CN112501209B - Method for packaging adeno-associated virus with controllable expression of exogenous gene - Google Patents
Method for packaging adeno-associated virus with controllable expression of exogenous gene Download PDFInfo
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- CN112501209B CN112501209B CN202011419528.4A CN202011419528A CN112501209B CN 112501209 B CN112501209 B CN 112501209B CN 202011419528 A CN202011419528 A CN 202011419528A CN 112501209 B CN112501209 B CN 112501209B
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Abstract
The invention relates to the field of molecular biology, in particular to an adeno-associated virus packaging method for controllably expressing exogenous genes. The adeno-associated virus packaging vector adopted by the method is provided with two ITR fragments and a gene expression cassette inserted between the two ITR fragments; the gene expression cassette comprises a promoter, a repressor operator and optionally a gene of interest, connected in sequence; the repressible operator is capable of repressing the expression of a gene of interest downstream thereof in the presence of a repressor. The invention creatively integrates the repressed operator into the adeno-associated virus vector, and can reduce the expression of exogenous genes in the virus packaging process, thereby eliminating the influence of exogenous genes and improving the yield of specific viruses.
Description
Technical Field
The invention relates to the field of molecular biology, in particular to an adeno-associated virus packaging method for controllably expressing exogenous genes.
Background
Viral vectors can bring genetic material into cells by exploiting the molecular mechanism by which viruses bind to cell surface receptors with high affinity and transfer their genome into certain cell types for infection. Is widely applied to basic research, gene therapy or vaccine.
Adeno-associated virus (AAV), also known as adeno-associated virus, belongs to the genus dependovirus of the family picoviridae, and is the simplest class of structurally single-stranded DNA-deficient viruses currently found, requiring helper virus (typically adenovirus) to participate in replication. It encodes cap and rep genes in inverted repeats (ITRs) at both ends. ITRs are decisive for viral replication and packaging. The cap gene encodes viral capsid proteins and the rep gene is involved in viral replication and integration. AAV can infect a variety of cells. In the presence of the rep gene product, viral DNA is readily integrated into human chromosome 19.
The adeno-associated virus has the characteristics of high safety, low immunogenicity, wide host range, capability of mediating long-term stable expression of exogenous genes in animals, and the like, and is one of the vectors with the most application prospect in the field of gene therapy. AAV has important roles and great demands in the fields of neuroscience research, gene therapy of diseases and the like. In medical research, AAV is used in the study of gene therapy for a variety of diseases (including in vivo, in vitro experiments); meanwhile, the gene transfer vector is used as a characteristic gene transfer vector and is also widely used in aspects of gene function research, disease model construction, gene knockout mouse preparation and the like.
At present, research on viral vectors is focused on infection capability, specificity, packaging capacity and the like, but little attention is paid to the influence of packaged foreign Genes (GOI) on viruses, and in practice, a significant part of foreign genes cannot obtain effective viral particles. This is because, in the viral packaging process, on the one hand, the foreign gene is usually driven by a strong promoter, and its expression has an unexpected effect on the cell state, the packaging of the virus, the virus production and the like. It is not uncommon for exogenous genes to fail to or be low in virulence, for example, during packaging of adeno-associated viruses that overexpress GPR78 (NM_080819), expression of GPR78 renders the vector incapable of obtaining viral particles.
Disclosure of Invention
The invention relates to an adeno-associated virus packaging vector, which is provided with two ITR fragments and a gene expression cassette inserted between the two ITR fragments;
the gene expression cassette comprises a promoter, a repressor operator and optionally a gene of interest, connected in sequence;
the repressible operator is capable of repressing the expression of a gene of interest downstream thereof in the presence of a repressor.
According to yet another aspect of the present invention, the present invention also relates to an adeno-associated virus packaging vector system comprising an adeno-associated virus packaging vector as described above and a vector carrying AAV rep, cap genes.
According to a further aspect of the invention, the invention also relates to a method of packaging an adeno-associated virus comprising transferring an adeno-associated virus packaging vector system as described above into a host cell and packaging in the presence of said repressor.
Compared with the prior art, the invention has the beneficial effects that:
the invention has the advantages that whether the exogenous gene affects the adeno-associated virus is not needed to be judged, and the repression system does not play a role and does not affect the expression of the target gene when no repressor exists. When the virus is found to be obviously lower than the average level, the virus packaging process of inhibiting the exogenous gene can be carried out by matching with an auxiliary plasmid containing the expression of the repressor protein, so that the influence of the exogenous gene is eliminated, the yield of the specific virus is improved, a new shuttle vector is not required to be reconstructed, and a large amount of time, manpower and material resources are saved.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the description of the embodiments or the prior art will be briefly described, and it is obvious that the drawings in the description below are some embodiments of the present invention, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.
