A method for providing immune cells with enhanced function.

Abstract

This invention provides a method for generating immune cells with enhanced function. [Solution] A method for enhancing the function of immune cells is disclosed herein, comprising 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. A method also disclosed herein, comprising modifying stem cells or progenitor cells that can differentiate 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. The use of immune cells or stem cells produced by this method, and immune cells in therapeutic treatments, is also disclosed herein.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to U.S. Provisional Patent Application No. 62 / 938,022, filed on November 20, 2019, and incorporates its entirety herein.

[0002] Technical fields of this disclosure This disclosure relates to methods for generating immune cells with enhanced function. More specifically, the disclosure herein includes methods for enhancing the function of immune cells, comprising 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. The disclosure herein also includes methods for modifying stem cells or progenitor cells that can differentiate 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. The use of immune cells or stem cells produced by these methods, and immune cells in therapeutic treatments, is also disclosed herein.

[0003] Inclusion by referencing the sequence list The sequence listing in the 10KB ASCII text file named 37830WO_ND201903_SequenceListing.txt, created on November 3, 2020, is incorporated herein by reference. [Background technology]

[0004] T cells expressing chimeric antigen receptors (CAR-T cells) have been shown to be highly effective in killing tumor cells in diseases such as acute lymphoblastic leukemia (ALL) and non-Hodgkin lymphoma (NHL). Approved products targeting the B cell antigen CD19 are generated by introducing CAR gene constructs into patient-derived ("autologous") T cells (Kershaw et al., Gene-engineered T cells for cancer therapy, Nat Rev Cancer, 2013, 13(8):525-41). Additional autologous products targeting other blood cell markers, such as B cell maturation antigen (BCMA) for other hematological malignancies, are under development (Sadelain et al., Therapeutic T cell engineering, Nature, 2017, 545(7655):423-431).

[0005] While the clinical outcomes of CAR-T cell therapy have been impressive in hematological cancers, similar results have not yet been achieved in the treatment of solid tumors. Several reasons contribute to the relative lack of efficacy against solid tumors, including limitations in reaching the tumor site, the immunosuppressive nature of the tumor microenvironment, and the lack of solid tumor-specific target antigens. Furthermore, the lack of persistence and "exhaustion" of administered CAR-T cells are consistently observed limitations (Newick et al., CAR T Cell Therapy for Solid Tumors, Annu Rev Med, 2017, 68:139-152).

[0006] Inhibitory receptors such as CTLA-4, PD-1, or LAG-3 can attenuate CAR-T cell activation and accelerate T cell exhaustion. Disruption of PD-1 by genome editing was predicted to improve the antitumor activity of T cells (Liu et al., CRISPR-Cas9-mediated multiplex gene editing in CAR-T cells, Cell Res, 2017, 27(1):154-157). However, the removal of PD-1 from T cells may increase susceptibility to exhaustion, shorten lifespan, and negate the improved antitumor effect (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, whether gene editing of T cells enhances antitumor activity needs to be evaluated on a case-by-case basis.

[0007] CRISPR / Cas9 is a key component of the bacterial immune system that enables bacteria to remember and destroy bacteriophages. In mammalian cells, CRISPR / Cas9 can be applied to gene editing, as well as other gene editing technologies such as TALENs and ZFNs. The CRISPR system comprises two main components: the Cas9 nuclease and a guide RNA. Specifically, the designed guide RNA forms a complex with the Cas9 nuclease-guided Cas9-gRNA ribonucleoprotein (RNP) complex at user-defined cleavage sites within the human genome. RNP breaks result in double-strand DNA breaks within the genome, which are repaired by an error-prone process called non-homologous end joining (NHEJ). In the NHEJ pathway, nucleotide deletions or insertions ("indels") result in gene disruption or knockout (Addgene, CRISPR 101: A Desktop Resource (2nd Edition), 2017). The on-target efficiency and off-target effects of the guide RNA determine the specificity and safety of CRISPR / Cas9 gene targeting applications. Consequently, specially designed guide RNAs play a crucial role in the success of gene disruption.

[0008] Recent studies using CRISPR have performed genome-wide loss-of-function screening of immunomodulators and found that removing 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 T Cells Reveal Key Regulators of Immune Function. Cell, 2018, 175(7):1958-1971, e15). However, short-term in vitro cytotoxicity limits the induction of effects of gene inhibition or gene deletion on function or lifespan in vivo. The potential effects that gene inhibition may have on the function of immune cells (including T cells, NK cells, NKT cells, etc.), including their activity or lifespan, need to be evaluated more extensively in vitro and in vivo.

[0009] Enhanced immune cells are a potential weapon against cancer, but challenges remain in numerically generating, scaling up, and characterizing immune cell products. Immune cells can be generated from pluripotent stem cells (PSCs). Therefore, pluripotent stem cell technology is theoretically a very promising technology because pluripotent stem cells provide an unlimited regenerative cell source. The ability to directly provide an effectively unlimited supply of enhanced immune cells from stem cells (e.g., induced pluripotent stem cells (iPSCs)), including a wide range of target recognition systems (TCRs / CARs / cytotoxic receptors) capable of responding to multiple pathogens and cancers, represents a significant commercial opportunity. This is therefore also related to understanding the effects of inhibiting specific target genes on the viability, self-renewal, proliferation, and ability of iPSCs to differentiate into immune cells. [Prior art documents] [Non-patent literature]

[0010] [Non-Patent Document 1] Kershaw et al., Gene-engineered T cells for cancer therapy, Nat Rev Cancer, 2013, 13(8):525-41 [Non-Patent Document 2] Sadelain et al.,Therapeutic T cell engineering,Nature,2017,545(7655):423-431 [Non-Patent Document 3] Newick et al.,CAR T Cell Therapy for Solid Tumors,Annu Rev Med,2017,68:139-152 [Non-Patent Document 4] Liu et al.,CRISPR-Cas9-mediated multiplex gene editing in CAR-T cells,Cell Res,2017,27(1):154-157 [Non-Patent Document 5] 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

Non-Patent Document 6

Non-Patent Document 7

Summary of the Invention

Problems to be Solved by the Invention

[0011] In this specification, it has been demonstrated that the inhibition of several genes enhanced the persistence and anti-tumor activity of cytotoxic cells in vivo.

Means for Solving the Problems

[0012] In one aspect, a method of enhancing the function of immune cells is provided herein. The method includes modifying immune cells to inhibit the function of at least one gene (i.e., one or more genes) selected from the group consisting of RC3H1, RC3H2, A2AR, FAS, TGFBR1, and TGFBR2.

[0013] In another aspect, provided herein is a method of modifying stem cells that can differentiate into immune cells. The method includes modifying the stem cells to inhibit the function of at least one gene selected from the group consisting of RC3H1, RC3H2, A2AR, FAS, TGFBR1, and TGFBR2. In some embodiments, the modified stem cells further differentiate into immune cells, and the function of the at least one gene is inhibited within the immune cells.

[0014] In some embodiments, inhibition of the gene function is achieved by reducing the mRNA level or function, optionally via small interfering RNA (siRNA), small hairpin RNA (shRNA), microRNA (miRNA), or antisense nucleic acid.

[0015] In some embodiments, inhibition of the gene function is achieved by reducing the level or activity of the protein encoded by the gene, optionally using an antibody or small molecule.

[0016] In some embodiments, inhibition of the gene function is achieved by a gene editing system. In some embodiments, the gene editing system is selected from the group consisting of CRISPR / Cas, TALEN, and ZFN. In some embodiments, the gene editing system is a CRISPR / Cas system including a guide RNA-nuclease complex. In some embodiments, the guide RNA targets a nucleotide sequence selected from the group consisting of SEQ ID NOs: 2 to 16. In some embodiments, the CRISPR / Cas system is 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 The system utilizes a guide RNA-dependent nuclease selected from the group consisting of 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.

[0017] In some embodiments, immune cells are selected from T cells (including cells such as NKT cells) or NK cells.

[0018] In some embodiments, the modified cells produced by the methods disclosed herein, for example, modified immune cells or modified stem cells, further comprise nucleic acids encoding chimeric antigen receptors (CARs).

[0019] In some embodiments, modified immune cells generated by the methods disclosed herein recognize one or more target antigens. In some embodiments, the target antigens are selected from the group consisting of TAG-72, CD19, CD20, CD24, CD30, CD47, folate receptor alpha (FRα), and BCMA.

[0020] In a further embodiment, immune cells generated by the method disclosed herein are provided herein.

[0021] In another embodiment, modified stem cells generated by the methods disclosed herein are provided herein.

[0022] In one embodiment, modified immune cells are provided herein in which the function of at least one gene is inhibited in the modified immune cells compared to unmodified immune cells, and at least one (i.e., one or more) genes are selected from the group consisting of RC3H1, RC3H2, A2AR, FAS, TGFBR1, and TGFBR2. In some embodiments, the RC3H1 gene is inhibited in the modified immune cells. In some embodiments, the RC3H2 gene is inhibited in the modified immune cells. In some embodiments, the A2AR gene is inhibited in the modified immune cells. In some embodiments, the FAS gene is inhibited in the modified immune cells. In some embodiments, the TGFBR1 gene is inhibited in the modified immune cells. In some embodiments, the TGFBR2 gene is inhibited in the modified immune cells. In some embodiments, multiple genes selected from RC3H1, RC3H2, A2AR, FAS, TGFBR1, and TGFBR2 are inhibited.

[0023] In some embodiments, inhibition of the function of a modified immune cell gene results from a decrease in the level or function of mRNA transcribed from the gene, or a decrease in the level or activity of the protein encoded by the gene.

[0024] In some embodiments, inhibition of gene function is due to alterations in the nucleic acid sequence of the gene.

[0025] In some embodiments, the modified immune cells are selected from T cells (including cells such as NKT cells) or NK cells.

[0026] In some embodiments, the modified immune cells express chimeric antigen receptors (CARs).

[0027] In some embodiments, the modified immune cells recognize one or more target antigens. In some embodiments, the target antigens are selected from the group consisting of TAG-72, CD19, CD20, CD24, CD30, CD47, folate receptor alpha (FRα), and BCMA.

[0028] In another embodiment, modified stem cells capable of differentiating into immune cells are provided herein, comprising modifications to the nucleic acid sequence of at least one gene, wherein the modifications inhibit the function of at least one gene, and the at least one gene is selected from the group consisting of RC3H1, RC3H2, A2AR, FAS, TGFBR1, and TGFBR2.

[0029] In some embodiments, the RC3H1 gene is inhibited in the modified stem cells. In some embodiments, the RC3H2 gene is inhibited in the modified stem cells. In some embodiments, the A2AR gene is inhibited in the modified stem cells. In some embodiments, the FAS gene is inhibited in the modified stem cells. In some embodiments, the TGFBR1 gene is inhibited in the modified stem cells. In some embodiments, the TGFBR2 gene is inhibited in the modified stem cells. In some embodiments, multiple genes selected from RC3H1, RC3H2, A2AR, FAS, TGFBR1, and TGFBR2 are inhibited.

[0030] In some embodiments, the modified stem cells are induced pluripotent stem cells.

[0031] In some embodiments, the modified stem cells contain nucleic acids encoding chimeric antigen receptors (CARs).

[0032] In a further embodiment, compositions for enhancing the function of immune cells are provided herein, comprising a guide RNA-nuclease complex capable of editing the sequence of a target gene, wherein the guide RNA targets a nucleotide sequence selected from the group consisting of SEQ ID NOs: 2 to SEQ ID NOs: 16.

[0033] In some embodiments, the nucleases are 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, It contains at least one protein selected from the group consisting of 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.

[0034] In another embodiment, a method is provided for treating a target condition, comprising administering modified immune cells as disclosed herein to the target. In some embodiments, the condition is cancer, infection, autoimmune disorder, organ fibrosis, or endometriosis.

[0035] This patent or application file includes at least one drawing made in color. A copy of this patent or patent application publication accompanied by the color drawing will be provided by the Patent Office upon request and payment of the required fees. [Brief explanation of the drawing]

