CN117683797B

CN117683797B - Plasmid system for packaging recombinant adeno-associated virus and application thereof

Info

Publication number: CN117683797B
Application number: CN202311646675.9A
Authority: CN
Inventors: 李华鹏; 韩靖鸾; 卜晔; 陈欢; 代志勇
Original assignee: Guangzhou Packgene Biotech Co ltd
Current assignee: Guangzhou Packgene Biotech Co ltd
Priority date: 2023-12-04
Filing date: 2023-12-04
Publication date: 2024-07-05
Anticipated expiration: 2043-12-04
Also published as: CN117683797A; WO2025118911A1

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

Plasmid system for packaging recombinant adeno-associated virus and application thereof

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 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, into which the site sequence recognized by the recombinase is inserted, and pAAVS.sup.1 into which the recombinase sequence is inserted. Preferably, the plasmid system comprises pGOI, pRep-Cap, pHelper with inserted loxP and pAAVS.sup.1 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 includes sequences from different serotypes, ITR sequences, and optimized sequences thereof. 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 plasmid pGOI-loxP, pRep-Cap and pHelper plasmids are used for rAAV production (+ Cre group), pGOI, pRep-Cap and pHelper three plasmids are used for rAAV production (CT group), and WPRE titer (a), ori titer (b) and anti-packing rate (c) of rAAV package. 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 pAAVS.sup.1 plasmid map.

FIG. 3 is a diagram of rAAV packaging with the addition of pAAVS/Cre plasmid;

In fig. 3, fig. a-c: pGOI-loxP, pRep-Cap, pHelper, pAAVS1/Cre four plasmids were used for rAAV production (+ pCre groups), pGOI-loxP, pRep-Cap, pHelper, pAAVS1 four plasmids were used for rAAV production (EV groups), 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 + pCre group compared to the 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 of loxP at Cre recombinase recognition site (ATAACTTCGTATAGCATACATTATACGAAGTTAT) and pGOI plasmid information (Addgene: 27970) (GOI-F: CAGCAACGCGGCCTTTTTACGGTTC; GOI-R: GGTTGCTTTGACGTATGCGGTGTGA).

The pGOI plasmid was then amplified using loxP-VB-F/loxP-VB-R, GOI-F/GOI-R, respectively. The amplification system was ：25μL 2×KOD OnePCR Master Mix(ToYoBo,#KMM-101)、1.0μL Forward primer(10μM)、1.0μL Reverse primer(10μM)、20ng DNA templates, and sterilized water was used to make up to 50. Mu.L. 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), thereby obtaining a GOI fragment and a 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 the vector backbone fragment, 100ng of the 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 selected and sent to the Optimazaceae for sequencing. Finally, a plasmid was 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 resulting plasmid was designated pGOI-loxP.

High-concentration and high-purity pGOI, pGOI-loxP, pRep-Cap and pHelper plasmids are extracted. Cre recombinase (NEW ENGLAND Biolabs, #M0298S) was added in vitro and the pGOI-loxP plasmid was recombined. The recombination reaction system is as follows: 5.0. Mu.L of 10X Cre Recombinase Buffer, 4.0. Mu. L Cre Recombinase, 1.0. Mu. g pGOI-loxP plasmid was filled 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, plated on 10-cm cell culture dishes at 6X 10 ⁶ cells/dish and incubated at 37℃under 5% CO ₂ to a cell confluency 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), pGOI-loxP, pRep-Cap, pHelper (+cre group) after recombination with Cre recombinase were mixed in a ratio of 2.0. Mu.g:2.5. Mu.g, 3 replicates per group, respectively. 0.5mL of DMEM was used to mix the plasmid DNA, 0.5mL of DMEM was used to mix the plasmid DNA with 8.4 mu L PEIpro, and then 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.

The pGOI-loxP and pRep-Cap and pHelper plasmids digested by Cre recombinase are added in vitro and are jointly used for rAAV production (+cre group), and compared with the rAAV production (CT group) of pGOI, pRep-Cap and pHelper three plasmids, the rate of the plasmid skeleton reverse-packing into rAAV capsids (reverse-packing 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 (Cre-F：ttacaaagacgatgacgataagATGTCCAATCTCCTGACTGTTCA;Cre-R：agcgagctctaggaattcttaTCAGTCACCATCTTCGAGCAGTC) for amplifying the Cre fragment and primers for amplifying the pAAVS 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 designed for the backbone (VB-F: TGATAAGAATTCCTAGAGCTCGCT; VB-R: CATCTTATCGTCATCGTCTTTGTAA).

The Cre gene fragment was amplified using Cre-F/Cre-R, and the pAAVS plasmid was amplified using VB-F/VB-R. The amplification system was ：25μL 2×KOD OnePCR Master Mix(TOYOBO,#KMM-101)、1.0μL Forward primer(10μM)、1.0μL Reverse primer(10μM)、20ng DNA templates, and sterilized water was used to make up to 50. Mu.L. 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), thereby obtaining the Cre gene fragment and pAAVS-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 the 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 the monoclonal was selected and sent to the qinghao for sequencing. Finally, a plasmid was obtained in which Cre coding frame sequence was inserted into pAAVS's 1 plasmid, and the plasmid was designated pAAVS/Cre.

High concentration, high purity pGOI-loxP, pRep-Cap, pHelper, pAAVS1, pAAVS1/Cre plasmids were extracted.

