A method for constructing ARHGAP9 gene knockout cells and its application in anti-tumor

Abstract

The present invention belongs to the field of biomedicine technology, and specifically relates to a method for constructing an ARHGAP9 gene knockout cell and its application in anti-tumor. The present invention first provides a method for constructing an ARHGAP9 gene knockout cell line, comprising: (1) determining the target site of a gene to be knocked out of a cell, wherein the gene to be knocked out is ARHGAP9, and the target site is an exon; (2) designing a gRNA targeting the exon; (3) constructing a plasmid with the gRNA, further using the constructed plasmid to package a lentivirus to infect cells, and flow-sorting positively infected cells.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and specifically relates to a method for constructing ARHGAP9 gene knockout cells and an application thereof in anti-tumor. Background Art

[0002] In recent years, cell therapies represented by CAR-T and CAR-NK have achieved great success in clinical cancer treatment. The full name of CAR-T therapy is chimeric antigen receptor T cell therapy. CAR-T cells are genetically engineered to express T cells with chimeric receptors targeting specific antigens. The expression of CAR molecules incubates T cells to recognize and eliminate tumor cells expressing the corresponding antigens to achieve the goal of treating tumors. At present, there are 9 CAR-T therapies that have been put on the market for clinical use, of which 6 target CD19 and 3 target BCMA, which are used for the treatment of B cell lymphoma and multiple myeloma respectively. In addition, a number of dual-target CAR-T cell products are also in clinical trials. CAR-NK therapy is similar to CAR-T therapy. It also uses genetic engineering methods to express CAR molecules on NK cells. In clinical trials for anti-tumor treatment, the response rate reached 73%, significantly prolonging the survival of patients. In addition, CAR-NK therapy has been reported to have better safety than CAR-T therapy, does not cause fatal cytokine storm and neurotoxicity, and can be prepared in advance without HLA matching (N Engl J Med. 2020 Feb 6; 382(6): 545-553.). In order to further improve the clinical efficacy of CAR-T therapy or CAR-NK therapy, there are currently a large number of reports on the combined use of immune checkpoint inhibitors such as pembrolizumab, nivolumab, and ipilimumab to block the PD-1 and CTLA-4 inhibitory receptors of CAR-T cells, or to use gene editing strategies to knock out immune checkpoint receptors PD1 and TIGIT in CAR-T cells, which can effectively enhance the anti-tumor effect of CAR-T therapy (Mol Cancer. 2022 Mar 18; 21(1): 78; Front Immunol. 2023 Jun 9: 14: 1175920.). However, the current effects of CAR-NK therapy and CAR-T therapy in solid tumors are still poor, mainly due to the immunosuppressive microenvironment of solid tumors, which causes the infused CAR-T or CAR-NK to infiltrate solid tumors and inhibit their function in the solid tumor microenvironment. Therefore, it is necessary to explore new strategies that may enhance the anti-tumor effect of CAR-T cell or CAR-NK cell therapy.

[0003] The Rho (RAS homolog) family is a class of Ras superfamily members containing a GTPase domain. Its activity is regulated by its GTPase domain, and it functions as a molecular switch by switching between inactive Rho-GDP and active Rho-GTP. Rho family proteins have complex functions and are involved in cell morphology, movement, division, gene expression, and cytoskeleton regulation. They are currently known to be related to cardiovascular diseases, tumor invasion and metastasis, autoimmune diseases, Parkinson's disease, and other diseases. The Rho-GAPs subfamily is an inhibitory molecule of Rho-GTP signaling, with 47 known members. ARHGAP9 (RhoGTPase activating protein 9) is one of the members of the Rho-GAPs subfamily. However, there are contradictions in the current reports on the functions of ARHGAP9 in cancer. Some articles report that ARHGAP9 is a pro-oncogene, including that ARHGAP9 can promote the proliferation and metastasis of colon cancer cells (Sun et al. Tissue cell. 2022); ARHGAP9 is highly expressed in ovarian cancer and negatively correlated with the patient's prognosis, and can be used as a prognostic indicator for ovarian cancer (Shen et al. Transl Cancer Res. 2021; 10:4440-4453.). However, there are also contrary reports suggesting that ARHGAP9 is a tumor suppressor gene, including bioinformatics analysis that found that compared with patients with low ARHGAP9 expression, patients with breast cancer and bladder cancer with high ARHGAP9 expression had longer survival and better prognosis (Chen et al. Oncol Lett. 2019; 18: 6017-6025.); ARHGAP9 inhibited the proliferation and migration of liver cancer cells (Zhang et al. Cell Death Dis. 2018; 9: 916.); ARHGAP9 knockout can promote lung cancer metastasis (Song et al. Genomics. 2023). Therefore, the role of ARHGAP9 in the occurrence and development of cancer is still controversial. However, there are no reports linking the expression of ARHGAP9 to the efficacy of CAR-T or CAR-NK. Summary of the invention