FIGS. 1 to 5 are sequential plasmid maps of pcDNA3.1-CymR, CMV-CuO, EF1a-CuO, SFH-CuO, and CAG-CuO vectors according to one embodiment of the present invention;
FIG. 6 is a graph showing the result of verification of the repression efficiency of CymR-CuO in lentiviral vectors according to one embodiment of the present invention;
FIG. 7 is a schematic diagram of a CMV-TrpO plasmid according to one embodiment of the invention;
FIG. 8 is a graph showing the result of verification of repression efficiency of tryptophan manipulation subsystem in lentiviral vectors according to one embodiment of the invention;
FIG. 9 is a diagram of the EF1a-ToxO plasmid according to one embodiment of the present invention;
FIG. 10 is a graph showing the results of verification of repression efficiency of a diphtheria toxin inhibitor regulatory system in lentiviral vectors according to one embodiment of the invention;
FIG. 11 is a map of SFH-LacO plasmid in one embodiment of the present invention;
FIG. 12 is a graph showing the results of verification of repression efficiency of lactose manipulation subsystem in lentiviral vectors according to one embodiment of the invention;
FIG. 13 is a schematic representation of the insertion position of the TrpO element relative to the TATA Box of CMV in one embodiment of the invention;
FIG. 14 is a graph showing the effect of different insertion positions of TrpO elements on repression efficiency according to one embodiment of the invention;
FIG. 15 is a schematic view showing the insertion position of a TATA Box of a CuO element relative to CMV in one embodiment of the invention;
FIG. 16 is a graph showing the effect of various insertion positions of CuO elements on repression efficiency in one embodiment of the present invention;
FIG. 17 shows the results of a viral titer assay for GPR78 expressed by an AAV viral vector according to one embodiment of the invention;
FIGS. 18-20 are plasmid maps of pAAV-CMV-GPR78-3 xFLAG-WPRE, pAAV-CMV-CuO-GPR78-3 xFLAG-WPRE, and pAAV-CMV-TrpO-GPR78-3 xFLAG-WPRE vectors, in order, according to one embodiment of the present invention;
FIG. 21 shows the results of a viral titer assay for Cdkn1a expressed by an AAV viral vector according to one embodiment of the invention;
FIGS. 22-24 are plasmid maps of pAAV-CMV-Cdkn1a-3 xFLAG-WPRE, pAAV-CMV-CuO-Cdkn1a-3 xFLAG-WPRE, and pAAV-CMV-TrpO-Cdkn1a-3 xFLAG-WPRE vectors in order according to one embodiment of the present invention;
fig. 25 is a schematic diagram of the principles of the present invention.
Detailed Description
Reference now will be made in detail to embodiments of the invention, one or more examples of which are described below. Each example is provided by way of explanation, not limitation, of the invention. Indeed, it will be apparent to those skilled in the art that various modifications and variations can be made to the present invention without departing from the scope or spirit of the invention. For example, features illustrated or described as part of one embodiment can be used on another embodiment to yield still a further embodiment.
Accordingly, it is intended that the present invention cover such modifications and variations as fall within the scope of the appended claims and their equivalents. Other objects, features and aspects of the present invention will be disclosed in or be apparent from the following detailed description. It is to be understood by one of ordinary skill in the art that the present discussion is a description of exemplary embodiments only, and is not intended as limiting the broader aspects of the present invention.
The invention relates to an adeno-associated virus packaging vector, which is provided with two ITR fragments and a gene expression cassette inserted between the two ITR fragments;
the gene expression cassette comprises a promoter, a repressor operator and optionally a gene of interest, connected in sequence;
the repressible operator is capable of repressing the expression of a gene of interest downstream thereof in the presence of a repressor.
The invention can control the expression of target gene by repressing operator, to restrain the expression of exogenous gene in the gland-related virus packing cell, to avoid the negative effect of exogenous gene on gland-related virus packing and production, to improve the production efficiency and titer of virus, while the expression level of exogenous gene is not reduced in target cell, to avoid affecting the treatment effect or research the function of exogenous gene.
The term "vector" as used herein refers to a nucleic acid vehicle into which a polynucleotide may be inserted. When a vector enables expression of a protein encoded by an inserted polynucleotide, the vector is referred to as an expression vector. The vector may be introduced into a host cell by transformation, transduction or transfection such that the genetic material elements carried thereby are expressed in the host cell. The vector is preferably a plasmid, but not limited to this, and may be a virus or the like, for example, as shown in the invention patent of international publication No. WO2017124588A1, whose international publication date is 2017, 7, 27.
In some embodiments, the repressible operator is selected from a tryptophan operator and/or a Cumate-CuO regulatable system.
In some embodiments, the repressible operator has one or more copies.
In some embodiments, the TrpO element in the amino acid operator is 18 nucleotides or less, e.g., 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 nucleotides, from the TATA BOX of the promoter at the insertion position of the gene expression cassette.
In some embodiments, the Cumate-CuO can regulate the insertion position of the CuO element in the system 40-50 nucleotides, e.g., 41, 42, 43, 44, 45, 46, 47, 48, 49 nucleotides, from the TATA BOX of the promoter.
The distance between the repressible operator element and the TATA BOX of the promoter is the number of nucleotides from the first nucleotide (not included) at the 5 'end of the element to the first nucleotide (not included) at the 3' end of the TATA BOX.
It is considered that within 8 nucleotides (including the 8 th nucleotide) after the TATA BOX belongs to the promoter core region. It is preferred that the distance between the TrpO element and the TATA BOX of the promoter is greater than 8 nucleotides and may be closer to the TATA BOX when the element has an overlapping sequence with the 8 nucleotides.
