An extracellular vesicle-based drug delivery carrier and its application

Abstract

The present invention relates to a drug delivery carrier based on extracellular vesicles and its application. The drug delivery carrier contains an extracellular vesicle scaffold protein and a recombinant membrane fusion protein. The recombinant membrane fusion protein includes a first fragment and a second fragment. The amino acid sequence of the first fragment is the amino acid sequence shown in SEQ ID NO:5, and the second fragment has n amino acids, and the sequence of the second fragment is the sequence of the first n amino acids at the N-terminus of the amino acid sequence shown in SEQ ID NO:7, where n is an integer between 50 and 200. The efficiency of releasing drug active molecules by the drug delivery carrier of the present invention before reaching lysosomes is significantly improved, and the efficacy of extracellular vesicle-based drugs can be enhanced.

Description

Technical Field

[0001] The present invention belongs to the technical field of molecular biology, and particularly relates to a drug delivery carrier based on extracellular vesicles and its application. Background Art

[0002] Extracellular vesicles are a group of vesicles with a lipid bilayer membrane structure secreted by living cells. According to their size, they can be divided into: exosomes, microvesicles, and apoptotic bodies. Extracellular vesicles originate from the endocytic pathway and are released extracellularly by the fusion of multivesicular bodies in cells with the plasma membrane. They can mediate the intercellular transfer of intracellular substances and thus can be used as drug delivery carriers in vivo. Among them, exosomes, as a kind of natural nanoparticles, have great potential as a drug delivery platform.

[0003] The key to determining the efficacy of extracellular vesicle-based drugs (drugs using extracellular vesicles as carriers) mainly lies in two aspects. The first aspect is the drug loading aspect. Loading more drug molecules on a single extracellular vesicle carrier can significantly improve the drug efficacy. Currently, in the field, the improvement of the drug loading aspect is mainly achieved by exploring efficient extracellular vesicle scaffold proteins. The second aspect is the release aspect. After extracellular vesicle-based drugs are endocytosed and taken up by target cells, they will reach lysosomes through endosomal structures and are finally degraded by various hydrolases in lysosomes. According to academic research evaluations, only less than one in ten thousand drug molecules can be prematurely released from endosomal structures into the cytoplasm to achieve drug efficacy, and the vast majority of drug molecules ultimately reach lysosomes and are degraded. Therefore, improving the efficiency of extracellular vesicle-based drugs released from endosomes and thus avoiding degradation by lysosomes can improve drug efficacy. Currently, in the field, generally membrane proteins derived from viruses are used and overexpressed on the surface of extracellular vesicle membranes to enhance the efficiency of extracellular vesicle-based drugs releasing drug active molecules before reaching lysosomes. However, the number of membrane proteins that can currently help extracellular vesicles release drug active molecules reported is very limited, and the VSVG membrane protein of vesicular stomatitis virus, which is mainly used, has a relatively low efficiency in helping extracellular vesicles release drug active molecules into the cytoplasm. Summary of the Invention

[0004] In order to solve the problems existing in the prior art, the present invention provides a drug delivery carrier based on extracellular vesicles and its application. By using a novel recombinant membrane fusion protein, the efficiency of extracellular vesicles releasing drug active molecules is improved, and finally the efficacy of extracellular vesicle-based drugs is enhanced.

[0005] In the first aspect of the present invention, a drug delivery carrier is provided. The drug delivery carrier includes extracellular vesicles, which contain extracellular vesicle scaffold proteins and recombinant membrane fusion proteins. The recombinant membrane fusion protein includes a first fragment and a second fragment. The amino acid sequence of the first fragment is the amino acid sequence shown in SEQ ID NO:5. The second fragment has n amino acids, and the sequence of the second fragment is the sequence of the first n amino acids at the N-terminus of the amino acid sequence shown in SEQ ID NO:7.

[0006] Further, n is an integer between 60 and 100, or an integer between 101 and 180.

[0007] In the embodiments of the present invention, the second fragment is connected to the C-terminus of the first fragment.

[0008] In some embodiments of the present invention, the recombinant membrane fusion protein consists of the first fragment and the second fragment.

[0009] Further, the recombinant membrane fusion protein further includes a third fragment, the third fragment has m amino acids, and the amino acid sequence of the third fragment is the sequence of m amino acids starting from the (n + 1)-th amino acid at the N-terminus of the amino acid sequence shown in SEQ ID NO:7. m is an integer greater than or equal to 2.

[0010] In some embodiments of the present invention, the third fragment is connected to the C-terminus of the second fragment.

[0011] In some embodiments of the present invention, the recombinant membrane fusion protein consists of the first fragment, the second fragment and the third fragment.

[0012] In some embodiments of the present invention, the total amino acid length n+m of the second fragment and the third fragment is 60-100 or 101-200 or 201-298.For example, the total amino acid length n + m of the second fragment and the third fragment is 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298.

[0013] According to the present invention, when referring to an amino acid sequence, unless otherwise specified, the mentioned amino acid sequence not only includes the exactly same sequence of this sequence but also includes those sequences that those skilled in the art can easily think of based on this sequence and can be expected to have equivalent effects (for example, those skilled in the art should know that sequences with high identity (such as more than 85%, especially more than 90%, particularly more than 95%)) usually have equivalent effects).

[0014] For example, the range referred to by the amino acid sequence shown in SEQ ID NO:5 not only includes the amino acid sequence that is exactly the same as SEQ ID NO:5, but also includes fragments or variants that have the same function of helping extracellular vesicles release drugs as the amino acid sequence shown in SEQ ID NO:5, such as amino acid sequences that have 85% - 90% identity or 91% - 95% identity or 96% - 100% identity with the amino acid sequence shown in SEQ ID NO:5. According to a preferred embodiment of the present invention, the sequence of the first fragment is the same as the VSVG protein (Vesicular Stomatitis Virus G protein).

[0015] For example, the amino acid sequence shown in SEQ ID NO:7 not only includes the amino acid sequence that is exactly the same as SEQ ID NO:7, but also includes fragments or variants that have the same function of being able to be used to modify the VSVG protein to enhance the function of extracellular vesicles releasing drugs as the amino acid sequence shown in SEQ ID NO:7, such as amino acid sequences that have 85% - 90% identity or 91% - 95% identity or 96% - 100% identity with the amino acid sequence shown in SEQ ID NO:7.

[0016] According to some other embodiments of the present invention, the recombinant membrane fusion protein is a VSVG protein modified with the sequence of an amino acid fragment truncated from the SDCBP protein (syndecan-binding protein), where the modification refers to connecting the sequence of the amino acid fragment truncated from the SDCBP protein (syndecan-binding protein) to the C-terminus of the VSVG protein, and the sequence of the amino acid fragment is preferably 60 amino acids starting from the N-terminus, or in addition to including 60 amino acids starting from the N-terminus, further includes more amino acids or amino acid sequences, for example, amino acids or amino acid sequences selected from the remaining fragments of the SDCBP protein or amino acid sequences that have 85% - 90% identity or 91% - 95% identity or 96% - 100% identity with them.

[0017] In the embodiments of the present invention, the extracellular vesicles are exosomes, microvesicles or apoptotic bodies.

