NKG2A and TGFBR2 double-gene knockout NK cell as well as construction method and application thereof

Abstract

The invention discloses an NK cell with double genes of NKG2A and TGFBR2 knocked out as well as a construction method and application of the NK cell, and belongs to the technical field of gene editing. According to the NKG2A and TGFBR2 double-gene knockout method for the NK cells, the NKG2A and TGFBR2 genes can be knocked out at the same time, the editing efficiency is larger than or equal to 90%, the NK cell amplification multiple reaches 1238-2934 times after culture is conducted for 14 days, the inhibition effect of NKG2A / HLA-E and TGF-beta / TGFBR2 channels on the activity of the NK cells is removed through efficient double-gene knockout, the activation channel of NKG2C / HLA-E is promoted, and the NKG2A and TGFBR2 double-gene knockout method for the NKG2A and TGFBR2 double-gene knockout is achieved. The killing activity on HLA-E positive tumor cells is obviously improved, and the clinical application prospect is wide.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of gene editing, and particularly relates to an NK cell with NKG2A and TGFBR2 double gene knockout and a construction method and application thereof. BACKGROUND

[0002] NK cells, as natural killer cells, are a core component of the innate immune system and the first line of defense against cancer and viral infections, and have the characteristics of no pre-sensitivity, rapid killing and relatively high safety. However, in the tumor microenvironment, tumor cells can secrete various immunosuppressive factors, such as TGF-β and IL-10, to directly inhibit the activation and function of NK cells. In addition, there are a large number of ligands in the tumor microenvironment that can bind to the inhibitory receptors on the surface of NK cells to inhibit the killing activity of NK cells. NKG2A is the most important inhibitory receptor on NK cells, and TGFBR2 is the receptor of the immunosuppressive factor TGF-β, both of which are related to tumor immune suppression and tumor escape.

[0003] NKG2A and NKG2C can both form heterodimers with CD94 to recognize HLA-E. NKG2A transduces inhibitory signals, and NKG2C transduces activating signals, but the affinity of NKG2A to HLA-E is 6 times stronger than that of NKG2C.

[0004] TGF-β has a certain inhibitory effect on various immune cells, can reduce IL-2 secretion, and inhibit the proliferation of immune cells. TGFBR2 is the receptor with the highest affinity to TGF-β. Studies have shown that knocking out TGFBR2 of CAR-T cells can reduce the induced Treg transformation, prevent the exhaustion of CAR-T cells, and has better tumor elimination effect in both cell line-derived xenograft tumors (lung cancer) and patient-derived xenograft solid tumors (pancreatic cancer) mouse models. Adopting NK cells with knockout of TGFBR2 to treat mice transplanted with glioblastoma stem cells (GSCs) can prevent GSCs-induced NK cell dysfunction and eliminate cancer cells.

[0005] Patent document CN110913870A provides a method for knocking out one or more genes of NK cell CBLB, NKG2A and TGFBR2 using the CRISPR / Cas system, and the knockout efficiency of NKG2A is 40-80%, and the average knockout efficiency of TGFBR2 is 88.1%.

[0006] Patent document CN118185875A provides a method for simultaneously editing the TIGIT, TGFBR2 and NKG2A genes or the PVRIG, TGFBR2 and NKG2A genes of NK cells using a base editor. The base edited NK cells have a high efficiency of killing tumor cells. After gene knockout, the NKG2A positive rate is still greater than 16.4%, and the expansion ratio of NK cells cultured for 14 days is only 100-150 times, which directly affects the subsequent application of the gene edited NK cells.

[0007] Based on the above facts, in order to solve the problems of low double gene knockout efficiency and poor NK cell expansion efficiency after gene knockout in the prior art, the present application provides a technical solution for double gene knockout of NKG2A and TGFBR2 of NK cells, which realizes simultaneous knockout of NKG2A and TGFBR2 genes, and the editing efficiency is ≥90%, and the NK cell expansion ratio after 14 days of culture reaches 1238-2934 times, which has a broad clinical application prospect. SUMMARY

[0008] The purpose of the present application is to construct NK cells with double gene knockout of NKG2A and TGFBR2, which removes the inhibitory effect of NKG2A / HLA-E and TGF-β / TGFBR2 pathways on NK cell activity, and plays a combined synergistic role in killing HLA-E positive tumor cells. At the same time, it is found through in vitro culture that the double gene knockout NK cells highly express NKG2C activating receptors, which promotes the activation of NKG2C / HLA-E pathway, further enhances the anti-tumor activity, especially the killing activity of HLA-E positive tumor cells.

[0009] The present application includes the following technical solutions: In a first aspect of the present application, the present application provides a genetically edited natural killer (NK) cell, characterized in that the genetic editing reduces or knocks out the expression of an NK cell activity inhibitory receptor.

[0010] In the present application, the NK cell refers to a human NK cell, which is an important component of the human innate immune system and plays a crucial role in the first line of defense against pathogens and cancer cells.

[0011] In some embodiments of the present application, the NK cell activity inhibitory receptor is selected from one or more than two combinations of NKG2A, TGFBR2.

[0012] Preferably, the NK cell activity inhibitory receptor is selected from a combination of NKG2A and TGFBR2.

