Exosome scaffold protein and use thereof

Abstract

The present invention relates to an exosome scaffold protein and the use thereof. PVR protein is used as an exosome scaffold protein to load effective substances, such that the loading capacity and the loading efficiency of the effective substances are improved. In addition, the exosome scaffold protein can also be used for loading larger and more structurally complex effective substances.

Description

Exosome scaffold proteins and their applications Technical Field

[0001] The present invention belongs to the technical field of molecular biology, and specifically relates to an exosome scaffold protein and applications thereof. Background Art

[0002] Exosomes are small vesicles secreted by cells, with a membrane structure composed of a phospholipid bilayer. They possess a variety of specific membrane proteins on their surface and carry a variety of important biological signaling substances such as proteins, RNA, and DNA. They are widely distributed in various body fluids, including blood, urine, cerebrospinal fluid, saliva, milk, and bile, and serve as important intermediaries for intercellular communication.

[0003] Exosomes are naturally occurring nanostructures with enormous potential as drug delivery platforms and have been extensively explored and studied as a new therapeutic modality across numerous disease areas. Compared to some traditional drug delivery methods, exosomes offer unique advantages in several areas. For example, exosomes derived from different cell sources have varying tissue and organ targeting properties due to differences in their surface physicochemical properties, demonstrating potential for precision medicine. Compared to synthetic lipid nanoparticles, exosomes are less immunogenic and have improved biosafety.

[0004] How to load therapeutic drugs, especially biomacromolecules such as interleukins, antibodies, and cytokines, onto exosomes is a core difficulty in the field of innovative exosome drugs. Existing methods include fusing biomacromolecules with exosome-enriched sequences through cell-level engineering modifications, so that the fusion proteins are enriched on exosomes. These exosome-enriched sequences, because they act as scaffolds, are also figuratively called exosome scaffold proteins. Currently used exosome scaffold protein sequences include glycosylphosphatidylinositol anchor sequences (GPI anchors), lipid anchor sequences (lipid anchors), and known exosome marker proteins including CD9, CD63, CD81, and LAMP2B. However, drugs developed based on these exosome scaffold proteins have low loading efficiency of active substances on exosomes, resulting in low biological activity.

[0005] Summary of the Invention

[0006] The problem to be solved by the present invention is to develop a new type of scaffold protein with higher loading efficiency and exosomes loaded with effective substances.

[0007] To solve the above problems, the present invention provides, in a first aspect, exosomes loaded with effective substances, comprising exosomes, a PVR protein overexpressed in the exosomes, and an effective substance connected to the PVR protein.

[0008] According to some specific embodiments of the present invention, the effective substance is loaded on the outer surface of the exosomes.

[0009] According to some specific embodiments of the present invention, the nucleotide sequence encoding the PVR protein is shown as SEQ ID NO: 1.

[0010] Those skilled in the art will understand that, unless otherwise specified, reference to a PVR protein encompasses not only the PVR protein itself but also fragments, variants, or derivatives thereof. A PVR protein, when a fragment, variant, or derivative thereof, typically has an amino acid sequence that is more than 70% identical to the full-length amino acid sequence of the PVR protein, and specifically, for example, may have an identity of 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%, or 100%.

[0011] According to some embodiments of the present invention, the active substance is linked directly or indirectly via a linker to the N-terminus or C-terminus of the PVR protein. Preferably, the active substance is linked to the N-terminus of the PVR protein to form a fusion protein, which facilitates the location of the active substance in the overexpressed fusion protein outside the phospholipid bilayer.

[0012] In the present invention, the effective substance can be linked to the N-terminus or C-terminus of the PVR protein via a polypeptide linker, which can be a cleavable linker or a flexible linker commonly used in the art.

[0013] According to some embodiments of the present invention, the effective substance includes one or more of proteins, polypeptides, carbohydrates, nucleic acids or small molecule compounds.

[0014] In the embodiments of the present invention, the proteins include, but are not limited to, various therapeutic proteins, immunogenic proteins, or functional proteins (e.g., gene editing system proteins, reporter gene proteins). They may also include various functional lipids, DNA or RNA editing protein complexes, RNA and DNA binding proteins, cytokines, toxins, and the like.

[0015] In the embodiments of the present invention, the polypeptides include but are not limited to various therapeutic polypeptides, targeting polypeptides or partial polypeptide fragments of the above proteins, such as enzymes, antibodies, interleukins, serum albumin, lectins, coagulation factors, etc.

[0016] 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, dsDNA, antisense oligonucleotides (ASOs), lncRNA, or siRNA.

[0017] In the present invention, the small molecule compounds include but are not limited to various functional small molecule compounds or small molecule drugs.

[0018] The second aspect of the present invention provides a recombinant expression vector, which is a plasmid vector containing a gene encoding a PVR protein, a plasmid vector containing both a gene encoding a PVR protein and a gene encoding an effective substance, or a plasmid vector containing both a gene encoding a PVR protein and a gene encoding a polypeptide linker connected to an effective substance.

[0019] The recombinant expression vectors of the present invention are used to introduce exogenous nucleic acid fragments encoding a fusion protein containing PVR and an active substance into cells for overexpression. Cells overexpressing the fusion protein then produce exosomes carrying the active substance. Therefore, the construction of the recombinant expression vector is crucial to the efficiency of exosome loading with the active substance.

[0020] According to some specific embodiments of the present invention, the nucleotide sequence of the gene encoding the PVR protein is shown in SEQ ID NO: 1.

[0021] According to some specific embodiments of the present invention, an antibiotic screening system is used to screen positive stably transfected cells for subsequent exosome production, so the recombinant expression vector contains an antibiotic encoding gene.

[0022] The antibiotic encoding gene in the present invention includes, but is not limited to, one or more of a puromycin encoding gene, a neomycin encoding gene, a kanamycin encoding gene, or a tetracycline encoding gene.

[0023] The expression vector used in the present invention can 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.

