Recombinant immune cell therapy

Abstract

The present disclosure relates, in part, to, among other things, recombinant immune cells that suppress a host immune response.

Description

[Technical field]

[0001] The present disclosure relates to recombinant immune cells that evade recognition and / or clearance by the host immune system.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63 / 157,332, filed March 5, 2021, the entire contents of which are incorporated herein by reference. [Background technology]

[0003] Autologous recombinant cell therapies, such as autologous chimeric antigen receptor T cell (CAR-T) therapies, have revolutionized the treatment of hematological cancers, but they are limited by production time and variability, the requirement for lymphodepletion, and side effects associated with cytokine release. Allogeneic cell therapies derived from gene-edited induced pluripotent stem cells (iPSCs) are being developed to address the challenges associated with autologous recombinant cell therapies. These "off-the-shelf" cell therapies contain specific edits designed to mitigate immune rejection and confer improved therapeutic properties and greater safety. However, efficient, traceless, biallelic targeting of defined loci in iPSCs remains technically challenging with current gene editing approaches.

[0004] Furthermore, although induced pluripotent stem cells (iPSCs) rapidly differentiate into a wide variety of cell types both in vitro and in vivo, developing directed differentiation procedures that reliably obtain pure populations of functional cells has proven challenging, particularly when differentiating into cells of lymphoid or myeloid lineages. Generating functional immune cells from iPSCs is of particular interest in supporting the development of off-the-shelf recombinant cell therapies for immuno-oncology applications.

[0005] There is a need for improved compositions and methods for generating cell therapies that can be engineered and producible in a practical manner. Summary of the Invention

[0006] Thus, the present disclosure relates to compositions and methods for cell therapy, e.g., recombinant immune cells that evade recognition and / or clearance by the host immune system and therefore have a therapeutic effect that is not diminished or lost due to the subject's immune response.

[0007] In one aspect, an isolated immune cell is provided that comprises a genetic disruption, e.g., optionally, loss of function in both alleles of the B2M gene, in the beta-2-microglobulin (B2M) gene, wherein the immune cell is selected from a lymphoid cell or a myeloid cell. Optionally, the lymphoid cell is a T cell, e.g., a cytotoxic T cell or a gamma-delta T cell; a NK cell; or a NK-T cell. Optionally, the myeloid cell is a macrophage, e.g., an M1 macrophage or an M2 macrophage. In an embodiment, the immune cell has downregulated MHC class I expression and / or activity. In an embodiment, the immune cell has reduced or eliminated sensitivity to cell killing by T cells or other immune cells compared to another immune cell that comprises a genetic disruption in the B2M gene. In an embodiment, the immune cell has reduced or eliminated immune cell fratricide, e.g., NK cell fratricide. In embodiments, the immune cells are stealth cells, e.g., the immune cells are substantially unrecognized by the immune system upon administration to a subject.

[0008] In some cases, the immune cells express a fusion protein that includes a B2M polypeptide and HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, and HLA-G polypeptides. The fusion protein can be expressed by inserting a repair template into a single- or double-stranded break in the B2M gene; in some cases, the repair template includes coding sequences for B2M and HLA genes. In particular, the fusion protein replaces the endogenous B2M and HLA pair expressed by the immune cells, making the immune cells less likely to be reduced or eliminated by the host's immune cells.

[0009] In embodiments, immune cells, optionally T cells or NK cells, are genetically engineered to express a recombinant chimeric antigen receptor (CAR), comprising an intracellular signaling domain, a transmembrane domain, and an extracellular domain comprising an antigen binding region. In embodiments, immune cells, optionally T cells or NK cells, are engineered to be directed to ROR1 and / or CD19.

[0010] In embodiments, the cells, e.g., B2M knockout immune cells, e.g., T cells, NK cells, or macrophages, have substantially reduced or absent self-killing and even self-activating activity (even in the absence of cytokines such as IL-2 and IL-15). Additionally, the cells, e.g., B2M knockout immune cells, e.g., T cells, NK cells, or macrophages, have tumoricidal activity (even in the absence of cytokines such as IL-2 and IL-15) and unexpected expansion and proliferation properties.

[0011] In another aspect, a method for producing a recombinant immune cell is provided, comprising: (a) reprogramming a somatic cell into an iPS cell, the reprogramming comprising contacting the iPS cell with ribonucleic acid (RNA) encoding one or more reprogramming factors; (b) disrupting a B2M gene in the iPS cell, the disrupting comprising genetically editing the cell by contacting the cell with RNA encoding one or more gene editing proteins; and (c) differentiating the iPS cell into an immune cell, the immune cell being selected from a lymphoid cell or a myeloid cell. In some cases, the lymphoid cell is a T cell, e.g., a cytotoxic T cell or a gamma-delta T cell; a NK cell; or a NK-T cell. In some cases, the myeloid cell is a macrophage, e.g., an M1 macrophage or an M2 macrophage.

[0012] In another aspect, a method of treating cancer is provided, comprising: (a) obtaining an isolated immune cell comprising a genetically engineered disruption in the B2M gene; and (b) administering the isolated immune cell to a subject in need thereof, wherein the immune cell is selected from a lymphoid cell or a myeloid cell. In some cases, the lymphoid cell is a T cell, e.g., a cytotoxic T cell or a gamma-delta T cell; a NK cell; or a NK-T cell. In some cases, the myeloid cell is a macrophage, e.g., an M1 macrophage or an M2 macrophage. The immune cell can be further genetically engineered to express a chimeric antigen receptor (CAR).

[0013] Further aspects and advantages of the present disclosure will become readily apparent to those skilled in the art from the following detailed description, which shows and describes only exemplary embodiments of the present disclosure. As will be understood, the present disclosure is capable of other and different embodiments, and its several details can be modified in various obvious respects, all without departing from the present disclosure. The drawings and description are to be regarded as illustrative in nature and not as restrictive. Any description herein of a specific composition and / or method can be applied to and used with any other specific composition and / or method disclosed herein. Furthermore, any composition disclosed herein can be applied to any method disclosed herein. In other words, any aspect or embodiment described herein can be combined with any other aspect or embodiment disclosed herein. [Brief description of the drawings]

[0014] [Figure 1A] FIG. 1 shows a non-limiting schematic diagram of the present disclosure's mRNA-based reprogramming and gene editing followed by differentiation. [Figure 1B] Shows differentiated cells that kill cancer cells. [Diagram 2]1 shows the design of a gene editing scheme for beta-2-microglobulin (B2M); the following sequences are shown: TCATCCATCCGACATTGA (SEQ ID NO:50), AGTTGACTTACTGAAG (SEQ ID NO:51), AATGGAGAGAGAGATTGAA (SEQ ID NO:52). [Diagram 3] 1 shows an RNA gel showing gene editing of B2M. [Figure 4] 1 shows a sequencing experiment showing a 14 base pair deletion from gene edit B2M; the following sequences are shown from bottom to top: ACATTGAAGAATGGAG (SEQ ID NO:55), ACATTGAAGTTGACTTACTGAAGAATGGAG (SEQ ID NO:54), and TGAATTGCTATGTGTCTGGGTTTCATCCATCCGACATTGAAGTTGACTTACTGAAGAATGGAGAGAGAATTGAAAAAGTGGAGCATTCAGACTTGT (SEQ ID NO:53). [Diagram 5] RNA levels of B2M are shown with or without IFN-gamma activation ("IFNY"; left two bars are B2M knockout, right two bars are naïve cells). [Figure 6] 1 shows a sequencing experiment demonstrating heterozygosity for CD16a (at G147D dbSNP:rs443082, Y158H dbSNP:rs396716, and F176V dbSNP:rs396991); the following sequences are shown from top to bottom: GKGRKYFHHNSDFHIPKATLKDS (SEQ ID NO:56), GKDRKYFHHNSDFYIPKATLKDS (SEQ ID NO:57), KDSGSYFCRGLFGSKNVSSETVN (SEQ ID NO:58), and KDSGSYFCRGLVGSKNVSSETVN (SEQ ID NO:59). [Figure 7] A and B show images of control (PMBC-isolated) NK cells in co-culture with K-562 tumor cells, demonstrating NK cytotoxicity of tumor cells (referred to as immunological thrombosis, or "clumping"). [Figure 8]A and B show images of gene edited and differentiated cells of the present disclosure (e.g., B2M knockout NK cells) in co-culture with K-562 tumor cells, demonstrating NK cytotoxicity of tumor cells (referred to as immunological thrombosis, or "clumping"). [Figure 9A] Results of a cytokine release assay using Luminex MAGPIX are shown. Unless otherwise indicated (i.e., "+IL2, IL15"), conditions were without the addition of IL-2 or IL-15. Additionally, cell ratios are shown (1:1, or 3:1). As elsewhere herein, PBMC-NK are control NK cells. Interferon gamma is shown. [Figure 9B] Results of a cytokine release assay using Luminex MAGPIX are shown. Unless otherwise indicated (i.e., "+IL2, IL15"), conditions were without the addition of IL-2 or IL-15. Additionally, cell ratios are shown (1:1, or 3:1). As elsewhere herein, PBMC-NK are the control NK cells. IL-2 is shown. [Figure 9C] Results of a cytokine release assay using Luminex MAGPIX are shown. Unless otherwise indicated (i.e., "+IL2, IL15"), conditions were without the addition of IL-2 or IL-15. Additionally, cell ratios are shown (1:1, or 3:1). As elsewhere herein, PBMC-NK are control NK cells. IL-7 is shown. [Figure 9D] Results of a cytokine release assay using Luminex MAGPIX are shown. Unless otherwise indicated (i.e., "+IL2, IL15"), conditions were without the addition of IL-2 or IL-15. Additionally, cell ratios are shown (1:1, or 3:1). As elsewhere herein, PBMC-NK are control NK cells. IL-13 is shown. [Figure 9E]Results of a cytokine release assay using Luminex MAGPIX are shown. Unless otherwise indicated (i.e., "+IL2, IL15"), conditions were without the addition of IL-2 or IL-15. Additionally, cell ratios are shown (1:1, or 3:1). As elsewhere herein, PBMC-NK are the control NK cells. MIP-1a is shown. [Figure 9F] Results of a cytokine release assay using Luminex MAGPIX are shown. Unless otherwise indicated (i.e., "+IL2, IL15"), conditions were without the addition of IL-2 or IL-15. Additionally, cell ratios are shown (1:1, or 3:1). As elsewhere herein, PBMC-NK are the control NK cells. MIP-1b is shown. [Figure 9G] Results of a cytokine release assay using Luminex MAGPIX are shown. Unless otherwise indicated (i.e., "+IL2, IL15"), conditions were without the addition of IL-2 or IL-15. Additionally, cell ratios are shown (1:1, or 3:1). As elsewhere herein, PBMC-NK are control NK cells. TNFa is shown. [Figure 9H] Results of a cytokine release assay using Luminex MAGPIX are shown. Unless otherwise indicated (i.e., "+IL2, IL15"), conditions were without the addition of IL-2 or IL-15. Additionally, cell ratios are shown (1:1, or 3:1). As elsewhere herein, PBMC-NK are control NK cells. GM-CSF is shown. [Figure 10A] 1 shows flow cytometry data for gene edited and differentiated cells (e.g., B2M knockout NK cells) of the present disclosure as described in the Examples. [Figure 10B] 1 shows flow cytometry data for gene edited and differentiated cells (e.g., B2M knockout NK cells) of the present disclosure as described in the Examples. [Figure 10C] 1 shows flow cytometry data for gene edited and differentiated cells (e.g., B2M knockout NK cells) of the present disclosure as described in the Examples. [Figure 10D] 1 shows flow cytometry data for gene edited and differentiated cells (e.g., B2M knockout NK cells) of the present disclosure as described in the Examples. [Figure 11A] The structure of the B2M-HLA-E repair template is shown. [Figure 11B] Ideal target sites for the B2M-HLA-A repair template (SEQ ID NO: 60: MSRSVALAVLALLSLSGLEAIQ; and SEQ ID NO: 61: ATGTCTCGCTCCGTGGCCTTAGCTGTGCTCGCGCTACTCTCTCTTTCTGGCCTGGAGGCTATCCAGCgtgagtctctcctaccctcccgctc) are shown. [Figure 11C] Additional target binding sites are shown (SEQ ID NO:60 and SEQ ID NO:61 are shown again). [Figure 11D] A gel with the size of two lines is shown, with the B2M-HLA-E repair template inserted. [Figure 11E] FIG. 11D includes a graph showing the signal intensities and ratios of the bands shown in FIG. [Figure 11F] A gel with the size of two lines is shown, with the B2M-HLA-E repair template inserted. [Figure 11G] FIG. 11F includes a graph showing the signal intensities and ratios of the bands shown in FIG. [Figure 11H] The relevant sequences in the B2M-HLA-E repair template are shown. [Figure 12] A and B show the target site sequence and repair template for replacing phenylalanine (F) with valine (V) at position 158 of CD16a. The relevant sequences are shown in these figures. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] This disclosure is based, in part, on the discovery that immune cells of lymphoid or myeloid lineage, such as T cells, NK cells, and macrophages, can be gene edited and differentiated using mRNA- and iPS-based methods to obtain therapeutic cells that are immunosuppressed, yet autoactive, proliferative, and anti-tumor.

[0016] Cytotoxic lymphocytes, including T cells and NK cells, have been developed as allogeneic "off-the-shelf" cell therapy for the treatment of blood and solid tumors. However, allogeneic lymphocyte therapy faces challenges including limited expansion potential and limited in vivo persistence due to host immune rejection. To address these challenges, the present disclosure relates, in part, to a method for producing mRNA reprogrammed iPSC lines with biallelic knockout of the beta-2-microglobulin (B2M) gene, a major component of MHC class I molecules, using an mRNA-encoded, chromatin context-dependent gene editing endonuclease. As disclosed herein, these B2M knockout iPSCs were then differentiated using a novel total suspension process that replaces dedicated micropatterned culture vessels with spheroid culture steps. Surface markers of the resulting lymphocytes were characterized by flow cytometry and incubated with cancer cells to assess tumor cell engagement and cytotoxicity. Notably, a consistently high yield of lymphocytes was obtained from the B2M knockout iPSC line compared to the parent wild-type iPSC line. After 24 hours, both wild-type and B2M knockout lymphocyte cells killed 75-90% of K562 cells (effector:target (E:T) ratio of 5:1). Interestingly, B2M knockout iPSC-derived cytotoxic lymphocytes showed greater K562 cell killing upon addition of IL15 and IL2, whereas killing by wild-type cells was not controlled by these activating cytokines. Cancer cell killing activity was maintained throughout cryopreservation, albeit at reduced levels (15-40% reduction in activity). Thus, the B2M knockout iPSCs of the present disclosure are an ideal source of cytotoxic lymphocytes for developing "off-the-shelf" allogeneic cell therapy without substantial host immune rejection for the treatment of cancer.

[0017] In one aspect, an isolated immune cell is provided, comprising a genetic disruption in the beta-2-microglobulin (B2M) gene, for example, optionally, loss of function in both alleles of the B2M gene, said immune cell being selected from a lymphoid cell or a myeloid cell. In some cases, the lymphoid cell is a T cell, for example, a cytotoxic T cell or a gamma-delta T cell; a NK cell; or a NK-T cell. In some cases, the myeloid cell is a macrophage, for example, an M1 macrophage or an M2 macrophage. In an embodiment, the immune cell is a NK cell.

[0018] The immune cells are sometimes referred to herein as "recombinant immune cells."

[0019] In another aspect, a method for producing a recombinant immune cell is provided, comprising: (a) reprogramming a somatic cell into an iPS cell, the reprogramming comprising contacting the iPS cell with ribonucleic acid (RNA) encoding one or more reprogramming factors; (b) disrupting a B2M gene in the iPS cell, the disrupting comprising genetically editing the cell by contacting the cell with RNA encoding one or more gene editing proteins; and (c) differentiating the iPS cell into an immune cell, the immune cell being selected from a lymphoid cell or a myeloid cell. In some cases, the lymphoid cell is a T cell, e.g., a cytotoxic T cell or a gamma-delta T cell; a NK cell; or a NK-T cell. In some cases, the myeloid cell is a macrophage, e.g., an M1 macrophage or an M2 macrophage.

[0020] In another aspect, a method of treating cancer is provided, comprising: (a) obtaining an isolated immune cell comprising a genetic disruption in the B2M gene; and (b) administering the isolated immune cell to a subject in need thereof, wherein the immune cell is selected from a lymphoid cell or a myeloid cell. In some cases, the lymphoid cell is a T cell, e.g., a cytotoxic T cell or a gamma-delta T cell; a NK cell; or a NK-T cell. In some cases, the myeloid cell is a macrophage, e.g., an M1 macrophage or an M2 macrophage.

[0021] immunosuppression In embodiments, the immune cells are engineered to evade recognition and / or clearance by the host immune system. In embodiments, the immune cells are stealth immune cells. In embodiments, the immune cells are substantially unrecognized by the immune system upon administration to a subject.

[0022] In embodiments, the immune cells have reduced or ablated sensitivity to cell killing by T cells compared to an immune cell that does not include a genetically engineered disruption in the B2M gene. In embodiments, the immune cells have reduced or ablated sensitivity to cell killing by other immune cells compared to another immune cell that includes a genetically engineered disruption in the B2M gene.

[0023] In embodiments, the immune cells are characterized by reduced or inhibited expression of B2M. In embodiments, the immune cells are characterized by reduced or inhibited function of B2M.

