CN117683797A - Plasmid system for packaging recombinant adeno-associated virus and application thereof - Google Patents
Plasmid system for packaging recombinant adeno-associated virus and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of biology, and discloses a plasmid system for packaging recombinant adeno-associated virus and application thereof. The present invention provides a plasmid system for rAAV packaging, the plasmid system comprising: a plasmid containing two terminal inverted repeat sequences, an adenovirus auxiliary plasmid and a rAAV recombinant packaging plasmid; at least one plasmid of the plasmids containing two terminal inverted repeat sequences, adenovirus auxiliary plasmids and rAAV recombinant packaging plasmids is inserted with a site sequence recognized by recombinase and/or a recombinase sequence of the sequence. The invention can effectively reduce the rate of non-rAAV related sequence being erroneously packaged into AAV viral capsids in the rAAV production process, and improve the purity of rAAV products, thereby greatly improving the drug property of the rAAV products.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a plasmid system for packaging recombinant adeno-associated virus and application thereof.
Background
The recombinant adeno-associated virus (rAAV) is used as a gene therapy vector, has the characteristics of high safety, strong transduction capability and the like, and currently, several gene therapy medicaments based on rAAV are marketed, and a plurality of gene therapy clinical tests based on rAAV are being developed. The rAAV production system mainly comprises a plasmid transfection production system based on HEK293 cells, a production system based on insect cells and baculovirus and a production system based on HEK293 cells and adenovirus. The plasmid transfection production system based on HEK293 cells can be used for adherent culture and suspension culture, and also comprises a plasmid or multi-plasmid transfection mode, is simple and quick, and is a widely adopted rAAV production system. Taking the classical three plasmid transfection production system as an example, this production system involves co-transfecting HEK293 cells with three plasmids: a helper plasmid pHelper, providing nucleic acid sequences capable of expressing adenovirus component proteins; a helper plasmid pRep-Cap providing nucleic acid sequences capable of expressing the Rep and Cap proteins of AAV, wherein the Rep proteins are responsible for replication of AAV genome and assist assembly of AAV genome particles, and the Cap proteins constitute AAV capsids; a plasmid comprising the sequence of interest, which may be abbreviated as pGOI, has a 5'ITR and 3' ITR sequence in the native AAV genome upstream and downstream of the sequence of interest.
In the rAAV production process, plasmid backbone nucleic acid sequences (ori nucleic acid sequences related to plasmid replication, resistance gene nucleic acid sequences related to plasmid screening and the like) can be erroneously packaged into AAV viral capsids by ITR/ITR-like mediation or in a random manner, so that the purity and titer of rAAV viruses are reduced, unpredictable side effects are brought, and risks are brought to in-vivo gene therapy.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide a plasmid system for packaging recombinant adeno-associated virus 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 invention provides a plasmid system for rAAV packaging, the plasmid system comprising: a plasmid containing two terminal inverted repeat sequences, an adenovirus auxiliary plasmid and a rAAV recombinant packaging plasmid; at least one plasmid of the plasmids containing two terminal inverted repeat sequences, adenovirus auxiliary plasmids and rAAV recombinant packaging plasmids is inserted with a site sequence recognized by recombinase and/or a recombinase sequence of the sequence.
The plasmid system of the invention adopts a recombinase system, can effectively reduce the rate of non-rAAV related sequence error package entering AAV viral capsids in the rAAV production process, can reduce plasmid skeleton nucleic acid sequence residues in rAAV, and improves the purity of rAAV products, thereby greatly improving the drug-forming property of the rAAV products.
As a preferred embodiment of the plasmid system of the present invention, the recombinase comprises a tyrosine recombinase or a serine recombinase. As a preferred embodiment of the plasmid system of the present invention, the tyrosine recombinase comprises at least one recombinase of any one of Cre-lox, FLP-FRT, dre-rox, R-RS; the serine recombinases include at least one recombinase in any one of CinH-RS2, parA-MRS, beta-six, gamma delta-res, phiC31, TP901-1, R4, bxb 1.
