CN117821394B - Exosome scaffold protein and application thereof - Google Patents
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- CN117821394B CN117821394B CN202410244625.6A CN202410244625A CN117821394B CN 117821394 B CN117821394 B CN 117821394B CN 202410244625 A CN202410244625 A CN 202410244625A CN 117821394 B CN117821394 B CN 117821394B
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
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Abstract
The invention relates to exosome scaffold protein and application thereof. The PVR protein is used as exosome scaffold protein to load effective substances, so that the loading capacity and the loading efficiency of the effective substances are improved, and the PVR protein can be used for loading the effective substances with larger structures and more complicated structures.
Description
Technical Field
The invention belongs to the technical field of molecular biology, and particularly relates to exosome scaffold protein and application thereof.
Background
Exosomes are small vesicles secreted by cells, with a membrane structure consisting of a phospholipid bilayer. The surface of the cell membrane has various specific membrane proteins, and meanwhile, important biological signal substances such as various proteins, RNA, DNA and the like are carried in the cell membrane, so that the cell membrane is widely distributed in various body fluids such as blood, urine, cerebrospinal fluid, saliva, milk, bile and the like, and is an important mediator for intercellular communication.
Exosomes are a naturally occurring nanostructure that has great potential as drug delivery platforms and has been deeply explored and studied in many disease areas as a new therapeutic modality. Exosomes have unique advantages over some traditional drug delivery approaches in several respects. For example, exosomes of different cell sources have different tissue and organ targeting due to differences in their surface physicochemical properties, thus exhibiting the potential for accurate medical treatment; compared with the artificially synthesized lipid nano-particles, the exosomes have lower immunogenicity and better biosafety.
How to load therapeutic drugs, especially biomacromolecule drugs such as interleukins, antibodies and cytokines, onto exosomes is a central difficulty in the field of exosome innovative drugs. Existing approaches include fusion of biological macromolecules with exosome enrichment sequences by means of cell-level engineering, thereby enriching the fusion proteins on exosomes. These exosome-rich sequences, which function as scaffolds (scaffold), are also visualized as exosome scaffold proteins. The exosome scaffold protein sequences currently used include glycosyl phosphatidylinositol anchor (GPI anchor), lipid anchor (lipid anchor), and known exosome marker proteins including CD9, CD63, CD81, and LAMP 2B. However, drugs developed based on these exosome scaffold proteins, in which the loading efficiency of the active substance on the exosomes is low, result in low bioactivity.
Disclosure of Invention
The invention aims to solve the problems of developing novel scaffold proteins with higher loading efficiency and exosomes loaded with effective substances.
To solve the above problems, the first aspect of the present invention provides an exosome loaded with an active substance, comprising an exosome, a PVR protein overexpressed in the exosome, and an active substance linked to the PVR protein.
According to some embodiments of the invention, the active substance is loaded on the outer surface of the exosome.
According to some embodiments of the invention, the nucleotide sequence encoding the PVR protein is shown in SEQ ID NO. 1.
It will be appreciated by those skilled in the art that when referring to a PVR protein, it encompasses not only the PVR protein itself, but also fragments, variants or derivatives thereof, without particular limitation. PVR proteins, when they are fragments, variants or derivatives, typically have an amino acid sequence which is more than 70% identical to the full length amino acid sequence of PVR proteins, and may in particular be, for example, 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% identical.
According to some embodiments of the invention, the active agent is directly or indirectly linked to the N-terminus or the C-terminus of the PVR protein via a linker. Preferably, the active agent is attached to the N-terminus of the PVR protein to form a fusion protein, and the active agent in the fusion protein is advantageously located outside the phospholipid bilayer membrane.
In the present invention, the active substance may be linked to the N-terminus or the C-terminus of the PVR protein through a polypeptide linker. The polypeptide linker may be a cleavable linker or a flexible linker conventionally used in the art.
According to some embodiments of the invention, the active substance comprises one or more of a protein, a polypeptide, a carbohydrate, a nucleic acid, or a small molecule compound.
In embodiments of the invention, the proteins include, but are not limited to, various therapeutic, immunogenic, or functional proteins (e.g., gene editing system proteins, reporter proteins). Various functional lipids, DNA or RNA editing protein complexes, RNA and DNA binding proteins, cytokines, toxins, etc. may also be included.
In embodiments of the invention, the polypeptides include, but are not limited to, various therapeutic polypeptides, targeting polypeptides, or partial polypeptide fragments of the above proteins. Examples of the enzyme include an enzyme, an antibody, an interleukin, serum albumin, lectin, and a coagulation factor.
In embodiments of the invention, the nucleic acid includes, but is not limited to, various functional nucleic acid molecules. For example, it may be a DNA or mRNA molecule encoding a protein or polypeptide, an RNA molecule having regulatory functions such as miRNA, dsDNA, antisense oligonucleotide ASO, lncRNA or siRNA.
In the present invention, the small molecule compounds include, but are not limited to, various functional small molecule compounds or small molecule drugs.
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 active substance, or a plasmid vector containing both a gene encoding a PVR protein and a gene encoding a polypeptide linker to which an active substance is linked.
The recombinant expression vector of the present invention is used to introduce an exogenous nucleic acid fragment encoding a fusion protein comprising PVR and an active agent into cells for overexpression, and then to produce exosomes with the active agent from the cells over-expressing the fusion protein. Thus, the construction of recombinant expression is related to the efficiency of exosomes loading with active substances.
According to some embodiments of the invention, the nucleotide sequence of the coding gene of the PVR protein is shown as SEQ ID NO. 1.
According to some embodiments of the invention, the invention relates to the use of an antibiotic screening system to screen positive stably transformed cells for subsequent exosome production, whereby the recombinant expression vector contains genes encoding antibiotics.
