ENGINEERED HUMAN IMMUNE CELLS, PREPARATION METHOD AND APPLICATION THEREOF

Abstract

Provided are engineered human immune cells, a preparation method, and an application thereof. The engineered human immune cells are immune killer lymphocytes induced by reprogramming human T cells, from which the BCL11B gene is deleted. The engineered human immune cells retain markers and functions of the T cells from which they are derived and have markers and functions of NK cells. The reprogrammed engineered human immune cells can be used to prepare drugs for treating tumors and infectious diseases.

Claims

1. Immune killer lymphocytes induced by reprogramming of human T cells, which retain markers and functions of human T cells from which the immune killer lymphocytes are derived and have markers and functions of NK cells, wherein the reprogramming of the human T cells involves deletion of a BCL11B gene.

2. The immune killer lymphocytes according to claim 1, wherein the human T cells are mature human T cells or a cell population comprising mature human T cells; preferably, the mature human T cells or the cell population comprising the mature human T cells are derived from human cord blood or human peripheral blood; and preferably, the mature human T cells or the cell population comprising the mature human T cells are derived from mature T cells or cell populations obtained by differentiation of pluripotent stem cells, embryonic stem cells or cord blood stem cells.

3. The immune killer lymphocytes according to claim 1, expressing functional TCR, CD3 and NKp30.

4. The immune killer lymphocytes according to claim 1, expressing a marker of NK cells selected from the following group consisting of CD11c, NKG2D and CD161; preferably, immunosuppression checkpoints PD-1, CTLA-4 or FOXP3 are of low expression or no expression in the immune killer lymphocytes; and preferably, NK-related markers CD127, CD16, KIRDL2, KIRDL3 and NKG2A are of low expression or no expression in the immune killer lymphocytes.

5. The novel immune killer lymphocytes according to claim 1, wherein compared with the T cells from which the immune killer lymphocytes are derived, expression of NOTCH is up-regulated

6. The immune killer lymphocytes according to claim 1, wherein compared with the T cells from which the immune killer lymphocytes are derived, expression of transcription factors LEF1 and TCF7 is down-regulated, and expression of NOTCH, AP1, ID2, TBX21 and NFIL3 is up-regulated.

7. The immune killer lymphocytes according to claim 1, wherein TCR-mediated signal transduction of the immune killer lymphocytes is enhanced; preferably, compared with the T cells from which the immune killer lymphocytes are derived, expression of genes of the cells, which are related to the TCR-mediated signal transduction and comprise CSF2, FOS, MAPK12, MAP3K8, IFNγ, NFKBIA, MAPK11, IL-10 and TEC, is up-regulated; and preferably, compared with NK cells, T cell recognition and TCR signal transduction of the immune killer lymphocytes are enhanced; and preferably, expression of CD3, CD4, CD8 and CD40LG is up-regulated.

8. The immune killer lymphocytes according to claim 1, wherein compared with the T cells from which the immune killer lymphocytes are derived, NK killing toxicity-related signal transduction of the immune killer lymphocytes is enhanced; and preferably, compared with the T cells from which the immune killer lymphocytes are derived, expression of genes of the immune killer lymphocytes, which are related to the NK killing toxicity-related signal transduction and comprise PRF1, CSF2, ICAM1, CD244, PLCG2, IFNG, FCER1G, GZMB, NCR2, NCR1, KIR2DL4 and SYK, is up-regulated.

9. The immune killer lymphocytes according to claim 1, comprising cell subsets CD8+NKp46+NKp44+NKp30+, CD4+NKp30+, and γδTCR+NKp46+NKp44+NKp30+T.

10. The immune killer lymphocytes according to claim 1, wherein the human T cells are mature human T cells, and the reprogramming of the human T cells comprises: 1) activating mature human T cells; 2) performing BCL11B gene knockout on the activated mature human T cells obtained in step 1); and 3) culturing the cells obtained in step 2) with a T cell culture medium.

11. The immune killer lymphocytes according to claim 10, wherein in step 1), the activation is performed using an anti-human CD3 antibody, an anti-human CD28 antibody, and an anti-human CD2 antibody; and preferably, magnetic beads of anti-human CD3 antibody, anti-human CD28 antibody and anti-human CD2 antibody are mixed with the mature human T cells in a ratio of 1:2 and incubated to activate the T cells.

12. The immune killer lymphocytes according to claim 10, wherein in step 2), the BCL11B gene knockout is performed by CRISPR/CAS9; preferably, a target of the gene knockout is at a second exon of the BCL11B gene; and preferably, the target of the gene knockout is at a third exon of the BCL11B gene.

13. The immune killer lymphocytes according to claim 10, wherein in step 3), the T cell medium comprises IL-2; and preferably, the cells obtained in step 2) are not co-cultured with OP9-DL1.

14. A method for preparing the immune killer lymphocytes according to claim 1, comprising: 1′) activating human T cells; 2′) performing BCL11B gene knockout on the activated human T cells obtained in step 1′); and 3′) culturing the cells obtained in step 2′) with a T cell culture medium.

15. The method according to claim 14, wherein the human T cells are mature human T cells or a cell population comprising mature human T cells; preferably, the mature human T cells or the cell population comprising the mature human T cells are derived from human cord blood or human peripheral blood; and preferably, the mature human T cells or the cell population comprising the mature human T cells are derived from mature T cells or cell populations obtained by differentiation of pluripotent stem cells, embryonic stem cells or cord blood stem cells.

16. The method according to claim 14, wherein in step 1′), the activation is performed using an anti-human CD3 antibody, an anti-human CD28 antibody, and an anti-human CD2 antibody; and preferably, magnetic beads of anti-human CD3 antibody, anti-human CD28 antibody and anti-human CD2 antibody are mixed with mature human T cells in a ratio of 1:2 and incubated to activate the T cells.

17. The method according to claim 14, wherein in step 2′), the BCL11B gene knockout is performed by CRISPR/CAS9; preferably, the gene knockout is performed at a second exon of a BCL11B gene; and preferably, the gene knockout is performed at a third exon of the BCL11B gene.

18. The method according to claim 1, wherein in step 3′), the T cell medium comprises IL-2; and preferably, the cells obtained in step 2′) are not co-cultured with OP9-DL1.

19. (canceled)

20. A method for treating tumors, AIDS and infectious diseases, comprising administering an effective amount of the immune killer lymphocytes according to claim 1 to subject in need thereof; and preferably, the infectious diseases are viral infectious diseases.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0041] FIG. 1 is a schematic diagram of a PX458-gBCL11B vector constructed in Example 1 of the present application.

