Engineered extracellular vesicles

Abstract

The present invention provides an engineered extracellular vesicle, a pharmaceutical composition comprising the engineered extracellular vesicle, and uses thereof, and also provides a method for constructing the engineered extracellular vesicle. The engineered extracellular vesicle can be used to encapsulate a drug in the vesicle for delivery.

Description

[0001] Join by reference

[0002] All publications, patents, and patent applications cited in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application were specifically set forth and individually incorporated herein by reference in its entirety. Technical Field

[0003] The present invention relates to the field of biomedicine, and in particular to an engineered extracellular vesicle, a preparation method thereof, and an application thereof. Background Art

[0004] Extracellular vesicles (EVs) are small, membrane-bound vesicles secreted by cells that can be taken up by recipient cells. They range in diameter from approximately 30 to 1000 nm. EVs act as vehicles for the intercellular transport of biomacromolecules such as proteins, RNA, and lipids, and are crucial mediators of cell-to-cell communication. Because EVs are widely present in various bodily fluids, they can serve as a sample source for liquid biopsies and are also considered a naturally occurring drug carrier.

[0005] EVs can serve as drug delivery vehicles to carry and transport therapeutically active peptides and proteins. For EV-carried cargo proteins to be effective, sufficient levels of the cargo proteins within the EVs are essential. However, non-exosomal cargo proteins heterologously expressed in cells are not actively sorted into extracellular vesicles.

[0006] According to existing reports, approximately 20 proteins have been reported to be used for loading cargo proteins into EVs (Theranostics, 2019; 9(4): 1015-1028). Patent applications such as WO2013084000A2, WO2014168548A2, WO2018015535A1, and WO2019040920A1 also describe methods for loading proteins into EVs. Summary of the Invention

[0007] The inventors of the present application unexpectedly discovered during their research that a polypeptide can carry and enrich the cargo protein fused with it into extracellular vesicles, and named it as the internalizing polypeptide. On this basis, the present invention was completed.

[0008] In its first aspect, the present invention provides a fusion protein comprising a cargo polypeptide and a cargo protein, wherein the cargo polypeptide is located at the N-terminus of the cargo protein and comprises one or more myristoylation sites and one or more palmitoylation sites. Myristoylation is a modification of eukaryotic proteins that typically occurs at the glycine residue at the N-terminus of the polypeptide chain. Palmitoylation is also a modification of eukaryotic proteins, with the most common forms of palmitoylation being S-palmitoylation and N-palmitoylation. S-palmitoylation occurs at cysteine residues, while N-palmitoylation occurs at the amino terminus or at the epsilon amino group of a lysine.

[0009] In some embodiments, the myristoylation site and the palmitoylation site are separated by 0 to 6 amino acids, eg, by 0, 1, 2, 3, 4, 5, or 6 amino acids.

[0010] In other embodiments, the loaded polypeptide comprises a fragment represented by formula (I),

[0011] MGX1 m CX2 n (I), where

[0012] The length of the fragment of formula (I) is not less than 10 amino acids, and X1 and X2 are each independently any amino acid;

[0013] m is an integer from 0 to 6, for example, 0, 1, 2, 3, 4, 5 or 6;

[0014] n is a positive integer, for example, an integer from 1 to 300, 2 to 200, 3 to 100, 4 to 80, 5 to 60, or 7 to 30;

[0015] Wherein, when m is greater than 1, the at least two X1s are each independently any amino acid; when n is greater than 1, the at least two X2s are each independently any amino acid.

[0016] In other embodiments, the loaded polypeptide comprises a fragment represented by formula (II),

[0017] MGX1 m CX3X4X5X6X7X8X9(II), where

[0018] m is an integer from 0 to 3, for example, 0, 1, 2 or 3;

[0019] X3 is selected from R, T, L and F, X4 is selected from Q, V, L, G, Y and P, X5 is selected from S, N, G and C, X6 is selected from S, A, N and P, X7 is selected from E, K, S, N and G, X8 is selected from E, D, T, K and M, and X9 is selected from K, R, E, T and S.

[0020] In other embodiments, the loaded polypeptide is selected from the following sequences:

[0021]

[0022]

[0023] In some preferred embodiments, the loaded polypeptide is selected from the following sequences:

[0024] GNAZ(1-10):MGCRQSSEEK(SEQ ID NO:12),

[0025] GNAI3(1-10):MGCTLSAEDK (SEQ ID NO: 11), and

[0026] SVIP(1-10):MGLCFPCPGE(SEQ ID NO:9).

[0027] In other embodiments, the loaded polypeptide comprises the first 10, first 15, first 20, first 25, first 32, first 42, or first 50 amino acids from the N-terminus of GNAZ, GNAI1, GNAI2, GNAI3, GNAO, GNAS1, GNAS2, GNAL, or SVIP. As used herein, the "first 10 amino acids from the N-terminus" refers to the first to tenth amino acids from the N-terminus; the "first 15 amino acids from the N-terminus" refers to the first to fifteenth amino acids from the N-terminus; and so on. GNAZ, GNAI1, GNAI2, GNAI3, GNAO, GNAS1, GNAS2, and GNAL are all members of the G-alpha family.

[0028] In other embodiments, the loaded polypeptide comprises a polypeptide segment carrying positively charged amino acid residues, wherein the polypeptide segment carrying positively charged amino acid residues forms an α-helix, with at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of the positively charged amino acid residues located on the same side of the α-helix. The term "α-helix" as used herein has the meaning understood by those skilled in the art and is a common protein secondary structure in which the main chain of the polypeptide chain spirals regularly around a central axis, with one turn every 3.6 amino acid residues, resulting in a very stable structure. The α-helix comprised in the loaded polypeptide of the present invention refers to a complete α-helix or a portion of an α-helix (α-helix fragment). The phrase "at least 70%, at least 80%, at least 90%, or at least 95% of the positively charged amino acid residues located on the same side of the α-helix" herein means that, on a longitudinal section through the central axis of the α-helix, at least 70%, at least 80%, at least 90%, or at least 95% of the positively charged amino acid residues of the α-helical peptide segment are located on one side of the section.

