WO2022116815A1 - 一种慢病毒稳定包装细胞系及其制备方法 - Google Patents

一种慢病毒稳定包装细胞系及其制备方法 Download PDF

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WO2022116815A1
WO2022116815A1 PCT/CN2021/130967 CN2021130967W WO2022116815A1 WO 2022116815 A1 WO2022116815 A1 WO 2022116815A1 CN 2021130967 W CN2021130967 W CN 2021130967W WO 2022116815 A1 WO2022116815 A1 WO 2022116815A1
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plasmid
cotetr
cell line
cells
pgagpol
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WO2022116815A9 (zh
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齐菲菲
鲁薪安
何霆
邓翠云
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Beijing Yimiaoyiliao Co Ltd
Beijing Immunochina Pharmaceuticals Co Ltd
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Beijing Yimiaoyiliao Co Ltd
Beijing Immunochina Pharmaceuticals Co Ltd
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Priority to JP2023557474A priority patent/JP7672114B2/ja
Priority to US18/039,472 priority patent/US20240124848A1/en
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Definitions

  • the invention relates to the fields of immunology and molecular biology, mainly to the field of cell therapy, and in particular to a lentivirus stable packaging cell line and a preparation method thereof.
  • Lentivirus is a gene therapy vector, including human immunodeficiency virus (HIV), equine infectious anemia virus (EIA), simian immunodeficiency virus (SIV), feline immunodeficiency virus (FIV), etc.
  • Lentiviral vectors unlike other retroviral vectors, can infect both dividing and non-dividing cells. Lentivirus can infect cardiomyocytes, tumor cells, neuronal cells, stem cells, hepatocytes, endothelial cells and other types of cells, accommodate large exogenous fragments, achieve stable expression of target genes, and have a small immune response, so as to achieve good gene expression. The therapeutic effect has broad application prospects.
  • lentiviruses are produced in most cases by transiently transfecting cells with packaging and expression plasmids.
  • the lentivirus produced by the current production process has a low titer, a large amount of residual impurities, and the amount of virus production between batches of viruses is also unstable. Therefore, it is necessary to construct a stable lentiviral packaging cell line, so that the produced lentivirus has a high titer and a low impurity content.
  • the purpose of the present invention is to propose a lentiviral stable packaging cell line and a preparation method thereof.
  • Stable packaging cell lines produce lentiviruses with high titers and low levels of impurities.
  • one aspect of the present invention provides a lentiviral stable packaging cell line, which comprises a packaging plasmid group consisting of pPuro.coTetR plasmid, pVSVG plasmid and pGagPol-RRE-NES-cINT plasmid ;
  • the pPuro.coTetR plasmid contains the CoTetR gene
  • the pVSVG plasmid contains the VSVG gene
  • the pGagPol-RRE-NES-cINT plasmid contains the GagPol and Rev genes.
  • the pPuro.coTetR plasmid further comprises a CMV promoter, a chimeric intron, a SV40 promoter, a puromycin resistance gene and a polyadenylation signal;
  • the pVSVG plasmid also comprises a hybrid promoter consisting of a CMV promoter and two tetracycline operons, an SV40 promoter, a bleomycin resistance gene and a polyadenylation signal;
  • the pGagPol-RRE-NES-cINT plasmid also contains a hybrid promoter composed of a CMV promoter and two tetracycline operons, a chimeric intron, a SV40 promoter, a hygromycin resistance gene, and a cPPT/CTS element , RRE elements and polyadenylation signals.
  • the sequence of the CoTetR gene is shown in SEQ ID NO: 1
  • the sequence of the VSVG gene is shown in SEQ ID NO: 2
  • the sequences of the GagPol and Rev genes As shown in SEQ ID NOs: 3 and 4, respectively.
  • the sequence of the pPuro.coTetR plasmid is shown in SEQ ID NO: 5
  • the sequence of the pVSVG plasmid is shown in SEQ ID NO: 6
  • the pGagPol-RRE- The sequence of the NES-cINT plasmid is shown in SEQ ID NO:7.
  • the cell line is selected from one of HEK293, HEK293-T, HEK293-SF, TE671, HT1080 or HeLa cell lines;
  • the cell line is the HEK293 cell line.
  • another aspect of the present invention provides a method for preparing the above-mentioned lentiviral stable packaging cell line, comprising the following steps:
  • polyethyleneimine is also added during the co-transfection of the linearized pPuro.coTetR plasmid and pVSVG plasmid into cells.
  • step 3 in the process of transfecting the linearized pGagPol-RRE-NES-cINT plasmid into the first positive monoclonal cell, polyethylenimine is also added, and the polyethylenimine and pGagPol-RRE -The mass ratio of NES-cINT plasmid is (1 ⁇ 8):1;
  • the mass ratio of the polyethyleneimine to the pGagPol-RRE-NES-cINT plasmid is (2-5):1;
  • the mass ratio of the polyethyleneimine to the pGagPol-RRE-NES-cINT plasmid is 4:1.
  • the dosages of the pPuro.coTetR plasmid and the pVSVG plasmid are both (0.2-1.0) ⁇ g/10 6 cells;
  • the dosages of the pPuro.coTetR plasmid and the pVSVG plasmid are both (0.4-0.9) ⁇ g/10 6 cells;
  • the dosages of the pPuro.coTetR plasmid and the pVSVG plasmid are both 0.8 ⁇ g/10 6 cells;
  • the dosage of the pGagPol-RRE-NES-cINT plasmid is (0.2-1.0) ⁇ g/10 6 first positive monoclonal cells;
  • the dosage of the pGagPol-RRE-NES-cINT plasmid is (0.4-0.9) ⁇ g/10 6 first positive monoclonal cells;
  • the dosage of the pGagPol-RRE-NES-cINT plasmid is 0.8 ⁇ g/10 6 first positive monoclonal cells.
  • the first antibiotic is a combination of puromycin and bleomycin, and the dosages thereof are 1-5 ⁇ g/mL and 300-500 ⁇ g/mL, respectively ; More preferably, the consumption of described puromycin and bleomycin is 2 ⁇ g/mL and 400 ⁇ g/mL respectively;
  • the second antibiotic is a combination of puromycin, bleomycin and hygromycin, and the dosages thereof are respectively 1 ⁇ 5 ⁇ g/mL, 300 ⁇ 500 ⁇ g/mL and 300 ⁇ 50 ⁇ g/mL. 500 ⁇ g/mL; more preferably, the dosages of the puromycin, bleomycin and hygromycin are 2 ⁇ g/mL, 400 ⁇ g/mL and 400 ⁇ g/mL, respectively.
  • another aspect of the present invention provides a lentivirus production method, wherein the above-mentioned lentivirus stable packaging cell line is transfected with a target plasmid.
  • the target plasmid was transfected into the lentiviral stable packaging cell line, and then an inducer was added to induce the lentiviral stable packaging cell line to produce lentivirus.
  • the dosage of the target plasmid is (0.2-0.8) ⁇ g/10 6 lentiviral stable packaging cell lines;
  • the dosage of the target plasmid is (0.3-0.5) ⁇ g/10 6 lentiviral stable packaging cell lines;
  • the dosage of the target plasmid is 0.4 ⁇ g/10 6 lentiviral stable packaging cell lines.
  • the inducer is doxycycline
  • the addition time of the inducer is 20-30 hours after transfection; more preferably, the addition time of the inducer is 24 hours after transfection;
  • the added concentration of the inducer is 1-5 ⁇ g/mL, more preferably, the added concentration of the inducer is 2 ⁇ g/mL.
  • an enhancer is also added during the lentivirus production process to increase the expression level of the lentivirus
  • Described enhancer is sodium butyrate
  • the added concentration of the enhancer is 5-15 mM; more preferably, the added concentration of the enhancer is 10 mM;
  • the adding time of the enhancer is 20-30h after transfection; more preferably, the adding time of the enhancer is 24h after transfection;
  • the liquid exchange time of the enhancer is 6 to 8 hours after adding the enhancer
  • the collection time of the lentivirus is 45-50 hours; preferably, the collection time of the lentivirus is 48 hours.
  • Figure 1 is the plasmid map of the packaging cell line;
  • Figure 1a is the plasmid map of the pPuro.coTetR plasmid
  • Figure 1b is the plasmid map of the pVSVG plasmid
  • Figure 1c is the plasmid map of the pGagPol-RRE-NES-cINT plasmid;
  • Fig. 2 Agar for semi-quantitative PCR identification of CoTetR, VSVG and GagPol genes of monoclonal cells Clone1, 5, 6, 7, 14, 17, 18, 19, 23, 24, 26, 27, 28, 31, 32 and 33 Glycogel electrophoresis;
  • Figure 3 is a bar graph of the target plasmid transfected into a lentiviral packaging cell line for verification of toxin production
  • Figure 4 is a bar graph of the results of the optimization experiment of Clone31 for transfection and induction of toxin production
  • Figure 5 is a bar graph of the results of the Clone31 passage stability experiment
  • Fig. 6 is the bar graph of Clone31 toxigenic stability experiment result
  • Figure 7 is a histogram of the results of the copy number stabilization experiment of Clone31 cells;
  • Figure 7a is a histogram of the results of the Clone31 VSVG gene copy number stabilization experiment;
  • Figure 7b is a histogram of the results of the Clone31 GagPol gene copy number stabilization experiment;
  • Figure 7c is Clone31 Histogram of the results of the Rev gene copy number stabilization experiment;
  • Figure 8 is a graph showing the comparison of virus titers between batches of Clone31 and 293 cells
  • Fig. 9 is the comparison chart of Clone31 and 293 cell virus titer VP (P24);
  • Figure 10 is an enlarged comparison diagram of Clone31 and 293 cells;
  • Figure 10a is a comparison diagram of the viability of E1000 transfected 24h and 48h;
  • Figure 10b is a comparison diagram of HCP residues transfected with E1000;
  • Figure 10c is a comparison diagram of VP transfected with E1000.
  • the experimental methods in the following examples are conventional methods unless otherwise specified.
  • the culture medium, reagent materials and kits used in the following examples are all commercially available products unless otherwise specified.
  • concentrations of antibiotics puromycin, bleomycin and hygromycin
  • inducer dicycline
  • enhancer sodium butyrate
  • concentration in the medium the final concentration all refer to the concentration in the medium the final concentration.
  • addition of sodium butyrate at a concentration of 5-15 mM means that the addition amount of sodium butyrate is such that the final concentration of sodium butyrate in the medium is 5-15 mM.
  • the lentiviral vector system is developed with lentivirus as the backbone and has a packaging structure and a vector structure.
  • the lentiviral packaging system generally consists of two parts: lentiviral packaging plasmid and lentiviral expression plasmid.
  • Lentiviral packaging plasmids consist of sequences encoding viral proteins, and lentiviral expression plasmids contain long terminal repeats, packaging signals, and other genetic information required for packaging, transfection, and stable integration.
  • the packaging plasmid and the expression plasmid are used to co-transfect cells, the virus is packaged in the cell and secreted into the extracellular medium to generate viral particles, which can be used for the infection of host cells.
  • lentiviruses are produced by transiently transfecting packaging plasmids and expression plasmids into 293 or 293T cells cultured in cell factories; the packaging plasmids contain long terminal repeats, packaging signals, etc.
  • the genetic information required for transfection and stable integration, and the expression plasmid contains the target gene.
  • transient transfection of 293 cells for large-scale production of lentivirus is feasible and shows good promise.
  • transient transfection requires further optimization in the production process.
  • the main bottleneck is that the transfection process requires a large amount of plasmid DNA, so the production cost is expensive, it is not easy to scale up, and the virus cannot be produced sustainably.
  • the virus produced by transient transfection has a lot of residual impurities, especially host protein and DNA residue, which increases the difficulty of the subsequent purification process, and the virus yield is also unstable between batches, which brings uncertainty to the downstream process arrangement. Therefore, it is necessary to construct a stable lentiviral packaging cell line, and stably integrate the vector elements required for packaging the virus into the genome of the host cell, so that it can be stably inherited, facilitated amplification, and achieve sustainable production.
