Extracellular vesicle, preparation method therefor, and use thereof

Abstract

An extracellular vesicle. The surface of the extracellular vesicle comprises at least part of a viral envelope glycoprotein or a variant thereof; the at least part of the viral envelope glycoprotein or the variant thereof does not bind to a receptor or the binding to a receptor is inhibited; and the extracellular vesicle is difficult to infect a cell by binding to a glycoprotein receptor. When the extracellular vesicle is used for delivering a target protein, the targeting performance of the extracellular vesicle is significantly improved. When an extracellular vesicle membrane expresses a hydrophobic protein, the expression and purification of the hydrophobic protein are facilitated.

Description

Extracellular vesicle and preparation method and application thereof TECHNICAL FIELD

[0001] The present application relates to the field of polypeptide carriers, in particular to an extracellular vesicle and a preparation method and application thereof. BACKGROUND

[0002] Extracellular vesicles (EVs) are a class of naturally occurring nanoscale particles with a double-membrane structure, a diameter of 30-200 nm (exosomes) or <1000 nm (microvesicles) actively secreted by cells. EVs, as an important cell communication carrier, are involved in the processes of immune regulation, inflammation, tumor, metabolic disorders, angiogenesis, and play an important role in the physiological and pathological processes of the body. EVs can avoid being cleared by the immune system due to their excellent natural biocompatibility, and are therefore considered as an ideal protein transport carrier and have attracted much attention.

[0003] However, naturally occurring EVs have limitations such as uneven size and unstable structure. Existing technologies use envelope virus glycoproteins such as VSV-G to transduce into production cells to make the produced EVs more uniform in size and more stable in structure.

[0004] However, wild-type VSV-G has high immunogenicity and can easily trigger an immune response in the subject; and wild-type VSV-G can bind to the endocytosis receptor LDL-R widely expressed on a variety of cells; therefore, EVs containing wild-type VSV-G on the surface can infect a variety of cells by binding to LDL-R when transporting the target protein, and the targeting of the target protein is not good.

[0005] On the other hand, the operation of large-scale production of EVs is complex and costly, and the need for simple and inexpensive large-scale production of EVs has not been met. SUMMARY

[0006] Therefore, in order to solve at least one of the above problems, the first aspect of the present application provides an extracellular vesicle, the surface of the extracellular vesicle comprising at least a portion of an envelope virus glycoprotein or a variant thereof,

[0007] (a) the at least a portion of the envelope virus glycoprotein or the variant thereof comprises a functional domain promoting budding of the envelope virus; and

[0008] (b) the at least a portion of the envelope virus glycoprotein or the variant thereof (i) is inhibited from binding to an envelope virus glycoprotein receptor or (ii) does not bind to an envelope virus glycoprotein receptor.

[0009] In some embodiments of the application, at least a portion of the envelope virus glycoprotein or variant thereof (i) is inhibited from binding to an envelope virus glycoprotein receptor or (ii) does not bind to an envelope virus glycoprotein receptor relative to a wild-type envelope virus glycoprotein or an intact envelope virus glycoprotein.

[0010] In some embodiments of the application, the surface of the extracellular vesicle comprises the envelope virus glycoprotein variant.

[0011] In some embodiments of the application, the envelope virus glycoprotein or variant thereof is selected from the group consisting of a Vesiculovirus strain glycoprotein or variant thereof, a NiV glycoprotein G or variant thereof, a Measles virus glycoprotein H or variant thereof, a Lentivirus glycoprotein or variant thereof, a Rabies virus glycoprotein (RVG) or variant thereof, a gibbon ape leukemia virus glycoprotein (GaLV) or variant thereof, an amphotropic murine leukemia virus glycoprotein (MLV-A) or variant thereof, a feline endogenous virus (RD114) glycoprotein or variant thereof, a fowlpox virus (FPV) glycoprotein or variant thereof, an Ebola virus (EboV) glycoprotein or variant thereof, and a Lymphocytic choriomeningitis virus (LCMV) glycoprotein or variant thereof.

[0012] The Vesiculovirus strain glycoprotein or variant thereof includes a Vesiculovirus Indiana strain glycoprotein or variant thereof, a Vesiculovirus Cocal strain glycoprotein or variant thereof, a Vesiculovirus Maraba strain glycoprotein or variant thereof, a Vesiculovirus Morreton strain glycoprotein or variant thereof, a Vesiculovirus Alagoas strain glycoprotein or variant thereof, a Vesiculovirus New Jersey strain glycoprotein or variant thereof, a Vesiculovirus Carajas strain glycoprotein or variant thereof, a Vesiculovirus Chandipura strain glycoprotein or variant thereof, a Vesiculovirus Eptesicus strain glycoprotein or variant thereof, a Vesiculovirus Isfahan strain glycoprotein or variant thereof, a Vesiculovirus Jurona strain glycoprotein or variant thereof, a Vesiculovirus Malpais strain glycoprotein or variant thereof, a Vesiculovirus Perinet strain glycoprotein or variant thereof, a Vesiculovirus Piry strain glycoprotein or variant thereof, a Vesiculovirus Radi strain glycoprotein or variant thereof, a Vesiculovirus Rhinolopus strain glycoprotein or variant thereof, and a Vesiculovirus Yug Bogdanovac strain glycoprotein or variant thereof.

[0013] Preferably, the at least a portion of the enveloped virus glycoprotein or variant thereof is at least a portion of the Indiana strain vesiculovirus glycoprotein or variant thereof or at least a portion of the Cocal strain vesiculovirus glycoprotein or variant thereof, and the enveloped virus glycoprotein receptor is the LDL-R.

[0014] In some embodiments of the application, the surface of the extracellular vesicle comprises a vesiculovirus Indiana strain glycoprotein variant or a vesiculovirus Cocal strain glycoprotein variant, the enveloped virus glycoprotein receptor is the LDL-R, and the vesiculovirus glycoprotein variant comprises a first mutation comprising at least one of the following mutations:

[0015] (a) a substitution or a deletion at amino acid position 8, a substitution or a deletion at amino acid position 9, a substitution or a deletion at amino acid position 10, a substitution or a deletion at amino acid position 47, a substitution or a deletion at amino acid position 50, a substitution or a deletion at amino acid position 51, a substitution or a deletion at amino acid position 183, a substitution or a deletion at amino acid position 179, a substitution or a deletion at amino acid position 180, a substitution or a deletion at amino acid position 182, a substitution or a deletion at amino acid position 184, a substitution or a deletion at amino acid position 209, a substitution or a deletion at amino acid position 347, a substitution or a deletion at amino acid position 350, a substitution or a deletion at amino acid position 352, a substitution or a deletion at amino acid position 353, a substitution or a deletion at amino acid position 354, a deletion of amino acid positions 1-18, a deletion of amino acid positions 19-36, a deletion of amino acid positions 37-51, a deletion of amino acid positions 314-384, a deletion of amino acid positions 321-374, a deletion of amino acid positions 331-364, a deletion of amino acid positions 344-354, a deletion of amino acid positions 345-353; and

[0016] (b) upon best global alignment with SEQ ID NO: 1 or SEQ ID NO: 2, a substitution or deletion at an amino acid corresponding to position 8, a substitution or deletion at an amino acid corresponding to position 9, a substitution or deletion at an amino acid corresponding to position 10, a substitution or deletion at an amino acid corresponding to position 47, a substitution or deletion at an amino acid corresponding to position 50, a substitution or deletion at an amino acid corresponding to position 51, a substitution or deletion at an amino acid corresponding to position 183, a substitution or deletion at an amino acid corresponding to position 179, a substitution or deletion at an amino acid corresponding to position 180, a substitution or deletion at an amino acid corresponding to position 182, a substitution or deletion at an amino acid corresponding to position 184, a substitution or deletion at an amino acid corresponding to position 209, a substitution or deletion at an amino acid corresponding to position 347, a substitution or deletion at an amino acid corresponding to position 350, a substitution or deletion at an amino acid corresponding to position 352, a substitution or deletion at an amino acid corresponding to position 353, a substitution or deletion at an amino acid corresponding to position 354, a deletion of amino acids 1-18, a deletion of amino acids 19-36, a deletion of amino acids 37-51, a deletion of amino acids 314-384, a deletion of amino acids 321-374, a deletion of amino acids 331-364, a deletion of amino acids 344-354, a deletion of amino acids 345-353 of SEQ ID NO: 1 or SEQ ID NO: 2.

[0017] In some embodiments of the application, the first mutation comprises at least one of the following mutations:

[0018] (a) a substitution or deletion at H8, a substitution or deletion at N9, a substitution or deletion at Q10, a substitution or deletion at K47, a substitution or deletion at K50, a substitution or deletion at A51, a substitution or deletion at S183, a substitution or deletion at S179, a substitution or deletion at N180, a substitution or deletion at 1182, a substitution or deletion at M184, a substitution or deletion at Y209, a substitution or deletion at 1347, a substitution or deletion at T350, a substitution or deletion at T352, a substitution or deletion at E353, a substitution or deletion at R354, a deletion of amino acids 1-18, a deletion of amino acids 19-36, a deletion of amino acids 37-51, a deletion of amino acids 314-384, a deletion of amino acids 321-374, a deletion of amino acids 331-364, a deletion of amino acids 344-354, a deletion of amino acids 345-353 of SEQ ID NO: 1;

[0019] (b) substitution or deletion of H8, substitution or deletion of N9, substitution or deletion of Q10, substitution or deletion of K47, substitution or deletion of K50, substitution or deletion of A51, substitution or deletion of S183, substitution or deletion of S179, substitution or deletion of N180, substitution or deletion of 1182, substitution or deletion of M184, substitution or deletion of Y209, substitution or deletion of 1347, substitution or deletion of T350, substitution or deletion of T352, substitution or deletion of E353, substitution or deletion of R354, deletion of amino acids 1-18, deletion of amino acids 19-36, deletion of amino acids 37-51, deletion of amino acids 314-384, deletion of amino acids 321-374, deletion of amino acids 331-364, deletion of amino acids 344-354, deletion of amino acids 345-353 of SEQ ID NO: 1 upon optimal global alignment with SEQ ID NO: 1;

[0020] (c) substitution or deletion of Q8, substitution or deletion of S9, substitution or deletion of Q10, substitution or deletion of K47, substitution or deletion of K50, substitution or deletion of A51, substitution or deletion of D183, substitution or deletion of A179, substitution or deletion of T180, substitution or deletion of V182, substitution or deletion of T184, substitution or deletion of Y209, substitution or deletion of 1347, substitution or deletion of S350, substitution or deletion of T352, substitution or deletion of E353, substitution or deletion of R354, deletion of amino acids 1-18, deletion of amino acids 19-36, deletion of amino acids 37-51, deletion of amino acids 314-384, deletion of amino acids 321-374, deletion of amino acids 331-364, deletion of amino acids 344-354, deletion of amino acids 345-353 of SEQ ID NO: 2; and

[0021] (d) upon optimal global alignment with SEQ ID NO: 2, substitution or deletion of Q8, substitution or deletion of S9, substitution or deletion of Q10, substitution or deletion of K47, substitution or deletion of K50, substitution or deletion of A51, substitution or deletion of D183, substitution or deletion of A179, substitution or deletion of T180, substitution or deletion of V182, substitution or deletion of T184, substitution or deletion of Y209, substitution or deletion of 1347, substitution or deletion of S350, substitution or deletion of T352, substitution or deletion of E353, substitution or deletion of R354, deletion of amino acids 1-18, deletion of amino acids 19-36, deletion of amino acids 37-51, deletion of amino acids 314-384, deletion of amino acids 321-374, deletion of amino acids 331-364, deletion of amino acids 344-354, deletion of amino acids 345-353 of SEQ ID NO: 2.

[0022] In some embodiments of the application, the first mutation comprises at least one of the following mutations:

[0023] (a) deletion of amino acids 331-364, deletion of amino acids 344-354, substitution or deletion of K47, substitution or deletion of R354 of SEQ ID NO: 1 or SEQ ID NO: 2; and

[0024] (b) upon optimal global alignment with SEQ ID NO: 1 or SEQ ID NO: 2, deletion of amino acids 331-364, deletion of amino acids 344-354, substitution or deletion of K47, substitution or deletion of R354 of SEQ ID NO: 1 or SEQ ID NO: 2;

[0025] Preferably, the first mutation comprises at least one of the following mutations:

[0026] (a) deletion of amino acids 331-364, deletion of amino acids 344-354, substitution of the amino acid at position 47 from lysine K to glutamine Q (K47Q) or deletion of K47, substitution of the amino acid at position 354 from arginine R to glutamine Q (R354Q) or deletion of R354 of SEQ ID NO: 1 or SEQ ID NO: 2; and

[0027] (b) upon optimal global alignment with SEQ ID NO: 1 or SEQ ID NO: 2, deletion of amino acids 331-364, deletion of amino acids 344-354, K47Q or deletion of K47, R354Q or deletion of R354 of SEQ ID NO: 1 or SEQ ID NO: 2.

