Method for improving efficiency of lentivirus transfection of macrophages

Abstract

The invention provides a method for improving lentivirus transfection efficiency of macrophages. The method comprises the following steps: extracting CD14 < + > mononuclear cells from peripheral blood, inducing the CD14 < + > mononuclear cells to be polarized into M1 type macrophages, then inhibiting expression of SAMHD1 genes in the macrophages from a transcription or translation level, treating the macrophages which reduce the expression of the SAMHD1 genes by dNTPs, and then performing lentivirus transfection. The positive rate and the cell viability after lentivirus transfection of macrophages are effectively improved by optimizing the protamine content of a culture medium, the transfection volume and the like during lentivirus transfection of the macrophages. On the basis, siRNA is adopted to interfere SAMHD1 gene expression and is combined with dNTPs treatment, so that the lentivirus transfection positive rate of the macrophages is increased, the M1 phenotype of the macrophages is maintained, the operation is simple and effective, the cost is low, and the method is suitable for industrial large-scale production.

Description

Technical Field

[0001] The present invention relates to the technical fields of biotechnology and immunocyte therapy, and specifically, to a method for improving the lentiviral transfection efficiency of macrophages. Background Art

[0002] In recent years, CAR-T (Chimeric Antigen Receptor T-Cell) therapy has made breakthrough progress in the treatment of hematological malignancies, making researchers realize the great potential of CAR-M (Chimeric Antigen Receptor Macrophages) therapy for solid tumors.

[0003] The tumor microenvironment (TME) provides a crucial niche for the occurrence and development of cancer, shaping the survival, proliferation, and invasion conditions of tumor cells. The infiltration of bone marrow monocytes, especially the high proportion of monocytes, macrophages, and dendritic cells, is one of the significant characteristics of the TME. However, macrophages are a double-edged sword and have dual potential in tumors. M1 macrophages can secrete pro-inflammatory factors, present antigens, and actively respond to immune responses. M2 macrophages, on the other hand, have anti-inflammatory effects, promote tumor growth, and immunosuppressive effects. Therefore, it is extremely important to control the differentiation of macrophages into M1 type, and CD80 and CD86 are the main markers for detecting M1 macrophages.

[0004] CAR-M therapy utilizes the characteristics of macrophages, mainly M1 macrophages, such as high infiltration in solid tumors, the ability to phagocytose cancer cells, secrete cytokines, and chemokines to recruit other immune cells, and endows them with specific anti-tumor capabilities through genetic engineering. Currently, two CAR-M therapies, CT-0508 from Carisma Therapeutics and MCY-M11 from MaxCyte, have been approved by the FDA (Food and Drug Administration) of the United States to enter the clinical trial stage, and are used to treat HER2-positive solid tumors and other refractory solid tumors respectively, which marks a crucial step for CAR-M therapy in the field of tumor immunotherapy. Although CAR-M therapy has made some progress, it is still in its infancy, especially facing the problem of difficult transfection of macrophages.

[0005] There are a large number of pattern recognition receptors (PRRs) on the cell membrane surface of macrophages, which can recognize "non-self components" and bind to the corresponding pathogen-associated molecular patterns (PAMPs), thereby initiating a series of immune responses, such as phagocytosis, clearance, killing, and antigen presentation. These characteristics make it difficult for foreign nucleic acids to enter the interior of macrophages (Martinez et al., 2008). In addition, SAMHD1 (sterile α motif domain and HD domain-containing protein 1) present in macrophages and dendritic cells, as a dNTPase (deoxynucleoside triphosphate hydrolase), can degrade dNTPs (deoxyribonucleoside triphosphates) into dNs (deoxyribonucleosides), thereby maintaining an extremely low level of intracellular dNTPs. This mechanism restricts virus replication, especially the infection of HIV-1 lentivirus (Goldstone DC et al, 2011; Hrecka K et al, 2011), which also causes difficulties in the lentivirus transfection process of macrophages.

[0006] Currently, lentivirus and adenovirus are mainly used to transfect macrophages. CN116463381A discloses a method of modifying the lentivirus four-plasmid packaging system by utilizing the characteristic of Vpx antagonizing SAMHD1, thereby significantly improving the infection efficiency of macrophages, increasing it from 2% to 20%. However, this method is complex to operate. After introducing VPX into the packaging plasmid, the accumulation of VPX will lead to a decrease in virus titer, and its safety still needs to be further verified. Referring to the effectiveness of CAR-T therapy, generally speaking, the infection efficiency needs to reach more than 30% to show a therapeutic effect. Therefore, improving the transfection efficiency of macrophages remains an important issue.