The site-specific recombinase can specifically recognize and mediate recombination between specific sites, so that the mutation such as gene knockout, insertion, turnover, ectopic and the like of the specific sites is realized. Cre (cyclization recombinase) is a recombinase from a P1 phage whose C-terminal end comprises catalytic activity capable of catalyzing recombination of a DNA molecule containing a loxP (locus of X-over in P1) site. The loxP site consists of two 13bp reverse palindromic sequences and 8bp intermediate spacer sequences, wherein the reverse palindromic sequences are Cre enzyme recognition and binding sites. Two identical-direction loxP site sequences are added in the region, close to the target DNA fragment, of the annular plasmid skeleton, and after the loxP sites are identified by Cre enzyme, the plasmids are recombined to generate two annular miniplasmids; the target DNA fragment is separated from the vector backbone, thereby reducing the probability of ITR/ITR-like mediated packaging of the vector backbone.
As a preferred embodiment of the plasmid system of the present invention, the plasmid system comprises pGOI, pRep-Cap and pHelper, into which the site sequences recognized by the recombinant enzyme are inserted. Preferably, the plasmid system comprises GOI, pRep-Cap and pHelper with inserted loxP.
As a preferred embodiment of the plasmid system of the present invention, the plasmid system comprises pGOI, pRep-Cap, pHelper, and pAAVS1, into which the site sequence recognized by the recombinase is inserted. Preferably, the plasmid system comprises pGOI, pRep-Cap, pHelper with inserted loxP and pAAVS1 with inserted Cre.
As a preferred embodiment of the plasmid system of the present invention, the plasmid system comprises pGOI, pRep-Cap, into which the site sequence recognized by the recombinase is inserted, and pHelper-into which the recombinase sequence is inserted. Preferably, the plasmid system comprises pGOI, pRep-Cap with inserted loxP and pHelper with inserted Cre.
The plasmid pGOI comprises sequences from different serotypes, ITRs and their optimizations. The plasmid pRep-Cap comprises nucleic acid sequences from different serotypes for expressing Rep proteins and for expressing Cap proteins and optimized nucleic acid sequences thereof. The plasmid pHelper includes nucleic acid sequences from different helper virus plasmids, expressing adenovirus original proteins, and optimized sequences thereof.
In a second aspect, the invention provides a cell for rAAV production that stably expresses a recombinase; said cell comprising said plasmid system; the plasmid system includes pGOI, pRep-Cap and pHelper inserted with a sequence recognizing the site recognized by the recombinase.
Preferably, the recombinase is Cre recombinase; the plasmid system includes pGOI, pRep-Cap and pHelper with inserted loxP.
In a third aspect, the invention provides a method for reducing residue of a plasmid backbone nucleic acid sequence in a rAAV, and transfecting packaging cells with the plasmid system.
As a preferred embodiment of the method of the invention, the packaging cells include at least one of HEK293, HEK293T, HEK293F, HEK293A, hela, vero, CHO, as well as other cells for rAAV production.
In a fourth aspect, the invention applies the plasmid system, the cell, in rAAV production.
Compared with the prior art, the invention has the beneficial effects that:
when the plasmid system adopts a recombinase system to carry out rAAV production, the proportion of the plasmid skeleton nucleic acid sequence (ori nucleic acid sequence related to plasmid replication, resistance gene nucleic acid sequence related to plasmid screening and the like) which is erroneously packaged into AAV viral capsids is greatly reduced, the residue of the plasmid skeleton nucleic acid sequence in the rAAV can be reduced, the purity of the rAAV product is effectively improved, and the drug-forming property of the rAAV product is greatly improved.
Drawings
FIG. 1 is an in vitro rAAV package after plasmid digestion with Cre recombinase;
in fig. 1, fig. a-c: the Cre recombinase is added in vitro to digest the plasmid pGOI-loxP, and the digested plasmids pGOI-loxP, pRep-Cap and pHelper are jointly used for rAAV production (+cre group), pGOI, pRep-Cap and pHelper are used for rAAV production (CT group), and the WPRE titer (a), ori titer (b) and anti-packing rate (c) of rAAV package are added in vitro. Graph d-f: WPRE titer fold (d), ori titer fold (e), anti-pack rate fold (f) for the +cre group compared to the control CT group.