[0029] In some embodiments, the loaded polypeptide comprises a sequence selected from the group consisting of:

[0030] GNAZ(1-32):MGCRQSSEEKEAARRSRRIDRHLRSESQRQRR(SEQ ID NO:1),

[0031] GNAI1(1-32):MGCTLSAEDKAAVERSKMIDRNLREDGEKAAR(SEQ ID NO:2),

[0032] GNAI2(1-32): MGCTVSAEDKAAAERSKMIDKNLREDGEKAAR(SEQ ID NO:3),

[0033] GNAI3(1-32):MGCTLSAEDKAAVERSKMIDRNLREDGEKAAK(SEQ ID NO:4),

[0034] GNAO(1-32):MGCTLSAEERAALERSKAIEKNLKEDGISAAK(SEQ ID NO:20),

[0035] GNAS2(1-32):MGCLGNSKTEDQRNEEKAQREANKKIEKQLQK(SEQ ID NO:21), and

[0036] GNAL(1-32):MGCLGGNSKTTEDQGVDEKERREANKKIEKQL (SEQ ID NO: 22).

[0037] In some preferred embodiments, the loaded polypeptide is selected from the following sequences:

[0038] GNAZ(1-32):MGCRQSSEEKEAARRSRRIDRHLRSESQRQRR(SEQ ID NO:1),

[0039] GNAI1(1-32):MGCTLSAEDKAAVERSKMIDRNLREDGEKAAR(SEQ ID NO:2),

[0040] GNAI2(1-32): MGCTVSAEDKAAAERSKMIDKNLREDGEKAAR(SEQ ID NO:3),

[0041] GNAI3(1-32):MGCTLSAEDKAAVERSKMIDRNLREDGEKAAK (SEQ ID NO:4), and

[0042] GNAS2(1-32):MGCLGNSKTEDQRNEEKAQREANKKIEKQLQK (SEQ ID NO:21).

[0043] In some embodiments, the cargo protein is a therapeutic peptide, a DNA binding protein, an RNA binding protein, a fluorescent protein, an enzyme, or a linker to a therapeutic compound.

[0044] In some specific embodiments, the therapeutic peptide is an antibody and / or a cytokine, for example, the cytokine is selected from a member of the human interleukin family (e.g., IL-2, IL-7, IL-10, IL-11, IL-12, IL-15 and IL-23), a member of the tumor necrosis factor family (e.g., TNF, LTA, LTB, FASLG, TNFSF8, TNFSF9, TNFSF10, TNFSF11, TNFSF12, TNFSF13, TNFSF14, TNFSF15, TNFSF18 and EDA), an interferon (INF-α, INF-β and INF-γ), a T cell Engagers (e.g., 4-1BB, OX40, CD28, CD40, CD40L, CD47, CD27, CD70, CD80, CD86, GITRL, ICOSL, CD155, CD112, TIM-3, BTLA), and other cytokines (e.g., G-CSF, EPO, TPO, GM-CSF, EGF, bFGF, FVIIa, ATIII, TNK, α-Glucosidase, BMP-2, hirudin).

[0045] In other specific embodiments, the RNA binding protein is selected from L7Ae, hnRNPA2B1, hnRNPC1, hnRNPG, hnRNPK, hnRNPQ, YBX1, HuR, AGO2, IGF2BP1, MEX3C, ANXA2, ALIX, NCL, FUS and MVP.

[0046] In other specific embodiments, the enzyme is a luciferase, such as firefly luciferase, Renilla luciferase, Luciferase, etc.

[0047] In a second aspect, the present invention provides an engineered extracellular vesicle comprising the fusion protein of the first aspect.

[0048] In some embodiments, the extracellular vesicle comprises two or more fusion proteins, for example, the two or more fusion proteins each independently comprise a different cargo protein.

[0049] In a third aspect, the present invention provides a pharmaceutical composition comprising the fusion protein of the first aspect or the extracellular vesicle of the second aspect and a pharmaceutically acceptable carrier.

[0050] In a fourth aspect, the present invention provides an engineered cell for producing the extracellular vesicles described in the second aspect.

[0051] In some embodiments, the cell comprises a polynucleotide encoding the fusion protein, preferably, the polynucleotide is integrated into the genome of the cell.

[0052] In a fifth aspect, the present invention provides a method for treating a disease, comprising administering the fusion protein of the first aspect, the extracellular vesicle of the second aspect, the pharmaceutical composition of the third aspect, or the cell of the fourth aspect to a subject in need thereof. The present invention also provides use of the fusion protein of the first aspect, the extracellular vesicle of the second aspect, the pharmaceutical composition of the third aspect, or the cell of the fourth aspect in the preparation of a medicament for treating a disease.

[0053] In a sixth aspect, the present invention provides an extracellular vesicle imaging method, comprising loading the fusion protein of the first aspect into the extracellular vesicle, wherein the fusion protein is a fluorescent protein or luciferase, preferably, the fluorescent protein is selected from GFP (Green fluorescent protein), RFP (Red fluorescent protein), YFP (Yellow fluorescent protein) and CP (Chromoprotein); preferably, the luciferase is selected from firefly luciferase, Renilla luciferase and Luciferase.

[0054] In a seventh aspect, the present invention provides a method for preparing the extracellular vesicles described in the second aspect, comprising:

[0055] 1) providing a cell comprising a polynucleotide encoding the fusion protein;

[0056] 2) culturing the cells under conditions suitable for expressing the fusion protein; and

[0057] 3) Isolating the extracellular vesicles. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0059] Figure 1 shows the levels of eGFP fusion protein in total cell proteins and extracellular vesicles in Example 1, wherein Figure 1A Shows the WB test results, Figure 1B The statistical results are shown based on total cell protein.

[0060] Figure 2 The statistical results of the number of eGFP-positive EVs detected by nanoflow cytometry in Example 1 are shown.

