Method for improving lentivirus titer
By using sodium heparin to shield the positive charge of viral envelope proteins during lentivirus production, the problems of cell fusion and reinfection were solved, and viral titer and infection efficiency were improved.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- 山东丽山生物科技有限公司
- Filing Date
- 2026-04-14
- Publication Date
- 2026-05-12
AI Technical Summary
The problem with low lentiviral titers is that the absence of the R peptide sequence leads to enhanced cell fusion activity, the formation of syncytia, and subsequent apoptosis. Furthermore, viral reinfection occurs, affecting viral yield and infection efficiency.
Heparin sodium was added to the culture medium after plasmid transfection. Its high negative charge density allowed it to bind to viral envelope proteins, shielding positive charges, reducing electrostatic adsorption between the virus and the cell membrane, inhibiting cell fusion and reinfection, and increasing viral titer.
It significantly improved lentiviral titers, increased viral yield, and enabled efficient infection of NK cells and γδT cells, with an infection efficiency exceeding 80%.
Smart Images
Figure CN122012416A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of virus utilization technology, and more particularly to a method for increasing lentivirus titers. Background Technology
[0002] Lentiviral BaEVless can efficiently infect both dividing and non-dividing cells, making it a widely used vector in gene editing. The lentiviral vector BaEVless-LV is a key raw material for cell therapies such as CAR-T and CAR-NK. Third-generation lentiviral vectors typically employ a four-plasmid system, including a shuttle plasmid carrying the target gene, pLP1 (which provides the viral core structural proteins and replicase Gag / Pol), pLP2 (a helper plasmid containing Rev, which promotes nuclear export and efficient packaging of viral RNA), and BaEV-Rless-LV or VSV-G-LV (which expresses BaEV-gp or VSV-G envelope glycoproteins, respectively, achieving viral pseudotypening and determining the host cell infection spectrum). By isolating the essential viral replication genes env and structural genes, the risk of viral recombination is significantly reduced, improving biosafety and vector consistency. These plasmids are co-transfected into the 293T production cell line, ultimately generating usable viral particles.
[0003] The aforementioned available viral particles can be used to transduce immune cells for targeted cell therapy. However, for difficult-to-transduce cells such as NK cells and γδT cells, the traditional vector VSV-G-LV has low infection efficiency. BaEV-Rless-LV is more efficient than the traditional vector VSV-G-LV, but its low titer makes it difficult to utilize.
[0004] The problems with low lentiviral titers are: first, the deletion of the R peptide sequence enhances fusion activity, leading to abnormal fusion of 293T cells during production, forming syncytia. After cell fusion, shedding and apoptosis significantly reduce viral yield; second, a "reinfection" effect is common in lentiviral production, meaning that newly generated viral particles can re-enter 293T cells, producing non-replicating progeny viruses lacking key genes such as gag / pol / env, resulting in a dilution of the available viral titer.
[0005] Therefore, there is a need to develop a method to increase lentivirus titers by inhibiting syncytial apoptosis and reducing viral reinfection. Summary of the Invention
[0006] To address the aforementioned problems, the present invention aims to provide a method for increasing lentivirus titers.
[0007] The method for increasing lentivirus titer is as follows: Step 1: Cells are spaced at a rate of (2~2.5) × 10⁻⁶ 5 cell / cm 2Cells were seeded at the specified density and cultured in a 37°C incubator. Step 2: After 24 hours, perform plasmid transfection. The amount of PEI transfection agent is 3 times the amount of plasmid. The total amount of plasmid used is 0.22~0.35 μg / cm³. 2 The plasmids included pCDH-GFP, pLP1, pLP2, and BaEVless in a mass ratio of 6:4:4:1. Step 3: When changing the medium 6 hours after plasmid transfection, add heparin sodium to the culture dish to make the final concentration of the medium 10~75U / mL. After 48~72 hours after plasmid transfection, take the supernatant to obtain usable lentivirus.
[0008] Furthermore, the cell density in step one is 2.2 × 10⁻⁶. 5 cell / cm 2 .
[0009] Furthermore, the total dosage in step two is 0.3 μg / cm³. 2 .
[0010] Furthermore, in step three, the heparin sodium concentration is 10-25 U / mL.
[0011] Furthermore, in step three, the heparin sodium concentration is 10 U / mL.
[0012] Further, in step three, the supernatant was collected 48 hours after plasmid transfection.
