Skip to main content
NCBI home page
Cytotechnology logoLink to Cytotechnology
. 2020 Oct 29;72(6):897–905. doi: 10.1007/s10616-020-00433-4

Production of lentiviral vectors in suspension cells using low proportion of supercoiled circular plasmid DNA

Xin-An Lu 2,#, Ting He 2,#, Zhihai Han 4,#, Yanping Ding 2, Liang Zhao 2, Guanghua Liu 2, Floris De Smet 3, Xiaojun Huang 1, Danqing Chen 2, Feifei Qi 2,✉, Xiangyu Zhao 1,✉
PMCID: PMC7695760  PMID: 33123933

Abstract

The supercoiled circular (SC) topology form of plasmid DNA has been regarded to be advantageous over open circular or linearized analogue in transfection and expression efficiency, and therefore are largely demanded in the biopharmaceutical manufacturing. However, production of high-purity SC plasmid DNA would result in high manufacturing cost. The effect of SC proportion in plasmid DNA on the quality of packaged lentiviral vectors has never been reported. In this study, we established an efficient system for production of high-titer lentiviral vectors using suspension HEK293SF cells in serum-free media, and the lentiviral titer was not associated with the proportion of SC plasmid DNA. Plasmids DNA with different proportion of SC, open-circular, and linearized forms were prepared using the thermal denaturation method, and were transfected to adherent HEK293T or suspension HEK293SF cells for packaging of lentiviral vectors. The titer of lentiviral vectors from HEK293T cells, but not from HEK293SF cells, was significantly impaired when the proportion of SC plasmid DNA decreased from 60–80% to 30–40%. Further decrease of SC plasmid proportion to 3% led to a dramatic reduction of lentiviral titer no matter the packaging cell line was. However, lentiviral vectors from HEK293SF cells still showed a high titer even when the proportion of SC plasmid DNA was 3%. This study demonstrated that extremely high proportion of SC plasmid DNA was not required for packaging of high-titer lentiviral vector in HEK293SF cells, at least under our manufacturing process.

Keywords: Supercoiled circular plasmid DNA, Proportion, Lentiviral vector, Titer, Suspension cell culture

Introduction

Adoptive transfer of genetically engineered cells has become a promising strategy for immunotherapy and gene therapy. Vectors (including both viral and non-viral vectors) are important vehicles for introduction of target genes into the host cells. Compared with non-viral vectors, viral vectors especially lentiviral and retroviral vectors showed advantages in high transduction efficiency, stable expression, convenient operation, and low cellular damage (Naldini et al. 1996; Blömer et al. 1997; Kafri et al. 1997). Currently, lentiviral and retroviral vectors have been widely used for preparation of genetically engineered cell therapeutics including chimeric antigen receptor T cells (CAR-T), T cell receptor T cells, chimeric antigen receptor natural killer cells (CAR-NK), and genetically modified hematopoietic stem cells. Lentiviral vector is superior to retroviral vector in safety, and therefore establishment of an efficient and low-cost lentiviral production platform is extremely important for the gene and cellular therapy industry.

Plasmids DNA including transfer plasmid, packaging plasmid, and envelope plasmid are essential raw materials for lentivirus production (Sena-Esteves and Gao 2018). There are three topology forms of plasmid DNA, including supercoiled circular (SC), open-circular (nicked-circular), and linear forms. Due to high transduction and expression efficiency, SC plasmid DNA has been regarded as the desirable type for manufacturing of viral vectors (Lahijani et al. 1996). Pharmaceutical-grade DNA products need to meet the criteria that over 90% of plasmids is the SC form (Epstein 1996). However, the effect of the proportion of SC plasmids DNA on the quality of packaged lentiviral vectors has never been reported. Production of high-purity SC plasmid DNA would result in high manufacturing cost since SC plasmid DNA can be degraded by heat, enzymes, chemicals, and shear force during the large-scale manufacturing (Levy et al. 1999, 2000; Ando et al. 1999). Meanwhile, a strict stock condition is required for long-term storage of SC plasmid DNA. Thus, it is important to find a suitable proportion of SC plasmids DNA for lentivirus packaging that will be a balance of the quality and manufacturing cost of lentiviral vectors.