[0024] According to some embodiments of the present invention, the expression vector used by the recombinant expression vector is a pAAVS1 plasmid, a pIRES plasmid, or a pLENTI plasmid.

[0025] A third aspect of the present invention provides a recombinant cell, wherein the recombinant cell contains an overexpressed PVR protein or an overexpressed fusion protein formed by the PVR protein and an effective substance. The exosomes produced by the recombinant cell contain the overexpressed PVR protein or the overexpressed fusion protein formed by the PVR protein and an effective substance, thereby enabling the effective substance to be enriched in the exosomes.

[0026] According to some embodiments of the present invention, the recombinant cell contains the above-mentioned recombinant expression vector.

[0027] According to some embodiments of the present invention, the recombinant cell also contains a gene editing system, and the exogenous nucleic acid fragment in the recombinant expression vector is inserted into the genome of the host cell through gene editing. The exogenous nucleic acid fragment includes a gene encoding a PVR protein, a gene encoding an active substance, or a gene encoding a polypeptide linker for connecting an active substance and a PVR protein, a gene encoding an antibiotic, and a promoter and transcription termination sequence conventionally used in the art.

[0028] In the present invention, the promoter sequence may contain a transcriptional regulatory sequence that mediates protein or polypeptide expression. The promoter may be any nucleic acid sequence that is transcriptionally active in the selected cell, including mutant, truncated, and hybrid promoters. Preferably, the promoter is a CAG promoter, an ACTB promoter, a Ubc promoter, a CMV promoter, an EF1A promoter, a PGK promoter, or a TRE promoter.

[0029] In the present invention, a transcription termination sequence is a sequence that is recognized by the host cell to terminate transcription. The transcription termination sequence is operably linked to the 3' end of the nucleic acid sequence encoding the protein or polypeptide. Any terminator that is functional in the selected host cell can be used in the present invention.

[0030] In the present invention, various methods known in the art can be used to introduce exogenous nucleic acid fragments into cells. The gene editing system is one or more of a transposon-mediated system, a loxP-Cre gene editing system, a CRISPR / Cas gene editing system, a TALEN gene editing system, or a ZFN gene editing system.

[0031] According to some embodiments of the present invention, the gene editing system is a CRISPR / Cas9 gene editing system.

[0032] In the present invention, the host cell used for the recombinant cell can be any suitable cell type, including mammalian cells and insect cells, preferably mammalian cells.

[0033] According to some embodiments of the present invention, the host cell used for the recombinant cell is a HEK293 cell, a CHO cell, or a SF9 cell.

[0034] A fourth aspect of the present invention provides a use of the above-mentioned recombinant expression vector or the above-mentioned recombinant cell in preparing exosomes loaded with effective substances.

[0035] A fifth aspect of the present invention provides a method for preparing exosomes loaded with effective substances, wherein the exosomes loaded with effective substances are separated from the cell culture fluid of the above-mentioned recombinant cells.

[0036] In the present invention, exosome 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.

[0037] According to some embodiments of the present invention, density gradient centrifugation is used to separate exosomes loaded with effective substances, and the preparation method involved includes the following steps:

[0038] Providing a cell culture fluid of the recombinant cell, and separating by a centrifuge to obtain a supernatant;

[0039] filtering the supernatant using a deep filtration system and / or a microfiltration membrane, and collecting the filtrate;

[0040] Concentrating the filtrate using a tangential flow concentration system to obtain a concentrate;

[0041] The concentrate is enzymatically hydrolyzed with nuclease in a water bath at 20°C to 40°C, the enzymatically hydrolyzed solution is centrifuged, and the precipitate is resuspended in a buffer solution to obtain a crude extract solution;

[0042] Density gradient centrifugation is used to separate and purify the crude extract to obtain the exosomes loaded with effective substances.

[0043] Preferably, the density gradient centrifugation uses a discontinuous density gradient layering solution, and the layering solution is iodixanol-sucrose buffer.

[0044] According to some embodiments of the present invention, the preparation method includes the step of transfecting host cells with the above-mentioned recombinant expression vector to obtain the recombinant cell, wherein the transfection of the recombinant expression vector and the gene editing system are carried out simultaneously or separately.

[0045] According to some embodiments of the present invention, the culture medium used to culture the recombinant cells is a serum-free culture medium, to which is added an antibiotic corresponding to the antibiotic encoding gene in the recombinant expression vector, so as to facilitate the screening of stably transfected cells during the culture process.

[0046] A sixth aspect of the present invention provides a pharmaceutical composition comprising the above-mentioned exosomes loaded with effective substances, and optionally comprising pharmaceutically acceptable excipients.

[0047] 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.

[0048] In an embodiment of the present invention, 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.

[0049] A seventh aspect of the present invention provides a kit comprising the above-mentioned exosomes loaded with effective substances, or the above-mentioned pharmaceutical composition.

[0050] In an eighth aspect, the present invention provides the use of PVR protein as a scaffold protein for loading effective substances onto exosomes, wherein the effective substances are biological macromolecules, small molecule compounds, or a combination of the two.

[0051] A ninth aspect of the present invention provides a method for preventing or treating a disease, comprising administering the exosomes loaded with an effective substance or the pharmaceutical composition to a subject.

[0052] In an embodiment of the present invention, it is preferred to determine the specific disease that the exosomes loaded with effective substances correspond to for detection, prevention or treatment based on the effective substances loaded on the exosomes, including but not limited to various tumor diseases or non-tumor diseases.