FIG. 2 is a pAAVS1 plasmid map.
FIG. 3 is a diagram of the addition of pAAVS1/Cre plasmid for rAAV packaging;
in fig. 3, fig. a-c: pGOI-loxP, pRep-Cap, pHelper, pAAVS1/Cre four plasmids were used for rAAV production (+pCre group), pGOI-loxP, pRep-Cap, pHelper, pAAVS four plasmids were used for rAAV production (EV group), WPRE titer (a), ori titer (b), and turn-up rate (c) of rAAV packaging. Graph d-f: WPRE titer fold (d), ori titer fold (e), anti-pack rate fold (f) for +pcre group compared to control EV group.
FIG. 4 is a drawing of rAAV packaging after addition of Cre recombinase expression cassette nucleic acid sequences on plasmid pHelper; in fig. 4, fig. a-c: pGOI-loxP, pRep-Cap, pHelper/Cre three plasmids were used for rAAV production (+cre group), pGOI-loxP, pRep-Cap, pHelper three plasmids were used for rAAV production (EV group), WPRE titer (a), ori titer (b), and turn-up rate (c) of rAAV packaging. Graph d-f: WPRE titer fold (d), ori titer fold (e), anti-packet rate fold (f) for the +cre group compared to the control EV group.
FIG. 5 is a diagram of rAAV packaging using a cell Cre-KI stably expressing Cre recombinase;
in fig. 5, fig. a-c: pGOI-loxP, pRep-Cap, pHelper three plasmids transfect cells Cre-KI stably expressing Cre recombinase for rAAV production (Cre-KI group), pGOI, pRep-Cap, pHelper three plasmids transfect cells 293T for rAAV production (293T group), WPRE titer (a), ori titer (b), anti-pack rate (c) of rAAV packaging. Graph d-f: WPRE titer fold (d), ori titer fold (e), anti-packet rate fold (f) for Cre-KI group compared to control 293T group.
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. The WPRE titer is the copy number of the target DNA fragment; the ori titer is the copy number of the vector backbone; the turn-up rate is the ratio of the number of copies of the backbone of the vector to the number of copies of the DNA fragment of interest.
Example 1: rAAV packaging after in vitro Cre recombinase digestion of plasmid
(1) Vector construction and plasmid extraction
A pair of chimeric primers (loxP-VB-F: tcacaccgcatacgtcaaagcaaccATAACTTCGTATAGCATACATTATACGAAGTTATATagtacgcgccctgtagcg; loxP-VB-R: gaaccgtaaaaaggccgcgttgctgATAACTTCGTATAATGTATGCTATACGAAGTTATgcgtttttccataggctccg) containing loxP site and used for amplifying the vector backbone was designed based on the sequence information (ATAACTTCGTATAGCATACATTATACGAAGTTAT) of the loxP site of Cre recombinase and pGOI plasmid information (Addgene: 27970), and primers (GOI-F: cagcaacgcggcctttttacggttc; GOI-R: ggttgctttgacgtatgcggtgtga) used for amplifying the GOI fragment were designed.
pGOI plasmid was then amplified using loxP-VB-F/loxP-VB-R, GOI-F/GOI-R, respectively. The amplification system is as follows: mu.L of 2X KOD OnePCR Master Mix (ToYoBo, # KMM-101), 1.0. Mu.L of Forward primer (10. Mu.M), 1.0. Mu.L of Reverse primer (10. Mu.M), 20ng of DNA template were supplemented to 50. Mu.L with sterilized water. The amplification conditions were: pre-denaturation at 95℃for 3min; denaturation at 98℃for 10sec, annealing at 58℃for 5sec, extension at 68℃for 10sec/kb (32 cycles); extending at 68℃for 3min. After completion of amplification, the target DNA was purified by electrophoresis on a 1.0% agarose gel, and the gel containing the target DNA fragment was excised and recovered by "HiPure Gel Pure DNA Micro Kits" (Megan, #D21111-03) to obtain GOI fragment and VB fragment.
