CN112501209A - Adeno-associated virus packaging method with controllable expression of exogenous gene - Google Patents
Adeno-associated virus packaging method with controllable expression of exogenous gene Download PDFInfo
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- CN112501209A CN112501209A CN202011419528.4A CN202011419528A CN112501209A CN 112501209 A CN112501209 A CN 112501209A CN 202011419528 A CN202011419528 A CN 202011419528A CN 112501209 A CN112501209 A CN 112501209A
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Abstract
The invention relates to the field of molecular biology, in particular to an adeno-associated virus packaging method capable of realizing controllable expression of exogenous genes. The adeno-associated virus packaging vector adopted by the method has two ITR fragments and a gene expression cassette inserted between the two ITR fragments; the gene expression cassette comprises a promoter, a repressible operator and an optional target gene which are connected in sequence; when a repressor is present, the repressible operator is capable of repressing the expression of the gene of interest downstream thereof. The invention creatively integrates the repression type 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 the 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 capable of realizing controllable expression of exogenous genes.
Background
Viral vectors can bring genetic material into cells by utilizing the molecular mechanisms by which viruses bind cell surface receptors with high affinity and transmit their genomes into certain cell types for infection. Widely applied to basic research, gene therapy or vaccines.
Adeno-associated virus (AAV), also known as adeno-associated virus, belongs to the genus dependovirus of the family parvoviridae, is the single-stranded DNA-deficient virus of the simplest structure currently found, and requires a helper virus (usually adenovirus) to participate in replication. It encodes the cap and rep genes in inverted repeats (ITRs) at both ends. ITRs are crucial for replication and packaging of viruses. The cap gene encodes the viral capsid protein, 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 readily integrates 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 foreign genes in animal bodies 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 neuroscience research, gene therapy of diseases, and other fields. 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 widely applied to the aspects of gene function research, disease model construction, gene knock-out mouse preparation and the like.
At present, the research of virus vectors focuses on the aspects of infection capacity, specificity, packaging capacity and the like, but the influence of packaged foreign Genes (GOI) on viruses is rarely concerned, and in practice, a considerable part of foreign genes cannot obtain effective virus particles. This is because, in the virus packaging process, the foreign gene is usually driven by a strong promoter, and its expression has an unexpected influence on the cell state, the packaging of the virus, the virus production, and other processes. It is not uncommon that the virus is not or is low virulent because of a foreign gene, for example, during packaging of adeno-associated virus overexpressing GPR78(NM — 080819), GPR78 expression renders the vector unavailable for virion production.
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 repressible operator and an optional target gene which are connected in sequence;
when a repressor is present, the repressible operator is capable of repressing the expression of the gene of interest downstream thereof.
According to a further aspect of the invention, the invention also relates to an adeno-associated virus packaging vector system, which comprises the adeno-associated virus packaging vector as described above and a vector carrying AAV rep, cap genes.
According to a further aspect of the present invention, the present invention also relates to a packaging method for adeno-associated virus, wherein the packaging method comprises transferring the adeno-associated virus packaging vector system into a host cell, and packaging the vector in the presence of the repressor.
Compared with the prior art, the invention has the beneficial effects that:
the present invention has the advantage that it is not necessary to judge whether the foreign gene has an effect on the adeno-associated virus, because the repression system does not function and the expression of the target gene is not affected when no repressor is present. When the toxicity is obviously lower than the average level, the virus packaging process for inhibiting the foreign gene can be carried out by matching with the auxiliary plasmid containing the expression of the repressor protein, so that the influence of the foreign gene is eliminated, the yield of the specific virus is improved, a new shuttle vector does not need to be reconstructed, and a large amount of time, labor 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 used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.
