Retrovirus packaging cell and use thereof

Abstract

Provided are a retroviral packaging cell and the use thereof. The retrovirus packaging cell comprises a capsid gene of an MLV virus and an envelope gene of an FeRV virus, and can stably produce the retrovirus with a high titer. Also provided are a method for producing a retrovirus by using the packaging cell, and the use thereof in the fields of gene therapy and cell therapy, etc.

Description

Retroviral packaging cells and uses thereof Technical Field

[0001] The invention relates to retrovirus packaging cells and applications thereof, and belongs to the field of biopharmaceuticals, in particular to the fields of gene therapy and cell therapy. Background Art

[0002] In recent years, cell therapy and gene therapy have developed very rapidly, and the demand for viral vectors as a gene transduction tool has been increasing. However, conventional viral vectors are still produced by transient packaging methods, which have the disadvantages of unstable titer, low yield, and the need to prepare three or four plasmids for transient transfection, and cannot meet the growing demand for viral vector production capacity. At the same time, due to the current development of gene editing technology and the fifth-generation universal "armed" CAR-T technology, viral vectors are required to provide a higher gene loading capacity. The gene loading capacity of commonly used lentiviral vectors and AAV vectors is less than 8kb, which cannot meet this demand. Therefore, it is necessary to construct a viral vector production cell line with stable production, high titer expression, and high gene loading capacity to further meet the needs of technological development.

[0003] Retroviruses are a class of viruses that reverse transcribe their RNA genome into DNA, which is then stably inserted into the host cell genome. A single virus can carry a gene load exceeding 11 kb. Modified retroviruses, double-stranded RNA viral vectors with high gene load and stable expression, have been used for nearly 40 years. By selecting different envelopes, these viral vectors can specifically infect human hematopoietic stem cells (HSCs), T cells, and natural killer (NK) cells, thereby introducing exogenous genes into these cells for genetic modification. They have been widely used in HSC and T cell genetic modification. Commonly used are gamma-retroviruses (hereinafter referred to as retroviruses) derived from Moloney murine leukemia virus (MLV) and lentiviruses derived from human immunodeficiency virus type 1 (HIV-1). Retroviruses typically consist of a capsid protein expressed from the gag-pol gene of MLV, reverse transcriptase, and integrase, along with other proteins. The envelope consists of envelope genes from other species (such as the GALV envelope gene). The core carries the viral RNA structure containing the target gene. It enters the target cell through cell membrane fusion, and releases and integrates the target gene into the target cell genome.

[0004] Numerous publications have demonstrated that retroviruses can be produced on a large scale using a stable packaging method, with successful commercial applications. For example, Yescarta and Teacartus utilize PG13 cells stably expressing retroviruses for viral vector production. However, the PG13 cells used in these production processes are genetically modified NIH / 3T3 cells, which are of murine origin. Without downstream purification of the retrovirus, the viral harvest contains exogenous factors, particularly immunogenic substances from murine proteins, which can lead to immunological side effects in patients during clinical use. Furthermore, the titers of viruses produced by PG13 cells are low, ranging from approximately 2 to 3E+06 TU / ml. Although some publications have reported titers exceeding 5E+06 TU / ml, these titers still fall short of meeting the growing demand for CAR-T applications. While the stable packaging cell line FLYRD 18 / LNC-hB7, derived from HT1080, is human, its toxin production is consistently below 1E+06 TU / ml, making it insufficient to meet the growing demand for viral vector production.

[0005] Summary of the Invention

[0006] In order to solve the problems existing in the prior art, the present invention provides a human packaging cell that stably produces high-titer retroviruses, in which the capsid gene sequence of MLV virus and the envelope gene sequence of FeRV virus are stably present in the host cell genome and can be stably expressed without being lost during cell passage.

[0007] In a first aspect, the present invention provides a retroviral packaging cell comprising a capsid gene of an MLV virus and an envelope gene of an FeRV virus.

[0008] In one embodiment, the capsid gene of the MLV virus has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, 99% or 100% sequence identity with the nucleic acid sequence shown in SEQ ID NO: 1.

[0009] In one embodiment, the envelope gene encoding the FeRV virus has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity with the nucleic acid sequence shown in SEQ ID NO:3.

