Stable lentiviral packaging cell lines and methods for producing same

Abstract

The present invention provides stable lentiviral packaging cell lines and methods for their production. The stable lentiviral packaging cell line contains a group of packaging plasmids consisting of pPuro.coTetR plasmid, pVSVG plasmid, and pGagPol-RRE-NES-cINT plasmid, where the pPuro.coTetR plasmid contains the CoTetR gene, the pVSVG plasmid contains the VSVG gene, and the pGagPol-RRE-NES-cINT plasmid contains the GagPol gene and the Rev gene. The stable lentiviral packaging cell line is relatively stable in terms of passage, virus production, and genetic copy number, and the lentivirus produced using the stable lentiviral packaging cell line has a high titer and low impurity content.

Description

[Technical field]

[0001] The present invention is in the field of immunology and molecular biology, in particular in the field of cell therapy. In particular, the present invention relates to a stable lentiviral packaging cell line and a method for producing the same. [Background technology]

[0002] Lentiviruses, which are gene therapy vectors, include human immunodeficiency virus (HIV), equine infectious anemia virus (EIAV), simian immunodeficiency virus (SIV), feline immunodeficiency virus (FIV), etc. Unlike other retroviral vectors, lentivirus vectors can infect both dividing and non-dividing cells. Lentiviruses can infect various types of cells, such as cardiomyocytes, tumor cells, neuronal cells, stem cells, hepatocytes, and endothelial cells. Lentiviruses provide good gene therapy efficacy because they can accommodate larger foreign fragments and achieve stable expression of target genes while eliciting a lower level of immune response. Thus, lentiviruses have a wider applicability.

[0003] Currently, lentiviruses are mostly produced by transiently transfecting packaging and expression plasmids into cells.However, the lentiviruses produced by existing production processes have low titers, excessive impurity residues, and unstable batch-to-batch virus yields.Therefore, there remains a need to construct a stable lentivirus packaging cell line that allows the production of lentiviruses with low impurity content and high titers. Summary of the Invention [Problem to be solved by the invention]

[0004] Therefore, the object of the present invention is to provide a stable lentivirus packaging cell line and a method for preparing the same, which is relatively stable in terms of passage, virus production, and copy number of genetic genes, and the lentivirus produced by using the stable lentivirus packaging cell line has a high titer and a low content of impurities. [Means for solving the problem]

[0005] To achieve the above objectives, a stable lentiviral packaging cell line according to one embodiment of the present invention comprises a packaging plasmid set consisting of pPuro.coTetR plasmid, pVSVG plasmid and pGagPol-RRE-NES-cINT plasmid.

[0006] The pPuro.coTetR plasmid contains the CoTetR gene, the pVSVG plasmid contains the VSVG gene, and the pGagPol-RRE-NES-cINT plasmid contains the GagPol gene and the Rev gene.

[0007] In a preferred embodiment of the invention, said pPuro.coTetR plasmid further comprises a CMV promoter, a chimeric intron, an SV40 promoter, a puromycin resistance gene and a polyadenylation signal.

[0008] The pVSVG plasmid further contains a hybrid promoter consisting of a CMV promoter and two tetracycline operators, an SV40 promoter, a bleomycin resistance gene, and a polyadenylation signal.

[0009] The pGagPol-RRE-NES-cINT plasmid further contains a hybrid promoter consisting of a CMV promoter and two tetracycline operators, a chimeric intron, an SV40 promoter, a hygromycin resistance gene, a cPPT / CTS element, an RRE element, and a polyadenylation signal.

[0010] In a preferred embodiment of the invention, the CoTetR gene has the sequence shown in SEQ ID NO:1, the VSVG gene has the sequence shown in SEQ ID NO:2, and the GagPol gene and Rev gene have the sequences shown in SEQ ID NO:3 and SEQ ID NO:4, respectively.

[0011] In a preferred embodiment of the invention, the pPuro.coTetR plasmid has the sequence shown in SEQ ID NO:5, the pVSVG plasmid has the sequence shown in SEQ ID NO:6 and the pGagPol-RRE-NES-cINT plasmid has the sequence shown in SEQ ID NO:7.

[0012] In a preferred embodiment of the invention, the cell line is one selected from the group consisting of HEK293 cell line, HEK293-T cell line, HEK293-SF cell line, TE671 cell line, HT1080 cell line and HeLa cell line.

[0013] Preferably, said cell line is the HEK293 cell line.

[0014] Based on the same inventive concept, a method for producing a stable lentiviral packaging cell line according to another aspect of the present invention comprises: (1) linearizing the pPuro.coTetR plasmid, the pVSVG plasmid, and the pGagPol-RRE-NES-cINT plasmid by enzymatic digestion, respectively; (2) co-transfecting the linearized pPuro.coTetR plasmid and the pVSVG plasmid into cells, and adding a first antibiotic for screening to obtain a first positive monoclonal cell; (3) transfecting the linearized pGagPol-RRE-NES-cINT plasmid into the first positive monoclonal cell and adding a second antibiotic for screening to obtain a second positive monoclonal cell, i.e., a stable lentiviral packaging cell line.

[0015] In a preferred embodiment of the present invention, in step (2), polyethyleneimine is further added during co-transfection of the linearized pPuro.coTetR plasmid and the pVSVG plasmid into cells. The mass ratio of polyethyleneimine to the two plasmids is polyethyleneimine:(pPuro.coTetR plasmid+pVSVG plasmid)=(1-8):1; Preferably, the mass ratio of polyethyleneimine to the two plasmids is polyethyleneimine:(pPuro.coTetR plasmid+pVSVG plasmid)=(2 to 5):1; More preferably, the mass ratio of polyethylenimine to the two plasmids is polyethylenimine:(pPuro.coTetR plasmid+pVSVG plasmid)=4:1; and / or In the step (3), during transfection of the linearized pGagPol-RRE-NES-cINT plasmid into the first positive monoclonal cell, polyethyleneimine is further added; The mass ratio of polyethyleneimine to the pGagPol-RRE-NES-cINT plasmid is (1 to 8): 1; Preferably, the mass ratio of polyethyleneimine to the pGagPol-RRE-NES-cINT plasmid is (2 to 5):1; More preferably, the mass ratio of polyethylenimine to the pGagPol-RRE-NES-cINT plasmid is 4:1.

[0016] In a preferred embodiment of the present invention, in the step (2), the amount of each of the pPuro.coTetR plasmid and the pVSVG plasmid is (0.2 to 1.0) μg / 10 cells. 6 and Preferably, the amount of the pPuro.coTetR plasmid and the pVSVG plasmid used is (0.4 to 0.9) μg / 10 cells. 6 and More preferably, the amount of the pPuro.coTetR plasmid and the pVSVG plasmid used is 0.8 μg / 10 cells. 6 and / or In the step (3), the amount of the pGagPol-RRE-NES-cINT plasmid used is (0.2 to 1.0) μg / 10 of the first positive monoclonal cells. 6 and Preferably, the amount of the pGagPol-RRE-NES-cINT plasmid used is (0.4 to 0.9) μg / 10 of the first positive monoclonal cells. 6 and More preferably, the amount of the pGagPol-RRE-NES-cINT plasmid used is 0.8 μg / 10 6 The first positive monoclonal cells.

