Method for enhancing virus transduction efficiency based on combination of hypoxia and HIF-1 inhibitor

Abstract

The invention discloses a method for enhancing virus transduction efficiency based on combination of hypoxia and an HIF-1 inhibitor. The method comprises the following steps: carrying out virus packaging on packaging cells under a low-oxygen condition, collecting purified viruses, and infecting the cells pretreated by the HIF-1 inhibitor by using the purified viruses; the oxygen concentration under the low-oxygen condition is not higher than 20%. According to the method disclosed by the invention, the virus packaging efficiency under the hypoxia condition is improved by 10%, and the virus transduction efficiency is improved by 20% under the synergistic effect of pretreating the cells to be transduction by using the HIF-1 inhibitor and the viruses packaged under the hypoxia condition.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a method for enhancing the transduction efficiency of lentivirus by regulating oxygen concentration and inhibiting the HIF-1 signaling pathway, and its applications in gene therapy and cell engineering. Background Art

[0002] Lentiviral vectors can effectively integrate foreign genes or foreign shRNAs into the host chromosome, thereby achieving the effect of persistent expression of the target sequence. In terms of infection ability, they can effectively infect various types of cells such as neuron cells, liver cells, cardiomyocytes, tumor cells, endothelial cells, and stem cells, thus achieving good gene therapy effects. For some cells that are difficult to transfect, such as primary cells, stem cells, and undifferentiated cells, the use of lentiviral vectors can greatly improve the transduction efficiency of the target gene or target shRNA, and the probability of the target gene or target shRNA integrating into the host cell genome is greatly increased, enabling the long-term and stable expression of the target gene or target shRNA to be achieved more conveniently and quickly. Therefore, in the research of in vitro and in vivo experiments, lentivirus has become one of the commonly used vector forms for expressing foreign genes or foreign shRNAs and is being applied more and more widely.

[0003] Lentiviral vectors are widely used in gene therapy, cell reprogramming, and basic research because they can stably integrate into the host genome. However, the existing lentivirus packaging and transduction technologies have the following problems: 1. Low transduction efficiency: Especially in cell lines that are difficult to transduce, the transduction efficiency is often not ideal. 2. Mismatch of oxygen concentration: The oxygen concentration in conventional cell culture conditions (18 - 21% O2) is much higher than the physiological oxygen level of human tissues (2 - 10% O2), which may lead to abnormal cell metabolism and affect the packaging and transduction efficiency of the virus. 3. Influence of the HIF-1 signaling pathway: Under hypoxic conditions, the activation of HIF-1α may inhibit the entry of the virus into cells, further reducing the transduction efficiency. 4. Toxicity of chemical enhancers: Although traditional methods (such as polybrene) can improve the efficiency, they are accompanied by a certain risk of cell toxicity.

[0004] Therefore, there is an urgent need to provide a method for improving the transduction efficiency of lentivirus and its applications to overcome problems such as low transduction efficiency in cell lines that are difficult to transduce and obstacles to the entry of the virus into cells. Summary of the Invention

[0005] In view of the deficiencies of the prior art and actual needs, the present invention provides a method for enhancing the transduction efficiency of the virus based on hypoxia combined with HIF-1 inhibitors, which solves problems such as low transduction efficiency in cell lines that are difficult to transduce, abnormal cell metabolism affecting the packaging and transduction efficiency of the virus, and obstacles to the entry of the virus into cells under hypoxic conditions.

[0006] To achieve the object of the present invention, the present invention adopts the following technical solutions:

[0007] In a first aspect, the present invention provides a method for preparing a lentivirus with high transduction efficiency, the method comprising: placing packaging cells under hypoxic conditions for virus packaging, and collecting and purifying the virus; the oxygen concentration of the hypoxic conditions is not higher than 20%.

[0008] The oxygen environment commonly used in cell culture will disrupt the redox homeostasis, increase the production of reactive oxygen species (ROS), and cause DNA damage, thereby leading to cellular oxidative stress. The oxygen concentration is not higher than 20%, simulating the physiological oxygen environment, and reducing and improving the virus packaging efficiency.

[0009] Preferably, the packaging cells include HEK-293T.

[0010] Preferably, the oxygen concentration of the hypoxic conditions is 1%-20% (such as 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%), preferably 5%-15% (such as 5%, 6%, 7%, 8%, 9%, 10%, 12%, 15%).

[0011] Preferably, the hypoxic conditions further include adding a hypoxia mimetic agent.