FIGS. 1 to 5 are plasmid maps of pcDNA3.1-CymR, CMV-CuO, EF1a-CuO, SFH-CuO, CAG-CuO vectors in sequence in one embodiment of the present invention;
FIG. 6 shows the results of verifying the repression efficiency of CymR-CuO in a lentiviral vector in one embodiment of the present invention;
FIG. 7 is a CMV-TrpO plasmid map in one embodiment of the present invention;
FIG. 8 shows the results of verifying the repression efficiency of a tryptophan manipulation subsystem in a lentiviral vector in accordance with one embodiment of the present invention;
FIG. 9 is a EF1a-ToxO plasmid map according to an embodiment of the present invention;
FIG. 10 shows verification of repression efficiency of a diphtheria toxin repressor regulation system in a lentiviral vector in accordance with one embodiment of the invention;
FIG. 11 is a map of an SFH-LacO plasmid according to an embodiment of the present invention;
FIG. 12 shows the results of validation of repression efficiency of a lactose manipulation subsystem in a lentiviral vector in accordance with one embodiment of the present invention;
FIG. 13 is a schematic representation of the insertion position of a TrpO element relative to the TATA Box of the CMV in one embodiment of the present invention;
FIG. 14 is a graph showing the effect of different insertion positions of a TrpO element on repression efficiency in one embodiment of the invention;
FIG. 15 is a schematic diagram of the insertion position of a CuO element relative to the TATA Box of CMV in one embodiment of the present invention;
FIG. 16 is a graph illustrating the effect of different insertion positions of CuO elements on the suppression efficiency in one embodiment of the present invention;
FIG. 17 shows the results of testing viral titers of AAV viral vector-expressed GPR78 in one embodiment of the invention;
FIGS. 18 to 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 this order, according to an embodiment of the present invention;
FIG. 21 shows the result of testing the virus titer of AAV viral vector expressing Cdkn1a in one embodiment of the present invention;
FIGS. 22 to 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 this order according to an embodiment of the present invention;
fig. 25 is a schematic diagram of the principle of the present invention.
Detailed Description
Reference will now 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. In fact, it will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the scope or spirit of the invention. For instance, features illustrated or described as part of one embodiment, can be used on another embodiment to yield a still further embodiment.
It is therefore intended that the present invention cover the modifications and variations of this invention provided they come within the scope of the appended claims and their equivalents. Other objects, features and aspects of the present invention are disclosed in or are 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 repressible operator and an optional target gene which are connected in sequence;
when a repressor is present, the repressible operator is capable of repressing the expression of the gene of interest downstream thereof.
The invention can control the expression of the target gene through the repression operator, thereby selectively inhibiting the expression of the exogenous gene in the adeno-associated virus packaging cell, avoiding the negative influence of the exogenous gene on the adeno-associated virus packaging and production, improving the production efficiency and titer of the virus, and simultaneously not reducing the expression level of the exogenous gene in the target cell, influencing the treatment effect or researching the function of the exogenous gene.
The term "vector" as used herein refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted. When a vector is capable of expressing 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, and the genetic material elements carried thereby are expressed in the host cell. The vector is preferably a plasmid, but is not limited thereto, and may be a virus or the like, for example, as shown in patent publication WO2017124588a1, international publication No. 7/27/2017.
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 inserted at a position less than or equal to 18 nucleotides, 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.
In some embodiments, the Cumate-CuO regulatable system has an insertion position of the CuO element from 40 to 50 nucleotides, such as 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 defined as 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 should be noted that it is generally considered that the promoter core region is within 8 nucleotides (including 8 th nucleotide) of TATA BOX. It is therefore preferred that the distance between the TrpO element and the TATA BOX of the promoter is greater than 8 nucleotides, and that the element may be closer to the TATA BOX when it has an overlapping sequence with the 8 nucleotides.
A "promoter" is a DNA sequence that directs the binding of RNA polymerase and thereby initiates RNA synthesis. The promoters used 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 limited variety of cell and tissue types, respectively. In particular embodiments, it may be desirable to use cell, cell type, cell lineage, or tissue specific expression control sequences 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 subpopulation of cell types, cell lineages, or tissues, or at a particular developmental stage).
