Cell line for lentivirus production and preparation method therefor

Abstract

A cell line for lentivirus production and a preparation method therefor. A lentivirus stable packaging cell line, comprising a packaging plasmid group, wherein the packaging plasmid group consists of a pREV-rtTAtTS-puro plasmid, a pVSVG-Hyg plasmid, a pGagPol-BSD plasmid and a CMV-PBtransposase plasmid. Also provided are a preparation method for the lentivirus stable packaging cell line and a lentivirus stable production cell line based on the lentivirus stable packaging cell line.

Description

A cell line for lentivirus production and preparation method thereof Technical Field

[0001] The present invention relates to the field of lentiviral vectors, more specifically to the field of lentiviral vector preparation, and in particular to a lentiviral stable production cell line and a preparation method thereof. Background Art

[0002] Lentiviruses primarily include human immunodeficiency virus (HIV), simian immunodeficiency virus (SIV), equine infectious anemia (EIA), and feline immunodeficiency virus (FIV). Unlike other retroviral vectors, lentiviral vectors can effectively infect a variety of cell types, including neurons, hepatocytes, cardiomyocytes, tumor cells, endothelial cells, and stem cells. They can also effectively integrate exogenous genes into host chromosomes, achieving persistent expression and thus achieving effective gene therapy. Therefore, they have broad application prospects.

[0003] Currently, lentiviral vectors are typically produced by transiently transfecting cells with a helper plasmid and a lentiviral vector plasmid. However, this production process produces lentiviruses with high residual impurities, low titers, inconsistent yields between batches, and high production costs. Therefore, there is an urgent need to provide efficient, stable, and large-scale lentiviral packaging and production cell lines, as well as methods for their preparation.

[0004] Summary of the Invention

[0005] The purpose of the present invention is to provide a packaging cell line and a production cell line that can be used for efficient, stable and large-scale production of lentiviruses and a preparation method thereof.

[0006] In a first aspect, the present invention provides a lentiviral stable packaging cell line, comprising a packaging plasmid group consisting of a pREV-rtTAtTS-puro plasmid, a pVSVG-Hyg plasmid, a pGagPol-BSD plasmid, and a CMV-PBtransposase plasmid;

[0007] The pREV-rtTAtTS-puro plasmid contains rtTA, tTS and REV genes, the pVSVG-Hyg plasmid contains the VSVG gene, the pGagPol-BSD plasmid contains the GagPol gene, and the CMV-PBtransposase plasmid contains the PBtransposase gene;

[0008] The sequence of the rtTA gene is shown in SEQ ID NO: 9, the sequence of the tTS gene is shown in SEQ ID NO: 8, and the sequence of the REV gene is shown in SEQ ID NO: 6;

[0009] The sequence of the VSVG gene is shown in SEQ ID NO: 11;

[0010] The sequence of the GagPol gene is shown in SEQ ID NO: 13; and

[0011] The sequence of the PBtransposase gene is shown in SEQ ID NO: 18.

[0012] In another preferred embodiment, the pREV-rtTAtTS-puro plasmid further comprises a cHS4 insulator gene, a CBh promoter, a CMV promoter, a Tight promoter, a puromycin resistance gene puroR and a polyadenylation signal;

[0013] The pVSVG-Hyg plasmid also contains the PiggyBac transposon 5'ITR sequence gene, 3'ITR sequence gene, cHS4 insulator core gene, Tight promoter, intron, hPGK promoter, hygromycin resistance gene HygR and polyadenylation signal;

[0014] The pGagPol-BSD plasmid further comprises a PiggyBac transposon 5'ITR sequence gene, a 3'ITR sequence gene, a cHS4 insulator core gene, a Tight promoter, an intron, an SV40 promoter, a blasticidin resistance gene BSDR, a cPPT / CTS element, an RRE element and a polyadenylation signal; and

[0015] The CMV-PB transposase plasmid also contains the cHS4 insulator core gene and a polyadenylation signal.

[0016] In another preferred example, the sequence of the cHS4 insulator gene is shown as SEQ ID NO: 5, the sequence of the codon-optimized puromycin resistance gene puroR is shown as SEQ ID NO: 7, the sequence of the cHS4 insulator core gene is shown as SEQ ID NO: 10, the sequence of the codon-optimized hygromycin resistance gene HygR is shown as SEQ ID NO: 12, the sequence of the blasticidin resistance gene BSDR is shown as SEQ ID NO: 14, the sequence of the PiggyBac transposon 5'ITR gene is shown as SEQ ID NO: 15, the sequence of the PiggyBac transposon 3'ITR gene is shown as SEQ ID NO: 16, and the sequence of the Tight promoter gene is shown as SEQ ID NO: 17.

[0017] In another preferred example, the sequence of the pREV-rtTAtTS-puro plasmid is shown as SEQ ID NO: 1, the sequence of the pVSVG-Hyg plasmid is shown as SEQ ID NO: 2, the sequence of the pGagPol-BSD plasmid is shown as SEQ ID NO: 3, and the sequence of the CMV-PBtransposase plasmid is shown as SEQ ID NO: 4.

[0018] In another preferred embodiment, the cell line is selected from one of HEK293, HEK293-T, HEK293-SF, TE671, HT1080 or HeLa cell lines.

[0019] In another preferred embodiment, the cell line is HEK293T cell line.

[0020] In another preferred embodiment, the cell line is a cell line with a deposit number of CGMCC No.: 45528.

[0021] In a second aspect, the present invention provides a method for preparing a lentiviral stable packaging cell line according to the first aspect of the present invention, comprising the following steps:

[0022] The recipient cells are subjected to steps (i) and (ii) in any order:

[0023] i) linearizing the pREV-rtTAtTS-puro plasmid, transfecting cells with the linearized pREV-rtTAtTS-puro plasmid, and adding a first antibiotic for screening to obtain a first positive mixed cell;

[0024] ii) co-transfecting the cells with the pVSVG-Hyg plasmid, the pGagPol-BSD plasmid, and the CMV-PB transposase plasmid; and adding a second antibiotic for selection to obtain a second positive mixed cell;

[0025] Wherein, when step i) is performed first, the first antibiotic is puromycin; the second antibiotic is a combination of puromycin, hygromycin and blasticidin; and

[0026] When step ii) is performed first, the second antibiotic is hygromycin and blasticidin; the first antibiotic is a combination of puromycin, hygromycin and blasticidin;

[0027] The cells whose screening results in step i) and step ii) are both positive are the lentiviral stable packaging cell lines.

[0028] In another preferred embodiment of step i) and step ii), the method further comprises the step of further sorting cells that are positive in both step i) and step ii) to obtain monoclonal cells as the lentivirus stable packaging cell line.

[0029] In another preferred embodiment, in step i) and / or step ii), the transfection is performed in the presence of a transfection reagent, preferably, the transfection reagent is polyethyleneimine.

[0030] In another preferred embodiment, in step i), the process of transfecting the linearized pREV-rtTAtTS-puro plasmid into cells is carried out in the presence of the transfection reagent polyethyleneimine, and the mass ratio of polyethyleneimine to plasmid is: polyethyleneimine: pREV-rtTAtTS-puro plasmid = (1 to 4): 1.

[0031] And / or, in step ii), the process of co-transfecting the pVSVG-Hyg plasmid, pGagPol-BSD plasmid and CMV-PBtransposase plasmid into the cells is carried out in the presence of the transfection reagent polyethyleneimine, and the mass ratio of the polyethyleneimine to the total amount of (pVSVG-Hyg plasmid + pGagPol-BSD plasmid + CMV-PBtransposase plasmid) is (1 to 4):1.

[0032] In another preferred embodiment, the mass ratio of the polyethyleneimine to the pREV-rtTAtTS-puro plasmid is (1-2):1, preferably 1.5:1.

[0033] In another preferred embodiment, the mass ratio of the polyethyleneimine to the total amount of (pVSVG-Hyg plasmid+pGagPol-BSD plasmid+CMV-PBtransposase plasmid) is (1-2):1, preferably 1.5:1.

[0034] In another preferred embodiment, in step i), the amount of the pREV-rtTAtTS-puro plasmid is (0.5-3) μg / 10 6 cells; preferably, (1-2) μg / 10 6 cells, more preferably, 1.5 μg / 10 6 cells.

[0035] In another preferred embodiment, in step ii), the total amount of the (pVSVG-Hyg plasmid + pGagPol-BSD plasmid + CMV-PBtransposase plasmid) is (0.5-4) μg / 10 6 cells, preferably (1-3) μg / 10 6 cells, more preferably, 2 μg / 10 6 cells.

[0036] In another preferred embodiment, in step ii), when the (pVSVG-Hyg plasmid + pGagPol-BSD plasmid + CMV-PB transposase plasmid) is co-transfected into the cells, the molar plasmid ratio of the transposon plasmid (pVSVG-Hyg plasmid + pGagPol-BSD plasmid) to the transposase plasmid (CMV-PB transposase plasmid) is transposon plasmid: transposase plasmid = (1 to 6):1, preferably (2.5 to 5):1, and more preferably 4:1.

[0037] In another preferred embodiment, in step ii), when the (pVSVG-Hyg plasmid + pGagPol-BSD plasmid + CMV-PBtransposase plasmid) are co-transfected into cells, the molar plasmid ratio of the pVSVG-Hyg plasmid to the pGagPol-BSD plasmid is pVSVG-Hyg plasmid: pGagPol-BSD plasmid = 1:(0.5-4), preferably 1:(0.5-3), more preferably 1:(1-2) or 1:1.

