CN119662733B - Plasmid system for high-yield rAAV and application - Google Patents
Plasmid system for high-yield rAAV and applicationInfo
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Abstract
本发明属于生物技术领域，公开了一种高产rAAV的质粒系统及应用。本发明的高产rAAV的质粒系统包括含两个末端反向重复序列的转基因质粒，包装质粒，辅助质粒和shRNA表达盒；所述shRNA表达盒包括能够靶向所述转基因质粒中的目标基因的序列。本发明的质粒系统在rAAV生产过程中，通过表达shRNA靶向转基因质粒上的目标基因编码区或者靶标序列，抑制目标基因的转录表达。经过精心设计的靶标序列，不同于人和哺乳动物来源基因编码区序列，可以广谱用于各种转基因质粒，从而提高rAAV的产量，可显著提高哺乳动物细胞rAAV生产效率，实现了提高了rAAV的成药性和降低生产成本的技术效果。
The present invention belongs to the field of biotechnology, and discloses a high-yield rAAV plasmid system and application. The high-yield rAAV plasmid system of the present invention includes a transgenic plasmid containing two terminal inverted repeat sequences, a packaging plasmid, an auxiliary plasmid and an shRNA expression cassette; the shRNA expression cassette includes a sequence capable of targeting the target gene in the transgenic plasmid. During the rAAV production process, the plasmid system of the present invention inhibits the transcriptional expression of the target gene by expressing shRNA to target the target gene coding region or target sequence on the transgenic plasmid. The carefully designed target sequence is different from the coding region sequence of the gene from humans and mammals, and can be widely used in various transgenic plasmids, thereby increasing the yield of rAAV, significantly improving the production efficiency of rAAV in mammalian cells, and achieving the technical effect of improving the drugability of rAAV and reducing production costs.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a plasmid system for high-yield rAAV and application thereof.
Background
The recombinant adeno-associated virus (rAAV) vector has the advantages of capability of stably expressing exogenous genes for a long time, no integration into host genome, capability of infecting cells in the division phase/non-division phase, wide host cell range, strong specificity, low immunogenicity and the like, so that the recombinant adeno-associated virus (rAAV) vector becomes a safe and effective gene therapy tool, and is also considered as one of the gene therapy vectors with the most development prospect at present.
The rAAV production system mainly comprises a plasmid transfection production system based on HEK293 cells, a production system based on insect cells and baculovirus, a packaging cell-based or inducible packaging cell method and the like. Plasmid transfection production systems based on HEK293 cells are currently the mainstay technology for clinical/research grade rAAV production. The system mainly adopts a mode of co-transfecting HEK293 cells by using a transgenic plasmid containing two terminal inverted repeat sequences, a packaging plasmid for encoding AAV replication protein (Rep) and capsid protein (Cap), an auxiliary plasmid for encoding auxiliary factors required by rAAV replication and the like, and the main process comprises the steps of plasmid preparation, production cell expansion, plasmid transfection production cell, crude virus liquid harvesting by lysis cells, virus purification and the like.
However, during rAAV production, the transcriptional expression of the gene of interest is driven by the promoter on the transgenic plasmid, which is active in HEK293 cells, and these gene expression products, especially those that negatively affect the cells, may lead to a reduced ability to package rAAV. Therefore, research and development can carry out gene silencing on target genes in the rAAV production process, so that the rAAV yield is improved, and the method has important significance in promoting the scale production of rAAV and rAAV-mediated gene therapy.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide a plasmid system for high-yield rAAV 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 high-yield rAAV, comprising a transgenic plasmid containing two inverted terminal repeats, a packaging plasmid, a helper plasmid and an shRNA expression cassette;
The shRNA expression cassette includes sequences capable of targeting a gene of interest in the transgenic plasmid.
In the rAAV production process, the plasmid system of the invention carries out gene silencing on the target gene on the transgenic plasmid by expressing shRNA, thereby improving the yield of rAAV, remarkably improving the production efficiency of the rAAV of mammalian cells, and realizing the technical effects of improving the drug property of the rAAV and reducing the production cost.