[0053] Due to the adoption of the above technical solution, the present invention has the following advantages compared with the prior art:

[0054] The present invention uses PVR protein as an exosome scaffold protein to load effective substances, thereby improving the loading amount and loading efficiency of effective substances. In addition, it can also be used to load larger and more complex effective substances. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] Figure 1 is a schematic diagram of the sequence elements of a gene expression cassette;

[0056] FIG2 is a growth curve of drug addition screening of stably transfected cells loaded with HSA in Example 1, Comparative Example 1 and Comparative Example 4;

[0057] FIG3 is a flow cytometry analysis of stably transfected cells loaded with HSA in Example 1, Comparative Example 1 and Comparative Example 4;

[0058] FIG4 is a growth curve diagram of drug addition screening of stably transfected cells loaded with IL-15 in Example 2, Comparative Example 2 and Comparative Example 5;

[0059] FIG5 is a flow cytometry analysis of stably transfected cells loaded with IL-15 in Example 2, Comparative Example 2 and Comparative Example 5;

[0060] FIG6 is a growth curve diagram of drug addition screening of stably transfected cells loaded with 4-1BBL in Example 3, Comparative Example 3 and Comparative Example 6;

[0061] FIG7 is a flow cytometry analysis of stably transfected cells loaded with 4-1BBL in Example 3, Comparative Example 3 and Comparative Example 6;

[0062] FIG8 is an NTA analysis chart of HSA-loaded exosomes of Example 1, Comparative Example 1, and Comparative Example 4;

[0063] FIG9 is a TEM analysis of HSA-loaded exosomes of Example 1, Comparative Example 1, and Comparative Example 4;

[0064] FIG10 is a WB analysis of HSA-loaded exosomes of Example 1, Comparative Example 1, and Comparative Example 4;

[0065] FIG11 is an ELISA quantitative analysis graph of HSA-loaded exosomes in Example 1, Comparative Example 1, and Comparative Example 4;

[0066] FIG12 is an NTA analysis chart of IL-15-loaded exosomes of Example 2, Comparative Example 2, and Comparative Example 5;

[0067] FIG13 is a TEM analysis of IL-15-loaded exosomes of Example 2, Comparative Example 2, and Comparative Example 5;

[0068] FIG14 is a WB analysis of IL-15-loaded exosomes of Example 2, Comparative Example 2, and Comparative Example 5;

[0069] FIG15 is an ELISA quantitative analysis graph of IL-15-loaded exosomes in Example 2, Comparative Example 2, and Comparative Example 5;

[0070] FIG16 is an NTA analysis chart of exosomes loaded with 4-1BBL in Example 3, Comparative Example 3, and Comparative Example 6;

[0071] FIG17 is a TEM analysis of exosomes loaded with 4-1BBL in Example 3, Comparative Example 3, and Comparative Example 6;

[0072] FIG18 is an ELISA quantitative analysis graph of exosomes loaded with 4-1BBL in Example 3, Comparative Example 3, and Comparative Example 6;

[0073] FIG19 is a comparison of the loading efficiency of exosomes loaded with effective substances in Examples 1 to 3 and Comparative Examples 1 to 6. DETAILED DESCRIPTION

[0074] Exosomes are cell-derived extracellular vesicles (EVs) with a diameter of 20 to 300 nm. They contain a membrane enclosing an internal space, which is a phospholipid bilayer structure. Exosomes can mediate the intercellular transfer of intracellular substances.

[0075] "Engineered exosomes" refer to modified exosomes, for example, where the membrane has been modified in terms of protein, lipid, small molecule, carbohydrate, etc. This includes exosomes engineered through genetic, physical, or chemical methods to enable effective substance loading and / or delivery.

[0076] “Loading” refers to connecting the scaffold protein with the effective substance and overexpressing it on the exosomes, thereby enriching the effective substance on the exosomes.

[0077] A "scaffold protein" refers to a protein molecule that can be used to anchor an active substance or any exogenous biologically active moiety of interest to exosomes. A fusion protein can be formed by fusing the scaffold protein with an active substance, a biologically active moiety of an active substance, or a polypeptide linker attached to the active substance. The scaffold protein portion of the fusion protein is located within the exosome membrane or lumen.

[0078] "PVR protein", "CD99 protein" and "PTGFRN protein" are all type I transmembrane proteins and are all scaffold proteins described in the present invention.

[0079] "Effective substance" refers to any biological macromolecule or small molecule compound with functional properties of interest (e.g., preventive, therapeutic, diagnostic, etc.). "Effective substance" is also often referred to as "cargo."

[0080] "Biomacromolecules" refer to macromolecules such as proteins, nucleic acids, polysaccharides, etc. with a molecular weight greater than or equal to 1000 that exist in the cells of organisms.

[0081] "Small molecule compounds" refer to compounds with a molecular size of less than 1000 molecular weight, including conventional organic compounds and inorganic compounds, as well as a variety of small molecule compounds with biological activity, such as vitamins, hormones, amino acids and their derivatives, peptides, nucleotides, etc. with small molecular weight.

[0082] "Nucleic acid" refers to nucleotides of any length, including deoxyribonucleotides, ribonucleotides, combinations thereof, and analogs thereof.

[0083] "Polypeptide" and "peptide" are used interchangeably to refer to polymers of amino acids of any length. Thus, polypeptides, oligopeptides, proteins, antibodies, and enzymes are all included within the definition of polypeptide.

[0084] A "fragment" refers to a portion of a sequence. For example, a fragment of a nucleic acid sequence refers to a portion of the nucleic acid sequence, and a fragment of an amino acid sequence refers to a portion of the amino acid sequence.

[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 are derived from a different source than the host cell, for example, nucleic acid sequences isolated from an organism other than the host cell, i.e., are exogenous nucleic acid fragments relative to the host cell.

[0086] An "expression vector" is a nucleic acid molecule capable of transporting another nucleic acid molecule to which it is attached. Examples of vectors include, but are not limited to, plasmids, viruses, bacteria, bacteriophages, and insertable DNA segments. A "plasmid" is a circular double-stranded DNA fragment that can accept exogenous nucleic acid segments and replicate in prokaryotic or eukaryotic cells.