[0061] Figure 3 shows the levels of eGFP fusion protein in total cell proteins and extracellular vesicles in Example 2, wherein Figure 3A Shows the WB test results, Figure 3B Nanoflow cytometry results are shown.

[0062] Figure 4 Shows amino acid sequence alignment.

[0063] Figure 5 Schematic diagram showing the distribution of positively charged amino acids at the N-terminus of GNAZ and GNAI3, where K, R, and H are positively charged amino acids.

[0064] Figure 6 The numbers of positively charged amino acids in each GNAZ truncation are shown. The shaded amino acids are the positively charged amino acids R and K.

[0065] Figure 7 The levels of the fusion proteins of various GNAZ truncations and eGFP in total cell proteins and extracellular vesicles were detected by WB in Example 4.

[0066] Figure 8 The statistical results of the number of eGFP-positive EVs detected by ELISA in Example 4 are shown.

[0067] Figure 9 The levels of GNAI3 (1-32) mutant and eGFP fusion protein in total cell proteins and extracellular vesicles were detected by WB in Example 5.

[0068] Figure 10 The statistical results of the number of eGFP-positive EVs detected by nanoflow cytometry in Example 5 are shown.

[0069] Figure 11The luciferase activity in Example 6 is shown.

[0070] Figure 12 Example 7 shows that the RNA-binding protein L7AE sorts mRNA into EVs.

[0071] Figure 13 The levels of SVIP fragment and eGFP fusion protein in total cell protein and extracellular vesicles were detected by WB in Example 8.

[0072] Figure 14 The levels of SVIP, GNA13, and GNAI3 fragments and eGFP fusion proteins in total cell proteins and extracellular vesicles were detected by WB in Example 9. DETAILED DESCRIPTION

[0073] definition

[0074] Unless otherwise specified, terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which they belong.

[0075] The term "extracellular vesicles (EVs)" used in this article refers to vesicles with a double-layer membrane structure that are shed from the cell membrane or secreted by cells, with diameters ranging from 40nm to 1000nm. The main forms are microvesicles (MVs) and exosomes (Exs). Extracellular vesicles are widely present in cell culture supernatants and various body fluids (blood, lymph, saliva, urine, semen, and milk). They carry a variety of proteins, lipids, DNA, mRNA, miRNA, etc. related to cell origin and participate in processes such as intercellular communication, cell migration, angiogenesis, and immune regulation. The term "engineered extracellular vesicles" refers to artificially synthesized extracellular vesicles, or extracellular vesicles produced by cells after human intervention, or extracellular vesicles produced by cells after genetic engineering. The term "unengineered extracellular vesicles" refers to extracellular vesicles secreted by natural, unmodified cells under normal conditions (such as physiological conditions).

[0076] The protein referred to herein includes its variants. A "variant" of a protein refers to a protein whose amino acid sequence is different from the amino acid sequence of the corresponding naturally occurring protein (sometimes also called wild type) but has the same or similar function as the wild type protein. A variant of a protein can be derived from a naturally occurring protein by deletion, addition and / or substitution of amino acids. The variant can also be a truncated form of a natural protein (sometimes also called a fragment thereof). Herein, "protein", "polypeptide" and "peptide" are used interchangeably.

[0077] As used herein, the term "cargo polypeptide" refers to a polypeptide that can carry a fused protein into an extracellular vesicle. The fusion protein is sometimes also referred to as luminal cargo. The term "cargo proteins" as used herein refers to proteins carried within an extracellular vesicle. Proteins carried by the cargo polypeptide include, but are not limited to, therapeutic peptides, DNA-binding proteins, RNA-binding proteins, fluorescent proteins, and the like.

[0078] As used herein, the term "exogenous sequence" refers to a sequence that is not naturally present in a cell but is artificially constructed. The amino acids herein may be natural or non-natural amino acids, represented by three letters or single letters known in the art, such as alanine (Ala or A), arginine (Arg or R), asparagine (Asn or N), aspartic acid (Asp or D), cysteine (Cys or C), glutamine (Gln or Q), glutamic acid (Glu or E), glycine (Gly or G), histidine (His or H), isoleucine (Ile or I), leucine (Leu or L), lysine (Lys or K), methionine (Met or M), phenylalanine (Phe or F), proline (Pro or P), serine (Ser or S), threonine (Thr or T), tryptophan (Trp or W), tyrosine (Tyr or Y), and valine (Val or V). "Similar amino acids" refer to amino acids with similar structures and properties. When these amino acids are replaced with each other, they often have little effect on the structure and properties of the protein and can be used interchangeably.

[0079] The term "therapeutic peptide" as used herein refers to a protein or variant thereof having therapeutic activity, including but not limited to antibodies or antigen-binding fragments thereof, receptors, ligands, cytokines, hormones, etc. In some embodiments of the present invention, the therapeutic peptide can be a member of the human interleukin family (e.g., IL-2, IL-7, IL-10, IL-11, IL-12, IL-15 and IL-23), a member of the tumor necrosis factor family (e.g., TNF, LTA, LTB, FASLG, TNFSF8, TNFSF9, TNFSF10, TNFSF11, TNFSF12, TNFSF13, TNFSF14, TNFSF15, TNFSF18 and EDA), an interferon (INF-α, INF-β and INF-γ), a T cell Engagers (e.g., 4-1BB, OX40, CD28, CD40, CD40L, CD47, CD27, CD70, CD80, CD86, GITRL, ICOSL, CD155, CD112, TIM-3, BTLA), and other cytokines (e.g., G-CSF, EPO, TPO, GM-CSF, EGF, bFGF, FVIIa, ATIII, TNK, α-Glucosidase, BMP-2, hirudin).

[0080] The term "targeting peptide" as used herein refers to a polypeptide that can be recognized and bound by a specific cell, that is, a polypeptide that can target a specific cell. In some embodiments of the present invention, the targeting peptide is a cell-targeting polypeptide, which can be an antibody or an antigen-binding fragment thereof, a cell surface receptor, or a ligand.

