CN117625691A

CN117625691A - Method for gene delivery based on exosomes and polypeptides containing nuclear localization sequences

Info

Publication number: CN117625691A
Application number: CN202311604073.7A
Authority: CN
Inventors: 张恒; 武佳燕; 吴理达; 顾雨春; 胡兰芳
Original assignee: Chengnuo Regenerative Medical Technology Beijing Co ltd
Current assignee: Chengnuo Regenerative Medical Technology Beijing Co ltd
Priority date: 2023-11-28
Filing date: 2023-11-28
Publication date: 2024-03-01

Abstract

The invention belongs to the field of biological medicine, and in particular relates to a gene delivery method based on exosomes and polypeptides containing nuclear localization sequences. Specifically, the invention provides a method for improving the expression level of an exogenous gene in a target cell through an exosome, which comprises the steps of co-incubating the exosome and the exogenous gene after mixing and co-electrotransformation; the method further comprises mixing and incubating the exogenous gene with a helper polypeptide and/or KK58 prior to electrotransformation, the helper polypeptide comprising a nuclear localization signal, the nuclear localization signal being linked to a DNA binding domain.

Description

Method for gene delivery based on exosomes and polypeptides containing nuclear localization sequences

Technical Field

The invention belongs to the field of biological medicine, and in particular relates to a gene delivery method based on exosomes and polypeptides containing nuclear localization sequences.

Background

Gene therapy refers to editing of a disease-causing defective gene at the DNA level, and has a function of curing a disease by correcting a gene defect or regulating the expression of a gene. How to use the vector to accurately and effectively introduce exogenous genes into target cells is a key technology for successful gene therapy, and in the current clinical experimental research, 80% of the current clinical experimental research adopts virus vectors such as retrovirus, adenovirus, adeno-associated virus and the like, and further comprises non-virus vectors such as receptor, liposome, lipid nanoparticle (Lipid Nanoparticle, LNP) and the like. The vector has high transfection efficiency, but has potential safety hazards such as immunogenicity, toxicity, carcinogenicity, host DNA insertion integration and the like, and can overcome the problems of immunogenicity, safety and the like of the natural intercellular information vector, has stronger targeting property, is easy to pass through a blood brain barrier and has huge application potential in the field of drug carriers.

Exosomes are multivesicular bodies with diameters of 40-100nm, which are formed by invagination of intracellular lysosome particles, and consist of lipid bilayers containing transmembrane proteins, cytoplasmic proteins and various nucleic acids. The exosomes can be secreted by various cells and body fluids in the human body, including endothelial cells, immune cells, platelets, smooth muscle cells, and the like. When secreted into the recipient cell by the host cell, the exosomes can modulate the biological activity of the recipient cell by the proteins, nucleic acids, lipids, etc. that they carry.

Compared with the existing most gene drug carriers, the exosome has the following advantages: (1) The exosome has high biocompatibility and low immunity, is safer to use in vivo, and can be administered for multiple times; (2) The exosomes can be used for transshipping and delivering various types of molecules, including nucleic acids, proteins, small molecules and the like, and have the potential of being developed into gene drugs, small RNA drugs, protein drugs and small molecule drug carriers; (3) The exosomes can pass through blood brain and blood eye barriers, so that the exosomes can be conveniently administered to the central nervous system and eyes through a peripheral blood way; (4) Exosomes have high engineering potential, and specific loading of different drug molecules and specific targeted delivery of different tissues can be achieved through extracellular or intracellular engineering of the exosomes.

Disclosure of Invention

In order to improve the efficiency of exosome delivery of exogenous DNA, the invention provides the following technical scheme:

in a first aspect, the present invention provides a method for increasing the expression level of an exogenous gene in a target cell by an exosome, the method comprising the step of co-incubating the exosome with the cell after co-electrotransformation with the exogenous gene;

the method further comprises any one or more of the following steps:

1) Before electrotransformation, mixing and incubating the exogenous gene with auxiliary polypeptide and/or KK58, wherein the auxiliary polypeptide is formed by connecting a nuclear localization signal and a nuclear localization signal with a DNA binding domain, and the sequence of the KK58 is shown as SEQ ID NO. 17;

2) Adding a lysosomal inhibitor into a cell co-incubation medium;

3) Before electrotransformation, mixing and incubating exogenous genes and HSP proteins;

4) Mixing and incubating exogenous genes and P3000 before electrotransformation;

5) Mixing and incubating exosomes with low concentration of PEI prior to electrotransformation;

6) The exosomes were incubated in mixture with PLL prior to electrotransformation.

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

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

Specifically, the increase in the expression level of an exogenous gene in a target cell by an exosome may also be referred to as an increase in exosome transfer efficiency.