The coding genes of the antibiotics in the invention comprise one or more of puromycin coding genes, neomycin coding genes, kanamycin coding genes or tetracycline coding genes.
The expression vector used in the present invention may be one conventionally used in the art, including viral plasmid vectors, eukaryotic expression plasmid vectors, preferably viral plasmid vectors.
According to some embodiments of the invention, the recombinant expression vector employs an expression vector that is a pAAVS plasmid, a pIRES plasmid, or a pLENTI plasmid.
In a third aspect, the present invention provides a recombinant cell comprising an overexpressed PVR protein or an overexpressed fusion protein formed of a PVR protein and an active agent, and an exosome produced by the recombinant cell comprising an overexpressed PVR protein or an overexpressed fusion protein formed of a PVR protein and an active agent, whereby the active agent can be enriched on the exosome.
According to some embodiments of the invention, the recombinant cell contains the recombinant expression vector described above.
According to some embodiments of the invention, the recombinant cell further comprises a gene editing system, wherein the exogenous nucleic acid fragment in the recombinant expression vector is inserted into the genome of the host cell by gene editing, wherein the exogenous nucleic acid fragment comprises a gene encoding a PVR protein, a gene encoding an active substance or a gene encoding a polypeptide linker for linking the active substance and PVR protein, a gene encoding an antibiotic, and promoters and transcription termination sequences conventionally used in the art.
In the present invention, the sequence of the promoter may be a transcription regulatory sequence which mediates the expression of the protein or polypeptide. The promoter may be any nucleic acid sequence that is transcriptionally active in the cell of choice, including mutant, truncated, and hybrid promoters. Preferably, the promoter is a CAG promoter, ACTB promoter, ubc promoter, CMV promoter, EF1A promoter, PGK promoter or TRE promoter.
In the present invention, the transcription termination sequence is a sequence that can be recognized by a 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 which is functional in the host cell of choice may be used in the present invention.
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.
According to some embodiments of the invention, the gene editing system is a CRISPR/Cas9 gene editing system.
In the present invention, the host cell used by the recombinant cell may be any suitable cell type, including mammalian cells, insect cells, preferably mammalian cells.
According to some embodiments of the invention, the host cell used by the recombinant cell is a HEK293 cell, CHO cell, or SF9 cell.
In a fourth aspect, the present invention provides the use of a recombinant expression vector as defined above or a recombinant cell as defined above for the preparation of an exosome loaded with an active substance.
In a fifth aspect, the present invention provides a method for producing an exosome loaded with an active substance, wherein the exosome loaded with an active substance is isolated from a cell culture solution of the recombinant cell.
In the present invention, methods of exosome separation and purification known in the art may be employed, including but not limited to ultracentrifugation, density gradient centrifugation, size exclusion chromatography, affinity chromatography, adsorption chromatography, bonded phase chromatography, filtration, polymer-based precipitation techniques, immunoisolation techniques, or sieve separation.
According to some embodiments of the present invention, the method of preparing an exosome loaded with an active substance using density gradient centrifugation involves the steps of:
Providing a cell culture solution of the recombinant cells, and separating the cell culture solution by a centrifugal machine to obtain a supernatant;
Filtering the supernatant by a deep filtration system and/or a microfiltration membrane, and collecting filtrate;
concentrating the filtrate by adopting a tangential flow concentration system to obtain concentrated solution;
carrying out enzymolysis on the concentrated solution by nuclease under the water bath condition of 20-40 ℃, centrifuging the solution after enzymolysis, and carrying out resuspension precipitation by using a buffer solution to obtain a crude extract solution;
And separating and purifying the crude extract solution by adopting density gradient centrifugation to obtain the exosome loaded with the effective substances.
Preferably, the density gradient centrifugation uses a layering solution of discontinuous density gradient, wherein the layering solution is iodixanol-sucrose buffer solution.
According to some embodiments of the invention, the method of preparing comprises the step of transfecting a host cell with the recombinant expression vector described above to obtain the recombinant cell, wherein the transfection of the recombinant expression vector and the gene editing system are performed simultaneously or separately.
According to some embodiments of the invention, the medium used to culture the recombinant cells is a serum-free medium to which an antibiotic corresponding to the gene encoding the antibiotic in the recombinant expression vector is added, facilitating selection of stably transfected cells during the culture process.
In a sixth aspect, the present invention provides a pharmaceutical composition comprising an exosome as described above loaded with an active substance, optionally with pharmaceutically acceptable excipients.
In embodiments of the present invention, the particular excipients may be selected according to the intended mode of administration and therapeutic application, including but not limited to pharmaceutical, nutraceutical, or physiologically acceptable carriers.
In embodiments 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.
In a seventh aspect, the present invention provides a kit comprising an exosome loaded with an active substance as described above, or a pharmaceutical composition as described above.
In an eighth aspect the invention provides the use of a PVR protein as a scaffold protein for loading an effective substance onto an exosome, wherein the effective substance is a biological macromolecule, a small molecule compound or a combination of both.
In a ninth aspect, the present invention provides a method for preventing or treating a disease, the method comprising administering to a subject the above-described exosomes loaded with an active substance or the above-described pharmaceutical composition.
In embodiments of the present invention, it is preferable to determine the specific diseases that the exosomes loaded with the active substances correspondingly detect, prevent or treat, including but not limited to various neoplastic or non-neoplastic diseases, based on the active substances loaded on the exosomes.
By adopting the technical scheme, compared with the prior art, the invention has the following advantages:
The PVR protein is used as exosome scaffold protein to load effective substances, so that the loading capacity and the loading efficiency of the effective substances are improved, and the PVR protein can be used for loading the effective substances with larger structures and more complicated structures.