The packaging cell line is also critical for the production efficiency and quality of lentiviral vectors. HEK293T (HEK293T/17), a transformed human embryonic kidney cell line, has been commonly used for package of high-titer lentiviral vectors due to the high efficiency of plasmid transfection and protein expression (Sena-Esteves and Gao 2018). However, the adherent and serum-dependent growth nature limits the application of HEK293T cells in large-scale production of GMP-grade lentiviral vectors. Recently, many investigations have focused on adapting HEK293T cells to grow in suspension using serum-free medium (Bauler et al. 2019), enabling highly efficient and low-cost manufacturing of lentiviral vectors. The suspension and serum-free lentivirus packaging system has become a promising platform for development of gene and cellular therapy.

In this study, we investigated whether the proportion of SC plasmids DNA was critical for the titer of packaged lentiviral vectors, and established an efficient system for production of high-titer lentiviral vectors using suspension HEK293SF cells in serum-free media. This study will provide a direction for optimization of the manufacturing technology of lentiviral vectors.

Results

Preparation of plasmids DNA with different proportion of SC forms

The third-generation lentivirus packaging system was based on four plasmids DNA, including the transfer plasmid pLenti6.3/V5 containing the gene of a CD19-directed chimeric antigen receptor (pLenti6.3/V5-CD19 CAR), the envelope plasmid pLP/VSVG, and the packaging plasmids pLP1/MDK and pLP2/RSK (Table 1). Four plasmids were respectively amplified in E. coli and purified. The concentration and purity of these plasmids were evaluated by detecting the absorbance of light at 230 nm, 260 nm and 280 nm (Table 2). The agarose gel electrophoresis showed that the majority of native plasmids were SC form (Fig. 1, lane 1, 4, 7, 10). Quantification of the gel map using Image J showed that the ratio of SC form was about 65–90% in four native plasmids (Table 2, Group A). To decrease the proportion of SC form, the plasmids were incubated at 50 °C for 7 days. Gel electrophoresis showed that most of the SC plasmids disappeared while the OC and linear forms dramatically increased after 7-day heat denaturation (Fig. 1, lane 2, 5, 8, 11). The 7-day heat denaturation treatment resulted in plasmid samples containing 0–5% of SC plasmids (Table 2, Group C). To prepare plasmid samples with a medium proportion of SC form, the native plasmids and heat-denatured plasmids were each mixed at the ratio of 1:1. The plasmid topology and level of SC form was confirmed using gel electrophoresis (Fig. 1, lane 3, 6, 9, 12). The ratio of SC form in the four mixed samples was about 30–41% (Table 2, Group B). Further heat denaturation by extending the incubation period to 10 days at 50 °C only slightly increased the proportion of OC and linear forms compared with the 7-day denatured samples (Fig. 2, lane 3, 7, 11, 15). The linear forms of four plasmids DNA were generated using single endonuclease digestion of the plasmids and were confirmed using gel electrophoresis (Fig. 2, lane 4, 8, 12, 16).

Table 1.

The information of four plasmids for lentivirus packaging

Plasmid type Plasmid name Size (bp)
Transfer plasmid pLenti6.3/V5-CD19 CAR 7912
Envelope plasmid pLP/VSVG 5823
Packaging plasmid pLP1/MDK 8824
Packaging plasmid pLP2/RSK 4114
Open in a new tab

Table 2.

Groups with different SC proportion of plasmid DNA

Groups Plasmid name SC (%) OC (%) Linear (%) A260 A280 A230 A260/A280 A260/A230 Concentration (ng/µL)
A1 pLP/VSVG 82.02 16.03 1.95 12.278 6.464 5.489 1.90 2.24 613.929
pLP1/MDK 65.09 34.91 0 18.101 4.292 3.543 1.89 2.29 405.071
pLP2/RSK 87.72 10.89 1.39 10.126 5.295 4.516 1.91 2.24 506.315
pLenti6.3/V5-CD19 CAR 75.90 16.86 7.23 8.941 4.674 3.973 1.91 2.25 447.081
B2 pLP/VSVG 35.90 60.66 3.43 11.676 6.192 5.246 1.89 2.23 583.800
pLP1/MDK 30.64 64.83 4.54 9.998 5.321 4.545 1.88 2.20 499.938
pLP2/RSK 40.92 56.09 2.99 12.254 6.454 5.572 1.90 2.20 612.749
pLenti6.3/V5-CD19 CAR 34.19 60.65 5.16 9.922 5.253 4.538 1.89 2.15 496.119
C3 pLP/VSVG 3.32 93.59 3.10 13.417 7.532 6.582 1.78 2.04 670.881
pLP1/MDK 0.64 99.36 0 18.377 10.167 8.522 1.81 2.16 918.869
pLP2/RSK 4.81 95.19 0 13.871 7.552 6.633 1.84 2.09 693.563
pLenti6.3/V5-CD19 CAR 0 100 0 11.643 6.366 5.614 1.83 2.07 582.194
Open in a new tab

1Group A, native plasmids

2Group B, a mixture of native plasmids and heat-denatured plasmids at the ratio of 1:1

3Group C, heat-denatured plasmids

Fig. 1.

