WO2022121855A1 - 外源基因可控表达的病毒包装载体及其包装方法 - Google Patents

外源基因可控表达的病毒包装载体及其包装方法 Download PDF

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WO2022121855A1
WO2022121855A1 PCT/CN2021/135817 CN2021135817W WO2022121855A1 WO 2022121855 A1 WO2022121855 A1 WO 2022121855A1 CN 2021135817 W CN2021135817 W CN 2021135817W WO 2022121855 A1 WO2022121855 A1 WO 2022121855A1
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gene
hek
packaging vector
viral packaging
promoter
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French (fr)
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杨兴林
杨佳丽
杨蕊菊
贾国栋
由庆睿
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和元生物技术（上海）股份有限公司
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Priority claimed from CN202011418490.9A external-priority patent/CN112501207B/zh
Priority claimed from CN202011419528.4A external-priority patent/CN112501209B/zh
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C12N15/864—Parvoviral vectors, e.g. parvovirus, densovirus
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material

Definitions

  • the present application relates to the field of biotechnology, and in particular, to a viral packaging vector with controllable expression of foreign genes and a packaging method thereof.
  • Viral vectors can bring genetic material into cells.
  • the principle is to use the molecular mechanism by which viruses bind to cell surface receptors with high affinity and transmit their genomes into certain types of cells for infection. Widely used in basic research, gene therapy or vaccines.
  • viruses commonly used as viral vectors include adeno-associated virus, adenovirus, and the like.
  • Adeno-associated virus also known as adeno-associated virus, belongs to the genus Dependovirus of the family Parvoviridae, and is the simplest single-stranded DNA-deficient virus discovered so far. virus) involved in replication. It encodes the cap and rep genes in two terminal inverted repeats (ITRs). ITRs play a decisive role in viral replication and packaging.
  • the cap gene encodes the viral capsid protein, and the rep gene is involved in the replication and integration of the virus.
  • AAV can infect a variety of cells. In the presence of the rep gene product, viral DNA readily integrates into human chromosome 19.
  • Adeno-associated virus has the characteristics of high safety, low immunogenicity, wide host range, and can mediate long-term stable expression of foreign genes in animals. It is one of the most promising vectors in the field of gene therapy.
  • AAV has an important role and great demand in fields such as neuroscience research and gene therapy of diseases.
  • AAV is used in gene therapy research for various diseases (including in vivo and in vitro experiments); at the same time, as a characteristic gene transfer vector, it is also widely used in gene function research, disease model construction, and gene preparation. Knockout mice, etc.
  • Adenovirus is particularly suitable for use as a viral vector.
  • Adenoviruses are non-enveloped viruses with a diameter of about 90 nm to 100 nm, containing a nucleocapsid and a linear double-stranded DNA genome.
  • the viral nucleocapsid contains a penton capsid and a hexon capsid.
  • a unique fiber is associated with each penton matrix and helps the virus attach to the host cell via the coxsackievirus-adenovirus receptor on the host cell surface. More than 50 strains of adenovirus serotypes have been identified, most of which cause respiratory infections, conjunctivitis, and gastroenteritis in humans.
  • Adenoviruses typically replicate as episomal components in the host cell nucleus rather than integrating into the host genome.
  • the genome of adenovirus contains 4 early transcription units (E1, E2, E3 and E4), which mainly have regulatory functions and prepare the host cell for replication of the virus.
  • the genome also contains 5 late transcription units (L1, L2, L3, L4, and L5) whose transcription includes penton (L2), hexon (L3), scaffold protein (L4), and fibrin (L5) of structural proteins under the control of a single promoter.
  • Each end of the genome contains inverted terminal repeat (ITR) sequences necessary for viral replication.
  • ITR inverted terminal repeat
  • Various exemplary embodiments disclosed in the present application provide a viral packaging vector having two ITR fragments and a gene expression cassette inserted between the two ITR fragments;
  • the gene expression cassette comprises a sequentially linked promoter, a repressor-type operator and an optional gene of interest;
  • the repressible operator can repress the expression of its downstream target gene.
  • the present application also relates to a viral packaging vector system comprising the viral packaging vector and the framework vector as described above.
  • the present application also relates to a method for packaging a virus, the method comprising:
  • Packaging takes place in the presence of the repressor.
  • the viral packaging vector of the present application does not need to determine whether the exogenous gene has an impact on the virus, because when there is no repressor, the repressor system will not function and will not affect the expression of the target gene.
  • the virus packaging process to suppress foreign genes can be carried out with the helper plasmid containing the repressor protein expression, thereby eliminating the influence of foreign genes and increasing the yield of specific viruses without rebuilding new ones.
  • the shuttle carrier saves a lot of time as well as manpower and material resources.
  • Figures 1 to 5 are the plasmid maps of pcDNA3.1-CymR, CMV-CuO, EF1a-CuO, SFH-CuO, and CAG-CuO vectors in sequence according to one embodiment of the application;
  • Fig. 6 is the verification result of the repression efficiency of CymR-CuO in lentiviral vector in one embodiment of the application;
  • Fig. 7 is the CMV-TrpO plasmid map in one embodiment of the application.
  • FIG. 8 is the verification result of the repression efficiency of the tryptophan operator subsystem in the lentiviral vector according to one embodiment of the application;
  • Fig. 9 is the EF1a-ToxO plasmid map in one embodiment of the application.
  • Figure 10 is the verification result of the repression efficiency of the diphtheria toxin suppressor regulatory system in a lentiviral vector according to an embodiment of the application;
  • Fig. 11 is the SFH-LacO plasmid map in one embodiment of the application.
  • Figure 12 is the verification result of the repression efficiency of the lactose operator subsystem in a lentiviral vector according to an embodiment of the application;
  • FIG. 13 is a schematic diagram of the insertion position of the TrpO element relative to the TATA Box of the CMV in one embodiment of the application;
  • Figure 14 shows the effect of different insertion positions of TrpO elements on repression efficiency in one embodiment of the present application
  • 15 is a schematic diagram of the insertion position of the CuO element relative to the TATA Box of the CMV in one embodiment of the application;
  • Figure 16 shows the effect of different insertion positions of CuO elements on repression efficiency in one embodiment of the present application
  • FIG. 17 is a graph showing the fluorescence result of NLS-iCre gene expressed by an adenovirus vector in one embodiment of the application.
  • Figure 18 is the titer detection result of adenovirus vector expressing NLS-iCre gene in one embodiment of the application;
  • Figures 19 to 21 are sequentially pShuttle-CMV-iCre-3 ⁇ Flag-P2A-sfGFP, pShuttle-CMV-TrpO-iCre-3 ⁇ Flag-P2A-sfGFP and pShuttle-CMV-CuO-iCre in one embodiment of the application -3 ⁇ Plasmid map of Flag-P2A-sfGFP;
  • Figure 22 is a graph showing the fluorescence results of adenovirus vector expressing AceD81S gene in one embodiment of the application;
  • Figure 23 is the titer detection result of adenovirus vector expressing AceD81S gene in one embodiment of the application;
  • Figures 24 to 26 are the plasmid maps of pShuttle-CMV-AceD81S-mOrange2, pShuttle-CMV-TrpO-AceD81S-mOrange2 and pShuttle-CMV-CuO AceD81S-mOrange2 in sequence according to one embodiment of the application;
  • Figure 27 is a schematic diagram of the principle of A) adenovirus packaging without repressor operon, B) adenovirus packaging with Cumate-CuO regulatable system and C) adenovirus packaging with tryptophan operon;
  • Fig. 28 is the virus titer detection result of adeno-associated virus vector expressing GPR78 in one embodiment of the application;
  • Figures 29 to 31 are sequentially pAAV-CMV-GPR78-3 ⁇ FLAG-WPRE, pAAV-CMV-CuO-GPR78-3 ⁇ FLAG-WPRE and pAAV-CMV-TrpO-GPR78-3 ⁇ FLAG in one embodiment of the application - Plasmid map of WPRE vector;
  • Figure 32 is the virus titer detection result of adeno-associated virus vector expressing Cdkn1a in one embodiment of the application;
  • Figures 33 to 35 are sequentially pAAV-CMV-Cdkn1a-3 ⁇ FLAG-WPRE, pAAV-CMV-CuO-Cdkn1a-3 ⁇ FLAG-WPRE and pAAV-CMV-TrpO-Cdkn1a-3 ⁇ FLAG in one embodiment of the application - Plasmid map of WPRE vector;
  • Figure 36 is a schematic diagram of the principle of A) adeno-associated virus packaging without repressor operon, B) adeno-associated virus packaging with tryptophan operon and C) adeno-associated virus packaging with Cumate-CuO regulatable system.
  • the present application relates to a viral packaging vector having two ITR fragments and a gene expression cassette inserted between the two ITR fragments;
  • the gene expression cassette comprises a sequentially linked promoter, a repressor-type operator and an optional gene of interest;
  • the repressible operator can repress the expression of its downstream target gene.
  • the expression of the target gene can be regulated through the repressor type operator, so as to selectively inhibit the expression of exogenous genes in adenovirus packaging cells, avoid the negative impact of exogenous genes on adenovirus packaging and production, and improve the performance of the virus.
  • the production efficiency and titer are not reduced in the target cells, and the expression level of the exogenous gene is not affected, and the therapeutic effect or the function of the exogenous gene is not affected.
  • vector in this application refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted.
  • the vector can express the protein encoded by the inserted polynucleotide, the vector is called an expression vector.
