CN111500634A

CN111500634A - Exosome-encapsulated AAV vector, AAV-target gene vector, and preparation method and application thereof

Info

Publication number: CN111500634A
Application number: CN202010395265.1A
Authority: CN
Inventors: 雷博; 王卫萍; 刘婧阳; 郝冰涛
Original assignee: HENAN INSTITUTE OF OPHTHALMOLOGY
Current assignee: HENAN INSTITUTE OF OPHTHALMOLOGY
Priority date: 2020-05-12
Filing date: 2020-05-12
Publication date: 2020-08-07
Anticipated expiration: 2040-05-12
Also published as: CN111500634B

Abstract

The invention belongs to the technical field of gene drug vectors, and discloses an exosome-encapsulated AAV vector, an AAV-target gene vector, a preparation method and application thereof. Culturing HEK-293T cells with a DMEM complete culture medium, carrying out transfection when the cells grow until the fusion degree reaches 60% -80%, and replacing the old culture medium with a DMEM basic culture medium 1-4 h before transfection; co-transfecting pAAV-ZsGreen plasmid or pAAV-target gene-ZsGreen plasmid, pRC2-miR342 plasmid and pHelper plasmid into HEK-293T cell culture solution, adding the fetal calf serum without exosomes after the co-transfection for 12-16 h at 37 ℃, continuously culturing for 48-72 h, collecting supernatant, and performing centrifugal separation to obtain the exosome-encapsulated AAV or AAV-target gene vector. The exosome encapsulates the application of the AAV-target gene vector in the preparation of the drug for treating the diseases corresponding to the target genes. Compared with AAV-target gene vector, the natural membrane structure of the exosome prepared by the invention can effectively enhance the transportation of AAV in retinal tissue and increase the transfection efficiency of the target gene.

Description

Exosome-encapsulated AAV vector, AAV-target gene vector, and preparation method and application thereof

Technical Field

The invention belongs to the technical field of gene drug vectors, and particularly relates to an exosome-encapsulated AAV vector, an AAV-target gene vector, a preparation method and application thereof.

Background

Adeno-associated virus (AAV) is a non-enveloped virus belonging to the family of the parvoviridae. AAV is not pathogenic to humans and can only replicate in the presence of helper viruses such as adenovirus or herpes virus. The AAV genome is a single-stranded linear DNA molecule of approximately 4.7 kb with Inverted Terminal Repeats (ITRs) of hairpin structure at each end, which function as the genomic replication initiation site and as packaging for virions. DNA fragments smaller than 2.5 kb in size can be inserted into AAV viruses. AAV vector has high gene transfection efficiency in vivo and good overall safety, and has become the first choice gene drug vector. Although AAV is the first choice drug carrier, clinical trial treatment effect is far inferior to animal experiment effect due to the existence of neutralizing antibody, blood brain barrier, blood eye barrier and other reasons in the organism itself. Thus, differences were made in consideration of the poor transfection efficiency of AAV.

Exosomes (exosomes) consist of lipid bilayers containing transmembrane, cytoplasmic proteins and various nucleic acids. The particle size of the composite is 40-200 nm, and the composite is mainly formed by multi-vesicular bodies (MVBs) which are formed by invagination of cell membranes and contain lysosome particles, and is released into extracellular matrix after the multi-vesicular body outer membranes are fused with cell membranes. Is a promising novel drug and gene delivery vector. Compared with the traditional AAV vector, the exosome as the gene drug vector has the following advantages:

1. the exosome has the advantages of small particle size, high biocompatibility, low immunity and the like;

2. exosomes can deliver gene drugs, proteins, small molecule drugs, and the like;

3. exosomes can cross blood-brain barrier and blood-eye barriers, and exhibit significant resistance to neutralizing antibodies in vivo;

4. an exosome-encapsulating AAV vector (exo-AAV) can bypass the process that requires AAV capsids to bind to cellular receptors into cells to improve transfection efficiency;

5. exosome vectors not only deliver gene drugs more efficiently to the target tissue, but also protect the contents from degradation.

Disclosure of Invention

In order to overcome the defects in the prior art, the invention aims to provide an exosome-encapsulated AAV vector, an AAV-target gene vector, a preparation method and an application thereof.