[0024] In embodiments, the immune cells are characterized by reduced or inhibited expression of MHC class I. In embodiments, the immune cells are characterized by reduced or inhibited function of MHC class I.

[0025] In an embodiment, the B2M gene is the human B2M gene (e.g., NCBI reference sequence: NG_012920). The sequence of the B2M gene in various embodiments is described in the Examples section herein. B2M is a light chain of the MHC class I molecule and is therefore an integral part of the major histocompatibility complex. In humans, B2M is encoded by the b2m gene located on chromosome 15. The human protein is composed of 119 amino acids and has a molecular weight of 11.8 kilodaltons (e.g., UniProtKB-P61769). The amino acid sequence of human beta-2-microglobulin (B2M) is as follows: MSRSVALAVLALLSLSGLEAIQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDM (SEQ ID NO: 1).

[0026] In embodiments, the immune cell has substantially all copies of the B2M gene disrupted by genetic modification. In embodiments, the immune cell has a loss of function of the B2M gene. In embodiments, the immune cell has a loss of function of both alleles of the B2M gene.

[0027] In an embodiment, the genetic disruption of the B2M gene is in exon 3 of human B2M. In an embodiment, the genetic disruption of the B2M gene is a deletion. In an embodiment, the deletion is about 10 to about 20 nucleotides. In an embodiment, the deletion is about nucleotides 500 to 550 of the human B2M gene. In an embodiment, the deletion is of the sequence TTGACTTACTGAAG (SEQ ID NO: 2), or a functional equivalent thereof.

[0028] In embodiments, the immune cells have downregulated MHC class I expression and / or activity.

[0029] In embodiments, genetic disruption of B2M comprises gene editing, where gene editing is caused by contacting a cell with RNA encoding one or more gene editing proteins.

[0030] In embodiments, the immune cells are engineered to be further immunosuppressed, for example in addition to B2M (MHC class I) disruption. In embodiments, the immune cells are engineered to be disrupted in the human MHC II transactivator (CIITA) gene (NCBI Reference Sequence: NG_009628.1).

[0031] In embodiments, the immune cells have downregulated MHC class II expression and / or activity.

[0032] In an embodiment, the immune cells are characterized by reduced or inhibited expression of CIITA. In an embodiment, the immune cells are characterized by reduced or inhibited function of CIITA.

[0033] In embodiments, the immune cells are characterized by reduced or inhibited expression of MHC class II. In embodiments, the immune cells are characterized by reduced or inhibited function of MHC class II.

[0034] In embodiments, the genetic disruption of CIITA comprises gene editing, where gene editing is caused by contacting a cell with RNA encoding one or more gene editing proteins.

[0035] In an embodiment, the immune cells are characterized by reduced or inhibited expression of B2M and CIITA. In an embodiment, the immune cells are characterized by reduced or inhibited function of B2M and CIITA.

[0036] In embodiments, the immune cells are characterized by reduced or inhibited expression of MHC class I and MHC class II. In embodiments, the immune cells are characterized by reduced or inhibited function of MHC class I and MHC class II.

[0037] In embodiments, disruption of B2M and CIITA by genetic engineering comprises gene editing, where gene editing is caused by contacting a cell with RNA encoding one or more gene editing proteins.

[0038] In embodiments, the immune cells comprise a genetic mutation in one or more genes selected from HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, and HLA-G.

[0039] In embodiments, the immune cells express a fusion protein that includes a B2M polypeptide and HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, and HLA-G polypeptides. The fusion protein can be expressed by inserting a repair template into a single- or double-stranded break in the B2M gene; in some cases, the repair template includes coding sequences for B2M and HLA genes. In particular, the fusion protein replaces the endogenous B2M and HLA pair expressed by the immune cells, making the immune cells less likely to be reduced or eliminated by the host's immune cells.

[0040] In embodiments, the immune cells do not contain genetic mutations in one or more genes selected from HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, and HLA-G.

[0041] In embodiments, the genetic alteration is a genetic reduction or elimination of expression and / or activity of one or more genes selected from HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, and HLA-G.

[0042] In an embodiment, the genetic alteration is a genetic increase in expression and / or activity of one or more genes selected from HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, and HLA-G.

[0043] In embodiments, genetic disruption of B2M is combined with expression of a genetic fusion of B2M or a fragment thereof with one or more genes and / or fragments thereof selected from HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, and HLA-G.

[0044] In an embodiment, the B2M or fragment thereof and the one or more genes and / or fragments thereof are separated by a linker region. In an embodiment, the linker is (G4S)3.

[0045] In an embodiment, the genetic alteration is a genetic increase in expression and / or activity of one or more genes selected from IL-2, IL-15, IL-21. In an embodiment, IL-15 contains an N72D mutation. In an embodiment, IL-15 is fused to the cytokine binding domain of IL-15Rα.

[0046] In an embodiment, the immune cells are characterized by reduced or inhibited expression of a negative regulator of IL-15 signaling. In an embodiment, the negative regulator of IL-15 signaling is a CISH protein. In an embodiment, reduction or inhibition of the negative regulator of IL-15 signaling is achieved by disruption of the CISH gene by genetic engineering. The cytokine-inducible SH2-containing protein (CISH) gene is found at gene ID: NG_023194.1.

[0047] In embodiments, recombinant disruption of CISH comprises gene editing, where gene editing is caused by contacting cells with RNA encoding one or more gene editing proteins.

[0048] immune cells In embodiments, the immune cells are of lymphoid or myeloid lineage.

[0049] In some cases, the lymphoid cell is a T cell, such as a cytotoxic T cell or a gamma-delta T cell.

[0050] In some cases, the lymphoid cell is a NK cell, e.g., a NK-T cell. The NK cell can be a human cell.

[0051] In some cases, the bone marrow cell is a macrophage, e.g., an M1 macrophage or an M2 macrophage.

[0052] In various embodiments, immune cells are reprogrammed from stem cells, such as iPSCs, to differentiate into immune cells.

[0053] In an embodiment, the immune cell has a disruption in its beta-2-microglobulin (B2M) gene.

[0054] In embodiments, the immune cell has a disruption in its beta-2-microglobulin (B2M) gene and expresses a fusion protein comprising a B2M polypeptide and an HLA polypeptide (e.g., HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, and HLA-G polypeptides).

[0055] In embodiments, immune cells are gene edited to express a high affinity variant of CD16a (see Figures 12A and 12B).

[0056] In embodiments, the bone marrow cells are cells that are or can be derived from common bone marrow progenitor cells. In embodiments, the bone marrow cells are megakaryocytes, erythrocytes, mast cells, or myeloblasts. In embodiments, the bone marrow cells are cells that are or can be derived from myeloblasts. In embodiments, the bone marrow cells are basophils, neutrophils, eosinophils, or mononuclear cells. In embodiments, the bone marrow cells are cells that are or can be derived from mononuclear cells. In embodiments, the bone marrow cells are macrophages. In embodiments, the bone marrow cells are dendritic cells.

[0057] In an embodiment, the immune cells are NK cells. In an embodiment, the NK cells are human cells. In an embodiment, the NK cells are derived from somatic cells of the subject. In an embodiment, the NK cells are derived from allogeneic or autologous cells. In an embodiment, the NK cells are derived from induced pluripotent stem (iPS) cells. In an embodiment, the iPS cells are derived by reprogramming somatic cells into iPS cells, the reprogramming comprising contacting the iPS cells with ribonucleic acid (RNA) encoding one or more reprogramming factors, optionally selected from Oct4, Sox2, cMyc, and Klf4. In an embodiment, the iPS cells are derived from allogeneic or autologous cells. In an embodiment, the NK cells express one or more of CD56 and CD16.

[0058] In embodiments, the NK cells express CD16a, which optionally binds to an antibody / antigen complex on the tumor cell, and / or the CD16a is optionally a high affinity variant, optionally homozygous or heterozygous for F158V (see Figures 12A and 12B).

[0059] In an embodiment, the NK cells do not express CD3.

[0060] In embodiments, the NK cells are CD56 bright CD16 dim / - In embodiments, the NK cells are dim In embodiments, the NK cells are optionally CD56bright NK cells, including CD27- CD11b- NK cells tolerant In embodiments, the NK cells are optionally CD56 dim NK cells, including CD11b+ CD27- NK cells cytotoxic In embodiments, the NK cells are optionally CD56 bright NK cells, including CD27+ NK cells regulatory In embodiments, the NK cells are natural killer T (NKT) cells.

[0061] In embodiments, the NK cells secrete one or more cytokines selected from interferon gamma (IFN-g), tumor necrosis factor alpha (TNF-a), tumor necrosis factor beta (TNF-b), granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-2 (IL-2), interleukin-7 (IL-7), interleukin-10 (IL-10), interleukin-13 (IL-13), macrophage inflammatory protein 1a (MIP-1a), and macrophage inflammatory protein 1b (MIP-1b).

[0062] In embodiments, the immune cells have reduced or eliminated immune cell fratricide, e.g., NK cell fratricide. For example, in embodiments, the recombinant NK cells surprisingly do not participate in NK cytotoxicity and thus can survive, e.g., destruction in beta-2-microglobulin (B2M).

[0063] In embodiments, the immune cells are capable of autoactivation. In embodiments, the immune cells are capable of activation without the need for extracellular signals (e.g., cytokines), including signals that may be provided exogenously. In embodiments, the immune cells do not require ex vivo stimulation for activity. In embodiments, the immune cells are capable of autoactivation in the absence of an interleukin, optionally selected from IL-2 and IL-15.

[0064] In an embodiment, the immune cells are capable of inducing tumor cytotoxicity. In an embodiment, the immune cells are capable of inducing tumor cytotoxicity in the absence of an interleukin, optionally selected from IL-2 and IL-15. Assays for evaluating tumor cytotoxicity include in vivo anticancer response evaluation and microscopic evaluation, such as calcein acetoxymethyl (AM) staining-based microscopic methods (see the Examples and Chava et al. J Vis Exp. 2020 Feb 22; (156): 10.3791 / 60714, the entire contents of which are incorporated by reference). In addition, NK cell-mediated cytotoxicity assays based on colorimetric lactate dehydrogenase (LDH) measurement can be used (see Chava et al. J Vis Exp. 2020 Feb 22; (156): 10.3791 / 60714, the entire contents of which are incorporated by reference).

[0065] Immune cells bearing chimeric antigen receptors (CARs) In embodiments, the immune cells (e.g., cells that have been gene edited and reprogrammed into immune cells) are engineered with a chimeric antigen receptor (CAR), e.g., the immune cells are CAR-NK cells or CAR-T cells.

[0066] In embodiments, immune cells, optionally NK cells or T cells, are genetically engineered to express a recombinant chimeric antigen receptor (CAR) comprising an intracellular signaling domain, a transmembrane domain, and an extracellular domain comprising an antigen-binding region. In embodiments, the intracellular signaling domain comprises at least one immunoreceptor tyrosine-based activation motif (ITAM)-containing domain.

[0067] In embodiments, the intracellular signaling domain is derived from one of CD3-zeta, CD28, CD27, CD134 (OX40), and CD137 (4-1BB).

[0068] In embodiments, the transmembrane domain is derived from one of CD28 or CD8.

[0069] In embodiments, the antigen binding region binds one antigen. In embodiments, the binding region binds two antigens.

[0070] In embodiments, the extracellular domain comprising the antigen-binding region comprises (a) a natural ligand or receptor, or a fragment thereof, or (b) an immunoglobulin domain, optionally a single chain variable fragment (scFv). In embodiments, the extracellular domain comprising the antigen-binding region comprises two of (a) a natural ligand or receptor, or a fragment thereof, or (b) an immunoglobulin domain, optionally a single chain variable fragment (scFv). In embodiments, the extracellular domain comprising the antigen-binding region comprises one each of (a) a natural ligand or receptor, or a fragment thereof, and (b) an immunoglobulin domain, optionally a single chain variable fragment (scFv).

[0071] In embodiments, the antigen binding region binds to a tumor antigen.

[0072] In embodiments, the antigen-binding region is selected from the group consisting of: (i) CD94 / NKG2a, which optionally binds to HLA-E on tumor cells; (ii) CD96, which optionally binds to CD155 on tumor cells; (iii) TIGIT, which optionally binds to CD155 or CD112 on tumor cells; (iv) DNAM-1, which optionally binds to CD155 or CD112 on tumor cells; (v) KIR, which optionally binds to HLA class I on tumor cells; and (vi) NKG2, which optionally binds to HLA class I on tumor cells. (vii) CD16 (e.g., CD16a or CD16b) that optionally binds to an antibody / antigen complex on a tumor cell, and / or CD16a is optionally a high affinity variant, optionally homozygous or heterozygous for F158V; (viii) NKp30 that optionally binds to B7-H6 on a tumor cell; (ix) NKp44; and (x) NKp46.

[0073] In embodiments, the antigen binding region comprises an immunoglobulin domain, optionally an scFv directed against HLA-E, CD155, CD112 HLA class I, NKG2D-L, or B7-H6, and any variants thereof.

[0074] In embodiments, the antigen binding region is selected from the group consisting of AFP, APRIL, BCMA, CD123 / IL3Ra, CD133, CD135 / FLT3, CD138, CD147, CD19, CD20, CD22, CD239 (BCAM), CD276 (B7-H3), CD30, CD314 / NKG2D, CD319 / CS1 / SLAMF7, CD326 / EPCAM / TROP1, CD37, CD38, CD44v6, CD5, CD7, CD70, CLD The antibody binds to an antigen selected from N18.2, CLDN6, cMET, EGFRvIII, EPHA2, FAP, FR alpha, GD2, GPC3, IL13R alpha 2, integrin B7, Lewis Y (LeY), MESO, MG7 antigen, MUC1, NECTIN4, NKG2DL, PSCA, PSMA / FOL1, ROBO1, ROR1, ROR2, TNFRSF13B / TACI, and TRBC1, as well as any variants thereof. In embodiments, AFP, APRIL, BCMA, CD123 / IL3Ra, CD133, CD135 / FLT3, CD138, CD147, CD19, CD20, CD22, CD239 (BCAM), CD276 (B7-H3), CD30, CD314 / NKG2D, CD319 / CS1 / SLAMF7, CD326 / EPCAM / TROP1, CD37, CD38, CD44v6, CD5, CD7, CD70, CLDN18.2, CLDN6, cMET, EGFRvIII, EP Antigens selected from HA2, FAP, FR alpha, GD2, GPC3, IL13R alpha 2, integrin B7, Lewis Y (LeY), MESO, MG7 antigen, MUC1, NECTIN4, NKG2DL, PSCA, PSMA / FOL1, ROBO1, ROR1, ROR2, TNFRSF13B / TACI, TRBC1, and any variants thereof, can be used as single targeting CARs, dual targeting CARs, mAbs, or any combination of any of these.

[0075] In embodiments, the antigen-binding region binds to two antigens, the antigens being (a) AFP, APRIL, BCMA, CD123 / IL3Ra, CD133, CD135 / FLT3, CD138, CD147, CD19, CD20, CD22, CD239 (BCAM), CD276 (B7-H3), CD30, CD314 / NKG2D, CD319 / CS1 / SLAMF7, CD326 / EPCAM / TROP1, CD37, CD38, CD44v6, CD5, C D7, CD70, CLDN18.2, CLDN6, cMET, EGFRvIII, EPHA2, FAP, FR alpha, GD2, GPC3, IL13R alpha 2, integrin B7, Lewis Y (LeY), MESO, MG7 antigen, MUC1, NECTIN4, NKG2DL, PSCA, PSMA / FOL1, ROBO1, ROR1, ROR2, TNFRSF13B / TACI, TRBC1, TRBC2, and TROP2, and and (b) any variant of AFP, APRIL, BCMA, CD123 / IL3Ra, CD133, CD135 / FLT3, CD138, CD147, CD19, CD20, CD22, CD239 (BCAM), CD276 (B7-H3), CD30, CD314 / NKG2D, CD319 / CS1 / SLAMF7, CD326 / EPCAM / TROP1, CD37, CD38, CD44v6, CD5, CD7, CD70, CLDN18. .2, CLDN6, cMET, EGFRvIII, EPHA2, FAP, FR alpha, GD2, GPC3, IL13R alpha 2, integrin B7, Lewis Y (LeY), MESO, MG7 antigen, MUC1, NECTIN4, NKG2DL, PSCA, PSMA / FOL1, ROBO1, ROR1, ROR2, TNFRSF13B / TACI, TRBC1, TRBC2, and TROP2, and any variants thereof.