[0013] In the present application, the gene editing reduces or knocks out the expression and function of NKG2A protein encoded by NKG2A gene and TGFBR2 protein encoded by TGFBR2 gene in NK cells, and the NK cells have higher anti-tumor activity compared with NK cells without gene editing.

[0014] NKG2A is the most important inhibitory receptor on NK cells, and TGFBR2 is the receptor of immunosuppressive factor TGF-β, both of which are related to tumor microenvironment immunosuppression and tumor escape. Therefore, the genetically edited NK cells of the present application enhance anti-tumor activity by at least one of the following mechanisms of action: 1) removing the inhibitory effect of NKG2A / HLA-E pathway on NK cell activity; 2) removing the inhibitory effect of TGF-β / TGFBR2 pathway on NK cell activity; 3) high expression of NKG2C activating receptor, promoting NKG2C / HLA-E activating pathway.

[0015] In some embodiments of the present application, the gene editing is performed by RNA-guided endonuclease; further, the gene editing is performed by CRISPR / Cas9 system.

[0016] In a specific embodiment of the present application, the gene editing comprises contacting the NK cell population with a Cas9-sgRNA ribonucleoprotein complex (RNP).

[0017] In the present application, the sgRNA forming the Cas9-sgRNA ribonucleoprotein complex (RNP) comprises sgRNA for knocking out NKG2A gene NKG2A and sgRNA for knocking out TGFBR2 gene TGFBR2 , wherein the sgRNA NKG2A consists of a first sgRNA and a second sgRNA, the sgRNA TGFBR2 consists of a third sgRNA and a fourth sgRNA.

[0018] In the present application, the first sgRNA and the second sgRNA are each independently selected from any one of the nucleotide sequences shown in SEQ ID NO. 1-11, and the first sgRNA and the second sgRNA are different; the third sgRNA and the fourth sgRNA are each independently selected from any one of the nucleotide sequences shown in SEQ ID NO. 12-21, and the third sgRNA and the fourth sgRNA are different.

[0019] In one embodiment of the present application, the sgRNAs forming the Cas9-sgRNA ribonucleoprotein complex (RNP) consist of the first sgRNA shown in SEQ ID NO. 1, the second sgRNA shown in SEQ ID NO. 2, the third sgRNA shown in SEQ ID NO. 12, and the fourth sgRNA shown in SEQ ID NO. 13.

[0020] In one embodiment of the present application, the sgRNAs forming the Cas9-sgRNA ribonucleoprotein complex (RNP) consist of the first sgRNA shown in SEQ ID NO. 3, the second sgRNA shown in SEQ ID NO. 4, the third sgRNA shown in SEQ ID NO. 12, and the fourth sgRNA shown in SEQ ID NO. 13.

[0021] In one embodiment of the present application, the sgRNAs forming the Cas9-sgRNA ribonucleoprotein complex (RNP) consist of the first sgRNA shown in SEQ ID NO. 3, the second sgRNA shown in SEQ ID NO. 4, the third sgRNA shown in SEQ ID NO. 14, and the fourth sgRNA shown in SEQ ID NO. 13.

[0022] In one embodiment of the present application, the sgRNAs forming the Cas9-sgRNA ribonucleoprotein complex (RNP) consist of the first sgRNA shown in SEQ ID NO. 5, the second sgRNA shown in SEQ ID NO. 11, the third sgRNA shown in SEQ ID NO. 15, and the fourth sgRNA shown in SEQ ID NO. 16.

[0023] In one embodiment of the present application, the sgRNAs forming the Cas9-sgRNA ribonucleoprotein complex (RNP) consist of the first sgRNA shown in SEQ ID NO. 6, the second sgRNA shown in SEQ ID NO. 7, the third sgRNA shown in SEQ ID NO. 17, and the fourth sgRNA shown in SEQ ID NO. 18.

[0024] In one embodiment of the present application, the sgRNAs forming the Cas9-sgRNA ribonucleoprotein complex (RNP) consist of the first sgRNA shown in SEQ ID NO. 8, the second sgRNA shown in SEQ ID NO. 9, the third sgRNA shown in SEQ ID NO. 19, and the fourth sgRNA shown in SEQ ID NO. 14.

[0025] In one embodiment of the present invention, the sgRNA forming the Cas9-sgRNA ribonucleoprotein complex (RNP) consists of the first sgRNA shown in SEQ ID NO.10, the second sgRNA shown in SEQ ID NO.3, the third sgRNA shown in SEQ ID NO.20, and the fourth sgRNA shown in SEQ ID NO.21.

[0026] In the preferred embodiment of the present invention, the optimal sgRNA for forming the Cas9-sgRNA ribonucleoprotein complex (RNP) consists of the first sgRNA shown in SEQ ID NO.3, the second sgRNA shown in SEQ ID NO.4, the third sgRNA shown in SEQ ID NO.14, and the fourth sgRNA shown in SEQ ID NO.13.

[0027] In a second aspect, the present invention provides a method for constructing gene-edited NK cells, characterized in that the method includes contacting the NK cells with an RNA-guided endonuclease.

[0028] Furthermore, the method includes contacting NK cells with the Cas9-sgRNA ribonucleoprotein complex (RNP).

[0029] In some embodiments of the present invention, the method includes: incubating sgRNA with Cas9 protein via RNP to form a Cas9-sgRNA ribonucleoprotein complex (RNP), and introducing it into NK cells to achieve joint knockout of the NKG2A gene and the TGFBR2 gene in NK cells.