Illustrative examples of tissue-specific promoters include, but are not limited to: b29 promoter (B cell expression), run transcription factor (CBFa2) 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), myodesmin promoter (muscle cell expression), elastase I promoter (pancreatic acinar cell expression), endoglin 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-2 b (ITGA2B) promoter (megakaryocytes), intracellular adhesion molecule 2(ICAM-2) promoter (endothelial cells), interferon-beta (IFN-beta) promoter (hematopoietic cells), keratin 5 promoter (keratinocyte expression), Myoglobin (MB) promoter (muscle cell expression), myogenic differentiation I (MYOD1) promoter (muscle cell expression), nephroprotein promoter (podocyte expression), bone gamma-carboxyglutamic acid protein 2(OG-2) promoter (osteoblast expression), 3-keto acid CoA transferase 2B (Oxct2B) promoter (haploid sperm cell expression), surface-activated 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, Cytomegalovirus (CMV) very early promoter, viral simian virus 40(SV40) (e.g., early or late), moloney murine leukemia virus (MoMLV) LTR promoter, Rous Sarcoma Virus (RSV) LTR, Herpes Simplex Virus (HSV) (thymidine kinase) promoter, H5, P7.5, and Pll promoter of vaccinia virus, elongation factor l- α (EFla) promoter, early growth response i (egrl), ferritin H (ferh), ferritin l (ferl), 3_ glyceraldehyde phosphate dehydrogenase (GAPDH), eukaryotic translation initiation factor 4A1 (EIF4A1), heat shock 70kDa protein 5(HSPA5), heat shock protein 90- β member 1(HSP90B1), heat shock protein 70kDa (HSP70), β -kinesin (β -KIN), human R0SA26 locus (irs et al, ion et al, seq. (2007) Nature Biotechnology25,1477-1482), the ubiquitin C promoter (UBC), the phosphoglycerate kinase-1 (PGK) promoter, the cytomegalovirus enhancer/chicken β -actin (CAG) promoter, and the β -actin promoter. The promoter without cell specificity can ensure that the gene expression cassette has better universality and expression efficiency.
In some preferred embodiments, the promoter is CMV, EF1a, SFH, CAG, CBh, UBC, SFFV, SV40, RSV, mCMV, GAPDH, PGK, CASI, SMVP, GUSB (hGBp), or UCOE.
In some embodiments, the repressible operator has a gene of interest inserted downstream;
and negatively affect viral packaging when the gene of interest is expressed.
In the present invention, "negatively affecting viral packaging" means reducing the packaging efficiency of the virus, slowing or preventing AAV virus virulence, or directly killing packaging cells. On the other hand, if the target gene can be transcribed and translated in a large amount, it will occupy excessive RNA transcription and protein translation related enzymes and resources of the cell, thereby indirectly inhibiting the transcription and translation of each functional gene of the recombinant virus. Both effects reduce the efficiency of recombinant virus production in packaging cells and do not allow sufficient virus titers to be obtained.
In some embodiments, the gene of interest is cytotoxic to the packaging cell.
In some embodiments, the gene of interest is selected from the group consisting of a suicide gene, an apoptotic gene (or programmed cell death gene), and an oncogene.
The common suicide gene systems can be a tk-GCV system, a CD-5-FC system, a gpt-6-TX system, a P4502 BI-CPA system and the like.
Apoptosis genes may also be replaced in this application with programmed cell death genes, the following being listed without limitation: bcl-2 gene, P53 gene, cytochrome C gene, apoptosis protein activator 1 gene (Apaf-1), Caspase family protein gene, etc.
Caspase family proteins can preferably be divided into initiation (Caspase8, 9, 10) and execution (Caspase 3, 6, 7) caspases.
The main 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 associated gene. Such as Caspase family protein genes and MAPK signaling pathway related genes.
In some embodiments, the gene of interest directly inhibits viral replication and/or assembly.
For example, the expressed product of the gene of interest can directly cleave, interfere with, knock out, or bind to a substance that inhibits a relevant element of viral replication and/or assembly (e.g., VA RNA gene of E2A, E4, rap and cap gene products, etc.), such as miRNA, siRNA, certain specific proteins or enzymes, etc.
In some embodiments, the gene of interest is selected from the group consisting of a GPR78 gene, a Cdkn1a gene, and an APOBEC gene.