[0042] FIG. 2 is a graph showing detection and verification by gene sequencing of whether BCL11B exons of T cells transduced with PX458-gBCL11B had been knocked out, and the control group was T cells transduced with PX458 empty vectors (Mock).

[0043] FIG. 3 is a graph showing detection and verification by Western Blotting of the expression level of BCL11B proteins in T cells transduced with PX458-gBCL11B to further confirm whether BCL11B proteins were deleted, and the control group was T cells transduced with PX458 empty vectors (Mock).

[0044] FIG. 4 is a scatter plot of flow cytometry results, showing that compared with Mock T cells and NK cells, the ITNK cells of the present application (i.e., PX458-gBCL11B-transduced T cells, indicated by PAX458 in the figure) simultaneously highly expressed the T cell marker CD3 and the NK cell markers CD56 and NKp46. All samples were derived from the same PBMC sample, among which Mock T cells were sorted from PBMC through Pan-T, so there was 7.2% to 7.8% CD3−CD56+NKp46+NK cell mixed population; PX458 cells were obtained by transfection of Mock T cells with PX458-gBCL11B, so there was 7.6% to 7.8% NK cell subset that was the same as that in Mock T cells; and NK cells were obtained by culturing and purification of PBMC through an NK cell culture medium, so there was a small amount of 10.4% NKT or γδ T cell mixed population of CD3+CD56+.

[0045] FIG. 5 is a scatter plot (A) of flow cytometry results, showing that compared with Mock T cells, T cell markers CD3 and NK cell markers NKp46, CD56, NKp30 and NKp44 were of high expression in cord blood-derived ITNK cells (i.e., PX458-gBCL11B-transduced T cells, indicated by PAX458 in the figure), and a corresponding cell percentage statistical graph (B).

[0046] FIG. 6 is a scatter plot (A) of flow cytometry results, showing that compared with Mock T cells, T cell markers CD3 and NK cell markers NKp46, CD56, NKp30 and NKp44 are of high expression in peripheral blood-derived ITNK cells (i.e., PPX458-gBCL11B-transduced T cells, indicated by PAX458 in the figure), and a corresponding cell percentage statistical graph (B).

[0047] FIG. 7 is transmission electron microscopic images of Mock T cells, NK cells and ITNK cells (i.e., PX458-gBCL11B-transduced T cells, indicated by PAX458 in the figure), showing that the nucleoplasm of ITNK cells was lower than the nucleoplasm of T cells; in which 1 indicates the core, 2 indicates mitochondria, 3 indicates endoplasmic reticulum, and 4 indicates granules, with a scale of 2 μm in the left images and a scale of 500 nm in the right images.

[0048] FIG. 8 shows the expression of NK receptors in T cell subsets CD4+, CD8+, CD3+CD4−CD8− of PX458-gBCL11B-transduced T cells (i.e., ITNK cells) derived from cord blood and peripheral blood, showing that the expression of NKp46 in CD8+ T cells was significantly higher than the expression of NKp46 in CD4+ T cells; in which n=6, indicating that the experiment was a duplicate of 6 independent healthy donors, ***P≤0.001, and a paired t test was adopted.

[0049] FIG. 9 shows flow cytometry analysis of PX458-gBCL11-transduced T cells, in which CD56+ cells in subsets CD4+, CD8+ and CD4−CD8− were sorted and subjected to DNA sequenced, which further confirmed that the BCL11B site in ITNK cells was knocked out.

[0050] FIG. 10 shows the sequencing of TCRβ diversity, showing that all TCRβ chain sequence trajectory variables had the same diversity in the same source of T cells and ITNK cells.

[0051] FIG. 11 shows the sequencing of TCRα diversity, showing that all TCRα chain sequence trajectory variables had the same diversity in the same source of T cells and ITNK cells.

[0052] FIG. 12 is t-SEN dot density graphs of mass cytometry clustering analysis, in which (A) shows t-SEN dot densities of CD45+ monocytes derived from cord blood and peripheral blood, respectively, where CD45+ monocytes were divided into three experimental groups: cells (Mock-T) transduced with PX458 empty vectors, cells (PX458-T) transduced with PX458-gBCL11B and cells (activated PX458-T) that were transduced with PX458-gBCL11B and then activated by K562 cells for 24 hours; and (B) and (C) show clustering differences and over-transformation (indicated by arrows respectively) between cord blood (CB)-derived Mock-T cells and CB-derived T cells with BCL11B knocked out (BCL11B-KO T), CB-derived T cells with BCL11B knocked out (BCL11B-KO T) and activated CB-derived T cells with BCL11B knocked out (Activated BCL11B-KO T), adult peripheral blood (PBMC)-derived Mock-T cells and PBMC-derived T cells with BCL11B knocked out (BCL11B-KO T), PBMC-derived T cells with BCL11B knocked out (BCL11B-KO T) and activated PBMC-derived T cells with BCL11B knocked out (Activated BCL11B-KO T), respectively.

[0053] FIG. 13 is t-SEN color enrichment cluster graphs of cord blood and peripheral blood CD45+ cells fusion, in which ITNK cells are marked as indicated, where the standardized expression of CD3, CD4, CD8, γδ TCR, CD56, NKp46, NKp30, NKp44 and CD11c on t-SEN graphs stained the cells.

[0054] FIG. 14 is t-SEN color enrichment cluster graphs of ITNK cell immunophenotypic analysis-cord blood and peripheral blood CD45+ cells fusion; in which (A) shows the expression levels of markers NKG2D and CD161, (B) shows the expression levels of markers CD25, CD127, CD16, KIRDL2, KIRDL3 and NKG2A, and (C) shows the expression levels of immune checkpoint markers PD-1, CTLA-4, FOXP3 and TIM-3.

[0055] FIG. 15 shows that according to the grouping frequency of immunocyte subsets of cord blood T cells, ITNK cells can be differentiated from CD4+ T cells, CD8+ T cells and γδTCR+ T cells, and all ITNK subtypes can be activated by HLA negative cells (K562).

[0056] FIG. 16 shows that according to the grouping frequency of immunocyte subsets of peripheral blood T cells, ITNK cells can be differentiated from CD4+ T cells, CD8+ T cells and γδTCR+ T cells, and all ITNK subtypes can be activated by HLA negative cells (K562).

[0057] FIG. 17 shows a heatmap of mass cytometry analysis results, which shows the expression of markers for each immune cell subset.

