Composition for improving lentivirus packaging titer, lentivirus packaging method and application thereof

Abstract

The invention provides a composition for improving lentivirus packaging titer, a lentivirus packaging method and application thereof, and belongs to the technical field of lentivirus packaging. The invention discloses a composition for improving lentivirus packaging titer. The composition comprises a three-plasmid system, a transfection reagent, a host cell, a transfection auxiliary reagent and a concentration reagent, the three-plasmid system comprises a plasmid for coding envelope protein, a plasmid for coding gag and pol and a shuttle plasmid containing a target gene in a mass ratio of (1.8-10.2): (1.8-10.2): (0.8-1.2); wherein the transfection reagent comprises PEI MAX; the host cells comprise suspension culture cells; the transfection auxiliary reagent comprises a supplement (Supplement) and an enhancer (Enhancer); the reagent for concentration comprises PEG (Polyethylene Glycol) 8000. The composition provided by the invention can be packaged to obtain a high-titer lentivirus, is suitable for in-vitro cell gene modification, and especially can efficiently infect HSPC.

Description

Technical Field

[0001] The present invention belongs to the technical field of lentivirus packaging, and in particular relates to a composition for improving lentivirus packaging titer, a lentivirus packaging method and applications thereof. Background Art

[0002] Hematopoietic stem and progenitor cells (HSPCs) can self-renew and differentiate into all blood cell lineages. They can produce a variety of blood / immune cells in vitro, including bone marrow cells and immune cells such as NK, B and T cells, through the regulation of certain factors. It is an important stage to induce iPSC to differentiate into NK cells / T cells. Genetic manipulation of HSPCs helps to deeply understand the biological mechanisms of the hematopoietic system and develop therapeutic strategies for blood system diseases.

[0003] Lentivirus has become an important tool for HSPC gene delivery due to its ability to efficiently integrate into the host genome and infect dividing and non-dividing cells. Currently, most lentiviral packaging systems used for HSPC gene delivery are three-plasmid systems, which are plasmids encoding envelope protein genes, plasmids encoding gag, pol, rev and other protein genes, and shuttle plasmids containing target genes, which are co-transfected into 293T cells to obtain recombinant lentivirus. However, the current lentiviral packaging system and infection method face problems such as low virus titer and poor infection efficiency, which restrict the smooth progress of subsequent experiments. Summary of the invention

[0004] In view of this, the object of the present invention is to provide a composition for improving the packaging titer of lentivirus, which can greatly improve the titer of packaged lentivirus by optimizing the type of transfection reagent suitable for lentivirus and the reagent combination that enhances the transfection efficiency.

[0005] The present invention provides a composition for improving the titer of lentivirus packaging, comprising a three-plasmid system, a transfection reagent, a host cell, a transfection auxiliary reagent and a concentration reagent;

[0006] The three-plasmid system includes a plasmid encoding an envelope protein, a plasmid encoding gag and pol, and a shuttle plasmid containing a target gene; the mass ratio of the plasmid encoding an envelope protein, the plasmid encoding gag and pol, and the shuttle plasmid containing a target gene is (1.8-10.2): (1.8-10.2): (0.8-1.2);

[0007] The transfection reagent includes PEI and / or PEI MAX;

[0008] The host cells include suspension culture cells and / or adherent culture cells;

[0009] The transfection auxiliary reagents include supplements and enhancers;

[0010] The concentration reagents include PEG8000 and / or 50KDa ultrafiltration tube.

[0011] Preferably, the mass ratio of the plasmid encoding the envelope protein, the plasmid encoding gag and pol, and the shuttle plasmid containing the target gene is (1.8-2.2): (1.8-2.2): (0.8-1.2).

[0012] Preferably, the suspension culture cells include LV293F cells; and the adherent culture cells include HEK293T cells.

[0013] The present invention provides a packaging method for improving the titer of lentivirus based on the composition, comprising the following steps:

[0014] Mixing the three plasmid systems in the composition to obtain a plasmid system;

[0015] The transfection reagent is diluted and incubated for 1 to 5 minutes, then mixed with the plasmid system and allowed to stand before being added to the host cells for transfection. Supplements are added at the same time. After culturing for 6 to 12 hours, an enhancer is added to continue culturing, and the lentivirus is concentrated and collected.

[0016] Preferably, during the transfection, the working concentration of the transfection reagent is 0.2-1%;

[0017] The working concentration of the supplement is 3% to 8%;

[0018] The working concentration of the enhancer is 3% to 6%.

[0019] Preferably, when the concentration reagent is PEG8000, the mass percentage of PEG8000 is 8% to 12%.

[0020] Preferably, when the host cell is a LV293F cell, the culture and the continued culture are both shaking cultures; the rotation speed of the shaking culture is 120-150 rpm.

[0021] The present invention provides application of the composition or the packaging method in preparing hematopoietic stem cells infected by lentivirus.

[0022] The present invention provides a method for infecting hematopoietic stem cells with a lentivirus in vitro, comprising the following steps:

[0023] The hematopoietic stem cells are inoculated on a cell culture plate coated with human fibronectin, and a lentiviral infection enhancer Lentiboost is added at a final concentration of 0.1 to 1 mg / mL and then cultured to obtain cells to be infected;

[0024] The cells to be infected are infected with the lentivirus obtained by the packaging method, and the infection system contains culture after centrifugation or non-centrifugation to obtain hematopoietic stem cells infected with the lentivirus.