[0012] Preferably, the hypoxia mimetic agent includes cobalt chloride and / or deferoxamine.

[0013] In a second aspect, the present invention provides the use of the method for preparing a lentivirus with high transduction efficiency described in the first aspect in improving the transduction efficiency of lentivirus.

[0014] In a third aspect, the present invention provides a method for improving the transduction efficiency of lentivirus, the method for improving the transduction efficiency of lentivirus comprising infecting with the virus prepared by using the method for preparing a lentivirus with high transduction efficiency described in the first aspect.

[0015] Preferably, the method for improving the transduction efficiency of lentivirus further includes pretreating the cells to be infected with an HIF-1 inhibitor and then performing virus infection.

[0016] Preferably, the HIF-1 inhibitor includes any one or a combination of at least two of PX-478, BAY87-2243 or KC7F2.

[0017] The method of the present invention packages the virus under hypoxic conditions and combines the pretreatment of cells with an HIF-1 inhibitor. The two work synergistically to further improve the transduction efficiency of lentivirus. Before virus infection, pretreat the cells with an HIF-1 inhibitor (such as PX-478) to overcome the barrier of virus entry into cells, especially in the application of difficult-to-transduce cell lines.

[0018] Preferably, the concentration of the HIF-1 inhibitor is 20 - 40 μM (such as 20 μM, 25 μM, 30 μM, 35 μM, 40 μM).

[0019] As a preferred technical solution, the method for improving the lentiviral transduction efficiency in the present invention comprises the following steps:

[0020] (1) Inoculate cells in DMEM medium containing fetal bovine serum and culture until the confluence reaches 70 - 80% (such as 70%, 75% or 80%);

[0021] (2) Mix packaging plasmid pPAX2, envelope plasmid VSV-G and transfer plasmid pCDH-EF1-mNG at a weight ratio of 1:1:(1 - 3), dissolve them in NaCl solution, incubate the transfection reagent with the DNA and then add it to the cells. The transfection reagent includes any one or a combination of at least two of polyethyleneimine, liposome or calcium phosphate;

[0022] (3) Place the cells in a closed incubator with an oxygen concentration of 1% - 20% and continuously culture for 40 - 80 h (such as 40 h, 60 h, 65 h, 70 h or 80 h);

[0023] (4) Collect the cell supernatant, centrifuge to remove cell debris, filter and then ultracentrifuge, and resuspend the virus particles;

[0024] (5) Pretreat the cells to be infected with the HIF-1 inhibitor for 10 - 20 h (such as 10 h, 20 h, 30 h or 40 h);

[0025] (6) Mix the virus particles with the pretreated cells to be infected and co-incubate, replace the fresh medium and continue to culture for 40 - 60 h (such as 40 h, 45 h, 50 h or 60 h).

[0026] Fourthly, the present invention provides an application of the method for improving the lentiviral transduction efficiency described in the third aspect in the preparation of cells or drugs for preventing and / or treating cancer.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] (1) By packaging the virus under hypoxic conditions and combining pretreatment of cells with the HIF-1 inhibitor, the method of the present invention significantly improves the lentiviral transduction efficiency. Before virus infection, pretreatment of cells with the HIF-1 inhibitor (such as PX-478) overcomes the barrier of virus entry into cells, especially in the application of difficult-to-transduce cell lines;

[0029] (2) The method of the present invention increases the amount of virus produced by the same amount of packaging cells and reduces the experimental cost;

[0030] (3) The virus packaged by the method of the present invention has higher infectivity under hypoxic conditions and is applicable to various cell types;

[0031] (4) The method of the present invention is not only applicable to laboratory research but also can be used for large-scale gene delivery in industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1A Fluorescence images of 293T cells packaged under normoxic (N) and hypoxic (L) conditions;

[0033] Figure 1B Fluorescence images of K562 cells transduced under normoxic (N) and hypoxic (L) conditions;

[0034] Figure 1C Flow cytometry analysis images under normoxic (N) and hypoxic (L) conditions;

[0035] Figure 1D Quantification result images of the proportion of fluorescent cells under normoxic (N) and hypoxic (L) conditions;

[0036] Figure 1E qPCR analysis result images of virus integration into the cell genome;

[0037] Figure 2A Fluorescence images of cells infected under normoxic (N), hypoxic (L), or hyperoxic (H) conditions;

[0038] Figure 2B Flow cytometry analysis images under normoxic (N), hypoxic (L), or hyperoxic (H) conditions;