[0058] FIG. 18 shows ITNK cells sorting for RNA-Seq. CD3+ T, CD3−NKp46+ NK, CD3+NKp46+ CB-ITNK and PBMC-ITNK were sorted from cord blood or adult peripheral blood for RNA sequencing and atac sequencing. The purity of sorted cells from Mock-T cells (n=6), ITNK cells (n=6) and NK cells (n=4) was 95.3±3.61%, 92.41±2.6% and 92.88±2.06%, respectively.

[0059] FIG. 19 shows the principal component analysis of RNA sequencing-cell subset gene expression data.

[0060] FIG. 20 shows RNA sequencing-KEGG enrichment pathway analysis of gene up-regulation of ITNK cells relative to T cells (cut-off: 2 times absolute logarithm, change≥1; adjusted P value≤0.05).

[0061] FIG. 21 shows RNA sequencing-KEGG enrichment pathway analysis of gene up-regulation of ITNK cells relative to NK cells (cut-off: 2 times absolute logarithm, change≥1; adjusted P value≤0.05).

[0062] FIG. 22 is a hierarchical clustering heatmap of RNA-Seq, in which the left graph shows the difference of gene transcription expression among T cells, ITNK cells and NK cells (log 2 absolute fold change≥1; adjusted P value≤0.05), and the right graph is heatmaps of differential expression of genes screened by RNA-seq analysis in T cells, ITNK cells and NK cells; and compared with T cells, the expression of NK signal gene in ITNK cells was up-regulated while the expression of TCF1 and LEF1 genes was down-regulated (log 2 absolute fold change≥1; adjusted P value≤0.05).

[0063] FIG. 23 is dynamic flow cytometric immunophenotype analysis images of ITNK cells; where the proportion of positive subsets NKp30 and NKp46 in subsets CD3+CD4+, CD3+CD8+ were detected by flow cytometry on day 0, day 5, day 10, day 15 and day 20 after BCL11B knockout in human T cells; the data represents three experiments; and NKp46 was detected on day 5 after BCL11B knockout and stabilized on day 10 to day 15.

[0064] FIG. 24(A) is a t-SNE dot plot of Sc-RNA seq analysis results, in which the upper graph shows the cell distribution of D0-D20, and the lower graph shows the clustering distribution of 4948 cells on Day D0 (2263), D5 (1565), D10 (498), D15 (204) and D20 (418) after PX458-gBCL11B gene-transduced T cells were subjected to BCL11B knockout, where 4948 cells were clustered into 11 cell subsets, and ITNK cells were mainly concentrated in subsets labeled by (red) circles; FIG. 24(B) shows the expression of NK cell markers in 4948 detected cells (11 subsets) through t-SNE dot plots; and FIG. (C) shows the expression of T cell markers, NK cell markers, immune checkpoints, transcription factors and apoptosis gene-related genes in 4948 cells (11 subsets) through t-SNE dot plots.

[0065] FIG. 25(A) is KEGG signaling pathway analysis, showing high expression of NK cytotoxic genes in ITNK cells; FIG. 25(B) is a violin plot, showing the expression profiles of NK cells-related KAR gene and NK cells-related MR gene in different subsets; and FIG. 25(C) is a violin plot, showing the expression profiles of genes related to T cell and NK cell development, where the expression of NK signal genes (ID2 and TBX21), NOTCH-related genes (MXI1, ZMIZ1, and RBPJ) and AP-1-related genes (FOS, JUN, JUNB, and JUND) was up-regulated in ITNK cells.

[0066] FIG. 26(A) is an unsupervised trajectory analysis of individual cells from subset 5 (CD8+ T), subset 0 (early CD8+ ITNK) and subset 1 (late CD8+ ITNK), showing a gradual transition from CD8+ T cells to CD8+ ITNK cells; and FIG. 26(B) is an unsupervised trajectory analysis of individual cells from subset 4 (CD4+ T), subset 5 (early CD4+ ITNK) and subset 7 (late CD4+ ITNK), showing a gradual transition from CD4+ T cells to CD4+ ITNK cells.

[0067] FIG. 27(A) is a bar graph showing up-regulation of the expression of NK-related transcription factors ID2 and TBX21 in ITNK cells; and FIG. (B) is a Western Blot analysis of TBX21 and ID2 in immune cell lysate, in which NK cells were in the left lane, ITNK cells were in the middle lane, T cells were in the right lane, and β-Tublin was used as the internal reference.

[0068] FIG. 28 is a histogram of the IFNγ secretion of T cells and ITNK cells after stimulated by anti-NKp30 antibody, anti-NKp46 antibody and anti-CD3/CD28 antibody (5 ug/ml), respectively, by Elisa assay, in which the data came from samples of three independent donors; the data were represented as mean±SD; **P≤0.01, and ***P≤0.001; and a paired t test was adopted.

[0069] FIG. 29 shows the cytokine secretion of T cells, ITNK cells and NK cells after stimulated by K562 cells, where specifically, T cells, ITNK cells and NK cells were incubated with K562 cells at 37° C. for 18 hours at an effector (E)-target (T) ratio of 1:1, respectively, the supernatant was collected for measuring the concentrations of cytokines (CSF2, CCL4, IFN γ, CCL3, IL13, IL2, TNF, CX3CL1, IL8, IL10, IL23, IL7, IL4, ILS, CXCL11, CCL20, IL6, IL17A, IL21, IL12, and IL1 β) by multiplex immunoassay; in which the value was expressed as the mean of three different donors.

[0070] FIG. 30 shows the specific cytotoxicity percentages of ITNK cells to HLA-negative K562 cells (A), HLA-positive Hela cells (B), HLA-positive A549 cells (C), and HLA-positive NALM-6 cells (D), in which the data were expressed as mean±standard deviation (SD); **P 0.01 0.01; an unpaired t-test was adopted.

[0071] FIG. 31 shows the protein and phosphorylation levels of Fyn, PLC-g2, Syk, Erk1/2 and mTOR in immune cell lysate, measured by western blot after K562 cells were stimulated for 6 hours, in which NK cells were in the left three lanes, ITNK cells were in the middle three lanes, T cells were in the right three lanes, BCL11B was used as gene editing control, and GAPDH was used as sample control.

