Lentivirus envelope plasmid combination and application thereof, lentivirus and packaging method thereof, and hematopoietic stem cell transduction method

Abstract

The invention relates to the technical field of stem cells, in particular to a lentivirus envelope plasmid combination and application thereof, a lentivirus and a packaging method thereof and a method for transduction of hematopoietic stem cells. The invention provides an envelope plasmid combination for lentivirus packaging, which is composed of lentivirus packaging plasmids containing VSVG glycoprotein and lentivirus packaging plasmids containing BaEV glycoprotein in a ratio of 3: 7. The invention further provides a method for transduction of the hematopoietic stem cells by the lentivirus, the method is simple and convenient to operate, the long-term dryness of the hematopoietic stem cells in vitro can be maintained, efficient and stable transduction of the hematopoietic stem cells can be realized, and the transduction rate is greater than 90%. The lentivirus transduction method hematopoietic stem cells can be used for hematopoietic stem cell gene therapy hematopoietic system genetic diseases, such as severe combined immunodeficiency, beta-thalassemia and sickle cell disease, Wiskott-Aldrich syndrome and the like, and the application prospect is good.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of stem cell technology, and particularly relates to a lentivirus envelope plasmid combination and application thereof, a lentivirus and a packaging method thereof, and a method for transducing hematopoietic stem cells. BACKGROUND

[0002] Hematopoietic stem cell gene therapy can separate and extract hematopoietic stem cells from the patient's own bone marrow or mobilized peripheral blood, and after in vitro genetic modification (lentivirus, gene editing, etc.) and large-scale expansion, the hematopoietic stem cells are retransplanted into the patient's body, so that the expression of mutant genes returns to normal levels, thereby achieving the purpose of treating diseases. Autologous hematopoietic stem cells after in vitro genetic modification have the advantages of no need for matching, no immune rejection, and one-time treatment for lifelong effectiveness, and show great application potential and value in the treatment of single-gene genetic diseases.

[0003] In the process of clinical application, the low efficiency of lentivirus infection on hematopoietic stem cells often seriously weakens the full play of the in vivo efficacy of the retransplanted hematopoietic stem cells. To solve this problem, researchers often modify hematopoietic stem cells by changing the lentivirus envelope or using gene editing technology to further improve the modification efficiency of hematopoietic stem cells and the stability of gene expression, and enhance the efficacy of single-gene genetic diseases. At present, in the research of HSC cell gene therapy products, commonly used viral vectors include lentivirus and retrovirus vectors, among which lentivirus vectors are widely used due to their better safety and technical maturity.

[0004] The most widely used lentivirus envelope is VSVG envelope, however, due to the undifferentiated state of hematopoietic stem cells, the expression of LDLR, the receptor of VSVG envelope, is low, and the infection efficiency of lentivirus with this envelope on hematopoietic stem cells is very low. In recent years, BaEV envelope lentivirus has good infection efficiency on hematopoietic stem cells, but the packaging of lentivirus is prone to syncytia, and the low packaging efficiency limits its application.

[0005] Researchers also improve the transduction efficiency of hematopoietic stem cells by optimizing the culture conditions of hematopoietic stem cell pre-treatment. Studies have shown that specific cytokine combinations activate the cell cycle progression of hematopoietic stem cells (HSCs), such as SCF (stem cell factor) which binds to the c-Kit receptor to promote the expression of lentivirus helper receptors (such as CXCR4) on the surface of HSCs, increasing the density of virus binding sites by 2-3 times; TPO (thrombopoietin) drives HSCs in the G0 phase into the G1 / S phase, removes the barrier of nuclear membrane to virus DNA into the nucleus, and increases the transduction efficiency by 40%-60%; FLT3L (FMS-like tyrosine kinase 3 ligand) inhibits the caspase-3 pathway, reducing the apoptosis rate of HSCs cultured in vitro from 25% to <10%. However, excessive stimulation of cytokines can lead to uncontrollable differentiation risk of hematopoietic stem cells, increased DNA replication pressure due to cell cycle activation, and impaired genomic stability.