  • the vector can be introduced into a host cell by transformation, transduction or transfection, so that the genetic material elements carried by it can be expressed in the host cell.
  • the vector is a plasmid, but is not limited thereto.
  • the viral packaging vector is an adenoviral packaging vector or an adeno-associated viral packaging vector.
  • the repressible operon is selected from the tryptophan operon and/or the Cumate-CuO regulatable system.
  • the repressible operator has one or more copies.
  • the TrpO element in the tryptophan operon is at the insertion position of the gene expression cassette and is less than or equal to 18 nucleotides from the TATA BOX of the promoter, such as 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 nucleotides.
  • the CuO element in the Cumate-CuO regulatable system is inserted 40-50 nucleotides away from the TATA BOX of the promoter, eg, 41, 42, 43, 44, 45, 46, 47 , 48, 49 nucleotides.
  • the distance between the insertion position of the above-mentioned repressive operator element and the TATA BOX of the promoter refers to the first nucleotide (excluding) at the 5' end of the element to the first nucleotide at the 3' end of the TATA BOX ( number of nucleotides without ).
  • the distance between the TrpO element and the TATA BOX of the promoter is greater than 8 nucleotides, and the element can be closer to the TATA BOX when the element has overlapping sequences with the 8 nucleotides.
  • a “promoter” is a DNA sequence that directs RNA polymerase to bind and thereby initiate RNA synthesis. Promoters as used herein allow expression in a wide variety of cell and tissue types; either may be cell-free specific promoters, or may be "cell-specific", “cell-type-specific”, “cell lineage-specific” specific” or “tissue-specific” promoters that allow expression in a restricted variety of cells 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. , a nucleic acid encoding a polypeptide is expressed only in a subpopulation of cell types, cell lineages or tissues, or at specific developmental stages).
  • a desired polynucleotide sequence e.g. , a nucleic acid encoding a polypeptide is expressed only in a subpopulation of cell
  • tissue-specific promoters include, but are not limited to: B29 promoter (B cell expression), runt transcription factor (CBFa2) promoter (stem cell specific expression), CD14 promoter (monocyte expression), CD43 promoter promoter (leukocyte and platelet expression), CD45 promoter (hematopoietic cell expression), CD68 promoter (macrophage expression), CYP4503A4 or ALB promoter (hepatocyte expression), desmin promoter (muscle cell expression), Elastase 1 promoter (expressed in pancreatic alveolar cells), endoglin promoter (expressed in endothelial cells), fibroblast-specific protein 1 promoter (FSP1) promoter (expressed in fibroblasts), fibronectin promoter ( fibroblast expression), fms-related tyrosine kinase 1 (FLT1) promoter (endothelial cell expression), glial fibrillary acidic protein (GFAP) promoter (astrocyte expression), insulin promoter (pancre
  • the promoter is a cell-free promoter.
  • exemplary cell-free specific promoters include, but are not limited to, cytomegalovirus (CMV) very early promoter, viral simian virus 40 (SV40) (eg, early or late), Moroni murine leukemia virus (MoMLV) LTR promoter, Rous sarcoma virus (RSV) LTR, herpes simplex virus (HSV) (thymidine kinase) promoter, H5, P7.5 and P11 promoters of vaccinia virus, elongation factor 1-alpha (EF1a) promoter , early growth response 1 (EGR1), ferritin H (FerH), ferritin L (FerL), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), eukaryotic translation initiation factor 4A1 (EIF4A1), heat shock 70kDa protein 5 (HSPA5), heat shock protein 90kDa- ⁇ member 1 (HS
  • the promoter is a CMV, EF1a, SFH, CAG promoter, CBh, UBC, SFFV, SV40, RSV, mCMV, GAPDH, PGK, CASI, SMVP, GUSB (hGBp) or UCOE.
  • a gene of interest is inserted downstream of the repressible operator
  • "negatively affecting viral packaging” refers to reducing the packaging efficiency of the virus, slowing down or hindering the virus shedding, or directly killing packaging cells.
  • the target gene can be transcribed and translated in large quantities, it will occupy too much RNA transcription and protein translation-related enzymes and resources in the cell, thereby indirectly inhibiting the transcription and translation of each functional gene of the recombinant virus. The two influences reduce the production efficiency of recombinant virus in packaging cells and cannot obtain sufficient virus titers.
  • the gene of interest is cytotoxic to packaging cells.
  • the gene of interest is selected from suicide genes, apoptosis genes (or programmed cell death genes) and oncogenes.
  • Common suicide gene systems are as follows: tk-GCV system, CD-5-FC system, gpt-6-TX system, P450 2BI-CPA system, etc.
  • Apoptosis genes can also be replaced by programmed cell death genes in this application, the following genes are listed without limitation: Bcl-2 gene, P53 gene, cytochrome C gene, apoptosis protein activator 1 gene ( apoptotic proteaseactivating factor 1, Apaf-1), Caspase family protein genes, etc.
  • Caspase family proteins can be divided into initiating caspases (Caspase 8, 9, 10) and executing caspases (Caspase 3, 6, 7).
  • oncogenes include: tyrosine kinases (such as src), other protein kinases (such as raf), G proteins (such as ras), growth factors (such as Sis), growth factor receptors (such as ErbB), and internal proteins (such as the transcription factor MYC).
  • tyrosine kinases such as src
  • other protein kinases such as raf
  • G proteins such as ras
  • growth factors such as Sis
  • growth factor receptors such as ErbB
  • internal proteins such as the transcription factor MYC
  • the gene of interest is a cell cycle-related gene.
  • a cell cycle-related gene For example, Caspase family protein genes and MAPK signaling pathway related genes.
  • the gene of interest directly inhibits viral replication and/or assembly.
  • the expressed product of the target gene can directly cut, interfere, knock out, or bind to substances that inhibit viral replication and/or assembly related elements (such as E2A, E4 VA RNA gene, rap and cap gene products, etc.), For example, miRNA, siRNA, certain specific proteins or enzymes, etc.
  • the gene of interest is selected from the group consisting of GPR78 gene, Cdkn1a gene and APOBEC.
  • APOBEC apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like protein
  • APOBEC apolipoprotein B messenger RNA editing enzyme catalytic polypeptide-like protein.
  • pyrimidine deaminase APOBEC1, APOBEC2, APOBEC3A-H and APOBEC4 families. It was found that APOBEC has inhibitory effect on adeno-associated virus.
  • the gene of interest is selected from iCre gene and Caspase 8 gene.
  • the viral packaging vector further comprises at least one of a reporter gene, an enhancer, an internal ribosome entry site, a terminator and a polyadenylation signal.
  • the present application also relates to a viral packaging vector system comprising the viral packaging vector and the framework vector as described above.
  • the viral packaging vector system is an adeno-associated viral packaging vector system.
  • the framework vector is a vector carrying AAV rep, cap genes.
  • the vectors carrying the AAV rep, cap genes can be located on the same vector or on different vectors, or they can be located on the same vector as the viral packaging vector described above.
  • the adeno-associated virus packaging vector system is any one of AAV1-13 adeno-associated virus packaging vector systems.
  • the adeno-associated virus packaging vector system further comprises a helper virus vector.
  • the gene for the repressor is packaged in the helper virus vector.
  • the helper virus vector is an adenovirus or herpes virus helper virus vector.
  • the viral packaging vector system is an adenoviral packaging vector system.
  • the viral packaging vector is used as a shuttle vector.
  • the adenovirus packaging vector system is based on the Adeasy system or the AdMAX system.
  • the AdMax system is generally a two-plasmid system consisting of a shuttle plasmid (eg pHBAd series, including types including overexpression, interference, etc.) and a backbone plasmid (eg pBHGlox(delta)E1,3Cre).
  • the AdMax system uses the Cre-loxP recombinase system to make the adenovirus vector shuttle plasmid and backbone plasmid (adenovirus genome plasmid) co-transfected into cells to produce recombinant adenovirus under the action of recombinase, and the obtained virus titer is higher .
  • the main feature of the AdEasy packaging system is to use the Cre/loxP system to complete the process of inserting foreign genes into the adenovirus genome in Escherichia coli, to obtain a circular recombinant adenovirus genome, and to have the necessary elements for replication in bacteria.
  • the recombinant adenovirus genome was digested and linearized and then transfected into 293 cells to avoid double plasmid co-transfection to obtain recombinant adenovirus.
  • the present application also relates to a virus packaging method, which comprises the above-described viral packaging vector system into a host cell, and packaging in the presence of the repressor.
  • the virus is an adenovirus or an adeno-associated virus.
  • the host cell is a HEK-293 cell or a derivative thereof capable of providing the E1 region of adenovirus.
  • the derivative strain is 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- At least one of 6E, HEK-293F, HEK-293FT, HEK-293FTM, AAV-293, and GP2-293.
  • CymR-CuO tryptophan operon
  • diphtheria toxin suppressor regulatory system diphtheria toxin suppressor regulatory system
  • lactose operon lactose operon
  • the vector is a shuttle vector, which can be used as a universal eukaryotic vector when transfecting cells alone, and its conclusion is not limited to lentivirus
  • the nuclear system has the effect of inhibiting the expression of foreign genes.
  • the method for plasmid transfection of 293T specifically includes:
  • CymR-CuO system is a kind of regulated expression system. The principle is as follows:
  • CymR protein can specifically bind to CuO element in the absence of Cumate (citric acid), thereby inhibiting gene transcription. In the presence of Cumate, the CymR bound to Cumate will detach from the CuO element, allowing the gene to be expressed normally.