[0072] FIG. 32(A) is a schematic diagram of the test for detecting the killing of tumor cells by ITNK cells in vivo; FIGS. 32(B) and (C) show the quantitative analysis of the total flux of luciferase activity in vivo in tested mice at specific time points by bioluminescence imaging (5 mice in each group), in which the results were mean±SD, **P≤0.01, and an unpaired t test was adopted; FIG. 32(D) is a plot of survival time of K562 tumor-bearing mice treated with PBS (n=10), Mock T cells (n=15), ITNK cells (n=15) and NK cells (n=5); and FIG. 32(E) shows the size of tumors in Hela tumor-bearing mice that were treated on day 7 and day 10 with PBS, Mock T cells, ITNK cells and NK cells respectively, measured at designated time points (5 mice in each group), in which the data were expressed as mean±standard deviation, ***P≤0.001, and an unpaired t test was adopted.

[0073] FIG. 33 shows the distribution and maintenance of ITNK cells in mice after ITNK cells were transplanted into NSI-strain immunodeficient mice lacking T cells, B cells and NK cells, in which, the top graph of (A) is a schematic diagram of injection of ITNK cells into NSI-strain mice and detection experiment, showing that ITNK cells were subjected to short-term analysis and long-term analysis on day 1, day 7, day 14, day 21 and month 6, respectively (n=3 in each group); the lower graph of (A) shows the percentage of ITNK cells in CD3+ T cells, analyzed by representative flow cytometry; and (B) shows the dynamic distribution of ITNK cells in peripheral blood (PB), bone marrow (BM), spleen, liver and lung of mice, showing that T cells and ITNK cells were not detected 6 months after the injection of ITNK cells.

DETAILED DESCRIPTION

[0074] To further elaborate on the technical means adopted and the effects achieved in the present application, the solutions of the present application are further described below through specific examples in conjunction with drawings, but the present application is not limited to the scope of the examples.

[0075] The present application is not limited to the relative arrangement, numeric expressions and numerical values of the components and steps set forth in these examples, unless otherwise noted. Techniques, methods and devices known to those of ordinary skill in the art may not be discussed in detail, but in appropriate circumstances, techniques, methods and devices should be regarded as part of the specification.

EXAMPLE 1

Preparation of Reprogrammed Natural Killer (ITNK) Cells of the Present Application

[0076] Construction of Gene Knockout Plasmid Vector

[0077] According to the selection rule of CRISP/CAS9 target sites: GN19NGG, where GN19 was a target site, N was still better G, and the target site can be on the antisense strand (i.e., the sequence order on the sense strand is CCN N19C), the following target sequences were selected and forward (F) and reverse (R) primers were designed as guideRNA (gRNA), respectively. The gRNA was annealed and ligated into the digested PX458 vectors to construct PX458-gBCL11B vectors (as shown in FIG. 1). PX458-gBCL11B was transfected in the 293 T cell line, and monoclones were selected. The knockout conditions such as base deletion and dislocation of BCL11B knockout targets were detected by gene sequencing (see FIG. 2), and the knockout efficiency was statistically calculated, as shown in Table 1 below.

TABLE-US-00001 TABLE 1 BCL11B target sequences and their corresponding gRNAs list Knoc kout Target Speci- effi- Location sequences Forward and reverse primers ficity ciency Second GACCCTGACCTGC F: caccGACCCTGACCTGCTCACCTG 58 39 exon TCACCTG (SEQ ID NO: 2) (SEQ ID NO: 1) R: aaacCAGGTGAGCAGGTCAGGGTC (SEQ ID NO: 3) Second GAAGCAGTGTGG F: caccGAAGCAGTGTGGCGGCAGCT 67 48 exon CGGCAGCT (SEQ (SEQ ID NO: 5) ID NO: 4) R: aaacAGCTGCCGCCACACTGCTTC (SEQ ID NO: 6) Second CAGGTGGTCATCT F: caccCAGGTGGTCATCTTCGTCGG 97 90 exon TCGTCGG (SEQ ID (SEQ ID NO: 8) NO: 7) R: aaacCCGACGAAGATGACCACCTG (SEQ ID NO: 9) Second GCAGGTGGTCATC F: caccGCAGGTGGTCATCTTCGTC 95 60 exon TTCGTC (SEQ ID (SEQ ID NO: 11) NO: 10) R: aaacCGACGAAGATGACCACCTG (SEQ ID NO: 12) Second GCTCAGGAAAGT F: caccGCTCAGGAAAGTGTCCGAGC 86 59 exon GTCCGAGC (SEQ (SEQ ID NO: 14) ID NO: 13) R: aaacGCTCGGACACTTTCCTGAG (SEQ ID NO: 15) Second GAGTCCCGTCACC F: caccGAGTCCCGTCACCCGAGACC 93 49 exon CGAGACC (SEQ ID (SEQ ID NO: 17) NO: 16) R: aaacGGTCTCGGGTGACGGGACT (SEQ ID NO: 18) Third GAAGTGATCACG F: caccGAAGTGATCACGGATGAGTG 81 55 exon GATGAGTG (SEQ (SEQ ID NO: 20) ID NO: 19) R: aaacCACTCATCCGTGATCACTT (SEQ ID NO: 21) Third GGTGACGGGACTC F: caccGGTGACGGGACTCAGGGTGA 62 69 exon AGGGTGA (SEQ ID (SEQ ID NO: 23) NO: 22) R: aaacTCACCCTGAGTCCCGTCAC (SEQ ID NO: 24) Third TGCAGCGCGCGCC F: caccTGCAGCGCGCGCCCGGTCTC 87 63 exon CGGTCTC (SEQ ID (SEQ ID NO: 26) NO: 25) R: aaacGAGACCGGGCGCGCGCTGCA (SEQ ID NO: 27) Fourth CACGAGAGCGAC F: caccCACGAGAGCGACCCGTCGCT 99 49 exon CCGTCGCT (SEQ (SEQ ID NO: 29) ID NO: 28) R: aaacAGCGACGGGTCGCTCTCGTG (SEQ ID NO: 30) Fourth GCGACGGGTCGCT F: caccGCGACGGGTCGCTCTCGTGG 97 69 exon CTCGTGG (SEQ ID (SEQ ID NO: 32) NO: 31) R: aaacCCACGAGAGCGACCCGTCG (SEQ ID NO: 33) Fourth TCCATGCTGAAGC F: caccTCCATGCTGAAGCTCGACTC 91 60 exon TCGACTC (SEQ ID (SEQ ID NO: 35) NO: 34) R: aaacGAGTCGAGCTTCAGCATGGA (SEQ ID NO: 36) Fourth ACGGGTCGCTCTC F: caccACGGGTCGCTCTCGTGGTGG 90 92 exon GTGGTGG (SEQ ID (SEQ ID NO: 38) NO: 37) R: aaacCCACCACGAGAGCGACCCGT (SEQ ID NO: 39) Fourth AGCCGCAACCGC F: caccAGCCGCAACCGCGAGAACGG 98 55 exon GAGAACGG (SEQ (SEQ ID NO: 41) ID NO: 40) R: aaacCCGTTCTCGCGGTTGCGGCT (SEQ ID NO: 42) Fourth GCAACTTGACGGT F: caccGCAACTTGACGGTGCACCGG 97 73 exon GCACCGG (SEQ ID (SEQ ID NO: 44) NO: 43) R: aaacCCGGTGCACCGTCAAGTTG (SEQ ID NO: 45) Fourth GAGCTGGGCCGCC F: caccGAGCTGGGCCGCCCGGGGCC 39 76 exon CGGGGCC (SEQ ID (SEQ ID NO: 47) NO: 46) R: aaacGGCCCCGGGCGGCCCAGCT (SEQ ID NO: 48) Third GGTCAGACGGAG F: caccGGTCAGACGGAGGCTCCCTT 61 65 exon GCTCCCTT (SEQ ID (SEQ ID NO: 50) NO: 49) R: aaacAAGGGAGCCTCCGTCTGACC (SEQ ID NO: 51)