[0036] [Figure 1] Figures 1A and 1B illustrate exemplary strategies for evaluating the antitumor activity of modified immune cells. (A) A schematic diagram of a strategy performed for evaluating CAR-T cells, including CRISPR knockout of an immunomodulatory gene, showing a typical timeline of lentiviral CAR transduction, gene targeting, and functional analysis of primary T cells used in the examples. (B) A typical timeline for generating modified NK-92 cells, in which CRISPR knockout of an immunomodulatory gene was followed by lentiviral CAR transduction and functional analysis of NK-92 cells. [Figure 2]Figures 2A and 2B show lentiviral transduction of primary human T cells to generate TAG-72 CAR-T cells. (A) Schematic diagram of the TAG-72-specific CAR construct used in this study. (B) Transduction efficiency of CAR in primary human T cells. Expression was investigated 10 days after transduction with a lentiviral vector. The values ​​embedded in each dot plot represent the frequency of CAR+ events as the percentage of viable single cells. (Representative data from T cells obtained from one donor are shown). [Figure 3] Figure 3 shows the proliferation curve of TAG-72 CAR-T cells after CRISPR / Cas9 RNP transfection (representative data of T cells obtained from one donor). NT: Untransduced T cell; TAG-72 CAR: T cell transduced with TAG-72 CAR; TAG-72 CAR / PD-1 KO T: T cell transduced with TAG-72 CAR and CRISPR / Cas9 RNP targeting PD-1; TAG-72 CAR / A2AR KO T: T cell transduced with TAG-72 CAR and CRISPR / Cas9 RNP targeting A2AR; TAG-72 CAR / FAS KO T: T cell transduced with TAG-72 CAR and CRISPR / Cas9 RNP targeting FAS; TAG-72 CAR / RC3H1 KO T: T cell transduced with TAG-72 CAR and CRISPR / Cas9 RNP targeting RC3H1; TAG-72 CAR / RC3H2 KO T: TAG-72 T cells transduced with a CAR and a CRISPR / Cas9 RNP targeting RC3H2; TAG-72 CAR / TGFBR1 KO T: T cells transduced with a TAG-72 CAR and a CRISPR / Cas9 RNP introducing a dominant-negative mutation into TGFBR1; TAG-72 CAR / TGFBR2 KO T: T cells transduced with a TAG-72 CAR and a CRISPR / Cas9 RNP introducing a dominant-negative mutation into TGFBR2. [Figure 4]Figures 4A to 4D show that transfection with guide RNA-forming RNPs introduces insertions and deletions (indels) into the open reading frames of specific genes within CAR-T cells. The frequency of indels was assessed by the Inference of CRISPR Edits (ICE) assay. (A) Sanger sequencing traces from RC3H2 gRNA-transfected CAR-T cells ("edited sample") show heterogeneous bases downstream of the cleavage sites, in contrast to untransfected CAR-T cells ("control sample") (SEQ ID NO: 17 shows 281–346 bp from the edited sample; SEQ ID NO: 18 shows 281–346 bp from the control sample). The black underlined region in the control sample represents the guide sequence, and the horizontal red dotted underlined region represents the associated PAM (Protospacer Adjacent Motif) site. The vertical black dotted lines on both traces represent the cleavage sites. (B) Relative percentage of contribution of each edited sequence (normalized) in genomic DNA derived from RC3H2 RNP-transfected CAR-T cells. Sequences 19, 20, 21, 22, 23, 24, 25, and 26 are shown from top to bottom. (C) Distribution of indel sizes in the entire edited population of RC3H2 RNP-transfected CAR-T cells. Out-of-frame indel percentage is the percentage of indels that exhibit frameshift or are longer than 21 bp. The R² value calculated by the Pearson correlation coefficient indicates the confidence level of the indel percentage. (D) Summary of ICE assay results for RNP-transfected CAR-T cells. The representative guide RNAs used in this study formed RNP complexes (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; TGBFBR1, SEQ ID NO: 11; TGFBR2, SEQ ID NO: 14; representative T cell data obtained from one donor). [Figure 5]Figures 5A to 5H show that gene knockout TAG-72 CAR-T cells mediate potent cell killing of TAG-72hi-expressing target cells (OVCAR-3 cell line) (Figures 5A, 5C, 5E, and 5G), but do not mediate potent cell killing of TAG-72-neg / low-cancer target cells (MES-OV cell line) (Figures 5B, 5D, 5F, and 5H). After target cells were attached to plates overnight, CAR-T cells were added in an effector-to-target ratio of 1:1. Untransduced T cells (NTs) were included in the killing assay as a control. Cell impedance (mean ± SD, expressed as normalized cell index (NCI)) was monitored for 20 hours. Target cell proliferation under normal proliferation conditions ("target cells only") was also monitored throughout. (Representative data for T cells obtained from one donor, performed in a technical triplicate, are shown). CAR-T (Figures 5A-5H): TAG-72 CAR-T cells; PD-1 (Figures 5A-5B): PD-1 knockout TAG-72 CAR-T cells; RC3H1 (Figures 5C and 5D): RC3H1 knockout TAG-72 CAR-T cells; RC3H2 (Figures 5C and 5D): RC3H2 knockout TAG-72 CAR-T cells; A2AR (Figures 5E and 5F): A2AR knockout TAG-72 CAR-T cells; FAS (Figures 5E and 5F): FAS knockout TAG-72 CAR-T cells; TGFBR1 (Figures 5G and 5H): TGFBR1 dominant-negative TAG-72 CAR-T cells; TGFBR2 (Figures 5G and 5H): TGFBR2 dominant-negative TAG-72 CAR-T cells. [Figure 6]Figure 6 shows the 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 1 × 10⁷ OVCAR-3 tumor cells (TAG-72 positive). When the tumors grew to approximately 150-200 mm³, two doses of 5 × 10⁶ T cells were adopted by intravenous injection at 5-day intervals. Values ​​and error bars represent the mean tumor size (mm³ ± SEM). NT: Untransduced T cells; TAG-72 CAR-T: T cells transduced to TAG-72 CAR; TAG-72 CAR / PD-1 KO T: PD-1 gene knockout TAG-72 CAR-T cells; Mean ± SEM; Representative data of T cells obtained from one donor is shown. [Figure 7] Figure 7 shows the antitumor activity of RC3H1 and / or RC3H2 gene knockout CAR-T cells in an OVCAR-3 ovarian tumor NSG mouse xenograft model. Four NSG mice per group were subcutaneously administered 1 × 10⁷ OVCAR-3 tumor cells (TAG-72 positive). When the tumors grew to approximately 150–200 mm³, two doses of 5 × 10⁶ T cells were adopted by intravenous injection at 5-day intervals. Values ​​and error bars represent the mean tumor size (mm³ ± SEM). NT: Untransduced T cells; TAG-72 CAR-T: T cells transduced to TAG-72 CAR; TAG-72 CAR / RC3H1 KO T: RC3H1 gene knockout TAG-72 CAR-T cells; TAG-72 CAR / RC3H2 KO T: RC3H2 gene knockout TAG-72 CAR-T cells; TAG-72 CAR / RC3H1, 2 KO T: RC3H1 and RC3H2 double gene knockout TAG-72 CAR-T cells. **p<0.01, mixed-effects analysis using Greisser-Greenhouse correction and Dunnett's multiple comparison one-way ANOVA, comparing the mean of all groups to the TAG-72 CAR-T control group. Representative data of T cells obtained from one donor are shown. [Figure 8]Figure 8 shows the antitumor activity of A2AR and FAS gene knockout CAR-T cells in an OVCAR-3 ovarian tumor NSG mouse xenograft model. Four NSG mice per group were subcutaneously administered 1 × 10⁷ OVCAR-3 tumor cells (TAG-72 positive). When the tumors proliferated to approximately 150–200 mm³, two doses of 5 × 10⁶ T cells were adopted by intravenous injection at 5-day intervals. Values ​​and error bars represent the mean tumor size (mm³ ± SEM). NT: Untransduced T cells; TAG-72 CAR-T: T cells transduced to TAG-72 CAR; TAG-72 CAR / A2AR KO T: A2AR gene knockout TAG-72 CAR-T cells; TAG-72 CAR / FAS KO T: FAS gene knockout TAG-72 CAR-T cells. *p<0.05, **p<0.01, #p<0.001, two-way ANOVA comparing the mean of all groups with the CAR-T control group, followed by Dunnett's multiple comparison study. Representative T cell data obtained from one donor are shown. [Figure 9] Figure 9 shows the antitumor activity of TGFBR1 and TGFBR2 dominant-negative gene mutant CAR-T cells in an OVCAR-3 ovarian tumor NSG mouse xenograft model. Four NSG mice per group were subcutaneously administered 1 × 10⁷ OVCAR-3 tumor cells (TAG-72 positive). When the tumors grew to approximately 150–200 mm³, two doses of 5 × 10⁶ T cells were adopted by intravenous injection at 5-day intervals. Values ​​and error bars represent the mean tumor size (mm³ ± SEM). NT: Untransduced T cells, TAG-72 CAR-T: T cells transduced with TAG-72 CAR, TAG-72 CAR / TGFBR1 KO T: TAG-72 CAR-T cells with TGFBR1 dominant-negative gene knockout, TAG-72 CAR / TGFBR2 KO T: TAG-72 CAR-T cells with TGFBR2 dominant-negative gene knockout. *p<0.05, **p<0.01, ***p<0.001. Two-way ANOVA comparing the mean of all groups with the CAR-T control group, followed by Dunnett's multiple comparison study. Representative data of T cells obtained from one donor are shown. [Figure 10]Figure 10 shows the antitumor activity of CD19 CAR-T cells knocked out by RC3H1 and / or RC3H2 genes 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, mice were treated with a single dose of 5 × 10⁶ CAR-T cells by intravenous injection. (A) Tumor size was monitored for 23 days. Values ​​and error bars represent mean tumor size (mm³ ± SEM). Multiple t-tests with Holm-Sidak correction were performed to compare the RC3H1 and / or RC3H2 gene knockout CD19 CAR-T cell population with untransfected CD19 CAR-T cells. (*p<0.005; **p<0.001) (B) Kaplan-Meier survival curves were analyzed using the Log-rank (Mantel-Cox) test. NT: Non-transduced T cells; CD19 CAR: CD19 CAR-T cells; CD19 CAR / RC3H1 KO: RC3H1 gene knockout CD19 CAR-T cells; CD19 CAR / RC3H2 KO: RC3H2 gene knockout CD19 CAR-T cells; CD19 CAR / RC3H1, 2 KO: RC3H1 and RC3H2 double gene knockout CD19 CAR-T cells. Representative data of T cells obtained from one donor is shown. [Figure 11] Figure 11 shows the expression of activation markers on CD19 CAR-T cells with or without RC3H1 and / or RC3H2 gene knockouts after continuous activation exposure. The graph shows the expression of activation markers CD25 and CD69 on CAR+ cells after 7 days of antigen exposure. CD19 CAR-T cells were generated from a single healthy donor. Results represent the mean ± SD of two technical series. [Figure 12]Figure 12 shows CRISPR knockout analysis of the RC3H1 and RC3H2 genes in single and double knockout T cells. Human activated T cells were transfected with RC3H1 and RC3H2 guide RNA-forming RNPs to generate RC3H1 or RC3H2 single knockouts (RC3H1 KO T cells or RC3H2 KO T cells) or RC3H1 and RC3H2 double knockout T cells (RC3H1, 2 KO T cells). Knockout efficiency was analyzed using ICE analysis. Out-of-frame indel percentage refers to the percentage of indels that exhibit a frameshift or are longer than 21 bp. [Figure 13] Figure 13 shows the effect of RC3H1 and / or RC3H2 knockout on the function of CAR-free T cells (CD8+, CD4+). T cells ± RC3H1 and / or RC3H2 knockout were maintained in T cell expansion culture medium for at least 92 hours in the presence of Dynabeads® Human T-Activator CD3 / CD28 beads (Thermofisher, Massachusetts, United States) (DB) at a 1:1 bead-to-cell ratio. The beads were magnetically removed before using the effector cells in the xCELLigence® assay. Effector cells were added to target cancer cells (in this case, OVCAR-3) at a 1:1 effector-to-target (E:T) ratio. NCI was monitored for 20 hours. In all conditions, target cell elimination (observed as a decrease in NCI) was observed. Importantly, after continuous CD3 / CD28-mediated activation, cells genetically deficient in the RC3H1 and / or RC3H2 genes were able to more effectively eliminate target cells in vitro. Results represent the mean ± SEM of the three series of biological and assay results. [Figure 14]Figure 14 shows CRISPR knockout analysis of the RC3H1 and RC3H2 genes in single and double knockout NK-92 cells. NK-92 cells were transfected with RC3H1 and RC3H2 guide RNA-forming RNPs to generate single KOs (RC3H1 KO NK-92 cells or RC3H2 KO NK-92) or double KO NK-92 cells (RC3H1, 2 KO NK-92). Knockout efficiency was analyzed using ICE analysis. Out-of-frame indel percentage refers to the percentage of indels exhibiting frameshift or exceeding 21 bp in length. The R² value, calculated by Pearson correlation coefficient, indicates the confidence level of the indel percentage. [Figure 15]Figure 15 shows the effect of RC3H1 and / or RC3H2 knockout (with and without TAG-72 CAR) on the function of NK-92 cells. The ability of NK cell lines, NK-92 ± RC3H1 KO (green), RC3H2 KO (purple), or RC3H1,2 KO (orange) ± TAG-72 CAR to eliminate cancer cells in vitro was evaluated using the real-time cell monitoring system, xCELLigence®. (A) RC3H1 and / or RC3H2 genes were deleted in NK-92 cell lines using CRISPR / Cas9. Target cancer cells (MES-OV (left panel) or OVCAR-3 (right panel)) were supplemented with the resulting RC3H1 and / or RC3H2 KO NK-92 effector cells in an E:T ratio of 1:1. NCI was monitored for 40 hours. In both conditions, target cell elimination (observed as a decrease in NCI compared to target cells only (blue)) was observed. Results represent the mean ± SEM of a technical triple. (B) Additional genetic manipulation of NK-92 cells was performed to introduce TAG-72 CAR. Lentiviral transduction was performed post-transfection. Transduction efficiency was assessed by flow cytometry approximately 72 hours into culture, where the value embedded in each dot plot represents the percentage of CAR+ cells as the frequency of viable single cells. Fluorescence-activated cell sorting was used to determine the resulting TAG-72 CAR / RC3H1 and / or RC3H2 KO NK-92 cells were isolated and their in vitro function was evaluated as previously described. (C)NCI was monitored for 40 hours. Results represent mean ± SEM, n=1–3. [Figure 16] Figure 16 shows the generation of CRISPR gene knockout induced pluripotent stem cells (iPSCs) as a cell source for adoptive cell therapy. The workflow for inducing gene knockout immune cells from iPSCs involves transfecting iPSCs to knock out the target gene. These cells are then sequenced to characterize and validate the knockout, and subsequently differentiated into CD34+ cells and immune cells. [Figure 17]Figures 17A and 17B demonstrate that RC3H1 and RC3H2 double knockout (RC3H1, 2 KO iPSCs) in iPSCs does not affect pluripotency. This was characterized by (A) a morphology without differentiated cells (scale bar = 200 μm) and (B) flow cytometry analysis for iPSC markers TRA-1-60, TRA-1-81, and SSEA-4. Dead cells, debris, and doublets were gated out so that the histogram plots show all viable cells in cultures from either untransfected iPSC samples or RC3H1, 2 KO iPSC samples. Over 99% of all viable cells express all iPSC markers. [Figure 18] Figures 18A to 18C show that transfection with RC3H1 and RC3H2 guide RNA-forming RNPs introduces insertions and deletions (indels) into the open reading frames of specific genes within iPSCs. Sanger sequencing traces from iPSCs co-transfected with RC3H1 gRNA and RC3H2 gRNA ("edited samples") show heterogeneous bases downstream of the cleavage sites of the RC3H1 gene (A) and RC3H2 gene (B), in contrast to untransfected iPSCs ("control samples") (in A, SEQ ID NO: 27 shows 184–249 bp from the edited sample, and SEQ ID NO: 28 shows 183–248 bp from the control sample; in B, SEQ ID NO: 29 shows 270–336 bp from the edited sample, and SEQ ID NO: 30 shows 272–337 bp from the control sample). In the control sample, the underlined black region represents the guide sequence, and the underlined red dotted horizontal region represents the associated PAM site. The vertical black dotted lines on both traces represent the cleavage sites. (C) CRISPR knockout analysis of iPSCs co-transfected with RC3H1 gRNA and RC3H2 gRNA. ICE analysis was used to evaluate the knockout efficiency of the RC3H1 and RC3H2 genes. The out-of-frame indel percentage is the percentage of indels that exhibit a frameshift or are longer than 21 bp. The R2 value calculated by the Pearson correlation coefficient indicates the confidence level of the indel percentage. [Figure 19]Figure 19 shows that in iPSCs, RC3H1 and RC3H2 double knockouts do not inhibit differentiation into iCD34+ cells. Unstained cells and cells stained with antibodies against CD34+ were analyzed by flow cytometry. Dead cells, debris, and doublets were gated out so that histogram plots show whole-viable CD34+ cells in cultures from either untransfected iPSC samples or RC3H1, 2 knockout iPSC samples. Deletions of both the RC3H1 and RCH32 genes did not hinder iPSC development into a subset of iCD34 cells. [Figure 20] Figure 20 shows that iPSCs containing RC3H1 and RC3H2 double knockouts can differentiate into CD56+ cells expressing NK cytotoxic receptors NKG2D and NKp46. Dead cells, debris, and doublets were gated out so that the CD56+ histogram shows all living cells in the generated culture. The NKp46 and NKG2D plots show CD56+ cells gated off. Unstained controls and isotype controls are shown to show positive staining of each antibody against each respective receptor. Co-expression of NK functional receptors (NKp46 or NKG2D) with CD56 indicates that CD56+ cells derived from RC3H1, 2 KO iPSCs have the potential to perform NK-mediated cytotoxic function. [Figure 21] Figures 21A and 21B show that A2AR knockout does not affect pluripotency in iPSCs. This was characterized by (A) a morphology without differentiated cells (scale bar = 200 μm) and (B) flow cytometry analysis for iPSC markers TRA-1-60, TRA-1-81, and SSEA-4. Dead cells, debris, and doublets were gated out so that the histogram plots show all viable cells in cultures from either untransfected iPSC samples or A2AR KO iPSC samples. Over 95% of all viable cells express all iPSC markers. [Figure 22]Figures 22A to 22C show that transfection with A2AR guide RNA-forming RNPs introduces insertions and deletions (indels) into the open reading frame of the A2AR gene within iPSCs. The frequency of indels was evaluated by ICE analysis. (A) Sanger sequencing traces from A2AR KO iPSCs ("edited samples") show heterogeneous bases downstream of the cleavage sites, in contrast to untransfected iPSCs ("control samples"). Sequence ID 31 shows 134–199 bp from the edited samples; Sequence ID 32 shows 137–202 bp from the control samples. The black underlined region in the control samples represents the guide sequence, and the horizontal red dotted underlined region is the associated PAM site. The vertical black dotted lines on both traces represent the cleavage sites. (B) Relative percentage of contribution of each edited sequence (normalized) in the genomic DNA derived from A2AR KO iPSCs. Sequence numbers 33, 34, 35, 36, 37, and 38 show the sequence from top to bottom. (C) Distribution of indel sizes across the entire edited population of RNP-transfected iPSCs. Out-of-frame indel percentage is the percentage of indels that exhibit frameshift or are longer than 21 bp. The R² value calculated by the Pearson correlation coefficient indicates the confidence level of the indel percentage. [Figure 23] Figure 23 shows that including A2AR KOs in iPSCs does not inhibit their differentiation into iCD34+ cells. Cells stained with antibodies against CD34 were analyzed by flow cytometry. Unstained cells and cells stained with isotype controls were included as controls. Dead cells, debris, and doublets were gated out so that the histogram plots show all living cells in cultures generated from either untransfected iPSC samples or A2AR KO iPSC samples. Including KOs does not inhibit the development of iCD34+ cell subtypes. [Figure 24]Figure 24 shows that A2AR KO iPSCs can differentiate into iNK cells. Unstained cells and cells stained with antibodies against NK cell markers were analyzed by flow cytometry. Dead cells, debris, and doublets were gated out so that the CD56+ histogram shows all living cells in cultures generated from either untransfected iPSC samples or A2AR KO iPSC samples. Unstained samples are shown to clearly show positive staining of each antibody against each respective receptor. Appropriate isotype controls were also performed and were negative. Expression of NK functional receptors (NKp46, NKp30, NKp44, and NKG2D) demonstrates that CD56+ cells derived from A2AR KO iPSCs are iNK cells and potentially possess cytotoxic function. [Figure 25] Figure 25 shows that A2AR KO iPSCs can differentiate into functional iNK cells with enhanced in vitro killing activity. iNK cells were derived from non-transfected iPSCs and A2AR KO iPSCs. The function of the resulting iNK cells was evaluated in vitro using a real-time cell monitoring system (xCELLigence®) with OVCAR-3 cells as the target. A 1:2 effector-to-target ratio was used. (A) Changes in NCI were recorded at 15-minute intervals over at least 10 hours of co-culture, where a decrease in NCI indicates target cell death. (B) Results obtained from (A), shown as the cytotoxicity rate of iNK cells compared to a control of target cells only in both co-cultures at 5 hours (left panel) and 10 hours (right panel). Cells were derived from a single iNK differentiation. Each data point represents technical replication. [Modes for carrying out the invention]