Preferably, the ratio of the copy number of the exogenous gene to the exosome is about 100-2500:1, 250-1000:1, 400-600:1.

Preferably, the exogenous gene copy number to exosome ratio is about 500:1.

Preferably, the mixed incubation in steps 3) -6) is performed at room temperature.

Preferably, said mixed incubation in said steps 3) -6) lasts at least 10 minutes, 20 minutes, 30 minutes or more.

In another aspect, the invention provides a composition comprising any one or more of the following: auxiliary polypeptide, KK58, lysosomal inhibitor, HSP protein, P3000, low concentration PEI, PLL, the sequence of KK58 is shown in SEQ ID NO. 17.

In another aspect, the invention provides the use of any one or more of the following combinations of products to increase the efficiency of exosome delivery of exogenous genes to a target cell: auxiliary polypeptide, KK58, lysosomal inhibitor, HSP protein, P3000, low concentration PEI, PLL, the sequence of KK58 is shown in SEQ ID NO. 17.

Preferably, the lysosomal inhibitor is chloroquine.

Preferably, the exosomes comprise exosome-like vesicles.

Preferably, the exogenous gene is present in a plasmid (vector).

Preferably, the plasmid may be a viral expression vector.

In particular, the viral expression vector includes a lentiviral vector, an adenoviral vector, an adeno-associated viral expression vector, or other types of viral vectors.

In some embodiments, the plasmid may comprise a variety of elements that control expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, the plasmid may also comprise an origin of replication. The vector may also include components that facilitate its entry into the cell, such as viral particles, liposomes, or protein shells, but not just these.

Preferably, the exosomes are co-electrotransferred in admixture with the exogenous gene and then incubated with the cells after being allowed to stand on ice for at least 10 minutes, 20 minutes, 30 minutes or longer.

Preferably, the exogenous gene sequence length is at least a sequence length of 100bp, 200bp, 300bp, 400bp, 500bp, 600bp, 700bp, 800bp, 900bp, 1000bp or more.

In a specific example, the nuclear localization signal may be derived from SV40 large T antigen (SEQ ID NO. 1), nucleoplasmin (SEQ ID NO. 2), nucleoplasmin (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.

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

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

Preferably, the helper polypeptide and the foreign gene are according to the polypeptide: pDNA (exogenous gene) copy number = 10-1000: 1. the mass ratio of the substances of 20-500:1 and 50-200:1 is mixed, and the most preferable ratio is 100:1.

in a specific embodiment, the chloroquine is used at a working concentration of 1-200. Mu.M, 100-150. Mu.M, 40-60. Mu.M.

Preferably, the working concentration of chloroquine is 50 μm.

In a specific embodiment, the HSP proteins are obtained by heat shock culturing cells, followed by centrifugation and re-suspending the pellet with a buffer.

Preferably, the duration of the heat shock incubation is 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.

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), physiological solutions (physiological solutions).

Preferably, the temperature of the heat shock culture is 42 ℃.

Preferably, the heat shock culture is carried out for a culture time of 1 to 20 hours, preferably, 3 to 16 hours at 42 ℃.

Preferably, HSP proteins obtained per 500 ten thousand cells are treated and then dissolved in 1mL of buffer, and incubated with 30. Mu.g of exogenous gene.

Preferably, the amount of P3000 is: 2. Mu.L/g pDNA.

Preferably, the ratio of PEI to foreign gene (pDNA) is 1:3.

Preferably, the PLL is an epsilon PLL.

Preferably, the PLL has the exogenous gene dosage proportion of 1-50:1; preferably 4-33.33:1 (400. Mu.g: 100. Mu.g-1 mg: 30. Mu.g).

Preferably, the ratio of KK58 (KK 58 polypeptide) to the amount of exogenous gene is KK58: exogenous gene copy number = 100:1.

preferably, the exosome concentration in the electrotransport system is 9X 10 ⁹ /mL-5×10 ¹⁰ /mL。

In another aspect, the invention provides the target cells prepared by the method and application thereof in preparing medicines.

The term "Exosomes" as used herein is a vesicle (Extracellular Vesicles, EVs) secreted by cells to the outside of cells, having a bilayer membrane structure and a tea-tray-like morphology, containing abundant contents (including nucleic acids, proteins, lipids, etc.), and involved in the transfer of molecules between cells. Exosomes are widely present in cell culture supernatants and in various body fluids, including blood, lymph, saliva, urine, semen, milk, etc., as well as in tissue samples, such as brain tissue, muscle tissue, adipose tissue, etc.

Preferably, the source of exosomes comprises biological fluid, biological tissue or cell culture fluid.

Preferably, the biological fluid comprises saliva, urine, synovial fluid, amniotic fluid, milk.

