Engineered migratory bodies, methods of making and uses thereof

Abstract

Provided is an engineered migratory body and methods of making and using the same, in particular, a method of making a migratory body, the method comprising causing a cell to undergo a relative displacement and thereby produce a migratory body derived from the cell. Also provided is a migratory body produced by the method, a migratory body produced in vitro or ex vivo, and a delivery system comprising the migratory body.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biomedicine, and in particular to an engineered migrans, a method for preparing the engineered migrans, a delivery system comprising the engineered migrans and a method for preparing the delivery system. BACKGROUND

[0002] The existing delivery systems in the art have many problems, for example. The delivery systems of nanoparticles and microparticles of biological origin have high biocompatibility and relatively good targeting, but production is relatively difficult; engineered exosomes have obvious limitations, such as low yield, purification process dependent on ultracentrifuge, and contents mainly being post-loaded nucleic acids; engineered red blood cells cannot be cultured and proliferated in vitro, so that blood samples are needed for each preparation; red blood cells also have strong limitations on the loading, and the loading is mainly nucleic acids; membrane-modified nanoparticles are difficult to be metabolized and cleared by the human body, and can accumulate in the body, which has potential toxicity; the drugs that can be delivered by viruses and virus-like particles are basically limited to nucleic acids and small molecular weight proteins, and the production and purification process of viruses and virus-like particles is relatively difficult and costly, and modification is relatively difficult. Therefore, there is a need in the art for a new delivery system that can overcome one or more defects of the existing drug delivery systems.

[0003] The present application finds that engineered migrans similar in structure to natural migrans can be induced by various methods, and the engineered migrans are different from the "hypotonic pressure-induced vesicles" known in the art. The present application also finds that the engineered migrans are of biological origin, have the advantages of low toxicity and low immunogenicity of biological origin; the engineered migrans are a brand new extracellular vesicle, have unique advantages in terms of transportable molecules and in vivo biodistribution; and the preparation method of the engineered migrans is simple and has high yield. Therefore, the engineered migrans provided by the present application have high application value in the fields of drug delivery and vaccine preparation and the like. SUMMARY

[0004] The present application provides an engineered migrans, a method for preparing the engineered migrans, a delivery system comprising the engineered migrans and a method for preparing the delivery system. The engineered migrans include but are not limited to the following advantages: low toxicity, low immunogenicity, transportable multiple exogenous loading molecules, suitable in vivo biodistribution, simple preparation method, and / or high yield. The engineered migrans provided by the present application can be used for drug delivery and vaccine preparation and the like, and have excellent effects.

[0005] In one aspect, the present application provides a method for preparing a migran, the method comprising causing a cell to undergo relative displacement and thereby produce a migran derived from the cell.

[0006] In the method of one embodiment, it further comprises isolating the migrasome produced by the cell.

[0007] In the method of one embodiment, it comprises subjecting the cell to a hypotonic treatment.

[0008] In the method of one embodiment, wherein the hypotonic treatment comprises placing the cell in a low-osmolarity buffer solution.

[0009] In the method of one embodiment, wherein the hypotonic treatment comprises placing the cell in a buffer solution, and reducing the osmolarity of the buffer solution to a low-osmolarity buffer.

[0010] In the method of one embodiment, wherein the reducing comprises linearly reducing and / or stepwise reducing.

[0011] In the method of one embodiment, wherein the osmolarity of the low-osmolarity buffer solution is less than about 305 mOsmol / L.

[0012] In the method of one embodiment, wherein the osmolarity of the low-osmolarity buffer solution is between about 10 mOsmol / L and about 274.5 mOsmol / L.

[0013] In the method of one embodiment, it comprises disrupting the cytoskeleton of the cell.

[0014] In the method of one embodiment, wherein disrupting the cytoskeleton of the cell comprises contacting the cell with a cytoskeleton-disrupting agent.

[0015] In the method of one embodiment, wherein the cytoskeleton-disrupting agent comprises a microfilament and / or microtubule depolymerizing agent.

[0016] In the method of one embodiment, wherein the cytoskeleton-disrupting agent comprises Latrunculin A, Latrunculin B, Cytochalasin A, Cytochalasin B, Cytochalasin C, Cytochalasin D, and / or Cytochalasin E.

[0017] In the method of one embodiment, it comprises inhibiting the cell volume regulation function of the cell.

[0018] In the method of one embodiment, wherein inhibiting the cell volume regulation function of the cell comprises reducing the number and / or function of volume-regulating proteins in the cell.

[0019] In the method of one embodiment, wherein the volume-regulating proteins comprise volume-regulating ion channels and / or transporters.

[0020] In a method of an embodiment, wherein the volume-regulated ion channel comprises a volume-regulated anion channel VRAC and / or a volume-regulated cation channel VRCC.

[0021] In a method of an embodiment, wherein the volume-regulated anion channel VRAC comprises SWELL1 or a functionally active fragment thereof.

[0022] In a method of an embodiment, wherein the volume-regulated cation channel VRCC comprises TRPV4, TRPM3 and / or a functionally active fragment thereof.

[0023] In a method of an embodiment, wherein the transporter comprises a symporter.

[0024] In a method of an embodiment, wherein the symporter comprises KCC1, KCC3 and / or KCC4.

[0025] In a method of an embodiment, wherein inhibiting the cell volume regulation function of the cell comprises placing the cell in a buffer solution with attenuated volume regulation capacity.

[0026] In a method of an embodiment, wherein the buffer contains increased cations.

[0027] In a method of an embodiment, wherein the cations comprise K + , Na + , Cs + , Li + , Ca 2+ , Mg 2+ , Ba 2+ , Mn 2+ , Fe 2+ , Ni 2+ , Zn 2+ , Al 3+ , Fe 3+ , CH3NH3 + , C2H5NH3 + , (CH3)2NH2 + , (C2H5)2NH2 + , (C2H5)3N + , ammonium ions and / or choline ions.

[0028] In a method of an embodiment, wherein the buffer contains increased anions.

[0029] In a method of an embodiment, wherein the anions comprise Br - , Cl - , I - , F -, OH - , HCO3 - , H2PO4 - , NO2 - , NO3 - , CN - , HPO4 2- , CO3 2- , SO4 2- and / or PO4 3- .

[0030] In a method of an embodiment, wherein the method comprises detaching the cell from its adherent surface.

[0031] In a method of an embodiment, wherein the method comprises causing a relative displacement between the cell membrane of the cell and its adherent surface.

[0032] In a method of an embodiment, further comprising increasing the number and / or function of tetraspanin proteins, functional fragments and / or functional variants thereof in the cell.

[0033] In a method of an embodiment, comprising causing the cell to overexpress the tetraspanin proteins, functional fragments and / or functional variants thereof.

[0034] In a method of an embodiment, wherein the tetraspanin proteins are selected from the group consisting of: Tspanl, Tspan2, Tspan3, Tspan4, Tspan5, Tspan6, Tspan7, Tspan8, Tspan9, TspanlO, Tspanl l, Tspanl2, Tspanl3, Tspanl4, Tspanl5, Tspanl6, Tspanl7, Tspanl8, Tspanl9, Tspan20 (UPK1B), Tspan21 (UPK1A), Tspan22 (PRPH2), Tspan23 (ROM1), Tspan24 (CD151), Tspan25 (CD53), Tspan26 (CD37), Tspan27 (CD82), Tspan28 (CD81), Tspan29 (CD9), Tspan30 (CD63), Tspan31, Tspan32 and Tspan33.

[0035] In a method of an embodiment, the method further comprises reducing the size of the migratory body.

[0036] In a method of an embodiment, wherein the reducing the size of the migratory body comprises using a filter or an extruder to extrude the migratory body.

[0037] In a method of an embodiment, wherein the pore size of the filter or the extruder is about 30 nm to about 10,000 nm.

[0038] In a method of an embodiment, wherein the size of the migrans is about 50 nm to about 8,000 nm.

[0039] In a method of an embodiment, wherein the migrans is generated from the contractile filaments of the cell.

[0040] In a method of an embodiment, wherein the membrane of the migrans is enriched with sodium / potassium ATPase and / or a functional fragment thereof.

[0041] In a method of an embodiment, wherein the membrane of the migrans is enriched with integrin and / or a functional fragment thereof.

[0042] In a method of an embodiment, wherein the membrane of the migrans is enriched with tetraspanin protein, a functional variant thereof and / or a functional fragment thereof.

[0043] In a method of an embodiment, wherein the membrane of the migrans is enriched with cholesterol.

[0044] In a method of an embodiment, wherein the migrans is enriched with membrane microdomains.

[0045] In a method of an embodiment, wherein the migrans is generated in vitro or ex vivo.

[0046] In a method of an embodiment, wherein the content of the migrans is at least partially reduced or absent compared to a native migrans generated by the corresponding cell.

[0047] In a method of an embodiment, wherein the at least partially reduced content comprises intraluminal vesicles.

[0048] In a method of an embodiment, which is an in vitro or ex vivo method.

[0049] In a method of an embodiment, wherein the cell is a cell cultured in vitro.

[0050] In a method of an embodiment, wherein the cell is a cell cultured in suspension or adherently.

[0051] In a method of an embodiment, wherein the cell comprises a primary cell.

[0052] In a method of an embodiment, wherein the primary cell comprises a tissue cell derived from an organism, which comprises a human, a monkey, a mouse, a rat, a rabbit, a chicken and / or an insect.

[0053] In a method of an embodiment, wherein the primary cells comprise hepatocytes, splenocytes, kidney cells, tissue macrophages, brain glial cells, osteoclasts, bone marrow cells, white blood cells, fibroblasts, and / or adipocytes.

[0054] In a method of an embodiment, wherein the white blood cells comprise B cells, T cells, NK cells, dendritic cells, neutrophils, and / or macrophages.

[0055] In a method of an embodiment, wherein the cells comprise tumor cells.

[0056] In a method of an embodiment, wherein the tumor cells comprise tumor cell lines, primary or finite passaged tumor cells derived from a patient, tumor stromal cells, and / or tumor organoids.

[0057] In a method of an embodiment, wherein the cells comprise CHO cells, CHO-K1 cells, HEK293 cells, HEK293T cells, HEK293FT cells, HEK293F cells, Vero cells, NRK cells, L929 cells, MC38 cells, 4T1 cells, DC2.4 cells, MGC803 cells, Jurkat cells, NK-92MI cells, BJ cells, and / or HepG2 cells.

[0058] In a method of an embodiment, wherein the cells comprise white blood cells, stem cells, and / or fibroblasts.

[0059] In a method of an embodiment, wherein the stem cells comprise mesenchymal stem cells.

[0060] A migrator body prepared in a method of an embodiment.

[0061] In another aspect, the present application provides a migrator body prepared in vitro or ex vivo, wherein the migrator body has a size of about 50 nm to about 8000 nm.

[0062] In a migrator body of an embodiment, which is generated in vitro from a contractile filament of a cell.

[0063] In a migrator body of an embodiment, wherein the membrane of the migrator body is enriched with sodium / potassium ATPase and / or a functional fragment thereof.

[0064] In a migrator body of an embodiment, wherein the membrane of the migrator body is enriched with integrin and / or a functional fragment thereof.

[0065] In a migrator body of an embodiment, wherein the membrane of the migrator body is enriched with tetraspanin proteins, functional variants thereof, and / or functional fragments thereof.

[0066] In an embodiment of the migrans, wherein the migrans is enriched in cholesterol on the membrane.

[0067] In an embodiment of the migrans, wherein the migrans is enriched in membrane microdomains.

[0068] In an embodiment of the migrans, wherein the content of the migrans is at least partially reduced or absent compared to a native migrans produced by the corresponding cell.

[0069] In an embodiment of the migrans, wherein the at least partially reduced content comprises intraluminal vesicles.

[0070] In another aspect, the present application provides a use of a migrans for delivering an exogenous load.

[0071] In an embodiment of the use, wherein the migrans comprises a migrans described herein.

[0072] In another aspect, the present application provides a delivery system comprising a migrans and one or more exogenous loads.

[0073] In an embodiment of the delivery system, wherein the exogenous load is directly or indirectly bound, linked or embedded to the membrane and / or interior of the migrans.

[0074] In an embodiment of the delivery system, wherein the migrans comprises a migrans described herein.

[0075] In an embodiment of the delivery system, wherein the migrans is derived from a cell.

[0076] In an embodiment of the delivery system, wherein the exogenous load comprises one or more targeting substances and / or therapeutically active substances.

[0077] In an embodiment of the delivery system, wherein the exogenous load comprises proteins, lipids, polynucleotides, small molecule compounds, complexes, polysaccharides, polymers, nanoparticles, microparticles and / or organelles.

[0078] In an embodiment of the delivery system, wherein the exogenous load comprises membrane proteins, soluble proteins and / or polypeptides.

[0079] In an embodiment of the delivery system, wherein the exogenous load comprises DNA and / or RNA.

[0080] In an embodiment of the delivery system, wherein the exogenous load comprises an antibody or an antigen-binding antibody fragment thereof, an integrin or a fragment thereof, an immunogenic protein, a cytokine, a chemokine, a receptor protein or a fragment thereof, an enzyme, an onco-suppressor gene product, an siRNA, a microRNA, an antisense oligonucleotide ASO, a mRNA, a DNA, a gene editing tool, and / or a cytotoxic agent.

[0081] In an embodiment of the delivery system, wherein the exogenous load comprises a PAMP, a DAMP, CD47, CD24, IL-12, IL-15, coagulation factor VII, coagulation factor VIII, coagulation factor IX, and / or a functionally active fragment thereof.

[0082] In an embodiment of the delivery system, wherein the exogenous load is directly or indirectly bound to the migrator by gene editing, exogenous expression, liquid-to-solid transition, membrane fusion, charge adsorption, physical adsorption, and / or chemical linkage.

[0083] In an embodiment of the delivery system, wherein the exogenous load is bound or embedded to the migrator by direct or indirect linkage to a membrane component of the migrator.

[0084] In an embodiment of the delivery system, wherein the membrane component of the migrator comprises a membrane protein, a cholesterol, a phospholipid, a sugar chain on a glycoprotein, and / or a polysaccharide.

[0085] In an embodiment of the delivery system, wherein the indirect linkage comprises linkage by a click chemistry reaction.

[0086] In an embodiment of the delivery system, wherein the indirect linkage comprises providing the exogenous load linked to a first member of a binding pair, and contacting it with the migrator, the membrane of which comprises a second member of the binding pair, wherein the first member is capable of binding to the second member.

[0087] In an embodiment of the delivery system, wherein the first and second members of the binding pair are selected from the group consisting of an antigen and its antibody; a receptor and its ligand; biotin and avidin; a HaloTag and its ligand; and CP05 and CD63.

[0088] In an embodiment of the delivery system, wherein the exogenous load is expressed as a membrane protein on the inner or outer surface of the membrane of the migrator.

[0089] In an embodiment of the delivery system, wherein the exogenous load is expressed as a fusion protein fused to a membrane protein or a portion thereof on the inner or outer surface of the membrane of the migrator.

[0090] In an embodiment of the delivery system, wherein the exogenous load is expressed on the inner or outer surface of the membrane of the migrator as a fusion protein fused to a membrane protein or a portion thereof by gene editing and / or exogenous expression.

[0091] In another aspect, the application provides a method of making a delivery system, the method comprising providing a migrator, and causing the migrator to carry an exogenous load.

[0092] In an embodiment of the method, wherein the migrator is an isolated or purified migrator.

[0093] In an embodiment of the method, wherein the causing the migrator to carry an exogenous load comprises directly or indirectly attaching or embedding the exogenous load to the membrane of the migrator and / or the interior of the migrator.

[0094] In an embodiment of the method, further comprising isolating or purifying the migrator from a cell.

[0095] In an embodiment of the method, comprising providing a complex of the exogenous load and a first member of a binding pair; causing a cell to produce a migrator comprising a second member of the binding pair; and contacting the migrator with the complex to form the delivery system.

[0096] In an embodiment of the method, wherein the migrator comprises a migrator described herein.

[0097] In an embodiment of the method, wherein the migrator is derived from a cell.

[0098] In an embodiment of the method, wherein the exogenous load comprises one or more targeting substances and / or therapeutically active substances.

[0099] In an embodiment of the method, wherein the exogenous load comprises a protein, a lipid, a polynucleotide, a small molecule compound, a complex, a polysaccharide, a polymer, a nanoparticle, a microparticle, and / or an organelle.

[0100] In an embodiment of the method, wherein the exogenous load comprises a membrane protein, a soluble protein, and / or a polypeptide.

[0101] In an embodiment of the method, wherein the exogenous load comprises DNA and / or RNA.

[0102] In an embodiment, the method wherein the exogenous cargo comprises an antibody or antigen-binding fragment thereof, an integrin or fragment thereof, an immunogenic protein, a cytokine, a chemokine, a receptor protein or fragment thereof, an enzyme, an onco-suppressor gene product, an siRNA, a microRNA, an antisense oligonucleotide ASO, a mRNA, a DNA, a gene editing tool, and / or a cytotoxic agent.

[0103] In an embodiment, the method wherein the exogenous cargo comprises a PAMP, a DAMP, CD47, CD24, IL-12, IL-15, coagulation factor VII, coagulation factor VIII, coagulation factor IX, and / or a functionally active fragment thereof.

[0104] In an embodiment, the method wherein the exogenous cargo is directly or indirectly bound to the migrator by gene editing, exogenous expression, liquid-to-solid transformation, membrane fusion, charge adsorption, physical adsorption, and / or chemical linkage.

[0105] In an embodiment, the method wherein the exogenous cargo is attached or embedded to the migrator by direct or indirect binding to a membrane component of the migrator.

[0106] In an embodiment, the method wherein the membrane component of the migrator comprises a membrane protein, cholesterol, a phospholipid, a sugar chain on a glycoprotein, and / or a polysaccharide.

[0107] In an embodiment, the method wherein the indirect binding comprises attachment by a click chemistry reaction.

[0108] In an embodiment, the method wherein the indirect binding comprises providing the exogenous cargo attached to a first member of a binding pair, and contacting it with the migrator, the membrane of which comprises a second member of the binding pair, wherein the first member is capable of binding to the second member.

[0109] In an embodiment, the method wherein the first and second members of the binding pair are selected from the group consisting of an antigen and its antibody; a receptor and its ligand; biotin and avidin; a HaloTag and its ligand; and CP05 and CD63.

[0110] In an embodiment, the method wherein the exogenous cargo is expressed as a membrane protein on the inner or outer surface of the membrane of the migrator.

[0111] In an embodiment, the method wherein the exogenous cargo is expressed as a fusion protein fused to a membrane protein or a portion thereof on the inner or outer surface of the membrane of the migrator.

[0112] In another aspect, the present application provides a method of making a delivery system, the method comprising: causing a cell to express mRNA; causing the cell to produce a migratory body, the migratory body comprising an mRNA-binding protein, and the mRNA being attached to the migratory body via the mRNA-binding protein.

[0113] In another aspect, the present application provides a method of making a delivery system, the method comprising: causing a cell to express an exogenous cargo on a cell membrane; and causing the cell to produce a migratory body, the migratory body comprising the exogenous cargo.

[0114] In an embodiment of the method, wherein the exogenous cargo is a protein.

[0115] In an embodiment of the method, wherein the protein is a membrane protein.

[0116] In an embodiment of the method, wherein the protein is a soluble protein, and it is fused with a membrane protein or a portion thereof to form a fusion protein.

[0117] In another aspect, the present application provides a composition comprising a migratory body described herein or a delivery system described herein.

[0118] Other aspects and advantages of the present application can be readily ascertained by one skilled in the art from the following detailed description. Only the preferred embodiments of the present application are shown and described in the following detailed description. As will be realized, the application is capable of modifications in various obvious aspects, all without departing from the spirit and scope of the present application. Accordingly, the drawings and description are to be regarded as illustrative in nature, and not restrictive. BRIEF DESCRIPTION OF DRAWINGS

[0119] The specific features of the application involved herein are shown in the appended claims. The features and advantages of the application involved herein can be better understood by reference to the following detailed description of exemplary embodiments and the attached drawings. A brief description of the drawings is as follows:

[0120] Figure 1 a-c: Effect of low osmotic pressure stimulation on engineered migratory body formation. a. Formation of engineered migratory bodies at different time points under 76.3 mOsmol / L osmotic pressure stimulation (scale bar 5 μm); b. Formation of engineered migratory bodies under different osmotic pressures; c. Statistical results of the diameters of engineered migratory bodies in b.

[0121] Figure 2a-b: Effect of different latrunculin A treatment on engineered migration body formation. a. Engineered migration body formation under different latrunculin A concentrations; b. Statistical results of the number of engineered migration bodies in a.

[0122] Figure 3 a-c: Effect of Lrrc8a knockdown of SWELL1 encoding gene in cells on engineered migration body formation. a. Knockdown efficiency of Lrrc8a in cells determined by qPCR; b. Engineered migration bodies produced by Lrrc8a knockdown cells under stepwise hypo-osmotic stress observed by laser confocal microscope; c. Statistical results of the diameter of engineered migration bodies in b.

[0123] Figure 4 a-b: Effect of Lrrc8a knockdown of SWELL1 encoding gene in cells on engineered migration body formation. a. Knockdown efficiency of Lrrc8a in cells determined by qPCR; b. Engineered migration bodies produced by Lrrc8a knockdown cells under stepwise hypo-osmotic stress observed by laser confocal microscope; c. Statistical results of the diameter of engineered migration bodies in b.

[0124] Figure 5 a-b: Effect of different cations on engineered migration body formation. a. Engineered migration body formation under different cations; b. Statistical results of the diameter of engineered migration bodies in a.

[0125] Figure 6 a-b: Effect of Tspan4 overexpression on the number of engineered migration bodies. a. Engineered migration body formation caused by Tspan4 overexpression; b. Statistical results of the number of engineered migration bodies in a.

[0126] Figure 7 a-b: MGC803-T4-GFP cells produce migration bodies in temperature-sensitive coating dishes. Photos of MGC803-T4-GFP cells producing migration bodies under different dishes and conditions (a) and quantification of cell detachment rate (b).

[0127] Figure 8 a-d: Engineered migration bodies induced in different cell lines. a. Engineered migration bodies induced in different rodent cell lines; b. Local magnification of a (scale bar 5 pm). c. Engineered migration bodies induced in different T4-GFP transfected human embryonic kidney cell lines. d. Engineered migration bodies induced in different human cell lines stained with WGA.

[0128] Figure 9 a-b: Suspension cultured NRK (a) and MC38 (b) cells with different cell concentrations produce engineered migration bodies under different inductions.

