed in the cytoplasm of 293T cells, with a transfection efficiency exceeding 90% after 24 hours. EGFP without MH-EP1 linkage was uniformly expressed throughout the cells (including the cytoplasm and nucleus), as shown in the image. Figure 2 As shown in Figure A; however, EGFP linked to MH-EP1 is only expressed in the cytoplasm and not in the nucleus, as shown in Figure A. Figure 2 As shown in B.

[0117] 3) Enrichment and purification of EGFP-overexpressing exosomes

[0118] Collection of supernatant from overexpression cells: The lentiviral expression plasmid extracted in step 2) was transfected into 293T cells cultured in T175. Six hours after transfection, the 293T cell culture medium was discarded, and the cells were washed twice with sterile PBS to remove the transfection complex and FBS from the 293T culture medium to the greatest extent possible. The medium was replaced with GDEV medium (product number GDSJ0040, Guodian (Beijing) Pharmaceutical Technology Co., Ltd.). Cell supernatant was collected three times at 24h, 48h and 72h after transfection. Exosomes were isolated from the cell culture supernatant using the aforementioned exosome enrichment and purification methods.

[0119] 4) Characterization and analysis of genetically engineered exosomes loaded with EGFP

[0120] The operation procedure of the Fuliu-Nanoflow Cytometer was adopted. The particle size and number of exosomes were detected by the nanoflow cytometer (U30, Xiamen Fuliu) according to the aforementioned corresponding methods.

[0121] Exosomes obtained from 293T cells after EGFP expression had a particle diameter mainly concentrated between 60-100 nm. Nanoflow cytometry analysis showed that the number of EGFP-loaded exosomes was 2.07 × 10⁻⁶. 10 The exosomes enriched and purified from the supernatant of untransfected 293T cells did not express EGFP, serving as a negative control. Nanoflow cytometry results showed that exosomes isolated from the supernatant of cells transfected with EGFP-linker-MH-EP1 expressed EGFP. + The exosome content was 23.1%. In contrast, as a positive control, exosomes isolated from the supernatant of cells transfected with EGFP-linker-PTGFRN-454 showed a lower EGFP content. + The exosomes accounted for only 16.6%, as shown in the results. Figure 3 As shown, the MH-EP1 of the present invention has a higher enrichment efficiency in exosomes than the truncated PTGFRN-454 of the prior art.

[0122] 5) Cells take up genetically engineered exosomes loaded with EGFP

[0123] 293T cells were used at 10 4Cells / wells were seeded in confocal slides and 500 μL of 10% FBS / DMEM complete medium was added. The cells were incubated at 37°C and 5% CO2 for 24 h. The DMEM medium in the wells was discarded. A fresh culture medium was prepared with EGFP-loaded genetically engineered exosomes at a ratio of 10000:1, and 500 μL / well was added. The cells were then incubated at 37°C and 5% CO2 for 24 h. The medium in the wells was discarded, and the cells were washed twice with PBS and replaced with complete medium. The cells were then photographed using a confocal microscope. The results are shown below. Figure 4 As shown, exosomes expressing green fluorescent protein can be detected in 293T cells, indicating that 293T cells can effectively take up exosomes expressing EGFP.

[0124] Example 2: Loading IL10 cytokine onto the outer surface of exosome membrane using MH-EP1

[0125] Using the MH-EP1 of this invention to link the exogenous protein IL10 as a therapeutic target protein, genetically engineered exosomes were prepared and exerted an immunomodulatory effect in an inflammatory disease model.

[0126] 1) Design of exosomes for loading IL10 cytokine target protein

[0127] IL-10 was sequentially linked to MH-EP1 via a linker, allowing IL-10 to accumulate on the exosome membrane surface. The structure of the fusion protein is as follows: Figure 5 Example where IL-10 is designed as a dimer via linkers.

[0128] IL10 amino acid sequence:

[0129]

[0130] Its DNA sequence:

[0131]

[0132] The amino acid sequence of the signal peptide is: MGRLASRPLLLALLSLALCRG (SEQ ID NO.7). Its DNA sequence is:

[0133]

[0134] Two IL-10 monomers were linked by GSGGSGGSGGSGGSG (SEQ ID NO.9), and their DNA sequence is as follows:

[0135]

[0136] Use the connector described in SEQ ID NO.3 to connect IL-10 to MH-EP1.

[0137] 2) Immunofluorescence staining to detect the expression of IL10 cytokine in cells.

[0138] Using 293T cells, the expression plasmid was transfected according to the aforementioned method. IL-10 was stained using immunofluorescence under a confocal microscope, and IL-10 expression in the cell plasma membrane was observed. The results are as follows: Figure 6 As shown.