Preferably, the biological fluid and the biological tissue comprise any organism, and the exosome can be derived from milk of any animal such as milk.

The source of the exosomes can be human pluripotent stem cells or Mesenchymal Stem Cells (MSC) culture supernatant derived from various tissues, exosome vesicles extracted from milk and milk products as raw materials, exosome vesicles extracted from poultry eggs as raw materials, or a mixture formed by any proportion and combination of the exosome vesicles and the poultry eggs.

Preferably, the cell culture broth is a culture broth (supernatant) of mesenchymal stem cells;

most preferably, the exosomes in embodiments of the invention are obtained from culture of the harvested supernatant in a 1:1 ratio of adipose-derived mesenchymal stem cells to cord blood-derived mesenchymal stem cells.

The target cells of the present invention may be any type of cells, preferably animal cells, more preferably human cells, including in particular germ cells, 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 gland, pineal gland cells; nervous system cells, including glial cells; cells of the respiratory system, including lung cells; cells of the circulatory system, including cardiomyocytes, pericytes; digestive system cells including gastric cells, goblet cells, panda 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 muscle cells; urinary system cells including podocytes, periglomerular cells, mesangial cells/mesangial cells, tubular brush border cells proximal to the kidney, and macular density cells; germ cells including sperm, podocytes, leigh cells, ova; and other cells.

The electric conversion is a mode of exosome exogenous loading. Electrotransformation, also known as cell electroporation (electroporation), is an important method for introducing exogenous macromolecular substances DNA, RNA, siRNA, proteins, etc., as well as some small molecules into the interior of cell membranes. Under the action of instantaneous strong electric field, cell membrane in the solution has certain permeability, and charged exogenous matter enters cell membrane in electrophoresis mode. Because the resistance of the cell membrane phospholipid bilayer is large, the cell bipolar voltage generated by the current field outside the cell is born by the cell membrane, the voltage separated in the cytoplasm is negligible, and almost no current exists in the cytoplasm, so that the cytotoxicity in the electrotransformation process in the normal range is also determined to be small. The specific parameters of the electric rotating can be adjusted according to the exosome state and the culture environment, and the fine adjustment of the electric rotating parameters is within the skill of the person skilled in the art.

Specifically, the exogenous gene of the present invention may have any naturally occurring or artificially constructed polynucleotide sequence. Specifically, the foreign gene may be DNA or RNA, and the DNA may encode any functional gene, for example, a functional gene in a gene therapy product such as ADA (adenosine deaminase), growth factor beta 1 (TGF-. Beta.1), beta-globin gene (beta.A-T87Q-globin gene), ABCD1 gene, etc. 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).

Drawings

FIG. 1 is a graph of the detection result of the electrical transformation efficiency of exosomes, A is a standard curve made by taking a gradient copy number plasmid as a template; b is the statistics of copy number differences between plasma and EXO+plasma-0V compared to the EXO group, C is the Meng Peaks: wpre.

Fig. 2 is a graph of the results of testing the effect of p3000 and chloroquine in promoting expression of exosome-delivered pDNA.

Fig. 3 is a graph of the results of detection of the effect of HSP in promoting exosome delivery of pDNA expression.

FIG. 4 is a graph of the results of testing the effect of PV-7, KK16 polypeptides in promoting exosome delivery of pDNA expression.

FIG. 5 is a graph showing the results of detection of the effect of KG-33, KK-34 polypeptides in promoting expression of exosomes delivering pDNA.

FIG. 6 is a graph showing the results of detection of the effects of PK-35 and PY-54 polypeptides in promoting exosome delivery of pDNA expression.

Fig. 7 is a graph of the results of testing the effect of KK58 polypeptide in promoting exosome delivery of pDNA expression.

FIG. 8 is a graph of the results of a test of the effect of NLS-DBD polypeptides in promoting the delivery of pDNA expression by PEI-EXO complexes at low PEI concentrations.

FIG. 9 is a graph of the results of a test of the effect of NLS-DBD polypeptides in promoting the delivery of pDNA expression by PEI-EXO complexes at lower PEI concentrations.

FIG. 10 is a graph of the results of testing the effect of the epsilon-PLL-EXO complex and NLS-DBD in promoting exosome delivery of pDNA expression, where epsilon-PLL is 400 μg and plasmid is 100 μg.

FIG. 11 is a graph showing the results of detection of the effects of the epsilon-PLL-EXO complex and NLS-DBD in promoting exosome delivery of pDNA expression, wherein epsilon-PLL is 400. Mu.g and plasma id is 30. Mu.g.

FIG. 12 is a graph showing the results of detection of the effects of the epsilon-PLL-EXO complex and NLS-DBD in promoting exosome delivery of pDNA expression, wherein epsilon-PLL is 1mg and plasma is 100. Mu.g.

