Methods for providing immune cells with enhanced functions

Abstract

The present disclosure relates to methods for generating immune cells with enhanced functions. More specifically, disclosed herein is a method for enhancing immune cell function, which includes modifying immune cells to inhibit the function of at least one gene selected from the group consisting of RC3H1, RC3H2, A2AR, FAS, TGFBR1, and TGFBR2. Also disclosed herein is a method that includes modifying stem cells or progenitor cells capable of differentiating into immune cells to inhibit the function of at least one gene selected from the group consisting of RC3H1, RC3H2, A2AR, FAS, TGFBR1, and TGFBR2. Also disclosed are immune cells or stem cells prepared by the methods of the present invention and the use of immune cells in therapeutic treatment.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Application No. 62 / 938,022, filed on November 20, 2019, the contents of which are incorporated herein in their entirety. Technical Field

[0003] The present disclosure relates to methods for producing immune cells with enhanced function. More specifically, disclosed herein is a method for enhancing immune cell function, comprising modifying an immune cell to inhibit the function of at least one gene selected from RC3H1, RC3H2, A2AR, FAS, TGFBR1, and TGFBR2. Also disclosed herein is a method comprising modifying a stem cell or progenitor cell capable of differentiating into an immune cell to inhibit the function of at least one gene selected from RC3H1, RC3H2, A2AR, FAS, TGFBR1, and TGFBR2. Also disclosed herein are immune cells or stem cells produced by the methods of the present invention, as well as the use of immune cells in therapeutic treatments.

[0004] Incorporation by Reference into the Sequence Listing

[0005] The sequence listing in an ASCII text file, named 37830WO_ND201903_sequencelisting.txt, is 10 KB in size, was created on November 3, 2020, and is incorporated herein by reference. Technical Background

[0006] T cells expressing chimeric antigen receptors (CAR-T cells) have been shown to be very effective in killing tumor cells in diseases such as acute lymphoblastic leukemia (ALL) and non-Hodgkin's lymphoma (NHL). Approved products targeting B cell antigen CD19 are produced by introducing CAR gene constructs into T cells of patient origin ("autologous") (Kershaw et al., Gene-engineered T cells for cancer therapy, Nat Rev Cancer, 2013, 13 (8): 525-41). Additional autologous products are under development that target other blood cell markers of other hematologic malignancies (such as multiple myeloma), such as B cell maturation antigen (BCMA) (Sadelain et al., Therapeutic T cell engineering, Nature, 2017, 545 (7655): 423-431).

[0007] Although the clinical results of using CAR-T cells in blood-based cancers are impressive, similar results have not occurred in the treatment of solid tumors. There are multiple reasons for the relative lack of efficacy in solid tumors, including limiting access to the tumor site, the immunosuppressive properties of the tumor microenvironment, and the lack of solid tumor-specific target antigens. In addition, the lack of persistence and "exhaustion" of the administered CAR-T cells are limitations that have been observed (Newick et al., CAR T Cell Therapy for Solid Tumors, Annu Rev Med, 2017, 68: 139-152).

[0008] Inhibitory receptors (such as CTLA-4, PD-1 or LAG-3) can weaken the activation of CAR-T cells and accelerate T cell exhaustion. After PD-1 is destroyed by genome editing, it is expected that the anti-tumor activity of T cells will increase (Liu et al., CRISPR-Cas9-mediated multiplex gene editing in CAR-Tcells, Cell Res, 2017, 27 (1): 154-157). However, the ablation of PD-1 on T cells can also increase susceptibility to exhaustion, reduce lifespan and fail to improve anti-tumor effects (Odorizzi et al., Genetic absence of PD-1 promotes accumulation of terminally differentiated exhausted CD8+T cells, J Exp Med, 2015, 212 (7): 1125-37). For these reasons, it is necessary to evaluate on a case-by-case basis whether gene editing in T cells enhances anti-tumor activity.

[0009] CRISPR / Cas9 is an important component of the bacterial immune system, which allows bacteria to remember and destroy phages. In mammalian cells, CRISPR / Cas9 can be used for gene editing, such as other gene editing technologies, such as TALEN and ZFN. The CRISPR system contains two main components, Cas9 nuclease and guide RNA. Specifically, the designed guide RNA forms a complex with the Cas9 nuclease, guiding the Cas9-gRNA ribonucleoprotein (RNP) complex to the user-defined cleavage site in the human genome. RNP cutting causes double-stranded DNA breaks in the genome, and double-stranded DNA breaks are repaired by a process prone to errors known as non-homologous end joining (NHEJ). In the NHEJ pathway, nucleotide deletion or insertion ("indel") causes gene disruption or knockout (Addgene, CRISPR 101: A Desktop Resource (2nd Edition), 2017). The on-target efficiency and off-target effects of guide RNA determine the specificity and safety of CRISPR / Cas9 gene targeting applications. Therefore, specially designed guide RNA plays a crucial role in the success of gene disruption.

[0010] Recent studies have used CRISPR to screen for genome-wide loss of function of immunomodulators and found that ablation of negative regulators (such as TCE2, SOCS1, RASA and CBLB) significantly increased T cell cytotoxicity in vitro (Shifrut et al., Genome-wide CRISPR Screens in Primary Human TCells Reveal Key Regulators of Immune Function. Cell, 2018, 175(7): 1958-1971, e15). However, short-term in vitro cytotoxicity provides limited guidance on the effects of gene suppression or deletion on in vivo function or lifespan. The potential effects of gene suppression on the function (including activity or lifespan) of immune cells (including T cells, NK cells, NKT cells, etc.) need to be evaluated more extensively in vitro and in vivo.

[0011] Although enhanced immune cells are a potential weapon against cancer, there are challenges in numerically generating, amplifying and characterizing immune cell products. Immune cells can be generated from pluripotent stem cells (PSC). Therefore, pluripotent stem cell technology is a very promising technology because, in theory, pluripotent stem cells provide an unlimited and renewable source of cells. The ability to effectively and infinitely supply immune cells (which have enhanced capabilities and also include a wide range of target recognition systems (TCR / CAR / cytotoxic receptors) that can respond to a variety of pathogens and cancers) directly from stem cells (such as induced pluripotent stem cells (iPSC)) represents a major commercial opportunity. Therefore, it is also important to understand the impact of the inhibition of specific target genes on iPSC viability, self-renewal, proliferation capacity and ability to differentiate into immune cells. Summary of the Invention

[0012] It has been demonstrated herein that knockdown of several genes enhances the persistence and antitumor activity of cytotoxic cells in vivo.

[0013] In one aspect, the present invention provides a method for enhancing immune cell function. The method comprises modifying the immune cell to inhibit the function of at least one gene (i.e., one or more genes) selected from RC3H1, RC3H2, A2AR, FAS, TGFBR1, and TGFBR2.

[0014] In another aspect, provided herein is a method for modifying a stem cell capable of differentiating into an immune cell. The method comprises modifying the stem cell to inhibit the function of at least one gene selected from RC3H1, RC3H2, A2AR, FAS, TGFBR1, and TGFBR2. In some embodiments, the modified stem cell further differentiates into an immune cell, wherein the function of the at least one gene is inhibited in the immune cell.

[0015] In some embodiments, inhibition of gene function is achieved by reducing the level or function of mRNA, optionally by small interfering RNA (siRNA), short hairpin RNA (shRNA), micro RNA (miRNA), or antisense nucleic acid.

[0016] In some embodiments, inhibition of gene function is achieved by reducing the level or activity of the protein encoded by the gene, optionally through the use of antibodies or small molecules.

[0017] In some embodiments, inhibition of gene function is achieved by a gene editing system. In some embodiments, the gene editing system is selected from CRISPR / Cas, TALEN, and ZFN. In some embodiments, the gene editing system is a CRISPR / Cas system comprising a guide RNA-nuclease complex. In some embodiments, the guide RNA targets a nucleotide sequence selected from SEQ ID NO: 2 to SEQ ID NO: 16. In some embodiments, the CRISPR / Cas system utilizes a guide RNA-dependent nuclease selected from the group consisting of: Cpf1, Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas100, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, and CSF4.

[0018] In some embodiments, the immune cell is selected from a T cell (including cells such as NKT cells) or a NK cell.

[0019] In some embodiments, the modified cells, e.g., modified immune cells or modified stem cells, produced by the methods disclosed herein further comprise a nucleic acid encoding a chimeric antigen receptor (CAR).

[0020] In some embodiments, the modified immune cells produced by the methods disclosed herein recognize one or more target antigens. In some embodiments, the target antigens are selected from TAG-72, CD19, CD20, CD24, CD30, CD47, folate receptor alpha (FRα), and BCMA.

[0021] In another aspect, provided herein are immune cells produced by the methods disclosed herein.

[0022] In another aspect, provided herein are modified stem cells produced by the methods disclosed herein.

[0023] On the one hand, the present invention provides a modified immune cell, wherein the function of at least one gene in the modified immune cell is suppressed relative to the unmodified immune cell, wherein the at least one (i.e., one or more) gene is selected from RC3H1, RC3H2, A2AR, FAS, TGFBR1 and TGFBR2. In some embodiments, the RC3H1 gene is suppressed in the modified immune cell. In some embodiments, the RC3H2 gene is suppressed in the modified immune cell. In some embodiments, the A2AR gene is suppressed in the modified immune cell. In some embodiments, the FAS gene is suppressed in the modified immune cell. In some embodiments, the TGFBR1 gene is suppressed in the modified immune cell. In some embodiments, the TGFBR2 gene is suppressed in the modified immune cell. In some embodiments, multiple genes selected from RC3H1, RC3H2, A2AR, FAS, TGFBR1 and TGFBR2 are suppressed.

[0024] In some embodiments, the inhibition of gene function in the modified immune cell is caused by a decrease in the level or function of mRNA transcribed from the gene, or the level or activity of the protein encoded by the gene.

[0025] In some embodiments, inhibition of gene function results from modification of the nucleic acid sequence of the gene.

[0026] In some embodiments, the modified immune cell is selected from a T cell (including cells such as NKT cells) or a NK cell.

[0027] In some embodiments, the modified immune cell expresses a chimeric antigen receptor (CAR).

[0028] In some embodiments, the modified immune cells recognize one or more target antigens. In some embodiments, the target antigens are selected from TAG-72, CD19, CD20, CD24, CD30, CD47, folate receptor alpha (FRα), and BCMA.

[0029] In another aspect, provided herein are modified stem cells capable of differentiating into immune cells, comprising a modification of the nucleic acid sequence of at least one gene, wherein the modification inhibits the function of the at least one gene, and wherein the at least one gene is selected from RC3H1, RC3H2, A2AR, FAS, TGFBR1, and TGFBR2.

[0030] In some embodiments, the RC3H1 gene is suppressed in the modified stem cells. In some embodiments, the RC3H2 gene is suppressed in the modified stem cells. In some embodiments, the A2AR gene is suppressed in the modified stem cells. In some embodiments, the FAS gene is suppressed in the modified stem cells. In some embodiments, the TGFBR1 gene is suppressed in the modified stem cells. In some embodiments, the TGFBR2 gene is suppressed in the modified stem cells. In some embodiments, multiple genes selected from RC3H1, RC3H2, A2AR, FAS, TGFBR1, and TGFBR2 are suppressed.

[0031] In some embodiments, the modified stem cells are induced pluripotent stem cells.

[0032] In some embodiments, the modified stem cell comprises a nucleic acid encoding a chimeric antigen receptor (CAR).

[0033] In another aspect, provided herein is a composition for enhancing immune cell function, comprising a guide RNA-nuclease complex capable of editing a target gene sequence, wherein the guide RNA targets a nucleotide sequence selected from SEQ ID NO: 2 to SEQ ID NO: 16.

[0034] In some embodiments, the nuclease comprises at least one protein selected from the group consisting of: Cpf1, Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas100, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, and Csf4.

[0035] In another aspect, a method for treating a condition in a subject is provided, comprising administering to the subject a modified immune cell disclosed herein. In some embodiments, the condition is cancer, infection, autoimmune disease, organ fibrosis, or endometriosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] This patent or application file contains at least one drawing executed in color. Copies of color drawings of this patent or patent application publication will be provided by the Office upon request and payment of the necessary fee.

[0037] Figures 1A-1B Exemplary strategies for evaluating the anti-tumor activity of modified immune cells. (A) Schematic diagram of a strategy for evaluating CAR-T cells (including CRISPR knockout of immunomodulatory genes), showing a representative timeline of lentiviral CAR transduction, gene targeting, and functional analysis in primary T cells used in the examples. (B) A representative timeline of generating modified NK-92 cells, wherein CRISPR knockout of immunomodulatory genes, followed by lentiviral CAR transduction and functional analysis in NK-92 cells.

[0038] Figures 2A-2B Lentiviral transduction of human primary T cells to generate TAG-72CAR-T cells. (A) Schematic diagram of the TAG-72-specific CAR construct used in this study. (B) Transduction efficiency of CAR in human primary T cells. Expression was examined 10 days after transduction with the lentiviral vector. The values ​​embedded in each dot plot represent the frequency of CAR+ events as a percentage of live single cells. (Representative data of T cells from one donor are shown).

[0039] Figure 3Growth curve of TAG-72 CAR-T cells after CRISPR / Cas9 RNP transfection (representative data of T cells from one donor are shown). NT: untransduced T cells; TAG-72CAR: T cells transduced with TAG-72CAR; TAG-72CAR / PD-1KO T: T cells transduced with TAG-72CAR and CRISPR / Cas9 RNP targeting PD-1; TAG-72CAR / A2ARKO T: T cells transduced with TAG-72CAR and CRISPR / Cas9 RNP targeting A2AR; TAG-72CAR / FASKO T: T cells transduced with TAG-72CAR and CRISPR / Cas9 RNP targeting FAS; TAG-72CAR / RC3H1KOT: T cells transduced with TAG-72CAR and CRISPR / Cas9 RNP targeting RC3H1; TAG-72CAR / RC3H2KO T: T cells transduced with TAG-72CAR and CRISPR / Cas9 RNP targeting RC3H2; TAG-72CAR / TGFBR1 KO T: T cells transduced with TAG-72CAR and CRISPR / Cas9 RNP that introduces a dominant-negative mutation into TGFBR1; TAG-72CAR / TGFBR2 KO T: T cells transduced with TAG-72CAR and CRISPR / Cas9 RNP that introduces a dominant-negative mutation into TGFBR2.

[0040] Figure 4A-4D guide RNA formation of RNPs will be inserted and deleted (indel) into the open reading frame of specific genes in CAR-T cells. The frequency of indel was assessed by CRISPR editing (ICE) assay. (A) Sanger sequencing tracks from CAR-T cells transfected with RC3H2 gRNA ("edited sample") show that compared with untransfected CAR-T cells ("control sample"), there is an uneven mixing of bases downstream of the cleavage site (SEQ ID NO: 17 shows 281 to 346bp from the edited sample; SEQ ID NO: 18 shows 281 to 346bp from the control sample). The black underlined area of ​​the control sample represents the guide sequence, and the horizontal red dotted underlined area is the relevant PAM (original spacer adjacent motif (ProtospacerAdjacent Motif)) site. The vertical black dotted lines on the two tracks represent the cleavage site. (B) The relative percentage (normalized) of the contribution of each edited sequence in the genomic DNA of CAR-T cells transfected with RC3H2 RNP. The sequences from top to bottom are shown in SEQ ID NOs: 19, 20, 21, 22, 23, 24, 25, and 26, respectively. (C) Distribution of indel sizes in the entire edited population of CAR-T cells transfected with RC3H2 RNP. The percentage of indels outside the frame is the proportion of indels that are frameshifted or longer than 21 bp. R was calculated by Pearson's correlation coefficient. 2 The values ​​indicate the confidence level of the indel percentage. (D) Summary of ICE assay results for CAR-T cells transfected with RNPs. RNP complexes were formed by representative guide RNAs used in this study (PD-1, SEQ ID NO: 1; RC3H1, SEQ ID NO: 2; RC3H2, SEQ ID NO: 4; A2AR, SEQ ID NO: 7; FAS, SEQ ID NO: 9; TGFBR1, SEQ ID NO: 11; TGFBR2, SEQ ID NO: 14); representative data for T cells from one donor are shown.