[0018] In an embodiment of the present invention, the extracellular vesicle scaffold protein is any scaffold protein capable of enriching and loading cargo into extracellular vesicles, including but not limited to scaffold proteins known in the art. For example, the extracellular vesicle scaffold protein includes one or more of BASP1 protein or its fragment, PTGFRN protein or its fragment, TSPAN2 protein or its fragment, TSPAN3 protein or its fragment, CD63 protein or its fragment, CD9 protein or its fragment, CD81 protein or its fragment, LEAP protein or its fragment, PVR protein or its fragment.

[0019] The second aspect of the present invention provides an extracellular vesicle loaded with cargo, where the cargo includes a pharmaceutically active molecule. The extracellular vesicle loaded with cargo includes the above-mentioned drug delivery carrier and the pharmaceutically active molecule, and the pharmaceutically active molecule is directly or indirectly connected to the extracellular vesicle scaffold protein through a linker.

[0020] In an embodiment of the present invention, the pharmaceutically active molecule is directly or indirectly connected to the N-terminus and / or C-terminus of the extracellular vesicle scaffold protein through a linker.

[0021] In the present invention, the pharmaceutically active molecules include active molecules of prophylactic drugs, active molecules of therapeutic drugs, and active molecules of diagnostic drugs.

[0022] In an embodiment of the present invention, the pharmaceutically active molecule includes one or more of protein molecules and their fragments, polypeptide molecules and their fragments, nucleic acids and their fragments, or small molecule compounds.

[0023] In the present invention, the connection between the pharmaceutically active molecule and the extracellular vesicle scaffold protein or between the pharmaceutically active molecule and the linker can utilize connection techniques such as gene fusion, chemical synthesis, enzyme-catalyzed ligation, self-assembly into complexes, click chemistry, protein engineering, and nanotechnology.

[0024] In an embodiment of the present invention, the linker is a polypeptide linker and / or a protein linker. The polypeptide linker can be a cleavable linker or a flexible linker that is conventionally used in the art and can form a fusion protein by connecting a protein-based cargo or a polypeptide-based cargo to the polypeptide. The protein linker can be a linker that is conventionally used in the art and can form a protein-nucleic acid complex with nucleic acids, a linker that can bind to small molecule compounds, etc.

[0025] In some embodiments of the present invention, the recombinant membrane fusion protein is overexpressed by a recombinant cell, and the extracellular vesicle scaffold protein and the protein-based cargo or polypeptide-based cargo are simultaneously overexpressed by the recombinant cell.

[0026] In some other embodiments of the present invention, the recombinant membrane fusion protein is overexpressed by recombinant cells, the extracellular vesicle scaffold protein and the linker are simultaneously overexpressed by the recombinant cells, and exogenous nucleic acid cargoes or small molecule compound cargoes are bound to the linker on the isolated extracellular vesicles.

[0027] The binding of nucleic acid cargoes or small molecule compound cargoes to the fusion polypeptide or fusion protein is achieved by conventional technical means in the art. For example, the small molecule compound cargo can be linked to the extracellular vesicle scaffold protein through the affinity adsorption between the small molecule compound cargo and the linker.

[0028] For example, when sgRNA needs to be delivered, Cas9 protein can be used as a protein linker, which is fused to the N-terminus and / or C-terminus of the scaffold protein. The sgRNA then forms an RNP complex with the Cas9 protein, so as to load the sgRNA into the extracellular vesicles for delivery.

[0029] For another example, when siRNA needs to be delivered, AGO2 protein can be used as a protein linker, which is fused to the N-terminus and / or C-terminus of the scaffold protein. The siRNA can form an RNP complex with the AGO2 protein, so as to load the siRNA into the extracellular vesicles for delivery.

[0030] In the embodiments of the present invention, the protein molecules include but are not limited to various therapeutic proteins, immunogenic proteins, or functional proteins (such as proteins for gene editing, reporter gene proteins), enzyme proteins, etc.

[0031] In the embodiments of the present invention, the proteins for gene editing include various proteins for gene editing technologies / engineering, including but not limited to various recombinases that can promote the recombination or rearrangement of specific sequences between DNA molecules and proteins related to gene editing systems (i.e., proteins used in various gene editing systems). Commonly known gene editing-related proteins include but are not limited to Cre recombinase, Dre recombinase, Flp recombinase, RadA protein, RAD51 protein, RecA protein, Cas protein, transposon protein, zinc finger protein, and TALE protein.

[0032] In the embodiments of the present invention, the polypeptide molecules include but are not limited to various therapeutic polypeptides, targeting polypeptides, or partial polypeptide fragments of the above-mentioned proteins. For example, they can be antibodies, cytokines, anti-inflammatory proteins or polypeptides, serum albumin, lectin, coagulation factors, etc.

[0033] In the embodiments of the present invention, the nucleic acid includes, but is not limited to, various functional nucleic acid molecules. For example, it can be a DNA or mRNA molecule encoding a protein or polypeptide, an RNA molecule with regulatory functions such as miRNA, an antisense oligonucleotide ASO or siRNA, an sgRNA of the CRISPR system, or even plasmid DNA, etc.

[0034] In the embodiments of the present invention, the pharmaceutically active molecule is a cytokine receptor protein, a protein for gene editing, a cytokine or an RNA.

[0035] In some embodiments of the present invention, the pharmaceutically active molecule is Cre recombinase, Dre recombinase, Flp recombinase, Cas protein, cGAS, srIκB, IL-10, TGF-β, IL-4, STAT6, A20, SIRPα, IL-1Ra, SGK1, KDM5B, 4-1BBL, TMPRSS2, CD20, CD133, CCR4, CCR7, CCR8, CXCR4, CXCR5, GIPR, GPRC5D, NaPi2b, sgRNA or siRNA.

[0036] In some embodiments of the invention, the pharmaceutically active molecule is a protein molecule and its fragment or a polypeptide molecule and its fragment, and the pharmaceutically active molecule is directly or through a polypeptide linker connected to the N-terminus and / or C-terminus of the extracellular vesicle scaffold protein.

[0037] In some embodiments of the invention, the pharmaceutically active molecule is a nucleic acid and its fragment or a small molecule compound, and the pharmaceutically active molecule and the linker combine to form a complex, and the linker is connected to the extracellular vesicle scaffold protein, preferably connected to the C-terminus of the extracellular vesicle scaffold protein.

[0038] The third aspect of the present invention provides a pharmaceutical composition, which includes the above-mentioned drug delivery carrier or the extracellular vesicle loaded with cargo, and the pharmaceutical composition may selectively include pharmaceutically acceptable excipients.

[0039] In the embodiments of the present invention, specific excipients can be selected according to the expected administration mode and therapeutic application, and the pharmaceutically acceptable excipients include, but are not limited to, pharmaceutically, nutritionally or physiologically acceptable carriers.

[0040] Furthermore, the pharmaceutical composition includes, but is not limited to, tablets, granules, pills, capsules, emulsions, ointments, gels, suspensions, solutions, powders, transdermal patches, sprays, suppositories or implants.

[0041] The fourth aspect of the present invention provides a method for preparing the above-mentioned drug delivery carrier, which is isolated from the cell culture solution of recombinant cells, and the recombinant membrane fusion protein and the extracellular vesicle scaffold protein are overexpressed in the recombinant cells.

[0042] Further, the preparation method includes the step of obtaining the recombinant cells by transfecting host cells with a recombinant expression vector, wherein the recombinant expression vector includes a first recombinant plasmid containing the coding gene of the recombinant membrane protein and a second recombinant plasmid containing the coding gene of the extracellular vesicle scaffold protein.