[0004] In view of this, the purpose of the present invention is to provide a method for constructing ARHGAP9 gene knockout cells and their application in anti-tumor, wherein the cells include immune cells and immune-engineered cells, and the specific technical scheme is as follows.

[0005] A method for constructing an ARHGAP9 gene knockout cell line comprises the following steps:

[0006] S01: determining the target site of a gene to be knocked out in a cell, wherein the gene to be knocked out is ARHGAP9, and the target site is an exon;

[0007] S02: Designing a gRNA targeting the exon, wherein the sequence of the gRNA includes SEQ ID NO.1, NO.2, NO.3, NO.4 or NO.5;

[0008] S03: Use the gRNA designed in S02 to construct a plasmid, further use the constructed plasmid to package lentivirus to infect cells, and flow cytometry select positively infected cells.

[0009] Furthermore, the cells include immune cells; the immune cells include T cells and / or NK cells.

[0010] Furthermore, the cells include immune-engineered cells; the immune-engineered cells include CAR-T cells and / or CAR-NK cells.

[0011] Further, the plasmid expresses Crispr-Cas9 and the gRNA; or, the Cas9 purified protein is transfected and the plasmid expresses the gRNA at the same time.

[0012] Furthermore, the method uses a Crispr single-base editor to edit the ARHGAP9 start codon or the first and last sites of the exon to achieve the effect of knocking out ARHGAP9.

[0013] As a preferred embodiment, the core elements used to construct the plasmid include U6-gRNA-scaffold and a CD19-CAR containing a GFP green light expression sequence (CD19-CAR-GFP); the sequence of the U6-gRNA-scaffold is shown in SEQ ID NO.7; the sequence of the CD19-CAR containing a GFP green light expression sequence is shown in SEQ ID NO.8.

[0014] Application of the ARHGAP9 gene knockout cell line prepared by the above construction method in the preparation of anti-tumor reagents or anti-tumor drugs.

[0015] Furthermore, the tumor includes blood tumor and solid tumor.

[0016] Furthermore, the tumor targets include CD19, BCMA, CD20, CD22, EGFRvIII, CD123, CD33, HER2, CD38, CD70, PSMA, B7-H3, GPC3, GCC, Claudin18.2, CEA, CD5, EpCAM, GD2 and / or Mesothelin, etc.

[0017] It is understandable that the above targets are common solid tumor or hematological tumor targets, and the experiments in the embodiments of the present invention have demonstrated that immune cells and immune-engineered cells with ARHGAP9 knockout are effective against both solid tumors and hematological tumors.

[0018] A kit for knocking out ARHGAP9 based on CRISPR-Cas 9, comprising:

[0019] (1) a gRNA for targeted knockout of ARHGAP9 gene exons, wherein the sequence of the gRNA includes SEQ ID NO.1, NO.2, NO.3, NO.4 or NO.5;

[0020] (2) a plasmid, wherein the plasmid expresses Crispr-Cas9 and the gRNA; or, transfecting Cas9 purified protein while the plasmid expresses the gRNA;

[0021] (3) viral capsid plasmid pMD2.G and / or pSPAX2 plasmid, transfection reagent and / or infection enhancing reagent;

[0022] Optional, including transfection reagent Lipo8000, infection enhancement reagent TransGA, 10% sucrose concentrate, RetroNectin, sterile PBS, etc.