[0087] "Promoter" refers to a nucleic acid sequence that controls the transcription of a coding sequence. Promoter sequences include specific sequences sufficient for RNA polymerase to recognize, bind, and initiate transcription. A promoter can affect the transcription of a gene on the same nucleic acid molecule as itself or a gene on a different nucleic acid molecule.

[0088] "Host cells" are also referred to as recipient cells, and include, but are 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 is understood that the progeny of a single parental cell may not necessarily be completely identical to the original parent in morphology or in genomic or total DNA complement due to natural, accidental, or deliberate mutations.

[0089] "HSA," the abbreviation for human serum albumin, is a plasma protein and one of the most abundant proteins in the human body. Human serum albumin is primarily synthesized by the liver and present in the blood, playing an important physiological role in systemic circulation. Human serum albumin maintains blood stability by maintaining normal osmotic pressure and blood volume in plasma. This protein can also bind and transport a variety of substances, such as drugs, hormones, and lipids, to aid their transport and distribution within the body. Human serum albumin also participates in the regulation of blood coagulation and fibrinolysis, playing a vital role in maintaining normal blood coagulation and fibrinolysis. Human serum albumin acts as a buffer in the body, helping to maintain acid-base balance in blood and tissues. It also participates in the regulation of immune responses and has nonspecific protection against foreign substances such as bacteria and viruses. It is primarily used to prevent measles and infectious hepatitis, enhance immunity, and treat hypoproteinemia.

[0090] "IL-15" stands for interleukin-15, an important cytokine. Its biological functions primarily include stimulating the differentiation of T cells, B cells, and natural killer (NK) cells, as well as promoting their proliferation. IL-15 has a wide range of cell sources and target cells, making it suitable for immunotherapy and anti-tumor applications.

[0091] "4-1BBL" is the abbreviation of 4-1BB ligand, which is a type II membrane protein of the TNF superfamily and is expressed on antigen-presenting cells. 4-1BB and 4-1BBL can induce T cell expansion, cytokine induction, differentiation and upregulation of anti-apoptotic genes, and protect T cells from activation-induced cell death (AICD). 4-1BBL stimulates T cell proliferation and induces effective anti-tumor immune responses. 4-1BBL is an immunostimulatory molecule that interacts with the 4-1BB high-affinity receptor during antigen presentation and directs the expression of NF-kB, c-Jun and p38 downstream pathways to CD4 T cells. + and CD8 + T cells provide co-stimulatory signals, triggering pleiotropic effects on the immune system. They are suitable for immunotherapy and anti-tumor applications.

[0092] Embodiments of the present invention provide exosomes loaded with an effective substance, comprising exosomes, a PVR protein overexpressed in the exosomes, and an effective substance linked to the PVR protein. The PVR protein is a type I transmembrane protein that can span the phospholipid bilayer membrane of the exosomes and can be used to load the effective substance onto the exosomes, including both the interior and exterior of the exosomes. To maintain the bilayer membrane structure of the exosomes, the effective substance is preferably loaded onto the outer surface of the exosomes via the PVR protein. This provides more space for loading the effective substance on the outer surface of the exosomes, facilitating the loading of more effective substances.

[0093] The technical solutions of the present invention are further described below in conjunction with specific embodiments, but the present invention is not limited to the following embodiments.

[0094] 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 generally those used in routine experiments. Instruments, raw materials, and reagents not otherwise specified can be obtained commercially.

[0095] Example 1

[0096] This embodiment provides exosomes loaded with human serum HSA albumin and a method for preparing the same. This embodiment utilizes overexpressed PVR protein as an exosome scaffold protein to load human serum HSA albumin onto the outer surface of the exosomes, including the following steps:

[0097] 1. Construction of expression vector

[0098] The expression vector used in this example was the pAAVS1 plasmid, with a gene expression cassette inserted at the AAVS1 site. The expression cassette was designed as follows: using the CAG promoter (sequence shown in SEQ ID NO:6) as the transcription initiation point, fusion of human serum HSA albumin (sequence shown in SEQ ID NO:3) to the N-terminus of the PVR protein (sequence shown in SEQ ID NO:1), using BgH polyA (sequence shown in SEQ ID NO:7) as the transcription endpoint, and also containing the puromycin (PURO) selection gene (sequence shown in SEQ ID NO:8).

[0099] The specific steps are as follows:

[0100] (1) PCR cloning: Using conventional methods in the art, upstream primers and downstream primers were designed according to the above expression cassette, and the upstream primers, downstream primers and template DNA were synthesized by Jinweizhi Biotechnology Co., Ltd. In a 0.2 mL EP tube, 25 μL DNA polymerase (2×Phanta Max Master Mix, Vazyme, P515-01), 2 μL upstream primer, 2 μL downstream primer, and 10 g template DNA were added. The volume was made up to 50 μL with RNase-free ddH2O (Vazyme, P071-01-AA), mixed well, and placed in a PCR instrument. The amplification program was designed according to the primer annealing temperature.

[0101] (2) PCR product purification: Purify the PCR product according to the kit instructions (Vazyme, DC301-01).

[0102] (3) Enzyme digestion: Add 5 μL rCutSmart to a 0.2 mL EP tube. TM Buffer (BioLabs, 136004s), 0.5 μL Pac I enzyme (BioLabs, R0547L), 0.5 μL Not I enzyme (New England BioLabs (NEB), R3189L), 2 μg of PCR-purified fragment, and 5 μg of pAAVS1 vector plasmid were added. The volume was made up to 10 μL with enzyme-free ddH2O (Vazyme, P071-01-AA), mixed, and placed in a PCR instrument for enzyme digestion at 37°C for 16 hours.

