Drug delivery carrier based on extracellular vesicles and application thereof

Abstract

The invention relates to a drug delivery carrier based on extracellular vesicles and application thereof.The drug delivery carrier contains extracellular vesicle scaffold protein and recombinant membrane fusion protein, the recombinant membrane fusion protein comprises a first fragment and a second fragment, the amino acid sequence of the first fragment is shown as SEQ ID NO: 5, and the amino acid sequence of the second fragment is shown as SEQ ID NO: 6; the second fragment has n amino acids, the sequence of the second fragment is the sequence of the first n amino acids at the N end of the amino acid sequence as shown in SEQ ID NO: 7, and n is an integer between 50 and 200. According to the drug delivery carrier disclosed by the invention, the efficiency of releasing drug active molecules before reaching lysosome is remarkably improved, and the drug effect of extracellular vesicle drugs can be improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of molecular biology, and in particular relates to a drug delivery carrier based on extracellular vesicles and an application thereof. 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 to the outside of the cell by the fusion of multivesicular bodies in the cell with the plasma membrane. They can mediate the intercellular transfer of intracellular substances and can therefore be used as drug delivery carriers in vivo. Among them, exosomes, as a natural nanoparticle, have great potential as a drug delivery platform.

[0003] The key to determining the efficacy of extracellular vesicle drugs (drugs with extracellular vesicles as carriers) lies mainly in two links. The first link is the drug loading link. Loading more drug molecules on a single extracellular vesicle carrier can significantly improve the efficacy. At present, the field mainly improves the drug loading link by exploring efficient extracellular vesicle scaffold proteins. The second link is the release link. After the extracellular vesicle drugs are internalized and taken up by the target cells, they will reach the lysosome through the endosomal structure and eventually be degraded by various hydrolases in the lysosome. According to academic research and evaluation, only less than one in ten thousand drug molecules can be released from the endosomal structure to the cytoplasm in advance to achieve efficacy, and most drug molecules eventually reach the lysosome and are degraded. Therefore, improving the efficiency of extracellular vesicle drugs released from endosomes, thereby avoiding degradation by lysosomes, can improve the efficacy. At present, the field generally uses viral-derived membrane proteins, which are overexpressed on the surface of extracellular vesicle membranes to improve the efficiency of extracellular vesicle drugs in releasing drug active molecules before reaching lysosomes. However, the number of membrane proteins that have been reported to help extracellular vesicles release drug-active molecules is very limited, and the VSVG membrane protein of vesicular stomatitis virus, which is mainly used, is less efficient 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, which improves the efficiency of extracellular vesicles in releasing active drug molecules by adopting a novel recombinant membrane fusion protein, thereby ultimately improving the efficacy of extracellular vesicle drugs.

[0005] A first aspect of the present invention provides a drug delivery vector, which includes an extracellular vesicle, the extracellular vesicle containing an extracellular vesicle scaffold protein and a recombinant membrane fusion protein, the recombinant membrane fusion protein including 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] Furthermore, n is an integer between 60 and 100, or an integer between 101 and 180.

[0007] In an embodiment 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] Furthermore, the recombinant membrane fusion protein also includes a third fragment, the third fragment has m amino acids, and the amino acid sequence of the third fragment is the same as the sequence of m amino acids starting from the n+1th amino acid at the N-terminus of the amino acid sequence shown in SEQ ID NO:7, where m is an integer greater than 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 22, 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, 1 81, 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 amino acid sequence referred to includes not only sequences that are completely identical to the sequence but also sequences that are easily conceivable by a person skilled in the art based on the sequence and can be expected to have equivalent effects (for example, a person skilled in the art should know that sequences with high identity (for example, more than 85%, particularly more than 90%, and especially more than 95%) usually have equivalent effects).

[0014] For example, the amino acid sequence shown in SEQ ID NO: 5 refers to not only an amino acid sequence that is completely consistent with SEQ ID NO: 5, but also a fragment or variant that has the same function of helping extracellular vesicles release drugs as the amino acid sequence shown in SEQ ID NO: 5, such as an amino acid sequence that has 85% to 90% identity, 91% to 95% identity, or 96% to 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 VSVG protein (Vesicular Stomatitis Virus G protein).

[0015] For example, the amino acid sequence shown in SEQ ID NO:7 not only includes an amino acid sequence that is completely consistent with SEQ ID NO:7, but also includes fragments or variants that have the same function as the amino acid sequence shown in SEQ ID NO:7 and can be used to modify VSVG protein to enhance the function of extracellular vesicle drug release, such as an amino acid sequence that has 85% to 90% identity, 91% to 95% identity, or 96% to 100% identity with the amino acid sequence shown in SEQ ID NO:7.

[0016] According to other embodiments of the present invention, the recombinant membrane fusion protein is a VSVG protein modified with a sequence of an amino acid fragment truncated from the SDCBP protein (syndecan binding protein), wherein 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, wherein the sequence of the amino acid fragment is preferably 60 amino acids from the N-terminus, or in addition to the 60 amino acids from the N-terminus, further comprises 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 having 85% to 90% identity or 91% to 95% identity or 96% to 100% identity with them.

[0017] In an embodiment 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 in 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 fragments thereof, PTGFRN protein or fragments thereof, TSPAN2 protein or fragments thereof, TSPAN3 protein or fragments thereof, CD63 protein or fragments thereof, CD9 protein or fragments thereof, CD81 protein or fragments thereof, LEAP protein or fragments thereof, and PVR protein or fragments thereof.