[0007] In addition, CN115449525A modifies the way of Ad5F35 adenovirus infecting macrophages. When the virus MOI = 300 (high infection dose), the transfection efficiency is successfully increased to about 90%. However, this method requires the use of a large amount of adenovirus, and the industrial cost is very high and it is difficult to produce quantitatively. Due to the characteristics of adenovirus, the expression time of foreign genes is short and cannot be continuously expressed, and it has strong immunogenicity, which is likely to cause inflammatory and immune responses in the body. These factors limit its wide application.

[0008] At present, there has not yet been a simple, low-cost method that can significantly improve the efficiency of lentivirus transfection of macrophages and maintain the M1 phenotype of macrophages, and is suitable for industrial production. Therefore, developing a macrophage transfection method that is efficient, simple, and has the potential for industrial production remains an urgent challenge to be solved. Summary of the Invention

[0009] The object of the present invention is to provide a method for improving the efficiency of lentivirus transfection of macrophages.

[0010] The present invention discovers that by optimizing conditions such as the serum content, protamine content, and transfection volume during lentivirus transfection of macrophages, the positive rate and cell viability of lentivirus transfection of macrophages are effectively improved. On this basis, the present invention further uses siRNA to interfere with the expression of the SAMHD1 gene and combines it with dNTPs treatment, thereby effectively increasing the positive rate of lentivirus transfection of macrophages and successfully maintaining the M1 phenotype of macrophages, with simple operation and remarkable effects. Thus, the present invention achieves the above object.

[0011] Based on this, in order to achieve the object of the present invention, the present invention provides a method for improving the efficiency of lentivirus transfection of macrophages, comprising the following steps:

[0012] 1) Extract CD14+ monocytes from peripheral blood, induce the polarization of CD14+ monocytes into M1 macrophages to obtain macrophages;

[0013] 2) Inhibit the expression of the SAMHD1 gene in macrophages at the transcriptional or translational level;

[0014] 3) The macrophages with reduced SAMHD1 expression obtained in step 2) are treated with dNTPs and then subjected to lentivirus transfection.

[0015] In the present invention, the reference sequence number of the SAMHD1 gene in NCBI is 25939 (Gene ID: 25939).

[0016] Furthermore, the method for inducing the polarization of CD14+ monocytes into M1 macrophages in step 1) includes: adding RPMI 1640 medium containing IFN-γ, GM-CSF, and 10% FBS to a cell culture plate with a cell seeding density of 0.25×10 6 ~0.5×10 6 cells / mL; wherein the final concentration of IFN-γ is 10 - 100 ng / mL, the final concentration of GM-CSF is 10 - 100 ng / mL, and the treatment time is 3 - 7 days.

[0017] Preferably, the final concentration of IFN-γ is 50 ng / mL, and the final concentration of GM-CSF is 50 ng / mL.

[0018] Further, in step 2), the expression of the SAMHD1 gene is reduced by using the siRNA in vitro interference method.

[0019] Preferably, macrophages are treated with siRNA targeting the SAMHD1 gene, and the nucleic acid sequence of the siRNA targeting the SAMHD1 gene is shown as any one of SEQ ID NO: 1-3;

[0020] SEQ ID NO: 1: 5′-GCUUAGUUAUAUCCAGCGA-3′

[0021] SEQ ID NO: 2: 5′-GCAGAUAAGUGAACGAGAU-3′

[0022] SEQ ID NO: 3: 5′-GACAUGGAAGCCUAUACUA-3′

[0023] Preferably, the nucleic acid sequence of the siRNA is shown as SEQ ID NO: 1.

[0024] Further, the method of treating macrophages with siRNA targeting the SAMHD1 gene includes: adding a transfection complex of Lipo3000 liposome transfection reagent and siRNA to the serum-free medium of macrophages, and the final concentration of siRNA for treating macrophages is 10-200 nM, and the treatment time is 2-10 days.

[0025] Preferably, the serum-free medium is ImmunoCult TM -SF Macrophage Medium.