[0041] Fig. 5 A-5H knockout TAG-72CAR-T cell mediation TAG-72hi expression target cell (OVCAR-3 cell line) effective cell killing (Fig. 5 A, 5C, 5E and 5G), but does not mediate TAG-72-neg / low cancer target cell (MES-OV cell line) (Fig. 5 B, 5D, 5F and 5H). Before adding CAR-T cells with a 1: 1 effector and target ratio, target cells are allowed to adhere to the plate overnight. In the killing assay, untransduced T cells (NT) are included as a control. Cell impedance (mean ± SD, expressed as normalized cell index (NCI)) is monitored within 20 hours. Target cell proliferation ("only target cells") under normal growth conditions is also monitored throughout the whole process. (representative data of T cells from a donor performed in triplicate with technology) are shown. CAR-T (Figures 5A-5H): TAG-72CAR-T cells; PD-1 (Figures 5A-5B): PD-1 knockout TAG-72CAR-T cells; RC3H1 (Figures 5C and 5D): RC3H1 knockout TAG-72CAR-T cells; RC3H2 (Figures 5C and 5D): RC3H2 knockout TAG-72CAR-T cells; A2AR (Figures 5E and 5F): A2AR knockout TAG-72CAR-T cells; FAS (Figures 5E and 5F): FAS knockout TAG-72CAR-T cells; TGFBR1 (Figures 5G and 5H): TGFBR1 dominant-negative TAG-72CAR-T cells; TGFBR2 (Figures 5G and 5H): TGFBR2 dominant-negative TAG-72CAR-T cells.

[0042] Figure 6 Tumor growth curve of OVCAR-3 ovarian tumors in a NOD scid gamma (NSG) mouse xenograft model. Four NSG mice per group were subcutaneously administered 1x10 7 OVCAR-3 tumor cells (TAG-72 positive). When the tumor grows to about 150-200 mm 3 Two doses of 5x10 6 T cells. Values ​​and error bars represent the average tumor size (mm 3 ± SEM). NT: non-transduced T cells; TAG-72CAR-T: T cells transduced with TAG-72CAR; TAG-72CAR / PD-1KO T: PD-1 gene knockout TAG-72CAR-T cells; mean ± SEM; representative data of T cells from one donor are shown.

[0043] Figure 7Antitumor activity of RC3H1 and / or RC3H2 knockout CAR-T cells in the OVCAR-3 ovarian tumor xenograft model in NSG mice. 1x10 7 OVCAR-3 tumor cells (TAG-72 positive). When the tumor grows to about 150-200 mm 3 Two doses of 5x10 6 T cells. Values ​​and error bars represent the average tumor size (mm 3 ±SEM). NT: untransduced T cells; TAG-72CAR-T: T cells transduced with TAG-72CAR; TAG-72CAR / RC3H1 KO T: RC3H1 knockout TAG-72CAR-T cells; TAG-72CAR / RC3H2KO T: RC3H2 knockout TAG-72CAR-T cells; TAG-72CAR / RC3H1,2KO T: RC3H1 and RC3H2 double knockout TAG-72CAR-T cells. **p < 0.01, using mixed effects analysis with Greisser-Greenhouse correction and Dunnett's multiple comparison one-way ANOVA test to compare all group means with the TAG-72CAR-T control group. Representative data for T cells from one donor are shown.

[0044] Figure 8 Antitumor activity of A2AR and FAS knockout CAR-T cells in the OVCAR-3 ovarian tumor xenograft model in NSG mice. 1x10 7 OVCAR-3 tumor cells (TAG-72 positive). When the tumor grows to about 150-200 mm 3 Two doses of 5x10 6 T cells. Values ​​and error bars represent the average tumor size (mm 3 ±SEM). NT: untransduced T cells; TAG-72CAR-T: T cells transduced with TAG-72CAR; TAG-72CAR / A2AR KO T: A2AR knockout TAG-72CAR-T cells; TAG-72CAR / FAS KO T: FAS knockout TAG-72CAR-T cells. *p<0.05, **p<0.01, #p<0.001, two-way ANOVA followed by Dunnett's multiple comparison test, comparing all group means to the CAR-T control group. Representative data from T cells from a single donor are shown.

[0045] Figure 9Antitumor activity of TGFBR1 and TGFBR2 dominant negative gene mutation CAR-T cells in the OVCAR-3 ovarian tumor xenograft model in NSG mice. 1x10 7 OVCAR-3 tumor cells (TAG-72 positive). When the tumor grows to about 150-200 mm 3 Two doses of 5x10 6 T cells. Values ​​and error bars represent the average tumor size (mm 3 ± SEM). NT: untransduced T cells, TAG-72CAR-T: T cells transduced with TAG-72CAR, TAG-72CAR / TGFBR1 KO T: TGFBR1 dominant-negative gene knockout TAG-72CAR-T cells, TAG-72CAR / TGFBR2 KO T: TGFBR2 dominant-negative gene knockout TAG-72CAR-T cells. *p<0.05, **p<0.01, ***p<0.001, two-way ANOVA followed by Dunnett's multiple comparison test, comparing all group means with the CAR-T control group. Representative data for T cells from one donor are shown.

[0046] Figure 10 Antitumor activity of CD19 CAR-T cells with RC3H1 and / or RC3H2 knockout in a Raji lymphoma tumor NSG mouse xenograft model. Four NSG mice per group were subcutaneously administered Raji tumor cells (CD19 positive). Three days after tumor inoculation, a single dose of 5x10 6 CAR-T cell-treated mice. (A) Tumor size was monitored for 23 days. Values ​​and error bars represent mean tumor size (mm 3 ± SEM). A multiple t-test with Holm-Sidak correction was performed to compare the RC3H1 and / or RC3H2 gene knockout CD19 CAR-T cell group with untransfected CD19CAR-T cells. (*p<0.005; **p<0.001) (B) Kaplan-Meier survival curves were analyzed using the log-rank (Mantel-Cox) test. NT: untransduced T cells; CD19 CAR: CD19 CAR-T cells; CD19 CAR / RC3H1 KO: RC3H1 gene knockout CD19 CAR-T cells; CD19CAR / RC3H2 KO: RC3H2 gene knockout CD19 CAR-T cells; CD19 CAR / RC3H1,2KO: RC3H1 and RC3H2 double gene knockout CD19 CAR-T cells. Representative data of T cells from one donor are shown.

[0047] Figure 11 Expression of activation markers on CD19 CAR-T cells with or without RC3H1 and / or RC3H2 gene knockout after sustained activation exposure. The graph shows the expression of activation markers CD25 and CD69 on CAR+ cells 7 days after antigen exposure. CD19 CAR-T cells were generated from a single healthy donor. Results represent the mean ± SD of technical replicates.

[0048] Figure 12 CRISPR knockout analysis of the RC3H1 and RC3H2 genes in single and double knockout T cells. RNPs formed by RC3H1 and RC3H2 guide RNAs were transfected into activated human T cells to generate RC3H1 or RC3H2 single KO T cells (RC3H1 KOT cells or RC3H2 KO T cells), or RC3H1 and RC3H2 double KO T cells (RC3H1,2KO T cells). Knockout efficiency was analyzed using ICE analysis. The percentage of out-of-frame indels is the proportion of indels that indicate frameshifts or are longer than 21 bp.

[0049] Figure 13 Effects of RC3H1 and / or RC3H2 KO on the function of T cells (CD8+, CD4+) without CAR. TM T cells ± RC3H1 and / or RC3H2 KO were maintained in T cell expansion medium for at least 92 h in the presence of human T-activator CD3 / CD28 beads (Thermofisher, MA, USA) (DB) at a beads to cell ratio of 1:1. Before using the effector cells in the assay, the beads were removed by magnetism. Effector cells were added to target cancer cells (in this example, OVCAR-3) at an effector-target ratio (E:T) of 1:1. NCI was monitored for more than 20 hours. Elimination of target cells was observed under all conditions (NCI reduction was observed). Importantly, after sustained CD3 / CD28-mediated activation, cells with gene-deleted RC3H1 and / or RC3H2 genes were able to eliminate target cells more effectively in vitro. The results represent the average value of triplicates within the biological and assays. + SEM.

[0050] Figure 14CRISPR knockout analysis of RC3H1 and RC3H2 genes in single and double knockout NK-92 cells. RNPs formed by RC3H1 and RC3H2 guide RNAs were transfected into NK-92 cells to generate RC3H1 or RC3H2 single KO NK-92 cells (RC3H1 KO NK-92 cells or RC3H2KO NK-92), or RC3H1 and RC3H2 double KO NK-92 cells (RC3H1,2KO NK-92). Knockout efficiency was analyzed using ICE analysis. The percentage of out-of-frame indels is the proportion of indels that indicate frameshift or are longer than 21 bp. R was calculated by Pearson correlation coefficient. 2 Values ​​indicate confidence in indel percentage.

[0051] Figure 15 Effects of RC3H1 and / or RC3H2 KO on the function of NK-92 cells (with and without TAG-72CAR). Using a real-time cell monitoring system The ability of NK cell lines NK-92 ± RC3H1 KO (green) or RC3H2 KO (purple) or RC3H1,2 KO (orange) ± TAG-72CAR to eliminate cancer cells in vitro was assessed. (A) RC3H1 and / or RC3H2 genes were deleted in NK-92 cell lines using CRISPR / Cas9. The resulting RC3H1 and / or RC3H2 KO NK-92 effector cells were added to target cancer cells (MES-OV (left panel) or OVCAR-3 (right panel) at an E:T ratio of 1:1. NCI was monitored for more than 40 hours. Target cell elimination was observed under all conditions (reduced NCI was observed compared to target cells alone (blue)). Results represent the mean ± SEM of three technical replicates. (B) NK-92 cells were further genetically manipulated to introduce TAG-72CAR. Lentiviral transduction was performed after transfection. Transduction efficiency was assessed by flow cytometry after approximately 72 hours of culture, where the values ​​inset in each dot plot represent the proportion of CAR+ cells as the frequency of viable single cells. The resulting TAG-72CAR / RC3H1 and / or RC3H2 KO NK-92 cells were isolated using fluorescence-activated cell sorting and their in vitro function was assessed as described above. (C) NCI was monitored for more than 40 hours. Results represent the mean ± SEM, n = 1-3.

[0052] Figure 16 Generate CRISPR knockout induced pluripotent stem cells (iPSCs) as a cell source for adoptive cell therapy. This workflow describes how to derive knockout immune cells from iPSCs. iPSCs are transfected to knock out the gene of interest. These cells are then sequenced to characterize and validate the knockout and then differentiated into CD34+ cells and immune cells.

[0053] Figures 17A-17B RC3H1 and RC3H2 double KO in iPSC (RC3H1,2KO iPSC) does not affect pluripotency. Characterized by (A) morphology (scale bar = 200 μm), in which undifferentiated cells are present, and (B) flow cytometry analysis of iPSC markers TRA-1-60, TRA-1-81, and SSEA-4. Dead cells, debris, and doublets are excluded, so the histogram shows all living cells in the culture from untransfected iPSC or RC3H1,2KO iPSC samples. More than 99% of all living cells express all iPSC markers.

[0054] Figures 18A-18C Transfection of RNPs formed by RC3H1 and RC3H2 guide RNAs introduces insertions and deletions (indels) into the open reading frames of specific genes in iPSCs. Sanger sequencing tracks from iPSCs co-transfected with RC3H1 and RC3H2gRNA ("edited samples") show an uneven mix of bases downstream of the RC3H1 gene (A) and RC3H2 gene (B) cleavage sites compared to untransfected iPSCs ("control samples") (in A, SEQ ID NO: 27 shows 184 to 249 bp from the edited sample, SEQ ID NO: 28 shows 183 to 248 bp from the control sample; in B, SEQ ID NO: 29 shows 270 to 336 bp from the edited sample, SEQ ID NO: 30 shows 272 to 337 bp from the control sample). The black underlined area of ​​the control sample represents the guide sequence, and the horizontal red dotted underlined area is the associated PAM site. The vertical black dashed lines on the two traces represent the cleavage sites. (C) CRISPR knockout analysis of iPSCs co-transfected with RC3H1 and RC3H2 gRNAs. The knockout efficiency of RC3H1 and RC3H2 genes was assessed using ICE analysis. The percentage of out-of-frame indels is the proportion of indels that indicate frameshifts or are longer than 21 bp. R was calculated using the Pearson correlation coefficient. 2 Values ​​indicate confidence in indel percentage.

[0055] Figure 19 Double knockout of RC3H1 and RC3H2 in iPSCs does not prevent differentiation into iCD34+ cells. Unstained cells and cells stained with antibodies against CD34+ were analyzed by flow cytometry. Dead cells, debris, and doublets were excluded, so the histogram shows all live CD34+ cells in cultures from untransfected iPSCs or RC3H1,2KO iPSC samples. Deletion of both the RC3H1 and RC3H2 genes does not prevent iPSCs from developing into the iCD34+ cell subset.

[0056] Figure 20 iPSCs containing RC3H1 and RC3H2 double KO are able to differentiate into CD56+ cells expressing NK cytotoxic receptors with NKG2D and NKp46. Dead cells, debris, and doublets are excluded, so the CD56+ histogram shows all living cells in the generated culture. NKp46 and NKG2D figures exclude CD56+ cells. Unstained controls and isotype controls are presented to show positive staining of each antibody for each corresponding receptor. Co-expression of NK functional receptors (NKp46 or NKG2D) with CD56 indicates that CD56+ cells derived from RC3H1, 2KO iPSCs have the potential to carry out NK-mediated cytotoxic function.

[0057] Figures 21A-21B A2AR KO in iPSCs does not affect pluripotency. Characterized by (A) morphology (scale bar = 200 μm), the presence of undifferentiated cells, and (B) flow cytometric analysis of iPSC markers TRA-1-60, TRA-1-81, and SSEA-4. Dead cells, debris, and doublets were excluded, so the histogram shows all viable cells in culture from untransfected iPSC or A2AR KO iPSC samples. More than 95% of all viable cells expressed all iPSC markers.

[0058] Figures 22A-22C Transfection of RNPs formed by A2AR guide RNA introduces insertions and deletions (indels) into the open reading frame of the A2AR gene in iPSCs. The frequency of indels was assessed by ICE analysis. (A) Sanger sequencing traces from A2AR KO iPSCs ("edited sample") show an uneven mix of bases downstream of the cleavage site compared to untransfected iPSCs ("control sample"). SEQ ID NO: 31 shows 134 to 199 bp of the edited sample; SEQ ID NO: 32 shows 137 to 202 bp from the control sample. The black underlined area of ​​the control sample represents the guide sequence, and the horizontal red dotted underlined area is the associated PAM site. The vertical black dotted lines on the two traces represent the cleavage site. (B) The relative percentage (normalized) of the contribution of each edited sequence in the genomic DNA from A2AR KO iPSCs. The sequences from top to bottom are shown in SEQ ID NO: 33, 34, 35, 36, 37 and 38, respectively. (C) Distribution of indel sizes across the edited population of RNP-transfected iPSCs. The percentage of out-of-frame indels represents the proportion of indels that are frameshifted or longer than 21 bp. R was calculated using the Pearson correlation coefficient. 2 Values ​​indicate confidence in indel percentage.

[0059] Figure 23 Inclusion of the A2AR KO in iPSCs did not prevent their differentiation into iCD34+ cells. Cells stained with antibodies against CD34 were analyzed by flow cytometry. Unstained cells and cells stained with an isotype control were included as controls. Dead cells, debris, and doublets were excluded, so the histogram shows all viable cells in cultures generated from untransfected iPSCs or A2AR KO iPSC samples. Inclusion of the KO did not prevent the development of the iCD34+ cell subtype.

[0060] Figure 24 A2AR KO iPSC can be differentiated into iNK cells. By flow cytometry analysis of unstained cells and cells stained with antibodies for NK cell markers. Dead cells, debris and doublets are excluded, so CD56+ histograms show all living cells in the culture generated from untransfected iPSC or A2AR KO iPSC samples. Unstained samples are presented to show the clear positive staining of each antibody to each corresponding receptor. Appropriate isotype controls were also run and were negative. The expression of NK functional receptors (NKp46, NKp30, NKp44 and NKG2D) proves that the CD56+ cells derived from A2AR KO iPSC are iNK cells, and potentially can have cytotoxic function.

