Methods and compositions for generating stem cell-derived immune cells with enhanced function - Patents.com

Abstract

The present disclosure relates to a method for generating stem cell-derived immune cells with enhanced function. Disclosed herein is a method for modifying stem cells or progenitor cells that can differentiate into immune cells, and inducing the differentiation of the stem cells or progenitor cells into enhanced immune cells, inhibiting the function of at least one gene selected from DGKα and DGKζ. Also disclosed herein is the immune cells or stem cells produced by this method, and the use of the immune cells in therapeutic treatment.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority from U.S. Provisional Application No. 63 / 147,933, filed February 10, 2021, and U.S. Provisional Application No. 63 / 236,828, filed August 25, 2021, the contents of which are incorporated herein in their entireties.

[0002] Field of Disclosure The present disclosure relates to a method for generating stem cell-derived immune cells with enhanced function. More specifically, disclosed herein is a method for modifying stem cells or progenitor cells that can differentiate into immune cells to inhibit the function of at least one gene selected from DGKα and DGKζ, and inducing the differentiation of the stem cells or progenitor cells into enhanced immune cells. Also disclosed herein is the immune cells or stem cells produced by this method, and the use of the immune cells in therapeutic treatment.

[0003] Incorporation by reference of sequence listing The sequence listing in the ASCII text file entitled 39095WO_SequenceListing.txt, created on January 27, 2022, of 8 KB, is incorporated herein by reference. [Background technology]

[0004] Reference in this specification to any prior publication (or information derived therefrom), or to any known matter, should not be construed as an admission or acknowledgement, or any form of suggestion, that the prior publication (or information derived therefrom) or known matter forms part of the common general knowledge in the field of activity to which this specification pertains.

[0005] Malignant tumors, or cancers, grow in an uncontrolled manner, invade normal tissues, and often metastasize, growing at sites distant from the tissue of origin. Generally, cancer originates from one or just a few normal cells that have undergone a poorly understood process called malignant transformation. Cancers can arise from almost any tissue in the body. Those of epithelial cell origin, called carcinomas, are the most common type of cancer. Sarcomas are malignant tumors of mesenchymal tissues that arise from cells such as fibroblasts, muscle cells, and fat cells. Solid malignant tumors of lymphoid tissues are called lymphomas, and malignant tumors of bone marrow and blood origin of lymphocytes and other hematopoietic cells are called leukemias.

[0006] Cancer is one of the three leading causes of death in industrialized countries. As the treatment of infectious diseases and the prevention of cardiovascular diseases are continually improving and their life expectancies are increasing, it is likely that cancer will become the most common fatal disease in these countries. Therefore, successful cancer treatment requires the elimination or destruction of all malignant cells without causing the patient's death. The ideal way to achieve this is to induce an immune response against the tumor that distinguishes between the tumor's cells and their normal cellular counterparts.

[0007] Immunotherapy is a new and emerging area of ​​treatment that uses the body's own immune system. The treatment may use cells, such as T cells, NK cells, NKT cells, macrophages, B cells, dendritic cells, obtained from living organisms or produced in a laboratory, which are enhanced and infused into the patient's body to help its immune system fight cancer, infections, and other diseases. Several types of immunotherapy are currently being developed.

[0008] Some of these treatments use T cells expressing chimeric antigen receptors (CAR-T cells), which have been shown to be highly effective in killing tumor cells in diseases such as acute lymphocytic leukemia (ALL) and non-Hodgkin's lymphoma (NHL). Approved products targeting the B cell antigen CD19 are produced 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 home-grown products targeting other blood cell markers, such as B cell maturation antigen (BCMA), for other hematological malignancies such as multiple myeloma are in development (Sadelain et al., Therapeutic T cell engineering, 2017, Nature, 545(7655): 423-431).

[0009] In other treatments, NK cells have been used successfully against hematological malignancies, and gene editing tools have been applied to NK cells and CAR-NK cells to enhance their therapeutic efficacy. Autocrine or membrane-bound expression of cytokines such as IL-15 and IL-2 can improve NK cell proliferation and persistence (Nagashima et al., Stable transduction of the interleukin-2 gene into human natural killer cell lines and their phenotypic and functional characterization in vitro and in vivo, Blood, 15 May 1998, Vol.91(10), pp.3850-61; Hoyos et al., Engineering CD19-specific T lymphocytes with interleukin-15 and a suicide gene to enhance their anti-lymphoma / leukemia effects and safety, Leukemia, 2010 Jun, Vol.24(6), pp.1160-70; Imamura et al., Autonomous growth and increased cytotoxicity of natural killer cells expressing membrane-bound interleukin-15, Blood, 14 August 2014, Vol.124(7), pp.1081-8). Targeting immune checkpoints, primarily studied in T cells, has also shown promising results in NK cells.Knocking out TIGIT, a checkpoint receptor for NK cells, has been shown to prevent NK cell exhaustion and promote antitumor activity (Zhang et al., Blockade of the checkpoint receptor TIGIT prevents NK cell exhaustion and elicits potent anti-tumor immunity, Nature Immunology, Jul 2018, Vol.19(7), pp.723-73). Furthermore, gene disruption of receptors for immunosuppressive cytokines from the tumor microenvironment (TME), such as A2AR and TGFβR2, has been shown to neutralize inhibition from the TME and restore NK cell effector function (Wang et al., SMAD4 promotes TGF-beta-independent NK cell homeostasis and maturation and antitumor immunity, The Journal of clinical investigation, 01 November 2018, Vol.128(11), pp.5123-5136; Rouce et al., The TGF-beta / SMAD pathway is an important mechanism for NK cell immune evasion in childhood B-acute lymphoblastic leukemia, Leukemia, Apr 2016, Vol.30(4), pp.800-811; Young et al., A2AR Adenosine Signaling Suppresses Natural Killer Cell Maturation in the Tumor Microenvironment, Cancer research, 15 February 2018, Vol.78(4), pp.1003-1016).

[0010] Compared with T cells and CAR-T cells, NK cell and CAR-NK cell-based treatments present several advantages such as (1) greater safety, especially with regard to potentially life-threatening adverse events such as cytokine release syndrome and graft-versus-host disease, (2) multiple mechanisms for producing cytotoxic activity, and (3) high feasibility for "off-the-shelf" manufacturing (Xie et al., CAR-NK cells: A proposable cellular immunotherapy for cancer, EBioMedicine, September 2020, Vol.59; Liu et al., Use of CAR-Transduced Natural Killer Cells in CD19-Positive Lymphoid Tumors, The New England journal of medicine, February 6, 2020, Vol.382(6), pp.545-553).

[0011] Although cell-based immunotherapy has shown impressive results in clinical trials for hematological cancers, similar results have not been achieved in the treatment of solid tumors. There are multiple reasons for the relative lack of efficacy in solid tumors, including limited access to the tumor site, the immunosuppressive nature of the tumor microenvironment, and the lack of solid tumor-specific target antigens.

[0012] Additionally, lack of persistence and "exhaustion" of administered CAR cells is a consistently observed limitation (Newick et al., CAR T Cell Therapy for Solid Tumors, Annu Rev Med,2017,68:139-152).

[0013] Inhibitory receptors such as CTLA-4, PD-1, or LAG-3 may weaken the activation of CAR-T cells and accelerate T cell exhaustion. After PD-1 was disrupted by genome editing, improvement of antitumor activity of T cells was expected (Liu et al., CRISPR-Cas9-mediated multiplex gene editing in CAR-T cells, Cell Res, 2017, 27(1): 154-157). However, removal of PD-1 on T cells may also increase susceptibility to exhaustion, shorten lifespan, and fail to improve antitumor efficacy (Odorizzi et al., Genetic absence of PD-1 promotes accumulation of terminally differentiated exhausted CD8+T cells, J Exp Med, 2015, 212(7): 1125-37). For these reasons, it is necessary to evaluate on a case-by-case basis whether gene editing in immune cells enhances antitumor activity.

[0014] Genetically engineered immune cells are clearly a potential weapon against cancer, and therefore one of the challenges is to generate, expand and maintain them numerically. Immune cells are generated from hematopoietic stem cells (HSCs), which are in turn generated from pluripotent stem cells (PSCs). Therefore, PSC technology is a very promising technology, as in theory PSCs provide an infinite, renewable source of cells. [Prior art documents] [Non-patent literature]

[0015] [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, 2017, Nature, 545(7655):423-431

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[0016] The present invention relates to compositions and methods for gene editing cells such as human PSCs and differentiating them into hematopoietic stem cell-like cells and ultimately into functional immune cells, which have advantages over comparable non-genetically edited immune cells. Those skilled in the art will appreciate that deleting one or more non-redundant cellular genes can significantly affect normal cellular function, and may actually render the cells non-viable, non-functional, or in the case of stem cells, unable to differentiate into functional cells. The present invention discloses gene knockout (KO) of DGKα and / or DGKζ genes in iPSCs, and surprisingly demonstrates that DGK KO iPSCs are viable and can differentiate into DGK KO immune cells, particularly NK cells and T cells. Furthermore, DGK KO NK cells are fully functional in vitro and in vivo, and in fact function better than NK cells without DGK KO. Similarly, iPSC-derived DGK KO T cells are fully functional in vitro and show improved function when compared to T cells without DGK KO.

[0017] In one aspect, a method for generating stem cell-derived immune cells with enhanced function is provided herein, the method comprising modifying stem cells to inhibit the function of at least one target gene selected from the group consisting of DGKα and DGKζ, wherein the modified stem cells retain the target gene inhibition of the modified stem cells and can differentiate into stem cell-derived immune cells comprising enhanced activity.

[0018] In some embodiments, the stem cells are pluripotent stem cells selected from the group consisting of induced pluripotent stem cells (iPSCs) or embryonic stem cells. In some embodiments, the stem cells are selected from the group consisting of pre-HSCs, hemogenic endothelial (HE) or hematopoietic stem cells (HSCs) or HSC-like cells. In some embodiments, the stem cells are derived from a triple homozygous HLA haplotype donor.

[0019] In some embodiments, the method further comprises selecting engineered stem cells generated in which both alleles of the target gene are inhibited.

[0020] In some embodiments, the method further comprises the steps of (i) contacting a modified stem cell generated herein, or a clonal cell generated from the modified stem cell, with the composition to obtain a mesodermal cell, (ii) contacting the mesodermal cell with the composition to obtain a CD34+ cell, and (iii) contacting the CD34+ cell with the composition to obtain a stem cell-derived immune cell.

[0021] In some embodiments, at least one target gene is DGKα. In some embodiments, at least one target gene is DGKζ. In some embodiments, both the DGKα gene and the DGKζ gene are target genes.

[0022] In some embodiments, the stem cell-derived immune cell is selected from a multipotent progenitor cell, a lymphoid progenitor cell (e.g., a common lymphoid progenitor cell), an early thymic progenitor cell, a pre-T cell progenitor cell, a pre-NK progenitor cell, a T cell progenitor cell, a NK cell progenitor cell, a myeloid progenitor cell (e.g., a common myeloid progenitor cell), a T cell, a NK cell, a NKT cell, a B cell, a macrophage, and a monocyte.

[0023] In some embodiments, the immune cell is a T cell that expresses at least one of the markers selected from CD2, CD5, CD7, CD4, CD8a, CD8b, CD3, TCR alpha beta, and TCR gamma delta.

[0024] In some embodiments, the immune cells are NK cells that express CD56+ and CD45+.

[0025] In some embodiments, the inhibition of the function of the target gene is achieved by a gene editing system. In some aspects, the gene editing system is selected from the group consisting of CRISPR / Cas, TALEN, and ZFN.

[0026] In some embodiments, the gene editing system is a CRISPR / Cas system comprising a guide RNA-nuclease complex. In some embodiments, the guide RNA targets a nucleotide sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 16.

[0027] In some embodiments, the CRISPR / Cas system is selected from the group consisting of Cpf1, Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, Cas12, Cas13, Cas100, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm 4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, CasX, CasY, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, and Csf4.

[0028] In some embodiments, the modified stem cells are further modified to contain a nucleic acid encoding a chimeric antigen receptor (CAR). In some embodiments, the modified stem cells express the CAR.

[0029] In some embodiments, the immune cells produced by the method are modified to contain a nucleic acid encoding a chimeric antigen receptor (CAR). In some embodiments, the immune cells produced by the method express a CAR.

[0030] In some embodiments, the immune cells produced by the method recognize one or more target antigens. In some embodiments, the immune cells produced by the method recognize tumor targets or infectious agent targets. In some embodiments, the target antigens are selected from the group consisting of TAG-72, CCR4, CD19, CD20, CD22, CD24, CD30, CD47, folate receptor alpha (FRα), BCMA, mesothelin, and Muc1.

[0031] In a further aspect, provided herein is an immune cell produced by the methods disclosed herein.

[0032] In another aspect, provided herein is a modified cell in which the function of at least one target gene selected from the group consisting of DGKα and DGKζ is inhibited, the modified cell retaining the gene inhibition and capable of differentiating into an immune cell comprising enhanced activity.

[0033] In some embodiments, the modified cells are stem cells selected from the group consisting of embryonic stem cells, umbilical cord stem cells, and induced pluripotent stem cells. In some embodiments, the modified cells are selected from the group consisting of hemogenic endothelial cells, hematopoietic progenitor cells, hematopoietic precursor cells, hematopoietic stem cells, or hematopoietic-like stem cells. In some embodiments, the modified cells are derived from triple homozygous HLA haplotype donors.

[0034] In some embodiments, both alleles of the target gene are inhibited. In some embodiments, at least one target gene is DGKα. In some embodiments, at least one target gene is DGKζ. In some embodiments, both DGKα and DGKζ genes are target genes.

[0035] In some embodiments, the engineered cells comprise a nucleic acid encoding a chimeric antigen receptor (CAR). In some embodiments, the engineered cells express the CAR.

[0036] In another aspect, provided herein is a composition for modifying a cell to inhibit the function of at least one target gene selected from the group consisting of DGKα and DGKζ, the composition comprising a guide RNA-nuclease complex capable of editing the sequence of the target gene, wherein the guide RNA targets a nucleotide sequence selected from the group consisting of SEQ ID NO:1 to SEQ ID NO:16.

[0037] In some embodiments, the nuclease is Cpf1, Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, Cas12, Cas13, 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, CasX, CasY, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, and Csf4.

[0038] In a further aspect, provided herein is a method of treating a condition in a subject comprising administering to the subject an immune cell disclosed herein. In some embodiments, the condition is cancer, an infectious disease, an autoimmune disorder, organ fibrosis, or endometriosis.

[0039] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee. [Brief description of the drawings]