[0076] In embodiments, the antigen-binding region binds to two antigens, the antigens being: (a) CD16, CD64, CD78, CD96, CLL1, CD116, CD117, CD71, CD45, CD71, CD123, and CD138, tumor-associated surface antigens (such as ErbB2 (HER2 / neu)), carcinoembryonic antigen (CEA), epithelial cell adhesion molecule (EpCAM), epidermal growth factor receptor (EGFR), EGFR variant III (EGFRv111), CD19, CD20, CD30, CD40, disialoganglioside GD2, ductal epithelial mucin, gp36, TAG-72, glycosphingolipid, glioma-associated antigen, β-human chorionic gonadotropin, alpha-fetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CA IX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxylesterase, hsp-70-2, M-CSF, prostase, prostase specific antigen (PSA), PAP, NY-ESO-1, LAGA-1a, p53, prostein, PSMA, survival and telomerase, prostate cancer tumor antigen 1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, ephrin B2, CD22, insulin growth factor (IGF1)-1, IGF-II, IGFI receptor, mesothelin, major histocompatibility complex (MHC) molecules presenting tumor-specific peptide epitopes, 5T4, RORl, Nkp30, NKG2D, tumor stromal antigen, extra domain A (EDA) and extra domain B (EDB) of fibronectin, and the Al domain of tenascin-C (TnC Al) and fibroblast associated proteins (FAPs); lineage-specific or tissue-specific antigens (e.g., CD3, CD4, CD8, CD24, CD25, CD33, CD34, CD133, CD138, CTLA-4, B7-1 (CD80), B7-2 (CD86), GM-CSF), cytokine receptors, endoglin, major histocompatibility complex (MHC) molecules, BCMA (CD269, TNFRSF 17), multiple myeloma or lymphocytic leukemia antigens (e.g., those selected from TNFRSF17, SLAMF7, GPRC5D, FKBP11, KAMP3, ITGA8, and FCRL5), virus-specific surface antigens (e.g., HIV-specific antigens (such as HIV gpl20));EBV-specific antigen, CMV-specific antigen, HPV-specific antigen, Lassa virus (Lasse (b) an antigen selected from CD16, CD64, CD78, CD96, CLL1, CD116, CD117, CD71, CD45, CD71, CD123, and CD138, tumor-associated surface antigens (such as ErbB2 (HER2 / neu)), carcinoembryonic antigen (CEA), epithelial cell adhesion molecule (EpCAM), epidermal growth factor receptor (EGFR), EGFR variant III (EGFRv111), CD19, CD20, CD30, CD40, disialoganglioside GD2, ductal epithelial mucin, gp36, TAG-72, glycosphingolipid, glioma-associated antigen, β-human chorionic gonadotropin, alpha-fetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CA IX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxylesterase, hsp-70-2, M-CSF, prostase, prostase specific antigen (PSA), PAP, NY-ESO-1, LAGA-1a, p53, prostein, PSMA, survival and telomerase, prostate cancer tumor antigen 1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, ephrin B2, CD22, insulin growth factor (IGFI)-I, IGF-II, IGFI receptor, mesothelin, major histocompatibility complex (MHC) molecules presenting tumor-specific peptide epitopes, 5T4, RORl, Nkp30, N KG2D, tumor stromal antigen, extra domain A (EDA) and extra domain B (EDB) of fibronectin, and Al domain of tenascin-C (TnC Al) and fibroblast-associated protein (FAP);lineage- or tissue-specific antigens (e.g., CD3, CD4, CD8, CD24, CD25, CD33, CD34, CD133, CD138, CTLA-4, B7-1 (CD80), B7-2 (CD86), GM-CSF), cytokine receptors, endoglin, major histocompatibility complex (MHC) molecules, BCMA (CD269, TNFRSF 17), multiple myeloma or lymphocytic leukemia antigens (e.g., those selected from TNFRSF17, SLAMF7, GPRC5D, FKBP11, KAMP3, ITGA8, and FCRL5), virus-specific surface antigens (e.g., HIV-specific antigens (such as HIV gpl20)); antigens selected from EBV-specific antigens, CMV-specific antigens, HPV-specific antigens, Lasse virus-specific antigens, influenza virus-specific antigens, and any variants thereof;

[0077] In embodiments, the extracellular domain of the recombinant CAR comprises the extracellular domain of a NK cell activating receptor or an scFv.

[0078] In embodiments, the NK cells comprise gene edits in one or more of IL-7, CCL17, CCR4, IL-6, IL-6R, IL-12, IL-15, NKG2A, NKG2D, KIR, TRAIL, TRAC, PD1, and HPK1.

[0079] In embodiments, gene editing in one or more of IL-7, CCL17, CCR4, IL-6, IL-6R, IL-12, IL-15, NKG2A, NKG2D, KIR, TRAIL, TRAC, PD1, and HPK1 is caused by contacting the cell with RNA encoding one or more gene editing proteins. In embodiments, gene editing causes a reduction or elimination of expression and / or activity of IL-6, NKG2A, NKG2D, KIR, TRAC, PD1, and / or HPK1. In embodiments, gene editing causes an increase in expression and / or activity of IL-7, CCL17, CCR4, IL-6R, IL-12, IL-15, and / or TRAIL.

[0080] In embodiments, the immune cell, e.g., a T cell, a NK cell, or a macrophage, further comprises one or more recombinant genes capable of encoding a suicide gene product, in embodiments, the suicide gene product comprises a protein selected from the group consisting of thymidine kinase and an apoptosis signaling protein.

[0081] Any immune cell disclosed herein (i.e., comprising a gene edit (e.g., in B2M), expressing a high affinity CD16a receptor, and / or expressing a fusion protein comprising a B2M polypeptide and an HLA polypeptide) can be further genetically engineered to express a CAR.

[0082] RNA modification In embodiments, the present disclosure relates to RNA-based modifications, such as reprogramming and / or gene editing. In some embodiments, the RNA molecule encodes a gene editing protein. In some embodiments, the RNA molecule encodes a reprogramming factor.

[0083] In embodiments, the RNA is an mRNA. In embodiments, the RNA is a modified mRNA. In embodiments, the modified mRNA comprises one or more non-standard nucleotides.

[0084] In some embodiments, non-standard nucleotides can be incorporated into RNA to increase the efficiency with which RNA can be translated into protein and to reduce the toxicity of the RNA. In embodiments, the RNA molecule comprises one or more non-standard nucleotides. In some embodiments, the nucleic acid comprises one or more non-standard nucleotide members of the 5-methylcytidine demethylation pathway. In some embodiments, the nucleic acid comprises at least one of 5-methylcytidine, 5-hydroxymethylcytidine, 5-formylcytidine, and 5-carboxycytidine, or derivatives thereof. In some embodiments, the nucleic acid comprises at least one of pseudouridine, 5-methylpseudouridine, 5-methyluridine, 5-methylcytidine, 5-hydroxymethylcytidine, N4-methylcytidine, N4-acetylcytidine, and 7-deazaguanosine, or derivatives thereof.

[0085] In embodiments, the non-standard nucleotide has one or more substitutions at positions selected from 2C, 4C, and 5C relative to pyrimidines, or 6C, 7N, and 8C relative to purines.

[0086] In embodiments, the non-standard nucleotides include one or more of 5-hydroxycytidine, 5-methylcytidine, 5-hydroxymethylcytidine, 5-carboxycytidine, 5-formylcytidine, 5-methoxycytidine, pseudouridine, 5-hydroxyuridine, 5-methyluridine, 5-hydroxymethyluridine, 5-carboxyuridine, 5-formyluridine, 5-methoxyuridine, 5-hydroxypseudouridine, 5-methylpseudouridine, 5-hydroxymethylpseudouridine, 5-carboxypseudouridine, 5-formylpseudouridine, and 5-methoxypseudouridine, optionally in an amount of at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90%, or 100% of the non-standard nucleotides.

[0087] In some embodiments, the one or more non-standard nucleotides are selected from 5-methyluridine and 5-methylcytidine, 5-methyluridine and 7-deazaguanosine, 5-methylcytidine and 7-deazaguanosine, 5-methyluridine, 5-methylcytidine and 7-deazaguanosine, and 5-methyluridine, 5-hydroxymethylcytidine and 7-deazaguanosine. In some embodiments, the RNA molecule comprises at least two of 5-methyluridine, 5-methylcytidine, 5-hydroxymethylcytidine and 7-deazaguanosine, or one or more derivatives thereof. In some embodiments, the RNA molecule comprises at least three of 5-methyluridine, 5-methylcytidine, 5-hydroxymethylcytidine and 7-deazaguanosine, or one or more derivatives thereof. In embodiments, the mRNA is selected from the group consisting of 2-thiouridine, 5-azauridine, pseudouridine, 4-thiouridine, 5-methyluridine, 5-methylpseudouridine, 5-aminouridine, 5-aminopseudouridine, 5-hydroxyuridine, 5-hydroxypseudouridine, 5-methoxyuridine, 5-methoxypseudouridine, 5-ethoxyuridine, 5-ethoxypseudouridine, 5-hydroxymethyluridine, 5-hydroxymethylpseudouridine, 5-carboxyuridine, 5-carboxypseudouridine, 5-formyluridine, 5-formylpseudouridine, 5-methyl-5-azauridine, 5-amino-5-azauridine, 5-hydroxy-5-azauridine, 5-methylpseudouridine, 5-aminopseudouridine, 5-hydroxypseudouridine, 4-thio-5-azauridine, 4-thiopseudouridine, 4-thio-5-methyluridine, 4-thio-5-aminouridine, 4-thio-5-hydroxyuridine, 4-thio-5-methyl-5-azauridine, 4-thio-5-amino-5-azauridine, 4-thio-5-hydroxy-5-azauridine, 4-thio-5-methylpseudouridine, 4-thio-5-aminopseudouridine, 4-thio-5-hydroxypseudouridine, 2-thiocytidine, 5-azacytidine, pseudoisocytidine, N4-methylcytidine, N4-aminocytidine, N4-hydroxycytidine, 5-methylcytidine,5-aminocytidine, 5-hydroxycytidine, 5-methoxycytidine, 5-ethoxycytidine, 5-hydroxymethylcytidine, 5-carboxycytidine, 5-formylcytidine, 5-methyl-5-azacytidine, 5-amino-5-azacytidine, 5-hydroxy-5-azacytidine, 5-methylpseudoisocytidine, 5-aminopseudoisocytidine, 5-hydroxypseudoisocytidine, N4-methyl-5-azacytidine, N4-methylpseudoisocytidine, 2-thio-5-azacytidine, 2-thiopseudoisocytidine, 2-thio-N4-methyl Cytidine, 2-thio-N4-aminocytidine, 2-thio-N4-hydroxycytidine, 2-thio-5-methylcytidine, 2-thio-5-aminocytidine, 2-thio-5-hydroxycytidine, 2-thio-5-methyl-5-azacytidine, 2-thio-5-amino-5-azacytidine, 2-thio-5-hydroxy-5-azacytidine, 2-thio-5-methylpseudoisocytidine, 2-thio-5-aminopseudoisocytidine, 2-thio-5-hydroxypseudoisocytidine, 2-thio-N4-methyl-5-azacytidine, 2-thio-N4-methylpseudoisocytidine doisocytidine, N4-methyl-5-methylcytidine, N4-methyl-5-aminocytidine, N4-methyl-5-hydroxycytidine, N4-methyl-5-methyl-5-azacytidine, N4-methyl-5-amino-5-azacytidine, N4-methyl-5-hydroxy-5-azacytidine, N4-methyl-5-methylpseudoisocytidine, N4-methyl-5-aminopseudoisocytidine, N4-methyl-5-hydroxypseudoisocytidine, N4-amino-5-azacytidine, N4-aminopseudoisocytidine, N4-amino-5-methylcytidine, N4-amino-5-aminocytidine, N4-amino-5-hydroxycytidine, N4-amino-5-methyl-5-azacytidine, N4-amino-5-amino-5-azacytidine, N4-amino-5-hydroxy-5-azacytidine, N4-amino-5-methylpseudoisocytidine, N4-amino-5-aminopseudoisocytidine, N4-amino-5-hydroxypseudoisocytidine, N4-hydroxy-5-azacytidine, N4-hydroxypseudoisocytidine, N4-hydroxy-5-methylcytidine, N4-hydroxy-5-aminocytidine,N4-hydroxy-5-hydroxycytidine, N4-hydroxy-5-methyl-5-azacytidine, N4-hydroxy-5-amino-5-azacytidine, N4-hydroxy-5-hydroxy-5-azacytidine, N4-hydroxy-5-methylpseudoisocytidine, N4-hydroxy-5-aminopseudoisocytidine, N4-hydroxy-5-hydroxypseudoisocytidine, 2-thio-N4-methyl-5-methylcytidine, 2-thio-N4-methyl-5-aminocytidine, 2-thio-N4-methyl-5-hydroxycytidine, 2-thio-N4-methyl 2-thio-N4-methyl-5-amino-5-azacytidine, 2-thio-N4-methyl-5-hydroxy-5-azacytidine, 2-thio-N4-methyl-5-methylpseudoisocytidine, 2-thio-N4-methyl-5-aminopseudoisocytidine, 2-thio-N4-methyl-5-hydroxypseudoisocytidine, 2-thio-N4-amino-5-azacytidine, 2-thio-N4-aminopseudoisocytidine, 2-thio-N4-amino-5-methylcytidine, 2-thio-N4-amino-5-aminocytidine, 2-thio 2-thio-N4-amino-5-hydroxycytidine, 2-thio-N4-amino-5-methyl-5-azacytidine, 2-thio-N4-amino-5-amino-5-azacytidine, 2-thio-N4-amino-5-hydroxy-5-azacytidine, 2-thio-N4-amino-5-methylpseudoisocytidine, 2-thio-N4-amino-5-aminopseudoisocytidine, 2-thio-N4-amino-5-hydroxypseudoisocytidine, 2-thio-N4-hydroxy-5-azacytidine, 2-thio-N4-hydroxypseudoisocytidine, 2-thio-N4-hydroxy-5-azacytidine, 2-thio-N4-hydroxypseudoisocytidine, 2-thio-N4-hydroxy- cy-5-methylcytidine, N4-hydroxy-5-aminocytidine, 2-thio-N4-hydroxy-5-hydroxycytidine, 2-thio-N4-hydroxy-5-methyl-5-azacytidine, 2-thio-N4-hydroxy-5-amino-5-azacytidine, 2-thio-N4-hydroxy-5-hydroxy-5-azacytidine, 2-thio-N4-hydroxy-5-methylpseudoisocytidine, 2-thio-N4-hydroxy-5-aminopseudoisocytidine, 2-thio-N4-hydroxy-5-hydroxypseudoisocytidine, N6-methyladenosine,N6-aminoadenosine, N6-hydroxyadenosine, 7-deazaadenosine, 8-azaadenosine, N6-methyl-7-deazaadenosine, N6-methyl-8-azaadenosine, 7-deaza-8-azaadenosine, N6-methyl-7-deaza-8-azaadenosine, N6-amino-7-deazaadenosine, N6-amino-8-azaadenosine, N6-amino-7-deaza-8-azaadenosine, N6-hydroxyadenosine The non-standard nucleotides include one or more non-standard nucleotides selected from N6-hydroxy-7-deazaadenosine, N6-hydroxy-8-azaadenosine, N6-hydroxy-7-deaza-8-azaadenosine, 6-thioguanosine, 7-deazaguanosine, 8-azaguanosine, 6-thio-7-deazaguanosine, 6-thio-8-azaguanosine, 7-deaza-8-azaguanosine, and 6-thio-7-deaza-8-azaguanosine.

[0088] In embodiments, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100% of the non-standard nucleotides comprise one or more of 5-hydroxycytidine, 5-methylcytidine, 5-hydroxymethylcytidine, 5-carboxycytidine, 5-formylcytidine, 5-methoxycytidine, pseudouridine, 5-hydroxyuridine, 5-methyluridine, 5-hydroxymethyluridine, 5-carboxyuridine, 5-formyluridine, 5-methoxyuridine, 5-hydroxypseudouridine, 5-methylpseudouridine, 5-hydroxymethylpseudouridine, 5-carboxypseudouridine, 5-formylpseudouridine, and 5-methoxypseudouridine.

[0089] In some embodiments, the RNA molecule comprises at least one of one or more uridine residues, one or more cytidine residues, and one or more guanosine residues, and comprises one or more non-standard nucleotides. In one embodiment, about 20% to about 80% of the uridine residues are 5-methyluridine residues. In another embodiment, about 30% to about 50% of the uridine residues are 5-methyluridine residues. In a further embodiment, about 40% of the uridine residues are 5-methyluridine residues. In one embodiment, about 60% to about 80% of the cytidine residues are 5-methylcytidine residues. In another embodiment, about 80% to about 100 of the cytidine residues are 5-methylcytidine residues. In a further embodiment, about 100% of the cytidine residues are 5-methylcytidine residues. In yet a further embodiment, about 20% to about 100% of the cytidine residues are 5-hydroxymethylcytidine residues. In one embodiment, about 20% to about 80% of the guanosine residues are 7-deazaguanosine residues. In another embodiment, about 40% to about 60% of the guanosine residues are 7-deazaguanosine residues. In a further embodiment, about 50% of the guanosine residues are 7-deazaguanosine residues. In one embodiment, about 20% to about 80%, or about 30% to about 60%, or about 40% of the cytidine residues are N4-methylcytidine and / or N4-acetylcytidine residues. In another embodiment, each cytidine residue is a 5-methylcytidine residue. In a further embodiment, about 100% of the cytidine residues are 5-methylcytidine residues, and / or 5-hydroxymethylcytidine residues, and / or N4-methylcytidine residues, and / or N4-acetylcytidine residues, and / or derivatives of one or more of these. In yet a further embodiment, about 40% of the uridine residues are 5-methyluridine residues, about 20% to about 100% of the cytidine residues are N4-methylcytidine and / or N4-acetylcytidine residues, and about 50% of the guanosine residues are 7-deazaguanosine residues. In one embodiment, about 40% of the uridine residues are 5-methyluridine residues, and about 100% of the cytidine residues are 5-methylcytidine residues. In another embodiment, about 40% of the uridine residues are 5-methyluridine residues, and about 50% of the guanosine residues are 7-deazaguanosine residues.In further embodiments, about 100% of the cytidine residues are 5-methylcytidine residues and about 50% of the guanosine residues are 7-deazaguanosine residues. In one embodiment, about 40% of the uridine residues are 5-methyluridine residues, about 100% of the cytidine residues are 5-methylcytidine residues, and about 50% of the guanosine residues are 7-deazaguanosine residues. In another embodiment, about 40% of the uridine residues are 5-methyluridine residues, about 20% to about 100% of the cytidine residues are 5-hydroxymethylcytidine residues, and about 50% of the guanosine residues are 7-deazaguanosine residues. In some embodiments, less than 100% of the cytidine residues are 5-methylcytidine residues. In other embodiments, less than 100% of the cytidine residues are 5-hydroxymethylcytidine residues. In one embodiment, each uridine residue in the RNA molecule is a pseudouridine residue or a 5-methylpseudouridine residue. In another embodiment, about 100% of the uridine residues are pseudouridine residues and / or 5-methylpseudouridine residues. In a further embodiment, about 100% of the uridine residues are pseudouridine residues and / or 5-methylpseudouridine residues, about 100% of the cytidine residues are 5-methylcytidine residues, and about 50% of the guanosine residues are 7-deazaguanosine residues.