[0019] The second aspect of the present invention provides an extracellular vesicle loaded with cargo, wherein 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 via a linker.

[0021] The pharmaceutical active molecules of the present invention include active molecules of preventive 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 fragments thereof, polypeptide molecules and fragments thereof, nucleic acids and fragments thereof, or small molecule compounds.

[0023] In the present invention, the connection between the active pharmaceutical molecule and the extracellular vesicle scaffold protein or between the active pharmaceutical molecule and the linker can be achieved by using connection technologies such as gene fusion, chemical synthesis, enzyme catalysis connection, self-assembly into a complex, click chemistry, protein engineering, nanotechnology, etc.

[0024] In the 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 conventionally used in the art that can connect a protein cargo or a polypeptide cargo to the polypeptide to form a fusion protein. The protein linker can be a linker conventionally used in the art that can form a protein-nucleic acid complex with a nucleic acid, a linker that can bind to a small molecule compound, 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 cargo or polypeptide cargo are overexpressed by the recombinant cell at the same time.

[0026] In 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 cargo or small molecule compound cargo is bound to the linker on the isolated extracellular vesicles.

[0027] The combination of nucleic acid cargo or small molecule compound cargo and fusion polypeptide or fusion protein is achieved by conventional technical means in the art. For example, the small molecule compound cargo can be connected to the extracellular vesicle scaffold protein through 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, and the Cas9 protein can be fused to the N-terminus and / or C-terminus of the scaffold protein. The sgRNA then forms an RNP complex with the Cas9 protein, thereby loading the sgRNA into extracellular vesicles for delivery.

[0029] For another example, when siRNA needs to be delivered, AGO2 protein can be used as a protein linker, and the AGO2 protein can be fused to the N-terminus and / or C-terminus of the scaffold protein. The siRNA can form an RNP complex with the AGO2 protein, thereby loading the siRNA into 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 used for gene editing, reporter gene proteins), enzyme proteins, etc.

[0031] In the embodiments of the present invention, the proteins used for gene editing include various proteins used in gene editing technology / engineering, including but not limited to various recombinases that can promote the recombination or rearrangement of specific sequences between DNA molecules and gene editing system-related proteins (i.e., proteins used in various gene editing systems). Known common 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 proteins, such as antibodies, cytokines, anti-inflammatory proteins or polypeptides, serum albumin, lectins, 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, such as DNA or mRNA molecules encoding proteins or polypeptides, RNA molecules with regulatory functions such as miRNA, antisense oligonucleotides ASO or siRNA, sgRNA of CRISPR system, and even plasmid DNA.

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

[0035] In some embodiments of the present invention, 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.

[0036] In some embodiments of the invention, the pharmaceutically active molecule is a protein molecule and a fragment thereof or a polypeptide molecule and a fragment thereof, and the pharmaceutically active molecule is directly or via 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 a fragment thereof or a small molecule compound, 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 drug delivery vector or the extracellular vesicles 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 intended mode of administration and therapeutic application. 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, pastes, 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 vector, wherein the drug delivery vector is separated from the cell culture fluid of a recombinant cell, wherein the recombinant cell overexpresses the recombinant membrane fusion protein and the extracellular vesicle scaffold protein.

[0042] Furthermore, the preparation method includes the step of transfecting a host cell with a recombinant expression vector to obtain the recombinant cell, wherein the recombinant expression vector includes a first recombinant plasmid containing a gene encoding the recombinant membrane protein and a second recombinant plasmid containing a gene encoding the extracellular vesicle scaffold protein.

[0043] In the embodiments of the present invention, the recombinant cells involved in the preparation method are 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, thereby fusing the extracellular vesicle scaffold protein with the cargo. Whether a linker is needed can be determined based on the structure, function, stability, etc. of the extracellular vesicle scaffold protein and the effective substance.

[0045] In other embodiments of the present invention, the cargo is a nucleic acid cargo (e.g., 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 the protein linker and the nucleic acid cargo form a complex through non-covalent interactions. There is no particular limitation on whether the cargo encoding gene is connected to the extracellular vesicle scaffold protein or the linker encoding gene, as long as it can be expressed in a recombinant cell.

[0046] In other embodiments of the present invention, when the cargo is a nucleic acid cargo or a small molecule compound cargo, the prepared extracellular vesicles overexpressing a 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 and the linker can be combined to form a complex.

[0047] In the embodiments of the present invention, the expression vector used may be an expression vector conventionally used in the art, including a viral plasmid vector and a eukaryotic expression plasmid vector, and a viral plasmid vector is 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 an "extracellular vesicle scaffold protein" or a fusion polypeptide or fusion protein of a "extracellular vesicle scaffold protein-linker" or a fusion protein of a "extracellular vesicle scaffold protein-cargo" or a fusion protein of a "extracellular vesicle scaffold protein-linker-cargo" or a nucleic acid-protein complex into cells for expression, thereby obtaining a fusion protein formed by simultaneously overexpressing the recombinant protein and the extracellular vesicle scaffold protein or a fusion protein formed by simultaneously overexpressing the recombinant protein and the extracellular vesicle scaffold protein and the linker or a fusion protein formed by the extracellular vesicle scaffold protein and the cargo.