[0043] In the embodiments of the present invention, the recombinant cells involved in the preparation method are all obtained by transfecting host cells with recombinant plasmids.

[0044] In some embodiments of the present invention, the cargo is a protein cargo or a polypeptide cargo, and the coding gene of the extracellular vesicle scaffold protein in the second recombinant plasmid is directly or indirectly connected to the coding gene of the cargo through a gene encoding a linker, so as to fuse the extracellular vesicle scaffold protein with the cargo. Whether a linker needs to be used can be determined according to the structure, function, stability, etc. of the extracellular vesicle scaffold protein and the active substance.

[0045] In some other embodiments of the present invention, the cargo is a nucleic acid cargo (such as RNA), and the nucleic acid cargo can exist in the form of a protein-nucleic acid complex with a linker, and the linker is connected to the extracellular vesicle scaffold protein to indirectly connect the nucleic acid to the extracellular vesicle scaffold protein. For example, a fusion protein is formed by the extracellular vesicle scaffold protein and the protein linker, and at the same time, the protein linker interacts with the nucleic acid cargo through non-covalent bonds to form a complex. There is no particular limitation on whether the coding gene of the cargo is connected to the coding gene of the extracellular vesicle scaffold protein or the linker, as long as it can be expressed in the recombinant cells.

[0046] In some other embodiments of the present invention, when the cargo is a nucleic acid cargo or a small molecule compound cargo, the extracellular vesicles prepared by overexpressing the fusion polypeptide or fusion protein formed by the extracellular vesicle scaffold protein and the linker can be directly mixed with the cargo, and the cargo binds to the linker to form a complex.

[0047] In the embodiments of the present invention, the expression vector used can be an expression vector commonly used in the art, including viral plasmid vectors and eukaryotic expression plasmid vectors, and viral plasmid vectors are preferably used.

[0048] In an embodiment of the present invention, the first recombinant plasmid is used to introduce an exogenous nucleic acid fragment encoding a "recombinant membrane protein" into cells for expression, and the second recombinant plasmid is used to introduce an exogenous nucleic acid fragment encoding a fusion polypeptide or fusion protein of "extracellular vesicle scaffold protein" or "extracellular vesicle scaffold protein-linker" or a fusion protein of "extracellular vesicle scaffold protein-cargo" or a fusion protein of "extracellular vesicle scaffold protein-linker-cargo" or a nucleic acid-protein complex into cells for expression, so as to obtain cells that overexpress the recombinant protein and the extracellular vesicle scaffold protein simultaneously, or cells that overexpress the recombinant protein and the fusion protein formed by the extracellular vesicle scaffold protein and the linker simultaneously, or the fusion protein formed by the extracellular vesicle scaffold protein and the cargo.

[0049] In an embodiment of the present invention, the expression vector used for the recombinant plasmid is pAAVS1 plasmid, pIRES plasmid, or pLENTI plasmid. The expression vectors used for the first recombinant plasmid and the second recombinant plasmid may be the same or different.

[0050] In an embodiment of the present invention, the host cell used may be any suitable cell type, including mammalian cells and insect cells, preferably mammalian cells, such as HEK293 cells, CHO cells, or SF9 cells.

[0051] In an embodiment of the present invention, the culture medium used for culturing the recombinant cells is a serum-free medium.

[0052] In an embodiment of the present invention, extracellular vesicle isolation and purification methods known in the art may be used, including but not limited to ultracentrifugation, density gradient centrifugation, size exclusion chromatography, affinity chromatography, adsorption chromatography, bonded-phase chromatography, filtration, polymer-based precipitation techniques, immunoisolation techniques, or sieving separation.

[0053] According to some embodiments of the present invention, the separation is carried out by centrifugation. First, the cell culture solution of the recombinant cells is centrifuged at a centrifugal force of less than 1000 g, and the supernatant is collected. Then, the supernatant is centrifuged at a centrifugal force of 100000 g to 200000 g, and the precipitate is collected, which is the drug delivery carrier or the extracellular vesicles loaded with cargo.

[0054] Further, first, the cell culture solution of the recombinant cells is centrifuged at a centrifugal force of 200 g to 500 g, and the supernatant is collected. Then, the supernatant is centrifuged at a centrifugal force of 120000 g to 150000 g, and the precipitate is collected, which is the extracellular vesicles loaded with cargo.

[0055] The fifth aspect of the present invention provides a kit, which includes the above-mentioned drug delivery carrier, or the above-mentioned extracellular vesicles loaded with cargo, or the above-mentioned drug composition.

[0056] The sixth aspect of the present invention provides a recombinant membrane fusion protein, which is the above-mentioned recombinant membrane fusion protein.

[0057] The seventh aspect of the present invention provides a nucleic acid fragment having a nucleotide sequence encoding the above-mentioned recombinant membrane fusion protein.

[0058] The eighth aspect of the present invention provides a recombinant plasmid containing a nucleic acid fragment having a nucleotide sequence encoding the above-mentioned recombinant membrane fusion protein.

[0059] The ninth aspect of the present invention provides a recombinant plasmid composition comprising the above-mentioned recombinant plasmid and a recombinant plasmid containing a nucleotide sequence encoding the extracellular vesicle scaffold protein or a recombinant plasmid containing a nucleotide sequence encoding the extracellular vesicle scaffold protein and a drug active molecule.

[0060] In the embodiments of the present invention, the plasmid vector used for the recombinant plasmid can be a plasmid vector commonly used in the art, including viral plasmid vectors and eukaryotic expression plasmid vectors, and preferably a viral plasmid vector is used.

[0061] In the embodiments of the present invention, the plasmid vector used for the recombinant plasmid is pAAVS1 plasmid, pIRES plasmid, or pLENTI plasmid.

[0062] The tenth aspect of the present invention provides a recombinant cell containing the above-mentioned recombinant plasmid composition.

[0063] In the embodiments of the present invention, the host cell used for the recombinant cell can be any suitable cell type, including mammalian cells and insect cells, and preferably mammalian cells, such as HEK293 cells, CHO cells, or SF9 cells.

[0064] The eleventh aspect of the present invention provides the application of the above-mentioned recombinant membrane fusion protein, and the recombinant membrane fusion protein and the extracellular vesicle scaffold protein are used together and simultaneously for drug delivery.

[0065] The twelfth aspect of the present invention provides a method for diagnosing, preventing or treating a disease, which includes administering the above-mentioned extracellular vesicles loaded with cargo or the above-mentioned drug composition to a subject.

[0066] In the embodiments of the present invention, preferably, the specific disease to be detected, prevented or treated corresponding to the extracellular vesicles loaded with cargo is determined according to the drug active molecule loaded on the extracellular vesicles, including but not limited to various tumor diseases or infectious diseases.