[0006] In addition to the above methods, researchers also improve the transduction efficiency of hematopoietic stem cells by optimizing the composition of lentivirus infection medium. By adding virus transduction enhancers (TEs) such as Polybrene during transduction, the electrostatic repulsion between virus membrane and cell membrane is neutralized, and the virus particle adsorption efficiency is increased by 5-8 times, effectively overcoming the obstacles of lentivirus transduction. However, there is also dose-dependent cytotoxicity, and excessive use can inhibit the colony-forming ability of hematopoietic stem cells. At the same time, Polybrene can also activate the complement system and induce infusion-related inflammatory reactions, so it is not GMP certified and is only limited to research use.

[0007] Therefore, it is of great significance to develop a method for efficiently and stably transducing hematopoietic stem cells with lentivirus. SUMMARY

[0008] In view of the defects of the prior art, the present application provides a lentivirus envelope plasmid combination and its application, a lentivirus and a packaging method thereof, and a method for transducing hematopoietic stem cells.

[0009] The present application provides a lentivirus packaging envelope plasmid combination, which is composed of a lentivirus packaging plasmid containing VSVG glycoprotein and a lentivirus packaging plasmid containing BaEV glycoprotein, and the mass ratio of the two is 3:7.

[0010] Preferably, the lentivirus packaging plasmid containing VSVG glycoprotein is selected from at least one of pMD2.G plasmid, pLP / VSVG plasmid, and pl_P / VSVG plasmid; and the lentivirus packaging plasmid containing BaEV glycoprotein is selected from at least one of pMD2.BaEV and pCMV-BaEV-Rless.

[0011] The application provides the application of the envelope plasmid combination of any one of the above to lentivirus packaging.

[0012] The application provides a lentivirus packaging method, which is a transduction method of the envelope plasmid of any one of the above.

[0013] Preferably, it is a transduction method of the envelope plasmid of any one of the above, the pMDLg / pRRE plasmid and the pRSV-Rev plasmid as packaging plasmids, the pCDH-EF1 as a carrier plasmid, and the mass ratio of the carrier plasmid, the pMDLg / pRRE plasmid, the pRSV-Rev plasmid and the envelope plasmid is 2.4-3.6:0.8-1.2:0.8-1.2:0.8-1.2.

[0014] Preferably, it comprises: infecting 293T cells with the carrier plasmid, the pMDLg / pRRE plasmid, the pRSV-Rev plasmid and the envelope plasmid.

[0015] The application provides a lentivirus, the envelope glycoprotein of which is a combination of VSVG glycoprotein and BaEV glycoprotein, and the mass ratio of the two is 3:7.

[0016] Preferably, it is prepared by the lentivirus packaging method of any one of the above.

[0017] The application provides a method for transducing hematopoietic stem cells with lentivirus, which comprises the following steps: Step 1, pre-stimulating hematopoietic stem cells in complete culture medium; Step 2, transducing the pre-stimulated hematopoietic stem cells with the lentivirus of claim 7 or 8; Step 3, collecting the transduced hematopoietic stem cells; Step 4, culturing the cells in step 3 in complete culture medium.

[0018] Preferably, the formula of the complete culture medium comprises the following combination of cytokines: 240-360 ng / mL SCF, 80-120 ng / mL TPO, 48-72 ng / mL IL-3, 240-360 ng / mL FLT-L3; And / or, the formula of the complete culture medium further comprises a basic culture medium, and the basic culture medium is selected from one of X-VIVO15 basic culture medium and SFEM II basic culture medium; And / or, the pre-stimulation time in step 1 is 12-48 hours; And / or, the source of the hematopoietic stem cells in step 1 is mobilized peripheral blood; And / or, in step 2, the transduction time is 8-24 hours. And / or, in step 2, the transduction is carried out in the presence of a transduction-promoting agent, and the transduction-promoting agent comprises at least one of Vectofusin-1 and recombinant fibronectin; The pre-coating density of the recombinant fibronectin in the transduction system is 2-10 μg / cm 2 The concentration of Vectofusin-1 in the transduction system is 2-20 μg / mL; And / or, in step 2, the transduction is carried out in the presence of a transduction-promoting agent, and the transduction-promoting agent comprises at least one of Vectofusin-1 and recombinant fibronectin; And / or, in step 4, the time for the expansion culture is 7-10 days.

