ShRNA for inhibiting FAS gene expression, immune cell and application thereof

Abstract

The invention relates to the technical field of biology, in particular to shRNA for inhibiting FAS gene expression, an immune cell and application of the shRNA. The invention provides a shRNA molecule and application thereof, the shRNA molecule targets FAS expression, and can continuously, stably, efficiently and specifically inhibit FAS gene expression; according to the invention, the FAS gene is silenced, so that the multiplication capacity and the apoptosis resistance of the immune cells in a complex environment are effectively improved, the treatment effect of the immune cells in tumor immunotherapy is further improved, and the promotion effect on the promotion and the improvement of the tumor immunotherapy effect is achieved.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly to shRNAs that inhibit FAS gene expression, immune cells, and their applications. Background Art

[0002] Gene-modified cell therapy is a novel treatment method that treats diseases by modifying the genomes of cells in patients. This technology uses genetic engineering techniques to introduce foreign genes or regulatory factors into immune cells, endowing these immune cells with new functions or stronger therapeutic potential. Gene-modified cell technology is widely applied to immune cells such as NK cells, T cells, NKT cells, γδT cells, and macrophages for immunotherapy.

[0003] Apoptosis is an active cell death process triggered by death signals or changes in the internal and external environments, as well as under gene regulation. It is an important mechanism for multicellular organisms to regulate body development, maintain internal environmental stability, and control cell senescence. The occurrence and progression of apoptosis are precisely regulated and have a unique and complex signal transduction system. The FAS / FASL system is a pair of glycoprotein molecules that mediate apoptosis in the body and plays an important role in maintaining immune homeostasis and the occurrence and development of tumors. In most tumor cells, the expression of FAS decreases, while the expression of FASL increases, inducing apoptosis of immune effector cells in the tumor microenvironment and directly or indirectly promoting tumors to evade the surveillance of the body's immune system, thus facilitating the invasion and metastasis of tumor cells.

[0004] The FAS / FASL pathway is a very effective NK cell-mediated cell killing mechanism. The FASL expressed by NK cells binds to FAS on target cells to transmit a "death signal" to the target cells, inducing rapid apoptosis of the target cells. FAS functions as a cell death receptor, and the ligand FASL expressed by immune cells interacts with FAS expressed by tumor cells to induce apoptosis of tumor cells. However, during the progression of cancer cells, the expression of FAS is downregulated and the expression of FASL is increased, activating the FAS / FASL apoptosis signal and inducing apoptosis of immune cells, enabling tumor cells to evade immune surveillance. Moreover, during the culture and expansion of immune cells, the cell death receptor FAS is highly expressed, which can lead to the reverse killing of immune cells by tumor cells or induction of apoptosis in the tumor microenvironment, and there may also be mutual killing or suicide mediated by the FAS / FASL apoptosis signal between immune cells. In immunotherapy, immune cells with high expression of FAS may also be rejected allogeneically, weakening the killing efficacy of immune cells and reducing the durability of the efficacy.

[0005] Therefore, there is an urgent need to further design and develop new immunotherapy strategies targeting FAS to improve the survival ability and durability of immune cells in the complex tumor microenvironment and stimulate the potential of immune cells to treat tumors. SUMMARY OF THE INVENTION

[0006] The present invention aims to solve at least one of the technical problems in the related art to some extent. To this end, an object of the present invention is to provide an shRNA molecule and its application. The shRNA molecule targets FAS expression and can continuously, stably, efficiently and specifically inhibit FAS gene expression; silencing the FAS gene effectively improves the proliferation ability of immune cells in a complex environment and the ability to resist apoptosis, thereby improving the therapeutic effect of immune cells in tumor immunotherapy, which plays a promoting role in the popularization and improvement of the effect of tumor immunotherapy.

[0007] To this end, the first aspect of the present invention provides an shRNA molecule. According to an embodiment of the present invention, the shRNA molecule includes a sense strand fragment and an antisense strand fragment, the sequences of the sense strand fragment and the antisense strand fragment are complementary, and the sequence of the sense strand fragment is as shown in SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 29.

[0008] The present invention provides an shRNA that specifically targets the FAS molecule, and the shRNA sequence with the best silencing effect is obtained by screening. The shRNA according to the embodiment of the present invention can target FAS expression in immune cells and continuously, stably, efficiently and specifically inhibit FAS gene expression; silencing the FAS gene effectively improves the proliferation ability of immune cells in a complex environment and the ability to resist apoptosis, thereby improving the therapeutic effect of immune cells in tumor immunotherapy.

[0009] According to an embodiment of the present invention, the shRNA molecule further includes a stem-loop structure, the stem-loop structure connects the sense strand fragment and the antisense strand fragment, and the stem-loop structure includes 4-11 nucleotides.

[0010] According to an embodiment of the present invention, the DNA sequence encoding the shRNA molecule is as shown in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 11.

[0011] The second aspect of the present invention provides an expression vector. According to an embodiment of the present invention, the expression vector includes a DNA sequence expressing the shRNA molecule described in the first aspect.