FIG. 13 is a graph showing the results of detection of the effects of the epsilon-PLL-EXO complex and NLS-DBD in promoting exosome delivery of pDNA expression, wherein epsilon-PLL is 1mg and plasma is 30. Mu.g.

Detailed Description

The present invention will be further described with reference to specific embodiments, but the scope of the present invention is not limited thereto, and any person skilled in the art, who is within the scope of the present invention, should make equivalent substitutions or modifications according to the technical scheme of the present invention and the inventive concept thereof, and should be covered by the scope of the present invention.

Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.

The experimental material information used in the invention is as follows:

experimental materials

Exosomes used in the specific examples of the present invention were derived from adipose-derived mesenchymal stem cells (ADSCs) and cord blood-derived mesenchymal stem cells according to 1:1, purifying and concentrating the obtained supernatant by a hollow fiber column.

In the specific example of the present invention, plasmid pAAV-SFFV-Luciferase-eGFP (6676 bp) was used as the foreign DNA, which will be hereinafter also referred to as plasmid DNA, pDNA, pD.

In the specific embodiment of the invention, HEK293T is taken as a target cell for experiments.

Example one, exosomes encapsulate exogenous DNA by electroporation

The 1mL system electric rotating cup using the cellcyte electric rotating instrument has an electric rotating program of "-4", and the specific electric rotating system is as follows: from electrified transfer liquid or DPBS as electrotransfer buffer, plasmid copy number: exosome vesicle number = 500:1, the exosome concentration in the exosome electrical rotator system used in the present invention is 9×10 ⁹ /mL to 5X 10 ¹⁰ /mL (NTA detection particle count).

The steps of the electric conversion are as follows:

1) The exosomes were incubated with plasmid pAAV-SFFV-Luciferase-eGFP for 15min at room temperature;

2) Electroporation of exosome-plasmid systems;

3) Digesting the remaining plasmids not electroporated into exosomes using DNase I;

4) Heat-inactivating DNase I at 75 ℃ for 30min, crushing exosomes at 95 ℃ for 10 min, qPCR (quantitative polymerase chain reaction) detecting plasmid copy number wrapped by exosomes (detection primers are universal primers of WPRE elements carried by plasmids), and calculating DNA copy number entering exosomes according to the used exosomes to represent electrotransformation efficiency.

In the experiment, exosomes without mixed plasmid DNA are used as qPCR templates (EXO-1, EXO-2) as negative parameters, plasmid DNA with different concentrations is used as a qPCR template to be used as copy number-cycle number standard curve, and DNase I digestion groups are used as templates (plasmid-1, plasmid-2) for the plasmids without exosomes in experiment group 1; exosomes from experimental group 2 were mixed with plasmid without electrotransformation, and digested with DNase i as templates (exo+plasmid-0V-1, exo+plasmid-0V-2); exosomes from experimental group 3 were mixed with plasmid and then electrotransformed, and digested with DNase i as templates (exo+plasma- (-4) -1, exo+plasma- (-4) -2).

Table 1, experimental set-up of example 1

TABLE 2 electric conversion efficiency of exosomes

As shown in table 2 and fig. 1, the electroporation method used in experiment group 3 can significantly increase the copy number of plasmid DNA entering the exosomes, and the DNA entering the exosomes is not degraded by the subsequent DNase i due to the protection of the exosome phospholipid bilayer membrane, compared with the reference group and experiment groups 1 and 2.

Example two lysosomal inhibitors promote exosome delivery of pDNA expression

Previous experiments by the inventors found that the exosomes loaded with plasmid DNA by electrotransformation described in example 1 were added to the HEK293T cell culture system with only a small expression of the reporter GFP in the cells. The inventors speculate that the possible reasons for this phenomenon are: (1) The drug carrier enters cells through endocytic pathway to form endosomes (endosomes), and the carrier-drug particles wrapped by the endosomes are degraded by lysosomes in the cells; (2) DNA released into the cytoplasm needs to be transferred into the nucleus to complete transcription, whereas pure exosome-loaded exogenous DNA lacks a mechanism to direct exogenous DNA into the nucleus of the target cell.

The inventors propose the following assumptions: expression of exosome-delivered plasmid DNA in target cells is promoted by adding to the system certain components that promote the entry of exogenous genes into the target cell nucleus, and/or inhibit the degradation of exosome-DNA by lysosomes in the target cells. P3000 is an adjunct component of the commercial cationic lipid transfection reagent Lipo3000 that is widely used today, whose chemical composition is unpublished, and functions to facilitate the entry of DNA delivered by cationic lipid transfected cells into the target nucleus.