As a preferred embodiment of the high-yield rAAV plasmid system, a target sequence is inserted into the transgenic plasmid at the 5 'end or the 3' end outside the coding region of the target gene, and the nucleotide of the target sequence is 5'-GGCGAGAATGAGCCTGCCTGTAAA-3'.
As a preferred embodiment of the high-yield rAAV plasmid system, the shRNA expression cassette comprises a promoter for driving the shRNA, a shRNA hairpin structure for targeting the target gene coding region or the target sequence on the transgenic plasmid and a terminator.
As a preferred embodiment of the high-yield rAAV plasmid system, the shRNA expression cassette is positioned on any one plasmid of the transgenic plasmid, the packaging plasmid and the auxiliary plasmid.
As a preferred embodiment of the high-yield rAAV plasmid system, the transgenic plasmid is pGOI plasmids, the packaging plasmid is pRep-Cap, and the helper plasmid is pHelper.
The pGOI plasmids comprise nucleic acid sequences from different serotypes and ITR sequences and optimized sequences thereof, the pRep-Cap comprises nucleic acid sequences from different serotypes and expressed Rep proteins and nucleic acid sequences expressed Cap proteins and optimized nucleic acid sequences thereof, and the plasmid pHelper encodes cofactors required for rAAV replication, including nucleic acid sequences from different helper virus plasmids and expressed adenovirus original proteins and optimized sequences thereof.
In a second aspect, the invention provides a cell for high-yielding rAAV, comprising the plasmid system described above.
As a preferred embodiment of the cells for high-yield rAAV, the cells are at least one of HEK293, HEK derived cells, CHO derived cells, heLa cells, vero cells and SF-9 cells.
In a third aspect, the invention uses the plasmid system, the cell in rAAV production.
In a fourth aspect, the invention provides a method for enhancing production of recombinant adeno-associated virus, transfecting packaging cells with the plasmid system, or culturing the cells.
As a preferred embodiment of the method of the present invention, the packaging cell is at least one of HEK293, HEK-derived cell, CHO-derived cell, hela cell, vero cell, SF-9 cell.
Compared with the prior art, the invention has the beneficial effects that:
in the rAAV production process, the plasmid system of the invention carries out gene silencing on the target gene on the transgenic plasmid by expressing shRNA, thereby improving the yield of rAAV, remarkably improving the production efficiency of the rAAV of mammalian cells, and realizing the technical effects of improving the drug property of the rAAV and reducing the production cost.
Drawings
FIG. 1 is a graph showing the effect of rAAV packaging in inhibiting expression of a target gene by targeting a gene coding region on pGOI;
FIG. 1, panel a, schematic representation of pGOI and pHelper-shScram, pHelper-shGFP constructed according to the invention, panel b, fluorescence intensity of rAAV packages using pGOI and pRep-Cap co-transfected with pHelper, pHelper-shScram, pHelper-shGFP, respectively, panel c, relative genomic titres of rAAV packages using pGOI and pRep-Cap (AAV 9) co-transfected with pHelper, pHelper-shScram, pHelper-shGFP, respectively, and Panel d, relative genomic titres of rAAV packages using pGOI and pRep-Cap (AAV 2) co-transfected with pHelper, pHelper-shScram, pHelper-shGFP, respectively.
FIG. 2 is a graph showing the effect of rAAV packaging in inhibiting expression of a gene of interest by targeting a 5' end target sequence of the gene on pGOI;
FIG. 2, panel a, schematic representation of pGOI-5'TS, pHelper-shScram, pHelper-shTS constructed according to the invention, panel b, fluorescence intensity of rAAV packages using pGOI-5' TS, pRep-Cap co-transfected with pHelper, pHelper-shScram, pHelper-shTS, respectively, panel c, relative genomic titres of rAAV packages using pGOI-5'TS, pRep-Cap (AAV 9) co-transfected with pHelper, pHelper-shScram, pHelper-shTS, respectively, and Panel d, relative genomic titres of rAAV packages using pGOI-5' TS, pRep-Cap (AAV 2) co-transfected with pHelper, pHelper-shScram, pHelper-shTS, respectively.