[0012] According to an embodiment of the present invention, the expression vector is selected from any one of a lentiviral vector, a retroviral vector, an adenoviral vector or an adeno-associated viral vector.

[0013] The third aspect of the present invention provides a microorganism. According to an embodiment of the present invention, the microorganism includes the expression vector described in the second aspect.

[0014] The fourth aspect of the present invention provides a host cell. According to an embodiment of the present invention, the host cell includes the expression vector described in the second aspect.

[0015] The fifth aspect of the present invention provides a pharmaceutical composition. According to an embodiment of the present invention, the pharmaceutical composition includes at least one of the shRNA molecule described in the first aspect, the expression vector described in the second aspect, and the microorganism described in the third aspect.

[0016] The sixth aspect of the present invention provides the use of the shRNA molecule described in the first aspect, the expression vector described in the second aspect, the microorganism described in the third aspect, the host cell described in the fourth aspect, and the pharmaceutical composition described in the fifth aspect in the preparation of a drug. According to an embodiment of the present invention, the drug is used for preventing and / or treating tumors.

[0017] According to an embodiment of the present invention, the tumors include solid tumors and hematological tumors.

[0018] According to an embodiment of the present invention, the solid tumors include liver cancer.

[0019] The seventh aspect of the present invention provides a method for reducing the expression of the FAS gene in a target cell. According to an embodiment of the present invention, the method includes:

[0020] introducing the shRNA molecule described in the first aspect and / or the expression vector described in the second aspect into the target cell,

[0021] or infecting the target cell with the microorganism described in the third aspect.

[0022] It should be noted that the target cells include, but are not limited to, NK cells, T cells, Tregs, γδT cells, macrophages, and various cells derived from iPSCs, etc.

[0023] The eighth aspect of the present invention provides the use of the shRNA molecule described in the first aspect, the expression vector described in the second aspect, and the microorganism described in the third aspect in the preparation of a reagent or kit. According to an embodiment of the present invention, the reagent or kit is used for reducing the expression of at least one of NKG2A, TIGIT, and TIM3 in a target cell, and / or increasing the expression of at least one of NKG2D and NKp30 in the target cell.

[0024] The ninth aspect of the present invention provides a method for enhancing the anti-apoptosis ability of immune cells. According to an embodiment of the present invention, the method includes:

[0025] Introduce the shRNA molecule described in the first aspect and / or the expression vector described in the second aspect into the immune cells, or infect the immune cells with the microorganism described in the third aspect, so that the FAS gene is lowly expressed in the immune cells.

[0026] The tenth aspect of the present invention provides a reagent or kit for enhancing the anti-apoptosis ability of immune cells. According to an embodiment of the present invention, the reagent or kit includes at least one of the shRNA molecule described in the first aspect, the expression vector described in the second aspect, and the microorganism described in the third aspect.

[0027] The eleventh aspect of the present invention provides a method for improving the proliferation ability of immune cells in a co-culture environment. According to an embodiment of the present invention, the method includes:

[0028] Introduce the shRNA molecule described in the first aspect and / or the expression vector described in the second aspect into the immune cells, or infect the immune cells with the microorganism described in the third aspect, so that the FAS gene is lowly expressed in the immune cells.

[0029] Inhibiting FAS expression in immune cells can significantly improve the proliferation ability of immune cells in a complex environment, as well as the ability to resist apoptosis and exhaustion, thereby improving the therapeutic effect of immune cells in tumor immunotherapy. The present invention provides a new idea and strategy for optimizing tumor immunotherapy with immune cells in the art, and plays a promoting role in the popularization of tumor immunotherapy and the improvement of its effect.

[0030] The twelfth aspect of the present invention provides a method for enhancing the resistance of immune cells to FASL-induced apoptosis. According to an embodiment of the present invention, the method includes:

[0031] Introduce the shRNA molecule described in the first aspect and / or the expression vector described in the second aspect into the immune cells, or infect the immune cells with the microorganism described in the third aspect, so that the FAS gene is lowly expressed in the immune cells.

[0032] The thirteenth aspect of the present invention provides a method for enhancing the killing ability of immune cells against tumor cells. According to an embodiment of the present invention, the method includes:

[0033] Introduce the shRNA molecule described in the first aspect and / or the expression vector described in the second aspect into the immune cells, or infect the immune cells with the microorganism described in the third aspect, so that the FAS gene is lowly expressed in the immune cells.

[0034] The fourteenth aspect of the present invention provides the use of the shRNA molecule described in the first aspect, the expression vector described in the second aspect, and the microorganism described in the third aspect in the following:

[0035] Enhance the anti-apoptotic ability of immune cells;

[0036] Improve the proliferation ability of immune cells in a co-culture environment;

[0037] Enhance the resistance of immune cells to FASL-induced apoptosis;

[0038] Enhance the ability of immune cells to resist exhaustion in the tumor microenvironment;

[0039] Enhance the killing ability of immune cells against tumor cells.