[0026] Further, the following four-plasmid system can be used for lentiviral transfection: pCDH-EF1-GFP-kana, pMD2.G, pMDLG, and pRSV plasmids;

[0027] Preferably, the mass ratio of the plasmids pCDH-EF1-GFP-kana, pMD2.G, pMDLG, and pRSV in the four-plasmid system is 4:3:1:1.

[0028] Preferably, the medium used for dNTPs treatment is a serum-free medium (such as ImmunoCult TM -SF Macrophage Medium), and the final concentration of dNTPs in the medium is 0.04-10 mM. Preferably, the final concentration of dNTPs in the medium is 2.5 mM.

[0029] When performing lentiviral transfection, protamine is added to the serum-free medium to a final concentration of 0.1-10 μg / mL.

[0030] Furthermore, when performing lentiviral transfection, it also includes the steps of adding protamine to the medium and optimizing the concentration of protamine in the medium and the volume of the medium during transfection.

[0031] Preferably, when performing lentiviral transfection, protamine is added to the medium to a final concentration of 6 μg / mL. Preferably, the volume of the medium during transfection is 500 μL.

[0032] By means of the above technical solution, the present invention has at least the following advantages and beneficial effects:

[0033] (1) By optimizing the transfection conditions, the present invention effectively improves the positive rate and cell viability after lentiviral transfection of macrophages.

[0034] (2) On the basis of optimizing the lentiviral transfection conditions of macrophages, by using siRNA to interfere with SAMHD1 in vitro combined with dNTPs treatment, the transfection positive rate can be significantly improved and the M1 phenotype of macrophages can be maintained, and the operation is simple and effective.

[0035] (3) Compared with adenovirus which requires MOI = 100-1000 to transfect macrophages, the infection index of lentivirus in the present invention is MOI = 1-2 and only needs to be transfected once, and the transfection positive rate can be significantly increased to about 60%. This optimization strategy greatly reduces the transfection cost, and at the same time has the potential for industrial production, promoting the feasibility of this method in clinical and industrial applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 It is a schematic diagram of the pCDH-EF1-GFP-kana plasmid structure in Example 1.

[0037] Figure 2 It is a flow cytometry result diagram of the proportion of GFP-positive cells after macrophages are transfected with GFP lentivirus under different MOI conditions in Example 4.

[0038] Figure 3 It is a flow cytometry result diagram of the proportion of live cells after macrophages are transfected with GFP lentivirus under different protamine contents in Example 4, where A-G represent different protamine contents.

[0039] Figure 4 It is a flow cytometry result diagram of the proportion of GFP-positive cells after macrophages are transfected with GFP lentivirus under different transfection volume conditions in Example 4.

[0040] Figure 5The picture of the qRT-PCR detection result after siRNA interfering with the SAMHD1 gene in Example 6.

[0041] Figure 6 The flow cytometry result diagram of the proportion of GFP-positive cells after macrophages were transfected with GFP lentivirus under the condition of MOI = 1.6 after siRNA interfering with the SAMHD1 gene in Example 7.

[0042] Figure 7 The flow cytometry result diagram of the proportion of GFP-positive cells after macrophages were transfected with GFP lentivirus under the condition of MOI = 1.6 after siRNA interfering with the SAMHD1 gene and combined with dNTPs treatment in Example 7.

[0043] Figure 8 The flow cytometry result diagram of the proportion of CD80-positive cells after macrophages transfected with GFP lentivirus after siRNA interference combined with dNTPs treatment in Example 7.

[0044] Figure 9 The flow cytometry result diagram of the proportion of CD86-positive cells after macrophages transfected with GFP lentivirus after siRNA interference combined with dNTPs treatment in Example 7. Detailed implementation manners

[0045] The present invention provides a method for improving the transfection positive rate and maintaining the M1 phenotype of macrophages by in vitro interfering with SAMHD1 with siRNA and combining with dNTPs treatment.