[0010] As used herein, "packaging cell" refers to a host cell that has been stably or transiently transfected or transduced with a nucleic acid molecule comprising a viral helper construct using a heterologous nucleotide sequence, wherein the construct is capable of providing stable or transient expression of packaging functions, such as proteins required for replication and encapsidation, which can be provided in trans to produce viral particles. The viral helper can be expressed constitutively or inducibly.

[0011] In one embodiment, the host cell of the packaging cells used in the present invention is selected from the HEK-293 cell line, such as 293T cells, 293T / 17 cells, 293T / 17SF cells, 293H cells, 293E cells, 293-6E cells, 293F cells, 293FT cells, 293FTM cells, 293S cells, 293SG cells, and 293SGGD cells, preferably 293T cells. Other packaging cells known in the art are also suitable for use in the present invention, such as the packaging cells described in U.S. Pat. Nos. 7,070,994, 6,995,919, 6,475,786, 6,372,502, 6,365,150, and 5,591,624.

[0012] In a second aspect, the present invention provides a method for preparing the packaging cell as described above, comprising: transferring the capsid gene of the MLV virus and the envelope gene of the FeRV virus into a host cell via a plasmid.

[0013] In one embodiment, the method for preparing the packaging cells comprises: (1) transferring a plasmid containing the capsid gene of the MLV virus and the envelope gene of the FeRV virus into a host cell to obtain a cell pool; and (2) obtaining double-positive cells by drug screening. Preferably, the drug screening is antibiotic screening. Preferably, the antibiotic is selected from puromycin and / or blasticidin.

[0014] In one embodiment, the capsid gene of the MLV virus and the envelope gene of the FeRV virus are present on the same plasmid. In another embodiment, the capsid gene of the MLV virus and the envelope gene of the FeRV virus are present on different plasmids.

[0015] In one embodiment, the capsid gene of the MLV virus and the envelope gene of the FeRV virus are integrated into the chromosome of the host cell.

[0016] In a third aspect, the present invention provides a set of retroviral packaging vectors comprising:

[0017] (a) a first plasmid comprising the capsid gene of the MLV virus;

[0018] (b) A second plasmid containing the envelope gene of the FeRV virus.

[0019] In one embodiment, the first plasmid further comprises a transcriptional regulatory element, preferably, the transcriptional regulatory element is a CMV promoter.

[0020] In one embodiment, the first plasmid further comprises a resistance gene, preferably, the resistance gene is a puromycin resistance gene.

[0021] In one embodiment, the first plasmid further comprises polyA.

[0022] In one embodiment, the second plasmid further comprises a transcriptional regulatory element, preferably, the transcriptional regulatory element is a CMV promoter.

[0023] In one embodiment, the first plasmid further comprises polyA.

[0024] In one embodiment, the second plasmid further comprises a resistance gene, preferably, the resistance gene is a blasticidin-resistance gene.

[0025] In one embodiment, the first plasmid further comprises polyA.

[0026] In a fourth aspect, the present invention provides a cell for producing a retrovirus, comprising: (a) a first plasmid comprising a capsid gene of an MLV virus; (b) a second plasmid comprising an envelope gene of an FeRV virus; and (c) a master plasmid comprising a gene of interest. Preferably, the master plasmid is introduced into a packaging cell by transient or stable transfection.

[0027] In one embodiment, the target gene comprises a gene encoding a therapeutic molecule. The therapeutic molecules described in the present invention include but are not limited to genes, high molecular weight DNA, plasmid DNA, antisense oligonucleotides, peptides, ribozymes, peptide nucleic acids, chemicals (such as chemotherapy molecules) or any large molecules, including but not limited to DNA, RNA, viral particles, growth factors, cytokines, immunomodulators and other proteins, including but not limited to proteins that stimulate or inhibit the immune system when expressed, such as the CAR molecules used in the present invention.

[0028] In one embodiment, the master plasmid comprises LTRs at both ends.

[0029] In one embodiment, the master plasmid does not comprise the gag, pol or env genes.

[0030] In a fifth aspect, the present invention provides a method for producing retroviruses, comprising the following steps: culturing the cells for producing retroviruses as described above, or transferring the master plasmid containing the target gene into the packaging cells as described above, culturing the cells, and producing retroviruses.

[0031] The present invention has the following beneficial effects:

[0032] (1) The capsid gene from MLV and the envelope gene from FeRV are stably inserted into HEK293T cells. In the subsequent virus packaging process, after the target gene is stably inserted into the production cells, the production plasmid is no longer needed, avoiding the risk of introducing plasmid residues.