[0023] (4) NK cell culture medium or T cell culture medium, antibody.

[0024] In some optional ways, the antibody can be selected from anti-CD3 antibody or anti-CD28 antibody to stimulate T cells and promote proliferation.

[0025] In one embodiment of the present invention, the components of the culture medium include RPMI1640, 10% FBS, 50 μM 2-mercaptoethanol, 1× sodium pyruvate, 1× non-essential amino acids, 1% penicillin / streptomycin, 25 mM HEPES, 50 U / mL mouse IL-2, 5 ng / mL mouse IL-7 and 25 ng / mL IL-15.

[0026] It is understood that the above cell culture medium is a conventional cell culture medium, and the components of the above cell culture medium are only examples, not limitations. Other culture media that can culture T cells or NK cells can also be used.

[0027] As a preferred embodiment, the core elements used to construct the plasmid include U6-gRNA-scaffold and a CD19-CAR containing a GFP green light expression sequence; the sequence of the U6-gRNA-scaffold is shown in SEQ ID NO.7; the sequence of the CD19-CAR containing a GFP green light expression sequence is shown in SEQ ID NO.8.

[0028] Beneficial technical effects

[0029] The present invention first screened potential immune checkpoints through the Crispr / gRNA whole genome library and found that ARHGAP9, a member of the Rho-GAPs subfamily, can inhibit NK and T cell activation and has the potential to inhibit NK and T cell-mediated anti-tumor immune responses. The in vitro experiments of the present invention confirmed that knocking out ARHGAP9 can effectively improve the ability of NK and T cells to release cytokines and enhance the killing activity of NK and T cells against various tumor cells. In addition, it was confirmed in NK and T cell conditional knockout mice that ARHGAP9 knockout can activate NK and CD8 in vivo. + T cells, NK and CD8 + The anti-tumor effect of T cells prolongs the survival of mice.

[0030] Furthermore, the present invention proposes to use Crispr / gRNA gene editing method to knock out the ARHGAP9 gene in CAR-T and CAR-NK cells, which is confirmed to improve the anti-tumor activity of CAR-T and CAR-NK cells.

[0031] The present invention designs and optimizes gRNA for ARHGAP9 exon 1 and exon 2, targeting different sites of the exons, and the preferred sequences include those shown in SEQ ID NO.1 (targeted editing Exon1: sites 97-103), NO.2 (targeted editing Exon1: sites 141-147), NO.3 (targeted editing Exon2: sites 21-27), NO.4 (targeted editing Exon2: sites 27-33) or NO.5 (targeted editing Exon2: sites 1-6), all of which can effectively knock out ARHGAP9. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the prior art descriptions are briefly introduced below. The drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative labor.

[0033] Figure 1 Screening of ARHGAP9 as an immunosuppressive molecule from the CRISPR / gRNA library and analysis of its expression profile;

[0034] Figure 2 Knockout of ARHGAP9 can activate T cells to release cytokines;

[0035] Figure 3 Knockout of ARHGAP9 can activate NK cells to kill various tumor cells;

[0036] Figure 4Conditional knockout mice confirmed that ARHGAP9 knockout can activate tumor-infiltrating NK and CD8 + T cell anticancer activity;

[0037] Figure 5 Map for gARH-hCD19-CAR-GFP All-in-one plasmid construction;

[0038] Figure 6 Gene editing to knock out ARHGAP9 enhances the anti-tumor effects of CAR-T and CAR-NK. DETAILED DESCRIPTION

[0039] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0040] Herein "and / or" includes any and all combinations of one or more of the associated listed items.

[0041] Herein, "plurality" means two or more than two, ie, it includes two, three, four, five, etc.

[0042] It should be noted that, in this article, the terms "include", "comprises" or any other variations thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device. In the absence of further restrictions, an element defined by the sentence "comprises a ..." does not exclude the existence of other identical elements in the process, method, article or device including the element.