Drawings
FIG. 1 is a schematic diagram of sequence elements of a gene expression cassette;
FIG. 2 is a graph showing the growth curves of HSA-loaded stably transfected cells of example 1, comparative example 1 and comparative example 4;
FIG. 3 is a flow chart of the cell flow analyses of HSA-loaded stably transfected cells of example 1, comparative example 1 and comparative example 4;
FIG. 4 is a graph of the growth curves of the drug-loaded screening of IL-15-loaded stably transfected cells of example 2, comparative example 2 and comparative example 5;
FIG. 5 is a graph of cell flow analyses of IL-15 loaded stably transfected cells of example 2, comparative example 2 and comparative example 5;
FIG. 6 is a graph of the growth curves of the drug addition screens of the stably transfected cells loaded with 4-1BBL of example 3, comparative example 3 and comparative example 6;
FIG. 7 is a graph of cell flow assays for 4-1BBL loaded stably transfected cells of example 3, comparative example 3 and comparative example 6;
FIG. 8 is a graph of NTA analysis of HSA-loaded exosomes of example 1, comparative example 1 and comparative example 4;
Fig. 9 is a TEM analysis diagram of HSA-loaded exosomes of example 1, comparative example 1 and comparative example 4;
FIG. 10 is a WB analysis chart of HSA-loaded exosomes of example 1, comparative example 1 and comparative example 4;
FIG. 11 is a graph showing quantitative analysis of HSA-loaded exosomes of example 1, comparative example 1 and comparative example 4 by ELISA;
FIG. 12 is a graph of NTA analysis of IL-15 loaded exosomes of example 2, comparative example 2 and comparative example 5;
FIG. 13 is a TEM analysis of IL-15 loaded exosomes of example 2, comparative example 2 and comparative example 5;
FIG. 14 is a WB analysis of IL-15-loaded exosomes of example 2, comparative example 2 and comparative example 5;
FIG. 15 is a graph showing ELISA quantitative analysis of IL-15-loaded exosomes of example 2, comparative example 2 and comparative example 5;
FIG. 16 is a NTA analysis chart of 4-1 BBL-loaded exosomes of example 3, comparative example 3 and comparative example 6;
FIG. 17 is a TEM analysis chart of 4-1 BBL-loaded exosomes of example 3, comparative example 3 and comparative example 6;
FIG. 18 is a graph showing ELISA quantitative analysis of 4-1 BBL-loaded exosomes of example 3, comparative example 3 and comparative example 6;
Fig. 19 is a graph showing comparison of the loading efficiencies of the exosomes loaded with the active substances of examples 1 to 3 and comparative examples 1 to 6.
Detailed Description
The term "exosomes" refers to cell-derived Extracellular Vesicles (EVs) having a diameter of 20-300 nm, which comprise a membrane enclosing an internal space, the membrane being of a phospholipid bilayer structure. Exosomes may mediate the intercellular transfer of intracellular substances.
By "engineered exosomes" is meant exosomes that are modified, e.g., membranes are modified in their protein, lipid, small molecule, carbohydrate, etc., composition. Including exosomes engineered by genetic engineering, physical or chemical means to enable loading and/or delivery of an effective substance.
"Loading" refers to the attachment of the scaffold protein to the active agent and overexpression on the exosomes, thereby enriching the active agent onto the exosomes.
"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 an exosome. The fusion protein may be formed by fusion of the scaffold protein with the active agent or a biologically active portion of the active agent or a polypeptide linker attached to the active agent, the scaffold protein portion of the fusion protein being located in the exosome membrane or lumen.
The PVR protein, the CD99 protein and the PTGFRN protein belong to type I transmembrane proteins, and belong to scaffold proteins.
An "active agent" refers to any biological macromolecule or small molecule compound of interest that is functional (e.g., prophylactic, therapeutic, diagnostic, etc.). The "active substance" is also commonly referred to as "cargo".
The term "biological macromolecule" refers to a macromolecule such as a protein, a nucleic acid, or a polysaccharide having a molecular weight of 1000 or more, which is present in a cell of an organism.
"Small molecule compounds" refers to compounds having a molecular size of less than 1000, including conventional organic and inorganic compounds, and also includes a wide variety of biologically active small molecule compounds, such as vitamins, hormones, amino acids and derivatives thereof, peptides, nucleotides, and the like, having a relatively small molecular weight.
"Nucleic acid" refers to nucleotides of any length, including deoxyribonucleotides, ribonucleotides, combinations thereof, and analogs thereof.
"Polypeptide" and "peptide" are used interchangeably and refer to a polymer of amino acids of any length. Thus, polypeptides, oligopeptides, proteins, antibodies and enzymes are included within the definition of polypeptide.
"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.
An "exogenous nucleic acid fragment" includes any gene of interest or fragment thereof, such as a gene encoding a scaffold protein or fragment thereof. The exogenous nucleic acid fragment is of a different source than the host cell, e.g., a nucleic acid sequence isolated from an organism different from the host cell, i.e., is an exogenous nucleic acid fragment relative to the host cell.
An "expression vector" refers to a nucleic acid molecule capable of transporting another nucleic acid molecule to which it is linked. Examples of vectors include, but are not limited to, plasmids, viruses, bacteria, phages and insertable DNA fragments. The term "plasmid" refers to circular double-stranded DNA capable of receiving an exogenous nucleic acid fragment and capable of replication in a prokaryotic or eukaryotic cell.