In order to achieve the purpose, the technical scheme adopted by the invention is as follows:

a preparation method of an exosome-encapsulated AAV vector comprises the following steps:

(1) culturing HEK-293T cells by using a DMEM complete culture medium, carrying out transfection when the cells grow until the fusion degree reaches 60% -80%, and replacing the old culture medium by using a DMEM basic culture medium 1-4 h before transfection;

(2) co-transfecting the pAAV-ZsGreen plasmid, the pRC2-miR342 plasmid and the pHelper plasmid into the HEK-293T cell culture solution obtained in the step (1), adding the exosome-free fetal calf serum after the co-transfection is carried out for 12-16 h at 37 ℃, continuously culturing for 48-72 h, collecting the supernatant, and carrying out centrifugal separation to obtain an exosome-encapsulated AAV vector.

Specifically, the process of step (2) is that 10-15 μ g of pAAV-ZsGreen plasmid, 10-15 μ g of pRC2-miR342 plasmid and 20-25 μ g of pHelper plasmid are added into 50-100 μ L CaCl₂The three plasmids were added in solution form in both solution and water at a concentration of 1-2. mu.g/. mu. L₂The concentration of the solution is 2-3M, the amount of water is 10 times of the total volume of the three-plasmid solution, the three-plasmid solution is evenly blown and evenly mixed, the evenly mixed three-plasmid solution is dripped into 500-1000 mu L2 × HeBS solution in a vortex state, after standing for 20-30 min at room temperature, the three-plasmid solution is dripped into HEK-293T cell culture solution obtained in the step (1) of 8-10M L, after cotransfection is carried out for 12-16 h at 37 ℃, 160-200 mu L exosome-free fetal bovine serum is added for continuous culture for 48-72 h, cell culture supernatant is collected, and the exosome-encapsulated AAV vector is obtained by centrifugal separation.

Specifically, in the step (2), the centrifugal separation process is as follows: firstly, centrifuging at 300-600 g and 600-5 ℃ for 10-15min, collecting the supernatant after the centrifugation is finished, then centrifuging the collected supernatant at 2000-2500 g and 3-5 ℃ for 10-15min, and collecting the supernatant after the centrifugation is finished; further centrifuging the collected supernatant at 3-5 ℃ for 1-1.5 h at 100000 g, removing the supernatant after centrifugation is finished, collecting bottom sediment, adding PBS for heavy suspension sediment, centrifuging at 3-5 ℃ for 1-1.5 h at 100000 g again, removing the supernatant after centrifugation is finished, collecting the bottom sediment, and obtaining the sediment, namely the exosome-encapsulated AAV vector.

An exosome-encapsulating AAV vector prepared by the preparation method.

A preparation method of an exosome-encapsulated AAV-target gene vector comprises the following steps:

(1) firstly carrying out EcoRI enzyme digestion on the pAAV-ZsGreen plasmid to obtain a linear pAAV-ZsGreen plasmid; then constructing the target gene into a linear pAAV-ZsGreen plasmid by a homologous recombination method, and extracting the pAAV-target gene-ZsGreen plasmid by a large endotoxin-free plasmid extraction kit;

(2) culturing HEK-293T cells by using a DMEM complete culture medium, carrying out transfection when the cells grow until the fusion degree reaches 60% -80%, and replacing the old culture medium by using a DMEM basic culture medium 1-4 h before transfection;

(3) co-transfecting the pAAV-target gene-ZsGreen plasmid, the pRC2-miR342 plasmid and the pHelper plasmid into the HEK-293T cell culture solution obtained in the step (2), adding the exosome-free fetal calf serum after the co-transfection for 12-16 h at 37 ℃, continuously culturing for 48-72 h, collecting the supernatant, and performing centrifugal separation to obtain an exosome-encapsulated AAV-target gene vector.

Preferably, in the step (1), the EcoRI enzyme digestion system is pAAV-ZsGreen plasmid solution with the concentration of 150-.

Specifically, the process of step (3) is that 10-15. mu.g of pAAV-target gene-ZsGreen plasmid, 10-15. mu.g of pRC2-miR342 plasmid and 20-25. mu.g of pHelper plasmid are added into 50-100. mu. L CaCl₂The three plasmids are added in the form of solution and the concentration of the solution is 1-2 mug /μL，CaCl₂The concentration of the solution is 2-3M, the amount of water is 10 times of the total volume of the three-plasmid solution, the three-plasmid solution is evenly blown and evenly mixed, the evenly mixed three-plasmid solution is dripped into 500-1000 mu L2 × HeBS solution in a vortex state, after standing for 20-30 min at room temperature, the three-plasmid solution is dripped into HEK-293T cell culture solution obtained in the step (2) of 8-10M L, after cotransfection is carried out for 12-16 h at 37 ℃, 160-200 mu L exosome-free fetal bovine serum is added for continuous culture for 48-72 h, cell culture supernatant is collected, and centrifugal separation is carried out to obtain the exosome-encapsulated AAV-target gene vector.