[0103] (4) Ligation and transformation: In a 0.2 mL EP tube, add 4 μL of the enzyme digestion solution obtained in step (3), 1 μL of T4 DNA ligase (Novagen Vazyme, N103-01-AA) and 1 μL of T4 DNA ligase buffer (Novagen Vazyme, N103-01-AC). After incubation at room temperature for 10 minutes, 5 μL was added to competent cells (DH5α, Novagen Vazyme, C502-03) and incubated on ice for 30 minutes, incubated at 42°C for 90 seconds, incubated on ice for 2 minutes, and then added with 1 mL of LB medium (BeyoPure TM , 04.05.ST156) were placed in a shaking incubator and cultured for 1 hour. After culture, the cells were centrifuged at 12000g for 1 minute, the supernatant was removed and 100 μL was kept for resuspending and added to a bacterial culture dish (Biosharp, BS-90-D). The cells were then evenly spread with a disposable plastic coating rod (Biosharp, BS-PS-A) and stored in a 37°C constant temperature incubator for later use.

[0104] 2. Construction of Stable Cell Lines

[0105] As shown in Figure 1, there is a homology arm sequence for the AAVS1 site on the backbone of the expression vector. Using a Cas9-gRNA complex targeting the AAVS1 site (Cas9 sequence is shown in SEQ ID NO: 9, and AAVS1sgRNA sequence is shown in SEQ ID NO: 10), a DNA strand break is introduced at this site, allowing the expression cassette to be fully inserted into the genome via homologous recombination-mediated repair (HDR).

[0106] The specific steps are as follows:

[0107] (1) Prepare cells for transfection

[0108] EXPI293F cells were seeded at 1.5E+6 / mL with a viability of over 95% in a 125 mL shake flask with a total volume of 25 mL of culture medium. The cells were cultured overnight in an 8% CO2 constant temperature shaker. Transfection was started the next day.

[0109] (2) PEI transfection

[0110] 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:

[0111] The target plasmid is extracted from the transformed competent cells preserved by the above-mentioned coating culture using conventional technical means in the art to obtain the constructed expression vector. Prepare two 15mL centrifuge tubes, add 1.25mL serum-free cell culture medium, 12.5μg CRISPR / Cas9 plasmid (commissioned by Jinweizhi Biotechnology Co., Ltd. for synthesis) and 12.5μg target plasmid to one of them, and vortex to mix; add 1.25mL serum-free cell culture medium and 125μL TA293 transfection reagent to the other, and vortex to mix. Transfer all the liquid in the centrifuge tube containing the transfection reagent to the centrifuge tube containing the plasmid and vortex to mix. Incubate at room temperature for 10 minutes to prepare the plasmid-vector complex. Remove 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 culture.

[0112] (3) Cell screening

[0113] 96 hours after transfection, the cell density and viability were recorded using a cell counter, the transfection efficiency was analyzed using a fluorescence microscope (Invitrogen, EVOS M5000), and antibiotic screening was started. The cells were cultured with a medium containing 2 μg / mL puromycin, the medium was changed every 2-3 days, and the cell density and viability were continuously recorded using a low-speed centrifugation of 120g / 4min. The cell viability will gradually decrease in the first week of screening. The final screening success criterion is that the cell viability recovers to about 95%, which can be regarded as a stably transfected cell. Stably transfected cells were frozen using serum-free cell freezing solution at a freezing density of 1E7 cells / tube.

[0114] 3. Isolation and purification of exosomes

[0115] The frozen stably transfected cells were cultured in serum-free medium (Aupuma Biotech, CD05) at 37°C, 8% CO2, and a 120 rpm horizontal shaker until the viable cell density was greater than 9 × 10 6Cell viability was greater than 90%. Centrifuge at 6000g for 20 min at 4°C, and collect the supernatant. Centrifuge at 16000g for 30 min at 4°C, and collect the supernatant. Filter the supernatant using a depth filtration system (Cobetter, CSCCD1070PCP) and a sterile filtration system (Cobetter, L10THSLESS1P). Filter the supernatant through a 0.45μm filter (Millipore) and a 0.22μm filter (Millipore). Concentrate the supernatant 10-fold using a tangential flow filtration (TFF) system (Repligen, model KR2i) to obtain a concentrate. Add MgCl2 solution (final concentration 1 mM) and universal nuclease (final concentration 20 U / mL; Novoprotein, GMP-1707) to the concentrate and incubate in a water bath at 25°C for 16 h or at 37°C for 3 h. The concentrated solution after nuclease treatment was centrifuged at 133900g for 60 min at 4°C. The precipitate was resuspended in PBS and repeatedly pipetted with a 1 mL syringe until the precipitate was completely dissolved to obtain a crude extract solution.

[0116] Sucrose buffer (250 mM Sucrose, 10 mM Tris HCl, 1 mM EDTA, pH 7.4) was prepared. Iodixanol solution (Merck, Sigma, D1556-250 mL) was diluted with the sucrose buffer to prepare a 17.5% (v / v) dilution (iodixanol solution / (iodixanol solution + sucrose buffer)). A 45% (v / v) dilution (iodixanol solution / (iodixanol solution + crude extract solution)) was prepared. PBS was added to the bottom of a centrifuge tube using a syringe. The 17.5% dilution and the 45% dilution were then added sequentially from the bottom. The tube was topped up with PBS and centrifuged at 150,000 g at 4°C for 16 h. Carefully remove the white layer that has migrated to the interface between PBS and the 17.5% diluent and transfer it to a new tube. Fill the tube with PBS and centrifuge at 20,000g at 4°C for 30 minutes to remove contaminating proteins. Transfer the supernatant to a new tube and centrifuge at 135,000g at 4°C for 3 hours. Resuspend the pellet (i.e., the isolated exosomes) in 100 μL of PBS and store at 4°C until ready to use.

[0117] The HSA-loaded exosomes obtained in this example are labeled: HSA-PVR.

[0118] Example 2

[0119] This example provides exosomes loaded with interleukin-15 and a method for preparing the same. This example utilizes overexpressed PVR protein as an exosome scaffold protein to load interleukin-15 onto the exosome surface. The preparation method is essentially the same as in Example 1, except that human serum HSA albumin is replaced with interleukin-15 (IL-15, sequence shown in SEQ ID NO: 4).