[0033] (2) The titer of the CAR gene viral vector can reach above 1.5E+07TU / ml. Under the same total amount of virus added during the CAR-T production process, the volume of single virus addition is reduced compared to 5E+06TU / ml virus (PG13 expression level). This reduces the total amount of exogenous impurities introduced and the probability of immune side effects in patients.

[0034] (3) The use of human cells avoids the introduction of mouse-derived impurities, further improving safety.

[0035] (4) The present invention is a monoclonal cell, which meets regulatory requirements and can be used in the GMP-grade retrovirus preparation process. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1: Experimental design for HEK293T-O2 cell line screening.

[0037] Figure 2: Schematic diagram of the plasmids used in the HEK293T-O2 cell line screening experiment, among which BH504 is the plasmid expressing the MLV capsid gene, BH506 is the plasmid expressing the RD114 envelope, pMSCV-GFP and pMSCV-anti-CD19CAR are the plasmids used to identify stable production cells, and VSVG is the envelope plasmid used to package the intermediate virus.

[0038] Figure 3: GFP positive rate detection and confirmation results of HEK293T-O2cell poll cells.

[0039] Figure 4: Toxin titer detection results after candidate cells were transiently transfected with anti-CD19CAR plasmid.

[0040] Figure 5: Virus production results after candidate cells were transduced with RV-CD19CAR-VSVG virus.

[0041] Figure 6: Growth curve of HEK293T-O2 cells.

[0042] Figure 7: Images of HEK293T-O2 cell monoclonal screening. DETAILED DESCRIPTION

[0043] The present invention will be described in detail below with reference to the embodiments. It should be noted that those skilled in the art will appreciate that the embodiments of the present invention are merely examples and do not constitute any limitation to the present invention. In the absence of any contradiction, the embodiments and features in the embodiments of this application may be combined with each other.

[0044] The experimental materials whose specific sources are not indicated in the present invention are all materials that can be directly purchased through commercial channels. The experimental methods whose specific steps are not specified in the present invention are all conventional molecular biology methods in the art.

[0045] In the following examples, the capsid gene and envelope gene were sequentially transfected into HEK293T cells, and pressure screening was performed on them. After selecting double-positive cells, several clones were obtained by monoclonal cloning, and the GFP gene or anti-CD19 CAR was transferred into the monoclonal cells. Monoclonal cells with high titers were selected, that is, human-derived packaging cell lines that stably produce high-titer retroviruses (Figure 1).

[0046] The plasmid map used in the following examples is shown in FIG2 .

[0047] Example 1 Preparation of HEK293T-O2 cell pool

[0048] 1.1 Preparation of HEK293T (capsid gene) cell pool

[0049] HEK293T cells were revived and expanded to prepare a total number of HEK293T cells ≥ 2E+06 cells.

[0050] The envelope gene (SEQ ID NO: 1), CMV promoter gene (SEQ ID NO: 7) and puromycin resistance gene (SEQ ID NO: 2) from MLV were cloned into the pUC57 plasmid vector to form the pUC57-K-MLV-gp-puro-BH504 plasmid (BH504 plasmid for short).

[0051] The day before the experiment, cells were plated at 1.0E+05 / cm 2 The cells were expanded to T25 culture flasks. On the day of the experiment, the cells to be transfected were cultured to a confluence of 70%-80% before BH504 plasmid transfection.

[0052] 1.1.1 Plasmid transfection

[0053] 0.2 μg / cm 2Dilute the plasmid to be transfected to 1 μg / 100 μl in Opti-MEM medium (Gibco, Catalog No. 31985-070) and mix thoroughly to obtain a plasmid dilution. Add HP transfection reagent (Roche, Catalog No. 04038377027593) to the plasmid dilution at a 3:1 mass ratio. Mix thoroughly and incubate at room temperature for 15 minutes to obtain a plasmid-transfection reagent complex. Add the plasmid-transfection reagent complex to the well containing cells to be transfected at 10% of the culture volume and gently shake to combine.

[0054] 1.1.2 Antibiotic screening

[0055] 20-28 hours after transfection, replace the culture medium with fresh D10 medium for antibiotic selection. Add 2 μg / ml, 10 μg / ml, and 20 μg / ml puromycin for selection. Select for 3-10 days, until cells begin to expand. This will yield a HEK293T cell pool containing the capsid gene, referred to as the HEK293T (capsid gene) cell pool.