[0038] In another preferred embodiment, in step i) and / or step ii), the concentrations of puromycin, hygromycin and blasticidin in the first and second antibiotics are independently 0.5-3 μg / mL, 100-300 μg / mL and 10-20 μg / mL, respectively. Preferably, the concentrations of puromycin, hygromycin and blasticidin are independently 1-2 μg / mL, 100-200 μg / mL and 10-20 μg / mL, respectively.

[0039] In another preferred embodiment, step i) is performed first and then step ii), or step ii) is performed first and then step i).

[0040] In a third aspect, the present invention provides a lentivirus stable producer cell line, which is obtained by a method comprising the following steps: transfecting a plasmid containing a viral packaging target gene and a transposase plasmid into the lentivirus stable packaging cell line as described in the first aspect of the present invention, and screening cells in which the target gene is stably integrated, thereby constructing a lentivirus stable producer cell line.

[0041] In a fourth aspect, the present invention provides a method for constructing a lentiviral stable producer cell line, comprising the steps of transfecting a plasmid containing a viral packaging target gene and a transposase plasmid into the lentiviral stable packaging cell line as described in the first aspect of the present invention and screening cells in which the target gene is stably integrated, thereby constructing a lentiviral stable producer cell line.

[0042] In a fifth aspect, the present invention provides a method for producing a lentivirus, comprising the steps of transfecting a plasmid containing a viral packaging target gene and a transposase plasmid into the lentivirus stable packaging cell line as described in the first aspect of the present invention and screening cells in which the target gene is stably integrated, thereby constructing a lentivirus stable producer cell line, and then adding an inducer to induce the lentivirus stable producer cell line to produce lentivirus.

[0043] In another preferred embodiment, the inducer is doxycycline.

[0044] In another preferred embodiment, the inducer is added 20 to 30 hours after cell inoculation, preferably 24 hours after transfection.

[0045] In another preferred embodiment, the concentration of the inducer is 1-5 μg / mL, preferably 2 μg / mL.

[0046] In another preferred embodiment, an enhancer is added during the lentivirus production process to increase the expression level of the lentivirus;

[0047] The enhancer is sodium butyrate;

[0048] And / or, the lentivirus is collected 42 to 62 hours after transfection.

[0049] In another preferred embodiment, the concentration of the enhancer added is 1-4 mM, preferably 1.5 mM.

[0050] In another preferred embodiment, the enhancer is added 20 to 30 hours after cell inoculation, preferably 24 hours after transfection.

[0051] In another preferred embodiment, the lentivirus is collected 48 hours after induction.

[0052] In a sixth aspect, the present invention provides a lentivirus stable production cell line, wherein the cell line is produced by transfecting a plasmid containing a viral packaging target gene and a transposase plasmid into the lentivirus stable packaging cell line as described in the first aspect of the present invention and screening cells in which the target gene is stably integrated, wherein

[0053] The plasmid containing the viral packaging target gene includes a pTol2-pLv-GOI-Bleo plasmid, and the transposase plasmid is a CMV-Tol2 transposase plasmid; or

[0054] The plasmid containing the virus packaging target gene includes the pSB-Lv-GOI-TAT-Bleo plasmid, and the transposase plasmid is the CMV-SB100X plasmid.

[0055] In another preferred embodiment, the plasmid containing the viral packaging target gene includes a pTol2-Lv-GOI-Bleo plasmid carrying the target gene, and the pTol2-Lv-GOI-Bleo plasmid sequence is shown in SEQ ID NO: 80, and a CMV-Tol2 transposase plasmid sequence is shown in SEQ ID NO: 20.

[0056] In another preferred embodiment, the pTol2-Lv-GOI-Bleo plasmid further comprises a 5'IR sequence gene, a 3'IR sequence gene, a CMV promoter, a viral packaging transcription cassette, a WPRE gene and a polyadenylation signal;

[0057] The CMV-Tol2 transposase plasmid also contains a CMV promoter and a polyadenylation signal.

[0058] In another preferred embodiment, the pSB-lv-GOI-TAT-Bleo plasmid further comprises a 5'IR sequence gene, a 3'IR sequence gene, a CMV promoter, a viral packaging transcription cassette, a WPRE gene, a TAT gene and a polyadenylation signal;

[0059] The CMV-SB100X plasmid also contains a CMV promoter and a polyadenylation signal.

[0060] In another preferred embodiment, the plasmid containing the viral packaging target gene includes the pSB-Lv-GOI-TAT-Bleo plasmid carrying the target gene, and the pSB-Lv-GOI-TAT-Bleo plasmid sequence is shown in SEQ ID NO: 78, and the CMV-SB100X plasmid sequence is shown in SEQ ID NO: 77.

[0061] In another preferred embodiment, the sequence of the 5'IR gene is shown as SEQ ID NO: 21, and the sequence of the 3'IR gene is shown as SEQ ID NO: 22.

[0062] In another preferred embodiment, the sequence of the CMV-Tol2 transposase plasmid is shown in SEQ ID NO: 20.

[0063] In another preferred embodiment, the target gene is GFP, the promoter is CMV, and the pTol2-Lv-GOI-Bleo plasmid packages the CMV-GFP gene to form the plasmid pTol2-Lv-CMV-GFP-Bleo, whose sequence is shown in SEQ ID NO: 19.

[0064] In another preferred embodiment, the sequence of the 5'IR gene is shown as SEQ ID NO: 74, the sequence of the 3'IR gene is shown as SEQ ID NO: 75; and the sequence of the TAT gene is shown as SEQ ID NO: 76.

[0065] In another preferred embodiment, the sequence of the CMV-SB100X plasmid is shown as SEQ ID NO: 77.

[0066] In another preferred embodiment, the target gene is GFP, the promoter is CMV, and the pSB-lv-GOI-TAT-Bleo (SEQ ID NO: 78) plasmid packages the CMV-GFP gene to form the plasmid pSB-Lv-CMV-GFP-TAT-Bleo, whose sequence is shown in SEQ ID NO: 79. In a seventh aspect, the present invention provides a method for preparing the lentiviral stable producer cell line according to the sixth aspect of the present invention, comprising the following steps: 1) co-transfecting the stable packaging cell line according to the first aspect of the present invention with the pTol2-lv-GOI-Bleo plasmid (including or not including the viral packaging target gene) and the CMV-Tol2 transposase plasmid or the pSB-Lv-GOI-TAT-Bleo plasmid (including or not including the viral packaging target gene) and the CMV-SB100X plasmid, and adding a third antibiotic for screening and sorting to obtain cloned cells, namely the lentiviral stable producer cell line; wherein the third antibiotic is a combination of puromycin, hygromycin, blasticidin, and bleomycin.

[0067] In another preferred embodiment, in step 1), polyethyleneimine is further added during the co-transfection of the pTol2-lv-GOI-Bleo plasmid and the CMV-Tol2transposase plasmid into the lentiviral stable packaging cell line, and the mass ratio of the polyethyleneimine to (pTol2-lv-GOI-Bleo plasmid + CMV-Tol2transposase plasmid) is (1-4):1.

[0068] In another preferred embodiment, the mass ratio of polyethyleneimine to plasmid is: polyethyleneimine: (pTol2-lv-GOI-Bleo plasmid + CMV-Tol2transposase plasmid) = (1-2): 1, preferably, 1.5:1.

[0069] In another preferred embodiment, in step 1), the total amount of the (pTol2-lv-GOI-Bleo plasmid + CMV-Tol2 transposase plasmid) is (0.5-4) μg / 10 6 The optimal value is (1-3) μg / 10 6 cells, more preferably 2 μg / 10 6 cells.

[0070] In another preferred embodiment, in step 1), when the (pTol2-lv-GOI-Bleo plasmid + CMV-Tol2transposase plasmid) is co-transfected into cells, the molar plasmid ratio of the transposon plasmid (pTol2-lv-GOI-Bleo) to the transposase plasmid (CMV-Tol2transposase plasmid) is transposon plasmid: transposase plasmid = 1:(1-6), preferably 1:(1-4), and more preferably 1:1.

[0071] In another preferred embodiment, for the production cell line transfected with pSB-Lv-GOI-TAT-Bleo / CMV-SB100X, the preferred conditions include one or more characteristics selected from the following group:

[0072] In step 1), polyethylenimine is added during the co-transfection of the pSB-lv-GOI-TAT-Bleo plasmid and the CMV-SB100X plasmid into the lentiviral stable packaging cell line, and the mass ratio of the polyethylenimine to (pSB-lv-GOI-TAT-Bleo plasmid + CMV-SB100X plasmid) is (1-4):1;

[0073] Preferably, the mass ratio of polyethyleneimine to plasmid is: polyethyleneimine: (pSB-lv-GOI-TAT-Bleo plasmid + CMV-SB100X plasmid) = (1-2): 1, preferably, 1.5: 1;

[0074] In step 1), the total amount of the (pSB-lv-GOI-TAT-Bleo plasmid + CMV-SB100X plasmid) is (0.25-1) μg / 10 6 cells, preferably (0.25-0.5) μg / 10 6 cells, more preferably 0.5 μg / 10 6 cells; and / or

[0075] In step 1), when the (pSB-lv-GOI-TAT-Bleo plasmid + CMV-SB100X plasmid) are co-transfected into cells, the molar plasmid ratio of the transposon plasmid (pSB-lv-GOI-TAT-Bleo plasmid) to the transposase plasmid (CMV-SB100X plasmid) is transposon plasmid:transposase plasmid = (1-10):1, preferably (2.5-10):1, and more preferably (5-8):1.