[0078] According to the knockout efficiency in Table 1, the gRNA gene knockout plasmid vectors at the second exon and the third exon were selected to be knocked out for the next experiment. In the present application, preferably, BCL11B gene knockout was performed in the second exon and the third exon, the third pair of gRNA with the highest knockout efficiency was knocked out by using a mixture of the first pair of gRNA with the lowest knockout efficiency and the second pair of gRNA with the lowest knockout efficiency at the second exon, and the gene knockout plasmids corresponding to the third pair of gRNA with the lowest knockout efficiency at the third exon and the mixture thereof could reprogram T cells to obtain the immune killing lymphocytes of the present application. In this example, BCL11B gene knockout plasmids were constructed by using gRNA of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 50 and SEQ ID NO: 51, respectively, and mixed for the next experiment.

[0079] Sorting and Activation of T Cells

[0080] T Cells were sorted and activated using the following method.

[0081] (1) Peripheral blood including human mature T cells and cord blood including human mature T cells were centrifuged at 300×g for 10 minutes, and plasma was collected and thermally inactivated at 56° C. for 30 minutes.

[0082] (2) The precipitated granular blood cells were suspended with 0.9% NaCl, and peripheral blood mononuclear cells (PBMC) were separated by Ficoll density gradient centrifugation.

[0083] (3) Negative sorting was performed with MACS Pan T Cell Isolation kit (produced by Miltenyi Biotec in Bergisch Gladbach, Germany) to enrich all T cells (Pan T) from the blood such as peripheral blood and cord blood.

[0084] The above (1) to (3) are the steps of isolating human mature T cells from peripheral blood and cord blood, and it should be noted that other T cell sources are also acceptable, such as the directed differentiation of pluripotent stem cells and hematopoietic stem cells. T cells from all sources were activated with a T cell activation kit (produced by Miltenyi Biotec). Magnetic beads coated with anti-human CD3, anti-human CD28 antibody and anti-human CD2 were mixed and incubated with T cells in a ratio of 1:2 (cell density: 2.5×10.sup.6 cells/ml, and the medium: T551-H3 medium (produced by Takara, Japan) containing 5% autologous plasma, hIL2 (100 IU/ml), gentamicin sulfate (20 μg/ml), 10 mm of HEPES, 2 mm of glutamine and 1% penicillin/streptomycin) and then activated. After activation for 24 hours to 48 hours, T cells were eluted from antibiotin MACS iBead™ granules for later use.

[0085] Induced Reprogramming

[0086] (1) CRISP/CAS 9 knockout vectors PX458-gBCL11B were transduced into the above-mentioned activated T cells by electrotransfer (T-023, LONZA Amaxa Nucleofector, Lonza).

[0087] (2) After 12 hours, T cells transduced with PX458-gBCL11B (such cells were referred to PX458-T) were centrifuged and cultured with T551-H3 (produced by Takara, Japan) medium (containing 5% autologous plasma or fetal bovine serum (FBS), 500 IU/ml hIL2 and gentamicin sulfate (20 μg/ml)).

[0088] (3) The fresh medium was changed every 3 days, and the cell density was kept in the range of 0.5×10.sup.6 cells/ml to 1×10.sup.6 cells/ml until day 14 after electrotransfer.

[0089] (4) Whether the second exon and the third exon of BCL11B of T cells transduced with PX458-gBCL11B were subjected to knockout such as site induced insertion or deletion, was detected and verified by gene sequencing. The control group was T cells transduced with PX458 empty vectors (Mock).

[0090] (5) The expression level of BCL11B proteins in T cells transduced with PX458-gBCL11B was detected and verified by Western Blotting to further confirm the deletion of BCL11B proteins, and the control group was T cells transduced with PX458 empty vectors (Mock). The results of Western Blotting are shown in FIG. 3.

[0091] Phenotype Identification of Reprogrammed Cells

[0092] As described above, after 14 days of electrotransfer of T cells, 19.5% to 68.7% of the resulting cells expressed both T cell markers such as CD3 and NK cell markers such as NKp46, CD56, NKp30 and NKp44, and thus it was determined that the human ITNK cells of the present application were obtained. NK cells only expressed NK cell markers such as NKp46 and CD56, but did not express T cell markers such as CD3. T cells subjected to electrotransfer of empty vectors expressed T cell markers such as CD3, but did not express NK cell markers. The expression of cell markers of T cells, NK cells and ITNK cells is shown in FIGS. 4 to 6, and the phenotypic differences of these cells are also summarized in Table 2 below.

TABLE-US-00002 TABLE 2 Phenotypic differences of ITNK cells, T cells and NK cells — T cells NK cells ITNK cells T cell marker CD3, etc., — CD3, etc., NK cell marker — High High expression expression of of NKp46 NKp46, (which is of no expression/ CD56, NKp30 low expression in and NKp44 CD4-positive ITNK cells), CD56, NKp30 and NKp44 BCL11B High Low Deletion expression expression expression

[0093] In addition, according to the observation from the confocal microscope, the cell morphology of ITNK cells reprogrammed from T cells was different from the cell morphology of T cells and similar to the cell morphology of NK cells, and the reprogrammed ITNK cells had small nucleus (the volume of T cell nucleus relative to the whole cell), plentiful intracellular matrix, large granules, abundant endoplasmic reticulum and high protein synthesis activity, indicating that reprogrammed ITNK cells were immune killer lymphocytes. The transmission electron microscopic images of T cells, NK cells and ITNK cells are shown in FIG. 7.