  • Cumate citric acid
  • the lentiviral vector with CuO element was co-transfected with pcDNA3.1 empty vector and pcDNA3.1-CymR in 293T cells, and the fluorescence was photographed 24 hours later. It can be seen that under the action of CymR protein, the expression efficiency of CMV-CuO, EF1a-CuO, SFH-CuO, and CAG-CuO promoters were significantly inhibited (Fig. 6).
  • TrpR Trp repressor
  • a series of promoter vectors such as CMV-TrpO, EF1a-TrpO, SFH-TrpO, and CAG-TrpO were constructed, and it was confirmed that the TrpO element combined with the TrpR vector (pcDNA3.1-TrpR) can effectively inhibit the presence of tryptophan. gene expression.
  • the CMV-TrpO plasmid map is shown in Figure 7, and the design method of the plasmid map of the EF1a-TrpO, SFH-TrpO, and CAG-TrpO promoter vectors can be referred to Figures 2 to 5 on the basis of Figure 7, and will not be repeated.
  • the vector with TrpO element was co-transfected with pcDNA3.1 empty vector and pcDNA3.1-TrpR in 293T cells, and 0.3 mM tryptophan was added at the same time, and the fluorescence was photographed 24 hours later. It can be seen that under the action of TrpR protein, the expression efficiency of CMV-TrpO, EF1a-TrpO, SFH-TrpO, and CAG-TrpO promoters were significantly inhibited (Fig. 8).
  • the diphtheria toxin repressor DtxR protein can specifically bind to the ToxO element, thereby inhibiting gene transcription.
  • a series of promoter vectors such as CMV-ToxO, EF1a-ToxO, SFH-ToxO, and CAG-ToxO have been constructed.
  • the vector with ToxO element was co-transfected into 293T cells with pcDNA3.1 empty vector and pcDNA3.1-DtxR, and ferrous ion was added. As a result, the target gene was not found to be significantly inhibited (Fig. 10).
  • the plasmid map of EF1a-ToxO is shown in Figure 9, and the design method of the plasmid map of CMV-ToxO, SFH-ToxO, and CAG-ToxO promoter vectors can be referred to Figures 2 to 5 on the basis of Figure 9, and will not be repeated.
  • the lactose operon repressor protein LacI can specifically bind to the LacO element, thereby inhibiting gene transcription. In the presence of IPTG, it binds to it and undergoes a conformational change, loses its binding ability, and the downstream genes begin to express.
  • the lactose operon is widely used for inducible expression of foreign genes in prokaryotic cells.
  • a series of promoter vectors such as CMV-LacO, EF1a-LacO, SFH-LacO, and CAG-LacO have been constructed.
  • the vector with LacO element was co-transfected into 293T cells with pcDNA3.1 empty vector and pcDNA3.1-LacI, respectively. As a result, the target gene was not found to be significantly inhibited (Fig. 12).
  • the plasmid map of SFH-LacO is shown in Figure 11, and the design method of the plasmid map of CMV-LacO, EF1a-LacO, and CAG-LacO promoter vectors can be referred to Figures 2 to 5 on the basis of Figure 11, and will not be repeated.
  • the distance between the TrpO element and the TATA Box of CMV is different.
  • the inventors designed two promoters. As shown in Figure 13, the plasmid was co-transfected with pcDNA3.1-TrpR (0.1 mM tryptophan was added), and the fluorescence was observed after 48 hours. , the experiment shows that different positions from the TATA Box will produce different inhibitory effects (see Figure 14). This application subsequently uses the TrpO v2 promoter sequence with better inhibitory effect.
  • the distance between the CuO element and the TATA Box of CMV is different.
  • Three promoters were designed. As shown in Figure 15, the plasmid was co-transfected with pcDNA3.1-CymR, and the fluorescence was observed after 48 hours. The experiment showed that the CuO element was far from the TATA Box. Different positions will produce different inhibitory effects (see Figure 16), and the CuO v1 promoter sequence with better inhibitory effect is subsequently used in this application.
  • Example 2 Packaging of adenovirus and expression of target gene
  • HEK-293-CMV-CymR and HEK-293-CMV-TrpR stable cell lines including:
  • the pcDNA3.1-CymR or pcDNA3.1-TrpR was linearized by double digestion with MfeI and BstBI (NEB company); after 4-6 hours of digestion, the linearized band was recovered using a recovery kit (QIAGEN company). , After the recovery is completed, the concentration is measured and confirmed by agarose gel;
  • HEK 293 cells in good condition one day before plasmid transfection, collect cells after digestion, and plate them in a 24-well plate at 2 ⁇ 10 5 cells/well after counting, shake well, and place in a 37°C, 5% CO 2 incubator for culture. Change the medium before transfection: 1 hour before plasmid transfection, take out the 24-well plate laid the day before from the incubator, discard the original medium, add 0.5 mL of pre-warmed Opti-MEM medium to each well, and then Put into a 5% CO2 incubator to be transfected.
  • G418 400ug/ml was added for screening, and the mixed clone cells were identified to express CymR or TrpR; the mixed clone cells were selected according to the limiting dilution method to stably express pcDNA3.1-CymR or pcDNA3.1-TrpR. HEK-293 monoclonal cells.
  • This embodiment is an adenovirus packaging method, which is an adenovirus AdEasy system, specifically including:
  • the shuttle plasmid carrying the exogenous gene (NLS-iCre or AceD81S) was recombined with the plasmid carrying most of the adenovirus genome (pAdEasy), and the recombinant adenovirus genome plasmid was obtained through resistance screening.
  • the shuttle plasmids are: pShuttle-CMV-iCre-3xFlag-P2A-sfGFP, pShuttle-CMV-CuO-iCre-3xFlag-P2A-sfGFP or pShuttle-CMV-TrpO-iCre-3xFlag- P2A-sfGFP; for the exogenous gene AceD81S, the shuttle plasmids are: pShuttle-CMV-AceD81S-mOrange2, pShuttle-CMV-TrpO-AceD81S-mOrange2 or pShuttle-CMV-CuO-AceD81S-mOrange2. Specific steps are as follows:
  • the shuttle plasmid (pShuttle-CMV-iCre-3xFlag-P2A-sfGFP, pShuttle-CMV-CuO-iCre-3xFlag-P2A-sfGFP or pShuttle-CMV-TrpO-iCre-3xFlag-P2A-sfGFP; pShuttle-CMV- AceD81S-mOrange2, pShuttle-CMV-TrpO-AceD81S-mOrange2 or pShuttle-CMV-CuO-AceD81S-mOrange2) were linearized with PmeI (NEB);
  • Plating Select HEK 293 cells in good condition or HEK-293 cells stably expressing pcDNA3.1-CymR or pcDNA3.1-TrpR one day before plasmid transfection, collect cells after digestion, and count them according to 1 ⁇ 10 6 cells/well Spread on a 6-well plate, shake well, and place in a 37°C, 5% CO 2 incubator for culture.
  • the medium was changed every 3 to 5 days, and the virus was observed to be out of the virus, and the virus was amplified after the out of virus; sfGFP or pShuttle-CMV-TrpO-AceD81S-mOrange2) need to be replaced with complete medium containing 0.3 mM tryptophan.
  • the adenovirus titer detection kit is used for adenovirus detection.
  • the principle is that adenovirus can infect and replicate in HEK293 cells, express a special coat protein Hexon, and use immunostaining to detect positive cells. Positive cells infected by the virus Will be stained brown, count positive cells, and use formula to calculate adenovirus titer. Specifically include:
  • Adenovirus vector expresses NLS-iCre gene
  • the plasmids used in this example are pShuttle-CMV-iCre-3 ⁇ Flag-P2A-sfGFP, pShuttle-CMV-TrpO-iCre-3 ⁇ Flag-P2A-sfGFP and pShuttle-CMV-CuO-iCre-3 ⁇ Flag-P2A
  • the plasmid maps of -sfGFP are shown in Figure 19, Figure 20, and Figure 21 in turn.
  • the plasmid maps of the plasmids pShuttle-CMV-AceD81S-mOrange2, pShuttle-CMV-TrpO-AceD81S-mOrange2 and pShuttle-CMV-CuO-AceD81S-mOrange2 used in this example are shown in Figure 24, Figure 25, and Figure 26 in turn.
  • Example 3 Packaging of adeno-associated virus and expression of target gene
  • a method for virus packaging using adeno-associated virus vector specifically including:
  • the viral vector plasmid to be transfected (serotype plasmid, Helper auxiliary plasmid and shuttle plasmid) in Opti-MEM medium, mix gently, and leave standing to obtain a plasmid dilution;
  • the serotype plasmid is pAAV9, pAAV9-CMV-CymR or pAAV9--CMV-TrpR;
  • Helper helper plasmid is pHelper, pHelper-CMV-CymR or pHelper-CMV-TrpR;
  • the shuttle plasmid is pAAV-CMV-GPR78-3 ⁇ FLAG-WPRE, pAAV-CMV-CuO-GPR78-3 ⁇ FLAG-WPRE or pAAV-CMV-TrpO-GPR78-3 ⁇ FLAG-WPRE; for the target gene Cdkn1a, the pAAV-CMV
  • the above samples were diluted and used as templates, and the titer of adeno-associated virus was determined by Real-time PCR detection method.
  • Real-time PCR was performed on an ABI7500 instrument.
  • the reagent SYBR Master Mixture from TAKARA was used.