[0076] In another preferred embodiment, in step 1), the dosage of puromycin, hygromycin, blasticidin and bleomycin in the third antibiotic is 1 μg / mL, 150 ug / ml, 10 ug / ml and 400-800 ug / ml, respectively; preferably, they are 1 μg / mL, 150 ug / ml, 10 ug / ml and 400-600 ug / ml, respectively, and more preferably, they are 1 μg / mL, 150 ug / ml, 10 ug / ml and 500 ug / ml, respectively.

[0077] In an eighth aspect, the present invention provides a method for producing a lentivirus, comprising: adding an inducer to the production cell described in the sixth aspect of the present invention to induce a lentivirus stable production cell line to produce lentivirus.

[0078] In another preferred embodiment, the inducer is doxycycline.

[0079] In another preferred embodiment, the inducer is added 20 to 30 hours after cell inoculation, preferably 24 hours after transfection.

[0080] In another preferred embodiment, the concentration of the inducer is 1-5 μg / mL, preferably 2 μg / mL.

[0081] In another preferred embodiment, an enhancer is added during the lentivirus production process to increase the expression level of the lentivirus;

[0082] The enhancer is sodium butyrate;

[0083] And / or, the lentivirus is collected 42 to 62 hours after transfection.

[0084] In another preferred embodiment, the concentration of the enhancer added is 1-4 mM, preferably 1.5 mM.

[0085] In another preferred embodiment, the enhancer is added 20 to 30 hours after cell inoculation, preferably 24 hours after transfection.

[0086] In another preferred embodiment, the lentivirus is collected 48 hours after induction.

[0087] In another preferred embodiment, for the production cell line transfected with pSB-Lv-GOI-TAT-Bleo / CMV-SB100X, the preferred conditions include one or more characteristics selected from the following group:

[0088] The inducer is added 36-60 hours after cell inoculation, preferably 48 hours after inoculation;

[0089] The concentration of the inducer is 0.2-2 μg / mL, preferably 1 μg / mL;

[0090] During the lentivirus production process, enhancers are also added to increase the expression level of the lentivirus;

[0091] The enhancer is sodium butyrate;

[0092] And / or, the lentivirus is collected 24-48 hours after induction, preferably 40 hours;

[0093] The concentration of the enhancer added is 1-4 mM, preferably 1.5 mM.

[0094] The enhancer is added 36-60 hours after cell inoculation, preferably 48 hours after inoculation.

[0095] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features described in detail below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be listed here one by one. BRIEF DESCRIPTION OF THE DRAWINGS

[0096] Figure 1 is a plasmid map of the packaging cell line construction; Figure 1a is a plasmid map of the pREV-rtTAtTS-puro plasmid, Figure 1b is a plasmid map of the pVSVG-Hyg plasmid, Figure 1c is a plasmid map of the pGagPol-BSD plasmid, and Figure 1d is a plasmid map of the CMV-PBtransposase plasmid;

[0097] Figure 2 shows the plasmid maps for the construction of the production cell line; Figure 2a shows the plasmid map of the pTol2-Lv-CMV-GFP-Bleo plasmid, Figure 2b shows the plasmid map of the CMV-Tol2transposase plasmid; Figure 2c shows the plasmid map of the pSB-Lv-CMV-GFP-TAT-Bleo plasmid, and Figure 2d shows the plasmid map of the CMV-100X plasmid.

[0098] FIG3 is a graph showing the effect of different transfection ratios of GAGPOL and VSVG plasmids on the toxin production titer of cell lines;

[0099] FIG4 is a graph showing the effect of the total amount of GAGPOL and VSVG plasmid transfection on the toxin titer of the cell line;

[0100] FIG5 is a graph showing the toxin production titer of different monoclonal cells of stable packaging cell lines;

[0101] Figure 6 is a graph showing the toxin production titers of different monoclonal cell lines in stable production cell lines;

[0102] FIG7 is a graph showing the effect of plasmid transfection on the toxin production titer of the Clone4 monoclonal cell line;

[0103] Figure 8 is a graph showing the stability of Clone4 monoclonal cell passages;

[0104] Figure 9 shows the results of the toxin production stability of Clone4 monoclonal cells;

[0105] Figure 10 is a graph showing the stability of gene copy number across generations in Clone4 monoclonal cells; Figure 10a is a graph showing the stability of REV gene copy number across generations in Clone4 monoclonal cells, Figure 10b is a graph showing the stability of VSVG gene copy number across generations in Clone4 monoclonal cells, and Figure 10c is a graph showing the stability of GAGPOL gene copy number across generations in Clone4 monoclonal cells. DETAILED DESCRIPTION

[0106] After extensive and in-depth research, and through extensive screening and testing, the present inventors have provided a packaging cell line and a production cell line based thereon that can be used for efficient, stable, and large-scale production of lentiviruses, as well as a method for preparing the same. The present inventors unexpectedly discovered that by placing the genetic elements for lentivirus packaging into a plasmid according to the specific method of the present invention and transfecting cells, the resulting packaging cell line and the production cell line based thereon have excellent toxin production capabilities, and can produce up to 10 7 The viral titer of TU / mL is very suitable for the production of lentiviral vectors. Based on this, the present invention was completed.

[0107] the term

[0108] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0109] As used herein, when used in reference to a specific recited value, the term "about" means that the value may vary by no more than 1% from the recited value. For example, as used herein, the expression "about 100" includes all values ​​between 99 and 101 (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0110] As used herein, the terms "comprising" or "including" may be open, semi-closed, or closed. In other words, the terms also include "consisting essentially of" or "consisting of."

[0111] As used herein, the term "room temperature" or "normal temperature" refers to a temperature of 4-40°C, preferably, 25±5°C.

[0112] Lentiviral stable packaging cell lines

[0113] A lentiviral stable packaging cell line, comprising a packaging plasmid group consisting of a pREV-rtTAtTS-puro plasmid, a pVSVG-Hyg plasmid, a pGagPol-BSD plasmid, and a CMV-PBtransposase plasmid;

[0114] The pREV-rtTAtTS-puro plasmid contains rtTA, tTS and REV genes, the pVSVG-Hyg plasmid contains the VSVG gene, the pGagPol-BSD plasmid contains the GagPol gene, and the CMV-PBtransposase plasmid contains the PBtransposase gene;

[0115] The sequence of the rtTA gene is shown in SEQ ID NO: 9, the sequence of the tTS gene is shown in SEQ ID NO: 8, and the sequence of the REV gene is shown in SEQ ID NO: 6;

[0116] The sequence of the VSVG gene is shown in SEQ ID NO: 11;

[0117] The sequence of the GagPol gene is shown in SEQ ID NO: 13; and

[0118] The sequence of the PBtransposase gene is shown in SEQ ID NO: 18.

[0119] The pREV-rtTAtTS-puro plasmid, pVSVG-Hyg plasmid, pGagPol-BSD and CMV-PBtransposase plasmids of the present invention can be constructed based on a commercial plasmid backbone and then inserting the target gene, or they can be obtained by total plasmid synthesis by combining the preferred specific gene of the present invention with commonly used plasmid elements in the art. For example, the specific genes in the pREV-rtTAtTS-puro plasmid include the REV gene (which encodes a viral particle protein expression regulatory factor), rtTA and tTS genes (tetracycline-inducible system stable opening and closing genes), the sequence of the REV gene is shown in SEQ ID NO: 6, the sequence of the rtTA gene is shown in SEQ ID NO: 9, and the sequence of the tTS gene is shown in SEQ ID NO: 8; the specific genes of the pVSVG-Hyg plasmid include the VSVG (vesicular stomatitis virus glycoprotein) gene, the sequence of the VSVG gene is shown in SEQ ID NO: 11; the specific genes of the pGagPol-BSD plasmid include GagPol (Gag gene encodes viral capsid protein, Pol gene encodes viral reverse transcriptase and gene integrase), the sequence of the GagPol gene is shown in SEQ ID NO: 13; the specific genes of the CMV-PBtransposase plasmid include PBtransposase (PiggyBac transposase), the sequence of the PBtransposase gene is shown in SEQ ID NO: 18.

[0120] Preferably, the pREV-rtTAtTS-puro plasmid further comprises a cHS4 insulator gene, a CBh promoter, a CMV promoter, a Tight promoter, a puromycin resistance gene puroR and a polyadenylation signal.

[0121] Preferably, the pVSVG-Hyg plasmid further comprises a PiggyBac transposon 5'ITR sequence gene, a 3'ITR sequence gene, a cHS4 insulator core gene, a Tight promoter, an intron, an hPGK promoter, a hygromycin resistance gene HygR and a polyadenylation signal.

[0122] Preferably, the pGagPol-BSD plasmid further comprises a PiggyBac transposon 5'ITR sequence gene, a 3'ITR sequence gene, a cHS4 insulator core gene, a Tight promoter, an intron, an SV40 promoter, a blasticidin resistance gene BSDR, a cPPT / CTS element, an RRE element and a polyadenylation signal.