[0028] In some embodiments of the application, the first mutation comprises a mutation at:

[0029] (a) K47 of SEQ ID NO: 1 or SEQ ID NO: 2; or

[0030] (b) K47 of SEQ ID NO: 1 or SEQ ID NO: 2, upon optimal global alignment with SEQ ID NO: 1 or SEQ ID NO: 2.

[0031] In some embodiments of the application, the surface of the extracellular vesicle comprises any of the foregoing enveloped virus glycoprotein variants, which further comprises a second mutation that enhances or abolishes the ability of the enveloped virus glycoprotein variant to be inactivated by complement.

[0032] In some embodiments of the application, the ability of the enveloped virus glycoprotein variant to be inactivated by complement is enhanced or abolished relative to a wild-type enveloped virus glycoprotein that does not have the second mutation.

[0033] In some embodiments of the application, the second mutation comprises a mutation at at least one of:

[0034] (a) amino acid 214 of SEQ ID NO: 1 or SEQ ID NO: 2;

[0035] (b) amino acid 214 of SEQ ID NO: 1 or SEQ ID NO: 2, upon optimal global alignment with SEQ ID NO: 1 or SEQ ID NO: 2;

[0036] (c) amino acid 352 of SEQ ID NO: 1 or SEQ ID NO: 2;

[0037] (d) amino acid 352 of SEQ ID NO: 1 or SEQ ID NO: 2, upon optimal global alignment with SEQ ID NO: 1 or SEQ ID NO: 2;

[0038] (e) amino acid 50 of SEQ ID NO: 1 or SEQ ID NO: 2;

[0039] (f) amino acid 50 of SEQ ID NO: 1 or SEQ ID NO: 2, upon optimal global alignment with SEQ ID NO: 1 or SEQ ID NO: 2;

[0040] (g) is at amino acid position 146 of SEQ ID NO: 1 or SEQ ID NO: 2; and

[0041] (h) is at the amino acid position corresponding to amino acid position 146 of SEQ ID NO: 1 or SEQ ID NO: 2 after optimal global alignment of SEQ ID NO: 1 or SEQ ID NO: 2;

[0042] Preferably, the mutation is selected from the group consisting of a substitution, a deletion, and an insertion of an amino acid.

[0043] More preferably, the mutation is a substitution of an amino acid.

[0044] In some embodiments of the application, the second mutation comprises a mutation at at least one of the following positions:

[0045] (a) T214N, T352A, K50T, S146T of SEQ ID NO: 1 ; and

[0046] (b) K214N, T352A, K50T, S146T of SEQ ID NO: 2; and

[0047] (c) is at amino acid position 214 of SEQ ID NO: 2 by lysine K to asparagine N (K214N), T352A, K50T, S146T of SEQ ID NO: 2; and

[0048] (d) is at the amino acid position corresponding to K214N, T352A, K50T, S146T of SEQ ID NO: 2 after optimal global alignment of SEQ ID NO: 2.

[0049] In some embodiments of the application, the second mutation comprises a combination of mutations at any one of the following positions:

[0050] (a) is a substitution of (i) T214 and T352; or (ii) T214, T352, K50 and S146 of SEQ ID NO: 1 ; and

[0051] (b) is a substitution of (i) T214 and T352; or (ii) T214, T352, K50 and S146 of SEQ ID NO: 1 after optimal global alignment of SEQ ID NO: 1 ; and

[0052] (c) substitutions at (i) K214 and T352 of SEQ ID NO: 2; or (ii) K214, T352, K50 and S146 of SEQ ID NO: 2; and

[0053] (d) substitutions at (i) K214 and T352 of SEQ ID NO: 2; or (ii) K214, T352, K50 and S146 of SEQ ID NO: 2; and

[0054] Preferably, the second mutation comprises any one of the following combinations of site mutations:

[0055] (a) (i) T214N and T352A; or (ii) T214N, T352A, K50T and S146T of SEQ ID NO: 1;

[0056] (b) (i) T214N and T352A; or (ii) T214N, T352A, K50T and S146T of SEQ ID NO: 1; after optimal global alignment;

[0057] (c) (i) K214N and T352A; or (ii) K214N, T352A, K50T and S146T of SEQ ID NO: 2; and

[0058] (d) (i) K214N and T352A; or (ii) K214N, T352A, K50T and S146T of SEQ ID NO: 2; after optimal global alignment.

[0059] In some embodiments of the application, the surface of the extracellular vesicle comprises the envelope virus glycoprotein variant, the envelope virus glycoprotein variant comprises any one of the amino acid sequences of SEQ ID NOs: 3-20, and the envelope virus glycoprotein receptor is the LDL-R.

[0060] In some embodiments of the application, the envelope virus glycoprotein variant comprises the amino acid sequence of SEQ ID NO: 3; SEQ ID NO: 3 comprises a deletion of K47 relative to SEQ ID NO: 1.

[0061] In some embodiments of the application, the envelope virus glycoprotein variant comprises the amino acid sequence of SEQ ID NO: 4; SEQ ID NO: 4 comprises R354Q relative to SEQ ID NO: 1.

[0062] In some embodiments of the application, the enveloped virus glycoprotein variant comprises an amino acid sequence as set forth in SEQ ID NO: 5; SEQ ID NO: 5 comprises a deletion of K47 relative to SEQ ID NO: 2.

[0063] In some embodiments of the application, the enveloped virus glycoprotein variant comprises an amino acid sequence as set forth in SEQ ID NO: 6; SEQ ID NO: 6 comprises R354Q relative to SEQ ID NO: 2.

[0064] In some embodiments of the application, the enveloped virus glycoprotein variant comprises an amino acid sequence as set forth in SEQ ID NO: 7; SEQ ID NO: 7 comprises a deletion of R354 relative to SEQ ID NO: 1.

[0065] In some embodiments of the application, the enveloped virus glycoprotein variant comprises an amino acid sequence as set forth in SEQ ID NO: 8; SEQ ID NO: 8 comprises a deletion of R354 relative to SEQ ID NO: 2.

[0066] In some embodiments of the application, the enveloped virus glycoprotein variant comprises an amino acid sequence as set forth in SEQ ID NO: 9; SEQ ID NO: 9 comprises a deletion of K47, T214N (relative to before the deletion of K47), and T352A (relative to before the deletion of K47) relative to SEQ ID NO: 1.

[0067] In some embodiments of the application, the enveloped virus glycoprotein variant comprises an amino acid sequence as set forth in SEQ ID NO: 10; SEQ ID NO: 10 comprises a deletion of K47, T214N (relative to before the deletion of K47), T352A (relative to before the deletion of K47), K50T (relative to before the deletion of K47), and S146T (relative to before the deletion of K47) relative to SEQ ID NO: 1.

[0068] In some embodiments of the application, the enveloped virus glycoprotein variant comprises an amino acid sequence as set forth in SEQ ID NO: 11; SEQ ID NO: 11 comprises R354Q, T214N, and T352A relative to SEQ ID NO: 1.

[0069] In some embodiments of the application, the enveloped virus glycoprotein variant comprises an amino acid sequence as set forth in SEQ ID NO: 12; SEQ ID NO: 12 comprises R354Q, T214N, T352A, K50T, and S146T relative to SEQ ID NO: 1.

[0070] In some embodiments of the application, the enveloped virus glycoprotein variant comprises an amino acid sequence as set forth in SEQ ID NO: 13; SEQ ID NO: 13 comprises a deletion of R354, T214N (relative to before the deletion of R354), and T352A (relative to before the deletion of R354) relative to SEQ ID NO: 1.

[0071] In some embodiments of the application, the enveloped virus glycoprotein variant comprises an amino acid sequence as set forth in SEQ ID NO: 14; SEQ ID NO: 14 comprises a deletion of R354, T214N (relative to before the deletion of R354), T352A (relative to before the deletion of R354), K50T (relative to before the deletion of R354), and S146T (relative to before the deletion of R354) relative to SEQ ID NO: 1.

[0072] In some embodiments of the application, the enveloped virus glycoprotein variant comprises an amino acid sequence as set forth in SEQ ID NO: 15; SEQ ID NO: 15 comprises a deletion of K47, K214N (relative to before the deletion of K47), and T352A (relative to before the deletion of K47) relative to SEQ ID NO: 2.

[0073] In some embodiments of the application, the enveloped virus glycoprotein variant comprises an amino acid sequence as set forth in SEQ ID NO: 16; SEQ ID NO: 16 comprises a deletion of K47, K214N (relative to before the deletion of K47), T352A (relative to before the deletion of K47), K50T (relative to before the deletion of K47), and S146T (relative to before the deletion of K47) relative to SEQ ID NO: 2.

[0074] In some embodiments of the application, the enveloped virus glycoprotein variant comprises an amino acid sequence as set forth in SEQ ID NO: 17; SEQ ID NO: 17 comprises R354Q, K214N, and T352A relative to SEQ ID NO: 2.

[0075] In some embodiments of the application, the enveloped virus glycoprotein variant comprises an amino acid sequence as set forth in SEQ ID NO: 18; SEQ ID NO: 18 comprises R354Q, K214N, T352A, K50T, and S146T relative to SEQ ID NO: 2.

[0076] In some embodiments of the application, the enveloped virus glycoprotein variant comprises an amino acid sequence as set forth in SEQ ID NO: 19; SEQ ID NO: 19 comprises a deletion of R354, K214N (relative to before the deletion of R354), and T352A (relative to before the deletion of R354) relative to SEQ ID NO: 2.

[0077] In some embodiments of the application, the enveloped virus glycoprotein variant comprises an amino acid sequence as set forth in SEQ ID NO: 20; SEQ ID NO: 20 comprises a deletion of R354, K214N (relative to before the deletion of R354), T352A (relative to before the deletion of R354), K50T (relative to before the deletion of R354), and S146T (relative to before the deletion of R354) relative to SEQ ID NO: 2.

[0078] In some embodiments of the application, the extracellular vesicle surface comprises at least a portion of an enveloped virus glycoprotein, the at least a portion of the enveloped virus glycoprotein comprising a Truncated enveloped virus glycoprotein.

[0079] A Truncated enveloped virus glycoprotein, such as a Truncated VSV-G, comprises a functional domain of its ectodomain tail, transmembrane domain, and endodomain (cytosolic tail) sufficient to promote budding of the enveloped virus, binding to its receptor, such as LDL-R, is thus inhibited or does not bind LDL-R, while retaining the ability of the virus / EVs to bud, not only rendering EVs comprising the Truncated enveloped virus glycoprotein non-infectious, but also significantly reducing the immunogenicity of the EVs while significantly increasing the targeting of the EVs to a protein of interest.

[0080] In some embodiments of the application, the Truncated enveloped virus glycoprotein is a Truncated Vesiculovirus Indiana strain glycoprotein or a Truncated Vesiculovirus Cocal strain glycoprotein, the Truncated enveloped virus glycoprotein comprising an amino acid sequence as set forth in SEQ ID NO: 21 or 22 or an amino acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence as set forth in SEQ ID NO: 21 or 22.

[0081] Truncated Vesiculovirus Indiana strain glycoprotein:

[0082] FEHPHIQDAASQLPDDESLFFGDTGLSKNPIELVEGWFSSWKSSIASFFFIIGLIIGLFLVLRVGIHLCIKLKHTKKRQIYTDIEMNRLGK (SEQ ID NO: 21)

[0083] Truncated Vesiculovirus Cocal strain glycoprotein:

[0084] FEHPHLAEAPKQLPEEETLFFGDTGISKNPVELIEGWFSSWKSTVVTFFFAIGVFILLYVVARIVIAVRYRYQGSNNKRIYNDIEMSRFRK (SEQ ID NO: 22)

[0085] In some embodiments of the application, at least a portion of the enveloped virus glycoprotein or variant thereof is at least a portion of a Nipah virus glycoprotein G (NiV-G) or variant thereof;

[0086] Preferably, the extracellular vesicle comprises a NiV-G variant, the enveloped virus glycoprotein receptor is ephrin-B2 or B3, and the NiV-G variant comprises a mutation at at least one of the following positions:

[0087] at position 501 of SEQ ID NO: 23 or, following optimal global alignment with SEQ ID NO: 23, at a position equivalent to position 501 of SEQ ID NO: 23:

[0088] (a) the amino acid at position 501 is replaced from glutamic acid to alanine (E501A);

[0089] (b) the amino acid at position 504 is replaced from tryptophan to alanine (W504A);

[0090] (c) the amino acid at position 530 is replaced from glutamine Q to alanine A (Q530A); and

[0091] (d) the amino acid at position 533 is replaced from glutamic acid E to alanine A (E533A);

[0092] More preferably, the NiV-G variant comprises an amino acid sequence as set forth in SEQ ID NO: 24; SEQ ID NO: 24 comprises E501A, W504A, Q530A, and E533A relative to SEQ ID NO: 23.