[0040] [Figure 1]Schematic diagram of iPSC gene editing and single cell cloning. CRISPR / Cas9 gene editing was used to knock-in (KI) the TAG-72 CAR (described in patents PCT / AU2020 / 050800 and WO2017 / 088012, incorporated herein by reference) into iPSCs or knock-out (KO) the DGK genes (DGKα and / or DGKζ), or knock-out KO DGK genes (DGKα and / or DGKζ) into CAR iPSC clones. iPSCs or CAR iPSC clones were allowed to recover from electroporation until the iPSCs were growing normally again. This is typically any time beyond 4-6 days after electroporation. Upon reaching 80% colony confluence, iPSCs were allowed to expand until day 11, followed by single cell sorting (phase I). Untransfected iPSCs are referred to as wild type. The edited iPSC or CAR iPSC clones at the end of Phase I are referred to as pre-sorted in the following examples. Single cell sorting of iPSCs was performed using a FACSAria™ Fusion cell sorter, and colony formation from single cells was allowed to proceed for 9-12 days. The clones were then expanded (Phase II) until the required cell number was reached, and genetic analysis was performed to determine the accuracy and purity of the clonal iPSCs. The iPSCs at the end of Phase II are referred to as single cell clones in the following examples. [Figure 2A-2C]CRISPR / Cas9 introduces insertions and deletions (indels) into the open reading frame of the DGKα gene in iPSCs. Guide RNA (gRNA)-forming ribonucleoprotein (RNP) targeting the target sequence of the DGKα gene (SEQ ID NO: 3) was transfected into iPSCs, and the frequency of indels was assessed by Inference of CRISPR Edits (ICE) analysis. (A) Sanger sequencing traces from RNP-transfected iPSCs ("edited sample") (SEQ ID NO: 17) of gRNA targeting the target sequence of the DGKα gene (SEQ ID NO: 3), and non-transfected iPSCs ("control sample") (SEQ ID NO: 18). The edited sample shows a heterogeneous mix of bases downstream of the cut site in contrast to the non-transfected control sample. The black underlined region of the control sample represents the guide sequence, and the horizontally dotted red underlined region is the associated (protospacer adjacent motif) PAM site. Vertical black dotted lines on both traces represent the cleavage sites. (B) Relative percentage of contribution of each edited sequence (normalized) in genomic DNA from RNP-transfected iPSCs of gRNA targeting the target sequence of DGKα gene (SEQ ID NO: 3). Indel+1: SEQ ID NO: 19; Indel0: SEQ ID NO: 20; Indel-1: SEQ ID NO: 21; Indel-8: SEQ ID NO: 22; Indel-11: SEQ ID NO: 23; Indel-11: SEQ ID NO: 24; Indel-2: SEQ ID NO: 25; Indel-11: (SEQ ID NO: 26); Indel-2: SEQ ID NO: 27. (C) Distribution of indel sizes in the total edited population of RNP-transfected iPSCs of gRNA targeting the target sequence of DGKα gene (SEQ ID NO: 3). Out-of-frame indel percentage is the proportion of indels that show frameshift or are longer than 21 bp in length. R2 value calculated by Pearson correlation coefficient indicates the confidence of the indel percentage. [Figure 3A-3B]CRISPR / Cas9 editing of the DGK gene is successfully mediated in several iPSC lines. The formed RNPs of gRNA targeting the target sequence of the DGKα gene (SEQ ID NO:3) and gRNA targeting the target sequence of the DGKζ gene (SEQ ID NO:11) were transfected into two different iPSC lines: iPSC line 1 (A) or iPSC line 2 (B), derived from different cell types and delivered using different methods provided by two independent companies, to generate DGKα or DGKζ single KO (DGKαKO or DGKζKO iPSCs), or DGKα and DGKζ double KO (DGKαζKO iPSCs). iPSC line 1 was derived from mononuclear cells using episomal delivery of iPSC reprogramming factors. iPSC line 2 was derived from cells obtained from an umbilical cord blood fraction and reprogrammed using Sendai virus delivery of iPSC factors. The KO efficiency of DGKα and DGKζ genes was analyzed using ICE. The out-of-frame indel percentage is the percentage of indels that show frameshift or are longer than 21 bp in length. The R2 value calculated by Pearson correlation coefficient indicates the confidence of the indel percentage. [Figure 4A-4D] gRNAs successfully mediate CRISPR / Cas9 editing of DGK genes in single-cell clones. The formed RNPs of gRNA targeting the target sequence of DGKα gene and gRNA targeting the target sequence of DGKζ gene (SEQ ID NO: 3 and SEQ ID NO: 11, respectively) were transfected into iPSCs, and the efficiency of KO of DGKα (A), KO of DGKζ (B), and KO of DGKαζ. Indel% of DGKα (C) and indel% of DGKζ (D) were analyzed using ICE in different single-cell clones. Out-of-frame indel percentage is the percentage of indels that show frameshift or are greater than 21 bp long. iPSC single-cell clones with out-of-frame indel percentage between 99% and 100% were selected as single-cell clone candidates. Clone names are indicated as gene name followed by a number. [Figure 5A-5B]Sanger sequencing traces of gRNA cleavage sites in the DGK gene in non-transfected (wild type), DGKαζ KO pre-sorted iPSCs and DGKαζ KO iPSC single cell clone 01. After RNP transfection, a mixture of bases was introduced downstream of the cleavage site in the DGK KO pre-sorted iPSC genome. After sorting and single cell cloning, a single cell iPSC clone (DGKαζ KO iPSC single cell clone 01) with one thymine (T) insertion (+1) was identified from the DGK KO pre-sorted iPSCs evidenced by the removal of the mixture of bases downstream of the cleavage site in the DGKα (A) and DGKζ (B) genes. The black underlined region of the control sample represents the guide sequence and the horizontal dotted red underlined region is the associated PAM site. The vertical dotted black lines on both traces represent the cleavage site. In (A), the following sequences are shown: Non-transfected (wild type) iPSC: SEQ ID NO: 28; DGKα and DGKζ RNP transfected bulk iPSC: SEQ ID NO: 29; DGKα and DGKζ RNP co-transfected iPSC single cell clone: ​​SEQ ID NO: 30. In (B) the following sequences are shown: Non-transfected (wild type) iPSC: SEQ ID NO: 31; DGKα and DGKζ RNP transfected bulk iPSC: SEQ ID NO: 32; DGKα and DGKζ RNP co-transfected iPSC single cell clone: ​​SEQ ID NO: 33. [Figure 6]CRISPR / Cas9 editing of DGK gene in TAG-72 CAR iPSC single cell clone is successfully mediated. TAG-72 CAR iPSC single cell clone was generated as described in Figure 1. Then, the formed RNP of gRNA targeting the target sequence of DGKα gene and gRNA targeting the target sequence of DGKζ gene (SEQ ID NO: 3 and SEQ ID NO: 11, respectively) was transfected into three different TAG-72 CAR iPSC single cell clones (TAG-72 CAR iPSC single cell clones B11, C4 and D7) to generate TAG-72 CAR clone / DGKαζ KO iPSC. The KO efficiency of DGKα and DGKζ genes in TAG-72 CAR clone / DGKαζ KO pre-selected iPSC was analyzed individually using ICE. The out-of-frame indel percentage is the percentage of indels that indicate a frameshift or are greater than 21 bp in length. The R2 value calculated by the Pearson correlation coefficient indicates the confidence of the indel percentage. [Figure 7] Successful generation of TAG-72 CAR / DGKαζ KO iPSC single cell clones. TAG-72 CAR iPSC single cell clones were generated as described in Figure 1. Subsequently, the formed RNPs of gRNA targeting the target sequence of DGKα gene and gRNA targeting the target sequence of DGKζ gene (SEQ ID NO: 3 and SEQ ID NO: 11, respectively) were transfected into TAG-72 CAR iPSC single cell clone B11 to generate TAG-72 CAR / DGKαζ KO iPSC single cell clones (1-12). The KO efficiency of DGKα gene and DGKζ gene were analyzed separately using ICE. Out-of-frame indel percentage is the percentage of indels that show frameshift or are more than 21 bp long. Clones with out-of-frame indel percentages of 99%-100% were selected as single cell clone candidates. Numbers 1-12 represent TAG-72 CAR / DGKαζ KO iPSC single cell clones, with highlighted ones representing successful KOs. [Figure 8A-8B]Genetic deletion of DGKα and / or DGKζ in iPSCs does not affect iPSC pluripotency. This was characterized by (A) colony morphology (scale bar = 1 mm), and (B) surface expression of pluripotency markers TRA-1-60 and SSEA-4. Using CRISPR / Cas9 editing, TAG-72 CAR KI, single gene (DGKα or DGKζ) and double gene (DGKαζ) KO of DGK isoforms in iPSCs was performed. Pluripotent stem cell markers (TRA-1-60 and SSEA-4) were analyzed by flow cytometry, where histograms (Figure 8B) represent the expression of each pluripotency marker on all live cells (dead cells via PI, debris and doublets were gated out). Unstained controls are included in the histograms to highlight positive antibody staining. [Figure 9A-9B] Genetic deletion of DGKα and / or DGKζ and insertion of TAG-72 CAR into iPSCs does not affect iPSC proliferation compared to non-transfected (NT) controls. Graphs represent the time course of iPSC expansion culture. (A) Direct comparison between KO of each allele. (B) KO of DGKαζ performed on two different CAR iPSC single cell clones; both on non-transfected iPSC (NT) lines. (A): Blue line (NT) represents non-transfected iPSCs; Red line (DGKα) represents DGKα KO iPSC single cell clone 16; Green line (DGKζ) represents DGKζ KO iPSC single cell clone 02; Purple line represents DGKαζ KO iPSC single cell clone 24. (B): Blue line (NT) represents non-transfected iPSCs; red line TAG-72 CAR (clone D7) represents TAG-72 CAR iPSC single cell clone D7 (no KO of DGKα and / or DGKζ); green line TAG-72 CAR clone / DGKαζ KO (clone D7) represents pre-sorted TAG-72 CAR clone D7 / DGKαζ KO iPSCs. [Figure 10]Schematic diagram of iPSC to iNK differentiation method. iPSC were lifted and differentiated into hematopoietic / hematopoietic lineage in phase 1. CD34+ cells were then differentiated into lymphoid progenitors in phase 2 and induced into iNK cells in phase 3. [Figure 11A-11B] TAG-72 CAR KI and DGK gene KO do not affect the differentiation of iPSCs into CD34+ cells. Using CRISPR / Cas9 editing, TAG-72 CAR KI, single gene (DGKα or DGKζ) or double gene (DGKαζ) KO of DGK isoforms in iPSCs was performed. iPSCs were then differentiated into CD34+ cells and analyzed by flow cytometry. Representative flow cytometry plots show the population frequency of CD34+ cells in the absence (A) or presence (B) of TAG-72 CAR. A: iPSC (non-transfected) represents CD34+ cells derived from non-transfected iPSC; DGKαKO (clone 16) represents CD34+ cells derived from DGKαKO iPSC single cell clone 16; DGζKO (clone 07) represents CD34+ cells derived from DGKζKO iPSC single cell clone 07; DGKαζKO (clone 24) represents CD34+ cells derived from DGKαζKO iPSC single cell clone 24. B: iPSC (non-transfected) represents CD34+ cells derived from non-transfected iPSCs in this experiment; TAG-72 CAR (clone D7) represents CD34+ cells derived from TAG-72 CAR iPSC single cell clone (clone D7); TAG-72 CAR (clone D7) / DGKαζ KO pre-sorted represents CD34+ cells derived from TAG-72 CAR clone D7 / DGKαζ KO pre-sorted iPSCs. Histogram representation of cell surface expression of CD34 on live cells in culture. Dead cells, debris and doublets were gated out. An unstained control (blue) is included in the histogram to highlight positive antibody staining. [Figure 12A-12B]CRISPR / Cas9 mediates KO of DGK gene in iNK with and without TAG-72 CAR. TAG-72 CAR iPSC single cell clones were generated as described in FIG. 1. The RNP formed of gRNA targeting the target sequence of DGKα gene (SEQ ID NO: 3) and gRNA targeting the target sequence of DGKζ gene (SEQ ID NO: 11) was used to generate TAG-72 CAR clones / DGKαζ KO preselected iPSCs (A). Similarly, the RNP formed of gRNA targeting the target sequence of DGKα gene (SEQ ID NO: 3) and gRNA targeting the target sequence of DGKζ gene (SEQ ID NO: 11) was used to generate DGKαζ KO iPSCs, and DGKαζ KO iPSC single cell clones were formed as described in FIG. 1 (B). TAG-72 CAR clone D7 / DGKαζ KO preselected iPSC and DGKαζ KO iPSC single cell clone 24 were then differentiated into iNK cells. The KO efficiency of DGKα and DGKζ genes in iNK cells was analyzed using ICE. Out-of-frame indel percentage is the percentage of indels that show frameshift or are greater than 21 bp in length. [Figure 13]TAG-72 CAR KI and DGK gene KO do not affect the number of iNK cells generated per iPSC. TAG-72 CAR KI, single gene (DGKα or DGKζ) or double gene (DGKαζ) KO of DGK isoforms in iPSCs was performed using CRISPR / Cas9 editing as previously discussed. iPSCs were then differentiated into iNK cells. Representative yields of iNK cells (CD56+ cells) differentiated from KO iPSC single cell clones with and without CAR constructs compared to baseline yields of iNK cells generated from non-transfected iPSCs are expressed as mean ± standard deviation (SD) from 2-5 samples. That is, a representative yield score of 1 in any of the gene-edited samples indicates that this sample generated the same number of iNK cells as those derived from non-transfected iPSCs, and thus gene editing does not affect iNK yield. iNK (non-transfected) represents iNK derived from non-transfected iPSC; iNK DGKαKO represents iNK derived from DGKαKO iPSC single cell clone; iNK DGKζKO represents iNK derived from DGKζKO iPSC single cell clone; iNK DGKαζKO represents iNK derived from DGKαζKO iPSC single cell clone; iNK TAG-72 CAR represents iNK derived from TAG-72 CAR iPSC single cell clone; iNK TAG-72 CAR / DGKαζKO represents iNK derived from pre-selected TAG-72 CAR clone / DGKαζKO iPSC. One-way ANOVA statistics was performed across all groups. No significant differences were identified. [Figure 14A-14B]TAG-72 CAR KI and DGK gene KO do not alter iNK phenotype. Using CRISPR / Cas9 editing, we performed KI, single gene (DGKα or DGKζ) or double gene (DGKαζ) KO of the TAG-72 CAR of DGK isoforms in iPSCs. iPSCs were then differentiated into iNK cells and phenotype was assessed by flow cytometry. Representative phenotypic analysis of iNK differentiated from KO iPSCs in the absence (A) or presence (B) of TAG-72 CAR. For flow analysis, dead cells, debris and doublets were gated out so that each histogram represents all live cells in culture. Unstained controls (blue) are included in the histograms to highlight positive antibody staining for NK receptors CD56, CD45, NKp46, NKG2D, Nkp44 and 2B4. iNK(untransfected) represents iNK derived from untransfected iPSCs; iNK DGKαKO(clone 16) represents iNK derived from DGKαKO iPSC single cell clone 16; iNK DGKζKO(clone 07) represents iNK derived from DGKζKO iPSC single cell clone 07; iNK DGKαζKO(clone 24) represents iNK derived from DGKαζKO iPSC single cell clone 24; iNK TAG-72 CAR(clone D7) represents iNK derived from TAG-72 CAR iPSC single cell clone D7; iNK TAG-72 CAR(clone D7) / DGKαζKO(preselected) represents iNK derived from TAG-72 CAR clone D7 / DGKαζKO preselected iPSCs; iNK TAG-72 CAR(clone C4) represents iNK derived from TAG-72 CAR represents iNK derived from iPSC single cell clone C4; iNK TAG-72 CAR (clone C4) / DGKαζ KO (preselected) represents iNK derived from TAG-72 CAR (clone C4) / DGKαζ KO preselected iPSCs. [Figures 15A-15C]KO of TAG-72 CAR KI and DGK genes enhances the cytotoxicity of iNK cells against OVCAR-3 tumor cells and at the same time enhances survival / longevity after repeated exposure to the same cells. KO of TAG-72 CAR KI and double gene (DGKαζ) of DGK isoform in iPSCs was performed using CRISPR / Cas9 editing. iPSCs were then differentiated into iNK cells and determined using a real-time cell monitoring system (xCELLigence®). Survival / longevity of iNK cells was evaluated in a repeated antigen exposure assay. The cytotoxic function of TAG-72 CAR iNK cells is shown as the killing efficiency of OVCAR-3 cells over 72 hours with and without KO of DGKαζ (A). Killing efficiency was calculated as the percentage of OVCAR-3 cells remaining after treatment relative to untreated OVCAR-3 control. In addition, the cytotoxic function of iNK cells pre-exposed to OVCAR-3 cells was assessed using xCELLigence and presented as the normalized cell index over a 20-h period (B). The normalized cell index was calculated according to the manufacturer's recommendations. (C) The number of iNK cells remaining in the culture after repeated exposure to OVCAR-3 cells for 72 h is also shown. iNK TAG-72 CAR (clone C4) represents iNK cells derived from TAG-72 CAR iPSC single cell clone (clone C4); iNK TAG-72 CAR (clone C4) / DGKαζ KO (preselected) represents iNK cells derived from TAG-72 CAR (clone C4) / DGKαζ KO preselected iPSCs; iNK TAG-72 CAR (clone D7) represents iNK cells derived from TAG-72 CAR iPSC single cell clone D7; iNK TAG-72 CAR (clone D7) / DGKαζ KO (preselected) represents iNK cells derived from TAG-72 CAR (clone D7) / DGKαζ KO preselected iPSCs. [Figure 16A-16B]DGKαζ KO iNK cells show improved killing capacity compared to non-transfected iNK controls. (A) Normalized cell index (CI) readings over 24 h for DGKαζ KO and non-transfected iNK cells measured by the xCELLigence® Real-Time Cell Monitoring System. Error bars = SEM, NGM = standard growth medium for OVCAR-3 cells. (B) Expression of the proliferation-promoting marker Ki67 is increased on DGKαζ KO iNK cells after 120 h of repeated antigen stimulation with OVCAR-3 cells or left cultured under normal NK growth conditions. Percentage of Ki67+CD56+ positive cells determined by flow cytometry at the end of 120 h of repeated antigen stimulation. Error bars = SEM. [Figure 17] Inclusion of DGKαζ KO in iNK cells confers a functional advantage in vivo compared to non-transfected iNK cells (iNK cells without DGKαζ KO) in reducing tumor size and prolonging survival in nod-skid-gamma (NSG) mice bearing subcutaneous ovarian cancer (OVCAR-3) tumors. iNK cells (with and without DGKαζ KO) were injected at 3x106 cells at 14-day intervals. Dotted lines indicate when each iNK injection was performed. iNK cells were harvested in NK growth medium in vitro and then injected for 7 days. No cytokine support was co-administered during the in vivo experiments. (n=4 mice / group). [Figures 18A-18C]DGKαζ gene deletion confers resistance to TGFβ immunosuppression to iNK. (A) iNK cells with and without DGKαζ KO targeted against ovarian cancer cells (OVCAR-3) in vitro were assessed for their cytotoxic function via xCELLigence in culture medium with or without the addition of 10 ng / mL TGFβ. (B) Normalized cell index (CI) readings measured by the xCELLigence® real-time cell monitoring system demonstrated that the addition of TGFβ inhibited non-transfected iNK cell function at a 2:1 (effector:target) ratio, but did not suppress iNK function in cells containing DGKαζ KO. xCELLigence curves represent the mean ± standard deviation (n=3). (C) TAG-72 CAR (clone D7) iNK cells with or without KO of DGKαζ were assessed for their cytotoxic function against ovarian cancer cells (OVCAR-3) in vitro in culture medium with or without the addition of 100 ng / mL TGFβ via xCELLigence. xCelligence® curves represent the mean ± standard deviation (n=3). [Figure 19] TGFβR1 / 2 gene editing results in loss of pluripotency and spontaneous differentiation of iPSCs. The morphology of iPSC colonies was examined by light microscopy after RNP transfection. In contrast to non-transfected (NT) iPSCs, TGFβR1 and TGFβR2 dominant negative knockout iPSC colonies showed spontaneous differentiation after targeted transfection.