A "promoter" is a DNA sequence that directs RNA polymerase binding and thereby initiating RNA synthesis. Promoters useful in the present invention allow expression in a wide variety of cell and tissue types; or may be a cell-free specific promoter, or may be a "cell-specific", "cell type-specific", "cell lineage-specific" or "tissue-specific" promoter, which allows expression in a restricted variety of cells and tissue types, respectively. In particular embodiments, it is desirable to use a cell, cell type, cell lineage, or tissue specific expression control sequence to achieve cell type-specific, cell lineage-specific, or tissue-specific expression of a desired polynucleotide sequence (e.g., to express a nucleic acid encoding a polypeptide only in a cell type, cell lineage, or subgroup of tissues, or at a particular developmental stage).
Illustrative examples of tissue-specific promoters include, but are not limited to: b29 promoter (B cell expression), runt transcription factor (CBFa 2) promoter (Stem cell specific expression), CD14 promoter (monocyte expression), CD43 promoter (leukocyte and platelet expression), CD45 promoter (hematopoietic cell expression), CD68 promoter (macrophage expression), CYP4503A4 or ALB promoter (hepatocyte expression), myoline protein promoter (muscle cell expression), elastase I promoter (pancreatic acinar cell expression), endothelial glycoprotein promoter (endothelial cell expression), fibroblast specific protein I promoter (FSPl) promoter (fibroblast expression), fibronectin promoter (fibroblast expression), fms-related tyrosine kinase I (FLTl) promoter (endothelial cell expression), glial Fibrillary Acidic Protein (GFAP) promoter (astrocyte expression), insulin promoter (pancreatic cell expression), integrin-alpha-2B (ITGA 2B) promoter (megakaryocyte), intracellular adhesion molecule 2 (ICAM-2) promoter (ICAM-2), interferon (ICAM-beta) promoter (human cell), myoglobin (myoglobin) and myoglobin (myoglobin) expression (myoglobin expression), myoglobin expression (myo) and (myoglobin expression (myo-beta) expression (myo-cell expression) promoter (myo-1) differentiation (myo-cell expression) Kidney disease protein promoter (podocyte expression), bone gamma-carboxyglutamic acid protein 2 (OG-2) promoter (osteoblast expression), 3-keto acid CoA transferase 2B (Oxct 2B) promoter (haploid sperm cell expression), surface active protein B (SP-B) promoter (lung cell expression), synaptoprotein promoter (nerve cell expression), wiskott-Aldrich syndrome protein (WASP) promoter (hematopoietic cell expression).
In some embodiments, the promoter is a cell-free specific promoter. Exemplary cell-free specific promoters include, but are not limited to, the Cytomegalovirus (CMV) very early promoter, the viral simian virus 40 (SV 40) (e.g., early or late), the Moloney murine leukemia virus (MoMLV) LTR promoter, the Rous Sarcoma Virus (RSV) LTR, the Herpes Simplex Virus (HSV) (thymidine kinase) promoter, the vaccinia virus H5, P7.5 and Pll promoters, the elongation factor L-alpha (EFla) promoter, the early growth response I (EGRl), ferritin H (FerH), ferritin L (FerL), 3-phosphoglyceraldehyde dehydrogenase (GAPDH), eukaryotic translation initiation factor 4A1 (EIF 4A 1), heat shock 70kDa protein 5 (HSPA 5), heat shock protein 90 kDa-. Beta.member 1 (HSP 90B 1), heat shock protein 70kDa (HSP 70), beta-kinesin), the human R0SA26 gene locus (Irionet, (Nathntect) 1, beta-5, the UBC 2, and the protein-phosphoglycerate promoter (PG1471). The cell-free specific promoter can enable the gene expression cassette to have better universality and expression efficiency.
In some preferred embodiments, the promoter is CMV, EF1a, SFH, CAG, CBh, UBC, SFFV, SV, RSV, mCMV, GAPDH, PGK, CASI, SMVP, GUSB (hGBp) or UCOE.
In some embodiments, the repressible operator is inserted downstream with a gene of interest;
and can negatively impact viral packaging when the gene of interest is expressed.
In the present invention, "negatively affecting viral packaging" refers to reducing the packaging efficiency of the virus, slowing or preventing AAV from virulence, or directly killing packaging cells. On the other hand, if the target gene can be transcribed and translated in a large amount, excessive RNA transcription and protein translation related enzymes and resources are occupied, so that transcription and translation of each functional gene of the recombinant virus are indirectly inhibited. The two aspects of the effect reduce the production efficiency of recombinant viruses in packaging cells and do not allow to obtain sufficient viral titres.
In some embodiments, the gene of interest is cytotoxic to packaging cells.
In some embodiments, the gene of interest is selected from the group consisting of a suicide gene, an apoptosis gene (or apoptosis gene), and an oncogene.
The following suicide gene systems are known as tk-GCV system, CD-5-FC system, gpt-6-TX system, P450 2BI-CPA system, etc.
Apoptosis genes can also be replaced in this application with apoptosis genes, the following genes are listed without limitation: bcl-2 gene, P53 gene, cytochrome C gene, apoptosis-protein activator 1 gene (apoptotic proteaseactivating factor, apaf-1), caspase family protein gene, etc.