  • Upstream primer (25 ⁇ M): 0.5 ⁇ l;
  • Genomic DNA 2.0 ⁇ l
  • the PCR program is:
  • Step 1 95.0°Cfor 00:15;
  • Step 1 95.0°Cfor 00: 05
  • Step 2 60.0°Cfor 00:34;
  • the procedure for making the melting curve is:
  • Step 1 95.0°Cfor 01:00
  • Step 1 55.0°Cfor 01:00;
  • Step 1 55.0°C-95.0°Cfor 00:30
  • Adeno-associated virus vector expresses GPR78 (glucose-regulated protein, 78kDa) gene
  • GRP78 is a type of molecular chaperone protein that assists in the folding of newly generated protein molecules into the correct three-dimensional configuration. In virus production, it was found that viral particles could not be obtained by constructing the GPR78 gene into a common AAV vector, which is consistent with existing reports.
  • the use of CMV-CuO and CMV-TrpO promoters in conjunction with the expression of inhibitory proteins can effectively solve the problem of poisoning, and the results of virus titer detection are shown in Figure 28.
  • the plasmid maps of the plasmids pAAV-CMV-GPR78-3 ⁇ FLAG-WPRE, pAAV-CMV-CuO-GPR78-3 ⁇ FLAG-WPRE and pAAV-CMV-TrpO-GPR78-3 ⁇ FLAG-WPRE used in this example are sequentially As shown in Figure 29, Figure 30, and Figure 31.
  • Adeno-associated virus vector expresses Cdkn1a gene
  • the plasmid maps of the plasmids pAAV-CMV-Cdkn1a-3 ⁇ FLAG-WPRE, pAAV-CMV-CuO-Cdkn1a-3 ⁇ FLAG-WPRE and pAAV-CMV-TrpO-Cdkn1a-3 ⁇ FLAG-WPRE used in this example are sequentially As shown in Figure 33, Figure 34, and Figure 35.

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Abstract

本申请涉及生物技术领域，具体而言，涉及一种外源基因可控表达的病毒包装载体及其包装方法，所述病毒包装载体具有两个ITR片段及插入两个ITR片段之间的基因表达盒；所述基因表达盒包含顺次连接的启动子、阻遏型操作子以及任选的目的基因；当有阻遏物存在时，所述阻遏型操作子能够阻遏其下游目的基因的表达。

Description

外源基因可控表达的病毒包装载体及其包装方法
相关申请的交叉引用
本申请要求于2020年12月7日提交中国专利局、申请号为202011418490.9、发明名称为“外源基因可控表达的腺病毒包装方法”以及2020年12月7日提交中国专利局、申请号为202011419528.4、发明名称为“外源基因可控表达的腺相关病毒包装方法”的中国专利申请的优先权，其全部内容通过引用并入本文。
技术领域
本申请涉及生物技术领域，具体而言，涉及一种外源基因可控表达的病毒包装载体及其包装方法。
背景技术
病毒载体可将遗传物质带入细胞，原理是利用病毒具有高亲和性结合细胞表面受体，并传送其基因组进入某些类型细胞，进行感染的分子机制。广泛应用于基础研究、基因疗法或疫苗。
目前，常作为病毒载体使用的病毒包括腺相关病毒、腺病毒等。
腺相关病毒(adeno-associated virus，AAV)，也称腺伴随病毒，属于微小病毒科依赖病毒属,是目前发现的一类结构最简单的单链DNA缺陷型病毒，需要辅助病毒(通常为腺病毒)参与复制。它编码两个末端的反向重复序列(ITR)中的cap和rep基因。ITRs对于病毒的复制和包装具有决定性作用。cap基因编码病毒衣壳蛋白，rep基因参与病毒的复制和整合。AAV能感染多种细胞。rep基因产物存在时，病毒DNA很容易整合到人类第19号染色体。腺相关病毒具有安全性高、免疫原性低、宿主范围广、能介导外源基因在动物体内长期稳定表达等特点，是基因治疗领域最具应用前景的载体之一。AAV在神经科学研究以及疾病的基因治疗等领域具有重要作用和巨大需求。在医学研究中，AAV被用于多种疾病的基因治疗的研究(包括体内、体外实验)；同时作为一种有特点的基因转移载体，还广泛用于基因功能研究、构建疾病模型、制备基因敲除鼠等方面。
腺病毒(adenovirus，ADV)特别适于用作病毒载体。腺病毒是无包膜病毒，直径约90nm～100nm，包含核衣壳和线性双链DNA基因组。病毒核衣壳包含五邻体衣壳体和六邻体衣壳体。一种独特的纤维与各五邻体基质相关，并且帮助病毒经由宿主细胞表面的柯萨奇病毒-腺病毒受体而附着到宿主细胞上。已识别出超过50种的腺病毒血清型菌株，其中大多数引起人类的呼吸道感染、结膜炎和胃肠炎。腺病毒通常作为宿主细胞核中的游离基因成分复制，而不是整合到宿主基因组中。腺病毒的基因组包含4个早期转录单位(E1、E2、E3和E4)，其主要具有调节功能，并为宿主细胞复制病毒作准备。基因组还包含5个晚期转录单位(L1、L2、L3、L4和L5)，其转录包括五邻体(L2)、六邻体(L3)、支架蛋白(L4)和纤维蛋白(L5)在内的结构蛋白，其中结构蛋白受单个启动子的控制。基因组的每一末端都包含病毒复制所必需的末端反向重复(ITR)序列。
目前病毒载体的研究都集中在感染能力、特异性、包装容量等方面，而对于包装的外源基因(gene of interest，GOI)对于病毒的影响关注很少，在实践中，有相当一部分外源基因是无法获得有效的病毒颗粒。这是因为，在病毒包装过程中，外源基因通常由强启动子驱动，其表达对细胞状态，病毒的包装，出毒等过程都有无法预料的影响。
发明内容
本申请公开的各种示例性实施方式提供一种病毒包装载体，具有两个ITR片段及插入两个ITR片段之间的基因表达盒；
所述基因表达盒包含顺次连接的启动子、阻遏型操作子以及任选的目的基因；
当有阻遏物存在时，所述阻遏型操作子能够阻遏其下游目的基因的表达。
根据本申请的再一方面，本申请还涉及一种病毒包装载体系统，其包含如上所述的病毒包装载体以及框架载体。
根据本申请的再一方面，本申请还涉及一种病毒的包装方法，所述方法包括：
将如上所述的病毒包装载体系统转入宿主细胞，以及
于所述阻遏物存在的前提下进行包装。
与传统的病毒包装载体相比，使用本申请的病毒包装载体无需判断外源基因是否对病毒产生影响，因为没有阻遏物的时候，阻遏系统不会发挥作用，也不会影响目的基因的表达。当发现出毒明显低于平均水平的时候，配合包含阻遏蛋白表达的辅助质粒即可进行抑制外源基因的病毒包装过程，从而排除外源基因的影响，提升特定病毒的产量，无需重新构建新的穿梭载体，节省了大量的时间以及人力和物力。
附图说明
为了更清楚地说明本申请具体实施方式或传统技术中的技术方案，下面将对具体实施方式或传统技术描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图是本申请的一些实施方式，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他的附图。
图1～图5依次为本申请一个实施方式中pcDNA3.1-CymR、CMV-CuO、EF1a-CuO、SFH-CuO、CAG-CuO载体的质粒图谱；
图6为本申请一个实施方式中CymR-CuO在慢病毒载体中的阻遏效率验证结果；
图7为本申请一个实施方式中CMV-TrpO质粒图谱；
图8为本申请一个实施方式中色氨酸操作子系统在慢病毒载体中的阻遏效率验证结果；
图9为本申请一个实施方式中EF1a-ToxO质粒图谱；
图10为本申请一个实施方式中白喉毒素抑制子调控系统在慢病毒载体中的阻遏效率验证结果；
图11为本申请一个实施方式中SFH-LacO质粒图谱；
图12为本申请一个实施方式中乳糖操作子系统在慢病毒载体中的阻遏效率验证结果；