The APOBEC (apolipoprotein B mRNA-inhibiting enzyme catalytic polypeptide-like protein) enzyme family is polypeptide-like protein catalyzed by apolipoprotein B messenger RNA ediase, and is a class of proteases which are found to have deamination in recent years and comprise AID (activation-induced cytosine deaminase), APOBEC1, APOBEC2, APOBEC3A-H and APOBEC4 families. It has been found in the prior art that APOBEC has inhibitory effect on adeno-associated virus (Hui Chen et al, Curret Biology, Volume 16, Issue 5,7March 2006, Pages 480-.
In some embodiments, the adeno-associated viral packaging vector further comprises at least one of a reporter, an enhancer, an internal ribosome entry site, a terminator and a polyadenylation signal.
According to a further aspect of the invention, the invention also relates to an adeno-associated virus packaging vector system, which comprises the 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, the vector is an adeno-associated viral packaging vector system according to 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 viral vector.
In some embodiments, the helper viral vector is an adenovirus or a herpesvirus helper viral vector.
According to a further aspect of the present invention, the present invention also relates to a packaging method for adeno-associated virus, wherein the packaging method comprises transferring the adeno-associated virus packaging vector system into a host cell, and packaging the vector in the presence of the repressor.
In some embodiments, the cell is a HEK-293 cell or a derivative thereof capable of providing an adenoviral E1 region.
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 with reference to examples.
Example 1 selection of repressible operators
The invention totally tests 4 transcription regulation systems: CymR-CuO, a tryptophan operator, a diphtheria toxin suppressor regulation system and a lactose operator.
Whether the effect of inhibiting the expression of the foreign gene in a eukaryotic system exists or not is tested by introducing cis-acting elements of the four systems into a lentiviral vector (the vector is a shuttle vector, and the single transfected cell can be used as a general eukaryotic vector, and the conclusion is not limited to lentivirus).
First, plasmid transfection
The method for transfecting 293T by using the plasmid specifically comprises the following steps:
1. the day before transfection, 293T cells were seeded into culture dishes; taking out the cell culture dish one hour before transfection, discarding the original cell culture medium, adding an Opti-MEM culture medium, and putting the cells back to the culture box; preparing a complex of a transfection reagent and a plasmid comprising the steps of:
2. and mixing one or more plasmids to be transfected in equal proportion according to requirements, dissolving in an Opti-MEM culture medium, gently mixing uniformly, and standing to obtain a plasmid diluent.
3. Dissolving the transfection reagent in an Opti-MEM culture medium, gently mixing uniformly, and standing to obtain a transfection reagent diluent; dripping the transfection reagent diluent into the plasmid diluent, gently mixing while adding, and then placing at room temperature for 15-25min to ensure that the DNA and the transfection reagent are fully combined 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 returning the cell culture dish to the incubator;
4. after 5-8 hours the medium was aspirated, washed with PBS solution and incubated with fresh complete medium, wherein the tryptophan handle group required replacement of the complete medium with 0.3mM tryptophan and the diphtheria toxin inhibitor subsystem required the addition of divalent iron ions.
Validation of two, different thwarting-type operating subsystems
CymR-CuO system
The CymR-CuO system is a controllable expression system. The principle is as follows:
the CymR protein can be specifically combined with a CuO element in the absence of Cumate (cumic acid), so that the transcription of genes is inhibited. In the presence of Cumate, CymR bound to Cumate will dissociate from the CuO element, allowing normal gene expression.
The invention constructs a series of promoter vectors of CMV-CuO, EF1a-CuO, SFH-CuO and CAG-CuO, and proves that the CuO element can effectively inhibit the expression of genes by matching with a CymR vector (pcDNA3.1-CymR). Wherein the plasmid map of pcDNA3.1-CymR is shown in figure 1, and the plasmid maps of CMV-CuO, EF1a-CuO, SFH-CuO and CAG-CuO promoter vectors are sequentially shown in figures 2-5.
The lentiviral vector with the original CuO was co-transformed with pcDNA3.1 empty vector and pcDNA3.1-CymR into 293T cells, and fluorescence photographed after 24 hours, and it was found that the expression efficiencies of CMV-CuO, EF1a-CuO, SFH-CuO, and CAG-CuO promoters were all significantly inhibited under the action of CymR protein (FIG. 6).