To verify the above concept, this example adds P3000 and lysosomal inhibitor chloroquine on the basis of the system of example 1, and examines its effect on the efficiency of exosome delivery of pDNA.

The specific operation steps are as follows:

(1) P3000 and pDNA are mixed and incubated for 15min at room temperature, and the dosage of P3000 is as follows: 2. Mu.L/g pDNA.

(2) Exosomes were co-electrotransferred with P3000-pDNA mixture, plasmid copy number: exosome vesicle number = 500:1, a step of; here, the exosomes were 500 μg, and the plasmids used were 30 μg;

(3) Standing the electrotransformed sample on ice for 30min, adding the electrotransformed sample into a high-sugar DMEM cell culture medium containing 10% fetal bovine serum, simultaneously adding 50 mu M chloroquine, and transferring the cells into a six-hole plate in a 6-hole cell culture plate for 24h before incubating with 100 ten thousand HEK293T cells for 48h, wherein each hole contains 30 ten thousand cells, and the cells are about 100 ten thousand cells after 24 h;

(4) The expression efficiency of reporter GFP in target cells was observed using a fluorescence microscope.

The experiment uses plasmid DNA alone plus P3000 group (pd+p3000) as negative control 1; plasmid DNA plus P3000 and chloroquine group (pD+P3000+CQ) as negative control 2; mixing the plasmid with P3000, adding exosome, mixing, and performing no electric transformation (EXO+ (pD+P3000) -0V) to obtain experimental group 1; mixing the plasmid with P3000, adding exosome, mixing without electrotransformation, and adding chloroquine (EXO+ (pD+P3000) -0V+CQ) to obtain experimental group 2; mixing the plasmid with P3000, adding exosome, mixing, and converting to experimental group 3 by electrotransformation (EXO+ (pD+P3000) -4V); the plasmid was mixed with P3000, then exosomes were added for mixing and electrotransformation, and chloroquine (EXO+ (pD+P3000) -4V+CQ) was added during cell incubation as experimental group 4.

Table 3, experimental set-up of example 2

	Name of the name	Plasmid(s)	P3000	Chloroquine (CQ)	Electric rotating device
						Negative control 1	pD+P3000	√	√
Negative control 2	pD+P3000+CQ	√	√	√
						Experiment group 1	EXO+(pD+P3000)-0V	√	√
Experiment group 2	EXO+(pD+P3000)-0V+CQ	√	√	√
						Experiment group 3	EXO+(pD+P3000)--4V	√	√	√
Experiment group 4	EXO+(pD+P3000)--4V+CQ	√	√	√	√

The experimental results show that when P3000 and chloroquine are added to the exosome-plasmid DNA delivery system simultaneously, the expression of the reporter gene is increased, which proves that promoting DNA nuclear localization and lysosome inhibition can improve the delivery expression efficiency of pDNA (fig. 2).

Example III Heat Shock Protein (HSP) promotes exosome delivery of pDNA expression

The use of P3000 has little effect on DNA expression to promote exosome delivery and its composition is ambiguous. The present invention contemplates the use of defined components in place of P3000, and in particular relates to the expression of plasmids in cells that utilize polypeptides containing nuclear localization sequences and capable of binding DNA to assist in exosome delivery. The nuclear localization signal is typically a short amino acid sequence that promotes entry into the nucleus by interacting with the nuclear pore complex. The proof of concept phase utilizes Heat Shock Proteins (HSP) containing Nuclear Localization Sequences (NLS) to co-incubate with pDNA, and then the HSP-pDNA complex is electrotransformed into exosomes to infect cells.

The method comprises the following specific steps:

(1) 500 ten thousand HEK293T cells were incubated at 42℃with 5% C0 ₂ Culturing for 3h/8h/16h respectively;

(2) Collecting the culture supernatant, centrifuging at 1500r and 4 ℃ for 15min, and re-suspending the HSP protein precipitate by 1mL of DPBS;

(3) Repeatedly freezing and thawing HSP protein for 3 times;

(4) 1mL of the obtained HSP protein was mixed with 30. Mu.g of pDNA at room temperature and incubated for 30min;

(5) Exosomes were co-electrotransferred with HSP-pDNA mixtures, plasmid copy number: exosome vesicle number = 500:1, a step of;

(6) Standing the electrotransformed sample on ice for 30min, adding the sample to 2mL of high-sugar DMEM cell culture medium containing 10% fetal bovine serum, and incubating the sample with 30 ten thousand HEK293T cells in a 6-hole cell culture plate for 48h;

(7) The expression efficiency of the reporter gene GFP was examined using a fluorescence microscope.