  • the purpose of the present invention is to construct a lentivirus stable packaging cell line, and realize the production of suspension cells (especially 293 cells) by amplifying process, so that the produced lentivirus has a high titer and a low impurity content.
  • one aspect of the present invention provides a lentiviral stable packaging cell line, which comprises a packaging plasmid group consisting of pPuro.coTetR plasmid, pVSVG plasmid and pGagPol-RRE-NES-cINT plasmid ;
  • the pPuro.coTetR plasmid contains the CoTetR gene
  • the pVSVG plasmid contains the VSVG gene
  • the pGagPol-RRE-NES-cINT plasmid contains the GagPol and Rev genes.
  • the packaging plasmid and the expression plasmid are simultaneously transfected into cells (such as 293 or 293T cells).
  • the amount of toxin produced between times is also unstable.
  • the packaging plasmid is first transfected into the cell line to obtain the lentivirus stable packaging cell line, that is, the pPuro.coTetR plasmid, the pVSVG plasmid and the pGagPol-RRE-NES-cINT plasmid are transfected into the cell line to obtain the lentivirus stable packaging cell line, and then the The target plasmid is used to transfect the lentivirus stable packaging cell line, so that the produced lentivirus has a high titer and a low impurity content.
  • the present invention conducts stability research on the constructed lentiviral stable packaging cell line, including passage stability, toxin production stability and copy number stability.
  • the experimental results show that the constructed lentiviral stable packaging cell line has no screening pressure (no screening pressure).
  • the viability rate can reach more than 97%, indicating that it can be passed on for more than 30 generations without screening pressure; cells without antibiotics can be passed on normally and have the ability to produce toxins; GagPol gene
  • the copy numbers of , Rev gene and VSVG gene were relatively stable. That is, the lentivirus stable packaging cell line constructed by the present invention is relatively stable in terms of passage, toxin production and genetic gene copy number.
  • the pPuro.coTetR plasmid, the pVSVG plasmid and the pGagPol-RRE-NES-cINT plasmid of the present invention are all constructed on the basis of the commercial plasmid backbone and then inserted into the target gene.
  • the pPuro.coTetR plasmid is constructed based on the pIRESpuro3 plasmid and inserted into the CoTetR gene.
  • the CoTetR gene is a codon-optimized TetR (tetracycline repressor) gene.
  • the sequence of the CoTetR gene is shown in SEQ ID NO: 1.
  • the pVSVG plasmid It is constructed by inserting VSVG gene on the basis of pcDNA4 plasmid.
  • the sequence of VSVG (vesicular stomatitis virus glycoprotein) gene is shown in SEQ ID NO: 2.
  • the pGagPol-RRE-NES-cINT plasmid is based on pcDNA5 plasmid. , constructed by inserting GagPol (Gag gene encodes viral capsid protein, Pol gene encodes viral reverse transcriptase and gene integrase) and Rev gene (which encodes viral particle protein expression regulator), the sequences of GagPol and Rev genes are respectively As shown in SEQ ID NOs: 3 and 4.
  • the pPuro.coTetR plasmid also contains a human cytomegalovirus (CMV) promoter, a chimeric intron (which increases protein expression levels), a simian virus 40 (SV40) promoter, puromycin resistance gene (Puro R ) and polyadenylation signal, the SV40 promoter is required for expression of the puromycin resistance gene.
  • CMV human cytomegalovirus
  • SV40 simian virus 40
  • Puro R puromycin resistance gene
  • the pPuro.coTetR plasmid contains the puromycin resistance gene, and after selection with puromycin, nearly all surviving cells stably express the CoTetR gene, reducing the need to screen large numbers of cells for functional clones.
  • the pVSVG plasmid also contains a hybrid promoter consisting of a human cytomegalovirus (CMV) promoter and two tetracycline operons (TetO 2 ), a simian virus 40 (SV40) promoter, bleomycin Resistance gene (Zeo R ) and polyadenylation signal, the SV40 promoter is required for expression of the bleomycin resistance gene.
  • CMV human cytomegalovirus
  • TetO 2 tetracycline operons
  • SV40 simian virus 40
  • Zeo R bleomycin Resistance gene
  • polyadenylation signal the sequence of the pVSVG plasmid is shown in SEQ ID NO:6.
  • the pGagPol-RRE-NES-cINT plasmid also contains a hybrid promoter composed of a human cytomegalovirus (CMV) promoter and two tetracycline operons (TetO 2 ), a chimeric intron (can Increase protein expression level), simian virus 40 (SV40) promoter, hygromycin resistance gene (Hygro R ), cPPT/CTS element, RRE element and polyadenylation signal, SV40 promoter is the expression of hygromycin resistance. Promoters required for sex genes.
  • the sequence of the pGagPol-RRE-NES-cINT plasmid is shown in SEQ ID NO:7.
  • the pGagPol-RRE-NES-cINT plasmid contains the Human Immunodeficiency Virus (HIV) rev response element (RRE), a retroviral export element that regulates the transport of RNA transcripts from the nucleus to the cytoplasm.
  • HIV Human Immunodeficiency Virus
  • RRE Human Immunodeficiency Virus
  • the pGagPol-RRE-NES-cINT plasmid contains cis-acting elements—central polypurine region (cPPT) and central termination sequence (CTS).
  • cPPT central polypurine region
  • CTS central termination sequence
  • the cPPT/CTS sequence can be the cPPT/CTS of HIV1, which can improve vector integration and transduction efficiency.
  • a hybrid promoter consisting of a human cytomegalovirus (CMV) promoter and two tetracycline operons (TetO 2 ) means that two TetO 2 operon sequences are inserted into the CMV promoter, conferring tetracycline regulation function of the promoter.
  • the TetO 2 sequence consists of 2 copies of the 19 nucleotide sequence (5'-TCCCTATCAGTGATAGAGA-3').
  • another aspect of the present invention provides a method for preparing the above-mentioned lentiviral stable packaging cell line, comprising the following steps:
  • the pPuro.coTetR plasmid and pVSVG plasmid of the present invention contain puromycin resistance gene and bleomycin resistance gene, respectively, in the process of screening the first positive cloned cells, puromycin and bleomycin are added to carry out resistance
  • the pGagPol-RRE-NES-cINT plasmid contains the hygromycin resistance gene, so in the process of screening the second positive cloned cells, the resistance screening is carried out by adding puromycin, bleomycin and hygromycin .
  • polyethyleneimine (PEI) needs to be added in the two transfection processes to improve the transfection efficiency, and the dosage of PEI and each plasmid can be controlled to obtain the lentivirus stable packaging cell line of the present invention.
  • another aspect of the present invention provides a lentivirus production method, wherein the above-mentioned lentivirus stable packaging cell line is transfected with a target plasmid (an expression plasmid containing a target gene).
  • the target plasmid was transfected into the lentiviral stable packaging cell line, and then an inducer was added to induce the lentiviral stable packaging cell line to produce lentivirus.
  • the lentivirus stable packaging cell line of the present invention is an induction system.
  • doxycycline (dox) needs to be added to induce toxin production, and it is found that adding sodium butyrate during toxin production can improve the yield of lentivirus.
  • the invention also optimizes the conditions for transfecting the target plasmid into the lentivirus stable packaging cell line and inducing toxin production.
  • dox doxycycline
  • the target plasmid is not limited, and various target plasmids can be transfected as required.
  • the target plasmid is a CAR expression plasmid.
  • CAR Chimeric Antigen Receptor
  • T cell immunotherapy which is a kind of adoptive immune cell therapy.
  • 1BB and other transmembrane segments composed of a series of molecules involved in T cell activation, and then the gene segment is transfected into T cells extracted from the peripheral blood of patients by lentivirus or retroviral gene transduction; genetically modified T cells After the cells are infused back into the patient, the expressed CAR receptors are used to bind to molecules on the surface of tumor cells, thereby generating internal signals to activate T cells to rapidly destroy tumor cells.
  • CAR-T cells integrate the recognition specificity of antibodies and the killing effect and memory function of T cells.
  • the CAR expression plasmid can be transfected into the above lentiviral stable packaging cell line for the treatment of relapsed/refractory CD19-positive non-Hodgkin lymphoma.
  • CAR chimeric antigen receptor
  • the CAR expression plasmid is transfected into the above-mentioned lentivirus stable packaging cell line and the prior art four plasmid transient transfection (pPuro.coTetR plasmid, pVSVG plasmid, pGagPol-RRE-NES-cINT plasmid and CAR expression plasmid are simultaneously transfected into 293 cells ) for comparison, including cell viability, virus titer VP (P24), host protein residue and virus titer stability between batches, etc.
  • the experimental results show that the virus titer VP of the lentivirus stable packaging cell line constructed by the present invention The high content and stable toxin production between batches are beneficial to the downstream purification process.
  • the CAR expression plasmid transfection of the above lentiviral stable packaging cell line and the prior art four plasmid transient transfection were compared under the scale-up process, including cell viability, virus titer VP and HCP (host protein) residues.
  • the experimental results show that the lentiviral stable packaging cell line has high cell viability after transfection, low residual virus HCP (host protein) and high virus titer VP, which can be amplified for virus production.
  • the VP in virus titer VP refers to a method for determining the virus titer - the virus particle number (VP) method - which uses OD260 to detect the number of virus particles, and P24 refers to the virus coat protein-P24 protein.
  • the P24 protein was assayed to detect virus titers.
  • the lentiviral stable packaging cell line and the preparation method thereof provided by the present invention are relatively stable in terms of passage, toxin production and genetic gene copy number, and can be scaled up for production Virus.
  • the lentiviral stable packaging cell line has high cell viability, high viral titer VP content, less host protein and stable toxin production between batches, which is beneficial to the downstream purification process.
  • the lentiviral stable packaging cell line constructed in this example consists of three plasmids, pPuro.coTetR (containing CoTetR gene), pVSVG (containing VSVG gene), pGagPol-RRE-NES-cINT (containing GagPol and Rev genes), which are The plasmid maps of , see Figure 1a, Figure 1b and Figure 1c, respectively.
  • the INT fragment (230bp) was obtained by primer TetR-WF-1F/TetR-1R-linker and template pIRESpuro3 (TaKaRa, Cat. No.: 631619), and the INT fragment (230bp) was obtained by primer TetR-2F-linker/TetR-2R-linker and template coTetR (the sequence of which is as follows: SEQ ID NO: 1) obtained the coTetR fragment (624bp), and obtained the PA+F1ori+SV40promoter fragment (1042bp) by using the primers TetR-3F-linker/TetR-WF-3R and the template pcDNA4 (Thermo, Cat. No.: V102020).
  • the primer TetR-WF-1F/TetR-2R-linker was used to obtain the fragment TNT+coTetR
  • the primer TetR-WF-1F/TetR-WF-3R was used to obtain the full-length fragment TNT+coTetR+PA+F1ori+SV40promoter.
  • the full-length fragment prepared above was inserted into the HindIII site of the pIRESpuro3 vector by seamless cloning, and transformed into competent stbl3 (full gold, catalog number: CD521).
  • the plasmid (Kangwei Century, product number: cw2105s) was extracted, and the constructed target plasmid was used for the subsequent experiment of packaging cell line construction.