"Promoter" refers to a nucleic acid sequence that can control transcription of a coding sequence. Promoter sequences include specific sequences sufficient for RNA polymerase recognition, binding, and transcription initiation. Promoters can affect transcription of genes that are located on the same nucleic acid molecule as themselves or genes that are located on different nucleic acid molecules as themselves.
"Host cell" is also referred to as a recipient cell, and includes, but is not limited to, an animal cell, a plant cell, an algae cell, a fungal cell, a yeast cell, or a bacterial cell. Exemplary host cells include human embryonic kidney cells HEK293. It is understood that the progeny of a single parent cell need not be identical, in morphology or in genomic or total DNA complement, to the original parent, due to natural, accidental, or deliberate mutation.
"HSA" is an abbreviation for human serum albumin, a plasma protein, one of the more abundant proteins in humans. Human serum albumin is synthesized mainly by the liver, is present in the blood, and plays an important physiological role in the systemic circulation. Human serum albumin maintains blood stability by maintaining normal osmotic pressure and blood volume in plasma. The protein can also bind and transport various substances such as medicines, hormones, lipids and the like to assist in the delivery and distribution of the protein in vivo. Human serum albumin also participates in the regulation of blood coagulation and fibrinolysis systems, and plays an important role in maintaining the normal coagulation and dissolution state of blood. The human serum albumin can play a role in buffering in vivo and help maintain the acid-base balance of blood and tissues. Meanwhile, the human serum albumin participates in the regulation of immune response, and has nonspecific resistance to external substances such as bacteria, viruses and the like. Is mainly used for preventing measles and infectious hepatitis, improving immunity of organisms and treating hypoalbuminemia.
"IL-15" is an abbreviation for interleukin-15, an important cytokine. The biological functions of L-15 mainly include stimulation of differentiation of T cells, B cells and natural killer cells (NK cells) and promotion of proliferation of T cells, B cells and NK cells. The IL-15 has wide cell source and target cell distribution, and is suitable for immunotherapy and tumor resistance.
"4-1BBL" is an abbreviation for 4-1BB ligand, which is a type II membrane protein of the TNF superfamily, expressed on antigen presenting cells. 4-1BB and 4-1BBL can induce T cell expansion, cytokine induction, differentiation, and up-regulation of anti-apoptotic genes, and protect T cells from activation-induced cell death (AICD). 4-1BBL stimulates T cell proliferation and induces an effective anti-tumor immune response. 4-1BBL is an immunostimulatory molecule that interacts with the 4-1BB high affinity receptor during antigen presentation to trigger pleiotropic effects on the immune system by activating NF-kB, c-Jun, and p38 downstream pathways to provide costimulatory signals to CD4 + and CD8 + T cells. Is suitable for immunotherapy and anti-tumor.
Embodiments of the present invention provide exosomes loaded with an active substance, comprising an exosome, a PVR protein overexpressed in the exosome, and an active substance linked to the PVR protein. PVR proteins belong to the class I transmembrane proteins, which can cross phospholipid bilayer membranes of exosomes, through which active substances can be loaded onto exosomes, including both inside and outside exosomes. In order to ensure the bilayer membrane structure of the exosome, the active substance is preferably loaded onto the outer surface of the exosome through PVR protein, and the space for loading the active substance on the outer surface of the exosome is larger, which is beneficial to loading more active substance.
The technical scheme of the present invention will be further described with reference to specific examples, but the present invention is not limited to the following examples.
The conditions employed in the following examples and comparative examples may be further adjusted according to specific requirements, and the conditions not specified are generally those in routine experiments. Instruments, materials and reagents are commercially available without specific reference.
Example 1
The present example provides an exosome loaded with human serum HSA albumin and a method of preparing the same. This example uses over-expressed PVR protein as an exosome scaffold protein to load human serum HSA albumin onto the outer surface of an exosome, comprising the steps of:
1. Construction of expression vectors
The expression vector used in this example was pAAVS plasmid, and a gene expression cassette was inserted into the AAVS1 site. The design of the expression frame is as follows: human serum HSA albumin (sequence shown as SEQ ID NO: 3) is fused at the N-terminal of PVR protein (sequence shown as SEQ ID NO: 1) by using CAG promoter (sequence shown as SEQ ID NO: 6) as transcription start point, bgH polyA (sequence shown as SEQ ID NO: 7) as transcription end point, and Puromycin (PURO) screening gene (sequence shown as SEQ ID NO: 8) is also carried in the expression frame.
The method comprises the following specific steps:
(1) And (3) PCR cloning: the upstream and downstream primers were designed according to the above expression cassette using a conventional method in the art, and Jin Weizhi Biotechnology Co., ltd was commissioned to synthesize the upstream primer, the downstream primer and the template DNA. Into a 0.2mL EP tube, 25. Mu.L of DNA polymerase (2X Phanta Max Master Mix, nuo-NZan Vazyme, P515-01), 2. Mu.L of upstream primer, 2. Mu.L of downstream primer, 10g of template DNA were added, and the mixture was supplemented to 50. Mu.L with RNase-free ddH 2 O, nuo-NZan Vazyme, P071-01-AA, and the mixture was placed in a PCR apparatus, and an amplification program was designed according to the annealing temperature of the primer.
(2) And (3) purifying a PCR product: the PCR product was purified according to the kit instructions (Nuo Wei Vazyme, DC 301-01).
(3) And (3) enzyme cutting: into a 0.2mL EP tube, 5. Mu. L rCutSmart TM buffer (BioLabs, 136004 s), 0.5. Mu.L Pac I enzyme (BioLabs, R0547L), 0.5. Mu.L Not I enzyme (NEW ENGLAND BioLabs (NEB), R3189L), 2. Mu.g of PCR-purified fragment, 5. Mu. g pAAVS1 vector plasmid were added. The mixture was supplemented with enzyme-free water (Rnase-free ddH 2 O, nuo Weizan Vazyme, P071-01-AA) to 10. Mu.L, and the mixture was subjected to digestion at 37℃for 16h in a PCR apparatus.