图13为本申请一个实施方式中TrpO元件相对于CMV的TATA Box的插入位置示意图；
图14为本申请一个实施方式中TrpO元件不同插入位置对阻遏效率的影响；
图15为本申请一个实施方式中CuO元件相对于CMV的TATA Box的插入位置示意图；
图16为本申请一个实施方式中CuO元件不同插入位置对阻遏效率的影响；
图17为本申请一个实施方式中腺病毒载体表达NLS-iCre基因的荧光结果图；
图18为本申请一个实施方式中腺病毒载体表达NLS-iCre基因的滴度检测结果；
图19～图21依次为本申请一个实施方式中pShuttle-CMV-iCre-3×Flag-P2A-sfGFP、pShuttle-CMV-TrpO-iCre-3×Flag-P2A-sfGFP和pShuttle-CMV-CuO-iCre-3×Flag-P2A-sfGFP的质粒图谱；
图22为本申请一个实施方式中腺病毒载体表达AceD81S基因的荧光结果图；
图23为本申请一个实施方式中腺病毒载体表达AceD81S基因的滴度检测结果；
图24～图26依次为本申请一个实施方式中pShuttle-CMV-AceD81S-mOrange2、pShuttle-CMV-TrpO-AceD81S-mOrange2和pShuttle-CMV-CuO AceD81S-mOrange2的质粒图谱；
图27为A)不具有阻遏型操作子的腺病毒包装，B)具有Cumate-CuO可调控系统的腺病毒包装和C)具有色氨酸操作子的腺病毒包装的原理示意图；
图28为本申请一个实施方式中腺相关病毒载体表达GPR78的病毒滴度检测结果；
图29～图31依次为本申请一个实施方式中pAAV-CMV-GPR78-3×FLAG-WPRE、pAAV-CMV-CuO-GPR78-3×FLAG-WPRE以及pAAV-CMV-TrpO-GPR78-3×FLAG-WPRE载体的质粒图谱；
图32为本申请一个实施方式中腺相关病毒载体表达Cdkn1a的病毒滴度检测结果；
图33～图35依次为本申请一个实施方式中pAAV-CMV-Cdkn1a-3×FLAG-WPRE、pAAV-CMV-CuO-Cdkn1a-3×FLAG-WPRE以及pAAV-CMV-TrpO-Cdkn1a-3×FLAG-WPRE载体的质粒图谱；
图36为A)不具有阻遏型操作子的腺相关病毒包装，B)具有色氨酸操作子的腺相关病毒包装和C)具有 Cumate-CuO可调控系统的腺相关病毒包装的原理示意图。
具体实施方式
现将详细地提供本申请实施方式的参考，其一个或多个实例描述于下文。提供每一实例作为解释而非限制本申请。实际上，对本领域技术人员而言，显而易见的是，可以对本申请进行多种修改和变化而不背离本申请的范围或精神。例如，作为一个实施方式的部分而说明或描述的特征可以用于另一实施方式中，来产生更进一步的实施方式。
因此，旨在本申请覆盖落入所附权利要求的范围及其等同范围中的此类修改和变化。本申请的其它对象、特征和方面公开于以下详细描述中或从中是显而易见的。本领域普通技术人员应理解本讨论仅是示例性实施方式的描述，而非意在限制本申请更广阔的方面。
除非另有定义，本文所使用的所有的技术和科学术语与属于本申请的技术领域的技术人员通常理解的含义相同。本文中在本申请的说明书中所使用的术语只是为了描述具体的实施例的目的，不是旨在于限制本申请。本文所使用的术语“和/或”包括一个或多个相关的所列项目的任意的和所有的组合。
本申请涉及一种病毒包装载体，具有两个ITR片段及插入两个ITR片段之间的基因表达盒；
所述基因表达盒包含顺次连接的启动子、阻遏型操作子以及任选的目的基因；
当有阻遏物存在时，所述阻遏型操作子能够阻遏其下游目的基因的表达。
本申请通过阻遏型操作子使得目的基因的表达可被调控，从而实现选择性地抑制外源基因在腺病毒包装细胞中的表达，避免外源基因对腺病毒包装与生产的负面影响，提高病毒的生产效率和滴度，同时在靶细胞中不降低外源基因的表达水平，不影响治疗效果或研究外源基因的功能。
在本申请中术语“载体(vector)”是指，可将多聚核苷酸插入其中的一种核酸运载工具。当载体能使插入的多核苷酸编码的蛋白获得表达时，载体称为表达载体。载体可以通过转化，转导或者转染导入宿主细胞，使其携带的遗传物质元件在宿主细胞中获得表达。在一个实施方式中，载体为质粒，但并不限于此。
在一些实施方式中，所述病毒包装载体是腺病毒包装载体或腺相关病毒包装载体。
在一些实施方式中，所述阻遏型操作子选自色氨酸操作子和/或Cumate-CuO可调控系统。
在一些实施方式中，所述阻遏型操作子具有一个或多个拷贝。
在一些实施方式中，所述色氨酸操作子中的TrpO元件在所述基因表达盒的插入位置距离所述启动子的TATA BOX小于或等于18个核苷酸，例如17、16、15、14、13、12、11、10、9、8、7、6、5、4、3、2、1个核苷酸。
在一些实施方式中，所述Cumate-CuO可调控系统中的CuO元件的插入位置距离所述启动子的TATA BOX40～50个核苷酸，例如41、42、43、44、45、46、47、48、49个核苷酸。
上述阻遏型操作子元件的插入位置和启动子的TATA BOX之间的距离是指该元件的5’端的第一个核苷酸(不含)到TATA BOX 3’端的第一个核苷酸(不含)的核苷酸个数。
需要说明的是，一般认为TATA BOX后8个核苷酸以内(含第8个核苷酸)都属于启动子核心区域。在一个实施方式中，TrpO元件与启动子的TATA BOX之间的距离大于8个核苷酸，当该元件与该8个核苷酸有重叠序列时可以距离TATA BOX更近。
“启动子”为指导RNA聚合酶结合并由此启动RNA合成的DNA序列。本申请所使用的启动子允许在各种各样的细胞和组织类型中表达；或者可以为无细胞特异性启动子，或者可为“细胞特异性”、“细胞类型特异性”、“细胞谱系特异性”或“组织特异性”启动子，其允许分别在种类受限的细胞和组织类型中表达。在具体实施方案中，可取的是使用细胞、细胞类型、细胞谱系或组织特异性表达控制序列，以实现所需多核苷酸序列的细胞类型特异性、细胞谱系特异性或组织特异性表达(例如，仅在细胞类型、细胞谱系或组织的子群中或在特定发育阶段表达编码多肽的核酸)。
组织特异性启动子的示例性实例包括但不限于：B29启动子(B细胞表达)、runt转录因子(CBFa2)启动子(干细胞特异性表达)、CD14启动子(单核细胞表达)、CD43启动子(白细胞和血小板表达)、CD45启动子(造血细胞表 达)、CD68启动子(巨噬细胞表达)、CYP4503A4或ALB启动子(肝细胞表达)、肌间线蛋白启动子(肌肉细胞表达)、弹性蛋白酶1启动子(胰腺泡细胞表达)、内皮糖蛋白启动子(内皮细胞表达)、成纤维细胞特异性蛋白1启动子(FSP1)启动子(成纤维细胞表达)、纤连蛋白启动子(成纤维细胞表达)、fms-相关的酪氨酸激酶1(FLT1)启动子(内皮细胞表达)、胶质纤维酸性蛋白(GFAP)启动子(星形胶质细胞表达)、胰岛素启动子(胰腺细胞表达)、整合蛋白-α-2b(ITGA2B)启动子(巨核细胞)、胞内粘着分子2(ICAM-2)启动子(内皮细胞)、干扰素-β(IFN-β)启动子(造血细胞)、角蛋白5启动子(角化细胞表达)、肌红蛋白(MB)启动子(肌肉细胞表达)、成肌分化1(MYOD1)启动子(肌肉细胞表达)、肾病蛋白启动子(足细胞表达)、骨γ-羧基谷氨酸蛋白2(OG-2)启动子(成骨细胞表达)、3-酮酸CoA转移酶2B(Oxct2B)启动子(单倍体精细胞表达)、表面活化蛋白B(SP-B)启动子(肺细胞表达)、突触蛋白启动子(神经细胞表达)、Wiskott-Aldrich综合征蛋白(WASP)启动子(造血细胞表达)。
在一些实施方式中，所述启动子为无细胞特异性的启动子。示例性的无细胞特异性的启动子包括但不限于巨细胞病毒(CMV)极早期启动子，病毒性猿猴病毒40(SV40)(例如，早期或晚期)、莫罗尼鼠白血病病毒(MoMLV)LTR启动子，劳氏肉瘤病毒(RSV)LTR、单纯疱疹病毒(HSV)(胸苷激酶)启动子，牛痘病毒的H5、P7.5和Pll启动子，延长因子1-α(EF1a)启动子，早期生长应答1(EGR1)、铁蛋白H(FerH)、铁蛋白L(FerL)、3_磷酸甘油醛脱氢酶(GAPDH)、真核生物翻译起始因子4A 1(EIF4A1)、热休克70kDa蛋白5(HSPA5)、热休克蛋白90kDa-β成员1(HSP90B1)、热休克蛋白70kDa(HSP70)、β-驱动蛋白(β-KIN)、人R0SA26基因座(Irions et al.,(2007)Nature Biotechnology25,1477-1482)、泛激素C启动子(UBC)，磷酸甘油酸激酶-1(PGK)启动子，巨细胞病毒增强子/鸡β-肌动蛋白(CAG)启动子，以及β-肌动蛋白启动子。无细胞特异性的启动子能使得所述基因表达盒具有更好的通用性及表达效率。
在一些实施方式中，所述启动子是CMV、EF1a、SFH、CAG启动子、CBh、UBC、SFFV、SV40、RSV、mCMV、GAPDH、PGK、CASI、SMVP、GUSB(hGBp)或UCOE。
在一些实施方式中，所述阻遏型操作子下游插入有目的基因；
且当所述目的基因表达时会对病毒包装产生负面影响。