[0025] Preferably, the coating concentration of the human fibronectin is 10-100 μg / mL.

[0026] The present invention provides a composition for improving the titer of lentivirus packaging, comprising a three-plasmid system, a transfection reagent, a host cell, a transfection auxiliary reagent and a concentration reagent; the three-plasmid system comprises a plasmid encoding an envelope protein, a plasmid encoding gag and pol, and a shuttle plasmid containing a target gene; the mass ratio of the plasmid encoding the envelope protein, the plasmid encoding gag and pol, and the shuttle plasmid containing the target gene is (1.8-10.2): (1.8-10.2): (0.8-1.2); the transfection reagent comprises PEI and / or PEI MAX; the host cell comprises a suspension culture cell and / or an adherent culture cell; the transfection auxiliary reagent comprises a supplement and an enhancer; the concentration reagent comprises PEG8000 and / or a 50KDa ultrafiltration tube. The composition provided by the present invention can prepare a high-titer lentivirus, and the lentivirus titer reaches 1.66E+09TU / mL. The composition provided by the present invention provides a basis for efficient lentivirus infection of hematopoietic stem cells (HSPC).

[0027] The composition provided by the present invention specifically optimizes the transfection ratio of the three-plasmid system, and at the same time optimizes the types of transfection reagents, the types of concentration reagents, and the culture types of host cells. Experiments show that PEI MAX can more effectively improve the transfection efficiency than PEI, thereby improving the lentivirus packaging titer; at the same time, the results of the three-plasmid transfection ratio optimization experiment show that the three-plasmid system transfects host cells according to the transfection ratio of 2:2:1, which can improve the transfection efficiency more than the 2:1:1 and 10:10:1 treatment groups; at the same time, the results of the optimization of the reagent for virus concentration show that PEG8000 concentration has a more obvious advantage than 50KDa ultrafiltration tube concentration, and the concentration effect is improved by more than 4 times. In addition, the present invention also uses LV293F cultured in suspension and HEK293T cultured in adherent culture as host cells for lentivirus packaging. The results show that LV293F cultured in suspension not only improves the lentivirus packaging titer, greatly improves the packaging efficiency, but also improves the infection activity of the packaged virus. The GFP positive rate of the lentivirus obtained by culturing the suspension system is nearly 4 times that of the adherent system. It can be seen that the composition provided by the present invention can improve the efficiency of lentivirus on hematopoietic stem cells and provide a basis for the genetic modification of HSPCs. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 The shuttle plasmid pCDH-MSCV-MCS-EF1α-CopGFP-T2A-Puro is shown;

[0029] Figure 2 is the map of the envelope plasmid PMD2.G;

[0030] Figure 3 The map of packaging plasmid psPAX2;

[0031] Figure 4 GFP fluorescence images of HEK293T cells re-infected with lentivirus after transfection of cells with different ratios of shuttle plasmid to packaging plasmid

[0032] Figure 5 Comparison of lentiviral titers obtained for suspension system and adherent system

[0033] Figure 6 GFP fluorescence image (a) and GFP positive ratio detection flow cytometry image (b) of HEK293T cells infected with lentivirus obtained by suspension system and adherent system packaging;

[0034] Figure 7 The flow cytometry diagrams are shown for detecting the GFP positive ratio after HSPCs were infected with different titers of lentivirus, where a is the 5MOI treatment group; b is the 10MOI treatment group; c is the 20MOI treatment group; and d is the 40MOI treatment group. DETAILED DESCRIPTION

[0035] The invention provides a composition for improving the titer of lentivirus packaging, comprising a three-plasmid system, a transfection reagent, a host cell, a transfection auxiliary reagent and a concentration reagent.

[0036] In the present invention, the three-plasmid system in the composition includes a plasmid encoding an envelope protein, a plasmid encoding gag and pol, and a shuttle plasmid containing a target gene. In the embodiment of the present invention, the shuttle plasmid containing a target gene is pCDH-MSCV-MCS-EF1α-CopGFP-T2A-Puro. GFP is used as a representative of the target gene to illustrate the lentiviral packaging effect. pCDH-MSCV-MCS-EF1α-CopGFP-T2A-Puro, spectrum see Figure 1, is an HIV-based dual-promoter vector that can efficiently transfect cells and construct stable cell lines. The vector uses the MSCV and EF1α promoters to drive high and medium levels of expression of target genes or reporter genes. The EF1α promoter performs better than the CMV promoter in hematopoietic cell lines. The MSCV promoter combined with the U3 segment of the HIV LTR can significantly increase the transgene expression level of CD34+ hematopoietic cells and reduce transcriptional silencing in embryonic stem cells. By fusing the HIV LTR to the CpG-deficient version of the MSCV LTR, the vector can achieve efficient expression of the target gene and the reporter gene after genomic integration. The 2A peptide element ensures that both proteins are fully expressed under the drive of a single promoter, avoiding the problems of promoter interference and expression imbalance. The sequence of the envelope plasmid PMD2.G is as shown in Figure 2 The spectrum of the gag-pol packaging plasmid psPAX2 is shown in Figure 3 The present invention has no special limitation on the preparation method of the three-plasmid system, and the construction method of the three-plasmid system known in the art can be used.