[0081] The term "affinity tag" as used herein refers to a short polypeptide that can bind to a corresponding affinity agent and is commonly used for protein separation and purification. In some embodiments of the present invention, the affinity tag can be a His tag, glutathione S-transferase (GST), S-peptide, ZZ domain, albumin binding domain (ABD), HA, Myc, FLAG™, maltose binding protein (MBP), calmodulin binding peptide (CBP), SUMO, Streptococcal protein G (Protein G) and Staphylococcus aureus protein A (Protein A).

[0082] As used herein, the term "linker for attaching a therapeutic compound" refers to a substance that attaches a therapeutic compound to a fusion partner.

[0083] As used herein, the term "pharmaceutically acceptable carrier" can be selected from the group consisting of water, aqueous buffered solutions, isotonic saline solutions such as PBS (phosphate buffered saline), glucose, mannitol, dextrose, lactose, starch, magnesium stearate, cellulose, magnesium carbonate, 0.3% glycerol, hyaluronic acid, ethanol, or polyalkylene glycols such as polypropylene glycol, triglycerides, and the like. The type of pharmaceutically acceptable carrier used depends, inter alia, on whether the composition according to the present invention is formulated for oral, nasal, intradermal, subcutaneous, intramuscular, or intravenous administration. The composition according to the present invention may contain lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring substances, flavoring substances, and / or aromatic substances as additives.

[0084] As used herein, the term "subject" refers to a mammal, such as a human, but may also be other animals, such as wild animals (e.g., herons, storks, cranes, etc.), domestic animals (e.g., ducks, geese, etc.), or experimental animals (e.g., gorillas, monkeys, rats, mice, rabbits, guinea pigs, woodchucks, ground squirrels, etc.).

[0085] The term "about" or "approximately" generally means within a range of error for a particular value as determined by one of ordinary skill, which depends in part on the manner in which the measurements are made, i.e., on the limitations of the measurement system. For example, "about" can refer to one or more standard deviations, depending on the practice in a particular field. In particular, "about" can refer to values that are within 20%, 10%, 5%, or 1% of a given value.

[0086] Although the numerical ranges and parameter approximations shown in the broad scope of the present invention, the numerical values shown in the specific embodiments are recorded as accurately as possible. However, any numerical value is necessarily contained in a certain error, which is caused by the standard deviation present in their respective measurements. In addition, all ranges disclosed herein should be understood to cover any and all sub-ranges contained therein. For example, a range of "1 to 10" should be considered to include any and all sub-ranges between a minimum value of 1 and a maximum value of 10 (including endpoints); that is, all sub-ranges starting with a minimum value of 1 or greater, such as 1 to 6.1, and sub-ranges ending with a maximum value of 10 or less, such as 5.5 to 10.

[0087] As used herein, unless otherwise expressly stated, a noun is not preceded by an article or is modified by "the" to indicate that the term can be one or more. It should also be noted that as used in this specification, a singular form includes a plural form of its referent unless it is clearly and unequivocally limited to one referent.

[0088] Terms such as "comprises," "comprising," "including," and "include" as used herein are not intended to be limiting. Furthermore, "or," "alternatively," means "and / or" unless otherwise stated.

[0089] Other aspects and advantages of the present invention will be readily apparent to those skilled in the art based on the detailed description below. It should be noted that the following detailed description (including the accompanying drawings) is intended to be illustrative only and not limiting of the present invention. It will be readily understood that the present invention may be implemented in various other embodiments by adjusting and varying certain specific details, without departing from the present invention.

[0090] The following is a clear and complete description of the technical solution in conjunction with the embodiments of the present invention. Obviously, the described embodiments are merely illustrative and not an exhaustive list of all possible implementations of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts are within the scope of protection of the present invention.

[0091] Example

[0092] Materials and methods

[0093] The materials used in the examples of the present invention and their sources are shown in Table 1.

[0094] Table 1

[0095]

[0096]

[0097] Experimental methods

[0098] 1. Cell Transfection

[0099] 1) Cell preparation

[0100] On the day of transfection, the suspension cultured Expi293 F cells were counted and the cell density was controlled at 2-5×10 6 / ml, viability>95%; dilute cells to 2×10 6 / ml, final volume 30ml.

[0101] 2) Transfection

[0102] Prepare Solution A: Pipette 30 μg of plasmid into a 15 ml centrifuge tube containing 1 ml of Opti-MEM, gently pipette to mix, and let stand for 5 minutes.

[0103] Prepare Solution B: Use a pipette to draw 60 μl of PEI (working solution concentration 1 μg / ul) into a 15 ml centrifuge tube containing 1 ml of Opti-MEM, gently pipette to mix, and let it stand for 5 min;

[0104] Use a 1ml pipette to add solution B to solution A, mix gently, and let it stand at room temperature for 20 minutes;

[0105] Add the mixed transfection reagent dropwise to the Expi293 F cells while gently shaking.

[0106] 3) Add liquid after transfection

[0107] 4-6 hours after transfection, sterilize the cell flask with 75% alcohol and transfer it into the biosafety cabinet;

[0108] Use a 100 μl pipette to draw 30 μl of Anti-clumping into the shake flask;

[0109] The suspension culture was carried out in an incubator set at 37°C, 5% or 8% CO2, and 110 rpm.

[0110] 4) Add KT Feed

[0111] 22-24 hours after transfection, sterilize the cell flask with 75% ethanol and transfer it into the biosafety cabinet. Add 0.6 ml of KTfeed

[0112] and 2 g / L glucose to the cell culture;

[0113] The incubator was set to 37°C, 5% or 8% CO2, and 110 rpm according to the Standard Operating Procedure for Use, Cleaning, and Maintenance of the 00-ZCZY-AS8V Shaking Incubator (SOP-EQ-082).

[0114] 5) Cell Harvest

[0115] Cells were harvested after 4 days of culture;

[0116] The cell suspension was transferred to a 50 ml sterile centrifuge tube and centrifuged at 800 × g at 4 °C for 10 min to separate the cells and the supernatant;

[0117] The cell supernatant was transferred to a new 50 ml sterile centrifuge tube and centrifuged at 4000 × g at 4°C for 10 min.

