A gene delivery method based on exosomes and peptides containing nuclear localization sequences

Abstract

The present invention belongs to the field of biomedicine, and specifically relates to a gene delivery method based on exosomes and polypeptides containing nuclear localization sequences. Specifically, the present invention provides a method for increasing the expression of exogenous genes in target cells through exosomes, the method comprising the steps of mixing exosomes with exogenous genes and co-electroporating them with cells; the method also comprises mixing and incubating the exogenous genes with auxiliary polypeptides and / or KK58 before electroporation, wherein the auxiliary polypeptides are composed of nuclear localization signals, nuclear localization signals and DNA binding domains.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and in particular relates to a gene delivery method based on exosomes and polypeptides containing nuclear localization sequences. Background Art

[0002] Gene therapy refers to editing disease-causing defective genes at the DNA level, which cures diseases by correcting gene defects or regulating gene expression. How to use vectors to accurately and effectively introduce exogenous genes into target cells is the key technology for the success of gene therapy. 80% of current clinical experimental studies use viral vectors such as retroviruses, adenoviruses, and adeno-associated viruses. In addition, non-viral vectors such as receptors, liposomes, and lipid nanoparticles (LNP) are also included. Although the above-mentioned vectors have high transfection efficiency, they still have safety hazards such as immunogenicity, toxicity, carcinogenicity, and host DNA insertion and integration. Exosomes, as natural intercellular information carriers, can overcome the problems of the former in immunogenicity and safety, and also have stronger targeting and are easy to pass through the blood-brain barrier. They have great application potential in the field of drug carriers.

[0003] Exosomes refer to multivesicular bodies with a diameter of 40-100 nm that are derived from the invagination of endolysosomal microparticles in cells. They are composed of a lipid bilayer containing transmembrane proteins, cytoplasmic proteins, and various nucleic acids. Exosomes can be secreted by a variety of cells and body fluids in the human body, including endothelial cells, immune cells, platelets, smooth muscle cells, etc. When they are secreted from host cells into receptor cells, exosomes can regulate the biological activity of receptor cells through the proteins, nucleic acids, lipids, etc. they carry.

[0004] Compared with most existing gene drug carriers, exosomes have the following advantages: (1) Exosomes have high biocompatibility and low immunogenicity, making them safer for in vivo use and allowing for multiple dosing; (2) Exosomes can carry and deliver a variety of molecules, including nucleic acids, proteins, and small molecules, and have the potential to be developed as carriers for gene drugs, small RNA drugs, protein drugs, and small molecule drugs; (3) Exosomes can cross the blood-brain and blood-eye barriers, facilitating drug delivery to the central nervous system and eyes through the peripheral blood; and (4) Exosomes have high potential for engineering modification. Exosomes engineered outside or inside cells can achieve specific loading of different drug molecules and specific targeted delivery to different tissues. Summary of the invention

[0005] In order to improve the efficiency of exosome delivery of exogenous DNA, the present invention provides the following technical solutions:

[0006] In a first aspect, the present invention provides a method for increasing the expression of exogenous genes in target cells by exosomes, the method comprising the steps of mixing exosomes and exogenous genes, co-electroporating, and then co-incubating with cells;

[0007] The method further comprises any one or more of the following steps:

[0008] 1) Before electroporation, the exogenous gene is mixed with an auxiliary polypeptide and / or KK58 and incubated, wherein the auxiliary polypeptide is a nuclear localization signal, a nuclear localization signal and a DNA binding domain connected, and the sequence of KK58 is shown in SEQ ID NO.17;

[0009] 2) Add lysosomal inhibitors to the culture medium in which cells are co-incubated;

[0010] 3) Before electroporation, the exogenous gene is mixed and incubated with the HSP protein;

[0011] 4) Before electroporation, mix the exogenous gene with P3000 and incubate;

[0012] 5) Before electroporation, the exosomes were mixed and incubated with low concentration of PEI;

[0013] 6) Before electroporation, the exosomes were mixed and incubated with PLL.

[0014] Preferably, the method comprises the above steps 1) and 5).

[0015] Preferably, the method comprises the above steps 2) and 4).

[0016] Specifically, increasing the expression level of exogenous genes in target cells by exosomes can also be referred to as improving the exosome delivery efficiency.

[0017] Preferably, the ratio of the exogenous gene copy number to exosomes is about 100-2500:1, 250-1000:1, or 400-600:1.

[0018] Preferably, the ratio of the exogenous gene copy number to exosomes is about 500:1.

[0019] Preferably, the mixed incubation in steps 3)-6) is carried out at room temperature.

[0020] Preferably, the mixed incubation in steps 3)-6) lasts for at least 10 minutes, 20 minutes, 30 minutes or longer.