[0041] TGFβR KO gRNA JPEG2024506067000001.jpg23155 [Figures 20A-20C]In vitro characterization of phenotype and function of gene-edited iNK cells. (A) Flow cytometry analysis of different iNK effector populations before in vivo administration. CAR and NK surface receptor expression is reflected as a percentage of the CD45+CD56+ double positive population. (B) ICE analysis of DGKα and ζ loci characterized by indel % performed on iNK cells generated from enriched TAG-72 CAR iPSC lines with DGKαζ KO (non-clonal derived) (n=2). (C) iNK functional killing assay: effectors were added to OVCAR-3 target cells at E:T ratios of 1:2 and 1:1 and monitored for 20 hours using the xCELLigence® system. NCI=Normalized cell index. [Figures 21A-21E] In vivo function of TAG-72 CAR and TAG-72 CAR / DGKαζ iNK cells. (A, B) Representative bioluminescence images (BLI) of immunocompromised NSG mice bearing luciferase-labeled OVCAR-3 tumor cells from two independent experiments. Images reflect several time points following administration of frozen-thawed NK cells, as indicated. (C) Quantification of tumor burden plotted as fold change in flux units (photons / sec) over time relative to baseline BLI readings on day -1 for each group of mice (shown as median BLI for each group). (D, E) Histograms showing individual mouse BLI signal (plotted as fold change relative to baseline) at each time point following treatment. n=4-5 mice / group, 3 independent experiments. Error bars represent the mean ± SEM for each group. [Figures 22A-22C]TAG-72 CAR+DGKαζ gene-edited iPSCs differentiated into iT cells. (A) Indel efficiency of DGKα and DGKζ in TAG-72 CAR iPSCs by TIDE analysis. (B) Flow cytometry analysis of T cell markers and CAR expression comparing non-edited iT cells to TAG-72 CAR+DGKαζ iT cells (non-clonally derived). (C) Cytotoxic function analysis of T cells isolated from peripheral blood mononuclear cell (PBMC) fraction of healthy donors compared to iT cells differentiated from TAG-72 CAR+DGKαζ KO iPSCs targeted against the ovarian cancer cell line OVCAR-3 (expressing TAG-72). [Figures 23A-23C] In vitro function of TAG-72 CAR iT / DGKαζ KO cells in the presence of TGFβ. (A) Schematic of the methodology implemented to evaluate the impact of TGFβ preconditioning on iT cell function in vitro. (B-C) iT cell in vitro cytotoxicity assay: Effectors ± TGFβ (B: 10 ng / mL, C: 100 ng / mL) were added to OVCAR-3 targets at a 1:1 E:T ratio and monitored for 40 hours using the real-time cell monitoring system xCELLigence®. The function of TAG-72 CAR-iT / DGKαζ KO cells (purple) was compared to the iT (non-transfected) control (grey). Target cells alone ± TGFβ (blue) were maintained and monitored in parallel to provide the growth kinetics of the OVCAR-3 cell line in the absence of effectors. Data were normalized to the time of effector cell addition and are presented as arbitrary units, normalized cell index. Each data point represents the mean ± SD of technical triplicates. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0042] Throughout this specification the word "comprise" or variations such as "comprises" or "comprising" will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

[0043] Throughout this specification and the claims, the terms "a" and "an" should be interpreted to mean "at least one" and not to exclude "two or more" unless the context clearly dictates otherwise.

[0044] "Nucleic acid construct" as used herein generally refers to a nucleic acid molecule that is artificially or recombinantly constructed or produced, and is also interchangeably referred to as a nucleic acid vector. For example, a nucleic acid construct can be produced to contain a nucleotide sequence of interest that is desired to be transcribed in a cell, in some instances to produce an RNA molecule of a desired function (e.g., antisense RNA, siRNA, miRNA, or gRNA), and in other instances to produce an mRNA that is translated into a protein of interest (e.g., a Cas protein). The nucleotide sequence of interest in the nucleic acid construct can be operably linked 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). The nucleic acid construct can be in a circular (e.g., a plasmid) or linear form, can be an integrated nucleic acid (i.e., can be integrated into the chromosome of a host cell, e.g., a viral vector, such as a lentiviral vector), or can remain episomal (e.g., a plasmid).

[0045] The term "about" or "approximately" should be understood as a ±10% variation from a given value.

[0046] The term "ex vivo" refers to a process in which cells are removed from a living organism and grown outside the body (eg, in a test tube).

[0047] The term "in vivo" refers to events that take place within a living organism (e.g., an animal, a plant, and / or a microorganism).

[0048] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0049] General Description Disclosed herein is a method for generating stem cell-derived immune cells with enhanced function. For example, it is demonstrated herein that the removal of one or more selected genes in stem cells using CRISPR / Cas9 gene editing does not destroy the ability of the stem cells to differentiate into immune cells. It is also demonstrated that the immune cells generated from these modified stem cells include enhanced persistence, antitumor activity in vitro, and resistance to the immunosuppressive effects of TGFβ. Thus, methods are provided by inhibiting the function of one or more selected genes in stem cells. Additionally, methods are provided for differentiating these stem cells into immune cells, such as NK cells and T cells. Also disclosed herein are modified stem cells and generated immune cells by the method, as well as the use of the immune cells in therapeutic treatments.

[0050] source cells "Source cell," as used herein, refers to a cell that is converted into a "derived cell" by reprogramming or differentiation. Examples of source cells suitable for use in the methods disclosed herein include stem cells.

[0051] The term "stem cell" should be understood to refer to any cell that, given its particular genetic makeup, exhibits the potential to develop along multiple lineages and then form a new organism or regenerate tissues or cell populations of an organism. The stem cells utilized in accordance with the present invention may be of any suitable type that can differentiate along two or more lineages, including, but not limited to, embryonic stem cells (ESCs), adult stem cells, umbilical cord stem cells, hematopoietic stem cells (HSCs), progenitor cells, progenitor cells, pluripotent cells, multipotent cells, or dedifferentiated somatic cells (such as induced pluripotent stem cells). "Pluripotent" means that the subject stem cells can self-renew and differentiate to form cells of any one of the three germ layers, which are, inter alia, ectoderm, endoderm, and mesoderm.

[0052] In some embodiments, the source cells also express at least one homozygous HLA haplotype. In some embodiments, the source cells express at least one homozygous HLA haplotype that is a major transplantation antigen and is preferably expressed by a significant proportion of the population, for example, at least 5%, at least 10%, at least 15%, at least 17%, at least 20%, or more of the population. If the homozygous HLA haplotype corresponds to a dominant MHC I or MHC II HLA type (for tissue rejection), the use of such cells significantly reduces the problem of tissue rejection in the broader population that receives the cells of the present invention in the context of a treatment regimen. In other embodiments, the source cells can be homozygous for two or more HLA antigens, for example, two, three, or more HLA antigens. HLA antigens of interest 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.

[0053] In some embodiments, the source cells are homozygous for the inhibited gene.

[0054] In some embodiments, the source cell is genetically modified in one or more genes identified herein such that function of the modified gene in a target cell differentiated from the genetically modified source cell is inhibited.

[0055] In some embodiments, the source cells are also genetically modified to contain a nucleic acid encoding a CAR (i.e., a chimeric antigen receptor). The nucleic acid encoding the CAR can be introduced into the source cells by methods known in the art, such as gamma-retroviral or lentiviral transduction, and CRISPR-Cas9, TALEN or ZFN-mediated gene editing (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; Sadelain et al., Therapeutic T cell engineering, 2017, Nature, 545: 423-431; Eyquem et al., Targeting a CAR to the TRAC locus with CRISPR / Cas9 enhances tumour rejection, 2017, Nature, 543: 113-17).

[0056] iPSC iPSCs are usually generated directly from somatic cells. iPSCs can in principle be derived from any nucleated cell, including mononuclear cells, for example from blood and skin cells. In some embodiments, iPSCs may be generated from fully differentiated T cells; or from precursor T cells (such as thymocytes), which have begun or even completed 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 (e.g., a rearranged TCR gene) against an antigenic determinant (e.g., a tumor antigenic determinant) of interest. In one embodiment, iPSCs are derived from cells expressing a rearranged TCR, preferably a rearranged αβTCR. In another embodiment, said cells express a rearranged γδTCR. 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 precursor T cells.

[0057] Methods for generating iPSCs from mature or differentiated cells (such as T cells or precursor T cells) are known to those skilled in the art. For example, iPSCs can be induced by introducing a specific set of pluripotency-associated genes or "reprogramming factors" into somatic cell types. A commonly used set of reprogramming factors (also known as Yamanaka factors) are the genes Oct4 (Pou5f1), Sox2, c-Myc, and Klf4. This combination may be the most conventional combination used to produce iPSCs, but each of the factors can be functionally replaced by unrelated genes such as related transcription factors, miRNAs, small molecules, or even lineage specification factors. For example, the induction of iPSCs after transfection of Oct3 / 4, Sox2, Klf4, and c-Myc using a retroviral system has also been achieved by transfection of Oct4, Sox2, Nanog, and Lin28 using a lentiviral system. The former set of transcription factors is known as Yamanaka factors, and the latter is commonly known as Thomson factors. As understood by those skilled in the art, extensive modifications have been made to basic reprogramming factor expression vectors, and new delivery modes have been designed to increase efficiency and minimize or remove vector sequences that may otherwise be integrated into the reprogrammed iPSC genome.These methods are well known to those skilled in the art, and include, but are not limited to, single cassette reprogramming vectors with Cre-Lox-mediated transgene excision, and reprogramming with non-integrating viruses such as adenovirus or Sendai virus.Alternatively, expression of reprogramming factors as proteins provides a means of generating iPSCs that have not undergone germline integration of introduced vector DNA.Non-viral reprogramming methods have also been developed.These include, but are not limited to, mRNA Transfection (Warren et al (2010)), miRNA Infection / Transfection Subramanyam et al (2011); PiggyBac (Kaji et al (2009); Woltjen et al (2009)); Minicircle Vectors (Narsinh et al (2011)) and Episomal Plasmids (Chuo et al (2011)). Various small molecules have been shown in the art to enhance reprogramming efficiency; see, for example, those listed in the table below. Compounds that increase iPSC reprogramming efficiency JPEG2024506067000002.jpg90156

[0058] In some embodiments, the source cells are induced pluripotent stem cells (iPSCs). In some embodiments, the iPSCs are derived by episomal or Sendai virus transformation.

[0059] In some embodiments, the iPSCs have a positivity rate of greater than 98% for SSEA-4 and TRA-1-60.

[0060] In some embodiments, the subject source cells are cells that are more differentiated toward immune cells as compared to pluripotent stem cells.

[0061] In some embodiments, stem cells can be genetically modified using non-viral methods. In some embodiments, iPSCs can be modified using non-viral methods, such as CRISPR / Cas editing systems.

[0062] In some embodiments, chimeric antigen receptors (CARs) can be introduced into iPSCs using non-viral editing systems, such as CRISPR-Cas9.

[0063] In some embodiments, knockout (KO) of genetic material can be engineered in iPSCs using non-viral editing systems, such as CRISPR-Cas9. In some embodiments, the KO of either DGKα and / or DGKζ genes in iPSCs is achieved using non-viral editing systems, such as CRISPR-Cas9. In some embodiments, iPSCs comprise a double KO of DGKαζ genes, and the genetic engineering is performed using non-viral editing systems, such as CRISPR-Cas9.

[0064] In some embodiments, the iPSC-source cells are genetically modified to inhibit expression of the DGKαζ gene (DGKαζ KO iPSCs).

[0065] In some embodiments, the iPSC-source cells are genetically modified to express a chimeric antigen receptor (CAR), wherein the receptor comprises an antigen recognition moiety for an antigenic determinant, the antigenic determinant being an antigenic determinant of the tumor-associated antigen TAG-72 (TAG-72 CAR iPSC).

[0066] In some embodiments, iPSC source cells are genetically modified to inhibit expression of the DGKαζ gene and to further express the TAG-72 CAR (TAG-72 CAR / DGK KO iPSC). In some embodiments, such genetically modified iPSCs maintain pluripotency and the ability to grow in culture.

[0067] In some embodiments, the iPSC source cells can differentiate into CD34+ cells. In some embodiments, both the DGK KO iPSCs and / or the TAG-72 CAR / DGK KO iPSCs can differentiate into CD34+ hematopoietic progenitor cells.

[0068] In some embodiments, the iPSC source cells can differentiate into CD45+ CD56+ NK cells. In some embodiments, the DGK KO iPSCs and / or TAG-72 CAR / DGK KO iPSCs can differentiate into mature NK cells expressing normal NK stimulatory receptors.

[0069] In some embodiments, the iPSC source cells can differentiate into precursor T cells. In some embodiments, both the DGK KO iPSCs and / or the TAG-72 CAR / DGK KO iPSCs can differentiate into mature CD3+ T cells with a predominantly CD8+ phenotype. In some embodiments, both the DGK KO iPSCs and / or the TAG-72 CAR / DGK KO iPSCs can differentiate into mature CD3+ T cells expressing either TCRαβ or TCRγδ.

[0070] In some embodiments, the CAR is introduced into the iPSCs using a non-viral editing system, such as CRISPR-Cas9. In some embodiments, the resulting gene-edited iPSCs can be differentiated into CD34+ cells and / or T cell progenitors.

[0071] In some embodiments, iPSC-derived CD34+ cells can be differentiated into NK cells (iNK cells). In some embodiments, both DGK KO iPSC-derived CD34+ cells and / or TAG-72 CAR / DGK KO iPSC-derived CD34+ cells can be differentiated into NK cells without significantly affecting the efficiency of the differentiation process.

[0072] In some embodiments, either the single DGKα / DGKζ or double DGKαζ KO in iPSCs and / or TAG-72 CAR iPSCs does not affect the normal phenotype of said iPSC-derived NK cells and / or TAG-72 CAR iPSC-derived NK cells following differentiation.

[0073] In some embodiments, either the single DGKα / DGKζ or double DGKαζ KO in iPSCs and / or TAG-72 CAR iPSCs does not affect the normal phenotype of said iPSC-derived T cells and / or TAG-72 CAR iPSC-derived T cells following differentiation.

[0074] mesodermal cells Three major cell populations (primary germ layers) emerge during embryonic development: mesoderm, ectoderm, and endoderm. These cell populations are formed through a process known as gastrulation, following which each primary germ cell layer generates a specific set of cell populations and tissues. For example, the mesendoderm or mesoderm population gives rise to the heart, blood vessels, and blood cells (e.g., immune cells).

[0075] To form immune cells, mesodermal cells are induced into multiple differentiation stages, each represented by a subpopulation of cells with distinct potential / phenotype that can be broadly classified into hemogenic endothelium (HE), hematopoietic stem cells (HSC), progenitor cells and immune cells. Each differentiation stage brings the cell closer to its final cell type, for example, by altering its gene expression that limits its potential to become a different cell type.

[0076] In some embodiments, the subject source cells are mesodermal cells that can differentiate into immune cells.

[0077] hematopoietic cells The term "hematopoietic cell" should be understood as any cell differentiated from mesodermal cells (which can be obtained from pluripotent cells), including, for example, HE, pre-HSC and HSC, and progenitor cells (e.g., pluripotent progenitor cells, lymphoid progenitor cells such as common lymphoid progenitor cells, early thymic progenitor cells, pre-T cell progenitor cells, pre-NK progenitor cells, T progenitor cells, NK progenitor cells, and myeloid progenitor cells such as common myeloid progenitor cells). The term "HE / HSC" is used herein to refer to a subclass of hematopoietic cells. HE / HSC is a CD34+ stem cell that can give rise to both myeloid (e.g., macrophages and monocytes) and lymphoid cell types (e.g., B cells, T cells or NK cells), including HE, pre-HSC and HSC. iCD34+ cells represent CD34+ expressing cells differentiated from iPSCs.

[0078] During embryogenesis, pluripotent mesodermal cells differentiate into an endothelial cell lineage. HE are a subset of this lineage that acquire hematopoietic potential and give rise to multilineage hematopoietic stem and progenitor cells in a process called the endothelium-to-hematopoietic transition. The processes that lead to the hematopoietic specification of endothelial cells and the generation of HSCs from hemogenic endothelium remain under debate.

[0079] HSCs are blood stem cells that theoretically have the ability to become any blood cell of the lymphoid and myeloid lineages through the hematopoietic process. HSCs can be found in adult bone marrow, peripheral blood, and umbilical cord blood. HSCs can be collected from bone marrow, peripheral blood, and umbilical cord blood by established techniques and are generally associated with CD34+ expression.

[0080] In some embodiments, human HSCs can be defined as CD34+ CD38- CD90+ CD45RA-. In some embodiments, human HSCs can be defined as CD34+ CD43+ CD45+. In some embodiments, human HSCs can be defined as CD34+ CD133+.

[0081] When HSCs are derived from pluripotent stem cells, they are often called iHSCs (induced hematopoietic stem cells).

[0082] "Pre-HSC" should be understood as cells that have differentiated from HE cells but do not express typical HSC markers, for example CD45- cells that express CD34.

[0083] In some embodiments, cells cultured from pluripotent stem cells (such as iPSCs) that have undergone some differentiation into immune cells in culture, but have not fully differentiated into immune cells, are used as source cells.

[0084] In some embodiments, hematopoietic cells that can differentiate into immune cells are used as source cells.In some embodiments, the hematopoietic cells have not yet differentiated into hematopoietic stem cells (HSCs), for example, hematopoietic endothelium (HE) or pre-HSCs.In some embodiments, the hematopoietic cells are HSCs.

[0085] In some embodiments, the subject source cells are myeloid progenitor cells, eg, common myeloid progenitor cells.

[0086] In some embodiments, the subject source cells are lymphoid precursor cells, such as multipotent progenitor cells, common lymphoid progenitor cells, early thymic progenitor cells, pre-T cell progenitor cells, pre-NK progenitor cells, T cell progenitor cells, or NK cell progenitor cells (Galen et al., The unfolded protein response governs integrity of the haemopoietic stem cell pool during stress, Nature, 2014, Vol.510(7504), p.268). In some embodiments, the subject source cells are immature T cells, such as thymocytes, or immature NK cells.

[0087] derived cells The stem cell-derived immune cells generated by the method disclosed herein include hematopoietic cells that can differentiate into immune cells, and immune cells. Examples of stem cell-derived immune cells are HE, pre-HSC, HSC, multipotent progenitor cells, lymphoid progenitor cells such as common lymphoid progenitor cells, early thymic progenitor cells, pre-T cell progenitor cells, pre-NK progenitor cells, T cell progenitor cells, NK cell progenitor cells, myeloid progenitor cells such as common myeloid progenitor cells, and immune cells.

[0088] "Immune cells", as used herein, should be understood to include cells of the mammalian immune system, such as lymphocytes (T cells, B cells and NK cells), neutrophils, and monocytes (including macrophages and dendritic cells), as well as cell lines derived from cells of the mammalian immune system.

[0089] The present disclosure provides immune cells derived from stem cells produced by differentiation, which have improved function. By "enhanced function" is meant that the immune cells provided as a result of the modifications or manipulations disclosed herein exhibit enhanced activity (e.g., cytotoxicity), proliferation, survival, persistence, resistance to immunosuppressive effects (e.g., TGFβ inhibition) and / or infiltration compared to control immune cells (i.e., immune cells without modification or manipulation). Immune cell cytotoxicity generally refers to the ability of immune cells to kill target cells via receptor-based mechanisms.

[0090] In some embodiments, stem cell derived immune cells according to the present methods can be differentiated from stem cells or other more differentiated cells (such as cells cultured and differentiated from stem cells).