[0090] Other non-standard nucleotides that can be used in place of or in combination with 5-methyluridine include, but are not limited to, pseudouridine and 5-methylpseudouridine (also known as "1-methylpseudouridine" or "N1-methylpseudouridine"), or one or more derivatives thereof. Other non-standard nucleotides that can be used in place of or in combination with 5-methylcytidine and / or 5-hydroxymethylcytidine include, but are not limited to, pseudoisocytidine, 5-methylpseudoisocytidine, 5-hydroxymethylcytidine, 5-formylcytidine, 5-carboxycytidine, N4-methylcytidine, N4-acetylcytidine, or one or more derivatives thereof. In certain embodiments, for example, when only one transfection is performed or when cells are transfected that are not particularly sensitive to transfection-associated toxicity or innate immune signaling, the fraction of non-standard nucleotides can be reduced. Reducing the fraction of non-standard nucleotides can be beneficial, in part, because reducing the fraction of non-standard nucleotides can reduce the cost of the nucleic acid. In certain situations, for example, when a minimally immunogenic nucleic acid is desired, the fraction of non-standard nucleotides can be increased.

[0091] In an embodiment, the RNA molecule comprises a 5' cap structure. In an embodiment, the RNA molecule comprises a 5'-UTR comprising a Kozak consensus sequence. In an embodiment, the RNA molecule comprises a 5'-UTR comprising a sequence that increases RNA stability in vivo. In an embodiment, the RNA molecule comprises a 3'-UTR comprising a sequence that increases RNA stability in vivo. In an embodiment, the 5'-UTR comprises an alpha globin or beta globin 5'-UTR sequence. In an embodiment, the 3'-UTR comprises an alpha globin or beta globin 3'-UTR sequence. In an embodiment, the RNA molecule comprises a 3' poly(A) tail.

[0092] Particular embodiments relate to nucleic acids, or derivatives thereof, comprising a 5' cap structure selected from cap0, cap1, cap2, and cap3. In one embodiment, the nucleic acid comprises one or more UTRs. In another embodiment, the one or more UTRs increase the stability of the nucleic acid. In a further embodiment, the one or more UTRs comprise a 5'-UTR of alpha globin or beta globin. In yet a further embodiment, the one or more UTRs comprise a 3'-UTR of alpha globin or beta globin. In yet a further embodiment, the RNA molecule comprises a 5'-UTR of alpha globin or beta globin and a 3'-UTR of alpha globin or beta globin. In one embodiment, the 5'-UTR comprises a Kozak sequence substantially similar to a Kozak consensus sequence. In another embodiment, the nucleic acid comprises a 3' poly(A) tail. In a further embodiment, the 3' poly(A) tail is about 20 nt to about 250 nt, or about 120 nt to about 150 nt in length. In further embodiments, the 3' poly(A) tail is about 20nt, or about 30nt, or about 40nt, or about 50nt, or about 60nt, or about 70nt, or about 80nt, or about 90nt, or about 100nt, or about 110nt, or about 120nt, or about 130nt, or about 140nt, or about 150nt, or about 160nt, or about 170nt, or about 180nt, or about 190nt, or about 200nt, or about 210nt, or about 220nt, or about 230nt, or about 240nt, or about 250nt in length.

[0093] In some embodiments, the RNA comprises a tail made up of multiple adenines including one or more guanines.

[0094] In embodiments, the RNA comprises (a) a sequence encoding a protein, and (b) a tail region comprising deoxyadenosine nucleotides and one or more other nucleotides.

[0095] In embodiments, the one or more other nucleotides comprise deoxyguanosine residues. In embodiments, the tail region comprises about 1%, about 2%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, or about 50% deoxyguanosine residues. In embodiments, the tail region comprises more than 50% deoxyguanosine residues.

[0096] In embodiments, the one or more other nucleotides comprise deoxycytidine residues. In embodiments, the tail region comprises about 1%, about 2%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, or about 50% deoxycytidine residues. In embodiments, the tail region comprises more than 50% deoxycytidine residues.

[0097] In embodiments, the one or more other nucleotides comprise deoxythymidine residues. In embodiments, the tail region comprises about 1%, about 2%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, or about 50% deoxythymidine residues. In embodiments, the tail region comprises more than 50% deoxythymidine residues.

[0098] In embodiments, the one or more other nucleotides include deoxyguanosine and deoxycytidine residues. In embodiments, the tail region includes about 99%, about 98%, about 95%, about 90%, about 85%, about 80%, about 75%, about 70%, about 65%, about 60%, about 55%, or about 50% deoxyadenosine residues. In embodiments, the tail region includes less than 50% deoxyadenosine residues.

[0099] In embodiments, the one or more other nucleotides comprises a guanosine residue.

[0100] In embodiments, the tail region comprises about 1%, about 2%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, or about 50% guanosine residues, hi embodiments, the tail region comprises more than 50% guanosine residues.

[0101] In embodiments, the one or more other nucleotides comprise a cytidine residue. In embodiments, the tail region comprises about 1%, about 2%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, or about 50% cytidine residues. In embodiments, the tail region comprises more than 50% cytidine residues.

[0102] In embodiments, the one or more other nucleotides comprise a uridine residue. In embodiments, the tail region comprises about 1%, about 2%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, or about 50% uridine residues. In embodiments, the tail region comprises more than 50% uridine residues.

[0103] In embodiments, the one or more other nucleotides include guanosine and cytidine residues.In embodiments, the tail region includes about 99%, about 98%, about 95%, about 90%, about 85%, about 80%, about 75%, about 70%, about 65%, about 60%, about 55%, or about 50% adenosine residues.

[0104] In embodiments, the tail region contains less than 50% adenosine residues.

[0105] In an embodiment, the tail is (A) 150 In an embodiment, the tail is (A 39 G)3(A) 30 In an embodiment, the tail is (A 19 G)7(A) 10 In an embodiment, the tail is (A9G) 15 It is.

[0106] In embodiments, the length of the tail region is from about 80% nucleotides to about 120 nucleotides, from about 120 nucleotides to about 160 nucleotides, from about 160 nucleotides to about 200 nucleotides, from about 200 nucleotides to about 240 nucleotides, from about 240 nucleotides to about 280 nucleotides, or from about 280 nucleotides to about 320 nucleotides.

[0107] In embodiments, the length of the tail region is greater than 320 nucleotides.

[0108] In embodiments, the RNA comprises a 5' cap structure. In embodiments, the RNA 5'-UTR comprises a Kozak consensus sequence. In embodiments, the RNA 5'-UTR comprises a sequence that increases RNA stability in vivo, and the 5'-UTR can comprise the 5'-UTR of alpha globin or beta globin.

[0109] In embodiments, the RNA 3'-UTR comprises a sequence that increases RNA stability in vivo, and the 3'-UTR can comprise an alpha globin or beta globin 3'-UTR. In embodiments, the RNA comprises a 3' poly(A) tail. In embodiments, the RNA 3' poly(A) tail is about 20 nucleotides to about 250 nucleotides in length.

[0110] In embodiments, the RNA is from about 200 nucleotides to about 5000 nucleotides in length.

[0111] In embodiments, the RNA is prepared by in vitro transcription. In embodiments, the RNA is synthetic.

[0112] Gene-editing proteins In an embodiment, the present disclosure relates to gene editing to effect genetic disruption in a gene, such as beta-2-microglobulin (B2M). In an embodiment, gene editing is performed using an RNA molecule that codes for a gene editing protein.

[0113] In embodiments, the gene editing protein is selected from a nuclease, a Transcription Activator-Like Effector Nuclease (TALEN), a RiboSlice, a zinc finger nuclease, a meganuclease, a nickase, a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) associated protein or a naturally occurring or recombinant variant, family member, ortholog, fragment or fusion construct thereof.

[0114] In embodiments, the gene editing protein comprises (i) a DNA binding domain comprising a plurality of repeat sequences, and (ii) a nuclease domain comprising a catalytic domain of a nuclease. In embodiments, at least one of the repeat sequences comprises the amino acid sequence LTPvQVVAIAwxyzα ​​(SEQ ID NO:3), optionally 36-39 amino acids in length, wherein: v is Q, D, or E; w is S or N; x is I, H, N, or I; y is D, A, I, N, H, K, S, G or absent; z is GGRPALE (SEQ ID NO:4), GGKQALE (SEQ ID NO:5), GGKQALETVQRLLPVLCQDHG (SEQ ID NO:6), GGKQALETVQRLLPVLCQAHG (SEQ ID NO:7), GKQALETVQRLLPVLCQDHG (SEQ ID NO:8), GKQALETVQRLLPVLCQAHG (SEQ ID NO:9), GGKQALETVQRLLPVLCQD (SEQ ID NO:10), or GGKQALETVQRLLPVLCQA (SEQ ID NO:11); α is four consecutive amino acids.

[0115] In embodiments, a comprises at least one glycine (G) residue. In embodiments, a comprises at least one histidine (H) residue. In embodiments, a comprises at least one histidine (H) residue at any one of positions 33, 34, or 35. In embodiments, a comprises at least one aspartic acid (D) residue. In embodiments, a comprises at least one, or two, or three of a glycine (G), a histidine (H), and an aspartic acid (D) residue.

[0116] In embodiments, a comprises one or more hydrophilic residues, optionally selected from polar, positively charged hydrophilic amino acids selected from arginine (R) and lysine (K); polar, neutrally charged hydrophilic amino acids optionally selected from asparagine (N), glutamine (Q), serine (S), threonine (T), proline (P), and cysteine ​​(C); polar, negatively charged hydrophilic amino acids optionally selected from aspartic acid (D) and glutamic acid (E), and aromatic, polar, positively charged hydrophilic amino acids optionally selected from histidine (H).

[0117] In some embodiments, a comprises one or more polar, positively charged hydrophilic amino acids selected from arginine (R) and lysine (K). In some embodiments, a comprises one or more polar, neutrally charged hydrophilic amino acids selected from asparagine (N), glutamine (Q), serine (S), threonine (T), proline (P), and cysteine ​​(C). In some embodiments, a comprises one or more polar, negatively charged hydrophilic amino acids selected from aspartic acid (D) and glutamic acid (E). In some embodiments, a comprises one or more aromatic, polar, positively charged hydrophilic amino acids selected from histidine (H).

[0118] In embodiments, α comprises one or more hydrophobic residues, optionally selected from hydrophobic aliphatic amino acids selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V), and optionally hydrophobic aromatic amino acids selected from phenylalanine (F), tryptophan (W), and tyrosine (Y). In some embodiments, α comprises one or more hydrophobic aliphatic amino acids selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V). In some embodiments, α comprises one or more aromatic amino acids selected from phenylalanine (F), tryptophan (W), and tyrosine (Y). In embodiments, the DNA binding domain comprises about 15, or about 16, or about 17, or about 18, or about 18.5 repeats.

[0119] In an embodiment, α is selected from the group consisting of GHGG (SEQ ID NO: 12), HGSG (SEQ ID NO: 13), HGGG (SEQ ID NO: 14), GGHD (SEQ ID NO: 15), GAHD (SEQ ID NO: 16), AHDG (SEQ ID NO: 17), PHDG (SEQ ID NO: 18), GPHD (SEQ ID NO: 19), GHGP (SEQ ID NO: 20), PHGG (SEQ ID NO: 21), PHGP (SEQ ID NO: 22), AHGA (SEQ ID NO: 23), LHGA (SEQ ID NO: 24), VHGA (SEQ ID NO: 25), IVHG (SEQ ID NO: 26), IHGM (SEQ ID NO: 27), LHGA (SEQ ID NO: 28), VHGA (SEQ ID NO: 29), IVHG (SEQ ID NO: 30), IHGM (SEQ ID NO: 31), IHGM (SEQ ID NO: 32), IHGM (SEQ ID NO: 33), IHGM (SEQ ID NO: 34), IHGM (SEQ ID NO: 35), IHGM (SEQ ID NO: 36), IHGM (SEQ ID NO: 37), IHGM (SEQ ID NO: 38), IHGM (SEQ ID NO: 39), IHGM (SEQ ID NO: 40), IHGM (SEQ ID NO: 41), IHGM (SEQ ID NO: 42), IHGM (SEQ ID NO: 43), IHGM (SEQ ID NO: 44), IHGM (SEQ ID NO: 45), IHGM (SEQ ID NO: 46), IHGM (SEQ ID NO: 47), IHGM (SEQ ID NO: 48), IHGM (SEQ ID NO: 49), IHGM ( sequence number 27), RHGD (SEQ ID NO:28), RDHG (SEQ ID NO:29), RHGE (SEQ ID NO:30), HRGE (SEQ ID NO:31), RHGD (SEQ ID NO:32), HRGD (SEQ ID NO:33), GPYE (SEQ ID NO:34), NHGG (SEQ ID NO:35), THGG (SEQ ID NO:36), GTHG (SEQ ID NO:37), GSGS (SEQ ID NO:38), GSGG (SEQ ID NO:39), GGGG (SEQ ID NO:40), GRGG (SEQ ID NO:41), and GKGG (SEQ ID NO:42).

[0120] In embodiments, the gene editing protein comprises a repeat variable dinucleotide (RVD) at residues 12 or 13 that targets the DNA binding domain to the target DNA molecule.

[0121] In an embodiment, the RVD recognises a single base pair in the nucleic acid molecule. In an embodiment, the RVD recognises a C residue in the nucleic acid molecule and is selected from HD, N (null), HA, ND and HI. In an embodiment, the RVD recognises a G residue in the nucleic acid molecule and is selected from NN, NH, NK, HN and NA. In an embodiment, the RVD recognises an A residue in the nucleic acid molecule and is selected from NI and NS. In an embodiment, the RVD recognises a T residue in the nucleic acid molecule and is selected from NG, HG, H (null) and IG.

[0122] In some embodiments, the RVD that recognizes a C residue in the nucleic acid molecule is HD. In some embodiments, the RVD that recognizes a C residue in the nucleic acid molecule is N (null). In some embodiments, the RVD that recognizes a C residue in the nucleic acid molecule is HA. In some embodiments, the RVD that recognizes a C residue in the nucleic acid molecule is ND. In some embodiments, the RVD that recognizes a C residue in the nucleic acid molecule is HI. In some embodiments, the RVD that recognizes a G residue in the nucleic acid molecule is NN. In some embodiments, the RVD that recognizes a G residue in the nucleic acid molecule is NH. In some embodiments, the RVD that recognizes a G residue in the nucleic acid molecule is NK. In some embodiments, the RVD that recognizes a G residue in the nucleic acid molecule is HN. In some embodiments, the RVD that recognizes a G residue in the nucleic acid molecule is NA. In some embodiments, the RVD that recognizes an A residue in the nucleic acid molecule is NI. In some embodiments, the RVD that recognizes an A residue in the nucleic acid molecule is NS. In some embodiments, the RVD that recognizes a T residue in the nucleic acid molecule is NG. In some embodiments, the RVD that recognizes a T residue in the nucleic acid molecule is HG. In some embodiments the RVD recognizing a T residue in the nucleic acid molecule is H (null). In some embodiments the RVD recognizing a T residue in the nucleic acid molecule is IG.

[0123] In embodiments, the gene editing protein has a DNA binding domain with at least one repeat of LTPEQVVAIAS*RVD*GGKQALETVQRLLPVLCQAGHGG (SEQ ID NO:43, where "*RVD*" corresponds to the dinucleotide "xy" in SEQ ID NO:3).

[0124] In some embodiments, the repeat sequence is 33 or 34 amino acids in length. In some embodiments, the repeat sequence is 36-39 amino acids in length. In some embodiments, the repeat sequence is 36 amino acids in length. In some embodiments, the repeat sequence is 37 amino acids in length. In some embodiments, the repeat sequence is 38 amino acids in length. In some embodiments, the repeat sequence is 39 amino acids in length.

[0125] In an embodiment, the nuclease domain comprises a catalytic domain of a nuclease. In an embodiment, the nuclease domain is capable of dimerizing with another nuclease domain. In an embodiment, the nuclease is selected from FokI, StsI, or a hybrid, repeat sequence thereof. In an embodiment, the catalytic domain is a hybrid of the catalytic domains of FokI and StsI, including the α1, α2, α3, α4, α5, α6, β1, β2, β3, β4, β5, and β6 domains of Fok1, and at least one of the domains of FokI, which are replaced in whole or in part by the α1, α2, α3, α4, α5, α6, β1, β2, β3, β4, β5, and β6 domains of StsI, and optionally includes at least one mutation.