[0094] In addition, the inventors also compared the expression profiles of these NK markers in the subsets of BCL11B-deficient T cells derived from cord blood and peripheral blood, and found that the percentages of CD8+NKP46+ cells and CD8+CD56+ cells were significantly higher than the percentages of CD4+NKP46+ cells and CD4+CD56+ cells, indicating that NKP46+CD3+ ITNK cells mainly derived from CD8+ T cells (see FIG. 8). Unlike CD8+ T cells, after the deletion of BCL11B, CD4+ T cells expressed NKp30 but didn't express NKp46 (see FIG. 8B).

[0095] CD4−CD8−NKp46+ subset expressed “TCRγδ” and was γδTCR+ ITNK cells (see FIG. 9). The deletion of BCL11B in CD4+ T cells, CD8+ T cells and γδTCR+ T cells was further verified by DNA sequencing (see FIG. 9).

EXAMPLE 2

Source Identification of ITNK Cells of the Present Application

[0096] TCRαβ sequencing: T cells derived from the same donor and ITNK cells obtained in Example 1 were subjected to RNA extraction and CDR3 region targeted amplification through human TCRαβ analysis kit to obtain TCR RNA. TCR RNA was subjected to sequencing on Hiseq 4000 platform to obtain a TCR library. Clustering combination analysis was performed with MiXCR(ref). The type of TCRαβ clone was derived with the parameter of “-chain” through the MiXCR clone derivation instruction. The diversity of TCR clones of T cells derived from the same donor and ITNK cells was compared by TCR sequencing, and it was found that the diversity of TCR clones was consistent (see FIGS. 10 and 11). Therefore, it was determined that the resulting ITNK cells were obtained by reprogramming of T cells with BCL11B deletion and maintained the TCR diversity of T cells, instead of being amplified from special and unknown small subsets of human T cells.

EXAMPLE 3

Single-Cell Immunophenotype Identification of ITNK Cells of the Present Application

[0097] ITNK cells obtained in Example 1 were subjected to single-cell immunophenotype analysis by mass cytometry (CyTOF) respectively, and the control group was T cells transduced with empty vectors.

[0098] Preparation and pretreatment of mass spectrometer samples: Cells from culture suspension were centrifuged, re-suspended with PBS containing 0.5% BSA and 0.02% NaN.sub.3, and incubated with anti-human CD16/32 monoclonal antibody at room temperature for 10 minutes to block the Fc receptor. Then, a mixture of metal-labeled antibodies against cell surface molecules was added and incubated on ice for further 20 minutes. The antibodies were pre-coupled antibodies (produced by Fluidigm) or were internally coupled using a mass spectrometry flow coupling kit (produced by Fluidigm) according to the instructions. 5 mM of cisplatin was added to the cells, and the cells were incubated and stained on ice in FBS (produced by Fluidigm) for 1 minute. After the cells were treated with a fixation/permeabilization buffer (produced by Thermo Fisher), the cells were mixed and incubated with the metal-labeled antibodies to label intracellular proteins. After the cells were cleaned, the cells were stained with 1 mL of 191/193Ir DNA intercalator (produced by Fluidigm) that was diluted at a ratio of 1:4000 (the intercalator was diluted with PBS containing 1.6% paraformaldehyde (produced by EMS)) and then stored at 4° C. Before detection, the cells were washed once with PBS containing 0.5% BSA and 0.02% NaN.sub.3 and once with ddH.sub.2O, then re-suspended and diluted to about 10.sup.6 cells/ml with ultrapure water (ddH.sub.2O). Subsequently, cell sample data were detected and collected using CyTOF2 device (produced by Fluidigm) at an event rate of <400 events/sec.

[0099] According to the cellular immunophenotypic differences of 40 markers, clustering analysis was performed through PhenoGraph clustering algorithm. ITNK cells derived from cord blood (hereinafter referred to as CB-ITNK), ITNK cells derived from peripheral blood (hereinafter referred to as PBMC-ITNK) and Mock-T cells were integrated and classified into 39 subsets, as shown in FIGS. 12, 13, 14, 15, 16 and 17. As can be seen from FIG. 12, PX458-T cells and Mock-T cells were separated before and after stimulation by K562 cells, and there was almost no overlap, indicating that there was a significant difference between PX458-T cells and Mock-T cells; there was obvious overlap between PX458-T cells before and after stimulation by K562 cells, and more new subsets were derived from activated PX458-T cells.

[0100] According to results of cell marker expression heterogeneity analysis by mass cytometry, the ITNK cells of the present application included CD3-negative cell subset of NO. 33, CD4+ cell subsets of NOs. 5 to 10, CD8+ cell subsets of NOs. 20 to 22 and 26 to 28, and TCRγδ+ cell subsets of NOs. 23 to 24, and all expressed NK-related markers such as CD56, NKp30, NKp44, NKp46 or CD11C, and compared with γδT cells, NKp46, NKp30 and NKp44 markers were of high expression in TCRγδ+ ITNK cells, i.e., (NKp46.sup.high NKp30.sup.high NKp44.sup.high) (as shown in FIGS. 13 and 14A). The ITNK cells of the present application differed from conventional NK cells in that the ITNK cells of the present application didn't express CD127, CD16, NKG2A and KIR2DL2 (see FIG. 14B). Moreover, immunosuppression checkpoints PD-1, CTLA-4 and FOXP3 were of low expression in the ITNK cells of the present application (as shown in FIG. 14C), and since the high expression of immunosuppression checkpoints will induce immune cells to form immunosuppressive states such as low function and failure, it is indicated that the ITNK cells of the present application had a strong immune effect and were not easily suppressed by immunosuppressive microenvironments such as tumors.

[0101] In addition, in ITNK cells derived from cord blood, the histogram as shown in FIG. 15 clearly shows the percentages of various ITNK cells in CD45+ hematopoietic cells and the dynamic transition from static ITNK cells to effector cells after stimulation. As shown in FIG. 16, the dynamic transition of ITNK cells derived from adult peripheral blood from resting state to effector state after stimulation was the same as the dynamic transition of ITNK cells derived from cord blood. As shown in FIG. 17, the mass cytometry heatmap shows the dynamic changes of immunophenotype of ITNK cells before and after activation, and the expression of CD25 increased after activation. To sum up, ITNK cells, after activated, still remain the expression of NK cell activation receptors and T cell markers.