2. Tryptophan operator system
In the presence of tryptophan, the protein Trp decompressor (TrpR) can be specifically combined with a TrpO element, thereby inhibiting the transcription of the gene. This system has been found in prokaryotic cells and has long been used as an explanation for gene regulation, but it has not been used in eukaryotic cells because tryptophan is essential in mammalian cell culture.
The invention constructs a series of promoter vectors such as CMV-TrpO, EF1a-TrpO, SFH-TrpO, CAG-TrpO and the like, and proves that the TrpO element can effectively inhibit the expression of genes by matching with a TrpR vector (pcDNA3.1-TrpR) in the presence of tryptophan. The CMV-TrpO plasmid map is shown in FIG. 7, and the design modes of the plasmid maps of the EF1a-TrpO, SFH-TrpO and CAG-TrpO promoter vectors can refer to FIGS. 2-5 on the basis of FIG. 7, and are not described again.
The vector with the original TrpO was cotransferred to 293T cells with the empty vector pcDNA3.1 and pcDNA3.1-TrpR, and 0.3mM tryptophan was added at the same time, and fluorescence was photographed after 24 hours, and it was found that the expression efficiencies of CMV-TrpO, EF1a-TrpO, SFH-TrpO, and CAG-TrpO promoters were significantly suppressed under the action of the TrpR protein (FIG. 8).
3. Diphtheria toxin inhibitor regulation system
The diphtheria toxin repressor DtxR protein can be specifically combined with a ToxO element, so that the transcription of genes is inhibited. 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 the promoter vectors are respectively cotransformed with pcDNA3.1 empty vector and pcDNA3.1-DtxR 293T cell and ferrous ions are added, no obvious inhibition effect on a target gene is found (figure 10).
Wherein the plasmid map of EF1a-ToxO is shown in FIG. 9, and the design mode of the plasmid maps of CMV-ToxO, SFH-ToxO and CAG-ToxO promoter vectors can refer to FIGS. 2-5 on the basis of FIG. 9, and is not described again.
4. Lactose handling subsystem
The lacI repressor protein can be specifically combined with LacO elements, so that the transcription of genes is inhibited. In the presence of IPTG, the IPTG binds to the protein and undergoes conformational change, the binding capacity is lost, and the downstream gene begins to express. Lactose operons 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 co-transfers the promoter vectors with pcDNA3.1 empty vector and pcDNA3.1-LacI to 293T cells respectively, and no obvious inhibition effect on target genes is found (figure 12).
The plasmid map of SFH-LacO is shown in FIG. 11, and the design mode of the plasmid maps of CMV-LacO, EF1a-LacO and CAG-LacO promoter vectors can refer to FIGS. 2-5 on the basis of FIG. 11, and is not described again.
Thirdly, optimization of insertion position of repressing type operator element
TrpO element
The TrpO element has different distances relative to the TATA Box of the CMV, two promoters are designed by the inventor, as shown in FIG. 13, the plasmid is co-transfected with pcDNA3.1-TrpR (0.1 mM tryptophan is added), fluorescence is observed after 48 hours, experiments show that different positions from the TATA Box can generate different inhibition effects (see FIG. 14), and the TrpO v2 promoter sequence with better inhibition effect is used subsequently in the invention.
CuO element
The distances of the CuO element relative to the TATA Box of the CMV are different, 3 promoters are designed, as shown in FIG. 15, the plasmid is co-transfected by matching pcDNA3.1-CymR, and 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 CuO v1 promoter sequences with better inhibition effects are used subsequently in the invention.
Example 2 AAV viral packaging and viral titer detection
AAV virus packaging
The method for virus packaging by using the adeno-associated virus (AAV) vector specifically comprises the following steps:
1. the day before transfection, 293T cells were seeded into culture dishes; taking out the cell culture dish one hour before transfection, discarding the original cell culture medium, adding an Opti-MEM culture medium, and putting the cells back to the culture box; preparing a complex of a transfection reagent and a plasmid comprising the steps of:
2. dissolving virus vector plasmids (serotype plasmids, Helper plasmids and shuttle plasmids) to be transfected into an Opti-MEM culture medium, gently mixing uniformly, and standing to obtain a plasmid diluent; the serotype plasmid is pAAV9, pAAV9-CMV-CymR or pAAV 9-CMV-TrpR; the Helper plasmid is 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-Cdkn1a-3 XFLAG-WPRE, pAAV-CMV-CuO-Cdkn1a-3 XFLAG-WPRE or pAAV-CMV-TrpO-Cdkn1a-3 XFLAG-WPRE).