[0021] On the other hand, the present invention provides a composition, comprising any one or more of the following: auxiliary polypeptide, KK58, lysosomal inhibitor, HSP protein, P3000, low concentration of PEI, PLL, and the sequence of KK58 is shown in SEQ ID NO.17.

[0022] On the other hand, the present invention provides the use of any one or more of the following product combinations for improving the efficiency of exosomes in delivering exogenous genes to target cells: auxiliary polypeptides, KK58, lysosome inhibitors, HSP proteins, P3000, low concentrations of PEI, and PLL, wherein the sequence of KK58 is shown in SEQ ID NO.17.

[0023] Preferably, the lysosomal inhibitor is chloroquine.

[0024] Preferably, the exosomes comprise exosome-like vesicles.

[0025] Preferably, the foreign gene is present in a plasmid (vector).

[0026] Preferably, the plasmid may be a viral expression vector.

[0027] Specifically, the viral expression vector includes a lentiviral vector, an adenoviral vector, an adeno-associated viral expression vector or other types of viral vectors.

[0028] In some embodiments, the plasmid may contain a variety of elements that control expression, including a promoter sequence, a transcription initiation sequence, an enhancer sequence, a selection element, and a reporter gene. In addition, the plasmid may also contain an origin of replication. The vector may also include components that facilitate its entry into cells, such as viral particles, liposomes, or protein shells, but not only these substances.

[0029] Preferably, the exosomes and exogenous genes are mixed and co-electroporated and then left to stand on ice for at least 10 minutes, 20 minutes, 30 minutes or longer before being co-incubated with the cells.

[0030] Preferably, the exogenous gene sequence length is at least 100 bp, 200 bp, 300 bp, 400 bp, 500 bp, 600 bp, 700 bp, 800 bp, 900 bp, 1000 bp or longer.

[0031] In a specific embodiment, the nuclear localization signal can be derived from SV40 large T antigen (SEQ ID NO.1), nucleoplasmic protein (SEQ ID NO.2), nucleocyanin (AVKRPAATKKAGQAKKKKLD, as shown in SEQ ID NO.13), EGL-13 (MSRRRKANPTKLSENAKKLAKEVEN, as shown in SEQ ID NO.14), c-Myc (PAAAKRVKLD as shown in SEQ ID NO.15) and TUS protein (KLKIKRPVK, as shown in SEQ ID NO.16).

[0032] In a specific embodiment, the DNA binding domain can be any one of the following: LDGSGSKPKRPRGRPKG (as shown in SEQ ID NO.8), LDGSGSKEKRGRGRPRK (as shown in SEQ ID NO.9), LDGSGSKPGRKPRGRPKK (as shown in SEQ ID NO.10), GSGSKEILNNHGKSKKCCENKEEKCCRK (as shown in SEQ IDNO.11), GSGSKLSRDDISKAAGMVKGVVDHLLLRLKCDSAFRGVGLLNKGSYY (as shown in SEQ ID NO.12).

[0033] Preferably, the sequence of the auxiliary polypeptide is shown in any one of SEQ ID NO.1-7.

[0034] Preferably, the auxiliary polypeptide and the exogenous gene are mixed in a material amount ratio of polypeptide:pDNA (exogenous gene) copy number = 10-1000:1, 20-500:1, 50-200:1, and the most preferred ratio is 100:1.

[0035] In a specific embodiment, the working concentration of chloroquine is 1-200 μM, 100-150 μM, or 40-60 μM.

[0036] Preferably, the working concentration of chloroquine is 50 μM.

[0037] In a specific embodiment, the HSP protein is obtained by subjecting cells to heat shock culture, centrifuging and resuspending the precipitate with a buffer.

[0038] Preferably, the heat shock culture is for at least 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours or more.

[0039] Preferably, the buffer is DPBS (Dulbecco's Phosphate-Buffered Saline), and the buffer may also be other common balanced salt solutions (Balanced Salt Solution, BSS) and physiological solutions (Physiological Solution).

[0040] Preferably, the culture temperature of the heat shock culture is 42°C.

[0041] Preferably, the heat shock culture is carried out for 1-20 hours, preferably 3-16 hours at 42°C.

[0042] Preferably, the HSP proteins obtained from 5 million cells are treated and dissolved in 1 mL of buffer and incubated with 30 μg of exogenous gene.

[0043] Preferably, the dosage of P3000 is: 2 μL / g pDNA.

[0044] Preferably, the usage ratio of PEI to exogenous gene (pDNA) is 1:3.

[0045] Preferably, the PLL is an ε-PLL.

[0046] Preferably, the ratio of the amount of PLL to the exogenous gene is 1-50:1; preferably, 4-33.33:1 (400 μg: 100 μg-1 mg: 30 μg).

[0047] Preferably, the ratio of KK58 (KK58 polypeptide) to exogenous gene is KK58:exogenous gene copy number=100:1.

