Double-plasmid packaging system for improving AAV2 virus titer

Abstract

According to the double-plasmid packaging system for improving the AAV2 virus titer, the packaging system is co-transfection of an AAV producing cell and two plasmids, and the two plasmids are shuttle plasmids which are provided with two AAV ITRs, are respectively inserted with FRT sequences on two sides and are loaded with target genes; the invention relates to a helper plasmid which is provided with rep-cap, can express different serotypes, is inserted into a human miRNA (micro Ribonucleic Acid) sequence and a CAG-Flpo-wpre-pa sequence and provides a helper gene separated from an adenovirus. According to the system disclosed by the invention, the AAV2 virus with the titer of 1.0 * 10 < 10 > can be packaged by using a double-plasmid co-transfection system. The difficulty of plasmid cotransfection is reduced, and a product route is provided for commercial production of AAV2 virus packaging.

Description

Technical Field

[0001] The present invention relates to the biological field, and in particular to a double-plasmid packaging system for improving the titer of AAV2 virus. Background Art

[0002] The most commonly used AAV virus packaging system is the AAV helper-free system, which can produce recombinant adeno-associated viruses without helper viruses. This system is a co-transfection of AAV production cells with three plasmids. The three plasmids are a shuttle plasmid carrying the target gene with AAV ITR, a packaging plasmid expressing different serotypes with rep-cap, and a helper plasmid that provides auxiliary genes (such as e2a, e4, etc.) separated from adenovirus. However, the commercial production process of this virus packaging system is very difficult and there are many problems. For example, the problem of plasmid backbone contamination: the contamination sequence may come from the shuttle plasmid backbone. Virus titer and stability issues: The three-plasmid packaging system is relatively complicated, and sometimes the virus titer produced is very low or even non-toxic. Or the reproducibility is poor, and the titer of each batch varies greatly. These problems greatly increase the production cost of the virus. It is crucial for the packaging production of the virus to improve the virus packaging titer by modifying the packaging system. Summary of the invention

[0003] The purpose of the present invention is to provide a dual-plasmid packaging system for improving the titer of AAV2 virus.

[0004] The present invention achieves the above-mentioned purpose through the following technical scheme: a dual-plasmid packaging system for improving the titer of AAV2 virus, the packaging system is the co-transfection of AAV production cells and two plasmids, the two plasmids are respectively a shuttle plasmid carrying a target gene with two AAV ITRs and an FRT sequence inserted on both sides, and an auxiliary plasmid with a rep-cap that can express different serotypes and inserts a human miRNA sequence and a CAG-Flpo-wpre-pa sequence to provide auxiliary genes separated from adenovirus.

[0005] Furthermore, the human miRNA sequence can express hsa-miR-342, hsa-miR-342.

[0006] Furthermore, the titer of the packaging system can reach 1.0×10 10 .

[0007] Furthermore, the packaging system was prepared by a dual-plasmid packaging method.

[0008] Furthermore, the shuttle plasmid carries two AAV ITRs and an FRT sequence is inserted on each side.

[0009] Furthermore, the helper plasmid carrying the rep-cap and human miRNA sequences was able to express the Flp recombinase.

[0010] Compared with the prior art, the dual-plasmid packaging system of the present invention for improving the titer of AAV2 virus is advantageous in that the system can use a dual-plasmid co-transfection system to package a titer of 1.0×10 10 AAV2 virus. It reduces the difficulty of plasmid co-transfection and provides a product route for the commercial production of AAV2 virus packaging. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 is a schematic diagram of the shuttle plasmid.

[0012] Figure 2 is a schematic diagram of the helper plasmid. DETAILED DESCRIPTION

[0013] See also Figure 1 to Figure 2 , a dual-plasmid packaging system for improving the titer of AAV2 virus, which is the co-transfection of AAV production cells with two plasmids. The two plasmids are a shuttle plasmid carrying the target gene with two AAV ITRs and an FRT sequence inserted on each side, and an auxiliary plasmid with a rep-cap that can express different serotypes and inserts human miRNA sequences and CAG-Flpo-wpre-pa sequences to provide auxiliary genes isolated from adenovirus.

[0014] An FRT sequence is inserted on both sides of the two ITR sequences of the shuttle plasmid; Flp is a recombinase that recognizes the FRT site. When the FRT is on the same DNA and in the same direction, the gene between the two FRT sites is deleted, thereby achieving the purpose of removing the shuttle plasmid skeleton.