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

[0050] In the embodiment of the present invention, the host cell used can be any suitable cell type, including mammalian cells, 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 to culture the recombinant cells is a serum-free culture medium.

[0052] In an embodiment of the present invention, extracellular vesicle separation and purification methods known in the art can 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 technology, immunoseparation technology, or sieving separation.

[0053] According to some embodiments of the present invention, the separation is carried out by centrifugation. The cell culture fluid of the recombinant cells is first centrifuged at a centrifugal force of less than 1000g to collect the supernatant, and then the supernatant is centrifuged at a centrifugal force of 100000g~200000g to collect the precipitate, which is the drug delivery vector or the extracellular vesicle loaded with cargo.

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

[0055] A fifth aspect of the present invention provides a kit, which comprises the above-mentioned drug delivery vector, or the above-mentioned extracellular vesicles loaded with cargo, or the above-mentioned pharmaceutical composition.

[0056] A 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, which contains 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, which includes 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 pharmaceutically active molecule.

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

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

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

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

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

[0065] A twelfth aspect of the present invention provides a method for diagnosing, preventing or treating a disease, the method comprising administering the above-mentioned cargo-loaded extracellular vesicles or the above-mentioned pharmaceutical composition to a subject.

[0066] In the embodiment of the present invention, it is preferred to determine the specific disease to be detected, prevented or treated by the extracellular vesicles loaded with cargo based on the active pharmaceutical molecules 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: The extracellular vesicle-based drug delivery vector of the present invention comprises extracellular vesicles that overexpress recombinant membrane fusion proteins, which can significantly improve the efficiency of extracellular vesicles in releasing active drug molecules before reaching lysosomes, thereby promoting the improvement of the efficacy of extracellular vesicle drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0068] Figure 1 This is a particle size distribution diagram of the exosomes loaded with cGAS pro-inflammatory protein in Example 1; Figure 2 This is the particle size distribution diagram of the exosomes loaded with cGAS pro-inflammatory protein in Comparative Example 1; Figure 3 This is a transmission electron micrograph of the exosomes loaded with cGAS pro-inflammatory protein in Example 1; Figure 4 This is a transmission electron microscopy image of the exosomes loaded with cGAS pro-inflammatory protein in Comparative Example 1; Figure 5 It is a comparison diagram of the cargo delivery effect of the exosomes loaded with cGAS pro-inflammatory protein of Example 1 and Comparative Example 1; Figure 6 This is the particle size distribution diagram of the exosomes loaded with Cas9 protein in Example 2; Figure 7 This is the particle size distribution diagram of the exosomes loaded with Cas9 protein in Comparative Example 2; Figure 8 This is a transmission electron micrograph of the exosomes loaded with Cas9 protein in Example 2; Fig. 9 This is a transmission electron microscopy image of exosomes loaded with Cas9 protein in Comparative Example 2; Fig.10 This is a comparison diagram of the cargo delivery effect of the exosomes of the Cas9 protein of Example 2 and Comparative Example 2; Fig.11 This is the particle size distribution diagram of the exosomes loaded with Cre recombinase in Example 3; Fig.12 This is the particle size distribution diagram of the exosomes loaded with Cre recombinase in Comparative Example 3; Fig.13 This is a transmission electron microscopy image of the exosomes loaded with Cre recombinase in Example 3; Fig.14 This is a transmission electron microscopy image of the exosomes loaded with Cre recombinase in Comparative Example 3; Fig.15 This is a comparison diagram of the cargo delivery effects of the exosomes loaded with Cre recombinase in Example 3 and Comparative Example 3; Fig.16 This is the particle size distribution diagram of the exosomes loaded with Cre recombinase in Example 4; Fig.17This is the particle size distribution diagram of the exosomes loaded with Cre recombinase in Comparative Example 4; Fig.18 This is a transmission electron microscopy image of the exosomes loaded with Cre recombinase in Example 4; Fig.19 This is a transmission electron microscopy image of the exosomes loaded with Cre recombinase in Comparative Example 4; Fig. 20 This is a comparison chart of the cargo delivery effects of exosomes loaded with Cre recombinase in Example 4 and Comparative Example 4. DETAILED DESCRIPTION

[0069] The key to achieving functional delivery of extracellular vesicles (such as exosomes) requires efficient endosomal escape to release the loaded drugs into the cytoplasm to avoid degradation by lysosomes. In this field, viral-derived membrane fusion proteins such as VSVG membrane proteins are generally 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. After a lot of research and experimental verification, the inventors of this application found that the recombinant protein obtained by modifying the VSVG membrane protein through the modified sequence can significantly enhance the release efficiency of the drug.

[0070] The modified sequence includes the amino acid sequence shown in SEQ ID NO:7 or a fragment thereof, or a sequence having more than 85% identity therewith. The recombinant protein after the VSVG protein is modified by this modified sequence and the exosome scaffold protein (not limited to the specific scaffold protein) are used together for drug delivery based on extracellular vesicles, which significantly improves the efficiency of loading and delivering Cre recombinase, Cas9 protein and cGAS pro-inflammatory protein to the cytoplasm. The first 60 amino acids at the N-terminus of the amino acid sequence shown in SEQ ID NO:7 constitute a domain responsible for interacting with the biological membrane. In the embodiment of the present invention, modified sequences of different lengths containing this domain can be used to modify VSVG protein to enhance drug release. In practical applications, similar sequences of appropriate length can be selected to modify VSVG protein according to the size of the cargo or the loading capacity requirements.