[0056] 1.1.3 Copy Number Detection

[0057] The capsid gene copy number was detected by qPCR in cells with puromycin resistance. The qPCR detection method is as follows:

[0058] Primers and probes were prepared in RNase-free ddH₂O to a final concentration of 10 μM working solution (see Table 1 for the reaction system). Genomic DNA was extracted from cells using QIAGEN DNeasy Blood & Tissue Kits (Cat. No. 69504) and diluted using Easy Dilution. Standards were diluted to six different concentrations using TaKaRa Easy Dilution (for Real-Time PCR).

[0059] Table 1 QPCR reaction system

[0060] Aliquot into a 96-well plate and add 4 μl / well of the standard, the test sample, and NTC (Easy Dilution). Repeat in duplicate. Incubate in a 20 μl reaction volume. The reaction program is: 95°C for 10 min; 95°C for 15 sec, 60°C for 35 sec, 50 cycles. The internal reference gene channel is VIC, and the target gene channel is FAM.

[0061] Data processing: Actual number of reaction cells = reference gene copies / μl ÷ 2; average vector copy number (copies / cell) = target gene copy number (copies / μl) / actual number of reaction cells.

[0062] Experimental establishment standard: amplification curve R 2 ≥0.99; amplification efficiency is 90%-110%; NTC Cq value is greater than STD6 Cq value.

[0063] The results of capsid gene copy number detection are shown in Table 2. The capsid genes were successfully integrated in all three cell polls. The HEK293T (capsid gene) cell pool (capsid gene copy number 24.5 copies / cell) resistant to 20 μg / ml puromycin was selected for further envelope gene insertion.

[0064] Table 2 Capsid gene copy number detection results of HEK293T (capsid gene) cell pool

[0065] 1.2 Inserting the envelope gene into HEK293T (capsid gene)

[0066] The RD114 gene (SEQ ID NO: 3) from FeLV, the CMV promoter (SEQ ID NO: 7) and the blasticidin-resistance gene (SEQ ID NO: 4) were cloned into the pUC57 plasmid vector to form the pUC57-K-RD114-blasticidin-BH506 plasmid (BH506 plasmid for short).

[0067] One day before the experiment, the HEK293T (capsid gene) cell pool obtained in Example 1.1 was heated to 14E+04 / cm 2 The cells were expanded to culture flasks and cultured to a confluence of 70%-80% on the day of the experiment, and then transfected with the BH506 plasmid. The transfection method was the same as in Example 1.1.1.

[0068] 20-28 hours after transfection, replace the culture medium with fresh D10 medium and add blasticidin (Shanghai MacLean, Cat. No. B861447) at doses of 2 μg / ml, 5 μg / ml, 10 μg / ml, 12 μg / ml, 15 μg / ml, and 20 μg / ml. Allow the cells to expand for 7-14 days to obtain a HEK293T-O2 cell pool.

[0069] Cells resistant to 15 μg / ml puromycin were selected and the capsid gene copy number and envelope gene copy number were detected by qPCR method in Example 1.1.3. The detection results are shown in Table 3. It can be seen that the envelope gene was successfully integrated into HEK293T (capsid gene) cells, and the copy number was close to the capsid gene copy number level. Table 3 Capsid gene and envelope gene copy number detection results

[0070] Example 2 GFP gene transduction test

[0071] 2.1 Monoclonal cell screening

[0072] The HEK293T-O2 cell pool cells obtained in Example 1.2 were cultured in D10 complete medium containing 20 μg / ml puromycin and 15 μg / ml blasticidin, with a cell count of ≥3×10 6 After the cells have recovered, subclones are plated using a flow cytometer with sorting technology at a density of 0.8 cells per well. A total of 100 96-well plates are plated. Incubate the cells in a 37°C, 5% CO2 incubator. Once cell colonies emerge, single clones are selected for positive clone selection.

[0073] 2.2 Preparation of RV-GFP intermediate virus

[0074] The VSV-G envelope gene (SEQ ID NO: 8) was cloned into the pUC57 plasmid vector to construct the pUC57-K-VSVG plasmid (hereinafter referred to as the VSVG plasmid). The GFP gene sequence (SEQ ID NO: 5) was cloned into the pMSCV plasmid vector (pMSCV plasmid vector construction method: amp in pMSCV-TAP (addgene #12570) was replaced with kana and the TAP sequence was removed) to construct the pMSCV-GFP destination plasmid.