[0067] Due to the adoption of the above technical solution, the present invention has the following advantages compared with the prior art:

[0068] The extracellular vesicle-based drug delivery carrier of the present invention comprises extracellular vesicles overexpressing a recombinant membrane fusion protein, which can significantly improve the efficiency of extracellular vesicles in releasing drug active molecules before reaching lysosomes and promote the enhancement of the efficacy of extracellular vesicle-based drugs. Description of the Drawings

[0069] Figure 1 Particle size distribution diagram of exosomes loaded with cGAS pro-inflammatory protein in Example 1;

[0070] Figure 2 Particle size distribution diagram of exosomes loaded with cGAS pro-inflammatory protein in Comparative Example 1;

[0071] Figure 3 Transmission electron micrograph of exosomes loaded with cGAS pro-inflammatory protein in Example 1;

[0072] Figure 4 Transmission electron micrograph of exosomes loaded with cGAS pro-inflammatory protein in Comparative Example 1;

[0073] Figure 5 Comparison diagram of cargo delivery effects of exosomes loaded with cGAS pro-inflammatory protein in Example 1 and Comparative Example 1;

[0074] Figure 6 Particle size distribution diagram of exosomes loaded with Cas9 protein in Example 2;

[0075] Figure 7 Particle size distribution diagram of exosomes loaded with Cas9 protein in Comparative Example 2;

[0076] Figure 8 Transmission electron micrograph of exosomes loaded with Cas9 protein in Example 2;

[0077] Figure 9 Transmission electron micrograph of exosomes loaded with Cas9 protein in Comparative Example 2;

[0078] Figure 10 Comparison diagram of cargo delivery effects of exosomes of Cas9 protein in Example 2 and Comparative Example 2;

[0079] Figure 11 Particle size distribution diagram of exosomes loaded with Cre recombinase in Example 3;

[0080] Figure 12 Particle size distribution diagram of exosomes loaded with Cre recombinase in Comparative Example 3;

[0081] Figure 13 Transmission electron micrograph of exosomes loaded with Cre recombinase in Example 3;

[0082] Figure 14Transmission electron micrograph of exosomes loaded with Cre recombinase for Comparative Example 3;

[0083] Figure 15 Comparison chart of cargo delivery effects of exosomes loaded with Cre recombinase in Example 3 and Comparative Example 3;

[0084] Figure 16 Particle size distribution chart of exosomes loaded with Cre recombinase in Example 4;

[0085] Figure 17 Particle size distribution chart of exosomes loaded with Cre recombinase for Comparative Example 4;

[0086] Figure 18 Transmission electron micrograph of exosomes loaded with Cre recombinase in Example 4;

[0087] Figure 19 Transmission electron micrograph of exosomes loaded with Cre recombinase for Comparative Example 4;

[0088] Figure 20 Comparison chart of cargo delivery effects of exosomes loaded with Cre recombinase in Example 4 and Comparative Example 4. Detailed implementation mode

[0089] The key to the functional delivery of extracellular vesicles (such as exosomes) is to achieve efficient endosomal escape, release the loaded drug into the cytoplasm, and avoid degradation by lysosomes. In the art, membrane fusion proteins derived from viruses such as the VSVG membrane protein are usually used to help extracellular vesicles release drugs before reaching lysosomes, but the efficiency of VSVG in helping extracellular vesicles release drugs still needs to be further improved. The inventors of this application have found through a large number of studies and experimental verifications that the recombinant protein obtained by modifying the VSVG membrane protein with a modified sequence can significantly enhance the drug release efficiency.

[0090] The modified sequence includes the amino acid sequence shown in SEQ ID NO:7 or its fragment, or a sequence having more than 85% identity with them. The recombinant protein obtained by modifying the VSVG protein with this modified sequence and the exosome scaffold protein (not limited to a specific scaffold protein) are jointly applied to drug delivery based on extracellular vesicles, significantly improving the efficiency of loading and delivering Cre recombinase, Cas9 protein, and cGAS pro-inflammatory protein into the cytoplasm. The first 60 amino acids at the N-terminus of the amino acid sequence shown in SEQ ID NO:7 form a domain responsible for interacting with biological membranes. In the examples of the present invention, modified sequences of different lengths containing this domain can be used to modify the VSVG protein to enhance drug release. In practical applications, similar sequences of appropriate lengths can be selected to modify the VSVG protein according to the cargo size or loading amount requirements.

[0091] Preferably, the sequence of the amino acid fragment is 60 amino acids starting from the N-terminus, or in addition to including 60 amino acids starting from the N-terminus, it further includes more amino acids or amino acid sequences. For example, amino acids or amino acid sequences selected from the remaining fragments of the SDCBP protein.

[0092] Unless otherwise stated or inconsistent with the context, the terms or expressions used herein should be read in combination with the overall content of this article and as understood by those of ordinary skill in the art. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art.

[0093] "Extracellular vesicles" are a group of vesicles with a lipid bilayer membrane structure secreted by living cells, which can be divided into exosomes, microvesicles, and apoptotic bodies according to their size.

[0094] "Exosomes" refer to extracellular vesicles (EVs) derived from cells with a diameter of 30 - 150 nm, which include a membrane enclosing an internal space, and the membrane has a phospholipid bilayer structure. Exosomes can mediate the intercellular transfer of intracellular substances.

[0095] "VSVG membrane protein" is the glycoprotein (G protein) of vesicular stomatitis virus, which is a single transmembrane protein composed of 511 amino acid residues.

[0096] "Loading" refers to connecting and enriching cargo to extracellular vesicles, including various forms such as the cargo being inside the lumen of extracellular vesicles, on the membrane of extracellular vesicles, or attached to the outside of the membrane of extracellular vesicles.

[0097] "Release" refers to releasing the cargo that has been connected and enriched to extracellular vesicles into the cytoplasm.

[0098] "Extracellular vesicle scaffold protein" refers to a linker used to connect cargo to extracellular vesicles, which is used to anchor any cargo of interest to extracellular vesicles.

[0099] "Cargo" mainly refers to "bioactive molecules", that is, functional (such as prophylactic, therapeutic, diagnostic, etc.) proteins and their fragments, polypeptides and their fragments, nucleic acids and their fragments, or small molecule compounds of any interest. "Cargo" is also often referred to as "functional cargo" or "active substance".

[0100] "Small molecule compound" refers to a compound with a molecular size less than 1000 molecular weight, including conventional organic compounds and inorganic compounds.

[0101] "Polypeptide" and "oligopeptide" are defined according to the length of the peptide chain. A peptide chain with a length of 10 - 100 amino acids is called a "polypeptide", and a peptide chain with a length less than 10 amino acids is called an "oligopeptide".

[0102] "Recombinase" refers to a class of enzymes that can promote the recombination or rearrangement of modified sequences between DNA molecules. Common recombinases can all be loaded and delivered by the method of the present invention. Common recombinases include, but are not limited to, Cre recombinase, Dre recombinase, Flp recombinase, RadA protein, RAD51 protein, and RecA protein.

[0103] "Cytokine" is a class of small molecular proteins with a wide range of biological activities synthesized and secreted by immune cells (such as monocytes, macrophages, T cells, B cells, NK cells, etc.) and certain non-immune cells (endothelial cells, epidermal cells, fibroblasts, etc.) upon stimulation. Cytokines generally regulate cell growth, differentiation, and effector functions by binding to corresponding receptors, regulate immune responses, and have multiple functions such as regulating innate and adaptive immunity, hematopoiesis, cell growth, and repair of damaged tissues. Common cytokines include, but are not limited to, pro-inflammatory cytokines, anti-inflammatory cytokines, interleukins, interferons, growth factors, and chemokines.

[0104] "Gene editing protein" refers to a class of proteins used in gene editing systems. Common gene editing proteins include Cas proteins (such as Cas9, Cas12, Cas13, Cas2, Cas3, Cas10, Cas14, Cas12f, CasX, CasY, or Csm), transposon proteins, zinc finger proteins, and TALE proteins.