[0040] Current studies show that the cell death receptor FAS molecule is highly expressed during the in vitro activation and expansion of NK cells. Therefore, when NK cells are in a complex tumor microenvironment, due to the stimulation of FASL expressed by tumor cells, apoptosis of immune cells such as NK cells occurs. Based on this, the inventor constructed an shRNA that silences the cell surface death receptor FAS for modifying NK cells. The present invention is also applicable to modifying immune cells such as T cells, NKT cells, Tregs, γδT cells, and macrophages for immunotherapy.

[0041] Based on this, the present invention provides an shRNA that specifically targets the FAS molecule, and the shRNA sequence with the best silencing effect is obtained through screening. The shRNA according to the embodiments of the present invention can inhibit the expression of the FAS molecule on immune cells, enhance the resistance of immune cells to FAS ligand FASL-induced apoptosis, and thereby improve the anti-tumor activity of immune cells. The inventor also unexpectedly found that when co-cultured with unmodified immune cells (such as NK cells), the immune cells with silenced FAS have a proliferation advantage. Additionally, it was unexpectedly found that the inhibitory receptors NKG2A, TIGIT, and TIM3 on the surface of immune cells with silenced FAS are also reduced, while the activating receptors NKG2D and NKp30 are increased in expression, enhancing the killing ability against tumor target cells and the ability to resist exhaustion. Therefore, knocking down FAS may improve the efficacy and durability of immune cells in clinical tumor treatment.

[0042] The shRNA targeting and silencing FAS expression screened in the present invention can maintain a highly efficient, specific, and stable silencing effect; silencing the FAS molecule significantly improves the proliferation ability of immune cells in a co-culture environment; silencing the FAS molecule significantly improves the anti-apoptotic ability of immune cells.

[0043] Additional aspects and advantages of the present invention will be given in part in the following description, become apparent in part from the following description, or be learned through the practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] The above and / or additional aspects and advantages of the present invention will become apparent and be readily understood from the following description of embodiments in conjunction with the accompanying drawings, wherein:

[0045] Figure 1 It shows the detection of the expression level of FAS protein in expanded NK cells in peripheral blood by flow cytometry in Example 1;

[0046] Figure 2 It shows the detection of the expression level of FAS mRNA in NK cells transfected with different FAS shRNAs by qRT-PCR in Example 1;

[0047] Figure 3 It shows the detection of the expression level of FAS protein in NK cells transfected with different FAS shRNAs by flow cytometry in Example 1. Among them, in the left figure, from bottom to top are the expression levels of FAS protein in NK cells transfected with NTC, FAS-sh01 to FAS-sh16, and the right side is the column statistical result chart;

[0048] Figure 4 It shows the proliferation curves of NK cells transfected with FAS-sh02 and NTC respectively in Example 2;

[0049] Figure 5 It shows the results of cell proliferation and survival when NK cells transfected with FAS-sh02 and NTC respectively are co-cultured with unmodified NK cells in Example 2;

[0050] Figures 6A - 6C It shows the apoptosis results of NK cells transfected with FAS-sh02 and NTC respectively, T cells (Jurkat cells) transfected with FAS-sh02, and unmodified Jurkat cells after being treated with FASL in Example 3;

[0051] Figure 7 It shows the detection of the expression of inhibitory receptors such as NKG2A, TIGIT, TIM3, LAG-3, PD-1 and activating receptors such as NKG2D, NKp30, NKp44, CD16 on the surface of NK cells transfected with FAS-sh02 and NTC respectively by flow cytometry in Example 4. Detailed Embodiments

[0052] The embodiments of the present invention are described in detail below. The following described embodiments are exemplary and are only used to explain the present invention, and should not be construed as a limitation to the present invention.

[0053] It should be noted that the terms "first" and "second" are only used for descriptive purposes and should not be construed as indicating or implying relative importance or implicitly specifying the quantity of the indicated technical features. Thus, features defined with "first" and "second" may explicitly or implicitly include one or more of such features. Further, in the description of the present invention, unless otherwise specified, "a plurality of" means two or more.

[0054] The endpoints and any values in the ranges disclosed herein are not limited to the exact ranges or values. These ranges or values should be understood to include values close to these ranges or values. For numerical ranges, the endpoint values of each range, between the endpoint values of each range, between the endpoint values of each range and individual point values, and between individual point values can be combined with each other to obtain one or more new numerical ranges, and these numerical ranges should be regarded as specifically disclosed herein.

[0055] To make the present invention easier to understand, certain technical and scientific terms are specifically defined below. Unless otherwise clearly defined elsewhere in this document, all other technical and scientific terms used herein have the meanings commonly understood by those of ordinary skill in the art to which the present invention pertains.

[0056] In this document, the term "comprising" or "including" is an open-ended expression, that is, it includes the content specified by the present invention, but does not exclude other aspects.

[0057] In this document, the terms "optionally", "optional" or "option" generally mean that the subsequent events or conditions may or may not occur, and this description includes the cases where such events or conditions occur and the cases where such events or conditions do not occur.