[0093] In some embodiments of the application, the extracellular vesicle is selected from at least one of an exosome and a microvesicle;

[0094] Preferably, the extracellular vesicle is an exosome.

[0095] In some embodiments of the application, the surface of the extracellular vesicle comprises a protein of interest; preferably, the protein of interest is selected from the group consisting of a hydrophilic protein and a hydrophobic protein.

[0096] In some embodiments of the application, the surface of the extracellular vesicle comprises a transmembrane domain, the protein of interest is directly or indirectly linked to the transmembrane domain;

[0097] Preferably, the transmembrane domain is selected from the group consisting of a transmembrane domain of:

[0098] CD28, CD2, CD4, CD8a, CD5, CD3e, CD3d, CD3z, CD9, CD16, CD22, CD25, CD27, CD33, CD37, CD40, CD45, CD64, CD79A, CD79B, CD80, CD86, CD95 (Fas), CD134 (OX40), CD137 (4-1BB), CD150 (SLAMF1), CD152 (CTLA4), CD154 (CD40L), CD200R, CD223 (LAG3), CD270 (HVEM), CD272 (BTLA), CD273 (PD-L2), CD274 (PD-L1), CD278 (ICOS), CD279 (PD-1), CD300, CD357 (GITR), A2aR, DAP10, FcRa, FcRp, FcRy, Fyn, GAL9, KIR, Lck, LAT, LRP, NKG2D, NOTCH1, NOTCH2, NOTCH3, NOTCH4, PTCH2, ROR2, Ryk, Slp76, SIRPa, pTa, TCRa, TCRp, TIM3, TRIM, LPA5, and Zap70;

[0099] More preferably, the transmembrane domain comprises a transmembrane domain of CD8a.

[0100] In some embodiments of the application, the protein of interest is indirectly linked to the transmembrane domain via a linker domain;

[0101] Preferably, the linker domain is selected from the group consisting of:

[0102] (a) an immunoglobulin hinge region selected from the group consisting of wild-type or modified IgGl, IgG2, IgG3, IgG4, IgA, and IgD hinge regions;

[0103] (b) a hinge region selected from the wild-type or modified hinge region of CD28, CD7, CD8, CD8a, CD8b, CD3, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD134, CD137, ICOS, and CD154;

[0104] (c) all or a portion of an Fc domain selected from one or more of a CHI domain, a CH2 domain, and a CH3 domain; and

[0105] (d) a stalk region of a type II C-lectin selected from the stalk region of CD23, CD69, CD72, CD94, NKG2A, and NKG2D;

[0106] More preferably, the linking domain comprises a hinge region of CD8a.

[0107] In some embodiments of the application, the protein of interest comprises at least one of WNT3A and VEGF.

[0108] In a second aspect, the present application also provides an expression vector composition for preparing an extracellular vesicle, the expression vector composition comprising a first expression vector and a second expression vector,

[0109] (a) the first expression vector comprises a polynucleotide encoding at least a portion of an enveloped virus glycoprotein or a variant thereof, the at least a portion of the enveloped virus glycoprotein or the variant thereof (i) comprising a functional domain that facilitates budding of the enveloped virus; and (ii) being inhibited from binding to or not binding to an enveloped virus glycoprotein receptor; and

[0110] (b) the second expression vector comprises a gene integration element comprising an element that facilitates integration of an exogenous transgene into a genome of a producer cell.

[0111] In some embodiments of the application, the at least a portion of the enveloped virus glycoprotein or the variant thereof is the at least a portion of any of the foregoing enveloped virus glycoprotein or the variant thereof.

[0112] In some embodiments of the application, the first expression vector comprises a polynucleotide encoding a variant of an enveloped virus glycoprotein;

[0113] Preferably, the variant of the enveloped virus glycoprotein is any of the foregoing variant of a VSV Indiana strain glycoprotein, a VSV Cocal strain glycoprotein, or a NiV-G variant.

[0114] More preferably, the enveloped virus glycoprotein variant comprises the amino acid sequence of any one of SEQ ID NOs: 3-20 or the amino acid sequence as set forth in SEQ ID NO: 24.

[0115] In some embodiments of the application, the first expression vector comprises a polynucleotide encoding any one of the aforementioned truncated enveloped virus glycoproteins;

[0116] Preferably, the truncated enveloped virus glycoprotein is any one of the aforementioned truncated Vesiculovirus Indiana strain glycoproteins or truncated Vesiculovirus Cocal strain glycoproteins;

[0117] More preferably, the truncated enveloped virus glycoprotein comprises the amino acid sequence as set forth in SEQ ID NO: 21 or SEQ ID NO: 22.

[0118] In some embodiments of the application, the second expression vector further comprises an exogenous transgene comprising a polynucleotide encoding a protein of interest (GOI); preferably, the protein of interest comprises a hydrophilic protein and / or a hydrophobic protein.

[0119] In some embodiments of the application, the exogenous transgene further comprises a polynucleotide encoding a transmembrane domain;

[0120] Preferably, the transmembrane domain is selected from the transmembrane domain of the following proteins:

[0121] CD28, CD2, CD4, CD8a, CD5, CD3e, CD3d, CD3z, CD9, CD16, CD22, CD25, CD27, CD33, CD37, CD40, CD45, CD64, CD79A, CD79B, CD80, CD86, CD95 (Fas), CD134 (OX40), CD137 (4-1BB), CD150 (SLAMF1), CD152 (CTLA4), CD154 (CD40L), CD200R, CD223 (LAG3), CD270 (HVEM), CD272 (BTLA), CD273 (PD-L2), CD274 (PD-L1), CD278 (ICOS), CD279 (PD-1), CD300, CD357 (GITR), A2aR, DAP10, FcRa, FcRp, FcRy, Fyn, GAL9, KIR, Lck, LAT, LRP, NKG2D, NOTCH1, NOTCH2, NOTCH3, NOTCH4, PTCH2, ROR2, Ryk, Slp76, SIRPa, pTa, TCRa, TCRp, TIM3, TRIM, LPA5, and Zap70.

[0122] More preferably, the transmembrane domain comprises a transmembrane domain of CD8a.

[0123] In some embodiments of the application, the exogenous transgene further comprises a polynucleotide encoding a linker domain through which the protein of interest is indirectly linked to the transmembrane domain;

[0124] Preferably, the linker domain is selected from the group consisting of:

[0125] (a) an immunoglobulin hinge region selected from the group consisting of wild-type or modified IgGl, IgG2, IgG3, IgG4, IgA, and IgD hinge regions;

[0126] (b) a hinge region selected from the group consisting of wild-type or modified hinge regions of CD28, CD7, CD8, CD8a, CD8b, CD3, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD134, CD137, ICOS, and CD154;

[0127] (c) all or a portion of an Fc domain selected from the group consisting of one or more of a CHI domain, a CH2 domain, and a CH3 domain; and

[0128] (d) a stalk region of a type II C-lectin selected from the group consisting of stalk regions of CD23, CD69, CD72, CD94, NKG2A, and NKG2D;

[0129] More preferably, the linker domain comprises a hinge region of CD8a.

[0130] In some embodiments of the application, the first expression vector and the second expression vector are plasmids.

[0131] In some embodiments of the application, the first expression vector is an envelope plasmid in a lentiviral vector packaging system.

[0132] In some embodiments of the application, the second expression vector is a transfer plasmid in a lentiviral vector packaging system, the transfer plasmid comprising the gene integration element and the GOI.

[0133] In some embodiments of the application, the second expression vector is a transfer plasmid in a lentiviral vector packaging system, the transfer plasmid comprising the gene integration element and any one of the foregoing exogenous polynucleotides provided herein.

[0134] In some embodiments of the application, the gene integration element comprises a lentiviral backbone gene.

[0135] In some embodiments of the present application, the extracellular vesicle is selected from at least one of exosome and microvesicle.

[0136] Preferably, the extracellular vesicle is exosome.

[0137] In a third aspect, the present application provides a producer cell comprising the expression vector composition of the second aspect of the present application.

[0138] Preferably, the producer cell is selected from CHO cell, BHK cell, MDCK cell, C3H-10T1 / 2 cell, FLY cell, Psi-2 cell, BOSC 23 cell, PA317 cell, WEHI cell, COS cell, BSC-1 cell, BSC-40 cell, BMT-10 cell, VERO cell, W138 cell, MRC5 cell, A549 cell, HT1080 cell, HEK-293 cell, B-50 cell, 3T3 cell, NIH3T3 cell, HepG2 cell, Saos-2 cell, Huh7 cell, HeLa cell, W163 cell and 211 cell.

[0139] More preferably, the producer cell is HEK-293T cell.

[0140] In a fourth aspect, the present application provides a method for preparing extracellular vesicle, the method comprising culturing any of the producer cells of the third aspect of the present application, and collecting the extracellular vesicle produced by the producer cell.

[0141] In a fifth aspect, the present application provides an extracellular vesicle prepared by the method of the fourth aspect of the present application.

[0142] In a sixth aspect, the present application provides use of the extracellular vesicle of the first aspect of the present application, the expression vector composition of the second aspect of the present application, the producer cell of the fourth aspect of the present application or the extracellular vesicle of the fifth aspect of the present application in expressing and / or transporting a protein of interest; preferably, the protein of interest comprises hydrophobic protein and / or hydrophilic protein.

[0143] In a seventh aspect, the present application provides a method for expressing and / or transporting a protein of interest, the method comprising expressing the protein of interest on the surface of the extracellular vesicle.

[0144] Preferably, the protein of interest comprises hydrophobic protein and / or hydrophilic protein.

[0145] Preferably, the method comprises transfecting the expression vector composition comprising the GOI provided by the second aspect of the present application into the production cell, culturing the production cell, and collecting the extracellular vesicle expressing the target protein.

[0146] In an eighth aspect, the present application provides use of the extracellular vesicle provided by the first aspect of the present application, the expression vector composition provided by the second aspect of the present application, the production cell provided by the fourth aspect of the present application, or the extracellular vesicle provided by the fifth aspect of the present application in the preparation of a medicament.

[0147] In a ninth aspect, the present application provides a composition comprising a pharmaceutically acceptable carrier or excipient and any one of the following: the extracellular vesicle provided by the first aspect of the present application, the expression vector composition provided by the second aspect of the present application, or the extracellular vesicle provided by the fifth aspect of the present application.

[0148] The beneficial effects of the present application include:

[0149] The extracellular vesicle provided by the present application has its envelope viral glycoprotein on the surface thereof mutated or truncated, so that its ability to bind to receptors is inhibited or does not bind to receptors, but at the same time retains the ability to promote the budding of EVs; therefore, relative to the extracellular vesicle comprising the wild-type envelope viral glycoprotein, the extracellular vesicle provided by the present application has significantly improved ability to target transport the target protein and not to infect other cells;

[0150] Moreover, the extracellular vesicle provided by the present application can efficiently load and express the target protein including hydrophobic protein and hydrophilic protein on the surface thereof through simple operation and low cost, and can realize large-scale production of the extracellular vesicle expressing various target proteins on the surface thereof at low cost; especially for hydrophobic proteins, the present application effectively solves the technical problem that hydrophobic proteins are difficult to synthesize and express in vitro by simply expressing them on the surface of the extracellular vesicle; and the purification of the extracellular vesicle can be realized by the conventional technical means in the art such as ultracentrifugation, so that the extracellular vesicle purified in vitro is easier to be purified, and the purpose of further purifying the hydrophobic protein expressed on the surface of the extracellular vesicle can be achieved.

[0151] Herein:

[0152] "Inhibit" : The term "inhibit" when used in reference to the ability of a variant or truncated envelope virus glycoprotein to bind its natural target, such as a viral glycoprotein receptor, includes a complete ablation of the binding of the variant or truncated envelope virus glycoprotein to its natural target, as well as a significant reduction in the binding of the variant or truncated envelope virus glycoprotein to its natural target. In particular embodiments, "significant reduction" means a reduction in the binding of the variant or truncated envelope virus glycoprotein to the natural target selected from the group consisting of at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, at least 60%, at least 55%, at least 50%, at least 45%, at least 40%, at least 35%, at least 30%, at least 25%, at least 20%, at least 15%, and at least 10% reduction relative to a wild-type envelope virus glycoprotein.