[0105] "Exogenous nucleic acid fragment" includes any gene or fragment thereof of interest, such as a gene encoding a scaffold protein or a fragment thereof. The exogenous nucleic acid fragment is from a different source than the host cell. For example, a nucleic acid sequence isolated from an organism different from the host cell is an exogenous nucleic acid fragment relative to the host cell.

[0106] "Plasmid" refers to a circular double-stranded DNA that can accept exogenous nucleic acid fragments and can replicate in prokaryotic or eukaryotic cells.

[0107] "Promoter" refers to a nucleic acid sequence that can control the transcription of a coding sequence. The promoter sequence includes a modified sequence sufficient to enable RNA polymerase to recognize, bind, and initiate transcription. The promoter can affect the transcription of a gene located on the same nucleic acid molecule as itself or a gene located on a different nucleic acid molecule from itself.

[0108] "Host cell", also known as recipient cell, includes, but is not limited to, animal cells, plant cells, algal cells, fungal cells, yeast cells, or bacterial cells. Exemplary host cells include human embryonic kidney cell HEK293. It should be understood that due to natural, accidental, or intentional mutations, the offspring of a single parental cell may not be exactly the same as the original parent in terms of morphology or in terms of genomic or total DNA complement.

[0109] "cGAS pro-inflammatory protein" is the main sensor of cytoplasmic DNA, which can activate STING, induce the expression of type I IFN and IFN-stimulated pro-inflammatory cytokines.

[0110] "Cas9 protein" is a nuclease in the CRISPR-Cas system that can specifically cleave DNA molecules.

[0111] "Cre recombinase" is a protein encoded by a 1029-bp sequence in the genome of bacteriophage P1. It consists of 343 amino acids with a molecular weight of 3815 kD. It is a type I topoisomerase that catalyzes site-specific recombination of DNA between loxP sites and is used for gene insertion and deletion in gene therapy.

[0112] The technical solution of the present invention will be further described below in conjunction with specific embodiments, but the present invention is not limited to the following embodiments.

[0113] The implementation conditions used in the following examples and comparative examples can be further adjusted according to specific requirements. The implementation conditions not specified are usually those in conventional experiments. Instruments, raw materials, and reagents without special instructions can be obtained through commercial purchase.

[0114] Example 1

[0115] This example provides an exosome loaded with cGAS pro-inflammatory protein. In this example, Basp1 protein is used as the exosome scaffold protein, and the VSVG membrane protein modified by the modified sequence helps the exosome release the cGAS pro-inflammatory protein. The preparation method of the exosome loaded with cGAS pro-inflammatory protein in this example is as follows:

[0116] 1. Construct plasmid

[0117] Using the pIRES plasmid as the expression vector and Basp1 protein as the exosome scaffold protein, a first plasmid is constructed. The design of the expression frame of the first plasmid is as follows: using the CMV promoter (sequence shown in SEQ ID NO:1) as the transcription start point, using SV40 polyA (sequence shown in SEQ ID NO:2) as the transcription end point, and connecting the cGAS pro-inflammatory protein (sequence shown in SEQ ID NO:4) to the C-terminus of the Basp1 protein (sequence shown in SEQ ID NO:3).

[0118] Using the pIRES plasmid as the expression vector, a second plasmid was constructed. The design of the expression cassette of the second plasmid was as follows: The CMV promoter (the sequence is as shown in SEQ ID NO: 1) was used as the transcription start point, and the SV40 polyA (the sequence is as shown in SEQ ID NO: 2) was used as the transcription end point. A modified sequence (the sequence is as shown in SEQ ID NO: 6, which is identical to the first 60 amino acid sequences at the N-terminus of the SDCBP protein with the sequence as shown in SEQ ID NO: 7) was ligated to the C-terminus of the VSVG membrane protein (the sequence is as shown in SEQ ID NO: 5).

[0119] The first plasmid and the second plasmid were prepared respectively according to the following specific steps:

[0120] (1) PCR cloning: Using conventional methods in the art, upstream primers and downstream primers were designed according to the expression cassette, and the upstream primer, downstream primer and template DNA were synthesized. In a 0.2 mL EP tube, 25 μL of DNA polymerase (2×Phanta Max Master Mix, Vazyme, P515-01), 2 μL of upstream primer, 2 μL of downstream primer, 10 μg of template DNA were added, and made up to 50 μL with enzyme-free water (Rnase-free ddH2O, Vazyme, P071-01-AA). After mixing, it was placed in a PCR instrument, and the amplification program was designed according to the primer annealing temperature.

[0121] (2) Purification of PCR products: Purify the PCR products according to the kit instructions (Vazyme, DC301-01).

[0122] (3) Enzyme digestion: In a 0.2 mL EP tube, 5 μL of rCutSmart TM buffer (BioLabs, 136004s), 0.5 μL of Pac I enzyme (BioLabs, R0547L), 0.5 μL of Not I enzyme (BioLabs, R3189L), 2 μg of the PCR purified fragment, 5 μg of the pAAVS1 vector plasmid were added. Made up to 10 μL with enzyme-free water (Rnase-free ddH2O, Vazyme, P071-01-AA). After mixing, it was placed in a PCR instrument and digested at 37 °C for 16 h.

[0123] (4)Ligation and transformation: In a 0.2 mL EP tube, add 4 μL of the digested solution obtained in step (3), 1 μL of T4 DNA ligase (Vazyme, N103-01-AA), and 1 μL of T4 DNA ligase buffer (Vazyme, N103-01-AC). After incubating at room temperature for 10 min, take 5 μL and add it to the competent cells (DH5α, Vazyme, C502-03). Incubate on ice for 30 min, incubate at 42 °C for 90 s, incubate on ice for 2 min, then add 1 mL of LB medium (BeyoPure™, 04.05.ST156), place it in an orbital shaker incubator for 1 h. After culturing, centrifuge at 12000 g for 1 min, discard the supernatant, leave 100 μL, resuspend it, add it to a bacterial culture dish (Biosharp, BS-90-D), and then spread it evenly with a disposable plastic spreader (Biosharp, BS-PS-A), and place it in a 37 °C constant temperature incubator for storage, for standby use.

[0124] Among them, in step (1), the upstream primer sequence of the first plasmid is as shown in SEQ ID NO:8, the downstream primer sequence is as shown in SEQ ID NO:9, and the template DNA sequence is as shown in SEQ ID NO:10. The upstream primer sequence of the second plasmid is as shown in SEQ ID NO:11, the downstream primer sequence is as shown in SEQ ID NO:12, and the template DNA sequence is as shown in SEQ ID NO:13.

[0125] 2. Cell transfection

[0126] The specific steps are as follows:

[0127] (1)Prepare transfected cells

[0128] Inoculate EXPI293F cells at a density of 1.5E+6 / mL with a viability of over 95% into a 125 mL shake flask with a total culture volume of 25 mL. Incubate overnight in an 8% CO2 constant temperature shaker and start transfection the next day.