The Caspase family proteins can be preferably classified into those that initiate caspases (caspases 8, 9, 10) and those that perform caspases (caspases 3, 6, 7).
Major types of oncogenes include: tyrosine kinases (e.g., src), other protein kinases (e.g., raf), G proteins (e.g., ras), growth factors (e.g., sis), growth factor receptors (e.g., erbB), and proteins located in the nucleus (e.g., transcription factor MYC).
In some embodiments, the gene of interest is a cell cycle related gene. Such as Caspase family protein genes and MAPK signaling pathway related genes.
In some embodiments, the gene of interest directly inhibits replication and/or assembly of the virus.
For example, the product expressed by the gene of interest can directly cleave, interfere with, knock out, or bind to substances that inhibit viral replication and/or assembly of related elements (e.g., VA RNA gene, rap and cap gene products of E2A, E4, etc.), such as mirnas, sirnas, certain specific proteins or enzymes, etc.
In some embodiments, the gene of interest is selected from the group consisting of GPR78 gene, cdkn1a gene, and apodec gene.
The apodec (apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like protein) enzyme family is an apolipoprotein B messenger RNA editing enzyme-catalyzed polypeptide-like protein, a class of proteases that have been found in recent years to have deamination, and includes AID (activation-induced cytosine deaminase), apodec 1, apodec 2, apodec 3A-H, and apodec 4 families. The prior art has found that apodec has an inhibitory effect on adeno-associated viruses (Hui Chen et al, curret Biology, volume 16,Issue 5,7March 2006,Pages 480-485).
In some embodiments, the adeno-associated virus packaging vector further comprises at least one of a reporter gene, an enhancer, an internal ribosome entry site, a terminator, and a polyadenylation signal.
According to yet another aspect of the present invention, the present invention also relates to an adeno-associated virus packaging vector system comprising an adeno-associated virus packaging vector as described above and a vector carrying AAV rep, cap genes.
The vectors carrying the AAV rep, cap genes may be located on the same vector or on different vectors, or they may be located on the same vector as the adeno-associated viral packaging vector described above.
In some embodiments, it is an adeno-associated virus packaging vector system of any one of AAV 1-13.
In some embodiments, it further comprises a helper viral vector.
In some embodiments, the gene for the repressor is packaged in the helper virus vector.
In some embodiments, the helper viral vector is an adenovirus or a herpes virus helper viral vector.
According to a further aspect of the invention, the invention also relates to a method of packaging an adeno-associated virus comprising transferring an adeno-associated virus packaging vector system as described above into a host cell and packaging in the presence of said repressor.
In some embodiments, the cell is a HEK-293 cell or a derivative thereof capable of providing the E1 region of an adenovirus.
In some embodiments, the derivative is selected from at least one of HEK-293A, HEK-293S, HEK-293SG, HEK-293SGGD, HEK-293T, HEK-293T/17SF, HEK-293H, HEK-293E, HEK-293 6E, HEK-293F, HEK-293FT, HEK-293FTM, AAV-293, and GP 2-293.
Embodiments of the present invention will be described in detail below with reference to examples.
Example 1 selection of repressible operators
The present invention tests 4 transcription control systems in total: cymR-CuO, tryptophan operator, diphtheria toxin inhibitor regulation system, lactose operator.
Firstly, by introducing cis-acting elements of the four systems on a lentiviral vector (the vector is a shuttle vector, and the single transfected cells can be used as a universal eukaryotic vector, the conclusion of which is not limited to lentivirus), whether the effect of inhibiting the expression of exogenous genes in a eukaryotic system is achieved or not is tested.
1. Plasmid transfection
The method for transfecting the 293T by the plasmid specifically comprises the following steps:
1. the day before transfection, 293T cells were seeded into petri dishes; taking out the cell culture dish one hour before transfection, discarding the original cell culture medium, adding the Opti-MEM culture medium, and placing the cells back into the incubator; preparing a complex of a transfection reagent and a plasmid, comprising the steps of:
2. and (3) mixing one or more plasmids to be transfected in an Opti-MEM culture medium according to the required equal proportion, gently mixing, and standing to obtain plasmid diluent.
3. Dissolving a transfection reagent in an Opti-MEM culture medium, gently mixing, and standing to obtain a transfection reagent diluent; dripping the transfection reagent into the plasmid diluent, mixing gently while adding, and standing at room temperature for 15-25min to fully combine the DNA and the transfection reagent to form a stable transfection complex; taking out the cell culture dish, adding the prepared DNA-transfection reagent complex into the cell culture dish, and putting back into the incubator;
4. after 5-8 hours the medium was aspirated, washed with PBS solution and incubated with fresh complete medium, wherein tryptophan control group was replaced with complete medium containing 0.3mM tryptophan and diphtheria toxin inhibition subsystem was supplemented with ferrous ions.
2. Verification of different repressed operating subsystems
CymR-CuO System
The CymR-CuO system is a class of regulatable expression systems. The principle is as follows:
the CymR protein can specifically bind to CuO elements in the absence of Cumate, thereby inhibiting transcription of genes. In the presence of Cumate, the CymR combined with Cumate will be detached from the CuO element, thereby allowing the gene to be expressed normally.