[0123] Preferably, the CMV-PB transposase plasmid further comprises a cHS4 insulator core gene and a polyadenylation signal.

[0124] Unless otherwise specified, the specific sequence of the gene of the present invention is known to those skilled in the art.

[0125] In another preferred example, the sequence of the pREV-rtTAtTS-puro plasmid is shown as SEQ ID NO: 1, the sequence of the pVSVG-Hyg plasmid is shown as SEQ ID NO: 2, the sequence of the pGagPol-BSD plasmid is shown as SEQ ID NO: 3, and the sequence of the CMV-PBtransposase plasmid is shown as SEQ ID NO: 4.

[0126] In another preferred embodiment, the cell line that can be used as the recipient cell of the present invention is preferably selected from HEK293, HEK293-T, HEK293-SF, TE671, HT1080 or HeLa cell lines, preferably HEK293T cell line.

[0127] In another preferred embodiment, the lentiviral stable packaging cell line of the present invention is an adherent culture cell or a suspension cell.

[0128] In another preferred embodiment, the lentiviral stable packaging cell line is a mixed cell or a monoclonal cell, preferably a monoclonal cell.

[0129] A particularly preferred lentiviral stable packaging cell line of the present invention is the cell line with a deposit number of CGMCC No.: 45528. Experiments have shown that the toxin titer of the cell is > 4×10 7 TU / mL.

[0130] The method for constructing a lentiviral stable packaging cell line of the present invention can be used to prepare various lentiviral stable packaging cell lines and produce corresponding viruses as needed. For example, lentiviruses include (but are not limited to): human immunodeficiency virus (HIV), simian immunodeficiency virus (SIV), equine infectious anemia (EIA), feline immunodeficiency virus (FIV), and other virus genera. When constructing a packaging cell line of a corresponding virus type, the corresponding genes in the packaging, envelope, and helper plasmids need to be replaced with genes of the corresponding virus type.

[0131] The present invention conducted stability studies on the constructed lentiviral stable packaging cell line, including passage stability, toxin production stability, and copy number stability. The experimental results showed that the constructed lentiviral stable packaging cell line can be stably passaged and inherited in the absence of selection pressure (without the addition of antibiotics), and can be stably passaged for more than 30 generations in the absence of selection pressure; the cells can be normally inherited and have toxin production ability when passaged without the addition of antibiotics; and the copy numbers of the GagPol gene, VSVG gene, and Rev gene are relatively stable. In other words, the constructed lentiviral stable packaging cell line has excellent properties in terms of passage, toxin production, and genetic gene copy number.

[0132] Preparation of a stable lentiviral packaging cell line

[0133] The present invention also provides a method for preparing the above-mentioned lentiviral stable packaging cell line, which comprises the following steps:

[0134] Subject recipient cells to steps (i) and (ii) in any order:

[0135] i) linearizing the pREV-rtTAtTS-puro plasmid, transfecting cells with the linearized pREV-rtTAtTS-puro plasmid, and adding a first antibiotic for screening to obtain a first positive mixed cell;

[0136] ii) co-transfecting the cells with the pVSVG-Hyg plasmid, the pGagPol-BSD plasmid, and the CMV-PB transposase plasmid; and adding a second antibiotic for selection to obtain a second positive mixed cell;

[0137] Wherein, when step i) is performed first, the first antibiotic is puromycin; the second antibiotic is a combination of puromycin, hygromycin and blasticidin; and

[0138] When step ii) is performed first, the second antibiotic is hygromycin and blasticidin; the first antibiotic is a combination of puromycin, hygromycin and blasticidin;

[0139] The cells whose screening results in step i) and step ii) are both positive are the lentiviral stable packaging cell lines.

[0140] Those skilled in the art will appreciate that the order of plasmid transfection can be adjusted as needed, and the antibiotic used for screening can be adjusted accordingly. Cells transfected with all plasmids can be obtained by performing step i) first and then step ii), or by performing step ii) first and then step i). In step ii), the plasmids can also be transfected separately or co-transfected in different combinations.

[0141] Typically, in step i) and / or step ii), the transfection is performed in the presence of a transfection reagent. The present invention has no particular requirements for the transfection reagent, and commonly used transfection reagents in the present invention, such as polyethyleneimine, can be used.

[0142] In step i), the linearization can be achieved by restriction endonucleases.

[0143] Lentiviral stable production cell line and construction method thereof, and lentivirus stable production method

[0144] The present invention also provides a lentivirus stable production cell line, which is obtained by a method comprising the following steps: transfecting a plasmid containing a viral packaging target gene and a transposase plasmid into the lentivirus stable packaging cell line as described in the first aspect of the present invention and screening cells in which the target gene is stably integrated, thereby constructing a lentivirus stable production cell line.

[0145] The present invention also provides a method for constructing a lentiviral stable production cell line, comprising the steps of transfecting a plasmid containing a viral packaging target gene and a transposase plasmid into the lentiviral stable packaging cell line as described in the first aspect of the present invention and screening cells in which the target gene is stably integrated, thereby constructing a lentiviral stable production cell line.

[0146] The present invention also provides a method for producing lentivirus using the above-mentioned lentiviral stable producer cell line, comprising the steps of transfecting a plasmid containing a viral packaging target gene and a transposase plasmid into the lentiviral stable packaging cell line as described in the first aspect of the present invention and screening cells in which the target gene has been stably integrated, thereby constructing a lentiviral stable producer cell line, and then adding an inducer to induce the lentiviral stable producer cell line to produce lentivirus.

[0147] Preferably, antibiotics can be added for screening according to the type of resistance gene to obtain target cells.

[0148] Whether the target gene is stably integrated can be evaluated by detecting whether the cells can stably express the corresponding gene, which is well known to those skilled in the art.

[0149] The lentivirus stable production cell line of the present invention comprises a tetracycline-inducible expression system. When producing lentivirus, doxycycline (dox) can be added to induce toxin production. It has also been found that adding sodium butyrate during the toxin production process can increase the yield of lentivirus.

[0150] The present invention provides a specific lentivirus stable production cell line, which is obtained by transfecting a plasmid containing a viral packaging target gene and a transposase plasmid into the lentivirus stable packaging cell line as described in the first aspect of the present invention and screening cells in which the target gene is stably integrated, wherein

[0151] The virus packaging target gene includes pTol2-Lv-GOI-Bleo plasmid, and the transposase plasmid is CMV-Tol2transposase plasmid, or

[0152] More preferably, the virus packaging target gene includes pSB-Lv-GOI-TAT-Bleo plasmid, and the transposase plasmid is CMV-SB100X plasmid.

[0153] The pTol2-Lv-GOI-Bleo plasmid is an empty plasmid that does not contain the target gene. When the target gene (and promoter) is packaged, the plasmid containing the virus-packaged target gene can be formed.

[0154] The pSB-Lv-GOI-TAT-Bleo plasmid is an empty plasmid that does not contain the target gene. When the target gene (and promoter) is packaged, the plasmid containing the virus-packaged target gene can be formed.

[0155] For example, a target gene commonly used in an experiment is the GFP (green fluorescent protein) gene, and the promoter is CMV. The sequence of the plasmid pTol2-Lv-CMV-GFP-Bleo formed after the pTol2-Lv-GOI-Bleo plasmid packages the CMV-GFP gene is shown in SEQ ID NO: 19.

[0156] For example, the target gene commonly used in an experiment is the GFP (green fluorescent protein) gene, and the promoter is CMV. The sequence of the plasmid pSB-Lv-CMV-GFP-TAT-Bleo formed after the pSB-Lv-GOI-TAT-Bleo plasmid packages the CMV-GFP gene is shown in SEQ ID NO: 78.

[0157] Typically, the lentiviral stable producer cell line comprising the pTol2-Lv-GOI-Bleo plasmid (a plasmid not comprising the target gene or a plasmid comprising the target gene) and the CMV-Tol2 transposase plasmid can be prepared by the following method, which comprises the following steps: 1) co-transfecting the pTol2-lv-GOI-Bleo plasmid and the CMV-Tol2 transposase plasmid into a mixture of stable packaging cell lines, and adding a third antibiotic for screening and sorting to obtain monoclonal cells, i.e., the lentiviral stable producer cell line; wherein the third antibiotic is a combination of puromycin, hygromycin, blasticidin, and bleomycin hydrochloride.

[0158] Typically, the lentivirus stable production cell line constructed using the pTol2-Lv-GOI-Bleo plasmid and the CMV-Tol2 transposase plasmid is induced with an inducer to stably produce the lentivirus. Preferably, the inducer is doxycycline.

[0159] The lentiviral stable producer cell line comprising the pSB-Lv-GOI-TAT-Bleo plasmid (a plasmid not comprising the target gene or a plasmid comprising the target gene) and the CMV-SB100X plasmid can be prepared by the following method, which comprises the following steps: 1) co-transfecting the pSB-lv-GOI-TAT-Bleo plasmid and the CMV-SB100X plasmid into a mixed stable packaging cell line, and adding a third antibiotic for screening and sorting to obtain monoclonal cells, i.e., the lentiviral stable producer cell line; wherein the third antibiotic is a combination of puromycin, hygromycin, blasticidin, and bleomycin hydrochloride.