(4) Ligation transformation: into a 0.2mL EP tube, 4. Mu.L of the enzyme-digested liquid obtained in step (3), 1. Mu.L of T4 DNA ligase (Nuo Zan Vazyme, N103-01-AA) and 1. Mu.L of T4 DNA ligase buffer (Nuo Zan Vazyme, N103-01-AC) were added. After 10min incubation at room temperature, 5 μl was added to competent cells (DH 5 se:Sup>A, norway Vazyme, C502-03) and incubated on ice for 30min, at 42℃for 90s, on ice for 2min, then 1m of B medium (BeyoPure ™,04.05. ST156) was added, and after 1h incubation in se:Sup>A shaking incubator, 12000g was centrifuged for 1min, 100 μl of supernatant was removed and resuspended and added to bacterial dishes (BAISHAYI Biosharp, BS-90-D), and then se:Sup>A disposable plastic coating rod (BAISHAYI Biosharp, BS-PS-A) was smeared and then placed evenly in se:Sup>A 37℃incubator for storage.
2. Construction of stably transformed cell lines
As shown in fig. 1, there is a homology arm sequence of AAVS1 site on the backbone of the expression vector. Using a Cas9-gRNA complex directed against the AAVS1 site (Cas 9 sequence shown in SEQ ID NO: 9 and AAVS1 sgRNA sequence shown in SEQ ID NO: 10), a DNA strand break was introduced at this site, allowing the entire insertion of the expression cassette into the genome with a homologous recombination mediated repair mechanism (HDR).
The method comprises the following specific steps:
(1) Preparation of transfected cells
EXPI293F cells were inoculated at 1.5E+6/mL with a viability of 95% or more in 125mL shake flasks and the total volume of medium was 25mL. The cells were incubated overnight at 8% CO 2 with constant temperature and transfection was started the next day.
(2) PEI transfection
Using the TA293 reagent (Margaritifer kai Biotechnology Co., ltd., K2001), the transfection procedure was as follows, taking a volume of 25mL of cell suspension as an example:
And extracting the target plasmid from the transformed competent cells subjected to the coating culture preservation by adopting a conventional technical means in the field, thereby obtaining the constructed expression vector. Two 15mL centrifuge tubes were prepared, 1.25mL serum-free cell culture medium, 12.5. Mu.g CRISPR/Cas9 plasmid (assigned Jin Weizhi to Biotechnology Co., ltd.) and 12.5. Mu.g destination plasmid were added to one of them, and vortexed and mixed well; the other was added with 1.25mL of serum-free cell culture medium and 125. Mu.L of TA293 transfection reagent, and vortexed. All liquid in the centrifuge tube containing the transfection reagent was transferred to the centrifuge tube containing the plasmid and vortexed. The mixture was allowed to stand at room temperature for 10 minutes to prepare a plasmid-vector complex. The EXPI293F cells were removed from the thermostatic shaker, the prepared plasmid-vector complex was added drop-wise while shaking, and then returned to the CO 2 thermostatic shaker for shake culture.
(3) Cell screening
Cell density and viability were recorded using a cell counter 96 hours after transfection, transfection efficiency was analyzed using a fluorescence microscope (Invitrogen, EVOS M, 5000), and antibiotic screening was started. Cells were cultured with medium containing 2. Mu.g/mL puromycin, and the medium was changed every 2-3 days, and low-speed centrifugation was performed using 120g/4min while continuously recording cell density and viability. The survival rate of the cells at the first week of the initial screening is gradually reduced, and the final screening success standard is recovered to about 95% of the survival rate of the cells, which can be regarded as stable cells. The serum-free cell cryopreservation solution is used for cryopreserving stable transformed cells, and the cryopreservation density is 1E7 cells/tube.
3. Separation and purification of exosomes
Frozen stable cells are taken and cultured by adopting a serum-free culture medium (Aomo Pu Mai organism, CD 05) at 37 ℃ and 8 percent CO 2 and 120 rpm level in a shaking way until the viable cell density is more than 9 multiplied by 10 6/mL, and the cell survival rate is more than 90 percent. 6000 g, centrifuging at 4 ℃ for 20min, and collecting the supernatant. 16000 g, centrifuging at 4 ℃ for 30 min, and collecting the supernatant. The supernatant was filtered through a depth filtration system (cobber Cobetter, CSCCD1070 PCP) and a sterile filtration system (cobber Cobetter, L10THSLESS P). The supernatant was filtered through a 0.45 μm filter (Millipore) and a 0.22 μm filter (Millipore). The supernatant was concentrated 10-fold by a Tangential Flow Filtration (TFF) system (Repride gold Repligen, model KR2 i) to give a concentrate. MgCl 2 solution (final concentration 1 mM) and omnipotent nuclease (final concentration 20U/mL; off-shore protein Novoprotein, GMP-1707) were added to the concentrate, either in a 25℃water bath 16 h or a 37℃water bath 3 h. The nuclease treated concentrate was centrifuged at 133900 g and at 4℃at 60 min. The pellet was resuspended in PBS and repeatedly blown with a1 mL syringe until the pellet was completely dissolved to give a crude extract solution.