[0043] As used in this specification, the term "about" typically means that the value + / -5%, more typically the value + / -4%, more typically the value of + / -3%, more typically the value of + / -2%, or even more typically + / -1%, or even more typically the value + / -0.5%.

[0044] In this specification, some embodiments may be disclosed in a format of being within a certain range. It should be understood that such description of "being within a certain range" is only for convenience and brevity, and should not be interpreted as a rigid limitation on the disclosed range. Therefore, the description of the range should be considered to have specifically disclosed all possible sub-ranges and independent numerical values ​​within this range. For example, the range The description of should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within this range, for example, 1, 2, 3, 4, 5, and 6. The above rules apply regardless of the breadth of the range.

[0045] Specific drawings:

[0046] Figure 1 A is the Crispr / gRNA whole genome library virus-infected NK92MI-Cas9 cells and OT-I-Cas9 cells (CD8+T cells that specifically recognize the chicken ovalbumin OVA antigen), which were then co-cultured with tumor cells, and NK and OT-I cells expressing the activation molecule CD69 were flow-sorted for high-throughput sequencing. Figure 1 B is a volcano plot of NK cell screening and enrichment of gRNA. Figure 1 C is a volcano plot of gRNA enrichment screening in OT-I cells. Figure 1 D is the analysis of ARHGAP9 expression profile in various cell types in human blood using the Protein Atlas human single-cell database. Figure 1 E is the qPCR detection of ARHGAP9 in human NK and CD8 + T, expression in various types of leukemia cells, solid tumor cells, and normal cell lines.

[0047] Figure 2 A is the Western blot detection of ARHGAP9 expression knocked out in Jurkat cells by Crispr / gRNA method; Figure 2 BC: qPCR detection of the effect of knocking out AHRGAP9 on the mRNA expression of factors in Jurkat cells.

[0048] Figure 3 A is a schematic diagram of the method of knocking out the ARHGAP9 gene in NK92MI cells using the Crispr / gRNA method; Figure 3 B is Western blot detection of ARHGAP9 expression knocked out in NK92MI cells by Crispr / gRNA method; Figure 3 C is the co-culture of control or AHRGAP9 knockout NK92MI cells with different tumor cells at different concentrations, and the cell lysis rate was detected by calcein; Figure 3D is flow cytometry detection of cytokine expression in control or AHRGAP9 knockout NK92MI cells; Figure 3 E is the effect of knocking out AHRGAP9 on the mRNA expression of NK92MI cytokines detected by qPCR.

[0049] Figure 4 A is the strategy for constructing ARHGAP9 conditional knockout mice; Figure 4 B is the genotype identification of ARHGAP9 conditional knockout mice; Figure 4 CD is the effect of ARHGAP9 conditional knockout mice against leukemia E.G7 cell transplants and colon cancer MC38 transplants; Figure 4 E is the flow cytometry detection of cytokine release by tumor-infiltrating NK cells in transplanted tumors; Figure 4 F is the flow cytometry detection of cytokine release by tumor-infiltrating T cells in transplanted tumors.

[0050] Figure 5 This is the All-in-One single plasmid gARH-hCD19-CAR-GFP map.

[0051] Figure 6 AB is the anticancer cell transplant tumor growth effect of CAR-T cells with ARHGAP9 knocked out by gene editing; Figure 6 CD shows the anticancer cell transplant tumor growth effect of CAR-NK cells with ARHGAP9 knocked out by gene editing.

[0052] Example 1

[0053] The research team of the present invention respectively identified CD8 + The Crispr / gRNA whole genome library was screened on T cells. After co-culture of NK and OT-I cells infected with the Crispr / gRNA whole genome library virus with mouse lymphoma cells (Yac-1) or mouse colon cancer cells (MC38-OVA) expressing OVA antigen, NK and OT-I cells expressing the activation molecule CD69 were flow cytometry sorted for high-throughput sequencing. The sequencing results showed that ARHGAP9 was significantly enriched in both NK and OT-I cell screening, suggesting that gRNA knockout of ARHGAP9 may activate NK cells and T cells ( Figure 1 AC), on the contrary, ARHGAP9 may inhibit the activation of NK cells and T cells. Single cell expression profiling analysis also found that ARHGAP9 is specifically highly expressed in NK and CD8 + T cells ( Figure 1 D). Furthermore, qPCR experiments were used to detect the expression of ARHGAP9 on various cells. The experiment found that ARHGAP9 was expressed in NK and CD8 +Highly expressed on T cells ( Figure 1 E).