[0129] Figure 10 Figure 8: The schematic diagram of the isolation and purification of the engineered migrans induced by the engineered NRK cells.

[0130] Figure 11 a-c: The morphological observation of the engineered migrans induced by the engineered NRK cells. a. The laser confocal microscope image of the engineered migrans; b. The negative staining transmission electron microscope photo of the engineered migrans; c. The cryo-EM photo of the engineered migrans.

[0131] Figure 12 Figure 10: The western blot of the isolated and purified engineered migrans.

[0132] Figure 13 a-b: The permeability of the engineered migrans to Cy5 and dextran-TMR over time observed by laser confocal microscope. a. The laser confocal microscope observation results of the engineered migrans; b. The statistical results of the permeability of the engineered migrans to dextran-TMR after being placed at room temperature for 1.5 h, 6 h, 12 h, 24 h, 48 h.

[0133] Figure 14 a-c: The stability of the engineered migrans. a. The morphological results of the engineered migrans observed by laser confocal microscope at day 0, 1, 2, 3, 5 and 7; b. The amount of chicken ovalbumin (OVA) and mCherry protein loaded in the engineered migrans analyzed by western blot after being placed at room temperature for different days; c. The OVA-specific antibody produced after the immunization of the mice with the engineered migrans placed at room temperature for different days.

[0134] Figure 15 Figure 15: The effect of the cholesterol extraction reagent MβCD on the stability of the engineered migrans observed by laser confocal microscope.

[0135] Figure 16 a-b: The schematic diagram of the loading process of the membrane proteins, soluble proteins and small molecules on the engineered migrans (a) and the schematic diagram of the loading of soluble proteins with OVA as an example (b).

[0136] Figure 17 Figure 18: The localization of various loaded membrane proteins on the engineered migrans observed by laser confocal microscope.

[0137] Figure 18 Figure 19: The localization of the spike protein on the engineered migrans observed by laser confocal microscope.

[0138] Figure 19 Figure 20: The map of the vector pB-Hygro-GFP.

[0139] Figure 20: Map of the vector pB-Hygro-mCherry.

[0140] Figure 21 : Map of the vector pB-Hygro-mCherry.

[0141] Figure 22 : Localization of t-STX2-OVA fusion protein on engineered migrans observed by laser confocal microscope.

[0142] Figure 23 : Co-localization of Tspan4-HaloTag-GFP and HaloTag ligand-TMR on engineered migrans observed by laser confocal microscope.

[0143] Figure 24 a-c: S-protein specific immune responses induced in mice immunized with engineered migrans (e-migrans) loaded with SARS-CoV-2 spike protein. a. Schematic of animal experiment design; b. Western blot of S1 protein in spike-e-migrans, control-e-migrans group and purified S1 protein as control. c. Concentration of spike (S) protein specific IgG in mice serum after immunization with different methods;

[0144] Figure 25 a-c: Characterization of engineered NRK cells producing migrans. a) NRK cells overexpressing Tspan4-GFP after hypotonic treatment, fixed with 2.5% glutaraldehyde and observed with scanning electron microscope. Scale bar: 20 μm in left panel and 2 μm in right panel. b) 4D imaging of the process of engineered migrans production from NRK cells overexpressing Tspan4-GFP by spinning-disk confocal microscope, which showed that the cell body swelled, the basal surface contracted and a large number of filamentous structures were produced during the hypotonic treatment; engineered migrans were grown on the filamentous structures. c) Electron micrograph of A-431 cells producing hypotonic vesicles and micrograph of the final output vesicles (Reference: Cohen S, Ushiro H, Stoscheck C, Chinkers M A native 170000 epidermal growth factor receptor-kinase complex from shed plasma membrane vesicles. J Biol Chem 257: 1523-1531.).

[0145] Figure 26a-h: Comparison of characteristics of migrasomes and engineered migrasomes (e-migrasomes). Left: a: NRK cells expressing TSPAN4-mCherry were cultured and observed under confocal microscope without treatment. Scale bar: 5 pm. b: Engineered migrasomes generated from NRK cells overexpressing Tspan4-GFP observed under laser confocal microscope. Scale bar: 5 pm. c: NRK cells overexpressing Tspan4-GFP and stained with WGA labeled with tetramethylrhodamine. The upper panel is the cells in isotonic solution, and the lower panel is the cells after hypotonic treatment. The pictures are Z-stack overlay images taken after laser confocal microscope layer scanning. Scale bar: 5 pm. d: Electron microscope images of migrasomes (Ma et al, Cell Res. 2015). e: Transmission electron microscope images of engineered migrasomes generated from NRK cells overexpressing T4-GFP. Scale bar: 1 pm. Comparison of 3 times of production and purification of e-migrasomes and control migrasomes after mass spectrometry analysis (f) and 25 proteins most enriched in engineered migrasomes and control migrasomes (g): The numbers in the table represent Log2(relative fold change of expression compared to the cell body). (h) ERM family and other various proteins are enriched in engineered migrasomes (lower row) compared to migrasomes (upper row). Left 3 columns (Tspan-4-GFP, Ezrin-mCherry imaging and image Z-stack merging of the two, respectively); right two are Fxyd5-mCherry imaging; right one is Atp1p1-mCherry imaging. (Reference: Ma L, Li Y, Peng J, Wu D, Zhao X, Cui Y, Chen L, Yan X, Du Y, Yu L. Discovery of the migrasome, an organelle mediating release of cytoplasmic contents during cell migration. Cell Res. 2015 Jan;25(l):24-38. doi: 10.1038 / cr.2014.135; Zhao X, Lei Y, Zheng J, Peng J, Li Y, Yu L, Chen Y. Identification of markers for migrasome detection. Cell Discov. 2019 May 21;5:27. doi: 10.1038 / s41421-019-0093-y. Erratum in: Cell Discov. 2022 Apr 6;8(l):32. PMID: 31123599; PMCID: PMC6527679.)

[0146] Figure 27a-f: Comparison of small vesicles / exosomes and engineered migrans produced by Tspan4-GFP overexpressing MC-38 cells. a. Small vesicles / exosomes purification procedure; b. NTA detection of MC-38 small vesicles / exosomes after purification; c. Transmission electron microscopy (TEM) detection of MC-38 small vesicles / exosomes; d. Western blot analysis of small vesicles / exosomes, engineered migrans and cell cytoplasts from the same cell source. e. Comparison of production and yield of small vesicles / exosomes and engineered migrans from the same cell source. f. In vivo distribution. Small vesicles / exosomes or engineered migrans produced by Tspan4-GFP overexpressing MC-38 cells were labeled with DiD and injected intravenously with equal amount of fluorescent dye, and tissues were collected at different time points for fluorescence imaging.

[0147] Figure 28a-c: Quantitative mass spectrometry analysis of engineered migrans and small vesicles / exosomes from the same cell source. Left: PCA analysis; Middle: Heatmap of signal pathway analysis; Note: The three data points of engineered migrans are consistent, and coincide in the figure. Right: List of the top 10 most enriched proteins in the prepared engineered migrans and small vesicles / exosomes. Note: The three data points of engineered migrans are consistent, and the top 10 most enriched proteins are completely consistent. DETAILED DESCRIPTION

[0148] The present application will be described in greater detail by way of specific embodiments, from which the other advantages and effects of the present application will become readily apparent to those who are skilled in this art and claim the benefits thereof on the basis of the disclosure made herein.

[0149] DEFINITIONS OF TERMS

[0150] In the present application, the term "relative displacement" generally refers to displacement relative to a reference point or a reference surface. It refers to the displacement of the cell center relative to the environment (e.g. the adherent surface), or in the case of the cell center being stationary, the relative displacement of the membrane surface relative to the surrounding microenvironment (e.g. the adherent solid surface, or the liquid phase in the microenvironment), or relative to the cell center (e.g. swelling, from flat to raised, etc.).

[0151] In the present application, the term "migran" generally refers to a structure produced by a cell when it undergoes relative displacement. For example, the migran can be a novel organelle. For example, a vesicular structure produced at the tip or intersection of the contractile filaments left behind during cell migration. For example, during cell migration, the cytoplasm continuously transports intracellular substances to the migran, and then the contractile filaments break, the migran is released, which can be transported to a distant tissue in the local environment or through body fluids such as blood, and then be taken up by the extracellular space or surrounding cells. For example, the migran can be involved in the transmission of intracellular substances and signals between cells, thereby mediating intercellular communication.

[0152] In the present application, the term "membrane microdomain" generally refers to a region having a biological membrane structure. For example, sphingolipid and cholesterol (Ch)-based microdomains of the cell membrane, including lipid rafts, Tetraspanin-enriched microdomains (TEM), and the like.

[0153] In the present application, the term "intraluminal vesicle" generally refers to a membranous vesicle (e.g., organelle or larger vesicle) that forms or exists within a lumen or space of a lumen or tubular structure. Lumen can also be used herein to describe the interior space of a cellular component or structure. For example, an intraluminal vesicle can be produced by an organelle. For example, an intraluminal vesicle can include an intraluminal vesicle in a migrans, or an intraluminal vesicle that is not currently in a migrans but is expected to be transferred into a migrans.

[0154] In the present application, the term "exogenous load" generally refers to a substance that does not naturally occur in a chromosome or host cell. For example, the substance is not produced, or is not produced in substantial amounts (e.g., expressed) by the cell itself. For example, the cell producing the substance is distinguished from a natural cell, e.g., the substance is structurally or functionally different.

[0155] In the present application, the term "hypotonic" generally refers to a lower osmotic pressure than that of an isotonic solution of a cell. An isotonic solution refers to a solution having an osmotic pressure equivalent to that of blood plasma.

[0156] In the present application, the term "cytoskeleton" generally refers to a protein fibrillar network system in a cell, e.g., a system composed of microtubules, microfilaments, and intermediate filaments.

[0157] In the present application, the term "Tetraspanin" is generally a tetraspanin superfamily of proteins that contain four transmembrane domains. These proteins can form so-called tetraspanin-enriched microdomains (TEMs) (Rubinstein, E. (2011). The complexity of tetraspanins. Biochem Soc Trans 39, 501-505.). TEMs are about 100 nanometers in size and are enriched in a series of proteins and lipid raft lipids such as cholesterol, etc. During the formation of the migratory body, many small TEMs will aggregate to form macrodomains of micron size, called tetraspanin-enriched macrodomains (TEMA), the formation of TEMA can be related to the growth of the migratory body on the contractile filament. For example, the Tetraspanin family can contain 33 members, including Tspanl, Tspan2, Tspan3, Tspan4, Tspan5, Tspan6, Tspan7, Tspan8, Tspan9, TspanlO, Tspanl l, Tspanl2, Tspanl3, Tspanl4, Tspanl5, Tspanl6, Tspanl7, Tspanl8, Tspanl9, Tspan20 (UPK1B), Tspan21 (UPK1A), Tspan22 (PRPH2), Tspan23 (ROM1), Tspan24 (CD151), Tspan25 (CD53), Tspan26 (CD37), Tspan27 (CD82), Tspan28 (CD81), Tspan29 (CD9), Tspan30 (CD63), Tspan31, Tspan32, and Tspan33. Data from biochemical studies and gene knockout mice suggest that these Tetraspanin family members play an important role in membrane biology.

[0158] In the present application, the terms "isolated" and "purified" are used interchangeably and generally mean that the migratory body is identified, separated and / or recovered from a component of its production environment, such that the "isolated or purified" migratory body is free or substantially free of other contaminant components from its production environment that can interfere with its therapeutic or diagnostic use. Contaminant components can include non-biological substances (including chemicals) or biological substances, such as organelles, nucleic acids, proteins (e.g., soluble proteins), lipids, or metabolites. Thus, an "isolated or purified" migratory body can be prepared by at least one purification step that removes or substantially removes these contaminant components.

[0159] In the present application, the term "and / or" should be understood to mean either one of the options or both of the options.

[0160] In the present application, the term "comprising" generally means including, but not limited to, the specifically recited members.

[0161] In the present application, the term "about" generally means a range of variation above or below the specified value of 0.5-10%, for example, a range of variation above or below the specified value of 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10%. DETAILED DESCRIPTION

[0163] In one aspect, the present application provides a method for preparing a migratory body, which can comprise causing a cell to undergo relative displacement and thereby produce a migratory body derived from the cell. For example, the relative displacement in the present application can refer to the displacement of the cell center relative to the environment (e.g., an adherent surface), or in the case of the cell center being stationary, the relative displacement of the membrane surface relative to the surrounding microenvironment (e.g., an adherent solid surface, or a liquid phase in the microenvironment), or relative to the cell center (e.g., swelling, deformation from flat to raised, etc.). For example, the production and / or extent of the relative displacement can be intervened by physical and / or chemical and / or biological means.

[0164] For example, in the method of the present application, it can further comprise isolating the migratory body produced by the cell.

[0165] For example, in the method of the present application, it can comprise subjecting the cell to hypotonic treatment, disrupting the cytoskeleton of the cell, inhibiting the cell volume regulation function of the cell, detaching the cell from its adherent surface, and / or causing the relative displacement between the cell membrane of the cell and its adherent surface, and can comprise increasing the number and / or function of tetraspanin proteins, functional fragments and / or functional variants thereof in the cell.

[0166] For example, in a method of the application, it can comprise disrupting the cytoskeleton of the cell, inhibiting the cell volume regulation function of the cell, detaching the cell from its adherent surface, and / or causing relative displacement between the cell membrane of the cell and its adherent surface, and it can comprise increasing the number and / or function of tetraspanin proteins, functional fragments and / or functional variants thereof in the cell. For example, in a method of the application, it can comprise subjecting the cell to a hypotonic treatment, inhibiting the cell volume regulation function of the cell, detaching the cell from its adherent surface, and / or causing relative displacement between the cell membrane of the cell and its adherent surface, and it can comprise increasing the number and / or function of tetraspanin proteins, functional fragments and / or functional variants thereof in the cell. For example, in a method of the application, it can comprise subjecting the cell to a hypotonic treatment, disrupting the cytoskeleton of the cell, detaching the cell from its adherent surface, and / or causing relative displacement between the cell membrane of the cell and its adherent surface, and it can comprise increasing the number and / or function of tetraspanin proteins, functional fragments and / or functional variants thereof in the cell. For example, in a method of the application, it can comprise subjecting the cell to a hypotonic treatment, disrupting the cytoskeleton of the cell, inhibiting the cell volume regulation function of the cell, and / or causing relative displacement between the cell membrane of the cell and its adherent surface, and it can comprise increasing the number and / or function of tetraspanin proteins, functional fragments and / or functional variants thereof in the cell. For example, in a method of the application, it can comprise subjecting the cell to a hypotonic treatment, disrupting the cytoskeleton of the cell, inhibiting the cell volume regulation function of the cell, and / or detaching the cell from its adherent surface, and it can comprise increasing the number and / or function of tetraspanin proteins, functional fragments and / or functional variants thereof in the cell.

[0167] For example, in a method of the application, the method can comprise inhibiting the cell volume regulation function of the cell, detaching the cell from its adherent surface, and / or causing relative displacement between the cell membrane of the cell and its adherent surface, and can comprise increasing the number and / or function of tetraspanin proteins, functional fragments and / or functional variants thereof in the cell. For example, in a method of the application, the method can comprise subjecting the cell to a hypotonic treatment, detaching the cell from its adherent surface, and / or causing relative displacement between the cell membrane of the cell and its adherent surface, and can comprise increasing the number and / or function of tetraspanin proteins, functional fragments and / or functional variants thereof in the cell. For example, in a method of the application, the method can comprise subjecting the cell to a hypotonic treatment, disrupting the cytoskeleton of the cell, and / or inhibiting the cell volume regulation function of the cell, and can comprise increasing the number and / or function of tetraspanin proteins, functional fragments and / or functional variants thereof in the cell.

[0168] For example, in a method of the application, the method can comprise subjecting the cell to a hypotonic treatment and / or causing relative displacement between the cell membrane of the cell and its adherent surface, and can comprise increasing the number and / or function of tetraspanin proteins, functional fragments and / or functional variants thereof in the cell. For example, in a method of the application, the method can comprise disrupting the cytoskeleton of the cell and / or causing relative displacement between the cell membrane of the cell and its adherent surface, and can comprise increasing the number and / or function of tetraspanin proteins, functional fragments and / or functional variants thereof in the cell. For example, in a method of the application, the method can comprise inhibiting the cell volume regulation function of the cell and / or causing relative displacement between the cell membrane of the cell and its adherent surface, and can comprise increasing the number and / or function of tetraspanin proteins, functional fragments and / or functional variants thereof in the cell.

[0169] For example, in a method of the application, the method can comprise subjecting the cell to a hypotonic treatment.

[0170] For example, in a method of the application, the hypotonic treatment can comprise placing the cell in a low-osmolarity buffer solution.

[0171] For example, in a method of the application, the hypotonic treatment can comprise placing the cell in a buffer solution and reducing the osmotic pressure of the buffer solution to a low-osmolarity buffer.

[0172] For example, in a method of the application, the reduction can comprise a linear reduction and / or a stepwise reduction.

[0173] For example, in the methods of the present application, the hypotonic buffered solution can have an osmolarity of less than about 305 mOsmol / L. For example, the hypotonic buffered solution can have an osmolarity of less than about 305 mOsmol / L, less than about 300 mOsmol / L, less than about 270 mOsmol / L, less than about 250 mOsmol / L, less than about 200 mOsmol / L, less than about 150 mOsmol / L, less than about 100 mOsmol / L, less than about 90 mOsmol / L, less than about 80 mOsmol / L, less than about 70 mOsmol / L, less than about 60 mOsmol / L, less than about 50 mOsmol / L, less than about 40 mOsmol / L, less than about 30 mOsmol / L, less than about 20 mOsmol / L, less than about 15 mOsmol / L, less than about 10 mOsmol / L, less than about 5 mOsmol / L, or less than about 2 mOsmol / L.

[0174] For example, in the methods of the present application, wherein the hypotonic buffered solution can have an osmolarity of about 10 mOsmol / L to about 274.5 mOsmol / L. For example, in the methods of the present application, wherein the hypotonic buffered solution can have an osmolarity of about 10 mOsmol / L to about 274.5 mOsmol / L.For example, the low-osmolarity buffer solution can have an osmolarity of about 10 mOsmol / L to about 300 mOsmol / L, about 20 mOsmol / L to about 300 mOsmol / L, about 30 mOsmol / L to about 300 mOsmol / L, about 50 mOsmol / L to about 300 mOsmol / L, about 70 mOsmol / L to about 300 mOsmol / L, about 100 mOsmol / L to about 300 mOsmol / L, about 150 mOsmol / L to about 300 mOsmol / L, about 200 mOsmol / L to about 300 mOsmol / L, about 250 mOsmol / L to about 300 mOsmol / L, about 10 mOsmol / L to about 250 mOsmol / L, about 20 mOsmol / L to about 250 mOsmol / L, about 30 mOsmol / L to about 250 mOsmol / L, about 50 mOsmol / L to about 250 mOsmol / L, about 70 mOsmol / L to about 250 mOsmol / L, about 100 mOsmol / L to about 250 mOsmol / L, about 150 mOsmol / L to about 250 mOsmol / L, about 200 mOsmol / L to about 250 mOsmol / L, about 10 mOsmol / L to about 200 mOsmol / L, about 20 mOsmol / L to about 200 mOsmol / L, about 30 mOsmol / L to about 200 mOsmol / L, about 50 mOsmol / L to about 200 mOsmol / L, about 70 mOsmol / L to about 200 mOsmol / L, about 100 mOsmol / L to about 200 mOsmol / L, about 150 mOsmol / L to about 200 mOsmol / L, about 10 mOsmol / L to about 150 mOsmol / L, about 20 mOsmol / L to about 150 mOsmol / L, about 30 mOsmol / L to about 150 mOsmol / L, about 50 mOsmol / L to about 150 mOsmol / L, about 70 mOsmol / L to about 150 mOsmol / L, about 100 mOsmol / L to about 150 mOsmol / L, about 10 mOsmol / L to about 100 mOsmol / L, about 20 mOsmol / L to about 100 mOsmol / L, about 30 mOsmol / L to about 100 mOsmol / L, about 50 mOsmol / L to about 100 mOsmol / L, about 70 mOsmol / L to about 100 mOsmol / L, about 10 mOsmol / L to about 30 mOsmol / L, about 20 mOsmol / L to about 30 mOsmol / L, about 30 mOsmol / L to about 90 mOsmol / L, or about 30 mOsmol / L to about 70 mOsmol / L.wherein the low osmotic pressure treatment comprises placing the cell in a buffered solution and reducing the osmotic pressure of the buffered solution to a hypotonic buffered solution.

[0175] For example, in the methods of the application, it can comprise disrupting the cytoskeleton of the cell.

[0176] For example, in the methods of the application, wherein disrupting the cytoskeleton of the cell can comprise contacting the cell with a cytoskeletal disrupting agent. For example, in the methods of the application, wherein the cytoskeletal disrupting agent can comprise a microfilament and / or microtubule depolymerizing agent. For example, in the methods of the application, wherein the cytoskeletal disrupting agent can comprise Latrunculin A, Latrunculin B, Cytochalasin A, Cytochalasin B, Cytochalasin C, Cytochalasin D, and / or Cytochalasin E.

[0177] For example, in the methods of the application, it can comprise inhibiting the cell volume regulation function of the cell.

[0178] For example, in the methods of the application, wherein inhibiting the cell volume regulation function of the cell can comprise reducing the number and / or function of a volume-regulated protein in the cell. For example, the number and / or function of a volume-regulated protein in the cell of the application that inhibits the cell volume regulation function of the cell is reduced by about 5%, reduced by about 10%, reduced by about 20%, reduced by about 50%, or reduced by about 100% relative to an unmodified cell.

[0179] For example, in the methods of the application, wherein the volume-regulated protein can comprise a volume-regulated ion channel and / or a transport protein. For example, in the methods of the application, wherein the volume-regulated ion channel can comprise a volume-regulated anion channel VRAC and / or a volume-regulated cation channel VRCC. For example, in the methods of the application, wherein the volume-regulated anion channel VRAC can comprise SWELL1 or a functionally active fragment thereof. For example, in the methods of the application, wherein the volume-regulated cation channel VRCC can comprise TRPV4, TRPM3, and / or a functionally active fragment thereof.

[0180] For example, in the methods of the application, wherein the transport protein can comprise a cotransport protein. For example, in the methods of the application, wherein the cotransport protein can comprise KCC1, KCC3, and / or KCC4.

