Method for preparing AAV based on cell transfection method of extracellular vesicles, recombinant AAV virus particles and application

Abstract

The invention belongs to the field of biotechnology and gene therapy, and particularly relates to a cell transfection method based on extracellular vesicles. The method comprises the following steps: (1) preparing a plasmid-cationic polymer compound; (2) assembling the core-shell type load vesicles; (3) purifying the loaded vesicles; and (4) cell transfection and AAV preparation. By adopting the method disclosed by the invention, the damage to the cells is remarkably reduced, and the survival rate of the transfected cells is improved, so that healthier host cells are provided for virus packaging, and finally, the yield of AAV viruses is improved. The problem of contradiction between transfection efficiency and cytotoxicity in a stage AAV preparation process is solved.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and gene therapy, specifically relating to a method for preparing AAV based on extracellular vesicle cell transfection, recombinant AAV virus particles and their applications. Background Technology

[0002] Adeno-associated virus (AAV) has become one of the most commonly used vectors in gene therapy due to its high safety, broad host range, and long-term stable expression. It is widely used in the treatment research and clinical application of hereditary diseases, tumors, and other diseases. Currently, the preparation of AAV mainly relies on cell transfection technology, among which chemical transfection methods (such as PEI and liposome-mediated transfection) have become the mainstream method for industrial production and laboratory research due to their simplicity and low cost.

[0003] However, existing chemical transfection techniques have the following key defects: (1) Strong cytotoxicity: After PEI and other cationic polymers form complexes with DNA, they will cause mechanical and chemical damage to the cell membrane during endocytosis, interfere with normal cell metabolism, and lead to cell death or growth arrest after transfection, significantly reducing the number of effective virus packaging cells; (2) Uneven plasmid delivery efficiency: AAV preparation requires multi-plasmid co-transfection (Rep-Cap plasmid, target gene plasmid, helper plasmid). Traditional chemical transfection is difficult to achieve simultaneous delivery of multiple plasmids in equal amounts. Some cells cannot complete virus assembly due to lack of key plasmids, reducing overall production efficiency; (3) Poor plasmid stability: The naked plasmid-polymer complex is easily degraded by nucleases in the extracellular environment, resulting in a reduction in the amount of effective plasmids actually involved in transfection, further affecting AAV production; (4) Insufficient quality of viral products: Deterioration of cell growth status will lead to a decrease in the integrity of AAV viral particles, and AAV prepared by traditional methods is easily recognized by the host immune system, posing an immunogenicity risk when applied to in vivo treatment.

[0004] To address the aforementioned issues, existing technologies have attempted to optimize transfection reagent ratios and improve cell culture conditions, but none have overcome the core bottleneck of the toxicity of the chemical carrier itself. Some studies have explored the use of extracellular vesicles as drug delivery carriers, but these are limited to encapsulating pre-prepared AAV viruses and do not involve a technical path that uses extracellular vesicles as transfection carriers to mediate plasmid delivery and subsequently prepare AAV in situ. This approach fails to resolve the contradiction between transfection efficiency and cytotoxicity during AAV preparation. Therefore, developing a biocompatible, stable transfection efficiency, and significantly reduced cytotoxicity method for AAV preparation has become an urgent technical need in this field. Summary of the Invention

[0005] To address the contradiction between transfection efficiency and cytotoxicity in the current AAV preparation process, this invention provides a method for preparing AAV based on extracellular vesicle cell transfection, which significantly reduces cell damage, improves cell survival rate after transfection, thereby providing healthier host cells for virus packaging and ultimately increasing AAV virus yield.