[0153] In some embodiments of the application, the binding of the VSV-G variant, Cocal-G variant, truncated Cocal-G, or truncated VSV-G to its natural target, LDL-R, is "inhibited" to include a complete ablation of the binding of the VSV-G variant, Cocal-G variant, truncated Cocal-G, or truncated VSV-G to LDL-R, as well as a significant reduction in the binding of the VSV-G variant, Cocal-G variant, truncated Cocal-G, or truncated VSV-G to LDL-R; "significant reduction" means a reduction in the binding of the VSV-G variant, Cocal-G variant, truncated Cocal-G, or truncated VSV-G to LDL-R selected from the group consisting of at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, at least 60%, at least 55%, at least 50%, at least 45%, at least 40%, at least 35%, at least 30%, at least 25%, at least 20%, at least 15%, and at least 10% reduction relative to a wild-type VSV-G or Cocal-G.

[0154] "Exogenous" : Refers to any molecule, including nucleic acids, proteins, polypeptides, or small molecule compounds, etc., that originates from outside the organism. In contrast, the term "endogenous" refers to any molecule that originates from inside the organism (i.e., naturally produced by the organism).

[0155] “Hydrophobic protein”: As used herein, a hydrophobic protein refers to a protein whose amino acid sequence has a high proportion of hydrophobic amino acid residues (e.g., valine, leucine, isoleucine, phenylalanine, methionine, alanine, tryptophan, etc.) or that has been post-translationally modified to introduce a hydrophobic group (e.g., fatty acid acylation, prenylation, palmitoylation, etc.), resulting in a protein that exhibits hydrophobic properties overall. In some embodiments of the application, a hydrophobic protein includes a protein that contains a large number of hydrophobic amino acid residues, where “a large number” includes a proportion of hydrophobic amino acid residues that is greater than 50% of the total amino acid residues. In some embodiments of the application, a hydrophobic protein includes a protein that has a grand average of hydropathicity (GRAVY) greater than 0.

[0156] “Hydrophilic protein”: As used herein, a hydrophilic protein refers to a protein whose amino acid sequence has a high proportion of hydrophilic amino acid residues (e.g., lysine, arginine, aspartic acid, glutamic acid, asparagine, glutamine, histidine, serine, threonine, etc.) and that has not been post-translationally modified to significantly enhance hydrophobicity, resulting in a protein that exhibits hydrophilic properties overall. In some embodiments of the application, a hydrophilic protein includes a protein that contains a large number of hydrophilic amino acid residues, where “a large number” includes a proportion of hydrophilic amino acid residues that is greater than 50% of the total amino acid residues. In some embodiments of the application, a hydrophobic protein includes a protein that has a grand average of hydropathicity (GRAVY) less than 0.

[0157] “Complement”: The complement system is composed of a series of proteins that are part of the innate immune system. Complement (C) is present in the serum, tissue fluid and cell membrane surface of normal humans and animals, and has enzymatic activity after activation, which can occur in a complex cascade. The complement system is activated by a series of enzymes that cut each other, and ultimately forms a membrane attack complex that resembles a hole on the target microorganism, causing the microorganism to rupture and die. Complement components can be activated by antigen-antibody complexes or antibodies, and exhibit corresponding biological functions by lysing, opsonizing, phagocytizing, and mediating inflammatory reactions to clear immune complexes. Complement is widely involved in the body’s defense against microbial infection and immune regulation, and also mediates immunopathological damage, making it an important effector system and effector method system in the body.

[0158] By introducing the second mutation in the envelope virus glycoprotein, such as VSV-G, thereby increasing the ability of the envelope virus glycoprotein variant to antagonize complement inactivation, the EVs comprising the envelope virus glycoprotein variant are more suitable for use in in vivo protein delivery.

[0159] “Lentivirus”: Lentiviruses are complex retroviruses that contain additional genes with regulatory or structural functions in addition to the usual retroviral genes gag, pol, and env. The higher complexity enables the virus to regulate its life cycle, as it does during the process of latent infection. Lentiviruses belong to the Retroviridae family that can infect both dividing and non-dividing cells. Examples of lentiviruses include, but are not limited to, HIV (human immunodeficiency virus, including HIV type I and HIV type II), equine infectious anemia virus, feline immunodeficiency virus (FIV), bovine immunodeficiency virus (BIV), and simian immunodeficiency virus (SIV).

[0160] “Lentiviral Vector (LVV)”: As used herein, the term “Lentiviral Vector (LVV)” is intended to mean a self-inactivating viral particle that includes a viral envelope, has one or more characteristics of a lentivirus, is capable of invading a target cell and delivering a gene of interest (GOI) of interest, and is not self-replicating.

[0161] LVVs can stably integrate exogenous payload genes, such as CAR genes, into the chromosome of target cells, allowing the target cells to express the delivered transgene for a long period of time, providing a great advantage for gene therapy. In addition, they do not transfer viral genes, thus avoiding the problem of producing transduced cells that can be destroyed by cytotoxic T cells. And they have a relatively large clonal capacity, sufficient to meet most expected clinical applications.

[0162] “Packaging system”: As used herein, refers to a vector system comprising one or more nucleic acid vectors that contain nucleic acids necessary for the production, assembly, and / or packaging of a lentiviral vector in a packaging cell / packaging cell line when introduced into the packaging cell / packaging cell line for packaging of the lentiviral vector. Illustratively, the packaging system comprises (a) nucleic acids encoding viral proteins necessary for the production, assembly, and / or packaging of a lentiviral vector in a packaging cell line and (b) signals that provide the necessary signals for key functions such as viral replication, packaging, reverse transcription, and integration.

[0163] Packaging systems for commonly used lentiviral vectors include packaging systems for so-called third-generation lentiviral vectors. Packaging systems for third-generation lentiviral vectors include four plasmids, typically including a transfer plasmid and three packaging plasmids: a transfer plasmid / master plasmid containing a transgene / shuttle gene of interest / GOI, such as a CAR gene, a GagPol plasmid, a Rev plasmid, and an envelope plasmid containing a viral glycoprotein gene such as VSV-G or a variant thereof or Cocal-G or a variant thereof.

[0164] Generally, a “transfer vector” contains the lentiviral backbone genes, signals required for viral replication, packaging, reverse transcription, and integration, and other key functions. Transfer vectors typically have one or more transgenes flanked by long terminal repeat (LTR) sequences, which facilitate integration of the transgenes contained by the transfer vector, such as a CAR gene, into the host genome. The LTRs are responsible for the reverse transcription and integration processes of the viral genome. Through these sequences, the lentivirus can integrate the transgene into the genome of the host cell. For safety reasons, transfer vectors are often designed so that the resulting viral vectors are unable to self-replicate, e.g., the transfer vector lacks the genetic elements necessary to produce infectious lentiviral vectors in a host cell. In addition, transfer vectors can be designed to have a 3’ LTR deleted, rendering the virus “self-inactivating.” The TAT gene is eliminated from third generation pseudotyped lentiviral vector packaging systems by adding a chimeric 5’ LTR fused to a heterologous promoter (e.g., CMV or RSV promoter) on the transfer vector. Transfer vectors typically contain a Ψ sequence (Psi sequence, also known as Ψ packaging signal) downstream of the 5’ LTR, which is responsible for packaging the transgene RNA into the viral vector. The Ψ sequence ensures that only RNA containing the transgene is packaged into the viral vector. Transfer vectors can also optionally contain an Internal Ribosome Entry Site (“IRES”) to allow for the translation of two or more open reading frames (ORFs) on one mRNA, enabling multi-gene expression. Some transfer vectors, such as the master plasmid / transfer vector used in some embodiments of the present application, can also contain a selectable marker gene, such as an antibiotic resistance gene (e.g., PuroR, which encodes puromycin resistance) or a fluorescent protein gene (e.g., GFP), for selection or tracking of transduced cells.

[0165] Transfer vectors for packaging systems of lentiviral vectors are known in the art, see Naldini et al. (1996) Science 272:263-7; Zufferey et al. (1998) J. Virol. 72:9873-9880; Dull et al. (1998) J. Virol. 72:8463-8471; U.S. Patent No. 6,013,516; and U.S. Patent No. 5,994,136, each of which is incorporated by reference herein in its entirety. Generally, a transfer vector contains the basic nucleic acid sequences configured to carry out the selection of cells containing the vector, the incorporation of foreign nucleic acid into lentiviral particles, and the transfer of nucleic acid to target cells.

[0166] Lentiviral backbone genes generally refer to the most basic, essential cis-acting elements that make up the lentiviral vector transfer plasmid. These sequences do not encode viral proteins, but rather provide the signals needed for viral replication, packaging, reverse transcription, and integration. Specifically, lentiviral backbone genes generally include: long terminal repeat sequences (LTRs): located at both ends of the genome, contain promoter, enhancer, and terminator functions, which regulate viral gene transcription and integration processes; packaging signal (Ψ): determines which RNA molecules can be recognized and packaged into viral particles; central polypurine tract (cPPT) and central termination signal (CTS): help improve reverse transcription efficiency and nuclear transport; Rev response element (RRE): binds to the Rev protein, regulating the transport of viral RNA from the nucleus to the cytoplasm.

[0167] In constructing the transfer plasmid, in order to ensure safety and efficiency, only these necessary backbone sequences are usually retained in the transfer plasmid, and the sequences encoding viral structural proteins and enzymes (such as gag, pol, env, etc.) are removed from it, which are provided by the packaging system in the helper plasmid. This design not only ensures the function of the vector, but also reduces the risk of generating replication-competent viruses.

[0168] In some embodiments of the present application, "backbone genes" are intended to include nucleic acids encoding lentiviral cis nucleic acid sequences required for genome packaging. The backbone genes can also encode other cis nucleic acid sequences that are beneficial for gene delivery, including, for example, cis sequences required for reverse transcription, provirus integration, or genome transcription. Thus, the exact composition of the backbone genes will depend on the genetic material desired to be introduced into the target cell. Thus, the backbone genes can encode, for example, additional polypeptides or functions in addition to those required for packaging, reverse transcription, integration, or transcription. Such functions typically include encoding cis elements required for expression of the target nucleic acid / shuttle gene.

[0169] The packaging system of the third generation lentiviral vector usually also includes three packaging plasmids: GagPol plasmid, Rev plasmid and envelope plasmid. The envelope plasmid usually carries the envelope viral glycoprotein gene, for example, wild type VSV-G or Cocal-G is one of the commonly used viral glycoproteins; the viral glycoprotein gene is operably linked to a promoter, and the promoter is usually a CMV promoter, which initiates transcription of the viral glycoprotein gene.

[0170] The packaging system of third generation lentiviral vectors also includes two packaging plasmids, one containing the genes encoding the Gag protein and the Pol protein (GagPol packaging plasmid), while the other contains the gene encoding the Rev protein (Rev plasmid) as a further safety feature, which is an improvement over the single packaging plasmid of the so-called second generation packaging system. The Gag gene encodes the Gag polyprotein precursor containing lentiviral structural proteins, including matrix, capsid, and nucleocapsid; the Pol gene encodes the Pol polyprotein precursor providing lentiviral enzymatic functions necessary for replication, including protease, reverse transcriptase, and integrase; the Rev gene encodes the Rev protein, which binds the Rev Response Element (RRE) to allow nuclear export of unspliced and singly spliced HIV RNAs during viral replication. The Gag and Pol polyprotein precursors are cleaved during viral vector production. The Rev protein binds the Rev Response Element (RRE) sequence on the viral RNA, facilitating the transport of incompletely spliced viral RNA from the nucleus to the cytoplasm by interacting with the nuclear export machinery of the host cell. These unspliced RNAs can be translated into viral structural proteins and enzymes in the cytoplasm, or assembled into new viral vectors.

[0171] Exemplary, the packaging plasmids include, but are not limited to, pMD2.G, pRSV-rev, pMDLG-pRRE, and pRRL-GOI.

[0172] In some embodiments, the present application provides an expression vector composition for preparing extracellular vesicles, wherein the first expression vector is any of the foregoing envelope plasmids, and the second expression vector is any of the foregoing transfer plasmids.

[0173] In some embodiments, the EVs provided by the present application are prepared by transfecting production cells with a defined ratio of transfer plasmid and envelope plasmid. In some embodiments, the ratio of each plasmid is determined by mass, which is not particularly limited as long as it can package EVs. In some embodiments, the mass of the transfer plasmid is higher than that of the envelope plasmid for packaging EVs. In some embodiments, the defined ratio of the transfer plasmid and the envelope plasmid is about 1:1 to about 10:4. In some embodiments, the defined ratio of the transfer plasmid and the envelope plasmid is about 9:2.