Fig. 1

Open in a new tab

Agarose gel electrophoresis of SC, OC and linear forms of four plasmids. Lane 1, native plasmid DNA of VSVG; lane 2, 7-day treatment at 50 °C for plasmid DNA of VSVG; lane 3, a mixture of native VSVG plasmid DNA and heat-treated VSVG plasmid DNA at ratio of 1:1; lane 4, native plasmid DNA of MDK; lane 5, 7-day treatment at 50 °C for plasmid DNA of MDK; lane 6, a mixture of native MDK plasmid DNA and heat-treated MDK plasmid DNA at ratio of 1:1; lane 7, native plasmid DNA of RSK; lane 8, 7-day treatment at 50 °C for plasmid DNA of RSK; lane 9, a mixture of native RSK plasmid DNA and heat-treated RSK plasmid DNA at ratio of 1:1; lane 10, native plasmid DNA of pLenti6.3/V5-CD19 CAR; lane 11, 7-day treatment at 50 °C for plasmid DNA of pLenti6.3/V5-CD19 CAR; lane 12, a mixture of native pLenti6.3/V5-CD19 CAR plasmid DNA and heat-treated pLenti6.3/V5-CD19 CAR plasmid DNA at ratio of 1:1

Fig. 2.

Fig. 2

Open in a new tab

Agarose gel electrophoresis of SC, OC and linear forms of four plasmids after heat or enzyme treatment. Lane 1, native plasmid DNA of RSK; lane 2, 7-day treatment at 50 °C for plasmid DNA of RSK; lane 3, 10-day treatment at 50 °C for plasmid DNA of RSK; lane 4, RSK plasmid DNA was digested with Xho I; lane 5, native plasmid DNA of VSVG; lane 6, 7-day treatment at 50 °C for plasmid DNA of VSVG; lane 7, 10-day treatment at 50 °C for plasmid DNA of VSVG; lane 8, VSVG plasmid DNA was digested with Hand III; lane 9, native plasmid DNA of MDK; lane 10, 7-day treatment at 50 °C for plasmid DNA of MDK; lane 11, 10-day treatment at 50 °C for plasmid DNA of MDK; lane 12, MDK plasmid DNA was digested with Mun I; lane 13, native plasmid DNA of pLenti6.3/V5-CD19 CAR; lane 14, 7-day treatment at 50 °C for plasmid DNA of pLenti6.3/V5-CD19 CAR; lane 15, 10-day treatment at 50 °C for plasmid DNA of pLenti6.3/V5-CD19 CAR; lane 16, pLenti6.3/V5-CD19 CAR plasmid DNA digested with Sal I

Production of lentiviral vectors using HEK293T cells and plasmids with different proportion of SC forms

To evaluate the effect of plasmid topology on the packaging of lentiviral vectors, adherent HEK293T cells were transfected with above four plasmids DNA containing different proportion of SC form (Group A, Group B, or Group C plasmids in Table 2). After the transfected cells were cultured for 48 hours, the supernatant was collected, ultra-filtered, and ultracentrifuged for harvest of lentiviral vectors. As the transfer plasmid contained the gene encoding a CD19-directed chimeric antigen receptor (CAR), the lentivirus titer was determined using flow cytometry analysis of the cell surface expression of CAR after transduction of HEK293T cells. The median titer of lentivirus from Group A, B, and C was 3.49 × 106, 8.20 × 105, and 4.58 × 105 TU/mL, respectively (Fig. 3). The titer of lentivirus yielded by Group A plasmids was 4.64 times higher than that of lentivirus from Group B (P = 0.009), and 8.31 times higher than that of lentivirus from Group C (P = 0.007). Meanwhile, the lentivirus packaged from Group B plasmids also showed a higher titer than that from Group C plasmids (P = 0.011). These results demonstrated that the lentivirus titer was associated with the proportion of SC plasmids in adherent HEK293T packaging system.

Fig. 3.