[0006] The technical solution of the present invention is as follows: In a first aspect, a method for preparing AAV based on extracellular vesicle cell transfection is provided, comprising the following steps: (1) Preparation of plasmid-cationic polymer complex: The recombinant plasmid required for AAV packaging is premixed with the cationic polymer at a mass ratio of 1:2-4 to form a stable complex; (2) Assembly of core-shell loaded vesicles: Mammalian extracellular vesicles are mixed with the complex in step (1) and extruded sequentially through 500-1000nm and 100-300nm polycarbonate membranes to obtain uniformly sized loaded vesicles. (3) Purification of loaded vesicles: The loaded vesicles from step (2) are centrifuged to remove free complexes and broken vesicles, and high-purity loaded vesicles are collected. (4) Cell transfection and AAV preparation: The high-purity loaded vesicles from step (3) are co-cultured with production cells, and recombinant AAV virus particles are obtained through transfection, virus assembly and separation and purification.

[0007] As a specific embodiment of the present invention, the mammalian cells mentioned in step (1) include 293T cells, Expi293F cells or CHO cells.

[0008] As a specific embodiment of the present invention, the preparation process of extracellular vesicles of mammals in step (1) includes: collecting mammalian cells after culturing, washing, freezing, ultrasonic disruption, differential centrifugation and ultracentrifugation to obtain extracellular vesicles.

[0009] As a specific embodiment of the present invention, the ultrasonic crushing conditions are 4℃±2℃, power 30%-50%, and crushing interval 1-2min; the differential centrifugation is 3000-5000G for 20-40min, and the ultracentrifugation is 80000-120000G for 30-60min.

[0010] As a specific embodiment of the present invention, the cationic polymer in step (1) includes PEI with a molecular weight of 30,000-50,000 and a premixing time of 15-30 min.

[0011] As a specific embodiment of the present invention, the plasmids include pAAV-GFP plasmid, pAAV2 / 1 plasmid, and pAdDeltaF6 helper plasmid.

[0012] As a specific embodiment of the present invention, the volume ratio of extracellular vesicles to complex in step (2) is 1:0.5-1:2, and the extrusion pressure is 0.1-0.5 MPa.

[0013] As a specific embodiment of the present invention, the centrifugation conditions in step (4) are 2000-4000G centrifugation for 5-15 minutes and centrifugation temperature of 4℃±2℃.

[0014] As a specific embodiment of the present invention, the production cells in step (5) are mammalian cells cultured in suspension or adherence, and the culture conditions are 37℃±3℃ and 5%-8% CO2; the separation and purification include one or more combinations of density gradient centrifugation, size exclusion chromatography and ultrafiltration centrifugation.

[0015] Secondly, recombinant AAV virus particles prepared by the above method are provided, wherein the AAV virus particles have a particle size of 80-250 nm, a cell viability of ≥90% after cell transfection, and a virus titer of ≥1×10⁻⁶. 12 vg / mL.

[0016] As a specific embodiment of the present invention, the surface of the virus particles is bound with extracellular vesicle membrane fragments, which increases the tolerance to nucleases by more than 1.5 times compared with AAV prepared by traditional chemical transfection.

[0017] Thirdly, the application of the aforementioned recombinant AAV virus particles in the preparation of gene therapy drugs is provided.

[0018] As a specific embodiment of the present invention, the gene therapy drug is used to treat hereditary diseases, tumors, or neurodegenerative diseases.

[0019] Beneficial effects of the present invention 1. Low cytotoxicity: Traditional direct use of synthetic chemical reagents (such as liposomes and cationic polymers) as carriers can increase cytotoxicity and reduce cell transfection efficiency. However, using cell vesicles prepared from the cells themselves as carriers has higher biocompatibility and can significantly reduce cytotoxicity.

[0020] 2. “In vitro pre-assembled” core-shell structure: Through a unique “premix-extrusion” process, the plasmid-PEI complex can be actively loaded into the vesicle to form a structurally similar “core-shell” vector. This ensures that the plasmid is efficiently encapsulated in the closed space of the vesicle, avoiding its degradation by nucleases during delivery and improving the stability and transfection efficiency of the plasmid.