[0058] According to a specific embodiment of the present invention, the present invention provides an shRNA molecule, comprising a sense strand fragment and an antisense strand fragment, the sequences of the sense strand fragment and the antisense strand fragment being complementary, and the sequence of the sense strand fragment being as shown in SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:29. The target nucleotide sequences of these shRNA molecules are as shown in SEQ ID NO:19, and they can inhibit the expression of the FAS gene.

[0059] According to a specific embodiment of the present invention, the shRNA molecule further comprises a stem-loop structure, the stem-loop structure connecting the sense strand fragment and the antisense strand fragment, and the stem-loop structure comprising 4-11 nucleotides. The sequences of those stem-loop structures commonly used in the art when designing shRNA molecules are all applicable to the shRNA molecules of the present invention and are all covered within the protection scope of the present invention.

[0060] According to a specific embodiment of the present invention, the DNA sequences encoding the shRNA molecules are as shown in SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:11.

[0061] According to a specific embodiment of the present invention, the present invention provides an expression vector comprising a DNA sequence expressing the aforementioned shRNA molecule.

[0062] According to a specific embodiment of the present invention, the expression vector further contains a promoter that drives the transcription of RNA polymerase III.

[0063] According to a specific embodiment of the present invention, the expression vector is selected from any one of a lentiviral vector, a retroviral vector, an adenoviral vector, and an adeno-associated viral vector.

[0064] It should be noted that when the expression vector is a lentiviral vector, the expression vector includes, but is not limited to, the PLKO.1-CMV-PURO lentiviral vector.

[0065] According to a specific embodiment of the present invention, the expression vector is a recombinant vector obtained by inserting the DNA sequence expressing the aforementioned shRNA molecule into a lentiviral vector.

[0066] According to a specific embodiment of the present invention, the present invention provides a microorganism comprising the aforementioned expression vector. The microorganism can be, for example, a recombinant bacterium or a recombinant virus. The recombinant virus can be, for example, a lentivirus or others, all of which are covered within the protection scope of the present invention.

[0067] It should be noted that the present invention has designed multiple DNA sequences expressing shRNA molecules for the target gene FAS, and the shRNA molecules expressed by these DNA sequences have different silencing efficiencies for the gene FAS. To further improve the silencing efficiency of the target gene, multiple vectors can also be introduced into the recombinant microorganism simultaneously, and these vectors carry the DNA sequences of different shRNA molecules respectively.

[0068] According to a specific embodiment of the present invention, the present invention provides a host cell comprising the aforementioned expression vector. The host cell can be, for example, various immune cells, such as NK cells, T cells, B cells, NKT cells, γδT cells, Tregs, macrophages, and various cells derived from iPSCs, etc.

[0069] According to a specific embodiment of the present invention, the present invention provides a pharmaceutical composition comprising at least one of the aforementioned shRNA molecule, the aforementioned expression vector, and the aforementioned microorganism.

[0070] The pharmaceutical composition provided by the present invention can be used for preventing and / or treating tumors by enhancing the survival and treatment persistence of immune cells.

[0071] According to a specific embodiment of the present invention, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier or excipient.

[0072] According to a specific embodiment of the present invention, the present invention provides the use of the aforementioned shRNA molecule, the aforementioned expression vector, the aforementioned microorganism, the aforementioned host cell, and the aforementioned pharmaceutical composition in the preparation of a drug for preventing and / or treating tumors.

[0073] According to a specific embodiment of the present invention, the tumors include solid tumors and hematological tumors.

[0074] According to a specific embodiment of the present invention, the solid tumors include, but are not limited to, liver cancer.

[0075] According to a specific embodiment of the present invention, the present invention provides a method for reducing the expression of the FAS gene in target cells, comprising:

[0076] introducing the aforementioned shRNA molecule and / or the aforementioned expression vector into the target cells,

[0077] or infecting the target cells with the aforementioned microorganism.

[0078] It should be noted that the target cells include NK cells, T cells, Tregs, γδT cells, macrophages, and various cells derived from iPSCs, etc.

[0079] According to a specific embodiment of the present invention, the present invention provides the use of the aforementioned shRNA molecule, the aforementioned expression vector, and the aforementioned microorganism in the preparation of a reagent or kit for reducing the expression of at least one of NKG2A, TIGIT, and TIM3 in target cells and / or increasing the expression of at least one of NKG2D and NKp30 in target cells.

[0080] According to a specific embodiment of the present invention, the present invention provides a method for enhancing the anti-apoptotic ability of immune cells, comprising:

[0081] Introduce the aforementioned shRNA molecule and / or the aforementioned expression vector into immune cells, or infect the immune cells with the aforementioned microorganism, so that the FAS gene is lowly expressed in the immune cells.

[0082] Inhibiting FAS expression in immune cells can significantly improve the proliferation ability of immune cells in a complex environment, as well as their ability to resist apoptosis and exhaustion, thereby improving the therapeutic effect of immune cells in tumor immunotherapy. The present invention provides a new idea and strategy for optimizing tumor immunotherapy with immune cells in the art, and plays a promoting role in the popularization and improvement of the effect of tumor immunotherapy.