[0181] For example, in the methods of the application, wherein inhibiting the cell volume regulation function of the cell can comprise placing the cell in a buffered solution with attenuated volume regulation capacity. For example, in the methods of the application, wherein the buffered solution can contain increased cations. For example, in the methods of the application, wherein the cations can comprise K +, Na + , Cs + , Li + , Ca 2+ , Mg 2+ , Ba 2+ , Mn 2+ , Fe 2+ , Ni 2+ , Zn 2+ , Al 3+ , Fe 3+ , CH3NH3 + , C2H5NH3 + , (CH3)2NH2 + , (C2H5)2NH2 + , (C2H5)3N + , an ammonium ion and / or a choline ion.

[0182] For example, in the methods of the application, wherein the buffer can contain an increased anion. For example, in the methods of the application, wherein the anion can comprise Br - , Cl - , I - , F - , OH - , HCO3 - , H2PO4 - , NO2 - , NO3 - , CN - , HPO4 2- , CO3 2- , SO4 2- , and / or PO4 3- .

[0183] For example, in the methods of the application, wherein the method can comprise detaching the cell from its adherent surface.

[0184] For example, in the methods of the application, wherein the method can comprise causing a relative displacement between the cell membrane of the cell and its adherent surface.

[0185] For example, in the methods of the application, it can further comprise increasing the number and / or function of tetraspanin proteins, functional fragments, and / or functional variants in the cell. For example, in the methods of the application, it can comprise causing the cell to overexpress the tetraspanin proteins, functional fragments, and / or functional variants. For example, in the methods of the application, wherein the tetraspanin proteins can be selected from the group consisting of: Tspanl, Tspan2, Tspan3, Tspan4, Tspan5, Tspan6, Tspan7, Tspan8, Tspan9, TspanlO, Tspanl l, Tspanl2, Tspanl3, Tspanl4, Tspanl5, Tspanl6, Tspanl7, Tspanl8, Tspanl9, Tspan20 (UPK1B), Tspan21 (UPK1A), Tspan22 (PRPH2), Tspan23 (ROM1), Tspan24 (CD151), Tspan25 (CD53), Tspan26 (CD37), Tspan27 (CD82), Tspan28 (CD81), Tspan29 (CD9), Tspan30 (CD63), Tspan31, Tspan32, and Tspan33.

[0186] For example, in the methods of the application, the method can further comprise reducing the size of the migratory body.

[0187] For example, in the methods of the application, wherein the reducing the size of the migratory body can comprise using a filter or an extruder to extrude the migratory body.

[0188] For example, in the methods of the application, wherein the pore size of the filter or extruder can be about 30 nm to about 10,000 nm. For example, the pore size of the filter or extruder can be about 30 nm to about 100 nm, about 30 nm to about 1,000 nm, about 30 nm to about 10,000 nm, about 50 nm to about 100 nm, about 50 nm to about 1,000 nm, about 50 nm to about 10,000 nm, about 100 nm to about 1,000 nm, about 100 nm to about 10,000 nm, or about 1,000 nm to about 10,000 nm.

[0189] For example, in the methods of the application, wherein the size of the migratory body can be about 50 nm to about 8,000 nm. For example, the size of the migratory body can be about 50 nm to about 100 nm, about 50 nm to about 1,000 nm, about 50 nm to about 10,000 nm, about 100 nm to about 1,000 nm, about 100 nm to about 10,000 nm, or about 1,000 nm to about 10,000 nm.

[0190] For example, in the methods of the present application, the migration body can be generated from the contractile filaments of the cell.

[0191] For example, in the methods of the present application, the membrane of the migration body can be enriched with sodium / potassium ATPase and / or functional fragments thereof. For example, in the methods of the present application, the membrane of the migration body can be enriched with integrin and / or functional fragments thereof. For example, in the methods of the present application, the membrane of the migration body can be enriched with tetraspanin proteins, functional variants thereof and / or functional fragments thereof. For example, in the methods of the present application, the membrane of the migration body can be enriched with cholesterol. For example, enrichment can refer to the density of the molecule or substance on the membrane of the migration body is higher than that on the membrane of the corresponding cell, or compared to the situation on the membrane of the corresponding cell before the migration body is generated. For example, enrichment refers to the density of the molecule or substance on the migration body of the present application is higher than that on the membrane of the cell from which the migration body is generated.

[0192] For example, in the methods of the present application, the migration body can be enriched with membrane microdomains. For example, membrane microdomains refer to sphingolipid and cholesterol (Ch)-based microdomains of the cell membrane, including lipid rafts, tetraspanin-enriched microdomains (TEM), etc.

[0193] For example, in the methods of the present application, the migration body can be generated in vitro or ex vivo.

[0194] For example, in the methods of the present application, the content of the migration body can be at least partially reduced or absent compared to the natural migration body generated by the same cell. For example, taking the engineered migration body generated by NRK cells as an example, compared with the natural migration body, there are 4000 or more migration bodies with protein loss, and the natural migration body has about 1350 proteins that are not present in the natural migration body (i.e., extra proteins), such as AMP3, Myh4, Gorasp2, Asz1, Lats2, Scn2b, Pacsin1, and Alb. For example, “natural migration body” can refer to a migration body generated spontaneously by a cell without changing the external culture conditions (e.g., hypotonicity, temperature, etc.); and “engineered migration body” can refer to a migration body generated by a cell after being induced to undergo relative displacement by external stimulation.

[0195] For example, in the methods of the present application, the at least partially reduced content can comprise intraluminal vesicles. For example, the engineered migratory bodies provided herein can have a reduced intraluminal vesicle content. The intraluminal vesicles can refer to vesicles having a phospholipid bilayer structure in an outer membrane structure.

[0196] For example, in the methods of the present application, the methods can be in vitro or ex vivo methods. For example, the preparation methods of the present application can be performed in vitro. For example, the preparation methods of the present application can be performed outside of a living subject.

[0197] For example, in the methods of the present application, the cells can be cells cultured in vitro.

[0198] For example, in the methods of the present application, the cells can be cells cultured in suspension or adherent cells.

[0199] For example, in the methods of the present application, the cells can comprise primary cells.

[0200] For example, in the methods of the present application, the primary cells can comprise tissue cells derived from an organism, which can comprise a human, a monkey, a mouse, a rat, a rabbit, a chicken, and / or an insect.

[0201] For example, in the methods of the present application, the primary cells can comprise hepatocytes, splenocytes, kidney cells, tissue macrophages, brain glial cells, osteoclasts, bone marrow cells, leukocytes, fibroblasts, and / or adipocytes. For example, the primary cells of the present application can comprise Kupffer cells. For example, a phagocytic cell located on the inner surface of the liver sinusoid, capable of removing foreign antigens, antigen-antibody complexes, and cellular debris and other materials from the blood.

[0202] For example, in the methods of the present application, the leukocytes can comprise B cells, T cells, NK cells, dendritic cells, neutrophils, and / or macrophages.

[0203] For example, in the methods of the present application, the cells can comprise tumor cells.

[0204] For example, in the methods of the present application, the tumor cells can comprise tumor cell lines, primary or finite pass tumor cells derived from a patient, tumor stromal cells, and / or tumor organoids.

[0205] For example, in the methods of the present application, the cells can include CHO cells, CHO-K1 cells, HEK293 cells, HEK293T cells, HEK293FT cells, HEK293F cells, Vero cells, NRK cells, L929 cells, MC38 cells, 4T1 cells, DC2.4 cells, MGC803 cells, Jurkat cells, NK-92MI cells, BJ cells, and / or HepG2 cells.

[0206] For example, in the methods of the present application, the cells can include white blood cells, stem cells, and / or fibroblasts.

[0207] For example, in the methods of the present application, the stem cells can include mesenchymal stem cells.

[0208] For example, the present application provides a migrasome prepared in the methods of the present application. For example, the present application provides a novel extracellular vesicle that can be engineered to produce, an engineered migrasome and / or a migrasome forced to be produced efficiently by cells through an engineering method, and / or a composition comprising any of the above migrasomes, characterized in that the engineered migrasome has a size of 50-8000 nanometers (nm), an enrichment of proteins such as integrins and Tetraspanin (Tspan) family members on the membrane, and is present by the presence of membrane microdomains, in some cases growing on filamentous structures around the cells, which can be spontaneously released or separated from the cells by artificial methods, resulting in an intact vesicular structure, and / or its biochemical composition, morphology, and structure are similar to the natural migrasomes produced by the cells; it can also be specifically adjusted according to the needs of the application to obtain vesicular structures that are quite different from the natural migrasomes of the cells in structure and biochemical composition. For example, the present application provides the use of migrasomes / engineered migrasomes as a drug-loaded delivery carrier in the treatment of different diseases (such as tumors, inflammatory / autoimmune diseases, cardiovascular diseases, neurodegenerative diseases, and other nervous system diseases, etc.) and in the field of vaccines.

[0209] Migrasome is a novel organelle that is generated at the tip or cross-over of contractile filaments left behind the cell body during cell migration. It is a single-membrane vesicle structure with a diameter of 0.5-3 pm. During cell migration, the cell body continuously transports intracellular materials to the migrasome. Subsequently, the contractile filaments break and the migrasome is released, which can be transported to a distant tissue at the site of injury or through a body fluid such as blood, and then be taken up by the extracellular space or surrounding cells. This indicates that the migrasome can be involved in the transmission of intracellular materials and signals between cells, thereby mediating intercellular communication (Liang Ma et. al., Discovery of the migrasome, an organelle mediating release of cytoplasmic contents during cell migration, Cell Res (2015) 25: 24-38).

[0210] Studies have shown that migrasomes play an important role in signal transmission in processes with active cell migration, such as embryonic development, immune response, tumor, angiogenesis, and tissue regeneration. Through the study of migrasomes in zebrafish embryonic development, it was found that migrasomes, as membrane-coated carriers of signaling molecules, determine the spatial and temporal distribution of signaling molecules, thereby playing a regulatory role in organ development (Jiang D et.al., Migrasomes provide regional cues for organ morphogenesis during zebrafish gastrulation, Nat Cell Biol, 2019, 21(8): 966-977). Migrasomes can mediate protein and mRNA transfer between cells, and by transferring proteins and mRNAs to recipient cells through migrasomes, the life activities of recipient cells can be changed (Zhu M et.al., Lateral transfer of mRNA and protein by migrasomes modifies the recipient cells, Cell Res, 2020, doi; 10.1038 / s41422-020-00415-3). Migrasomes can regulate mitochondrial quality by removing damaged mitochondria, thereby maintaining mitochondrial homeostasis in cells (Jiang H et.al., Mitocytosis, a migrasome-mediated mitochondrial quality control process, Cell Press, doi: 10.1016 / j.cell.2021.04.027). Migrasomes have a regulatory effect on cancer cells in the tumor microenvironment, such as in pancreatic cancer cells, where migrasomes can induce an inhibitory immune microenvironment, thereby promoting tumor growth (Zhang Ronghua, Study on the mutual regulation of pancreatic cancer cell migrasomes on the phenotype and function of cancer cells and related immune cells in the tumor microenvironment, 2020).

[0211] Detached migrasomes are a type of extracellular vesicle that has many differences from known extracellular vesicles. For example, differences between migrasomes and exosomes: 1) different structures: migrasomes are attached to contractile filaments before being released and exhibit a structure of small vesicles contained within a large vesicle; while exosomes do not have this structure; 2) different sizes: exosomes are about 50-150 nm in diameter, while migrasomes are about 0.5-3 pm in diameter; 3) significantly different protein compositions: only 27% of the protein composition is the same between migrasomes and exosomes, for example, NDST1 (bifunctional heparan sulfate N-deacetylase / N-sulfotransferase 1), PIGK (phosphatidylinositol glycan anchor biosynthesis class K), CPQ (carboxypeptidase Q), and EOGT (EGF domain-specific O-linked N-acetylglucosamine transferase) are enriched on migrasomes and are not present in exosomes (Zhao X, Lei Y, Zheng J, Peng J, Li Y, Yu L, Chen Y. Identification of markers for migrasome detection. Cell Discov. 2019 May 21; 5: 27); 4) different genetic pathways regulate them, completely different biogenesis processes: exosomes are first produced as multivesicular bodies (MVBs) of vesicles, exosomes are released when MVBs fuse with the plasma membrane; while migrasomes are assembled from large domains on the plasma membrane (Huang Y, Zucker B, Zhang S, Elias S, Zhu Y, Chen H, Ding T, Li Y, Sun Y, Lou J, Kozlov MM*, Yu L*. Migrasome formation is mediated by assembly of micron-scale tetraspanin macrodomains. Nat Cell Biol. 2019 Aug; 21(8): 991-1002).

[0212] The present application found that engineered migrasomes, which are structurally similar to natural migrasomes, can be induced to produce by a variety of methods. Engineered migrasomes are different from the “hypotonic pressure-induced vesicles” known in the art (e.g., Cohen S, Ushiro H, Stoscheck C, Chinkers M, A native 170 000 epidermal growth factor receptor-kinase complex from shed plasma membrane vesicles. J Biol Chem 257: 1523-1531, 1982; and U.S. Patent No. 9, 105, 223, which are incorporated herein by reference in their entirety). Figure 1The vesicles shown), at least the following differences exist: 1) the site of vesicle production is different: engineered migrators are produced on the contractile filaments around the cell; while the vesicles produced by Cohen et al. are produced on the surface of the cell; 2) the size of the vesicles is different: the size of the engineered migrators is microns, and the diameter rarely exceeds 5 μm; while the vesicles induced by Cohen et al. using low osmotic pressure can be as large as 20 μm; 3) unlike the method used by Cohen et al. using low osmotic pressure, low osmotic pressure is not a necessary condition for the production of engineered migrators in this application, and engineered migrators can be induced by other methods.

[0213] Engineered extracellular vesicles are currently widely used in important biomedical fields such as drug delivery and vaccine preparation. For example, engineered exosomes (Kamerkar S, LeBleu VS, Sugimoto H, Yang S, Ruivo CF, Melo SA, Lee JJ, Kalluri R. Exosomes facilitate therapeutic targeting of oncogenic KRAS in pancreatic cancer. Nature. 2017 Jun 22; 546(7659): 498-503. doi: 10.1038 / nature22341), the main advantages of which mainly include: natural biological membrane source, low toxicity; low immunogenicity, not easily cleared by the immune system, thus longer circulation time in vivo; stable properties, can be stored at -80°C for a long time. However, it also has obvious limitations, for example, low yield; the purification process relies on a super-speed centrifuge; the contents are mainly post-loaded nucleic acids, etc.

[0214] The engineered migrators discovered in this application are biologically derived, have the advantages of low toxicity and low immunogenicity of biologically derived extracellular vesicles such as exosomes; they are a completely new type of extracellular vesicle, and have unique characteristics in terms of molecules that can be transported and biological distribution in vivo; at the same time, the preparation method is simple and the yield is high. In summary, the engineered migrators discovered in this application have extremely high application value in the field of biomedicine such as drug delivery and vaccine preparation.

[0215] A method for preparing engineered migrators, comprising artificially inducing cells to produce engineered migrators, and isolating and / or purifying the engineered migrators; and / or forcing the cell membrane to migrate with the culture environment (including the attached solid surface and the surrounding liquid environment), including but not limited to natural cell migration methods or relative displacement of the cell membrane with the environment at a sub-cellular scale.

[0216] In another aspect, the present application provides a migration body prepared in vitro or ex vivo, wherein the size of the migration body can be about 50 nm to about 8000 nm.

[0217] For example, in the migration body of the present application, the contractile filaments of the cell can be produced in vitro.

[0218] For example, in the migration body of the present application, the membrane of the migration body can be enriched with sodium / potassium ATPase and / or a functional fragment thereof.

[0219] For example, in the migration body of the present application, the membrane of the migration body can be enriched with integrin and / or a functional fragment thereof.

[0220] For example, in the migration body of the present application, the membrane of the migration body can be enriched with tetraspanin protein, a functional variant thereof and / or a functional fragment thereof.

[0221] For example, in the migration body of the present application, the membrane of the migration body can be enriched with cholesterol.

[0222] For example, in the migration body of the present application, the migration body can be enriched with membrane microdomains.

[0223] For example, in the migration body of the present application, the content of the migration body can be at least partially reduced or absent compared to the natural migration body produced by the corresponding cell.

[0224] For example, in the migration body of the present application, the at least partially reduced content can comprise intraluminal vesicles.

[0225] In another aspect, the present application provides a use of a migration body for delivering an exogenous load. For example, the "exogenous load" can refer to a substance that is not produced by the cell itself, is not produced (e.g., expressed) in a substantial amount, or the substance is produced by the cell itself, but the exogenous load is structurally or functionally distinguished from the substance produced by the cell itself.

[0226] For example, in the use of the present application, the migration body can comprise the migration body described in the present application.

[0227] In another aspect, the present application provides a delivery system, which can comprise a migration body and one or more exogenous loads.

[0228] For example, in the delivery system of the present application, the exogenous load can be directly or indirectly bound, linked or embedded to the membrane and / or the interior of the migration body.

[0229] For example, in the delivery system of the present application, the migration body can comprise the migration body described in the present application.

[0230] For example, in the delivery system of the present application, wherein the migrator can be derived from a cell.

[0231] For example, in the delivery system of the present application, wherein the exogenous load can comprise one or more targeting substances and / or therapeutically active substances.

[0232] For example, in the delivery system of the present application, wherein the exogenous load can comprise proteins, lipids, polynucleotides, small molecule compounds, complexes, polysaccharides, polymers, nanoparticles, microparticles and / or organelles.

[0233] For example, in the delivery system of the present application, wherein the exogenous load can comprise membrane proteins, soluble proteins and / or polypeptides.

[0234] For example, in the delivery system of the present application, wherein the exogenous load can comprise DNA and / or RNA.

[0235] For example, in the delivery system of the present application, wherein the exogenous load can comprise antibodies or antigen-binding antibody fragments thereof, integrins or fragments thereof, immunogenic proteins, cytokines, chemokines, receptor proteins or fragments thereof, enzymes, tumor suppressor gene products, siRNAs, microRNAs, antisense oligonucleotides (ASOs), mRNAs, DNA, gene editing tools and / or cytotoxic agents. For example, the gene editing tools can comprise nucleases, such as Cas proteins, CRISPR-Cas systems, Cre recombinase, zinc finger endonucleases, transcription activator-like effector nucleases and gene epigenetic editing tools.

[0236] For example, in the delivery system of the present application, wherein the exogenous load can comprise PAMPs, DAMPs, CD47, CD24, IL-12, IL-15, coagulation factor VII, coagulation factor VIII, coagulation factor IX and / or functionally active fragments thereof.

[0237] For example, in the delivery system of the present application, wherein the exogenous load can be directly or indirectly bound to the migrator by gene editing, exogenous expression, liquid-to-solid phase transition, membrane fusion, charge adsorption, physical adsorption and / or chemical linkage. For example, the exogenous expression can comprise plasmid expression, such as establishing a cell line for transient or stable overexpression of a certain gene.

[0238] For example, in the delivery system of the present application, wherein the exogenous load can be bound or embedded to the migrator by direct or indirect linkage to membrane components of the migrator.

[0239] For example, in a delivery system of the present application, wherein the membrane component of the migratory body can comprise a membrane protein, cholesterol, a phospholipid, a sugar chain on a glycoprotein, and / or a polysaccharide.

[0240] For example, in a delivery system of the present application, wherein the indirect linkage can comprise linkage via a click chemistry reaction.

[0241] For example, in a delivery system of the present application, wherein the indirect linkage can comprise providing the exogenous cargo linked to a first member of a binding pair, and contacting it with the migratory body, the membrane of which comprises a second member of the binding pair, wherein the first member is capable of binding to the second member.

[0242] For example, in a delivery system of the present application, wherein the first and second members of the binding pair can be selected from the group consisting of an antigen and its antibody; a receptor and its ligand; biotin and avidin; a HaloTag and its ligand; and CP05 and CD63.

[0243] For example, in a delivery system of the present application, wherein the exogenous cargo can be expressed on the inner or outer surface of the membrane of the migratory body as a membrane protein.

[0244] For example, in a delivery system of the present application, wherein the exogenous cargo can be expressed on the inner or outer surface of the membrane of the migratory body as a fusion protein fused to a membrane protein or a portion thereof.

[0245] For example, in a delivery system of the present application, wherein the exogenous cargo can be expressed on the inner or outer surface of the membrane of the migratory body as a fusion protein fused to a membrane protein or a portion thereof via gene editing and / or exogenous expression.

[0246] In another aspect, the present application provides a method of preparing a delivery system, which can comprise providing a migratory body, and causing the migratory body to carry an exogenous cargo. For example, the causing the migratory body to carry an exogenous cargo can comprise reversibly carrying an exogenous cargo. For example, the causing the migratory body to carry an exogenous cargo can comprise irreversibly carrying an exogenous cargo.

[0247] For example, in a method of the present application, wherein the migratory body can be an isolated or purified migratory body.

[0248] For example, in a method of the present application, wherein the causing the migratory body to carry an exogenous cargo can comprise directly or indirectly linking or embedding the exogenous cargo to the membrane of the migratory body and / or the interior of the migratory body.

[0249] For example, in a method of the present application, which can further comprise isolating or purifying the migratory body from a cell.

[0250] For example, in the methods of the present application, it can comprise providing a complex of the exogenous load and a first member of a binding pair; allowing a cell to produce a migratory body comprising a second member of the binding pair; and allowing the migratory body to contact the complex to form the delivery system.

[0251] For example, in the methods of the present application, wherein the migratory body can comprise a migratory body described herein.

[0252] For example, in the methods of the present application, wherein the migratory body can be derived from a cell.

[0253] For example, in the methods of the present application, wherein the exogenous load can comprise one or more targeting substances and / or therapeutically active substances.

[0254] For example, in the methods of the present application, wherein the exogenous load can comprise a protein, a lipid, a polynucleotide, a small molecule compound, a complex, a polysaccharide, a polymer, a nanoparticle, a microparticle, and / or an organelle.

[0255] For example, in the methods of the present application, wherein the exogenous load can comprise a membrane protein, a soluble protein, and / or a polypeptide.

[0256] For example, in the methods of the present application, wherein the exogenous load can comprise DNA and / or RNA.

[0257] For example, in the methods of the present application, wherein the exogenous load can comprise an antibody or antigen-binding antibody fragment thereof, an integrin or fragment thereof, an immunogenic protein, a cytokine, a chemokine, a receptor protein or fragment thereof, an enzyme, an onco-suppressor product, an siRNA, a microRNA, an antisense oligonucleotide (ASO), an mRNA, a DNA, a gene editing tool, and / or a cytotoxic agent.