FIG. 3 is a graph showing the effect of rAAV packaging in inhibiting expression of a gene of interest by targeting a target sequence at the 3' end of the gene on pGOI;
FIG. 3, panel a, schematic representation of pGOI-3'TS, pHelper-shScram, pHelper-shTS constructed according to the invention, panel b, fluorescence intensity of rAAV packages using pGOI-3' TS, pRep-Cap co-transfected with pHelper, pHelper-shScram, pHelper-shTS, respectively, panel c, relative genomic titres of rAAV packages using pGOI-3'TS, pRep-Cap (AAV 9) co-transfected with pHelper, pHelper-shScram, pHelper-shTS, respectively, and Panel d, relative genomic titres of rAAV packages using pGOI-3' TS, pRep-Cap (AAV 2) co-transfected with pHelper, pHelper-shScram, pHelper-shTS, respectively.
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, and the materials, reagents, etc. used, unless otherwise specified, are commercially available.
Example 1 rAAV packaging Effect of inhibiting expression of target Gene by targeting the Gene coding region on pGOI
(1) Plasmid construction and preparation
The 3' -end of the eGFP gene was inserted into the pAAV-GFP plasmid (Addgene: 32395) by using molecular biology techniques such as gene synthesis, PCR, gibson Assembly, sequencing analysis, etc., from the cleaved polypeptide P2A(Kim JH et.al.High cleavage efficiency of a 2A peptide derived from porcine teschovirus-1in human cell lines,zebrafish and mice.PLoS One.2011,6(4):e18556.doi:10.1371/journal.pone.0018556.) and CASP4 gene (GenBank accession: NM-001225), to obtain the pGOI plasmid shown in FIG. 1a.
The EF1a promoter (Qin JY et.al.Systematic comparison of constitutive promoters and the doxycycline-inducible promoter.PLoS One.2010,5(5):e10611.doi:10.1371/journal.pone.0010611.)、shRNA was inserted into a pHelper plasmid (GeneBank: AF 369965) against the hairpin structure (5'-tgtttgaatgaggcttcagtactttacagaatcgttgcctgcacatcttggaaacacttgctgggattacttcgacttcttaacccaacagaaggctcgagaaggtatattgctgttgacagtgagcgGCCTAAGGTTAAGTCGCCCTCGtagtgaagccacagatgtaCGAGGGCGACTTAACCTTAGGTtgcctactgcctcggacttcaaggggctagaattcgagcaattatcttgtttactaaaactgaataccttgctatctctttgatacatttttacaaagctgaattaaaatggtataaattaaatcacttt-3')、WPRE-SV40pA fragment (Choi JH et.al.Optimization of AAV expression cassettes to improve packaging capacity and transgene expression in neurons.Mol Brain.2014,7:17.doi:10.1186/1756-6606-7-17.Carswell S,Alwine JC.Efficiency of utilization of the simian virus 40late polyadenylation site:effects of upstream sequences.Mol Cell Biol.1989,9(10):4248-58.doi:10.1128/mcb.9.10.4248-4258.1989.) of shScarm to obtain a pHelper-shScram plasmid as shown in FIG. 1 a.
The fragment of the EF1a promoter, shGFP hairpin (5'-tgtttgaatgaggcttcagtactttacagaatcgttgcctgcacatcttggaaacacttgctgggattacttcgacttcttaacccaacagaaggctcgagaaggtatattgctgttgacagtgagcgTGCACAAGCTGGAGTACAACTAtagtgaagccacagatgtaTAGTTGTACTCCAGCTTGTGCCtgcctactgcctcggacttcaaggggctagaattcgagcaattatcttgtttactaaaactgaataccttgctatctctttgatacatttttacaaagctgaattaaaatggtataaa ttaaatcacttt-3')、WPRE-SV40pA targeting the GFP coding region was inserted into the pHelper plasmid to obtain the pHelper-shGFP plasmid as shown in FIG. 1 a.