[0083] According to a specific embodiment of the present invention, the present invention provides a reagent or kit for enhancing the anti-apoptotic ability of immune cells, and the reagent or kit includes at least one of the aforementioned shRNA molecule, the aforementioned expression vector, and the aforementioned microorganism.

[0084] According to a specific embodiment of the present invention, the present invention provides a method for improving the proliferation ability of immune cells in a co-culture environment, and the method includes:

[0085] Introduce the aforementioned shRNA molecule and / or the aforementioned expression vector into immune cells, or infect the immune cells with the aforementioned microorganism, so that the FAS gene is lowly expressed in the immune cells.

[0086] According to a specific embodiment of the present invention, the present invention provides a method for enhancing the resistance of immune cells to FASL-induced apoptosis, including:

[0087] Introduce the aforementioned shRNA molecule and / or the aforementioned expression vector into immune cells, or infect the immune cells with the aforementioned microorganism, so that the FAS gene is lowly expressed in the immune cells.

[0088] According to a specific embodiment of the present invention, the present invention provides a method for enhancing the killing ability of immune cells against tumor cells, including:

[0089] Introduce the aforementioned shRNA molecule and / or the aforementioned expression vector into immune cells, or infect the immune cells with the aforementioned microorganism, so that the FAS gene is lowly expressed in the immune cells.

[0090] According to a specific embodiment of the present invention, the present invention provides the use of the aforementioned shRNA molecule, the aforementioned expression vector, and the aforementioned microorganism in the following:

[0091] Enhancing the anti-apoptotic ability of immune cells;

[0092] Improve the proliferation ability of immune cells in a co - culture environment;

[0093] Enhance the resistance of immune cells to FASL - induced apoptosis;

[0094] Enhance the ability of immune cells to resist exhaustion in the tumor microenvironment;

[0095] Enhance the killing ability of immune cells against tumor cells.

[0096] The sequence description involved in the present invention is shown in Table 1 in detail.

[0097] Table 1: Amino acid / nucleotide sequence description table

[0098]

[0099]

[0100]

[0101]

[0102] The following will explain the solution of the present disclosure in combination with the embodiments. Those skilled in the art will understand that the following embodiments are only used to illustrate the present disclosure and should not be regarded as limiting the scope of the present disclosure. For those not specified in the embodiments about specific techniques or conditions, they shall be carried out according to the techniques or conditions described in the literature in this field or according to the product specifications. For reagents or instruments not specified about the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0103] Example 1 Design and screening of shRNA sequences for silencing the FAS gene

[0104] 1. Design of RNAi target sequences

[0105] In the NCBI website, the gene number of Homo sapiens Fas cell surface death receptor (FAS) is NM_000043.6, and the coding sequence (Coding sequence, CDS) of FAS mRNA is shown as SEQ ID NO:18. In the coding sequence of the FAS gene (nucleotide sequence shown as SEQ ID NO:19), 15 RNAi target sequences were designed for screening respectively.

[0106] According to the designed RNAi target sequence, design the shRNA sequence for constructing the shRNA lentiviral vector. The designed coding sequences of FAS shRNA (FAS-sh01 to FAS-sh15, nucleotide sequences are shown in SEQ ID NO: 2 to 16) and the non-sense control sequence (Non-targeting control, NTC, nucleotide sequence is shown in SEQ ID NO: 17) have the structure of sense, loop, antisense, and termination sequences. According to the restriction enzyme sites of the PLKO.1-CMV-PURO lentiviral vector, select the AgeI and EcoRI restriction enzyme sites to insert the shRNA sequence respectively, and the obtained plasmids are named pLKO.1-FAS-sh01 to pLKO.1-FAS-sh15 and PLKO.1-NTC respectively.

[0107] 2. Packaging of Lentivirus and Concentration of Virus Solution

[0108] Take 5×10 6 cells in the logarithmic growth phase and inoculate them into a 10 cm cell culture dish, add 10 mL of DMEM medium, and culture overnight in a 37 °C, 5% CO2 incubator. When the cell density in the cell culture dish reaches 80% - 90%, replace it with 10 mL of fresh DMEM medium, and continue to place the cell culture dish in the incubator for standby. Prepare the lentivirus packaging system and add the lentivirus packaging mixture to the 293T cell culture dish. After 24 h, change the medium, put the culture dish back into the 37 °C, 5% CO2 incubator, and after 48 h, collect the cell supernatant respectively, centrifuge at 400×g for 5 min to remove cell debris, filter the supernatant through a 0.45 μm syringe filter into a 50 mL centrifuge tube to obtain the virus solutions of different FAS shRNA (FAS-sh01 to sh15) and the non-sense control sequence (NTC) lentiviruses respectively, and concentrate the virus solutions. The concentrated virus is used to infect cells or stored in an -80 °C refrigerator.