[0120] The interleukin-15 loaded exosomes obtained in this example are labeled: IL15-PVR.

[0121] Example 3

[0122] This example provides exosomes loaded with the immunostimulatory molecule 4-1BBL protein and a method for preparing the same. This example utilizes overexpressed PVR protein as an exosome scaffold protein to load the immunostimulatory molecule 4-1BBL protein onto the exosome surface. The preparation method is essentially the same as in Example 1, except that human serum HSA albumin is replaced with the immunostimulatory molecule 4-1BBL protein (sequence shown in SEQ ID NO: 5).

[0123] The exosomes loaded with the immunostimulatory molecule 4-1BBL protein obtained in this example are labeled as: 41BBL-PVR.

[0124] Comparative Example 1

[0125] This comparative example provides another exosome loaded with human serum HSA albumin, which uses overexpressed CD99 protein as an exosome scaffold protein to load human serum HSA albumin onto the exosome surface. The preparation method is basically the same as that of Example 1, except that the PVR protein is replaced with CD99 protein (sequence shown in SEQ ID NO: 2).

[0126] The HSA-loaded exosomes obtained in this comparative example are labeled as: HSA-CD99.

[0127] Comparative Example 2

[0128] This comparative example provides another exosome loaded with interleukin-15, which uses overexpressed CD99 protein as an exosome scaffold protein to load interleukin-15 onto the exosome surface. The preparation method is basically the same as that of Comparative Example 1, except that human serum HSA albumin is replaced with interleukin-15 (abbreviated as IL-15, the sequence is shown in SEQ ID NO: 4).

[0129] The IL-15-loaded exosomes obtained in this comparative example were labeled as IL15-CD99.

[0130] Comparative Example 3

[0131] This comparative example provides another exosome containing an immunostimulatory molecule 4-1BBL protein, which uses overexpressed CD99 protein as an exosome scaffold protein to load the immunostimulatory molecule 4-1BBL protein onto the exosome surface. The preparation method is basically the same as that of Comparative Example 1, except that human serum HSA albumin is replaced with the immunostimulatory molecule 4-1BBL protein (sequence shown in SEQ ID NO: 5).

[0132] The exosomes loaded with 4-1BBL protein obtained in this comparative example are labeled as: 41BBL-CD99.

[0133] Comparative Example 4

[0134] This comparative example provides another exosome loaded with human serum HSA albumin, which uses overexpressed PTGFRN protein as an exosome scaffold protein to load human serum HSA albumin onto the exosome surface. The preparation method is basically the same as that in Example 1, except that the PVR protein is replaced with the PTGFRN protein of Codiak Biosciences (reference patents PCT / US2018 / 048026, US20200222556A1).

[0135] The HSA-loaded exosomes obtained in this comparative example were labeled as: HSA-PTGFRN.

[0136] Comparative Example 5

[0137] This comparative example provides another exosome loaded with interleukin-15, which uses overexpressed PTGFRN protein as an exosome scaffold protein to load interleukin-15 onto the exosome surface. The preparation method is basically the same as that of Comparative Example 4, except that human serum HSA albumin is replaced with interleukin-15 (abbreviated as IL-15, the sequence is shown in SEQ ID NO: 4).

[0138] The IL-15-loaded exosomes obtained in this comparative example were labeled as IL15-PTGFRN.

[0139] Comparative Example 6

[0140] This comparative example provides another exosome containing an immunostimulatory molecule 4-1BBL protein, which uses overexpressed PTGFRN protein as an exosome scaffold protein to load the immunostimulatory molecule 4-1BBL protein onto the exosome surface. The preparation method is basically the same as that of Comparative Example 4, except that human serum HSA albumin is replaced with the immunostimulatory molecule 4-1BBL protein (sequence shown in SEQ ID NO: 5).

[0141] The exosomes loaded with 4-1BBL protein obtained in this comparative example are labeled as: 41BBL-PTGFRN.

[0142] During the cell screening phase, the growth curve of HSA-loaded stably transfected cells following drug addition is shown in Figure 2. The screening process took an average of 3-4 weeks to complete (viability >95%). Flow cytometry analysis of HSA-loaded stably transfected cells is shown in Figure 3, demonstrating that the HSA protein expression positivity rate in all stable cell lines was >95%.

[0143] The growth curve of the drug-addition screening of stably transfected cells loaded with IL-15 is shown in Figure 4. The screening process took an average of 3-4 weeks to complete (viability >95%). Flow cytometric analysis of the stably transfected cells loaded with IL-15 is shown in Figure 5. The positive expression rate of IL-15 protein in all stably transfected cell lines was >95%.

[0144] The growth curve of the drug-dosing screening process for the stably transfected cells loaded with 4-1BBL is shown in Figure 6. The screening process took an average of 3-4 weeks to complete (viability >95%). Flow cytometric analysis of the stably transfected cells loaded with the 4-1BBL functional cargo is shown in Figure 7. The positive expression rate of the 4-1BBL protein in the stably transfected cell lines was >95%.

[0145] Analysis of the effective substance loading capacity of the engineered exosomes of Examples 1 to 3 and Comparative Examples 1 to 6

[0146] The exosomes were analyzed by NTA using a Nano-FCM instrument (Fuliu Biotechnology, Cat. No. N30E). Transmission electron microscopy (TEM) was used to analyze the exosomes. Western blot analysis was performed using electrophoresis. The amount of active substance loaded into the exosomes was quantitatively analyzed using ELISA.

[0147] The results of NTA analysis of HSA-loaded exosomes are shown in Figure 8. The particle sizes of HSA-PVR, HSA-CD99, and HSA-PTGFRN were relatively uniform, concentrated in the 130-160 nm range. The exosome yield per milliliter of cell culture medium reached 2E9 particles.