[0054] Example 2

[0055] In vitro knockout of ARHGAP9 can activate T cells to release cytokines.

[0056] ARHGAP9 was knocked out in T cells in vitro, and the control group used 1 Crispr / gRNA plasmid to package lentivirus, and the knockout group used 5 Crispr / gRNA plasmids to package lentivirus, infecting Jurkat cells (human T lymphocyte leukemia cells) to construct an ARHGAP9 knockout cell line. The gRNA information involved in this example is shown in Table 1.

[0057] Table 1

[0058]

[0059] Western blot was used to detect the knockout effect. The gRNAs shown in SEQ ID NO.1-5 in Table 1 can effectively knock out ARHGAP9 expression in Jurkat cells ( Figure 2 A). Furthermore, qPCR was used to detect changes in cytokine expression in knockout cell lines. The results showed that knockout of ARHGAP9 by gRNAs shown in SEQ ID NO.1-5 could significantly enhance the expression levels of IFNγ and TNFα in Jurkat cells ( Figure 2 BC). This experiment demonstrated that knocking out ARHGAP9 in T cells can activate T cells to release cytokines.

[0060] Example 3

[0061] In vitro knockout of ARHGAP9 can activate NK cells to kill a variety of cancer cells.

[0062] Furthermore, ARHGAP9 was knocked out in NK cells in vitro. The control group used one Crispr / gRNA plasmid to package lentivirus, and the knockout group used three Crispr / gRNA plasmids to package lentivirus, which infected NK92MI cells (NK cells from patients with malignant non-Hodgkin's lymphoma) to construct an ARHGAP9 knockout cell line. Three monoclones were selected for the control group and six monoclones were selected for the treatment group. Western blot was used to verify the knockout effect. The results showed that the ARHGAP9 knockout cell line of NK92MI was successfully constructed. Monoclonal cells including three control clones (Con gRNA1-1, Con gRNA1-2, Con gRNA1-3), gARH#3-3, gARH#4-4, and gARH#5-3 were used for subsequent experiments ( Figure 3AB). The gRNA information involved in this embodiment is shown in Table 2. The specific sequence information is as follows.

[0063] Table 2

[0064]

[0065] The results showed that gRNAs of SEQ ID NO.3, NO.4 and NO.5 could effectively knock out ARHGAP9 expression in NK92MI cells ( Figure 3 AB). Calcein-AM was used to stain acute leukemia cells (THP1, U937, Molm-13, Mono-Mac-6), large B-cell lymphoma cells (DB), non-small cell lung cancer cells (NCI-H1299), prostate cancer (DU-145), ovarian cancer (OVCAR3), breast cancer cells (MCF7), and melanoma cells (Colo-858). The above cells were co-cultured with the constructed ARHGAP9 knockout NK92MI cells. Among them, the ARHGAP9 knockout NK92MI cells are effector cells (Effector, E) and the tumor cells are target cells (Target, T).

[0066] Detecting the Calcein fluorescence released into the culture medium after target cell death can reflect the target cell death.

[0067] The results showed that ARHGAP9 knockout could significantly increase the killing effect of NK92MI cells on target cancer cells, with a good dose-effect relationship ( Figure 3 C), where E represents NK92MI cells with ARHGAP9 knocked out; T represents tumor cells. Flow cytometry analysis further confirmed that ARHGAP9 knockout increased the expression of NK cell activation markers (including TNFα, IFNγ, GZMB, and Perforin) ( Figure 3 D). qPCR detection of mRNA expression of NK cell activation markers also confirmed that compared with NK92MI cells without ARHGAP9 knockout, ARHGAP9 knockout significantly increased the release of cytokines by NK92MI cells, including TNFα, IFNγ, GZMB, and Perforin ( Figure 3 E).