[0160] Typically, the lentivirus stable production cell line constructed using the pSB-Lv-GOI-TAT-Bleo plasmid and the CMV-SB100X plasmid is induced with an inducer to stably produce the lentivirus. Preferably, the inducer is doxycycline.

[0161] The toxicity value of the lentivirus stable production cell line constructed using the lentivirus stable production cell line of the present invention can usually reach 8*10 6 -3*10 8 TU / ml.

[0162] The lentiviral vectors produced by the present invention can efficiently and stably integrate target genes into the genome of infected cells. The types of target genes that can be used in the present invention are not particularly limited and can be selected as needed. Typically, target genes can be used for (but are not limited to) gene overexpression, gene knockdown or knockout, reporter gene expression, cell fate regulation, gene therapy, and immunotherapy.

[0163] The main advantages of the present invention include:

[0164] The lentiviral stable production cell line constructed by the present invention can directly induce cells to produce lentiviral vectors without the need for plasmids and transfection reagents, greatly reducing the cost of viral vector production;

[0165] The stable lentivirus production cell line constructed by the present invention has a stable process for producing lentivirus, and the lentivirus product has small differences between batches;

[0166] The stable lentivirus producing cell line constructed by the present invention is easy to scale up when producing lentivirus, and has high yield.

[0167] The present invention will be further described below in conjunction with specific implementations. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. The experimental methods in the following examples, for which specific conditions are not specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are calculated by weight.

[0168] Example 1: Plasmid construction method

[0169] 1.1 Construction of pREV-rtTAtTS-puro, pVSVG-Hyg, pGagPol-BSD, and CMV-PBtransposase plasmids

[0170] The lentiviral stable packaging cell line constructed in this example is composed of four plasmids: pREV-rtTAtTS-puro plasmid (containing REV, rtTA, and tTS genes), pVSVG-Hyg plasmid (containing VSVG gene), pGagPol-BSD plasmid (containing gagpol gene), and CMV-PBtransposase plasmid (containing PiggyBac transposase PBtransposase gene). Their plasmid maps are shown in Figures 1a, 1b, 1c, and 1d, respectively.

[0171] 1.1.1 Construction of pREV-rtTAtTS-puro plasmid: The entire plasmid was synthesized (Qingke Biosynthesis).

[0172] 1.1.2 Construction of pGagPol-BSD plasmid: The KANA-ori gene fragment was obtained using primers 3ITR-ORI-F and 5ITR-Amppro-R and the template pREV-rtTAtTS-puro plasmid. The CHS4-3ITR gene fragment was obtained using primers bgPA-CHS4-F and Ori-3ITR-R and the template pPB-ITR-CHS4-Tight plasmid (Golden Weichi Biosynthesis). The 5ITR-Tight gene fragment was obtained using primers Amppro-5ITR-F and Intron-Tight-R and the template pPB-ITR-CHS4-Tight plasmid (Qingke Biosynthesis). The above fragments were ligated between the FspI and BamHI restriction sites of the plasmid pGAGPOL-KANA (Golden Weichi Biosynthesis) by homologous recombination to construct the plasmid pGagPol-BSD-1 and sequence verification. The SV40-BSD gene fragment was obtained using primers bgPA-sv40pro-F and CHS4-sv40PA-R and the template psv40-BSD plasmid (Qingke Biosynthesis). The SV40-BSD gene fragment was ligated between the FspI restriction site of the plasmid pGagPol-BSD-1 by homologous recombination to construct the plasmid pGagPol-BSD and sequence verification.

[0173] 1.1.3 Construction of the pVSVG-Hyg Plasmid: The pGagPol-BSD and pVSVG-KANA plasmids were digested with BamHI and FspI, respectively, and the corresponding fragments were recovered. The recovered fragments were ligated with T4 ligase to construct the pVSVG-1 plasmid and verified by sequencing. The PGK-Hyg-sv40PA gene fragment was obtained using primers bgPA-PGK-F and 3ITR-sv40PA-R and the template plasmid pPGK-Hyg-sv40PA (Qingke Biosynthesis). The PGK-Hyg-sv40PA gene fragment was ligated into the NotI and XbaI restriction sites of the pVSVG-1 plasmid by homologous recombination to construct the pVSVG-Hyg plasmid and verified by sequencing.

[0174] 1.1.4 Construction of CMV-PB transposase plasmid: The KANA-ori gene fragment was obtained using primers ORI-F and Apmpro-R and the template pREV-rtTAtTS-puro plasmid. The CHS4-rPolr2A gene fragment was obtained using primers Apmpro-CHS4-F and sv40Intron-R and the template Super PiggyBac Transposase plasmid (SBI-PB200PA-1). The transposase gene fragment was obtained using primers sv40Intron-transposase-F and ORI-sv40PA-R and the template Super PiggyBac Transposase plasmid (SBI-PB200PA-1). The three fragments were ligated by homologous recombination to construct the CMV-PB transposase plasmid and verified by sequencing.

[0175] 1.2 Construction of pTol2-Lv-GOI-Bleo plasmid and CMV-Tol2 transposase plasmid

[0176] The lentiviral stable production cell line constructed in this example is composed of two plasmids, the pTol2-Lv-CMV-GFP-Bleo plasmid (containing the lentiviral genome transcription and packaging target gene GFP) and the CMV-Tol2transposase plasmid (containing the Tol2 transposase Tol2transposase gene). Their plasmid maps are shown in Figures 2a and b, respectively.

[0177] 1.2.1 Construction of the pTol2-Lv-CMV-GFP-Bleo plasmid: The KANA-ori gene fragment was obtained using primers 3LTR-ORI-F and 5IR-Amppro-R and the template pREV-rtTAtTS-puro plasmid. The 5IR gene fragment was obtained using primers Amppro-5IR-F and CMV-5IR-R and the template pminiTol2 plasmid (ADDGENE-31829). The CMV-PAI gene fragment was obtained using primers 5IR-CMV-F and RRE-PAI-R and the template pTK945 plasmid (Qingke Biosynthesis). These fragments were ligated into the ApaI and NotI restriction sites of the plasmid pTK945 (Qingke Biosynthesis) by homologous recombination to construct the plasmid pLv-GOI-1 and sequence verification. The bgPA gene fragment was obtained using primers 3LTR-bgPA-F and sv40pro-bgPA-R and the template pTK945 plasmid (Qingke Biosynthesis). The SV0pro-Bleo-sv40PA gene fragment was obtained using primers bgPA-sv40pro-F2 and 3IR-sv40PA-R and the template pSV0pro-Bleo-sv40PA plasmid (Qingke Biosynthesis). The 3IR gene fragment was obtained using primers sv40pro-3IR-F and ORI-3IR-R and the template pminiTol2 plasmid (ADDGENE-31829). The above gene fragments were ligated between the ApaI restriction sites of the plasmid pLv-GOI-1 by homologous recombination to construct the plasmid pTol2-Lv-CMV-GFP-Bleo and sequenced for verification.

[0178] 1.2.2 Construction of CMV-Tol2 transposase plasmid: The KANA-ori gene fragment was obtained using primers sv40PA-ORI-F and CMV-Amppro-R and the template pREV-rtTAtTS-puro plasmid. The above fragment was ligated into the SphI restriction site of the plasmid pCMV-Tol2 plasmid (ADDGENE-31823) by homologous recombination to construct the plasmid CMV-Tol2 transposase and sequence verification.

[0179] Example 2: Construction of a stable lentiviral packaging cell line

[0180] 2.1 PB transposon transfection gagpol and VSVG plasmid transfection conditions

[0181] 2.1.1 Transfection ratio of gagpol and VSVG plasmids

[0182] This embodiment uses the PB transposon tool to stably integrate the gagpol and VSVG genes into the cell genome. There is an optimal transfection ratio of gagpol and VSVG during transfection, which affects the toxicity value of the cell line. 293T cells were inoculated into a 6-well plate (Corning, Catalog No.: 3516), with 8E+05 cells per well. After overnight culture, each well was transfected with different ratios of pVSVG-Hyg and pGagPol-BSD plasmids, as shown in Table 1. The ratio of plasmid to transfection reagent PEI (polyethyleneimine) (Polyplus, Catalog No.: 115-010) during transfection was total plasmid amount (ug): PEI = 1:1.5. After 48 hours of transfection, the transfected cells were digested and inoculated into a T75 culture flask (Corning, Catalog No.: 430641) with an inoculation density of 4E+04 / cm 2 24 hours after inoculation, the culture medium was replaced with a final concentration of 15 μg / mL BSD (blasticidin) (YEASEN, Catalog No. 60218ES10) and 200 μg / mL Hygromycin (YEASEN, Catalog No. 60224ES03) for pressure selection. The selection medium was changed every 3-4 days. After 2 weeks of selection, the cells were seeded into 6-well plates and transfected with pREV-rtTAtTS-puro and pTol2-Lv-CMV-GFP-Bleo plasmids to package the virus. 48 hours after transfection, the virus titers were harvested and analyzed. The toxicity of cell lines constructed with different transfection ratios of gagpol and VSVG plasmids was compared. The results are shown in Figure 3, indicating that a 1:1 VSVG:gagpol transfection ratio resulted in the highest toxicity of the cell line constructed.