High-concentration and high-purity pGOI, pRep-Cap (AAV 9), pRep-Cap (AAV 2) and pHelper, pHelper-shSram, pHelper-shGFP plasmids were extracted.
(2) RAAV packaging
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%.
The Control group plasmids pGOI, pRep-Cap (AAV 9 or AAV 2), pHelper are set, the shScram group plasmids pGOI, pRep-Cap (AAV 9 or AAV 2), pHelper-shScram are set, and the shGFP group plasmids pGOI, pRep-Cap (AAV 9 or AAV 2) and pHelper-shGFP are set.
On the day of transfection, fresh DMEM medium was changed. pGOI, pRep-Cap and pHelper were mixed in a ratio of 2.0. Mu.g to 2.5. Mu.g to 2.3. Mu.g, respectively, and each group was repeated 3 times. 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.
On the third day after transfection, eGFP fluorescence was observed under a fluorescence microscope. The fluorescence photographing results showed that the fluorescence intensity of shGFP group was significantly reduced compared to Control and shScram groups (fig. 1 b).
(3) Genome titre assay
To the transfected dishes, 100. Mu.L of cell lysate was added, and after 30min standing at room temperature, 50. Mu.L of supernatant was taken to determine genome titer. The harvested supernatant was digested with DNaseI, and the genome titer was determined using qRT-PCR, and a standard curve was prepared with a gradient diluted plasmid standard. Genome titer of rAAV samples was calculated from the standard curve.
The results of the genome titer test showed 5.91-fold (FIG. 1 c) and 2.22-fold increases in genome titer of the shGFP-fold (FIG. 1 d) of AAV9 and AAV2 groups, respectively, compared to the Control group.
Example 2 rAAV packaging Effect of inhibiting expression of a Gene of interest by targeting the 5' end target sequence of the Gene on pGOI
(1) Plasmid construction and preparation
All nucleotide sequences encoding PACKGENE amino acid sequences are generated by using a bioinformatics method, all sequences are arranged, potential shRNA target sequences are designed by using GPP Web Portal online program, and then the target sequence (5'-GGCGAGAATGAGCCTGCCTGTAAA-3') with the highest score is selected for testing. By comparison, the target sequence is different from the coding region sequence of the genes of human and mammal sources, so that the designed shRNA can not target and influence human or animal genes.
The pGOI-5'TS plasmid shown in FIG. 2a was obtained by inserting a 24bp target sequence (TS, 5'-GGCGAGAATGAGCCTGCCTGTAAA-3') into the 5' end of the eGFP gene on pGOI plasmids using molecular biology techniques such as gene synthesis, PCR, gibson Assembly, sequencing analysis, etc.
The fragment of the EF1a promoter, shTS hairpin (5'-tgtttgaatgaggcttcagtactttacagaatcgttgcctgcacatcttggaaacacttgctgggattacttcgacttcttaacccaacagaaggctcgagaaggtatattgctgttgacagtgagcgTGAGAATGAGCCTGCCTGTAAAtagtgaagccacagatgtaTTTACAGGCAGGCTCATTCTCGtgcctactgcctcggacttcaaggggctagaattcgagcaattatcttgtttactaaaactgaataccttgctatctctttgatacatttttacaaagctgaattaaaatggtataaattaaatcacttt-3')、WPRE-SV40pA targeting TS sequence was inserted onto the pHelper plasmid to obtain the pHelper-shTS plasmid as shown in FIG. 2 a.
High concentration, high purity pGOI' TS, pRep-Cap (AAV 9), pRep-Cap (AAV 2), pHelper, pHelper-shSram, pHelper-shTS plasmids were extracted.