[0091] In some embodiments, the stem cell-derived immune cell is a T cell. In some embodiments, the T cell is a NKT cell. In some embodiments, the stem cell-derived immune cell is a NK cell. In other embodiments, the stem cell-derived immune cell is a macrophage or a macrophage lineage cell (e.g., monocyte, dendritic cell).

[0092] T cells Reference to "T cells" should be understood as a reference to any cell that contains a T cell receptor. In this regard, the T cell receptor may include any one or more of α, β, γ or δ chains. As will be appreciated by those skilled in the art, NKT cells also express T cell receptors, and thus target antigen-specific NKT cells can also be generated in accordance with the present invention (understood to be included in the definition of T cells). While the present invention is not intended to be limited to any particular subclass of T cells, in one embodiment, the subject T cells express an α / β TCR dimer. In some embodiments, the T cells are CD4+ helper T cells, CD8+ killer T cells, or NKT cells. Without limiting the present invention to any one theory or mechanism of action, CD8+ T cells are also known as cytotoxic cells. As a major part of the adaptive immune system, CD8+ T cells primarily scan the intracellular environment to target and destroy infected cells. Small peptide fragments derived from the intracellular contents are processed and transported to the cell surface where they are presented in the context of MHC class I molecules. However, in addition to responding to viral infections, CD8+ T cells also provide an additional level of immune surveillance by monitoring and eliminating damaged or abnormal cells, including cancer. CD8+ T cell recognition of MHC I-presented peptides usually results in either the release of cytotoxic granules or lymphokines, or the activation of apoptotic pathways via FAS / FASL interactions to destroy the targeted cells. On the other hand, CD4+ T cells generally recognize peptides presented by antigen-presenting cells in the context of MHC class II, resulting in the release of cytokines designed to regulate B cell and / or CD8+ T cell immune responses. CD4+ T cells with cytotoxic activity have also been observed in a variety of immune responses.Furthermore, CD4+ CAR-T cells exhibit comparable cytotoxicity to CD8+ CAR-T cells in vitro and outperform CD8+ CAR-T cells in vivo with longer antitumor activity (see, e.g., Wang et al., Glioblastoma-targeted CD4+ CAR T cells mediate superior antitumor activity, JCI Insight, 2018, 3(10):e99048; Yang et al., TCR engagement negatively affects CD8 but not CD4 CAR T cell expansion and leukemic clearance, Science Translation Medicine, 2017 Nov, 22; 9(417), eaag1209).

[0093] In some embodiments, the stem cell-derived immune cells are cytotoxic immune cells, e.g., cytotoxic lymphocytes.

[0094] In some embodiments, the stem cell-derived immune cells are lymphoid cells. In some embodiments, the lymphoid cells are T cells. In some embodiments, the T cells are CD4+ helper T cells, CD8+ killer T cells, or NKT cells.

[0095] In some embodiments, the stem cell-derived T cells disclosed herein exhibit anti-tumor activity in vitro. In some embodiments, iPSC-derived TAG-72 CAR / DGKαζ KO T cells exhibit on-target activity against ovarian cancer cell lines in vitro. In some embodiments, TAG-72 CAR / DGKαζ KO T cells exhibit enhanced killing of a human ovarian cancer cell line (OVCAR-3) compared to non-transfected iPSC-derived T cells and non-transfected T cells isolated from PBMCs.

[0096] In some embodiments, iPSC-derived T cells retain function in the immunosuppressive microenvironment of tumors. In some embodiments, iPSC-derived DGKαζ KO T cells have reduced sensitivity to the suppressive effects of tumors, as demonstrated, for example, by such cells retaining in vitro cytotoxic function against OVCAR-3 cells in the presence of TGFβ, i.e., one of the key mediators of immunosuppression in the tumor microenvironment.

[0097] NK Cells Natural killer T cells (also called NKT or T / NK cells) are a specialized population of T cells that express the semi-invariant T cell receptor (TCRαβ) and surface antigens typically associated with natural killer cells. The TCR on NKT cells is unique in that it generally recognizes glycolipid antigens presented by the MHC I-like molecule CD1d. Most NKT cells express the invariant TCRα chain and one of a small number of TCRβ chains. The TCR present on type I NKT cells generally recognizes the antigen α-galactosylceramide (α-GalCer). Within this group, CD4 + CD8 - cells, CD4 - CD8 + Cells and CD4 - CD8 - Distinguishable subpopulations, including type II NKT cells, have been identified. Type II NKT cells (or non-invariant NKT cells) express a broader range of TCRα chains and do not recognize the α-GalCer antigen. NKT cells produce cytokines that have multiple, often opposing effects, for example, promoting inflammation or inducing immunosuppression, including tolerance. As a result, they can contribute to antibacterial and antiviral immune responses, promote tumor-associated immune surveillance, and inhibit or promote the development of autoimmune diseases. Like natural killer cells, NKT cells can also induce perforin-, Fas-, and TNF-related cytotoxicity. Thus, reference to T cells should be understood to include reference to NKT cells.

[0098] Natural killer (NK) cells are a type of cytotoxic lymphocyte that form part of the innate immune system. NK cells provide a rapid response against virus-infected cells, acting about 3 days after infection, and also respond to tumor formation. Typically, immune cells such as T cells detect major histocompatibility complexes (MHC) presented on the infected or transformed cell surface, triggering cytokine release, resulting in lysis or apoptosis of the target cell. However, NK cells have the ability to recognize stressed cells in the absence of antibodies or MHC, allowing for a much faster immune response. This role is particularly important, since harmful cells that are deficient or have lower than normal levels of MHC I markers 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 usually express the surface markers CD16 (FcγRIII) and CD56. In some embodiments, the stem cell-derived immune cells are NK cells.

[0099] In some embodiments, the present disclosure provides stem cell-derived NK cells with enhanced function. In some embodiments, iPSC-derived DGKαζ KO NK cells show enhanced functionality and survival time in long-term co-culture with target cancer cells in vitro compared to their respective non-transfected iPSC-derived NK control cells. The importance of DGKαζ KO to the enhanced survival and cytotoxic antitumor function of iPSC-derived NK cells is established herein.

[0100] In some embodiments, iPSC-derived TAG-72 CAR / DGKαζ KO NK cells exhibit enhanced killing of tumor cells in vitro (compared to unedited iPSC-derived NK cells and NK cells isolated from the peripheral blood mononuclear cell (PBMC) fraction of healthy adult donors).

[0101] In some embodiments, disclosed herein are stem cell-derived NK cells with improved proliferation capacity. In some embodiments, disclosed herein are iPSC-derived DGKαζ KO NK cells that exhibit improved proliferation capacity compared to non-transfected iPSC-derived NK cells.

[0102] In some embodiments, iPSC-derived NK cells retain function in the immunosuppressive microenvironment of a tumor. Disclosed herein, in some embodiments, are iPSC-derived DGKαζ KO NK cells that have reduced sensitivity to the suppressive effects of a tumor, as demonstrated, for example, by such cells retaining in vitro cytotoxic function in the presence of TGFβ, one of the key mediators of immunosuppression in the tumor microenvironment.

[0103] In some additional embodiments, disclosed herein are iPSC-derived TAG-72 / DGKαζ KO NK cells that retain function in the immunosuppressive microenvironment of a tumor. In some embodiments, the iPSC-derived TAG-72 / DGKαζ KO NK cells disclosed herein are capable of inducing the killing of tumor cells, such as the OVCAR-3 cell line, under conditions representative of an immunosuppressive microenvironment. In some embodiments, the iPSC-derived TAG-72 / DGKαζ KO NK cells retain in vitro cytotoxic function in the presence of TGFβ.

[0104] In some embodiments, the stem cell-derived NK cells disclosed herein exhibit anti-tumor activity in vivo. It is disclosed herein that treatment of tumor-bearing mice with iPSC-derived DGKαζ KO NK cells resulted in reduced tumor size and improved survival compared to treatment with unedited (non-transfected) iPSC-derived NK cells, with or without cytokine co-administration.

[0105] In some embodiments, the stem cell-derived TAG-72 CAR / DGKαζ KO NK cells disclosed herein exhibit long-term anti-tumor activity in vivo. It is shown herein that treatment of tumor-bearing mice with iPSC-derived TAG-72 CAR / DGKαζ KO NK cells results in lower average tumor burden and superior long-term efficacy compared to either PBMC-NK cells, unedited (non-transfected) iPSC-derived NK cells, or TAG-72 CAR iPSC-derived NK cells.

[0106] Inhibited genes According to the present disclosure, stem cell-derived immune cells with enhanced function can be generated by modifying a source cell to inhibit the function of one or more genes identified herein and differentiating the modified source cell into a stem cell-derived immune cell.

[0107] As used herein, "inhibition of the function of a gene" means that the level and / or activity of the protein encoded by the gene is ultimately reduced or eliminated. Thus, the function of a gene can be inhibited as a result of manipulation or modification of the genomic DNA sequence of the gene (e.g., resulting in gene disruption), as a result of inhibiting mRNA (e.g., by inhibiting transcription or translation, thereby reducing the level or function of mRNA), or as a result of inhibiting protein (e.g., by reducing the level or activity of protein). In some embodiments, the degree of inhibition is at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more when the level and / or activity of the protein encoded by the gene in modified cells is compared with the level and / or activity of the protein in unmodified cells.

[0108] According to the present disclosure, the gene whose function is inhibited is selected from the group consisting of DGKα and DGKζ.

[0109] DGKα and DGKζ Diacylglycerol kinase (DGK) is an enzyme that phosphorylates diacylglycerol (DAG) to phosphatidic acid (PA). There are multiple isoforms, two of which, DGKα and DGKζ, have strong functional links to immune cells. In both T and NK cells, DAG is one of the main messengers of activation and function, and DGKα and DGKζ act as a novel class of immune “checkpoints” that can reduce DAG-dependent activation (Riese et al., Diacylglycerol Kinases (DGKs): Novel Targets for Improving T Cell Activity in Cancer, Frontiers in cell and developmental biology, 2016, Vol.4, pp.108; Noessner, DGK-alpha: A Checkpoint in Cancer-Mediated Immuno-Inhibition and Target for Immunotherapy, Frontiers in cell and developmental biology, 2017, Vol.5, pp.16). DAG interacts with essential proteins involved in CD3 signaling, such as protein kinase C (PKC) and Ras activating protein (RasGRP1). Thus, activation of DGKs is thought to result in the downregulation of TCR-distal molecules, including extracellular signal-related kinase 1 / 2 (ERK1 / 2).DGKα and DGKζ are predominantly expressed in T cells and NK cells, and their functions do not appear to be completely overlapping, as their expression and activation are regulated in different manners.

[0110] According to the present disclosure, stem cells in which the function of at least one of the DGKα and DGKζ genes is inhibited are still capable of differentiation, and stem cell-derived immune cells differentiated from the modified stem cells include enhanced function.

[0111] Inhibition of gene function Inhibition of gene function can be achieved by a variety of approaches, for example, through gene editing, inhibition of translation via, for example, RNA interference or antisense oligonucleotides, or through the use of compounds such as small molecules or antibodies that directly antagonize the protein product.

[0112] Inhibition by gene editing In some embodiments, inhibition of gene function is achieved through the use of a gene editing system to modify the genomic sequence of the gene.

[0113] A gene editing system typically includes a DNA-binding protein or a DNA-binding nucleic acid coupled with a nuclease. The DNA-binding protein or the DNA-binding nucleic acid specifically binds or hybridizes to a target region of a gene, and the nuclease makes one or more double-strand breaks and / or one or more single-strand breaks in the target region of the gene. The target region may be a coding region of a gene, for example, an exon, near the N-terminal part of the coding region (e.g., within the first or second exon). The double-strand or single-strand break may undergo repair through a cellular repair process, such as by non-homologous end joining (NHEJ) or homology-directed repair (HDR). In some examples, the repair process introduces an insertion, deletion, missense mutation, or frameshift mutation (including, for example, biallelic frameshift mutation), leading to gene disruption and inhibition of gene function.

[0114] Examples of gene editing systems include fusions that comprise DNA binding proteins and nucleases, such as zinc finger nucleases (ZFN) or TAL effector nucleases (TALEN), or RNA-guided nucleases, such as clustered regularly interspaced short palindromic nucleic acid (CRISPR) / Cas systems.However, the present invention should not be limited to these types of gene editing systems.Rather, any type of inhibitor known or identified in the art can be used to inhibit the function of genes.

[0115] ZFPs and TALENs In some embodiments, inhibition of gene function is achieved by utilizing a gene editing system that includes one or more zinc finger proteins (ZFPs) or DNA binding proteins such as transcription activator-like proteins (TALs) fused to endonucleases. Examples include ZFNs, TALEs, and TALENs.

[0116] The DNA binding domain 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 or TALE protein can be modified to direct binding to a predetermined DNA sequence. Criteria for rational 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, WO98 / 53058, WO98 / 53059, WO98 / 53060, WO02 / 016536, WO03 / 016496, and U.S. Patent Application Publication No. 20110301073 A1.

[0117] In some embodiments, the DNA binding protein comprises a zinc finger protein (ZFP) or one or more zinc finger domains of a ZFP. A ZFP or a domain thereof binds to DNA in a sequence-specific manner through one or more "zinc fingers" (a region of amino acids in a binding domain whose structure is stabilized through the coordination of a zinc ion). The sequence specificity of naturally occurring ZFPs can be altered by amino acid substitutions at specific positions on the zinc finger recognition helix. In addition, many engineered gene-specific zinc fingers are commercially available (see, for example, the CompoZr platform for zinc finger construction developed by Sangamo Biosciences (Richmond, CA, USA) in partnership with Sigma-Aldrich (St. Louis, MO, USA)). Thus, in some embodiments, a ZFP is engineered to bind to a target sequence within a gene identified herein to be inhibited. Exemplary target sequences include exons, regions near the N-terminal region of the coding sequence (e.g., first exon, second exon), and 5'regulatory regions (promoter or enhancer regions). ZFPs are fused to an endonuclease or DNA cleavage domain to form zinc finger nucleases (ZFNs). Examples of DNA cleavage domains include the DNA cleavage domains of type IIS restriction enzymes.

[0118] In some embodiments, ZFN is introduced into cells (e.g., stem cells) via transfection of a nucleic acid construct (e.g., a plasmid, an mRNA or a viral vector) that contains a nucleic acid sequence encoding ZFN.The ZFN is then expressed in cells from the construct, resulting in the editing and destruction of target genes.In some embodiments, ZFN is introduced into cells in its protein form.

[0119] In some embodiments, the DNA binding protein comprises a naturally occurring or engineered transcription activator-like protein (TAL) DNA binding domain, such as in a transcription activator-like protein effector (TALE) protein. See, for example, US 20110301073 A1, which is incorporated herein by reference. A TALE DNA binding domain is a polypeptide that comprises one or more TALE repeats, each repeat being 33-35 amino acids in length and containing one or two DNA binding residues. It has been determined that the HD sequence at positions 12 and 13 of the TAL repeat results in binding to cytosine (C), NG binds to T, NI binds to A, and NN binds to G or A. See US 20110301073 A1. In some embodiments, TALEs can be designed to have an array of TAL repeats with specificity for a target DNA sequence of interest within a gene identified herein to be inhibited. Custom-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 nucleic acid target sequence within the gene identified herein to be inhibited.

[0120] In some embodiments, TALEN is introduced into cells (e.g., stem cells) through transfection of nucleic acid constructs (e.g., plasmids, mRNA or viral vectors) that contain the nucleic acid sequence encoding TALEN.Then, TALEN is expressed from constructs in cells, leading to the editing and destruction of target genes.In some embodiments, TALEN is introduced into cells in its protein form.

[0121] CRISPR / Cas In some embodiments, inhibition of gene function is achieved by utilizing the CRISPR (short for "Clustered Regularly Interspaced Short Palindromic Repeats") / Cas (short for "CRISPR-associated nuclease") system for gene editing. CRISPR / Cas is well known in the art, and reagents and protocols are readily available (Mali et al., RNA-Guided Human Genome Engineering via Cas9, Science, Feb 15, 2013, Vol.339(6121), p.823(4); Hsu et al., Development and applications of CRISPR-Cas9 for genome engineering, Cell, 05 June 2014, Vol.157(6), pp.1262-1278; Jiang et al., RNA-guided editing of bacterial genomes using CRISPR-Cas systems, Nature Biotechnology, 2013, Vol.31(3), p.233; Anzalone et al., Search-and-replace genome editing without double-strand breaks or donor DNA, Nature, December 2019, Vol.576(7785), pp.149-157; Komor et al., Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage, 2016, Nature 533: 420-424; Gaudelli et al., Programmable base editing of A·T to G·C in genomic DNA without DNA cleavage, Nature, 2017, Vol.551(7681), p.464).Exemplary CRISPR / Cas gene editing protocols are described in Jennifer Doudna, and Prashant Mali, CRISPR-Cas: A Laboratory Manual, 2016 (CSHL Press, ISBN: 978-1-621821-30-4) and Ran, et al., Genome engineering using the CRISPR-Cas9 system, Nature Protocols, 2013, Vol.8(11), p.2281.

[0122] CRISPR / Cas systems generally include two components: (1) an RNA-dependent DNA nuclease, also referred to herein as CRISPR endonuclease or Cas protein, e.g., Cas9, Cas12, or other alternative nucleases; and (2) a non-coding short "guide RNA" that includes either a duplex RNA including crRNA ("CRISPR RNA") and tracrRNA ("trans-activating crRNA") or a single-stranded full-length guide RNA, and includes a targeting sequence that directs the nuclease to a target site in the genome. The guide RNA (gRNA) directs the nuclease to the target site, where the nuclease generates a double-strand break (DSB) in the DNA at the target site. The resulting DSB is then repaired by one of two general repair pathways: the non-homologous end joining (NHEJ) pathway and the homology-directed repair (HDR) pathway. The NHEJ repair pathway is the most active repair mechanism that can rapidly repair DSBs, but frequently results in small nucleotide insertions or deletions (indels) at the DSB site, resulting in frameshift mutations that lead to the production of non-functional gene products. The HDR pathway is less efficient but has high fidelity. If the CRISPR endonuclease is provided with a DNA template homologous to the cut region, the double-stranded break is repaired using the homologous DNA template via HDR. The HDR pathway allows the insertion of large gene inserts into cells along with the RNP.