[0071] The sequence of the amino acid fragment is preferably 60 amino acids starting from the N-terminus, or in addition to the 60 amino acids starting from the N-terminus, it further comprises more amino acids or amino acid sequences, for example, amino acids or amino acid sequences selected from the remaining fragments of the SDCBP protein.

[0072] Unless otherwise specified or contradictory to the context, the terms or expressions used herein should be read in conjunction 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.

[0073] "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.

[0074] "Exosomes" refer to cell-derived extracellular vesicles (EVs) with a diameter of 30~150nm, which include a membrane that encloses the internal space. The membrane is a phospholipid bilayer structure. Exosomes can mediate the intercellular transfer of intracellular substances.

[0075] "VSVG membrane protein" is the glycoprotein (G protein) of vesicular stomatitis virus, which is a single-transmembrane protein composed of 511 amino acid residues.

[0076] “Loading” refers to the attachment and enrichment of cargo into extracellular vesicles, including various forms of cargo in the lumen of extracellular vesicles, on the membrane of extracellular vesicles, or attached to the outside of the membrane of extracellular vesicles.

[0077] “Release” refers to the release of cargo attached and enriched in extracellular vesicles into the cytoplasm.

[0078] "Extracellular vesicle scaffold protein" refers to a linker used to connect a cargo to an extracellular vesicle, which is used to anchor any cargo of interest to an extracellular vesicle.

[0079] "Cargo" mainly refers to "pharmaceutically active molecules", i.e., proteins and fragments thereof, polypeptides and fragments thereof, nucleic acids and fragments thereof, or small molecule compounds of any functional interest (e.g., preventive, therapeutic, diagnostic, etc.). "Cargo" is also often referred to as "functional cargo" or "effective substance".

[0080] "Small molecule compound" refers to a compound with a molecular size less than 1000 molecular weight, including conventional organic compounds and inorganic compounds.

[0081] "Polypeptide" and "oligopeptide" are defined according to the length of the peptide chain. Those with a length of 10 to 100 amino acids are called "polypeptides", and those with a length of less than 10 amino acids are called "oligopeptides".

[0082] "Recombinase" is a class of enzymes that can promote the recombination or rearrangement of modified sequences between DNA molecules. Common recombinases can be loaded and delivered using 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.

[0083] "Cytokines" are a class of small molecule proteins with a wide range of biological activities that are 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 effects by binding to corresponding receptors, and regulate immune responses. They have multiple functions such as regulating innate immunity and adaptive immunity, hematopoiesis, cell growth, and damaged tissue repair. Common cytokines include, but are not limited to, pro-inflammatory cytokines, anti-inflammatory cytokines, interleukins, interferons, growth factors, and chemokines.

[0084] "Gene editing proteins" are 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.

[0085] "Exogenous nucleic acid fragments" include any gene or fragment thereof of interest, such as a gene or fragment thereof encoding a scaffold protein. Exogenous nucleic acid fragments have a different origin from the host cell, for example, nucleic acid sequences isolated from an organism different from the host cell, i.e., are exogenous nucleic acid fragments relative to the host cell.

[0086] "Plasmid" refers to a circular double-stranded DNA that is capable of accepting foreign nucleic acid fragments and replicating in prokaryotic or eukaryotic cells.

[0087] "Promoter" refers to a nucleic acid sequence that can control the transcription of a coding sequence. The promoter sequence includes a modified sequence that is sufficient to allow RNA polymerase to recognize, bind and initiate transcription. A 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.

[0088] "Host cell" is also called recipient cell, including but not limited to animal cells, plant cells, algae cells, fungal cells, yeast cells, or bacterial cells. Exemplary host cells include human embryonic kidney cells HEK293. It should be understood that the offspring of a single parental cell may not be completely identical to the original parent in morphology or in terms of genome or total DNA complement due to natural, accidental or intentional mutations.

[0089] The "cGAS pro-inflammatory protein" is a major sensor of cytoplasmic DNA and a pro-inflammatory cytokine that can activate STING, induce type 1 IFN expression and IFN stimulation.

[0090] "Cas9 protein" is a nuclease in the CRISPR-Cas system that can specifically cut DNA molecules.

[0091] "Cre recombinase" is a protein encoded by a 1029bp sequence in the genome of bacterial phage P1. It consists of 343 amino acids and has a molecular weight of 3815kD. 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.

[0092] The technical solution of the present invention is further described below in conjunction with specific embodiments, but the present invention is not limited to the following embodiments.

[0093] The implementation conditions used in the following examples and comparative examples can be further adjusted according to specific requirements, and the implementation conditions not specified are usually the conditions in conventional experiments. The instruments, raw materials and reagents not specifically specified can be obtained commercially.

[0094] Example 1 This embodiment provides an exosome loaded with cGAS pro-inflammatory protein. In this embodiment, Basp1 protein is used as the exosome scaffold protein, and VSVG membrane protein modified by modification sequence helps exosomes release cGAS pro-inflammatory protein. The preparation method of exosomes loaded with cGAS pro-inflammatory protein in this embodiment is as follows: 1. Construction of Plasmid The first plasmid was constructed using pIRES plasmid as an expression vector and Basp1 protein as an exosome scaffold protein. The expression frame of the first plasmid was designed as follows: CMV promoter (sequence shown in SEQ ID NO: 1) was used as the transcription start point, SV40 polyA (sequence shown in SEQ ID NO: 2) was used as the transcription end point, and cGAS pro-inflammatory protein (sequence shown in SEQ ID NO: 4) was connected to the C-terminus of Basp1 protein (sequence shown in SEQ ID NO: 3).