[0037] In the present invention, the mass ratio of the plasmid encoding the envelope protein, the plasmid encoding gag and pol, and the shuttle plasmid containing the target gene is (1.8-10.2): (1.8-10.2): (0.8-1.2). In order to optimize the transfection efficiency, in one embodiment of the present invention, the plasmid encoding the envelope protein, the plasmid encoding gag and pol, and the shuttle plasmid containing the target gene are transfected at a ratio of 2:1:1, 2:2:1, and 10:10:1, respectively. The results show that the GFP-positive ratio of cells in the 2:2:1 treatment group is the highest, followed by 10:10:1, and the 2:1:1 treatment group has the worst transfection effect.

[0038] In the present invention, the transfection reagent in the composition includes PEI and / or PEI MAX, preferably PEI MAX. In one embodiment of the present invention, PEI and PEI MAX were used as transfection reagents for lentivirus packaging, and the results showed that the virus titer of the group transfected with PEIMAX was higher, indicating that the transfection reagent PEI MAX is more advantageous than PEI in producing lentivirus.

[0039] In the present invention, the host cells in the composition include suspension culture cells and / or adherent culture cells, preferably suspension culture cells. The suspension culture cells preferably include LV293F cells; the adherent culture cells preferably include HEK293T cells. In the embodiment of the present invention, lentivirus packaging was performed using the LV293F cell suspension culture system and the HEK293T cell adherent culture system, respectively. The results showed that the lentivirus titer obtained by suspension culture was 21.6 times higher than that obtained by adherent culture. This shows that the suspension system significantly increases the lentivirus titer compared with the adherent system.

[0040] In the present invention, the concentration reagent in the composition includes PEG8000 and / or a 50KDa ultrafiltration tube, preferably PEG8000. In an embodiment of the present invention, in order to further optimize the virus concentration method, PEG8000 and 50KDa ultrafiltration tubes were used for virus concentration, and the results showed that after PEG8000 concentration, the lentivirus titer could reach 1.62E+09TU / mL, and the virus was concentrated by about 54 times. Using a 50KDa ultrafiltration tube for concentration, the lentivirus titer could reach 3.83E+08TU / mL, and the virus was concentrated by about 13.5 times. Therefore, the concentration effect of PEG8000 for the lentivirus obtained in the suspension system is significantly better than that of the 50KD ultrafiltration tube.

[0041] In the present invention, the transfection auxiliary reagent in the composition includes a supplement and an enhancer, both of which have the function of enhancing the transfection efficiency.

[0042] The present invention provides a packaging method for improving the titer of lentivirus based on the composition, comprising the following steps:

[0043] Mixing the three plasmid systems in the composition to obtain a plasmid system;

[0044] The transfection reagent is diluted and incubated for 1 to 5 minutes, then mixed with the plasmid system and allowed to stand before being added to the host cells for transfection. Supplements are added at the same time. After culturing for 6 to 12 hours, an enhancer is added to continue culturing, and the lentivirus is concentrated and collected.

[0045] In the present invention, the solvent for mixing the three-plasmid system is OPTI-MEM. The volume ratio of the total mass of the three-plasmid system to the transfection reagent is preferably 1:(2.4-4). The mixing and standing time is preferably 12-17 min, and can be 15 min. During the transfection, the working concentration of the transfection reagent is 0.2-1%, preferably 0.6%. The present invention has no special restrictions on the transfection method, and the transfection steps known in the art can be used. The working mass concentration of the supplement is 3%-8%, preferably 5%. The working mass concentration of the enhancer is 3-6%, preferably 4%. When the concentration reagent is PEG8000, the mass percentage of PEG8000 is preferably 8%-12%. When the host cell is LV293F cell, the culture and continued culture are both shaking culture; the rotation speed of the shaking culture is preferably 120-150 rpm, and can be 130-140 rpm.

[0046] The present invention provides application of the composition or the packaging method in preparing hematopoietic stem cells infected by lentivirus.

[0047] In the present invention, the hematopoietic stem cells preferably include CD34+ cells differentiated from umbilical cord blood, bone marrow, peripheral blood or PSC (pluripotent stem cells).

[0048] The present invention provides a method for infecting hematopoietic stem cells with a lentivirus in vitro, comprising the following steps:

[0049] The hematopoietic stem cells are inoculated on a cell culture plate coated with human fibronectin, and after culture, a lentiviral infection enhancer with a final concentration of 0.1 to 1 mg / mL is added to obtain cells to be infected;

[0050] The cells to be infected are infected with the lentivirus obtained by the packaging method, and the infection system contains hematopoietic stem cells infected with the lentivirus after culturing with or without centrifugation.

[0051] The invention inoculates hematopoietic stem cells on a cell culture plate coated with human fibronectin, adds a lentiviral infection enhancer with a final concentration of 0.1-1 mg / mL, and then cultures the cells to be infected.

