CN117660532A - Helper plasmid for reducing rcAAV residues in recombinant adeno-associated virus and application - Google Patents
Helper plasmid for reducing rcAAV residues in recombinant adeno-associated virus and application Download PDFInfo
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Abstract
The invention belongs to the technical field of genetic engineering, and discloses an auxiliary plasmid for reducing rcAAV residues in recombinant adeno-associated virus and application thereof. The auxiliary plasmid for reducing rcAAV residues in recombinant adeno-associated virus comprises a skeleton plasmid and a recombinant sequence; the recombinant sequence comprises: rep, cap protein coding sequence, E2A, E, VA RNA sequence, at least one DA' sequence and at least one promoter sequence. The helper plasmid of the invention can obviously reduce the residue of rcAAV, can improve the purity of AAV products and reduce the treatment risk of product acceptors.
Description
Technical Field
The invention relates to the technical field of genetic engineering, in particular to an auxiliary plasmid for reducing rcAAV residues in recombinant adeno-associated virus and application thereof.
Background
Adeno-associated virus (AAV) is an icosahedral enveloped virus of about 22nm diameter belonging to the genus Paramyxoviridae. The genome is approximately 4.7kb and comprises four Open Reading Frames (ORFs) encoding the Rep protein, capsid protein (VP 1/VP2/VP 3), assembly Activating Protein (AAP) and Membrane Associated Accessory Protein (MAAP), flanked by 145bp inverted terminal repeats, which fold to form a T-hairpin structure. Recombinant adeno-associated virus (rAAV) is the most commonly used delivery vehicle in clinical gene therapy, has been widely used in more than 150 clinical trials, and has good safety.
As increasingly stringent examination of viral formulations for use in transformation medicine has revealed the presence of non-nucleic acid contaminants of interest in rAAV formulations, the introduction of non-therapeutic nucleic acids can increase the immunogenicity or other risk of rAAV therapy. rAAV vector rcAAV contamination is a major safety concern, packaging of rAAV can recombinantly produce replication-competent adeno-associated virus (i.e., replication-competent adeno-associated virus, rcAAV), which is packaged by AAV capsid particles flanked by the rep and cap genomes of ITRs, which can replicate in the presence of helper virus, and rcAAV may promote immunotoxicity by an incompletely understood mechanism. Rep may induce DNA fragmentation due to its helicase and endonuclease activities, while Cap expression may trigger an immune response. It is therefore particularly important to reduce rcAAV to the lowest levels achievable in rAAV vectors prepared for clinical trial product development.
Because rcAAV and rAAV vectors are very similar, the inability to isolate them by purification process steps presents a more difficult challenge to the clinical production of rAAV. In systems that use AAV/Ad to produce AAV, studies have reported that the production of rcAAV can be reduced by deleting homologous sequences from AAV packaging vectors and replacing promoters, yet the presence of rcAAV can still be detected significantly during mass production. Based on the method, splitting the Rep and the Cap on the helper plasmid into two expression cassettes with opposite directions can reduce the probability that the Rep and the Cap are assembled simultaneously to form rcAAV, but the yield of rAAV is reduced by about 40%. In addition, there are studies to reduce rcAAV production by limiting the helper genes Rep and Cap to the cytoplasm using poxviruses, and this system uses adenovirus for rAAV production, potentially causing adenovirus contamination to induce an immune response in the body. In order to inhibit the immune toxicity of residual rcAAV in rAAV to the body, it has been studied to inhibit the production of rcAAV capsid protein by adding a microRNA binding cassette to the end of helper plasmid Cap. . There is no good method for removing rcAAV, so there is a need to develop better production systems to reduce rcAAV production.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide an auxiliary plasmid for reducing rc AAC residues in recombinant adeno-associated viruses and application thereof.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:
in a first aspect, the present invention provides a helper plasmid for reducing replication competent adeno-associated virus residues in a recombinant adeno-associated virus, the helper plasmid comprising a backbone plasmid and a recombinant sequence; the recombinant sequence comprises:
rep, cap protein coding sequence, E2A, E, VA RNA sequence, at least one DA' sequence and at least one promoter sequence.
The helper plasmid of the present invention can significantly reduce rcAAV residues. Can be used for reducing rcAAV residual AAV production system, can improve the purity of AAV products and reduce the treatment risk of product acceptors.
As a preferred embodiment of the helper plasmid of the present invention, the E2A, E, VA RNA sequence is located downstream of the Rep, cap protein coding sequence.
As a preferred embodiment of the helper plasmid according to the invention, the DA' sequence is located downstream of the E2A, E, VA RNA sequence.
As a preferred embodiment of the helper plasmid of the present invention, the promoter sequence is located downstream of the E2A, E, VA RNA sequence.