[0174] “Production cell line”: The production / host / packaging cells that can be used to prepare the EVs provided by the present application include, but are not limited to, human embryonic kidney (HEK) 293 cells and their derivatives. The production cells can be adherent cell lines such as HEK-293T production cells, or suspension cell lines such as HEK-293T / 17SF production cells.

[0175] Exemplary, the packaging cell / producer cell is selected from the group consisting of CHO cells, BHK cells, MDCK cells, C3H-10T1 / 2 cells, FLY cells, Psi-2 cells, BOSC 23 cells, PA317 cells, WEHI cells, COS cells, BSC-1 cells, BSC-40 cells, BMT-10 cells, VERO cells, W138 cells, MRC 5 cells, A549 cells, HT1080 cells, HEK-293 cells, B-50 cells, 3T3 cells, NIH 3T3 cells, HepG2 cells, Saos-2 cells, Huh7 cells, HeLa cells, W163 cells, and 211 cells;

[0176] Preferably, the packaging cell / producer cell is a HEK-293T cell.

[0177] “Functional domain”: i.e. a region of a protein that has a specific biological function.

[0178] “Variant”: refers to a protein or polypeptide that has one or more amino acid alterations compared to a wild-type protein or polypeptide sequence. Variants are typically formed as a result of changes in the coding sequence due to point mutations, insertions, or deletions in the gene sequence, which alter the amino acid sequence of the protein or polypeptide.

[0179] “Pharmaceutically acceptable excipient or carrier”: pharmaceutically acceptable excipients or carriers include, but are not limited to, diluents, solubilizers, emulsifiers, preservatives, preservatives, and / or adjuvants. The excipient is preferably non-toxic or essentially non-toxic to recipients at the dosages and concentrations employed. Such excipients include, but are not limited to, saline, buffers, dextrose, water, glycerol, ethanol, and combinations thereof. In certain embodiments, the pharmaceutical composition can contain substances for improving, maintaining or preserving, for example, the pH, osmolarity, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution or release rate, absorption, or permeability of the composition. The optimal pharmaceutical composition will be determined by the intended route of administration, delivery means, and desired dosage.

[0180] “and / or”: is to be taken to mean either one or both alternatives.

[0181] “Comprise”: herein, unless the context requires otherwise, the word “comprise”, and variations such as “comprises” or “comprising”, will be understood to imply the inclusion of a stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements. In some embodiments of the application, the terms “include”, “has”, “have”, and “comprise” are used synonymously.

[0182] "Embodiment": Reference throughout this specification to "some embodiments", "some examples", means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the application. Thus, appearances of the foregoing phrases, as well as appearances of the phrases "in one embodiment" or "in an embodiment" or "in some embodiments" in various places throughout this specification are not necessarily intended to refer to the same embodiment.

[0183] "Sequence identity": Generally, "sequence identity" refers to the exact correspondence of nucleotides and nucleotides or amino acids and amino acids, respectively, of two polynucleotide or polypeptide sequences. Typically, techniques for determining sequence identity include determining the nucleotide sequence of a polynucleotide and / or determining the amino acid sequence encoded thereby, and comparing these sequences to a second nucleotide or amino acid sequence. Two or more sequences (polynucleotide or amino acid) can be compared by determining their "percent identity". Whether nucleic acid or amino acid sequences, the percent identity of two sequences is the number of exact matches between two aligned sequences divided by the length of the shorter sequence, multiplied by 100. Sequence information can also be compared using the Advanced BLAST computer program, available from the National Institutes of Health, to determine percent identity. The BLAST program is based on the following alignment method: Karlin and Altschul, Proc. Natl. Acad. Sci. USA 87:2264-2268 (1990) and discussed in Altschul et al., J. Mol. Biol. 215:403-410 (1990); Karlin and Altschul, Proc. Natl. Acad. Sci. USA 90:5873-5877 (1993); and Altschul et al., Nucleic Acids Res. 25:3389-3402 (1997). Briefly, the BLAST program defines identity as the number of identical aligned symbols (typically nucleotides or amino acids) divided by the total number of symbols in the shorter of the two sequences. The program can be used to determine percent identity over the entire length of the proteins being compared.

[0184] "Transduction": As used herein, the terms "transfection", "transformation", and "transduction" are used synonymously to refer to the process of introducing or incorporating exogenous nucleic acid into a producer cell. A "transfected", "transformed", or "transduced" cell is one which has been transfected, transformed, or transduced with exogenous nucleic acid. The cell includes the original recipient cell and its progeny.

[0185] All publications, literature and patent documents mentioned herein are hereby incorporated in their entirety by reference as if each individual publication, document or patent was specifically and individually incorporated in its entirety by reference herein. In case of conflict, the present application, including any definitions herein, will control. However, any reference, article, publication, patent, patent publication, and patent application cited herein is not intended to be, and is not, admitted to be prior art with respect to the present application.

[0186] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. BRIEF DESCRIPTION OF DRAWINGS

[0187] Figure 1: Schematic diagram of the exogenous transgene and gene integration element contained in the transfer plasmid 1.

[0188] Figure 2: Western Blot detection results of Alix and WNT3A in EVs-WNT3A, CTR1- EVs and CTR3-EVs.

[0189] Figure 3: Schematic diagram of the exogenous transgene and gene integration element contained in the transfer plasmid 2.

[0190] Figure 4: Western Blot detection results of Alix and VEGF in EVs-VEGF, CTR1-EVs and CTR2-EVs.

[0191] Figure 5: Plasmid map of the VSV-G variant 1.

[0192] Figure 6: Plasmid map of the transfer plasmid 1.

[0193] Figure 7: Plasmid map of the transfer plasmid 2.

[0194] Figure 8: Plasmid map of the membrane anchor-free transfer plasmid 1.

[0195] Figure 9: Plasmid map of the membrane anchor-free transfer plasmid 2.

[0196] Figure 10: Plasmid map of the truncated VSV-G.

[0197] Figure 11: Western Blot detection results of Alix and WNT3A in tEVs-WNT3A, CTR1- tEVs.

[0198] Figure 12: Western Blot detection results of Alix and VEGF in tEVs-VEGF, CTR1- tEVs. DETAILED DESCRIPTION

[0199] The concept and the technical effects of the present application will be described clearly and completely in combination with the embodiments below, so as to fully understand the purposes, features and effects of the present application. Obviously, the described embodiments are only some of the embodiments of the present application, but not all the embodiments; based on the embodiments of the present application, other embodiments obtained by those skilled in the art without creative labor are within the scope of protection of the present application.

[0200] The experimental methods not specified in the following embodiments are selected according to the conventional methods and conditions known in the art, or according to the commodity instruction. The reagents and raw materials not specified in the present application are commercially available. The methods used in the present application, such as Western Blot, are the methods known in the art, which can be described by textbooks or related literatures, and will not be described here. The methods described in the present application are described in the present application.

[0201] Example 1

[0202] 1. Transfect and culture HEK-293T cells, collect cell supernatant

[0203] A. Preparation of EV production system:

[0204] (a) Preparation of HEK-293T cell culture system: DMEM / high glucose (brand: GIBCO, item number: C12430500BT) + 10% FBS (brand: EXCELL, item number: #FSP500) + 1x pen-strep mixture;

[0205] (b) Preparation of expression vector composition for preparing EVs:

[0206] (i) The first expression vector: VSV-G variant 1 plasmid; the VSV-G variant 1 plasmid comprises a polynucleotide encoding VSV-G variant 1, which comprises an amino acid sequence as shown in SEQ ID NO: 9; the VSV-G variant 1 comprises a first mutation K47 deletion and a second mutation T214N+T352A (relative to before K47 deletion) relative to wild-type VSV-G comprising an amino acid sequence as shown in SEQ ID NO: 1; the VSV-G variant 1 plasmid is synthesized by conventional molecular cloning method, which is well known to those skilled in the art, and the plasmid map of the VSV-G variant 1 plasmid is shown in Figure 5;

[0207] Wild-type VSV-G:

[0208] Wild-type VSV-G full-length protein:

[0209] wherein the sequence MKCLLYLAFLFIGVNC contained in sites 1-16 is the signal peptide amino acid sequence of wild-type VSV-G.

[0210] VSV-G variant 1:

[0211] (ii) a second expression vector: transfer plasmid 1; the transfer plasmid 1 comprises an exogenous polynucleotide 1 comprising: a polynucleotide encoding WNT3A (a target gene 1), a polynucleotide encoding a hinge region of CD8a, and a polynucleotide encoding a transmembrane region of CD8a; the transfer plasmid 1 comprises gene expression elements as shown in Figure 1; the plasmid map of the transfer plasmid 1 is shown in Figure 6;

[0212] The exogenous polynucleotide 1 comprises, in order from 5' to 3': a polynucleotide encoding WNT3A, a polynucleotide encoding a hinge region of CD8a, a polynucleotide encoding a transmembrane region of CD8a;

[0213] The WNT3A protein comprises an amino acid sequence as shown in SEQ ID NO: 25;

[0214] The CD8a hinge region comprises an amino acid sequence as shown in SEQ ID NO: 26;

[0215] The CD8a transmembrane region comprises an amino acid sequence as shown in SEQ ID NO: 27;

[0216] B. Transfecting HEK-293T cells using the expression vector composition:

[0217] At Day 0, HEK-293T cells were seeded at 4E6 cells per 10 cm dish, and after about 48 h when the cells reached 80-90% confluency, 2 ug of the VSV-G variant 1 plasmid and 9 ug of the transfer plasmid 1 were transfected into the HEK-293T cells by PEI reagent (Polyethylenimine transfection reagent), and the medium was changed after 6-8 h of transfection, and the culture supernatant was collected at 48 h post-transfection.

[0218] 2. Isolation of EVs-WNT3A

[0219] EVs-WNT3A expressing the target gene 1 WNT3A were isolated from the collected culture supernatant by ultracentrifugation, and the specific implementation is as follows:

[0220] After the culture supernatant was filtered using a 0.45 um filter membrane, the EVs-WNT3A were resuspended using 5% trehalose (Dawo) and stored at -80°C for long-term preservation after centrifugation at 50000g for 2.5h using an ultracentrifuge (JXP-26).

[0221] 3. Identification of EVs-WNT3A

[0222] A. Detection of EVs positive marker protein and WNT3A expression using Western Blot

[0223] (a) Preparation of control group EVs

[0224] (i) Preparation of control group 1 EVs transfected with VSV-G variant 1 plasmid only

[0225] Referring to the method for preparing EVs-WNT3A described above, control group 1 EVs (CTR1-EVs) were prepared by the following method: only the VSV-G variant 1 plasmid was transfected into HEK-293T cells, the transfer plasmid 1 was not transfected, the HEK-293T cells were cultured, and the CTR1-EVs were collected and separated.

[0226] (ii) Preparation of EVs transfected with VSV-G variant 1 plasmid and membrane anchor-free transfer plasmid

[0227] Referring to the method for preparing EVs-WNT3A described above, control group 3 EVs (CTR3-EVs) were prepared by the following method: the VSV-G variant 1 plasmid and the membrane anchor-free transfer plasmid 1 were transfected into HEK-293T cells, the HEK-293T cells were cultured, and the CTR3-EVs were collected and separated.

[0228] The membrane anchor-free transfer plasmid 1 only contains a polynucleotide encoding WNT3A, and does not contain a polynucleotide encoding a CD8a hinge region and a CD8a transmembrane region; the plasmid map of the membrane anchor-free transfer plasmid 1 is shown in Figure 8.

[0229] (b) Detection of Alix and WNT3A in EVs-WNT3A and control group EVs

[0230] The expression of Alix (EVs positive marker protein) and WNT3A in EVs-WNT3A, CTR1-EVs and CTR3-EVs was detected using Western Blot, and the results are shown in Figure 2.

[0231] As shown in FIG. 2, Alix was detected in EVs-WNT3A, CTR1-EVs and CTR3-EVs, however, only CTR3-EVs and EVs-WNT3A detected the expression of WNT3A; and, since in EVs-WNT3A, WNT3A was connected with CD8a transmembrane region through CD8a hinge region to express on the surface membrane of EVs, therefore, the expression of target gene WNT3A in EVs-WNT3A was higher than that in CTR3-EVs which did not contain membrane anchoring structure.