[0258] For example, in the methods of the present application, wherein the exogenous load can comprise a PAMP, a DAMP, CD47, CD24, IL-12, IL-15, coagulation factor VII, coagulation factor VIII, coagulation factor IX, and / or a functionally active fragment thereof.

[0259] For example, in the methods of the present application, wherein the exogenous load can be directly or indirectly associated with the migratory body by gene editing, exogenous expression, liquid-to-solid transformation, membrane fusion, charge adsorption, physical adsorption, and / or chemical linkage.

[0260] For example, in the methods of the present application, wherein the exogenous load can be attached or embedded to the migratory body by direct or indirect association with a membrane component of the migratory body.

[0261] For example, in the methods of the application, wherein the membrane component of the migrator can comprise a membrane protein, cholesterol, a phospholipid, a sugar chain on a glycoprotein, and / or a polysaccharide.

[0262] For example, in the methods of the application, wherein the indirect conjugation can comprise linking via a click chemistry reaction.

[0263] For example, in the methods of the application, wherein the indirect conjugation can comprise providing the exogenous cargo conjugated to a first member of a binding pair, and contacting it with the migrator, the membrane of which comprises a second member of the binding pair, wherein the first member is capable of binding to the second member.

[0264] For example, in the methods of the application, wherein the first and second members of the binding pair can be selected from the group consisting of an antigen and its antibody; a receptor and its ligand; biotin and avidin; a HaloTag and its ligand; and CP05 and CD63.

[0265] For example, in the methods of the application, wherein the exogenous cargo can be expressed as a membrane protein on the inner or outer surface of the migrator membrane.

[0266] For example, in the methods of the application, wherein the exogenous cargo can be expressed as a fusion protein fused to a membrane protein or a portion thereof on the inner or outer surface of the migrator membrane.

[0267] In another aspect, the present application provides a method of making a delivery system, which can comprise: causing a cell to express an mRNA; causing the cell to produce a migrator, the migrator comprising an mRNA-binding protein, and the mRNA being linked to the migrator via the mRNA-binding protein. For example, the mRNA-binding protein can comprise a protein forming complex for binding the mRNA, for example, the RNA-binding protein comprises an RNA recognition motif. The RNA-binding protein comprises a native or inactivated nuclease activity. The RNA-binding protein or a functional domain thereof can comprise a Cys2-His2, Gag-knuckle, Treble-clet, zinc ribbon, Zn2 / Cys6-like motif. Exemplary mRNA-binding proteins can comprise, but are not limited to, RNA binding proteins (RBPs).

[0268] In another aspect, the present application provides a method of making a delivery system, which can comprise: causing a cell to express an exogenous cargo on a cell membrane; and causing the cell to produce a migrator, the migrator comprising the exogenous cargo. The expression of the exogenous cargo can be achieved by gene editing or exogenous expression, for example, plasmid expression, etc.

[0269] For example, in the methods of the application, wherein the exogenous cargo can be a protein.

[0270] For example, in the methods of the application, the protein can be a membrane protein.

[0271] For example, in the methods of the application, the protein can be a soluble protein, and it can be fused with a membrane protein or a portion thereof to form a fusion protein.

[0272] In another aspect, the application provides a composition, which can comprise a migratory body described herein or a delivery system described herein.

[0273] 1. A method of preparing an engineered migratory body, comprising

[0274] (a) subjecting a cell to one or more of the following treatments to induce the cell to produce an engineered migratory body:

[0275] (1) a hypotonic treatment;

[0276] (2) disrupting the cytoskeleton of the cell;

[0277] (3) inhibiting the cell volume regulation function of the cell; and

[0278] (4) overexpressing in the cell one or more proteins selected from the Tetraspanin family members; and

[0279] (5) inducing rapid detachment of the cell from an adherent surface or inducing relative displacement of the cell membrane and the adherent surface.

[0280] (b) isolating and / or purifying the engineered migratory body.

[0281] 2. The method according to technical solution 1, wherein in step (a), at least one of (2)-(5) is performed, at least two of (1)-(5) is performed, at least three of (1)-(5) is performed, at least four of (1)-(5) is performed, or all five of (1)-(5) is performed.

[0282] 3. The method according to technical solution 1 or 2, wherein step (a) comprises subjecting the cell to a hypotonic treatment.

[0283] 4. The method according to any one of technical solutions 1-3, wherein the hypotonic treatment is performed by placing the cell in a low-osmolarity buffer solution having an osmolarity of 30.5-274.5 mOsmol / L, such as 30.5-150 mOsmol / L.

[0284] 5. The method according to any one of technical solutions 1-3, wherein the hypotonic treatment is performed by placing the cell in a buffer solution and reducing the osmolarity of the buffer solution to 30.5-274.5 mOsmol / L, such as 30.5-150 mOsmol / L.

[0285] 6. The method according to item 5, wherein reducing the osmolarity of the buffer solution comprises a linear reduction or a stepwise reduction.

[0286] 7. The method according to any one of items 1-6, wherein step (a) comprises disrupting the cytoskeleton of the cell.

[0287] 8. The method according to item 7, wherein the cytoskeleton of the cell is disrupted by contacting the cell with an agent that disrupts the cytoskeleton.

[0288] 9. The method according to item 8, wherein the agent that disrupts the cytoskeleton comprises a microfilament depolymerizing agent, such as selected from the group consisting of Latrunculin A, Latrunculin B, Cytochalasin A, Cytochalasin B, Cytochalasin C, Cytochalasin D, and Cytochalasin E.

[0289] 10. The method according to any one of items 1-9, wherein step (a) comprises inhibiting the cell volume regulation function of the cell.

[0290] 11. The method according to item 10, wherein the cell volume regulation function of the cell is inhibited by inhibiting the expression or activity of a protein that regulates cell volume of the cell.

[0291] 12. The method according to item 11, wherein the protein that regulates cell volume is selected from the group consisting of ion channels and transporters.

[0292] 13. The method according to item 12, wherein the protein that regulates cell volume is selected from the group consisting of volume-regulated anion channels (VRAC), such as SWELL 1, volume-regulated cation channels (VRCC), such as TRPV4 and TRPM3, and co-transporters, such as KCC1, KCC3, and KCC4.

[0293] 14. The method according to item 10, wherein the cell volume regulation function of the cell is inhibited by placing the cell in a buffer solution comprising cations or anions with attenuated ability to regulate changes in cell volume.

[0294] 15. The method according to item 14, wherein the cations are selected from the group consisting of K + , Na + , Cs + , Li + , Ca 2+ , Mg 2+ , Ba 2+ , Mn 2+ , Fe 2+ , Ni2+ Zn 2+ Al 3+ Fe 3+ CH3NH3 + C2H5NH3 + (CH3)2NH2 + (C2H5)2NH2 + (C2H5)3N + one or more of an ammonium ion and / or a choline ion.

[0295] 16. The method according to technical solution 14, wherein the anion is selected from one or more of Br - Cl - I - F - OH - HCO3 - H2PO4 - NO2 - NO3 - CN - HPO4 2- CO3 2- SO4 2- and / or PO4 3-

[0296] 17. The method according to any one of technical solutions 1-16, wherein step (a) comprises causing the cell to overexpress one or more proteins selected from the Tetraspanin family of members.

[0297] 18. The method according to technical solution 17, wherein the Tetraspanin family member comprises Tspanl, Tspan2, Tspan3, Tspan4, Tspan5, Tspan6, Tspan7, Tspan8, Tspan9, TspanlO, Tspanl l, Tspanl2, Tspanl3, Tspanl4, Tspanl5, Tspanl6, Tspanl7, Tspanl8, Tspanl9, Tspan20 (UPK1B), Tspan21 (UPK1A), Tspan22 (PRPH2), Tspan23 (ROM1), Tspan24 (CD151), Tspan25 (CD53), Tspan26 (CD37), Tspan27 (CD82), Tspan28 (CD81), Tspan29 (CD9), Tspan30 (CD63), Tspan31, Tspan32, and Tspan33.

[0298] ​19. The method according to technical solution 17, wherein step (a) comprises causing the cell to overexpress Tspan4.

[0299] 20. The method according to any one of technical solutions 1-19, wherein the method further comprises reducing the size of the engineered migratory body.

[0300] 21. The method according to technical solution 20, wherein the size of the engineered migratory body is reduced by extruding the engineered migratory body through a filter of a specific pore size.

[0301] Optionally, the size of the reduced engineered migratory body is nanoscale, e.g., 50-200 nm.

[0302] 22. The method according to any one of technical solutions 1-21, wherein the cell is a normal cell or a cancer cell.

[0303] 23. An engineered migratory body prepared by the method according to any one of technical solutions 1-22.

[0304] 24. A delivery system comprising an isolated or purified migratory body and a payload directly or indirectly linked to the membrane of the migratory body.

[0305] 25. The delivery system according to technical solution 24, wherein the migratory body is selected from the group consisting of a naturally occurring migratory body and an engineered migratory body artificially induced.

[0306] 26. The delivery system according to technical solution 25, wherein the engineered migratory body is produced by the method according to any one of technical solutions 1-22.

[0307] 27. The delivery system according to any one of technical solutions 24-26, wherein the payload is selected from the group consisting of a protein, e.g., a membrane protein and a soluble protein, a peptide, a nucleic acid, e.g., DNA and RNA, and a small molecule compound.

[0308] 28. The delivery system according to any one of technical solutions 24-26, wherein the payload is selected from the group consisting of a therapeutic protein, an immunogenic protein, a cytokine, an enzyme, an siRNA, a microRNA, an antisense oligonucleotide (ASO), an mRNA, a CRISPR system, a cytotoxic agent, a therapeutic small molecule, and a targeting molecule.

[0309] 29. The delivery system according to technical solution 28, wherein the targeting molecule is selected from the group consisting of an antibody or an antigen-binding fragment thereof; an integrin; a find-me / eat me signal, e.g., a PAMP and a DAMP; and a don’t-eat-me signal, e.g., CD47 and CD24.

[0310] 30. The delivery system according to any one of items 24-29, wherein the cargo is attached or embedded to the membrane of the migrator by physical adsorption or chemical linkage.

[0311] 31. The delivery system according to any one of items 22-29, wherein the cargo is attached or embedded to the membrane of the migrator by a means selected from the group consisting of:

[0312] (1) attachment to a membrane component of the migrator, such as a membrane protein or cholesterol;

[0313] (2) attachment to a protein or peptide attached to the membrane of the migrator, preferably the protein or peptide is attached to the membrane of the migrator by click chemistry; and

[0314] (3) attachment to a first member of a binding pair, wherein the membrane of the migrator comprises a second member of the binding pair, the cargo is attached to the membrane of the migrator by the binding of the first and second members.

[0315] 32. The delivery system according to item 31, wherein the binding pair comprises a receptor-ligand binding pair.

[0316] 33. The delivery system according to item 32, wherein the binding pair is a HaloTag and its ligand, or CP05 and CD63.

[0317] 34. The delivery system according to item 31, wherein the cargo is mRNA, and the membrane of the migrator comprises an mRNA binding protein, the mRNA is attached to the membrane of the migrator by its binding to the mRNA binding protein through a protein binding site of the 3'-UTR of the mRNA.

[0318] 35. The delivery system according to any one of items 24-29, wherein the cargo is expressed on the surface of the membrane of the migrator as a membrane protein.

[0319] 36. The delivery system according to any one of items 24-29, wherein the cargo is expressed on the surface of the membrane of the migrator as a fusion protein fused to a membrane protein or a portion thereof.

[0320] 37. A method of producing a delivery system comprising an isolated or purified migrator and a cargo selected from the group consisting of a protein, a peptide, a nucleic acid (e.g., DNA and RNA), and a small molecule compound, wherein the method comprises:

[0321] isolating or purifying a migrator from a cell, the migrator being a naturally produced migrator or an engineered migrator produced by artificial induction; and

[0322] attaching the cargo directly or indirectly to the membrane of the migrator, thereby producing the delivery system.

[0323] 38. The method according to technical solution 37, wherein the cargo is attached to the membrane of the migrator by physical adsorption or chemical linkage.

[0324] 39. The method according to technical solution 37, wherein the cargo is attached to the membrane of the migrator by a means selected from the group consisting of:

[0325] (1) linking the cargo to a membrane component of the migrator, such as a membrane protein or cholesterol;

[0326] (2) linking the cargo to a protein or peptide attached to the membrane of the migrator, preferably the protein or peptide is attached to the membrane of the migrator by click chemistry.

[0327] 40. A method of producing a delivery system comprising an engineered migrator and a cargo selected from the group consisting of a protein, a peptide, a nucleic acid (such as DNA and RNA) and a small molecule compound, wherein the cargo is attached to a first member of a binding pair and the membrane of the migrator comprises a second member of the binding pair, wherein the method comprises:

[0328] causing the cell to express the second member of the binding pair on the cell membrane;

[0329] producing from the cell an engineered migrator comprising the second member of the binding pair on the membrane; and

[0330] contacting the cargo in complex with the first member of the binding pair with the engineered migrator, thereby producing the delivery system by binding of the first member to the second member.

[0331] 41. The method according to technical solution 40, wherein the binding pair comprises a receptor-ligand binding pair.

[0332] 42. The method according to technical solution 41, wherein the binding pair is a HaloTag and its ligand, or CP05 and CD63.

[0333] 43. A method of producing a delivery system comprising an engineered migrator and a cargo, the cargo being an mRNA and the membrane of the migrator comprising an mRNA binding protein, the mRNA being bound to the mRNA binding protein through a protein binding site of its 3’-UTR, wherein the method comprises:

[0334] causing the cell to express the mRNA,

[0335] prior to, after or simultaneously with the above step, causing the cell to express the mRNA binding protein on the cell membrane; and

[0336] producing, from the cell, a delivery system comprising the engineered migrans and an mRNA, wherein the mRNA is linked to the membrane of the engineered migrans by binding to the mRNA-binding protein.

[0337] 44. A method of producing a delivery system comprising an engineered migrans and a cargo, the cargo being a protein and expressed on the surface of the membrane of the engineered migrans, wherein the method comprises:

[0338] expressing the protein on the cell membrane by the cell; and

[0339] producing, from the cell, a delivery system comprising the engineered migrans and the protein expressed on the surface of the membrane of the engineered migrans.

[0340] 45. The method according to item 44, wherein the protein is a membrane protein.

[0341] 46. The method according to item 44, wherein the protein is a soluble protein and expressed on the surface of the membrane of the migrans as a fusion protein fused with a membrane protein or a portion thereof.

[0342] 47. The method according to any one of items 37-46, wherein the engineered migrans is produced by the method according to any one of items 1-22.

[0343] Compared with drug conjugated delivery systems and drug delivery systems from non-biological sources, the migrans delivery system of the present application has higher biocompatibility, more types of drugs can be delivered, the capacity is larger, and the membrane modification is easier to increase or change the targeting. Compared with engineered exosomes and engineered red blood cells, the migrans delivery system retains high biocompatibility and easy modification characteristics, and has a larger volume than traditional small vesicles (such as exosomes), which can more effectively deliver drugs, especially macromolecular proteins and nucleic acids. At the same time, the migrans delivery system has high modifiability, and the size of the migrans and the substances loaded on the membrane can be adjusted according to the drugs to be delivered and the cells to be targeted. The migrans also has the advantage of low toxicity, making it easier to modify and modify the membrane components. In addition, the preparation of engineered migrans does not require ultracentrifugation, nor does it require the extraction of blood samples each time, greatly reducing the preparation time and cost.

[0344] Therefore, compared with existing drug delivery systems, the migrans delivery system of the present application has the characteristics of low toxicity, high loading capacity, easy modification, fast production, low cost, and adjustable antigenicity, and has a unique antigen presentation function, which can be used as a vaccine delivery platform.

[0345] The present applicant has found that by subjecting cells to one or more of the following treatments: hypotonic treatment, disruption of the cytoskeleton of the cells, inhibition of the cell volume regulation function of the cells and overexpression of Tetraspanin family members in the cells, the cells can be induced to produce engineered migrans.

[0346] Although migrans have a vesicular structure, they are not identical to extracellular vesicles and exosomes. Migrans can not belong to the strict sense of extracellular vesicles. Although migrans that have detached from cells are a type of extracellular vesicle, many of the functions of migrans are performed before they detach from the cell body. This is the reason why migrans are considered organelles and can not be a type of extracellular vesicle. The production of extracellular vesicles (detached migrans) is only one of the many functions of migrans.

[0347] The detached migrasome is an extracellular vesicle, but it can have many differences from exosomes. For example, 1) the two structures are different: migrasomes are attached to contractile filaments before being released and exhibit a structure of small vesicles contained within a large vesicle; while exosomes do not have this structure; 2) the sizes of the two are different: the diameter of exosomes is about 50-150 nm, while the diameter of migrasomes is about 0.5-3 pm; 3) the protein compositions of the two are significantly different: only 27% of the protein compositions are the same between migrasomes and exosomes, for example, NDST1 (bifunctional heparan sulfate N-deacetylase / N-sulfotransferase 1), PIGK (phosphatidylinositol glycan anchor biosynthesis K), CPQ (carboxypeptidase Q), and EOGT (EGF domain-specific O-linked N-acetylglucosamine transferase) are enriched in migrasomes and are not present in exosomes (Zhao X, Lei Y, Zheng J, Peng J, Li Y, Yu L, Chen Y. Identification of markers for migrasome detection. Cell Discov. 2019 May 21; 5: 27); 4) the two are regulated by different genetic pathways and have completely different biogenesis processes: exosomes are first produced as vesicles of multivesicular bodies (MVBs), and exosomes are released when MVBs fuse with the plasma membrane; while migrasomes are formed by assembly of large domains on the plasma membrane (Huang Y, Zucker B, Zhang S, Elias S, Zhu Y, Chen H, Ding T, Li Y, Sun Y, Lou J, Kozlov MM*, Yu L*. Migrasome formation is mediated by assembly of micron-scale tetraspanin macrodomains. Nat Cell Biol. 2019 Aug; 21(8): 991-1002).

[0348] The term "engineered migrasome" as used herein refers to a migrasome produced by engineering and / or artificially inducing a cell. It should be noted that "engineered migrasome" can not imply that it is synthetic, it can still be of cellular origin.

[0349] The engineered migrators of the present application can be different from the "hypotonic pressure-induced vesicles" known in the art (e.g., Cohen S, Ushiro H, Stoscheck C, Chinkers M An native 170 000 epidermal growth factor receptor-kinase complex from shed plasma membrane vesicles. J Biol Chem 257: 1523-1531 Figure 1 The engineered migrators of the present application can be different from the "hypotonic pressure-induced vesicles" known in the art (e.g., Cohen S, Ushiro H, Stoscheck C, Chinkers M An native 170 000 epidermal growth factor receptor-kinase complex from shed plasma membrane vesicles. J Biol Chem 257: 1523-1531

[0350] Unless otherwise expressly stated, migrators herein can include naturally occurring migrators and artificially induced engineered migrators.

[0351] In some embodiments, an isolated or purified migrator can be free of detectable contaminant components, or the level or amount of contaminant components is equal to or less than an acceptable level or amount.

[0352] In some embodiments, engineered migrators can be isolated and / or purified by centrifugation. For example, engineered migrators can be isolated and / or purified by centrifugation at 300- 17000 x g. In some embodiments, engineered migrators can be isolated and / or purified by filtration.

[0353] The present applicant has discovered that hypotonic treatment of cells induces rapid formation of a large number of micron-sized vesicles on the cell's contractile filaments, which are artificially induced engineered migrans. Accordingly, in some embodiments of the present engineered migran preparation method, inducing cells to produce engineered migrans can comprise hypotonic treatment of the cells. In some embodiments, the hypotonic treatment can be performed by placing the cells in a hypotonic buffer solution having an osmotic pressure of 10-90%, e.g., 15-85%, 20-80%, 25-75%, 30-70%, 35-65%, 40-60%, 50-55%, or any value or sub-range therebetween, e.g., 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, of the osmotic pressure of an isotonic buffer solution. In other embodiments, the hypotonic treatment can be performed by placing the cells in a buffer solution and reducing the osmotic pressure of the buffer solution. In some embodiments, reducing the osmotic pressure of the buffer solution comprises linearly reducing or stepwise reducing. For example, in some embodiments, the salt concentration of the buffer solution can be stepwise reduced at predetermined time intervals. For example, the salt concentration of the buffer solution can be stepwise reduced at least 3 times (e.g., 3-5 times), each time by 1 / 6-1 / 2 of the salt concentration.

[0354] The present applicant has also discovered that the formation of engineered migratory units is significantly enhanced by disrupting the cytoskeleton of the cells, causing the cells to contract, which causes the cell edges to retract towards the center, while the cells adhere at various points to the bottom of the culture plate via focal adhesions, anchoring the plasma membrane in place as the anchor points for the formation of membrane tethers. The contraction of the cells causes the formation of a large number of membrane tethers on the area previously occupied by the cells prior to contraction, which results in a significant enhancement of the formation of engineered migratory units. Accordingly, in some embodiments, inducing cells to produce engineered migratory units can comprise disrupting the cytoskeleton of the cells. In some embodiments, the cytoskeleton of the cells can be disrupted by contacting the cells with an agent that disrupts the cytoskeleton. In some embodiments, the agent that disrupts the cytoskeleton comprises, for example, a microfilament and / or microtubule depolymerizing agent, such as selected from the group consisting of Latrunculin A, Latrunculin B, Cytochalasin A, Cytochalasin B, Cytochalasin C, Cytochalasin D, and Cytochalasin E. In some embodiments, the agent that disrupts the cytoskeleton is Latrunculin A. In some embodiments, the concentration of the agent that disrupts the cytoskeleton can be any suitable concentration. For example, the concentration of the microfilament and / or microtubule depolymerizing agent can be at least 0.01 mM, at least 0.1 mM, at least 0.2 mM, at least 0.5 mM, at least 1 mM, at least 2 mM, at least 3 mM, at least 4 mM, at least 5 mM, at least 6 mM, at least 7 mM, at least 8 mM, at least 9 mM, at least 10 mM.

[0355] Cells can counteract changes in osmotic pressure through regulated changes in cell volume. Accordingly, inhibiting the cell volume regulation function of a cell can facilitate the formation of engineered migratory units. In some embodiments, inducing cells to produce engineered migratory units can comprise inhibiting the cell volume regulation function of the cells. In some embodiments, the cell volume regulation function of the cells can be inhibited by inhibiting the expression or activity of a protein that regulates cell volume of the cells. For example, the protein that regulates cell volume can be selected from the group consisting of ion channels and transporters. In some embodiments, the protein that regulates cell volume can be selected from the group consisting of volume-regulated anion channels (VRAC), such as SWELL 1; volume-regulated cation channels (VRCC), such as TRPV4 and TRPM3; and cotransporters, such as KCC1, KCC3, and KCC4. The expression or activity of the protein can be inhibited by any method or agent known in the art, such as, for example, disrupting the genetic sequence encoding the protein, RNAi, inhibitors of protein activity, and the like.