[0061] Figure 25: A2AR KO iPSCs can differentiate into functional iNK cells with enhanced in vitro cytotoxicity. iNK cells were derived from untransfected iPSCs and A2AR KO iPSCs. Real-time cell monitoring system The function of the obtained iNK cells was evaluated in vitro, where OVCAR-3 cells were used as targets. A 1:2 effect-target ratio was used. (A) During a co-culture period of at least 10 hours, changes in NCI were recorded every 15 minutes, where a decrease in NCI indicated target cell death. (B) The results of (A) are shown as the percentage of cytotoxicity of iNK cells relative to target cells when co-cultured for 5 hours (left panel) and 10 hours (right panel). Cells were derived from a single iNK differentiation. Each data point represents a technical replicate. Detailed Description of the Invention

[0063] Throughout this specification, the word "comprises" or variations such as "comprises" or "comprising" will be understood to imply the inclusion of stated elements, integers or steps, or groups of elements, integers or steps, but not the exclusion of any other elements, integers or steps, or groups of elements, integers or steps.

[0064] In the specification and claims, unless the context clearly indicates otherwise, the terms "a" and "an" should be understood to mean "at least one" and should not be understood to exclude "two or more".

[0065] As used herein, " nucleic acid construct " generally refers to the nucleic acid molecule of artificial or recombinant construction or preparation, and is also referred to as nucleic acid vector interchangeably.For example, nucleic acid construct can be made to include target nucleotide sequence, it is expected that the target nucleotide sequence is transcribed in the cell, and in some cases, RNA molecules (such as antisense RNA, siRNA, miRNA or gRNA) with desired function are produced, and in other cases, the mRNA translated into target protein (such as Cas protein) is produced. The target nucleotide sequence in nucleic acid construct can be operably connected to 5' regulatory region (for example, promoter such as heterologous promoter) and / or 3' regulatory region (for example, 3' untranslated region (UTR) such as heterologous 3' UTR). Nucleic acid construct can be circular (such as plasmid) or linear form, can be integrated nucleic acid (that is, can be integrated into the chromosome of host cell, for example, viral vector such as lentiviral vector) or can keep free (for example, plasmid).

[0066] General Description

[0067] Disclosed herein is a method for providing immune cells with enhanced function by inhibiting the function of one or more selected genes. For example, it has been demonstrated herein that ablation of one or more selected genes using CRISPR / Cas9 gene editing technology enhances the persistence and anti-tumor activity of cytotoxic lymphocytes in vivo. Therefore, a method is provided by inhibiting the function of one or more selected genes in immune cells or stem cells capable of differentiating into immune cells. Also disclosed herein are immune cells or stem cells prepared by the methods of the present invention, as well as the use of immune cells in therapeutic treatments.

[0068] immune cells

[0069] As used herein, "immune cells" should be understood to include cells of the mammalian immune system (e.g., lymphocytes (T cells, B cells, NK cells, and NKT cells), neutrophils, and monocytes (including macrophages and dendritic cells)) and cell lines derived from cells of the mammalian immune system. Immune cells can be isolated from a mammalian subject, collected from a cell line culture of immune cells derived from a mammalian subject, or produced by differentiation from stem cells.

[0070] The present disclosure is intended to provide immune cells with enhanced function. "Enhanced function" means that the immune cells provided as a result of the modification or manipulation disclosed herein exhibit enhanced activity (e.g., cytotoxicity), proliferation, survival, persistence, and / or infiltration compared to control immune cells (i.e., immune cells that have not been modified or manipulated). The cytotoxicity of an immune cell refers to the ability of an immune cell to kill a target cell, typically through a receptor-based mechanism.

[0071] In some embodiments, the immune cell is a cytotoxic immune cell, such as a cytotoxic lymphocyte.

[0072] In some embodiments, the immune cell is a T cell. In some embodiments, the T cell is a NKT cell. In some embodiments, the immune cell is a NK cell.

[0073] Reference to "T cells" should be understood as reference to any cell comprising a T cell receptor. In this regard, the T cell receptor may comprise any one or more of α, β, γ or δ chains. As will be appreciated by those skilled in the art, NKT cells also express T cell receptors, and therefore target antigen-specific NKT cells may also be generated according to the present invention. The present invention is not intended to be limited to any specific T cell subclass, although in one embodiment, the subject's T cells express α / β TCR dimers. In some embodiments, the T cells are CD4+ helper T cells, CD8+ killer T cells or NKT cells. Without limiting the present invention to any theory or mode of action, CD8+ T cells are also referred to as cytotoxic cells. As a major part of the adaptive immune system, CD8+ T cells scan the intracellular environment, primarily to target and destroy infected cells. Small peptide fragments derived from intracellular contents are processed and transported to the cell surface, where they are presented in the context of MHC class I molecules. However, in addition to responding to viral infections, CD8+ T cells also provide additional immune surveillance by monitoring and removing damaged or abnormal cells (including cancer). CD8+T cell recognition of MHC I presented peptides usually results in the release of cytotoxic granules or lymphokines, or activates apoptosis pathways to destroy subject cells by FAS / FASL interactions. On the other hand, CD4+T cells usually recognize peptides presented by antigen presenting cells in the context of MHC II class, resulting in the release of cytokines intended to regulate B cells and / or CD8+T cell immune responses. CD4+T cells with cytotoxic activity have also been observed in various immune responses. In addition, CD4+CAR-T cells show cytotoxicity comparable to CD8+CAR-T cells in vitro, and even for longer-lasting anti-tumor activity, are superior to CD8+CAR-T cells in vivo (see, for example, Wang et al., JCI Insight.2018; 3(10): e99048; Yang et al., Sci Transl Med.2017Nov22; 9(417), eaag1209).

[0074] Natural killer T cells (also known as NKT or T / NK cells) are a population of specialized T cells that express a semi-invariant T cell receptor (TCRα-β) and surface antigens typically associated with natural killer cells. The TCR on NKT cells is unique in that it typically recognizes glycolipid antigens presented by the MHC I-like molecule CD1d. Most NKT cells express an invariant TCRα chain and one of a few TCRβ chains. The TCR present on type I NKT cells typically recognizes the antigen α-galactosylceramide (α-GalCer). Within this group, distinguishable subpopulations have been identified, including CD4 + CD8 - cells, CD4 - CD8+ cells and CD4 - CD8 - Cells. Type II NKT cells (or non-invariant NKT cells) express a wider range of TCRα chains and do not recognize α-GalCer antigens. NKT cells produce cytokines with a variety of generally opposite effects (e.g., promoting inflammation or inducing immunosuppression (including tolerance)). Therefore, they can contribute to antibacterial and antiviral immune responses, promote tumor-associated immune surveillance, and inhibit or promote the development of autoimmune diseases. Like natural killer cells, NKT cells can also induce perforin, FAS, and TNF-related cytotoxicity. Therefore, reference to T cells should be understood to include reference to NKT cells.

[0075] Natural killer (NK) cells are a type of cytotoxic lymphocytes that form a part of the innate immune system. NK cells provide a rapid response to cells infected with viruses, work about 3 days after infection, and also react to tumor formation. Typically, immune cells (such as T cells) detect the major histocompatibility complex (MHC) present on the surface of infected or transformed cells, triggering cytokine release and causing target cell lysis or apoptosis. However, NK cells have the ability to recognize stress cells in the absence of antibodies or MHC, allowing faster immune responses. This effect is particularly important because harmful cells lacking MHC I markers cannot be detected and destroyed by other immune cells (such as T cells). Compared with NKT cells, NK cells do not express TCR or CD3, but they typically express surface markers CD16 (FcγRIII) and CD56.

[0076] In some embodiments, immune cells to be modified or manipulated according to the methods of the present invention can be isolated from a mammalian subject, including, for example, blood (whole blood, serum, or plasma), bone marrow, thymus, lymph nodes.

[0077] In some embodiments, immune cells to be modified or manipulated according to the methods of the present invention can be collected from a cell line culture of immune cells (eg, a T cell line) derived from a mammalian subject.

[0078] In some embodiments, the immune cells to be modified or manipulated according to the present invention can be differentiated from stem cells or other progenitor cells (e.g., cells cultured and differentiated from stem cells). Methods for differentiating stem cells into immune cells, particularly into T cells or NK cells, are known in the art (Li et al., Human iPSC-Derived Natural Killer Cells Engineered with Chimeric Antigen Receptors Enhance Anti-tumor Activity, Cell Stem Cell, 2018, 23(2): 181-192e5; Themeli et al., Generation of tumor-targeted human T lymphocytes from induced pluripotent stem cells for cancer therapy, Nat Biotechnol, 2013, 31(10): 928-33; Maeda et al., Regeneration of CD8alphabeta T Cells from T-cell-Derived iPSC Imparts Potent Tumor Antigen-Specific Cytotoxicity, Cancer Res, 2016, 76(23): 6839-6850).

[0079] stem cells

[0080] As used herein, "source cells" refer to cells that are converted into "derivative cells" by reprogramming or differentiation. Examples of source cells suitable for the methods disclosed herein include stem cells. Examples of "derivative cells" include immune cells, such as T cells, NKT cells, and NK cells.

[0081] The term "stem cell" should be understood to refer to cells that can self-renew and show the potentiality of developing to multiple lineage directions given its specific phenotype, and thus form new organisms or regenerate tissues or cell mass of an organism. The stem cell utilized according to the present invention is multipotent (pluripotent) and pleiotropic (multipotent) and can be differentiated along two or more lineages, and includes but is not limited to embryonic stem cells (ESC), adult stem cells, umbilical cord stem cells, hematopoietic stem cells (HSC), progenitor cells, precursor cells, multipotent cells, pleiotropic cells or dedifferentiated somatic cells (such as induced pluripotent stem cells). "Multipotent" means that subject stem cells can be differentiated to form, especially any one of the three germ layers, which are ectoderm, endoderm and mesoderm.

[0082] In some embodiments, the source cells also express at least one homozygous major HLA genotype. In some embodiments, the source cells express at least one homozygous HLA genotype, which is a major transplant antigen and is preferably expressed by a significant proportion of the population, such as at least 5%, at least 10%, at least 15%, at least 17%, at least 20% or more of the population. When the homozygous HLA genotype corresponds to a dominant MHC I or MHC II HLA type (in terms of tissue rejection), using such cells in the context of a treatment regimen results in a significantly reduced tissue rejection problem in a wider population receiving cells of the present invention. In other embodiments, the source cells may be homozygous for more than one HLA antigen, such as two, three or more HLA antigens. The target HLA antigen may be selected from, for example, HLA A1, B8, C7, DR17, DQ2, or HLA A2, B44, C5, DR4, DQ8, or HLA A3, B7, C7, DR15, DQ6.

[0083] In some embodiments, the source cell is homozygous for the suppressor gene.

[0084] In some embodiments, the source cell has been genetically modified in one or more genes identified herein such that the function of the modified gene is suppressed in derivative cells differentiated from the genetically modified source cell.

[0085] In some embodiments, the source cell has also been genetically modified to include nucleic acid encoding CAR (i.e., chimeric antigen receptor). The nucleic acid encoding CAR can be introduced into the source cell by methods known in the art.

[0086] In some embodiments, the source cell is a stem cell. In some embodiments, the source cell is an induced pluripotent stem cell (iPSC).

[0087] In some embodiments, progenitor cells capable of differentiating into immune cells are used to be modified; for example, cells cultured from pluripotent stem cells (eg, iPSCs) that have undergone some differentiation into immune cells in culture but have not yet fully differentiated into immune cells.

[0088] iPSC

[0089] iPSC is usually generated directly from somatic cells. iPSC can be induced from any nucleated cell in principle, including, for example, monocytes and skin cells from blood. In some embodiments, iPSC can be generated by fully differentiated T cells; or generated from precursor T cells (such as thymocytes, these precursor T cells have begun or even completed the rearrangement of their TCR and show target antigen specificity). In another embodiment, iPSC is transfected with one or more nucleic acid molecules encoding TCR (such as rearranged TCR genes) for target antigenic determinants (for example, tumor antigen determinants). In one embodiment, iPSC is derived from cells expressing rearranged TCR, preferably rearranged α β TCR. In another embodiment, the cells express rearranged γ δ TCR. Examples of cells suitable for generating iPSC of the present invention include, but are not limited to, CD4+ T cells, CD8+ T cells, NKT cells, thymocytes, or other forms of precursor T cells.

[0090] In another embodiment, the iPSCs are derived from another type of immune cell, such as a NK cell.

[0091] Methods for generating iPSCs from mature or differentiated cells (eg, T cells or precursor T cells) are known to those skilled in the art (Themeli, Kloss et al. 2013, Li, Hermanson et al. 2018).

[0092] In some embodiments, the source cell is an induced pluripotent stem cell (iPSC).

[0093] In some embodiments, source cells are generated from umbilical cord blood PBMCs (peripheral blood mononuclear cells).

[0094] In some embodiments, the subject source cell is a cell that is more differentiated toward an immune cell than a pluripotent stem cell.

[0095] The derived immune cells generated by the methods disclosed herein include hematopoietic lineage cells and specific types of immune cells that can differentiate into immune cells. Examples of derived immune cells are HE, pre-HSC, HSC, multipotent progenitor cells, common lymphocyte progenitor cells, early thymic progenitor cells, pre-T cell progenitor cells, pre-NK progenitor cells, T progenitor cells, NK progenitor cells, macrophages and other immune cells (e.g., T cells, NK-T cells, and NK cells).

[0096] The present disclosure relates to providing immune cells with enhanced function or derived immune cells produced by differentiation. "Enhanced function" means that the immune cells provided as a result of the modification or manipulation disclosed herein exhibit enhanced activity (e.g., cytotoxicity), proliferation, survival, persistence, and / or infiltration compared to control immune cells (i.e., immune cells that have not been modified or manipulated). The cytotoxicity of an immune cell refers to the ability of an immune cell to kill a target cell, typically through a receptor-based mechanism.

[0097] Genes to be suppressed

[0098] According to the present disclosure, inhibition of the function of one or more genes identified herein can enhance the function of immune cells.

[0099] As used herein, "inhibition of gene function" means that the level and / or activity of the protein encoded by the gene is ultimately reduced or eliminated. Thus, the function of a gene can be inhibited due to manipulation or modification of the genomic DNA sequence of the gene (e.g., resulting in the destruction of the gene), due to inhibition of mRNA (e.g., reducing the level or function of mRNA, such as by inhibiting transcription or translation), or due to inhibition of protein (e.g., by reducing the level or activity of the protein). In some embodiments, when the level and / or activity of the protein encoded by the gene in the modified cell is compared with the level and / or activity of the protein in the unmodified cell, the degree of inhibition is at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more.

[0100] In some embodiments, the gene whose function is to be inhibited is selected from RC3H1, RC3H2, A2AR, FAS, TGFBR1, and TGFBR2. In some embodiments, the inhibition is directed against a single gene selected from RC3H1, RC3H2, A2AR, FAS, TGFBR1, and TGFBR2; for example, a single gene selected from RC3H1, RC3H2, A2AR, FAS, TGFBR1, or TGFBR2. In some embodiments, the inhibition is directed against a single gene selected from RC3H1, RC3H2, A2AR, FAS, TGFBR1, and TGFBR2, combined with the inhibition of at least one other gene. In some embodiments, the inhibition is directed against two or more genes selected from RC3H1, RC3H2, A2AR, FAS, TGFBR1, and TGFBR2, for example, the RC3H1 and RC3H2 genes, the TGFBR1 and TGFBR2 genes, or the TGFBR1 and RC3H2 genes; and optionally combined with the inhibition of at least one other gene.

[0101] As described below, members of the genomes of RC3H1, RC3H2, A2AR, FAS, TGFBR1 and TGFBR2 are known in the art to relate to immune cell functions. However, it is not known in the art whether suppressing the function of these genes individually or in combination produces adverse consequences. In particular, it is expected that the function of completely removing these genes (e.g., by gene editing) may have an adverse effect on important cell functions, thereby causing cell viability or replication capacity to decrease. In addition, it is expected that removing the function of these genes in stem cells (e.g., iPSC) may have an adverse effect on cell functions, such as viability, self-renewal, pluripotency, the ability to differentiate into specific cell types (e.g., immune cells) and making these cell types work. Those skilled in the art will recognize that maintaining these key cell functions is a key feature of the present invention.

[0102] RC3H1, RC3H2

[0103] RC3H1 is also known as RC3H1, Roquin-1, RING finger and CCCH-type domain 1, RING finger and CCCH-type zinc finger domain-containing protein 1, RING finger and C3H zinc finger protein 1, RING finger and CCCH-type zinc finger domain 1, ROQ1, RNF198, or RING finger protein 198.

[0104] RC3H2 is also known as Roquin-2, Roquin2, RING finger and CCCH-type domain 2, RING finger and CCCH-type zinc finger domain-containing protein 2, RING finger and CCCH-type zinc finger domain 2, MNAB, ROQ2, RNF164, or RING finger protein 164.