[0052] In the present invention, the method for preparing a cell culture plate coated with human fibronectin preferably comprises the following steps:

[0053] Dilute human fibronectin to the coating concentration with sterile PBS to obtain human fibronectin coating solution;

[0054] Add the human fibronectin coating solution to each well in the cell culture plate, seal the plate with a sealing film, and let it stand to obtain a cell culture plate coated with human fibronectin. The coating concentration of the human fibronectin preferably includes 10 to 100 μg / mL, which can be 20 μg / mL, 30 μg / mL, 40 μg / mL, 50 μg / mL, 60 μg / mL, 70 μg / mL, 80 μg / mL, and 90 μg / mL. The standing condition is preferably placed at 4°C overnight or incubated at 37°C for 2 hours. The inoculation concentration of the hematopoietic stem cells is 1E+05 cells / well. The function of the human fibronectin is to promote lentiviral infection.

[0055] After obtaining the cells to be infected, the present invention infects the cells to be infected with the lentivirus packaged by the packaging method, and the infection system contains hematopoietic stem cells infected with the lentivirus after being cultured with or without centrifugation to obtain

[0056] In the present invention, the multiplicity of infection of the lentivirus is 5 to 30 MOI, preferably 10 to 20 MOI. Centrifugation or non-centrifugation of the infection system after inoculation of the lentivirus has little effect on the infection efficiency.

[0057] The experiments of the present invention show that the lentivirus prepared by the packaging method of the present invention can not only improve the infection efficiency of hematopoietic stem cells, but also has little effect on the cell viability of hematopoietic stem cells.

[0058] The following is a detailed description of a composition for improving lentivirus packaging titer, a lentivirus packaging method and applications provided by the present invention in conjunction with the examples, but they should not be construed as limiting the scope of protection of the present invention.

[0059] The experimental methods in the following examples, unless otherwise specified, all adopt conventional techniques. The experimental materials used in the examples, unless otherwise specified, were purchased from conventional biochemical reagent companies.

[0060] LV-MAX TM Lentiviral production system (Gibico, 2822826 / 2853684); Thermo Nalgene TM PETG disposable sterile flat-bottom conical flask: sterile-250mL (ThermoFisher, 1376037); Thermo Nalgene TM PETG disposable sterile flat-bottom conical flask: sterile-125 mL (ThermoFisher, 1353699); polyethylene glycol 8000 (PEG8000) (Sigma, P2139); (TAKARA, T100AC); PEI MAX (Polysciences, 24765-1); PEI (MCE, HY-K2014); Lentiboost (Revvity, LentiBOOST-P).

[0061] Example 1

[0062] Transfection reagent optimization experiment

[0063] 1) Cell recovery: Take out the frozen HEK293T cells from liquid nitrogen and thaw them quickly in a 37°C water bath.

[0064] 2) Cell culture

[0065] a. Take 20 μL of the resuscitated cell suspension and count the cells. The total number of thawed cells is about 5E+06.

[0066] b. Add 10 mL of fresh 10% DMEM medium to a 10 cm culture dish and inoculate all the cell suspension into the dish.

[0067] c. Place the culture dish in the incubator, mix it in a 'M' pattern, and then culture it.

[0068] d. After 2 days of adherent culture, HEK293T cells were digested with trypsin and inoculated into three 10 cm culture dishes (10 mL 10% DMEM medium) at a cell density of 0.2-0.4E+06 cells / mL. After 2 generations of stable culture in an incubator, the cells were plated for virus packaging.

[0069] e. After 2 days of adherent culture, HEK293T cells were digested with trypsin and seeded into 4 10 cm culture dishes (10 mL 6% DMEM medium) at a cell density of 0.6-0.8E+06 cells / mL. After culturing in an incubator for 24 hours, transfection was performed.

[0070] 3) Transfection

[0071] HEK293T cells were plated in 10 cm culture dishes and transfected when the density reached about 80%. Two transfection reagents, PEI and PEI MAX, were used for transfection. The working concentration of the two transfection reagents was 1 mg / mL, and the transfection process was performed strictly according to the manufacturer's instructions. The three plasmids used included the shuttle plasmid pCDH-MSCV-MCS-EF1α-CopGFP-T2A-Puro, the auxiliary plasmid psPAX2 and pMD2.G, and the transfection mass ratio was set to 2:2:1. In each 10 cm culture dish, the total transfection amount of plasmid was 25 μg. After 48 hours of transfection, the lentiviral supernatant was collected, filtered with a 0.45 μm filter membrane, and concentrated with PEG8000. After centrifugation of the precipitated lentiviral precipitate, it was resuspended in 200 μL PBS to obtain concentrated lentivirus.

[0072] Lentiviral titer detection method

[0073] 1) Cell culture and counting: HEK293T cells were plated in a 24-well plate at 1E+05 cells / well and cultured for 12-24 hours before lentiviral infection. Before infection, cells in one well were digested and counted, which was the initial cell number.

[0074] 2) Virus dilution and infection: The concentrated virus solution was diluted 10 times with PBS, and then diluted 3 times to obtain 10-fold, 30-fold, 90-fold, 270-fold, and 810-fold diluted virus solutions. 10 μL of virus solution with different dilutions was added to each cell well. Lentiboost was added at a final concentration of 1 mg / mL to promote infection.