[0013] The present invention can bring the following beneficial effects: Heparin sodium, as a highly sulfated glycosaminoglycan, possesses a dense molecular structure containing sulfonic acid and carboxylic acid groups, giving it a high negative charge density. Due to its strong polyanionic properties, heparin sodium preferentially recognizes and binds to locally positively charged regions exposed on the surface of viral envelope proteins. In this invention, this binding effectively shields the positive charge on the surface of viral particles, thereby weakening unnecessary electrostatic adsorption between the virus and the production cell membrane, and enhancing heparin sulfate-dependent binding.
[0014] On the one hand, the above mechanism helps reduce the "reinfection" of production cells by the produced lentivirus BaEVless and increase the viral titer; on the other hand, during the production process, sodium heparin can significantly reduce apoptosis after the production cells are fused, increase cell survival time, and increase viral yield.
[0015] It should be noted that, based on experimental results, the optimal implementation parameters of this invention can increase the crude viral titer of the lentivirus BaEVless to 1.1 × 10⁻⁶. 7TU / mL; and the lentivirus BaEVless produced using optimal implementation parameters can efficiently infect NK cells and γδT cells, with an infection efficiency >80%. Attached Figure Description
[0016] The accompanying drawings, which are included to provide a further understanding and demonstration of the invention, are provided in the drawings. In the drawings: Figure 1 The effect of different compounds and concentration gradients on the crude virus titer harvested at 48 h of lentivirus (****, p <0.0001); Figure 2 The viral titers of crude lentiviruses harvested after 48 hours for different concentrations of heparin sodium groups were determined (ns, p>0.05.*, p<0.05.**, p<0.01.). Figure 3 The viral titers of crude lentiviruses harvested at 72 h for different concentrations of heparin sodium groups were determined (ns, p>0.05.*, p<0.05.**, p<0.01.***, p<0.001.). Figure 4 Comparison of the viral titers of crude virus harvested at 48h and 72h for different concentrations of heparin sodium groups; Figure 5 The GFP fluorescence expression and syncytial formation of lentivirus at 48h and 72h were observed in different concentrations of heparin sodium groups. Figure 6 The effect of different concentrations of heparin sodium on the infection efficiency of lentivirus in 293T cells (***, p<0.001. ****, p<0.0001). Figure 7 Infection efficiency of NK cells by lentiviruses produced in the heparin sodium system (MOI=10). Figure 8 Infection efficiency of lentiviruses produced in the heparin sodium system for infecting γδT cells (MOI=5). Detailed Implementation
[0017] To more clearly illustrate the overall concept of the present invention, the overall solution of the present invention will be described in detail below by way of embodiments; in the following description, numerous specific details are set forth in order to provide a more thorough understanding of the present invention; however, it will be apparent to those skilled in the art that the present invention may be practiced without one or more of these details; in other instances, some technical features known in the art have not been described in order to avoid confusion with the present invention.
[0018] The lentivirus of this invention is a BaEVless lentivirus containing the green fluorescent protein (GFP) gene for labeling, and its functional titer can be detected by flow cytometry. The CAS Registry Numbers for Poly-L-Lysine, Quercetin, Heparin, Trehalose, and Hyaluronic Acid are: 25988-63-0, 117-39-5, 9041-08-1, 99-20-7, and 9004-61-9, respectively. Virus packaging was performed in 10cm cell culture dishes; virus titer was detected using 293T cells; cell plating was performed in 24-well plates, with 2.2 × 10⁻⁶ cells per well. 5 cells; The cells used for cell plating were 293T cells; the rehydration solution used in the experiment was DMEM complete medium; for detailed steps of cell plating, please refer to: Cell Plating (Detailed Steps) - Experimental Steps, Operation Methods - Dingxiang Experiment https: / / www.biomart.cn / lab-web / method / 343c382go4ntf.html.
[0019] Unless otherwise specified, all raw material components in the following examples can be purchased commercially, the experimental instruments used are all conventional laboratory instruments, and the performance testing methods are known in the art.
[0020] The implementation method and experimental verification are as follows: Example 1: The following methods can be used to increase lentivirus titers: Step 1: Before plating, evenly spread 0.5 mL / cm² of the solution into two sets of culture dishes. 2 5.0 μg / mL poly-L-lysine and 20.0 μg / mL poly-L-lysine were applied, and the mixture was incubated at 37°C for 30 min. The liquid was then discarded, and the mixture was incubated at 2.2 × 10⁻⁶. 5 cell / cm 2 Cells were seeded at specific densities; the remaining groups were prepared using untreated dishes at 2.2 × 10⁻⁶ cells / mL. 5 cell / cm 2 Cells were seeded at the specified density and cultured in a 37°C incubator.