[0030] In some embodiments of the present invention, (2-10)×10 6 For gene editing of NK cells, the required molar mass ratio of Cas9 protein to sgRNA is (10-200) pmol:(20-400) pmol, and the preferred molar mass ratio is (20-200) pmol:(40-400) pmol.

[0031] In specific embodiments of the present invention, the molar mass ratio of the required Cas9 protein to sgRNA composition includes, but is not limited to, 200 pmol:400 pmol, 100 pmol:200 pmol, 50 pmol:100 pmol, 20 pmol:40 pmol, or 10 pmol:20 pmol.

[0032] The importation methods include electroporation transfection, liposome transfection, and viral transfection. In a specific embodiment of the present invention, the importation method is electroporation transfection, and the electroporation program is selected from CA138, CM137, CM158, EN138, or CM189.

[0033] In this invention, the sgRNA that forms the Cas9-sgRNA ribonucleoprotein complex (RNP) is as described in the first aspect of this invention.

[0034] In the most preferred embodiment of the present invention, the optimal sgRNA for forming the Cas9-sgRNA ribonucleoprotein complex (RNP) consists of the first sgRNA shown in SEQ ID NO.3, the second sgRNA shown in SEQ ID NO.4, the third sgRNA shown in SEQ ID NO.14, and the fourth sgRNA shown in SEQ ID NO.13.

[0035] Those skilled in the art will understand that sgRNA is a key factor affecting gene knockout efficiency. The sgRNA provided by this invention can reduce the transcription of target genes NKG2A and TGFBR2 by approximately 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or more (inclusive of any value listed). In the preferred embodiment of this invention, the target genes NKG2A and TGFBR2 are completely knocked out, making the transcription of the target genes undetectable.

[0036] In a third aspect, the present invention provides a pharmaceutical composition, characterized in that the pharmaceutical composition comprises the gene-edited NK cells described in the first aspect of the present invention.

[0037] Furthermore, the pharmaceutical composition also includes a pharmaceutically acceptable carrier or excipient.

[0038] In a fourth aspect, the present invention provides the use of gene-edited NK cells as described in the first aspect of the present invention or the pharmaceutical composition as described in the third aspect of the present invention in the preparation of a medicament for treating HLA-E positive tumors.

[0039] In some embodiments of the present invention, the HLA-E positive tumors include pancreatic cancer, liver cancer, colorectal cancer, ovarian cancer, glioblastoma, lymphoma, chronic lymphocytic leukemia, esophageal cancer, gastric cancer, kidney cancer, head and neck tumors, lung cancer, and melanoma.

[0040] In a fifth aspect, the present invention provides an sgRNA composition comprising sgRNA that specifically targets the NKG2A gene on NK cells. NKG2Aand sgRNA that specifically targets the TGFBR2 gene on NK cells TGFBR2 , wherein the sgRNA NKG2A It consists of a first sgRNA and a second sgRNA, wherein the sgRNA TGFBR2 It consists of a third sgRNA and a fourth sgRNA.

[0041] In this invention, the first sgRNA and the second sgRNA are each independently selected from any one of the nucleotide sequences shown in SEQ ID NO.1-11, and the first sgRNA and the second sgRNA are different; the third sgRNA and the fourth sgRNA are each independently selected from any one of the nucleotide sequences shown in SEQ ID NO.12-21, and the third sgRNA and the fourth sgRNA are different.

[0042] In one embodiment of the present invention, the sgRNA composition comprises a first sgRNA shown in SEQ ID NO.1, a second sgRNA shown in SEQ ID NO.2, a third sgRNA shown in SEQ ID NO.12, and a fourth sgRNA shown in SEQ ID NO.13.

[0043] In one embodiment of the present invention, the sgRNA composition comprises a first sgRNA shown in SEQ ID NO.3, a second sgRNA shown in SEQ ID NO.4, a third sgRNA shown in SEQ ID NO.12, and a fourth sgRNA shown in SEQ ID NO.13.

[0044] In one embodiment of the present invention, the sgRNA composition comprises a first sgRNA shown in SEQ ID NO.3, a second sgRNA shown in SEQ ID NO.4, a third sgRNA shown in SEQ ID NO.14, and a fourth sgRNA shown in SEQ ID NO.13.

[0045] In one embodiment of the present invention, the sgRNA composition comprises a first sgRNA shown in SEQ ID NO. 5, a second sgRNA shown in SEQ ID NO. 11, a third sgRNA shown in SEQ ID NO. 15, and a fourth sgRNA shown in SEQ ID NO. 16.

[0046] In one embodiment of the present invention, the sgRNA composition comprises a first sgRNA shown in SEQ ID NO. 6, a second sgRNA shown in SEQ ID NO. 7, a third sgRNA shown in SEQ ID NO. 17, and a fourth sgRNA shown in SEQ ID NO. 18.

[0047] In one embodiment of the present invention, the sgRNA composition comprises a first sgRNA shown in SEQ ID NO. 8, a second sgRNA shown in SEQ ID NO. 9, a third sgRNA shown in SEQ ID NO. 19, and a fourth sgRNA shown in SEQ ID NO. 14.

[0048] In one embodiment of the present invention, the sgRNA composition comprises a first sgRNA shown in SEQ ID NO.10, a second sgRNA shown in SEQ ID NO.3, a third sgRNA shown in SEQ ID NO.20, and a fourth sgRNA shown in SEQ ID NO.21.