[0019] The lentivirus packaging is obtained from a packaging plasmid, a packaging plasmid, and a vector plasmid.

[0020] The pMD2.BaEV plasmid is a lentivirus packaging plasmid containing BaEV glycoprotein, which is obtained by replacing the VSVG sequence (SEQ ID NO. 1) in the pMD2.G plasmid with the BaEV sequence (SEQ ID NO. 2).

[0021] The envelope of the lentivirus is composed of a lipid bilayer and a glycoprotein, and the innovation of the present application lies in the proportion of the glycoprotein.

[0022] The present application provides a lentivirus packaging method for significantly promoting the titer of lentivirus by screening the components and amounts of lentivirus envelope plasmid combinations; further, by screening the pretreatment methods of hematopoietic stem cells, transduction reagents and other conditions, a method for lentivirus transduction of hematopoietic stem cells is provided, which is simple in operation, can maintain the long-term stemness of hematopoietic stem cells in vitro, can realize efficient and stable transduction of hematopoietic stem cells, and the transduction rate is greater than 90%. The hematopoietic stem cells prepared by the lentivirus transduction method of the present application can be used for hematopoietic stem cell gene therapy of hematopoietic system genetic diseases such as severe combined immunodeficiency disease (SCID), β-thalassemia and sickle cell disease, Wiskott-Aldrich syndrome (WAS), etc., and has good application prospect.

[0023] Obviously, according to the above content of the present application, according to the ordinary technical knowledge and conventional means in the art, other various forms of modifications, replacements or changes can be made without departing from the above basic technical ideas of the present application.

[0024] The above content of the present application will be further described in detail through the following embodiment form. However, it should not be understood that the scope of the above subject matter of the present application is limited to the following examples. Any technology realized based on the above content of the present application belongs to the scope of the present application. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1 Flow chart for lentivirus transduction of hematopoietic stem cells; Figure 2 Flow chart for lentivirus packaging for transduction of hematopoietic stem cells; Figure 3 Flow cytometry results for detection of positive rate of hematopoietic stem cells after CD34 sorting; wherein, the vertical axis SSC-H in the flow cytometry graph represents the intensity of side scatter light, and the horizontal axis CD34-BV421 represents the fluorescence intensity of cell surface CD34-BV421; Figure 4 Flow cytometry results for detection of positive rate of hematopoietic stem cells after lentivirus transduction; wherein, Figure 4 A is the flow cytometry results for detection of positive rate of hematopoietic stem cells after lentivirus transduction for 2 days, Figure 4 B is the flow cytometry results for detection of positive rate of hematopoietic stem cells after lentivirus transduction for 5 days; wherein, the vertical axis SSC-H in the flow cytometry graph represents the intensity of side scatter light, and the horizontal axis EGFP-FITC represents the fluorescence intensity of EGFP; Figure 5 Survival rate of hematopoietic stem cells after lentivirus transduction; wherein, MOI represents multiplicity of infection, i.e. the ratio of virus to cell number during infection; Figure 6 CFU results of hematopoietic stem cells after lentivirus transduction in Example 1; Figure 7 Lentivirus titer detection results under different plasmid ratios during lentivirus packaging; wherein, Figure 7 A is the flow cytometry results when the plasmid ratio is 3:1:1:1:0 in Experimental Example 1, Figure 7 B is the flow cytometry results when the plasmid ratio is 3:1:1:0:1 in Experimental Example 1, Figure 7 C is the flow cytometry results when the plasmid ratio is 3:1:1:0.25:0.75 in Experimental Example 1, Figure 7 D is the flow cytometry results when the plasmid ratio is 3:1:1:0.3:0.7 in Experimental Example 1, Figure 7 E is the flow cytometry results when the plasmid ratio is 3:1:1:0.5:0.5 in Experimental Example 1, Figure 7 F is the flow cytometry results when the plasmid ratio is 3:1:1:0.1:0.9 in Experimental Example 1; wherein, the vertical axis SSC-A in the flow cytometry graph represents the intensity of side scatter light, and the horizontal axis FITC-A represents the fluorescence intensity of FITC corresponding wavelength; Figure 8 Results of syncytia formation of double envelope lentivirus and single envelope lentivirus; Figure 9 Results of the effect of pretreatment of hematopoietic stem cells with different concentrations of factor combinations on stemness maintenance; Figure 10 Figure 4 shows the results of the effect of adding a transduction-promoting agent on the transduction efficiency of hematopoietic stem cells, where *** indicates p < 0.001, **** indicates p < 0.0001, and ns indicates p ≥ 0.05, no significant difference. DETAILED DESCRIPTION