The invention constructs a series of promoter vectors of CMV-CuO, EF1a-CuO, SFH-CuO and CAG-CuO, and proves that the CuO element is matched with the CymR vector (pcDNA3.1-CymR) to effectively inhibit the expression of genes. Wherein the plasmid map of pcDNA3.1-CymR is shown in figure 1, and the plasmid map of CMV-CuO, EF1a-CuO, SFH-CuO and CAG-CuO promoter vectors are shown in figures 2-5 in sequence.
When the lentiviral vector with CuO original was co-transferred to 293T cells with pcDNA3.1 empty vector and pcDNA3.1-CymR respectively, fluorescent photographing was performed after 24 hours, and it was found that under the action of CymR protein, the expression efficiency of CMV-CuO, EF1a-CuO, SFH-CuO and CAG-CuO promoters was significantly inhibited (FIG. 6).
2. Tryptophan handling subsystem
In the presence of tryptophan, trp repressor (TrpR) protein can specifically bind to TrpO elements, thereby inhibiting transcription of genes. This system was found in prokaryotic cells and has long been used as an demonstration of gene regulation, but it has not been used in eukaryotic cells because tryptophan is necessary in mammalian cell culture.
A series of promoter vectors such as CMV-TrpO, EF1a-TrpO, SFH-TrpO, CAG-TrpO and the like are constructed, and the fact that TrpO elements are matched with a TrpR vector (pcDNA3.1-TrpR) can effectively inhibit gene expression in the presence of tryptophan is proved. The design mode of the plasmid patterns of the CMV-TrpO plasmid patterns shown in FIG. 7 and the plasmid patterns of the EF1a-TrpO, SFH-TrpO and CAG-TrpO promoter vectors can be referred to in FIGS. 2-5 on the basis of FIG. 7, and will not be repeated.
The vector with TrpO original was co-transferred with pcDNA3.1 empty vector and pcDNA3.1-TrpR 293T cells, and 0.3mM tryptophan was added at the same time, and fluorescent photographing was performed after 24 hours, which revealed that under the action of TrpR protein, the expression efficiencies of CMV-TrpO, EF1a-TrpO, SFH-TrpO and CAG-TrpO promoters were significantly inhibited (FIG. 8).
3. Diphtheria toxin inhibitor regulation and control system
The diphtheria toxin inhibitor DtxR protein can specifically bind to ToxO elements, thereby inhibiting transcription of the gene. The invention constructs a series of promoter vectors such as CMV-ToxO, EF1a-ToxO, SFH-ToxO, CAG-ToxO and the like, and under the condition that 293T cells are respectively co-transformed with pcDNA3.1 empty vector and pcDNA3.1-DtxR and divalent iron ions are added, no obvious inhibition effect on target genes is found (figure 10).
The plasmid patterns of EF1a-ToxO are shown in FIG. 9, and the design modes of the plasmid patterns of CMV-ToxO, SFH-ToxO and CAG-ToxO promoter vectors can be referred to in FIGS. 2-5 on the basis of FIG. 9, and are not repeated.
4. Lactose operation subsystem
Lactose operator inhibitor LacI can specifically bind to LacO elements, thereby inhibiting transcription of genes. In the presence of IPTG, it binds to and undergoes a conformational change, losing binding capacity and the downstream gene begins to express. Lactose operators are widely used in prokaryotic cells for the inducible expression of foreign genes.
The invention constructs a series of promoter vectors such as CMV-LacO, EF1a-LacO, SFH-LacO, CAG-LacO and the like, and the vectors are respectively co-transformed with pcDNA3.1 empty vector and pcDNA3.1-LacI into 293T cells, and no obvious inhibition effect on target genes is found (figure 12).
The plasmid patterns of SFH-LacO are shown in FIG. 11, and the design modes of the plasmid patterns of CMV-LacO, EF1a-LacO and CAG-LacO promoter vectors can be referred to in FIGS. 2-5 on the basis of FIG. 11, and are not repeated.
3. Insertion position optimization of repressible operator elements
TrpO element
The inventors designed two promoters at different distance of TATA Box relative to CMV, as shown in FIG. 13, the plasmids were co-transfected with pcDNA3.1-TrpR (0.1 mM tryptophan was added), fluorescence was observed after 48 hours, and experiments showed that different inhibition effects were generated at different positions from TATA Box (see FIG. 14), and TrpO v2 promoter sequences with better inhibition effects were used later in the invention.
CuO element
The distance between the CuO element and the TATA Box of CMV is different, 3 promoters are designed, as shown in FIG. 15, plasmids are co-transfected with pcDNA3.1-CymR, fluorescence is observed after 48 hours, experiments show that different inhibition effects can be generated due to different positions of the CuO element from the TATA Box (see FIG. 16), and the CuO v1 promoter sequence with better inhibition effects is used later in the invention.