[0021] 3. The final product of this invention is a pre-assembled, ready-to-use cell vesicle formulation. This formulation is convenient, time-saving, and stable to use. It can be directly added to cells without the need for traditional transfection steps, thus reducing the time and effort required for transfection. Attached Figure Description

[0022] Figure 1 A map of the recombinant plasmid used in the specific implementation method; Figure 2 This is a schematic diagram of the particle size of EVs prepared by nanoparticle tracking analysis technology in Specific Example 1; Figure 3 A transmission electron microscope image of the EVs prepared in specific embodiment 2; Figure 4 A schematic diagram of cell viability determination by CCK8 assay for EVs prepared in Specific Example 3; Figure 5 A schematic diagram showing the cell transfection results of EVs prepared in Specific Example 3; Figure 6 A viral titer statistical chart of AAV was prepared for specific Example 4. Detailed Implementation

[0023] Experimental materials Cell line: 293T cells (ATCC number: CRL-3216); Plasmids: pAAV-GFP plasmid, pAAV2 / 1 plasmid, pAdDeltaF6 helper plasmid (all purchased from Addgene); Reagents: Polysciences PEI transfection reagent (molecular weight 40,000, Polysciences catalog number: 24765-100), Gibco™ DMEM high glucose medium (Gibco, catalog number C11995500BT), Gibco™ fetal bovine serum (Gibco, catalog number: 10099141C), Servicebio PBS buffer (Servicebio, catalog number: G4207-500ML), Yeasen Cell Counting Kit (CCK-8) CCK-8 kit (Yeasen, catalog number: 40203ES60 100 T), iodixanol (Sigma, catalog number: D1556); Instruments: cryogenic ultrasonic homogenizer, ultracentrifuge, nanoparticle tracking analyzer (NTA), transmission electron microscope (TEM), microplate reader, fluorescence microscope, cell culture incubator, QPCR instrument.

[0024] Example 1 1. Preparation of extracellular vesicles 1.1 Cell Culture: 293T cells were cultured in DMEM medium containing 10% inactivated fetal bovine serum and cultured in large quantities in 15 cm cell culture dishes at 37°C and 5% CO2 for 24 h. 1.2 Cell collection: Remove the supernatant culture medium, wash once with pre-cooled PBS, gently scrape the cells with a cell scraper, resuspend in 10 mL of PBS, centrifuge at 1000 rpm for 5 min, discard the supernatant, and collect the cell pellet; 1.3 Freeze-thaw and disruption: The cell pellet was resuspended in pre-cooled PBS and frozen at -80℃ for 12 h. After freezing, the pellet was thawed on ice, pipetted and mixed, and then placed in a cryosonic disruptor. The parameters were: 4℃, 1 second on, 2 seconds off, for a total of 1.5 minutes, 40% power, to obtain a clear cell solution. 1.4 Centrifugation purification: Centrifuge the cell solution at 4℃ and 4000G for 30 min to remove cell debris, cell nuclei and organelles, and collect the supernatant; centrifuge the supernatant at 4℃ and 100000G for 45 min, discard the supernatant, resuspend the precipitate in 1 mL PBS to obtain extracellular vesicles, and store them at -80℃ for later use.

[0025] 2. Preparation of plasmid-PEI complex The plasmid was mixed with PEI (40000) at a mass ratio of 1:3 and premixed for 20 minutes to form a stable plasmid-PEI complex.

[0026] 3. Preparation and purification of core-shell loaded vesicles (1) Mixing reaction: Take 100 μL of the extracellular vesicles prepared in step 1, add 110 μL of the plasmid-PEI complex prepared in step 2, and gently invert to mix. (2) Extrusion process: The mixture is passed through 800nm ​​and 200nm polycarbonate membranes in sequence to obtain primary vesicles loaded with plasmid-PEI; (3) Purification: Centrifuge the primary vesicles at 4℃ and 3000G for 8 minutes, discard the precipitate (free complex and broken vesicles), and collect the supernatant, which is the high-purity loaded vesicle.