(2) RAAV packaging
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%.
Set Control group plasmids pGOI ' TS, pRep-Cap (AAV 9 or AAV 2), pHelper, shScram group plasmids pGOI-5' TS, pRep-Cap (AAV 9 or AAV 2), pHelper-shScram, shTS group plasmids pGOI-5' TS, pRep-Cap (AAV 9 or AAV 2), pHelper-shTS.
On the day of transfection, fresh DMEM medium was changed. pGOI, pRep-Cap and pHelper were mixed in a ratio of 2.0. Mu.g to 2.5. Mu.g to 2.3. Mu.g, respectively, and each group was repeated 3 times. 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.
On the third day after transfection, eGFP fluorescence was observed under a fluorescence microscope. The fluorescence photographing result showed that the fluorescence intensity of shTS group was slightly decreased compared to Control and shScram groups (fig. 2 b).
(3) Genome titre assay
To the transfected dishes, 100. Mu.L of cell lysate was added, and after 30min standing at room temperature, 50. Mu.L of supernatant was taken to determine genome titer. The harvested supernatant was digested with DNaseI, and the genome titer was determined using qRT-PCR, and a standard curve was prepared with a gradient diluted plasmid standard. Genome titer of rAAV samples was calculated from the standard curve.
The results of the genome titer test showed 5.29-fold and 3.08-fold increases in genome titer of the shTS-fold groups of AAV9 and AAV2 (fig. 2 c) and 3.08-fold groups, respectively, compared to the Control group.
Example 3 rAAV packaging Effect of inhibiting expression of a Gene of interest by targeting the 3' -terminal target sequence of the Gene on pGOI
(1) Plasmid construction and preparation
By using molecular biology techniques such as gene synthesis, PCR, gibson Assembly, sequencing analysis, etc., a 24bp Target Sequence (TS) was inserted into the 3 'end of the eGFP gene on pGOI plasmids, to obtain pGOI-3' TS plasmids as shown in FIG. 3 a.
High concentration, high purity pGOI-3' TS, pRep-Cap (AAV 9), pRep-Cap (AAV 2), pHelper, pHelper-shSram, pHelper-shTS plasmids were extracted.
(2) RAAV packaging
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%.
Set Control group plasmids pGOI-3' TS, pRep-Cap (AAV 9 or AAV 2), pHelper, shScram group plasmids pGOI-5' TS, pRep-Cap (AAV 9 or AAV 2), pHelper-shScram, shTS group plasmids pGOI-3' TS, pRep-Cap (AAV 9 or AAV 2), pHelper-shTS.
On the day of transfection, fresh DMEM medium was changed. pGOI, pRep-Cap and pHelper were mixed in a ratio of 2.0. Mu.g to 2.5. Mu.g to 2.3. Mu.g, respectively, and each group was repeated 3 times. 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.
On the third day after transfection, eGFP fluorescence was observed under a fluorescence microscope. The fluorescence photographing results showed that the fluorescence intensity of shTS group was significantly reduced compared to Control and shScram groups (fig. 3 b).
(3) Genome titre assay
To the transfected dishes, 100. Mu.L of cell lysate was added, and after 30min standing at room temperature, 50. Mu.L of supernatant was taken to determine genome titer. The harvested supernatant was digested with DNaseI, and the genome titer was determined using qRT-PCR, and a standard curve was prepared with a gradient diluted plasmid standard. Genome titer of rAAV samples was calculated from the standard curve.
The results of the genome titer test showed 8.68-fold and 6.23-fold increases in genome titer of the shTS groups of AAV9 and AAV2 (fig. 3 c) compared to the Control group, respectively.
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.
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| Rescue of Adeno-Associated Virus Production by shRNA Cotransfection;Maria C. Guimaro et al.;《HUMAN GENE THERAPY》;20201231;第31卷(第19和20期);1069页左栏第2-3段，第1070页最后一段至1071页第3段，第1072段左栏第2段 * |
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