[0148] TEM analysis of HSA-loaded exosomes is shown in Figure 9. Clear protein corona structures were observed on the surfaces of both HSA-PVR and HSA-PTGFRN, while the surface of HSA-CD99 was relatively smooth. This suggests that PVR and PTGFRN, as scaffold proteins, can load HSA onto the exosome surface at a high density, whereas CD99, as a scaffold protein, exhibits a low density of HSA loading.

[0149] The results of Western blotting analysis of HSA-loaded exosomes are shown in Figure 10. Under the same loading conditions, HSA-PVR exhibited the brightest band, followed by HSA-PTGFRN, and HSA-CD99, indicating that the brighter the band, the greater the loading capacity of the exosomes.

[0150] The results of ELISA quantitative analysis of HSA-loaded exosomes are shown in Figure 11. The order of HSA cargo loading from high to low is: HSA-PVR, HSA-PTGFRN, and HSA-CD99.

[0151] The results of NTA analysis of IL-15-loaded exosomes are shown in Figure 12. The particle sizes of IL15-PVR, IL15-CD99, and IL15-PTGFRN are relatively uniform, concentrated in the range of 120-160 nm.

[0152] The TEM analysis results of IL-15-loaded exosomes are shown in Figure 13. Due to the low expression level of IL-15 itself, the loading density on the exosome surface was relatively low, and no prominent protein corona structure similar to that of HSA exosomes was observed.

[0153] The results of Western blotting analysis of IL-15-loaded exosomes are shown in Figure 14. Under the same loading conditions, the IL15-PVR band has the brightest intensity, followed by IL15-PTGFRN, and IL15-CD99 has the weakest intensity. A brighter band indicates a greater loading capacity and a higher effective substance content on the exosomes.

[0154] The results of ELISA quantitative analysis of IL-15-loaded exosomes are shown in Figure 15. The order of IL-15 loading from high to low is: IL15-PVR, IL15-PTGFRN, and IL15-CD99.

[0155] NTA analysis results for 4-1BBL-loaded exosomes are shown in Figure 16. The particle sizes of 41BBL-PVR, 41BBL-CD99, and 41BBL-PTGFRN were relatively uniform, concentrated in the 140-160 nm range. The exosome yield per milliliter of cell culture medium was approximately 5E9 particles.

[0156] The TEM analysis results of 4-1BBL-loaded exosomes are shown in Figure 17. Due to the low expression level of 4-1BBL itself, the loading density on the exosome surface was relatively low, and no obvious protein corona structure similar to that of HSA exosomes was observed.

[0157] The results of ELISA quantitative analysis of exosomes loaded with 4-1BBL are shown in Figure 18. The order of 4-1BBL loading from high to low is: 41BBL-PVR, 41BBL-PTGFRN, and 41BBL-CD99.

[0158] The results of the analysis of the effective substance loading efficiency of the exosomes of Examples 1 to 3 and Comparative Examples 1 to 6 are shown in Figure 19. Compared with PTGFRN and CD99 from Codiak Biosciences, the exosomes using PVR as a scaffold protein showed a higher effective substance loading capacity.

[0159] The PVR protein used in the present invention is 417 aa in length, while the PTGFRN protein is 879 aa in length. The PVR protein is shorter and has the following obvious advantages as an exosome scaffold protein:

[0160] 1. Higher gene editing efficiency. In most cases, shorter genes can lead to higher knock-in efficiency and lower editing difficulty at the cellular level, facilitating cell engineering.

[0161] 2. Applicable to a wider range of vectors. For some viral vectors, the genome length that can be packaged is relatively limited. For example, the currently mainstream AAV vector generally has a packaging capacity of no more than 5kbp. Therefore, the shorter the length of the scaffold protein, the longer the length of the drug protein that can be delivered, which can significantly expand the delivery capacity and drug development potential of vectors such as AAV.

[0162] 3. Protein synthesis is more efficient and suitable for the delivery of more active substances. In most cases, shorter proteins are more efficient in cellular synthesis, and shorter scaffold proteins can often carry larger and more complex active substances.

[0163] Example 4

[0164] This example provides a formulation for extending the half-life of exosomes in the blood, comprising the HSA-PVR resuspension of Example 1. After the exosomes are modified with HSA protein, HSA can interact with the FcRn receptor, preventing endocytosed HSA exosomes from being degraded by lysosomes and allowing them to be directly recycled outside the cell, thereby extending the half-life of the modified exosomes in the blood.

[0165] Example 5

[0166] This example provides a formulation for inhibiting solid tumor growth, comprising the IL15-PVR resuspension of Example 2, for direct intratumoral injection into solid tumors. Cytokines such as IL15 can promote the proliferation and activation of immune cells such as T cells, B cells, and NK cells, thereby transforming "cold" tumors into "hot" tumors and inhibiting tumor growth. Furthermore, the enhanced permeability and retention (EPR) effect of IL15-PVR exosomes within the tumor allows IL15 to remain within the tumor microenvironment, thereby reducing IL15's systemic toxicity.

[0167] Example 6

[0168] This embodiment provides a preparation for in vitro expansion of T cells / NK cells, the formula of which is: the 41BBL-PVR resuspension of Example 3 is added to the culture medium of T cells / NK cells at a ratio of at least 1000:1 exosomes: cells. The 41BBL on the surface of the exosomes directly interacts with the 4-1BB high-affinity receptor on the cell surface, activating the NF-kB, c-Jun and p38 downstream pathways to CD4 + / CD8 + T cells / NK cells provide co-stimulatory signals, which can significantly improve the expansion and activation efficiency of T cells / NK cells.

[0169] 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 scope of protection of the present invention. The present invention is not limited to the above embodiments. Any equivalent changes or modifications made based on the spirit of the present invention should be included in the scope of protection of the present invention.

Claims

1. An exosome loaded with an effective substance, characterized in that: The invention comprises exosomes, PVR proteins overexpressed in the exosomes, and effective substances connected to the PVR proteins.