[0075] 3) Virus infection and GFP detection: 2 days after lentiviral infection, all well cells were collected, GFP expression was detected by flow cytometry, and the data of the wells with GFP positive rate of 15-35% were recorded.

[0076] 4) Titer calculation: Based on the collected GFP positive rate data, the lentiviral titer was calculated according to the following formula I:

[0077] Titer (TU / mL) = (initial cell number × GFP positive rate % × dilution factor) × 10 3 / 10Formula I.

[0078] The titer test results are shown in Table 1. The virus titer of the group transfected with PEI MAX is higher, which suggests that this transfection reagent has more advantages in producing lentivirus.

[0079] Table 1 Comparison of lentiviral titers obtained with different transfection reagents

[0080] Transfection reagents PEI PEIMAX EF1α-GFP virus titer TU / mL 5.29E+07 8.74E+07

[0081] Example 2

[0082] Optimization of the transfection ratio of three plasmids:

[0083] The transfection mass ratios of the three-plasmid shuttle plasmid (pCDH-MSCV-MCS-EF1α-CopGFP-T2A-Puro, hereinafter referred to as: EF1α-GFP), psPAX2 and pMD2.G used to transfect HEK293T cells were set to 2:1:1, 2:2:1 and 10:10:1, respectively, and the total amount of plasmid transfection was fixed at 2 μg. HEK293T cells were plated on a 6-well plate and transfected when their density reached about 80%. During transfection, PEI MAX was used to strictly follow the manufacturer's instructions for the transfection process. After 48 hours of transfection, the lentiviral supernatant was collected, filtered through a 0.45 μm filter membrane, and 200 μL of the virus solution was used to infect HEK293T cells pre-plated on a 12-well plate, and polybrene at a final concentration of 10 μg / mL was added to promote infection. After 48 hours of infection, the GFP fluorescence expression in each well was observed under a fluorescence microscope.

[0084] The observation results show that Figure 4 As shown in the figure, the group transfected with a ratio of 2:2:1 had the highest GFP positive ratio, which suggests that this plasmid transfection ratio has obvious advantages in producing efficient and active lentivirus. Based on this result, this advantageous three-plasmid transfection ratio can be applied to the lentivirus packaging process of the suspension system.

[0085] Example 3

[0086] Optimization of incubation time of transfection reagent after dilution

[0087] In order to further optimize the transfection conditions, the effect of the incubation time of the transfection reagent after dilution before mixing with the plasmid on the virus yield was explored. Transfection was performed according to the method of Example 2, and the lentivirus titers obtained under different transfection reagent incubation times (1-5 min, 5-10 min) were compared.

[0088] The results are shown in Table 2 below:

[0089] Table 2 Comparison of lentiviral titers obtained after different dilutions and incubation times of transfection reagents

[0090]

[0091] It can be seen that when the incubation time of the diluted transfection reagent is less than 5 minutes, the average titer of the lentivirus can reach 1.154E+09TU / mL. However, if the incubation time exceeds 5 minutes, the virus titer is significantly reduced to about 6.935E+07TU / mL. The virus titer of the former is about 16.6 times that of the latter. This phenomenon may be caused by the poor stability of the diluted transfection reagent. Therefore, during the transfection process, the incubation time of the diluted transfection reagent must be strictly controlled to ensure that it does not exceed 5 minutes to maintain a high virus titer.

[0092] Example 4

[0093] Virus concentration method optimization

[0094] Comparison of two commonly used virus concentration methods: PEG8000 concentration and ultrafiltration tube concentration. The specific operation methods are as follows:

[0095] 1) Ultrafiltration tube concentration

[0096] a. Use sterile ddH 2 O rinse the ultrafiltration tube, transfer 15 mL of virus solution to the upper layer of the ultrafiltration tube, and centrifuge at 3000 g for 30 min.

[0097] b. During the centrifugation process, the ultrafiltration tube was removed for observation. When the volume of the lower layer of virus liquid was about 7.5 mL, the lower layer of liquid was discarded, and 7.5 mL of 250 mM high salt buffer (lentivirus stock solution) was added to the upper layer, and centrifugation was continued for 30 min.

[0098] c. When half of the upper layer of liquid is filtered, discard the lower layer of liquid, transfer the unconcentrated lentiviral solution to the upper layer, and centrifuge again. Repeat this step until all the lentiviral solution is ultrafiltered.

[0099] d. After concentration, the volume of the lentivirus is about 500 μL, which is dispensed into 1.5 mL sterile EP tubes, 100 μL per tube, and stored at -80°C.

[0100] 2) PEG8000 Concentration

[0101] a. Add 5×PEG8000 lentivirus concentrate (10% PEG8000) to the filtered lentivirus solution at a ratio of 1:4, invert and mix 8 to 10 times, and place at 4°C overnight, inverting and mixing from time to time.

[0102] b. Take out the lentiviral concentrate, centrifuge at 3000g for 30 min at 4°C, discard the supernatant, resuspend the virus precipitate with 300μL PBS, divide into 1.5mL sterile EP tubes, 50μL per tube, and store at -80°C.