[0037] Throughout this specification, variations of the word “comprise,” “comprises,” or “comprising” shall be understood to mean that they include the elements, integers, or processes, or groups of elements, integers, or processes described, but do not exclude other elements, integers, or processes, or groups of elements, integers, or processes.

[0038] Throughout the specification and claims, the terms “a” and “an” should be interpreted as meaning “at least one,” and not as excluding “two or more” unless otherwise explicitly indicated in the context.

[0039] As used herein, “nucleic acid constructs” generally refer to nucleic acid molecules that are artificially or recombinantly constructed or produced, and are also referred to without distinction as nucleic acid vectors. For example, a nucleic acid construct may contain a target nucleotide sequence that is desired to be transcribed in a cell, and in some examples it may be constructed to produce an RNA molecule with a desired function (e.g., antisense RNA, siRNA, miRNA, or gRNA), and in other examples it may be constructed to produce mRNA that is translated into a target protein (e.g., Cas protein). The target nucleotide sequence in a nucleic acid construct may be operably ligated to a 5' regulatory region (e.g., a promoter such as a heterologous promoter) and / or a 3' regulatory region (e.g., a 3' untranslated region (UTR) such as a heterologous 3'UTR). A nucleic acid construct may be in a circular form (e.g., a plasmid) or a linear form, may be an integrated nucleic acid (i.e., a viral vector such as a lentiviral vector that can be integrated into the chromosome of a host cell), or may remain in an episome (e.g., a plasmid).

[0040] General explanation This specification discloses a method for providing immune cells with enhanced function by inhibiting the function of one or more selected genes. For example, this specification demonstrates that the removal of one or more selected genes using CRISPR / Cas9 gene editing technology enhances the persistence and antitumor activity of cytotoxic lymphocytes in vivo. Thus, the method is provided by inhibiting the function of one or more selected genes in immune cells or stem cells that can differentiate into immune cells. The use of immune cells or stem cells produced by this method, and the use of immune cells in therapeutic procedures, are also disclosed herein.

[0041] immune cells As used herein, “immune cells” should be understood to include cells of the mammalian immune system, such as lymphocytes (T cells, B cells, NK cells, and NKT cells), neutrophils and monocytes (including macrophages and dendritic cells), as well as cell lines derived from cells of the mammalian immune system. Immune cells may be isolated from mammalian subjects, recovered from cultures of cell lines derived from immune cells of mammalian subjects, or generated by differentiation from stem cells.

[0042] This disclosure relates to providing immune cells having 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 invasion compared to control immune cells (i.e., immune cells without modification or manipulation). Cytotoxicity of immune cells generally refers to the ability of immune cells to kill target cells, typically through receptor-based mechanisms.

[0043] In some embodiments, the immune cells are cytotoxic immune cells, such as cytotoxic lymphocytes.

[0044] In some embodiments, the immune cells are T cells. In some embodiments, the T cells are NKT cells. In some embodiments, the immune cells are NK cells.

[0045] References to “T cells” should be understood as references to any cell containing a T cell receptor. In this regard, a T cell receptor may contain one or more of the α, β, γ, or δ chains. As will be understood by those skilled in the art, NKT cells also express T cell receptors, and therefore, target antigen-specific NKT cells can also be generated according to the present invention. The present invention is not intended to be limited to any particular subclass of T cells, but in one embodiment, the target T cell expresses an α / β TCR dimer. In some embodiments, the T cell is a CD4+ helper T cell, a CD8+ killer T cell, or an NKT cell. The present invention is not intended to be limited to any one theory or mode of action, but CD8+ T cells are also known 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 relation to MHC class I molecules. However, CD8+ T cells also provide an additional level of immune surveillance by monitoring and eliminating damaged or abnormal cells, including cancer cells, in addition to simply responding to viral infections. CD8+ T cell recognition of MHC I-presented peptides typically results in either the release of cytotoxic granules or lymphokines, or the activation of an apoptotic pathway via FAS / FASL interactions to destroy target cells. CD4+ T cells, on the other hand, generally recognize peptides presented by antigen-presenting cells in relation to MHC class II, resulting in the release of cytokines designed to modulate the immune response of B cells and / or CD8+ T cells. CD4+ T cells with cytotoxic activity have also been observed in a variety of immune responses. Furthermore, CD4+CAR-T cells exhibited comparable cytotoxicity to CD8+CAR-T cells in vitro and performed better than CD8+CAR-T cells in vivo due to their longer-lasting antitumor activity (see, for example, Wang et al., JCI Insight. 2018;3(10):e99048; Yang et al., Sci Transl Med. 2017 Nov 22;9(417),eaag1209).

[0046] Natural killer T cells (also known as NKT or T / NK cells) are a special population of T cells that express semi-invariant T cell receptors (TCRα-β) and surface antigens typically associated with natural killer cells. The TCRs on NKT cells are unique in that they commonly recognize glycolipid antigens presented by the MHC I-like molecule CD1d. Most NKT cells express the invariant TCR alpha chain and one of a few TCR beta chains. The TCRs present on type I NKT cells generally recognize the antigen alpha-galactosylceramide (alpha-GalCer). Within this group, CD4 + CD8 - cells, CD4 - CD8 + Cells and CD4 - CD8 - Identifiable subpopulations, including T cells, have been identified. Type II NKT cells (or non-invariant NKT cells) express a broader range of TCR α chains and do not recognize the alpha-GalCer antigen. NKT cells produce cytokines that have multiple, often opposing, effects, such as promoting inflammation or inducing immunosuppression, including tolerance. As a result, they can contribute to antimicrobial 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-associated cytotoxicity, FAS-associated cytotoxicity, and TNF-associated cytotoxicity. Therefore, references to T cells should be understood to include references to NKT cells.

[0047] Natural killer (NK) cells are a type of cytotoxic lymphocyte that forms part of the innate immune system. NK cells provide a rapid response to virus-infected cells, acting approximately three days after infection and also responding to tumorigenesis. Typically, immune cells such as T cells detect major histocompatibility complexes (MHC) presented on the surface of infected or transformed cells, triggering cytokine release and leading to lysis or apoptosis of target cells. However, NK cells have the ability to recognize stressed cells in the absence of antibodies or MHC, enabling a much faster immune response. This role is particularly important because harmful cells lacking the MHC I marker cannot be detected and destroyed by other immune cells such as T cells. In contrast to NKT cells, NK cells do not express TCR or CD3, but typically express the surface markers CD16 (FcγRIII) and CD56.

[0048] In some embodiments, immune cells modified or manipulated according to this method can be isolated from mammalian subjects, including, for example, blood (whole blood, serum, or plasma), bone marrow, thymus, and lymph nodes.

[0049] In some embodiments, the immune cells modified or manipulated according to this method can be collected from cell lines derived from mammalian immune cells, such as cultures of T cell lines.

[0050] In some embodiments, immune cells modified or manipulated according to this method can be differentiated from stem cells or other progenitor cells (such as cells cultured and differentiated from stem cells). Methods for differentiating stem cells into immune cells, particularly 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):181-192 e5; 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).

[0051] stem cells As used herein, “source cells” refers to cells that are converted into “induced cells” by reprogramming or differentiation. Examples of source cells suitable for use in the methods disclosed herein include stem cells. Examples of “induced cells” include immune cells such as T cells, NKT cells, and NK cells.

[0052] The term “stem cell” should be understood as a reference to any cell that is capable of self-regeneration and, given its particular phenotype, develops in the direction of multiple lineages, and thus exhibits the potential to form a new organism or regenerate the tissue or cell population of an organism. Stem cells utilized in accordance with the present invention are pluripotent and multipotent, and can differentiate along two or more lineages, and include, but are not limited to, embryonic stem cells (ESCs), adult stem cells, umbilical cord stem cells, hematopoietic stem cells (HSCs), progenitor cells, precursor cells, pluripotent cells, multipotent cells or dedifferentiated cells (such as induced pluripotent stem cells). “Pluripotent” means that the stem cell in question can differentiate to form cells of any one of the three germ layers, which are, among other things, the ectoderm, endoderm, and mesoderm.

[0053] 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 that is the major transplant antigen and is preferably expressed by a significant proportion of the population, e.g., at least 5%, at least 10%, at least 15%, at least 17%, at least 20%, or more of the population. If the homozygous HLA genotype corresponds to a dominant MHC I or MHC II HLA type (with respect to tissue rejection), the use of such cells would significantly reduce the problem of tissue rejection in a relatively broad population administered with the cells of the present invention in the context of a therapeutic regimen. In other embodiments, the source cells may be homozygous with respect to multiple HLA antigens, e.g., two, three, or more HLA antigens. The target HLA antigen can 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.

[0054] In some embodiments, the source cells are homozygous with respect to the inhibited gene.

[0055] In some embodiments, the source cells are genetically modified in one or more genes identified herein such that the function of the modified genes in induced cells differentiated from the genetically modified source cells is inhibited.

[0056] In some embodiments, the source cells are also genetically modified to contain nucleic acids encoding CARs (i.e., chimeric antigen receptors). The nucleic acids encoding CARs can be introduced into the source cells by methods known in the art.

[0057] In some embodiments, the source cells are stem cells. In some embodiments, the source cells are induced pluripotent stem cells (iPSCs).

[0058] In some embodiments, progenitor cells capable of differentiating into immune cells, such as cells cultured from pluripotent stem cells (iPSCs, etc.), which have undergone some differentiation in culture into immune cells but have not fully differentiated into immune cells, are used for modification.

[0059] iPSC iPSCs are typically generated directly from somatic cells. In principle, iPSCs can be derived from any nucleated cell, including, for example, mononucleocytes derived from blood and skin cells. In some embodiments, iPSCs may be generated from fully differentiated T cells or from progenitor T cells, such as thymocytes, which have initiated or even completed the rearrangement of their TCRs and exhibit the desired antigen specificity. In another embodiment, iPSCs are transfected with one or more nucleic acid molecules encoding a TCR (such as a rearranged TCR gene) directed to the desired antigenic determinant (e.g., a tumor antigen determinant). In one embodiment, the iPSCs are derived from cells expressing a rearranged TCR, preferably a rearranged αβ TCR. In another embodiment, the cells express a rearranged γδ TCR. Examples of cells suitable for use in generating iPSCs of the present invention include, but are not limited to, CD4+ T cells, CD8+ T cells, NKT cells, thymocytes, or other forms of progenitor T cells.

[0060] In another embodiment, iPSCs are derived from another type of immune cell, such as NK cells.

[0061] Methods for generating iPSCs from mature or differentiated cells (e.g., T cells or progenitor T cells) are known to those skilled in the art (Themeli, Kloss et al. 2013, Li, Hermanson et al. 2018).

[0062] In some embodiments, the source cells are induced pluripotent stem cells (iPSCs).

[0063] In some embodiments, the source cells are generated from umbilical cord blood PBMCs (peripheral blood mononuclear cells).

[0064] In some embodiments, the target source cells are cells that have further differentiated towards immune cells rather than pluripotent stem cells.

[0065] The induced immune cells produced by the methods disclosed herein include hematopoietic cells that can differentiate into immune cells, and certain types of immune cells. Examples of induced immune cells include HE, pre-HSC, HSC, pluripotent progenitor cells, common lymphoid progenitor cells, early thymic progenitor cells, pre-T cell progenitor cells, pre-NK progenitor cells, T progenitor cells, NK progenitor cells, macrophages, as well as other immune cells such as T cells, NK-T cells, and NK cells.