[0048] Preferably, the exosome concentration in the electroporation system is 9×10 9 / mL-5×10 10 / mL.

[0049] On the other hand, the present invention provides target cells prepared by the aforementioned method and use thereof in preparing drugs.

[0050] The term "exosomes" in the present invention refers to a type of vesicle (EVs) secreted by cells outside the cell, which has a double-layer membrane structure and a saucer-shaped morphology, contains rich inclusions (including nucleic acids, proteins and lipids, etc.), and participates in the molecular transfer between cells. Exosomes are widely present in cell culture supernatants and various body fluids, including blood, lymph, saliva, urine, semen, milk, etc., and also in tissue samples, such as brain tissue, muscle tissue, adipose tissue, etc.

[0051] Preferably, the source of the exosomes includes biological fluid, biological tissue or cell culture fluid.

[0052] Preferably, the biological fluid comprises saliva, urine, synovial fluid, amniotic fluid, and breast milk.

[0053] Preferably, the biological fluid and biological tissue include any organism, and the exosomes may be derived from milk of any animal such as cow's milk.

[0054] The source of the exosomes referred to in the present invention can be the culture supernatant of human pluripotent stem cells or mesenchymal stem cells (MSC) from various tissue sources, or exosome-like vesicles extracted from milk and dairy products, or exosome-like vesicles extracted from poultry eggs, or a mixture thereof in any proportion and combination.

[0055] Preferably, the cell culture medium is a culture medium (supernatant) of mesenchymal stem cells;

[0056] Most preferably, the exosomes in the specific embodiment of the present invention are obtained from the supernatant harvested from culturing adipose-derived mesenchymal stem cells and umbilical cord blood-derived mesenchymal stem cells in a 1:1 ratio.

[0057] The target cells of the present invention can be any type of cells, preferably animal cells, more preferably human cells, specifically including germ cells and somatic cells. Non-limiting examples of somatic cells include, but are not limited to, lymphocytes, such as B cells, T cells, natural killer cells, cytokine-induced killer cells (CIK) cells; bone marrow cells, such as granulocytes, monocytes / macrophages, erythrocytes, mast cells, platelets / megakaryocytes, dendritic cells; cells from the endocrine system, including thyroid, parathyroid, adrenal, pineal cells; nervous system cells, including glial cells; respiratory system cells, including lung cells; circulatory system cells, including cardiomyocytes, pericytes; digestive system cells, including gastric cells, goblet cells, Paneth cells, etc. cells, S cells; enteroendocrine cells, including enterochromaffin cells, APUD cells, hepatocytes, cartilage / bone / muscle; bone cells, including osteoblasts, osteocytes, osteoclasts, teeth; chondrocytes, including chondroblasts, chondrocytes; skin cells, including hair cells, keratinocytes, melanocytes; muscle cells, including myocytes; urinary system cells, including podocytes, juxtaglomerular cells, glomerular mesangial cells / extraglomerular mesangial cells, renal proximal tubule brush border cells, macular density cells; reproductive system cells, including sperm, podocytes, Leydig cells, eggs; and other cells.

[0058] The electroporation of the present invention is a method of exogenous loading of exosomes. Electroporation is also called cell electroporation, which is an important method for introducing exogenous macromolecules such as DNA, RNA, siRNA, proteins, etc. and some small molecules into the cell membrane. Under the action of a strong instantaneous electric field, the cell membrane in the solution has a certain permeability, and the charged exogenous substances enter the cell membrane in a manner similar to electrophoresis. Due to the large resistance of the cell membrane phospholipid bilayer, the voltage of the cell poles generated by the external current field of the cell is borne by the cell membrane, and the voltage distributed in the cytoplasm can be ignored. There is almost no current inside the cytoplasm, which also determines that the cytotoxicity in the electroporation process within the normal range is very small. The specific parameters of the electroporation can be adjusted according to the exosome state and the culture environment, and the subtle adjustment of the electroporation parameters is within the skill range of those skilled in the art.

[0059] Specifically, the exogenous gene of the present invention may have any naturally occurring or artificially constructed polynucleotide sequence. Specifically, the exogenous gene may be DNA or RNA, and the DNA may encode any functional gene, such as ADA (adenosine deaminase), growth factor β1 (TGF-β1), β-globin gene (βA-T87Q-globin gene), ABCD1 gene and other functional genes in gene therapy products. The RNA includes coding RNA such as mRNA, non-coding RNA such as tRNA, rRNA, snoRNA, snRNA, miRNA and lncRNA or artificially synthesized RNA such as siRNA (small interferring RNA), shRNA (short hairpin RNA), sgRNA (small guide RNA), ASO (antisense oligonucleotide). BRIEF DESCRIPTION OF THE DRAWINGS

[0060] Figure 1 It is a graph showing the detection result of exosome electroporation efficiency. A is a curve based on the gradient copy number plasmid as a template; B is the statistical difference in the copy number of plasmid and EXO+plasmid-0V compared with the EXO group; C is Melting Peaks: wpre.