The two DNA fragments obtained by purification were ligated and recovered using the Gibson Assembly method. The reaction system is as follows: mu.L of 3/4X Gibson Assembly Master Mix, 100ng of vector backbone fragment, 100ng of GOI fragment were made up to 20. Mu.L with sterile water. After mixing, the mixture was incubated at 50℃for 30min. The ligated DNA was transformed into XL10-Gold Chemically Competent Cell (Shanghai Wei, #DL 1050), screened with ampicillin-containing LB medium, and then the monoclonal was selected and sent to the Optimazaceae for sequencing. Finally, plasmids were obtained in which loxP sites were added to the 5 'end of the 5' ITR and the 3 'end of the 3' ITR of pGOI plasmid, respectively, and the plasmids were named pGOI-loxP.
High-concentration and high-purity pGOI, pGOI-loxP, pRep-Cap and pHelper plasmids are extracted. The pGOI-loxP plasmid was recombined by adding Cre recombinase (New England Biolabs, #M0298S) in vitro. The recombination reaction system is as follows: mu.L of 10X Cre Recombinase Buffer, 4.0. Mu. L Cre Recombinase, 1.0. Mu.g pGOI-loxP plasmid was made up to 50. Mu.L with sterile water. The enzyme digestion conditions are as follows: incubation was performed at 37℃for 50min and at 70℃for 10min. After completion of the reaction, purification was performed using "HiPure Gel Pure DNA Micro Kits".
(2) rAAV packaging and titer determination
The day before transfection, 293T cells were counted after suspension in DMEM medium at 6X 10 6 The individual cells/dishes were spread evenly on a 10-cm cell culture dish and placed at 37℃in 5% CO 2 The conditions were cultured to a cell confluence of about 80%.
Setting CT group plasmid: pGOI, pRep-Cap, pHelper; +cre group plasmid: pGOI-loxP, pRep-Cap and pHelper after Cre recombinase recombination.
On the day of transfection, fresh DMEM medium was changed. pGOI, pRep-Cap, pHelper (CT group), added with Cre recombinase, recombined pGOI-loxP, pRep-Cap, pHelper (+cre group) were mixed in a ratio of 2.0. Mu.g:2.5. Mu.g, 3 replicates per group. 0.5mL of DMEM was mixed with the plasmid DNA, 0.5mL of DMEM was mixed with 8.4. Mu.L of PEIpro, and the PEIpro mixture was added to the DNA mixture and mixed well, and allowed to stand at room temperature for 15min. Adding the DNA-PEIpro mixed solution into the grown cells, gently mixing, and then placing the mixture into an incubator for culture.
72h after transfection, cells were lysed by addition of 1% chloroform and collected into 50mL centrifuge tubes. The rAAV was purified by concentration using PEG8000 after digestion with nuclease. The purified rAAV was digested with DNaseI and then subjected to qRT-PCR to determine WPRE titer and ori titer, and a standard curve was prepared from the gradient diluted plasmid standard. WPRE and ori titers of rAAV samples were calculated from the standard curve.
pGOI-loxP, pRep-Cap and pHelper plasmids digested by Cre recombinase are added in vitro and are jointly used for rAAV production (+cre group), compared with the case that pGOI, pRep-Cap and pHelper three plasmids are used for rAAV production (CT group), the rate of the plasmid skeleton reverse package entering rAAV capsid (reverse package rate) is reduced to 0.4 times of that of the control CT group.
Example 2: rAAV packaging by adding pAAVS1/Cre plasmid
(1) Vector construction and plasmid extraction
The gene synthesis was performed after obtaining Cre recombinase (GenBank accession: AY 056050) sequence information from the GeneBank database query. Primers designed for amplification of the Cre fragment (Cre-F: ttacaaagacgatgacgataagATGTCCAATCTCCTGACTGTTCA; cre-R: agcgagctctaggaattcttaTCAGTCACCATCTTCGAGCAGTC) and for amplification of the pAAVS1 plasmid (the plasmid map of which is shown in FIG. 2 and the nucleotide sequence of which is shown in SEQ ID No. 1) were used as primers for the backbone (VB-F: tgataagaattcctagagctcgct; VB-R: catcttatcgtcatcgtctttgtaa).