The negative control group is an HSP-free electrotransformation group (EXO+pD-4); experiment group 1 is exosome non-electrotransformation group (EXO+ (pD+HSP-16 h) -0) of HSP recovered after HEK293T heat shock culture for 16h; experiment group 2 is exosome electrotransformation group (EXO+ (pD+HSP-3 h) - -4) of HSP collected after HEK293T is added for heat shock culture for 3 h; experiment group 3 is exosome electrotransformation group (EXO+ (pD+HSP-8 h) - -4) of HSP collected after HEK293T is added for heat shock culture for 8h; experimental group 4 is exosome electrotransformation group (EXO+ (pD+HSP-16 h) - -4) of HSP harvested after 16h of heat shock culture with HEK 293T.

Table 4, experimental set-up of example 3

	Name of the name	Electric rotating device	Heat shock incubation time
				Negative control	EXO+pD--4	√
Experiment group 1	EXO+(pD+HSP-16h)-0		16
				Experiment group 2	EXO+(pD+HSP-3h)--4	√	3
Experiment group 3	EXO+(pD+HSP-8h)--4	√	8
				Experiment group 4	EXO+(pD+HSP-16h)--4	√	16

The results show that in the absence of HSP added to the system, the electroporation exosome delivery plasmid had little expression in the target cells, and in the electroporation exosome system, the addition of heat shock proteins allowed expression of exosome delivered DNA in part of the target cells (fig. 3).

Collecting culture medium centrifugal precipitation containing HSP generated by HEK293T heat stress, incubating with pDNA, co-electrotransferring negative control with exosomes, and performing no eGFP expression on the experimental group 1 without electrotransferring, wherein a small amount of eGFP expression is generated, and the eGFP expression is increased along with the extension of HEK293T heat stress time of the experimental groups 2-4.

Example IV, NLS/NLS-DBD promotes exosome delivery of pDNA expression

In order to assist in the expression of exosome-delivered DNA using the defined polypeptides, 7 polypeptides were designed containing a nuclear localization sequence (Nuclear localization signal, nuclear localization sequence, NLS) and/or a DNA Binding Domain (DBD) with the polypeptide sequences shown in table 5.

TABLE 5 polypeptide sequences

Note that: the underlined sequence in the NLS-DBD sequence is the DBD sequence.

In addition to the 7 polypeptides above, other polypeptide sequences can be designed based on other nuclear localization signals and DNA binding domains, and sequences that enhance pDNA recognition, localization, expression can also be linked. Different polypeptides can act in combination and complement each other.

The method comprises the following specific steps:

1) According to the mass concentration ratio of the substances (note: not mass ratio), polypeptide (accessory polypeptide): pDNA copy number = 100:1. pDNA copy number: exosome number = 500:1, preparing a sample according to the proportion;

2) Mixing and incubating the polypeptide and pDNA for 30min at room temperature;

3) Co-electrotransport of exosomes with the polypeptide-pDNA mixture;

4) Standing the electrotransformed sample on ice for 30min, adding the sample into a cell culture medium, and incubating the sample with 100 ten thousand HEK293T cells for 48h;

5) The expression efficiency of the reporter gene in the target cells and the proportion of positive cells were detected using a fluorescence microscope and a flow cytometer, respectively.

As a result, as shown in FIGS. 4 to 6, the plasmid delivered by directly electroporated exosomes was not efficiently expressed in the target cells, whereas the plasmid delivered by exosome electroporation after the pre-mixing of NLS/NLS-DBD and plasmid DNA was expressed in part of the target cells.

Example five, NLS and KK58 polypeptides promote exosome delivery of pDNA expression

The present invention detects KK58 polypeptide ((KH) ₆ ) For use in combination with NLS/NLS-DBD, the KK58 polypeptide sequence is: KHKHKHKHKKKHKHKHKHKKHKHKHKHKKKHKHKHKHKKHK HKHKHK KKHKHKHKHKK (SEQ ID NO. 17).

Taking PV-7 NLS as an example, adding KK58 polypeptide into an exosome-pDNA+NLS sample after electrotransformation, mixing thoroughly, and incubating for 30min; KK58 polypeptide: pDNA copy number = 100:1 followed by incubation of 48H in 100 ten thousand H EK293T, fluorescence microscopy and flow detection of reporter gene expression in target cells.

The results indicate that the combination KK58 polypeptide can further promote the efficiency of exosome delivery of pDNA (fig. 7). Example six NLS-DBD polypeptide promotes Gene delivery efficiency of exosome-PEI Mixed vector with lower concentration of PEI

In the method reported by Munagala, R et al, 2021, (doi: 10.1016/j. Canlet. 2021.02.011), pDNA was delivered using an exosome and Polyethylenimine (PEI) complex, with PEI levels up to 50% of the exosome, with excessive concentrations causing significant cytotoxicity and overall delivery efficiency.