[0026] In the following examples and experimental examples, reagents and materials not specifically mentioned are commercially available.

[0027] Example 1: A method for lentivirus transduction of hematopoietic stem cells This example provides a method for lentivirus transduction of hematopoietic stem cells, as shown in Figure 1 The specific steps are as follows: S1. Use suspended 293T cells to package lentivirus, and determine the lentivirus infection titer after concentration.

[0028] Specifically, the process of obtaining lentivirus includes 293T cell culture, plasmid transfection of 293T cells, lentivirus liquid collection, lentivirus concentration, lentivirus infection of target cells, and flow detection analysis to determine the lentivirus titer.

[0029] S1.1. Culture 293T cells to an appropriate density for lentivirus packaging.

[0030] The packaging cells are suspended 293T cells, and the suspended cells are cultured using serum-free medium. Compared with adherent cells, the lentivirus yield is higher and the impurities are fewer. Before lentivirus packaging, the density of 293T cells is adjusted to 1 x 10 6 / mL S1.2. Configure the lentivirus packaging system and transfect 293T cells.

[0031] As shown in Figure 2 , the plasmids are mixed in the following mass ratio: pCDH-EF1 expression plasmid containing EGFP structural sequence: pMDLg / pRRE plasmid: pRSV-Rev plasmid: pMD2.G plasmid: pMD2.BaEV plasmid = 3:1:1:0.3:0.7. The plasmid is configured at 1 μg / mL, mixed uniformly by blowing, and then left to stand for 5 minutes. A PEI solution with a concentration of 1 μg / μL is configured, and it is mixed uniformly with the plasmid at a mass ratio of 2:1. After standing for 30 minutes, it is added to the suspended culture 293T cells.

[0032] pCDH-EFl expression plasmid is pCDH-EFl-copGFP-T2A-Puro, purchased from Miro Biosciences, and the item number is P36206; pMD2.G plasmid is a lentivirus packaging plasmid of VSVG glycoprotein, purchased from Miro Biosciences, and the item number is P0262; pMD2.BaEV plasmid is a lentivirus packaging plasmid containing BaEV glycoprotein, which is modified from pMD2.G plasmid, specifically, the VSVG sequence (SEQ ID NO. 1) is replaced by the BaEV sequence (SEQ ID NO. 2).

[0033] VSVG sequence (SEQ ID NO. 1): BaEV sequence (SEQ ID NO. 2): S1.3, after plasmid transfection, the supernatant of the lentivirus is collected after the cells produce enough virus, and part of the impurities is removed, and the lentivirus is concentrated.