[0232] Example 2

[0233] 1. Preparation of EVs-VEGF containing VEGF protein on the surface

[0234] Referring to the method for preparing, collecting and isolating EVs-WNT3A in Example 1, EVs-VEGF containing VGEF protein on the surface was prepared, collected and isolated; wherein the transfer plasmid 1 was replaced by a transfer plasmid 2 containing an exogenous polynucleotide 2 comprising: a polynucleotide encoding VEGF (target gene 2), a polynucleotide encoding the hinge region of CD8a and a polynucleotide encoding the transmembrane region of CD8a; the transfer plasmid 2 comprises gene expression elements as shown in FIG. 3; the plasmid map of the transfer plasmid 2 is shown in FIG. 7;

[0235] The exogenous polynucleotide 2 comprises, from 5' end to 3' end: a polynucleotide encoding VEGF, a polynucleotide encoding the hinge region of CD8a, a polynucleotide encoding the transmembrane region of CD8a.

[0236] The VEGF protein comprises an amino acid sequence as shown in SEQ ID NO: 29.

[0237] 2. Preparation of control group EVs

[0238] Referring to the preparation method of CTR3-EVs in Example 1, CTR2-EVs were prepared, and the specific operation method was as follows: the VSV-G variant 1 plasmid and the membrane anchoring-free transfer plasmid 2 were transfected into HEK-293T cells, the HEK-293T cells were cultured, and CTR2-EVs were collected and isolated.

[0239] The membrane anchoring-free transfer plasmid 2 only comprises a polynucleotide encoding VEGF, and does not comprise polynucleotides encoding the hinge region of CD8a and the transmembrane region of CD8a; the plasmid map of the membrane anchoring-free transfer plasmid 2 is shown in FIG. 9.

[0240] 3. Detection of Alix and target gene 2 VEGF

[0241] The expression of Alix and the gene of interest 2 VEGF in EVs-VEGF, CTR2-EVs and CTR1-EVs was detected by Western Blot, and the results are shown in Figure 4.

[0242] As shown in Figure 4, Alix was detected in EVs-VEGF, CTR2-EVs and CTR1-EVs, however, the expression of VEGF was only detected in CTR2-EVs and EVs-VEGF; and, since in EVs-VEGF, VEGF is connected to CD8a transmembrane region through CD8a hinge region to express on the surface membrane of EVs, the expression of the gene of interest VEGF in EVs-VEGF is higher than that in CTR2-EVs which does not contain a membrane-anchoring structure.

[0243] Example 3

[0244] 1. Preparation of EVs (truncated-EVs, "tEVs") containing truncated VSV-G on the surface.

[0245] The truncated VSV-G contains the tail end of the extracellular domain, the transmembrane region and the intracellular domain of VSV-G, retains the ability to promote virus / EVs budding, while its extracellular domain does not contain most of the sites that bind to LDL-R, so the binding to LDL-R is inhibited or does not bind to LDL-R; at the same time, the immunogenicity of the truncated VSV-G is also significantly reduced;

[0246] Referring to the preparation method of EVs-WNT3A or EVs-VEGF described in Examples 1 and 2, the EVs containing the truncated VSV-G and expressing the gene of interest 1 WNT3A or the gene of interest 2 VEGF, tEVs-WNT3A or tEVs-VEGF, are prepared, isolated and collected;

[0247] Wherein, the VSV-G variant 1 plasmid is replaced by a truncated VSV-G plasmid containing a polynucleotide encoding the truncated VSV-G, and the truncated VSV-G contains an amino acid sequence as shown in SEQ ID NO: 21; the plasmid map of the truncated VSV-G plasmid is shown in Figure 10;

[0248] 2. Preparation of control group EVs

[0249] Referring to the preparation method of CTR1-EVs described in Example 1, CTR1-tEVs are prepared; the specific operation method is as follows:

[0250] HEK-293T cells were transfected with the truncated VSV-G plasmid only, cultured, collected and isolated CTR1-tEVs;

[0251] 3. Detection of Alix and the gene of interest

[0252] The expression of Alix and WNT3A in tEVs-WNT3A and CTR1-tEVs was detected by Western Blot, respectively, and the results are shown in Figure 11.

[0253] As shown in Figure 11, Alix was detected in both tEVs-WNT3A and CTR1-tEVs, however, the expression of WNT3A was only detected in tEVs-WNT3A.

[0254] The expression of Alix and the gene of interest 2 VEGF in tEVs-VEGF and CTR1-tEVs was detected by Western Blot, respectively, and the results are shown in Figure 12.

[0255] As shown in Figure 12, Alix was detected in both tEVs-VEGF and CTR1-tEVs, however, the expression of VEGF was only detected in tEVs-VEGF.

Claims

1. Extracellular Vesicles (EVs) comprising, the surface of the extracellular vesicle comprises at least a portion of an enveloped virus glycoprotein or a variant thereof, (a) the at least a portion of the enveloped virus glycoprotein or a variant thereof comprises a functional domain that facilitates Budding of the enveloped virus; and (b) the at least a portion of the enveloped virus glycoprotein or a variant thereof (i) is inhibited for binding to an enveloped virus glycoprotein receptor or (ii) does not bind to an enveloped virus glycoprotein receptor.

2. Extracellular vesicle according to claim 1, characterized in that, the enveloped virus glycoprotein or a variant thereof is selected from the group consisting of a Vesiculovirus strain glycoprotein or a variant thereof, a NiV glycoprotein G or a variant thereof, a Morbillivirus glycoprotein H or a variant thereof, a Lentivirus glycoprotein or a variant thereof, a Rabies virus glycoprotein (RVG) or a variant thereof, a gibbon ape leukemia virus glycoprotein (GaLV) or a variant thereof, an amphotropic murine leukemia virus glycoprotein (MLV-A) or a variant thereof, a feline endogenous virus (RD114) glycoprotein or a variant thereof, a fowlpox virus (FPV) glycoprotein or a variant thereof, an Ebola virus (EboV) glycoprotein or a variant thereof, and a Lymphocytic choriomeningitis virus (LCMV) glycoprotein or a variant thereof; the Vesiculovirus strain glycoprotein or a variant thereof comprises a Vesiculovirus Indiana strain glycoprotein or a variant thereof, a Vesiculovirus Cocal strain glycoprotein or a variant thereof, a Vesiculovirus Maraba strain glycoprotein or a variant thereof, a Vesiculovirus Morreton strain glycoprotein or a variant thereof, a Vesiculovirus Alagoas strain glycoprotein or a variant thereof, a Vesiculovirus New Jersey strain glycoprotein or a variant thereof, a Vesiculovirus Carajas strain glycoprotein or a variant thereof, a Vesiculovirus Chandipura strain glycoprotein or a variant thereof, a Vesiculovirus Eptesicus strain glycoprotein or a variant thereof, a Vesiculovirus Isfahan strain glycoprotein or a variant thereof, a Vesiculovirus Jurona strain glycoprotein or a variant thereof, a Vesiculovirus Malpais strain glycoprotein or a variant thereof, a Vesiculovirus Perinet strain glycoprotein or a variant thereof, a Vesiculovirus Piry strain glycoprotein or a variant thereof, a Vesiculovirus Radi strain glycoprotein or a variant thereof, a Vesiculovirus Rhinolopus strain glycoprotein or a variant thereof, and a Vesiculovirus Yug Bogdanovac strain glycoprotein or a variant thereof; Preferably, the at least a portion of the enveloped virus glycoprotein or a variant thereof is at least a portion of a Vesiculovirus Indiana strain glycoprotein (VSV-G) or a variant thereof or at least a portion of a Vesiculovirus Cocal strain glycoprotein (Cocal-G) or a variant thereof, and the enveloped virus glycoprotein receptor is the LDL-R.

3. Extracellular vesicle according to claim 1 or 2, characterized in that, the surface of the extracellular vesicle comprises a VSV-G variant or a Cocal-G variant, the envelope virus glycoprotein receptor is LDL-R, the VSV-G variant or the Cocal-G variant comprises a first mutation, the first mutation comprises at least one of the following mutations: (a) a substitution or a deletion at amino acid position 8, a substitution or a deletion at amino acid position 9, a substitution or a deletion at amino acid position 10, a substitution or a deletion at amino acid position 47, a substitution or a deletion at amino acid position 50, a substitution or a deletion at amino acid position 51, a substitution or a deletion at amino acid position 183, a substitution or a deletion at amino acid position 179, a substitution or a deletion at amino acid position 180, a substitution or a deletion at amino acid position 182, a substitution or a deletion at amino acid position 184, a substitution or a deletion at amino acid position 209, a substitution or a deletion at amino acid position 347, a substitution or a deletion at amino acid position 350, a substitution or a deletion at amino acid position 352, a substitution or a deletion at amino acid position 353, a substitution or a deletion at amino acid position 354, a deletion of amino acid positions 1-18, a deletion of amino acid positions 19-36, a deletion of amino acid positions 37-51, a deletion of amino acid positions 314-384, a deletion of amino acid positions 321-374, a deletion of amino acid positions 331-364, a deletion of amino acid positions 344-354, a deletion of amino acid positions 345-353 of SEQ ID NO: 1 or SEQ ID NO: 2; and (b) a substitution or a deletion at amino acid position 8, a substitution or a deletion at amino acid position 9, a substitution or a deletion at amino acid position 10, a substitution or a deletion at amino acid position 47, a substitution or a deletion at amino acid position 50, a substitution or a deletion at amino acid position 51, a substitution or a deletion at amino acid position 183, a substitution or a deletion at amino acid position 179, a substitution or a deletion at amino acid position 180, a substitution or a deletion at amino acid position 182, a substitution or a deletion at amino acid position 184, a substitution or a deletion at amino acid position 209, a substitution or a deletion at amino acid position 347, a substitution or a deletion at amino acid position 350, a substitution or a deletion at amino acid position 352, a substitution or a deletion at amino acid position 353, a substitution or a deletion at amino acid position 354, a deletion of amino acid positions 1-18, a deletion of amino acid positions 19-36, a deletion of amino acid positions 37-51, a deletion of amino acid positions 314-384, a deletion of amino acid positions 321-374, a deletion of amino acid positions 331-364, a deletion of amino acid positions 344-354, a deletion of amino acid positions 345-353 after optimal global alignment with SEQ ID NO: 1 or SEQ ID NO: 2; preferably, the first mutation comprises at least one of the following mutations: (a) a deletion of amino acids 331-364, a deletion of amino acids 344-354, a substitution or deletion of K47, a substitution or deletion of R354, located in SEQ ID NO: 1 or SEQ ID NO: 2; and (b) a deletion of amino acids 331-364, a deletion of amino acids 344-354, a substitution or deletion of K47, a substitution or deletion of R354, located in SEQ ID NO: 1 or SEQ ID NO: 2, upon optimal global alignment of SEQ ID NO: 1 or SEQ ID NO: 2; More preferably, the first mutation comprises at least one of the following mutations: (a) a deletion of amino acids 331-364, a deletion of amino acids 344-354, a substitution of the 47thamino acid from lysine K to glutamine Q (K47Q) or a deletion of K47, a substitution of the 354thamino acid from arginine R to glutamine Q (R354Q) or a deletion of R354, located in SEQ ID NO: 1 or SEQ ID NO: 2; and (b) a deletion of amino acids 331-364, a deletion of amino acids 344-354, a substitution or deletion of K47, a substitution or deletion of R354, located in SEQ ID NO: 1 or SEQ ID NO: 2, upon optimal global alignment of SEQ ID NO: 1 or SEQ ID NO:

2.

4. Extracellular vesicle according to claim 3, characterized in that, The first mutation comprises the following mutation: (a) a deletion of K47, located in SEQ ID NO: 1 or SEQ ID NO: 2; or (b) a deletion of K47, located in SEQ ID NO: 1 or SEQ ID NO: 2, upon optimal global alignment of SEQ ID NO: 1 or SEQ ID NO:

2.

5. Extracellular vesicle according to claim 3 or 4, characterized in that, The first mutation renders the VSV-G variant or Cocal-G variant (i) inhibited for binding to LDL-R or (ii) not binding to LDL-R.

6. Extracellular vesicle according to any one of claims 1 -5, characterized in that, The surface of the extracellular vesicle comprises the envelope virus glycoprotein variant, which further comprises a second mutation that enhances or does not inactivate the ability of the envelope virus glycoprotein variant to antagonize complement inactivation.

7. Extracellular vesicle according to claim 6, characterized in that, The second mutation comprises a mutation of at least one of the following positions: (a) the 214thamino acid, located in SEQ ID NO: 1 or SEQ ID NO: 2; (b) the 214thamino acid, located in SEQ ID NO: 1 or SEQ ID NO: 2, upon optimal global alignment of SEQ ID NO: 1 or SEQ ID NO: 2; (c) the 352ndamino acid, located in SEQ ID NO: 1 or SEQ ID NO: 2; (d) the 352ndamino acid, located in SEQ ID NO: 1 or SEQ ID NO: 2, upon optimal global alignment of SEQ ID NO: 1 or SEQ ID NO: 2; (e) the 50thamino acid, located in SEQ ID NO: 1 or SEQ ID NO: 2; (f) at amino acid position 50 of SEQ ID NO: 1 or SEQ ID NO: 2 following the best global alignment of SEQ ID NO: 1 or SEQ ID NO: 2; (g) at amino acid position 146 of SEQ ID NO: 1 or SEQ ID NO: 2; and (h) at amino acid position 146 of SEQ ID NO: 1 or SEQ ID NO: 2 following the best global alignment of SEQ ID NO: 1 or SEQ ID NO: 2; Preferably, the mutation is selected from the group consisting of substitution, deletion and insertion of an amino acid; More preferably, the mutation is a substitution of an amino acid.