[0075] The day before the experiment, cells were plated at 1E+06 cells / cm 2 The cells were expanded to T25 culture flasks and cultured to a confluence of 70%-80% on the day of the experiment before plasmid transfection.

[0076] According to 0.2 μg / cm 2 Take VSVG plasmid and pMSCV-GFP (mass ratio 1:1), dilute to 1μg / 100μl with Opti-MEM medium (Gibco, catalog number: 31985-070), mix well, and obtain plasmid dilution solution. Add HP transfection reagent (Roche, catalog number: 04038377027593) to the plasmid dilution solution at a mass ratio of 3:1. Mix well and incubate at room temperature for 15 minutes to obtain the transfection complex. Add the transfection complex to the cell well to be transfected at 10% of the culture volume and shake gently. Replace with fresh D10 medium 20h-28h after transfection. After the medium is replaced, place at 32°C and 5% CO2. Collect the culture supernatant every 24h and replace with fresh D10 medium. After 72h, collect the culture supernatant to obtain the RV-GFP intermediate virus.

[0077] 2.3 Infection of Monoclonal Cells with RV-GFP Intermediate Virus

[0078] Monoclonal cells were infected with the RV-GFP intermediate virus as follows: RetroNectin solution (Takara, Catalog No. T100B) was diluted to 20 μg / ml in DPBS (Mei Lun, Catalog No. PWL052). 0.3 ml / well was added to a 24-well non-treated cell culture plate and incubated overnight at 2-8°C. After coating, the RetroNectin solution was aspirated and the cells were washed twice with 2 ml / well of DPBS. Blocking buffer (1 ml / well) in DPBS supplemented with 5% FBS (Gibco, Catalog No. 10099-141C) was added for 30 minutes at room temperature. The blocking buffer was aspirated and the cells were washed twice with 1 ml / well of DPBS. RV-GFP virus was added at 1 ml / well and the cells were centrifuged at 2000 g for 2 hours at 32°C. The supernatant was discarded and supplemented with 1 ml of D10 medium. After infection, cells were plated at 7E+04 cells / well into a 96-well plate and incubated at 37°C in a 5% CO2 incubator for 20-28 hours. D10 medium was then replaced with 0.5 ml / well of culture medium and cultured for an additional 48-72 hours. Cell surface GFP expression was determined, and clones with a GFP-positive rate of ≥10% were designated as positive. A total of 18 positive cell lines were obtained, including 13E5, 13F11, 15G2, 18D8, 22C4, 22C6, 23C2, 23D11, 23D12, 23E4, 23G3, 25B3, 25D1, 25F2, 41D11, 44F4, 45F2, and 48C9 (NC represents a negative control). The results are shown in Figure 3.

[0079] According to the results in Figure 3 , six cell lines with higher GFP positivity rates, 18D8, 22C4, 22C6, 23D11, 23E4, and 25D1, were selected for virus titer detection.

[0080] Virus titer detection method:

[0081] (1) Cell plating: 293T cells in the logarithmic growth phase were prepared in D10 complete medium. A cell suspension was plated in a six-well plate at a concentration of 5E+06 cells per 2 mL. The cells were cultured at 37°C, 5% CO₂ for 24 h. The supernatant was discarded and the cells were washed once with PBS before infection.

[0082] (2) Cell counting: Before loading the virus, first select two wells, digest the cells, and count them. The average value is used to obtain the total number of cells C in each well.

[0083] (3) Virus dilution: Based on the total number of cells, the assumed value n, the virus loading volume is 100 μL, and the sample is diluted with 1% BSA-PBS to obtain different MOIn.

[0084] (4) Virus loading: Each MOIn corresponds to one cell well, with a loading volume of 100 μL. At the same time, a negative well (1% BSA-PBS, 100 μL) was set up. The cells were cultured at 37°C and 5% CO2 for 2 h, and shaken every 15 min. After the incubation, the culture medium was aspirated and washed once with PBS. 2 mL of D10 complete medium was added and the cells were cultured in a 37°C, 5% CO2 incubator for 48 h.