The Cre gene fragment was amplified using Cre-F/Cre-R and the pAAVS1 plasmid was amplified using VB-F/VB-R. The amplification system is as follows: mu.L of 2X KOD OnePCR Master Mix (TOYOBO, # KMM-101), 1.0. Mu.L of Forward primer (10. Mu.M), 1.0. Mu.L of Reverse primer (10. Mu.M), 20ng of DNA template were supplemented to 50. Mu.L with sterilized water. The amplification conditions were: pre-denaturation at 95℃for 3min; denaturation at 98℃for 10sec, annealing at 58℃for 5sec, extension at 68℃for 10sec/kb (32 cycles); extending at 68℃for 3min. After amplification, the gel containing the target DNA fragment was excised by electrophoresis on a 1.0% agarose gel, and the target DNA was recovered and purified by "HiPure Gel Pure DNA Micro Kits" (Megan, #D21111-03) to obtain the Cre gene fragment and pAAVS1-VB fragment.
The two DNA fragments obtained by purification were ligated and recovered using the Gibson Assembly method. The reaction system is as follows: 7.5. Mu.L of 3/4X Gibson Assembly Master Mix, 100ng of Cre gene fragment, 100ng of pAAVS1-VB fragment were made up to 10. Mu.L with sterile water. After mixing, the mixture was incubated at 50℃for 30min. The ligated DNA was transformed into XL10-Gold Chemically Competent Cell, screened with ampicillin-containing LB medium, and then the monoclonal was selected and sent to the qinghao for sequencing. Finally, a plasmid was obtained in which the Cre coding frame sequence was inserted into the pAAVS1 plasmid, and the plasmid was named pAAVS1/Cre.
High-concentration and high-purity pGOI-loxP, pRep-Cap, pHelper, pAAVS1 and pAAVS1/Cre plasmids are extracted.
(2) rAAV packaging and titer determination
The day before transfection, 293T cells were counted after suspension in DMEM medium at 6X 10 6 The individual cells/dishes were spread evenly on a 10-cm cell culture dish and placed at 37℃in 5% CO 2 The conditions were cultured to a cell confluence of about 80%.
Setting EV group plasmids: pGOI-loxP, pRep-Cap, pHelper, pAAVS1; +cre group plasmid: pGOI-loxP, pRep-Cap, pHelper, pAAVS1/Cre.
On the day of transfection, fresh DMEM medium was changed. pGOI-loxP, pRep-Cap, pHelper, pAAVS1 (EV group), pGOI-loxP, pRep-Cap, pHelper, pAAVS1/Cre (+cre group) were mixed in a ratio of 2.0. Mu.g:2.5. Mu.g:0.5. Mu.g, respectively, and 3 replicates were performed in each group. 0.5mL of DMEM was used to mix the plasmid DNA, 0.5mL of DMEM was used to mix with 9.0. Mu.L of PEIpro, and the PEIpro mixture was added to the DNA mixture and mixed well, and allowed to stand at room temperature for 15min. Adding the DNA-PEIpro mixed solution into the grown cells, gently mixing, and then placing the mixture into an incubator for culture.
72h after transfection, cells were lysed by addition of 1% chloroform and collected into 50mL centrifuge tubes. The rAAV was purified by concentration using PEG8000 after digestion with nuclease. After purified rAAV was digested with DNaseI, WPRE titer and ori titer were determined by qRT-PCR, and standard curves were made with gradient diluted plasmid standards. WPRE and ori titers of rAAV samples were calculated from the standard curve.