[0118] The supernatant was taken, sterilized by filtration using a 0.22 μm filter membrane, and the cell supernatant was transferred to a sterile 50 ml centrifuge tube for use.

[0119] 2. EV Extraction

[0120] The cell supernatant was added to an ultracentrifuge tube and centrifuged at 16,000 × g for 2 h at 4°C. The supernatant was collected.

[0121] Transfer the supernatant obtained in the previous step to a clean ultracentrifuge tube, centrifuge at 100,000 × g for 2 h at 4°C, and discard the supernatant;

[0122] After centrifugation, the EV pellet was resuspended in PBS;

[0123] The resuspended EVs were added to a 100 kD ultrafiltration tube and centrifuged at 4000 × g for 10 min. EVs were collected into a 1.5 mL centrifuge tube and total protein concentration was determined by BCA assay.

[0124] 3. Western blotting (WB)

[0125] Take EV with 10μg of total protein or cell lysate with 10μg of total protein, add appropriate amount of 5× loading buffer to the final concentration of 1× loading buffer, and heat at 95℃ for 10min. Prepare running buffer (1L) according to the following formula: 100mL 10× running buffer (Kangwei Century) + 900mL ddH2O. Mix gently and set aside. Install the precast gel (Biyuntian) of appropriate concentration in the running gel tank. After confirming that the assembled running gel tank is leak-proof, pour the prepared running buffer into the running gel tank so that the running buffer covers the gel surface. Use a pipette to take an appropriate amount of heat-denatured sample and carefully add it to the gel well of the SDS-PAGE gel (be careful not to use too much force when loading the sample to avoid contamination of the sample in one well by the sample in another well). After correctly connecting the running gel tank power supply, run the gel at 80V voltage. After the sample enters the separation gel, adjust the voltage to 120V and continue running the gel. Stop after the protein is separated.

[0126] Take a nitrocellulose membrane (NC membrane) of appropriate size and soak it in methanol for at least 20 seconds. Then, rinse it with an appropriate amount of pre-chilled trans buffer and set aside. (Prepare trans buffer as follows: 100 mL 10× trans buffer (Conway Century) + 200 mL methanol + 700 mL ddH2O. Pre-chill.) Remove the run gel and soak it in pre-chilled trans buffer. Assemble the sandwich in the following order: black side - cotton - filter paper - gel - NC membrane - filter paper - cotton - white side. Then, place the sandwich in the transfer chamber in the following order: black side - black side, white side - white side. Add two ice cubes that have been frozen overnight at -80°C to the transfer chamber. Pour transfer buffer into the transfer chamber to soak the entire sandwich. Install the transfer chamber and transfer the membrane at a constant current of 350 mA for 1 hour. Blocking: Prepare 5% BSA blocking solution (dissolved in 1× TBST). Remove the transferred membrane and place it in the blocking solution. Block for 1-2 hours at room temperature on a horizontal shaker. Primary antibody incubation: After blocking, wash three times with 1×TBST, prepare the primary antibody solution in TBST according to the proportion, and incubate for the appropriate time. Secondary antibody incubation: After blocking, wash three times with 1×TBST, prepare the secondary antibody solution in TBST according to the proportion, and incubate for the appropriate time. ECL chemiluminescence color development: After the secondary antibody incubation, wash three times with 1×TBST. Before color development, mix the ECL chemiluminescence solution AB in a 1:1 ratio and develop the membrane on the Tianneng chemiluminescence gel imaging system Tanon-5200Multi.

[0127] 4. Quantitative detection of cargo protein (fusion protein), ELISA

[0128] Take 1.2μg of EV total protein and add it to 250μl of RIPA lysis buffer (containing protease inhibitors). Mix thoroughly and lyse at 4℃ for 1h. Centrifuge at low speed to remove insoluble matter. 30min before the ELISA experiment, remove the GFP ELISA kit (AbCAM, ab171581) and let it equilibrate at room temperature. Prepare standards of different concentrations according to the instructions and dilute the samples with the diluent provided by the kit. Incubate the samples at 37℃ for 90min, then wash them four times with a plate washer. Add 100μl of antibody and incubate at 37℃ for 60min. Wash the plate four times with a plate washer, then add 100μl of enzyme conjugate and incubate at 37℃ for 30min. After washing four times with a plate washer, color development is carried out for 15min. Add stop solution and read using an enzyme reader.

[0129] 5. EV concentration measurement (nanoflow cytometry)

[0130] The concentration and size of EVs were detected using a Flow NanoAnalyzer N30E (NanoFCM Inc., Xiamen, China) equipped with a 488 nm laser. The instrument was first calibrated with quality control microspheres (250 nm SiNPs) and mixed microspheres (68-155S16-Exo) for identification. Then, EVs were diluted to 1 × 10 8 ~6×10 8 The concentration range of EV particles / mL (total particle count detected was 2000–12000 particles / min) was used, and sample data were collected with an acquisition time of 60 s. The concentration and size distribution of EVs were analyzed using Flow NanoAnalyzer software (NF Profession version 2.0).

[0131] Example 1: The N-terminal fragment of GNAZ can carry cargo proteins into extracellular vesicles

[0132] The inventors unexpectedly discovered that the first 32 amino acids at the N-terminus of the GNAZ protein can carry cargo proteins fused to its C-terminus into extracellular vesicles. The sequence of the first 32 amino acids at the N-terminus of the GNAZ protein (abbreviated as GNAZ(1-32)) is: MGCRQSSEEKEAARRSRRIDRHLRSESQRQRR (SEQ ID NO: 1).

[0133] Plasmid construction: Two plasmids were designed. The positive plasmid expressed the fusion protein GNAZ(1-32)-Flag-eGFP, while the control plasmid only expressed the eGFP protein. The vector used was pcDNA3.4.