[0105] The ROQUIN family of proteins includes ROQUIN1 (encoded by RC3H1) and ROQUIN2 (encoded by RC3H2), which are RNA-binding proteins that play an important role in the innate and adaptive immune systems (Athanasopoulos, V., RR Ramiscal and CG Vinuesa, ROQUIN signaling pathways in innate and adaptive immunity. Eur J Immunol, 2016, 46(5): p. 1082-90). Rc3h1 mutations in mice (sanroque mice) lead to increased ICOS expression in T cells, which causes a lupus-like autoimmune syndrome in mice (Yu, D. et al., Roquin represses autoimmunity by limiting inducible T-cell co-stimulator messenger RNA. Nature, 2007, 450(7167): p. 299-303). Although knockout of either RC3H1 or RC3H2 alone does not produce autoantibodies and lacks autoimmunity in mice, mice deficient for both RC3H1 and RC3H2 display an immunophysiological phenotype similar to that of sanroque mice. To date, no human disease has been found to harbor mutations in RC3H1 or RC3H2 (Athanasopoulos, V., RR Ramiscal, and CG Vinuesa, ROQUIN signaling pathways in innate and adaptive immunity. Eur J Immunol, 2016, 46(5): p. 1082-90). Prior to this disclosure, the role of RC3H1 and RC3H2 genes in human T cells, particularly their function in cytotoxic cells, was unknown.

[0106] According to the present disclosure, inhibition of the function of one or both of the RC3H1 and RC3H2 genes enhances the function of immune cells.

[0107] A2AR

[0108] A2AR is also known as ADORA2A, adenosine A2a receptor, adenosine receptor A2a, ADORA2, adenosine receptor subtype A2a, or RDC8.

[0109] Extracellular adenosine produced by tumor cells is a key immunosuppressive metabolite that limits the activation of cytotoxic lymphocytes and inhibits antitumor immune responses through the adenosine 2A receptor (A2AR).

[0110] According to the present disclosure, inhibition of the function of the A2AR gene, such as by gene editing (eg, mediated by CRISPR / Cas9 based on specifically designed guide RNAs), enhances the function of immune cells.

[0111] FAS

[0112] FAS is also known as Fas cell surface death receptor, APT1, CD95, FAS1, APO-1, FASTM, ALPS1A, or TNFRSF6.

[0113] The FAS receptor (also known as CD95 and APO-1) induces apoptosis and terminal differentiation of cytotoxic T cells. Engagement of FAS with its ligand FASL may inhibit the anti-tumor activity of CAR-T cells.

[0114] According to the present disclosure, inhibition of the function of the FAS gene, such as by gene editing (eg, mediated by CRISPR / Cas9), enhances the function of immune cells.

[0115] TGFBR1 and TGFBR2

[0116] TGFBR1 is also known as TGFRBRI, TGFB receptor 1, TGF-β receptor 1, AAT5, ALK5, ESS1, LDS1, MSSE, SKR4, TBRI, ALK-5, LDS1A, LDS2A, TBR-1, TGFR-1, ACVRLK4, tβR-1, transforming growth factor beta receptor 1, or transforming growth factor beta receptor 1.

[0117] TGFBR2 is also known as TGFBRII, AAT3, FAA3, LDS2, MFS2, RIIC, LDS1B, LDS2B, TAAD2, TBRII, TBR-ii, TGFR-2, TGFβ-RII, transforming growth factor beta receptor 2, or transforming growth factor beta receptor II.

[0118] TGF-β exerts systemic immunosuppressive effects and inhibits host immune surveillance, and is considered to be one of the main factors of the immunosuppressive microenvironment in tumors.

[0119] According to the present disclosure, inhibition of the function of the TGFBR1 and / or TGFBR2 genes, for example by gene editing (e.g., mediated by CRISPR / Cas9 based on specifically designed guide RNAs), enhances the function of immune cells.

[0120] According to the present disclosure, inhibition of the function of at least one gene selected from RC3H1, RC3H2, A2AR, FAS, TGFBR1, and TGFBR2, combined with inhibition of at least another gene, enhances the function of immune cells.

[0121] Inhibit gene function

[0122] Inhibition of gene function can be achieved by a variety of methods, such as by gene editing (inhibition of translation by, for example, RNA interference or antisense oligonucleotides), or by the use of compounds (such as small molecules or antibodies that directly antagonize the protein product).

[0123] Inhibition through gene editing

[0124] In some embodiments, inhibition of gene function is achieved by using a gene editing system that modifies the genomic sequence of a gene.

[0125] Gene editing systems typically include DNA binding proteins or DNA binding nucleic acids coupled to nucleases. DNA binding proteins or DNA binding nucleic acids specifically bind or hybridize to the targeted region of a gene, and the nuclease produces one or more double-strand breaks and / or one or more single-strand breaks in the targeted region of a gene. The targeted region can be the coding region of a gene, such as in an exon, near the N-terminal portion of the coding region (for example, in the first or second exon). Double-stranded or single-stranded breaks can be repaired by a cellular repair process, such as by non-homologous end joining (NHEJ) or homology-directed repair (HDR). In some cases, the repair process introduces insertions, deletions, missense mutations, or frameshift mutations (including, for example, biallelic frameshift mutations), resulting in the destruction of a gene and the suppression of gene function.

[0126] Examples of gene editing systems include fusions comprising a DNA-binding protein and a nuclease, such as zinc finger nucleases (ZFNs) or TAL effector nucleases (TALENs), or RNA-guided nucleases, such as the clustered regularly interspaced short palindromic nucleic acids (CRISPR)-Cas system.

[0127] ZFPs and TALENs

[0128] In some embodiments, inhibition of gene function is achieved by utilizing a gene editing system that includes a DNA binding protein fused to an endonuclease, such as one or more zinc finger proteins (ZFPs) or transcription activator-like proteins (TALs). Examples include ZFNs, TALEs, and TALENs.

[0129] The DNA binding domains of ZFPs and TALs can be "engineered" to bind to a target DNA sequence of interest. For example, one or more amino acids in the recognition helical region of a naturally occurring zinc finger or TALE protein can be modified to bind directly to a predetermined DNA sequence. The criteria for rational design are described in, for example, U.S. Patent 6,140,081, U.S. Patent 6,453,242, U.S. Patent 6,534,261, WO 98 / 53058, WO 98 / 53059, WO 98 / 53060, WO 02 / 016536, WO 03 / 016496, and U.S. Patent Publication No. 20110301073A1.

[0130] In some embodiments, the DNA binding protein comprises one or more zinc finger domains of zinc finger proteins (ZFPs) or ZFPs. ZFPs or their domains bind to DNA in a sequence-specific manner through one or more "zinc fingers" (amino acid regions within the binding domain whose structure is stabilized by the coordination of zinc ions). The sequence specificity of naturally occurring ZFPs can be changed by amino acid substitutions at certain positions on the zinc finger recognition helix. In addition, many engineered gene-specific zinc fingers are commercially available (see, for example, the CompoZr platform for zinc finger construction, developed in collaboration with Sangamo Biosciences (Richmond, California, USA) and Sigma-Aldrich (St. Louis, Missouri, USA). Therefore, in some embodiments, ZFPs are engineered to bind to target sequences within genes identified herein as to be inhibited. Typical target sequences include exons, regions near the N-terminal region of the coding sequence (e.g., first exon, second exon) and 5' regulatory regions (promoter or enhancer regions). ZFPs are fused to endonucleases or DNA cleavage domains to form zinc finger nucleases (ZFNs). Examples of DNA cleavage domains include the DNA cleavage domains of type IIS restriction enzymes.

[0131] In some embodiments, ZFNs are introduced into cells (e.g., immune cells or stem cells) by transfecting a nucleic acid construct comprising a nucleic acid sequence encoding the ZFN. The ZFN is then expressed from the construct in the cell and results in editing and disruption of the target gene. In some embodiments, the ZFN is introduced into the cell in its protein form.

[0132] In some embodiments, the DNA binding protein comprises a naturally occurring or engineered transcription activator-like protein (TAL) DNA binding domain, such as in a transcription activator-like protein effector (TALE) protein. See, for example, US20110301073 A1, incorporated herein by reference. The TALE DNA binding domain is a polypeptide comprising one or more TALE repeats, each of which is 33-35 amino acids in length and comprises 1 or 2 DNA binding residues. It has been determined that the HD (histamine-aspartic acid) sequence at positions 12 and 13 of the TAL repeats results in binding to cytosine (C), NG (asparagine-glycine) binds to T, NI (asparagine-isoleucine) binds to A, and NN (asparagine-asparagine) binds to G or A. See, for example, US 20110301073 A1. In some embodiments, a TALE can be designed to have an array of TAL repeats that are specific for a target DNA sequence of interest within a gene identified herein as being to be inhibited. Custom-designed TALE arrays are also commercially available from Cellectis Bioresearch (Paris, France), Transposagen Biopharmaceuticals (Lexington, Kentucky, USA), and Life Technologies (Grand Island, New York, USA). In some embodiments, the TAL DNA binding domain is fused to an endonuclease to form a TALE-nuclease (TALEN), which cleaves a nucleotide sequence at a target site within the gene identified herein to be inhibited.

[0133] In some embodiments, TALEN is introduced into cells by transfection of a nucleic acid construct (e.g., plasmid, mRNA, or lentiviral vector) comprising a nucleic acid sequence encoding TALEN. TALEN is then expressed from the construct in the cell and results in editing and destruction of the target gene. In some embodiments, TALEN is introduced into cells in its protein form.

[0134] CRISPR / Cas

[0135] In some embodiments, inhibition of gene function is achieved by utilizing the CRISPR (for "clustered regularly interspaced short palindromic repeats") / Cas (for "CRISPR-associated nuclease") system for gene editing. CRISPR / Cas is well known in the art, with reagents and protocols readily available (Mali et al., 2013, Science, 339(6121), 823-826; Hsu et al., 2014, Cell, 157.6: 1262-1278; Jiang et al., 2013, Nature Biotechnology, 31, 233-239; Anzalone et al., Nature (2019) doi: 10.1038 / s41586-019-1711-4; Komor et al., Nature 533: 420–424, 2016; Gaudelli et al., Nature 551: 464-471 (2017)). Exemplary CRISPR-Cas gene editing protocols are described in Jennifer Doudna and Prashant Mali, 2016, "CRISPR-Cas: A Laboratory Manual" (CSHL Press, ISBN: 978-1-621821-30-4) and Ran et al. 2013, Nature Protocols, 8(11): 2281-2308.

[0136] The CRISPR / Cas system typically comprises two components: (1) an RNA-dependent DNA nuclease, also referred to herein as a CRISPR endonuclease or Cas protein, such as Cas9, Cas12, or other alternative nucleases; and (2) a non-coding short “guide RNA” comprising a dual RNA or single-stranded full-length guide RNA containing crRNA (“CRISPR RNA”) and tracrRNA (“trans-activating crRNA”), and comprising a targeting sequence that directs the nuclease to a target site in the genome. The guide RNA (gRNA) directs the nuclease to the target site, where the nuclease generates a double-strand break (DSB) in the DNA at the target site. The resulting DSB is then repaired by one of two general repair pathways: the non-homologous end joining (NHEJ) pathway and the homology-directed repair (HDR) pathway. The NHEJ repair pathway is the most active repair mechanism, capable of rapidly repairing DSBs, but often results in small nucleotide insertions or deletions (Indels) at the DSB site, leading to frameshift mutations that knock out functional genes. The HDR pathway is less efficient but has high fidelity. When the CRISPR endonuclease is provided with a DNA template that is homologous to the break region, the double-strand break is repaired via HDR using the homologous DNA template. The HDR pathway allows large genetic inserts to be inserted into cells along with RNPs.

[0137] The design or selection of gRNA sequences comprising sequences that target a target site in a target gene has been described in the art. The target site can include regulatory region sequences (e.g., promoters and enhancers), or sequences within a coding region (e.g., exons, e.g., exons near the 5' end, or exons encoding a specific domain or region of a protein). In some embodiments, the target site is selected based on its position immediately adjacent to the 5' position of the PAM sequence, e.g., typically NGG or NAG.

[0138] The guide sequence is designed to include a targeting sequence that is complementary to the target sequence (the nucleotide sequence at the target site). Perfect complementarity is not necessarily required, as long as there is sufficient complementarity to cause specific hybridization between the guide sequence and the target sequence and promote the formation of the CRISPR complex at the target site. In some embodiments, the degree of complementarity between the targeting sequence of the gRNA and the target sequence is at least 80%, 85%, 90%, 95%, 98%, 99% or more (e.g., 100% or perfect complementarity).

[0139] In some embodiments, the guide sequence is at least 15 nucleotides in length, e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, or 75 or more nucleotides. In some embodiments, the guide sequence is no more than 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, or 20 nucleotides in length. In some embodiments, the targeting sequence portion of the guide sequence is about 20 nucleotides in length. Truncated gRNAs with shorter regions of target complementarity (<20 nucleotides) have been described as being effective in improving target specificity (see, e.g., Fu et al., Nature Biotechnol., 32(3):279-284, 2014). In some embodiments, the targeting sequence of the guide RNA is 17, 18, 19 or 20 nucleotides in length. In some embodiments, the targeting sequence of the guide RNA is fully complementary to the nucleotide sequence at the target site. In some embodiments in which the targeting sequence of the guide RNA is not fully complementary to the nucleotide sequence at the target site, the targeting sequence portion (also referred to as the seed region) near the PAM sequence in the genome is fully complementary to the nucleotide sequence at the target site. In other words, some variations of the nucleotide 5' of the guide sequence (i.e., non-seed region) are allowed. For example, the guide sequence can be designed to comprise a targeting portion that is at least 17 nucleotides in length (e.g., 17, 18, 19 or 20 nucleotides in length), having a seed region of at least 17 nucleotides that are fully complementary to at least 17 nucleotides in the target sequence.

[0140] Examples of target sequences in specific genes are provided in Table 1. In some embodiments, the guide sequence comprises a targeting sequence of 17-20 nucleotides, wherein at least 17 nucleotides in the seed region (the 3' portion of the targeting sequence) are fully complementary to at least 17 nucleotides in the target sequence, e.g., fully complementary to the 17 nucleotides at the 3' end of the target sequence.

[0141] Table 1

[0142]

[0143]

[0144] A gRNA database for CRISPR genome editing is publicly available, providing exemplary sgRNA target sequences in constitutive gene exons of the human genome or mouse genome (see, for example, the gRNA database provided by GenScript and MIT; also see Sanjana et al. (2014) Nat. Methods, 11: 783-4). In some embodiments, the gRNA sequence is or comprises a sequence that has minimal off-target binding to non-target genes.

[0145] Examples of Cas proteins or CRISPR endonucleases suitable for use herein include Cpf1 (Zetsche et al., Cell (2015) 163(3):759-771), Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas100, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2 , Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3 or Csf4, or a functional derivative thereof (i.e., a mutant form or derivative of a naturally occurring CRISPR endonuclease, such as a fragment thereof, which substantially retains the RNA-dependent endonuclease activity of the naturally occurring form). See, e.g., US20180245091A1 and US20190247517A1). In some embodiments, the Cas protein is Cas9, such as Cas9 from Streptococcus pyogenes (S. pyogenes), Staphylococcus aureus (S. aureus), or Streptococcus pneumoniae (S. pneumoniae). In some embodiments, the Cas protein is a Cas9 protein from Streptococcus pyogenes having an amino acid sequence provided in the SwissProt database with accession number Q99ZW2.

[0146] In some embodiments, the inhibition of gene function is achieved by CRISPR-mediated gene editing, which includes introducing a first nucleic acid encoding a Cas nuclease and a second nucleic acid encoding a guide RNA (gRNA) that is specific for a target sequence in a gene identified herein as being to be inhibited into a cell (e.g., an immune cell or stem cell). These two nucleic acids can be contained in a nucleic acid construct (or vector) or provided on different constructs (or vectors) to achieve expression of the Cas protein and gRNA in the cell. The expression of Cas nuclease and gRNA in the cell guides the formation of a CRISPR complex at the target sequence, which results in DNA cutting.