  • TetR-WF-1F 5 ACTCACTATAGGGAGACCCA GTGAGTACTCCCTCTCAAAA-3’
  • TetR-1R-linker 5 TTGGACTTGTCCAGCCTAGACATCTGTGGAGAAAGGCAAAGTGGAT-3’
  • TetR-2F-linker 5 ATCCACTTTGCCTTTCTCTCCACAGATGTCTAGGCTGGACAAGTCCAA-3’
  • TetR-2R-linker 5 -ATGGCTGGCAACTAGAAGGCACAGTCAGCTGCCGCTTTCGCACTTGA-3’
  • TetR-3F-linker 5 TetR-3F-linker 5’-TCAAGTGCGAAAGCGGCAGCTGACTGTGCCTTCTAGTTGCCAGCCAT-3’
  • TetR-WF-3R 5 -CTTGTGGGTTGTGGCAAGCT TTTGCAAAAGCCTAGGCCTC-3’
  • the VSVG fragment (1536bp) was obtained by primer VSVG-WF-F/VSVG-WF-R and template VS-SC (its sequence is shown in SEQ ID NO: 8), and the fragment prepared above was inserted into pcDNA4 vector by seamless cloning method
  • the XhoI site was transformed into competent stbl3. After the sequencing was correct, the plasmid was extracted using the Kangwei endotoxin-free intermediate extraction kit, and the constructed target plasmid was used for subsequent packaging cell line construction experiments.
  • Primer sequence VSVG-WF-F 5’-ATATCCAGCACAGTGGCGGCCGCATGAAGTGCCTTTTGTACTT-3’
  • VSVG-WF-R 5 AACGGGGCCCTCTAGACTCGATTACTTTCCAAGTCGGTTCA-3’
  • GagPol fragment (4307bp) was obtained by primer pGagPol-XhoI-WF/pGagPol-XhoI-WR and template MDK-SC (its sequence is shown in SEQ ID NO: 9), and the fragment prepared above was inserted into pcDNA5 by seamless cloning method
  • the XhoI site of the vector (Thermo, catalog number: V103320) was transformed into competent stbl3. After the sequencing was correct, the plasmid was extracted using the Kangwei endotoxin-free intermediate extraction kit, and the constructed target plasmid pGagPol was used for subsequent vector construction.
  • the RRE-NES fragment (1705 bp) was obtained by primers pGagPol-REV-Xba I-WF/pGagPol-REV-Xba I-WR and template AX (also known as psPAX2 vector, whose sequence is shown in SEQ ID NO: 10), The fragment prepared above was inserted into the XbaI site of the pGagPol vector by seamless cloning, and transformed into competent stbl3. NES was used for subsequent vector construction.
  • the cINT fragment (1122bp) was obtained by primer pGagPol-RN-cINT--Not I-QF/pGagPol-INT-Not I-WR and template AX, and the fragment prepared above was inserted into pGagPol-RRE-NES vector by seamless cloning method
  • the NotI site was transformed into competent stbl3.
  • the plasmid was extracted using the Kangwei endotoxin-free middle extraction kit, and the constructed target plasmid pGagPol-RRE-NES-cINT was used for subsequent vector construction.
  • Example 1 The three plasmids constructed in Example 1 were linearized with Fsp I (Thermo, Cat. No.: FD1224), and then dephosphorylated, and used to transfect 293 cells.
  • Fsp I Thermo, Cat. No.: FD1224
  • the linearized pPuro.coTetR and pVSVG plasmids were co-transfected with PEI (polyethyleneimine) (Polysciences, Cat. No. 23966) in 293 suspension cells.
  • Polyclonal PV cells were plated by limiting dilution in 96-well plates (Thermo, Cat. No. 167008) with 200 ⁇ L of medium per well. Then microscopically, the wells with only single cells were picked out, labeled, and incubated in an incubator for about 20-30 days.
  • the surviving monoclonal cells in the 96-well plate were expanded and cultured using 48-well plate, 24-well plate, 12-well plate, 6-well plate, and shake flask E125, respectively.
  • the PV monoclonal cells were taken to extract RNA, and after reverse transcription into cDNA, semi-quantitative PCR was performed to identify whether the target genes CoTetR and VSVG were contained, and ⁇ -actin was used as the internal reference gene.
  • the correctly identified PV monoclonal cells were transfected with PEI (polyethyleneimine) to the linearized plasmid pGagPol-RRE-NES-cINT, and the amount of pGagPol-RRE-NES-cINT plasmid was 0.8 ⁇ g/10 6 during transfection
  • PEI amount: pGagPol-RRE-NES-cINT plasmid amount 4:1, after using 2 ⁇ g/mL Puro (puromycin), 400 ⁇ g/mL Zeo (bleomycin), 400 ⁇ g/mL mL HYG (hygromycin) (Invitrogen, product number: 10687010) antibiotics were subjected to pressurized screening for 2 weeks to obtain a drug-resistant positive stable cell pool, that is, polyclonal PVGR cells.
  • Polyclonal PVGR cells were plated by limiting dilution in 96-well plates with 200 ⁇ L of medium per well. Then microscopically, the wells with only single cells were picked out, labeled, and incubated in an incubator for about 20-30 days. The monoclonal cells that survived in the 96-well plate were numbered Clone1, 5, 6, 7, 14, 17, 18, 19, 23, 24, 26, 27, 28, 31, 32 and 33.
  • the surviving monoclonal cells in the 96-well plate were expanded and cultured using 48-well plate, 24-well plate, 12-well plate, 6-well plate, and shake flask E125, respectively.
  • the PVGR monoclonal cells were taken to extract RNA, and after reverse transcribed into cDNA, semi-quantitative PCR was performed to identify whether they contained the target genes CoTetR, VSVG and GagPol.
  • ⁇ -actin was used as an internal reference gene. Specific steps are as follows:
  • the extracted RNA was reverse transcribed into cDNA with 2 ⁇ g each, and the first strand cDNA synthesis kit (Thermo Scientific TM , Cat: K1612) was used for the operation method as described in the instructions.
  • PCR amplification of the target gene system add 10 ⁇ L of 2 ⁇ phanta Max master mix (Nanjing Novizan, product number: P515-02), add 1 ⁇ L of forward and reverse primers, add 7 ⁇ L of ddH 2 O, and add 1 ⁇ L of cDNA template.
  • PCR reaction program 95°C for 10 min, 95°C for 30s, 56°C for 30s, 72°C for 30s, 72°C for 10 min, 30 cycles.
  • the semi-quantitative PCR primers are as follows:
  • VSVG forward primer 5'-ccgctcgagatgaagtgccttttgtactt-3'
  • GagPol forward primer 5'-ccggccataaagcaagagtt-3'
  • GagPol reverse primer 5'-ccgcagatttctatgagtat-3'
  • ⁇ -actin forward primer 5′-CTCCATCCTGGCCTCGCTGT-3′
  • ⁇ -actin reverse primer 5'-GCTGTCACCTTCACCGTTCC-3'
  • the CAR molecule of this example comprises a signal peptide, an extracellular binding region, an optional hinge region, a transmembrane region, and an intracellular signal region.
  • the nucleotide sequence of the extracellular binding region is in the antigen binding region of the anti-CD19 chimeric antigen receptor (named here as anti-CD19 scFv-S0, abbreviated as scFv-S0, which is derived from mice, see J. Immunother.2009September;32(7):689-702.) based on the nucleotide sequence obtained by humanization.
  • the preparation steps of the nucleotide sequence of the CAR molecule are specifically described.
  • primer sequences used in this example are as follows:
  • 6-1 5'-CTAGACTAGTATGCTTCTCCTGGTGACAAGCC-3'
  • the corresponding CAR molecular parts were cloned by PCR, which are GMCSF, CD28-TM+CD28-signal (the two parts are linked) and CD3 ⁇ -signal. Then, the GMCSF+scFv fragment was obtained by bridging primers 4-1 and 4-2, and the CD28-TM+CD28-signal+CD3 ⁇ -signal fragment was obtained by bridging primers 5-1 and 5-2, followed by bridging primers 6-1 and 6.
  • -2 Obtain the nucleotide sequence of the complete CAR molecule, and the restriction sites are SpeI and MluI.
  • the nucleotide sequence of the CAR molecule prepared above was double digested by SpeI (Fermentas) and MluI (Fermentas), ligated with T4 ligase (Fermentas) and inserted into the SpeI-MluI site of the lentiviral IM19 vector, and transformed into competent E. .coli (DH5 ⁇ ), after the sequencing was correct, the plasmid was extracted and purified using the plasmid purification kit of Qrigene Company, and the constructed target plasmid (CAR expression plasmid) was used for the subsequent toxin production experiments.
  • the PVGR monoclonal cells identified correctly are used to verify the toxin production, that is, the PVGR monoclonal cells of Example 3 are transiently transfected with the target plasmid constructed in Example 4 to verify the toxin production. Specific steps are as follows:
  • PVGR monoclonal cells were plated in a 12-well plate the day before, and 1 mL of SMM293 TI+0.1% F68 medium was used to plate 8 ⁇ 10 5 cells/mL per well, and the target plasmid was transfected the next day. 24h after transfection, the inducer dox (doxycycline) was added at a final concentration of 2 ⁇ g/mL. 48h after transfection, the virus was collected, centrifuged at 3000rpm, 4°C for 20min. The virus was infected with Jurkat cells, and the flow rate was detected after 48 h, and the virus titer (TU/mL) was calculated.
  • inducer dox dioxycycline
  • Example 6 Experiments on the optimization of conditions for transfection of target plasmids into stable packaging cell lines and induction of toxin production
  • the monoclonal Clone31 with relatively high toxigenic titer was selected for further study.
  • Clone31 was inoculated into E125 shake flasks (Corning, Cat: 431145), 8 ⁇ 10 5 cells/mL, 10 mL each, and 10 mL of SMM293 TI medium.
  • the factors include: plasmid amount (target plasmid), PEI (polyethyleneimine) amount, concentration of sodium butyrate, time of adding sodium butyrate, sodium butyrate The time of medium change, time of addition of dox (doxycycline), concentration of added dox (doxycycline) and time of virus harvest, determine the effects of curvature and factors.
  • the second time a central composite design was used to determine the optimal experimental scheme. 48h after transfection, the virus was collected, centrifuged at 3000rpm, 4°C for 20min, and the virus titer TU was detected.
  • Example 7 Stable packaging cell line passage, toxin production, copy number stability verification
  • the lentiviral stable packaging cell line Clone31 was used for further stability studies, including passage stability, toxigenic stability and copy number stability.
  • the Clone31 monoclonal cells were passaged in E125 shake flasks with and without antibiotics, and cultured 10 mL each time, and the cell number and viability were counted.
  • inoculate Clone31 in E125 shake flasks (Corning, Cat: 431145), respectively, inoculate 8 ⁇ 10 5 cells/mL, each with 10 mL, respectively use 10 mL SMM293 TI medium (without antibiotics), 10 mL SMM293 TI+ 0.6 ⁇ g/mL Puro + 50 ⁇ g/mL Zeo + 50 ⁇ g/mL Hyg. Passage every other day, and count the cell number and viability. Look at its generation stability.
  • inoculate Clone31 into E125 shake flasks (Corning, Cat: 431145) respectively, inoculate 1.4 ⁇ 10 6 cells/mL each, 10 mL each, and use 10 mL SMM293 TI medium (without antibiotics), 10 mL SMM293 TI + 0.6 ⁇ g/mL Puro (puromycin) + 50 ⁇ g/mL Zeo (bleomycin) + 50 ⁇ g/mL Hyg (hygromycin).
  • transfection with the target plasmid (constructed in Example 4) was performed. Toxicity was verified by transfection every few generations.
  • Clone31 monoclonal cells were verified for copy number stability with and without antibiotics every few passages. Using the Cell Genome Extraction Kit (Tiangen Biochemical Technology Co., Ltd., Cat. No.: DP304-03), the genomic DNA of Clone31 monoclonal cells with and without antibiotics was extracted respectively, and then the Taqman-qPCR method was used to amplify the Clone31 monoclonal cells. Copy number of GagPol, Rev and VSVG in the genome of cloned cells.
  • inoculate Clone31 in E125 shake flasks (Corning, Cat: 431145), respectively, inoculate 8 ⁇ 10 5 cells/mL, each with 10 mL, respectively use 10 mL SMM293 TI medium (without antibiotics), 10 mL SMM293 TI+ 0.6 ⁇ g/mL Puro (puromycin) + 50 ⁇ g/mL Zeo (bleomycin) + 50 ⁇ g/mL Hyg (hygromycin). Cellular genomic DNA was extracted every few generations.