[0049] In the preferred embodiment of the present invention, the preferred sgRNA composition consists of the first sgRNA shown in SEQ ID NO.3, the second sgRNA shown in SEQ ID NO.4, the third sgRNA shown in SEQ ID NO.14, and the fourth sgRNA shown in SEQ ID NO.13.

[0050] In a sixth aspect of the present invention, an RNP-mediated CRISPR / Cas9 gene editing system is provided, wherein the gene editing system achieves joint knockout of the NKG2A gene and the TGFBR2 gene in NK cells, characterized in that the gene editing system is composed of the sgRNA composition described in the fourth aspect of the present invention and the Cas9 protein.

[0051] In a seventh aspect, the present invention provides the use of the sgRNA composition described in the fifth aspect or the RNP-mediated CRISPR / Cas9 gene editing system described in the sixth aspect of the present invention in at least one of the following: 1) Application in knocking out NKG2A and TGFBR2 genes in NK cells; 2) Application in the preparation of products for knocking out the NKG2A and TGFBR2 genes in NK cells.

[0052] The products include, but are not limited to, reagents, kits, chips, or gene editing platforms.

[0053] In some embodiments of the present invention, the product for knocking out the NKG2A gene and the TGFBR2 gene in NK cells is a kit, the kit comprising the sgRNA composition described in the fifth aspect of the present invention or the RNP-mediated CRISPR / Cas9 gene editing system described in the sixth aspect of the present invention.

[0054] Furthermore, the kit also includes conventional reagents used by those skilled in the art for NK cell isolation, NK cell activation, and electroporation.

[0055] The technical solution provided by this invention has the following advantages: This invention develops a highly efficient method for simultaneously knocking out the NKG2A and TGFBR2 genes in NK cells based on RNP-mediated CRISPR / Cas9 gene editing technology. Specifically, it includes: screening and obtaining sgRNA compositions capable of efficiently targeting the NKG2A and TGFBR2 genes in NK cells; constructing a CRISPR / Cas9 gene editing system based on this sgRNA composition; and transfecting the CRISPR / Cas9 gene editing system into NK cells in a combined form to achieve simultaneous knockout of both genes. The optimal knockout rate for both the NKG2A and TGFBR2 genes was found to be 100%.

[0056] In the field of gene editing technology, sgRNA is a key factor and foundation affecting gene knockout efficiency. This invention is based on the analysis of the NKG2A and TGFBR2 gene sequences in NK cells, and according to the design principle of CRISPR / Cas9 target site recognition, it screens target gene sites and designs sgRNAs targeting these sites. The sgRNAs of the NKG2A or TGFBR2 genes are repeatedly transfected into NK cells in pairs using electroporation. Subsequently, the optimal sgRNA composition is screened and verified at both DNA and protein expression levels, ultimately obtaining the sgRNA composition with the highest knockout efficiency.

[0057] For in vitro validation of antitumor activity, this invention first adds TGF-β1 to the culture medium to simulate the tumor microenvironment conditions of NK cells, and then uses various HLA-E positive tumor cells (hematologic malignancies and solid tumors) for in vitro killing, which significantly improves antitumor activity compared with unmodified NK cells.

[0058] Furthermore, the present invention unexpectedly discovered that the NK cells with NKG2A and TGFBR2 dual gene knockout constructed by the present invention can achieve a high gene knockout rate while also ensuring a high cell expansion efficiency. After 14 days of culture, the expansion fold reached 1238-2934 times, which is significantly higher than the existing technology. Moreover, it breaks through the traditional technical barrier that the higher the gene knockout rate, the lower the cell expansion efficiency, laying the foundation for subsequent clinical applications. Attached Figure Description

[0059] Figure 1 NK purity detection results based on flow cytometry.

[0060] Figure 2 Results of CD16 positivity detection based on flow cytometry.

[0061] Figure 3 Results of NKG2A positivity rate detection based on flow cytometry.

[0062] Figure 4 Results of Smad2 / 3 phosphorylation level detection based on flow cytometry.

[0063] Figure 5 TGFBR2 TA clone detection results.

[0064] Figure 6 NKG2A TA clone detection results.

[0065] Figure 7 Results of NK cell killing rate against U937 cells at an effector-to-target ratio of 1:1.

[0066] Figure 8 Results of NK cell killing rate against U937 cells at an effector-to-target ratio of 5:1.

[0067] Figure 9 Results of NK cell killing rate against various adherent tumor cell lines at an effector-to-target ratio of 1:1.

[0068] Figure 10 Results of NKG2A positivity rate detection after RNP dosage optimization.

[0069] Figure 11 Results of Smad2 / 3 phosphorylation level detection after RNP dosage optimization.

[0070] Figure 12 Results of NKG2C positivity rate detection based on flow cytometry.