在本申请中，“对病毒包装产生负面影响”是指降低病毒的包装效率，减缓或阻碍病毒出毒，或直接杀伤包装细胞。另一方面，目的基因如果能大量转录与翻译也会占用细胞过多的RNA转录、蛋白质翻译相关的酶类与资源，从而间接抑制了重组病毒各功能基因的转录与翻译。两个方面的影响降低了重组病毒在包装细胞中的生产效率，无法获得足够的病毒滴度。
在一些实施方式中，所述目的基因对包装细胞产生细胞毒性。
在一些实施方式中，所述目的基因选自自杀基因、细胞凋亡基因(或称细胞程序性死亡基因)和癌基因。
常见的自杀基因系统有以下几种可以为tk-GCV系统、CD-5-FC系统、gpt-6-TX系统、P450 2BI-CPA系统等。
细胞凋亡基因在本申请中也可用细胞程序性死亡基因所替代，以下非限制性地列出下述基因：Bcl-2基因、P53基因、细胞色素C基因、凋亡蛋白激活因子1基因(apoptotic proteaseactivating factor 1,Apaf-1)、Caspase家族蛋白基因等。
Caspase家族蛋白可以分为启动Caspase(Caspase8、9、10)和执行Caspase(Caspase 3、6、7)。
癌基因主要的类型包括：酪氨酸激酶(如src)、其他蛋白激酶(如raf)、G蛋白(如ras)、生长因子(如Sis)、生长因子受体(如ErbB)，以及位于细胞核内的蛋白(如转录因子MYC)。
在一些实施方式中，所述目的基因为细胞周期相关基因。例如Caspase家族蛋白基因和MAPK信号通路相关基因。
在一些实施方式中，所述目的基因直接抑制病毒的复制和/或组装。
例如所述目的基因所表达的产物能够直接切割、干扰、敲除、或结合抑制病毒复制和/或组装的相关元件(如E2A、E4的VA RNA基因、rap和cap基因产物等)的物质，例如miRNA、siRNA、某些特定的蛋白或酶等。
在一些实施方式中，所述目的基因选自GPR78基因、Cdkn1a基因和APOBEC。APOBEC(apolipoprotein B  mRNA-editing enzyme catalytic polypeptide-like protein)酶家族为载脂蛋白B信使RNA编辑酶催化多肽样蛋白，是近年来发现具有脱氨基作用的一类蛋白酶，它包括AID(活化诱导胞嘧啶脱氨酶)、APOBEC1、APOBEC2、APOBEC3A-H以及APOBEC4家族。有人发现APOBEC对腺相关病毒具有抑制作用。
在一些实施方式中，所述目的基因选自iCre基因和Caspase 8基因。
在一些实施方式中，所述的病毒包装载体还包含报告基因、增强子、内部核糖体进入位点、终止子和多腺苷酸化信号中的至少一种。
根据本申请的再一方面，本申请还涉及一种病毒包装载体系统，其包含如上所述的病毒包装载体以及框架载体。
在一些实施方式中，所述病毒包装载体系统是腺相关病毒包装载体系统。在其中一个实施方式中，所述框架载体是携带AAV rep、cap基因的载体。携带AAV rep、cap基因的载体可位于相同载体上也可位于不同载体上，或者它们可与所述如上所述的病毒包装载体位于同一载体上。在其中一个实施方式中，腺相关病毒包装载体系统为AAV1～13中任意一种的腺相关病毒包装载体系统。在其中一个实施方式中，腺相关病毒包装载体系统还包含辅助病毒载体。在其中一个实施方式中，所述阻遏物的基因包装于所述辅助病毒载体中。在其中一个实施方式中，所述辅助病毒载体为腺病毒或疱疹病毒辅助病毒载体。
在一些实施方式中，所述病毒包装载体系统是腺病毒包装载体系统。所述的病毒包装载体作为穿梭载体使用。在其中一个实施方式中，所述腺病毒包装载体系统基于Adeasy系统或AdMAX系统。AdMax系统通常为两质粒系统，组成为穿梭质粒(例如pHBAd系列，包括包括过表达、干扰等类型)和骨架质粒(例如pBHGlox(delta)E1,3Cre)。AdMax系统通过Cre-loxP重组酶系统，使共转染到细胞中的腺病毒载体穿梭质粒和骨架质粒(腺病毒基因组质粒)在重组酶的作用下产生重组腺病毒，获得的病毒滴度更高。AdEasy包装系统主要的特点是利用Cre/loxP系统在大肠杆菌中完成外源基因插入腺病毒基因组的过程，获得环状的重组腺病毒基因组，并且具有在细菌中复制的必需元件。将重组腺病毒基因组酶切线性化后转染293细胞，避免了双质粒共转染得到重组腺病毒。
根据本申请的再一方面，本申请还涉及一种病毒的包装方法，所述方法将包含如上所述的病毒包装载体系统转入宿主细胞，以及于所述阻遏物存在的前提下进行包装。
在一些实施方式中，所述病毒为腺病毒或腺相关病毒。
在一些实施方式中，所述宿主细胞为HEK-293细胞或其能够提供腺病毒E1区的衍生株。
在一些实施方式中，所述衍生株选自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以及GP2-293中的至少一种。
下面将结合实施例对本申请的实施方案进行详细描述。
实施例1阻遏型操作子的选择
本申请共测试了4种转录调控系统：CymR-CuO、色氨酸操作子、白喉毒素抑制子调控系统、乳糖操作子。
首先通过在慢病毒载体(该载体为穿梭载体，单独转染细胞使用可作为通用真核载体使用，其结论不局限于慢病毒)上引入这四个系统的顺式作用元件，测试是否在真核系统中有抑制外源基因表达的效果。
一、质粒转染
质粒转染293T的方法，具体包括：
1、转染前一天，将293T细胞接种到培养皿中；转染前一小时取出细胞培养皿，弃去原有细胞培养基，加入Opti-MEM培养基，将细胞放回培养箱；制备转染试剂和质粒的复合物，包括以下步骤：
2、将待转染的一种或多种质粒根据需要等比例混合溶于Opti-MEM培养基，轻轻混匀，静置后得到质粒稀释液。
3、将转染试剂溶于Opti-MEM培养基，轻轻混匀，静置后得到转染试剂稀释液；将转染试剂稀释液滴加到质粒稀释液中，边加边轻轻混匀后在室温放置15-25min，使DNA和转染试剂充分结合形成稳定的转染复合体；取出细胞培养皿，将准备好的DNA-转染试剂复合体加入到细胞培养皿中，放回培养箱；
4、5-8小时后吸去培养基，用PBS溶液洗涤后加入新鲜完全培养基培养，其中色氨酸操作子组需更换含有0.3mM色氨酸的完全培养基，白喉毒素抑制子系统需要加入二价铁离子。
二、不同阻遏型操作子系统的验证
1.CymR-CuO系统
CymR-CuO系统是一类可调控的表达系统。其原理如下：
CymR蛋白在Cumate(枯酸)不存在的情况下，可以特异性的和CuO元件结合，从而抑制基因的转录。在Cumate存在的情况下，结合了Cumate的CymR将脱离CuO元件，从而使基因正常表达。
本申请构建了CMV-CuO、EF1a-CuO、SFH-CuO、CAG-CuO一系列启动子载体，证实了CuO元件配合CymR载体(pcDNA3.1-CymR)可以有效抑制基因的表达。其中pcDNA3.1-CymR的质粒图谱如图1所示，CMV-CuO、EF1a-CuO、SFH-CuO、CAG-CuO启动子载体的质粒图谱依次如图2～5所示。
将带有CuO元件的慢病毒载体分别与pcDNA3.1空载体和pcDNA3.1-CymR共转293T细胞，24小时后荧光拍照。可以看到，在CymR蛋白的作用下，CMV-CuO、EF1a-CuO、SFH-CuO、CAG-CuO启动子的表达效率均有明显的抑制(图6)。
2.色氨酸操作子系统
在Trp repressor(TrpR)蛋白在色氨酸存在的情况下，可以特异性的和TrpO元件结合，从而抑制基因的转录。该系统在原核细胞中发现，长期以来用作基因调控的讲解示范，但是由于哺乳动物细胞培养中色氨酸是必须的，所以该系统并没有在真核细胞中得以应用。
本申请构建了CMV-TrpO、EF1a-TrpO、SFH-TrpO、CAG-TrpO等一系列启动子载体，证实了TrpO元件配合TrpR载体(pcDNA3.1-TrpR)在色氨酸的存在下可以有效抑制基因的表达。其中CMV-TrpO质粒图谱如图7所示，EF1a-TrpO、SFH-TrpO、CAG-TrpO启动子载体的质粒图谱的设计方式可在图7的基础上参考图2～5，不再赘述。
带有TrpO元件的载体分别与pcDNA3.1空载体和pcDNA3.1-TrpR共转293T细胞，同时添加0.3mM的色氨酸，24小时后荧光拍照。可以看到，在TrpR蛋白的作用下，CMV-TrpO、EF1a-TrpO、SFH-TrpO、CAG-TrpO启动子的表达效率均有明显的抑制(图8)。
3.白喉毒素抑制子调控系统
白喉毒素抑制子DtxR蛋白可以特异性的和ToxO元件结合，从而抑制基因的转录。本申请构建了CMV-ToxO、EF1a-ToxO、SFH-ToxO、CAG-ToxO等一系列启动子载体。带有ToxO元件的载体分别与pcDNA3.1空载体和pcDNA3.1-DtxR共转293T细胞并加入二价铁离子。结果，没有发现目的基因受到明显的抑制作用(图10)。
其中EF1a-ToxO的质粒图谱如图9所示，CMV-ToxO、SFH-ToxO、CAG-ToxO启动子载体的质粒图谱的设计方式可在图9的基础上参考图2～5，不再赘述。
4.乳糖操作子系统
乳糖操作子抑制蛋白LacI可以特异性的和LacO元件结合，从而抑制基因的转录。在IPTG存在的情况下则与之结合并发生构象变化，丧失结合能力，下游基因开始表达。乳糖操作子在原核细胞中广泛用于外源基因的诱导表达。