[0126] In some embodiments, specific fragments of the endonuclease cleavage domain are used, including fragments that are truncated at the N-terminus, fragments that are truncated at the C-terminus, fragments with internal deletions, and fragments that combine N-terminus, C-terminus, and / or internal deletions and maintain some or all of the catalytic activity of the complete endonuclease cleavage domain. Determining whether a fragment can maintain some or all of the catalytic activity of the complete domain can be achieved, for example, by synthesizing a gene editing protein containing the fragment according to the methods of the invention, inducing a cell to express the gene editing protein according to the methods of the invention, and measuring the efficiency of gene editing. In some embodiments, the measurement of gene editing efficiency is used to determine whether any specific fragment maintains some or all of the catalytic activity of the complete endonuclease cleavage domain. Certain embodiments therefore relate to biologically active fragments of the endonuclease cleavage domain. In one embodiment, the endonuclease cleavage domain is selected from FokI, StsI, StsI-HA, StsI-HA2, StsI-UHA, StsI-UHA2, StsI-HF, and StsI-UHF, or a naturally occurring or recombinant variant thereof or a biologically active fragment thereof, or a hybrid or chimera thereof.

[0127] In an embodiment, the gene editing protein comprises a linker. In another embodiment, the linker connects the DNA binding domain to the nuclease domain. In a further embodiment, the linker is about 1 to about 10 amino acids in length. In some embodiments, the linker is about 1, about 2, or about 3, or about 4, or about 5, or about 6, or about 7, or about 8, or about 9, or about 10 amino acids in length. In one embodiment, the gene editing protein is capable of generating a nick or double-stranded break in the target DNA molecule.

[0128] In embodiments, the gene editing protein is any of those described in International Patent Publication No. WO2014 / 071219, or U.S. Provisional Application No. 63 / 023,678, which are incorporated by reference in their entireties.

[0129] Formulation / Administration In some embodiments, the disclosure relates to a composition described herein in the form of a pharmaceutical composition.

[0130] In various embodiments, the invention relates to a pharmaceutical composition comprising the immune cells described herein and a pharma- ceutically acceptable carrier or excipient. In some embodiments, the invention relates to a pharmaceutical composition comprising the immune cells.

[0131] Lipids / cell contact / transfection In an embodiment, the present invention relates to lipid-mediated delivery of the present RNA molecules. In an embodiment, the present mRNAs encoding gene-editing proteins and / or reprogramming factors are delivered by lipid.

[0132] In embodiments, the lipid is a compound of formula (I):

[0133] [ka] [In the formula, Q1, Q2, Q3, and Q4 are independently an atom or group capable of bearing a positive charge, A1 and A2 are independently absent, H, or optionally substituted C1-C6 alkyl; L1, L2, and L3 are independently absent, a bond, (C1-C 20 )Alkanediyl, (halo)(C1-C 20 )Alkanediyl, (hydroxy)(C1-C 20 )Alkanediyl, (alkoxy)(C1-C 20 ) alkanediyl, arylene, heteroarylene, cycloalkanediyl, heterocycle-diyl, or any combination of the above, optionally linked by one or more of ether, ester, anhydride, amide, carbamate, secondary amine, tertiary amine, quaternary ammonium, thioether, urea, carbonyl, or imine; R1, R2, R3, R4, R5, R6, R7, and R8 are independently selected from the group consisting of: absent, H, (C1-C 60 )Alkyl, (Halo)(C1-C 60 )Alkyl, (hydroxy)(C1-C 60 )Alkyl, (alkoxy)(C1-C 60 )Alkyl, (C2-C 60 ) alkenyl, (halo)(C2-C 60 )Alkenyl, (hydroxy)(C2-C 60 ) alkenyl, (alkoxy)(C2-C 60 ) alkenyl, (C2-C 60 )alkynyl, (halo)(C2-C 60 )Alkynyl, (hydroxy)(C2-C 60 )Alkynyl, (alkoxy)(C2-C 60 ) alkynyl, wherein at least one of R1, R2, R3, R4, R5, R6, R7, and R8 contains at least two unsaturated bonds, and x, y, and z are independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15.

[0134] In embodiments, the lipid is a compound of formula (II):

[0135] [ka] wherein R9, R10, R11, R12, R13, R14, R15, R16, R17, R18, R19, R20, R21, R22, R23, R24, R25, R26, R27, and R28 are independently H, halo, OH, (C1-C6)alkyl, (halo)(C1-C6)alkyl, (hydroxy)(C1-C6)alkyl, (alkoxy)(C1-C6)alkyl, aryl, heteroaryl, cycloalkyl, or heterocyclo; i, j, k, m, s, and t are independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15.

[0136] In embodiments, the lipid is a compound of formula (III):

[0137] [ka] [In the formula, L4, L5, L6, and L7 are independently a bond, (C1-C 20 )Alkanediyl, (halo)(C1-C 20 )Alkanediyl, (hydroxy)(C1-C 20 )Alkanediyl, (alkoxy)(C1-C 20 )Alkanediyl, arylene, heteroarylene, cycloalkanediyl, heterocycle-diyl, -(CH2) v1 -C(O)-, -((CH2) v1 -O) v2 - or -((CH2) v1 -C(O)-O) v2 - and R 29 , R 30 , R 31 , R 32 , R 33 , R 34 , and R 35 are independently H, (C1-C 60 )Alkyl, (Halo)(C1-C 60 )Alkyl, (hydroxy)(C1-C 60 )Alkyl, (alkoxy)(C1-C 60 )Alkyl, (C2-C 60 ) alkenyl, (halo)(C2-C 60 )Alkenyl, (hydroxy)(C2-C 60 ) alkenyl, (alkoxy)(C2-C 60 ) alkenyl, (C2-C 60 )alkynyl, (halo)(C2-C 60 )Alkynyl, (hydroxy)(C2-C 60 )Alkynyl, (alkoxy)(C2-C 60 ) alkynyl, and R 29 , R 30 , R 31 , R 32 , R 33 , R 34 , and R35 at least one of which contains at least two unsaturated bonds; v, v1, and v2 are independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15.

[0138] In embodiments, the lipid is a compound of formula (IV):

[0139] [ka] [wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15.]

[0140] In embodiments, the lipid is a compound of formula (V):

[0141] [ka]

[0142] In embodiments, the lipid is a compound of formula (VI):

[0143] [ka]

[0144] In embodiments, the lipid is a compound of formula (VII):

[0145] [ka]

[0146] In embodiments, the lipid is a compound of formula (VIII):

[0147] [ka]

[0148] In embodiments, the lipid is a compound of formula (IX):

[0149] [ka]

[0150] In embodiments, the lipid is a compound of formula (X):

[0151] [ka]

[0152] In an embodiment, the lipid is a compound of formula (XI):

[0153] [ka]

[0154] In an embodiment, the lipid is a compound of formula (XII):

[0155] [ka]

[0156] In an embodiment, the lipid is a compound of formula (XIII):

[0157] [ka]

[0158] In embodiments, the lipid is a compound of formula (XIV):

[0159] [ka]

[0160] In embodiments, the lipid is a compound of formula (XV):

[0161] [ka] [wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15.]

[0162] In an embodiment, the lipid is a compound of formula (XVI):

[0163] [ka]

[0164] In embodiments, the compounds (e.g., of Formulas I-XVI) are components of pharmaceutical compositions, and / or lipid assemblies, and / or lipid carriers, and / or lipid-nucleic acid complexes, and / or liposomes, and / or lipid nanoparticles.

[0165] In embodiments, the compounds (e.g., of Formulas I-XVI) are components of pharmaceutical compositions, and / or lipid assemblies, and / or lipid carriers, and / or lipid-nucleic acid complexes, and / or liposomes, and / or lipid nanoparticles, without the need for additional lipids or helper lipids. In embodiments, the compound (e.g., of formula I-XVI) is a component of a pharmaceutical composition, and / or a lipid assembly, and / or a lipid carrier, and / or a lipid-nucleic acid complex, and / or a liposome, and / or a lipid nanoparticle, further comprising a neutral lipid (e.g., dioleoylphosphatidylethanolamine (DOPE), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), or cholesterol), and / or an additional cationic lipid (e.g., N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), 1,2-bis(oleoyloxy)-3-3-(trimethylammonium)propane (DOTAP), or 1,2-dioleoyl-3-dimethylammonium-propane (DODAP)).

[0166] In embodiments, the lipid is any of those described in International Patent Publication No. WO2021 / 003462, the entirety of which is incorporated herein by reference.

[0167] In an embodiment, the lipid is any of those in Table A.

[0168] [Table 1-1] [Table 1-2]

[0169] Method of preparation In an aspect, the present disclosure provides a method of making a recombinant immune cell, comprising: (a) reprogramming a somatic cell into an iPS cell, the reprogramming comprising contacting the iPS cell with ribonucleic acid (RNA) encoding one or more reprogramming factors; (b) disrupting a beta-2-microglobulin (B2M) gene in the iPS cell, the disrupting comprising genetically editing the cell by contacting the cell with RNA encoding one or more gene editing proteins; and (c) differentiating the iPS cell into an immune cell, the immune cell being selected from a lymphoid cell or a myeloid cell. In some cases, the lymphoid cell is a T cell, e.g., a cytotoxic T cell or a gamma-delta T cell; a NK cell; or a NK-T cell. In some cases, the myeloid cell is a macrophage, e.g., an M1 macrophage or an M2 macrophage.

[0170] In an embodiment, the method further comprises disrupting the CIITA gene in an iPS cell, said disrupting comprising genetically editing the cell by contacting the cell with RNA encoding one or more gene-editing proteins.

[0171] In an embodiment, the immune cell is a NK cell.

[0172] In an embodiment, the somatic cells are fibroblasts or keratinocytes.

[0173] In embodiments, the methods result in an increased proliferation rate of iPS cells compared to the rate of iPS cells without disruption of the B2M gene.

[0174] In embodiments, the methods result in an increased proliferation rate of differentiated cells along lymphoid lineage cells compared to the rate of iPS cells without disruption of the B2M gene.

[0175] In embodiments, the methods result in increased expansion of differentiated cells along lymphoid lineage cells compared to the rate of iPS cells without disruption of the B2M gene.

[0176] In embodiments, the differentiation comprises embryoid body-based hematopoietic commitment. In embodiments, the differentiation comprises enrichment for CD34+ cells. In embodiments, the differentiation comprises differentiation into CD5+ / CD7+ general lymphoid progenitor cells.

[0177] In embodiments, the method comprises: dim yields CD16+ NK cells.

[0178] In embodiments, the RNA is associated with one or more lipids selected from and / or formulas I-XVI.

[0179] Treatment In an aspect, the disclosure provides a method of treating cancer comprising: (a) obtaining an isolated immune cell that comprises a genetic disruption in a beta-2-microglobulin (B2M) gene; and (b) administering the isolated immune cell to a subject in need thereof, wherein the immune cell is selected from a lymphoid cell or a myeloid cell.

[0180] In some cases, the lymphoid cell is a T cell, such as a cytotoxic T cell or a gamma-delta T cell; a NK cell; or a NK-T cell.

[0181] In some cases, the bone marrow cell is a macrophage, e.g., an M1 macrophage or an M2 macrophage.

[0182] In an embodiment, the immune cell is a NK cell.

[0183] In an embodiment, the cancer is a blood cancer. In an embodiment, the cancer is a solid tumor. In an embodiment, the cancer is a basal cell carcinoma, biliary tract cancer; bladder cancer; bone cancer; brain and central nervous system cancer; breast cancer; peritoneal cancer; cervical cancer; choriocarcinoma; colon and rectal cancer; connective tissue cancer; digestive system cancer; endometrial cancer; esophageal cancer; eye cancer; head and neck cancer; gastric cancer (including digestive cancer); glioblastoma; hepatocellular carcinoma; liver cancer; intraepithelial neoplasia; kidney or renal cancer; laryngeal cancer; leukemia; liver cancer; lung cancer (e.g., small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma); melanoma; myeloma; neuroblastoma; oral cancer (lips, tongue, mouth, and pharynx); ovarian cancer; pancreatic cancer; prostate cancer; retinoblastoma ;rhabdomyosarcoma;rectal cancer;cancer of the respiratory system;salivary gland cancer;sarcoma (e.g., Kaposi's sarcoma);skin cancer;squamous cell carcinoma;gastric cancer;testicular cancer;thyroid cancer;uterine or endometrial cancer;cancer of the urinary system;vulvar cancer;lymphomas, including Hodgkin's lymphoma and non-Hodgkin's lymphoma, and B-cell lymphomas (low-grade / follicular non-Hodgkin's lymphoma (NHL);small lymphocytic (SL) NHL;intermediate-grade / follicular NHL;intermediate-grade diffuse NHL;high-grade immunoblastic NHL;high-grade lymphoblastic NHL;high-grade small non-cleaved cell NHL (high and Waldenstrom's macroglobulinemia), chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); hairy cell leukemia; chronic myeloblastic leukemia; and other carcinomas and sarcomas; and post-transplant lymphoproliferative disorder (PTLD), and abnormal blood vessel proliferation associated with phakomatosis, edema (e.g., associated with brain tumors), and Meigs' syndrome.

[0184] The immune cells of the disclosure can be administered systemically (e.g., through a vein or artery) or can be introduced at or near a tumor.

[0185] definition Unless otherwise defined, all terms of the art, notations, and other technical and scientific terms or terminology used herein are intended to have the same meaning as commonly understood by those skilled in the art to which the claimed subject matter belongs. In some cases, terms having commonly understood meanings are defined herein for clarity and / or ready reference, and the inclusion of such definitions herein should not necessarily be construed as indicating a substantial difference to what is commonly understood in the art. The terms used herein are for the purpose of describing particular cases only and are not intended to be limiting.

[0186] As used in this specification and claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0187] As used herein, the terms "at least one," "one or more," and "and / or" are open-ended expressions that are both conjunctive and disjunctive in operation. For example, the phrases "at least one of A, B, and C," "at least one of A, B, or C," "one or more of A, B, and C," "one or more of A, B, or C," and "A, B, and / or C" mean A only, B only, C only, A and B, A and C, B and C, or A, B, and C, respectively.

[0188] As used herein, "or" can mean "and," "or," or "and / or," and can be used both exclusively and inclusively. For example, the term "A or B" can mean "A or B," "A but not B," "B but not A," and "A and B." In some cases, the context may dictate a particular meaning.

[0189] As used herein, the term "about" a number means a number that is ±10% of the number and / or within one standard deviation (±) of the number. The term "about" a range means the range minus 10% of its minimum value and plus 10% of its maximum value, and the range minus one standard deviation of its minimum value and plus one standard deviation of its maximum value.

[0190] Throughout this application, various embodiments may be presented in a range format. Descriptions in range format should be understood merely for convenience and simplicity, and should not be construed as inflexible limitations on the scope of the present disclosure. Thus, descriptions of ranges should be considered to have all possible subranges specifically disclosed, as well as individual numerical values ​​within that range. For example, descriptions of ranges such as 1-6 should be considered to have specifically disclosed subranges such as 1-3, 1-4, 1-5, 2-4, 2-6, 3-6, as well as individual numerical values ​​within that range, such as 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

[0191] When used anywhere in this disclosure and / or claims, the terms "comprise," "comprising," "contain," "containing," "including," "includes," "having," "has," "involving," or variations thereof are intended to be inclusive in the same manner as the term "comprising."

[0192] "Preventing" means at least avoiding the occurrence of a disease and / or reducing the likelihood of contracting the disease. Treating means at least alleviating or avoiding the effects of the disease, including alleviating the signs or symptoms of the disease.

[0193] The term "substantially" generally means to a significant degree or essentially means. In other words, the term "substantially" can mean nearly exact to a desired characteristic or slightly different from the exact characteristic. "Substantially" may be indistinguishable from the desired characteristic. "Substantially" may be distinguishable from the desired characteristic, but the difference is insignificant or negligible.

[0194] As used herein, the terms "increased," "increasing," or "increase" generally refer to a statistically significant increase compared to a reference level. In some embodiments, the terms "increased" or "increase" refer to an increase of at least 10% compared to a reference level, for example, an increase of at least about 10%, at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or an increase of 100% or less, or any increase between 10 and 100% compared to a reference level. Other examples of "increase" include an increase of at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, or at least 1000-fold or more compared to a reference level.

[0195] The terms "decreased", "reducing", or "reducing" are generally used herein to mean a decrease in value compared to a reference level. In some embodiments, "decreased" or "reducing" means a decrease of at least 10% compared to a reference level, such as at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or up to 100% or less (e.g., no level or an undetectable level compared to a reference level), or any decrease between 10% and 100% compared to a reference level.

[0196] Any aspect or embodiment described herein may be combined with any other aspect or embodiment disclosed herein.

[0197] Embodiment Embodiment 1. A composition comprising an isolated immune cell comprising a genetic disruption in the beta-2-microglobulin (B2M) gene, wherein said immune cell is selected from a lymphoid cell or a myeloid cell.

[0198] Embodiment 2. The composition of embodiment 1, wherein said immune cells comprise a genetic disruption of substantially all copies of the B2M gene.

[0199] Embodiment 3. The composition of embodiment 1 or 2, wherein the immune cells have a loss of function of the B2M gene.