Fig. 3

Open in a new tab

Effect of the SC plasmid DNA proportion on the functional titer of lentiviral vectors produced by HEK293T cells. Functional titer was measured by transduction units (TU). Lentivirus samples in three groups were harvested individually. Error bars represent the standard deviation of three independent infections. *P < 0.05; **P < 0.01

Production of lentiviral vectors using HEK293SF cells and plasmids with different proportion of SC forms

To further evaluate the effect of plasmid topology on the titer of lentiviral vectors packaged in suspension HEK293SF cells, plasmids DNA with different proportion of SC forms (Group A, Group B, or Group C plasmids in Table 2) were transfected into HEK293SF cells, and lentiviral vectors were harvested 48 hours later. Flow cytometry analysis of the transduction efficiency showed that the lentivirus titer was 1.67 × 107, 1.46 × 107, and 8.71 × 106 TU/mL in Group A, B, and C, respectively (Fig. 4). The titer of lentivirus from Group A plasmids was 1.92 folds higher than that of lentivirus from Group C plasmids (P = 0.002, Fig. 4), but was similar to that of lentivirus from Group B plasmids (P = 0.307, Fig. 4). Furthermore, the lentivirus vector from Group B plasmids showed a 1.68-fold higher titer compared with that from Group C plasmids (P = 0.015). These results suggested that the suspension lentivirus packaging system was much more efficient than the adherent system, and most importantly, extremely high proportion of SC plasmids was not necessary for achieving high lentivirus titer in the suspension packaging cells.

Fig. 4.

Fig. 4

Open in a new tab

Effect of the SC plasmid DNA proportion on the functional titer of lentiviral vectors produced by HEK293SF cells. Functional titer was measured by transduction units (TU). Lentivirus samples in three groups were harvested individually. Error bars represent the standard deviation of three independent experiments. *P < 0.05; **P < 0.01; NS, no significant difference

Discussion

The curved helix axis of DNA naturally exists in human genome, rather than linear form. This well packaged DNA form, usually called SC form, can largely store potential energy for DNA replication and transcription, and therefore are demanded in the biopharmaceutical manufacturing such as lentivirus production (Schleef and Schmidt 2004; Cherng et al. 1999). Regulatory agencies such as Food and Drug Administration (FDA) recommend that the product homogeneity of plasmid DNA must be higher than 90% in SC form (Schleef and Schmidt 2004). During large-scale plasmid production, plasmid DNA can be maintained in a supercoiled form by enzymes within the host cells (Drlica 1992). However, the SC form of plasmid DNA can easily be degraded during experimental processes such as the scale-up culture of the E. coli cells, purification of plasmid DNA and long-term storage, resulting in the generation of OC or linear form of plasmid DNA once one strand or both strands became nicked (Prazeres et al. 1998; Evans et al. 2000; Walther et al. 2003). The limitations in stability lead to high cost for production of high-purity SC plasmid DNA. Previous studies have shown that storage at − 20 °C is mandatory to avoid the denaturation of SC plasmid DNA during the first 8 weeks after alkaline lysis (Freitas et al. 2007). Our results showed that incubation of SC plasmid DNA at 50 °C for 7 days could result in plasmid denaturation to some extent (Fig. 1). However, elongation of the incubation period to 10 days could not further increase the denaturation level (Fig. 2), indicating the relative stability of plasmid DNA upon thermal treatment over a certain period of time. Decreasing the proportion of SC form in plasmid DNA would be a strategy for lowering the manufacturing cost. However, it has never been reported whether the proportion of SC form could affect the quality of final biomedical products. In this study, we took lentivirus packaging as an example, and investigated the effect of SC plasmid DNA level on the titer of lentiviral vectors using the 7-day thermal treatment to prepare plasmid DNA samples with different proportion of SC forms.

By agarose gel electrophoresis, we observed that thermal treatment could lead to substantial decrease of low molecular weight band (the SC form) and dramatic increase of high molecular weight band (the OC, linear, or other forms of DNA, Fig. 1). This phenomenon may be caused by failure of DNA base pair after a single strand DNA nick or melting of large region of the DNA. Meanwhile, the agarose gel result showed that a small population of the plasmid DNA was resistant to the thermal treatment, indicating that the native plasmid DNA was not entirely in the double strand DNA form.