Example 2 AAV viral packaging and viral titre detection
1. AAV viral packaging
A method for viral packaging using an adeno-associated viral vector (AAV), comprising:
1. the day before transfection, 293T cells were seeded into petri dishes; taking out the cell culture dish one hour before transfection, discarding the original cell culture medium, adding the Opti-MEM culture medium, and placing the cells back into the incubator; preparing a complex of a transfection reagent and a plasmid, comprising the steps of:
2. dissolving viral vector plasmids (serotype plasmids, helper plasmids and shuttle plasmids) to be transfected in an Opti-MEM culture medium, gently mixing, and standing to obtain plasmid diluent; the serotype plasmid is pAAV9, pAAV9-CMV-CymR or pAAV 9-CMV-TrpR; helper plasmids are pHelper, pHelper-CMV-CymR or pHelper-CMV-TrpR; the shuttle plasmid is pAAV-CMV-GPR78-3 xFLAG-WPRE, pAAV-CMV-CuO-GPR78-3 xFLAG-WPRE or pAAV-CMV-TrpO-GPR78-3 xFLAG-WPRE (pAAV-CMV-Cdkn 1a-3 xFLAG-WPRE, pAAV-CMV-CuO-Cdkn1a-3 xFLAG-WPRE or pAAV-CMV-TrpO-Cdkn1a-3 xFLAG-WPRE).
3. Dissolving a transfection reagent in an Opti-MEM culture medium, gently mixing, and standing to obtain a transfection reagent diluent; dripping the transfection reagent into the plasmid diluent, mixing gently while adding, and standing at room temperature for 15-25min to fully combine the DNA and the transfection reagent to form a stable transfection complex; taking out the cell culture dish, adding the prepared DNA-transfection reagent complex into the cell culture dish, and putting back into the incubator;
4. after 5-8 hours the medium was aspirated, washed with PBS solution and fresh complete medium was added for cultivation, wherein the tryptophan operon group (pAAV-CMV-TrpO-GPR 78-3 xFLAG-WPRE or pAAV-CMV-TrpO-Cdkn1a-3 xFLAG-WPRE) was replaced with complete medium containing 0.3mM tryptophan.
5. After 60-72 hours of transfection, the cells were repeatedly blown with a gun head, all cells were completely detached from the dish, and all cell samples and supernatants were collected.
6. Repeatedly freezing and thawing the collected cell sample at-80 ℃ and 37 ℃, centrifuging, collecting the supernatant, and removing cell fragments by using a PVDF filter with the diameter of 0.45 μm; and then purifying the collected AAV by using an AAV purification kit to obtain AAV, and preserving the AAV in a refrigerator at-80 ℃.
2. AAV viral titer detection
1. Extracting AAV virus DNA;
taking 20 mu L of concentrated virus liquid, adding 1 mu L of RNase-free DNase, mixing uniformly, incubating for 30min at 37 ℃, centrifuging at 10000rpm for 10min, taking 20 mu L of supernatant, adding 80 mu L of dilution Buffer into another sterile tube, mixing uniformly, and reacting for 10min in a metal bath at 100 ℃. Naturally cooling to room temperature, adding 3 mu L of proteinase K, incubating at 37 ℃ for 60min, reacting in a metal bath at 100 ℃ for 10min, and cooling to room temperature.
2. Real-time PCR detection titer
The samples were diluted and used as templates to determine AAV virus titers using the Real-time PCR assay (using primers F: ctatgtggatacgctgcttta R: cataaagagacagcaaccagg). Real-time PCR was performed on an ABI7500 instrument. Reagent SYBR Master Mixture was used from TAKARA corporation.
1. The reaction system was configured in the following proportions:
SYBR premix ex taq：10μl；
ROX：0.4μl；
upstream primer (25. Mu.M): 0.5 μl;
downstream primer (25. Mu.M): 0.5 μl;
Genomic DNA：2.0μl；
6.6 μl of water;
2. the program was set to two-step Real-Time quantification. Pre-denaturation at 95 ℃,15S, followed by denaturation at 95 ℃,5S, annealing extension at 60 ℃,34S for 40 cycles. The absorbance was read each time during the extension phase.
The PCR procedure was:
Cycle 1：(1X)
Step 1：95.0℃for 00：15；
Cycle 2：(40X)
Step 1：95.0℃for 00：05
Step 2：60.0℃for 00：34；
data collection and real-time analysis are enabled.
3. And (5) preparing a melting curve. After the PCR was completed, the sample was denatured at 95℃for 1min. Then cooled to 55℃to allow the DNA double strand to bind well. Starting from 55 ℃ to 95 ℃, each step was increased by 0.5 ℃ and held for 30S while the absorbance was read.
The procedure for preparing the dissolution profile is:
Cycle 3：(1X)
Step 1：95.0℃for 01:00；
Cycle 4：(1X)
Step 1：55.0℃for 01:00；
Cycle 5：(81X)
Step 1：55.0℃-95.0℃for 00:30
after 2 cycles the set point temperature was raised by 0.5 ℃.
Example 3 AAV viral vectors express GPR78 (glucose-regulated protein,78 kDa)
Gene
GRP78 is one of the chaperones that functions to assist in folding the newly formed protein molecule into the correct three-dimensional configuration. In viral production we found that the construction of the GPR78 gene into normal AAV vectors was essentially unavailable as a viral particle, consistent with current reports (Maria C.Guimaro et al Human Gene Therapy, DOI: 10.1089/hum.2019.249); the use of CMV-CuO and CMV-TrpO promoters in combination with inhibition of protein expression can effectively solve the problem of toxicity (FIG. 17). Plasmid maps of the plasmids pAAV-CMV-GPR78-3 xFLAG-WPRE, pAAV-CMV-CuO-GPR78-3 xFLAG-WPRE, and pAAV-CMV-TrpO-GPR78-3 xFLAG-WPRE used in this example are shown in FIGS. 18, 19 and 20 in this order.