[0039] Figure 2C Quantification result images of the proportion of infected cells under normoxic (N), hypoxic (L), or hyperoxic (H) conditions;

[0040] Figure 3A CCK-8 experiment result images of PX-478-treated K562 cells;

[0041] Figure 3B Fluorescence images of infected cells with or without PX-478 pretreatment under hypoxic (L) and normoxic (N) conditions;

[0042] Figure 3C Flow cytometry analysis images of infected cells with or without PX-478 pretreatment under hypoxic (L) and normoxic (N) conditions;

[0043] Figure 3D Quantification result images of the proportion of infected cells with or without PX-478 pretreatment under hypoxic (L) and normoxic (N) conditions;

[0044] Figure 3E qPCR analysis result graph for virus integration into the cellular genome;

[0045] Figure 4A Fluorescence graph of infected cells after the synergistic effect of hypoxic, normoxic packaging and PX-478 pretreatment;

[0046] Figure 4B Flow cytometry analysis graph of infected cells after the synergistic effect of hypoxic, normoxic packaging and PX-478 pretreatment;

[0047] Figure 5 Quantification result graph of the proportion of fluorescent cells after the synergistic effect of hypoxic, normoxic packaging and PX-478 pretreatment;

[0048] Figure 6 qPCR analysis graph of virus integration into the cellular genome after the synergistic effect of hypoxic, normoxic packaging and PX-478 pretreatment;

[0049] Figure 7A Fluorescence graph of infected cells at different hypoxic concentrations;

[0050] Figure 7B Flow cytometry analysis graph of infected cells at different hypoxic concentrations;

[0051] Figure 7C Quantification result graph of the proportion of fluorescent cells of infected cells at different hypoxic concentrations;

[0052] Figure 8A CCK-8 experiment result graph of KC7F2-treated K562 cells;

[0053] Figure 8B Fluorescence graph of infected cells with or without KC7F2 pretreatment under normoxic conditions;

[0054] Figure 8C Flow cytometry analysis graph of infected cells with or without KC7F2 pretreatment under normoxic conditions;

[0055] Figure 8D Quantification result graph of the proportion of infected cells with or without KC7F2 pretreatment under normoxic conditions. Detailed implementation manners

[0056] To further elaborate on the technical means and effects adopted by the present invention, the present invention will be further described below in conjunction with embodiments and the accompanying drawings. It can be understood that the specific implementation manners described herein are only used to explain the present invention, rather than limiting the present invention.

[0057] In the examples where specific techniques or conditions are not indicated, they shall be in accordance with the techniques or conditions described in the literature in this field or in accordance with the product specifications. For reagents or instruments whose manufacturers are not indicated, they are all conventional products that can be obtained through regular commercial channels.

[0058] To make the present invention more easily understood, certain terms are first defined. As used in the present invention, unless otherwise defined, all technical terms and scientific terms used in the present invention have the same meanings as those commonly understood by those skilled in the art to which the present invention pertains. All patents and publications mentioned in the present invention are incorporated herein by reference in their entirety.

[0059] The term "HIF-1" refers to hypoxia-inducible factor 1, which regulates the adaptive response of cells to a hypoxic environment.

[0060] The term "PX-478" refers to an HIF-1α inhibitor that can inhibit the translation and activity of HIF-1α.

[0061] The term "PEI" refers to polyethyleneimine, a commonly used transfection reagent.

[0062] Example 1

[0063] This example detects the effects of oxygen concentration on virus packaging and transduction efficiency.

[0064] 1. Virus packaging

[0065] Cell culture: HEK-293T cells were seeded in a 10 cm culture dish and cultured in DMEM medium containing 10% fetal bovine serum at 37 °C and 5% CO2 until 80% confluence.

[0066] Plasmid transfection: Packaging plasmid (pPAX2), envelope plasmid (VSV-G), and transfer plasmid (pCDH-EF1-mNG) were mixed at a weight ratio of 4:4:5 and dissolved in 150 mM NaCl solution. PEI 40K (1 mg / mL) was mixed with DNA at an N / P ratio of 18 and incubated at room temperature for 20 min, then added to the cells.

[0067] Culturing under different oxygen concentration conditions: After transfection, the cells were placed in a sealed incubator, and three different oxygen concentration conditions were set (normoxia: 21% O2; hypoxia: 10% O2; hyperoxia: 30% O2). The oxygen concentration was monitored in real time using a gas detector (ADKS-1), and the cells were continuously cultured for 72 h.