[0129] (2)PEI transfection

[0130] Using TA293 reagent (Zhuhai Kairui Biotechnology Co., Ltd., K2001), taking 25 mL of cell suspension as an example, the transfection process is as follows:

[0131] The target plasmids were extracted from the above-transformed competent cells preserved by coating and culturing using conventional technical means in the art, namely, the constructed first plasmid and second plasmid were obtained, and they were mixed in equal mass for standby. Prepare two 15 mL centrifuge tubes. Add 1.25 mL of serum-free cell culture medium, 25 μg of the mixture of the first plasmid and the second plasmid to one of them, and vortex to mix evenly; add 1.25 mL of serum-free cell culture medium and 125 μL of TA293 transfection reagent to the other one, and vortex to mix evenly. Transfer all the liquid in the centrifuge tube containing the transfection reagent to the centrifuge tube containing the plasmid, and vortex to mix evenly. Incubate at room temperature for 10 minutes to prepare the plasmid-vector complex. Take out the EXPI293F cells from the constant temperature shaker, add the prepared plasmid-vector complex drop by drop while shaking, and then put them back into the CO2 constant temperature shaker and shake culture for 72 hours.

[0132] 3. Isolate and purify exosomes

[0133] The cell culture solution after shaking culture for 72 hours was first centrifuged at 300 g and 4 °C at low speed for 5 min, the supernatant was collected, and then the supernatant was ultracentrifuged at 133900 g and 4 °C for 1 h, and the precipitate was collected. The precipitate (i.e., the isolated exosomes) was resuspended with 100 μL of PBS and stored at 4 °C for standby.

[0134] Comparative Example 1

[0135] This comparative example provides exosomes loaded with cGAS pro-inflammatory protein. In this comparative example, Basp1 protein was used as the exosome scaffold protein, and the VSVG membrane protein was used to help the exosomes release cGAS pro-inflammatory protein. In this comparative example, the design of the expression frame of the first plasmid was the same as that in Example 1. The modified sequence connected to the C-terminus of the VSVG membrane protein in the expression frame of the second plasmid in Example 1 was deleted to obtain the expression frame of the second plasmid in this comparative example. The preparation method of this comparative example was the same as that of Example 1 except that the upstream primer (sequence shown in SEQ ID NO:11), downstream primer (sequence shown in SEQ ID NO:14), and template DNA (sequence shown in SEQ ID NO:15) of the second plasmid were redesigned according to the adjusted expression frame, and finally exosomes loaded with cGAS pro-inflammatory protein were obtained.

[0136] Example 2

[0137] This example provides exosomes loaded with Cas9 protein. In this example, Basp1 protein is used as the exosome scaffold protein, and the modified VSVG membrane protein through sequence modification helps the exosomes release Cas9 protein. Refer to the preparation method of Example 1 to prepare exosomes loaded with Cas9 protein. The second plasmid in this example is the same as the second plasmid in Example 1. Replace the cGAS pro-inflammatory protein with the amino acid sequence shown in SEQ ID NO: 4 in the expression frame of the first plasmid in Example 1 with the Cas9 protein with the amino acid sequence shown in SEQ ID NO: 16. Except for redesigning and synthesizing the upstream primer (sequence shown in SEQ ID NO: 8), downstream primer (sequence shown in SEQ ID NO: 17), and template DNA (sequence shown in SEQ ID NO: 18) of the first plasmid according to the adjusted expression frame, the rest refer to Example 1, and finally obtain exosomes loaded with Cas9 protein.

[0138] Comparative Example 2

[0139] This comparative example provides exosomes loaded with Cas9 protein. In this comparative example, Basp1 protein is used as the exosome scaffold protein, and the VSVG membrane protein helps the exosomes release Cas9 protein. The first plasmid in this comparative example is the same as the first plasmid in Example 2, and the second plasmid is the same as the second plasmid in Comparative Example 1. Except for using the upstream primer, downstream primer, and template DNA of the first plasmid designed in Example 2 and using the upstream primer, downstream primer, and template DNA of the second plasmid designed in Comparative Example 1, the rest refer to Example 1, and finally obtain exosomes loaded with Cas9 protein.

[0140] Example 3

[0141] This example provides exosomes loaded with Cre recombinase. In this example, Basp1 protein is used as the exosome scaffold protein, and the modified VSVG membrane protein through sequence modification helps the exosomes release Cre recombinase. Refer to the preparation method of Example 1 to prepare exosomes loaded with Cre recombinase. The second plasmid in this example is the same as the second plasmid in Example 1. Replace the cGAS pro-inflammatory protein with the amino acid sequence shown in SEQ ID NO: 4 in the expression frame of the first plasmid in Example 1 with the Cre recombinase with the amino acid sequence shown in SEQ ID NO: 19. Except for redesigning and synthesizing the upstream primer (sequence shown in SEQ ID NO: 8), downstream primer (sequence shown in SEQ ID NO: 20), and template DNA (sequence shown in SEQ ID NO: 21) of the first plasmid according to the adjusted expression frame, the rest refer to Example 1, and finally obtain exosomes loaded with Cre recombinase.

[0142] Comparative Example 3

[0143] This comparative example provides an exosome loaded with Cre recombinase, in which Basp1 protein is used as an exosome scaffold protein, and VSVG membrane protein is used to help the exosome release Cas9 protein. The first plasmid in this comparative example is the same as the first plasmid in Example 3, and the second plasmid is the same as the second plasmid in Comparative Example 1. Except for using the upstream primer, downstream primer and template DNA of the first plasmid designed in Example 3 and the upstream primer, downstream primer and template DNA of the second plasmid designed in Comparative Example 1, the rest is referred to Example 1, and finally exosomes loaded with Cre recombinase are obtained.

[0144] Example 4

[0145] This embodiment provides another exosome loaded with Cre recombinase. In this embodiment, PTTG1IP protein is used as an exosome scaffold protein, and the VSVG membrane protein modified by the modified sequence helps the exosome release the Cre recombinase. Exosomes loaded with Cre recombinase are prepared with reference to the preparation method of Example 1. The second plasmid in this embodiment is the same as the second plasmid in Example 1, and the cGAS pro-inflammatory protein with an amino acid sequence as shown in SEQ ID NO:4 in the expression frame of the first plasmid in Example 1 is replaced with the Cre recombinase shown in the amino acid sequence SEQ ID NO:19, and the Basp1 protein with an amino acid sequence as shown in SEQ ID NO:3 is replaced with the PTTG1IP protein shown in the amino acid sequence SEQ ID NO:22. In addition to redesigning and synthesizing the upstream primer (sequence as shown in SEQ ID NO:23), the downstream primer (sequence as shown in SEQ ID NO:20) and the template DNA (sequence as shown in SEQ ID NO:24) of the first plasmid according to the adjusted expression frame, the rest is referred to Example 1, and finally another exosome loaded with Cre recombinase is obtained.

[0146] Comparative Example 4

[0147] This comparative example provides another exosome loaded with Cre recombinase, in which PTTG1IP protein is used as an exosome scaffold protein, and VSVG membrane protein is used to help exosomes release Cas9 protein. The first plasmid in this comparative example is the same as the first plasmid in Example 4, and the second plasmid is the same as the second plasmid in Comparative Example 1. Except for using the upstream primer, downstream primer and template DNA of the first plasmid designed in Example 4 and the upstream primer, downstream primer and template DNA of the second plasmid designed in Comparative Example 1, the rest is referred to Example 1, and finally exosomes loaded with Cre recombinase are obtained.

[0148] Example 5

[0149] This example provides an exosome loaded with sgRNA. The sequence of the sgRNA is as shown in SEQ ID NO:25. The sgRNA is mixed with the exosome loaded with Cas9 protein in Example 2. Using Cas9 protein as a protein linker, the sgRNA and Cas9 protein form an RNP complex, thereby loading the sgRNA onto the exosome to obtain an exosome loaded with sgRNA.