[0109] 3. Detection of FAS Protein Expression Level in Peripherally Blood-Amplified NK Cells by Flow Cytometry

[0110] Isolate NK cells from the donor's peripheral blood using the NK cell sorting and purification kit, and appropriately amplify the sorted and purified NK cells in vitro. Detect the expression level of FAS protein in NK cells by flow cytometry. Transfer the NK cells into a flow tube, add 3 mL of 1×PBS solution, centrifuge at 100×g for 5 min, discard the supernatant, flick the NK cell pellet to resuspend it, and wash it again with 1×PBS solution. After resuspending the NK cell pellet, add APC-labeled anti-human FAS antibody (Biolegend) for staining, and incubate at room temperature for 30 min. Then add 1×PBS to wash twice, and resuspend the NK cells and load them onto the flow cytometer for detection. The results are asFigure 1 As shown, the expanded NK cells in peripheral blood express a relatively high level of FAS protein.

[0111] 4. Detection of the silencing efficiency of different FAS shRNAs

[0112] NK cells were infected with lentiviruses carrying different FAS shRNAs (FAS-sh01 to FAS-sh15) and a nonsense control sequence (NTC). For the expanded NK cells, an appropriate number of NK cells were taken, centrifuged at 100×g for 5 min to harvest the NK cells, resuspended in an appropriate amount of NK cell medium, and the NK cell density was adjusted to 5×10 5 cells / mL. 5×10 5 NK cells, 0.2 mL of the virus concentrate prepared in "2. Packaging of lentivirus and concentration of virus solution" in Example 1 of this embodiment, 0.8 mL of NK cell medium, and a pro-infection reagent were added to each well of a 24-well plate and mixed evenly. The 24-well plate with the evenly mixed solution was placed in an incubator at 37°C and 5% CO2 for culture. After 24 h, the status of the NK cells was observed, and a fresh NK cell medium with a volume twice that of the original was added. The NK cells were transferred to a cell culture flask and continued to be cultured. Fresh medium was added every 2 days, and growth factors such as IL2 that promote NK cell growth were added.

[0113] After the NK cells were infected with lentiviruses carrying different FAS shRNAs (FAS-sh01 to FAS-sh15) and the nonsense control sequence (NTC), puromycin screening was performed. On the 7th day after infection, the expression of FAS in the infected NK cells was detected by quantitative real-time PCR (qPCR) and flow cytometry, respectively.

[0114] qRT-PCR: The infected NK cells were transferred to a 15 mL centrifuge tube, centrifuged at 300×g for 5 min, the supernatant was discarded, the cell pellet was resuspended and washed with PBS, centrifuged at 300×g for 5 min, the supernatant was discarded, 1 mL of Trizol reagent was added to lyse the cell pellet, and the mixture was transferred to a 1.5 mL RNase Free EP tube. Total cellular RNA was extracted and reverse transcribed into cDNA, and then real-time fluorescence quantitative PCR was performed using FAS-specific primers to detect the expression level of the mRNA transcribed from the FAS gene.

[0115] The results are as Figure 2As shown, compared with uninfected NK cells or the nonsense control sequence group (NTC), the expression levels of FAS mRNA in cells infected with different FAS shRNAs (FAS-sh01 to FAS-sh15) lentiviruses vary. Some shRNAs show significant silencing effects, while some show insignificant or even no silencing effects. Among them, the sequences of FAS-sh01, sh02, sh06, sh07, sh08, and sh10 have a relatively high silencing efficiency, up to about 70%, and can maintain a stable silencing effect.

[0116] Flow cytometry: Transfer the infected NK cells into a flow cytometry tube, add 3 mL of 1×PBS solution, centrifuge at 100×g for 5 min, discard the supernatant, flick the precipitate of the infected NK cells, and wash again with 1×PBS solution. After resuspending the precipitate of the infected NK cells, add APC-labeled anti-human FAS antibody (Biolegend) for staining and incubate at room temperature for 30 min. Then wash twice with 1×PBS solution, resuspend the precipitate of the infected NK cells, and perform flow cytometry detection on the machine.

[0117] Respectively, count the mean fluorescence intensity (MFI) of FAS in the NK cell control group (NTC) cells and the NK cells with silenced FAS (FAS-sh01 to FAS-sh15), and calculate the FAS silencing efficiency.

[0118] The results show that compared with uninfected NK cells or the nonsense control sequence group (NTC), the expression levels of FAS in cells infected with different FAS shRNAs (FAS-sh01 to FAS-sh15) lentiviruses vary. Some shRNAs show significant silencing effects, while some show insignificant silencing effects or even lead to a slight increase in the FAS expression level. For specific results, see Figure 3 . Among them, the sequences of FAS-sh01, sh02, sh06, sh07, sh08, and sh10 have a relatively high silencing efficiency, around 70% - 80%, and can maintain a stable knockdown effect. For specific results, see Figure 3 .

[0119] Example 2 Silencing FAS Promotes the Proliferation of NK Cells in a Co-Culture Environment

[0120] The inventors verified that silencing FAS does not affect the proliferation of NK cells. The specific method is as follows: Inoculate the same number of cells (1×10 5 ) of NK cells and NK cells with silenced FAS into 12-well plates, and perform trypan blue staining cell counting every 48 h. Observe and culture until the 8th day, and draw a cell proliferation curve.