[0017] In a preferred embodiment of the present invention, in the step (2), the first antibiotic is a combination of puromycin and bleomycin, and the amounts used are 1 to 5 μg / mL and 300 to 500 μg / mL, respectively, more preferably 2 μg / mL and 400 μg / mL, respectively; and / or In step (3), the second antibiotic is a combination of puromycin, bleomycin and hygromycin, and the amounts used are 1 to 5 μg / mL, 300 to 500 μg / mL and 300 to 500 μg / mL, respectively, and more preferably 2 μg / mL, 400 μg / mL and 400 μg / mL, respectively.

[0018] Based on the same inventive concept, a method for producing a lentivirus according to another aspect of the present invention comprises transfecting a target plasmid into said lentivirus packaging cell line.

[0019] In a preferred embodiment of the present invention, the method for producing a lentivirus comprises the steps of: transfecting the stable lentiviral packaging cell line with the target plasmid; and adding an inducer to induce the stable lentiviral packaging cell line to produce lentivirus; Includes.

[0020] In a preferred embodiment of the invention, further comprising adding polyethyleneimine during transfection of said target plasmid into said stable lentiviral packaging cell line, The mass ratio of polyethyleneimine to the target plasmid is polyethyleneimine:target plasmid=(1 to 8):1; Preferably, the mass ratio of polyethyleneimine to the target plasmid is polyethyleneimine:target plasmid=(2 to 5):1; More preferably, the mass ratio of polyethyleneimine to the target plasmid is polyethyleneimine:target plasmid=4:1.

[0021] In a preferred embodiment of the present invention, the amount of the target plasmid used is (0.2 to 0.8) μg / 10 per stable lentiviral packaging cell line. 6 and Preferably, the amount of the target plasmid used is (0.3 to 0.5) μg / 10 per stable lentivirus packaging cell line. 6 and More preferably, the amount of the target plasmid used is 0.4 μg / 10 per stable lentiviral packaging cell line. 6 It is.

[0022] In a preferred embodiment of the invention, the inducer is doxycycline; Preferably, the inducer is added 20 to 30 hours after transfection, more preferably, the inducer is added 24 hours after transfection, Preferably, the inducer is added at a concentration of 1 to 5 μg / mL, and more preferably, the inducer is added at a concentration of 2 μg / mL.

[0023] In a preferred embodiment of the present invention, an enhancer is further added during the production of the lentivirus to increase the lentivirus expression level, the enhancer is sodium butyrate; Preferably, the enhancer is added at a concentration of 5 to 15 mM, more preferably, the enhancer is added at a concentration of 10 mM; Preferably, the enhancer is added 20 to 30 hours after transfection, more preferably, the enhancer is added 24 hours after transfection, Preferably, the enhancer is replaced 6 to 8 hours after addition of the enhancer, and / or The lentivirus is harvested after 45 to 50 hours, preferably after 48 hours. [Brief description of the drawings]

[0024] [Figure 1] FIG. 1 shows the plasmid maps of the packaging cell lines, where FIG. 1a is the plasmid map of the pPuro.coTetR plasmid, FIG. 1b is the plasmid map of the pVSVG plasmid, and FIG. 1c is the plasmid map of the pGagPol-RRE-NES-cINT plasmid. [Diagram 2] FIG. 2 shows agarose gel electrophoresis charts of the CoTetR gene, the VSVG gene, and the GagPol gene of monoclonal cell clones 1, 5, 6, 7, 14, 17, 18, 19, 23, 24, 26, 27, 28, 31, 32, and 33 identified by semi-quantitative PCR. [Diagram 3] FIG. 3 is a histogram showing validation of virus production by lentiviral packaging cell lines transfected with target plasmids. [Figure 4] FIG. 4 is a histogram showing the experimental results of optimizing the transfection and induction of viral production of clone 31 cells. [Diagram 5] FIG. 5 is a histogram showing the results of a passage stability assay of clone 31 cells. [Figure 6] FIG. 6 is a histogram showing the results of a virus production stability assay of clone 31 cells. [Figure 7] FIG. 7 is a histogram showing the results of a copy number stability assay of clone 31 cells, FIG. 7a is a histogram showing the results of a VSVG gene copy number stability assay of clone 31 cells, FIG. 7b is a histogram showing the results of a GagPol gene copy number stability assay of clone 31 cells, and FIG. 7c is a histogram showing the results of a Rev gene copy number stability assay of clone 31 cells. [Figure 8] FIG. 8 is a comparative chart of viral titers between batches of clone 31 cells and 293 cells. [Figure 9] FIG. 9 is a comparison chart of viral titer VP(P24) in clone 31 cells and 293 cells. [Figure 10] FIG. 10 is a scale-up comparison chart between Clone 31 cells and 293 cells, FIG. 10a is a comparison chart of viability 24 and 48 hours after transfection in E1000, FIG. 10b is a comparison chart of HCP residues after transfection in E1000, and FIG. 10c is a comparison chart of VP after transfection in E1000. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0025] Unless otherwise defined, technical or scientific terms used in one or more instances herein have the common meaning as understood by a person skilled in the art to which this disclosure belongs.

[0026] The experimental methods used in the following examples are conventional methods unless otherwise specified. The culture media, reagent materials and kits used in the following examples are all commercially available products unless otherwise specified.

[0027] The concentrations of antibiotics (puromycin, bleomycin, and hygromycin), inducers (doxycycline), and enhancers (sodium butyrate) used herein all refer to their final concentrations in the culture medium. For example, a concentration of sodium butyrate to be added of 5-15 mM means that the final concentration of sodium butyrate in the culture medium is 5-15 mM.

[0028] Lentiviral vector system is developed with lentivirus as the backbone, and includes packaging structure and vector structure. Lentiviral packaging system generally consists of two parts: lentiviral packaging plasmid and lentiviral expression plasmid. The lentiviral packaging plasmid consists of a base sequence that codes for viral proteins. The lentiviral expression plasmid contains long terminal repeats, packaging signals, and other genetic information required for packaging, transfection, and stable integration. The packaging plasmid and the expression plasmid are co-transfected into cells. The virus is packaged in cells and secreted into the extracellular medium to produce viral particles that can be used to infect host cells.

[0029] In most cases, existing lentiviruses are produced by transient transfection of 293 or 293T cells grown in cell factories with a packaging plasmid containing the long terminal repeats, packaging signal and other genetic information required for packaging, transfection and stable integration, and an expression plasmid containing the target gene.

[0030] Large-scale lentivirus production by transient transfection of 293 cells is feasible and shows good prospects. However, transient transfection needs to be further optimized in the production process. The main obstacle is that the transfection process requires a large amount of plasmid DNA, which leads to high costs, difficulty in scaling up, and the inability to sustain virus production. The virus produced by transient transfection, which contains excess residual impurities (especially host cell proteins and DNA residues), will cause further difficulties in the subsequent purification process and also lead to uncertainty in the downstream process arrangement by making the virus yield unstable between batches. Therefore, it is necessary to construct a stable lentivirus packaging cell line that can stably integrate the vector elements required for virus packaging into the genome of the host cell, is genetically stable and convenient for mass production, and therefore can ensure sustainable virus production.

[0031] The objective of the present invention is to obtain lentivirus with low impurity content and high titer by constructing a stable lentivirus packaging cell line that allows for large-scale production of suspension cells, particularly 293 cells.

[0032] To achieve the above objectives, a stable lentiviral packaging cell line according to one embodiment of the present invention comprises a packaging plasmid set consisting of pPuro.coTetR plasmid, pVSVG plasmid and pGagPol-RRE-NES-cINT plasmid.