[0066] This disclosure relates to providing immune cells generated by differentiation or induced immune cells having 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 invasion compared to control immune cells (i.e., immune cells without modification or manipulation). Cytotoxicity of immune cells generally refers to the ability of immune cells to kill target cells, typically through receptor-based mechanisms.

[0067] inhibited genes According to this disclosure, inhibition of the function of one or more genes identified herein may enhance the function of immune cells.

[0068] As used herein, “inhibition of gene function” means that the level and / or activity of the protein encoded by that gene is ultimately reduced or eliminated. Therefore, gene function can be inhibited as a result of manipulation or modification of the gene's genomic DNA sequence (e.g., resulting in gene disruption), as a result of mRNA inhibition (e.g., by inhibiting transcription or translation, thereby reducing the level or function of mRNA), or as a result of protein inhibition (e.g., by reducing the level or activity of a protein). In some embodiments, when comparing the level and / or activity of a gene-encoded protein in a modified cell with the level and / or activity of an unmodified protein in a cell, the degree of inhibition is at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more.

[0069] In some embodiments, the gene whose function is inhibited is selected from the group consisting of RC3H1, RC3H2, A2AR, FAS, TGFBR1, and TGFBR2. In some embodiments, the inhibition targets a single gene selected from the group consisting of RC3H1, RC3H2, A2AR, FAS, TGFBR1, and TGFBR2, for example, a single gene that is RC3H1, RC3H2, A2AR, FAS, TGFBR1, or TGFBR2. In some embodiments, the inhibition targets a single gene selected from the group consisting of RC3H1, RC3H2, A2AR, FAS, TGFBR1, and TGFBR2 in combination with the inhibition of at least another gene. In some embodiments, the inhibition may, in combination with the inhibition of at least one other gene, target two or more genes selected from the group consisting of RC3H1, RC3H2, A2AR, FAS, TGFBR1, and TGFBR2, for example, the RC3H1 gene and the RC3H2 gene, the TGFBR1 gene and the TGFBR2 gene, or the TGFBR1 gene and the RC3H2 gene.

[0070] As described below, members of the gene group consisting of RC3H1, RC3H2, A2AR, FAS, TGFBR1, and TGFBR2 are known in the art to be involved in immune cell function. However, it is not known in the art whether inhibition of the function of these genes, individually or in combination, may have adverse consequences. In particular, complete removal of the function of these genes (e.g., by gene editing) may be predicted to adversely affect important cellular functions, thereby resulting in cells with reduced viability or replication capacity. Furthermore, removal of the function of these genes within stem cells (e.g., iPSCs) may be predicted to adversely affect cellular function, such as viability, self-renewal, pluripotency, the ability to differentiate into specific cell types (e.g., immune cells), and the functional nature of those cell types. It will be recognized by those skilled in the art that maintaining these important cellular functions is a key feature of the present invention.

[0071] RC3H1, RC3H2 RC3H1 is also known as RC3H1, Roquin-1, Ring Finger And CCCH-Type Domains 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 Domains 1, ROQ1, RNF198, or RING Finger Protein 198.

[0072] RC3H2 is also known as Roquin-2, Roquin2, Ring finger and CCCH-type domains 2, Ring finger and CCCH-type zinc finger domain-containing protein 2, Ring finger and CCCH-type zinc finger domains 2, MNAB, ROQ2, RNF164, or RING Finger Protein 164.

[0073] The ROQUIN family of proteins includes ROQUIN1 (encoded by RC3H1) and ROQUIN2 (encoded by RC3H2), which are RNA-binding proteins that play important roles in both the innate and adaptive immune systems (Athanasopoulos, V., RRRamiscal, and CGVinuesa, ROQUIN signalling pathways in innate and adaptive immunity. Eur J Immunol, 2016, 46(5): p.1082-90). In mice (Sanroque mice), Rc3h1 mutations increase ICOS expression in T cells, which leads to 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). In mice, RC3H1 knockout or RC3H2 knockout alone did not develop autoantibodies and lacked autoimmunity, while RC3H1 and RC3H2 double knockout mice exhibited a similar immunophysiological phenotype to San Roque mice. To date, no humans have been found with disease-causing mutations in RC3H1 or RC3H2 (Athanasopoulos, V., RRRamiscal, and CGVinuesa, ROQUIN signalling pathways in innate and adaptive immunity. Eur J Immunol, 2016, 46(5):p.1082-90). The roles of the RC3H1 and RC3H2 genes in human T cells, particularly their function in cytotoxic cells, were unknown prior to this disclosure.

[0074] According to this disclosure, inhibiting the function of either or both of the RC3H1 and RC3H2 genes enhances the function of immune cells.

[0075] A2AR A2AR is also known as ADORA2A, Adenosine A2a Receptor, Adenosine Receptor A2a, ADORA2, Adenosine Receptor Subtype A2a, or RDC8.

[0076] Extracellular adenosine produced by tumor cells is an important immunosuppressive metabolite that limits the activation of cytotoxic lymphocytes and inhibits the anti-tumor immune response via the adenosine 2A receptor (A2AR).

[0077] According to this disclosure, for example, inhibiting the function of the A2AR gene by gene editing (e.g., mediated by CRISPR / Cas9 based on a specifically designed guide RNA) enhances the function of immune cells.

[0078] FAS FAS is also known as Fas cell surface death receptor, APT1, CD95, FAS1, APO-1, FASTM, ALPS1A, or TNFRSF6.

[0079] The FAS receptor (also known as CD95 and APO-1) induces apoptosis and terminal differentiation of cytotoxic T cells. Binding of FAS to its ligand FASL may, in some cases, weaken the antitumor activity of CAR-T cells.

[0080] According to this disclosure, for example, inhibiting the function of the FAS gene by gene editing (e.g., mediated by CRISPR / Cas9) enhances the function of immune cells.

[0081] TGFBR1 and TGFBR2 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-I, TGFR-1, ACVRLK4, tbetaR-I, Transforming Growth Factor Beta Receptor 1, or Transforming Growth Factor Beta Receptor I.

[0082] TGFBR2 is also known as TGFBRII, AAT3, FAA3, LDS2, MFS2, RIIC, LDS1B, LDS2B, TAAD2, TBRII, TBR-ii, TGFR-2, TGF beta-RII, Transforming Growth Factor Beta Receptor 2, or Transforming Growth Factor Beta Receptor II.

[0083] TGF-β exerts systemic immunosuppression, inhibits host immune surveillance, and is considered one of the major factors in the immunosuppressive microenvironment within tumors.

[0084] According to this disclosure, for example, inhibiting the function of the TGFBR1 and / or TGFBR2 genes by gene editing (e.g., mediated by CRISPR / Cas9 based on a specifically designed guide RNA) enhances the function of immune cells.

[0085] According to this disclosure, inhibition of the function of at least one gene selected from the group consisting of RC3H1, RC3H2, A2AR, FAS, TGFBR1, and TGFBR2, in combination with inhibition of at least another gene, enhances the function of immune cells.

[0086] Inhibition of gene function Inhibition of gene function can be achieved by various methods, such as gene editing, such as RNA interference or inhibition of translation via antisense oligonucleotides, or by using compounds such as small molecules or antibodies that directly antagonize protein products.

[0087] Inhibition by gene editing In some embodiments, inhibition of gene function is achieved by using gene editing systems that modify the genomic sequence of the gene.

[0088] Gene editing systems typically involve a DNA-binding protein or DNA-binding nucleic acid conjugated with a nuclease. The DNA-binding protein or DNA-binding nucleic acid specifically binds to or hybridizes to a target region of a gene, and the nuclease results in one or more double-strand breaks and / or one or more single-strand breaks within the target region of the gene. The target region may be the coding region of a gene, for example, within an exon (e.g., within the first or second exon), near the N-terminal portion of the coding region. Double-strand or single-strand breaks can be repaired via cellular repair processes, such as non-homologous end joining (NHEJ) or homologous recombination repair (HDR). In some cases, the repair process introduces insertions, deletions, missense mutations, or frameshift mutations (e.g., including bi-allele frameshift mutations), resulting in gene disruption and inhibition of gene function.

[0089] Examples of gene editing systems include fusions of DNA-binding proteins with nucleases, such as zinc finger nucleases (ZFNs) or TAL effector nucleases (TALENs), or RNA guide nucleases, such as clustered, regularly spaced, short-interval palindromic nucleic acid (CRISPR)-Cas systems.

[0090] ZFP and TALEN In some embodiments, inhibition of gene function is achieved by utilizing a gene editing system that includes a DNA-binding protein, such as one or more zinc finger proteins (ZFPs) or transcription activator-like proteins (TALs) fused to an endonuclease. Examples include ZFNs, TALEs, and TALENs.

[0091] 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 helix region of a naturally occurring zinc finger protein or TALE protein can be modified to facilitate binding to a given DNA sequence. Criteria for reasonable design are described, for example, in U.S. Patent No. 6,140,081, U.S. Patent No. 6,453,242, U.S. Patent No. 6,534,261, International Publication No. 98 / 53058, International Publication No. 98 / 53059, International Publication No. 98 / 53060, International Publication No. 02 / 016536, International Publication No. 03 / 016496, and U.S. Patent Publication No. 20110301073.

[0092] In some embodiments, the DNA-binding protein comprises a zinc finger protein (ZFP), or one or more zinc finger domains of a ZFP. The ZFP or its domains bind to DNA in a sequence-specific manner via one or more "zinc fingers" (regions of amino acids within the binding domain whose structure is stabilized by the coordination of zinc ions). The sequence specificity of naturally occurring ZFPs can be altered by performing amino acid substitutions at specific positions on the zinc finger recognition helix. Furthermore, many engineered gene-specific zinc fingers are commercially available (see, for example, the CompoZr platform for zinc finger construction, developed by Sangamo Biosciences (Richmond, Calif., USA) in collaboration with Sigma-Aldrich (St. Louis, Mo., USA). Therefore, in some embodiments, the ZFP is engineered to bind to a target sequence within a gene identified herein to be inhibited. Typical target sequences include exons, regions near the N-terminus of coding sequences (e.g., the first and second exons), and 5' regulatory regions (promoter or enhancer regions). To form zinc finger nucleases (ZFNs), ZFPs are fused to endonuclease or DNA cleavage domains. Examples of DNA cleavage domains include those of IIS-type restriction enzymes.

[0093] In some embodiments, ZFNs are introduced into cells (e.g., immune cells or stem cells) by transfection with a nucleic acid construct (e.g., plasmid, mRNA, or viral vector) containing the nucleic acid sequence encoding the ZFN. The ZFN is then expressed in the cell from the construct, resulting in editing and disruption of the target gene. In some embodiments, ZFNs are introduced into cells in their protein form.

[0094] In some embodiments, the DNA-binding protein includes a naturally occurring or engineered activator-like protein (TAL) DNA-binding domain, such as an activator-like protein effector (TALE) protein. See, for example, U.S. Patent No. 20110301073, incorporated herein by reference. The TALE DNA-binding domain is a polypeptide comprising one or more TALE repeats, each repeat being 33–35 amino acids long and containing one or two DNA-binding residues. It has been shown that the HD (Histadine-Aspartate) sequences at positions 12 and 13 of the TAL repeat result in binding to cytosine (C), NG (Asparagine-Glycine) to T, NI (Asparagine-Isoleucine) to A, and NN (Asparagine-Asparagine) to G or A. See, for example, U.S. Patent No. 20110301073. In some embodiments, TALE may be designed to have an array of TAL repeats that are specific to a target DNA sequence of interest within a gene identified herein to be inhibited. Specially designed TALE arrays are also commercially available through Cellectis Bioresearch (Paris, France), Transposagen Biopharmaceuticals (Lexington, Ky., USA) and Life Technologies (Grand Island, NY, 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 a gene identified herein to be inhibited.

[0095] In some embodiments, TALENs are introduced into cells (e.g., immune cells or stem cells) by transfection with a nucleic acid construct (e.g., plasmid, mRNA, or viral vector) containing the nucleic acid sequence encoding the TALEN. The TALEN is then expressed in the cell from the construct, resulting in editing and disruption of a target gene. In some embodiments, TALENs are introduced into cells in their protein form.

[0096] CRISPR / CAS In some embodiments, inhibition of gene function is achieved by utilizing the CRISPR ("clustered, regularly spaced, short-palindromic repeats") / Cas ("CRISPR-related nuclease") system for gene editing. CRISPR / Cas is well known in the art using readily available reagents and protocols (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.

[0097] The CRISPR / Cas system generally comprises two elements: (1) an RNA-dependent DNA nuclease, also known herein as a CRISPR endonuclease or Cas protein, e.g., Cas9, Cas12, or other alternative nucleases; and (2) a non-coding short "guide RNA," comprising either a duplex RNA containing crRNA ("CRISPR RNA") and tracrRNA ("transactivating crRNA"), or a single-stranded full-length guide RNA, which contains a targeting sequence that guides the nuclease to a target site in the genome. The guide RNA (gRNA) guides 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 common repair pathways: the non-homologous end joining (NHEJ) pathway and the homologous recombination repair (HDR) pathway. The NHEJ repair pathway is the most active repair mechanism capable of rapidly repairing double-strand breaks (DSBs), but it 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 highly fidelity. When a homologous DNA template is provided to the cleavage region for CRISPR endonucleases, the double-strand break is repaired via HDR using the homologous DNA template. The HDR pathway, along with RNPs, allows for the insertion of large gene insertions into cells.

[0098] In the art, the design or selection of gRNA sequences containing sequences that target a target site within a gene of interest is described. The target site may include sequences in a regulatory region (such as a promoter and an enhancer) or sequences within a coding region (such as an exon, e.g., an exon near the 5' end, or an exon that codes for a specific domain or region of a protein). In some embodiments, the target site is selected based on its position immediately 5' to a PAM sequence, typically such as NGG or NAG.

[0099] 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 induce specific hybridization between the guide sequence and the target sequence and to promote the formation of the CRISPR complex at the target site. In some embodiments, the degree of complementarity between the targeting sequence and the target sequence of the gRNA is at least 80%, 85%, 90%, 95%, 98%, 99%, or higher (e.g., 100% or perfectly complementary).

[0100] In some embodiments, the guide sequence is at least 15 nucleotides long, for example, 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 nucleotides or longer. In some embodiments, the guide sequence is 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, or 20 nucleotides or shorter. In some embodiments, the targeting sequence portion of the guide sequence is approximately 20 nucleotides long. It has been reported that cleaved gRNAs with even shorter target complementarity regions (<20 nucleotides) have improved target specificity and effectiveness (see, for example, Fu et al., Nature Biotechnol., 32(3):279-284, 2014). Therefore, in some embodiments, the targeting sequence of the guide RNA is 17, 18, 19, or 20 nucleotides long. In some embodiments, the targeting sequence of the guide RNA is perfectly complementary to the nucleotide sequence of the target site. In some embodiments where the targeting sequence of the guide RNA is not perfectly complementary to the nucleotide sequence of the target site, the portion of the targeting sequence close to the PAM sequence in the genome (also called the seed region) is perfectly complementary to the nucleotide sequence of the target site. In other words, some variation in the 5' nucleotide of the guide sequence (i.e., the non-seed region) is acceptable. For example, the guide sequence can be designed to include a targeting portion of at least 17 nucleotides long (e.g., 17, 18, 19, or 20 nucleotides long) which has a seed region of at least 17 nucleotides that is perfectly complementary to at least 17 nucleotides in the target sequence.