[0046] The present invention adopts the following technical solutions:

[0047] The present invention provides a method for improving the efficiency of lentivirus transfection of macrophages, comprising the following steps:

[0048] (1) Viral packaging and virus collection of HEK293T cells;

[0049] (2) Viral titer determination;

[0050] (3) Extraction of peripheral blood CD14+ monocytes;

[0051] (4) Polarization of monocytes towards M1 macrophages;

[0052] (5) Optimization of the serum content, protamine, and culture medium volume in the culture medium during lentivirus transfection of macrophages;

[0053] (6) Flow cytometry detection of macrophages: The macrophages after lentivirus transfection were digested, centrifuged, and the cell precipitate was collected, resuspended with PBS and filtered through a 70μm sieve, and the positive rate was detected by flow cytometry;

[0054] (7) Design of siRNA targeting SAMHD1 gene;

[0055] (8) Treatment of macrophages with siRNA: Macrophages were treated with siRNA. After treatment, cellular RNA was extracted, reverse transcribed into cDNA, and then the interference efficiency of siRNA was detected by qRT-PCR (fluorescent quantitative PCR);

[0056] (9) Treatment of macrophages with dNTPs;

[0057] (10) Lentiviral transfection of macrophages;

[0058] (11) Detection of macrophages by flow cytometry.

[0059] As a preferred technical solution of the present invention, in step (1), the mass ratio of the lentiviral four-plasmid system plasmids pCDH-EF1-GFP-kana:pMDLG:pMD2.G:pRSV is 4:3:1:1.

[0060] As a preferred technical solution of the present invention, in step (2), THP-1 is a cell growing in suspension, no serum is added to the medium during the transfection process, and the titer is selected within the confidence range of a positive rate of 20% or less.

[0061] As a preferred technical solution of the present invention, in step (4), the cell seeding density is 0.25×10 6 ~0.5×10 6 cells / mL, the medium is RPMI 1640 medium supplemented with a final concentration of 10% FBS, the IFN-γ concentration is 50 ng / mL, the GM-CSF concentration is 50 ng / mL, and the treatment time is 3 to 7 days.

[0062] As a preferred technical solution of the present invention, in step (5), the serum content is 0 to 10%, the final concentration of protamine is 0.1 to 10 μg / mL, the medium volume is 0.25 to 2 mL, and the medium is RPMI 1640.

[0063] As a preferred technical solution of the present invention, in step (7), the siRNA sequences are as shown in SEQ ID NO:1 to SEQ ID NO:3 (synthesized by Beijing Zixi Biotechnology Co., Ltd.).

[0064] SEQ ID NO:1: 5′-GCUUAGUUAUAUCCAGCGA-3′

[0065] SEQ ID NO:2: 5′-GCAGAUAAGUGAACGAGAU-3′

[0066] SEQ ID NO:3: 5'-GACAUGGAAGCCUAUACUA-3'

[0067] As a preferred technical solution of the present invention, in step (8), the siRNA transfection reagent is Lipo3000, the final concentration of siRNA for treating macrophages is 10-200 nM, the treatment time is 2-10 days, and the culture medium is serum-free medium ImmunoCult TM -SF Macrophage Medium.

[0068] As a preferred technical solution of the present invention, the primer sequences for qRT-PCR detection in step (8) are shown as SEQ ID NO:4 and 5.

[0069] SEQ ID NO:4: 5'-atcccaaattgaaagacgcac-3'

[0070] SEQ ID NO:5: 5'-aaaatcttcagccttcagtttcac-3'

[0071] As a preferred technical solution of the present invention, in step (9), the final concentration of dNTPs is 0.04-10 mM, and the culture medium used for dNTPs treatment is serum-free medium, such as ImmunoCult TM -SF Macrophage Medium.

[0072] As a preferred technical solution of the present invention, in steps (6) and (11), the detection time is the 5th day after lentiviral transfection.

[0073] The following examples are used to illustrate the specific applications of the present invention, but do not limit the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well-known to those skilled in the art, and the raw materials used are all commercially available products.

[0074] The FBS and RPMI 1640 culture medium used in the following examples were all purchased from Cytiva, cat. no.: SH30396.02, SH30605.01.

[0075] Example 1 Lentivirus Packaging and Virus Titer Detection

[0076] S1. Lentivirus packaging: When HEK 293T cells are resuscitated and passaged until the confluence reaches 70 - 80%, lentivirus packaging is carried out. First, Opti-MEM (Thermo Fisher Scientific, catalog number: 11058021) and PEI (Sigma-Aldrich (Shanghai) Trading Co., Ltd., catalog number: 26658 - 46 - 8) are mixed and incubated for 20 min. Subsequently, the above mixture and four plasmids (pCDH-EF1-GFP-kana( Figure 1 ))、pMD2.G、pMDLG、pRSV) are mixed and incubated again for 20 min. Then, the above mixture is added to DMEM medium containing 5% FBS (GE Healthcare Life Sciences, catalog number: SH30243.FS) and mixed well. Subsequently, the old medium in the HEK 293T cell culture flask is replaced with the above medium containing plasmids and PEI. After 6 h, it is replaced with DMEM medium containing 10% FBS. After 48 h, the medium is collected and filtered through a 0.22 μm filter membrane to obtain a virus suspension.