[0121] This example exemplarily shows the results of NK cells with silenced FAS. The results show that the proliferation ability of NK cells with silenced FAS is comparable to that of the control group NK cells. For details, see Figure 4 . The above test results indicate that silencing FAS does not affect the proliferation of NK cells.

[0122] The inventors further verified the proliferation effect of silencing FAS on NK cells in a co-culture system. The specific method is as follows: Stably stain NK cells and NK cells with silenced FAS with the cell dyes CTV and CFSE respectively. Incubate the dyes with the cells in a 37 °C incubator for 20 min. The dyes enter the cells. Then add 5-10 times PBS to the cell culture medium, incubate in the dark at 4 °C for 10 min, then centrifuge at 300×g for 5 min to wash away the unbound dyes, and resuspend the cell pellet with complete medium. Flow cytometry is used to detect and determine that the cells have different fluorescence staining. Then co-incubate NK cells (CTV-stained) and NK cells with silenced FAS (CFSE-stained) with the same cell number (1×10 5 ) at a ratio of 1:1, and detect the cell ratio changes at 0 h, 24 h, and 48 h respectively to determine the cell number changes in the co-culture system. After co-culturing for 48 h, use a cell apoptosis detection kit (Annexin V-APC / 7-AAD Apoptosis Kit) to detect the apoptosis situation. Perform the staining operation according to the kit instructions, and then detect the apoptosis situation by flow cytometry.

[0123] This example exemplarily shows the results of NK cells with silenced FAS. The results show that NK cells with silenced FAS have a proliferation advantage in the co-culture system, and the apoptosis rate of NK cells with silenced FAS is lower. For details, see Figure 5 . The above test results indicate that NK cells with silenced FAS have a proliferation advantage when co-cultured with NK cells. This proliferation advantage is due to the fact that after silencing FAS, it can avoid the suicide or mutual killing of NK cells caused by the FAS / FASL signal. Other shRNA sequences with high silencing efficiency also have the same efficacy improvement. Similarly, for T cells, there is also a similar efficacy of avoiding suicide or mutual killing.

[0124] Example 3 Silencing FAS to Prevent FasL-Induced Cell Apoptosis

[0125] The inventors further verified that silencing FAS can prevent FasL-mediated cell apoptosis. The specific method is as follows: Take the same number (2×10 5Uninfected NK cells and NK cells with silenced FAS (FAS-sh02) were added to a 12-well plate, and 100 ng / mL FASL protein (AdipoGen Life Sciences) was added thereto to induce apoptosis. Similarly, the same number (2×10 5 ) of uninfected T cells (Jurkat cells) and Jurkat cells with silenced FAS (FAS-sh02) were added to a 12-well plate, and 100 ng / mL FASL protein was added to induce apoptosis. The apoptosis situation was monitored using an apoptosis detection kit (Annexin V-APC / 7-AAD Apoptosis Kit) at different time points. The staining operation was performed according to the kit instructions, and then the apoptosis situation was detected by flow cytometry.

[0126] This example exemplarily shows the results of immune cells with silenced FAS preventing FASL-induced apoptosis. The results show that NK cells or T cells with silenced FAS can prevent FASL-mediated apoptosis, as specifically shown in Figures 6A - 6C . Other shRNA sequences with high silencing efficiency also have the same efficacy improvement.

[0127] Example 4 Detection of the expression of inhibitory and activating receptors of NK cells with silenced FAS

[0128] The inventors used flow cytometry to detect and compare the expression levels of FAS in NK cells before and after FAS knockdown. The results are shown in Figure 3 . It was found that after gene knockdown by the shRNA technique, the level of FAS expression in NK cells was significantly decreased. The inventors observed whether silencing FAS would affect the expression of surface activating and inhibitory receptors when NK cells contacted tumor target cells and whether it would affect the sensitivity of NK cells to tumor microenvironment-induced functional exhaustion. After co-incubating NK cells with hepatoma cells PLC-PRF-5 at a ratio of 1:1 for 72 hours, the NK cells were collected, and flow cytometry was used to detect the expression of inhibitory receptors such as NKG2A, TIGIT, TIM3, LAG3, PD-1 and activating receptors NKG2D, NKp30, NKp44 and CD16 on the surface of NK cells. The results are shown in Figure 7。The results showed that, compared with NK cells, the expression of inhibitory receptors NKG2A, TIGIT, and TIM3 on the surface of NK cells with silenced FAS (FAS-sh02 sequence) was significantly reduced, while there was no significant change in LAG3 and PD-1; the expression of activating receptors NKG2D and NKp30 was significantly increased, and the expression of NKp44 and CD16 showed no obvious change. Therefore, the present invention unexpectedly found that silencing the expression of FAS on the surface of NK cells by the method of the present invention would weaken the expression of inhibitory receptors such as NKG2A, TIGIT, and TIM3, while enhancing the expression of activating receptors such as NKG2D and NKp30, promoting the balance of activating and inhibitory receptors on NK cells to tilt towards the activating direction, thereby enhancing the activation and function of NK cells and enhancing the resistance of NK cells to exhaustion induced by the tumor microenvironment. For details, see Figure 7 Other shRNA sequences with high silencing efficiency also have the same effect, and also have a similar effect of resisting immune exhaustion on T cells.