Sucrose (Sucrose) buffer (250 mM Sucrose, 10 mM Tris HCl, 1mM EDTA, pH 7.4) was prepared, iodixanol (Iodixanol) solution (merck Sigma, D1556-250 ML) was diluted with Sucrose buffer, 17.5% (V/V) was prepared (Iodixanol solution/(Iodixanol solution+sucrose buffer)), and 45% (V/V) was prepared (Iodixanol solution/(Iodixanol solution+crude extract solution)). The PBS was added to the bottom of the centrifuge tube with a syringe, 17.5% of the diluent and 45% of the diluent were then added sequentially from the bottom, the uppermost layer was filled with PBS, and the tube was centrifuged at 150000 g and 4℃for 16: 16 h. The white interface layer that migrated between PBS and 17.5% dilution was carefully removed and transferred to a fresh tube, the tube was filled with PBS, centrifuged at 20000 g, 4℃for 30min, and the contaminating proteins removed. The supernatant was transferred to a new tube and centrifuged at 135000 g at 4℃for 3: 3h. The pellet (i.e., isolated exosomes) was resuspended in 100 μl PBS and stored at 4 ℃ for later use.
The HSA-loaded exosomes obtained in this example were labeled: HSA-PVR.
Example 2
The present example provides an exosome loaded with interleukin-15 and a method of preparing the same. This example uses over-expressed PVR protein as exosome scaffold protein to load interleukin-15 onto the surface of exosomes. The preparation method is basically the same as in example 1, except that human serum HSA albumin is replaced by interleukin-15 (IL-15 for short, the sequence is shown as SEQ ID NO: 4).
The interleukin-15-loaded exosome markers obtained in this example were: IL15-PVR.
Example 3
The embodiment provides an exosome loaded with immune stimulating molecule 4-1BBL protein and a preparation method thereof. This example uses over-expressed PVR protein as exosome scaffold protein to load immunostimulatory molecule 4-1BBL protein onto the surface of the exosome. The preparation method is basically the same as in example 1, except that human serum HSA albumin is replaced with immunostimulatory molecule 4-1BBL protein (sequence shown as SEQ ID NO: 5).
The exosome markers of the immunostimulatory molecule 4-1BBL protein loaded obtained in this example were: 41BBL-PVR.
Comparative example 1
This comparative example provides another exosome loaded with human serum HSA albumin, which uses the over-expressed CD99 protein as an exosome scaffold protein to load human serum HSA albumin onto the exosome surface, with the exception that the PVR protein is replaced with a CD99 protein (sequence shown in SEQ ID NO: 2) in substantially the same manner as example 1.
The HSA-loaded exosomes obtained in this comparative example were labeled: HSA-CD99.
Comparative example 2
This comparative example provides another interleukin-15-loaded exosome which uses over-expressed CD99 protein as exosome scaffold protein to load interleukin-15 onto the surface of exosome, and its preparation method is basically the same as comparative example 1, except that human serum HSA albumin is replaced with interleukin-15 (abbreviated as IL-15, sequence shown as SEQ ID NO: 4).
The IL-15 loaded exosomes obtained in this comparative example were labeled: IL15-CD99.
Comparative example 3
This comparative example provides an exosome of another immunostimulatory molecule 4-1BBL protein, which uses an over-expressed CD99 protein as an exosome scaffold protein to load the immunostimulatory molecule 4-1BBL protein onto the exosome surface, with the exception that human serum HSA albumin is replaced with the immunostimulatory molecule 4-1BBL protein (sequence shown as SEQ ID NO: 5) in substantially the same manner as comparative example 1.
The exosome markers loaded with 4-1BBL protein obtained in this comparative example were: 41BBL-CD99.
Comparative example 4
This comparative example provides another exosome loaded with human serum HSA albumin, which uses the over-expressed PTGFRN protein as exosome scaffold protein to load human serum HSA albumin onto the exosome surface, and the preparation method is basically the same as example 1, except that PVR protein is replaced with a PTGFRN protein of Codiak Biosciences company (see patent PCT/US2018/048026, US20200222556 A1).
The HSA-loaded exosomes obtained in this comparative example were labeled: HSA-PTGFRN.
Comparative example 5
This comparative example provides another interleukin-15-loaded exosome which uses an over-expressed PTGFRN protein as an exosome scaffold protein to load interleukin-15 onto the surface of the exosome, with the exception that human serum HSA albumin is replaced with interleukin-15 (abbreviated as IL-15, sequence shown in SEQ ID NO: 4) in substantially the same manner as in comparative example 4.
The IL-15 loaded exosomes obtained in this comparative example were labeled: IL15-PTGFRN.
Comparative example 6
This comparative example provides an exosome of another immunostimulatory molecule 4-1BBL protein, which uses an overexpressed PTGFRN protein as an exosome scaffold protein to load the immunostimulatory molecule 4-1BBL protein onto the exosome surface, with the exception that human serum HSA albumin is replaced with the immunostimulatory molecule 4-1BBL protein (sequence shown as SEQ ID NO: 5).
The exosome markers loaded with 4-1BBL protein obtained in this comparative example were: 41BBL-PTGFRN.
In the cell screening phase, HSA-loaded stably transformed cells were screened for growth by drug addition as shown in FIG. 2, taking 3-4 weeks on average to complete the screening (activity > 95%). The results of the cell flow analysis of the HSA-loaded stable transgenic cells are shown in FIG. 3, and the expression positive rate of the HSA protein of the stable transgenic cell strain is more than 95%.
The dosing screen growth curve of IL-15 loaded stably transfected cells is shown in FIG. 4. On average, screening was completed in 3-4 weeks (activity > 95%). The results of the cell flow analysis of the stable transgenic cells loaded with IL-15 are shown in FIG. 5, and the expression positive rate of the IL-15 protein of the stable transgenic cell strain is more than 95%.