[0034] The lentivirus supernatant is collected at 48 hours and 72 hours after plasmid transfection, filtered through a 0.22 μm filter membrane, 5x PEG8000 is added, shaken uniformly, and then placed at 4℃ overnight; centrifuged at 4℃, 3900 rpm for 40 minutes, the supernatant was discarded, and the lentivirus precipitate was resuspended in culture medium to obtain the lentivirus concentrate.

[0035] S1.4, the titer of the lentivirus concentrate is determined, the lentivirus concentrate is used to transduce specific target cells, and the positive rate of the cells is determined by flow cytometry after infection, and then the titer of the lentivirus is calculated.

[0036] Jurkat cells are used as target cells for determining the titer, and the Jurkat cells are plated into a 24-well plate at 5x10 5 per well, 400 μL of medium is added to each well, the lentivirus is gradient diluted, the final volume is 100 μL, and the lentivirus is added to the cells. 24 hours later, the liquid is supplemented, and 65 hours after infection, the cells are collected, the positive rate is detected by flow cytometry, and the titer of the lentivirus is calculated. The calculation method is: lentivirus titer (TU / ml) = number of cells at the time of infection * positive rate / volume of lentivirus concentrate (μL) * 1000.

[0037] The present application also provides that before, during and / or after transduction, the cells can be cultured in a culture medium suitable for cell maintenance, growth or proliferation, and the culture medium includes X-VIVO15 medium, SFEM II medium.

[0038] S2, sort the mobilized peripheral blood hematopoietic stem cells and determine the sorting efficiency.

[0039] Specifically, after obtaining human peripheral blood cells, CD34 immunomagnetic beads are used for sorting to enrich hematopoietic stem cells, and flow cytometry is used to confirm the sorting efficiency.

[0040] S2.1, CD34 immunomagnetic beads are added to the peripheral blood cells, and hematopoietic stem cells are enriched by column separation.

[0041] First, 30 μm cell screen is wetted with DPBS, and the obtained mononuclear cells are separated by filtration to remove cell clumps; cell counting is performed using a cell counter, and the number of columns used is determined according to the number of cells; the filtered mononuclear cells are centrifuged at 2000 r / min for 4 min; the supernatant is discarded, and the cells are resuspended with 300 μl of hematopoietic stem cell sorting buffer; 1x10 8Cells plus 100 μl of FcR blocker and 100 μl of CD34 magnetic beads; after mixing, incubate in a 4-8°C refrigerator for 30 min; 1 x 10 8 Cells plus 5-10 mL of hematopoietic stem cell sorting buffer; centrifuge at 2000 r / min for 10 min at room temperature; discard the supernatant, and resuspend 1 x 10 8 Resuspend the cells in 500 μl of buffer; install the sorting column on the sorter, rinse the sorting column with hematopoietic stem cell sorting buffer, add the cell suspension to the sorting column, and collect the unmarked cells that flow through the sorting column; rinse the sorting column with 1 ml of hematopoietic stem cell sorting buffer three times, collect the unmarked cells that flow through the sorting column, remove the sorting column from the sorter and place it in a suitable collection tube, add an appropriate 1 ml equivalent of hematopoietic stem cell sorting buffer to the sorting column, and immediately push the piston into the sorting column with uniform force to push the target cells out (to improve purity, the cells can be sorted twice through the column).

[0042] S2.2, use flow cytometry to stain the sorted hematopoietic stem cells, and determine the positive rate by flow cytometry analysis.

[0043] As shown in Figure 3 , CD34 + cells are enriched from human mobilized peripheral blood, and the sorted cells are labeled with BV421 anti-human CD34 antibody and subjected to flow cytometry analysis. The purity of the sorted CD34 + cells is >95%.

[0044] S3, use the lentivirus packaged in S1 to transduce the hematopoietic stem cells obtained in S2.

[0045] First, the hematopoietic stem cells are pretreated, the lentivirus is mixed with the transduction reagent, and then the mixture is added to the hematopoietic stem cells to achieve transduction of the hematopoietic stem cells.