[0071] Figure 13 Amplification status of edited and unedited cell cultures. Detailed Implementation

[0072] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0073] NK cell NKG2A and TGFBR2 dual gene knockout protocol and efficacy validation 1. Test Methods 1.1 Design of sgRNAs targeting NKG2A and TGFBR2 genes Human NKG2A and TGFBR2 gene sequences were analyzed. Based on the design principle of CRISPR / Cas9 target site recognition, target sites for the target genes (NKG2A and TGFBR2) were screened, and multiple sgRNAs were designed for the screened target sites. After preliminary screening, sgRNAs targeting the NKG2A gene were obtained as shown in SEQ ID NO. 1-11, and sgRNAs targeting the TGFBR2 gene were obtained as shown in SEQ ID NO. 12-21. In this embodiment, each target gene was edited using a pair of sgRNAs. The designed sgRNAs were combined in pairs and repeatedly screened using electroporation transfection to obtain sgRNA pairs with high editing efficiency for later use. As shown in the table below, the sgRNA pairs targeting NKG2A selected in this invention are shown in the left column, and the sgRNA pairs targeting TGFBR2 selected in this invention are shown in the right column. Furthermore, in order to achieve dual gene knockout, the present invention further combines the screened sgRNAs (pairs) targeting the two target genes in pairs to form sgRNA combinations consisting of 4 sgRNAs, and then screens them again through multiple electroporation transfection methods to obtain the optimal sgRNA combination for achieving dual gene knockout.

[0074] Table 1 sgRNA Combinations 1.2 NK cell isolation (1) Collect 40ml of peripheral blood (or umbilical cord blood) from one healthy person; (2) Place the blood in a sterile 50ml centrifuge tube, centrifuge at 500g for 10 minutes; (3) Extract the upper plasma layer and dilute it with sodium chloride injection or PBS in a 1:1 ratio to the lower blood layer; (4) Add NK purification reagent and mix gently. Incubate at room temperature for 20 min. (5) Take another 50ml centrifuge tube and add 20ml of Ficoll; (6) Gently spread the incubated blood onto Ficoll, 20 ml per tube; (7) Centrifuge at 500g for 30 minutes; (8) Remove the upper layer of liquid; (9) Aspirate NK cells from the middle white membrane layer into a new centrifuge tube; (10) Add sodium chloride injection or PBS, and centrifuge at 500g for 10 minutes; (11) Discard the upper clear; (12) Repeat steps (10) and (11) once.

[0075] 1.3 Gene knockout (1) After the NK cells were separated, they were gently blown and sampled for AO / PI counting. Gene editing was performed according to the grouping in Table 1. (2) Take (2-10)×10 for each group 6 Centrifuge each cell at 500g for 10 minutes; (3) After aspirating the supernatant, add serum-free culture medium, PBS, or sodium chloride injection to resuspend the solution, 500g, for 10 minutes; (4) Discard the supernatant, add 82 μl of P3 Primary and 18 μl of supplement electroporation buffer (LONZA) and gently resuspend the cells; (5) Mix 200 pmol of Cas9 protein and 400 pmol of sgRNA (100 pmol of each of the four sgRNAs) and incubate at room temperature for 10-20 minutes; (6) Mix the incubated RNP mixture with the cell suspension and let it stand at room temperature for 5-10 minutes; (7) Transfer to the electro-rotation cup and perform electro-rotation using the CA138 electro-rotation program; (8) After electroporation, the cells were transferred to preheated complete immune cell culture medium (immune cell culture medium + 3-10% autologous plasma / 3-10% fetal bovine serum / 2-5% serum substitute, containing 500-1000 IU / ml IL2), and 0.3-3 times the number of K562 feeder cells (expressing IL21 and 41BBL) were added. The cells were then cultured in a 37℃ 5% CO2 incubator, and cell counts were performed every 2-3 days. On day 7, 0.1 to 1 times the number of K562 feeder cells (expressing IL21 and 41BBL) were added.

[0076] 1.4 Edit Rate Detection Samples were taken from NK cells cultured for 14-20 days for flow cytometry analysis to detect NK cell purity, NKG2A positivity rate, NKG2C positivity rate, and Smad2 / 3 phosphorylation level in the TGFBR2 downstream signaling pathway. Separate samples were taken for genomic analysis and TA cloning.

[0077] 1.5 Killing Activity Detection (1) Killing effect of NK cells on U937 suspension cells: NK cells were stimulated with 10-200 ng / ml TGF-β1 for 48-72 hours, and target cells U937 (human histiocytic lymphoma cells) were stimulated with 300-2000 IU / ml IFN-γ for 24 hours. Killing experiments were conducted with effector-target ratios of 1:1 and 5:1. The killing rate was detected by flow cytometry 15 minutes after 7-AAD staining 4 hours after killing.

[0078] (2) Killing effect of NK cells on adherent tumor cells: The three groups with the highest editing rate and the unedited group of NK cells were selected for the killing experiment of adherent tumor cells. NK cells were stimulated with 10-200 ng / ml TGF-β1 for 48-72 hours, and target cells were stimulated with 300-2000 IU / ml IFN-γ for 24 hours. The effector-to-target ratio was 1:1. The target cells were A549 (human lung adenocarcinoma cells), PANC1 (human pancreatic cancer cells), OSRC2 (human renal cancer cells), U251 (human glioma cells), HepG2 (human liver cancer cells), SK-OV-3 (human ovarian cancer cells), and SW-480 (human colon adenocarcinoma cells). Real-time cell analysis was used for detection.