[0147] In some embodiments, inhibition of gene function is achieved by CRISPR-mediated gene editing, which includes introducing a combination or complex of gRNA and Cas nuclease into cells. In some embodiments, the Cas protein / gRNA combination or complex can be delivered to cells by methods such as electroporation, gene gun, calcium phosphate transfection, cell compression or squeezing, liposomes, nanoparticles, microinjection, naked DNA plasmid transfer, protein transduction domain-mediated transduction or viral mediation (including integrating viral vectors (e.g., retroviruses and lentiviruses) and non-integrating viral vectors (e.g., adenovirus, AAV, HSV, vaccinia)).

[0148] Regardless of the specific gene editing method used, to confirm that the gene sequence has been modified and gene function has been inhibited, a variety of assays can be performed, including, for example, by examining DNA or mRNA by Southern and Northern blotting, PCR including RT-PCR, or nucleic acid sequencing, or by detecting the presence or activity of specific proteins or peptides by, for example, immunological means (ELISA and Western blotting).

[0149] In some embodiments, the function of at least one of the RC3H1, RC3H2, A2AR, and FAS genes is inhibited by introducing an indel into an early exon of at least one of these genes using the CRISPR / Cas9 system, resulting in a frameshift mutation in at least one of these genes, such that a functional protein is not translated from the edited gene. In some embodiments, the function of two or more of the RC3H1, RC3H2, A2AR, and FAS genes is inhibited by introducing an indel into an early exon of two or more of these genes using CRISPR / Cas9, resulting in a frameshift mutation in two or more of these genes, such that a functional protein is not translated from the edited gene. In some embodiments, two or more of the RC3H1, RC3H2, A2AR, and FAS genes comprise RC3H2 in combination with another gene, such as RC3H2 and RC3H1.

[0150] In some embodiments, the function of at least one of the TGFBR1 and TGFBR2 genes is inhibited by introducing indels into an exon and upstream of the codon for the start amino acid residue of the intracellular signaling domain of at least one of these genes using the CRISPR / Cas9 system, resulting in a frameshift mutation that eliminates the intracellular signaling domain, which is a dominant negative mutation. In some embodiments, the function of both the TGFBR1 and TGFBR2 genes is inhibited by introducing indels into an exon and upstream of the codon for the start amino acid residue of the intracellular signaling domain of at least one of these genes using CRISPR / Cas9, resulting in a frameshift mutation that eliminates the intracellular signaling domain, which is a dominant negative mutation.

[0151] By using a nickase (i.e., Cas9 nickase) and a high-fidelity enzyme, the CRISPR / Cas system can also be used without double-strand breaks or donor DNA. See, for example, Anzalone, A et al., Nature (2019) doi: 10.1038 / s41586-019-1711-4; Komor et al., Nature 533: 420–424, 2016; Gaudelli et al., Nature 551: 464-471 (2017).

[0152] Inhibit by reducing or eliminating the level or function of mRNA

[0153] In some embodiments, inhibition of gene function is achieved by reducing or eliminating the level or function of mRNA transcribed from the gene (i.e., inhibiting mRNA). Unlike inhibition by gene editing systems, inhibition of mRNA is temporary.

[0154] In some embodiments, inhibition of mRNA can be achieved by using, for example, antisense nucleic acids, ribozymes, small interfering RNA (siRNA), short hairpin RNA (shRNA), miRNA (microRNA) or its precursors, or nucleic acid constructs that can be transcribed in cells to produce antisense RNA, siRNA, shRNA, miRNA or their precursors.

[0155] Antisense-antisense technology is well-known method.Antisense RNA is to be complementary to the full length or part of endogenous mRNA and blocks the RNA molecule of translation from endogenous mRNA by forming duplex with endogenous mRNA.Antisense RNA can be prepared synthetically and introduced in target cell (such as immune cell), or prepared by transcribing the nucleic acid construct introduced from exogenous source in target cell, to realize the inhibition of target gene expression.Antisense RNA need not be complementary to the full length mRNA from target gene.However, the length of antisense RNA should be enough to form duplex with target mRNA and block the translation based on target mRNA.Usually, the length of antisense RNA is at least 15 nucleotides, for example 15,16,17,18,19,20,21,22,23,24,25,26,27,30,35,40,50,75,100,200,300,400,500 nucleotides or more. In some embodiments, the antisense RNA is no more than 500, 400, 300, 200, 100, 75, or 50 nucleotides in length.Antisense molecules can also be DNA, DNA analogs, and RNA analogs.

[0156] Ribozymes-ribozymes (i.e., catalytic RNA) can be designed to specifically pair with target RNA and cut the phosphodiester backbone at a specific position, thereby functionally inactivating the target RNA. See, for example, U.S. Patent No. 6,423,885, U.S. Patent No. 5,254,678 and Perriman et al., PNAS 92 (13): 6175-6179 (1995). Ribozymes can be prepared synthetically and introduced into target cells (e.g., immune cells), or prepared in target cells by transcription of a nucleic acid construct imported from an exogenous source.

[0157] RNAi (RNA interference) - The inhibition of gene expression or translation by RNAi is known in the art and can be achieved using RNA molecules such as siRNA ("small interfering RNA"), shRNA ("short hairpin RNA") and miRNA ("micro RNA"). siRNA and shRNA are known to participate in the RNA interference pathway and interfere with the expression of specific genes. siRNA is a small (typically 20-25 nucleotides in length) double-stranded RNA and can be designed to contain a sequence homologous or complementary to a target mRNA (i.e., an mRNA transcribed from a target gene) or a portion of a target mRNA. shRNA is cleaved by the ribonuclease DICER to produce siRNA. Given the sequence of the target gene, siRNA or shRNA can be synthetically designed and prepared and introduced into a target cell (e.g., an immune cell), or prepared in a target cell (e.g., an immune cell) from an exogenously introduced nucleic acid construct encoding such an RNA. miRNA is also a small RNA molecule (typically about 21-22 nucleotides) processed from a long precursor transcribed from a non-protein coding gene, and interrupts translation by imprecise base pairing with the target mRNA. MiRNA or its precursor (pri-miRNA or pre-miRNA) can be prepared synthetically and introduced into target cells (e.g., immune cells), or prepared in target cells (e.g., immune cells) from exogenously introduced nucleic acid constructs encoding miRNA or its precursor.

[0158] In some embodiments, the inhibition of mRNA can be achieved using a modified form of the CRISPR / Cas system, in which a Cas molecule that is an enzyme-inactivated nuclease is used in combination with a gRNA targeting the target gene. The target site can be in the 5' regulatory region (e.g., a promoter or enhancer region) of the gene. In some embodiments, the Cas molecule is an enzyme-inactivated Cas9 molecule that comprises a mutation that eliminates or significantly reduces DNA cleavage activity, such as a point mutation (see, e.g., WO2015 / 161276). In some embodiments, the enzyme-inactivated Cas9 molecule is fused directly or indirectly to a transcriptional repressor protein.

[0159] Suppress by other means

[0160] The present invention includes other methods known in the art for inhibiting gene function, including reducing the level or activity of the protein encoded by the gene, such as by introducing compounds (e.g., small molecules, antibodies, etc.) that directly inhibit the activity of the protein encoded by the gene into cells (e.g., immune cells).

[0161] CAR

[0162] In some embodiments, the cells (e.g., immune cells or stem cells) modified to suppress one or more selected genes are also modified to contain a nucleic acid encoding a chimeric antigen receptor (or "CAR").

[0163] In some embodiments, the nucleic acid encoding CAR can be introduced into the cell before, simultaneously or after the function of the selected gene is modified in the cell. In the embodiment where the inhibition is transient (for example, by antisense RNA or RNAi), preferably the nucleic acid encoding CAR is introduced into the cell before the cell is modified to achieve inhibition. In the embodiment where the inhibition is permanent (for example, by gene editing), the nucleic acid encoding CAR can be introduced into the cell before, simultaneously or after the cell is modified to achieve inhibition. In some embodiments, the nucleic acid encoding CAR is designed to allow the target site of gene editing to be inserted by HDR after introducing DSB, that is, the gene is destroyed by knocking in or inserting the nucleic acid encoding CAR.

[0164] In some embodiments, the CAR gene can be introduced into cells through a variety of techniques, including lentiviral or retroviral vectors, transposon systems, CRISPR-Cas9, or TALEN-mediated gene knock-in.

[0165] The term "chimeric antigen receptor" ("CAR", also known as "artificial T cell receptor", "chimeric T cell receptor" and "chimeric immune receptor") should be understood to refer to an engineered receptor that is transplanted onto an immune cell by an antigen recognition portion. Generally speaking, CAR is composed of an antigen recognition portion specific for a target antigen, a transmembrane domain and an intracellular / cytoplasmic signaling domain of a receptor naturally expressed on immune cells, which are operably connected to each other. "Operably connected" means that each domain is connected to each other so that after the antigen recognition portion is bound to the target antigen, a signal is induced by the intracellular signaling domain to activate cells (e.g., T cells or NK cells) expressing CAR and to activate its effector function.

[0166] The antigen recognition portion of the CAR is the extracellular portion of the receptor that recognizes and binds to the epitope of the target antigen. The antigen recognition portion is typically, but not limited to, an scFv.

[0167] The intracellular domain of CAR can include the primary cytoplasmic signaling sequence of the naturally occurring receptor of immune cells and / or the secondary or costimulatory sequence of the naturally occurring receptor of immune cells.The example of primary cytoplasmic signaling sequence includes those derived from TCRζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3 epsilon, CD5, CD22, CD79a, CD79b and CD66d. In some embodiments, the intracellular signaling domain of CAR includes the cytoplasmic signaling sequence of CD3-ζ. In some embodiments, the intracellular signaling domain of CAR can include the cytoplasmic signaling sequence of CD3-ζ combined with the costimulatory signaling sequence of costimulatory molecules. The example of suitable costimulatory molecules includes CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, TIM3, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, etc. In some embodiments, the cytoplasmic domain of the CAR is designed to contain the signaling domain of CD3-ζ and the signaling domain of CD28.

[0168] The membrane spanning domain of CAR is typically a typical hydrophobic alpha helix, which spans the membrane and can be derived from any membrane-bound protein or transmembrane protein. The membrane spanning domain can be derived from natural sources or synthetic sources. In the case where the source is natural, the domain can be derived from any membrane-bound protein or transmembrane protein. For example, the membrane spanning region can be derived from α, β or ζ chains of T cell receptors, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, or from immunoglobulins such as IgG4. Alternatively, the membrane spaning domain can be synthetic, in which case it mainly comprises hydrophobic residues, such as leucine and valine.

[0169] The term "target antigen" should be understood to refer to any protein or non-protein molecule expressed by a cell that is intended to be targeted by an immune cell (e.g., a T cell or NK cell) expressing a receptor. The target antigen can be a "self" molecule (a molecule expressed in the patient's body) or a non-self molecule (e.g., from an infected microorganism). The target antigen referred to herein is not limited to molecules that are naturally capable of eliciting a T or B cell immune response; on the contrary, "target antigen" refers to any protein or non-protein molecule intended to be targeted. In some embodiments, the target antigen is expressed on the surface of a cell. It should be understood that the target antigen can be expressed only by target cells, or it can be expressed by non-target cells. In some embodiments, the target antigen is a non-self molecule or a molecule expressed only by cells intended to be targeted or by cells intended to be targeted at a level significantly higher than that of normal cells. Non-limiting examples of target antigens include the following: differentiation antigens (e.g., MART-1 / MelanA (MART-I), gp100 (Pmel 17), tyrosinase, TRP-1, TRP-2) and tumor-specific multi-lineage antigens (e.g., MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, p15); overexpressed glycoproteins, such as MUC1 and MUC16; overexpressed embryonic antigens, such as CEA; overexpressed oncogenes and mutated tumor suppressor genes, such as p53, Ras, HER-2 / neu; unique tumor antigens generated by chromosomal translocations; for example, BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR; and viral antigens, such as the Epstein-Barr virus antigen EBVA and the human papillomavirus (HPV) antigens E6 and E7.Other tumor-associated antigens include folate receptor α (FRα), EGFR, CD47, CD24, TSP-180, MAGE-4, MAGE-5, MAGE-6, RAGE, NY-ESO, p1 85erbB2, p180erbB-3, cMet, nm-23H1, PSA, CA19-9, CAM 17.1, NuMa, K-ras, β-catenin, CDK4, Mum-1, p15, p16, 43-9F, 5T4, 791Tgp72, α-fetoprotein, β-HCG, BCA225, BTAA, CA125, CA 15-3\CA27.29\BCAA, CA 195, CA 242, CA-50, CAM43, CD68, P1, CO-029, FGF-5, G250, Ga733, EpCAM, HTgp-175, M344, MA-50, MG7-Ag, NB / 70K, NY-CO-1, RCAS 1, SDCCAG16, TA-90, Mac-2 binding protein, cyclophilin C-related protein, TAAL6, TAG-72, TLP, TPS, PSMA, mesothelin, or BCMA.

[0170] In some embodiments, the target antigen is a tumor-associated antigen, particularly a protein, glycoprotein, or non-protein tumor-associated antigen.

[0171] In some embodiments, the target antigen is selected from CD47, folate receptor alpha (FRα), and BCMA.

[0172] In some embodiments, the target antigen is a tumor-associated antigen, such as the tumor-associated antigen TAG-72.

[0173] In other embodiments, the target antigen is a surface protein, such as CD24, and in another embodiment, a surface protein useful for tumor targeting, such as CD19 or CD20.

[0174] Pharmaceutical compositions and therapeutic uses of modified cells

[0175] In another aspect, provided herein are compositions comprising cells produced by the methods disclosed herein, ie, modified cells in which the function of one or more selected genes has been inhibited.

[0176] In some embodiments, provided herein is a pharmaceutical composition, which comprises the cell produced herein and a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers include solvents, dispersion media, isotonic agents, etc. The example of a carrier includes oil, water, saline solution, gel, lipid, liposome, resin, porous matrix, preservative, etc., or a combination thereof. In some embodiments, the pharmaceutical composition is prepared and formulated for use to the patient, for example, for adoptive cell therapy, typically in unit dose injectable form (solution, suspension, emulsion). In some embodiments, the pharmaceutical composition can adopt a delivery system of timed release, delayed release, and sustained release.

[0177] In some embodiments, the pharmaceutical composition comprises an amount of cells effective to treat or prevent a disease or condition, e.g., a therapeutically effective amount or a prophylactically effective amount. In some embodiments, the pharmaceutical composition comprises a modified cell disclosed herein in an amount of about 1 million to about 100 billion cells, e.g., at least 1, 5, 10, 25, 50, 100, 200, 300, 400, or 500 million cells, up to about 10, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 100 billion cells.

[0178] In some embodiments, the pharmaceutical composition further comprises another active agent or drug, such as a chemotherapeutic agent.

[0179] In another aspect, provided herein are methods and uses of the modified cells disclosed herein, such as methods and uses in adoptive cell therapy.

[0180] In some embodiments, the methods comprise administering a modified cell disclosed herein, or a composition comprising a modified cell disclosed herein, to a subject having a disease or disorder or at risk of developing a disease or disorder.

[0181] In some embodiments, the disease or condition is a neoplastic condition (i.e., cancer), a microbial or parasitic infection (e.g., HIV, STD, HCV, HBV, CMV, COVID-19, or antibiotic-resistant bacteria), an autoimmune disease (e.g., rheumatoid arthritis (RA), type I diabetes, systemic lupus erythematosus (SLE), inflammatory bowel disease, psoriasis, scleroderma, autoimmune thyroid disease, Grave's disease, Crohn's disease, multiple sclerosis, asthma), organ fibrosis (e.g., heart, lung, liver, etc.), or endometriosis.

[0182] In some embodiments, neoplastic disorders include central nervous system tumors, retinoblastoma, neuroblastoma, pediatric tumors, head and neck cancer (e.g., squamous cell carcinoma), breast and prostate cancer, lung cancer (small cell and non-small cell lung cancer), kidney cancer (e.g., renal cell adenocarcinoma), esophagogastric cancer, hepatocellular carcinoma, pancreaticobiliary tumors (e.g., adenocarcinomas and islet cell tumors), colorectal cancer, cervical and anal cancer, uterine cancer and other reproductive tract cancers, urinary tract cancer (e.g., ureter and bladder), germ cell tumors (e.g., testicular germ cell tumors or ovarian germ cell tumors), ovarian cancer (e.g., ovarian epithelial carcinoma), cancer of unknown primary, human immunodeficiency-associated malignancies (e.g., Kaposi's sarcoma), lymphoma, leukemia, malignant melanoma, sarcoma, endocrine tumors (e.g., thyroid), mesothelioma and other pleural or peritoneal tumors, neuroendocrine tumors, and carcinoid tumors.