[0033] The pPuro.coTetR plasmid contains the CoTetR gene, the pVSVG plasmid contains the VSVG gene, and the pGagPol-RRE-NES-cINT plasmid contains the GagPol gene and the Rev gene.

[0034] As known in the prior art, the method of producing lentivirus includes co-transfecting packaging plasmid and expression plasmid into cells (e.g., 293 cells or 293T cells). The main drawback of this method is that it produces lentivirus with low titer, high impurity content, and unstable batch-to-batch virus yield. The method of the present invention includes first transfecting packaging plasmid into cell line to obtain stable lentivirus packaging cell line, i.e. transfecting pPuro.coTetR plasmid, pVSVG plasmid and pGagPol-RRE-NES-cINT plasmid into cell line to obtain stable lentivirus packaging cell line, and then transfecting target plasmid into said stable lentivirus packaging cell line to produce lentivirus with low impurity content and high titer.

[0035] The stable lentivirus packaging cell line constructed according to the present invention was studied for stability, including passage stability, virus production stability and copy number stability. Experimental results showed that the stable lentivirus packaging cell line constructed herein could be stably inherited from generation to generation in the absence of selection pressure (without the addition of antibiotics) with a survival rate of 97% or more, suggesting that the constructed cells could be stably inherited for more than 30 generations in the absence of selection pressure. The cells inherited without antibiotics can be normally inherited, have the ability to produce lentivirus, and have stable copy numbers of GagPol gene, Rev gene and VSVG gene. In other words, the stable lentivirus packaging cell line constructed herein can maintain a stable state in terms of passage, virus production, and copy number of inherited genes.

[0036] The pPuro.coTetR, pVSVG and pGagPol-RRE-NES-cINT plasmids of the present invention are all constructed by inserting a target gene using a commercially available plasmid backbone as a base. For example, the pPuro.coTetR plasmid is constructed by inserting the CoTetR gene using the pIRESpuro3 plasmid as a base. The CoTetR gene is a codon-optimized TetR (tetracycline repressor) gene and has the sequence shown in SEQ ID NO:1. The pVSVG plasmid is constructed by inserting the VSVG (vesicular stomatitis virus glycoprotein) gene using the pcDNA4 plasmid as a base. The VSVG gene has the sequence shown in SEQ ID NO:2. The pGagPol-RRE-NES-cINT plasmid is constructed by using the pcDNA5 plasmid as a basis and inserting the GagPol gene (wherein the Gag gene encodes the viral capsid protein, and the Pol gene encodes the viral reverse transcriptase and gene integrase) and the Rev gene (encoding a regulator of viral particle protein expression). The GagPol gene and the Rev gene have the sequences shown in SEQ ID NOs: 3 and 4, respectively.

[0037] As shown in FIG. 1a, the pPuro.coTetR plasmid contains a human cytomegalovirus (CMV) promoter, a chimeric intron (to increase protein expression), a Simian Virus 40 (SV40) promoter, and a puromycin resistance gene (Puro R) and a polyadenylation signal. The SV40 promoter is required for expression of the puromycin resistance gene. The pPuro.coTetR plasmid contains a puromycin resistance gene. After screening with puromycin, almost all surviving cells stably express the CoTetR gene, so there is no need to screen a large number of cells for functional clones. When using the pPuro.coTetR plasmid, the antibiotic exerts selection pressure over the entire expression cassette. Thus, a high dose of antibiotic selects for cells expressing a high level of the CoTetR gene. This selection pressure also ensures that the expression of the CoTetR gene remains stable. The pPuro.coTetR plasmid has the sequence shown in SEQ ID NO:5.

[0038] As shown in FIG. 1b, the pVSVG plasmid contains a hybrid promoter consisting of a human cytomegalovirus (CMV) promoter and two tetracycline operators (TetO2), a simian virus 40 (SV40) promoter, and a bleomycin resistance gene (Zeo R ) and a polyadenylation signal. The SV40 promoter is required for expression of the bleomycin resistance gene. The pVSVG plasmid has the sequence shown in SEQ ID NO:6.

[0039] As shown in FIG. 1c, the pGagPol-RRE-NES-cINT plasmid contains a hybrid promoter consisting of a human cytomegalovirus (CMV) promoter and two tetracycline operators (TetO2), a chimeric intron, a simian virus 40 (SV40) promoter, and a hygromycin resistance gene (Hygro R ), a cPPT / CTS element, an RRE element and a polyadenylation signal. The SV40 promoter is required for expression of the hygromycin resistance gene. The pGagPol-RRE-NES-cINT plasmid has the sequence shown in SEQ ID NO:7.

[0040] 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.

[0041] The pGagPol-RRE-NES-cINT plasmid contains cis-acting elements, i.e., a central polypurine tract (cPPT) and a central termination sequence (CTS). The cPPT / CTS base sequence can be the cPPT / CTS of HIV1, which can improve vector integration and transduction efficiency.

[0042] The hybrid promoter consisting of the human cytomegalovirus (CMV) promoter and two tetracycline operators (TetO2) means that two TetO2 operator sequences are inserted into the CMV promoter to confer the function of tetracycline to regulate the promoter. The TetO2 sequence consists of two copies of a 19-nucleotide sequence (5'-TCCCTATCAGTGATAGAGA-3').

[0043] Based on the same inventive concept, a method for producing a stable lentiviral packaging cell line according to another aspect of the present invention comprises: (1) linearizing the pPuro.coTetR plasmid, the pVSVG plasmid, and the pGagPol-RRE-NES-cINT plasmid by enzymatic digestion, respectively; (2) co-transfecting the linearized pPuro.coTetR plasmid and the pVSVG plasmid into cells, and adding a first antibiotic for screening to obtain a first positive monoclonal cell; (3) transfecting the linearized pGagPol-RRE-NES-cINT plasmid into the first positive monoclonal cell and adding a second antibiotic for screening to obtain a second positive monoclonal cell, i.e., a stable lentiviral packaging cell line.

[0044] The pPuro.coTetR plasmid and pVSVG plasmid of the present invention contain puromycin resistance gene and bleomycin resistance gene, respectively, so that puromycin and bleomycin are added during the screening process of the first positive clone cells to perform resistance screening. Meanwhile, the pGagPol-RRE-NES-cINT plasmid contains hygromycin resistance gene, so that puromycin, bleomycin, and hygromycin are added during the screening process of the second positive clone cells to perform resistance screening. In addition, polyethyleneimine (PEI) needs to be added between the two transfection processes to improve transfection efficiency, and the stable lentivirus packaging cell line of the present invention can be obtained by controlling the amount of PEI and each plasmid.

[0045] Based on the same inventive concept, a method for producing a lentivirus according to another embodiment of the present invention comprises transfecting a target plasmid (an expression plasmid containing a target gene) into the lentivirus packaging cell line.

[0046] In a preferred embodiment of the present invention, the method for producing a lentivirus comprises the steps of: transfecting the target plasmid into the stable lentiviral packaging cell line; and adding an inducer to induce the stable lentiviral packaging cell line to produce lentivirus; Includes.