本申请构建了CMV-LacO、EF1a-LacO、SFH-LacO、CAG-LacO等一系列启动子载体。带有LacO元件的载体分别与pcDNA3.1空载体和pcDNA3.1-LacI共转293T细胞。结果，没有发现目的基因受到明显的抑制作用(图12)。
其中SFH-LacO的质粒图谱如图11所示，CMV-LacO、EF1a-LacO、CAG-LacO启动子载体的质粒图谱的设计方式可在图11的基础上参考图2～5，不再赘述。
三、阻遏型操作子元件的插入位置优化
1.TrpO元件
TrpO元件相对于CMV的TATA Box距离不同，发明人设计了两个启动子，如图13所示，质粒配合pcDNA3.1- TrpR共同转染(加入0.1mM色氨酸)，48小时后观察荧光，实验显示，距离TATA Box的位置不同，会产生不同的抑制效果(见图14)，本申请后续使用了抑制效果更好的TrpO v2启动子序列。
2.CuO元件
CuO元件相对于CMV的TATA Box距离不同，设计了3个启动子，如图15所示，质粒配合pcDNA3.1-CymR共同转染，48小时后观察荧光，实验显示，CuO元件距离TATA Box的位置不同，会产生不同的抑制效果(见图16)，本申请后续使用了抑制效果更好的CuO v1启动子序列。
实施例2腺病毒的包装及目的基因的表达
一、腺病毒包装与病毒滴度检测
1、HEK-293-CMV-CymR和HEK-293-CMV-TrpR稳定细胞株的构建
此实施例构建了HEK-293-CMV-CymR和HEK-293-CMV-TrpR稳定细胞株，具体包括：
将pcDNA3.1-CymR或pcDNA3.1-TrpR利用MfeI和BstBI(NEB公司)进行双酶切线性化；酶切4～6小时后，利用回收试剂盒(QIAGEN公司)对线性化条带进行回收，回收完成后，测浓度及琼脂糖凝胶确认；
质粒转染前一天选取状态良好的HEK 293细胞，消化后收集细胞，计数后按照2×10 5个/孔铺于24孔板，摇匀，放置37℃、5％CO 2培养箱培养。转染前换液：质粒转染前1小时将前一天铺好的24孔板从培养箱中拿出，弃去原培养基，每孔加入0.5mL预热好的Opti-MEM培养基，然后放入5％CO 2培养箱待转染。将待转染的线性化的质粒(pcDNA3.1-CymR或pcDNA3.1-TrpR)溶于Opti-MEM培养基，轻轻混匀，静置；将转染试剂与Opti-MEM培养基，轻轻混匀，静置；将转染试剂混匀液滴加到质粒混匀液中，边加边轻轻混匀后在室温放置15-25分钟；取出细胞培养板，将准备好的DNA-转染试剂复合体加入到细胞培养板中，避免细胞吹起，放回培养箱；
转染48小时后，加入G418(400ug/ml)进行筛选，经鉴定混合克隆细胞表达CymR或TrpR；将此混合克隆细胞按照有限稀释法挑选pcDNA3.1-CymR或pcDNA3.1-TrpR稳定表达的HEK-293单克隆细胞。
2、腺病毒包装
此实施例为一种腺病毒的包装方法，为腺病毒AdEasy系统，具体包括：
(1)穿梭质粒重组
将带有外源基因(NLS-iCre或AceD81S)的穿梭质粒与携带了腺病毒大部分基因组的质粒(pAdEasy)进行重组，经抗性筛选获得重组腺病毒基因组质粒。对于外源基因NLS-iCre，所述穿梭质粒为：pShuttle-CMV-iCre-3xFlag-P2A-sfGFP、pShuttle-CMV-CuO-iCre-3xFlag-P2A-sfGFP或者pShuttle-CMV-TrpO-iCre-3xFlag-P2A-sfGFP；对于外源基因AceD81S，所述穿梭质粒为：pShuttle-CMV-AceD81S-mOrange2、pShuttle-CMV-TrpO-AceD81S-mOrange2或者pShuttle-CMV-CuO-AceD81S-mOrange2。具体步骤如下：
a.将穿梭质粒(pShuttle-CMV-iCre-3xFlag-P2A-sfGFP、pShuttle-CMV-CuO-iCre-3xFlag-P2A-sfGFP或者pShuttle-CMV-TrpO-iCre-3xFlag-P2A-sfGFP；pShuttle-CMV-AceD81S-mOrange2、pShuttle-CMV-TrpO-AceD81S-mOrange2或者pShuttle-CMV-CuO-AceD81S-mOrange2)用PmeI(NEB公司)进行线性化酶切；
b.酶切4～6小时后，利用回收试剂盒(QIAGEN公司)对线性化条带进行回收，回收完成后，测浓度及琼脂糖凝胶确认；
c.将BJ5183感受态细胞从-80℃拿出，迅速置于冰上，5分钟待菌块融化后，将目的质粒(1.1和1.2处理得到线性化后的穿梭质粒)与pAdEasy共同加入BJ5183感受态细胞中，轻轻混匀后，冰上静置25-30分钟，42℃水浴热激90s，立即放置冰上2分钟；向离心管中加入800ul不含抗生素的无菌培养基，37℃，200rpm振荡培养60分钟；3000rpm离心1分钟收集菌体，留取100μl上清重悬菌体，均匀涂布于含有卡纳(Kana)抗性的筛选平板上，正面放置10min，待菌液完全被培养基吸收后倒置培养皿，37℃培养箱培养12-16小时；
d.挑取10-20个单克隆菌落，小量质粒抽提后进行Pac I酶切鉴定，鉴定到重组阳性的克隆即为目的重组质粒。
e.将重组好的质粒参考1.1和1.2的进行线性化处理用于下步的转染
(2)重组后质粒的转染
a.铺板：质粒转染前一天选取状态良好的HEK 293或者pcDNA3.1-CymR或pcDNA3.1-TrpR稳定表达的HEK-293细胞，消化后收集细胞，计数后按照1×10 6个/孔铺于6孔板，摇匀，放置37℃、5％CO 2培养箱培养。
b.转染前换液：质粒转染前1小时将前一天铺好的6孔板从培养箱中拿出，弃去原培养基，每孔加入1.5mL预热好的Opti-MEM培养基，然后放入5％CO 2培养箱待转染。
c.配置转染体系：(6孔板的每个孔)
将待转染的线性化的病毒穿梭质粒溶于Opti-MEM培养基，轻轻混匀，静置；将转染试剂与Opti-MEM培养基，轻轻混匀，静置；将转染试剂混匀液滴加到质粒混匀液中，边加边轻轻混匀后在室温放置15-25分钟；
d.取出细胞培养板，将准备好的DNA-转染试剂复合体加入到细胞培养板中，避免细胞吹起，放回培养箱；
(3)病毒扩增
将转好后的细胞，3～5天更换一次培养基，注意观察病毒出毒，出毒后进行病毒扩增；其中色氨酸操作子组(pShuttle-CMV-TrpO-iCre-3xFlag-P2A-sfGFP或pShuttle-CMV-TrpO-AceD81S-mOrange2)需更换含有0.3mM色氨酸的完全培养基。
(4)病毒收毒及纯化
准备好50ml离心管，用1ml的移液器吸取细胞上清液来回吹打皿细胞，使细胞完全脱落，将细胞及上清全部收集到50ml的离心管中，利用液氮进行反复冻融后，4000rpm离心10分钟，取上清，用0.22μm的滤器过滤病毒，分装到病毒管中，-80℃保存。
3、腺病毒滴度检测
采用腺病毒滴度检测试剂盒进行腺病毒检测，其原理是腺病毒可以感染并且在HEK293细胞中复制，表达一种特殊的外壳蛋白Hexon，利用免疫染色方法检测阳性细胞，被病毒感染的阳性细胞会被染成褐色，计数阳性细胞，利用公式计算腺病毒滴度。具体包括：
a.选取状态良好的HEK293细胞，铺板，37℃、5％CO 2培养1小时；
b.准备好10倍梯度稀释的病毒样品，每孔逐滴加入100μl病毒样品，混匀后置于37℃、5％CO 2培养箱感染2天；
c.轻轻的去除培养液，沿着24孔板侧壁缓缓加入预冷的甲醇，-20℃固定20分钟；
d.使用PBS轻轻的冲洗细胞3次，每次5分钟；
e.使用1％BSA的PBS，37℃封闭1小时；
f.加入anti-Hexon抗体溶液至每个孔中，37℃孵育1小时；
g.使用PBS轻轻的冲洗细胞3次，每次5min；
h.加入辣根过氧化物酶标记的二抗至每孔，37℃孵育1小时；
i.使用PBS轻轻的冲洗细胞3次，每次5min。
j.加入1×DAB工作液至每孔，室温孵育5-10分钟
k.弃DAB，使用PBS清洗2次，每孔加入1ml PBS。
l.每孔随机选择5个视野，使用光学显微镜10×物镜下计算阳性细胞个数。
m.计算每孔阳性细胞的平均个数和腺病毒滴度。
二、腺病毒载体表达NLS-iCre基因
在病毒生产中，发现NLS-iCre基因构建到普通ADV载体中得到的病毒颗粒无法进行有效的出毒扩增。使用CMV-CuO和CMV-TrpO启动子配合抑制蛋白表达的稳定株(原理示意图见图27)，可以有效解决出毒问题(图17、图18)。
本实施例所采用的质粒pShuttle-CMV-iCre-3×Flag-P2A-sfGFP、pShuttle-CMV-TrpO-iCre-3×Flag-P2A-sfGFP和pShuttle-CMV-CuO-iCre-3×Flag-P2A-sfGFP的质粒图谱依次如图19、图20、图21所示。
三、腺病毒载体表达AceD81S基因
在病毒生产中，发现AceD81S基因构建到普通ADV载体中得到的病毒颗粒无法进行有效的出毒扩增。使用 CMV-CuO和CMV-TrpO启动子配合抑制蛋白表达的稳定株(原理示意图见图27)，可以有效解决出毒问题(图22、图23)。