As a further preferred embodiment of the helper plasmid according to the invention, the promoter sequence is located downstream of the DA' sequence.
As a preferred embodiment of the helper plasmid of the present invention, the recombinant sequence comprises, in order from 5 'to 3': the Rep, cap protein coding sequence, the E2A, E, VA RNA sequence, the DA' sequence and the promoter sequence.
In a second aspect, the invention provides a plasmid set for reducing replication competent adeno-associated virus residues in recombinant adeno-associated virus, comprising the helper plasmid and an AAV plasmid. As a preferred embodiment of the plasmid set of the present invention, the AAV plasmid is an AAV plasmid of a fluorescent protein driven by a CAG promoter.
Preferably, the AAV plasmid is AAV plasmid pAAV. CAG. EGFP vector of fluorescent protein driven by CAG promoter.
In a third aspect, the invention provides an AAV production system with low rcAAV residues, which is obtained by transforming host cells with the helper plasmid.
Preferably, the host cell is a HeLa, HEK293 or insect Sf9 cell.
In a fourth aspect, the present invention provides a method for producing AAV with reduced rcAAV residues, using the AAV production system described above.
In a fifth aspect, the present invention provides use of the above plasmid set, the above AAV production system, in AAV production.
Compared with the prior art, the invention has the beneficial effects that:
the helper plasmid of the invention can obviously reduce the residue of rcAAV. Can be used for reducing rcAAV residual AAV production system, can improve the purity of AAV products and reduce the treatment risk of product acceptors.
Drawings
FIG. 1 is a schematic diagram of the experimental design for studying P5, DA' functions in the examples.
FIG. 2 is a schematic diagram of structural elements of a plasmid backbone in examples.
Detailed Description
For a better description of the objects, technical solutions and advantages of the present invention, the present invention will be further described with reference to the following specific examples. It will be appreciated by persons skilled in the art that the specific embodiments described herein are for purposes of illustration only and are not intended to be limiting.
The test methods used in the examples are conventional methods unless otherwise specified; the materials, reagents and the like used, unless otherwise specified, are all commercially available.
Example 1: construction of helper plasmid pRCHelper
pRCHelper helper vector (FIG. 1) was constructed based on backbone plasmid sequences by conventional molecular cloning methods by designing combinations of placement positions containing DA' sequences, P5 promoters, rep2 gene sequences (SEQ ID NO: 4), cap gene sequences, E2A gene sequences, E4 gene sequences, and VA RNA sequences, and then comparing the residuals of rcAAV in AAV produced by the two helper vectors in parallel.
Wherein, the plasmid skeleton (shown in figure 2) has a sequence shown in SEQ ID NO:1 is shown in the specification; the sequence of the P5 promoter is shown in SEQ ID NO:2 is shown in the figure; DA' has the sequence shown in SEQ ID NO:3 is shown in the figure; the sequence of the Rep2 gene is shown as SEQ ID NO. 4; cap is from type 9 adeno-associated virus, and the sequence of the Cap is shown in SEQ ID NO:5 is shown in the figure; the E2A gene sequence is shown as SEQ ID NO. 6; e4 sequence is shown as SEQ ID NO. 7; the VA RNA sequence is shown as SEQ ID NO. 8. The helper plasmids A and B in FIG. 1 were constructed as follows:
(1) The RepCap gene sequence, DA' sequence, P5 sequence and E2A/E4/VA RNA gene sequence are amplified respectively by using the plasmid containing the RepCap gene as a template.
The PCR reaction system is shown in Table 1:
TABLE 1PCR reaction System
The primer sequences used for plasmid a are shown below:
forward primer 1: ATGGCTGCCGATGGTTATC;
reverse primer 1: acgtaatccgtagatgtacctgg;
forward primer 2: aggtacatctacggattacgtAAGCCGAATTCTGCAGATATCC;
reverse primer 2: gtgacctctaatacaggacctAGCTCCCCCGATACCGTC;
forward primer 3: aggtcctgtattagaggtcacg;
reverse primer 3: ACCATCGGCAGCCATacctgatttaaatcatttattgttcaaagatg.
The primer sequences used for plasmid B are shown below:
forward primer 1: CCCCCTCGATCGAGGATGCCGGGGTTTTACGAGAT;
reverse primer 1: CCTCCCACCAGATCACCATC;
forward primer 2: aggtacatctacggattacgtACCTGCAAGGAACCCCTAGT;
reverse primer 2: gagttgggtaccggatccGTTCAACTGAAACGAATCAACCG;
forward primer 3: ggatccggtacccaactcca;
reverse primer 3: acgtaatccgtagatgtacctgg.