The dosing screening growth curve for the whole process of stable cell construction loaded with 4-1BBL is shown in FIG. 6, taking 3-4 weeks on average to complete screening (activity > 95%). The results of the cell flow analysis of the stably transformed cells loaded with 4-1BBL functional goods are shown in FIG. 7, and the expression positive rate of the stably transformed cell strain 4-1BBL protein is more than 95%.
Analysis of the effective substance Loading Capacity of engineered exosomes of examples 1 to 3 and comparative examples 1 to 6
NTA analysis was performed on the exosomes using a Nano-FCM instrument (Fu Living beings, cat. N30E). TEM analysis was performed on the exosomes using transmission electron microscopy. And (3) performing WB analysis on the exosomes by adopting electrophoresis. The number of the above exosomes loaded with the active substances was quantitatively analyzed by ELISA.
The results of the HSA-loaded exosome NTA analysis are shown in FIG. 8. The particle sizes of HSA-PVR, HSA-CD99 and HSA-PTGFRN are relatively uniform, and are intensively distributed in the range of 130-160 nm. The exosome yields available per ml of cell culture fluid can reach the order of 2E9 particle.
The results of HSA-loaded exosomes TEM analysis are shown in FIG. 9. The clear protein crown structure was seen on both the surface of HSA-PVR and HSA-PTGFRN, while the surface of HSA-CD99 was relatively smooth. PVR and PTGFRN were shown to be able to load HSA to the surface of the exosomes and the exosome surface loaded HSA density was higher, whereas CD99 as a scaffold protein the exosome surface loaded HSA density was lower.
The results of the HSA-loaded exosome WB analysis are shown in FIG. 10. Under the condition of loading the same total number of particles, the band brightness of the HSA-PVR is the strongest, the HSA-PTGFRN is the weakest, and the stronger the band brightness is, the larger the loading capacity of the effective substances on the exosome is, and the higher the loading capacity is.
The results of quantitative analysis of HSA-loaded exosome ELISA are shown in FIG. 11. The HSA cargo loading quantity is sequentially from high to low: HSA-PVR, HSA-PTGFRN, HSA-CD99.
The results of the IL-15 loaded exosome NTA analysis are shown in FIG. 12. The particle sizes of the IL15-PVR, the IL15-CD99 and the IL15-PTGFRN are relatively uniform, and are intensively distributed in the range of 120-160 nm.
The results of the IL-15 loaded exosome TEM analysis are shown in FIG. 13. Because of the low expression level of IL-15 itself, the loading density on the surface of the exosomes was low, and no particularly pronounced protein corona structure resembling the HSA exosomes was seen.
The results of the IL-15 loaded exosome WB assay are shown in FIG. 14. Under the condition of loading the same total number of particles, the brightness of the IL15-PVR band is the strongest, and the IL15-PTGFRN and the IL15-CD99 are the weakest, wherein the stronger the brightness of the band is, the greater the loading capacity of the effective substances on the exosomes is, and the greater the loading capacity is.
The results of ELISA quantitative analysis of IL-15-loaded exosomes are shown in FIG. 15. The loading amount of IL-15 is sequentially from high to low: IL15-PVR, IL15-PTGFRN, IL15-CD99.
The NTA analysis results of the 4-1 BBL-loaded exosomes are shown in FIG. 16. The particle sizes of the 41BBL-PVR, the 41BBL-CD99 and the 41BBL-PTGFRN are relatively uniform and are intensively distributed in the range of 140-160 nm. The exosome yield that can be obtained per ml of cell culture fluid is on the order of 5E9 particle.
TEM analysis results of 4-1 BBL-loaded exosomes are shown in FIG. 17. Because of the low expression level of 4-1BBL itself, the loading density on the surface of the exosomes was low, and no particularly pronounced protein corona structure resembling the HSA exosomes was seen.
The results of ELISA quantitative analysis of 4-1 BBL-loaded exosomes are shown in FIG. 18. The loading quantity of the 4-1BBL is sequentially from high to low: 41BBL-PVR, 41BBL-PTGFRN, 41BBL-CD99.
The results of the analysis of the effective substance loading efficiencies of the exosomes of examples 1 to 3 and comparative examples 1 to 6 are shown in fig. 19. The exosomes using PVR as scaffold protein exhibited a higher loading capacity for active substances than PTGFRN and CD99 of Codiak Biosciences.
The PVR protein adopted in the invention has the length of 417aa, the PTGFRN protein has the length of 879aa, and the PVR protein has the shorter length, so the PVR protein has the following obvious advantages as an exosome scaffold protein:
1. the gene editing efficiency is higher. In most cases, the gene is shorter, the knock-in efficiency is higher, the editing difficulty at the cell level is lower, and the engineering transformation of cells is facilitated.
2. Is suitable for more carriers. For some viral vectors, the length of the genome that can be packaged is relatively limited, e.g., currently mainstream AAV vectors, typically packaging capacities of no more than 5kbp. Thus, the shorter the length of the scaffold protein, the longer the length of the drug protein that can be delivered, which can significantly extend the delivery capacity and the patent drug potential of AAV such vectors.
3. Protein synthesis is more efficient and is suitable for the delivery of more active substances. In most cases, the length of the protein is shorter, the cell synthesis efficiency is higher, and shorter scaffold proteins tend to be loaded with larger, more structurally complex active substances.
Example 4
The present example provides a formulation for extending the half-life of exosomes in blood, comprising: HSA-PVR resuspension of example 1. After the HSA protein is modified on the surface of the exosome, HSA can interact with FcRn receptor, so that the HSA exosome endocytosed into the cell is not degraded by lysosomes, but directly recovered to the outside of the cell, thereby prolonging the half-life of the modified exosome in blood.