[0123] Design or selection of gRNA sequence comprising sequence that targets target site in gene of interest has been described in the art.Target site can include sequences of regulatory regions (such as promoters and enhancers) and sequences in coding regions (such as exons, e.g. exons near the 5' end or exons that code for specific domains or regions of protein).In some embodiments, target site is selected based on its position, typically immediately 5' of protospacer adjacent motif (PAM) sequence such as NGG or NAG.

[0124] The guide sequence is designed to include a targeting sequence that has complementarity with the target sequence (the base sequence of the target site). Perfect complementarity is not necessarily required, as long as there is sufficient complementarity between the guide sequence and the target sequence to cause hybridization and promote the formation of CRISPR complexes at the target site. In some embodiments, the degree of complementarity between the targeting sequence of the gRNA and the target sequence is at least 80%, 85%, 90%, 95%, 98%, 99% or more (e.g., 100% or perfect complementarity).

[0125] In some embodiments, the guide sequence is at least 15 nucleotides, e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, or 75 or more nucleotides in length. In some embodiments, the guide sequence is 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, or 20 nucleotides in length or less. In some embodiments, the targeting sequence portion of the guide sequence is about 20 nucleotides in length. Truncated gRNAs with shorter regions of target complementarity (less than 20 nucleotides) have been described as effective with improved target specificity (see, for example, Fu et al., Improving CRISPR-Cas nuclease specificity using truncated guide RNAs, Nature Biotechnology, 2014, Vol.32(3), p.279). Thus, 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 fully complementary to the nucleotide sequence at the target site. In some embodiments where the targeting sequence of the guide RNA is not fully complementary to the nucleotide sequence at the target site, the portion of the targeting sequence close to the PAM sequence in the genome (also called the seed region) is fully complementary to the nucleotide sequence at the target site. In other words, some variation in the nucleotides 5' of the guide sequence (i.e., the non-seed region) is tolerated. For example, a guide sequence can be designed to include a targeting portion that is at least 17 nucleotides in length (e.g., 17, 18, 19, or 20 nucleotides in length).

[0126] Examples of target sequences in specific genes are provided in Table 1.

[0127] In some embodiments, the guide sequence comprises a targeting sequence of 17-20 nucleotides, wherein at least 17 nucleotides in the seed region (the 3' portion of the targeting sequence) are perfectly complementary to at least 17 nucleotides in the target sequence, e.g., 17 nucleotides from the 3' end of the target sequence. [Table 1]

[0128] A gRNA database for CRISPR genome editing is publicly available, which provides exemplary sgRNA target sequences in the constitutive exons of genes in the human or mouse genome (see, for example, the gRNA database provided by GenScript and the Massachusetts Institute of Technology; see also Sanjana et al., Improved vectors and genome-wide libraries for CRISPR screening, Nature Methods, 2014, Vol. 11(8), p. 783). In some embodiments, the gRNA sequence is or includes a sequence with minimal off-target binding to non-target genes.

[0129] Examples of Cas proteins or CRISPR endonucleases suitable for use herein include Cpf1 (Zetsche et al., Cpf1 Is a Single RNA-Guided Endonuclease of a Class 2 CRISPR-Cas System, Cell (Cambridge), 22 October 2015, Vol.163(3), pp. 759-771), Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas12, Cas13, 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, CasX, CasY, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, or Csf4, or functional derivatives thereof (i.e., mutant forms or derivatives of naturally occurring CRISPR endonucleases, e.g., fragments thereof, that substantially retain the RNA-dependent endonuclease activity of the naturally occurring form). See, e.g., US20180245091 A1 and US20190247517 A1. In some embodiments, the Cas protein is Cas9, e.g., Cas9 from S. pyogenes, S. aureus, or S. pneumoniae. In some embodiments, the Cas protein is the Cas9 protein from S. pyogenes, which has an amino acid sequence provided in the SwissProt database under accession number Q99ZW2. Many Cas proteins identified after CRISPR-Cas9 provide desirable features. For example, CRISPR-Cas12 can perform staggered cuts and edit the epigenome (chemical compounds that can turn genes on or off). Cas13 affects gene expression by targeting RNA instead of DNA.CRISPR-CasX is smaller than Cas9 and can be used to control gene expression as well as edit genes, while CasY acts like Cas9 but consists of a completely different protein structure, allowing it to function in different conditions.

[0130] In some embodiments, the inhibition of the function of the gene is achieved by CRISPR-mediated gene editing, which includes introducing into a cell (e.g., a pluripotent stem cell, or an iPSC, or an HE, or an HSC, or a progenitor cell) a first nucleic acid encoding a Cas nuclease and a second nucleic acid encoding a guide RNA (gRNA) specific to a target sequence in the gene identified herein to be inhibited. The two nucleic acids can be included in one nucleic acid construct (or vector) or provided on different constructs (or vectors) to achieve expression of the Cas protein and gRNA in the cell. Expression of the Cas nuclease and gRNA in the cell leads to the formation of a CRISPR complex at the target sequence, which results in DNA cleavage.

[0131] In some embodiments, the inhibition of gene function is achieved by CRISPR-mediated gene editing, comprising the introduction of a combination or complex of gRNA and Cas nuclease into cells.In some embodiments, the combination or complex of Cas protein / gRNA can be delivered to cells via, for example, electroporation, particle gun, calcium phosphate transfection, cell compaction or squeezing, liposome, nanoparticle, microinjection, naked DNA plasmid transfer, protein transduction domain-mediated transduction or virus-mediated (including integrative viral vectors such as retrovirus and lentivirus, and non-integrative viral vectors such as adenovirus, AAV, HSV, vaccinia).

[0132] Regardless of the particular gene editing method used, a variety of assays can be performed to confirm that the gene sequence has been altered and gene function inhibited, including, for example, by examining DNA or mRNA via Southern and Northern blotting, PCR including RT-PCR, or nucleic acid sequencing, or by detecting the presence or activity of specific proteins or peptides, for example, via immunological means (ELISA and Western blots).

[0133] In some embodiments, the function of at least one of the DGKα and DGKζ genes is inhibited by introducing an indel(s) into at least one early exon of these genes via the CRISPR / Cas9 system, which results in a frameshift mutation(s) in at least one of these genes so that no functional protein is translated from the edited gene. In some embodiments, the function of two genes is inhibited by introducing an indel into the early exon of the two genes using CRISPR / Cas9, which results in a frameshift mutation in the two genes so that no functional protein is translated from the edited gene. In some embodiments, the inhibition of one of the two genes is performed in combination with the inhibition of another gene.

[0134] The CRISPR / Cas system can also be used without double-strand breaks or donor DNA by using nickases (i.e., CAS9 nickase) and high fidelity enzymes. See, for example, Anzalone, A et al., Search-and-replace genome editing without double-strand breaks or donor DNA, Nature, December 2019, Vol.576(7785), pp.149-157; Komor et al., Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage, Nature, 2016, 533: 420-424; Gaudelli et al., Programmable base editing of A·T to G·C in genomic DNA without DNA cleavage, Nature, 2017, Vol.551(7681), p.464.

[0135] Inhibition by reducing or eliminating mRNA levels 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 inhibiting mRNA. Unlike inhibition by gene editing systems, inhibition of mRNA is transient.

[0136] In some embodiments, inhibition of mRNA can be achieved, for example, by the use of antisense nucleic acids, ribozymes, small interfering RNA (siRNA), small hairpin RNA (shRNA), miRNA (microRNA) or precursors thereof, or nucleic acid constructs that can be transcribed within a cell to produce antisense RNA, siRNA, shRNA, miRNA or precursors thereof.

[0137] Antisense-Antisense technology is a well-known method.Antisense RNA is an RNA molecule that is complementary to the full length or part of endogenous mRNA and blocks translation from endogenous mRNA by forming a double strand with endogenous mRNA.Antisense RNA can be synthetically produced and introduced into a cell of interest (e.g., stem cell), or can be produced in a cell of interest via transcription from an exogenously introduced nucleic acid construct to achieve inhibition of expression of a gene of interest.Antisense RNA does not need to be complementary to the full length mRNA from a gene of interest.However, antisense RNA should be long enough to form a double strand with target mRNA and block translation based on target mRNA. Typically, the antisense RNA is at least 15 nucleotides in length, e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 30, 35, 40, 50, 75, 100, 200, 300, 400, 500 or more nucleotides in length. In some embodiments, the antisense RNA is less than 500, 400, 300, 200, 100, 75 or 50 nucleotides in length.

[0138] Ribozymes- Ribozymes (i.e., catalytic RNAs) can be designed to specifically pair with target RNA and cleave the phosphodiester backbone at a specific position, thereby functionally inactivating the target RNA.See, for example, U.S. Patent No. 6,423,885, U.S. Patent No. 5,254,678, and Perriman et al., Effective ribozyme delivery in plant cells, Proceedings of the National Academy of Sciences of the United States of America, June 20, 1995, Vol.92(13), pp.6175-6179. Ribozymes can be synthetically produced and introduced into cells of interest (e.g., stem cells), or produced in cells of interest via transcription from exogenously introduced nucleic acid constructs.

[0139] RNAi (RNA interference) - Inhibition of gene expression or translation by RNAi is known in the art and can be achieved utilizing RNA molecules such as siRNA ("small interfering RNA"), shRNA ("short hairpin RNA"), and miRNA ("microRNA"). siRNA and shRNA are known to be involved in the RNA interference pathway and interfere with the expression of specific genes. siRNAs are small (typically 20-25 nucleotides long) double-stranded RNAs that can be designed to contain sequences that are homologous or complementary to a target mRNA (i.e., mRNA transcribed from a gene of interest) or a portion of a target mRNA. shRNAs are cleaved by DICER, a ribonuclease, to produce siRNAs. Given the sequence of the target gene, siRNAs or shRNAs can be synthetically designed and produced and introduced into a cell of interest (e.g., stem cells) or can be produced in a cell of interest (e.g., stem cells) from an exogenously introduced nucleic acid construct encoding such an RNA. miRNAs are also 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 imprecise base pairing with target mRNAs. miRNAs or their precursors (pri-miRNAs or pre-miRNAs) can be synthetically produced and introduced into a cell of interest (e.g., stem cells), or can be produced in a cell of interest (e.g., stem cells) from an exogenously introduced nucleic acid construct encoding either the miRNA or its precursor.

[0140] In some embodiments, inhibition of mRNA 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 that targets the gene of interest. The target site can be in the 5'regulatory region of the gene (e.g., promoter or enhancer region). In some embodiments, the Cas molecule is an enzymatically inactive Cas9 molecule that contains a mutation, e.g., a point mutation, that eliminates or substantially reduces DNA cleavage activity (see, e.g., WO2015 / 161276). In some embodiments, the enzymatically inactive Cas9 molecule is directly or indirectly fused to a transcriptional repressor protein.

[0141] Interference by other means The present invention includes other methods known in the art for inhibiting the function of a gene, including, for example, reducing the level or activity of a protein encoded by a gene by introducing into a cell (e.g., a stem cell) a compound (e.g., a small molecule, an antibody, among others) that directly inhibits the activity of the protein encoded by the gene.

[0142] CAR In some embodiments, cells (e.g., stem cells) that have been modified to have inhibition of one or more selected genes are also modified to contain a nucleic acid encoding a chimeric antigen receptor (or "CAR").

[0143] In some embodiments, the nucleic acid encoding the CAR can be introduced into the cell before, simultaneously, or after the cell is modified to inhibit the function of the 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 inhibition. In embodiments where the inhibition is permanent (e.g., through gene editing), the nucleic acid encoding the CAR can be introduced into the cell before, simultaneously, or after the cell is modified to achieve inhibition. In some embodiments, the nucleic acid encoding the CAR is designed to allow insertion by HDR at the target site of gene editing after introduction of a DSB, i.e., the gene is disrupted by knock-in or insertion of the nucleic acid encoding the CAR.

[0144] In some embodiments, a cell (e.g., a hematopoietic cell or an immune cell) derived from a cell (e.g., a stem cell) that has been modified to have inhibition of one or more selected genes is also modified to contain a nucleic acid encoding a chimeric antigen receptor (or "CAR").

[0145] The term "chimeric antigen receptor" (also known as "CAR", "artificial T cell receptor", "chimeric T cell receptor" and "chimeric immune receptor") should be understood as a reference to an engineered receptor that grafts an antigen recognition portion onto an immune cell. Generally speaking, a CAR is composed of an antigen recognition portion specific for a target antigen, a transmembrane domain, and an intracellular / cytoplasmic signaling domain of a receptor naturally expressed on an immune cell, operably linked to each other. "Operably linked" means that the individual domains are linked to each other such that when the antigen recognition portion binds to the target antigen, a signal can be induced through the intracellular signaling domain to activate the cell expressing the CAR (e.g., a T cell or NK cell) and activate its effector function.

[0146] The antigen recognition portion of a CAR is the extracellular portion of a receptor that recognizes and binds to an epitope of a target antigen. The antigen recognition portion is usually, but is not limited to, an scFv.

[0147] The intracellular domain of the CAR may comprise a primary cytoplasmic signaling sequence of a naturally occurring receptor of an immune cell, and / or a secondary or costimulatory sequence of a naturally occurring receptor of an immune cell. 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 the CAR comprises a cytoplasmic signaling sequence from CD3 zeta. In some embodiments, the intracellular signaling domain of the CAR may comprise a cytoplasmic signaling sequence from CD3 zeta in combination with a costimulatory signaling sequence of a costimulatory molecule. Examples of suitable costimulatory 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, B7-H3, and the like. In some embodiments, the cytoplasmic domain of the CAR is designed to contain the signaling domain of CD3 zeta and the signaling domain of CD28.

[0148] The transmembrane domain of CAR is generally a typical hydrophobic alpha helix that spans the membrane and can be derived from any membrane-bound or transmembrane protein. The transmembrane domain can be derived from either natural or synthetic sources. If the source is natural, the domain can be derived from any membrane-bound or transmembrane protein. For example, the transmembrane region can be derived from the alpha, beta or zeta chain of the T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, or immunoglobulins such as IgG4. Alternatively, the transmembrane domain can be synthetic, in which case it mainly contains hydrophobic residues such as leucine and valine.

[0149] The term "target antigen" should be understood as a reference to any proteinaceous or non-proteinaceous molecule expressed by a cell that is sought to be targeted by a receptor-expressing immune cell, such as a T cell or a NK cell. The target antigen may be a "self" molecule (a molecule expressed in the patient's body) or a non-self molecule (e.g., from an infectious microorganism). The target antigen referred to herein is not limited to a molecule that can naturally induce a T cell or B cell immune response; rather, the "target antigen" is a reference to any proteinaceous or non-proteinaceous molecule that is sought to be targeted. In some embodiments, the target antigen is expressed on a cell surface. It should be understood that the target antigen may be expressed exclusively by the target cell or may also be expressed by a non-target cell. In some embodiments, the target antigen is a non-self molecule, or a molecule that is expressed exclusively by the cell that is sought to be targeted, or that is expressed by the cell that is sought to be targeted at a significantly higher level compared to a normal cell. Non-limiting examples of target antigens include: differentiation antigens such as MART-1 / MelanA (MART-1), gplOO (Pmel 17), tyrosinase, TRP-1, TRP-2, and tumor-specific multilineage antigens such as MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, pl5; overexpressed glycoproteins such as MUC1 and MUC16; overexpressed embryonic antigens such as CEA; overexpressed oncogenes and mutated tumor suppressor genes such as p53, Ras, HER-2 / neu; unique tumor antigens resulting from chromosomal translocations such as BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR; and viral antigens such as Epstein-Barr virus antigen EBVA and human papilloma virus (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, pl 85erbB2, pl 180erbB-3, cMet, nm-23 Hl, PSA, CA19-9, CAM17.1, NuMA, K-ras, beta-catenin, CDK4, Mum-1, p15, p16, 43-9F, 5T4, 791Tgp72, alpha-fetoprotein, beta-HCG, BCA225, BTAA, CA125, CA15-3\CA 27,29\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-CO1, RCAS1, SDCCAG16, TA-90\Mac-2 binding protein\cyclophilin C-related protein, TAAL6, TAG-72, TLP, TPS, PSMA, mesothelin, or BCMA.

[0150] In some embodiments, the target antigen is a tumor-associated antigen, in particular a protein, glycoprotein or non-protein tumor-associated antigen.

[0151] In some embodiments, the target antigen is selected from the group consisting of CD47, folate receptor alpha (FRα) and BCMA.

[0152] In some embodiments, the target antigen is a tumor-associated antigen, for example, the tumor-associated antigen TAG-72.

[0153] In other embodiments, the target antigen is a surface protein, such as CD24, and in further embodiments, a surface protein that can be used for tumor targeting, such as CD19 or CD20.

[0154] In some aspects, the source cells comprise one or more nucleic acid molecules encoding a chimeric antigen receptor ("CAR"), said receptor comprising an antigen recognition moiety directed to an antigenic determinant. In some embodiments, the source cells express at least one CAR.

[0155] In other embodiments, the derivative cells comprise one or more nucleic acid molecules encoding a chimeric antigen receptor ("CAR"), said receptor comprising an antigen recognition moiety for an antigenic determinant. In some embodiments, the derivative cells express at least one CAR.

[0156] It will be appreciated by those of skill in the art that the mechanism by which a genetic modification (e.g., introduction of a nucleic acid molecule encoding a chimeric antigen receptor ("CAR")) is introduced into a cell can take any suitable form that is well known and understood by those of skill in the art. For example, genetic material is commonly conveniently introduced into a cell through the use of an expression construct.

[0157] CARs can be introduced by transfection of plasmid DNA or mRNA; transduction of viral vectors including gamma-retrovirus, lentivirus and adeno-associated virus; and CRISPR-Cas9, TALEN or ZFN-mediated gene editing.

[0158] Differentiation method The present invention relates generally to methods and compositions for modifying stem cells, particularly iPSCs, and further differentiating them into stem cell-derived immune cells that contain enhanced activity.