[0027] 4. Cell transfection and AAV preparation (1) Cell seeding: 293T cells were seeded into 10cm cell culture dishes, with 1×10 cells per dish. 7 Cells were added to DMEM medium containing 10% inactivated fetal bovine serum and cultured for 24 hours. (2) Transfection operation: Add 30 μL of the loaded vesicles prepared in Example 3 to each plate, gently shake the culture plate to distribute the vesicles evenly, and continue to culture at 37°C and 5% CO2. (3) Virus collection: 48 h after transfection, centrifuge at 4℃ and 1000G for 10 min to collect all the precipitated cells. Add 5 ml of PBS and collect the crude virus extract by repeated freeze-thaw cycles three times.

[0028] (4) Virus purification: Add the crude extract to an iodixanol density gradient centrifuge tube, centrifuge at 4°C and 100,000G for 2 hours, collect the virus band, add it to a 100KD ultrafiltration tube, centrifuge at 4°C and 3,000G for 30 minutes three times, and further concentrate and purify to obtain recombinant AAV-GFP virus concentrate.

[0029] Implementation results: I. Vesicle Analysis: 1. Particle size: The obtained empty cell vesicles were divided into two parts: Part A: extruded sequentially through 800nm ​​and 200nm polycarbonate membranes to obtain homogenized empty vesicles. Part B: 50μl of 400ng / μl pAAV-GFP plasmid, 50μl of 400ng / μl pAAV2 / 1 plasmid, 150μl of 400ng / μl pAdDeltaF6 plasmid, and 300μl of PEI were mixed to obtain a pAAV-GFP plasmid-PEI complex. The empty vesicles were then mixed with the pAAV-GFP plasmid-PEI complex, resulting in 300μl empty vesicles and 300μl pAAV-GFP plasmid-PEI complex. This mixture was then processed through the same extrusion procedure to obtain vesicles loaded with pAAV-GFP plasmid-PEI. The empty vesicles, the extruded empty vesicles, and the vesicles loaded with the three plasmids-PEI were then analyzed using a nanoparticle tracking analyzer. Figure 1 It can be seen that the particle size of the unextruded empty EVs prepared by nanoparticle tracking analysis technology is between 100-400 nm, while the particle size of the empty EVs after extrusion is concentrated between 100-200 nm. The particle size of the EVs carrying the three plasmid-PEI complex is also between 100-200 nm.

[0030] 2. Appearance Transmission electron microscopy (TEM) was used to image homogenized empty vesicles and vesicles loaded with the three plasmid-PEI complex prepared in Example 1. These were dropped onto a copper grid, negatively stained with phosphotungstic acid solution, and allowed to air dry before being observed and imaged under a TEM at 10,000x magnification. Figure 2 As shown, unloaded empty vesicles exhibit a typical "black heart" structure after negative staining, indicating that the empty vesicles have a hollow internal structure. Vesicles loaded with the triplasmid-PEI complex prepared by the method of this invention appear light-colored or white in the image, indicating that the vesicles are loaded with the triplasmid-PEI complex.

[0031] II. CCK8 assay for cell viability 1. Digest, centrifuge, and count the cultured 293T cells. Seed 100 μl of 5000 cells / well in a 96-well plate and divide into 3 groups. The first group is the normal control group (NC), the second group is conventional PEI transfection (PEI), and the third group is cell vesicle transfection (EVs). Each group is further divided into three subgroups according to the transfection time: 24h, 48h, and 72h. Each subgroup has 5 replicates and is cultured at 37℃, 5% CO2, and 90% humidity for 24, 48, and 72 hours.

[0032] 2. Once cells have fully adhered to the culture vessel, transfection can begin. The transfection system is as follows: PEI group: 0.4 μg pAAV-GFP plasmid, 0.4 μg pAAV2 / 1 plasmid, 1.2 μg pAdDeltaF6 plasmid, and 6 μg PEI per well. Mix the PEI and plasmid thoroughly and add to each well along with 100 μl of serum-free medium. EVs group: Add vesicles loaded with the three plasmid-PEI complex (prepared according to Example 1) to each well, equal to the total plasmid amount. Add to each well along with 100 μl of serum-free medium. NC group: No treatment is applied to each well; add 100 μl of serum-free medium. After 12 hours, replace the serum-free medium in all three groups with complete medium. At the planned transfection time, add 10 μL of CCK-8 solution to each well. 3. After incubating at 37℃, 5% CO2, and 90% humidity for 1.5 hours, measure the absorbance at 450 nm using a microplate reader. (For example...) Figure 4 As shown, the CCK-8 assay revealed that after 48 and 72 hours of transfection, the cell viability in the PEI group was only 70%, indicating that PEI had a severe toxic effect on cells. In contrast, using the cell vesicles described in this invention, with an equivalent amount of plasmid, the cell viability in the EVs group was as high as 100%.