[0079] 1.6 RNP Usage Optimization NK cells were isolated, activated, and gene-edited according to the experimental procedures in 1.2 and 1.3, using the sgRNA combination from combination 3 for gene editing. The amounts of Cas9 protein and sgRNA were optimized, reduced to 100 pmol / 200 pmol (group A), 50 pmol / 100 pmol (group B), 20 pmol / 40 pmol (group C), and 10 pmol / 20 pmol (group D), respectively. NK cells were cultured for 8-20 days after gene editing, and samples were taken for flow cytometry analysis to determine the editing rate.

[0080] 2. Experimental Results 2.1 Flow cytometry analysis of NK cell purity and CD16 positivity rate After culturing NK cells for 14-20 days, take 1×10⁻⁶ cells. 6 Flow cytometry was used to stain individual cells to detect NK cell purity and CD16 positivity rate. The results showed that the purity of NK cells cultured using this method was higher than 99%. Figure 1 The CD16 positivity rate was higher than 91% ( Figure 2 ).

[0081] 2.2 Flow cytometry detection of NKG2A positivity rate The NKG2A positivity rate test results showed that the NKG2A positivity rate in unedited cells was as high as 74.52%, while the NKG2A positivity rate in edited cells decreased significantly. The NKG2A positivity rates of combinations 2 and 3 were only 0.38% and 0.33%, respectively, with editing rates reaching 99.49% and 99.56%. This indicates that the optimal sgRNA composition targeting NKG2A is the first sgRNA shown in SEQ ID NO.3 and the second sgRNA shown in SEQ ID NO.4. Figure 3 ).

[0082] 2.3 Flow cytometry detection of Smad2 / 3 phosphorylation level, a downstream signaling pathway of TGFBR2. TGF-β signals through type I and type II TGF-β receptors (TGFBR1 and TGFBR2). After binding to TGFBR2, TGF-β recruits and phosphorylates TGFBR1. This phosphorylation process continues downstream, recruiting and phosphorylating intracellular Smad. The Smad complex enters the nucleus and interacts with other transcription factors to transmit transcription and translation signals downstream. Detecting the phosphorylation level of downstream Smad2 / 3 after TGFBR2 knockout can indirectly reflect the TGFBR2 knockout efficiency. Compared with unedited cells, the phosphorylation level of edited cells decreased, with combination 3 showing the largest decrease. Figure 4 This indicates that the optimal sgRNA composition for targeting TGFBR2 is the third sgRNA shown in SEQ ID NO.14 and the fourth sgRNA shown in SEQ ID NO.13.

[0083] 2.4 TA cloning and sequencing to detect the knockout rates of TGFBR2 and NKG2A Combination 3, which had the highest editing rate detected by streaming cytometry, was selected for TA cloning. The results showed that the editing rate of TGFBR2 was 100%. Figure 5 ), NKG2A has an edit rate of 100% ( Figure 6 ).

[0084] 2.5 Killing Activity Detection (1) Killing effect of NK cells on suspension cells U937 Using effector-to-target ratios of 1:1 and 5:1, NK cells stimulated with TGF-β1 showed only 4.41% and 6.55% kill rates for unedited cells at 1:1 and 5:1 ratios (target cells were HLA-E-positive U937 cells), respectively. In contrast, combination 3, with the highest edited rate, achieved a kill rate of 37.53% at a 1:1 ratio and 70.35% at a 5:1 ratio. These results indicate that knocking out TGFBR2 and NKG2A genes significantly enhances the killing effect of NK cells on HLA-E-positive tumor cells in the tumor microenvironment. Figure 7 andFigure 8 ).

[0085] (2) Killing effect of NK cells on adherent tumor cells The results of killing assays on seven adherent tumor cell lines showed that, compared with unedited NK cells, the killing effect of NK cells was significantly increased after knocking out the NKG2A and TGFBR2 genes, indicating that knocking out the NKG2A and TGFBR2 genes can enhance the anti-tumor effect of NK cells. Figure 9 ).

[0086] 2.6 Detection of NKG2A Positive Rate and Smad2 / 3 Phosphorylation Level after RNP Dosage Optimization Gene editing was performed using the sgRNA combination of combination 3. After the amounts of Cas9 protein and sgRNA were reduced to 100 pmol / 200 pmol (group A), 50 pmol / 100 pmol (group B), 20 pmol / 40 pmol (group C), and 10 pmol / 20 pmol (group D), respectively, the NKG2A positivity rate in groups A, B, and C was still <1% (knockout rates were 99.38%, 99.36%, and 99.22%, respectively), while the NKG2A positivity rate in group D was 13.01%, and the editing rate decreased to 85.42%.

[0087] Flow cytometry analysis of Smad2 / 3 phosphorylation levels in the TGFBR2 downstream signaling pathway showed that the TGFBR2 downstream signaling pathway was almost completely blocked in groups A, B, and C, indicating that the TGFBR2 gene knockout was thorough. In group D, some cells still had Smad2 / 3 phosphorylation, indicating that some TGFBR2 gene function remained. Figure 10 and Figure 11 ).

[0088] The above results indicate that sgRNAs SEQ ID NO.3 and SEQ ID NO.4 targeting NKG2A and sgRNAs SEQ ID NO.14 and SEQ ID NO.13 targeting TGFBR2 have extremely high editing efficiency. Even when the amount of Cas9 protein and sgRNA is reduced to 20 pmol / 40 pmol, the editing rate can still reach over 90%.