[0021] Step 2: After 24 hours, perform plasmid transfection. The amount of PEI transfection agent is 3 times the amount of plasmid. The total amount of plasmid used is 0.3 μg / cm³. 2 The plasmids included pCDH-GFP, pLP1, pLP2, and BaEVless in a mass ratio of 6:4:4:1. Step 3: When changing the medium 6 hours after plasmid transfection, add different components to each culture dish to make the final concentrations 40 μM quercetin, 80 μM quercetin, 50 U / mL sodium heparin, 500 U / mL sodium heparin, 10 U / mL hyaluronic acid, 20 U / mL hyaluronic acid, 0.1% trehalose aqueous solution and 1% trehalose aqueous solution respectively. Collect the supernatant 48 hours after plasmid transfection to obtain usable lentivirus.
[0022] The functional titer of the virus in the cells containing the obtained lentivirus was detected by flow cytometry. The experimental results are as follows: Figure 1 As shown, the groups with added quercetin, hyaluronic acid, and poly-L-lysine did not show a significant increase in BaEVless lentivirus titer compared to the control group (P>0.5). A 1% trehalose aqueous solution caused cell suspension and detachment, and while a 0.1% trehalose aqueous solution did not affect cell state, it also failed to increase viral titer. However, 50 U / mL heparin sodium effectively increased BaEVless lentivirus titer (P<0.0001). Therefore, heparin sodium was selected as the preferred compound from poly-L-lysine, quercetin, heparin sodium, trehalose, and hyaluronic acid. Subsequent experiments will further explore the optimization of methods for increasing BaEVless lentivirus titer.
[0023] Example 2: The following methods can be used to increase lentivirus titers: Step 1: According to 2.2×10 5 cell / cm 2 Cells were seeded at the specified density and cultured in a 37°C incubator. Step 2: After 24 hours, perform plasmid transfection. The amount of PEI transfection agent is 3 times the amount of plasmid. The total amount of plasmid used is 0.3 μg / cm³. 2 The plasmids included pCDH-GFP, pLP1, pLP2, and BaEVless in a mass ratio of 6:4:4:1. Step 3: When changing the medium 6 hours after plasmid transfection, add heparin sodium to each culture dish to make the final concentrations 0 U / mL, 10 U / mL, 25 U / mL, 50 U / mL, 75 U / mL, and 100 U / mL respectively. Collect the supernatant 48 hours and 72 hours after plasmid transfection to obtain usable lentivirus.
[0024] The functional titer of the virus in the cells containing the obtained lentivirus was detected by flow cytometry. The experimental results are as follows: Figure 2 , Figure 3 As shown, the results at 48h harvest are as follows: Figure 2As shown, the 25 U / mL titer increased by 469.1% compared to the 0 U / mL group, similar to the increase in titer of the 10 U / mL group, and by 43.34% compared to the 50 U / mL group, 48.53% compared to the 75 U / mL group, and 219.64% compared to the 100 U / mL group. At 72 hours of harvest, the results were as follows... Figure 3 As shown, the 10 U / mL titer increased by 665.6% compared to the 0 U / mL group, 17.5% compared to the 25 U / mL group, 77.1% compared to the 50 U / mL group, 127.8% compared to the 75 U / mL group, and 329.2% compared to the 100 U / mL group. For this invention, the preferred dosage of heparin sodium is 10 U / mL-25 U / mL, and the optimal dosage is 10 U / mL.
[0025] Example 3: Example 2 was repeated, and the functional titer of the virus in the cells containing the obtained lentivirus was detected by flow cytometry. The virus harvesting time was compared, and the experimental results are as follows: Figure 4 As shown, the titer at 48 hours is generally higher than that at 72 hours, and the best harvest time is 48 hours.
[0026] Example 4: Example 2 was repeated, and the cells containing the obtained lentivirus were photographed and their fluorescence intensity was detected to verify the effect of the method of the present invention on reducing syncytial apoptosis in producing cells; the experimental results are as follows. Figure 5 As shown, compared with the group without heparin sodium, the cells in the heparin sodium group showed increased GFP fluorescence, reduced cell fusion, and reduced syncytial shedding; the addition of heparin sodium can significantly reduce the formation of syncytial cells in 293T cells, reduce cell shedding, and thus increase lentivirus yield.