  • the logarithm of the standard copy number is the abscissa, and the CT value is the ordinate.
  • the qPCR reaction system (Takara, product number: RR390A) is: 2 ⁇ Premix Ex Taq (Probe qPCR) plus 10 ⁇ L, F primer (10 ⁇ mol/L) plus 0.4 ⁇ L, R primer (10 ⁇ mol/L) plus 0.4 ⁇ L, TaqMan Probe (5 ⁇ mol/L) /L) add 0.4 ⁇ L, ROX Reference DyeII (50 ⁇ ) add 0.4 ⁇ L, template add 2 ⁇ L, ddH 2 O add 6.4 ⁇ L; the F primer and R primer are as follows:
  • VSVG-q-R 5'-CCTGGGTTTTTAGGAGCAAGATAG-3'
  • GagPol-q-F 5’-GCAGTTCATGTAGCCAGTGGATAT-3’
  • GagPol-q-R 5’-TGGTGAAATTGCTGCCATTG-3’
  • the qPCR reaction program was: 95°C for 30s, 95°C for 5s, 55°C for 15s, 72°C for 35s, 40 cycles. Substitute the obtained CT value into the scale to calculate the copy number, and then according to the mass, calculate the copy number per cell.
  • the copy number of Clone31 VSVG gene is shown in Figure 7a
  • the copy number of Clone31 GagPol gene is shown in Figure 7b
  • the copy number of Clone31 Rev gene is shown in Figure 7c.
  • the experimental results showed that the copy numbers of GagPol, Rev and VSVG genes in Clone31 monoclonal cells were relatively stable.
  • Clone31 monoclonal cells were transfected and induced with optimized conditions.
  • 293 cells were transfected with transient conditions: the pPuro.coTetR plasmid, pVSVG plasmid and pGagPol-RRE-NES-cINT plasmid constructed in Example 1 and the target plasmid constructed in Example 4 were simultaneously transfected into 293 cells, and the amount of transiently transfected plasmid It has little effect on the titer of the virus within a certain range.
  • the total plasmid amount is 0.8 ⁇ g to 1.0 ⁇ g/10 6 cells.
  • pVSVG The mass ratio of NES-cINT plasmid and target plasmid was 1:1:1: 2 , and the total plasmid amount was 0.8 ⁇ g/106 cells (i.e.
  • Virus titer stability and virus titer VP(P24) were compared between batches after transfection (see Figures 8 and 9).
  • the Clone31 monoclonal cells were expanded from E125 shake flasks to E1000 (Corning, Cat. No.: 431147), and then the target plasmid (constructed in Example 4) was transfected into Clone31 monoclonal cells for toxin production verification, and 293 cells were transfected for control, Clone31
  • the transfection conditions of monoclonal cells and 293 cells were the same as in Example 8. Comparisons of cell viability, virus titer VP (Takara, Cat. No. 632200) and HCP (Host Protein) (Cygnus, Cat. No. F650) residues were performed (see Figure 10).
  • the experimental results of Figure 10a show that the cell viability of Clone31 monoclonal cells is higher than that of 293 cells at 24h and 48h of transfection; the experimental results of Figure 10b show that the HCP (host protein) residue of Clone31 monoclonal cells is 1/3 of that of 293 cells, and that of Clone31 cells Monoclonal cells have less HCP (host protein) residues; the experimental results in Figure 10c show that the viral titer VP (P24) of Clone31 monoclonal cells is 10-20 times that of 293 cells, and the viral titer VP (P24) of Clone31 monoclonal cells ) high content.

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Abstract

提供了一种慢病毒稳定包装细胞系及其制备方法，该慢病毒稳定包装细胞系包含由pPuro.coTetR质粒、pVSVG质粒和pGagPol-RRE-NES-cINT质粒组成的包装质粒组；其中，pPuro.coTetR质粒包含CoTetR基因，pVSVG质粒包含VSVG基因，pGagPol-RRE-NES-cINT质粒包含GagPol和Rev基因。该慢病毒稳定包装细胞系在传代、产毒和遗传基因拷贝数方面都比较稳定，而且使用该慢病毒稳定包装细胞系生产的慢病毒滴度高，杂质含量少。

Description

一种慢病毒稳定包装细胞系及其制备方法 技术领域
本发明涉及免疫学和分子生物学领域，主要是细胞治疗领域，尤其涉及一种慢病毒稳定包装细胞系及其制备方法。
背景技术
慢病毒(Lentivirus)是一种基因治疗载体，包括人类免疫缺陷病毒(HIV)、马传染性贫血病毒(EIA)、猴免疫缺陷病毒(SIV)、猫免疫缺陷病毒(FIV)等。慢病毒载体与其它逆转录病毒载体不同，可以感染分裂细胞和非分裂细胞。慢病毒可以感染心肌细胞、肿瘤细胞、神经元细胞、干细胞、肝细胞、内皮细胞等多种类型的细胞，容纳外源片段大，实现目的基因的稳定表达，免疫反应小，从而达到良好的基因治疗效果，具有广阔的应用前景。
目前的慢病毒在大多数情况下是通过将包装质粒和表达质粒瞬时转染细胞产生的。但是目前的生产工艺生产的慢病毒滴度低，杂质残留多，且病毒批次间产毒量也不稳定。因此，需要构建稳定的慢病毒包装细胞系，使生产的慢病毒滴度高，杂质含量少。
发明内容
有鉴于此，本发明的目的是提出一种慢病毒稳定包装细胞系及其制备方法，该慢病毒稳定包装细胞系在传代、产毒和遗传基因拷贝数方面都比较稳定，而且使用该慢病毒稳定包装细胞系生产的慢病毒滴度高，杂质含量少。
基于上述目的，本发明的一个方面提供了一种慢病毒稳定包装细胞系，其中，包含包装质粒组，所述包装质粒组由pPuro.coTetR质粒、pVSVG质粒和pGagPol-RRE-NES-cINT质粒组成；
所述pPuro.coTetR质粒包含CoTetR基因，所述pVSVG质粒包含VSVG基因，所述pGagPol-RRE-NES-cINT质粒包含GagPol和Rev基因。
在本发明的优选的实施方案中，其中，所述pPuro.coTetR质粒还包含CMV启动子、嵌合体内含子、SV40启动子、嘌呤霉素抗性基因和多腺苷酸化信号；
所述pVSVG质粒还包含由CMV启动子和两个四环素操纵子组成的杂合启动子、SV40启动子、博来霉素抗性基因和多腺苷酸化信号；
所述pGagPol-RRE-NES-cINT质粒还包含由CMV启动子和两个四环素操纵子组成的杂合启动子、嵌合体内含子、SV40启动子、潮霉素抗性基因、cPPT/CTS元件、RRE元件和多腺苷酸化信号。
在本发明的优选的实施方案中，其中，所述CoTetR基因的序列如SEQ ID NO：1所示，所述VSVG基因的序列如SEQ ID NO：2所示，所述GagPol和Rev基因的序列分别如SEQ ID NO：3和4所示。
在本发明的优选的实施方案中，其中，所述pPuro.coTetR质粒的序列如SEQ ID NO：5所示，所述pVSVG质粒的序列如SEQ ID NO：6所示，所述pGagPol-RRE-NES-cINT质粒的序列如SEQ ID NO：7所示。
在本发明的优选的实施方案中，其中，所述细胞系选自HEK293、HEK293-T、HEK293-SF、TE671、HT1080或HeLa细胞系中的一种；
优选地，所述细胞系为HEK293细胞系。
基于相同的发明构思，本发明的另一个方面提供了一种制备上述的慢病毒稳定包装细胞系的方法，其中，包括以下步骤：
1)将pPuro.coTetR质粒、pVSVG质粒和pGagPol-RRE-NES-cINT质粒分别线性化酶切；
2)将线性化的pPuro.coTetR质粒和pVSVG质粒共转染细胞，并加入第一抗生素进行筛选，得到第一阳性单克隆细胞；
3)用线性化的pGagPol-RRE-NES-cINT质粒转染第一阳性单克隆细胞，并加入第二抗生素进行筛选，得到第二阳性单克隆细胞，即为慢病毒稳定包装细胞系。
在本发明的优选的实施方案中，其中，在步骤2)中，将线性化的pPuro.coTetR质粒和pVSVG质粒共转染细胞的过程中还加入聚乙烯亚胺，所述聚乙烯亚胺与质粒的质量比为：聚乙烯亚胺：(pPuro.coTetR质粒+pVSVG质粒)＝(1～8):1；
优选地，所述聚乙烯亚胺与质粒的质量比为：聚乙烯亚胺：(pPuro.coTetR质粒+pVSVG质粒)＝(2～5):1；
更优选地，所述聚乙烯亚胺与质粒的质量比为：聚乙烯亚胺：(pPuro.coTetR质粒+pVSVG质粒)＝4:1；