[0047] The stable lentiviral packaging cell line of the present invention is an inducible system. During the production of the lentivirus, it is necessary to add doxycycline (dox) to induce virus production, and it has been found that adding sodium butyrate during the virus production process can increase the yield of lentivirus. The present invention also optimizes the conditions for transfecting the target plasmid into the stable lentiviral packaging cell line to induce virus production. The optimal conditions are when the amount of the target plasmid is 0.4 μg / 10 cells. 6 The ratio of the amount of PEI to the amount of target plasmid is 4:1, the concentration of sodium butyrate is 10 mM, the time to add sodium butyrate is 24 hours after transfection, the time to replace sodium butyrate is 6 to 8 hours after adding sodium butyrate, the time to add dox is 24 hours, the concentration of dox is 2 μg / mL, and the time to collect lentivirus is 48 hours. In the method of producing lentivirus by transient transfection with four plasmids in the prior art, it is not necessary to add doxycycline (dox) and sodium butyrate.

[0048] In the present invention, the target plasmid is not limited. Various target plasmids can be used for transfection as needed. Preferably, the target plasmid is a CAR expression plasmid.

[0049] Chimeric antigen receptor (CAR) T cell immunotherapy is an adoptive immune cell therapy, in which scFv fragments capable of recognizing tumor-specific antigens are genetically engineered and fused with transmembrane segments consisting of molecules involved in the activation of T cells, such as CD28, 4-1BB, and then the genetic segments are transfected into T cells extracted from the patient's peripheral blood by lentivirus or retrovirus gene transduction, and after the genetically modified T cells are reinfused into the patient, they use the expressed CAR receptor to bind to molecules on the surface of tumor cells, thereby generating an internal signal to activate the T cells and rapidly kill the tumor cells. The CAR-T cells integrate the recognition specificity of antibodies, as well as the killing effect and memory function of T cells. Therefore, they can recognize tumor antigens without being restricted by MHC, and can recognize a wider range of targets than natural T cells. Based on the actual needs, CAR expression plasmids can be transfected into the stable lentivirus packaging cell lines for the treatment of relapsed / refractory CD19-positive non-Hodgkin's lymphoma. For the design and vector construction of CAR, reference can be made to the publication of Chinese Patent Application No. 201510324558 (especially Examples 1 and 2), which designs a chimeric antigen receptor (CAR) including a signal peptide, an extracellular binding region, an optional hinge region, a transmembrane region and an intracellular signal region (sequentially linked), and constructs a vector (such as IM19 plasmid).

[0050] In the present invention, the transfection of CAR expression plasmid into said stable lentivirus packaging cell line is compared with the transient transfection of four plasmids known in the prior art (i.e., pPuro.coTetR plasmid, pVSVG plasmid, pGagPol-RRE-NES-cINT plasmid and CAR expression plasmid are co-transfected into 293 cells) in terms of cell viability, viral titer VP (P24), host cell protein (HCP) residue and batch-to-batch viral titer stability.The experimental results show that the stable lentivirus packaging cell line constructed herein has high viral titer VP content, stable batch-to-batch viral yield, which is favorable for downstream purification process.

[0051] In addition, the stable lentivirus packaging cell line transfected with CAR expression plasmid is compared with 293 cells transiently transfected with four plasmids known in the prior art in terms of cell viability, virus titer VP, and host cell protein (HCP) residue in a scale-up process.The experimental results show that the stable lentivirus packaging cell line after transfection has high cell viability, low host cell protein (HCP) residue in the produced virus, and high virus titer VP, so it can be used for large-scale virus production.

[0052] The VP in viral titer VP(P24) refers to the viral particle count (VP) method, which is a method for measuring viral titer, and uses OD260 to detect the number of viral particles. P24 refers to the viral capsid protein, i.e., the P24 protein. The viral titer is detected by measuring the P24 protein.

[0053] As is clear from the above disclosure, the present invention provides a stable lentivirus packaging cell line and a method for its construction.The stable lentivirus packaging cell line is relatively stable in terms of passage, virus production, and genetic gene copy number, and the stable lentivirus packaging cell line is suitable for use in large-scale virus production.Compared with 293 cells transiently transfected with four plasmids, the stable lentivirus packaging cell line has higher cell viability, higher virus titer VP content, lower host cell protein residue, and stable batch-to-batch virus yield, which is favorable for downstream purification process. EXAMPLES

[0054] The technical solutions provided by the present invention are further described below in conjunction with specific embodiments. The following examples are only used to illustrate the present invention and do not limit the protection scope of the present invention.

[0055] [Example 1] Construction of pPuro.coTetR plasmid, pVSVG plasmid, and pGagPol-RRE-NES-cINT plasmid

[0056] The stable lentiviral packaging cell line constructed in this example consists of three plasmids, pPuro.coTetR (containing the CoTetR gene), pVSVG (containing the VSVG gene), and pGagPol-RRE-NES-cINT (containing the GagPol and Rev genes), which have plasmid maps shown in Figure 1a, Figure 1b, and Figure 1c, respectively.

[0057] Construction of pPuro-coTetRn vector An INT fragment (230 bp) was obtained using TetR-WF-1F / TetR-1R-linker as a primer and pIRESpuro3 (Takara Bio Inc., catalog number: 631619) as a template, a TecoTetR fragment (624 bp) was obtained using TetR-2F-linker / TetR-2R-linker as a primer and coTetR (having the nucleotide sequence shown in SEQ ID NO: 1) as a template, and a PA+F1ori+SV40 promoter fragment (1042 bp) was obtained using TetR-3F-linker / TetR-WF-3R as a primer and pcDNA4 (Thermo, catalog number: V102020) as a template. Then, by bridge PCR, the fragment TNT+coTetR was obtained using the primers TetR-WF-1F / TetR-2R-linker, and the full-length fragment TNT+coTetR+PA+F1ori+SV40 promoter was obtained using the primers TetR-WF-1F / TetR-WF-3R.

[0058] The full-length fragment constructed above was inserted into the HindIII site of pIRESpuro3 vector by seamless cloning method and transformed into competent stbl3 (Full Gold, Catalog No.: CD521). After sequencing to confirm correct insertion, the plasmid was extracted with EndoFree Plasmid Midi Kit (CWBio, Catalog No.: cw2105s). The constructed target plasmid was used in the subsequent experiment to construct packaging cell lines.

[0059] [Table 1]

[0060] Construction of pVSVG vector A VSVG fragment (1536 bp) was obtained using VSVG-WF-F / VSVG-WF-R as primers and VS-SC (having the sequence shown in SEQ ID NO: 8) as a template. The fragment prepared above was inserted into the XhoI site of pcDNA4 vector by seamless cloning method and transformed into competent stbl3. After sequencing to confirm correct insertion, the plasmid was extracted using EndoFree Plasmid Midi Kit (available from CWBio). The constructed target plasmid was used in the subsequent experiment to construct a packaging cell line.

[0061] [Table 2]

[0062] Construction of pGagPol-RRE-NES-cINT vector A GagPol fragment (4307 bp) was obtained using pGagPol-XhoI-WF / pGagPol-XhoI-WR as primers and MDK-SC (having the sequence shown in SEQ ID NO: 9) as template. The fragment prepared above was inserted into the XhoI site of pcDNA5 vector (Thermo, Cat. No.: V103320) by seamless cloning method and transformed into competent stbl3. After sequencing to confirm correct insertion, the plasmid was extracted using EndoFree Plasmid Midi Kit (available from CWBio). The constructed target plasmid pGagPol was used for the subsequent vector construction.

[0063] Using pGagPol-REV-Xba I-WF / pGagPol-REV-Xba I-WR as primers and AX (also known as psPAX2 vector with sequence shown in SEQ ID NO: 10) as template, RRE-NES fragment (1705 bp) was obtained. The fragment prepared above was inserted into the XbaI site of pGagPol vector by seamless cloning method and transformed into competent stbl3. After sequencing to confirm correct insertion, the plasmid was extracted using EndoFree Plasmid Midi Kit (CWBio). The constructed target plasmid pGagPol-RRE-NES was used for the subsequent vector construction.