[0159] Methods for differentiating source cells (e.g., pluripotent stem cells) into immune cells (e.g., 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).

[0160] They typically follow three major steps: 1) generation and expansion of CD34+ cells from PSCs, 2) differentiation of progenitor / immune cells from CD34+ cells, and 3) expansion of progenitor / immune cells.

[0161] Preparation of pluripotent cells In some embodiments, PSCs may be prepared and / or selected prior to differentiation into mesodermal cells. The selection process may be based on one or more genes or one or more markers.

[0162] After reprogramming to generate iPSCs, cells can be characterized based on embryo-like morphology, transgene silencing after reprogramming, pluripotency assessment by alkaline phosphatase assay, or detection of pluripotency and regeneration markers such as TRA-1-60, TRA-1-81, Nanog, and Oct4. Differentiation potential is monitored by embryoid body formation and / or teratoma formation. Karyotype analysis, identity matching, and sterility are also usually assessed (Huang et al., Human iPSC banking: barriers and opportunities, Journal of Biomedical Science, Oct 28, 2019, Vol.26(1)).

[0163] Various methods are used for the maintenance and preparation of human iPSCs, including feeder cell-dependent culture using inactivated mouse embryonic fibroblast (MEF) cells, culture of iPSCs using MEF-conditioned medium, generation of embryoid bodies from iPSCs, and matrix-dependent growth using serum-free and feeder-free growth medium or MEF-conditioned medium. iPSCs are passaged using enzymatic or mechanical passaging methods, generally when colonies become too large / dense or when increased differentiation occurs. Optimal iPSC colonies are those observed to have clear edges and uniform morphology across the colony.

[0164] Transcription Activator-Like Effector Nucleases (TALENs) and CRISPR / Cas9 are the most common for genome editing of PSCs. Generally, TALE arrays or CRISPR guide RNAs are designed and cloned using a cloning vector. Single-cell human PSCs are transfected or transduced with the vectors, and targeted cells can be selected by FACS via fluorescent reporters or by antibiotic dissection via selection markers. One to two weeks after enrichment, PSCs are harvested and expanded for genomic DNA analysis and targeted clone recovery and propagation. Both gene editing approaches have been used to generate knock-in and / or knock-out iPSC lines (Hendricks et al., Genome Editing in Human Pluripotent Stem Cells: Approaches, Pitfalls, and Solutions, Cell stem cell, 07 January 2016, Vol.18(1), pp.53-65).

[0165] In some embodiments, iPSCs are lifted out of the adherent state as single cells in solution using Accutase or similar, and then electroporated in the presence of an RNP complex containing a guide RNA for a specific gene KO, and / or a plasmid containing a sequence that may be desired to be knocked into the cell at the site of the gene of interest.

[0166] In some embodiments, after gene editing, the iPSCs are left to stabilize and returned to normal culture before any further manipulation or testing in functional studies, such as differentiation into NK cells.

[0167] Generation and expansion of CD34+ cells from pluripotent stem cells Most methods in the art begin with differentiating PSCs into mesodermal cells, which can then be subsequently differentiated into HE / HSCs, which can also be expanded simultaneously.

[0168] PSC differentiation methods can follow various approaches including embryoid body (EB) formation, feeder cell co-culture, two-dimensional extracellular matrix-coated culture, and programming or reprogramming using transcription factor transduction (Lim et al., Hematopoietic cell differentiation from embryonic and induced pluripotent stem cells Stem cell research & therapy, 18 June 2013, Vol.4(3), pp.71; Tajer et al., Ex Vivo Expansion of Hematopoietic Stem Cells for Therapeutic Purposes: Lessons from Development and the Niche, Cells, 18 February 2019, Vol.8(2)). These methods can produce hematopoietic progenitor cells. Increasing evidence suggests that hemogenic endothelial (HE) cells are transient intermediates that contribute to the de novo production of pluripotent HSCs.

[0169] Three-dimensional EBs of PSC differentiation mimic in vivo embryonic development, therefore, several methods using EB formation have been developed for hematopoietic differentiation of PSCs. These include spontaneous EB formation, hanging drop EB formation and spin EB formation. To specifically induce the hematopoietic system, single-cell suspensions of EBs are directed into methylcellulose culture medium, which functions to support hematopoietic development in the presence of hematopoietic cytokines and growth factors. Differentiation to HSCs based on the spin EB method may require 4 to 11 days of culture.

[0170] Feeder co-culture is a method in which a layer of feeder cells is cultured with PSCs to support them towards hematopoietic lineage development in an appropriate culture medium. Stromal cell co-culture is used to obtain HE / HSCs. OP9 stromal cells, stromal cells derived from the aorta-gonad-mesonephros region, fetal liver-derived stromal cells such as S17 and M210 and bone marrow-derived stromal cells, as well as AFT04 stromal cells are exemplary lines used in co-culture. Stromal cell co-culture methods are usually animal-derived and can be either serum-dependent or independent.

[0171] Two-dimensional culture in culture dishes coated with extracellular matrices such as collagen and fibronectin are used as monolayer cultures to differentiate PSCs. Matrices using human fibronectin or collagen IV are mainly utilized to generate hematopoietic progenitor cells. PSCs also differentiate into mesodermal cells in the presence of matrix components such as laminin, collagen I, entactin and heparin-sulfate proteoglycans as well as growth factors and some other undefined compounds. These mesodermal cells can induce hematopoietic cells after replacement with hematopoietic cocktail culture medium.

[0172] Some cell-extrinsic factors are required in hematopoietic cocktail culture medium for PSC differentiation into HE / HSC.The combination of chemicals, cytokines and growth factors includes but is not limited to bFGF, BMP4, activin A, SB431542, DKK, CHIR99021, VEGF, IL-6, IGF-1, IL-11, SCF, EPO, TPO, IL-3 and FLT3-L.

[0173] Direct programming or reprogramming of endothelial cells to HE / HSCs via transcription factor transduction has been used to differentiate PSCs to HSCs. Transcription factors include, but are not limited to, ERG, HOXA5, HOXA9, HOXA10, LCOR, RUNX1, SPI1, FOSB, GFI1, ETV2 and GATA2.

[0174] Hematopoietic cocktails are used to expand CD34+ cells / HSCs. These include, but are not limited to, those mentioned above. Commercial kits of hematopoietic cocktails are also available for the expansion of CD34+ cells.

[0175] Other small molecules, proteins and chemicals can also be used in combination with the above cytokines / growth factors for the expansion of HSC / CD34+ cells. These include, but are not limited to, tetraethylenepentamine, HOXB4, prostaglandin E2, stemreginin 1, UM729, UM171, Notch ligand, angiopoietin-like 5, IGFBP2, pleiotrophin, valproic acid, and small molecule inhibitors of histone deacetylase and DNA methyltransferase. Three-dimensional nanofiber scaffolds have also been used for HSC expansion.

[0176] Differentiation of HE / HSC into lymphoid / myeloid lineage cells In addition to bone marrow, the main source of HSC / CD34+ hematopoietic cell precursors (HSCs) is blood. Specifically, they can be isolated from peripheral blood or umbilical cord blood. iPSC cell lines are also a source of these cells. Enrichment of HSC / CD34+ hematopoietic precursor cells can be performed using anti-CD34 immunomagnetic particles / beads.

[0177] CD34+ progenitor cells can be differentiated and expanded using a proliferation cocktail containing various cytokines, growth factors and / or small molecules. Successful differentiation is monitored using antibody staining and flow cytometric analysis of marker expression. Hematopoietic potential is monitored by clonogenic colony forming unit assays. Relevant hematopoietic gene expression is monitored via PCR, transcriptome analysis or other molecular biology-based methods. Engraftment can be tested in animals such as NOD / SCID mice.

[0178] Combinations of cytokines and growth factors are used to promote differentiation of HSCs into immune cells, for example, IL-3, SCF, IL-15 and FLT3-L are used in combination with IL-2, IL-15 and IL-7 for the generation of NK cells by EBs in the presence or absence of feeder cells such as EL081D2.

[0179] To induce lymphoid commitment, OP9 cell lines expressing the Notch ligand delta-like-1 (OP9-DLL1) or OP9-DLL4 were utilized in the presence of SCF, FLT3L and IL-7 or IL-15 to generate NK cells without CD34 enrichment or spin EB formation.

[0180] Expansion of NK cells derived from PSC / HSC can be performed using feeder cells such as K562 with membrane-bound IL-5 and 4-1BB ligands. Other cell lines used for NK cell expansion include membrane-bound IL-21 artificial antigen-presenting cells. Commercially available kits for differentiating HSC into NK cells are available, such as StemSpan™ NK Cell Generation Kit (STEMCELL Technologies).

[0181] T cells can also be induced by culturing OP9 and OP9-DLL1 in the presence of FLT3L, IL-7 and IL-2, followed by stimulation with anti-CD3 anti-CD28. Commercially available kits for differentiating HSCs into T cells are also available, e.g., StemSpan™ T Cell Generation Kit (STEMCELL Technologies).

[0182] Monocytes and macrophages can be generated using, for example, the SC17 cell line or stromal cell-based methods using spin-EB methodology and subsequent culture in the presence of IL-3, CSF-1 and M-CSF.

[0183] Pharmaceutical compositions and therapeutic uses of modified cells In a further aspect, provided herein are compositions containing cells produced by the methods disclosed herein, i.e., modified cells in which the function of one or more of the selected genes is inhibited, and cells derived therefrom.

[0184] In some embodiments, provided herein is a pharmaceutical composition comprising the cells produced herein and a pharma- ceutically acceptable carrier. Pharmaceutically acceptable carriers include solvents, dispersion media, isotonic agents, and the like. Examples of carriers include oils, water, saline, gels, lipids, liposomes, resins, porous matrices, preservatives, and the like, or 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 a unit dose injectable form (solution, suspension, emulsion). In some embodiments, the pharmaceutical composition can use time-release, delayed release, and sustained release delivery systems.

[0185] In some embodiments, the pharmaceutical composition comprises an amount of cells effective to treat or prevent a disease or condition, e.g., a therapeutically effective amount or a prophylactically effective amount. In some embodiments, the pharmaceutical composition comprises the modified cells disclosed herein in an amount of about 1 million to about 100 billion cells, e.g., at least 1 million, 5 million, 10 million, 25 million, 50 million, 100 million, 200 million, 300 million, 400 million, or 500 million cells 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.

[0186] In some embodiments, the pharmaceutical composition further comprises another active agent or drug, such as a chemotherapeutic agent.

[0187] In another aspect, provided herein are methods and uses of the modified cells disclosed herein, such as therapeutic methods and uses in adoptive cell therapy.

[0188] In some embodiments, the methods include administration of modified cells disclosed herein or compositions comprising modified cells disclosed herein to a subject having a disease or condition or at risk of developing a disease or condition.

[0189] In some embodiments, the disease or condition is a neoplastic condition (i.e., cancer), or a microbial or parasitic infection (e.g., HIV, STD, HCV, HBV, CMV, or antibiotic-resistant bacteria), or 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).

[0190] In some embodiments, neoplastic conditions include central nervous system tumors, retinoblastoma, neuroblastoma, childhood tumors, head and neck cancer (e.g., squamous cell carcinoma), breast and prostate cancer, lung cancer (both small cell and non-small cell lung cancer), kidney cancer (e.g., renal cell carcinoma), esophagogastric cancer, hepatocellular carcinoma, pancreatic and biliary neoplasms (e.g., adenocarcinoma and islet cell carcinoma), colorectal cancer, cervical and anal cancer, uterine and other reproductive tract cancers, urinary tract cancer (e.g., ureteral and bladder cancer), germ cell tumors (e.g., testicular germ cell tumors or ovarian germ cell tumors), ovarian cancer (e.g., ovarian epithelial cancer), cancer of unknown primary, human immunodeficiency-associated malignancies (e.g., Kaposi's sarcoma), lymphoma, leukemia, malignant melanoma, sarcoma, endocrine tumors (e.g., thyroid tumors), mesothelioma and other pleural or peritoneal tumors, neuroendocrine tumors, and carcinoid tumors.

[0191] In some embodiments, the method of the present invention provides for the treatment of a condition, i.e., the reduction or amelioration of a condition or any one or more symptoms of a condition, for example, by inhibiting tumor growth and / or metastasis in the context of treating cancer, or by reducing viral load and / or spread in the context of treating viral infection. The term "treatment" does not necessarily mean complete recovery. In some embodiments, the method provides for the prevention of a condition, i.e., preventing a condition, reducing the risk of developing a condition, or delaying the onset of a condition. Similarly, "prevention" does not necessarily mean that a subject will not eventually suffer from the condition.

[0192] In some embodiments, the subject, e.g., a patient, to which the cells or compositions are administered is a mammal, typically a primate such as a human.

[0193] In some embodiments, the cells or compositions comprising the cells are administered parenterally. As used herein, the term "parenteral" includes intravenous, intramuscular, subcutaneous, and intraperitoneal administration.

[0194] The desired dosage of the derivative cells or compositions comprising the derivative cells can be delivered by a single administration, by multiple administrations, or by continuous infusion of the composition. Therapeutic or prophylactic efficacy can be monitored by periodic evaluation of the treated subject.

[0195] In some embodiments, source cells are generated from cells isolated from a subject, then modified (to inhibit the function of one or more genes) according to the methods disclosed herein, further differentiated, and then administered to the same subject.

[0196] In some embodiments, adoptive cell therapy is performed by allogeneic transfer, where the source cells are generated from cells derived from a donor subject that is different from the subject receiving the cell therapy (the recipient subject). In some embodiments, the donor subject and the recipient subject express the same HLA class or supertype. EXAMPLES

[0197] The following examples, without being limited thereto, illustrate that CRISPR / Cas9 gene editing technology was used to eliminate two DGK genes to generate stem cell-derived immune cells with enhanced function for tumor therapy. Gene editing efficiency was examined by quantification based on genomic DNA sequencing. iPSCs were lifted as single cells using Accutase™, and then the Cas9 nuclease complex with specifically designed guide RNA was transfected into these iPSCs to eliminate immune regulator genes. The iPSCs were then differentiated into CD34+ cells. The CD34+ cells were further differentiated into iNK cells. Cytotoxicity was monitored in vitro.

[0198] Example 1 – Generation of TAG-72 CAR iPSC single cell clones Stem cells such as iPSCs can self-renew 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 already been generated from iPSCs for cancer therapy (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 Grants Potent Tumor Antigen-Specific Cytotoxicity, Cancer Res, 2016, 76(23): 6839-6850). CAR-T cells or CAR-NK cells can be derived from lentiviral CAR-transduced iPSCs following similar methods. To generate TAG-72 CAR iPSCs, the TAG-72 CAR expression cassette was introduced into the AAVS1 safe harbor site via the non-viral CRISPR / Cas9 gene knock-in method described in WO2017 / 088012 and PCT / AU2020 / 050800, which are incorporated herein by reference. TAG-72 CAR-positive iPSCs were sorted to generate purified TAG-72 CAR iPSC single cell clones (Figure 1). CAR expression on the surface of iPSCs was characterized by flow cytometry.

[0199] Single Cell Cloning For single cell cloning, 96-well plates were first coated with CellAdhere™ Laminin-521 in PBS for 2 hours at 37°C. TAG-72 CAR iPSCs were sorted and seeded into 96-well plates at an average density of 1 cell / well. Transfected iPSCs were lifted as single cells using Accutase®, followed by counting, washing, and resuspending for staining with the relevant antibody cocktail as required. Cells were stained for 15 minutes at 4°C and then sorted on a BD FACSAria™ Fusion. Propidium iodide (PI) staining was used for dead cell exclusion. Cells were gated to remove debris, doublets, and dead cells, and then sorted into 96-well plates. Plates were immediately placed in a 37°C cell incubator for 48 hours, with daily medium changes if cells were suitable for passaging.

[0200] Example 2 – Generation of DGK KO iPSCs (pre-selected single cell clones) Both DGKα and DGKζ have multiple transcripts. To remove all of the major isoforms of DGKα and DGKζ, a list of guide RNAs was designed to target the DGKα and DGKζ genes (Table 1). Thus, out-of-frame indels could be introduced into the early exons of their open reading frames (ORFs) to disrupt the translation of DGKα and DGKζ.

[0201] To generate CRISPR DGKα and / or DGKζ gene double knockout iPSCs, RNP complexes formed by representative gRNAs (SEQ ID NO:3; SEQ ID NO:11) were transfected into iPSCs using the Lonza 4D-Nucleofector™ system. First, 12-well plates were coated with CellAdhere™ Laminin-521 (STEMCELL Technologies) in PBS and incubated at 37°C for 2 h. Prior to transfection, iPSCs were pre-incubated with mTeSR Plus™ medium (STEMCELL Technologies) containing RevitaCell™ supplement (Life Technologies) for 2 h. RNPs were prepared by combining full-length guide RNA (gRNA) with Lonza P3 buffer and Cas9. The RNP mixture was then incubated at room temperature for 10–20 min. After preincubation, iPSCs were harvested as single cells using Accutase® (Life Technologies) and cultured at 1 × 10 per reaction for electroporation. 6 cells were obtained. To generate gene knockout iPSCs, cells and RNP mixture in Lonza P3 buffer were combined in a PCR tube; to generate gene knock-in iPSCs, cells in Lonza P3 buffer were combined with RNP mixture and donor DNA. Cells with RNP mixture were loaded into a Lonza 4D-Nucleofector™ for electroporation. After this, mTeSR Plus™ with CloneR™ medium was added to the reaction and incubated at room temperature for 10 minutes. After incubation, cells were added to laminin-521 pre-coated plates in mTeSR Plus™ with CloneR™ medium. Daily medium changes with mTeSR Plus™ were performed for 72 hours and cells were passaged when they reached approximately 80% confluency (6-7 days after electroporation). Cells at the end of this stage are referred to as DGK KO pre-sorted iPSCs.

[0202] After RNP transfection and expansion, genomic DNA was extracted from iPSCs 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)). DGKα gene editing efficiency analysis from DGKα gRNA (SEQ ID NO: 3) iPSC transfected samples is shown here as a representative result of ICE analysis (Figure 2A-2C). In addition, it has also been shown that DGKα gRNA (SEQ ID NO: 3) and DGKζ gRNA (SEQ ID NO: 11) can introduce indels into multiple iPSC lines (Figure 3A-3B).