[0101] Table 1 shows examples of target sequences within specific genes. In some embodiments, the guide sequence includes a targeting sequence of 17–20 nucleotides, and at least 17 nucleotides in the seed region (the 3' portion of the targeting sequence) are perfectly complementary to at least 17 nucleotides in the target sequence, for example, to 17 nucleotides from the 3' end of the target sequence. [Table 1]

[0102] Publicly available gRNA databases for CRISPR genome editing provide exemplary sgRNA target sequences within constitutive exons of genes in the human or mouse genome (see, e.g., GenScript and the gRNA databases provided by the Massachusetts Institute of Technology; also see Sanjana et al. (2014) Nat. Methods, 11:783-4). In some embodiments, the gRNA sequence is or includes a sequence with minimal off-target binding to non-target genes.

[0103] Examples of Cas proteins or CRISPR endonucleases suitable for use in this specification 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, and Cmr4. Examples include Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3 or Csf4 or their functional derivatives (i.e., mutant forms or derivatives, e.g., fragments thereof, of naturally occurring CRISPR endonucleases that substantially retain the RNA-dependent endonuclease activity of the naturally occurring form). See, for example, U.S. Patent No. 20180245091 and U.S. Patent No. 20190247517. In some embodiments, the Cas protein is Cas9, e.g., Cas9 derived from S. pyogenes, S. aureus or S. pneumoniae. In some embodiments, the Cas protein is a Cas9 protein derived from S. pyogenes, having the amino acid sequence provided in the SwissProt database under accession number Q99ZW2.

[0104] In some embodiments, inhibition of gene function is achieved by CRISPR-mediated gene editing, which involves introducing a first nucleic acid encoding a Cas nuclease and a second nucleic acid encoding a guide RNA (gRNA) specific to a target sequence within the gene identified herein to be inhibited, into a cell (e.g., an immune cell or stem cell). The two nucleic acids may be contained in a single nucleic acid construct (or vector) or provided on different constructs (or vectors) to achieve expression of the Cas protein and gRNA in the cell. Intracellular expression of the Cas nuclease and gRNA leads to the formation of a CRISPR complex at the target sequence, which results in DNA cleavage.

[0105] In some embodiments, inhibition of gene function is achieved by CRISPR-mediated gene editing, which involves introducing a combination or complex between gRNA and Cas nuclease into cells. In some embodiments, the Cas protein / gRNA combination or complex may be delivered to cells by, for example, electroporation, particle gun, calcium phosphate transfection, cell compression or squeezing, liposomes, nanoparticles, microinjection, naked DNA plasmid transfer, protein transduction domain-mediated transduction, or virus-mediated transduction (including integrated viral vectors, e.g., retroviruses and lentiviruses, as well as non-integrated viral vectors, e.g., adenoviruses, AAVs, HSVs, and vaccinia).

[0106] Regardless of the specific gene editing method used, various assays may be performed to confirm that the gene sequence has been modified and gene function has been inhibited. These assays may include, for example, examining DNA or mRNA via Southern blotting and Northern blotting, PCR including RT-PCR, or nucleic acid sequencing, or detecting the presence or activity of a specific protein or peptide via immunological means (ELISA and Western blotting).

[0107] In some embodiments, the function of at least one of the RC3H1, RC3H2, A2AR, and FAS genes is inhibited by introducing an indel into the early exon of at least one of these genes via the CRISPR / Cas9 system, thereby resulting in a frameshift mutation in at least one of these genes, preventing the functional protein from being 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 indels into the early exons of two or more of these genes using CRISPR / Cas9, resulting in a frameshift mutation in two or more of these genes, preventing the functional protein from being translated from the edited gene. In some embodiments, two or more of the RC3H1, RC3H2, A2AR, and FAS genes include another gene, for example, RC3H2 in combination with RC3H1.

[0108] In some embodiments, the function of at least one of the TGFBR1 and TGFBR2 genes is inhibited by introducing an indel upstream of the exon and codon related to the starting amino acid residue of at least one of the intracellular signaling domains of these genes via the CRISPR / Cas9 system, resulting in a dominant-negative frameshift mutation that removes the intracellular signaling domain. In some embodiments, the function of both the TGFBR1 and TGFBR2 genes is inhibited by introducing an indel upstream of the exon and codon related to the starting amino acid residue of each of the intracellular signaling domains of these genes using CRISPR / Cas9, resulting in a dominant-negative frameshift mutation that removes the intracellular signaling domain.

[0109] The CRISPR / Cas system can also be used without double-strand breaks or donor DNA by using nickase (i.e., Cas9 nickase) and a high-fidelity enzyme. 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).

[0110] Inhibition by reduction or elimination of mRNA level or function. 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., by mRNA inhibition. Unlike inhibition by gene editing systems, mRNA inhibition is transient.

[0111] In some embodiments, mRNA inhibition can be achieved, for example, by using antisense nucleic acids, ribozymes, small interfering RNA (siRNA), small 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 its precursors.

[0112] Antisense techniques are well-known methods. Antisense RNA is an RNA molecule that is complementary to the full length or a portion of endogenous mRNA and blocks translation from the endogenous mRNA by forming a double helix with it. Antisense RNA can be synthetically produced and introduced into target cells (e.g., immune cells) to achieve inhibition of the expression of the target gene, or it can be produced in target cells via transcription from exogenously introduced nucleic acid constructs. Antisense RNA does not need to be complementary to the full-length mRNA of the target gene. However, antisense RNA should be long enough to form a double helix with the target mRNA and block translation based on the target mRNA. Typically, antisense RNA is at least 15 nucleotides long, 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 longer. In some embodiments, antisense RNA is 500, 400, 300, 200, 100, 75, or 50 nucleotides or less in length. Antisense molecules can also be DNA, DNA analogues, and RNA analogues.

[0113] Ribozymes (i.e., catalytic RNAs) can be designed to specifically pair with target RNA, cleave the phosphodiester backbone at a specific site, and thereby functionally inactivate 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 synthesized and introduced into target cells (e.g., immune cells), or they can be produced in target cells via transcription from exogenously introduced nucleic acid constructs.

[0114] RNA interference (RNAi) – the inhibition of gene expression or translation by RNAi is well known in the art and can be achieved using RNA molecules such as siRNA ("small interfering RNA"), shRNA ("small hairpin RNA"), and miRNA ("microRNA"). siRNA and shRNA are known to be involved in the RNA interference pathway and to interfere with the expression of specific genes. siRNA is a small (typically 20-25 nucleotides long) double-stranded RNA that can be designed to contain a sequence homologous or complementary to the target mRNA (i.e., mRNA transcribed from the gene of interest) or a portion of the target mRNA. shRNA is cleaved by the ribonuclease DICER to produce siRNA. Given the sequence of the target gene, either siRNA or shRNA can be synthetically designed and produced and introduced into the cell of interest (e.g., immune cells), or it can be produced in the cell of interest (e.g., immune cells) from an exogenously introduced nucleic acid construct encoding such RNA. Furthermore, miRNAs are small RNA molecules (generally about 21-22 nucleotides) that are processed from long precursors transcribed from non-protein-coding genes and interfere with translation through inaccurate base pairing with target mRNA. miRNAs or their precursors (pri-miRNAs or pre-miRNAs) can be synthesized and introduced into target cells (e.g., immune cells), or they can be synthesized in target cells (e.g., immune cells) from exogenously introduced nucleic acid constructs encoding either miRNAs or their precursors.

[0115] In some embodiments, mRNA inhibition can be achieved using a modified version of the CRISPR / Cas system in which an enzymatically inactive nuclease Cas molecule is used in combination with a gRNA targeting the gene of interest. The target site may be within the gene's 5' regulatory region (e.g., promoter or enhancer region). In some embodiments, the Cas molecule is an enzymatically inactive Cas9 molecule containing a mutation, such as a point mutation, that eliminates or substantially reduces DNA cleavage activity (see, for example, International Publication No. 2015 / 161276). In some embodiments, the enzymatically inactive Cas9 molecule is fused directly or indirectly to a transcriptional repressor protein.

[0116] Inhibition by other means The present invention includes, for example, other methods known in the art for inhibiting gene function, such as reducing the level or activity of a gene-encoded protein, by introducing a compound (e.g., particularly small molecules, antibodies) that directly inhibits the activity of a gene-encoded protein into a cell (e.g., an immune cell).

[0117] CAR In some embodiments, cells (e.g., immune cells or stem cells) that have been modified to inhibit one or more selected genes are also modified to contain nucleic acids encoding chimeric antigen receptors (or "CARs").

[0118] In some embodiments, the nucleic acid encoding the CAR may be introduced into the cell simultaneously with or following the modification of the cell to inhibit the function of a selected gene. In embodiments where the inhibition is transient (e.g., via antisense RNA or RNAi), the nucleic acid encoding the CAR is preferably introduced into the cell before the cell is modified to achieve the inhibition. In embodiments where the inhibition is permanent (e.g., via gene editing), the nucleic acid encoding the CAR may be introduced into the cell simultaneously with or following the modification of the cell to achieve the inhibition. In some embodiments, the nucleic acid encoding the CAR is designed to allow HDR insertion at the target site of gene editing after the introduction of a DSB, i.e., the gene is disrupted by knock-in or insertion of the nucleic acid encoding the CAR.

[0119] In some embodiments, CAR genes can be introduced into cells via a variety of techniques, including lentiviral or retroviral vectors, transposon systems, CRISPR-Cas9, or TALEN-mediated gene knock-in.

[0120] The term “chimeric antigen receptor” (also known as “artificial T cell receptor,” “chimeric T cell receptor,” and “chimeric immune receptor” or “CAR”) should be understood as a reference to an engineered receptor in which an antigen-recognizing portion is grafted onto an immune cell. Generally speaking, a CAR consists of a target antigen-specific antigen-recognizing portion, a transmembrane domain, and an intracellular / cytoplasmic signaling domain of a receptor naturally expressed on immune cells, all of which are operably linked to one another. “Operatably linked” means that the individual domains are linked to one another so that when the antigen-recognizing portion binds to the target antigen, a signal is induced via the intracellular signaling domain, enabling the activation of the cell expressing the CAR (e.g., a T cell or NK cell) and activating its effector function.

[0121] The antigen-recognition portion of a CAR is the extracellular part of the receptor that recognizes and binds to the epitope of the target antigen. While not limited to a specific group, the antigen-recognition portion is typically scFv.

[0122] The intracellular domain of CAR may include the primary cytoplasmic signaling sequence of a naturally occurring receptor on immune cells, and / or the secondary or co-stimulatory sequence of a naturally occurring receptor on immune cells. Examples of primary cytoplasmic signaling sequences include those derived from TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d. In some embodiments, the intracellular signaling domain of CAR includes a cytoplasmic signaling sequence derived from CD3-zeta. In some embodiments, the intracellular signaling domain of CAR may include a cytoplasmic signaling sequence derived from CD3-zeta in combination with the co-stimulatory signaling sequence of a co-stimulatory molecule. Examples of suitable co-stimulatory molecules include CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, TIM3, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, and B7-H3. In some embodiments, the cytoplasmic domain of the CAR is designed to include a CD3-zeta signaling domain and a CD28 signaling domain.

[0123] The transmembrane domain of a CAR is generally a typical hydrophobic alpha-helix spanning the membrane and can originate from any membrane-bound or transmembrane protein. The transmembrane domain can originate from either a natural or synthetic source. If the source is natural, the domain can originate from any membrane-bound or transmembrane protein. For example, the transmembrane region may originate from the alpha, beta, or zeta chain of a T cell receptor, or from immunoglobulins such as CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, or IgG4. Alternatively, the transmembrane domain may be synthetic, in which case it mainly consists of hydrophobic residues such as leucine and valine.

[0124] The term “target antigen” should be understood as a reference to any protein or non-protein molecule expressed by cells that are to be targeted by receptor-expressing immune cells such as T cells or NK cells. A target antigen may be a “self” molecule (a molecule expressed in the patient’s body) or a non-self molecule (e.g., derived from an infectious microorganism). The target antigens referred to herein are not limited to molecules that can spontaneously trigger an immune response in T cells or B cells. Rather, “target antigen” is a reference to any protein or non-protein molecule that is to be targeted. In some embodiments, the target antigen is expressed on the cell surface. It should be understood that the target antigen may be expressed exclusively by target cells or also by non-target cells. In some embodiments, the target antigen is a non-self molecule, or a molecule expressed exclusively by cells that are to be targeted at significantly higher levels than normal cells, or expressed by cells that are to be targeted. Non-limiting examples of target antigens include: differentiation antigens, e.g., MART-1 / MelanA (MART-I), gplOO (Pmel 17), tyrosinase, TRP-1, TRP-2, and tumor-specific multisystem antigens, e.g., MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, pl5; overexpressed glycoproteins, e.g., MUC1 and MUC16; overexpressed embryonic antigens, e.g., CEA; overexpressed oncogenes and mutant tumor suppressor genes, e.g., p53, Ras, HER-2 / neu; unique tumor antigens arising from chromosomal translocations, e.g., BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR; and viral antigens, e.g., Epstein-Barr virus antigen (EBVA), and human papillomavirus (HPV) antigens E6 and E7.Other tumor-associated antigens include folate receptor alpha (FRα), EGFR, CD47, CD24, TSP-180, MAGE-4, MAGE-5, MAGE-6, RAGE, NY-ESO, pl85erbB2, pl80erbB-3, cMet, nm-23Hl, PSA, CA19-9, CAM17.1, NuMa, K-ras, beta-catenin, CDK4, Mum-1, p15, p16, 43-9F, 5T4, 791Tgp72, alpha-fetoprotein, beta-HCG, BCA225, and BTA. A, CA125, CA15-3\CA27.29\BCAA, CA195, CA242, CA-50, CAM43, CD68\P1, CO-029, FGF-5, G250, Ga733\EpCAM, HTgp-175, M344, MA-50, MG7-Ag, NB / 70K, NY-CO-1, RCAS1, SDCCAG16, TA-90\Mac-2 binding protein\Cyclophyllin C-related protein, TAAL6, TAG-72, TLP, TPS, PSMA, Mesothelin, or BCMA are included.

[0125] In some embodiments, the target antigen is a tumor-associated antigen, particularly a protein-based tumor-associated antigen, a glycoprotein-based tumor-associated antigen, or a non-protein-based tumor-associated antigen.