Specifically, in the step (3), the centrifugal separation process is as follows: firstly, centrifuging at 300-600 g and 600-5 ℃ for 10-15min, collecting the supernatant after the centrifugation is finished, then centrifuging the collected supernatant at 2000-2500 g and 3-5 ℃ for 10-15min, and collecting the supernatant after the centrifugation is finished; further centrifuging the collected supernatant for 1-1.5 h at 3-5 ℃ at 100000 g, removing the supernatant after centrifugation is finished, collecting bottom sediment, adding PBS for heavy suspension sediment, centrifuging for 1-1.5 h at 3-5 ℃ at 100000 g again, removing the supernatant after centrifugation is finished, collecting the bottom sediment, and obtaining the sediment, namely the exosome-encapsulated AAV-target gene vector.

An exosome-encapsulating AAV-target gene vector prepared by the preparation method.

An application of the AAV-target gene vector encapsulated by the exosome in preparing the medicine for treating the diseases corresponding to the target gene.

In the present invention, the target gene includes, but is not limited to, the following genes: human Retinoschisin 1, CHM, BEST1, RPE65, ND 1-6, SOD2 and HIF-1. Correspondingly, the diseases include, but are not limited to, the following diseases: congenital retinoschisis, retinitis pigmentosa, optic neuropathy, pulselessness, Best vitelliform macular dystrophy, congenital amaurosis.

Compared with the prior art, the invention has the following advantages and effects:

1. according to the invention, an exosome is selected to construct and encapsulate an AAV (adeno-associated virus) delivery vector, and by utilizing the natural property of the exosome, compared with the AAV vector, the exosome has the advantages of small particle size, high biocompatibility, low immunity and the like, can pass through barriers such as blood brain barriers and blood eyes, and can show obvious resistance to neutralizing antibodies in vivo; the AAV-target gene vector encapsulated by the exosome can bypass the process of needing AAV capsid to combine with cell receptor to enter the cell, thereby improving AAV transfection efficiency;

2. the invention realizes the loading of the therapeutic gene by using the mode that the exosome wraps the AAV-target gene vector, has simple and efficient operation, and compared with the AAV-target gene vector, the natural membrane structure of the exosome can effectively enhance the transportation of the AAV in the retinal tissue and increase the transfection efficiency of the target gene.

Drawings

FIG. 1: synthesis scheme (a) of pAAV-RS1-ZsGreen plasmid, horizontal gel electrophoresis (B) and partial base sequence test (C), where M: marker, lanes 1-6: pAAV-RS1-ZsGreen colony.

FIG. 2: an electron transmission microscope image (A) of exo-AAV2-RS1-ZsGreen vector, a nano-tracker analysis particle size distribution image (B) and a western blotting image (C).

FIG. 3: AAV2-RS1-ZsGreen vector and exo-AAV2-RS1-ZsGreen vector transfected HEK-293T cell fluorescence microscopy (A), RT-PCR (B, C) and western blotting (D, E).

FIG. 4: AAV2-RS1-ZsGreen vector and exo-AAV2-RS1-ZsGreen vector infected mouse retina frozen section immunofluorescence image.

FIG. 5: AAV2-RS1-ZsGreen vector and exo-AAV2-RS1-ZsGreen vector infected mouse retina RT-PCR map (A, B) and western blotting map (C, D).

Detailed Description

The present invention will be further described with reference to the following specific examples. It should be understood that the following examples are illustrative only and are not intended to limit the scope of the present invention.