Example 5
The present example provides a formulation for inhibiting the growth of solid tumors comprising: IL15-PVR resuspension of example 2, intratumoral injection was directly performed on solid tumors. Since cytokines such as IL15 can promote proliferation and activation of immune cells such as T cells, B cells and NK cells, it turns a "cold" tumor into a "hot" tumor, inhibiting tumor expansion. In addition, IL15 is retained in the tumor microenvironment by the EPR (enhanced permeability and retention) effect of IL15-PVR exosomes within the tumor, thereby reducing the systemic toxicity of IL 15.
Example 6
The present example provides a formulation for in vitro expansion of T cells/NK cells, the formulation of which is: the 41BBL-PVR suspension of example 3, was prepared at a rate of at least 1000: exosomes of 1: the ratio of cells was added to the T cell/NK cell broth. Through the direct interaction of 41BBL on the surface of an exosome and a 4-1BB high-affinity receptor on the surface of a cell, NF-kB, c-Jun and p38 downstream channels are activated to provide co-stimulation signals for CD4 +/CD8+ T cells/NK cells, and the expansion and activation efficiency of the T cells/NK cells can be remarkably improved.
The present invention has been described in detail in order to make those skilled in the art understand the present invention and implement it, but the present invention is not limited to the above embodiments, and all equivalent changes or modifications according to the spirit of the present invention should be included in the scope of the present invention.
Claims (23)
1. A pharmaceutical composition comprising an active agent-loaded exosome, optionally comprising pharmaceutically acceptable excipients, said active agent-loaded exosome comprising an exosome, a PVR protein overexpressed in said exosome, and an active agent linked to said PVR protein, the nucleotide sequence encoding said PVR protein being shown in SEQ ID No. 1, said active agent being loaded on the outer surface of said exosome, said active agent being a prophylactic, therapeutic, diagnostic biological macromolecule or small molecule compound.
2. The pharmaceutical composition of claim 1, wherein the biological macromolecule is a protein, polypeptide, saccharide, or nucleic acid.
3. The pharmaceutical composition of claim 1, wherein the active agent is HSA, IL-15 or 4-1BBL.
4. The pharmaceutical composition of claim 1, wherein the active agent is directly or indirectly linked to the N-terminus or the C-terminus of the PVR protein via a linker.
5. The pharmaceutical composition of claim 1, wherein the active agent is linked to the N-terminus or the C-terminus of the PVR protein via a polypeptide linker.
6. The pharmaceutical composition of claim 1, wherein the pharmaceutical composition is a tablet, granule, pill, capsule, emulsion, paste, gel, suspension, solution, powder, transdermal patch, spray, suppository, or implant.
Use of PVR protein as scaffold protein for the preparation of a pharmaceutical composition according to any one of claims 1 to 6, characterized in that the PVR protein as scaffold protein loads the active substance onto the outer surface of the exosome.
8. The use according to claim 7, wherein the active agent-loaded exosomes are isolated from a cell culture broth of recombinant cells containing over-expressed PVR protein and/or over-expressed fusion protein formed of PVR protein and active agent.
9. The use according to claim 8, wherein the recombinant cells contain therein 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 active substance, or a plasmid vector containing both a gene encoding a PVR protein and a gene encoding a polypeptide linker linked to an active substance.
10. The use according to claim 9, wherein the recombinant expression vector contains a gene encoding an antibiotic.
11. The use according to claim 10, wherein the gene encoding the antibiotic is one or more of the puromycin gene, the neomycin gene, the kanamycin gene, or the tetracycline gene.
12. The use according to claim 9, wherein the expression vector used for the recombinant expression vector is a viral plasmid vector or a eukaryotic expression plasmid vector.
13. The use according to claim 12, wherein the recombinant expression vector is a pAAVS plasmid, a pIRES plasmid or a pLENTI plasmid.
14. The use of claim 8, wherein the recombinant cell comprises a gene editing system therein.
15. The use of claim 14, wherein 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.
16. The use according to claim 8, wherein the host cell used by the recombinant cell is a mammalian cell or an insect cell.
17. The use according to claim 8, wherein the host cell used by the recombinant cell is HEK293, CHO or SF9 cell.
18. The use according to any one of claims 8 to 17, wherein the method for preparing the active substance-loaded exosomes comprises the steps of:
providing a cell culture solution of the recombinant cells, and separating the cell culture solution by adopting a centrifugal machine to obtain a supernatant;
Filtering the supernatant by a deep filtration system and/or a microfiltration membrane, and collecting filtrate;
concentrating the filtrate by adopting a tangential flow concentration system to obtain concentrated solution;
carrying out enzymolysis on the concentrated solution by nuclease under the water bath condition of 20-40 ℃, centrifuging the solution after enzymolysis, and carrying out resuspension precipitation by using a buffer solution to obtain a crude extract solution;
And separating and purifying the crude extract solution by adopting density gradient centrifugation to obtain the exosome loaded with the effective substances.
19. The use of claim 18, wherein the density gradient centrifugation uses a discontinuous density gradient of a layering liquid which is iodixanol-sucrose buffer.
20. The use of claim 18, wherein the method of preparing further comprises the step of transfecting a host cell with the recombinant expression vector and the gene editing system to obtain the recombinant cell, wherein the transfection of the recombinant expression vector and the gene editing system are performed simultaneously or separately.
21. The use according to claim 18, wherein the stable transformed cells are selected using an antibiotic selection system, and an antibiotic corresponding to the gene encoding the antibiotic in the recombinant expression vector is added to the cell culture broth used to culture the recombinant cells.
22. The use according to claim 18, wherein the cell culture broth used for culturing the recombinant cells is serum-free medium.
23. A kit comprising the pharmaceutical composition of any one of claims 1 to 6.
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