[0183] In some embodiments, the methods of the present invention result in the treatment of a condition, i.e., the alleviation or improvement of the condition or any one or more symptoms of the condition, for example, by inhibiting tumor growth and / or metastasis in the context of treating cancer or by reducing viral load and / or spread in the context of treating viral infection. The term "treatment" does not necessarily mean complete recovery. In some embodiments, the methods of the present invention result in the prevention of a condition, i.e., preventing, reducing the risk of developing, or delaying the onset of a condition. Similarly, "prevention" does not necessarily mean that the subject will ultimately not have the condition.

[0184] In some embodiments, the subject (eg, patient) to whom the cells or compositions are administered is a mammal, typically a primate, such as a human.

[0185] In some embodiments, the cells or compositions comprising the cells are administered parenterally.As used herein, the term "parenteral" includes intravenous, intramuscular, subcutaneous, and intraperitoneal administration.

[0186] The desired dose of the modified cells or composition comprising the modified cells can be delivered by a single administration, multiple administrations, or continuous infusion of the composition.Therapeutic or prophylactic efficacy can be monitored by regular assessment of the treated subject.

[0187] In some embodiments, adoptive cell therapy is carried out by autologous transfer. Immune cells (such as T cells) are separated and / or otherwise prepared from a subject to be treated with cell therapy or from a sample derived from such a subject. In some embodiments, immune cells (such as T cells or NK cells) are separated from a subject, modified according to the methods disclosed herein (to inhibit the function of one or more genes) and then administered to the same subject.

[0188] In some embodiments, adoptive cell therapy is performed by allogeneic transfer, wherein cells are isolated and / or otherwise prepared from a donor subject that is different from the subject to be treated with the cell therapy (recipient subject).In some embodiments, the donor and recipient subjects express the same HLA class or supertype. Example

[0189] In the following examples, it has been described but not limited to that, in order to enhance the function of CAR-T cells, T cells, NK cells and derivative cells (such as iNK cells) for tumor treatment, CRISPR / Cas9 gene editing technology is used to eliminate the negative immunomodulators of these immune cells. In the case of T cells containing CAR, cells are first transduced by lentiviral CAR vectors after activation, and then the Cas9 nuclease complex with specially designed guide RNA is transfected into CAR-T cells to ablate immunomodulatory genes (Fig. 1A). In the case of NK-92 cells containing CAR, cells are first transfected with Cas9 nuclease complex with specially designed guide RNA to ablate immunomodulatory genes, and then lentiviral CAR vectors are used for transduction (Fig. 1B). Gene editing efficiency is checked by quantitative analysis based on genomic DNA sequencing. Cytotoxicity and amplification rate are then monitored during the in vitro expansion of cells. In order to evaluate persistence in vivo, CAR- cells (CAR-T cells in the following examples) are adoptively transferred into mouse xenograft tumor models ( Figures 1A-1B ).

[0190] Example 1 - Generation of Second Generation TAG-72 CAR-T Cells

[0191] TAG-72 is a confirmed adenocarcinoma tumor marker and is also the target of CAR-T cells in some solid tumors. The generation of second-generation TAG-72CAR-T cells as described in WO2017 / 088012 is incorporated herein by reference. The TAG-72CAR expression cassette includes a kappa leader sequence as a signal peptide, an anti-TAG-72scFv as a tumor antigen binding portion, a hinge region and a transmembrane region from human CD8, and a cytoplasmic activation signal transduction domain of 4-1BB and CD3ζ. P2A is a signal sequence for instructing proteolytic cleavage, which releases EGFP as a fluorescent reporter protein expressed by CAR (Fig. 2A). Therefore, after lentiviral transduction, GFP flow cytometry can be used to detect CAR transduction efficiency and expression level (Fig. 2B) in T cells.

[0192] Human T cell isolation and culture

[0193] Primary human T cells were isolated from fresh whole blood or buffy coats obtained from the Australian Red Cross Blood Service from healthy human donors (substandard / discarded material was not suitable for clinical purposes). All patients and healthy donors provided informed consent. The cells were centrifuged using Leucosep® (GE Healthcare, Illinois, USA) using Ficoll-Paque® according to the manufacturer's instructions. TM Peripheral blood mononuclear cells (PBMCs) were isolated using 4% paraformaldehyde (PNA) tubes (Greiner, Kremsmünster, Austria). PBMCs were cryopreserved before use. For transduction and transfection, PBMCs were thawed and used Human T-activator CD3 / CD28 beads (Thermofisher, Massachusetts, USA) separate and activate T cells. Cells and beads are incubated at room temperature in a ratio of 1:3 for 1 hour while continuing gentle mixing. Unbound cells are then removed by placing the cell-bead suspension on a magnet for 1-2 minutes. The supernatant is removed and the cell-bead mixture is incubated at 37°C 5% CO2 in T cell culture medium: TexMACS culture medium (Miltenyi Biotech, Bergisch Gladbach, Germany) with 5% human AB serum (Sigma-Aldrich, Missouri, USA) and 100U / mL IL-2 for approximately 65 hours. T cells are collected by mixing 20-50x dissociation cell-bead complexes, immediately placed on a magnet for 1-2 minutes and the supernatant containing the cells is collected. In MUSE TM The isolated T cell suspension was counted on a cell counter (Merck-Millipore, MA, USA) and prepared for transfection.

[0194] Lentiviral transduction

[0195] Activated human CD3+ T cells were transduced using lentiviral CAR vectors as described in WO2017 / 088012, incorporated herein by reference. To generate lentiviral CAR-T cells, activated human CD3+ / CD28+ T cells were transduced into (Takara Bio Inc)-coated plates were incubated with lentiviral particles for 48 h.

[0196] Flow cytometry of CAR expression.

[0197] To detect the expression of CAR constructs in lentiviral-transduced CAR-T cells, Flow cytometry analysis was performed on a Flow Cytometry Analyzer 10 (Miltenyi Biotec, Bergisch Gladbach, Germany). GFP expression was analyzed. Propidium iodide solution (Miltenyi Biotec) or Viobility 405 / 520 dye was used to distinguish live from dead cells.

[0198] Example 2 - Generation of gene-edited TAG-72 CAR-T cells using CRISPR

[0199] To generate CRISPR knockout (KO) CAR-T cells, 48 ​​hours after lentiviral TAG-72CAR transduction on day 5, representative guide RNAs (PD1 KO, SEQ ID NO: 1; RC3H1 KO, SEQ ID NO: 2; RC3H2 KO, SEQ ID NO: 4; A2AR KO, SEQ ID NO: 7; FAS KO, ​​SEQ ID NO: 9; TGBFBR1 KO, SEQ ID NO: 11; TGFBR2 KO, SEQ ID NO: 14) were transfected into T cells (Figures 1A to 3). Although electroporation-induced cell death occurred, the use of the protocol disclosed herein ( Figure 3 ) CAR-T cells transfected with RNP and untransfected CAR-T cells can be recovered and amplified. Four days after RNP transfection, the genomic DNA of CAR-T cells was extracted for quantitative analysis of gene editing. The gene editing efficiency was analyzed using ICE (Inference of CRISPR Edits) assay (Hsiau et al., Inference of CRISPR Edits from Sanger Trace Data.bioRxiv, 2018, 10.1101 / 251082 (251082)). This article shows the representative results of RC3H2 gene editing efficiency analysis as an ICE assay ( Figure 4A 4C). RC3H2 gRNA (SEQ ID NO: 4) showed high activity in introducing indels (total indel frequency = 92%) into early exons of the RC3H2 gene. In addition, it caused a high frequency of frameshifts in the open reading frame (out-of-frame indel frequency = 91%), thereby disrupting the translation of functional RC3H2 protein ( Figure 4Band 4C). In this study, for all CRISPR gene-edited CAR-T cells, very high gene editing efficiency (total indel percentage = 89% to 96%) and efficient gene knockout results (out-of-frame indel frequency = 61% to 91%) were achieved (Figure 4D). Taken together, these results indicate that the gRNA used in the study has been verified to have high activity in disrupting the expression of the corresponding gene in CAR-T cells without interfering with the in vitro expansion of CAR-T cells.

[0200] CRISPR gene editing of CAR-T cells

[0201] Two days after lentiviral TAG-72CAR transduction, T cells were washed with dPBS for Cas9 RNP transfection. CrRNA and tracrRNA (Synthego or IDT) were annealed to form full-length guide RNA. Cas9 RNP was prepared by incubating Cas9 protein with full-length gRNA for 10 to 20 minutes at room temperature before transfection at a ratio of 1:2. To transfect Cas9 RNP, T cells were electroporated with a Neon transfection device (Thermofisher) or a 4D-Nucleofector device (Lonza, Basel, Switzerland).

[0202] Quantitative assessment of genome editing

[0203] The efficacy and mutation spectrum of CRISPR / Cas9 genome editing efficiency were analyzed by ICE assay (Hsiau et al., Inference of CRISPR Edits from Sanger Trace Data.bioRxiv, 2018, 10.1101 / 251082 (251082)). According to the manufacturer's instructions, genomic DNA was extracted from cells using ISOLATE II genomic DNA kit (Bioline) 4 days after electroporation. PCR amplicons spanning gRNA genomic target sites were generated using high-fidelity Taq polymerase (New England Biolabs). Purified PCR products were subjected to Sanger sequencing, and sequence chromatograms were analyzed using online available ICE software.

[0204] Example 3 - In vitro function of CRISPR gene-edited TAG-72 CAR-T cells

[0205] During the expansion phase of gene-edited TAG-72CAR-T cells, prior to in vivo evaluation, Real-time assay to evaluate the tumor killing ability of cells. As described in Examples 1 and 2, gene-edited TAG-72 CAR-T cells were generated and validated.

[0206] T cell cytotoxicity assay in vitro

[0207] Using a real-time cell monitoring system To determine the killing efficiency of CAR-T cells in vitro. 10,000 target cells / 100 μL (such as ovarian cancer cell line OVCAR-3) are resuspended in a culture medium (such as RPMI-1640 basal medium) supplemented with 10%-20% fetal bovine serum and bovine insulin and deposited into an RTCA plate. The target cells are kept at 37 ° C, 5% CO2 for 3-20 hours to allow cell attachment. After the target cells attach, TAG-72CAR-T effector cells are added with various target ratios of 1: 5 to 5: 1. In some cases, effector cells are separated by FACS based on GFP expression before use. In parallel, untransfected T cells are co-cultured with target cells to demonstrate the background functionality of T cells in vitro. All co-cultures are kept under optimal growth conditions for at least 20 hours. Throughout the process, cell impedance is monitored; a decrease in impedance indicates cell detachment and eventual cell death.

[0208] To compare the initial ability of gene-edited lentiviral TAG-72 CAR-T cells to lyse tumor cells, tumor cells with high or low TAG-72 expression were incubated with gene-edited CAR-T cells or other negative control effector T cells (untransfected) and the cells were expressed by In vitro cytotoxicity was monitored. All of these gene-edited TAG-72CAR-T cells killed TAG-72 high tumor cells (OVCAR-3) as efficiently as TAG-72CAR-T cells (Figures 5A, 5C, 5E, and 5G), while no lysis of TAG-72 low tumor cells (MES-OV) was observed (Figures 5B, 5D, 5F, and 5H). These results indicate that the gene-edited TAG-72CAR-T cells generated using the CRISPR program disclosed herein retain the tumor killing ability and specificity of TAG-72CAR-T cells.

[0209] Example 4 - In vivo function of CRISPR gene-edited TAG-72 CAR-T cells

[0210] Recent studies have shown that TAG-72CAR-T cells can reduce ovarian tumor burden in vivo, but cannot continuously prevent tumor recurrence (Murad, JP et al., Effective Targeting of TAG72(+) Peritoneal Ovarian Tumors via Regional Delivery of CAR-Engineered T Cells. Front Immunol, 2018, 9: p. 2268). The efficacy of TAG-72CAR-T cells generated and validated as described in Examples 1, 2, and 3 was evaluated in an in vivo mouse solid tumor (xenograft) model. For this model, approximately 1x10 7 Human TAG-72-positive OVCAR-3 cancer cells are injected subcutaneously into the flanks of 6- to 10-week-old mice. Human tumor cell lines are grown on the flanks of NSG mice. Within 7 to 9 weeks, fully formed 150-200 mm tumors develop at the injection site. 3 Once the tumors reached this volume, the groups were randomized for treatment. CAR-T cells with different edited genes were administered intravenously to mice for a total of 2 injections of 5x10 6 T cells / injection. Tumor volume, body weight, and clinical scores were monitored after CAR-T cell infusion. Tumors with a size of 800 mm were culled according to animal ethics approval. 3 Up to 1000mm 3 In this ovarian cancer tumor model, treatment with second-generation TAG-72 CAR-T cells initially reduced tumor size, but tumor recurrence was observed approximately 30 days after CAR-T cell administration ( Figure 6 ). Gene-edited TAG-72CAR-T cells were generated according to the methods described in Examples 1 and 2, and in vivo efficacy was evaluated in the same model. PD-1 gene knockout TAG-72CAR-T cells did not improve the anti-tumor activity or persistence of TAG-72CAR-T cells ( Figure 6 ). However, knockout of the RC3H1 and / or RC3H2 genes resulted in significantly improved anti-tumor activity and durability of TAG-72CAR-T therapy. In addition, RC3H1 and RC3H2 double-knockout TAG-72CAR-T cells (TAG-72CAR / RC3H1,2KO T cells) showed the best anti-tumor activity and durability among these groups, as demonstrated by complete prevention of tumor recurrence in TAG-72CAR / RC3H1,2KO T cell-treated mice during the monitoring period ( Figure 7A2AR and FAS gene knockout also improved the anti-tumor efficacy and durability of TAG-72CAR-T therapy, which delayed tumor recurrence in the NSG mouse xenograft model ( Figure 8 CRISPR-directed dominant-negative mutations of TGFβ receptors 1 and 2 also enhanced the persistence of TAG-72CAR-T cells, as demonstrated by more durable control of tumor volume 60 days after CAR-T treatment ( Figure 9 ).

[0211] Example 5 - Generation of RC3H1 and / or RC3H2 Gene-Edited CD19 CAR-T Cells Using CRISPR and In Vivo Function

[0212] CD19 CAR-T cell therapy is the first successful CAR-T treatment approved for B cell malignancies (Porter et al., N Engl J Med, 2011.365 (8): 725-33 pages). In order to verify that the anti-tumor activity of CAR-T cells enhanced by CRISPR gene knockout is not limited to OVCAR-3 tumor models, TAG-72 antigens or TAG-72CAR-T cells, CD19 CAR-T cells with RC3H1 and / or RC3H2 gene knockout are also generated for in vivo functional evaluation. As previously mentioned, construct CD19 scFv-4-1BB-CD3ζCAR expression cassette (Porter et al., N Engl J Med, 2011.365 (8): 725-33 pages; Milone et al., Mol Ther, 2009.17 (8): 1453-64 pages; See also WO2017088012). As described in Example 1, CD19 scFv-4-1BB-CD3ζCAR lentiviral vectors were prepared and transduced into human activated T cells to generate CD19CAR-T cells, and then transfected with RNP complexes formed by RC3H1 gRNA (SEQ ID NO: 2) and / or RC3H2 gRNA (SEQ ID NO: 4) as described in Example 2 to generate CRISPR RC3H1 and / or RC3H2 gene knockout CD19CAR-T cells.

[0213] In vivo cytotoxicity assay of CD19 CAR-T cells

[0214] The in vivo efficacy of T cells was evaluated in a Burkitt lymphoma xenograft model. For this model, 5x10 5 CD19-positive Raji lymphoma cells were injected subcutaneously into the flank of 6- to 10-week-old NSG mice. Three days after tumor inoculation, each mouse was intravenously injected with a single dose of 5 × 10 6CAR-T cells. Tumor volume, body weight, and clinical scores were monitored after CD19 CAR-T cell infusion. Tumors were culled at a size of 800 mm according to animal ethics approval. 3 Up to 1000mm 3 In this lymphoma tumor model, compared with CD19 CAR-T cell treatment, CD19CAR / RC3H1,2KOT cell treatment significantly delayed tumor growth in mice and increased the median survival of tumor-bearing mice ( Figures 10A-10B The results indicate that knockout of the RC3H1 and RC3H2 genes enhanced the in vivo anti-tumor activity of CD19 CAR-T cells, similar to that observed using TAG-72 CAR-T cells.