EXAMPLE 4

RNA-Seq Transcription Profile Analysis of ITNK Cells of the Present Application

[0102] In order to study the entire gene expression profile of ITNK cells, the inventors performed RNA sequencing analysis on T cells derived from 4 cord blood samples and 3 adult peripheral blood samples, ITNK cells derived from 4 cord blood samples and 3 adult peripheral blood samples, and NK cells derived from 2 cord blood samples and 2 adult peripheral blood samples. The sorting operation is as follows: flow cytometry analysis or sorting was performed by flow cytometers Canto, FACS Fortessa (BD), FACSAriaII, etc. Cell-surface receptor labeling: the cells and antibody were mixed in 50 μl of flow buffer (PBS solution containing 2% FBS) and incubated at 4° C. for 30 minutes in the dark. Cell intracellular labeling: the cells were subjected to permeable treatment with Foxp3/transcription factor staining buffer (produced by eBioscience), after the buffer was eluted, blocked with mouse serum or rabbit serum, incubated with antibodies at 4° C. for 30 minutes in the dark, washed with flow buffer and then suspended for subsequent flow cytometry analysis or sorting. Cell sorting strategy and sorting purity were verified (as shown in FIG. 18).

[0103] Principal component analysis (PCA) was performed on RNA sequencing results of 18 samples for similarity evaluation, and it was found that ITNK cells were different from T cells and NK cells according to transcriptome analysis (as shown in FIG. 19). Compared with T cells, 776 genes were increased (data were not shown), including NK specific transcription factors (such as ID2, TBX21, NFIL3, and IRF8), NK cell activation and inhibition receptors/proteins (such as IL2RB, IFNG, PRF1, GZMB, TNFRSF4, NCR1, NCR2, and PLCG2), and histone genes (HIST1H1D, HIST1H2AC/D/E/G/M, and HIST2H2A3/4) (as shown in FIGS. 22 and 20). On the contrary, compared with T cells, 592 genes such as TCF-1/TCF-7, LEF-1, IL-7R, MYC, PD-L1, FOXP3, etc. were down-regulated in ITNK cells (as shown in Table 3). Compared with NK cells, the expression of 666 genes in ITNK cells increased (data were not shown), and most of these genes were enriched in T cell recognition and TCR signal transduction (CD3, CD4, CD8, CD40LG) (FIG. 21). Interestingly, compared with NK cells, the expression of KIR genes KIR2DL1, KIR2DL3, KIR3DL1 and KIR3DL2 in ITNK was down-regulated (as shown in Table 4), while KIR2DL1, KIR2DL3, KIR3DL1 and KIR3DL2 were NK cell inhibition receptors and mediated the inhibition of immune cells, and their low expression indicates that ITNK cells had resistance to immunosuppressive microenvironments. The results of whole transcriptome sequencing analysis show that the expression of NK cell marker genes IL2RB, ID2 and NFIL3 was up-regulated in ITNK cells (shown in the right image of FIG. 22).

TABLE-US-00003 TABLE 3 Expression of related genes of ITNK cells relative to NK cells ENTREZ Basic log2 fold ID Marker value change lfcSE stat P value padj 6932 TCF7 6118.39588 −1.7050171 0.20634066 −8.2631176  1.4192E−16 3.4959E−14 51176 LEF1 4011.12315 −1.1451791 0.15917866 −7.194300 36.2782E−13 6.9436E−11 3575 IL7R 1810.03157 −3.9931208 0.40305169 −9.9072177  3.8728E−23 6.2964E−20 4609 MYC 1160.22897 −2.6861929 0.28569782 −9.4022167  5.3425E−21 4.1361E−18 50943 FOXP3  769.302246 −2.0215289 0.58426298 −3.459964 0.00054025 0.00415092

TABLE-US-00004 TABLE 4 Expression of related genes of ITNK cells relative to NK cells ENTREZ Basic log2 fold ID Marker value change lfcSE stat P value padj 3811 KIR3DL1 365.099198 −7.1254794 0.75146844 −9.482074 2.4928E−21  3.971E−18 3804 KIR2DL3 473.412488 −4.8525337 0.89072948 −5.4478198 5.0991E−08 2.9645E−06 3812 KIR3DL2 373.96061 −3.0917182 0.86143947 −3.5890139 0.00033193 0.00378501 3802 KIR2DL1 282.000446 −4.7696308 0.99147586 −4.8106373 1.5045E−06 5.0993E−05

EXAMPLE 5

Single-Cell Transcriptome Sequencing Analysis of ITNK Cells of the Present Application

[0104] Flow cytometry shows that CD8+CD3+NKp46+ ITNK cells and CD4+CD3+NKp30+ ITNK cells appeared on day 5 after BCL11B was knocked out (as shown in FIG. 23). In order to further explain the cell fate transition during reprogramming of T cells to ITNK cells, through micropore-based single-cell transcriptome sequencing (scRNA-seq), the gene expression profiles of T cells and ITNK cells in CD3+ cord blood samples at different time points after BCL11B knockout were studied.