[0095] The pIRES plasmid was used as an expression vector to construct a second plasmid. The expression frame of the second plasmid was designed as follows: using a CMV promoter (sequence as shown in SEQ ID NO: 1) as the transcription start point, using SV40 polyA (sequence as shown in SEQ ID NO: 2) as the transcription end point, and connecting a modified sequence (sequence as shown in SEQ ID NO: 6, which is consistent with the first 60 amino acids of the N-terminal of the SDCBP protein as shown in SEQ ID NO: 7) to the C-terminus of the VSVG membrane protein (sequence as shown in SEQ ID NO: 5).

[0096] The first plasmid and the second plasmid were prepared according to the following specific steps: (1) PCR cloning: Use conventional methods in the art to design upstream primers and downstream primers according to the expression frame, and synthesize upstream primers, downstream primers and template DNA. In a 0.2mL EP tube, add 25μL DNA polymerase (2×Phanta MaxMaster Mix, Vazyme, P515-01), 2μL upstream primer, 2μL downstream primer, 10g template DNA, and add enzyme-free water (RNase-free dd H2O, Vazyme, P071-01-AA) to 50μL, mix well, and put into the PCR instrument, and design the amplification program according to the primer annealing temperature.

[0097] (2) Purification of PCR products: Purify the PCR products according to the kit instructions (Vazyme, DC301-01).

[0098] (3) Enzyme digestion: Add 5 μL rCutSmart TM Buffer (BioLabs, 136004s), 0.5μL Pac I enzyme (BioLabs, R0547L), 0.5μL Not I enzyme (BioLabs, R3189L), 2μg PCR purified fragment, 5μg pAAVS1 vector plasmid. Add enzyme-free water (RNase-free ddH2O, Vazyme, P071-01-AA) to 10μL, mix well, and place in a PCR instrument for enzyme digestion at 37℃ for 16h.

[0099] (4) Ligation transformation: In a 0.2 mL EP tube, add 4 μL of the digestion 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 out 5 μL and add it to 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 LB medium (BeyoPure™, 04.05.ST156) and culture in a shaking incubator for 1 h. After culture, centrifuge at 12000g for 1 min, remove the supernatant and keep 100 μL, resuspend and add to the bacterial culture dish (White Shark Biosharp, BS-90-D), then use a disposable plastic coating rod (White Shark Biosharp, BS-PS-A) to spread evenly and store in a 37°C constant temperature incubator for later use.

[0100] Wherein, in step (1), the upstream primer sequence of the first plasmid is shown as SEQ ID NO: 8, the downstream primer sequence is shown as SEQ ID NO: 9, and the template DNA sequence is shown as SEQ ID NO: 10. The upstream primer sequence of the second plasmid is shown as SEQ ID NO: 11, the downstream primer sequence is shown as SEQ ID NO: 12, and the template DNA sequence is shown as SEQ ID NO: 13.

[0101] 2. Cell Transfection The specific steps are as follows: (1) Prepare cells for transfection EXPI293F cells were inoculated into a 125 mL shake flask at 1.5E+6 / mL with a survival rate of more than 95%, and the total volume of the culture medium was 25 mL. The cells were placed in an 8% CO2 constant temperature shaker for overnight culture, and transfection was started the next day.

[0102] (2) PEI transfection Using TA293 reagent (Zhuhai Kairui Biotechnology Co., Ltd., K2001), taking a volume of 25 mL of cell suspension as an example, the transfection process is as follows: The target plasmid is extracted from the transformed competent cells preserved by the above-mentioned coating culture by conventional technical means in the art, that is, the constructed first plasmid and the second plasmid are obtained, and they are mixed in equal mass for standby use. Prepare two 15mL centrifuge tubes, add 1.25mL serum-free cell culture medium, 25μg of the first plasmid and the second plasmid mixture to one of them, and vortex mix; add 1.25mL serum-free cell culture medium and 125μL TA293 transfection reagent to the other, and vortex mix. Transfer all the liquid in the centrifuge tube containing the transfection reagent to the centrifuge tube containing the plasmid and vortex mix. 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 dropwise while shaking, and then put it back into the CO2 constant temperature shaker for shaking and culture for 72 hours.

[0103] 3. Isolation and purification of exosomes After 72 hours of shaking culture, the cell culture fluid was first centrifuged at 300g, 4°C, for 5 minutes, and the supernatant was collected. The supernatant was then ultracentrifuged at 133900g, 4°C for 1 hour to collect the precipitate. The precipitate (i.e., the isolated exosomes) was resuspended in 100 μL PBS and stored at 4°C for later use.