3. Dissolving the transfection reagent in an Opti-MEM culture medium, gently mixing uniformly, and standing to obtain a transfection reagent diluent; dripping the transfection reagent diluent into the plasmid diluent, gently mixing while adding, and then placing at room temperature for 15-25min to ensure that the DNA and the transfection reagent are fully combined 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 returning the cell culture dish to the incubator;
4. after 5-8 hours the medium was aspirated, washed with PBS solution and cultured with fresh complete medium, the tryptophan operational group (pAAV-CMV-TrpO-GPR78-3 XFLAG-WPRE or pAAV-CMV-TrpO-Cdkn1a-3 XFLAG-WPRE) being replaced with complete medium containing 0.3mM tryptophan.
5. And after 60-72 hours of transfection, repeatedly blowing and beating the cells by using a gun head to ensure that all the cells completely fall off from the culture dish, and collecting all cell samples and supernatant.
6. Subjecting the collected cell sample to repeated freeze thawing at-80 deg.C and 37 deg.C, centrifuging, collecting supernatant, and removing cell debris with 0.45 μm PVDF filter; subsequently, the collected AAV is purified by an AAV purification kit to obtain AAV, and the AAV is stored in a refrigerator at-80 ℃.
Second, AAV virus titer detection
1. Extracting AAV virus DNA;
and (3) taking 20 mu L of concentrated virus solution, adding 1 mu L of RNase-free DNase, uniformly mixing, incubating for 30min at 37 ℃, centrifuging for 10min at 10000rpm, taking 20 mu L of supernatant, adding 80 mu L of diluted Buffer into another sterile tube, uniformly mixing, and carrying out metal bath reaction for 10min at 100 ℃. Naturally cooling to room temperature, adding 3 μ L proteinase K, incubating at 37 deg.C for 60min, reacting at 100 deg.C in metal bath for 10min, and cooling to room temperature.
2. Real-time PCR detection titer
The above samples were diluted and used as templates to determine AAV viral titers by the Real-time PCR assay (using primers: F: ctatgtggatacgctgcttta R: cataaagagacagcaaccagg). Real-time PCR was performed on an ABI7500 instrument. The reagent SYBR Master mix was used from TAKARA.
1. The reaction system is prepared according to the following proportion:
SYBR premix ex taq：10μl；
ROX：0.4μl；
upstream primer (25 μ M): 0.5 mul;
downstream primer (25 μ M): 0.5 mul;
Genomic DNA：2.0μl；
6.6 mul of water;
2. the procedure was set to two step Real-Time quantitation. Pre-denaturation 95 ℃ for 15S, followed by denaturation 95 ℃ for 5S, annealing extension 60 ℃ for 34S in each step, for a total of 40 cycles. Each time reading the absorbance value 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. A melting curve was prepared. After the PCR was completed, the mixture was denatured at 95 ℃ for 1 min. Then cooled to 55 ℃ to allow the DNA double strands to be fully bound. Starting at 55 ℃ to 95 ℃, each step was increased by 0.5 ℃ and held for 30S while absorbance was read.
The dissolution curve procedure was:
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
the set point temperature was increased by 0.5 ℃ after 2 cycles.
Example 3 AAV viral vector expressing GPR78(glucose-regulated protein,78kDa)
Gene
GRP78 is a type of chaperone protein that functions to assist the newly formed protein molecule in folding into the correct three-dimensional configuration. We found that the GPR78 Gene was constructed into viral particles that are essentially unavailable in common AAV vectors in virus production, consistent with existing reports (Maria C. Guimaro et al, Human Gene Therapy, DOI: 10.1089/hum.2019.249); the toxicity problem can be effectively solved by using CMV-CuO and CMV-TrpO promoters in combination with inhibiting the expression of proteins (FIG. 17). The 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 FIG. 18, FIG. 19 and FIG. 20 in this order.