[0027] Example 5: The following methods can be used to increase lentivirus titers: Step 1: According to 2.2×10 5 cell / cm 2 Cells were seeded at the specified density and cultured in a 37°C incubator. Step 2: After 24 hours, perform plasmid transfection. The amount of PEI transfection agent is 3 times the amount of plasmid. The total amount of plasmid used is 0.3 μg / cm³. 2 The plasmids included pCDH-GFP, pLP1, pLP2, and BaEVless in a mass ratio of 6:4:4:1. Step 3: When changing the medium 6 hours after plasmid transfection, add heparin sodium to each culture dish to make the final concentrations 0 U / mL, 10 U / mL, and 25 U / mL respectively. Take the supernatant 48 hours after plasmid transfection to obtain usable lentivirus.
[0028] The proportion of GFP-positive cells in the cells containing the obtained lentivirus was detected by flow cytometry to verify that heparin sodium inhibits viral infection of producing cells and reduces the re-entry of BaEVless viral particles into 293T cells; the experimental results are as follows. Figure 6 As shown, the GFP positivity rate in the 10 U / mL and 25 U / mL heparin sodium treatment groups was significantly lower than that in the control group (P < 0.005). This indicates that heparin sodium can effectively inhibit the re-infection of 293T cells by BaEVless-GFP lentivirus. In the process of producing BaEVless lentivirus using 293T cells in this invention, the addition of heparin sodium can prevent newly generated lentivirus particles from re-infecting the production cells, thereby increasing the lentivirus titer.
[0029] Example 6: The following methods can be used to increase lentivirus titers: Step 1: According to 2.2×10 5 cell / cm 2 Cells were seeded at the specified density and cultured in a 37°C incubator. Step 2: After 24 hours, perform plasmid transfection. The amount of PEI transfection agent is 3 times the amount of plasmid. The total amount of plasmid used is 0.3 μg / cm³. 2 The plasmids included pCDH-GFP, pLP1, pLP2, and BaEVless in a mass ratio of 6:4:4:1. Step 3: When changing the medium 6 hours after plasmid transfection, add heparin sodium to the culture dish to make the final concentration 10 U / mL. 48 hours after plasmid transfection, take the supernatant to obtain usable lentivirus.
[0030] The obtained lentivirus was tested by infecting NK cells and γδT cells, and the positive rate was detected. The results are as follows: Figure 7 and Figure 8 As shown, the infection efficiency can reach over 80%, proving that BaEVless lentivirus produced with added heparin sodium can efficiently infect NK cells and γδT cells.
[0031] The above description is merely an embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention can have various modifications and variations. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principle of the present invention should be included within the scope of the claims of the present invention.
Claims
1. A method for increasing lentivirus titers, characterized in that, The method for increasing lentivirus titer is as follows: Step 1: Cells are spaced at a rate of (2~2.5) × 10⁻⁶ 5 cell / cm 2 Cells were seeded at the specified density and cultured in a 37°C incubator. Step 2: After 24 hours, perform plasmid transfection. The amount of PEI transfection agent is 3 times the amount of plasmid. The total amount of plasmid used is 0.22~0.35 μg / cm³. 2 The plasmids included pCDH-GFP, pLP1, pLP2, and BaEVless in a mass ratio of 6:4:4:
1. Step 3: When changing the medium 6 hours after plasmid transfection, add heparin sodium to the culture dish to make the final concentration of the medium 10~75U / mL. After 48~72 hours after plasmid transfection, take the supernatant to obtain usable lentivirus.
2. The method for increasing lentivirus titer according to claim 1, characterized in that, The cell density in step one is 2.2 × 10⁻⁶. 5 cell / cm 2 .
3. The method for increasing lentivirus titer according to claim 1, characterized in that, The total dosage in step two is 0.3 μg / cm³. 2 .
4. The method for increasing lentivirus titer according to claim 1, characterized in that, In step three, the heparin sodium concentration is 10-25 U / mL.
5. The method for increasing lentivirus titer according to claim 1, characterized in that, Step 3: The heparin sodium concentration is 10 U / mL.
6. The method for increasing lentivirus titer according to claim 1, characterized in that, Step 3: Collect the supernatant 48 hours after plasmid transfection.