[0068] Example 4

[0069] Conditional knockout mice confirmed in vivo that ARHGAP9 knockout can activate NK cells and T cells to release cytokines, inhibit tumor growth, and prolong mouse survival.

[0070] To further verify whether ARHGAP9 has an immunosuppressive effect, this example constructed ARHGAP9fl / fl Mice were bred with NCR1-Cre and CD4-Cre mice to obtain offspring NK cell conditional knockout ARHGAP9 mice (ARHGAP9 fl / fl NCR1-Cre and T cell conditional knockout ARHGAP9 mice (ARHGAP9 fl / fl CD4-Cre). Knockout mouse construction strategy is as follows ( Figure 4 A) shows that the knockout mice were successfully constructed by genotyping verification ( Figure 4 B).

[0071] Using ARHGAP9 + / + NCR1-Cre mice and ARHGAP9 + / + CD4-Cre mice were used as negative controls. Mice were subcutaneously inoculated with colon cancer cells MC38 and leukemia cells E.G7, and the volume of transplanted tumors and the survival time of mice were recorded every other day. The results showed that conditional knockout of ARHGAP9 in NK or T cells could significantly inhibit the growth of transplanted tumors in mice and prolong the survival time of mice ( Figure 4 CD), as can be seen in the figure, as time goes by, the tumor size decreases and the survival rate of mice increases. It is worth noting that the method used in this example to record the change in tumor size of mice after inoculation of leukemia cells E.G7 is in line with the technical operation specifications in this field.

[0072] Furthermore, the infiltrating lymphocytes in the transplanted tumor tissue were separated by Percoll separation, and NK and CD8 + T cell and immune activation markers. The results showed that ARHGAP9 can significantly increase the proportion of tumor-infiltrating NK cells expressing IFNγ, TNFα, and GZMB after NK cell knockout ( Figure 4 E). At the same time, ARHGAP9 can also increase tumor infiltrating CD8 + T and CD4 + T cells express IFNγ, TNFα ( Figure 4 F).

[0073] In summary, the results of this example can confirm that ARHGAP9 is an immunosuppressive molecule, and knocking out ARHGAP9 can activate NK and T cells to release cytokines and enhance anti-tumor immunity.

[0074] Example 5

[0075] The previous examples have confirmed that ARHGAP9 is found in NK and CD8 + The expression of ARHGAP9 in T cells is much higher than that in tumor cells and normal cell lines, and knockout of ARHGAP9 can effectively activate NK and CD8 +Therefore, this example continues to verify the effect of knocking out ARHGAP9 in enhancing the activity of CAR-T and CAR-NK cells.

[0076] The gRNA shown in SEQ ID NO.5 shown in Table 1 was used to construct the plasmid, and the U6-gRNA-scaffold was fused with the CD19-CAR-GFP sequence to construct an All-in-One single plasmid gARH-hCD19-CAR-GFP plasmid. The core elements of the plasmid are as follows Figure 5 The core element sequence of the gARH-hCD19-CAR-GFP plasmid is shown in Table 3.

[0077] Table 3

[0078]

[0079]

[0080] The plasmids in Table 3 were used to package lentivirus, and then infect CAR-T and CAR-NK cells. The detailed steps are as follows.

[0081] (1) Day 1: 293FT cells were plated. The healing rate was about 70% on the second day. The culture medium formula was DMEM + 10% FBS + 1% BSA.

[0082] (2) Day 2: Co-transfect hCD19-CAR-GFP plasmid and viral capsid plasmid, the plasmid ratio is shown in Table 4. Carefully replace the 293FT cell culture medium, add 10 mL of fresh culture medium, drop the mixture of liposomes and plasmids into the culture plate, and return to the incubator for 48 h.

[0083] Table 4

[0084]

[0085] (3) Day 3: Add NK92MI or CD8 + T cells were added to each well at 1 × 10 5 Cells were cultured overnight for subsequent titer detection.