[0077] S2. Lentivirus titer detection: The cultured THP-1 cells (Cell Bank of the Chinese Academy of Sciences, catalog number: SCSP-567) are replaced with serum-free RPMI 1640 medium, and the cell density is adjusted to 0.5×10 6 cells / mL. 0.25×10 6 cells are seeded into each well of a 48-well plate. Subsequently, 0.25 μL of protamine (Sigma-Aldrich (Shanghai) Trading Co., Ltd., catalog number: 53597 - 25 - 4, storage concentration: 10 mg / mL) is added to each well, mixed with the medium, and then the virus is added. After 6 h, RPMI 1640 medium containing 30% FBS (GE Healthcare Life Sciences, catalog number: SH30255.01) with half the volume of the transfection volume is supplemented, and it is placed in an incubator at 37 °C and 5% CO2 for culture.

[0078] S3. Flow cytometry detection: After culturing for 48 h, the positive rate of GFP (FITC channel) is detected by flow cytometry. Detection result: The titer of GFP lentivirus is 1.614×10 8 TU / mL.

[0079] Example 2 Extraction of peripheral blood CD14+ monocytes

[0080] S1. PBMC Isolation: Centrifuge peripheral blood at 700 g for 20 min, collect the upper serum into a clean 50 mL centrifuge tube. Place the serum in a 56 °C water bath for 30 min, then centrifuge at 3000 g for 10 min, and take the supernatant for later use. Dilute the lower blood cell suspension with PBS at a ratio of 1:1. Slowly add the diluted blood cell solution to the upper layer of lymphocyte separation medium (Shanghai Beyotime Biotechnology Co., Ltd., product number: C0025-200 mL). Operate according to the instructions of the lymphocyte separation medium to isolate mononuclear cells and count them.

[0081] S2. Extraction of CD14+ Monocytes from Peripheral Blood: Centrifuge the PBMC obtained above at 300 g for 10 min, discard the supernatant, resuspend it with CD14 magnetic bead separation medium, and perform cell sorting according to the instructions of the CD14+ positive selection kit (Miltenyi Biotec GmbH, product number: 130-050-201).

[0082] Example 3 Polarization of Peripheral Blood Monocytes into M1 Macrophages

[0083] S1. Culture of Monocytes: Centrifuge the CD14+ cells obtained in Example 2 above at 300 g for 10 min, resuspend the cell pellet with RPMI 1640 medium containing 10% autologous serum, and adjust the cell density to 1×10 6 cells / mL. Inoculate 2×10 6 cells per well into a 6-well plate and place it in an incubator at 37 °C and 5% CO2 for culture.

[0084] S2. Tendency of Monocytes to Polarize into M1 Macrophages: Aspirate the supernatant of the CD14+ cells plated the previous day, digest them with cell digestive solution (Sigma-Aldrich (Shanghai) Trading Co., Ltd., product number: A6964). After centrifugation, resuspend the cell pellet with RPMI 1640 medium containing 10% autologous serum, IFN-γ with a final concentration of 50 ng / mL (Suzhou Novoprotein Science & Technology Co., Ltd., product number: DC014), and GM-CSF (Suzhou Novoprotein Science & Technology Co., Ltd., product number: DC040). Adjust the cell density, and inoculate 0.5×10 6 cells per well into a 24-well plate, and continue to culture in the incubator, changing the culture medium every 2 days.