本实施例所采用的质粒pShuttle-CMV-AceD81S-mOrange2、pShuttle-CMV-TrpO-AceD81S-mOrange2和pShuttle-CMV-CuO-AceD81S-mOrange2的质粒图谱依次如图24、图25、图26所示。
实施例3腺相关病毒的包装及目的基因的表达
一、腺相关病毒包装与病毒滴度检测
1、腺相关病毒包装
利用腺相关病毒载体(AAV)进行病毒包装的方法，具体包括：
(1)转染前一天，将293T细胞接种到培养皿中；转染前一小时取出细胞培养皿，弃去原有细胞培养基，加入Opti-MEM培养基，将细胞培养皿放回培养箱；制备转染试剂和质粒的复合物，包括以下步骤：
(2)将待转染的病毒载体质粒(血清型质粒、Helper辅助质粒和穿梭质粒)溶于Opti-MEM培养基，轻轻混匀，静置后得到质粒稀释液；所述血清型质粒为pAAV9、pAAV9-CMV-CymR或pAAV9--CMV-TrpR；Helper辅助质粒为pHelper、pHelper-CMV-CymR或pHelper-CMV-TrpR；对于目的基因GPR78，所述穿梭质粒为pAAV-CMV-GPR78-3×FLAG-WPRE、pAAV-CMV-CuO-GPR78-3×FLAG-WPRE或pAAV-CMV-TrpO-GPR78-3×FLAG-WPRE；对于目的基因Cdkn1a，所述pAAV-CMV-Cdkn1a-3×FLAG-WPRE、pAAV-CMV-CuO-Cdkn1a-3×FLAG-WPRE或pAAV-CMV-TrpO-Cdkn1a-3×FLAG-WPRE。
(3)将转染试剂溶于Opti-MEM培养基，轻轻混匀，静置后得到转染试剂稀释液；将转染试剂稀释液滴加到质粒稀释液中，边加边轻轻混匀后在室温放置15-25min，使DNA和转染试剂充分结合形成稳定的转染复合体；取出细胞培养皿，将准备好的DNA-转染试剂复合体加入到细胞培养皿中，放回培养箱；
(4)5-8小时后吸去培养基，用PBS溶液洗涤后加入新鲜完全培养基培养，其中色氨酸操作子组(pAAV-CMV-TrpO-GPR78-3×FLAG-WPRE或者pAAV-CMV-TrpO-Cdkn1a-3×FLAG-WPRE)需更换含有0.3mM色氨酸的完全培养基。
(5)转染60～72小时后，用枪头反复吹打细胞，使所有细胞从培养皿上完全脱落下来，收集所有细胞样品及上清。
(6)将收集到的细胞样品在-80℃和37℃进行反复的冻融，离心，收集上清，用0.45μm的PVDF滤器去除细胞碎片；随后，再利用AAV纯化试剂盒对收集的腺相关病毒进行纯化来获取腺相关病毒，-80℃冰箱保存。
2、腺相关病毒滴度检测
(1)腺相关病毒DNA抽提；
取20μL浓缩病毒液，加1μL RNase-free DNase，混匀，37℃，孵育30min，10000rpm离心10min，取20μL上清加80μL稀释Buffer至另一个无菌管中，混匀，100℃金属浴反应10min。自然冷却至室温，加入3μL蛋白酶K，37℃孵育60min，100℃金属浴反应10min，冷却至室温。
(2)Real-time PCR检测滴度
将上述样品稀释后用做模板，采用Real-time PCR检测法测定腺相关病毒滴度。Real-time PCR在ABI7500仪器上完成。使用试剂SYBR Master Mixture来自TAKARA公司。
a.按下列比例配置反应体系：
SYBR premix ex taq：10μl；
ROX：0.4μl；
上游引物(25μM)：0.5μl；
下游引物(25μM)：0.5μl；
Genomic DNA：2.0μl；
水6.6μl；
b.设定程序为两步法Real-Time定量。预变性95℃，15S，之后每一步变性95℃，5S，退火延伸60℃，34S， 共进行40个循环。每次在延伸阶段读取吸光值。
PCR程序为：
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；
启用数据收集和实时分析。
c.制作熔解曲线。PCR结束后，在95℃变性1min。然后冷却至55℃，使DNA双链充分结合。从55℃开始到95℃，每一步增加0.5℃，保持30S，同时读取吸光值。
制作溶解曲线程序为：
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
Cycle 5中每循环2次后将设定点温度提高0.5℃。
二、腺相关病毒载体表达GPR78(glucose-regulated protein,78kDa)基因
GRP78是分子伴侣蛋白的一种，作用是协助新生成的蛋白质分子折叠成正确的三维构型。在病毒生产中，发现将GPR78基因构建到普通AAV载体中基本无法获得病毒颗粒，这与现有的报道一致。使用CMV-CuO和CMV-TrpO启动子配合抑制蛋白的表达(原理示意图见图36)，可以有效解决出毒问题，病毒滴度检测结果如图28所示。本实施例所采用的质粒pAAV-CMV-GPR78-3×FLAG-WPRE、pAAV-CMV-CuO-GPR78-3×FLAG-WPRE以及pAAV-CMV-TrpO-GPR78-3×FLAG-WPRE的质粒图谱依次如图29、图30、图31所示。
三、腺相关病毒载体表达Cdkn1a基因
在病毒生产中，发现将Cdkn1a基因构建到普通AAV载体中获得的病毒颗粒总量非常低。使用CMV-CuO和CMV-TrpO启动子配合抑制蛋白的表达(原理示意图见图36)，可以有效解决出毒问题，病毒滴度检测结果如图32所示。本实施例所采用的质粒pAAV-CMV-Cdkn1a-3×FLAG-WPRE、pAAV-CMV-CuO-Cdkn1a-3×FLAG-WPRE以及pAAV-CMV-TrpO-Cdkn1a-3×FLAG-WPRE的质粒图谱依次如图33、图34、图35所示。
以上所述实施例的各技术特征可以进行任意的组合，为使描述简洁，未对上述实施例中的各个技术特征所有可能的组合都进行描述，然而，只要这些技术特征的组合不存在矛盾，都应当认为是本说明书记载的范围。
以上所述实施例仅表达了本申请的几种实施方式，其描述较为具体和详细，但并不能因此而理解为对发明专利范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本申请构思的前提下，还可以做出若干变形和改进，这些都属于本申请的保护范围。因此，本申请专利的保护范围应以所附权利要求为准。

Claims (16)

  1. 一种病毒包装载体，具有两个ITR片段及插入两个ITR片段之间的基因表达盒；
    所述基因表达盒包含顺次连接的启动子、阻遏型操作子以及任选的目的基因；
    当有阻遏物存在时，所述阻遏型操作子能够阻遏其下游目的基因的表达。
  2. 根据权利要求1所述的病毒包装载体，其中，所述病毒包装载体是腺病毒包装载体或腺相关病毒包装载体。
  3. 根据权利要求2所述的病毒包装载体，其中，所述阻遏型操作子选自色氨酸操作子和/或Cumate-CuO可调控系统。
  4. 根据权利要求3所述的病毒包装载体，其中，所述色氨酸操作子中的TrpO元件在所述基因表达盒的插入位置距离所述启动子的TATA BOX小于或等于18个核苷酸。
  5. 根据权利要求3所述的病毒包装载体，其中，所述Cumate-CuO可调控系统中的CuO元件的插入位置距离所述启动子的TATA BOX 40～50个核苷酸。
  6. 根据权利要求2所述的病毒包装载体，其中，所述启动子为CMV、EF1a、SFH、CAG、CBh、UBC、SFFV、SV40、RSV、mCMV、GAPDH、PGK、CASI、SMVP、GUSB或UCOE。
  7. 根据权利要求1～6中任一项所述的病毒包装载体，其中，所述阻遏型操作子下游插入有目的基因；
    且当所述目的基因表达时会对病毒包装产生负面影响。
  8. 根据权利要求7所述的病毒包装载体，其中，所述目的基因对包装细胞产生细胞毒性。
  9. 根据权利要求8所述的病毒包装载体，其中，所述目的基因选自自杀基因、细胞凋亡基因和癌基因。
  10. 根据权利要求7所述的病毒包装载体，其中，所述目的基因为细胞周期相关基因。
  11. 根据权利要求7所述的病毒包装载体，其中，所述目的基因直接抑制病毒的复制和/或组装。
  12. 根据权利要求1～11中任一项所述的病毒包装载体，其中，所述病毒包装载体还包含报告基因、增强子、内部核糖体进入位点、终止子和多腺苷酸化信号中的至少一种。
  13. 一种病毒包装载体系统，其包含根据权利要求1～11中任一项所述的病毒包装载体以及框架载体。
  14. 一种病毒的包装方法，包括：
    将根据权利要求13所述的病毒包装载体系统转入宿主细胞，以及
    于阻遏物存在的前提下进行包装。
  15. 根据权利要求14所述的包装方法，所述宿主细胞为HEK-293细胞或其能够提供腺病毒E1区的衍生株。
  16. 根据权利要求15所述的包装方法，所述衍生株选自HEK-293A、HEK-293S、HEK-293SG、HEK-293SGGD、HEK-293T、HEK-293T/17 SF、HEK-293H、HEK-293E、HEK-293-6E、HEK-293F、HEK-293FT、HEK-293FTM、AAV-293以及GP2-293中的至少一种。
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