[0086] (4) Day 4: Collect the virus, disinfect the ultracentrifuge cap, sleeve, and ultracentrifuge tube with 70% ethanol, wash the ultracentrifuge tube twice with PBS to remove the residual ethanol, dry it, and disinfect it under ultraviolet for 30 minutes for later use. Collect the virus supernatant with a syringe and filter it into an ultracentrifuge tube using a 0.45μm filter membrane. Add 8mL of lentivirus culture medium to the cells and culture them for 24 hours for secondary virus collection. Pipette 2.5mL of 10% sucrose concentrate (100mM NaCl, 0.5mM EDTA 2Na2 H 2 O, 10% sucrose, 50mM Tris-HCl (pH7.4), carefully add to the bottom of the centrifuge tube. Put the balance into the biosafety cabinet, add sterile PBS to ensure that the weight difference of the 6 tubes is less than 0.1g, insert the sleeve, and tighten the lid. Then centrifuge at 10000g and 4℃ for 4h. Unscrew the lid in the biosafety cabinet, take out the centrifuge tube with tweezers, pour out all the supernatant, evaporate for 3min, resuspend the virus with 200μL sterile PBS (without calcium and magnesium), and freeze it at -80℃ after 50μL aliquoting.

[0087] (5) Leave a small amount of virus, take 10 μL and add it to 90 μL PBS, mix well, take another 10 μL and add it to 90 μL PBS, and dilute it 10 times (1, 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 The diluted virus was added to a 96-well plate in which 293FT was cultured at 10 μL / well, and 4 μL of infection enhancement reagent TransGA (Gene Gene) was added to each well and cultured overnight for determination of virus titer.

[0088] (6) Day 5: Take out the 96-well plate, count the number of GFP-positive cells under a microscope, and calculate the virus titer based on the virus dilution factor. -4 If there is one positive GFP cell in the well, the virus titer is: 1 / 10 -4 =1×10 4 TU / μL=1×10 7 TU / ml.

[0089] (7) Infecting target cells: Isolating CD8 + T cells, the operation method is based on EasySep Mouse CD8 + T Cell Isolation Kit instructions (Stem cell #19853). Isolated T cells were stimulated with CD3 / CD38 antibodies in vitro for 2 days. The culture medium components were: RPMI1640, 10% FBS, 50 μM 2-mercaptoethanol, 1× sodium pyruvate, 1× non-essential amino acids, 1% penicillin / streptomycin, 25 mM HEPES, 50 U / mL mouse IL2, 5 ng / mL mouse IL-7 and 25 ng / mL IL-15.

[0090] The culture conditions of the target cell Cas9-expressing NK92MI cells were: α-MEM supplemented with 10% FBS, 0.1 mM 2-mercaptoethanol, 1% penicillin / streptomycin, 0.2 mM inositol, and 0.02 mM folic acid.

[0091] (8) Day 6: Add 50 μg / mL RetroNectin (recombinant human fibronectin, Takara, T100A) into a 6-well plate and coat the plate overnight.

[0092] (9) Day 7: Pipette out RetroNectin, add lentivirus to the plate at an MOI of 10 based on the measured virus titer, and centrifuge at 3200 rpm and 32°C for 2 h. Discard the supernatant and wash the plate once with PBS. + T and NK92MI cells were collected after centrifugation at 1200 rpm for 5 min, and 1×10 5 Each cell was added to a 6-well plate and cultured for 3 days using T cell or NK cell culture medium.

[0093] (10) Day 10: Collect cells by centrifugation, select GFP-positive cells by flow cytometry, and count 3×10 6 The dose of 10 cells / mouse was adopted into mice that had been subcutaneously inoculated with colon cancer MC38-hCD19 or Daudi cells through the tail vein. CAR-T was adopted once. CAR-NK was adopted once every 3 days, for a total of 4 times. Subsequently, the growth of the transplanted tumor was monitored every other day and the survival time of the mice was recorded. The transplanted tumor volume was >2000mm 3 Set as ethical death endpoint.