Example 4 AAV viral vectors express Cdkn1a Gene
In virus production, we find that the total amount of virus particles obtained by constructing the Cdkn1a gene into a common AAV vector is very low. The CMV-CuO promoter and the CMV-TrpO promoter are matched with the expression of the suppressor protein, so that the toxicity problem can be effectively solved, and the detection result of the virus titer is shown in figure 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 FIG. 22, FIG. 23 and FIG. 24 in this order.
The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
Claims (19)
1. An adeno-associated virus packaging vector, which has two ITR fragments and a gene expression cassette inserted between the two ITR fragments;
the gene expression cassette comprises a promoter, a repressible operator and an optional target gene which are connected in sequence;
when a repressor is present, the repressible operator is capable of repressing the expression of the gene of interest downstream thereof.
2. The adeno-associated virus packaging vector according to claim 1, wherein the repressible operator is selected from the group consisting of a tryptophan operator and/or a Cumate-CuO regulatable system.
3. The adeno-associated virus packaging vector according to claim 2 wherein the TrpO element in the amino acid operator is inserted at a position less than or equal to 18 nucleotides from the TATA BOX of the promoter at the position of the gene expression cassette insertion.
4. The adeno-associated virus packaging vector according to claim 2, wherein the Cumate-CuO controllable system comprises a CuO element inserted 40-50 nucleotides from the TATA BOX of the promoter.
5. The adeno-associated viral packaging vector according to claim 1 wherein the promoter is CMV, EF1a, SFH, CAG, CBh, UBC, SFFV, SV40, RSV, mCMV, GAPDH, PGK, CASI, SMVP, GUSB or UCOE.
6. The adeno-associated virus packaging vector according to any one of claims 1 to 5 wherein a gene of interest is inserted downstream of the repressive operator;
and negatively affect viral packaging when the gene of interest is expressed.
7. The adeno-associated virus packaging vector according to claim 6 wherein the gene of interest is cytotoxic to the packaging cell.
8. The adeno-associated virus packaging vector according to claim 7 wherein the gene of interest is selected from the group consisting of a suicide gene, an apoptotic gene and an oncogene.
9. The adeno-associated viral packaging vector according to claim 6 wherein the gene of interest is a cell cycle associated gene.
10. The adeno-associated viral packaging vector according to claim 6 wherein the gene of interest directly inhibits viral replication and/or assembly.
11. The adeno-associated viral packaging vector according to claim 6 wherein the gene of interest is selected from the group consisting of the GPR78 gene, the Cdkn1a gene and the APOBEC gene.
12. The adeno-associated viral packaging vector according to any one of claims 1 to 5 and 7 to 11 further comprising at least one of a reporter gene, an enhancer, an internal ribosome entry site, a terminator and a polyadenylation signal.
13. An adeno-associated virus packaging vector system comprising the adeno-associated virus packaging vector according to any one of claims 1 to 12 and a vector carrying AAV rep and cap genes.
14. The adeno-associated virus packaging vector system according to claim 13, which is an adeno-associated virus packaging vector system according to any one of AAV 1-13.
15. The adeno-associated viral packaging vector system according to claim 13 or 14 further comprising a helper viral vector.
16. The adeno-associated virus packaging vector system according to claim 15 wherein the helper viral vector is an adenovirus or herpes virus helper viral vector.
17. A method for packaging an adeno-associated virus, comprising transferring the adeno-associated virus packaging vector system according to any one of claims 13 to 16 into a host cell, and packaging the vector in the presence of the repressor.
18. The method for packaging adeno-associated virus according to claim 17, wherein the host cell is a HEK-293 cell or a derivative thereof capable of providing the E1 region of adenovirus.
19. The method for packaging adeno-associated virus according to claim 18, wherein the derivative is at least one selected from the group consisting 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.
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Denomination of invention: Packaging method for adenovirus with controllable expression of exogenous genes Granted publication date: 20240213 Pledgee: Industrial Commercial Bank of China Ltd. Shanghai Zhangjiang science and Technology Branch Pledgor: OBIO TECHNOLOGY (SHANGHAI) Corp.,Ltd. Registration number: Y2024980041310 |