[0134] EV preparation: See the Materials and Methods section. Both plasmids were transfected into HEK293 cells to construct mixed clones. EVs were then extracted from the cell supernatant after culturing and harvesting. The cells were then lysed and the lysate was collected for later use.

[0135] Western blot (WB): See the Materials and Methods section. Two groups of samples, the GNAZ(1-32) group (expressing the GNAZ(1-32)-Flag-eGFP fusion protein) and the eGFP group, as well as a wild-type HEK293 (WT) control, were subjected to Western blot analysis with equal protein loading. The expression of eGFP in each sample was analyzed (Figure 1). Figure 1A The results showed that eGFP was expressed in cells and EVs that were fused with GNAZ (1-32) and expressed eGFP, and the abundance of eGFP in EVs was much higher than that in cells; eGFP was expressed in cells and EVs that were only fused with eGFP, but the abundance of eGFP in cells was much higher than that in EVs; eGFP was not expressed in wild-type 293 (WT) cell lysates or EVs. Figure 1AThe eGFP signal intensity in the WT was quantified, and the results are shown in Figure 1B After fusion with GNAZ(1-32), the amount of eGFP sorted into EVs increased by about 6.8 times.

[0136] Nanoflow cytometry: See the Materials and Methods section. Nanoflow cytometry was used to detect the fluorescence of EVs harvested from GNAZ (1-32) and eGFP as well as wild-type HEK293 cells, and the eGFP positive rate was analyzed. The results showed that ( Figure 2 ), compared with the eGFP group, the number of eGFP-positive EVs in the GNAZ(1-32) group increased by about 3.7 times.

[0137] Example 2: N-terminal fragments of other proteins in the same family also have the function of carrying cargo proteins into extracellular vesicles

[0138] The expression plasmid was constructed based on the N-terminal 1-32 amino acids of the GNAZ homologous proteins GNAI1, GNAI2, and GNAI3 reported in the prior art.

[0139] Plasmid construction: The Flag-eGFP coding sequence was connected to the N-terminal 32 amino acid coding sequences of GNAI1, GNAI2, and GNAI3, respectively, to express the fusion proteins GNAI1(1-32)-Flag-eGFP, GNAI2(1-32)-Flag-eGFP, and GNAI3(1-32)-Flag-eGFP.

[0140] GNAI1 (1-32) amino acid sequence: MGCTLSAEDKAAVERSKMIDRNLREDGEKAAR (SEQ ID NO: 2);

[0141] GNAI2 (1-32) amino acid sequence: MGCTVSAEDKAAAERSKMIDKNLREDGEKAAR (SEQ ID NO: 3);

[0142] GNAI3 (1-32) amino acid sequence: MGCTLSAEDKAAVERSKMIDRNLREDGEKAAK (SEQ ID NO: 4).

[0143] WB: The method was the same as in Example 1. The results (3A) showed that the N-terminal fragments of GNAI1, GNAI2 and GNAI3 could all improve the efficiency of eGFP fusion protein sorting into EVs.

[0144] Nanoflow cytometry: The method was the same as in Example 1. The results (3B) showed that the EV positive rate in the GNAI1, GNAI2 and GNAI3 experimental groups also increased by nearly 3 times.

[0145] Example 3: Structural Characterization of the N-Terminus of GNAZ Homologous Proteins

[0146] Structural analysis of the N-terminal 32 amino acids of GNAZ, GNAI1, GNAI2, and GNAI3 (using SwissPalm software, https: / / www.swisspalm.org) revealed that these proteins possess both myristoylation and palmitoylation sites at their N-termini. Following these sites lies an α-helix with positively charged amino acids concentrated on one side. Myristoylation and palmitoylation increase the protein's lipophilicity; the α-helix stabilizes the structure, and the positively charged amino acids clustered on one side form a contact surface that interacts with negatively charged lipid membranes. The α-helix itself can also embed into the lipid membrane, enhancing the protein's stability on the EV membrane.

[0147] The N-terminal 32 amino acid sequence alignment of GNAZ, GNAI1, GNAI2, and GNAI3 can be found in Figure 4 .

[0148] Schematic diagram of the distribution of positively charged amino acids at the N-terminus of GNAZ and GNAI3. Figure 5 .

[0149] Example 4: N-terminal truncations and mutants of GNAZ still have internalization function

[0150] Plasmid construction: Expression plasmids for fusion proteins of different lengths of GNAZ N-terminal fragments with eGFP were constructed and named GNAZ(1-10), GNAZ(1-18), GNAZ(1-32), and GNAZ(1-356). GNAZ(1-10) expresses a fusion protein of the N-terminal 1-10 amino acids of GNAZ with eGFP; GNAZ(1-18) expresses a fusion protein of the N-terminal 1-18 amino acids of GNAZ with eGFP; GNAZ(1-32) expresses a fusion protein of the N-terminal 1-32 amino acids of GNAZ with eGFP; and GNAZ(1-356) expresses a fusion protein of the full-length GNAZ with eGFP.

[0151] GNAZ (1-356) full-length sequence:

[0152] MGCRQSSEEKEAARRSRRIDRHLRSESQRQRREIKLLLLGTSNSGKSTIVKQMKIIHSGGFNLEACKEYKPLIIYNAIDSLTRIIRALAALRIDFHNPDRAYDAVQLFALTGPAESKGEITPELLGVMRRLWADPGAQACFSRSSEYHLEDNAAYYLNDLERIAAADYIPTVEDILRSR DMTTGIVENKFTFKELTFKMVDVGGQRSERKKWIHCFEGVTAIIFCVELSGYDLKLYEDNQTSRMAESLRLFDSICNNNWFINTSLILFLNKKDLLAEKIRRIPLTICFPEYKGQNTYEEAAVYIQRQFEDLNRNKETKEIYSHFTCATDTSNIQFVFDAVTDVIIQNNLKYIGLC(SEQ ID NO:5).