(2) RAAV packaging and titer determination

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 of each group were performed. 0.5mL of DMEM was used to mix the plasmid DNA, 0.5mL of DMEM was used to mix the plasmid DNA with 9.0 mu L PEIpro, and then 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 pGOI-loxP, pRep-Cap, pHelper, pAAVS1/Cre four plasmids were used for rAAV production (+cre group), and the rate of reverse-packing of the plasmid backbone into rAAV capsids (reverse-packing rate) was reduced to 0.05-fold that of the control EV group, compared to pGOI-loxP, pRep-Cap, pHelper, pAAVS four plasmids were used for rAAV production (EV group), and rAAV produced by +cre group.

Example 3: rAAV packaging after adding Cre recombinase expression cassette nucleic acid sequence to plasmid pHelper

(1) Vector construction and plasmid extraction

Primers designed for amplification of a fragment comprising 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 was ：25μL2×KOD OnePCR Master Mix(ToYoBo,#KMM-101)、1.0μL Forward primer(10μM)、1.0μL Reverse primer(10μM)、20ng DNA templates, and sterilized water was used to make up to 50. Mu.L. 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 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 XL 10-Gold Chemically Competent Cell, screened with ampicillin-containing LB medium, and 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, pRep-Cap, pHelper, pHelper/Cre plasmid was extracted.

(2) RAAV packaging and titer determination

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 used to mix the plasmid DNA, 0.5mL of DMEM was used to mix the plasmid DNA with 8.4 mu L PEIpro, and then PEIpro mixture was added to the DNA mixture 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.

The pGOI-loxP, pRep-Cap, pHelper/Cre three plasmids were used for rAAV production (+cre group), and the rate of repacking of the plasmid backbone into rAAV capsids (repacking rate) was reduced to 0.08-fold that of the control EV group, compared to pGOI-loxP, pRep-Cap, pHelper three plasmids used for rAAV production (EV group), +cre group produced rAAV.

Example 4: rAAV packaging Using Cre-KI cells

(1) Vector construction and plasmid extraction

SgAAVS1 primers were designed to target the AAVS1 site (sgAAVS 1-F: CACCTAAGGAATCTGCCTAACAGG; sgAAVS-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) was 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 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".

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 were supplemented to 10. Mu.L with sterilized water. 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 the monoclonal was selected and sent to the qinghao for sequencing. The final result was insertion sgAAVS of the target site on the pX330 plasmid, designated pX330/sgAAVS1.

High-concentration and high-purity pGOI, pGOI-loxP, pRep-Cap, pHelper, pAAVS/Cre and pX330/sgAAVS1 plasmids are extracted.

(2) Cell transfection and screening

The day before transfection, 293T cells were suspended and counted using DMEM medium and plated at 1X 10 ⁶/well uniformly in 6-well cell culture plates. On the day of transfection, pAAVS/Cre plasmid was mixed with pX330/sgAAVS1 plasmid at 2.0. Mu.g:1.0. Mu.g plasmid, 6.0. Mu. L P3000 reagent and 125. Mu.L Opti-MEMI were added and mixed. 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 is added into a 6-hole cell culture plate, and after being gently mixed, the mixture is placed at 37 ℃ and cultured for about 48 to 72 hours under the condition of 5 percent CO ₂.

Transfected cells were collected, suspended and counted using DMEM. Then, 1X 10 ⁶/well was inoculated into a 6-well cell culture plate, and 1.0. Mu.g/mL Puro was added thereto, followed by culturing and screening at 37℃under 5% CO ₂ for about 1 to 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 suspended in DMEM medium and counted, plated on 10-cm cell culture dishes at 6X 10 ⁶ cells/dish and incubated at 37℃under 5% CO ₂ to a cell confluency 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 used to mix the plasmid DNA, 0.5mL of DMEM was used to mix the plasmid DNA with 8.4 mu L PEIpro, and then 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 group). 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

1. A plasmid system for packaging of recombinant adeno-associated virus, said plasmid system comprising any one of:

a. pGOI plasmid containing two ITRs inserted with loxP sequence, adenovirus auxiliary plasmid inserted with Cre enzyme sequence and rAAV recombinant packaging plasmid;

b. pGOI plasmids containing two ITRs, adenovirus helper plasmids, rAAV recombinant packaging plasmids and pAAVS plasmids inserted with Cre enzyme sequences, wherein the pGOI plasmids are inserted with loxP sequences;

the loxP site is added to the 5 'end of the 5' ITR and the 3 'end of the 3' ITR of the pGOI plasmid respectively;

the nucleotide sequence of pAAVS plasmid is shown as SEQ ID No. 1.

2. The plasmid system of claim 1, comprising pGOI, pRep-Cap, pHelper with inserted loxP sequences and pAAVS a with inserted Cre enzyme sequences.

3. The plasmid system of claim 1, comprising pGOI, pRep-Cap with loxP inserted and pHelper with Cre enzyme inserted.

4. A cell for rAAV production, wherein the cell stably expresses Cre enzyme; the cell comprising the plasmid system of claim 1.

5. A method for reducing residue of a plasmid backbone nucleic acid sequence in a rAAV, comprising transfecting a packaging cell with the plasmid system of any one of claims 1-3.

6. The method of claim 5, wherein the packaging cells comprise at least one of HEK293, HEK293T, HEK293F, HEK293A, hela, vero, CHO.

7. Use of the plasmid system of any one of claims 1-3, the cell of claim 4 in rAAV production.