[0126] In some embodiments, the target antigen is selected from the group consisting of CD47, folate receptor alpha (FRα), and BCMA.

[0127] In some embodiments, the target antigen is a tumor-associated antigen, such as the tumor-associated antigen TAG-72.

[0128] In other embodiments, the target antigen is a surface protein, such as CD24, and in yet another embodiment, it is a surface protein that can be used for tumor targeting, such as CD19 or CD20.

[0129] Pharmaceutical compositions and therapeutic uses of modified cells In a further embodiment, compositions are provided herein that contain cells produced by the methods disclosed herein, i.e., modified cells in which the function of one or more of selected genes is inhibited.

[0130] In some embodiments, pharmaceutical compositions comprising cells generated herein and a pharmaceutically acceptable carrier are provided herein. The pharmaceutically acceptable carrier includes solvents, dispersion media, isotonic agents, and the like. Examples of carriers include oils, water, saline, gels, lipids, liposomes, resins, porous matrices, preservatives, and combinations thereof. In some embodiments, the pharmaceutical composition is prepared and formulated for administration to a patient, for example, for adoptive cell therapy, typically in an injectable form (solution, suspension, emulsion) of a unit dose. In some embodiments, the pharmaceutical composition may be delivered via time-release, delayed-release, and sustained-release systems.

[0131] In some embodiments, the pharmaceutical composition contains an amount of cells effective in treating or preventing a disease or condition, for example, a therapeutically effective amount or a preventively effective amount. In some embodiments, the pharmaceutical composition contains modified cells disclosed herein in amounts ranging from about 1 million to about 100 billion cells, for example, at least 1 million, 5 million, 10 million, 25 million, 50 million, 100 million, 200 million, 300 million, 400 million or 500 million cells, and up to about 1 billion, 5 billion, 10 billion, 20 billion, 30 billion, 40 billion, 50 billion, 60 billion, 70 billion, 80 billion, 90 billion or 100 billion cells.

[0132] In some embodiments, the pharmaceutical composition further comprises another active agent or drug, such as a chemotherapeutic agent.

[0133] In another aspect, methods and uses of modified cells disclosed herein, such as therapeutic methods and uses in adoptive cell therapy, are provided herein.

[0134] In some embodiments, the method involves administering modified cells as disclosed herein, or compositions comprising modified cells as disclosed herein, to a subject who has a disease or condition, or who is at risk of developing a disease or condition.

[0135] In some embodiments, the disease or condition is a neoplasm (i.e., cancer), a microbial or parasitic infection (such as HIV, STD, HCV, HBV, CMV, COVID-19, or antibiotic-resistant bacteria), an autoimmune disease (e.g., rheumatoid arthritis (RA), type 1 diabetes, systemic lupus erythematosus (SLE), inflammatory bowel disease, psoriasis, scleroderma, autoimmune thyroid disease, Graves' disease, Crohn's disease, multiple sclerosis, asthma), an organ fibrosis (e.g., heart, lung, liver, etc.), or endometriosis.

[0136] In some embodiments, neoplastic conditions include central nervous system tumors, retinoblastoma, neuroblastoma, pediatric tumors, head and neck cancers (e.g., squamous cell carcinoma), breast and prostate cancers, lung cancers (both small cell and non-small cell lung cancers), renal cancers (e.g., renal cell adenocarcinoma), esophageal and gastric cancers, hepatocellular carcinomas, pancreatic and biliary duct neoplasms (e.g., adenocarcinoma and islet cell tumors), colorectal cancers, cervical and anal cancers, uterine and other reproductive system cancers, urinary tract cancers (e.g., of the ureters and bladder), germ cell tumors (e.g., testicular germ cell tumors or ovarian germ cell tumors), ovarian cancers (e.g., ovarian epithelial carcinoma), carcinomas of unknown primary human immunodeficiency-associated malignancies (e.g., Kaposi's sarcoma), lymphomas, leukemias, malignant melanomas, sarcomas, endocrine tumors (e.g., of the thyroid), mesotheliomas and other pleural or peritoneal tumors, neuroendocrine tumors, and carcinoid tumors.

[0137] In some embodiments, the method results in the treatment of a disease, i.e., reduction or improvement of the disease, or reduction or improvement of any one or more symptoms of the disease, for example, by inhibiting tumor growth and / or metastasis in the context of treating cancer, or by reducing the amount and / or spread of the virus in the context of treating a viral infection. The term “treatment” does not necessarily mean complete recovery. In some embodiments, the method results in the prevention of a disease, i.e., prevention of the disease, reduction of the risk of developing the disease, or delay of the onset of the disease. Similarly, “prevention” does not necessarily mean that the subject will never ultimately develop the disease.

[0138] In some embodiments, the subject to which the cells or composition are administered, for example, a patient, is a mammal, typically a primate, such as a human.

[0139] In some embodiments, cells, or compositions containing cells, are administered parenterally. As used herein, the term “parenteral” includes intravenous, intramuscular, subcutaneous, and intraperitoneal administration.

[0140] The desired dose of the modified cells, or a composition containing modified cells, may be delivered by a single dose, multiple doses, or continuous infusion. The therapeutic or prophylactic effect can be monitored by periodic evaluation of the treated subject.

[0141] In some embodiments, adoptive cell therapy is performed by autotransfer. Immune cells (such as T cells) are isolated and / or prepared from the subject receiving cell therapy or from a sample derived from such a subject. In some embodiments, immune cells (e.g., T cells or NK cells) are isolated from the subject, modified according to a method disclosed herein (to inhibit the function of one or more genes), and then administered to the same subject.

[0142] In some embodiments, adoptive cell therapy is performed by allogeneic transfer, in which case the cells are isolated and / or prepared from a donor subject different from the subject to receive cell therapy (recipient subject). In some embodiments, the donor subject and recipient subject express the same HLA class or HLA supertype. [Examples]

[0143] The following examples, though not limited to those described, demonstrate the use of CRISPR / Cas9 gene editing technology to eliminate negative immunomodulatory factors in CAR-T cells, T cells, NK cells, and induced cells (e.g., iNK cells) for tumor therapy, in order to enhance the function of these immune cells. For CAR-containing T cells, the cells were first transfected with a lentiviral CAR vector after activation, and then transfected with a Cas9 nuclease complex containing a specifically designed guide RNA to remove the immunomodulatory factor genes (Figure 1A). For CAR-containing NK-92 cells, the cells were first transfected with a Cas9 nuclease complex containing a specifically designed guide RNA to remove the immunomodulatory factor genes, and then transfected using a lentiviral CAR vector (Figure 1B). Gene editing efficiency was investigated by quantification based on genomic DNA sequencing. Cytotoxicity and expansion culture rates were then monitored during in vitro cell proliferation. To evaluate in vivo persistence, CAR cells (CAR-T cells in the following examples) were adopted into a mouse xenograft tumor model (Figures 1A-1B).

[0144] Example 1 - Generation of second-generation TAG-72 CAR-T cells TAG-72 is an established tumor marker for adenocarcinoma and is also a target for CAR-T cells in certain solid tumors. Second-generation TAG-72 CAR-T cells were generated as described in International Publication No. 2017 / 088012, incorporated herein by reference. The TAG-72 CAR expression cassette included a kappa reader sequence as a signal peptide, anti-TAG-72 scFv as a tumor antigen binding moiety, a human CD8-derived hinge and transmembrane region, and cytoplasmic activation signaling domains of 4-1BB and CD3 zeta. P2A is a signal sequence that leads to proteolytic cleavage and releases EGFP as a fluorescent reporter for CAR expression (Figure 2A). Therefore, CAR transduction efficiency and expression levels in T cells could be detected using GFP flow cytometry after lentiviral transduction (Figure 2B).

[0145] Isolation and culture of human T cells Primary human T cells were isolated from healthy human donors, either from fresh whole blood or from buffy coat (unsuitable / discarded material not clinically viable) obtained from the Australian Red Cross Blood Service. All patients and healthy donors provided informed consent. Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll-Paque (GE Healthcare, Illinois, United States) centrifugation using Leucosep® tubes (Greiner, Kremsmunster, Austria) according to the manufacturer's instructions. PBMCs were cryopreserved before use. For transduction and transfection, PBMCs were thawed, and T cells were isolated and activated using Dynabeads® Human T-Activator CD3 / CD28 beads (Thermofisher, Massachusetts, United States). Cells and beads were incubated at a 1:3 ratio for 1 hour at room temperature with continuous, gentle mixing. Next, unbound cells were removed by placing the cell bead suspension on a magnet for 1-2 minutes. The supernatant was removed, and the cell bead mixture was incubated with 5% human AB serum (Sigma-Aldrich, Missouri, United States) and 100 U / mL IL-2 in T cell medium: TexMACS Medium (Miltenyi Biotech, Bergisch Gladbach, Germany) at 37°C and 5% CO2 for approximately 65 hours. T cells were collected by dissociating the cell bead complex by mixing 20-50 times, and the cell-containing supernatant was immediately collected by placing the mixture on a magnet for 1-2 minutes. The isolated T cell suspension was counted using a MUSE® cell counter (Merck-Millipore, Massachusetts, United States) and prepared for transfection.

[0146] Lentiviral phenotype introduction Activated human CD3+ T cells were transduced using a lentiviral CAR vector, as described in International Publication No. 2017 / 088012, incorporated herein by reference. To generate lentiviral CAR-T cells, activated human CD3+ / CD28+ T cells were incubated with lentiviral particles for 48 hours in a RetroNectin® (Takara Bio Inc) coated plate.

[0147] Flow cytometry of CAR expression. Flow cytometry analysis was performed using MACSQuant® Analyzer 10 (Miltenyi Biotec, Bergisch Gladbach, Germany) to detect the expression of CAR constructs in CAR-T cells transduced by lentivirus. GFP expression was analyzed. Live and dead cells were distinguished using propidium iodide solution (Miltenyi Biotec) or Viobility 405 / 520 dye.

[0148] Example 2 - Generation of gene-edited TAG-72 CAR-T cells using CRISPR To generate CRISPR gene knockout (KO) CAR-T cells, RNP complexes formed by 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 48 hours after lentiviral TAG-72 CAR transduction on day 5 (Figures 1A-3). Despite electroporation-induced cell death, RNP-transfected CAR-T cells could be recovered and expanded cultured as untransfected CAR-T cells using the protocol disclosed herein (Figure 3). Four days after RNP transfection, genomic DNA from CAR-T cells was extracted for quantitative analysis of gene editing. Gene editing efficiency was analyzed using the ICE (Inference of CRISPR Edits) assay (Hsiau et al., Inference of CRISPR Edits from Sanger Trace Data. bioRxiv, 2018, 10.1101 / 251082 (251082)). The RC3H2 gene editing efficiency analysis is shown here as a representative result of the ICE assay (Figures 4A-4C). The gRNA (SEQ ID NO: 4) showed high activity in introducing indels into the early exons of the RC3H2 gene (total indel frequency = 92%). Furthermore, this resulted in a high frequency of frameshifts in the open reading frame (out-of-frame indel frequency = 91%), which disrupted the translation of the functional RC3H2 protein (Figures 4B and 4C). In this study, extremely 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 in all CRISPR gene-edited CAR-T cells (Figure 4D). In summary, these results demonstrate that the gRNA used in the study has high activity in disrupting the expression of the corresponding gene within CAR-T cells without disrupting in vitro expansion culture of CAR-T cells.

[0149] CRISPR gene editing in CAR-T cells Two days after lentivirus TAG-72 CAR 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 RNPs were prepared before transfection by incubating Cas9 protein with full-length gRNA in a 1:2 ratio at room temperature for 10–20 minutes. T cells were electroporated using a Neon transfection device (Thermofisher) or a 4D-Nucleofector device (Lonza, Basel, Switzerland) to transfect with the Cas9 RNPs.

[0150] Quantitative evaluation of genome editing The effectiveness and mutation spectrum of CRISPR / Cas9 genome editing were analyzed using the ICE assay (Hsiau et al., Inference of CRISPR Edits from Sanger Trace Data. bioRxiv, 2018, 10.1101 / 251082(251082)). Genomic DNA was extracted from cells 4 days after electroporation using the ISOLATE II Genomic DNA Kit (Bioline) according to the manufacturer's instructions. PCR amplicons spanning gRNA genome target sites were generated using High-Fidelity Taq polymerase (New England Biolabs). Purified PCR products were Sanger sequenced, and sequence chromatograms were analyzed using online available ICE software.

[0151] Example 3 - In vitro function of CRISPR gene-edited TAG-72 CAR-T cells During the expansion culture phase of gene-edited TAG-72 CAR-T cells, the tumor-killing capacity of the cells was evaluated in vitro using the xCELLigence® real-time assay prior to in vivo evaluation. Gene-edited TAG-72 CAR-T cells were generated and validated as described in Examples 1 and 2.

[0152] T cell in vitro cytotoxicity assay The killing efficiency of CAR-T cells was determined in vitro using a real-time cell monitoring system (xCELLigence®). 10,000 target cells / 100 μL (e.g., ovarian cancer cell line OVCAR-3) were resuspended in culture medium supplemented with 10%–20% fetal bovine serum and bovine insulin (e.g., RPMI-1640 basic medium) and placed in RTCA plates. Target cells were maintained at 37°C and 5% CO2 for 3–20 hours to allow cell adhesion. After target cell adhesion, TAG-72 CAR-T effector cells were added in various effector-to-target ratios ranging from 1:5 to 5:1. In some cases, effector cells were isolated based on GFP expression by FACS before use. In parallel, untransfected T cells were co-cultured with target cells to demonstrate the background functionality of T cells in vitro. The entire co-culture was maintained under optimal growth conditions for at least 20 hours. Cell impedance was monitored throughout. A decrease in impedance indicates cell detachment and ultimately cell death.

[0153] 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 in vitro cytotoxicity was monitored by xCELLigence®. All of these gene-edited TAG-72 CAR-T cells killed TAG-72 high tumor cells (OVCAR-3) as efficiently as TAG-72 CAR-T cells (Figure 5A, C, E, and G), but no lysis of TAG-72 low tumor cells (MES-OV) was observed (Figure 5B, D, F, and H). These results demonstrate that gene-edited TAG-72 CAR-T cells generated using the CRISPR procedure disclosed herein retain the tumor-killing ability and specificity of TAG-72 CAR-T cells.