[0033] III. Cell Transfection Transfection was performed in 6-well plates. Healthy cells (e.g., HEK-293T cells) were seeded into 6-well plates and randomly divided into three groups: Group 1: Traditional PEI transfection (PEI+DNA): Using the traditional method, plasmids and PEI transfection reagent were mixed at a mass ratio of 1:3. 0.4 μg pAAV-GFP plasmid, 0.4 μg pAAV-2 / 1 plasmid, 1.2 μg pAdDeltaF6 plasmid, and 6 μg PEI were added to each well. After 30 min, the resulting complex was mixed with 1.5 ml of serum-free cell culture medium and added to the well. Group 2: Vesicle transfection (EVs+DNA group, this invention): A total amount of vesicles loaded with the three plasmid-PEI complex (prepared according to Example 1) and 100 μl of serum-free culture medium were added to each well. Group 3: Normal group (NC): An equal volume of serum-free cell culture medium without plasmids was added as a blank control. Then, at 12 hours, the serum-free medium in the three wells was replaced with complete medium, and transfection images were taken under a fluorescence microscope at 24 hours, 48 ​​hours, and 72 hours post-transfection. The results are as follows: Figure 5 As shown, compared with the traditional PEI transfection method, the cell vesicle delivery system provided by this invention can significantly overcome the cytotoxicity problem of PEI while achieving the same high transfection efficiency, effectively maintaining the number and activity of the cell population. Figure 5 As shown, the experimental results indicate that, compared with the traditional PEI transfection method, the traditional PEI method causes cell death due to its own toxicity, resulting in a significant decrease in cell density in the field of view. The cell vesicle delivery system provided by this invention can significantly maintain the number and activity of the cell population while achieving similar transfection efficiency.

[0034] IV. AAV Preparation Transfection was performed in 10 10cm cell culture dishes. Healthy cells (e.g., HEK-293T cells) were seeded into 10cm cell culture dishes and randomly divided into two groups: PEI group: Plasmids and PEI transfection reagent were mixed at a mass ratio of 1:3. Each dish contained 3 μg of pAAV-GFP plasmid, 3 μg of pAAV-2 / 1 plasmid, 9 μg of pAdDeltaF6 plasmid, and 45 μg of PEI. After 30 minutes, the resulting complex was added to each dish at a rate of 15 μg of plasmid mixed with 10 ml of serum-free cell culture medium. EVs group (this invention): Cell vesicles loaded with the three plasmid-PEI complex (prepared according to the method in Example 1) were added to each dish in an equal amount of plasmid-loaded vesicles, and then mixed with 10 ml of serum-free cell culture medium. 24 hours after transfection, the serum-free culture medium in the 10 cell culture dishes was replaced with complete culture medium. After transfection and culture for approximately 48-72 hours, cells were scraped off with a cell scraper, centrifuged at 1000G for 10 min at 4°C, and all cells were collected. The crude virus extract was collected by repeating freeze-thaw cycles three times. The crude extract was added to iodixanol density gradient centrifuge tubes, centrifuged at 100,000G for 2 h at 4°C, and the viral bands were collected. The virus was then added to 100KD ultrafiltration tubes and centrifuged three times at 3000G for 30 min at 4°C for further concentration and purification. The viral concentrate was then analyzed for viral titer using a QPCR instrument. The results are as follows: Figure 6 As shown, compared with the PEI group, the viral titer of the EVs group was significantly increased. The new strategy of using cell vesicles to deliver plasmids for AAV virus packaging can significantly improve the final viral yield without changing the core production process.