[0085] Example 4 Transfection of Macrophages with GFP Lentivirus

[0086] According to the virus titer measured in Example 1, the virus volume required for macrophages to be transfected with GFP lentivirus was calculated. After rinsing the macrophages cultured for 3 to 7 days once with serum-free RPMI 1640 medium, 0.5 to 1 mL of serum-free RPMI 1640 medium was added to each well, and then 0.1 to 0.5 μL of protamine and the corresponding volume of virus were added to each well. After careful mixing, the cells were cultured in an incubator. After 6 h, half of the volume of RPMI 1640 medium containing 30% autologous serum was added to terminate the transfection. After culturing in a 37 °C, 5% CO2 incubator for 5 days, the cells were digested, centrifuged to collect the cells, and the proportion of GFP-positive cells was detected by flow cytometry. The results are as Figures 2 to 4 shown. Under the transfection conditions of serum-free, protamine content of 0.3 μL, and medium volume of 500 μL, the positive infection rate and viability of macrophages were both increased by about 20%.

[0087] It can be seen from the above experimental results that after optimizing the transfection conditions, the positive infection rate and cell viability of macrophages transfected with GFP lentivirus can be significantly improved.

[0088] Example 5 Transfection of siRNA into Macrophages

[0089] Design siRNA targeting SAMHD1 and transfer it into macrophages to interfere with the expression of the SAMHD1 gene, thereby reducing its restrictive effect on the infection of macrophages by HIV-1 lentivirus.

[0090] S1. Formation of siRNA transfection complex: Dilute 1.5 μL of siRNA with a final concentration of 20 μM (any one of the sequences shown in SEQ ID NO: 1-3) with 50 μL of Opti-MEM, gently pipette and mix well. Then dilute 3 μL of Lipofectamine 3000 transfection reagent (Thermo Fisher Scientific, catalog number: L3000015) with 50 μL of Opti-MEM, gently pipette and mix well, and let it stand at room temperature for 5 min. After standing, mix the siRNA mixture with the transfection reagent mixture and let it stand at room temperature for 20 min.

[0091] S2. Transfection of siRNA into macrophages: After rinsing the macrophages cultured for 3 to 7 days once with serum-free medium ImmunoCult TM -SF Macrophage Medium (Stemcell, catalog number 10961), add 400 μL of serum-free medium to each well. Mix the above siRNA complex evenly with the serum-free medium at 100 μL / well and culture it in a 37 °C, 5% CO2 incubator. After 48 - 72 h, detect the interference effect of siRNA.

[0092] Example 6 Detection of siRNA Interference Results

[0093] In macrophages, the expression of the SAMHD1 gene was interfered with by siRNA (SEQ ID NO: 1 - 3). After 48 h, macrophage RNA was extracted using the Tiangen RNAprep Pure Micro Kit (Tiangen Biochemical Technology (Beijing) Co., Ltd., product number: DP420, RT411). After reverse transcribing the RNA into cDNA (Nanjing Novoprotein Biotechnology Co., Ltd., product number: R412 - 01), qRT - PCR detection was performed (the primer sequences used for qRT - PCR detection are shown in SEQ ID NO: 4 - 5 and were synthesized by Beijing Tsingke Biotechnology Co., Ltd.). The detection results are as follows Figure 5 shown. The siRNA treatment significantly reduced the expression level of the SAMHD1 gene, and the inhibition efficiency reached 70% - 90%, indicating that the siRNA has a good gene silencing effect.

[0094] Example 7 siRNA interference combined with dNTPs treatment to improve the positive rate of lentiviral transfection in macrophages

[0095] After confirming the decrease in SAMHD1 gene expression by siRNA interference, a lentiviral transfection experiment was further carried out. GFP lentivirus was transfected under the condition of MOI = 1.6, and it was detected that the GFP positive rate only increased by 4% - 13% ( Figure 6 ). The study found that the half - life of the SAMHD1 protein is relatively long. The siRNA treatment can only inhibit the newly synthesized SAMHD1 protein, while the remaining undegraded SAMHD1 protein in the cells can still exert its dNTPase activity, thus continuing to limit lentiviral infection.

[0096] S1. Treat macrophages with siRNA interference combined with dNTPs: Replace the medium of the macrophages treated with siRNA in Example 5, and add dNTPs (Beyotime Biotechnology Co., Ltd., product number: D7366) with a final concentration of 2.5 mM to the serum - free medium after replacement, and treat for 4 h.