[0061] Figure 2 This is a graph showing the results of testing the effects of p3000 and chloroquine in promoting the expression of pDNA delivered by exosomes.

[0062] Figure 3 This is a graph showing the results of testing the effect of HSP in promoting the expression of pDNA delivered by exosomes.

[0063] Figure 4 This is a graph showing the results of testing the effects of PV-7 and KK16 polypeptides in promoting the expression of pDNA delivered by exosomes.

[0064] Figure 5 This is a graph showing the results of testing the effects of KG-33, KK-33, and KK-34 polypeptides in promoting the expression of pDNA delivered by exosomes.

[0065] Figure 6 This is a graph showing the test results of the effects of PK-35 and PY-54 polypeptides in promoting the expression of pDNA delivered by exosomes.

[0066] Figure 7 This is a graph showing the effect of KK58 polypeptide in promoting the expression of pDNA delivered by exosomes.

[0067] Figure 8 This is a graph showing the results of testing the effect of NLS-DBD polypeptide in promoting pDNA expression delivered by PEI-EXO complexes at low PEI concentrations.

[0068] Fig. 9 This is a graph showing the results of testing the effect of NLS-DBD polypeptide in promoting pDNA expression delivered by PEI-EXO complexes at lower PEI concentrations.

[0069] Fig.10 This is a graph showing the results of testing the effects of ε-PLL-EXO complex and NLS-DBD in promoting the expression of pDNA delivered by exosomes, where ε-PLL is 400 μg and plasmid is 100 μg.

[0070] Fig.11 This is a graph showing the results of testing the effects of ε-PLL-EXO complex and NLS-DBD in promoting the expression of pDNA delivered by exosomes, where ε-PLL is 400 μg and plasmid is 30 μg.

[0071] Fig.12 This is a graph showing the results of testing the effects of ε-PLL-EXO complex and NLS-DBD in promoting the expression of pDNA delivered by exosomes, where ε-PLL is 1 mg and plasmid is 100 μg.

[0072] Fig.13 This is a graph showing the results of testing the effects of ε-PLL-EXO complex and NLS-DBD in promoting the expression of pDNA delivered by exosomes, where ε-PLL is 1 mg and plasmid is 30 μg. DETAILED DESCRIPTION

[0073] The present invention is further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes based on the technical solution and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

[0074] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0075] The experimental material information used in the present invention is as follows:

[0076] Experimental Materials

[0077]

[0078] The exosomes used in the specific embodiment of the present invention are obtained by culturing adipose-derived mesenchymal stem cells (ADSCs) and umbilical cord blood-derived mesenchymal stem cells in a ratio of 1:1, and are purified and concentrated by hollow fiber columns.

[0079] In the specific embodiment of the present invention, plasmid pAAV-SFFV-Luciferase-eGFP (size 6676 bp) is used as exogenous DNA, hereinafter also referred to as plasmid DNA, pDNA, and pD.

[0080] In the specific embodiment of the present invention, HEK293T cells were used as target cells for experiments.

[0081] Example 1: Exosomes encapsulate exogenous DNA via electroporation

[0082] The 1 mL system electroporation cup of the cellcyte electroporator was used, and the electroporation program was "-4". The specific electroporation system was as follows: the electroporation solution provided by the electroporator or DPBS was used as the electroporation buffer, the number of plasmid copies: the number of exosome vesicles = 500:1, and the exosome concentration in the exosome electroporation system used in the present invention was 9×10 9 / mL to 5×10 10 / mL (NTA detected particle number).

[0083] The steps for electroporation are as follows:

[0084] 1) Exosomes were mixed with plasmid pAAV-SFFV-Luciferase-eGFP and incubated at room temperature for 15 min;

[0085] 2) Electroporation of the exosome-plasmid system;

[0086] 3) Use DNase I to digest the remaining plasmid that was not electroporated into exosomes;

[0087] 4) DNaseⅠ was heat-inactivated by treatment at 75°C for 30 min, and exosomes were disrupted by treatment at 95°C for 10 min. The number of copies of the plasmid encapsulated by the exosomes was detected by qPCR (the detection primers were universal primers for the WPRE element carried by the plasmid). The number of DNA copies entering the exosomes was calculated based on the amount of exosomes used to indicate the electroporation efficiency.

[0088] In the experiment, exosomes without plasmid DNA were used as qPCR templates (EXO-1, EXO-2) as negative references, and plasmid DNA of different concentrations was used as qPCR templates for copy number-cycle number calibration curves. Experimental group 1 used plasmids without exosomes and DNaseⅠ digestion group as templates (plasmid-1, plasmid-2); experimental group 2 used exosomes and plasmids mixed without electroporation and used DNaseⅠ digestion as templates (EXO+plasmid-0V-1, EXO+plasmid-0V-2); experimental group 3 used exosomes and plasmids mixed after electroporation and used DNaseⅠ digestion as templates (EXO+plasmid-(-4)-1, EXO+plasmid-(-4)-2).