The ratio of the reverse inclusion of the plasmid backbone into the rAAV capsid (reverse inclusion rate) was reduced to 0.05-fold that of the control EV group by the use of pGOI-loxP, pRep-Cap, pHelper, pAAVS1/Cre four plasmids for rAAV production (+cre group) versus pGOI-loxP, pRep-Cap, pHelper, pAAVS four plasmids for rAAV production (EV group) and +cre group production of rAAV.
Example 3: rAAV packaging after adding Cre recombinase expression cassette nucleic acid sequence to plasmid pHelper
(1) Vector construction and plasmid extraction
Primers were designed to amplify fragments containing the Cre expression cassette (pHel-Cre-F: acgtcgacgtttaaaccaCGTTACATAACTTACGGTAAATGG; pHel-Cre-R: gcctttgagtgagctgatcatatgTCCCCAGCATGCCTGCTATTCTC).
pAAVS1/Cre was amplified using pHel-Cre-F/pHel-Cre-R. The amplification system is as follows: mu.L of 2X KOD OnePCR Master Mix (ToYoBo, # KMM-101), 1.0. Mu.L of Forward primer (10. Mu.M), 1.0. Mu.L of Reverse primer (10. Mu.M), 20ng of DNA template were supplemented to 50. Mu.L with sterilized water. The amplification conditions were: pre-denaturation at 95℃for 3min; denaturation at 98℃for 10sec, annealing at 58℃for 5sec, extension at 68℃for 10sec/kb (32 cycles); extending at 68℃for 3min. After completion of amplification, the gel containing the target DNA fragment was excised by electrophoresis on a 1.0% agarose gel, and the target DNA was recovered and purified by using "HiPure Gel Pure DNA Micro Kits" (Megan, D21111-03) to obtain a TelN expression cassette fragment.
The pHelper plasmid (GenBank: AF 369965) was digested with NdeI (ThemoFisher Scientific, # FD 0583). The enzyme digestion system is as follows: mu.L of 10X FastDigest buffer, 2.0. Mu.L of NdeI enzyme, 2.0. Mu.g of pHelper plasmid were made up to 50. Mu.L with sterile water. The enzyme digestion conditions are as follows: incubation was performed at 37℃for 30min and at 65℃for 5min. After cleavage, purification was performed using "HiPure Gel Pure DNA Micro Kits".
The Cre expression cassette fragment was ligated to the digested pHelper using the Gibson Assembly method. The reaction system is as follows: mu.L of 3/4X Gibson Assembly Master Mix, 20ng of the digested pHelper, 10ng of Cre expression cassette fragment were made up to 20. Mu.L with sterile water. After mixing, the mixture was incubated at 50℃for 30min. The ligated DNA was transformed into XL10-Gold Chemically Competent Cell, screened with ampicillin-containing LB medium, and then the monoclonal was selected and sent to the qinghao for sequencing. Finally, a plasmid was obtained in which the Cre expression cassette was inserted into the pHelper plasmid, and the plasmid was designated pHelper/Cre.
High-concentration and high-purity pGOI-loxP and pRep-Cap, pHelper, pHelper/Cre plasmids are extracted.
(2) rAAV packaging and titer determination
The day before transfection, 293T cells were counted after suspension in DMEM medium at 6X 10 6 The individual cells/dishes were spread evenly on a 10-cm cell culture dish and placed at 37℃in 5% CO 2 The conditions were cultured to a cell confluence of about 80%.
Setting EV group plasmids: pGOI-loxP, pRep-Cap, pHelper; +cre group plasmid: pGOI-loxP, pRep-Cap, pHelper/Cre.
On the day of transfection, fresh DMEM medium was changed. pGOI-loxP, pRep-Cap, pHelper (EV group), pGOI-loxP, pRep-Cap, pHelper/Cre (+cre group) were mixed in a ratio of 2.0. Mu.g:2.5. Mu.g, 3 replicates per group, respectively. 0.5mL of DMEM was mixed with the plasmid DNA, 0.5mL of DMEM was mixed with 8.4. Mu.L of PEIpro, and the PEIpro mixture was added to the DNA mixture, and the mixture was allowed to stand at room temperature for 15 minutes. Adding the DNA-PEIpro mixed solution into the grown cells, gently mixing, and then placing the mixture into an incubator for culture.