[0068] Virus collection and purification: Collect the cell supernatant, centrifuge at 5000×g for 10 min to remove cell debris, filter through a 0.45-μm filter membrane, then ultracentrifuge at 50,000×g for 2 h. Resuspend the virus particles in PBS and store in aliquots at -80°C.

[0069] 2. Cell pretreatment and virus infection

[0070] Seed K562 cells at a concentration of 5×10 5 cells / mL in a 6-well plate and culture for 16 h.

[0071] Virus infection: Add the virus particles obtained above and incubate in three groups (normoxia: 21% O2; hypoxia: 10% O2; hyperoxia: 30% O2) for 8 h, then replace with fresh medium and continue culturing for 48 h.

[0072] Detection and analysis of results:

[0073] Flow cytometry: Collect the cells, wash with PBS and resuspend in PBS containing 0.5% BSA, and detect the proportion of mNeonGreen-positive cells.

[0074] qPCR: Extract genomic DNA, design specific primers for mNG, and quantify the virus genome integration efficiency. ns: no significance; *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.

[0075] Results: The fluorescence images of 293T cells packaged under normoxia (N) and hypoxia (L) conditions are as Figure 1A shown. The results of flow cytometry analysis under normoxia (N) and hypoxia (L) conditions are as Figure 1C shown. The virus packaged under hypoxia has a stronger fluorescence signal in K562 cells ( Figure 1B ), flow cytometry quantification shows that the infection efficiency is increased by 10% ( Figure 1D ), and qPCR confirms a significant increase in the genome integration rate ( Figure 1E ).

[0076] The fluorescence images of cells infected under normoxia (N), hypoxia (L), or hyperoxia (H) conditions are as Figure 2A shown. Hypoxia significantly inhibits the infection efficiency during the virus entry stage (0 - 8 h) ( Figure 2C ), and hyperoxia cannot reverse this inhibition ( Figure 2B ).

[0077] Example 2

[0078] In this example, the HIF-1α inhibitor PX-478 was used to reverse hypoxia-induced inhibition and enhance the detection of virus genome integration.

[0079] The method of this example refers to Example 1. The difference from Example 1 is that the virus obtained by packaging under normoxic conditions is used, and the infected cells are pretreated with different concentrations of the HIF-1α inhibitor PX-478 (0 μM, 10 μM, 20 μM, 40 μM, 60 μM, 80 μM, 120 μM) under normoxic (N) and hypoxic (L) infection conditions, and other experimental operations and conditions are the same.

[0080] Under hypoxic (L) and normoxic (N) infection conditions, the fluorescence images of the infected cells with or without PX-478 pretreatment are shown in Figure 3B as follows, and the flow cytometry analysis images are shown in Figure 3C as follows. Pretreatment with the HIF-1α inhibitor PX-478 restored and increased the infection efficiency of the hypoxic group by 10% ( Figure 3D ), and showed a dose-dependence ( Figure 3A ), and at the same time increased the relative expression level of the mNeonGreen gene ( Figure 3E ).

[0081] Example 3

[0082] This example examines the synergistic effect of hypoxic packaging and PX-478 pretreatment.

[0083] The method of this example refers to Example 1. The difference from Example 1 is that the virus obtained by packaging under normoxic (N) conditions is used, and the infected K562 cells are pretreated with different concentrations of the HIF-1α inhibitor PX-478 (0 μM, 20 μM) under normoxic (N) and hypoxic (L) infection conditions, and other experimental operations and conditions are the same.

[0084] The fluorescence images of the infected cells after the synergistic effect of hypoxic, normoxic packaging and PX-478 pretreatment are shown in Figure 4A as follows, and the flow cytometry analysis images of the infected cells are shown in Figure 4B as follows. The combination of hypoxic packaging and PX-478 pretreatment increased the infection efficiency by 20% ( Figure 5 ), and the genomic integration rate increased by 0.5 times ( Figure 6 ).

[0085] Example 4

[0086] This example examines the detection of the infection efficiency of viruses packaged under different hypoxic concentrations.

[0087] The method of this example refers to Example 1. The difference from Example 1 is that HEK-293T cells are cultured under hypoxic (L) packaging conditions with oxygen concentration contents of 5%, 10% and 15%, and other experimental operations and conditions are the same.

[0088] The fluorescence signals of the viruses packaged under different hypoxic concentrations in K562 cells are shown in Figure 7Aand Figure 7B As shown, the flow cytometry quantification results Figure 7C show that a 5% oxygen concentration reduces the packaging efficiency, and a 15% oxygen concentration increases the infection efficiency by 3%.