The invention uses NLS-DBD polypeptide, can obviously improve the efficiency of exosome delivery of pDNA and reduce the dosage of PEI, prevents cytotoxicity, and has a positive rate of reporter gene expression of more than 70%.

The method comprises the following specific steps:

(1) According to the polypeptide (helper polypeptide): pdna=100: 1, pDNA: exosome = 500:1, preparing a sample according to the proportion;

(2) Mixing and incubating the polypeptide and pDNA for 30min at room temperature;

(3) Exosomes were incubated with PEI for 5min at room temperature, PEI dose: 10 mug

(4) Co-electrotransport of PEI-exosomes with the polypeptide-pDNA mixture;

(5) Standing the sample subjected to electric transformation on ice for 30min, adding 1/3 of the sample into a cell culture medium, and incubating 48 and h with cells;

(6) The expression efficiency was examined.

PEI was used in the electrotransport system at a dose of 10. Mu.g (original concentration of reagent: 1. Mu.g/. Mu.L, added 10. Mu.g/. Mu.L), pDNA at 30. Mu.g, and the ratio of exosome to pDNA copy number was 1:500 (250. Mu.g of exosomes used in this experiment), 1/3 of the sample was taken and 10 was added during cell incubation ⁷ In the cells, the amount of PEI required for the systemic delivery of equivalent plasmids was 1.9% and 0.95% of the prior art, and the positive rate of reporter gene expression was significantly higher than that of the prior art (FIGS. 8 and 9).

Example seven, epsilon-PLL promoted exosome delivery of pDNA expression

Polylysine (PLL) can be classified into a-PLL and epsilon-PLL according to the amino groups forming peptide bonds between monomers. At present, amino acid polymers such as polylysine or polyhistidine are taken as nucleic acid vectors, which are generally unsuccessful, and the main problem is that the delivery efficiency of the vectors used independently is low, and various practical application scenes cannot be met.

The present invention uses epsilon-PLL to deliver plasmid DNA in a mixed fashion with exosomes, in particular 400 μg and 1mg epsilon-PLL, respectively, is used instead of PEI.

epsilon-PLL and plasmid were shocked alone without exosomes (PLL+pD-0V) as controls, and experiment 1 was first incubated with epsilon-PLL and exosomes in mixture, plasmid with KK34 polypeptide in mixture ((PLL-EXO) + (pD+KK34) -0V); experiment group 2 was first incubated with epsilon-PLL and exosomes, plasmid and KK34 polypeptides, followed by electroporation ((PLL-EXO) + (pD+KK34) - -4). The method comprises the following specific steps:

1) According to the mass concentration of the substance, the polypeptide: pdna=100: 1. pDNA: exosome = 500:1, preparing a sample according to the proportion;

3) Exosomes were incubated with epsilon-PLL for 5min at room temperature, epsilon-PLL amounts: 400 μg and 1mg;

4) Co-electrotransport of epsilon-PLL-exosomes with the polypeptide-pDNA mixture;

5) Standing the electrotransformed sample on ice for 30min, adding the sample into a cell culture medium, and incubating the sample and cells for 48h;

6) Fluorescence microscopy and flow detection of expression efficiency.

Table 6, experimental set-up of example 7

The results in FIGS. 10-13 show that both the different doses of epsilon-PLL and plasmid experimental groups showed that the "-4" electrotransformed group, pDNA-eGFP, had better expression efficiency than the non-electrotransformed group and epsilon-PLL+plasma group, and that epsilon-PLL 1mg was better than 400. Mu.g. It has also been demonstrated that the mixed incubation of epsilon-PLL with exosomes and the combination of NLS-DBD polypeptides also increases the efficiency of exosome delivery of pDNA, and can be used for in vivo large gene drug delivery.

Claims

1. A method for increasing the expression level of an exogenous gene in a target cell by an exosome, the method comprising the step of co-incubating the exosome with the cell after co-electrotransformation with the exogenous gene;

the method further comprises any one or more of the following steps:

1) Before electrotransformation, mixing and incubating the exogenous gene with auxiliary polypeptide and/or KK58, wherein the sequence of KK58 is shown as SEQ ID NO.17, and the auxiliary polypeptide is a nuclear localization signal or is formed by connecting the nuclear localization signal with a DNA binding domain;

2) Adding a lysosomal inhibitor into a cell co-incubation medium;

5) Mixing and incubating exosomes with PEI before electrotransformation;