[0356] It is known that cations and anions can regulate cell volume, but different ions have different abilities to regulate cell volume changes. Thus, ions with weaker abilities to regulate cell volume changes also have weaker abilities to counteract osmotic pressure changes, and thus can promote the formation of migrasomes. Thus, in some embodiments, the cell volume regulation function of a cell can be inhibited by placing the cell in a buffer solution comprising a cation or anion with a weakened ability to regulate cell volume changes. The term "weakened ability to regulate cell volume changes" as used herein is relative to a cation (e.g., sodium ion) or anion that enables a cell to have normal cell volume regulation ability.

[0357] In some embodiments, inducing a cell to produce an engineered migrasome can comprise causing the cell to overexpress one or more proteins selected from the group consisting of Tetraspanin family members or key enzymes and structural proteins of the migrasome production pathway or stimulation with a migrasome production agonist. For example, Tetraspanin family members can include Tspanl, Tspan2, Tspan3, Tspan4, Tspan5, Tspan6, Tspan7, Tspan8, Tspan9, TspanlO, Tspanl l, Tspanl2, Tspanl3, Tspanl4, Tspanl5, Tspanl6, Tspanl7, Tspanl8, Tspanl9, Tspan20 (UPK1B), Tspan21 (UPK1A), Tspan22 (PRPH2), Tspan23 (ROM1), Tspan24 (CD151), Tspan25 (CD53), Tspan26 (CD37), Tspan27 (CD82), Tspan28 (CD81), Tspan29 (CD9), Tspan30 (CD63), Tspan31, Tspan32, and Tspan33. In some embodiments, a cell can be induced to produce an engineered migrasome by causing the cell to overexpress one or more Tspan proteins.

[0358] In some embodiments, cells can be induced to produce migrasomes by regulating cell attachment properties to cause rapid detachment of cells, to reduce the contact area of cells with the culture surface, or to cause relative displacement of the cell membrane from the surface. The cells can be subjected to hypotonic treatment at 4, 8, 16, 24, 37 degrees Celsius, or not subjected to hypotonic treatment, and the cell detachment process can be performed at 4, 8, 16, 24 degrees Celsius. And the cell package and migrasome can be separated.

[0359] In some embodiments, the method of producing the engineered migratory bodies of the present application further comprises reducing the size of the engineered migratory bodies. The size of the engineered migratory bodies can be reduced by any means known in the art, for example, by using a filter of a specific pore size or by extruding the engineered migratory bodies to reduce the size of the engineered migratory bodies. In some embodiments, the size of the engineered migratory bodies can be reduced by processing the engineered migratory bodies using an extruder containing a filter membrane of a specific pore size. In some embodiments, the pore size of the filter membrane or extruder can be 30 nm - 10,000 nm, for example, 50 nm - 8,000 nm, 50 nm - 1,000 nm, 50 nm - 10,000 nm, 100 nm - 1,000 nm, 100 nm - 10,000 nm, 1,000 nm - 10,000 nm, 10 - 400 nm, 20 - 300 nm, 30 - 200 nm, 40 - 100 nm, 50 - 80 nm, or any value or sub-range therebetween. In some embodiments, the size of the reduced engineered migratory bodies can be nanoscale, for example, 1 - 1,000 nm, 10 - 900 nm, 20 - 300 nm, 30 - 200 nm, 40 - 100 nm, 50 - 80 nm, or any value or sub-range therebetween.

[0360] In this context, the cells used to produce the engineered migratory bodies can be any cells, for example, cells cultured in vitro or cells in vivo; suspension or adherent cultured cell lines / strains, normal cells, primary cells, or disease-derived cells including cancer cells, in suspension or adherent state, with or without modification. In addition, the cells used to produce the engineered migratory bodies can be derived from any cell line suitable for in vitro proliferation, modification, and expression of exogenous molecules and production of engineered migratory bodies. In some embodiments, the cells can be animal cells, particularly mammalian cells, including murine and human cells. Examples of suitable cells include, but are not limited to, normal rat kidney (NRK) cells, mouse fibroblast cells such as NIH 3T3 cells, mouse breast cancer 4T1 cells, mouse colon cancer MC38 cells, commonly used human embryonic kidney (HEK) cell lines / strains such as HEK293 or HEK293FT cells, human gastric cancer MGC-803 cells, human T lymphocyte Jurkat cells, human skin fibroblast BJ cells, Chinese hamster ovary (CHO) cells, mesenchymal stem cells (MSCs), or any other suitable cells.

[0361] The term "payload" as used herein can refer to any substance that can be loaded onto a migratory body to be effectively delivered. For example, the payload can be delivered to a target cell by the interaction of surface molecules of the migratory body with the target cell. One or more of the payloads of the present application can be loaded onto the membrane of the engineered migratory body during or after formation of the engineered migratory body, or directly onto the membrane of a naturally occurring migratory body. Examples of payloads include, but are not limited to, therapeutic agents, such as synthetic biologically active compounds, natural biologically active compounds, anti-bacterial compounds, anti-viral compounds, proteins or peptides (e.g., enzymes or antibodies), nucleotides (e.g., nucleotides comprising a detectable moiety or a toxin or nucleotides that disrupt transcription), nucleic acids (e.g., DNA or mRNA molecules encoding polypeptides such as enzymes, immunogenic proteins, cytokines, tumor suppressor genes, antibodies, etc., or RNA molecules with regulatory functions such as microRNAs, dsDNA, antisense oligonucleotides (ASOs), IncRNAs, and siRNAs), genome editing systems, lipids, carbohydrates, small molecules (e.g., small molecule drugs and toxins), targeting molecules, polysaccharides, complexes, organelles, nano- and microparticles, or any combination thereof.

[0362] In some embodiments, the payload can be a microRNA or siRNA, such as a microRNA or siRNA that specifically binds to a transcript encoding a mutant or non-mutant oncogene. Binding of the microRNA or siRNA can block mRNA translation and protein synthesis. Such genes include, but are not limited to, ABL1, BLC1, BCL6, CBFA1, CBL, CSFIR, ERBA, ERBB, EBRB2, ETS1, ETS1, ETV6, FGR, FOX, FYN, HCR, HRAS, JUN, KRAS, LCK, LYN, MDM2, MLL, MYB, MYC, MYCL1, MYCN, NRAS, PIM1, PML, RET, SRC, TAL1, TCL3, YES, VEGF, FGF, G-CSF, CXCR4, etc.

[0363] In some embodiments, the payload can be a genome editing system. Genome editing systems include, but are not limited to, meganuclease systems, zinc finger nuclease (ZFN) systems, transcription activator-like effector nuclease (TALEN) systems, and clustered regularly interspaced short palindromic repeat (CRISPR) systems. In some embodiments, the payload can be a CRISPR system. In some embodiments, the CRISPR system can be a CRISPR-Cas9 system. The CRISPR-Cas9 system comprises a nucleotide sequence encoding a Cas9 protein, a nucleotide sequence encoding a CRISPR RNA that hybridizes to a target sequence (crRNA), and a nucleotide sequence encoding a trans-activating CRISPR RNA (tracrRNA). The crRNA and the tracrRNA can be fused into a guide RNA. The CRISPR-Cas9 system can further comprise a nuclear localization signal (NLS). The migrators loaded with the CRISPR-Cas9 system can be used to alter gene expression and function for disease treatment, regenerative medicine, and tissue engineering.

[0364] In some embodiments, the payload can be a therapeutic protein or fragment thereof, such as an antibody or fragment thereof. In some embodiments, the payload can be a protein (including an antibody or antibody fragment, an immunogenic protein, a cytokine, an enzyme, a tumor suppressor product, etc.) or fragment thereof, such as chicken ovalbumin (OVA), a spike protein of SARS-CoV-2, or an S1 fragment thereof.

[0365] In some embodiments, the cargo can be a targeting molecule. The term "targeting molecule" as used herein refers to a molecule that is capable of specifically binding to another molecule (a target molecule). For example, a targeting molecule can be used to specifically localize a migratory body presenting the targeting molecule on the surface to a certain entity, such as a tissue or cell expressing the target molecule, thereby improving the targeting specificity of the delivery system. In some embodiments, the delivery system of the present application can comprise at least two different targeting molecules, thereby further improving the targeting specificity or otherwise improving the targeting to a target cell or a target tissue. For example, the targeting molecule can specifically bind to a surface protein overexpressed on a cancer cell, such as an antibody or an antigen-binding fragment thereof. In some embodiments, the targeting molecule can be selected from the group consisting of an antibody or an antigen-binding fragment thereof; an integrin; a cytokine, a chemokine, and / or a cytokine, chemokine receptor; a polysaccharide; a find-me / eat me signal, such as a PAMP and a DAMP; and a don't-eat-me signal, such as CD47 and CD24. In some embodiments, an antibody or an antigen-binding fragment thereof can deliver a migratory body loaded with the antibody or the antigen-binding fragment thereof to a target cell expressing the corresponding antigen by binding to its corresponding antigen. In some embodiments, an integrin or other targeting molecule can achieve the targeting of a migratory body loaded with the integrin or other targeting molecule to a specific organ by pairing with the extracellular matrix specific to the tissue.

[0366] The term "find-me / eat me signal" as used herein refers to a signal exposed or released by an apoptotic cell to initiate phagocytic uptake, which in turn activates a tolerogenic pathway to prevent an immune response against self antigens. In this context, the find-me / eat me signal is loaded on the migratory body, which find-me / eat me signal is recognized by macrophages and the like, such that the migratory body is phagocytosed by specific cells. Exemplary find-me / eat me signals include, for example, pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs). A "pathogen-associated molecular pattern (PAMP)" refers to molecular structures that are present on the surface of pathogenic microorganisms and are not found in the human host, but are shared by many related microorganisms, are structurally conserved and evolutionarily conserved. PAMPs recognized by innate immunity are often major parts on which pathogenic microorganisms depend for survival, and thus change less, such as double-stranded RNA of viruses and lipopolysaccharide of bacteria, for which pathogenic microorganisms are difficult to mutate to escape the action of innate immunity. PAMPs can be expressed on the surface of pathogenic microorganisms or free outside immune cells, or can be present in the cytosol of immune cells, and in various intracellular compartments carrying pathogenic microorganisms, such as endosomes and phagolysosomes. PAMPs mainly include two types. The first type is mainly composed of bacterial cell wall components such as lipopolysaccharide, peptidoglycan, lipoteichoic acid, mannose, lipids, lipoarabinomannan, lipoprotein, and flagellin. Among them, the most common and representative include lipopolysaccharide (LPS) produced by gram-negative bacteria; proteoglycan produced by gram-positive bacteria; glycolipid produced by mycobacteria; and mannose produced by yeast. The second type is viral products and bacterial nuclear components, such as non-methylated oligonucleotide CpG DNA, single-stranded RNA, and double-stranded RNA. A "damage-associated molecular pattern (DAMP)" is a type of substance released into the intercellular space or blood circulation after tissues or cells are stimulated by factors such as damage, hypoxia, stress, and the like, which can induce autoimmunity or immune tolerance through pattern recognition receptors such as Toll-like receptors, RIG-1-like receptors, or NOD-like receptors, and plays an important role in the occurrence and development of diseases such as arthritis, atherosclerosis, tumors, systemic lupus erythematosus, and the like. DAMPs exist in the nucleus, cytoplasm (for example, high mobility group protein box (HMGB) 1, S100 protein), extracellular matrix (for example, hyaluronic acid), and plasma (for example, complement C3a, C4a, C5a), or as exosomes (for example, heat shock proteins). Non-protein forms of DAMPs include adenosine triphosphate, uric acid, heparan sulfate, RNA, and DNA. These proteins and non-proteins are limited within cells under healthy conditions, and are released outside the cells when the cells are damaged.

[0367] The term "don't-eat-me signal" as used herein refers to a signal expressed on the surface of a tumor cell for binding to a ligand on the surface of an immune cell, thereby inhibiting killing of the tumor cell by the immune cell. Exemplary don't-eat-me signals include, for example, CD47 and CD24. In this context, the don't-eat-me signal is loaded on the migrator by gene editing, membrane fusion, etc., so that the migrator evades clearance by the immune system, prolonging the circulation time in the blood, achieving better tissue infiltration.

[0368] For example, the migrator can have a monolayer membrane structure, the membrane of which is derived from cell membrane and intracellular vesicles of cells. Compared with cell membrane, the membrane of the migrator is specifically enriched with some proteins (such as Tetraspanin) and lipids (such as cholesterol, sphingomyelin).

[0369] The load of the present application can be directly or indirectly connected or embedded to the membrane of the migrator and / or inside the migrator by any means known in the art.

[0370] In some embodiments, the load can be connected or embedded to the membrane of the migrator and / or inside the migrator by liquid-solid conversion, membrane fusion, charge adsorption, physical adsorption or chemical connection.

[0371] In other embodiments, the load can be connected or embedded to the membrane of the migrator and / or inside the migrator by means selected from the group consisting of: (1) connection to components of the membrane of the migrator and / or inside the migrator, such as membrane proteins, cholesterol, phospholipids, sugar chains on glycoproteins or polysaccharides; (2) connection to proteins or peptides, sugar chains on glycoproteins or polysaccharides, phospholipids, cholesterol, etc. connected or embedded to the membrane of the migrator and / or inside the migrator, preferably the proteins or peptides, sugar chains on glycoproteins or polysaccharides, phospholipids, cholesterol, etc. are connected or embedded to the membrane of the migrator and / or inside the migrator by click chemistry; and (3) connection with the first member of a binding pair, wherein the membrane of the migrator and / or inside the migrator comprises the second member of the binding pair, and the load is connected or embedded to the membrane of the migrator and / or inside the migrator by the binding of the first member and the second member.

[0372] In some embodiments, the cargo is linked or embedded to the membrane of the migrator and / or inside the migrator by linking to a membrane and / or internal components of the migrator, such as a membrane protein or a lipid. In some embodiments, the cargo is linked or embedded to the membrane of the migrator and / or inside the migrator by linking to a membrane protein of the migrator, such as a Tetraspanin. In some embodiments, the cargo is linked or embedded to the membrane of the migrator and / or inside the migrator by linking to a Tetraspanin protein. In some embodiments, the cargo is linked or embedded to the membrane of the migrator and / or inside the migrator by linking to a membrane lipid (e.g., cholesterol and sphingomyelin), a protein or peptide, a sugar chain on a glycoprotein, or a polysaccharide of the migrator.

[0373] In some embodiments, the binding pair comprises an antigen-antibody, a receptor-ligand, biotin-avidin, a HaloTag and its ligand, etc. For example, the binding pair is a HaloTag and its ligand, or CP05 and CD63.

[0374] In some embodiments, the cargo is an mRNA, and the membrane of the migrator and / or inside the migrator comprises an mRNA binding protein, and the mRNA is linked or embedded to the membrane of the migrator and / or inside the migrator by its protein binding site to the mRNA binding protein. The mRNA binding protein and its protein binding site can be those known in the art. For example, in some embodiments, the mRNA binding protein is L7Ae, and the protein binding site is a C / D Box. In other embodiments, the mRNA binding protein is MS2BP, and the protein binding site is a MS2 stem loop (MS2SL).

[0375] In some embodiments, the cargo can be expressed on the inner or outer surface of the migrator membrane as a membrane protein. In other embodiments, the cargo can be expressed on the inner or outer surface of the migrator membrane as a fusion protein fused with a membrane protein or a portion thereof. Any membrane protein known in the art can be used as a membrane anchoring protein fused with a soluble protein. Examples of membrane proteins fused with a soluble protein include, but are not limited to, cell receptors, ion channels, transport proteins, etc., such as Tspan-4, CD81, CD9, CD63, PDGFR, Lamp2b, Synaptosomal-Associated Protein 2 (STX2), etc. In some embodiments, the membrane protein fused with a soluble protein can be STX2. In some embodiments, the membrane protein fused with a soluble protein can be a truncated STX2 (t-STX2). For example, the N-terminus of STX2 can be engineered to remove its intracellular end function to obtain t-STX2. A soluble protein can then be linked to the C-terminus of t-STX2 that is extracellular to form a soluble protein-t-STX2 fusion protein, so that the soluble protein is expressed on the migrator membrane as a man-made plasma membrane localization fusion protein.

[0376] In another aspect, the present application relates to a method of producing a delivery system comprising an isolated or purified migratory body and a payload selected from the group consisting of a protein, a peptide, a nucleic acid (e.g., DNA and RNA), a lipid, a small molecule compound, a polysaccharide, a complex, a nano / microparticle, an organelle, or a simultaneous loading of more than one of the above payloads, and the like, wherein the method comprises: isolating or purifying a migratory body from a cell, the migratory body being a naturally produced migratory body or an artificially induced engineered migratory body; and directly or indirectly linking or embedding the payload to the membrane of the migratory body and / or inside the migratory body, thereby producing the delivery system. In some embodiments of the method of producing a delivery system of the present application, the payload is linked or embedded to the membrane of the migratory body and / or inside the migratory body by liquid-to-solid transition, membrane fusion, charge adsorption, physical adsorption, or chemical linkage.

[0377] In other embodiments of the method of producing a delivery system of the present application, the payload is linked to or embedded into the membrane of the migratory body and / or inside the migratory body by a means selected from the group consisting of: (1) linking the payload to a membrane and / or inside the migratory body component of the migratory body, such as a membrane protein, cholesterol, a phospholipid, a sugar chain on a glycoprotein, or a polysaccharide; and (2) linking the payload to a protein or peptide linked to the membrane of the migratory body and / or inside the migratory body, a sugar chain on a glycoprotein or a polysaccharide, a phospholipid, cholesterol, and the like, preferably the protein or peptide, a sugar chain on a glycoprotein or a polysaccharide, a phospholipid, cholesterol, and the like is linked to the membrane of the migratory body and / or inside the migratory body by click chemistry.

[0378] In some embodiments, a naturally produced migratory body can be isolated or purified from a cell by methods known in the art. In some embodiments, an artificially induced engineered migratory body is produced by the methods of producing an engineered migratory body disclosed herein. In some embodiments, a payload can be one or more of the payloads described elsewhere herein.

[0379] In another aspect, the present application relates to a method of producing a delivery system comprising an engineered migratory body and a payload selected from the group consisting of a protein, a peptide, a nucleic acid (e.g., DNA and RNA), a lipid, a small molecule compound, a polysaccharide, a complex, a nano / microparticle, an organelle, or a simultaneous loading of more than one of the above payloads, and the like, wherein the payload is linked to a first member of a binding pair and the membrane of the migratory body and / or inside the migratory body comprises a second member of the binding pair, wherein the method comprises: expressing the second member of the binding pair on the cell membrane of the cell; producing an engineered migratory body from the cell comprising the second member of the binding pair on the membrane; and contacting the complex of the payload and the first member of the binding pair with the engineered migratory body, thereby producing the delivery system by the binding of the first member to the second member.

[0380] In some embodiments of the method of producing a delivery system disclosed herein, the method comprises introducing into a cell a nucleotide sequence comprising a coding sequence for a second member of a binding pair, such that the cell expresses the second member of the binding pair on the cell membrane. In some embodiments, the method comprises introducing into a cell a nucleotide sequence comprising a coding sequence for a second member of a binding pair, and culturing the cell under conditions that allow the cell to express the second member of the binding pair, such that the cell expresses the second member of the binding pair on the cell membrane.

[0381] In some embodiments, the method comprises: a) introducing into a cell a nucleotide sequence comprising a coding sequence for a second member of a binding pair; b) culturing the cell under conditions that allow the cell to express the second member of the binding pair; c) producing from the cell an engineered migratory unit comprising the second member of the binding pair on a membrane; d) linking the payload to a first member of the binding pair to form a complex; and e) contacting the engineered migratory unit with the complex to thereby produce the delivery system. In some embodiments, step d) above is performed before, after, or simultaneously with step a).

[0382] In some embodiments, the second member of the binding pair is a membrane protein or a soluble protein. In some embodiments, the second member of the binding pair is a membrane protein and is expressed on the inner surface or the outer surface of the engineered migratory unit membrane. In some embodiments, the second member of the binding pair is a soluble protein and is expressed on the inner surface or the outer surface of the engineered migratory unit membrane as a fusion protein fused to a membrane protein or a portion thereof.

[0383] In some embodiments, the binding pair comprises an antigen-antibody, a receptor-ligand, a biotin-avidin, a HaloTag and its ligand, etc. In some embodiments, the binding pair is a HaloTag and its ligand, or CP05 and CD63.

[0384] In some embodiments, the engineered migratory unit is produced by a method of producing an engineered migratory unit disclosed herein. In some embodiments, the payload can be one or more of the payloads described elsewhere herein.

[0385] In yet another aspect, the present application relates to a method of producing a delivery system comprising an engineered migratory body and a cargo, the cargo being an mRNA, and the membrane of the migratory body and / or the interior of the migratory body comprising an mRNA-binding protein, the mRNA being bound to the mRNA-binding protein via its protein-binding site, wherein the method comprises: causing the cell to express the mRNA; prior to, after or simultaneously with the above step, causing the cell to express the mRNA-binding protein on the cell membrane; and producing, from the cell, a delivery system comprising an engineered migratory body and an mRNA, wherein the mRNA is linked to the membrane of the engineered migratory body and / or the interior of the migratory body via binding to the mRNA-binding protein.

[0386] In some embodiments of the method of producing a delivery system of the present application, the method comprises introducing into the cell a nucleotide sequence comprising a coding sequence of an mRNA comprising a protein-binding site, to cause the cell to express the mRNA.

[0387] In some embodiments, the method comprises introducing into the cell a nucleotide sequence comprising a coding sequence of an mRNA-binding protein, to cause the cell to express the mRNA-binding protein on the cell membrane.

[0388] In some embodiments, the method comprises introducing into the cell a nucleotide sequence comprising a coding sequence of an mRNA-binding protein, to cause the cell to express the mRNA-binding protein on the cell membrane.

[0389] In some embodiments, the mRNA-binding protein is a membrane protein or a soluble protein. In some embodiments, the mRNA-binding protein is a membrane protein and is expressed on the inner surface or the outer surface of the engineered migratory body membrane. In some embodiments, the mRNA-binding protein is a soluble protein and is expressed on the inner surface or the outer surface of the engineered migratory body membrane as a fusion protein fused to a membrane protein or a portion thereof.