Example 1

A preparation method of an exosome encapsulating AAV vector, namely exo-AAV2-ZsGreen vector, comprises the following steps:

(1) 5 × 10⁶The HEK-293T cells were plated on 10 cm dishes, cultured in 10 m L DMEM complete medium, the medium was changed when the cells grew to 80% confluency, and 10 m L DMEM high-sugar basic medium (Hy) without serum and antibody was usedClone, SH 30022.01) for 2 h; wherein, the preparation of DMEM complete medium: DMEM high-glucose basal medium (HyClone, SH 30022.01) was supplemented with 10% (v/v) fetal bovine serum (FBS; Corning, 35-081-CV) and 1% (v/v) antibody (Solarbio, P1400) to obtain DMEM complete medium, and stored at 4 ℃;

(2) co-transfection, 12. mu.g of pAAV-ZsGreen plasmid (Takara, 6231), 12. mu.g of pRC2-miR342 plasmid (Takara, 6234) and 25. mu.g of pHelper plasmid (Takara, 6234) were added to 59. mu. L CaCl₂In the solution and the sterile aqueous solution, the three plasmids are added in the form of solution, the concentration of the solution is 1 mu g/mu L-ZsGreen plasmid solution, the addition amount of the solution is 12 mu L2-miR 342 plasmid solution is 12 mu L, and the addition amount of the pHelper plasmid solution is 25 mu L₂The concentration of the solution is 2M, the using amount of sterile water is 490 mu L, the mixture is blown up gently and mixed evenly, the mixed three plasmid solution is dripped into 590 mu L × HeBS solution under the vortex state, the mixture is stood for 20 min at room temperature, the three plasmid solution after being stood at room temperature is dripped into 10M L HEK-293T cell culture solution obtained in the step (1), the cotransfection is carried out for 16 h at 37 ℃, then 200 mu L non-exosome fetal bovine serum (Absin, abs 993) is supplemented for further culture for 72 h, the supernatant is collected, the supernatant is centrifuged for 10 min at 300 g at 4 ℃, the supernatant is collected after the centrifugation is finished, the collected supernatant is centrifuged for 10 min at 4 ℃, the supernatant is collected after the centrifugation is discarded, the collected supernatant is further centrifuged for 1h at 100000 g by a super-speed centrifuge (CP 100, Japan) for 1h, the supernatant is removed after the centrifugation is collected, the bottom sediment is added into 1M sterile PBS, the test tube is resuspended in 100000 h at 4 ℃, the test tube is the test tube, the test tube is used for resuspension, the white PBS is the test tube, the AAV supernatant is added, the test tube is resuspended in the test tube, the.

Example 2

The X-related retinoschisis (X L RS) is caused by the mutation of a Retinoschisin 1(RS1) gene, RS1 protein is mainly synthesized by bipolar cells and photoreceptor cells and secreted to each cell layer of retina, RS1 gene mutation causes the deletion of RS1 protein, further causes retinal cavity, synaptic dysfunction and reduced visual sensitivity, and the RS1 gene is taken as a target gene for example, which indicates that an exosome-encapsulated AAV-target gene vector, namely exo-AAV2-RS1-ZsGreen vector, is prepared.

An exo-AAV2-RS1-ZsGreen vector preparation method comprises the following steps:

(1) the preparation method of the pAAV-RS1-ZsGreen plasmid comprises the following steps: firstly carrying out EcoRI enzyme digestion on the pAAV-ZsGreen plasmid to obtain a linear pAAV-ZsGreen plasmid; then constructing a target gene Retinoschisin 1 into a linear pAAV-ZsGreen plasmid by a homologous recombination method, and extracting the pAAV-RS1-ZsGreen plasmid by an endotoxin-free plasmid large-scale extraction kit (TIANGEN, DP 117);

the EcoRI enzyme digestion system is pAAV-ZsGreen plasmid solution with the concentration of 150 ng/mu L of 5 mu L, EcoRI enzyme 1 mu L1, 10L 0Buffer solution of 2 mu L2 and water is supplemented to the total system of 20 mu L, the enzyme digestion conditions are 16 h at 37 ℃ and 5min at 80 ℃, the construction system is linear pAAV-ZsGreen plasmid solution with the concentration of 150 ng/mu L of 1 mu L, RS1 cDNA solution with the concentration of 30 ng/mu L of 2 mu L, 2 × SoSoo solution (Qingke, TSV-S1) of 5 mu L and water is supplemented to the total system of 10 mu L, and the construction conditions are 15min at 50 ℃ and infinity at 4 ℃;

(2) preparation of AAV2-RS1-ZsGreen vector and exo-AAV2-RS1-ZsGreen vector:

(2a) 5 × 10⁶Inoculating HEK-293T cells into a 10 cm culture dish, culturing in 10 m L DMEM complete medium, changing the medium when the cells grow to reach 80% confluency, and continuously culturing for 2 h by using 10 m L DMEM high-glucose basal medium (HyClone, SH 30022.01) without serum and antibody;