[0183] Table 1

[0184] 2.1.2. Transfection amount of gagpol and VSVG plasmids

[0185] 293T cells were seeded into 6-well plates, with 8E+05 cells per well. After overnight culture, each well was transfected with different amounts of CMV-PBTransposase, pVSVG-Hyg, and pGagPol-BSD plasmids, as shown in Table 2. The ratio of plasmid to transfection reagent PEI (polyethyleneimine) during transfection was 1:1.5: total plasmid (μg): PEI. 48 hours after transfection, the transfected cells were digested and seeded into T75 culture flasks at a seeding density of 4E+04 / cm 224 hours after inoculation, the culture medium containing a final concentration of 15 μg / mL BSD (blasticidin) and 200 μg / mL Hyg (hygromycin) antibiotics was replaced for pressure selection. The selection medium was replaced every 3-4 days. After 2 weeks of cell selection, the cells were seeded into 6-well plates and transfected with pREV-rtTAtTS-puro and pTol2-Lv-CMV-GFP-Bleo plasmids to package the virus. The virus titer was measured 48 hours after transfection. The toxicity value of cell lines constructed by transfecting different amounts of CMV-PBTransposase, pVSVG-Hyg, and pGagPol-BSD plasmids was compared. The results are shown in Figure 4. It shows that increasing the amount of plasmid transfection will increase the toxicity value of the cell line, but further increasing the amount of transfection after reaching 2 μg / 1E+06 cells does not significantly increase the titer value.

[0186] Table 2

[0187] 2.2 Preparation of stable lentiviral packaging cell lines

[0188] 293T cells were seeded in T75 culture flasks at a seeding density of 6E+06 / flask. After overnight culture, the pREV-rtTAtTS-puro plasmid constructed in Example 1 was linearized and dephosphorylated using pvuI (NEB, catalog number: #R3150S). The linearized pREV-rtTAtTS-puro plasmid and PEI (polyethyleneimine) were co-transfected into 293T cells. The amount of pREV-rtTAtTS-puro plasmid used during transfection was 1.5 μg / 10 6 cells, and the PEI dosage was pREV-rtTAtTS-puro plasmid:PEI=1:1.5. 48 hours after transfection, the cells were re-seeded into T175 culture flasks (Corning, catalog number: 431080) at a seeding density of 4E+04 / cm 2 After the cells adhered overnight, the culture medium was replaced with Puromycin (YEASEN, Cat. No. 60210ES25) at a final concentration of 2 μg / mL for pressure selection. The selection medium was changed every 3-4 days. After 10 days of cell selection, a pool of drug-resistant stable transfected cells was obtained, the first positive mixed cell line. The first positive cells were seeded into 6-well plates and transfected with pVSVG-Hyg, pGagPol-BSD, and pTol2-Lv-CMV-GFP-Bleo plasmids to package the virus. The virus was harvested 48 hours after transfection and the titer was measured.

[0189] After titer detection confirmed that the REV gene was stably integrated into the cells and could be stably expressed, the first positive mixed cells were seeded in a 6-well plate with 8E+05 cells per well. After overnight culture, the pVSVG-Hyg, pGagPol-BSD and CMV-PBtransposase plasmids and PEI (polyethyleneimine) were co-transfected into the first positive mixed cells. The plasmid dosage during transfection (pVSVG-Hyg+pGagPol-BSD+CMV-PBtransposase) was 2 μg / 10 6 The PEI ratio was 1:1.5 (pVSVG-Hyg + pGagPol-BSD + CMV-PB transposase) for each cell. Forty-eight hours after transfection, cells were reseeded into T75 culture flasks at a seeding density of 4E+04 / cm2. After the cells adhered overnight, the culture medium was replaced with a final concentration of 2μg / mL Puro (puromycin), 15μg / mL BSD (blasticidin), and 200μg / mL Hygromycin for pressure selection. The selection medium was changed every 3-4 days. After two weeks of selection, a pool of drug-resistant, stably transfected cells was obtained, i.e., a lentiviral stably packaged cell pool. The polyclonal lentiviral stably packaged cell pool was plated using limiting dilution in 96-well plates (Falcon, Cat. No. 353072) at 0.5 cells / 100μL of culture medium per well. The cells were then observed under a microscope, and wells containing single cells were identified and marked. The cells were then cultured in an incubator for approximately 20-30 days. The single clones that survived the 96-well plate were numbered Clone 2, 4, 11, 34, 53, 54, 68, 72, 81, 82, 90, 102, 110, 112, 132, 138, 145, and 150. Titer testing of the different clones is shown in Figure 5. Clone 4, which produced the highest virus titer and exhibited excellent cell stability, has been deposited with the China General Microbiology Center under the China General Microbiology Center, with the deposit number CGMCC No. 45528.

[0190] Example 3: Construction of a stable lentiviral production cell line

[0191] 3.1 Transgenic plasmid transfection conditions

[0192] This example uses the Tol2 transposon tool to stably integrate the viral packaging transcription cassette and the packaging target gene GFP into the cell genome. During transfection, there is an optimal transfection ratio of the Tol2 transposon to the transposase and the optimal total amount of plasmid transfection, which affects the virulence value of the cell line. Stable packaging cell line clone 4 cells were inoculated into 6-well plates, with 8E+05 cells per well. After overnight culture, each well was transfected with different ratios and total amounts of pTol2-Lv-CMV-GFP-Bleo and CMV-Tol2 transposase plasmids, as shown in Table 3. The ratio of plasmid to transfection reagent PEI (polyethyleneimine) during transfection was 1:1.5 total plasmid (μg):PEI. 48 hours after transfection, the transfected cells were digested and inoculated into T75 culture flasks with an inoculation density of 4E+04 / cm2. 24 hours after inoculation, the culture medium containing 1μg / mL Puro (puromycin), 10μg / mL BSD (blasticidin), 150μg / mL Hyg (hygromycin) and 500ug / ml Zeocin (zeocin hydrochloride) (YEASEN, catalog number: 60216ES60) antibiotics was replaced for pressure screening. The screening medium was replaced every 3-4 days. After 2 weeks of cell screening, the cells were inoculated into 6-well plates and induced to produce toxins. The toxins were collected 48 hours after induction and the titer was detected. The toxicity of cell lines constructed with different ratios and total amounts of pTol2-Lv-CMV-GFP-Bleo and CMV-Tol2transposase plasmids was compared. The results showed that the transfection ratio of pTol2-Lv-CMV-GFP-Bleo and CMV-Tol2transposase was 1:1, and the total transfection amount was 2ug / 10 6 The cell line constructed with 10 cells has the highest toxic effect value.

[0193] Table 3

[0194] 3.2 Preparation of Stable Lentivirus Producer Cell Lines

[0195] The stable packaging cell line clone4 cells were seeded in 6-well plates at a seeding density of 6E+06 / bottle. After overnight culture, the pTol2-Lv-CMV-GFP-Bleo plasmid and CMV-Tol2transposase plasmid were co-transfected with PEI (polyethyleneimine) into the first positive mixed cells. The amount of plasmid used during transfection (pTol2-Lv-CMV-GFP-Bleo + CMV-Tol2transposase) was 2 μg / 10 6The transfection ratio of pTol2-Lv-CMV-GFP-Bleo and CMV-Tol2transposase plasmids was 1:1, and the PEI dosage was (pTol2-Lv-CMV-GFP-Bleo + CMV-Tol2transposase) plasmid: PEI = 1:1.5. After 48 hours of transfection, the cells were re-inoculated into T75 culture flasks at a seeding density of 4E+04 / cm 2 After the cells have attached overnight, the culture medium containing a final concentration of 1 μg / mL Puro (puromycin), 10 μg / mL BSD (blasticidin), 150 μg / mL Hyg (hygromycin), and 500 μg / ml Zeocin (bleomycin hydrochloride) is replaced for pressure selection. The selection medium is replaced every 3-4 days. After 2 weeks of cell screening, a pool of drug-resistant positive stably transfected cells is obtained, namely, a lentiviral stable production mixed cell. The polyclonal lentiviral stable packaging mixed cell is plated in a 96-well plate by limiting dilution, with 0.5 cells / 100 μL culture medium per well. Then observe under a microscope, pick out the wells with only single cells, mark them, and culture in an incubator for about 20-30 days. The monoclonal cells that survived in the 96-well plate were numbered Clone 4, 8, 15, 28, 33, 38, 56, 60, 70, 71, 85, 92, 100, 103, 121, 124, 136, 145, 159, 170, 181, and 183. The titers of different clones were tested, and the results are shown in Figure 6.

[0196] Example 4: Optimization of production conditions for monoclonal lentiviral stable production cell lines

[0197] The monoclonal clone Clone33 with a relatively high virus production titer in Figure 6 was picked for further study. Clone33 was inoculated in a 6-well plate with 1E+06 cells per well. After 24 hours, the virus production conditions were set using DoE design (experimental design). The factors included: the concentration of sodium butyrate, the time of adding sodium butyrate, the time of adding dox (doxycycline), the concentration of adding dox (doxycycline), and the time of virus harvesting. If it is shown that the optimal experimental scheme is: the concentration of sodium butyrate (Sigma, catalog number: 303410-100G) is 1.5mM, the time of adding sodium butyrate is 24 hours after cell inoculation, the time of adding dox (Sigma, catalog number: doxycycline) (D3072-1ML) is 24 hours after cell inoculation, that is, when the sodium butyrate solution is changed, the concentration of dox (doxycycline) added is 2μg / mL, and the time of virus harvesting is 48 hours after the addition of the inducer. The virus titer produced under the optimal conditions is 4.5E+07 (i.e. 4.5×10 7 )TU / mL.