[0097] S2. Transfect macrophages with lentivirus: After 4 h, replace it with a new serum - free medium, add 0.5 μL of protamine and mix gently. Calculate the virus dosage according to the GFP virus titer in Example 1, add the GFP lentivirus to the medium and mix well, and culture in an incubator at 37 °C and 5% CO2 for 5 days, and then detect the proportion of GFP - positive cells. As Figure 7As shown, under the condition of MOI = 1.6, the improvement of the infection efficiency by single siRNA treatment is limited, while the combined treatment of siRNA interference and dNTPs significantly improves the transfection efficiency of lentivirus, increasing the positive rate to ~60%. Further research shows that after the macrophages are treated with siRNA interference and then infected with lentivirus, the decrease in the markers CD80 and CD86 slows down, and the M1 macrophage phenotype is maintained( Figure 8 and Figure 9 ).

[0098] Although the present invention has been described in detail above with general descriptions and specific embodiments, based on the present invention, some modifications or improvements can be made, which are obvious to those skilled in the art. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection required by the present invention.

[0099] References:

[0100] Martinez, F.O., Sica, A., Mantovani, A., & Locati, M. (2008). Macrophage activation and polarization.

[0101] Frontiers in bioscience: a journal and virtual library, 13, 453–461.

[0102] Goldstone DC, Ennis-Adeniran V, Hedden JJ, et al. HIV-1 restriction factor SAMHD1 is a deoxynucleoside triphosphate triphosphohydrolase. Nature. 2011;480(7377):379-382. Hrecka K, Hao C, Gierszewska M, et al. Vpx relieves inhibition of HIV-1 infection of macrophages mediated by the SAMHD1 protein. Nature. 2011;474(7353):658-661。

Claims

1. A method for improving the efficiency of lentivirus transfection of macrophages, characterized in that, It includes the following steps: 1) Extract CD14+ monocytes from peripheral blood, induce the polarization of CD14+ monocytes into M1 macrophages to obtain macrophages; 2) Inhibit the expression of SAMHD1 gene in macrophages at the transcriptional or translational level; 3) The macrophages with reduced SAMHD1 expression obtained in step 2) are treated with dNTPs and then subjected to lentiviral transfection.

2. The method according to claim 1, characterized in that The method for inducing CD14+ monocytes to polarize into M1 macrophages in step 1) includes: adding RPMI 1640 medium containing IFN-γ, GM-CSF, and 10% FBS to a cell culture plate with a cell seeding density of 0.25×10 6 ~0.5×10 6 cells / mL; wherein the final concentration of IFN-γ is 10-100 ng / mL, the final concentration of GM-CSF is 10-100 ng / mL, and the treatment time is 3-7 days; Preferably, the final concentration of IFN-γ is 50 ng / mL, and the final concentration of GM-CSF is 50 ng / mL.

3. The method according to claim 1, wherein In step 2), the siRNA in vitro interference method is used to reduce the expression of SAMHD1 gene.

4. The method according to claim 3, wherein Macrophages are treated with siRNA targeting the SAMHD1 gene, and the nucleic acid sequence of the siRNA targeting the SAMHD1 gene is shown in any one of SEQ ID NO:1-3; Preferably, the nucleic acid sequence of the siRNA is shown in SEQ ID NO:

1.

5. The method according to claim 4, characterized in that, Treating macrophages with siRNA targeting the SAMHD1 gene includes: adding a transfection complex of Lipo3000 liposome transfection reagent and siRNA to the serum-free medium of macrophages. The final concentration of siRNA for treating macrophages is 10-200 nM, and the treatment time is 2-10 days.

6. The method according to claim 1, characterized in that, The following four-plasmid system is used for lentiviral transfection: pCDH-EF1-GFP-kana, pMD2.G, pMDLG, and pRSV plasmids.

7. The method according to claim 6, wherein The mass ratio of the plasmids pCDH-EF1-GFP-kana, pMD2.G, pMDLG, and pRSV in the four-plasmid system is 4:3:1:

1.

8. The method according to claim 1, wherein The medium used for dNTPs treatment is a serum-free medium, and the final concentration of dNTPs in the medium is 0.04-10 mM.

9. The method according to any one of claims 1-8, characterized in that, When performing lentiviral transfection, it also includes the step of adding protamine to the medium and optimizing the concentration of protamine in the medium and the volume of the medium during transfection.

10. The method according to claim 9, characterized in that, When performing lentiviral transfection, protamine is added to the medium to a final concentration of 0.1-10 μg / mL; the volume of the medium during transfection is 500 μL.

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