[0203] To generate single / double knockout iPSCs and single-cell cloning of the CRISPR DGKα and DGKζ genes, two phases are required (Figure 1). First, phase I consists of CRISPR / Cas9 gene editing, including electroporation of iPSCs and their recovery, followed by the subsequent expansion culture of transgenic iPSCs. Phase II consists of single-cell cloning of the transfected iPSCs, which includes single-cell sorting and the subsequent expansion culture of the clonal iPSCs.

[0204] Single Cell Cloning For single cell cloning, 96-well plates were first coated with CellAdhere™ Laminin-521 in PBS for 2 hours at 37°C. DGKα (SEQ ID NO:3) and / or DGKζ (SEQ ID NO:11) RNP-transfected iPSCs were sorted and seeded at a density of 1 cell / well in 96-well plates and cultured until colonies formed (e.g., 5-9 days). Transfected iPSCs were removed as single cells using Accutase®, subsequently counted, washed, and, if necessary, resuspended for staining with the relevant antibody cocktail. Cells were stained for 15 minutes at 4°C and then sorted on a BD FACSAria™ Fusion. PI staining was used for dead cell exclusion. Cells were gated to remove debris, doublets, and dead cells and subsequently sorted into 96-well plates. Plates were immediately placed in a 37°C cell incubator for 48 hours, with daily medium changes until cells were suitable for passaging. After single-cell cloning, the genotypes of the single-cell clones were analyzed using Sanger sequencing and ICE assay. Single-cell clones with 99%-100% out-of-frame frequency were selected as KO iPSC single-cell clones (Figure 4A-4D). The genotypes of these selected clones were further compared with wild-type Sanger sequencing traces. A representative genotyping result of a DGK double gene KO clone (DGKαζ KO iPSC single-cell clone 01) from DGKα and DGKζ RNP co-transfected iPSCs is shown. The pre-sorted DGKα and DGKζ RNP co-transfected iPSCs were purified after single-cell cloning, which was evidenced by the removal of a heterogeneous mixture of bases downstream of the cleavage site and the identification of a thymine (T) insertion (+1) in the single clones compared to the non-transfected (wild-type) cells (Figure 5A-5B). In summary, these results demonstrated that DGKα gRNA and DGKζ gRNA could introduce indels into the DGK gene, resulting in efficient frameshift mutations in iPSCs and generating DGK KO iPSCs.

[0205] TGFβ exerts systemic immunosuppression, inhibits host immune surveillance, and is believed to be one of the major factors of the immunosuppressive microenvironment in tumors. Knockout of TGFβ receptors and direct inhibition of TGFβ signaling in embryonic stem cells results in loss of pluripotency (Watabe and Miyazono, Roles of TGF-beta family signaling in stem cell renewal and differentiation, Cell Research, Jan 2009, Vol.19(1), pp.103-15). The inventors used CRISPR / CAS9 technology (described in International Application PCT / AU2020 / 051243, incorporated herein by reference) to generate dominant-negative TGFβ1 and TGFβR2 iPSCs. However, TGFβR1 and TGFβR2 dominant-negative mutations in iPSCs lost their pluripotency and began to differentiate spontaneously after gene editing (Figure 19). This result indicates that gene knockout or direct inhibition of TGFβ receptors in iPSCs cannot generate source cells for immune cell therapy.

[0206] Example 3 – Pre-screening of TAG-72 CAR clones / DGK KO iPSCs and generation of TAG-72 CAR / DGK KO iPSC single cell clones To generate pre-selected TAG-72 CAR clones / DGK KO iPSCs, TAG-72 CAR iPSC single cell clones were first generated as described in Example 1. Then, the RNP complexes formed by representative gRNAs were transfected into TAG-72 CAR iPSCs using the Lonza 4D-Nucleofector™ system described in Example 2. ICE analysis results show that the gRNAs of DGKα and DGKζ can introduce indels into the DGK gene, resulting in frameshift mutations in TAG-72 CAR iPSCs at high frequencies (Figure 6). Furthermore, TAG-72 CAR / DGK KO iPSC single cell clones can also be derived using the method described in Example 2 (Figure 7).

[0207] Example 4 – Differentiation of DGK KO iPSC single cell clones and TAG-72 CAR clones / DGK KO preselected iPSCs into CD34+ cells Stem cells, such as induced pluripotent stem cells (iPSCs), can self-renew indefinitely and differentiate into a variety of cell types, including hematopoietic stem cells (HSCs) and immune cells.

[0208] CD34+ cells can be generated using a range of published methods, such as those described in U.S. Pat. No. 9,260,696 B2 (Kaufman, Knorr) and by Li et al. (Stem Cell, 23 (2018) 181-197), or using commercially available culture systems, such as the STEMdiff™ Hematopoietic Kit (STEMCELL Technologies). In one form of these methods, induction of differentiation of iPSCs into CD34+ hematopoietic progenitor cells is facilitated by embryoid body (EB) formation using AggreWell™ 400 6-well plates. AggreWell™ plates can be pretreated with Anti-adherence Rinsing Solution (STEMCELL Technologies) and washed once with warm mTeSR Plus™ before use. mTeSR Plus™ with RevitaCell™ supplement is then added at 2.5 mL per well. iPSC cultures are grown until they reach 70% confluency, at which point the iPSCs are dissociated into single cells using Accutase® and re-seeded into AggreWell™ plates, and mTeSR Plus™ with RevitaCell™ supplement is added to the medium for a total of 5 mL per well. The AggreWell™ plates are then centrifuged at 100×g for 3 minutes to pull the cells into each microwell and incubated at 37° C., 5% CO 2After the incubation period, the medium is removed with a serological pipette and 5 mL of STEMdiff™ Hematopoietic Medium A (STEMCELL Technologies) is added per well. This represents day 0 of hematopoietic specification. On day 2, a half medium change is performed, where 2.5 mL of Medium A is removed and replaced with 2.5 mL of fresh Medium A. On day 3, a full medium change is performed, where Medium A is removed and replaced with 5 mL of STEMdiff™ Hematopoietic Medium B (STEMCELL Technologies). On day 5, EBs are collected by firmly pipetting the culture medium against the surface of each well to dislodge the EBs. The suspension is passed through a 40 μm filter. The filter is then inverted and the EBs collected on the surface of the filter are washed with 2.5 mL of Medium B into a new 50 mL plastic tube. The EBs are then seeded into wells of a non-tissue culture treated 6-well plate in 2.5 mL of Medium B. On day 7, 2.5 mL of Medium B is added per well to remove the EBs. Add Medium B. On days 9 and 11, perform half-medium changes with Medium B as outlined above. On day 12, collect both adherent and non-adherent cell fractions and sort CD34+ cells using a CD34 MicroBead Kit (Miltenyi Biotec) according to the manufacturer's instructions.

[0209] TAG-72 KI, DGK KO and cloning were performed as described in the above examples. DGK KO in iPSC single cell clones and TAG-72 CAR clones / DGK KO pre-sorted iPSCs were then differentiated into CD34+ cells. The sorting purity and hematopoietic progenitor cell profile of the generated CD34+ cells were determined by flow cytometry for CD34 expression. Flow cytometry analysis of hematopoietic progenitor cells identified from DGK KO iPSCs or TAG-72 CAR / DGK KO iPSCs showed expression of CD34 comparable to non-transfected controls, indicating that neither DGK KO alone nor the combination of TAG-72 CAR KI and DGK KO affected the differentiation of iPSCs into CD34+ hematopoietic progenitor cells (Figures 11A-11C).

[0210] Example 5 - Differentiation of DGK KO CD34+ (with or without TAG-72 CAR) into iNK cells iNK cells can be generated using a series of published methods, such as those described in U.S. Pat. No. 9,260,696 B2 (Kaufman, Knorr) and by Li et al. (Stem Cell, 23 (2018) 181-197), or using the commercially available culture system StemSpan™ NK Cell Generation Kit (STEMCELL Technologies). In one form of these methods, sorted (e.g., as described in Example 4) CD34+ hematopoietic progenitor cells derived from either DGK KO iPSC single cell clones or pre-sorted TAG-72 CAR clones / DGK KO iPSCs are replated in StemSpan™ Lymphoid Progenitor Cell Growth Medium (STEMCELL Technologies) on plates coated with StemSpan™ Lymphoid Differentiation Coating Material (STEMCELL Technologies). This marks day 0 of NK cell specification. On day 3, one volume of growth medium is added to the culture vessel. On days 7 and 10, half-medium exchanges are performed with fresh growth medium. On day 14, non-adherent lymphoid progenitor cells are collected and centrifuged at 300×g for 5 minutes. The cells are then resuspended in StemSpan™ NK cell differentiation medium supplemented with 1 μM UM729. On day 17, one volume of NK differentiation medium is added to the culture vessel. On days 21 and 24, half-medium exchanges are performed with fresh NK differentiation medium. Differentiation into NK cells is complete by day 28. The efficiency of NK cell differentiation can be enhanced by supplementing the medium with a combination of cytokines, including IL-15, FLT3 and IL-7.

[0211] DGK KO CD34+ (with or without TAG-72 CAR) were differentiated into iNK cells and analyzed for DGK KO, NK cell phenotype, and NK cell function. ICE analysis confirmed that iNK±TAG-72 CARs had indels at both the α and ζ loci at a percentage comparable to their parental DGK KO iPSC±TAG-72 CAR (Figure 12A-12B). Furthermore, it was shown that single or double gene DGK KO in iPSCs with or without TAG-72 CAR KI did not affect the yield of NK cells obtained from the differentiation process (Figure 13). Flow cytometry analysis for NK cell markers CD56, CD45, NKp46, NKG2D, NKp44, and 2B4 showed comparable expression levels between untransfected and transgenic iNKs, indicating that single or double DGK KO did not affect the iNK cell phenotype in the presence or absence of TAG-72 CAR KI (Figures 14A-14B).

[0212] Example 6 –TAG-72 CAR KO iNK cells and in vitro function of KO iNK cells A real-time cell monitoring system (xCELLigence®) was used to determine the killing efficiency of iNK cells in vitro. 10,000 cells / 100 μL of target cells (e.g., ovarian cancer cell line OVCAR-3) were resuspended in culture medium (e.g., RPMI-1640 basal medium) supplemented with 20% fetal bovine serum and bovine insulin and placed in a Real Time Cell Analysis microtiter ePlate compatible with the xCELLigence® system. Target cells were incubated at 37° C., 5% CO 2The cells were maintained at RT for 3-20 hours to allow cell attachment. After target cell attachment, iNK effector cells with or without TAG-72 CAR or DGKαζ KO were added at an effector-to-target (E:T) ratio of 1:1. All co-cultures were maintained at optimal growth conditions for at least 20 hours. Cell impedance was monitored throughout; a decrease in impedance indicates target cell detachment and ultimately cell death. DGKαζ KO was performed on iPSC clones with or without TAG-72 CAR insertion as described in Examples 1, 2 and 3. These cells were then sequenced and differentiated into iNK cells. iNK cells with and without TAG-72 CAR and DGKαζ KO were placed into the xCELLigence® assay (as described above) targeting OVCAR-3 cells at an effector-to-target ratio of 1:1 to assess baseline iNK function prior to chronic exposure to OVCAR-3 cells. For antigen exposure assays, OVCAR-3 cells (routinely cultured according to ATCC recommendations) were cultured at 8 × 10 4 Cells / well were seeded and left to attach to the plate for 6 hours. Then, 8 × 10 5 iNK cells were placed into OVCAR-3-containing wells and incubated for a total of 72 hours. Every 24 hours (i.e., 24, 48, and 72 hours of co-culture), the non-adherent fraction of the cultures containing iNK cells and any dead OVCAR-3 cells was collected, centrifuged at 300×g for 10 minutes, resuspended in fresh iNK culture medium, and placed into wells containing uncontacted OVCAR-3 cells. The remaining OVCAR-3 cells not killed by iNK (i.e., the remaining adherent fraction) were enzymatically separated using a trypsin-EDTA solution and counted using a MUSE® Cell Counter. These cell counts were utilized to calculate the iNK killing efficiency, as shown in FIG. 15A. iNK cells at the end of the antigen exposure assay were also assessed for cytotoxic function using xCELLigence® as previously described.

[0213] iNK cells with CAR KI and DGKαζ KO showed enhanced long-term cytotoxicity function against OVCAR-3 tumor cells in vitro (Figure 15A). The effect of including DGKαζ KO with CAR was evident after 72 h of repeated exposure to cancer cell lines. These data demonstrated the inclusion of a novel function in DGKαζ KO in iPSCs that confers functional benefits in iNK cells (Figure 15A). After 72 h of repeated exposure to OVCAR-3, the time it took to eliminate target cells increased (15-20 h) compared to baseline (~5 h) (Figure 15B). However, when comparing the function of iNK cells exposed to OVCAR-3 cells for 72 h before assessing killing capacity, TAG-72 CAR / DGKαζ KO iNK showed improved function (Figure 15B). Furthermore, the total number of iNK cells that survived long-term antigen exposure assays was improved in TAG-72 CAR / DGKαζ KO iNK clones compared to their respective non-transfected iNK controls (Figure 15C). Taken together, this indicates that DGKαζ KO is important for enhancing iNK survival and cytotoxic function against tumor cells.

[0214] Example 7 – In vitro function of DGKαζ KO iNK cells The killing efficiency of iNK cells in vitro was determined using a real-time cell monitoring system (xCELLigence®) as previously described in Example 6 above. For long-term antigen exposure assays, OVCAR-3 cells (routinely cultured according to ATCC recommendations) were plated at 2.5×10 per well in 48-well plates. 4 Cells were seeded and left to attach to the plate for 4-5 hours. Then, 5 × 10 5iNK cells (E:T 20:1) were placed on OVCAR-3 wells and incubated for 24 hours. After 24 hours, the non-adherent fraction of the cultures containing iNK cells as the only viable cells was collected, centrifuged at 300g for 10 minutes, resuspended in fresh iNK culture medium, and counted using a Guava® MUSE® Cell Analyser. These remaining iNKs were then placed into new wells containing OVCAR-3 cells freshly seeded 4 hours earlier. The remaining OVCAR-3 cells not killed by iNK were picked up via trypsin-EDTA and counted using a Guava® MUSE® Cell Analyser. This process was repeated at each 24 hour interval for a total of 5 days. iNK cells at the end of the antigen exposure assay were also placed into the xCELLigence® assay to demonstrate the effect of iNK cytotoxic function after 120 hours of exposure to OVCAR-3 cells. At the experimental endpoint, samples of iNK cells were also subjected to flow cytometric analysis to assess expression of the proliferative nuclear marker Ki67.

[0215] iNK cells with DGKαζ KO showed enhanced long-term cytotoxicity function against OVCAR-3 tumor cells in vitro (Figure 16A). After 120 h of repeated antigen exposure, DGKαζ KO iNK cells demonstrated enhanced killing capacity compared to non-transfected iNK cells as measured by xCELLigence® assay (Figure 16A). These data demonstrate the novel function of including DGKαζ KO in iPSCs that provides functional benefits in iNK cells (Figure 16A). Furthermore, flow cytometry analysis after repeated antigen exposure suggested an increased proportion of cells expressing the proliferation marker Ki67 in DGKαζ KO iNK compared to non-transfected iNK (Figure 16B). This trend toward increased Ki67 expression is a hint to the improved proliferation capacity in DGKαζ KO iNK.

[0216] The functional advantage of DGK KO in iNK cells in the tumor microenvironment was modeled by an in vitro cytotoxicity function assay described in Example 6 in the presence of TGFβ. As a general phenomenon, solid tumor microenvironments are intensively infiltrated with various immune checkpoint inhibitors and soluble factors such as TGFβ, which causes tumor escape and resistance to immune cells (Alsina-Sanchis, Elisenda et al., TGFβ Controls Ovarian Cancer Cell Proliferation, International journal of molecular sciences, 2017-07-30, Vol.18 (8), p.1658). For example, previous studies have shown that TGFβ can suppress natural killer cell function through multiple signaling pathways, resulting in downregulation of NK activating receptors such as natural killer group 2 member D (NKG2D) (reviewed by Yang, Li et al., TGF-β and immune cells: an important regulatory axis in the tumor microenvironment and progression, 2010, Vol.31 (6), p.220-227), impairing killing capacity here. The response of non-transfected iNK cells and DGKαζ KO iNK cells targeted against OVCAR-3 at an effector-to-target ratio of 2:1 in either standard NK culture medium or supplemented with 10 ng / mL TGFβ was investigated in Figures 18A-18B. OVCAR-3 cell proliferation was monitored in culture medium alone with or without TGFβ (no iNK cells) as an internal control within the assay. In this study, inclusion of DGKαζ KO in iNK cells allowed them to resist or circumvent the suppressive effect of TGFβ on cytotoxic function against OVCAR-3 cells compared to non-transfected iNK cells, which had a reduced killing response in TGFβ-supplemented medium.Since TGFβ KO cannot be performed directly in iPSCs without triggering spontaneous differentiation (as shown in FIG. 19 ), the ability of DGKαζ KO to enable downstream benefits in iNK cells by minimizing the inhibitory effects in TGFβ-related pathways demonstrates a novel advantage of DGKαζ KO in iPSCs and iNK cells.

[0217] The functional benefit of DGKαζ KO was further evaluated in vitro in combination with TAG-72 CAR. In this assay, TAG-72 CAR (clone D7) iNK cells with or without DGKαζ KO were used as effector cells in an xCELLigence® assay against OVCAR-3 cells under more stringent conditions, where the effector-to-target ratio was lowered to 1:5 and supplementation of TGFβ in the NK medium was increased to 100 ng / mL. In Figure 18C, the immunosuppressive impact of TGFβ on effector cells was greater in TAG-72 CAR iNK cells than in TAG-72 CAR / DGKαζ KO iNK cells. Addition of TGFβ at 100 ng / mL abolished the cytotoxic function of TAG-72 CAR iNK cells against OVCAR-3 cells. Under the same conditions, the cytotoxicity of TAG-72 CAR / DGKαζ KO iNK was only slightly reduced by the addition of TGFβ. These data support that TAG-72 CAR iNK cells are sensitive to immunosuppressive factors, including TGFβ, that mimic aspects of the endogenous tumor microenvironment. Inclusion of DGKαζ KO in iPSC cells and downstream retention in iNK cells after differentiation reduces the impact of TGFβ on the cytotoxic function of iNK against OVCAR-3 cells.