The lentivirus production requires plasmids DNA including transfer plasmid, packaging plasmid, and envelope plasmid. Both the second-generation and third-generation packaging systems which are different in the tat gene expression in the packaging plasmids have shown great potential for production of high-titer lentivirus. Differences in the lentiviral vector components may lead to variations in lentivirus titer. For example, Lee and Cobrinik reported that replacing the RSV promoter with CMV promoter in the transfer plasmid pLKO.1 could increase the lentivirus titer by 3 folds (Lee and Cobrinik 2020). In our study, we used the third-generation packaging system and the transfer plasmid pLenti6.3/V5 that contained the EF1α promoter for efficient expression of the target gene. Using the packaging plasmid system, we found that the functional lentivirus titer was around 1 × 107 TU/mL that seemed to be lower than previous reports (Lee and Cobrinik 2020; Sena-Esteves and Gao 2018; Bauler et al. 2019; Ferreira et al. 2019). The variation is likely attributed to differences in the size and DNA sequences of the target gene, and the different methods for evaluation of the lentivirus titer. Most of previous studies designed fluorescent molecule such as GFP in the transfer plasmid that was smaller than CD19 CAR in size, and calculated the lentivirus titer by detecting the expression of GFP. In our study, we evaluated the titer by flow cytometry analysis of cell surface expression of CD19 CAR protein in Jurkat cells using an antibody that recognized the extracellular fragment of CAR protein. Nevertheless, our lentivirus packaging system did not affect following evaluation of the effect of SC plasmid proportion on lentivirus titer.

Besides plasmids, the packaging cell line is also critical for the lentivirus titer and yield. Adherent HEK293T cells are most commonly used for packaging of lentiviral vectors in research laboratories due to the high proliferation rate, high transfection efficiency, and innate immune competence although expressing the SV40 large T-antigen (Sena-Esteves and Gao 2018; Ferreira et al. 2019). A suspension growing type of HEK293T cells have been developed for large-scale production of clinical-grade lentiviral vectors (Backliwal et al. 2008a, b; Bauler et al. 2019). Thus, we investigated the effect of SC plasmid proportion on lentivirus titer in both adherent HEK293T cells and suspension HEK293SF cells. We found that the plasmid topology profiles are relatively critical for the transfection efficiency and lentivirus titer in the HEK293T cell packaging system (Fig. 3). Transfection of HEK293SF cells with a very low proportion of SC plasmid DNA resulted in similar lentivirus titer compared with that of HEK293T cells with high proportion of SC plasmid (Figs. 3 and 4). Our result supported that the suspension cell packaging system was much more efficient than the adherent system, and was independent of high SC plasmid proportion. The variations are likely associated with the transfection efficiency of plasmids and cell growing conditions. HEK293T cells are required to reach 90% confluency for high transfection efficiency when using PEI or other chemically defined reagents. Further cell culture after transfection may lead to high cell density and contact inhibition that gradually reduced the cell growth rate, poor uptake of foreign nucleic acids and final gene expression efficiency. Thus, cell density is a limitation for adherent HEK293T cells which are primarily applied in small-scale lentivirus production (Segura et al. 2013). Suspension HEK293SF cells were cultured in orbital shaker incubator under agitation which allowed cell densities up to 5 × 106 cells/mL (Muller et al. 2005). The cells can proliferate and grow well in the three-dimensional culture system that is beneficial for production of more lentiviral vectors. Meanwhile, the whole cell membrane of suspension cells can contact with the transfection reagent-plasmid DNA complexes to improve the binding opportunity. Therefore, the HEK293SF system did not require high level of SC plasmids.

In conclusion, the suspension HEK293SF platform is suitable for large-scale production of lentiviral vectors, and does not require high proportion of SC plasmids. Our study provided strong evidence for lowering the level of SC plasmid DNA in the packaging of lentiviral vectors, an alternative way to decrease the manufacturing cost.

Materials and methods

Cells lines

HEK293T cells (HEK293T/17, ATCC) were cultured in high glucose-containing Dulbecco’s modified Eagle’s medium (ThermoFisher Scientific, Waltham, MA, USA) supplemented with 10% fetal calf serum (ThermoFisher Scientific, Waltham, MA, USA) at 37 °C with 5% of CO2. HEK293SF cells (HEK293T/17 SF, ATCC) were grown in FreeStyle 293 Expression Medium (ThermoFisher Scientific, Waltham, MA, USA). HEK293SF cells were maintained on an orbital shaker platform (INFORS Celltron, Swiss) at 130 revolutions per minute (rpm) in a humidified incubator (ThermoFisher Scientific, Waltham, MA, USA). Cell growth was monitored manually using a hemocytometer. HEK293T cells were passaged every two days when cells reached 80–90% of confluency. Adherent cells were digested using trypsin/EDTA solution, centrifuged at 400g for 5 min, resuspended in fresh medium and seeded at a 1:3 dilution. HEK293SF cells were passaged every 2 days to a starting cell density of 6 × 105 cells/mL in fresh medium.