[0046] Specifically, the pretreatment method is as follows: After the hematopoietic stem cells are resuscitated, X-VIVO15 medium is used, and a combination of cytokines at the following concentrations (300 ng / mL SCF, 100 ng / mL TPO, 60 ng / mL IL3, and 300 ng / mL FIL-3L) is added for pretreatment for 24 hours for the following transduction.

[0047] Under long-term culture conditions in medium containing the combination of cytokines at the above concentrations, the stemness markers of the hematopoietic stem cells are detected by flow cytometry, and the results show that the stemness of the hematopoietic stem cells can be well maintained within 7 days.

[0048] Specifically, the transduction method is as follows: Dissolve 48 μg of recombinant fibronectin (fibronectin is purchased from Yixing Biological, item number: 40113ES03) with 1-2 mL of PBS solution, add to a 6-well plate well, coat at 37°C for 1 h, remove all liquid, and wait for use. Resuspend the cells obtained in S2.1 with complete culture medium, inoculate 1 mL at 5E5 / mL in the well plate, and culture at 37°C under 5% CO2 for 24 h. Mix 4E7 TU of lentivirus in step S1 with 10 μg of Vectofusin-1 (purchased from Miltenyi, item number: 130-111-163), incubate for 5 min, add to the cells (multiplicity of infection MOI is 80), mix, and culture at 37°C under 5% CO2 for 16 h. Collect all cells, centrifuge at 300xg for 5 min, resuspend the cells with complete culture medium, and culture at 37°C under 5% CO2.

[0049] As shown in Figure 4 , the transduction efficiency of hematopoietic stem cells after 2 days and 5 days of transduction is analyzed, and EGFP positive cells are analyzed by flow cytometry. The positive rate of cells after transduction can reach 95%.

[0050] As shown in Figure 5 , the viability of hematopoietic stem cells after 2 days and 5 days of transduction is analyzed. The viability of cells after transduction can be maintained at more than 95% for a long time.

[0051] As shown in Figure 6 , the stemness of hematopoietic stem cells after transduction is analyzed based on CFU experiment. After lentivirus transduction, the number and proportion of CFU do not change significantly compared with cells without transduction.

[0052] The above method of lentivirus transduction of hematopoietic stem cells is simple to operate and can realize efficient and stable transduction of hematopoietic stem cells.

[0053] The above embodiment only expresses one embodiment of the present application, which is described in detail, but it cannot be understood as a limitation on the scope of the patent. It should be noted that for ordinary skilled persons in the art, several modifications and improvements can be made without departing from the concept of the present application, which are within the protection scope of the present application. Therefore, the protection scope of the present application patent should be subject to the appended claims.

[0054] The technical solutions of the present application are further described through experiments below.

[0055] Experimental Example 1: Screening of combination of envelope plasmids for lentivirus packaging I. Experimental method Adjust the density of the cultured suspension 293T cells to 1x10 6 / mL, the expression plasmid containing the EGFP structural sequence, the pMDLg / pRRE plasmid, the pRSV-Rev plasmid, the lentivirus packaging plasmid containing the VSVG glycoprotein, and the lentivirus packaging plasmid containing the BaEV glycoprotein are mixed in the proportions shown in Table 1 below, the plasmid is configured at 1 μg / mL, and after being mixed uniformly by blowing, it is left to stand for 5 minutes; the PEI solution is configured, and it is mixed uniformly with the plasmid at a ratio of 2:1, left to stand for 30 minutes, and then added to the cell suspension culture 293T cells.

[0056] Specifically, the lentivirus supernatant is collected 48 hours after plasmid transfection, filtered through a 0.22 μm filter membrane, 5×PEG8000 is added, shaken uniformly, left to stand at 4°C overnight, centrifuged at 4°C, 3900 rpm for 40 minutes, the supernatant is discarded, and the lentivirus precipitate is resuspended in the culture medium to obtain a lentivirus concentrate, which is used to infect target cells, and the lentivirus titer is detected by flow cytometry 48 hours later.