[0150] Example 6

[0151] This example provides an exosome loaded with siRNA. The sense strand sequence of the siRNA is as shown in SEQ ID NO:26, and the antisense strand sequence is as shown in SEQ ID NO:27. Using AGO2 protein (sequence as shown in SEQ ID NO:28) as a protein linker, first prepare an exosome loaded with AGO2 protein according to the method of Example 1, and then mix the siRNA with the exosome loaded with AGO2. The siRNA and AGO2 protein form an RNP complex, thereby loading the siRNA onto the exosome to obtain an exosome loaded with siRNA.

[0152] Example 7

[0153] This example provides an exosome loaded with a small molecule compound (rapamycin). Using FKBP12 protein (sequence as shown in SEQ ID NO:29) as a protein linker, first prepare an exosome loaded with FKBP12 protein according to the method of Example 1, and then mix rapamycin with the exosome loaded with FKBP12 protein. Rapamycin and FKBP12 protein form a complex, thereby loading rapamycin onto the exosome to obtain an exosome loaded with a small molecule compound.

[0154] Example 8

[0155] This example provides a pharmaceutical composition for enhancing immune effect, which is formulated with the exosome loaded with cGAS pro-inflammatory protein in Example 1 and physiological saline into an injection with a particle concentration of 1.0E12 particles / mL, and administered to experimental mice by intramuscular injection. Since the exosome loaded with cGAS pro-inflammatory protein in Example 1 can effectively activate the immune response mediated by the STING pathway and promote the secretion of various inflammatory factors, the pharmaceutical composition in this example can enhance the anti-tumor, anti-viral and other immune effects of the body, and can be used as a vaccine adjuvant to improve vaccine efficacy.

[0156] (1) Exosome identification:

[0157] The NTA analysis of the exosome resuspension of the above-mentioned examples and comparative examples was performed using a ZetaView instrument (Particle Metrix of Flowing Bio, product number N30E). The particle size distribution of the exosomes loaded with the cGAS pro-inflammatory protein in Example 1 is shown in Figure 1 , and the particle size distribution of the exosomes loaded with the cGAS pro-inflammatory protein in Comparative Example 1 is shown in Figure 2 , the particle size distribution of the exosomes loaded with the Cas9 protein in Example 2 is shown in Figure 6 , and the particle size distribution of the exosomes of the Cas9 protein in Comparative Example 2 is shown in Figure 7 , the particle size distribution of the exosomes loaded with the Cre recombinase in Example 3 is shown in Figure 11 , and the particle size distribution of the exosomes loaded with the Cre recombinase in Comparative Example 3 is shown in Figure 12 , the particle size distribution of the exosomes loaded with the Cre recombinase in Example 4 is shown in Figure 6 , and the particle size distribution of the exosomes loaded with the Cre recombinase in Comparative Example 4 is shown in Figure 17 , and the average particle size and median particle size are statistically shown in Table 1.

[0158]

[0159] Table 1 shows that the particle sizes of the exosomes loaded with cargo in Examples 1 to 4 and Comparative Examples 1 to 4 are all about 120 to 150 nm.

[0160] (2) Observe the structure of the exosomes prepared in the above-mentioned examples and comparative examples under a transmission electron microscope (TEM). As shown in Figure 3 , Figure 4 , Figure 8 , Figure 9 , Figure 13 , Figure 14 , Figure 18 and Figure 19 show that the exosomes loaded with cargo in Examples 1 to 4 and Comparative Examples 1 to 4 all show typical exosome vesicle structures (cup-shaped or saucer-shaped) under the electron microscope.

[0161] (3) Analysis of exosome cargo delivery efficiency:

[0162] The exosomes loaded with the cGAS pro-inflammatory protein prepared in Example 1 and Comparative Example 1 were added to peripheral blood mononuclear cells (PBMCs) in equal amounts (the same number of exosome particles) respectively. Under normal circumstances, PBMC cells do not have an inflammatory response and do not secrete inflammatory cytokines; while for PBMC cells with successful delivery of the cGAS pro-inflammatory protein, the STING pathway is activated, resulting in an inflammatory response, so PBMC cells secrete inflammatory cytokines such as TNF-α and IL-6. As shown in Figure 5As shown, after PBMC cells received the exosomes of Example 1, the amounts of TNF-α and IL-6 secreted were significantly higher than those of Comparative Example 1, and the exosomes of Example 1 had a higher delivery efficiency for the cGAS pro-inflammatory protein. Since the same exosome scaffold protein was used in Example 1 and Comparative Example 1, the influence of loading efficiency on delivery efficiency was excluded. Compared with the VSVG membrane protein, the VSVG membrane protein modified with the modified sequence could help the exosomes release more cGAS pro-inflammatory protein into the cytoplasm, thus making the exosomes of Example 1 have a higher delivery efficiency for the cGAS pro-inflammatory protein.

[0163] The exosomes loaded with Cas9 protein prepared in Example 2 and Comparative Example 2 were added to the reporter cells for detecting Cas9 protein in equal amounts (the same number of exosome particles). Under normal circumstances (when Cas9 protein was not delivered to the reporter cells), the reporter cells only expressed red fluorescent protein; while for the reporter cells with successful delivery of Cas9 protein, the GFP gene with a random +1 or +2 frameshift mutation was corrected, and the reporter cells expressed green fluorescent protein. As Figure 10 shown, the expression of green fluorescent protein in the cells using the exosomes of Example 2 to deliver Cas9 protein was significantly better than that of Comparative Example 2, and the exosomes of Example 2 had a higher delivery efficiency for Cas9 protein. Since the same exosome scaffold protein was used in Example 2 and Comparative Example 2, the influence of loading efficiency on delivery efficiency was excluded. Compared with the VSVG membrane protein, the VSVG membrane protein modified with the modified sequence could help the exosomes release more Cas9 protein into the cytoplasm, thus making the exosomes of Example 2 have a higher delivery efficiency for Cas9 protein.

[0164] The exosomes loaded with Cre recombinase of Example 3 and Comparative Example 3 were added to the reporter cells for detecting Cre recombinase in equal amounts (the same number of exosome particles). The genome of the reporter cells for detecting Cre recombinase contained both green fluorescent protein and red fluorescent protein, among which the expression of red fluorescent protein was silenced, and only green fluorescent protein was expressed before delivering Cre recombinase. The recombination mediated by Cre recombinase would activate the expression of red fluorescent protein, and the reporter cells with successful delivery of Cre recombinase would express red fluorescent protein located in the nucleus. As Figure 15 shown, the expression of red fluorescent protein in the cells using the exosomes of Example 3 to deliver Cre recombinase was significantly better than that of Comparative Example 3, and the exosomes of Example 3 had a higher delivery efficiency for Cre recombinase. Since the same exosome scaffold protein was used in Example 3 and Comparative Example 3, the influence of loading efficiency on delivery efficiency was excluded. Compared with the VSVG membrane protein, the VSVG membrane protein modified with the modified sequence could help the exosomes release more Cre recombinase into the cytoplasm, thus making the exosomes of Example 3 have a higher delivery efficiency for Cre recombinase.