[0089] 2.7 Effect of NKG2A knockout on NKG2C positivity rate in NK cells Both NKG2A and NKG2C can form heterodimers with CD94 to recognize HLA-E. NKG2A transduces inhibitory signals, while NKG2C transduces activating signals. This invention further verifies the positivity rate of NKG2C in NKG2A knockout NK cells.

[0090] As in this invention Figure 3As shown, for NKG2A knockout NK cells, groups 2 and 3 had the lowest NKG2A positivity rates, only 0.38% and 0.33% respectively, but groups 2 and 3 had the highest NKG2C positivity rates, at 61.22% and 61.22% respectively. Figure 12 The inventors believe that knocking out the NKG2A gene in NK cells blocks the inhibitory pathway of NKG2A / HLA-E, increases NKG2C expression, promotes the activation pathway of NKG2C / HLA-E, and thus enhances the anti-tumor effect of NK cells.

[0091] 2.8 Effects of double knockout of NKG2A and TGFBR2 genes on cell proliferation in NK cells Those skilled in the art will know that electroporation gene knockout damages cells. Generally, samples with higher gene knockout efficiency have lower cell viability, thus affecting subsequent cell proliferation. [1] Furthermore, literature indicates that NKG2A is a crucial checkpoint for maintaining the proliferative capacity of NK cells. Knockout of NKG2A weakens NK cell proliferation and increases activation-induced cell death, thereby affecting their proliferative capacity. [2] Based on this, the present invention tested the in vitro culture expansion of gene knockout cells in groups 1-7, and the expansion of edited and unedited cells after 19 days of culture was as follows: Figure 13 After 14 days of amplification, the fold increase in each group ranged from 1238 to 2934 times, and after 19 days, the fold increase ranged from 7397 to 14076 times. It can be seen that knocking out the NKG2A and TGFBR2 genes in NK cells using the method of this invention did not affect the fold increase of NK cells; all groups of cells could expand by more than 7000 times.

[0092] The above data show that, in the examples of NK cell target gene editing provided by this invention, the Cas9-sgRNA ribonucleoprotein complex formed by the sgRNA combinations shown in Groups 1-7 can achieve highly efficient gene editing of selected targets while maintaining the cell's efficient expansion capacity.

[0093] The above specific embodiments are merely illustrative of the invention and do not represent a limitation thereof. Those skilled in the art will recognize that other variations of the specific structure of this invention are possible.

[0094] [1] Lin H, Ye S, Zhang S, Ge T, Li D, Huang L, Zhu L, Mu W.Optimizing the procedure for manufacturing clinical-grade geneticallymanipulated natural killer cells for adoptive immunotherapy. Cytotherapy.2025 Feb;27(2):247-257. [2] Kaulfuss M, Mietz J, Fabri A, Vom Berg J, Münz C, Chijioke O. TheNK cell checkpoint NKG2A maintains expansion capacity of human NK cells. SciRep. 2023 Jun 29;13(1):10555.

Claims

1. A gene-edited NK cell, characterized in that, The gene editing reduces or knocks out the expression of the NK cell activity inhibitory receptor, which is selected from a combination of NKG2A and TGFBR2; The gene editing reduced or knocked out the expression and function of NKG2A protein encoded by the NKG2A gene and TGFBR2 protein encoded by the TGFBR2 gene in NK cells, resulting in higher anti-tumor activity compared with NK cells that had not undergone gene editing. The gene editing was performed using an RNA-guided endonuclease. The gene editing was performed using the CRISPR / Cas9 system; The gene editing involves contacting a population of NK cells with a Cas9-sgRNA ribonucleoprotein complex; The sgRNAs forming the Cas9-sgRNA ribonucleoprotein complex include sgRNAs used to knock out the NKG2A gene. NKG2A and sgRNA for knocking out the TGFBR2 gene TGFBR2 , wherein the sgRNA NKG2A It consists of a first sgRNA and a second sgRNA, wherein the sgRNA TGFBR2 It consists of a third sgRNA and a fourth sgRNA; The first sgRNA and the second sgRNA are each independently selected from any one of the nucleotide sequences shown in SEQ ID NO. 1-11, and the first sgRNA and the second sgRNA are different; the third sgRNA and the fourth sgRNA are each independently selected from any one of the nucleotide sequences shown in SEQ ID NO. 12-21, and the third sgRNA and the fourth sgRNA are different.