The PCR reaction conditions are shown in Table 2:
TABLE 2PCR reaction conditions
(2) The PCR amplified bands were detected by agarose gel electrophoresis, and the target fragment was recovered using a gel recovery kit.
(3) Multi-fragment ligation was performed using a seamless cloning kit.
The seamless cloning reaction system is shown in table 3:
TABLE 3 reaction system
| Reaction components | Volume (mu L) |
| 2×Assembly Mix | 5 |
| Linearized vector | 1 |
| Inserts | n |
| Nuclease-free Water | to 10 |
The reaction conditions were 50℃for 1h.
E.coli is transformed into the product after the reaction, the product is coated on a plate with kana resistance, colony PCR identification is carried out by picking clones every other day, positive clones are sent to Guangzhou Jinwei corporation for sequencing, and the required correct plasmid is selected.
Example 2: AAV production by double plasmid transfection
(1) Spreading 293T cells in about 5E+06 to 15cm cell culture dish with high sugar DMEM medium containing 10% new born calf serum and 1% Penicillin/Streptomycin at 37deg.C and 5% CO 2 Culturing in a cell culture box for about 48 hours, wherein the cell density is about 60-70% during transfection;
(2) The helper plasmids A and B (pRCHelper) constructed in example 1 and AAV plasmid pAAV. CAG. EGFP vector of fluorescent protein driven by CAG promoter were added to 0.5mL DMEM medium at 1. Mu.g/0.5. Mu.g, followed by adding PEI 3. Mu.L (1. Mu.g/. Mu.L), vortexing, standing at room temperature for 10min, and then adding to 25mL transfection medium, vortexing. The medium in the dish was aspirated, the transfection mixture medium was added, and the mixture was returned to the 37℃cell incubator (5% CO2 concentration) for cultivation.
(3) After 72 hours of culture, 25. Mu.L of cell lysate was added and the cells and supernatant were collected in a centrifuge bottle. AAV virus is purified by iodixanol gradient ultra-high speed centrifugation, the virus titer is measured to be proper titer between 1E+12GC/mL and 1E+13GC/mL, and the AAV virus is placed at the temperature of minus 80 ℃ for standby.
Example 3 replication-complete AAV (rcAAV) detection
(1) Laying 293T cells about 1E+07 in T75 cell culture flask, using high sugar DMEM medium containing 10% new born calf serum, 1% Penicillin/Streptomycin, at 37deg.C, 5% CO 2 The cells were cultured overnight in a cell incubator.
(2) The test pieces (AAV produced by transfection of helper plasmids A and B in example 2) were diluted to 1E+11vg/mL with DMEM, respectively, wtAAV2 work (ATCC-derived AAV2 standard: ATCC VR-680) was diluted to 1E+13IU/mL, and the Ad5 work (TCC-derived Ad5 standard: ATCC VR-1516) was diluted 4-fold.
(3) The cells were removed from the incubator, the medium was aspirated, rinsed once with PBS, and then the prepared working solution was added and incubated for 4h at 37 ℃. The loading system is shown in Table 4:
TABLE 4 sample addition System
(4) After 4h, 7mL of 2 Xcomplete medium was added per flask, and the mixture was placed at 37℃with 5% CO 2 The cell incubator continues to incubate overnight. Then the complete culture medium is used for liquid exchange, each bottle is 14mL, and the mixture is continuously put into 37 ℃ and 5 percent CO 2 The cells were cultured overnight in a cell incubator. After the culture, the cells were collected in 15mL centrifuge tubes, centrifuged at 3000g for 3min, the supernatant was aspirated, and 1mL PBS was added to resuspend the cells and transferred to 1.5mL centrifuge tubes, frozen at-80℃for 2h, and thawed at 37℃for 10min. The virus was inactivated in a water bath at 56℃for 60min after repeated freeze thawing. The whole inactivated supernatant was collected by centrifugation at 10000g for 5min in a 1.5mL centrifuge tube as a second round of infection samples.
(5) Laying 293T cells about 1E+07 in T75 cell culture flask, using high sugar DMEM medium containing 10% new born calf serum, 1% Penicillin/Streptomycin, at 37deg.C, 5% CO 2 The cells were cultured overnight in a cell incubator.
(6) The cells were removed from the incubator, the medium was aspirated, rinsed once with PBS, and then the prepared working solution was added and incubated for 4h at 37 ℃. The loading system is shown in Table 5:
TABLE 5 sample addition System
| Group of | DMEM(mL) | Ad5 work (mu L) | Inactivating the supernatant |
| Blank control | 6 | 4 | All inactivated supernatant |
| Test article | 6 | 4 | All inactivated supernatant |
| Reference control | 6 | 4 | All inactivated supernatant |
| Positive control | 6 | 4 | All inactivated supernatant |
(7) After 4h, 7mL of 2 Xcomplete medium was added per flask, and the mixture was placed at 37℃with 5% CO 2 The cell culture was continued for 44 hours in the incubator.