[0139] 3) Enrichment, purification and identification of IL10-loaded cytokine exosomes

[0140] Using the aforementioned methods, exosomes were collected, purified, and characterized. The diameter of the exosomes loaded with IL10 cytokine was mainly concentrated between 50-100 nm, and the number of exosomes was 2.63 × 10⁻⁶. 10 cells / ml (e.g.) Figure 7 A). Nanoflow cytometry results showed that the positivity rate of IL10 on the surface of engineered exosomes overexpressing IL10 was 9.3% (e.g., Figure 7 B), the positive rate of exosomal CD63 was 22.5% (e.g. Figure 7 C) indicates that IL10 can be effectively enriched on the outer surface of exosome membranes via MH-EP1.

[0141] Immunoblot technology was used to detect 293T cells overexpressing IL10 and IL10-loaded exosomes, respectively. The results are as follows: Figure 7 As shown in Figure D, IL10 expression was detected in both cell lysis buffer and exosome lysis buffer. Exosome positive marker proteins (such as CD63, CD9, TSG101, Alix, Syntenin-1, and EEA1) were detected in both cell lysis buffer and exosome lysis buffer, while the exosome negative marker protein Caln was only detected in cell lysis buffer and not expressed in exosome lysis buffer. This further verifies that IL10 is expressed in cells and can be effectively enriched on the exosome membrane surface by the polypeptide MH-EP1 of this invention.

[0142] 4) Immunomodulatory role in LPS-induced acute lung injury disease model

[0143] An animal model of acute lung injury in mice was established using LPS intratracheal instillation. IL-10 engineered exosomes were then administered to the model. The modeling and administration procedures are as follows: Figure 8 As shown.

[0144] Acute lung injury modeling protocol in mice: Healthy mice aged 8-10 weeks were selected as experimental mice. After intraperitoneal injection of afodin (0.1ml / 10g), the mice were fixed on the operating table. The neck was routinely disinfected, and the skin of the neck was cut along the midline of the neck. The muscles and thyroid gland were separated layer by layer to expose the trachea. 50μl of LPS or the same volume of physiological saline was dripped into the trachea of ​​the mouse using a 1ml insulin syringe. Then, 50μl of air was injected. The mouse was then upright and rotated vertically to ensure that the drug was evenly distributed in both lungs.

[0145] Three hours after modeling, mice were administered 10 μg of engineered IL-10 exosomes via nebulization. At 24, 48, and 72 hours, the mice were again administered 10 μg of exosomes via nebulization. Two hours after the last administration, the mice were sacrificed, and bronchoalveolar lavage fluid was collected for analysis of cellular components and cytokines. Results showed that compared to normal mice (untreated group in the figure), the total white blood cell count in the bronchoalveolar lavage fluid of LPS-infused mice (PBS group in the figure, nebulized PBS treatment control group) was significantly increased, indicating the establishment of a good LPS-induced acute lung injury model. Treatment with IL-10 exosomes in LPS-infused mice significantly reduced the total white blood cell count in bronchoalveolar lavage fluid, especially reducing the total number of neutrophils recruited from peripheral blood through chemotaxis (e.g., ...). Figure 9 A and 9B), but there was no significant difference in alveolar macrophages (e.g., Figure 9 C).

[0146] Further analysis of bronchoalveolar lavage fluid using Luminex revealed that, compared to the PBS-treated group and the 293T natural exosome treatment group (results not shown), the IL10 exosome group showed significantly lower levels of TNFα, CCL2, and CCL3, and lower levels of GM-CSF, IL-2, IL-6, IL-1β, and CXCL10, but these levels did not show significant differences (e.g., Figure 10 This indicates that IL-10 engineered exosomes can exert an immunomodulatory effect by reducing the inflammatory response in LPS-induced acute lung injury.

[0147] Example 3: Using the MH-EP1 of the present invention to load therapeutic NA-1 peptide and neuroprotective protein ATAD1 into exosomes

[0148] Using MH-EP1 tandemly with NA-1 and ATAD1 as therapeutic target proteins, genetically engineered exosomes were prepared to exert neuroprotective functions in a neurological injury disease model.

[0149] 1) Design of exosomes loaded with dual exogenous proteins NA-1 and ATAD1

[0150] The N-terminus of MH-EP1 was linked to the NA-1 peptide via a linker, and the C-terminus was directly linked to ATAD1 after the signal peptide was deleted. The structural diagram of the fusion protein is shown below. Figure 11 As shown.