[0094] Experimental results: Figure 6 As shown, ARHGAP9 knocked out CAR-T and CAR-NK cells have stronger tumor suppression ability. Compared with CAR-T and CAR-NK cells without ARHGAP9 knockout, ARHGAP9 knocked out CAR-T and CAR-NK cells significantly inhibited the growth of MC38-hCD19 or Daudi cell transplanted tumors and prolonged the survival of mice. The results in this part show that the gene editing method provided by the present invention can knock out ARHGAP9 in CAR-T and CAR-NK cells, thereby effectively enhancing the anti-tumor effect of CAR-T and CAR-NK cell therapy.

[0095] The embodiments of the present invention are described above in conjunction with the accompanying drawings, but the present invention is not limited to the above-mentioned specific implementation modes, which are merely illustrative rather than restrictive. Under the guidance of the present invention, ordinary technicians in this field can also make many forms without departing from the scope of protection of the present invention and the claims, all of which are within the protection of the present invention.

Claims

1. A method for constructing an ARHGAP9 gene knockout cell line, characterized in that: The following steps are involved: S01: determining a target site of a gene to be knocked out in a cell, wherein the gene to be knocked out is ARHGAP9, and the target site is an exon; the cell is an immune cell, and the immune cell is a T cell and / or a NK cell; S02: Designing a gRNA targeting the exon, wherein the sequence of the gRNA is shown in SEQ ID NO.1, NO.2, NO.3, NO.4 or NO.5; S03: Use the gRNA designed in S02 to construct a plasmid, further use the constructed plasmid to package the lentivirus to infect the immune cells, and flow-sort the positively infected cells; when the cells used for ARHGAP9 gene knockout are T cells, the sequence of the gRNA is shown in any one of SEQ ID NO.1-5; when the cells used for ARHGAP9 gene knockout are NK cells, the sequence of the gRNA is shown in any one of SEQ ID NO.3-5.

2. The construction method according to claim 1, characterized in that: The cells are immune engineered cells; the immune engineered cells are CAR-T cells and / or CAR-NK cells.

3. The construction method according to claim 1, characterized in that: The plasmid expresses Crispr-Cas9 and the gRNA; alternatively, the Cas9 purified protein is transfected and the plasmid expresses the gRNA at the same time.

4. The construction method according to claim 1, characterized in that: The core elements used to construct the plasmid include U6-gRNA-scaffold and CD19-CAR containing a GFP green light expression sequence; the sequence of the U6-gRNA-scaffold is shown in SEQ ID NO.7; the sequence of the CD19-CAR containing a GFP green light expression sequence is shown in SEQ ID NO.

8.

5. Use of the ARHGAP9 gene knockout cell line prepared by the construction method of any one of claims 1 to 4 in the preparation of anti-tumor agents or anti-tumor drugs.

6. The use according to claim 5, characterized in that The tumors include blood tumors and solid tumors.

7. The use according to claim 6, characterized in that The tumor targets include CD19, BCMA, CD20, CD22, EGFRvIII, CD123, CD33, HER2, CD38, CD70, PSMA, B7-H3, GPC3, GCC, Claudin18.2, CEA, CD5, EpCAM, GD2 and / or Mesothelin.

8. A kit for knocking out ARHGAP9 based on CRISPR-Cas 9, characterized in that: include: (1) a gRNA for targeted knockout of an exon of the ARHGAP9 gene, wherein the sequence of the gRNA is shown in SEQ ID NO.1, NO.2, NO.3, NO.4 or NO.5; (2) a plasmid, wherein the plasmid expresses Crispr-Cas9 and the gRNA; or, the Cas9 purified protein and the plasmid expresses the gRNA; (3) Virus capsid plasmid pMD2.G and / or pSPAX2 plasmid, transfection reagent and / or infection enhancing reagent; (4) NK cell culture medium or T cell culture medium, antibodies.

9. The kit according to claim 8, characterized in that The core elements used to construct the plasmid include U6-gRNA-scaffold and CD19-CAR containing a GFP green light expression sequence; the sequence of the U6-gRNA-scaffold is shown in SEQ ID NO.7; the sequence of the CD19-CAR containing a GFP green light expression sequence is shown in SEQ ID NO.8.

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