[0215] Example 6 - Activation markers of CD19 CAR / RC3H1 and / or RC3H2 KO T cells after sustained activation exposure

[0216] RC3H1 and / or RC3H2 knockout CD19 CAR-T cells were generated as described in Example 5.

[0217] Evaluation of CD19 CART cells + Differences in activation markers after antigen exposure between RC3H1 and / or RC3H2 KO ( Figure 11 The engineered CD19 overexpressing cell line OVCAR-3 (CD19) was irradiated (30 Gy) and seeded at 80,000 cells / mL / well in 24-well tissue culture plates. On day 0, 1×10 6 CAR-T cells (with and without RC3H1 and / or RC3H2 KO) were added to each well; these CAR-T cells were then transferred daily to untouched irradiated OVCAR-3 (CD19) cell monolayers over a period of 7 days. After 7 days of continuous antigen exposure, effector cells were washed once by centrifugation and the expression of activation markers CD69 and CD25 was assessed. These markers are associated with early and late activation, respectively, where expression is associated with TCR engagement. To detect the expression of these activation markers on CAR-T cells, a 400 μM ELISA kit was used. Flow cytometry analysis was performed using an analyzer 10. CAR expression was detected indirectly by detecting co-expressed GFP. Cell surface staining for CD69 and CD25 was performed using a standard protocol, in which cells were incubated with fluorescently conjugated antibodies for 15 minutes at 4°C, protected from light. Cells were washed twice with FACS buffer before analysis. Propidium iodide solution was used to distinguish live from dead cells. Flow Logic TM Data analysis was performed using the BIOMEDIC® software (Miltenyi Biotec).

[0218] Following sustained antigen exposure, CD19 CAR / RC3H1 and / or RC3H2 KO T cells lacking one or both genes showed evidence of a higher frequency of CAR+ / CD25+ / CD69+ expressing cells. Although the increase was not statistically significant, this was consistent across all three KO T cells, indicating increased activation compared to untransfected CD19 CAR-T cells. Figure 11 ).

[0219] Example 7 - Generation of RC3H1 and / or RC3H2 KO T cells using CRISPR and in vitro function

[0220] To demonstrate that the method used to generate gene-knockout immune cells is not limited to CAR-T cells, equivalent CRISPR gene knockouts were also performed in normal T cells.

[0221] To generate CRISPR T cells, human T cells were isolated and activated using CD3 / CD28 beads as described in Example 1. Three days after activation and in vitro expansion, activated human T cells were transfected with RNP complexes formed by RC3H1 gRNA (SEQ ID NO: 2) and / or RC3H2 gRNA (SEQ ID NO: 4) as described in Example 2.

[0222] The CRISPR indel frequency and gene knockout efficiency of transfected T cells were analyzed by ICE assay as described in Example 2. The ICE assay results showed that these guide RNAs also showed high activity to introduce indels (including out-of-frame indels) in activated human T cells ( Figure 12 ).

[0223] In vitro killing by prolonged T cell activation ( Figure 13 )

[0224] To determine whether the effect of KO was limited to CAR-T cells, normal T cells were polyclonally activated through their TCR and CD28 co-helper molecules ( Figure 13). RC3H1 and / or RC3H2 KO T cells were maintained at a 1:1 beads to cell ratio for at least 92 hours in the presence of αCD3 / αCD28 beads. Cell counts were performed approximately every 24 hours, with fresh beads added accordingly. After sustained activation, RC3H1 and / or RC3H2 KO T cells showed improved in vitro function compared to untransfected (NT) T cells over a 20-hour monitoring period. Although the differences were not statistically significant, each of the three KO T cells was more effective in killing target tumor cells than untransfected T cells, indicating that prolonged activation of KO T cells may not result in "exhaustion" of the killing function.

[0225] Example 8 - Generation of RC3H1 and / or RC3H2 KO NK-92 cells (with and without CAR) using CRISPR

[0226] In order to prove that the method for generating gene knockout immune cells is not limited to T cells, equivalent CRISPR gene knockout (Fig. 1B) is also performed in NK-92 cells. NK-92 is a natural killer (NK) cell line with high cytotoxicity to cancer targets. NK-92 function (Klingemann et al., Front Immunol, 2016.7: Page 91) can be improved by genetic modification (including CAR expression). NK-92 cell line is maintained and amplified in RPMI-1640 culture medium with 200U / mL IL-2 and fetal bovine serum.

[0227] To generate RC3H1 and RC3H2 knockout NK-92 cells (RC3H1 KO NK-92 cells and RC3H2KO NK-92 cells, respectively), NK-92 cells were transfected with RNP complexes formed by RC3H1 gRNA (SEQ ID NO: 2) and / or RC3H2 gRNA (SEQ ID NO: 4) as described in Example 2. The CRISPR indel frequency and gene knockout efficiency of the transfected NK-92 cells were also analyzed by ICE assay as described in Example 2. The ICE assay results showed that these guide RNAs can also introduce indels (including out-of-frame indels) at a high frequency in NK-92 cells ( Figure 14 ).

[0228] To generate TAG-72CAR / RC3H1 and / or RC3H2 KO NK-92 cells, RC3H1 and / or RC3H2 KO NK-92 cells were transduced with TAG-72CAR lentiviral vectors as described in Example 1.

[0229] Example 9 - In vitro function of RC3H1 and / or RC3H2 KO NK-92 and TAG-72CAR / RC3H KO NK-92 cells

[0230] As described in Example 8, the resulting RC3H1 and / or RC3H2 KO NK-92 cells were transduced using a lentiviral TAG-72 CAR vector. RC3H1 and / or RC3H2 KO NK-92 ± CAR cells were generated in RPMI-1640 supplemented with 10% FBS and 100 U / mL IL-2 L-glutamine and routinely maintained in culture. After at least 3 days of culture, transduction efficiency was assessed by flow cytometry. In addition, the ability of RC3H1 and / or RC3H2 KO NK-92 ± CAR cells to eliminate cancer cells was evaluated in vitro.

[0231] Using a real-time cell monitoring system Determine the in vitro killing efficiency of RC3H1 and / or RC3H2 KO NK-92 cells. Target cells (10,000 target cells / 100uL) (e.g., ovarian cancer cell lines MES-OV or OVCAR-3) are resuspended in a culture medium (e.g., McCoy's 5a or RPMI-1640 basal medium) supplemented with 10%-20% FBS with (OVCAR-3) or without (MES-OV) bovine insulin and are distributed into RTCA plates. Target cells are kept at 37°C, 5% CO2 for at least 5 hours to allow cell attachment. After target cell attachment, RC3H1 and / or RC3H2KO NK-92 effector cells are added at an E:T ratio of 1:1. In parallel, untransfected NK-92 cells are co-cultured with target cells to demonstrate the background functionality of NK-92 cells in vitro. All co-cultures are kept under optimal growth conditions for at least 40 hours. Cell impedance is monitored throughout the process.

[0232] To compare the ability of RC3H1 and / or RC3H2 KO NK-92 cells to lyse tumor cells, tumor cells were incubated with RC3H1 and / or RC3H2 KO NK-92 cells or untransfected NK-92 cells and then Monitor in vitro cytotoxicity.When co-cultured with MES-OV cells, all NK-92 cells (Figure 15 A, left figure) all show inhibitory cell effect.When compared with untransfected NK-92 controls, RC3H2 KO NK-92 cells and RC3H1,2KO NK-92 cells are used to improve the effect, demonstrating the enhancement of in vitro function. In addition, when co-cultured with OVCAR-3 cells, all NK-92 cells (Figure 15 A, right figure) all show cytotoxic effect, as demonstrated by NCI reduction. Compared with untransfected NK-92 cell conditions, RC2H2KO NK-92 cells and RC2H1 / 2KO NK-92 cells are used to improve the effect respectively, demonstrating the enhancement of in vitro function.

[0233] Similar determinations were performed using TAG-72CAR NK-92 cells. In order to confirm that TAG-72CAR NK-92 cells were transduced into RC3H1 and / or RC3H2 KO NK-92 cells, flow analysis (as described in Example 1) was performed, wherein GFP was used as a surrogate for integration and expression of CAR (Figure 15B). Values ​​represent CAR (GFP)%, expressed as a percentage of living cells, excluding fragments and doublets in the parental gate.

[0234] To compare the ability of RC3H1 and / or RC3H2 knockout TAG-72CAR-NK-92 cells to lyse tumor cells, cancer cell lines (in this case OVCAR-3) were co-cultured with TAG-72CAR NK-92 cells + RC3H1 and / or RC3H2 KO and the cells were expressed by In vitro cytotoxicity was monitored. When co-cultured with OVCAR-3 cells, all NK-92 cells with TAG-72CAR ( Figure 15C ) all had cytotoxic effects, as demonstrated by a plateau or decrease in NCI. Within 40 hours of co-culture, the effects were significantly greater using TAG-72CAR / RC3H1 KO NK-92 cells, TAG-72CAR / RC3H2 KO NK-92 cells, and TAG-72CAR / RC3H1,2KO NK-92 cells, demonstrating enhanced in vitro function.

[0235] Example 10 - Generation of gene KO iPSCs using CRISPR

[0236] Stem cells (e.g., induced pluripotent stem cells (iPSCs)) can self-renew indefinitely and differentiate into various cell types, including hematopoietic stem cells (HSCs) and immune cells. Immune cells (e.g., T cells and NK cells) have previously been generated from iPSCs for cancer therapy (Themeli et al., Nat Biotechnol, 2013.31(10): p. 928-33; Li et al., Cell Stem Cell, 2018.23(2): p. 181-192e5). Following a similar approach, CRISPR gene knockout T or NK cells can be derived from iPSCs ( Figure 16 ). To generate CRISPR RC3H1 and RC3H2 double knockout (RC3H1,2KO iPSC) and A2AR knockout iPSC (A2AR KO iPSC), the RNP complex formed by representative gRNA (RC3H1, SEQ ID NO: 2; RC3H2, SEQ ID NO: 4; A2AR, EQ ID NO: 7) was transfected into iPSC using the Lonza 4D Nucleofector system. First, 12-well plates were coated with laminin-521 (STEMCELL Technologies) in PBS and incubated at 37°C for 2 hours. Before transfection, iPSCs were incubated with a RevitaCell TM mTeSRPlus supplement (Life Technologies) TM Pre-incubate with culture medium (STEMCELL Technologies) for 2 hours. RNPs were prepared by combining full-length gRNA with Lonza P3 buffer and Cas-9. The RNP mixture was then incubated at room temperature for 10-20 minutes. After pre-incubation, iPSCs were isolated as single cells (Life Technologies), and 1 x 10 cells were obtained per reaction. 6 To generate knockout iPSCs, cells and RNP mixture in Lonza P3 buffer were combined into PCR tubes and then loaded into the Lonza 4D Nucleofector for electroporation. TM mTeSR Plus culture medium (STEMCELL Technologies) TM Add to the reaction and incubate at room temperature for 10 minutes. After incubation, cells were added to TM mTeSR Plus culture medium TM Laminin-521 pre-coated plates were used.TM Daily medium changes were performed for 72 hours, and cells were passaged after reaching approximately 80% confluence (6-7 days after electroporation).After transfection, RC3H1,2KO iPSC and A2ARKO iPSC colonies with pluripotent stem cell-like morphology were maintained in culture (Figures 17A and 21A).

[0237] Untransfected and transfected iPSCs were cultured in mTeSR Plus TM were cultured on laminin-521 and used Bright field microscopy was used to image the image at 10x magnification. The cells were lifted and collected as single cells. They were then stained using antibodies targeting TRA-1-60 (Miltenyi Biotec), TRA-1-81 (STEMCELL Technologies) and SSEA-4 (Miltenyi Biotec) as recommended by the manufacturer. TRA-1-60, TRA-1-81 and SSEA-4 are surface receptors expressed on pluripotent stem cells and are a common practice considered for characterizing iPSCs (Baghbaderani et al. 2015, Stem Cell Reports). Cells, along with unstained samples and appropriate isotype controls, were analyzed by flow cytometry (Miltenyi Biotec). Dead cells (by PI staining), debris, and doublets were excluded; flow cytometry was performed using FlowLogic TM Generate a histogram ( Figures 17A-17B , RC3H1,2KO and Figures 21A-21B , A2AR KO). iPSCs with or without KO showed nearly identical TRA-1-60, TRA-1-81, and SSEA-4 pluripotency markers, with all markers co-expressed at >95% (Figures 17B and 21B). There were no visual differences in iPSC morphology (Figures 17A and 21A), indicating that RC3H1 and RC3H2 double KO or A2AR KO had no negative impact on iPSC maintenance and pluripotency.

[0238] As described in Example 2, the CRISPR indel frequency and gene knockout efficiency of transfected iPSCs were analyzed by ICE assay. The ICE assay results showed that gRNAs generated indels (including out-of-frame indels) at a high frequency in iPSCs ( Figures 18A-18C , RC3H1,2KO and Figures 22A-22C , A2AR KO).

[0239] In summary, our data demonstrate that the gene-edited iPSCs described herein can be differentiated into CD34+ / HE / HSCs and then into immune cells (e.g., NK, NKT, or T cells) using known methods for subsequent cancer therapy.

[0240] Example 11 - Differentiation of RC3H1 and RC3H2 KO (RC3H1,2KO) iPSCs into iNK cells (edited iNK cells).

[0241] iCD34+ cells

[0242] Receptor CD34 is expressed on HE and HSC, and it is the stem cell source that forms a platform to produce immune cells. The differentiation of iPSC to CD34+ cells is a prerequisite and necessary condition for being able to produce iPSC-derived immune cells (Sturgeon et al. Nature Biotechnology, 2014 Vol. 32 (6) pp. 554-561, Knorr et al. STEM CELLS Translational Medicine vol 2 (4) pp. 274-283, Zeng et al., Stem Cell Reports, 2017 Vol. 9 (6) pp. 1796-1812). Characterizing CD34+ expression as an intermediate cell type between iPSC and immune cells is considered to be common practice, and is a key step in demonstrating that including gene-KO in iPSC does not destroy any potential differentiation pathways during initial development.

[0243] Following the manufacturer's instructions, use STEMdiff TM Untransfected and transfected iPSCs (containing RC3H1, 2KO) were differentiated into iCD34+ cells using a hematopoietic cell differentiation kit (STEMCELL Technologies). Cells were isolated and stained using an antibody targeting CD34 (Miltenyi Biotec) according to the manufacturer's recommendations. Cells were analyzed by flow cytometry (Miltenyi Biotec) using unstained samples and appropriate isotype controls. Dead cells (by PI staining), debris, and doublets were excluded; FlowLogic TM Perform data analysis.

[0244] iPSCs containing or not containing RC3H1,2KO ( Figure 19 These data demonstrate the successful generation of iCD34+ cells from RC3H1,2KO iPSCs and suggest that the key developmental pathways required for the conversion of iPSCs through all intermediate phenotypes into a cell population containing CD34-expressing cells remain intact.

[0245] iNK cells

[0246] iPSCs containing RC3H1,2KO can differentiate into iNK immune cells.

[0247] The knockout iCD34+ cells (derived from RC3H1,2KO iPSCs) were further differentiated into iNK cells driven by a combination of cytokines including IL-15, FLT3, and IL-7. iNK cells can be cultured using published methods (e.g., methods described in U.S. Pat. No. 9,260,696B2 (Kaufman, Knorr), Li et al. (Stem Cell, 23 (2018) 181-197)) or using the commercially available culture system StemSpan TM NK cell generation kit (Stem Cell Technologies) was used for preparation.

[0248] Differentiated cells were isolated and stained using antibodies targeting CD56 (Miltenyi Biotec), NKp46 (Miltenyi Biotec), and NKG2D (Miltenyi Biotec) according to the manufacturer's recommendations. TM Differentiated cells were analyzed by flow cytometry (Miltenyi Biotec) using unstained samples and appropriate isotype controls. Dead cells (by PI staining), debris, and doublets were excluded using FlowLogic TM Perform data analysis ( Figure 20 The expression of NK functional receptors (NKp46 and NKG2D) supports the NK-specific cytotoxic function of iCD56+ cells.

[0249] Example 12 - Differentiation of A2AR KO iPSCs into iNK cells (edited iNK cells).