[0153] Each truncation contains positively charged amino acids such as Figure 6 As shown, GNAZ (1-10) contains 2 positively charged amino acids (MGCRQSSEEK, SEQ ID NO: 12); GNAZ (1-18) contains 6 positively charged amino acids (MGCRQSSEEKEAARRSRR, SEQ ID NO: 19); GNAZ (1-32) contains 11 positively charged amino acids (SEQ ID NO: 1).

[0154] EV preparation: The method is the same as Example 1.

[0155] WB: The method is the same as in Example 1. The results show that ( Figure 7 ), although the expression level of the full-length GNAZ fusion protein in cells is low and almost no signal is detected in cell lysates (cell lysis), a signal can be detected in EVs samples (at approximately 75kDa), indicating that the full-length GNAZ has the function of carrying cargo proteins into EVs. The three truncated fusion proteins have similar expression levels in cells and can all enrich the fusion proteins into EVs. Among them, GNAZ (1-32) has the highest abundance in EVs. Although the ability of the eGFP fusion protein to be actively sorted into exosomes gradually decreases with the decrease in the number of retained positively charged amino acids, GNAZ (1-10) still has a significant EV enrichment effect.

[0156] Nanoflow cytometry: The method is the same as Example 1. The number of EVs in each sample was detected.

[0157] The results are as follows Figure 8As shown in Figure 3, as the number of retained positively charged amino acids decreases, the ability of the eGFP fusion protein to be actively sorted into exosomes gradually decreases. In contrast, the sorting ability of the full-length GNAZ fusion protein is far inferior to that of its truncated form.

[0158] Example 5: Effect of site mutations on the sorting ability of loaded polypeptides

[0159] The inventors found that the first 32 amino acids of the sequences of GNAI1~GNAI3 and GNAZ have high homology, and there is also a myristoylation site and a palmitoylation site. In order to determine the key sites affecting the loading capacity of the fragment, mutation experiments were performed based on GNAI3 (1-32).

[0160] Plasmid construction: eGFP fusion protein expression plasmids were constructed. Wild-type GNAI3(1-32) expressing GNAI3(1-32)-Flag-eGFP; depalmitoylated GNAI3 (C3N), in which the third amino acid C was mutated to N; demyristoylated GNAI3 (G2N), in which the second amino acid G was mutated to N; and GNAI3(4R), in which four positively charged amino acids R were added between amino acids 6 and 7; and control plasmid eGFP.

[0161] EV preparation: The method is the same as Example 1.

[0162] WB: The method is the same as in Example 1. The results show that ( Figure 9 ), both depalmitoylation and demyristoylation significantly reduced the efficiency of eGFP active sorting into EVs, indicating that both actions are required for optimal sorting. Adding a positively charged amino acid to the N-terminus did not significantly affect the sorting efficiency of the fusion protein, presumably because the existing number of positively charged amino acids was sufficient, and thus adding a further positively charged amino acid would not have an additional effect.

[0163] Nanoflow cytometry: The method is the same as in Example 1. Results ( Figure 10 ) Similar to the results of western blot, both depalmitoylation and demyristoylation significantly reduced the efficiency of eGFP being actively sorted into EVs.

[0164] Example 6: Fusion with the loaded polypeptide does not affect the biological activity of the cargo protein

[0165] Luciferase (also abbreviated herein as nanoluc) as a cargo protein to detect whether the polypeptide contained in the present invention affects its function. Luciferase-related patent (ZL 2010 80019477.4).

[0166] The amino acid sequence of NanoLuc is as follows:

[0167] MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILA (SEQ ID NO: 6).

[0168] Plasmid construction: GNAZ(1-32)-Flag-nanoluc

[0169] EV preparation: The method is the same as Example 1.

[0170] Activity detection: EVs of different concentrations were treated as follows: Group (1) Triton X-100 lysis; Group (2) Proteinase K digestion; Group (3) lysis and digestion; then substrate was added to detect chemiluminescence. Triton X-100 treatment can lyse EVs, releasing EV contents, including GNAZ (1-32) -Flag-nanoluc; Proteinase K can enzymatically hydrolyze proteins that are in direct contact with it, but cannot enzymatically hydrolyze proteins contained in intact EVs. Luciferase substrate can penetrate the EV membrane, react with luciferase and emit a signal, which can be detected. This example uses Luciferase reporter gene assay system (Promega, N1110).

[0171] Fluorescence signal results are as follows Figure 11 As shown. The results of group (1) show that fusion with the internalized polypeptide GNAZ (1-32) does not affect the function of luciferase. The results of groups (2) and (3) show that nanoluc is indeed expressed inside the EV cell, rather than in a free form or displayed outside the EV membrane. When the EV membrane is intact, proteinase K cannot digest luciferase; after EV lysis, the luciferase released from the cavity is digested by proteinase K and cannot react with the substrate.

[0172] Example 7: High-abundance sorting of mRNA by loaded polypeptides

[0173] The loaded polypeptide of the present invention can also achieve efficient sorting of mRNA. By fusing the loaded polypeptide with an RNA binding protein, the specific RNA can be carried into the EV cavity through the RNA binding protein binding to the specific RNA. The RNA binding protein used in this embodiment is L7AE, which can bind to RNA with a C / D box (box C / D). The amino acid sequence of L7AE used in this embodiment is:

[0174] MYVRFEVPEDMQNEALSLLEKVRESGKVKKGTNETTKAVERGLAKLVYIAEDVDPPEIVAHLPLLCEEKNVPYIYVKSKNDLGRAVGIEVPCASAAIINEGELRKELGSLVEKIKGLQK (SEQ ID NO: 7).

[0175] Plasmid construction: (1) P496-mRNA plasmid, expressing nanoluc mRNA with a C / D box; (2) P497-GNAZ(1-32)-GSGSGS-Flag-GGSS-L7AE, expressing GNAZ(1-32)-Flag-L7AE fusion protein.

[0176] EV preparation: The method is the same as Example 1.

[0177] Activity detection: Using actin as the internal reference gene, the relative expression levels of nanoluc mRNA in the EVs of the above four samples were detected by qPCR.