(2b) and co-transfection, namely adding 12 mu g of pAAV-RS1-ZsGreen plasmid, 12 mu g of pRC2-miR342 plasmid and 25 mu g of pHelper plasmid into 59 mu L CaCl₂In the solution and the sterile aqueous solution, the three plasmids are added in the form of solution, the concentrations of the solutions are 1 mu g/mu L-RS 1-ZsGreen plasmid solution, the addition amount of the plasmid solution is 12 mu L2-miR 342, the addition amount of the plasmid solution is 12 mu L, and the addition amount of the pHelper plasmid solution is 25 mu L₂The concentration of the solution is 2M, the using amount of sterile water is 490 mu L, the mixture is lightly blown and evenly mixed, the evenly mixed triplasmid solution is dripped into 590 mu L2 × HeBS solution under the vortex state, and the solution is placed in a roomStanding at room temperature for 20 min, dripping the three-plasmid solution standing at room temperature into the 10 m L HEK-293T cell culture solution obtained in the step (2 a), performing cotransfection at 37 ℃ for 16 h, then supplementing 200 mu L exosome-free fetal calf serum for continuous culture for 72 h, and respectively collecting cells and cell supernatants;

(2c) preparing a constant-temperature water bath kettle at 37 ℃ and liquid nitrogen, repeatedly freezing and thawing the collected cells in liquid nitrogen and a water bath at 37 ℃ for three times, centrifuging at 4 ℃ for 5min at 2000 ℃, removing cell fragments, collecting supernatant, purifying and concentrating according to an adeno-associated virus purification kit to obtain an AAV2-RS1-ZsGreen vector, adding 200 mu L sterile PBS for resuspension, and freezing and storing at-80 ℃;

(2d) and (2 b) centrifuging the cell supernatant obtained in the step (2 b) for 10 min at 300 g at 4 ℃, collecting the supernatant after centrifugation is finished, centrifuging the collected supernatant for 10 min at 4 ℃ and 2000 g at 4 ℃, collecting the supernatant after centrifugation is finished, discarding the precipitate, further centrifuging the collected supernatant for 1h at 100000 g at 4 ℃ by using a super high speed centrifuge (CP 100WX, Japan), removing the precipitate at the bottom of a test tube after centrifugation is finished, adding 1 m L sterile PBS to resuspend the precipitate at the bottom of the test tube, centrifuging for 1h at 100000 g at 4 ℃, sucking the PBS in the test tube by using a gun head, and observing that the white precipitate is at the bottom of the test tube, namely the exo-AAV2-RS1-ZsGreen vector, adding 200 mu L sterile PBS to resuspend, and storing the cell supernatant at-80 ℃ until use.

FIG. 1 is a schematic diagram of the synthesis of pAAV-RS1-ZsGreen plasmid (A), horizontal gel electrophoresis (B) and partial base sequence test (C), in which M: marker, lanes 1-6: pAAV-RS1-ZsGreen colony. As can be seen from fig. 1: the cDNA of RS1 (675 bp in length) was successfully constructed into the pAAV-ZsGreen plasmid, and the base sequence of RS1 was forward, and the two were completely identical by comparing the CDS of RS1 at NCBI.

Identification of exo-AAV2-RS1-ZsGreen vector

The exo-AAV2-RS1-ZsGreen vector PBS resuspension obtained in step (2 d) of example 2, 50. mu. L was resuspended in 50. mu. L sterile PBS, which was then negatively stained with 2% phosphotungstic acid and spread on a grid, and transmission images were obtained using a transmission electron microscope.

The exo-AAV2-RS1-ZsGreen vector PBS re-suspension obtained in step (2 d) of example 2 was diluted 100. mu. L in 50M L sterile PBS and subjected to nanoparticle tracking analysis using a NanoSight L M10 instrument.

The exo-AAV2-RS1-ZsGreen vector PBS resuspension obtained in step (2 d) of example 2 was Western blotted on a 10% SDS-PAGE gel protein was blotted onto a polyvinyl fluoride membrane using CD63 antibody (Novus, NBP 2-32830; volume ratio, antibody: antibody dilution (Solambio, A1800) = 1: 1000) incubated at 4 ℃ for 16 h. the blot was visualized with HRP-conjugated secondary antibody (CST, 7076P 2; volume ratio, antibody: antibody dilution (Solambio, A1800) = 1: 10000) and EC L detection reagent (Millipor, WBK L S0050) and imaged on a bio-RAD instrument.