Preparation of plasmid DNA

The transfer plasmid pLenti6.3/V5-CD19 CAR was constructed by ligating the PCR product of CD19 CAR to the third-generation EF1α promoter-based lentiviral transfer plasmid pLenti6.3/V5 (ThermoFisher, Waltham, MA, USA). The packaging plasmids (pLP1/MDK and pLP2/RSK, ThermoFisher, Waltham, MA, USA) and envelop plasmid (pLP/VSVG, ThermoFisher, Waltham, MA, USA) were each diluted in sterile water. The frozen competent E. coli Stbl3 (ThermoFisher Scientific, Waltham, MA, USA) were thawed out on ice. About 100 µL of the competent E. coli cell suspension was transferred into a sterile tube, and incubated with one of the plasmids (around 50 ng) on ice for 30 min. Then four tubes were placed in a 42 °C water bath and incubated for exactly 90 seconds without shaking. The transformed reaction was plated out on LB agar plates with antibiotics and transferred into an incubator at 37 °C. The next day, single colony was inoculated in 100 mL of LB containing the antibiotic and incubated in the shaking incubator at 300 rpm overnight at 37 °C until the solution became quite turbid. The next day, the plasmid DNA was extracted using the AxyPrep Plasmid Midiprep Kit (Axygen, Corning Incorporated, USA). The plasmid DNA concentration was determined with a spectrophotometer (Allsheng Instruments, China). To confirm the SC proportion in each plasmid DNA, agarose gel electrophoresis was used to examine the extracted plasmid DNA.

Denaturation of plasmid DNA

Temperature is one of the important factors that affect DNA denaturation, exposure to high temperature for a long period of time was utilized to create more OC form plasmid DNA from the native plasmid DNA. The temperature of water bath was adjusted at 50 °C in advance, and plasmid DNA was maintained at 50 °C for 7 days or 10 days. Agarose gel electrophoresis was applied to examine the heat denaturation of plasmid DNA.

Packaging of lentiviral vectors

Lentivirus particles were packaged by transfecting the HEK293T or HEK293SF cells with the pLenti6.3/V5-CD19 CAR, pLP1/MDK, pLP2/RSK, and pLP/VSVG plasmids. For the HEK293T cell system, cells were inoculated into flasks at the density of 3 × 105 cells/mL, and cultured in DMEM supplemented with 10% FCS. The next day, the culture medium was replaced with fresh DMEM without FCS. The amount of total plasmid DNA used in the transfection was 1 µg per 106 cells, and the ratio of polyethylenimine (PEI) to total plasmid DNA were 4:1 (v:w). Four plasmids (pLP/VSVG, pLP1/MDK, pLP2/RSK, and pLenti6.3/V5-CD19 CAR) were diluted in Opti-MEM medium at a ratio of 1:1:1:2, and incubated for 5 minutes at room temperature. The transfection reagent PEI dilution and plasmid DNA dilution were mixed, incubated for 20 minutes at room temperature and transferred to each well drop-wisely. Four hours post-transfection, the medium was replaced with fresh growth medium. After 48 hours, the virus-containing supernatant was harvested and ultracentrifuged to determine the lentivirus titer.

As for the HEK 293SF cell system, cells were seeded into a 125 mL flask at 1 × 106 cells/mL in a total 10 mL of growth medium. The next day, HEK 293SF cells were transfected with the four plasmids (pLP/VSVG, pLP1/MDK, pLP2/RSK, and pLenti6.3/V5-CD19 CAR) in the similar way as described above. Forty-eight hours post-transfection, the virus-containing supernatant was harvested by ultracentrifugation, and subjected to lentivirus titer determination.

Agarose gel electrophoresis

To evaluate the denaturation of plasmid DNA, plasmid samples were mixed with 6 × MassRuler DNA loading dye (ThermoFisher Scientific, Waltham, MA, USA). Then the extracted plasmid DNA solutions were loaded onto a 1% agarose gel, electrophoresed at 80 V for 45 minutes, and visualized on a U.V. trans-illuminator. The gray value of the agarose gels was further analyzed using Image J software.