[0390] The mRNA-binding protein and its protein-binding site can be those known in the art. For example, in some embodiments, the mRNA-binding protein is L7Ae and the protein-binding site is C / D Box. In other embodiments, the mRNA-binding protein is MS2BP and the protein-binding site is MS2 stem loop (MS2 SL).

[0391] In some embodiments, the engineered migratory body is produced by the method of producing an engineered migratory body disclosed herein.

[0392] In another aspect, the present application relates to a method of producing a delivery system comprising an engineered migratory body and a cargo, the cargo being a protein and expressed on an inner surface or an outer surface of a membrane of the engineered migratory body, wherein the method comprises: causing the cell to express the protein on a cell membrane; and producing, from the cell, a delivery system comprising an engineered migratory body and a protein expressed on an inner surface or an outer surface of a membrane of the engineered migratory body.

[0393] In some embodiments, the method comprises introducing into a cell a nucleotide sequence comprising a protein coding sequence to cause the cell to express the protein on a cell membrane. In some embodiments, the method comprises introducing into a cell a nucleotide sequence comprising a protein coding sequence and culturing the cell under conditions that allow the cell to express the protein to cause the cell to express the protein on a cell membrane.

[0394] In some embodiments, the method comprises: introducing into a cell a nucleotide sequence comprising a protein coding sequence; culturing the cell under conditions that allow the cell to express the protein; and producing, from the cell, a delivery system comprising an engineered migratory body and a protein expressed on an inner surface or an outer surface of a membrane of the engineered migratory body.

[0395] In some embodiments, the protein is a membrane protein. In other embodiments, the protein is a soluble protein and is expressed on the surface of the migratory body membrane as a fusion protein fused to a membrane protein or a portion thereof.

[0396] Examples of membrane proteins fused as soluble proteins include, but are not limited to, cell receptors, ion channels, transport proteins, etc., such as Tspan-4, CD81, CD9, CD63, PDGFR, Lamp2b, Synaptophysin 2 (STX2), etc., as described above.

[0397] In some embodiments, the engineered migratory body is produced by the engineered migratory body production method disclosed herein.

[0398] The present application also provides a pharmaceutical or diagnostic composition comprising the engineered migratory body of the present application, in combination with one or more pharmaceutically acceptable excipients, diluents or carriers. Accordingly, the present application also provides the use of the engineered migratory body of the present application in the manufacture of a pharmaceutical composition.

[0399] The application also provides a method for preparing a pharmaceutical or diagnostic composition comprising adding and mixing the engineered migratory bodies of the application together with one or more pharmaceutically acceptable excipients, diluents or carriers. The engineered migratory bodies can be the sole active ingredient in the pharmaceutical or diagnostic composition, or can be accompanied by other active ingredients such as steroids or other pharmaceutical molecules. The composition can be administered to a patient individually or can be administered in combination (e.g., simultaneously, consecutively, or separately) with other agents, drugs, or hormones.

[0400] A pharmaceutical composition can comprise a therapeutically effective amount of the engineered migratory bodies of the application. In the present application, the term "therapeutically effective amount" refers to the amount of a therapeutic agent required to treat, ameliorate, or prevent a targeted disease or condition or to exhibit a detectable therapeutic or preventative effect. For any engineered migratory body disclosed, a therapeutically effective amount can be estimated initially in cell culture assays or in animal models, usually in rodents, rabbits, dogs, pigs, or primates. The animal model can also be used to determine the appropriate concentration range and route of administration. Such information can then be used to determine effective doses and routes of administration in humans. The precise therapeutically effective amount will depend upon the severity of the disease condition, the overall health status of the subject, the age, weight and gender of the subject, diet, time and frequency of administration, drug combination(s), reaction sensitivities and tolerance / response to therapy.

[0401] A pharmaceutical composition of the application can be administered by a variety of routes, including, but not limited to, oral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, intraventricular, transdermal, transcutaneous, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, intravaginal or rectal routes. The pharmaceutical compositions of the application can also be administered using a needle-free injector. Typically, the therapeutic compositions can be prepared as injectables, either as liquid solutions or suspensions. Solid forms suitable for dissolving or suspending in liquid vehicles prior to injection can also be prepared. The pharmaceutical compositions comprising a therapeutic dose of the engineered migratory bodies of the application do not exhibit significant toxicological effects in vivo.

[0402] Without wishing to be bound by any theory, the examples below are merely intended to illustrate the engineered migratory bodies, methods of preparation and uses of the application, and are not intended to limit the scope of the application.

[0403] Examples

[0404] The present application is further illustrated by the following specific examples. It should be understood that these examples are intended to illustrate the present application and are not intended to limit the scope of the present application. The experimental methods in the following examples, for which no specific conditions are noted, are carried out according to the conditions described in the art, for example, Sambrook and Rusself et al., Molecular Cloning: A Laboratory Manual (3rd edition) (2001), CSHL Press, or according to the conditions suggested by the manufacturer. Unless otherwise specified, the experimental materials and reagents used in the following examples are commercially available.

[0405] Reagents

[0406] ddH2O (solarbio), KCl (sigma), KH2PO4 (sangon), Na2HPO4-7H2O (sangon), BSA (VWR), Latrunculin A (cayman), human fibronectin (invitrogen or sigma), PBS (Gibco), RPMI1640 (Gibco), FBS (BI), WGA-AF488 / AF594 / AF647 (invitrogen), BCA kit (invitrogen).

[0407] Preparation of 10x KDPBS (500 ml): weigh 50 g KCl, 2.5 g KH2PO4, 0.5 g Na2HPO4-7H2O, add ddH2O to 500 ml, filter with 0.45 um filter membrane.

[0408]

[0409]

[0410] Preparation of 100x BSA (50 ml): weigh 5 g BSA, add ddH2O to 50 ml, filter with 0.45 um filter membrane.

[0411] Example 1

[0412] Inducing cells to produce engineered migrans

[0413] Example 1: engineered migrans generated by hypotonic treatment

[0414] The inventors found that hypotonic treatment of cells leads to cell body swelling, basal shrinkage and generation of a large number of filamentous structures (shrinkage filaments) on which migran-like structures grow. In order to visually observe the engineered migrans, a NRK cell line stably expressing Tspan4-GFP was established, and the formation of engineered migrans was observed by the signal of Tspan4, one of the markers of migrans. NRK cells overexpressing Tspan4-GFP were treated with 25% DPBS (corresponding to osmotic pressure 76.3 mOsmol / L), and the results are shown in Figure 1Results show that 30 seconds after the low-osmotic treatment, Tspan4-GFP signals began to enrich on the contractile filaments, and then form micron-sized vesicles; after reaching their peak intensity, Tspan4-GFP signals began to diffuse from the vesicles, accompanied by the shrinking of the vesicles; 460 seconds after the low-osmotic treatment, most of the vesicle structures induced by low-osmotic pressure disappeared. The process of the formation of these micron-sized vesicles and their attachment on the contractile filaments are similar to those of natural migrasomes.

[0415] To further confirm whether the vesicle structures induced by low-osmotic pressure are engineered migrasomes, the cells were stained with fluorescently labeled wheat germ agglutinin (WGA). WGA is a lectin that specifically binds to sialic acid and N-acetyl-D-glucosamine. Fluorescently labeled WGA can be used to label migrasomes in cells and is a specific probe for detecting migrasomes (Chen et al., WGA is a probe for migrosomes, Cell Discovery (2019) 5: 13).

[0416] NRK cells overexpressing Tspan4-GFP were subjected to isotonic treatment (100% DPBS, 305 mOsmol / L) and low-osmotic treatment with 25% DPBS (76.3 mOsmol / L), respectively, and then stained with tetramethylrhodamine-labeled WGA and observed under a laser confocal microscope. The results are shown in Figure 26c Results show that the vesicle structures induced by overexpression of Tspan4 in NRK cells are specifically stained by WGA, indicating that the induced vesicle structures are engineered migrasomes.

[0417] To determine the effect of the size of osmotic pressure on the formation of engineered migrasomes, NRK cells expressing Tspan4-GFP were subjected to isotonic treatment (100% DPBS, 305 mOsmol / L) or low-osmotic treatment with different strengths (50%, 25%, and 17% DPBS, corresponding to osmotic pressures of 152.5, 76.3, and 50.8 mOsmol / L, respectively). The results are shown in Figure 1 b, and the statistical results of the diameters of the engineered migrasomes are shown in Figure 1 c. It can be found that the size of the osmotic pressure is negatively correlated with the size of the engineered migrasomes, and the lower the osmotic pressure, the larger the size of the induced engineered migrasomes.

[0418] Based on the above results, the inventors established a step-by-step low-osmotic pressure reduction program and found that step-by-step application of low-osmotic pressure significantly increased the duration of engineered migrasomes, and in cells subjected to 5-step low-osmotic treatment, the induced engineered migrasomes still existed 20 minutes after low-osmotic treatment.

[0419] Example 2 Engineered migrators generated by latrunculin A treatment

[0420] Since the number of engineered migrators generated by hypo-osmotic induction depends on the number of contractile filaments, and the majority of the plasma membrane that serves as the source of the engineered migrator membrane is on the cell body, it was hypothesized that if the cell was contracted by disrupting the cytoskeleton, the contraction would cause the cell edge to retract towards the center, while the cell adhered at various points to the culture plate bottom through focal adhesions, thereby allowing the plasma membrane to adhere in situ as the anchor points for the membrane tether generation. If this was indeed the case, the contraction of the cell would generate a large number of membrane tubes in a manner similar to the formation of contractile filaments during migration.

[0421] To test this hypothesis, NRK cells expressing Tspan4-GFP were incubated with isotonic buffer solution DPBS containing different concentrations of the microfilament depolymerizing agent latrunculin A (0, 0.25, 0.5, and 1 mM) for 10 min, and then subjected to three consecutive hypo-osmotic stress by stepwise addition of water to reduce the salt concentration by 1 / 6 every 2 min. The results after stepwise hypo-osmotic treatment are shown in Figure 2 a, and the statistical results of the number of engineered migrators generated by each cell are shown in Figure 2 b.

[0422] As expected, it was found that treating the cells with latrunculin A caused the cells to contract, and it was also observed that a large number of membrane tethers were formed on the area previously occupied by the cells before contraction, and these newly formed membrane tethers led to a significant enhancement of engineered migrator formation. Therefore, Latrunculin A was able to significantly promote the increase in the number of engineered migrators, and this promotion was dose-dependent. This suggests that disrupting the cytoskeleton can promote the formation of engineered migrators.

[0423] Example 3 Engineered migrators generated by inhibiting the cell volume regulation function

[0424] Cells can counteract changes in osmotic pressure through regulated volume changes. To test whether regulated volume changes would affect engineered migrator formation, the key component of the volume-regulated anion channel SWELL 1, which maintains constant cell volume by responding to changes in osmotic pressure, was knocked down or knocked out.

[0425] The gene Lrrc8a encoding SWELL 1 was knocked down in NRK cells expressing Tspan4-GFP, and the efficiency of Lrrc8a knockdown in the cells was verified by qPCR, which showed that the gene Lrrc8a encoding SWELL 1 was successfully knocked down to about 15% of the wild-type cells Figure 3a). Lrrc8a-knockdown cells (Lrrc8a-KD) and non-knockdown control cells (NC) were placed in DPBS, and subjected to three consecutive hypotonic shocks by stepwise addition of water to reduce the salt concentration by 1 / 3 every 2 min, and then observed by laser confocal microscopy. The results are shown in Figure 3 b. The statistical results of the engineered migratory body size are shown in Figure 3 c.

[0426] The gene Lrrc8a encoding SWELL 1 was knocked out in NRK cells expressing Tspan4-GFP, and the knock-out of Lrrc8a in the cells was verified by Western blotting. No expression of SWELL 1 was observed in the knock-out cells (Lrrc8a-KO), indicating that the gene Lrrc8a encoding SWELL 1 was successfully knocked out Figure 4 a). Lrrc8a-KO cells (KO14# cell line and KO18# cell line) and non-knockout control cells (WT) were placed in DPBS, and subjected to five consecutive hypotonic shocks by stepwise addition of water to reduce the salt concentration by 1 / 6 every 1 min, and then observed by laser confocal microscopy. The results are shown in Figure 4 b.

[0427] The results show that the engineered migratory body size produced by Lrrc8a-knockdown cells is significantly larger than that of non-knockdown cells Figure 3 b and 3c) after hypotonic treatment; the engineered migratory body size produced by Lrrc8a-knockout cells is also significantly larger than that of non-knockout cells Figure 4 b). This indicates that knocking down or knocking out SWELL 1 significantly enhances the formation of engineered migratory bodies, suggesting that the formation of engineered migratory bodies can be enhanced by reducing the ability of cells to adjust their volume during osmotic pressure changes.

[0428] It is known that cations can regulate cell volume. If different cations have different abilities to regulate cell volume during osmotic pressure changes, the regulated cell volume changes can be weakened by cation replacement, thereby promoting the formation of engineered migratory bodies. Next, the effects of different cations on the formation of engineered migratory bodies were tested. Sodium chloride in DPBS was replaced with equimolar concentrations of potassium chloride, cesium chloride or choline chloride to prepare isotonic buffers containing different cations, and the cells were incubated in these isotonic buffers. The cells were subjected to five consecutive hypotonic shocks by stepwise addition of water to reduce the salt concentration by 1 / 6 every 2 min in the corresponding buffer. The results after stepwise hypotonic treatment are shown in Figure 5 a, and the statistical results of the engineered migratory body size are shown in Figure 5 b.

[0429] The results show that different cations have different abilities to promote the formation of engineered migratory bodies, among the tested cations, the ability of sodium ion to promote the formation of engineered migratory bodies is relatively weak, while the ability of potassium ion, cesium ion and choline ion to promote the formation of migratory body-like structures is significantly stronger.

[0430] Example 4 Producing engineered migratory bodies by overexpressing Tspan4 in cells

[0431] Tspan4 is a key protein for promoting the formation of migratory bodies, in order to test whether Tspan4 can promote the formation of engineered migratory bodies, NRK cells overexpressing only mCherry-Kras or NRK cells overexpressing both Tspan4-GFP and mCherry-Kras were subjected to stepwise hypotonic stimulation. After incubating NRK cells in KDPBS containing 2 mM latrunculin A for 10 min, three consecutive hypotonic stimulations were performed by reducing the salt concentration by 1 / 6 every 2 min interval, and then the formation of engineered migratory bodies was observed by differential interference contrast microscopy (DIC). Figure 6 a, and the statistical results of the number of engineered migratory bodies produced by each cell are shown in Figure 6 b.

[0432] The results show that the overexpression of Tspan4 has a significant promoting effect on the number of engineered migratory bodies.

[0433] Example 5 Producing migratory bodies by rapid cell detachment from a petri dish temperature-sensitive coating

[0434] MGC803-T4-GFP cells were cultured on a temperature-sensitive petri dish, after cytoskeleton disruption treatment, the petri dish was left still at room temperature or 37 degrees for 45 minutes after hypotonic treatment or without hypotonic treatment. More than 80% of the cells detached from the bottom of the dish and formed migratory bodies, and part of the migratory bodies detached from the dish with the cells (see Figure 7 ). After collecting the supernatant, the residual cells and migratory bodies were blown and collected.

[0435] Example 6 Producing engineered migratory bodies in various cell lines

[0436] To test whether engineered migrans can be induced in different species and genetic backgrounds of cell lines, hypotonic induction was performed on three different rodent cell lines overexpressing Tspan4 (normal rat kidney cells (NRK), mouse breast cancer cell line (4T1), and mouse colon cancer cell line (MC38)), two commonly used human embryonic kidney cell lines / strains (HEK-293T and HEK-293FT), three different human cell lines (human gastric cancer MGC-803 cells, human T lymphocyte Jurkat cells, human skin fibroblast BJ cells). NRK cells were incubated in K-DPBS containing 2 mM latrunculin A for 10 min, and then subjected to three consecutive hypotonic shocks with 1 / 6 reduction of salt concentration every 2 min; 4T1 cells were incubated in K-DPBS containing 2 mM latrunculin A for 20 min, and then subjected to three consecutive hypotonic shocks with 1 / 4 reduction of salt concentration every 2 min; MC38 cells were incubated in K-DPBS containing 2 mM latrunculin A for 45 min, and then subjected to three consecutive hypotonic shocks with 1 / 4 reduction of salt concentration every 2 min. The results are shown in FIGS. 1A-1C. Figure 8 As can be seen, vesicle structures were produced in different cell lines.

[0437] Further, the above-mentioned hypotonic-treated cell lines were stained with tetramethyl rhodamine-labeled WGA, a specific probe for migrans, and then observed under a laser confocal microscope. The results are shown in FIGS. 2A-2D. Figure 8 b. It was also tested that human cell lines (human embryonic kidney cells (HEK293T, HEK293FT; Figure 8 c), human gastric cancer cells (MGC803), human skin fibroblasts (BJ), and human peripheral blood leukemia T cells (Jurkat); Figure 8 d), and it was found that engineered migrans can be produced by the method of embodiments 1-5. The results show that the vesicle structures produced by the eight cell lines after induction were stained with WGA, a specific probe for migrans, indicating the production of engineered migrans.

[0438] The above results show that engineered migrans can be produced by the method of embodiments 1-5 in different cell lines.

[0439] Example 7 Separation, purification and characterization of engineered migrans Figure 9 a-b)

[0440] a. Production of engineered migrans by stimulating adherent cells

[0441] I. Coating culture flasks, plating cells

[0442] Coat the bottom of the culture flask with 2 μg / ml human fibronectin in PBS for more than one hour at 37°C, then seed cells at 1 x 10 7

[0443] II. Inducing production of engineered migrans

[0444] 1. After 14-16 hours of cell culture, discard the medium and wash the cells once with PBS;

[0445] 2. Add K-DPBS containing 2 μM latrunculin A and incubate at 37°C (the incubation time should be adjusted according to the sensitivity of the cell line to latrunculin A, for example, 10 minutes for NRK cells and 45-60 minutes for MC38 cells; the cells should show a wrinkled appearance and a large number of reticular structures around the cell body, similar to contractile filaments);

[0446] 3. Place the culture flask on a horizontal shaker in the cell culture incubator at 40 rpm, and add water every three minutes for a total of three times (the volume of water added at each step is determined by the hypotonic induction gradient and the initial volume of liquid in the culture flask; the hypotonic induction gradient varies depending on the cell line, for example, for MC38 cells, the salt concentration is reduced by 1 / 4 at each step, the initial volume is 15 ml, and the volumes of water added at each step are 5 ml, 6.5 ml, and 8.5 ml; for NRK cells, the initial volume is 15 ml, and the volumes of water added at each step are 3 ml, 3.6 ml, and 4.4 ml);

[0447] 4. Adjust the speed to 60 rpm and shake for 5 minutes.

[0448] b. Generating engineered migratory bodies by stimulating suspended cells

[0449] Take the cells (the number varies depending on the cell line), wash them once with PBS, and discard the supernatant

[0450] Resuspend the cells in the culture flask with K-DPBS containing 2 μM latrunculin A and incubate at 40 rpm on a horizontal shaker (the incubation time varies depending on the cell line)

[0451] Add sterile water to the culture flask to reduce the osmotic pressure (the reduction ratio varies depending on the cell line)

[0452] Adjust the speed to 60 rpm and shake for 5 minutes.

[0453] ​To test whether engineered migrans could be induced in cell lines in suspension, hypotonic induction was performed on two different rodent cell lines that overexpress Tspan4 (normal rat kidney cells (NRK), and mouse colon carcinoma cells (MC38)). The results are shown in Figures 9a and 9b. Vesicle structures were produced in both cell lines in suspension, and immunoblotting showed enrichment of membrane proteins characteristic of engineered migrans and lack of intracellular material in the vesicles purified from both cells. Figure 9 a and 9b. Vesicle structures were produced in both cell lines in suspension, and immunoblotting showed enrichment of membrane proteins characteristic of engineered migrans and lack of intracellular material in the vesicles purified from both cells.

[0454] III. Isolation and purification of engineered migrators Figure 10 )

[0455] a. Purification of engineered migrans from adherent cells

[0456] 1. Discard supernatant, wash cells twice with hypotonic KDPBS (h-KDPBS; the salt concentration is similar to that of the solution system at the end of the hypotonic induction; for MC38 cells, h-KDPBS is 40% KDPBS; for NRK cells, h-KDPBS is 60% KDPBS), and discard supernatant;

[0457] 2. Add h-KDPBS containing 1 mg / ml BSA (h-KDPBS-BSA), shake at 130 rpm for 3 min;

[0458] 3. Collect supernatant in a 50 ml centrifuge tube;

[0459] 4. Add h-KDPBS-BSA, gently pipette the bottom of the culture flask, and combine the collected liquid with the supernatant from step 3;

[0460] 5. Centrifuge at 300 x g for 10 min at 4°C, and retain the supernatant;

[0461] 6. Centrifuge at 500 x g for 10 min at 4°C, and retain the supernatant;

[0462] 7. Filter the supernatant through a parylene filter with a pore size of 8 um into a 50 ml low-adsorption tube;

[0463] 8. Centrifuge at 17,000 x g for 45-60 min at 4°C, and discard the supernatant;

[0464] 9. Resuspend the pellet with h-KDPBS-BSA, transfer to an EP tube (centrifuge tube 1), and add an equal volume of PBS-BSA; at this point, take a small amount of liquid (about 1 / 50 of the total volume) in another centrifuge tube (centrifuge tube 2) for measuring the protein concentration;

[0465] 10. 4°C, 17000x g centrifugation for 15-20 min, discard the supernatant, add PBS, resuspend the precipitate before injection, to obtain the engineered transmigrators resuspended in PBS.

[0466] b. Purification method of engineered transmigrators produced by suspended cells

[0467] 1. Collect the supernatant into a 15 ml centrifuge tube, gently blow with a pipette;

[0468] 5. 4°C, 300x g centrifugation for 10 min, retain the supernatant;

[0469] 6. 4°C, 500x g centrifugation for 10 min, retain the supernatant;

[0470] 7. Filter the supernatant into a low adsorption tube with a parylene filter membrane with a pore size of 8 μm;

[0471] 8. 4°C, 17000x g centrifugation for 45-60 min, discard the supernatant;

[0472] 9. Resuspend the precipitate with PBS, transfer to an EP tube, to obtain the engineered transmigrators resuspended in PBS.

[0473] IV. Characterization of engineered migrans

[0474] 1. Determination of total protein

[0475] Centrifuge the liquid in the centrifuge tube 2 at 4°C, 17000x g for 5 min, wash the precipitate once with PBS after discarding the supernatant, lyse the precipitate with 30 μl 2% SDS, and boil the sample in a 95°C metal bath. Determine the protein concentration by the BCA method, and calculate the total protein amount in the centrifuge tube 1, and the corresponding resuspension volume according to the injection dose.