和/或，在步骤3)中，将线性化的pGagPol-RRE-NES-cINT质粒转染第一阳性单克隆细胞的过程中还加入聚乙烯亚胺，所述聚乙烯亚胺与pGagPol-RRE-NES-cINT质粒的质量比为(1～8):1；
优选地，所述聚乙烯亚胺与pGagPol-RRE-NES-cINT质粒的质量比为(2～5):1；
更优选地，所述聚乙烯亚胺与pGagPol-RRE-NES-cINT质粒的质量比为4:1。
在本发明的优选的实施方案中，其中，在步骤2)中，所述pPuro.coTetR质粒和pVSVG质粒的用量均为(0.2～1.0)μg/10 6个细胞；
优选地，所述pPuro.coTetR质粒和pVSVG质粒的用量均为(0.4～0.9)μg/10 6个细胞；
更优选地，所述pPuro.coTetR质粒和pVSVG质粒的用量均为0.8μg/10 6个细胞；
和/或，在步骤3)中，所述pGagPol-RRE-NES-cINT质粒的用量为(0.2～1.0)μg/10 6个第一阳性单克隆细胞；
优选地，所述pGagPol-RRE-NES-cINT质粒的用量为(0.4～0.9)μg/10 6个第一阳性单克隆细胞；
更优选地，所述pGagPol-RRE-NES-cINT质粒的用量为0.8μg/10 6个第一阳性单克隆细胞。
在本发明的优选的实施方案中，其中，在步骤2)中，所述第一抗生素为嘌呤霉素和博来霉素的组合物，其用量分别为1～5μg/mL和300～500μg/mL；更优选地，所述嘌呤霉素和博来霉素的用量分别为2μg/mL和400μg/mL；
和/或，在步骤3)中，所述第二抗生素为嘌呤霉素、博来霉素和潮霉素的组合物，其用量分别为1～5μg/mL、300～500μg/mL和300～500μg/mL；更优选地，所述嘌呤霉素、博来霉素和潮霉素的用量分别为2μg/mL、400μg/mL和400μg/mL。
基于相同的发明构思，本发明的另一个方面提供了一种慢病毒的生产方法，其中，将目的质粒转染上述的慢病毒稳定包装细胞系。
在本发明的优选的实施方案中，其中，包括：
将目的质粒转染慢病毒稳定包装细胞系，然后加入诱导剂诱导慢病毒稳定包装细胞系产生慢病毒。
在本发明的优选的实施方案中，其中，将目的质粒转染慢病毒稳定包装细胞系的过程 中还加入聚乙烯亚胺，所述聚乙烯亚胺与目的质粒的质量比为：聚乙烯亚胺：目的质粒＝(1～8):1；
优选地，所述聚乙烯亚胺与目的质粒的质量比为：聚乙烯亚胺：目的质粒＝(2～5):1；
更优选地，所述聚乙烯亚胺与目的质粒的质量比为：聚乙烯亚胺：目的质粒＝4:1。
在本发明的优选的实施方案中，其中，所述目的质粒的用量为(0.2～0.8)μg/10 6个慢病毒稳定包装细胞系；
优选地，所述目的质粒的用量均为(0.3～0.5)μg/10 6个慢病毒稳定包装细胞系；
更优选地，所述目的质粒的用量为0.4μg/10 6个慢病毒稳定包装细胞系。
在本发明的优选的实施方案中，其中，所述诱导剂为强力霉素；
优选地，所述诱导剂的加入时间为转染后20～30h；更优选地，所述诱导剂的加入时间为转染后24h；
优选地，所述诱导剂的加入浓度为1～5μg/mL，更优选地，所述诱导剂的加入浓度为2μg/mL。
在本发明的优选的实施方案中，其中，在慢病毒产生过程中还加入增强剂提高慢病毒的表达量；
所述增强剂为丁酸钠；
优选地，所述增强剂的加入浓度为5～15mM；更优选地，所述增强剂的加入浓度为10mM；
优选地，所述增强剂的加入时间为转染后20～30h；更优选地，所述增强剂的加入时间为转染后24h；
优选地，所述增强剂的换液时间为加入增强剂后6～8h；
和/或，所述慢病毒收集的时间为45～50h；优选地，所述慢病毒收集的时间为48h。
附图说明
图1为包装细胞系的质粒图谱；其中图1a为pPuro.coTetR质粒的质粒图谱，图1b为pVSVG质粒的质粒图谱，图1c为pGagPol-RRE-NES-cINT质粒的质粒图谱；
图2为半定量PCR鉴定单克隆细胞Clone1、5、6、7、14、17、18、19、23、24、26、27、28、31、32和33的CoTetR、VSVG和GagPol基因的琼脂糖凝胶电泳图；
图3为目的质粒转染慢病毒包装细胞系进行产毒验证的柱状图；
图4为Clone31进行转染和诱导产毒的优化实验结果的柱状图；
图5为Clone31传代稳定性实验结果的柱状图；
图6为Clone31产毒稳定性实验结果的柱状图；
图7为Clone31细胞的拷贝数稳定实验结果的柱状图；其中图7a为Clone31 VSVG基因拷贝数稳定实验结果的柱状图；图7b为Clone31 GagPol基因拷贝数稳定实验结果的柱状图；图7c为Clone31 Rev基因拷贝数稳定实验结果的柱状图；
图8为Clone31和293细胞批次间病毒滴度对比图；
图9为Clone31和293细胞病毒滴度VP(P24)对比图；
图10为Clone31和293细胞放大对比图；其中图10a为E1000转染24h和48h活率对比图；图10b为E1000转染HCP残留对比图；图10c为E1000转染VP对比图。
具体实施方式
需要说明的是，除非另外定义，本说明书一个或多个实施例使用的技术术语或者科学 术语应当为本公开所属领域内具有一般技能的人士所理解的通常意义。
下述实施例中的实验方法，如无特殊说明，均为常规方法。下述实施例中所用的培养基、试剂材料和试剂盒等，如无特殊说明，均为市售购买产品。
需要说明的一点，本发明中的抗生素(嘌呤霉素、博来霉素和潮霉素)、诱导剂(强力霉素)和增强剂(丁酸钠)的浓度均指的是在培养基中的终浓度。例如，丁酸钠的加入浓度为5～15mM指的是，丁酸钠的加入量为使丁酸钠在培养基中的终浓度为5～15mM。
慢病毒载体系统以慢病毒为骨架发展起来，有包装结构和载体结构。慢病毒包装系统一般由两个部分组成：慢病毒包装质粒和慢病毒表达质粒。慢病毒包装质粒由编码产生病毒蛋白的序列组成，慢病毒表达质粒含有长末端重复系列、包装信号等包含了包装、转染、稳定整合所需要的遗传信息。利用包装质粒和表达质粒时共转染细胞，病毒在细胞中包装并分泌到细胞外培养基，产生病毒颗粒，可以用于宿主细胞的感染。
目前的慢病毒在大多数情况下是通过将包装质粒和表达质粒瞬时转染在细胞工厂中培养的293或293T细胞产生的；其中包装质粒含有长末端重复系列、包装信号等包含了包装、转染、稳定整合所需要的遗传信息，表达质粒含有目的基因。
利用293细胞瞬时转染大规模生产慢病毒是可行的，并显示出良好的前景。然而，瞬时转染在工艺生产中还需要进一步的优化，其主要瓶颈是转染过程需要大量的质粒DNA，因此生产成本昂贵，不易放大，不可持续生产病毒。瞬时转染生产的病毒，杂质残留多，特别是宿主蛋白、DNA残留等，增加后续纯化工艺难度，且病毒批次间产毒量也不稳定，这给下游工艺安排带来不确定性。因此，需要构建稳定的慢病毒包装细胞系，将包装病毒所需的载体元件稳定的整合在宿主细胞的基因组中，使其可以稳定的遗传，方便放大，实现可持续生产。
本发明的目的是构建一种慢病毒稳定包装细胞系，实现悬浮细胞(特别是293细胞)放大工艺生产，使生产的慢病毒滴度高，杂质含量少。
基于上述目的，本发明的一个方面提供了一种慢病毒稳定包装细胞系，其中，包含包装质粒组，所述包装质粒组由pPuro.coTetR质粒、pVSVG质粒和pGagPol-RRE-NES-cINT质粒组成；
所述pPuro.coTetR质粒包含CoTetR基因，所述pVSVG质粒包含VSVG基因，所述pGagPol-RRE-NES-cINT质粒包含GagPol和Rev基因。
现有技术中慢病毒的生产方法中是将包装质粒和表达质粒同时转染细胞(例如293或293T细胞)，其存在的主要问题在于生产的慢病毒滴度低，杂质含量高，且病毒批次间产毒量也不稳定。本发明先将包装质粒转染细胞系得到慢病毒稳定包装细胞系，即将pPuro.coTetR质粒、pVSVG质粒和pGagPol-RRE-NES-cINT质粒转染细胞系得到慢病毒稳定包装细胞系，然后再将目的质粒转染慢病毒稳定包装细胞系，使生产的慢病毒滴度高，杂质含量少。
本发明对构建的慢病毒稳定包装细胞系进行了稳定性研究，包括传代稳定性、产毒稳定性和拷贝数稳定性，实验结果表明，构建的慢病毒稳定包装细胞系在无筛选压力(不加抗生素)时能稳定传代遗传，并且活率可达97％以上，说明其在无筛选压力时能稳定传代30代以上；在不加抗生素传代的细胞能正常遗传，具有产毒能力；GagPol基因、Rev基因和VSVG基因的拷贝数相对稳定。即本发明构建的慢病毒稳定包装细胞系在传代、产毒和遗传基因拷贝数方面都比较稳定。
本发明的pPuro.coTetR质粒、pVSVG质粒和pGagPol-RRE-NES-cINT质粒均是以商业化的质粒骨架为基础，然后插入目的基因而构建的。例如pPuro.coTetR质粒是以 pIRESpuro3质粒为基础，插入CoTetR基因而构建的，CoTetR基因是密码子优化后的TetR(四环素阻遏物)基因，CoTetR基因的序列如SEQ ID NO：1所示，pVSVG质粒是以pcDNA4质粒为基础，插入VSVG基因而构建的，VSVG(水泡性口炎病毒糖蛋白)基因的序列如SEQ ID NO：2所示，pGagPol-RRE-NES-cINT质粒是以pcDNA5质粒为基础，插入GagPol(Gag基因编码病毒的壳蛋白，Pol基因编码病毒的反转录酶和基因整合酶)和Rev基因(其编码病毒颗粒蛋白表达调节因子)而构建的，GagPol和Rev基因的序列分别如SEQ ID NO：3和4所示。
如图1a所示，pPuro.coTetR质粒还包含人类巨细胞病毒(CMV)启动子、嵌合体内含子(可提高蛋白的表达水平)、猿猴病毒40(SV40)启动子、嘌呤霉素抗性基因(Puro R)和多腺苷酸化信号，SV40启动子是表达嘌呤霉素抗性基因所需的启动子。pPuro.coTetR质粒含有嘌呤霉素抗性基因，用嘌呤霉素选择后，几乎所有存活的细胞都会稳定表达CoTetR基因，从而减少了筛选大量细胞以寻找功能性克隆的需要。当使用pPuro.coTetR质粒时，抗生素会在整个表达盒上施加选择性压力。因此，高剂量的抗生素将选择表达高水平CoTetR基因的细胞。这种选择压力还确保了CoTetR基因的表达将保持稳定。所述pPuro.coTetR质粒的序列如SEQ ID NO：5所示。
如图1b所示，pVSVG质粒还包含由人类巨细胞病毒(CMV)启动子和两个四环素操纵子(TetO 2)组成的杂合启动子、猿猴病毒40(SV40)启动子、博来霉素抗性基因(Zeo R)和多腺苷酸化信号，SV40启动子是表达博来霉素抗性基因所需的启动子。所述pVSVG质粒的序列如SEQ ID NO：6所示。
如图1c所示，pGagPol-RRE-NES-cINT质粒还包含由人类巨细胞病毒(CMV)启动子和两个四环素操纵子(TetO 2)组成的杂合启动子、嵌合体内含子(可提高蛋白的表达水平)、猿猴病毒40(SV40)启动子、潮霉素抗性基因(Hygro R)、cPPT/CTS元件、RRE元件和多腺苷酸化信号，SV40启动子是表达潮霉素抗性基因所需的启动子。所述pGagPol-RRE-NES-cINT质粒的序列如SEQ ID NO：7所示。
pGagPol-RRE-NES-cINT质粒含有人类免疫缺陷病毒(HIV)rev应答元件(RRE)，该RRE元件为逆转录病毒输出元件，调节RNA转录物从细胞核到细胞质的转运。
pGagPol-RRE-NES-cINT质粒含有顺式作用元件——中央多聚嘌呤区(cPPT)和中心终止序列(CTS)，cPPT/CTS序列可以是HIV1的cPPT/CTS，能够提高载体整合和转导效率。
在本发明中，由人类巨细胞病毒(CMV)启动子和两个四环素操纵子(TetO 2)组成的杂合启动子是指两个TetO 2操纵子序列插入到CMV启动子中，赋予四环素调节该启动子的功能。TetO 2序列由19个核苷酸序列(5'-TCCCTATCAGTGATAGAGA-3')的2个拷贝组成。
基于相同的发明构思，本发明的另一个方面提供了一种制备上述的慢病毒稳定包装细胞系的方法，其中，包括以下步骤：