[0129] In the description of this specification, the description with reference to terms such as "one embodiment", "some embodiments", "example", "specific example", "some implementation manners" or "some examples" means that the specific features, structures, materials or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner. In addition, without contradiction, those skilled in the art can combine and combine the different embodiments or examples described in this specification and the features of different embodiments or examples.

[0130] Although the embodiments of the present invention have been shown and described above, it can be understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those of ordinary skill in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A shRNA molecule, characterized in that, It includes a sense strand fragment and an antisense strand fragment, the sequences of the sense strand fragment and the antisense strand fragment are complementary, and the sequence of the sense strand fragment is as shown in SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:

29.

2. The shRNA molecule according to claim 1, wherein The shRNA molecule further includes a stem-loop structure that connects the sense strand fragment and the antisense strand fragment, and the stem-loop structure includes 4-11 nucleotides.

3. The shRNA molecule according to claim 1, wherein The DNA sequence encoding the shRNA molecule is as shown in SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:

11.

4. An expression vector, characterized in that, It includes a DNA sequence that expresses the shRNA molecule described in any one of claims 1-3.

5. The expression vector according to claim 4, wherein, The expression vector is selected from any one of lentiviral vectors, retroviral vectors, adenoviral vectors, and adeno-associated viral vectors.

6. A microorganism, characterized in that, It includes the expression vector described in claim 4 or 5.

7. A host cell, characterized in that, It includes the expression vector described in claim 4 or 5.

8. A pharmaceutical composition, characterized in that, It includes at least one of the shRNA molecule described in any one of claims 1-3, the expression vector described in claim 4 or 5, and the microorganism described in claim 6.

9. Use of the shRNA molecule according to any one of claims 1-3, the expression vector according to claim 4 or 5, the microorganism according to claim 6, the host cell according to claim 7, and the pharmaceutical composition according to claim 8 in the preparation of a drug, characterized in that, The drug is used for preventing and / or treating tumors.

10. The use according to claim 9, characterized in that, The tumors include solid tumors and hematological tumors; Optionally, the solid tumor includes liver cancer.

11. A method for reducing the expression of the FAS gene in target cells, characterized in that, It includes: Introducing the shRNA molecule described in any one of claims 1-3 and / or the expression vector described in claim 4 or 5 into the target cell, or infecting the target cell with the microorganism described in claim 6.

12. Use of the shRNA molecule according to any one of claims 1-3, the expression vector according to claim 4 or 5, and the microorganism according to claim 6 in the preparation of a reagent or a kit, characterized in that, The reagent or kit is used for reducing the expression of at least one of NKG2A, TIGIT, and TIM3 in the target cell, and / or increasing the expression of at least one of NKG2D and NKp30 in the target cell.

13. A method for enhancing the anti-apoptosis ability of immune cells, characterized in that, The method includes: Introducing the shRNA molecule described in any one of claims 1-3 and / or the expression vector described in claim 4 or 5 into the immune cell, or infecting the immune cell with the microorganism described in claim 6, so that the FAS gene is lowly expressed in the immune cell.

14. A reagent or kit for enhancing the anti-apoptotic ability of immune cells, characterized in that, The reagent or kit includes at least one of the shRNA molecule described in any one of claims 1-3, the expression vector described in claim 4 or 5, and the microorganism described in claim 6.

15. A method for improving the proliferation ability of immune cells in a co-culture environment, characterized in that, The method includes: Introducing the shRNA molecule described in any one of claims 1-3 and / or the expression vector described in claim 4 or 5 into the immune cell, or infecting the immune cell with the microorganism described in claim 6, so that the FAS gene is lowly expressed in the immune cell.

16. A method for enhancing the resistance of immune cells to FASL-induced apoptosis, characterized in that, The method includes: Introducing the shRNA molecule described in any one of claims 1-3 and / or the expression vector described in claim 4 or 5 into the immune cell, or infecting the immune cell with the microorganism described in claim 6, so that the FAS gene is lowly expressed in the immune cell.

17. A method for enhancing the killing ability of immune cells against tumor cells, characterized in that, The method includes: Introduce the shRNA molecule described in any one of claims 1-3 and / or the expression vector described in claim 4 or 5 into the immune cells, or infect the immune cells with the microorganism described in claim 6, such that the FAS gene is lowly expressed in the immune cells.

18. Use of the shRNA molecule described in any one of claims 1-3, the expression vector described in claim 4 or 5, and the microorganism described in claim 6 in the following: Enhancing the anti-apoptosis ability of immune cells; Improving the proliferation ability of immune cells in a co-culture environment; Enhancing the resistance of immune cells to FASL-induced apoptosis; Enhancing the ability of immune cells to resist exhaustion in the tumor microenvironment; Enhancing the killing ability of immune cells against tumor cells.

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