8. Extracellular vesicle according to claim 7, characterized in that, The second mutation comprises at least one mutation at the following positions: (a) at amino acid position 214 of SEQ ID NO: 1 is substituted from threonine T to asparagine N (T214N), at amino acid position 352 of SEQ ID NO: 1 is substituted from threonine T to alanine A (T352A), at amino acid position 50 of SEQ ID NO: 1 is substituted from lysine K to threonine T (K50T), at amino acid position 146 of SEQ ID NO: 1 is substituted from serine S to threonine T (S146T); (b) at T214N, T352A, K50T, S146T of SEQ ID NO: 1 following the best global alignment of SEQ ID NO: 1 ; (c) at amino acid position 214 of SEQ ID NO: 2 is substituted from lysine K to asparagine N (K214N), T352A, K50T, S146T; and (d) at K214N, T352A, K50T, S146T of SEQ ID NO: 2 following the best global alignment of SEQ ID NO:

2.

9. Extracellular vesicle according to claim 8, characterized in that, The second mutation comprises a combination of mutations at any of the following positions: (a) at (i) T214N and T352A of SEQ ID NO: 1 ; or (ii) T214N, T352A, K50T and S146T of SEQ ID NO: 1 ; (b) at (i) T214N and T352A of SEQ ID NO: 1 ; or (ii) T214N, T352A, K50T and S146T of SEQ ID NO: 1 following the best global alignment of SEQ ID NO: 1 ; (c) at (i) K214N and T352A of SEQ ID NO: 2; or (ii) K214N, T352A, K50T and S146T of SEQ ID NO: 2; and (d) at (i) K214N and T352A of SEQ ID NO: 2; or (ii) K214N, T352A, K50T and S146T of SEQ ID NO: 2 following the best global alignment of SEQ ID NO:

2.

10. Extracellular vesicle according to claim 1, characterized in that, The surface of the extracellular vesicle comprises the envelope virus glycoprotein variant, the envelope virus glycoprotein variant comprising any of the amino acid sequences of SEQ ID NOs: 3-20, and the envelope virus glycoprotein receptor is LDL-R.

11. Extracellular vesicle according to claim 1, characterized in that, The surface of the extracellular vesicle comprises at least a portion of an enveloped virus glycoprotein, the at least a portion of the enveloped virus glycoprotein comprising a Truncated enveloped virus glycoprotein.

12. Extracellular vesicle according to claim 11, characterized in that, The Truncated enveloped virus glycoprotein is a Truncated VSV-G or a Truncated Cocal-G, the enveloped virus glycoprotein receptor is LDL-R; Preferably, the Truncated VSV-G or Truncated Cocal-G (a) comprises an amino acid sequence as set forth in SEQ ID NO: 21 or 22 or (b) an amino acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid sequence as set forth in SEQ ID NO: 21 or 22.

13. Extracellular vesicle according to claim 1, characterized in that, The at least a portion of the enveloped virus glycoprotein or variant thereof is at least a portion of a NiV-G or variant thereof; Preferably, the extracellular vesicle comprises a NiV-G variant, the enveloped virus glycoprotein receptor is ephrin-B2 or B3, the NiV-G variant comprises at least one mutation at the following positions: in SEQ ID NO: 23 or, after optimal global alignment with SEQ ID NO: 23, in the equivalent position of SEQ ID NO: 23: (a) the amino acid at position 501 is replaced from glutamic acid to alanine (E501A); (b) the amino acid at position 504 is replaced from tryptophan to alanine (W504A); (c) the amino acid at position 530 is replaced from glutamine Q to alanine A (Q530A); and (d) the amino acid at position 533 is replaced from glutamic acid E to alanine A (E533A); More preferably, the NiV-G variant comprises an amino acid sequence as set forth in SEQ ID NO: 24; SEQ ID NO: 24 comprises E501A, W504A, Q530A and E533A relative to SEQ ID NO:

23.

14. Extracellular vesicle according to any one of claims 1 - 13, characterized in that, The surface of the extracellular vesicle further comprises a protein of interest; preferably, the protein of interest is selected from at least one of a hydrophilic protein and a hydrophobic protein.

15. Extracellular vesicle according to claim 14, characterized in that, The surface of the extracellular vesicle comprises a transmembrane domain, the protein of interest is directly or indirectly linked to the transmembrane domain; Preferably, the transmembrane domain is selected from a transmembrane domain of a protein selected from the group consisting of: CD28, CD2, CD4, CD8a, CD5, CD3e, CD3d, CD3z, CD9, CD16, CD22, CD25, CD27, CD33, CD37, CD40, CD45, CD64, CD79A, CD79B, CD80, CD86, CD95 (Fas), CD134 (OX40), CD137 (4-1BB), CD150 (SLAMF1), CD152 (CTLA4), CD154 (CD40L), CD200R, CD223 (LAG3), CD270 (HVEM), CD272 (BTLA), CD273 (PD-L2), CD274 (PD-L1), CD278 (ICOS), CD279 (PD-1), CD300, CD357 (GITR), A2aR, DAP10, FcRa, FcRp, FcRy, Fyn, GAL9, KIR, Lck, LAT, LRP, NKG2D, NOTCH1, NOTCH2, NOTCH3, NOTCH4, PTCH2, ROR2, Ryk, Slp76, SIRPa, pTa, TCRa, TCRp, TIM3, TRIM, LPA5, and Zap70; More preferably, the transmembrane domain comprises a transmembrane domain of CD8a.

16. Extracellular vesicle according to claim 15, characterized in that The protein of interest is indirectly linked to the transmembrane domain via a linker domain; Preferably, the linker domain is selected from the group consisting of: (a) an immunoglobulin hinge region selected from the group consisting of wild-type or modified IgGl, IgG2, IgG3, IgG4, IgA, and IgD hinge regions; (b) a hinge region selected from the group consisting of wild-type or modified hinge regions of CD28, CD7, CD8, CD8a, CD8b, CD3, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD134, CD137, ICOS, and CD154; (c) all or a portion of an Fc domain selected from the group consisting of one or more of a CHI domain, a CH2 domain, and a CH3 domain; and (d) a stalk region of a type II C-lectin selected from the group consisting of stalk regions of CD23, CD69, CD72, CD94, NKG2A, and NKG2D; More preferably, the linker domain comprises a hinge region of CD8a.

17. Extracellular vesicle according to any one of claims 1 - 16, characterized in that, The extracellular vesicle is selected from the group consisting of at least one of an exosome and a microvesicle; Preferably, the extracellular vesicle is an exosome.

18. An expression vector composition for preparing an extracellular vesicle, characterized by, The expression vector composition comprises a first expression vector and a second expression vector, (a) the first expression vector comprises a polynucleotide encoding at least a portion of an enveloped virus glycoprotein or variant thereof, the at least a portion of the enveloped virus glycoprotein or variant thereof (i) comprising a domain that facilitates budding of the enveloped virus; and (ii) inhibited from binding to or does not bind to an enveloped virus glycoprotein receptor; and (b) the second expression vector comprises Integration Elements comprising elements that facilitate integration of an exogenous transgene into the genome of a Production Cell.

19. The expression vector composition of claim 18, wherein, The enveloped virus glycoprotein or variant thereof is selected from the group consisting of a Vesiculovirus strain glycoprotein or variant thereof, a NiV glycoprotein G or variant thereof, a Morbillivirus glycoprotein H or variant thereof, a Lentivirus glycoprotein or variant thereof, a Rabies virus glycoprotein (RVG) or variant thereof, a gibbon ape leukemia virus glycoprotein (GaLV) or variant thereof, an amphotropic murine leukemia virus glycoprotein (MLV-A) or variant thereof, a feline endogenous virus (RD114) glycoprotein or variant thereof, a fowlpox virus (FPV) glycoprotein or variant thereof, an Ebola virus (EboV) glycoprotein or variant thereof, and a Lymphocytic choriomeningitis virus (LCMV) glycoprotein or variant thereof; The Vesiculovirus strain glycoprotein or variant thereof includes a Vesiculovirus Indiana strain glycoprotein or variant thereof, a Vesiculovirus Cocal strain glycoprotein or variant thereof, a Vesiculovirus Maraba strain glycoprotein or variant thereof, a Vesiculovirus Morreton strain glycoprotein or variant thereof, a Vesiculovirus Alagoas strain glycoprotein or variant thereof, a Vesiculovirus New Jersey strain glycoprotein or variant thereof, a Vesiculovirus Carajas strain glycoprotein or variant thereof, a Vesiculovirus Chandipura strain glycoprotein or variant thereof, a Vesiculovirus Eptesicus strain glycoprotein or variant thereof, a Vesiculovirus Isfahan strain glycoprotein or variant thereof, a Vesiculovirus Jurona strain glycoprotein or variant thereof, a Vesiculovirus Malpais strain glycoprotein or variant thereof, a Vesiculovirus Perinet strain glycoprotein or variant thereof, a Vesiculovirus Piry strain glycoprotein or variant thereof, a Vesiculovirus Radi strain glycoprotein or variant thereof, a Vesiculovirus Rhinolopus strain glycoprotein or variant thereof, and a Vesiculovirus Yug Bogdanovac strain glycoprotein or variant thereof.

20. The expression vector composition of claim 19, wherein, The at least a portion of the enveloped virus glycoprotein or variant thereof is at least a portion of VSV-G or a variant thereof or at least a portion of Cocal-G or a variant thereof, and the enveloped virus glycoprotein receptor is LDL-R.

21. The expression vector composition of claim 20, wherein, The first expression vector comprises a polynucleotide encoding the VSV-G variant or Cocal-G variant, the VSV-G variant or Cocal-G variant comprising a first mutation comprising at least one of the following mutations: (a) a substitution or deletion at amino acid position 8, a substitution or deletion at amino acid position 9, a substitution or deletion at amino acid position 10, a substitution or deletion at amino acid position 47, a substitution or deletion at amino acid position 50, a substitution or deletion at amino acid position 51, a substitution or deletion at amino acid position 183, a substitution or deletion at amino acid position 179, a substitution or deletion at amino acid position 180, a substitution or deletion at amino acid position 182, a substitution or deletion at amino acid position 184, a substitution or deletion at amino acid position 209, a substitution or deletion at amino acid position 347, a substitution or deletion at amino acid position 350, a substitution or deletion at amino acid position 352, a substitution or deletion at amino acid position 353, a substitution or deletion at amino acid position 354, a deletion of amino acids 1-18, a deletion of amino acids 19-36, a deletion of amino acids 37-51, a deletion of amino acids 314-384, a deletion of amino acids 321-374, a deletion of amino acids 331-364, a deletion of amino acids 344-354, a deletion of amino acids 345-353 of SEQ ID NO: 1 or SEQ ID NO: 2; and (b) a substitution or deletion at amino acid position 8, a substitution or deletion at amino acid position 9, a substitution or deletion at amino acid position 10, a substitution or deletion at amino acid position 47, a substitution or deletion at amino acid position 50, a substitution or deletion at amino acid position 51, a substitution or deletion at amino acid position 183, a substitution or deletion at amino acid position 179, a substitution or deletion at amino acid position 180, a substitution or deletion at amino acid position 182, a substitution or deletion at amino acid position 184, a substitution or deletion at amino acid position 209, a substitution or deletion at amino acid position 347, a substitution or deletion at amino acid position 350, a substitution or deletion at amino acid position 352, a substitution or deletion at amino acid position 353, a substitution or deletion at amino acid position 354, a deletion of amino acids 1-18, a deletion of amino acids 19-36, a deletion of amino acids 37-51, a deletion of amino acids 314-384, a deletion of amino acids 321-374, a deletion of amino acids 331-364, a deletion of amino acids 344-354, a deletion of amino acids 345-353 after optimal global alignment with SEQ ID NO: 1 or SEQ ID NO:

2.