Titration of lentivirus

Titration of CAR-expressing lentiviral vector was carried out by transduction of Jurkat cells. Briefly, 6 × 105 Jurkat cells were plated onto the 12-well plate and cultured in 1 mL of SMM293-TI medium per well. Four plasmid DNA was diluted with RPMI 1640 medium by five and ten folds, respectively. Subsequently, Jurkat cells were transduced with these dilutions of plasmid DNA. Forty-eight hours post transduction, the CAR expressing cells were stained with a phycoerythrin-labeled antibody that recognized the single chain fragment variant of the CD19 CAR (Immunochina Pharmaceuticals, Beijing, China), and analyzed using fluorescent activated cell sorting assay. The lentivirus titer was calculated based on following formula: average cell number per well × %fluorescent cells × dilution ratio of plasmid DNA / 0.02.

Acknowledgements

This work was supported by the Beijing Municipal Science & Technology Commission (Nos. Z171100001017098, Z181100006218036, Z181100002218040, Z191100001119097), the Scientific Research Foundation for Capital Medicine Development (No. 2018-2-4084), the Peking University Clinical Scientist Program (No. BMU2019LCKXJ003), the Clinical Medicine Plus X-Young Scholars Project of Peking University (No. PKU2020LCXQ015), Peking University People’s Hospital Research and Development Funds (No. RDX2019-14), the Zhongguancun Science Park Management Committee (No. 201805028-1), and the Fundamental Research Funds for the Central Universities in China.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Footnotes

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Xin-An Lu, Ting He and Zhihai Han are co-first authors.

Contributor Information

Feifei Qi, Email: qifeifei@immunochina.com.

Xiangyu Zhao, Email: zhao_xy@bjmu.edu.cn.