[0035] It should be noted that the embodiments described above are only for explaining the present invention and do not constitute any limitation on the present invention. The present invention has been described with reference to typical embodiments, but it should be understood that the words used therein are descriptive and explanatory terms, not limiting terms. Modifications can be made to the present invention within the scope of the claims, and revisions can be made to the present invention without departing from the scope and spirit of the present invention. Although the present invention described herein relates to specific methods, materials, and embodiments, it does not mean that the present invention is limited to the specific examples disclosed herein; on the contrary, the present invention can be extended to all other methods and applications with the same function.

Claims

1. A method for preparing AAV based on extracellular vesicle cell transfection, recombinant AAV viral particles, and their applications, characterized in that... Includes the following steps: (1) Preparation of plasmid-cationic polymer complex: The plasmid required for AAV packaging is premixed with the cationic polymer at a mass ratio of 1:2-4 to form a stable complex; (2) Assembly of core-shell loaded vesicles: Mammalian extracellular vesicles are mixed with the complex in step (1) and extruded sequentially through 500-1000nm and 100-300nm polycarbonate membranes to obtain uniformly sized loaded vesicles. (3) Purification of loaded vesicles: The loaded vesicles from step (2) are centrifuged to remove free complexes and broken vesicles, and high-purity loaded vesicles are collected. (4) Cell transfection and AAV preparation: The high-purity loaded vesicles from step (3) are co-cultured with production cells, and recombinant AAV virus particles are obtained through transfection, virus assembly and separation and purification.

2. The method according to claim 1, characterized in that, The mammalian cells mentioned in step (1) include 293T cells, Expi293F cells, or CHO cells; And / or, the preparation process of extracellular vesicles of mammals in step (1) includes: collecting mammalian cells after culturing, washing, freezing, ultrasonic disruption, differential centrifugation and ultracentrifugation to obtain extracellular vesicles; Preferably, the ultrasonic crushing conditions are 4℃±2℃, power 30%-50%, and crushing time 1-2 min; the differential centrifugation is 3000-5000G for 20-40 min, and the ultracentrifugation is 80000-120000G for 30-60 min.

3. The method according to claim 1, characterized in that, The cationic polymer mentioned in step (1) includes PEI with a molecular weight of 30,000-50,000 and a premixing time of 15-30 min; And / or, the plasmids include pAAV-GFP plasmid, pAAV2 / 1 plasmid, and pAdDeltaF6 helper plasmid.

4. The method according to claim 1, characterized in that, The volume ratio of extracellular vesicles to complex in step (2) is 1:0.5-1:2, and the extrusion pressure is 0.1-0.5 MPa.

5. The method according to claim 1, characterized in that, The centrifugation conditions described in step (4) are 2000-4000G centrifugation for 5-15 minutes and centrifugation temperature of 4℃±2℃.

6. The method according to claim 1, characterized in that, The production cells mentioned in step (5) are mammalian cells cultured in suspension or adherence, and the culture conditions are 37℃±3℃ and 5%-8% CO2; the separation and purification include one or more combinations of density gradient centrifugation, size exclusion chromatography and ultrafiltration centrifugation.

7. A recombinant AAV virus particle prepared by the method according to any one of claims 1-6, characterized in that, The AAV virus particles have a diameter of 80-250 nm, and the cell survival rate after transfection is ≥90%, with a virus titer ≥1×10¹² vg / mL.

8. The recombinant AAV virus particle according to claim 7, characterized in that, The virus particles have extracellular vesicle membrane fragments bound to their surface, which increases their tolerance to nucleases by more than 1.5 times compared to AAV prepared by traditional chemical transfection.

9. The use of the recombinant AAV virus particles according to claim 7 in the preparation of gene therapy drugs.

10. The application according to claim 9, characterized in that, The gene therapy drugs are used to treat hereditary diseases, tumors, or neurodegenerative diseases.

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