After the culture is finished, collecting cells into a 15mL centrifuge tube, centrifuging for 3min at 3000g, sucking the supernatant, adding 2mL PBS to resuspend the cells, equally dividing into 2 tubes and 1.5mL centrifuge tubes, centrifuging for 3min at 3000g, sucking the supernatant, and harvesting cell sediment.
(8) Cell DNA was extracted and DNA concentration was determined using Nanodrop.
(9) Fluorescent quantitative PCR detection of each group of samples, preparing 11+6n reaction systems, wherein n is the number of the samples to be detected, subpackaging 15 μl of reaction solution into PCR tubes, adding 5 μl of reaction template into each tube, and respectively obtaining blank control DNA, sample DNA, reference control DNA, positive control DNA and H 2 O (NTC), 3 wells each, the reaction solution system is shown in Table 6 below:
TABLE 6 reaction liquid System
10 After the sample addition is completed, a matched cover is covered, the mixture is mixed by slight shaking, and the mixture is rapidly centrifuged for 10 seconds and then is placed into a fluorescent quantitative PCR instrument. The reporter fluorophore is FAM and the probe is REP. The procedure for the set-up reaction is shown in table 7 below:
TABLE 7 reaction liquid System
rcAAV assay results are shown in Table 8:
TABLE 8 detection results
Comparison of rcAAV residues in the two purified plasmid packages shows that compared with the P5 promoter before Rep2, the rcAAV residues can be reduced by placing the promoter sequence after Rep/Cap protein coding sequence and E2A/E4/VA RNA and under the condition of adding DA' sequence.
Helper plasmids of a particular serotype (Rep 2Cap 9) are employed in the examples, and it will be understood by those skilled in the art that the invention is not limited to this particular serotype, but may be practiced with other serotypes of helper plasmids that are currently known and that may continue to be discovered in the future.
Finally, it should be noted that the above embodiments are only for illustrating the technical solution of the present invention and not for limiting the scope of the present invention, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solution of the present invention may be modified or substituted equally without departing from the spirit and scope of the technical solution of the present invention.
Claims (10)
1. A helper plasmid for reducing replication competent adeno-associated virus residues in a recombinant adeno-associated virus, wherein the helper plasmid comprises a backbone plasmid and a recombinant sequence; the recombinant sequence comprises:
rep, cap protein coding sequence, E2A, E, VA RNA sequence, at least one DA' sequence and at least one promoter sequence.
2. The helper plasmid of claim 1, wherein said E2A, E, VA RNA sequence is located downstream of said Rep, cap protein coding sequence; the DA' sequence is located downstream of the E2A, E, VA RNA sequence.
3. The helper plasmid according to claim 1, wherein said promoter sequence is located downstream of said E2A, E, VA RNA sequence.
4. A helper plasmid according to claim 3, wherein said promoter sequence is located downstream of said DA' sequence.
5. The helper plasmid according to claim 1, wherein said recombination sequences comprise, in order from 5 'to 3': the Rep, cap protein coding sequence, the E2A, E, VA RNA sequence, the DA' sequence and the promoter sequence.
6. A plasmid set for reducing replication competent adeno-associated virus residue in a recombinant adeno-associated virus comprising the helper plasmid of any of claims 1-5 and an AAV plasmid.
7. The plasmid of claim 6, wherein the AAV plasmid is an AAV plasmid of a CAG promoter-driven fluorescent protein.
8. An AAV production system for reducing rcAAV residues, comprising transforming a host cell with a helper plasmid according to any one of claims 1-5.
9. A method of producing AAV with reduced rcAAV residues, wherein the AAV is produced using the AAV production system of claim 8.
10. Use of the plasmid set of any one of claims 1-5, the plasmid set of claim 6 or 7, the AAV production system of claim 8 in AAV production.
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| TW202200790A (en) * | 2020-06-19 | 2022-01-01 | 日商Ｊｃｒ製藥股份有限公司 | Nucleic acid molecule for manufacturing recombinant AAV virion improving the production efficiency of a recombinant AAV particle |
| CN117043344A (en) * | 2021-03-29 | 2023-11-10 | 株式会社钟化 | Vector, method for producing linear covalently-closed DNA using the same, method for producing parvoviral vector, and parvoviral vector-producing cell |
| CN115197967A (en) * | 2021-04-08 | 2022-10-18 | 广州派真生物技术有限公司 | Helper plasmid for preparing recombinant adeno-associated virus and application thereof |
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