72h after transfection, cells were lysed by addition of 1% chloroform and collected into 50mL centrifuge tubes. The rAAV was purified by concentration using PEG8000 after digestion with nuclease. The purified rAAV was digested with DNaseI and then subjected to qRT-PCR to determine WPRE titer and ori titer, and a standard curve was prepared from the gradient diluted plasmid standard. WPRE and ori titers of rAAV samples were calculated from the standard curve.
The ratio of the plasmid backbone to the rAAV capsid (turn-up rate) was reduced to 0.08 times that of the control EV group when pGOI-loxP, pRep-Cap, pHelper/Cre three plasmids were used for rAAV production (+cre group) compared to when pGOI-loxP, pRep-Cap, pHelper three plasmids were used for rAAV production (EV group), +cre group production.
Example 4: rAAV packaging Using Cre-KI cells
(1) Vector construction and plasmid extraction
The sgAAVS1 primer was designed targeting the AAVS1 site (sgAAVS 1-F: CACCtaaggaatctgcctaacagg; sgAAVS1-R: AACcctgttaggcagattcctta). Preparing a primer annealing system: 2.0. Mu.L Forward primer (10. Mu.M), 2.0. Mu.L Reverse primer (10. Mu.M) were supplemented to 20. Mu.L with sterile water. Incubate at 98℃for 5min and cool naturally to room temperature.
The pX330 plasmid (Addgene: 42230) was digested with BpiI (ThemoFisher Scientific, # FD 1014). The enzyme digestion system is as follows: mu.L of 10X FastDigest buffer, 2.0. Mu.L of BpiI enzyme, 2.0. Mu.g of pX330 plasmid were supplemented to 50. Mu.L with sterile water. The enzyme digestion conditions are as follows: incubation was performed at 37℃for 30min and at 65℃for 5min. After cleavage, purification was performed using "HiPure Gel Pure DNA Micro Kits".
Preparing a DNA ligation reaction system: 1.0. Mu.L of 10 XT 4 DNA Ligase Buffer, 1.0. Mu. L T4 DNA Ligase (TaKaRa, # 6022), 0.5. Mu.L of pX330 cleavage product, 2.0. Mu.L of annealed primer, and 10. Mu.L of sterilized water were used. Mixing, and incubating at 16deg.C for about 2h. The ligated DNA was transformed into XL10-Gold Chemically Competent Cell, screened with ampicillin-containing LB medium, and then the monoclonal was selected and sent to the qinghao for sequencing. Finally, the insertion of the sgAAVS1 target site on the pX330 plasmid was obtained and designated as pX330/sgAAVS1.
High-concentration and high-purity pGOI, pGOI-loxP, pRep-Cap, pHelper, pAAVS1/Cre, pX330/sgAAVS1 plasmids were extracted.
(2) Cell transfection and screening
The day before transfection, 293T cells were suspended and counted using DMEM medium at 1X 10 6 The wells were plated uniformly in 6-well cell culture plates. On the day of transfection, pAAVS1/Cre plasmid was mixed with pX330/sgAAVS1 plasmid at 2.0. Mu.g:1.0. Mu.g plasmid, and 6.0. Mu. L P3000 reagent was addedAnd 125. Mu.L Opti-MEMI. Into another centrifuge tube, 3.75. Mu.L Lipofectamin 3000, 125. Mu.L Opti-MEMI were added and mixed well. Then adding the DNA-P3000 mixed solution into the Lipofectamin 3000 mixed solution, uniformly mixing, and standing at room temperature for 15min. The transfection mixture was added to a 6-well cell culture plate, gently mixed, and placed at 37℃in 5% CO 2 Culturing for about 48-72 h.
Transfected cells were collected, suspended and counted using DMEM. Then at 1X 10 6 Inoculating into 6-well cell culture plate, adding 1.0 μg/mL Puro, standing at 37deg.C and 5% CO 2 The conditioned medium is screened for about 1-2 weeks. The monoclonal is then isolated in 96-well cell culture plates and expanded, with the cells labeled Cre-KI.