[0105] About 5000 cells were detected and analyzed in all experimental groups. Different time-point groups of cell samples were detected by scRNA-seq, 2000 to 4000 genes per cell were detected, and a total of 20000 human genes were detected among all cells. In the t-distributed random neighbor-embedded (t-SNE) analysis of transcription profiles, the cells were projected into two dimensions, which provided a visual representation of the cell fate transition in the process of ITNK cell reprogramming. The results of the unbiased t-SEN analysis show that the cells from day 0 to day 20 after knockout could be clustered into 11 subsets (as shown in FIG. 24). According to the expression of marker genes in T cells and NK cells, it is determined that ITNK cells were mainly concentrated in subset 6 (CD4+ ITNK), subset 1 (CD8+ ITNK) and subset 10 (γδTCR+ ITNK) (as shown in FIG. 24), and these subsets completely coincided with the BCL11B-deleted subset, further indicating that ITNK cells were reprogrammed by knocking out BCL11B in T cells. KEGG enrichment analysis shows that NK marker genes and related genes were of specific high expression in ITNK cells (as shown in FIG. 25). As can be seen from FIGS. 24C and 25C, NOTCH1, NOTCH2, ZMIZ1 (NOTCH1 cofactor) and RBPJ (NOTCH downstream transcription factor) were up-regulated in human ITNK cells, indicating that the NOTCH signaling pathway played a role in reprogramming of ITNK cells. FOS, JUN, JUNB, the three AP-1 transcription factor subunits were of low expression in the early stage of ITNK reprogramming, but gradually up-regulated in the late stage of ITNK reprogramming (see FIG. 25C). As can be seen, the NOTCH signal and the AP-1 signal were up-regulated after ITNK reprogramming T cells and ITNK cells were closely aggregated in the t-SNE dot plots, indicating that the transition from T cells to NK cells was almost synchronous. As can be seen from the analysis of the trajectory of NK marker genes of CD8+ T (subset 5), early CD8+ ITNK (subset 0) and late CD8+ ITNK (subset 1), there was a gradual transition from CD8+ T cells to CD8+ ITNK cells (see FIG. 26A). Similarly, the analysis of the trajectory of NK marker genes of CD4+ T cells and ITNK cells also shows the gradual transition from T cells to ITNK cells (FIG. 26B). Consistent with the results of dynamic flow cytometric immunophenotype analysis of ITNK cells (FIG. 23), T cells began to reprogram into ITNK cells on day 5 after knockout of BCL11B. It is found that the expression of transcription factor genes (TBX2, ID2, etc.) was significantly up-regulated during the reprogramming of T cells to ITNK cells, which was further verified by western blotting (as shown in FIG. 27).

EXAMPLE 6

Ability of ITNK Cells of the Present Application to Recognize and Kill MHCI-Positive/Negative Tumor Cells In Vitro

[0106] In order to determine whether the NK cell receptor (NCR) and T cell receptor (TCR) expressed on ITNK cells of the present application are functional, the ITNK cells were stimulated with anti-NKp30 monoclonal antibody, anti-NKp46 monoclonal antibody and anti-CD3/CD28 monoclonal antibody, respectively. It is found that the secretion of interferon (IFN) in ITNK cells increased after the ITNK cells were stimulated with anti-NKp30 antibody and anti-NKp46 antibody, while the secretion of IFN in T cells of the control group did not increase (as shown in FIG. 28); and the secretion of interferon in ITNK cells increased after the ITNK cells were stimulated with anti-CD3/CD28 antibody, while the secretion of interferon in T cells of the control group did not increase (as shown in FIG. 28). It is indicated that the NCR and the TCR in ITNK cells were functional.

[0107] Similar to NK cells, ITNK cells of the present application could secrete a variety of cytokines, including GM-CSF, IFN, and TNF (as shown in FIG. 29), and recognize and kill MHC-I negative K562 cells (as shown in FIG. 30A). In addition, ITNK cells could effectively kill Hela cells and A549 cells (as shown in FIGS. 30B and 30C), and the two cells are ligands with high expression of NK-activated receptors and are MHC I-positive; and as shown in Table 2, since NK cell KIR receptors (KIR2DL1, KIR2DL3, KIR3DL1 and KIR3DL2) which mediated immunosuppression were of low expression in ITNK cells, ITNK cells had a better tumor-killing effect than NK cells. However, NALM-6 did not express the NCR ligand and highly expressed MHC-I molecules, and ITNK cells and NK cells had no significant killing effect on NALM-6 (as shown in FIG. 30D). Then, the inventors stimulated ITNK cells, NK cells and T cells with K562 cells, respectively, and it is found that the expression levels of phosphorylated Fyn, PLC-γ2, Syk, Erk and m-TOR in ITNK cells and NK cells were similar, but higher than the expression levels in T cells (as shown in FIG. 31). These results indicate that ITNK cells had similar NK cell functions in NCR activation, cytokine secretion, cytotoxicity and signaling pathway.

EXAMPLE 7

Ability of ITNK Cells of the Present Application to Inhibit Tumor Growth In Vivo

[0108] The inventors also evaluated whether the ITNK cells of the present application could inhibit the growth of xenograft tumors. Specifically, K562 cells labeled with luciferase were implanted into NSI mice to construct K562 tumor-bearing mouse models, and then ITNK cells, NK cells or T cells were injected in a single time (FIG. 32A). The survival status of K562 cells in mice in vivo was detected at specific time points by living imaging equipment. Compared with the group injected with T cells (negative control) and the group injected with PBS (blank control), K562 tumor burden in the experimental mice treated with ITNK cells and NK cells significantly reduced after 28 days of injection (FIGS. 32B and 32C), and these mice survived longer (FIG. 32D). In addition, the inventors also transplanted Hela cells into NSI mice and treated Hela xenograft mice with ITNK cells, NK cells or T cells, respectively. The results show that the growth rate of Hela tumor in tumor-bearing mice treated with ITNK cells was significantly slower than the growth rate in the group treated with NK cells, the group treated with T cells or the group treated with PBS (FIG. 32E). As can be seen, the ITNK cells of the present application were effective killers of tumor cells in vivo and could prevent tumor progression.

EXAMPLE 8

In Vivo Safety Assessment of ITNK Cells of the Present Application

[0109] In order to verify the distribution and maintenance ability of ITNK cells in vivo, ITNK cells were transplanted into NSI-strain immunodeficient mice lacking T cells, B cells and NK cells, and the percentage of ITNK cells in peripheral blood (PB), spleen (SP), bone marrow (BM), liver, and lung was measured on day 1, day 7, day 14, day 21 and day 180 after transplantation (FIGS. 33A and 33B). The proportion of ITNK cells peaked on day 21 after transplantation, then gradually decreased, and could not be detected after 6 months (FIG. 33B). As can be seen from FIG. 33B, ITNK cells had better maintenance ability in vivo than T cells. The case where ITNK cells attack the host or amplified without restriction was not observed.

[0110] In order to evaluate the possible off-target mutation induced by PX458-gBCL11B, T cells electroporated with PX458-gBCL11B were subjected to the whole genome sequencing of high coverage. Compared with wild-type T cells, it is found from two independent experiments that there were very few off-target mutations caused by nuclease in T cells edited by PX458-gBCL11B.

[0111] The applicant has stated that although the detailed method of the present application is described through the examples described above, the present application is not limited to the detailed method described above, which means that implementation of the present application does not necessarily depend on the detailed method described above. It should be apparent to those skilled in the art that any improvements made to the present application, equivalent replacements of raw materials of the product of the present application, additions of adjuvant ingredients to the product of the present application, and selections of specific manners, etc., all fall within the protection scope and the disclosed scope of the present application.