2. The exosomes loaded with effective substances according to claim 1, characterized in that The effective substance is loaded on the outer surface of the exosomes.

3. The exosomes loaded with effective substances according to claim 1, characterized in that The nucleotide sequence encoding the PVR protein is shown in SEQ ID NO:

1.

4. The exosomes loaded with effective substances according to claim 1, characterized in that The effective substance is directly or indirectly connected to the N-terminus or C-terminus of the PVR protein through a linker.

5. The exosomes loaded with effective substances according to claim 1, characterized in that The effective substance is connected to the N-terminus or C-terminus of the PVR protein via a polypeptide linker.

6. The exosomes loaded with effective substances according to claim 1, characterized in that The effective substances include one or more of proteins, polypeptides, carbohydrates, nucleic acids or small molecule compounds.

7. A recombinant expression vector, characterized in that: The recombinant expression vector is a plasmid vector containing a gene encoding PVR protein, a plasmid vector containing both a gene encoding PVR protein and a gene encoding an effective substance, or a plasmid vector containing both a gene encoding PVR protein and a gene encoding a polypeptide linker connected to an effective substance.

8. The recombinant expression vector according to claim 7, characterized in that The nucleotide sequence of the gene encoding the PVR protein is shown in SEQ ID NO:

1.

9. The recombinant expression vector according to claim 7, characterized in that The effective substances include one or more of proteins, polypeptides, carbohydrates, nucleic acids or small molecule compounds.

10. The recombinant expression vector according to claim 7, characterized in that The recombinant expression vector contains the antibiotic coding gene.

11. The recombinant expression vector according to claim 10, characterized in that The antibiotic encoding gene is one or more of a puromycin encoding gene, a neomycin encoding gene, a kanamycin encoding gene or a tetracycline encoding gene.

12. The recombinant expression vector according to claim 7, characterized in that The expression vector used by the recombinant expression vector is a viral plasmid vector or a eukaryotic expression plasmid vector.

13. The recombinant expression vector according to claim 7, characterized in that The expression vector used by the recombinant expression vector is pAAVS1 plasmid, pIRES plasmid or pLENTI plasmid.

14. A recombinant cell, characterized in that The recombinant cell contains overexpressed PVR protein and / or overexpressed fusion protein formed by PVR protein and effective substance.

15. The recombinant cell according to claim 14, characterized in that The recombinant cell contains the recombinant expression vector according to any one of claims 7 to 13.

16. The recombinant cell according to claim 14, characterized in that The recombinant cell contains a gene editing system.

17. The recombinant cell according to claim 16, characterized in that The gene editing system is one or more of a transposon-mediated system, a loxP-Cre gene editing system, a CRISPR / Cas gene editing system, a TALEN gene editing system, and a ZFN gene editing system.

18. The recombinant cell according to claim 14, characterized in that The host cells used in the recombinant cells are mammalian cells or insect cells.

19. The recombinant cell according to claim 14, characterized in that The host cells used in the recombinant cells are HEK293 cells, CHO cells or SF9 cells.

20. Use of the recombinant expression vector according to any one of claims 7 to 13 or the recombinant cell according to any one of claims 14 to 19 in preparing exosomes loaded with effective substances.

21. A method for preparing exosomes loaded with effective substances, characterized in that: Exosomes loaded with effective substances are isolated from the cell culture fluid of the recombinant cell according to any one of claims 14 to 19.

22. The preparation method according to claim 21, characterized in that It includes the following steps: Providing a cell culture fluid of the recombinant cell according to any one of claims 14 to 19, wherein the supernatant is obtained by separation using a centrifuge; filtering the supernatant using a deep filtration system and / or a microfiltration membrane, and collecting the filtrate; Concentrating the filtrate using a tangential flow concentration system to obtain a concentrate; The concentrate is enzymatically hydrolyzed with nuclease in a water bath at 20°C to 40°C, the enzymatically hydrolyzed solution is centrifuged, and the precipitate is resuspended in a buffer solution to obtain a crude extract solution; Density gradient centrifugation is used to separate and purify the crude extract to obtain the exosomes loaded with effective substances.

23. The preparation method according to claim 22, characterized in that The density gradient centrifugation uses a discontinuous density gradient layering liquid, and the layering liquid is iodixanol-sucrose buffer.

24. The preparation method according to claim 22, characterized in that The preparation method further comprises the step of using the recombinant expression vector according to any one of claims 7 to 13 and the gene editing system to transfect host cells to obtain the recombinant cells, wherein the transfection of the recombinant expression vector and the gene editing system is performed simultaneously or separately.

25. The preparation method according to claim 24, characterized in that An antibiotic screening system is used to screen stably transfected cells, and an antibiotic corresponding to the antibiotic coding gene in the recombinant expression vector is added to the cell culture medium used to culture the recombinant cells.

26. The preparation method according to claim 22, characterized in that The cell culture medium used to culture the recombinant cells is a serum-free medium.

27. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the exosomes loaded with effective substances according to any one of claims 1 to 6, and may optionally comprise pharmaceutically acceptable excipients.

28. The pharmaceutical composition according to claim 27, characterized in that The pharmaceutical composition is in the form of tablets, granules, pills, capsules, emulsions, ointments, gels, suspensions, solutions, powders, transdermal patches, sprays, suppositories or implants.

29. A kit, characterized in that The kit comprises the exosomes loaded with an effective substance according to any one of claims 1 to 6, or the pharmaceutical composition according to claim 27 or 28.

30. Use of PVR protein as a scaffold protein for loading effective substances onto exosomes, wherein the effective substances are biological macromolecules, small molecule compounds, or a combination of the two.

31. The use according to claim 30, characterized in that The effective substance includes one or more of proteins, polypeptides, and nucleic acids.

32. A method for preventing or treating a disease, characterized in that: The method comprises administering the exosomes loaded with an effective substance according to any one of claims 1 to 6 or the pharmaceutical composition according to claim 27 or 28 to a subject.

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