[0103] During the ultrafiltration process, due to factors such as culture medium protein and high-titer lentivirus, a large number of protein clumps will be generated, and they cannot be dissolved using high-salt solutions. For this reason, the clumps are removed by flash centrifugation, and the virus supernatant is taken for subsequent titer determination and infection testing. The experimental results show that PEG8000 concentration is significantly superior to ultrafiltration concentration in terms of titer and homogeneity of virus liquid.

[0104] Table 3 Comparison of lentiviral titers obtained by different concentration methods

[0105]

[0106] After PEG8000 concentration, the lentivirus titer can reach 1.62E+09TU / mL, and the virus is concentrated by about 54 times. However, when concentrated using a 50KDa ultrafiltration tube, the lentivirus titer is only 3.83E+08TU / mL, and the virus is concentrated by about 13.5 times. Therefore, the concentration effect of PEG8000 for lentivirus obtained in the suspension system is significantly better than that of a 50KDa ultrafiltration tube.

[0107] Example 5

[0108] Optimization of transfection efficiency when packaging lentivirus based on suspension LV293F cells

[0109] 1) Cell recovery: Take out the frozen LV293F cells from liquid nitrogen and thaw quickly in a 37°C water bath.

[0110] 2) Cell culture

[0111] a. Take 20 μL of thawed cell suspension for cell counting. The total number of cells is about 1E+07, and the cell viability is above 95%.

[0112] b. Add 30 mL of fresh LV-MAX medium to a 125 mL culture flask and inoculate all the cell suspension into the culture flask.

[0113] c. Place the culture flask at 37°C and 8% CO 2 The culture was shaken in an incubator at a rate of 125 rpm (revolutions per minute).

[0114] d. After 3-4 days, the suspended cells were inoculated into a 125 mL culture flask (30 mL LV-MAX medium) at a cell density of 0.3-0.5E+06 cells / mL and placed in an incubator for shaking culture.

[0115] e. After 3-4 days, the suspended cells were inoculated into a 250 mL culture flask (60 mL LV-MAX medium) at a cell density of 0.3-0.5E+06 cells / mL and placed in an incubator for shaking culture.

[0116] f. On the 3rd to 4th day after culture, when the cell density reaches 3.5-5.5E+06 cells / mL, the cells are subcultured at a cell density of 3.5E+06 cells / mL and cultured in an incubator with shaking for 24 hours.

[0117] g. On the next day, the cell density was adjusted to 4.7E+06 cells / mL and the cells were dispensed into 125 mL culture flasks (30 mL / flask), and 1.5 mL of supplement (5% of the total volume) was added to each flask.

[0118] 3) Transfection

[0119] Preparation of aA solution: In 1.5 mL OPTI-MEM medium, lentiviral packaging shuttle plasmid (EF1α-GFP), psPAX2, and pMD2.G were mixed in a ratio of 2:2:1, with a total plasmid amount of 75 μg.

[0120] Preparation of bB solution: Take another 1.5mL OPTI-MEM medium, add 180μL supplement solution, and gently invert to mix. The incubation time of B solution should not exceed 5min.

[0121] c. Slowly add solution B to solution A, invert and mix, and let stand at room temperature for 10 minutes. Then, gently add the transfection complex to the cell suspension in 2)g. and place at 37°C and 8% CO 2 Incubate in an incubator with shaking at 125 rpm.

[0122] d. After 6-12 hours, add 1.2 mL of enhancer (4% of the total volume) to each bottle and continue shaking culture.

[0123] 4) Harvesting the virus

[0124] 48 h after transfection, the cell suspension was transferred to a 50 mL centrifuge tube and centrifuged at 400 g for 5 min. The viral supernatant was harvested and the cell debris was removed using a 0.45 μm filter. The filtrate was then concentrated using PEG8000.

[0125] Example 6

[0126] A method for infecting HSPC with lentivirus

[0127] 1) Coating of lentiviral infection detection plate:

[0128] a. Prepare Retronectin coating solution: dilute human fibronectin Retronectin with sterile PBS to a final concentration of 20 μg / mL.

[0129] b. Add 20 μg / mL Retronectin coating solution to a 24-well plate at a volume of 300 μL / well. Seal the plate with sealing film and incubate the plate at 4°C overnight or at 37°C for 2 hours.

[0130] c. Before use, remove the plate from 4°C and equilibrate it at room temperature for 30 minutes.

[0131] 2) Lentivirus infection of HSPCs:

[0132] To further optimize the lentiviral infection conditions, the lentivirus obtained by the method of Example 5 was used to infect HSPCs at the same multiplicity of infection (40 MOI), and the infected cell plates were centrifuged at 300 g for 1 h or not. After 24 h, the positive rate of GFP protein was detected by flow cytometry.

[0133] The results are detailed in Table 4 below.

[0134] Table 4 Effects of centrifugation and non-centrifugation on the efficiency of lentivirus infection of HSPCs

[0135] Whether centrifugal Centrifugation Not centrifugal GFP% 84.68 80.54

[0136] The results showed that after centrifugation, the percentage of GFP-positive cells was 84.68%, while without centrifugation, the percentage of GFP-positive cells was 80.54%, and the difference between the two was not significant. Therefore, it is possible to consider infecting HSPCs without centrifugation, which can not only simplify the operation process, but also help to expand the scale of viral infection.