[0476] 2. Morphological observation

[0477] Take 1 μl of the engineered transmigrators sample obtained in step III, dilute to 10 μl (to observe the morphology of the vesicles and the phase separation on the membrane surface, WGA dye can be added in the diluent according to a ratio of 1:500-1:1000), drop onto a confocal dish precoated with 10 μg / ml fibronectin, stand at room temperature for more than 1 h, and observe under a laser confocal microscope. The results are shown in Figure 11 a.

[0478] In addition, the engineered transmigrators obtained in step III are observed by negative staining transmission electron microscopy and cryo-electron microscopy. The results are shown in Figure 11 b and 11c, respectively.

[0479] 3. Flow cytometry

[0480] Take 1 μl of the liquid at step III-9 or 10, dilute to 100 μl. If the vesicles show double-color fluorescence, dilute directly with PBS; if the vesicles only show single-color fluorescence, stain with 1:500 WGA dye to improve the effect of grouping.

[0481] 4. Western blot

[0482] Take a small amount of liquid in the supernatant of step III-1, centrifuge at 100x g, and then lyse the precipitate with 2% SDS as the cell body sample; the sample obtained in step IV-1 is the engineered migratory body sample. Load the cell sample and the engineered migratory body sample with equal amounts of protein for western-blot, and detect the classic markers of various organelles: nucleus (histone H3), mitochondria (Tim23), endoplasmic reticulum (calnexin), lysosome (Lamp2), cytoplasm (GAPDH), cell membrane (Na+-K+-ATPase), cell membrane focal adhesion (integrin a5), Tspan4-GFP (GFP), and the results are shown in Figure 12

[0483] The results show that the engineered migratory bodies obtained by separation and purification are highly enriched in cell membrane proteins such as Tspan4, integrin a5, and Na+-K+-ATPase, and almost no or little contamination from intracellular organelles and soluble proteins.

[0484] 5. Permeability evaluation

[0485] Drop the engineered migratory bodies obtained by separation and purification into a confocal dish, and add Cy5 (a fluorescent dye that cannot pass through the intact membrane) and dextran-TMR (Dex-TMR) to the buffer to indicate the permeability of the vesicles. Use a laser confocal microscope to take long-term photos of the droplets, and the results are shown in Figure 13 a. It can be observed that the engineered migratory bodies are almost completely permeable to Cy5 (MW < 1 kDa) at the beginning at room temperature, and show slow permeability to dextran-TMR (MW = 40 kDa) with a larger molecular weight. The statistical results of the permeability of dextran-TMR after the engineered migratory bodies are placed at room temperature for 1.5 h, 6 h, 12 h, 24 h, and 48 h are shown in Figure 13 b.

[0486] 6. Stability evaluation

[0487] To explore the stability of the engineered migratory bodies at room temperature, drop the engineered migratory bodies obtained by separation and purification into a confocal dish, and observe the morphology on days 0, 1, 2, 3, 5, and 7, respectively, and the results are shown in Figure 14 ​Six samples were kept in parallel and used for Western blotting to detect the loaded OVA and mCherry (14b) at day 0, 3, 7, 14, and for immunization of mice to detect the OVA-specific antibody concentration in serum (14c). Figure 14 c).

[0488] The results showed that the engineered migrasomes were stable in vesicle morphology, protein loading, and immunogenicity as a vaccine during the 7-14 days of room temperature storage, indicating that the engineered migrasomes were very stable.

[0489] 7. Effect of cholesterol on the stability of engineered migrasomes

[0490] It has been previously reported that cholesterol is essential for the formation of migrasomes (Huang Y, Zucker B, Zhang S, Elias S, Zhu Y, Chen H, Ding T, Li Y, Sun Y, Lou J, Kozlov MM, Yu L. Migrasome formation is mediated by assembly of micron-scale tetraspanin macrodomains. Nat Cell Biol. 2019 Aug;21(8):991-1002). To investigate the effect of cholesterol on the formation of engineered migrasomes, the isolated and purified engineered migrasomes were treated with methyl-β-cyclodextrin (MβCD), which can selectively extract cholesterol from the plasma membrane. Cholesterol extraction reagent MβCD (10 mM) was added to the droplets, and then laser confocal microscopy was performed, and the results are shown in Figure 15 It was found that most of the engineered migrasomes were severely deformed and damaged within 10 min, indicating that cholesterol is essential for the stability of engineered migrasomes.

[0491] Drug loading of engineered migrans

[0492] Example 8 Loading of membrane proteins on engineered migrasomes

[0493] During the preparation of engineered migrasomes, part of the cell membrane can be converted into the migrasome membrane, therefore, the delivery of membrane proteins can be achieved by directly transferring the plasmid encoding the target gene and Tspan4 into the production cells Figure 16a) The overexpression of Tspan4, together with other steps for the preparation of engineered migratory bodies, can greatly increase the production efficiency of engineered migratory bodies, and the overexpressed membrane proteins will be enriched on the produced engineered migratory bodies, thereby achieving the loading of membrane proteins on the engineered migratory bodies. The loadable membrane proteins include various cell receptors (such as various GPCRs, PD-1, VEGFR, etc.), extracellular enzymes (such as CD36, CD73), ion channels, transport proteins, and various antigens (such as S protein), etc.

[0494] As examples, the loading of three typical plasma membrane proteins DAG1, Tgfbr1 and PDCD1, one membrane-bound protein Kras( Figure 17 ), and the spike (S) protein of SARS-CoV-2( Figure 18 ) on engineered migratory bodies was verified.

[0495] For the membrane proteins DAG1, Tgfbr1, PDCD1 and Kras, plasmids containing Tspan4-GFP and the target gene sequence were transfected into the production cells NRK cells. To facilitate the observation of the subcellular localization and expression amount of the target protein, the target gene fragment was fused to the mCherry tag through a linker. The amino acid sequences and vector information of each fusion protein are shown below.

[0496] Tspan4-Linker-GFP (vector: pB-Hygro-GFP (vector map as shown in Figure 19 ), insertion site: BsrGI+BamHI): (SEQ ID NO: 1)

[0497]

[0498]

[0499] The amino acid sequence of Tspan4 is underlined; the amino acid sequence of GFP is in bold italic; and the amino acid sequence between Tspan4 and GFP is the linker sequence (PG).

[0500] DAG1-Linker-mCherry (vector: pmCherry-N1 (vector map as shown in Figure 20 ), insertion site: EcoRI+KpnI): (SEQ ID NO: 2)

[0501]

[0502] The amino acid sequence of DAG1 is underlined; the amino acid sequence of mCherry is in bold italic; and the amino acid sequence between DAG1 and mCherry is the linker sequence (GDPPVAT).

[0503] PDCD1-linker-mCherry (vector: pB-Hygro-mCherry (vector map as shown in Figure 21 , insertion site: BsrGI + MluI): (SEQ ID NO: 3)

[0504]

[0505]

[0506] The amino acid sequence of PDCD1 is underlined; the amino acid sequence of mCherry is in bold italic; the amino acid sequence between PDCD1 and mCherry is the linker sequence (TVPRARDPPVAT).

[0507] Tgfbr1-linker-mCherry (vector: pmCherry-N1 (vector map as shown in Figure 20 , insertion site: EcoRI + KpnI): (SEQ ID NO: 4)

[0508]

[0509] The amino acid sequence of Tgfbr1 is underlined; the amino acid sequence of mCherry is in bold italic; the amino acid sequence between Tgfbr1 and mCherry is the linker sequence (TVPRARDPPVAT).

[0510] mCherry-linker-Kras (vector: pB-Hygro-mCherry (vector map as shown in Figure 21 , insertion site: BsrGI + MluI): (SEQ ID NO: 5)

[0511]

[0512] The amino acid sequence of mCherry is underlined; the amino acid sequence of Kras is in bold italic; the amino acid sequence between mCherry and Kras is the linker sequence (SGLRSRG).

[0513] After transfection, the cells were subjected to drug killing screening against the resistance carried on the plasmid, and the cell lines stably expressing various membrane proteins marked by mCherry were established. Then the cell lines were induced to produce engineered migratory bodies carrying the corresponding membrane proteins by the hypotonic treatment conditions for NRK cells in Example 5, and then laser confocal microscopy observation was performed. The results showed that the four target membrane proteins were correctly positioned on the engineered migratory bodies Figure 17 ), indicating that the membrane proteins can be integrated on the engineered migratory bodies.

[0514] For S protein, the plasmid containing Tspan4-GFP and the sequence of the target gene was transfected into the production cell MC38 cell. To facilitate the observation of the subcellular localization and expression amount of the target protein, the mCherry tag was fused after the target gene fragment. After transfection, the cells were subjected to drug killing screening against the resistance on the plasmid, and the cell line stably expressing the S protein with mCherry marker was established. Then the cell line was induced to produce the engineered migratory body with the transfected S protein under the hypotonic treatment condition for MC38 cells in Example 5, and then laser confocal microscopy observation was performed. The results showed that the S protein was correctly positioned on the engineered migratory body Figure 18 ), indicating that the S protein can be effectively integrated on the engineered migratory body.

[0515] Example 9 Loading of soluble protein on the engineered migratory body

[0516] Since the engineered migratory body is leaky, it can not be used for direct delivery of cytosolic soluble proteins. To achieve the delivery of soluble proteins, the soluble protein can be anchored on the membrane by making the production cell express a fusion protein of the target protein-membrane protein, thereby preventing the leakage of the soluble protein Figure 16 a and Figure 16 b). For example, the N-terminal of the membrane protein syntaxin-2 (STX2) can be engineered to remove its intracellular end function to obtain a truncated STX2 (t-STX2). Then the soluble protein is connected to the C-terminal of t-STX2 outside the cell to form a soluble protein-t-STX2 fusion protein Figure 16 b), so that the soluble protein is expressed as a man-made plasma membrane localization fusion protein. The topology of t-STX2 itself makes the soluble target protein finally hang outside the cell membrane, thereby, for example, more conducive to the recognition of the protein as a complete antigen by the immune system.

[0517] In addition to STX2, a variety of other membrane proteins are often used as membrane anchor proteins, such as Tetraspanin-4, CD81, CD9, CD63, PDGFR, Lamp2b, etc.

[0518] As an example, the loading of soluble protein OVA on the engineered migratory body was verified. The plasmid containing Tspan4-GFP and t-STX2-OVA-mCherry coding sequence was transfected into the production cell MC38 cell. The amino acid sequence and vector information of the fusion protein of t-STX2 and OVA are as follows.

[0519] t-STX2-Linker-OVA-Linker-mCherry (vector pmCherry-N1 (vector map as Figure 20HindIII+BamHI): (SEQ ID NO: 6)

[0520]

[0521] The amino acid sequence of t-STX2 is shown in bold; the amino acid sequence of OVA is shown underlined; the amino acid sequence of mCherry is shown in bold italic.

[0522] After transfection, the cells were subjected to drug selection against the resistance carried on the plasmid to establish a cell line stably expressing the t-STX2-OVA fusion protein marked with mCherry. Then the cell line was induced to produce the engineered migratory bodies carrying the t-STX2-OVA fusion protein using the hypotonic treatment conditions for MC38 cells in Example 5, and then subjected to laser confocal microscope observation, with the results shown in Figure 22

[0523] The results show that the t-STX2-OVA fusion protein is correctly positioned on the engineered migratory bodies, indicating that the soluble protein OVA is successfully loaded on the engineered migratory bodies.

[0524] Example 10 Loading of other molecules of interest on engineered migratory bodies

[0525] The loading of small molecule drugs, small nucleic acid drugs, peptide segments, etc. on engineered migratory bodies can be achieved by modification of the antigen-antibody, receptor-ligand, biotin-avidin binding system for the molecules of interest and the producer cells. Figure 16 ​a) For example, the receptor-ligand interaction of HaloTag with its synthetic ligand can be utilized to achieve the loading of a molecule of interest onto the engineered migratory bodies (Los, G. V., Encell, L. P., McDougall, M. G., Hartzell, D. D., Karassina, N., Zimprich, C, Wood, M. G., Learish, R., Ohana, R. F., Urh, M., Simpson, D., Mendez, J., Zimmerman, K., Otto, P., Vidugiris, G., Zhu, J., Darzins, A., Klaubert, D. H., Bulleit, R. F., & Wood, K. V. (2008). HaloTag: a novel protein labeling technology for cell imaging and protein analysis. ACS chemical biology, 3(6), 373-382.). First, a plasmid encoding a fusion protein of the membrane protein Tspan4 and the receptor protein HaloTag, Tspan4-HaloTag, is constructed. Then, the plasmid is transfected into the engineered migratory body production cells to achieve the modification of the production cells. The modified production cells are induced to produce engineered migratory bodies, and the purified engineered migratory bodies contain the receptor protein HaloTag on the membrane. By coupling the molecule of interest with the ligand of HaloTag, a molecule of interest-ligand conjugate is formed, thereby achieving the modification of the molecule of interest. Then, the molecule of interest-ligand conjugate is co-incubated with the engineered migratory bodies containing HaloTag on the membrane in vitro, and the molecule of interest is fixed to the membrane of the engineered migratory bodies and / or inside the migratory bodies through the interaction of HaloTag with its ligand. The covalent binding of HaloTag with its ligand is specific, efficient and irreversible.

[0526] In addition to Tspan4, other membrane proteins, such as those described elsewhere herein, can also be used as membrane anchor proteins for loading of molecules of interest on engineered migrators. In addition to HaloTag and its ligand, other antigen-antibody, receptor-ligand, biotin-avidin, etc. binding systems can also be used for loading of molecules of interest on engineered migrators, such as the CP05 and CD63 binding system (X. Gao, N. Ran, X. Dong, B. Zuo, R. Yang, Q. Zhou, H. M. Moulton, Y. Seow, H. Yin, Anchor peptide captures, targets, and loads exosomes of diverse origins for diagnostics and therapy, Sci. Transl. Med. 10 (2018)), in which the receptor CD63 is first loaded onto the membrane of the engineered migrator, and then the molecule of interest-CP05 conjugate is loaded onto the engineered migrator membrane containing CD63 by taking advantage of the binding between CD63 and its ligand CP05. When selecting antigen-antibody, receptor-ligand, biotin-avidin binding systems, one can try to avoid using receptors that have a large number of natural ligands in the body (such as PD-1).

[0527] To conceptually validate this loading method, plasmids containing Tspan4-HaloTag-GFP coding sequences were transfected into producer cells NRK cells, and plasmids containing Tspan4-GFP coding sequences were transfected as controls. The amino acid sequence of the fusion protein of Tspan4 and HaloTag and the vector information are shown below.

[0528] Tspan4-Linker-GFP-Linker-Halo (vector: pB-Hygro-GFP (vector map as shown in Figure 19 , insertion site: BsrGI + MluI): (SEQ ID NO: 7)

[0529]

[0530]

[0531] The amino acid sequence of Tspan4 is shown in bold; the amino acid sequence of GFP is shown underlined; the amino acid sequence of Halo is shown in bold italic.

[0532] After transfection, the cells were subjected to drug selection against the resistance carried on the plasmid to establish a stable expression cell line. Then the cell line was induced to produce engineered migrators under the hypotonic treatment conditions for NRK cells in Example 5. The engineered migrators were added with fluorescent HaloTag ligand-TMR dye, and after 15 min of co-incubation at room temperature, they were washed twice with dye-free buffer, and then dropped into a confocal dish and left at room temperature for 5 h before laser confocal microscopy observation. The results are shown in Figure 23 .

[0533] The results show that HaloTag ligand-TMR co-localizes with Tspan4-HaloTag-GFP on the engineered migrators, indicating that the target molecules can be loaded onto the engineered migrators through the interaction of HaloTag and its ligand, and this loading is efficient, specific and irreversible.

[0534] In addition to the above antigen-antibody, receptor-ligand, biotin-avidin coupling methods, small nucleic acid drugs can also be loaded onto the membrane of engineered migrators through various other modification methods. For example, chemically modified siRNA or antisense oligonucleotide (ASO) can be coupled with cholesterol, and small nucleic acid drugs can be loaded onto the membrane of engineered migrators through the compatibility of cholesterol with cell membranes (S.S. Yerneni, S. Lathwal, P. Shrestha, H. Shirwan, K. Matyjaszewski, L. Weiss, E.S. Yolcu, P.G. Campbell, S.R. Das, Rapid on-demand extracellular vesicle augmentation with versatile oligonucleotide tethers, ACS Nano 13 (2019) 10555-10565). This method does not require additional modification of the migrators and is a fast and economical loading method. siRNA conjugates can also be fixed to the membrane through click chemistry (T. Tian, H.X. Zhang, C.P. He, S. Fan, Y.L. Zhu, C. Qi, N.P. Huang, Z.D. Xiao, Z.H. Lu, B.A. Tannous, J. Gao, Surface functionalized exosomes as targeted drug delivery vehicles for cerebral ischemia therapy, Biomaterials 150 (2018) 137-149). This method requires coupling siRNA with a peptide segment first, and then coupling the peptide segment to the membrane through click chemistry.

[0535] Loading of mRNA drugs into engineered transporters can be achieved through mRNA-binding proteins. Commonly used mRNA-binding proteins include L7Ae (Kojima, R., Bojar, D., Rizzi, G., Hamri, GC, El-Baba, MD, Saxena, P., S., Tan, K. R., & Fussenegger, M. (2018). Designer exosomes produced by implanted cells intracerebrally deliver therapeutic cargo for Parkinson's disease treatment. Nature communications, 9(1), 1305; Zhitnyuk, Y., Gee, P., Lung, M., Sasakawa, N., Xu, H., Saito, H., & Hotta, A. (2018). Efficient mRNA delivery system utilizing chimeric VSVG-L7Ae virus-like particles. Biochemical and biophysical research communications, 505(4), 1097-1102) and MS2BP (Prel, A., Caval, V., Gayon, R., Ravassard, P., Duthoit, C., Payen, E., Maouche-Chretien, L., Creneguy, A., Nguyen, T. H., Martin, N., Piver, E., Sevrain, R., Lamouroux, L., Leboulch, P., Deschaseaux, F., Bouille, P., Sensébe, L., & Pagès, J. C. Highly efficient in vitro and in vivo delivery of functional RNAs using new versatile MS2-chimeric retrovirus-like particles, Mol. Ther. - Meth. Clin. Dev. 2 (2015), 15039). First, a plasmid encoding a membrane protein-mRNA binding protein fusion protein is constructed, such as Tspan4-L7Ae or Tspan4-MS2. Next, an mRNA expression plasmid is constructed, containing a protein binding site, the protein binding site for L7Ae is C / D Box, and the protein binding site for MS2BP is MS2 Stem Loop (MS2SL). Then, the fusion protein plasmid and the mRNA plasmid are co-transfected into production cells, and the production cells are induced to produce engineered migrators, and the purified migrators contain the target mRNA molecules on their membranes.

[0536] Example 11 Engineered migrators loaded with SARS-CoV-2 spike protein induce immune responses in mice in vivo

[0537] The experimental procedure is shown in Figure 24 a. The experimental animals were 8-week-old female mice of C57BL / 6 strain, 5 in each group. The specific grouping was as follows:

[0538] I. 50 pg S1 recombinant protein (Sino Biological) + aluminum adjuvant (thermo scientific), intraperitoneal injection (i.p.);

[0539] II. 10 pg S1 recombinant protein, intravenous injection (i.v.);

[0540] III. Engineered migrators expressing only Tspan4-GFP (NC-eMig) with a total protein of 20 pg, tail vein injection, as a negative control;

[0541] IV. Engineered migrators co-expressing Tspan4-GFP and S protein-mCherry (S-eMig) with a total protein of 20 pg, tail vein injection.

[0542] The S1 protein content in the S-eMig group was semi-quantitatively analyzed by Western blotting Figure 24 b). The first 6 lanes from left to right are 1, 2, 5, 10, 20 and 50 ng of S1 recombinant protein, and the 7th-10th lanes are 1 pg of total protein of NC-eMig or S-eMig (eMig-1 and eMig-2 represent samples from two independent experiments). By comparing the brightness of the bands in each lane, it can be calculated that the S1 protein in one dose (20 pg) of S-eMig is about 100 ng, which is much less than the amount of S1 recombinant protein used in the aluminum adjuvant group (50 pg).

[0543] The mice received 1 immunization on day 0 and were sacrificed on day 14, and the peripheral blood was taken to detect the concentration of S protein-specific IgG antibodies in the serum by ELISA, and the results are shown in Figure 24 c. It was found that both the S1 protein injection group alone (group II) and the NC-eMig negative control group (group III) could not promote antibody production; the S1 protein + aluminum adjuvant group (group I) and the S-eMig group (group IV) could effectively promote antibody production, and the immune effects of the two were roughly similar. This shows that compared with the traditional adjuvant plus immunogenic protein immunization method, the engineered migrators can cause similar antibody responses without adjuvant and with a small amount of protein, and are more efficient delivery vectors that can effectively promote immune responses.

[0544] Comparison of engineered migrans to known extracellular vesicles

[0545] Example 12 Comparison of engineered and native migrans

[0546] Control migrans (production and purification process see Ma et al, Cell Res. 2015) and engineered migrans (production and purification process see Figure 9 ) from the same cell source were produced and purified, and compared in many aspects.

[0547] NRK cells overexpressing Tspan4-GFP were fixed with 2.5% glutaraldehyde after hypotonic treatment and observed by scanning electron microscope Figure 25 a). 4D imaging of the process of engineered migrans production by NRK cells overexpressing Tspan4-GFP using spinning-disk confocal microscope showed that during the process of hypotonic treatment, the cell body swelled, the basal surface contracted and a large number of filamentous structures were produced; engineered migrans grew on the filamentous structures Figure 25 b).