[0200] Embodiment 4. The composition of embodiments 1-3, wherein the immune cells optionally have a loss of function of both alleles of the B2M gene caused by contacting the immune cells with RNA encoding one or more gene-editing proteins.

[0201] Embodiment 5. The composition of any one of embodiments 1 to 4, wherein said recombinant disruption of the B2M gene is in exon 3 of human B2M.

[0202] Embodiment 6. The composition of any one of embodiments 1 to 5, wherein the disruption of the B2M gene by genetic modification is a deletion.

[0203] Embodiment 7. The composition of embodiment 6, wherein the deletion is about 10 to about 20 nucleotides.

[0204] Embodiment 8. The composition of embodiment 7, wherein the deletion is about 14 nucleotides.

[0205] Embodiment 9. The composition of embodiment 7 or embodiment 8, wherein the deletion is located at approximately nucleotides 500-550 of the human B2M gene.

[0206] Embodiment 10. The composition of embodiment 9, wherein the deletion is of the sequence TTGACTTACTGAAG (SEQ ID NO: 2), or a functional equivalent thereof.

[0207] Embodiment 11. The composition of any one of embodiments 1 to 10, wherein the immune cells have downregulated MHC class I expression and / or activity.

[0208] Embodiment 12. The composition of any one of embodiments 1 to 11, wherein the immune cells are substantially unrecognized by the host immune system upon administration to a subject.

[0209] Embodiment 13. The composition of any one of embodiments 1-12, wherein the immune cells have reduced or ablated sensitivity to cell killing by host T cells compared to immune cells that do not contain a genetic disruption in the B2M gene.

[0210] Embodiment 14. The composition of any one of embodiments 1-13, wherein the immune cell has reduced or ablated sensitivity to cell killing by other host immune cells compared to another immune cell that comprises a genetic disruption in the B2M gene.

[0211] Embodiment 15. A composition described in any one of embodiments 1 to 14, wherein the immune cells are stealth cells.

[0212] Embodiment 16. The composition of any one of embodiments 1 to 15, wherein the immune cells have reduced or eliminated host immune cell fratricide, e.g., NK cell fratricide.

[0213] Embodiment 17. The composition of any one of embodiments 1 to 16, wherein the immune cells are capable of autoactivation.

[0214] Embodiment 18. The composition of embodiment 17, wherein the immune cells are capable of autoactivation in the absence of an interleukin selected from interleukin-2 (IL-2) and interleukin-15 (IL-15).

[0215] Embodiment 19. The composition of any one of embodiments 1 to 18, wherein the immune cells are capable of inducing tumor cell cytotoxicity.

[0216] Embodiment 20. The composition of any one of embodiments 1 to 19, wherein the immune cells are capable of inducing tumor cell cytotoxicity in the absence of an interleukin selected from IL-2 and IL-15.

[0217] Embodiment 21. The composition of any one of embodiments 1 to 20, wherein the immune cells further comprise a genetic disruption in the MHC II transactivator (CIITA) gene.

[0218] Embodiment 22. The composition of embodiment 21, wherein the immune cells have downregulated MHC class II expression and / or activity.

[0219] Embodiment 23. The composition of any one of embodiments 1 to 22, wherein the immune cells comprise a genetically engineered mutation in one or more genes selected from HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, and HLA-G.

[0220] Embodiment 24. The composition of any one of embodiments 1 to 23, wherein the immune cell expresses a fusion protein comprising a B2M polypeptide and HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, and HLA-G polypeptides.

[0221] Embodiment 25. The composition of embodiment 24, wherein the fusion protein is expressed by inserting a repair template into a single- or double-stranded break in the B2M gene; and the repair template comprises coding sequences for the B2M and HLA genes.

[0222] Embodiment 26. The composition of embodiment 24 and embodiment 25, wherein the fusion protein replaces the endogenous B2M and HLA pair expressed by an immune cell, thereby making the immune cell less likely to be depleted or eliminated by host immune cells.

[0223] Embodiment 27. The composition of any one of embodiments 1 to 26, wherein the immune cells do not contain a genetically modified mutation in one or more genes selected from HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, and HLA-G.

[0224] Embodiment 28. A composition described in any one of embodiments 1 to 27, wherein the mutation due to genetic recombination is a reduction or elimination, due to genetic recombination, of the expression and / or activity of one or more genes selected from HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, and HLA-G.

[0225] Embodiment 29. A composition described in any one of embodiments 1 to 27, wherein the mutation due to genetic recombination is an increase in expression and / or activity of one or more genes selected from HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, and HLA-G due to genetic recombination.

[0226] Embodiment 30. The composition of any one of embodiments 1 to 29, wherein the immune cells, optionally NK cells, are genetically engineered to express a recombinant chimeric antigen receptor (CAR), comprising an intracellular signaling domain, a transmembrane domain, and an extracellular domain comprising an antigen-binding region.

[0227] Embodiment 31. The composition of embodiment 30, wherein the intracellular signaling domain comprises at least one immunoreceptor tyrosine-based activation motif (ITAM)-containing domain.

[0228] Embodiment 32. The composition of any one of embodiments 30 or 31, wherein the intracellular signaling domain is derived from one of CD3-zeta, CD28, CD27, CD134 (OX40), and CD137 (4-1BB).

[0229] Embodiment 33. The composition of any one of embodiments 30 to 32, wherein the transmembrane domain is derived from one of CD28 or CD8.

[0230] Embodiment 34. The composition of any one of embodiments 30 to 33, wherein the antigen-binding region binds to one antigen.

[0231] Embodiment 35. The composition of any one of embodiments 30 to 33, wherein the antigen-binding region binds to two antigens.

[0232] Embodiment 36. The extracellular domain comprising an antigen-binding region is a. a natural ligand or receptor, or a fragment thereof, or b. Immunoglobulin domains, optionally single chain variable fragments (scFv) 36. The composition of any one of embodiments 30 to 35, comprising:

[0233] Embodiment 37. The extracellular domain comprising an antigen-binding region is a. a natural ligand or receptor, or a fragment thereof, or b. Immunoglobulin domains, optionally single chain variable fragments (scFv) The composition of any one of embodiments 30 to 35, comprising two of:

[0234] Embodiment 38. The extracellular domain comprising an antigen-binding region is a. a natural ligand or receptor, or a fragment thereof, and b. Immunoglobulin domains, optionally single chain variable fragments (scFv) 36. The composition of any one of embodiments 30 to 35, comprising one of each of:

[0235] Embodiment 39. The composition of any one of embodiments 30 to 38, wherein the antigen-binding region binds to a tumor antigen.

[0236] Embodiment 40. The antigen-binding region comprises: a. CD94 / NKG2a, which optionally binds to HLA-E on tumor cells; b. CD96, optionally binding to CD155 on tumor cells; c. TIGIT optionally binds to CD155 or CD112 on tumor cells; d. DNAM-1, which optionally binds to CD155 or CD112 on tumor cells; e. KIR that optionally binds to HLA class I on tumor cells; f. NKG2D, which optionally binds to NKG2D-L on tumor cells; g. optionally, CD16a that binds to an antibody / antigen complex on a tumor cell, and / or the CD16a is optionally a high affinity variant, optionally homozygous or heterozygous for F158V; h. NKp30, which optionally binds to B7-H6 on tumor cells; i.NKp44; and j.NKp46 40. The composition of any one of embodiments 30-39, comprising one or more of:

[0237] Embodiment 41. The composition of any one of embodiments 30 to 40, wherein the antigen-binding region comprises an immunoglobulin domain, optionally an scFv directed against HLA-E, CD155, CD112 HLA class I, NKG2D-L, or B7-H6.

[0238]

[0039] Embodiment 42. The antigen-binding region is selected from the group consisting of AFP, APRIL, BCMA, CD123 / IL3Ra, CD133, CD135 / FLT3, CD138, CD147, CD19, CD20, CD22, CD239 (BCAM), CD276 (B7-H3), CD30, CD314 / NKG2D, CD319 / CS1 / SLAMF7, CD326 / EPCAM / TROP1, CD37, CD38, CD44v6, CD5, CD7, CD70, CLDN18.2, CLD 42. The composition of any one of embodiments 30 to 41, which binds to an antigen selected from N6, cMET, EGFRvIII, EPHA2, FAP, FR alpha, GD2, GPC3, IL13R alpha 2, integrin B7, Lewis Y (LeY), MESO, MG7 antigen, MUC1, NECTIN4, NKG2DL, PSCA, PSMA / FOL1, ROBO1, ROR1, ROR2, TNFRSF13B / TACI, TRBC1, TRBC2, and TROP2.

[0239] Embodiment 43. The antigen-binding region binds to two antigens, and the antigens are: a.AFP, APRIL, BCMA, CD123 / IL3Ra, CD133, CD135 / FLT3, CD138, CD147, CD19, CD20, CD22, CD239(BCAM), CD276(B7 -H3), CD30, CD314 / NKG2D, CD319 / CS1 / SLAMF7, CD326 / EPCAM / TROP1, CD37, CD38, CD44v6, CD5, CD7, CD70, CLDN18. 2, CLDN6, cMET, EGFRvIII, EPHA2, FAP, FR alpha, GD2, GPC3, IL13R alpha 2, integrin B7, Lewis Y (LeY), MESO, MG7 antigen, MUC1, NECTIN4, NKG2DL, PSCA, PSMA / FOL1, ROBO1, ROR1, ROR2, TNFRSF13B / TACI, TRBC1, TRBC2, and TROP2, and b.AFP, APRIL, BCMA, CD123 / IL3Ra, CD133, CD135 / FLT3, CD138, CD147, CD19, CD20, CD22, CD239(BCAM), CD276(B 7-H3), CD30, CD314 / NKG2D, CD319 / CS1 / SLAMF7, CD326 / EPCAM / TROP1, CD37, CD38, CD44v6, CD5, CD7, CD70, CLDN1 8.2, an antigen selected from CLDN6, cMET, EGFRvIII, EPHA2, FAP, FR alpha, GD2, GPC3, IL13R alpha 2, integrin B7, Lewis Y (LeY), MESO, MG7 antigen, MUC1, NECTIN4, NKG2DL, PSCA, PSMA / FOL1, ROBO1, ROR1, ROR2, TNFRSF13B / TACI, TRBC1, TRBC2, and TROP2. The composition of any one of embodiments 30 to 42, wherein

[0240] Embodiment 44. The composition of any one of embodiments 30 to 43, wherein the extracellular domain of the recombinant CAR comprises the extracellular domain of a NK cell activating receptor or an scFv.

[0241] Embodiment 45. The composition of any one of embodiments 30-44, wherein the immune cells comprise gene edits in one or more of IL-7, CCL17, CCR4, IL-6, IL-6R, IL-12, IL-15, NKG2A, NKG2D, KIR, TRAIL, TRAC, PD1, and HPK1.

[0242] Embodiment 46. The composition of embodiment 45, wherein the gene editing in one or more of IL-7, CCL17, CCR4, IL-6, IL-6R, IL-12, IL-15, NKG2A, NKG2D, KIR, TRAIL, TRAC, PD1, and HPK1 is caused by contacting the cell with RNA encoding one or more gene editing proteins.

[0243] Embodiment 47. The composition of embodiment 46, wherein the gene editing causes a reduction or elimination of expression and / or activity of IL-6, NKG2A, NKG2D, KIR, TRAC, PD1, and / or HPK1.

[0244] Embodiment 48. The composition of embodiment 46, wherein the gene editing causes increased expression and / or activity of IL-7, CCL17, CCR4, IL-6R, IL-12, IL-15, and / or TRAIL.

[0245] Embodiment 49. The composition of any one of embodiments 1 to 48, wherein the lymphoid cells are T cells.

[0246] Embodiment 50. The composition of embodiment 49, wherein the T cells are gamma-delta T cells.

[0247] Embodiment 51. The composition of any one of embodiments 1 to 48, wherein the lymphoid cells are NK cells.

[0248] Embodiment 52. The composition of embodiment 51, wherein the NK cells are NK-T cells.

[0249] Embodiment 53 The composition of embodiment 51, wherein the NK cells are human cells.

[0250] Embodiment 54. The composition of any one of embodiments 51 to 53, wherein the NK cells are derived from the subject's somatic cells.

[0251] Embodiment 55. The composition of any one of embodiments 51-54, wherein the NK cells are derived from allogeneic or autologous cells.

[0252] Embodiment 56. The composition of any one of embodiments 51 to 55, wherein the NK cells are derived from induced pluripotent stem (iPS) cells.

[0253] Embodiment 57. The composition of embodiment 56, wherein the iPS cells are derived by reprogramming somatic cells into iPS cells, and the reprogramming comprises contacting the iPS cells with ribonucleic acid (RNA) encoding one or more reprogramming factors, optionally selected from Oct4, Sox2, cMyc, and Klf4.

[0254] Embodiment 58. The composition of embodiment 57, wherein said reprogramming comprises contacting said iPS cells with one or more RNAs encoding each of Oct4, Sox2, cMyc, and Klf4.

[0255] Embodiment 59. The composition of any one of embodiments 56 or 57, wherein the iPS cells are derived from allogeneic or autologous cells.

[0256] Embodiment 60. The composition of any one of embodiments 1 to 59, wherein the disruption of B2M by genetic engineering comprises gene editing, and the gene editing is caused by contacting the cell with RNA encoding one or more gene editing proteins.

[0257] Embodiment 61. The composition of any one of embodiments 1 to 60, wherein the NK cells express one or more of CD56 and CD16.

[0258] Embodiment 62. The composition of embodiment 61, wherein the NK cells optionally express CD16a that binds to an antibody / antigen complex on tumor cells, and / or the CD16a is optionally a high affinity variant, optionally homozygous or heterozygous for F158V.

[0259] Embodiment 63. The composition of any one of embodiments 1 to 62, wherein the NK cells do not express CD3.

[0260] Embodiment 64. The composition of any one of embodiments 1 to 63, wherein the NK cells are CD56bright CD16dim / -.

[0261] Embodiment 65. The composition of any one of embodiments 1 to 64, wherein the NK cells are CD56dim CD16+.

[0262] Embodiment 66. The composition of any one of embodiments 1 to 65, wherein the NK cells are optionally NKtolerant cells, including CD56bright NK cells, or CD27- CD11b- NK cells.

[0263] Embodiment 67. The composition of any one of embodiments 1 to 65, wherein the NK cells are NKcytotoxic cells, optionally including CD56dim NK cells, or CD11b+ CD27- NK cells.

[0264] Embodiment 68. The composition of any one of embodiments 1 to 65, wherein the NK cells are NK regulatory cells, optionally including CD56bright NK cells, or CD27+ NK cells.

[0265] Embodiment 69. The composition of any one of embodiments 1 to 65, wherein the NK cells are natural killer T (NKT) cells.

[0266] Embodiment 70. The composition of any one of embodiments 1 to 69, wherein the NK cells secrete one or more cytokines selected from interferon gamma (IFN-g), tumor necrosis factor alpha (TNF-a), tumor necrosis factor beta (TNF-b), granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-2 (IL-2), interleukin-7 (IL-7), interleukin-10 (IL-10), interleukin-13 (IL-13), macrophage inflammatory protein 1a (MIP-1a), and macrophage inflammatory protein 1b (MIP-1b).

[0267] Embodiment 71. The composition of any one of embodiments 1 to 70, wherein the NK cells further comprise one or more recombinant genes capable of encoding a suicide gene product.

[0268] Embodiment 72. The composition of embodiment 71, wherein the suicide gene product comprises a protein selected from the group consisting of thymidine kinase and apoptosis signaling proteins.

[0269] Embodiment 73. The composition of any one of embodiments 60 to 72, wherein the gene editing protein is selected from a nuclease, a Transcription Activator-Like Effector Nuclease (TALEN), a RiboSlice, a zinc finger nuclease, a meganuclease, a nickase, a clustered regularly interspaced short palindromic repeats (CRISPR) associated protein or a naturally occurring or recombinant variant, family member, ortholog, fragment or fusion construct thereof.

[0270] Embodiment 74. A composition described in any one of embodiments 2 to 73, wherein the RNA is mRNA.

[0271] Embodiment 75. The composition of embodiment 74, wherein the RNA is a modified mRNA.

[0272] Embodiment 76 The composition of embodiment 75, wherein the modified mRNA comprises one or more non-standard nucleotides.

[0273] Embodiment 77. The composition of embodiment 76, wherein the non-standard nucleotide has one or more substitutions at positions selected from 2C, 4C, and 5C relative to pyrimidines, or 6C, 7N, and 8C relative to purines.

[0274] Embodiment 78. The composition of any one of embodiments 76 or 77, wherein the non-standard nucleotides optionally comprise one or more of 5-hydroxycytidine, 5-methylcytidine, 5-hydroxymethylcytidine, 5-carboxycytidine, 5-formylcytidine, 5-methoxycytidine, pseudouridine, 5-hydroxyuridine, 5-methyluridine, 5-hydroxymethyluridine, 5-carboxyuridine, 5-formyluridine, 5-methoxyuridine, 5-hydroxypseudouridine, 5-methylpseudouridine, 5-hydroxymethylpseudouridine, 5-carboxypseudouridine, 5-formylpseudouridine, and 5-methoxypseudouridine, in an amount of at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90%, or 100% of the non-standard nucleotides.

[0275] Embodiment 79. The composition of any one of embodiments 2 to 78, wherein the RNA comprises a 5' cap structure.