[0151] The amino acid sequence of the 3×NA-1 polypeptide is: KLSSIESDVGKLSSIESDVGKLSSIESDV (SEQ ID NO. 11), and its DNA sequence is:

[0152]

[0153] The amino acid sequence of ATAD1 (named N40-ATAD1) after deleting the signal peptide is as follows:

[0154]

[0155] Its DNA sequence:

[0156]

[0157] The amino acid sequence of the signal peptide is: MGRLASRPLLLALLSLALCRG (SEQ ID NO.7). Its DNA sequence is: ATGGGGCGCCTGGCCTCGAGGCCGCTGCTGCTGGCGCTCCTGTCGTTGGCTCTTTGCCGAGGG (SEQ ID NO.8)

[0158] The connector uses the G4S cascaded Flag sequence: GGGGS DYKDDDDK GGGGS (SEQ ID NO.15), its DNA sequence:

[0159]

[0160] 2) Identification of exosomes loaded with dual exogenous proteins NA-1 and ATAD1

[0161] Using the aforementioned method, exosomes targeting both NA-1 and N40-ATAD1 were collected, purified, and characterized. The diameters of the exosomes loaded with both NA-1 and N40-ATAD1 were mainly concentrated between 50 and 100 nm, and the number of exosome particles was 2.79 × 10⁻⁶. 10 cells / ml (e.g.) Figure 12 A) The positive rate of exosome flag tags by nanoflow cytometry was 8.2% (e.g., Figure 12 B), the positive rate of CD63 in exosomes stained by nanoflow cytometry was 16.4% (e.g. Figure 12C). Immunoblot technology was used to detect 293T cells and exosomes overexpressing dual targets NA-1 and N40-ATAD1, respectively. The results are shown in Figure (e.g.). Figure 12 As shown in D), Flag tag expression was detected in both cell lysate and exosome lysate, indicating that the dual targets NA-1 and N40-ATAD1 were overexpressed in cells and could be effectively enriched on the exosome membrane via MH-EP1.

[0162] 3) The role of neuronal oxygen-glucose deprivation in OGD

[0163] Isolation of primary cortical neurons: Female mice at 13.5 days of gestation were euthanized by dislocation, immersed in 75% ethanol for 3 minutes, their abdomens were cut open, the fetal chain was removed, and the cells were placed in a 10cm culture dish. The fetal chain was severed using ophthalmic scissors, and the brain tissue was dissected and placed under a dissecting microscope. Under the microscope, the two cerebral hemispheres and cerebellum were dissected and separated. The cerebellum was discarded, and the meninges on the outer surface of the cerebral cortex were carefully dissected using microforceps. After dissection, the cortical layer beneath the meninges was removed using microscissors and placed in centrifuge tubes. The cerebral cortex was dissected sequentially, and the meninges were separated. Cells were digested with trypsin, counted by pipetting, and seeded into 96-well culture plates at 20,000 cells per well. The plates were incubated at 37°C in a 5% CO2 incubator. After 4 hours of culture, the medium was replaced with Neurobasalmedium + B27 neuronal culture medium. Healthy neurons developed synapses after 24 hours and began to extend and interweave. On the 3rd day of culture, the medium was replaced with Neurobasal medium + B27 + 5F2DU (30μM) to inhibit the growth of glial cells. Half of the medium was replaced every 3-4 days.

[0164] Primary cortical neurons isolated and cultured for 7 days were then added to the culture medium along with bifunctional exosomes loaded with NA-1 and ATAD1 (exosome particle count 10). 5 Continue culturing for 12 hours. Wash neurons three times with pre-warmed PBS. Replace with CSS sugar-free medium and place the culture plate in an OGD floating box for 120 minutes of hypoxia and sugar-free intervention. Dilute the Hoechst 33342 / PI working concentration staining solution 30 minutes in advance. Add to the culture plate for staining (Hoechst 33342 staining for 30 minutes, PI staining for 10 minutes). Acquire images using a confocal microscope.

[0165] The results showed that, compared with the control, neurons loaded with bifunctional exosomes of NA-1 and ATAD1 experienced significantly reduced cell death during OGD, indicating that exosome-treated neurons can play a neuroprotective role in OGD-induced neuronal death.

[0166] The embodiments of the present invention have been described above. However, the present invention is not limited to the above embodiments. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A fusion protein, characterized in that, The fusion protein comprises a polypeptide with the amino acid sequence shown in SEQ ID NO.1, and two IL-10 molecules are linked in series through a linker structure to form a dimer at the N-terminus of the polypeptide. The amino acid sequence of the linker is GSGGSGGSGGSGGSG, and the amino acid sequence of the IL-10 is shown in SEQ ID NO.

5. The IL-10 dimer is linked to the N-terminus of the polypeptide through the linker GGGGSGGGGS.

2. A nucleic acid, characterized in that: The nucleic acid encodes the fusion protein of claim 1.

3. An expression vector, characterized in that: The expression vector comprises the nucleic acid as described in claim 2.

4. A host cell, characterized in that: The host cell contains the expression vector of claim 3 or its genome in which the nucleic acid of claim 2 is integrated.

5. An engineered exosome, characterized in that: The exosomes comprise the fusion protein of claim 1.

6. The method for preparing engineered exosomes according to claim 5, characterized in that: Culture the host cells as described in claim 4, and isolate and collect exosomes.

7. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the engineered exosomes as described in claim 5.

8. The application of the engineered exosomes according to claim 5 in the preparation of drugs, characterized in that: The drug is used to treat or improve inflammatory responses.

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