6) Mixing and incubating exosomes with PLL prior to electrotransformation;

preferably, the method comprises steps 1) and 5) above;

preferably, the ratio of the copy number of the exogenous gene to the exosome is 100-2500:1, 250-1000:1, 400-600:1;

preferably, the ratio of the copy number of the exogenous gene to the exosome is 500:1;

preferably, said mixed incubation in steps 3) -6) is performed at room temperature;

preferably, said mixed incubation in said steps 3) -6) lasts at least 10 minutes, 20 minutes, 30 minutes or more;

preferably, the exosomes comprise exosome-like vesicles;

preferably, the exosomes are co-electrotransferred in admixture with the exogenous gene and allowed to stand on ice for at least 10 minutes, 20 minutes, 30 minutes or longer before being co-incubated with the cells;

preferably, the lysosomal inhibitor is chloroquine;

preferably, the working concentration of the chloroquine is 1-200 mu M, 100-150 mu M and 40-60 mu M;

preferably, the working concentration of chloroquine is 50 μm;

preferably, the amount of the P3000 is 2. Mu.L/g exogenous gene;

preferably, the dosage ratio of PEI to exogenous gene is 1:3;

preferably, the ratio of KK58 to the copy number of the foreign gene is 100:1, a step of;

preferably, the PLL has the exogenous gene dosage proportion of 1-50:1; preferably, 4-33.33:1;

2. The method of claim 1, wherein the sequence of the nuclear localization signal is set forth in SEQ ID No.1-2 or 13-16;

preferably, the sequence of the DNA binding domain is shown in SEQ ID NO. 8-12;

preferably, the sequence of the auxiliary polypeptide is shown in any one of SEQ ID NO. 1-7;

preferably, the helper polypeptide and the foreign gene are according to the polypeptide: exogenous gene copy number = 10-1000: 1. the mass ratio of the substances of 20-500:1 and 50-200:1 is mixed, and the most preferable ratio is 100:1.

3. the method of claim 1, wherein the HSP protein is obtained by subjecting cells to heat shock culture, centrifuging and re-suspending the pellet with a buffer;

preferably, the duration of the heat shock incubation is 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;

preferably, the temperature of the heat shock culture is 42 ℃;

preferably, the buffer is DPBS.

4. The method of claim 1, wherein the exogenous gene is present in a plasmid;

preferably, the plasmid is a viral expression vector;

preferably, the viral expression vector comprises a lentiviral vector, an adenoviral vector, an adeno-associated viral expression vector;

preferably, the exogenous gene sequence length is at least 100bp, 200bp, 300bp, 400bp, 500bp, 600bp, 700bp, 800bp, 900bp, 1000bp or longer in sequence length;

preferably, the exogenous gene is DNA, RNA, or a hybrid thereof;

preferably, the RNA comprises coding RNA such as mRNA, non-coding RNA or synthetic RNA.

5. The method of claim 1, wherein the source of exosomes comprises biological fluid, biological tissue or cell culture fluid;

preferably, the biological fluid comprises fluid, saliva, urine, synovial fluid, amniotic fluid, or milk;

preferably, the cell culture fluid is a culture fluid of mesenchymal stem cells;

most preferably, the exosomes are obtained from the harvested supernatant by culturing the adipose-derived mesenchymal stem cells in a 1:1 ratio to cord blood-derived mesenchymal stem cells.

6. The target cell prepared by the method of claim 1.

7. Use of the target cell of claim 6 in the preparation of a gene therapy drug.

8. Use of an agent for increasing the efficiency of exosome delivery of a foreign gene to a target cell, said agent being any one or more of the following combinations: accessory polypeptides, KK58, lysosomal inhibitors, HSP proteins, P3000, PEI, PLL;

preferably, the agent is a helper polypeptide, the helper polypeptide being a nuclear localization signal, or the helper polypeptide being a combination of a nuclear localization signal and a DNA binding domain;

preferably, the sequence of the nuclear localization signal is shown as SEQ ID NO.1-2 or 13-16;

preferably, the sequence of the DNA binding domain is shown in SEQ ID NO. 8-12;

preferably, the agent is a combination of a helper polypeptide and KK58, the KK58 having the sequence shown in SEQ ID NO. 17;

preferably, the agent is a combination of accessory polypeptide and PEI.

9. A composition comprising any of a plurality of: auxiliary polypeptide, KK58, lysosomal inhibitor, HSP protein, P3000, PEI, PLL, wherein the sequence of KK58 is shown in SEQ ID NO. 13;

preferably, the composition is a combination of a helper polypeptide and any one or more of the following: KK58, lysosomal inhibitors, HSP proteins, P3000, PEI, PLL;

preferably, the composition is a combination of a helper polypeptide and KK 58;

preferably, the composition is a combination of a helper polypeptide and PEI.

10. The composition of claim 9, wherein the auxiliary polypeptide is a nuclear localization signal or wherein the auxiliary polypeptide is formed by linking a nuclear localization signal to a DNA binding domain;

preferably, the sequence of the DNA binding domain is shown in SEQ ID NO. 8-12;