1)将pPuro.coTetR质粒、pVSVG质粒和pGagPol-RRE-NES-cINT质粒分别线性化酶切；
2)将线性化的pPuro.coTetR质粒和pVSVG质粒共转染细胞，并加入第一抗生素进行筛选，得到第一阳性单克隆细胞；
3)用线性化的pGagPol-RRE-NES-cINT质粒转染第一阳性单克隆细胞，并加入第二抗生素进行筛选，得到第二阳性单克隆细胞，即为慢病毒稳定包装细胞系。
由于本发明的pPuro.coTetR质粒和pVSVG质粒分别含有嘌呤霉素抗性基因和博来霉 素抗性基因，因此在筛选第一阳性克隆细胞的过程中通过加入嘌呤霉素和博来霉素来进行抗性筛选；同时pGagPol-RRE-NES-cINT质粒含有潮霉素抗性基因，因此在筛选第二阳性克隆细胞的过程中通过加入嘌呤霉素、博来霉素和潮霉素来进行抗性筛选。另外，在两次转染过程中需要加入聚乙烯亚胺(PEI)以提高转染效率，并且控制PEI与各个质粒的用量可获得本发明的慢病毒稳定包装细胞系。
基于相同的发明构思，本发明的另一个方面提供了一种慢病毒的生产方法，其中，将目的质粒(含有目的基因的表达质粒)转染上述的慢病毒稳定包装细胞系。
在本发明的优选的实施方案中，其中，包括：
将目的质粒转染慢病毒稳定包装细胞系，然后加入诱导剂诱导慢病毒稳定包装细胞系产生慢病毒。
本发明的慢病毒稳定包装细胞系是诱导系统，在产生慢病毒时，需要加入强力霉素(dox)进行诱导产毒，同时发现在产毒过程中加入丁酸钠可提高慢病毒的产量。本发明还对目的质粒转染慢病毒稳定包装细胞系和诱导产毒的条件进行了优化，最优的条件为：目的质粒量为0.4μg/10 6个细胞，PEI量：目的质粒量＝4:1，丁酸钠的浓度为10mM，加入丁酸钠的时间为转染后24h，丁酸钠换液时间为加入丁酸钠后6-8小时，加入dox时间为24h，加入dox的浓度为2μg/mL，病毒收获时间为48h。现有技术中四质粒瞬时转染产生慢病毒的方法中无需添加强力霉素(dox)和丁酸钠。
在本发明中，对目的质粒并不作限制，可以根据需要转染多种目的质粒。优选的，目的质粒为CAR表达质粒。
CAR(Chimeric Antigen Receptor)-T全称即嵌合抗原受体T细胞免疫疗法，是一种过继免疫细胞疗法；将能够识别肿瘤特异性抗原的scFv片段通过基因工程改造，融合在由CD28、4-1BB等一系列参与T细胞激活的分子组成的跨膜链段上，然后将该基因片段通过慢病毒或逆转录病毒基因转导方式转染患者外周血中提取的T细胞；经基因修饰的T细胞回输患者体内后，利用表达的CAR受体结合肿瘤细胞表面的分子，从而产生内部信号激活T细胞快速摧毁肿瘤细胞。CAR-T细胞整合了抗体的识别特异性和T细胞的杀伤效应及记忆性功能，识别肿瘤抗原无需MHC限制，较天然T细胞可识别更广泛的目标。基于实际需要，可将CAR表达质粒转染上述慢病毒稳定包装细胞系，用于治疗复发/难治CD19阳性的非霍奇金淋巴瘤。CAR的设计及载体构建可参考中国发明专利申请201510324558.X的公开文本，特别是其实施例1和2，设计包含顺序连接的信号肽、胞外结合区、任选的铰链区、跨膜区和胞内信号区的嵌合抗原受体(CAR)，并进行载体(例如IM19质粒)构建。
本发明对CAR表达质粒转染上述慢病毒稳定包装细胞系和现有技术四质粒瞬时转染(pPuro.coTetR质粒、pVSVG质粒、pGagPol-RRE-NES-cINT质粒和CAR表达质粒同时转染293细胞)进行比较，包括细胞活率、病毒滴度VP(P24)、宿主蛋白残留和批次间病毒滴度稳定性等，实验结果表明，本发明构建的慢病毒稳定包装细胞系的病毒滴度VP含量高和批次间产毒稳定，有利于下游纯化工艺。
另外，在放大工艺下比较了CAR表达质粒转染上述慢病毒稳定包装细胞系和现有技术四质粒瞬时转染，包括细胞活率、病毒滴度VP和HCP(宿主蛋白)残留。实验结果表明慢病毒稳定包装细胞系在转染后细胞活率高，生产的病毒HCP(宿主蛋白)残留低，病毒滴度VP高，可进行放大生产病毒。
病毒滴度VP(P24)中的VP是指测定病毒滴度的一种方法——病毒颗粒数(VP)法——是利用OD260检测病毒颗粒数，P24是指病毒外壳蛋白-P24蛋白，通过对P24蛋 白进行测定，从而检测病毒滴度。
从上面所述可以看出，本发明提供的慢病毒稳定包装细胞系及其制备方法，该慢病毒稳定包装细胞系在传代、产毒和遗传基因拷贝数方面都比较稳定，而且可进行放大生产病毒。该慢病毒稳定包装细胞系和293细胞四质粒瞬时转染对比，其细胞活率高、病毒滴度VP含量高、宿主蛋白少和批次间产毒稳定，有利于下游纯化工艺。
下面结合具体的实施例对本发明提供的技术方案做进一步的描述。下述实施例仅用于对本发明进行说明，并不会对本发明的保护范围进行限制。
实施例1pPuro.coTetR质粒、pVSVG质粒和pGagPol-RRE-NES-cINT质粒的构建
本实施例构建的慢病毒稳定包装细胞系，由三个质粒构成，pPuro.coTetR(包含CoTetR基因)，pVSVG(包含VSVG基因)，pGagPol-RRE-NES-cINT(包含GagPol和Rev基因)，它们的质粒图谱分别参见图1a，图1b和图1c。
pPuro-coTetR载体构建：
通过引物TetR-WF-1F/TetR-1R-linker和模板pIRESpuro3(TaKaRa，货号：631619)获得INT片段(230bp)，通过引物TetR-2F-linker/TetR-2R-linker和模板coTetR(其序列如SEQ ID NO：1所示)获得coTetR片段(624bp)，通过引物TetR-3F-linker/TetR-WF-3R和模板pcDNA4(Thermo，货号：V102020)获得PA+F1ori+SV40promoter片段(1042bp)。再通过搭桥PCR，引物TetR-WF-1F/TetR-2R-linker获得片段TNT+coTetR，引物TetR-WF-1F/TetR-WF-3R获得全长片段TNT+coTetR+PA+F1ori+SV40promoter。
将上述制备的全长片段通过无缝克隆的方法插入pIRESpuro3载体的HindⅢ位点，转化感受态stbl3(全式金，货号：CD521)，经测序正确后，使用康为无内毒素中提试剂盒提取质粒(康为世纪，货号：cw2105s)，将构建的目的质粒用于后续的包装细胞系构建实验。
引物名称 引物序列
TetR-WF-1F 5’-ACTCACTATAGGGAGACCCA GTGAGTACTCCCTCTCAAAA-3’
TetR-1R-linker 5’-TTGGACTTGTCCAGCCTAGACATCTGTGGAGAGAAAGGCAAAGTGGAT-3’
TetR-2F-linker 5’-ATCCACTTTGCCTTTCTCTCCACAGATGTCTAGGCTGGACAAGTCCAA-3’
TetR-2R-linker 5’-ATGGCTGGCAACTAGAAGGCACAGTCAGCTGCCGCTTTCGCACTTGA-3’
TetR-3F-linker 5’-TCAAGTGCGAAAGCGGCAGCTGACTGTGCCTTCTAGTTGCCAGCCAT-3’
TetR-WF-3R 5’-CTTGTGGGTTGTGGCAAGCT TTTGCAAAAGCCTAGGCCTC-3’
pVSVG载体构建：
通过引物VSVG-WF-F/VSVG-WF-R和模板VS-SC(其序列SEQ ID NO：8所示)获得VSVG片段(1536bp)，将上述制备的片段通过无缝克隆的方法插入pcDNA4载体的XhoⅠ位点，转化感受态stbl3，经测序正确后，使用康为无内毒素中提试剂盒提取质粒，将构建的目的质粒用于后续的包装细胞系构建实验。
引物名称 引物序列
VSVG-WF-F 5’-ATATCCAGCACAGTGGCGGCCGCATGAAGTGCCTTTTGTACTT-3’
VSVG-WF-R 5’-AACGGGCCCTCTAGACTCGATTACTTTCCAAGTCGGTTCA-3’
pGagPol-RRE-NES-cINT载体构建：
通过引物pGagPol-XhoⅠ-WF/pGagPol-XhoⅠ-WR和模板MDK-SC(其序列如SEQ ID NO：9所示)获得GagPol片段(4307bp)，将上述制备的片段通过无缝克隆的方法插入pcDNA5载体(Thermo，货号：V103320)的XhoⅠ位点，转化感受态stbl3，经测序正确后，使用康为无内毒素中提试剂盒提取质粒，将构建的目的质粒pGagPol用于后续的载体构建。
通过引物pGagPol-REV-Xba Ⅰ-WF/pGagPol-REV-Xba Ⅰ-WR和模板AX(也称为psPAX2载体，其序列如SEQ ID NO：10所示)获得RRE-NES片段(1705bp)，将上述制备的片段通过无缝克隆的方法插入pGagPol载体的XbaⅠ位点，转化感受态stbl3，经测序正确后，使用康为无内毒素中提试剂盒提取质粒，将构建的目的质粒pGagPol-RRE-NES用于后续的载体构建。
通过引物pGagPol-RN-cINT--Not Ⅰ-QF/pGagPol-INT-Not Ⅰ-WR和模板AX获得cINT片段(1122bp)，将上述制备的片段通过无缝克隆的方法插入pGagPol-RRE-NES载体的NotⅠ位点，转化感受态stbl3，经测序正确后，使用康为无内毒素中提试剂盒提取质粒，将构建的目的质粒pGagPol-RRE-NES-cINT用于后续的载体构建。
引物名称 引物序列
pGagPol-XhoⅠ-WF 5’-TCCAGCACAGTGGCGGCCGCATGGGTGCGAGAGCGTCAGT-3’
pGagPol-XhoⅠ-WR 5’-AACGGGCCCTCTAGACTCGATTAATCCTCATCCTGTCTAC-3’
pGagPol-REV-XbaⅠ-WF 5’-AGGATGAGGATTAATCGAGTCACATGGAATTCTGCAACAA-3’
pGagPol-REV-XbaⅠ-WR 5’-GGTTTAAACGGGCCCTCTAGAGCCAGAAGTCAGATGCTCA-3’
pGagPol-RN-cINT--NotⅠ-QF 5’-CAGATATCCAGCACAGTGGCGGAGTCGCTGCGCGCTGCCT-3’
pGagPol-INT-NotⅠ-WR 5’-GCTCTCGCACCCATGCGGCCCTCTCACCAGTCGCCGCC-3’
实施例2慢病毒稳定包装细胞系的制备
将实施例1构建的三个质粒都经过Fsp I(Thermo，货号：FD1224)线性化酶切，然后去磷酸化后，用于转染293细胞。
线性化的pPuro.coTetR和pVSVG质粒，用PEI(聚乙烯亚胺)(Polysciences，货号：23966)共转染293悬浮细胞，转染时pPuro.coTetR和pVSVG质粒用量均为0.8μg/10 6个细胞，PEI量：(pPuro.coTetR质粒量+pVSVG质粒量)＝4:1，经过用2μg/mL Puro(嘌呤霉素)(InVivogen，货号：ant-pr-1)，400μg/mL Zeo(博来霉素)(Invitrogen，货号：R25001)抗生素进行加压筛选2周，得到抗药的阳性稳转细胞池，即多克隆PV细胞。将多克隆PV细胞进行96孔板(Thermo，货号：167008)有限稀释法铺细胞，每孔200μL培养基。然后显微镜观察，挑出只有单个细胞的孔，并标记，培养箱培养约20-30天。
将96孔板中存活下来的单克隆细胞进行扩大培养，分别用48孔板、24孔板、12孔板、6孔板、摇瓶E125。取PV单克隆细胞进行提取RNA，经过反转录成cDNA后，进行半定量PCR鉴定是否含有目的基因CoTetR和VSVG，用β-actin作为内参基因。