[0154] Example 4 - In vivo function of CRISPR gene-edited TAG-72 CAR-T cells. Recent studies have shown that TAG-72 CAR-T cells could reduce in vivo ovarian tumor burden, but could not persist to 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). TAG-72 CAR-T cells generated and validated as described in Examples 1, 2, and 3 were evaluated for their effects in an in vivo mouse solid tumor (xenograft) model. In this model, approximately 1 × 10⁻⁶ 7 Human tumor cell lines were proliferated in the flanks of NSG mice by subcutaneous injection of human-derived TAG-72-positive OVCAR-3 cancer cells into the flanks of 6-10 week old mice. Within 7-9 weeks, fully formed tumors of 150-200 mm were observed. 3Tumors developed at the injection site. After the tumors reached this volume, the groups were randomized for treatment. CAR-T cells with various edited genes were administered intravenously to mice, with a total of two injections of 5×10 6 individual T cells per injection. After CAR-T cell injection, tumor volume, body weight, and clinical score were monitored. In accordance with animal ethics approval, mice with a tumor size of 800 mm 3 to 1000 mm 3 and significant weight loss or poor clinical score were euthanized. In this ovarian cancer tumor model, second-generation TAG-72 CAR-T cell treatment initially reduced tumor size, but tumor recurrence was observed at approximately 30 days after CAR-T cell administration (Figure 6). Genetically edited TAG-72 CAR-T cells were generated according to the methods described in Examples 1 and 2 and evaluated for in vivo effects in the same model. PD-1 gene knockout TAG-72 CAR-T cells did not improve the antitumor activity or persistence of TAG-72 CAR-T cells (Figure 6). However, knockout of the RC3H1 and / or RC3H2 genes resulted in a significant improvement in the antitumor activity and persistence of TAG-72 CAR-T therapy. Furthermore, RC3H1 and RC3H2 double gene knockout TAG-72 CAR-T cells (TAG-72 CAR / RC3H1,2 KO T cells) showed the best antitumor activity and persistence in these groups, as demonstrated by the complete prevention of tumor recurrence in TAG-72 CAR / RC3H1,2 KO T cell-treated mice over the monitoring period (Figure 7). A2AR and FAS gene knockout also improved the antitumor effect and durability of TAG-72 CAR-T therapy, thereby delaying tumor recurrence in the NSG mouse xenograft model (Figure 8). Dominant negative mutations of TGFβ receptors 1 and 2 by CRISPR also enhanced the persistence of TAG-72 CAR-T cells, as demonstrated by more permanent control of tumor volume starting 60 days after CAR-T treatment (Figure 9).

[0155] Example 5 - Generation of RC3H1 and / or RC3H2 gene-edited CD19 CAR-T cells using CRISPR and in vivo functionality CD19 CAR-T cell therapy was the first successful CAR-T therapy approved for B-cell malignancies (Porter et al., N Engl J Med, 2011. 365(8): p.725-33). To verify that the antitumor activity of CAR-T cells, enhanced by CRISPR gene knockout, is not limited to OVCAR-3 tumor models, CD19 CAR-T cells with TAG-72 antigen or TAG-72 CAR-T cells, and RC3H1 and / or RC3H2 gene knockouts were also generated for in vivo functional evaluation. The CD19 scFv-4-1BB-CD3ζ CAR expression cassette was constructed as previously described (Porter et al., N Engl J Med, 2011.365(8):p.725-33; Milone et al., Mol Ther, 2009.17(8):p.1453-64; see also International Publication No. 2017088012). The CD19 scFv-4-1BB-CD3ζ CAR lentiviral vector was generated and transduced into human activated T cells to generate CD19 CAR-T cells as described in Example 1, and then transfected with an RNP complex formed by RC3H1 gRNA (SEQ ID NO: 2) and / or RC3H2 gRNA (SEQ ID NO: 4) to generate CRISPR RC3H1 and / or RC3H2 gene knockout CD19 CAR-T cells as described in Example 2.

[0156] CD19 CAR-T cell in vivo cytotoxicity assay The in vivo effect of T cells was evaluated in a Burkitt lymphoma xenograft model. In this model, 5 × 10¹⁶ T cells were implanted in the flank of 6-10 week old NSG mice. 5 Individual CD19-positive Raji lymphoma cells were subcutaneously injected. Three days after tumor inoculation, 5 × 10⁶ cells were administered. 6A single dose of CD19 CAR-T cells was administered intravenously to each mouse. Tumor volume, body weight, and clinical score were monitored after CD19 CAR-T cell infusion. In accordance with animal ethical approval, 800 mm 3 ~1000mm 3 Mice with tumor size, significant weight loss, or poor clinical scores were culled. In this lymphoma tumor model, CD19 CAR / RC3H1,2 KO T cell treatment significantly delayed tumor growth in mice and improved median survival time of tumor-bearing mice compared to CD19 CAR-T cell treatment (Figures 10A-10B). These results indicate that knockout of the RC3H1 and RC3H2 genes improves the antitumor activity of CD19 CAR-T cells in vivo, similar to what was observed with TAG-72 CAR-T cells.

[0157] Example 6 - Activation markers of CD19 CAR / RC3H1 and / or RC3H2 KO T cells after continuous activation exposure As described in Example 5, RC3H1 and / or RC3H2 gene knockout CD19 CAR-T cells were generated.

[0158] Differences in activation markers after antigen exposure were evaluated for CD19 CAR T cells ± RC3H1 and / or RC3H2 KO (Figure 11). Manipulated CD19 overexpressing cell lines, OVCAR-3(CD19), were irradiated (30 Gy) and seeded in wells of 80,000 cells / mL / 24-well tissue culture plates. 1 × 10 6Aliquots of CAR-T cells (with and without RC3H1 and / or RC3H2 knockout) were added to each well on day 0. These CAR-T cells were then transferred daily to an untouched monolayer of irradiated OVCAR-3 (CD19) cells for 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 evaluated. These markers are associated with early and late activation, respectively, which are related to TCR ligation. Flow cytometry analysis was performed using MACSQuant® Analyzer 10 to detect the expression of these activation markers on CAR-T cells. CAR expression was indirectly detected by detecting co-expressed GFP. Cells were incubated with fluorescently conjugated antibodies at 4°C for 15 minutes and then stained on the cell surface for CD69 and CD25 using a standard protocol protected from light. Cells were washed twice with FACS buffer before analysis. Live and dead cells were identified using propidium iodide solution. Data analysis was performed using FlowLogic® software (Miltenyi Biotec).

[0159] Following continuous antigen exposure, CD19 CAR / RC3H1 and / or RC3H2 knockout T cells lacking either or both genes showed evidence of a higher frequency of CAR+ / CD25+ / CD69+ expression. Although this increase was not statistically significant, it was consistent across all three knockout T cells and showed increased activation compared to untransfected CD19 CAR-T cells (Figure 11).

[0160] Example 7 - Generation of RC3H1 and / or RC3H2 KO T cells using CRISPR and in vitro functionality To demonstrate that methods for generating gene knockout immune cells are not limited to CAR-T cells, equivalent CRISPR gene knockout was also performed on normal T cells.

[0161] 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, the 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) and expanded in vitro as described in Example 2.

[0162] The CRISPR indel frequency and gene knockout efficiency of transfected T cells were also analyzed by ICE assay as described in Example 2. The results of the ICE assay showed that these guide RNAs exhibited high activity in introducing indels, including out-of-frame indels, into activated human T cells (Figure 12).

[0163] In vitro killing of T cells by long-term activation (Figure 13) To determine whether the KO effect was limited to CAR-T cells, normal T cells were polyclonally activated with their TCR and CD28 co-auxiliary molecules (Figure 13). RC3H1 and / or RC3H2 KO T cells were maintained in the presence of αCD3 / αCD28 beads in a 1:1 bead-to-cell ratio for at least 92 hours. Cell counting was performed approximately every 24 hours, and fresh beads were added accordingly. After continuous activation, RC3H1 and / or RC3H2 KO T cells showed improved function in vitro compared to untransfected (NT) T cells over a 20-hour monitoring period. Although the difference was not statistically significant, each of the three knockout T cells was more efficient at killing target tumor cells than non-transfected T cells, suggesting that long-term activation of knockout T cells may not "exhaust" their killing function.

[0164] Example 8 - Generation of RC3H1 and / or RC3H2 KO NK-92 cells (with and without CAR) using CRISPR To demonstrate that the method for generating gene knockout immune cells is not limited to T cells, equivalent CRISPR gene knockout was also performed on NK-92 cells (Figure 1B). NK-92 is a natural killer (NK) cell line with high cytotoxicity against cancer targets. NK-92 function can be improved by genetic modification, including CAR expression (Klingemann et al., Front Immunol, 2016.7:p.91). NK-92 cell lines were maintained and expanded in RPMI-1640 medium containing 200 U / mL IL-2 and fetal bovine serum.

[0165] To generate RC3H1 and RC3H2 gene knockout NK-92 cells (RC3H1 KO NK-92 cells and RC3H2 KO 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 results of the ICE assay showed that these guide RNAs could introduce indels, including out-of-frame indels, to NK-92 cells at a high frequency (Figure 14).

[0166] To generate TAG-72 CAR / RC3H1 and / or RC3H2 KO NK-92 cells, RC3H1 and / or RC3H2 KO NK-92 cells were transduced using the TAG-72 CAR lentiviral vector as described in Example 1.

[0167] Example 9 - In vitro function of RC3H1 and / or RC3H2 KO NK-92 and TAG-72 CAR / RC3H KO NK-92 cells Using the lentiviral TAG-72 CAR vector, the resulting RC3H1 and / or RC3H2 KO NK-92 cells were transduced as described in Example 8. RC3H1 and / or RC3H2 KO NK-92±CAR cells were generated and maintained adjunctally in RPMI-1640 containing L-glutamine, supplemented with 10% FBS and 100 U / mL IL-2. Transduction efficiency was assessed by flow cytometry at least 3 days after culture. Furthermore, the ability of RC3H1 and / or RC3H2 KO NK-92±CAR cells to eliminate cancer cells was evaluated in vitro.

[0168] The killing efficiency of RC3H1 and / or RC3H2 KO NK-92 cells was determined in vitro using a real-time cell monitoring system (xCELLigence®). Target cells (10,000 target cells / 100uL) (e.g., ovarian cancer cell lines MES-OV or OVCAR-3) were resuspended in culture medium supplemented with 10-20% FBS, either containing (OVCAR-3) or not containing (MES-OV) bovine insulin (e.g., McCoy's 5a or RPMI-1640 basic medium), and dispensed into RTCA plates. Target cells were maintained at 37°C and 5% CO2 for at least 5 hours to allow cell adhesion. After target cell adhesion, RC3H1 and / or RC3H2 KO NK-92 effector cells were added in a 1:1 E:T ratio. In parallel, untransfected NK-92 cells were co-cultured with target cells to demonstrate the background functionality of NK-92 cells in vitro. The entire co-culture was maintained under optimal growth conditions for at least 40 hours. Cellular impedance was monitored throughout.

[0169] 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 in vitro cytotoxicity was monitored by xCELLigence®. All NK-92 cells (Figure 15A, left) showed a cell proliferation inhibitory effect when co-cultured with MES-OV cells. This effect was improved in RC3H2 KO NK-92 cells and RC3H1,2 KO NK-92 cells compared to untransfected NK-92 controls, demonstrating enhanced function in vitro. Furthermore, all NK-92 cells (Figure 15A, right) showed a cytotoxic effect when co-cultured with OVCAR-3 cells, as demonstrated by a reduction in NCI. This effect was improved in RC2H2 KO NK-92 cells and RC2H1 / 2 KO NK-92 cells, respectively, compared to non-transfected NK-92 control conditions, demonstrating enhanced function in vitro.

[0170] A similar assay was performed using TAG-72 CAR NK-92 cells. Flow analysis was performed (as described in Example 1) to confirm the transduction of TAG-72 CAR NK-92 cells into RC3H1 and / or RC3H2 KO NK-92 cells, using GFP as an alternative for CAR integration and expression (Figure 15B). The value represents %CAR(GFP) as the percentage of viable cells, and debris and doublets were excluded by parent gate.

[0171] To compare the ability of RC3H1 and / or RC3H2 gene knockout TAG-72 CAR-NK-92 cells to lyse tumor cells, cancer cell lines (in this case, OVCAR-3) were co-cultured with TAG-72 CAR NK-92 cells ± RC3H1 and / or RC3H2 KO, and in vitro cytotoxicity was monitored by xCELLigence®. All NK-92 cells with TAG-72 CAR (Figure 15C) exhibited cytotoxic effects when co-cultured with OVCAR-3 cells, as demonstrated by a plateau or decrease in NCI. This effect was significantly greater within 40 hours of co-culture in TAG-72 CAR / RC3H1 KO NK-92 cells, TAG-72 CAR / RC3H2 KO NK-92 cells, and TAG-72 CAR / RC3H1,2 KO NK-92 cells, demonstrating enhanced function in vitro.

[0172] Example 10 - Generation of gene knockout iPSCs using CRISPR Stem cells, such as induced pluripotent stem cells (iPSCs), can regenerate indefinitely and differentiate into various cell types, including hematopoietic stem cells (HSCs) and immune cells. Immune cells such as T cells and NK cells have previously been generated from iPSCs for cancer treatment (Themeli et al., Nat Biotechnol, 2013.31(10):p.928-33; Li et al, Cell Stem Cell, 2018.23(2):p.181-192 e5). CRISPR gene knockout T cells or NK cells can be obtained from iPSCs following a similar method (Figure 16). To generate CRISPR RC3H1 and RC3H2 gene double knockout (RC3H1, 2 KO iPSCs) and A2AR gene knockout iPSCs (A2AR KO iPSCs), iPSCs were transfected with RNP complexes formed by representative gRNAs (RC3H1, SEQ ID NO: 2; RC3H2, SEQ ID NO: 4; A2AR, SEQ ID NO: 7) using the Lonza 4D Nucleofector system. First, 12-well plates were coated with a PBS solution of Laminin-521 (STEMCELL Technologies) and incubated at 37°C for 2 hours. Prior to transfection, iPSCs were pre-incubated for 2 hours with mTeSR Plus (STEMCELL Technologies) medium containing RevitaCell® Supplement (Life Technologies). 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 are detached as single cells using Accutase® (Life Technologies) and 1 × 10⁶ cells per reaction for electroporation. 6100 cells were obtained. To generate gene knockout iPSCs, the cells were combined in Lonza P3 buffer and RNP mixture and placed in PCR tubes, then added to a Lonza 4D Nucleofector for electroporation. After this, mTeSR Plus™ containing CloneR™ medium (STEMCELL Technologies) was added to the reaction mixture and incubated at room temperature for 10 minutes. After incubation, the cells were added to Laminin-521 precoated plates in mTeSR Plus™ containing CloneR™ medium. Daily medium changes with mTeSR Plus™ were performed for 72 hours until approximately 80% confluence was reached, after which the cells were passaged (6-7 days after electroporation). After transfection, RC3H1, 2 KO iPSC colonies and A2AR KO iPSC colonies with pluripotent stem cell-like morphology were maintained in culture (Figure 17A and Figure 21A).