[0057] II. Experimental results The results are shown in Figure 7 and Table 1. The amount of components in the VSVG and BaEV plasmid combination during lentivirus packaging has a significant effect on the lentivirus titer, and in particular, when the VSVG:BaEV ratio is 0.3:0.7, the lentivirus titer is significantly improved.

[0058] Table 1 Lentivirus titer detection results under different plasmid ratios during lentivirus packaging Experimental Example 2 Comparison of Double Envelope Lentivirus and BaEV Single Envelope Lentivirus This experimental example provides a comparison of the results of the formation of syncytia by double envelope lentivirus and BaEV single envelope lentivirus.

[0059] Specifically, the double envelope lentivirus or BaEV single envelope lentivirus is packaged according to the plasmid ratio in Example 1, and after transfection of 293T cells for 48 hours, a fluorescence microscope is used to observe the cell syncytia.

[0060] The results are shown in Figure 8 . The BaEV single envelope lentivirus produces more syncytia when formed, irreversibly damages the cells, and affects the virus production efficiency. Therefore, the double envelope lentivirus according to the preferred ratio of the present application is not prone to produce syncytia, and has high packaging efficiency.

[0061] Experimental Example 3 Screening of Hematopoietic Stem Cell Pretreatment Methods I. Experimental methods After the hematopoietic stem cells were recovered, the percentage of CD34 positive cells, CD38- cells, hematopoietic stem cells (HSCs), MPPs (multipotent progenitor cells), and MLPs (multilineage progenitor cells) in total cells was determined by flow cytometry under the condition of long-term culture (7 days) of the hematopoietic stem cells using X-VIVO 15 medium and a combination of factors at the concentrations shown in Table 2.

[0062] Table 2. Pretreatment factor combinations II. Experimental results The results are shown in Table 3. Figure 9 The effects of pretreatment of hematopoietic stem cells on maintaining stemness were different, and under the condition of long-term culture of the hematopoietic stem cells at the factor concentrations in combination 2, the stemness of the hematopoietic stem cells was well maintained within 7 days.

[0063] Experimental Example 4. Effect of transduction-promoting reagent on transduction efficiency of hematopoietic stem cells I. Experimental methods 1. Experimental grouping This experimental example provides three kinds of transduction-promoting reagents, which are (1) fibronectin, (2) Vectofusin-1, and (3) fibronectin + Vectofusin-1. Fibronectin was pre-coated in the culture bottle at a concentration of 5 μg / cm 2 , and the final concentration of Vectofusin-1 was 5 μM.

[0064] 2. Determination method According to the grouping, the hematopoietic stem cells (HSCs) and the lentivirus were transduced according to the method of Example 1, and the transduction was determined by flow cytometry.

[0065] This experimental example also determined the transduction of MPPs (multipotent progenitor cells) and MLPs (multilineage progenitor cells) and total cells by lentivirus under the same method. Among them, MPPs, MLPs, and total cells were separated and enriched from human peripheral blood cells using immunomagnetic beads. The total cells were CD34 positive cells.

[0066] II. Experimental results The results are shown in Table 3. Figure 10 The combination of fibronectin + Vectofusin-1 as a transduction-promoting reagent can effectively improve the transduction efficiency of hematopoietic stem cells by 20%-40%, and the transduction efficiency of hematopoietic stem cells is increased to 90% by flow cytometry.

[0067] It can be seen from the above examples and experimental examples that the application provides a lentivirus packaging method for significantly promoting lentivirus titer by screening components and amounts of lentivirus envelope plasmid combinations; further, by screening hematopoietic stem cell pretreatment methods, transduction reagents and other conditions, a lentivirus transduction method for hematopoietic stem cells is provided, the method is simple to operate, can maintain long-term stemness of hematopoietic stem cells in vitro, can realize efficient and stable transduction of hematopoietic stem cells, and the transduction rate is greater than 90%. The lentivirus transduction method for hematopoietic stem cells can be used for hematopoietic stem cell gene therapy for hematopoietic system genetic diseases, such as severe combined immunodeficiency disease (SCID), beta-thalassemia and sickle cell disease, Wiskott-Aldrich syndrome (WAS) and the like, and has a good application prospect.