Example 4 expression of Cdkn1a Gene by AAV viral vectors
In viral production we found that the total amount of viral particles obtained by constructing the Cdkn1a gene into a common AAV vector was very low. The use of CMV-CuO and CMV-TrpO promoters in combination with the expression of the inhibitor protein can effectively solve the problem of virus production, and the detection result of the virus titer is shown in FIG. 21. The plasmid maps of the plasmids pAAV-CMV-Cdkn1a-3 xFLAG-WPRE, pAAV-CMV-CuO-Cdkn1a-3 xFLAG-WPRE and pAAV-CMV-TrpO-Cdkn1a-3 xFLAG-WPRE used in this example are shown in FIGS. 22, 23 and 24.
The technical features of the above-described embodiments may be arbitrarily combined, and all possible combinations of the technical features in the above-described embodiments are not described for brevity of description, however, as long as there is no contradiction between the combinations of the technical features, they should be considered as the scope of the description.
The above examples illustrate only a few embodiments of the invention, which are described in detail and are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.
Claims (19)
1. An adeno-associated virus packaging vector system for transferring into host cells, comprising an adeno-associated virus packaging vector and a vector carrying AAV rep and cap genes, wherein the adeno-associated virus packaging vector is provided with two ITR fragments and a gene expression cassette inserted between the two ITR fragments;
the gene expression cassette comprises a promoter, a repressor operator and optionally a gene of interest, connected in sequence; the desired gene, when expressed, can negatively impact viral packaging;
the repressed operator is capable of repressing expression of a gene of interest downstream thereof in the presence of a repressor;
the repressed operator is selected from TrpO elements in a tryptophan operation subsystem or CuO elements in a Cumate-CuO controllable system;
the TrpO element in the tryptophan operator system is 18 nucleotides or less from the TATABOX of the promoter at the insertion position of the gene expression cassette; the sequence of the TrpO element is gaactagttaactagtacg;
the insertion position of a CuO element in the Cumate-CuO adjustable system is 40-50 nucleotides away from the TATABOX of the promoter; the sequence of CuO elements is aacaaacagacaatctggtctgtttgta;
wherein the host cell is a HEK-293 cell or a derivative thereof capable of providing an adenovirus E1 region;
the adeno-associated virus packaging vector system further comprises a helper virus vector in which the gene for the repressor is packaged.
2. The adeno-associated virus packaging vector system according to claim 1, wherein the derivative capable of providing the adenovirus E1 region is selected from at least one of HEK-293A, HEK-293S, HEK-293SG, HEK-293SGGD, HEK-293T, HEK-293T/17SF, HEK-293H, HEK-293E, HEK-293-6E, HEK-293F, HEK-293FT, HEK-293FTM, AAV-293 and GP 2-293.
3. The adeno-associated virus packaging vector system of claim 2, wherein the TrpO element in the tryptophan operator is 4 nucleotides from the TATABOX of the promoter at the insertion site of the gene expression cassette.
4. The adeno-associated virus packaging vector system of claim 2, wherein the CuO element in the Cumate-CuO regulatable system is 45 nucleotides from the TATA BOX of the promoter at the insertion site of the gene expression cassette.
5. The adeno-associated virus packaging vector system of claim 1, wherein the promoter is CMV, EF1a, SFH, CAG, CBh, UBC, SFFV, SV, RSV, mCMV, GAPDH, PGK, CASI, SMVP, GUSB, or UCOE.
6. The adeno-associated virus packaging vector system of claim 1, wherein the adeno-associated virus packaging vector comprises a sequence of CMV-TrpO v2 or CMV-CuO v 1;
wherein the CMV-TrpO v2 sequence is as follows:
cgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgccaatagggactttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgaccttatgggactttcctacttggcagtacatctacgtattagtcatcgctattaccatggtgatgcggttttggcagtacatcaatgggcgtggatagcggtttgactcacggggatttccaagtctccaccccattgacgtcaatgggagtttgttttggcaccaaaatcaacgggactttccaaaatgtcgtaacaactccgccccattgacgcaaatgggcggtaggcgtgtacggtgggaggtctatataagcagaactagttaactagtacgtcagatcgcctggagacgccatccacgctgttttgacctccatagaagacaccgactctac；
the CMV-CuO v1 sequence is as follows:
Cgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgccaatagggactttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgaccttatgggactttcctacttggcagtacatctacgtattagtcatcgctattaccatggtgatgcggttttggcagtacatcaatgggcgtggatagcggtttgactcacggggatttccaagtctccaccccattgacgtcaatgggagtttgttttggcaccaaaatcaacgggactttccaaaatgtcgtaacaactccgccccattgacgcaaatgggcggtaggcgtgtacggtgggaggtctatataagcagagctcgtttagtgaaccgtcagatcgcctggagacgttgaaaacaaacagacaatctggtctgtttgtattataagtaaggactagt。
7. the adeno-associated virus packaging vector system according to claim 1, wherein the gene of interest is cytotoxic to packaging cells.