[0173] Untransfected and transfected iPSCs were cultured in mTeSR Plus® on Laminin-521 and imaged at 10x magnification using an EVOS® bright-field microscope. Cells were detached with Accutase® and recovered 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) according to manufacturer recommendations. TRA-1-60, TRA-1-81, and SSEA-4 are surface receptors expressed on pluripotent stem cells and are considered common practice for characterizing iPSCs (Baghbaderani et al. 2015, Stem Cell Reports). Cells were analyzed using a MACSQuant® flow cytometer (Miltenyi Biotec) with unstained samples and appropriate isotype controls. Dead cells (by PI staining), debris, and doublets were gated out. Histogram plots were created using FlowLogic® (Figures 17A-17B for RC3H1, 2 KOs, and Figures 21A-21B for A2AR KO). iPSCs, with and without KOs, showed nearly identical pluripotency markers for TRA-1-60, TRA-1-81, and SSEA-4, all of which were co-expressed in >95% of cells (Figures 17B and 21B). There were no visual differences in iPSC morphology (Figures 17A and 21A), indicating that RC3H1 and RC3H2 double KOs, or A2AR KOs, did not adversely affect iPSC maintenance or pluripotency.

[0174] The CRISPR indel frequency and gene knockout efficiency of transfected iPSCs were analyzed by ICE assay as described in Example 2. The results of the ICE assay showed that gRNA induced indels at a high frequency, including out-of-frame indels, in iPSCs (Figures 18A-18C for RC3H1, 2 KO and Figures 22A-22C for A2AR KO).

[0175] In summary, our data demonstrate that the gene-edited iPSCs described herein can differentiate into CD34+ / HE / HSCs and subsequently into immune cells such as NK cells, NKT cells, or T cells using known methods for subsequent use in cancer treatment.

[0176] Example 11 - Differentiation of RC3H1 and RC3H2 KO (RC3H1,2 KO) iPSCs into iNK cells (edited iNK cells). iCD34+ cells The receptor CD34 is expressed on HE and HSCs, which are stem cell sources that form a platform for generating immune cells. Differentiation of iPSCs into CD34+ cells is a prerequisite and essential for generating iPSC-derived immune cells (Sturgeon et al. Nature Biotechnology, 2014 vol 32(6) p554-561, Knorr et al. STEM CELLS Translational Medicine vol 2(4) p274-283, Zeng et al. Stem Cell Reports, 2017 vol 9(6) p1796-1812). Characterizing CD34+ expression as an intermediate cell type between iPSCs and immune cells is considered common practice and is an important step in demonstrating that including gene knockouts in iPSCs does not disrupt potential differentiation pathways during early development.

[0177] Untransfected and transfected iPSCs (including RC3H1, 2 KOs) were differentiated into iCD34+ cells using the STEMdiff® Hematopoietic Kit (STEMCELL Technologies) according to the manufacturer's instructions. Cells were isolated and stained with a CD34-targeting antibody (Miltenyi Biotec) according to the manufacturer's recommendations. Cells were analyzed using a MACSQuant® flow cytometer (Miltenyi Biotec) with unstained samples and appropriate isotype controls. Dead cells (by PI staining), debris, and doublets were gated out. Data analysis was performed using FlowLogic®.

[0178] iPSCs with or without RC3H1,2 knockout (Figure 19) were differentiated into iCD34+ cells. These data demonstrate the success of generating iCD34+ cells from RC3H1,2 knockout iPSCs and show that the important developmental pathways necessary for transitioning from iPSCs through any intermediate phenotype to a population of CD34-expressing cells remain intact.

[0179] iNK cells iPSCs containing RC3H1,2 KO can differentiate into iNK immune cells.

[0180] Gene knockout iCD34+ (derived from RC3H1, 2 KO iPSCs) was further differentiated into iNK cells promoted by a combination of cytokines including IL-15, FLT3, and IL-7. iNK cells can be produced using publicly available methods such as those described in U.S. Patent No. 9,260,696 by Li et al. (Kaufman, Knorr) (Stem Cell, 23(2018) 181-197), or using the commercially available culture system StemSpan® NK Generation Kit (Stem Cell Technologies).

[0181] Differentiated cells were isolated and stained using antibodies targeting CD56 (Miltenyi Biotec), NKp46 (Miltenyi Biotec), and NKG2D (Miltenyi Biotec) according to manufacturing recommendations. Differentiated cells were analyzed using a MACSQuant® flow cytometer (Miltenyi Biotec) with unstained samples and appropriate isotype controls. Dead cells (by PI staining), debris, and doublets were gated out, and data analysis was performed using FlowLogic® (Figure 20). Expression of NK functional receptors (NKp46 and NKG2D) supports the idea that iCD56+ cells possess NK-specific cytotoxic function.

[0182] Example 12 - Differentiation of A2AR KO iPSCs into iNK cells (edited iNK cells). iCD34+ cells Untransfected and transfected iPSCs (including A2AR KOs) were differentiated into iCD34+ cells using the STEMdiff® Hematopoietic Kit (STEMCELL Technologies) according to the manufacturer's instructions. Cells were isolated and stained with a CD34-targeting antibody (Miltenyi Biotec) according to the manufacturer's recommendations. Cells were analyzed using a MACSQuant® flow cytometer (Miltenyi Biotec) with unstained samples and appropriate isotype controls. Dead cells (by PI staining), debris, and doublets were gated out. Data analysis was performed using FlowLogic®.

[0183] iPSCs with or without A2AR knockout (Figure 23) successfully differentiated into iCD34+ cells. These data demonstrate the generation of iCD34+ cells from A2AR knockout iPSCs and show that the essential developmental pathways necessary for transitioning from iPSCs through any intermediate phenotype to a population of CD34-expressing cells remain intact.

[0184] iNK cells iPSCs containing A2AR knockout cells can differentiate into iNK immune cells.

[0185] Non-transfected iCD34 (derived from non-transfected iPSCs) and gene knockout iCD34+ (derived from gene knockout iPSCs) were further differentiated into iNK cells promoted by a combination of cytokines including IL-15, FLT3, and IL-7. iNK cells can be produced using publicly available methods such as those described in U.S. Patent No. 9,260,696 by Li et al. (Kaufman, Knorr) (Stem Cell, 23(2018) 181-197), or using the commercially available culture system StemSpan® NK Generation Kit (Stem Cell Technologies).

[0186] Differentiated cells were evaluated for NK cell marker expression by flow cytometry. Dead cells, debris, and doublets were gated out so that the CD56+ histogram shown in Figure 24 represents all viable cells in cultures generated from either untransfected or transfected iPSC samples. Unstained samples are shown to clearly demonstrate positive staining of each antibody against each respective receptor. Appropriate isotype controls were negative. Expression of NK functional receptors (NKp46, NKp30, NKp44, and NKG2D) confirms that iCD56+ cells are iNK cells with cytotoxic function.

[0187] Example 13 - Function of edited iNK cells A2AR KO iPSCs were generated (Example 10) and differentiated into edited iNK cells (Example 12). The iNK cells were then collected after 20–40 days and used for subsequent functional assays.

[0188] The ability of iNK cells to kill cancer cells was evaluated in vitro using a real-time cell monitoring system (xCELLigence®). Target cells (10,000 cells / 100uL) (e.g., ovarian cancer cell line OVCAR-3) were resuspended in culture medium supplemented with 10-20% FBS and bovine insulin (e.g., RPMI-1640 containing L-glutamine basal medium) and dispensed into RTCA plates. Target cells were maintained at 37°C and 5% CO2 for at least 5 hours to allow cell adhesion. After target cell adhesion, iNK effector cells were added in a 1:1 E:T ratio. In parallel, iPSC-derived iNK cells were co-cultured with target cells to demonstrate the background functionality of untransfected iNK cells in vitro. The entire co-culture was maintained under optimal growth conditions for at least 10 hours. Throughout the process, cell impedance was monitored and presented herein as NCI, which normalization occurred by the time effector cells were added. The cytotoxicity rate (% cytotoxicity) of iNK±A2AR KO effector cells (test) compared to target cells alone (control) was calculated after 5 and 10 hours of co-culture using the following formula: ((Normalized cell index) 対照 - Normalized cell index 試験 ) / Normalized cell index 対照 ) × 100

[0189] 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 in vitro cytotoxicity was monitored using xCELLigence®. NT iNK cells showed cytotoxic effects when co-cultured with OVCAR-3 target cells (Figure 25A). This effect was improved in A2AR KO iNK cells compared to untransfected controls, demonstrating enhanced function in vitro. Furthermore, A2AR KO iNK cells showed even higher cytotoxicity after both 5 hours of co-culture (Figure 25B, left) and 10 hours of co-culture (Figure 25B, right). In summary, these data indicate that A2AR KO can enhance antitumor activity not only in T cells but also in iNK cells compared to untransfected control cells.

[0190] Various publications, including patents, patent applications, published patent applications, accession numbers, technical papers, and academic papers, are cited throughout this Specified Publication. Each of these cited publications, in its entirety, is incorporated herein by reference for any purpose. The present invention includes the following embodiments. [1] A method for enhancing the function of immune cells, comprising 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. [2] A method for modifying stem cells that can differentiate into immune cells, comprising modifying the stem cells to inhibit the function of at least one gene selected from the group consisting of RC3H1, RC3H2, A2AR, FAS, TGFBR1, and TGFBR2. [3] The method according to [2], further comprising differentiating the modified stem cells into immune cells, wherein the function of the at least one gene is inhibited within the immune cells. [4] The method according to any one of [1] to [3], wherein inhibition of gene function is achieved by a gene editing system. [5] The method according to [4], wherein the gene editing system is selected from the group consisting of CRISPR / Cas, TALEN, and ZFN. [6] The method according to [4], wherein the gene editing system is a CRISPR / Cas system including a guide RNA-nuclease complex. [7] The method according to [6], wherein the guide RNA targets a nucleotide sequence selected from the group consisting of SEQ ID NOs: 2 to 16. [8] The method according to [6], 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. [9] The method according to any one of [1] to [8], wherein the immune cells are selected from T cells, NK cells, NKT cells, or macrophages.

[10] The method according to [1] or [2], wherein inhibition of gene function is achieved by reducing the level or function of mRNA, sometimes via small interfering RNA (siRNA), small hairpin RNA (shRNA), microRNA (miRNA), or antisense nucleic acid.

[11] The method according to [1] or [2], wherein inhibition of gene function is achieved by reducing the level or activity of the protein encoded by the gene, sometimes by using an antibody or small molecule.

[12] The method according to any one of [1] to

[11] , wherein the modified cells produced by the method further comprise nucleic acids encoding chimeric antigen receptors (CARs).

[13] The method according to any one of [1] to

[12] , wherein the modified immune cells generated by the method described above recognize one or more target antigens.

[14] The method according to

[13] , wherein the target antigen is selected from the group consisting of TAG-72, CD19, CD20, CD24, CD30, CD47, folate receptor alpha (FRα), and BCMA.

[15] The method according to any one of [1] to

[14] , wherein at least one of the genes is RC3H1.

[16] The method according to any one of [1] to

[14] , wherein at least one of the genes is RC3H2.

[17] The method according to any one of [1] to

[14] , wherein at least one of the genes is A2AR.

[18] The method according to any one of [1] to

[14] , wherein at least one of the genes is FAS.

[19] The method according to any one of [1] to

[14] , wherein at least one of the genes is TGFBR1.

[20] The method according to any one of [1] to

[14] , wherein at least one of the genes is TGFBR2. [twenty one] Immune cells generated by the method described in any one of items [1] or [3] to

[20] , or differentiated from modified stem cells generated by the method described in any one of items [2] or [4] to

[20] . [twenty two] Modified immune cells in which the function of at least one gene is inhibited within the modified immune cells, wherein the at least one gene is selected from the group consisting of RC3H1, RC3H2, A2AR, FAS, TGFBR1, and TGFBR2. [twenty three] The modified immune cells according to

[22] , wherein the inhibition of gene function results in a decrease in the level or function of mRNA transcribed from the gene, or a decrease in the level or activity of the protein encoded by the gene. [twenty four] The modified immune cell according to

[22] , wherein the inhibition of gene function is due to a modification in the nucleic acid sequence of the gene. [twenty five] Modified immune cells as described in any one of the following paragraphs,

[22] to

[24] , selected from T cells, NK cells, NKT cells, or macrophages.

[26] Modified immune cells expressing a chimeric antigen receptor (CAR), as described in any one of the items

[22] to

[25] .

[27] A modified immune cell according to any one of the items

[22] to

[26] that recognizes one or more target antigens.

[28] The modified immune cells described in

[27] , wherein the target antigen is selected from the group consisting of TAG-72, CD19, CD20, CD24, CD30, CD47, folate receptor alpha (FRα), and BCMA.

[29] A modified immune cell according to any one of

[22] to

[28] , wherein at least one of the genes is RC3H1.

[30] A modified immune cell according to any one of

[22] to

[28] , wherein at least one of the genes is RC3H2.

[31] A modified immune cell according to any one of the following paragraphs

[22] to

[28] , wherein at least one of the genes is A2AR.

[32] A modified immune cell according to any one of the following paragraphs

[22] to

[28] , wherein at least one of the genes is FAS.

[33] A modified immune cell according to any one of

[22] to

[28] , wherein at least one of the genes is TGFBR1.

[34] A modified immune cell according to any one of

[22] to

[28] , wherein at least one of the genes is TGFBR2.

[35] A modified stem cell capable of differentiating into immune cells, comprising a modification in the nucleic acid sequence of at least one gene, wherein the modification inhibits the function of the at least one gene, and the at least one gene is selected from the group consisting of RC3H1, RC3H2, A2AR, FAS, TGFBR1, and TGFBR2.

[36] A modified stem cell described in

[35] , which is an induced pluripotent stem cell.

[37] The modified stem cells described in

[36] , wherein the induced pluripotent stem cells are generated from homozygous donor cells with respect to three HLA genotypes.

[38] A modified stem cell according to any one of the items

[35] to

[37] , further comprising a nucleic acid encoding a chimeric antigen receptor (CAR).

[39] A modified stem cell according to any one of the following paragraphs

[35] to

[38] , wherein at least one of the genes is RC3H1.

[40] A modified stem cell according to any one of the following paragraphs

[35] to

[38] , wherein at least one of the genes is RC3H2.

[41] A modified stem cell according to any one of the following paragraphs

[35] to

[38] , wherein at least one of the genes is A2AR.

[42] A modified stem cell according to any one of the following paragraphs

[35] to

[38] , wherein at least one of the genes is FAS.

[43] A modified stem cell according to any one of the following paragraphs

[35] to

[38] , wherein at least one of the genes is TGFBR1.

[44] A modified stem cell according to any one of the following paragraphs

[35] to

[38] , wherein at least one of the genes is TGFBR2.

[45] A composition for enhancing the function of immune cells, comprising a guide RNA-nuclease complex capable of editing the sequence of a target gene, A composition wherein the guide RNA targets a nucleotide sequence selected from the group consisting of SEQ ID NOs: 2 to 16.

[46] The composition according to

[45] , wherein the nuclease comprises at least one protein 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.

[47] A method for treating a target condition, comprising administering modified immune cells as described in any one of the items

[21] to

[34] .

[48] The method according to

[47] , wherein the condition is cancer, infection, autoimmune disorder, organ fibrosis, or endometriosis.

Claims

[Claim 1] The invention described in the specification.

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