22. The expression vector composition of claim 21, wherein, The first mutation comprises at least one of the following mutations: (a) a deletion of amino acids 331-364, a deletion of amino acids 344-354, a substitution or deletion of K47, a substitution or deletion of R354 of SEQ ID NO: 1 or SEQ ID NO: 2; and (b) a substitution or deletion at amino acid position 8, a substitution or deletion at amino acid position 9, a substitution or deletion at amino acid position 10, a substitution or deletion at amino acid position 47, a substitution or deletion at amino acid position 50, a substitution or deletion at amino acid position 51, a substitution or deletion at amino acid position 183, a substitution or deletion at amino acid position 179, a substitution or deletion at amino acid position 180, a substitution or deletion at amino acid position 182, a substitution or deletion at amino acid position 184, a substitution or deletion at amino acid position 209, a substitution or deletion at amino acid position 347, a substitution or deletion at amino acid position 350, a substitution or deletion at amino acid position 352, a substitution or deletion at amino acid position 353, a substitution or deletion at amino acid position 354, a deletion of amino acids 1-18, a deletion of amino acids 19-36, a deletion of amino acids 37-51, a deletion of amino acids 314-384, a deletion of amino acids 321-374, a deletion of amino acids 331-364, a deletion of amino acids 344-354, a deletion of amino acids 345-353 after optimal global alignment with SEQ ID NO: 1 or SEQ ID NO:

2. (b) upon optimal global alignment with SEQ ID NO: 1 or SEQ ID NO: 2, is located at a deletion of amino acids 331-364, a deletion of amino acids 344-354, a substitution or deletion of K47, or a substitution or deletion of R354 of SEQ ID NO: 1 or SEQ ID NO: 2; Preferably, the first mutation comprises at least one of the following mutations: (a) is located at a deletion of amino acids 331-364, a deletion of amino acids 344-354, a substitution or deletion of K47, or a substitution or deletion of R354 of SEQ ID NO: 1 or SEQ ID NO: 2; and (b) upon optimal global alignment with SEQ ID NO: 1 or SEQ ID NO: 2, is located at a deletion of amino acids 331-364, a deletion of amino acids 344-354, a substitution or deletion of K47, or a substitution or deletion of R354 of SEQ ID NO: 1 or SEQ ID NO:

2.

23. The expression vector composition of claim 22, wherein, The first mutation comprises a mutation at: (a) a deletion of K47 of SEQ ID NO: 1 or SEQ ID NO: 2; or (b) upon optimal global alignment with SEQ ID NO: 1 or SEQ ID NO: 2, is located at a deletion of K47 of SEQ ID NO: 1 or SEQ ID NO:

2.

24. The expression vector composition of any one of claims 18-23, wherein, The first expression vector comprises a polynucleotide encoding the envelope virus glycoprotein variant, the envelope virus glycoprotein variant comprising a second mutation that enhances or does not inactivate the ability of the envelope virus glycoprotein variant to antagonize complement inactivation.

25. The expression vector composition of claim 24, wherein, The second mutation comprises a mutation at at least one of the following positions: (a) is located at amino acid 214 of SEQ ID NO: 1 or SEQ ID NO: 2; (b) upon optimal global alignment with SEQ ID NO: 1 or SEQ ID NO: 2, is located at amino acid 214 of SEQ ID NO: 1 or SEQ ID NO: 2; (c) is located at amino acid 352 of SEQ ID NO: 1 or SEQ ID NO: 2; (d) upon optimal global alignment with SEQ ID NO: 1 or SEQ ID NO: 2, is located at amino acid 352 of SEQ ID NO: 1 or SEQ ID NO: 2; (e) is located at amino acid 50 of SEQ ID NO: 1 or SEQ ID NO: 2; (f) upon optimal global alignment with SEQ ID NO: 1 or SEQ ID NO: 2, is located at amino acid 50 of SEQ ID NO: 1 or SEQ ID NO: 2; (g) is located at amino acid 146 of SEQ ID NO: 1 or SEQ ID NO: 2; and (h) is located at amino acid 214 of SEQ ID NO: 1 or SEQ ID NO:

2. (h) upon a global optimal alignment with SEQ ID NO: 1 or SEQ ID NO: 2, is at a position equivalent to amino acid 146 of SEQ ID NO: 1 or SEQ ID NO: 2; Preferably, the mutation is selected from the group consisting of a substitution, a deletion, and an insertion of an amino acid; More preferably, the mutation is a substitution of an amino acid.

26. The expression vector composition of claim 25, wherein, The second mutation comprises a mutation at at least one of the following positions: (a) T214N, T352A, K50T, S146T of SEQ ID NO: 1 ; (b) upon a global optimal alignment with SEQ ID NO: 1, is at a position equivalent to T214N, T352A, K50T, S146T of SEQ ID NO: 1 ; (c) K214N, T352A, K50T, S146T of SEQ ID NO: 2; and (d) upon a global optimal alignment with SEQ ID NO: 2, is at a position equivalent to K214N, T352A, K50T, S146T of SEQ ID NO:

2.

27. The expression vector composition of claim 26, wherein, The second mutation comprises a combination of mutations at any one of the following positions: (a) (i) T214N and T352A; or (ii) T214N, T352A, K50T, and S146T of SEQ ID NO: 1 ; (b) upon a global optimal alignment with SEQ ID NO: 1, is at a position equivalent to (i) T214N and T352A; or (ii) T214N, T352A, K50T, and S146T of SEQ ID NO: 1 ; (c) (i) K214N and T352A; or (ii) K214N, T352A, K50T, and S146T of SEQ ID NO: 2; and (d) upon a global optimal alignment with SEQ ID NO: 2, is at a position equivalent to (i) K214N and T352A; or (ii) K214N, T352A, K50T, and S146T of SEQ ID NO:

2.

28. The expression vector composition of claim 18, wherein, The first expression vector comprises a polynucleotide encoding the envelope virus glycoprotein variant, the envelope virus glycoprotein variant comprising the amino acid sequence of any one of SEQ ID NOs: 3-20, the envelope virus glycoprotein receptor is LDL-R.

29. The expression vector composition of claim 18, wherein, The first expression vector comprises a polynucleotide encoding at least a portion of the envelope virus glycoprotein, the at least a portion of the envelope virus glycoprotein comprising a truncated envelope virus glycoprotein; Preferably, the truncated envelope virus glycoprotein is a truncated VSV-G or a truncated Cocal-G, the envelope virus glycoprotein receptor is LDL-R; more preferably, the truncated envelope virus glycoprotein comprises an amino acid sequence as set forth in SEQ ID NO: 21 or 22 or an amino acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence as set forth in SEQ ID NO: 21 or 22.

30. The expression vector composition of claim 18, wherein, at least a portion of the envelope virus glycoprotein or the variant thereof is at least a portion of a glycoprotein G of a Nipah virus or a variant thereof; Preferably, the first expression vector comprises a polynucleotide encoding a NiV-G variant, the envelope virus glycoprotein receptor is ephrin-B2 or B3, the NiV-G variant comprises at least one mutation at the following positions: at position 501 amino acid is replaced by alanine from glutamic acid (E501A); (b) at position 504 amino acid is replaced by alanine from tryptophan (W504A); (c) at position 530 amino acid is replaced by alanine A from glutamine Q (Q530A); and (d) at position 533 amino acid is replaced by alanine A from glutamic acid E (E533A); More preferably, the NiV-G variant comprises an amino acid sequence as set forth in SEQ ID NO: 24; SEQ ID NO: 24 comprises E501A, W504A, Q530A and E533A relative to SEQ ID NO:

23. The second expression vector further comprises an exogenous transgene, the exogenous transgene comprises a polynucleotide encoding a protein of interest (GOI); preferably, the protein of interest is selected from at least one of a hydrophilic protein and a hydrophobic protein.

31. The expression vector composition of any one of claims 18-30, wherein, The exogenous transgene further comprises a polynucleotide encoding a transmembrane domain, the protein of interest is directly or indirectly linked to the transmembrane domain; 32. The expression vector composition of claim 31, wherein, Preferably, the transmembrane domain is selected from the transmembrane domain of the following proteins: CD28, CD2, CD4, CD8a, CD5, CD3e, CD3d, CD3z, CD9, CD16, CD22, CD25, CD27, CD33, CD37, CD40, CD45, CD64, CD79A, CD79B, CD80, CD86, CD95 (Fas), CD134 (OX40), CD137 (4-1BB), CD150 (SLAMF1), CD152 (CTLA4), CD154 (CD40L), CD200R, CD223 (LAG3), CD270 (HVEM), CD272 (BTLA), CD273 (PD-L2), CD274 (PD-L1), CD278 (ICOS), CD279 (PD-1), CD300, CD357 (GITR), A2aR, DAP10, FcRa, FcRp, FcRy, Fyn, GAL9, KIR, Lck, LAT, LRP, NKG2D, NOTCH1, NOTCH2, NOTCH3, NOTCH4, PTCH2, ROR2, Ryk, Slp76, SIRPa, pTa, TCRa, TCRp, TIM3, TRIM, LPA5, and Zap70; More preferably, the transmembrane domain comprises the transmembrane domain of CD8a. ​ 33. The expression vector composition of claim 32, wherein, the exogenous transgene further comprises a polynucleotide encoding a linker domain through which the protein of interest is indirectly linked to the transmembrane domain; Preferably, the linker domain is selected from the group consisting of: (a) an immunoglobulin hinge region selected from the group consisting of wild-type or modified IgGl, IgG2, IgG3, IgG4, IgA, and IgD hinge regions; (b) a hinge region selected from the group consisting of wild-type or modified hinge regions of CD28, CD7, CD8, CD8a, CD8b, CD3, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD134, CD137, ICOS, and CD154; (c) all or a portion of an Fc domain selected from the group consisting of one or more of a CHI domain, a CH2 domain, and a CH3 domain; and (d) a stalk region of a type II C-lectin selected from the group consisting of a stalk region of CD23, CD69, CD72, CD94, NKG2A, and NKG2D; More preferably, the linker domain comprises a hinge region of CD8a.

34. The expression vector composition of any one of claims 18-33, wherein, The first expression vector and the second expression vector are plasmids; The first expression vector comprises an envelope plasmid in a lentiviral vector packaging system.

35. The expression vector composition of any one of claims 31-34, wherein, The second expression vector comprises a transfer plasmid (Transfer Plasimid) in a lentiviral vector packaging system, the transfer plasmid comprising the genetic integration element and the GOI; Preferably, the genetic integration element comprises a lentiviral backbone gene.

36. The expression vector composition of any one of claims 18-35, wherein, The extracellular vesicle is selected from at least one of the group consisting of an exosome and a microvesicle; Preferably, the extracellular vesicle is an exosome.

37. A producer cell, wherein, The producer cell comprises the expression vector composition of any one of claims 18-36; Preferably, the producer cell is selected from the group consisting of a CHO cell, a BHK cell, an MDCK cell, a C3H-10T1 / 2 cell, a FLY cell, a Psi-2 cell, a BOSC 23 cell, a PA317 cell, a WEHI cell, a COS cell, a BSC-1 cell, a BSC-40 cell, a BMT-10 cell, a VERO cell, a W138 cell, a MRC5 cell, an A549 cell, an HT1080 cell, a HEK-293 cell, a B-50 cell, a 3T3 cell, a NIH3T3 cell, a HepG2 cell, a Saos-2 cell, a Huh7 cell, a HeLa cell, a W163 cell, and a 211 cell; More preferably, the producer cell is a HEK-293T cell.

38. A method of preparing an extracellular vesicle, characterized by, The method comprises culturing the producer cell of claim 37, and collecting the extracellular vesicle produced by the producer cell.

39. An extracellular vesicle, characterized in that, The extracellular vesicle is prepared by the method of claim 38.

40. Use of the extracellular vesicle of any one of claims 1-17, the expression vector composition of any one of claims 18-36, the producer cell of claim 37, or the extracellular vesicle of claim 39 in expressing and / or transporting a protein of interest; preferably, the protein of interest is selected from at least one of a hydrophobic protein and a hydrophilic protein.

41. A method of expressing and / or transporting a protein of interest, comprising, The method comprises expressing the protein of interest on the surface of the extracellular vesicle; Preferably, the protein of interest is selected from at least one of a hydrophobic protein and a hydrophilic protein; Preferably, the method comprises transfecting the expression vector composition of any one of claims 32-36 into a producer cell, culturing the producer cell, and collecting the extracellular vesicle expressing the protein of interest.

42. Use of the extracellular vesicle of any one of claims 1-17, the expression vector composition of any one of claims 18-36, the producer cell of claim 37, or the extracellular vesicle of claim 39 in the manufacture of a medicament.

43. A composition comprising, The composition comprises a pharmaceutically acceptable carrier or excipient and any one of the following: the extracellular vesicle of any one of claims 1-17, the expression vector composition of any one of claims 18-36, or the extracellular vesicle of claim 39.

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