[0064] Using pGagPol-RN-cINT-Not I-QF / pGagPol-INT-Not I-WR as primers and AX as template, a cINT fragment (1122 bp) was obtained. The fragment prepared above was inserted into the Not I site of pGagPol-RRE-NES vector by seamless cloning method and transformed into competent stbl3. After sequencing to confirm correct insertion, the plasmid was extracted using EndoFree Plasmid Midi Kit (available from CWBio). The constructed target plasmid pGagPol-RRE-NES-cINT was used for subsequent vector construction.

[0065] [Table 3]

[0066] Example 2: Creation of stable lentivirus packaging cell lines

[0067] The three plasmids constructed in Example 1 were linearized by digestion with Fsp I (Thermo, catalog number: FD1224), then dephosphorylated, and transfected into 293 cells.

[0068] The linearized pPuro.coTetR plasmid and the linearized pVSVG plasmid were co-transfected into 293 suspension cells using PEI (polyethyleneimine) (Polysciences, catalog number: 23966). The amounts of pPuro.coTetR and pVSVG plasmids used in the transfection were both 0.8 μg / 10 cells. 6 The ratio of PEI amount to (pPuro.coTetR plasmid amount + pVSVG plasmid amount) was 4:1. Pressure selection was performed by using two antibiotics, 2 μg / mL Puro (puromycin) (InVivogen, Catalog No.: ant-pr-1) and 400 μg / mL Zeo (bleomycin) (Invitrogen, Catalog No.: R25001), for two weeks to obtain a stably transfected drug-resistant positive cell pool, i.e., polyclonal PV cells. The polyclonal PV cells were seeded on a 96-well plate (Thermo, Catalog No.: 167008) at a density of 200 μL per well by limiting dilution. The cells were then observed under a microscope. Wells with only single cells were selected, labeled, and cultured in an incubator for about 20 to 30 days.

[0069] The monoclonal cells that survived in the 96-well plate were amplified and cultured in 48-well plate, 24-well plate, 12-well plate, 6-well plate and shake flask E125, respectively. The PV monoclonal cells were harvested and RNA was extracted. After reverse transcription to cDNA, semi-quantitative PCR was performed to identify the presence of the target genes CoTetR and VSVG. β-actin was used as an internal reference gene.

[0070] The linearized plasmid pGagPol-RRE-NES-cINT was transfected into the PV monoclonal cells identified as correct using PEI (polyethyleneimine). The amount of pGagPol-RRE-NES-cINT plasmid used in the transfection was 0.8 μg / 10 of the first positive monoclonal cells. 6The ratio of PEI amount to pGagPol-RRE-NES-cINT plasmid amount was set to 4:1. Pressurized selection was performed by using three antibiotics, 2 μg / mL Puro (puromycin), 400 μg / mL Zeo (bleomycin), and 400 μg / mL HYG (hygromycin) (Invitrogen, Catalog No.: 10687010), for two weeks to obtain a stably transfected drug-resistant positive cell pool, i.e., polyclonal PV cells. The polyclonal PV cells were seeded on a 96-well plate (Thermo, Catalog No.: 167008) at a density of 200 μL per well by limiting dilution. The cells were then observed under a microscope. Wells with only single cells were selected, labeled, and cultured in an incubator for about 20 to 30 days. The monoclonal cells that survived in the 96-well plates were numbered as clone 1, clone 5, clone 6, clone 7, clone 14, clone 17, clone 18, clone 19, clone 23, clone 24, clone 26, clone 27, clone 28, clone 31, clone 32 and clone 33.

[0071] [Example 3] Identification of stable packaging cell lines by semi-quantitative PCR

[0072] The monoclonal cells that survived in the 96-well plate were amplified and cultured in 48-well plate, 24-well plate, 12-well plate, 6-well plate and shake flask E125, respectively. The PVGR monoclonal cells were harvested and RNA was extracted. After reverse transcription to cDNA, semi-quantitative PCR was performed to identify the presence of the target genes CoTetR, VSVG and GagPol. β-actin was used as an internal reference gene. The specific steps are as follows:

[0073] One day before the experiment, PVGR monoclonal cells were cultured at 3 × 10 per well. 5Cells / mL were seeded into 12-well plates and cultured in medium containing 1 mL of SMM293TI (Sino Biological, Catalog No. M293TI) and 0.1% F68 (Gibco, Catalog No. 24040-032). 2 μg / mL of dox (Sigma, Catalog No. D9891) was added on day 2 and cells were harvested on day 3 for RNA extraction. RNA was extracted using TRIzol™ Reagent (Invitrogen™, Catalog No. 15596026) as described in the manufacturer's instructions.

[0074] 2 μg of extracted RNA each was reverse transcribed into cDNA using a First Strand cDNA Synthesis Kit (Thermo Scientific™, Catalog No.: K1612) according to the manufacturer's instructions.

[0075] 2 μg of reverse transcribed cDNA was used to amplify the target genes CoTetR, VSVG and GagPol by PCR, respectively. Agarose gel electrophoresis was performed using β-actin as the internal reference gene. The system for amplifying the target genes by PCR included 10 μL of 2×phanta Max master mix (Nanjing Vazyme Co., Ltd., Cat. No.: P515-02), 1 μL of forward primer, 1 μL of reverse primer, 7 μL of ddH2O and 1 μL of cDNA template. The PCR reaction program was 95°C for 10 min and 30 cycles of 95°C for 30 s, 56°C for 30 s, 72°C for 30 s and 72°C for 10 min. The primers for semi-quantitative PCR were as follows: CoTetR forward primer: 5'-atccactttgcctttctctccacagatgtctaggctggacaagtccaa-3' CoTetR reverse primer: 5'-atggctggcaactagaaggcacagtcagctgccgctttcgcacttga-3' VSVG forward primer: 5'-ccgctcgagatgaagtgccttttgtactt-3' VSVG reverse primer: 5'-tgcattttccgttgatgaac-3' GagPol forward primer: 5'-ccggccataaagcaagagtt-3' GagPol reverse primer: 5'-ccgcagatttctatgagtat-3' β-actin forward primer: 5'-CTCCATCCTGGCCTCGCTGT-3' β-actin reverse primer: 5'-GCTGTCACCTTCACCGTTCC-3'

[0076] The experimental results are shown in Figure 2. As shown in Figure 2, clone 1, clone 18, clone 19, clone 23, clone 28, clone 31, clone 32 and clone 33 all contain the target fragments CoTetR, VSVG and GagPol.

[0077] [Example 4] Construction of target plasmid (CAR expression plasmid)

[0078] 4.1 Generation of the nucleotide sequence of the CAR molecule

[0079] The CAR molecule of this example includes a signal peptide, an extracellular binding region, an optional hinge region, a transmembrane region, and an intracellular signal region. Based on the nucleotide sequence of the antigen-binding region of an anti-CD19 chimeric antigen receptor (herein referred to as anti-CD19scFv-S0; see J Immunother. 2009 September; 32(7): 689-702, mouse-derived scFv-S0), the nucleotide sequence of the extracellular binding region was obtained by humanization manipulation. The process of creating the nucleotide sequence of the CAR molecule will be specifically described.