[0137] The specific steps of lentiviral infection of HSPC are as follows:

[0138] a. Hematopoietic stem cells (HSPCs) from different sources were plated at a density of 1E+05 cells / well in a 24-well plate coated with Retronectin, followed by infection.

[0139] b. Add Lentiboost (lentiviral infection enhancer) at a final concentration of 1 mg / mL to each well and shake gently to mix.

[0140] c. Add the corresponding volume of lentivirus solution at the target MOI (multiplicity of infection) and shake gently to mix.

[0141] d. Place the plate in a 37°C incubator and continue culturing for 24 hours. You can then test the infection efficiency or conduct subsequent experiments.

[0142] Example 7

[0143] Effects of suspension packaging system and adherent packaging system on lentiviral titer

[0144] Suspended LV293F lentiviral packaging system: The suspended LV293F cells in the above embodiment were revived and suspended and shaken using LV-MAX medium. The cells in good condition were diluted at a density of 0.5E+06 cells / mL and continued to be shaken for 3-4 days. After that, the cells were further diluted to 3.5E+06 cells / mL, and the suspension and shaking culture was continued for 1 day, and then the virus was packaged. The conditions for virus packaging are: cell density is 4.7E+06 cells / mL, plasmid dosage is 75μg, plasmid ratio is shuttle plasmid (EF1α-GFP): Pol / gag: psPAX2:PMD2.G=2:2:1, and the ratio of transfection reagent to plasmid DNA is 2.4:1 (V / W). After transfection for 6 hours, an enhancer with a final concentration of 4% was added, and the cell culture supernatant was collected 48 hours later, PEG8000 was used to concentrate the lentivirus, and the lentiviral titer was determined.

[0145] HEK293T adherent lentiviral packaging system: HEK293T adherent cells were revived and inoculated in a culture dish containing DMEM medium (supplemented with 10% fetal bovine serum and 1% double antibody) at 37°C and 5% CO 2 After the cells recovered, they were cultured for 2 generations to allow the cells to grow to a good state. The cells were digested with trypsin, and a cell suspension was prepared. The cell suspension was inoculated into three new 10 cm culture dishes at a density of 6E+06 cells / mL and continued to be cultured at 37°C and 5% CO. 2 Culture in an incubator for 24 hours. The cell density should reach about 80%. Ensure that the cells are in the best condition before virus packaging. Use Polysciences PEI MAX for transfection. The specific steps are as follows: First, prepare the required plasmids, including shuttle plasmid (EF1α-GFP), psPAX2 and PMD2.G, and mix them in a ratio of 2:2:1. The total amount of plasmid per 10 cm culture dish is 25 μg. Then, add PEI MAX reagent to the plasmid mixture, and incubate for 15 minutes at a ratio of 4:1 (V / W) to the total amount of PEI MAX and plasmid. Finally, add the transfection complex to the cell culture medium and place it at 37°C and 5% CO 2The cells were cultured in an incubator at 4 °C. After 6 h of transfection, fresh medium containing a final concentration of 4% enhancer was added and continued to be cultured. After 48 h, the cell culture supernatant was collected and filtered using a 0.45 μm filter to remove cell debris and other impurities, and then the filtered supernatant was concentrated with PEG8000 for lentivirus. Finally, the lentivirus titer was determined to evaluate the effect of virus packaging.

[0146] In order to compare the lentiviral packaging effects of the suspension and adherent systems, four lentiviral packagings were performed and the lentiviral titers were tested. The lentiviral titer test results are shown in Table 5.

[0147] Table 5 Comparison of lentiviral titers obtained by suspension system and adherent system

[0148]

[0149] The results showed that the average lentiviral titer obtained in the suspension system was 1.66E+09TU / mL, while the average lentiviral titer obtained in the adherent system was 7.68E+07TU / mL. Figure 5 As shown in the figure, the difference in lentiviral titers obtained by the suspension system and the adherent system is extremely significant. The virus titer obtained by the suspension system is about 21.6 times that of the adherent system, indicating that the suspension system significantly improves the lentiviral titer compared with the adherent system. These experimental data further confirm the significant advantages of the suspension system in improving the efficiency of lentiviral packaging and provide a more efficient method for the production of lentivirus.

[0150] Example 8

[0151] 1 μL of each of the lentiviral solution obtained by the suspension packaging system and the adherent packaging system disclosed in Example 7 was used to infect HEK293T cells. For specific methods, see Example 6. After 24 hours of infection, the expression of GFP protein was observed by fluorescence microscopy to determine the infection efficiency. Subsequently, the HEK293T cells were digested and the proportion of GFP-positive cells was detected by flow cytometry.

[0152] like Figure 6 As shown in a and b, compared with the adherent system, the expression of GFP protein in HEK293T cells was significantly enhanced after the lentivirus obtained by the suspension system infected the cells. The flow cytometry results further showed that the GFP positive rate of the suspension system reached 97.89%, while the GFP positive rate of the adherent system was only 24.75%. This shows that compared with the adherent system, the titer of the lentivirus obtained by the suspension system was significantly improved.