8. The adeno-associated virus packaging vector system according to claim 7, wherein the gene of interest is selected from the group consisting of suicide genes, apoptosis genes and oncogenes.
9. The adeno-associated virus packaging vector system according to claim 1, wherein the gene of interest is a cell cycle-associated gene, and the cell cycle-associated gene is a Caspase family protein gene or a MAPK signal pathway-associated gene.
10. The adeno-associated virus packaging vector system according to claim 1, wherein the gene of interest directly inhibits replication and/or assembly of the virus.
11. The adeno-associated viral packaging vector system according to claim 1, wherein the gene of interest is selected from the group consisting of GPR78 gene, cdkn1a gene and apodec gene.
12. The adeno-associated virus packaging vector system according to any one of claims 1-11, further comprising at least one of a reporter gene, an enhancer, an internal ribosome entry site, a terminator and a polyadenylation signal.
13. The adeno-associated viral packaging vector system according to claim 10, wherein the expressed product of the gene of interest is a substance capable of directly cleaving, interfering, knocking out, or binding to a relevant element inhibiting viral replication and/or assembly, which is the VA RNA gene of E2A or E4 or is the rap or cap gene product.
14. The adeno-associated virus packaging vector system according to any one of claims 1-11, which is an adeno-associated virus packaging vector system of any one of AAV 1-13.
15. The adeno-associated virus packaging vector system according to any one of claims 1-11 or claim 14, wherein the repressor operator is a TrpO element in the tryptophan operator system and the repressor is a TrpR protein; alternatively, the repressed operator is a CuO element in the Cumate-CuO controllable system, and the repressor is a CymR protein.
16. The adeno-associated virus packaging vector system according to any one of claims 1-11, wherein the helper virus vector is an adenovirus or a herpes virus helper virus vector.
17. A method of packaging an adeno-associated virus comprising transferring an adeno-associated virus packaging vector system according to any one of claims 1 to 16 into a host cell and packaging in the presence of said repressor;
wherein, when selecting TrpO element in the tryptophan operating subsystem, the repressor is TrpR protein, and tryptophan exists when packaging adeno-associated virus; or when CuO elements in the Cumate-CuO adjustable system are selected, the repressor is CymR protein, and the cumic acid is not present when the adeno-associated virus is packaged;
the host cell is a HEK-293 cell or a derivative thereof capable of providing the E1 region of the adenovirus.
18. The method of claim 17, wherein said adenovirus packaging vector system is transfected into said host cell.
19. The method of packaging adeno-associated virus according to claim 17, wherein the derivative is at least one selected from HEK-293A, HEK-293S, HEK-293SG, HEK-293SGGD, HEK-293T, HEK-293T/17SF, HEK-293H, HEK-293E, HEK-293-6E, HEK-293F, HEK-293FT, HEK-293FTM, AAV-293 and GP 2-293.
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Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107466325A (en) * | 2015-03-03 | 2017-12-12 | 泰莱托恩基金会 | Multi-carrier system and its application |
| WO2019144186A1 (en) * | 2018-01-23 | 2019-08-01 | Southern Eye Equipment | Expression vector and method |
| CN110484565A (en) * | 2019-09-03 | 2019-11-22 | 和元生物技术（上海）股份有限公司 | A kind of suicide gene system and its application based on gland relevant viral vector transformation |
| CN110892064A (en) * | 2017-07-25 | 2020-03-17 | 牛津遗传学有限公司 | Adenoviral vectors |
| WO2020132059A1 (en) * | 2018-12-21 | 2020-06-25 | Lonza Walkersville, Inc. | Adeno-associated virus (aav) producer cell line and related methods |
| WO2020161484A1 (en) * | 2019-02-05 | 2020-08-13 | Oxford Genetics Limited | Inducible aav system comprising cumate operator sequences |
| CN111808175A (en) * | 2020-09-01 | 2020-10-23 | 和元生物技术（上海）股份有限公司 | Recombinant adeno-associated virus particles and application thereof |
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|---|---|---|---|---|
| US10392622B2 (en) * | 2014-08-01 | 2019-08-27 | The Trustees Of The University Of Pennsylvania | Compositions and methods for self-regulated inducible gene expression |
-
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Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107466325A (en) * | 2015-03-03 | 2017-12-12 | 泰莱托恩基金会 | Multi-carrier system and its application |
| CN110892064A (en) * | 2017-07-25 | 2020-03-17 | 牛津遗传学有限公司 | Adenoviral vectors |
| WO2019144186A1 (en) * | 2018-01-23 | 2019-08-01 | Southern Eye Equipment | Expression vector and method |
| WO2020132059A1 (en) * | 2018-12-21 | 2020-06-25 | Lonza Walkersville, Inc. | Adeno-associated virus (aav) producer cell line and related methods |
| WO2020161484A1 (en) * | 2019-02-05 | 2020-08-13 | Oxford Genetics Limited | Inducible aav system comprising cumate operator sequences |
| CN110484565A (en) * | 2019-09-03 | 2019-11-22 | 和元生物技术（上海）股份有限公司 | A kind of suicide gene system and its application based on gland relevant viral vector transformation |
| CN111808175A (en) * | 2020-09-01 | 2020-10-23 | 和元生物技术（上海）股份有限公司 | Recombinant adeno-associated virus particles and application thereof |
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