[0250] iCD34+ cells

[0251] Following the manufacturer's instructions, use STEMdiff TM Untransfected and transfected iPSCs (containing RC3H1, 2KO) were differentiated into iCD34+ cells using a hematopoietic cell differentiation kit (STEMCELL Technologies). Cells were isolated and stained using an antibody targeting CD34 (Miltenyi Biotec) according to the manufacturer's recommendations. Cells were analyzed by flow cytometry (Miltenyi Biotec) using unstained samples and appropriate isotype controls. Dead cells (by PI staining), debris, and doublets were excluded; FlowLogic TM Perform data analysis.

[0252] iPSCs with or without A2AR KO ( Figure 23 These data demonstrate the successful generation of iCD34+ cells from A2AR KO iPSCs and suggest that the key developmental pathways required for the conversion of iPSCs through all intermediate phenotypes into a cell population containing CD34-expressing cells remain intact.

[0253] iNK cells

[0254] iPSCs containing A2AR KO are able to differentiate into iNK immune cells.

[0255] Untransfected iCD34 (derived from untransfected iPSC) and knockout iCD34+ (derived from knockout iPSC) were further differentiated into iNK cells driven by a combination of cytokines including IL-15, FLT3, and IL-7. iNK cells can be cultured using published methods (e.g., methods described in U.S. Patent 9,260,696B2 (Kaufman, Knorr), Li et al. (Stem Cell, 23 (2018) 181-197)) or using the commercially available culture system StemSpan TM NK cell generation kit (Stem Cell Technologies) was used for preparation.

[0256] Differentiated cells were evaluated for expression of NK cell markers by flow cytometry. Dead cells, debris, and doublets were excluded. Figure 24 The CD56+ histograms presented in Figure 2 show all viable cells in cultures generated from untransfected or transfected iPSC samples. Unstained samples are presented to show clear positive staining of each antibody for each corresponding receptor. Appropriate isotype controls are negative. The expression of NK functional receptors (NKp46, NKp30, NKp44, and NKG2D) confirms that iCD56+ cells are iNK cells with cytotoxic function.

[0257] Example 13 - Function of edited iNK cells

[0258] A2AR KO iPSCs were generated (Example 10) and differentiated into edited iNK cells (Example 12). iNK cells were then collected after 20-40 days and used for subsequent functional assays.

[0259] Using a real-time cell monitoring system The ability of iNK cells to kill cancer cells was assessed in vitro. Target cells (10,000 / 100uL) (e.g., ovarian cancer cell line OVCAR-3) were resuspended in a culture medium (e.g., RPMI-1640 and L-glutamine basal medium) supplemented with 10%-20% FBS and bovine insulin and distributed to RTCA plates. Target cells were kept at 37°C, 5% CO2 for at least 5 hours to allow cell attachment. After target cell attachment, iNK effector cells were added at an E:T ratio of 1:1. In parallel, iPSC-derived iNK cells were co-cultured with target cells to demonstrate the background functionality of untransfected iNK cells in vitro. All co-cultures were kept under optimal growth conditions for at least 10 hours. Throughout the process, cell impedance was monitored and presented herein as NCI, with normalization occurring at the time of adding effector cells. The following equation was used to calculate the iNK effector activity after 5 and 10 hours of co-culture. + Percent cytotoxicity (% cytotoxicity) of A2AR KO effector cells (test) relative to target cells alone (control):

[0260] ((Normalized cell index 对照 – Normalized cell index 测试 ) / normalized cell index 对照 )x 100

[0261] To compare the ability of A2AR KO iPSC-derived iNK cells (A2AR KO iNK cells) to lyse tumor cells, tumor cell lines were incubated with A2AR KO iNK cells or NT iNK cells and the cells were cultured by Monitor in vitro cytotoxicity.NT iNK cells show cytotoxic effects when co-cultured with OVCAR-3 target cells (Figure 25 A). Compared with untransfected controls, A2AR KO iNK cells were used to improve the effect, demonstrating the enhancement of in vitro function. In addition, the cytotoxicity after co-culturing 5 hours (Figure 25 B, left figure) and co-culturing 10 hours (Figure 25 B, right figure) in A2AR KO iNK all shows higher cytotoxicity. In short, these data show that, compared with untransfected control cells, A2AR KO can enhance anti-tumor activity not only in T cells but also in iNK cells.

[0262] Throughout this specification, various publications are cited, including patents, patent applications, published patent applications, accession numbers, technical articles, and scholarly articles. Each of these cited publications is incorporated herein by reference in its entirety and for all purposes. Sequence Listing <110> CARTHERICS PTY. LTD. <120> Methods for providing immune cells with enhanced function <130> 37830WO (ND201903) <150> 62 / 938,022 <151> 2019-11-20 <160> 38 <170> PatentIn version 3.5 <210> 1 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Oligonucleotides <400> 1 gtctgggcgg tgctacaact 20 <210> 2 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Oligonucleotides <400> 2 tgcctgtaca agctccacaa 20 <210> 3 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Oligonucleotides <400> 3 gagaggaaat ccgtccattg 20 <210> 4 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Oligonucleotides <400> 4 tgtgaacaac ctaaactgat 20 <210> 5 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Oligonucleotides <400> 5 tgcctgtgca ggcagctcaa 20 <210> 6 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Oligonucleotides <400> 6 agcttccaca atgcctgtgc 20 <210> 7 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Oligonucleotides <400> 7 ctccaccgtg atgtacaccg 20 <210> 8 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Oligonucleotides <400> 8 ctcctcggtg tacatcacgg 20 <210> 9 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Oligonucleotides <400> 9 gtgactgaca tcaactccaa 20 <210> 10 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Oligonucleotides <400> 10 ggagttgatg tcagtcactt 20 <210> 11 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Oligonucleotides <400> 11 ctcgatggtg aatgacagtg 20 <210> 12 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Oligonucleotides <400> 12 ggtgaatgac agtgcggttg 20 <210> 13 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Oligonucleotides <400> 13 ccatcgagtg ccaaatgaag 20 <210> 14 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Oligonucleotides <400> 14 gcttctgctg ccggttaacg 20 <210> 15 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Oligonucleotides <400> 15 ttgaactcag cttctgctgc 20 <210> 16 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Oligonucleotides <400> 16 gcagaagctg agttcaacct 20 <210> 17 <211> 66 <212> DNA <213> Artificial sequence <220> <223> Oligonucleotide, "N" represents any nucleotide <220> <221> misc_feature <222> (37) <223> n is a, c, g, or t <220> <221> misc_feature <222> (43)..(43) <223> n is a, c, g, or t <220> <221> misc_feature <222> (56)..(56) <223> n is a, c, g, or t <220> <221> misc_feature <222> (59)..(60) <223> n is a, c, g, or t <220> <221> misc_feature <222> (66)..(66) <223> n is a, c, g, or t <400> 17 ataatgaatt tgatgagaat gtgcacaaac ccatcangtt aangttgttc acacantgnn 60 tgcaan 66 <210> 18 <211> 66 <212> DNA <213> Artificial sequence <220> <223> Oligonucleotide, "N" represents any nucleotide <400> 18 ataatgaatt tgatgagaat gtgcacaaac ccatcagttt aggttgttca cacactgttt 60 gcaaga 66 <210> 19 <211> 75 <212> DNA <213> Artificial sequence <220> <223> Oligonucleotide, "N" represents any nucleotide <220> <221> misc_feature <222> (26) <223> n is a, c, g, or t <400> 19 tgatgagaat gtgcacaaac ccatcnagtt taggttgttc acacactgtt tgcaagacct 60 gcttgaataa acttc 75 <210> 20 <211> 49 <212> DNA <213> Artificial sequence <220> <223> Oligonucleotide, "N" represents any nucleotide <400> 20 tgatgagaat gtgcaacact gtttgcaaga cctgcttgaa taaacttca 49 <210> twenty one <211> 73 <212> DNA <213> Artificial sequence <220> <223> Oligonucleotide, "N" represents any nucleotide <400> twenty one tgatgagaat gtgcacaaac ccatctttag gttgttcaca cactgtttgc aagacctgct 60 tgaataaact tca 73 <210> twenty two <211> 74 <212> DNA <213> Artificial sequence <220> <223> Oligonucleotide, "N" represents any nucleotide <400> twenty two tgatgagaat gtgcacaaac ccatcgttta ggttgttcac acactgtttg caagacctgc 60 ttgaataaac ttca 74 <210> twenty three <211> 70 <212> DNA <213> Artificial sequence <220> <223> Oligonucleotide, "N" represents any nucleotide <400> twenty three tgatgagaat gtgcacaaac ccatcaggtt gttcacacac tgtttgcaag acctgcttga 60 ataaacttca 70 <210> twenty four <211> 75 <212> DNA <213> Artificial sequence <220> <223> Oligonucleotide, "N" represents any nucleotide <400> twenty four tgatgagaat gtgcacaaac ccatcagttt aggttgttca cacactgttt gcaagacctg 60 cttgaataaa cttca 75 <210> 25 <211> 56 <212> DNA <213> Artificial sequence <220> <223> Oligonucleotide, "N" represents any nucleotide <400> 25 tgatgagaat gtgcacaaac ccacactgtt tgcaagacct gcttgaataa acttca 56 <210> 26 <211> 72 <212> DNA <213> Artificial sequence <220> <223> Oligonucleotide, "N" represents any nucleotide <400> 26 tgatgagaat gtgcacaaac ccatcttagg ttgttcacac actgtttgca agacctgctt 60 gaataaactt ca 72 <210> 27 <211> 65 <212> DNA <213> Artificial sequence <220> <223> Oligonucleotide, "N" represents any nucleotide <400> 27 ttgtaactcc agaagaatgc ctgtacaagc tccaacaagg gacggatttc ccctccggcc 60 cattt 65 <210> 28 <211> 65 <212> DNA <213> Artificial sequence <220> <223> Oligonucleotide, "N" represents any nucleotide <400> 28 ttgtaactcc agaagaatgc ctgtacaagc tccacaatgg acggatttcc tctcctgccc 60 aattt 65 <210> 29 <211> 66 <212> DNA <213> Artificial sequence <220> <223> Oligonucleotide, "N" represents any nucleotide <400> 29 ataatgaatt tgatgagaat gtgcacaaac ccatcaagtt taggttgttc acacactgtt 60 tgcaaa 66 <210> 30 <211> 66 <212> DNA <213> Artificial sequence <220> <223> Oligonucleotide, "N" represents any nucleotide <400> 30 ataatgaatt tgatgagaat gtgcacaaac ccatcagttt aggttgttca cacactgttt 60 gcaaga 66 <210> 31 <211> 67 <212> DNA <213> Artificial sequence <220> <223> Oligonucleotide, "N" represents any nucleotide <400> 31 ggccatgccc atcatgggct cctcggttgt acatcacggt ggagctggcc attgctgtgc 60 tggccat 67 <210> 32 <211> 66 <212> DNA <213> Artificial sequence <220> <223> Oligonucleotide, "N" represents any nucleotide <400> 32 ggccatgccc atcatgggct cctcggtgta catcacggtg gagctggcca ttgctgtgct 60 ggccat 66 <210> 33 <211> 75 <212> DNA <213> Artificial sequence <220> <223> Oligonucleotide, "N" represents any nucleotide <220> <221> misc_feature <222> (26) <223> n is a, c, g, or t <400> 33 atcatgggct cctcggtgta catcancggt ggagctggcc attgctgtgc tggccatcct 60 gggcaatgtg ctggt 75 <210> 34 <211> 63 <212> DNA <213> Artificial sequence <220> <223> Oligonucleotide, "N" represents any nucleotide <400> 34 atcatgggct cctcggtgga gctggccatt gctgtgctgg ccatcctggg caatgtgctg 60 gtg 63 <210> 35 <211> 75 <212> DNA <213> Artificial sequence <220> <223> Oligonucleotide, "N" represents any nucleotide <400> 35 atcatgggct cctcggtgta catcacggtg gagctggcca ttgctgtgct ggccatcctg 60 ggcaatgtgc tggtg 75 <210> 36 <211> 57 <212> DNA <213> Artificial sequence <220> <223> Oligonucleotide, "N" represents any nucleotide <400> 36 atcatgggct cctcggttgg ccattgctgt gctggccatc ctgggcaatg tgctggt 57 <210> 37 <211> 64 <212> DNA <213> Artificial sequence <220> <223> Oligonucleotide, "N" represents any nucleotide <400> 37 atcatgggct cctcggttgg agctggccat tgctgtgctg gccatcctgg gcaatgtgct 60 ggtg 64 <210> 38 <211> 66 <212> DNA <213> Artificial sequence <220> <223> Oligonucleotide, "N" represents any nucleotide <400> 38 atcatgggct cctcggtggt ggagctggcc attgctgtgc tggccatcct gggcaatgtg 60 ctggtg 66

Claims

1. A modified stem cell capable of differentiating into immune cells, wherein the function of at least one gene is inhibited in the modified stem cell, and wherein the at least one gene is A2AR, and the modified stem cell is an induced pluripotent stem cell.

2. The modified stem cell according to claim 1, wherein the function of one or more genes selected from FAS, TGFBR1, and TGFBR2 is further inhibited.

3. The modified stem cell according to claim 1 or 2, wherein the inhibition of the gene function is caused by a decrease in the level or function of the mRNA transcribed from the gene, or by a decrease in the level or activity of the protein encoded by the gene.

4. The modified stem cell according to claim 1 or 2, wherein the inhibition of the gene function is caused by modification of the nucleic acid sequence of the gene.

5. The modified stem cell according to claim 1 or 2, wherein the induced pluripotent stem cell is generated from donor cells homozygous for three HLA genotypes.

6. The modified stem cell according to claim 1 or 2, which further comprises a nucleic acid encoding a chimeric antigen receptor (CAR).

7. The modified stem cell according to claim 6, wherein the CAR comprises an antigen recognition portion directed against a target antigen selected from the group consisting of TAG-72, CD19, CD20, CD24, CD47, tissue factor, folate receptor alpha (FRα), and BCMA.

8. An in vitro method of modifying induced pluripotent stem cells capable of differentiating into immune cells, comprising: modifying the induced pluripotent stem cells to inhibit the function of at least one gene, and wherein the at least one gene is A2AR.

9. The method according to claim 8, which further comprises: modifying the induced pluripotent stem cells to inhibit the function of one or more genes selected from FAS, TGFBR1, and TGFBR2.

10. The method according to claim 8 or 9, which further comprises differentiating the modified induced pluripotent stem cells into immune cells, wherein the function of the at least one gene is inhibited in the immune cells.

11. The method according to claim 8 or 9, wherein the inhibition of the gene function is achieved by a gene editing system.

12. The method according to claim 11, wherein the gene editing system is selected from CRISPR / Cas, TALEN, and ZFN.

13. The method according to claim 12, wherein the gene editing system is a CRISPR / Cas system comprising a guide RNA-nuclease complex.

14. The method according to claim 13, wherein the guide RNA targets a nucleotide sequence selected from the following: SEQ ID NO: 7-8 for A2AR, SEQ ID NO: 9-10 for FAS, SEQ ID NO: 11-13 for TGFBR1, and SEQ ID NO: 14-16 for TGFBR2.

15. The method according to claim 13, wherein the CRISPR / Cas system utilizes a guide RNA-dependent nuclease selected from the group consisting of: Cpf1, Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, Cas100, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3 and Csf4.

16. The method according to claim 8 or 9, wherein the inhibition of gene function is achieved by reducing the level or function of mRNA.

17. The method according to claim 8 or 9, wherein the inhibition of gene function is achieved by reducing the level or function of mRNA by small interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA) or antisense nucleic acid.

18. The method according to claim 8 or 9, wherein the inhibition of gene function is achieved by reducing the level or activity of the protein encoded by the gene.

19. The method according to claim 8 or 9, wherein the inhibition of gene function is achieved by reducing the level or activity of the protein encoded by the gene by virtue of using an antibody or a small molecule.

20. The method according to claim 8 or 9, wherein the immune cells are selected from T cells, NK cells, NKT cells or macrophages.

21. The method according to claim 8 or 9, wherein the modified cells produced by the method further comprise a nucleic acid encoding a chimeric antigen receptor (CAR).

22. The method according to claim 8 or 9, wherein the modified immune cells produced by the method recognize one or more target antigens.

23. The method according to claim 22, wherein the target antigen is selected from TAG-72, CD19, CD20, CD24, CD30, CD47, tissue factor, folate receptor alpha (FRα) and BCMA.

24. Use of the modified cells according to any one of claims 1-7 or the modified cells produced by the method according to any one of claims 8-23 in the preparation of a medicament for treating a disease in a subject, wherein the disease is ovarian cancer or lymphoma.

Images