[0089] Table 1. Experimental group settings of Example 1

[0090]

[0091]

[0092] Table 2. Exosome electroporation efficiency

[0093]

[0094] The results are shown in Table 2 and Figure 1 As shown, compared with the Yin ginseng group and experimental groups 1 and 2, the electroporation method in experimental group 3 can significantly increase the copy number of plasmid DNA entering the exosomes. The DNA entering the exosomes is protected by the exosome phospholipid bilayer membrane and is not degraded by subsequent DNaseⅠ.

[0095] Example 2: Lysosomal inhibitors promote exosome delivery of pDNA expression

[0096] The inventors found in their previous experiments that when the exosomes loaded with plasmid DNA by electroporation as described in Example 1 were added to the HEK293T cell culture system, the reporter gene GFP carried by the plasmid was only slightly expressed in the cells. The inventors speculated that the possible reasons for this phenomenon are: (1) the drug carrier forms endosomes after entering the cell through the endocytic pathway, and the carrier-drug particles encapsulated in the endosomes are degraded by the lysosomes in the cells; (2) the DNA released into the cytoplasm needs to be transferred to the nucleus to complete transcription, and the exogenous DNA loaded by the exosomes alone lacks a mechanism to guide the exogenous DNA into the nucleus of the target cell.

[0097] The inventors proposed the following idea: by adding certain components to the system that promote the entry of exogenous genes into the target cell nucleus and / or inhibit the degradation of exosome-DNA by lysosomes in target cells, the expression of plasmid DNA delivered by exosomes in target cells is promoted. P3000 is an auxiliary component of the currently widely used commercial cationic lipid transfection reagent Lipo3000, whose chemical composition has not been disclosed, and its function is to promote the entry of DNA delivered by cationic lipid transfection cells into the target cell nucleus.

[0098] To verify the above concept, this example added P3000 and lysosomal inhibitor chloroquine to the system of Example 1 to investigate their effects on the efficiency of exosome delivery of pDNA.

[0099] The specific steps are as follows:

[0100] (1) P3000 and pDNA were mixed and incubated at room temperature for 15 min. The dosage of P3000 was 2 μL / g pDNA.

[0101] (2) Co-electroporation of the exosomes and P3000-pDNA mixture, with the ratio of plasmid copy number to exosome vesicle number = 500:1; here, 500 μg of exosomes and 30 μg of plasmid were used;

[0102] (3) The electroporated samples were placed on ice for 30 min and then added to high-glucose DMEM cell culture medium containing 10% fetal bovine serum. 50 μM chloroquine was added and incubated with 1 million HEK293T cells in a 6-well cell culture plate for 48 h. The cells were transferred to the six-well plate 24 h before incubation, with 300,000 cells per well and about 1 million cells after 24 h);

[0103] (4) Use a fluorescence microscope to observe the expression efficiency of the reporter gene GFP in target cells.

[0104] In this experiment, the plasmid DNA plus P3000 group (pD+P3000) was used as negative control 1; the plasmid DNA plus P3000 and chloroquine group (pD+P3000+CQ) was used as negative control 2; the plasmid was mixed with P3000 and then added to the exosomes without electroporation (EXO+(pD+P3000)-0V) was used as experimental group 1; the plasmid was mixed with P3000 and then added to the exosomes without electroporation and incubated Chloroquine was added to the cells (EXO+(pD+P3000)-0V+CQ) as experimental group 2; the plasmid was mixed with P3000 and then exosomes were added and mixed and electroporated (EXO+(pD+P3000)--4V) as experimental group 3; the plasmid was mixed with P3000 and then exosomes were added and mixed and electroporated, and chloroquine was added when incubating the cells (EXO+(pD+P3000)--4V+CQ) as experimental group 4.

[0105] Table 3. Experimental group settings of Example 2

[0106] name Plasmids P3000 Chloroquine (CQ) Electroporation Negative control 1 pD+P3000 √ √ Negative control 2 pD+P3000+CQ √ √ √ Experimental Group 1 EXO+(pD+P3000)-0V √ √ Experimental Group 2 EXO+(pD+P3000)-0V+CQ √ √ √ Experimental Group 3 EXO+(pD+P3000)--4V √ √ √ Experimental Group 4 EXO+(pD+P3000)--4V+CQ √ √ √ √

[0107] The experimental results showed that when P3000 and chloroquine were added to the exosome-plasmid DNA delivery system at the same time, the expression of the reporter gene increased, proving that promoting DNA nuclear localization and lysosomal inhibition can improve the delivery and expression efficiency of pDNA ( Figure 2 ).