[0104] Comparative Example 1 This comparative example provides an exosome loaded with cGAS pro-inflammatory protein. In this comparative example, Basp1 protein is used as an exosome scaffold protein, and VSVG membrane protein is used to help exosomes release cGAS pro-inflammatory protein. In this comparative example, the design of the expression frame of the first plasmid is the same as in Example 1. The expression frame of the second plasmid in this comparative example is obtained by deleting the modified sequence connected to the C-terminus of the VSVG membrane protein in the expression frame of the second plasmid in Example 1. In addition to redesigning the upstream primer (sequence as shown in SEQ ID NO: 11), the downstream primer (sequence as shown in SEQ ID NO: 14) and the template DNA (sequence as shown in SEQ ID NO: 15) of the second plasmid according to the adjusted expression frame, the rest of the preparation method of this comparative example is referred to Example 1, and finally exosomes loaded with cGAS pro-inflammatory protein are obtained.

[0105] Example 2 This embodiment provides an exosome loaded with Cas9 protein. In this embodiment, Basp1 protein is used as an exosome scaffold protein, and the VSVG membrane protein modified by the modified sequence helps the exosome release the Cas9 protein. Exosomes loaded with Cas9 protein 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 Cas9 protein with an amino acid sequence as shown in SEQ ID NO:16. In addition to redesigning and synthesizing the upstream primer (sequence as shown in SEQ ID NO:8), the downstream primer (sequence as shown in SEQ ID NO:17) and the template DNA (sequence as shown in SEQ ID NO:18) of the first plasmid according to the adjusted expression frame, the rest is referred to Example 1, and finally exosomes loaded with Cas9 protein are obtained.

[0106] Comparative Example 2 This comparative example provides an exosome loaded with Cas9 protein. In this comparative example, Basp1 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 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 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 Cas9 protein are obtained.

[0107] Example 3 This embodiment provides an exosome loaded with Cre recombinase. In this embodiment, Basp1 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. In addition to redesigning and synthesizing the upstream primer (sequence as shown in SEQ ID NO:8), the downstream primer (sequence as shown in SEQ ID NO:20) and the template DNA (sequence as shown in SEQ ID NO:21) of the first plasmid according to the adjusted expression frame, the rest is referred to Example 1, and finally exosomes loaded with Cre recombinase are obtained.

[0108] Comparative Example 3 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.

[0109] Example 4 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.

[0110] Comparative Example 4 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.

[0111] Example 5 This embodiment provides an exosome loaded with sgRNA, the sequence of sgRNA is shown in SEQ ID NO: 25, the sgRNA is mixed with the exosome loaded with Cas9 protein of Example 2, the Cas9 protein is used as a protein linker, the sgRNA and the Cas9 protein form an RNP complex, so that the sgRNA is loaded on the exosome, and the exosome loaded with sgRNA is obtained.

[0112] Example 6 This embodiment provides an exosome loaded with siRNA, wherein the sense strand sequence of the siRNA is shown in SEQ ID NO: 26, the antisense strand sequence is shown in SEQ ID NO: 27, and the AGO2 protein (sequence is shown in SEQ ID NO: 28) is used as a protein linker. The exosome loaded with the AGO2 protein is first prepared by referring to the method of Example 1, and then the siRNA is mixed with the exosome loaded with AGO2, and the siRNA and the AGO2 protein form an RNP complex, thereby loading the siRNA onto the exosome, and obtaining the exosome loaded with siRNA.

[0113] Example 7 This embodiment 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 preparing exosomes loaded with FKBP12 protein with reference to the method of Example 1, and then mixing rapamycin with the exosomes loaded with FKBP12 protein, so that rapamycin and FKBP12 protein form a complex, thereby loading rapamycin onto the exosomes, and obtaining exosomes loaded with the small molecule compound.

[0114] Example 8 This embodiment provides a pharmaceutical composition for improving the immune effect, which is prepared from the exosomes loaded with cGAS pro-inflammatory protein in Example 1 and normal saline into an injection solution with a particle concentration of 1.0E12 particles / mL, and administered to experimental mice by intramuscular injection. Because the exosomes 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 multiple inflammatory factors, the pharmaceutical composition of this embodiment can enhance the body's anti-tumor, anti-viral and other immune effects, and can be used as a vaccine adjuvant to improve the efficacy of vaccines.

[0115] (1) Exosome identification: The exosomes resuspended in the above examples and comparative examples were subjected to NTA analysis using a ZetaView instrument (Fuliu Bio Particle Metrix, catalog number N30E). The particle size distribution of the exosomes loaded with cGAS pro-inflammatory protein in Example 1 is shown in Figure 1 The particle size distribution of the exosomes loaded with cGAS pro-inflammatory protein in Comparative Example 1 is shown in Figure 2 The particle size distribution of the exosomes loaded with Cas9 protein in Example 2 is shown in Figure 6 The particle size distribution of the exosomes of Cas9 protein in comparative example 2 is shown in Figure 7 The particle size distribution of the exosomes loaded with Cre recombinase in Example 3 is shown in Fig.11 The particle size distribution of the exosomes loaded with Cre recombinase in comparative example 3 is shown in Fig.12 The particle size distribution of the exosomes loaded with Cre recombinase in Example 4 is shown in Figure 6 The particle size distribution of the exosomes loaded with Cre recombinase in Comparative Example 4 is shown in Fig.17 The average particle size and median particle size are shown in Table 1.

[0116]

[0117] Table 1 shows that the particle sizes of the cargo-loaded exosomes of Examples 1 to 4 and Comparative Examples 1 to 4 are all around 120 to 150 nm.