[0015] Inserting human miRNA sequences into the helper plasmid can increase the titer of AAV2 virus production. Specifically, hsa-miR-342 can be expressed, hsa-miR-342 can be expressed, hsa-miR-342 is a human miRNA that can increase the titer of AAV2 virus in the virus preparation system. Compared with the pRC2 Vector that generally only expresses the Rep gene and the Cap gene, the virus titer can be increased by 2 times. Insert the rep2 and cap2 genes into the helper plasmid. These genes encode the replication (Rep) and capsid (Cap) proteins of AAV2.

[0016] Inserting the CAG-Flpo-wpre-pa sequence into the auxiliary plasmid can express the Flpo recombinase. The modified shuttle plasmid can be cut to remove the shuttle plasmid skeleton, and the ITR sequence is exposed after cutting. ITR plays a key role in the replication and packaging of the virus, and is involved in the integration and escape of the viral genome on the host genome. The exposed ITR sequence is more conducive to improving the packaging efficiency.

[0017] The packaging method is as follows:

[0018] 1) Cell culture: AAV-293 cells were cultured in T25 culture flasks;

[0019] 2) Co-transfection: The shuttle plasmid and the auxiliary plasmid are co-transfected into AAV-293 cells. The molar ratio of shuttle plasmid to auxiliary plasmid is 1:3 to 1:7.

[0020] 3) Virus collection: After about 48-72 hours of production time, collect cells and culture supernatant containing rAAV into 50 mL centrifuge tubes.

[0021] 4) Purification: After low-speed centrifugation at 4000 rpm for 5 minutes, the supernatant in the 50 mL centrifuge tube was transferred to a 50 mL syringe and filtered through a 0.22 um filter into a 15 mL ultrafiltration tube.

[0022] 5) Concentration: The ultrafiltration tube was leveled and placed in a high-speed refrigerated centrifuge rotor, and centrifuged at 4°C, 8000 rpm for 18 minutes; the virus solution in the ultrafiltration tube was slowly blown up and down 6-8 times with a pipette to mix, and then the virus solution was transferred to a 1.5 mL sterile cryotube with the pipette.

[0023] 6) Titer determination: Quantify the number of rAAV particles by qPCR. The titer can reach 1.0×10 10 .

[0024] The system of the present invention can use a double plasmid co-transfection system to package the titer at 1.0×10 10 AAV2 virus. It reduces the difficulty of plasmid co-transfection and provides a product route for the commercial production of AAV2 virus packaging.

[0025] The above shows and describes the basic principles and main features of the present invention and the advantages of the present invention. It is obvious to those skilled in the art that the present invention is not limited to the details of the above exemplary embodiments, and the present invention can be implemented in other specific forms without departing from the spirit or basic features of the present invention. Therefore, no matter from which point of view, the embodiments should be regarded as exemplary and non-restrictive. The scope of the present invention is defined by the attached claims rather than the above description, and it is intended that all changes falling within the meaning and scope of the equivalent elements of the claims are included in the present invention. Any figure mark in the claims should not be regarded as limiting the claims involved.

[0026] In addition, it should be understood that although the present specification is described according to implementation modes, not every implementation mode contains only one independent technical solution. This description of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment may also be appropriately combined to form other implementation modes that can be understood by those skilled in the art.

Claims

1. A dual-plasmid packaging system for improving the titer of AAV2 virus, characterized in that: The packaging system is a co-transfection of AAV production cells with two plasmids. The two plasmids are a shuttle plasmid carrying the target gene with two AAV ITRs and an FRT sequence inserted on each side, and an auxiliary plasmid with a rep-cap that can express different serotypes and inserts human miRNA sequences and CAG-Flpo-wpre-pa sequences to provide auxiliary genes isolated from adenovirus.

2. The dual-plasmid packaging system for improving AAV2 virus titer according to claim 1, characterized in that: The human miRNA sequence can express hsa-miR-342, hsa-miR-342.

3. The dual-plasmid packaging system for improving AAV2 virus titer according to claim 1, characterized in that: The titer of the packaging system can reach 1.0×10 10 .

4. The dual-plasmid packaging system for improving AAV2 virus titer according to claim 1, characterized in that: The packaging system was prepared by a dual-plasmid packaging method.

5. The dual-plasmid packaging system for improving AAV2 virus titer according to claim 1, characterized in that: The shuttle plasmid carries two AAV ITRs and is flanked by an FRT sequence.

6. The dual-plasmid packaging system for improving AAV2 virus titer according to claim 1, characterized in that: The helper plasmid carries the rep-cap and human miRNA sequences and is able to express the Flp recombinase.

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