[0276] Embodiment 80. The composition of any one of embodiments 2 to 79, wherein the RNA 5'-UTR comprises a Kozak consensus sequence.

[0277] Embodiment 81. The composition of embodiment 80, wherein the RNA 5'-UTR comprises a sequence that increases RNA stability in vivo, and the 5'-UTR can comprise an alpha globin or beta globin 5'-UTR.

[0278] Embodiment 82. The composition of any one of embodiments 2 to 81, wherein the RNA 3'-UTR comprises a sequence that increases RNA stability in vivo, and the 3'-UTR can comprise an alpha globin or beta globin 3'-UTR.

[0279] Embodiment 83. The composition of any one of embodiments 2 to 82, wherein the RNA comprises a 3' poly(A) tail.

[0280] Embodiment 84. The composition of embodiment 83, wherein the RNA 3' poly(A) tail is from about 20 nucleotides to about 250 nucleotides in length.

[0281] Embodiment 85. The composition of any one of embodiments 2 to 84, wherein the RNA is from about 200 nucleotides to about 5000 nucleotides in length.

[0282] Embodiment 86. The composition of any one of embodiments 2 to 85, wherein the RNA is prepared by in vitro transcription.

[0283] Embodiment 87. The composition of any one of embodiments 1 to 86, wherein the bone marrow cells are macrophages.

[0284] Embodiment 88. The composition of embodiment 87, wherein the macrophages are M1 macrophages or M2 macrophages.

[0285] Embodiment 89. A pharmaceutical composition comprising an isolated NK cell according to any of the preceding embodiments.

[0286] Embodiment 90. A method for producing recombinant immune cells, comprising: a. reprogramming a somatic cell into an iPS cell, said reprogramming comprising contacting said iPS cell with ribonucleic acid (RNA) encoding one or more reprogramming factors; b. disrupting a B2M gene in the iPS cells, the disrupting comprising genetically editing the cells by contacting the cells with RNA encoding one or more gene-editing proteins; c. differentiating the iPS cells into immune cells, d. The method, wherein the immune cell is selected from a lymphoid cell or a myeloid cell.

[0287] Embodiment 91 The method of embodiment 90, wherein the immune cells are NK cells.

[0288] Embodiment 92 The method of embodiment 91, wherein the NK cells are NK-T cells.

[0289] Embodiment 93 The method of embodiment 91 or 92, wherein the NK cells are human cells.

[0290] 94. The method of embodiment 90, wherein the lymphoid cell is a T cell.

[0291] Embodiment 95 The method of embodiment 94, wherein the T cells are gamma-delta T cells.

[0292] Embodiment 96 The method of embodiment 90, wherein the bone marrow cells are macrophages.

[0293] Embodiment 97. The method of embodiment 96, wherein the macrophages are M1 macrophages or M2 macrophages.

[0294] Embodiment 98. The method of any one of embodiments 90 to 97, wherein the somatic cells are fibroblasts or keratinocytes.

[0295] Embodiment 99. The method of any one of embodiments 90 to 98, wherein the method results in an increased proliferation rate of iPS cells compared to the rate of iPS cells without disruption of the B2M gene.

[0296] Embodiment 100. The method of any one of embodiments 90 to 99, wherein the method results in an increased proliferation rate of differentiated cells along lymphoid lineage cells compared to the rate of iPS cells without disruption of the B2M gene.

[0297] Embodiment 101. The method of any one of embodiments 90 to 100, wherein the method results in an increased expansion of differentiated cells along lymphoid lineage cells compared to the rate of iPS cells without disruption of the B2M gene.

[0298] Embodiment 102. The method of any one of embodiments 90 to 101, wherein said differentiation comprises embryoid body-based hematopoietic commitment.

[0299] Embodiment 103. The method of any one of embodiments 90 to 102, wherein said differentiation comprises enrichment of CD34+ cells.

[0300] Embodiment 104. The method of any one of embodiments 90 to 103, wherein the differentiation comprises differentiation into CD5+ / CD7+ general lymphoid progenitor cells.

[0301] Embodiment 105. The method of any one of embodiments 90 to 104, wherein said method results in CD56dim CD16+ NK cells.

[0302] Embodiment 106. The method of any one of embodiments 90 to 105, wherein the RNA is associated with one or more lipids selected from Table A and / or formulas I-XVI.

[0303] Embodiment 107. The method according to any one of embodiments 90 to 106, wherein the immune cells are cells according to any one of embodiments 1 to 86.

[0304] Embodiment 108. A method for treating cancer, comprising: a. obtaining isolated immune cells that contain a genetic disruption in the B2M gene; b. administering the isolated immune cells to a subject in need thereof; c. The method, wherein the immune cell is a lymphoid cell or a CAR myeloid cell.

[0305] Embodiment 109. The method of embodiment 108, wherein the immune cells are T cells, e.g., cytotoxic T cells or gamma-delta T cells; NK cells, e.g., NK-T cells; or macrophages, e.g., M1 macrophages or M2 macrophages, and NK cells.

[0306] Embodiment 110. The method of any one of embodiments 108 or 109, wherein the cancer is a hematological cancer.

[0307] Embodiment 111. The method of any one of embodiments 108 or 109, wherein the cancer is a solid tumor.

[0308] Embodiment 112. The cancer is selected from the group consisting of basal cell carcinoma, biliary tract cancer, bladder cancer, bone cancer, brain and central nervous system cancer, breast cancer, peritoneal cancer, cervical cancer, choriocarcinoma, colon and rectal cancer, connective tissue cancer, digestive system cancer, endometrial cancer, esophageal cancer, eye cancer, head and neck cancer, gastric cancer (including digestive cancer), glioblastoma, hepatocellular carcinoma, liver cancer, intraepithelial neoplasia, renal or kidney cancer, laryngeal cancer, leukemia, liver cancer, lung cancer (e.g., small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma), melanoma, myeloma, neuroblastoma, oral cancer (lips, tongue, mouth, and pharynx), ovarian cancer, pancreatic cancer, prostate cancer, retinoblastoma, and / or thyroid cancer. cystic cyst;rhabdomyosarcoma;rectal cancer;cancer of the respiratory system;salivary gland cancer;sarcoma (e.g., Kaposi's sarcoma);skin cancer;squamous cell carcinoma;gastric cancer;testicular cancer;thyroid cancer;uterine or endometrial cancer;cancer of the urinary system;vulvar cancer;lymphomas, including Hodgkin's lymphoma and non-Hodgkin's lymphoma, and B-cell lymphomas (low-grade / follicular non-Hodgkin's lymphoma (NHL); small lymphocytic (SL) NHL; intermediate-grade / follicular NHL; intermediate-grade diffuse NHL; high-grade immunoblastic NHL; high-grade lymphoblastic NHL; high-grade small non-cleaved cell NHL (high The method of any one of embodiments 108 to 111, wherein the patient is selected from chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); hairy cell leukemia; chronic myeloblastic leukemia; and other carcinomas and sarcomas; and post-transplant lymphoproliferative disorder (PTLD), and the abnormal blood vessel proliferation associated with phakomatosis, edema (e.g., associated with brain tumors), and Meigs syndrome.

[0309] Embodiment 113. The method according to any one of embodiments 108 to 112, wherein the immune cells are cells according to any one of embodiments 1 to 86.

[0310] Embodiment 114. A composition comprising an isolated immune cell comprising a gene edit in the CD16a gene, wherein the immune cell is selected from a lymphoid cell or a myeloid cell.

[0311] Embodiment 115. The composition of embodiment 114, wherein the gene editing transforms the CD16a into a high affinity variant of CD16a.

[0312] Embodiment 116. The composition of embodiment 114 or embodiment 115, wherein the gene editing introduces a phenylalanine to valine substitution at position 158 (F158V).

[0313] Embodiment 117. The composition of embodiment 116, wherein the cells are homozygous or heterozygous for F158V.

[0314] The invention is further illustrated by the following non-limiting examples. EXAMPLES

[0315] The following examples are included for illustrative purposes only and are not intended to limit the scope of the invention.

[0316] Example 1: Cell preparation method and results Figure 1A shows a schematic diagram of the cell production method used in this example. We used mRNA-based cell reprogramming (fibroblast to iPS cell) and gene editing (beta-2-microglobulin (B2M) knockout). Furthermore, the edited cells were differentiated into cytotoxic lymphoid cells. Figure 1B shows the differentiated cytotoxic lymphoid cells that kill cancer cells.

[0317] Fibroblasts were obtained from a human subject and reprogrammed into iPS cells using mRNA-based reprogramming.

[0318] Efficient targeting of defined loci in iPSCs was performed using messenger RNA (mRNA)-encoded gene editing endonucleases that contain a DNA binding domain containing a novel linker region (e.g., a gene editing protein that contains a DNA binding domain with at least one repeat of LTPEQVVAIAS*RVD*GGKQALETVQRLLPVLCQAGHGG (SEQ ID NO: 43, where "*RVD*" corresponds to the dinucleotide "xy" in SEQ ID NO: 3). Exon 3 of B2M, a major component of the MHC class I molecule, was targeted and targeted editing was confirmed in 10 of 12 lines (6 of 12 lines contain the desired biallelic deletion). Gene knockout in iPSCs was confirmed by RT-PCR and immunofluorescence in terms of B2M upregulation after exposure to interferon-γ.

[0319] More specifically, the following beta-2-microglobulin (B2M) genes were targeted:

[0320] [Table 2] And the following primers were used: B2M_Fwd: CAGAGAAAGGCTCTTAAAAATGCAGCGCAATCTCCAG (SEQ ID NO: 45) and B2M_Rev: CACTTAACTATCTTGGGCTGTGACAAAGTCACATGGTTCACAC (SEQ ID NO: 46) and B2M_Rev_RC: GTGTGAACCATGTGACTTTGTCACAGCCCAAGATAGTTAAGTG (SEQ ID NO: 47). See Figure 2.

[0321] In the above sequence, from top to bottom, the sequence is characterized by: Bold: exons. Unmarked: Intron Underlined (single line): gene editing protein binding site on the left. Underlined (double line): gene editing protein binding site on the right. Uppercase letters. Separation region between the gene editing protein binding site and the cleavage region. Underlined (dotted): amplification primer binding site.

[0322] Figure 3 shows successful gene editing. 5 iPSCs were electroporated and plated in conditioned medium onto rhLaminin-521-coated 24-well plates and grown for 48 hours. Cells were passaged onto rhLaminin-521-coated 6-well plates. Cells were cultured for an additional 4 days. Cells were split between genomic DNA extractions to yield 2.0 × 10 4 Cells were seeded into wells of a 6-well plate coated with rhLaminin-521. The length of the B2M amplicon was 587 bp, edited band 1 was 416 bp (see "*"), and edited band 2 was 171 bp (see "*"). Sequencing confirmed a 14 base pair deletion in B2M (see Figure 4).

[0323] FIG. 5 shows RNA levels of B2M with or without IFN-gamma activation ("IFNY"; the left two bars are B2M knockouts and the right two bars are naive cells). 4 iPSCs were plated in conditioned medium on rhLaminin-521 coated 24-well plates and grown for 24 hours. The medium was then replaced with conditioned medium with or without IFN gamma at a concentration of 25ng / mL (t=0). Medium was changed daily until cells were harvested at t=72 hours. RNA was extracted, quantified, and normalized for RT-qPCR analysis. The housekeeping gene was GAPDH and the test gene was B2M.

[0324] We developed a scalable 3D culture system to direct human iPSCs to differentiate into functional NK cells. The process involved a short embryoid body-based hematopoietic commitment step performed either in micropatterned wells or a tailored version of the process in multi-layer culture vessels (StemDiff™ T / NK Cell Kit → StemSpan T / NK Differentiation Kit). Hematopoietic commitment was followed by expansion, enrichment for CD34+ cells, and differentiation into CD5+ / CD7+ general lymphoid progenitors. The process then went through a 14-day NK cell differentiation phase to obtain functional CD56dim / CD16+ NK cells, or a 22-day T cell differentiation phase to obtain CD8+ T cells.

[0325] The table below gives an overview of the cells generated:

[0326] [Table 3]

[0327] The table also shows that B2M knockout cells proliferated robustly compared to wild type. B2M- / - iPSC lines, carrying a 14 bp deletion at the target site (shown in FIG. 4), showed increased proliferation rates both as iPSCs and during differentiation along the lymphoid lineage when compared to wild type iPSC lines.

[0328] Further, the resulting NK cells, as an exemplary differentiated cell type, were characterized for CD16a (UniProtKB P08637 (FCG3A_HUMAN)) and were determined to be heterozygous for G147D dbSNP:rs443082, Y158H dbSNP:rs396716, and F176V dbSNP:rs396991. F176V dbSNP:rs396991 shows high binding capacity for IgG1, IgG3, and IgG4. See FIG. 6. gDNA was amplified with a two-step PCR: Kappa HiFi HotStart (35× / 64° C. extension): primers were F: CTGATCTAGAACTTACTGTGAATCCTTGTCACCTGCCAC (SEQ ID NO: 48), and R: GATAAGAGGAGGCCAGCACGATAGGAACATATGACAC (SEQ ID NO: 49).

[0329] This example shows, inter alia, a scalable 3D process for the differentiation of both wild-type and recombinant iPSCs into functional NK cells as an exemplary differentiated cell type. The 3D process described herein is also useful for the differentiation of both wild-type and recombinant iPSCs into other functional immune cells of lymphoid or myeloid lineage, including, but not limited to, T cells, e.g., cytotoxic T cells or gamma-delta T cells; NK-T cells; and macrophages, e.g., M1 macrophages or M2 macrophages. This process supports the development of next-generation cell therapy for immuno-oncology applications.

[0330] The methods disclosed herein are enhanced when the transfected RNA is associated with one or more lipids selected from Table A and / or Formulas I-XVI.

[0331] Example 2: Cell Characterization Methods and Results The cells of Example 1 were evaluated using phenotypic and functional characterization assays.

[0332] Phenotypic characterization used flow cytometry staining of surface markers, specifically CD56 / CD16 (e.g., gated on CD56). CD56 / NKG2D, CD56 / CD45, CD56 / CD3, CD56 / CD244, CD56 / CD94 / NKG2A, CD56 / NKp46, CD56 / NKp44, CD56 / KIR, CD56 / TRAIL, and CD56 / FASL (e.g., gated on CD56) were also evaluated. See Figures 10A-C and the table below:

[0333] The data in this first table characterize PBMC-isolated NK cells and iPSC-derived NK cells at the first round of suspension:

[0334] [Table 4]

[0335] The data in this second table characterize iPSC-derived NK cells-AggreWell™ and iPSC-derived NK cells in second round suspension.

[0336] [Table 5]

[0337] Exemplary B2M-edited, differentiated cell types, i.e., NK cells, were CD45+, CD56+, CD16-, NKG2D-, KIR2DL4-, KIR2DL1-, and CD8-. See also FIG. 10.

[0338] Functional characterization involved the evaluation of cytotoxicity, activation measurements, as well as cytokine release assays and proliferation assays against an exemplary differentiated cell type, namely NK cells.

[0339] NK cytotoxicity was measured using target cells loaded with calcein AM, a cell-permeable dye used to measure cell viability in most eukaryotic cells. In live cells, non-fluorescent calcein AM is converted to green fluorescent calcein after acetoxymethyl ester hydrolysis by intracellular esterases. Various effector (NK cell): target (K-562 cell) ratios (E:T ratios) were tested and tumor killing was observed. K-562 cells are a cancer cell line derived from a 53-year-old woman with chronic myeloid leukemia (CML) in end-stage blast crisis (more than 30% immature cells in bone marrow (BM), peripheral blood, large concentrations of blasts in BM, or presence of extramedullary infiltration by blasts). These cells are generally used in cytotoxicity assays because they lack the MHC complexes necessary to inhibit NK activity. Experiments also include effector cells incubated with and without a cytokine cocktail (IL-15 and IL-2).

[0340] K-562 cells were loaded with Calcein AM for 1 hour, washed with complete RPMI-1640 containing 10% FBS, and then co-cultured with NK cells. Cells were co-cultured for 18 hours, imaging every 30 minutes using an Operatta high content imager. At the end of the run, the cell suspension was centrifuged and the media was collected. Conditioned media was tested with Luminex MAGPIX to detect and measure the concentrations of IFNg and TNFa. Cells were resuspended and stained for CD56 / CD16, and CD56 / CD1074a.

[0341] Activation was assessed by measuring the activation marker CD107a by flow cytometry using methods described herein and / or known in the art.

[0342] For cytokine release assays, following cytotoxicity measurements, media was collected and assayed for cytokine release (IFNg and TNFa) using methods described herein and / or known in the art.

[0343] For proliferation, cells were incubated in the presence of the activating cytokine IL-15 and the medium was changed every 3 days. Every 3 days, samples were pelleted, washed, re-seeded in fresh medium containing the activating cytokine, and counted to track cell number and viability over time. Cells were tracked for 28 days. During this experiment, medium was saved for cytokine evaluation by Luminex immunopanel.

[0344] Cells remaining from this treatment were seeded into untreated 96-well plates with known cell numbers and imaged, cells were counted, and media was replaced with IL-15 every 3 days for 28 days.