References

  1. Ando S, Putnam D, Pack DW, Langer R. PLGA microspheres containing plasmid DNA: preservation of supercoiled DNA via cryopreparation and carbohydrate stabilization. J Pharm Sci. 1999;88:126–130. doi: 10.1021/js9801687. [DOI] [PubMed] [Google Scholar]
  2. Backliwal G, Hildinger M, Hasija V, Wurm FM. High-density transfection with HEK-293 cells allows doubling of transient titers and removes need for a priori DNA complex formation with PEI. Biotechnol Bioeng. 2008;99:721–727. doi: 10.1002/bit.21596. [DOI] [PubMed] [Google Scholar]
  3. Backliwal G, Hildinger M, Chenuet S, Wulhfard S, De Jesus M, Wurm FM. Rational vector design and multi-pathway modulation of HEK 293E cells yield recombinant antibody titers exceeding 1 g/l by transient transfection under serum-free conditions. Nucleic Acids Res. 2008;36:e96. doi: 10.1093/nar/gkn423. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bauler M, Roberts J, Wu C, Fan B, Ferrara F, Yip B, Diao S, Kim Y, Moore J, Zhou S, Wielgosz M, Ryu B, Throm R. Production of lentiviral vectors using suspension cells grown in serum-free media. Mol Ther Methods Clin Dev. 2019;17:58–68. doi: 10.1016/j.omtm.2019.11.011. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Blömer U, Naldini L, Kafri T, Trono D, Verma IM, Gage FH. Highly efficient and sustained gene transfer in adult neurons with a lentivirus vector. J Virol. 1997;71:6641–6649. doi: 10.1128/JVI.71.9.6641-6649.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Cherng JY, Schuurmans-Nieuwenbroek NM, Jiskoot W, Talsma H, Zuidam NJ, Hennink WE, Crommelin DJ. Effect of DNA topology on the transfection efficiency of poly((2-dimethylamino)ethyl methacrylate)–plasmid complexes. J Control Release. 1999;60:343–353. doi: 10.1016/S0168-3659(99)00089-9. [DOI] [PubMed] [Google Scholar]
  7. Drlica K. Control of bacterial DNA supercoiling. Mol Microbiol. 1992;6:425–433. doi: 10.1111/j.1365-2958.1992.tb01486.x. [DOI] [PubMed] [Google Scholar]
  8. Epstein S. Addendum to the points to consider in human somatic cell and gene therapy (1991) Hum Gene Ther. 1996;7:1181–1190. doi: 10.1089/hum.1996.7.9-1181. [DOI] [PubMed] [Google Scholar]
  9. Evans RK, Xu Z, Bohannon KE, Wang B, Bruner MW, Volkin DB. Evaluation of denaturation pathways for plasmid DNA in pharmaceutical formulations via accelerated stability studies. J Pharm Sci. 2000;89:76–87. doi: 10.1002/(SICI)1520-6017(200001)89:1&#x0003c;76::AID-JPS8&#x0003e;3.0.CO;2-U. [DOI] [PubMed] [Google Scholar]
  10. Ferreira C, Sumner R, Rodriguez-Plata M, Rasaiyaah J, Milne R, Thrasher A, Qasim W, Towers G. Lentiviral vector production titer is not limited in HEK293T by induced intracellular innate immunity. Mol Ther Methods Clin Dev. 2019;17:209–219. doi: 10.1016/j.omtm.2019.11.021. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Freitas SS, Azzoni AR, Santos JA, Monteiro GA, Prazeres DM. On the stability of plasmid DNA vectors during cell culture and purification. Mol Biotechnol. 2007;36:151–158. doi: 10.1007/s12033-007-0028-y. [DOI] [PubMed] [Google Scholar]
  12. Kafri T, Blomer U, Peterson DA, Gage FH, Verma IM. Sustained expression of genes delivered directly into liver and muscle by lentiviral vectors. Nat Genet. 1997;17:314–317. doi: 10.1038/ng1197-314. [DOI] [PubMed] [Google Scholar]
  13. Lahijani R, Hulley G, Soriano G, Horn NA, Marquet M. High-yield production of pBR322-derived plasmids intended for human gene therapy by employing a temperature-controllable point mutation. Hum Gene Ther. 1996;7:1971–1980. doi: 10.1089/hum.1996.7.16-1971. [DOI] [PubMed] [Google Scholar]
  14. Lee S, Cobrinik D. Improved third-generation lentiviral packaging with pLKO.1C vectors. Biotechniques. 2020;68:349–352. doi: 10.2144/btn-2019-0155. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Levy MS, Ciccolini LAS, Yim SSS, Tsai JT, Titchener-Hooker N, Ayazi Shamlou P, Dunnill P. The effects of material properties and fluid flow intensity on plasmid DNA recovery during cell lysis. Chem Eng Sci. 1999;54:3171–3178. doi: 10.1016/S0009-2509(98)00358-3. [DOI] [Google Scholar]
  16. Levy MS, O’Kennedy RD, Ayazi-Shamlou P, Dunnill P. Biochemical engineering approaches to the challenges of producing pure plasmid DNA. Trends Biotechnol. 2000;18:296–305. doi: 10.1016/S0167-7799(00)01446-3. [DOI] [PubMed] [Google Scholar]
  17. Muller N, Girard P, Hacker DL, Jordan M, Wurm FM. Orbital shaker technology for the cultivation of mammalian cells in suspension. Biotechnol Bioeng. 2005;89:400–406. doi: 10.1002/bit.20358. [DOI] [PubMed] [Google Scholar]
  18. Naldini L, Blömer U, Gallay P, Ory D, Mulligan R, Gage FH, Verma IM, Trono D. In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector. Science. 1996;272:263–267. doi: 10.1126/science.272.5259.263. [DOI] [PubMed] [Google Scholar]
  19. Prazeres DM, Schluep T, Cooney C. Preparative purification of supercoiled plasmid DNA using anion-exchange chromatography. J Chromatogr A. 1998;806:31–45. doi: 10.1016/S0021-9673(97)01254-5. [DOI] [PubMed] [Google Scholar]
  20. Schleef M, Schmidt T. Animal-free production of ccc-supercoiled plasmids for research and clinical applications. J Gene Med. 2004;6:S45–S53. doi: 10.1002/jgm.511. [DOI] [PubMed] [Google Scholar]
  21. Segura MM, Mangion M, Gaillet B, Garnier A. New developments in lentiviral vector design, production and purification. Expert Opin Biol Ther. 2013;13:987–1011. doi: 10.1517/14712598.2013.779249. [DOI] [PubMed] [Google Scholar]
  22. Sena-Esteves M, Gao G. Production of high-titer retrovirus and lentivirus vectors. Cold Spring Harb Protoc. 2018;2018:273–280. doi: 10.1101/pdb.prot095687. [DOI] [PubMed] [Google Scholar]
  23. Walther W, Stein U, Voss C, Schmidt T, Schleef M, Schlag PM. Stability analysis for long-term storage of naked DNA: impact on nonviral in vivo gene transfer. Anal Biochem. 2003;318:230–235. doi: 10.1016/S0003-2697(03)00244-6. [DOI] [PubMed] [Google Scholar]

Articles from Cytotechnology are provided here courtesy of Springer Science+Business Media B.V.

RESOURCES