[0218] Example 8 – In vivo function of DGKαζ KO iNK cells In this model, approximately 1 × 10 7 Human tumor cell lines were grown in the flanks of NSG mice by subcutaneous injection of human-derived TAG-72-positive OVCAR-3 cancer cells into the flanks of 6- to 10-week-old mice. Within 6–7 weeks, fully formed tumors of approximately 100 mm 3Tumors of 1 × 10 developed at the injection site. Once tumors reached this volume, the groups were randomized for treatment. iNK cells derived from either unedited (i.e., non-transfected) iPSC sources or DGKαζ KO iPSC cells were injected at 1 × 10 per injection. 6 Mice were intravenously administered with 800 mm iNK cells. Prior to injection, iNK cells were cultured in vitro under normal NK growth conditions for 7 days. No exogenous cytokines were co-administered to the mice with iNK cells. Tumor volume, body weight and clinical scores were monitored after iNK cell injection. 3 From 1000mm 3 Mice with tumor sizes > 100%, significant weight loss, or poor clinical scores were culled in accordance with animal ethics approval.

[0219] Administration of human cytokines has been demonstrated to support human NK cell survival and function in NSG mice. However, in this study, human cytokines were not co-administered with iNK cells, and the iNK cells still demonstrated anti-tumor activity. Mice treated with iNK cells carrying DGKαζ KO demonstrated reduced tumor size at multiple time points throughout the assay, indicating improved survival (Figure 17).

[0220] Example 9 –In vitro and in vivo characterization of TAG-72 CAR and TAG-72 CAR / DGKαζ KO iNK cells Prior to in vivo administration, effector cell preparations were pre-evaluated by a set of optimized in vitro assays, including flow cytometric analysis of NK cell purity, phenotypic characterization of relevant NK stimulatory receptors, TAG-72 CAR expression, DGKαζ KO status, as well as in vitro OVCAR-3 killing activity.

[0221] Flow cytometry analysis of TAG-72 CAR / DGKαζ KO iNK cells demonstrated a high percentage of double-positive CD45+ CD56+ cells, suggesting a relatively pure population of NK cells (Figure 20A). Of these double-positive TAG-72 CAR / DGKαζ KO cells, over 60% expressed the TAG-72 CAR, suggesting that CAR retention was not affected by the manufacturing and editing process (within acceptable limits). The NK stimulatory receptors NKG2D, NKp46, and 2B4, as well as the early activation marker CD69, were also expressed at significant levels on TAG-72 CAR / DGKαζ KO cells, with expression comparable to that observed in TAG-72 CAR iNK (Figure 20A). Functional assessment using xCELLigence® impedance-based technology demonstrated that TAG-72 CAR / DGKαζ KO iNK cells enhanced OVCAR-3 cell killing compared to unedited iNK and NK cells isolated from healthy adult donor peripheral blood mononuclear cells (PBMCs) at both effector:target ratios of 1:2 and 1:1 (Figure 20C shows 1:2 ratio, data for 1:1 ratio not presented). Furthermore, TAG-72 CAR / DGKαζ KO iNKs showed similar killing ability to TAG-72 CAR alone iNKs. DGKα and DGKζ indel efficacy was analyzed using the ICE tool developed by Synthego (Figure 20B). This demonstrated an indel efficiency of approximately 60% and 75% for the DGKα and DGKζ loci, respectively, suggesting that the cytotoxic effects observed both in vitro (Figures 20A-20C) and in vivo (Figures 21A-21E) were primarily mediated by cells containing the DGKαζ gene KO.

[0222] A luciferase-based bioluminescence imaging (BLI) xenograft model was used to determine the in vivo efficacy of TAG-72 CAR and TAG-72 CAR / DGKαζ KO iNK cells generated as described in Examples 4 and 5 above. The OVCAR-3 human ovarian cancer cell line was selected as a xenograft based on its known expression of the target antigen TAG-72, efficient engraftment of OVCAR-3 cells into NSG mice, and the sensitivity of OVCAR-3 cells to TAG-72 CAR and / or TAG-72 CAR DGKαζ KO iNK cells in vitro (see Example 6). The experimental details are as follows: 4 days prior to effector cell administration (day -4), 8-week-old female immunodeficient NSG mice were injected with 2×10 5 luciferase-labeled OVCAR-3 cells were injected intraperitoneally (i.p.). One day prior to NK cell administration (day -1), mice were measured for baseline tumor burden using an AMI-HTX animal imager (Spectral Instruments Imaging) and placed into comparable luciferase-expressing groups. On day 0, 1 × 10 7 Cryopreserved iNK cells were thawed and injected i.p. into mice. To support the survival and proliferation of human iNK effector cells, all groups received 1 x 10 cells administered every 2-3 days in the first 3 weeks of the experiment. 4 They received 1 U of interleukin (IL)-2 (for 21 days) and / or 10 ng of IL-15 administered daily (for 7 days). Tumor burden was monitored and quantified weekly by BLI until the end of the study (day 84).

[0223] In this model, mice treated with TAG-72 CAR iNK or TAG-72 CAR / DGKαζ KO iNK cells displayed measurably lower tumor burdens compared to PBS, PBMC-NK control and unedited iNK treated mice over the 84 day observation period (Figure 21A-C). Beginning on day 70, a progressive increase in bioluminescence signal reflecting tumor cell proliferation was observed in TAG-72 CAR iNK treated mice but not in TAG-72 CAR / DGKαζ KO iNK treated mice (Figure 21C). Analysis of individual time points revealed a strong trend suggesting that inclusion of DGKαζ KO in iNK resulted in superior long-term in vivo efficacy as reflected by a consistently lower mean tumor burden compared to TAG-72 CAR alone iNK (Figure 21D,E).

[0224] Example 10 – Differentiation of DGK KO CD34+ (with or without TAG-72 CAR) into iT cells iT cells can be generated using a variety of published methods, including, for example, through induction of iT cells by Delta-like ligand 1 (DLL1) (Themeli et al., Generation of tumor-targeted human T lymphocytes from induced pluripotent stem cells for cancer therapy, Nat Biotechnol., Oct 2013, Vol 31(10), pp. 928-933) or Delta-like ligand 4 (DLL4) (Flippe et al., Rapid and Reproducible Differentiation of Hematopoietic and T Cell Progenitors From Pluripotent Stem Cells, Front. Cell Dev. Biol., 20 Oct 2020, Vol 8, Article 577464), or through Notch ligand signaling bound to a supporting structure in suspension (US20200399599A1, Method for generating cells of the T cell It has been described that the expression of mouse stromal support cells (OP9) in the presence of cytokines such as IL-7, FLT3, SCF, etc. is commonly utilized, or the commercially available culture system STEMdiff™ T Cell Kit (STEMCELL Technologies) is used.

[0225] The iPSC source can be either unedited or contain at least one of any combination of DGKα KO, DGKζ KO, DGKαζ KO or CAR knock-in, and can be either clone-derived, enriched population, or unpurified bulk gene-edited population. These iPSC sources are differentiated into CD34+ cells. Pre-sorted CD34+ cells are re-seeded on plates coated with StemSpan™ Lymphoid Differentiation Coating Material (STEMCELL Technologies) in StemSpan™ Lymphoid Progenitor Cell Growth Medium (STEMCELL Technologies). This is marked as day 0 of T cell differentiation. After 14 days, medium is changed twice a week, and progenitor T cells are collected and placed in StemSpan™ T Cell Progenitor Maturation Medium (STEMCELL Technologies) for the recommended period. T cells are collected and then activated in CD8 SP T Cell Maturation Medium (STEMCELL Technologies) using either ImmunoCult™ Human CD3 / CD28 / CD2 T Cell Activator (STEMCELL Technologies) or Human T-Activator CD3 / CD28 Dynabeads (Thermo Fisher) to generate mature CD3+ T cells with a predominantly CD8+ phenotype.

[0226] A CAR construct targeting TAG-72 was first knocked into the safe harbor locus of iPSCs using CRISPR / Cas9 gene editing, and then both DGKα and DGKζ were knocked out using CRISPR / Cas9 (as described in Examples 1-2). Cells were cloned after gene editing, and homozygous DGKαζ KO and CAR KI were selected for differentiation (see Example 3). The indel efficiency of TAG-72 CAR iPSC enriched lines was 90% for DGKα and 99% for DGKζ (Figure 22A). These iPSC lines were successfully differentiated into iT cells, where hallmark markers of bona fide T cells were characterized by flow cytometry (Figure 22B). Importantly, cell populations co-expressing TCRαβ and TCRγδ were obtained at comparable levels with or without CAR and DGKαζ KO. This supports the finding that inclusion of DGKαζ KO in iPSCs does not block the development of iPSCs into iT cells. Co-expression of CAR with CD3 further confirms that iT cells explicitly retain the CAR.

[0227] As shown in Figure 22C, iPSC-derived TAG-72 CAR+DGKαζ KO iT cells show on-target CAR-mediated activity against the TAG-72-expressing ovarian cancer cell line OVCAR-3 as shown by xCELLigence® in vitro. Conversely, compared to OVCAR-3 controls, non-transfected iT cells and T cells isolated from PBMCs without CAR constructs do not show comparable function. Importantly, potent cytotoxicity function is retained in iT cells derived from iPSC clones with DGKαζ KO and CAR KI gene editing, thus demonstrating functional CAR iT cells with DGKαζ KO.

[0228] Example 11 –In vitro function of TAG-72 CAR / DGK KO iT cells in the presence of TGFβ iT cells can be generated as described in Example 10.

[0229] The functional benefit of DGK KO in iT cells in the tumor microenvironment was modeled using an in vitro cytotoxicity functional assay (outlined above in Example 7) in the presence of TGFβ, as shown in FIG. 23A. Briefly, iT cells with or without CAR and DGKαζ KO were preconditioned with either 0 ng / mL, 10 ng / mL or 100 ng / mL of TGFβ in optimal growth medium for at least 8 hours, after which the killing efficacy of iT cells in vitro was determined using a real-time cell monitoring system (xCELLigence®). 10,000 cells / 100 μL of target cells (e.g., ovarian cancer cell line OVCAR-3) were resuspended in culture medium (e.g., RPMI-1640 basal medium) supplemented with 20% fetal bovine serum and bovine insulin, and placed in a Real Time Cell Analysis microtiter ePlate compatible with the xCELLigence® system. Target cells were incubated at 37° C., 5% CO 2 Co-cultures were maintained at 1:1 E:T for 3–10 h to allow cell attachment. After target cell attachment, iT effector cells with or without TAG-72 CAR and DGKαζ KO, and with or without exposure to TGFβ, were added at 1:1 E:T. All co-cultures were maintained in optimal growth conditions ± TGFβ for at least 40 h. Additionally, target cells alone ± TGFβ were maintained in parallel. Throughout, cell impedance was monitored at 15 min intervals. A decrease in impedance indicates target cell detachment and ultimately cell death.

[0230] In the absence of TGFβ preconditioning, cytotoxic function is observed in iT (non-transfected) cells (Figure 23B and Figure 23C, filled symbols), with nearly complete target cell death observed within a 40-h monitoring period, as indicated by a normalised cell index of 0. In contrast, preconditioning and maintenance of iT (non-transfected) cells in either 10 ng / mL TGFβ (Figure 23B) or 100 ng / mL TGFβ (Figure 23C) resulted in a decrease in in vitro cytotoxic function. Such inhibitory effects of TGFβ on in vitro cytotoxicity were not observed in TAG-72 CAR-iT cells with DGKαζ KO, indicating that deletion (knockout) of DGKαζ rendered these cells less sensitive to the suppressive effects of TGFβ on the cytotoxic function of iT cells.

Claims

1. A modified cell in which the function of at least one target gene selected from the group consisting of DGKα and DGKζ is inhibited, said modified cell retaining said gene inhibition and capable of differentiating into an immune cell having enhanced activity.

2. The modified cell of claim 1, wherein the modified cell is a stem cell selected from the group consisting of embryonic stem cells, umbilical cord stem cells and induced pluripotent stem cells, or a cell selected from the group consisting of hemogenic endothelial cells, hematopoietic progenitor cells, hematopoietic stem cells or hematopoietic-like stem cells, and optionally the modified cell is a cell derived from a triple homozygous HLA haplotype donor.

3. The modified cell described in claim 1 or 2, characterized in that the modified cell contains a nucleic acid encoding a chimeric antigen receptor (CAR), and the immune cell differentiated from the modified cell expresses the CAR.

4. 1. A method for generating stem cell-derived immune cells with enhanced function, the method comprising: (I) A method for producing a modified stem cell by modifying the stem cell to inhibit the function of at least one target gene selected from the group consisting of DGKα and DGKζ, The method, wherein the modified stem cells are capable of differentiating into stem cell-derived immune cells that retain the targeted gene inhibition of the modified stem cells and have enhanced activity.

5. 5. The method of claim 4, wherein the stem cells are pluripotent stem cells selected from the group consisting of induced pluripotent stem cells (iPSCs) or embryonic stem cells, or stem cells selected from the group consisting of pre-HSCs, hemogenic endothelial (HE), and hematopoietic stem cells (HSCs), and optionally the stem cells are stem cells derived from a triple homozygous HLA haplotype donor.

6. 6. The method of claim 4 or 5, further modified to include a nucleic acid encoding a chimeric antigen receptor (CAR), optionally wherein the immune cells produced by the method express the CAR.

7. The method further comprises, in addition to step (I), (i) contacting the modified stem cells obtained in step (I) or cloned cells generated from the modified stem cells with a composition to obtain mesodermal cells; (ii) contacting the mesodermal cells with a composition to obtain CD34+ cells; and (iii) contacting the CD34+ cells with a composition to obtain stem cell-derived immune cells. The method according to any one of claims 4 to 6.

8. The method according to any one of claims 4 to 7, characterized in that the inhibition of the function of the target gene in step (I) is achieved by a gene editing system, optionally wherein the gene editing system is selected from the group consisting of CRISPR / Cas, TALEN and ZFN.

9. The gene editing system is a CRISPR / Cas system comprising a guide RNA-nuclease complex, and optionally the CRISPR / Cas system is selected from the group consisting of Cpf1, Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, Cas12, Cas13, Cas100, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Csm7, Csm8, Csm9, Csm10, Csm11, Csm12, Csm13, Csm14, Csm15, Csm16, Csm17, Csm18, Csm19, Csm20, Csm21, Csm22, Csm23, Csm24, Csm25, Csm26, Csm27, Csm28, Csm29, Csm30, Csm31, Csm32, Csm33, Csm34, Csm35, Csm36, Csm37, Csm38, Csm39, Csm31, Csm31, Csm32, Csm33, Csm34, Csm35, Csm36, Csm37, Csm38, Csm39, Csm39, Csm31, Csm31, Csm31, Csm32, Csm33, Csm34, Csm35, Csm36, Csm37, Csm38, Csm39, Csm39, Csm39, Csm39, Csm39, Csm39, Csm 9. The method of claim 8, utilising a guide RNA-dependent nuclease selected from the group consisting of mr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, CasX, CasY, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3 and Csf4, preferably wherein the guide RNA targets a nucleotide sequence selected from the group consisting of SEQ ID NO:1 to SEQ ID NO:

16.

10. The method of any one of claims 4 to 9, wherein both alleles of the target gene are inhibited.

11. The method according to any one of claims 4 to 10, wherein both the DGKα gene and the DGKζ gene are inhibited.

12. 12. The method of any one of claims 4 to 11, wherein the immune cell is selected from a multipotent progenitor cell, a common lymphoid progenitor cell, an early thymic progenitor cell, a pre-T cell progenitor cell, a pre-NK progenitor cell, a T cell progenitor cell, a NK cell progenitor cell, a T cell, a NK cell, a NKT cell, a B cell, a common myeloid progenitor cell, a macrophage, and a monocyte.

13. The method of any one of claims 4 to 12, wherein the immune cells recognize one or more target antigens, preferably the target antigens are selected from the group consisting of TAG-72, CCR4, CD19, CD20, CD22, CD24, CD30, CD47, folate receptor alpha (FRα), BCMA, mesothelin, and Mucl.

14. A stem cell-derived immune cell produced by the method described in any one of claims 4 to 13.

15. The stem cell-derived immune cell of claim 14, wherein both alleles of the target gene are inhibited.

16. The stem cell-derived immune cell of claim 15, wherein both the DGKα gene and the DGKζ gene are inhibited.

17. The stem cell-derived immune cell according to any one of claims 14 to 16, wherein the immune cell is selected from a multipotent progenitor cell, a common lymphoid progenitor cell, an early thymic progenitor cell, a pre-T cell progenitor cell, a pre-NK precursor cell, a T precursor cell, a NK precursor cell, a T cell, a NK cell, a NKT cell, a B cell, a common myeloid precursor cell, a macrophage, and a monocyte.

18. The stem cell-derived immune cell of any one of claims 14 to 17, wherein the immune cell recognizes one or more target antigens, preferably the target antigens are selected from the group consisting of TAG-72, CCR4, CD19, CD20, CD22, CD24, CD30, CD47, folate receptor alpha (FRα), BCMA, mesothelin, and Mucl.

19. A composition for modifying a cell to inhibit the function of at least one target gene selected from the group consisting of DGKα and DGKζ, the composition comprises a guide RNA-nuclease complex capable of editing a sequence of a target gene; The composition, wherein the guide RNA targets a nucleotide sequence selected from the group consisting of SEQ ID NO:1 to SEQ ID NO:16; Optionally, the nuclease is Cpf1, Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, Cas12, Cas13, 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, CasX, CasY, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, and Csf4.

20. 19. The stem cell-derived immune cell of any one of claims 14 to 18, for use in treating a pathology in a subject, optionally wherein the pathology is cancer, an infectious disease, an autoimmune disorder, organ fibrosis, or endometriosis.

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