[0085] (5) Primary antibody incubation: resuspend the cells in 1 mL of 5% FBS-PBS, incubate at room temperature for 30 min, centrifuge and discard the supernatant; add 0.5 mL of rabbit anti-human scFv polyclonal antibody to each tube, incubate at 4°C for 60 min, centrifuge and wash twice with 1% BSA-PBS.

[0086] (6) Secondary antibody incubation: Add 0.5 mL of PE-labeled goat anti-rabbit IgG secondary antibody to each tube, incubate at 4°C for 60 min, centrifuge, and wash once with 1% BSA-PBS.

[0087] (7) Flow cytometer assay: Add 0.5 mL of PBS to each tube, mix thoroughly, and then analyze on a flow cytometer. Use the negative tube to set the dividing line between the negative and positive zones, and then record the percentage of positive cells (R) in each tube (i.e., the percentage of positive cells corresponding to each MOIn).

[0088] (8) Data fitting: With MOIn as the horizontal axis and the positive percentage as the vertical axis, curve fitting is performed to calculate the titer value m. The actual infection titer is m = avg (R × C × MOIn × dilution).

[0089] For titer detection, the total number of infected cells was 100,000 cells, the virus added was 10 μl, and the virus was diluted 4-fold before addition.

[0090] The titer test results are shown in Table 4. As can be seen, all six cell lines were positive for cytotoxicity. Among them, the cell lines corresponding to clones 22C4 and 22C6 had higher cytotoxicity titers and were designated as candidate cell lines.

[0091] Table 4 RV-GFP virus production confirmation results

[0092] Example 3 Anti-CD19CAR gene expression verification

[0093] 3.1 Verification of transient expression of anti-CD19 CAR gene

[0094] The gene sequence encoding anti-CD19CAR (SEQ ID NO: 6) was cloned into the pMSCV plasmid vector to construct the pMSCV-CD19CAR plasmid.

[0095] The pMSCV-CD19CAR plasmid was transiently transfected into 22C4 and 22C6 monoclonal cells to prepare a retrovirus containing the anti-CD19CAR gene and test the toxin titer. The preparation method is as follows: According to the method in Example 1.1.1, the anti-CD19CAR gene plasmid-transfection reagent complex was prepared. The cells were plated at 1E+06 cell / cm 2 Amplify the culture into a T25 culture flask, add the plasmid-transfection reagent complex and place it in a 37°C, 5% CO2 incubator for 20h-28h, then replace with D10 medium at 3ml / flask, continue culturing for 48-72h, and collect the supernatant to detect the virus titer.

[0096] The results of virus titer detection are shown in Figure 4. It can be seen that both 22C4 and 22C6 cell lines can package retrovirus containing the anti-CD19CAR gene. The highest virus titer is the titer of the virus produced by the 22C6 clone at 72 hours, which is 1.24E+07TU / ml.

[0097] 3.2 Verification of stable expression of anti-CD19 CAR gene

[0098] According to the method for preparing the intermediate virus in Example 2.2, pMSCV-GFP was replaced with pMSCV-CD19CAR to obtain RV-CD19CAR-VSVG virus.

[0099] According to the method of infecting monoclonal cells with the intermediate virus in Example 2.3, the RV-GFP virus was replaced with the RV-CD19CAR-VSVG virus, and 22C4 and 22C6 cells were transduced with the RV-CD19CAR-VSVG virus. Fresh D10 culture medium was replaced 24 hours after transduction. After the cells grew to a certain number, the virus was produced, and the virus produced at 72 hours and 96 hours was collected for titer determination. The results are shown in Figure 5. It can be seen that all cells can produce the virus, and the infectious titer of the 22C6 clone virus at different time points is greater than 1.5E+07TU / ml. Therefore, the 22C6 clone was selected as a human cell line for stable production of high-titer retrovirus and named HEK293T-O2 cells.

[0100] Example 4 Cell Growth Curve, Monoclonal Origin and Titer Detection

[0101] 4.1 Cell growth curve test

[0102] HEK293T-O2 cells were cultured at 4E+04 cells / cm 2 Cells were seeded into 6-well plates, and one well was trypsinized every 22-26 hours. The cells were resuspended in 1 ml of complete D10 medium and counted. A cell growth curve was plotted (Figure 6) with incubation time (h) as the horizontal axis and total cell density (E+06 cells) as the vertical axis. The growth curve shows that the logarithmic growth phase of HEK293T-O2 cells lasts from 24 to 96 hours after inoculation; the cell doubling period during the logarithmic growth phase is approximately 24 hours, meeting the expected quality target (cell doubling period ≤ 48 hours; cell doubling period DT = t*[log2 / (logNt - lgN0)]).