[0080] First, primers are designed. The base sequences of the primers used in this example are listed below: 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'

[0081] Using human cDNA library as template, 1-1 and 1-2, 2-1 and 2-2, 3-1 and 3-2 as primers, respectively, GMCSF, CD28-TM+CD28-signal (two parts linked) and CD3-signal were cloned by PCR. Bridge primers 4-1 and 4-2 were used to obtain GMCSF+scFv fragment, bridge primers 5-1 and 5-2 were used to obtain CD28-TM+CD28-signal+CD3-signal fragment, and bridge primers 6-1 and 6-2 were used to obtain the nucleotide sequence of the complete CAR molecule. The digestion sites were SpeI and MluI.

[0082] 4.2 Construction of viral vectors containing the nucleic acid sequence of the CAR molecule

[0083] The nucleotide sequence of the CAR molecule constructed above was double digested with SpeI (Fermentas) and MluI (Fermentas), ligated with T4 ligase (Fermentas), inserted into the SpeI-MluI site of the lentiviral IM19 vector, and transformed into competent E. coli (DH5α). After sequencing to confirm correct insertion, the plasmid was extracted and purified with a plasmid purification kit (OriGene Technologies). The constructed target plasmid (CAR expression plasmid) was used for subsequent virus production experiments.

[0084] [Example 5] Verification of virus production by stable packaging cell lines

[0085] In this example, the PVGR monoclonal cells identified as correct were verified for virus production. That is, to verify virus production, the target plasmid constructed in Example 4 was transiently transfected into the PVGR monoclonal cells of Example 3. The specific steps are as follows:

[0086] The day before the experiment, PVGR monoclonal cells were plated in 12-well plates at 8 x 10 cells per well. 5 The cells were seeded at a density of 1000 cells / mL and cultured in a medium containing 1 mL of SMM293 TI and 0.1% F68. The target plasmid was used for transfection on the second day. 24 hours after transfection, the inducer dox (doxycycline) was added at a final concentration of 2 μg / mL. 48 hours after transfection, the virus was harvested and centrifuged at 3000 rpm for 20 min at 4 °C. Jurkat cells were infected with the virus and analyzed by flow cytometry after 48 hours to calculate the virus titer (TU / mL). The experiment results showed that clone 1, clone 18, clone 19, clone 23, clone 28, clone 31, clone 32, and clone 33 all had the ability to produce virus, and clone 31 produced the highest virus titer of 2.68E+06 (i.e., 2.68 × 10 6) TU / mL (see FIG. 3). Clone 31 was selected as the test cell in subsequent experiments.

[0087] Example 6: Experiments to optimize conditions for transfection of stable packaging cell lines with target plasmids and induction of virus production

[0088] Monoclonal clone 31 cells with relatively high virus titers were selected for further study. 10 mL of clone 31 cells were placed in shake flasks E125 (Corning, Cat. No. 431145) at 8 × 10 5 The cells were seeded at a density of 1000 cells / mL and cultured in medium containing 10 mL of SMM293 TI. Design of Experiments (DOE) was used. First, a screening design experiment was used. Factors included the amount of plasmid (target plasmid), the amount of PEI (polyethyleneimine), the concentration of sodium butyrate, the time of adding sodium butyrate, the time to replace sodium butyrate, the time of adding dox (doxycycline), the concentration of dox (doxycycline) added, and the time to harvest lentivirus. The curvature and the effect of the factors were determined. Then, a central composite design was used to determine the optimal experimental scheme. The virus was harvested 48 hours after transfection and centrifuged at 3000 rpm for 20 minutes at 4 °C to determine the viral titer TU.

[0089] Finally, the optimal experimental scheme is one in which the amount of plasmid (target plasmid) is 0.4 μg / 10 cells. 6It can be concluded that the ratio of the amount of PEI (polyethyleneimine) to the amount of plasmid (target plasmid) is 4:1, the concentration of sodium butyrate (Sigma, Catalog No.: B5887) is 10 mM, the time to add sodium butyrate is 24 hours after transfection, the time to replace sodium butyrate is 6-8 hours after adding sodium butyrate, the time to add dox (doxycycline) is 24 hours, the concentration of added dox (doxycycline) is 2 μg / mL, and the time to harvest the virus is 48 hours. Jurkat cells were infected with the harvested virus. After 48 hours, the cells were analyzed by flow cytometry to calculate the virus titer. The final optimized virus titer of clone 31 in shake flask E125 was 4E+06 (i.e., 4 × 10 6 ) TU / mL (see Figure 4).

[0090] [Example 7] Verification of passage stability, virus production stability, and copy number stability of stable packaging cell lines

[0091] The stable lentiviral packaging cell line clone 31 was further used to perform stability studies, including passage stability, virus production stability and copy number stability.

[0092] 7.1 Passage stability

[0093] Clone 31 monoclonal cells were passaged in E125 shake flasks with and without antibiotics, each time in a volume of 10 mL. The cell number and viability were calculated, respectively. In particular, Clone 31 cells were passaged in E125 shake flasks (Corning, Cat. No. 431145) at 8 × 10 5 Cells / mL and volume were seeded at 10 mL. 10 mL of SMM293 TI medium (without antibiotics) and 10 mL of SMM293 TI + 0.6 μg / mL Puro + 50 μg / mL Zeo + 50 μg / mL Hyg were used, respectively. Cells were passaged once every 2 days. Cell count and viability were calculated, respectively. Passage stability was observed.

[0094] Experiments showed that the cell number of clone 31 monoclonal cells without the addition of antibiotics (in the absence of selective pressure) was not significantly different from that with the addition of antibiotics, and that clone 31 monoclonal cells were stably passaged without the addition of antibiotics (in the absence of selective pressure), had a viability of more than 97%, and could be stably passaged for more than 30 generations in the absence of selective pressure (see Figure 5).

[0095] 7.2 Viral yield stability

[0096] The virus yield stability of clone 31 monoclonal cells with and without antibiotics was examined. The target plasmid (constructed in Example 4) was transfected into clone 31 every few generations for both the cases of antibiotics and no antibiotics, and the virus titer was measured. In particular, on the day before the experiment, clone 31 cells were transfected at 1.4 × 10 6 Cells / mL and a volume of 10 mL were seeded into E125 shake flasks (Corning, Cat. No. 431145). 10 mL of SMM293 TI medium (without antibiotics) and 10 mL of SMM293 TI + 0.6 μg / mL Puro (puromycin) + 50 μg / mL Zeo (bleomycin) + 50 μg / mL Hyg (hygromycin) were used, respectively. On day 2, the target plasmid (constructed in Example 4) was transfected into the cells. Transfections were performed every few generations to verify virus production.

[0097] The experimental results showed that the cells had the ability to produce viruses when no antibiotics were added, and the passaged cells were able to inherit normally without the addition of antibiotics (see Figure 6).