[0089] Example 5

[0090] In this example, the effect of the HIF-1α inhibitor KC7F2 on the virus transfection efficiency was detected.

[0091] The difference between this example and Example 1 is that the virus obtained under normoxic (N) packaging conditions was used, and K562 cells were pretreated with different concentrations of the HIF-1α inhibitor KC7F2 (0 μM, 5 μM, 10 μM, 20 μM, 40 μM, 80 μM) under normoxic (N) infection conditions, and other experimental operations and conditions were the same.

[0092] The CCK-8 assay results of KC7F2-treated K562 cells are shown as Figure 8A shown. The HIF-1α inhibitor KC7F2 with different mechanisms of action can also improve the virus transfection efficiency ( Figure 8B , Figure 8C , Figure 8D ).

[0093] In summary, the efficiency of virus packaging by the method of the present invention under hypoxic conditions is increased by 10%, and the synergistic effect of HIF-1α inhibitor pretreatment and hypoxic conditions increases the virus transduction efficiency by 20%.

[0094] The applicant declares that the present invention uses the above examples to illustrate the detailed method of the present invention, but the present invention is not limited to the above detailed method, that is, it does not mean that the present invention must rely on the above detailed method to be implemented. Those skilled in the art should understand that any improvement to the present invention, the equivalent substitution of the raw materials of the products of the present invention, the addition of auxiliary components, the selection of specific methods, etc., all fall within the protection scope and the disclosure scope of the present invention.

Claims

1. A method for preparing lentivirus with high transduction efficiency, characterized in that, The method includes: placing packaging cells under hypoxic conditions for virus packaging, and collecting and purifying the virus; the oxygen concentration of the hypoxic conditions is not higher than 20%.

2. The method for preparing lentivirus with high transduction efficiency according to claim 1, wherein The packaging cells include HEK-293T.

3. The method for preparing lentivirus with high transduction efficiency according to claim 1 or 2, characterized in that, The oxygen concentration of the hypoxic conditions is 1%-20%, preferably 5%-15%.

4. The method for preparing lentivirus with high transduction efficiency according to any one of claims 1-3, characterized in that, The hypoxic conditions further include adding a hypoxia mimetic agent.

5. The method for preparing lentivirus with high transduction efficiency according to claim 4, characterized in that, The hypoxia mimetic agent includes cobalt chloride and / or deferoxamine.

6. Use of the method for preparing lentivirus with high transduction efficiency according to any one of claims 1-5 in improving the transduction efficiency of lentivirus.

7. A method for improving the transduction efficiency of lentivirus, characterized in that, The method for improving the transduction efficiency of lentivirus includes infecting with the virus prepared by using the method for preparing lentivirus with high transduction efficiency according to any one of claims 1-5.

8. The method for improving the lentiviral transduction efficiency according to claim 7, wherein The method for improving the transduction efficiency of lentivirus further includes pretreating the cells to be infected with an HIF-1 inhibitor and then performing virus infection; Preferably, the HIF-1 inhibitor includes any one or a combination of at least two of PX-478, BAY87-2243 or KC7F2; Preferably, the concentration of the HIF-1 inhibitor is 20-40 μM.

9. The method for improving the lentiviral transduction efficiency according to claim 7 or 8, characterized in that, The method for improving the transduction efficiency of lentivirus includes the following steps: (1) Inoculating cells in DMEM medium containing fetal bovine serum and culturing until 70-80% confluence; (2) Mixing packaging plasmid pPAX2, envelope plasmid VSV-G and transfer plasmid pCDH-EF1-mNG in a weight ratio of 1:1:(1-3) and dissolving them in NaCl solution, incubating the transfection reagent with the DNA and then adding it to the cells, and the transfection reagent includes any one or a combination of at least two of polyethyleneimine, liposome or calcium phosphate; (3) Placing the cells in a closed incubator with an oxygen concentration of 1%-20% and continuously culturing for 40-80 h; (4) Collecting the cell supernatant, centrifuging to remove cell debris, filtering and then ultracentrifuging, and resuspending the virus particles; (5) Pretreating the cells to be infected with an HIF-1 inhibitor for 10-20 h; (6) Mixing the virus particles with the pretreated cells to be infected and co-incubating, and replacing the fresh medium and continuing to culture for 40-60 h.

10. Use of the method for improving the transduction efficiency of lentivirus according to any one of claims 7-9 in the preparation of cells or drugs for preventing and / or treating cancer.

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