[0103] 4.2 Confirmation of Monoclonal Origin

[0104] Starting from the first day after subcloning, images were taken using a Clone Select Imager to continuously record cell status. Figure 7 shows images taken on days 1, 6, and 15 (the last day before cell expansion) after subcloning. It can be seen that HEK293T-O2 cells were grown from a single cell. This confirms their monoclonal origin, ensuring the genetic uniformity of all subsequent progeny cells.

[0105] 4.3 Titer detection

[0106] 22C6 monoclonal cells were cryopreserved in a cryopreservation solution composed of 90% D10 medium + 10% DMSO. Three vials (1 ml / vial) were cryopreserved at a cryopreservation density of 6.48E+06 cells / ml. After freezing at 4°C for 8 hours, -20°C for 8 hours, and -80°C for 12 hours, the cells were transferred to liquid nitrogen for storage. One vial of 22C6 (HEK293T-O2) cells was then revived, expanded to five T175 cells, trypsinized, resuspended in cryopreservation solution, and aliquoted at 1 ml / vial. Ten vials were used as the PCB primary cell bank, and 20 vials were used as the RCB research cell bank.

[0107] After cryopreservation, one RCB cell bank was tested for viability using trypan blue staining, resulting in a viability of 93.53%, meeting the requirements for bank establishment. Three HEK293T-O2-RCB cells were tested for sterility, mycoplasma, and bacterial endotoxins. The results, shown in Table 5, showed that sterility, mycoplasma, and bacterial endotoxins all met the requirements of the current edition of the Chinese Pharmacopoeia.

[0108] Table 5 HEK293T-O2-PCB and RCB library construction information

[0109] It should be noted that the above are only preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention may be modified and varied in various ways. It is understood by those skilled in the art that any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention shall be included in the scope of protection of the present invention.

Claims

1. A packaging cell comprising the capsid gene of MLV virus and the envelope gene of FeRV virus.

2. The packaging cell according to claim 1, wherein the capsid gene of the MLV virus has at least 80% identity with the nucleic acid sequence shown in SEQ ID NO:

1.

3. The packaging cell according to claim 1 or 2, wherein the envelope gene of the FeRV virus has at least 80% identity with the nucleic acid sequence shown in SEQ ID NO:

3.

4. The packaging cell according to any one of claims 1 to 3, wherein the host cell is selected from the HEK-293 cell line.

5. The packaging cell according to any one of claims 1 to 4, wherein the capsid gene of the MLV virus and the envelope gene of the FeRV virus are integrated into the chromosome of the host cell.

6. The method for preparing the packaging cell according to any one of claims 1 to 5, comprising: The capsid gene of MLV virus and the envelope gene of FeRV virus are transferred into the host cells through plasmid.

7. The preparation method according to claim 6, comprising: (1) transferring the capsid gene of MLV virus and the envelope gene of FeRV virus into host cells via plasmids to obtain a cell pool; (2) Obtain double-positive cells using drug screening.

8. The preparation method according to claim 6 or 7, wherein the capsid gene of the MLV virus and the envelope gene of the FeRV virus are present on the same or different plasmids.

9. A set of packaging plasmids, comprising: (a) a first plasmid comprising the capsid gene of the MLV virus; and (b) A second plasmid comprising the envelope gene of the FeRV virus.

10. The packaging plasmid according to claim 9, wherein the first plasmid further comprises a transcriptional regulatory element and / or a resistance gene, preferably, the transcriptional regulatory element is a CMV promoter, and the resistance gene is a puromycin resistance gene.

11. The packaging plasmid according to claim 9 or 10, wherein the second plasmid further comprises a transcriptional regulatory element and / or a resistance gene, preferably, the transcriptional regulatory element is a CMV promoter, and the resistance gene is a blasticidin resistance gene.

12. A cell for producing retrovirus, comprising: (a) a first plasmid containing the capsid gene of MLV virus; (b) a second plasmid containing the envelope gene of FeRV virus; and (c) a main plasmid containing a target gene.

13. A method for producing a retrovirus, comprising the following steps: Cultivate the cell of claim 12, or transfer the main plasmid containing the target gene into the packaging cell of any one of claims 1 to 5, and culture the cell.

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