[0098] 7.3 Copy number stability

[0099] The stability of copy number was verified every several generations for clone 31 monoclonal cells with and without antibiotics. Genomic DNA was extracted from clone 31 monoclonal cells with and without antibiotics using a cell genome extraction kit (TIANGEN BIOTECH (BEIJING), catalog number: DP304-03), and then the copy numbers of GagPol, Rev, and VSVG in the genome of clone 31 monoclonal cells were amplified by Taqman-qPCR. In particular, clone 31 cells were cultured in E125 shake flasks (Corning, catalog number 431145) at 8 × 10 5 The cells were inoculated at 10 cells / mL and a volume of 10 mL. 10 mL of SMM293 TI medium (without antibiotics) and 10 mL of SMM293 TI + 0.6 μg / mL Puro (puromycin) + 50 μg / mL Zeo (bleomycin) + 50 μg / mL Hyg (hygromycin) were used, respectively. Cell genomic DNA was extracted every few generations. 2 × 10 cells were used for genomic DNA extraction using a Cell Genome Extraction Kit (TIANGEN BIOTECH (BEIJING) Co., Ltd., Cat. No.: DP304-03). 6 Cells / mL were harvested. For detailed operation, please refer to the kit manufacturer's instruction manual. Cell genomic DNA was diluted to approximately 5ng / μL for qPCR. The Taqman-qPCR probe method includes the following operation steps: Three standard plasmid DNAs were each diluted at 1 × 10 6 Copies / µL, 1 x 10 5 Copies / µL, 1 x 10 4 Copies / µL, 1 x 10 3 Copies / µL, and 1 x 10 2Dilute with nucleic acid diluent to copies / μL. Three genes, GagPol, Rev and VSVG, were amplified, respectively. Three standard curves were plotted by taking the logarithm of the copy number of the standard product as the abscissa and the C value as the ordinate. The qPCR reaction system (Takara Bio Inc., Catalog No.: RR390A) included 10 μL of 2× Premix Ex Taq (probe qPCR), 0.4 μL of F primer (10 μmol / L), 0.4 μL of R primer (10 μmol / L), 0.4 μL of TaqMan probe (5 μmol / L), 0.4 μL of ROX Reference Dye II (50×), 2 μL of template, and 6.4 μL of ddH2O. The F primer and R primer are listed below. (1) qPCR primers for VSVG gene: VSVG-qF:5'-AAGAAACCTGGAGCAAATCAGA-3' VSVG-qR:5'-CCTGGGTTTTTAGGAGCAAGATAG-3' VSVG-Taqman probe: 5'-FAM-CGGGTCTTCCAATCTCTCCAGTGGATCT-TAMRA-3' (2) qPCR primers for GagPol gene: GagPol-qF:5'-GCAGTTCATGTAGCCAGTGGATAT-3' GagPol-qR:5'-TGGTGAAATTGCTGCCATTG-3' GagPol-Taqman probe: 5'-FAM-CAGAGACAGGGCAAGAAACAGCATACTTCC-TAMRA-3' (3) qPCR primers for Rev gene: Rev-qF:5'-CCTTGGAATGCTAGTTGGAGTAATAA-3' Rev-qR:5'-TGTTAATTTCTCTGTCCCACTCCAT-3' Rev-Taqman probe: 5'-FAM-TCTCTGGAACAGATTTGGAATCACACGACC-TAMRA-3'

[0100] The qPCR reaction procedure was 95° C. for 30 s and 40 cycles of 95° C. for 5 s, 55° C. for 15 s, and 72° C. for 35 s. The obtained C values ​​were inserted into a standard curve to calculate the copy number, and the copy number of each cell was calculated based on the mass.

[0101] The copy numbers of the VSVG gene for clone 31 are shown in Figure 7a, the copy numbers of the GagPol gene for clone 31 are shown in Figure 7b, and the copy numbers of the Rev gene for clone 31 are shown in Figure 7c. The experimental results showed that the copy numbers of the GagPol, Rev and VSVG genes in clone 31 monoclonal cells were relatively stable.

[0102] [Example 8] Comparison of stable packaging cell lines with transfected 293 cells

[0103] Clone 31 monoclonal cells and 293 cells were transfected and compared. Three batches were made, each containing three parallel groups. Clone 31 monoclonal cells were transfected and induced under optimal conditions, and 293 cells were transfected under transient transfection conditions (PEI amount:plasmid amount=4:1) to compare the transfection results. Specifically, 293 cells and clone 31 cells were transfected at 8 × 10 5 Cells / mL and a volume of 10 mL were seeded into E125 shake flasks (Corning, Cat. No: 431145). 10 mL of SMM293 TI + 0.1% F68 medium was used. Cells were seeded in three E125 shake flasks, i.e., three groups, in parallel. Transfection of clone 31 monoclonal cells was performed and induced under optimized conditions. The experimental scheme is as follows: the amount of plasmid was 0.4 μg / 10 cells; 6the ratio of PEI (polyethyleneimine) amount to plasmid amount was 4:1, the concentration of sodium butyrate was 10 mM, the time to add sodium butyrate was 24 hours after transfection, the time to replace sodium butyrate was 6 to 8 hours after adding sodium butyrate, the time to add dox (doxycycline) was 24 hours, the concentration of added dox (doxycycline) was 2 μg / mL, and the time to collect the virus was 48 hours.

[0104] The conditions for the transient transfection of 293 cells include co-transfecting the pPuro.coTetR plasmid, pVSVG plasmid, and pGagPol-RRE-NES-cINT plasmid constructed in Example 1 with the target plasmid constructed in Example 4 into 293 cells. For example, the total amount of plasmids is 0.8 μg to 1.0 μg / 10 6 The amount of plasmid within a certain range for transient transfection has little effect on the virus titer as long as the range of cells is within the range. In this example, the transfected cells were pPuro.coTetR plasmid, pVSVG plasmid, pGagPol-RRE-NES-cINT plasmid and target plasmid in a mass ratio of 1:1:1:2, and the total amount of plasmid was 0.8 μg / 10 cells. 6 (i.e., 0.16 μg / 10 pPuro.coTetR plasmid per cell) 6 , pVSVG plasmid was 0.16 μg / 10 cells 6 , pGagPol-RRE-NES-cINT plasmid was added at 0.16 μg / 10 cells. 6 , and 0.32 μg / 10 target plasmid 6 Cells) and a ratio of PEI (polyethyleneimine) amount to plasmid (Puro.coTetR plasmid + pVSVG plasmid + pGagPol-RRE-NES-cINT plasmid + target plasmid) amount of 4:1. Viral titer stability and viral titer VP(P24) were compared between batches after transfection (see Figures 8 and 9).

[0105] As can be seen from the experimental results in Figure 8, clone 31 monoclonal cells had better batch-to-batch viral titer stability than 293 cells, and as can be seen from the experimental results in Figure 9, clone 31 monoclonal cells had a much higher viral titer VP(P24) than 293 cells, and clone 31 monoclonal cells had a higher viral titer VP(P24) content.

[0106] [Example 9] Scale-up process of stable packaging cell lines

[0107] Clone 31 monoclonal cells were scaled up from E125 to E1000 shake flasks (Corning, Catalog No.: 431147), and then the target plasmid (constructed in Example 4) was transfected into Clone 31 monoclonal cells to verify virus production. Meanwhile, 293 cells were transfected as a control, and the conditions for transfecting Clone 31 monoclonal cells and 293 cells were the same as those described in Example 8. The transfected cells were compared in terms of their cell viability, virus titer VP (Takara Bio Inc., Catalog No.: 632200) and HCP (host cell protein) (Cygnus, Catalog No.: F650) residues (see Figure 10).

[0108] The experimental results in Figure 10a show that the clone 31 monoclonal cells have higher cell viability than the 293 cells 24 and 48 hours after transfection. The experimental results in Figure 10b show that the clone 31 monoclonal cells have one-third of the HCP (host cell protein) residues of the 293 cells, i.e., they have fewer HCP (host cell protein) residues than the 293 cells. The experimental results in Figure 10c show that the viral titer VP(P24) of the clone 31 monoclonal cells is 10-20 times higher than that of the 293 cells, i.e., the viral titer VP(P24) content of the clone 31 monoclonal cells is higher.