将鉴定正确的PV单克隆细胞，用PEI(聚乙烯亚胺)转染线性化的质粒pGagPol-RRE-NES-cINT，转染时pGagPol-RRE-NES-cINT质粒的用量为0.8μg/10 6个第一阳性单克隆细胞，PEI量：pGagPol-RRE-NES-cINT质粒量＝4:1，经过用2μg/mL Puro(嘌呤霉素)，400μg/mL Zeo(博来霉素)，400μg/mL HYG(潮霉素)(Invitrogen，货号：10687010)抗生素进行加压筛选2周，得到抗药的阳性稳转细胞池，即多克隆PVGR细胞。将多克隆PVGR细胞进行96孔板有限稀释法铺细胞，每孔200μL培养基。然后显微镜观察，挑出只有单个细胞的孔，并标记，培养箱培养约20-30天。在96孔板中存活下来的单 克隆细胞编号为Clone1、5、6、7、14、17、18、19、23、24、26、27、28、31、32和33。
实施例3稳定包装细胞系半定量PCR鉴定
将96孔板中存活下来的单克隆细胞进行扩大培养，分别用48孔板、24孔板、12孔板、6孔板、摇瓶E125。取PVGR单克隆细胞进行提取RNA，经过反转录成cDNA后，进行半定量PCR鉴定是否含有目的基因CoTetR、VSVG和GagPol。用β-actin作为内参基因。具体步骤如下：
前一天将PVGR单克隆细胞铺于12孔板中，用1mL SMM293 TI(Sino Biological，货号：M293TI)+0.1％F68(Gibco，货号：24040-032)培养基，每孔铺3×10 5个/mL，第二天加2μg/mL dox(强力霉素)(Sigma，货号：D9891)，第三天收获细胞，用于提取RNA。用TRIzol TM试剂(Invitrogen TM，货号：15596026)提取RNA，提取方法按照说明书操作。
将提取的RNA各用2μg进行反转录成cDNA，用第一链cDNA合成试剂盒(Thermo Scientific TM,Cat:K1612)操作方法见说明书。
将反转录的cDNA进行PCR扩增目的基因CoTetR、VSVG和GagPol，各用2μg。用β-actin作为内参基因，然后进行琼脂糖凝胶电泳。PCR扩增目的基因体系：2×phanta Max master mix(南京诺唯赞，货号：P515-02)加10μL，正向和反向引物各加1μL，ddH 2O加7μL，cDNA模板1μL。PCR反应程序：95℃10min，95℃30s，56℃30s，72℃30s，72℃10min，30个循环。半定量PCR引物如下：
CoTetR正向引物：
5'-atccactttgcctttctctccacagatgtctaggctggacaagtccaa-3'
CoTetR反向引物：
5'-atggctggcaactagaaggcacagtcagctgccgctttcgcacttga-3'
VSVG正向引物：5'-ccgctcgagatgaagtgccttttgtactt-3'
VSVG反向引物：5'-tgcattttccgttgatgaac-3'
GagPol正向引物：5'-ccggccataaagcaagagtt-3'
GagPol反向引物：5'-ccgcagatttctatgagtat-3'
β-actin正向引物：5′-CTCCATCCTGGCCTCGCTGT-3′
β-actin反向引物：5′-GCTGTCACCTTCACCGTTCC-3’
实验结果见图2所示。根据图2可知，其中Clone1、18、19、23、28、31、32和33都有目的片段CoTetR、VSVG和GagPol。
实施例4目的质粒(CAR表达质粒)的构建
4.1CAR分子的核苷酸序列的制备
本实施例的CAR分子包含信号肽、胞外结合区、任选的铰链区、跨膜区和胞内信号区。所述胞外结合区的核苷酸序列是在抗CD19的嵌合抗原受体的抗原结合区(在此命名为anti-CD19scFv-S0，简称为scFv-S0，其来源于小鼠，参见J Immunother.2009September；32(7):689–702.)的核苷酸序列的基础上进行人源化改造而获得的。具体说明CAR分子的核苷酸序列的制备步骤。
首先进行引物设计，本实施例中使用的引物序列如下：
1-1：5’-ATGCTTCTCCTGGTGACAAG-3’
1-2：5’-TGGGATCAGGAGGAATGCTG-3’
2-1：5’-TACATCTGGGCGCCCTTGGCCGG-3’
2-2：5’-GGAGCGATAGGCTGCGAAGTCGCG-3’
3-1：5’-AGAGTGAAGTTCAGCAGGAGCG-3’
3-2：5’-TTAGCGAGGGGGCAGGGCCT-3’
4-1：5’-ATGCTTCTCCTGGTGACAAGCC-3’
4-2：5’-TGAGGAGACGGTGACTGAGGTTCCTTGG-3’
5-1：5’-GCGGCCGCAATTGAAGTTATGTA-3’
5-2：5’-TTAGCGAGGGGGCAGGGCCTGC-3’
6-1：5’-CTAGACTAGTATGCTTCTCCTGGTGACAAGCC-3’
6-2：5’-CGACGCGTTTAGCGAGGGGGCAGGGCCTGC-3’
以人的cDNA文库为模板，分别以1-1和1-2、2-1和2-2、3-1和3-2为引物，通过PCR克隆出相应的CAR分子部分，分别为GMCSF、CD28-TM+CD28-signal(这两部分是相连的)和CD3ζ-signal。再通过搭桥引物4-1和4-2获得GMCSF+scFv片段，通过搭桥引物5-1和5-2获得CD28-TM+CD28-signal+CD3ζ-signal片段，随后通过搭桥引物6-1和6-2获得完整的CAR分子的核苷酸序列，酶切位点为SpeI和MluI。
4.2包含CAR分子的核酸序列的病毒载体的构建
将上述制备的CAR分子的核苷酸序列经SpeI(Fermentas)和MluI(Fermentas)双酶切、经T4连接酶(Fermentas)连接插入慢病毒IM19载体的SpeI-MluI位点，转化到感受态E.coli(DH5α)，经测序正确后，使用Qrigene公司的质粒纯化试剂盒提取并纯化质粒，将构建的目的质粒(CAR表达质粒)用于后续的产毒实验。
实施例5稳定包装细胞系产毒验证
在本实施例中，将鉴定正确的PVGR单克隆进行产毒验证，即用实施例4构建的目的质粒瞬时转染实施例3的PVGR单克隆细胞进行产毒验证。具体步骤如下：
前一天将PVGR单克隆细胞铺于12孔板中，用1mL SMM293 TI+0.1％F68培养基，每孔铺8×10 5个/mL，第二天进行目的质粒转染。转染24h后，加终浓度为2μg/mL诱导剂dox(强力霉素)。转染48h后收集病毒，3000rpm，4℃，离心20min。将病毒感染Jurkat细胞，48h后检测流式，计算出病毒滴度(TU/mL)。实验结果表明，Clone1、18、19、23、28、31、32和33均能产毒，而且Clone31的病毒滴度最高为2.68E+06(即2.68×10 6)TU/mL(参见图3)。后续实验选择Clone31作为实验细胞。
实施例6目的质粒转染稳定包装细胞系和诱导产毒的条件优化实验
挑取产毒滴度比较高的单克隆Clone31进行进一步的研究。将Clone31分别接种于E125摇瓶中(Corning，Cat:431145)，各接种8×10 5个/mL，各10mL，用10mL SMM293 TI培养基。使用DoE设计(试验设计)，第一次使用筛选设计实验，因子包括：质粒量(目的质粒)、PEI(聚乙烯亚胺)量、丁酸钠的浓度、加入丁酸钠时间、丁酸钠换液时间、加入dox(强力霉素)时间、加入dox(强力霉素)的浓度和病毒收获时间，确定曲率和因子的影响。第二次使用中心复合设计，确定最优的实验方案。转染48h后收集病毒，3000rpm，4℃，离心20min，检测病毒滴度TU。
最终得出，最优的实验方案为：质粒量(目的质粒)为0.4μg/10 6个细胞，PEI(聚乙烯亚胺)量：质粒量(目的质粒)＝4:1，丁酸钠(Sigma，货号：B5887)的浓度为10mM，加入丁酸钠的时间为转染后24h，丁酸钠换液时间为加入丁酸钠后6-8小时，加入dox(强 力霉素)时间为24h，加入dox(强力霉素)的浓度为2μg/mL，病毒收获时间为48h。将收获的病毒用于感染Jurkat细胞，48h后检流式，计算出病毒滴度。Clone31在E125摇瓶中，最终优化的病毒滴度为4E+06(即4×10 6)TU/mL(见图4)。
实施例7稳定包装细胞系传代、产毒、拷贝数稳定性验证
慢病毒稳定包装细胞系Clone31进行进一步的稳定性研究，包括传代稳定性、产毒稳定性和拷贝数稳定性。
7.1传代稳定性
将Clone31单克隆细胞分别在加抗生素和不加抗生素时，进行E125摇瓶传代，每次各培养10mL，分别计细胞数和活率。具体为：将Clone31分别接种于E125摇瓶中(Corning，Cat:431145)，各接种8×10 5个/mL，各10mL，分别用10mL SMM293 TI培养基(不加抗生素)，10mL SMM293 TI+0.6μg/mL Puro+50μg/mL Zeo+50μg/mL Hyg。每隔一天传代一次，并计细胞数和活率。看其传代稳定性。
实验证明，在不加抗生素(无筛选压力)时，Clone31单克隆细胞与加抗生素时，细胞数差别不大，且Clone31单克隆细胞在不加抗生素(无筛选压力)时，其能稳定传代遗传，并且活率可达97％以上，说明其在无筛选压力时能稳定传代30代以上(见图5)。
7.2产毒稳定性
探索Clone31单克隆细胞在加和不加抗生素时的产毒稳定。在抗生素和不加抗生素下，每隔几代，将目的质粒(实施例4构建)分别转染Clone31，检测病毒滴度。具体为：前一天，将Clone31分别接种于E125摇瓶中(Corning，Cat:431145)，各接种1.4×10 6个/mL，各10mL，分别用10mL SMM293 TI培养基(不加抗生素)，10mL SMM293 TI+0.6μg/mL Puro(嘌呤霉素)+50μg/mL Zeo(博来霉素)+50μg/mL Hyg(潮霉素)。第二天，进行目的质粒(实施例4构建)转染。每隔几代进行转染验证产毒情况。
实验结果表明，在不加抗生素时，其具有产毒能力，说明不加抗生素传代的细胞能正常遗传(见图6)。
7.3拷贝数稳定性
Clone31单克隆细胞，每隔几代，在加和不加抗生素时进行拷贝数稳定验证。用细胞基因组提取试剂盒(天根生化科技有限公司，货号：DP304-03)，分别提取在加和不加抗生素下，Clone31单克隆细胞的基因组DNA，然后用Taqman-qPCR方法，扩增Clone31单克隆细胞基因组中的GagPol、Rev和VSVG的拷贝数。具体为：将Clone31分别接种于E125摇瓶中(Corning，Cat:431145)，各接种8×10 5个/mL，各10mL，分别用10mL SMM293 TI培养基(不加抗生素)，10mL SMM293 TI+0.6μg/mL Puro(嘌呤霉素)+50μg/mL Zeo(博来霉素)+50μg/mL Hyg(潮霉素)。每隔几代提取细胞基因组DNA。取2×10 6个/mL，用于提取细胞基因组DNA，用细胞基因组提取试剂盒(天根生化科技(北京)有限公司，货号：DP304-03)，具体操作见试剂盒说明书。将细胞基因组DNA稀释成大约5ng/μL，用于qPCR。Taqman-qPCR探针法操作步骤：用核酸稀释液分别将3个标准品质粒DNA稀释至1×10 6copies/μL，1×10 5copies/μL，1×10 4copies/μL，1×10 3copies/μL，1×10 2copies/μL。分别扩3个基因GagPol，Rev，VSVG，制作3个标准曲线，标准品拷贝数对数为横坐标，CT值为纵坐标。qPCR反应体系(Takara，货号：RR390A)为：2×Premix Ex Taq(Probe qPCR)加10μL，F引物(10μmol/L)加0.4μL，R引物(10μmol/L)加0.4μL，TaqMan Probe(5μmol/L)加0.4μL，ROX Reference DyeII(50×)加0.4μL，模板加2μL，ddH 2O加6.4μL；其中F引物和R引物如下所示：
(1)VSVG基因qPCR引物：
VSVG-q-F：5’-AAGAAACCTGGAGCAAAATCAGA-3’