Claims

1. An envelope plasmid assembly for lentivirus packaging, characterized in that: The plasmid combination consists of a lentiviral packaging plasmid containing VSVG glycoprotein and a lentiviral packaging plasmid containing BaEV glycoprotein, with a mass ratio of 3:

7.

2. The envelope plasmid assembly for lentivirus packaging according to claim 1, characterized in that: The lentiviral packaging plasmid containing VSVG glycoprotein is selected from at least one of pMD2.G plasmid, pLP / VSVG plasmid, and p1_P / VSVG plasmid; the lentiviral packaging plasmid containing BaEV glycoprotein is selected from at least one of pMD2.BaEV and pCMV-BaEV-Rless.

3. The envelope plasmid assembly for lentiviral packaging as described in claim 1 or 2 is used for lentiviral packaging applications.

4. A lentivirus packaging method, characterized in that: It is a transduction method that uses the envelope plasmid combination for lentivirus packaging as described in claim 1 or 2 as the envelope plasmid.

5. The lentivirus packaging method according to claim 4, characterized in that: It is a transduction method that uses the envelope plasmid combination for lentivirus packaging as described in claim 1 or 2 as the envelope plasmid, pMDLg / pRRE plasmid and pRSV-Rev plasmid as the packaging plasmid, and pCDH-EF1 as the vector plasmid. The mass ratio of the vector plasmid, pMDLg / pRRE plasmid, pRSV-Rev plasmid and envelope plasmid is 2.4-3.6:0.8-1.2:0.8-1.2:0.8-1.

2.

6. The lentivirus packaging method according to claim 5, characterized in that, It includes: The vector plasmid, pMDLg / pRRE plasmid, pRSV-Rev plasmid, and envelope plasmid were used to infect 293T cells.

7. A lentivirus, characterized in that: Its envelope glycoprotein is a combination of VSVG glycoprotein and BaEV glycoprotein, with a mass ratio of 3:

7.

8. The lentivirus according to claim 7, characterized in that: It is prepared by the lentivirus packaging method according to any one of claims 4 to 6.

9. A method for lentivirus transduction of hematopoietic stem cells, characterized in that, Includes the following steps: Step 1: Inoculate hematopoietic stem cells into a complete culture medium for pre-stimulation; Step 2: Use the lentivirus pre-stimulated hematopoietic stem cells as described in claim 7 or 8; Step 3: Collect transduced hematopoietic stem cells; Step 4: Expand the cells from Step 3 in a complete culture medium.

10. The method for lentivirus transduction of hematopoietic stem cells according to claim 9, characterized in that, The complete culture medium is formulated with a combination of the following cytokines: 240-360 ng / mL SCF, 80-120 ng / mL LTPO, 48-72 ng / mL IL-3, and 240-360 ng / mL FLT-L3; And / or, the formulation of the complete culture medium further includes a basal culture medium selected from X-VIVO15 basal culture medium and SFEM II basal culture medium; And / or, the pre-stimulation time described in step 1 is 12-48 hours; And / or, the source of the hematopoietic stem cells in step 1 is mobilized peripheral blood; And / or, in step 2, the transduction time is 8-24 hours; And / or, in step 2, the transduction is carried out in the presence of a transduction-promoting agent, which includes at least one of Vectofusin-1 and recombinant fibronectin. The pre-coating density of the recombinant fibronectin in the transduction system is 2-10 μg / cm³. 2 The concentration of Vectofusin-1 in the transduction system was 2-20 μg / mL; And / or, in step 2, the transduction occurs 1-3 times; And / or, in step 4, the amplification culture time is 7-10 days.

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