Claims

1. pPuro. Contains a packaging plasmid set consisting of 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, and the pGagPol-RRE-NES-cINT plasmid contains the GagPol gene and the Rev gene; A stable lentivirus packaging cell line, characterized in that the pPuro.coTetR plasmid has the sequence shown in SEQ ID NO:5, the pVSVG plasmid has the sequence shown in SEQ ID NO:6, and the pGagPol-RRE-NES-cINT plasmid has the sequence shown in SEQ ID NO:

7.

2. The pPuro.coTetR plasmid further comprises a CMV promoter, a chimeric intron, an SV40 promoter, a puromycin resistance gene, and a polyadenylation signal; The pVSVG plasmid further comprises a hybrid promoter consisting of a CMV promoter and two tetracycline operators, an SV40 promoter, a bleomycin resistance gene, and a polyadenylation signal; 2. The stable lentiviral packaging cell line of claim 1, wherein the pGagPol-RRE-NES-cINT plasmid further comprises a hybrid promoter consisting of a CMV promoter and two tetracycline operators, a chimeric intron, an SV40 promoter, a hygromycin resistance gene, a cPPT / CTS element, an RRE element, and a polyadenylation signal.

3. 3. The stable lentiviral packaging cell line of claim 1 or 2, wherein the CoTetR gene has the sequence shown in SEQ ID NO:1, the VSVG gene has the sequence shown in SEQ ID NO:2, and the GagPol gene and Rev gene have the sequences shown in SEQ ID NO:3 and SEQ ID NO:4, respectively.

4. 4. The stable lentiviral packaging cell line of any one of claims 1 to 3, wherein the cell line is one selected from the group consisting of HEK293 cell line, HEK293-T cell line, HEK293-SF cell line, TE671 cell line, HT1080 cell line and HeLa cell line.

5. (1) linearizing the pPuro.coTetR plasmid, the pVSVG plasmid, and the pGagPol-RRE-NES-cINT plasmid by enzymatic digestion, respectively; (2) co-transfecting the linearized pPuro.coTetR plasmid and the pVSVG plasmid into cells, and adding a first antibiotic for screening to obtain a first positive monoclonal cell; (3) transfecting the linearized pGagPol-RRE-NES-cINT plasmid into the first positive monoclonal cell and adding a second antibiotic for screening to obtain a second positive monoclonal cell, i.e., a stable lentiviral packaging cell line; The first antibiotic is a combination of puromycin and bleomycin, and the second antibiotic is a combination of puromycin, bleomycin and hygromycin. A method for producing a stable lentiviral packaging cell line according to any one of claims 1 to 4.

6. In the step (2), polyethyleneimine is further added during co-transfection of the linearized pPuro.coTetR plasmid and the pVSVG plasmid into cells; The mass ratio of polyethyleneimine to the two plasmids is polyethyleneimine:(pPuro.coTetR plasmid+pVSVG plasmid)=(1-8):1; and / or In the step (3), during transfection of the linearized pGagPol-RRE-NES-cINT plasmid into the first positive monoclonal cell, polyethyleneimine is further added; The method according to claim 5, wherein the mass ratio of polyethylenimine to the pGagPol-RRE-NES-cINT plasmid is (1-8):

1.

7. The method described in claim 6, wherein the mass ratio of polyethyleneimine to the two plasmids is polyethyleneimine: (pPuro.coTetR plasmid + pVSVG plasmid) = (2-5):

1.

8. The method described in claim 7, wherein the mass ratio of polyethyleneimine to the two plasmids is polyethyleneimine:(pPuro.coTetR plasmid + pVSVG plasmid) = 4:

1.

9. The method according to claim 6, wherein the mass ratio of polyethyleneimine to the pGagPol-RRE-NES-cINT plasmid is (2-5):

1.

10. The method of claim 9, wherein the mass ratio of polyethyleneimine to the pGagPol-RRE-NES-cINT plasmid is 4:

1.

11. In the step (2), the amount of the pPuro.coTetR plasmid and the pVSVG plasmid used was (0.2 to 1.0) μg / 10 ６ Individual cells, and / or In the step (3), the amount of the pGagPol-RRE-NES-cINT plasmid used is (0.2 to 1.0) μg / 10 ６ The method of claim 5, wherein the first positive monoclonal cell is a single positive monoclonal cell.

12. The method according to claim 11, wherein the amount of each of the pPuro.coTetR plasmid and the pVSVG plasmid is (0.4 to 0.9) μg / 10 6 cells.

13. The method according to claim 12, wherein the amount of each of the pPuro.coTetR plasmid and the pVSVG plasmid is 0.8 μg / 10 6 cells.

14. The method according to claim 11, wherein the amount of the pGagPol-RRE-NES-cINT plasmid used is (0.4-0.9) μg / 10 6 first positive monoclonal cells.

15. The method according to claim 14, wherein the amount of the pGagPol-RRE-NES-cINT plasmid used is 0.8 μg / 10 6 first positive monoclonal cells.

16. In the step (2), the first antibiotic is a combination of puromycin and bleomycin, the amounts used being 1-5 μg / mL and 300-500 μg / mL, respectively; and / or The method according to claim 5, wherein in the step (3), the second antibiotic is a combination of puromycin, bleomycin and hygromycin, and the amounts used are 1 to 5 μg / mL, 300 to 500 μg / mL and 300 to 500 μg / mL, respectively.

17. A method for producing a lentivirus, comprising transfecting a target plasmid into the stable lentivirus packaging cell line according to any one of claims 1 to 4.

18. transfecting the stable lentiviral packaging cell line with the target plasmid; and adding an inducer to induce the stable lentiviral packaging cell line to produce lentivirus; 20. The method of claim 17, comprising:

19. further adding polyethylenimine during transfection of the target plasmid into the stable lentiviral packaging cell line; The method according to claim 17 or 18, wherein the mass ratio of polyethyleneimine to the target plasmid is polyethyleneimine:target plasmid=(1-8):

1.

20. The method described in claim 19, wherein the mass ratio of polyethyleneimine to the target plasmid is polyethyleneimine:target plasmid = (2 to 5):

1.

21. The method described in claim 20, wherein the mass ratio of polyethyleneimine to the target plasmid is polyethyleneimine:target plasmid = 4:

1.

22. The amount of the target plasmid used is (0.2 to 0.8) μg / 10 ６ The method of claim 17 or 18, wherein the cell line is a stable lentiviral packaging cell line.

23. The method according to claim 22, wherein the amount of the target plasmid used is (0.3-0.5) μg / 10 6 stable lentiviral packaging cell lines.

24. The method of claim 23, wherein the amount of the target plasmid used is 0.4 μg / 10 6 stable lentiviral packaging cell lines.

25. the inducer is doxycycline; The method of claim 18, wherein the inducer is added 20 to 30 hours after transfection at a concentration of 1 to 5 μg / mL.

26. further adding an enhancer during production of the lentivirus to increase lentivirus expression levels; the enhancer is sodium butyrate; and / or The method of claim 17 or 18, wherein the lentivirus is harvested at 45 to 50 hours.

27. ​​The method described in claim 26, wherein the enhancer is added at a concentration of 5 to 15 mM 20 to 30 hours after transfection.

28. The method of claim 26, wherein the enhancer is replaced 6 to 8 hours after addition of the enhancer.

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