FIG. 2 is an electron transmission microscope image (A), a nanotrap analysis particle size distribution image (B) and a western blotting image (C) of exo-AAV2-RS1-ZsGreen vector. As can be seen from fig. 2: the extracted exosome obviously has a phospholipid bilayer membrane structure and a tea-saucer-like shape (A); the particle size distribution range of the exosome is 50-200 nm, and the particle size is mainly 120 nm (B); CD63 is a main marker protein of an exosome, and Western blotting detects a CD63 membrane protein (C) in the exosome.

Titer determination of AAV2-RS1-ZsGreen vector and exo-AAV2-RS1-ZsGreen vector

Respectively taking the AAV2-RS1-ZsGreen vector PBS resuspension obtained in the step (2 c) and the exo-AAV2-RS1-ZsGreen vector PBS resuspension liquid obtained in the step (2 d) 3 mu L to add into a DNaseI enzyme reaction system comprising DNaseI enzyme 10 mu L, buffer solution 6 mu L and sterile water 41 mu L2-RS 1-ZsGreen vector PBS resuspension liquid or exo-AAV2-RS1-ZsGreen vector PBS resuspension liquid 3 mu L and total system 60 mu L, and carrying out a reaction procedure comprising water bath at 37 ℃ for 1h and water bath at 100 ℃ for 10 min for degrading the genome attached to the virus vector shell and the exosome vector shell in the extraction process, adding protease K2 mu L, water bath at 55 ℃ for 1h and water bath at 100 ℃ for 10 min for completely cracking the virus vector protein and the exosome protein, completely releasing the subsequent genome to obtain the complete encapsidated genomePreparation is made for detecting genomic copy number. The standard pAAV-RS1-ZsGreen plasmid was diluted with DEPC water in a gradient of 5 concentrations, each 10⁹、10⁸、10⁷、10⁶、10⁵Gene copy number/mu L, the gene group extracted from AAV2-RS1-ZsGreen vector, the gene group extracted from exo-AAV2-RS1-ZsGreen vector and 5 gradient standard pAAV-RS1-ZsGreen plasmid are subjected to RT-PCR detection, each sample is tested repeatedly for 3 times, the reaction program comprises the following steps of 50 ℃ for 2 min, 95 ℃ for 15 s, 60 ℃ for 15 s, 72 ℃ for 1 min, and 40 cycles, the results are shown in Table 1, and the gene copy numbers of AAV2-RS1-ZsGreen vector and exo-AAV2-RS1-ZsGreen vector respectively reach 10¹²gc/mL，10¹¹gc/mL。

In vitro transfection efficiency determination of AAV2-RS1-ZsGreen vector and exo-AAV2-RS1-ZsGreen vector

HEK-293T cells were seeded in 6-well plates at 1.2 × 10 per well⁶The method comprises the steps of completely culturing cells and 2 m L DMEM in a culture box at 37 ℃ for 24 hours, then discarding culture medium in wells, adding 1 m L DMEM high-glycosyl basal medium without serum and antibody to a Ctrl group, namely a blank control group, adding 2 mu L step (2C) obtained AAV2-RS1-ZsGreen vector PBS resuspension or step (2D) obtained exo-AAV2-RS1-ZsGreen vector PBS resuspension, and 1 m L serum and antibody-free DMEM high-glycosyl basal medium, continuing transfection for 6 hours, adding 20 mu L fetal calf serum, continuing incubation for 72 hours, detecting the fluorescence intensity of ZsGreen by an inverted fluorescence microscope (O L YMPUS 73, Japan), extracting cell nucleic acid and protein respectively, carrying out RT-PCR and western blot experiments, detecting the difference between the fluorescence intensity of different groups, the nucleic acid and the protein, and carrying out RT-PCR and western blot experiments, and detecting the difference between AAV 2-Ex RS 27-RS 4629-AAV RS-293 DNA and AAV RS 465-PCR expression of AAV RS-RS 1-AAV RS-RS vector in-RT 465, and AAV-RS vector DNA, and AAV-PCR expression of the AAV-PCR expression profile (AAV-RS 465) of the different groups, and AAV-mRNA-PCR, wherein the difference results show that the difference of different groups are obtainedThe expression level of the neutralizing protein is significantly higher than that of the AAV2-RS1-ZsGreen vector (FIG. 3B, C, D, E).