[0118] (2) The structures of the exosomes prepared in the above examples and comparative examples were observed under a transmission electron microscope (TEM). Figure 3 , Figure 4 , Figure 8 , Fig. 9 , Fig.13 , Fig.14 , Fig.18 and Fig.19 As shown, the cargo-loaded exosomes of Examples 1 to 4 and Comparative Examples 1 to 4 all showed typical exosome vesicle structures (cup-shaped or saucer-shaped) under an electron microscope.

[0119] (3) Analysis of exosome cargo delivery efficiency: The exosomes loaded with cGAS pro-inflammatory protein prepared in Example 1 and Comparative Example 1 were added to peripheral blood mononuclear cells (PBMC) in equal amounts (same number of exosome particles). Under normal circumstances, PBMC cells do not have an inflammatory response and do not secrete inflammatory cytokines; however, in PBMC cells where cGAS pro-inflammatory protein is successfully delivered, the STING pathway is activated and an inflammatory response is produced, so PBMC cells secrete inflammatory cytokines such as TNF-α and IL-6. Figure 5 As shown, after the PBMC cells received the exosomes of Example 1, the amount of TNF-α and IL-6 secreted was significantly higher than that of Comparative Example 1, and the exosomes of Example 1 had a higher delivery efficiency for the cGAS pro-inflammatory protein. Because Example 1 and Comparative Example 1 used the same exosome scaffold protein, the effect of loading efficiency on delivery efficiency was eliminated. Compared with VSVG membrane protein, the VSVG membrane protein modified by the modified sequence can help exosomes release more cGAS pro-inflammatory proteins into the cytoplasm, thereby making the exosomes of Example 1 more efficient in delivering cGAS pro-inflammatory proteins.

[0120] 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 (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 the reporter cells to which Cas9 protein was successfully delivered randomly corrected the GFP gene with +1 or +2 frameshift mutations, and the reporter cells expressed green fluorescent protein. Fig.10 As shown, the expression of green fluorescent protein in the cells using the exosomes of Example 2 to deliver the Cas9 protein is significantly better than that of Comparative Example 2, and the exosomes of Example 2 have a higher delivery efficiency for the Cas9 protein. Because Example 2 and Comparative Example 2 use the same exosome scaffold protein, the effect of loading efficiency on delivery efficiency is eliminated. Compared with VSVG membrane protein, the VSVG membrane protein modified by the modified sequence can help exosomes release more Cas9 protein into the cytoplasm, thereby making the exosomes of Example 2 more efficient in delivering the Cas9 protein.

[0121] The exosomes loaded with Cre recombinase in Example 3 and Comparative Example 3 were added to the reporter cells for detecting Cre recombinase in equal amounts (same number of exosome particles). The genome of the reporter cells for detecting Cre recombinase contains both green fluorescent protein and red fluorescent protein, wherein the expression of red fluorescent protein is silent. Before the delivery of Cre recombinase, only green fluorescent protein is expressed. Cre recombinase-mediated recombination 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 cell nucleus. Fig.15As shown, the expression of red fluorescent protein in the cells using the exosomes of Example 3 to deliver Cre recombinase is significantly better than that of Comparative Example 3, and the exosomes of Example 3 have a higher delivery efficiency for Cre recombinase. Because Example 3 and Comparative Example 3 use the same exosome scaffold protein, the effect of loading efficiency on delivery efficiency is eliminated. Compared with VSVG membrane protein, the VSVG membrane protein modified by the modified sequence can help exosomes release more Cre recombinase into the cytoplasm, thereby making the exosomes of Example 3 more efficient in delivering Cre recombinase.

[0122] The exosomes loaded with Cre recombinase of Example 4 and Comparative Example 4 were added to the reporter cells for detecting Cre recombinase in equal amounts (same number of exosome particles). The genome of the reporter cells for detecting Cre recombinase contains both green fluorescent protein and red fluorescent protein, wherein the expression of red fluorescent protein is silent. Before the delivery of Cre recombinase, only green fluorescent protein is expressed. Cre recombinase-mediated recombination will activate the expression of red fluorescent protein, and 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 the scaffold proteins used in Example 3 and Comparative Example 3, there are differences in the loading efficiency of Cre recombinase in Example 4 and Example 3, but as Fig. 20 As shown, the expression of red fluorescent protein in the cells in which the exosomes of Example 4 delivered Cre recombinase was significantly better than that in Comparative Example 4, indicating that compared with Comparative Example 4, the exosomes of Example 4 had a higher delivery efficiency for Cre recombinase. This comparison result was consistent with the comparison results of Example 3 and Comparative Example 3, indicating that for exosomes loaded with cargo using different scaffold proteins, the VSVG membrane proteins modified by the modified sequence can help improve the efficiency of exosome release of cargo.

[0123] The above embodiments and comparative examples show that the present invention modifies the VSVG membrane protein by modifying the sequence, and adds the modified sequence to the end of VSVG to obtain a recombinant membrane fusion protein, which can help improve the efficiency of exosomes in releasing cargo, thereby improving the efficiency of exosomes in delivering cargo to the cytoplasm, and further improving the efficacy of exosome drugs.

[0124] The above detailed description of the present invention is intended to enable those skilled in the art to understand the contents of the present invention and implement them, but it does not limit the protection scope of the present invention. The present invention is not limited to the above embodiments, and any equivalent changes or modifications made according to the spirit 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 an extracellular vesicle, the extracellular vesicle 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, the second fragment has n amino acids, and the amino acid 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.