[0345] NK-92 cells are an interleukin 2 (IL-2)-dependent natural killer cell line derived from peripheral blood mononuclear cells (PBMCs) of a 50-year-old Caucasian man with aggressive non-Hodgkin's lymphoma. NK-92 cells are used as control cells for NK cytotoxicity experiments to demonstrate functional killing of tumor cells. Herein, NK-92 cells are used in cytotoxicity assays using calcein AM. Upon engagement of NK cells, they secrete cytokines and histones into the medium. Histones are critically involved in inflammatory and coagulation mechanisms, known as the development of "immunological thrombosis", observable as "clumping" of cells.

[0346] For cytotoxicity assays, activation, and cytokine release plate layouts, samples were run in triplicate; cell mixtures were tested with and without cytokine cocktail (IL-15+IL-2); PBMC isolated NK cells from a single donor were used as control; PBMC isolated NK cells and 3D B2M- / - NK cells (generated by the disclosed method) were tested at two different E:T ratios (20,000 NK cells vs. 20,000 K-562 cells (1:1 E:T ratio) and 30,000 NK cells vs. 20,000 K-562 cells (3:1 E:T ratio)); 2D wild type and 2D B2M- / - NK cells (generated by the disclosed method) were tested at 1:1 E:T ratio; and 3D wild type was not tested and plated for expansion.

[0347] The NK cytotoxicity assay is shown in Figures 7A-B and 8A-B (time course is 5 frames per second). 5 frames corresponds to 2.5 hours. Note that for orientation, K-562 cells are larger in the figures here than NK cells. Figures 7A-B show PBMC-isolated NK cells (i.e., control cells) co-cultured with K-562 (3:1 E:T) without cytokine cocktail at 0 and 18 hours. Clumping of K-562 cells due to NK cell attack was observed, indicating that the assay was performing as expected. Figures 8A-B show 3D B2M- / - NK cells co-cultured with K-562 (3:1 E:T) without cytokine cocktail. Clumping of K-562 cells is observed as NK cell attack and at levels surprisingly much higher than those of PMBC-isolated NK cells (compare Figures 7B and 8B: note more clumping and less unengaged NK cells).

[0348] Results of the cytokine release assay using Luminex MAGPIX are shown in Figures 9A-9H. Unless otherwise indicated (i.e., "+IL2, IL15"), conditions were without the addition of IL-2 or IL-15. Additionally, cell ratios are shown (1:1, or 3:1). As elsewhere herein, wild-type PBMC-derived NK are control NK cells. Figure 9A shows interferon gamma. Figure 9B shows IL-2. Figure 9C shows IL-7. Figure 9D shows IL-13. Figure 9E shows MIP-1a. Figure 9F shows MIP-1b. Figure 9G shows TNFa. Figure 9H shows GM-CSF.

[0349] In summary, but without limitation, the data herein demonstrate the generation of B2M knockout immune cells that are self-activating rather than self-killing (even in the absence of cytokines such as IL-2 and IL-15). Furthermore, these B2M knockout cells are capable of killing tumor cells (even in the absence of cytokines such as IL-2 and IL-15) and have unexpected expansion and growth properties.

[0350] Example 3: Insertion of B2M-HLA-E In this example, a repair template (B2M-HLA-E repair template) containing B2M coding sequence and HLA-E (major histocompatibility complex class I, E) coding sequence was inserted into beta-2-microglobulin (B2M) editing, where iPSCs with edited B2M gene disclosed herein are contacted with a repair template containing coding sequence for HLA-E. Alternatively, unedited iPSCs are contacted with gene editing components to edit the B2M gene together with the repair template containing coding sequence for HLA-E. In both cases, the resulting cells (either in iPSCs or upon differentiation into immune cells of lymphoid or myeloid lineage) will have the edited B2M gene and will express HLA-E where it was altered.

[0351] As shown in Figure 11A, the B2M signal peptide sequence (B2M_sp), contained entirely within exon 1 of B2M, is included in the B2M-HLA-E repair template. Without wishing to be bound by theory, editing exon 1 of B2M to include the entire B2M CDS provides the most direct route to generating the fusion.

[0352] The ideal insertion of the B2M-HLA-E repair template is located at the exon 1-intron 1 boundary of B2M, as shown in Figure 11B. Additional binding sites, including solid lines 1 / 1 and 2 / 2, that have been gene-edited to incorporate the B2M-HLA-E repair template into their genome are shown in Figure 11C.

[0353] Using the method disclosed herein, cells are genetically edited to insert a repair template into the genome. Figure 11D shows a gel with the size of two lines with B2M-HLA-E repair template (about 1.5 kb) that inserts the repair template into the genome at 1 / 1 and 2 / 2 positions in Figure 11C. In this example, mesenchymal stem cell (MSC) cells are genetically edited. Figure 11E shows the intensity of the signal from the band shown in Figure 11D and its ratio.

[0354] Using the method disclosed herein, cells are genetically edited and repair template is inserted into genome. Figure 11F shows the size gel of one line with B2M-HLA-E repair template (about 1.5kb), which inserts repair template into genome at 2 / 2 position of Figure 11C. In this example, iPSCs are genetically edited. Figure 11G shows the intensity of the signal from the band shown in Figure 11F and its ratio.

[0355] FIG. 11H shows the relevant sequences in the B2M-HLA-E repair template.

[0356] In particular, other cells, e.g., differentiated cells as described herein, can be gene edited to insert a repair template that includes a coding sequence of interest, e.g., an HLA-E coding sequence.

[0357] According to the method of this example, the repair template causes the cell to express B2M, e.g., as a fusion protein, with HLA-E, which is required to make B2M functional. Thus, the method "stealths" the cell by disrupting the native endogenous B2M gene and preventing other HLA from becoming functional.

[0358] The methods disclosed herein are enhanced when the transfected RNA is associated with one or more lipids selected from Table A and / or Formulas I-XVI.

[0359] Example 4: Insertion of high affinity CD16a In this example, C16a is edited and replaced with the coding sequence of a high affinity CD16a variant. As shown in Figures 12A and 12B, the phenylalanine (F) at position 158 of CD16a is targeted by gene editing to replace F with valine (V). The relevant sequences are shown in these figures.

[0360] As shown in FIG. 12B, the NheI site does not appear in the amplicon for CD16a, so the presence of a band at approximately 2127 / 912 bp after NheI digestion indicates successful correction by CD16_NheI_ssODN_81 and CD16_NheI_ssODN_81_PT.

[0361] The methods disclosed herein are enhanced when the transfected RNA is associated with one or more lipids selected from Table A and / or Formulas I-XVI.

[0362] Example 5: Gene Editing and Differentiation Cells are engineered with a Chimeric Antigen Receptor (CAR) In this example, immune cells of the present disclosure, e.g., T cells or NK cells, generated by the methods of the present disclosure (e.g., by disrupting the B2M gene by gene editing and differentiating cells from stem cells into immune cells of lymphoid or myeloid lineage), are genetically engineered to express a recombinant chimeric antigen receptor (CAR). A CAR comprises an intracellular signaling domain, a transmembrane domain, and an extracellular domain that comprises an antigen binding region. In embodiments, the immune cells of the present disclosure are engineered to target ROR1 and / or CD19. Methods for genetically engineering cells to express recombinant CARs are well known in the art, and any such well-known methods can be used with the immune cells of the present disclosure.

[0363] In some cases, the intracellular signaling domain of the CAR comprises at least one immunoreceptor tyrosine-based activation motif (ITAM)-containing domain.

[0364] In some cases, the intracellular signaling domain of the CAR is derived from one of CD3-zeta, CD28, CD27, CD134 (OX40), and CD137 (4-1BB).

[0365] In some cases, the transmembrane domain of the CAR is derived from one of CD28 or CD8.

[0366] In some cases, the antigen binding region binds one antigen. In embodiments, the binding region binds two antigens.

[0367] In some cases, the extracellular domain comprising the antigen-binding region comprises (a) a natural ligand or receptor, or a fragment thereof, or (b) an immunoglobulin domain, optionally a single chain variable fragment (scFv). In embodiments, the extracellular domain comprising the antigen-binding region comprises two of (a) a natural ligand or receptor, or a fragment thereof, or (b) an immunoglobulin domain, optionally a single chain variable fragment (scFv). In embodiments, the extracellular domain comprising the antigen-binding region comprises one each of (a) a natural ligand or receptor, or a fragment thereof, and (b) an immunoglobulin domain, optionally a single chain variable fragment (scFv).

[0368] In some cases, the antigen-binding region comprises one or more of CD94 / NKG2a, which optionally binds to HLA-E on tumor cells; CD96, which optionally binds to CD155 on tumor cells; TIGIT, which optionally binds to CD155 or CD112 on tumor cells; DNAM-1, which optionally binds to CD155 or CD112 on tumor cells; KIR, which optionally binds to HLA class I on tumor cells; NKG2D, which optionally binds to NKG2D-L on tumor cells; CD16a, which optionally binds to an antibody / antigen complex on tumor cells, and / or where CD16a is optionally a high affinity variant, optionally homozygous or heterozygous for F158V; NKp30, which optionally binds to B7-H6 on tumor cells; NKp44; and NKp46.

[0369] Example 6: Cancer Treatment In this example, the immune cells of the disclosure are used to treat cancer.

[0370] The method of treating cancer includes obtaining an isolated immune cell that contains a genetic disruption in the B2M gene, and administering the isolated immune cell to a subject in need thereof.In this method, the immune cell is a lymphoid lineage or a myeloid cell.Optionally, the immune cell is a T cell, such as a cytotoxic T cell or a gamma-delta T cell; a NK cell, such as a NK-T cell; or a macrophage, such as an M1 macrophage or an M2 macrophage, and a NK cell.

[0371] In some cases, in addition or alternatively, the immune cells express the high affinity CD16a receptor.

[0372] In some cases, the immune cells express a fusion protein that includes a B2M polypeptide and HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, and HLA-G polypeptides. The fusion protein can be expressed by inserting a repair template into a single- or double-stranded break in the B2M gene; in some cases, the repair template includes coding sequences for B2M and HLA genes. In particular, the fusion protein replaces the endogenous B2M and HLA pair expressed by the immune cells, making the immune cells less likely to be reduced or eliminated by the host's immune cells.

[0373] In some cases, the immune cells are further genetically modified to express a chimeric antigen receptor (CAR).

[0374] The cancer may be a hematological cancer.

[0375] The cancer may be a solid tumor.

[0376] Cancers include: basal cell carcinoma, biliary tract cancer; bladder cancer; bone cancer; brain and central nervous system cancer; breast cancer; peritoneal cancer; cervical cancer; choriocarcinoma; colon and rectal cancer; connective tissue cancer; digestive system cancer; endometrial cancer; esophageal cancer; eye cancer; head and neck cancer; gastric cancer (including digestive cancer); glioblastoma; hepatocellular carcinoma; liver cancer; intraepithelial neoplasia; kidney or renal cancer; laryngeal cancer; leukemia; liver cancer; lung cancer (e.g., small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma); melanoma; myeloma; neuroblastoma; oral cancer (lip, tongue, mouth, and pharynx); ovarian cancer; pancreatic cancer; prostate cancer; retinoblastoma; rhabdomyosarcoma. Myosarcoma;rectal cancer;cancer of the respiratory system;salivary gland cancer;sarcoma (e.g., Kaposi's sarcoma);skin cancer;squamous cell carcinoma;gastric cancer;testicular cancer;thyroid cancer;uterine or endometrial cancer;cancer of the urinary system;vulvar cancer;lymphomas, including Hodgkin's lymphoma and non-Hodgkin's lymphoma, and B-cell lymphomas (low-grade / follicular non-Hodgkin's lymphoma (NHL); small lymphocytic (SL) NHL; intermediate-grade / follicular NHL; intermediate-grade diffuse NHL; high-grade immunoblastic NHL; high-grade lymphoblastic NHL; high-grade small non-cleaved cell NHL (high and Waldenstrom's macroglobulinemia), chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); hairy cell leukemia; chronic myeloblastic leukemia; and other carcinomas and sarcomas; and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal blood vessel proliferation associated with phakomatosis, edema (e.g., associated with brain tumors), and Meigs' syndrome.

[0377] Equivalent While the invention has been described in relation to specific embodiments thereof, it is understood that further modifications are possible, and this application is intended to cover any variations, uses, or adaptations of the invention in accordance with the principles of the invention in general, including departures from the present disclosure as come within known or customary practice in the art to which this invention pertains, as well as departures from the present disclosure as may be applied to the essential features described herein and as fall within the scope of the appended claims.

[0378] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments specifically described herein which equivalents are intended to be encompassed within the scope of the following claims.

[0379] Incorporation by Reference All patents and publications referenced herein are incorporated by reference in their entirety.

[0380] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein should be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention.

[0381] As used herein, all headings are for organizational purposes only and are not intended to limit the disclosure in any way. The contents of any individual section may apply equally to all sections.

Claims

1. A method for producing a recombinant immune cell, comprising: a. reprogramming a somatic cell into an iPS cell, said reprogramming comprising contacting said somatic cell with ribonucleic acid (RNA) encoding one or more reprogramming factors; b. disrupting a B2M gene in the iPS cell to generate a gene-edited iPS cell, the disrupting comprising contacting the iPS cell with RNA encoding one or more gene-editing proteins to gene-edit the iPS cell; c. Differentiating the gene-edited iPS cells into recombinant immune cells, wherein the recombinant immune cells are myeloid or lymphoid cells; The method.

2. The method of claim 1 , wherein the lymphoid cell is a NK cell.

3. The method further comprises: ｄｉｍ The method of claim 2, which results in CD16+ NK cells.

4. The method of claim 2 or 3, wherein the NK cells have reduced or eliminated NK cell fratricide compared to corresponding NK cells not involving disruption of the B2M gene.

5. The method of claim 1, wherein the lymphoid cell is a T cell.

6. A method according to any one of claims 1 to 5, wherein the somatic cells are fibroblasts or keratinocytes.

7. The method of claim 1, wherein the method results in an increased proliferation rate of the gene-edited iPS cells compared to the proliferation rate of corresponding iPS cells without disruption of the B2M gene.

8. A method according to any one of claims 1 to 7, wherein the method results in an increased proliferation rate of differentiated cells along the lymphoid lineage compared to the proliferation rate of differentiated cells generated from corresponding iPS cells without disruption of the B2M gene.

9. 9. The method of any one of claims 1 to 8, wherein the RNA is associated with one or more lipids selected from Table A and / or formulas I-XVI.

10. A method according to any one of claims 1 to 9, wherein the RNA is mRNA.

11. The method described in claim 10, wherein the mRNA is a modified mRNA.

12. The method described in claim 11, wherein the modified mRNA contains one or more non-standard nucleotides.

13. The method of claim 12, wherein the one or more non-standard nucleotides have one or more substitutions at positions selected from 2C, 4C, and 5C for pyrimidines, or 6C, 7N, and 8C for purines.

14. The method of claim 12 or 13, wherein the one or more non-standard nucleotides optionally comprise one or more of 5-hydroxycytidine, 5-methylcytidine, 5-hydroxymethylcytidine, 5-carboxycytidine, 5-formylcytidine, 5-methoxycytidine, pseudouridine, 5-hydroxyuridine, 5-methyluridine, 5-hydroxymethyluridine, 5-carboxyuridine, 5-formyluridine, 5-methoxyuridine, 5-hydroxypseudouridine, 5-methylpseudouridine, 5-hydroxymethylpseudouridine, 5-carboxypseudouridine, 5-formylpseudouridine, and 5-methoxypseudouridine in an amount of at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90%, or 100% of the non-standard nucleotides.

15. A method according to any one of claims 1 to 14, wherein the differentiation includes embryoid body-based hematopoietic commitment.

16. The method of claim 1, wherein the differentiation comprises enrichment of CD34 + cells.

17. The method of claim 1, wherein the differentiation comprises differentiation of the gene-edited iPS cells into CD5+ / CD7+ general lymphoid progenitor cells.

18. The method of any one of claims 1 to 17, wherein the one or more gene editing proteins are selected from a nuclease, a transcription activator-like (TAL) effector nuclease, a RiboSlice, a zinc finger nuclease, a meganuclease, a nickase, a clustered regularly interspaced short palindromic repeats (CRISPR) associated protein or a natural variant, recombinant variant, family member, ortholog, fragment or fusion construct thereof.

19. The method of any one of claims 1 to 18, wherein the recombinant immune cell expresses a fusion protein comprising a B2M polypeptide and an HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, or HLA-G polypeptide.

20. The method of claim 19, wherein the fusion protein is expressed by inserting a repair template into a single-stranded or double-stranded break in the B2M gene, the repair template comprising a coding sequence for the fusion protein.

21. The method of any one of claims 1 to 20, wherein the recombinant immune cells are genetically engineered to express a recombinant chimeric antigen receptor (CAR) comprising an intracellular signaling domain, a transmembrane domain, and an extracellular domain comprising an antigen-binding region.

22. The method of any one of claims 1 to 21, wherein the method comprises genetically editing the recombinant immune cells to increase expression and / or activity of a cytokine selected from the group consisting of interleukin (IL)-2, IL-7, IL-12, IL-15, and any combination thereof.

23. The method of any one of claims 2 to 22, wherein the NK cells secrete one or more cytokines selected from the group consisting of IL-2, IL-7, IL-10, and any combination thereof.

24. A pharmaceutical composition comprising: (a) a recombinant immune cell produced by the method of any one of claims 1 to 23; and (b) a pharma- ceutically acceptable excipient.

25. Use of the pharmaceutical composition of claim 24 in the manufacture of a medicament for treating cancer.

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