In vivo transfection efficiency assay for vector and exo-AAV2-RS1-ZsGreen vector

Bovine serum albumin and Triton-X100 were dissolved in 1 × PBS to prepare a blocking solution, the concentration of bovine serum albumin in the blocking solution was 5% (g/m L) and the concentration of Triton-X100 in the blocking solution was 0.3% (v/v), and Tween 20 was dissolved in 1 × PBS to prepare a washing solution, and the concentration of Tween 20 in the washing solution was 0.1% (v/v).

Male C57B L/6J mice (vitamin River L biological technologies Co. L td, Beijing) 4-5 weeks old were randomized into two groups (n = 6) (1) single eye injection of 1 μ L AAV2-RS1-ZsGreen vector 1 × 10⁸gc/eye (gene copy number/eye, same below), control eye injected with 1. mu. L PBS, (2) single eye injection of 1. mu. L exo-AAV2-RS1-ZsGreen vector 1 × 10⁸gc/eye, control eye 1 μ L PBS injection 28 days later, mice inhaled excess CO₂And (6) killing.

The mouse eye is picked up and washed three times in PBS.the anterior section of cornea is cut off under a binocular stereomicroscope (XT L-2300, caikang), the crystal is taken out to leave an eye cup which is fixed in 4% paraformaldehyde solution (v/v) for 4 h at room temperature, then 10% (g/m L) sucrose solution and 20% (g/m L) sucrose solution are dehydrated for 2 h at room temperature, finally 30% (g/m L) sucrose solution is dehydrated for 4 ℃ overnight-20 ℃ O.C.T frozen section embedding agent (SAKURA, 4583) is embedded in a microtome (L eica CM 3050S, Germany) the obtained retina frozen section is subjected to immunofluorescence staining, the obtained retina frozen section is placed in a cassette for blocking 1h at room temperature, the washing liquid is washed three times to wash blocking liquid, then the blocking liquid is diluted by the blocking liquid for one time to wash the blocking liquid for 1h at room temperature, the blocking liquid is washed three times, the blocking liquid is diluted by the blocking liquid for one time, then the blocking liquid is diluted by the blocking liquid, the blocking liquid diluted by the blocking liquid, the blocking liquid diluted by the AAV sRS 7 sRS 19 RS 80, the fluorescent protein is added in the AAV sRS 19 RS gene, the AAV sRS gene, the fluorescent protein is added to obtain the fluorescent protein expressed in the AAV sRS 19S-RS 19S, the AAV sRS 19 RS, the AAV sRS 19S, the fluorescent protein, the fluorescent protein is observed under the fluorescent protein, the fluorescent protein is observed under the fluorescent protein, the fluorescent protein is observed under the fluorescent protein, the fluorescent protein is observed under the fluorescent protein expressed in the fluorescent protein, the fluorescent protein fluorescent.

Mice were sacrificed and the excised retinas were first minced with a pestle, a portion of the tissues was lysed with Trizol (Thermo Fisher, 10296028) at room temperature, RNA was extracted sequentially with chloroform, isopropanol, 75% absolute ethanol and finally dissolved in DEPC water (Beyotim, R0021), PrimeScript RT reverse kit (Takara, RR 037A) for reverse transcription of RNA to cDNA, SRBR Green premix (Thermo Fisher, 4472913) for real-time fluorescence quantification, another portion of the tissues was added with lysate containing protein inhibitors (MCE, HY-K0010) (Thermo Fisher, 89901), centrifuged 15min at 12000 g at 4 ℃ and the supernatant was transferred to a new enzyme-free Ep tube, SDS-PAGE isolate protein, polyvinylidene difluoride membrane transfer protein, incubated with 4 ℃ RS1 (Sigma, SAB 1401345; Milli. blocking antibody: 1: CSTR = 1), anti-protein transfection assay 1000, PCR 400: 1000: AAV RS 4676, AAV RS 76, AAV-K-RT 32. Grassyping test DNA expression in AAV vector III PCR-S5, AAV-K630, AAV DNA expression test-DNA expression test, AAV DNA test, AAV test DNA test, AAV test paper, test paper.