[0108] Example 3: Heat shock protein (HSP) promotes exosome delivery of pDNA expression

[0109] The use of P3000 has a low effect on promoting the expression of DNA delivered by exosomes, and its components are unclear. The present invention attempts to use a clear component to replace the effect of P3000, specifically involving the use of a polypeptide containing a nuclear localization sequence and capable of binding to DNA to assist the expression of a plasmid delivered by exosomes in cells. The nuclear localization signal is usually a short amino acid sequence that promotes nuclear entry by interacting with the nuclear pore complex. In the proof-of-concept stage, a heat shock protein (HSP) containing a nuclear localization sequence (NLS) was co-incubated with pDNA, and then the HSP-pDNA complex was electroporated into exosomes and infected cells.

[0110] The specific steps are as follows:

[0111] (1) 5 million HEK293T cells were cultured at 42°C, 5% CO2 for 3 h / 8 h / 16 h respectively;

[0112] (2) Collect the culture supernatant, centrifuge at 1500 r, 4°C for 15 min, and resuspend the HSP protein precipitate in 1 mL of DPBS;

[0113] (3) The HSP protein was repeatedly frozen and thawed three times;

[0114] (4) 1 mL of the obtained HSP protein was mixed with 30 μg of pDNA and incubated at room temperature for 30 min;

[0115] (5) Co-electroporation of the exosomes and HSP-pDNA mixture, with the ratio of plasmid copy number to exosome vesicle number = 500:1;

[0116] (6) After the electroporated samples were placed on ice for 30 min, they were all added to 2 mL of high-glucose DMEM cell culture medium containing 10% fetal bovine serum and incubated with 300,000 HEK293T cells in a 6-well cell culture plate for 48 h;

[0117] (7) Use fluorescence microscopy to detect the expression efficiency of the reporter gene GFP.

[0118] The negative control group was a group without HSP electroporation (EXO+pD--4); experimental group 1 was an exosome non-electroporation group of HSP collected after 16 hours of heat shock culture with HEK293T (EXO+(pD+HSP-16h)-0); experimental group 2 was an exosome electroporation group of HSP collected after 3 hours of heat shock culture with HEK293T (EXO+(pD+HSP-3h)--4); experimental group 3 was an exosome electroporation group of HSP collected after 8 hours of heat shock culture with HEK293T (EXO+(pD+HSP-8h)--4); experimental group 4 was an exosome electroporation group of HSP collected after 16 hours of heat shock culture with HEK293T (EXO+(pD+HSP-16h)--4).

[0119] Table 4. Experimental group settings of Example 3

[0120] name Electroporation Heat shock culture time Negative control EXO+pD--4 √ Experimental Group 1 EXO+(pD+HSP-16h)-0 16 Experimental Group 2 EXO+(pD+HSP-3h)--4 √ 3 Experimental Group 3 EXO+(pD+HSP-8h)--4 √ 8 Experimental Group 4 EXO+(pD+HSP-16h)--4 √ 16

[0121] The results showed that when HSP was not added to the system, the electroporated exosome delivery plasmid was almost not expressed in the target cells. Adding heat shock protein to the electroporated exosome system could achieve the expression of exosome-delivered DNA in some target cells ( Figure 3 ).

[0122] The culture medium containing HSP produced by heat stress of HEK293T was collected by centrifugation and precipitated. After incubation with pDNA, it was co-electroporated with exosomes. The negative control group had no eGFP expression. The non-electroporated group had a small amount of eGFP expression. In experimental groups 2-4, the eGFP expression increased as the heat stress time of HEK293T was prolonged.

[0123] Example 4: NLS / NLS-DBD promotes exosome delivery of pDNA expression

[0124] In order to use polypeptides with defined components to assist the expression of DNA delivered by exosomes, 7 polypeptides containing nuclear localization signal (NLS) and / or DNA binding sequence (DBD) were designed. The polypeptide sequences are shown in Table 5.

[0125] Table 5. Peptide sequences

[0126]

[0127] Note: The underlined sequence in the above NLS-DBD sequence is the DBD sequence.

[0128] In addition to the above 7 polypeptides, other polypeptide sequences can be designed based on other nuclear localization signals and DNA binding domains, and sequences that improve pDNA recognition, localization, and expression can also be connected. Different polypeptides can work together and complement each other.

[0129] The specific steps are as follows:

[0130] 1) Prepare samples according to the ratio of substance concentration (note: not mass ratio), polypeptide (auxiliary polypeptide): pDNA copy number = 100:1, pDNA copy number: exosome particle number = 500:1;

[0131] 2) The peptide and pDNA were mixed and incubated at room temperature for 30 min;

[0132] 3) Co-electroporation of the exosomes and the peptide-pDNA mixture;

[0133] 4) Place the electroporated sample on ice for 30 min, then add it to the cell culture medium and incubate it with 1 million HEK293T cells for 48 h;

[0134] 5) Fluorescence microscopy and flow cytometry were used to detect the expression efficiency of the reporter gene in target cells and the proportion of positive cells.