[0198] Example 5: Gene copy number saturation test of monoclonal lentiviral stable packaging cell line

[0199] The stable packaging cell line clone4 was inoculated into a 6-well plate with 8E5 cells per well. 24 hours later, each well was transfected with 0.8ug pREV-rtTAtTS-puro and 1.6ug pTol2-Lv-CMV-GFP-Bleo, 0.8ug pVSVG-Hyg and 1.6ug pTol2-Lv-CMV-GFP-Bleo, 0.8ug pGagPol-BSD and 1.6ug pTol2-Lv-CMV-GFP-Bleo plasmids, respectively. 48 hours after transfection, the viral supernatants were collected and the titers were tested. The changes in the cell toxicity after the stable packaging cell line clone4 was transfected with the supplemented plasmids were compared with the changes in the toxicity of the original cell line. The results are shown in Figure 7.

[0200] The results showed that after supplementing the plasmids of various elements required for transfection virus packaging, the cell toxicity value did not change significantly, indicating that the ratio of integrated genes in the stable packaging cell line has reached the optimal level.

[0201] Example 6: Stability Verification of Monoclonal Lentivirus Stable Packaging Cell Line

[0202] Further stability studies were conducted on the lentiviral stable packaging cell line Clone4, including cell passage stability and differences in cell toxin production between batches.

[0203] 6.1 Stability of cell culture

[0204] 6.1.1 Cell Growth Stability

[0205] We tested the cell growth stability of the Clone4 monoclonal cell line with and without antibiotics. Specifically, we seeded Clone4 cells into T75 culture flasks and supplemented them with either 1μg / mL Puro (puromycin), 10μg / mL BSD (blasticidin), and 150μg / mL Hygromycin (hygromycin) at a final concentration, or with antibiotic-free medium. We passaged the cells every other day, counting cell counts and viability, maintaining the same seeding density between passages. This was done to assess cell stability.

[0206] Experiments have shown that when no antibiotics are added (no selection pressure), the cell growth rate and cell viability of Clone4 monoclonal cells remain basically the same when antibiotics are added or not, indicating that the cells can be stably passaged for more than 30 generations in the absence of selection pressure (see Figure 8).

[0207] 6.1.2 Toxin production stability

[0208] The stability of toxin production by the Clone4 monoclonal cell line was tested with and without antibiotics. Clone4 cells were seeded into T75 culture flasks and supplemented with either medium containing a final concentration of 1 μg / mL Puro (puromycin), 10 μg / mL BSD (blasticidin), and 150 μg / mL Hygromycin, or with medium without antibiotics. Cells were passaged every other day, and cell number and viability were counted. The cells were seeded at the same seeding density between passages. Cells were cryopreserved at passages 1, 2, 4, 8, 12, 16, 20, 24, and 30, and thawed at the same time. After passage one, the cells were transfected with the pLv-CMV-GFP-Bleo plasmid to induce toxin production. The stability of toxin production at different passages was compared. The experiment demonstrated that the toxin production of the Clone4 monoclonal cell line remained consistent when antibiotics were added or not, and that toxin production remained stable for 30 passages without selective pressure (see Figure 9).

[0209] 6.1.3 Copy Number Stability

[0210] The gene passage stability of Clone4 monoclonal cells with and without antibiotics was tested. The genomic DNA of Clone4 monoclonal cells with and without antibiotics was extracted using a cell genome extraction kit (Tiangen Biochemical Technology Co., Ltd., catalog number: DP304-02). The genomic DNA of Clone4 monoclonal cells was then extracted using the qPCR method to amplify the copy numbers of GagPol, Rev, and VSVG in the genome of Clone4 monoclonal cells. Specifically, Clone4 cells were inoculated into T75 culture flasks, and culture medium containing a final concentration of 1 μg / mL Puro (puromycin), 10 μg / mL BSD (blasticidin), and 150 μg / mL Hyg (hygromycin) antibiotics or antibiotic-free culture medium was added. The cells were passaged every other day, and the cell number and viability were counted. The cells were inoculated at the same inoculation density during passage. Cells of the 5th, 10th, 20th, and 30th generations were collected and frozen. 5×10 5 Extract genomic DNA from frozen cells of each passage using a cell genome extraction kit. Refer to the kit instructions for specific procedures. Dilute the cell genomic DNA to approximately 10-50 ng / μL for qPCR quantification. The specific steps are as follows: Dilute 5 standard plasmid DNAs to 1×10 5 copies / μL, 1×10 4 copies / μL, 1×10 3 copies / μL, 1×10 2 copies / μL, 1×10 1PCR amplified GagPol, Rev, VSVG, and ALB, a total of four genes, respectively. The same genomic DNA sample was added to all four genes tested. The qPCR reaction system (Thermo, Catalog No. A25742) is as follows:

[0211] The F primer and R primer are as follows:

[0212] The qPCR reaction program is shown in the table below. At the same time, the instrument default melting curve program is set.

[0213] After the PCR reaction is completed, the total number of copies of each gene to be tested is calculated based on the standard curve. Since the genomic DNA added to the reactions of different genes to be tested is the same sample and the addition amount is the same, and the copy number of the ALB gene in the test sample is known to be 2 copies / cell, the gene copy number / cell of other genes is calculated according to the following formula:

[0214] The number of copies of the gene to be tested / cell = 2 × the total number of copies of the gene to be tested / the total number of copies of the ALB gene

[0215] The single-cell copy number results of each gene in Clone4 monoclonal cells are shown in Figure 10. The results show that the copy numbers of GagPol, Rev, and VSVG genes in Clone4 monoclonal cells are relatively stable.

[0216] Example 7: Plasmid construction method

[0217] 7.1 Construction of pSB-Lv-GOI-TAT-Bleo and CMV-SB100X Plasmids

[0218] The lentiviral stable production cell line constructed in this example is composed of two plasmids, the pSB-Lv-CMV-GFP-TAT-Bleo plasmid (containing the lentiviral genome transcription and packaging target gene GFP) and the CMV-100X plasmid (containing the SB transposase gene). Their plasmid maps are shown in Figures 2c and 2d, respectively.

[0219] 7.1.1 Construction of pSB-Lv-CMV-GFP-TAT-Bleo plasmid: The 3IR-KANA-ori-5IR gene fragment was obtained using primers SV40PA-3IR-F and CMV-5IR-R and the template pSBtet-GP (ADDGENE-60495). The BGPA gene fragment was obtained using primers 5LTR-BGPA-F and EF1-BGPA-R and the template pTK945 plasmid (Qingke Biosynthesis). The E gene fragment was obtained using primers BGPA-EF1-F and TAT-EF1-R and the template 881-3-SJ19 plasmid (Qingke Biosynthesis). The F1 gene fragment was obtained by using primers EF1-TAT-F and Bleo-TAT-R and the template PUC19-TAT plasmid (Qingke Biosynthesis), and the Bleo-SV40PA gene fragment was obtained by using primers TAT-Bleo-F and 3IR-SV40PA-R and the template pTK945 plasmid (Qingke Biosynthesis). The above fragments were ligated between the pmeI and MluI restriction sites of the plasmid pTK945 plasmid (Qingke Biosynthesis) by homologous recombination to construct the plasmid pSB-Lv-CMV-GFP-TAT-Bleo and sequenced for verification.

[0220] 7.1.2 Construction of CMV-SB100X Plasmid: The SB100X gene fragment was obtained using primers BGPA-SB100X-F and INTRON-SB100X-R and the template plasmid PC-Sleeping Beauty (ADDGENE-65487). The above fragment was ligated into the EcoRI restriction site of plasmid pMD2.G (ADDGENE-12259) by homologous recombination to construct plasmid CMV-SB100X and sequence verification.

[0221] Example 8: Construction of a stable lentiviral production cell line

[0222] 8.1.1 Transgenic Plasmid Transfection Conditions

[0223] This example uses the Sleeping Beauty transposon tool to stably integrate the viral packaging transcription cassette and the packaging target gene GFP gene into the cell genome. During transfection, there is an optimal transfection ratio of SB transposon and transposase and the optimal total amount of plasmid transfection, which affects the virulence value of the cell line. Stable packaging cell line clone 4 cells were inoculated into 6-well plates, with 8E+05 cells per well. After overnight culture, each well was transfected with different ratios and total amounts of pSB-Lv-CMV-GFP-TAT-Bleo and CMV-SB100X plasmids, as shown in Table 4. The ratio of plasmid to transfection reagent PEI (polyethyleneimine) during transfection was total plasmid amount (ug): PEI = 1:1.5. 48 hours after transfection, the transfected cells were digested and inoculated into T75 culture flasks with an inoculation density of 4E+04 / cm2. 24 hours after inoculation, the culture medium containing 1μg / mL Puro (puromycin), 10μg / mL BSD (blasticidin), 150μg / mL Hyg (hygromycin) and 500ug / ml Zeocin (zeocin hydrochloride) (YEASEN, catalog number: 60216ES60) antibiotics was replaced for pressure screening. The screening medium was replaced every 3-4 days. After 2 weeks of cell screening, the cells were inoculated into 6-well plates and induced to produce toxins. The toxins were collected 48 hours after induction and the titer was detected. The cytotoxicity of cell lines constructed with different ratios and total amounts of pSB-Lv-CMV-GFP-TAT-Bleo and CMV-SB100X plasmids was compared. The results showed that the transfection ratio of pSB-Lv-CMV-GFP-TAT-Bleo and CMV-SB100X was 1:6, and the total transfection amount was 0.5ug / 10 6 The cell line constructed with 10 cells has the highest toxic effect value.