[0153] Example 9

[0154] The suspended LV293F cells in Example 8 above were revived and suspended and shaken in culture using LV-MAX medium. After the cells were in good condition, they were diluted at a density of 0.5E+06cells / mL and continued to be shaken for 3-4 days. Subsequently, the cell density was diluted to 3.5E+06cells / mL, and virus packaging was performed after 1 day of suspension and shaking culture. The virus packaging conditions are: the density of lentivirus packaging cells is 4.7E+06cells / mL, and 5% of supplements are added to the cell suspension. The amount of plasmid is 75μg, the shuttle plasmid (EF1α-GFP): psPAX2:PMD2.G=2:2:1, and the ratio of transfection reagent to plasmid DNA is 2.4:1 (V / W). After 6h of transfection, an enhancer with a final concentration of 4% was added, and the cell culture medium supernatant was collected 48h after transfection, and PEG8000 was used to concentrate the lentivirus, and the lentivirus titer was determined. The titer test results showed that the lentivirus titer was 1.24E+09TU / mL. Figure 7 As shown in a, b, c, and d, HSPCs were infected with purified lentivirus at MOIs of 5, 10, 20, and 40, respectively, and the corresponding GFP positive rates were 8.44%, 18.01%, 38.97%, and 79.81%, respectively. The cell viability before and after lentivirus infection was detected, and the results are shown in Table 6.

[0155] Table 6 HSPC cell viability before and after lentiviral infection

[0156]

[0157] As the MOI increases, the overall cell viability does not change much, which indicates that the suspension lentivirus packaging and concentration system of the present invention has little effect on the viability of HSPCs.

[0158] It can be seen from the results of the above examples that the lentivirus titer obtained by using the suspension lentivirus packaging and concentration system of the present invention is high, reaching 1.24E+09TU / mL. When the virus infects HSPC, the GFP positive rate is high, and the viability of HSPC after virus infection is not significantly affected. These results further confirm the high efficiency of the optimized lentivirus packaging and concentration system and HSPC infection system of the present invention.

[0159] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A composition for improving the titer of lentivirus packaging, characterized in that: It includes a three-plasmid system, a transfection reagent, a host cell, a transfection auxiliary reagent, and a concentration reagent; The three-plasmid system includes a plasmid encoding an envelope protein, a plasmid encoding gag and pol, and a shuttle plasmid containing a target gene; the mass ratio of the plasmid encoding an envelope protein, the plasmid encoding gag and pol, and the shuttle plasmid containing a target gene is (1.8-10.2): (1.8-10.2): (0.8-1.2); The transfection reagent includes PEI and / or PEI MAX; The host cells include suspension culture cells and / or adherent culture cells; The transfection auxiliary reagents include supplements and enhancers; The concentration reagents include PEG8000 and / or 50KD ultrafiltration tube.

2. The composition according to claim 1, characterized in that The mass ratio of the plasmid encoding the envelope protein, the plasmid encoding gag and pol, and the shuttle plasmid containing the target gene is (1.8-2.2): (1.8-2.2): (0.8-1.2).

3. The composition according to claim 1 or 2, characterized in that The suspension culture cells include LV293F cells; the adherent culture cells include HEK293T cells.

4. A packaging method for increasing the titer of a lentivirus based on the composition according to any one of claims 1 to 3, characterized in that: The following steps are involved: Mixing the three plasmid systems in the composition to obtain a plasmid system; The transfection reagent is diluted and incubated for 1 to 5 minutes, then mixed with the plasmid system and allowed to stand before being added to the host cells for transfection. Supplements are added at the same time. After culturing for 6 to 12 hours, an enhancer is added to continue culturing, and the lentivirus is concentrated and collected.

5. The packaging method according to claim 4, characterized in that: During the transfection, the working concentration of the transfection reagent is 0.2-1%; The working concentration of the supplement is 3% to 8%; The working concentration of the enhancer is 3% to 6%.

6. The packaging method according to claim 4, characterized in that: When the concentration reagent is PEG8000, the mass percentage of the PEG8000 is 8% to 12%.

7. The packaging method according to claim 4, characterized in that: When the host cell is LV293F cell, the culture and the continued culture are both shaking culture; the rotation speed of the shaking culture is 120-150 rpm.

8. Use of the composition according to any one of claims 1 to 3 or the packaging method according to any one of claims 4 to 7 in preparing hematopoietic stem cells infected with a lentivirus.

9. A method for infecting hematopoietic stem cells with a lentivirus in vitro, characterized in that: The following steps are involved: The hematopoietic stem cells are inoculated on a cell culture plate coated with human fibronectin, and a lentiviral infection enhancer Lentiboost is added at a final concentration of 0.1 to 1 mg / mL and then cultured to obtain cells to be infected; The cells to be infected are infected with the lentivirus obtained by the packaging method according to any one of claims 4 to 7, and the infection system contains hematopoietic stem cells infected with the lentivirus after culturing with or without centrifugation.

10. The method according to claim 9, characterized in that: The coating concentration of the human fibronectin is 10-100 μg / mL.

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