[0135] The results are as follows Figure 4-6 As shown, the plasmid delivered by exosomes directly electroporated could not be effectively expressed in the target cells, while the plasmid delivered by exosome electroporation after premixing NLS / NLS-DBD and plasmid DNA could be expressed in some target cells.

[0136] Example 5: NLS and KK58 polypeptides promote exosome delivery of pDNA expression

[0137] The present invention detected the use of KK58 polypeptide ((KH)6) in combination with NLS / NLS-DBD. The KK58 polypeptide sequence is: KHKHKHKHKKKHKHKHKHKHKHKHKHKHKHKHKHKHKHKHKHKHKHK HKHKHK KKHKHKHKHKK (SEQ ID NO.17).

[0138] Taking PV-7 NLS as an example, KK58 polypeptide was added to the electroporated exosome-pDNA+NLS sample and fully mixed and incubated for 30 min; KK58 polypeptide: pDNA copy number = 100:1, and then incubated in 1 million HEK293T for 48 h, and the expression of reporter genes in target cells was detected by fluorescence microscopy and flow cytometry.

[0139] The results showed that the combination with KK58 peptide could further promote the efficiency of exosome delivery of pDNA ( Figure 7Example 6: NLS-DBD polypeptide promotes gene delivery efficiency of exosome-PEI hybrid vectors containing lower concentrations of PEI

[0140] In the method reported by Munagala, R et al. in 2021 (doi: 10.1016 / j.canlet.2021.02.011), pDNA was delivered using a complex of exosomes and polyethyleneimine (PEI). The content of PEI used reached 50% of the exosomes. Excessive concentration caused obvious cytotoxicity and the overall delivery efficiency was low.

[0141] The present invention uses NLS-DBD polypeptide, which can significantly improve the efficiency of exosome delivery of pDNA and reduce the amount of PEI, thereby preventing the occurrence of cytotoxicity, and the positive rate of reporter gene expression can reach more than 70%.

[0142] The specific steps are as follows:

[0143] (1) Prepare samples according to the ratio of polypeptide (auxiliary polypeptide): pDNA = 100:1, pDNA: exosomes = 500:1;

[0144] (2) The peptide and pDNA were mixed and incubated at room temperature for 30 min;

[0145] (3) Exosomes were incubated with PEI at room temperature for 5 min. PEI dosage: 10 μg

[0146] (4) co-electroporation of the PEI-exosomes and peptide-pDNA mixture;

[0147] (5) After the electroporated sample was placed on ice for 30 min, 1 / 3 of the sample was added to the cell culture medium and incubated with the cells for 48 h.

[0148] (6) Detect expression efficiency.

[0149] The dosage of PEI used in this electroporation system was 10 μg (the original concentration of the reagent was 1 μg / μL, and 10 μg was added to μL), pDNA was 30 μg, and the copy number ratio of exosomes to pDNA was 1:500 (250 μg of exosomes were used in this experiment). When incubating cells, 1 / 3 of the sample was added with 10 7 In cells, the amount of PEI required for the delivery of equal amounts of plasmids in this system was 1.9% and 0.95% of that in the existing schemes, and the positive rate of reporter gene expression was significantly higher than that in the existing schemes ( Figure 8 , 9 ).

[0150] Example 7: ε-PLL promotes exosome-delivered pDNA expression

[0151] Polylysine (PLL) can be divided into α-PLL and ε-PLL according to the different amino groups that form peptide bonds between monomers. Currently, the use of amino acid polymers such as polylysine or polyhistidine as nucleic acid carriers has been generally unsuccessful. The main problem is that the delivery efficiency of such carriers alone is low and cannot meet various practical application scenarios.

[0152] The present invention uses ε-PLL mixed with exosomes to deliver plasmid DNA, and in particular, 400 μg and 1 mg of ε-PLL are used to replace PEI, respectively.

[0153] The ε-PLL and plasmid were electroporated alone without exosomes (PLL+pD-0V) as the control group. Experimental group 1 was first mixed with ε-PLL and exosomes, and then mixed with plasmid and KK34 polypeptide ((PLL-EXO)+(pD+KK34)-0V); experimental group 2 was first mixed with ε-PLL and exosomes, and then mixed with plasmid and KK34 polypeptide, and then electroporated ((PLL-EXO)+(pD+KK34)--4). The specific steps are as follows:

[0154] 1) Prepare samples according to the molar concentration of peptide:pDNA=100:1, pDNA:exosome=500:1;

[0155] 2) The polypeptide and pDNA were mixed and incubated at room temperature for 30 minutes;

[0156] 3) Exosomes were incubated with ε-PLL at room temperature for 5 min, and the dosage of ε-PLL was 400 μg and 1 mg;

[0157] 4) co-electroporating the ε-PLL-exosomes and the peptide-pDNA mixture;

[0158] 5) Place the electroporated sample on ice for 30 min, then add it to the cell culture medium and incubate with the cells for 48 h;

[0159] 6) Expression efficiency was detected by fluorescence microscopy and flow cytometry.