[0165] The exosomes loaded with Cre recombinase in Example 4 and Comparative Example 4 were added to the reporter cells for detecting Cre recombinase in equal amounts (the same number of exosome particles). The genome of the reporter cells for detecting Cre recombinase contains both green fluorescent protein and red fluorescent protein, among which the expression of red fluorescent protein is silenced. Only green fluorescent protein is expressed before the delivery of Cre recombinase. The recombination mediated by Cre recombinase will activate the expression of red fluorescent protein. The reporter cells with successful delivery of Cre recombinase will express red fluorescent protein localized in the nucleus. Since the scaffold proteins used in Example 4 and Comparative Example 4 are different from those used in Example 3 and Comparative Example 3, there are differences in the loading efficiency of Cre recombinase between Example 4 and Example 3. However, as Figure 20 shown, the expression of red fluorescent protein in the cells with exosomes delivering Cre recombinase in Example 4 is significantly better than that in Comparative Example 4, indicating that the exosomes in Example 4 have a higher delivery efficiency of Cre recombinase compared to Comparative Example 4. This comparison result is consistent with the comparison result between Example 3 and Comparative Example 3. It can be seen that for exosomes loaded with cargo using different scaffold proteins, the VSVG membrane protein modified by the modified sequence can help improve the efficiency of exosome cargo release.

[0166] The above examples and comparative examples show that the present invention modifies the VSVG membrane protein by modifying the sequence, and a recombinant membrane fusion protein is obtained by adding a modified sequence to the end of VSVG. This recombinant membrane fusion protein can help improve the efficiency of exosome cargo release, thereby improving the efficiency of exosome delivering cargo into the cytoplasm, and further improving the efficacy of exosome drugs.

[0167] The above has made a detailed description of the present invention, aiming to enable those skilled in the art to understand the content of the present invention and implement it. However, it cannot be used to limit the protection scope of the present invention. Moreover, the present invention is not limited to the above examples. Any equivalent changes or modifications made according to the spirit and essence of the present invention should be covered within the protection scope of the present invention.

Claims

1. A drug delivery carrier, characterized in that, The drug delivery carrier includes extracellular vesicles, which contain extracellular vesicle scaffold proteins and recombinant membrane fusion proteins. The recombinant membrane fusion protein consists of a first fragment and a second fragment. The amino acid sequence of the first fragment is the amino acid sequence shown in SEQ ID NO:5, and the amino acid sequence of the second fragment is the sequence of the first 60 to 100 amino acids at the N-terminus of the amino acid sequence shown in SEQ ID NO:

7. The second fragment is connected to the C-terminus of the first fragment.

2. The drug delivery carrier according to claim 1, wherein, The extracellular vesicles are exosomes, microvesicles or apoptotic bodies.

3. The drug delivery carrier according to claim 1, wherein The extracellular vesicle scaffold proteins include one or more of BASP1 protein, PTGFRN protein, TSPAN2 protein, TSPAN3 protein, CD63 protein, CD9 protein, CD81 protein, LEAP protein or PVR protein.

4. The drug delivery carrier according to claim 1, characterized in that, The amino acid sequence of the second fragment is the sequence of the first 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79 or 80 amino acids at the N-terminus of the amino acid sequence shown in SEQ ID NO:

7.

5. The pharmaceutical delivery vehicle according to claim 1, characterized in that, The amino acid sequence of the second fragment is the sequence of the first 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100 amino acids at the N-terminus of the amino acid sequence shown in SEQ ID NO:

7.

6. An extracellular vesicle loaded with cargo, wherein the cargo comprises a pharmaceutically active molecule, characterized in that, The extracellular vesicles loaded with cargo include the drug delivery carrier according to any one of claims 1 to 5 and the drug active molecule, and the drug active molecule is directly or indirectly connected to the extracellular vesicle scaffold protein through a linker.

7. The extracellular vesicle for loading cargo according to claim 6, characterized in that, The drug active molecules include one or more of protein molecules and their fragments, polypeptide molecules and their fragments, nucleic acids and their fragments or small molecule compounds.

8. The extracellular vesicles loaded with cargo according to claim 7, wherein, The drug active molecule is a cytokine receptor protein, a protein for gene editing, a cytokine or RNA.

9. The extracellular vesicles loaded with cargo according to claim 8, wherein, The drug active molecule is Cre recombinase, Dre recombinase, Flp recombinase, Cas protein, cGAS, srIκB, IL-10, TGF-β, IL-4, STAT6, A20, SIRPα, IL-1Ra, SGK1, KDM5B, 4-1BBL, TMPRSS2, CD20, CD133, CCR4, CCR7, CCR8, CXCR4, CXCR5, GIPR, GPRC5D, NaPi2b, sgRNA or siRNA.

10. The extracellular vesicles for loading cargo according to claim 7, wherein, The drug active molecule is a protein molecule and its fragment or a polypeptide molecule and its fragment, and the drug active molecule is directly or connected to the N-terminus and / or C-terminus of the extracellular vesicle scaffold protein through a polypeptide linker; and / or, the drug active molecule is a nucleic acid and its fragment or a small molecule compound, and the drug active molecule and the linker form a complex, and the linker is connected to the N-terminus and / or C-terminus of the extracellular vesicle scaffold protein.

11. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the drug delivery carrier according to any one of claims 1 to 5 or the cargo-loaded extracellular vesicles according to any one of claims 6 to 10, and the pharmaceutical composition may optionally comprise a pharmaceutically acceptable excipient.

12. The pharmaceutical composition according to claim 11, characterized in that, The pharmaceutical composition is a tablet, granule, pill, capsule, emulsion, ointment, gel, suspension, solution, powder, transdermal patch, spray, suppository or implant.

13. The preparation method of the drug delivery carrier according to any one of claims 1 to 5, characterized in that, The drug delivery carrier is isolated from the cell culture solution of recombinant cells, and the recombinant membrane fusion protein and the extracellular vesicle scaffold protein are overexpressed in the recombinant cells.

14. The preparation method of the drug delivery carrier according to claim 13, wherein, The cell culture solution for culturing the recombinant cells uses a serum-free medium; and / or, the isolation is carried out by centrifugation. First, the cell culture solution of the recombinant cells is centrifuged at a centrifugal force of less than 1000 g to collect the supernatant, and then the supernatant is centrifuged at a centrifugal force of 100000 g to 200000 g to collect the precipitate, which is the drug delivery carrier.

15. A kit, characterized in that, The kit comprises the drug delivery carrier according to any one of claims 1 to 5, or the cargo-loaded extracellular vesicles according to any one of claims 6 to 10, or the pharmaceutical composition according to claim 11 or 12.

16. A recombinant membrane fusion protein, characterized in that, The recombinant membrane fusion protein is the recombinant membrane fusion protein according to any one of claims 1 to 5.

17. A nucleic acid fragment, characterized in that, The nucleic acid fragment has a nucleotide sequence encoding the recombinant membrane fusion protein according to claim 16.

18. A recombinant plasmid, characterized in that, The recombinant plasmid contains the nucleic acid fragment according to claim 17.

19. A recombinant plasmid composition, characterized in that, It comprises the recombinant plasmid according to claim 18 and a recombinant plasmid containing a nucleotide sequence encoding the extracellular vesicle scaffold protein or a recombinant plasmid containing a nucleotide sequence encoding the extracellular vesicle scaffold protein and a drug active molecule.

20. A recombinant cell, characterized in that, The recombinant cells contain the recombinant plasmid composition according to claim 19.

21. Use of the recombinant membrane fusion protein according to claim 16, characterized in that, The recombinant membrane fusion protein and the extracellular vesicle scaffold protein are used together and simultaneously for preparing a drug delivery system.

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