2. The drug delivery carrier according to claim 1, characterized in that The n is an integer between 60 and 100 or an integer between 101 and 180.

3. The drug delivery carrier according to claim 1, characterized in that The second fragment is connected to the C-terminus of the first fragment; And / or, the recombinant membrane fusion protein consists of the first fragment and the second fragment.

4. The drug delivery carrier according to claim 1, characterized in that The recombinant membrane fusion protein also includes a third fragment, which has m amino acids. The amino acid sequence of the third fragment is a sequence of m amino acids starting from the n+1th amino acid at the N-terminus of the amino acid sequence shown in SEQ ID NO:7, where m is an integer greater than 2.

5. The drug delivery carrier according to claim 4, characterized in that The third fragment is connected to the C-terminus of the second fragment; And / or, the recombinant membrane fusion protein consists of the first fragment, the second fragment and the third fragment; And / or, the total amino acid length n+m of the second fragment and the third fragment is 60-100 or 101-200 or 201-298.

6. The drug delivery carrier according to claim 1, characterized in that The extracellular vesicles are exosomes, microvesicles or apoptotic bodies.

7. The drug delivery carrier according to claim 1, characterized in that The extracellular vesicle scaffold protein includes one or more of BASP1 protein or a fragment thereof, PTGFRN protein or a fragment thereof, TSPAN2 protein or a fragment thereof, TSPAN3 protein or a fragment thereof, CD63 protein or a fragment thereof, CD9 protein or a fragment thereof, CD81 protein or a fragment thereof, LEAP protein or a fragment thereof, and PVR protein or a fragment thereof.

8. An extracellular vesicle loaded with cargo, wherein the cargo includes a pharmaceutically active molecule, characterized in that: The cargo-loaded extracellular vesicle comprises the drug delivery carrier according to any one of claims 1 to 7 and the pharmaceutically active molecule, wherein the pharmaceutically active molecule is directly or indirectly connected to the extracellular vesicle scaffold protein through a linker.

9. The cargo-loaded extracellular vesicle according to claim 8, characterized in that The pharmaceutically active molecules include one or more of protein molecules and fragments thereof, polypeptide molecules and fragments thereof, nucleic acids and fragments thereof or small molecule compounds.

10. The cargo-loaded extracellular vesicle according to claim 9, characterized in that The pharmaceutically active molecule is a cytokine receptor protein, a protein for gene editing, a cytokine or RNA.

11. The cargo-loaded extracellular vesicle according to claim 10, characterized in that The active drug 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.

12. The cargo-loaded extracellular vesicle according to claim 9, characterized in that The pharmaceutically active molecule is a protein molecule and a fragment thereof or a polypeptide molecule and a fragment thereof, and the pharmaceutically active molecule is connected to the N-terminus and / or C-terminus of the extracellular vesicle scaffold protein directly or through a polypeptide linker; And / or, the pharmaceutically active molecule is a nucleic acid and a fragment thereof or a small molecule compound, the pharmaceutically active molecule and the linker combine to form a complex, and the linker is connected to the N-terminus and / or C-terminus of the extracellular vesicle scaffold protein.

13. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the drug delivery vector according to any one of claims 1 to 7 or the cargo-loaded extracellular vesicle according to any one of claims 8 to 12, and the pharmaceutical composition may optionally comprise a pharmaceutically acceptable excipient.

14. The pharmaceutical composition according to claim 13, characterized in that The pharmaceutical composition is in the form of tablets, granules, pills, capsules, emulsions, pastes, gels, suspensions, solutions, powders, transdermal patches, sprays, suppositories or implants.

15. The method for preparing a drug delivery carrier according to any one of claims 1 to 7, characterized in that: The drug delivery carrier is separated from the cell culture fluid of the recombinant cells, and the recombinant cells overexpress the recombinant membrane fusion protein and the extracellular vesicle scaffold protein.

16. The method for preparing a drug delivery carrier according to claim 15, characterized in that: The cell culture medium used to culture the recombinant cells is a serum-free medium; And / or, the separation is carried out by centrifugation, first centrifuging the cell culture fluid of the recombinant cells at a centrifugal force of less than 1000g, collecting the supernatant, and then centrifuging the supernatant at a centrifugal force of 100000g~200000g to collect the precipitate, which is the drug delivery vector.

17. A kit, characterized in that: The kit comprises the drug delivery vector according to any one of claims 1 to 7, or the cargo-loaded extracellular vesicle according to any one of claims 8 to 12, or the pharmaceutical composition according to claim 13 or 14.

18. A recombinant membrane fusion protein, characterized in that: The recombinant membrane fusion protein is the recombinant membrane fusion protein described in any one of claims 1 to 7.

19. A nucleic acid fragment, characterized in that The nucleic acid fragment has a nucleotide sequence encoding the recombinant membrane fusion protein according to claim 18.

20. A recombinant plasmid, characterized in that: The recombinant plasmid contains the nucleic acid fragment according to claim 19.

21. A recombinant plasmid composition, characterized in that: It comprises the recombinant plasmid according to claim 20 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 pharmaceutically active molecule.

22. A recombinant cell, characterized in that The recombinant cell contains the recombinant plasmid composition according to claim 21.

23. The use of the recombinant membrane fusion protein according to claim 18, characterized in that: The recombinant membrane fusion protein and the extracellular vesicle scaffold protein are used together and simultaneously for drug delivery.

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