[0160] Table 6. Experimental group settings of Example 7

[0161]

[0162]

[0163] Figure 10-13The results showed that the pDNA-eGFP expression efficiency of the "-4" electroporation group was better than that of the non-electroporation group and the ε-PLL+plasmid group, and ε-PLL 1mg was better than 400μg. This also proves that the mixed incubation of ε-PLL with exosomes and combined with NLS-DBD peptide can also improve the efficiency of exosome delivery of pDNA, which can be used for large gene drug delivery in vivo.

Claims

1. A method for increasing the expression of exogenous genes in target cells by exosomes, the method comprising any one of the following: 1) The plasmid containing the exogenous gene is mixed with the auxiliary polypeptide and incubated, and then co-electroporated with the exosomes. After electroporation, the KK58 polypeptide is added and co-incubated with the cells. The sequence of the KK58 is shown in SEQ ID NO.17; the ratio of the copy number of the KK58 to the plasmid is 100:1; 2) The plasmid containing the exogenous gene is mixed and incubated with the auxiliary polypeptide, and co-electroporated with the PEI-exosome mixture, and then co-incubated with the cells after electroporation; the PEI-exosome mixture is obtained by co-incubating PEI and exosomes; the dosage ratio of the PEI to the plasmid is 1:3; 3) The plasmid containing the exogenous gene is mixed and incubated with the auxiliary polypeptide, and co-electroporated with the ε-PLL-exosome mixture, and then co-incubated with the cells after electroporation; the ε-PLL-exosome mixture is obtained by co-incubating ε-PLL and exosomes; the usage ratio of the ε-PLL to the plasmid is 4-33.33:1; The sequence of the auxiliary polypeptide is shown in SEQ ID NO.1-6; the auxiliary polypeptide and the plasmid are mixed according to the amount ratio of auxiliary polypeptide: plasmid copy number = 100:1; The ratio of the plasmid copy number to the exosomes was 500:

1.

2. The method of claim 1, wherein the co-electroporation is performed by standing on ice for at least 10 minutes before incubating with the cells.

3. The method of claim 1, wherein the co-electroporation is performed by standing on ice for at least 20 minutes before incubating with the cells.

4. The method of claim 1, wherein the co-electroporation is performed by standing on ice for at least 30 minutes before incubating with the cells.

5. The method of claim 1, wherein the exosomes comprise exosome-like vesicles. The method according to claim 1 , wherein the plasmid is a viral expression vector.

7. The method of claim 6, wherein the viral expression vector is selected from a lentiviral vector, an adenoviral vector, and an adeno-associated viral expression vector.

8. The method of claim 1, wherein the exogenous gene sequence has a length of at least 100 bp.

9. The method of claim 1, wherein the exogenous gene sequence has a length of at least 200 bp.

10. The method according to claim 1, wherein the exogenous gene sequence has a sequence length of at least 300 bp.

11. The method of claim 1, wherein the exogenous gene sequence has a sequence length of at least 400 bp.

12. The method of claim 1, wherein the exogenous gene sequence has a length of at least 500 bp.

13. The method of claim 1, wherein the exogenous gene sequence has a length of at least 600 bp.

14. The method of claim 1, wherein the exogenous gene sequence has a length of at least 700 bp.

15. The method of claim 1, wherein the exogenous gene sequence has a length of at least 800 bp.

16. The method of claim 1, wherein the exogenous gene sequence has a length of at least 900 bp.

17. The method of claim 1, wherein the exogenous gene sequence has a sequence length of at least 1000 bp.

18. The method according to claim 1, wherein the exogenous gene is DNA, RNA or a hybrid thereof.

19. The method of claim 18, wherein the RNA comprises coding RNA, non-coding RNA or artificially synthesized RNA.

20. The method of claim 1, wherein the source of the exosomes comprises biological fluid or biological tissue.

21. The method of claim 20, wherein the biological fluid comprises saliva, urine, synovial fluid, amniotic fluid, or breast milk.

22. The method of claim 1, wherein the exosomes are obtained from the supernatant harvested from culturing adipose-derived mesenchymal stem cells and umbilical cord blood-derived mesenchymal stem cells in a 1:1 ratio.

23. Use of the method according to claim 1 in improving the efficiency of exosomes in delivering exogenous genes to target cells.

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