VSVG-q-R：5’-CCTGGGTTTTTAGGAGCAAGATAG-3’
VSVG-Taqman-Probe：
5’-FAM-CGGGTCTTCCAATCTCTCCAGTGGATCT-TAMRA-3’
(2)GagPol基因qPCR引物：
GagPol-q-F:5’-GCAGTTCATGTAGCCAGTGGATAT-3’
GagPol-q-R:5’-TGGTGAAATTGCTGCCATTG-3’
GagPol-Taqman-Probe:
5’-FAM-CAGAGACAGGGCAAGAAACAGCATACTTCC-TAMRA-3’
(3)Rev基因qPCR引物：
Rev-q-F:5’-CCTTGGAATGCTAGTTGGAGTAATAA-3’
Rev-q-R:5’-TGTTAATTTCTCTGTCCCACTCCAT-3’
Rev-Taqman-Probe:
5’-FAM-TCTCTGGAACAGATTTGGAATCACACGACC-TAMRA-3’
qPCR反应程序为：95℃30s，95℃5s，55℃15s，72℃35s，40个循环。将得出的CT值代入标曲，计算出拷贝数，然后根据质量，计算出每个细胞拷贝数。
Clone31 VSVG基因拷贝数见图7a，Clone31 GagPol基因拷贝数见图7b；Clone31 Rev基因拷贝数见图7c。实验结果表明，Clone31单克隆细胞中的GagPol、Rev和VSVG基因的拷贝数相对稳定。
实施例8稳定包装细胞系和293细胞转染对比
将Clone31单克隆细胞和293细胞进行转染比较，做3批，每批各3个平行。Clone31单克隆细胞用转染和诱导优化后的条件，293细胞用瞬时转染条件(PEI量：质粒量＝4:1)，进行转染比较。具体为：将293细胞和Clone31分别接种于E125摇瓶中(Corning，Cat:431145)，各接种8×10 5个/mL，各10mL，用10mL SMM293 TI+0.1％F68培养基，各三个E125摇瓶，即三个平行。Clone31单克隆细胞用转染和诱导优化后的条件，实验方案为：质粒量为0.4μg/10 6个细胞，PEI(聚乙烯亚胺)量：质粒量＝4:1，丁酸钠的浓度为10mM，加入丁酸钠的时间为转染后24h，丁酸钠换液时间为加入丁酸钠后6-8小时，加入dox(强力霉素)时间为24h，加入dox(强力霉素)的浓度为2μg/mL，病毒收获时间为48h。
293细胞用瞬时转染条件：将实施例1构建的pPuro.coTetR质粒、pVSVG质粒和pGagPol-RRE-NES-cINT质粒以及实施例4构建的目的质粒同时转染293细胞，瞬时转染的质粒量在一定范围内对病毒的滴度影响不大，例如总质粒量为0.8μg～1.0μg/10 6个细胞均可，在本实施例中，按照pPuro.coTetR质粒、pVSVG质粒、pGagPol-RRE-NES-cINT质粒和目的质粒的质量比为1：1：1：2，总质粒量为0.8μg/10 6个细胞(即pPuro.coTetR质粒为0.16μg/10 6个细胞、pVSVG质粒为0.16μg/10 6个细胞、pGagPol-RRE-NES-cINT质粒为0.16μg/10 6个细胞和目的质粒为0.32μg/10 6个细胞)，PEI(聚乙烯亚胺)量：质粒量(pPuro.coTetR质粒+pVSVG质粒+pGagPol-RRE-NES-cINT质粒+目的质粒)＝4:1，进行转染比较。比较转染后批次间病毒滴度稳定性和病毒滴度VP(P24)(参见图8和9)。
由图8的实验结果可以看出，Clone31单克隆细胞的批次间病毒滴度稳定性好于293细胞；由图9的实验结果可以看出，Clone31单克隆细胞的病毒滴度VP(P24)远远高于293细胞，Clone31单克隆细胞的病毒滴度VP(P24)含量高。
实施例9稳定包装细胞系放大工艺
将Clone31单克隆细胞由E125摇瓶扩大到E1000(Corning，货号：431147)，然后将目的质粒(实施例4构建)转染Clone31单克隆细胞进行产毒验证，同时转染293细胞进行对照，Clone31单克隆细胞和293细胞的转染条件和实施例8相同。进行各项比较细胞活率、病毒滴度VP(Takara，货号：632200)和HCP(宿主蛋白)(Cygnus，货号：F650)残留(参见图10)。
图10a的实验结果表明Clone31单克隆细胞在转染24h和48h细胞活率均高于293细胞；图10b的实验结果表明Clone31单克隆细胞的HCP(宿主蛋白)残留是293细胞1/3，Clone31单克隆细胞的HCP(宿主蛋白)残留少；图10c的实验结果表明Clone31单克隆细胞的病毒滴度VP(P24)是293细胞的10-20倍，Clone31单克隆细胞的病毒滴度VP(P24)含量高。

Claims (15)

  1. 一种慢病毒稳定包装细胞系，其中，包含包装质粒组，所述包装质粒组由pPuro.coTetR质粒、pVSVG质粒和pGagPol-RRE-NES-cINT质粒组成；
    所述pPuro.coTetR质粒包含CoTetR基因，所述pVSVG质粒包含VSVG基因，所述pGagPol-RRE-NES-cINT质粒包含GagPol和Rev基因。
  2. 根据权利要求1所述的慢病毒稳定包装细胞系，其中，所述pPuro.coTetR质粒还包含CMV启动子、嵌合体内含子、SV40启动子、嘌呤霉素抗性基因和多腺苷酸化信号；
    所述pVSVG质粒还包含由CMV启动子和两个四环素操纵子组成的杂合启动子、SV40启动子、博来霉素抗性基因和多腺苷酸化信号；
    所述pGagPol-RRE-NES-cINT质粒还包含由CMV启动子和两个四环素操纵子组成的杂合启动子、嵌合体内含子、SV40启动子、潮霉素抗性基因、cPPT/CTS元件、RRE元件和多腺苷酸化信号。
  3. 根据权利要求1或2所述的慢病毒稳定包装细胞系，其中，所述CoTetR基因的序列如SEQ ID NO：1所示，所述VSVG基因的序列如SEQ ID NO：2所示，所述GagPol和Rev基因的序列分别如SEQ ID NO：3和4所示。
  4. 根据权利要求1-3之一所述的慢病毒稳定包装细胞系，其中，所述pPuro.coTetR质粒的序列如SEQ ID NO：5所示，所述pVSVG质粒的序列如SEQ ID NO：6所示，所述pGagPol-RRE-NES-cINT质粒的序列如SEQ ID NO：7所示。
  5. 根据权利要求1-4之一所述的慢病毒稳定包装细胞系，其中，所述细胞系选自HEK293、HEK293-T、HEK293-SF、TE671、HT1080或HeLa细胞系中的一种；
    优选地，所述细胞系为HEK293细胞系。
  6. 一种制备如权利要求1-5任一项所述的慢病毒稳定包装细胞系的方法，其中，包括以下步骤：
    1)将pPuro.coTetR质粒、pVSVG质粒和pGagPol-RRE-NES-cINT质粒分别线性化酶切；
    2)将线性化的pPuro.coTetR质粒和pVSVG质粒共转染细胞，并加入第一抗生素进行筛选，得到第一阳性单克隆细胞；
    3)用线性化的pGagPol-RRE-NES-cINT质粒转染第一阳性单克隆细胞，并加入第二抗生素进行筛选，得到第二阳性单克隆细胞，即为慢病毒稳定包装细胞系。
  7. 根据权利要求6所述的方法，其中，在步骤2)中，将线性化的pPuro.coTetR质粒和pVSVG质粒共转染细胞的过程中还加入聚乙烯亚胺，所述聚乙烯亚胺与质粒的质量比为：聚乙烯亚胺：(pPuro.coTetR质粒+pVSVG质粒)＝(1～8):1；
    优选地，所述聚乙烯亚胺与质粒的质量比为：聚乙烯亚胺：(pPuro.coTetR质粒+pVSVG质粒)＝(2～5):1；
    更优选地，所述聚乙烯亚胺与质粒的质量比为：聚乙烯亚胺：(pPuro.coTetR质粒+pVSVG质粒)＝4:1；
    和/或，在步骤3)中，将线性化的pGagPol-RRE-NES-cINT质粒转染第一阳性单克隆细胞的过程中还加入聚乙烯亚胺，所述聚乙烯亚胺与pGagPol-RRE-NES-cINT质粒的质量比为(1～8):1；
    优选地，所述聚乙烯亚胺与pGagPol-RRE-NES-cINT质粒的质量比为(2～5):1；
    更优选地，所述聚乙烯亚胺与pGagPol-RRE-NES-cINT质粒的质量比为4:1。
  8. 根据权利要求6或7所述的方法，其中，在步骤2)中，所述pPuro.coTetR质粒和pVSVG质粒的用量均为(0.2～1.0)μg/10 6个细胞；
    优选地，所述pPuro.coTetR质粒和pVSVG质粒的用量均为(0.4～0.9)μg/10 6个细胞；
    更优选地，所述pPuro.coTetR质粒和pVSVG质粒的用量均为0.8μg/10 6个细胞；
    和/或，在步骤3)中，所述pGagPol-RRE-NES-cINT质粒的用量为(0.2～1.0)μg/10 6个第一阳性单克隆细胞；
    优选地，所述pGagPol-RRE-NES-cINT质粒的用量为(0.4～0.9)μg/10 6个第一阳性单克隆细胞；
    更优选地，所述pGagPol-RRE-NES-cINT质粒的用量为0.8μg/10 6个第一阳性单克隆细胞。
  9. 根据权利要求6-8之一所述的方法，其中，在步骤2)中，所述第一抗生素为嘌呤霉素和博来霉素的组合物，其用量分别为1～5μg/mL和300～500μg/mL；更优选地，所述嘌呤霉素和博来霉素的用量分别为2μg/mL和400μg/mL；
    和/或，在步骤3)中，所述第二抗生素为嘌呤霉素、博来霉素和潮霉素的组合物，其用量分别为1～5μg/mL、300～500μg/mL和300～500μg/mL；更优选地，所述嘌呤霉素、博来霉素和潮霉素的用量分别为2μg/mL、400μg/mL和400μg/mL。
  10. 一种慢病毒的生产方法，其中，将目的质粒转染权利要求1-5之一所述的慢病毒稳定包装细胞系。
  11. 根据权利要求10所述的生产方法，其中，包括：
    将目的质粒转染慢病毒稳定包装细胞系，然后加入诱导剂诱导慢病毒稳定包装细胞系产生慢病毒。
  12. 根据权利要求10或11所述的方法，其中，将目的质粒转染慢病毒稳定包装细胞系的过程中还加入聚乙烯亚胺，所述聚乙烯亚胺与目的质粒的质量比为：聚乙烯亚胺：目的质粒＝(1～8):1；
    优选地，所述聚乙烯亚胺与目的质粒的质量比为：聚乙烯亚胺：目的质粒＝(2～5):1；
    更优选地，所述聚乙烯亚胺与目的质粒的质量比为：聚乙烯亚胺：目的质粒＝4:1。
  13. 根据权利要求10-12之一所述的方法，其中，所述目的质粒的用量为(0.2～0.8)μg/10 6个慢病毒稳定包装细胞系；
    优选地，所述目的质粒的用量均为(0.3～0.5)μg/10 6个慢病毒稳定包装细胞系；
    更优选地，所述目的质粒的用量为0.4μg/10 6个慢病毒稳定包装细胞系。
  14. 根据权利要求10-13之一所述的方法，其中，所述诱导剂为强力霉素；
    优选地，所述诱导剂的加入时间为转染后20～30h；更优选地，所述诱导剂的加入时间为转染后24h；
    优选地，所述诱导剂的加入浓度为1～5μg/mL，更优选地，所述诱导剂的加入浓度为2μg/mL。
  15. 根据权利要求10-14之一所述的方法，其中，在慢病毒产生过程中还加入增强剂提高慢病毒的表达量；
    所述增强剂为丁酸钠；
    优选地，所述增强剂的加入浓度为5～15mM；更优选地，所述增强剂的加入浓度为10mM；
    优选地，所述增强剂的加入时间为转染后20～30h；更优选地，所述增强剂的加入时间为转染后24h；
    优选地，所述增强剂的换液时间为加入增强剂后6～8h；
    和/或，所述慢病毒收集的时间为45～50h；优选地，所述慢病毒收集的时间为48h。
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