(3) rAAV packaging and titer determination
The day before transfection, 293T, cre-KI cells were counted after suspension in DMEM medium at 6X 10 6 The individual cells/dishes were spread evenly on a 10-cm cell culture dish and placed at 37℃in 5% CO 2 The conditions were cultured to a cell confluence of about 80%.
On the day of transfection, fresh DMEM medium was changed. pGOI, pRep-Cap, pHelper (293T group), pGOI-loxP, pRep-Cap, pHelper (Cre-KI group) were mixed in a ratio of 2.0. Mu.g:2.5. Mu.g, 3 replicates per group, respectively. 0.5mL of DMEM was mixed with the plasmid DNA, 0.5mL of DMEM was mixed with 8.4. Mu.L of PEIpro, and the PEIpro mixture was added to the DNA mixture and mixed well, and allowed to stand at room temperature for 15min. Adding the DNA-PEIpro mixed solution into the grown cells, gently mixing, and then placing the mixture into an incubator for culture.
72h after transfection, cells were lysed by addition of 1% chloroform and collected into 50mL centrifuge tubes. The rAAV was purified by concentration using PEG8000 after digestion with nuclease. The purified rAAV was digested with DNaseI and then subjected to qRT-PCR to determine WPRE titer and ori titer, and a standard curve was prepared from the gradient diluted plasmid standard. According to the standard curve, the WPRE titer and ori titer of the rAAV sample are calculated respectively.
pGOI-loxP, pRep-Cap, pHelper three plasmids were transfected into cells Cre-KI stably expressing Cre recombinase for rAAV production (Cre-KI panel). pGOI, pRep-Cap, pHelper three plasmids transfected cells 293T for rAAV production (293T panel). The rAAV produced by Cre-KI group had a reduced rate of reverse inclusion of the plasmid backbone into the rAAV capsid (reverse inclusion rate) to 0.12 fold that of the control 293T group.
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 plasmid system for packaging of recombinant adeno-associated virus, the plasmid system comprising: a plasmid containing two terminal inverted repeat sequences, an adenovirus auxiliary plasmid and a rAAV recombinant packaging plasmid; at least one plasmid of the plasmids containing two terminal inverted repeat sequences, adenovirus auxiliary plasmids and rAAV recombinant packaging plasmids is inserted with a site sequence recognized by recombinase and/or a recombinase sequence of the sequence.
2. The plasmid system of claim 1 wherein the recombinase comprises a tyrosine recombinase or a serine recombinase.
3. The plasmid system of claim 2 wherein the tyrosine recombinase comprises at least one recombinase of any one of Cre-lox, FLP-FRT, dre-rox, R-RS; the serine recombinases include at least one recombinase in any one of CinH-RS2, parA-MRS, beta-six, gamma delta-res, phiC31, TP901-1, R4, bxb 1.
4. The plasmid system of claim 1 or 2, comprising pGOI, pRep-Cap and pHelper inserted with a site sequence recognized by the recombinase.
5. The plasmid system of claim 1 or 2, comprising pGOI, pRep-Cap, pHelper inserted with a site sequence recognized by the recombinase and pAAVS1 inserted with the recombinase sequence.
6. The plasmid system of claim 1 or 2, comprising pGOI, pRep-Cap inserted with a site sequence recognized by the recombinase and pHelper inserted with the recombinase sequence.
7. A cell for rAAV production, wherein the cell stably expresses a recombinase; the cell comprising the plasmid system of claim 4.
8. A method for reducing residue of a plasmid backbone nucleic acid sequence in a rAAV, wherein a packaging cell is transfected with the plasmid system of any one of claims 1-6.
9. The method of claim 8, wherein the packaging cells comprise at least one of HEK293, HEK293T, HEK293F, HEK293A, hela, vero, CHO.
10. Use of the plasmid system of any one of claims 1 to 6, the cell of claim 7 in rAAV production.
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