[0224] Table 4

[0225] 8.1.2 Preparation of Stable Lentivirus Producer Cell Lines

[0226] The stable packaging cell line clone 4 cells were seeded into 6-well plates at a seeding density of 6E+06 / bottle. After overnight culture, the pSB-Lv-CMV-GFP-TAT-Bleo plasmid and CMV-SB100X plasmid were co-transfected with PEI (polyethyleneimine) into the first positive mixed cells. The plasmid dosage during transfection (pSB-Lv-CMV-GFP-TAT-Bleo + CMV-SB100X) was 0.5 μg / 10 6The transfection ratio of pSB-Lv-CMV-GFP-TAT-Bleo + CMV-SB100X plasmid was 1:5, and the PEI dosage was (pSB-Lv-CMV-GFP-TAT-Bleo + CMV-SB transposase) plasmid: PEI = 1:1.5. After 48 hours of transfection, the cells were re-inoculated into T75 culture flasks at a seeding density of 4E+04 / cm 2 After the cells have attached overnight, the culture medium containing a final concentration of 1 μg / mL Puro (puromycin), 10 μg / mL BSD (blasticidin), 150 μg / mL Hyg (hygromycin), and 500 μg / ml Zeocin (bleomycin hydrochloride) is replaced for pressure screening. The screening medium is replaced every 3-4 days. After 2 weeks of cell screening, a drug-resistant positive stably transfected cell pool is obtained, i.e., a lentivirus stable production mixed cell line.

[0227] Example 9: Optimization of lentiviral production conditions in a mixed cell pool of stable lentiviral production cell lines

[0228] The mixed cells of the lentivirus stable production cell line were inoculated in a 6-well plate with 8E+05 cells per well. After 48 hours, the DoE design (experimental design) was used to set the virus production conditions. The factors included: the concentration of sodium butyrate, the time of adding sodium butyrate, the time of adding dox (doxycycline), the concentration of adding dox (doxycycline) and the time of virus harvesting. If it is shown that the optimal experimental scheme is: the concentration of sodium butyrate (Sigma, catalog number: 303410-100G) is 1.5mM, the time of adding sodium butyrate is 48 hours after cell inoculation, the time of adding dox (Sigma, catalog number: doxycycline) (D3072-1ML) is 48 hours after cell inoculation, that is, when the sodium butyrate solution is changed, the concentration of dox (doxycycline) added is 1μg / mL, and the time of virus harvesting is 40 hours after the addition of the inducer. The virus titer produced under the optimal conditions is 2.5E+08 (i.e. 2.5×10 8 )TU / mL.

[0229] It can be seen that compared with the stable lentiviral production cell line of Example 4, the virus titer produced by the production cell line transfected with pSB-Lv-GOI-TAT-Bleo / CMV-SB100X is increased by an order of magnitude (about 10 times), and has significantly better application prospects.

[0230] strain preservation

[0231] [Corrected 02.07.2024 according to Rule 91] The lentiviral stable packaging cell line 293T-GPVG-R (2-1-4) of the present invention has been deposited in the China General Microbiological Culture Collection Center (CGMCC, Beijing, China), with a deposit date of March 31, 2023, and a deposit number of CGMCC No.: 45528.

[0232] All documents mentioned in this application are incorporated herein by reference, just as if each document were incorporated herein by reference individually. It should also be understood that after reading the above teachings of the present invention, those skilled in the art may make various changes or modifications to the present invention, and that such equivalents also fall within the scope of the claims appended hereto.

Claims

1. A lentiviral stable packaging cell line, wherein: It comprises a packaging plasmid group, wherein the packaging plasmid group consists of a pREV-rtTAtTS-puro plasmid, a pVSVG-Hyg plasmid, a pGagPol-BSD plasmid and a CMV-PBtransposase plasmid; The pREV-rtTAtTS-puro plasmid contains rtTA, tTS and REV genes. The pVSVG-Hyg plasmid contains the VSVG gene, The pGagPol-BSD plasmid contains the GagPol gene, and The CMV-PBtransposase plasmid contains the PBtransposase gene; Wherein, the sequence of the rtTA gene is shown in SEQ ID NO: 9, The sequence of the tTS gene is shown in SEQ ID NO: 8, The sequence of the REV gene is shown in SEQ ID NO: 6; The sequence of the VSVG gene is shown in SEQ ID NO: 11; The sequence of the GagPol gene is shown in SEQ ID NO: 13; and The sequence of the PBtransposase gene is shown in SEQ ID NO:

18.

2. The lentiviral stable packaging cell line according to claim 1, characterized in that The sequence of the pREV-rtTAtTS-puro plasmid is shown in SEQ ID NO: 1, the sequence of the pVSVG-Hyg plasmid is shown in SEQ ID NO: 2, the sequence of the pGagPol-BSD plasmid is shown in SEQ ID NO: 3, and the sequence of the CMV-PBtransposase plasmid is shown in SEQ ID NO:

4.

3. The lentiviral stable packaging cell line according to claim 1, characterized in that The cell line is a cell line with a deposit number of CGMCC No.:45528.

4. A method for preparing a lentiviral stable packaging cell line as claimed in claim 1, wherein: The following steps are involved: Subject recipient cells to steps (i) and (ii) in any order: i) linearizing the pREV-rtTAtTS-puro plasmid; transfecting the linearized pREV-rtTAtTS-puro plasmid into cells, and adding a first antibiotic for screening to obtain a first positive mixed cell; ii) co-transfecting the first positive mixed cells with the pVSVG-Hyg plasmid, the pGagPol-BSD plasmid and the CMV-PBtransposase plasmid, and adding the second antibiotic for screening to obtain the second positive mixed cells; Wherein, when step i) is performed first, the first antibiotic is puromycin; the second antibiotic is a combination of puromycin, hygromycin and blasticidin; and When step ii) is performed first, the second antibiotic is hygromycin and blasticidin; the first antibiotic is a combination of puromycin, hygromycin and blasticidin; The cells whose screening results in step i) and step ii) are both positive are the lentiviral stable packaging cell lines.

5. A lentivirus stable production cell line is obtained by a method comprising the following steps: transfecting a plasmid containing a virus packaging target gene and a transposase plasmid into the lentivirus stable packaging cell line as claimed in claim 1 and screening cells in which the target gene is stably integrated, thereby constructing a lentivirus stable production cell line.

6. The lentiviral stable production cell line according to claim 5, wherein: The cell line is obtained by transfecting a plasmid containing a viral packaging target gene and a transposase plasmid into the lentiviral stable packaging cell line according to claim 1, wherein The plasmid containing the virus packaging target gene includes a pTol2-Lv-GOI-Bleo plasmid, and the transposase plasmid is a CMV-Tol2 transposase plasmid; or The plasmid containing the virus packaging target gene includes pSB-Lv-GOI-TAT-Bleo plasmid, and the transposase plasmid is CMV-SB100X plasmid.

7. The lentiviral stable production cell line according to claim 6, wherein The plasmid containing the virus packaging target gene includes a pTol2-Lv-GOI-Bleo plasmid carrying the target gene, and the pTol2-Lv-GOI-Bleo plasmid sequence is as shown in SEQ ID NO: 80, and a CMV-Tol2transposase plasmid sequence is as shown in SEQ ID NO: 20; or The plasmid containing the virus-packaged target gene includes a pSB-Lv-GOI-TAT-Bleo plasmid carrying the target gene, and the pSB-Lv-GOI-TAT-Bleo plasmid sequence is shown in SEQ ID NO: 78, and a CMV-SB100X plasmid sequence is shown in SEQ ID NO:

77.

8. A method for constructing a lentiviral stable production cell line as claimed in any one of claims 5 to 7, comprising the steps of transfecting a plasmid containing a viral packaging target gene and a transposase plasmid into the lentiviral stable packaging cell line as claimed in claim 1 and screening cells in which the target gene is stably integrated, thereby constructing a lentiviral stable production cell line.

9. A method for constructing a lentiviral stable production cell line as claimed in claim 8, wherein: The following steps are involved: 1) co-transfecting the stable packaging cell line according to claim 1 with pTol2-lv-GOI-Bleo plasmid and CMV-Tol2transposase plasmid or pSB-lv-GOI-TAT-Bleo plasmid and CMV-SB100X plasmid, and adding a third antibiotic for screening and sorting to obtain cloned cells, which are lentivirus stable production cell lines; wherein the third antibiotic is a combination of puromycin, hygromycin, blasticidin and bleomycin.

10. A method for producing a lentivirus, comprising the steps of transfecting a plasmid containing a virus packaging target gene and a transposase plasmid into the lentivirus stable packaging cell line as claimed in claim 1 and screening cells in which the target gene is stably integrated, thereby constructing a lentivirus stable production cell line, and then adding an inducer to induce the lentivirus stable production cell line to produce lentivirus.

11. A method for producing a lentivirus according to claim 10, comprising: Adding an inducer to the production cell line according to any one of claims 5 to 7 induces the lentivirus stable production cell line to produce lentivirus.

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