2. The gene-edited NK cells according to claim 1, characterized in that... The sgRNA forming the Cas9-sgRNA ribonucleoprotein complex consists of the first sgRNA shown in SEQ ID NO.1, the second sgRNA shown in SEQ ID NO.2, the third sgRNA shown in SEQ ID NO.12, and the fourth sgRNA shown in SEQ ID NO.13; or, The sgRNA forming the Cas9-sgRNA ribonucleoprotein complex consists of the first sgRNA shown in SEQ ID NO.3, the second sgRNA shown in SEQ ID NO.4, the third sgRNA shown in SEQ ID NO.12, and the fourth sgRNA shown in SEQ ID NO.13; or, The sgRNA forming the Cas9-sgRNA ribonucleoprotein complex consists of the first sgRNA shown in SEQ ID NO.3, the second sgRNA shown in SEQ ID NO.4, the third sgRNA shown in SEQ ID NO.14, and the fourth sgRNA shown in SEQ ID NO.13; or, The sgRNAs forming the Cas9-sgRNA ribonucleoprotein complex consist of the first sgRNA shown in SEQ ID NO. 5, the second sgRNA shown in SEQ ID NO. 11, the third sgRNA shown in SEQ ID NO. 15, and the fourth sgRNA shown in SEQ ID NO. 16; or, The sgRNAs forming the Cas9-sgRNA ribonucleoprotein complex consist of the first sgRNA shown in SEQ ID NO. 6, the second sgRNA shown in SEQ ID NO. 7, the third sgRNA shown in SEQ ID NO. 17, and the fourth sgRNA shown in SEQ ID NO. 18; or, The sgRNAs forming the Cas9-sgRNA ribonucleoprotein complex consist of the first sgRNA shown in SEQ ID NO. 8, the second sgRNA shown in SEQ ID NO. 9, the third sgRNA shown in SEQ ID NO. 19, and the fourth sgRNA shown in SEQ ID NO. 14; or, The sgRNA that forms the Cas9-sgRNA ribonucleoprotein complex consists of the first sgRNA shown in SEQ ID NO.10, the second sgRNA shown in SEQ ID NO.3, the third sgRNA shown in SEQ ID NO.20, and the fourth sgRNA shown in SEQ ID NO.

21.

3. The method for constructing gene-edited NK cells according to any one of claims 1-2, characterized in that, The method includes contacting NK cells with an RNA-guided endonuclease; The method includes contacting NK cells with a Cas9-sgRNA ribonucleoprotein complex; The method includes: incubating sgRNA with Cas9 protein via RNP to form a Cas9-sgRNA ribonucleoprotein complex, introducing it into NK cells to achieve joint knockout of the NKG2A gene and the TGFBR2 gene in NK cells, and forming the sgRNA of the Cas9-sgRNA ribonucleoprotein complex as described in claim 1 or 2.

4. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the gene-edited NK cells as described in any one of claims 1-2, and a pharmaceutically acceptable carrier or excipient.

5. The use of the gene-edited NK cells according to any one of claims 1-2 or the pharmaceutical composition according to claim 4 in the preparation of a medicament for treating HLA-E positive tumors.

6. An sgRNA composition comprising sgRNA that specifically targets the NKG2A gene on NK cells. NKG2A and sgRNA that specifically targets the TGFBR2 gene on NK cells TGFBR2 ,in, The sgRNA NKG2A It consists of a first sgRNA and a second sgRNA, wherein the sgRNA TGFBR2 It consists of a third sgRNA and a fourth sgRNA; The first sgRNA and the second sgRNA are each independently selected from any one of the nucleotide sequences shown in SEQ ID NO. 1-11, and the first sgRNA and the second sgRNA are different; the third sgRNA and the fourth sgRNA are each independently selected from any one of the nucleotide sequences shown in SEQ ID NO. 12-21, and the third sgRNA and the fourth sgRNA are different.

7. The sgRNA composition according to claim 6, characterized in that, The sgRNA composition comprises the first sgRNA shown in SEQ ID NO.1, the second sgRNA shown in SEQ ID NO.2, the third sgRNA shown in SEQ ID NO.12, and the fourth sgRNA shown in SEQ ID NO.13; or The sgRNA composition comprises the first sgRNA shown in SEQ ID NO. 3, the second sgRNA shown in SEQ ID NO. 4, the third sgRNA shown in SEQ ID NO. 12, and the fourth sgRNA shown in SEQ ID NO. 13; or The sgRNA composition comprises the first sgRNA shown in SEQ ID NO. 3, the second sgRNA shown in SEQ ID NO. 4, the third sgRNA shown in SEQ ID NO. 14, and the fourth sgRNA shown in SEQ ID NO. 13; or The sgRNA composition comprises the first sgRNA shown in SEQ ID NO. 5, the second sgRNA shown in SEQ ID NO. 11, the third sgRNA shown in SEQ ID NO. 15, and the fourth sgRNA shown in SEQ ID NO. 16; or The sgRNA composition comprises the first sgRNA shown in SEQ ID NO. 6, the second sgRNA shown in SEQ ID NO. 7, the third sgRNA shown in SEQ ID NO. 17, and the fourth sgRNA shown in SEQ ID NO. 18; or The sgRNA composition comprises the first sgRNA shown in SEQ ID NO. 8, the second sgRNA shown in SEQ ID NO. 9, the third sgRNA shown in SEQ ID NO. 19, and the fourth sgRNA shown in SEQ ID NO. 14; or The sgRNA composition consists of the first sgRNA shown in SEQ ID NO.10, the second sgRNA shown in SEQ ID NO.3, the third sgRNA shown in SEQ ID NO.20, and the fourth sgRNA shown in SEQ ID NO.

21.

8. An RNP-mediated CRISPR / Cas9 gene editing system, wherein the gene editing system achieves dual gene knockout of NKG2A and TGFBR2 genes in NK cells, characterized in that, The gene editing system comprises the sgRNA composition as described in claim 6 or 7 and the Cas9 protein.

9. The use of the sgRNA composition of claim 6 or 7 or the RNP-mediated CRISPR / Cas9 gene editing system of claim 8 in at least one of the following: 1) Application in knocking out NKG2A and TGFBR2 genes in NK cells; 2) Application in the preparation of products for knocking out the NKG2A and TGFBR2 genes in NK cells, including but not limited to reagents, kits, chips or gene editing platforms.

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