[0548] Native migrans were produced due to the displacement of the cell center point, the contractile filaments attached to the migrans were only located at the tail end of cell migration, and the number of migrans was small (Fig. 26a); engineered migrans were induced by different ways to induce the relative displacement of the cell membrane, and the contractile filaments attached to the migrans were distributed in all directions of the cell, and the number of migrans on each contractile filament was significantly higher than that of control migrans (Fig. 26b). After staining with tetramethyl rhodamine-labeled WGA, NRK cells overexpressing Tspan4-GFP could clearly see the difference between control migrans Figure 26c (top) and engineered migrans Figure 26c (bottom). Under electron microscope, native migrans had different numbers of secretory vesicles inside (Fig. 26d; excerpted from Ma et al, Cell Res. 2015), while engineered migrans had limited or lacked contents under electron microscope (Fig. 26e). Mass spectrometry analysis was performed on engineered migrans and control migrans (control migrans can also be referred to as non-hypotonic migrans or native migrans) from the same cell source. Compared with native migrans, nearly 4000 proteins were missing in engineered migrans (Fig. 26f), some of which were significantly enriched in native migrans compared with the cell body (Fig. 26g); in addition, the expression of 1350 proteins missing in engineered migrans was higher in some engineered migrans, including ERM family member proteins such as Ezrin and Fxyd5, Atp1β1 protein Figure 26h(Reference: Ma L, Li Y, Peng J, Wu D, Zhao X, Cui Y, Chen L, Yan X, Du Y, Yu L. Discovery of the migrasome, an organelle mediating release of cytoplasmic contents during cell migration. Cell Res. 2015 Jan;25(l):24-38. doi: 10.1038 / cr.2014.135.)

[0549] Example 13 Engineered migrasomes are distinguished from other hypotonic vesicles

[0550] Engineered migrasomes produced by NRK cells under scanning electron microscope Figure 25 a) Distinct from reported hypotonic vesicles Figure 25 c) The main differences in process and properties are as follows:

[0551] 1) Engineered migrasomes are produced on filaments around the cell, rather than on the cell surface as suggested by Cohen et al.

[0552] 2) Engineered migrasomes are micrometer-sized, with few larger than 5 pm in diameter. The vesicles produced by Cohen et al. using hypotonicity can be as large as 20 pm.

[0553] 3) The hypotonic induction process for engineered migrasomes is a gentle stepwise reduction (in several cell lines tested, the final osmolarity is not less than 40%), while Cohen et al. used a very drastic 5% PBS treatment, and a high-osmolarity buffer (118%) for vesiculation. (Reference: Cohen S, Ushiro H, Stoscheck C, Chinkers M A native 170 000 epidermal growth factor receptor-kinase complex from shed plasma membrane vesicles. J Biol Chem 257: 1523-1531.)

[0554] Example 14 Engineered migrasomes are distinguished from extracellular small vesicles / exosomes

[0555] Migrasomes and exosomes are both extracellular vesicles, but the mechanisms of production and the sizes of the vesicles (exosomes are generally 50-150 nm) are different. The extracellular small vesicles (sEVs) / exosomes (production and purification process shown in Figure 27a) and engineered migrasomes (production and purification process shown in Figure 27b) from the same cell source were compared.Figure 9 ) for production, purification and comparison of different aspects. NTA detection (Fig. 27b) and transmission electron microscopy (TEM) observation (Fig. 27c) of the purified exosomes confirmed the size and morphology of the exosomes consistent with the literature description. In addition, Western blotting experiments confirmed that the purified exosomes were enriched in recognized extracellular vesicle / exosome markers such as tsg101, Alix, CD63, CD81, while the engineered migrasomes were not enriched in these extracellular vesicle / exosome markers. At the same time, the engineered migrasomes were more enriched in migrasome markers such as Na / K ATPase, Lamp2, etc. (Fig. 27d), indicating that the effective production purified migrasomes and exosomes of the same cell origin, while the proteomic composition and markers of the two were significantly different. Figure 27d

[0556] Engineered migrasome production method:

[0557] The MC-38 cells overexpressing Tspan4-GFP were plated in the cell culture dishes treated with fibronectin one day in advance;

[0558] On the experimental day, the supernatant was discarded, and the cells were washed with 40% KDPBS; 1 mg / ml BSA was added to h-KDPBS (h-KDPBS-BSA), and the cells were shaken at 130 rpm for 3 min; the supernatant was collected; h-KDPBS-BSA was added to blow the bottom of the culture bottle, and the collected liquid was combined with the supernatant of the previous step; the supernatant was centrifuged at 4°C and 300x g for 10 min, and the supernatant was retained; the supernatant was centrifuged at 4°C and 500x g for 10 min, and the supernatant was retained; the supernatant was filtered through a parylene filter membrane with a pore size of 8 um into a 50 ml low adsorption tube; the supernatant was centrifuged at 4°C and 17000x g for 45-60 min, and the supernatant was discarded; the precipitate was resuspended with h-KDPBS-BSA and transferred to an EP tube (centrifuge tube 1), and an equal volume of PBS-BSA was added; at this time, about 1 / 50 of the total volume was taken in another centrifuge tube (centrifuge tube 2) for measuring the protein concentration; the precipitate was resuspended with PBS after centrifugation at 4°C and 17000x g for 15-20 min, and the precipitate was resuspended in PBS to obtain the engineered migrasomes.

[0559] Production and purification of extracellular vesicles:

[0560] ​MC-38 cell line overexpressing mouse Tspan4-GFP protein was seeded at a confluency of ~20% and cultured for 72 hours; the culture medium was collected, and the cell bodies were removed by centrifugation at 4000 x rcf for 10 min, and the supernatant was collected; the supernatant was filtered using a 0.45-micron filter membrane; the filtered supernatant was concentrated using a 70-kD ultrafiltration tube; 1 mM MgCl2 and 20 U / mL of Benzonase were added to the concentrated supernatant, and the mixture was treated at room temperature overnight; the mixture was filtered using a 0.22-um filter membrane; the filtered liquid was centrifuged at 140,000 x g at 4°C for 1 hour using an ultracentrifuge; the supernatant was discarded, and the precipitate was resuspended in PBS; the resuspended precipitate was mixed with 60% iodixanol, and the mixture was added to the bottom of an ultracentrifuge tube; 9 mL of 30% iodixanol, 5 mL of 23% iodixanol, and 6 mL of 18% iodixanol were sequentially added to the ultracentrifuge tube; the mixture was centrifuged at 150,000 x g for 16 hours, and the components between the density gradients (a total of 4 components, F1-F4) were collected by gentle aspiration; the collected exosome components were mixed with 30 mL of PBS, centrifuged at 16,000 x g for 1 hour, and the supernatant was retained; the supernatant was filtered using a 0.2-um filter membrane, and the mixture was centrifuged at 150,000 x g at 4°C for 2 hours, and the pellet was resuspended to 1 x 1010 13 particles / mL for cryopreservation.

[0561] By producing and purifying extracellular small vesicles / exosomes and engineered migrators in the same cell line, the present applicant found that the unit yield of engineered migrators was much higher than that of extracellular small vesicles, and the production speed was greatly increased without the need for ultracentrifugation, ultracentrifugal density gradient centrifugation, and the like. When an equal amount of cells was used for culture, the yield of engineered migrators was about 35 times that of extracellular small vesicles / exosomes (Fig. 27e).

[0562] At the same time, after tail vein injection of C57BL / 6 mice with an equal amount of dye of extracellular small vesicles and engineered migrators for 5 minutes, the accumulation of engineered migrators and extracellular small vesicles in each organ was almost the same. However, with the increase of time, the accumulation of engineered migrators in the liver, spleen, and lung was significantly increased compared with that of extracellular small vesicles at 4 hours and 24 hours after injection, which also indicated that the metabolism and tissue accumulation of the two in the body were different, and the delivery effect of engineered migrators was better under the condition of carrying the same amount of dye (Fig. 27f).

[0563] Mass spectrometric analysis of extracellular small vesicles / exosomes and engineered migrators in the same cell line showed that PCA analysis showed that the three engineered migrator samples were highly similar and obviously different from the three extracellular small vesicle / exosome samples (Fig. 28a), and signal pathway analysis heat map (Fig. 28b) and the top 10 most enriched proteins ( Figure 28c ) showed that there was a significant difference between extracellular small vesicles / exosomes and engineered migrators.

[0564] The foregoing detailed description has been presented for purposes of illustration and description. It is not intended to be exhaustive or to limit the claims to the precise form disclosed. Many modifications and variations are possible in light of the above teaching. It is intended that the scope of claims and the patent be limited not with the details of the description, but rather by the language of the claims and the equivalents thereof.

Claims

1. A method of preparing a migratory body, the method comprising subjecting a cell to a hypotonic treatment, causing the cell to generate a relative displacement and in turn a migratory body derived from the cell.

2. The method of claim 1, further comprising isolating the migratory body generated by the cell.

3. The method of claim 1, wherein the hypotonic treatment comprises placing the cell in a hypotonic buffer solution.

4. The method of claim 1, wherein the hypotonic treatment comprises placing the cell in a buffer solution and reducing the osmolarity of the buffer solution to a hypotonic buffer solution, wherein the osmolarity of the hypotonic buffer solution is lower than the osmolarity of an isotonic solution.

5. The method of claim 4, wherein the reduction is a linear reduction and / or a stepwise reduction.

6. The method of claim 4, wherein the osmolarity of the hypotonic buffer solution is lower than about 305 mOsmol / L.

7. The method of claim 4, wherein the osmolarity of the hypotonic buffer solution is from about 10 mOsmol / L to about 274.5 mOsmol / L.

8. The method of claim 1, further comprising one or more of the following steps: (a) disrupting the cytoskeleton of the cell; (b) inhibiting the cell volume regulation function of the cell; (c) detaching the cell from its adherent surface; (d) increasing the number and / or function of tetraspanin proteins, functional fragments and / or functional variants thereof in the cell; and (e) reducing the size of the migratory body.

9. The method of claim 8, wherein disrupting the cytoskeleton of the cell comprises contacting the cell with a cytoskeleton-disrupting agent.

10. The method of claim 9, wherein the cytoskeleton-disrupting agent is a microfilament and / or microtubule depolymerizing agent.

11. The method of claim 10, wherein the cytoskeleton-disrupting agent is selected from the group consisting of: Latrunculin A, Latrunculin B, Cytochalasin A, Cytochalasin B, Cytochalasin C, Cytochalasin D and / or Cytochalasin E.

12. The method of claim 8, wherein inhibiting the cell volume regulation function of the cell comprises decreasing the number and / or function of volume-regulating proteins in the cell.

13. The method of claim 12, wherein the volume-regulating proteins are volume-regulating ion channels and / or transport proteins.

14. The method of claim 13, wherein the volume-regulating ion channels are volume-regulating anion channels VRAC and / or volume-regulating cation channels VRCC.

15. The method of claim 14, wherein the volume-regulating anion channels VRAC are SWELL1 or functionally active fragments thereof.

16. The method of claim 14, wherein the volume-regulating cation channels VRCC are TRPV4, TRPM3 and / or functionally active fragments thereof.

17. The method of claim 13, wherein the transport proteins comprise symport proteins.

18. The method of claim 17, wherein the co-transporter comprises KCC1, KCC3, and / or KCC4.

19. The method of claim 8, wherein inhibiting the cell volume regulation function of the cell comprises placing the cell in a buffered solution with attenuated volume regulation ability.

20. The method of claim 19, wherein the buffered solution comprises a potassium ion, cesium ion, or choline ion substituted sodium ion buffered solution, wherein the buffered solution comprises a DPBS buffer.

21. The method of claim 8, comprising causing the cell to overexpress the tetraspanin protein, functional fragment, and / or functional variant thereof.

22. The method of claim 21, wherein the tetraspanin protein is selected from the group consisting of: Tspanl, Tspan2, Tspan3, Tspan4, Tspan5, Tspan6, Tspan7, Tspan8, Tspan9, TspanlO, Tspanl l, Tspanl2, Tspanl3, Tspanl4, Tspanl5, Tspanl6, Tspanl7, Tspanl8, Tspanl9, Tspan20 (UPK1B), Tspan21 (UPK1A), Tspan22 (PRPH2), Tspan23 (ROM1), Tspan24 (CD151), Tspan25 (CD53), Tspan26 (CD37), Tspan27 (CD82), Tspan28 (CD81), Tspan29 (CD9), Tspan30 (CD63), Tspan31, Tspan32, and Tspan33.

23. The method of claim 8, wherein the reducing the size of the migratory body comprises extruding the migratory body using a filter or an extruder.

24. The method of claim 23, wherein the filter or extruder has a pore size of about 30 nm to about 10,000 nm.

25. The method of any one of claims 1-24, further comprising characterizing the obtained migratory body as having one or more of the following characteristics: (a) the migratory body has a size of about 50 nm to about 8,000 nm; (b) the migratory body is generated from a contractile filament of the cell; (c) the migratory body has enriched on its membrane sodium / potassium ATPase and / or a functional fragment thereof, integrin and / or a functional fragment thereof, tetraspanin, a protein functional variant thereof and / or a functional fragment thereof, cholesterol, and / or membrane microdomains; (d) the content of the migratory body is at least partially reduced or absent as compared to a native migratory body generated by the corresponding cell.

26. The method of any one of claims 1-24, wherein the migratory body is generated in vitro or ex vivo.

27. The method of claim 25, wherein the at least partially reduced content comprises intraluminal vesicles.

28. The method of any one of claims 1-24, which is an in vitro or ex vivo method.

29. The method of any one of claims 1-24, wherein the cell is a cell cultured in vitro.

30. The method of any one of claims 1-24, wherein the cell is a cell cultured in suspension or adherently.

31. The method of any one of claims 1-24, wherein the cell is a primary cell.

32. The method of claim 31, wherein the primary cell is a tissue cell derived from an organism selected from the group consisting of a human, a monkey, a mouse, a rat, a rabbit, a chicken, and / or an insect.

33. The method of claim 31, wherein the primary cell is selected from the group consisting of a hepatocyte, a spleen cell, a kidney cell, a tissue macrophage, a brain glial cell, an osteoclast, a bone marrow cell, a white blood cell, a fibroblast, and / or an adipocyte.

34. The method of claim 33, wherein the white blood cell is selected from the group consisting of a B cell, a T cell, a NK cell, a dendritic cell, a neutrophil, and / or a macrophage.

35. The method of any one of claims 1-24, wherein the cell is a tumor cell.

36. The method of claim 35, wherein the tumor cell is selected from the group consisting of a tumor cell line, a primary or finite pass tumor cell derived from a patient, a tumor stromal cell, and / or a tumor organoid.

37. The method of any one of claims 1-24, wherein the cell is selected from the group consisting of a CHO cell, a CHO-K1 cell, a HEK293 cell, a HEK293T cell, a HEK293FT cell, a HEK293F cell, a Vero cell, a NRK cell, a L929 cell, a MC38 cell, a 4T1 cell, a DC2.4 cell, a MGC803 cell, a Jurkat cell, a NK-92MI cell, a BJ cell, and / or a HepG2 cell.

38. The method of any one of claims 1-24, wherein the cell is selected from the group consisting of a white blood cell, a stem cell, and / or a fibroblast.

39. The method of claim 38, wherein the stem cell is a mesenchymal stem cell.

40. A migratory body prepared by the method of any one of claims 1-39.

41. The migratory body of claim 40, wherein the migratory body has one or more of the following characteristics: (a) the migratory body has a size of about 50 nm to about 8000 nm; (b) the migratory body is generated from a contractile filament of the cell; (c) the migratory body has enriched on its membrane sodium / potassium ATPase and / or a functional fragment thereof, integrin and / or a functional fragment thereof, tetraspanin, a protein functionally variant thereof and / or a functional fragment thereof, cholesterol, and / or a membrane microdomain; (d) the content of the migratory body is at least partially reduced or absent as compared to a native migratory body generated by the corresponding cell.

42. The migrator of claim 41, wherein the at least partially reduced content comprises intraluminal vesicles.

43. Use of a migrator for delivering an exogenous load, wherein the migrator is the migrator of any one of claims 40-42.

44. A delivery system comprising a migrator and one or more exogenous loads, wherein the migrator is the migrator of any one of claims 40-42.

45. The delivery system of claim 44, wherein the exogenous load is directly or indirectly bound, linked or embedded to the membrane and / or interior of the migrator.

46. The delivery system of claim 44, wherein the migrator is derived from a cell.

47. The delivery system of claim 44, wherein the exogenous load comprises one or more targeting agents and / or therapeutically active agents.

48. The delivery system of any one of claims 44-47, wherein the exogenous load comprises a protein, a lipid, a polynucleotide, a small molecule compound, a complex, a polysaccharide, a polymer, a nanoparticle, a microparticle and / or an organelle.

49. The delivery system of claim 48, wherein the exogenous load comprises a membrane protein, a soluble protein and / or a polypeptide.

50. The delivery system of claim 48, wherein the exogenous load is DNA and / or RNA.

51. The delivery system of claim 48, wherein the exogenous load is selected from an antibody or an antigen-binding antibody fragment thereof, an integrin or a fragment thereof, an immunogenic protein, a cytokine, a chemokine, a receptor protein or a fragment thereof, an enzyme, a tumor suppressor product, an siRNA, a microRNA, an antisense oligonucleotide (ASO), an mRNA, a DNA, a gene editing tool and / or a cytotoxic agent.

52. The delivery system of claim 48, wherein the exogenous load is selected from a PAMP, a DAMP, CD47, CD24, IL-12, IL-15, coagulation factor VII, coagulation factor VIII, coagulation factor IX and / or a functionally active fragment thereof.

53. The delivery system of any one of claims 44-47, wherein the exogenous load is directly or indirectly bound to the migrator by gene editing, exogenous expression, liquid-to-solid transition, membrane fusion, charge adsorption, physical adsorption and / or chemical linkage.

54. The delivery system of any one of claims 44-47, wherein the exogenous load is bound or embedded to the migrator by direct or indirect linkage to a membrane component of the migrator.

55. The delivery system of claim 54, wherein the membrane component of the migrator is selected from a membrane protein, a cholesterol, a phospholipid, a sugar chain on a glycoprotein and / or a polysaccharide.

56. The delivery system of claim 54, wherein the indirect linkage is by a click chemistry reaction.

57. The delivery system of claim 56, wherein the indirect linkage comprises providing the exogenous load linked to a first member of a binding pair and contacting it with the migratory body, which comprises on its membrane a second member of the binding pair, wherein the first member is capable of binding to the second member.

58. The delivery system of claim 57, wherein the first and second members of the binding pair are selected from the group consisting of an antigen and its antibody; a receptor and its ligand; biotin and avidin; a HaloTag and its ligand; and CP05 and CD63.

59. The delivery system of any one of claims 44-47, wherein the exogenous load is expressed as a membrane protein on the inner or outer surface of the membrane of the migratory body.

60. The delivery system of any one of claims 44-47, wherein the exogenous load is expressed as a fusion protein fused to a membrane protein or a portion thereof on the inner or outer surface of the membrane of the migratory body.

61. The delivery system of any one of claims 44-47, wherein the exogenous load is expressed as a fusion protein fused to a membrane protein or a portion thereof on the inner or outer surface of the membrane of the migratory body by gene editing and / or exogenous expression.

62. A method of making a delivery system, the method comprising providing a migratory body by the method of any one of claims 1-39, and carrying the migratory body with an exogenous load.

63. The method of claim 62, wherein the migratory body is an isolated or purified migratory body.

64. The method of claim 62, wherein the carrying the migratory body with an exogenous load comprises linking or embedding the exogenous load directly or indirectly to the membrane of the migratory body and / or the interior of the migratory body.

65. The method of claim 62, further comprising isolating or purifying the migratory body from a cell.

66. The method of any one of claims 62-65, comprising providing a complex of the exogenous load and a first member of a binding pair; causing a cell to produce a migratory body comprising a second member of the binding pair; and contacting the migratory body with the complex to form the delivery system.

67. The method of any one of claims 62-65, wherein the exogenous load is one or more targeting substance and / or therapeutically active substance.

68. The method of any one of claims 62-65, wherein the exogenous load is selected from the group consisting of a protein, a lipid, a polynucleotide, a small molecule compound, a complex, a polysaccharide, a polymer, a nanoparticle, a microparticle, and / or an organelle.

69. The method of claim 68, wherein the exogenous load is selected from the group consisting of a membrane protein, a soluble protein, and / or a polypeptide.

70. The method of claim 68, wherein the exogenous load is DNA and / or RNA.

71. The method of claim 68, wherein the exogenous payload is selected from an antibody or an antigen-binding antibody fragment thereof, an integrin or a fragment thereof, an immunogenic protein, a cytokine, a chemokine, a receptor protein or a fragment thereof, an enzyme, an onco-suppressor product, an siRNA, a microRNA, an antisense oligonucleotide (ASO), an mRNA, a DNA, a gene editing tool, and / or a cytotoxic agent.

72. The method of claim 68, wherein the exogenous payload is selected from a PAMP, a DAMP, CD47, CD24, IL-12, IL-15, coagulation factor VII, coagulation factor VIII, coagulation factor IX, and / or a functionally active fragment thereof.

73. The method of any one of claims 62-65, wherein the exogenous payload is directly or indirectly bound to the migrator by gene editing, exogenous expression, liquid-to-solid transition, membrane fusion, charge adsorption, physical adsorption, and / or chemical linkage.

74. The method of any one of claims 62-65, wherein the exogenous payload is attached or embedded to the migrator by direct or indirect binding to a membrane component of the migrator.

75. The method of claim 74, wherein the membrane component of the migrator is selected from a membrane protein, a cholesterol, a phospholipid, a sugar chain on a glycoprotein, and / or a polysaccharide.

76. The method of claim 74, wherein the indirect binding is by a click chemistry reaction.

77. The method of claim 74, wherein the indirect binding comprises providing the exogenous payload attached to a first member of a binding pair, and contacting it with the migrator, which comprises a second member of the binding pair on its membrane, wherein the first member is capable of binding to the second member.

78. The method of claim 77, wherein the first and second members of the binding pair are selected from an antigen and its antibody; a receptor and its ligand; biotin and avidin; a HaloTag and its ligand; and CP05 and CD63.

79. The method of any one of claims 62-65, wherein the exogenous payload is expressed as a membrane protein on the inner or outer surface of the membrane of the migrator.

80. The method of any one of claims 62-65, wherein the exogenous payload is expressed as a fusion protein fused to a membrane protein or a portion thereof on the inner or outer surface of the membrane of the migrator.

81. A method of making a delivery system, the method comprising: causing a cell to express an mRNA; producing a migrator from the cell by the method of any one of claims 1-39, the migrator comprising an mRNA-binding protein, and the mRNA being attached to the migrator by the mRNA-binding protein.

82. A method of making a delivery system, the method comprising: causing a cell to express an exogenous payload on a cell membrane; and producing a migrator from the cell by the method of any one of claims 1-39, the migrator comprising the exogenous payload.

83. The method of claim 82, wherein the exogenous payload is a protein.

84. The method of claim 83, wherein the protein is a membrane protein.

85. The method of claim 83, wherein the protein is a soluble protein and is fused to a membrane protein or portion thereof to form a fusion protein.

86. A composition comprising the migratory body of any one of claims 40-42 or the delivery system of any one of claims 44-61.

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