[0178] The results show that ( Figure 12 ), exosomes transfected with P497 alone, like wild-type exosomes, did not express nanoluc mRNA. Transfection with P496 alone allowed EVs to carry a small amount of nanoluc mRNA, while cells transfected with both P496 and P497 showed a significant increase in the expression of nanoluc mRNA actively sorted into EVs due to the interaction between L7AE and C / DBOX.

[0179] Example 8: Other loaded polypeptides

[0180] The SVIP (Small VCP / p97-interacting protein) protein has not only a myristoylation site at its N-terminus, but also two predicted palmitoylation sites. Furthermore, four positively charged amino acids are located on the same side of the α-helix near the N-terminus. This peptide fragment was hypothesized to have an endogenous protein-binding function, and the following verification was performed.

[0181] Plasmid construction: Two plasmids were designed. The positive plasmid expressed the fusion protein SVIP(1-43)-Flag-eGFP, while the control plasmid expressed only the eGFP protein. SVIP(1-43) amino acid sequence: MGLCFPCPGESAPPTPDLEEKRAKLAEAAERRQKEAASRGILD (SEQ ID NO: 8).

[0182] Among them, the G at position 2 is a myristoylation site, the C at positions 4 and 7 are palmitoylation sites, and positions 18 to 38 are α-helices.

[0183] EV preparation: The method is the same as Example 1.

[0184] WB: The method is the same as in Example 1. The results show that ( Figure 13 ), SVIP(1-43) can also be used as an internalized polypeptide to significantly improve the efficiency of cargo proteins fused with it being sorted into EVs.

[0185] Example 9: Built-in polypeptide consisting of 10 amino acids

[0186] The results of Example 4 showed that the first 10 amino acids at the N-terminus of GNAZ can carry cargo proteins into EVs. To verify whether the first 10 amino acids at the N-terminus of other members of the GNAZ family can also serve as cargo polypeptides, plasmids expressing eGFP fusion proteins were constructed as follows: SVIP(1-10), GNA13(1-10), and GNAI3(1-10). The N-termini of the fusion proteins carry the first 10 amino acids at the N-terminus of SVIP, GNA13, and GNAI3, respectively.

[0187] The N-terminal first 10 amino acid sequence of SVIP: MGLCFPCPGE (SEQ ID NO: 9);

[0188] The first 10 amino acids of the N-terminus of GNA13 are: MADFLPSRSV (SEQ ID NO: 10);

[0189] The N-terminal first 10 amino acid sequence of GNAI3 is: MGCTLSAEDK (SEQ ID NO: 11).

[0190] EV preparation: The method is the same as Example 1.

[0191] WB: The method is the same as in Example 1. The results show that ( Figure 14 ), the first 10 amino acids at the N-terminus of SVIP and GNAI3 significantly enriched the fusion protein into EVs, demonstrating its ability to carry peptides; whereas GNA13 (1-10) showed no significant EV enrichment effect. Sequence comparison revealed that the first 10 amino acids at the N-terminus of SVIP and GNAI3 contain a myristoylation site (G) and a palmitoylation site (C), while the first 10 amino acids at the N-terminus of GNA13 do not contain either a myristoylation site or a palmitoylation site. This result further confirms that the presence of both a myristoylation site and a palmitoylation site at the N-terminus significantly improves the efficiency of fusion protein sorting into EVs.

[0192] Table 2 lists the sequences of GNA family members and similar proteins whose first 10 amino acids at the N-terminus contain a myristoylation site (G) and a palmitoylation site (C).

[0193] Table 2

[0194] Protein name The first 10 amino acids at the N-terminus SEQ ID NO: GNAZ <![CDATA[M GC RQSSEEK]]> 12 GNAI1 <![CDATA[M GC TLSAEDK]]> 13 GNAI2 <![CDATA[M GC TVSAEDK]]> 14 GNAI3 <![CDATA[M GC TLSAEDK]]> 11 GNAO <![CDATA[M GC TLSAEER]]> 15 GNAS2 <![CDATA[M GC LGNSKTE]]> 16 GNAL <![CDATA[M GC LGGNSK]]> 17 GNAS1 <![CDATA[M G VRN C LIE]]> 18 SVIP <![CDATA[M G L C FPCPGE]]> 9

[0195] Table 3 lists the first 32 amino acids at the N-terminus of some GNA family members, i.e., the sequence containing the first α-helix.

[0196] Table 3

[0197]

[0198]

[0199] The above description is only part of the embodiments of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention are included in the scope of protection of the present invention.

Claims

1. A fusion protein comprising a loaded polypeptide and a cargo protein, wherein the loaded polypeptide is located at the N-terminus of the cargo protein, the amino acid sequence of the loaded polypeptide is as shown in SEQ ID NO: 9, and the cargo protein is a therapeutic peptide, a DNA-binding protein, an RNA-binding protein, a fluorescent protein or an enzyme. The fusion protein according to claim 1 , wherein the therapeutic peptide is an antibody and / or a cytokine.

3. The fusion protein of claim 1, wherein the RNA-binding protein is selected from the group consisting of L7Ae, hnRNPA2B1, hnRNPC1, hnRNPG, hnRNPK, hnRNPQ, YBX1, HuR, AGO2, IGF2BP1, MEX3C, ANXA2, ALIX, NCL, FUS, and MVP. An engineered extracellular vesicle comprising the fusion protein according to any one of claims 1 to 3. The extracellular vesicle according to claim 4 , comprising two or more fusion proteins, each of which independently comprises a different cargo protein. 6 . A pharmaceutical composition comprising the fusion protein according to any one of claims 1 to 3 or the extracellular vesicle according to claim 4 and a pharmaceutically acceptable carrier.

7. An engineered cell for producing the extracellular vesicles according to claim 4, wherein the cell comprises a polynucleotide encoding the fusion protein.

8. A method for preparing the extracellular vesicles according to claim 4, comprising 1) providing a cell comprising a polynucleotide encoding the fusion protein; 2) culturing the cells under conditions suitable for expressing the fusion protein; and 3) Isolating the extracellular vesicles.

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