Claims

1. A preparation method of an exosome-encapsulated AAV vector is characterized by comprising the following steps:

2. The method for producing an exosome-encapsulating AAV vector according to claim 1, wherein the specific process in the step (2) comprises adding 10-15 μ g of pAAV-ZsGreen plasmid, 10-15 μ g of pRC2-miR342 plasmid and 20-25 μ g of pHelper plasmid to 50-100 μ L CaCl₂The three plasmids were added in solution form in both solution and water at a concentration of 1-2. mu.g/. mu. L₂The concentration of the solution is 2-3M, the amount of water is 10 times of the total volume of the three-plasmid solution, the three-plasmid solution is evenly blown and evenly mixed, the evenly mixed three-plasmid solution is dripped into 500-1000 mu L2 × HeBS solution in a vortex state, after standing for 20-30 min at room temperature, the three-plasmid solution is dripped into HEK-293T cell culture solution obtained in the step (1) of 8-10M L, after cotransfection is carried out for 12-16 h at 37 ℃, 160-200 mu L exosome-free fetal bovine serum is added for continuous culture for 48-72 h, cell culture supernatant is collected, and the exosome-encapsulated AAV vector is obtained by centrifugal separation.

3. The method for producing an exosome-encapsulating AAV vector according to claim 1, wherein in step (2), the specific process of centrifugation is: firstly, centrifuging at 300-600 g and 600-5 ℃ for 10-15min, collecting the supernatant after the centrifugation is finished, then centrifuging the collected supernatant at 2000-2500 g and 3-5 ℃ for 10-15min, and collecting the supernatant after the centrifugation is finished; further centrifuging the collected supernatant at 3-5 ℃ for 1-1.5 h at 100000 g, removing the supernatant after centrifugation is finished, collecting bottom sediment, adding PBS for heavy suspension sediment, centrifuging at 3-5 ℃ for 1-1.5 h at 100000 g again, removing the supernatant after centrifugation is finished, collecting the bottom sediment, and obtaining the sediment, namely the exosome-encapsulated AAV vector.

4. An exosome-encapsulating AAV vector prepared by the preparation method according to any one of claims 1 to 3.

5. A preparation method of an exosome-encapsulated AAV-target gene vector is characterized by comprising the following steps:

6. The method for preparing an exosome-encapsulating AAV-target gene vector according to claim 5, wherein in step (1), the EcoRI digestion system comprises pAAV-ZsGreen plasmid solution 3-5 μ L with a concentration of 150-.

7. The method for preparing an exosome-encapsulating AAV-target gene vector according to claim 5, wherein the specific process of the step (3) comprises adding 10-15 μ g pAAV-target gene-ZsGreen plasmid, 10-15 μ g pRC2-miR342 plasmid and 20-25 μ g pHelper plasmid to 50-100 μ L CaCl₂The three plasmids were added in solution form in both solution and water at a concentration of 1-2. mu.g/. mu. L₂The concentration of the solution is 2-3M, the amount of water is 10 times of the total volume of the three-plasmid solution, the three-plasmid solution is evenly blown and evenly mixed, the evenly mixed three-plasmid solution is dripped into 500-1000 mu L2 × HeBS solution in a vortex state, after standing for 20-30 min at room temperature, the three-plasmid solution is dripped into HEK-293T cell culture solution obtained in the step (2) of 8-10M L, after cotransfection is carried out for 12-16 h at 37 ℃, 160-200 mu L exosome-free fetal bovine serum is added for continuous culture for 48-72 h, cell culture supernatant is collected, and centrifugal separation is carried out to obtain the exosome-encapsulated AAV-target gene vector.

8. The method for producing an exosome-encapsulating AAV-target gene vector according to claim 5, wherein in step (3), the specific process of centrifugation is: firstly, centrifuging at 300-600 g and 600-5 ℃ for 10-15min, collecting the supernatant after the centrifugation is finished, then centrifuging the collected supernatant at 2000-2500 g and 3-5 ℃ for 10-15min, and collecting the supernatant after the centrifugation is finished; further centrifuging the collected supernatant for 1-1.5 h at 3-5 ℃ at 100000 g, removing the supernatant after centrifugation is finished, collecting bottom sediment, adding PBS for heavy suspension sediment, centrifuging for 1-1.5 h at 3-5 ℃ at 100000 g again, removing the supernatant after centrifugation is finished, collecting the bottom sediment, and obtaining the sediment, namely the exosome-encapsulated AAV-target gene vector.

9. An exosome-encapsulating AAV-target gene vector prepared by the preparation method according to any one of claims 5 to 8.

10. Use of the exosome-encapsulating AAV-target gene vector according to claim 9 in the preparation of a medicament for treating a disease to which a target gene corresponds.